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Teenage and Young Adult Cancer: research priorities
Research Priorities for Teenage and Young Adult Cancer
Final report of the James Lind Alliance Teenage and Young Adult Cancer Priority Setting Partnership (TYA
PSP)
Executive Summary
The Top 10 research priorities for teenage and young adult cancers as agreed at the final workshop of
the Teenage and Young Adult Cancer Priority Setting Partnership are detailed here. Following the
national consultation, fourteen professionals, seven patients, and four parents prioritized the final 30
questions identified as unanswered research questions by young people, carers, significant others, and
professionals. During the workshop, questions were considered, debated, and deliberated throughout
the day as the final list was named. The final questions reflect the breadth of the cancer experience for
young people, from early diagnosis through to targeted treatments based on the biology of the disease;
access to clinical trials, psychosocial care, and support after treatment; the impact of cancer on the
wider support networks for young people such as parents and siblings; and also recognize that not all
cancers in young people are curable.
Top 10 research priorities
1 What psychological support package improves psychological well-being, social functioning and mental
health during and after treatment?
2 What interventions, including self-care, can reduce or reverse adverse short and long-term effects of
cancer treatment?
3 What are the best strategies to improve access to clinical trials?
4 What General Practitioner or young person strategies, such as awareness campaigns and education,
improve early diagnosis for young people with suspected cancer?
5 What are the best ways of supporting a young person who has incurable cancer?
6 What are the most effective strategies to ensure that young people who are treated outside of a
young person’s Principal Treatment Centre receive appropriate practical and emotional support?
7 What interventions are most effective in supporting young people when returning to education or
work?
8 How can parents/carers/siblings/partners be best supported following the death of a young person
with cancer?
9 What is the best method of follow-up and timing which causes the least psychological and physical
harm, while ensuring relapse/complications are detected early?
10 What targeted treatments are effective and have fewer short and long term side-effects?
Foreword
Our philosophy is clear – young people with cancer should always be treated as young people first,
cancer patient second. Teenage Cancer Trust is naturally delighted at how this partnership has given the
opportunity to ensure that young people’s priorities for research are heard loud and clear. Organizations
like ours and decision-makers across the country must always make sure that young people’s voices are
front and center as we strive to improve their lives and experiences of care. Young people here are
saying that effective psychological support is top of the list. Every single one of these top ten areas is
vital, but it is increasingly clear that young people are wanting more support to handle the devastating,
long-lasting, and wide-ranging psychological impact that can be felt long after treatment. We know that
Teenage Cancer Trust Nurses and Youth Support Coordinators play a vital role in improving
psychological well-being during and after treatment – but right now they can’t reach everyone. Going
forward we will be working with researchers to develop evidence of the impact of this support and
make sure we can in helping every young person get back on track after cancer.
Kate Collins Chief Executive
Being part of the James Lind Alliance Teenage and Young Adult Cancer Priority Setting Partnership (TYA
PSP) and helping to identify the top 10 research priorities for teenage and young adult cancers has been
a privilege for CLIC Sargent. Developing quality research and knowledge on the issues important to
young cancer patients and their families is core to CLIC Sargent and what we do. The top 10 research
priorities identified by young people, parents, and professionals will provide valuable insight into how to
shape the future evidence base on teenage and young adult cancers and make changes for young cancer
patients and their families.
Kate Lee Chief Executive Officer
Children with Cancer UK is delighted to have been involved in the James Lind Alliance Teenage and
Young Adult Cancer Priority Setting Partnership (TYA PSP). We know very well through our own work the
importance of being responsive to the needs and priorities of young people and their families. At
Children with Cancer UK, we have a broad remit to investigate the cause, treatment, prevention, and
survival of cancer in children and young people. We hope that the top 10 research priorities established
by the PSP will be used to shape future policy and research, for the benefit of all young people living
with cancer.
Denis Henshaw Scientific Director
…‘I think it speaks volumes we have psychological support as our top research priority. Physical health is
so important but the impact mental health issues can have is monumental so I’m delighted we agreed
this is our utmost priority. Having patient input in this process is fundamental - we have a different
perspective of what works and what doesn’t and where there’s room for research and improvements to
be made. Having a priority that names ways to improve GPs’ knowledge of TYA cancer is essential in
empowering young people to feel able to attend their GPs if they are noticing changes in their body.’
Table of Contents
List of Figures 5
Abbreviations 5
Background 6
Management and scope 7-8
Establishing the partnership 7
Steering group 7
The Scope 8
Partners 8
Process 9-23
Stage 1 - Gathering the questions 9-12
l First survey results 9
Stage 2 - Refining the questions 13-14
l Organising the questions 13
l Removing out of scope questions 13
l Formatting questions 14
l Searching for evidence 14
Stage 3 - Prioritising the questions 15-19
l Interim survey 15
Stage 4 - Agreeing the Top 10 20-23
l Decision making: prioritising the Top 10 20
TOP 10 Research Priorites 23
The remaining 20 priorities 24
Next steps 25
For research funders 25
For researchers 25
For Charities 25
For the TYA PSP 25
Acknowledgements 25
Reference 25
Appendix 1: Partners 26
Appendix 2: Overview of TYA PSP methodology and results 27
Appendix 3: Initial survey 28-32

Appendix 4: Answered questions 33
Appendix 5: Ongoing studies 34-35
Appendix 6: Unanswered questions included in the interim survey 36-40
Appendix 7: Unanswered questions not included in the interim survey 41-43
List of Figures
Figure 1 Gender distribution of respondents (round 1) 9
Figure 2 Ethnicity of respondents (round 1) 10
Figure 3 Age, in years, of respondents (round 1) 10
Figure 4 Geographical distribution of respondents (round 1) 11
Figure 5 Cancer distribution in young people, carers, and significant
others (round 1)
11
Figure 6 Cancer timeline of respondents (round 1) 12
Figure 7 Distribution of carers/significant others (round 1) 12
Figure 8 Distribution of professionals (round 1) 12
Figure 9 Gender distribution of respondents (round 2) 16
Figure 10 Ethnicity of respondents (round 2) 16
Figure 11 Age of respondents (round 2) 17
Figure 12 Geographical distribution of respondents (round 2) 17
Figure 13 Cancer distribution in young people, carers, and significant
others (round 2)
18
Figure 14 Cancer timeline of respondents (round 2) 18
Figure 15 Distribution of carers/significant others (round 2) 19
Figure 16 Distribution of professionals (round 2) 19
Abbreviations
GP General Practitioner
JLA James Lind Alliance
NCRI National Cancer Research Institute

PSP Priority Setting Partnership
TYA Teenage and Young Adult
TYAC Teenage and Young Adults with Cancer Professional Organisation
Background
The Teenage and Young Adult Cancer Priority Setting Partnership (TYA PSP) was established in 2014 and
emerged from the National Cancer Research Institute’s (NCRI) Teenage and Young Adult Health Services
Research Subgroup and the Teenagers and Young Adults with Cancer (TYAC) Research and Registration
Group, two independent but linked research groups. The research agenda for young people with cancer,
broadly those aged 13-24 years at diagnosis, has typically been set by professionals caring for young
people with cancer and researchers. In the United Kingdom, we have previously asked young people to
prioritize research themes identified by professionals as key areas for research however, we had not
explored working with young people and carers/significant others in an equal partnership to undertake
a research priority-setting exercise in this field. This is an important step, as there is increasing evidence
that research questions and outcomes identified by professionals as important may not be the same as
those experiencing the disease, particularly for young people with cancer who present with several rare
diseases during a time of enormous physical and psychological growth (Chalmers et al. 2013). Working
with the James Lind Alliance (JLA), using an established methodology, we aimed to address this
imbalance to generate a list of the Top 10 research priorities for young people with cancer agreed upon
by multiple stakeholders affected by the disease. Employing rigorous methodology we have been able to
bring patients, carers/significant others and professionals together to prioritise unanswered research
questions in teenage and young adult cancer research.
Management and scope
Establishing the partnership
The project was funded by Teenage Cancer Trust, CLIC Sargent, and Children with Cancer UK, with
support from the University of Surrey and the NCRI. The coordinating team oversaw and directed the
project to completion.
In the first instance, we needed to identify the range of professionals involved in the treatment and care
of young people with cancer. We aimed to reflect the breadth of professionals involved in the
multidisciplinary care of young people in the steering group. We also contacted several specialist
teenage and young adult cancer treatment centers and relevant charity partners asking for patient
representatives. Due to the time commitment required to be part of the steering group, establishing
one with the right mix of patients and professionals took longer than initially planned. We had aimed to
have a GP (General Practitioner) as part of the group; a GP joined the start but unfortunately due to
retirement was unable to continue their involvement and we were unable to recruit another GP to join
us in the time given.
Several young people who were involved at the start of the project left midway. This is expected for
projects which are carried out over several years, as young people will leave as they transition back to
their previous lives before their cancer diagnosis and move for education or employment purposes.
Three young people remained with the project for the duration and they were joined by two who
replaced those who had left and remained to project completion.
Steering group
The project was managed by a steering group, led by an independent chair from the JLA, Sheela
Upadhyaya. The steering group included young people with a previous cancer diagnosis, healthcare and
allied health professionals involved in treatment delivery as well psychosocial support. Two of the
funding partners were part of the steering group but were not involved in the final prioritization
exercise. The steering group approved the aims and objectives of the process, approved all decisions
relating to the project, tested and ensured the surveys were accessible to a wide range of people, and
provided expert opinions on the evidence checking.
Patient representatives:
Amy Callaghan Glasgow
Leila Hamrang Manchester
Demi McGeachy Glasgow
Lara Veitch London
Max Williamson London
The Partnership and the priority setting process coordinating team:
Sheela Upadhyaya (JLA Adviser)
Susie Aldiss (Research Fellow)
Lorna Fern (NCRI TYA CSG Teenage and Young Adult Researcher and Patient/Public Involvement Lead)
Faith Gibson (Professor of Child Health and Cancer Care)
Steering Group professional representatives:
Bob Phillips (Clinical Lead of the TYA PSP; Paediatric Oncology Consultant)
Karen Dyker (Consultant Clinical Oncologist)
Mike Groszmann (Consultant Child and Adolescent Psychiatrist)
Rachael Hough (Consultant Haematologist)
Sue Morgan MBE (Teenage Cancer Trust Nurse Consultant)
Sam Smith (Head of Nursing and Clinical Services, Teenage Cancer Trust)
Helen Veitch (Head of Youth Support Coordinators, Teenage Cancer Trust)
Helen Gravestock (Head of Research, Policy, and Campaigning, CLIC Sargent)
Jeremy Whelan (Consultant Medical Oncologist)

Management and scope
The Scope
The treatment and care of young people with cancer is complex, Young people present with a range of
cancer types occurring during a period of unique physical and psychological growth, superimposed on
the social and cultural dimensions of teenage and young adult development. This is further complicated
by an extended network of significant others, such as parents, siblings, partners, and peers.
Consequently, the scope of the project was kept broad allowing participants to ask questions about
prevention, causes of cancer, diagnosis, treatment, care, follow-up, survivorship, relapse, and end-of-life
care.
The final aim of the TYA PSP was:
‘To identify gaps and unanswered questions in research, the answers to which may reduce the individual
and societal burden of young peoples cancer.’
Partners
The steering group identified potential partner organizations. This was achieved through peer
knowledge, consultations, and steering group member networks. Potential partners were contacted
about the TYA PSP and asked to join the partnership and contribute to disseminating the surveys and
results through their contacts and networks, the partner list can be seen in Appendix 1.
Process
The TYA PSP followed the methodology described in the JLA Guidebook – http://www.jla.nihr.ac.uk/jla-
guidebook/. The full protocol is available here http://www.jla.nihr.ac.uk/priority-setting-
partnerships/teenage-and-young-adult-cancer/. An overview of the process is shown in Appendix 2.
Stage 1: Gathering the questions
Questions were gathered in an online public survey (Appendix 3) which was launched in October 2016
and remained open until 31st December 2016.
The following groups of people were invited to participate:
• People diagnosed or treated for cancer between the ages of 13 and 24 years old;
• Relatives/friends/partners/carers of someone who has been diagnosed or treated for cancer between
the ages of 13 and 24 years old;
• Professionals working with teenagers/young adults with cancer. The survey was built using Bristol
Online Survey software. The wording and design of the survey were piloted with five young people, nine
parents, and five professionals outside the steering group and adapted to incorporate their feedback.
On the day of the survey launch, a press release was issued and the partner organizations which had
previously agreed to help to disseminate the survey were notified that the survey was open. Some of
these partner organizations added a link to the survey on their website, mentioned the project in their
newsletter, or sent an email to their members to alert them. The survey was promoted at conferences,
including a conference for young people with cancer. Social media was used throughout the three
months to promote the survey, including a bespoke Twitter handle @TYAPSP.
Respondents were invited to submit up to five questions about any aspect of teenage and young adult
cancer they considered to be unanswered. Basic demographic data were requested and a box was
provided for any additional comments respondents wished to add.
First survey results
A total of 292 respondents answered the first survey submitting 855 potentially unanswered questions.
The proportion of respondents was similar across the three groups, patients (n=108; 36%), carers
(n=101; 34%), and professionals (n=83; 30%). Across all groups, more females responded than males, as
is often typical with survey research (Figure 1).
Figure 1: Gender distribution of respondents (round 1)
Process - Stage 1: Gathering the questions
Figure 2: Ethnicity of respondents (round 1)
The majority of respondents across all groups described themselves as white, in particular
parents/carers (Figure 2). For young people, the proportion of respondents from other ethnic groups
was slightly less than the incidence cases reported by Public Health England (PHE), where approximately
84% of cancers occur in white European people compared to around 90% of respondents answering the
survey (PHE Data: personal communication).
Figure 3: Age, in years, of respondents (round 1)
The majority of young people answering the survey were aged 19-24 years, followed by those aged 25-
34 years, and for parents/relatives/friends/partners the majority were aged 45-54 years, followed by 55-
64 years, most professionals were aged 35-54 years (Figure 3)
Figure 4: Geographical distribution of respondents (round 1)
The geographical distribution of respondents was broadly similar to the proportion of incidence cases
from the four devolved nations in the United Kingdom (Figure 4): the majority of young people are
diagnosed in England, followed by Scotland, Wales, and Northern Ireland.
Figure 5: Cancer distribution in young people, carers, and significant others (round 1)
The representativeness of cancer types in young people responding was broadly similar to those
occurring in the 13-24 age group (Figure 5). For young people, most of the respondents had been
diagnosed with Hodgkin’s Disease (27%); Hodgkin’s Disease represents around a third of all newly
diagnosed cases in this age group. This was followed by leukemia (17%) and bone tumors (9%).
Testicular cancers were underrepresented with only 4% of respondents despite this being the most
common cancer in young males with around 27% of incidence cases
(http://www.cancerresearchuk.org/healthprofessional/cancer-statistics/teenagersand-young-adults-
cancers/incidence). The reasons for this may be two-fold, a) lower male participation in survey research
in general and b) location of care for patients with testicular cancer as many do not receive their care in
a specialist TYA center and therefore may not be linked into the TYA networks the survey was circulated
through.
Figure 6: Cancer timeline of respondents (round 1)
The cancer types of carers/significant others were also similar to the distribution of cancers expected.
With the notable exception of brain tumors which were overrepresented in the carer/significant other
cohort and under-represented in the young people’s group; this may be due to the lower survival rates
of this group. The young people on the steering group identified several timeline points that young
people would consider themselves to be on (Figure 6). Respondents could select more than one timeline
point. The majority of young people, 64%, described themselves as ‘survivor/follow up care’. Just under
one-third of carers/significant others were bereaved. Differences were observed for those who
described themselves as ‘on treatment’, only 5% of young people said they were on treatment
compared to 19% of carers/significant others. We know from other research that during the treatment
phase young people will approach their treatment team for answers to questions and also, it is when
nearing the end of treatment that young people start to consider additional questions.
Figure 7: Distribution of carers/significant others (round 1)
Figure 8: Distribution of professionals (round 1)
The composition of the carer/significant other group can be seen in Figure 7 with the majority of
respondents describing themselves as a parent/carer, and around 10% describing themselves as a
relative or friend; a smaller proportion of partners responded (<5%). A broad range of professionals
responded to the first survey, as would be expected considering the multidisciplinary care of young
people with cancer. Figure 8 illustrates the distribution between medical, nursing, allied health
professionals, and ‘other’. ‘Other' included several professionals such as people from the third sector
and academic researchers.
Process
Stage 2: Refining the questions
All the submitted questions were extracted from Bristol Online Survey into an Excel spreadsheet.
Multiple questions written in the same box were separated. The comments sections were checked for
further questions. In total, 855 questions were submitted.
Organizing the questions
Initial coding of the questions was carried out by coordinating team members LF, SA, and FG using the
International Cancer Research Partnership Common Scientific Outline ( CSO,
https://www.icrpartnership.org/cso ): this served two purposes. Firstly, we were able to group the
questions into themes to make them easier to review and discuss. Secondly, we will be able to compare
research funding allocation to the priorities identified by young people, carers, and healthcare
professionals. The CSO is an internationally recognized classification system organized around six broad
areas of scientific interest in cancer research:
CSO1: Biology;
CSO2: Etiology;
CSO3: Prevention;
CSO4: Early diagnosis, detection, and prognosis;
CSO5: Treatment;
CSO6: Cancer control, survivorship, outcomes.
The six main categories contain numbered sub-categories and these sub-categories all have several
points within them. The bullet points were numbered and used as sub-codes. For example:
CSO6: Cancer control, survivorship, outcomes
CSO6.1 Patient Care and Survivorship Issues
1. Research into patient-centered outcomes
2. Quality of life When the question being coded did not fit into an already existing sub-code, additional
sub-codes were created; this was mainly for questions specific to young people such as getting back to
education, or the role of youth support coordinators. Each question in the spreadsheet was assessed by
the three coordinating team members and coded one by one. Sometimes the bullet points (sub-codes)
were not used and coding was at the sub-category level. This was for questions that seemed to fit all the
sub-codes in that particular category. For example, ‘Why did a healthy young adult get cancer?’ was
coded as:
CSO2: Etiology
CSO2.1 Exogenous Factors in the Origin and Cause of Cancer
CSO2.2 Endogenous Factors in the Origin and Cause of Cancer
CSO2.3 Interactions of Genes and/or Genetic Polymorphisms with Exogenous and/or Endogenous
Factors.
As demonstrated by the above example, some questions were coded in more than one sub-category or
category. Differences in opinion about which codes to use were resolved by discussion between FG, LF,
and SA. When consensus could not be reached, the question was added to a list of queries to be
discussed by the steering group. Once all the questions had been coded, questions in the same category
were grouped together and categories separated into different tabs within the Excel spreadsheet to
assist with data management.
Removing out of scope questions
During the coding, LF, SA and FG identified questions that were potentially ‘out of scope’. Questions
were out of scope if they were deemed not to be reducing the individual and societal burden of young
peoples’ cancer or could not be answered by research. The steering group agreed the following criteria
to identify out-of-scope questions, an example is given under each criterion:
1. It did not fit the scope of reducing the individual and societal burden of young peoples’ cancer or
could not be answered by research.
Can a cancer sufferer become an organ donor?

2. It was a statement rather than a question (and no specific question could be identified from the
statement).
Can the late effects Drs stop telling us how BAD outcomes are and focus just a little on some of the
POSITIVE outcomes
3. The question was ambiguous, was interpreted in different ways by steering group members and the
meaning could not be resolved following discussion.
Supportive care
4. The focus was on research methods rather than a research topic.
How should we collect information about the late-onset side effects of cancer treatment in TYA?
5. The question related to a specific person’s situation/issue.
Who can I talk to about my worries for my child?
6. It was a political statement.
Should medical professionals routinely explain to patients that there may be more up-to-date
treatments available in other parts of the world, which may increase the patient’s chances of survival?
Questions identified as out of scope were discussed face-to-face with the wider steering group. The
steering group also reviewed the full list of submitted questions to identify any further out-of-scope
questions. Identification of out of scope questions was an iterative process. All out-of-scope questions
were checked and agreed upon by the steering group. In total, 326 questions were identified as out of
scope. There is a commitment from the organizations represented on our steering group to review and
consider how to make the best use of these questions.
Formatting questions
During two steering group meetings, members were grouped depending on their area of expertise and
worked in small groups to review questions in a number of CSO categories. During this process, similar
questions were merged and were then converted into an overarching research question. Five hundred
and twenty nine questions were merged, resulting in 208 unique questions. The submitted questions
took several forms which had to be organized into the recognized research question structure of
Population, Intervention/Exposure, Comparison, and Outcomes (PICO). It was noted that most questions
either stated or assumed two elements of this: the population was generally ‘young people with cancer’,
the comparison was ‘young people without cancer’, or ‘cancer in older or younger populations. The
intervention and outcome were varied, for example, interventions in the 'time to diagnosis' question
below included ‘education’ and ‘awareness campaigns’. Outcomes included cancer incidence, survival,
relapse, psychological well-being, social functioning, and mental health. For example, 17 questions were
submitted in the survey about improving time to diagnosis, including: How can we get earlier stage
diagnosis for young patients? How can we improve doctor diagnosis/early referral of cancer in young
people? How do we improve the diagnostic pathway for all young people to ensure they receive a timely
diagnosis? Do you think GPs require more training to raise awareness of the possibility of cancer when
young people go to their Dr? The early diagnosis I feel is difficult. How can the medical profession get
better at this with young adults? Should cancer awareness be taught in school and be a compulsory part
of the curriculum- signs/symptoms, self-examination?
These questions were all incorporated into the research question: What GP or young person strategies,
such as awareness campaigns and education, improve early diagnosis for young people with suspected
cancer?
Searching for evidence
A data assessment group consisting of members of the steering group including young people and
experts in evidence synthesis was established to oversee the evidence searches. A search strategy was
produced by the data group and discussed with the steering group. Searches were limited to evidence
published in the last five years (since January 2012), to ensure evidence was up-to-date. Only
publications which brought evidence from multiple studies together (such as systematic reviews and
qualitative meta-synthesis) were considered. As the evidence was unlikely to be restricted to the 13 to
24 age range, evidence that included participants between these ages along with older/younger
participants was also considered. When reviewing the evidence, the data assessment group and steering
group discussed whether further work focusing specifically on 13 to 24-year-olds was needed. Searches
were also carried out for ongoing studies. This involved personal communication with experts in the
field and checking clinical trial databases. Steering group members provided keywords for the evidence
searches within their area of expertise. Searches were carried out in August and September 2017 by LF
and SA. For many questions, no reviews were identified. In some cases, the identified reviews only partly
answered the question; these questions were recorded as unanswered. When any evidence was found,
it was initially reviewed by SA/LF and BP. Seven questions were identified as already answered
(Appendix 4). All were discussed with the steering group to ensure consensus that the question had
been answered. Sixteen questions were the focus of studies currently underway (Appendix 5). One
hundred and eighty-five unanswered questions remained.
Stage 3: Prioritising the questions
A steering group meeting was held to discuss the prioritization of the 185 unanswered questions. As this
number of questions was too many to ask people to consider in the interim survey, the steering group
decided that all questions which had been asked by more than one person should be included (64
questions) and the remaining 121 questions would be rated by the steering group in an online survey;
the top scoring questions would be included.
When carrying out the rating, steering group members were asked to, ‘identify the questions which
would have the most impact if funded, on either the teenage and young adult cancer community as a
whole or young people with that particular cancer type’. Questions were presented in random order and
rated as to whether answering them would have: a high impact (score 4), moderate impact (score 3),
low impact (score 2), or negligible impact (score 1). Total scores for each question were calculated and
questions were ordered from highest to lowest score. It was originally intended that the 36 top-rated
questions would be included in the interim survey (to give a total of 100 questions in the survey).
However, the steering group decided that the top 40 questions would go through to the survey as 13
questions at the cut-off point had scored the same, if these were excluded only 27 questions would go
through and the steering group wanted to take it as many questions as possible to the public vote. A
further preparation step which involved ensuring all questions were understandable to the public was
carried out by LF and SA and checked by the young people on the steering group.
Interim survey
The interim survey was created using Qualtrics online survey software and launched in November 2017;
it was open for three weeks. Responses were invited from the same groups of people as the first survey.
The opportunity to take part was publicized through the same partner organizations as the first survey
and on social media. Everyone who had requested to stay involved in the project and provided their
email address in the first survey was sent the survey link directly.
Respondents were invited to rate the 104 questions (Appendix 6) according to how much of a priority
they thought it was for research to be undertaken to answer that particular question. The options for
rating were: very low priority (score 1), low priority (score 2), high priority (score 3), very high priority
(score 4), and no opinion (no score). Respondents were asked to select ‘no opinion’ if they were unsure
about whether a question was a priority for research to answer or did not have an opinion about that
question. Questions on similar topics were grouped into sections. Each respondent was presented with
the sections in a random order to minimize the chance of survey fatigue:
1. Causes of cancer, prevention, and diagnosis
2. Treatments and therapies
3. Short and long-term side effects
4. Information and support
5. Psychological support
6. How cancer impacts daily life
7. Impact of cancer on families, friends, and partners
8. End of treatment and follow up
9. Healthcare delivery
10. End-of-life care
Ratings were submitted by 174 people. The demographics of those responding can be seen in Figures 9
to 16. As there was some missing data, average scores for each question were calculated for the three
groups of respondents: patients/former patients, family members/friends/partners, and professionals.
The questions were then ordered from highest to lowest scores for each group. The steering group
reviewed how the ratings were compared between respondent groups. As the distribution of
respondents from the three groups was not equal, an overall ranking that combined scores would give
more weight to professionals’ views and so this method was discarded. Instead, the average ranks
across the three groups were calculated to give a shared ranked order. The top 30 questions were
shortlisted for prioritization at the workshop. The 81 unanswered questions not included in the interim
survey are shown in Appendix 7.
Process - Stage 3: Prioritising the questions

Figure 9: Gender distribution of respondents (round 2)
A higher proportion of males responded for young people in the second round of the survey (28%
compared to 18%), however taking into account the smaller number of respondents in the second round
the actual number of males responding had decreased (Figure 9). A smaller proportion of male
professionals responded in the second round, 18% compared to 30% in round 1.
Figure 10: Ethnicity of respondents (round 2)
The ethnic distribution of those answering the survey in round 2 was similar to round 1 (Figure 10).
Figure 11: Age of respondents (round 2)
The age distribution of the young people answering the survey in Round 2 was similar to that observed
in Round 1, the majority of respondents were aged 19-24 years, followed by 25-34 years (Figure 11). The
age demographics of carers/significant others answering in round 2 were also similar with the majority
of respondents aged 45-55 years.
Figure 12: Geographical distribution of respondents (round 2)
The geographical distribution of respondents answering the survey in Round 2 was similar to Round 1
(Figure 12).
Figure 13: Cancer distribution in young people, carers, and significant others (round 2)
There were slight differences in the distribution of cancers in round 2 (Figure 13). Similar to round 1 the
most frequently occurring cancer for young people was Hodgkin’s disease/lymphoma, followed by
leukemia, however for carers/significant others this was slightly reversed with a higher proportion of
Hodgkin’s lymphoma compared to leukemia respondents in round 1. Overall, a higher proportion of
respondents had brain tumors, however, given the lower numbers in round 2 the actual number of
young people with a brain tumor responding was the same (n=8)
Figure 14: Cancer timeline of respondents (round 2)
Where young people considered themselves to be in their cancer timeline was similar to round 1, as
were the responses of the carers/significant others (Figure 14)
Figure 16: Distribution of professionals (round 2)
Figure 15: Distribution of carers/significant others (round 2)
The majority of carers/significant others responding in round 2 were parents/carers which is similar to
round 1 (Figure 15).A greater proportion of nurses compared to doctors completed the round 2 survey,
the actual number of nurses responding was similar to round 1 and the differences were due to lower
numbers of doctors in round (Figure 16).
Process
Stage 4: Agreeing the Top 10
A final prioritisation workshop took place in central London on 19th January 2018 to identify the Top 10
unanswered research questions for teenage and young adult cancer. The workshop was attended by 25
participants: seven young people who had experienced cancer, four parents of a young person with
cancer, and 14 professionals who work with young people who have cancer.
The professionals were from a wide range of backgrounds including nurses, oncologists, social workers,
youth support coordinators, q and physiotherapists. Seven participants were members of the steering
group. The other participants were invited as they had indicated they would like to attend when
completing the interim survey or they were suggested by steering group members due to their
professional role. Young people were also recruited through the BRIGHT LIGHT study Young Advisory
Panel. The workshop was also advertised to parents through CLIC Sargent’s parent Facebook page.
Participants were asked to individually rank the 30 questions in order of importance before the
workshop; this was used as a starting point for discussion. Biographies of participants were also
circulated before the day.
The workshop was chaired and facilitated by Sheela Upadhyaya with support from two co-facilitators
from the JLA, Katherine Cowan and Toto Gronlund. The participants were divided into three groups
which had been pre-arranged to ensure a balance of professionals from different disciplines, young
people, and parents. Each group was given a set of 30 questions on A4 cards, which were laid out on a
table. For the first step, each person was asked to tell their group the three questions they had ranked
highest and lowest in their ranking. Discussion followed and the groups were asked to place the 30
questions in a collective order of importance. Each participant was encouraged to share their views and
give consideration to other people’s opinions. At lunchtime, the ranking of the 30 questions from the
three groups was combined. In the afternoon session, in new group compositions, the consensus
ranking was the starting point for discussion. Following this second round of discussion, the group
rankings were again collated, and the participants came together as one group to agree on the Top 10
and debate their order.
Decision making: prioritizing the Top 10
In this section we aim to give an overview of the discussions in the workshop and how the Top 10 were
decided upon. What was striking was the similarities between the three groups within the workshop
who independently developed very similar strategies to decide which questions should be in the Top 10.
Their strategies involved:
Ensuring all parts of the pathway of care are covered
All three groups wanted to ensure the Top 10 questions covered topics relating to diagnosis and
treatment, through to end-of-life care and reflected the range of experiences of young people with
cancer.
Ensuring all the themes within the questions were represented The groups tried to cluster the questions
into similar themes, such as support, treatment, or side effects, they aim to include each ‘theme’ in the
Top 10. For example, the groups wanted either, ‘What GP or young person strategies, such as awareness
campaigns and education, improve early diagnosis for young people with suspected cancer?’ or, ‘What
are the most effective strategies for engaging primary care professionals (e.g. GPs) to listen to young
people?’ to be included in the Top 10, but not both as they felt this would be a wasted opportunity for
answering a question in a different area. Opting for questions that could include other questions
/overlap
Linked to the above point regarding themes, the groups considered which questions overlapped and
could cover other questions. For example, they thought, ‘What are the best ways of supporting a young
person who has incurable cancer?’ could include, ‘For young people with incurable cancer, how should
parents/carers communicate with them to improve quality of life and experience?’ and ‘For young
people with incurable cancer, how should healthcare professionals communicate with them to improve
quality of life and patient experience?’ as good communication is part of providing support.
Process - Stage 4: Agreeing on the top 10
Opting for questions that could have a wide impact
Initially, the majority of participants selected their top three questions based on what was relevant to
their personal experience or focused on the area they worked within. However, during the discussion
that followed their opinions changed and the groups decided that the Top 10 questions should be
generic and not focus on one type of cancer (e.g. ‘What is the best treatment for brain cancers to
increase survival and decrease toxicity?’) or one type of treatment (e.g. ‘What are the long-term physical
effects of stem cell transplants, how long do they last and how could they be reduced?’). Reducing side
effects was important and again the groups opted for a broad question, ‘What interventions, including
self-care, can reduce or reverse adverse short and long-term effects of cancer treatment?’ rather than
the questions which focused on one side effect (‘chemobrain’ and fatigue). They reasoned that not
enough is known about how to reduce all side effects to single out a particular side effect in the Top
10.Including a question that is focused on support for families
Support for families was a high priority for the participants; they wanted to ensure a question was
included that focused on this. The gaps in support following the death of a young person were discussed
and, ‘How can parents/carers/siblings/partners be best supported following the death of a young person
with cancer?’ was included. Although the following question does not specifically mention families,
‘What psychological support package improves psychological well-being, social functioning, and mental
health during and after treatment?’ the participants thought research on this should include supporting
families as part of the young person’s support network.
Opting for questions focussed on intervention rather than description
The groups were clear that although it is useful to describe a problem, it is activated through an
intervention that is required to improve young people’s and families' experiences. Therefore, ‘What
psychological support package improves psychological well-being, social functioning, and mental health
during and after treatment?’ was chosen rather than, ‘How common is psychological distress and/or
mental health problems in young people following treatment?’ Similarly, ‘How can
parents/carers/siblings/partners be best supported following the death of a young person with cancer?’
was selected rather than, ‘What are the support needs of the family following the death of a young
person with cancer?’
Excluding questions considered to be too subjective to be answered by research
‘What are the factors that should determine stopping treatment when the young person cannot be
cured?’ was considered to be difficult to research as it is too dependent on an individual’s situation. ‘For
young people with incurable cancer, how should healthcare professionals communicate with them to
improve quality of life and patient experience?’ was also considered to be subjective, with participants
feeling it is important but it is a difficult skill to teach to professionals and also was a wider social
problem around our views on dying and death. From the very start of the workshop when the
participants were asked to give their top three questions, some questions were a high priority for many
and stayed high in the Top 10 throughout the workshop. The question ranked as a top priority, ‘What
psychological support package improves psychological well-being, social functioning, and mental health
during and after treatment?’ was the top priority for the two groups after the first group discussion and
in second place for the third group. Following the second group discussion, it was top for all three
groups. The young people present discussed the importance of support being available after treatment
as this can be lacking and can be when young people need it the most. Reducing the burden of side
effects of treatment was also fundamental. ‘What interventions, including self-care, can reduce or
reverse adverse short and long-term effects of cancer treatment?’, was placed at number two in the
final Top 10, it featured as number four for two groups after the first discussion and number five for the
third group. After the second discussion, it was in second place for all three groups.
There were a few questions that featured in the groups’ initial Top 10s which were not included in the
final Top 10. One example is, ‘What are the long-term physical effects of a cancer diagnosis and
treatment and how long do they last?’ Following the group discussions, this question had an average
ranking of number three across the groups. During the whole group discussion at the end of the
workshop, the decision was made to move it out of the Top 10. The participants agreed that the long-
term effects are known in practice. There was consensus that it is more important to focus on
interventions to reduce side effects and treatments that have fewer side effects. Moving the question
out of the Top 10 made room for, ‘What targeted treatments are effective and have fewer short and
long-term side-effects’ to be included instead. ‘What is the best method of follow-up and timing which
causes the least psychological and physical harm, while ensuring relapse/complications are detected
early?’ had only featured in one group’s Top 10 however, during the whole group discussion, there was
a push to include it in the final Top 10. The inclusion of this question meant there were questions across
the pathway of care. Although it was recognized that this is a broad question to answer which is
dependent on a young person’s cancer and treatment, it was considered to be important that
professionals get follow-up right for young people. As this population moves around more (e.g. to
university), follow-up that is tailored to their needs is important.
Rewording of questions
The workshop participants requested that three questions be reworded:
1) ‘What are the best ways to support young people getting back to a ‘normal’ life after
treatment?’ There was consensus that the word ‘normal’ should not be used as the participants
Process - Stage 4: Agreeing on the Top 10

said that life does not go completely back to ‘normal’ and what is ‘normal’ is unclear. This question was
reworded to, ‘What are the best ways to support young people getting back to an ‘everyday’ life after
treatment?’ 2) ‘What are the most effective strategies to ensure that young people who are treated in
non-specialist hospitals receive appropriate practical and emotional support?’ Non-specialist was
changed to ‘outside of a young person’s Principal Treatment Centre’, What are the most effective
strategies to ensure that young people who are treated outside of a young person’s Principal Treatment
Centre receive appropriate practical and emotional support?’ 3) The young people at the workshop
asked for ‘including self-care to be added to the following question, ‘What interventions, including self-
care, can reduce or reverse adverse short and long-term effects of cancer treatment?’ They thought
‘intervention’ referred to something that is ‘done to’ a person and was not aware that interventions
could include self-care. They wanted this to be made clear as it was important to them to know what
they could do to help their recovery and reduce the side effects of treatment. By adding ‘self-care’ to
the question, they felt it also encompassed, ‘What can young people do to help their recovery after
chemotherapy or radiotherapy?’
The Workshop Group
This photograph shows the people present at the workshop including young people, parents,
professionals, and the coordinating team.
TOP 10 Research Priorities
The Top 10 priorities for teenage and young adult cancer research were agreed as:
1 What psychological support package improves psychological well-being, social functioning, and mental
health during and after treatment?
2 What interventions, including self-care, can reduce or reverse adverse short and long-term effects of
cancer treatment?
3 What are the best strategies to improve access to clinical trials?
4 What General Practitioner or young person strategies, such as awareness campaigns and education,
improve early diagnosis for young people with suspected cancer?
5 What are the best ways of supporting a young person who has incurable cancer?
6 What are the most effective strategies to ensure that young people who are treated outside of a
young person’s Principal Treatment Centre receive appropriate practical and emotional support?
7 What interventions are most effective in supporting young people when returning to education or
work?
8 How can parents/carers/siblings/partners be best supported following the death of a young person
with cancer?
9 What is the best method of follow-up and timing which causes the least psychological and physical
10 What targeted treatments are effective and have fewer short and long-term side effects?
The remaining 20 priorities
The following 20 questions were also discussed at the workshop but not placed in order of priority:
DIAGNOSIS
. What factors affect the time to diagnosis and what outcomes are affected?
. What are the most effective strategies for engaging primary care professionals (e.g. GPs) to listen to
young people?
TREATMENT
. What key factors (both cancer and individual) determine whether a treatment plan for children or
adults will give better outcomes?
. What is the best treatment for brain cancers to increase survival and decrease toxicity?
. What are the factors that predict life-threatening chemotherapy side effects?
AFTER TREATMENT
. What can young people do to help their recovery after chemotherapy or radiotherapy?
. What are the long-term physical effects of a cancer diagnosis and treatment and how long do they
last?
. What are the long-term physical effects of stem cell transplants, how long do they last and how could
they be reduced?
. How common are psychological distress and/or mental health problems in young people following
treatment?
. What causes problems with cognitive functioning (‘chemobrain’), how long do they last and what are
the most effective treatments and strategies?
. At the end of treatment and during long-term follow-up, what support services improve psychological
well-being, social functioning, and mental health?
. What are the best ways to support young people getting back to ‘everyday’ life after treatment?
. What interventions are most effective in supporting young people who are experiencing
fatigue/tiredness when returning to work or education?
. What are the best strategies for detecting and treating second primary cancers early?
RELAPSE
. What is the most effective way of supporting young people with relapsed cancer?
END OF LIFE
. What are the factors that should determine stopping treatment when the young person cannot be
cured?
. For young people with incurable cancer, what methods, techniques, or strategies for communication
can help them to talk with their family and friends about their situation?
. For young people with incurable cancer, how should healthcare professionals communicate with them
to improve their quality of life and patient experience?
. For young people with incurable cancer, how should parents/carers communicate with them to
improve their quality of life and experience?
. What are the support needs of the family following the death of a young person with cancer?
Next steps / Acknowledgements / Reference
For research funders
The Top 10 list provides major national research funders such as the National Institute for Health
Research (NIHR), Medical Research Council (MRC) and Economic and Social Research Council (ESRC) with
clear guidance on what questions are important to young people, carers/significant others and
professionals. The findings will enable existing funders to target their funds to prioritize what matters
most to those involved in the care of young people with cancer.
For Researchers
A common question in reviewers’ comments and applications for funding includes whether it is a) an
important question b) whether it has already been answered. This Top 10 will allow researchers to tailor
their research questions and strategies to develop a portfolio of studies relevant to young people with
cancer based on priorities agreed upon by multiple stakeholders. The long list of questions will be made
publically available via the James Lind Alliance website.
For Charities
Charitable funders within their research and policy teams can refer to the TYA PSP to demonstrate the
need for research funding in priority areas for young people. The out-of-scope questions may be able to
inform where more information resources are required to describe services and service provision in
local areas.
For the TYA PSP
The survey returned 855 potential research questions, many of which included comments and questions
which did not fit within the scope of the TYA PSP. We will look at how these questions, statements, and
service inquiries can be best used to improve outcomes for young people, carers/significant others, and
the professionals who care for them.
Acknowledgments
This project was supported and funded by CLIC Sargent, Teenage Cancer Trust, and Children with Cancer
UK. We would like to thank Dr. Sabine Best, Marie Curie, and Prof Helen Roberts, UCL Great Ormond
Street Institute of Child Health, for their contribution to the data assessment group. We would also like
to thank previous Steering Group member Dr. Ken Lawton. With thanks to the following people from
CLIC Sargent for their contribution Caroline Weston, Anna Carnegie, Beccie Platt, and Jane Darragh. The
TYA Cancer PSP would like to thank everyone who took the time to send in their questions and rate their
importance of them. Thank you also to the young people, parents, and professionals who attended the
final workshop.
Reference
Chalmers I, Atkinson P, Fenton M, Firkins L, Crowe S, Cowan K.J (2013) Tackling treatment uncertainties
together: the evolution of the James Lind Initiative, 2003-2013. Journal of the Royal Society of Medicine,
106(12):482-91.
Appendix 1
Partners
The TYA Cancer PSP is grateful for the support of all of our Partners who helped to distribute the surveys
and Top 10 priorities:
. Antony Nolan
. Bloodwise
. Bone Cancer Research Trust
. Brain Tumour Charity
. Cancer Research UK
. Children’s Cancer and Leukaemia Group (CCLG)
. Coppafeel!
. Ellen MacArthur Cancer Trust
. Find Your Sense of Tumour Steering Committee
. Guy Francis Bone Cancer Research Fund
. JTV Cancer Support
. Royal College of Nursing
. Marie Curie
. Macmillan Cancer Support
. National Cancer Research Institute (NCRI) - All relevant NCRI groups including: the Teenage, Young
Adult, and Germ
Cell Tumour Clinical Study Group
. Sarcoma UK
. Target Ovarian Cancer
. Teens Unite
. Trekstock
. TYAC – Teenagers and Young Adults with Cancer Professional Organisation
. Young People’s Health Special Interest Group (YPHSIG)
Appendix 2
Overview of TYA PSP Methodology and Results
Initial survey
292 respondents submitted
855 questions
326 questions removed as ‘out of scope’
Questions put into research question format duplicates combined
208 questions
Questions checked against current evidence
7 answered
16 ongoing studies
185 unanswered questions
Interim prioritization
174 respondents prioritized the 104 questions
Workshop
25 participants prioritized the
30 questions
Top 10 Priorities published
Appendix 3
Initial survey
James Lind Alliance
Teenage and Young Adult Cancer Priority Setting Partnership Survey
Do you have unanswered questions about Teenage and Young Adult Cancer?
We are aiming to identify questions that patients, families, friends, carers, and professionals think are
important about Teenage and Young Adult Cancer but which have not yet been answered by research.
The questions will be made available to research funders to ensure that they are aware of what matters
most to patients, their friends and families, and professionals. We would like to invite you to complete
this survey if you:
. have been diagnosed or treated for cancer when you were between the ages of 13 and 24 years old (it
does not matter what age you are now)
. are a relative/friend/partner/carer of someone who has been diagnosed or treated for cancer between
the ages of 13 and 24 years old
. are a professional working with teenagers/young adults with cancer
This survey is being overseen by the Teenage and Young Adult Priority Setting Partnership (TYA PSP)
Steering Group; led by the James Lind Alliance in partnership with Teenage Cancer Trust, CLIC Sargent,
and Children with Cancer UK. You can find more information about the TYA PSP and this survey here
(www.tyac.org.uk/News/ have-you-or-someone-you-know-beenaffected-by-cancer-as-a-teenager-
oryoung-adult), by emailing tyapsp@gmail.com or by telephoning XXXXX. For further information about
the James Lind Alliance please go to http://www.jla.nihr.ac.uk/.
Consent
As part of this process, the question(s) that you suggest when taking part in this survey may be
published on the James Lind Alliance website. By participating in this survey you are agreeing to allow us
to publish your question(s). Your name will not be published in association with your question(s). For
more information, or to see what this will look like, please go to http://www.jla.nihr.ac.uk/top-10-
priorities/.
Appendix 3 - Initial survey
Information about you
It would be really helpful to know some information about you; this will enable us to check whether we
have responses from a wide range of people or whether any groups are missing. If there are any
questions you would prefer not to answer, please select ‘prefer not to answer’.
Who are you?
● Patient or former patient
● Parent/carer of a teenager or young adult who has been diagnosed or treated for cancer
● Relative of someone who has been diagnosed or treated for Teenage and Young Adult Cancer
● Friend of someone who has been diagnosed or treated for Teenage and Young Adult Cancer
● Partner of someone who has been diagnosed or treated for Teenage and Young Adult Cancer
● Professional working with teenagers/young adults with cancer
How old are you?
● Under 13 (option for family members/friends/partners/carers only)
● 13-15 (option for patients/former patients and family members/friends/partners/carers only)

● 16 – 18
● 19 – 24
● 25 – 34
● 35 – 44
● 45 – 54
● 55 – 64
● 65+
● Prefer not to answer
What is your gender?
● Male
● Female
● Other
If you selected Other please specify:
What is your ethnic group?
● White
● Mixed/multiple ethnic groups
● Asian or Asian British
● Black African, Black Caribbean, or Black British
● Other
How old are you?
● Under 13
(option for family members/friends/partners/carers only)
● 13-15
(option for patients/former patients and family members/
friends/partners/carers only)
● 16 – 18
● 19 – 24
● 25 – 34
● 35 – 44
● 45 – 54
● 55 – 64
● 65+
Please select the country you live in.
(Patients/former patients and family members/ friends/ partners/carers)
Please select the country you work in.
(Professionals)
● England
● Scotland
● Wales
● Northern Ireland
● Other
Which cancer were you first diagnosed with?
(Patients/former patients)
Which cancer was your relative/friend/partner first diagnosed with?
(Family members/ friends/ partners/carers)
● Leukaemia
● Hodgkin's disease/lymphoma
● Brain or spinal cord
● Bone tumor (sarcoma)
● Soft tissue tumor (sarcoma)
● Non-Hodgkin’s Lymphoma
● Testicular
● Ovarian
● Thyroid
● Colorectal (bowel cancer)
● Breast
● Cervical
● Melanoma (skin cancer)
● Not sure
● Other
Which of the following best describes your current situation?
(Patients/former patients)
Which of the following best describes your relationship/friend/partner’s current situation?
(Family members/friends/partners/carers)
You can select more than one response if you wish.
● On treatment
● End of treatment
● Relapsed
● Survivor/Follow-up care
● Palliative and end-of-life care
● Deceased
(option for family members/friends/partners/carers only)
● Not sure
What is your profession?
(Professionals)
● Doctor
● Nurse
● Allied Health Professional
● Other
Please enter your job title:
Your questions
In your experience of Teenage and Young Adult Cancer, you may have had questions that you think have
not yet been answered. What questions would you like to see answered by research? To help you, here
are some examples of the questions that have been submitted for other health conditions:
. What are the most effective ways of supporting carers of people with dementia living at home?
. Are counseling/psychological strategies (e.g. talking therapies) effective to promote the mental health
of children and young people with neuro disabilities?
. What are the best ways to help people come to terms with the long-term consequences of stroke?
. Which treatment is more effective for vitiligo: steroid creams/ointments or light therapy?
. Is high-dose Loperamide safe and effective in the treatment of diarrhoea in Inflammatory Bowel
Disease?
. What treatments are helpful for reducing balance problems and falls in people with Parkinson’s?
Your questions can be about any aspect of Teenage and Young Adult Cancer during:
Pre-diagnosis Diagnosis Referral
Treatment
End of Treatment Follow-up Relapse
Survivorship
Palliative care and end of life
Such as:
■ Communication ■ Care ■ Education ■ Health ■ Long-term effects
■ Relationships ■ Family ■ Work ■ Social life ■ Side effects
Please suggest a maximum of five questions. Your questions can be written in any
order of importance.
1 Please write your first question here:
2 What is your next question? (Please leave blank if you do not have any further questions)
Do you have any additional comments you would like to share with us?
Next steps
Would you like to be involved in the next stage of the project, which is to vote for the questions you
think are most important of all those submitted. This may involve voting online or attending a workshop.
If you are 16 years old or over and would like to be involved please provide your details for your
preferred method of contact:
If you are under 16 years old please ask a parent/carer to contact us with your details. Please email
tyapsp@gmail.com and put ‘TYA PSP Contact’ in the subject line. All contact details will be kept
confidential and secure, following the Data Projection Act. We will not publish your details and they will
not be linked to your responses on this form. We will not use your details for any purpose other than
inviting you to take part in the next stage.
Name:
Email address:
Address:
Thank you for completing this survey
These are our contact details:
Tel: XXXX
Email: tyapsp@gmail.com
Completing this survey may have made you feel that you would like further support or information
about cancer, if so, you could speak to a health professional involved in your care or you may find the
following links helpful:
Teenage Cancer Trust: https://www.teenagecancertrust.org/gethelp
Macmillan Cancer Support: http://www.macmillan.org.uk/informationand-support/index.html
CLIC Sargent: http://www.clicsargent.org.uk/content/help-and-support
Appendix 4
Answered questions
Question
Question submitted by
Evidence found
Evidence web link
Comment
1. How can young people with osteosarcoma of the leg be successfully treated without amputation?
1 x patient
Salang K, Foocharoen T, Laopaiboon M, Jirarattanaphochai K. Limb salvage for treating pathological

fracture at diagnosis in children and adolescents with localized high-grade osteosarcoma (Protocol).
Cochrane Database of Systematic Reviews 2016, Issue 4. Art. No.: CD012146. DOI:
10.1002/14651858.CD012146; Yin K, Liao Q, Zhong D, Ding J, Niu B, Long Q, Ding D. Meta-analysis of
limb salvage versus amputation for treating high-grade and localized osteosarcoma in patients with
pathological fracture. Experimental and Therapeutic Medicine.2012;4(5):889-894.
http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD012146/full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493816/
Correspondence with Sarcoma expert - Most young people are treated successfully without amputation.
Amputation rates ~ 10%
2. Does the use of low-level laser therapy reduce the incidence of mucositis in highly mucositis-
provoking chemotherapy regimens for young people with cancer?
1 x professional
Oberoi S, Zamperlini-Netto G, Beyene J, Treister NS, Sung L. Effect of prophylactic low-level laser therapy
on oral mucositis: a systematic review and meta-analysis. PLOS ONE.2014;9(9):e107418.
http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0107418&type=printable
3. What are the side effects of chemotherapy in young people with cancer?
1 x patient
Ahmad S, Reinius M, Hatcher H, Ajithkumar T. Anticancer chemotherapy in teenagers and young adults:
managing long-term side effects. BMJ 2016; 354 doi: https://doi.org/10.1136/bmj.i4567 BMJ
2016;354:i4567 Bukowinski AJ, Burns KC, Parsons K, Perentesis JP, O'Brien MM.Semin Oncol Nurs. 2015
Toxicity of Cancer Therapy in Adolescents and Young Adults (AYAs). Aug;31(3):216-26. doi:
10.1016/j.soncn.2015.05.003. Epub 2015 May 7.
http://www.bmj.com/content/354/bmj.i4567.long https://www.ncbi.nlm.nih.gov/pubmed/26210200
4. What are the survival rates for young people with cancer?
1 x patient
Trends in five-year survival for teenagers and adults with cancer in the UK, Public Health England.
Pattern of deaths in the year following diagnosis in cancer patients aged 15-24 years in England Authors:
Tony Moran, Debasree Purkayastha, Catherine O’Hara Date: 15.4.2013.
http://www.ncin.org.uk/view?rid=2751 http://www.ncin.org.uk/view?rid=2134
5. What is the incidence of new primary and secondary cancers following cancer treatment as a young
person?
2 x parent/carer
1 x patient
Second cancers among survivors of teenager and young adult cancer, National Cancer Intelligence
Network Data Briefing.
Franklin J, Eichenauer DA, Becker I, Monsef I, Engert A. Optimisation of chemotherapy and radiotherapy
for untreated Hodgkin lymphoma patients concerning second malignant neoplasms, overall and
progression-free survival: individual participant data analysis. Cochrane Database of Systematic Reviews
2017, Issue 9. Art. No.: CD008814. DOI: 10.1002/14651858.CD008814.pub2. Ongoing study: Teenage
and Young Adult Cancer Survivor Study
http://www.ncin.org.uk/view?rid=1606
http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD008814.pub2/epdf
http://www.birmingham.ac.uk/research/activity/mds/projects/HaPS/PHEB/CCCSS/TYACSS/index.aspx
This question was discussed by the steering group and agreed that it was answered with more data
collection underway from the ongoing Teenage and Young Adult Cancer Survivor Study.
Specific to Hodgkin’s disease – currently unanswered but should be answered by the Teenage and Young
Adult Cancer Survivor Study.
6. What are the outcomes associated with physical activity during treatment for young people with
cancer on managing fatigue?
1 x professional
Tomlinson D1, Diorio C, Beyene J, Sung L. Am J Phys Med Rehabil. 2014 Aug;93(8):675-86. doi:
10.1097/PHM.0000000000000083. Effect of exercise on cancer-related fatigue: a meta-analysis.
https://www.ncbi.nlm.nih.gov/pubmed/24743466
7. What are the barriers to clinical trials for young people with cancer?
1 x professional
Eric Tai, Natasha Buchanan, Lauren Westervelt, Dena Elimam, Silvana Lawvere Pediatrics June 2014,
VOLUME 133 / ISSUE Supplement 3. Treatment Setting, Clinical Trial Enrollment, and Subsequent
Outcomes Among Adolescent
Appendix 5
Ongoing studies
Question
Question submitted by
Evidence found
Evidence web link
Comments
1. What is the genetic fingerprint of Ewing’s Sarcoma?
1 x parent/carer
Correspondence with Sarcoma expert - work on this question is ongoing in several labs. Publications
exist from Delattre/Ladhanyi.
2. What targeted therapies are effective for young people with Ewing’s Sarcoma?
1 x parent/carer
Correspondence with Sarcoma expert - early phase studies are currently underway.
3. Do PD1 pathway-affecting drugs have a role in the treatment of young people with bone and soft
tissue sarcomas?
1 x professional
Correspondence with Sarcoma expert - work on this question is ongoing and some trials have been
reported in the abstract.
4. How do education outcomes for young people with cancer compare to young people without cancer?
3 x professional,
1 x patient
Review protocol: Andrew Thomas, Defne Saatchi, Alastair Sutcliffe.
Educational progression of survivors of cancer diagnosed in childhood: a systematic review. PROSPERO

2017:CRD42017057501
Ongoing study: Study at University College London looking at this in children’s Educational Outcomes
Following Childhood Cancer in England: A Population-Based Linkage Study.
http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42017057501
5. How do protocol adherence and toxicity impact the prognosis for young people with cancer?
1 x parent/carer
Systematic review protocol in this area is in development. Correspondence with a pediatric expert - A
protocol for a systematic review in this area is in development.
6. Do specialist cancer services for young people improve outcomes and patient experience?
5 x professional Ongoing study: BRIGHT LIGHT is the overarching name for a collection of research
projects designed to answer a single question: Do specialist services for teenagers and young adults add
value?
http://www.brightlightstudy.com/
7. Is the life expectancy of teenagers and young adults with cancer shorter compared to the general
population?
3 x patient
1 x partner
Ongoing study: Teenage and Young Adult Cancer Survivor Study
Correspondence with Chief Investigator – confirmed this question will be answered by this ongoing
study.
8. Are young people with germ cell tumors more likely to develop leukemia?
2 x parent/carer Ongoing study: Teenage and Young Adult Cancer Survivor Study
study.
9. What treatment is most effective for relapsed leukaemia in young people?
1 x professional
Search of clinicaltrials.gov database found 138 studies ongoing or set up (September 2017).
https://clinicaltrials.gov/ct2/results?term=relapse&type=Intr&rslt=&recrs=b&recrs=a&recrs=f&recrs=e&
age_v=&gndr=&cond=Leukemia&intr=&titles=&outc=&spons=&lead=&id=&cntry
1=&state1=&cntry2=&state2=&cntry3=&state3=&locn=&phase=2&sfpd_s=&sfpd_e=&lupd_s=&lupd_e=
10. Do young people with Hodgkin’s disease have a higher incidence of second cancers than young
people with other cancers?
1 x patient
Second cancers among survivors of teenager and young adult cancer, National Cancer Intelligence
Network Data Briefing
http://www.ncin.org.uk/view?rid=1606
study
11 For young people with incurable cancer, what are the barriers and facilitators to accessing
hospice/specialist palliative care?
2 x professional Review protocol:
Johanna Taylor, Lorna Fraser, Bryony Beresford, Bob Phillips, Alison Booth, Kath Wright, Stuart Jarvis.
Specialist pediatric palliative care for children and young people with malignancies: a mixed methods
systematic review. PROSPERO 2017:CRD42017064874
http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42017064874
12. What are the incidence of heart disease and second cancers in young people following ABVD?
1 x parent/carer
Henson KE, Reulen RC, Winter DL, et al. Cardiac Mortality Among 200000 Five-Year Survivors of Cancer
Diagnosed at 15 to 39 Years of Age: The Teenage and Young Adult Cancer Survivor Study. Circulation.
2016;134(20):1519-1531. doi:10.1161/CIRCULATIONAHA.116.022514. (see Table 4)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5106083/
13. What is the best model of survivorship care based on risk stratification and individual needs?
7 x professionals 3 x patients 1 x relative
Guidelines on models of care for childhood and adolescent cancer survivors are in development.
Correspondence with expert- guidelines on models of care for childhood and adolescent cancer
survivors are in development. We looked for evidence concerning survivors treated for cancer up until
25 years of age.
14. What fertility preservation techniques are effective for young women with cancer?
2 x patients 3 x professionals 1 x parent/carer
British Fertility Society Policy and Practice Guideline is under development.
Correspondence with fertility expert - British Fertility Society Policy and Practice Guideline is under
development.
15. Is the use of Vincristine and Dexamethasone pulses required during maintenance treatment in young
people with cancer?
1 x patient Ongoing study:
United Kingdom Trial for Children and young adults with Acute Lymphoblastic Leukaemia and
Lymphoma 2011.
Correspondence with expert hematologist - This question is being studied as a randomized question in
the current ALL trial which includes TYA patients up to their 25th birthday, UKALL2011.
16. Do the benefits outweigh the risks of continuing to use steroids in maintenance as part of the
treatment protocol for young people with leukemia?
1 x patient
Ongoing study: United Kingdom Trial for Children and young adults with Acute Lymphoblastic Leukaemia
and Lymphoma 2011
Correspondence with expert hematologist - This question is being studied in the current ALL trial which
includes TYA patients up to 25th birthday, UKALL,2011.
Appendix 6
Unanswered questions included in the interim survey
SECTION: CAUSES OF CANCER, PREVENTION AND DIAGNOSIS
Why do young people develop cancer? (This question was asked about all cancers and specifically:
Hodgkin’s disease/
lymphoma, cancer of the appendix, bowel cancer, leukemia, and brain tumors)
Are the children of young people who have had cancer more likely to develop cancer compared to
those whose parents have
not had cancer?
How does cancer biology in young people differ from cancer biology in children and adults? (This
question was asked about all
cancers and specifically: brain tumors and Hodgkin’s disease/lymphoma)
What GP or young person strategies, such as awareness campaigns and education, improve early
diagnosis for young people
with suspected cancer?
What factors affect the time to diagnosis and what outcomes are affected?
How can cancer awareness campaigns be used/adapted to address the needs of young people without
creating alarm?
What written information do young people with cancer want at diagnosis to meet their needs without
overloading them?
SECTION: TREATMENTS AND THERAPIES
What impact does eating well during treatment have on patient outcomes?
What targeted treatments are effective and have fewer short and long-term side effects?
What is the role and how effective is cannabis oil in the treatment of young people with cancer?
What is the best treatment for brain cancers to increase survival and decrease toxicity?
What are the best strategies to improve access to clinical trials?
What are the most effective ways to ensure that young people follow their treatment plan?
Are there effective less toxic treatments for corticosteroids for young people with cancer?
Is proton radiotherapy more effective than photon radiotherapy for young people with brain tumors?
What are the key factors that impact outcomes in osteosarcoma / soft tissue tumors?
What key factors (both cancer and individual) determine whether a treatment plan for children or
adults will give better outcomes?
What complementary or relaxation therapies improve quality of life?
SECTION: SHORT AND LONG-TERM SIDE EFFECTS
What causes problems with cognitive functioning (chemobrain), how long do they last and what are the
most effective treatments and strategies?
What are the long-term physical effects of stem cell transplants, how long do they last and how could
they be reduced?
Appendix 6 - Unanswered questions included in the interim survey
What are the long-term physical effects of a cancer diagnosis and treatment and how long do they last?
(This question was
asked about all cancers and specifically about leukemia, thyroid cancer, and brain tumors. It was also
asked about these treatments: spinal radiation, bone marrow transplant, mercaptopurine, and
methotrexate and about pain conditions/nerve pain)
Following cancer treatment, do young people have different cognitive functioning (‘chemobrain’)
compared to young people without cancer?
What factors influence poorer health and psychological outcomes?
What cancers and treatments cause avascular necrosis, how does it develop, how common is it, what
are the physical and psychological effects, and what can be done to improve early diagnosis and
treatment?
What types of cancer and treatment lead to fatigue/tiredness, how long does it last and what are the
most effective interventions (apart from exercise) for overcoming cancer-related fatigue/tiredness?
What are the differences in outcomes when febrile neutropenia/neutropenia is managed as outpatient
compared to inpatient?
What interventions can reduce or reverse the adverse short and long-term effects of cancer treatment?
What is the long-term impact of different cancer treatments on the fertility of women who are treated
as young adults?
What is the psychological impact on young people whose fertility has been affected by treatment?
How can the short and long-term negative effects of radiotherapy be reduced?
What are the factors that predict life-threatening chemotherapy side effects?
SECTION: INFORMATION AND SUPPORT
What is the best approach to prepare young people for treatment and long-term side effects?
What are the best ways to provide information about services and treatment and when do young
people want this information?
How are young people best supported to talk about their cancer diagnosis with families, friends, and
schools?
What is the most effective way of supporting young people with relapsed cancer?
What factors influence young people with a recent cancer diagnosis to decline first-line treatment and
how are they best supported?
What is the most effective way of providing peer support for young people during and after treatment?
What outcomes are affected and how much does it cost?
What are the best ways of communicating information about treatment to young people with cancer
who have learning difficulties or social communication disorders?
Do young people and parents who use a decision aid make more informed choices about treatment and
side effects compared to people who do not use a decision aid?
What are the most effective approaches to communicating prognosis?
What prevents and helps young people access the support they need following a cancer diagnosis?
What information and support are required to improve patient experience and psychological well-being
in young people with cancer exploring fertility preservation options?
SECTION: PSYCHOLOGICAL SUPPORT
What is the role of social media and/or online support in supporting young people during and after
treatment?
What psychological support package improves psychological well-being, social functioning, and mental
health during and after treatment? (This question was asked about all cancers and specifically brain
tumors and Hodgkin’s disease/lymphoma)
Are the holistic care needs of young people with cancer being adequately assessed and met?
In young people with cancer, how does the quality of family relationships impact acceptance of the
diagnosis, adherence to therapies, and health outcomes? What individual and family-based
interventions improve these outcomes?
Do internet-based psychological support programs delivered to young people and families during and
off-treatment improve their mental health and well-being?
What is the best intervention to support young women psychologically when their fertility has been
affected by cancer?
Do young people receiving mental health therapies do better than those receiving physical treatment
only?
SECTION: HOW CANCER IMPACTS ON DAILY LIFE
What interventions are most effective in supporting young people who are experiencing
fatigue/tiredness when returning to education or work?
What interventions are most effective in supporting young people when returning to education or
work?
What methods of support from education/school for young people improve well-being, participation,
and mental health? (This question was asked about all cancers and specifically about brain tumors)
What interventions are most effective in supporting young people to maintain their education whilst on
treatment?
How are career choices and prospects affected by a cancer diagnosis and are some groups more at risk
of encountering issues than others?
What interventions can reduce the potential negative impact of a cancer diagnosis on a young person’s
employment and career prospects?
How can schools and teachers better support young people with memory problems following cancer?
How are young people best supported to reintegrate with their peers when returning to school?
What interventions are most effective in supporting young people to maintain their social lives whilst
on treatment?
How does cancer and its treatment impact a young person’s friendships?
What interventions best support young people in developing and maintaining romantic and sexual
relationships during and after treatment?
What are the best ways to support young people getting back to a 'normal' life after treatment? (This
question was asked about all cancers and specifically about brain tumors)
How effective is rehabilitation in assisting recovery from cancer?
What are the barriers and motivators to exercise during and after treatment?

SECTION: IMPACT OF CANCER ON FAMILIES, FRIENDS, AND PARTNERS
How are families and partners best included in communications and supported during and after
treatment?
What is the most effective way of supporting families at relapse?
What is the impact of a cancer diagnosis in a young person on the family?
What are the best ways for families to re-establish normal life after treatment?
How can families best support young people through the emotional and social impact of a cancer
diagnosis?
What type of psychological support do families and partners want during and after treatment?
What are the most effective interventions to improve mental health in families and friends of young
people with cancer?
SECTION: END OF TREATMENT AND FOLLOW UP
What are the best strategies for detecting and treating second primary cancers early?
What are the health outcomes of long term follow-up and screening?
Are long-term follow-up and screening cost-effective?
What are the best strategies to help healthcare professionals to coordinate care to address survivorship
needs raised by a young person?
What are the most effective approaches to communicating end-of-treatment information to young
people?
What is the ideal period of psychological support after treatment?
What can young people do to help their recovery after chemotherapy or radiotherapy?
What are the most effective strategies for achieving long-term health behavior change with young
people following cancer?
What impact does attending an end-of-treatment clinic have on the health behavior of young people in
the first 12 months after treatment has ended successfully compared to those who do not attend a
clinic?
What are the best strategies to empower young people to get their survivorship needs discussed and
addressed by healthcare professionals?
What are the survivorship needs of people diagnosed with cancer as a teenager or young adult
compared with people who had cancer as a child or older adult?
How does having cancer as a teenager or young adult affects a person socially and emotionally in later
life?
What is the best method of follow-up and timing which causes the least psychological and physical
At the end of treatment and during long-term follow-up, what support services improve psychological
well-being, social functioning, and mental health?
How common are psychological distress and/or mental health problems in young people following
treatment? (This question was asked about all distress and specifically about post-traumatic stress
disorder and depression)
What are the rates of relapse, predictive factors, and survival outcomes?
SECTION: END-OF-LIFE CARE
What are the best ways of supporting a young person who has incurable cancer?
How can parents/carers/siblings/partners be best supported following the death of a young person with
cancer?
For young people with incurable cancer, how should healthcare professionals communicate with them
to improve their quality of life and patient experience?
For young people with incurable cancer, how should parents/carers communicate with them to improve
their quality of life and experience?
What are the support needs of the family following the death of a young person with cancer?
What are the factors that should determine stopping treatment when the young person cannot be
cured?
For young people with incurable diseases, what methods, techniques, or strategies for communication
can help them to talk with their family and friends about their situation?
SECTION: HEALTHCARE DELIVERY
What is the most effective model of care for the delivery of regional teenage and young adult cancer
services?
How can patient-reported outcome measures be used to improve care for young people?
How can communication methods between healthcare professionals and young people be enhanced in
the current age of digital communication?
What are the barriers for health professionals in engaging with the teenage and young adult multi-
professional team and regional teenage and young adult service?
What is the structure of the ideal multi-professional team caring for young people at diagnosis, during
treatment, end of treatment, long-term survivorship, and end of life?
What are the barriers to transition to adult services for young people and what are the most effective
strategies to improve transition?
What are the most effective strategies to support nurses caring for young people with cancer?
What are the most effective strategies for engaging primary care professionals (e.g. GPs) to listen to
young people?
How do socio-economic factors influence where a young person chooses to be cared for (for example
choosing a local hospital versus a specialist hospital)?
When do young people want family present in consultations with healthcare professionals and when
would they prefer family not to be present?
What are the most effective strategies to ensure that young people who are treated in non-specialist
hospitals receive appropriate practical and emotional support?
Appendix 7
Unanswered questions not included in the interim survey
In young people with cancer, how do we enable new technologies (molecular profiling) to be integrated
into standard management approaches to monitor, adapt and target treatment?
What is the best treatment for young people with relapsed bone and soft tissue sarcomas?
How should we investigate novel targeted therapies in rare cancers?
How does having cancer as a young person shape identity across the life course?
What is the best strategy for young people to restart fitness and exercise after treatment?
How many young people return to their parent’s home during treatment and how does this impact their
mental health and quality of life compared to those who continue independent living?
What are the financial stresses on young people with cancer and their families?
What are the long-term effects on fertility for males who received high-intensity chemotherapy?
What is the most effective hormone support for post-transplant female survivors?
What are the outcomes associated with physical activity during treatment for young people with cancer
on the side effects of chemotherapy, including managing social isolation and returning to school, leisure,
and work?
What are the best ways to provide peer support for families of young people with cancer?
What is the most effective strategy to embed end-of-treatment summaries into routine cancer care for
young people with cancer?
What proportion of young people with recurrence are offered clinical trials?
How can electronic health records empower young people with cancer?
What factors contribute to poorer survival outcomes in young people with breast cancer compared to
older women?
In young people with cancer, would screening all patients for cancer predisposition syndromes impact
the patient and family and their children to improve uptake of appropriate screening /early detection of
related cancers?
Does appropriate physical challenge improve confidence post-treatment in young people with cancer?
How do disease and treatment impact patient experience of symptoms for young people with cancer?
In young people with cancer which 'holistic care assessment', best delineates baseline psychological
functioning, social functioning, and mental health?
Does emergency admission to an adult ward impact young people’s compliance with treatment?
What are the most effective strategies to empower young people to engage with follow-up?
Do clinical trial accrual rates vary across the UK for young people with cancer?
How effective is pre-habitation in assisting recovery from cancer?
In young people with cancer, what information should be delivered and by whom to improve knowledge
and satisfaction, and well-being?
In young people with cancer who are themselves parents, what emotional/social/psychological support
services improve psychological functioning social functioning, and mental health for the young person
and their children)?
Are there regional differences in survival rates for acute myeloid leukemia (AML) and acute
lymphoblastic leukemia (ALL)?
Appendix 7 - Unanswered questions not included in the interim survey
What evidence is there of the effective end-of-treatment transition onto community services?
How do young people with incurable diseases choose their preferred place of death?
What are the most effective ways of supporting a friend with an incurable disease?
What is the psychological impact of hospital isolation (hospitalization in single rooms) whether short-
term or long term when hospitalized?
In young people with cancer treatment, what safety netting (information/support/education) can be
given to improve the appropriate management and reduce toxic complications?
What is the educational trajectory of young people with cancer from 6 months pre-diagnosis up to age
18? (This question was asked about all cancers and specifically about brain tumors and leukaemia)
What are the most effective ways of supporting young people with long term adverse psychosocial
impact of cancer in addition to previous life events/trauma?
Is there an association between early psychosocial adversity (individual and family) and cancer
aetiology?
What lifestyle changes can young people make to prevent cancer in young people?
Why is Ewing’s sarcoma drug resistant in some young people?
Does a cancer diagnosis and treatment in young people during puberty affect survival?
In young people who are cancer survivors, what is the prevalence of anxiety about their future children
developing cancer and about dying early after having children?
How can sleep be improved in young people undergoing cancer treatment?
What are the most effective ways of supporting a friend undergoing treatment?
How do young people manage their experience of their friend being diagnosed with cancer?
What is the experience of parents of young people with cancer as their child transitions into adulthood
and discussions about treatment and management are now directed at their child?
What is the interaction between pre-existing immune system disorders and lymphoma?
Is ABVD (adriamycin; bleomycin; vinblastine; dacarbazine) equally effective if bleomycin is omitted?
What are the survival outcomes of a second stem cell transplant for young people with cancer?
How can GI (gastrointestinal) side effects be diagnosed and managed more effectively in young people
with cancer?
What is the impact of uncertainty and changes on a young person and their family following a brain
tumor diagnosis?
What are the barriers to accessing radiotherapy for young people with brain tumors?
How does EBV (Epstein-Barr Virus) interact with T-cells and NK cells?
How do young people describe the impact of a cancer diagnosis once treatment is completed at one
year and two years?
In young people exposed to ototoxic agents, how long should hearing tests/assessments be undertaken
in follow-up?
In young people who have anticancer treatments, are they at increased risk of autoimmune disease in
later life?
Does early detection and treatment of GVHD (graft-versus-host disease) impact recovery time for young
people with cancer?
How can we minimize the vascular effects of cisplatin in young people with cancer?
Appendix 7 - Unanswered questions not included in the interim survey
In young people with cancer who have an eating disorder, do interventions need to be modified to take
account of factors that arise uniquely from the 'cancer journey'?
Does participation in support activities for young people with cancer differ by gender?
How does treatment for lymphoma affect outcomes for immune system diseases?
What are the most effective treatments for rare cancers in young people such as Sertoli cell tumors?
Is it safe to use NSAIDs (non-steroidal anti-inflammatory drugs) as an analgesic in young people

undergoing chemotherapy treatment?
How can hair growth be stimulated following radiotherapy in young people with cancer?
How do the needs of lesbian, gay, bisexual, or transgender (LGBT) young people with cancer differ from
the general population of young people with cancer?
Is cardiac screening post anthracyclines more effective if carried out by a cardiac specialist in the late
effects of cancer?
Why is the incidence of brain cancer in young people high in comparison to older adults?
Is there an association between young people’s cancer and eating disorders?
What alternatives are there to loperamide for treating chemotherapy-induced diarrhea?
What is the relationship between daunorubicin and cytarabine doses and bowel problems?
How can grandparents who have a grandchild with cancer talk with them about their cancer?
What are the most effective ways of offering psychological support to young people with lymphedema?
What proportion of young people who have had leukemia relapse more than once?
What are the survival rates for young people with leukemia who have Down syndrome?
Are the early warning indicators of sepsis the same in children, young people, and adults?
What support do young people with cancer and their families want from Grandparents?
What are the recurrence rates for melanoma?
Do young people with posterior fossa tumors have higher rates of Autistic Spectrum Diagnoses than
young people with other cancers/general population?
How many liver cancers in young people are not related to lifestyle choices (diet, health, drinking)?
Would taking T3 alongside T4 after a thyroidectomy help with tiredness even when blood is normal
when taking T4 alone?
What are the recurrence rates of thyroid cancer after five years?
What is the incidence of mixed leukemia?
Does taking blood thinners long-term cause health problems?
Is there a link between HPV (human papillomavirus) vaccines and blood cancers?
Would young people prefer their nurse specialists to wear a uniform or not?
This report should be cited as:

Aldiss S., Fern L.A., Phillips B., Gibson F, on behalf of the Teenage and Young Adult Cancer Priority
Setting Partnership Steering Group (2018)
Teenage and Young Adult Cancer: Research Priorities. Final Report of the James Lind Alliance Priority
Setting Partnership.
http://www.jla.nihr.ac.uk/priority-setting-partnerships/teenage-and-young-adult-cancer/
Contact us:
Email: tyapsp@gmail.com Twitter: @TYAPSP
Breast Cancer Awareness at the Community Level among Women in Delhi, India
Article in Asia Pacific journal of cancer prevention: APJCP · July 2015
4 authors, including:
Some of the authors of this publication are also working on these related projects:
Tribals and Migration in India View project
Portraying Breast Cancer Survival in Delhi - Bridging Knowledge Gaps about Breast Cancer
Survivors and Survivorship in India View project
Subhojit Dey
Disha Foundation
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Public Health Foundation of India
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RESEARCH ARTICLE
Breast Cancer Awareness at the Community Level among Women in Delhi, India
Subhojit Dey1*, Arti Mishra1, Jyotsna Govil2, Preet K Dhillon3
Abstract Background: To assess women’s awareness from diverse sections of society in Delhi
regarding various aspects of breast cancer (BC) – perceptions, signs and symptoms, risk
factors, prevention, screening, and treatment. Materials and Methods: Community-level survey
was undertaken in association with the Indian Cancer Society (ICS), Delhi from May 2013-
March 2014. Women attending BC awareness workshops by ICS were given self-administered
questionnaires before the workshop in the local language to assess BC literacy. Information
provided by 2017 women was converted into awareness scores (aware=1) for analysis using
SPSS. Awareness scores were dichotomized with median score=19 as the cut-off, creating
more aware and less aware categories. Bivariate and multivariate analysis provided P-values,
odds ratios (ORs), and 95% confidence intervals (CIs). Results: Broadly, 53.4% women were
aware about various aspects of BC. Notably, 49.1% women believed that BC was incurable and
73.9% women believed pain to be an initial BC symptom. Only 34.9% women performed breast
self-examination (BSE) and 6.9% women had undergone clinical breast-
examination/mammography. 40.5% women had higher awareness (awareness score > median
score of 19), which was associated with education [graduates (OR=2.31; 95%CI=1.78, 3.16),
post-graduates (OR=7.06; 95%CI=4.14, 12.05) compared to ≤ high school] and socio-economic
status (SES) [low-middle (OR=4.20; 95%CI=2.72, 6.49), middle (OR=6.00; 95%CI=3.82, 9.42)
and upper (OR=6.97; 95%CI=4.10, 11.84) compared to low SES]. Conclusions: BC awareness
of women in Delhi was suboptimal and was associated with low SES and education. Awareness
must be drastically increased via community outreach and use of media as a first step in the
fight against BC.
Keywords: Breast cancer - awareness - cancer prevention - breast self-examination - Delhi –

India
Introduction
Breast cancer (BC) is one of the most common cancers in women across the world accounting
for 23% of all cancer cases (Jemal et al., 2011). According to GLOBOCAN 2012 (Ferlay et al.,
2013), almost 1.7 million women were diagnosed with BC in 2012 globally. Since 2008, BC
incidence has increased by more than 20%, while mortality has increased by 14%. BC is also
the most common cause of death among women (522000 deaths in 2012) and the most
frequently diagnosed cancer among women in 140 of 184 countries worldwide (Ferlay et al.,
2013). GLOBOCAN 2012 also reveals striking patterns of cancer in women and highlights that
priority should be given to cancer prevention and control measures for breast and cervical
cancers globally (Ferlay et al., 2013). Incidence of BC has been increasing in most regions of
the world, but there are huge differences between rich and poor countries (Ferlay et al., 2013).
In many low- and middle-income countries (LMICs), the incidence of BC is now rising sharply
due to changes in reproductive factors, lifestyle, and increased life expectancy (Benson and
Jatoi2012). Today, more than half of incident cases occur in the developing world (Shulman et
al., 2010). In India around 145,000 new cases of BC (27% of all cancer cases in both genders)
are diagnosed every year with around 70,000 deaths due to BC every year, both of which are
higher than any cancer among both genders (Ferlay et al., 2013). Published reports from
different cancer registries in India indicate rising trends in BC incidence and declining trends in
cervical cancer (Asthana et al., 2014).In developing countries, women most commonly present
at late stages of BC with this figure being around 70% for India (Sankaranarayanan et al.,
2010). 5-year survival rate was only around 60% in the 2005-09 period and hadn’t increased
much from 1995 (Allemani et al., 2014) mostly due to limited access to early detection and
treatment in addition to late presentation. Low levels of cancer awareness are a very important
risk factor for delay in presentation by the patient (Ramirez et al., 1999; McDonald et al., 2004).
Studies have shown that few women in LMICs have the right knowledge or perform regular
breast self-examination (BSE) (Yadav et al., 2010; Shallawani et al., 2010; Shalini et al., 2011;
Loh and Chew, 2011; Wu et al., 2012; Yoo et al., 2012;
Indian Institute of Public Health-Delhi, 3

Centre for Control of Chronic Conditions, Public Health Foundation of India, Gurgaon, 2
Honorary Additional Secretary, Indian Cancer Society, New Delhi, India *For correspondence:
subhojit.dey@iiphd.org
Subhojit Dey et al
5244 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
Radi, 2013; Karadag et al., 2014). The reasons for the low rate of BSE and sometimes
unwillingness to undergo screening among these women include the fear of finding that they
have BC, inadequate knowledge regarding how to perform BSE, and lack of awareness about
what to do if a lump is found (Wu et al., 2012; Sreedevi et al., 2014). Given the high and
increasing burden of disease due to late presentation of BC, early detection and subsequent
prompt treatment are the only ways to ensure long-term survival, and awareness of BC and
BSE seems to be an essential option for the early detection of BC (Serey et al., 2011;
Norsa’adah et al., 2012). Available studies depict that the majority of the research on cancer in
LMICs is related to treatment with minuscule amounts of research devoted to BC prevention,
awareness, early detection, and palliation (Lodge and Corbex, 2011). A quick review of the
literature in India strongly suggests that BC prevention, awareness, and early detection remain
neglected areas in our health system. Compared to hospital-based studies we have very weak
community-based studies to assess BC awareness (Parmeshwari et al., 2013). BC mortality
rates being much higher in LMICs such as India compared to high-income countries (Parkin and
Fernandez, 2006), BC awareness and prevention must be given due importance. In light of the
above and to fulfill the gaps in BC awareness research, the present study attempted to assess
the awareness of various aspects of BC among the diverse society of women in Delhi, India. by
filling out the questionnaire. To make it comprehensible, English and Hindi, which are commonly
used languages in Delhi, were used. The questionnaire was pilot tested before being
administered to participants in the study. Results of the pilot test were used to modify the
wording of questions to maximize their easy comprehension. Cronbach’s alpha coefficient was
calculated to check the internal consistency among the different questions available in the
questionnaire. The value of Cronbach’s alpha was 0.821 which shows a high level of internal
consistency or reliability of the study tool. A participant identification number was assigned to
each participant and additional information like name, address, contact numbers, and email ID
(if available) was collected to identify every participant. We adapted items for use in this study
by including the response, “Don’t know” in addition to the yes (agree) and no (disagree). The
questionnaire used in this study had a total of 40 questions, of which 7 assessed knowledge
about general perception of BC, 11 assessed knowledge related to BC epidemiology and risk
factors, 4 assessed BC symptoms, and 18 assessed BC detection methods and treatment. A
socio-demographic section of the questionnaire was used to acquire participants’ information
such as gender, age, education, marital status, religion, family type, and total monthly
expenses. Based on previous studies, we hypothesized that these factors could potentially
influence BC knowledge among the participants. Examining these variables can also help in
identifying women groups lacking BC awareness. The survey also intended to identify the
source of information of these women and hence the most suitable medium for future activities
to raise their awareness levels.
Materials and Methods

With the collaboration of the Indian Cancer Society (ICS), a community-level survey was
designed to evaluate the level of BC awareness among women from diverse sections of society
in Delhi from May 2013-March 2014. In particular, we focused on perceptions about the causes
of BC, risk factors associated with BC, knowledge of BSE, clinical breast examination (CBE)
and mammography, and knowledge of BC treatment. Women participants were drawn from
people who came to attend a BC awareness campaign organized by the ICS. As a part of this
campaign, workshops were purposively organized in various parts of Delhi by ICS. Ethical
permission to conduct the study was granted by the Public Health Foundation of India’s (PHFI)
Institutional Ethics Committee (TRC-IEC-165/13). At participating institutions, a trained research
assistant described the purpose of the study and emphasized the confidentiality and anonymity
of the responses. Verbal consent was taken and participants who freely agreed to participate
were enrolled in this study. A total of 2017 volunteered and completed the survey. Study tool
We used a semi-structured self-administered questionnaire to collect the data. Questionnaires
were administered before the workshops. Only women, 16 years or older were included in the
survey. We did not have any refusals to participate although a small proportion of women did
provide incomplete information. In the case where participants were unable to fill out the
questionnaire (due to illiteracy), an interviewer assisted the respondent.
Data management and statistical analysis
The data collected were entered in Excel 2010. Different numerical codes were given for
different responses and categories. The self-reported average monthly family expense was
used to assess the socio-economic status (SES) of the respondent. Respondents who reported
average monthly family expenses 10 to 25,000 per month and 26 to 50,000 per month, were
respectively classified in lower middle and middle SES categories while who reported average
monthly expenses <10,000 and >50,000 were respectively classified as low and upper-middle
SES categories. The SES classification was based on quartiles. Regarding questions related to
BC awareness, each correct answer on the questionnaire was assigned a score of 1 (aware),
while an incorrect answer was awarded a score of 0 (not aware). A total score for each
participant was computed by summing the number of correct answers. We had a total of 40
questions and the median awareness score (19) was used as a cut-off point. Based on cut-off
points participants were categorized as more aware (scores above 19) and less aware (scores
of 19 and less). Also, for each response, the proportion of women who knew the right answer
(or were aware) was calculated to give us the prevalence of awareness. After cleaning and
checking for consistency, data were exported into analytical software IBM Statistical Product
and Service Solutions (SPSS) version 20 for analysis. Pearson Chi-square tests were used to
examine the association between socio-demographic variables and BC awareness score.
Statistical significance was assessed at P<0.05. Associations of BC awareness score with
various factors were investigated using bivariate analysis and multivariate logistic regression
which provided odds ratios (OR) and 95% confidence intervals (CIs).
Results
Socio-demographic profile
A total of 2017 women from diverse sections of society participated in the study and provided
information for an assessment of awareness related to various aspects of BC. The age group of
women ranged from 14-75 years (Mean=30.7 years, SD=10.5) (Table 1). A majority (86.5%) of
women were below 50 years. Most of the women were married (62.6%) and Hindu (75.8%) by
religion. Most women were graduates (51.2%), employed (51.6%), and belonged to nuclear
families (57.2%). Women were almost equally distributed in the lower-middle and lower
categories (35%). Regarding the average number of visits to health care providers, the majority
(61.5%) of the respondents had reported 0 to 2 visits per year, and most (78.3%) of the
respondents reported good quality of life (Table 1).
Associations of BC Awareness and Socio-demographic

factors
Table 1 also explores the associations between awareness scores and various socio-
demographic factors. 817 (40.5%) women were more aware (awareness score > median score
of 19). Awareness status was found to be associated with different age groups (P < 0.0001),
religions (P=0.005), education status (P < 0.0001), occupations (P < 0.0001), average monthly
expenses (P < 0.0001), visits to health care provider (P < 0.0001) and self-assessed quality of
life (P < 0.0001) (Table 1). Prevalence of awareness regarding various aspects of BCGeneral
perception about BC: Regarding general perception, it was noticeable that 49.1% of women
thought
Table 1. Distribution of Socio-demographic Factors and their Association with Awareness
Scores Among 2017
Women in Delhi
Socio-demographic factors Awareness status* P-value#
Total (N = 2017) More aware Less aware
(N = 817; 40.5%) (N = 1200; 59.5%)
N (%) n (%) n (%)
Age (30.71±10.48)
<18 166 (8.2) 70 (8.6) 96 (8.0) <0.0001
18 to <30 789 (39.1) 257 (31.5) 532 (44.3)
30 to <50 956 (47.4) 431 (52.8) 525 (43.8)
≥ 50 106 (5.3) 59 (7.2) 47 (3.9)
Marital status
Married 1262 (62.6) 525 (64.3) 737 (61.4) 0.43
Unmarried 670 (33.2) 259 (31.7) 411 (34.2)
Other 85 (4.2) 33 (4.0) 52 (4.3)
Religion
Hindu 1529 (75.8) 639 (77.8) 893 (74.4) 0.005
Muslim 215 (10.7) 65 (8.0) 150 (12.5)
Other 273 (13.5) 116 (14.2) 157 (13.1)
Education
High school and less 863 (42.8) 200 (24.5) 663 (55.2) <0.0001
Graduation 1032 (51.2) 532 (65.1) 500 (41.7)
Post-Graduation 122 (6.0) 85 (10.4) 37 (3.1)
Occupation
Employed 1041 (51.6) 486 (59.5) 555 (46.2) <0.0001
Housewife 485 (24.0) 143 (17.5) 342 (28.5)
Student 491 (24.3) 188 (23.0) 303 (25.2)
Family type
Nuclear 1119 (57.2) 435 (55.0) 684 (58.6) 0.112
Joint 839 (42.8) 356 (45.0) 483 (41.4)
Average monthly expense (in INR)
Low (<10,000) 392 (19.4) 37 (4.5) 355 (29.6) <0.0001
Lower-middle (10-25,000) 706 (35.0) 289 (35.4) 417 (34.8)
Middle (26-50,000) 704 (34.9) 359 (43.9) 345 (28.8)
Upper-middle (>50,000) 215 (10.7) 132 (16.2) 83 (6.9)
Visit to health care provider
0-2 1140 (61.5) 554 (73.2) 586 (53.5) <0.0001
<5 429 (23.2) 121 (16.0) 308 (28.1)
≥5 284 (15.3) 82 (10.8) 202 (18.4)
Self-assessed quality of life
Poor to average 398 (21.7) 117 (15.2) 281 (26.4) <0.0001
Good 1436 (78.3) 652 (84.8) 784 (73.6)
BC is incurable and 60.4% of women thought that BC is inheritable. However, very few women
thought that because was due to supernatural causes related to disregard of religious or cultural
factors (93.4% aware) (Table 2). Knowledge related to BC epidemiology & risk factors70.9% of
women reported BC as an uncommon disease in India (Table 2). Most of the women were
aware that dietary and lifestyle-related factors affect BC risk (70.3%). Although most women
also thought that having a risk factor for BC meant having BC (69.2%). 70.9% of women thought
that family history is the main risk factor for BC which is consistent with our earlier finding of
most women considering BC to be inheritable. Most of the women (66.4%) fallaciously reported
breast augmentation and wearing underwire bras can cause BC. Interestingly 75.3% of women
were found aware of breastfeeding as a preventive measure of BC (Table 2).
Knowledge about BC symptoms
Although 84.5% of women were aware of BC usually presenting as a lump, 73.9% women
spuriously believed pain to be an initial sign of BC. Most women were also not aware that BC
can also present without a lump (62.9%) (Table 2).
Table 2. Number and Proportion of Women Aware about Various Aspects of Breast Cancer
(BC) among 2017
Women in Delh
Items Aware Not aware Total % of
n (%)* n (%)* N 2017#
General Perception about BC
Breast cancer is an incurable disease. [Cannot be cured] 1007(50.9) 970(49.1) 1977 98
Breast cancer is contagious / communicable / infectious 1727(87.6) 244(12.4) 1971 98
Breast cancer is usually inherited. 777(39.6) 1183(60.4) 1960 97
Breast cancer can be caused by,
supernatural causes of due to disregard to religious or cultural factors 1848(93.4) 130(6.6)
1978 98
Certain breast conditions, like harmless breast lump, can predispose to BC 1548(80.0) 388(20)
1936 96
Cancer cannot be cured 1007(50.9) 970(49.1) 1977 98
Cancer cannot be prevented 1007(50.9) 970(49.1) 1977 98
Knowledge related to BC epidemiology and risk factors
Breast cancer is uncommon in India 581(29.1) 1417(70.9) 1998 99
Only women get breast cancer 656(33.1) 1315(66.9) 1981 98
Breast cancer occurs more commonly in older women, aged 40 years and older 975(49.3)
1004(50.7) 1979 98
Chance of breast cancer is increased by,
dietary and lifestyle related factors, e.g. sedentary, lifestyle high fat diet, drinking 1390(70.3)
588(29.7) 1978 98
Having a risk factor for breast cancer means I will get breast cancer 600(30.8) 1349(69.2) 1949
97
A family history of breast cancer is the main risk factor for getting breast cancer 571(29.1)
1394(70.9) 1965 97
Breast augmentation, under wire bras can cause breast cancer 653(33.6) 1292(66.4) 1945 96
If I do certain chest /breast exercises, I will never get breast cancer 649(33.5) 1288(66.5) 1937
96
Being physically active early in life may reduce the risk of developing breast cancer 1263(65.0)
680(35) 1943 96
Obesity [ being very fat] increases one’s risk of getting breast cancer 973(49.9) 976(50.1) 1949
97
Breast feeding is protective against breast cancer 1464(75.3) 480(24.7) 1944 96
Knowledge about BC symptoms
Breast cancer usually presents as a lump 1647(84.5) 303(15.5) 1950 97
Pain is one of the initial sign of breast cancer. 508(26.1) 1440(73.9) 1948 97
All breast lumps are breast cancer 1516(77.9) 429(22.1) 1945 96
Breast cancer can also present without any palpable breast lump 719(37.1) 1217(62.9) 1936 96
Knowledge of BC detection methods
With appropriate technology, breast cancer can be detected in early stage 831(42.7) 1117(53.3)
1948 97
Early detection improves one’s survival from breast cancer. 1755(90.1) 193(9.9) 1948 97
Observing one’s breast is useful in early detection of breast cancer 1730(89.0) 214(11) 1944 96
Have you heard of: Clinical Breast Examination (CBE) 959(50.1) 955(49.9) 1914 95
Have you heard of breast self-examination (BSE) 1118(58.5) 793(41.5) 1911 95
Have you heard of mammography 1101(57.3) 820(42.7) 1921 95
Detection practice
Do you perform BSE currently? 692(34.9) 1290(65.1) 1982 98
Have you undergone CBE / Mammography in last two years? 140(6.9) 1877(93.1) 2017 100
If you were told that CBE / Mammography is helpful in early detection of breast cancer would
you undergo CBE / mammography?
1294(66.1) 664(33.9) 1958 97
If you were told that CBE / Mammography is helpful in early detection of breast cancer would
you undergo CBE / mammography?
1294(66.1) 664(33.9) 1958 97
Knowledge of BC treatment
Breast cancer is curable when detected early and treated appropriately and adequately.
1729(89.1) 211(10.9) 1940 96
Surgery / operation for breast cancer means removal of entire breast. 507(26.2) 1428(73.8)
1935 96
Alternative healthcare modalities like yoga/ayurveda etc. have been proven to cure breast
cancer
402(20.9) 1523(79.1) 1925 95
What would you do if you find a lump in your breast? 1684(89.4) 199(10.6) 1883 93
*Row percentages of actual total for each item. #Percentage of the full sample size of 2017 - to
depict missing information for each item
Knowledge of BC detection methods
Almost all women (90%) were found to be aware of the importance of early detection of BC
(Table 2). But this awareness was lacking regarding the specifics related to early detection of
BC. Only about half of the women (49.9%) were aware of clinical breast examination (CBE).
Even less (41.2%) were aware of BSE and mammography (42.7%). When asked about the
source of information for CBE/BSE/Mammography majority of women reported media (25%),
health worker (19%) and friends or family (14%) as a source of information (Figure 1).
Figure 1. Source of Information for Breast Cancer Detection Methods
Table 3. Unadjusted and Adjusted Odds Ratios (ORs) and 95% Confidence Intervals (CIs) of
Awareness Scores in Association with Various Factors among 2017 Women in Delhi
Socio-demographic factors Unadjusted Adjusted*
OR 95% CI OR 95% CI
Age (30.71 ± 10.48)
<18 1 - 1 -
18 to <30 0.66 0.47-0.93 0.26 0.16-0.42
30 to <50 1.13 0.81-1.57 0.38 0.22-0.67
≥ 50 1.72 1.05-2.82 0.45 0.22-0.91
Religion
Hindu 1 - 1 -
Muslim 0.61 0.45-0.83 1.88 1.25-2.81
Other 1.04 0.8-1.35 0.87 0.63-1.2
Education
High school and less 1 - 1 -
Graduation 3.53 2.89-4.31 2.38 1.78-3.17
Post-Graduation 7.62 5.02-11.6 7.06 4.14-12.1
Occupation
Employed 1 - 1 -
Housewife 0.48 0.38-0.6 0.79 0.59-1.07
Student 0.71 0.57-0.88 0.49 0.33-0.72
Average monthly expense
<10,000 1 - 1 -
10-25,000 6.65 4.59-9.63 4.2 2.72-6.5
26-50,000 9.98 6.9-14.5 6 3.82-9.42
>50,000 15.3 9.87-23.6 6.97 4.11-11.8
Visit to health care provider
0-2 1 - 1 -
<5 0.42 0.33-0.53 0.53 0.4-0.7
≥5 0.43 0.32-0.57 0.78 0.55-1.1
Self-assessed quality of life
Poor to average 1 - 1 -
Good 2 1.57-2.54 1.72 1.29-2.28
*Adjusted for all variables in the table using an unconditional logistic regression model
Detection practice
In contrast to knowing “BC detection methods”, detection practice was woefully lacking (Table
2). Only 34.9% of women reported that they performed BSE and very few (6.9%) women were
found to have undergone CBE/ mammography, although most women (66.1%) women were
willing to undergo early detection (Table 2).
Knowledge of BC treatment
The majority of women (89.1%) are aware of BC being curable if detected early and treated
appropriately and adequately (Table 2). On investigating specific areas of BC treatment though
a majority (73.8%) of women believed that surgery/operation for BC means removal of the entire
breast. 79.1% of women also thought that alternative healthcare modalities like yoga/ayurveda
could result in a BC cure. Also, on asking what women would do if they found a lump, most
women (89.4%) responded that they would visit an allopathic doctor (Table 2). Socio-
demographic profile and BC awareness status Table 3 depict the associations between BC
awareness scores and various sociodemographic variables using unconditional logistic
regression. BC awareness was found to increase with increasing age compared to the lowest
age group [18 to < 30 (OR=0.26; CI=0.16, 0.42), 30 to < 50 (OR=0.38; 95% C I=0.22, 0.67) and
≥ 50 (OR=0.45; 95%CI=0.22, 0.91)] but remained lower than the youngest age group.
Compared to Hindus, Muslim women were more aware (OR=1.88; CI=1.25; 2.81). Predictably
BC awareness increased with education. On comparing with high school or less education,
graduates (OR=2.38; 95%CI=1.78, 3.17) and post-graduates (OR=7.06; CI 4.14, 12.1) were
more aware. Compared to employed women, students (OR=0.49; 95%CI=0.33, 0.72) were less
aware. Awareness increased with average monthly expenditure compared to the lowest levels
(<10,000) [10-25,000 (OR=4.2; 95%CI=2.72, 6.5), 26-50,000 (OR=6; 95%CI=3.82, 9.42) and
>50,000 (OR=6.97; 95%CI=4.11, 11.8)]. Increasing visits to the health provider did not increase
awareness [< 5 visits per year (OR=0.53; 95%CI=0.4, 0.7) compared to 0-2 visits]. Awareness
increased with higher self-assessed quality of life [good (OR=1.72; 95%CI=1.29, 2.28).
Discussion
To summarize our findings, in our sample of 2017 women from the city of Delhi, most of whom
were below 50, Hindu, married, and belonging to lower-middle and lower socioeconomic
classes with good quality of life, we discovered that around half of the women thought cancer
was incurable. Around 70% of women were aware of the dietary and lifestyle risk factors of BC,
thought BC to be inheritable, and feared getting BC if they had a risk factor for it. More than
70% of women also knew about the protective effects of breastfeeding. While more than 80% of
women knew that BC presented as a lump, more than 70% also thought pain to be an initial
symptom of BC. While 90% of women were aware of the importance of detecting BC early, less
than half knew about CBE, BSE, or mammography. Prevalence of practicing early detection
methods was much lower, with only around 35% of women currently conducting BSE and a
dismal 7% having undergone any CBE/mammogram in the last 2 years. Women had some
misconceptions regarding BC treatment with more than 70% of women believing that the entire
breast is removed during surgery and almost 80% believing that alternative therapies held a
cure for BC. Associations with various sociodemographic factors depicted that BC awareness
tended to increase with age, was higher among Muslim women, increased with education,
higher among employed women, increased with income was less with higher visits to healthcare
providers and is better in those women with a higher quality of life. To the best of our
knowledge, this is the largest community-based study from India to assess BC awareness in
women. The findings indicated that the level of awareness of BC is sub-optimal among the
women participants of Delhi. Our overall results are consistent with studies done in other parts
of India (Somdatta and Baridalyne, 2008; Gupta, 2009; Khokhar, 2009; Yadav and Jaroli, 2010;
Shalini et al., 2011; Grosse et al., 2013; Parameshwari et al., 2013; Sathian et al., 2014;
Sreedevi et al., 2014) and the world (Okobia et al., 2006; Elsie et al., 2010; Forbes et al., 2011;
Samganje and Mafuvadze, 2012; Radi, 2013; Karadag et al., 2014; Kratzke et al., 2014; Asif et
al., 2014). Among the few weak studies that have been carried out in India to assess awareness
about BC and its prevention, Shalini et al. (2011) discovered that in a sample of 40 young
college-going girls, approximately 72% had an average idea of BSE and only 1 out of 40 girls
performed BSE. In another small study by Yadav et al. (2010) done in the North-Indian state of
Rajasthan, all college-going women were aware of BC although 28% did not know about BSE.
In a cross-sectional study of 441 school teachers in Delhi regarding BC awareness only 36.1%
of teachers had heard about BSE and very few knew about the details of doing a BSE and none
had ever done a BSE (Khokhar, 2009). In our study, 41% of women knew about BSE but only
around 35% practiced it. Half of the women in our study were graduates and employed which
might explain a higher prevalence of BSE practice compared to school teachers. BSE practice
is high if women are employed especially in health-related professions (Sreedharan et al., 2010;
Andsoy and Gul 2014). Apart from BSE, participation in other early detection/screening
practices was quite low in this sample. Only 6.9% of women had undergone
CBE/mammography in the previous 2 years. Thus although half of the women in the sample
were graduates and employed, early screening/early detection behaviour did not correlate with
it. This is however in the context of India where BC screening/early detection is not provided at
the population level by the health system. Even in countries such as Malaysia, where screening
and early detection are standard provisions in the health system, studies discovered 10.5%
(Rosmawati et al., 2010) to 13.6% (Parsa and Kandiah, 2010) to 19% (Kanaga et al., 2011)
women having had undergone mammography with slightly higher numbers for CBE underlining
the need for increasing awareness. In our study, we also discovered that college-going and
younger women had the highest levels of awareness. Awareness in higher ages was lower than
in the lowest age group but kept on increasing with age. These findings are similar to other
studies which found higher awareness among college-going young women (Shalini et al., 2011).
However other studies have depicted an increase in awareness with age such as a study on
Nigerian women which has found that older women (≥50) appear to have higher awareness
scores compared with younger women (Okobia et al., 2006). Had only 10.7% Muslim women in
our study. Multivariate results showed that Muslims had better awareness regarding BC
compare to Hindu, a finding which was absent in unadjusted results. On deeper investigation it
was revealed that participants from the area of residence of Muslim women had benefitted from
BC screening program organized by ICS earlier, consequently they were found more aware
compare to Hindu and other religion’s participants. This was a consequence of the purposive
nature of selecting areas for conducting workshops where ICS had a better community
presence, which might be a potential limitation. In our study, it was observed that women
belonging to high SES had better knowledge about BC, and it’s in agreement with the study
done by Samina Khokher et al., (2011) which reveals that BSE frequency is highest among
professional women on the job (57%) and most educated women groups (46.9%). A recent
study was done on awareness of BC warning signs and screening methods among female
residents of Pokhara Valley in Nepal by Sathian et al. (2014) also supported the finding by
adding that high SES women are exposed to health-related issues through mass media,
internet, and a better socioeconomic status enables support of screening services. Similar
associations with SES were observed by Kanaga et al. in Malaysia (Kanaga et al., 2011) and
Amin et al. in Saudi Arabia when enquiring about BC gene testing awareness (Amin et al.,
2012). It’s evident from different studies done on BC awareness (Gupta, 2009; Al-Naggar et al.,
2011; Rasu et al., 2011; Kanaga et al., 2011; Khokher et al., 2011; Kumar et al., 2011; Grosse
et al., 2013; Karadag et al., 2014; Sreedevi et al., 2014) that there has been positive association
between BC awareness and educational status, including the present study. Grosse et al.
(2013) has emphasized that education programs, aiming to increase BC awareness should
target less educated women to increase their knowledge about BC. In our study, about 30% of
women belonged to low-income level and we endeavored to include more women from this
section of society in our study and the BC awareness workshops. Parallel to our findings, the
study done by Samina Khokher et al., (2011) revealed that only ~29% of respondents correctly
identified “family history” as a risk factor for the BC. Unlike previous studies, 75.3% of Delhi
women were found aware of breastfeeding as a preventive measure of BC. While Aswathy
Sreedevi et al., (2005) and Sambanje et al. (2012) have noted in their studies that less than
9.85% and 8% respectively were aware of breastfeeding in reducing BC risk. This might be
because we had urban women in our sample, almost half of whom were graduates and
employed. Regarding BC symptoms majority (73.9%) of women in the current study reported
pain as the initial sign of BC. This is similar to the figures reported by Powe et al., (2005) and is
a widespread misconception as most people associate pain with the occurrence of cancer. The
fact is that pain is not necessarily an early sign of BC (Sambanje and Mafuvadze, 2012). A
study done by Ukwenya et al. (2008) reported from Nigeria that a majority of BC patients cited
ignorance of the seriousness of a painless lump as a reason for the prolonged delay before
seeking medical advice. A large percentage of women (90.1% and 89.1% respectively) were
found aware of the fact that early detection improves one’s survival from BC and that BC is
curable if detected early and treated appropriately and adequately. This is in agreement with the
studies done by Khokher et al., (2011) and Okobia et al., (2006) in which most Pakistani women
(81%) and a lesser number of Nigerian women (41%) were respectively found aware of life-
saving benefits of early detection. 80.7% of women in Saudi Arabia were also aware that cancer
is curable if detected early (Ravichandran et al., 2010). However, we must also note that the
awareness about ‘cancer being curable if detected early’ is in conjunction with the fact that
simultaneously nearly half of the women think BC to be incurable. This is probably because very
few women had seen other women cured of BC and survive the disease. While there have been
widespread campaigns in India that have spread the message that cancer is curable if detected
early, when it comes to the specifics nearly 74% of women also thought that surgery for BC
meant the removal of the entire breast. Maybe it is this fear of treatment that led nearly 80% of
women to believe that alternative healthcare modalities have a cure for BC, which are known to
have fewer side effects and no surgery or chemotherapy. Previous studies have found an
association between having advanced BC and belief in alternative medicine (Norsa’adah et al.,
2012). The high belief in alternative medicine is because more than 89% of women replied
rightly that they will visit an allopathic doctor if they found a breast lump. Some of these
contradictions in perceptions need to be investigated more deeply to further dissect the various
complex aspects related to BC awareness. As far as the source of information is concerned,
media (25%) was found to be one of the most important sources of information for BC
awareness. This is in agreement with the study done in Delhi (Somdatta, 2008) where 80% of
women reported television as one of the most important sources of information for BC
awareness. A study done in an educational institute in Lahore, Pakistan also reported television
(14%) and hearsay (17%) as the most common sources of information (Khokher, 2011). Similar
evidence exists about electronic media and television being the most important sources of
information on BC (Montazeri et al., 2010; Ravichandran et al., 2010; Yoo et al., 2012) followed
by relatives and friends for less educated women (Hatefnia et al., 2010). Also, the odds of being
aware decreased with increasing visits to the health provider. This concurs with the known
evidence and is contrary to the expected prevalent opinion. In general, healthcare providers are
not known to be knowledgeable about screening/early detection techniques, nor do they
encourage women to apply screening/early detection behavior (Akhigbe and Omuemu, 2009;
Harirchi et al., 2009). In a study in Korea by Yoo et al. only 17.2% of women had been
recommended by medical staff to practice BSE (Yoo et al., 2012). The most important strength
of this study was the large sample size of more than 2000 women. To the best of our
knowledge, this is larger than any other study on BC awareness in India so far. However, this
study also had some limitations. We had organized this study around workshops organized by
ICS which were purposive. Although we involved many sections of society in this study, the
sample of our study may not be strictly random. Also, we had nearly 50% of women were
graduates or employed. As such, the awareness levels we found in our study might be
representative of urban areas of India but cannot be generalized to other parts of India. Also for
the same reason, it is quite possible that the prevalence of awareness observed in our study is
higher than what might be expected in other parts of India. As such, it can be expected that BC
awareness might be much lower in less urban parts of India which is a matter of concern. This
warrants further investigation and might require more intense interventions if BC awareness is
to be increased in less urban and non-urban populations. In conclusion, the present study
covers a diverse section of women at the community level in Delhi, the capital of India. The
findings of the study revealed that BC awareness of women in Delhi was suboptimal and was
associated with low SES and education levels. Less than half of the women were aware of BC
detection methods but the prevalence of practice was much lower, especially CBE or
mammography. There is an urgent need to increase the awareness of women regarding BC and
BSE so that BSE may become a routine practice among women. In our previous studies in
other LMICs, we have depicted that regular practice of BSE increases early presentation among
women substantially (Stapleton et al., 2010). There are lots of myths and stigmas related to BC
in society and awareness needs to be drastically increased as a first step in the fight against
BC. Lack of evidence on the interventions to promote cancer awareness and improve early
presentation has been dampening the development of policy and action especially in LMICs
(Austoker et al., 2009). Active steps should be taken to generate more evidence regarding BC
awareness and the necessary interventions to increase awareness such as BC awareness
campaigns via community outreach - especially in low socioeconomic sections of society,
educating healthcare providers, and use of electronic media to reduce the BC burden.
Acknowledgments
This work was supported by a Welcome Trust Capacity Strengthening Strategic Award to the
Public Health Foundation of India and a consortium of UK universities (research grant number
F012/RG/SD/15).
References
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OTUD4-mediated GSDME deubiquitination enhances radiosensitivity in nasopharyngeal

carcinoma by inducing pyroptosis
Muping Di1,2,3†, Jingjing Miao1,2†, Qiuzhong Pan1,3†, Zonglong Wu4†, Boyu Chen1,2, Muru
Wang5, Jingjing Zhao3, Huageng Huang2, Jiewen Bai1, Qijing Wang3, Yan Tang3, Yongqiang
Li3, Jia He3, Tong Xiang1,3, Desheng Weng3, Lin Wang1,2*, Jianchuan Xia1,3* and Chong
Zhao1,2*
Abstract
Background: Radioresistance is the primary cause of nasopharyngeal carcinoma (NPC)
treatment failure. Previous studies have focused on the deficits in cellular apoptosis as a
mechanism for radioresistance; however, additional potential death modes involved in
modulating the radiosensitivity of NPC have not been explored.
Methods: Pyroptosis was assessed by phase-contrast imaging, LDH release assays, live cell
imaging, and Western blotting. In vitro and in vivo assays were used to investigate the function
of gastrin E (GSDME) and ovarian tumor family deubiquitinase 4 (OTUD4). NPC tissues were
analyzed using Western blotting, immunohistochemistry, and real-time PCR. The molecular
mechanism was determined using immunoprecipitation assays and mass spectrometry.
Results: Live cell imaging revealed that 40—75% of irradiation-induced dead NPC cells were
pyroptotic cells. Furthermore, irradiation-induced pyroptosis is triggered by GSDME, which is
cleaved by activated caspase-3 in the intrinsic mitochondrial pathway. Additionally, GSDME was
significantly downregulated in radioresistant NPC specimens. Low GSDME expression was a
predictor of worse prognosis and conferred NPC radioresistance both in vitro and in vivo.
Mechanistically, OTUD4 deubiquitinated and stabilized GSDME, enhancing the radiosensitivity
of NPC cells by promoting pyroptosis. Clinically, OTUD4 was significantly correlated with
GSDME in NPC biopsies, and patients with low expression of both OTUD4 and GSDME
suffered the worst radiotherapy response and survival.
Conclusions: GSDME-dependent pyroptosis is a critical determinant of radiosensitivity in NPC,
and is modulated by OTUD4 via deubiquitinating and stabilizing GSDME. These findings reveal
a promising novel direction to investigate radioresistance and suggest potential therapeutic
targets for sensitizing NPC to radiotherapy.
Keywords: OTUD4, GSDME, Pyroptosis, Radioresistance, Nasopharyngeal carcinoma
Background
Radiotherapy (RT) is the most common treatment for nasopharyngeal carcinoma (NPC), a
malignant tumor which derives from the nasopharyngeal epithelium [1]. Due to the widespread
application of intensity-modulated radiation therapy (IMRT), the locoregional disease control and
overall survival of patients with NPC have considerably improved [2]. Nevertheless, 10–17% of
NPC patients sufer local recurrence, even in early-stage NPC
The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution
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in this article unless otherwise stated in a credit line to the data.
Muping Di, Jingjing Miao, Qiuzhong Pan, and Zonglong Wu these authors contributed equally to
this work.
*Correspondence: wangl1@sys
ucc.org.cn3; xiajch@mail.sysu.edu.cn; zhaoch@mail.sysu.edu.cn
Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China,
Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China’s Full list of
author information is available at the end of the article
3, 4]. Radioresistance is a major barrier to the successful treatment of NPC [5]. Therefore,
uncovering the underlying mechanisms of radioresistance in NPC is crucial. Pyroptosis, an
inflammatory programmed cell death, is executed by the gasdermin protein family [6–10]. Gas-
determine family members contain a similar amino (N)-terminal domain structure, possessing
pore-forming activity when cleaved by cleaved-caspases or granzymes [11, 12]. Apoptosis has
been widely recognized and studied as a mechanism for regulating cell death in antitumor
treatment responses over the past few decades [13, 14]. Recently, growing evidence has
confirmed the antineoplastic effects of pyroptosis in chemotherapy [9, 15–19], targeted therapy
[20], and immunotherapy [21]. Pyroptosis differs from apoptosis in that it involves cytolysis, as
indicated by large bubbles erupting from a swollen cell membrane, which liberates cell contents,
including various inflammatory molecules, resulting in the activation of antitumor immunity [10,
22]. Moreover, pyroptosis triggered by chemotherapy drugs and radiotherapy was found to be
associated with the toxicity of chemotherapy drugs and radiation injury in normal tissues [9, 23–
25]. Pyroptosis and apoptosis are two different ways of cell death [26]. The proportion of dead
cells after irradiation represents the radiosensitivity of cancer cells. Tus, pyroptosis probably
affects the radiosensitivity of NPC cells. Nevertheless, the role of pyroptosis in NPC
radiotherapy remains elusive. Ubiquitination and deubiquitination are vital post-translational
modifications regulating various critical cellular processes [27]. Deubiquitination is the
functionally reciprocal process of ubiquitination that prevents protein proteasomal degradation
through deubiquitinating enzymes (DUB)-mediated removal of ubiquitin chains from target
proteins [28]. Ovarian tumor family deubiquitinase 4 (OTUD4), a member of the OTU family,
preferentially cleaves K48-conjugated polyubiquitin chains and participates in the
deubiquitination of multiple proteins [29–31]. Viral infection causes the IRF3/7-dependent
induction of OTUD4, which binds to MAVS to cleave K48-conjugated ubiquitin chains, stabilizing
MAVS and activating antiviral innate immune responses [31]. Recently, OTUD4 has been
identified to be down-regulated in multiple human tumor types, and lower OTUD4 expression
indicates a poor prognosis [32]. However, the expression and functional role of OTUD4 in NPC
remains largely unknown. In this study, we identified that radiotherapy-induced gastrin E
(GSDME)-dependent pyroptosis in NPC cells through the intrinsic mitochondrial apoptotic
pathway. Low GSDME expression could predict worse prognosis and was associated with
radioresistance both in vitro and in vivo. In addition, we demonstrated that OTUD4
deubiquitinated and stabilized GSDME, enhancing radiosensitivity in NPC by promoting
GSDME-dependent pyroptosis. Tese novel findings indicate a possible direction to investigate
radiosensitivity, and open new avenues to sensitize NPC to radiotherapy by targeting
pyroptosis.
Materials and methods
Cell culture
Sun Yat-sen University Cancer Center (State Key Laboratory of Oncology in South China,
Guangzhou, China) provided the NPC cell lines HK1, SUNE1, HONE1, HNE1, 5-8F, 6-10B and
CNE1, melanoma cell lines A375 and SK-MEL-30, esophageal cancer cell lines KYSE30 and
KYSE250, prostate cancer cell lines PC3 and 22RV1, as well as breast cancer cell lines 159
and 231. Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium
(Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (HyClone, Logan,
UT, USA) plus 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). HEK293T cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA)
supplemented with 10% FBS. All cells were cultured and maintained in a humidified atmosphere
with 5% CO2 incubator at 37 °C.
Clinical tissue samples and patient information
The Institutional Review Board of Sun Yat-Sen University Cancer Center approved the present
study (Approval No. GZR2020-193). Between 2011 and 2015, 150 paraffin-embedded tumor
specimens were obtained from pathologically diagnosed, non-metastatic NPC patients who
were treated with radical radiotherapy or combined chemoradiotherapy at the Sun Yat-Sen
University Cancer Center. Serum LDH levels were measured by the clinical laboratory of Sun
Yat-sen University Cancer Center. Nasopharyngoscopy images and magnetic resonance
images were provided by the Nasopharyngeal Carcinoma Department and Medical Imaging
Department, respectively. Radiosensitive NPC patients were outlined as ones with complete
regression after radical radiotherapy or without relapse after the end of radiation therapy.
Radioresistant NPC patients were outlined as ones with incomplete regression of lesions after
radical radiotherapy; residual tumors at more than six weeks after the end of radiation therapy;
or relapse after radical radiation therapy. The fresh NPC samples were quickly frozen in liquid
nitrogen and stored at -80 °C until use. The detailed clinical information of the samples is
summarized in Supplementary Table S1.
Immunohistochemistry (IHC)
Immunohistochemical staining was performed using GSDME antibody (#ab175614, Abcam) and
OTUD4 antibody (#HPA036623, Sigma) on 150 paraffin-embedded NPC tissue sections. The
stained tissues were examined by two independent pathologists. Evaluation Principles included
the proportion of tumor cells and staining intensity. The proportion of tumor cells was scored as
follows: 0 (no positive tumor cells); 1 (<10% positive tumor cells); 2 (10–35% positive tumor
cells); 3 (35–75% positive tumor cells); 4 (>75% positive tumor cells). The staining intensity was
graded according to the following criteria: 1 (absent or weak staining=light yellow); 2 (moderate
staining=yellow brown); 3 (strong staining=brown). The staining index (SI; values 0‐12)=the
proportion of tumor cells staining intensity. Tissues with an SI≥6 were divided into high
expression, while tissues with an SI score below 6 were divided into low expression. We used
the Kaplan–Meier method to estimate locoregional recurrence-free survival (LRRFS) and
progression-free survival (PFS); the log-rank test to assess survival differences between
groups.
Plasmids, transfection, and siRNA knockdown
NPC cells were infected using lentivirus generated by the pEGFP-N1 vector for 72 h, and the
GFP-positive cells were collected on MoFlo Astrios (Beckman Coulte). Polymerase chain
reaction (PCR) was used to amplify the cDNA fragments of GSDME and OTUD4, which were
then cloned into vector pLVX-IRES-Hyg. Short-hairpin RNAs (shRNAs) targeting GSDME and
OTUD4 were cloned into the vector pSuper-retro-neo. Stable cell lines with GSDME/OTUD4
overexpression and GSDME/OTUD4 knockdown were generated via infection of retrovirus
packaged from HEK293T cells. The viral supernatants were infected into target cells for 72 h,
and then corresponding antibiotics were used to select stable cell lines for 9 days. Specific
siRNAs (5′-GATGATGGAGTATCTGATCTT3′ and 5′-GGAUUGAUGAGGAGGAAUUTT-3′) were
used to knock down GSDME and GSDMD expression respectively, following the manufacturer’s
protocol. The efficiency of depletion was confirmed by Western blotting. shRNA target
sequences are listed in Supplementary Table S4.
Microscopy
To observe the morphology of pyroptotic, apoptotic, and surviving cells, cells were seeded in
dishes at an appropriate density and received the indicated treatments. Cell images were
acquired using an Olympus IX73 microscope. To capture the dynamic process of cell death,
EGFP-expressing cells were seeded up to appropriate density into 35 mm glass-bottom culture
dishes, and then videos were taken using a CV1000 Confocal Scanner System (Yokogawa,
Tokyo, Japan) in 50 randomly selected fields of view. Pyroptotic, apoptotic, and surviving cells
were counted and analyzed using CV1000 Software.
Flow cytometry
Cells were subjected to the indicated treatments and then were collected, and stained with the
Annexin V-Alexa647 and propidium iodide (PI) Apoptosis Kit (Yishan Biotech) following the
manufacturer’s instructions. Stained cells were analyzed on a FACSort instrument (BD
Biosciences) with CellQuest software.
Cell viability assay
The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) kit (Sigma) was
used to measure cell viability following the manufacturer’s instructions.
Western blotting
Cells and tissue samples were lysed in RIPA buffer with proteinase inhibitors (Sigma) and
phosphatase inhibitors (Sigma). Cellular fractionation was performed using the Qproteome Cell
Compartment Kit (Qiagen). Proteins were detected using the following primary antibodies:
GSDME (#ab215191, Abcam), GSDMD (#ab210070, Abcam), Caspase-3 (#9665, CST), PARP
(#9532, CST),
See figure on next page.)
Fig. 1 Radiotherapy induces GSDME-dependent pyroptosis in NPC cells through the intrinsic
mitochondrial apoptotic pathway. A-B Six NPC cell lines were treated with or without 6 Gy
irradiation. 72 h later, (A) cell morphological changes and (B) LDH release was measured. C
Typical processes of cell pyroptosis, apoptosis, and survival in HNE1 cells are shown.
Brightfield and fluorescent images of stable EGFP-overexpressing NPC cells were taken at
specific time points post-6 Gy irradiation. D Percentage of pyroptotic, apoptotic, and viable cells
in six NPC cell lines within 72 h after 6 Gy irradiation is indicated. E GSDME silencing inhibited
pyroptosis (left), LDH release (upper right), and cell death (bottom right) in HONE1 cells at 72 h
after 6 Gy irradiation. F Western blotting revealed the levels of indicated proteins in six NPC cell
lines at 72 h after 6 Gy irradiation. Cyto C: cytochrome c. G Cell morphological changes (top),
LDH release assay (bottom left), live cell imaging (bottom right), and (H) the levels of indicated
proteins were assessed in HONE1 cell lines at the indicated time points after irradiation (6 Gy).
(I) Cell morphological changes (top), LDH release assay (bottom left), live cell imaging (bottom
right), and (J) the levels of indicated proteins were assessed in HONE1 cell lines at 72 h after
the indicated irradiation dose. GAPDH was used to normalize the amount of protein loaded. All
data are presented as the mean SD of three independent experiments. ns, No significant
difference. ***P<0.001, ****P<0.0001
cytochrome c (#11,940 S, CST), OTUD4 (#SAB3500018, Sigma), Myc tag (#2278S, CST), Flag
tag (#14793S, CST), HA tag (#3724, CST), and GAPDH (#5174, CST). The secondary
antibodies used were horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies
(GE Healthcare). The secondary antibody anti-rabbit IgG light chain specific (#211–032-171,
Jackson Immune Research Laboratories) was used for immunoprecipitation assays to weaken
the signals from the heavy chain.
Immunoprecipitation (IP) assays
Whole-cell lysates were prepared using IP lysis buffer (Beyotime, Wuhan, China) and
precipitated with antiFlag-tag beads (Sigma-Aldrich) or anti-Myc-tag beads (Biolegend)
overnight at 4 °C. Precipitates were washed 6 times with cold IP wash buffer. Elutes were
further subjected to Western blotting or mass spectrometry (MS) analysis.
LDH release assay
Following the indicated treatments, the release of LDH was determined using an LDH assay kit
(Solarbio) according to the manufacturer’s protocol.
Mouse models
Male BALB/c nude mice (3–4 weeks) were purchased from the Experimental Animal Center of
Sun Yat-sen University (Guangzhou, China). Mice were divided into four groups randomly. 1×
106 indicated cells were then subcutaneously injected into the nude mice. When tumor volumes
reached 50–100 mm3, the mice in the radiotherapy group was irradiated with 12 Gy (2 Gy/day
for 6 days). Tumor size was monitored every 4 days, and the tumor volume was calculated as
volume=1/2×L× W2. Blood samples were collected, and serum LDH concentrations were
evaluated pre-radiotherapy and following the third and sixth radiotherapy treatments. The tumor
burdens of nude mice were measured using IVIS Lumina II with (Caliper) Living Image Software
on day 42. Tumors were then excised from euthanized mice, weighed, and paraffin-embedded.
Above paraffin-embedded samples were sectioned for IHC and hematoxylin–eosin (H&E)
staining. All the animal experiments in this study were authorized by the Institutional Animal
Care and Use Committee of the Sun Yat-sen University Cancer Center (SYSUCC) (Approval
No. L102012021110I).
Statistical analysis
SPSS version 19.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism software were applied
in the statistical analysis. The statistical tests for data analysis included the χ2 test, two-tailed
Student’s t-test log-rank test, Spearman-rank correlation test, and one-way or two-way ANOVA.
Data are expressed as the mean±SD. A significant difference was defined by *p≤0.05, **p≤0.01,
***p≤0.001, and ****p≤0.0001.
Results
Radiotherapy induces GSDME-dependent pyroptosis in NPC cells through the intrinsic
mitochondrial apoptotic pathway
It is generally accepted that radiation destroys cancer cells primarily by inducing apoptosis [13].
In the present study, we observed that radiation caused the morphological characteristics of
pyroptosis in NPC cells, as evidenced by swollen cells and the emergence of enormous bubbles
from the plasma (Fig. 1A). The nature of cell death was further identified by increased LDH
release (Fig. 1B). The necroptosis inhibitor GSK’872 could not reduce cell death, suggesting
that pyroptosis instead of necroptosis was triggered by irradiation (Supplementary Figure S1A).
Furthermore, the NPC cells stably expressing EGFP were irradiated at a dose of 6 Gy, and
imaged using a CV1000 Confocal Scanner System. Supplementary Video 1–3 and Fig. 1C
show typical processes of cellular pyroptosis, apoptosis and survival. In six NPC cell lines, the
proportions of pyroptotic cells after irradiation ranged from 4 to 24% (Fig. 1D). To identify which
gasdermin family member is responsible for mediating radiation-induced pyroptosis in NPC
cells, we knocked down the expression of GSDME and
(See figure on next page.)
Fig. 2 Upregulation of GSDME enhances pyroptosis and radiosensitivity in NPC cells in vitro. A
The ratio of pyroptotic cells to dead cells in six NPC cell lines (HNE1, 5-8F, 6-10B, SUNE-1,
HONE1, and HK1) within 72 h after 6 Gy irradiation, as calculated using live cell imaging data
(left). Correlation analysis between the ratio of pyroptotic cells to dead cells and surviving
fraction was performed (right). B Correlation analysis between relative GSDME-N levels and the
surviving fraction in NPC cell lines within 72 h after 6 Gy irradiation was performed. C Western
blotting showed GSDME expression in seven NPC cells and eight other cancer cells. MM:
melanoma, EC: esophageal cancer, PCa: prostate cancer, BC: breast cancer. A375 cells were
included as a positive control. D-M Stable GSDME-overexpressing SUNE1 cells (D, left upper)
and stable GSDME-knockdown HK1 cells (E, left upper) were established. These cells were
exposed to the indicated irradiation dose, and then phase-contrast cell imaging (D, E, right),
LDH release assay (D, E, left lower), Western blotting analysis of GSDME-N (F, H), live cell
imaging (G, I), Annexin/PI assay (J, K), and colony formation assay (L, M, left) were performed
at designated time points. The survival curves of stable cell lines are indicated (L, M, right).
GAPDH was used to determine the amount of loading proteins. All data are presented as the
mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
GSDMD by siRNAs in HONE1 cells (Supplementary Figure S1B and C). Knockdown of GSDME
(Fig. 1E), but not GSDMD (Supplementary Figure S1D), attenuated typical morphological
changes of pyroptosis, as well as the release of LDH and reduction in cell viability. Consistently,
cleavage of GSDME was observed in NPC cells after radiation, accompanied by cleavage of
caspase-3 and poly-(ADP-ribose) polymerase (PARP; a marker for cell apoptosis), and release
of cytochrome c from mitochondria (Fig. 1F). In addition, after radiation exposure, the
proportion of pyroptotic cells, the release of LDH, and the cleavage of GSDME, PARP, and
caspase-3 increased in a time- and dose-dependent manner (Fig. 1G-J; Supplementary Figure
S1E-H). The above data suggest that radiotherapy results in GSDME-dependent pyroptosis
through the intrinsic mitochondrial apoptotic pathway.
Upregulation of GSDME enhances pyroptosis and radiosensitivity in NPC cells in vitro
Live cell imaging analysis showed that pyroptotic cells accounted for a large proportion of dead
cells (≥ 40%) induced by radiation (Fig. 2A, left), and the proportion of pyroptotic cells/dead
cells was inversely proportional to the surviving fraction (Y=-0.5855*X+113.66, R2=0.6315)
(Fig. 2A, right). Additionally, the relative protein level of GSDME-N was negatively correlated
with the surviving fraction of NPC cell lines following 6 Gy irradiation (Y=-1.8698*X+89.85,
R2=0.8316) (Fig. 2B). GSDME is silenced in most tumors due to promoter methylation[33–35].
However, in this study, GSDME was found to be more highly expressed in NPC cell lines
compared to the A375 melanoma cell line, which is known to express GSDME [36]. As
representatives of radiosensitive tumors, NPC cells universally exhibited higher GSDME
expression than other cancer cells (Fig. 2C).
To further assess the effect of GSDME on the radiosensitivity of NPC cells, we constructed
SUNE1 and 6-10B cells that stably overexpress GSDME (Fig. 2D, left upper; Supplementary
Figure S2A, left upper). Concurrently, we silenced endogenous GSDME expression in HK1 and
HONE1 cells using shRNA (Fig. 2E, left upper; Supplementary Figure S2B, left upper).
Upregulation of GSDME in SUNE1 and 6-10B cells led to more obvious pyroptotic bubbles (Fig.
2D, right; Supplementary Figure S2A, right), increased the release of LDH (Fig. 2D, left lower;
Supplementary Figure S2A, left lower), enhanced the generation of GSDME-N fragments
(Fig. 2F; Supplementary Figure S2C), resulted in a higher proportion of pyroptotic cells
(Fig. 2G; Supplementary Figure S2D), and resulted in a higher proportion of dead cells after
irradiation (Fig. 2J; Supplementary Figure S2K). On the other hand, downregulation of GSDME
in HK1 and HONE1 cells resulted in the opposite effects (Fig. 2E, H, I, K; Supplementary
Figure S2B, E, F, H). Furthermore, overexpression of GSDME enhanced the radiosensitivity of
NPC cells (Fig. 2L; Supplementary Figure S2I), while knockdown of GSDME diminished
radiosensitivity (Fig. 2M; Supplementary Figure S2J). Additionally, overexpression of GSDME
increased the oligomerization level of GSDME-N in NPC cells after irradiation, while GSDME
knockdown decreased the oligomerization level (Supplementary Figure S2K). These data
demonstrate that upregulation of GSDME enhances pyroptosis and radiosensitivity in NPC cells
by increasing the oligomerization level of GSDME-N in vitro.
GSDME-dependent pyroptosis sensitizes NPC to radiotherapy in vivo
To explore whether GSDME could affect the radiosensitivity of NPC in vivo, we established
xenograft mouse models. 1× 106 luciferases expressing NPC cells (SUNE1-Vector, SUNE1-
GSDME, HONE1-Scramble, and HONE1-shGSDME) were subcutaneously injected into nude
mice. After 14 days, the mice were randomly assigned to two groups (control group and
radiotherapy group). Tumors in the pre-radiotherapy group was irradiated at a dose of 2 Gy per
day (12 Gy in total). The serum LDH concentrations were detected pre-radiotherapy and after
the third and sixth radiotherapy treatments. Tumor diameters were measured every four days
(Fig. 3A). In the absence of radiation, no significant difference was observed in the growth rate
(Fig. 3B)
Fig. 3 GSDME-dependent pyroptosis sensitizes NPC to radiotherapy in vivo. A Schematic view
of the radiotherapy xenograft model. Cells stably expressing luciferase, SUNE1-Vector, SUNE1-
GSDME, HONE1-Scramble, or HONE1-shGSDME, were subcutaneously injected into the nude
mice (n=28 mice per group). When tumor volumes reached 50–100 mm.3, mice bearing each
type of tumor were randomized into a control group and a radiotherapy group (n=14 in each
subgroup). The mice in the radiotherapy group were irradiated with 12 Gy (2 Gy/day for 6 days).
B Tumor size was monitored every 4 days (n=5). C Representative bioluminescence images of
tumors were taken on day 42. D Statistical analysis of photon fux (n=5). E Images of resected
tumors. F Tumor weight on day 42 (n=5). G Serum LDH concentrations were determined pre-
radiotherapy and following the third and sixth radiotherapy treatments (n=3). H Representative
images of HE (top) and IHC (bottom) staining of the tumor sections are shown. The dashed
lines circumscribe the areas of tumor necrosis. Western blotting showed the level of GSDME-N
in resected tumor tissues. GAPDH was used to normalize the amount of protein loaded. Each
bar represents the mean±SD of three independent experiments. ns, No significant difference.
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
fluorescence signal (Fig. 3C and D), size (Fig. 3E), and weight (Fig. 3F) of tumors with
upregulated or silenced GSDME compared to the controls. However, following irradiation
treatment, GSDME overexpressing tumors grew distinctly slower (Fig. 3B) and exhibited
weaker fluorescence signal (Fig. 3C and D), smaller size (Fig. 3E), and lower weight (Fig. 3F)
compared with the vector group, while GSDME knockdown in HONE1 cells led to the opposite
results. In addition, the serum LDH concentrations in the GSDME-overexpressing group were
significantly higher after radiation treatment than those in the vector group, whereas the
GSDME-knockdown group displayed lower serum LDH concentrations (Fig. 3G). H&E staining
of sectioned tumors showed that overexpression of GSDME significantly increased the area of
inflammatory necrosis of the tumor after irradiation, and silencing of GSDME decreased the
area (Fig. 3H). Consistently, Western blotting analysis of resected tumor tissues revealed
increased cleavage of GSDME in GSDME-overexpressing mice after irradiation while silencing
GSDME decreased the cleavage of GSDME (Fig. 3I). Tese results collectively suggest that
GSDME enhances radiosensitivity of NPC cells by mediating pyroptosis in vivo.
Low GSDME expression correlates with radioresistance and poor prognosis in NPC
To investigate whether GSDME is involved in the radiotherapy response, the level of GSDME in
NPC was analyzed in 10 fresh frozen tissues and 150 paraffin-embedded tissues (Fig. 4A,
Supplementary Table S1). The radioresistant NPC tissues consistently demonstrated lower
protein levels of GSDME compared with radiosensitive NPC tissues (Fig. 4B). According to the
scoring system described in Materials and Methods, 70.0% (105/150) of patients exhibited high
GSDME expression, while 30.0% (45/150) exhibited low GSDME expression (typical low and
high expression of GSDME IHC staining was presented in Fig. 4C) (Fig. 4D). The IHC score of
GSDME in the radioresistant tissues was dramatic lower than that in the radiosensitive tissues
(P=0.0002, Fig. 4E). Subsequent analysis revealed that there was a lower percentage of
radiosensitive NPC cases in the GSDME-low expression group than in the GSDME-high
expression group (P=0.0016, Fig. 4F). Importantly, lower GSDME expression significantly
correlated with poorer PFS (P=0.0034) and LRRFS (P=0.0224) in all NPC cases (Fig. 4G). In
addition, low GSDME expression was recognized to be a poor independent prognostic factor for
5-year PFS (P=0.007) and 5-year LRRFS (P=0.031) in NPC by multivariate Cox regression
analysis (Fig. 4H and Supplementary Table S2). These data demonstrate that GSDME is
commonly expressed in NPC, and suggest that downregulated GSDME in NPC tissues may
result in radiotherapy resistance and lead to poor clinical outcomes.
Similar to previous studies on EGFR inhibitors [20], serum LDH levels were elevated after
radiotherapy in a fraction of NPC subjects with high GSDME expression who showed
nasopharyngoscopy-verified (Fig. 4I, patient I & Supplementary Figure S3A, patients V, VI, who
received radiotherapy alone) or MR-verified (Fig. 4J, patient III & Supplementary Figure S3B,
patient VIII, who received chemoradiotherapy) rapid cancer regression, representing
radiosensitive cases. However, the serum LDH concentration of patients with low GSDME
expression showed no apparent upward trend after radiotherapy, and nasopharyngoscopy
assessment showed a slow regression of the tumor, representing radioresistance (Fig. 4I,
patient II & Supplementary Figure S3A, patient VII, who received radiotherapy alone; Fig. 4J,
patient IV & Supplementary Figure S3B, patients IX, X, who received chemoradiotherapy).
Terefore, GSDME expression could serve as a prospective indicator of radiosensitivity in NPC
patients, and the changes in serum LDH concentrations after radiotherapy could be used to
monitor the efficacy of radiotherapy in these patients dynamically.
OTUD4 deubiquitinates and stabilizes GSDME
To further explore the mechanism by which GSDME is downregulated in radioresistant NPC,
real-time PCR was
Fig. 4 Low GSDME expression correlates with radioresistance and poor prognosis in NPC. A
Schematic of specimens obtained from NPC via nasopharyngoscopy. B GSDME expression
was analyzed by Western blotting in radiosensitive and radioresistant NPC tissues collected
from 10 patients. GAPDH was used to normalize the amount of protein loaded. C
Representative immunohistochemical images of GSDME low- and high-expression in NPC
tissues (left and right, respectively). The magnified inset area is shown at the bottom. Scale bars
represent 50 μm. D Percentage of GSDME low expression and high expression in NPC tissues
(n=150). Low GSDME expression, GSDME_L; High GSDME expression, GSDME_H. E The
IHC score of GSDME in the radiosensitive and radioresistant tissues. F The correlation between
GSDME expression and radiation therapy response. RS, radiosensitive; RR, radioresistant. G
Association of GSDME expression with 5-year progression-free survival and 5-year locoregional
recurrence-free survival was analyzed by Kaplan–Meier and Log Rank test. H Cox regression
analysis of potential prognostic factors, including GSDME levels and other clinical
characteristics. Representative NPC cases received radiotherapy alone, showing the
relationship between GSDME expression, serum LDH, and cancer regression. The yellow
dashed lines indicate tumors in nasopharyngoscopy images. Scale bars represent 50 μm. RT,
radiotherapy. J Representative NPC cases received chemoradiotherapy showing the
relationship between GSDME expression, serum LDH, and cancer regression. The red lines
indicate tumor tissue in magnetic resonance images. **P<0.01, ***P<0.001
performed to analyze the mRNA expression of GSDME in radiosensitive and radioresistant NPC
specimens. The analysis exhibited no noticeable difference in the mRNA expression of GSDME
between radiosensitive and radioresistant NPC tissues (Supplementary Figure S4A). This
finding indicates that GSDME downregulation may occur at the post-transcriptional level in
radioresistant NPC. The ubiquitin–proteasome protein degradation pathway plays a critical role
in the regulation of protein levels[37]. Interestingly, MS analysis of GSDME-interacting proteins
concentrated on the deubiquitinase OTUD4 (Fig. 5A and B). Moreover, OTUD4 protein
expression was distinctly down-regulated in radioresistant NPC cells (Fig. 5C). Therefore, we
hypothesized that OTUD4 deubiquitylates and maintains GSDME stability. Interaction between
OTUD4 and GSDME was confirmed by co-immune-precipitation (co-IP) assays (Fig. 5D). To
determine the regions that mediate the interaction between OTUD4 and GSDME, we made
OTUD4 truncations and GSDME truncations (Fig. 5E). IP assays revealed that only truncated
OTUD4 containing the OTU domain bound to GSDME, indicating that OTU domain was
essential for GSDME (Fig. 5F). Moreover, the N-terminal region of GSDME was responsible for
its interaction with OTUD4 (Fig. 5G). As expected, the upregulation of OTUD4 distinctly
increased, while OTUD4 knockdown reduced, the GSDME protein level (Fig. 5H). Upregulation
of OTUD4 significantly prolonged the half-life of endogenous GSDME, and silencing OTUD4
diminished the degradation half-life of GSDME in NPC cells (Fig. 5I and J). In agreement with
these findings, the overexpression of OTUD4 attenuated GSDME polyubiquitination (Fig. 5K).
These results indicate that OTUD4 promotes the deubiquitination and stability of GSDME in
NPC.
OTUD4 enhances radiosensitivity in NPC cells by promoting GSDME-dependent pyroptosis
To further study the impact of OTUD4 on pyroptosis and radiosensitivity in NPC cells, HONE1
and 5-8F cells with or without OTUD4 overexpression were transfected with GSDME-shRNA,
and we then examined their response to ionizing radiation (Fig. 6A). OTUD4 overexpression in
HONE1 and 5-8F cells led to more distinct pyroptotic bubbles (Fig. 6B), increased release of
LDH (Fig. 6C), a higher proportion of pyroptosis (Fig. 6D), and a higher proportion of dead
cells after irradiation (Fig. 6E; Supplementary Figure S5A). In addition, the colony formation
assay demonstrated that OTUD4 overexpression sensitized HONE1 and 5-8F cells to irradiation
(Fig. 6F and G). However, the phenotype of OTUD4 overexpression was reversed by GSDME
knockdown. In vivo experiments showed that upregulating OTUD4 or silencing GSDME did not
significantly affect xenograft tumor growth without ionizing radiation (Supplementary Figure
S6A-E). Nonetheless, OTUD4 overexpression sensitized xenograft tumors to irradiation, and
silencing GSDME successfully impaired the effect of OTUD4 on radiosensitivity in vivo
(Fig. 6H-L). In addition, serum LDH concentrations were higher in the OTUD4-overexpressing
group following radiation therapy than in the control group, but silencing GSDME restored the
aforementioned trend (Fig. 6M). Furthermore, IHC displayed higher GSDME protein levels in
OTUD4-overexpressing tumor xenografts (Fig. 6N). More importantly, upregulating OTUD4
increased the oligomerization level of GSDMEN in NPC cells after irradiation. However, the
effect of OTUD4 overexpression was reversed by GSDME knockdown (Supplementary Figure
S7A). These results demonstrate that OTUD4 promotes GSDME-dependent pyroptosis and
enhances radiosensitivity in NPC cells by upregulating the protein level of GSDME and the
oligomerization level of GSDME-N.
The clinical relevance of the OTUD4/GSDME axis and radiotherapy response in NPC
We next examined whether OTUD4 downregulation is of clinical significance and whether
OTUD4 and GSDME are clinically relevant. We measured the protein level of OTUD4 in the
same tissues of the 150 NPC patients. Typical low and high expression of GSDME IHC staining
was presented in Fig. 7A. 41.3% (62/150) of specimens showed high OTUD4 expression, while
58.7% (88/150)
Fig. 5 OTUD4 deubiquitinates and stabilizes GSDME. A Representative images of silver-stained
gels of immunoprecipitation proteins using anti-IgG and anti-Flag affinity beads. B Upper panel:
list of candidate proteins from tandem affinity purification-coupled mass spectrometry. Lower
panel: MS profiles of OTUD4. C Upper panel: schematic of protein extraction from parental or
radioresistant SUNE1 and HK1 cells. Lower panel: Western blotting showed OTUD4 protein
expression in parental or radioresistant SUNE1 and HK1 cells. D Interaction of GSDME with
OTUD4. Co-IP was performed using cell lysates obtained from HEK293T cells co-transfected
with Flag-GSDME and Myc-OTUD4. E Upper panel: schematic of OTUD4 truncations. Lower
panel: schematic of GSDME truncations. F HEK293T cells were transfected with Flag-GSDME
and indicated Myc-OTUD4 truncations. Lysates were then IP with Myc-beads for Western
blotting. G HEK293T cells were transfected with Myc-OTUD4 and indicated Flag-GSDME
truncations. Lysates were then IP with Flag-beads for Western blotting. H Indicated cells were
transfected with OTUD4-expressing plasmid, or infected with lentiviral mediated shRNA against
OTUD4 (#1 and #2). Whole-cell extracts were collected for Western blotting analysis. I Cells
were harvested for Western blotting analysis at indicated time points after cycloheximide (CHX)
treatment. J Quantification of GSDME protein levels normalized to GAPDH. K SUNE1 and HK1
cells were transfected with HA-Ub and Flag-GSDME with or without Myc-OTUD4. After
treatment with MG132 (10 µM, 6 h) GSDME ubiquitination was measured. Results are
presented as mean±SD from three independent experiments
exhibited low OTUD4 expression (Supplementary Figure S8A). The radioresistant group
showed a lower IHC score of OTUD4 than the radiosensitive group (P=0.0063, Fig. 7B).
Subsequent analysis revealed that there was a lower percentage of radiosensitive NPC cases in
the OTUD4-low expression group than in the OTUD4-high expression group (P=0.0216,
Fig. 7C). Importantly, lower OTUD4 expression significantly correlated with poorer PFS
(P=0.0278) in all NPC cases (Fig. 7D). These data demonstrate that downregulated OTUD4 in
NPC tissues may result in radioresistance and lead to poor clinical outcomes.
More importantly, patients with low OTUD4 expression (Fig. 7E, biopsy III, IV) showed a lower
level of GSDME, no significant upward trend of serum LDH concentration, and a slower
regression of the tumor than patients with high OTUD4 expression (Fig. 7E, biopsy I, II). Further
analysis demonstrated a positive relationship
between OTUD4 and GSDME expression (P<0.0001,
Fig. 7F, and Supplementary Table S3), validating the clinical relevance of the OTUD4/GSDME
axis. More significantly, patients with both low expression of OTUD4 and low expression of
GSDME had the worst radiotherapy response (66.7% vs. 81.8% vs. 94.6%, P=0.0018, Fig. 7G),
PFS, and LRRFS (Fig. 7H). In brief, these data further confirm that OTUD4 stabilizes and
upregulates GSDME and that the downregulation of OTUD4 inhibits GSDME-dependent
pyroptosis, leading to radioresistance and poor prognosis in NPC.
Discussion
Radiotherapy is the major therapeutic method for NPC; however, radioresistance is the major
cause of treatment failure [38]. Previous research has focused on apoptotic deficits as a
mechanism for radioresistance in NPC. In this study (summarized in Fig. 7I), we evaluated the
effect of pyroptosis on the radiosensitivity of NPCs. First, we observed that pyroptosis, mediated
by caspase-3 cleavage of GSDME in the mitochondrial death pathway, also plays a critical role
in the radiation antitumor process in NPC. Radioresistant NPC tissues showed lower GSDME
expression than radiosensitive NPC tissues. In addition, reduced GSDME expression predicted
a poorer outcome and conferred NPC radioresistance both in vitro and in vivo. Furthermore, we
identified GSDME as a substrate of deubiquitinase OTUD4. OTUD4 deubiquitinated and
stabilized GSDME, thereby increasing GSDME-dependent pyroptosis and boosting
radiosensitivity in NPC cells. More significantly, we confirmed the clinical relevance of the
OTUD4/GSDME axis and the radiosensitivity of NPC tissues.
Pyroptosis occurs as a result of several antitumor therapies and contributes to the antineoplastic
efficacy of these treatment modalities [9, 15–21]. For example, lobaplatin triggers pyroptosis to
eliminate colon cancer cells via the ROS/JNK/Bax pathway [16]. Targeted drugs against KRAS-,
EGFR- or ALK-driven lung cancer induces apoptosis and pyroptosis simultaneously [20].
Therefore, it is of great significance to investigate the potential role that pyroptosis plays in the
anti-tumor activity of radiotherapy in NPC. Here we show that pyroptosis occurs in a time- and
dose-dependent manner after radiotherapy. Similar to its role in the anticancer activity of other
treatments [16, 17, 19, 20, 36], we suggest that pyroptosis may be a nonapoptotic death
mechanism with significant clinical value in enhancing the radiosensitivity of NPC.
The gasdermin family members are indispensable pyroptosis executors [39]. In the present
study, we demonstrate that GSDME establishes the foundation for robust activation of
pyroptosis in NPC following radiotherapy, building on the premise that radiotherapy generates
considerable caspase-3 cleavage via the mitochondrial death pathway. Although GSDME is
silenced in most human malignancies due to promoter methylation [33–35], GSDME is
ubiquitously expressed in the majority of NPC tissues and cell lines, particularly in radiosensitive
NPC. The prevalence of high GSDME expression may explain why radiotherapy is effective for
the majority of NPC patients. Nevertheless, among these 150 patients, 30% of patients
exhibited low expression of GSDME, indicating a poor prognosis following radiotherapy. Indeed,
despite treatment with combined chemotherapy or targeted therapy with radical radiotherapy,
10% of patients with NPC have a poor prognosis and suffer from recurrence because of primary
radioresistance [3, 4]. Importantly, patients with high GSDME expression exhibited elevated
LDH

Fig. 6 OTUD4 enhances radiosensitivity in NPC cells by promoting GSDME-dependent
pyroptosis. Western blotting showed the protein levels of OTUD4 and GSDME in HONE1 and 5-
8F cells transfected with or without OTUD4 and shGSDME. B-G The above cells were exposed
to the indicated irradiation dose, and then (B) phase-contrast cell imaging, (C) LDH release
assay, (D) live cell imaging, (E) Annexin/PI assay, and (F) colony formation assay was
performed at designated time points. G The dose-survival curves of the above cells are
indicated. All data are presented as the mean±SD of three independent experiments. H
Representative bioluminescence images of tumors were taken on day 42. I Statistical analysis
of photon fux (n=5). J Tumor size was monitored every 4 days (n=5). K Images of resected
tumors. L Tumor weight on day 42 (n=5). MSerum LDH concentrations were determined pre-
radiotherapy and following the third and sixth radiotherapy treatments (n=3). H Representative
images of IHC staining of the tumor sections are shown. Scale bars represent 50 μm. ns, No
significant difference. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
levels and rapid tumor regression after radiotherapy, as verified by nasopharyngoscopy or MRI.
Consistent with previous reports [20], we discovered that cleaved GSDME and serum LDH
release by radiotherapy may represent the extent of pyroptosis; these parameters may serve as
potential biomarkers for predicting radiosensitivity and improving treatment strategies. Following
previous reports [20], we demonstrate that the intrinsic mitochondrial apoptotic pathway is
upstream of apoptosis and GSDME-dependent pyroptosis, and can simultaneously trigger both
processes. The final mode of cell death following caspase-3 cleavage is probably determined by
the availability and activity of downstream effectors. However, prior research did not assess the
relative contribution of pyroptosis and apoptosis to antitumor effects. In this study, live cell
imaging analysis revealed that 40—75% of radiation-induced dead cells were pyroptotic cells,
which contradicts the conventional view and identifies pyroptosis as a critical mode of
radiotherapy-triggered tumor cell death in NPC. Notably, the ratio of pyroptotic cells to dead
cells was negatively correlated with the surviving fraction, indicating that a significant pyroptosis
response in NPC was associated with higher radiosensitivity. The ectopic upregulation of
GSDME increased the proportion of pyroptotic cells after radiotherapy and the radiosensitivity of
NPC cells. Nanoparticles that selectively deliver decitabine directly into tumor cells reduce DNA
methylation modification and elevate GSDME expression, eventually increasing chemotherapy-
induced pyroptosis [40]. The identification of mechanisms that modulate GSDME expression to
improve the radiotherapy response in NPC is of great significance. Since there were no
significant variations in GSDME mRNA levels across radiosensitive and radioresistant patient
tissues, we hypothesized that GSDME expression in NPC is regulated post-translationally. As
anticipated, the mass spectrometry analysis of GSDME-interacting proteins focused on the
deubiquitinase OTUD4. As a member of the OTU family, OTUD4 encodes a protein containing
an OTU domain that exerts its functions through its deubiquitination activity [41]. OTUD4
maintains MAVS stability and enhances antiviral innate immune responses through cleaving
K48-conjugated ubiquitin chains from MAVS [31]. The expression of OTUD4 is significantly
down-regulated in various solid tumor tissues and the upregulation of OTUD4 impedes the
proliferation, migration, and invasion of liver, breast, and lung cancer cells by suppressing the
AKT pathway [32]. In the current work, OTUD4 protein expression was revealed to be down-
regulated in radioresistant NPC cells and tissues. Based on the OTU domain, pOTUD4 could
bind to GSDME, mediating GSDME deubiquitination and stabilization. Upregulation of OTUD4
enhances radiosensitivity in NPC cells by promoting GSDME-dependent pyroptosis. Finally, we
confirmed the clinical relevance of the OTUD4/GSDME axis, and patients with low OTUD4
expression also had reduced GSDME levels. The low expression of OTUD4/GSDME is closely
associated with a poor radiotherapy response. Identifying pyroptotic cell death as the critical
radiotherapy-induced cell death mechanism in NPC offers novel insights into radioresistance in
NPC and suggests that the expression of GSDME influences radiosensitivity. Moreover, this
study uncovered the first posttranslational regulation of GSDME, which is triggered by OTUD4.
Therefore, targeting OTUD4/ GSDME axis may be a promising strategy for sensitizing NPC to
radiotherapy independent of apoptosis. In addition, as a mode of pro-inflammatory cell death,
pyroptosis may activate the antitumor immune response [10, 21], which could also be related to
enhanced radiosensitivity of NPC cells and radiotherapy-induced abscopal effects [42].
Furthermore, elucidating specific antigens and inflammatory cytokines released from
radiotherapy-triggered pyroptotic tumor cells and subsequent immune responses will help guide
the combined application of immunotherapy in NPC. Finally, the discovery of GSDME as a
crucial mediator of pyroptosis suggests that other potential effector proteins of pyroptosis
remain to be discovered that may also be involved in modulating the response of NPC to
radiotherapy
Fig. 7 The clinical relevance of the OTUD4/GSDME axis and radiotherapy response in NPC.
Representative immunohistochemical images of OTUD4 low- and high-expression in NPC
tissues (left and right, respectively). The magnified inset area is shown at the bottom. Scale bars
represent 50 μm. B The IHC score of OTUD4 in the radiosensitive and radioresistant tissues. C
The association between OTUD4 expression and radiotherapy response, χ2 test. RS,
radiosensitive; RR, radioresistant. D 5-year PFS for NPC was calculated by Kaplan–Meier
analysis, compared using the Log Rank test, and stratified by low and high OTUD4 levels.
Progression-free survival, PFS. E Representative NPC cases received radiotherapy alone,
showing the relationship between OTUD4 expression, GSDME expression, serum LDH, and
cancer regression. The yellow dashed lines indicate tumors in nasopharyngoscopy images.
Scale bars represent 50 μm. RT, radiotherapy. F The correlation between OTUD4 expression
and GSDME expression. G The correlation between OTUD4/GSDME expression and
radiosensitivity. H Comparison of the progression-free survival and locoregional recurrence-free
survival between 39 OTUD4low/GSDMElow, 56 OTUD4high/GSDME.high, and 55 others.
Actuarial probabilities were analyzed by Kaplan–Meier (Log Rank test). (I) The proposed model
shows that GSDME-dependent pyroptosis is identified as a critical determinant of
radiosensitivity in NPC. In addition, OTUD4/GSDME interaction inhibits OTUD4-mediated
GSDME ubiquitination and stability, thereby promoting GSDME-dependent pyroptosis and
enhancing radiosensitivity in NPC. *P<0.05, **P<0.01, ****P<0.0001
Conclusions
In summary, we reveal that radiotherapy elicits GSDME-dependent pyroptosis in NPC cells
through the intrinsic mitochondrial apoptotic pathway, and it is a critical determinant of
radiosensitivity in NPC. Additionally, OTUD4 deubiquitinates and stabilizes GSDME, resulting in
increased levels of GSDME protein and radiation-induced pyroptosis, ultimately enhancing the
radiosensitivity of NPC. Low expression of OTUD4 or GSDME is correlated with poor
radiotherapy response and survival, while the combination of low OTUD4 and GSDME
expression is associated with a more significant poor prognosis. Therefore, targeting
OTUD4/GSDME axis to induce pyroptosis is a novel strategy for sensitizing NPC to
radiotherapy.
Abbreviations
NPC: Nasopharyngeal carcinoma; GSDME: Gasdermin E; OTUD4: Ovarian tumor family
deubiquitinase 4; RT: Radiotherapy; IMRT: Intensity-modulated radiation therapy; DUB:
Deubiquitinating enzymes; RPMI: Roswell Park Memorial Institute; FBS: Fetal bovine serum;
DMEM: Dulbecco’s modified Eagle’s medium; IHC: Immunohistochemistry; LRRFS:
Locoregional recurrence-free survival; PFS: Progression-free survival; shRNAs: Short-hairpin
RNAs; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; H&E:
Hematoxylin–eosin; PARP: Poly-(ADP-ribose) polymerase.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s13046-
022-02533-9.
Additional file 1: Figure S1. Radiotherapy induces GSDME-dependent pyroptosis in NPC cells
through the intrinsic mitochondrial apoptotic pathway. Figure S2. Upregulation of GSDME
enhances pyroptosis and radiosensitivity in NPC cells in vitro. Figure S3. Low GSDME
expression correlates with radioresistance and poor prognosis in NPC. Figure S4. No significant
difference is found in the mRNA expression of GSDME between radiosensitive and
radioresistant NPC specimens. Figure S5. Knockdown GSDME significantly reverses cell dead
promotion induced by overexpression OTUD4 after irradiation. Figure S6. Upregulating or
silencing OTUD4/GSDME results in no significant difference in xenograft tumor growth in the
absence of ionizing radiation. Figure S7. Upregulating OTUD4 increases the oligomerization
level of GSDME-N in NPC cells after irradiation, which is reversed by GSDME knockdown.
Figure S8. Percentage of OTUD4 low expression and high expression in NPC tissues. Table
S1. Clinicopathological characteristics and tumor-specific expression of GSDME in NPC. Table
S2. Univariate and multivariate analysis of factors associated with PFS and LRRFS in 150 NPC
patients. Table S3. Relationship between OTUD4 expression and patient clinicopathological
features. Table S4.shRNA target sequences used in this study.
Additional file 2: Video 1. Representative processes of cell pyroptosis induced by 6 Gy X-ray
irradiation in HNE1 cells. For time-lapse microscopy, HNE1 cells were grown on a 35-mm glass
bottom dishes (Nest). DIC images were acquired with a CV1000 Confocal Scanner System after
irradiation.
Additional file 3: Video 2. Representative processes of cell apoptosis induced by 6 Gy X-ray
irradiation in HNE1 cells. For time-lapse microscopy, HNE1 cells were grown on 35-mm glass
bottom dishes (Nest). DIC images were acquired with a CV1000 Confocal Scanner System after
irradiation.
Additional file 4: Video 3. Representative processes of cell survival after 6 Gy of X-ray irradiation
in HNE1 cells. For time-lapse microscopy, HNE1 cells were grown on 35-mm glass bottom
dishes (Nest). DIC images were acquired with a CV1000 Confocal Scanner System after
irradiation.
Acknowledgments
We thank all members of Xia’s laboratory for their advice and technical assistance.
Authors’ contributions
M.D., Z.W., and C.Z. conceived the project. M.D. and Z.W. designed the experiments. M.D.,
Z.W., B.C., M.W., and J.B. performed the experiments. M.D., J.M., Q.P, J.Z., H.H., Q.W., and
Y.T. contributed to the data analysis and interpretation. Y.L., J.H., D.W. and L.W. provided
advice and technical assistance. C.Z., J.X. and Q.P. contributed to the revision of the
manuscript. M.D. and Z.W. wrote the paper. All authors read and approved the final version of
the manuscript.
Funding
This work was supported by the National Key R&D Program of China (grant number
2018YFC1313400) and the National Natural Science Foundation of China (grant numbers
81773110, 81872469, 82073330).
Availability of data and materials
The data used and/or analyzed in the present study are available from the corresponding author
upon reasonable request.
Declarations
Ethics approval and consent to participate
The animal experiments were approved by the Animal Research Committee of Sun
Yat-sen University Cancer Center and were performed following established guidelines
(Approval No. L102012021110I). The use of human NPC tissues was reviewed and approved
by the ethical committee of Sun Yat-sen University
Cancer Center and informed consent were obtained from patients (Approval No. GZR2020-
193). The samples were retrospectively acquired from the Department of nasopharyngeal
carcinoma archives of Sun Yat-sen University Cancer Center.
Consent for publication
All the authors agree to the content of the paper and are listed as co-author of the paper.
Competing interests
The authors declare that they have no competing interests.
Author Details
1 Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China,
Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China.
2 Department of Nasopharyngeal Carcinoma, Guangdong Key Laboratory of Nasopharyngeal
Carcinoma Diagnosis and Therapy, Sun Yat-Sen University Cancer Center, Guangzhou
510060, China.
3 Department of Biotherapy, Sun Yat-Sen University Cancer Center, Guangzhou 510060,
China.
4 Department of Urology, Peking University Third Hospital, Beijing 100000, China.
5 Division of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430030, China. Received: 11 August 2022 Accepted: 6
November 2022
Published online 21st of November
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Teenage and Young Adult Cancer: research priorities

Research Priorities for Teenage and Young Adult Cancer

Top 10 research priorities

3 What are the best strategies to improve access to clinical trials?

Kate Collins Chief Executive

Kate Lee Chief Executive Officer

Denis Henshaw Scientific Director

Establishing the partnership 7

Stage 1 - Gathering the questions 9-12

l First survey results 9

Stage 2 - Refining the questions 13-14

l Organising the questions 13

l Removing out of scope questions 13

l Searching for evidence 14

Stage 3 - Prioritising the questions 15-19

Stage 4 - Agreeing the Top 10 20-23

l Decision making: prioritising the Top 10 20

TOP 10 Research Priorites 23

The remaining 20 priorities 24

For research funders 25

For the TYA PSP 25

Appendix 2: Overview of TYA PSP methodology and results 27

Appendix 3: Initial survey 28-32

Appendix 5: Ongoing studies 34-35

Appendix 6: Unanswered questions included in the interim survey 36-40

Appendix 7: Unanswered questions not included in the interim survey 41-43

Figure 1 Gender distribution of respondents (round 1) 9

Figure 2 Ethnicity of respondents (round 1) 10

Figure 3 Age, in years, of respondents (round 1) 10

Figure 4 Geographical distribution of respondents (round 1) 11

Figure 5 Cancer distribution in young people, carers, and significant

Figure 6 Cancer timeline of respondents (round 1) 12

Figure 7 Distribution of carers/significant others (round 1) 12

Figure 8 Distribution of professionals (round 1) 12

Figure 9 Gender distribution of respondents (round 2) 16

Figure 10 Ethnicity of respondents (round 2) 16

Figure 11 Age of respondents (round 2) 17

Figure 12 Geographical distribution of respondents (round 2) 17

Figure 13 Cancer distribution in young people, carers, and significant

Figure 14 Cancer timeline of respondents (round 2) 18

Figure 15 Distribution of carers/significant others (round 2) 19

Figure 16 Distribution of professionals (round 2) 19

JLA James Lind Alliance

NCRI National Cancer Research Institute

TYA Teenage and Young Adult

TYAC Teenage and Young Adults with Cancer Professional Organisation

Management and scope

Establishing the partnership

Amy Callaghan Glasgow

Leila Hamrang Manchester

Demi McGeachy Glasgow

Lara Veitch London

Max Williamson London

The Partnership and the priority setting process coordinating team:

Sheela Upadhyaya (JLA Adviser)

Susie Aldiss (Research Fellow)

Faith Gibson (Professor of Child Health and Cancer Care)

Steering Group professional representatives: