Breast Cancer Panel Research Articles

Abstract B01: Profiling of serum and plasma angiogenic biomarkers in breast cancer and colon cancer using Luminex xMAP technology-based human angiogenesis multiplex immunoassay panels

Abstract Tumors require neovascularization for growth. Consequently, the angiogenesis process is key to understanding tumor development and progression, as well as diagnosis and treatment. Measuring the levels of pro- and anti-angiogenic factors in plasma or serum in cancer patients may provide prognostic information concerning disease processes and mechanisms and the elucidation of potential biomarkers for epidemiological studies or clinical trials. The Luminex xMAP technology, a bead-based multiplex immunoassay for the simultaneous measurement of multiple analytes in a small sample size, has been widely used in cancer biomarker discoveries. We report here the use of two newly developed multiplex human angiogenesis panels to profile the circulating biomarkers commonly used in the angiogenesis pathway analysis. The two panels are 1) MILLIPLEX MAP Human Angiogenesis Panel 1, a 17-plex multiplex immunoassay for the simultaneous quantification of the following 17 angiogenesis biomarkers: Angiopoietin-2, BMP-9, EGF, Endoglin, Endothelin-1, FGF-1, FGF-2, Follistatin, G-CSF, HB-EGF, HGF, IL-8, Leptin, PLGF, VEGF-A, VEGF-C, and VEGF-D, and 2) MILLIPLX MAP Human Angiogenesis Panel 2, a 20-plex multiplex immunoassay for simultaneous quantitation of the following 20 human angiogenesis biomarkers: Angiostatin, soluble E-Selectin, Osteopontin, PDGF-AB/BB, PECAM-1/sCD31, Tenascin C, Thrombospondin-2 (TSP-2), soluble AXL, soluble c-Kit, soluble EGFR, soluble Her2/ErbB2, soluble Her3/ErbB3, soluble HGFR/c-Met, soluble IL-6Rα, soluble Neuropilin-1, soluble Tie-2, soluble urokinase receptor (suPAR), soluble VEGFR1, soluble VEGFR2, and soluble VEGFR3. In this study, we report the use of these 37 angiogenic, growth factor and soluble receptor biomarkers to profile serum samples and plasma samples from a panel of breast cancer and colon cancer patients, with a similar number of normal serum and plasma samples, as a corresponding set, to generate a tumor profile of angiogenic factors using the Luminex xMAP platform. The results of this study demonstrated the utility of these human angiogenesis panels in studying cancer development and cancer biomarker discovery, as well as its potential application in translational research. Citation Format: Wen-Rong Lie, Deborah Droll, Jehangir Mistry. Profiling of serum and plasma angiogenic biomarkers in breast cancer and colon cancer using Luminex xMAP technology-based human angiogenesis multiplex immunoassay panels. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B01.

NCCN Guidelines Insights Breast Cancer, Version 1.2016.

These NCCN Guideline Insights highlight the important updates to the systemic therapy recommendations in the 2016 NCCN Guidelines for Breast Cancer. In the most recent version of these guidelines, the NCCN Breast Cancer Panel included a new section on the principles of preoperative systemic therapy. In addition, based on new evidence, the panel updated systemic therapy recommendations for women with hormone receptor-positive breast cancer in the adjuvant and metastatic disease settings and for patients with HER2-positive metastatic breast cancer. This report summarizes these recent updates and discusses the rationale behind them.

Decreased CDK10 expression correlates with lymph node metastasis and predicts poor outcome in breast cancer patients - a short report.

Cyclin-dependent kinase 10 (CDK10) has recently been identified as a tumor suppressor and, concordantly, its encoding gene has frequently been found to be inactivated in various human cancers. Here, we examined the expression status of CDK10 in a panel of primary human breast cancers and evaluated its correlation with clinicopathological parameters and clinical outcome. Western blotting was used to assess CDK10 protein levels in 20 paired breast cancer tissues and adjacent noncancerous tissues. In addition, immunohistochemistry was performed in 128 formalin-fixed, paraffin-embedded tumor tissues. Associations of CDK10 expression with various clinicopathological parameters were evaluated and Kaplan-Meier survival analyses and Cox proportional hazards models were used to estimate its effect on patient survival. We found that CDK10 protein expression was markedly decreased in cancer tissues compared to adjacent noncancerous tissues. Immunohistochemistry revealed decreased CDK10 levels in 65/128 (50.8%) of the primary breast cancer tissues tested. These decreased levels were found to be significantly associated with lymph node metastasis (P = 0.003), advanced tumor stage (P < 0.001) and unfavorable overall survival (P < 0.001). Furthermore, multivariate analyses indicated that CDK10 expression may serve as an independent prognostic factor for survival (P = 0.001). Down-regulated CDK10 expression frequently occurs in breast cancers and correlates with disease progression and poor survival. CDK10 may serve as a prognostic biomarker for breast cancer.

Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells.

BackgroundMany cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks.ResultsWe report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay, based on the substrate competitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks and an increase in permissive marks at the promoter and transcription start site of re-expressed genes, while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3. The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2.ConclusionsWe have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0118-9) contains supplementary material, which is available to authorized users.

8p deletion is strongly linked to poor prognosis in breast cancer

Deletions of chromosome 8p occur frequently in breast cancers, but analyses of its clinical relevance have been limited to small patient cohorts and provided controversial results. A tissue microarray with 2,197 breast cancers was thus analyzed by fluorescence in-situ hybridization using an 8p21 probe in combination with a centromere 8 reference probe. 8p deletions were found in 50% of carcinomas with no special type, 67% of papillary, 28% of tubular, 37% of lobular cancers and 56% of cancers with medullary features. Deletions were always heterozygous. 8p deletion was significantly linked to advanced tumor stage (P < 0.0001), high-grade (P < 0.0001), high tumor cell proliferation (Ki67 Labeling Index; P < 0.0001), and shortened overall survival (P < 0.0001). For example, 8p deletion was seen in 32% of 290 grade 1, 43% of 438 grade 2, and 65% of 427 grade 3 cancers. In addition, 8p deletions were strongly linked to amplification of MYC (P < 0.0001), HER2 (P < 0.0001), and CCND1 (p = 0.001), but inversely associated with ER receptor expression (p = 0.0001). Remarkably, 46.5% of 8p-deleted cancers harbored amplification of at least one of the analyzed genes as compared to 27.5% amplifications in 8p-non-deleted cancers (P < 0.0001). In conclusion, 8p deletion characterizes a subset of particularly aggressive breast cancers. As 8p deletions are easy to analyze, this feature appears to be highly suited for future DNA based prognostic breast cancer panels. The strong link of 8p deletion with various gene amplifications raises the possibility of a role for regulating genomic stability.

Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.

Simultaneous inhibition of deubiquitinating enzymes (DUBs) and autophagy synergistically kills breast cancer cells.

Breast cancer is one of the leading causes of cancer death among women in the United States. Patients expressing the estrogen and progesterone receptor (ER and PR) and human epidermal growth factor 2 (HER-2) tumor markers have favorable prognosis and efficacious therapeutic options. In contrast, tumors that are negative for these markers (triple-negative) have a disproportionate share of morbidity and mortality due to lack of a validated molecular target. Deubiquitinating enzymes (DUBs) are a critical component of ubiquitin-proteasome-system degradation and have been shown to be differentially expressed and activated in a number of cancers, including breast, with their aberrant activity linked to cancer prognosis and clinical outcome. We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines. Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress. Treatment with sub-optimal doses of DUB and lysosome inhibitors synergistically kills TNBC cells. This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.

In vitro activity of the mTOR inhibitor everolimus, in a large panel of breast cancer cell lines and analysis for predictors of response.

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Breast cancer risk associated with CHEK2 mutations.

Most efforts to identify individuals who have a hereditary predisposition for developing breast cancer had focused on the BRCA1 and BRCA2 genes. Less common susceptibility genes also are associated with increased risk for developing breast cancer, but until recently have often gone undetected. With the advent of next generation sequencing (NGS), many families with suspected hereditary risk are undergoing testing for multiple genes associated with increased cancer risk (Mahon, 2013a). One gene that is commonly included on NGS hereditary breast cancer panels is CHEK2. Increasingly, oncology nurses will encounter patients and families affected with mutations on this gene and need to understand the implications it has for screening and treatment.

Abstract 4076: Characterizing tumor-specific memory stem like T cells in blood and bone marrow of breast cancer patients

Abstract The identification of memory T cells with stem cell-like properties has been reported recently in both humans and mice. Such stem cell-like memory T cells (Tscm) upon adoptive transfer into a humanized mouse model had superior self-renewal, proliferation and mediated potent anti tumor immunity. However, very little is known about their specificities, physiological function and contribution to anti-tumor immune responses in cancer patients. We investigated the presence of Tscm cells in the peripheral blood (PB) and bone marrow (BM) of breast cancer patients by multicolor flow cytometry analyses with a panel of human T cell markers to distinguish naïve (Tn), central memory (Tcm), effector memory (Tem) and Tscm (CCR7+, CD45RO-, CD127+, CD45RA+, CD95+, CXCR3+, IL2Rβ+) population. Tscm cells were detected in both CD4+ and CD8+ T cells in the PB and BM of breast cancer patients. Interestingly, we observed a significant increase (3-fold) in the number of Tscm cells among the CD4+ and CD8+ T cells in breast cancer patients as compared to healthy donors. This was significantly higher among the CD8+ T cells as compared to CD4+ T cells. We further evaluated the expression of exhaustion markers (PD-1 &amp; LAG3) and found that majority of Tscm cells (&amp;gt;95%) did not express PD-1 &amp; LAG3 and comprised the least exhausted T cell compartment as compared to Tn, Tem and Tcm. To investigate the contribution of different subsets of memory T cells to tumor-specific T cells in breast cancer patients, we used a panel of breast cancer associated MHC class-I pentamers against human epidermal growth factor receptor 2 (HER2348-356 , HER2435-443), Mucin-1 (MUC112-20) and Carcinoembryonic antigen (CEA571-579). Cumulative analyses of tumor-specific pentamer+ CD8+ T cells in PB and BM showed that 20-25% of total tumor-specific T cells were of Tscm phenotype. We observed higher percentages of tumor-specific pentamer+CD8+ T cells among the total Tscm cells in BM as compared to PB, however this difference was not significant. We performed ex vivo re-stimulation assays using autologous dendritic cells loaded with several breast cancer-specific peptides with PB and BM of breast cancer patients to further assess the frequency and function of tumor-specific Tscm cells. Cumulative analyses showed that 20-23% of tumor-specific IFN-γ+ CD8+ and 26-32% IL-2+ CD4+ had a Tscm phenotype. In summary, we report the presence of Tscm cells in the PB and BM of breast cancer patients and a significant increase in their number as compared to healthy donors. Importantly, we could show that Tscm contribute to the population of functional and tumor reactive CD8+ and CD4+ T cells in PB and BM of breast cancer patients. Our further aim is to investigate the therapeutic potential of Tscm cells in adoptive T cell experiments with humanized mouse model of breast cancer. Citation Format: Mudita Pincha, Paranchai Boonsawat, Christoph Domschke, Philipp Beckhove. Characterizing tumor-specific memory stem like T cells in blood and bone marrow of breast cancer patients. [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 4076. doi:10.1158/1538-7445.AM2014-4076

Abstract 1992: Y-box protein-associated acidic protein promotes breast cancer progression and is a potential molecular target

Abstract The Y-Box protein- associated acidic protein (YBAP1), also known as Complement component 1, q subcomponent binding protein, is a multifunctional protein which is involved in in the regulation of complement activation, pre-mRNA splicing and mitochondrial function. As YBAP1 has been implicated in tumorigenesis, this study evaluates its role in breast cancer metastasis. YBAP1 mRNA expression was analysed by quantitative real time RT-PCR and YBAP1 protein by Western blot, immunocytochemical and immunofluorescence staining in a panel of breast cancer cell lines. Down-regulation of the YBAP1 gene in MDA-MB-231 breast cancer cells was performed by siRNA-mediated silencing and overexpression of the gene by transfection with the pCI-neo-YBAP1 plasmid, before analysis of the effects on cell migration and invasion. YBAP1 gene profiling was also performed in samples from the commercially available TissueScan Human Breast Cancer Panel 1 (Origene, Rockville, MD). YBAP1 immunohistochemical staining was carried out in breast cancer tissue microarrays constructed from 168 patients diagnosed with invasive ductal breast carcinoma. The YBAP1 immunostaining was performed using rabbit polyclonal anti-YBAP1 (RIKEN) at a dilution of 1:500. The percentage and intensity of YBAP1 immunopositive staining in malignant epithelial cells were recorded. YBAP1 was observed to be expressed in all the breast cancer cell lines (MCF-7, ZR-75, T47D and MDA-MB-231) examined. The protein was observed to be localized in the cytoplasm of breast cancer cells. Overexpression of YBAP1 increased cell migration and invasion in the highly malignant MDA-MB-231 breast cancer cells. Conversely silencing of the YBAP1 gene had the opposite effect, resulting in reduced cell migration and invasion. YBAP1 gene expression was also found to be up-regulated in the more advanced stages of breast cancer (grouped as Stage 0, I and IIa and compared with Stage II b, IIIa and IIIc; P &amp;lt;0.05). For the tissue microarrays, a positive correlation was observed between YBAP1 immunostaining and axillary lymph node status. YBAP1 augments the spread of breast cancer by enhancing cell migration and invasion. Hence, YBAP1 is a potential metastatic biomarker and targeting YBAP1 could be a strategy to combat breast cancer metastasis. Citation Format: Olivia J. Scully, Aye Aye Thike, George Wai-Cheong Yip, Puay Hoon Tan, Ken Matsumoto, Boon Huat Bay. Y-box protein-associated acidic protein promotes breast cancer progression and is a potential molecular target. [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 1992. doi:10.1158/1538-7445.AM2014-1992

Abstract 3403: Circadian fluctuations in “housekeeping” gene expression measured by RNA-seq

Abstract A housekeeping gene is described as a gene that is stably expressed across tissues under normal conditions. These genes are extensively used in gene expression studies, particularly those involving quantitative real-time PCR, as a means to control for overall RNA abundance. Previous studies have reported extensive circadian variation at the transcriptional level, with as many as 10% of all expressed transcripts cycling over the course of a day. To determine the influence of circadian fluctuation on housekeeping gene stability, and the corresponding implications for estimates of gene expression, we evaluated time-course RNA-seq data from primary human mammary epithelial cells. These data demonstrate substantial time-dependent variability in the expression of several commonly used housekeeping genes. Of the 12 housekeeping genes evaluated, 8 had greater than 15% coefficient of variation (CV) with b-actin and GAPDH having CVs of 33.9% and 36.9%, respectively. In order to assess the impact of housekeeping gene variability on expression estimates of target genes which may use housekeeping transcript levels for the purposes of normalization, we stratified the 12 housekeeping genes into three categories: high variability, moderate variability, and low variability, and analyzed the effect of time-dependent fluctuations in several clinically-relevant targets, including ERBB2 and the genes used in a breast cancer panel to predict likelihood of recurrence. ERBB2, a proto-oncogene, is over-expressed in 15-30% of breast tumors and is associated with increased disease recurrence and a poor prognosis. Evaluating ERBB2 expression in our RNA-Seq data, which is normalized internally using both transcript length and total reads, we find that expression of ERBB2 is relatively stable over time (average FPKM = 23.28, SD= 2.91). However, when expression is normalized to genes in the high variability housekeeping set, expression fluctuates by as much as 50% above or below the mean. In contrast, normalization to genes in the low variability set provides an accurate depiction of expression across all time points. We also evaluated all genes in a commonly used clinical assay for predicting recurrence in newly diagnosed, early stage breast cancer patients. We find that a majority of the 16 genes included in the assay vary considerably over time when normalized to the 5 housekeeping genes identified by the assay developer, which could have important implications for result interpretation. Our RNA-Seq time-course data suggests that there is circadian variability in housekeeping gene expression and that it is important to consider circadian fluctuations during the experimental design phase in order to identify appropriate genes for RNA content normalization. Citation Format: Tracy L. Peters, Elizabeth J. Ferree, Yaou Sheng, Aaron E. Hoffman. Circadian fluctuations in “housekeeping” gene expression measured by RNA-seq. [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 3403. doi:10.1158/1538-7445.AM2014-3403

Gene expression profiling reveals epithelial mesenchymal transition (EMT) genes can selectively differentiate eribulin sensitive breast cancer cells.

ObjectivesEribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. Eribulin is a mechanistically unique inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with eribulin activity compared to paclitaxel, which stabilizes microtubules, based on gene expression profiling of cell line panels of breast, endometrial, and ovarian cancer in vitro.ResultsWe determined the sets of genes that were differentially altered between eribulin and paclitaxel treatment in breast, endometrial, and ovarian cancer cell line panels. Our unsupervised clustering analyses revealed that expression profiles of gene sets altered with treatments were correlated with the in vitro antiproliferative activities of the drugs. Several tubulin isotypes had significantly lower expression in cell lines treated with eribulin compared to paclitaxel. Pathway enrichment analyses of gene sets revealed that the common pathways altered between treatments in the 3 cancer panels were related to cytoskeleton remodeling and cell cycle regulation. The epithelial-mesenchymal transition (EMT) pathway was enriched in genes with significantly altered expression between the two drugs for breast and endometrial cancers, but not for ovarian cancer. Expression of genes from the EMT pathway correlated with eribulin sensitivity in breast cancer and with paclitaxel sensitivity in endometrial cancer. Alteration of expression profiles of EMT genes between sensitive and resistant cell lines allowed us to predict drug sensitivity for breast and endometrial cancers.ConclusionGene expression analysis showed that gene sets that were altered between eribulin and paclitaxel correlated with drug in vitro antiproliferative activities in breast and endometrial cancer cell line panels. Among the panels, breast cancer provided the strongest differentiation between eribulin and paclitaxel sensitivities based on gene expression. In addition, EMT genes were predictive of eribulin sensitivity in the breast and endometrial cancer panels.

Breast cancer and aging: results of the U13 conference breast cancer panel.

Breast cancer is predominantly a disease of older women, yet there is a knowledge gap due to the persisting misalignment between the age distribution of women with breast cancer and the age distribution of participants in clinical trials. The purpose of this report is to state the U13 conference breast cancer panel's recommendations regarding therapeutic clinical trials that will fill gaps in knowledge regarding the care of older patients with breast cancer. The U13 conference was a collaboration between the Cancer and Aging Research Group and the National Institute on Aging and the National Cancer Institute (NCI). Clinical trials should be developed for frail and vulnerable patients who would not enroll on the standard phase III trials, as well as efforts need to be made to increase enrollment of fit older patients on standard phase III trials. As a result of this conference, panel members are working with the NCI and cooperative groups to address these knowledge gaps. With the aging population and increasing incidence of breast cancer with age, it is essential to study the feasibility, toxicity, and efficacy of cancer therapy in this at-risk population.

Breast Cancer Version 3.2014

Breast cancer is the most common malignancy in women in the United States and is second only to lung cancer as a cause of cancer death. The overall management of breast cancer includes the treatment of local disease with surgery, radiation therapy, or both, and the treatment of systemic disease with cytotoxic chemotherapy, endocrine therapy, biologic therapy, or combinations of these. The NCCN Guidelines specific to management of large clinical stage II and III tumors are discussed in this article. These guidelines are the work of the members of the NCCN Breast Cancer Panel. Expert medical clinical judgment is required to apply these guidelines in the context of an individual patient to provide optimal care. Although not stated at every decision point of the guidelines, patient participation in prospective clinical trials is the preferred option of treatment for all stages of breast cancer.

An Investigation of Genetic Counselors’ Testing Recommendations: Pedigree Analysis and the Use of Multiplex Breast Cancer Panel Testing

Genetic testing recommendations for hereditary breast and ovarian cancer involve pedigree analysis and consultation of testing guidelines. The testing landscape for hereditary cancer syndromes is shifting as multiplex panel tests become more widely integrated into clinical practice. The purpose of the current study was to assess how genetic counselors utilize pedigrees to make recommendations for genetic testing, to determine consistency of these recommendations with National Comprehensive Cancer Network (NCCN) Guidelines and to explore current use of multiplex panel testing. Sixty-nine genetic counselors were recruited through the National Society of Genetic Counselors Cancer Special Interest Group's Discussion Forum. Participation involved pedigree analysis and completion of an online questionnaire assessing testing recommendations and use of multiplex panel testing. Pedigree analysis and test recommendations were scored for consistency with NCCN guidelines. The average score was 12.83/15 indicating strong consistency with NCCN guidelines. Participants were more likely to consider multiplex testing when pedigrees demonstrated highly penetrant dominant inheritance but were not indicative of a particular syndrome. Participant concerns about multiplex panel testing include limited guidelines for both testing eligibility and medical management. This study demonstrates high utilization of pedigree analysis and raises new questions about its use in multiplex genetic testing.

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

Abstract B20: Prediction of lung cancer survival by genes in the PAM50 breast cancer panel.

Abstract Accumulating evidence shows that lung tumors are responsive to the steroid hormones β-estradiol and progesterone, and variables related to hormone receptor status have been reported to affect lung cancer survival. These findings suggest that there may be similarities in how hormonal pathways control tumor progression between lung cancer and breast cancer. We examined the relationship between disease-free survival (DFS) and expression of genes included in three published breast cancer survival signatures in a cohort of 104 early-stage (IA and IB) non-small cell lung cancers. Cases were selected from the University of Pittsburgh Lung Cancer SPORE Tissue Bank based on the following criteria: tumor tissue originated from a completely resected primary T1N0 or T2N0 adenocarinoma or squamous cell lung cancer; fresh-frozen lung cancer tissue was available; no neo-adjuvant therapy given; clinical variables (smoking history, age at diagnosis, sex, size of tumor, pleural invasion, site of recurrence) and outcome were known. RNA isolated from banked frozen tumor tissue was used to assess mRNA expression with the Illumina Human HT-12 v4 BeadChip; all mRNA analyzed had passed quality checks. Data were also subjected to background subtraction and quantile normalization. The Illumina BeadChip contains 16 out of 16 published Oncotype DX genes, 61 out of 66 MammaPrint genes, and 49 of 50 PAM50 genes. Supervised Principal Component Analysis was conducted to evaluate the ability to predict disease-free survival (DFS) for each gene panel as a group and also by using the top genes from the gene panels that showed differential expression in the lung cancer cohort. All results were subjected to 10-fold cross-validation. Genes from the Oncotype DX panel showed no differential expression in the cohort and had no ability to separate cases based on DFS. For the MammaPrint genes, three probes (ALDH4A1, FLT1, RECQL5) showed significant differences in expression in the cohort (p &amp;lt; 0.05), and the gene panel showed a relationship to DFS (HR 1.72, p = 0.07). Examination of the top differentially expressed genes did not improve prediction for the MammaPrint genes. For the PAM50 gene panel, three probes (CXXC5, FGFR4, FOXC1) showed significant differential expression (p &amp;lt; 0.05) and 23 probes showed differential expression at p &amp;lt; 0.1. The PAM50 gene panel as a group was able to separate lung cancer cases based on DFS (HR 1.9, p = 0.03). The PAM50 genes with differential expression at p &amp;lt; 0.1 also separated the cohort into four prognosis groups (p = 0.008 for trend). The worst prognosis group had a HR of 4.76 (p = 0.002). These genes were then analyzed in a subset of 44 cases in the Stage I cohort for whom ERβ protein expression status was known from immunohistochemistry (IHC) analysis. Comparing ERβ high (Allred score of &amp;gt;4, N= 20) and ERβ low (Allred score of 4 or less, N = 24) cases, the top PAM50 genes were more informative in ERβ high cases (HR 11.7, p = 0.0007) compared to ERβ low cases (HR 3.38, p = 0.045). For ERβ high cases, Kaplan-Meier survival curves showed that 50% of the high-risk group relapsed by 22 months, while 50% relapse was not reached in the low-risk group at 60 months. Similar analyses in the MammaPrint and Oncotype DX gene panels did not improve DFS prediction. We next validated the top PAM50 genes in a different cohort of 64 NSCLC cases, which included all stages and histologies, and had available ERβ IHC data, expression data from the Illumina Human HT-12 v4 BeadChip, and survival information. The top PAM50 genes were also able to predict DFS in this cohort: HR 2.19, p = 0.034 in all cases and HR 3.24, p = 0.042 in ERβ high cases. Pathway analysis indicated that the informative PAM50 genes describe a network that contains the estrogen and progesterone receptors, HER2, and cyclin E. The top canonical pathways identified were HER2/HER3 signaling and estrogen signaling. These results suggest that genes involved in interactions between hormonal and HER signaling may be predictive of lung cancer survival, especially in early-stage lung cancer, and provide further evidence for the importance of hormonal pathways in the biology of lung cancer. Supported by NCI SPORE in Lung Cancer P50 040990 and by the University of Pittsburgh Medical Center Genomics Initiative. Citation Format: Jill M. Siegfried, Yan Lin, Sanja Dacic, Brenda Diergaarde, Laura P. Stabile, Hui-Min Lin, Marjorie Romkes. Prediction of lung cancer survival by genes in the PAM50 breast cancer panel. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B20.

Breast cancer and aging: results of the U13 conference breast cancer panel

Breast cancer and aging: results of the U13 conference breast cancer panel

Mutations in EGFR, BRAF and RAS are rare in triple-negative and basal-like breast cancers from Caucasian women

Basal-like and triple-negative breast cancers usually display a high level of genomic instability and often carry TP53 mutations. Mutations in EGFR have been reported in about 10% triple-negative tumours from Chinese women, and there is some evidence that triple-negative and basal-like tumours might carry additional mutations against which targeted therapies are available. We, therefore, sought to determine the frequency of 238 targetable mutations in 19 oncogenes (including EGFR) in a panel of basal-like and triple-negative breast cancers from Caucasian women. We used the OncoCarta panel to screen for 238 mutations across 19 common oncogenes in 107 basal-like and triple-negative breast cancers from Caucasian women. Mutations were then verified using Sanger sequencing or primer extension by iPLEX. We identified and validated 10 mutations across five genes. Most of the mutations were observed in the PIK3CA gene (18/107, 16.8%), while mutations in KRAS, NRAS, MET and AKT1 were present in only one tumour each (1/107, 0.9%). Among the missense substitutions in PIK3CA the point mutation resulting in the amino acid change H1047R was the most frequent (8/18, 44%). All mutations were mutually exclusive, apart from one basal-like breast tumour which harboured mutations in both MET (p.T992I) and PIK3CA (p.H1047R). We did not identify any mutations in the EGFR gene. In conclusion, we found that with the exception of mutations in PIK3CA, these actionable oncogenic mutations on the Oncocarta panel are rare in basal-like and triple-negative breast cancers from Caucasian women. Custom panels, designed to detect mutations identified by exome sequencing of basal-like and triple-negative breast cancers, are, therefore, needed to identify women who might be eligible for targeted treatment.