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David dabbs

A History of Hormone Receptors in Breast Cancer: Lessons for the future

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(Second abstract and handout , see below)

Estrogen receptor alpha (ERa) and progesterone receptor (PgR) are prognostic and predictive biomarkers which play a major role in determining the therapy of patients with invasive breast cancer (IBC). ERa and PgR are weak prognostic factors but very strong predictive factors of response to endocrine therapies.  It is currently mandatory to evaluate ERa and PgR in all IBCs for the purpose of predicting therapeutic response.  In current practice, immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue (FFPET) samples is the primary method used to evaluate ERa and PgR.

The American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) recently jointly published guidelines for ERa and PgR testing in breast cancer recommending that specific IHC assays must be rigorously standardized and validated to be utilized in routine clinical practice .  Adherence to these guidelines is now mandatory for laboratory accreditation by the CAP, which also provides many educational and support materials to facilitate compliance.

Estrogen Receptor-alpha
ERa expression has been evaluated in IBCs for almost 40 years.  During the first 25 years it was primarily measured by biochemical ligand-binding assays (LBAs) on whole tissue extracts prepared from fresh-frozen tumor samples, which was costly and difficult.  Many studies using LBAs in large randomized clinical trials demonstrated that ERa was a weak prognostic factor but a very strong predictive factor for response to endocrine therapies such as tamoxifen.  Tamoxifen binds ERa and inhibits the estrogen-stimulated growth of tumor cells, which significantly reduces cancer recurrences and prolongs survival in patients with ERa-positive IBCs of all stages.  More recently, tamoxifen, has also been shown to reduce subsequent breast cancer in patients with ERa-positive ductal carcinoma in situ (DCIS), and in patients who are cancer-free but at high risk for developing breast cancer.  The clinical response to newer types of endocrine therapies, such as the aromatase inhibitors, which suppress the production of estrogen, is also dependent on the status of ERa, and only positive tumors benefit.

Although the clinical utility of assessing ERa was initially based almost entirely on studies using standardized LBAs, beginning in the early 1990s, laboratories around the world abandoned LBAs in favor of IHC, which is used for nearly all testing today.  There are advantages to using IHC over LBAs, especially its ability to measure ERa on routine FFPET samples, eliminating the need for fresh-frozen samples and the burdensome infrastructure required to provide it.  Other advantages include lower cost, higher safety, and superior sensitivity and specificity (providing it is done correctly) because assessment of ERa expression is restricted to tumor cells under direct microscopic visualization - independent of the numbers of tumor cells present, or the presence of receptor-positive benign epithelium, which are problematical for LBAs.  Several head-to-head comparisons have demonstrated that assessing ERa by IHC can be equivalent or better than LBAs in predicting response to endocrine therapy, which is comforting, since IHC replaced LBA before such proof was available.

IHC was approved more than a decade ago by the CAP and ASCO for routine clinical testing of ERa  and PgR.  Despite these approvals, there were significant problems with the technical and clinical validation of IHC that persist today, resulting in inaccurate interpretations (i.e. positive vs. negative) in 20% or more of cases.  Most of the errors are false-negatives, which is potentially catastrophic because the patients involved will usually not get the endocrine therapy that would greatly improve their outcome.

There are many causes and no easy solutions to the problem of inaccurate testing, although there are useful guidelines and recommendations intended to help avoid mistakes including, in particular, those recently published by ASCO and CAP.  Surprisingly, there are relatively few IHC assays for ERa or PgR that entirely satisfy all of these guidelines and recommendations, although a few come close.  The strategy published by Harvey and colleagues was among the first to be well validated.  It is based on a highly specific and sensitive primary antibody to ERa  (mouse monoclonal 6F11), a quantitative and reproducible method of scoring results (the so-called Allred Score, and a definition of positive results calibrated to clinical outcome in several large studies, including randomized clinical trials.  The latter involved patients with all stages of breast cancer treated with tamoxifen or aromatase inhibitors in adjuvant, neoadjuvant, and advanced disease settings.  It is extremely difficult to standardize and validate IHC assays for ERa and PgR in a comprehensive manner, but any laboratory can utilize assays that have already been validated.

Studies evaluating ERa by IHC in breast cancer collectively demonstrate that about 75-80% express ERa, that it is almost entirely nuclear in location, and that there is tremendous variation of expression on a continuum ranging from 0% to nearly 100% positive cells.  More importantly, they show a direct correlation between the likelihood of clinical response to endocrine therapies and the level of ERa expression.  Surprisingly, the gradient is skewed such that tumors expressing even very low levels show a significant benefit far above that of entirely ERa-negative tumors, which are essentially unresponsive.  This evidence provides support for laboratories adopting ³1% positive staining tumor cells as the definition of “ERa-positive”, which has now been validated in several other comprehensive studies, and is endorsed by the ASCO/CAP guidelines.

Two studies have reported an essentially bimodal (either entirely negative or strongly positive) distribution of ERa assessed by IHC in IBCs, leading some to conclude that reporting results as simply positive or negative is sufficient, but these assays do not reflect the quantitative continuum that is expected for a proper technically validated assay.  There does appear to be a recent shift towards an increasing incidence of ER-positive IBCs, which may be partially due to earlier detection before additional genetic alterations are acquired resulting in loss of expression.  

Several strategies based on technologies other than IHC have been developed to assess multiple prognostic and predictive biomarkers simultaneously.  For example, one promising strategy evaluates RNA expression of 21 genes which are important in breast cancer (including ER and PgR) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) on FFPET samples, and it appears to be very powerful in predicting clinical outcome in several settings. 

Progesterone Receptor

PgR is also routinely assessed by IHC in IBCs.  ERa regulates the expression of PgR, so the presence of PgR usually indicates that the estrogen-ERa pathway is functionally intact.  PgR is activated by the hormone progesterone to help regulate several normal cellular functions, including proliferation which, like estrogen and ERa, is detrimental to patients with breast cancer.  Most of the discussion regarding the historical assessment of ERa in IBCs also applies to PgR.  It was measured by standardized LBAs for nearly two decades and shown to be a weak prognostic factor but a relatively strong predictive factor for response to endocrine therapy.  LBAs for PgR were replaced by IHC beginning in mid-1990s and IHC was eventually approved by the CAP and ASCO for routine clinical use, despite persistent shortcomings.

 

Breast Cancer Molecular Profiling: Promises and Limitations

Handout

Traditionally breast cancers have been classified broadly into ductal and lobular types based on their ability to form ducts and cellular cohesiveness. A smaller percentage of tumors are classified as “special subtype carcinomas”. Another important component of morphologic classification is tumor grading that incorporates tubule formation in the tumor, nuclear pleomorphism and a measure of tumor cell proliferation by counting the number of mitotic figures per 10 high power fields. This classification system has been developed and improved over a number of years and provides useful prognostic information. Long term follow up studies have shown an excellent prognosis for Nottingham grade I tumors and a poor survival rate for Nottingham grade III tumors. Although incredibly cheap and extremely useful, the morphologic classification does have several drawbacks. First and foremost, there is no difference in disease free and overall survival between ductal and lobular tumors. Secondly, investigators have used different criteria to define special subtype tumors and most criteria are arbitrary. Last, but not the least, the most important part of morphologic classification i.e. grading suffers from poor inter-observer reproducibility, especially when breast tumors are graded by non-breast pathologists. All the above factors plus the desire to identify new prognostic and predictive factors and the availability of gene expression profiling assays prompted the new molecular classification of breast carcinoma.

 In the past decade, apart from intrinsic gene set based molecular classification, several other multi-gene prediction assays were also described and a few are already in clinical use. A brief summary of the two most prominent tests that are in clinical use is summarized here.

 Seventy gene profile (Mammaprint®): The 70-gene good versus poor outcome model was developed by Van de Vijver et al and Van’t Veer et al. The authors used oligonucleotide array to identify genes that predict prognosis in breast cancer. They estimated that an odds ratio for metastasis among tumors with “good prognosis” gene signature as compared to “poor prognosis” gene signature was approximately 15 using cross validation procedures. The poor prognosis signature consisted of genes regulating cell cycle, invasion, metastasis and angiogenesis. They further studied 295 cases of breast cancers from young patients including pT1 and pT2 cases with (n=144) or without (n=151) lymph node metastasis. Of the 295 cases 180 showed “poor prognosis” and 115 showed “good prognosis” profile and the mean (SE) overall 10 year survival rates were 54.6% (+/- 4.4%) and 94.5% (+/-2.6%) respectively. The estimated hazard ratio for distant metastases with the “poor prognosis” signature as compared to the group with “good prognosis” signature was 5.1. This ratio remained significant, when the groups were analyzed with respect to the lymph node status. This assay has now formed the basis of a commercial test called MammaPrint® (Agendia BV, Amsterdam, The Netherlands). The test was recently cleared by the US Food and Drug Administration for clinical use however ASCO guidelines committee for tumor markers in breast cancer judged that more evidence is required for advocating use in clinical practice. In the meantime a clinical trial assessing the usefulness of MammaPrint® assay is underway. The trial is called MINDACT (Microarray In Node-negative and 1 to 3 positive lymph node Disease may Avoid ChemoTherapy). The trial is a prospective randomized study comparing the 70-gene signature with the common clinical-pathological criteria in selecting patients for adjuvant chemotherapy in breast cancer with 0-3 positive nodes. The trial began in 2007 and involves patients from 9 different European countries. The results are not expected until 2013. Another problem with the test is requirement of fresh frozen tissue containing at least 30% of the invasive tumor and must reach the company in their kit within 5 days of obtaining the tissue. Recent advances have resolved the issue of the need for frozen tissue-the test can now be performed on FFPE.

Recurrence score model (oncotype DX®): Recurrence score model is better known as oncotype DX®, which is a commercially available RT-PCR based assay that provides a Recurrence Score (RS) and has been shown to provide prognostic and predictive information in estrogen receptor-positive lymph node-negative breast cancers. The test analyzes the expression of 21 genes (16 cancer related and 5 control genes) to give a distant disease Recurrence Score (RS) ranging from 0-100. The RS score was created using training sets and a proprietary analytic method. The oncotype DX™ RS was originally validated in 668 lymph node-negative, ER-positive breast cancer patients receiving tamoxifen in NSABP trial B-14, where a multivariate analysis of patient age, tumor size, tumor grade, HER2 status, hormone receptor status, and RS demonstrated that only tumor grade and recurrence score were significant predictors of distant recurrence, and the RS was also significantly correlated with the relapse-free interval and overall survival. The RS was subsequently validated as a predictive marker for response to chemotherapy and tamoxifen in 651 patients on NSABP B-20 and 645 patients on NSABP B-14. Combination of  portions of the training sets and validation sets is a significant reason why the test cannot meet FDA approval (J. Levin, FDA, personal communication).

 The 16 genes analyzed by the test can be categorized as the Estrogen group (ER, PGR, BCL2, SCUBE2), HER2 group (GRB7, HER2), Proliferation group (KI67, STK15, Survivin, CCNB1, MYBL2), Invasion group (MMP11, CTSL2), and Others (GSTM1, CD68, BAG1). The unscaled RS (RSU) is derived from the quantitative levels of these gene expression products that are fitted into an equation (RSU = +0.47 x GRB7group score–0.34 x ERgroup score +1.04 x proliferation group score +0.10 x invasion group score +0.05 x CD68–0.08 x GSTM1–0.07 xBAG1). The commercial oncotype DX® assay report actually gives the recurrence score which ranges from 0-100 where an increasing score represents the increasing risk of recurrence over 10 years for hormone receptor positive, lymph node negative patients who had been administered five years of tamoxifen therapy. The RS is stratified into low risk  (RS <18; group average 7% recurrence risk over ten years), intermediate risk (RS 18-30; group average 14% risk of recurrence over ten years) and high risk (RS ≥ 31; group average 31% risk of recurrence over ten years). Oncologists offer chemotherapy to patients that have high RS and avoid chemotherapy in the low risk group. The decision in patients with intermediate risk RS is more problematic and is dependent on several other factors such as patient preference, co-morbid conditions etc.. The test is currently endorsed by American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) for clinical decision making in estrogen receptor positive lymph node negative breast cancer patients. It is also the test which is being used in the clinical trial titled Trial Assigning IndividuaLized Options for Treatment (Rx), or TAILORx. The TAILORx trial is sponsored by the National Cancer Institute (NCI), and is coordinated by the Eastern Cooperative Oncology Group (ECOG). The study will randomized more than 10,000 patients at 900 sites in the United States and Canada. Women diagnosed with hormone-receptor positive, HER2-negative breast cancers that had not yet spread to the lymph nodes will be eligible for the study. The trial started in mid-2006 and is well on its way to target accrual. The trial was mainly designed to address the intermediate risk RS category (i.e. RS range 18-30). However, it is important to note that this group was arbitrarily narrowed to include patients with RS ranging from 11-25, i.e. patient with RS >25 were offered chemotherapy and patients with less than 11 were advised against chemotherapy. The patients with RS 11-25 are being randomized to receive either hormonal therapy alone or receive hormonal and chemotherapy. The primary aim of the trial is to compare the distant recurrence-free interval, recurrence-free interval, and overall survival of patients with an RS of 11-25 treated with these regimens. The secondary aim is to determine if adjuvant hormonal therapy alone is sufficient treatment (i.e., 10-year distant disease-free survival of at least 95%) for patients with an RS of ≤ 10 and to compare the outcomes projected at 10 years using classical pathologic information, including tumor size, hormone receptor status, and histologic grade, with those made by the Genomic Health oncotype DX® test in patients treated with these regimens. 

 The problems with commercial molecular assays for breast cancer:
 
1.      oncotype Dx:

·         Laboratory developed test- not FDA approved, yet has major impact on patients lives. The actual benefit or lack of benefit for each individual patient is completely unknown.
·         Proprietary-corporate structures control the science or lack thereof.
·         Her2 gene of the test is unreliable.   Others??
·         Impact on clinical outcomes unknown.
 2.      Mammaprint
·         Excellent data set provides for stratification of patients into low risk and high risk types-but technically a prognostic test, not a predictive test.
·         Impact on clinical utility unknown.

References

Arpino G, Bardou VJ, Clark GM, Elledge RM. Infiltrating lobular carcinoma of the breast: tumor characteristics and clinical outcome. Breast Cancer Res. 2004;6:R149-156.

Baehner FL, Yoshizawa C, Shak S. Accurate assessment of human epidermal growth factor receptor 2. J Clin Oncol. 2012 May 10;30(14):1727-8; author reply
Bhargava R, Dabbs DJ. Oncotype DX test on unequivocally HER2-positive cases: potential for harm. J Clin Oncol. 2012 Feb 10;30(5) Evaluation of Genomic Applications in Practice and Prevention Working Group Genetics in Medicine 2009 11(1):66-73
Dabbs DJ, Klein ME, Mohsin SK, Tubbs RR, Shuai Y, Bhargava R. High false-negative rate of HER2 quantitative reverse transcription polymerase chain reaction of the Oncotype DX test: an independent quality assurance study. J Clin Oncol. 2011
Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19:403-410.
Esposito NN, Acs G, Dabbs DJ, Flanagan MB, Laronga C, Bhargava R. Validation of the Magee Study Equation in Prediction of Breast Cancer Recurrence Risk Category by oncotype Dx™. Mod Pathol. 2010;23 (Suppl 1):Abstract 192.
Flanagan MB, Dabbs DJ, Brufsky AM, Breriwal S, Bhargava R. Histopathologic variables predict Oncotype DX™ recurrence score. Mod Pathol. 2008;21:1255-1261.
Ma XJ, Hilsenbeck SG, Wang W, et al. The HOXB13:IL17BR expression index is a prognostic factor in early-stage breast cancer. J Clin Oncol. 2006;24:4611-4619.
Ma XJ, Salunga R, Dahiya S, et al. A five-gene molecular grade index and HOXB13:IL17BR are complementary prognostic factors in early stage breast cancer. Clin Cancer Res. 2008;14:2601-2608.
Mersin H, Yildirim E, Gulben K, Berberoglu U. Is invasive lobular carcinoma different from invasive ductal carcinoma? Eur J Surg Oncol. 2003;29:390-395.
Molland JG, Donnellan M, Janu NC, Carmalt HL, Kennedy CW, Gillett DJ. Infiltrating lobular carcinoma--a comparison of diagnosis, management and outcome with infiltrating duct carcinoma. Breast. 2004;13:389-396.
Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004;351:2817-2826.
Paik S, Tang G, Shak S, et al. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol. 2006;24:3726-3734.
Rakha EA, El-Sayed ME, Lee AH, et al. Prognostic significance of Nottingham histologic grade in invasive breast carcinoma. J Clin Oncol. 2008;26:3153-3158.
Rakha EA, El-Sayed ME, Menon S, Green AR, Lee AH, Ellis IO. Histologic grading is an independent prognostic factor in invasive lobular carcinoma of the breast. Breast Cancer Res Treat. 2008;111:121-127.

CV

Dr. Dabbs attended medical school at the Medical College of Ohio, where he also did a Post-sophomore Fellowship in Pathology. It was this Fellowship that helped him decide that he wanted to become a Pathologist. He performed his Residency and Fellowship in Anatomic Pathology at the University of Washington Affiliated Hospitals in Seattle, Washington.

Dr. Dabbs has held academic appointments at East Carolina University, Case Western Reserve University in Cleveland, Penn State University at Hershey Medical Center and now at the University of Pittsburgh.
 
Currently, Dr. Dabbs is Professor and Chief of Pathology at Magee-Womens Hospital of the University of Pittsburgh and is Chief Diagnostic Consultant for US Labs.
 
Dr. Dabbs is well known for his book, Diagnostic Immunohistochemistry, now in its Third Edition, with a Fourth Edition due next year.
 
Dr. Dabbs is also editor and co-author of a new release book Breast Pathology (Elsevier, 2012).
 
He has an active consultation practice, especially in tumor pathology and in breast/gynecologic pathology.
 
e-mail: ddabbs@upmc.edu
 
Twitter: @DAVIDJDABBSMD

 

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