Citations

Abstract Background: It has been shown that t(14;18), a translocation involving IgH and BCL-2 genes that is present in over 80% of all cases of follicular lymphomas, is detectable in the peripheral blood of up to 52% of normal Caucasian patients from Germany and up to 16% of normal individuals from Japan (Yasukawa et al, Blood 2001). The fact that follicular lymphoma is one of the two most common lymphomas in the USA and Germany and much less common in Japan, demonstrates that this molecular alteration is perhaps an early event in the development of this malignancy which is not sufficient for its causation. There have not been any studies evaluating the prevalence of this translocation in healthy African American (AA) subjects. We speculated that the prevalence of this translocation will be lower in healthy AA subjects given the findings of lower prevalence of IgH translocations in AA patients with multiple myeloma compared with Caucasian patients, which may explain the better outcome of myeloma in AA subjects (Baker et al, Blood 2013). This effort to evaluate the prevalence of t (14; 18) in AA subjects, was part of a pilot study in which we brought an educational program on the importance of cancer research together with concomitant access to a translational research project to AA churches utilizing a mobile research unit in an attempt to overcome some of the known barriers to AA subject participation in clinical research (Colon-Otero et al, Cancer2008; Colon-Otero et al, J Cancer Educ 2012). Methods: IRB approval of the project was obtained and peripheral blood samples collected from consented subjects during educational sessions in Northeast Florida AA Churches. Caucasian subjects were identified from the Mayo Clinic Bio-bank. DNA was isolated from the buffy coat fractions and a PCR quality control assay targeting a 293bp fragment of an HLA class II gene was used to determine acceptable DNA quality and quantity. Next, a two-step semi-nested PCR was performed on the t (14;18) major and minor breakpoint cluster regions using ~500ng of DNA for each assay. A consensus JH primer was used in combination with primers complementary to BCL-2 sequences for both, the major and minor breakpoint cluster regions. PCR products were run on a 1% agarose gel. In order to confirm the positive results, positive PCR products were analyzed by direct sequence by Sanger method and subsequently assembled to the human genome using BLAST tool. Results: A total of 110 blood samples from AA subjects were analyzed for the presence of t (14;18). A total of 13 of the samples were positive for the major breakpoint site (12%) and 4 were positive for the minor breakpoint site (4%), for a total of 17 positive samples (15%). In 167 Caucasian patients samples, a total of 22 (13%) were positive for the major breakpoint site and 5 (3%) were positive for the minor breakpoint site for a total of 27 positive samples (16%). All cases were confirmed by Sanger sequencing. Conclusions: The prevalence of t(14;18) is not significantly different among African American and Caucasian subjects from the USA based on our findings. This represents the first report of the prevalence of t(14;18) in an African American population. The prevalence found in our Caucasian subjects is significantly lower than what had been described in previous population series. A plausible explanation is the high heterogeneity of approaches used across studies, significantly affecting the assay sensitivity. Additional studies with larger series may reveal minor differences in the prevalence of t(14;18) among these populations. Citation Format: Scott Van Wier, Gerardo Colon-Otero, Gregory Ahmann, Esteban Braggio, Monica Albertie, Sikander Ailawadhi, Rafael Fonseca. Similar prevalence of t(14,18) in African American and Caucasian subjects. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B48.

Abstract African American Hereditary Prostate Cancer Study (AAHPC) was developed as a national collaboration to explore the role of genetics in the causation of hereditary prostate cancer (HPC) in African American (AA) men. AAHPC is in partnership with the International Consortium for Prostate Cancer Genetics (ICPCG), which conducts collaborative studies of HPC genetics in multiplex families. As part of an ICPCG sequencing study of 539 affected individuals from 366 HPC pedigrees, we performed whole exome sequencing in 21 ICPCG AA families, of which there were 14 AAHPC affected men from 11 pedigrees. The combined ICPCG AA cohort consisted of N=26 affected members. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth < 10, Quality Score < 20, Quality Score: Read Depth Ratio < 0.5, Call Rate < 0.75. Variants were additionally filtered by MAF based on the NHLBI ESP650051-V2 exomes variant frequencies for the AA population using a MAF threshold of 5%. Following QC, 176/299 SNVs and 20/39 INDELs remained for further analysis. In these analyses, we focused on 13 known cancer genes (MSR1, AR, BRCA1, BRCA2, BTNL2, EPHB2, CDH1, RNASEL, ELAC2, HOXB13, CHEK2, TP53 and NBN). Three sequenced families had > 1 affected members sequenced (2 or 3 per family) and the remaining 18 families had one member sequenced. Under the dominant model, our preliminary results show that no rare variants in the 13 candidate genes were found in 3/3 affecteds in two families. Rare SNVs in seven candidate genes (AR, CDH1, ELAC2, HOXB13, RNASEL, BRCA2 and EPHB2) were found in 2/3 affecteds for two families and 2/2 affecteds in one family. Several of the remaining 18 affected men (1 sequenced per pedigree) shared the same rare SNV in these candidate genes. For INDELs, rare variants in three candidate genes were found in pedigrees with ≥ 2 affecteds. Several of the remaining 18 affected men (one sequenced per pedigree) shared the same rare INDEL. Additional QC is underway to validate these variants and bioinformatic analyses are being used to predict effects of the variants in an effort to unravel the complex genetic heterogeneity of HPC in AA. Citation Format: Cheryl D. Cropp, Shannon K. McDonnell, Sumit Middha, Melissa DeRycke, Danielle M. Karyadi, Daniel Schaid, Stephen N. Thibodeau, William B. Isaacs, Elaine A. Ostrander, Janet Stanford, Kathleen A. Cooney, Joan E. Bailey-Wilson, John D. Carpten. Rare variant discovery in known cancer genes from whole-exome sequencingof African American hereditary prostate cancer families. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B40.

Abstract Background: The rising incidence of hepatocellular carcinoma in the US is partly due to increased immigration from sub-Saharan Africa. Viral hepatitis is prevalent in sub-Saharan Africa, resulting in ~20% of all hepatocellular carcinoma cases worldwide. Community-based education and screening efforts aimed at identifying viral hepatitis cases among African immigrants will likely reduce the burden of hepatocellular carcinoma. Aim: To determine the knowledge, attitudes and behaviors (KAB) of Liberian and Kenyan immigrants residing in Minnesota about hepatitis B virus and hepatitis C virus screening, vaccination, acquisition, and disease management. Methods: A community-engaged research framework was used for the development of this cross-sectional study. The team created a survey using validated items designed to assess the KAB of viral hepatitis measured on a 1 to 20 scale with 1 being low and 20 being high. Using community-engaged methodology, participants who were ≥18 years of age and self-identified either as Liberian and Kenyan were recruited from churches, community centers, and community events. The survey was administered between June 2014 and February 2015. Spearman's rho correlation was used to identify associations among scores while the Mann-Whitney U-test was used for comparisons between Liberians and Kenyans. Results: Of 80 distributed surveys, 73 were returned, achieving a response rate of 91%. Respondents were 50 (64%) Liberians and 23 (29%) Kenyans; 42 (54%) were female; the mean age was 42 years (range 18-90); most were college educated (57%) and had health insurance (83%). 55% reported previous screening for hepatitis B, 51% had completed hepatitis B vaccination and 38% reported being screened for hepatitis C. Mean scores ± SD were 4.6±2.8 for knowledge, 4.7±1.0 for attitude, and 4.2±1.9 for behavior, resulting in poor knowledge and adequate attitude and behavioral levels. Significant and positive linear correlation was observed between attitude and behavior (r=.385, p<.001), while there were no significant correlations between knowledge and attitude (r=-.115, p=.33) or knowledge and behavior (r=-.11, p=.36). When Liberians and Kenyans were compared, knowledge was higher in Liberians (6.2±1.9 vs1.5±1.0; p<.001); there were no significant differences observed in attitude or behavior scores (p>.05 for both). Conclusions: There is lack of awareness among African immigrants of the health risks associated with viral hepatitis which contributes to disparities in healthcare seeking behaviors. It is important to implement comprehensive education and screening programs on viral hepatitis in these communities in order to reduce the burden of viral hepatitis and hepatocellular carcinoma development. Citation Format: Hassan M. Shaleh, Nasra H. Giama, Essa A. Mohamed, Hager F. Ahmed Mohammed, Linda M. Kerandi, Abdul M. Oseini, Abdiwahab O. Ali, Ibrahim A. Waaeys, Jonggi Choi, Henry M. Kerandi, Safra A. Mohamed, Hamdi A. Ali, Hawa M. Ali, Joyce E. Balls-Berry, Lewis R. Roberts. A cross-sectional assessment of knowledge, attitudes, and behaviors about viral hepatitis and hepatocellular carcinoma among Kenyan and Liberian immigrants living in Minnesota. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A46.

Abstract There is a paucity of data on African Americans in community-based research secondary to difficulties in recruitment. These challenges include transportation, outreach barriers, fear and mistrust of researchers, lack of knowledge about disorder/issue under study, and lack of incentives. We hypothesized that we could improve recruitment in rural African American women by engaging community centers such as churches, community health centers, and doctor's offices. Over a 1 month period, we sought to recruit 40 women for a community based research project with a primary outcome of increasing activity in rural African American women (45-70 years old) with knee pain. The outlets used to recruit these women included: five churches with a community liaison, many primary care physicians' offices, and a newspaper ad. In 1 month, 67 women expressed interest in our program by initiating contact. Of those 67 women, 42 were eligible and 37 were enrolled. Of the 42 eligible women, 24 were recruited from churches, 11 through word of mouth, 3 from physicians' offices, and 3 from the newspaper ad. Lack of literature on how to recruit in rural African American communities is a significant barrier to research participation among African Americans. In this project, we provide a reproducible recruitment process for engaging rural African American women in community based research. While our primary focus was knee pain, this recruitment process can easily be translated into other areas of community based research including cancer and cardiovascular disease among others. Citation Format: Michelle Scott, Tamara Huff, Monica Albertie, Gerardo Colon-Otero. Recruitment methods for engaging rural African American populations in community-based research. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A27.

Abstract Purpose: The objective of this study is to characterize the function of porcupine inhibitors in osteosarcoma and to determine the contribution of the porcupine enzyme to osteosarcoma cell growth. Each year, approximately 1000 children and adults in the United States are diagnosed with osteosarcoma (OS), the most common primary bone malignancy among children. Although overall survival is relatively good in OS patients, the need for aggressive surgery (often involving limb amputation) to prevent relapse significantly impacts the quality of life of survivors. OS has been well characterized to display aberrantly activated Wnt signaling. The necessity for active Wnt signaling pathways in OS patient tissue and cultured cell lines has been well established. Recently, a group of Wnt pathway inhibitors targeting the porcupine enzyme have been developed and used in treatment of pancreatic adenocarcinoma, BRAF mutant colorectal cancer and other tumors reported to exhibit aberrant Wnt signaling. Porcupine is a membrane bound O-acyl transferase which lipid-modifies the Wnt proteins prior to their secretion. This processing step regulates their release and paracrine or autocrine activity in stimulating both canonical and non-canonical Wnt signaling. These drugs have proven effective in initial studies which have applied them in vitro, in mouse models of Wnt- dependent cancers, and a recent phase 1 clinical trial. Our data show the first report of increased porcupine enzyme expression in human OS in vitro cell lines, compared to primary human osteoblasts. These findings have been confirmed by quantitative real-time PCR (qRT-PCR) analysis of PPN mRNA expression. Surprisingly, we found that inhibition of the porcupine enzyme with newly developed porcupine inhibitors significantly increased OS cell viability. While we observed only slight decreases in canonical Wnt pathway signaling with PPN inhibitor treatment (as measured by TOPflash reporter assay), we found that both NFATc1 and Cyclin D1 mRNA expression was upregulated. These data indicate that treatment with PPN inhibitors may stimulate a pro-tumorigenic response that is mediated primarily through non-canonical Wnt signaling. Continuing work is being done to establish the mechanism of action of these drugs, as well as unique roles that PPN-mediated non-canonical Wnt signaling may play in promoting OS cell growth. In conclusion, this study is aimed to provide insight into the function of a group of novel Wnt-targeting therapies in osteosarcoma, a disease which is well established to exhibit aberrant Wnt signaling. We aim to utilize these insights to rationally exploit Wnt signaling as a therapeutic measure for osteosarcoma. Methods: Western blotting was performed according to accepted methods using BioRad Criterion™ 10% gel and an antibody polyclonal to known porcupine splice variants (anti-rabbit) obtained from GeneTex. Cell viability was determined by CellTiter 96 ® (Promega) proliferation assay. C59 (Novartis) was purchased from Cayman Chemical and reconstituted in DMSO. Quantitative PCR performed using an Applied Biosystems ABI 7900HT Fast Real Time PCR system. FuGENE® HD utilized for transfection of TOPflash components. Citation Format: Carl T. Gustafson, Avudai Maran, Michael J. Yaszemski. Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B11.

Abstract Background ER+ BC is associated with activated CDK4/6. The CDK4/6 inhibitor palbociclib (P) markedly improves time to progression in advanced ER+HER2- BC. We conducted a neoadjuvant phase II trial to determine the activity of P in primary breast cancer as a prelude to adjuvant studies. Methods To assess molecular changes induced by anastrozole (A) or P+A, patients (pts) were treated initially with A alone (1mg PO daily) for 28 days in cycle 0 (C0) before the addition of P (125mg PO daily on D1-21 each cycle) on C1D1. P+A was administered for 4 28-day cycles followed by C5 with A alone for 2-4 weeks (wks) before surgery. P was added in C5 for 10-12 days immediately prior to surgery in the last 20 pts enrolled to assess molecular changes induced by A, either alone or in combination with P immediately prior to surgery, in resected tumor. Goserelin was added in premenopausal pts. Research tumor biopsies were obtained at baseline, C1D1, and C1D15. Central Ki67 analysis was performed at all timepoints, those with Ki67 >10% at C1D15 went off study treatment. The primary endpoint was complete cell cycle arrest (CCA), defined as Ki67 <2.7%, at C1D15. Patient stratification was based on PIK3CA mutation status with an initial focus on PIK3CA wild type (WT) disease. Pts with PIK3CA mutant (Mut) tumors enrolled to a separate cohort. A sample size of 33 pts in the PIK3CA WT cohort was chosen based on the Fleming's single-stage phase II design to test the hypothesis that P+A leads to > 50% improvement over A in CCA rate on C1D15 biopsy (44% with A alone based on historical data, vs 66% with P+A, power = 0.8, alpha=0.05). The primary endpoint is met if >20 pts achieved CCA in this cohort. Correlative endpoints included assessment of markers of proliferation, apoptosis, senescence, Rb, gene expression microarray, intrinsic subtype, and next generation sequencing of 83-gene panels, which will be reported at the meeting. Results Between 4/23/2013 and 4/24/2015, 50 pts (33 PIK3CA WT, 11 PIK3CA Mut, 2 pending, 4 tissue quantity or quality not sufficient for sequencing (QNS)) were enrolled to the study. Median age was 57.5 (range: 34.1–79.6) years. Four pts, all with WT PIK3CA, went off study due to Ki67 >10% on C1D15 biopsy, 26 pts completed treatment and surgery, 1 refused surgery, 3 withdrew study treatment in C1, and 16 continued to receive study drug (2 in C0, 3 in C1, 4 in C2, 5 in C3, 1 in C4, and 1 in C5). Among the 40 pts currently evaluable for the primary endpoint (C1D15 Ki67), CCA occurred in 34 (85%) pts, including 9 of 9 (100%) PIK3CA Mut, 22 of 28 (78.5%) WT, and 3 of 3 QNS pts. Preliminary analysis of available data indicated a significantly lower Ki67 value after 2 wks of P+A (C1D15) compared to that on A alone (C1D1) (p=0.034, n=18). Conclusion This study met the primary endpoint demonstrating that P+A is a highly effective anti-proliferative combination. The sequential biopsy design clearly demonstrated that P+A increased cell cycle control over A alone. P+A was effective regardless of PIK3CA mutation status and these results support the evaluation of this combination in the adjuvant setting for ER+HER2- BC. Citation Format: Ma CX, Gao F, Northfelt D, Goetz M, Forero A, Naughton M, Ademuyiwa F, Suresh R, Anderson KS, Margenthaler J, Aft R, Hobday T, Moynihan T, Gillanders W, Cyr A, Eberlein TJ, Hieken T, Krontiras H, Hoog J, Han J, Guo Z, Vij K, Mardis E, Al-Kateb H, Sanati S, Ellis MJ. A phase II trial of neoadjuvant palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, in combination with anastrozole for clinical stage 2 or 3 estrogen receptor positive HER2 negative (ER+HER2-) breast cancer (BC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S6-05.

Abstract Background The clinical results of NSABP B-35, phase III trial comparing 1 mg/day A to 20 mg/day tam, each given for 5 years, were reported at ASCO 2015. B-35 demonstrated a statistically significant benefit in breast cancer free interval for women assigned to A, primarily in women <60 years. A secondary endpoint of B-35 was quality of life (QOL) and symptom (SX) outcomes in the two treatment groups. The primary hypotheses of the PRO study were that there would be no differences in QOL between the two treatments, and that patients receiving A would report higher rates of hot flashes compared to patients receiving tam. Other SX comparisons were secondary endpoints. Methods QOL and SX were assessed at baseline (prior to randomization), and every 6 months thereafter for 5 years of treatment and in the following 12 months. QOL was measured with the SF-12 Physical Component Summary (PCS) and Mental Component Summary (MCS). SX were measured with selected scales from the BCPT symptom-checklist, and other standardized instruments. Stratification was by age (<60 v ≥60) as in the main trial. Study hypotheses and endpoints were examined by comparing PROs in the two treatment arms using a mixed model for repeated measures analysis with adjustment for the baseline scores, time point and age category, using an intention-to-treat principle and including only patients who completed the baseline and at least one follow-up questionnaire. Patients with protocol events were censored. Only data through 60 months are reported here. The accrual goal for the sub-study was 1,150 consecutive patients. Results Between January 6, 2003 and June 15, 2006, a total of 3,104 patients were entered and randomly assigned to NSABP Protocol B-35. Accrual to the PRO study of B-35 closed on December 28, 2004, at which time 1,275 patients were entered, with 1,193 patients included in this analysis. There were no medical or demographic differences between patients assigned to A or tam in the PRO sub-study, and they reflected the characteristics of the parent trial. Adherence to data collection across the 60 months was 87%. There were no significant differences in QOL outcomes by treatment for the PCS (p=0.16) or the MCS (p=0.38). SX subscales: hot flash scale was greater in tam group and this difference varied over time (p=0.001); musculoskeletal pain was significantly greater in A group for time points 6-24 months (all p<.001); vaginal problems were greater in A group (p=0.03). Hot flash and vaginal problems were significantly worse in women <60 years. Additional SX outcomes (depression, fatigue, sexual function) will be reported at presentation. Conclusion In this large, double-blind, placebo-controlled trial comparing A to tam in patients with DCIS, there was no significant difference in QOL between the two treatments. However, there were important treatment differences in SX outcomes, which should be considered as part of treatment decision-making discussions, along with the clinical breast cancer outcome results. Support: CA-180868, 180822, 189867, 196067, 114732; AstraZeneca Pharmaceuticals LP. Citation Format: Ganz PA, Cecchini RS, Julian TB, Margolese RG, Costantino JP, Vallow LA, Albain KS, Whitworth PW, Cianfrocca ME, Brufsky A, Gross HM, Soori GS, Hopkins JO, Fehrenbacher L, Sturtz K, Wozniak TF, Seay TE, Mamounas EP, Wolmark N. Patient-reported outcome (PRO) results, NRG Oncology/NSABP B-35: A clinical trial of anastrozole (A) vs tamoxifen (tam) in postmenopausal patients with DCIS undergoing lumpectomy plus radiotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S6-04.

Abstract Background: Mutations in BRCA1 and BRCA2 influence the molecular pathogenesis of breast cancer. However, little is known about the association between mutations, response to therapy, and prognosis. We therefore analysed these associations in triple negative breast cancer (TNBC) patients in the neoadjuvant GeparQuinto Study. Methods: The GeparQuinto study recruited 1956 patients with HER2 negative disease including a prespecified group of 671 TNBC patients with untreated breast cancer. Patients were randomized to receive four cycles EC (90/600 mg/m2; q3w) followed by four cycles docetaxel (100 mg/m2; q3w), with or without bevacizumab (15 mg/kg). Here we present the analysis for both randomization arms combined. Sufficient blood was available of 482 TNBC patients. BRCA1 and BRCA2 genotyping was successful in 469 patients with available germline DNA and was conducted by SureSelect custom capture and sequencing on HiSeq 2500. Mutation status was correlated with pathological complete response rates (pCR) and disease free survival (DFS). Results: A total of 74 (15.8%) mutations in BRCA1 (n=61) and BRCA2 (n=13) were detected after initial sequencing. A pCR (ypT0/ypN0) was observed in 50% (n=37) of mutation carriers but only 31.1% (n=123) of patients without mutations (p=0.002). Similar results were observed for pCR (pT0is/pN0) (52.7% vs. 36.5%; p=0.010). pCR (ypT0/ypN0) was predictive of disease free survival (DFS) in all patients (Hazard Ratio, HR=0.23; 95%CI: 0.15-0.37; p<0.001) and in patients without BRCA mutations pCR (ypT0/ypN0) (HR= 0.20; 95%CI: 0.11-0.34; p<0.001). However in mutation carriers this effect was reduced (HR=0.48; 95%CI: 0.18 1.27; p=0.129). Conclusion: TNBC Patients with BRCA mutations showed a higher frequency of pCR than patients without BRCA mutations after treatment with epirubicin, cyclophosphamide and docetaxel (+/ bevacizumab), suggesting that BRCA mutations may influence pCR in response to treatment regimens that do not include platin chemotherapy. pCR as a surrogate marker for DFS was also confirmed in patients without BRCA mutations. In addition, the effect of pCR on prognosis seemed to be smaller among the mutation carriers, although the number of mutation carriers was too low to test for differences between mutation carriers and wildtype patients. The project has partly been funded within NIH-NIGMS U19 GM61388 and the Breast Cancer Research Foundation. Citation Format: Fasching PA, Loibl S, Eidtmann H, Tesch H, Untch M, Hilfrich J, Schem C, Rezai M, Gerber B, Costa SD, Blohmer JU, Fehm TN, Huober J, Liedtke C, Müller V, Nekljudova V, Weber K, Rack B, Rübner M, Wang L, Ingle JN, Weinshilboum RM, von Minckwitz G, Couch F. BRCA mutations, therapy response and prognosis in the neoadjuvant GeparQuinto study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S5-06.

Abstract Background: Germline mutations in the BRCA1 and BRCA2 genes lead to hereditary breast cancers that are defective in homologous recombination (HR) repair and sensitive to DNA damaging agents. HR deficiency (HRD) also occurs in sporadic breast cancers, but its incidence and etiology are unclear. Genomic signatures of HRD recently were employed as biomarkers in clinical trials with modest success. We posited that sporadic breast cancers displaying functional HR deficiency would harbor genetic alterations affecting HR DNA repair genes. To test this hypothesis, we applied a functional assay to define lack of competent HR DNA repair and sequenced the exomes of consecutive sporadic breast cancers. Methods: We developed an assay to assess the ability of cancer cells to localize RAD51 into sub nuclear foci in response to ex-vivo irradiation (IR) in fresh sporadic breast cancer tissue specimens from 60 patients. RAD51 focus formation was compared between mock and IR conditions to determine relative fold induction. Twenty-nine tumors with sufficient DNA underwent whole-exome sequencing. Structural genomic signatures of HRD (i.e. large state transitions (LST), telomeric imbalance (NtAI), loss-of-heterozygositiy (LOH)) and a previously reported mutational signature related to BRCA1/2 hereditary breast cancers were assessed. HR deficient tumors were defined as those with both RAD51 foci defects and a genomic signature of HR deficiency (LST>15 or presence of a BRCA mutational signature). Somatic, germ-line, and copy number changes in HR genes were investigated. BRCA1 methylation status was determined. Results: Seventeen of 60 (28%) tumors displayed defective RAD51 recruitment following ex-vivo IR (RAD51-DEF). RAD51-DEF was seen in all breast cancer subtypes , including 7 of 33 (21%) ER+/HER2-, 4 of 14 (29%) HER2+, and 6 of 13 (48%) triple-negative cases. Of the 29 sequenced tumors, 13 (45%) were RAD51-DEF and 16 (55%) were competent for inducing RAD51 foci. LST was elevated in 10 tumors (LST >15) and associated with RAD51-DEF (p=0.02), whereas NtAI (p=0.10) and LOH (p=0.052) did not show a significant association with RAD51-DEF. The BRCA1/2 mutational signature was evident in 4 tumors, all were RAD51-DEF (p=0.03) and 2 were BRCA2 mutated. Nine of 29 (31%) sequenced tumors were determined to have HRD by the RAD51 assay and presence of a genomic scar. Eight of these 9 (88%) cases with HRD had a genetic alteration of both alleles of a bona fide HR gene due to a pathogenic mutation (somatic or germline) coupled with loss of heterozygosity or a homozygous deletion compared to 1 (5%) tumor without HRD (p<0.001). Both alleles of a gene were affected for BRCA2 (n=4), FAAP100 (n=2), CHEK2 (n=1), TP53BP1 (n=1) and BRCA1 (n=1). BRCA1 gene promoter methylation was found not to be significantly associated with HRD. Conclusion: Combined functional and genomic analyses of breast tumors demonstrated that genetic loss of an HR gene may underpin HRD in sporadic breast cancers. Our findings warrant further comprehensive genetic assessment (somatic, germline, and copy number) of HR genes as potential biomarker for HR-directed therapies. Citation Format: Powell SN, Riaz N, Mutter RW, Ng CKY, Delsite R, Piscuoglio S, King TA, Martelotto L, Sakr R, Brogi E, Edelweiss M, Lim R, Higginson D, Weigelt B, Lee W, Reis-Filho JS. A functional assay for homologous recombination (HR) DNA repair and whole exome sequencing reveal that HR-defective sporadic breast cancers are enriched for genetic alterations in DNA repair genes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-03.

Abstract Fusion transcript 24analysis by five groups, each using "in house" analytical workflow to analyze RNA-seq data from 813 breast tumors from The Cancer Genome Atlas project detected 2514 high confidence RNA fusions (2 or more junction spanning reads detected by 2 or more groups). A subset of tumors with fusions were evaluated for chromosomal rearrangement using whole genome sequence data (WGS), and about half of the high confidence fusions could be validated, with the remainder likely subclonal and therefore below the limits of detection by WGS at the relatively low depth of coverage (30X) available. The majority of tumors contained one or more fusion transcript (range 0-24 fusions/tumor), but recurrence of specific chimaeric transcripts was low. Only ESR1->CCDC170, C20orf3->ACSS1, and USP22->MYH10 fusions were detected in 10 or more tumors. Additional ESR1 fusions were detected with AKAP12, BNC2, C6orf211, GNAS, POLH, USP25, and UTRN, within 19 tumors, primarily of the Luminal B subtype. RARA was the second most common 5' fusion partner with 17 tumors, predominately of the HER2-enriched subtype, expressing out of frame RARA fusions to 19 different 3' fusion partners. Other common 5' fusion partners included FBXL20, USP22, USP32, BCAS3, MED1, BPTF, ERBB2, MED24, and PPF1A1 (expressed in 10 or more tumors each). The most common 3' fusion partners were ERBB2, CCDC170, PITPNC1, ACSS1, MYH10, IKZF3, and RAB6A (10 or more tumors each). HER2-enriched tumors expressed significantly more fusions than other subtypes (4.9 fusions/tumor vs 3.3 for Basal-like, 1.3 for LumA, and 3.2 for LumB). Almost all HER2-enriched tumors expressed fusions (97%), whereas 85% of Basal-like tumors and 79% of Luminal B tumors expressed one or more fusions. Among Luminal A tumors, only 48% expressed one or more fusion transcript. Those Luminal A tumors with multiple fusion transcripts contained significantly more copy number aberrations and were enriched for expression of mitotic cell cycle genes (p=3.07E-18), characteristic of the recently described high risk CNH subclass of Luminal A tumors. We conclude that, consistent with other observations, breast tumors generally exhibit a high level of genomic instability, associated with multiple copy number alterations and multiple fusion transcripts. However, recurrence of specific fusion transcripts is low. About half of Luminal A tumors express fusion transcripts; and these tumors exhibit multiple copy number events, consistent with high level genomic instability, as well as enriched expression of mitotic cell cycle genes. These tumors appear to be related to the relatively high risk Luminal A CNH subclass, and express high levels of aurora kinases and polo-like kinases, which might be considered as therapeutic targets. Given the increasing emphasis on transcript profiling of clinical samples and the clinical significance of high risk Luminal A tumors, it may be timely to consider inclusion of fusion transcripts in such analyses as surrogate markers of genomic instability and potential therapeutic and/or prognostic indicators. Citation Format: Thompson EA, Asmann YW, Su X, Ellis MJ, Shao J, Hu Y, White KP, Cherniack AD, Hoadley KA, Serie DJ, Perez EA, Perou CM. A comprehensive analysis of fusion transcripts in breast cancer reveals associations between number of fusion transcripts, copy number events, gene expression profiles, and potentially clinical outcome. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-02.