Y-Y Chen

P2-10-01: Extracellular Matrix Stiffness and Mammographic Density in the Human Breast.

Introduction: Mammographic density (MD) is associated with greater risk to malignancy. MD is also correlated to high collagen content in the extra cellular matrix (ECM). Data from our group and others have highlighted the importance of mechanical cues from the ECM in breast tissue homeostasis and tumor progression to invasion [1; 2; reviewed in 3]. Whether the stiffness of the ECM could also initiate breast cancer and if so how remains unknown. Because elevated collagen levels increases ECM stiffness, we hypothesize that MD increases breast cancer risk because the ECM is stiffer. Materials and Methods: We studied breast tissues obtained through prophylactic mastectomy from women with low (BIRADS 1) versus high MD (BIRADS 4). From each surgically excised breast, samples of 0.5cm x 0.5cm x 1cm dimension were removed from the retroareolar region and from 4 peripheral quadrants. Sample sections were subjected to biophysical, morphological and biochemical analysis. Biophysical analysis included the application of Atomic Force Microscopy to obtain an extensive force map of distinct anatomical regions of the ECM associated with the intra-lobular and inter-lobular ECM. Topological analysis of ECM architecture was performed using two photons and SIM-POL imaging coupled with picrosirius staining, polarized light imaging and image quantification. Biochemical and morphological analysis consisted of immunohistochemistry for markers that detect mechano-signaling in the epithelium and stromal fibroblasts, and HE71(24 Suppl):Abstract nr P2-10-01.

Abstract P3-07-49: Residual cancer burden (RCB) with veliparib/carboplatin in the I-SPY2 trial

Background: I-SPY2 is a multicenter phase 2 trial in high risk stage II/III breast cancer (BC) using adaptive randomization within biomarker subtypes to evaluate novel agents added to standard neoadjuvant chemotherapy. The first regimen to graduate based on the predicted probability of a higher pCR rate within predefined subsets was veliparib/carboplatin + paclitaxel (VC+T→AC vs T→AC) in triple negative BC (TNBC). In TNBC the residual cancer burden (RCB) is prognostic, whether as a continuous index or grouped into classes, with pCR (RCB-0) and RCB-I classes having identical survival. Therefore, we evaluated the use of RCB to further discriminate between investigational and control arms. Methods: Site pathologists reported RCB for 99% of subjects in the primary efficacy analysis based on pCR (n=114/115). We compared the distribution of RCB reported as a continuous index in each treatment-subset combination to matched concurrently randomized controls using the Wilcoxon rank sum test for RCB index, and Fisher9s Exact test for RCB classes (RCB-0/I vs RCB-II/III). The statistics are descriptive rather than inferential, and given the small sample size have no claim on generalizability. We modified the Bayesian model used to compute the estimated probability of success in a future, randomized, phase 3 trial of 300 subjects, if response were defined by either pCR or RCB-I (RCB0/I), or separately if it were defined by pCR alone. Results: VC+T→AC led to a significantly lower RCB index than T→AC in TNBC (p=0.0021), with a near-significant trend when those with pCR were excluded (p=0.06). There was no significant difference in RCB distributions in the other breast cancer subtypes treated. In TNBC, the odds ratio (OR) for achieving RCB-0/I in the VC+T→AC arm vs control was 8.2 (95% confidence interval (CI): 2.1–35), whereas the OR for achieving pCR was 4.56 (95% CI: 1.25–19.53). The simulations using response information from I-SPY2 to predict the probability of success for VC+T→AC for TNBC in a future phase 3 trial estimated this probability to be 0.99 if modeled using RCB-0/I as the response endpoint, and 0.90 if modeled using pCR as the response endpoint. Conclusions: Use of RCB index and classes provided additional insight into the effect of adding VC to T, appearing to magnify the improved treatment response that had been observed with pCR rates in TNBC. It will be important to test in randomized trials whether a decrease in the RCB index relative to controls, and/or increased rates of RCB-0/I class, are predictive of survival benefit in TNBC. Citation Format: Liu MC, Symmans WF, Yau C, Chen Y-Y, Rugo HS, Olopade OF, Datnow B, Chen B, Feldman M, Kallakury B, Hasteh F, Tickman R, Ritter J, Troxel M, Mhawech-Fauceglia P, Duan X, Berry D, Esserman L, DeMichele A. Residual cancer burden (RCB) with veliparib/carboplatin in the I-SPY2 trial. [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 P3-07-49.

Abstract P4-05-06: Host inflammation and breast cancer molecular subtypes: Updated results from a TCGA sub-analysis

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.

Abstract P4-05-15: Breast cancers with BRCA1 and BRCA2 mutations are associated with specific pathologic features and molecular profiles: Results from a TCGA sub-analysis

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 study9s 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.

Abstract P4-05-10: PIK3CA mutations are enriched in invasive lobular carcinomas and invasive mammary carcinomas with lobular features: Results from a TCGA sub-analysis

Background: Aberrant signaling via the PI3K pathway is a common alteration in breast cancer (BC), with frequent activating mutations in the PIK3CA gene helical (exon 9) and catalytic (exon 20) domains. These mutations occur across all BC subtypes with an overall incidence of 36%, with the highest frequency (∼45%) in luminal A/ER+ tumors. Lobular phenotype is common among luminal A tumors. We examined associations between lobular histology and molecular features among BC samples submitted for comprehensive molecular analyses for The Cancer Genome Atlas (TCGA). Design: Experts in breast pathology reviewed digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA. Tumors were graded, subtyped and scored for additional histopathologic features. We tested pairwise associations between lobular features and components of grade, PAM50-derived molecular subtype and mutational status for BRAC1/2, PIK3CA, TP53 and CDH1 by performing Chi-Square analysis for comparisons with a categorical variable and the Mann-Whitney test for comparisons with an ordinal variable Results: A total of 1132 images were scored from 589 unique cases in TCGA. For cases with multiple scorers (43% of cases), we summarized scores by taking the median (for ordinal variables) or the consensus diagnosis (for categorical variables). A total of 567 cases had a consensus diagnosis for lobular features, all of which had pathological information on components of histologic grade and 540 of which had data for TP53, CDH1, and PIK3CA mutations. 110/567 (19%) of cases were classified as invasive lobular or invasive mammary carcinoma with lobular features. The lobular cases had significantly less nuclear pleomorphism (p = 3.3 e -12), lower mitotic index (p = 3.4e-16), less tubule formation (p = 3.9e-8), increased association with lobular carcinoma in situ (p < 2.2 e-16), decreased stromal inflammation (p = 1.5e-7), and decreased necrosis (p = 4.4e-11) compared with cases without lobular features. Cases with lobular features were highly enriched for CDH1 mutations with 19% of cases with lobular features having CDH1 mutations, compared with only 1% of cases without lobular features (p = 2.4 e-14). The lobular features cases were more likely to have PIK3CA mutations (p = 0.01), with 33% of the lobular features cases having PIK3CA mutations, compared with 21% of the non-lobular cases. The lobular features cases were less likely to have TP53 mutations (p = 0.02), with 13% of lobular features cases having TP53 mutations as compared with 24% of the non-lobular feature cases. Lobular status was associated with PAM50 molecular subtype (Chi-square p = 0.002) with the lobular cases significantly less likely to be basal molecular subtype and more likely to be Luminal-A. Conclusions: PIK3CA mutations are enriched in invasive lobular carcinomas and invasive mammary carcinomas with lobular features. These associations point to the possibility that PIK3CA mutations as well as CDH1 alterations are important drivers of invasive lobular carcinomas. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-10.

Abstract P5-16-04: Predictive biomarkers for invasion on final pathology in patients with preoperative diagnosis of ductal carcinoma in situ of the breast by needle biopsy

Background Predictors of synchronous invasive breast cancer in patients diagnosed with only ductal carcinoma in situ (DCIS) in pre-operative needle biopsies have not been well-defined. Establishing such predictors of invasion has potential to significantly alter management by identifying those patients for whom surgery may be avoidable en lieu of conservative management. This study aims to identify clinicopathologic factors from pre-operative needle biopsies that are predictive of invasive cancer on subsequent surgical excision. Methods The study population consisted of 69 breasts from 67 patients initially diagnosed with only DCIS on needle biopsy (core needle or mammotome) at St. Luke9s International Hospital, Japan from 2006 until 2008. Parameters analyzed included presenting clinical features, DCIS nuclear grade and morphologic pattern, and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), epidermal growth factor receptor-2 (HER2), Ki-67 antigen, p16, p53 and cyclooxygenase-2 (COX2) in biopsy specimens. These immunohistochemical markers were previously identified to foretell invasive carcinoma subsequent to DCIS (Kerlikowske, JNCI 2010). Associations between clinical, pathological, and immunohistochemical findings in initial biopsy specimens and the presence of invasive cancer on subsequent excision were analyzed for significance using univariate and multivariate analysis. Results Of 69 breasts with only DCIS on initial needle biopsy, subsequent surgical excision revealed pure DCIS in 46 (66.7%), microinvasive carcinoma in 4 (5.8%), and invasive carcinoma in 19 (27.5%) cases. Sentinel node biopsy was performed in 57 (82.6%) of 69 cases, and 53 (93.0%) of these showed no evidence of lymph node metastases. All 4 cases with lymph node metastases revealed invasive carcinoma in surgical excisions. By univariate analysis, pre-operative factors significantly associated with invasion on surgical excision included detection of a lump by palpation (p Conclusion If confirmed in a larger sample, predictive clinical and biomarker parameters can help identify those patients diagnosed with only DCIS in needle biopsy who are at high risk of harboring unsampled invasive cancer on final pathology. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-16-04.

P1-03-01: ECM Stiffness and Breast Tumor Histology and Treatment Phenotype.

Currently, the extent of local surgery and radiation for breast cancer is not guided by biological or anatomical principles, and the optimal volume of treatment is not defined. Local tumor progression is strongly influenced by a dynamic interplay between the genetically-modified epithelium and the associated cellular and non-cellular stroma (Butcher, 2009; Levental, 2009). The extracellular matrix (ECM) is modified in breast tumors and data indicate that the ECM stiffens progressively as mammary tumors evolve and enhancing ECM stiffness promotes mammary tumorigenesis while inhibiting ECM stiffening reduces tumor progression. However, the degree and extent of local stromal changes, and their clinical impact in breast cancer evolution and treatment remain unclear. This has led us to predict that the desmoplastic stroma surrounding breast tumors reflects tumor phenotype and behavior and can be characterized by biophysical metrics of the ECM stroma and molecular signatures of the breast epithelium. Accordingly, we hypothesize that these features may be integrated into a stromal phenotype that can be developed as a predictive tool. To test this concept we used a rigorous biomechanical and biochemical analysis of mastectomy specimens from women with various stages and histology phenotypes of treated and non-treated breast cancer to explore the relationship between ECM stiffness, mechanotransduction and tumor behavior. The goal of the study was to characterize stromal changes as a function of tumor stage and histology phenotype as well as before and after chemotherapy by measuring biophysical and topological features of the ECM and biochemical and molecular signatures of the mammary epithelium. The objective was to determine if there is a significant association with tumor histology phenotype that could be integrated to generate a stromal phenotype. Egan sections of were acquired from breast tissue containing benign and invasive ER positive and negative carcinoma and from treated and non-treated patients. All tissues were subjected to IHC histological and mechanosignaling analysis (HE 397FAK; Lysyl Oxidase; phosphomyosin) and biomechanical assessment (Atomic Force Microscopy; Structured Illumination Polarized Imaging; Two Photon Imaging). Preliminary data were consistent with prior in vitro and in vivo studies and showed that there was a significant increase in ECM stiffness as the tissues transitioned from normal to an invasive lesion with the highest stiffness being located at the tumor edge (∼2-4 folds greater). Intriguingly, we observed that ER negative tumors were substantially stiffer than ER positive tumors (50% increase in upper 10-percentile) and that there were tracks of ECM stiffness that correlated with orientated parallel collagen fibers and ECM birefringence. Moreover, we quantified a significant decrease in ECM stiffness following treatment (40% lower) with the most striking reduction in ECM tension being noted in ER negative patient tissues who demonstrated the most robust response. This study should lead to a deeper understanding of the nature of breast cancer stroma and its role in tumor phenotype and response to therapy. Supported by: NCI SPORE P50 CA058207 to VMW, CP, & SH; U01 ES019458-02 to ZW; NCI R01 CA138818-01A1 to VMW; & U54 CA143836-01 to VMW & JL. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-01.

P5-01-04: FOXP3 Positive Regulatory T Lymphocytes and Epithelial FOXP3 Expression in Synchronous Normal, Ductal Carcinoma In Situ and Invasive Cancer of the Breast.

Background: FOXP3 expressing regulatory T cells (Tregs) have been associated with worse outcome in a number of malignancies, including breast cancer. In this study we evaluate the number of FOXP3 positive T lymphocytes as well as quantitating FOXP3 expression of epithelium in synchronous normal, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) in order to determine whether FOXP3 expression increases with cancer progression. Materials and Methods: 32 cases containing concurrent normal, DCIS and IDC lesions of the breast were stained for FOXP3 (e-Bioscience) and CD3 (DAKO) using the Envision G/2 Double stain System (DAKO). Multiplex stains were separated by imaging using the CRi9s Nuance imager. The ratio of colocalized FOXP3 and CD3 pixels (Treg lymphocytes)/CD3 pixels (all lymphocytes) in adjacent stroma was used as a measure of FOXP3 positive Tregs. Epithelial FOXP3 expression was quantitated by Image J analysis. A subset of the cases (n= 13) were also stained by standard immunohistochemistry for FOXP3. The number of FOXP3 positive lymphocytes/total lymphocytes and the epithelial intensity were scored. Results: The mean patient age was 52 years. ER, PR and HER2 positivity in the invasive component was 94%, 84% and 16% respectively. One case was triple negative. The median fraction of FOXP3 lymphocytes increased from normal to DCIS to IDC (0.005, 0.019 and 0.030 respectively). Significant differences were seen for normal vs. IDC (p= For epithelial FOXP3 staining intensity, the median increased significantly from normal to DCIS (0.130 to 0.175, p= FOXP3 lymphocyte count had a positive correlation with FOXP3 epithelial intensity in the normal and invasive components (Spearman r=0.4343, p=0.0130 for normal and Spearman r=0.5456, p=0.0012 for IDC). FOXP3 lymphocyte fraction and FOXP3 epithelial intensity did not vary significantly with size of invasive tumor, nodal status, or stage of disease. Discussion: FOXP3 is expressed both by tumor infiltrating T lymphocytes as well as tumor epithelium. The significant increase in FOXP3 T regulatory lymphocytes and FOXP3 epithelial expression from normal to invasive cancer suggests a role of FOXP3 in breast carcinogenesis and progression. The significant correlation of FOXP3-T regulatory lymphocytes and FOXP3 epithelial expression in the normal and invasive components suggests expression of one may influence the other early in the breast cancer progression pathway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-04.

Expression levels of histone deacetylases and acetylation of histone H4 and tubulin associated with breast cancer progression.

Abstract #2051 Background: Excess histone deacetylase (HDAC) activity can induce global hypoacetylation of histone and non-histone protein substrates, altering gene expression patterns and cell behavior potentially associated with malignant transformation. However, HDAC expression and protein acetylation have not previously been studied in the context of breast cancer progression.
 Materials and Methods: We performed immunohistochemistry (IHC) to assess expression levels of acetylated histone H4 (acH4), acH4K12, acTubulin, HDAC1, HDAC2 and HDAC6 in formalin fixed paraffin embedded sections with synchronous normal epithelium (N), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components from 58 archived samples at UCSF. Staining was assessed on the basis of H-score, defined as the sum of the intensity of staining (0-3) multiplied by % of stained cells for each intensity (score range 0-300). H-score differences between groups were tested for significance using either the Mann-Whitney test or the Wilcoxon signed rank test for paired samples.
 Results: From N to DCIS, there was a marked reduction in histone acetylation (acH4, acH4K12). Tubulin acetylation, a marker of non-histone protein acetylation, showed a smaller reduction. Expression of all three HDACs was also reduced, but to a lesser extent than histone acetylation. Moreover, from DCIS to IDC, the same pattern of changes in acetylation and HDAC expression was seen, although these differences were of smaller magnitude than between N and DCIS. All markers were significantly different between N and DCIS (all p 50).
 Conclusions: Overall there was a pattern of hypoacetylation associated with malignant progression, with normal epithelium exhibiting the highest acH4, acH4K12, and acTubulin levels and IDC exhibiting the lowest levels. Paradoxically, expression of HDAC 1, 2 and 6 was not increased; thus, our data support that histone hypoacetylation is not regulated by HDAC expression but rather by modulation of HDAC activity. Outcome studies and clinical trials are needed to assess the prognostic significance of these findings and to determine how effectively HDAC inhibitors could reverse the hypoacetylation observed in progression from N to DCIS and IDC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2051.