Cooperative Breast Cancer Tissue Resource Research Articles

Abstract 2272: Association of G-protein estrogen receptor (GPER) in primary breast cancer in Caucasian and Black women by tumor immunophenotype and menopausal status

Abstract Background: Assignment of breast cancer therapies is largely defined by immunohistochemical analyses that evaluate steroid hormone and growth factor receptor status in primary tumor biopsies. Patients whose tumors are devoid of these receptors have fewer treatment options, and poor survival. This outcome is more commonly measured among Black versus Caucasian patients, and in patients with intact ovarian function. The G-protein coupled estrogen receptor, GPER, holds significance for breast cancer biology and treatment as 17β-estradiol and ER antagonists trigger the release of epidermal growth factor (EGF)-ligands and GPER associates directly with clinicopathological factors that predict advanced disease. Experimental design: GPER was measured by immunohistochemical analysis and a semi-quantitative scoring method in 521 primary breast tumors obtained from the NCI Cooperative Breast Cancer Tissue Resource and the Nashville Breast Health Study, including tumor specimens derived from 389 Caucasian and 108 Black patients. GPER was correlated with disease progression variables and evaluated by race, menopausal status and tumor immunophenotype. Results: GPER is expressed in more than two-thirds of invasive breast carcinomas in this study with no difference observed between Caucasian and Black women (p = 0.69). Coexpression of GPER and ER was measured in less than half of all tumors (48%). Additionally, GPER is commonly expressed in ER-negative tumors (107/171; 62%) and in triple negative breast cancer (TNBC) (42/56; 75%) and did not differ by race for this newly defined measure of estrogen responsiveness (p = 0.42). Moreover, while ER-negative tumors are more common in pre- (79/153; 52%) versus postmenopausal women (92/336; 27%), GPER expression is not influenced by menopausal status. GPER is positively associated with tumor size (particularly in premenopausal women) in both studies, and with frank metastases at first diagnosis in the NCI population. No significant association was seen with nodal invasion. Survival data in NBHS patients found that women who died of breast cancer are more likely to have GPER-positive tumors than survivors. Conclusion: GPER is expressed in the majority of invasive breast tumors (> 60%) at diagnosis and is commonly retained in patients with ER-negative or triple-negative disease regardless of race or menopause. Unlike ER, which varies inversely with variables that predict advanced disease, GPER is positively associated with these same prognostic parameters. These data support GPER as an independent marker of estrogen responsiveness and breast cancer progression. Most importantly, our results suggest that GPER represents a promising therapeutic target since its expression is constant across multiple types of invasive breast cancer, including patients with TNBC and in tumors from pre- and postmenopausal women. Citation Format: Sandra L. Deming-Halverson, Carl Graeber, Jason Machan, Edmond Sabo, Wei Zheng, Edward J. Filardo. Association of G-protein estrogen receptor (GPER) in primary breast cancer in Caucasian and Black women by tumor immunophenotype and menopausal status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2272.

Evaluation of Lymphangiogenic Factors, Vascular Endothelial Growth Factor D and E-Cadherin in Distinguishing Inflammatory From Locally Advanced Breast Cancer

BackgroundInflammatory breast cancer (IBC) is an aggressive form of breast cancer that on presentation resembles locally advanced breast cancer (LABC). This study identified molecular features of IBC and LABC to investigate pathogenesis. Materials and MethodsThis study involved 100 IBC cases identified in a national IBC registry and 107 non-IBC LABC cases from the National Cancer Institute's Cooperative Breast Cancer Tissue Resource (CBCTR). Vascular endothelial growth factor D (VEGF-D) and E-cadherin levels and lymphatic vessel density (LVD) measured by podoplanin staining were examined by immunohistochemistry on paraffin-embedded tumor specimens. Intralymphatic tumor emboli (ILTE) were assessed in IBC and non-IBC tumors. IBC cases diagnosed by clinicians but not meeting the case definitions of the American Joint Committee on Cancer (AJCC) or the Surveillance, Epidemiology and End Results (SEER) Program of the National Cancer Institute (NCI)(designated atypical IBC) were compared with AJCC- and/or SEER-defined cases (designated classic IBC). ResultsE-cadherin levels were significantly higher in classic IBC cases compared with non-IBC cases (P = .031), whereas compared with classic IBC, patients with non-IBC LABC had significantly higher LVD (P = .0017) and VEGF-D levels (P < .0001). ILTE was marginally greater in classic IBC than in non-IBC (P = .046). The profile of laboratory values in atypical IBC cases more closely resembled those fitting classic IBC than LABC. ConclusionE-cadherin levels, LVD, VEGF-D expression, and to a lesser extent, ILTE differed between classic IBC and non-IBC LABC. The similarity of laboratory results between atypical IBC and classic IBC vs. LABC suggests the need for broadening both the AJCC and SEER case definitions for this disease.

Abstract 698: Overexpression of the aryl hydrocarbon receptor correlates with high tumor grade in human breast invasive carcinomas

Abstract The aryl hydrocarbon receptor (AhR) is a ligand-activated basic helix-loop-helix transcription factor which binds environmental poly aromatic hydrocarbons (PAH) and mediates their carcinogenic effects. Recent reports, including data from our laboratory implicate AhR in breast cancer development and progression independent of the receptor occupancy by PAH. In this study we investigated the potential of AhR as a stage specific marker of breast cancer. We examined the expression of AhR by immunohistochemistry in tissue microarrays (TMA) containing 192 specimens of clinically defined three stages of invasive breast cancer: node-negative (NN), node-positive (NP) and metastatic (Met) carcinoma. The TMA were obtained from the National Cancer Institute Cooperative Breast Cancer Tissue Resource [NCI-CBCTR] and were stained for AhR using high affinity antibody. The AhR staining was then scored by three evaluators, including two pathologists who were blinded to the study. Statistical analysis showed a significant correlation between the AhR expression and the carcinoma case type (NN, NP or Met) (p-value of ANOVA is &amp;lt; 0.0003), and although there is a significant difference of AhR expression among carcinoma case type of white women (p&amp;lt; 0.0017) there is no significant difference for black women (p&amp;lt;0.0968). There is also a strong positive correlation between the receptor expression and the tumor grade (p &amp;lt;0.0001). While there is no correlation with the status of estrogen or progesterone receptors (p= 0.1643 and 0.1884, respectively), there is strong correlation of high AhR expression and invasive breast carcinoma in women over 50 years old (p&amp;lt;0.0043). In conclusion, our findings identify the AhR as a new predictive clinical marker for metastatic breast cancer and a unique target for the design of novel selective inhibitors for therapeutic intervention of metastatic breast cancer. More importantly, the AhR overexpression identifies a subset of patients who could benefit from therapy targeting this receptor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 698. doi:1538-7445.AM2012-698

P2-12-25: Blinded Multi-Site Validation of a Pathology-Based Prognostic Marker Profile for Operable Hormone Receptor-Positive Breast Cancer.

Abstract Background We previously reported development and preliminary validation of a marker profile with a risk of recurrence algorithm for patients with operable hormone receptor (HR)-positive breast cancer using tissue microarrays. The profile includes ER, PR, HER2, EGFR, BCL2, and p53 (assessed by IHC) and MYC/8q24 (assessed by FISH). It also directly incorporates the standard clinicopathologic risk factors tumor size, tumor grade, and nodal status to the extent that their prognostic value is not replaced by the molecular markers. Here, we demonstrate validation of the profile in a blinded multi-site study conducted on whole sections. Material and methods: The study was conducted in a blinded fashion using an independent data management firm (Synteract, Carlsbad, CA). Eligible patients were females diagnosed between 1985 and 1997 with HR-positive stage I-IIIA breast cancer treated only with hormone therapy after definitive surgery for whom sufficient tumor samples were available for testing. Slides with FFPE tumor tissue sections and associated clinicopathologic, treatment, and outcome data were provided by four clinical sites through the NCI-funded Cooperative Breast Cancer Tissue Resource program: Fox-Chase Cancer Center (Philadelphia, PA) (n=106), Kaiser Permanente Northwest (Portland, OR) (n=165), University of Miami (FL) (n=55), and Washington University St. Louis (MO) (n=74). Marker assays, scoring, and calculation of risk scores using a predefined algorithm were conducted at Clarient, a CLIA-certified laboratory. The algorithm assigned a risk score to each patient on a scale of 0 to 10+, and a predefined threshold of 3.8 was used to separate patients into low and high risk groups. Results: Complete data for all seven markers was obtained in 349 of the 400 patients. The algorithm defined 28% of the patients (n=99) as high-risk and 72% of the patients (n=250) as low-risk. The high-risk and low-risk patient groups had 10-year distant metastasis rates of 34% and 9%, respectively, resulting in a hazard ratio of 4.7 (95% CI, 2.3−7.8, p&amp;lt;0.0001) through 10 years without an intermediate-risk group. Similar results were achieved when using disease-specific survival (death with evidence of breast cancer recurrence) as the outcome. Using a standard threshold score of 3.4, the Nottingham Prognostic Index (NPI) also stratified the patients (n=349) into low and high risk groups (p=0.03), but only 10% of the patients were in the low-risk category, and the profile further risk stratified patients in the NPI intermediate and high risk categories similar to the overall patient set (p&amp;lt;0.001). In multivariate Cox proportional hazards analysis with clinicopathologic factors, only the profile and tumor size were significant (Wald statistic p&amp;lt;0.001 for both). Discussion: This blinded study, using a predefined algorithm and threshold, validates the prognostic and clinical utility of the multi-marker profile to help guide the appropriate level of adjuvant treatment in breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-25.

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Transcription factors Stat5a and Stat5b: Favorable prognostic markers in breast cancer

22071 Background: We have previously reported that presence of nuclear localized, tyrosine phosphorylated Stat5 in breast carcinoma cells is associated with a highly favorable prognosis in a material of 209 patients with node-negative breast cancer. This initial analysis was performed on tissue array sections and not on whole sections. Furthermore, the initial study did not distinguish between Stat5a and Stat5b, since the phosphotyrosine-motif recognized by the antibody is the same in Stat5a and Stat5b. Stat5a and Stat5b are highly homologous transcription factors with overlapping but distinct regulation and biological effects. It has remained unclear whether both Stat5a and Stat5b contribute to the favorable prognosis associated with positive anti-phospho-Stat5 status. Methods: We have now performed a retrospective follow-up analysis of nuclear localized, tyrosine phosphorylated Stat5 in whole sections of breast cancer from an independent material of 400 node-negative breast cancer specimens with outcome data provided by the National Cancer Institute's Cooperative Breast Cancer Tissue Resource (CBCTR). We also analyzed levels of Stat5a and Stat5b individually and performed survival analyses. Results: Nuclear localized, tyrosine phosphorylated Stat5 remained a significant favorable prognostic marker in this independent follow-up material of human breast cancer, validating our previous data. Furthermore, nuclear localized Stat5a and Stat5b both correlated with favorable prognosis. In contrast, nuclear localized phosphorylated Stat3 did not significantly correlate with prognosis. Conclusions: Levels of nuclear localized, tyrosine phosphorylated Stat5a/b, alone or in conjunction with levels of nuclear Stat5a or Stat5b proteins, could be a cost-effective test identifying a subset of node-negative breast cancer patients with excellent prognosis who may be managed less aggressively following surgery. No significant financial relationships to disclose.

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Interferon Regulatory Factor 1 (IRF-1) and IRF-2 Expression in Breast Cancer Tissue Microarrays

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine with potent antitumor effects, both in vitro and in vivo. The antitumor activity of IFN-gamma is mediated in part through IFN regulatory factor-1 (IRF-1) and may be blocked by IRF-2. To test our hypothesis that some tumors escape the antitumor effects of IFN-gamma by cellular changes reflected in IRF-1 and IRF-2 expression, we examined IRF-1 and IRF-2 expression in tissue microarrays (TMA) containing 187 specimens of clinically defined invasive breast carcinoma. TMAs (Cooperative Breast Cancer Tissue Resource [CBCTR], National Cancer Institute [NCI]) were stained and then scored by three evaluators blinded to the patients' clinical status. After final scoring, the CBCTR provided the available clinical data for each patient. Whether sorted by carcinoma type or for all data together, statistical analysis showed a significant positive correlation between IRF-1 and IRF-2 expression (p = 0.01) and a negative correlation between IRF-1 expression and tumor grade (p = 0.005). IRF-1 expression is consistent with its role as a tumor suppressor; high-grade breast carcinomas were less likely to maintain expression of IRF-1, a finding consistent with a role for IRF-1 as a tumor suppressor. Further, tumors maintained expression of IRF-2 if there was coincident expression of IRF-1. These data support a model in which alterations of the expression of intracellular effectors of IFN-gamma signaling may diminish the immune-mediated tumor control mechanisms of IFN-gamma.

Breast carcinoma malignancy grading by Bloom–Richardson system vs proliferation index: reproducibility of grade and advantages of proliferation index

Questions of reproducibility and efficacy of histologic malignancy grading relative to alternative proliferation index measurements for outcome prediction remain unanswered. Microsections of specimens from the Cooperative Breast Cancer Tissue Resource (CBCTR) were evaluated by seven pathologists for reproducibility of grade and classification. Nuclear figure classification was assessed using photographs. Grade was assigned by the Bloom–Richardson method, Nottingham modification. Proliferation index was evaluated prospectively by deoxyribose nucleic acid precursor uptake with thymidine (autoradiographic) or bromodeoxyuridine (immunohistochemical) labeling index using fresh tissue from 631 node-negative breast cancer patients accessioned at St Luke’s Hospital. A modified Nottingham–Bloom–Richardson grade was derived from histopathologic data. Median post-treatment observation was 6.4 years. Agreement on classification of nuclear figures (N=43) was less than good by kappa statistic (κ=0.38). Grade was moderately reproducible in four trials (N=10,10,19, 10) with CBCTR specimens (κ=0.50–0.59). Of components of Bloom–Richardson grade, agreement was least for nuclear pleomorphism (κ=0.37–0.50), best for tubularity (κ=0.57–0.83), and intermediate for mitotic count (κ=0.45–0.64). Bloom–Richardson grade was a univariate predictor of prognosis in node-negative St Luke’s patients, and was improved when mitotic count was replaced by labeling index (low, mid, or high). When labeling index was added to a multivariate model containing tumor size and vessel invasion, grade was no longer a significant predictor of tumor-specific relapse-free or overall survival. Mitotic index predicted best when intervals were lowered to 0–2, 3–10, and >10 mitotic figures per ten 0.18 mm2 high-power fields. We conclude that Nottingham–Bloom–Richardson grades remain only modestly reproducible. Prognostically useful components of grade are mitotic index and tubularity. The Nottingham–Bloom–Richardson system can be improved by lowering cutoffs for mitotic index and by counting 20–30 rather than 10 high-power fields. Measurement of proliferation index by immunohistochemically detectable markers will probably give superior prognostic results in comparison to grade.

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