Next-generation Sequencing-based Assay Research Articles

Clinical use of measurable residual disease in adult ALL: recommendations from a panel of U.S. experts.

Measurable residual disease (MRD) is a powerful predictor of clinical outcomes in acute lymphoblastic leukemia (ALL). In addition to its clear prognostic importance, MRD information is increasingly used in clinical decision algorithms to guide therapeutic interventions. While it is well-established that achievement of MRD-negative remission is an important endpoint of ALL therapy, the prognostic and therapeutic implications of MRD in an individual patient are influenced by both disease-related factors (e.g. cytomolecular risk) and assay-related factors (e.g. sensitivity, specimen source, and timing of assessment), which add complexity to MRD-guided treatment decisions. In this review, we discuss the data supporting the use of MRD assessment in adult ALL and how this information can rationally inform clinical decisions, including selection of patients for MRD-directed therapies or allogeneic hematopoietic stem cell transplantation. We also discuss important interpretative challenges related to novel high sensitivity next-generation sequencing-based MRD assays, which are becoming increasingly used in clinical practice.

Prognostic Performance of the Next-Generation Sequencing-Based Multigene Assay in Early Breast Cancer Patients Treated According to the 21-Gene Assay Results.

Multigene assays guide treatment decisions in early-stage hormone receptor-positive breast cancer. OncoFREE, a next-generation sequencing assay using 179 genes, was developed for this purpose. This study aimed to evaluate the concordance between the Oncotype DX (ODX) Recurrence Score (RS) and the OncoFREE Decision Index (DI) and to compare their performance. We retrospectively collected tumor blocks from patients who underwent ODX and treatment between 2012 and 2022 at four tertiary hospitals and performed OncoFREE on these samples. Distant metastasis-free survival (DMFS) was compared using RS and DI, with score cut-offs of 25 and 20, respectively. Among 838 patients, a strong correlation was observed between RS and DI (Pearson correlation coefficient 0.83). At a median follow-up of 54 months, patients with high DI had significantly worse DMFS compared to those with low DI (log-rank p < 0.001, hazard ratio [HR] 5.73, 95% confidence interval [CI] 1.87-17.57; multivariable p=0.048, HR 3.45, 95% CI 1.01-11.76). In 513 patients aged ≤50 years, DMFS was significantly different as a function of DI (p=0.035, HR 3.98, 95% CI 1.00-15.89) but not RS (p=0.792). Among 376 patients aged ≤50 years who avoided chemotherapy based on low RS, 64 with high DI had worse DMFS (p=0.015, HR 5.91, 95% CI 1.17-29.78). OncoFREE showed strong concordance with ODX and effectively identified high-risk patients, particularly in younger individuals. It could be an affordable alternative to ODX for guiding treatment in hormone receptor-positive early breast cancer.

Spatially Resolved Single-Cell Omics: Methods, Challenges, and Future Perspectives.

Overlaying omics data onto spatial biological dimensions has been a promising technology to provide high-resolution insights into the interactome and cellular heterogeneity relative to the organization of the molecular microenvironment of tissue samples in normal and disease states. Spatial omics can be categorized into three major modalities: (a) next-generation sequencing-based assays, (b) imaging-based spatially resolved transcriptomics approaches including in situ hybridization/in situ sequencing, and (c) imaging-based spatial proteomics. These modalities allow assessment of transcripts and proteins at a cellular level, generating large and computationally challenging datasets. The lack of standardized computational pipelines to analyze and integrate these nonuniform structured data has made it necessary to apply artificial intelligence and machine learning strategies to best visualize and translate their complexity. In this review, we summarize the currently available techniques and computational strategies, highlight their advantages and limitations, and discuss their future prospects in the scientific field.

Urine Biopsy as Dynamic Biomarker to Enhance Clinical Staging of Bladder Cancer in Radical Cystectomy Candidates.

There is significant interest in identifying complete responders to neoadjuvant chemotherapy (NAC) before radical cystectomy (RC) to potentially avoid removal of a pathologically benign bladder. However, clinical restaging after NAC is highly inaccurate. The objective of this study was to develop a next-generation sequencing-based molecular assay using urine to enhance clinical staging of patients with bladder cancer. Urine samples from 20 and 44 patients with bladder cancer undergoing RC were prospectively collected for retrospective analysis for molecular correlate analysis from two clinical trials, respectively. The first cohort was used to benchmark the assay, and the second was used to determine the performance characteristics of the test as it correlates to responder status as measured by pathologic examination. First, to benchmark the assay, known mutations identified in the tissue (MT) of patients from the Accelerated Methotrexate, Vinblastine, Doxorubicin, Cisplatin trial (ClinicalTrials.gov identifier: NCT01611662, n = 16) and a cohort from University of California-San Francisco (n = 4) were cross referenced against mutation profiles from urine (MU). We then determined the correlation between MU persistence and residual disease in pre-RC urine samples from a second prospective clinical trial (The pT0 trial; ClinicalTrials.gov identifier: NCT02968732). Residual MU status correlated strongly with residual disease status (pT0 trial; n = 44; P = .0092) when MU from urine supernatant and urine pellet were assessed separately and analyzed in tandem. The sensitivity, specificity, PPV, and NPV were 91%, 50%, 86%, and 63% respectively, with an overall accuracy of 82% for this second cohort. MU are representative of MT and thus can be used to enhance clinical staging of urothelial carcinoma. Urine biopsy may be used as a reliable tool that can be further developed to identify complete response to NAC in anticipation of safe RC avoidance.

Multi-centre analytical performance verification of an IVD assay to quantify donor-derived cell-free DNA in solid organ transplant recipients.

Donor-derived cell-free DNA (dd-cfDNA) has been widely studied as biomarker for non-invasive allograft rejection monitoring. Earlier rejection detection enables more prompt diagnosis and intervention, ultimately improving patient treatment and outcomes. This multi-centre study aims to verify analytical performance of a next-generation sequencing-based dd-cfDNA assay at end-user environments. Three independent laboratories received the same experimental design and 16 blinded samples to perform cfDNA extraction and the dd-cfDNA assay workflow. dd-cfDNA results were compared between sites and against manufacturer validation to evaluate concordance, reproducibility, repeatability and verify analytical performance. A total of 247 sample libraries were generated across 18 runs, with completion time of <24 h. A 96.0% first pass rate highlighted minimal failures. Overall observed versus expected dd-cfDNA results demonstrated good concordance and a strong positive correlation with linear least squares regression r2 = 0.9989, and high repeatability and reproducibility within and between sites, respectively (p > 0.05). Manufacturer validation established limit of blank 0.18%, limit of detection 0.23% and limit of quantification 0.23%, and results from independent sites verified those limits. Parallel analyses illustrated no significant difference (p = 0.951) between dd-cfDNA results with or without recipient genotype. The dd-cfDNA assay evaluated here has been verified as a reliable method for efficient, reproducible dd-cfDNA quantification in plasma from solid organ transplant recipients without requiring genotyping. Implementation of onsite dd-cfDNA testing at clinical laboratories could facilitate earlier detection of allograft injury, bearing great potential for patient care.

Practical Guidance for Clinical Microbiology Laboratories: Microbiologic diagnosis of implant-associated infections.

SUMMARYImplant-associated infections (IAIs) pose serious threats to patients and can be associated with significant morbidity and mortality. These infections may be difficult to diagnose due, in part, to biofilm formation on device surfaces, and because even when microbes are found, their clinical significance may be unclear. Despite recent advances in laboratory testing, IAIs remain a diagnostic challenge. From a therapeutic standpoint, many IAIs currently require device removal and prolonged courses of antimicrobial therapy to effect a cure. Therefore, making an accurate diagnosis, defining both the presence of infection and the involved microorganisms, is paramount. The sensitivity of standard microbial culture for IAI diagnosis varies depending on the type of IAI, the specimen analyzed, and the culture technique(s) used. Although IAI-specific culture-based diagnostics have been described, the challenge of culture-negative IAIs remains. Given this, molecular assays, including both nucleic acid amplification tests and next-generation sequencing-based assays, have been used. In this review, an overview of these challenging infections is presented, as well as an approach to their diagnosis from a microbiologic perspective.

Non-invasive rejection surveillance in stable heart transplantation using local laboratory-run donor-derived cell-free DNA assay: two-centre European feasibility study

Abstract Background Monitoring adverse events is crucial after heart transplantation (HTx). Allograft rejection is a major etiological factor of graft dysfunction, hospitalisation, and death posttransplant. Endomyocardial biopsy (EMB) is the standard method of monitoring rejection; however, there are risks and limitations associated with it. The ISHLT guidelines for the care of HTx recipients have included non-invasive surveillance such as testing for donor-derived cell-free DNA (dd-cfDNA). While major studies have been conducted with patient cohorts and centralised testing in the USA, hereby we explored a European two-site cohort where dd-cfDNA was quantified at local centres by next-generation sequencing-based CE-marked assay. Purpose Our objective was to assess feasibility of assay and evaluate dd-cfDNA values in clinically stable HTx recipients. Methods HTx patients were enrolled at their scheduled visits in this cross-sectional study in 2 European HTx centres. Serial data after HTx, including clinical status, maintenance immunosuppression therapy and drug levels, diagnostic tests (blood tests, echocardiograms, coronary angiograms, EMBs), and adverse events were collected. Venous blood specimens were drawn in Streck cfDNA blood collection tubes for quantification of dd-cfDNA levels. We analysed dd-cfDNA fraction in plasma using AlloSeq cfDNA assay. For analysis, clinical stability was defined as asymptomatic patients &amp;gt;28 days post-HTx with normal graft function, without cytomegalovirus infection, history of rejection, or allograft vasculopathy. Results Between July 2021 and December 2022, 50 HTx recipients were enrolled at a median of 274 (112–395) days posttransplant. The median age at enrolment was 55.5 years. Of the total 82 dd-cfDNA results, 34 samples were excluded as they did not meet stability criteria. The median dd-cfDNA value of stable patients was 0.11% (95% CI 0.08%–0.13%). Median levels of dd-cfDNA did not differ between samples drawn in the first year after HTx (n=40) and at later time (n=8) [0.11% vs. 0.11%; p=0.41]. Of the 48 dd-cfDNA samples, 7 had values at risk based on the dd-cfDNA cut-off values from previous clinical validation trials: 4 at the injury cut-off of ≥0.20% and 3 at the severe injury threshold of ≥0.35%. Comparing clinical variables of samples with dd-cfDNA ≥0.20% (n=7) and &amp;lt;0.20% (n=41), we observed a 3-fold, non-significant increase in rate of de novo DSA formation (33.3% vs. 10.8%; p=0.19). Conclusions Our data show feasibility to analyse dd-cfDNA at the local level. In our two-site European reference cohort, dd-cfDNA is present at low levels (median 0.11%) which is within former reported ranges for clinically stable HTx recipients. Levels of dd-cfDNA do not vary beyond the first post-HTx year. The fact that in patients with de novo DSA higher dd-cfDNA levels are found; although, not statistically significant, suggests that dd-cfDNA may be a marker of subclinical rejection and requires further study.

Diagnostic miRNA Signatures in Paired Tumor, Plasma, and Urine Specimens From Renal Cell Carcinoma Patients.

The incidence of patients diagnosed with renal cell carcinoma (RCC) is increasing. There are no approved biofluid biomarkers for routine diagnosis of RCC patients. This retrospective study aims to identify cell-free microRNA (cfmiR) signatures in urine samples that can be utilized as biomarkers for early diagnosis of sporadic RCC patients. Tissue, plasma, and urine samples (n = 221) from 56 sporadic RCC patients and respective normal healthy donors were profiled for 2083 microRNAs (miRs) using the next-generation sequencing-based HTG EdgeSeq miR Whole Transcriptome Assay. DESeq2 (FC |1.2|, false discovery rate <0.05) was performed to identify differentially expressed miRs. Data from RCC tissue samples of The Cancer Genome Atlas database were used for miR validation. We found a 10-miR signature that distinguished RCC tissues from remote normal kidney tissue or benign kidney lesion samples. Additionally, we identified subtype-specific miRs (miR-122-5p, miR-210-3p, and miR-21-3p) and miRs specific for all RCC subtypes (miR-106b-3p, miR-629-5p, and miR-885-5p). We observed that miR-155-5p was associated with tumor size. Using The Cancer Genome Atlas data sets, we validated the miRs found in RCC tissue samples. In plasma or urine analysis, we found cfmiRs that were consistently and significantly upregulated in RCC tissue samples. A 15-cfmiR signature was proposed in urine samples of RCC patients, of which miR-1275 was consistently upregulated in tissue, plasma, and urine samples. This integrative study found diagnostic miRs/cfmiRs for RCC patients, which were validated using The Cancer Genome Atlas data sets. Distinctive cfmiR signatures found in urine may have clinical utility for the diagnosis of RCC.

Local laboratory-run donor-derived cell-free DNA assay for rejection surveillance in heart transplantation-first six months of clinical experience.

Donor-derived cell-free DNA (dd-cfDNA) is a blood biomarker detecting graft injury with high negative predictive value. While non-invasive strategies for heart transplant (HTx) rejection surveillance are widely adopted in the United States with centralized testing, data on the feasibility of dd-cfDNA assay at the local level are lacking. Here, we report the first 6 months of experience with a local laboratory-run dd-cfDNA assay in the routine clinical surveillance setting. Twenty-six HTx patients with stable graft function were transitioned from endomyocardial biopsy-based (EMB) to dd-cfDNA-led rejection surveillance using a commercially available next-generation sequencing-based assay. In the 90 samples analyzed, dd-cfDNA fraction remained continuously low in most patients, thus 88% of surveillance EMBs could be safely avoided. In the case of ≥.25% dd-cfDNA, EMB was performed. There was no missed rejection. Our data show the feasibility to analyze dd-cfDNA at the local level and successful implementation of this non-invasive surveillance method into clinical practice, thus considerably reducing the frequency of invasive surveillance EMBs.

STRAP binds to and promotes the repair of N1-methyldeoxyadenosine in DNA

N1-methyladenosine (m1A) is an important RNA modification that functions in various biological processes by interacting with cellular proteins. However, the binding proteins of N1-methyldeoxyadenosine (1mdA) in DNA remain largely unknown. Herein, we employed a quantitative proteomics strategy to identify the potential binding proteins of 1mdA in human cells. Our results revealed that serine‑threonine kinase receptor-associated protein (STRAP) can bind to 1mdA-carrying DNA. We further demonstrated that STRAP participates in alkylating agent-induced DNA damage response and can promote the repair of 1mdA embedded in DNA. Moreover, we investigated the effects of STRAP on 1mdA-induced perturbation in transcription using a shuttle vector- and next-generation sequencing-based assay, and found that STRAP is involved in the transcriptional bypass of 1mdA in human cells. Together, our study revealed STRAP as a novel 1mdA-binding protein in human cells and provided new insight into the biological implications of STRAP and 1mdA modification in human diseases.

Clinical validation of next-generation sequencing-based clonality assays for minimal residual disease tracking in multiple myeloma.

e20037 Background: The prognosis of multiple myeloma (MM) patients is dependent on minimal residual disease (MRD) status after treatment. Multicolor flow cytometry (MFC) as a clinical MRD evaluation method has certain limitations regarding sample requirement, sensitivity and specificity. Next-generation sequencing (NGS)-based B cell receptor (BCR) clonality assessment has an edge in lymphoid clone MRD detection, with easier sample preparation and higher sensitivity. NGS-based BCR clonality assays were performed to evaluate curative effect and track MRD of MM patients, and its performance in testing samples with limited DNA contents was explored. Methods: In this retrospective study, newly diagnosed or relapsed MM patients with paired sample collected before and after initial treatment were included, and OriMIRACLE L NGS assay were used for BCR clonality detection in genomic DNA (gDNA) extracted from clinical residual marrow smear samples. The quantitative accuracy of NGS was compared to MFC. The concordance of MRD between MFC, NGS and clinical response assessment were analyzed. Results: From December 2016 to April 2022, 29 patients were included with a median age of 65 years old. The gDNA input of the sample collected before treatment was 100.0 ng, and the median DNA content of the sample collected after treatment was 1673.4 ±594.8 ng. The quantitative results of NGS and MFC were consistent in samples with a frequency of cancer cells of above 1x10-4 among all nucleated cells. BCR clonality detection in 23 newly diagnosed and 6 relapsed cases showed a 100% sensitivity at diagnosis. For post-treatment monitoring, patients with positive MRD by both MFC and NGS (n = 18), the MRD results were consistent with clinical response assessment. One of the patients achieved complete response (CR), while the remaining patients were unable to achieved CR. The CR patient was relapsed after 134 days. The clinical responses of 11 cases with negative MRD by MFC and positive MRD by NGS were reviewed. There were 7 cases with CR, 3 cases with very good partial response or partial response, and one case with stable disease. Among those CR patients, except for one patient who was lost to follow-up, 4 patients who underwent continuous treatment were still CR and 2 patients who had treatment discontinued recurred at 67 and 134 days. Conclusions: OriMIRACLE L NGS assay showed the higher sensitivity and specificity of MRD evaluation than MFC. For patients with negative MRD by MFS and positive MRD by NGS, recurrence might occur in case of treatment discontinuation. It is recommended to consider carefully that whether maintenance treatment is required in such cases.

Epigenotype-genotype-phenotype correlations in SETD1A and SETD2 chromatin disorders.

Germline pathogenic variants in two genes encoding the lysine-specific histone methyltransferase genes SETD1A and SETD2 are associated with neurodevelopmental disorders (NDDs) characterized by developmental delay and congenital anomalies. The SETD1A and SETD2 gene products play a critical role in chromatin-mediated regulation of gene expression. Specific methylation episignatures have been detected for a range of chromatin gene-related NDDs and have impacted clinical practice by improving the interpretation of variant pathogenicity. To investigate if SETD1A and/or SETD2-related NDDs are associated with a detectable episignature, we undertook targeted genome-wide methylation profiling of > 2M CpGs using a next-generation sequencing-based assay. A comparison of methylation profiles in patients with SETD1A variants (n = 6) did not reveal evidence of a strong methylation episignature. A review of the clinical and genetic features of the SETD2 patient group revealed that, as reported previously, there were phenotypic differences between patients with truncating mutations (n = 4, Luscan-Lumish syndrome; MIM:616831) and those with missense codon 1740 variants [p.Arg1740Trp (n = 4) and p.Arg1740Gln (n = 2)]. Both SETD2 subgroups demonstrated a methylation episignature, which was characterized by hypomethylation and hypermethylation events, respectively. Within the codon 1740 subgroup, both the methylation changes and clinical phenotype were more severe in those with p.Arg1740Trp variants. We also noted that two of 10 cases with a SETD2-NDD had developed a neoplasm. These findings reveal novel epigenotype-genotype-phenotype correlations in SETD2-NDDs and predict a gain-of-function mechanism for SETD2 codon 1740 pathogenic variants.

Abstract CT216: Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial

Abstract Background: 1L pembro + platinum-based chemo has shown clinical activity in metastatic NSCLC regardless of tissue tumor mutational burden (tTMB) status. bTMB assessed using circulating tumor DNA in plasma may be a surrogate for tTMB. The single-arm, phase 2 KEYNOTE-782 study (NCT03664024) evaluated the correlation of bTMB with efficacy of 1L pembro + chemo in nonsquamous NSCLC. Methods: Eligible patients (pts) had histologically or cytologically confirmed stage IV nonsquamous NSCLC with measurable disease per RECIST v1.1 and were not eligible for EGFR-, BRAF-, ROS1-, or ALK-directed therapy. Pts were not previously treated for advanced or metastatic disease, had an ECOG PS of 0 or 1, and had an evaluable biopsy sample for biomarker analysis. All pts received intravenous pembro 200 mg Q3W + platinum chemo doublet Q3W (pemetrexed 500 mg/m2 + 4 cycles of carboplatin AUC 5 mg/mL/min or cisplatin 75 mg/m2). The primary objective was to evaluate the association of baseline bTMB with ORR per RECIST v1.1 by investigator assessment. Secondary objectives were to determine safety and the association of baseline bTMB with PFS per RECIST v1.1 by investigator assessment and OS. A study-specific next-generation sequencing-based assay using a 1.9 Mb/654-gene cancer panel, which includes specific lung cancer-associated gene targets, was used to measure bTMB (continuous scale) in cell-free DNA extracted from baseline plasma samples. Paired white blood cell DNA sequencing was also performed to eliminate potential clonal hematopoiesis-derived somatic mutations. Database cutoff: November 5, 2021. Results: 117 pts were enrolled; median age was 64.0 years (range, 37-85), and the majority were male (60.7%), had an ECOG PS of 1 (69.2%), and no brain metastases at baseline (92.3%). Median time from first dose to data cutoff was 19.3 months (range, 1.0-35.5). ORR was 40.2% (95% CI, 31.2-49.6; 6 CRs, 41 PRs), median PFS was 7.2 months (95% CI, 5.6-9.8), and median OS was 18.1 months (95% CI, 13.5-25.6). Treatment-related adverse events (TRAEs) occurred in 113 pts (96.6%) and grade 3-5 TRAEs occurred in 56 (47.9%). Eight pts (6.8%) died due to a TRAE (febrile neutropenia and pneumonitis [n = 2 each], and septic shock, pulmonary sepsis, general physical health deterioration, and neutropenia [n = 1 each]). bTMB data were available for 101 pts. The area under the receiver operating curve for bTMB as a continuous variable to discriminate response was 0.47 (95% CI, 0.36-0.59). The posterior probabilities for a positive association of bTMB with PFS and OS were 16.8% and 7.8%, respectively. Conclusions: Baseline bTMB showed no evidence of an association with ORR, PFS, or OS in pts with nonsquamous NSCLC treated with 1L pembro + chemo. These findings indicate no clinical utility of bTMB in this patient population and treatment setting. No new safety signals were observed. Citation Format: Jair Bar, Emilio Esteban, Delvys Rodríguez-Abreu, Santiago Ponce Aix, Zsuzsanna Szalai, Enriqueta Felip, Maya Gottfried, Mariano Provencio Pulla, Andrew Robinson, Andrea Fülöp, Suman B. Rao, D. Ross Camidge, Giovanna Speranza, Steven M. Townson, Julie Kobie, Mark Ayers, Elisha J. Dettman, Robert McDaniel, Byoungsok Jung, David Burkhardt, Ruth Mauntz, Tibor Csőszi. Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT216.

Tumor mutational burden as a potential predictive marker for the efficacy of immunotherapy in advanced gastric cancer.

324 Background: Tumor mutational burden (TMB) is known as an independent predictive factor in predicting treatment response of immunotherapy. We aimed to evaluate the efficacy of immunotherapy in advanced gastric cancer (AGC) according to TMB and other markers. Methods: The patients with AGC, aged ≥ 18, who received pembrolizumab or nivolumab from October 1, 2020, to July 27, 2021, at Samsung medical center in Korea, were analyzed retrospectively. TMB was measured by next-generation sequencing-based assays, and PD-L1 is tested using immunohistochemical assay 22C3 pharmDx. Referring to the results of previous studies, TMB-high was defined as 14.31 mutations/megabase (mt/Mb) or above. Results: A total of 54 AGC patients who were treated with immunotherapy, pembrolizumab or nivolumab, were analyzed. Seven patients were TMB-high, and 47 patients were TMB-low. Compared to TMB-low group, TMB-high group showed higher overall response rate (ORR) (42.9% vs 6.4%, p=0.023), and disease control rate (66.7% vs. 22.9%; p=0.033). The median OS was longer in the TMB-high group with 22.4 months (95% CI, 19.738-25.115), while the TMB-low group with 3.6 months (95% CI, 2.109-5.104; p=0.020). In addition to TMB, microsatellite status was statistically significantly associated with the efficacy of immunotherapy in AGC. Microsatellite instability group had better ORR (n=3/4, 75%) compared to microsatellite stable group (n=4/50, 8.0%; p=0.005). Other than that, the ORR of the patients whose PD-L1 CPS was 1 or above did not differ from the patients whose PD-L1 CPS was lower than 1. Although there was no statistical significance, among TMB-low patients, PD-L1 CPS ≥1 group numerically exhibited a higher overall response rate (13.0% vs 4.0%; p=0.338). Conclusions: In our analysis, the cut-off value of TMB was set to 14.31mb/Mt, and TMB-high was associated with the better ORR and OS of immunotherapy in AGC. TMB could be a potential predicting marker for a therapeutic response of immunotherapy in AGC.[Table: see text]

Deep-learning generative models enable design of synthetic orthologs of a signaling protein.

Understanding the relationships between protein sequence and function is critical for rational protein engineering. We have developed a deep learning model based on a variational autoencoders (VAE) to perform data-driven learning of these relationships within homologous protein families. In an application to the C-terminal SH3 domain of the high osmolarity signaling protein Sho1 (Sho1-SH3) in budding yeast, our trained VAE latent space accurately discriminates Sho1-SH3 orthologs and paralogs and enables the generative design of novel synthetic proteins predicted to possess high osmosensing function. Experimental gene synthesis and in vivo functional assays by a quantitative next-generation sequencing-based selective assay demonstrates the capability of the model to design more than 500 novel synthetic proteins possessing similar or superior function to natural sequences while sharing at minimum 60% sequence similarity with any known natural SH3 domains. Our results demonstrate the potential of deep learning models to learn the rules of protein design from extant natural proteins and use these rules to rationally design novel synthetic proteins with high functionality.

Epithelioid fibrous histiocytoma: three diagnostically challenging cases with novel ALK gene fusions, unusual storiform growth pattern, and a prominent spindled morphology.

Epithelioid fibrous histiocytoma (EFH) is a distinctive cutaneous neoplasm with a relatively variable morphological appearance. Recently, it has been shown that this tumor is molecularly characterized by ALK gene fusions. We report three EFHs with unusual histological presentation represented by a prominent/predominant spindle cell proliferation arranged in a variably storiform/whirling architectural pattern with or without stromal sclerosis. One of the cases closely resembled cellular fibrous histiocytoma. All three cases were immunohistochemically ALK-positive and were analyzed for ALK gene rearrangements using a next-generation sequencing-based assay (FusionPlex Sarcoma Kit, ArcherDx). Three novel fusions, namely AP3D1::ALK, COL1A::ALK, and LRRFIP2::ALK, were detected and further confirmed by FISH in all 3 cases and RT-PCR in 1 case. All patients were elderly (62-63years) and presented with a solitary polypoid lesion on the extremities. The awareness of these morphological variants is important since it entertains a wide and slightly different differential diagnosis than conventional EFH. We also presented evidence that a clear separation of EFH from BFH in all cases may not be as straightforward as previously thought. The consistent ALK immunoexpression and the continually expanding scale of ALK gene rearrangements provide a useful tool to distinguish EFH from its histologic mimics.

Real-world evidence of the intrinsic limitations of PCR-based EGFR mutation assay in non-small cell lung cancer

Detection of driver gene mutations is important in advanced NSCLC. The cobas EGFR mutation test is a mutant allele-specific real-time PCR assay with limitation owing to its primer design. Next-generation sequencing-based assay has a higher mutation detection coverage; however, its clinical impact remains unclear. We retrospectively collected the records of stage IV NSCLC patients with wild-type EGFR tested by cobas test. FoundationOne CDx was used for comprehensive genomic profiles. We then evaluated the missed EGFR mutations by the cobas test. We studied 62 patients. The median age was 60 (range: 35–86 years). Most patients were male and 58.1% were smokers. 91.9% were adenocarcinomas. Of the 62 samples, 7 (11.3%) were detected with EGFR mutations by NGS. Among these overlooked EGFR mutations, five were exon 20 insertions, and two were exon 19 deletions. Two patients received EGFR TKIs and showed durable response with PFS 5.9 months and 10.1 months, respectively. Using NGS as the standard, the false-negative rate of the cobas EGFR mutation test was 11.3%—in a population with a high prevalence of EGFR mutations. The most overlooked mutations were exon 20 insertions. A comprehensive EGFR mutation assay can provide significant benefits to patients with NSCLC.

Next-Generation Sequencing-Based Analysis of the Effects of N1- and N6-Methyldeoxyadenosine Adducts on DNA Transcription.

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric N1- and N6-methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription. Our results revealed that 1mdA can induce multiple types of mutant transcripts and strongly inhibit DNA transcription, whereas 6mdA is a nonmutagenic DNA adduct that can exhibit a subtle but significant inhibitory effect on DNA transcription in vitro and in human cells. Moreover, our results demonstrated that the transcription-coupled nucleotide excision repair pathway is dispensable for the removal of 1mdA and 6mdA from the template DNA strand in human cells. These findings provided new important insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.

Comparison of methylation episignatures in KMT2B- and KMT2D-related human disorders.

Aim & methods: To investigate peripheral blood methylation episignatures in KMT2B-related dystonia (DYT-KMT2B), the authors undertook genome-wide methylation profiling of ∼2M CpGs using a next-generation sequencing-based assay and compared the findings with those in controls and patients with KMT2D-relatedKabuki syndrome type 1 (KS1). Results: A total of 1812 significantly differentially methylated CpG positions (false discovery rate <0.05) were detected in DYT-KMT2B samples compared with controls. Multi-dimensional scaling analysis showed that the 10 DYT-KMT2B samples clustered together and separately from 29 controls and 10 with pathogenic variants in KMT2D. The authors found that most differentially methylated CpG positions were specific to one disorder and that all (DYT-KMT2B) and most (Kabuki syndrome type 1) methylation alterations in CpG islands were gain of methylation events. Conclusion: Using sensitive methylation profiling methodology, the authors replicated recent reports of a methylation episignature for DYT-KMT2B. These findings will facilitate the development of episignature-based assays to improve diagnostic accuracy.

Driver Mutation Variant Allele Frequency in Circulating Tumor DNA and Association with Clinical Outcome in Patients with Non–Small Cell Lung Cancer and EGFR- and KRAS-Mutated Tumors

Clinical implementation of mutational analysis for next-generation sequencing-based assays has largely been of a binary nature, with pathogenic mutations being reported as either positive or negative. Actionability on the continuous output of variant allele frequency (VAF) has not been well characterized in the clinical setting, thus limiting its use. In this study, analytical validity and performance of the short variant VAF based on a next-generation sequencing-based liquid biopsy assay, FoundationOne Liquid CDx, were evaluated through assessment of precision, analytical accuracy, limit of blank, and limit of detection. Analytical validation of VAF values measured by using FoundationOne Liquid CDx supports that these values are accurate, precise, robust, and linear with the true VAF value. The association of allele frequency of clinically relevant short variants in circulating-free DNA and the association with overall survival for patients using real-world data were also assessed. The results of the association analysis indicate that VAF of the predictive biomarker mutation negatively correlates with overall survival of patients with non-small cell lung cancer.