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This review on in situ gel was written to fill in any gaps in the current body of knowledge, with a special emphasis on in-situ gelling systems that prolong the residence time of drugs at their targets. Drugs with very short half-lives and high solubility are especially interested in in-situ gels. The use of in-situ gelling systems as drug delivery systems for both local and systemic effects has recently been developed. The advantages of in-situ polymeric delivery systems, such as ease of administration, decreased frequency of administration, enhanced patient compliance, and comfort, have drawn the most attention in current in-situ gelling system development. The studies that evaluated the use and performance of these systems are summarized in this review. This review paper is prepared to highlight formulation considerations employed during the development of an in-vivo drug delivery system, pertinent biodegradable polymers for the sol-gel transition, characterization, assessment criteria, and commercial formulations of the in-vivo polymeric system.

Background: Copy Number Variations (CNVs) are prominent features of cancer cells. From a clinical standpoint, their accurate detection at a low cost is a priority. With regular increases in the number of markers to be tested, the cost effectiveness and practicality of gold standard techniques like Fluorescence In Situ Hybridization (FISH) are slowly decreasing. Cost-efficient Next Generation Sequencing (NGS) targeted gene panels can be scaled up but accurately detecting CNVs from the resulting data remains challenging. We demonstrate large amounts of data and machine learning can help bridge the gap between the two techniques. Methods: We collected the sequencing data of 6,277 patients tested using a custom amplicon based NGS assay designed to detect somatic alterations in 297 hematological cancer relevant genes such that at least one concurrent FISH test was also performed. FISH results were used to infer the gain, loss, or normality information for each corresponding gene. The annotated genes were then used to curate a training set by extracting 20 features per gene from the alignment results. A 3-class random forest classifier was trained using this dataset. The selected model was evaluated on a distinct set of 2,738 patients. Results: Evaluation results are provided in Table 1 for 8 genes for which the FISH probe used to infer the gain, loss, or normality information directly spanned the gene region. The predicted CNVs are almost a perfect match with FISH for 6 of these genes with a limit of detection at 20% abnormal cells. In most cases, the model reduces discordant calls by over 50% compared to using existing CNV detection software only. Conclusion: We show the CNV detection capabilities of a targeted NGS assay can closely match the gold-standard FISH technique by analytically correcting the biases introduced by the targeting procedures. The model presented here is used to detect CNVs in ALL patients after a successful formal validation in our laboratory. Table 1. Evaluation results. Gene FISH Positive Cases (Gain/Loss) FISH Negative Cases (Normal) All FISH Cases Total Concordant Sensitivity Total Concordant Specificity Total Concordant Accuracy ATM 72 72 100.00% 641 629 98.13% 713 701 98.32% CBFB 23 22 95.65% 517 506 97.87% 540 528 97.78% EGR1 171 169 98.83% 1,541 1,538 99.81% 1,712 1,707 99.71% KMT2A 27 25 92.59% 474 472 99.58% 501 497 99.20% MET 113 113 100.00% 1,593 1,582 99.31% 1,706 1,695 99.36% NF1 15 15 100.00% 908 908 100.00% 923 923 100.00% TERT 10 10 100.00% 1,723 1,709 99.19% 1,733 1,719 99.19% TP53 76 73 96.05% 1,567 1,556 99.30% 1,643 1,629 99.15% Citation Format: Christophe N. Magnan, Hyunjun Nam, Shashikant Kulkarni, Segun C. Jung. Bridging the gap between targeted NGS and FISH gene-level CNV detection capabilities in hematologic malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4294.

Background: Acute lymphoblastic leukemia (ALL) can be classified to subtypes by molecular signatures correlating to prognosis and treatment regimen, making accurate molecular profiling of interest for both clinical and research communities. NGS technology allow for identification of a wide array of molecular events (e.g. structural variants and single nucleotide variants (SNVs)), and integrate these with global profiles of gene expression. Most tests screen either DNA or RNA to provide a piece of a complex molecular puzzle, resulting in higher cost, processing time and amounts of input material. We report a single tube NGS assay that uses total nucleic acid as input for simultaneous screening of DNA and RNA (allowing us to profile gene expression, fusions and multiple variants), establishing a work frame for comprehensive molecular profiling in a single test. Methods: 243 clinical ALL samples (206 bone marrow and 37 peripheral blood) were used in this study, split in to two cohorts, 102 for initial investigation and 141 for reproducibility testing. Libraries were done using a custom Qiagen Multimodal NGS panel of 302 DNA and 229 RNA targets. Amplicon libraries were sequenced with UDI on Illumina NovaSeq 6000. Data were analyzed by in-house bioinformatic pipeline. Expression values were normalized to GUSB. Previous in-house studies were used to establish cut-offs for upregulation of 31 genes associated with ALL. Variant enrichment for genes was done by normalizing the number of variants to the size of the gene and genes with elevated variant loads were identified. Pathway enrichment analysis was done using gProfiler2. Results: A set of 102 ALL samples were used to study expression patterns within the cohort. Principal component analysis (PCA) on the 229 RNA targets was used to identify clusters. We then investigated whether the predetermined 31 over-expressed (OE) genes could be driving the clusters. 3 clusters were observed based on OE quantification, low OE degree (0-1 genes), medium (8-11), and high (>17 genes). EPOR OE is characteristic of the high-degree cluster, while Ph+ samples are most common in the medium cluster. Pathway enrichment on the OE genes suggest that the medium cluster is enriched for genes linked with missregulation of transcription, and the high cluster shows enrichment of kinase activation. We then repeated the analysis with a second set of 141 samples, to assess the reproducibility of these findings and confirming our results. Finally, we investigated SNVs’ role in our data. We observed that KMT2D was enriched in variants in our cohort, underscoring that epigenetics is an important component of the disease landscape. Conclusions: We demonstrate the use of a single tube multimodal NGS assay for comprehensive genomic profiling. Our analysis shows that this powerful and cost-effective tool can help identify molecular signatures, guiding diagnostic and therapeutic management for ALL patients. Citation Format: Lina Zelinger, Samuel Koo, Charmeine Ko, Francys Alarcon, Kenneth B. Thomas, Segun C. Jung, Fernando J. Lopez Diaz, Fei Ye, Shashikant Kulkarni. Single-tube NGS profiling allows identification of molecular signature in ALL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 219.

Background: WHO recognizes 23 genomic rearrangements or fusions which define subclasses of AML, MDS/MPN and related neoplasms, and their detection is essential for patient management. Discerning true fusions from artificial calls in RNAseq-based tests is challenging due to biological and technical noise. We previously developed a method to identify fusion transcripts by a single-tube NGS assay capable of concurrent analysis of DNA and RNA alterations in ALL patients. We expanded the assay with an improved fusion calling algorithm and used it to study the landscape of myeloid RNA fusions in the clinical setting. Methods: Total nucleic acid (TNA) from bone marrow or peripheral blood was analyzed in our clinical laboratory by a CLIA grade custom amplicon-based multimodal NGS assay, targeting 302 genes by DNA-seq and 185 genes by RNA-seq. Libraries were sequenced on a NovaSeq6000 instrument, and fusions were called from RNA: de-duplicated and error-corrected UMI reads were processed by an in-house developed BI pipeline leveraging machine learning, to assign a final confidence score (F1). Deidentified patient data was used according to an approved IRB. Results: Distribution of F1 scores was used to improve the discrimination between technical noise and real fusion calls. Analytical validation of RNA fusion calling against FISH and Sanger-seq in 74 hematologic disorder samples demonstrated 98.2% specificity and 97.0% sensitivity. Data from 789 patients was used to study the distribution of myeloid fusion events in community cases. 17% of patients had fusions involving genes from WHO/NCCN recommendations. Frequencies for most common fusions were 2.3% (18/789) for KMT2A, 2% for PML::RARA, 1.9% for BCR::ABL1, 0.8% for RUNX1::RUNX1T1 and CBFB::MYH11 and 0.6% for NUP98. Fusions of PDGFRA, ETV6, ZNF384, FGFR1 and other genes were also observed and BCR::ABL1 fusions were seen not only in CML patients but also in a patient with AML. For KMT2A, 25% (2/8) fusions detected by NGS were confirmed by Sanger-seq but missed by FISH, which correlates with higher sensitivity of the NGS assay. Novel fusions were called in ~8% of patients. This included an AML patient with a CCND2::MGP fusion, resulting in cyclin D2 (CCND2), frequently activated by DNA mutations in AML, fused to matrix Gla protein, a highly expressed gene in hematopoietic progenitor cells. The fusion was confirmed by Sanger-seq, and shown to lack ex5 of CCND2, which contains Thr280, a residue required for ccnd2 degradation. This fusion is thus predicted to generate high cellular levels of oncogenic ccnd2-mgp. Conclusions: Frequencies of well-known fusions in real world data obtained by a robust low-noise RNA fusion assay were similar to other studies done in academic setting. Reliable detection of bona-fide RNA fusions with this clinical test is invaluable for patient care and novel fusion identification. Citation Format: Michal Krawczyk, Chaugiang Duong, Lina Zelinger, Fei Ye, Hyunjun Nam, Brad Thomas, Vincent Funari, Shashikant Kulkarni, Fernando Lopez-Diaz. Landscape of known and novel myeloid neoplasia fusions identified by a multimodal comprehensive genomic profiling test in 789 patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1401.

Ependymomas are neuroepithelial tumors that differentiate along the ependymal cell lineage, a lining of the ventricles of the brain and the central canal of the spinal cord. They are rare in adults, but account for around 9% of brain tumors in children, where they usually have an aggressive course. Efficient stratification could lead to improved care but remains a challenge even in the genomic era. Recent studies proposed a multivariate classification system based on tumor location, age, and broad genomic findings like global patterns of methylation and copy number variants (CNVs). This system shows improved prognostic utility, but is relatively impractical in the routine clinical setting because it necessitates multiple diagnostic tests. We analyzed 13 intracranial grade II and III ependymoma specimens on a DNA microarray to identify discrete CNVs that could support the existing classification. The loss of chr22 and the gain of 5p15.31 were common throughout our cohort (6 and 11 cases, respectively). Other CNVs correlated well with the previously proposed classification system. For example, gains of chr20 were unique to PF-EPN-B tumors of the posterior fossa and may differentiate them from PF-EPN-A. Given the ease of detecting CNVs using multiple, clinically validated methods, these CNVs should be further studied to confirm their diagnostic and prognostic utility, for incorporation into clinical testing algorithms.

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. While these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (e.g., population frequency, functional and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation and importantly impacts patient care. Therefore, it’s essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium (CGC), and the Variant Interpretation for Cancer Consortium (VICC) used a consensus approach to develop a Standard Operating Procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification, and has been validated on 94 somatic variants in ten common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of a somatic variant.

Whole genome sequencing (WGS) shows promise as a first-tier diagnostic test for patients with rare genetic disorders. However, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading health care and research organizations in the US and Canada, was formed to expand access to high quality clinical WGS by convening experts and publishing best practices. Here, we present best practice recommendations for the interpretation and reporting of clinical diagnostic WGS, including discussion of challenges and emerging approaches that will be critical to harness the full potential of this comprehensive test.

The severity and perseverance of inflammation have been demonstrated in many health conditions. The limitations of existing medications suggest the need for new alternative anti-inflammatory medications. In our earlier studies, we demonstrated the topical anti-inflammatory potential of the crude ethanolic extract of Tetrastigma sulcatum leaves and its fractions. In the present study, we further explored the anti-inflammatory activity of T. sulcatum extract, fractions, pure compound and its derivatives using in vitro and in vivo bioassay techniques. We attempted to isolate a pure compound from the leaf extract and identified it as a Friedelan-3β-ol (CI). Furthermore, Friedelinol acetate (C II) and friedelinol methyl ether (C III), derivatives of Friedelan-3β-ol (CI) were synthesised. LPS-induced inflammatory RAW 264.7 macrophages were used as in vitro model to study anti-inflammatory and anti-oxidative effects. Inflammation-induced oxidative damage was found to be restricted significantly (P 0.001), with scavenging activity and increased SOD activity of crude extract and fractions. Treatment with crude extract (TSETOH) and fractions (TSHEX, TSTOL) significantly reduced (P 0.001) the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and nitric oxide (NO) production in LPS-stimulated inflammation in RAW 264.7 cells in a dose-dependent manner. Likewise, compounds CI and CIII showed a similar pattern of significant inhibition (P 0.001) of pro-inflammatory cytokines and NO production in a dose-dependent manner. An in vivo study in a carrageenan-induced mouse paw oedema model demonstrated reduced paw oedema and pro-inflammatory cytokines in a dose-dependent manner upon treatment with the extract, its fractions, pure compound (CI), and their derivatives (CII, and CIII). The present study confirmed the anti-inflammatory activity of T. sulcatum, suggesting that Friedelan-3β-ol is an active component of the crude extract.

Interactions between the bone marrow microenvironment and MDS tumor clones play a role in pathogenesis and response to treatment. We hypothesized G-CSF and plerixafor may enhance sensitivity to azacitidine in MDS. 28 patients with MDS were treated with plerixafor, G-CSF and azacitidine with a standard 3+3 design. Subjects received G-CSF 10 mcg/kg D1–D8, plerixafor D4–D8, and azacitidine 75 mg/m2 D4–D8, but the trial was amended to reduce G-CSF dose to 5 mcg/kg for 5 days after 2 patients had significant leukocytosis. Plerixafor was dose escalated to 560mcg/kg/day without dose limiting toxicity. Two complete responses and 6 marrow responses were seen for an overall response rate (ORR) of 36% in evaluable patients, and ORR of 53% in patients receiving the triplet. Evidence of mobilization correlated with a higher ORR, 60% vs 17%. Plerixafor, G-CSF and azacitidine appears tolerable when given over 5 days and has encouraging response rates.

Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendations on clinical WGS analytical validation for the diagnosis of individuals with suspected germline disease with a focus on test development, upfront considerations for test design, test validation practices, and metrics to monitor test performance. This work also provides insight into the current state of WGS testing at each member institution, including the utilization of reference and other standards across sites. Importantly, members of this initiative strongly believe that clinical WGS is an appropriate first-tier test for patients with rare genetic disorders, and at minimum is ready to replace chromosomal microarray analysis and whole-exome sequencing. The recommendations presented here should reduce the burden on laboratories introducing WGS into clinical practice, and support safe and effective WGS testing for diagnosis of germline disease.

In its landmark paper about Standards and Guidelines for the Interpretation of Sequence Variants, the American College of Medical Genetics and Genomics (ACMG), and Association for Molecular Pathology (AMP) did not address how to use tumor data when assessing the pathogenicity of germline variants. The Clinical Genome Resource (ClinGen) established a multidisciplinary working group, the Germline/Somatic Variant Subcommittee (GSVS) with this focus. The GSVS implemented a survey to determine current practices of integrating somatic data when classifying germline variants in cancer predisposition genes. The GSVS then reviewed and analyzed available resources of relevant somatic data, and performed integrative germline variant curation exercises. The committee determined that somatic hotspots could be systematically integrated into moderate evidence of pathogenicity (PM1). Tumor RNA sequencing data showing altered splicing may be considered as strong evidence in support of germline pathogenicity (PVS1) and tumor phenotypic features such as mutational signatures be considered supporting evidence of pathogenicity (PP4). However, at present, somatic data such as focal loss of heterozygosity and mutations occurring on the alternative allele are not recommended to be systematically integrated, instead, incorporation of this type of data should take place under the advisement of multidisciplinary cancer center tumor-normal sequencing boards.

Harmonization of cancer variant representation, efficient communication, and free distribution of clinical variant‐associated knowledge are central problems that arise with increased usage of clinical next‐generation sequencing. The Clinical Genome Resource (ClinGen) Somatic Working Group (WG) developed a minimal variant level data (MVLD) representation of cancer variants, and has an ongoing collaboration with Clinical Interpretations of Variants in Cancer (CIViC), an open‐source platform supporting crowdsourced and expert‐moderated cancer variant curation. Harmonization between MVLD and CIViC variant formats was assessed by formal field‐by‐field analysis. Adjustments to the CIViC format were made to harmonize with MVLD and support ClinGen Somatic WG curation activities, including four new features in CIViC: (1) introduction of an assertions feature for clinical variant assessment following the Association of Molecular Pathologists (AMP) guidelines, (2) group‐level curation tracking for organizations, enabling member transparency, and curation effort summaries, (3) introduction of ClinGen Allele Registry IDs to CIViC, and (4) mapping of CIViC assertions into ClinVar submission with automated submissions. A generalizable workflow utilizing MVLD and new CIViC features is outlined for use by ClinGen Somatic WG task teams for curation and submission to ClinVar, and provides a model for promoting harmonization of cancer variant representation and efficient distribution of this information.

Organ shortage is the greatest challenge facing the field of organ transplantation today. We aimed to study the attitude and knowledge toward organ donation among health-care professionals (HCPs) in rural India. The study was conducted in a rural town in Konkan region of Maharashtra in India. A questionnaire testing knowledge and attitude about various aspects of organ donation was distributed to HCPs. One hundred percent of the respondents were aware about organ donation. Nearly 40.6% and 21.9% believed that a healthy person and a cardiac dead person can be donors, respectively. Fifty percent believed that a brain dead person can be a donor and 3.1% clearly stated as to be having no idea regarding the health status of a donor. Almost 37.5% were ready to believe a heart beating person declared as "brain dead" as dead. Nearly 15.6% were ready to accept a brain dead person as "legally" dead. Highest awareness was observed regarding eye donation, i.e., 87.5%. High awareness was also observed regarding liver, kidney, heart, skin, and body donations, i.e., 78.1%, 65.6%, 37.5%, 31.3%, and 25.0%, respectively. Awareness regarding organ donation of other tissues and organs was poor. Nearly 46.9% HCPs stated that they felt need for an educational session on organ donation. Awareness regarding concept of organ donation among HCPs in rural India is high. Awareness regarding details of organ donation needs further awareness drives. There is a lack of understanding regarding various aspects of brain death and its importance in organ donation.

Clinical significance of somatic gene variants needs to be comprehensively characterized for their diagnostic, prognostic and/or therapeutic actionability in patient management. However, challenges remain due to discrepancies in interpretation and reporting of these somatic variants among different testing labs. Therefore, standardized curation, clinical interpretation and reporting of somatic variants in hematologic cancers is critical. To address this issue, the Hematologic Cancer Taskforce (HCT), composed of 52 multi-disciplinary experts including oncologists, molecular pathologists, lab directors, genomic scientists and biocurators, was formed in January 2020 within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematologic malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, HCT members curate, edit, and verify Evidence Items for each variant extracted from peer-reviewed publications. Monthly discussions based on these Evidence Items lead to the preparation of variant Assertions, which summarize the state of the field consensus variant interpretation and include tiering based on the AMP/ASCO/CAP guidelines (PMID: 27993330). FMS-like tyrosine kinase 3 (FLT3) encodes a class III receptor tyrosine kinase expressed in hematopoietic cells. FLT3 mutations, including both internal tandem duplication (ITD) and mutations in the tyrosine kinase domain (TKD), are the most common mutations in acute myeloid leukemia (AML), occurring in approximately 30% of all AML cases. Implementing FLT3 tyrosine kinase inhibitors (TKIs) in different treatment regimens for FLT3 mutated AML patients has led to significantly improved overall survival. Type I FLT3 inhibitors, including midostaurin, gilteritinib, sunitinib, lestaurtinib, and crenolanib, bind to the ATP-binding site when the receptor is in active conformation. Type II FLT3 inhibitors, including sorafenib, ponatinib, and quizartinib, interact with a hydrophobic region directly adjacent to the ATP-binding domain that is only accessible when the receptor is inactive, which prevents receptor activation. Generally in AML cells, type I FLT3 inhibitors prevent activity for both ITD and TKD mutations, while Type II inhibitors target ITD but lack efficiency against TKD mutations. The development of TKD mutations in AML cells with ITD have proved to be a mechanism of acquired, or secondary resistance to Type II FLT3 inhibitors. The HCT is piloting curation assessments of FLT3 alterations, including ITD, TKD and non-TKD variants, in AML. So far, the HCT has curated 75 evidence items for FLT3 somatic variants. FLT3-ITD, as well as D835 and I836 were asserted as tier 1 level A variants based on the prediction of response to gilteritinib in relapsed/refractory AML (PMIDs: 27993330, 31665578, 28645776, 28516360, 27908881). Recent curation activities are focused on FLT3 D839G and N676K, as clinical trials using large AML patient cohorts are lacking in their ability to validate drug response/resistance associations of these two TKD variants due to their low frequency. Functional studies showed both variants result in increased proliferation and protection from apoptosis, supporting the oncogenic potential of these two variants (PMIDs: 26891877, 2468088). FLT3 D839G combined with ITD confers resistance to pexidartinib and ponatinib, both Type II FLT3 inhibitors (PMIDs: 25847190, 23430109). FLT3 N676K predicts response to the Type I FLT3 inhibitor, gilteritinib, when N676K is present alone or in combination with ITD. Interestingly, FLT3 N676K in the absence of ITD predicts response to sorafenib, a Type II FLT3 inhibitor (PMIDs: 32040554, 32984009). However, these results are mostly derived from in vitro studies. Two separate Tier II, Level D Assertions have been submitted for FLT3-ITD&D839G for its response to pexidartinib and ponatinib, and more evidence is being collected to form an Assertion for FLT3 N676K. The complexity of the prediction of response/resistance associated with FLT3 D839G and N676K supports the importance of evidence-based curation and collection for these variants in the context of the overall mutation profile, disease context and specific FLT3 TKIs to clearly define their clinical impact. Disclosures Pullarkat: Stemline Therapeutics: Honoraria.

Childhood cancers are driven by unique profiles of somatic genetic alterations, with a significant contribution from predisposing germline variants. Understanding the genomic landscape of pediatric cancers is complicated by their rarity, the heterogeneity of variation within a given disease, and the complex forms of structural variation they contain. Variants in childhood disease may differ from those in adult versions of the same cancer type, or may have different clinical significance. Currently, pediatric variants are underrepresented in cancer variant databases, and an urgent need exists for their publicly available expert curation. To address this, the Pediatric Cancer Taskforce (PCT) was formed within the Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group (CDWG) (https://www.clinicalgenome.org/working-groups/somatic/). The PCT is a multi-institutional group of 39 members with broad experience in childhood cancer and variant curation, whose work consists of standardization and classification of genetic variants in pediatric cancers. The CIViC knowledgebase (www.civicdb.org) is a freely available resource for Clinical Interpretation of Variants in Cancer, which leverages public curation and expert moderation to address the problem of annotating the large volume of clinically actionable cancer variants. PCT curators work together with PCT expert members and the CIViC team on variant curation, and have submitted over 230 Evidence Items and over 10 Assertions to CIViC. To further address issues specific to pediatric curation, the PCT is working with CIViC to develop new pediatric-specific CIViC features and modifications of the data model that will aid in pediatric curation. A pediatric user interface, as well as representation of large scale structural and copy number variation are being developed for version two of CIViC, expected to be released in 1-2 years, which will enable curation of a new class of structural variants often encountered in pediatric cancer. A novel standard operating procedure for childhood cancer curation in CIViC is being developed by PCT experts, curators and the CIViC team. This SOP will cover topics including curation of structural variants, as well as pediatric-specific variant tiering guidelines which take into account the sparse nature of evidence in pediatric cases. A companion resource, CIViCmine (http://bionlp.bcgsc.ca/civicmine/), will be further developed to incorporate pediatric data. These and other joint efforts of the PCT and CIViC will significantly enhance pediatric variant representation for public use, to support the care of children with cancer. Citation Format: Arpad Danos, Wan-Hsin Lin, Jason Saliba, Angshumoy Roy, Alanna J. Church, Shruti Rao, Deborah Ritter, Kilannin Krysiak, Alex Wagner, Erica Barnell, Lana Sheta, Adam Coffman, Susanna Kiwala, Joshua F. McMichael, Laura Corson, Kevin Fisher, Heather E. Williams, Matthew Hiemenz, Katherine A. Janeway, Jianling Ji, Kesserwan A. Chimene, Laura Fuqua, Lisa Dyer, Huiling Xu, Jeffrey Jean, Laveniya Satgunaseelan, Liying Zhang, Ted W. Laetsch, Donald W. Parsons, Ryan Schmidt, Lynn M. Schriml, Kristen L. Sund, Shashikant Kulkarni, Subha Madhavan, Xinjie Xu, Rashmi Kanagal-Shamana, Marian Harris, Yasmine Akkari, Nurit Paz Yacov, Panieh Terraf, Malachi Griffith, Obi L. Griffith, Gordana Raca. Advancing knowledgebase representation of pediatric cancer variants through ClinGen/CIViC collaboration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 210.

Background: Although genomic changes identified by clinical tumor sequencing may be present in the germline, precision oncology trials have generally not incorporated germline testing and reporting. We describe our experience with clinical reporting of matched tumor and germline results from the NCI-COG Pediatric Molecular Analysis for Therapy Choice (MATCH) trial (NCT03155620). Design/Method: Patients age 1 to 21 years old with treatment-refractory solid tumors, non-Hodgkin lymphomas, or histiocytic disorders were eligible. DNA and RNA extracted from FFPE tumors were sequenced using Oncomine Assay v.3 with reporting of potentially actionable fusions, amplifications, and mutations, including loss of function variants in tumor suppressor genes. Germline reporting focused on 36 genes from the same DNA panel that convey adult or pediatric cancer susceptibility but does not include all cancer susceptibility genes (e.g. APC, DICER1). Deletions and splice site variants were not reported. Results: As of June 2020, 1009 patients had been enrolled from 132 COG sites, with tumor and germline testing complete for 868 patients to date. Overall, 62 (7.1%) germline reports included a pathogenic or likely pathogenic germline variant (termed germline findings) in 18 cancer susceptibility genes. This frequency was similar in patients with solid tumors (46/633, 7.3%), CNS tumors (14/204, 6.9%), and lymphomas/histiocytoses (2/31, 6.5%). Variant(s) of potential germline significance were identified in 25% of tumor reports; of these, 74% (163/221) had no germline findings. The proportion of germline findings for genes with >10 reported tumor variants varied: 32% (7/22) in NF1, 25% (3/12) in RB1, 16% (17/108) in TP53 and 0% in ALK (0/21) and PTEN (0/12). Of note, 82% (14/17) of tumor variants in breast cancer susceptibility genes (BRCA1/2, CHEK2 and PALB2) had germline findings. On study intake forms, 25% of patients with germline findings had a “known genetic disease” reported. Oncologists caring for patients with a germline finding were then queried about prior knowledge of a genetic diagnosis. Of 24 respondents, 13 (54%) reported no prior molecular diagnosis of the identified condition. Conclusions: Coordinated germline and tumor testing revealed clinically relevant cancer susceptibility variants across a spectrum of genes in 7% of pediatric patients with treatment-refractory cancers. This is likely an underestimate of germline findings given test limitations. The parallel tumor/normal reporting approach minimized the need for targeted reflex genetic testing in 19% of study participants and provided new information on cancer susceptibility in germline positive patients to half of the responding oncologists. The NCI-COG Pediatric MATCH trial reporting process can serve as a model for precision oncology trials and clinical tumor profiling. Citation Format: Sarah Scollon, Sharon E. Plon, Steven Joffe, Jaclyn A. Biegel, Shashikant Kulkarni, George Miles, David Patton, Brent Coffey, Paul M. Williams, Gregory J. Tsongalis, Mark J. Routbort, Julie M. Gastier-Foster, Lauren Saguilig, Jin Piao, Todd A. Alonzo, Katherine A. Janeway, Peter C. Adamson, Margaret Mooney, James V. Tricoli, Nita L. Seibel, Donald W. Parsons. Germline cancer predisposition results from the National Cancer Institute - Children's Oncology Group (NCI-COG) Pediatric MATCH Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 631.

To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses.

Widespread clinical laboratory implementation of next-generation sequencing–based cancer testing has highlighted the importance and potential benefits of standardizing the interpretation and reporting of molecular results among laboratories. A multidisciplinary working group tasked to assess the current status of next-generation sequencing–based cancer testing and establish standardized consensus classification, annotation, interpretation, and reporting conventions for somatic sequence variants was convened by the Association for Molecular Pathology with liaison representation from the American College of Medical Genetics and Genomics, American Society of Clinical Oncology, and College of American Pathologists. On the basis of the results of professional surveys, literature review, and the Working Group's subject matter expert consensus, a four-tiered system to categorize somatic sequence variations based on their clinical significances is proposed: tier I, variants with strong clinical significance; tier II, variants with potential clinical significance; tier III, variants of unknown clinical significance; and tier IV, variants deemed benign or likely benign. Cancer genomics is a rapidly evolving field; therefore, the clinical significance of any variant in therapy, diagnosis, or prognosis should be reevaluated on an ongoing basis. Reporting of genomic variants should follow standard nomenclature, with testing method and limitations clearly described. Clinical recommendations should be concise and correlate with histological and clinical findings.

Venetoclax is an oral, highly selective, BCL2 inhibitor approved by the FDA for use in chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia. Despite favorable responses, multiple biological mechanisms lead to treatment resistance. One such mechanism includes somatic mutations in the BCL2 gene. Multiple lines of evidence suggest that hot-spot mutations in BCL2 such as Gly101Val induce treatment resistance by disrupting the binding of BCL2 to the BCL2 inhibitors such as venetoclax. Further, widespread use of high-throughput NGS technologies has identified multiple BCL2 mutations and additional concurrent molecular alterations at various variant allele frequencies in patients with progression while undergoing venetoclax therapy. In order to understand and determine the clinical significance of each of these mutations, careful expert curation and integration into somatic variant annotation AMP/ASCO/CAP guidelines is needed. Further, curation of those somatic variants that may not have sufficient functional evidence in literature may benefit from additional tools such as in silico analysis. To address these issues, we have undertaken an effort to integrate the contributions of a multidisciplinary expert panel (clinical laboratory diagnosticians, oncologists, biomedical informaticians and lab-based researchers) for curation of BCL2 variants in hematological malignancies under the umbrella of ClinGen, an NIH/NHGRI funded consortium to establish standards and centralized resources for assessing the clinical significance of gene variants. Within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) ((https://www.clinicalgenome.org/working-groups/somatic/), the somatic hematological malignancy taskforce has identified 56 peer-reviewed publications on BCL2 inhibitors (Jan 2014 to June 2020). The functional evidence contained within these publications was curated using CIViC (Clinical Interpretation of Variants in Cancer, civicdb.org), an open access, crowdsourced aggregation of expert curated evidence. Only a fraction of the somatic variants identified in BCL2 has established functional evidence on variant induced disruption to Venetoclax inhibition. Curation of these remaining variants of unknown significance (VUS) only have in silico functional assays to provide evidence on their potential resistance to Venetoclax. The current curation guidelines do not consider in silico prediction as a strong line of evidence for the interpretation of somatic sequence variants, however this recommendation is meant to interpret generalized in silico predictors and not robust computational models of specific protein function. The latter are more comparable to an experimental functional assay, and provide curators with more trustworthy computational assessments of disruption to protein specific functions. In order to assess their potential to integrate and supplement experimental evidence, the interaction of Ventoclax with several drug resistant BCL2 variants was simulated using AutoDock Vina (J Comput Chem. 2010;31(2):455-61). Facilitated by the SNP2SIM workflow (BMC Bioinformatics. 2019;20(1):171), the relative impact on binding energy was compared to the wildtype system. The in silico binding assay accurately predicted resistance (Fig 1), and demonstrates the utility of applying these methods to the large number of VUS in BCL2. In conclusion, the evidence-based expert curation of BCL2 variants provides a standardized approach for reporting and interpretation across all labs. For those variants (Tier 3) with limited published evidence, computational models that can predict specific changes to functional protein interactions can provide additional tools to the expert curators. Development and incorporation of these tools into curation guidelines requires the refinement of the predictive models through focused validation studies. Disclosures No relevant conflicts of interest to declare.

Many successful deep learning networks for object detection have been proposed in recent years, but their direct comparison is difficult due to different resolutions, training and validation data and frameworks used to train these networks. Moreover, suitability of these object detectors in context of autonomous driving is not compared or studied extensively. Autonomous driving has a wide operational domain containing diverse open world scenarios like different weather, daylight and road conditions etc. Most of the object detection datasets are from specific operational domains, e.g. KITTI dataset is mostly urban scenarios. In this work, we aim to first club many open source datasets (simulated and real) available for object detection in autonomous driving scenario. Clubbing the datasets makes them diverse by including different weather, lighting, time of the day and road conditions. Secondly, by clubbing most of the publicly available object detection datasets, we show the generalization ability of object detection networks in diverse scenarios. We also provide comparative study of accuracy vs resolution and the results of DNN based object detection on real images by training only on simulated data. Additionally, range of detection is really important for autonomous driving basic functionality like highway driving, and market trend is to move towards higher resolution images, yet most of the well known DNN based object detectors operate at very small image resolution which restricts range of detection. We demonstrate that the use of ROI instead of entire image can help in increasing detection range. Finally, in the context of autonomous driving these DNNs needs to run on low powered Edge devices. We measure the deployability of these DNNs by measuring their run time performance on Nvidia Drive PX2.

Current non-invasive prenatal screening is targeted toward the detection of chromosomal abnormalities in the fetus1,2. However, screening for many dominant monogenic disorders associated with de novo mutations is not available, despite their relatively high incidence3. Here we report on the development and validation of, and early clinical experience with, a new approach for non-invasive prenatal sequencing for a panel of causative genes for frequent dominant monogenic diseases. Cell-free DNA (cfDNA) extracted from maternal plasma was barcoded, enriched, and then analyzed by next-generation sequencing (NGS) for targeted regions. Low-level fetal variants were identified by a statistical analysis adjusted for NGS read count and fetal fraction. Pathogenic or likely pathogenic variants were confirmed by a secondary amplicon-based test on cfDNA. Clinical tests were performed on 422 pregnancies with or without abnormal ultrasound findings or family history. Follow-up studies on cases with available outcome results confirmed 20 true-positive, 127 true-negative, zero false-positive, and zero-false negative results. The initial clinical study demonstrated that this non-invasive test can provide valuable molecular information for the detection of a wide spectrum of dominant monogenic diseases, complementing current screening for aneuploidies or carrier screening for recessive disorders.

The genomic events responsible for the pathogenesis of relapsed adult B-lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole-genome, whole-exome, custom capture, whole-transcriptome (RNA-seq), and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. Although the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched-unrelated donor stem cell transplantation. The patient remains in complete remission more than 4 years later. Analysis of this patient's relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate a novel therapeutic intervention that could alter outcome.

Hematological malignancies comprise a genetically heterogeneous spectrum of diseases caused by abnormal proliferation or maturation of a variety of hematological cell lineages. Genomic abnormalities including chromosomal translocations, copy number variations and sequence level gene mutations underlie the pathogenesis of these disorders and frequently serve as important diagnostic, prognostic and/or therapeutic markers. However, the substantial discrepancy in interpretation and reporting of these genomic abnormalities among testing labs creates challenges for patient management. Therefore, standardizing the curation, clinical interpretation and reporting of somatic alterations within the context of their diagnostic, prognostic and therapeutic significance in hematological cancers is critical. In January 2020, the ClinGen Somatic Cancer Clinical Domain working group formed the Hematological Cancer Taskforce (HCT) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematological malignancies. The HCT has recruited 32 multi-disciplinary experts including oncologists, molecular pathologists, clinical lab directors, genomic scientists and biocurators with expertise in hematological malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, variants from peer-reviewed publications are curated with editor review for clinical utility as evidence items. Monthly discussions based on these evidence items lead to the creation of summary variant assertions using the AMP/ASCO/CAP guidelines (Li M, et al., Journal of Molecular Diagnostics, 2017). The HCT is currently focused on expert curation and clinical interpretation of somatic variants in FLT3 (internal tandem duplication, tyrosine kinase domain and non tyrosine kinase domain variants) in acute myeloid leukemia (AML). Expert curation of gene fusions in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) in collaboration with the ClinGen somatic pediatric cancer taskforce is currently underway. To date, the HCT has curated 45 evidence items from clinical and pre-clinical studies on the aforementioned genes/variants. In addition, three AMP Tier I, level A variant assertions of FLT3-ITD, D835 and I836, which predict response to Gilteritinib, an FDA-approved drug for relapsed or refractory AML, have been curated. In the future, the HCT plans to extend its focus on curation of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia (CML). Based on the initial pilot curation phase, the HCT will develop gene-specific recommendations to standardize the reporting and interpretation of somatic variants to better assist clinical decisions and apply to become official ClinGen Somatic Expert Panels in each of these gene-disease domains. Disclosures Blombery: Novartis: Consultancy; Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria.

Somatic variants in cancer-relevant genes are interpreted from multiple partially overlapping perspectives. When considered in discovery and translational research endeavors, it is important to determine if a particular variant observed in a gene of interest is oncogenic/pathogenic or not, as such knowledge provides the foundation on which targeted cancer treatment research is based. In contrast, clinical applications are dominated by diagnostic, prognostic, or therapeutic interpretations which in part also depends on underlying variant oncogenicity/pathogenicity. The Association for Molecular Pathology, the American Society of Clinical Oncology, and the College of American Pathologists (AMP/ASCO/CAP) have published structured somatic variant clinical interpretation guidelines which specifically address diagnostic, prognostic, and therapeutic implications. These guidelines have been well-received by the oncology community. Many variant knowledgebases, clinical laboratories/centers have adopted or are in the process of adopting these guidelines. The AMP/ASCO/CAP guidelines also describe different data types which are used to determine oncogenicity/pathogenicity of a variant, such as: population frequency, functional data, computational predictions, segregation, and somatic frequency. A second collaborative effort created the European Society for Medical Oncology (ESMO) Scale for Clinical Actionability of molecular Targets to provide a harmonized vocabulary that provides an evidence-based ranking system of molecular targets that supports their value as clinical targets. However, neither of these clinical guideline systems provide systematic and comprehensive procedures for aggregating population frequency, functional data, computational predictions, segregation, and somatic frequency to consistently interpret variant oncogenicity/pathogenicity, as has been published in the ACMG/AMP guidelines for interpretation of pathogenicity of germline variants. In order to address this unmet need for somatic variant oncogenicity/pathogenicity interpretation procedures, the Variant Interpretation for Cancer Consortium (VICC, a GA4GH driver project) Knowledge Curation and Interpretation Standards (KCIS) working group (WG) has developed a Standard Operating Procedure (SOP) with contributions from members of ClinGen Somatic Clinical Domain WG, and ClinGen Somatic/Germline variant curation WG using an approach similar to the ACMG/AMP germline pathogenicity guidelines to categorize evidence of oncogenicity/pathogenicity as very strong, strong, moderate or supporting. This SOP enables consistent and comprehensive assessment of oncogenicity/pathogenicity of somatic variants and latest version of an SOP can be found at https://cancervariants.org/wg/kcis/. Citation Format: Peter Horak, Malachi Griffith, Arpad Danos, Beth A. Pitel, Subha Madhavan, Xuelu Liu, Jennifer Lee, Gordana Raca, Shirley Li, Alex H. Wagner, Shashikant Kulkarni, Obi L. Griffith, Debyani Chakravarty, Dmitriy Sonkin. A standard operating procedure for the interpretation of oncogenicity/pathogenicity of somatic mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5707.

The Clinical Genome (ClinGen) Resource is a US National Human Genome Research Institute (NHGRI)-funded program dedicated to building an expert curated and freely available central resource that defines the clinical relevance of genes and variants for use in precision medicine. Teams of experts in various clinical domains come together as working groups within ClinGen to facilitate the interpretation, annotation and utilization of genes-variants for clinical application. Somatic Cancer is one such ClinGen Clinical Domain Working Group (CDWG) that consists of over 90 members worldwide, including clinicians, clinical laboratory diagnosticians, genomic scientists and bioinformaticians. Members of this CDWG identify high priority somatic variants in different cancer types that require expert curation and consensus in their clinical interpretation. In order to accurately implement practice guidelines/standards for variant interpretation, the Somatic Cancer CDWG recently established a somatic Variant Curation Expert Panel (VCEP) approval process. Based on their interest and clinical domain expertise, a subset of the CDWG members formed somatic VCEPs to perform authoritative curation on the shortlisted genes-somatic variants within the context of a disease, therapeutic indication or biological pathway. Expert curation within these VCEPs is performed by utilizing guidelines such as those recommended by AMP/ASCO/CAP (Li et al. 2017) and the Somatic Cancer CDWG (Ritter et al. 2016). Wherever necessary, the somatic VCEPs will develop: 1) gene- and disease-specific modifications to address gaps in existing variant assessment guidelines, 2) quantitative approaches for variant interpretation, and 3) implement standardized protocols for annotating somatic variants in genes for a specific disease, drug or biological pathway. Furthermore, ClinGen recently formed the Cancer Variant Interpretation (CVI) committee to provide support, review and feedback on the provisional somatic variant interpretation proposals developed by the VCEPs. The CVI provides somatic VCEPs with a preliminary approval before the final approval by the ClinGen CDWG oversight committee and ultimately ‘expert panel' status in ClinVar, an NCBI-maintained database of clinically relevant gene variants. NTRK fusions in cancer is the first Somatic VCEP going through the ClinGen Somatic Expert Panel approval process. Alterations in the FGFR pathway in GU cancers is the second somatic VCEP under consideration. The Somatic CDWG uses the CIViC (Clinical Interpretation of Variants in Cancer) platform for curation of somatic variants. To date, the CDWG has curated 268 evidence items relating to cancer variants in CIViC, 6 assertions, and 33 evidence source suggestions. The ultimate goal of the Somatic CDWG is to enhance the usability, dissemination and implementation of cancer somatic changes in the ClinGen resource and other cancer variant knowledgebases. Citation Format: Shruti Rao, Deborah Ritter, Arpad Danos, Gordana Raca, Angshumoy Roy, Kilannin Krysiak, Wan-Hsin Lin, Erica Barnell, Matthew McCoy, Beth Pitel, Dmitriy Sonkin, Jue Wang, Seyed Ali Hosseini, Shamini Selvarajah, Ian King, Rashmi Kanagal-Shamana, Xinjie Xu, Jeremy L. Warner, Funda Meric-Bernstam, Jason D. Merker, Marilyn Li, Alex H. Wagner, Malachi Griffith, Obi L. Griffith, Shashikant Kulkarni, Subha Madhavan. ClinGen somatic cancer working group: Disseminating standardized cancer molecular diagnostic data and evidence through global collaboration and expert curation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3215.