Slettedahl, Seth W.

Abstract Background: Discovery of methylated DNA markers (MDM) of esophageal squamous cell carcinoma (ESCC) has sparked interest in assessing these markers in tissue. We evaluated MDMs in ESCC from three geographically and ethnically distinct populations, and explored the feasibility of assaying MDMs from DNA obtained by swallowed balloon devices. Methods: MDMs were assayed in ESCC and normal tissues obtained from the populations of United States, Iran, and China, and from exfoliative cytology specimens obtained by balloons in a Chinese population. Areas under the receiver operating curve (AUC) of MDMs discriminating ESCC from normal tissues were calculated. Random forest prediction models were built, trained on U.S. cases and controls, and calibrated to U.S.-only controls (model 1) and three-country controls (model 2). Statistical tests were used to assess the relationship between dysplasia and MDM levels in balloons. Results: Extracted DNA from 333 ESCC and 322 normal tissues was analyzed, in addition to archival DNA from 98 balloons. For ESCC, model 1 validated in Iranian and Chinese tissues with AUCs of 0.90 and 0.87, and model 2 yielded AUCs of 0.99, 0.96, and 0.94 in tissues from the United States, Iran, and China, respectively. In Chinese balloons, MDMs showed a statistically significant trend of increasing levels with increasing grades of dysplasia (P < 0.004). Conclusions: MDMs accurately discriminate ESCC from normal esophagus in tissues obtained from high- and low-incidence countries. Preliminary data suggest that levels of MDMs assayed in DNA from swallowed balloon devices increase with dysplasia grade. Larger studies are needed to validate these results. Impact: MDMs coupled with minimally invasive collection methods have the potential for worldwide application in ESCC screening.

INTRODUCTION: Nonendoscopic Barrett's esophagus (BE) screening may help improve esophageal adenocarcinoma outcomes. We previously demonstrated promising accuracy of methylated DNA markers (MDMs) for the nonendoscopic diagnosis of BE using samples obtained from a capsule sponge-on-string (SOS) device. We aimed to assess the accuracy of these MDMs in an independent cohort using a commercial grade assay. METHODS: BE cases had ≥ 1 cm of circumferential BE with intestinal metaplasia; controls had no endoscopic evidence of BE. The SOS device was withdrawn 8 minutes after swallowing, followed by endoscopy (the criterion standard). Highest performing MDMs from a previous study were blindly assessed on extracted bisulfite-converted DNA by target enrichment long-probe quantitative amplified signal (TELQAS) assays. Optimal MDM combinations were selected and analyzed using random forest modeling with in silico cross-validation. RESULTS: Of 295 patients consented, 268 (91%) swallowed the SOS device; 112 cases and 89 controls met the pre-established inclusion criteria. The median BE length was 6 cm (interquartile range 4–9), and 50% had no dysplasia. The cross-validated sensitivity and specificity of a 5 MDM random forest model were 92% (95% confidence interval 85%–96%) and 94% (95% confidence interval 87%–98%), respectively. Model performance was not affected by age, gender, or smoking history but was influenced by the BE segment length. SOS administration was well tolerated (median [interquartile range] tolerability 2 [0, 4] on 10 scale grading), and 95% preferred SOS over endoscopy. DISCUSSION: Using a minimally invasive molecular approach, MDMs assayed from SOS samples show promise as a safe and accurate nonendoscopic test for BE prediction.

6072 Background: Effective screening tests for ovarian cancer (OC) are lacking; most cases present at advanced stage and portend poor prognosis. DNA methylation is an early event in carcinogenesis and can be detected in blood plasma samples from cancer patients. In DNA extracted from tissues, we first discovered, then validated discriminant methylated DNA marker (MDM) candidates for OC and subsequently tested independent plasma from women with and without OC. Methods: For discovery, DNA from 67 frozen tissues (18 high grade serous (HGS), 18 endometrioid, 15 clear cell (CC), 6 mucinous OCs; 10 benign fallopian tube epithelium (FT); and 19 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with OC. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent FFPE tissues from OCs (36 HGS, 22 endometrioid, 21 CC, and 14 mucinous) and 29 FT. Top performing MDMs in tissue were tested using long-probe quantitative amplified signal assays in independent pre-treatment plasma samples from women newly-diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. Results: After RRBS discovery and biological validation, 33 MDMs showed marked methylation fold changes (10 to > 1000) across all OC histologies vs FT. The top 11 MDMs ( GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, DSCR6, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (76 (84%) HGS) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95%CI 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86 - 0.96)). Among HGS, the panel correctly identified 83%, including 5/6 stage I/II, and the majority of other subtypes (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded outstanding candidate MDMs for OC that performed with promisingly high sensitivity and specificity in plasma. Larger plasma-based OC MDM testing studies, with larger numbers of non-HGS histologies are warranted. [Table: see text]

Abstract Abstract 2692 Background: Massive splenomegaly is occasionally encountered in patients with Non-Hodgkin's Lymphoma (NHL) and can be associated with abdominal symptoms, anemia and thrombocytopenia. The spleen may also be a primary focus of the disease itself. Splenectomy offers the benefit of localized treatment of the disease as well as relief of symptoms from splenomegaly and associated cytopenias. The safety and long term clinical outcomes of splenectomy for massive splenomegaly (>1500 gm) in patients with various histologies of NHL was analyzed. Methods: Data was obtained from clinical records of 90 patients with a diagnosis of NHL who underwent a splenectomy at Mayo Clinic Rochester from January 1998 to December 2007. Demographics, previous treatments, histology, post- surgical complications were collected and analyzed for the entire cohort. Additionally, median overall survival, improvement in symptoms, anemia and thrombocytopenia were analyzed by NHL type. Results: Median age at splenectomy was 67 years (range 42–84 yrs.) and 56 (62%) of patients were male. Average time from diagnosis to splenectomy was 3 months (range 0–217 months). Splenectomy was the first treatment in 56 (62%) patients; the other 34 patients were treated with chemotherapy with an average of two previous regimens. Most patients had involvement outside of the spleen, 65 (72%) with concurrent bone marrow involvement and 35 (50%) with lymph node involvement. Various different NHL histologies were represented: marginal zone (MZ) (34%), mantle cell (MC) (26%), diffuse large B-cell lymphoma (DLBCL) (10%), follicular (FL) (11%), lymphoplasmacytic (LP) (6%) and T cell NOS (4%). With a median follow-up time of 25 months, the median overall survival differed by histology type of NHL (Table 1). Indolent lymphomas such as FL, LP and MZ had more favorable survival compared with more aggressive lymphomas such as DLBCL and T cell NOS. Splenectomy was safe and well tolerated with only one treatment related death. There were no reported post-splenectomy septic events. Two patients did develop a portal vein thrombosis. 95% of patients had relief in pressure and abdominal symptoms post-splenectomy. With the exception of patients with DLBCL and T-cell NHL, a majority of patients had improvement in anemia and thrombocytopenia related to splenomegaly (Table 2). Conclusions: This represents the largest reported series of patients with lymphoma treated with splenectomy. Our results indicate that median overall survival after splenectomy is determined by the underlying type of NHL. Patients with LP, FL, MZ and MCL have a favorable outcomes following splenectomy. Splenectomy remains a safe and effective treatment option for massive splenomegaly in selected patients during the course of their disease management. Disclosures: No relevant conflicts of interest to declare.

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation‐specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele‐specific real‐time target and signal amplification assays on independent plasma‐extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long‐probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross‐validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six‐marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957 , endothelin‐converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C‐type lectin domain containing 11A [CLEC11A]) normalized by beta‐1,3‐galactosyltransferase 6 (B3GALT6) level yielded a best‐fit AUC of 0.96 (95% CI, 0.93‐0.99) with HCC sensitivity of 95% (88%‐98%) at specificity of 92% (86%‐96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha‐fetoprotein (AFP) was 0.80 (0.74‐0.87) compared to 0.94 (0.9‐0.97) for the cross‐validated MDM panel (P < 0.0001). Conclusion : MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.

Abstract Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls. Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls. Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity. Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Abstract Background: Guidelines recommend germline mutation testing of breast cancer predisposition genes in triple negative (TN) breast cancer cases with a family history of breast or ovarian cancer or when diagnosed under age 60. However, the prevalence of mutations in these genes among TN cases unselected for family history of breast or ovarian cancer is not known. Methods: To assess the frequency of mutations in 16 predisposition genes in TN cases we screened a large cohort of TN patients (n=1824) unselected for family history of breast or ovarian cancer from 12 centers and 824 study matched unaffected controls for mutations using a panel-based sequencing approach. Results: Deleterious mutations were identified in 15% of TN patients: 8.5% had BRCA1, 2.7% had BRCA2, and 3.6% had mutations in 12 other genes. Mutations in non-BRCA1/2 genes encoding proteins implicated in homologous recombination repair of DNA double strand breaks were detected at the same frequency as in breast cancer families. TN cases with mutations had high-grade tumors and were diagnosed at an earlier age than non-mutated cases. However, 10% of TN cases diagnosed at ≥60 years and 5% with no family history of cancer were also found to carry mutations. Inactivating mutations in non-BRCA1/2 predisposition genes were associated with moderate to high risks of TN breast cancer. Conclusions: National Comprehensive Cancer Network (NCCN) guidelines support clinical genetic testing of breast cancer predisposition genes in 95% of TN breast cancer patients carrying mutations in susceptibility genes. In contrast, National Institute of Health and Care Excellence (NICE) guidelines in the U.K. do not support genetic testing of a substantial proportion of TN patients with predisposing alleles. Frequency tables for inherited mutations in known predisposition genes based on age of diagnosis and family history of cancer will allow for selection of TN patients most likely to carry mutations in the predisposition genes. Citation Format: Fergus J Couch, Steven N Hart, Priyanka Sharma, Amanda Ewart Toland, Penelope Miron, Janet E Olson, Andrew Godwin, Vernon S Pankratz, Curtis Olswold, Seth Slettedahl, Lucia Guidugli, Matthias W Beckmann, Brigitte Rack, Arif B Ekici, Irene Konstantopoulou, Florentia Fostira, George Fountzilas, Liisa M Pelttari, Song Yao, Judy Garber, Angela Cox, Hiltrud Brauch, Christine Ambrosone, Heli Nevanlinna, Drakoulis Yannoukakos, Susan L Slager, Celine M Vachon, Diana M Eccles, Peter A Fasching. Triple-negative breast cancer: Frequency of inherited mutations in breast cancer susceptibility genes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-03.

Abstract Recent studies have shown that associations between common genetic variation and gene expression in breast tumors provide insight into the functional mechanisms underlying breast cancer risk loci. However, the relationship between genetic variation, gene expression, and risk of triple negative (TN) breast cancer remains largely unexplored. We performed a genome-wide expression quantitative trait locus (eQTL) analysis of TN breast cancer using 668 formalin-fixed paraffin embedded TN tumors from the Triple Negative Breast Cancer Consortium (TNBCC) to identify novel genes relevant to TN breast cancer risk and to further explore the biology underlying known TN risk loci. Cis-eQTLs were defined as correlations between single nucleotide polymorphisms (SNP) and expression of genes within 1Mb, and trans-eQTLs were defined as the remainder of all SNP-gene correlations. In a genome-wide analysis, we identified 68,012 cis-eQTLs, representing correlations between 42,193 SNPs and 2,092 genes that were significant at a 10% false discovery rate (FDR). We also identified 70 trans-eQTLs across two unique genes at the same FDR threshold. Five cis-eQTLs involved SNPs previously associated with TN breast cancer risk (p&lt;5.0x10-5) in 3,700 TN cases and 4,700 controls from the TNBCC. Three of these, located on chromosomes 5p (cis-eQTL p=4.8x10-34; risk p=1.1x10-7), 7p (cis-eQTL p=6.1x10-10; risk p=1.3x10-7), and 17q (cis-eQTL p=1.8x10-15; risk p=2.9x10-7), have previously been reported in public databases of normal tissue. The remaining two TN breast cancer cis-eQTLs, located on chromosomes 8q (cis-eQTL p=6.3x10-6, risk p=8.3x10-6) and 14q (cis-eQTL p=3.1x10-6, risk p=1.2x10-5), have not been reported as eQTLs in other tissues. In addition, 12 of 25 known TN breast cancer risk loci (PEX14, MDM4, 2q31.1, ESR1, 11q13.1, 11q24.3, 12p13.1, PTHLH, NTN4, 12q24, 19p13.1, MLK1), contained at least one significant cis-eQTL. In summary, we have identified five cis-eQTLs that may identify novel risk loci for TN breast cancer. Citation Format: Kristen S. Purrington, Drakoulis Yannoukakos, Jane Carpenter, Heli Nevanlinna, Angela Cox, Gianluca Severi, Christine Ambrosone, Amanda Ewart Toland, Andrew K. Godwin, Hiltrud Brauch, Peter A. Fasching, Penelope Miron, Jenny Chang-Claude, Nicholas G. Martin, Grant W. Montgomery, Vessela Kristensen, Hoda Anton-Culver, Paul Goodfellow, Janet E. Olson, Hugues Sicotte, Naresh Prodduturi, Daniel W. Visscher, Jeanette E. Eckel-Passow, S. Keith Anderson, Seth Slettedahl, Curtis Olswold, Xianshu Wang, V. Shane Pankratz, Susan Slager, Wei Zheng, Arto Mannermaa, Ute Hamann, Diana M. Eccles, Celine M. Vachon, Fergus J. Couch. Expression quantitative trait locus analysis of triple negative breast cancer. [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 3266. doi:10.1158/1538-7445.AM2014-3266

Purpose Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. Patients and Methods Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. Results Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. Conclusion Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives.