Breast Cancer Panel Research Articles

The molecular detection of germline mutations in BRCA1 and BRCA2 genes in a small group of patients from Romania

Objective: The purpose of this study was to identify and classify the spectrum of small polymorphisms found in BRCA1 and BRCA2 genes in breast cancer risk-assessed patients. Methods and results: The detection of germline mutations was performed by targeted amplification of 47 samples, followed by NGS and mutation data analysis. We identified 62 short polymorphisms that were classified according to their clinical significance reported in ClinVar database: three pathogenic, four with conflicting interpretations of pathogenicity, five likely benign, 46 benign, two without clinical evaluation and two not previously reported. Conclusions: Among the detected polymorphisms a few are shared by most patients and might be prevalent in the Romanian population, a hypothesis that requires further investigation. The detection of only three pathogenic mutations suggests the need to extend the panel of breast cancer predisposing genes. It is conceivable that a mutational screening based primarily on risk factor evaluation would detect a lower number of pathogenic mutations than in confirmed breast cancer cases. Our results contribute to the setup of a BRCA1 and BRCA2 mutations database for the Romanian population.

Targeted next-generation sequencing assays using triplet samples of normal breast tissue, primary breast cancer, and recurrent/metastatic lesions

BackgroundNext-generation sequencing (NGS) has shown that recurrent/metastatic breast cancer lesions may have additional genetic changes compared with the primary tumor. These additional changes may be related to tumor progression and/or drug resistance. However, breast cancer-targeted NGS is not still widely used in clinical practice to compare the genomic profiles of primary breast cancer and recurrent/metastatic lesions.MethodsTriplet samples of genomic DNA were extracted from each patient’s normal breast tissue, primary breast cancer, and recurrent/metastatic lesion(s). A DNA library was constructed using the QIAseq Human Breast Cancer Panel (93 genes, Qiagen) and then sequenced using MiSeq (Illumina). The Qiagen web portal was utilized for data analysis.ResultsSuccessful results for three or four samples (normal breast tissue, primary tumor, and at least one metastatic/recurrent lesion) were obtained for 11 of 35 breast cancer patients with recurrence/metastases (36 samples). We detected shared somatic mutations in all but one patient, who had a germline mutation in TP53. Additional mutations that were detected in recurrent/metastatic lesions compared with primary tumor were in genes including TP53 (three patients) and one case each of ATR, BLM, CBFB, EP300, ERBB2, MUC16, PBRM1, and PIK3CA. Actionable mutations and/or copy number variations (CNVs) were detected in 73% (8/11) of recurrent/metastatic breast cancer lesions.ConclusionsThe QIAseq Human Breast Cancer Panel assay showed that recurrent/metastatic breast cancers sometimes acquired additional mutations and CNV. Such additional genomic changes could provide therapeutic target.

Screening of cytochrome 4Z1 expression in human non-neoplastic, pre-neoplastic and neoplastic tissues.

Background:Cytochromes P450 (CYPs) constitute an enzyme family involved in the oxidative metabolism of a wide variety of endogenous and exogenous compounds, including anti-cancer drugs and carcinogens. Unlike other human CYPs, CYP4Z1 is highly expressed in human breast carcinoma and is associated with poor prognosis. As a result, CYP4Z1 was hypothesised to be a potential biomarker or drug target for the discovery and development of promising anti-cancer therapies.Materials and methods:CYP4Z1 expression was immunohistochemically studied in a set of 100 different human tissues, including normal, benign, malignant and metastatic tissues, which originated from 27 anatomical sites. As a tumour model for CYP4Z1 expression, a panel of different breast cancers was evaluated for CYP4Z1 expression and its relation to histopathological features and prognostic immunohistochemical markers.Results:The immunohistochemical results revealed that CYP4Z1 was expressed in only one (4.3%) of the normal tissues from the mammary glands, while the expression of the enzyme was positive in 1 (11%), 12 (19%) and 2 (40%) of the benign, malignant and metastatic tissues, respectively. Interestingly, several tumour entities showed prominent expressions of CYP4Z1, including carcinomas of adrenal cortex, squamous cells of oesophagus, lung and cervix, as well as seminoma, astrocytoma, melanoma and lastly endometrial adenocarcinoma. In breast cancers, CYP4Z1 was expressed in 82% of the cases. Its expression was significantly associated with the pathology of tumour, histological grade and status of lymph node metastasis. Importantly, it was also significantly associated with the expressions of Her2, P53 and Ki-67.Conclusion:These findings greatly support future plans for the use of CYP4Z1 as a biomarker or target for anti-cancer drugs. However, large-scale validation studies are needed to better delineate the potential use of CYP4Z1 for therapeutic purposes.

Abstract 1360: Understanding genetic variation in cancer using targeted nanopore long read sequencing

Abstract Structural variations (SV), a hallmark of genomic instability in cancer can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types. They are pervasive in nature however, next-generation sequencing (NGS) is mostly blind to SVs. They lack sensitivity and exhibit very high false positive rates (up to 89%) in SV detection. Long-read, single-molecule sequencing platforms like Pacific Biosciences and Oxford Nanopore Technologies (ONT) can address larger variations as they typically generate read lengths of tens of thousands of bases and have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage offered by whole genome long read sequencing makes it infeasible to conduct large-scale genomic studies. Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population of cells. It will facilitate large population SV analysis, which will help define the landscape of such variants in the population and help identify regions of therapeutic or diagnostic interest. However, a lack of efficient long-read compatible targeting techniques makes it difficult to study specific regions of interest on existing long-read platforms. To address this, we are evaluating CRISPR/Cas-based systems of targeted long-read sequencing to enrich for specific regions of the cancer genome (panel of 12 genes, including BRCA1 and BRCA2) in two breast cell lines – MCF 10A and SK-BR-3. Using the ONT Cas9-mediated PCR-free enrichment system we targeted 12 genes of interest in two breast cell lines – MCF 10A and SK-BR-3. Initially, we used guides that targeted a 200kb region around the BRCA1 gene in SK-BR-3 and generated a 198kb read spanning the entire BRCA1 region. However, the number of reads on target were not sufficient to accurately call SVs. To address the issue of low on-target reads, we included an Affinity-based Cas-9-Mediated Enrichment (ACME) step, that uses a HisTag-based isolation and pulldown of background/non-target reads. By targeting all 11 genes together in a single reaction, our ACME worked extremely well, since each reaction now had over 10 times as many guides as opposed to targeting a single gene per reaction. This gave us more cut sites and Cas9-bound non-targets, increasing the enrichment efficiency of the HisTag pulldown, giving us a 75-fold enrichment of the BRCA2 region and close to 100x coverage of the entire 95kb target. We observed an increase in enrichment and coverage of the other genes on the panel as well, with enrichment as high as 4000-fold for some genes. Our goal is to develop a long-read breast cancer panel to facilitate large population SV analysis, which will help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest. Citation Format: Shruti V. Iyer, Sara Goodwin, Melissa Kramer, W. Richard McCombie. Understanding genetic variation in cancer using targeted nanopore long read sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1360.

PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer.

Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.

Abstract P4-09-07: UltraSEEK breast cancer panel for low frequency mutation detection in breast cancer brain metastasis

Abstract Background: Breast cancer brain metastases (BCBM) are a growing clinical problem and a significant cause of morbidity and mortality. The genomic landscape of BCBM can be divergent from that of the primary breast cancer (BC) and extracranial metastasis. Identifying key and targetable genomic aberrations within BCBM will be key in developing novel therapeutic strategies to treat this area of unmet and to need improve patient outcomes. Identifying rare and low-level mutations due to tumour heterogeneity in cancer samples and/or poor quality DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues requires tools that offer both low frequency detection and efficient use of starting material. This study aims to assess paired primary BC and brain metastasis (BM) for aberrations within known BC drivers: TP53, PIK3CA, ERBB2, ESR1 and AKT utilising the MassARRAY® UltraSEEK™ technology (Agena Bioscience, San Diego, USA), that provides a targeted, multiplexed method for detecting rare events and with sensitivity as low as 0.1%, developed to support DNA isolated from FFPE tissues, plasma and cerebrospinal fluid. Methods: DNA was isolated (Qiagen GeneRead FFPE kit) from of 32 BC and their matched BM cases and screened using the custom UltraSEEK™ BC Panel (Agena Bioscience, San Diego, USA). The Panel tests 44 mutations across 5 oncogenes in 8 multiplex assays; AKT1 (E17K, L52R), ERBB2 (G309E, G309A, S310F, L755R, L755S, L755_T759del, D769H, D769Y, V777L, V777L, L869R), ESR1 (A283V, K303R, E380Q, V392I, S436P, V534E, L536R, L536Q, L536Q, Y537N, Y537S, Y537C, D538G, S576L), PIK3CA (N345K, C420R, E542K, E545K, E545Q, E545A, H1047R, H1047L, E542K), TP53 (R175H, R213*, Y220C, R248W, R248W, R248Q, R273C, R273H). Results: Preliminary data analysis using the automated Typer software v4.0.26.74 (Agena Bioscience) identified more than one mutations in 29/32 (90.6%) primary BCs and 26/32 (81.3%) BM. TP53, ESR1, PIK3CA were mutated in 82.7%, 41.4% and 27.6% respectively of primary cases and in 80.8%, 34.6% and 23.1% respectively of BCBM. Of note, the pattern of mutations was similar in only 13/32 (40.6%) of the paired cases. The commonest mutations identified in both the primary and BCBM were TP53 (R175H, R273H, R248Q, R213X), PIK3CA (H1047L, E545K, E542K). The ESR1 (S576L) was the commonest ESR1 mutation identified in both BC and BM but in only one paired case, whereas the Y537N was present in only 2 BM cases (2/9, 22.2% of BM cases harbouring ESR1 mutations) cases. In addition, the AKT1-E17K was present in 3 patients; a paired case of BC and BM, one BC and one BM. The ERBB2-L755S was present in 1 BC and the ERBB2-L755_T759del only in a BM. As this is a custom BC panel, further analysis and validation is required. Conclusion: This study provides further evidence that the genomic landscape of the BCBM can differ from the primary, that BCBM contain potential actionable mutations and that they harbour ESR1 mutations. Such BCBM specific genomic data will be key to developing rationale therapeutic studies to develop treatments for this area of unmet need. The UltraSEEK™ technology provides a powerful tool to investigate low abundance mutations without compromising analytical accuracy and sensitivity. Citation Format: Athina Giannoudis, Lee Eastoe, Christopher Charlton, Nicholas Hickson, Rasheed Zakaria, Angela Platt-Higgins, Philip S Rudland, Darryl Irwin, Alexander Sartori, Michael D Jenkinson, Carlo Palmieri. UltraSEEK breast cancer panel for low frequency mutation detection in breast cancer brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-07.

Abstract 3512: AZD7648, a potent and selective inhibitor of DNA-PK, potentiates the activity of ionising radiation and doxorubicin in vitro and causes tumour regression in xenograft models

Abstract DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex that is a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of DNA double strand breaks (DSB). Cancer therapies such as ionising radiation (IR) or topoisomerase II inhibitors (doxorubicin) generate DSB which can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). It can therefore be hypothesised a DNA-PK inhibitor would potentiate the activity of these agents. We have developed a highly potent and selective inhibitor of DNA-PK, AZD7648, which inhibits IR-induced DNA-PK S2056 auto phosphoryalation with an IC50 = 92 nM in A549 non-small cell lung cancer (NSCLC) cells. AZD7648 is a potent radiosensitiser where treatment in combination with IR led to a concentration-dependent reduction of the colony survival capacity of A549 and H1299 NSCLC cells (DEF37 at 100 nM = 1.7 and 2.5, respectively). In A549 cells, AZD7648 (≥1 µM) in combination with 2Gy IR for 48 hours led to a significant accumulation of cells arrested in the G2/M of the cell cycle, a 4-fold increase in micronuclei formation, and 3-fold induction of γH2AX, pATM S1981 and 53BP1 foci formation compared with IR alone. AZD7648 was also found to combine synergistically with doxorubicin in a panel of ovarian and triple negative breast cancer (TNBC) cell lines in cell growth inhibition assays when applying the Loewe additivity model (synergy scores 4 - 35). In vivo the combination of AZD7648 with IR (5x 2Gy) induced tumour regression in H1299 and A549 NSCLC xenografts in a dose-dependent manner (84 and 11% regression respectively), while monotherapy treatment only achieved tumour growth inhibition. In these two models the increased activation by IR of three primary DNA-PK pharmacodynamic markers, pDNAPK (S2056), pRPA32 (S4/8) and γH2AX, was inhibited by AZD7648 treatment (70-90% inhibition 2 h after IR + AZD7648). Similarly, liposomal doxorubicin (2.5 mg/kg weekly) in combination with AZD7648 (37.5 mg/kg bid) induced tumour regressions in the BT474c ER+ breast cancer xenograft model and in a TNBC PDX model (63% and 33% regression respectively), while monotherapy treatments only achieved tumour growth inhibition. These data confirm that DNA-PK inhibition with AZD7648 enhances the efficacy of a range of DSB inducing agents in vitro and in vivo, providing a clear rationale for its clinical investigation. Citation Format: Jacqueline H. Fok, Antonio Ramos-Montoya, Neil James, Mercedes Vazquez-Chantada, Valeria Follia, Ankur Karmokar, Anna Staniszewska, Lenka Oplustil O’Connor, Emma Dean, Simon J. Hollingsworth, Barry R. Davies, Elaine B. Cadogan. AZD7648, a potent and selective inhibitor of DNA-PK, potentiates the activity of ionising radiation and doxorubicin in vitro and causes tumour regression in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3512.

Abstract 837: Circulating cell-free DNA methylation assay: Towards early detection of multiple cancer types

Abstract Background: Alterations in DNA methylation play a key role in tumor initiation, and methylation markers are well established for detecting various cancer types. Cancer-specific DNA methylation patterns of cell-free DNA (cfDNA) isolated from blood samples is a non-invasive method to obtain representative epigenetic information from solid tumors. In the present study, we identified and validated cancer-specific methylation markers for diagnosis of liver, breast and colorectal cancers with high sensitivity and specificity. Methods: Three blinded validation studies were performed in order to evaluate individual panels of DNA methylation markers developed for the detection of liver, breast or colorectal cancers. For the liver cancer panel study, samples drawn from 154 subjects were analyzed, including healthy donors (30) and subjects diagnosed with benign liver disease (10), liver cancer stage I-IV (60), or another cancer type (30). For the breast cancer panel study, samples drawn from 151 subjects were analyzed, including healthy donors (39) and subjects diagnosed with benign breast disease (15), breast cancer stage I-IV (65), or another cancer type (32). For the colorectal cancer panel study, samples drawn from 154 subjects were analyzed, including healthy donors (42) and subjects diagnosed with benign colorectal disease (14), colorectal cancer stage I-IV (68), or another cancer type (30). Cell-free DNA was extracted from all samples, bisulfite converted, and DNA methylation was quantified at target sites by using the IvyGene Platform for each of the three cancer-specific marker panel. Results: By quantifying DNA methylation at the target sites, the cancer-specific markers were able to differentiate subjects diagnosed with cancer from both healthy donors and subjects with benign diseases. The liver cancer cfDNA methylation panel showed an overall sensitivity of 95% and specificity of 97.5%. Whereas, the breast cancer cfDNA methylation panel correctly identified the breast cancer samples for an overall calculated sensitivity of 89% and specificity of 96%. The colorectal cancer cfDNA methylation panel also showed high overall sensitivity of 93% and specificity of 100%. Conclusions: These results demonstrate the high diagnostic potential of cfDNA methylation markers isolated from blood for the detection of multiple cancers at various stages with high sensitivity and specificity. In addition, a quantitative analysis of cfDNA methylation signature provides an opportunity for monitoring of disease. The benefits of a sensitive and specific assay to identify and monitor cancer is anticipated to aid disease management and ultimately, improve patient outcomes. Citation Format: Dhruvajyoti Roy, David Taggart, Lianghong Zheng, Dan Liu, Gen Li, Mingzhen Li, Kang Zhang, Richard A. Van Etten. Circulating cell-free DNA methylation assay: Towards early detection of multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 837.

Results of NGS panel of hereditary breast and ovarian cancer in Lebanese women.

e13045 Background: Hereditary breast (BC) and ovarian cancer (OC) genetic alterations are considered the most prevalent among familial cancer. To date, four studies have exposed the mutations related to hereditary BC predisposition in the Lebanese population , with percentage of BRCA-related pathogenic mutations ranging between 5 % and 15 %. Methods: Between 2017 and 2019, 117 patients with high risk hereditary breast and ovarian cancer were referred to undergo the testing at the Unité de Génétique médicale (UGM) of Saint-Joseph University of Beirut, Lebanon. The sequencing was accomplished by using the 21-panel Next-Generation Sequencing (NGS) method for all of our patients, to which we also added the MLPA technique followed by the Sanger sequencing for validation whenever a genetic alteration was found. Results: From 117 Lebanese women with high-risk hereditary breast and ovarian cancer predisposition, 19 pathogenic mutations were identified in this study: 11 BRCA1, 1 BRCA2, 2 PALB2, 1 ATM, 1 CDH1, 1 MSH6, 1 RAD51C, and 1 BRIP1. Among those, 13 patients had BC, one had OC and five were healthy individuals. Five similar mutations were found within the BRCA1 gene, the p.C44F mutation, accounting for 45.4 %, thus suggesting a founder effect. Average age at diagnosis in the BC patients carrying a mutation was 41 years and 38.5% had a triple negative BC. Conclusions: The overall pathogenic mutation rate was equal to 16.2% while the BRCA deleterious mutation rate was 10.3% lower to those reported in the literature. The p.C44F mutation appeared five times suggesting a founder effect. [Table: see text]

TP53, EGFR and PIK3CA gene variations observed as prominent biomarkers in breast and lung cancer by plasma cell-free DNA genomic testing

Use of plasma cell-free DNA genomic testing, also know as liquid biopsy, reveals information for early detection and monitoring of solid tumors. Our study reports the analysis of 113 lung and 18 breast cancer patients using commercially available platforms. Lung and breast cancer panel hotspot regions on the genes were investigated. There was a significant increase in isolation efficiency with very fresh blood samples of at least 15 millilitres which were processed in minutes. TP53 gene variations were detected in both types of tumors. Additionally, associations were found for EGFR variations in lung tumors and PIK3CA variations in breast tumors. Mutation assessment of these three genes are recommended as useful biomarkers for predictive studies, to follow up tumor growth and for personalized treatment. Mutations observed in this study warrant further investigation for follow up studies and may justify expression studies. However, in our subsequent studies, we intensify our tumor profiling strategy with other methods. However in terms of true personalized medicine,future plans would include repeating these studies with ctDNA size analysis and methylation analysis of the non-coding region in the individual tumors.

Inhibition of Copper Transport Induces Apoptosis in Triple-Negative Breast Cancer Cells and Suppresses Tumor Angiogenesis.

Treatment of advanced breast cancer remains challenging. Copper and some of the copper-dependent proteins are emerging therapeutic targets because they are essential for cell proliferation and survival, and have been shown to stimulate angiogenesis and metastasis. Here, we show that DCAC50, a recently developed small-molecule inhibitor of the intracellular copper chaperones, ATOX1 and CCS, reduces cell proliferation and elevates oxidative stress, triggering apoptosis in a panel of triple-negative breast cancer (TNBC) cells. Inhibition of ATOX1 activity with DCAC50 disrupts copper homeostasis, leading to increased copper levels, altered spatial copper redistribution, and accumulation of ATP7B to the cellular perinuclear region. The extent and impact of this disruption to copper homeostasis vary across cell lines and correlate with cellular baseline copper and glutathione levels. Ultimately, treatment with DCAC50 attenuates tumor growth and suppresses angiogenesis in a xenograft mouse model, and prevents endothelial cell network formation in vitro Co-treatment with paclitaxel and DCAC50 enhances cytotoxicity in TNBC and results in favorable dose reduction of both drugs. These data demonstrate that inhibition of intracellular copper transport targets tumor cells and the tumor microenvironment, and is a promising approach to treat breast cancer.

SAT-334 Proinflammatory Cytokines Modulate Resistin Expression in Breast Cancer Cells

Resistin is a proiflammatory adipokine produced by the white adipose tissue macrophages and adipocytes. Resistin expression is upregulated in breast cancer tissue and positively correlates with tumor stage, tumor size and lymph node metastasis. Previous studies found that resistin increases breast cancer invasiveness, stimulates epithelial to mesenchymal transition (EMT) and induces acquisition of cancer stem cell properties in breast cancer cells. Breast cancer cells express a variety of cytokines and their receptors, and resistin mRNA expression was previously detected in HCT-116 colorectal cancer cells. By using qRT-PCR analysis, we amplified resistin mRNA and compared its relative expression levels in a panel of breast cancer and breast non-carcinogenic cell lines, including MCF-7, T47D, BT474, HCC1806, MDA-MB-468, MDA-MB-231, and MCF-10A cells. We found that MCF-7, T47D, and HCC1806 cells express detectable levels of resistin mRNA. Using MCF-7 cells, we further investigated whether this expression can be modulated by treatment with different cytokines (resistin, TNFα, or TGFβ). Our results indicated that resistin (in concentration of 12.5 ng/ml), TNFα and TGFβ (in concentrations of 10 ng/ml) upregulated the expression of resistin mRNA in a time-dependent manner. In conclusion, we report that breast cancer cells express resistin mRNA, and that this expression is upregulated after treatment with resistin, TNFα, and TGFβ. To our best knowledge, this is the first study describing resistin mRNA expression in breast cancer cells and its alteration by proinflammatory factors. Further experiments are in progress.

Abstract P5-09-03: Expanded panel testing superior to BRCA1/2 and breast cancer panel in patients with breast cancer

Abstract Background: The testing of hereditary breast and ovarian cancer (HBOC) patients for BRCA1/2 only was established years ago to identify patients with clinically actionable variants and limit the economic burden. However, the cost of genetic testing has plummeted, and the number of breast cancer-risk genes with management guidelines has expanded. We created a community-based registry to test all breast cancer patients. A primary objective of this registry included accruing and comparing patients who did and did not meet NCCN guidelines and determining if providing all breast cancer patients with comprehensive multi-gene panel testing yields additional clinical value than testing BRCA1/2 alone. Methods: An IRB-approved multicenter prospective registry was initiated with 20 community-based and academic breast sites, selected to insure geographic and ethnic diversity. Consecutive patients ages 18-90 with current or prior breast cancer were offered testing with an 80-gene panel (Invitae, San Francisco, CA). HIPAA-compliant case report forms collected patient diagnosis, test results, and physician recommendations made after test results. Results: Over 1,000 patients were enrolled and data on 911 have been analyzed to date. Median age of patients is 60.5 (range 22 to 93). 56.0% were recently diagnosed with breast cancer. Of these patients, 50.54% met NCCN criteria, and 49.5% did not. 10.9% had history of a prior non-breast cancer. The pathogenic/likely pathogenic (P/LP) variant rate for patients on a comprehensive 80-gene panel was 8.9%. When restricted to a guidelines-based 11-gene breast cancer panel (BRCA1/2, ATM, CDH1, CHEK2, NBN, NF1, PTEN, STK11, TP53, PALB2), 4.9% had P/LP variants; when limited to BRCA1/2, 1.6% had P/LP variants. Of all patients with P/LP findings, 93% had variants in cancer-risk genes with established management recommendations (Table 1) and 80% had germline variants conferring eligibility for precision medicine-based cancer treatments, such as PARP inhibitors, through actively enrolling clinical trials. Conclusions: This study demonstrates that comprehensive panel testing of breast cancer patients provides a higher yield of clinically actionable P/LP variants than BRCA1/2 testing alone. Limited panels may miss clinically relevant P/LP variants, leaving risk for preventable cancers undiscovered and unnecessarily restricting patients' treatment options. These results also suggest that variants in tumor suppressor genes, not previously thought related to breast cancer, may contribute to its etiology. A comprehensive panel strategy reveals untapped clinical utility and can impact breast cancer patient care by informing implementation of precision medicine treatment interventions and guiding long-term medical management and surveillance for patients and their family members. PatientsVariantsWith breast cancer management guidelines (including variants ATM*, BRCA1*, BRCA2*, CHEK2*, NBN*, NF1, PALB2*, TP53*)45 (56%)46 (55%)With cancer guidelines and clinical management implications (including variants BARD1*, FH, MITF, MSH6*, MUTYH*, PTCH1, RAD50*, RAD51C*, RAD51D*, RB1, RET, VHL)31 (38%)33 (39%)Evidence of actionability accruing (including variants BLM, DIS3L2, RECQL4)5 (6%)5 (6%)Totals8184*P/LP variants in these genes confer potential clinical trial eligibility, e.g. NCT02401347. Citation Format: Beitsch P, Whitworth P, Baron P, Rosen B, Compagnoni G, Simmons R, Smith LA, Holmes D, Brown E, Gold L, Clark P, Coomer C, Grady I, Barbosa K, Riley L, Kinney M, Lyons S, MacDonald H, Kahn S, Ruiz A, Patel R, Curcio L, Esplin E, Yang S, Michalski S. Expanded panel testing superior to BRCA1/2 and breast cancer panel in patients with breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-09-03.

Abstract P5-09-06: Underdiagnosis of HBOC in breast cancer patients: Are genetic testing guidelines a tool or an obstacle?

Abstract Background: Pathogenic genetic variants are estimated to occur in 10-15% of all breast cancer patients, with BRCA 1/2 accounting for 40-50% of pathogenic/likely pathogenic (P/LP) variants. However, it is estimated that <30% of breast cancer patients harboring a BRCA 1/2 variant have been identified, with the percentage being much less for ˜20 other breast cancer associated genes. Reasons for this are multifactorial and include complicated and restrictive testing guidelines developed at a time when the cost of testing was high and guidelines for management were limited. Today, cost has plummeted and there are definitive management guidelines for a broader range of genes. We created a community based Registry to determine the incidence of P/LP variants in breast cancer patients who meet and who do not meet the NCCN 2017 genetic testing criteria. Methods: An IRB-approved multicenter prospective registry was initiated with 20 community and academic sites experienced incancer genetic testing and counseling. Eligibility criteria included patients with a breast cancer diagnosis who had not been previously tested. Consecutive patients aged 18-90 were consented and underwent an 80 gene panel test (Invitae –Multi-Cancer Panel). The non-inferiority study was powered to detect a difference in P/LP variant rate of 4 percentage points with statistical significance (p<0.05, Fisher's exact test). HIPAA compliant electronic case report forms collected information on patient diagnosis, test results, and physician recommendations made after test results were received. Results: Over 1000 patients were enrolled and data from 910 subjects analyzed to date. 50.4% met NCCN criteria and 49.5% did not. Median age for the enrolled patients is 60.5 and ranged from 22-93. 56.0% of patients were recently diagnosed with breast cancer. 10.9% of patients had a history of a prior non breast cancer. Overall, 8.9% of patients had a pathogenicvariant. 9.6% of patients who met NCCN criteria with test results had a P/LP variant. 8.2% of patients who did not meet criteria had a P/LP variant. The difference of positive cases among the two groups is not statistically significant (P = 0.49) 4.9% of patients had pathogenic variants if only an 11 gene standard breast cancer panel was considered. The spectrum of mutated genes varied between the two groups, with some overlap. Conclusions: There was no statistically significant difference in the number of pathogenic/likely pathogenic variants between those patients who met and those who did not meet NCCN guidelines. Expanded panel testing yields more medically actionable P/LP variants than testing BRCA 1/2 alone or breast cancer panels with 11 genes. This study demonstrates that there will be a significant number of patients with P/LP variants are missed if NCCN guidelines are required for genetic testing. Current NCCN guidelines for the genetic testing of breast cancer patients are an obstacle to identifying patients with P/LP variants and should be removed. Universal BC Genetic Testing RegistryNCCN Criteria (910 patients analyzed)#/% who have P/LP variants#/% who do not have P/LP variantsPatients who meet guidelines44/459 (9.6%)415/459 (90.4%)Patients who do not meet guidelines37/451 (8.2%)414/451 (91.8%) Citation Format: Beitsch P, Whitworth P, Baron P, Rosen B, Compagnoni G, Simmons R, Smith LA, Holmes D, Brown E, Gold L, Clark P, Coomer C, Grady I, Barbosa K, Riley L, Kinney M, Lyons S, MacDonald H, Kahn S, Ruiz A, Patel R, Curcio L, Esplin E, Yang S. Underdiagnosis of HBOC in breast cancer patients: Are genetic testing guidelines a tool or an obstacle? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-09-06.

Spatially constrained tandem bromodomain inhibition bolsters sustained repression of BRD4 transcriptional activity for TNBC cell growth

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.

Abstract 5472: Basal-like breast cancer subtype is characterized by deregulated glutamine metabolism and is sensitive to GLS inhibition

Abstract Tumor cells display enhanced requirements for energy and building blocks such as amino acids, nucleic acids and lipids to sustain their proliferation. Metabolic demands of cancer cells are often met in the context of limited access to nutrients and the need to maintain redox balance. Glutamine catabolism is considered critical to cancer cell survival by not only delivering carbon and nitrogen atoms needed for nucleic acid and amino acid precursors but also by serving as the source of antioxidants such as NADPH and glutathione. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS).In this study, we confirmed the significance of GLS and enhanced glutamine utilization in the basal subtype of breast cancer as evidenced by higher GLS to GLUL (Glutamine synthetase) ratio in a panel of breast cancers in The Cancer Genome Atlas (TCGA) database. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines led to profound tumor growth inhibition in vitro and in vivo. This anti-tumor effect of GLS knockdown was rescued either using shRNA resistant cDNAs encoding both GLS isoforms or by addition of an a-ketoglutarate analog thus confirming the critical role of GLS in TNBC. Along with these observations, we have found that pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) coupled with an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). In line with the observed impact on mTOR pathway we found that GLS inhibition synergizes with mTOR inhibition. In conclusion, our preliminary investigation revealed that glutaminolysis is deregulated in a majority of TNBC cancer patients. Our target credentialing studies confirmed that GLS is essential for the survival of TNBC cell lines and xenografts tumors. The synergistic activity of GLS and mTOR inhibitors in this specific breast cancer subtype suggests a new therapeutic modality that may bring benefit to TNBC patients with high unmet need. Citation Format: Christophe Henry, Michael Lampa, Timothy He, Beatriz Ospina, Bailin Zhang, Gejing Deng, Claude Barberis, Dietmar Hoffmann, Jack Pollard, Adrian Francisco, Heike Arlt, Jason Reeves, Joshua Murtie, Christopher Winter, Victoria Richon, Hong Cheng, Carlos Garcia-Echeverria, Dmitri Wiederschain, Lakshmi Srinivasan. Basal-like breast cancer subtype is characterized by deregulated glutamine metabolism and is sensitive to GLS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5472.

PO-401 Detection of mutational patterns associated to HR deficiency from low counts of mutations

Introduction A common source of tumorigenesis in several cancer types such as breast and ovarian is a defect in the homologous recombination (HR) machinery. HR defect can be detected by identifying single nucleotide variants (SNVs), deletions, duplications, and other errors generated by the alternative double-strand break repair machineries. However, current diagnostic tools for HR defect relies on copy number changes with reduced sensitivity and specificity, or on the existence of BRCA mutations. While, recent efforts to detect HR defect using mutational signature require a large number of SNVs, beyond what is typically obtained from panel sequencing which is widely used in clinics. Material and methods We developed a computational software called SigMA to identify signatures of HR defect even from low SNV counts. SigMA carries out a multivariate analysis to isolate the effect of a single biological process in the presence of multiple mutagenic processes. The multivariate analysis includes likelihood estimations, which we suggest as novel and sensitive measures, together with commonly used measures such as cosine similarity, and signature amplitudes. First, we determine the signature composition of SNVs in 720 whole-genome sequenced (WGS) breast cancer samples, with non-negative matrix factorization (NMF). Then, a subset of mutations from each sample is selected based on whether they fall within the coverage of the sequencing technique of interest. We optimise our algorithm using the simulated data and considering NMF results as a baseline measurement, and we validate its performance on panel data of 890 breast tumours. Results and discussions Whereas previous attempts in detection of mutational signatures from panel data were limited to cases with a high mutation burden, SigMA can be applied to a large fraction of samples with panel data. On a panel consisting of 410 genes, we were able to dramatically improve the detection of mutational signature of HR defect, with 0.75 (0.5) sensitivity and 0.1 (0.01) false positive rate. SigMA detected 213 cases with HR defect in 890 breast cancer panels. For exome and whole-genome data, the performance of the tool is further improved. Conclusion We developed a tool which extends the applicability of mutational signatures analysis to low counts of mutations. Application of our SigMA algorithm to detect HR defect and other mutational signatures in panel sequencing data will increase the number of cases that may benefit therapeutic agents that target specific classes of genomic alterations.

Frequency of 14 genetic variants associated with breast cancer risk and treatment in a Colombian population

Introducción: las variaciones genéticas se han relacionado con el riesgo y la eficacia del tratamiento. Es sabido que muchos polimorfismos en cáncer de mama influyen en la susceptibilidad, el riesgo de cáncer y el resultado del tratamiento. Los polimorfismos varían entre las poblaciones, y por tanto, es necesario realizar estudios locales.Objetivo: establecer la frecuencia de polimorfismos asociados al riesgo de cáncer de mama y la farmacogenómica del tratamiento en un grupo de individuos colombianos.Métodos: los datos de los perfiles de microarreglos, incluidos los polimorfismos genéticos asociados con el tratamiento del cáncer de mama, se obtuvieron de forma retrospectiva (Pathway Genomics®). Se estudiaron la frecuencia del marcador CYP2D6 rs3892097 y un panel de cáncer de mama (CAS8 rs1045485, CHEK21100delC, ESR1 rs2046210, FGFR2 rs1219648, intergenic_2q35rs13387042, intergenic_8q24 rs13281615, MSRP30 rs10941679, TNRC9 rs3803662, AKAP9 rs6964587, LSP1 rs3817198, MAP3K1rs889312, PALBS1592 delT, ESR1 rs3020314).Resultados: se analizaron los datos de microarreglos de 68 hombres y 92 mujeres. Todos los polimorfismos siguieron el equilibrio Hardy-Weinberg. Las frecuencias fenotípicas de CYP2D6 rs3892097 C/T, CAS8 rs1045485 G/C, y aquellas de los genes incluidos en un panel de cáncer de mama (CAS8 rs1045485, CHEK21100delC, FGFR2rs1219648, intergenic_2q35rs13387042, intergenic_8q24 rs13281615, MSRP30 rs10941679, TNRC9 rs3803662, LSP1 rs3817198, MAP3K1rs889312, PALBS1592 del T, ESR1rs3020314) no difirieron significativamente de los datos publicados previamente. ESR1 rs2046210, con frecuencias alélicas de C = 0,04 y T = 0,02, y AKAP9 rs6964587, con una frecuencia de A = 0,005, se determinaron como raras.

Cancer Panel Sequencing to Guide Personalized Therapy of Breast and Gynecologic Cancers [32A

INTRODUCTION: Recent genomic studies have demonstrated significant heterogeneity in breast and gynecologic cancers, characterized by distinctive genomic profiles in individual patients that could affect treatment responses. The aim of this study was to determine what percentage of patients may have findings that are of potential clinical consequence. METHODS: We analyzed 56 tumor samples from 32 breast cancers and 24 gynecologic cancers (ovarian, uterine, cervical) using the Ion AmpliSeq Hotspot Panel v2 or the Oncomine Comprehensive Assay v2 to identify somatic mutations. We also generated copy number alteration and RNA-sequencing data to further characterize the tumors. RESULTS: TP53 was the most frequently mutated genes in this cohort, consistent with genomic sequencing studies by TCGA and ICGC. Mutations with therapeutic implications were identified in many cases. PI3-Kinase pathway is likely activated in 31% and 42% of the breast and gynecologic cancers respectively due to PIK3CA activating mutations, PTEN loss and/or TSC1 loss. We also observed EGFR exon-20 insertions in 3 breast tumor samples, representing a novel mechanism of activating the ERK pathway in breast cancers. CONCLUSION: Given that everolimus has been approved by the FDA for post-menopausal, advanced hormone-receptor positive, HER2-negative breast cancers, and in light of recent promising results from several clinical trials of PI3-Kinase pathway inhibitors in breast, ovarian, and uterine cancers, breast and gynecologic cancer panel sequencing in precision oncology holds promise for more directed therapies, clinical trial recruitment and future research.

Predictors of next-generation sequencing panel selection using a shared decision-making approach

The introduction of next-generation sequencing panels has transformed the approach for genetic testing in cancer patients, however, established guidelines for their use are lacking. A shared decision-making approach has been adopted by our service, where patients play an active role in panel selection and we sought to identify factors associated with panel selection and report testing outcomes. Demographic and clinical data were gathered for female breast and/or ovarian cancer patients aged 21 and over who underwent panel testing. Panel type was classified as ‘breast cancer panel’ (BCP) or ‘multi-cancer panel’ (MCP). Stepwise multiple logistic regression analysis was used to identify clinical factors most predictive of panel selection. Of the 265 included subjects, the vast majority selected a broader MCP (81.5%). Subjects who chose MCPs were significantly more likely to be ≥50 years of age (49 vs. 31%; p < 0.05), Chinese (76 vs. 47%; p < 0.001) and have a personal history of ovarian cancer (41 vs. 8%; p < 0.001) with the latter two identified as the best predictors of panel selection. Family history of cancer was not significantly associated with panel selection. There were no statistically significant differences in result outcomes between the two groups. In summary, our findings demonstrate that the majority of patients have a preference for interrogating a larger number of genes beyond those with established testing guidelines, despite the additional likelihood of uncertainty. Individual factors, including cancer history and ethnicity, are the best predictors of panel selection.