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Targeting critical epigenetic regulators reverses aberrant transcription in cancer, thereby restoring normal tissue function1–3. The interaction of menin with lysine methyltransferase 2A (KMT2A), an epigenetic regulator, is a dependence in acute leukaemia caused by either rearrangement of KMT2A or mutation of the nucleophosmin 1 gene (NPM1)4–6. KMT2A rearrangements occur in up to 10% of acute leukaemias and have an adverse prognosis, whereas NPM1 mutations occur in up to 30%, forming the most common genetic alteration in acute myeloid leukaemia7,8. Here, we describe the results of the first-in-human phase 1 clinical trial investigating revumenib (SNDX-5613), a potent and selective oral inhibitor of the menin–KMT2A interaction, in patients with relapsed or refractory acute leukaemia (ClinicalTrials.gov, NCT04065399). We show that therapy with revumenib was associated with a low frequency of grade 3 or higher treatment-related adverse events and a 30% rate of complete remission or complete remission with partial haematologic recovery (CR/CRh) in the efficacy analysis population. Asymptomatic prolongation of the QT interval on electrocardiography was identified as the only dose-limiting toxicity. Remissions occurred in leukaemias refractory to multiple previous lines of therapy. We demonstrate clearance of residual disease using sensitive clinical assays and identify hallmarks of differentiation into normal haematopoietic cells, including differentiation syndrome. These data establish menin inhibition as a therapeutic strategy for susceptible acute leukaemia subtypes.

Supplementary Figure 1 depicts copy number alterations in the described cohort

Supplementary Table 1 shows point mutations found in DSRCT samples

Supplementary Figure 3 provides a breakdown of the structural variant types found via whole genome sequencing in DSRCT samples

Supplementary Figure 2 depicts whole genome sequencing findings

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1–WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1–WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1–WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study.Implications:These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Supplementary Figure 4 shows data related to FGFR4 inhibition in DSRCT and control models

Supplementary Table 2 shows the raw data further described in Figure 3

Purpose:Although multimodal chemotherapy has improved outcomes for patients with osteosarcoma, the prognosis for patients who present with metastatic and/or recurrent disease remains poor. In this study, we sought to define how often clinical genomic sequencing of osteosarcoma samples could identify potentially actionable alterations.Experimental Design: We analyzed genomic data from 71 osteosarcoma samples from 66 pediatric and adult patients sequenced using MSK-IMPACT, a hybridization capture-based large panel next-generation sequencing assay. Potentially actionable genetic events were categorized according to the OncoKB precision oncology knowledge base, of which levels 1 to 3 were considered clinically actionable.Results:We found at least one potentially actionable alteration in 14 of 66 patients (21%), including amplification of CDK4 (n = 9, 14%: level 2B) and/or MDM2 (n = 9, 14%: level 3B), and somatic truncating mutations/deletions in BRCA2 (n = 3, 5%: level 2B) and PTCH1 (n = 1, level 3B). In addition, we observed mutually exclusive patterns of alterations suggesting distinct biological subsets defined by gains at 4q12 and 6p12-21. Specifically, potentially targetable gene amplifications at 4q12 involving KIT, KDR, and PDGFRA were identified in 13 of 66 patients (20%), which showed strong PDGFRA expression by IHC. In another largely nonoverlapping subset of 14 patients (24%) with gains at 6p12-21, VEGFA amplification was identified.Conclusions:We found potentially clinically actionable alterations in approximately 21% of patients with osteosarcoma. In addition, at least 40% of patients have tumors harboring PDGFRA or VEGFA amplification, representing candidate subsets for clinical evaluation of additional therapeutic options. We propose a new genomically based algorithm for directing patients with osteosarcoma to clinical trial options.

A. Tumors were undetectable at 4 weeks by IVIS imaging when b-AP15 was administered from day 1 after injecting A673 cells expressing GFP-luciferase. NSG mice bearing A673 xenografts were treated with DMSO, bortezomib or b-AP15 after they reached 80-100mm3 and weekly tumor size measurements were taken. B. b-AP15 slowed the growth of the established A673 xenografts and C. prolonged survival of A673 tumor bearing mice compared to bortezomib. D. VLX-1570 and b-AP15 treatment did not cause significant weight loss in xenograft models. (two way Anova with Tukey's multiple comparison test).

KDR immunohistochemical stain in OS with 4q12 amplification

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525–34. ©2016 AACR.

Cell lines were treated with the indicated compounds for 96 h and then growth determined. Results represent the mean {plus minus} SD of 2-4 experiments in which each condition was assayed in triplicate.

Supplementary Tables S1-S8

Results and analysis of a genome-wide functional genomic screen using the Sigma-Aldrich shRNA Human Genome Library consisting of 63,093 shRNAs targeting 11,748 human genes.

Supplementary Figure 1 legend

A. Pair�wise expression profile comparisons of EWS cell lines treated with the known proteasome inhibitors bortezomib and MG�262 demonstrated highly significant overlap with profiles treated with compounds EWS-P and EWS-W. B. Overlap included 13 core genes established through published studies as being upregulated by 20S inhibitors such as bortezomib and MG-262. C. EWS cell lines TC-71 and CHP100 show demonstrate an increase in polyubiquitinated proteins following treatment with proteasome inhibitors MG-262, bortezomib, EWS-P, and EWS-W.

General Procedures for synthesis of 2,6-Bis-arylidenepiperidone scaffold

Ingenuity Pathway Analysis of positive hits identifies enrichment of genes involved in the ubiquitin-proteasome pathway.

72 hour IC50 growth inhibition levels (μM) of 23 compounds (labeled EWS-A through EWS-X) against five EWS cell lines and 11 non-EWS cell lines

Purpose: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT).Experimental Design: We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing.Results: Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively.Conclusions: In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies. Clin Cancer Res; 18(3); 748–57. ©2011 AACR.

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Panel upper left: CHP100 cells exposed 3 hours to different concentrations of b-AP15. Extracts were labeled with HA-Ub-VS and subjected to western blot. Filters probed with HA antibody. Note the lack of inhibition of any of the DUBs labeled by HA-Ub-VS. Panel upper right: CHP100 cells exposed 3 hours to 1 uM b-AP15 Extracts were labeled with Ub-VS and subjected to western blot. Filters probed with USP14, UCHL5 or UCHL1 antibodies. b-AP15 treated extracts demonstrate inhibition of USP14, weak inhibition of UCHL5, and no inhibition of UCHL1. Middle Panels: Purified 26S proteasomes (5 nM) were treated with indicate concentrations for b-AP15 and DUB activity was determined by cleavage of Ub-AMC. Note the reduction in 26S associated proteasome activity following treatment with b-AP15. Lower Panels: Recombinant UCHL1 or UCHL3 was treated with b-AP15 and DUB activity was monitored using Ub-AMC cleavage as above. No inhibition of either UCHL1 or UCHL3 was observed.

Supplementary Table 1 and Figures 1-4 from TFE3 Fusions Activate MET Signaling by Transcriptional Up-regulation, Defining Another Class of Tumors as Candidates for Therapeutic MET Inhibition

Supplementary Methods and Figure Legends 1-4 from TFE3 Fusions Activate MET Signaling by Transcriptional Up-regulation, Defining Another Class of Tumors as Candidates for Therapeutic MET Inhibition

Specific chromosomal translocations encoding chimeric transcription factors are considered to play crucial oncogenic roles in a variety of human cancers but the fusion proteins themselves seldom represent suitable therapeutic targets. Oncogenic TFE3 fusion proteins define a subset of pediatric renal adenocarcinomas and one fusion (ASPL-TFE3) is also characteristic of alveolar soft part sarcoma (ASPS). By expression profiling, we identified the MET receptor tyrosine kinase gene as significantly overexpressed in ASPS relative to four other types of primitive sarcomas. We therefore examined MET as a direct transcriptional target of ASPL-TFE3. ASPL-TFE3 binds to the MET promoter and strongly activates it. Likewise, PSF-TFE3 and NONO-TFE3 also bind this promoter. Induction of MET by ASPL-TFE3 results in strong MET autophosphorylation and activation of downstream signaling in the presence of hepatocyte growth factor (HGF). In cancer cell lines containing endogenous TFE3 fusion proteins, inhibiting MET by RNA interference or by the inhibitor PHA665752 abolishes HGF-dependent MET activation, causing decreased cell growth and loss of HGF-dependent phenotypes. MET is thus a potential therapeutic target in these cancers. Aberrant transcriptional up-regulation of MET by oncogenic TFE3 fusion proteins represents another mechanism by which certain cancers become dependent on MET signaling. The identification of kinase signaling pathways transcriptionally up-regulated by oncogenic fusion proteins may reveal more accessible therapeutic targets in this class of human cancers. [Cancer Res 2007;67(3):919–29]

PURPOSE Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS ( P = .04). The most frequently altered genes in the primary cohort were TP 53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort ( P = .015) and the validation cohort ( P = .012). CONCLUSION The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Effective reinduction regimens are needed for children with relapsed and refractory acute myeloid leukemia (AML), as outcomes remain poor. Therapeutic options are limited in this heavily pretreated patient population, many of whom have reached lifetime recommended doses of anthracycline chemotherapy. The development of effective non-anthracycline–based salvage regimens is crucial to these patients who are at significant risk of life-threatening cardiotoxicity. We previously reported results of a phase 2 trial of a clofarabine-based regimen with topotecan, vinorelbine, and thiotepa (TVTC) in patients with relapsed acute leukemias. Here we report on an expanded bicenter cohort of 33 patients

PURPOSE The US Food and Drug Administration–expanded access program (EAP) uses a single patient use (SPU) mechanism to provide patient access to investigational agents in situations where no satisfactory or comparable therapy is available. Genomic profiling of de novo and relapsed or refractory childhood cancer has led to increased identification of new drug targets in the last decade. The aim of this study is to examine the SPU experience for genomically targeted therapies in patients with pediatric cancer. PATIENTS AND METHODS All genomically targeted therapeutic SPUs obtained over a 5-year period were evaluated at four large pediatric cancer programs. Data were collected on the type of neoplasm, agents requested, corresponding molecularly informed targets, and clinical outcomes. RESULTS A total of 45 SPUs in 44 patients were identified. Requests were predominantly made for CNS and solid tumors (84.4%) compared with hematologic malignancies (15.6%). Lack of an available clinical trial was the main reason for SPU initiation (64.4%). The median time from US Food and Drug Administration submission to approval was 3 days (range, 0-12 days) and from Institutional Review Board submission to approval was 5 days (range, 0-50 days). Objective tumor response was seen in 39.5% (15 of 38) of all evaluable SPUs. Disease progression was the primary reason for discontinuation of drug (66.7%) followed by toxicity (13.3%). CONCLUSION SPU requests remain an important mechanism for pediatric access to genomically targeted agents given the limited availability of targeted clinical trials for children with high-risk neoplasms. Furthermore, this subset of SPUs resulted in a substantial number of objective tumor responses. The development of a multi-institutional data registry of SPUs may enable systematic review of toxicity and clinical outcomes and provide evidence-based access to new drugs in rare pediatric cancers.

Key Points BVB was an effective and tolerable retrieval regimen for pediatric patients with R/R HL and resulted in minimal toxicity.Stem cell mobilization and collection was successful in patients before autologous stem cell transplant., Visual Abstract, In patients with relapsed/refractory (R/R) Hodgkin lymphoma (HL), achieving a complete metabolic response (CMR) after salvage therapy is associated with superior outcomes, and optimal treatments must be identified. The combination of brentuximab vedotin and bendamustine (BVB), although highly active in adult patients, has not been extensively evaluated in pediatric patients with R/R HL. We performed a multicenter, retrospective review of pediatric patients

PURPOSE The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) can be abnormally activated in neuroblastoma, and somatic ALK mutations occur in 6%-10% of patients. The differential clinical impact of these mutations has not been clearly elucidated. METHODS Data on patients with neuroblastoma harboring ALK mutations were retrospectively analyzed. ALK sequencing was performed by whole-genome sequencing, hybrid-based capture of targeted exomes, or hotspot ALK mutation profiling. The differential impact of ALK mutation site on clinical characteristics, response to treatment, and survival was analyzed. In a subgroup of patients with locoregional neuroblastoma diagnosed after 2014, the impact of all ALK mutations was compared with wild-type ALK. RESULTS Of 641 patients with neuroblastoma with ALK status analyzed on at least one tumor sample, 103 (16%) had tumors harboring ALK mutations. Mutations existed across all ages (birth to 67.8 years), stages (30% locoregional and 70% metastatic), and risk groups (20%, 11%, and 69% with low-, intermediate-, and high-risk disease, respectively). Mutation sites included F1174 (51%), R1275 (29%), R1245 (10%), and others (10%). Mutation site was not prognostic for progression-free survival or overall survival in the entire cohort, high-risk subgroup, or locoregional subgroup. Locoregional tumors with any ALK mutation were generally invasive: L2 by International Neuroblastoma Research Group staging in 30/31 patients with a 2-year progression-free survival (59%, 95% CI, 37.4 to 80.5) that was inferior to historical controls. This observation was corroborated in the post-2014 subgroup in which gross total resection was less likely for ALK-mutated tumors. CONCLUSION Somatic ALK mutations are present across all stages and risk groups of neuroblastoma. No specific mutation carries differential prognostic significance. Locoregional neuroblastoma has an invasive phenotype when harboring somatic ALK mutations in this population.

High-risk neuroblastoma is generally metastatic and often lethal. Using genomic profiling of 470 sequential and spatially separated samples from 283 patients, we characterize subtype-specific genetic evolutionary trajectories from diagnosis, through progression and end-stage metastatic disease. Clonal tracing timed disease initiation to embryogenesis. Continuous acquisition of structural variants at disease defining loci (MYCN, TERT, MDM2-CDK4) followed by convergent evolution of mutations targeting shared pathways emerged as the predominant feature of progression. At diagnosis metastatic clones were already established at distant sites where they could stay dormant, only to cause relapses years later and spread via metastasis-to-metastasis and polyclonal seeding after therapy.

Ewing sarcoma (ES) is a primitive sarcoma defined by EWSR1-ETS fusions as the primary driver alteration. To better define the landscape of cooperating secondary genetic alterations in ES, we analyzed clinical genomic profiling data of 113 patients with ES, a cohort including more adult patients (18 years) and more patients with advanced stage at presentation than previous genomic cohorts.The data set consisted of patients with ES prospectively tested with the US Food and Drug Administration-cleared Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets large panel, hybrid capture-based next-generation sequencing assay. To assess the functional significance ofNovel subsets were defined by recurrent secondary alterations inOur findings open avenues to new insights into ES pathobiology and to novel therapeutic approaches in a subset of patients with ES.

Myeloid/lymphoid neoplasm with eosinophilia (MLN-Eo) is a World Health Organization (WHO) established category of hematologic malignancies primarily arising in adults. We discuss an 8-month-old infant who presented with clinical features similar to those of juvenile myelomonocytic leukemia (JMML) but who was diagnosed with MLN-Eo driven by an ETV6-FLT3 fusion. Results of patient-derived leukemia ex vivo studies demonstrated increased sensitivity to type I FLT3 inhibitors as compared with type II inhibitors. Treatment with the type I inhibitor gilteritinib resulted in complete immunophenotypic and cytogenetic remission. This patient subsequently underwent a hematopoietic stem cell transplant and remains in complete remission 1 year later. This is the youngest patient reported with an ETV6-FLT3 fusion and adds to the mounting reports of FLT3-rearranged MLN-Eo, supporting its addition to the WHO classification. Furthermore, this case highlights the clinical utility of ex vivo drug testing of targeted therapies.

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1–WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1–WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1–WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study.Implications:These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Whole genome sequencing (WGS) enables the identification of all cancer associated biomarkers in a patient’s tumor genome. Whilst fresh frozen (FF) derived WGS data provides optimal data quality, the majority of clinical biospecimens are from formalin fixed paraffin embedded (FFPE) tissue which results in DNA damage and an increase in artifactual mutation calls. Development of analytical frameworks tailored to FFPE derived WGS data can unlock the potential of genome profiling in clinical oncology. We performed comprehensive WGS analysis on 58 matched FF/FFPE specimens derived from 3 cancer centers. Consensus calling detected high-confidence somatic mutations across variant classes including: single nucleotide variants (SNVs), insertions/deletions (indels), structural variants (SVs) and copy number aberrations (CNAs). For each sample, genome-wide mutational patterns including tumor mutational burden (TMB), SNV/indel signatures, and homologous recombination deficiency (HRD) scores were estimated. We developed a random forest based framework using 33 features to learn mutation patterns associated with FFPE artifacts and implemented a filtration strategy for FFPE derived WGS data within a clinical prototype analytical workflow. We identified a high degree of concordance (~91%, n=192/210) for oncogenic variants between FF/FFPE WGS data. Comparison of small mutation calls presented an average 2-fold increase in FFPE samples with a range up to 152x for SNVs and 43x for indels. However, this was not the case for SVs: -0.4x (range -0.8-1.4). We demonstrate that genome-wide mutation patterns were significantly affected, impacting estimates for TMB, HRD and signature contributions. On average, TMB was overestimated in FFPE (median=10.3, range: 1.4-94) versus FF (median=3.4, range:0.04-29.6). For 7 patients with evidence of HRD in FF, HRD scores did not reach statistical significance in FFPE. Mutational signatures in FFPE were enriched for COSMIC signatures 37 and 5. Our artifact classification model achieved ROC AUC of 97.5% and precision-recall of 98.9%. Post artifact filtration, precision in SNV/indel calling was increased from 49.3% to 93.4% and 61.8% to 82.7% respectively with no effect on driver alterations. This increased global signal concordance drastically, with comparable TMB scores (median 2.4; range .03-26.1) and improved cosine similarity for SNV/indel signatures (median 0.98; range 0.40-1). HRD was successfully detected in 7/7 patients from FFPE derived data post filtering with probability scores ranging from 0.76-1. We demonstrate that FFPE specimens harbor a variable increase in artifactual mutation burden in SNV/indels but not in SVs. We propose an effective filtering procedure which successfully removes FFPE related artifacts enabling accurate profiling of clinically relevant driver mutations and genome-wide mutation patterns from readily available FFPE-derived tumor specimens. Citation Format: Dylan Domenico, Gunes Gundem, Max F. Levine, Juanes E. Arango-Ossa, Pauline Robbe, Georgios Asimomitis, Cassidy Cobbs, Emily Stockfisch, Janine Senz, Dawn Cochrane, Neeman Mohibullah, Neerav Shukla, Sohrab P. Shah, Andrew McPherson, Anna Schuh, Andrew L. Kung, Elli Papaemmanuil. Enabling whole genome sequencing analysis from FFPE specimens in clinical oncology [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 3142.