Hybrid Capture Assay Research Articles

Association of elevated ctDNA burden following one cycle of chemotherapy with inferior outcomes for patients with metastatic Ewing sarcoma: A report from the Children’s Oncology Group (COG).

10018 Background: All patients with Ewing sarcoma (EWS) currently receive the same intensive chemotherapy and novel risk-adapted approaches to therapy are needed. We have previously shown that elevated baseline ctDNA burden is prognostic among patients with EWS. Here we investigate the prognostic impact of on-treatment ctDNA burden in patients with metastatic EWS treated on COG AEWS1221. Methods: AEWS1221 was a randomized phase 3 trial evaluating ganitumab plus interval compressed chemotherapy (vincristine/doxorubicin/cyclophosphamide alternating with ifosfamide/etoposide given every 2 weeks) for patients with newly diagnosed metastatic EWS. We evaluated 680 serial blood samples and data from 273 patients enrolled to AEWS1221. We utilized a custom NGS hybrid-capture assay for ctDNA quantification that detects EWSR1 and FUSfusions. We used log rank tests to compare event-free survival (EFS) from the designated timepoint according to ctDNA burden post cycle 1 of VDC (planned day 14) and post cycle 2 VDC+IE (planned day 28) of chemotherapy. We dichotomized patients by ≥0.5% ctDNA at each timepoint. A P-value of ≤0.05 was considered evidence of a significant association. Results: Of 298 eligible patients with metastatic EWS, 273 provided blood samples for ctDNA analysis. Three patients had non-FET-ETS family fusions ( CIC::DUX4, EWSR1::WT1 and EWSR1::GTDC1) and were excluded from further analysis. Eighty-five percent of patients were < 21, 56% were male, 33% had pelvic bone primary tumors, and 40% had lung only metastatic disease. ctDNA was ≥0.5% in 86% of patients at baseline (median 25%, range 0-113%, n = 264), in 53% (median 0.53%, range 0-15%, n = 34) after 1 cycle (median day 14, range 11-20), and in 14% (median 0, range 0-53%, n = 130) after 2 cycles (median day 29, range 25-36). Baseline ctDNA burden ≥0.5% was associated with an increased risk of EFS-event (3-year EFS 35% [95% CI 29-41%] vs. 61% [95% CI 43-76%], HR = 2.2, P= 0.002). ctDNA ≥0.5% after cycle 1 was also associated with an increased risk of EFS-event (3-year EFS 6% [95% CI 0.4-22%] vs. 56% [95% CI 29-76%], HR = 6.0, P< 0.0001). ctDNA ≥0.5% after cycle 2 was not associated with an increased risk of EFS-event (28% [95% CI 10-49%] vs. 34% [95% CI 25-43%], HR = 1.2, P= 0.55). Conclusions: ctDNA burden ≥0.5% following one cycle of chemotherapy identifies patients highly likely to relapse, albeit in a small cohort with available data at that timepoint. Conversely, ctDNA at a cutpoint of ≥0.5% after two cycles of chemotherapy was not prognostic, suggesting either more sensitive assays are needed or the prognostic value of ctDNA burden is diminished following additional therapy. These findings will enable novel trials of risk-adapted therapy focused on baseline and early ctDNA burden.

Abstract PO2-04-06: Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib

Abstract Background: Advanced HER2-positive breast cancer remains an incurable and highly morbid condition. One of the key challenges has been treatment of central nervous system (CNS) metastases, commonly seen in those with HER2-positive disease. Tucatinib is a recently approved treatment HER2-targetting tyrosine kinase inhibitor with proven CNS activity. There has been little analysis of potential genomic mechanisms of drug resistance. Method: We retrospectively identified patients that were treated with tucatinib, capecitabine and trastuzumab. We collated baseline clinical data and treatment history and all tumour and plasma samples available were obtained for testing. DNA was extracted from plasma samples using the QIAamp DNA Mini Kit (Qiagen) on 4ml plasma samples. Targeted next generation sequencing (NGS) of serial tumour and plasma samples was performed using a custom hybrid capture assay that covered 180 genes known to be recurrently mutated in breast cancer focusing on the known genomic landscape of metastatic disease as well as the inclusion of specific actionable targets. Survival analyses was performed using Cox regression models. Results: We identified 11 patients at our institution. Plasma samples were available for all 11 patients with a total of 107 serial samples across multiple time points, with a median of 8 (range 2-19) samples per patient. All 11 patients had baseline and End of Treatment (EOT) samples. In addition, 6/11 (54%) patients had tumour sequenced on the same NGS platform prior to commencement of therapy, with 12 individual tumour samples tested. The median PFS of our cohort was 9.6 months (4.0 - NR) with a median OS of 36.2 months (10.4-NR). 7/11 (64%) patients had CNS disease at baseline with their median PFS being 13.5 months (3.9-24.4) and median OS of 36.2months (10.4-NR). Overall, we observed an average of 18.4 (4-35) mutations per patient in tumour, 5.1 (1-15) in baseline plasma and 5.8 (2-14) in EOT plasma. The most frequent mutations seen in baseline tumour samples were ERBB2 (92%), KMT2C (67%) and NCOR1 (58%) compared with KMT2C (64%), NOTCH4 (36%) and PIK3CA (27%) in baseline plasma. Patients with a lower number of ctDNA mutations at baseline showed a trend towards improved survival (PFS 14 months vs 7.5 months). In this small cohort of patients we identified a higher than expected rates of KMT2C mutations. A KMT2C mutation was identified in 67% of samples which is higher than previously described rates of 7-15% in TCGA and METABRIC cohorts respectively. The presence of a concordant ctDNA KMT2C mutation in baseline and progression samples was a poor prognostic indicator (mPFS 7.5 months) when compared with no KMT2C mutation (mPFS 15.5 months) p =0.14 suggesting a possible mechanism of resistance to tucatinib. Conclusions: In this retrospective analysis of patients treated with Tucatinib, Trastuzumab and Capecitabine we established the genomic landscape of heavily pre-treated, advanced HER2-positive disease is complex. We observed there may be a correlation with KMT2C mutations and poorer clinical outcomes, however given the very small number of patients this data remains hypothesis generating, requiring validation with larger datasets. Citation Format: Elizabeth Blackley, Courtney van Geelen, Yi-An Ko, Stephen Wong, Miriam Yeung, Stephen Luen, Sarah-Jane Dawson, Sherene Loi. Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-06.

Abstract LB105: Sensitive urothelial cancer detection via high volume urine DNA analysis

Abstract Background: The detection of cancer-associated DNA in urine samples has significant potential in urothelial cancer (UC) detection and surveillance. Every year millions of patients with urinary symptoms undergo cystoscopy, an examination of the bladder using a flexible scope. Only 10% of these invasive examinations result in a cancer diagnosis. Cystoscopy is also used in post-operative surveillance, with many UC patients requiring life-long cystoscopies. Methods: We developed a high-volume urine DNA collection kit and laboratory platform and prospectively analyzed 433 urine samples collected from diagnostic and post-operative timepoints from 167 UC patients and 95 negative controls from January 2021 to September 2023. We used a hybridization capture assay targeting the coding regions of 21 commonly mutated genes in urothelial tumors, as well as 8 frequently copy number altered loci. Samples were sequenced using Illumina NovaSeq instruments. Mutations, copy number alterations, and chromosomal rearrangements were computationally identified. Key Findings: In the diagnostic settings our assay achieved a sensitivity of 100% for pT1+ bladder cancer, 97% for pTa bladder cancer, and 91% for upper tract urothelial cancer (UTUC) (100% for pT1+ or high grade) in an all-comers UC cohort, at a specificity of 93%. Post-operative urine DNA surveillance informed by preoperative urine mutations detected 28/30 (93%) residual cancer cases found in cystoscopy or surgery, with two pTa low grade recurrences missed. Using a secondary, highly stringent urine tumor DNA detection threshold (100% specificity for UC), we show that urine DNA testing in the diagnostic setting may allow up to 68% of UC patients to be scheduled directly to operating room procedures, reducing hospital workload and expediting care. In addition, we demonstrate the feasibility of urine DNA for detecting underlying Lynch syndrome in UC patients by identifying three UTUC patients who carried germline defects in mismatch repair genes MSH2 or MSH6, indicative of Lynch syndrome. In two of these patients our assay also found a somatic second hit and high mutation load. Conclusions and Clinical Implications: These results show promise in urine DNA based cancer diagnostics even in patients with low grade tumors. The assay is designed to offer a non-invasive and cost-effective alternative for cystoscopies. Citation Format: Jussi Nikkola, Lauri Ryyppö, Juuso Vuorinen, Heini Kallio, Hanna Selin, Pyry Jämsä, Jonne Åkerla, Tarmo Pekkarinen, Antti Kaipia, Matti Nykter, Thea Veitonmäki, Matti Annala. Sensitive urothelial cancer detection via high volume urine DNA analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB105.

Abstract 949: A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity

Abstract Introduction: In many instances in Precision Oncology Medicine fragmented DNA from formalin-fixed paraffin-embedded (FFPE) samples is the only option. The majority of such degradedsamples need to be sequenced using next generation sequencing (NGS) [1]. Standardizedguidelines highlight that a minimum of 500 × coverage depth is required for tissue analyses [2].However, coverage isn’t the only metric required for a successful NGS analysis. Highly uniformDNA libraries allow clinical labs to generate more hits from their screens, saving both time andmoney across the course of diagnosis therefore ultimately benefits patient care. The commerciallyavailable single stranded DNA library prep kit tested in this study renders all input moleculessingle-stranded before ligation therefore it recovers template molecules containing DNA nicks andlabile lesions thus maximizing library complexity. Method: 10 solid tumor FFPE derived DNA samples went through three different NGS workflows:100ng of DNA input in an amplicon-based assay 325 gene panel (Assay A), 60ng input for a 500gene mechanical sheared-based double strand DNA library preparation with hybridization captureassay (~500 gene panel, Assay B), 50ng and 10ng input for a enzymatic shear-based singlestranded DNA library preparation with hybridization capture assay (WES with 1000+ enhancedgene content, Assay C). Data from assays A and C was processed with in-house bioinformaticpipeline, while Assay B’s data was processed with a commercially available bioinformatic pipelinethat was bundled with the assay. NGS QC metrics were compared across the three datasets. Results: This novel single stranded DNA library preparation with hybridization capture workflowshowed superior QC metrics compared to the other two workflows, especially on coverageuniformity and percent target above 100X, with as low as 10ng input. Conclusion: Employing a novel commercially available DNA library preparation workflow in anNGS assay, NeoGenomics was able to rescue poor quality samples that had previously failed tomeet our internal uniformity metric on other NGS assays and generate robust and reportableresults with as low as 10ng DNA input.[1] Nagahashi et al., J Surg Res. 2017 Dec; 220: 125-132.[2] Cappello et al., J Pers Med. 2022 May; 12(5): 750. Citation Format: Jieying Chu, Rachel Schell, Faqiang Wu, Michal Krawczyk, Daniel Whang, Segun Jung, Hyunjun Nam, Kenneth B. Thomas, Fernando J. Lopez-Diaz, Vincent Funari, Julie A. Mayer, Shashi Kulkarni, Jiannan Guo, Steven P. Lau-Rivera. A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 949.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

NSCLC Digital PCR Panel Returns Low-Input Sample Results Where Sequencing Fails

Molecular diagnostics has drastically improved the survival rate of patients diagnosed with non-small cell lung cancer (NSCLC) over the last 10 years. Despite advancements in molecular testing, targeted therapies, and national guideline recommendations, more than half of NSCLC patients in the United States either never receive testing or patient care is not informed via molecular testing. Here, we sought to explore the relationship between DNA/RNA input, the molecular testing method, and test success rates. On a shared set of low-input reference test materials (n = 3), we ran both a hybrid capture-based, next-generation sequencing (NGS) assay and a multiplexed digital PCR (dPCR) panel. The dPCR panel was highly sensitive and specific for low-input samples in dilution studies ranging from 40 to 1 ng DNA and from 20 to 2.5 ng RNA, while NGS had up to an 86% loss in sensitivity as contrived sample inputs were serially diluted. The dPCR panel also demonstrated a high PPA (>95%) at diluted inputs as low as 15/7.5 ng DNA/RNA on 23 banked clinical samples with the same NGS hybrid capture assay at a high input. These data suggest that digital PCR is an accurate and effective way of identifying clinically relevant NSCLC mutations at low nucleotide input and quality.

Abstract LB_A03: Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples

Abstract Activating KRAS mutations, homologous recombination deficiency (HRD), deficient mismatch repair (dMMR), and other prognostic or predictive biomarkers are seen in fewer than half of pancreatic ductal adenocarcinoma (PDAC) cases. Structural variants (SV), such as gene fusions, translocations, deletions, duplications, etc, are known to play a role in oncogenesis, metastasis, and/or treatment resistance. In solid tumors, SV discovery and validation has been limited, often due to a fresh or live tissue requirement in cytogenomic analyses. Recent innovations in processing formalin-fixed paraffin-embedded (FFPE) samples for “next-generation” chromatin capture and long-range sequencing platforms (i.e. Hi-C) have overcome this barrier. Hi-C is a genome-wide technology which surveys rearrangements, generating genomic contiguity data from highly fragmented chromatin. Unlike targeted (e.g. hybrid capture) assays, Hi-C can survey the landscape of genomic aberrations in an unbiased manner. Archived FFPE has previously been an obstacle to Hi-C analysis of solid tumors. We present here, to our knowledge, the first Hi-C analysis of FFPE PDAC samples using a previously described, proprietary Hi-C platform. Thirty-one samples from 20 unique patients diagnosed and treated at the University of Colorado Comprehensive Cancer Center between 2014-2022 were processed and analyzed. Twenty-two samples were surgical resections paired with later biopsies of metastatic recurrences. Nine were unpaired surgical resections or metastatic biopsies. Seven of these cases had somatic KRAS mutations, five had germline mutations in DNA repair genes, and none had evidence of dMMR or MSI-high. Fifty percent of samples had sufficient tumor content for processing and successful library generation. Notably, two paired samples from 2014 were successfully processed and sequenced demonstrating the applicability of this technology to archival samples. In successfully analyzed samples, the vast majority of SVs observed in metastases were also seen in paired primary tumors. Several duplications and deletions recurred across two or more patients. Furthermore, at least three SVs from this cohort were observed in other tumor types from a separate cohort analysis using this technology. Cases with relatively more SVs demonstrated a longer time to metastatic recurrence than cases with fewer SVs. Cases with germline-mutated cancer-predisposing genes demonstrated a similar SV signature and time to metastatic recurrence as some cases with or without unknown germline and somatic tumor mutation status. Overall, our findings show that this novel platform can successfully profile archived FFPE PDAC samples. Some paired samples show a shared SV signature suggesting the presence of metastatic clones at the time of initial diagnosis. Following these results, we plan to process and analyze a larger cohort of PDAC cases enriched for wild-type KRAS and HRD carriers. We plan to compare our findings to Hi-C results from other tumors and further focus on any SVs recurrent across tumor types. Citation Format: Abhishek Pandey, Stephen Eacker, Adrie Van Bokhoven, Kathleen Torko, Christopher H Lieu. Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A03.

A low-cost, paper-based hybrid capture assay to detect high-risk HPV DNA for cervical cancer screening in low-resource settings.

Cervical cancer is a leading cause of cancer death for women in low-resource settings. The World Health Organization recommends that cervical cancer screening programs incorporate HPV DNA testing, but available tests are expensive, require laboratory infrastructure, and cannot be performed at the point-of-care. We developed a two-dimensional paper network (2DPN), hybrid-capture, signal amplification assay and a point-of-care sample preparation protocol to detect high-risk HPV DNA from exfoliated cervical cells within an hour. The test does not require expensive equipment and has an estimated cost of <$3 per test without the need for batching. We evaluated performance of the paper HPV DNA assay with short synthetic and genomic HPV DNA targets, HPV positive and negative cellular samples, and two sets of clinical samples. The first set of clinical samples consisted of 16 biobanked, provider-collected cervical samples from a study in El Salvador previously tested with careHPV and subsequently tested in a controlled laboratory environment. The paper HPV DNA test correctly identified eight of eight HPV-negative clinical samples and seven of eight HPV-positive clinical samples. We then performed a field evaluation of the paper HPV DNA test in a hospital laboratory in Mozambique. Cellular controls generated expected results throughout field testing with fully lyophilized sample preparation and 2DPN reagents. When evaluated with 16 residual self-collected cervicovaginal samples previously tested by the GeneXpert HPV assay ("Xpert"), the accuracy of the HPV DNA paper test in the field was reduced compared to testing in the controlled laboratory environment, with positive results obtained for all eight HPV-positive samples as well as seven of eight HPV-negative samples. Further evaluation showed reduction in performance was likely due in part to increased concentration of exfoliated cells in the self-collected clinical samples from Mozambique compared with provider-collected samples from El Salvador. Finally, a formal usability assessment was conducted with users in El Salvador and Mozambique; the assay was rated as acceptable to perform after minimal training. With additional optimization for higher cell concentrations and inclusion of an internal cellular control, the paper HPV DNA assay offers promise as a low-cost, point-of-care cervical cancer screening test in low-resource settings.

Final Analysis of Consolidative Use of Radiotherapy to Block (CURB) Oligoprogression Trial - A Randomized Study of Stereotactic Body Radiotherapy for Oligoprogressive Metastatic Lung and Breast Cancers

Final Analysis of Consolidative Use of Radiotherapy to Block (CURB) Oligoprogression Trial - A Randomized Study of Stereotactic Body Radiotherapy for Oligoprogressive Metastatic Lung and Breast Cancers

Genomic hybrid capture assay to detect Borrelia burgdorferi: an application to diagnose neuroborreliosis in horses.

Antemortem diagnosis of neuroborreliosis in horses has been hindered by both the low sensitivity of PCR testing for Borrelia burgdorferi in CSF and the low specificity of serum:CSF ELISA ratios used to determine intrathecal antibody production against the bacterium. PCR testing of the CSF of an adult horse with acute neurologic disease for the B. burgdorferi flagellin gene was negative. However, we enriched B. burgdorferi DNA through nucleic acid hybrid capture, followed by next-generation sequencing, and identified B. burgdorferi in the CSF of the horse, confirming a diagnosis of neuroborreliosis.

Detection of M. tuberculosis in the environment as a tool for identifying high-risk locations for tuberculosis transmission

Tuberculosis (TB) remains a leading cause of infectious mortality globally, yet most cases cannot be epidemiologically linked even with extensive contact investigations and whole genome sequencing. Consequently, there remain major gaps in our understanding of where and when M. tuberculosis (Mtb) exposures occur. We aimed to investigate whether Mtb can be detected in environments where TB patients were recently present, which could serve as a tool for characterizing exposure risk. We collected 389 environment surface (ES) swabs from two high TB burden prisons in Brazil, sampling 41 (n = 340) cells occupied by individuals with active TB and 7 (n = 49) cells from individuals without TB. In a subset of pooled swabs (n = 6) and a swab from a cigarette lighter from the cell with active TB patients, we enriched Mtb DNA using RNA-bait hybrid capture assays and performed whole genome sequencing. In prison cells, Mtb DNA was detected in 55/340 (16 %) of ES swabs from cells occupied by active TB patients and none (0/49) from cells in which no active TB patients were present. Mtb was detected in 13/16 (81 %) prison cells occupied by the individuals with high/medium sputum Xpert Mtb load and 8/25 (32 %) with low/very low sputum Mtb load (p = 0.003). Seven hybrid capture samples had a median genomic coverage of 140×. rpoB mutations conferring high-level rifampin resistance were detected in 3/7 ES swabs. Mtb was frequently detectable in environments recently occupied by individuals with active TB. This approach could be applied in congregate environments to identify and characterize high-risk settings for Mtb exposure.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

Human papillomavirus bowel colonization in inflammatory bowel disease: A comparative case control study.

ABSTRACTBackground and Aims:Although much of the research on the plausible environmental triggers for inflammatory bowel disease (IBD) has focused on bacterial pathogens, the relationship between bowel colonization with human papillomavirus (HPV) and IBD has not been previously explored. In this study, we aimed to investigate the association between HPV ileocolonic colonization and IBD.Patients and Methods:We performed a cross-sectional study involving consecutive patients with established IBD who were referred for endoscopic evaluation. During endoscopy, mucosal biopsies were obtained from the most inflamed colonic or ileal segments in cases and from the rectosigmoid region for controls. A hybrid capture assay was used to detect tissue HPV. The prevalence of HPV colonization was determined for cases and controls and was compared using Fisher’s exact test.Results:A total of 201 patients, including 104 patients with IBD and 97 non-IBD controls, were prospectively included. Females comprised 55.5% of the study participants (58% vs. 55.2% for controls, P = 0.94). Fifty-seven (54.8%) patients had ulcerative colitis, and 45 (43.2%) had Crohn’s disease. The mean age was 43.2 +-18.2 years. Endoscopically active disease was documented in 56 cases (56%). HPV colonization was detected in four (4.1% subjects in controls vs. none in the cases, P = 0.05).Conclusions:There was no evidence of HPV ileocolonic colonization in this cohort of patients with IBD, regardless of disease activity. HPV colonization does not appear to be linked to IBD diagnosis or disease severity.

The FORCE Panel: An All-in-One SNP Marker Set for Confirming Investigative Genetic Genealogy Leads and for General Forensic Applications.

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3931 autosomal SNPs for extended kinship analysis, and no clinically relevant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict first to fifth degree relationships with strong statistical support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5000 SNP panel for forensic applications.

Evaluating the Performance of Hybrid Capture 2 Test as a Primary Screening Test from Studies Conducted in Low and Middle-Income Country Settings- Special Focus India.

Among the screening tests for cervical cancer, advantages of screening with second generation molecular Hybrid Capture 2 (HC2) test is the high sensitivity and negative predictive value that makes it easy to implement as a cervical cancer screening policy necessitating less screening rounds. High income countries are now implementing HC2 test in their national cervical cancer screening program. Since the acceptance of any screening test depends on the sensitivity of the test, the current study was carried out to evaluate the sensitivity of HC2 test reported from Low- and Middle-income countries (LMIC) which share major burden of cervical cancer globally and to establish if HC2 test could be used as a primary screening test in India. Materials and methods:The population based cross sectional studies from LMICs which evaluated HC2 test as a primary screening modality to diagnose Cervical intraepithelial neoplasm grade 2 and above (CIN2+) lesions were included. Results:A total of 18 studies from LMIC involving 1,13,086 women were reviewed for sensitivity of HC2 as a primary screening test. The overall average sensitivity and specificity to diagnose CIN2+ lesions were 79.84% (95% CI-71.01,86.73) and 85.63% (95% CI- 84.37,86.92) respectively. India demonstrated an average sensitivity and specificity of 65% (95% CI 57,77) and 93% (95% CI- 92,94) respectively. Conclusion:Results from LMIC demonstrate a comparably low sensitivity of HC2 test to diagnose CIN2+ lesions as compared to that reported from High income countries. Sensitivity of HC2 was substantially low for India. The current study discusses issues of HC2 assay and the role of untreated Reproductive tract infections as probable causes for low sensitivity of the test. This needs further research in an attempt to improve the sensitivity of the test in an era of self-sampling and low-cost HPV test on horizon to improve the coverage for cervical cancer.

Digital Hybridization Human Papillomavirus Assay with Attomolar Sensitivity without Amplification.

Detection of nucleic acid without amplification can avoid problems associated with thermal cycling such as labor-intensiveness and aerosol pollution. Here we develop a droplet-based digital microfluidic hybridization assay for nucleic acid detection with attomolar sensitivity. This assay provides a clinically useful sensitivity for detecting human papillomavirus (HPV) without amplification. The sensitivity is accomplished using femtoliter-sized droplet microfluidics for concentrating enzyme-catalyzed fluorescent products into a detectable signal and magnetic beads for accelerating reaction time. Meanwhile, using magnetic beads and droplet microfluidic chips, we can improve the sampling efficiency over conventional methods. We characterized the sensitivity, selectivity, detection range, stability, and accuracy of our assay. Our assay is 50-fold more sensitive than the traditional hybrid capture assay. The assay without amplification avoids problems of complex handling procedures and aerosol pollution. The direct and sensitive detection of nucleic acid using a droplet microfluidic system provides an early disease diagnosis tool.

Abstract 2187: Circulating tumor DNA (ctDNA) gene sequencing results from prospective screening of patients with newly diagnosed metastatic gastric and gastroesophageal adenocarcinoma (GEA) for the bemarituzumab FIGHT randomized trial using a plasma NGS assay

Abstract Background: Metastatic GEA is a molecularly heterogeneous disease with limited therapeutic options. It is the third most common cause of cancer death globally. Targeted therapies against HER2 positive and microsatellite instability-high (MSI-H) GEA have improved overall survival. There has not been a prospective evaluation of the prevalence of potentially actionable molecular targets in a global population with newly diagnosed advanced-stage GEA. Methods: As part of the global FIGHT study of bemarituzumab (NCT03694522), patients with newly diagnosed advanced stage GEA that was not known to be HER2 positive were screened using the PGDx elioTM Plasma Resolve Investigation Use Only assay. This next generation sequencing (NGS) hybrid-capture assay was developed to include coding and select noncoding regions of 33 cancer-related genes. Circulating cell free DNA (cfDNA) extracted from plasma samples was used as input. FGFR2-amplification positive clinical samples were identified in a primary report for enrollment in the trial. Variant calls for translocations, amplifications and SNVs/indels were reported for informational use only. Results: Of the 721 samples submitted for analyses, 661 had sufficient cfDNA to proceed with the assay, 51 samples failed to generate sufficient sequencing data to be interpreted based upon quality cutoffs. Of the 610 remaining samples 5% were amplified for FGFR2. Of these 610 samples, 496 (82%) individual patient samples were tested for additional molecular changes with 29 failing to generate sufficient sequencing data; leaving 467 patient samples with evaluable data. 81% of patients had at least one gene aberration. Of the 8 genes evaluated for amplification, the most common to least commonly amplified were MYC (10%), EGFR (7%), ERBB2 (7%), MET (7%), FGFR2 (5%), CCND1 (4%), and CD274 (1%). No KIT amplification was reported. The most frequently mutated genes among the 33 examined were p53, ARID1A, PIK3CA, BRCA2, KRAS, CDH1, APC, ATM, BRCA1 and FGFR2. Other identified mutations in genes of interest for which targeted therapies exist include mutations in NTRK1 (n=8) and ROS1 (n=15). Screening for 5 gene fusions identified fusions in 15 patients with 12 involving FGFR2. Conclusions: This is the largest prospective global survey of genetic abnormalities using targeted ctDNA testing in newly-diagnosed advanced-stage GEA in patients whose tumors were not known to be HER2 positive. Using a multi-gene panel NGS approach to identify patients with FGFR2 amplification by ctDNA, other potentially clinically actionable genetic abnormalities were identified. Citation Format: Daniel Catenacci, Yoon-Koo Kang, Peter Enzinger, Giovanni G. Cardellino, Raquel Guardeno Sanchez, Aaron Q. Xu, Allie Tappe, Michelle Y. Sun, Siddhartha Mitra, Derek Murphy, Ian Misner, Steven Smith, Zev Wainberg. Circulating tumor DNA (ctDNA) gene sequencing results from prospective screening of patients with newly diagnosed metastatic gastric and gastroesophageal adenocarcinoma (GEA) for the bemarituzumab FIGHT randomized trial using a plasma NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2187.

High-Resolution Architecture and Partner Genes of MYC Rearrangements in Lymphoma with DLBCL Morphology

High-Resolution Architecture and Partner Genes of MYC Rearrangements in Lymphoma with DLBCL Morphology

Different Methods in HPV Genotyping of Anogenital and Oropharyngeal Lesions: Comparison between VisionArray® Technology, Next Generation Sequencing, and Hybrid Capture Assay

(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray® (κ = 0.802), as well as between HC II and VisionArray® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results.

Abstract A55: Circulating tumor DNA in newly diagnosed intermediate-risk rhabdomyosarcoma

Abstract Background: Patients with intermediate-risk rhabdomyosarcoma (IR RMS) have approximately 60-70% chance of survival with current therapies. However, biomarkers of outcome and response to therapy are lacking for these patients. Circulating tumor DNA (ctDNA) has been shown to be prognostic in a wide range of cancer types, but it is unknown whether patients with IR RMS have detectable levels of ctDNA. Objective: To study ctDNA prevalence and prognostic impact in newly diagnosed IR RMS, we utilized two next-generation sequencing (NGS) approaches that detect the presence of somatic copy-number changes and oncogenic translocations. Methods: Cell-free DNA was extracted from serum samples obtained from patients with newly diagnosed IR RMS who enrolled on the COG studies ARST0531 and D9803, including patients with embryonal (ERMS) and alveolar (ARMS) histology subtypes. While both subtypes are characterized by frequent somatic copy-number variants (CNVs), ARMS is also defined by recurrent translocations. To detect CNVs, we performed ultra-low passage whole-genome sequencing (ULP-WGS). Translocations were detected with a validated custom hybrid capture assay (TranSS-Seq). Results: Serum samples were analyzed from 132 patients with IR RMS, including 75 with ERMS and 57 with ARMS. ctDNA was detected by CNVs in 70% (92/132) of IR RMS patients with similar detection rates in each histology: 65% (49/75) in ERMS and 75% (43/57) in ARMS. Among the ARMS samples sequenced, only 37% (18/49) were positive by TranSS-Seq. Furthermore, copy-number events resulting in amplifications of the somatic translocation frequently resulted in miscalculation of ctDNA content by the TranSS-Seq method. Estimates of event-free and overall survival were lower in patients with detectable ctDNA, though the differences were not statistically significant. Conclusion: Patients with IR RMS frequently have detectable levels of ctDNA that can be measured by NGS assays designed to detect somatic structural events. This study relied on previously banked serum samples and a relatively small retrospective analysis. These findings provide justification for our current efforts to utilize a large prospective study, COG ARST1431, to collect pretreatment and serial blood samples using procedures optimized for ctDNA assays. Sequencing of matched tumor samples is ongoing to understand the differences in sensitivity between ULP-WGS and TranSS-Seq for ctDNA detection in patients with ARMS. Citation Format: Samuel Abbou, David Hall, Donald A. Barkauskas, Kelly Klega, Anwesha Nag, Aaron R. Thorner, Mark Krailo, Steven Dubois, Douglas S. Hawkins, Brian D. Crompton. Circulating tumor DNA in newly diagnosed intermediate-risk rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A55.