Next-generation Sequencing Assay Research Articles

Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review.

The female genital tract microbiome has become a particular area of interest in improving assisted reproductive technology (ART) outcomes with the emergence of next-generation sequencing (NGS) technology. However, NGS assessment of microbiomes currently lacks uniformity and poses significant challenges for accurate and precise bacterial population representation. As multiple NGS platforms and assays have been developed in recent years for microbiome investigation-including the advent of long-read sequencing technologies-this work aimed to identify current trends and practices undertaken in female genital tract microbiome investigations. Areas like sample collection and transport, DNA extraction, 16S amplification vs. metagenomics, NGS library preparation, and bioinformatic analysis demonstrated a detrimental lack of uniformity. The lack of uniformity present is a significant limitation characterised by gap discrepancies in generation and interpretation of results. Minimal consistency was observed in primer design, DNA extraction techniques, sample transport, and bioinformatic analyses. With third-generation sequencing technology highlighted as a promising tool in microbiota-based research via full-length 16S rRNA sequencing, there is a desperate need for future studies to investigate and optimise methodological approaches of the genital tract microbiome to ensure better uniformity of methods and results interpretation to improve clinical impact.

Rapid detection of SARS-CoV-2 variants by molecular-clamping technology-based RT-qPCR.

We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

Clinical and Technical Validation of OncoIndx® Assay-A Comprehensive Genome Profiling Assay for Pan-Cancer Investigations.

Comprehensive next-generation sequencing (NGS) assays enable the identification of clinically relevant mutations, enhancing the capability for targeted therapeutic interventions. In addition, genomic alterations driving the oncogenic roadmap and leading to resistance mechanisms are reshaping precision oncology. We report the workflow and clinical and technical validation of the OncoIndx® NGS platform-a comprehensive genomic profiling (CGP)-based assay for pan-cancer investigation. We evaluated the concordance between the OncoIndx® test findings and clinically established hotspot detection using SeraSeq reference standards. OncoIndx is a hybridization capture-based NGS assay for the targeted deep sequencing of all exons and selected introns of 1080 cancer-related genes. We show the outcome in the form of tier I and tier II single nucleotide variants (SNVs), copy number alterations (CNAs), and specific gene fusions. OncoIndx® also informs genome-wide tumor mutational burden (TMB), microsatellite instability (MSI), homologous recombination deficiency (HRD), and genomic loss of heterozygosity (gLOH). A total of 63 samples were utilized for validation with reference standards, clinical samples, and orthogonal assessment for genomic alterations. In addition, 49 cross-laboratory samples were validated for microsatellite instability (MSI), and for the tumor mutation burden (TMB), 18 samples as reference standards, 6 cross-laboratory samples, and 29 TCGA samples were utilized. We show a maximum clinical sensitivity of 98% and a positive predictive value (PPV) of 100% for the clinically actionable genomic variants detected by the assay. In addition, we demonstrate analytical validation with the performance of the assay, limit of detection (LoD), precision, and orthogonal concordance for various types of SVs, CNAs, genomic rearrangements, and complex biomarkers like TMB, MSI, and HRD. The assay offers reliable genomic predictions with the high-precision detection of actionable variants, validated by established reference standards.

B-191 Genomic Profile of Circulating Tumor DNA in Advanced Lung Cancer: Comparison with Tissue DNA

Abstract Background The most common cancer in Korea is thyroid cancer, followed by lung cancer, and the most common cancer in men is lung cancer. The cancer with the highest mortality rate is lung cancer. Recently, the precision medical approach is becoming a major treatment strategy for lung cancer. For precision medical approach, the usefulness of next-generation sequencing (NGS) analysis targeting circulating tumor DNA (ctDNA) is being emphasized. Therefore, in this study, NGS analysis was performed on blood and tissues of advanced lung cancer patients to compare and analyze genetic mutation patterns to confirm the usefulness of ctDNA NGS assay. Methods A total of 101 patients with advanced lung cancer (stage 3 or 4) were recruited, and ctDNA NGS assays were performed on their plasma using a Pan100 panel (Dxome). Tissues from primary lung cancer, metastatic lymph nodes, or metastatic organs of 22 patients were analyzed using ONCOaccuPanel (NGeneBio). All variants were classified into four tiers based on the level of clinical significance, according to the standards and guidelines established by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. Results In the ctDNA NGS assay of 101 patients, 70 (69.3%) harbored 154 tier 1 or 2 variants. Mutations in TP53 (45, 29.2%) and EGFR (23, 14.9%) were the most frequently detected, followed by mutations in RB1, CHEK2, ATM, JAK2, ATR, DNMT3A, and KRAS. Among the 22 patients whose tissue was examined, tier 1 or 2 variants were detected in both the plasma and tissue of 14 patients and only in the tissue of six patients. All genes for these variants were targeted using both methods. Identical variants were detected in the plasma and tissue of six of the 14 patients, and discordant results were observed in the remaining eight patients. Twenty copy number variations were detected in 13 of the 101 patients, with copy number gain of EGFR being the most common (6, 30%). Fusion genes were detected in five of the 101 patients, two of which were not detected in the tissue. Overall, 74 (73.3%) of the 101 patients had positive ctDNA NGS assay results. Conclusions Although the NGS assay with ctDNA is less sensitive than that with tissue DNA, some genetic alterations are detected only in ctDNA NGS assays; therefore, it is useful to apply the ctDNA NGS assay as a complement.

B-232 One Year Post-Implementation Experience Using a Custom Designed DNA Contamination Detection Module for a Clinical Myeloid Neoplasm Next Generation Sequencing Assay

Abstract Background In complex automated clinical laboratory Next Generation Sequencing (NGS) workflows, numerous opportunities exist for contamination events to occur. For oncology testing applications, detection of these events is crucial for accurate results reporting. A contamination detection module (MICon) using microhaplotype (MH) regions and an in-house designed analysis model was developed for use in a NGS assay for myeloid neoplasms (NGSHM) [1]. Variant detection for NGSHM has a validated analytical sensitivity of 2% variant allele frequency (VAF). Gross contamination events can potentially cause erroneous false positive variant detection whereas true low VAF mutations may be masked and evade detection. The following study was performed to evaluate MICon module performance on samples processed over the course of a year. Methods MH regions contain highly conserved tandem single nucleotide polymorphisms (SNPs) with high global heterogeneity occurring within a 300-nucleotide span. From the work of Kidd et al [2], 27 MH regions covering 92 SNPs and spanning 16 chromosomes were selected for inclusion in the 47 gene NGSHM target-capture panel. Sequencing was performed on a NovaSeq 6000 and analyzed through an internal bioinformatics pipeline, which incorporates the MICon module. MICon uses a binary classification model incorporating VAFs from MH regions, number of MH genotypes, and the contamination estimation score from verifyBamID to compute a single value on a 0-100 scale. Scores <50 are classified as non-contaminated and scores ≥50 require further investigation. Results The dataset was comprised of 10,990 samples. A total of 10,232 patient cases (93.1%) had non-contaminated MICon scores <50. Control replicates accounted for 261 samples (2.4%). There were 497 patient cases (4.5%) with MICon scores ≥50. Of the 497 patient cases, 137 (27.6%) had undergone hematopoietic stem cell transplantation (HSCT) and 114 (22.9%) contained gross chromosomal abnormalities, representing iatrogenic or tumor biological factors. An additional 135 (27.2%) of cases were contaminated from laboratory processing errors, 45 (9.1%) occurred due to chemistry failures during run processing, and 66 (13.2%) had an undetermined cause for a high score, representing laboratory-related and unknown factors. Overall, the 135 laboratory processing errors detected accounted for 1.3% of patient cases analyzed by the laboratory. These cases were repeated, and the non-contaminated results were accurately reported. Conclusions Herein it was shown the implementation of MICon has improved patient safety over the course of a year. The 1.3% of patient cases identified as arising from laboratory processing errors were successfully reprocessed. While the overall rate of laboratory processing errors appears low, the absolute number can be significant in a high-volume test setting, despite reliance on highly automated processes. Value is added by MICon through identifying errors that could result in the release of inaccurate patient results. By detecting previously unrealized errors, MICon has become an invaluable asset to the NGSHM assay by enhancing patient laboratory testing safety.

Solar elastosis correlates with high tumor mutation burden and better 5-year disease-specific survival in patients with stage II/III melanoma

ObjectiveTo evaluate the relation between solar elastosis and tumor mutation burden (TMB) in a large clinically annotated cohort of stage II and III melanoma patients. MethodsPrimary cutaneous melanomas from 469 AJCC (8th edition) stage II and III patients with clinical annotation including outcome at 5 years of diagnosis were histopathologically evaluated for solar elastosis. Next-generation sequencing assay MSK-IMPACTTM was employed to determine TMB. Analysis by Fisher’s exact test, chi-square, and Kruskal-Wallis were performed, as well as uni- and multivariate logistic regression. ResultsTumors stratified by low and high TMB showed marked and statistically significant differences in presence and extent of associated solar elastosis. Lower risk patient stage (II versus III by AJCC 8th edition) as well as better 5-year melanomaspecific survival (as binary variable of controls-survivors versus cases-dead of disease at 5 years of diagnosis) were associated with severe solar elastosis. On univariate and multivariate logistic regression models, severe solar elastosis predicted significantly decreased odds of dying of melanoma within 5 years of diagnosis (OR 0.60, 95% CI 0.39-0.89; and OR 0.42, 95% CI 0.20-0.83, respectively; both p<0.05) ConclusionThe association of solar elastosis to TMB and 5-year melanoma specific survival points to its potential as a biomarker of clinical relevance that can be assessed by routine histopathology.

Comparison of commercial next-generation sequencing assays to conventional culture methods for bacterial identification and antimicrobial susceptibility of samples obtained from clinical cases of canine superficial bacterial folliculitis.

Bacterial identification and antimicrobial susceptibility testing is an important step in timely therapeutic decisions for canine superficial bacterial folliculitis (SBF), commonly caused by Staphylococcus pseudintermedius. Next-generation sequencing (NGS) offers the appeal of potentially expedited results with complete detection of bacterial organisms and associated resistance genes compared to culture. Limited studies exist comparing the two methodologies for clinical samples. To compare and contrast genotypic and phenotypic methods for bacterial identification and antimicrobial susceptibility from cases of canine SBF. Twenty-four client-owned dogs with lesions consistent with SBF were enrolled. A sterile culturette swab was used to sample dogs with SBF lesions. The swab was rinsed in 0.9 mL of sterile phosphate-buffered saline and vortexed to create a homogenous solution. Two swabs for NGS laboratories (Labs) and one swab for culture (Culture Lab) were randomly sampled from this solution and submitted for bacterial identification and antimicrobial susceptibility. No statistical difference regarding turnaround time for NGS Labs compared to Culture Lab was found. NGS Lab 1 identified more organisms than NGS Lab 2 and Culture Lab, which were both statistically significant. There was no statistical difference in detection frequency for Staphylococcus spp. among all laboratories. There was poor agreement for the presence of meticillin resistance and most antimicrobials among all laboratories. Utilisation of NGS as a replacement for traditional culture when sampling canine SBF lesions is not supported at this time.

Advancing transplantation monitoring and disease insight: A Next-Generation Sequencing Assay for detecting HLA loss

Advancing transplantation monitoring and disease insight: A Next-Generation Sequencing Assay for detecting HLA loss

A Novel Next-Generation Sequencing Assay for the Identification of BCR::ABL1 Transcript Type and Accurate and Sensitive Detection of TKI-Resistant Mutations.

The clinical management of chronic myeloid leukemia (CML) patients requires the identification of the type of BCR::ABL1 transcript at diagnosis and the monitoring of its expression and potential tyrosine kinase inhibitor (TKI) resistance mutations during treatment. Detection of resistant mutation requires transcript type-specific amplification of BCR::ABL1 from RNA. In this study, a custom RNA-based next-generation sequencing (NGS) assay (Dup-Seq BCR::ABL1) that enables (a) the identification of BCR::ABL1 transcript type and (b) the detection of resistance mutations from common and atypical BCR::ABL1 transcript types was developed and validated. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification for error correction. The custom data analysis pipeline enables breakpoint determination and overlapped mutation calling from duplicates, which minimizes the low-level mutation artifacts. This study demonstrates that this novel assay achieves high accuracy (positive percent agreement (PPA) for fusion: 98.5%; PPA and negative percent agreement (NPA) for mutation at 97.8% and 100.0%, respectively) and sensitivity (limit of detection (LOD) for mutation detection at 3% from 10 000 copies of BCR::ABL1 input). The Dup-Seq BCR::ABL1 assay not only allows for the identification of BCR::ABL1 typical and atypical transcript types and accurate and sensitive detection of TKI-resistant mutations but also simplifies molecular testing work flow for the clinical management of CML patients.

Systemic ALK-negative anaplastic large cell lymphoma with NPM1::TYK2 rearrangement.

Anaplastic large cell lymphoma (ALCL) is a rare subtype of non-Hodgkin lymphoma, with most cases harboring ALK gene rearrangement (ALK + ALCL); however, 20-50% of ALCLs do not have the rearrangement (ALK- ALCL) but exhibit distinct genetic alterations. In this report, we present an unusual case of systemic ALK- ALCL with NPM1::TYK2 fusion. Diagnosis of this case was challenging prior to the NGS findings. A comprehensive panel of immunohistochemical and in-situ hybridization studies was conducted. FISH assays were utilized to target the rearrangements of DUSP22 and TP63 genes. Moreover, next-generation sequencing (NGS) assays were performed to detect clonal rearrangements of IGH and TRG genes, somatic mutations, and potential fusions. The lymphoma cells in this case are negative for all hematolymphoid markers stained, except for CD30 expression and focal and weak CD43 expression. However, NGS studies detected clonal TRG rearrangement and NPM1::TYK2 rearrangement, which aid in the diagnosis of ALK- ALCL. NPM1::TYK2 rearrangement is a rare genetic alteration that has been reported in rare cases of primary cutaneous ALCL, mycosis fungoides, and lymphomatoid papulosis. To the best of our knowledge, this is the first reported instance of such rearrangement in systemic ALK- ALCL.

Genomic landscape of malignant phyllodes tumors reveals multiple targetable opportunities.

Malignant phyllodes tumors (MPT) are rare fibroepithelial breast cancers with no known effective systemic therapy; metastatic progression portends a dismal prognosis. We sought to describe the genomic landscape of MPTs through genomic profiling and immunotherapeutic biomarker analysis. Cases of sequenced MPT were identified from a Clinical Laboratory Improvement Amendments-certified, College of American Pathologists-accredited laboratory (Foundation Medicine). All cases underwent genomic profiling using adaptor ligation-based, next-generation sequencing assay of 324 genes. Tumor agnostic immunotherapy biomarkers, microsatellite instability, tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) expression were evaluated. Fisher's Exact Tests and analysis of variance were used to test for differences between groups and for continuous variables as appropriate. Of 135 MPT cases identified; 94 (69.6%) were localized/locally recurrent and 41 (30.4%) were metastatic. Median age was 54 years (range 14-86). The median TMB was 2.5 mut/Mb and 3 were TMB-high (≥10 mut/Mb). 21.4% were PD-L1+ via Dako 22C3 assay (CPS ≥1). Most commonly altered genes included TERT-promoter (69.7%), CDKN2A (45.9%), TP53 (37.8%), NF1 (35.6%), CDKN2B (33.3%), MED12 (28.9%), MTAP (27.7%), KMT2D (22.2%), PIK3CA (20.0%), PTEN (18.5%), and RB1 (18.5%). Several tumors harboring genomic alterations with US Food and Drug Administration-approved indications in other tumor types were found including NF1, PIK3CA, EGFR Exon 19/20 insertions, and BRAF V600E mutations. In the largest genomic evaluation of MPT to date, multiple clinically actionable mutations were found. Routine sequencing of metastatic MPT may provide additional information to guide treatment decisions and clinical trial enrollment.

Next-generation sequencing-based evaluation of the actionable landscape of genomic alterations in solid tumors: the "MOZART" prospective observational study.

The identification of the most appropriate targeted therapies for advanced cancers is challenging. We performed a molecular profiling of metastatic solid tumors utilizing a comprehensive next-generation sequencing (NGS) assay to determine genomic alterations' type, frequency, actionability, and potential correlations with PD-L1 expression. A total of 304 adult patients with heavily pretreated metastatic cancers treated between January 2019 and March 2021 were recruited. The CLIA-/UKAS-accredit Oncofocus assay targeting 505 genes was used on newly obtained or archived biopsies. Chi-square, Kruskal-Wallis, and Wilcoxon rank-sum tests were used where appropriate. Results were significant for P < .05. A total of 237 tumors (78%) harbored potentially actionable genomic alterations. Tumors were positive for PD-L1 in 68.9% of cases. The median number of mutant genes/tumor was 2.0 (IQR: 1.0-3.0). Only 34.5% were actionable ESCAT Tier I-II with different prevalence according to cancer type. The DNA damage repair (14%), the PI3K/AKT/mTOR (14%), and the RAS/RAF/MAPK (12%) pathways were the most frequently altered. No association was found among PD-L1, ESCAT, age, sex, and tumor mutational status. Overall, 62 patients underwent targeted treatment, with 37.1% obtaining objective responses. The same molecular-driven treatment for different cancer types could be associated with opposite clinical outcomes. We highlight the clinical value of molecular profiling in metastatic solid tumors using comprehensive NGS-based panels to improve treatment algorithms in situations of uncertainty and facilitate clinical trial recruitment. However, interpreting genomic alterations in a tumor type-specific manner is critical.

The Impact of Li-Fraumeni and Germline Retinoblastoma Mutations on Leiomyosarcoma Initiation, Outcomes, and Genetic Testing Recommendations.

Leiomyosarcomas (LMS) are clinically and molecularly heterogeneous, occurring mostly in sporadic but also syndromic settings. The role of pathogenic germline variants (PGV) as LMS drivers and their impact on outcomes remains uncertain. We performed a comprehensive clinicopathologic and molecular analysis using a tumor-normal DNA next-generation sequencing assay (Memorial Sloan Kettering-Integrated Mutational Profiling of Actionable Cancer Targets) of germline-associated LMS compared with sporadic LMS. Among 285 LMS [120 soft-tissue LMS (STLMS) and 165 uterine LMS (ULMS)] with germline testing, 78 (27%, 43 STLMS and 35 ULMS) cases harbored PGV, with 35/78 (45%) of PGV carriers showing biallelic inactivation of the corresponding gene in the tumor (26 STLMS and nine ULMS). The most frequent germline predispositions were TP53 (Li-Fraumeni syndrome; 17 patients, 16 in STLMS) and RB1 (retinoblastoma; 13 patients, 11 in STLMS). Germline TP53 and somatic RB1 alterations often co-occurred in the tumor andvice versa. Other biallelically inactivated PGV were enriched in DNA damage repair-related genes: CHEK2, MSH2, MSH6, RAD51D, BRCA2, and FANCA. Monoallelic PGV were mostly in ULMS and associated with co-occurring TP53 and RB1 somatic alterations. Patients with STLMS with biallelic but not monoallelic PGV were significantly younger than patients with sporadic STLMS (median ages 38 vs. 52 vs. 58 years). No differences in disease-specific or progression-free survival were observed in germline-associated versus sporadic LMS regardless of biallelic status. Although patients with ULMS had a relatively low proportion of PGV, a high percentage of patients with STLMS with PGV had tumor biallelic status, indicating that PGV drive tumorigenesis in these individuals. These findings have significant implications for genetic testing recommendations.

Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett's Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device.

Barrett's esophagus (BE) is a known precursor to esophageal adenocarcinoma (EAC). Guidelines recommend BE screening in populations with multiple risk factors, for which non-endoscopic esophageal cell collection with biomarker testing is considered as an acceptable alternative to esophagogastroduodenoscopy (EGD). The aim of this study was to evaluate analytical performance characteristics of EsoGuard® (EG), a DNA methylation biomarker assay, as a laboratory-developed test (LDT) in esophageal samples collected with the swallowable EsoCheck® (EC) device. EG is a next-generation sequencing (NGS) assay that evaluates methylated vimentin (VIM) and cyclin A1 (CCNA1), clinically validated biomarkers for the detection of BE and EAC. The studies were conducted according to standards of College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA), and New York (NY) state requirements for the analytical validation of molecular assays. Comparison to Sanger sequencing showed that EG was 100% accurate at all 31 CpG sites evaluated by the assay. The analytical sensitivity, specificity, and accuracy of the assay were 89%, 100%, and 96%, respectively. Intra- and inter-assay precision was 100%. The limit of detection (LOD) was 1 in 400 methylated cells, and the reference range was 84%. In summary, EsoGuard demonstrates high analytical accuracy, repeatability, and reproducibility in samples collected using the EsoCheck device.

Enhancing mutation detection in multiple myeloma with an error-corrected ultra-sensitive NGS assay without plasma cell enrichment

BackgroundRisk stratification in multiple myeloma (MM) patients is crucial, and molecular genetic studies play a significant role in achieving this objective. Enrichment of plasma cells for next-generation sequencing (NGS) analysis has been employed to enhance detection sensitivity. However, these methods often come with limitations, such as high costs and low throughput. In this study, we explore the use of an error-corrected ultrasensitive NGS assay called positional indexing sequencing (PiSeq-MM). This assay can detect somatic mutations in MM patients without relying on plasma cell enrichment.MethodDiagnostic bone marrow aspirates (BMAs) and blood samples from 14 MM patients were used for exploratory and validation sets.ResultsPiSeq-MM successfully detected somatic mutations in all BMAs, outperforming conventional NGS using plasma cells. It also identified 38 low-frequency mutations that were missed by conventional NGS, enhancing detection sensitivity below the 5% analytical threshold. When tested in an actual clinical environment, plasma cell enrichment failed in most BMAs (14/16), but the PiSeq-MM enabled mutation detection in all BMAs. There was concordance between PiSeq-MM using BMAs and ctDNA analysis in paired blood samples.ConclusionThis research provides valuable insights into the genetic landscape of MM and highlights the advantages of error-corrected NGS for detecting low-frequency mutations. Although the current standard method for mutation analysis is plasma cell-enriched BMAs, total BMA or ctDNA testing with error correction is a viable alternative when plasma cell enrichment is not feasible.

Circulating tumor DNA analysis in patients with esophageal cancer treated with neoadjuvant chemoradiotherapy followed by surgery.

69 Background: The aim of this prospectively study is to investigate circulating tumor DNA (ctDNA) as a prognostic marker for survival in locally advanced esophageal squamous cell cancer (ESCC) patients treated with neoadjuvant chemoradiotherapy (nCRT) and surgery. Methods: Serial plasma samples were collected from 68 ESCC patients treated with neoadjuvant chemoradiotherapy before surgery. ctDNA was detected before treatment, after neoadjuvant chemoradiotherapy and after surgery by personalized, next-generation sequencing assay. Cox regression analyses were used to evaluate the prognostic significance of ctDNA for recurrence-free survival (RFS) and overall survival (OS). Results: ctDNA was detectable in 89.7%, 17.2%, and 6.9% of pretreatment, post-neoadjuvant chemoradiotherapy and post-surgery plasma samples, respectively. The conversion of ctDNA status from positive at baseline to negative at 4-6 weeks after neoadjuvant chemoradiotherapy was significantly associated with pathological complete response (pCR) ( P = 0.02). Significant worse RFS was related to detectable ctDNA after neoadjuvant chemoradiotherapy ( P = 0.008) or after surgery ( P = 0.001). Detectable ctDNA at baseline ( P = 0.01) and after surgery ( P = 0.008) were associated with worse OS. In multivariate analysis, detectable postoperative ctDNA was significantly associated with RFS ( P = 0.012) and OS ( P = 0.025) after adjusting for other clinicopathological risk factors. Conclusions: ctDNA status is a strong prognostic factor of recurrence in locally advanced ESCC patients. Consequently, ctDNA could potentially improve pre- and post-treatment risk assessment and facilitate individual therapy for ESCC patients.

Single-Base Substitution Causing Dual-Exon Skipping Event in PKD2 Gene: Unusual Molecular Finding from Exome Sequencing in a Patient with Autosomal Dominant Polycystic Kidney Disease.

Background: Pathogenic variants in the Polycystic Kidney Disease 2 (PKD2) gene are associated with Autosomal Dominant Polycystic Kidney Disease (ADPKD) in approximately 30% of cases. In recent years, the high-throughput sequencing techniques have significantly increased the number of variants identified in affected patients. Here, we described the peculiar effect of a PKD2 splicing variant, the c.1717-2A>G, identified in an Italian male patient with ADPKD. This variant led to the unusual and rare skipping of two consecutive exons, causing a large in-frame deletion. Methods: The genetic evaluation of the patient was performed using the Next-Generation Sequencing (NGS) assay Clinical Exome Solution® (SOPHiA Genetics). Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). Prediction of pathogenicity was carried out by integrating several in silico tools. RNA evaluation was performed to test the effect of the variant on the PKD2 splicing using a Reverse-Transcription PCR coupled with cDNA sequencing. Results: NGS revealed the presence of the PKD2 c.1717-2A>G variant that lies in the canonical splice site of intron 7. This rare variant was predicted to have a significant impact on the splicing, proved by the RNA-based analysis. We identified the presence of a transcript characterised by the simultaneous skipping of exons 8 and 9, with a retained reading frame and the merging of exons 7-10. Conclusions: We described for the first time a dual-exon skip event related to the presence of a single-base substitution in the PKD2 gene in an ADPKD-affected patient. We assumed that the molecular basis of such a rare mechanism lies in the specific order of intron removal. The finding represents novel evidence of an alternative and unusual splicing mechanism in the PKD2 gene, adding insights to the pathogenesis of the ADPKD.

51 Epigenetic regulation of ribosomal RNA transcription and processing by the tumor suppressor SETD2

Abstract Background The methyltransferase SETD2 was originally identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), and ccRCC with SETD2 mutations have increased metastatic potential and poor progression free survival. A known epigenetic regulator, SETD2 interacts with actively transcribing RNA polymerase II (RNAPII) and tri-methylates Histone 3 at Lysine 36 (H3K36me3), a modification that correlates with active gene transcription and is recognized by mRNA methylation and splicing factors. Despite these known roles in mRNA transcription, loss of SETD2 does not alter genic transcription, raising the question of whether or how SETD2 regulates RNA biology. Methods Given that SETD2 influences RNA processing, we sought to identify RNA-protein complexes that are regulated by SETD2. To do this in an unbiased and high-throughput manner, we used orthogonal organic phase separation (OOPS) coupled with mass spectrometry to identify changes in RNA binding proteins and pathways between control and SETD2-knockout cells. Results Corroborating previous findings, we identified that RNA binding proteins governing mRNA splicing and cellular metabolism were dysregulated in SETD2 mutant cells. However, the most robust changes in SETD2 knockout cells occurred in pathways regulated ribosome biogenesis (See Figure, orange highlights), resulting in a significant decrease in binding of ribosomal RNA (rRNA) processing factors to RNA. We confirm these alterations lead to defects in rRNA transcription, processing, and ribosome biogenesis. We identify that loss of SETD2 catalytic activity phenocopies these rRNA processing defects, suggesting H3K36me3 is essential for rRNA processing. Disruptions occur to both the large (60S) and small (40S) ribosome subunits, modifying protein translation. Lastly, we identify synthetic lethality between SETD2 deletion or inhibition and small molecules that interfere with rRNA transcription. Conclusions While the majority of research emphasizes the role SETD2 in the regulation of mRNA, our data identify that SETD2 and its catalytic activity play a critical role in the transcription and processing of rRNA. Importantly, rRNA comprises 80-90% of cellular RNA yet is typically overlooked and understudied. Next-generation RNA sequencing or transcriptional assays investigate mRNA or genic transcription while ignoring rRNA, demonstrating why SETD2-mediated rRNA processing has been unexplored. This may also have important implications for prospective clinical trials that utilized RNA-sequencing to identify optimal therapeutic regimens. Our work also identified that SETD2-mutated cells are more sensitive to compounds that inhibit rRNA transcription, potentially uncovering a new therapeutic vulnerability of ccRCC with mono- and bi-allelic loss of SETD2. Alterations in ribosome biogenesis leads to epithelial-to-mesenchymal transition and metastasis in many tumor types, and our future goal will be to investigate the role of ribosomes and rRNA transcription in transformation and metastasis in ccRCC as well as the molecular mechanisms connecting SETD2 to ribosome fidelity. DOD CDMRP Funding: yes

The Next Chapter in Cancer Diagnostics: Advances in HPV-Positive Head and Neck Cancer.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), is an increasingly prevalent pathology worldwide, especially in developed countries. For diagnosing HPV in HNSCC, the combination of p16 immunohistochemistry (IHC) and polymerase chain reaction (PCR) offers high sensitivity and specificity, with p16 IHC being a reliable initial screen and PCR confirming HPV presence. Advanced techniques like next-generation sequencing (NGS) and RNA-based assays provide detailed insights but are primarily used in research settings. Regardless of HPV status, standard oncological treatments currently include surgery, radiation, and/or chemotherapy. This conventional approach does not account for the typically better prognosis of HPV-positive HNSCC patients, leading to increased chemo/radiation-induced secondary morbidities and reduced quality of life. Therefore, it is crucial to identify and detect HPV positivity and other molecular characteristics of HNSCC to personalize treatment strategies. This comprehensive review aims to summarize current knowledge on various HPV detection techniques and evaluate their advantages and disadvantages, with a focus on developing methodologies to identify new biomarkers in HPV-positive HNSCC. The review discusses direct and indirect HPV examination in tumor tissue, DNA- and RNA-based detection techniques, protein-based markers, liquid biopsy potentials, immune-related markers, epigenetic markers, novel biomarkers, and emerging technologies, providing an overall insight into the current state of knowledge.

The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable EGFR Variants in Non-Small-Cell Lung Cancer.

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the EGFR gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable EGFR variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for EGFR variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of EGFR variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all EGFR variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of EGFR exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more EGFR variants in both assays. Additionally, we observed TP53 to be a frequently co-mutated gene in EGFR-positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in EGFR variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.