Clinical Next-generation Sequencing Research Articles

TP53-specific mutations serve as a potential biomarker for homologous recombination deficiency in breast cancer: a clinical next-generation sequencing study.

TP53 mutations and homologous recombination deficiency (HRD) occur frequently in breast cancer. However, the characteristics of TP53 pathogenic mutations in breast cancer patients with/without HRD are not clear. Clinical next-generation sequencing (NGS) of both tumor and paired blood DNA from 119 breast cancer patients (BRCA-119 cohort) was performed with a 520-gene panel. Mutations, tumor mutation burden (TMB), and genomic HRD scores were assessed from NGS data. NGS data from 47 breast cancer patients in the HRD test cohort were analyzed for further verification. All TP53 pathogenic mutations in patients had somatic origin, which was associated with the protein expression of estrogen receptor and progestogen receptor. Compared to patients without TP53 pathologic mutations, patients with TP53 pathologic mutations had higher levels of HRD scores and different genomic alterations. The frequency of TP53 pathologic mutation was higher in the HRD-high group (HRD score≥42) relative to that in the HRD-low group (HRD score<42). TP53 has different mutational characteristics between the HRD-low and HRD-high groups. TP53-specific mutation subgroups had diverse genomic features and TMB. Notably, TP53 pathogenic mutations predicted the HRD status of breast cancer patients with an area under the curve (AUC) of 0.61. TP53-specific mutations, namely HRD-low mutation, HRD-high mutation, and HRD common mutation, predicted the HRD status of breast cancer patients with AUC values of 0.32, 0.72, and 0.58, respectively. Interestingly, TP53 HRD-high mutation and HRD common mutation combinations showed the highest AUC values (0.80) in predicting HRD status. TP53-specific mutation combinations predict the HRD status of patients, indicating that TP53 pathogenic mutations could serve as a potential biomarker for poly-ADP-ribose polymerase (PARP) inhibitors in breast cancer patients .

Abstract 5054: Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications

Abstract Formalin-fixed paraffin-embedded (FFPE) biopsies are highly valuable and widely used tissue specimens for clinical diagnostics. However, obtaining sufficient and high-quality nucleic acid material from limited FFPE samples presents a challenge for downstream molecular analysis, such as next-generation sequencing (NGS). We present an optimized sequential extraction method that generates high-quality DNA and RNA from a single set of input tissues that is automatable and operation-friendly. This workflow performs well with reduced FFPE tissue input and efficiently supports various high-throughput clinical NGS applications. The DNA/RNA yield, quality, purity, and impacts on NGS assay performances were on par or better than an existing validated comparator extraction method. With comparable FFPE input, the new method demonstrated a superior extraction performance with significantly higher yield, quality, and purity. For DNA, NGS libraries were made with two different library preparation methods: a TA-ligation-based method and a single-strand-based method, followed by hybrid capturing and sequencing. The DNA from the new extraction method demonstrated a superior library conversion rate and improved target enrichment uniformity with both chemistries. This provides the potential of reduced input requirements, allowing very limited tissue, such as fine needle aspirate or core needle biopsy, for clinical NGS testing. The sequencing results were highly concordant between the existing and new extraction methods when the extracted DNA was subjected to a validated comprehensive genomic profiling (CGP) clinical test, demonstrating the quality and robustness of the extraction. For RNA, NGS libraries were made following the validated CGP clinical test with hybrid capturing, and the results were comparable between the two extraction methods. In conclusion, an FFPE extraction method was optimized to allow more clinical biopsy samples to be tested with different NGS workflows, providing a better diagnostic value for patient care. Citation Format: Rebecca Hong, Jieying Chu, Steven Hsiao, Daniel Whang, Steven P. Rivera, Heng Xie, Jiannan Guo. Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications [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 5054.

Abstract 6469: The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers

Abstract Background: Benefit from single-agent immune checkpoint inhibitor has been observed in 5-10% of the patients with microsatellite stable (MSS) gastrointestinal (GI) cancers. However, a reliable predictive marker to immune checkpoint inhibitor has not been discovered in this patient population. We sought to assess the incidence of mismatch repair (MMR) gene alterations and their association with tumor mutational burden (TMB) in MSS GI cancers. Methods: This is a retrospective cohort study of patients with advanced GI cancer who had molecular profiling of the tumor using either Guardant360 blood-based or Tempus tissue next generation sequencing (NGS) platforms between 2020 and 2023. We assessed four MMR genes including MLH1, MSH2, MSH6 and PMS2 for alterations. Continuous variables were summarized as median and interquartile range (IQR) and compared using Mann-Whitney test. Categorical variables were summarized as counts and proportions and compared using Fisher’s exact test. Results: A total of 2920 patients with Guardant360 were included with a median age of 65 (IQR: 56-73) years. 870 (30%) patients had pancreas cancer; 846 (29%) patients had colorectal cancer; 688 (24%) patients had biliary tract cancers; and the rest had other GI cancers. Among patients evaluable for microsatellite instability (MSI) status, 58 patients (2%) had MSI-high GI cancers, 31 (53%) of whom had MMR gene alterations. 2445 (98%) patients had MSS GI cancers, 130 (5.3%) of whom had MMR gene alterations. Patients with MSS GI cancers bearing MMR gene alterations had a median age of 70 (IQR: 62-75) years, significantly older than those without MMR gene alterations (p&amp;lt;0.001). In addition, patients with MMR gene alterations had significantly higher median blood TMB of 11 (IQR: 7.4-16) mut/Mb compared to 7.7 (IQR: 4.8-11) mut/Mb in patients without MMR gene alterations. In MSS GI cancers, MMR gene alterations are more commonly associated with the presence of alterations in APC (p=0.03), TP53 (p=0.01), CTNNB1 (p&amp;lt;0.001) and SMAD4 (p=0.01). 1453 patients with GI cancer and Tempus tissue NGS data were included as a validation cohort. They had comparable demographics with the Guardant360 cohort. 14 (1.6%) of 896 patients with MSS GI cancers had MMR gene alterations, significantly fewer (p&amp;lt;0.001) than those found in the Guardant360 cohort. Patients with MMR gene alterations and wild type POLE had significantly higher (p=0.001) median TMB of 9.8 (IQR: 4.8-14) mut/Mb compared to 4.7 (IQR: 3.2-6.3) mut/Mb in patients without MMR gene alterations. Conclusions: MMR gene alterations can be identified by clinical NGS platforms in a small proportion of patients with MSS GI cancers. They are associated with elevated TMB, which may suggest a hypermutated profile serving as a basis for potential role of immune checkpoint inhibitor in MSS GI cancers. Citation Format: Conor D. O'Donnell, Mojun Zhu, Hao Xie. The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers [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 6469.

Abstract 4631: Evaluation of the impact of homozygous MTAP truncations on the activity and selectivity of MTA-cooperative PRMT5 inhibitors

Abstract Homozygous deletion of the MTAP gene occurs in 10-15% of all human cancers. To benefit this large and diverse patient population, MTA-cooperative PRMT5 inhibitors, including TNG908 and TNG462, have been developed to leverage the synthetic lethal relationship between MTAP deletion and PRMT5 inhibition. MTA-cooperative PRMT5 inhibitors selectively bind the PRMT5-MTA complex driving selective inhibition of PRMT5 in MTAP-deleted cancers while sparing normal, MTAP-proficient cells. Our PRMT5 inhibitors are currently in Phase I/II clinical trials (NCT05275478 and NCT05732831), and eligibility is restricted to patients with tumors with confirmed MTAP loss either detected by next-generation sequencing (NGS) or immunohistochemistry. MTAP gene loss occurs in cancers because of its chromosomal proximity to one of the most common genetically altered tumor suppressor genes, CDKN2A, but the chromosomal 9p breakpoints for the co-deletion are not uniform. Indeed, while clinical NGS testing and preclinical data confirm that homozygous intragenic MTAP breakpoints occur, the functional consequence of any given breakpoint on MTAP enzymatic activity and protein function remains unknown. Given the potential implications for homozygous intragenic MTAP deletions to impact the clinical response to MTA-cooperative PRMT5 inhibitors, we have started to evaluate the loss-of-function phenotype of various MTAP truncations to determine whether they retain MTAP activity. Here, we present our initial functional genomics analysis of this important diagnostic biomarker using in vitro cDNA reconstitution approaches for MTAP activity combined with analysis of PRMT5 inhibitor sensitivity. Ultimately, these data may help refine patient enrollment on clinical trials to drive the maximum benefit for patients with MTAP-deleted cancers. Citation Format: Matthew R. Tonini, Andre A. Mignault, Douglas A. Whittington, Steven A. Lombardo, Binzhang Shen, Hannah Stowe, Satoshi Yoda, Shangtao Liu, Minjie Zhang, Kevin M. Cottrell, Samuel R. Meier, Heidi Rego, Jennifer Morawiak, Ellen Hooper, Yi Yu, Heather DiBenedetto, Adam S. Crystal, Teng, Kimberly Briggs. Evaluation of the impact of homozygous MTAP truncations on the activity and selectivity of MTA-cooperative PRMT5 inhibitors [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 4631.

Validation of LymphGen classification on a 400-gene clinical next-generation sequencing panel in diffuse large B-cell lymphoma: real-world experience from a cancer center.

Not available.

Identification of pathogenic germline variants in a large Chinese lung cancer cohort by clinical sequencing.

Genetic factors play significant roles in the tumorigenicity of lung cancer; however, there is lack of systematic and large-scale characterization of pathogenic germline variants for lung cancer. In this study, germline variants in 146 preselected cancer-susceptibility genes were detected in 17 904 Chinese lung cancer patients by clinical next-generation sequencing. Among 17 904 patients, 1738 patients (9.7%) carried 1840 pathogenic/likely pathogenic (P/LP) variants from 87 cancer-susceptibility genes. SBDS (SBDS ribosome maturation factor) (1.37%), TSHR (thyroid stimulating hormone receptor) (1.20%), BLM (BLM RecQ like helicase) (0.62%), BRCA2 (BRCA2 DNA repair associated) (0.62%), and ATM (ATM serine/threonine kinase) (0.45%) were the top five genes with the highest overall prevalence. The top mutated pathways were all involved in DNA damage repair (DDR). Case-control analysis showed SBDS c.184A>T(p.K62*), TSHR c.1574T>C(p.F525S), BRIP1 (BRCA1 interacting helicase 1) c.1018C>T(p.L340F), and MUTYH (mutY DNA glycosylase) c.55C>T(p.R19*) were significantly associated with increased lung cancer risk (q value < 0.05). P/LP variants in certain genes were associated with early onset of lung cancer. Our study indicates that Chinese lung cancer patients have a higher prevalence of P/LP variants than previously reported. P/LP variants are distributed in multiple pathways and dominated by DNA damage repair-associated pathways. The association between identified P/LP variants and lung cancer risk requires further studies for verification.

Pangenome graphs improve the analysis of structural variants in rare genetic diseases

Rare DNA alterations that cause heritable diseases are only partially resolvable by clinical next-generation sequencing due to the difficulty of detecting structural variation (SV) in all genomic contexts. Long-read, high fidelity genome sequencing (HiFi-GS) detects SVs with increased sensitivity and enables assembling personal and graph genomes. We leverage standard reference genomes, public assemblies (n = 94) and a large collection of HiFi-GS data from a rare disease program (Genomic Answers for Kids, GA4K, n = 574 assemblies) to build a graph genome representing a unified SV callset in GA4K, identify common variation and prioritize SVs that are more likely to cause genetic disease (MAF < 0.01). Using graphs, we obtain a higher level of reproducibility than the standard reference approach. We observe over 200,000 SV alleles unique to GA4K, including nearly 1000 rare variants that impact coding sequence. With improved specificity for rare SVs, we isolate 30 candidate SVs in phenotypically prioritized genes, including known disease SVs. We isolate a novel diagnostic SV in KMT2E, demonstrating use of personal assemblies coupled with pangenome graphs for rare disease genomics. The community may interrogate our pangenome with additional assemblies to discover new SVs within the allele frequency spectrum relevant to genetic diseases.

The diagnostic utility of methylthioadenosine phosphorylase immunohistochemistry for pancreatic ductal adenocarcinoma in FNA and small biopsy specimens.

Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC. In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases. Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n=13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n=13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n=4). Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples.

TP53 mutations and Their Impact on Survival in Patients with Myeloproliferative Neoplasms

Introduction: TP53 mutations in patients with myeloproliferative neoplasms (MPN) are associated with poor prognosis, including progression to blast phase MPN. However, low variant allele fraction (VAF) TP53 mutations have been reported to remain stable over years in chronic phase MPN. A major unmet clinical need in MPN is the ability to discriminate patients with TP53-mutant MPN who are at high-risk of secondary AML (sAML) and warrant immediate intervention from those who are at lower risk of sAML in whom active surveillance can be employed. Therefore, we sought to identify parameters associated with leukemic transformation and overall survival in the context of MPN with genetic aberrations in TP53. Materials and Methods: We retrospectively analyzed a cohort of 947 MPN patients from the Dana-Farber Cancer Institute Hematologic Malignancies Data Repository (HMDR) with at least one clinical next-generation sequencing (NGS) panel performed. Patient characteristics such as age at MPN diagnosis, gender, MPN subtype and driver mutations were recorded. Furthermore, information about the course of disease was extracted including occurrence of sAML and overall survival (Figure 1). We also analyzed type and number of additional mutations as well as cytogenetics. With respect to TP53-specific parameters, we evaluated the number of TP53 mutations, TP53 VAF, loss of heterozygosity (LOH) at the TP53 locus, phenotypic annotations of TP53 (i.e. PHANTM score) and 17p deletion. We defined “multi-hit” TP53 as the presence of two or more TP53 mutations, TP53 VAF higher than 50%, TP53 mutation plus 17p deletionor TP53 mutation and documented LOH. Results: A total of 947 patients were analyzed, of which 40 harbored at least one detectable TP53 mutation. A total of 13 patients were found to have a multi-hit TP53 mutations defined by &amp;gt; 50% VAF in 6 patients, two or more TP53 mutations in 5 patients and TP53 mutation + 17p deletion in 5 patients. The MPN diagnosis at time of TP53 mutation detection was post ET/PV myelofibrosis (secondary MF) (n=23, 58%), primary myelofibrosis (MF) (n=7, 18%), pre-fibrotic MF (n=2, 5%), essential thrombocythemia (ET) (n=6, 15%) and polycythemia vera (PV) (n=2, 5%). Two patients with ET and one patient with PV did not have a concurrent in-house bone marrow biopsy performed at the time the TP53 mutation was detected. Two patients with ET developed sAML within 12 months of TP53 mutation detection, without prior mention of fibrosis. Age at first MPN diagnosis was not significantly different between patients with or without TP53 mutation. The average time from initial MPN diagnosis to detection of the first TP53 mutation was 9 years (range: 0-33 years). The most common MPN driver mutation among patients with TP53 mutations was JAK2 (75%), followed by CALR (13%)and MPL (5%). Out of all TP53-mutated patients, 8% showed a triple negative status. The most frequent additional mutations among patients with TP53 mutations were TET2 (25%), U2AF1 (15%), ASXL1 (13%), and DNMT3A (10%). There was no significant difference between single-hit and multi-hit TP53 status regarding MPN subtype, driver mutations and co-mutations (Table 1). Seven patients (single-hit: 15%, multi-hit: 23%) with a TP53 mutation developed sAML during the course of their disease, compared with only 3% of all patients without a TP53 mutation and 50% (single-hit: 41%, multi-hit: 69%) were deceased at the time of the last follow-up compared to 18% of all patients without a TP53 mutation. We focused on overall survival from the initial MPN diagnosis and considered whether patients developed bone marrow fibrosis during their disease course (Figure 1). Survival did not differ significantly between single-hit TP53 and patients with multi-hit TP53 (p=0.2), but survival did differ significantly between multi-hit TP53 patients and TP53 wildtype patients with MF/prefibrotic MF/Secondary MF (p=0.02) as well as compared to all MPN patients without a TP53 mutation (p&amp;lt;0.001). Survival was not significantly different between single-hit TP53 and TP53 wildtype MF/prefibrotic MF/Secondary MF patients (p=0.4). Conclusions: In a large cohort of 947 molecularly characterized MPN patients, 4% of the cohort developed a TP53 mutation during their course of disease. 18% of all TP53-mutant patients developed sAML with an adverse effect on overall survival for patients with multi-hit but not single-hit TP53 mutations.

Mutations in Histone Lysine Methyltransferase Genes Are Associated with Autoimmune Cytopenias

Introduction Epigenetic mechanisms are important in regulation of gene expression by modifying chromatin structure to facilitate DNA accessibility (Lau et al Nat Immunol. 2018). DNA methylation and histone modifications are critical for the pathogenesis of hematologic malignancies and autoimmune diseases (Surace et al Front Immunol. 2019) Alterations in enzymes involved in histone modification are associated with immunodeficiencies, including Kabuki syndrome (KS), a rare genetic disorder associated with autoimmune cytopenias, such as immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (Margot et al J Am Coll Med Genet 2020) caused by loss-of-function KMT2D and KDM6A mutations. Somatic mutations in the KMT2D gene have also been observed in patients with Cold Agglutinin Disease. (Małecka et al Br J Haematol. 2018). Aims: The aims of this study are: 1) to identify the frequency of mutations in histone methyltransferase genes (MT) KMT2D, KMT2A, KMT2C and KDM6A in patients with autoimmune cytopenias, and 2) to compare clinical, laboratory and immunological characteristics, and response to therapy in patients with or without mutation in MT and autoimmune cytopenias (AIC). Methods Data mining software from the electronic medical record was used to identify patients ≥ 18 years with a diagnosis of AIC, including ITP, warm and cold AIHA, autoimmune neutropenia, and Evans Syndrome (ES), who had Next Generation Sequencing (NGS) performed. Cytopenias due to malignancy, nutritional deficiency, medications, pregnancy, or infection were excluded. Patients were categorized into two groups based on the presence or absence of mutation in the MT genes (MT+, MT-) with variant allelic frequency (VAF) &amp;gt; 2%. Demographic, laboratory, and clinical characteristics, treatment outcomes, and NGS sequencing data were collected and compared between groups. Next, KMT2 mutations seen in patients with AIC were cross-referenced against the 1000 Genome Project (1000GP) population database to evaluate the frequency of polymorphisms in the general population (Auton et al Nature 2015). CADD in silico prediction algorithm was used to score the potential deleterious effect of MT mutations (Kirchner Nat Genet. 2014). Chi-Square test was used for categorical variables. Statistical analysis was completed in R software. Results Among 495 patients who underwent NGS, 79 (16%) had a mutation in MT genes. These mutations were present in 36% of patients with AIC as compared to 13% in those without AIC (p=0.0005). Among 55 patients with AIC, mutations in the MT genes were present in 20 patients (MT+), 35 patients did not have these mutations (MT-). KMT2C mutations were seen in 10 (50%) patients, KMT2D mutations in 9 patients (45%, one patient had two KMT2D mutations), 1 patient (5%) had both KMT2C &amp; KMT2D variants (Figure 1). The median VAF was 39.5% (IQR, 15.45 - 49.80). The MT+ group had a significantly higher proportion of Hispanic (60% vs 31%) and non-Hispanic White patients (25% vs 14%), p=0.038, Table 1. The median age at diagnosis for the MT+ cohort was younger compared to MT- (45.5 vs 58 years old, respectively), though not statistically significant. AIC diagnoses in both groups were ITP (44%) wAIHA (22%) cAIHA (9%) Autoimmune Neutropenia (2%), and ES (24%). Compared to the MT- group, the MT+ group had lower IgG levels (p=0.0041) and absolute lymphocyte count (p=0.041). MT+ patients with AIHA were more likely to have positive Direct Coombs and complement involvement. No significant differences were found in median nadir hemoglobin and platelet counts, need for therapy, or median number of lines of therapy. Best overall response to therapy was similar in the two groups (93%), with more complete responses in MT- group (55% vs 33%) though not statistically significant. Next, we assessed KMT2 polymorphisms in the general population. Most of the polymorphisms were not present in the 1000GP database and none had a frequency &amp;gt; 0.001. The average Phred score was 19.8; 12/20 patients had Phred score &amp;gt; 20, suggesting that most mutations are in the top 1% of the deleterious variants. Conclusion We report for the first time that patients with autoimmune cytopenias have a high frequency of variants in MT genes. Median allelic frequency close to 50% suggests potential germline predisposition to immune dysregulation and autoimmunity however further studies are needed to better understand the impact of these observations.

Clinical Consequences of Rare Germline Variation in Genes Associated with Coagulation across 451,958 Individuals

Introduction Despite deep biological insights over the preceding decades, our ability to assess for inherited hypercoagulable states remains limited. In light of these challenges and the increasing relevance of precision medicine and next-generation sequencing (NGS) to clinical practice, the identification of rare germline variants in coagulation-focused NGS panels is increasingly becoming accepted practice. However, the discovery of variants of unknown significance (VUS) remains a common clinical conundrum in molecular testing, particularly because evidence for the thrombosis risk associated with many genes derives from small, uncontrolled studies, case reports, and pedigree analysis. Large-scale evaluation of the clinical consequences associated with loss of function in individual coagulation-related genes would contribute to the clinical assessment of patients presenting with heterozygous VUS in poorly characterized genes. Objectives Determine the risk of four thrombosis phenotypes associated with rare, germline loss-of-function variants in a major clinical coagulation-focused NGS panel. Methods The UK Biobank (UKBB) contains paired clinical and whole exome sequencing data for over 450,000 participants. We identified rare (minor allele frequency &amp;lt;0.1%) germline variants that were predicted in silico to alter protein activity (functional impact score ≥0.8) within the 54 evaluable genes of the Yale NGS coagulation panel. For each gene, parallel multivariable Firth's logistic regression models were generated to assess the association between rare qualifying variants and four thrombotic phenotypes as well as coagulopathic bleeding in a gene-level collapsing analysis (N=270 independent models). We adjusted for age, sex, ancestry, and sequencing batch. Phenotypes of interest included venous thromboembolism (VTE), myocardial infarction (MI), stroke (CVA), peripheral artery disease (PAD), and coagulopathic bleeding. The Bonferroni corrected p-value for each phenotype was 0.001. We validated functional impact score predictions in seven secreted proteins measured in a subset of 47,923 UKBB participants who underwent plasma proteomic assessment (Olink Explore 1536 panel). Results A total of 451,958 individuals in the UKBB with whole exome sequencing data were evaluated. Among the 54 genes in the Yale NGS coagulation panel, there were 18,975 qualifying variants (98.4% heterozygous). Function-altering variants in 4 of 54 genes were associated with a statistically significant increased risk of thrombosis after adjusting for multiple comparisons: PROC (OR = 5.7, 95% CI: 3.2 - 9.6, p = 1.6x10 -7), SERPINC1 (OR = 5.0, 95% CI: 2.7 - 8.7, p = 4.0x10 -6), PROS1 (OR = 3.2, 95% CI: 2.0 - 4.8, p = 6.0x10 -6), and STAB2 (OR = 1.4, 95% CI: 1.2 - 1.7, p = 5.3x10 -4) (Figure 1). Nominal significance was achieved for 7 genes in the VTE phenotype, 1 gene in CVA, 3 genes in MI, 5 genes in PAD, and 0 genes in coagulopathic bleeding. Variants in a total of 37 genes did not appear to be significantly associated with any of the evaluated phenotypes. To assess the reliability of variant functional impact scores derived in silico, plasma levels for 13 of the 53 coagulation genes were analyzed. Of these, 8 were identified as secreted proteins amenable to assessment by plasma levels, including ADAMTS13 ( ADAMTS13), factor IX ( F9), factor VII ( F7), tissue plasminogen activator ( PLAT), urokinase ( PLAU), protein C ( PROC), plasminogen activator inhibitor 1 ( SERPINE1), and von Willebrand factor ( VWF). For these proteins, average plasma levels consistently demonstrated a negative correlation with functional impact score (Pearson r = -0.98, P=0.0009), indicating that our in silico assessments in this context are broadly reliable (Figure 2). Conclusion Within the commonly encountered clinical context of heterozygous loss of function, most genes contained in coagulation-focused NGS panels likely have fairly weak effects on disease risk that are limited to venous thrombosis. However, our findings do not rule out the possibility of more profound effects from homozygous or compound heterozygous loss of function in these same genes. By defining the clinical consequence of heterozygous loss of function in specific coagulation-related genes across a large population, our findings may help inform the interpretation of molecular testing results in which patients are found to have a VUS.

Unlike TET2 and ASXL1, Co-Occurring DNMT3A Mutations Are Not Associated with Increased Age in JAK2-Mutant Myeloproliferative Neoplasms (MPNs)

MPNs including essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) are characterized by JAK2, CALR and MPL mutations. Additional mutations outside these driver genes also co-occur. To better understand interactions between phenotypic driver mutations and concomitant mutations, we evaluated patterns of somatic mutation acquisition in 1301 MPN (535 ET, 392 PV, 331 MF, and 43 MPN NOS) patients who underwent clinical next generation sequencing (NGS) at our institution. For this analysis, we focused on a single NGS panel obtained closest to MPN diagnosis. Median age at MPN diagnosis was 59 years, with MF patients older than ET/PV patients (p&amp;lt;4e-11). 67%, 17%, and 4.5% of patients had mutations in JAK2, CALR, or MPL. The average variant allele fraction (VAF) for JAK2, CALR and MPL was 48%, 34%, and 40%. 52% of patients had pathogenic mutations in an additional gene (range 0-7), most frequently TET2, DNMT3A, and ASXL1. PV patients were more likely to have a TET2 mutation compared with JAK2-mutated ET patients (42% vs 25%). Mutation combinations that occurred together more frequently than expected (p&amp;lt;1e-3) included DNMTA-TET2, ASXL1-TET2, ASXL1-DNMT3A, and ASXL1-SRSF2. We then investigated how these concomitant mutations interacted with the MPN driver mutation and each other. JAK2-mutatedMPN patients with no concomitant mutations were younger than JAK2-mutated patients with TET2 or ASXL1 mutations. In contrast, age distributions were similar between JAK2-only patients and those with JAK2 and DNMT3A mutations (Fig 1A). This observation held when looking within PV and ET, and whether patients with single or multiple concomitant mutations were considered. Since DNMT3A, TET2 and ASXL1 clonal hematopoiesis is associated with increasing age, the similar age distribution of JAK2-only and JAK2-DNMT3A co-mutant patients was an unexpected finding, and worthy of further study. We evaluated the fraction of JAK2-mutated patients with a given concomitant mutation as a function of age. The fraction of patients with TET2 and ASXL1 mutations increased with age, but not the fraction of patients with DNMT3A mutations (95% CI of slope: 4e-3 to 7e-3 for TET2, 0 to 3e-3 for DNMT3A). Similarly, when we evaluated the fraction of JAK2-mutated patients with a given concomitant mutation as a function of JAK2 VAF, the fraction of patients with TET2 and ASXL1 mutations increased as JAK2 VAF increased, but the fraction of patients with DNMT3A mutations decreased (Fig 1B; 95% CI of slope: 1e-3 to 3e-3 for TET2, -2e-3 to 0 for DNMT3A). One potential explanation for this is that JAK2 and DNMT3A mutations occur as independent clonal acquisitions and that the JAK2-mutant clone “out-competes” the DNMT3A-mutant clone over time. At JAK2 VAFs of 50% and 100%, the ratio of TET2:JAK2 VAFs clustered at 1.0 and 0.5, suggesting frequent presence of a dominant JAK2-TET2 clone in all cells with heterozygous or homozygous JAK2 mutations. ASXL1 mutations occurred mostly in patients with JAK2 VAF &amp;gt; 50%, consistent with it being a subclonal acquisition. In contrast, DNMT3A concomitant mutations were more likely to occur in patients with JAK2 VAF&amp;lt;50% vs &amp;gt;50%, again suggesting that DNMT3A clones diminish as JAK2 clones expand. 6/45 DNMT3A mutations that occurred as the sole concomitant mutation were R882 hotspot mutations. However, DNMT3A was more likely to co-occur at JAK2 VAF&amp;gt;50% in patients with &amp;gt;1 concomitant mutation (most commonly TET2). This suggests that in the context of JAK2-mutant driven MPNs, DNMT3A subclones may require the presence of an additional mutation to expand. In summary, we found DNMT3A mutations behave differently from TET2 and ASXL1 mutations in JAK2-mutant MPNs. Patients with JAK2-mutant MPN are less likely to have DNMT3A mutations as JAK2 VAF increases. Compared with TET2 or ASXL1, DNMT3A appears more likely to exist as an independent clone in JAK2-mutant MPN, with our data suggesting the JAK2-mutant clone exhibits increased fitness compared to the DNMT3A-mutantclone over time. However, DNMT3A mutations persist when accompanied by other concomitant mutations (particularly TET2). These results raise interesting questions regarding clonal competition in MPN. Since all our analyses were performed from single clinical NGS panels, our findings require additional validation, including with multi-gene single-cell genotyping, which we are currently pursuing.

Crebbp Mutations Are Associated with Slow Minimal Residual Response to Upfront Induction Chemotherapy in Pediatric High-Hyperdiploid B-Cell Precursor Acute Lymphoblastic Leukemia Treated in the AIEOP-BFM ALL 2009 Protocol

Introduction High-hyperdiploidy (HHD) represents the most common cytogenetic abnormality found in B-cell precursor acute lymphoblastic leukemia (B-ALL). Despite the overall good outcome of this entity, up to 15% of patients suffer from disease relapse. Genome-wide ALL studies with next-generation sequencing (NGS) approaches identified a distinct pattern of somatic mutations in HHD ALL, enriched in mutations occurring in genes activating the RAS pathway and in CREBBP gene, especially in relapsed patients. Although several studies have attempted to explore the prognostic significance of these mutations that are potentially susceptible of targeted therapies, their impact in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus on their clinical significance. In this study, we aimed to investigate the frequency of recurrent mutations and to define their prognostic role in the setting of newly diagnosed HHD B-ALL, by using end of induction minimal residual disease (MRD) and event-free survival (EFS). Methods We included 101 consecutive patients, aged between 1-17 years, with newly diagnosed HHD B-ALL and enrolled in the AIEOP-BFM ALL 2009 protocol (NCT01117441). HHD was defined by the presence of a DNA index ≥1.16 and lack of ETV6::RUNX1, BCR::ABL1, KMT2A::AFF1 translocations. Diagnostic leukemia samples of 89 patients were sequenced using a validated clinical NGS panel profiling 39 genes involved in leukemia pathogenesis. Mutations affecting RAS pathway ( NRAS, KRAS, PTPN11, NF1, BRAF) and mutations affecting CREBBP gene were included. In addition, digital multiplex ligation-dependent probe amplification (digital MLPA) analysis for the identification of IKZF1 deletion and IKZF1plus phenotype was evaluable for 67 patients. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. MRD was assessed by an IG/TR PCR assay at day +33 of induction (timepoint 1, TP1) and at day +78 (timepoint 2, TP2). EFS was estimated by using Kaplan and Meier method and differences between groups were tested using the log-rank test. Resistance to treatment, relapse, death or second malignant neoplasm were considered as events, whichever occurred first. Results Patients' characteristics according to the presence of studied mutations are shown in Table 1. Mutations affecting RAS pathway and CREBBP occurred in 57.3% and 12.3% of HHD B-ALL patients, respectively. Clonal RAS pathway mutations, defined by variant allele frequency (VAF) ≥25%, were found in 16.8% of cases. Among the 11 patients with mutations of CREBBP, 7 had also a mutation affecting KRAS and the association was significant (p=0.03). Seven patients (10.4%) had deletion of IKZF1, of whom 5 (7.4%) were IKZF1plus. There was no association between any mutations or IKZF1 status and patient presenting characteristics, as including sex, age and WBC counts. MRD was evaluable in 84 patients at the end of induction (TP1). MRD level ≥5x10 -4 was found in 36.4% of patients with CREBBP mutations and in 9.6% of those without, and this association was statistically significant (p=0.02). Additionally, MRD negativity was found in 9.1% and 38.4%, respectively. At TP2, MRD was still detectable in 45.5% of patients with CREBBP mutations compared to 20.6% of those with CREBBP wildtype (p=0.08). Similarly, we observed an association between RAS pathway mutations and MRD at TP1 (p=0.01) with a marked difference in MRD negative patients (21.3% vs 51.4% in patients with and without mutations). No association was observed between MRD level and clonal RAS pathway mutations or IKZF1 status. Despite the association between CREBBP mutations and high MRD at TP1, no significant difference in final high-risk group was found (27.3% vs 11.5%, p=0.17). A total of 8 events were observed, of which 6 were relapses. No impact of any mutations or IKZF1 status in term of 5-years EFS was found in our cohort. Conclusion Our study shows that CREBBP mutations in HHD B-ALL are associated with inferior early response to treatment in terms of MRD level at the end of induction (TP1) but not with worse EFS with our whole current chemotherapy backbone. Therefore, the inclusion of CREBBP mutated patients in very low risk protocols should be carefully evaluated. The validation of these findings is ongoing through the analysis of patients treated by other international study groups adopting different chemotherapy backbones.

2303P Protein functional interpretation of gene variants observed in clinical next-generation sequencing (NGS) for pleural mesothelioma

2303P Protein functional interpretation of gene variants observed in clinical next-generation sequencing (NGS) for pleural mesothelioma

Clinico-Genomic Profiling of Conventional and Dedifferentiated Chondrosarcomas Reveals TP53 Mutation to Be Associated with Worse Outcomes.

Chondrosarcomas are the most common primary bone tumor in adults. Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are prevalent. We aimed to assess the clinico-genomic properties of IDH mutant versus IDH wild-type (WT) chondrosarcomas as well as alterations in other genes. We included 93 patients with conventional and dedifferentiated chondrosarcoma for which there were available clinical next-generation sequencing data. Clinical and genomic data were extracted and compared between IDH mutant and IDH WT chondrosarcomas and between TP53 mutant and TP53 WT chondrosarcomas. IDH1 and IDH2 mutations are prevalent in chondrosarcoma (50.5%), more common in chondrosarcomas arising in the extremities, associated with higher age at diagnosis, and more common in dedifferentiated chondrosarcomas compared with grades 1-3 conventional chondrosarcoma. There was no difference in survival based on IDH mutation in univariate and multivariate analyses. TP53 mutation was the next most prevalent (41.9%) and is associated with worse overall survival and metastasis-free survival in both univariate and multivariate analyses. TP53 mutation was also associated with higher risk of recurrence following curative-intent surgery and worse survival among patients that presented with de novo metastatic disease. IDH mutations are prevalent in chondrosarcoma though were not associated with survival outcomes in this cohort. TP53 mutations were the next most common alteration and were associated with worse outcomes.

The success rates of clinical cancer next-generation sequencing based on pathologic diagnosis: Experience from a single academic laboratory.

This article aims to establish the relationship between pathologic diagnosis and the rate of success in cancer next-generation sequencing testing. Clinical next-generation sequencing results performed for solid tumors were reviewed. The rate of success was analyzed in the context of tumor type and accompanying variables. Out of 683 total specimens, 533 (78.0%) underwent successful sequencing. The rate of success was 91.8% for ovarian carcinomas, 87.5% for lung non-small cell carcinomas, 82.0% for colorectal adenocarcinomas, 78.3% for melanomas, 75.9% for breast carcinomas, and 64.7% for pancreatic adenocarcinomas. For specimens that successfully underwent sequencing, pancreatic adenocarcinomas had the lowest median tumor proportion and somatic RAS and TP53 mutation allele fractions compared with other tumor types. Cytology specimens had a 33.3% success rate for pancreatic adenocarcinomas (5 of 15) and a 93.3% success rate for lung carcinomas (14 of 15). Compared with tissue from primary sites, tissue from metastatic sites showed a higher success rate for pancreatic adenocarcinomas and lower success rates for colorectal adenocarcinomas and melanomas. The success rate of cancer next-generation sequencing testing is dependent on pathologic diagnosis, tissue site, and diagnostic procedure. Understanding which specimens are at higher risk for failing molecular testing may help pathologists and clinical care teams optimize tissue acquisition and usage for patient care.

Clinical next-generation sequencing assay combining full-length gene amplification and shotgun sequencing for the detection of CMV drug resistance mutations

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads <500IU/mL and volumes <0.5 mL - conditions otherwise unacceptable by reference laboratories. Here, we describe the development and implementation of a robust NGS LDT that offers greater testing flexibility and sensitivity to accommodate a more diverse patient population.

Computational pharmacogenotype extraction from clinical next-generation sequencing.

Next-generation sequencing (NGS), including whole genome sequencing (WGS) and whole exome sequencing (WES), is increasingly being used for clinic care. While NGS data have the potential to be repurposed to support clinical pharmacogenomics (PGx), current computational approaches have not been widely validated using clinical data. In this study, we assessed the accuracy of the Aldy computational method to extract PGx genotypes from WGS and WES data for 14 and 13 major pharmacogenes, respectively. Germline DNA was isolated from whole blood samples collected for 264 patients seen at our institutional molecular solid tumor board. DNA was used for panel-based genotyping within our institutional Clinical Laboratory Improvement Amendments- (CLIA-) certified PGx laboratory. DNA was also sent to other CLIA-certified commercial laboratories for clinical WGS or WES. Aldy v3.3 and v4.4 were used to extract PGx genotypes from these NGS data, and results were compared to the panel-based genotyping reference standard that contained 45 star allele-defining variants within CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, CYP4F2, DPYD, G6PD, NUDT15, SLCO1B1, TPMT, and VKORC1. Mean WGS read depth was >30x for all variant regions except for G6PD (average read depth was 29 reads), and mean WES read depth was >30x for all variant regions. For 94 patients with WGS, Aldy v3.3 diplotype calls were concordant with those from the genotyping reference standard in 99.5% of cases when excluding diplotypes with additional major star alleles not tested by targeted genotyping, ambiguous phasing, and CYP2D6 hybrid alleles. Aldy v3.3 identified 15 additional clinically actionable star alleles not covered by genotyping within CYP2B6, CYP2C19, DPYD, SLCO1B1, and NUDT15. Within the WGS cohort, Aldy v4.4 diplotype calls were concordant with those from genotyping in 99.7% of cases. When excluding patients with CYP2D6 copy number variation, all Aldy v4.4 diplotype calls except for one CYP3A4 diplotype call were concordant with genotyping for 161 patients in the WES cohort. Aldy v3.3 and v4.4 called diplotypes for major pharmacogenes from clinical WES and WGS data with >99% accuracy. These findings support the use of Aldy to repurpose clinical NGS data to inform clinical PGx.

Abstract 6: Applying Next-Generation Sequencing to Improve Pediatric Cancer Diagnosis in Mexico

Abstract Purpose: In Mexico, cancer is the second leading cause of mortality among children between 4 to 15 years old. The overall cure rates remain below 50%, compared to &amp;gt;90% in the United States. This discrepancy is in part explained by the limited access to methods for accurate diagnosis and identification of appropriate therapies in Mexico. Only 48% of large hospitals across Mexico report having the capacity to perform some of the complex testing necessary for subclassification of pediatric cancers. Pan-cancer next generation sequencing (NGS) panels are not available in Mexico. We developed a strategy to perform clinical NGS in pediatric cancer patients in Mexico for identification of single nucleotide variants, copy number changes, and fusion events that are useful for risk stratification and adjusted therapy. Methods: We established collaborative agreements between UCSF and the National Institute for Genomic Medicine (INMEGEN) in Mexico City. We developed a pediatric cancer focused panel (IMOP) encompassing 200 genes and created a cloud-based bioinformatic and analysis pipeline for remote data interpretation. We established concordance by comparing the sequencing results of clinical cases obtained with the validated UCSF500 panel and the IMOP panel. Results: The results of 8 clinical samples previously sequenced using the clinically validated UCSF500 panel were compared to results obtained using the IMOP panel. Clinically relevant SNVs and fusions were successfully detected by the IMOP panel at similar variant allelic frequencies. The IMOP panel was shipped to INMEGEN where two samples with prior sequencing results were successfully sequenced. Re-sequencing of the 8 clinical samples to establish concordance at INMEGEN is pending. Conclusion: Our ongoing pilot proof-of-concept study shows feasibility of a strategy to implement clinical NGS testing in a middle-income country. We hypothesize that implementation of centralized NGS for pediatric cancers at INMEGEN will improve diagnostic accuracy. Moving forward, we plan to complete the clinical validation of the IMOP panel for routine sequencing of pediatric cancer patients. We will use the RE-AIM implementation science framework to assess the effectiveness of the assay in refining clinical diagnosis and treatment planning for pediatric cancer cases in Mexico. Citation Format: Roberto Ruiz-Cordero, Ela Alcantara, Ana Quintanar-Alfaro, Carlos A Espinoza-Mendez, Katherine Van Loon, Lindsay Breithaupt, Jorge Melendez Zajgla, Alejandro Sweet-Cordero. Applying Next-Generation Sequencing to Improve Pediatric Cancer Diagnosis in Mexico [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 6.

Integration of clinical sequencing and immunohistochemistry for the molecular classification of endometrial carcinoma

PurposeUsing next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data. Experimental designAll patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution. ResultsUtilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949–0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC. ConclusionsThe integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification.