Large-scale Genomic Screening Research Articles

Preferences of parents from diverse backgrounds on genomic screening of apparently healthy newborns.

Genomic sequencing has been proposed as a strategy to expand newborn screening. Perspectives on genomic newborn screening from parents of diverse racial, ethnic, and socioeconomic backgrounds are needed to shape equitable implementation of this modality. We conducted 20 semi-structured interviews (15 English, 5 Spanish) and seven focus groups (4 English, 3 Spanish) with parents from diverse backgrounds to assess their perspectives regarding which disorders and variants might be screened, data privacy, and barriers to pursuing specialized care. Parents felt that genomic newborn screening would provide them with improved understanding of their children's health and had the potential to yield health and personal benefits. Themes that became evident included: interest in childhood and family health risks, the value of emotional preparation and personal planning, understanding of uncertain and low-risk results, concerns regarding data privacy, and concerns about support following the receipt of a positive newborn screening result. The expected benefits and concerns expressed by parents of diverse backgrounds regarding genomic newborn screening should guide future policy decisions. Their preferences should be considered prior to the implementation of large-scale genomic newborn screening programs.

Large T antigen mediated target gene replication improves site-specific recombination efficiency.

With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >107 diversity, from which GFP variants with enhanced fluorescence were isolated. We expect the underlying principle of target gene amplification can be applicable to other SSR processes and gene editing approaches in general for directed evolution and large-scale genomic screening in mammalian cells.

Interpreting the effect of mutations to protein binding sites from large-scale genomic screens

Predicting the functionality of missense mutations is extremely difficult. Large-scale genomic screens are commonly performed to identify mutational correlates or drivers of disease and treatment resistance, but interpretation of how these mutations impact protein function is limited. One such consequence of mutations to a protein is to impact its ability to bind and interact with partners or small molecules such as ATP, thereby modulating its function. Multiple methods exist for predicting the impact of a single mutation on protein–protein binding energy, but it is difficult in the context of a genomic screen to understand if these mutations with large impacts on binding are more common than statistically expected. We present a methodology for taking mutational data from large-scale genomic screens and generating functional and statistical insights into their role in the binding of proteins both with each other and their small molecule ligands. This allows a quantitative and statistical analysis to determine whether mutations impacting protein binding or ligand interactions are occurring more or less frequently than expected by chance. We achieve this by calculating the potential impact of any possible mutation and comparing an expected distribution to the observed mutations. This method is applied to examples demonstrating its ability to interpret mutations involved in protein–protein binding, protein-DNA interactions, and the evolution of therapeutic resistance.

Establishment and application of a root wounding-immersion method for efficient virus-induced gene silencing in plants.

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Prospective, multi-site study of healthcare utilization after actionable monogenic findings from clinical sequencing

As large-scale genomic screening becomes increasingly prevalent, understanding the influence of actionable results on healthcare utilization is key to estimating the potential long-term clinical impact. The eMERGE network sequenced individuals for actionable genes in multiple genetic conditions and returned results to individuals, providers, and the electronic health record. Differences in recommended health services (laboratory, imaging, and procedural testing) delivered within 12months of return were compared among individuals with pathogenic or likely pathogenic (P/LP) findings to matched individuals with negative findings before and after return of results. Of 16,218 adults, 477 unselected individuals were found to have a monogenic risk for arrhythmia (n= 95), breast cancer (n= 96), cardiomyopathy (n= 95), colorectal cancer (n= 105), or familial hypercholesterolemia (n= 86). Individuals with P/LP results more frequently received services after return (43.8%) compared to before return (25.6%) of results and compared to individuals with negative findings (24.9%; p<0.0001). The annual cost of qualifying healthcare services increased from an average of $162 before return to $343 after return of results among the P/LP group (p<0.0001); differences in the negative group were non-significant. The mean difference-in-differences was $149 (p<0.0001), which describes the increased cost within the P/LP group corrected for cost changes in the negative group. When stratified by individual conditions, significant cost differences were observed for arrhythmia, breast cancer, and cardiomyopathy. In conclusion, less than half of individuals received billed health services after monogenic return, which modestly increased healthcare costs for payors in the year following return.

Genome sequencing-based transcriptomic analysis reveals novel genes in Peucedanum praeruptorum

BackgroundPeucedanum praeruptorum Dunn, a traditional Chinese herbal medicine, contains coumarin and volatile oil components that have clinical application value. However, early bolting often occurs in the medicinal materials of Apiaceae plants. The rhizomes of the medicinal parts are gradually lignified after bolting, resulting in a sharp decrease in the content of coumarins. At present, the link between coumarin biosynthesis and early bolting in P. praeruptorum has not been elucidated.ResultsCombining the genome sequencing and the previous transcriptome sequencing results, we reanalyzed the differential transcripts of P. praeruptorum before and after bolting. A total of 62,088 new transcripts were identified, of which 31,500 were unknown transcripts. Functional classification and annotation showed that many genes were involved in the regulation of transcription, defense response, and carbohydrate metabolic processes. The main domains are the pentatricopeptide repeat, protein kinase, RNA recognition motif, leucine-rich repeat, and ankyrin repeat domains, indicating their pivotal roles in protein modification and signal transduction. Gene structure analysis showed that skipped exon (SE) was the most dominant alternative splicing, followed by the alternative 3’ splice site (A3SS) and the alternative 5’ splice site (A5SS). Functional enrichment of differentially expressed genes showed that these differentially expressed genes mainly include transmembrane transporters, channel proteins, DNA-binding proteins, polysaccharide-binding proteins, etc. In addition, genes involved in peroxisome, hexose phosphate pathway, phosphatidylinositol signaling system, and inositol phosphate metabolism pathway were greatly enriched. A protein-protein interaction network analysis discoverd 1,457 pairs of proteins that interact with each other. The expression levels of six UbiA genes, three UGT genes, and four OMT genes were higher during the bolting stage. This observation suggests their potential involvement in the catalytic processes of prenylation, glycosylation, and methylation of coumarins, respectively. A total of 100 peroxidase (PRX) genes were identified being involved in lignin polymerization, but only nine PRX genes were highly expressed at the bolting stage. It is worth noting that 73 autophagy-related genes (ATGs) were first identified from the KEGG pathway-enriched genes. Some ATGs, such as BHQH00009837, BHQH00013830, and novel8944, had higher expression levels after bolting.ConclusionsComparative transcriptome analysis and large-scale genome screening provide guidance and new opinions for the identification of bolting-related genes in P. praeruptorum.

Genomic newborn screening for rare diseases.

Rare diseases are a leading cause of infant mortality and lifelong disability. To improve outcomes, timely diagnosis and effective treatments are needed. Genomic sequencing has transformed the traditional diagnostic process, providing rapid, accurate and cost-effective genetic diagnoses to many. Incorporating genomic sequencing into newborn screening programmes at the population scale holds the promise of substantially expanding the early detection of treatable rare diseases, with stored genomic data potentially benefitting health over a lifetime and supporting further research. As several large-scale newborn genomic screening projects launch internationally, we review the challenges and opportunities presented, particularly the need to generate evidence of benefit and to address the ethical, legal and psychosocial issues that genomic newborn screening raises.

Large-scale clinico-genomic profile of non-small cell lung cancer with KRAS G12C: Results from LC-SCRUM-Asia study

IntroductionKRAS G12C is an oncogenic driver mutation, accounting for approximately 14% of Caucasian patients with non-small cell lung cancer (NSCLC). Recently, several KRAS G12C-targeted drugs have been developed; however, the clinico-genomic characteristics of NSCLC patients with KRAS G12C remain unclear. Materials and MethodsBased on the large-scale prospective lung cancer genomic screening project (LC-SCRUM-Asia) database, the clinico-genomic characteristics and therapeutic outcomes of NSCLC patients with KRAS G12C were evaluated. ResultsFrom March 2015 to March 2021, 10,023 NSCLC patients were enrolled in LC-SCRUM-Asia. KRAS mutations were detected in 1258 patients (14 %), including G12C in 376 (4.0 %), G12D in 289 (3.1 %) and G12V in 251 (2.7 %). The proportions of males and smokers were higher in patients with KRAS G12C than in those with KRAS non-G12C mutations (males: 73 % vs 63 %, p < 0.001; smokers: 89 % vs 76 %, p < 0.001). KRAS G12C-positive tumors showed a higher tumor mutation burden (TMB) (mean, 8.1 mut/Mb, p < 0.001) and a higher percentage of tumors with programmed cell death ligand-1 (PD-L1) expression ≥50 % (52 %, p = 0.08). The overall survival in patients with KRAS G12C (median, 24.6 months) was not different between patients with other mutation subtypes (G12V: 18.2 months, p = 0.23; G12D: 20.6 months, p = 0.65; other KRAS mutations: 18.3 months, p = 0.20). Among KRAS-mutated patients who received immune checkpoint inhibitors (ICIs), the progression-free survival in G12C-positive patients (median, 3.4 months) was similar to that in G12V-positive patients (4.2 months, p = 0.90), but significantly longer than that in G12D- (2.0 months, p = 0.02) and other KRAS mutation-positive patients (2.5 months, p = 0.02). ConclusionsThe frequencies of KRAS G12C were lower in Asian than in Caucasian NSCLC patients. Among the KRAS-mutated NSCLC patients, G12C-positive tumors showed increased immunogenicity, such as high TMB and high PD-L1 expression, and potential sensitivity to ICIs.

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

Characterisation of a Novel Cell Line (ICR-SS-1) Established from a Patient-Derived Xenograft of Synovial Sarcoma.

Synovial sarcoma is a rare translocation-driven cancer with poor survival outcomes, particularly in the advanced setting. Previous synovial sarcoma preclinical studies have relied on a small panel of cell lines which suffer from the limitation of genomic and phenotypic drift as a result of being grown in culture for decades. Patient-derived xenografts (PDX) are a valuable tool for preclinical research as they retain many histopathological features of their originating human tumour; however, this approach is expensive, slow, and resource intensive, which hinders their utility in large-scale functional genomic and drug screens. To address some of these limitations, in this study, we have established and characterised a novel synovial sarcoma cell line, ICR-SS-1, which is derived from a PDX model and is amenable to high-throughput drug screens. We show that ICR-SS-1 grows readily in culture, retains the pathognomonic SS18::SSX1 fusion gene, and recapitulates the molecular features of human synovial sarcoma tumours as shown by proteomic profiling. Comparative analysis of drug response profiles with two other established synovial sarcoma cell lines (SYO-1 and HS-SY-II) finds that ICR-SS-1 harbours intrinsic resistance to doxorubicin and is sensitive to targeted inhibition of several oncogenic pathways including the PI3K-mTOR pathway. Collectively, our studies show that the ICR-SS-1 cell line model may be a valuable preclinical tool for studying the biology of anthracycline-resistant synovial sarcoma and identifying new salvage therapies following failure of doxorubicin.

Targeting the DNA damage response beyond poly(ADP-ribose) polymerase inhibitors: novel agents and rational combinations.

Poly(ADP-ribose) polymerase (PARP) inhibitors have transformed treatment paradigms in multiple cancer types defined by homologous recombination deficiency (HRD) and have become the archetypal example of synthetic lethal targeting within the DNA damage response (DDR). Despite this success, primary and acquired resistance to PARP inhibition inevitability threaten the efficacy and durability of response to these drugs. Beyond PARP inhibitors, recent advances in large-scale functional genomic screens have led to the identification of a steadily growing list of genetic dependencies across the DDR landscape. This has led to a wide array of novel synthetic lethal targets and corresponding inhibitors, which hold promise to widen the application of DDR inhibitors beyond HRD and potentially address PARP inhibitor resistance. In this review, we describe key synthetic lethal interactions that have been identified across the DDR landscape, summarize the early phase clinical development of the most promising DDR inhibitors, and highlight relevant combinations of DDR inhibitors with chemotherapy and other novel cancer therapies, which are anticipated to make an impact in rationally selected patient populations. The DDR landscape holds multiple opportunities for synthetic lethal targeting with multiple novel DDR inhibitors being evaluated on early phase clinical trials. Key challenges remain in optimizing the therapeutic window of ATR and WEE1 inhibitors as monotherapy and in combination approaches.

Co-occurring gene alterations associated with efficacy of osimertinib in EGFR-mutated lung cancer: Based on a large-scale genomic screening project (LC-SCRUM-Asia).

9103 Background: Osimertinib is a standard drug for first-line treatment of patients with advanced non–small cell lung cancer (NSCLC) harboring EGFR mutations (mt). While tumor mutational burden (TMB)-high and co-occurring genetic alterations (alt) have been reported to be negatively associated with the efficacy of other EGFR-TKIs, the impact of co-occurring genetic alt with EGFR major mt on the efficacy of osimertinib remains unclear. Methods: In a multi-institutional genomic screening project (LC-SCRUM-Asia), we have analyzed lung cancer patients for genomic alt by a targeted next-generation sequencing (NGS) system, Oncomine Comprehensive Assay and Genexus/OPA. We retrospectively evaluated the association between the genomic profile and efficacy of first-line osimertinib for EGFR-mutated NSCLC based on the LC-SCRUM-Asia database. Results: Between March 2015 and January 2022, 12,705 NSCLC patients were enrolled in the LC-SCRUM-Asia database, and EGFR mt was detected in 2,232 patients. Of these, 324 patients, including 171 with ex19del (53%) and 153 with L858R (47%), received first-line treatment with osimertinib. The patient characteristics were as follows: median age, 69 years (range 31-97); females, 64%; never-smokers, 57%; adenocarcinoma, 97%; and performance status 0-1, 99%. The frequency of compound EGFR mt and TMB were higher in the L858R (LR) group than in the ex19del (Ex19) group (compound mt (%), 12 vs. 4; mean TMB (mt/Mb), 3.4 vs. 2.5). There were no differences in the frequencies of other co-occurring genetic alt between the two groups. Higher TMB, alt of genes encoding receptor tyrosine kinase (RTK), including FGFR1, RET, MET etc., and amp of cell-cycle related genes were significantly associated with shorter progression-free survival (PFS) in the entire group (median PFS: TMB &gt; 3 vs. ≤3 mt/Mb = 11.4 vs. 17.1 months; p = 0.023; RTK gene alt+ vs. alt- = 9.7 vs. 15.2 months; p = 0.014; cell-cycle gene amp+ vs. amp- = 10.6 vs. 15.6 months, p = 0.001). EGFR subgroup analysis showed that a higher TMB was significantly associated with a shorter PFS in the LR group (&gt; 3 vs. ≤3 mt/Mb = 10.0 vs. 17.1 months, p &lt; 0.001), but not in the Ex19 group. On the other hand, alt of genes encoding RTK and amp of cell-cycle related genes were significantly associated with a shorter PFS in the Ex19 group (RTK gene alt+ vs. alt- = 8.4 vs. 17.8 months, p = 0.008; cell-cycle gene amp+ vs. amp- = 10.6 vs. 17.5 months, p = 0.003), but not in the LR group. Multivariate analysis identified RTK gene alt in the Ex19 group and higher TMB in the LR group as being independently associated with a shorter PFS. Conclusions: First-line osimertinib treatment was less effective in NSCLC patients harboring Ex19 with other RTK gene alt or LR with a higher TMB, indicating that co-occurring genetic alt affecting the efficacy of osimertinib differ between NSCLC patients harboring Ex19 and LR.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence &lt;em&gt;In Vitro&lt;/em&gt;

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

In This Issue

In This Issue

An In Vivo CRISPR Screening Platform to Identify New Therapeutic Targets in AML

First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing.We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery.Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically.Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML.Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. DisclosuresDharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Clinico-pathological and genomic features of NRAS- or HRAS-mutated non-small cell lung cancer (NSCLC) identified in large-scale genomic screening project (LC-SCRUM-Asia).

9054 Background: RAS ( KRAS, NRAS and HRAS) is a targetable oncogene family in several cancers, including NSCLC, and the clinical development of various RAS-targeted therapies are ongoing. However, the clinical relevance of uncommon RAS mutations, such as NRAS and HRAS mutations, in NSCLC patients (pts) remains unclear. Methods: In a large-scale genomic screening project (LC-SCRUM-Asia), we have prospectively analyzed lung cancer pts for genomic alterations by a targeted next-generation sequencing (NGS) system, Oncomine Comprehensive Assay. We evaluated clinico-pathological and genomic characteristics in NRAS- or HRAS-mutated NSCLC pts comparing with those in KRAS-mutated pts based on the LC-SCRUM-Asia database. Results: Since March 2015 to December 2020, 9131 NSCLC pts were enrolled in the LC-SCRUM-Asia, and 8374 of them (92%) were successfully analyzed by NGS. The RAS mutation frequencies were 1134 KRAS (14%), 50 NRAS (0.6%), and 15 HRAS (0.2%). The most frequent variant of NRAS and HRAS mutations was Q61X (78%) and G13X (80%), respectively, whereas that of KRAS was G12X (84%). Patient characteristics were summarized in Table. Male was significantly frequent in NRAS- than in KRAS-group (p=0.03), and smokers were frequent in all the three groups (overall, 79%). The majority of NRAS (70%) and KRAS mutations (89%) were detected in adenocarcinoma (Ad), whereas 60% of HRAS mutations were in squamous cell carcinoma (Sq). Tumor mutation burden (TMB) was significantly higher in NRAS-mutated tumors than in KRAS-mutated tumors (p=0.03). Concomitant TP53 mutations were significantly frequent in HRAS-mutated pts than in KRAS-mutated pts (53% vs. 30%, p=0.05), and STK11 mutations were also tended to be frequent in HRAS-mutated pts than in KRAS-mutated pts (20 vs. 7%, p=0.10). Therapeutic efficacy of PD-1/PD-L1 inhibitors was not different among the three groups in the current follow-up data, but HRAS-mutated tumors did not respond to PD-1/PD-L1 inhibitors (response rate, 0%; median PFS, 1.6 months). Conclusions: NRAS- or HRAS-mutated NSCLCs were different from KRAS-mutated NSCLCs in clinico-pathological and genomic profiles. In particular, the immunotherapies were not effective for HRAS-mutated NSCLCs.[Table: see text]

Systems Approaches to Unravel T Cell Function and Therapeutic Potential in Autoimmune Disease

Producing Ag-specific immune responses constrained to target tissues or cells that can be engaged or disengaged at will is predicated on understanding the network of genes governing immune cell function, defining the rules underlying Ag specificity, and synthesizing the tools to engineer them. The successes and limitations of chimeric Ag receptor (CAR) T cells emphasize this goal, and advances in high-throughput sequencing, large-scale genomic screens, single-cell profiling, and genetic modification are providing the necessary data to bring it to fruition-including a broader application into the treatment of autoimmune diseases. In this review, we delve into the implementation of these developments, survey the relevant works, and propose a framework for generating the next generation of synthetic T cells informed by the principles learned from these systems approaches.

Returning negative results from large-scale genomic screening: Experiences from the eMERGE III network.

Population-based genomic screening has the potential to improve health outcomes by identifying genetic causes of disease before they occur. While much attention has been paid to supporting the needs of the small percentage of patients who will receive a life-altering positive genomic screening result that requires medical attention, little attention has been given to the communication of negative screening results. As there are currently no best practices for returning negative genomic screening results, we drew on experiences across the electronic medical records and genomics (eMERGE) III Network to highlight the diversity of reporting methods employed, challenges encountered in reporting negative test results, and "lessons learned" across institutions. A 60-item survey that consisted of both multiple choice and open-ended questions was created to gather data across institutions. Even though institutions independently developed procedures for reporting negative results, and had very different study populations, we identified several similarities of approach, including but not limited to: returning results by mail, placing results in the electronic health record via an automated process, reporting results to participants' primary care provider, and providing genetic counseling to interested patients at no cost. Differences in procedures for reporting negative results included: differences in terminology used to describe negative results, definitions of negative results, guidance regarding the meaning of negative results for participants and their family members, and recommendations for clinical follow up. Our findings highlight emerging practices for reporting negative genomic screening results and highlight the need to create patient education and clinical support tools for reporting negative screening results.