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Background: Patients with androgen receptor (AR) positive triple-negative breast cancer (TNBC) may derive a significant benefit from anti-androgen therapy. While AR positivity is often defined by protein expression by immunohistochemistry (IHC), the benefit of anti-androgen therapy in patients who have the Luminal Androgen Receptor (LAR) molecular subtype as defined by genomic profiling is unclear. Our aim was to study the clinical, pathologic and molecular profiles of AR+ tumors by IHC in a diverse set of TNBC. Methods: Tissue microarrays from two separate, well-annotated institutional cohorts (Case Western Reserve University, Yale University) of early-stage TNBC were evaluated for AR expression by IHC (clone SP107, Ventana Benchmark Ultra). AR positivity was defined as greater than or equal to 10% staining in tumor nuclei. AR expression was correlated with clinical and pathologic features such as age, race, grade, and stage within and between the two cohorts. Gene expression was analyzed with the DASL assay (Illumina) on RNA extracted from formalin-fixed paraffin-embedded material using the Ambion RecoverAll kit (Applied Biosystems AM1975). Pietenpol TNBC molecular subtypes were calculated using the online TNBC type tool. Co-expression of AR with other hormone-related proteins including gross cystic disease fluid protein-15 (GCDFP-15, clone EP1582Y, Ventana Benchmark Ultra) and GATA transcription factor 3 (GATA3, clone L50-823, Bondmax Leica) were also assessed. Results: Overall, 22% of cases (n=192) were AR+ by IHC. There was no association between AR expression and age, race, grade or stage within or between the two cohorts. Gene expression data was available on 88 tumors with AR staining results and demonstrated that AR positivity by IHC was not exclusive for the TNBC LAR molecular subtype. Three of five (3/5) tumors that were classified and LAR were in fact AR+ by IHC. Tumors that were AR+ by IHC were also identified in the basal-like 2 (BL2), mesenchymal stem-like (MSL), immunomodulatory (IM), and unstable molecular TNBC categories with low frequency. Notably, of cases that were rejected as ER+ by gene expression profiling despite being clinically TNBC, three of five (3/5) were in fact AR+ by IHC. Examination of other hormone-related proteins in a subset of these patients revealed that AR expression was significantly associated with GCDFP-15 expression (p=0.0007) and was borderline significant for GATA3 expression (p=0.068). Conclusions: AR protein expression levels by IHC were similar in two separate institutional archival cohorts of TNBC and did not correlate with age, race, grade or stage. Comparison of AR IHC data with genomic data suggests that the AR+ phenotype is not unique to LAR subtype of TNBC by gene expression profiling. AR protein expression may be seen in clinically TNBC patients who have a more luminal subtype as evidenced by co-expression of GCDFP-15 and GATA3. Our results suggest that AR staining by IHC may be necessary to capture all patients who may benefit from anti-androgen therapy. Citation Format: Hannah Gilmore, Vinay Varadan, Nicole Williams, Cheryl L Thompson, Stephanie Kim, Peter Hsu, Kristy Miskimen, Aditi Palkar, Shaveta Vinayak, Robert Lindner, Lyndsay Harris. Androgen receptor expression in triple negative breast cancer [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 P3-04-07.

Background. Clinically, it is becoming increasingly important to assess genomic alterations in breast cancers using Next Generation Sequencing to find potential targets for personalized treatment. However, care paths for using such results to guide treatment have not yet been established. Previously reported studies suggest that developing an interdisciplinary review board to evaluate genomic findings can help to guide patient care. We report on a prospective qualitative study of a multidisciplinary genomic tumor board (MGTB) developed to discuss breast cancer patients’ genomic test results in the context of the clinical history and current practice guidelines. Our goal was to observe and describe the establishment of the MGTB and its members’ perceptions of a tumor board approach to interpreting genomic test results. Materials and Methods. After IRB approval, we used qualitative case study methodology to study the MGTB and its participants, which include medical, surgical, and radiation oncologists, pathologists, geneticists, epidemiologists, bioinformaticians, pharmacologists, nurses, and bioethicists. MGTB meets on a monthly basis. Cases are anonymously presented by the treating physician. Genomic tumor findings from commercial vendors and in-house studies are discussed. Treatment recommendations are made by consensus, with level of evidence assigned based on Simon-Paik-Hayes biomarker guidelines. After obtaining consent from MGTB members, MGTB proceedings and interviews were recorded and transcribed for thematic analysis. Social dynamics within the MGTB, which includes interaction among MGTB members regarding interpretation and communication of genomic findings, were analyzed. Results. MGTB began meeting monthly in 10/2013. As of 6/2014, 24 cases have been presented. All were female with metastatic breast cancer; 16/24 (67%) hormone receptor positive (ER or PR >1%), 7/24 (29%) triple negative, and 2/24 (9%) her2 positive. Analysis of meeting and interview transcripts revealed that though inherently multidisciplinary, the MGTB’s recommendations seem to be driven by medical oncologists with input from geneticists and pharmacologists when present. MGTB recommendations have become increasingly consistent over time. Based on genomic findings, the board often recommends referring patients to clinical trials (18/24, 75%) and to Medical Genetics (9/24, 38%) for evaluation of suspected germline abnormalities over other options such as drug therapies approved by the FDA for use in other tumor types. Discussion and Conclusion. MGTB is a unique forum to discuss genomic test results and departs from typical tumor boards by involving non-clinicians. This case study suggests the feasibility of standardizing interdisciplinary evaluation and level of evidence recommendations for genomic results. The findings of this qualitative case study reveal MGTB members’ discomfort with recommending off-label use of FDA-approved therapies. Hence, on an institutional level, team dynamics will determine how testing and recommendations are established. Further study is required to assess institutional and professional variability in offering genomic testing and making treatment recommendations on the basis of genomic findings. Citation Format: Michelle McGowan, Shaveta Vinayak, Steven Maximuk, Roselle Ponsaran, Patricia Marshall, Lyndsay Harris, Paula Silverman. An academic cancer center's experience in establishing a breast cancer-focused multidisciplinary genomic tumor board [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 P2-03-12.

Purpose: This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2+ trastuzumab breast cancer trial. Experimental Design: This analysis included 1,736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray sections containing three biopsies per patient or whole tissue sections using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC+) if the nuclear 3+ staining percentage was more than 30%. Results: Five hundred and seventy-four (33%) tumors were MYC+. MYC+ was associated with hormone receptor positivity (χ2, P = 0.006), tumors 2 cm or more (χ2, P = 0.02), and a higher rate of nodal positivity (χ2, P < 0.001). HRs for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (P = 0.006) and 0.65 (P = 0.006) for patients with MYC+ and MYC− tumors, respectively (Pinteraction = 0.40). For Arm B versus A, HRs for patients with MYC+ and MYC− tumors were 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (Pinteraction = 0.71). For Arm C versus B, HRs for patients with MYC+ and MYC− tumors were 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (Pinteraction = 0.17). Conclusions: Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab. Clin Cancer Res; 19(20); 5798–807. ©2013 AACR.

Purpose It has been suggested that PTEN, a negative regulator of PI3K/AKT signaling, is involved in tumor sensitivity to trastuzumab. We investigated the association between tumor PTEN protein expression and disease-free survival (DFS) of patients randomly assigned to receive chemotherapy alone (arm A) or chemotherapy with sequential (arm B) or concurrent trastuzumab (arm C) in the phase III early-stage human epidermal growth factor receptor 2 (HER2) –positive trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block (n = 1,286) or in whole tissue sections (WS; n = 516) by using standard immunohistochemistry (138G6 monoclonal antibody). Tumors were considered positive for PTEN (PTEN-positive) if any core or WS had any invasive cells with ≥ 1+ staining. Median follow-up was 6.0 years. Results Of 1,802 patients included in this analysis (of 3,505 patients registered to N9831), 1,342 (74%) had PTEN-positive tumors. PTEN positivity was associated with hormone receptor negativity (χ2 P < .001) and nodal positivity (χ2 P = .04). PTEN did not have an impact on DFS within the various arms. Comparing DFS of arm C to arm A, patients with PTEN-positive and PTEN-negative tumors had hazard ratios (HRs) of 0.65 (P = .003) and 0.47 (P = .005), respectively (interaction P = .16). For arm B versus arm A, patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 (P = .009) and 0.85 (P = .44), respectively (interaction P = .47). Conclusion In contrast to selected preclinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors, our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer, independent of tumor PTEN status.

To identify a group of patients who might benefit from the addition of weekly paclitaxel to conventional anthracycline-containing chemotherapy as adjuvant therapy of node-positive operable breast cancer. The predictive value of PAM50 subtypes and the 11-gene proliferation score contained within the PAM50 assay were evaluated in 820 patients from the GEICAM/9906 randomized phase III trial comparing adjuvant FEC to FEC followed by weekly paclitaxel (FEC-P). Multivariable Cox regression analyses of the secondary endpoint of overall survival (OS) were performed to determine the significance of the interaction between treatment and the (1) PAM50 subtypes, (2) PAM50 proliferation score, and (3) clinical and pathological variables. Similar OS analyses were performed in 222 patients treated with weekly paclitaxel versus paclitaxel every 3 weeks in the CALGB/9342 and 9840 metastatic clinical trials. In GEICAM/9906, with a median follow up of 8.7 years, OS of the FEC-P arm was significantly superior compared to the FEC arm (unadjusted HR = 0.693, p = 0.013). A benefit from paclitaxel was only observed in the group of patients with a low PAM50 proliferation score (unadjusted HR = 0.23, p

Triple negative breast cancer (TNBC) is characterized by high proliferation, poor differentiation and a poor prognosis due to high rates of recurrence. Despite lower overall incidence African American (AA) patients suffer from higher breast cancer mortality in part due to the higher proportion of TNBC cases among AA patients compared to European Americans (EA). It was recently shown that the clinical heterogeneity of TNBC is reflected by distinct transcriptional programs with distinct drug response profiles in preclinical models. In this study, gene expression profiling and immunohistochemistry were used to elucidate potential differences between TNBC tumors of EA and AA patients on a molecular level. In a retrospective cohort of 136 TNBC patients, a major transcriptional signature of proliferation was found to be significantly upregulated in samples of AA ethnicity. Furthermore, transcriptional profiles of AA tumors showed differential activation of insulin-like growth factor 1 (IGF1) and a signature of BRCA1 deficiency in this cohort. Using signatures derived from the meta-analysis of TNBC gene expression carried out by Lehmann et al., tumors from AA patients were more likely of basal-like subtypes whereas transcriptional features of many EA samples corresponded to mesenchymal-like or luminal androgen receptor driven subtypes. These results were validated in The Cancer Genome Atlas mRNA and protein expression data, again showing enrichment of a basal-like phenotype in AA tumors and mesenchymal subtypes in EA tumors. In addition, increased expression of VEGF-activated genes together with elevated microvessel area determined by the AQUA method suggest that AA patients exhibit higher tumor vascularization. This study confirms the existence of distinct transcriptional programs in triple negative breast cancer in two separate cohorts and that these programs differ by racial group. Differences in TNBC subtypes and levels of tumor angiogenesis in AA versus EA patients suggest that targeted therapy choices should be considered in the context of race.

The erbB-2 receptor plays an important role in the prognosis of breast cancer and is expressed at high levels in nearly 30% of tumors in breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in human breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. The discovery of heregulin has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2/4 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance, and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in human breast cancer. Preliminary studies in vitro have shown that heregulin induces a biphasic growth effect on cells with erbB-2 overexpression. Interestingly, we observed that expression of heregulin correlates with a more aggressive/invasive, vimentin-positive phenotype in breast cancer cells lines. Therefore, we have postulated that heregulin is involved in breast cancer tumor progression. We have shown that heregulin induces in vitro chemoinvasion and chemotaxis of breast cancer cells as well as growth in an anchorage dependent and independent manner. Interestingly, a heregulin neutralizing antibody inhibits chemotaxis and results in cell growth inhibition and blockade of the invasive phenotype. Strikingly, genetically engineered cells which constitutively express heregulin demonstrate critical phenotypic changes that are associated with a more aggressive phenotype. Specifically, these cells are no longer dependent on estrogen for growth and are resistant to tamoxifen in vitro and in vivo, and moreover these cells metastasize to lymph nodes in athymic nude mice. These tumors appear to have lost bcl-2 expression as compared with the control tumors. In addition, presumably by activation/regulation of topoisomerase II, the heregulin-transfected cells become exquisitely sensitive to doxorubicin and VP-16. Clearly, mechanistic aspects of the erbB-2/4 and heregulin interaction need to be understood from a therapeutic standpoint which could provide additional insights into synergistic treatments for certain patients, or improve treatment regimens for a large number of women. The study of heregulin and its co-expression with erbB-2/4 receptor and the assessment of its involvement in the progression from the in situ stage of breast tumors to the invasive one will additionally increase the relevance of heregulin as a prognostic/diagnostic factor. We believe that our studies provide new insights into breast cancer diagnosis, prognosis, and treatment.

Background: Preclinical data demonstrates that activation of RAS/MEK/ERK pathway in basal-like breast cancer (BLBC) leads to resistance to chemotherapy. Inhibiting MEK pathway was highly effective in BLBC models with intact PTEN. Conversely activated PI3K/AKT pathway (loss of PTEN which occurs in ∼ 30% of TNBC) led to resistance to MEK inhibition. We proposed to test the hypothesis that single agent trametinib (T) will demonstrate efficacy in a subset of TNBC and that addition of AKTi, GSK2141795 (G) will overcome resistance to T. Methods: This is a single arm, multicenter study through the ETCTN. Eligible patients (pts) with advanced TNBC, measurable disease with 1-3 prior chemotherapies received mandatory research biopsy and entered Part I of the study with T alone (2 mg Q day-28 days). At the point of progression pts received a mandatory second biopsy and moved to PART II of study with 1.5 mg of T + 50 mg of G. Optional research biopsy was performed at the point of progression on the combination. Restaging scans were done every 2 cycles. Blood samples were collected at baseline, cycle 2 and at progression. Results: 33 pts with median age 55 (35-71) from six cancer centers were enrolled to Part I (T alone) and 17 pts entered part II (T +G). Most common toxicities for pts on T alone included Gr1: diarrhea, rash, transaminitis, Gr 2: fatigue; Gr 3: thromboembolism. Of 31 evaluable pts on T alone, two pts (1 still on study after 8 cycles) had a partial response (PR) and one pt has stable disease after 8 cycles and remains on study. Of the sixteen evaluable patients on part II (T+G) one patient has an unconfirmed PR (34% reduction on RECIST). Common toxicities for the combination include: Gr1: diarrhea, nausea, vomiting, myelosuppression, rash, hypertension, transaminitis, Gr2: Rash, fatigue, mucositis, diarrhea, Gr 3: diarrhea. Eleven patients died on study (10-progression, 1-adverse event). Blood and tissue samples are being analyzed for whole genome sequencing, IHC for PTEN, Quantitative targeted absolute proteomics (QTAP) for kinome assay and RPPA for PTEN, PI3KCA, AKT, ERK and MEK. This study is currently closed to accrual for interim analysis for efficacy on part II. Conclusion: Trametinib alone and in combination with AKTi, GSK2141795 has limited efficacy in TNBC. Proposed correlatives in serial biopsies may identify biomarkers in the few responders and help identify other novel targets. Citation Format: Bhuvaneswari Ramaswamy, Ewa Mrozek, Maryam Lustberg, Robert Wesolowski, Rachel Layman, Mahmoud Abdel-Rasoul, Cynthia Timmers, Robyn Patrick, Jennifer Sexton, Erin Macrae, Charles Shapiro, Thomas Budd, Lyndsay Harris, Claudine Isaacs, Roohi Ismail-Khan, Claire Dees, Andrew Poklepovic, Micheal Grever, Helen Chen, Miguel Villalona, William Carson. NCI 9455: Phase II study of trametinib followed by trametinib plus AKT inhibitor,GSK2141795 in patients with advanced triple negative breast cancer. [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 LB-216.

Background: HER2 tumors are heterogeneous at both the clinical and molecular levels (Harris et al 2007, Varadan, AACR 2015). We and others have identified a distinct molecular subtype termed ‘HER2 basal’ that demonstrates lower response to preoperative trastuzumab and is characterized by overexpression of the ‘basal-like’ signature genes (Harris, CCR 2007; Carey, JCO 2015). We sought to better understand the molecular underpinnings of the HER2 basal phenotype by performing whole exome sequencing (WES) in a cohort of HER2 tumors treated with trastuzumab-containing preoperative therapy. Methods: A multicenter trial (BrUOG 211B) was conducted to determine predictors of pathologic complete response (pCR) to trastuzumab (T)-containing preoperative therapy. Sixty HER2+ Stage II-III patients were enrolled and assigned to either a single dose of T or nab-paclitaxel (N) followed by T+N+carboplatin (TNC). Fresh tumor core biopsies were taken at baseline and 2 weeks after the initial dose of T or N. PAM50 subtyping was performed using gene expression data from patient tumor biopsies and tumors were classified into HER2-Enriched, HER2-Luminal and HER2-Basal subtypes based on ER/PR IHC values and relative expression of the proliferation-associated genes within the PAM50 gene list. WES was performed on a total of 86 samples (49 baseline and 37 from the post brief-exposure timepoint), with each sample sequenced at an average depth of 90X. Somatic mutations (single nucleotide variants and short indels) were assessed using Genome Analysis Toolkit (GATK) UnifiedGenotyper followed by a robust pipeline comparing multiple databases to eliminate germline variants. Copy-number estimation was performed on the WES data using a novel algorithm, ‘ENVE’ (Varadan, Genome Med, 2015) that robustly identifies somatic copy-number alterations. Results: WES profiles of HER2 basal tumors (n = 6) revealed lower average copy number for HER2 (2.5 vs 5.8) and were less likely to have high-level amplification (>4 copies per cell) of other amplicons (e.g. 11q13, 20q13) with the exception of 8q24. The so-called ‘Myc’ amplicon was co-amplified in all subtypes relatively equally (3/6 in HER2-B, 11/19 in HER2-E, 10/23 in HER2-L). HER2-B tumors were also characterized by higher frequency of mutations in Rb (2/6 versus 1/42; P = 0.0376) and HER3 (4/6 vs 2/42; P = 0.0011) compared with other subtypes and frequent alteration of p53 (4/6 versus 17/42; P = 0.22). pCR to preop TNC was HER2-B 3/8, HER2-E 10/20, HER2-L 6/21, not significant in this small cohort. The presence of HER3 mutations suggests a potential mechanism for the previously reported poor clinical response to preoperative anti-HER2 therapy in HER2 basal tumors. Conclusions: The HER2 basal subtype is more likely to exhibit mutations in HER3 and is less likely to respond to trastuzumab-based therapy. Larger cohorts are required to confirm these findings and trials of other HER2 targeted agents in this subtype should be considered. Citation Format: Vinay Varadan, Salendra Singh, Hannah Gilmore, Shikha Parsai, George Somlo, Maysa Abu-Khalaf, William Sikov, Lyndsay N. Harris. HER2 basal tumors have frequent mutation of HER3 and are more resistant to HER2-targeted therapy. [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 304.

Purpose/Objective: Breast cancer is a collection of diseases with distinct molecular subtypes with differing prognoses. Clinically, there is a group of therapy resistant breast cancers characterized by resistance to conventional chemo- and radiotherapy with short overall survival and poor prognosis, which we termed primary refractory breast cancer (PRBC). We hypothesized that PRBC is characterized by specific genomic changes that may contribute to therapy resistance. The aim of this study was to identify changes in gene expression profiles of PRBC compared to triple negative (TNBC) and ER+/-PR positive (HR) breast cancer cohorts. Methods: PRBC was defined as patients with an initial diagnosis of Stage I-III breast cancer who either I) developed metastatic disease within 6 months of completing treatment or II) died of breast cancer within two years of initial diagnosis. Formalin-fixed paraffin-embedded (FFPE) tissue was obtained under an IRB approved protocol 01-13-43c. RNA was extracted from macro-dissected FFPE tumor tissue. Gene expression profiling was performed with Affymetrix Human Transcriptome Array 2.0 and gene signatures were analyzed using a single sample gene set enrichment methodology. Gene signatures of PRBC patients were compared to signatures of two TNBC and HR cohorts with gene expression data generated by identical methodology. Results: A total of nineteen samples from eight patients had sufficient tumor material to obtain RNA. The median age at diagnosis was 44.3 years, 62.5% were triple negative. At initial diagnosis the stage ranged from IA to IIIC. Sixty-three percent received neoadjuvant chemotherapy. The median time to relapse was 8.4 months and median time to death 1.7 years. Genomic analysis identified highly significant (p Conclusions: PRBC is characterized by significant upregulation of multiple gene expression signatures important in drug resistance including ABC transporters, hypoxia, AKT and DNA repair pathways. Confirmation of these results would allow the development of alternative treatment strategies for PRBC to help improve the devastatingly poor prognosis of this subgroup of patients. Citation Format: Charlene Kan, Shikha Parsai, Stefanie Avril, Janice Lyons, Hannah Gilmore, Junran Zhang, Lyndsay Harris. Drug resistance gene expression pathways in primary refractory breast cancer. [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 169.

Background: Recent discoveries of recurrent and targetable gene fusions in breast cancer suggest the need to characterize the functional significance of such genomic aberrations within larger cohorts. We quantified fusion transcript expression in patient samples using RNASeq to identify recurrent gene fusion events in breast cancer as well as study the fusions post-brief exposure to mono-therapy. Methods: We sequenced transcriptomes of core biopsy RNA from 130 breast tumors obtained from brief-exposure preoperative clinical trials BrUOG 211A/211B. HER2- patients were treated with brief exposure to bevacizumab (B) or nab-paclitaxel (nP) followed by treatment with B/nP/carboplatin while HER2+ patients received brief exposure to trastuzumab (T) or nP followed by T/nP/carboplatin. Paired-end sequencing on 75 baseline biopsies and 55 post-exposure biopsies using amplified total RNA yielded 55 million reads on average perlsample. Fusion transcript abundance was evaluated using 2 pipelines, TopHat-Fusion and deFuse, due to their complementary strategies in fusion detection. We eliminated gene-pseuodogene fusion pairs as likely false positives arising due to alignment artifacts. Fusions that met 1 or more of the following 3 criteria were considered high confidence: i) Called by both deFuse and TopHat. ii) Called by deFuse with probability >95% iii) Called by TopHat with > 15 reads supporting the fusion. Results: We identified high confidence gene fusions, detected by both TopHat and deFuse, in 73 of the 75 baseline biopsies with 16 fusions on average per sample. We looked for modulation of gene fusions upon brief exposure to therapy in 55 patients that had post exposure biopsy data and found that out of the 545 high confidence fusions detected across these patients, 62 (11.37%) of the fusions were found to be still present after the therapy exposure. For the recurrent fusion analyses, we considered the 75 baseline samples. We found a total of 1158 unique candidate fusions. Out of these, 116 (10%) were recurrent in more than 1 patient. After further filtering, we were able to narrow down to 9 (0.77%) fusions that were reliable since they were predicted by both the algorithms in different patients. 2 of these 9 fusions involved GAS5 as a partner gene. GAS5 have been studied to have a role in apoptosis and its down-regulation has been associated with cell proliferation, which makes it a very interesting fusion candidate. Conclusions: We find that gene fusions in breast cancer are highly heterogeneous but are enriched with cancer-related pathway genes. This is the first study to report 2 novel gene-lincRNA fusion transcripts: MDN1-GAS5 and GABRB3-GAS5. Both these fusions are called in the baseline & post-therapy for atleast 1 patient (different patients each). GAS5 has been found as participating in a fusion in B-cell lymphoma. We are currently in the process of validating the fusion calls using qRT-PCR. The heterogeneity of detected fusions suggests that multiple mechanisms could underlie the selective advantage of tumor cells expressing fusion transcripts. The brief-exposure preoperative paradigm provides a unique opportunity to evaluate modulation of fusion transcripts that can shed light on their functional importance. Citation Format: Agrawal V, Varadan V, Banerjee N, Miskimen K, Vadodkar A, Abu-Khalaf M, Sikov W, Harris L, Dimitrova N. Novel recurrent lncRNA fusions detected in breast cancer using RNA-Seq technology in a neoadjuvant setting. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-08.

The detection and quantification of fusion transcripts has both biological and clinical implications. RNA sequencing provides a means for unbiased and high resolution characterization of fusion transcript information in tissue samples. We evaluated two gene fusion detection algorithms, TopHat-Fusion and deFuse, using transcriptomes derived from breast cancer biopsies and cell line. The two algorithms use different strategies to reduce false positive fusion calls: TopHat-Fusion uses a cascade of heuristically designed filters while deFuse relies on statistical methodologies to assess and prune potential fusions. We evaluated the concordance of gene fusion calls between the two algorithms and characterized the evidence generated by each methodology for the common fusions. Our results show that the concordance between the two algorithms is about 6% at comparable number of fusions detected. deFuse tends to be more sensitive in detecting fusions when compared to TopHat-Fusion. On average, deFuse identifies proportionally more reads than TopHat-Fusion as evidence for concordant fusions. While deFuse provides a more quantitative summary of the reliability of the fusion calls, TopHat-Fusion is able to detect additional fusion transcripts that were missed by deFuse. Further research is required to develop algorithms that can detect fusions with high sensitivity and specificity.

The purpose of this study is to explore the relationship between tumor hypoxia assessed by CA IX protein expression and loss of BRCA1 function in triple negative breast cancer (TNBC). Protein expression of CA IX and BRCA1 was evaluated by AQUA™ technology on two breast cancer cohorts: an unselected cohort of 637 breast cancer patients and a TNBC cohort of 120 patients. Transcriptional profiling was performed on FFPE samples from the TNBC cohort to evaluate a gene expression signature associated with BRCA1 mutation (van’t Veer et al., Nature 415(6871):530–536, 2002). CA IX is expressed in 7 % of the unselected breast cancer cohort and in 25 % of the TNBCs and is significantly associated with the triple negative phenotype. CA IX protein expression and BRCA1 protein expression are inversely correlated in both cohorts. Patients expressing high levels of CA IX show significantly worse overall survival (p = 0.02). Importantly, high CA IX protein expression occurs in patients who show the BRCA1 mutant signature and low levels of BRCA1 protein. These data suggest that elevated CA IX protein in TNBC is associated with a BRCA1 mutant signature and loss of BRCA1 function. CA IX may be a useful biomarker to identify triple negative patients with defective homologous recombination, who might benefit from PARP inhibitor therapy.

Since circulating tumor cells were first reported in 1955, the field has seen major advances in their detection and has established their prognostic impact. Here we review the current evidence for the prognostic and predictive value of circulating tumor cells in metastatic breast cancer. We then evaluate the role of CTCs and DTCs in early stage breast cancer. The weight of the evidence supports the role of CTCs and DTCs as prognostic indicators, however their role in therapy prediction remains unclear. Ongoing trials may provide answers and newer detection methods which improve sensitivity and specificity may have greater impact. At this point, the data does not support incorporation into clinical practice for early breast cancer patients.

The insulin-like growth factor (IGF) axis is fundamentally important in cell growth, development and cancer. We used genomic technologies to better characterize the activity of the IGF axis in human breast cancer and to identify predictors of response to IGF targeted therapies. Analysis of the gene expression patterns and pathway analysis in 204 clinically annotated primary breast cancers were performed and compared to levels of mRNA for IGF ligands and receptors. Pathway activation scores were calculated by Pearson correlation (+1, −1). Network analysis was performed using Ingenuity software. IGF-1 ligand levels were strongly negatively correlated (P < 10−6) with a published IGF-IR activation signature. A signature of high IGF-1 ligand was associated with better prognosis (P = 0.025–1.5 × 10−8) in several public datasets. Pathway analysis revealed upregulation of pathways associated with breast differentiation (adipocyte growth factors, PPAR-gamma) and down-regulation of proliferation pathways (AKT/MAPK) in the IGF-1 ligand high group. Of note, the IGF-1 ligand signature was anti-correlated with IGFIR receptor levels (P = 0.07). In conclusion, a breast tumor-derived signature of high IGF-1 ligand is associated with favorable outcome, in contrast to a previously reported IGF-IR activation signature. The prognostic value of the IGF-I ligand signature is validated in three independent datasets. These signatures should be applied in study of IGF1-R targeted therapy.

Hypoxia, a pathological feature of many solid tumours, is caused by an imbalance between tumour proliferation and angiogenesis (Dang and Semenza, 1999; Dewhirst et al, 2008). Hypoxic regions are defined by an oxygen tension (pO2) of ⩽10 mm Hg. They have been identified in up to 50% of locally advanced breast tumours (Vaupel et al, 2002), and the presence of hypoxia is known to be a negative predictor of survival in cancer patients as it may contribute to more aggressive tumour phenotypes, increased invasiveness, and metastasis (Harris, 2002). The transmembrane glycoprotein carbonic anhydrase IX (CA IX) has been identified as a potentially important marker of hypoxia in breast tumours. Known to catalyse the transformation of carbon dioxide to carbonic acid, CA IX may contribute to acidification of the extracellular microenvironment in a variety of tumours (Pastorek et al, 1994; Lindskog, 1997). Expression of CA IX is dependent on the transcription factor hypoxia-inducible factor-1 (HIF-1) (Wykoff et al, 2000), and the presence or absence of CA IX is correlated with microelectrode measurements of tumour oxygenation in cervical carcinoma (Loncaster et al, 2001). Immunohistochemical studies demonstrate that CA IX co-localises with pimonidazole, a bioreductive marker of hypoxia (Olive et al, 2001). Although CA IX is widely accepted as a marker of tumour hypoxia, its prognostic significance remains the subject of significant debate (Chia et al, 2001; Bartosova et al, 2002; Span et al, 2003; Brennan et al, 2006). The differing conclusions in these studies may be explained by the fact that heterogeneous patient populations have been treated with different combinations of surgery, radiation, and chemotherapy. Breast cancer is the most common malignancy affecting women today aside from non-melanoma skin cancer, and it trails only lung cancer as the most common cause of cancer death in females (Jemal et al, 2008). Although standard surgical and radiotherapy techniques have resulted in over 95% local control of primary breast tumours (Bartelink et al, 2001), the 10-year overall survival (OS) rate still hovers just above 80% because of failures of local and systemic therapies (Navalta et al, 2010). Although surgery remains the mainstay of early-stage breast cancer treatment, approaches involving adjuvant chemotherapy are increasing. In early stage disease, adjuvant chemotherapy provides only a modest survival benefit while causing significant systemic toxicity and patient suffering. Thus, there is considerable interest in identifying predictive markers of response to chemotherapy to enable clinicians to select agents most likely to benefit a given patient and avoid ineffectual treatments. Hypoxia is also widely accepted to have a role in resistance to radiotherapy and chemotherapy in a variety of human tumours. Anthracyclines, a group of chemotherapeutic agents that inhibit topoisomerase IIα, are the most common chemotherapeutic agents used worldwide to treat breast cancer. Under hypoxic conditions, cancer cells experience a large pH gradient across the cellular membrane, maintaining acidic extracellular and basic intracellular environments (Svastova et al, 2004). The acidic extracellular environment can decrease the uptake of anthracyclines by cells, because these drugs are weak bases, which ionise at low pH (Gerweck and Seetharaman, 1996). Furthermore, one of the mechanisms by which anthracyclines mediate cellular death is through iron-mediated generation of reactive oxygen species (ROS). As ROS formation is dependent on the presence of oxygen within the tumour microenvironment, anthracyclines may be less effective in hypoxic tumours. To address this potential disparity in therapeutic effectiveness, we gathered a set of early-stage breast cancer biopsies from patients who received anthracycline-based chemotherapeutic regimens and had >10 years of follow-up. Using immunohistochemical detection of CA IX, we demonstrate that tumour hypoxia is correlated with worse outcome for early-stage breast cancer patients treated with doxorubicin. Further, we found CA IX to predict outcome, independent of human epidermal growth factor receptor 2 (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification.

144 Background: Adjuvant chemotherapy for breast cancer has undergone many changes over the past 10 yrs. The objective of the study was to review prescribing trends in adjuvant chemotherapy regimens for early stage breast cancer in a single institution, over a decade. Methods: A retrospective chart review was conducted of patients (pts) with early stage breast cancer treated at YNHH from 2002 to 2011.Total charts analyzed was 1097 and 308 pts had complete data. Results: The HER 2 positive group accounted for 64 pts. The most commonly used regimen was ACTH (doxorubicin and cyclophosphamide 4-14 d cycles, paclitaxel and trastuzumab weekly for 12 weeks, and trastuzumab for 1 yr). This regimen was utilized in 35% of pts and was most likely to be used in node positive (N+ve) pts compared to node negative (N-ve) pts (OR 2.9 (95 % CI 0.97-9.1)).TCH (docetaxel, carboplatin, trastuzumab,6-21 d cycles and trastuzumab for 1 year) was the second most common regimen used in 21% of pts and predominantly in N-ve pts(OR 12.4 (95% CI 02.45-63.2)). Majority of the study group (184 pts) were ER+PR+ and HER 2- .The predominant chemo regimen was DD AC T (dose dense AC followed by paclitaxel in 4 -14d cycles each) in 50% of pts, and TC (Docetaxel, cyclophosphamide4-21d cycles) utilized in 48 pts (26.09 %). Majority of the N-ve pts were treated with TC (OR 12.4(95% CI 2.4-63.27) whereas N+ve pts were predominately treated with DDACT (OR 2.98(95% CI 0.97-9.16). The Triple negative group included a total of 57 pts .The most frequent chemo regimen through the years was DDACT (54.39% of pts) followed by TC (21.05% of pts). N+ve pts were more likely to be treated with DDACT (OR 8.91(95% CI (2.65-29.94)) whereas N-ve pts mostly received TC (OR 8.75(95% CI (1.70-45.00)) The methotrexate and 5 fluorouracil based regimens were utilized more frequently in earlier years of study and less frequently in later years. Conclusions: Anthracycline and taxane based regimens have gained precedence over older regimens. HER 2 +ve pts were most commonly treated with ACTH, and TCH. N+ve HER 2+ pts were most likely to be treated with ACTH and N-ve HER2+ pts with TCH. In Her 2 negative and triple negative pts, DDACT was more utilized in N+ve pts whereas, TC regimen in the N-ve pts.

Human epidermal growth factor receptor 2 amplified (HER2+) breast cancer is common and aggressive. Trastuzumab is a targeted therapy to the HER2 cell surface receptor and has greatly improved prognosis for HER2+ breast cancer patients, but trastuzumab-resistance is a significant problem. Few biomarkers of trastuzumab resistance of response have been found, and there has been tremendous difficulty identifying in vitro biomarkers that transfer to the in vivo HER2+ breast cancer setting.We identified gene expression signatures of trastuzumab-response and trastuzumab-resistance in HER2+ breast cancer cell lines across 24-hour exposure to trastuzumab. A set of 180-genes changed significantly in a trastuzumab-sensitive HER2+ cell line (BT474) and 58 genes changed significantly across treatment in a trastuzumab-resistant HER2+ cell line (UACC812).To demonstrate that these signatures have clinical relevance, we applied them in vivo to gene expression profiles from early stage HER2+ breast tumors treated with a single dose of trastuzumab. We found that the BT474 trastuzumab-responsive signature was enriched among the changes in expression across treatment for responsive patients. The expression change for the BT474 signature genes was also able to partially cluster responsive and resistant breast tumors by outcome. A subset of the UACC812 trastuzumab-resistance signature was also enriched in the differential between responsive and resistant tumors prior to trastuzumab treatment.These results show for the first time that cell line-derived gene signatures can be useful in identifying whether breast cancer patients will ultimately benefit from trastuzumab treatment. This approach might one day be applied to enhance clinical treatment decisions.

Introduction: Biomarkers, such as Estrogen Receptor, are used to determine therapy and prognosis in breast carcinoma. Immunostaining assays of biomarker expression have a high rate of inaccuracy; for example, estimates are as high as 20% for Estrogen Receptor. Biomarkers have been shown to be heterogeneously expressed in breast tumors and this heterogeneity may contribute to the inaccuracy of immunostaining assays. Currently, no evidencebased standards exist for the amount of tumor that must be sampled in order to correct for biomarker heterogeneity. The aim of this study was to determine the optimal number of 20X fields that are necessary to estimate a representative measurement of expression in a whole tissue section for selected biomarkers: ER, HER-2, AKT, ERK, S6K1, GAPDH, Cytokeratin, and MAP-Tau. Methods: Two collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using the Automated Quantitative Analysis (AQUA) method of quantitative immunofluorescence. Simulated sampling of various numbers of fields (ranging from one to thirty five) was performed for each marker. The optimal number was selected for each marker via resampling techniques and minimization of prediction error over an independent test set. Results: The optimal number of 20X fields varied by biomarker, ranging between three to fourteen fields. More heterogeneous markers, such as MAP-Tau protein, required a larger sample of 20X fields to produce representative measurement. Conclusions: The optimal number of 20X fields that must be sampled to produce a representative measurement of biomarker expression varies by marker with more heterogeneous markers requiring a larger number. The clinical implication of these findings is that breast biopsies consisting of a small number of fields may be inadequate to represent whole tumor biomarker expression for many markers. Additionally, for biomarkers newly introduced into clinical use, especially if therapeutic response is dictated by level of expression, the optimal size of tissue sample must be determined on a marker-by-marker basis.

Summary Background We previously identified a functional variant in a let-7 microRNA (miRNA) complementary site in the 3′-untranslated region of the KRAS oncogene (rs61764370) which is associated with cancer. We aimed to investigate the association of this KRAS variant with breast cancer and tumour biology. Methods We assessed frequency distributions of the KRAS variant in 415 patients with histologically confirmed breast cancer and 457 controls from Connecticut, USA (study group 1) and association of this variant with breast-cancer subtypes in 690 Irish women with known oestrogen receptor (ER), progesterone receptor (PR), and HER2 statuses, and 360 controls (study group 2). We pooled data for study groups 1 and 2 with a cohort of 140 women with triple-negative breast cancer and 113 controls to assess the association of the KRAS variant with triple-negative breast cancer risk, and genome-wide mRNA and specific miRNA expression in patients with triple-negative breast cancer. Findings Although frequency distributions of the KRAS variant in study group 1 did not differ between all genotyped individuals, eight (33%) of 24 premenopausal women with ER/PR-negative cancer had the KRAS variant, compared with 27 (13%) of 201 premenopausal controls (p=0·015). In study group 2, the KRAS variant was significantly enriched in women with triple-negative breast cancer (19 [21%] of 90 cases) compared with 64 (13%) of 478 for luminal A, 13 (15%) of 87 for luminal B, and two (6%) of 35 for HER2-positive subgroups (p=0·044). Multivariate analysis in the pooled study groups showed that the KRAS variant was associated with triple-negative breast cancer in premenopausal women (odds ratio 2·307, 95% CI 1·261–4·219, p=0·0067). Gene-expression analysis of triple-negative breast-cancer tumours suggested that KRAS -variant positive tumours have significantly altered gene expression, and are enriched for the luminal progenitor and BRCA1 deficiency signatures. miRNA analysis suggested reduced levels of let-7 miRNA species in KRAS -variant tumours. Interpretation The KRAS variant might be a genetic marker for development of triple-negative breast cancer in premenopausal women, and altered gene and miRNA expression signatures should enable molecular and biological stratification of patients with this subgroup of breast cancer. Funding US National Institutes of Health.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Introduction HER2 and estrogen receptor (ER) are important in breast cancer and are therapeutic targets of trastuzumab (Herceptin) and tamoxifen, respectively. Retinoids inhibit breast cancer growth, and modulate signaling by HER2 and ER. We hypothesized that treatment with retinoids and simultaneous targeting of HER2 and/or ER may have enhanced anti-tumor effects. Methods The effects of retinoids combined with trastuzumab or tamoxifen were examined in two human breast cancer cell lines in culture, BT474 and SKBR3. Assays of proliferation, apoptosis, differentiation, cell cycle distribution, and receptor signaling were performed. Results In HER2-overexpressing/ER-positive BT474 cells, combining all-trans retinoic acid (atRA) with tamoxifen or trastuzumab synergistically inhibited cell growth, and altered cell differentiation and cell cycle. Only atRA/trastuzumab-containing combinations induced apoptosis. BT474 and HER2-overexpressing/ER-negative SKBR3 cells were treated with a panel of retinoids (atRA, 9-cis-retinoic acid, 13-cis-retinoic acid, or N-(4-hydroxyphenyl) retinamide (fenretinide) (4-HPR)) combined with trastuzumab. In BT474 cells, none of the single agents except 4-HPR induced apoptosis, but again combinations of each retinoid with trastuzumab did induce apoptosis. In contrast, the single retinoid agents did cause apoptosis in SKBR3 cells; this was only modestly enhanced by addition of trastuzumab. The retinoid drug combinations altered signaling by HER2 and ER. Retinoids were inactive in trastuzumab-resistant BT474 cells. Conclusions Combining retinoids with trastuzumab maximally inhibits cell growth and induces apoptosis in trastuzumab-sensitive cells. Treatment with such combinations may have benefit for breast cancer patients.

Oncotype DX (ODX) is a 21 panel gene-expression based assay for predicting whether patients with estrogen receptor-positive (ER+) breast cancer (BCa) are candidates for adjuvant chemotherapy. However, the time and expense associated with genomic assays suggests the need for a non-invasive, imaging-based, pre-therapeutic tool for assessment of disease risk and selection of an appropriate treatment regimen. The objective of this research was to determine whether (a) computer extracted image features on T2-weighted (T2w) MRI and H&E stained histopathology are independently able to distinguish ER+ BCa with low and high ODX recurrence scores (RS) and (b) to determine whether there is a correlation between MRI and histologic features identified as being predictive of low and high ODX risk categories. A total of 11 ER+ BCa patients were considered in this study, based on availability of in vivo 1.5 Tesla T2w MRI. For each study, the corresponding formalin-fixed paraffin-embedded H&E stained tissue specimens were digitized at 20x (0.5 μm/pixel) using a whole-slide scanner. Of the 11 patients, 8 were identified in the low ODX (RS < 18) and 3 in the high ODX (RS > 30) risk categories. Each dataset was accompanied by expert annotations of (a) the lesion ROI on MRI and (b) boundaries of epithelial nuclei from a representative field-of-view on the digitized histology slide. For each MRI study, a multi-scale, multi-orientation Gabor filter bank was convolved with the annotated lesion area providing a set of 192 texture features (FMRI). For each corresponding histology image, 471 features (FHIST) were extracted describing both nuclear morphology (NM) and Laws texture (LT) within the nuclear regions. Independent 2-sample t-tests were used to identify salient features in FMRI and FHIST that are able to distinguish low and high ODX risk categories. We found that, for the MRI dataset, Gabor texture features at several scales and orientations yielded salient features (p < 0.05) while on histopathology, nuclear texture and convexity (shape) features were identified as the top discriminative features (p < 0.01). Relationships between significant features were evaluated via Spearman's rank correlation test (see table), where high correlations were observed between lesion texture on T2w MRI and nuclear texture and shape on histology. Correlation of histologic and MRI features able to distinguish low and high ODX RSHistologic feature correlated with ODXMRI feature correlated with ODXCorrelation coefficient (ρ)p-valueLT: 70 Mean HSVGF: Scale 2: Orientation 3: min/max-0.85450.0008NM: ConvexityGF: Scale 5: Orientation 6: mean-0.85450.0008LT: 70 Mean HSVGF: Scale 2: Orientation 3: min/max-0.83640.0013LT: 70 Mean HSVGF: Scale 3: Orientation 8: mean-0.83640.0013LT: 70 Mean HSVGF: Scale 3: Orientation 2: mean-0.81820.0021 Our results suggest that quantitative features extracted on both T2w MRI and histopathology can independently distinguish between low and high risk ODX classes. Moreover, some of these MRI and histologic features appear to be significantly correlated, suggesting that information regarding tumor biology is reflected in both MRI and histologic image features. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-03-01.

The worse outcomes in African-American (AA) breast cancer patients have been attributed to a variety of factors, including compliance/variations with treatment. We evaluated a large cohort of AA patients treated with breast conservation therapy (BCT) in an effort to determine if compliance with radiation or if choice of chemotherapy regimen could be a contributing factor for the worse outcomes reported in AA patients. Our two cohorts consisted of 221 AA and 2170 white patients treated with BCT. Chart reviews were conducted to document dates of treatment, treatment breaks, dose delivered, and chemotherapy delivered. The data were analyzed to detect differences between the two cohorts. The median radiation dose delivered (including cone-down) was 64 Gy in both cohorts (p = 0.9910). The median number of treatment days was: AA, 45 (range 21-71 days) versus white, 45 (range 14-90 days; p = 0.8465). The chemotherapy regimens administered were: adriamycin/cytoxan (17% AA versus 19% white, respectively), adriamycin/cytoxan/taxol (13% versus 5%), cytoxan/methotrexate/5-FU (53% versus 59%), cytoxan/methotrexate/5-FU/vincristine (0% versus 3%), cytoxan/adriamycin/5-FU (12% versus 11%), high dose/transplant (0% versus 1%), and other (4% versus 5%), p = 0.113. Our large institutional series suggests that compliance with radiation therapy, once patients are seen in consultation and simulated, does not differ significantly between AA and white patients. In our large cancer center environment, AA patients referred for radiation therapy appear to receive the same radiation doses in a comparable time frame to white patients. Further exploration of compliance for adjuvant chemotherapy regimens in AA patients is warranted.

Pegylated liposomal doxorubicin has activity in both breast and ovarian cancer. Preclinical data noted that ZD1839 acts synergistically with chemotherapy. Given the lack of cross-resistance between these two agents, a phase I trial was initiated examining the safety and efficacy of the combination of liposomal doxorubicin and ZD1839 in patients with recurrent gynecologic or metastatic breast cancer. Dose-limiting toxicity (DLT) was defined within the first two cycles of treatment. Escalating doses of liposomal doxorubicin were administered every 4 weeks with ZD1839. Pharmacokinetic analysis and correlative studies were performed. Thirty-five patients were enrolled in this study: six in each cohort. One DLT (febrile neutropenia) was observed in cohort 2. Dose level 3 was determined to be the maximum tolerated dose (MTD), and an additional ten patients were accrued. Serious adverse events (SAEs) included one patient with mental status changes believed secondary to disease progression and two central nervous system (CNS) bleeds believed to be unrelated to the combination of study agents. Toxicities were generally mild except for skin and gastrointestinal toxicity. No cardiac toxicity was observed. The best response to therapy included four partial responses and 20 patients with stable disease. Liposomal doxorubicin with ZD1839 is an active regimen but is associated with increased skin toxicity in patients with advanced breast and gynecologic cancer.

Although trastuzumab (Herceptin) is an important advance in the treatment of breast cancer, a significant proportion of patients do not respond to trastuzumab either alone or in combination with chemotherapy. In this study, we observe that epidermal growth factor receptor (EGFR) and HER3 expression is substantially increased after long-term trastuzumab exposure of HER2-positive breast carcinoma–derived cell lines that show primary resistance to trastuzumab. Furthermore, long-term trastuzumab exposure of trastuzumab-resistant cell lines induces de novo sensitivity to the EGFR-targeted agents gefitinib or cetuximab in two of three cell lines accompanied by increased EGFR expression. Together, these results indicate that primary trastuzumab resistance is not synonymous with lack of responsiveness to trastuzumab and, importantly, suggest that trastuzumab priming may sensitize trastuzumab-resistant tumors to other HER family-directed therapeutics. [Cancer Res 2009;69(6):2191–4]

BACKGROUND. African–American (AA) and white patients with early–stage disease who were treated with breast conservation therapy (BCT) were examined to detect differences in clinicopathologic features and outcomes as a function of race. METHODS. Clinical data from the charts of 2164 white and 207 AA patients treated with BCT, and p53 expression status on 444 patients (from an existing tissue database), were analyzed to detect differences between the 2 cohorts. RESULTS. The median follow-up was 7 years. There were no differences in the method of tumor detection, lymph nodes excised, surgical margin status, or chemotherapy/radiotherapy delivered, reflecting similar screening and treatment policies for AA women in the study community. Despite this, AA patient presented at a younger age, with higher T and N classifications, and more estrogen and progesterone negative and “triple negative” tumors (all P values

Although techniques such as RT-PCR and in situ hybridization (ISH) can give information about gene expression, they are limited in scope as typically one gene product is evaluated with each assay. The advent of transcriptional profiling using DNA microarray has revolutionized the field of molecular medicine as measurement of thousands of genes simultaneously in a given sample provide a vast amount of data for new disease classifications and biomarker discoveries. DNA microarray-based gene expression profiling relies on nucleic acid polymers, immobilized on a solid surface, which act as probes for complementary gene sequences.1 Microarrays typically contain several thousand single-stranded DNA sequences, which are “arrayed” at specific locations on a synthetic “chip” through covalent linkage. These DNA fragments provide a matrix of probes for fluorescently labeled complementary RNA (cRNA) derived from the sample of interest. The expression of each gene in the sample is quantified by the intensity of fluorescence emitted from a specific location on the array matrix which is proportional to the amount of that gene product (Figure 10.1).2 Open image in new window Figure 10.1 Reprinted from Sauter G, Simon R. Predictive molecular pathology. N Engl J Med 2002;347:1995–1996. (Copyright 2002 Massachusetts Medical Society)

BACKGROUND. Previous reports based on small patient numbers suggested that changes in serum HER-2/neu levels may predict response or lack of response to trastuzumab-based therapies in metastatic breast cancer (MBC). The objectives of this study were to pool data from 307 patients with MBC from 7 medical institutions to validate that the serum HER-2/neu profile predicts patient resistance to trastuzumab and to establish a clinically relevant cutoff. METHODS. This was an international, multicenter, retrospective analysis of individual pooled data from 307 patients with MBC who were treated with first-line trastuzumab-based therapy. Serum was collected at baseline and 30 to 120 days after the initiation of trastuzumab therapy. A serum HER-2/neu decrease ≥20% (receiver operating curve analysis) was defined as a significant HER-2/neu change. RESULTS. Of the 307 patients with MBC, 191 patients (62%) had a significant decline (>20%) in serum HER-2/neu and 116 patients (38%) did not. The objective response rate was 57% for patients who achieved this decline in serum HER-2/neu (>20%) compared with 28% for patients who did not. Patients who achieved this decline in serum HER-2/neu also had a significantly longer time to disease progression (320 days vs 180 days; P < .0001), longer duration of response (369 days vs 230 days; P = .008), and longer overall survival (898 days vs 593 days; P < .018). CONCLUSIONS. In this pooled analysis of 307 patients with MBC, individuals who did not achieve a significant decline (≥20%) in serum HER-2/neu levels had decreased benefit from trastuzumab-based therapy, and these patients should be considered for clinical trials evaluating additional HER-2/neu-targeted interventions. Cancer 2008. © 2008 American Cancer Society.

It is accepted that preoperative chemotherapy can result in increased breast preservation for breast cancers greater than 4 cm. The benefits of preoperative chemotherapy are less clear, however, for patients who present with smaller tumors and are already candidates for breast-preserving surgery. The goal of this study is to assess the effect of preoperative chemotherapy on breast cancers between 2 and 4 cm diameter.A retrospective chart review was conducted of patients diagnosed with new breast cancer at the Yale-New Haven Breast Center for the years 2002-2007. Patients were included in the study if their breast cancer was between 2 and 4 cm and their initial surgical treatment had been completed. Patients with distant metastases were excluded.There were 156 new cancers that met study requirements. Forty-seven patients underwent preoperative chemotherapy, and 109 patients had their surgery first, usually followed by chemotherapy. Initial surgery was lumpectomy for 31 out of 47 patients (66%) in the preoperative chemotherapy group compared with 62 out of 109 patients (57%) in the surgery group. For patients with lumpectomies, 2 out of 31 patients (6%) in the preoperative group had positive margins and required re-excision compared with 20 out of 62 patients (37%) in the surgery-first group (P0.01).We conclude that, for tumors between 2 and 4 cm, preoperative chemotherapy is associated with a significantly decreased rate of re-excision following lumpectomy. This not only results in fewer mastectomies, but also avoids the morbidity and inferior cosmetic results associated with a re-excision lumpectomy.

Background Detection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance. However, background expression of 'tumor specific markers' in peripheral blood mononuclear cells (PBMC) may confound these studies. The goal of this study was to identify the origin of Cytokeratin 19 (CK19) and HER-2 signal in PBMC and suggest an approach to enhance techniques involved in detection of CTC in breast cancer patients. Methods PBMC from healthy donors were isolated and fractionated into monocytes, lymphocytes, natural killer cells/granulocytes and epithelial populations using immunomagnetic selection and fluorescent cell-sorting for each cell type. RNA isolated from each fraction was analyzed for CK19, HER2 and Beta 2 microglobulin (B2M) using real-time qRT-PCR. Positive selection for epithelial cells and negative selection for NK/granulocytes were used in an attempt to reduce background expression of CK19 and HER2 markers. Results In normal PBMC, CK19 was expressed in the lymphocyte population while HER-2 expression was highest in the NK/granulocyte population. Immunomagnetic selection for epithelial cells reduced background CK19 signal to a frequency of Conclusion We present a novel method to improve the specificity of the traditional method of detecting CTC by identifying the source of the background signals and reducing them by negative immunoselection. Further studies are warranted to improve sensitivity and specificity of methods of detecting CTC will prove to be useful tools for clinicians in determining prognosis and monitoring treatment responses of breast cancer patients.

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.

Gene expression analysis has identified biologically relevant subclasses of breast cancer. However, most classification schemes do not robustly cluster all HER2+ breast cancers, in part due to limitations and bias of clustering techniques used. In this article, we propose an alternative approach that first separates the HER2+ tumors using a gene amplification signal for Her2/neu amplicon genes and then applies consensus ensemble clustering separately to the HER2+ and HER2− clusters to look for further substructure. We applied this procedure to a microarray data set of 286 early-stage breast cancers treated only with surgery and radiation and identified two basal and four luminal subtypes in the HER2− tumors, as well as two novel and robust HER2+ subtypes. HER2+ subtypes had median distant metastasis-free survival of 99 months [95% confidence interval (95% CI), 83–118 months] and 33 months (95% CI, 11–54 months), respectively, and recurrence rates of 11% and 58%, respectively. The low recurrence subtype had a strong relative overexpression of lymphocyte-associated genes and was also associated with a prominent lymphocytic infiltration on histologic analysis. These data suggest that early-stage HER2+ cancers associated with lymphocytic infiltration are a biologically distinct subtype with an improved natural history. [Cancer Res 2007;67(22):10669–76]

Background Neoadjuvant chemotherapy precludes the accurate pre-surgical pathologic measurement of tumor size. The purpose of this study is to review imaging studies performed in patients who received preoperative chemotherapy prior to surgery and determine whether MRI, ultrasound (US) or physical exam best predicted final pathologic tumor size. Methods Stage I, II, and III breast cancer patients were treated with neoadjuvant therapy on trial. As part of the trials, women underwent MRI, US, and physical exam prior to the start of therapy and 1 week after completion of neoadjuvant chemotherapy. Results Of the 68 patients with MRI data, the correlation coefficient (r) of MRI to pathologic size of tumor was r = 0.749. Among the 52 patients who had an US assessment the correlation of US to pathology was r = 0.612. Sixty-two patients had physical exam data, and the correlation of examination to pathology size was r = 0.439. MRI correctly predicted 8 of 11 complete responders and accurately evaluated the size of non-responders to neoadjuvant therapy (r = 0.869) Conclusions In a select group of women undergoing neoadjuvant therapy for invasive breast cancer, MRI best predicted pathology response. The use of MRI in neoadjuvant therapy may allow for accurate prediction of patients eligible for breast conservation. J. Surg. Oncol. 2007;96:474–480. © 2007 Wiley-Liss, Inc.

Purpose: To assess pathologic complete response (pCR), clinical response, feasibility, safety, and potential predictors of response to preoperative trastuzumab plus vinorelbine in patients with operable, human epidermal growth factor receptor 2 (HER2)–positive breast cancer. Experimental Design: Forty-eight patients received preoperative trastuzumab and vinorelbine weekly for 12 weeks. Single and multigene biomarker studies were done in an attempt to identify predictors of response. Results: Eight of 40 (20%) patients achieved pCR (95% confidence interval, 9-36%). Of 9 additional patients recruited for protocol-defined toxicity analysis, 8 were evaluable; 42 of 48 (88%) patients had clinical response (16 patients, clinical complete response; 26 patients, clinical partial response). T1 tumors more frequently exhibited clinical complete response (P = 0.05) and showed a trend to exhibit pCR (P = 0.07). Five (13%) patients experienced grade 1 cardiac dysfunction during preoperative treatment. Neither HER2 nor estrogen receptor status changed significantly after exposure to trastuzumab and vinorelbine. RNA profiling identified three top-level clusters by unsupervised analysis. Tumors with extremes of response [pCR (n = 3) versus nonresponse (n = 3)] fell into separate groups by hierarchical clustering. No predictive genes were identified in pCR tumors. Nonresponding tumors were more likely to be T4 stage (P = 0.02) and express basal markers (P < 0.00001), growth factors, and growth factor receptors. Insulin-like growth factor-I receptor membrane expression was associated with a lower response rate (50% versus 97%; P = 0.001). Conclusions: Preoperative trastuzumab plus vinorelbine is active and well tolerated in patients with HER2-positive, operable, stage II/III breast cancer. HER2-overexpressing tumors with a basal-like phenotype, or with expression of insulin-like growth factor-I receptor and other proteins involved in growth factor pathways, are more likely to be resistant to this regimen.

Background: HER2-positive (HER2+) breast cancer is biologically heterogeneous; no consistent biomarkers of response to T have been identified. Recent data suggest intrinsic subtypes and immune infiltration estimates applied to HER2 cohorts are able to predict response to T-based therapy. Two multicenter trials (DFCI 03-311 and BrUOG 211B) were conducted to determine if exposure to T alone prior to combination T-based chemotherapy could provide predictors of pathologic complete response (pCR). Design: In both trials, fresh tumor core biopsies were taken at baseline and a 2 week time point after a single dose of T. Eighty HER2+ early breast cancer (EBC) patients enrolled in the 03-311 trial of T>T+C. A total of 122 samples (50 matched pairs and 22 single timepoints) produced useable gene expression data using the Illumina HT12v3 platform. Similarly, in the BrUOG 211B trial, 60 HER2+ EBC patients were enrolled and randomized to either a single dose of T or a single dose of nab-paclitaxel (N) followed by T+N+C. RNA from a total of 74 samples (33 matched pairs and 8 single timepoints) was profiled using a novel targeted RNA-sequencing platform that included a total of 950 genes, including the predefined PAM50 and Immune Score (ESTIMATE) signatures. Results: PAM50 subtypes were largely stable after one dose of therapy in both the 03-311 and 211B trials. PAM50 subtypes evaluated at baseline in the 03-311 trial showed an increase in pCR in the HER2-enriched subtype (10/26; 38.4%) as compared to Luminal B (1/9; 11.11%) and HER2-basal (5/29; 17.2%). This was confirmed in 211B, with the HER2-enriched subtype exhibiting the highest pCR rate (4/8; 50%) as compared to Luminal B (4/15; 26.6%) or HER2-basal (2/9; 22.2%). After a single dose of T, a significant increase in a 140-gene expression-based index of immune infiltration (Immune Score) was observed only in the HER2-enriched subtype in both the 03-311 (p = 0.04) and the 211B (p = 0.016) trials. Interestingly, the Immune Score distinguished between responders and non-responders after a single dose of T in both the 03-311 (p = 0.03) and the 211B (p = 0.05) trials but not at baseline in either trial. In contrast, brief-exposure to nab-paclitaxel did not show either of the above associations. Conclusions: The HER2-enriched subtype has the highest response rate to preoperative trastuzumab, confirmed by two brief-exposure preoperative clinical trials. Strikingly, both trials also revealed that the Immune Score was discriminative of response after a single dose of trastuzumab and not at baseline suggesting a unique role of immune infiltration within the HER-enriched subtype. The brief-exposure paradigm adopted in our study provides novel insights into tumor biology mediating response, while also allowing for the discovery of novel biomarkers predictive of pCR. Citation Format: Vinay Varadan, Kristy L.S. Miskimen, Shikha Parsai, Ian E. Krop, Eric P. Winer, Veerle Bossuyt, Maysa Abu-Khalaf, William Sikov, Lyndsay N. Harris. A discovery and validation set of breast cancer preoperative clinical trials show an increase in immune score upon brief-exposure to trastuzumab (T) and the HER2-enriched subtype predict response to combination chemotherapy and T. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2836. doi:10.1158/1538-7445.AM2015-2836

2530 Background: TNBC are high grade tumors which have a poor prognosis. SLC1A5 is a neutral amino acid transporter found in these tumors and high levels correlate with aggressive biological behavior. By targeting SLC1A5 function, tumor cell growth can be inhibited and autophagy activated which should result in improved survival benefit for patients with TNBC. We evaluated the role of SLC1A5 on tumor death rate in patients who were treated with chemotherapy for early TNBC. Methods: A retrospective, cohort study of patients with TNBC was performed to evaluate survival compared with the level of expression of SLC1A5. A histogram was constructed to determine a median point which could be used to define a “high” versus “low” score. Five was used as the average cut-point. Thereafter to determine overall survival, Kaplan-Meier product-moment technique (K-M) was used, with comparisons between groups made by using the log rank test. Statistical significance was taken as p < 0.05. Results: The cohort consisted of ...

1592 Background: A number of lifestyle factors have been associated with risk of breast cancer. Studies have suggested that risk factors for triple negative breast cancer (TNBC) are different from ...

619 Background: Increased TILs are prognostic and predictive of therapy response in HER2+ breast cancer (BC). BrUOG-211B, a phase II neoadjuvant trial included early stage HER2+ BC pts treated with...

617 Background: ALTTO (NCT00490139) is a large adjuvant breast cancer (ABC) study evaluating lapatinib, alone and in combinations with trastuzumab and taxanes. Characterization of hepatic abnormali...

Background: CTCs are associated with clinical outcomes in metastatic breast cancer irrespective of ER/PR/HER2 status. Some data support the prognostic relevance of serial CTC enumeration relative to adjuvant chemotherapy in early stage breast cancer. However, data from a large scale study focused on HER2 directed therapy for HER2+ disease have been lacking. We therefore sought to prospectively evaluate the effect of trastuzumab +/- lapatinib on CTCs and assess the prognostic/predictive value of CTC monitoring in HER2+ early stage breast cancer patients (pts). Methods: The Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO; NCT00490139) Trial is an international, randomized, open-label phase III study of two targeted agents for HER2+ breast cancer. From June 2007 to July 2011, 8381 pts were randomised from 946 sites in 44 countries to 1 of 4 arms with sequential or concurrent chemotherapy: (i) 52 wks of trastuzumab (T); (ii) 52 wks of oral lapatinib (L); (iii) 12 or 18 wks of T followed by a washout and then 34 or 38 wks of L; or (iv) 52 wks of L+T. 540 (6%) pts provided optional informed consent and up to 30 mL peripheral blood suitable for CTC analyses at baseline with additional collections at 13 or 19 wks, 52 wks, 18 mos, 24 mos, and recurrence. CTC analyses are being conducted in three laboratories (Mayo Clinic Rochester, n=431; Institut Jules Bordet and University of Munich, n=109). 2-3 x 10 mL CellSave™ samples are pooled and processed at each time point for CTC enumeration and HER2 expression using the immunomagnetic/immunofluorescence assay (CellSearch™). A round-robin concordance project was done between Mayo Clinic Rochester and Institut Jules Bordet before embarking on the primary correlative work. Results: At baseline, 20% pts had detectable (i.e., ≥1) EpCAM+/CK+/DAPI+/CD45- CTCs, and 16% pts had detectable EpCAM+/CK+/DAPI+/CD45-/HER2+ CTCs. Correlative analyses with clinical outcome are ongoing with plans for completion by Fall 2014. Conclusions: CTCs were detected in 20% of pts with HER2+ early stage breast cancer. This is similar to the frequency of detection in mixed early stage breast cancer populations relative to ER/PR and HER2 status. Concordance of enumeration and HER2 assessments between the two experienced laboratories, and correlation between disease free survival and CTC findings (from serial samples collected at baseline, during the course of HER2 directed therapy, and at set intervals of follow-up) will be reported. Citation Format: Minetta C Liu, Brigitte Rack, Amylou C Dueck, David W Hillman, Michael B Campion, Monica M Reinholz, Kevin C Halling, Christos Sotiriou, Françoise Rothé, Marion Maetens, Ghizlane Rouas, Wolfgang Janni, Antonio C Wolff, Lyndsay N Harris, Julie R Gralow, Kathleen I Pritchard, Susan Ellard, Nguyet A Le-Lindqwister, Frances Boyle, Evandro De Azambuja, Martine J Piccart-Gebhart, Michail Ignatiadis, Edith A Perez. Circulating tumor cell (CTC) enumeration and HER2 assessment as predictors of breast cancer outcomes in the ALTTO (BIG 2-06, Alliance N063D) Trial [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 P4-01-01.

Background: Estrogen receptor (ER) inhibition is an important treatment option for advanced breast cancer (BC) pts. Recent studies describe recurring somatic mutations in codons 537 and 538 within the ligand-binding domain (LBD) of ∼ 20% of ER+ metastatic disease, but not treatment-naïve ER+ cancer or ER-negative disease, that render the ER constitutively active and confer partial resistance to endocrine agents. Few studies have described the clinicopathologic and genomic covariates that accompany the ESR1 genomic alterations (GAs). Correlating genomic events as captured by FoundationOne in pts with ESR1-mutated BCs with clinical history may provide clinical insight into these alterations. Methods: Hybridization capture of 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage using FoundationOne (Foundation Medicine, Cambridge, MA). Results: 176 of the 2,208 (7.9%) BC pt cases harbored ESR1 alterations. 1127 short variants (SV) were detected in 176 patient samples for an average of 6.4 GA/sample. 16.5% SV (186/1127) were ESR1 GAs with an average of 1.0 ESR1 GAs/sample. ESR1 GAs consisted of base substitutions (77%) and amplifications (20%, median copy number 9X, range 6-28). Base substitutions occurred at codons 538 (71/145) and 537 (70/145) and at two other novel sites, 341 (2/145) and 563 (1/145). A patient-derived xenograft study suggested that tumors refractory to ER inhibitors and harboring ESR1 amplification could be responsive to higher doses of estradiol. (Li et al, Cell Reports, 2013). The most frequently co-occurring GAs were PIK3CA (37.5%), GATA3 (22.7%), TP53 (24.4%), MAP3K1 (10.2%) and CDH1 (7.9). Collection of data on tissue sites sequenced, treatments and outcomes on these pts with ESR1 GAs are ongoing. Of note, one pt had recurrent disease following adjuvant tamoxifen and letrozole. FoundationOne testing of her recurrent bony disease revealed an ESR1 Y537 alteration along with mutations in PIK3CA, APC, and RAD51. She has been on fulvestrant monotherapy with stable metastatic disease 13 + months. Conclusions: We found through FoundationOne testing that ESR1 GAs are reasonably common in advanced BCs, with base substitutions accounting for 77% of ESR1 GAs and amplifications for 20%. Identification and characterization of ESR1-mutated advanced BC pts by comprehensive genomic profiling capable of detecting both base substitutions and copy number changes may identify clinically relevant GAs. Clinical correlation is pending. Citation Format: Norma A Palma, Siraj Ali, Garrett Frampton, Kai Wang, Hannah Gilmore, Julio Peguero, Lyndsay N Harris, Massimo Cristofanilli, Juliann Chmielecki, Jeffrey S Ross, Deborah Morosini, Vincent A Miller, Phil J Stephens, Gary Palmer, Joyce O'Shaughnessy. Profiling of ESR1-mutated metastatic breast cancers by FoundationOne® allows a broad genomic understanding for potential clinical implications [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 PD6-5.

PurposeTo determine the prognostic significance of triple negative breast cancers with respect to locoregional relapse and distant metastasis in conservatively managed breast cancer patients.Patients and MethodsA database of conservative managed (conservative surgery followed by radiation) patients, in whom all three markers (estrogen receptor, progesterone receptor, and HER2/neu) were available, was reviewed. Patients were classified as triple negative if they tested negative for all three markers. Of 482 patients with all three markers available, 117 were classified as triple negative.ResultsAs of September 2005, with a median follow-up time of 7.9 years, of the 482 patients in the study, there have been 53 in-breast relapses, 10 nodal relapses, 77 distant relapses, and 69 deaths. At 5 years, the triple negative cohort had a poorer distant metastasis-free rate compared with the other subtypes (67% v 82%, respectively; P = .002). Triple negative subtype was an independent predictor of distant metastasis (hazard ratio = 2.14; 95% CI, 1.31 to 3.53; P = .002) and cause-specific survival (hazard ratio = 1.79; 95% CI, 1.03 to 3.22; P = .047). There was no significant difference in local control between the triple negative and other subtypes (83% v 83%, respectively). Of 99 BRCA-tested patients in this cohort, 10 had deleterious mutations in BRCA1, and seven had mutations in BRCA2. Of 10 BRCA1 patients, eight were triple negative, whereas only one of seven BRCA2 patients was triple negative (P < .001).ConclusionPatients classified as triple negative have a poor prognosis. However, there was no evidence that these patients are at higher risk for local relapse after conservative surgery and radiation. Patients with BRCA1 mutations develop predominantly triple negative tumors.

Our objectives were to ascertain the safety (cardiotoxicity) and efficacy of non-pegylated liposomal doxorubicin [Myocet (M)] compared with conventional doxorubicin (A) in patients with metastatic breast cancer (MBC) who had received adjuvant anthracycline treatment and were at high risk of developing iatrogenic cardiomyopathy. This retrospective analysis is based on data pooled from two prospective phase III comparative randomized clinical trials comparing Myocet versus conventional doxorubicin in combination with cyclophosphamide and as single agents, respectively, for the treatment of MBC. The outcome measures reviewed in this analysis were overall response, time to treatment failure, time to disease progression, overall survival and cardiotoxicity. The analysis was carried out by strata according to patients' previous exposure to adjuvant anthracyclines. Kaplan-Meier, log-rank chi-test, Cox proportional-hazards and Cochran-Mantel-Haenszel statistics were used for the analysis. Sixty-eight patients were included in this analysis: 29 and 39 patients from Studies 1 and 2, respectively, had received adjuvant anthracycline treatment. Study 1, with n=297, compared M 60 mg/m (M60) plus cyclophosphamide (C) 600 mg/m (C600) versus A 60 mg/m (A60) plus C600 as first-line treatment for MBC. Twenty-nine patients had received prior adjuvant doxorubicin, of whom, after randomization, 14 received M60+C600 and 15 received A60+C600 for the treatment of MBC. Study 2, with n=224, compared M 75 mg/m (M75) with A 75 mg/m (A75) as first-line treatment for MBC disease. Thirty-nine patients had received prior adjuvant doxorubicin, of whom, after randomization, 18 received M75 and 21 received A75 for their MBC. Hence, 32 patients received M-containing regimens and 36 received A-containing regimens for the treatment of MBC. Median age in both groups was 54 years. The groups were well balanced in terms of demographic characteristics. Overall response rates were 31% and 11% for M-treated patients and A-treated patients, respectively (Cochran-Mantel-Haenszel P=0.04, odds ratio=4.0). Median time to progression was 4.5 versus 3.4 months [log-rank P=0.66, hazard ratio (HR)=1.14], median time to treatment failure was 4.2 versus 2.1 months (log-rank P=0.01, HR=2.06) and median survival time was 16 versus 15 months (log-rank P=0.71, HR=1.12). Cardiac events occurred in 22% of M-treated patients [one congestive heart failure (CHF)] versus 39% of A-treated patients (three CHFs) (log-rank, P=0.001). Median lifetime dose at onset of cardiotoxicity was 780 mg/m for M versus 580 mg/m for A (log-rank P=0.001, HR=4.8). This retrospective analysis shows that treatment based on non-pegylated liposomal doxorubicin (Myocet) significantly reduced the risk of cardiotoxicity in patients with MBC who had received prior adjuvant doxorubicin. Furthermore, anti-tumor activity and time to treatment failure were significantly improved compared with patients who received treatment based on conventional doxorubicin for their MBC. This analysis revisits the therapeutic option of including doxorubicin in the treatment of MBC patients who have had prior adjuvant anthracycline exposure.

Several reports have suggested that breast cancer patients with elevated serum levels of interleukin-6 (IL-6) have a worse prognosis than patients with lower levels. We have studied IL-6 in breast cancer cell lines and have shown that autocrine production of IL-6 can confer multi-drug resistance in vitro by inducing multidrug resistance gene-1 transcription with subsequent overexpression of P-glycoprotein (PGP). Both IL-6 and PGP expression can be measured in malignant cells using immunohistochemical (IHC) techniques. We hypothesized that patients whose tumors expressed higher amounts of IL-6 or PGP would be less likely to respond to paclitaxel, an agent affected by the PGP pathway. If so, then IL-6 could serve as a predictive factor for paclitaxel sensitivity. Both IL-6 and PGP expression were measured in patients treated in a randomized trial that compared three doses of single agent paclitaxel (175, 210, and 250 mg/m(2) over 3 h every 3 weeks) in 469 women with metastatic breast cancer (CALGB 9342). No difference in complete and partial response was found among the three treatment arms. Tissue blocks in this trial were analyzed for IL-6 (154 patients) and PGP (149 patients) in paraffin-embedded sections from tumor samples; clinical characteristics of these patients were similar to the total sample of 469 patients. There were no significant differences among IL-6 or PGP scores whether measured as continuous or dichotomous variables, or by other scoring, and response to paclitaxel. In multivariate analysis neither IL-6 nor PGP was a significant predictor of time to progression or overall survival. IHC expression of IL-6 and PGP levels in tumor cells is not a predictive marker for response to paclitaxel in women with metastatic breast cancer.

Purpose Cancer and Leukemia Group B Protocol 9342 was initiated to determine the optimal dose of paclitaxel administered as a 3-hour infusion every 3 weeks to women with metastatic breast cancer. Patients and Methods Four hundred seventy-four women with metastatic breast cancer who had received one or no prior chemotherapy regimens were randomly assigned to one of three paclitaxel dosing regimens—175 mg/m2, 210 mg/m2, or 250 mg/m2—each administered as a 3-hour infusion every 3 weeks. Women completed self-administered quality of life and symptom assessment questionnaires at baseline and after three cycles of treatment. Results No evidence of a significant dose-response relationship was demonstrated over the dose range assessed. Response rates were 23%, 26%, and 21% for the three regimens, respectively. A marginally significant association (P = .04) was seen between dose and time to progression; however, in a multivariate analysis, the difference was even less apparent. No statistically significant difference was seen in survival. Neurotoxicity and hematologic toxicity were more severe on the higher dose arms. There was no significant difference in quality of life on the three arms. Conclusion Higher doses of paclitaxel administered as a 3-hour infusion to women with metastatic breast cancer did not improve response rate, survival, or quality of life. There was a slight improvement in time to progression with higher dose therapy, which was offset by greater toxicity. When a 3-hour infusion of paclitaxel is administered every 3 weeks, 175 mg/m2 should be considered the optimal dose.

Purpose: The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors. Experimental Design: We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis. Results: The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile. Conclusions: REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.