Citations

Abstract Background: Breast tumors exhibit intratumor heterogeneity resulting in targeted therapy resistance and other challenges in disease management. To address the sources of heterogeneity, we performed a unique, in-depth analysis of clonal architecture in primary chemoradiation-naïve breast cancers. We combined DNA content-based flow cytometry and ploidy analysis with aCGH and whole exome next-generation sequencing (NGS) in multiple biopsies from the tumors and involved lymph nodes (LNs). Material and methods: We sorted nuclei from distinct populations of diploid, tetraploid, and aneuploid cells in surgical tumor samples from three chemoradiation-naïve patients. Each sorted tumor cell population was interrogated with aCGH and whole exome NGS. In Patient #1, we sorted and interrogated the genomes of tumor populations from 12 fresh frozen sections morphologically mapped from within a HER2+, ER+, PR- primary invasive ductal carcinoma (IDC) of histological grade 3 with LN involvement and 2-3 sections from 2 out of 5 LNs. In Patient #2, 11 morphologically mapped fresh frozen sections were analyzed from a grade 2, ER+, PR+, HER2+, BRCA2 mutant LN- IDC. In parallel, matching samples were processed for IHC assays. In Patient #3 biopsies from a grade 2 ER+, PR+ primary tumor were profiled. Results: The 18 primary and LN biopsies from Patient #1 fell into 6 distinct ploidy groups albeit with aberrant but homogenous aCGH profiles, characterized by SARC amplification and homozygous deletions in ROBO1 and ROBO2. In contrast a dominant ploidy was identified throughout 10 biopsies in Patient #2 but with heterogeneous aCGH profiles. Patient #3 had a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Mutation profiles obtained through exome sequencing further confirmed that ploidy was the main driver in Patient #1 whereas copy number aberrations played the key role in Patient #2 with the BRCA2 mutation (R3129X). A dendrogram based on exome variant calls of the aneuploid populations in Patient #1 strongly correlated with ploidy group and further revealed the specific clonal population characterized by a 5N ploidy and homozygous mutations in TP53 and PIK3CA as the progenitor to the ploidies present in the distant LNs. Strikingly, patients #1 and #2 were HER2 wild type across their clonal populations, contradicting IHC staining in a single core biopsy. Conclusions:Rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying clonal populations and then interrogating their genomes, provides an objective method of exploring the sources and clinical significance of tumor heterogeneity. Our approach of clonal analysis has broad implications in the study of tumor heterogeneity and the identification of drivers in breast and other solid tumors that can advance more effective treatment and clinical management of patients with this disease. Citation Format: MARIACARLA ANDREOZZI, Princy Francis, Elizabeth Lenkiewicz, Mia Champion, Brady Laughlin, Karen Anderson, Heather Cunliffe, Ann E. McCullough, Michael T. Barrett, Barbara Pockaj. Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in breast cancer. [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 LB-175. doi:10.1158/1538-7445.AM2015-LB-175

Abstract Background: Next generation sequencing (NGS) has revealed that the genetic profiles of individual tumors are highly heterogeneous due to their subclonal composition. Tumor heterogeneity has profound clinical implications affecting differences in treatment response and therapeutic resistance. Methods: An estrogen receptor positive, Her2 normal breast cancer patient underwent definitive surgical treatment with modified radical mastectomy. Multiple samples were obtained from the primary cancer and metastatic lymph nodes. Flow cytometry based methods were used to isolate and classify distinct cell subpopulations according to their ploidy, and samples were processed for whole exome sequencing (WES). Comparative Genomic Hybridization (CGH) methods were used to identify somatic unique copy-number alterations (CNAs). WES-Single Nucleotide Variations (SNVs) were used to infer subclonal phylogenetic relationships using Maximum Parsimony methods and annotated with variation-cluster-barcodes (vc-barcodes) generated using a variation Bayesian mixture model (VBMM) approach that focuses on copy-number neutral sequence variations for subclone identification (SciClone). Our comparative phylogenetic-VBMM clustering method was used to identify CNA and/or SNV drivers that are key in differentiating subclonal populations at multiple tumor sites. Results: CNA-neutral WES-SNVs from 25 subclonal populations (isolated from 4 distinct sites) clustered into 6 major groups and were used to generate vc-barcodes. A phylogenetic reconstruction of subclonal architecture annotated with vc-barcode information expedited identification of key variations underlying the differentiation of subclones at distinct tumor sites. For example, we identified an amplification event involving the Anaplastic Lymphoma Kinase (ALK) gene that was more frequent in primary biopsy subclones (12/17, ∼71%) in comparison to metastatic subclones (2/6, ∼34%). As the ALK amplification is lost, we find that most of the metastatic subclones also acquire a predicted damaging mutation in this oncogene (4/6, ∼67%). In addition, we identified a potential driver metastatic cell lineage that carries the ALK amplification in the absence of the nonsynonymous mutation. Conclusions: We developed a hybrid clustering methodology and used it to reconstruct the subclonal architecture in primary and metastatic tumors from a single breast cancer patient. Our methods have identified several CNAs and/or SNVs underlying subclonal differentiation, as well as potential biomarkers of disease progression and indicators of emerging resistance. Application of these methods to larger cohorts and types of tumors should be conducted to ascertain more precise estimates of the predictive accuracy of our processes. Citation Format: Mia D. Champion, Princy Francis, Barbara A. Pockaj, Michael T. Barrett. Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes. [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 4861. doi:10.1158/1538-7445.AM2015-4861

Abstract Introduction: Overexpression and amplification of PD-L1 has been reported in a subset of cancers, including lymphoma, and gastric cancer. We have recently described amplification of chromosome 9p24 targeting PD-L1 in a subset of triple negative breast cancers (TNBC), as well as 1-3% of colorectal carcinomas and glioblastomas. The clinical impact of these amplifications is not known. Methods: Tumor samples from 41 subjects with TNBC were evaluated for copy number aberrations. We performed DNA-based flow cytometry to sort nuclei from diploid, tetraploid, and aneuploid populations of tumor cells. The genomes of each sorted tumor cell population were then interrogated with oligonucleotide CGH arrays. Copy number intervals were identified and their genomic boundaries mapped using a step gram algorithm. Clinical data was available on 36 of 45 (80%) patients. Results: A recurring top ranked high level (log2ratio >1) amplicon that spanned 9p24.1 was detected in 12/41 (29.3%) of TNBCs. In contrast this amplicon was absent in ER+ (n = 8) and Her2+ (N = 15) breast tumors. The shortest region of overlap (SRO) spanned 777 kb and always included the PD-1 ligands PD-L1, PD-L2, and Janus kinase 2 (JAK2) loci, termed the “PDJ” amplicon. Additional genes in this amplicon include RCL1, MIR101-2, INSL6, INSL4, RLN2, RLN1, and C9orf46. The height of the PDJ amplicon included mean log2ratios of >4, consistent with genomic drivers such as Her2 and Myc. Patients with PD-L1 amplification (n = 8) were noted to have larger tumors (mean 3.9 vs. 1.9 cm, p = 0.04) and a higher incidence of lymph node metastases (75% vs. 26%, p = 0.01). Lymphocytic infiltration was only noted in 4 patients, all without PDJ amplification. Almost all patients received chemotherapy after definitive surgical therapy. The recurrence rate at 5 years was 62.5% in the PDJ amplified patients, and 25% in the unamplified patients (p = 0.02). Overall survival at 5 years was 37.5% in the PDJ amplified patients, compared with 78.2% in the unamplified patients (p = 0.02). Conclusion: We identified a focal amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 in a significant proportion of TNBCs. The high level of amplification and inverse correlation with survival suggests that the PDJ amplicon is a genetic driver event in these cancers, and warrants confirmation in larger studies. Citation Format: Karen S. Anderson, Christine L. Klassen, Amylou C. Dueck, Ann E. McCullough, Donald W. Northfelt, Mariacarla Andreozzi, Barbara A. Pockaj, Michael T. Barrett. High-level amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with worse DFS and OS in triple negative breast cancer. [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 2861. doi:10.1158/1538-7445.AM2015-2861

Abstract Background: While the biological functions and clinical importance of ERα are well understood in breast cancer, much less is known about its most closely related family member, ERβ, particularly in the setting of triple negative disease. Additionally, the ability to therapeutically target ERβ in triple negative breast cancer (TNBC) has not been fully explored. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in a cohort of 71 TNBCs. To further define the biological functions of ERβ in TNBC, novel ERβ expressing triple negative cell lines (MDA-MB-231 and Hs578T) were developed and comprehensively characterized at the level of global gene expression profiling, modulation of important biological pathways, cellular proliferation and response to targeted therapies. Results: In TNBCs from 71 patients, nuclear and cytoplasmic ERβ was detected at moderate to high levels in 24% and 32% of cases respectively. This moderate to high expression of both nuclear and cytoplasmic ERβ was associated with higher levels of Ki67. Of the 17 tumors expressing ERβ, 13 (76%) were negative for androgen receptor expression. In the triple negative MDA-MB-231-ERβ and Hs578T-ERβ cell lines, expression of ERβ led to inhibition of proliferation in response to both estrogen and multiple ERβ specific agonists. Microarray analysis and RT-PCR profiling of these cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors, while suppressing the expression of many interleukins. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells inhibited the proliferation rates and blocked TGFβ signaling in non-ERβ expressing TNBC cells, effects that were completely reversed following depletion of cystatins from the conditioned media. Conclusions: ERβ is expressed in a substantial proportion of TNBCs, and most do not express the androgen receptor. In TNBC, where targeted therapies are lacking, our data suggest that estrogen or ERβ specific agonists would be expected to elicit anti-tumor effects when ERβ is expressed. These tumor suppressive effects of ERβ appear to be mediated in part through the actions of cystatins, and their inhibition of TGFβ signaling, suggesting that this family of secreted proteins may represent novel biomarkers for monitoring ERβ specific drug responsiveness and/or patient outcomes. Citation Format: Jordan M Reese, Malayannan Subramaniam, Vera J Suman, Xianglin Wu, Vivian Negron, Anne Gingery, Kevin S Pitel, Sejal S Shah, Heather E Cunliffe, Ann E McCullough, Barbara A Pockaj, Thomas C Spelsberg, Matthew P Goetz, James N Ingle, John R Hawse. Therapeutic targeting of ERβ 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 P6-03-06.

Abstract Background: The development of novel chemotherapeutic agents has significantly improved the prognosis and survival of patients with breast cancer. ER positive or luminal tumors represent around two thirds of all breast cancers and these cancers are comprised of different histologies including differing gene expression and mutational profiles. This study examined biomarkers involved in immune evasion including PD-L1 and its association with other biological pathways as potential treatment options for luminal breast cancer patients. Methods: We analyzed 1311 breast samples using a multiplatform approach including whole genome mRNA expression (HumanHT-12 v4 BeadChip Illumina Inc., San Diego, CA), protein expression (immunohistochemistry), gene copy number changes (in situ hybridization) and gene sequencing and an additional 304 breast samples were tested for PD-1 and PD-L1 by IHC. The mRNA expression data was based on whole tumor and represents cell type heterogeneity. Heat map analysis was done to look at differential gene expression between the PD-L1 high vs low luminal breast cancers. Results: Based on expression of ER, PR and HER2 by IHC, we subdivided the data into sub- cohorts. Elevated mRNA expression of immune markers including PD-L1, CTLA4, B7H-3, and IDO1 was noted in the ER+HER2- luminal population including ER+ PR- and ER+PR+ cohort. Positive correlation was found between PD-L1 and other immune regulators including CTLA-4, B7-H3 and IDO1 (Spearman correlations of 0.49, 0.36 and 0.46). Protein expression of PD-L1 was found to be specific to the HER2 negative cohort with no expression in the HER2 positive cohort regardless of ER status. Within the ER+HER2- cohort, PD-L1 expression was 5% {ER+PR+ was 5% (4/81) and the ER+PR- was 6% (2/34)}. In contrast, PD-L1 expression was higher in the triple negative cohort, 17% (13/75). The expression of PD-1 on the other hand was present throughout the different cohorts. PD-1 expression ranged from 43% in the ER+HER2- cohort {41% (14/34) in ER+PR-HER2-; 43% (35/81) in ER+PR+HER2-} to 33% in the ER+HER2+ cohort {44% (4/9) in ER+PR-Her2+; and 17% (1/6) in ER+PR+HER2+}. In the ER-HER2+ cohort PD-L1 expression was 75%{6/8 in ER-PR-HER2+ and 0/0 in ER-PR+HER+} and 63%(47/75) in the triple negative cohort. Pathway analysis of the PD-L1 negative vs positive luminal population identified 127 genes involved in various cancer pathways including EGFR and VEGF signaling network (P=7.47e-09). Conclusions: The expression of immune regulatory targets in the breast cancer population suggests that immune- targeted therapies with anti PD-1/PD-L1, CTLA4, B7-H3 and IDO1 may be effective especially in the luminal cohort. Our study further shows that ER+HER2- breast cancer patients may be better candidates for immunotherapy with checkpoint inhibitors as compared to ER+HER2+ due to lack of PD-L1 expression in the HER2 positive cohort. Further validation of our findings is ongoing. Citation Format: Gargi D Basu, Anatole Ghazalpour, Randal Vader, Sandeep Reddy, Karen Anderson, Ann McCullough, Barbara Pockaj. Expression of novel immunotherapeutic targets in luminal breast cancer patients [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 P5-04-08.

Abstract Background: Breast tumors exhibit intratumor heterogeneity resulting in targeted therapy resistance and other challenges in disease management. To address the sources of heterogeneity, we performed a unique, in-depth analysis of clonal architecture in primary chemoradiation-naïve breast cancers. We combined DNA content-based flow cytometry and ploidy analysis with aCGH and next-generation sequencing (NGS) in multiple biopsies from the tumors and involved lymph nodes (LNs). Material and methods: We used DNA content-based flow sorting to isolate nuclei from distinct populations of diploid and aneuploid tumor cells in surgical tumor samples from two chemoradiation-naïve patients. Each sorted tumor cell population was interrogated with aCGH and exome NGS. In Patient #1, we used 12 fresh frozen sections morphologically mapped from within a HER2+, ER+, PR- primary invasive ductal carcinoma (IDC) of histological grade 3 with LN involvement and 2-3 sections from 2 out of 5 LNs. In Patient #2, 11 morphologically mapped fresh frozen sections were analyzed from a grade 2, ER+, PR+, HER2+, BRCA2 mutant LN- IDC. In parallel, matching samples were processed for IHC assays. Results: We identified multiple co-existing aneuploid populations within the biopsies. The 17 primary and LN biopsies from Patient #1 fell into 6 distinct ploidy groups albeit with aberrant but homogenous aCGH profiles, characterized by SARC amplification and homozygous deletions in ROBO1 and ROBO2. In contrast a dominant ploidy was identified throughout Patient #2 but with heterogeneous aCGH profiles. Mutation profiles obtained through exome sequencing further confirmed that ploidy was the main driver in Patient #1 whereas copy number aberrations played the key role in Patient #2 with the BRCA2 mutation (R3129X). A dendrogram based on exome variant calls of the aneuploid populations in Patient #1 strongly correlated with ploidy group and further revealed the specific clonal population characterized by a 5N ploidy and homozygous mutations in TP53 and PIK3CA as the progenitor to the ploidies present in the distant LNs. Strikngly, both patients had no HER2 amplification or mutation across their clonal populations, contradicting the initial IHC staining in a single core biopsy. Conclusions Rather than inferring the presence of distinct tumor cell populations, our novel flow-sorting based approach of first identifying the clonal populations and then interrogating their genomes, provides an objective method of exploring the sources and clinical significance of tumor heterogeneity. Our approach of clonal analysis has broad implications in the study of tumor heterogeneity and the identification of drivers in breast and other solid tumors that can advance more effective treatment and clinical management of patients with this disease. Citation Format: Princy Francis, Lucretia M Alvarez, Elizabeth Lenkiewicz, Mia Champion, Lisa Evers, Karen L Anderson, Ann E McCullough, Michael T Barrett, Barbara Pockaj. Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in 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 P2-03-07.

Abstract Background: Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity that make the management and treatment of these diseases rather difficult. In order to study clinically relevant tumor heterogeneity in detail we performed an in-depth analysis of the clonal architecture of primary chemoradiation-naive breast cancers and their lymph node metastases. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes and transcriptomes of multiple biopsies from the tumors and involved lymph nodes. Thus rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying and defining the clonal populations and then interrogating their genomes provides a highly objective method of exploring the clinical significance of tumor heterogeneity in solid tumors. Material and methods: We used DAPI-based DNA content measurements and flow sorting to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples and distant lymph nodes from chemoradiation naive patients. In parallel, matching samples were processed for pathological, transcriptome, and IHC based assays. Each distinct sorted tumor cell population was then interrogated with aCGH and next generation sequencing. For each patient, the detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present in the primary tumor and lymph. In one detailed case, we used 12 fresh frozen sections morphologically mapped from within a chemo-naïve primary invasive ductal carcinoma of histological grade 3 with lymph node involvement, 2-3 fresh frozen sections from 2 out of 5 lymph nodes with metastatic carcinoma, formalin-fixed paraffin-embedded (FFPE) tissue sections from 2 more of the involved lymph nodes and 5 morphologically mapped FFPE samples of normal tissue adjacent to the tumor. Results: We identified multiple coexisting aneuploid populations within biopsies from individual patients. Our clonal analysis determined the genomic aberrations arising in lineages that spread throughout the primary tumor and later bifurcated to distinct lymph nodes, revealing distinct therapeutic targets in the trunk and branches of the tumor. We identified driver events including high-level focal amplifications (e.g. SARC) and homozygous deletions (e.g. ROBO1 and ROBO2), that converge on distinct cellular signaling pathways. Conclusion: The approach highlighted here has broad implications in the study of tumor heterogeneity in breast and other solid tumors by providing a unique ultra-high resolution view of tumor heterogeneity that can advance more effective treatment and clinical management of patients with this disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-7.

Abstract Background: Estrogen receptor beta (ERβ), unlike ERα, classically functions as a tumor suppressor in vitro. However, ERβ's biological functions in vivo and predictive/prognostic value in breast cancer are controversial. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in the following 3 cohorts: 1) a cohort with all breast cancer subtypes (n = 182), 2) a prospective NCCTG adjuvant tamoxifen trial for postmenopausal women with ERα positive breast cancer with long-term follow-up (n = 198) and 3) a cohort of 80 triple negative breast cancers (TNBCs). To elucidate the biological functions of ERβ in breast cancer, novel ERβ expressing MCF7 and MDA-MB-231 cell lines were developed and characterized using multiple techniques and were examined for responsiveness to various ERβ targeted therapies. Results: About one-third of all breast tumors, regardless of sub-type, were shown to express nuclear ERβ and this expression was independent of ERα or HER2. In the NCCTG 89-30-52 cohort, breast cancer recurrence rates were significantly associated with ERβ protein expression with 10 year recurrence rates of 25%, 15% and 4% for zero, low or high levels of ERβ expression respectively. Interestingly, in TNBCs, nuclear ERβ was expressed at intermediate or high levels in 24% of tumors. In the triple negative MDA-MB-231 cell line, expression of ERβ led to inhibition of proliferation and induction of apoptosis in response to estrogen and multiple ERβ specific agonists. Conversely, these same treatments induced proliferation of ERβ-expressing MCF7 cells which endogenously express ERα. However, ERβ expression sensitized MCF7 cells to the anti-proliferative effects of anti-estrogens. Microarray analysis and RT-PCR profiling of MDA-MB-231-ERβ cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors which function as tumor suppressors, and potently inhibited canonical TGFβ signaling. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells suppressed the proliferation rates and inhibited TGFβ signaling in other TNBC cell lines, effects that were completely reversed following the depletion of cystatins from the conditioned media. Conclusions: These data demonstrate that ERβ is expressed in a substantial proportion of breast cancers and may have value as a predictive and/or prognostic biomarker. Therapeutic targeting of ERβ may have clinical benefit in multiple breast cancer sub-types; however, the specific drug of choice may vary based on ERα status. Specifically, we have demonstrated that ERβ expression in ERα+ MCF7 cells sensitizes them to the effectiveness of anti-estrogens, an observation that was confirmed in women enrolled in a prospective adjuvant tamoxifen trial. In TNBCs, where targeted therapies are lacking, our data suggest that targeting ERβ with either estrogen or ERβ specific agonists will elicit anti-tumor effects through the induction of cystatins and inhibition of TGFβ signaling resulting in tumor regression and improved patient outcomes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-03.

Abstract We report that administration of celecoxib, a specific cyclooxygenase-2 (COX-2) inhibitor, in combination with a dendritic cell-based cancer vaccine significantly augments vaccine efficacy in reducing primary tumor burden, preventing metastasis, and increasing survival. This combination treatment was tested in MMTV-PyV MT mice that develop spontaneous mammary gland tumors with metastasis to the lungs and bone marrow. Improved vaccine potency was associated with an increase in tumor-specific CTLs. Enhanced CTL activity was attributed to a significant decrease in levels of tumor-associated IDO, a negative regulator of T cell activity. We present data suggesting that inhibiting COX-2 activity in vivo regulates IDO expression within the tumor microenvironment; this is further corroborated in the MDA-MB-231 human breast cancer cell line. Thus, a novel mechanism of COX-2-induced immunosuppression via regulation of IDO has emerged that may have implications in designing future cancer vaccines.