Search

Introduction: Molecular profiling has become essential in the management of patients with biliary tract cancer (BTC). The aim of this study was to evaluate the practices of French genetics platforms in the management of BTCs., Methods: A survey was developed by a multidisciplinary group and distributed to each of the 28 French genetics platforms over a one-month period., Results: Twenty-one platforms answered the survey (75%). A majority (62%) had performed more than 50 analyses for BTCs over the last two years, with an average turnaround time for results evaluated between 11 and 15 days for 62% of them. Three quarters (76%) of the platforms performed both DNA and RNA analysis, while a quarter (24%) performed RNA analysis only. A commercial panel was used by 50% of platforms for DNA analysis, and 80% for RNA. Panels included between 10 and 50 genes for 76% of platforms. All responding platforms systematically tested for IDH1 mutations, FGFR2 fusions and BRAF mutations. A majority systematically tested for HER2 amplification, MSI status and TP53 mutation (88%, 81% and 69% respectively)., Discussion: This national survey of French genetics platforms shows good performance and compliance with recommendations for molecular analysis. However, many medical, financial and organizational obstacles remain upstream of these platforms., (Copyright © 2024 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: Multitarget stool DNA (MT-sDNA) tests (here, Cologuard®) are currently used in average-risk patients as a primary method of screening for colorectal cancer. However, MT-sDNA testing has also been used in patients who previously underwent colonoscopy who wish to avoid repeat colonoscopy. Here, in a large primary care practice setting, our aim was to evaluate the diagnostic performance of MT-sDNA testing in patients with a previously normal colonoscopy., Methods: This retrospective cohort study included 5827 patients from 35 different primary locations in South Carolina. Patients aged 45 and above with a previously documented normal, high-quality colonoscopy prior to the MT-sDNA test date were included. High-risk patients and those with a previous negative MT-sDNA result were excluded., Results: Of 5827 ordered MT-sDNA tests, 248 patients had a prior normal colonoscopy. The average time from initial colonoscopy to MT-sDNA testing was 7.3 years. Of the 63 patients who had a positive MT-sDNA test, 41 patients (65%) completed follow-up colonoscopy and 40 patients had complete colonoscopy data. Of these 40 patients, 12 patients (30%) had advanced adenomas and none had colorectal cancer. Compared to patients without a previous colonoscopy, patients with prior colonoscopies had fewer adenomas of all types (1.6 vs 2.4) and fewer advanced adenomas (1.4 vs 2.0)., Conclusion: Patients with a previously negative colonoscopy and subsequent positive MT-sDNA test were found to have a high rate of advanced adenomas on follow-up colonoscopy (30%). Thus, in patients with a previously negative colonoscopy, MT-sDNA testing may be a reasonable alternative screening option., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Flow-cytometry (FC) is a powerful tool that can assist in lymphoma diagnosis in lymph node (LN) specimens. Although lymphoma diagnosis and classification are mainly based on tumor cell characteristics, surrounding cells are less employed in this process. We retrospectively investigated alterations in the ploidy status, proliferative cell fraction (PF) and the percentages of surrounding immune cells in 62 consecutive LN specimens with B-Cell Non-Hodgkin Lymphoma (B-NHL) that were submitted for FC evaluation between 2019-2022. Compared with indolent B-NHLs, aggressive B-NHLs show increased DNA aneuploidy and PF, increased monocytes, immature-granulocytes, mature granulocytes, CD8 + T-cells, Double-Negative-T-cells and Double-Positive-T-cells, and decreased total CD45 + cells, total lymphocytes, CD4 + T-cells and CD4/CD8 ratio. Receiver operating characteristic analysis determined PF > 6.8% and immature-granulocytes > 0.9% as optimal cutoffs with highest specificity and sensitivity in differentiating aggressive and indolent B-NHLs. These findings further strength the diagnostic value of DNA content analysis by FC and suggest the utilization of tumor surrounding immune cells in NHL diagnosis and classification., (© 2024. The Author(s).)

Background: Colorectal cancers (CRCs) arise from adenomas, which can produce fecal occult blood and can be detected endoscopically, or sessile serrated lesions (SSLs), which rarely bleed and may be more challenging to detect. Models informing CRC screening policy should reflect both pathways, accounting for uncertainty., Methods: Novel decision-analytic model of the adenoma and serrated pathways for CRC (ANSER) to compare current and emerging screening strategies, accounting for differential test sensitivities for adenomas and SSLs, and uncertainty. Strategies included colonoscopy every 10 years, stool-DNA/FIT (sDNA-FIT) every 1-3 years, or fecal immunochemical testing (FIT) every year from age 45 to 75 years. Outcomes included CRC cases and deaths, cost-effectiveness (cost/quality-adjusted life-year [QALY] gained), and burden-benefit (colonoscopies/life-year gained), with 95% uncertainty intervals (UIs)., Results: ANSER predicted 62.5 (95% UI = 58.8-66.3) lifetime CRC cases and 24.1 (95% UI = 22.5-25.7) CRC deaths/1000 45-year-olds without screening, and 78%-87% CRC mortality reductions with screening. The tests' outcome distributions overlapped for QALYs gained but separated for required colonoscopies and costs. All strategies cost less than $100 000/QALY gained vs no screening. Colonoscopy was the most effective and cost-effective, costing $9300/life-year gained (95% UI = $500-$21 900) vs FIT. sDNA-FIT cost more than $500 000/QALY gained vs FIT. As more CRCs arose from SSLs, colonoscopy remained preferred based on clinical benefit and cost-effectiveness, but cost-effectiveness improved for a next-generation sDNA-FIT., Conclusion: When the serrated pathway is considered, modeling suggests that colonoscopy is cost-effective vs FIT. In contrast, modeling suggests that sDNA-FIT is not cost-effective vs FIT despite its greater sensitivity for SSLs, even if a substantial minority of CRCs arise from SSLs., (© The Author(s) 2024. Published by Oxford University Press.)

The prognostic significance of positive peritoneal cytology still varied between cancer types and geographical origin. However, because of the lack of sensitivity of this biomarker, conventional cytology is not routinely performed in every country. Here, we wanted to test a new biomarker, peritoneal tumour DNA, using NGS technique, in order to compare it with the historical one, in patients having peritoneal metastases of gastrointestinal or ovarian cancer., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Goals: The aim was to assess patient adherence to multitarget stool DNA testing as well as factors associated with adherence., Background: In the United States, disparities in colorectal cancer screening exist along racial and socioeconomic lines. While some studies suggest that stool-based screening tests may help reduce the screening gap, the data for multitarget stool DNA testing is unclear., Study: We conducted a single-center retrospective cohort study on multitarget stool DNA testing ordered between April 2020 and July 2021. We calculated the proportion of patients who completed testing and used multivariate logistic regression to identify covariates associated with test adherence., Results: Among 797 patients ordered for multitarget stool DNA testing, 481 patients (60.4%) completed testing. Adherence rates by patient subgroups ranged from 35.8% to 78.1%. Higher test adherence was found in Asian patients (odds ratio 2.65, 95% CI 1.36-5.18) and those who previously completed colorectal cancer screening (OR 1.45, 95% CI 1.01-2.09), while Black patients (OR 0.58, 95% CI 0.39-0.87), patients with resident primary care physicians (OR 0.34, 95% CI 0.21-0.56), and patients contacted through an outreach program (OR 0.47, 95% CI 0.25-0.87) had lower adherence., Conclusions: A significant proportion of patients ordered for multitarget stool DNA testing did not complete testing. Differences in adherence rates among patient subgroups may be reflective of underlying disparities in health care access., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The detection of cancer-associated nucleic acids and mutations through liquid biopsy has emerged as a highly promising non-invasive approach for early cancer detection and monitoring. In this study, we report the development of primer exchange reaction (PER) based signal amplification strategy that enables the rapid, sensitive and specific detection of nucleic acids bearing cancer specific single nucleotide mutations using flow cytometry. Using micrometer size beads as support for immobilizing oligonucleotides and programmable PER assembly for target oligonucleotide recognition and fluorescence signal amplification, we demonstrated the versatile detection of target nucleic acids including KRAS oligonucleotide, fragmented mRNAs, and miR-21. Moreover, our detection system can discriminate single base mutations frequently occurred in cancer-associated genes including KRAS, PIK3CA and P53 from cell extracts and circulating tumor DNAs (ctDNAs). The detection is highly sensitive, with a limit of detection down to 27 fM without pre-amplification. In view of a clinical application, we demonstrate the detection of single mutations after extraction and pre-amplification of ctDNAs from the plasma of breast cancer patients. Importantly, our detection strategy enabled the detection of single KRAS mutation even in the presence of 1000-fold excess of wild type (WT) DNA using multi-color flow cytometry detection approach. Overall, our strategy holds immense potential for clinical applications, offering significant improvements for early cancer detection and monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies., Methods: AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene., Results: We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable., Conclusion: Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Detection of peritoneal dissemination (PD) in gastric cancer (GC) patients remains challenging. The feasibility of tumor-guided cell-free DNA (cfDNA) detection in prospectively collected peritoneal fluid (ascites and peritoneal lavage) was investigated and compared to conventional cytology in 28 patients. Besides conventional cytology, next generation sequencing was performed on primary tumor DNA and cell-free DNA from peritoneal fluid. Patients were retrospectively grouped into: a positive group (with PD) and a negative group (without PD). Detectable mutations were found in the primary tumor of 68% (n = 19). Sensitivity of PD detection by tumor-guided cfDNA analysis was 91%, compared to 64% by conventional cytology. Within the positive group (n = 11), tumor-guided cfDNA was detected in all patients with ascites samples (4/4, 100%) and in 86% (6/7) of the lavage samples, opposed to 4/4 (100%) patients with ascites and 43% (3/7) with lavage by conventional cytology. Within the negative group (n = 8), conventional cytology was negative for all samples. In two patients, tumor-guided cfDNA was detected in peritoneal lavage fluid. Interestingly, these 2 patients developed PD within 6 months, suggesting a prognostic value of tumor-guided cfDNA detection. This study showed that tumor-guided cfDNA detection in peritoneal fluids of GC patients is feasible and superior to conventional cytology in detecting PD., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue ( p -value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp ( p -value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.

Purpose: Gastric cancers commonly spread to the peritoneum. Its presence significantly alters patient prognosis and treatment-intent; however, current methods of peritoneal staging are inaccurate. Peritoneal tumor DNA (ptDNA) is tumor-derived DNA detectable in peritoneal lavage fluid. ptDNA positivity may indicate peritoneal micrometastasis and may be more sensitive than cytology in staging the peritoneum. In this meta-analysis, we evaluated the prognostic potential of ptDNA in gastric cancer., Methods: PubMed, Embase, Scopus, and Web of Science databases were searched using PRISMA guidelines. Studies published between January 1, 1990, and April 30, 2023, containing quantitative data relating to ptDNA in gastric cancer were meta-analyzed., Results: Six studies were analyzed. Of the total 757 patients with gastric adenocarcinoma, 318 (42.0%) were stage I, 311 (41.0%) were stage II/III, 116 (15.3%) were stage IV, and 22 (2.9%) were undetermined. Overall, ptDNA detected cytology-positive cases with a sensitivity and specificity of 85.2% (95% CI, 66.5 to 100.0) and 91.5% (95% CI, 86.5 to 96.6), respectively. Additionally, ptDNA was detected in 54 (8.5%) of 634 cytology-negative patients. The presence of ptDNA negatively correlated with pathological stage I (relative risk [RR], 0.29 [95% CI, 0.13 to 0.66]) and positively correlated with pathological stage IV (RR, 8.61 [95% CI, 1.86 to 39.89]) disease. Importantly, ptDNA positivity predicted an increased risk of peritoneal-specific metastasis (RR, 13.81 [95% CI, 8.11 to 23.53]) and reduced 3-year progression-free (RR, 5.37 [95% CI, 1.39 to 20.74]) and overall (hazard ratio, 4.13 [95% CI, 1.51 to 11.32]) survival., Conclusion: ptDNA carries valuable prognostic information and can detect peritoneal micrometastases in patients with gastric cancer. Its clinical utility in peritoneal staging for gastric cancer deserves further investigation.

Introduction: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage., Methods: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis., Results: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens., Conclusion: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis., (© 2024 S. Karger AG, Basel.)

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Objective: Evaluate the performance of different DNA image cytometry (DNA-ICM) ploidy parameters in the categorisation of DNA-ICM results and identification of high-grade cervical intraepithelial neoplasia or worse (≥ CIN2)., Methods: Cervical samples from 232 women were collected for DNA-ICM analysis and biopsy confirmation. Five DNA parameters were used to define DNA aneuploidy: number of cells with exceeding events (EE) over 2.5cEE, 4cEE, 5cEE and 9cEE, and aneuploid stemlines. DNA-ICM results were categorised as normal, suspicious, and abnormal., Results: For individual DNA ploidy parameters, sensitivity values for 50 cells with 2.5cEE, 45 cells with 4cEE, 1 cell with 9cEE and aneuploid stemline were 72.95%. 54.1%, 69.67% and 54.1%, while specificity values were 80.0%, 90.0%, 89.09% and 95.45%, respectively. For the 5cEE parameter, the sensitivity values for 1, 2, 3, 4 and 5 cells were 93.44%, 85.25%, 81.97%, 77.87% and 75.41%, while specificity values were 46.36%, 63.64%, 74.55%, 76.36% and 80.91%, respectively. For categorised DNA-ICM results, a suspicious result showed superior sensitivity than an abnormal result (87.70% vs 82.79%, P = 0.031), but lower specificity (54.55% vs 75.45%, P &lt; 0.001). Both types of DNA-ICM result were statistically significantly different from a normal result (P &lt; 0.05)., Conclusion: For prognostic purposes, 1 cell with 9cEE, 45 cells with 4cEE and aneuploid stemline are the best parameters with which to categorise an abnormal DNA-ICM result, followed by 50 cells with 2.5cEE and 4 cells with 5cEE. For screening purposes, 10 cells with 2.5cEE, 10 cells with 4cEE, and 2 cells with 5cEE are suitable parameters with which to categorise a suspicious DNA-ICM result., (© 2022 John Wiley & Sons Ltd.)

In theory, any plant tissue providing intact nuclei in sufficient quantity is suitable for nuclear DNA content estimation using flow cytometry (FCM). While this certainly opens a wide variety of possible applications of FCM, especially when compared to classical karyological techniques restricted to tissues with active cell division, tissue selection and quality may directly affect the precision (and sometimes even reliability) of FCM measurements. It is usually convenient to first consider the goals of the study to either aim for the highest possible accuracy of estimates (e.g., for inferring genome size, detecting homoploid intraspecific genome size variation, aneuploidy, among others), or to decide that histograms of reasonable resolution provide sufficient information (e.g., ploidy level screening within a single model species). Here, a set of best practices guidelines for selecting the optimal plant tissue for FCM analysis, sampling of material, and material preservation and storage are provided. In addition, factors potentially compromising the quality of FCM estimates of nuclear DNA content and data interpretation are discussed., (© 2021 International Society for Advancement of Cytometry.)

Objective: Morphological indicators of chromosomal instability (CI), including multipolar mitoses, chromatin bridges (CB), strings, nuclear buds (NB), micronuclei (MN), and deoxyribonucleic acid (DNA) ploidy analysis help in prognostication of breast carcinoma. The present study was done to evaluate CI in breast carcinoma and correlate with DNA ploidy and tumor grade., Study Design: Fifty cases of carcinoma breast diagnosed by fine-needle aspiration cytology were included. Robinson's grading method was used on smears to grade breast carcinoma. To assess the morphological features of CI, the best May-Grünwald Giemsa stained smear was chosen. At least 1,000 epithelial cells on oil immersion magnification (×100 objective) were counted. DNA ploidy on the aspirates was done by flow cytometry., Results: All the patients were female, diagnosed as infiltrating ductal carcinoma on cytology. Eight tumors were grade I, 32 were grade II, and 10 were grade III. MN was seen in 48 cases, NB in 45, and CB in 12 cases. Mean MN, NB, and CB scores in aneuploid (24) cases were 9.96 ± 8.42, 5.29 ± 4.71, and 1.08 ± 1.84 while 6.19 ± 6.67, 1.92 ± 1.79, and 0.11 ± 0.33 were seen in diploid (26) cases. Statistically significant positive correlation was observed between CI and DNA ploidy., Conclusions: Morphological evaluation of CI by light microscopy on routinely stained breast aspirates is feasible, although a meticulous search is required. Cytomorphological features of CI and ploidy have a positive correlation with increasing tumor grade., (© 2022 S. Karger AG, Basel.)

Objective: To assess health care provider (HCP) preferences related to colorectal cancer (CRC) screening overall, and by HCP and patient characteristics., Participants and Methods: We developed a survey based on the Theoretical Domains Framework to assess factors associated with CRC screening preferences in clinical practice. The survey was administered online November 6 through December 6, 2019, to a validated panel of HCPs drawn from US national databases and professional organizations. The final analysis sample included 779 primary care clinicians (PCCs) and 159 gastroenterologists (GIs)., Results: HCPs chose colonoscopy as their preferred screening method for average-risk patients (96.9% (154/159) for GIs, 75.7% (590/779) for PCCs). Among PCCs, 12.2% (95/779) preferred multi-target stool DNA (mt-sDNA), followed by fecal immunochemical test (FIT), (7.3%; 57/779) and guaiac-based fecal occult blood test (gFOBT) (4.8%; 37/779). Preference among PCCs and GIs generally shifted toward noninvasive screening options for patients who were unable to undergo invasive procedures; concerned about taking time from work; unconvinced about need for screening; and refusing other screening recommendations. Among PCCs, preference for mt-sDNA over FIT and gFOBT was less frequent in larger compared with smaller clinical practices. Additionally, preference for mt-sDNA over FIT was more likely among PCCs with more years of clinical experience, higher patient volumes (> 25/day), and practice locations in suburban and rural settings (compared to urban)., Conclusion: Both PCCs and GIs preferred colonoscopy for CRC screening of average-risk patients, although PCCs did so less frequently and with approximately a quarter preferring stool-based tests (particularly mt-sDNA). PCCs' preference varied by provider and patient characteristics. Our findings underscore the importance of informed choice and shared decision-making about CRC screening options., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt-290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing., (© 2021. The Author(s).)

The implementation of precision medicine and next-generation sequencing technologies in the field of oncology is a novel approach being more widely studied and used in cases of high-risk primary and recurrent malignancies. Leukemias are the second most common cause of cancer-related mortality in children and the sixth most in adults. Relapsed leukemia represents a major component of the population that may benefit from genomic sequencing. However, ethical and analytic challenges arise when considering sequencing of biologic samples obtained from patients with relapsed leukemia following allogeneic hematopoietic stem-cell transplantation. Blood from the recipient after transplantation would include donor-derived cells and thus, genomic sequencing of recipient blood will interrogate the donor germline in addition to the somatic genetic profile of the leukemia cells and the recipient germline. This is a situation for which the donor will not have typically provided consent and may be particularly problematic if actionable secondary or incidental findings related to the donor are uncovered. We present the challenges raised in this scenario and provide strategies to mitigate this risk., Competing Interests: Joerg KruegerConsulting or Advisory Role: Novartis, Kite/Gilead, SOBISpeakers' Bureau: NovartisTravel, Accommodations, Expenses: Novartis Elizabeth A. StephensonConsulting or Advisory Role: Medtronic, Abbott Diabetes, Insulet CorporationResearch Funding: Bank of Montreal David AllanOther Relationship: Canadian Blood Services Patrick SullivanHonoraria: BayerConsulting or Advisory Role: Bayer David MalkinConsulting or Advisory Role: BayerNo other potential conflicts of interest were reported.

Purpose: This study aimed to determine if both ubiquitous and heterogeneous somatic mutations could be detected in circulating cell-free DNA (cfDNA) in patients with esophageal squamous cell carcinoma (ESCC)., Methods: Paired multi-regional tumor tissues, cfDNA, and white blood cells (WBCs) were collected from five ESCC patients before treatment, as part of an ongoing prospective study (NCT02395705). Samples from Cohort 1 were sequenced by whole-exome sequencing and samples from Cohort 2 were sequenced by targeted capture sequencing. Somatic single-nucleotide variations (SNVs) were detected by comparing solid tumor or cfDNA with matched WBCs, with a minimum variant allele frequency (VAF) of 0.1% and P value <0.05., Results: Genomic DNA (gDNA) and plasma-derived cfDNA from 26 samples were sequenced successfully. In Cohort 1, a significant linear relationship between the tumor and cfDNA VAFs (R 2 = 0.78, P < 0.0001) was found. In Cohort 2, cfDNA could recover an average of 60.7% (31/51; range, 35.7-76.2%) of somatic mutations present in matched solid tumors. There was a significant positive correlation in VAFs between cfDNA and matched solid tumor tissues (R 2 = 0.92, P < 0.0001)., Conclusions: Both sequencing approaches revealed high intratumoral heterogeneity in ESCC, and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2021. Published by Elsevier Inc.)

BACKGROUND. Noninvasive tests for colorectal cancer (CRC) screening and prevention limit the need for invasive colonoscopy to follow up positive test results. However, the relative performance characteristics of available noninvasive tests have not yet been adequately compared. OBJECTIVE. We performed a systematic review and meta-analysis to compare the diagnostic performance of the available noninvasive CRC screening tests, including multitarget stool DNA (mt-sDNA) testing, fecal immunochemical testing (FIT), and CT colonography (CTC), with an emphasis on comparison of PPV and detection rate (DR) for advanced neoplasia (AN; encompassing cases of advanced adenomas and CRC). EVIDENCE ACQUISITION. After systematic searches of MEDLINE and Google Scholar databases, 10 mt-sDNA, 27 CTC, and 88 FIT published screening studies involving 25,132, 33,493, and 2,355,958 asymptomatic adults, respectively, were included. Meta-analysis with hierarchic Bayesian modeling was conducted in accordance with Cochrane Collaboration and PRISMA guidelines to determine test positivity rates (TPRs) leading to optical colonoscopy, as well as PPVs and DRs for both AN and CRC. Different positivity thresholds were considered for FIT and CTC. EVIDENCE SYNTHESIS. Point estimates (with 95% credible intervals) from pooled Bayesian meta-analysis combining all thresholds for FIT and stratifying CTC results by a polyp size threshold of 6 mm or larger (CTC6) and 10 mm or larger (CTC10) were calculated. TPR was 13.5% (10.9-16.6%) for mt-sDNA testing, 6.4% (5.8-7.2%) for FIT, 13.4% (11.4-15.6%) for CTC6, and 6.6% (5.2-7.7%) for CTC10. AN PPV was 26.9% (95% credible interval, 21.8-33.2%) for mt-sDNA testing, 31.8% (29.3-34.5%) for FIT, 34.4% (27.2-41.0%) for CTC6, and 61.0% (54.0-70.0%) for CTC10. CRC PPV was 2.4% (1.5-3.9%) for mt-sDNA testing, 4.9% (4.3-5.3%) for FIT, 3.5% (2.5-4.8%) for CTC6, and 6.0% (4.3-8.0%) for CTC10. The DR for AN was 3.4% (95% credible interval, 2.5-4.8%) for mt-SDNA, 2.0% (1.8-2.3%) for FIT, 4.8% (4.0-6.5%) for CTC6, and 4.0% (3.0-4.6%) for CTC10. When FIT is restricted to a lower threshold (< 10 μg Hb/g feces), its performance profile is similar to that of mt-sDNA testing, although available data are limited. AN PPV odds ratios (relative to CTC10 as the reference) were 0.24 (95% credible interval, 0.17-0.33) for mt-sDNA testing, 0.30 (0.24-0.45) for FIT, and 0.33 (0.25-0.47) for CTC6. CONCLUSION. Among noninvasive CRC screening tests, CTC with a polyp size threshold of 10 mm or larger most effectively targets AN, preserving detection while also decreasing unnecessary colonoscopies compared with mt-sDNA testing and FIT. CLINICAL IMPACT. CTC performed with a polyp size threshold for colonoscopy referral set at 10 mm or larger represents the most effective and efficient noninvasive screening test for CRC prevention and detection.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

To evaluate the applicability of bisulfate conversion-free methylation assay based on enzyme digestion in fecal screening for colorectal cancer (CRC). Stool samples were collected from a total of 1142 participants with intestinal abnormalities, including 180 positive cases, 60 advanced adenomas, and 902 negative cases. DNA from reference cell lines and clinical samples was extracted and digested with an enzyme to detect the methylation of CRC markers SEPT9, SDC2, NDRG4, SFRP2, and BMP3 genes. Statistical analysis was then used to determine the ability of the markers, both individually and in combination, to detect CRC and adenoma. Our results showed that the enzyme digestion method could suitably detect DNA marker methylation in as low as 1% of the cell lines. BMP3 had a considerably low detection rate in all clinical samples, with only 6 positive cases detected out of 180 cancer samples. Our findings showed that the combination of SEPT9, SDC2, and SFRP2 had an area under the receiver operation curve of 0.937, sensitivity of 94.11%, and specificity of 89.21% for detecting CRC. Moreover, the detection sensitivity of adenoma can also reach 38.33%. After innovatively utilizing bisulfate conversion-free methylation assay for CRC screening, this study verified the potential clinical applicability of combining multiple biomarkers for CRC screening in a large number of samples., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Chong Liu et al.)

Purpose: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study., Experimental Design: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non-small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms., Results: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed., Conclusions: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network., (©2020 American Association for Cancer Research.)

Background: The standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis., Methods and Findings: We applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4-38.9; p = 0.02) compared to utDNA MRD-negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point., Conclusions: utDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.A.C. has patent filings related to cancer biomarkers, and has served as a consultant/advisor to Roche, Tempus, Geneoscopy, NuProbe, Daiichi Sankyo, AstraZeneca, Fenix Group International and Guidepoint; A.A.C. has stock options in Geneoscopy, research support from Roche, and ownership interests in Droplet Biosciences. V.K.A. has received research funding from ORIC Pharmaceuticals and currently serves as an employee of Bristol Myers Squibb; V.K.A. has stock options in both companies. Z.L.S. serves as a consultant/advisor for Photocure, outside of the submitted work. B.C.B. discloses honoraria from Mevion Medical Systems and consulting work for Regeneron/Sanofi, outside of the submitted work. R.K.B. is employed as a hospital medicine physician for SSM Health St. Louis. F.Q. has stock options in Centene, Gilead, and Horizon Therapeutics. No potential conflicts of interest were disclosed by the other authors.

Purpose: Monitoring mutation status in circulating free DNA (cfDNA) during target therapy could hold significant clinical importance in non-small cell lung cancer (NSCLC). Our aim is to establish if EGFR mutational status change on cfDNA has predictive value that can impact clinical management of NSCLC patients care., Methods: This study included 30 patients with EGFR-mutated NSCLC. Blood samples were collected at diagnosis (T0) and in 19 patients during therapy (T1)., Results: Concordance between T0 and T1 EGFR mutation status for patients evaluable for both samples (n = 19) was 79%, with a sensitivity of 100% (95% CI: 55.5-100.0) and specificity of 60.0% (95% CI: 26.2-86.8). For the patients in oncological therapy with targeted drug and with T1 sample available (n = 18), survival outcomes were evaluated. For both mutation-negative T0 and T1 patients, 12-month progression-free survival (PFS) was 66.7% (95% CI: 27.2-100.0) and 12-month overall survival (OS) was 100% (95% CI: 1.00-1.00); for patients mutated both at T0 and T1, PFS was 22.2% (95% CI: 6.5-75.4%) and OS was 55.6% (95% CI: 20.4-96.1%)., Conclusion: EGFR mutation status can be assessed using cfDNA for routine purposes and longitudinal assessment of plasma mutation is an easy approach to monitor the therapeutic response or resistance onset.

Purpose: To test whether analyzing DEPArray (Menarini Silicon Biosystems) isolated single B cells from the vitreous fluid can reveal crucial genomic and clinicopathological features to distinguish patients with vitreoretinal lymphoma (VRL) from those with chronic inflammation using immunoglobulin heavy chain (IGH), disease biomarker myeloid differentiation primary response 88 (MYD88) L265P mutation, and copy number profiling., Design: A single-center, retrospective study., Participants: Remnant vitreous biopsies from 7 patients with VRL and 4 patients with chronic inflammation were acquired for molecular analysis., Methods: Vitreous fluid samples were prefixed in PreservCyt (Hologic) and underwent cytologic analysis and immunohistochemistry examination. Single cells were isolated using the DEPArray NxT system, followed by downstream genomic analysis., Main Outcome Measures: The frequencies of the dominant IGH and MYD88 L265P mutation and the genome-wide copy number aberration (CNA) profiles of individual vitreous-isolated B cells were characterized., Results: An average of 10 to 13 vitreous B cells were used in the single-cell IGH and MYD88 analyses. Higher frequencies of dominant IGH (88.8% ± 13.2%) and MYD88 L265P mutations (35.0% ± 31.3%) were detected in patients with VRL than in patients with chronic inflammation (65.9% ± 13.4% and 1.5% ± 2.6% for IGH and MYD88 L265P , respectively). In a cytology-proven VRL case, all 15 vitreous isolated B cells were derived from the same clone with 100% paired IGH: immunoglobulin light chain (IGK) sequences. Genome-wide copy number profiling revealed a high degree of similarity between B cells from the same patient with VRL, with extensive gains and losses at the same areas across the whole genome. In addition, 14 of 15 B cells showed a BCL2/JH t(14;18) translocation, confirming cellular malignancy with a clonal origin. Clustering analysis of the copy number profiles revealed that malignant B cells derived from different patients with VRL had no common genome-wide signatures., Conclusions: Single B-cell genomic characterization of the IGH, MYD88 L265P mutation, and copy number profile enables VRL diagnosis. Because our study involved only a small cohort, these meaningful proof-of-concept data now warrant further investigation in a larger patient cohort., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Due to its poor prognosis and the late stage at which it is typically diagnosed, early detection of pancreatic cancer is a pressing clinical problem. Advances in genomic analysis of human pancreatic tissue and other biospecimens such as pancreatic cyst fluid, pancreatic juice and blood have opened the possibility of DNA-based molecular approaches for early detection of pancreatic cancer. In this Review, we discuss and focus on the pathological and molecular features of precancerous lesions of the pancreas, including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm and mucinous cystic neoplasm, which are target lesions of early detection approaches. We also discuss the most prevalent genetic alterations in these precancerous lesions, including somatic mutations in the oncogenes KRAS and GNAS as well as tumour suppressor genes CDKN2A, TP53 and SMAD4. We highlight the latest discoveries related to genetic heterogeneity and multifocal neoplasia in precancerous lesions. In addition, we review specific approaches, challenges and clinically available assays for early detection of pancreatic cancer using DNA-based molecular techniques. Although detection and risk stratification of precancerous pancreatic neoplasms are difficult problems, progress in this field highlights the promise of molecular approaches for improving survival of patients with this disease.

Purpose: The purpose of this study was to research and validate techniques for extracting DNA from human genomes, explore the sensitivity and specificity of known nucleic acid markers of intestinal malignancy in Chinese patients with early colorectal cancer. We also tried to find adenoma-specific biomarkers in human DNA in feces., Methods: We compared the ability of fecal DNA testing, Fecal Occult Blood Testing (FOBT) and serum tumor markers to diagnose different types of polyps, and DNA testing was significantly superior to the other two methods. We also found a dominant expression of NDRG12b methylation in multi-target DNA testing, which may be a promising marker for detection of colorectal precancerosis., Results: The sensitivity of NDRG4 12b methylation was 85.7% for advanced adenomatous polyp (AP), and 62.6% for non-advanced AP, respectively, with specificity of 70.8%. The diagnostic efficacy of NDRG4 12b methylation for detecting advanced AP was significantly higher than FOBT (sesitivity: 85.7% vs. 42.9%, p<0.05). The receiver operating characteristics (ROC) curve for NDRG4 12b methylation in detecting AP showed a relatively high area under the curve (AUC = 0.807)., Conclusions: Our results indicate that highly sensitive fecal DNA testing of NDRG4 12b methylation is a promising marker for detection of colorectal precancerosis, especially in detecting adenomatous polyp.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with predilection for peritoneal dissemination. Accurate peritoneal staging is imperative for treatment recommendations, as one-third of patients develop peritoneal recurrence after resection. Because >90% of PDAC tumors harbor mutant KRAS (mKRAS), we sought to determine feasibility of mKRAS DNA detection in peritoneal lavage (PL) fluid using droplet-digital polymerase chain reaction (ddPCR) via a prospective trial., Study Design: Patients with nonmetastatic PDAC undergoing staging laparoscopy with PL were included. PL fluid was sent for cytologic examination, CA19-9/CEA levels, and mKRAS ddPCR assay. Clinically positive laparoscopy was defined as gross metastases or positive cytology. PL mKRAS status was compared with gross findings, cytology, and CA19-9/CEA levels., Results: There were 136 patients enrolled; 70 of 136 (51%) patients received neoadjuvant therapy before PL, and 32 of 136 (24%) patients had clinically positive laparoscopy. Cytology was positive in 17 of 136 (13%) patients, and 22 of 136 (16%) patients had gross metastases. Of patients with gross metastases, only 8 of 22 (36%) had positive cytology; 97 of 136 (71%) patients had mKRAS in PL. PL mKRAS was present in 27 of 32 (84%) clinically positive laparoscopies, with higher mean copy number in clinically positive patients (643 vs 10, p = 0.02). Peritoneal mKRAS was positive in an additional 70 clinically negative patients., Conclusions: This prospective study establishes the feasibility of PL mKRAS detection. Clinically positive disease was identified in 1 in 4 staging laparoscopies. Although PL mKRAS was highly associated with clinically positive findings, many clinically negative laparoscopies had detectable PL mKRAS, suggesting that standard staging may be inadequate. Longer follow-up will elucidate utility of this promising molecular assay., (Copyright © 2021 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Introduction: Significant variability between colonoscopy operators contributes to postcolonoscopy colorectal cancers (CRCs). We aimed to estimate postcolonoscopy colorectal neoplasia (CRN) detection by multi-target stool DNA (mt-sDNA), which has not previously been studied for this purpose., Methods: In a retrospective cohort of patients with +mt-sDNA and completed follow-up colonoscopy, positive predictive value (PPV) for endpoints of any CRN, advanced adenoma, right-sided neoplasia, sessile serrated polyps (SSP), and CRC were stratified by the time since previous colonoscopy (0-9, 10, and ≥11 years). mt-sDNA PPV at ≤9 years from previous average-risk screening colonoscopy was used to estimate CRN missed at previous screening colonoscopy., Results: Among the 850 studied patients with +mt-sDNA after a previous negative screening colonoscopy, any CRN was found in 535 (PPV 63%). Among 107 average-risk patients having +mt-sDNA ≤9 years after last negative colonoscopy, any CRN was found in 67 (PPV 63%), advanced neoplasia in 16 (PPV 15%), right-sided CRN in 48 (PPV 46%), and SSP in 20 (PPV 19%). These rates were similar to those in 47 additional average risk persons with previous incomplete colonoscopy and in an additional 68 persons at increased CRC risk. One CRC (stage I) was found in an average risk patient who was mt-sDNA positive 6 years after negative screening colonoscopy., Discussion: The high PPV of mt-sDNA 0-9 years after a negative screening colonoscopy suggests that lesions were likely missed on previous examination or may have arisen de novo. mt-sDNA as an interval test after negative screening colonoscopy warrants further study., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2 , and THSD4 , had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. SIGNIFICANCE: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2612/F1.large.jpg., (©2021 American Association for Cancer Research.)

Purpose: Somatic mutations in the BRAF gene are common in several types of cancer, especially in ovarian serous cancer (OSC). Normally, the BRAF protein is switched on and off in response to signals that control cell growth and development., Methods: To investigate the correlation between the mutation of BRAF gene and the expression of BRAF protein in OSC, pyrosequencing was performed to detect the mutation of the 600th codon in BRAF gene (written as Val600Glu or V600E) in 23 cases of high-grade serous ovarian cancer (HGSC), 28 cases of low-grade serous ovarian cancer (LGSC) and 72 cases of serous borderline ovarian tumors (SBT). Meanwhile, immunohistochemistry which stained with the specific antibody VE1 were used to clarified the expression level of BRAF V600E mutant protein., Results: Finally, we found that V600E mutation in LGSC and SBT of occurred in 2 of 23 (7.1%) and 21of 72 (29.2%), respectively. The V600E mutation was not detected in 23 cases of HGSC. One case of HGSC (1, 4.3%), 3 cases of LGSC (3 of 28, 10.7%) and 25 cases of SBT (25 of 72, 34.7%) were positive expression detected by immunohistochemistry. Compared with BRAF gene mutation, the sensitivity, specificity and consistency of BRAF V600E protein were 91.3%, 92% and 91.9%, respectively., Conclusion: Our findings indicate that BRAF mutations in LGSC and SBT, which are closely related to tumor staging. The specific antibody VE1 could be used as a preliminary screening for the mutation of BRAF gene.

TRAF2 and TRAF3 genes of tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family are involved in diverse cell signaling, and function as both tumor suppressor gene and oncogene. Alterations of TRAF2 and TRAF3 in colon cancer (CC) along with their regional difference and microsatellite instability (MSI) are largely unknown. In the present study, we analyzed TRAF2 and TRAF3 frameshift mutations in 168 sporadic CCs (100 high MSI (MSI-H) and 68 microsatellite-stable (MSS) CCs). We identified TRAF2 and TRAF3 frameshift mutations in 4 (4%) and 3 CCs (3%) with MSI-H, respectively, but none in 68 cases of MSS CCs. Of the 168 CCs, we analyzed the mutations in multi-regions for 39 CCs (16 MSI-H and 23 MSS CCs), and discovered that 12.5% (2/16) and 6.3% (1/16) of MSI-H CCs exhibited regional difference in TRAF2 and TRAF3 mutations, respectively. In the multi-region samples of 23 MSS CCs, neither TRAF2 nor TRAF3 frameshift mutation was found. In 40% of CCs, both TRAF2 and TRAF3 expressions were increased compared to normal colon cells. Our data indicate that TRAF2 and TRAF3 frameshift mutations and their regional difference as well as altered expressions are present in MSI-H CCs, which could contribute to MSI-H cancer development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moon, Son, Choi, Yoo and Lee.)

Introduction: We evaluated the off-label use of multitarget stool DNA (mt-sDNA) testing in the primary care setting., Methods: We reviewed all mt-sDNA orders between July 1, 2018, and June 30, 2019, to determine the frequency of off-label mt-sDNA orders., Results: Nine hundred two patients with mt-sDNA orders were evaluated, of which 160/902 patients (17.7%) met at least 1 criterion for off-label mt-sDNA order. Increasing age was associated with off-label order (Odds Ratio [OR] 2.32 [95% CI, 1.86-2.89] for every 10-year increase in age, P < 0.0001). On multivariate analysis, increased age (OR 1.04 [1.02-1.06], P = 0.001) and need for diagnostic colonoscopy (OR 2.9 [1.01-8.34], P = 0.048) were associated with a positive mt-sDNA result., Discussion: Off-label mt-sDNA testing is common, and further efforts are needed to educate patients and providers on appropriate use of mt-sDNA for colorectal cancer screening.

High-specificity colorectal cancer screening is desirable to triage patients <50 years for colonoscopy; however, most endorsed colorectal cancer screening tests have not been rigorously evaluated in younger populations. This prospective cross-sectional study determined the specificity of the multitarget stool DNA (mt-sDNA) test in an average-risk screening population of 45 to 49 year-olds. Specificity was the primary outcome and was measured in participants without colorectal cancer or advanced precancerous lesions [APL- advanced adenomas (AA), and sessile serrated lesions ≥10 mm], and in the subgroup of participants with negative colonoscopic findings. APL sensitivity was a secondary outcome. The evaluable cohort included those who completed the study without protocol deviations and had a usable mt-sDNA test. Of 983 enrolled participants, 816 formed the evaluable cohort, with a mean age of 47.8 (SD, 1.5) years; 47.7% were women. No participants had colorectal cancer, 49 had APL, 253 had nonadvanced adenomas (NAA), and 514 had negative colonoscopic findings. mt-sDNA test specificity was 95.2% (95% CI, 93.4-96.6) in participants with NAA or negative findings [96.3% (confidence interval (CI), 94.3%-97.8%)] in those with negative findings, and did not differ by sex ( P = 0.75) or race ( P = 0.36) in participants with NAA or negative findings. Sensitivity for APL was 32.7% (CI, 19.9-47.5%), with most APL (83.7%) measuring 10-19 mm and none having high-grade dysplasia. The area under the ROC curve for discriminating between APL and lesser findings was 0.72 (CI, 0.64-0.81). mt-sDNA's high specificity would help minimize risk from unnecessary diagnostic procedures in this age group. This study shows that mt-sDNA has high specificity among average-risk 45 to 49-year olds, supporting its use as a noninvasive option for colorectal cancer screening. PREVENTION RELEVANCE: This study shows that mt-sDNA has high specificity among average-risk 45-49 year olds, supporting its use as a non-invasive option for colorectal cancer screening., (©2021 American Association for Cancer Research.)

Purpose: DNA ploidy measurement has earlier been suggested as a potentially powerful prognostic tool in many cancer types, but the role in renal tumors is still unclear., Methods: To clarify its prognostic impact, we analyzed the DNA content of 1320 kidney tumors, including clear cell, papillary and chromophobe renal cell carcinoma (RCC) as well as renal oncocytoma and compared these data with clinico-pathological parameters and patient prognosis., Results: A non-diploid DNA content was seen in 37% of 1276 analyzable renal tumors with a striking predominance in chromophobe carcinoma (74.3% of 70 cases). In clear cell carcinoma, a non-diploid DNA content was significantly linked to high-grade (ISUP, Fuhrman, Thoenes; p < 0.0001 each), advanced tumor stage (p = 0.0011), distant metastasis (p < 0.0001), shortened overall survival (p = 0.0010), and earlier recurrence (p < 0.0001). In papillary carcinoma, an aberrant DNA content was significantly linked to high Fuhrman grade (p = 0.0063), distant metastasis (p = 0.0138), shortened overall survival (p = 0.0010), and earlier recurrence (p = 0.0003)., Conclusion: In summary, the results of our study identify a non-diploid DNA content as a predictor of an unfavorable prognosis in clear cell and papillary carcinoma.

Prostate cancer is the most common malignancy affecting men. Prostate biopsy remains the key clinical tool for selecting appropriate treatment options. The process of specimen collection and diagnosis is multistep and vulnerable to human error along every stage. Specimen provenance testing (SPT) aims to provide certainty that biopsy results can be trusted when recommending life changing treatments and has emerged as a necessary tool in medicine to counteract human error and specimen contamination. In this study we report our practice's experience using the Know Error test to verify prostate biopsy specimens. In this study, we retrospectively reviewed the results of a specific SPT known as Know Error which is used in our institution for specimen verification during prostate biopsy. Over a period of 16 months, we identified 445 patients with a total of 921 specimens. The percentage of patients who had 1, 2 or 3 specimens analyzed was 29%, 38%, and 30%, respectively. Our cohort's rate of specimen verification was 92.8% with a 2.8% contamination rate. The pathology reports for 445 patients were then examined to determine Gleason Grade Group (GG) showing 180 GG1 and 148 GG2 patients. Cross reference of pathology reports and Know Error reports showed 8 GG1 and 9 GG2 patients had contaminated biopsy specimens. Specimen provenance complications such as contamination can negatively impact patient counselling and treatment modalities leading to unnecessary intervention and detrimental patient outcomes.

This trial assessed the utility of applying tumor DNA sequencing to treatment selection for patients with advanced, refractory cancer and somatic mutations in one of four signaling pathways by comparing the efficacy of four study regimens that were either matched to the patient's aberrant pathway (experimental arm) or not matched to that pathway (control arm)., Materials and Methods: Adult patients with an actionable mutation of interest were randomly assigned 2:1 to receive either (1) a study regimen identified to target the aberrant pathway found in their tumor (veliparib with temozolomide or adavosertib with carboplatin [DNA repair pathway], everolimus [PI3K pathway], or trametinib [RAS/RAF/MEK pathway]), or (2) one of the same four regimens, but chosen from among those not targeting that pathway., Results: Among 49 patients treated in the experimental arm, the objective response rate was 2% (95% CI, 0% to 10.9%). One of 20 patients (5%) in the experimental trametinib cohort had a partial response. There were no responses in the other cohorts. Although patients and physicians were blinded to the sequencing and random assignment results, a higher pretreatment dropout rate was observed in the control arm (22%) compared with the experimental arm (6%; P = .038), suggesting that some patients may have had prior tumor mutation profiling performed that led to a lack of participation in the control arm., Conclusion: Further investigation, better annotation of predictive biomarkers, and the development of more effective agents are necessary to inform treatment decisions in an era of precision cancer medicine. Increasing prevalence of tumor mutation profiling and preference for targeted therapy make it difficult to use a randomized phase II design to evaluate targeted therapy efficacy in an advanced disease setting., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Shivaani KummarStock and Other Ownership Interests: PathomIQ, Arxeon Consulting or Advisory Role: Corvus Pharmaceuticals, MedTree, Nodus Therapeutics, Genentech, ShangPharma Innovation, Seattle Genetics, Bayer, Boehringer Ingelheim, Mundipharma EDO GMBH, Harbour BioMed, Cadila Pharmaceuticals Research Funding: Bristol Myers Squibb, Dynavax Technologies, Pfizer, Loxo, Corvus Pharmaceuticals, Plexxikon, Jounce Therapeutics, ADC Therapeutics, Advenchen Laboratories, Incyte, Taiho Pharmaceutical, Bayer, Astex Pharmaceuticals, Seattle Genetics, Amgen, Genome & Company Travel, Accommodations, Expenses: BayerNancy MoorePatents, Royalties, Other Intellectual Property: Nestle NutritionP. Mickey WilliamsResearch Funding: Illumina Patents, Royalties, Other Intellectual Property: I was a co-inventor of the DLBCL cell of origin patent recently filed by the NIHKanwal P. S. RaghavConsulting or Advisory Role: AstraZeneca, Bayer, Eisai, Daiichi SankyoFunda Meric-BernstamEmployment: MD Anderson Cancer Center Honoraria: Mayo Clinic, Rutgers Cancer Institute of New Jersey Consulting or Advisory Role: Genentech, Inflection Biosciences, Samsung Bioepis, Spectrum Pharmaceuticals, Aduro Biotech, OrigiMed, Xencor, Debiopharm Group, Mersana, Seattle Genetics, Silverback Therapeutics, Immunomedics, IBM, Roche, PACT Pharma, eFFECTOR Therapeutics, Jackson Laboratory for Genomic Medicine, Kolon Life Sciences, Parexel International, Pfizer, Tyra Biosciences, Zymeworks, Puma Biotechnology, Zentalis, Alkermes Speakers' Bureau: Chugai Pharma Research Funding: Novartis, AstraZeneca, Taiho Pharmaceutical, Genentech, Calithera Biosciences, Debiopharm Group, Bayer, Aileron Therapeutics, Puma Biotechnology, CytomX Therapeutics, Jounce Therapeutics, Zymeworks, Curis, Pfizer, eFFECTOR Therapeutics, Abbvie, Boehringer Ingelheim, Guardant Health, Daiichi Sankyo, GlaxoSmithKline, Seattle Genetics, Millennium Travel, Accommodations, Expenses: Taiho Pharmaceutical, Beth Israel Deaconess Medical CenterStephen LeongEmployment: Merck Sharp & Dohme Stock and Other Ownership Interests: Antares Pharmaceuticals, Spectrum Pharmaceuticals Consulting or Advisory Role: Bristol Myers Squibb Research Funding: Deciphera, Karyopharm Therapeutics, Bristol Myers Squibb, Lilly Travel, Accommodations, Expenses: Genentech/RocheSaiama WaqarResearch Funding: Spectrum Pharmaceuticals, Lilly, Pfizer, Genentech/Roche, Daiichi Sankyo, Newlink Genetics, EMD Serono, Puma Biotechnology, Novartis, Xcovery, Synermore Biologics, Celgene, Vertex, Bristol Myers Squibb, Stemcentrx, Hengrui Therapeutics, Checkpoint Therapeutics, Ignyta, AstraZeneca, ARIAD, Roche, MerckBiswajit DasResearch Funding: IlluminaChris KarlovichStock and Other Ownership Interests: Clovis Oncology Research Funding: Illumina Travel, Accommodations, Expenses: IlluminaChih-Jian LihEmployment: Exelixis Stock and Other Ownership Interests: ExelixisEric PolleyResearch Funding: GRAILRichard SimonConsulting or Advisory Role: AbbVie, Amgen, Janssen, Bristol Myers Squibb, Pfizer, Onco-Nano Travel, Accommodations, Expenses: Amgen No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Aim: During the last few years, determination of microstatellite instability (MSI) status has become a routine part of clinical practice, essentially to detect Lynch syndrome. Recently, MSI testing has increased with the development of immunotherapy and has expanded to a large panel of solid tumours. The aim of our work was to evaluate a fully automated system developed by Biocartis, the Idylla MSI Test, which performs an MSI analysis within 150 min., Methods: A comparison between pentaplex PCR, immunohistochemistry and Idylla MSI Test was performed in 53 colorectal carcinoma samples, 7 small intestine adenocarcinomas, 15 duodenal and pancreatic adenocarcinomas, 16 gastric tumours, 15 endometrial adenocarcinomas, 5 ovarian carcinomas and 4 cases of urinary tract tumours using extracted DNA. Limit-of-detection (LOD) experiment was also done using a commercial DNA known to harbour MSI phenotype., Results: The overall sensitivity was 94% and the overall specificity was 100%. Two invalid and three false-negative results were observed. Our experiments showed that the amount of DNA loaded into the cartridge was decisive and should be superior to 25 ng. LOD comprised between 4% and 8%., Conclusion: Overall, we have demonstrated that the Idylla MSI Test is a rapid and valid option to detect MSI phenotype which can be used in a large panel of solid tumours., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Pediatric papillary thyroid carcinoma (PTC) is clinically and biologically distinct from adult PTC. We sequenced a cohort of clinically annotated pediatric PTC cases enriched for high-risk tumors to identify genetic alterations of relevance for diagnosis and therapy., Methods: Tumor DNA and RNA were extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation using a custom 124-gene hybridization capture panel and the 75-gene Archer Oncology Research Panel, respectively. NGS libraries were sequenced on an Illumina MiSeq., Results: Thirty-six pediatric PTC cases were analyzed. Metastases were frequently observed to cervical lymph nodes (29/36, 81%), with pulmonary metastases less commonly found (10/36, 28%). Relapsed or refractory disease occurred in 18 patients (18/36, 50%). DNA sequencing revealed targetable mutations in 8 of 31 tumors tested (26%), most commonly BRAF p.V600E (n = 6). RNA sequencing identified targetable fusions in 13 of 25 tumors tested (52%): RET (n = 8), NTRK3 (n = 4), and BRAF. Mutually exclusive targetable alterations were discovered in 15 of the 20 tumors (75%) with both DNA and RNA analyzed. Fusion-positive PTC was associated with multifocal disease, higher tumor staging, and higher American Thyroid Association risk levels. Both BRAF V600E mutations and gene fusions were correlated with the presence of cervical metastases., Conclusions: Targetable alterations were identified in 75% of pediatric PTC cases with both DNA and RNA evaluated. Inclusion of RNA sequencing for detection of fusion genes is critical for evaluation of these tumors. Patients with fusion-positive tumors were more likely to have features of high-risk disease., (© 2020 Wiley Periodicals LLC.)

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A , SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A , SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.