Fl Baehner

Breast cancer prognostic classification in the molecular era: the role of histological grade

Breast cancer is a heterogeneous disease with varied morphological appearances, molecular features, behavior, and response to therapy. Current routine clinical management of breast cancer relies on the availability of robust clinical and pathological prognostic and predictive factors to support clinical and patient decision making in which potentially suitable treatment options are increasingly available. One of the best-established prognostic factors in breast cancer is histological grade, which represents the morphological assessment of tumor biological characteristics and has been shown to be able to generate important information related to the clinical behavior of breast cancers. Genome-wide microarray-based expression profiling studies have unraveled several characteristics of breast cancer biology and have provided further evidence that the biological features captured by histological grade are important in determining tumor behavior. Also, expression profiling studies have generated clinically useful data that have significantly improved our understanding of the biology of breast cancer, and these studies are undergoing evaluation as improved prognostic and predictive tools in clinical practice. Clinical acceptance of these molecular assays will require them to be more than expensive surrogates of established traditional factors such as histological grade. It is essential that they provide additional prognostic or predictive information above and beyond that offered by current parameters. Here, we present an analysis of the validity of histological grade as a prognostic factor and a consensus view on the significance of histological grade and its role in breast cancer classification and staging systems in this era of emerging clinical use of molecular classifiers.

S4-6: A Quantitative Multigene RT-PCR Assay for Predicting Recurrence Risk after Surgical Excision Alone without Irradiation for Ductal Carcinoma In Situ (DCIS): A Prospective Validation Study of the DCIS Score from ECOG E5194.

Background: We have previously reported the results of surgical excision without irradiation for selected patients with DCIS in ECOG E5194, where the 5-year rates of local recurrence varied with age, grade, and lesion size (Hughes et al. J Clin Oncol 27:5319, 2009). New methods are needed to provide more accurate and reproducible assessment of recurrence risk. Methods: ECOG E5194 included 670 eligible patients with DCIS treated with surgical excision (≥ 3 mm negative margins) without irradiation, 228 of whom received tamoxifen. Patients had low or intermediate grade DCIS ≤ 2.5 cm, or high grade DCIS ≤ 1 cm. The Oncotype DX® assay was performed by quantitative RT-PCR using formalin fixed paraffin embedded tumor specimens from 327 patients (49% of the parent study). Recurrence Score® (RS) was calculated using the published algorithm. A new, prespecified DCIS Score™ was designed to predict recurrence using an optimized gene expression algorithm. The primary objective was to determine whether there was a significant association between the risk of an ipsilateral breast event (IBE) and the continuous DCIS Score in Cox models. 46 patients had an IBE (defined as ipsilateral local recurrence of DCIS [n=20] or invasive cancer [n=26]). Median follow-up was 8.8 years. Results: The 10-year IBE rates were 15.4% for low/intermediate grade DCIS and 15.1% for high-grade DCIS (as determined by central pathology review), and for invasive IBE, 5.6% and 9.8%, respectively. Comparison between local and expert grading showed substantial disagreement. Continuous DCIS Score was significantly associated with IBE (HR 2.34 per 50 units; 95% CI 1.15, 4.59; p=0.02) when adjusted for tamoxifen use (prespecified primary analysis) and with invasive IBE (HR 3.73; CI 1.34, 9.82; p=0.01). DCIS Score was significantly associated with outcome when evaluated by the prespecified risk groups (see Table). Similar results were observed with and without adjustment for tamoxifen use or for negative margin width. Features associated with IBE in multivariate models included menopausal status (HR 0.49; 95% CI 0.27, 0.90; p=0.02), tumor size (HR 1.52 per 5 mm; 95% CI 1.11, 2.01; p=0.01), and continuous DCIS Score (HR 2.41; 95% CI 1.15, 4.89; p=0.02). The standard RS, which is calculated using thresholding of many genes unlike the DCIS Score, was not associated with IBE or invasive IBE (p > 0.6). Conclusions: We have prospectively validated a multigene assay that quantifies recurrence risk and complements traditional clinical and pathologic factors in selected patients with DCIS treated with surgical excision without irradiation. The DCIS Score provides a new clinical tool for individualized selection of treatment for patients with DCIS. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S4-6.

Elevated Levels of Proliferating and Recently Migrated Tumor-associated Macrophages Confer Increased Aggressiveness and Worse Outcomes in Breast Cancer

PurposeMacrophages play a major role in inflammatory processes and have been associated with poor prognosis in a variety of cancers, including breast cancer. Previously, we investigated the relationship of a subset of tumor-associated macrophages (PCNA+ TAMs) with clinicopathologic characteristics of breast cancer. We reported that high PCNA+ TAM counts were associated with hormone receptor (HR)-negative, high-grade tumors and early recurrence. To further understand the significance of elevated PCNA+ TAMs and the functionality of TAMs, we examined the expression of S100A8/S100A9 with the antibody Mac387. The heterodimeric S100A8/S100A9 complex plays a role in inflammation and is increased in several cancer types.MethodsWe performed immunohistochemistry using the Mac387 antibody on 367 invasive human breast cancer cases. Results were compared to previous PCNA+ TAM counts and were correlated with patient outcomes adjusting for HR status and histologic grade.ResultsLike PCNA+ TAMs, high Mac387 counts were associated with HR negativity, high tumor grade, younger age, and decreased recurrence-free survival. Mac387, however, appears to identify both a subset of macrophages and a subset of tumor cells. The concordance between Mac387 and PCNA+ TAM counts was low and cases that had both high Mac387 and high PCNA+ TAMs counts had a stronger association with early recurrence.ConclusionsThe presence of high numbers of PCNA+ TAMs and Mac387-positive cells in breast cancers with poor outcomes may implicate a subset of TAMs in breast cancer pathogenesis, and may ultimately serve to develop potential cellular targets for therapeutic interventions.

Characterizing the immune microenvironment in high-risk ductal carcinoma in situ of the breast.

PurposeThe recent increase in the incidence of ductal carcinoma in situ (DCIS) has sparked debate over the classification and treatment of this disease. Although DCIS is considered a precursor lesion to invasive breast cancer, some DCIS may have more or less risk than is realized. In this study, we characterized the immune microenvironment in DCIS to determine if immune infiltrates are predictive of recurrence.MethodsFifty-two cases of high-grade DCIS (HG-DCIS), enriched for large lesions and a history of recurrence, were age matched with 65 cases of non-high-grade DCIS (nHG-DCIS). Immune infiltrates were characterized by single- or dual-color staining of FFPE sections for the following antigens: CD4, CD8, CD20, FoxP3, CD68, CD115, Mac387, MRC1, HLA-DR, and PCNA. Nuance multispectral imaging software was used for image acquisition. Protocols for automated image analysis were developed using CellProfiler. Immune cell populations associated with risk of recurrence were identified using classification and regression tree analysis.ResultsHG-DCIS had significantly higher percentages of FoxP3+ cells, CD68+ and CD68+PCNA+ macrophages, HLA-DR+ cells, CD4+ T cells, CD20+ B cells, and total tumor infiltrating lymphocytes compared to nHG-DCIS. A classification tree, generated from 16 immune cell populations and 8 clinical parameters, identified three immune cell populations associated with risk of recurrence: CD8+HLADR+ T cells, CD8+HLADR− T cells, and CD115+ cells.ConclusionThese findings suggest that the tumor immune microenvironment is an important factor in identifying DCIS cases with the highest risk for recurrence and that manipulating the immune microenvironment may be an efficacious strategy to alter or prevent disease progression.

Abstract S5-04: A large prospectively-designed study of the DCIS score: Predicting recurrence risk after local excision for ductal carcinoma in situ patients with and without irradiation

Background: DCIS patients need better tools to align the aggressiveness of treatment with the aggressiveness of their disease. The DCIS Score (DS) was validated as a predictor of ipsilateral breast recurrence (IBR; DCIS or invasive) in 327 E5194 patients treated by breast-conserving surgery (BCS) without radiation (RT) (Solin,2013). This Ontario population based DCIS study of 3335 women with DCIS from 1994 to 2003 (Rakovitch,2013) was conducted to test the DCIS Score as a predictor of recurrence risk in patients treated with BCS alone and in patients treated with BCS+RT. Methods: REMARK guidelines were followed. Breast pathologists centrally reviewed all HE 718 received BCS without RT (N=571 with CM) and 846 received BCS+RT (N=689 with CM). Median follow-up was 9.4 years. Among 1260 pts with CM, 100 pts treated with BCS alone had an IBR (DCIS, N=44; invasive, N=57); 86 pts treated with BCS+RT had an IBR (DCIS, N=32; invasive, N=55). In the primary analysis, among 571 patients treated by BCS alone with CM the continuous DS was significantly associated with IBR in ER+ patients (HR 2.26; 95%CI 1.41,3.59; P=0.001) and in all patients (HR 2.15; 95%CI 1.43,3.22; P= Conclusions: DCIS Score quantifies recurrence risk for DCIS patients treated by BCS with or without RT. Integrating the DCIS Score with established risk factors, such as multifocality, age, and tumor size, can help identify DCIS patients treated with BCS alone with low 10 year risk ( Citation Format: Eileen Rakovitch, Sharon Nofech-Mozes, Wedad Hanna, Frederick L Baehner, Refik Saskin, Steven M Butler, Alan Tuck, Sandip Sengupta, Leela Elavathil, Prashant A Jani, Michel Bonin, Martin C Chang, Elzbieta Slodkowska, Joseph M Anderson, Farid Jamshidian, Diana B Cherbavaz, Steven Shak, Lawerence Paszat. A large prospectively-designed study of the DCIS score: Predicting recurrence risk after local excision for ductal carcinoma in situ patients with and without irradiation [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S5-04.

Fluvastatin has biologic effects on stage 0 and 1 breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4122 Introduction: Statins are safe, reduce cardiovascular risk, and impact pathways critical to cancer progression. We and others have shown lipophilic statins cause apoptosis and growth suppression in vitro and in vivo, and though epidemiologic data are mixed, statin effect appears most evident in estrogen receptor (ER) negative or grade 3 disease. To look for a direct biologic effect of lipophilic statins, we conducted a perioperative pilot window trial in women with breast cancer (BC). Methods: 40 subjects with stage 0,1 BC were randomized to high dose (80mg/day) or low dose (20mg/day) fluvastatin for 3-6 weeks prior to surgery. Paired tissue (core biopsy and surgical specimen), peripheral blood and MRI were obtained. Primary endpoint was Ki-67 (proliferation) change. Secondary endpoints included cleaved caspase-3 (CC3, apoptosis), longest diameter (LD) by MRI, and C-reactive protein (CRP) change. Subgroup analyses was planned by grade (3 vs. 1,2), statin dose; and ER status. Immunohistochemistry (IHC) on paraffin tissue used standard streptavidin biotin methods. A single breast pathologist reviewed all slides; a single radiologist read all MRIs, both blinded to timepoint. Results: Median serum cholesterol decreased by 16% (-23% and -12% for high and low dose, respectively p=0.012), indicating drug effect and compliance. 29 patients had sufficient tumor for paired IHC, 14 and 15 were grade 3 and 1,2, and 10 and 19 were ER - and +, respectively. In grade 3 (73% of which were ER-) vs. 1,2 tumors, there was a significant decrease in Ki-67, -7.2% (interquartile range (IQR) -13.4%, 0% ) vs. -0.3% (IQR -3%, .8%), respectively, p=0.04. CC3 (apoptosis) increased, 60% vs. 13% for grade 3 vs. 1,2 tumors, respectively, p=0.015. ER- and ER+ cases had a similar reduction in Ki67 with a median drop of 2% (IQR -13.4%, 1%) and 1.2% (IQR -6.6%,0.8%), respectively, p=0.56. While CC3 was increased in ER- vs. + (55% vs. 29%), the difference was not statistically significant. There was no dose dependent effect on Ki-67or CC3.There was no evidence of Ki67 or CC3 change when all grades were analyzed together (median drop 1.2%) and no change in CRP. Of 14 subjects with paired MRIs, 4 grade 3 cases showed a significant decrease in LD, marked ductal dilatation and increased necrosis.with statin exposure. Conclusions: A lipophilic statin, fluvastatin, reduced cholesterol and had measurable biologic changes (reduced proliferation, size and increased apoptosis) in stage 0,1 BC after only 3-6 weeks of exposure, specifically in the grade 3 subset. Results support the study of statins for chemoprevention for women at risk for or with stage 0 grade 3 BC, where new agents are needed. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4122.

Gene Expression Profiling of Phenotypically-Defined Hormone-Receptor Positive Breast Cancer: Evidence for Increased Transcriptional Activity of the Insulin Growth Factor Receptor Pathway and Other Pathways.

Background: Approximately 70% of all breast cancers are hormone receptor (HR)-positive tumors that are sensitive to endocrine therapy, but some patients have recurrence despite adjuvant endocrine therapy. We performed an exploratory analysis of gene expression in HR-pos operable breast cancer in order to identify potential novel therapeutic targets and biomarkers associated with recurrence. Methods: RNA was extracted from primary tumor samples obtained from 776 patients with stage I-III breast cancer treated with adjuvant chemohormonal therapy in trial E2197 (JCO 2008; 26: 4092-4099), of whom 458 had HR-pos disease (defined in a central lab; JCO 2008; 26: 2473). We evaluated RNA expression patterns (by quantitative RT-PCR using a panel of 371 rationally selected genes) in HR-pos cases compared with the HR-neg cases using weighted T statistics, and determined which genes in the HR-pos, HER2-neg group were associated with recurrence (using Cox proportional hazards model score test, Korn9s adjusted P value Results: The top 10 genes exhibiting significantly higher expression in the HR-pos group (p≤ 6.17e-160) included ESR1 plus 5 estrogen regulated genes, confirming our approach of evaluating gene expression in phenotypically-defined subsets. Other pathways that exhibited higher expression in the HR-pos group (among the 40 top genes with higher expression, p IRS1, IGFR1, IGFB2 ), Ras ( RhoB, RhoC, RAB27B, GGPS1 ), and HER pathways ( ERBB2, ERBB3, ERBB4 ), and other genes involved in apoptosis ( BCL2, BCL2L1, BAG1, NME6, BBC3 ), signaling ( MAPK3, SEMA3F, RXRA ), mismatch repair ( MSH3 ), cell cycle regulation ( CCND1 ), stress response ( HSPB1 ), and tumor suppressor genes ( TP53BP1, APC ). These patterns were similar in HER2-pos cases. Pathway analysis (Ingenuity) revealed substantial interconnectivity among these genes, especially between IGFR1 , ERB2/3/4 , MAPK3 , BCL2 , and CCND1 , but not RhoB/RhoC . Genes for which increased expression was associated with increased recurrence included those associated with proliferation ( TOP2A, AURKB, PLK1 ) and apoptosis ( BIRC5 - survivin). Conclusions: This exploratory analysis reveals several pathways that exhibit higher transcriptional expression in HR-pos disease, some of which are also associated with a higher risk of recurrence, suggesting that they may be potential therapeutic targets. This provides rationale for testing agents currently available in the clinic that inhibit the IGF and other pathways. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5165.

Abstract S1-06: Stromal tumor-infiltrating lymphocytes(S-TILs): In the alliance N9831 trial S-TILs are associated with chemotherapy benefit but not associated with trastuzumab benefit

Background: Tumor-infiltrating lymphocytes (TILs) at diagnosis are reported to be prognostic in triple-negative breast cancer (BC). Analysis of a small subset of 209 HER2+ patients (pts) with 49 events concluded that higher levels of S-TILs are associated with increased trastuzumab benefit (Loi, 2014). Here we report the largest study to date evaluating S-TILs and their prognostic and predictive association with clinical outcome in N9831 pts treated with either chemotherapy or chemotherapy plus trastuzumab. Methods: Samples assessed were from primary tumors of pts on N9831 arm A (standard AC→T chemotherapy) and arm C (concurrent chemotherapy with trastuzumab) (Perez, 2011). S-TILs were evaluated on HE ≥60% S-TILs was used for the categorical cutoff (Denkert, 2010). The association between S-TILs, treatment (tx) and recurrence-free survival (RFS) was studied and the interaction between S-TILs, trastuzumab benefit and RFS was calculated. Results: 489 pts from arm A (chemo) and 456 pts from arm C (chemo with trastuzumab) were assessed and were similar to pts in the overall trial; all had RFS information and a median follow-up of 4.4yr. Tumors from 54% of pts in arms A and C were HR+; 14% were node-negative. Tumors with high S-TILs were more likely to be hormone receptor-negative (p Conclusions: In exploratory analyses from this subset HER2+ population from N9831, S-TILs were associated with RFS in patients treated with chemotherapy alone, and were not shown to be associated with RFS in patients treated with chemotherapy plus trastuzumab. Citation Format: Edith A Perez, Karla V Ballman, S Keith Anderson, E Aubrey Thompson, Sunil S Badve, Helen Bailey, Frederick L Baehner. Stromal tumor-infiltrating lymphocytes(S-TILs): In the alliance N9831 trial S-TILs are associated with chemotherapy benefit but not associated with trastuzumab benefit [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-06.

Abstract P6-13-01: Local excision without radiation for ductal carcinoma in situ: 12-year results from the ECOG E5194 study

Background: The ECOG E5194 study was a prospective trial designed to evaluate surgical excision (lumpectomy) without radiation for selected women with ductal carcinoma in situ (DCIS) of the breast with low risk clinical and pathologic features. Methods: Eligible patients were enrolled on two study cohorts (not randomized): (1) low or intermediate grade DCIS, tumor size Results: Median patient age was 60 years and 58 years for Cohort 1 and Cohort 2, respectively. Tumor size was 5 mm for 64% and 69% of patients, respectively. There were 99 IBE’s, of which 51 (52%) were an invasive IBE. The IBE and invasive IBE rates increased over time in both cohorts (see Table). The 12-year rates of an IBE were 14.4% for Cohort 1 and 24.6% for Cohort 2 (p = 0.003), and for an invasive IBE, 7.5% and 13.4%, respectively (p = 0.08). No difference was seen for the 12-year rates of overall survival (84.0% vs 82.8%; p = .96) or contralateral breast events (6.7% vs 12.0%; p = 0.16). On multivariate analysis, study cohort (hazard ratio = 1.81; p = 0.01) and tumor size (p = 0.01) were statistically significant for an IBE, and study cohort was borderline statistically significant for an invasive IBE (p = 0.08). On central pathology review (75% of cases), neither grade nor comedo necrosis was associated with the risk of an IBE or invasive IBE (all p > 0.15). Salvage treatment at the time of an IBE included mastectomy for 42% (31/74) and 64% (16/25) of the patients, respectively. Conclusions: For these selected patients with favorable DCIS based on clinical and pathologic characteristics treated with surgical excision without radiation, the rates of an IBE and an invasive IBE continued to increase through at least 12 years of follow-up. Citation Format: Lawrence J Solin, Robert Gray, Lorie L Hughes, William C Wood, Mary Ann Lowen, Sunil Badve, Frederick L Baehner, James N Ingle, Edith A Perez, Abram Recht, Joseph Sparano, Kathy Miller, Nancy E Davidson. Local excision without radiation for ductal carcinoma in situ: 12-year results from the ECOG E5194 study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-13-01.

Abstract PD1-5: Characterizing the Tumor Immune MicroEnvironment (TIME) in high-risk ductal carcinoma in situ

Background: Ductal carcinoma in situ (DCIS) of the breast is a premalignant condition. Although DCIS is treated as an obligate precursor of invasive ductal carcinoma, the rate and latency of progression from DCIS to invasive breast cancer (IBC) in the absence of treatment are unknown. DCIS is not one condition, but rather a spectrum of disease and although DCIS itself is not a lethal condition, women with DCIS are at higher risk of developing subsequent IBC over a time period of 1-20 years depending on DCIS subtype. Features of DCIS that are associated with high risk of recurrence include large size (> 5cm), high grade, comedo necrosis, palpable mass, hormone receptor (HR) negativity, and HER2 positivity. The objective of this study was to characterize the tumor immune microenvironment (TIME) of these high-risk DCIS lesions. Methods: Forty-eight cases of high grade DCIS, enriched for large, confluent lesions and history of recurrence were age matched with 64 cases of non-high grade DCIS. IHC analyses were performed as single or two-color stains for the following antigens: CD68, CD8, CD4, CD20, HLA-DR, CD115, FoxP3, PCNA, Mac387, MRC1, ALDH, CD24, CD44, Ki-67, and HER2. HR status was determined from ER and PR staining results in pathology reports. A Nuance multispectral imaging system was used to image and spectrally unmix each stain. Protocols for automated image analysis were developed using CellProfiler software. Associations between immune cell populations and clinical parameters (tumor palpability, recurrence, HR status, HER2 status, and Van Nuys score [12-point scale: margins, age, size, grade]) were identified with non-parametric Spearman correlation tests. Results: We found a high macrophage infiltrate associated with a high Van Nuys score, palpability, and high Ki-67. High CD115 (CSF-1 receptor) was associated with HER2+, high Ki-67, and recurrence. Mac387+ cells and FoxP3+ regulatory T cells (Treg) were associated with high Van Nuys score, comedo necrosis, high Ki-67, HR- and HER2+. Interestingly, both Mac387 and CD115 were expressed on tumor cells as well as macrophages and high CD115 staining on tumor cells was associated with recurrence. The presence of CD8+HLA-DR-negative T cells throughout a section was associated with high Van Nuys score, HR-, HER2+, and recurrence. In contrast, CD8+ T cells within the nests of tumor cells were negatively associated with Van Nuys score, palpability, and comedo necrosis. A tumor immune microenvironment score (TIME score) was developed based on the proportions of various immune cell populations. A high TIME score was significantly associated with high Van Nuys scores as well as with recurrence. Summary: These results demonstrate that high risk DCIS features (palpability, high Van Nuys score, high proliferation, HR-, HER2+, and increased recurrence) are associated with a suppressed tumor immune micro-environment (high FoxP3+ cells, CD68+Mac387+ cells, CD8+HLA-DR-neg T cells, and upregulated CD115). These high risk lesions truly represent an opportunity to prevent cancer. Identifying these high risk lesions with the help of tumor immune microenvironment markers and manipulating the DCIS TIME via local or systemic immunotherapeutic strategies may represent an ideal preventative intervention. Citation Format: Michael J Campbell, Rita Mukhtar, Ekene Obi-Okoye, Booyeon Han, Vickram Tandon, Sarah Zheng, Zelos Zhu, Max Endicott, Max Wicha, Linda Lindstrom, Alfred Au, Frederick Baehner, Joe Gray, Laura Esserman. Characterizing the Tumor Immune MicroEnvironment (TIME) in high-risk ductal carcinoma in situ [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-5.