Next-generation Sequencing Assay Research Articles

Reduced representation sequencing accurately quantifies relative abundance and reveals population-level variation in Pseudo-nitzschia spp.

Certain species within the genus Pseudo-nitzschia are able to produce the neurotoxin domoic acid (DA), which can cause illness in humans, mass-mortality of marine animals, and closure of commercial and recreational shellfisheries during toxic events. Understanding and forecasting blooms of these harmful species is a primary management goal. However, accurately predicting the onset and severity of bloom events remains difficult, in part because the underlying drivers of bloom formation have not been fully resolved. Furthermore, Pseudo-nitzschia species often co-occur, and recent work suggests that the genetic composition of a Pseudo-nitzschia bloom may be a better predictor of toxicity than prevailing environmental conditions. We developed a novel next-generation sequencing assay using restriction site-associated DNA (2b-RAD) genotyping and applied it to mock Pseudo-nitzschia communities generated by mixing cultures of different species in known abundances. On average, 94% of the variance in observed species abundance was explained by the expected abundance. In addition, the false positive rate was low (0.45% on average) and unrelated to read depth, and false negatives were never observed. Application of this method to environmental DNA samples collected during natural Pseudo-nitzschia spp. bloom events in Southern California revealed that increases in DA were associated with increases in the relative abundance of P. australis. Although the absolute correlation across time-points was weak, an independent species fingerprinting assay (Automated Ribosomal Intergenic Spacer Analysis) supported this and identified other potentially toxic species. Finally, we assessed population-level genomic variation by mining SNPs from the environmental 2bRAD dataset. Consistent shifts in allele frequencies in P. pungens and P. subpacifica were detected between high and low DA years, suggesting that different intraspecific variants may be associated with prevailing environmental conditions or the presence of DA. Taken together, this method presents a potentially cost-effective and high-throughput approach for studies aiming to evaluate both population and species dynamics in mixed samples.

Heterogeneity and viral replication fitness of HSV-1 clinical isolates with mutations in the thymidine kinase and DNA polymerase.

Prolonged antiviral therapy in immunocompromised individuals can result in the emergence of (multi)drug-resistant herpes simplex virus 1 (HSV-1) infections, forming a therapeutic challenge. To evaluate spatial and temporal differences in drug resistance of HSV-1 samples from a HSCT recipient and to determine the effect of resistance mutations on viral replication fitness. Five HSV-1 isolates were recovered from a HSCT recipient who suffered from persistent HSV-1 lesions, consecutively treated with aciclovir, foscarnet, cidofovir and a combination of ganciclovir and cidofovir. Spatial and temporal differences in HSV-1 drug resistance were evaluated genotypically [Sanger sequencing and next-generation sequencing (NGS) of the viral thymidine kinase (TK) and DNA polymerase (DP)] and phenotypically (plaque reduction assay). Viral replication fitness was determined by dual infection competition assays. Rapid evolution to aciclovir and foscarnet resistance was observed due to acquisition of TK (A189V and R222H) and DP (L778M and L802F) mutations. Virus isolates showed heterogeneous populations, spatial virus compartmentalization and minor viral variants in three out of five isolates (detectable by NGS but not by Sanger sequencing). Mutations in the TK and DP genes did not alter replication fitness without drug pressure. TK and/or DP mutants influenced replication fitness under antiviral pressure and showed increased fitness under pressure of the drug they showed resistance to. The use of NGS and dual infection competition assays revealed rapid evolution of HSV-1 drug resistance in a HSCT recipient with spatial and temporal compartmentalization of viral variants that had altered replication fitness under antiviral pressure.

Comparison of Homologous Recombination Repair Gene Next-Generation Sequencing Analysis in Patients With Metastatic Castration-Resistant Prostate Cancer Between Local and Central Laboratories in Korea.

Following success of the phase III PROfound trial, the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib was approved by the US Food and Drug Administration in May 2020 for adult patients with deleterious homologous recombination repair (HRR) gene-mutated metastatic castration-resistant prostate cancer (mCRPC). As locally adopted multigene panel next-generation sequencing (NGS) assays for selecting PARP inhibitor candidates have not been thoroughly evaluated, we compared the analytical performance of the FoundationOne CDx (Foundation Medicine, Inc., Cambridge, MA, USA) (central laboratory) and other NGS assays (local laboratory) with samples from the PROfound trial in Korea. One hundred PROfound samples (60 HRR mutation [HRRm] cases and 40 non-HRRm cases) were analyzed. The results of HRR gene mutation analysis were compared between the FoundationOne CDx and two other NGS assays [SureSelect Custom Design assay (Agilent Technologies, Inc., Santa Clara, CA, USA) and Oncomine Comprehensive assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA)]. The positive percent agreement for single nucleotide variants (SNVs) and insertion/deletions (indels) between the central laboratory and local laboratory was 98.7%-100.0%. The negative percent agreement and overall percent agreement (OPA) for SNVs and indels between central and local laboratories were both 100%. Compared with that of the FoundationOne CDx assay, the OPA for copy number variations of the Oncomine Comprehensive and SureSelect Custom assays reached 99.8%-100%. Most mCRPC patients harboring a deleterious genetic variant were successfully identified with both local laboratory assays. The NGS approach at a local laboratory showed comparable analytical performance for identifying HRRm status to the FoundationOne CDx assay used at the central laboratory.

Real-World Data on Ivosidenib in Patients with Previously Treated Isocitrate Dehydrogenase 1-Mutated Intrahepatic Cholangiocarcinomas: An Early Exploratory Analysis.

The results of the phase III ClarIDHy trial have led to US FDA approval of ivosidenib as a therapeutic option for patients with locally advanced or metastatic cholangiocarcinoma (CCA) harboring isocitrate dehydrogenase 1 (IDH1) mutations. In this study, we report the first real-world experience including eight patients with previously treated locally advanced or metastatic IDH1-mutated CCA treated with ivosidenib. Patients treated with ivosidenib as second and third line for advanced CCA were collected with the aim of evaluating the survival outcomes. A molecular study has been performed by next-generation sequencing assay. After a median follow up of 9.4 months, median progression-free survival (PFS) from the start of treatment with ivosidenib was 4.4 months (95% confidence interval [CI] 3.3-5.8), whereas median overall survival (OS) was not reached. The disease control rate was 62.5%, with two patients achieving a partial response (25%); 12.5% of patients experienced a treatment-related adverse event (AE), but no grade 3 or higher AEs were reported. The observed grade 2 AEs were prolonged QT interval and hypomagnesemia (25% of the sample). Molecular profiling was performed on six of eight patients, highlighting TP53, BAP1, CDKN2A and CDKN2B as the most common co-altered genes in these patients. Efficacy outcomes were consistent with those reported in the ClarIDHy trial. Real-world experiences on larger samples are needed in order to confirm our results.

1048P Molecular predictors of immunotherapy efficacy in lung squamous-cell carcinoma (LSCC): Results from the randomized prospective SQUINT trial

Immunotherapy as single agent or in combinationis the standard of care for metastatic non-small-cell lung cancer (NSCLC), including LSCC. The SQUINT trial was a randomized, prospective, phase II trial which assesses the efficacy of nivolumab plus investigator-choice platinum-based chemotherapy (N-CT) versus the combination of nivolumab and ipilimumab (NI) in patients with advanced/metastatic LSCC. All eligible patients were randomly assigned to either NI or N-CT. Nivolumab was administered at the standard dose of 360 mg every 3 weeks, while ipilimumab was administered at the dose of 1 mg/kg every 6 weeks. The primary endpoint was overall survival (OS) at 12 months. Availability of an adequate tumor sample obtained before trial inclusion was mandatory. All samples were analyzed using the Vela Diagnostics’ OncoKey SL 525 Plus Panelthat is a next-generation sequencing (NGS) assay that enables comprehensive genomic profiling of tumor samples. The panel contains 525 cancer-related genes that can be used to assess pathogenic single-nucleotide variants (SNV) and multiple-nucleotide variants, gene fusions, splice variants, copy number variants (CNVs), MSI, TMB, and ten oncogenic pathogens with both DNA and RNA simultaneously from FFPE samples. From September 2017 to February 2022, a total of 91 patients were included in the study; 45 were assigned to NI and 46 to N-CT. With a median follow-up of 18 months, 1-year OS rate was 59.4% with NI and 56.7% with N-CT. Median OS and median PFS were 14.7 and 3.8 months in the NI arm and 12.9 and 6.3 months in the N-CT arm. Response rate was 24.4% with NI and 34.8% with N-CT. In the NI arm median was 13.9, not reached and 16.3 months in PD-L1 negative, PD-L1 low and PD-L1 high, respectively. In the N-CT arm median OS was 12.1, 13.8 and 11.7 months in PD-L1 negative, PD-L1 low and PD-L1 high, respectively. Biomarker analyses are ongoing and results are not available at the time of this analysis. Results of the SQUINT trial showed no difference between NI and N-CT for any clinical end-point. Correlative biomarker analyses will be presented at the meeting.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work...

925P Analytical performance of a next-generation sequencing (NGS) assay kit for assessing homologous recombination deficiency (HRD) from solid tumor samples

HRD status is predictive of response to PARP inhibitors (PARPi) in ovarian cancer. Developing reliable methods to assess HRD status is important to enable genomic profiling of tumor types with PARPi indications. Here we describe the analytical performance of the TruSight™ Oncology 500 HRD (TSO 500 HRD) research assay, an in-development NGS kit for assessing HRD using DNA from solid tumor samples. The TSO 500 HRD assay was developed by adding an HRD probe pool targeting genome-wide single nucleotide polymorphisms (SNPs) to the TruSight Oncology 500 (TSO 500) assay to enable Genomic Instability Score (GIS) assessment. For each sample, the DNA library was split into two hybridization reactions, one with TSO 500 probes and the other with HRD probes. Both libraries were pooled for sequencing with 8 samples per NextSeq™ 550 run. The GIS algorithm was licensed from Myriad, re-implemented as part of the DRAGEN Bio-IT software suite and integrated into the TSO 500 analysis workflow. Forty nanograms of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples was used as assay input. Samples were also tested with a reference assay as the orthogonal test. Testing of serial dilutions of 8 ovarian cancer samples showed the limit of detection for small variant calling in BRCA1/2 genes is at 5-10% variant allele frequency (VAF), for large rearrangement variant calling in BRCA1/2 genes is at 30-50% VAF, and for GIS is at 30%-40% tumor content. The false positive rate for GIS status and BRCA variants is 0%. Testing of ovarian cancer samples and control samples with multiple operators, reagent lots, and library preparation start days showed >95% concordance in BRCA variant calling and GIS status. The GIS scores were highly concordant with the reference method assay (R = 0.98). The results were robust on both NextSeq 550 and NovaSeq™ 6000 with SP flowcells. The TSO 500 HRD assay demonstrated high analytical sensitivity and specificity in detecting BRCA variants and GIS status for HRD status assessment in ovarian cancer samples. TSO 500 HRD enables high resolution comprehensive genomic profiling alongside HRD assessment to maximize biomarker detection from limited amount of sample.

Circulating Tumor DNA After Neoadjuvant Chemotherapy in Breast Cancer Is Associated With Disease Relapse.

Detection of circulating tumor DNA (ctDNA) after neoadjuvant chemotherapy in patients with early-stage breast cancer may allow for early detection of relapse. In this study, we analyzed ctDNA using a personalized, tumor-informed multiplex polymerase chain reaction-based next-generation sequencing assay. Plasma samples (n = 157) from 44 patients were collected before neoadjuvant therapy (baseline), after neoadjuvant therapy and before surgery (presurgery), and serially postsurgery including a last follow-up sample. The primary end point was event-free survival (EFS) analyzed using Cox regression models. Thirty-eight (86%), 41 (93%), and 38 (86%) patients had baseline, presurgical, and last follow-up samples, respectively. Twenty patients had hormone receptor-positive/human epidermal growth factor receptor 2-negative, 13 had triple-negative breast cancer, and 11 had human epidermal growth factor receptor 2-positive disease. Baseline ctDNA detection was observed in 22/38 (58%) patients and was significantly associated with Ki67 > 20% (P = .036) and MYC copy-number gain (P = .0025, false discovery rate = 0.036). ctDNA detection at presurgery and at last follow-up was observed in 2/41 (5%) and 2/38 (5%) patients, respectively. Eight relapses (seven distant and one local) were noted (median follow-up 3.03 years [range, 0.39-5.85 years]). After adjusting for pathologic complete response (pCR), ctDNA detection at presurgery and at last follow-up was associated with shorter EFS (hazard ratio [HR], 53; 95% CI, 4.5 to 624; P < .01, and HR, 31; 95% CI, 2.7 to 352; P < .01, respectively). Association between baseline detection and EFS was not observed (HR, 1.4; 95% CI, 0.3 to 5.9; P = .67). The presence of ctDNA after neoadjuvant chemotherapy is associated with relapse in early-stage breast cancer, supporting interventional trials for testing the clinical utility of ctDNA monitoring in this setting.

Detection of BRCA1, BRCA2, and ATM Alterations in Matched Tumor Tissue and Circulating Tumor DNA in Patients with Prostate Cancer Screened in PROfound.

Not all patients with metastatic castration-resistant prostate cancer (mCRPC) have sufficient tumor tissue available for multigene molecular testing. Furthermore, samples may fail because of difficulties within the testing procedure. Optimization of screening techniques may reduce failure rates; however, a need remains for additional testing methods to detect cancers with alterations in homologous recombination repair genes. We evaluated the utility of plasma-derived circulating tumor DNA (ctDNA) in identifying deleterious BRCA1, BRCA2 (BRCA), and ATM alterations in screened patients with mCRPC from the phase III PROfound study. Tumor tissue samples were sequenced prospectively at Foundation Medicine, Inc. (FMI) using an investigational next-generation sequencing (NGS) assay based on FoundationOne®CDx to inform trial eligibility. Matched ctDNA samples were retrospectively sequenced at FMI, using an investigational assay based on FoundationOne®Liquid CDx. 81% (503/619) of ctDNA samples yielded an NGS result, of which 491 had a tumor tissue result. BRCA and ATM status in tissue compared with ctDNA showed 81% positive percentage agreement and 92% negative percentage agreement, using tissue as reference. At variant-subtype level, using tissue as reference, concordance was high for nonsense (93%), splice (87%), and frameshift (86%) alterations but lower for large rearrangements (63%) and homozygous deletions (27%), with low ctDNA fraction being a limiting factor. We demonstrate that ctDNA can greatly complement tissue testing in identifying patients with mCRPC and BRCA or ATM alterations who are potentially suitable for receiving targeted PARP inhibitor treatments, particularly patients with no or insufficient tissue for genomic analyses.

Viral Metagenomics for the Identification of Emerging Infections in Clinical Samples with Inconclusive Dengue, Zika, and Chikungunya Viral Amplification.

Viral metagenomics is increasingly being used for the identification of emerging and re-emerging viral pathogens in clinical samples with unknown etiology. The objective of this study was to shield light on the metavirome composition in clinical samples obtained from patients with clinical history compatible with an arboviral infection, but that presented inconclusive results when tested using RT-qPCR. The inconclusive amplification results might be an indication of the presence of an emerging arboviral agent that is inefficiently amplified by conventional PCR techniques. A total of eight serum samples with inconclusive amplification results for the routinely tested arboviruses—dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) obtained during DENV and CHIKV outbreaks registered in the state of Alagoas, Northeast Brazil between July and August 2021—were submitted to metagenomic next-generation sequencing assay using NextSeq 2000 and bioinformatic pipeline for viral discovery. The performed bioinformatic analysis revealed the presence of two arboviruses: DENV type 2 (DENV-2) and CHIKV with a high genome coverage. Further, the metavirome of those samples revealed the presence of multiple commensal viruses apparently without clinical significance. The phylogenetic analysis demonstrated that the DENV-2 genome belonged to the Asian/American genotype and clustered with other Brazilian strains. The identified CHIKV genome was taxonomically assigned as ECSA genotype, which is circulating in Brazil. Together, our results reinforce the utility of metagenomics as a valuable tool for viral identification in samples with inconclusive arboviral amplification. Viral metagenomics is one of the most potent methods for the identification of emerging arboviruses.

High-Sensitivity Mutation Analysis of Cell-Free DNA for Disease Monitoring in Endometrial Cancer.

We sought to determine whether sequencing analysis of circulating cell-free DNA (cfDNA) in patients with prospectively accrued endometrial cancer captures the mutational repertoire of the primary lesion and allows for disease monitoring. Peripheral blood was prospectively collected from 44 newly diagnosed patients with endometrial cancer over a 24-month period (i.e., baseline, postsurgery, every 6 months after). DNA from the primary endometrial cancers was subjected to targeted next-generation sequencing (NGS) of 468 cancer-related genes, and cfDNA to a high-depth NGS assay of 129 genes with molecular barcoding. Sequencing data were analyzed using validated bioinformatics methods. cfDNA levels correlated with surgical stage in endometrial cancers, with higher levels of cfDNA being present in advanced-stage disease. Mutations in cfDNA at baseline were detected preoperatively in 8 of 36 (22%) patients with sequencing data, all of whom were diagnosed with advanced-stage disease, high tumor volume, and/or aggressive histologic type. Of the 38 somatic mutations identified in the primary tumors also present in the cfDNA assay, 35 (92%) and 38 (100%) were detected at baseline and follow-up, respectively. In 6 patients with recurrent disease, changes in circulating tumor DNA (ctDNA) fraction/variant allele fractions in cfDNA during follow-up closely mirrored disease progression and therapy response, with a lead time over clinically detected recurrence in two cases. The presence of ctDNA at baseline (P < 0.001) or postsurgery (P = 0.014) was significantly associated with reduced progression-free survival. cfDNA sequencing analysis in patients with endometrial cancer at diagnosis has prognostic value, and serial postsurgery cfDNA analysis enables disease and treatment response monitoring. See related commentary by Grant et al., p. 305.

Targeted Next-Generation Sequencing for Comprehensive Testing for Selected Vector-Borne Pathogens in Canines.

The standard for detecting vector-borne pathogens is real-time PCR (rtPCR). However, this requires many individual tests to obtain an accurate diagnosis. The purpose of this study was to develop and validate a targeted next-generation sequencing (NGS) assay for vector-borne pathogens. Pathogen target regions were amplified via PCR using two primer pools that were developed in conjunction with ThermoFisher Scientific, and barcoded DNA libraries were prepared and sequenced with the Ion Torrent S5 system. Data were assembled using SPAdes and mapped to a reference file containing sequences from the pathogens. The raw reads were analyzed to confirm the results. Test feasibility and analytical specificity were evaluated with type strains or validated positive clinical samples from dogs. The analytical sensitivity of the method was compared to Ct values obtained by rtPCR testing. Diagnostic sensitivity and specificity were assessed with a set of known positive and negative clinical samples based on rtPCR testing. Positive and negative percent agreements and Cohen’s kappa were calculated. The primer sets were specific for the intended targets, based on sequence analysis of the amplified products, and the method detected 17 different pathogens. Analytical sensitivity was equivalent to an rtPCR Ct value of approximately 35–36. The positive percent agreement was 92%, and the negative percent agreement was 88%. Cohen’s kappa was 0.804, which indicates almost perfect agreement between the rtPCR assays and the targeted NGS assay. Using a targeted method reduces the costs associated with NGS sequencing and allows for a 2–3 day turn-around time, making this a viable method for detection of vector-borne pathogens in canine whole blood samples.

Prevalence of BRCA1 and BRCA2 pathogenic variants in 8627 unselected patients with breast cancer: stratification of age at diagnosis, family history and molecular subtype.

Comprehensively analyzing the prevalence of BRCA1/2 germline pathogenic variants (PVs) in a large cohort of unselected Chinese patients with breast cancer has great clinical importance. Germline pathogenic variants in full-length BRCA1/2 genes were determined through next-generation sequencing and/or Sanger sequencing assays in 8627 unselected Chinese patients with breast cancer who were treated at the Breast Center of Peking University Cancer Hospital. The prevalence of BRCA1/2 PVs was further stratified by age at diagnosis, family history of cancer and molecular subtype. We found that the overall prevalence of BRCA1/2 PVs was 6.0% in the entire cohort, 2.4% in BRCA1 and 3.7% in BRCA2. The prevalence of BRCA1/2 PVs in patients with early-onset breast cancer (age at diagnosis ≤ 40years) was significantly higher than that in patients over the age of 40 (9.7% vs. 5.1%). The prevalence rates of BRCA1/2 PVs in patients with a family history of breast, ovarian, pancreatic, and prostate cancer were 19.5%, 39.0%, 11.1%, and 12.8%, respectively. Moreover, the number of relatives affected by breast cancer was associated with a higher prevalence of BRCA1/2 PVs. Molecular subtypes were associated with the prevalence of BRCA1/2 PVs. Patients with the triple-negative phenotype had the highest prevalence of BRCA1/2 PVs (13.3%) among the three molecular groups, followed by the HR + and HER2- group (5.9%), and the lowest was in the HER2 + group (2.5%). Our study provides the most comprehensive information to date on the prevalence of BRCA1/2 PVs in unselected Chinese patients with breast cancer.

Targeted NGS analysis of the canonical genes in 274 Indian patients with suspected myeloproliferative neoplasms: An Indian diagnostic laboratory’s perspective

Myeloproliferative neoplasms (MPNs) are caused by somatic pathogenic variants that stimulate increased production and clonal expansion of CD34 multipotent hematopoietic stem cells. Recent World Health Organization (WHO) diagnostic criteria for the diagnosis of Philadelphia chromosome (Ph) negative MPNs includes detection of mutations in the Janus Kinase 2 (), myeloproliferative leukemia (), and calreticulin () genes. The purpose of this study was to demonstrate the clinical utility of an in-house next-generation sequencing (NGS) assay targeting only these canonical genes for the molecular diagnosis of patients with Ph-negative MPNs. We tested 274 samples of patients clinically suspected of having Ph-negative MPNs using an in-house developed NGS panel. The assay consists of two parts, a multiplexed PCR and a highly multiplexed NGS workflow capable of handling diverse samples. The assay is capable of simultaneously detecting mutations in exons 12 and 14, exon 9, and exon 10. Of the 274 samples tested, 49 samples harbored mutations in the gene (48 for the V617F and 1 for exon 12), 31 harbored mutations in the gene, and two harbored mutations in the gene. One sample harbored a mutation each in the and genes. Here, we present the distribution of mutations in an Indian cohort of 274 patients from India with Ph-negative MPNs. Moreover, we have successfully demonstrated the clinical utility of our in-house multiplexed NGS assay for the molecular diagnosis of MPNs with varying mutation depths

Clinicopathologic Characteristics and Outcomes for Patients With KRAS G12D-Mutant NSCLC

IntroductionCo-occurring mutations in KRAS-mutant NSCLC are associated with discrete biological properties and modulate therapeutic susceptibilities. As G12D-specific inhibitors are expected to enter the clinic, we sought to investigate the characteristics and outcomes of patients with KRAS G12D-mutant NSCLC. MethodsThis was a retrospective single-institution study. Patients with NSCLC and KRAS G12D mutations detected by the Massachusetts General Hospital SNaPshot next-generation sequencing assay were identified. Clinical and pathologic characteristics were collected by chart review. ResultsA total of 107 patients with KRAS G12D-mutant NSCLC were identified. Most patients were former smokers (80, 74.8%) and had tumors with adenocarcinoma pathologic subtype (93, 86.9%). Among 56 patients evaluated for programmed death-ligand 1 expression, tumor proportion score was less than 50% in 43 (76.8%). Concomitant mutations were identified in STK11 (17 of 107, 15.9%), KEAP1 (10 of 58, 17.2%), TP53 (36 of 107, 33.6%), and SMARCA4 (11 of 107, 10.3%). Among 57 patients treated with first-line therapy, patients with STK11 co-mutations had shorter progression-free survival (1.2 mo, 95% confidence interval [CI]: 0.6–2.9 versus 4.1 mo, 95% CI: 2.5–6.0, p = 0.0235) and overall survival (4.3 mo, 95% CI: 1.2–10.6 versus 17.9 mo, 95% CI: 8.6–31.1, p = 0.0018) compared with wild type. Patients with KEAP1 co-mutations had shorter overall survival (4.6 mo, 95% CI: 1.2–10.6 versus 17.9 mo, 95% CI: 7.1–30.1, p = 0.0125) than those without. TP53 co-mutations exerted no influence on survival. ConclusionsCo-occurring mutations were common in patients with KRAS G12D-mutant NSCLC. STK11 and KEAP1 co-mutations were associated with worse clinical outcomes, whereas co-occurring TP53 did not affect survival.

Molecular and pathologic data can guide selection of endometrioid endometrial cancer patients for ovarian preservation (239)

<b>Objectives:</b> To investigate the association of The Cancer Genome Atlas (TCGA) molecular subtype, <i>CTNNB1</i> (B-catenin) hotspot mutation, and pathologic factors with concurrent or recurrent ovarian disease in endometrial cancer (EC) patients (pts). <b>Methods:</b> EC pts aged ≤50 years were grouped by those electing oophorectomy (BO) versus ovarian preservation (OP) at staging surgery (January 2010-June 2021). ECs were classified as TCGA subtypes using a clinical tumor-normal next-generation sequencing (NGS) assay and immunohistochemistry data: <i>POLE</i> hotspot mutated, microsatellite instability-high (MSI-H), copy number high (CN-high), or copy number low (CN-low). <i>CTNNB1</i> hotspot mutations were classified. Germline data identified Lynch syndrome pts. Associations between molecular/pathologic features and concurrent ovarian disease (synchronous or metastatic) in BO pts were compared with Wilcoxon rank-sum and Fisher's exact tests. Associations with isolated ovarian recurrences in OP pts were examined with time to event outcomes. <b>Results:</b> Of 420 pts, 317 underwent BO, and 103 OP followed institutional guidelines (Table 1). Among BO pts with NGS (<i>n</i>=90), 14 (16%) had <i>POLE,</i> 22 (24%) MSI-H, six (7%) CN-high and 48 (53%) CN-low EC. Pathogenic <i>CTNNB1</i> mutations were present in 30/90 (33%) ECs. Of 102 pts with germline testing, 28 (28%) had Lynch syndrome. TCGA classification for OP pts with NGS (<i>n</i>=37) included six (16%) <i>POLE</i>, one (3%) MSI-H, one (3%) CN-high and 29 (78%) CN-low EC. Pathogenic <i>CTNNB1</i> mutations were present in 19/37 (51%) ECs. One of 23 (4%) pts with germline testing had Lynch syndrome. Of 317 BO pts, 27 (9%) had a concurrent ovarian disease. Among BO pts with NGS, concurrent ovarian tumors were diagnosed in 0/14 (0%) <i>POLE</i>, 2/48 (4%) CN-low, 10/22 (45%) MSI-H and 3/6 (50%) CN-high ECs (p<0.001). Concurrent ovarian disease was present in 1/37 (3%) hotspot <i>CTNNB1</i> mutated versus 9/58 (16%) wildtype/<i>CTNNB1</i> non-hotspot mutated ECs (p=0.15). Seven of 28 (25%) Lynch versus 7/74 (10%) non-Lynch syndrome pts had concurrent ovarian tumors (p=0.06). In BO pts, concurrent ovarian disease was associated with FIGO grade 2-3 versus grade 1 EC (20% vs 5%; p<0.001), positive versus negative lymphovascular space invasion (LVSI) (20% vs 7%; p=0.004), positive versus negative washings (28% vs 7%; p=0.016) and ≥50% versus <50% myoinvasion (24% vs 7%; p=0.006). Of 103 OP pts, four (4%) had isolated ovarian recurrences over a median follow-up of 17 months. Two pts had high-risk pathologic features and two had high-risk molecular features (CN-high EC and Lynch syndrome). <b>Conclusions:</b> Molecular and pathologic data improve risk stratification of pre-menopausal EC pts and enhance patient selection for ovarian preservation. In addition to Lynch syndrome, concurrent ovarian disease was seen more frequently in CN-high and MSI-H tumors or those with grade 2-3, LVSI, deep myoinvasion, and positive washings. Isolated ovarian recurrences may be independently associated with high-risk molecular features.

Detection of molecular signatures of homologous recombination deficiency in endometrial cancer (180)

<b>Objectives:</b> Homologous recombination repair deficiency (HRD) is observed in endometrial cancer (EC). Tumors harboring HRD are candidates for poly-ADP-ribose polymerase inhibitor (PARP inhibitor), and several PARP inhibitor trials in EC are underway. However, the prevalence and mechanism of HRD in endometrial cancer are less characterized. <b>Methods:</b> Ninety-five patients with endometrial cancer with a median follow-up of 55 months were included in this study. The homologous repair status was determined using genomics scar scores (including loss-of-heterozygosity, large-scale transitions, number of telomeric allelic imbalances, and ploidy). In addition, microsatellite instability (MSI), tumor mutation burden (TMB), and genomic mutation landscape were obtained using a panel-based next-generation sequencing assay. <b>Results:</b> Among 95 patients, we detected 15/95 (15.8%) patients with HRD (HRD score > 0). Homologous repair pathway and DNA damage pathway alterations did not contribute to HRD. Patients with HRD were associated with increased <i>TP53</i> mutation (HRD vs HRP, 92.3% vs 15.4%, p < 0.0001) and lower TMB (HRD vs HRP; 2.8 vs 26.3, p=0.02). Although patients with HRD presented significantly higher stages (p = 0.03), only marginal p values were found related to recurrence rate (p = 0.08). Median PFS was numerically higher in HRD (20 months, 95% CI: 14.1 - 25.9) versus HRP (40 months, 95% CI: 7.1 -72.9). Additionally, the HRD score was not related to the clinical outcomes of. platinum-based chemotherapy (<i>n</i> = 45) or immunotherapy (<i>n</i> = 20) for the treatment of recurrence. <b>Conclusions:</b> A subset of endometrial tumors has genomic features suggestive of HRD and has a tendency towards a worse prognosis. However, EC patients with HRD may be more likely to benefit from therapies, such as PARP inhibitors.

Fetal Disseminated Malignant Rhabdoid Tumor.

Fetal Disseminated Malignant Rhabdoid Tumor.

Prospective Clinical Genomic Profiling of Ewing Sarcoma: ERF and FGFR1 Mutations as Recurrent Secondary Alterations of Potential Biologic and Therapeutic Relevance.

PURPOSEEwing sarcoma (ES) is a primitive sarcoma defined by EWSR1-ETS fusions as the primary driver alteration. To better define the landscape of cooperating secondary genetic alterations in ES, we analyzed clinical genomic profiling data of 113 patients with ES, a cohort including more adult patients (> 18 years) and more patients with advanced stage at presentation than previous genomic cohorts.METHODSThe data set consisted of patients with ES prospectively tested with the US Food and Drug Administration–cleared Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets large panel, hybrid capture-based next-generation sequencing assay. To assess the functional significance of ERF loss, we generated ES cell lines with increased expression of ERF and lines with knockdown of ERF. We assessed cell viability, clonogenic growth, and motility in these ES lines and performed transcriptomic and epigenetic analyses. Finally, we validated our findings in vivo using cell line xenografts.RESULTSNovel subsets were defined by recurrent secondary alterations in ERF, which encodes an ETS domain transcriptional repressor, in 7% of patients (five truncating mutations, one deep deletion, and two missense mutations) and in FGFR1 in another 2.7% (one amplification and two known activating mutations). ERF alterations were nonoverlapping with STAG2 alterations. In vitro, increased expression of ERF decreased tumor cell growth, colony formation, and motility in two ES cell lines, whereas ERF loss induced cellular proliferation and clonogenic growth. Transcriptomic analysis of cell lines with ERF loss revealed an increased expression of genes and pathways associated with aggressive tumor biology, and epigenetic, chromatin-based studies revealed that ERF competes with EWSR1-FLI1 at ETS-binding sites.CONCLUSIONOur findings open avenues to new insights into ES pathobiology and to novel therapeutic approaches in a subset of patients with ES.

Next-generation sequencing and PCR technologies in monitoring the hospital microbiome and its drug resistance.

The hospital environment significantly contributes to the onset of healthcare-associated infections (HAIs), which represent one of the most frequent complications occurring in healthcare facilities worldwide. Moreover, the increased antimicrobial resistance (AMR) characterizing HAI-associated microbes is one of the human health’s main concerns, requiring the characterization of the contaminating microbial population in the hospital environment. The monitoring of surface microbiota in hospitals is generally addressed by microbial cultural isolation. However, this has some important limitations mainly relating to the inability to define the whole drug-resistance profile of the contaminating microbiota and to the long time period required to obtain the results. Hence, there is an urgent need to implement environmental surveillance systems using more effective methods. Molecular approaches, including next-generation sequencing and PCR assays, may be useful and effective tools to monitor microbial contamination, especially the growing AMR of HAI-associated pathogens. Herein, we summarize the results of our recent studies using culture-based and molecular analyses in 12 hospitals for adults and children over a 5-year period, highlighting the advantages and disadvantages of the techniques used.