State of the Art REVIEW

Cancer of unknown primary

BMJ: first published as 10.1136/bmj.m4050 on 7 December 2020. Downloaded from http://www.bmj.com/ on 8 December 2020 by guest. Protected by copyright.
Michael S Lee,1 Hanna K Sanoff2
A BST RAC T

1
Cancers of unknown primary (CUPs) are histologically confirmed, metastatic
Department of Gastrointestinal
Medical Oncology, University malignancies with a primary tumor site that is unidentifiable on the basis of standard
of Texas MD Anderson Cancer
Center, Houston, TX, USA
evaluation and imaging studies. CUP comprises 2-5% of all diagnosed cancers
2
Division of Hematology/ worldwide and is characterized by early and aggressive metastasis. Current standard
Oncology, Department of
Medicine, University of North evaluation of CUP requires histopathologic evaluation and identification of favorable
Carolina at Chapel Hill, Chapel
Hill, NC, USA
risk subtypes that can be more definitively treated or have superior outcomes.
Correspondence to: M S Lee Current standard treatment of the unfavorable risk subtype requires assessment of
mslee2@mdanderson.org
Cite this as: BMJ 2020;371:m4050
prognosis and consideration of empiric chemotherapy. The use of molecular tissue
http://dx.doi.org/10.1136/bmj.m4050 of origin tests to identify the likely primary tumor site has been extensively studied,
Series explanation: State of the
Art Reviews are commissioned
and here we review the rationale and the evidence for and against the use of such
on the basis of their relevance tests in the assessment of CUPs. The expanding use of next generation sequencing
to academics and specialists
in the US and internationally. in advanced cancers offers the potential to identify a subgroup of patients who have
For this reason they are written
predominantly by US authors. actionable genomic aberrations and may allow for further personalization of therapy.

Introduction range of 6-16 per 100 000 person years.7-9 Studies

Cancers of unknown primary (CUPs) are defined as
histologically confirmed, metastatic malignancies
with a primary tumor site that cannot be identified
on standard baseline evaluation.1 These cancers
are typically characterized by aggressive and early
metastasis and unpredictable patterns of spread.2 CUP
is not a single disease; rather, it is a heterogeneous
grouping comprising diverse primary tumor types
that elude identification with standard evaluation. A
systematic review of 884 patients with CUP reported
in 12 autopsy cohort studies conducted between 1944
and 2000 reported that a primary tumor site could
be identified by autopsy in 644 (73%) patients, with
the most common sites being lung (27%), pancreas
(24%), hepatobiliary (8%), kidneys (8%), bowel (7%),
genitourinary (7%), and stomach (6%).3 The inability
to identify such primary sites before death presents a
diagnostic and therapeutic dilemma for patients and
their physicians. Tissue of origin tests that identify
primary tumor type on the basis of transcriptomic
analysis with good sensitivity and specificity have
been studied and commercialized. However, despite
the divergence of chemotherapy and molecularly
targeted therapy in most solid tumors, whether
identification of the primary tumor site and receipt of
site directed treatment improves patients’ outcomes
in a meaningful way remains, surprisingly, unclear.
Here, we will review the management of CUP and the
data on the utility and accuracy of tissue of origin tests
and additional next generation sequencing assays in
the evaluation of CUP.
Incidence and prevalence
CUPs are estimated to comprise 2-5% of all
diagnosed cancers worldwide,4-6 with an incidence
of national cancer registries show a peak in
incidence of CUP about 1980 in the US and in the
1990s in Europe and Australia, with subsequent
declines in age adjusted incidence rates,4 10-12
which may reflect both better diagnostic imaging
and evolution of standard pathologic assessments
such as immunohistochemistry, as well as true
declines in incidence rates. However, registry
based studies of incidence and prevalence are
hampered by inadequate specificity in recording
diagnoses, and more detailed auditing may result
in reclassification of up to 32% of cases of CUP to
an alternative primary tumor.8 In the US, a higher
proportion of CUP is found in patients who are
older, female, or black or reside in less affluent
or less educated counties.10 These data, however,
cannot fully distinguish whether this reflects
a distinct biology of CUP in these communities
or a disparity in the extent of the diagnostic
investigation.
Sources and selection criteria
We searched PubMed and Medline for articles
written in English in 2005-19, using search terms
“occult primary”, “cancer of unknown primary”,
and “cancer of unknown origin”. We selected
articles for inclusion on the basis of study quality
and design, prioritizing randomized controlled
trials and prospective and retrospective studies of
sufficient size. Given the ongoing pace of clinical
research, including recent reports of important
randomized clinical trials, we have also included
additional sources relevant to this review such as
meeting abstracts describing final results of relevant
randomized clinical trials.

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 State of the Art REVIEW
Presentation and initial evaluation of CUP
Because of the heterogeneity of CUP, patients can
present with any number of signs and symptoms that
are related to the site of malignant involvement. In the
absence of a radiographically detected, or clinically
suspected, primary site, a biopsy should be taken
from the site that can most safely undergo at least
core needle biopsy to ensure that adequate tissue is
available for histopathologic, and possibly molecular
and genetic, analyses. Immunohistochemical stains
are routinely used to attempt to identify the most
likely primary site and are done in a tiered fashion
to seek to identify the broad type of malignancy
(carcinoma, melanoma, lymphoma, or sarcoma)
and then identify the subtype and primary site.13-15
In most cases of CUP, histopathology is sufficient to
determine epithelial source but cannot further define
a primary tumor site or histology. About half of
cases of CUP have pathologic findings of metastatic
adenocarcinoma, 30% have undifferentiated or
poorly differentiated carcinoma, and 15% have
squamous cell carcinoma. Approximately 5% of
CUPs cannot be classified beyond undifferentiated
neoplasm, a grouping that comprises a mixture of
neuroendocrine carcinomas, lymphomas, germ
cell tumors, melanomas, sarcomas, and embryonal
malignancies.16
If the site of origin remains uncertain after routine
histopathologic analyses, further evaluation should
follow the consensus guidelines developed by leading
cancer agencies. Figure 1 summarizes subsequent
management. The National Comprehensive Cancer
Network (NCCN), the European Society of Medical
Oncology (ESMO), and the National Institute for
Health and Care Excellence (NICE) recommend a
basic evaluation in all patients, as described in box
1, with additional investigation considered given
details of clinical presentation.17-19 For example,
upper endoscopy may be useful in selected patients
with suspicious signs, symptoms, or laboratory
abnormalities suggestive of an upper gastrointestinal
primary.
Positron emission tomography (PET) scans are not
routinely recommended in evaluation of CUPs in the
NCCN, ESMO, or NICE guidelines. Select scenarios
exist, however, in which PET should be considered.
Specifically, the ESMO and NICE guidelines highlight
the potential utility of PET scans in patients with
isolated squamous cell carcinoma in cervical
lymph nodes. A meta-analysis of 246 patients with
biopsy confirmed carcinoma of cervical nodes
with occult primary included in seven studies of 18
F-fluorodeoxyglucose PET-computed tomography
(PET-CT) for detection of primary tumor site showed
a primary tumor detection rate of 44% (95%
confidence interval 31% to 58%), with excellent
sensitivity of 97% (63% to 99%) but specificity of
only 68% (49% to 83%).20 In other CUPs, PET-CTs
may also be informative of primary tumor site.
However, the overall rate of primary detection with
PET scans has been shown be quite low for patients
with CUP in other anatomic sites. A meta-analysis of
2
PET-CT in 433 CUP patients from 11 studies found
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a primary tumor detection rate of 37%, with 84%
(78% to 88%) sensitivity and 84% (78% to 89%)
specificity.21 A more recent meta-analysis including
1942 patients in 20 studies found a primary tumor
detection rate of 40.9% (39.0% to 42.9%).22
However, prospective clinical trials comparing
PET-CT with conventional imaging modalities such
as conventional computed tomography scans are
lacking. In a prospective study of 136 patients
newly diagnosed as having CUP with extra-cervical
metastases, patients underwent PET-CT along with
administration of intravenous and oral contrast
to facilitate interpretation of diagnostic computed
tomography images. Imaging results were correlated
with a standard of reference established by a
multidisciplinary team to determine the most likely
primary tumor site. PET-CT determined the primary
tumor site in 38/135 (28%), whereas conventional
computed tomography determined the primary tumor
site in 43/135 (32%); no significant differences in
sensitivity, specificity, or accuracy were seen.23 Given
the lack of prospective trial data showing the clinical
benefit of PET-CTs compared with conventional
computed tomography imaging, guidelines do not
recommend PET-CT scans as standard.
Notably, identifying patients with germ cell tumors
and lymphomas or other hematologic malignancies,
which have potential for curative therapy, is of para­
mount importance. NCCN guidelines also prioritize
identification of thyroid carcinomas, neuroendocrine
tumors, and sarcomas, which have markedly different
treatment options and prognoses.
Identification and management of favorable and
unfavorable risk groups of CUP
Favorable risk groups
Distinct subsets exist within the heterogeneous
grouping of CUP, and their recognition is important
as their treatment differs considerably from that
of undifferentiated CUP. These more favorable risk
subsets encompass approximately 10-20% of cases
of CUP and are distinguished on the basis of clinical
and pathologic features that provide ample evidence
to suggest the site of origin even if it is not actually
detected,18 24 as described in table 1.
Isolated localized lymph node metastases to
the neck or to the axilla in a woman are important
subsets of CUP, and patients treated using loco­
regional paradigms of head and neck cancer or
breast cancer, respectively, have the opportunity for
potentially curative therapy. A systematic review of
24 retrospective studies found that 321/446 (72%)
women with isolated axillary nodal metastases, even
if no breast primary is found on initial evaluation,
are ultimately found to have an occult breast primary
when treated definitively with surgical resection.25
Given this high rate of primary tumor detection,
these patients should receive therapy with curative
intent as indicated for stage II breast cancer.
Similarly, identifying diseases that would have
a better prognosis if disease specific therapy is
doi: 10.1136/bmj.m4050 | BMJ 2020;371:m4050 | the bmj
 State of the Art REVIEW

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Fig 1 | Summary of recommended evaluation for cancer of unknown primary (CUP). ECOG PS=Eastern Cooperative Oncology Group performance
status; LDH=lactate dehydrogenase

administered is important. For example, in a

retrospective study of 42 patients with CUP who
were deemed to have a colorectal primary, of whom
Box 1: Recommended management of cancer of unknown primary17-19
32 received first line or second line therapy with a
Initial evaluation for essentially all patients colorectal cancer specific regimen, median survival
• Thorough medical history, particularly regarding past biopsies or malignancies, among patients receiving a colorectal site specific
removed lesions, and spontaneously regressing lesions regimen was 27 months. The retrospective nature
• Complete physical examination, including head and neck, skin, lymph node, rectal, of the study makes it subject to reporting bias, but
genitourinary, pelvic, and breast examination these data may suggest favorable prognosis in this
• Laboratory studies including complete blood count, electrolytes, liver function tests, subgroup with identifiable colorectal cancer.26
creatinine, calcium, lactate dehydrogenase, and urinalysis Identifying a site of origin would allow for
• Computed tomography scans of thorax, abdomen, and pelvis, with intravenous administration of optimal evidence based, disease
contrast unless contraindicated specific treatments to patients, so it is reasonable
• Mammography for female patients* to assume that many patients would have impro­
• Serum prostate specific antigen for men over aged ≥40, especially with ved outcomes if a site specific regimen can be
adenocarcinoma bone metastases* administered. However, this has not been definitively
• Fecal occult blood test demonstrated for CUP more broadly.
• Biopsy (optimally core needle biopsy) of most accessible site
Additional recommended investigations in specific clinical scenarios Unfavorable risk groups
Following evaluation, most patients (80-90%) do not
Isolated cervical lymph nodes
fit any of the favorable risk subgroups as outlined
• Head and neck computed tomography. Consider PET-CT (to identify primary head
in table 1 and thus comprise an unfavorable subset
and neck site)
of disease, with poor prognosis. A retrospective
Supraclavicular/axillary lymph nodes in women, or other histopathologic evidence for analysis of 49 patients with unfavorable subset CUP
breast cancer and liver metastases showed a median survival of
• Breast magnetic resonance imaging and/or ultrasonography, if mammogram 10 (95% confidence interval 7 to 13) months, and
unremarkable a systematic review of four additional published
Mediastinum series in similar cohorts showed a range of median
• β-hCG and α-fetoprotein (to evaluate germ cell tumor) survival from 1.7 to 7.2 months.27 Patient prognosis
Retroperitoneal mass must be incorporated into any decisions made on
• β-hCG, α-fetoprotein, testicular ultrasonography if male <65 years use of chemotherapy and choice of chemotherapy.
hCG=human chorionic gonadotropin; PET-CT=positron emission tomography-computed tomography
A prognostic model, developed from 150 unselected
*National Institute for Health and Care Excellence guidelines specifically do not recommend these tests unless patients with CUP and then validated with an
compatible clinical and/or pathologic features are presents external dataset of 116 patients, established poor

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State of the Art REVIEW

Table 1 | Favorable risk subtypes of cancer of unknown primary (CUP)

CUP subtype/characteristics
Poorly differentiated neuroendocrine
carcinoma of unknown primary
Well differentiated neuroendocrine tumor
of unknown primary
Peritoneal adenocarcinomatosis of serous papillary
histology in females
Isolated axillary nodal metastases in females
Squamous cell carcinoma involving cervical lymph nodes
Squamous cell carcinoma involving inguinal lymph nodes
with unknown primary
Poorly differentiated carcinoma involving mediastinum or
retroperitoneum in young males, especially with elevated
β-hCG or α-fetoprotein
CUP with colorectal IHC (CK20+, CDX2+, CK7–)
Single metastatic lesion of unknown primary
Blastic bone metastases with IHC/serum
PSA expression in males
ER=estrogen receptor; hCG=human chorionic gonadotropin; HER2=human epidermal growth factor receptor 2; IHC=immunohistochemistry; PR=progesterone receptor; PSA=prostate specific
antigen.
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Potential treatment Analogous tumor type
Platinum-etoposide chemotherapy Poorly differentiated neuroendocrine
carcinoma
Somatostatin analogs, everolimus, sunitinib, peptide receptor Well differentiated neuroendocrine tumor
radiotherapy
Optimal surgical debulking, followed by platinum-taxane Stage III ovarian cancer, primary peritoneal
chemotherapy cancer
Axillary nodal dissection, mastectomy or breast radiation therapy, Stage II breast cancer
and adjuvant therapy per ER/PR and HER2 status
Neck dissection and/or radiation therapy of bilateral neck and Head and neck squamous cell cancer
head-neck axis
Inguinal lymph node dissection and/or radiation therapy Urogenital squamous cell carcinoma
Testicular or germ cell platinum based chemotherapy regimen Extragonadal germ cell tumor
Colorectal cancer based systemic treatment Metastatic colorectal cancer
Resection and/or radiation therapy +/− systemic therapy Oligometastatic disease
Androgen deprivation therapy +/− radiation therapy Prostate cancer

performance status (Eastern Cooperative Oncology
Group performance status (ECOG PS) 2-3) and
elevated serum lactate dehydrogenase concentration
as key independent prognostic variables. A poor risk
group was defined as having poor performance or
elevated lactate dehydrogenase, with 11% one year
survival and 3.9 months median survival (compared
with 45% one year survival and 11.7 months
median survival in the good prognostic group).28
ESMO guidelines recommend consideration of two
drug chemotherapy combinations for patients with
ECOG PS 0-1 and normal lactate dehydrogenase,
and weighing chemotherapy versus best supportive
care for patients with ECOG PS 2 or greater, elevated
lactate dehydrogenase, or both.18
For patients with unfavorable subset CUP with
adequate performance status, the mainstay of
treatment is empiric chemotherapy. Multiple chemo­
therapy regimens have been tested in phase II studies,
but no single regimen has shown superiority. A meta-
analysis of 543 patients with unfavorable subset CUP
treated with a total of 16 regimens in 10 randomized
controlled trials found that no single regimen had
a significant benefit compared with any others,
with wide confidence intervals. The chemotherapy
regimens in these studies included platinum agents,
taxanes, vinca alkaloids, fluoropyrimidines, and
irinotecan.29 Thus, various suggested or preferred
chemotherapy regimens for patients with unfavorable
subset CUP are listed in NCCN and ESMO guidelines
and are summarized in table 2.
Tissue of origin tests
The molecular underpinnings of diverse types of
human cancers are increasingly understood, as
genomic, transcriptomic, and epigenetic analyses of
a range of common and rare tumor types are ongoing.
These studies show that distinct tumor types have
recognizable differences in gene expression and other
molecular features, suggesting that the site of origin
of CUP might be elucidated from a tumor sample by
analyzing how a given tumor’s genetic and molecular
profile compares with the common patterns seen in
cancers with known sites of origin. For example, the
Cancer Genome Atlas has characterized genomic and
gene expression variations within multiple tumor
types and is studying differences and similarities
across cancer types in the Pan-Cancer Atlas.30 In
an updated analysis, 11 286 tumor samples from
33 cancer types were assayed for aneuploidy, DNA
methylation, mRNA, and microRNA profiles. Among
10 165 tumors analyzed by mRNA expression profiles,
25 groupings were identified, with tumor type
being a primary driving factor for these groupings.
Additionally, tumors with similar histology, such as
squamous morphology inclusive of cervical, head and
neck, and lung primaries, clustered together. Clusters
were also identified on the basis of similar tissue or
organ system of origin, including neuroendocrine
and glioma, melanomas of skin and eye, clear cell
and papillary renal carcinomas, hepatocellular and
cholangiocarcinomas, a gastrointestinal group of
colorectal and stomach adenocarcinomas, and a
digestive system group of pancreatic and stomach
adenocarcinomas.31 Thus, molecular features such
as gene expression or microRNA profiles differ
among tumor tissue types and may be used to
determine the primary tumor tissue type. Several
platforms have been developed and commercialized
to apply a classifier to identify the tissue of origin in
CUP samples. Although several studies to validate
the sensitivity and specificity of these assays have
been performed, as will be described in the following
sections, no clear gold standard exists to determine
the true tissue of origin, so these limitations should
be considered in analyzing these results.
92 gene real time polymerase chain reaction based
assay
Accuracy
A 92 gene set (CancerTYPE ID, Biotheranostics,
San Diego, CA, USA) was initially developed using

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 State of the Art REVIEW

Table 2 | Recommended treatment regimens for cancer of unknown primary (CUP)

Suggested chemotherapy regimens
Cisplatin-gemcitabine
Cisplatin-etoposide
Paclitaxel-carboplatin
Docetaxel-carboplatin
Irinotecan-oxaliplatin
Capecitabine-oxaliplatin
Gemcitabine-irinotecan
Fluorouracil-oxaliplatin
Fluorouracil-irinotecan
Paclitaxel-cisplatin
Cisplatin-fluorouracil
Gemcitabine-docetaxel
Docetaxel-cisplatin
Irinotecan-carboplatin
Capecitabine
Fluorouracil
Paclitaxel, carboplatin, etoposide
Fluorouracil, irinotecan, oxaliplatin
Docetaxel, cisplatin, fluorouracil
ECOG PS=Eastern Cooperative Oncology Group performance status; ESMO=European Society of Medical Oncology; NCCN=National Comprehensive Cancer
Network.
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ESMO guidelines NCCN guidelines
Yes Yes (adenocarcinoma, preferred); yes (squamous cell carcinoma)
Yes
Yes Yes (adenocarcinoma, preferred); yes (squamous cell carcinoma, preferred)
Yes Yes (adenocarcinoma); yes (squamous cell carcinoma)
Yes -
Yes Yes (adenocarcinoma, preferred)
Yes Yes (adenocarcinoma, if ineligible to receive platinum based chemotherapy)
- Yes (adenocarcinoma, preferred); yes (squamous cell carcinoma, preferred)
- Yes (adenocarcinoma, preferred)
- Yes (squamous cell carcinoma)
- Yes (squamous cell carcinoma)
- Yes (adenocarcinoma)
- Yes (adenocarcinoma); yes (squamous cell carcinoma)
- Yes (adenocarcinoma)
- Yes (adenocarcinoma); yes (squamous cell carcinoma)
- Yes (adenocarcinoma); yes (squamous cell carcinoma)
- Yes (adenocarcinoma: only with ECOG PS 0-1)
- Yes (adenocarcinoma, if presumed gastrointestinal primary site)
- Yes (squamous cell carcinoma: only with ECOG PS 0-1)

a training set of 578 tumors representing 39 tumor
classes, including a range of epithelial and non-
epithelial tumor types. This gene set included 87
genes differentially expressed in the distinct tumor
types and five reference genes expressed at a relatively
invariable level across disease types. The genes
selected were enriched in DNA binding transcription
factors and in cell surface receptor proteins. This
panel resulted in 87% classification accuracy in
a validation cohort.32 A subsequent expansion of
the training set to 2206 specimens representing 30
tumor types and 54 histological subtypes was used
to enhance the classifier. Comparing a sample of
unknown origin against the gene expression profiles
for the reference tumors of each subtype within the
database provides a probability that the unknown
sample is classified as a known tumor type. In an
internal validation study, this classifier resulted in
85% sensitivity for tumor type and 87% sensitivity
for histological subtype.33 In a separate test set of
187 formalin fixed, paraffin embedded (FFPE) tumor
samples, the 92 gene assay had an 83% sensitivity
for tumor type.33 A validation study performed in
three independent laboratories used the assay on
790 FFPE tumor samples encompassing more than
50 subtypes and found overall sensitivity of 87%
(84% to 89%) and specificity ranging from 98%
to over 99% for tumor types. For tumor subtyping
analysis, sensitivity was 82% (79% to 85%), with
specific ranging from 98% to over 99%. No significant
decrease in performance was seen on the basis of
metastatic tumors, histological grade, or cases with
limited tissue.34
Multiple retrospective studies of the 92 gene set
classifier have been performed to determine the
sensitivity of the assay and correlate results to clinical
actionability. A retrospective, multicenter analysis
was performed on 501 patients deemed to have CUP,
of whom 38 were subsequently found to have a latent
primary site that became manifest during routine
care. Of these 38 patients with a “gold standard” for
the primary site, 28 had sufficient tissue to perform
the 92 gene real time polymerase chain reaction (RT-
PCR) assay, and 20/28 had sufficient RNA quality
and quantity for the 92 gene classifier to predict a
site of origin. The assay result was correct in 15/20
(75%) of cases, was incorrect in 3/20 (15%), and was
unclassifiable in 2/20 (10%). By comparison, clinical
features and immunohistochemistry suggested a
primary site in six cases, five of which were correct,
and suggested two or more primary sites in 13 cases,
eight of which included the correct primary site.35
Although the number of cases was very limited, this
study provided early evidence that a more definitive
primary tumor site could be identified using the
92 gene classifier compared with standard clinical
assessment.
A subsequent study built on this experience by
pooling these 20 patients with an additional 151
patients with CUP prospectively studied using the 92
gene classifier. Of the 171 patients, 144 successfully
had a single tumor type determined using the 92 gene
classifier. The most common tumor sites determined
included colorectal in 26/171 (15%), non-small
cell lung in 18/171 (11%), breast in 15/171 (9%),
hepatocellular in 10/171 (6%), ovary in 9/171 (5%),
and pancreas in 9/171 (5%). As an update to the
previous test, 18/24 (75%) patients who eventually
had a latent primary tumor identified had the tumor
type correctly identified by the 92 gene classifier.
Additionally, of 52 patients who had a likely primary
tumor site identified by immunohistochemistry,
the 92 gene panel was concordant in 40 (77%).36
Thus, these studies show that compared with a gold
standard method of identifying a primary tumor, the
92 gene classifier is accurate in 75-77% of cases.
The accuracy of the 92 gene classifier was compared
against standard immunohistochemical pathology
assessment in a prospectively defined, blinded study
of 131 high grade, predominantly metastatic tumors

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State of the Art REVIEW
with known reference diagnoses, 122 of which were
evaluable. FFPE slides were provided to blinded
pathologists to perform immunohistochemistry and
also used to determine cancer type by the 92 gene
classifier. The 92 gene assay had an accuracy of 79%
(96/122; 95% confidence interval 71% to 85%)
for tumor type, compared with an accuracy of 69%
(84/122; 60% to 76%) for immunohistochemistry.
The P value for difference in sensitivity was 0.019.37
Clinical utility
The clinical utility of the 92 gene classifier has been
evaluated in a few prospective studies. A multicenter,
prospective phase II trial enrolled patients with CUP
and sufficient tissue for testing with the 92 gene
assay. Patients waited two to three weeks before
starting therapy, pending results of the 92 gene assay,
at which time they were assigned to receive protocol
prescribed treatment based on the predicted tissue
of origin. The regimens included were standard for
the era of enrollment from October 2008 through
December 2011, although therapy for many of these
diseases has since evolved.38
The primary objective of the study was to assess
the efficacy of classifier guided therapy for patients
with CUP, as measured by the overall survival
compared with historical controls. In total, 289
patients were enrolled, of whom 252 (87%) had
the assay successfully performed. Of the patients
who had the assay successfully performed, 247/252
(98%) had a tissue of origin predicted; the most
common sites predicted were biliary tract in 52/252
(21%), urothelium in 31 (12%), colorectum in 28
(11%), non-small cell lung in 27 (11%), pancreas
in 12 (5%), and breast in 12 (5%), with other sites
having less than 5% each. Of the 252 patients, 223
(87%) received treatment in the study, of whom 194
(87%) or 67% of the enrolled population received
assay directed therapy. The median overall survival
for these 194 patients was 12.5 (95% confidence
interval 9.1 to 15.4) months. The median survival
was 13.4 months in patients with tumor types
deemed to have higher rates of response to treatment
(colorectal, breast, ovary, kidney, prostate, bladder,
non-small cell lung, germ cell, poorly differentiated
neuroendocrine, lymphoma, and small cell lung),
compared with 7.6 months for those with tumor types
with lower rates of response (biliary tract, pancreas,
gastroesophageal, liver, sarcoma, cervix, carcinoid,
endometrium, mesothelioma, melanoma, skin,
thyroid, head and neck, and adrenal) (P=0.04).38
This was thought to reflect improved outcomes
compared with median survival of less than 10
months in historical controls receiving empiric
therapy for CUP. Of course, systemic therapy options
for distinct disease types have evolved markedly
since that era, in particular with the evolution of
predictive biomarkers for targeted therapies and now
immune checkpoint inhibitors, so outcomes in the
current landscape of disease need to be studied.
A more recent prospective multicenter observational
trial was performed to determine clinical impact,
6
as measured by the composite primary outcome
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of changes in the patient’s treatment, narrowing
of treatment options, or elimination of a treatment
option based on the results of the 92 gene assay.
The primary outcome was assessed through a survey
completed by ordering medical oncologists (n=73)
and attending pathologists (n=34). In the study, 444
patients were enrolled from February 2013 through
October 2014, and 397 (89%) had sufficient RNA for
analysis. Of these, 379 (95%) had tumor type and
histological subtype determined by the 92 gene assay.
Among 271 assay results from medical oncologists,
the 92 gene assay either confirmed or narrowed the
diagnosis in a significant proportion of patients but
often provided a result that was not initially suspected.
In 79/271 (29%) cases, a single site was clinically
suspected, and this site was confirmed in 60% of
cases, but a previously unsuspected site was indicated
by the assay in 39% of cases. In 80/271 (30%) cases,
two or more sites were suspected in the differential
diagnosis, and the assay narrowed the diagnosis in
66% of cases and provided a previously unsuspected
site in 27% of cases. In the remainder of the 112 cases
that the oncologist reported were CUP without an a
priori suspected primary site, the assay provided a
tumor type prediction in 97% of cases. Only 203 of
the patients included in this study went on to receive
therapy, and among these patients the oncologists
reported that the 92 gene assay changed the planned
treatment regimen in 47% of cases,39 suggesting a
considerable effect of testing on patient care.
Microarray based tissue of origin test
The Tissue of Origin Test (Cancer Genetics) uses gene
expression profiling via microarrays to determine the
similarity of a tumor sample’s gene expression profile
to 15 known tumor types. This test uses a 1550 gene
expression profile obtained from microarrays and
reports a similarity score, ranging from 0 to 100, for
each of the 15 potential tissue types. The assay was
trained using 2039 human tumor samples from 15
tissues of origin.40 The analytic performance of this
assay was studied at four independent laboratories
that each performed the assay on 60 frozen tissue
specimens from metastatic and primary tumors and
found high reproducibility, with overall concordance
of 89.4% (range 87.0-92.5%).41 A subsequent blinded
validation study processed 547 frozen tumor samples
at four laboratories, with 258 (47%) samples derived
from metastatic tissue and the remainder from poorly
differentiated or undifferentiated primary tumor
tissue. The overall agreement with the reference
diagnosis was 87.8% (480/547; 95% confidence
interval 84.7% to 90.4%), with a sensitivity of 87.8%
(84.7% to 90.4%) and a specificity of 99.4% (98.3%
to 99.9%).40 A subsequent retrospective study of
21 fresh frozen CUP samples found that 16 (76%)
samples resulted in a positive result in a single tissue,
with an indeterminate result in five (24%). This study
was limited by lack of a gold standard given that the
CUP samples by definition did not have a known
primary tumor type.42
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Subsequently, validation studies for this assay
were done using FFPE specimens. Microarray
data for 462 metastatic, poorly differentiated,
or undifferentiated FFPE tissues that all had a
reference diagnosis were obtained, and an 88.5%
(85.3% to 91.3%) concordance was found. The
reproducibility was established in three independent
laboratories, with 89.3% (133/149) concordance.43
A blinded, multicenter, prospective study sought to
compare the accuracy of immunohistochemistry and
pathologists’ interpretation of tumor tissues against
the microarray assay. FFPE metastatic tumor samples
from 160 cases with known reference were prepared
and sent to blinded pathologists for standard
immunohistochemical analysis and for microarray
analysis. The microarray result was concordant
with the reference diagnosis in 89.2% of samples,
compared with 83.3% per immunohistochemical
assessment (odds ratio 2.9, 1.2 to 6.7). In 51
poorly differentiated and undifferentiated tumors,
the accuracy of the microarray assay was 94.1%,
compared with immunohistochemical accuracy of
79.1% (P=0.016), although results were similar in well
and moderately differentiated tumors (microarray
accuracy 85.3% versus immunohistochemical
accuracy 86.8%; P=0.52).44
Summary of investigational assays
Various additional assays have been studied to
identify tissue of origin for CUPs,45 46 and these are
summarized in table 3. These additional assays are
not currently commercially available, but they show
the potential of tissue of origin testing. These include
a microRNA based assay, initially developed using
quantitative RT-PCR,47-49 and subsequently refined
using a custom designed microarray identifying 42
tumor types with overall assay sensitivity of 85% and
specificity of more than 99%.50 The second generation
microRNA assay also had 70% (59/84) concordance
with initial clinical diagnosis at presentation and
92% (77/84) agreement with final clinical diagnosis
in patients who clinically were deemed to have
CUP.51 The microarray gene expression database
that was used to develop the 92 gene classifier was
also used by an independent group to develop a
distinct classifier for adenocarcinoma of unknown
primary, which identified 70/84 (83%) tumors of
known origin.52 Finally, a 10 gene quantitative
RT-PCR assay was developed that can identify six
tumor types (lung, breast, colon, ovary, pancreas,
and prostate)53 and successfully assigned a tissue of
origin in 63/104 (61%) patients with CUP, although
Table 3 | Summary of tissue of origin tests
Assay method Assay name and manufacturer
92 gene quantitative RT-PCR to assay mRNA expression CancerTypeID (Biotheranostics)
mRNA gene expression microarray assaying 1550 genes (frozen tissue) Tissue Of Origin (TOO) (previously Pathwork, now
or 2000 genes (FFPE tissue) Cancer Genetics)
64 microRNA expression microarray miRview mets2 (Rosetta Genomics, now defunct)
1900 gene gene expression microarray CupPrint (Agendia BV)
10 gene quantitative RT-PCR CUP Assay (Veridex)
FFPE=formalin fixed, paraffin embedded; RT-PCR=real time polymerase chain reaction.
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State of the Art REVIEW
most samples were identified as lung, pancreas, or
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colon.54 However, categorizing only six tumor types
would not be sufficient to successfully identify
primary tumor type in the current era, especially as
fairly commonly misidentified tumor types such as
biliary and gastroesophageal were not included in
the panel.
Prospective randomized clinical trials using tissue
of origin testing
The first prospective randomized study reported was
performed at multiple sites in Japan and randomized
patients with CUP 1:1 to receive empiric carboplatin-
paclitaxel or site specific therapy on the basis of
the results of tissue of origin testing.55 However,
the molecular classifier used was not one of the
commercialized, previously validated classifiers
that is available for current use. The molecular assay
required a fresh frozen tumor specimen, from which
RNA was extracted, and an Affymetric microarray
was run. Training data were developed using 1024
tumors of known origin obtained either from publicly
available Gene Expression Omnibus datasets or
from the Department of Genome Biology at Kindai
University, and a tenth of the data was randomly set
aside for use as a validation cohort to determine the
proportion of true classifications. This procedure was
repeated 10 times, resulting in an average 78.6%
cross validated estimate of accuracy. However,
this microarray classifier has not been validated
on additional datasets. In the trial, 130 patients
were randomized, with gene expression profiling
successfully performed for all of them. Twenty nine
patients did not proceed to study treatment, 19 of
whom had a primary site subsequently identified,
leaving 101 patients for efficacy analysis. Of these,
26 (26%) had pancreas, 23 (23%) had gastric, 11
(11%) had lymphoma, eight (8%) had urothelial,
seven (7%) had cervical, and six (6%) had ovarian
sites predicted.
The one year survival rate was 44.0% in the site
specific arm and 54.9% in the empiric carboplatin-
paclitaxel arm (P=0.264). At a median follow-up
time of 9.7 (range 1.2-83.9) months for the site
specific arm and 12.5 (0.9-66.5) months for the
empiric carboplatin-paclitaxel arm, the median
overall survival was 9.8 (95% confidence interval
5.7 to 13.8) months for the site specific arm and 12.5
(8.9 to 16.1) months for the empiric chemotherapy
arm (stratified log rank P=0.896), with a stratified
Cox hazard ratio of 1.03 (0.68 to 1.56).55 Several
limitations of the study must be considered, however,
No of tumor types identified
30 tumor types; 54 histological subtypes
15 tumor types
42 tumor types
48 tumor subtypes
6 tumor types
7
State of the Art REVIEW
before concluding that site specific therapy does not
improve outcomes. Firstly, this study did not enroll
a sufficient number of patients in whom efficacy
could be evaluated to satisfy its initial statistical
analysis, which called for a total sample size of 114
patients, possibly resulting in an underpowered
study. More importantly, the molecular assay that
was the integral biomarker for this study was not
robustly validated with independent samples,
so whether this microarray based assay has the
requisite sensitivity and specificity for clinical
use remains unclear, and we can thus not be sure
whether the data from this study can be generalized
to other molecular classifiers. Finally, the patient mix
was quite different from the previous retrospective
studies, and the site specific therapies may not be
optimal for the most common disease types. Given
that the prognosis and treatment paradigms differ
markedly with lymphoma, any patients suspected to
have lymphoma should optimally be treated under
a different paradigm and generally would not be
considered for empiric carboplatin-paclitaxel.
Although the Japanese study has several key
flaws, the lack of a significant survival improvement
from site of origin testing was also seen with the
presentation of the results of the GEFCAPI-04
randomized trial at the ESMO congress in 2019. This
trial also showed no significant survival benefit of
using molecular classifiers to provide site specific
therapy compared with empiric chemotherapy. In
this study, patients with CUP were randomized 1:1
to receive either empiric gemcitabine-cisplatin or to
have gene expression testing followed by site specific
treatment. The classifiers used were extensively
studied in previous publications, including the
92 gene CancerTYPE ID classifier (n=222) and the
Tissue Of Origin Pathwork test (n=21). The primary
endpoint was progression-free survival. Patients
were enrolled from March 2012 through February
2018, and 243 patients were randomized. The
most commonly reported primary tumor types from
molecular classifiers included pancreaticobiliary
cancer in 19%, squamous cell carcinoma in 11%,
kidney cancer in 8%, and lung cancer in 8%. Tailored
site specific treatment was given in 91/123 patients
randomized to site specific treatment. No significant
difference was seen in median progression-free
survival, with a hazard ratio of 0.95 (0.72 to 1.25)
on central radiologic review and 0.80 (0.60 to 1.06)
on local radiologic review. The secondary endpoint
of overall survival was also similar in the overall
population, with a hazard ratio of 0.92 (0.69 to 1.23)
and a median of 10 months versus 10.7 months.56
The results of the Japanese trial and GEFCAPI-04
indicate that site specific therapy does not improve
outcomes such as median progression-free or overall
survival compared with empiric chemotherapy.
However, treatment paradigms are markedly different
and non-chemotherapy centered in some cancer
types, such as renal cell carcinoma. In these subsets
of patients, it is plausible that treatment with empiric
chemotherapy could result in inferior outcomes,
8
so current clinical practice and future trials should
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focus on identifying patients in whom identifying a
primary disease site would markedly affect treatment
paradigms.
Next generation sequencing
Carcinomas of unknown primary commonly have
potentially targetable genomic alterations and
mutations. Identifying genomic aberrations for
which aberration specific therapies are available
results in clinically meaningful changes in outcomes
for patients with a variety of solid tumors, including
non-small cell lung cancer, colorectal cancer, and
melanoma. Available data suggest that carcinomas
of unknown primary similarly harbor clinically
meaningful rates of actionable mutations.
A retrospective study of 200 carcinomas of
unknown primary that had next generation sequen­
cing (NGS) performed by Foundation Medicine found
that 192 (96%) had at least one alteration identified,
with 169 (85%) having at least one clinically relevant
alteration, and found 26 alterations for which
targeted therapies approved in a known tumor type
are available.57 These included six (3%) with EGFR
substitution, six (3%) with ERBB2 amplification,
11 (6%) with BRAF substitution, and two (1%) with
ALK substitution.57 Importantly, cases were also
described of patients treated with targeted therapies,
such as crizotinib for MET amplification or ALK
fusion, in which patients had significant responses,57
suggesting that identifying actionable alterations is
clinically meaningful.
A second retrospective study of 150 patients who
had NGS performed at Memorial Sloan Kettering
Cancer Center found that 137 (91%) had at least one
alteration detected and 45 (30%) had potentially
targetable alterations. These included six (4%) with
BRAF V600E mutation, seven (5%) with ERBB2
amplification, and four (3%) with FGFR2/3 fusion. Of
these patients, 15 (10%) received targeted therapies,
with time to treatment failure ranging from less than
one month to 14 months; the longest durations of
treatment (of five to more than 14 months) were seen
in patients with genomic rearrangements, including
ALK fusion, RET fusion, FGFR2 fusion, and NTRK1
fusion.58
Use of circulating tumor DNA (ctDNA) to facilitate
assessment of genomic alterations via a liquid
biopsy is an emerging platform. Analyses of ctDNA
in patients with CUP have shown rates of actionable
mutations comparable to the two previous series
using tumor tissue. In a study from the University
of California San Diego, 442 patients with CUP had
ctDNA testing using the Guardant assay between
2014 and 2016, during which time 54-70 genes
were tested using NGS. Of the samples from 442
patients, 353 (80%) had ctDNA alterations detected
and 290 (66%) had at least one characterized,
likely pathogenic alteration. Targetable alterations
included nine (2%) patients with BRAF V600E, four
(1%) with EGFR L858R, and three (1%) with RET
fusion. A total of 282/442 (64%) had an alteration
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theoretically actionable by an agent approved by the
US Food and Drug Administration.59 This may thus
serve as a potential novel platform for testing.
When interpreting these retrospective reports of
genomic tumor testing, it is important to note that the
definition of a “potentially actionable” alteration is a
matter of debate. For example, although the ctDNA
study reported that 64% of patients were found to
have a potentially actionable mutation, many of
those are truly only theoretically actionable, such as
the 164 patients with a TP53 mutation. These studies
also do not clearly describe the number of patients
who have therapy changed as a consequence of
results of testing, and nor do they robustly show
clinical benefit of mutation specific testing. Finally,
even if a “potentially actionable” alteration is
detected, attaining access to targeted therapies may
be challenging as no targeted agents have regulatory
approval in CUP. Given this, enrollment in clinical
trials is recommended, as this will allow patients
access to targeted therapies while generating data to
determine the efficacy and safety of these therapies.
However, no difference exists in the rationale for
performing large, multigene NGS in CUP compared
with other advanced solid tumors. Recent regulatory
approvals of biomarker selected therapies agnostic of
primary tumor site, including treatments for cancers
with NTRK fusions or microsatellite instability, show
that sequencing results may affect standard therapy
choices, even in carcinoma of unknown primary.
However, additional prospective clinical trials would
ideally be conducted to show a clinically meaningful
benefit in other biomarker defined CUP populations,
although such studies are unlikely to be conducted.
The benefit is likely limited to the small proportion
of patients who are found to have a clearly validated
alteration that serves as a predictive biomarker for a
targeted therapy.
Potential utility of NGS results to identify tissue of
origin
Many genomic aberrations or mutation patterns that
are detected by NGS are strongly associated with
certain primary tumor sites and thus may prove
useful to identify a primary tumor site. For example,
APC loss of function mutations are highly associated
with colorectal cancers, and mutational signatures
consistent with ultraviolet light exposure are highly
associated with cutaneous melanomas. These
patterns may be discernible using machine learning
to result in enhanced ability to determine a primary
tumor type. Recently, NGS data from Memorial Sloan
Kettering were used to develop a genomics based
classifier, using 7791 samples in 22 cancer types
as a training set, and the model then underwent
cross validation on training data along with an
independent test set. On cross validation, 5748/7791
(73.8%) tumor types were successfully identified,
and the classifier successfully identified tumor type
in 8623/11 644 (74.1%) cases in an independent
test set. The probability of each prediction varied in
different tumor types—it exceeded 95% in 43.5% of
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State of the Art REVIEW
cases, whereas most of the inaccurately classified
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samples had a probability below 50%. This classifier
successfully identified a likely tissue of origin with
a probability greater than 50% in 95/141 (67%)
patients who clinically had CUP and occasionally
resulted in changes in clinical management of
patients.60 With increasingly widespread use of
NGS in metastatic carcinomas, these classifiers may
provide additional opportunities to identify tumor
type in CUP. Caris has developed an analysis based on
a machine learning algorithm trained and validated
on more than 40 000 tumor samples with NGS data
to identify primary tumor site, with tumor lineage
classifier accuracy ranging from 82% to 96%.61 This
assay, dubbed the MI Genomic Profiling Similarity
(GPS) score, is now commercially available. Overall,
these early results seem to be comparable to the
accuracy of previous tissue of origin tests, although
whether these result in clinically meaningful
improvements in outcome remains unknown.
Potential immunologic biomarkers
An appreciable proportion of CUPs have biomarkers
suggestive of likely response to immune check­
point inhibitors, including microsatellite instability,
tumor mutation burden, and high programmed
death-ligand 1 (PD-L1) expression. A retrospective
analysis of 389 cases of CUP used NGS to elucidate
total mutational load (TML) and microsatellite
instability and immunohistochemistry to determine
PD-L1 expression. High microsatellite instability was
found in 7/389 (1.8%) cases and was confirmed by
immunohistochemistry, showing loss of a mismatch
repair protein in six cases. High TML (≥17 mutations/
Mb) was found in 46/389 (11.8%) samples, and PD-
L1 expression (using SP142 antibody) on at least
5% of tumor cells was found in 82/365 (22.5%)
samples.62 These findings present the hypothesis that
a subpopulation of CUPs may respond to immune
checkpoint inhibitors, most notably the group with
high microsatellite instability tumors, who have the
potential for a profound difference in their prognosis
with the use of checkpoint inhibitors. Further
assessment of the optimal biomarker to use for
selection of patients for checkpoint immunotherapy
is important for all diseases, including CUP.
Prospective clinical trials of mutation testing in CUP
Given the clearly demonstrated benefit of treating
patients whose cancer is found to harbor one of
several genomic aberrations that can be detected
in a range of malignancies, clinical trials are now
prospectively assessing genomic aberrations and
assigning patients to a therapy option based on
the underlying aberration. High profile trials such
as NCI-MATCH and TAPUR enroll patients with a
variety of cancers including CUP. The CUPISCO study
(clinicaltrials.gov identifier NCT03498521) is a
randomized phase II trial focused on CUP that enrolls
previously untreated patients with adenocarcinoma
or poorly differentiated carcinoma of unknown
primary. CUPISCO compares molecularly directed
9
State of the Art REVIEW
therapy based on detected genomic aberrations with
platinum based chemotherapy. In this study, patients
receive three cycles of induction chemotherapy with
the investigator’s choice of carboplatin-paclitaxel,
carboplatin-gemcitabine, or cisplatin-gemcitabine.
Archival tissue is tested using hybrid capture based
comprehensive genomic profiling, and microsatellite
instability, TML, and genomic loss of heterozygosity
are calculated. Additionally, tumor expression of
PD-L1 is assessed by immunohistochemistry using
DAKO 22C3 antibody. Patients with complete or
partial response or stable disease following induction
chemotherapy are then randomized 3:1 to receive
molecularly directed therapy based on the counsel of
a molecular tumor board and the final decision of the
treating investigator, or otherwise to continue with
three additional cycles of chemotherapy. Patients
with progressive disease will receive molecularly
directed therapy as per the molecular tumor board
counsel. The primary endpoint of this study is
investigator assessed progression-free survival, with
accrual planned for 790 patients.63
The study is still open and enrolling, and interim
feasibility results were presented at the 2019 ESMO
congress. Of 303 patients whose data were examined
retrospectively, 96 (32%) would have been matched
to a molecularly directed therapy arm. Notably, key
genomic alterations included HER2 (7%), PIK3CA
(6%), NF1 (6%), NF2 (5%), BRAF (4%), PTEN (4%),
FGFR2 (4%), EGFR (4%), and MET (4%). Gene
fusions were also observed, including ALK (1%), RET
(1%), and ROS1 (1%). High TML (≥20 mut/Mb) was
found in 9%, and 1% had microsatellite instability.
Additionally, 14% had high PD-L1 with a tumor
proportion score of 50% or higher.64
Additional studies of novel molecularly targeted
systemic therapies in CUP are ongoing. For example,
the CUPem study is enrolling patients with CUP
who have received at least one previous regimen of
chemotherapy to receive the PD-1 antibody pembro­
lizumab. Correlative studies, including archival
tissue at baseline to document PD-L1 expression, are
planned, but there is no intrinsic biomarker selection
for the study (NCT03752333). The results of the
NivoCUP study were presented at the 2020 ASCO
virtual meeting and showed an overall response
rate of 10/45 (22%, 95% confidence interval 11%
to 37%) among patients who had received previous
treatment and 2/11 (18%, 2% to 52%) among
previously untreated patients with CUP.65
Emerging assays
Most of the published studies using tissue of
origin testing with commercially available tests
use relatively older technologies to assess gene
expression profiles. The proliferation of cancer
genome data published has allowed for additional
analyses of transcriptomics, genomics, and epigeno­
mics to identify tissue of origin, which may offer more
accurate results. A 154 gene expression signature
correctly classified 97.1% of 9626 primary tumor
samples and 92% of 1248 poorly differentiated
10
or metastatic samples.66 An ensemble of neural
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networks was trained using data from the Cancer
Genome Atlas and used to retrospectively determine
primary tumor type from RNAseq data from 201
metastatic tumors of known tumor types; it had an
overall mean accuracy of 86%.67 A microarray DNA
methylation signature, dubbed EPICUP, was also
trained to identify 38 tumor types and in a validation
study showed 97.7% (96.1% to 99.2%) sensitivity
and 99.6% (99.5% to 99.7%) specificity.68
Guidelines
Guidelines from NCCN, ESMO, and NICE for
management of cancers of unknown or occult
primary are highlighted throughout this review.
The Spanish society of medical oncology (Sociedad
Espanola de Oncologia Medica; SEOM) published
clinical guidelines for cancer of unknown primary in
2017,69 which are generally concordant with NCCN
and ESMO guidelines. NCCN guidelines specify that
“tumor sequencing and gene signature profiling for
tissue of origin is not recommended for standard
management at this time,” noting that “there
may be diagnostic benefit, though not necessarily
clinical benefit.” However, this recommendation was
category 3, indicating that major disagreement exists
within the NCCN as to whether the intervention is
appropriate.17 SEOM guidelines also state that “the
impact on clinical benefit of targeted treatment based
on molecular studies remains controversial and the
level of evidence and degree of recommendation is
low.” 69 ESMO guidelines similarly state that tissue of
origin tests “may aid in the diagnosis of the putative
primary tumour site in some patients… However,
their impact on patient outcome via administration
of primary site-specific therapy remains questionable
and unproven in randomized trials.” 18 Finally, NICE
guidelines also direct: “Do not use gene-expression-
based profiling to identify primary tumours in
patients with provisional CUP.” 19 All guidelines thus
stress the need for randomized phase II and III studies
to show the utility of tissue of origin testing. Future
versions of the guidelines will likely incorporate the
final results of GEFCAPI-04 and CUPISCO, and final
publication will be eagerly awaited.
Conclusion
Modern oncologic treatment increasingly stresses
personalization of therapy, as novel predictive
biomarkers and targeted therapies are identified.
Current management of unfavorable subtypes of
CUP focuses on empiric chemotherapy. Studies to
date have not shown disease specific chemotherapy
to result in a significant improvement in outcomes,
with the exception of the small number of clinical
scenarios in which therapy with curative intent is
feasible. Just as is the case with patients in whom
the site of origin is known, future management of
CUPs will emphasize identification of subgroups
of patients who have diseases that are susceptible
to targeted therapies and immunotherapies on the
basis of a predictive biomarker.
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 State of the Art REVIEW

16  Pavlidis N, Briasoulis E, Hainsworth J, Greco FA. Diagnostic and

Research questions

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