Evaluation of HR[ER/PgR] status and correlation with Ki67 expression in BC Gayatri Gogoi, MD Assistant Professor Deptt of Pathology, Assam Medical College, India id

Co-Authors  M Borgohain, MD Professor of Pathology  Manash P Baruah, DM, Consultant Endocrinologist  S A Fazal, MS Associate Professor of Surgery

Background  Breast cancer[BC] is a leading cause of death in women. Hormonal Receptor status is the most important prognostic and predictive marker for BC.  It has been used in management of breast cancer since 1970s as an indicator of endocrine responsiveness and prognostic factor for recurrences

Management depends For comprehensive management testing of BC panel is a must, namely Estrogen Receptor, Progesterone Receptor, Her2/neu  ER-positive (ER+) tumors typically respond to hormone therapy,  while HER2+ tumors respond to anti-HER2 therapy, and  no targeted therapy is currently available for widespread use for ER−/HER− tumors

Molecular Classification  Breast cancers are classified into 5 molecular subtypes  [1] Luminal A, is defined by as only Estrogen receptor[ER] with or without Progesterone Receptor[PR] expression  [2] Luminal B, is defined by ER with or without PR as well as Her2/neu expresssion

Molecular classification  3. Normal breast like  4. Her2neu enriched  5.Basal Like including Triple negative BC[TNBC]  ER,PR,Her2,all 3 markers negative is TNBC.  BC expressing basal markers CK5/6 are called basal like

Hormonal receptors are ER & PR  Luminal BC means arising from luminal cells which are positive for cytokeratin 8 & 18.  It can be either Luminal A or B  Markers of proliferation, and specifically Ki-67- labelling index, is also considered important for the determination of prognosis. This was the back ground knowledge I had at beginning of this study in

Aim and Objective  To evaluate the hormonal[ER/PR] receptor status in invasive BC and study the proliferative behavior of both Luminal A and B by Ki67 expression.  Correlate the findings with prognostic parameters

Setting and Design  A prospective analysis of 112 BC cases, was undertaken to study the histomorphological features followed by immunohistochemical study in Department of Pathology.  The mastectomy specimens were received from Department of Surgery and private hospitals of surrounding area.

Material and Methods:  The present study was approved by the institutional ethical committee for Human Research.  It was carried out in the Department of Pathology, in Histopathology and Immunohistochemistry Sections, Assam Medical College & Hospital, Assam, from July 2011 to June 2012.

Material and Methods:  All specimens were fixed in 10% buffered formalin( P H ) in an adequate volume of 10 fold immediately after operation and allowed to stay between 12—24 hours (ASCO guidelines 6—72 hours)  Specimens were grossed on the same day mostly or next day,, with special emphasis to the site, size of the tumour, margins and lymph node sampling of the lesion and sections from representative area.  The sections were processed under standardized conditions for paraffin embedding.

Material and Methods:  Histopathologic features were determined prior to IHC examination.  Histopathologic types were ascertained  Histological grade was assessed using Bloom and Richardson's method, modified by Eltson and Ellis. by evaluation of tubule formation, nuclear pleomorphism, mitotic count.  Involvement of lymph nodes and numbers.  Comment on surgical margins, presence of DCIS, LymphoVascular invasion or perineural involvement etc Bloom HJG, Richardson WW. Histologic grading and prognosis in breast cancer: a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer. 1957;11:359– 377

 Interpretation of IHC results were done according to ASCO and CAP Guideline Recommendations for IHC Testing of ER and PR in Breast Cancer.  According to the guidelines it is recommended that ER & PR assays be considered positive if at least 1% positive tumour nuclei in the sample on testing in presence of expected reactivity of internal control (normal epithelial element) and external control. Interpretation of IHC Results:

IHC scoring for ER,PgR

Her2+ More than 30% of cells showing strong complete membrane + in invasive BC component to be interpreted as over expressed

Ki 67 or Mib 1 Antibody :  Only showing distinct nuclear staining by Ki67 of invasive carcinoma component were selected for counting.  Though various authors used different criteria for counting in various malignancies but purpose of breast, it was counted like mitosis counting formula of BRG, so that it is easily comparable to mitotic count and can be analyzed effectively (Trihia H et al, 2003).   Trihia H, Murray S, Price K, et al: Ki-67 expression in breast carcinoma. Cancer 97: ,2003.

Results & observations Table 1.1: Age

Table–1.1 shows more than 66.08% of the invasive breast carcinoma cases belonged to younger than 50 years of age group out of which 33.04% from 40—49 years age range and 26% from less than 39 years age range. The youngest case was 24 years and the oldest one was 75 years with a mean age of presentation is 

Histology Grade I [BRG]

IDC with apocrine change BRG II

IDC –BRG III

Table 1.2 Bloom Richardson Grade

 The histological grading showed that 44.64% of invasive breast carcinoma cases belonged to grade II category.  Next 32.12% cases of breast carcinoma having poorly differentiated features belonged to grade III. Last 23.21% cases show well differentiated feature with histologic grade I.  So it is seen that best prognostic category have least numbers of cases in the study.

Table 1.3 ER/PgR profile

ER/PgR profile This study of 112 invasive BC immunohistochemical evaluation showed 47.32% are ER positive and 46.58% are PgR positive. There are ER+ cases where histologic grades are high, Her2 overexpressed, Ki67 high. We tagged them as Luminal B

HR positive : Luminal A

Micropapillary type

Her2 +

ER+, Allred score 8/8

PgR+ but low 2-5% cells

Ki 67 high grade- Luminal B

Luminal B example  Micropapillary case belonged to Luminal B.  ER + strong 100%  PR + low 2-5%,  overexpression of her2/neu  High expression of Ki67

Table 1.4 Luminal A and Luminal B

Total Hormonal Receptor positive in 47.29% of cases, but pure ER and or PR positive cases are only 31.25% which is grouped as Luminal A category. The subgroup with Her2neu over expression is 16% which is categorized as luminal B according to available literature

Table 1.5 ER+ groups correlation to Ki67 expression

Table :1.7 Luminal A, B with Ki67

Correlation in subgroups significant  ER+ expression profiles were correlated, no significant correlation found.  But when correlated in subgroup as luminal A and B seperately, it shows a statistically significant relationship with P value<.005, high expression in Luminal B type  Luminal A group show mostly low and moderate Ki67 expression

Table 1.6 :Lymph node in ER + groups

Lymph node status in ER + groups  Luminal A have maximum node negative breast cancer, which is statistically significantWith P value less than.005  Luminal B have maximum number node positive cases of 1-3 node followed by more than 4 nodes.

Discussion & review literature  we have found in Luminal B cases are generally  High histologic grades,  High expression of ki67,  PR score is low or absent  more likelihood of axillary node positive  More commonly seen in less than 50 years woman.  Besides Her2 overexpression which is criteria to include that category.

PR role in clinics  PR another molecular marker that may be used in the clinic, as loss of PR in ER+ tumors is thought to be predictive for lack of response to hormone therapy.  In our study while ER+ subgrouping were done with help of protein expression taking the criteria of ER & Her2 +, or ER, PR&her2+  In that situation, PR downregulation was not taken into consideration for favour of Luminal B as most literature or consensus for clinical setting doesnot talk much on it.   -Cui X, Schiff R, Arpino G, Osborne CK, Lee AV. Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy. J Clin Oncol. 2005;23(30):7721–7735. [PubMed]PubMed

Predictive value of PR  The predictive value of PR has long been attributed to the dependence of PR expression on ER activity, with the absence of PR reflecting a nonfunctional ER and resistance to hormonal therapy.  However, recent clinical and laboratory evidence suggests that ER-positive/PR-negative breast cancers may be specifically resistant to selective ER modulators,  whereas they may be less resistant to estrogen withdrawal therapy with aromatase inhibitors, which is a result inconsistent with the nonfunctional ER theory

Pathophysiology of Luminal B  The designation of luminal B first came about with the early gene expression profiling studies of breast cancer near the start of the millennium, it has long been understood that there exists a more aggressive form of ER+ breast cancer.  Earlier studies had noted that ∼ 30% of ER+ invasive breast cancers showed no benefit from hormone therapy, suggesting that these cancers either augment or entirely bypass the classical estrogen- stimulated mitogenic pathway  . Allred DC, Brown P, Medina D. The origins of estrogen receptor alpha-positive and estrogen receptor alpha-negative human breast cancer. Breast Cancer Res. 2004;6(6):240–245. [PMC free article] [PubMedPMC free articlePubMed

ER modulator resistance  Novel alternative molecular mechanisms potentially explaining SERM resistance in ER- positive/PR-negative tumors have been suggested by recent experimental indications that growth factors may downregulate PR levels.  Thus, the absence of PR may not simply indicate a lack of ER activity, but rather may reflect hyperactive cross talk between ER and growth factor signaling pathways that downregulate PR even as they activate other ER functions

Alternate pathway of ER function Loss of PR expression is thought to represent a surrogate for a more aggressive disease phenotype that is less dependent upon estrogen signaling; in correlative studies PR loss has been associated with lower ER levels, more positive nodes, aneuploidy, larger tumor size, higher proliferation, and expression of growth factor receptors (GFRs) including EGFR and HER2

 Assays for diagnosing luminal B subset of ER+ in the clinic are currently available, though it will likely take some time for these assays to become routine as new clinical practices are often gradually adopted. Eg- Oncotype Dx, Mammaprint etc. Cost?  PR is one marker that is frequently measured in the clinic along with ER, in order to further subdivide ER+ breast cancers by prognosis or anticipated therapeutic response.  . Cui X, Schiff R, Arpino G, Osborne CK, Lee AV. Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy. J Clin Oncol. 2005;23(30):7721–7735. [PubMedPubMed Diagnosis of Luminal B

Down PR,up Her2, EGFR?  the association of PR with hormone response has not been observed everywhere, which may be an indicator of a single biomarker having insufficient information as compared to a biomarker panel  Clinical variables such as grade or Ki-67 can help distinguish the subset of ER+ breast tumors with expected worse outcome.  Her2 over expression is well established but EGFR expression in Luminal B require further study – Cheang MC, Chia SK, Voduc D, et al. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst. 2009;101(10):736–750. [PMC free article] [PubMedPMC free articlePubMed

Clinical potential and future research  At present, luminal B breast cancer is routinely treated as an ER+, hormone-sensitive disease.  While often considered as simply a more aggressive form of the ER+ subtype, luminal B might be considered more in the future as a type of breast cancer that is entirely distinct in many ways from luminal A. Prat A, Ellis MJ, Perou CM. Practical implications of gene-expression- based assays for breast oncologists. Nat Rev Clin Oncol. 2011;9(1):48–57. [PMC free article] [PubMed]PMC free articlePubMed

What best can be done in clinical setting?  Having that background knowledge, we can sum up  ER+ positive BC have two different entities  pathophysiologic, treatment, molecular profiling point of view,  What best can be done in our clinical setting respect to our limited facility ?

Stratification ER+ type of BC  Ki67 expression can be used document proliferative behavior as the previous studies applied to test aggressive behavior in long term management of early BC.  This ki67 is also can be used to tag Luminal B in clinical setting and managment   Molecular profile of Luminal B cancer, Chad J Creighton, Biologics 2012, 6:

Ki67 in BC clinics Ki67 can be used in ER+ Breast Cancer sub grouping In Clinical Setting Our study too find its higher expression is directly proportional to poor prognostic parameters.

Conclusion & Future perspective Ki67 expression can significantly add into understanding of aggressive behaviour of BC It is a valuable tool in stratification of ER+[Luminal tumours] into likely SERM responsive or resistant or Luminal A or B It also has a definitive role in long term management of early BC. Testing of Ki67 in both primary tumour and lymph node together may help in understanding its prognostic role.

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Acknowledgement: Assam Medical College &Hospital Srimanta Sankardev University& Health Science Assam, India But the Beginning of the Brown Era of IHC. But the Beginning of the Brown Era of IHC At AMCH At AMCH So, everyone of us to give the best to the realize the Dream

References Molecular profile of Luminal B cancer, Chad J Creighton, Biologics 2012, 6: Cui X, Schiff R, Arpino G, Osborne CK, Lee AV. Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy. J Clin Oncol. 2005;23(30):7721–7735. [PubMed]PubMed. Sørlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001;98(19):10869– [PMC free article] [PubMed]PMC free articlePubMed