Edwards, David K.

Abstract The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case–control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). Prevention Relevance: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93

63 Background: Population-based colorectal cancer (CRC) screening can reduce mortality by detecting and removing advanced precancerous lesions (APL) and early-stage invasive disease. One guideline-included strategy is the multi-target stool DNA test (mt-sDNA), which combines detection of methylation DNA markers (MDMs), KRAS mutations, and fecal hemoglobin. Since the mt-sDNA pivotal study was conducted, novel biomarkers have been discovered. A panel of highly discriminant MDMs ( LASS4, LRRC4, PPP2R5C, and reference marker ZDHHC1) was identified through a blinded, case-control study of archival specimens. Here, we evaluated the performance of this novel mt-sDNA panel, combined with fecal hemoglobin, in archival stool samples weighted to early-stage CRC and prospectively collected APL, simulating a screening population. Methods: The verification study featured 777 samples—210 cases (112 CRC [49 stage I, 38 stage II, 17 stage III, and 8 stage IV] and 98 APL) and 567 controls (176 non-APL and 391 colonoscopy-negative)—from three trials (NCT01397747, NCT01260168, and NCT02503631). Median APL size was 12 mm (interquartile range: 10 mm to 15 mm), with 86.7% adenomas (n = 85) and 13.3% sessile serrated polyps (SSPs; n = 13). The average age was 65.5 years for cases (57% men) and 63.2 for controls (47% men). Samples were processed through homogenization, targeted MDM capture, bisulfite conversion, and MDM quantitation using Long‐probe Quantitative Amplified Signal (LQAS). Fecal hemoglobin was quantified using enzyme-linked immunosorbent assay (ELISA). Samples were split into stratified 75%/25% training-testing sets and model outcomes were cross-validated 1,000 times. Results: Mean performance from the cross-validation analysis is summarized in the table below. Overall sensitivity was 95.2% for CRC and 57.2% for APL, with specificities of 89.8% (no CRC/APL) and 92.4% (no neoplasia). Subgroup analyses showed high sensitivity for early-stage CRC, with 93.4% at stage I and 94.2% at stage II. By APL subtype, sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSPs. Conclusions: These data support high sensitivity and specificity for a second-generation mt-sDNA panel. A multicenter pivotal trial evaluating the panel is underway (NCT04144738). [Table: see text]

Introduction Population-level screening has been shown to reduce the incidence and mortality of colorectal cancer (CRC). Unfortunately, adherence to screening recommendations among eligible US adults remains below national goals. A relatively new non-invasive screening modality, the Food and Drug Administration–approved multi-target stool DNA (mt-sDNA) assay (commercialised as Cologuard), which combines the detection of haemoglobin and DNA abnormalities, has been completed by more than 3 million individuals. Given mt-sDNA’s recent availability, the effectiveness of mt-sDNA screening with respect to CRC incidence and mortality reduction has not yet been established. Methods and analysis Through an academic–industry collaboration, a prospective cohort study (Voyage) was designed with an initial enrolment target of 150 000 individuals with mt-sDNA ordered by their healthcare provider for CRC screening. Consented participants will be asked to complete a baseline questionnaire to collect sociodemographic and health information. Additional questionnaires will be provided after 1 year, and every 3 years thereafter, to collect data regarding CRC screening follow-up in order to estimate rates of CRC incidence and other health outcomes. Linkage to the National Death Index will be used to estimate mortality rates. Ethics and dissemination The Voyage study will be conducted in accordance with international guidelines and local regulatory requirements and laws. Data will be stored and retained at Mayo Clinic. Only limited data elements required for research purposes will be transmitted between Mayo Clinic and Exact Sciences Laboratories. Results of the Voyage study will be disseminated through scientific presentations and publications. Trial registration number NCT04124406.

Abstract The gene KDM6A/UTX was recently identified as a putative tumor suppressor (van Haaften et al). The gene is frequently lost in a variety of malignancies but most commonly in esophageal squamous cell carcinomas (8%) and multiple myeloma (10%). The initial characterization of UTX abnormalities in multiple myeloma utilized a mixture of 58 cell lines and patient samples. To establish the frequency of UTX inactivation in multiple myeloma we studied the copy number profiling dataset (n=239) available from the Multiple Myeloma Genomics Initiative (MMGI) and our internal dataset of 68 human myeloma cell lines which were characterized by array-based CGH (Agilent 400k), whole exome sequencing, and mRNA sequencing. We identified homozygous deletions in 2.5% of patients and 26.5% of cell lines. There are no mutations in the available whole genome and exome sequencing data from 38 patients in the MMGI that partially overlaps the 239 copy number samples sequencing. However, in the cell lines another 14.3% of samples showed loss of UTX through a variety of mechanisms (missense, nonsense, frameshifts, single exon deletions and insertions of LINE elements) detected by exome and mRNA sequencing, bringing the total percentage of cell lines with UTX inactivation to 40.8%. The increased frequency of UTX inactivation in cell lines likely suggests that it is associated with a more aggressive disease or promotes the growth of the tumor outside of the normal bone marrow microenvironment. As part of the cell line characterization phase of the project we found that ARD has a homozygous deletion encompassing the entire UTX locus while its isogenic counterparts, ARP-1 and CAG, express wild-type UTX at a level comparable to other cell lines without UTX abnormalities. To ensure these isogenic cell lines represent the same tumor not different tumors from the same individual we cloned the immunoglobulin receptor rearrangements in all there lines, which confirmed these are derived from the same original tumor. To develop this model system we first generated single cell clones from each of the three cell lines. Six clones from each were randomly selected and their DNA content was determined by flow cytometry. In all cases the clones matched the parental population in ploidy (ARD 3.2N, ARP 3.3N, CAG 3.8N). We randomly select one subclone from each cell line to characterize in detail using spectral karyotyping, array-based CGH, whole exome sequencing, mRNA sequencing, and CHIP-seq for H3K4me3, H3K27ac, H3K27me1, H3K27me2, and H3K27me3. The detailed characterization of this isogenic model system will make it an excellent platform for future work aimed at understanding how UTX loss contributes to myelomagenesis. Citation Format: Kristi Allen, Venkata Yellapantula, Quan Zhao, David Edwards, Marta Chesi, Leif Bergsagel, John Carpten, Jonathan Keats. Characterization of an isogenic model system for KDM6A/UTX loss in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5209. doi:10.1158/1538-7445.AM2013-5209