CLASSFICATION OF BREAST CANCER ON THE BASIS OF

IMMUNOHISTOCHEMISTERY

Assiengment

Name ; Muhammad wajid

Disp; MS MLS 2 nd semester

INTRODUCTION

Breast cancer is the most common type of cancer in women worldwide, including
in the Republic of Macedonia. (1). It's also the leading cause of cancer-related deaths
among women. Factors like tumor size, type, grade, and lymph node status help predict
how the cancer will progress. Newer factors like hormone receptor presence, HER-2
receptor status, and Ki67 levels also play a role. These factors help doctors determine
the best treatment plan. Different combinations of these factors classify breast cancer
into subtypes, like Luminal A, Luminal B, HER2 positive, HER2 enriched, and triple
negative. Research looks at how these subtypes vary across studies and if they relate
to the cancer's stage. (2)
Material and Methods
During 2014, 290 patients treated for breast cancer at the University Clinic for Thoracic
Surgery in Skopje, Republic of Macedonia, underwent thorough analysis with complete
medical histories and all relevant parameters. This included standard histological
examinations using H&E staining, as well as immunohistochemical staining for ER,
PgR, HER-2, and Ki-67 on tissue samples from primary tumors. Path histological tests
were conducted in three accredited laboratories, and specific protocols were followed
for staining and scoring. The classification of breast cancer subtypes was based on
established criteria, and statistical analysis was performed using Statistical 7, including
descriptive analyses, χ² tests, and ANOVA tests for variance analysis. (3)

RESULTS

We analyzed patients aged 18-90, averaging 57.6 years, with primary tumor sizes
averaging 30.27 + 18.3 mm and 59% showing axillary lymph nodes metastases. Using
the new St. Galen classification system, we categorized breast cancer subtypes into five
groups, with the distribution as follows: 77 (26.55%) Luminal A, 91 (31.38%) Luminal B
HER2 negative, 70 Luminal B HER2 positive, 25 (8.62%) HER2 enriched, and 27
(9.31%) Basal-like (or triple negative) patients.

DISCUSION

Breast cancer is a varied condition with diverse clinical and histopathological features, each
demonstrating unique gene expressions across different subtypes and molecular profiles. This
variety contributes to varied predictive and prognostic factors among patients. While DNA
microarrays are frequently used for gene expression analysis, their cost can be prohibitive,
leading to the widespread acceptance of immunohistochemical marker analysis as an
alternative method for subtype classification in breast cancer. The 2011 St. Gallon consensus
conference delineated five subtypes based on receptor presence on tumor cell surfaces and
Ki67 measurement values. Estrogen receptors, situated on tumor cell surfaces, play a crucial
role in stimulating cell growth and division upon estrogen binding, making drugs that block
estrogen receptors or synthesis effective in inhibiting tumor growth. Similarly, overexpressed or
gene-amplified HER-2 neu receptors, found in about 20% of breast cancer cases, drive cell
proliferation and anti-apoptosis signals, significantly contributing to breast cancer development
and progression. HER-2 neu receptors are detected on the surface of malignant cells using
immunohistochemical assays. Recognizing that receptor presence or absence on breast cancer
cell surfaces is influenced by gene mutations and overexpression, subtypes can be identified
through gene expression assessment, leading to the term genotype of breast cancer mentioned
in the literature. The most common subtype, Luminal A, accounts for 50-72% of breast cancer
cases, characterized by a favorable prognosis with a low proliferative index, good differentiation,
and minimal risk of local recurrence and relapse (4). However, varying rates for this subtype are
documented in the literature, such as 34% in Italy and . Saudi Arabia 3.9%, China 65.3%, and
Japan 71% . The recommended treatment for these patients includes third-generation
aromatase inhibitors in postmenopausal women, selective estrogen receptor modulators (like
tamoxifen), and selective estrogen receptor modulators (like fulverstone) . In our analysis,
Luminal A type was identified in only 26.55% of the cases. Luminal B subtype is characterized
by positive estrogen receptors, with positive or negative HER2 receptors, and a higher Ki-67
value exceeding 14%, all of which contribute to a worse prognosis compared to Luminal A
subtype. Luminal B type is present in 10-20% of breast cancer patients, with HER-2 negativity
detected in 31.38% and HER-2 positivity in 24.14% of the cases in our study. References in the
literature indicate rates such as 36% in Italy and 24.6% in Egypt . This indicates a
predominance of aggressive tumors in our cohort. Luminal B subtype is notably more
aggressive than Luminal A, characterized by poor differentiation, increased frequency of bone
metastases, and a poorer prognosis. Many authors note that patients in this subtype are
typically younger, with larger tumors, positive nodal status, and a higher N stage [16]. Given the
prevalence of larger tumors and advanced disease stages, Luminal B findings are more
frequent in our study. Although there was no age discrepancy between Luminal A and Luminal
B types in our study, Luminal B was more common in older patients . The recommended
treatment includes tamoxifen, along with chemotherapy in neoadjuvant and adjuvant regimens .
HER-2 enriched subtype is identified in 15-20% of patients, but only 8.62% in our group. This
subtype is characterized by a high proliferative index, poor differentiation, and frequent p53
mutations. It represents a highly aggressive tumor type, with a 10-year survival rate of only 12%
. Targeted HER-2 therapy with monoclonal antibody trastuzumab (Herceptin) is recommended,
in combination with chemotherapy in the neoadjuvant and adjuvant setting. Trastuzumab
treatment in combination with DM1 is also an option . Basal-like subtype (triple negative) is
characterized by larger tumors, poor differentiation, high mitotic index, and tumor necrosis,
occurring in 10-20% of patients. A similar subgroup, Claudine-low, differs only in EGFR
expression and is found in 12-14% of patients. Both subgroups have a poor prognosis, high
mitotic index, and often exhibit metastases in visceral organs, lungs, and CNS. Triple-negative
breast cancer has the worst prognosis, with frequent relapse within the first three years, and
p53 mutations are common. In our study, triple negative cases accounted for 9.31% of the total.
The recommended treatment includes chemotherapy, as well as PARP inhibitors like olaparib
for detected BRCA1 or BRCA2 mutations (5). The normal breast cell-like subtype, identified in
5-10% of patients, does not respond to neoadjuvant therapy but only to adjuvant chemotherapy.
It is typically well-differentiated, with a low proliferation index and a median overall survival rate .
The distribution of subtype frequencies in our study varies from those reported in the literature,
highlighting the global heterogeneity of breast cancer. Certain subtypes may be more prevalent
in specific populations, such as triple negative breast cancer being more common among
African-American women . Understanding subtype prevalence can aid in designing tailored
therapeutic strategies . There were no significant age differences observed among breast
cancer subgroups in our study, with patients typically ranging between 52.72 and 58.83 years
old, except for Luminal B HER negative and HER-enriched subgroups (P = 0.0255). Patient age
distribution across breast cancer subgroups is depicted in Figure 1. Tumor size varies among
subgroups, ranging from 27.18 to 35 mm, with triple-negative subtype having the largest tumors.
However, there were no significant differences observed across the entire group or between
subgroups. Tumor size, defined by T stage, also did not vary significantly among subgroups.
High-grade differentiation (G3-low differentiation) of malignant cells was observed in 35.16% of
Luminal B subtype patients, whereas only 14.8% of triple negative subtype patients exhibited
G3 differentiation, contrary to some literature findings . Regarding lymph vascular invasion, the
highest rates were found in Luminal B HER-2 negative subtype, at 49.45%, indicating an
invasive and aggressive breast cancer subtype with a propensity for lymphatic spread and
higher axillary lymph node involvement (69.23%). Interestingly, triple-negative subtype exhibited
relatively low lymph vascular invasion rates (44.44%), with lower axillary lymph node
involvement (62.96%), aligning with literature trends. This suggests alternative pathways for
malignant cell dissemination beyond the lymphatic system, considering the aggressive nature of
triple-negative breast cancer . Ki67 values, indicating tumor proliferative activity and aggression,
were higher than 14% in Luminal B HER negative subtype (across all patients) and in HER-2
enriched and triple negative subtypes. In contrast, Luminal A subtype did not exhibit Ki67 values
exceeding 14%, indicating a less aggressive nature (6) . The distribution patterns of estrogen,
progesterone, and HER-2 neu receptor-positive cells varied significantly among subgroups,
reflecting their crucial role in defining breast cancer subtypes . Identifying breast cancer
subtype, alongside histological type and TNM stage, aids in predicting disease prognosis,
detecting metastasis sites, and guiding therapeutic decisions . Since all factors determining
breast cancer subtypes can be assessed from core biopsy materials prior to treatment initiation,
certain subtypes, especially aggressive ones, can be targeted with appropriate drugs in a
neoadjuvant protocol . In summary, subtype identification in breast cancer is vital for prognosis
and treatment planning, necessitating routine determination of the molecular subtype in
histopathological assays. (7)

We observed variations in frequencies among our subgroups compared to those documented in

the literature, highlighting the diverse nature of breast cancer worldwide. Literature mentions
certain subtypes being more prevalent in specific ethnicities (such as triple negative being more
common in African-American women) . Understanding subtype prevalence in a population
allows for a more tailored therapeutic strategy . Age did not significantly differ across breast
cancer subgroups, with patients typically aged between 52.72 and 58.83 years, except for
Luminal B HER negative and HER enriched subgroups (P = 0.0255). Patient age distribution in
breast cancer subgroups is illustrated in Figure 1. Tumor sizes varied across subgroups,
ranging from 27.18 to 35 mm, with the triple negative subgroup exhibiting the largest tumors.
However, there were no significant differences observed overall or between subgroups
regarding tumor size. The same trend applied to tumor size categorized by T stage, with no
significant variations between subgroups. High-grade differentiation (G3-low differentiation) of
malignant cells was noted in 35.16% of Luminal B subtype patients, whereas only 14.8% of
triple negative subtype patients showed G3 differentiation, contradicting findings from many
literature studies . Figure 1: Age distribution among breast cancer subgroups Regarding
lymphovascular invasion, the highest rates were found in Luminal B HER-2 negative subtype, at
49.45% of patients, indicating an invasive and aggressive breast cancer subtype with a
tendency for lymphatic spread and increased axillary lymph node involvement (69.23%).
Interestingly, triple negative subtype showed relatively lower lymph vascular invasion rates
(44.44%), with less axillary lymph node involvement (62.96%), consistent with literature findings.
This suggests alternative pathways for malignant cell dissemination beyond the lymphatic
system, considering the aggressive nature of triple-negative breast cancer . Ki67 values,
reflecting tumor proliferative activity and aggression, exceeded 14% in Luminal B HER negative
subtype (in all patients), as well as in HER-2 enriched and triple negative subtypes. (8) In
contrast, no values above 14% were found in Luminal A subtype, indicating a less aggressive
nature . The distribution patterns of cells positive for estrogen, progesterone, and HER-2 neu
receptors differed significantly among subgroups, which is expected given their role in defining
breast cancer subtypes . Understanding breast cancer subtype, along with histological type and
TNM stage, aids in predicting disease prognosis, identifying metastasis sites, and guiding
treatment decisions (9) . Since all factors determining breast cancer subtypes can be assessed
from core biopsy materials before treatment initiation, certain subtypes, particularly aggressive
ones, can be targeted with appropriate drugs in a neoadjuvant protocol . In conclusion,
identifying breast cancer subtype is crucial for prognosis and treatment planning. Therefore,
routine determination of the molecular subtype of breast cancer is essential in histopathological
assessments.

REFRENCES

1. clasification of breast cancer on the basis of immunohistochemistery . Justo N, Wilking N, Jönsson B,

Luciani S, Cazap E. A Review of. 2013, pp. 50-60.

2. Breast cancer classification according to immunohistochemical markers: clinicopathologic features

and short-term survival analysis in a population-based study from the South of Switzerland. A. Bordoni,
P. Mazzola, M.P. Curado, B. Edwards, H.R. Shin, et al. 2002.

3. Molecular Subtyping of Triple‐Negative Breast Cancers by Immunohistochemistry: Molecular Basis and

Clinical Relevance. Bauer KR, Brown M, Cress RD et al. Descriptive analysis of estrogen receptor (ER)‐
negative, progesterone receptor (PR)‐negative, and HER2‐negative invasive breast cancer, the so‐
called triple‐negative phenotype: A population‐based study from the Cal. 2020.

4. Practical and Robust Identification of Molecular Subtypes in Colorectal Cancer by

Immunohistochemistry . Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012
and 62:10–29. 2017.

5. Molecular classification of breast cancer: A retrospective cohort study. Almuhanna, R., …, Makhtoum,
T. 2020.

6. Pathologic diagnosis, immunohistochemistry, multigene assays and breast cancer treatment: progress
toward “precision” cancer therapy. DG Hicks, B Turner. 2014.

7. Immunohistochemical versus molecular (BluePrint and MammaPrint) subtyping of breast carcinoma.

Outcome results from the EORTC 10041/BIG 3-04 MINDACT trial. Viale, G.

8. Breast cancer biomarkers in clinical testing: analysis of a UK national external quality assessment
scheme for immunocytochemistry and in situ hybridisation database containing results from 199 300
patients. Andrew Dodson, Suzanne Parry, Merdol Ibrahim, John MS Bartlett, Sarah Pinder, Mitch
Dowsett, Keith Miller. 2018.

9. Intratumoral heterogeneity as a source of discordance in breast cancer biomarker classification. Emma

H. Allott, Joseph Geradts, Xuezheng Sun. 2016.

11. Breast cancer biomarkers in clinical testing: analysis of a UK national external quality assessment
scheme for immunocytochemistry and in situ hybridisation database containing results from 199 300
patients. Andrew Dodson, Suzanne Parry, Merdol Ibrahim,. 2018.