Combined Next-generation Sequencing Research Articles

Abstract 6399: Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay

Abstract Introduction: Comprehensive molecular profiling with combined DNA-RNA next-generation sequencing (NGS) on tumor tissue is now increasingly being used as standard-of-care for sequence therapy selection to improve cancer survival. Combined DNA-RNA NGS is also considered standard-of-care for central nervous system (CNS) tumor classification. Despite the genetic heterogeneity and diversity of cancers, most commercial combined DNA/RNA NGS assays are validated in Western populations, resulting in knowledge gaps in patient outcomes. We report here for the first time a range of actionable outcomes from a comprehensive DNA/RNA tissue NGS assay designed specifically to probe Asian-prevalent cancers and biomarkers in an international multicenter real-world study in Asia. Methods: 1,002 formalin-fixed paraffin-embedded tissue samples from 995 Asian cancer patients across >70 centers in 7 countries and/or territories underwent real-world DNA/RNA NGS testing in a CAP-accredited CLIA-certified laboratory. Genomic alterations including SNVs, INDELS, gene fusions, CNVs, TMB, and MSI were analyzed using an Asian-centric pan-cancer hybrid-capture NGS panel that comprehensively profiles 572 cancer-related genes and 91 RNA fusion partners (UNITED). Biomarkers were considered actionable if the therapeutic indication in any solid tumor or hematological cancer was FDA-approved, guidelines-recommended, or supported by well-powered phase III clinical trials. Results: 27 cancer types were represented in 1,002 tissue samples, with lung, breast, colorectal, and prostate cancers comprising 45.5% of clinical volume. 79.5% of samples harbored ≥1 actionable biomarker, with the most frequently altered genes being TP53, KRAS, PIK3CA, EGFR, and NF1. Overall, 42.6% of samples had ≥1 biomarker with an indication in the patient’s cancer type, including 81.5% of lung, 59.0% of breast, 61.8% of colorectal, and 42.1% of prostate cancers. Across the pan-cancer cohort, 13.3% was TMB-high and 1.9% was MSI-high. 26.3% of samples were from cancer types outside the original assay design; the top 3 were pancreatic cancer (7.3%), sarcoma (3.3%), and CNS tumor (3.1%). Cancers of unknown primary (CUP) also constituted a significant proportion (3.8%). In CNS tumors, 38.7% harbored an actionable mutation in IDH1, BRAF, or H3-3A. In CUP, 78.8% harbored an actionable mutation; 50% of samples harbored biomarkers that additionally informed potential tumor origin, including mutations in EGFR, MET, ALK, ROS1, and BRCA1. Conclusion: The use of an Asian-focused pan-cancer DNA/RNA NGS panel resulted in detection of actionable biomarkers in 79.5% of clinical cases. These findings support the utility of DNA/RNA NGS assays in informing standard-of-care therapeutic decisions in Asian cancer patients for maximizing cancer survival. Citation Format: Jason Yongsheng Chan, Jing Yi Lee, Zi Yi Wan, Abner Herbert Lim, Tun Kiat Ko, Cedric Chuan-Young Ng, Donald Poon, Jens Samol, Tsz Him So, Su Pin Choo, Ravindran Kanesvaran, Cheng-Vai Hui, Joseph Siu-Kie Au, Aya El Helali, Timothy Yip, Vikneswari Rajasegaran, Sandy Lim, Ruifen Weng, Puay Hoon Tan, Bin Tean Teh, Min-Han Tan, Jonathan Poh. Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6399.

The genetic landscape of mitochondrial diseases in the next-generation sequencing era: a Portuguese cohort study.

Introduction: Rare disorders that are genetically and clinically heterogeneous, such as mitochondrial diseases (MDs), have a challenging diagnosis. Nuclear genes codify most proteins involved in mitochondrial biogenesis, despite all mitochondria having their own DNA. The development of next-generation sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MDs. In this new genetic era, using the NGS approach, we aimed to identify the genetic etiology for a suspected MD in a cohort of 450 Portuguese patients. Methods: We examined 450 patients using a combined NGS strategy, starting with the analysis of a targeted mitochondrial panel of 213 nuclear genes, and then proceeding to analyze the whole mitochondrial DNA. Results and Discussion: In this study, we identified disease-related variants in 134 (30%) analyzed patients, 88 with nuclear DNA (nDNA) and 46 with mitochondrial DNA (mtDNA) variants, most of them being pediatric patients (66%), of which 77% were identified in nDNA and 23% in mtDNA. The molecular analysis of this cohort revealed 72 already described pathogenic and 20 novel, probably pathogenic, variants, as well as 62 variants of unknown significance. For this cohort of patients with suspected MDs, the use of a customized gene panel provided a molecular diagnosis in a timely and cost-effective manner. Patients who cannot be diagnosed after this initial approach will be further selected for whole-exome sequencing. Conclusion: As a national laboratory for the study and research of MDs, we demonstrated the power of NGS to achieve a molecular etiology, expanding the mutational spectrum and proposing accurate genetic counseling in this group of heterogeneous diseases without therapeutic options.

AI/ML combined with next-generation sequencing of VHH immune repertoires enables the rapid identification of de novo humanized and sequence-optimized single domain antibodies: a prospective case study.

Introduction: In this study, we demonstrate the feasibility of yeast surface display (YSD) and nextgeneration sequencing (NGS) in combination with artificial intelligence and machine learning methods (AI/ML) for the identification of de novo humanized single domain antibodies (sdAbs) with favorable early developability profiles. Methods: The display library was derived from a novel approach, in which VHH-based CDR3 regions obtained from a llama (Lama glama), immunized against NKp46, were grafted onto a humanized VHH backbone library that was diversified in CDR1 and CDR2. Following NGS analysis of sequence pools from two rounds of fluorescence-activated cell sorting we focused on four sequence clusters based on NGS frequency and enrichment analysis as well as in silico developability assessment. For each cluster, long short-term memory (LSTM) based deep generative models were trained and used for the in silico sampling of new sequences. Sequences were subjected to sequence- and structure-based in silico developability assessment to select a set of less than 10 sequences per cluster for production. Results: As demonstrated by binding kinetics and early developability assessment, this procedure represents a general strategy for the rapid and efficient design of potent and automatically humanized sdAb hits from screening selections with favorable early developability profiles.

Variability in donor leukocyte counts confound the use of common RNA sequencing data normalization strategies in transcriptomic biomarker studies performed with whole blood

Gene expression data generated from whole blood via next generation sequencing is frequently used in studies aimed at identifying mRNA-based biomarker panels with utility for diagnosis or monitoring of human disease. These investigations often employ data normalization techniques more typically used for analysis of data originating from solid tissues, which largely operate under the general assumption that specimens have similar transcriptome composition. However, this assumption may be violated when working with data generated from whole blood, which is more cellularly dynamic, leading to potential confounds. In this study, we used next generation sequencing in combination with flow cytometry to assess the influence of donor leukocyte counts on the transcriptional composition of whole blood specimens sampled from a cohort of 138 human subjects, and then subsequently examined the effect of four frequently used data normalization approaches on our ability to detect inter-specimen biological variance, using the flow cytometry data to benchmark each specimens true cellular and molecular identity. Whole blood samples originating from donors with differing leukocyte counts exhibited dramatic differences in both genome-wide distributions of transcript abundance and gene-level expression patterns. Consequently, three of the normalization strategies we tested, including median ratio (MRN), trimmed mean of m-values (TMM), and quantile normalization, noticeably masked the true biological structure of the data and impaired our ability to detect true interspecimen differences in mRNA levels. The only strategy that improved our ability to detect true biological variance was simple scaling of read counts by sequencing depth, which unlike the aforementioned approaches, makes no assumptions regarding transcriptome composition.

The value of metagenomic next-generation sequencing for the diagnosis of pulmonary tuberculosis using bronchoalveolar lavage fluid.

The aim of this study was to compare metagenomic next-generation sequencing (mNGS) with other methods, including Xpert MTB/RIF, Mycobacterium tuberculosis (MTB) culture, and acid-fast bacillus (AFB) staining in the diagnosis of pulmonary tuberculosis (PTB) using bronchoalveolar lavage fluid (BALF). The data of 186 patients with suspected PTB were retrospectively collected from January 2020 to May 2021 at Tongji Hospital. BALF samples were collected from all patients and analyzed using AFB staining, MTB culture, Xpert MTB/RIF, and mNGS. Of the 186 patients, 38 patients were ultimately diagnosed as PTB. Metagenomic next-generation sequencing exhibited a sensitivity of 78.95%, which was higher than AFB staining (27.59%) and MTB culture (44.12%) but similar to Xpert MTB/RIF (72.73%). Utilization of combined methods demonstrates improvement for PTB diagnosis. In support of this, the area under the receiver operating characteristic curve for the combination of mNGS and MTB culture (0.933, 95% CI: 0.871, 0.995) was larger than those of mNGS, Xpert MTB/RIF, MTB culture, and the combination of Xpert MTB/RIF and MTB culture. The sensitivity of mNGS in the diagnosis of PTB using BALF specimen is similar to Xpert MTB/RIF.Metagenomic next-generation sequencing in combination with MTB culture may further improve the diagnosis of pulmonary tuberculosis.

Identification of genetic alterations in couples and their products of conceptions from recurrent pregnancy loss in North Indian population.

Background: Recurrent pregnancy loss (RPL) is one of the most common pregnancy-related complications, which can be stressful and emotionally draining for a couple. Genetic alterations, which are responsible for RPL, can be present in either of the three genomes: mother, father, or their fetuses. In addition, environmental factors interacting with these three genomes can affect germline cells. With this aim, the present study was conducted to understand the underlying etiology of RPL using Next-generation sequencing (NGS; couple exome and TRIO exomes) in combination with cytogenetic tests [karyotyping and chromosomal microarray (CMA)]. Material & Methods: In present study we recruited 61 couples with RPL (history of ≥ 2 abortions) and 31 products of conceptions (POCs). For all couples karyotyping was done at the time of recruitment, followed by collection of POC samples and parental blood samples. Before processing POC samples for CMA, they were checked for maternal cell contamination (MCC) by QF-PCR. In POC samples with no pathogenic variant, TRIO exome sequencing was done. Further, in case of unavailability of POC sample, couple exome sequencing was done for RPL couples. Results: In six individuals out of 61 couples (5%), abnormality in karyotypes was detected. Among 116 normal karyotypes, there were 11 heteromorphisms (9.5%), for which the couples had to be counselled and reassured. Out of the 31 POCs, 10 were excluded because of MCC (around 30%) and one had major aneuploidy. CMA in POCs identified pathogenic copy number variations (CNVs) in 25% of cases (5/20) and variant of unknown significance (VUS) in 20% of cases (4/20). Autosomal trisomy was the most frequent chromosomal abnormality diagnosed. NGS was performed to establish single-gene causes of RPL. Couple exome sequencing was performed in 20 couples, and 14 were found to be carriers for autosomal recessive conditions. A total of 50 potential disease-causing variants in 40 genes were identified in 33 of 40 individuals (82.5%). Putative causative variants were identified in 37.5% of the TRIO cases (3/8). Mutations in few important genes (SRP54, ERBB4, NEB, ALMS, ALAD, MTHFR, F5, and APOE), which are involved in vital pathways, early embryonic development, and fetal demise, were identified in the POCs. Conclusion: It enhances our understanding of prenatal phenotypes of many Mendelian disorders. These mutated genes may play an auxiliary role in the development of treatment strategies for RPL. There was no correlation of the number of abortions with etiological yield of any technique to detect the cause of RPL. This study shows the utilization of combination of techniques in improving our understanding of the cause of early embryonic lethality in humans.

First proficiency testing for NGS-based and combined NGS- and FISH-based detection of FGFR2 fusions in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma harbours druggable genetic lesions including FGFR2 gene fusions. Reliable and accurate detection of these fusions is becoming a critical component of the molecular work-up, but real-world data on the performance of fluorescence in situ hybridisation (FISH) and targeted RNA-based next-generation sequencing (NGS) are very limited. Bridging this gap, we report results of the first round robin test for FGFR2 fusions in cholangiocarcinoma and contextualise test data with genomic architecture. A cohort of 10 cholangiocarcinoma (4 fusion positive and 6 fusion negative) was tested by the Institute of Pathology, University Hospital Heidelberg, Germany. Data were validated by four academic pathology departments in Germany. Fusion-positive cases comprised FGFR2::BICC1, FGFR2::DBP, FGFR2::TRIM8, and FGFR2::ATE1 fusions. In a second step, a round robin test involving 21 academic and non-academic centres testing with RNA-based NGS approaches was carried out; five participants performed FISH testing in addition. Thirteen of 16 (81%) centres successfully passed the NGS only and 3 of 5 (60%) centres passed the combined NGS + FISH round robin test. Identified obstacles were bioinformatic pipelines not optimised for the detection of FGFR2 fusions and assays not capable of detecting unknown fusion partners. This study shows the benefit of targeted RNA-NGS for the detection of FGFR2 gene fusions. Due to the marked heterogeneity of the genomic architecture of these fusions, fusion partner agnostic (i.e. open) methodological approaches that are capable of identifying yet unknown fusion partners are superior. Furthermore, we highlight pitfalls in subsequent bioinformatic analysis and limitations of FISH-based tests.

Highly sensitive liquid biopsy Duplex sequencing complements tissue biopsy to enhance detection of clinically relevant genetic variants.

Liquid biopsy (LB) is a promising complement to tissue biopsy for detection of clinically relevant genetic variants in cancer and mosaic diseases. A combined workflow to enable parallel tissue and LB analysis is required to maximize diagnostic yield for patients. We developed and validated a cost-efficient combined next-generation sequencing (NGS) workflow for both tissue and LB samples, and applied Duplex sequencing technology for highly accurate detection of low frequency variants in plasma. Clinically relevant cutoffs for variant reporting and quantification were established. We investigated assay performance characteristics for very low amounts of clinically relevant variants. In plasma, the assay achieved 100% sensitivity and 92.3% positive predictive value (PPV) for single nucleotide variants (SNVs) and 91.7% sensitivity and 100% PPV for insertions and deletions (InDel) in clinically relevant hotspots with 0.5-5% variant allele frequencies (VAFs). We further established a cutoff for reporting variants (i.e. Limit of Blank, LOB) at 0.25% VAF and a cutoff for quantification (i.e. Limit of Quantification, LOQ) at 5% VAF in plasma for accurate clinical interpretation of analysis results. With our LB approach, we were able to identify the molecular cause of a clinically confirmed asymmetric overgrowth syndrome in a 10-year old child that would have remained undetected with tissue analysis as well as other molecular diagnostic approaches. Our flexible and cost-efficient workflow allows analysis of both tissue and LB samples and provides clinically relevant cutoffs for variant reporting and precise quantification. Complementing tissue analysis by LB is likely to increase diagnostic yield for patients with molecular diseases.

Spring is associated with increased total and allergenic fungal concentrations in house dust from a pediatric asthma cohort in New York City

IntroductionAsthma and allergy symptoms vary seasonally due to exposure to environmental sources of allergen, including fungi. However, we need an improved understanding of seasonal influence on fungal exposures in the indoor environment. We hypothesized that concentrations of total fungi and allergenic species in vacuumed dust vary significantly by season. ObjectiveAssess seasonal variation of indoor fungi with greater implications related to seasonal asthma control. MethodsWe combined next-generation sequencing with quantitative polymerase chain reaction (qPCR) to measure concentrations of fungal DNA in indoor floor dust samples (n = 298) collected from homes participating in the New York City Neighborhood Asthma and Allergy Study (NAAS). ResultsTotal fungal concentration in spring was significantly higher than the other three seasons (p ≤ 0.005). Mean concentrations for 78% of fungal species were elevated in the spring (26% were significantly highest in spring, p < 0.05). Concentrations of 8 allergenic fungal species were significantly (p < 0.5) higher in spring compared to at least two other seasons. Indoor relative humidity and temperature were significantly highest in spring (p < 0.05) and were associated with total fungal concentration (R2 = 0.049, R2 = 0.11, respectively). ConclusionThere is significant seasonal variation in total fungal concentration and concentration of select allergenic species. Indoor relative humidity and temperature may underlie these associations.

Molecular classification of endometrial carcinomas: a singleinstitution review (084)

<b>Objectives:</b> To describe endometrial carcinoma (EC) molecular classification utilizing clinical next-generation sequencing (NGS) and immunohistochemistry (IHC) and to assess the clinical, pathologic, and survival outcomes based on molecular data. <b>Methods:</b> We identified all primary and recurrent EC samples of 341-505 cancer-related genes undergoing NGS (MSK-Integrated Mutation Profiling of Actionable Cancer Targets; MSK-IMPACT) from test inception in 2014 through 2020. Samples were classified into the Cancer Genome Atlas (TCGA) molecular subtypes utilizing MSK-IMPACT results for <i>POLE</i> hotspot mutations, <i>TP53</i> mutations, and MSIsensor score; and mismatch repair (MMR) and p53 IHC results collected from pathology reports. Tumors not classified per TCGA based on available NGS and IHC data were excluded from the analysis. Clinical, pathologic, and survival data were obtained for primary ECs undergoing initial staging surgery or treatment at our institution after staging surgery. Only NGS performed prior to documented recurrence was used for survival analysis, to avoid ascertainment bias. Appropriate statistical tests were applied. <b>Results:</b> Clinical MSK-IMPACT sequencing was performed on 2174 ECs. Except for 40 unclassifiable cases, 2134 tumors corresponded to 2066 unique patients, including 1712 (80%) primary and 409 (19%) recurrent tumors. Molecular classification was distributed as 5% <i>POLE</i> (<i>n</i>=113), 22% microsatellite instability-high (MSI-H; <i>n</i>=479), 38% copy-number high (CN-H) (<i>n</i>=818), and 34% CN-low (<i>n</i>=724). MMR-deficiency by IHC was found in 16% of <i>POLE</i>, and IHC-based aberrant p53 expression in 12% of <i>POLE</i> and 2% of MSI-H ECs. Due to the hierarchy of our hybrid classification algorithm, this did not affect molecular designation. Otherwise, all available MMR and p53 IHC results were concordant with NGS results to determine classification. CN-H was 91% non-endometrioid with 67% FIGO G3 within the endometrioid type, while <i>POLE</i>, MSI, and CN-L were 10-32% and 17-21%, respectively (p<0.05). Criteria for survival analysis using primary tumor NGS results were met by 1118 patients. The median follow-up was 22.5 months (0.5-214 months). Three-year progression-free survival was 97% for <i>POLE</i> (SE 2.0), 90% CN-L (SE 2.1), 80% MSI-H (SE 3.1), and 46% CN-H (SE 3.5), (p<0.01). Three-year overall survival was 100% for <i>POLE</i>, 94% MSI-H (SE 2.1), 93% CN-L (SE 2.2), and 71% CN-H (SE 3.6), (p<0.01). CN-H classification remained prognostic for recurrence outcomes when adjusting for age, stage, and histology (HR: 6.2, 95% CI: 1.5-26.6; p=0.04). <b>Conclusions:</b> Our approach to molecular classification involves a hybrid strategy utilizing combined NGS and IHC analysis as a surrogate for whole-exome sequencing employed by TCGA. Our current database is mostly CN-H, which may reflect the referral-based nature of our institution. When applying appropriate criteria for survival analysis, CN-H portends the worst outcomes. Future studies should consider the histological heterogeneity of CN-H when considering treatment options.Fig. 1

Analysis of secondary metabolite gene clusters and chitin biosynthesis pathways of Monascus purpureus with high production of pigment and citrinin based on whole-genome sequencing.

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.

Endometriosis Associated-miRNome Analysis of Blood Samples: A Prospective Study.

The aim of our study was to describe the bioinformatics approach to analyze miRNome with Next Generation Sequencing (NGS) of 200 plasma samples from patients with and without endometriosis. Patients were prospectively included in the ENDO-miRNA study that selected patients with pelvic pain suggestive of endometriosis. miRNA sequencing was performed using an Novaseq6000 sequencer (Illumina, San Diego, CA, USA). Small RNA-seq of 200 plasma samples yielded ~4228 M raw sequencing reads. A total of 2633 miRNAs were found differentially expressed. Among them, 8.6% (n = 229) were up- or downregulated. For these 229 miRNAs, the F1-score, sensitivity, specificity, and AUC ranged from 0–88.2%, 0–99.4%, 4.3–100%, and 41.5–68%, respectively. Utilizing the combined bioinformatic and NGS approach, a specific and broad panel of miRNAs was detected as being potentially suitable for building a blood signature of endometriosis.

Usefulness of combined NGS and QF-PCR analysis for product of conception karyotyping.

PurposeSince chromosomal abnormalities can be detected in more than half of miscarriages, cytogenetic testing of the product of conception (POC) can provide important information when preparing for a subsequent pregnancy. Conventional karyotyping is the common diagnostic method for a POC but can be problematic due to the need for cell culture.MethodsWe here conducted shallow whole‐genome sequencing (sWGS) using next‐generation sequencing (NGS) for alternative POC cytogenomic analysis. Since female euploidy samples can include 69,XXX triploidy, additional QF‐PCR was performed in these cases.ResultsWe here analyzed POC samples from miscarriages in 300 assisted reproductive technology (ART) pregnancies and detected chromosomal abnormalities in 201 instances (67.0%). Autosomal aneuploidy (151 cases, 50.3%) was the most frequent abnormality, consistent with prior conventional karyotyping data. Mosaic aneuploidy was detected in seven cases (2.0%). Notably, the frequency of triploidy was 2.3%, 10‐fold lower than the reported frequency in non‐ART pregnancies. Structural rearrangements were identified in nine samples (3%), but there was no case of segmental mosaicism.ConclusionsThese data suggest that NGS‐based sWGS, with the aid of QF‐PCR, is a viable alternative karyotyping procedure that does not require cell culture. This method could also assist with genetic counseling for couples who undergoes embryo selection based on PGT‐A data.

Oncomine™ Comprehensive Assay v3 vs. Oncomine™ Comprehensive Assay Plus.

Simple SummaryThe detection of genetic alterations in cancer is important to obtain knowledge of the underlying mutational tumor composition. Knowing the mutational profile can assist oncologists on tailoring optimal personalized treatments. Moreover, obtaining additional information from a broader cancer-related gene panel, without compromising performance, can benefit both current and future patients. In this study, we assessed the performance of gene mutations identified from sequencing using the newly Oncomine™ Comprehensive Assay Plus (OCA-Plus). The assessment was performed in comparison to gene mutations identified from sequencing using the Oncomine™ Comprehensive Assay v3 (OCAv3), currently used in our routine clinical setting. Therefore, an investigation of their performance was conducted on intersecting nucleotide positions within overlapping genes covered by both the OCA-Plus and the OCAv3. We show here that there is a 91% concordance between identified pathogenic and likely pathogenic classified variants. The usage of next generation sequencing in combination with targeted gene panels has enforced a better understanding of tumor compositions. The identification of key genomic biomarkers underlying a disease are crucial for diagnosis, prognosis, treatment and therapeutic responses. The Oncomine™ Comprehensive Assay v3 (OCAv3) covers 161 cancer-associated genes and is routinely employed to support clinical decision making for a therapeutic course. An improved version, Oncomine™ Comprehensive Assay Plus (OCA-Plus), has been recently developed, covering 501 genes (144 overlapping with OCAv3) in addition to microsatellite instability (MSI) and tumor mutational burden (TMB) assays in one workflow. The validation of MSI and TMB was not addressed in the present study. However, the implementation of new assays must be validated and confirmed across multiple samples before it can be introduced into a clinical setting. Here, we report the comparison of DNA sequencing results from 50 ovarian cancer formalin-fixed, paraffin-embedded samples subjected to OCAv3 and OCA-Plus. A validation assessment of gene mutations identified using OCA-Plus was performed on the 144 overlapping genes and 313,769 intersecting nucleotide positions of the OCAv3 and the OCA-Plus. Our results showed a 91% concordance within variants classified as likely-pathogenic or pathogenic. Moreover, results showed that a region of PTEN is poorly covered by the OCA-Plus assay, hence, we implemented rescue filters for those variants. In conclusion, the OCA-Plus can reflect the mutational profile of genomic variants compared with OCAv3 of 144 overlapping genes, without compromising performance.

OAB-005: Update of safety and efficacy of Isatuximab short-duration fixed-volume infusion plus Bortezomib, Lenalidomide, and Dexamethasone combined therapy for NDMM ineligible/with no immediate intent for ASCT

<h3>Introduction</h3> One standard-of-care treatment for patients with newly diagnosed multiple myeloma (NDMM) not eligible for autologous stem cell transplant (ASCT) is the combination of bortezomib (V), lenalidomide (R), and dexamethasone (d). Isatuximab (Isa), a monoclonal antibody targeting a specific epitope of CD38, is approved in combination with pomalidomide/carfilzomib plus d for the treatment of adults with relapsed/refractory multiple myeloma. The objective of this study was to evaluate the efficacy and safety of the approved short-duration fixed-volume infusion of Isa, combined with VRd (Isa-VRd) in patients with NDMM ineligible/with no immediate intent for ASCT. <h3>Methods</h3> In Part A of this Phase 1b study (NCT02513186), a weight-based infusion of Isa-VRd was effective and well tolerated (median infusion duration at first infusion, 3.7 hours). Here, we present results from Part B, where Isa (10 mg/kg) was administered as a fixed-volume infusion of 250 mL with standard doses of VRd (Ocio Blood 2020). The primary endpoint is the complete response (CR) rate of Isa-VRd. <h3>Results</h3> Of 46 patients in Part B, 30 (65.2%) were receiving study treatment at data cutoff (March 17, 2021). Median patient age was 70.0 years (range, 49–87), and 8 (17.4%) had high-risk cytogenetics. There were 13 (28.3%) patients eligible, but with no immediate intent, for ASCT; of these, 7 (53.8%) proceeded to mobilization and 6 (46.2%) performed apheresis (median 8.1×10⁶ CD34+ cells/kg collected). The median duration of Isa infusion decreased to 1.3 hours from the third infusion onward. The overall response rate was 97.8%, including a CR/stringent CR (sCR) rate of 35.6% and very good partial response rate of 55.6%. After implementation of the SEBIA Hydrashift Isa immunofixation assay assessing serum M-protein without Isa interference, the adjusted CR/sCR rate increased to 53.3%. Investigation of minimal residual disease (MRD) negativity by combined next-generation flow cytometry or sequencing methods at a threshold of 10-5 revealed 23/45 (51.1%) of response-evaluable patients with MRD negativity. All patients experienced ≥1 treatment-emergent adverse event (TEAE), 32 (69.6%) had Grade ≥3 TEAEs, 20 (43.5%) had serious TEAEs, and 6 (13.0%) had TEAEs leading to death. Infusion reactions were observed in 13 (28.3%) of patients; none were of Grade ≥3. The most frequently reported TEAEs were constipation (69.6%), asthenia (67.4%), diarrhea (56.5%), and peripheral sensory neuropathy (50.0%). The most common Grade ≥3 hematologic abnormalities were lymphopenia (76.1%) and neutropenia (41.3%). <h3>Conclusions</h3> These results confirm the feasibility, efficacy, and safety of the approved short-duration fixed-volume infusion method of Isa in combination with VRd in patients with NDMM ineligible/with no immediate intent for ASCT. Isa-VRd is under investigation in ongoing Phase 3 studies. <h3>Funding</h3> Sanofi.

Genetic screening and prenatal diagnosis in high-risk families with tuberous sclerosis complex syndrome

To carry out genetic testing and prenatal diagnosis for 29 Chinese pedigrees affected with tuberous sclerosis complex (TSC) and assess efficacy of combined next generation sequencing (NGS) and multiple ligation-dependent probe amplification (MLPA) for the diagnosis. NGS and MLPA were used in conjunct to detect variants of TSC1 and TSC2 genes among the probands of the pedigrees. Paternity test was carried out to exclude maternal DNA contamination. Prenatal diagnosis was provided to 14 couples based on the discoveries in the probands. Twenty-seven variants were identified in the TSC1 and TSC2 genes among the 29 pedigrees, which yielded a detection rate of 93.1%. Respectively, 5 (18.5%) and 22 (81.5%) variants were identified in the TSC1 and TSC2 genes. Twelve variants were unreported previously. Prenatal diagnosis showed that five fetuses were affected with TSC, whilst the remaining nine were unaffected. Above finding has expanded the spectrum of TSC1 and TSC2 gene variants. Combined NGS and MLPA has enabled diagnosis of TSC with efficiency and accuracy.

Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset

About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2–c.378+1G>T) in the first patient and a nonsense mutation (DSG2–p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.

Technologies enabling rapid crop improvements for sustainable agriculture: example pennycress (Thlaspi arvense L.)

Growing concerns over food insecurity and ecosystems health related to population growth and climate change have challenged scientists to develop new crops, employing revolutionary technologies in combination with traditional methods. In this review, we discuss the domestication of the oilseed-producing cover crop pennycress, which along with the development of other new crops and improvements to farming practices can provide sustainable solutions to address malnutrition and environmental impacts of production agriculture. We highlight some of the new technologies such as bioinformatics-enabled next-generation sequencing and CRISPR genome editing in combination with traditional mutation breeding that has accelerated pennycress development as a new crop and a potential model system. Furthermore, we provide a brief overview of the technologies that can be integrated for improving pennycress and other crops and the status of pennycress development using these technologies.

Population-based Screening for BRAF V600E in Metastatic Colorectal Cancer Reveals Increased Prevalence and Poor Prognosis.

BRAF V600E mutations portend poor prognosis in metastatic colorectal cancer (mCRC); however, the true prevalence and prognosis are unknown, as unwell patients may not undergo BRAF sequencing. We reviewed a population-based cohort of 1,898 patients with colorectal cancer that underwent reflexive IHC mismatch repair (MMR) and BRAF V600E testing. Outcomes among IHC-detected BRAF V600E mCRC (BRAF IHC) were compared with patients with next-generation sequencing (NGS)-identified BRAF V600E-mutated mCRC from two institutions (BRAF NGS) with patients spanning from 2004 to 2018. All-stage population prevalence of BRAF V600E was 12.5% (238/1,898) and did not differ between early and metastatic stages (P = 0.094). Prevalence among mCRC was 10.6% (61/575), of whom 51 (83.6%) were referred to oncology and 26 (42.6%) had NGS testing. BRAF IHC had worse median overall survival (mOS) than BRAF NGS [5.5 vs. 20.4 months; HR, 2.90; 95% confidence interval (CI), 1.89-4.45; P < 0.0001], which persisted in multivariate analysis (P < 0.0001). Across a combined NGS and IHC cohort, BRAF V600E tumors with deficient MMR showed worse mOS compared with MMR proficient tumors (8.9 vs. 17.2 months; HR, 1.46; 95% CI, 0.96-2.27; P = 0.043). In this combined cohort, first-line progression-free survival was 5.9 months, with minimal differences between regimens. Within the population-based cohort, attrition between treatment lines was high with only 60.7% receiving first-line chemotherapy and 26.2% receiving second line. Patients with BRAF V600E-mutated mCRC have a worse prognosis than previously suggested, potentially arising from referral bias for testing. High attrition between lines of therapy suggests efficacious therapies need to be prioritized early for patients to benefit.

Real world data analysis of next generation sequencing and protein expression in metastatic breast cancer patients

Next generation sequencing (NGS) together with protein expression analysis is back bone of molecularly targeted therapy in precision medicine. Our retrospective study shows our experience with NGS of 324 genes in combination with protein expression in patients with advanced breast cancer (aBC). The primary purpose was to analyze the prevalence of individual genetic alterations combined with protein expression to define potential targets for an individualized therapy. Between April 2018 and September 2019, 41 patients with aBC were offered a NGS test. The test was used to detect clinically relevant genomic alterations and to support further targeted therapy decisions. Hormone receptors, ERBB2 of tumors and PD-L1 was stained by immunohistochemistry. The data was recorded up to September 2019. After prior consent 41 results were available for further analysis. The most common BC subtypes were triple-negative (n = 16), HR+/ERBB2− (n = 15), and ERBB2+ (n = 9), with one missing data of the primary tumor. 27 patients had more than one genetic alteration. The most common alterations were PIK3CA (n = 14) and ERBB2 alterations (n = 11). Followed by ESR1 (n = 10), FGFR1 (n = 7) and PTEN (n = 7). 68% of the alterations were clinically relevant (tier I and II of ESCAT classification). The most common treatment recommendation was ERBB2-directed therapy (single or double blockade, trastuzumab emtansine and lapatinib) followed by alpelisib in combination with fulvestrant. Comprehensive genomic profiling combined with protein expression analysis in aBC allowed a guided personalized therapy for half of our patients. So far there are no well-defined tools allowing interpretations of genomic alterations detected by NGS in combination with protein expression and other factors.