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RNAseq gene expression changes in BRCA1-I26A mutant MEFs (Oct1 CRISPR vs control).

Supp. Figure S1 Differential metabolite levels in BRCA1 I26A compared to normal MEFs. Supp. Figure S2 Control immunofluorescence microscopy images using Oct1-deficient MEFs. Supp. Figure S3 Small increase in Oct1 proteins levels in MEFs with a C-terminal BRCA1 mutation. Supp. Figure S4 Equivalent infection rates in Oct1 and control CRISPR infection rates in BRCA1-I26A MEFs. Supp. Figure S5 Quality-control and validation of RNAseq. Supp. Figure S6 CRISPR-mediated Oct1 loss in either BRCA1-I26A MEFs or MCF-7 cells minimally affects HIF-1α and c-Myc levels. Supp. Figure S7 Equivalent infection rates in Oct1 and control CRISPR infection rates in MCF-7 cells. Supp. Figure S8 MG-132 treatment reveals ubiquitylated Oct1 bands in the presence of urea. Supp. Figure S9 O2 consumption rate (OCR) was assessed in MCF-7 cells transduced with either empty vector control (EV), or viruses expressing WT or K9/403R Oct1. Supp. Figure S10 Oct1 protein stability in WT and I26A MEFs. Supp. Figure S11 Immunoprecipitating in vitro ubiquitylation assay products with control rabbit IgG antibodies results in recovery of background ubiquitylation. Supp. Figure S12 The same as in Figure 6A except with individual datapoints (Supplemental Table S4) superimposed. Supp. Figure S13 Elevated Oct1 (Pou2f1) mRNA levels correlate with poor patient outcome in gastric but not breast cancer.

RNAseq analysis of dysregulated genes from Oct1-KO and control BRCA1-I26A MEFs.

The tumor suppressor BRCA1 regulates the DNA damage response (DDR) and other processes that remain incompletely defined. Among these, BRCA1 heterodimerizes with BARD1 to ubiquitylate targets via its N-terminal E3 ligase activity. Here, it is demonstrated that BRCA1 promotes oxidative metabolism by degrading Oct1 (POU2F1), a transcription factor with proglycolytic and tumorigenic effects. BRCA1 E3 ubiquitin ligase mutation skews cells toward a glycolytic metabolic profile while elevating Oct1 protein. CRISPR-mediated Oct1 deletion reverts the glycolytic phenotype. RNA sequencing (RNAseq) confirms deregulation of metabolic genes downstream of Oct1. BRCA1 mediates Oct1 ubiquitylation and degradation, and mutation of two ubiquitylated Oct1 lysines insulates the protein against BRCA1-mediated destabilization. Oct1 deletion in MCF-7 breast cancer cells does not perturb growth in standard culture, but inhibits growth in soft agar and xenograft assays. In primary breast cancer clinical specimens, Oct1 protein levels correlate positively with tumor aggressiveness and inversely with BRCA1. These results identify BRCA1 as an Oct1 ubiquitin ligase that catalyzes Oct1 degradation to promote oxidative metabolism and restrict tumorigenicity. Mol Cancer Res; 16(3); 439–52. ©2018 AACR.

Legends for Supplementary Figures S1-S13, and Tables S1-S4

Original and normalized GC-MS metabolite readings from control and BRCA1-I26A mutant MEFs.

Oct1 and BRCA1 staining in breast cancer tissues.

Supplemental Figure Legend

Supplemental Figures 1-7 include a graphical outline of the study, graphs depicting probe performance, correlations between MHCII and TIL scores, ROC statistics, and additional Kaplan Meier curves.

Supplemental Figure: Description of sample sets for the DNAseq and RNAseq experiments.

Equations to calculate sample size in a homozygous discordant design. Supplemental Table: Sample size requirements (n in each group)

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916–25. ©2016 AACR.

Approximately 40% of patients with stage I–III triple-negative breast cancer (TNBC) recur after standard treatment, whereas the remaining 60% experience long-term disease-free survival (DFS). There are currently no clinical tests to assess the risk of recurrence in TNBC patients. We previously determined that TNBC patients with MHC class II (MHCII) pathway expression in their tumors experienced significantly longer DFS. To translate this discovery into a clinical test, we developed an MHCII Immune Activation assay, which measures expression of 36 genes using NanoString technology. Preanalytical testing confirmed that the assay is accurate and reproducible in formalin-fixed paraffin-embedded (FFPE) tumor specimens. The assay measurements were concordant with RNA-seq, MHCII protein expression, and tumor-infiltrating lymphocyte counts. In a training set of 44 primary TNBC tumors, the MHCII Immune Activation Score was significantly associated with longer DFS (HR = 0.17; P = 0.015). In an independent validation cohort of 56 primary FFPE TNBC tumors, the Immune Activation Score was significantly associated with longer DFS (HR = 0.19; P = 0.011) independent of clinical stage. An Immune Activation Score threshold for identifying patients with very low risk of relapse in the training set provided 100% specificity in the validation cohort. The assay format enables adoption as a standardized clinical prognostic test for identifying TNBC patients with a low risk of recurrence. Correlative data support future studies to determine if the assay can identify patients in whom chemotherapy can be safely deescalated and patients likely to respond to immunotherapy.Significance:The MHCII Immune Activation assay identifies TNBC patients with a low risk of recurrence, addressing a critical need for prognostic biomarker tests that enable precision medicine for TNBC patients.

Supplementary Methods, Figures 1-7 from Protein Arginine Methyltransferase 5 Accelerates Tumor Growth by Arginine Methylation of the Tumor Suppressor Programmed Cell Death 4

Background: Breast tumor subtyping has failed to provide impact in susceptibility genetics. The PAM50 assay categorizes breast tumors into: Luminal A, Luminal B, HER2-enriched and Basal-like. However, tumors are often more complex than simple categorization can describe. The identification of heritable tumor characteristics has potential to decrease heterogeneity and increase power for gene finding. Methods: We used 911 sporadic breast tumors with PAM50 expression data to derive tumor dimensions using principal components (PC). Dimensions in 238 tumors from high-risk pedigrees were compared with the sporadic tumors. Proof-of-concept gene mapping, informed by tumor dimension, was performed using Shared Genomic Segment (SGS) analysis. Results: Five dimensions (PC1-5) explained the majority of the PAM50 expression variance: three captured intrinsic subtype, two were novel (PC3, PC5). All five replicated in 745 TCGA tumors. Both novel dimensions were significantly enriched in the high-risk pedigrees (intrinsic subtypes were not). SGS gene-mapping in a pedigree identified a 0.5 Mb genome-wide significant region at 12q15. This region segregated through 32 meioses to 8 breast cancer cases with extreme PC3 tumors (P = 2.6 × 10−8). Conclusions: PC analysis of PAM50 gene expression revealed multiple independent, quantitative measures of tumor diversity. These tumor dimensions show evidence for heritability and potential as powerful traits for gene mapping. Impact: Our study suggests a new approach to describe tumor expression diversity, provides new avenues for germline studies, and proposes a new breast cancer locus. Similar reparameterization of expression patterns may inform other studies attempting to model the effects of tumor heterogeneity. Cancer Epidemiol Biomarkers Prev; 27(6); 644–52. ©2018 AACR.

Purpose: The sentinel lymph node (SLN) is biopsied during breast conservation surgery (BCS) since it is the first site of metastasis for breast cancer. If malignant, axillary lymph node dissection (ALND) may be performed to remove additional nodes in and around the affected breast, limiting the spread of the malignancy. Often, ALND results in hardship for the patient in the form of further surgeries, having nodes removed that may not be malignant, and in debilitating side effects. Developing a method to analyze SLNs intraoperatively would eliminate additional surgeries and the patient's associated suffering. This research aims to develop the use of high-frequency (HF) ultrasound (20-80 MHz) as a real-time analysis method to determine SLN status during BCS. Background: HF ultrasonic data from BCS tissue specimens were gathered from 73 patients at the Huntsman Cancer Institute, Salt Lake City, UT, immediately following surgery. In addition to 349 margin specimens, data were collected from 78 SLNs, with initial results displaying high statistical measures. A limitation of the SLN analyses, however, was that pathology results were provided only on a per sample basis, whereas the ultrasonic method often tested multiple positions on a node (max = 4, avg = 1.26). Because of the mismatch between the ultrasonic measurements (per position basis) and pathology results (per specimen basis), an ambiguity existed in how to best analyze the data. The aim of this study was to examine the scope of this ambiguity by determining the differences in statistical measures obtained by analyzing the SLN data on a per position basis versus a per specimen basis given the current data available. Method: HF ultrasound parameters extracted from the data were peak density (the number of peaks in the ultrasonic spectra) and attenuation. Both parameters correlate to tissue malignancy, and were used in a multivariate analysis to provide the final results. The statistical measures for the ultrasonic test results were calculated as follows: (1) per position basis: the pathology of each position was determined by the pathology results for the entire specimen; (2) per specimen basis: only one measurement position on each node was selected, based on the highest peak density value, to correlate to the specimen pathology. Results: The analyses revealed that the HF ultrasonic data yielded an accuracy, sensitivity, and specificity of 79.6%, 76.9%, and 80.0%, respectively, for the per position basis, and 84.6%, 87.5%, and 84.3%, respectively, for the per specimen basis. The results indicate that HF ultrasound provides intraoperative detection capabilities competitive with many SLN evaluation methods currently in use, including imprint cytology, frozen-section analysis, and qRT-PCR. Detailed analyses of the SLN pathology slides from the 73-patient study are currently being conducted to improve the correlations between the ultrasound results and histopathology. Image analysis methods are being used to quantify the extent of the malignant tissue in each SLN. This will provide pathology results on a per position basis, and thus more accurate, one-to-one correlations. These correlations would significantly further the development of HF ultrasound for real-time SLN evaluation. Citation Format: Khelfa S, Factor RE, Sanjinez DA, Zambrana AF, Doyle TE. Evaluation of sentinel lymph nodes with high-frequency ultrasound: Correlations to histopathology [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD2-05.

BACKGROUND: A decrease in Ki67 has been shown to be a predictor of response to tamoxifen. Previous trials have shown a decreased Ki67 proliferation index in breast tumors with as little as 2 weeks of preoperative tamoxifen. However, shortening the preoperative treatment time in window of opportunity studies increases patient acceptance for trial participation. The POWERPIINC trial examined the effect of 7 days of preoperative tamoxifen on breast tumor proliferation and patient symptoms. METHODS: Adult women with untreated stage I or II invasive breast cancer that was ER positive (>1%) planning on breast surgery with no contraindications to tamoxifen were enrolled. Women received 20mg of tamoxifen for 7 days up to the day of surgery and for 14 days afterwards. Proliferation was assessed by Ki67 immunohistochemistry before and after 7 days of tamoxifen. The proliferation genes from the PAM50 were also assessed by RT-PCR. Symptoms and QOL were assessed by the FACT-ES, MENQoL, and BMQ. RESULTS: 52 women were enrolled, and 44 were evaluable for Ki67. The median age was 58.5 years, and the median tumor diameter was 1.2cm. Most women (73%) were post-menopausal. Most tumors were PR positive (88%). Only 8% of tumors were HER2-positive. The Ki67 decreased by a geometric mean of 40% (95% CI 29%-63%), and 73% (95% CI 57%-85%) of women had tumors with decreased proliferation after 7 days of tamoxifen (p=0.0001 by paired t-test). No correlation was seen between the change in Ki-67 and change in FACT-ES or MENQoL scores. Women reported minimal to no bother from psychosocial or physical symptoms at baseline or on the day of surgery. Expression level of individual proliferation genes did not change after 7 days of tamoxifen. CONCLUSION: Seven days of tamoxifen showed a similar relative decrease in the Ki67 proliferation index as that reported for longer courses. Therefore, short window of opportunity trials can be informative. Citation Format: Cohen AL, Factor RE, Mooney K, Wade M, Serpico V, Salama M, Nelson E, Porretta J, Matsen C, Ostrander E, Bernard P, Boucher K, Neumayer L. PowerPIINC trial: Changes in tumor proliferation index and quality of life with 7 days of preoperative tamoxifen [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-10-05.

Background: Predicting the risk of relapse in patients with non-metastatic breast cancer is important for medical decisions and patient counseling. For patients who receive neoadjuvant chemotherapy, pathologic response and especially pathologic complete response (pCR) has been associated with risk of relapse; however this association is imperfect. Our prior work in patient-derived xenograft (PDX) models indicated that tumor engraftment in mice correlated with risk of recurrence. To understand further the prognostic utility of PDX, we designed a prospective clinical trial to determine the correlation between PDX generation with residual disease following neoadjuvant chemotherapy and relapse. Preliminary data are presented. Methods: Women with newly diagnosed non-metastatic hormone receptor low-positive (HR-low, ER and/or PR ≤ 10%) or negative breast cancer planned to receive systemic chemotherapy prior to definitive surgery were eligible. Tumor tissue at diagnosis was orthotopically implanted in NOD/SCID mice. The primary objective of the study is to correlate the ability of a tumor to engraft in mice with pathologic responses and clinical outcomes. Results: Between 12/2016 and 2/2020, 58 patients enrolled (triple negative breast cancer (TNBC), n=37; HR-low or negative/Her2+, n=11; or HR-low/Her2-, n=8; mixed, n=1). PDXs were successfully established from 16 patients. Patients uniformly received intensive preoperative chemotherapy per standard of care. Among the 12 patients whose tumors engrafted in mice (PDX(+)) and underwent surgery, the pCR rate was 41.7%. In the subgroup of patients with postoperative follow up >6 months (n=9), 3 patients had achieved a pCR, 1 of whom recurred; and 6 patients had residual disease, 3 of whom recurred (overall relapse rate 44.4%). All patients who relapsed (TNBC n=3; HR-low/Her2- n=1), experienced a very early relapse ( Among the 38 patients whose tumors did not engraft (PDX(-)) and underwent surgery, the overall pCR rate was 60.6%. In the subgroup of patients with postoperative follow up >6 months (n=30), 18 patients had achieved a pCR of whom none recurred, and 12 had residual disease of whom 1 patient (with TNBC) had locoregional recurrence 35.1 months after her definitive surgery (relapse rate 3.3%). She underwent repeat surgery followed by radiation therapy and 5.5 months after her recurrence, she remains disease-free. Achievement of pCR was not statistically different between the PDX(+) and PDX(-) group. In the entire cohort, the presence of residual disease was associated with high risk of relapse (22.2%) as compared to the achievement of pCR (4.8%, p = 0.16). However, successful tumor engraftment was associated not only with a higher risk of relapse (44.4% vs 3.3%, p = 0.0068, odds ratio 20.45), but also an early and exceptionally aggressive relapse. Conclusion. Our functional studies not only identify a patient population with a high risk of relapse with greater precision than the achievement of pCR, but also identify patients whose relapse has a particularly aggressive natural history. We established models that recapitulate this aggressive disease and in-depth genomic studies are underway. These studies will lead us to better patient stratification on the basis of risk of relapse and novel therapeutic strategies focused on patients with exceptionally aggressive breast cancers that our current diagnostic methods cannot identify. Citation Format: Christos Vaklavas, Zhengtao Chu, Rachel E Factor, Alana L Welm, Bryan E Welm, Cindy B Matsen. Towards personalized medicine - patient-derived breast tumor grafts as predictors of relapse and response to therapy. Preliminary results [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-01.

Background: The PAM50, a gene expression assay to categorize breast tumors into intrinsic subtypes, has not been previously used to examine short- and long-term prognostication in a population-based cohort where treatment patterns and time of initial follow-up vary. Methods: In a stratified case–cohort design of 1,691 women from the LACE and Pathways breast cancer survivor cohorts, we used PAM50 to categorize tumors into Luminal A (LumA), Luminal B (LumB), HER2-enriched (HER2-E), Basal-like and Normal-like, and to examine risk of early and late recurrence and mortality by Cox proportional hazards regression. Results: Compared with LumA, cumulative risk of recurrence and breast cancer death was higher for LumB, HER2-E, and Basal-like tumors at 2, 5, and 10 years. However, HR of breast cancer death varied over time [ 5 years (late)] for both Basal-like (HR, 6.23 early vs. HR, 0.63 late) and HER2-E tumors (HR, 2.97 early vs. HR, 0.73 late) but not for LumB tumors where risk was elevated consistently (HR, 2.67 early vs. HR, 1.47 late). The contrast between LumB, HER2-E, and Basal-like compared with LumA on early recurrence was stronger when subtype was defined by PAM50 than by immunohistochemistry (IHC) markers. Conclusions: The PAM50 categorized intrinsic subtypes in a manner that more accurately predicts recurrence and survival, especially for luminal tumors, compared with commonly used methods that rely on traditional IHC clinical markers. Impact: The PAM50 is robust for use in epidemiologic studies and should be considered when archived tumor tissues are available. Cancer Epidemiol Biomarkers Prev; 23(5); 725–34. ©2014 AACR.

Basal-like intrinsic subtype and poor prognosis are characteristic of breast cancers in BRCA1 families, but other genes also confer inherited predisposition to breast cancer, and subtype associations may differ. The objective of this study was to examine whether family history of breast cancer is associated with basal-like subtype and/or poor prognosis in a population-based sample of women with breast cancer. METHODS: The study population was the Life After Cancer Epidemiology (LACE) cohort, diagnosed with breast cancer in 1996-2000, and the Pathways cohort, diagnosed with breast cancer in 2006-2008. History of breast cancer among first-degree relatives was obtained by self-report at study enrollment, and women were followed for breast cancer recurrence and mortality. Primary breast cancer tissue was obtained for a case-cohort sample. Intrinsic subtypes were classified based on RT-PCR assay of 50 genes (PAM50). Sample-weighted subtype distributions were compared by family history. Hazard ratios (HR) for recurrence and mortality were estimated from Cox proportional hazards models. RESULTS: From 4,256 women in the parent cohorts who responded to the family history question, 20.5% reported a first-degree family history of breast cancer. Among women with family history, 80.6% were diagnosed at ages 50 and older, compared with 76.3% of women without family history, p = 0.007. Among cases with family history, 79.2% were non-Hispanic whites, compared with 72.3% of cases without family history, p < 0.001. Within the subcohort with PAM50 results (n = 1,319), cases with and without family history had 8.5% and 10.2% prevalence of basal-like tumors, respectively, p = 0.27. For cases diagnosed before age 50, the prevalences of basal-like subtype for women with and without family history were 18.6% and 15.9%, respectively, p = 0.62. Women with family history had similar recurrence risk to those without, HR 0.82 (95% CI 0.61, 1.11; age- and race-adjusted). Women with family history had a suggestive reduced risk of death from all causes, HR 0.75 (95% 0.55, 1.02). Family history was not significantly associated with recurrence or survival within any intrinsic subtype group. DISCUSSION: Although BRCA1 is known to predispose to basal-like subtype, we found no association between family history of breast cancer and basal-like subtype or worse prognosis in this population-based cohort. Mutation status for BRCA1 and other cancer susceptibility genes are unknown for cohort members. First-degree family history of breast cancer may represent the presence of one of the several inherited breast cancer susceptibility genes, or chance aggregation of sporadic cases. Particularly for a woman diagnosed at an older age, her mother and sisters would also have reached ages when sporadic breast cancer incidence is high. Among women diagnosed before age 50, family history was associated with a small and non-significant excess of basal-like tumors. Implications of this study are that for women from the general population diagnosed with breast cancer, a first-degree family history of breast cancer indicates neither a higher probability of a basal-like tumor nor worse prognosis. Support: NIH CA129059 and CA105274. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-01.

The use of high-frequency (HF) ultrasound (20-80 MHz) to determine tissue pathology is an inexpensive and real-time tool for differentiation of surgical margin specimens from breast conservation surgery (BCS). The development of this method as an intraoperative tool for BCS would greatly reduce the rate of re-excisions due to positive breast tissue margins. HF ultrasound was previously used in a 17-patient study to determine differences in breast tissue pathology of 34 surgical margins. Results from this pilot study demonstrated high accuracy, specificity, and sensitivity in the HF ultrasound data, and showed the immense potential of this method as a breast cancer detection tool. A large-scale validation study of this method was subsequently conducted using 349 margin specimens taken from 73 patients during BCS. Specimens ranged from 1-5 cm in length and width, 0.2-1.6 cm in thickness, and did not require any additional procedures or resection that affected the patient or surgical outcome. Each specimen was tested with HF ultrasound immediately following resection and then forwarded to pathology. Through-transmission data were collected from the specimens using two broadband, single-element transducers with a 50-MHz peak frequency (Olympus NDT, V358-SU), a HF square-wave pulser/receiver (UTEX, UT340), and a 1-GHz digital oscilloscope (Agilent, DSOX3104A). Peak density (the number of peaks and valleys in the 20-80 MHz frequency spectra range) and attenuation data were calculated from the ultrasonic spectra and waveforms, respectively. Peak density and attenuation correlate directly to tissue malignancy, and thresholds for these two parameters for differentiating benign vs. malignant tissue were determined using Fisher's Exact Test applied to the previously collected pilot study data. The peak density and attenuation results were then combined using a multivariate analysis. Since the ultrasonic measurements were collected on a per position basis (1-5 positions per margin), but the histopathology results were reported on a per specimen basis, the statistical measures for the ultrasonic results were calculated using two methods. First, the statistical measures were calculated based on a per position basis, where the pathology of each position was determined by the pathology results for the entire specimen. Second, the statistical measures were calculated based on a per specimen basis, where only one measurement position on each margin was selected (based on the highest peak density value) to correlate to the specimen pathology. The results of the first approach (per position basis) showed a sensitivity of 82.6%, a specificity of 72.3%, and an accuracy of 72.7%. The results of the second approach (per specimen basis) showed a sensitivity of 82.6%, a specificity of 61.7%, and an accuracy of 63.0%. The results of this study show potential for a rapid and inexpensive method of determining pathology of breast tissue surgical margins during BCS. This work was supported by funds from the Elsa U. Pardee Foundation, the Eppley Foundation for Research, the Western Alliance for Expanding Student Opportunities, theGovernor's Office of Economic Development of the State of Utah, the University of Utah, and Utah Valley University. Citation Format: Carter C, Neumayer LA, Factor RE, Doyle TE. Using high-frequency ultrasound (20-80 MHz) to differentiate malignant vs benign breast tissue in surgical margins [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-03-10.

Introduction: The updated 2013 ASCO/CAP HER2 guidelines proposed revised scoring with the aim of capturing all patients eligible for targeted therapy. The current guidelines also address intratumoral heterogeneity using immunohistochemistry (IHC) as a platform to identify and target heterogeneously staining areas for FISH analysis. We wanted to determine if identifying the most intense areas of staining in IHC equivocal / 2+ cases would help identify genetic heterogeneity and potentially more FISH amplified patients. Materials and Methods: Our lab offers HER2 immunohistochemistry by HercepTest™ (Dako) or 4B5 (Ventana), and dual probe FISH (Abbot Molecular or Dako IQ). We offer HER2 testing as IHC or FISH only, and also as a reflex FISH following equivocal (2+) IHC. HER2 tests performed and interpreted after 10/2013, following the implementation of the ASCO/CAP 2013 guidelines, to 5/2015 were included in this study. Both IHC and FISH were manually read following ASCO/CAP 2013 guidelines by one and two observers respectively. Equivocal (2+) cases with heterogeneous staining showing areas with ≥ 10% weak or moderate circumferential membrane staining were circled by a pathologist and reflexed to HER2 FISH. In those cases where samples were received for FISH testing only, the pathologist circled the entire area of invasive tumor on H&E slides. All circled tumor areas were evaluated before signal enumeration of the FISH probes. Results: 1805 HER2 FISH test requests received in our laboratory during the study period had interpretable FISH results. 1210 of these cases did not have prior HER2 IHC testing performed in our laboratory ("FISH only"), 595 had reflex FISH testing following equivocal (2+) IHC ("IHC2+/reflex FISH"). Amplification rates between the "IHC2+/reflex FISH" group of patients with targeted areas for FISH and the "FISH only" group where the entire invasive carcinoma was marked on H&E slide and scored were similar (16.3% and 20% respectively). 10/595 (1.7%) "IHC2+/reflex FISH", and 15/1210 (1.4%) "FISH only" groups showed genetic heterogeneity by FISH with discrete population of amplified tumor cells respectively (p=0.4) FISH Testing Utilizing Entire Tumor Area * n=1210FISH Testing Utilizing Most Intense Staining Areas (following equivocal IHC) n=595Genetic Heterogeneity15 (1.4%)10 (1.7%)Amplified242 (20.0%)97 (16.3%)*includes equivocal IHC, and FISH as first line of testing Conclusion: The updated HER2 ASCO/CAP guidelines recommend that intratumoral heterogeneity for HER2 may be easier to detect with IHC which can be used to guide FISH enumeration. In our study, identifying areas of more intense staining on IHC slides for targeting FISH analysis did not result in a difference in identification of genetic heterogeneity. Citation Format: Gulbahce HE, Factor RE, Geiersbach KB, Downs-Kelly E. Does HER2 immunohistochemistry-guided targeted FISH analysis help identify intratumoral heterogeneity in breast cancer?. [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 P1-01-13.

Background: There is increasing evidence that the presence of a host inflammatory response to breast cancer may influence outcomes. Utilizing inflammation scores on the histology of breast cancer samples submitted for comprehensive molecular analyses for The Cancer Genome Atlas (TCGA), we provide an updated look at associations between the presence of host inflammation and breast cancer molecular and pathologic features. Design: Experts in breast pathology reviewed the digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA and scored each case for the level of inflammation present (high/moderate vs mild/minimal). We tested pairwise associations between host inflammation and molecular subtypes (DNA copy-number, RNA expression, RPPA defined subtypes, miRNA subtypes, methylation subtypes) and pathological features by performing Chi-Square analyses. Multiple hypothesis testing correction was performed using the Bonferroni method. Results: 598 breast cancer cases with TCGA molecular profiling data were scored by the expert breast pathologists for morphological features (including inflammation). 195 (33%) of these were scored as high/moderate inflammation. Cases with inflammation had a significantly higher rate of TP53 mutations (p = 9.0e-8) with 64 of 118 (54.2%) p53 mutant cases with inflammation. Inflammation was also significantly associated with PAM50 molecular subtypes (p = 2.2e-11), with the greatest enrichment among basal-like (64.5% of 70 basal-like cases had inflammation) and the greatest depletion among Luminal A (18.1% of 166 Luminal A cases had inflammation). Cases with inflammation were significantly less likely to be lobular (p = 1.5e-7), had less tubule formation (p = 0.0006), increased mitoses (p Conclusions: There are strong associations between breast cancer molecular and pathological features and the host inflammatory response. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-06.

Background: Previous studies have found that particular pathologic features are more common in breast cancers arising in BRCA mutation carriers. However, the biologic and molecular bases for the morphologic associations are not clear. This study is conducted to analyze pathologic and molecular features in tumors stratified by BRCA1 or BRCA2 mutation status using the breast cancer samples that have comprehensive molecular portraits characterized by the Cancer Genome Atlas (TCGA). Methods: The digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA were reviewed by expert breast pathologists, who were unaware of the BRCA status or other molecular signatures. Each tumor was evaluated and scored for histologic type, nuclear pleomorphism, tubule formation, mitosis, stromal inflammation, and necrosis. 562 cases had both pathology and tumor exome sequencing data available and constituted the current study population. We determined the association of somatic BRCA1 and BRCA2 mutation status with pathologic features and molecular characteristics (mutation of PIK3CA and TP53, and molecular subtypes defined by PAM50 mRNA data) using the Fisher exact test for categorical variables and the Wilcoxon test for ordinal variables. Results: Of the 562 tumors, 514 had no BRCA1 or BRCA2 mutation, while 48 (8.5%) of tumors were found to harbor a BRCA1 mutation (n = 16, 3%), BRCA2 mutation (n = 30, 5%), or mutation in both (n = 2, 0.3%). BRCA1 and BRCA2 mutational status showed no significant association with lobular features, tubule formation, nuclear pleomorphism, or stromal inflammation (all p > 0.05), although there was a trend for increased nuclear pleomorphism in BRCA2 mutant cases (p = 0.07). The lack of significant association of BRCA1/2 mutational status with these features may be due to our study's relatively small number of BRCA1/2 mutant cases. Both BRCA1 and BRCA2 mutations were associated with a higher mitotic count (p = 0.03 and 0.04, respectively). BRCA2 mutation showed no association with necrosis (p = 1), while BRCA1 mutation status was associated with increased necrosis (OR = 2.7, p = 0.04). BRCA2 mutation status showed no significant association with PAM50 subtype (p = 0.37), while BRCA1 mutation status was significantly associated with PAM50 molecular subtype (p = 0.005), with the greatest enrichment among Basal-like (7/70 Basal-like with BRCA1 mutation, 10%) and depletion among Luminal-B (0/79 Luminal-B with BRCA1 mutation, 0%). Neither BRCA1 nor BRCA2 mutations were significantly association with PIK3CA mutations (p = 0.39, 0.08, respectively). BRCA2 mutation status was not associated with TP53 mutations (p = 0.65), while BRCA1 mutation status was associated with increased TP53 mutations (OR = 4.0, p = 0.005). Conclusion: Tumors with BRCA1 and BRCA2 alterations are associated with specific pathologic and molecular features. However, there is molecular and morphologic heterogeneity within these cancers. These factors need to be considered when designing algorithms for BRCA testing and targeted therapy in BRCA-related cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-15.

Background: ER expression in normal breast epithelium (NBR) is higher in women with a history of breast cancer (BC) compared to controls. In adjuvant setting, metanalysis showed effective Tamoxifen treatment was restricted to ER positive cancers. However, it is also known prophylactic oophorectomy (a form of estrogen suppression) significantly reduces the incidence of BC in BRCA1 carriers. This is in contrast with the 80% rate of ER negative tumors in BRCA1 patients. The aim of this study is to quantify ER expression in NBR away from tumor in women with BC and to correlate it with BC subtypes. Methods: 204 consecutive patients were identified for whom NBR away from tumor was available. 27 reduction mammoplasty (RM) tissues from women with no history of BC were used as controls. Tissue microarrays were constructed and slides were stained with ER and scanned using Aperio XT Scan Scope. Normal terminal duct lobular epithelium was manually circled on scanned images and annotations were recorded in separate digital layers. ER staining was quantitated in marked areas of the electronic image using an optimized scoring nuclear IHC algorithm (Aperio technologies, Inc., Vista, CA). Clinical information and tumor characteristics (menopausal status, ER, HER2 expression, grade, size, number of positive nodes, stage) were recorded based on pathology reports and tumor registry data. Results: The mean ER positivity in NBR was 16 ± 12.4 % (range: 0-5-5.7%) for all patients with BC, 20.8±13.9% for postmenopausal (n=74) and 13.7 ±10.9% for peri+ premenopausal (n=170) subgroups. ER positivity was higher in patients with BC compared to those undergoing RM with no history of BC. ER positivity in NBR did not vary by tumor size, positive lymph nodes status, tumor grade, or stage in post-menopausal, and pre + perimenopausal women. Older age at diagnosis was significantly associated (p Estrogen receptor expression in normal terminal duct lobular units of post-menopausal women with breast cancer in relation to tumor subtypes. Postmenopausal (N=74) NMean % (SD)P ValueTumor ER Status 0.05 Negative1427.4 (13.9) Positive6019.3 (13.6) Tumor HER2 Status 0.34 Negative5721.8 (14.9) Positive817.7 (5.7) Tumor Triple Negative 0.05 No5319.7 (13.6) Yes1228.5 (14.7) HER2 status was not known in some cases and was excluded from statistical calculations. Conclusion: This computer assisted image analysis study confirms ER expression in NBR increases with age and menopausal status in women with BC. We report, for the first time, a significant association between ER expression in normal breast epithelium with ER negative and triple negative cancers in post-menopausal women. Our study suggests that ER expression in normal epithelium may play a role in development of hormone receptor negative breast cancers. Citation Format: H Evin Gulbahce, Cindy Blair, Carol Sweeney, Rachel Factor, Mohamed Salama. Digital quantification of estrogen receptor expression in normal breast in post-menopausal women with breast cancer and association with tumor subtypes [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 P5-02-06.

Introduction: In 2013 ASCO/CAP published new guidelines for HER2 testing to decrease false negatives. We retrospectively classified cases submitted to a national reference laboratory for HER2 testing using the new guidelines in order to 1) see the overall effect of the 2013 vs 2007 guidelines and 2) predict shifts in interpretation of different HER2 testing methods in the future. M&M: Our laboratory offers HER2 immunohistochemistry (IHC) by HercepTest (Dako) or 4B5 (Ventana) and HER2 dual probe FISH (Abbott Molecular). HER2 IHC and/or FISH tests performed between 1/2010-8/2013, originally scored with the 2007 guidelines, were reclassified with the 2013 guidelines. For IHC, the guideline scores of 0, 1+, 2+ and 3+ were recorded along with intensity (weak, moderate, strong) and % circumferential staining. For FISH, the HER2 and CEP 17 copy number per cell, and HER2:CEP 17 ratio were recorded. Results: 2358 samples submitted for HER2 FISH were available for review. Using the 2007 guidelines, 246 (10.4%) were amplified (Amp), 62 (2.7%) equivocal (Eq), and 2050 (86.9%) were non-amplified (NA). 29/62 (46.8%) FISH Eq cases had HER2:CEP17 ratio between 2.0-2.2, 1 (1.6%) had HER2 copy number >6 per cell. Therefore 30/2358 (1.3%) of all FISH tests previously classified as Eq would be reclassified as Amp following new guidelines increasing the overall FISH Amp rate by 12.5% to 11.7%. Over the same time period, 4043 HER2 IHC tests were performed using old guidelines 1097/4043 (27.1%) of which were Eq (2+). 731/1097 (66.6%) had reflex FISH requested on 2+ IHC cases. 24/731 (3.3%) did not have an iterpretable FISH result. Using the 2007 guidelines, 49 (6.9%) of the 2+ cases were FISH Amp, 23 (3.3%) remained Eq, 635 (89.8%) were NA. Using the new guidelines, an additional 8 (1.1%) of these 707 reflex FISH would be classified as Amp (7 cases due to HER2:CEP17 ratio 2.0 to 2.2 and one case for HER2 copy number >6). Also 8 NA cases would be reclassified as Eq due to a HER2 copy number between 4 and 5.9. With the new guidelines, the overall FISH Amp rate identified from reflex 2+ IHC is 8.1%. 507/707 (71.7%) of HER2 Eq (2+) cases reflexed to FISH had 10-30% circumferential staining (8 strong; 136 moderate; 363 weak). HER2 Amp rates for these are shown in Table 1. HER2 Amplification Status of IHC Equivocal (2+) Cases by Percent Membrane StainingIHC 2+FISH Amplified (2013 Guidelines) (%)2+ Cases (All)57/707 (8.1%)2+ Cases, 10-30% Membrane Staining37/507 (7.3%)-------Strong-------1/8 (12.5%)-------Moderate-------16/136 (11.8%)-------Weak-------20/363 (5.5%) Conclusion: 2013 guidelines will identify a higher % (1.1% for FISH) of eligible patients for targeted therapy. In this series, 2+ IHC cases with reflex FISH testing also had higher Amp rates by 2013 guidelines (8.1% vs 6.9%). Evaluating these rates over time will help assess the effect of the change in the scoring criteria. Amp rates for Eq (2+) IHC cases with 10-30% strong and moderate membrane staining are similar, although only the former group is classified as positive (3+) following new guidelines and would not be retested by FISH. Cases with less than 30% membrane staining may represent a biological spectrum that is not well represented by current IHC testing guidelines, and FISH confirmation on these cases may be justified. Citation Format: H Evin Gulbahce, Rachel E Factor, Erinn Downs-Kelly, Margaret Coppin, Katherine B Geiersbach. Effect of the new 2013 ASCO / CAP guidelines on HER2 reporting [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 P5-02-07.

Background: Obesity is associated with breast cancer (BC) in postmenopausal women, but its relationship with intrinsic subtype is unclear. We examined associations of body mass index (BMI) with intrinsic subtype and expression of selected genes among women with invasive BC. Methods: A case-cohort of 1,676 BC patients was sampled from two prospective Kaiser Permanente Northern California cohorts: LACE (AJCC stage I (≥1 cm), II, IIIA diagnosed 1997-2000) and Pathways (invasive stage ≥0.5 cm diagnosed 2006-2008). The PAM50 quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assay was used to: a) classify tumors into intrinsic subtype (Luminal A, Luminal B, Basal-like, HER2-E, Normal-like) and b) assess gene expression levels for ESR1, PGR, ERBB2, and a composite of 10 proliferation genes. BMI was from self-reported weight and height within one year prior to BC diagnosis and categorized as: normal ( Results: The cohort was comprised of 52% Luminal A, 21% Luminal B, 10% Basal-like, 14% HER2-E, and 3% Normal-like subtypes. Basal-like was more prevalent in the highly obese (19%), compared with the other BMI groups (8%-10%), whereas Luminal A was less common in the highly obese (36%), compared with the other groups (49%-57%). Greater expression of proliferation genes was seen with increasing BMI (p Discussion: Among postmenopausal women with breast cancer, those who were highly obese, but not mildly obese, had increased odds of Basal-like and Luminal B subtypes and had tumors with greater expression of proliferation genes. Tumor subtypes may vary by level of obesity. Citation Format: Marilyn L. Kwan, Candyce H. Kroenke, Carol Sweeney, Philip S. Bernard, Erin Weltzien, Adrienne Castillo, Rachel E. Factor, Kaylynn Shakespear, Inga J. Stijleman, Charles P. Quesenberry, Laurel A. Habel, Lawrence H. Kushi, Bette J. Caan. Association of high obesity with PAM50 breast cancer intrinsic subtypes and gene expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3263. doi:10.1158/1538-7445.AM2014-3263

BACKGROUND The PAM50 gene expression signature for identifying intrinsic subtypes of breast cancer has been shown to add significant clinical information beyond standard molecular biomarkers (ER, PR, Her2). Previous studies with the PAM50 have focused primarily on retrospective clinical trials in which the drug regimens and enrollment criteria are relatively homogenous. We assess the prognostic significance of subtyping within the community setting using therapies appropriate at the time of diagnosis. METHODS Two prospective studies of breast cancer survivors, Life After Cancer Epidemiology (LACE) and Pathways, were subtyped by the PAM50 using quantitative RT-PCR. LACE participants were diagnosed from 1997-2000 with tumors ≥1 cm; while Pathways enrolled subjects at time of diagnosis (2006-2008) with tumors ≥0.5 cm. Both studies included AJCC stages I-III. LACE participants were enrolled on average 2 years post-diagnosis, such that early recurrences were excluded. In addition, women in Pathways were diagnosed after 2006 when Herceptin treatment was available. Formalin-fixed, paraffin-embedded tumor blocks were centrally reviewed by a pathologist and 1mm directed punches of invasive cancer were taken for RNA extraction and expression profiling. A published algorithm was used for subtyping and provided a continuous gene expression score for proliferation, ESR1/ER, PGR/PR, and ERBB2/Her2 (Bastien et al, BMC Med Genomics. 2012 Oct 4;5:44). Subtype classification was correlated with disease-free survival, estimated using Kaplan-Meier plots and log-rank testing. Multivariable delayed entry Cox regression analyses determined the prognostic significance of subtypes in the context of standard clinical indicators of survival. RESULTS The PAM50 subtype distribution for the cohort was Luminal A (52%), Luminal B (20%), HER2-E (14%), Basal-like (10%), and Normal-like (4%). Approximately 7% of Luminal tumors (A and B) were called ER negative by immunohistochemistry (IHC). Luminal A tumors were more likely to be PR positive (82%) compared to those classified as Luminal B (61%). Only 2% of Luminal B tumors were low proliferation. The HER2-E subtype contained 66% clinically Her2+ tumors and the Basal-like tumors were 90% triple negative. In univariate analyses, women with non-Luminal A subtypes were all at higher risk of recurrence. Controlling for tumor size and positive nodes, those with Basal-like subtype still had a significantly higher risk of recurrence (HR=2.34, 95% CI 1.37, 3.98). CONCLUSIONS There are proportionally more Luminal A patients within the population-based studies compared to clinical trials that have focused on higher risk patients. The PAM50 subtype remains a significant prognostic indicator of recurrence in the context of standard therapies used in community practice today. Citation Format: Philip Bernard, Erin Weltzien, Inge Stijleman, Candyce H. Kroenke, Carole Davis, Marilyn L. Kwan, Charles Quesenberry, Adrienne Castillo, Laurel Habel, Rachel Factor, Larry Kushi, Carol Sweeney, Bette Caan. PAM50 breast cancer subtyping and risk of recurrence in a population-based cohort. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3650. doi:10.1158/1538-7445.AM2013-3650

Background: Lifestyle and reproductive factors known to influence estrogen levels have been associated with breast cancer (BC) risk and prognosis. However, it is unknown whether associations vary by tumor molecular subtype at diagnosis. Methods: A case-cohort study population of 1,439 women with invasive BC was sampled from two prospective cohort studies: LACE (AJCC stage I (≥1 cm), II, IIIA diagnosed 1997-2000), and Pathways (any invasive stage ≥0.5 cm diagnosed 2006-2008). Molecular subtype (luminal A, luminal B, HER2-enriched, basal-like) was determined by RT-PCR of RNA extracted from samples from formalin-fixed, paraffin-embedded primary tumor blocks and assayed for expression of PAM50 genes. Information on hormone replacement therapy (HRT), oral contraceptive use (OC), reproductive factors (age at menarche, parity, age at first birth, breastfeeding), alcohol intake, and body mass index (BMI) were obtained from baseline participant questionnaires, and an index of lifetime hormonal exposure [low (0-1, reference), medium (2-4), high (5-6)] was created based on their expected influence on circulating estrogen. Specifically, any factor associated with more estrogen exposure was assigned a score of 1 (e.g., HRT yes, OC no, BMI≥25 kg/m2) while less exposure was assigned a score of 0 (e.g., HRT no, OC yes, BMI Results: There were 370 recurrences and 501 total deaths (269 BC-related) over a median 6.6±3.9 years. In the overall study population, a higher hormonal exposure index was associated with an increased risk of breast cancer death (p for trend=0.02). By molecular subtype, the highest tertile of the hormonal index was associated with both recurrence (HR=3.8; 95% CI: 1.0, 14.5) and BC-specific death (HR=7.3; 95% CI: 1.7, 31.7), compared with a low hormonal index, among the luminal A subtype. In contrast, no associations of the hormonal index with recurrence and BC-specific death were observed in the luminal B, HER2-enriched, or basal-like subtypes (p for homogeneity=0.01). A high hormonal index was not associated with all-cause mortality overall and by subtype. Discussion: Traditional hormonal risk factors for BC showed differing associations with prognosis and survival depending on tumor molecular subtype of the primary BC. Subtypes that are treated similar based on conventional prognostic indicators such as ER status (luminal A and luminal B) may have differing associations between risk factors and prognosis. Funded by NCI R01 CA129059. Citation Format: Marilyn L. Kwan, Carol Sweeney, Laurel A. Habel, Erin Weltzien, Adrienne Castillo, Carole Davis, Rachel E. Factor, Candyce H. Kroenke, Kaylynn Shakespear, Charles P. Quesenberry, Inge J. Stijleman, Philip S. Bernard, Lawrence H. Kushi, Bette J. Caan. Hormonal risk factors and breast cancer prognosis and survival by PAM50 molecular subtype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-29. doi:10.1158/1538-7445.AM2013-LB-29

Background: African-Americans have poorer breast cancer (BC) prognosis, and Asians and Hispanics have better prognosis, compared with Whites. The Black-White difference is unexplained and has been attributed to diagnosis with less treatable tumor subtypes in African-Americans. However, little research has examined whether race-survival differences exist by breast cancer subtype. Therefore, we examined associations between race and BC survival by PAM50 breast cancer subtypes (luminal A, luminal B, basal-like, HER2 enriched) in a prospective cohort of 1,282 breast cancer survivors from the Life After Cancer Epidemiology and Pathways cohorts. Methods: Self-reported race was obtained at study entry from mailed questionnaires. 1 mm punches were obtained from areas of representative tumor in formalin-fixed, paraffin-embedded tumor blocks. Expression of the PAM50 genes for molecular subtype was determined by RT-PCR of extracted RNA. After a median 6.3 years of follow-up (range 0.3-15.5 years), 213 deaths, with 118 from breast cancer, were reported. Delayed entry Cox proportional hazard models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) of BC mortality, controlling for time from diagnosis to enrollment, socioeconomic status, BC severity, BC subtype, treatment, and other known prognostic factors. Logistic regression was used to evaluate associations between race and subtype. Survival analyses stratified by subtype were adjusted for age, time from diagnosis to enrollment, BC severity, and BC treatment. Results: Consistent with previous research, adjusted for stage and breast cancer treatment, BC mortality was significantly higher in African-Americans (HR=2.90, 95% CI: 1.74-4.86) and lower in Latinas and Asians (HR=0.51, 95% CI: 0.26-0.99), compared with Whites. In addition, compared with Whites, African-Americans had a lower likelihood of the luminal A (OR=0.61, 95% CI: 0.38-0.98) and luminal B (OR=0.43, 95% CI: 0.23-0.82) subtypes and a greater likelihood of the less treatable basal subtype (OR=2.93, 95% CI: 1.94-4.44). Asians were less likely to be diagnosed with the basal subtype (OR=0.41, 95% CI: 0.23-0.71) and somewhat more likely to be diagnosed with the HER2-enriched subtype (OR=1.47, 95% CI: 0.97-2.21). Stratified by subtype, African-Americans had poorer prognosis among those with luminal A (HR=2.64, 95% CI: 0.93-7.49), luminal B (HR=2.20, 95% CI: 0.43-11.26), basal-like (HR=1.66, 95% CI: 0.78-3.54), and HER2 enriched (HR=3.25, 95% CI: 1.04-10.15) subtypes than Whites. Conclusion: African-Americans had worse breast cancer survival than other racial/ethnic groups and had less treatable types of breast cancer. However, breast cancer mortality was higher in African-Americans across tumor subtypes, suggesting that the Black-White survival difference may not be attributable to differential diagnosis by subtype. Citation Format: Candyce Kroenke, Marilyn Kwan, Philip Bernard, Adrienne Castillo, Carole Davis, Rachel Factor, Laurel Habel, Larry Kushi, Charles Quesenberry, Kaylynn Shakespear, Inge Stijleman, Carol Sweeney, Erin Weltzien, Bette Caan. Race and breast cancer prognosis by PAM50 subtype in the LACE and Pathways studies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 131. doi:10.1158/1538-7445.AM2013-131

BACKGROUND Gene expression profiling has been shown to improve prognostication in clinical trials, but data are lacking on expression-based molecular subtypes in context of variables of interest for epidemiology. We provide interim data on the distribution of subtypes determined by PAM50 assay in relation to standard immunohistochemical (IHC) markers and to subject characteristics in a breast cancer cohort. METHODS The study population combines women from the Life After Cancer Epidemiology (LACE) study with invasive breast cancer stages I (>=1 cm), II, or IIIA, diagnosed in 1997-2000 and the Pathways Study with invasive breast cancer at any stage (>= 0.5 cm) diagnosed in 2006-2008. IHC results for estrogen and progesterone receptors (ER, PR) and her2-neu (HER2) were obtained from records. Race, ethnicity, and breast cancer risk factors were from self-report. For a stratified sample of cases, 1 mm punches were obtained from areas of representative tumor in formalin-fixed, paraffin-embedded tissue blocks. Expression of the PAM50 genes was determined by RT-PCR of extracted RNA. Random blind duplicate tissue punches were assayed. A published centroid-based algorithm (Parker, J Clin Oncol 2009;27:1160-7) was used to generate, for each sample, five continuous-scale normalized subtype scores and a predicted subtype classification. We estimated sample-weighted subtype distributions, Pearson correlations among subtype scores, and age-adjusted odds ratios (OR). RESULTS PAM50 subtype predictions for 702 breast tumors from a community cohort were 55% Luminal (Lum) A, 19% LumB, 11% HER2-enriched, 10% Basal-like, and 5% Normal-like. Among the 72% of tumors categorized as ER+ or PR+, HER2- by IHC, PAM50 subtypes were: 72% LumA, 20% LumB, 2% HER2-enriched, 1% Basal-like, and 5% Normal-like. Continuous-scale LumA, LumB, HER2-enriched, and Normal-like scores were mutually positively correlated. Basal-like score was inversely correlated with other subtypes. 32 of 34 (94%) blind duplicate pairs were concordant for subtype. Compared to non-Hispanic whites, African-American women were less likely to have LumB subtype, OR 0.1 (95% CI 0.03 - 0.4), and somewhat more likely to have Basal-like subtype, OR 2.6 (95% CI 0.8-8.5) and Hispanics more often had non-LumA subtypes. Women with a family history of breast cancer had a lower frequency of HER2-enriched tumors, OR 0.3 (95% CI 0.2-0.7). DISCUSSION Molecular subtyping by PAM50 shifted 23% of tumors from the low-risk IHC-based ER+ or PR+, HER2- category to predicted higher-risk subtypes. Correlations among subtype scores and (infrequent) discordance of duplicate tissue punches may be accounted for by borderline phenotypes and/or heterogeneous cancers. Interim results showing PAM50 subtype differences by race and ethnicity and by breast cancer risk factors indicate that molecular subtyping is a promising tool for describing etiologic heterogeneity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1670. doi:1538-7445.AM2012-1670