Jaime Shamonki

FOXC1 Is a Potential Prognostic Biomarker with Functional Significance in Basal-like Breast Cancer

Gene expression signatures for a basal-like breast cancer (BLBC) subtype have been associated with poor clinical outcomes, but a molecular basis for this disease remains unclear. Here, we report overexpression of the transcription factor FOXC1 as a consistent feature of BLBC compared with other molecular subtypes of breast cancer. Elevated FOXC1 expression predicted poor overall survival in BLBC (P = 0.0001), independently of other clinicopathologic prognostic factors including lymph node status, along with a higher incidence of brain metastasis (P = 0.02) and a shorter brain metastasis-free survival in lymph node-negative patients (P < 0.0001). Ectopic overexpression of FOXC1 in breast cancer cells increased cell proliferation, migration, and invasion, whereas shRNA-mediated FOXC1 knockdown yielded opposite effects. Our findings identify FOXC1 as a theranostic biomarker that is specific for BLBC, offering not only a potential prognostic candidate but also a potential molecular therapeutic target in this breast cancer subtype.

Microbial dysbiosis is associated with human breast cancer.

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.

Assessment of DNA methylation status in early stages of breast cancer development.

Background:Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development.Methods:To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARβ2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n=52), ductal hyperplasia (n=23), atypical ductal hyperplasia (n=31), ductal carcinoma in situ (DCIS, n=95) and AJCC stage I invasive ductal carcinoma (IDC, n=34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed.Results:Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, <0.0001 and <0.0001 for MINT17, MINT31, RARβ2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics.Conclusion:DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential.

Personalizing breast cancer staging by the inclusion of ER, PR, and HER2

IMPORTANCE Nonanatomic factors, such as histologic grade and biomarkers, can guide breast cancer management but are not included in the current TNM staging system. OBJECTIVE To use as an example the triple-negative phenotype (TNP) defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) to examine whether such inclusion improves the prognostic accuracy of TNM staging for breast cancer. DESIGN, SETTING, AND PARTICIPANTS Women diagnosed with primary invasive ductal breast cancer from January 1, 1991, through December 31, 2008, were identified from a prospective institutional database. Excluded were patients who received neoadjuvant therapy, those whose staging information was incomplete, or those whose tumor lacked ER, PR, and HER2 data. Breast cancers were categorized by TNM stage and by the presence or absence of TNP. MAIN OUTCOMES AND MEASURES Overall survival at 5 years. RESULTS Database review identified 1842 consecutive eligible patients with breast cancer. When patients were stratified by TNM stage, overall survival curves for those with TNP breast cancer matched those for patients whose non-TNP breast cancer was 1 TNM stage higher. Multivariable analysis showed that TNP status was a powerful prognostic variable, and the likelihood ratio test revealed that the prognostic accuracy of the TNM staging system that incorporated TNP was superior to the current TNM staging system (P< .001). A TNM staging system that incorporated TNP reduced early-stage compression by 15%. CONCLUSIONS AND RELEVANCE The internationally recognized and easily reproducible examination of ER, PR, and HER2 status exemplifies how nonanatomic factors can improve the prognostic accuracy of breast cancer staging.

Cross-platform comparison of independent datasets identifies an immune signature associated with improved survival in metastatic melanoma

Platform and study differences in prognostic signatures from metastatic melanoma (MM) gene expression reports often hinder consensus arrival. We performed survival/outcome-based pairwise comparisons of three independent MM gene expression profiles using the threshold-free algorithm rank-rank hypergeometric overlap analysis (RRHO). We found statistically significant overlap for genes overexpressed in favorable outcome (FO) groups, but no overlap for poor outcome (PO) groups. This “favorable outcome signature” (FOS) of 228 genes coinciding on all three overlapping gene lists showed immune function predominated in FO MM. Surprisingly, specific cell signature-enrichment analysis showed B cell-associated genes enriched in FO MM, along with T cell-associated genes. Higher levels of B and T cells (p<0.05) and their relative proximity (p<0.05) were detected in FO-to-PO tumor comparisons from an independent MM patients cohort. Finally, expression of FOS in two independent Stage III MM tumor datasets correctly predicted clinical outcome in 12/14 and 44/70 patients using a weighted gene voting classifier (area under the curve values 0.96 and 0.75, respectively). This RRHO-based, cross-study analysis emphasizes the RRHO approach power, confirms T cells relevance for prolonged MM survival, supports a favorable role for B cells in anti-melanoma immunity, and suggests B cells potential as means of intervention in melanoma treatment.

Abstract LB-267: Comparison of tumor-infiltrating lymphocytes from breast cancer to T cells from adjacent tumor tissue

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA While T cells contribute to the eradication of several cancers, including melanoma, the role of T cells present in healthy-appearing, tumor-adjacent tissue is less well described. Memory T cells contain distinct populations of effector memory and central memory, distinguished by their surface phenotype and functions. Effector T cells are thought to be terminally differentiated. We hypothesize that T cells present in healthy-appearing breast tissue contribute to anti-tumor immunity and immune surveillance. Here, we studied breast cancer patients, comparing tumor-infiltrating T cells in breast cancer to T cells from healthy-appearing, tumor-adjacent breast tissue. Since the amount of tissue available for study from excised primary tumors is limited, we utilized a previously established three-dimensional explant method to expand resident T cells from fresh breast tissue. T cell differentiation phenotypes were defined for ten breast cancer specimens and nine tumor-adjacent breast specimens. Six of the tumor/tumor-adjacent tissue specimens were paired. T cell phenotype and function were studied using multiparameter flow cytometry. Tumor-infiltrating CD4 effector T cells (TEFF CD45RO-, CD62L-) were more highly enriched compared to T cells derived from tumor-adjacent breast tissue (mean 21% vs. 6%, p=0.05). In contrast, effector memory CD4 T cells (CD45RO+, CD62L-) were enriched in the healthy adjacent lymphocytes (mean 71% vs. 54%, p<0.05) whereas central memory CD4 T cell (CD45RO+, CD62L+) frequency was equivalent (23% vs. 24%, p=0.83). Functionally, T cells (CD4 and CD8) derived from tumor tissue spontaneously released granules at a higher level than tumor-adjacent T cells as indicated by CD107a expression (1.8% vs 0.8%, p<0.001). In contrast, we detected a higher frequency of mitogen-induced CCL4-producing CD4 T cells from tumor-adjacent tissue vs. tumor (62% vs. 51%, p<0.05), as well as a higher frequency of IFN-γ-producing CD8 T cells (80% vs. 65%, p<0.05). These studies demonstrate the feasibility of using this three-dimensional explant approach to investigate the role of T cells in immune surveillance against breast cancer. Our findings indicate that T cell phenotypes differ in breast tissue; tumor-adjacent T cells are enriched in effector memory cells whereas TILs are characterized by terminally differentiated effector T cells. The data further suggest that breast TILs, through their comparatively reduced frequency of CCL4- and IFN-γ-producing cells have a diminished functional capacity to restrict tumor growth and metastasis. Citation Format: Yi Guo, Jaime Shamonki, Michelle Kinnaird, Maggie DiNome, Maureen Chung, Peter Sieling, Delphine J. Lee. Comparison of tumor-infiltrating lymphocytes from breast cancer to T cells from adjacent tumor tissue. [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 LB-267. doi:10.1158/1538-7445.AM2014-LB-267

Papillary lesions of the breast: Can larger core needle biopsy samples identify patients who may avoid surgical excision?

44 Background: Papillary lesions of the breast are frequently diagnosed on core needle biopsy (CNB). The ability to distinguish benign from atypical/malignant papillary lesions is limited by the representative nature of the biopsy method; thus follow-up excision is usually recommended. We aimed to determine if larger CNB samples can more reliably predict the true benign nature of a papillary lesion, thereby sparing certain patients a formal surgical excision. METHODS We reviewed medical records of 53 female patients diagnosed with histologically benign papillary lesions on CNB from 2000 to 2010, who subsequently underwent surgical excision. Pathology slides of the CNB were reviewed to document the benign histologic features of the papilloma, the number of cores sampled and the area of tissue biopsied (mm2). Statistical analysis was performed to identify the characteristics of the CNB that were associated with retention of benign histology on excision. RESULTS Atypical ductal hyperplasia (ADH) and carcinoma were identified in 6% (3/53) and 8% (4/53) of papillary lesions, respectively, when excised. Clinical and radiographic characteristics did not distinguish the ADH/malignant lesions from benign papillomas. The CNB needle sizes ranged from 9- to 18-gauge (median 14). The number of cores sampled ranged from 3-16 (mean 4.5). Patients with benign excisions had a significantly larger area of tissue sampled by CNB than those found to have ADH/malignant lesions on excision (mean ± SD: 95.6 ± 101.2 vs. 41.7 ± 21.9, respectively, p=0.003). By logistic regression, CNB tissue samples consisting of ≥7 cores, or measuring >96 mm2 in aggregate, had a negative predictive value for ADH/malignancy of 100% (AUC of 0.69 and 0.68, respectively). CONCLUSIONS Although no clinical or radiologic features distinguished benign from pathologically significant papillary lesions, larger sample sizes significantly improved the predictive value of benign histology on CNB. A papilloma sampled by ≥ 7 cores or > 96 mm2 showing benign histology at CNB, retained benign features upon excision. Close surveillance may be a reasonable option for patients whose benign papillomas are generously sampled at the time of CNB.

Abstract 3742: FOXC1 is a potential prognostic marker with functional significance in basal-like breast cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Although basal-like breast cancer (BLBC) is a distinct molecular subtype, it has no defining gene or protein marker that can be used as the basis for therapy. Estrogen receptor (ER) and HER2 receptor guide treatment of luminal and HER2 breast cancer subtypes, respectively, but chemotherapy is still the only systemic treatment available for BLBC. In view of its poor clinical outcomes, high proliferation rates and preferential metastasis to the brain, there is an urgent need for effective targeted therapy of BLBC. In a previous study using cDNA microarray analyses and immunohistochemistry of archival breast cancer tissue specimens, we showed consistent and exclusive overexpression of FOXC1 in BLBC versus other molecular subtypes. FOXC1 overexpression was significantly associated with poor overall survival and brain metastasis. In the present study, we show that overexpression of FOXC1 in breast cancer cells with low endogenous FOXC1 levels increased cellular proliferation, migration, and invasion, and induced epithelial-mesenchymal transition. Knockdown of FOXC1 by shRNA in breast cancer cells with high endogenous FOXC1 expression had the opposite effect. Cell signaling studies showed that FOXC1 activated the NF-κB pathway by upregulating the peptidyl-prolyl isomerase Pin1 and downregulating the ubiquitin ligase SOCS-1. Pin1 binds to p65, inhibits the p65 interaction with IκBα, and thus enhances p65 nuclear localization and protein stability. SOCS-1 directly interacts with p65 and promotes its degradation. FOXC1 knockdown reduced p65 protein levels and NF-κB activity. The importance of the NF-κB pathway for FOXC1 function was confirmed by the increased sensitivity to NF-κB inhibitors in cells that overexpressed FOXC1. Because BLBC under-expresses ER, FOXC1 was inversely associated with ER, as expected. Interestingly, FOXC1 repressed ER transcription and activity via NF-κB signaling, and FOXC1 overexpression switched MCF-7 breast cancer cell growth from estrogen-dependent to estrogen-independent. This may explain why ER is not normally detected in BLBC. These results suggest that FOXC1 may be a specific diagnostic and prognostic biomarker for BLBC and may play an important role in regulating aggressive traits associated with BLBC. It might also serve as a potential molecular therapeutic target for BLBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3742.