PIK3R1 Mutations Research Articles

Abstract P3-08-04: The genomic and transcriptomic landscape of PIK3R1 mutated breast cancers

Abstract Background: Human breast cancers may harbor pathogenic somatic alterations in PIK3R1, which encodes the regulatory subunit—p85a—of the PI3K signaling complex. Prior studies developed an isogenic cellular system lacking p85a and investigated therapeutic approaches for breast cancers that lack functional p85a. For instance, somatic loss of PIK3R1 may sensitize breast cancer cells to MEK inhibition; work in patient-derived xenograft models confirmed this observation. Here, we wanted to investigate the significance of PIK3R1 mutations (PIK3R1MUT) in human breast cancer by using real-world data to characterize the genomic landscape of breast cancer patients with PIK3R1MUT. Additionally, our methodology allows us to assess the effect of PIK3R1MUT on corresponding mRNA expression-levels, tumor mutational burden (TMB), and microsatellite instability (MSI) to better understand the molecular-level effects of this important gene. Methods: We used the Tempus LENS platform to retrospectively analyze next-generation sequencing (NGS) data from 3400 HER2-negative (HER2-) advanced or metastatic breast cancer patients with confirmed hormone receptor status (HR+/-). Our cohort consisted of molecularly profiled, deidentified breast cancer cases using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). This assay assesses mutations in both germline and somatic tissue, and characterizes nucleotide variants, insertions/deletions, and copy number variations. Results: The prevalence of PIK3R1MUT in HER2- breast cancer was 2.3% (77/3400). PIK3R1MUT occurred more frequently than expected in HR- breast cancer relative to the proportion we observed for wild-type PIK3R1 (Chi-squared test, p<0.001). In the HR+ subtype, high TMB (defined as 10 mutations/MB) occurred more frequently in PIK3R1MUT tumors than in samples with wild-type PIK3R1 (PIK3R1WT) status (Fisher’s exact test, p=0.039). Further, MSI-high status was absent in PIK3R1MUT tumors but was observed in PIK3R1WT samples. We assessed co-mutational patterns and found that pathogenic or likely pathogenic mutations in PTEN, TP53. and NF1 were more frequent in PIK3R1MUT samples, whereas mutations in PIK3CA were more frequent in the PIK3R1WT cohort (Table 1). At the transcriptional-level, PIK3R1 expression was similar in HR+ samples regardless of PIK3R1 mutational status but PIK3R1 transcript expression was significantly higher among PIK3R1MUT samples (Wilcoxon rank-sum test, p<0.001). Conclusions: Our study used real-world evidence to build on previous pre-clinical studies, and illustrates the importance of PIK3R1MUT in breast cancer. We found that certain mutations associated with poor outcomes and endocrine therapy resistance (e.g., PTEN, and NF1) were more frequent in PIK3R1MUT tumor samples. Interestingly, we did not find evidence that PIK3R1MUT results in decreased PIK3R1 mRNA expression but instead observed that in the HR- subtype PIK3R1MUT gene expression was significantly higher. Overall, this study shows that PIK3R1 may be an important therapeutic target in breast cancer. Table 1.- Co-mutations and PIK3R1 transcript abundance in PIK3R1MUT vs PIK3R1WT samples.Correlation of Gene Co-mutations with PIK3R1MUTGenesPIK3R1MUT (n=77)n (%)PIK3R1WT (n=3323)n (%)p-valuePositively correlatedPTEN21 (27%)176 (5.3%)<0.001NF113 (17%)134 (4.0%)<0.001TP5344 (57%)1289 (39)0.001Negatively CorrelatedPIK3CA9 (12%)861 (26%)0.005PIK3R1 gene expressionSub-typeNormalized expressionNormalized expressionp-valueHR+3.34 (n=30)3.28 (n=1720)0.2HR-3.45 (n=33)3.23 (n=678)<0.001 Citation Format: Melody Cobleigh, Emmanuel Okeke, Brett Mahon, Elizabeth Mauer, Alex Barrett, Abde Abukhdeir. The genomic and transcriptomic landscape of PIK3R1 mutated breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-04.

Abstract GS2-09: Tamoxifen instigates uterine cancer development by activating PI3K signaling and supersedes PIK3CA driver mutations

Abstract Tamoxifen is widely used in the adjuvant treatment of estrogen receptor–positive (ER+) breast cancer and is an important drug for pre-menopausal women and post-menopausal patients who cannot tolerate aromatase inhibitors. Despite the clear clinical benefit in improving relapse-free and overall survival in these patients, an adverse effect of tamoxifen is a 2- to 7-fold increased risk of uterine cancer (UC) after 2-5 years of treatment. To date, the mechanism of tamoxifen-driven tumorigenesis is not well understood, and preventive approaches are lacking. Here, to molecularly characterize tamoxifen-associated uterine cancers (TA-UCs) and gain insights into their unique evolution, we performed whole-exome sequencing of 21 TA-UCs (discovery cohort) and droplet digital PCR (ddPCR) of an additional 40 TA-UCs (validation cohort) obtained from the ‘Tamoxifen Associated Malignancies: Aspects of Risk’ (TAMARISK) study. In addition, we used in vivo mouse models to: (i) further investigate tamoxifen-activated molecular pathways that may be involved in TA-UC tumorigenesis; and (ii) offer mechanistic insights. Overall, we discovered that TA-UCs were genomically similar to non–TA-UCs from The Cancer Genome Atlas (TCGA) project, with one profound exception: TA-UCs are characterized by a lower-than-expected frequency of mutations in two highly prevalent UC driver genes in the PI3K pathway: PIK3CA (14% [3/21] vs 48% [265/554] in non–TA-UC; P=0.003, Fisher’s exact test; Q=0.02, Benjamini-Hochberg FDR) and PIK3R1 (0%, [0/21] vs 31% [174/554]; P=0.001; Q=0.01). We used ddPCR in the independent TA-UC validation cohort and confirmed the low frequency of mutations in PIK3CA (7.5% [3/40] vs 21% [144/685] in control UCs from the Dana-Farber contribution to the AACR GENIE project; P=0.04). We next performed mouse in vivo studies and demonstrated that tamoxifen activated the PI3K pathway and increased cell proliferation in normal mouse uterine tissue through paracrine and autocrine effects, both of which were abrogated by the PI3K inhibitor alpelisib. Taken together, we describe a distinct and novel pathway of carcinogenesis in which tamoxifen acts as a driver event in the uterus and promotes tumor development in a mutation-independent manner. Indeed, tamoxifen may increase the risk of UC by activating the PI3K pathway, which can substitute for the early acquisition of oncogenic PIK3CA or PIK3R1 mutations observed in non–TA-UC tumors. Furthermore, the ability of a PI3K inhibitor to reduce cell proliferation in our mouse model raises the possibility that downregulating the PI3K pathway may prevent or significantly reduce TA-UC development, offering a potential future therapeutic and prevention strategy for specific high-risk patients undergoing tamoxifen therapy. Citation Format: Kirsten Kubler, Agostina Nardone, Shankara Anand, Daniel Gorvich, Marjolein Droog, Francisco Hermida-Prado, Tara Akshi, Avery S Feit, Gabriella Cohen, Gwen Dackus, Matthew Pun, Yanan Kuang, Justin Cha, Mendy Miller, William J Gibson, Cloud P Paweletz, Eliezer M Van Allen, Flora E van Leeuwen, Petra Nederlof, Harry Hollema, Quang-Dé Nguyen, Marian JE Mourits, Ignaty Leshchiner, Chip Stewart, Ursula A Matulonis, Wilbert Zwart, Yosef E Maruvka, Gad Getz, Rinath Jeselsohn. Tamoxifen instigates uterine cancer development by activating PI3K signaling and supersedes PIK3CA driver mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS2-09.

Genomic landscape of TCRαβ and TCRγδ T-large granular lymphocyte leukemia

AbstractLarge granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 T-cell receptor αβ [TCRαβ] T-LGL and 12 TCRγδ T-LGL). Seventy-six mutations were observed in 3 or more patients in the cohort, and out of those, STAT3, KMT2D, PIK3R1, TTN, EYS, and SULF1 mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole-exome cohort. Hotspot mutations in STAT3, PIK3R1, and FAS were detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2D and TET2 were observed. Moreover, STAT3 mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2D and SETD1B (P < .01 and P < .05, respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower absolute neutrophil count values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3 coiled-coil domain were characterized. STAT3-mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferon-γ signaling and decreased phosphatidylinositol 3-kinase–Akt signaling for STAT3-mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.

Suppression of PI3K/Akt/mTOR/c-Myc/mtp53 Positive Feedback Loop Induces Cell Cycle Arrest by Dual PI3K/mTOR Inhibitor PQR309 in Endometrial Cancer Cell Lines.

Gene mutations in PIK3CA, PIK3R1, KRAS, PTEN, and PPP2R1A commonly detected in type I endometrial cancer lead to PI3K/Akt/mTOR pathway activation. Bimiralisib (PQR309), an orally bioavailable selective dual inhibitor of PI3K and mTOR, has been studied in preclinical models and clinical trials. The aim of this study is to evaluate the anticancer effect of PQR309 on endometrial cancer cells. PQR309 decreased cell viability in two-dimensional and three-dimensional cell culture models. PQR309 induced G1 cell cycle arrest and little cell death in endometrial cancer cell lines. It decreased CDK6 expression and increased p27 expression. Using the Proteome Profiler Human XL Oncology Array and Western blot assay, the dual inhibitor could inhibit the expressions of c-Myc and mtp53. KJ-Pyr-9, a c-Myc inhibitor, was used to prove the role of c-Myc in endometrial cancer survival and regulating the expression of mtp53. Knockdown of mtp53 lowered cell proliferation, Akt/mTOR pathway activity, and the expressions of c-Myc. mtp53 silence enhanced PQR309-inhibited cell viability, spheroid formation, and the expressions of p-Akt, c-Myc, and CDK6. This is the first study to reveal the novel finding of the PI3K/mTOR dual inhibitor in lowering cell viability by abolishing the PI3K/Akt/mTOR/c-Myc/mtp53 positive feedback loop in endometrial cancer cell lines.

Gene Expression and Mutation Analysis of Early-Stage Recurrent Endometrial Cancer

Purpose/Objective(s)As currently under investigation in PORTEC-4a, an integrated molecular model of endometrial cancer is postulated to risk stratify women to better identify those more likely to benefit from adjuvant radiation therapy following hysterectomy for early-stage disease. Stratification of tumor biology is based on several features, including: p53 mutation, POLE mutation, CTNNB1 mutation, microsatellite instability (MSI), LICAM expression and the presence of LVSI. TCGA data evaluating a large cohort of early and advanced stage endometrial tumors suggests that gene expression profiles may also distinguish between favorable and unfavorable biology of endometrial tumors. We aimed to determine whether recurrent early-stage endometrial tumors have distinct gene-expression patterns from early-stage tumors that did not recur.Materials/MethodsUsing the TCGA-UCEC dataset available via the Genomic Data Commons, 394 Stage I-II tumors’ gene expression data were evaluated and analyzed using R (R Core Team, 2020). Of these cases, 17 were confirmed as relapsed endometrial cancer (10 endometrioid and 7 serious/mixed) and 370 were tumors without documentation of a recurrence. Gene expression analysis was performed with limma, gene set variation analysis with GSVA, and gene enrichment with Metascape. The frequency of several common mutations was evaluated in the recurrent tumor subset.ResultsThere were 860 differentially expressed genes in recurrent versus not recurrent early-stage tumors identified from the TCGA-UCEC dataset. The most significant of which include DDIT3, THAP12, EMSY, CLNS1A, and AAMDC (log[Fold Change] > 1000, adjusted P-value < 0.001). Differentially expressed genes show pathway enrichment in GO Biologic Processes and Reactome Gene Sets, specifically, mitochondrion organization, proteolysis, organelle disassembly, translation, response to endoplasmic reticulum stress, proteinases, cellular component disassembly, covalent chromatin modification, and intrinsic apoptotic signaling pathway. Non-overlapping mutations within the recurrent tumors include, 9 of 17 recurrent tumors with p53 mutation, 3 of 17 with CTNNB1 mutation, 4 of 17 with PIK3R1, PIK3CR and PIK3CB mutation, and 1 of 17 a mutation in POLE.ConclusionOf recurrent early-stage tumors within the TCGA-UCEC dataset, of which as least a majority classify as intermediate or unfavorable according to PORTEC-4a criteria, differential gene expression in recurrent tumors compared to not recurrent tumors demonstrate relative increased expression of genes involved in catabolic and cell cycle processes. PORTEC-4a does not employ gene expression signatures to distinguish amongst favorable, intermediate, and unfavorable subtypes of early-stage endometrial tumors. However, further investigation may prove gene expression incorporation into stratification beneficial. As currently under investigation in PORTEC-4a, an integrated molecular model of endometrial cancer is postulated to risk stratify women to better identify those more likely to benefit from adjuvant radiation therapy following hysterectomy for early-stage disease. Stratification of tumor biology is based on several features, including: p53 mutation, POLE mutation, CTNNB1 mutation, microsatellite instability (MSI), LICAM expression and the presence of LVSI. TCGA data evaluating a large cohort of early and advanced stage endometrial tumors suggests that gene expression profiles may also distinguish between favorable and unfavorable biology of endometrial tumors. We aimed to determine whether recurrent early-stage endometrial tumors have distinct gene-expression patterns from early-stage tumors that did not recur. Using the TCGA-UCEC dataset available via the Genomic Data Commons, 394 Stage I-II tumors’ gene expression data were evaluated and analyzed using R (R Core Team, 2020). Of these cases, 17 were confirmed as relapsed endometrial cancer (10 endometrioid and 7 serious/mixed) and 370 were tumors without documentation of a recurrence. Gene expression analysis was performed with limma, gene set variation analysis with GSVA, and gene enrichment with Metascape. The frequency of several common mutations was evaluated in the recurrent tumor subset. There were 860 differentially expressed genes in recurrent versus not recurrent early-stage tumors identified from the TCGA-UCEC dataset. The most significant of which include DDIT3, THAP12, EMSY, CLNS1A, and AAMDC (log[Fold Change] > 1000, adjusted P-value < 0.001). Differentially expressed genes show pathway enrichment in GO Biologic Processes and Reactome Gene Sets, specifically, mitochondrion organization, proteolysis, organelle disassembly, translation, response to endoplasmic reticulum stress, proteinases, cellular component disassembly, covalent chromatin modification, and intrinsic apoptotic signaling pathway. Non-overlapping mutations within the recurrent tumors include, 9 of 17 recurrent tumors with p53 mutation, 3 of 17 with CTNNB1 mutation, 4 of 17 with PIK3R1, PIK3CR and PIK3CB mutation, and 1 of 17 a mutation in POLE. Of recurrent early-stage tumors within the TCGA-UCEC dataset, of which as least a majority classify as intermediate or unfavorable according to PORTEC-4a criteria, differential gene expression in recurrent tumors compared to not recurrent tumors demonstrate relative increased expression of genes involved in catabolic and cell cycle processes. PORTEC-4a does not employ gene expression signatures to distinguish amongst favorable, intermediate, and unfavorable subtypes of early-stage endometrial tumors. However, further investigation may prove gene expression incorporation into stratification beneficial.

TCL-232: Integrated Genomic and Transcriptomic Sequencing Reveals the Landscape of T, NK, and Gamma-Delta Large Granular Lymphocyte Leukemia

<h3>Context</h3> Large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder of primarily CD8+ T or natural killer (NK) cell lineage whose molecular landscape is poorly characterized. <h3>Objective</h3> In this study, we defined the landscape of non-synonymous somatic variants in LGL leukemia exomes, asked whether these mutational profiles differed across LGL leukemia subtypes, and tested whether mutational groups were associated with clinical features. <h3>Design</h3> We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia study to date, which included 116 patients (93 TCRαβ T-LGL, 11 NK-LGL, and 12 TCRγδ T-LGL). <h3>Patients</h3> The study was conducted under IRB-approved protocols for the LGL Leukemia Registry at the University of Virginia School of Medicine. 116 LGL patients meeting diagnosis criteria by the World Health Organization (evidence of clonality, persistence of chronic expansion >6 months) were recruited for this study. <h3>Results</h3> The mutational landscape of LGL leukemia identified ARHGAP25, CCL22, CDH8, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using IntOGen bioinformatic driver analysis. The majority of gene mutations spanned all LGL subtypes, with the notable exception of CCL22, which was mutated exclusively within NK-LGL (4/11; 36.4%; p=0.0008). Moreover, there was a predilection for co-occurrence of STAT3 mutations with epigenetic modifier KMT2D (p < 0.01). Hotspot mutations in PIK3R1 and FAS were detected in both T-LGL subtypes, hinting at domain-specific mechanisms. STAT3 mutant patients exhibited increased non-silent mutation burden and a mutational signature associated with increased cell division. Gene expression analysis revealed enrichment of interferon-alpha signaling for STAT3 mutant patients, while increased PI3K-Akt signaling was observed for STAT3 WT patients. Finally, STAT3 mutations were associated with reduced neutrophil counts for only the D661Y and N647I variants but not Y640F, whereas all three of these mutation groups were associated with anemia. <h3>Conclusions</h3> These findings highlight the clinical and molecular heterogeneity of this rare disorder. LGL leukemia research is supported by NCI R01CA178393, P30CA044579, T32LM012416, T32GM007267, F30CA225046. Additional support is provided by Bess Family Charitable Fund, LGL Leukemia Foundation, Dr. Charles and Katharine Hutton Tweedy, William J. Branch, Dr. Szabolcs Szentpetery, and a generous anonymous donor.

Whole-genome profiling of primary cutaneous anaplastic large cell lymphoma.

Primary cutaneous anaplastic large cell lymphoma (pcALCL), a hematological neoplasm caused by skin-homing CD30+ malignant T cells, is part of the spectrum of primary cutaneous CD30+ lymphoproliferative disorders. To date, only a small number of molecular alterations have been described in pcALCL and, so far, no clear unifying theme that could explain the pathogenetic origin of the disease has emerged among patients. In order to clarify the pathogenetic basis of pcALCL, we performed high-resolution genetic profiling (genome/transcriptome) of this lymphoma (n=12) by using whole-genome sequencing, whole-exome sequencing and RNA sequencing. Our study, which uncovered novel genomic rearrangements, copy number alterations and small-scale mutations underlying this malignancy, revealed that the cell cycle, T-cell physiology regulation, transcription and signaling via the PI-3-K, MAPK and G-protein pathways are cellular processes commonly impacted by molecular alterations in patients with pcALCL. Recurrent events affecting cancer-associated genes included deletion of PRDM1 and TNFRSF14, gain of EZH2 and TNFRSF8, small-scale mutations in LRP1B, PDPK1 and PIK3R1 and rearrangements involving GPS2, LINC-PINT and TNK1. Consistent with the genomic data, transcriptome analysis uncovered upregulation of signal transduction routes associated with the PI-3-K, MAPK and G-protein pathways (e.g., ERK, phospholipase C, AKT). Our molecular findings suggest that inhibition of proliferation-promoting pathways altered in pcALCL (particularly PI-3-K/AKT signaling) should be explored as potential alternative therapy for patients with this lymphoma, especially, for cases that do not respond to first-line skin-directed therapies or with extracutaneous disease.

Disorders Related to PI3Kδ Hyperactivation: Characterizing the Clinical and Immunological Features of Activated PI3-Kinase Delta Syndromes.

Phosphoinositide-3-kinase δ (PI3Kδ) is found in immune cells and is part of the PI3K/AKT/mTOR/S6K signalling pathway essential to cell survival, growth and differentiation. Hyperactivation of PI3Kδ enzyme results in Activated PI3-kinase delta syndrome (APDS). This childhood onset, autosomal dominant, combined immunodeficiency, is caused by heterozygous gain of function (GOF) mutations in PIK3CD (encodes PI3Kδ catalytic subunit p110δ), mutations in PIK3R1 (encodes PI3Kδ regulatory subunit p85α) or LOF mutations in PTEN (terminates PI3Kδ signalling) leading to APDS1, APDS2 and APDS-Like (APDS-L), respectively. APDS was initially described in 2013 and over 285 cases have now been reported. Prompt diagnosis of APDS is beneficial as targeted pharmacological therapies such as sirolimus and potentially PI3Kδ inhibitors can be administered. In this review, we provide an update on the clinical and laboratory features of this primary immunodeficiency. We discuss the common manifestations such as sinopulmonary infections, bronchiectasis, lymphoproliferation, susceptibility to herpesvirus, malignancy, as well as more rare non-immune features such as short stature and neurodevelopmental abnormalities. Laboratory characteristics, such as antibody deficiency and B cell and T cell, phenotypes are also summarised.

Clinical Manifestations and Outcomes of Activated Phosphoinositide 3-Kinase δ Syndrome from the USIDNET Cohort

Activated phosphoinositide 3-kinase δ syndrome is a combined primary immunodeficiency characterized by gain-of-function mutations in PIK3CD and PIK3R1. Activated phosphoinositide 3-kinase δ syndrome demonstrates a large range of phenotypes including respiratory and herpesvirus infections, lymphadenopathy, autoimmunity, and developmental delay. To describe clinical phenotypes and disease outcomes of a large activated phosphoinositide 3-kinase δ syndrome cohort from the United States Immunodeficiency Network Registry. A total of 38 patients were enrolled in the United States Immunodeficiency Network Registry, and 2 additional patients were obtained from the Clinical Immunology Division at Mount Sinai Hospital. Each patient's demographic characteristics, disease complications, genetic studies, laboratory data, therapeutic interventions, and clinical outcomes were reviewed. There was a high frequency of respiratory infections (70.0% pneumonia) and herpesvirus infections (37.5%). Bronchiectasis was observed in 45.0% of patients. Lymphadenopathy was common (52.5%), and 12.5% of patients developed lymphoma. Neurological and developmental findings were common: 20.0% had developmental delay, 15.0% had seizures, and 10.0% had dysmorphic features. Asthma was more common in PIK3CD compared with PIK3R1 patients (63.6% vs 14.3%). More subjects with PIK3CD had CD3 lymphopenia compared with the PIK3R1 cohort. Seven patients underwent hematopoietic stem cell transplantation. One patient died from infectious complications. This is the first cohort comparing clinical manifestations in PIK3CD and PIK3R1 patients from the USIDNET Registry. Similar frequencies of respiratory and herpesvirus infections, lymphadenopathy, and developmental delay were observed compared with previous cohort studies. However, a higher frequency of asthma and CD3 lymphopenia in the PIK3CD cohort compared with the PIK3R1 cohort was observed.

Genomic Analysis and Pathway Characterization of Genes with Somatic Variants in Infant Acute Lymphoblastic Leukemia

IntroductionInfant acute lymphoblastic leukemia (ALL) is an aggressive cancer with poor relapse-free and overall survival. Other than MLL (KMT2A ) rearrangement (MLL -r), infant ALL contains remarkably few genomic lesions. We previously reported results of whole genome sequencing (WGS) on 14 matched diagnosis-remission-relapse (DX-RM-RL) MLL -r infant ALL trios (cohort A) treated on Children's Oncology Group (COG) trial AALL0631 (Kostadinov, et al. Blood 2016, 128(22):1735).Here, we examine the presence of somatic variants in both MLL -r (n=12) and non- MLL -r (n=15) infant ALL cases (COG AALL0631), which, to date, have not relapsed (cohorts B and C, respectively). For MLL -r cases, MLL partners were 6 AF4 (AFF1 ), 5 ENL (MLLT1 ), and 1 unspecified. For all cases, paired RM samples served as germline controls. In addition, we manually curated genes with somatic variants into functional pathway categories to determine whether any pathways were recurrently altered amongst all 3 cohorts.MethodsWe performed WGS on 27 paired DX-RM blood or bone marrow samples using an Illumina Hiseq 4000 or 2500 to a minimum depth of 90Gb. Alignment and variant calling were performed using BWA and GATK. Variants present in the DX sample and absent in the paired RM sample were considered somatic. Filters included an allelic depth threshold of 4 reads, minor allele frequency ≤0.1%, and a variant annotation of known pathogenic, likely pathogenic, or uncertain significance per RUNES (Saunders, et al. Sci Transl Med 2012, 4(154):154). Next, somatic variant genes (SVG) in these 2 cohorts, as well as cohort A (n=25 at DX and 156 at RL), were categorized into 1 or more of 41 functional pathways using publically available protein databases (UniProt, GenAtlas, BioGPS) and published literature. We then compared the prevalence of pathways affected across all 3 cohorts, specifically examining relapsed versus non-relapsed cases, MLL -r versus non- MLL -r, DX versus RL, and MLL - AF4 versus MLL - ENL , to identify any differences.ResultsWe found 23 non-synonymous somatic variants (NSSV) across 22 genes in cohort B; 4 were small (<40 bp) insertions/deletions (indels) and 19, single nucleotide variants (SNVs), of which 17 were missense, 1 nonsense, and 1 splicing. The mean variant allele frequency (VAF) was 0.42 ± 0.12 (range (r) 0.14-0.58). The mean number of NSSV per case at DX was 1.92 ± 1.73 (r 0-6). Mutations in KRAS (n=2), PIK3R1 (n=1), and NRAS (n=1) were observed. In cohort C, 29 NSSV across 28 genes were identified; 3 were small indels and 26, SNVs: 23 missense, 1 nonsense, and 2 splicing. The mean VAF was 0.37 ± 0.13 (r 0.13-0.67). The mean number of NSSV per case at DX was 2.0 ± 1.73 (r 0-6). Mutations in KRAS (n=2) and JAK2 (n=1) were observed. Using the same analysis filters for cohort A, we found that the mean number of NSSV per case was 2.1 at DX, and 11.5 at RL. The most frequently mutated genes included NRAS and FLT3 (n=3 cases each) at DX and KRAS , NRAS , and TP53 ,at RL (n=2 cases each).Pathway classification revealed that 2 categories-signal transduction and transcriptional regulation-contained the greatest number of SVG (n≥8) in each cohort at DX. Other categories with a high number of SVG (n≥4) across cohorts at DX included: cell cycle, metabolism, and developmental regulation. Within the signal transduction pathway group, the RAS/MAPK/ERK and PI3K/AKT/mTOR signaling pathways contained the greatest number of SVG; interestingly, these were more likely to be found in MLL-AF4 cases (n=9 and 10, respectively, of 14 total) than MLL - ENL (n=0 and 3, respectively, of 10 total). SVG involved in NF-kappa-B signaling were only found in MLL -r cases (n=2 of 26) versus non- MLL -r (n=0 of 15). SVG involved in WNT signaling and ion transport were more common at DX in cases that eventually relapsed (n=3 and 4, respectively, of 14 total) than those that did not (n=0 and 1, of 12 total). Of all the SVG gained at relapse (n=136), most fell into the signal transduction and transcriptional regulation categories.ConclusionsOverall, the mean number of NSSV per case at DX for each cohort was low. Functional pathways containing the highest numbers of SVG were also largely similar across cohorts, though we have highlighted some potentially important differences in subsets of cases. Further correlation with the type of variant found, as well as the expression of SVG across infant ALL cases, is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Genomic Landscape of Primary Cutaneous Diffuse Large B Cell Lymphoma, Leg Type

Primary cutaneous diffuse large B-cell lymphoma, leg type (DLBCL-LT) is an aggressive subtype of cutaneous B cell lymphoma (CBCL) with a poor prognosis. It is most often diagnosed on the legs of the elderly, frequently relapses, and has a tendency towards extracutaneous dissemination. Current treatment strategies are ineffective and revolve around surgical excision, multiagent chemotherapy (e.g. R-CHOP) and radiation therapy, with estimated 5-year survival rates of ~50%. Identifying genetic mutations and altered pathways specific for DLBCL-LT is crucial for the diagnosis and development of effective targeted therapies for this condition.We performed whole exome capture and massively parallel sequencing on 13 tumor samples of DLBCL-LT (4 matched and 9 unmatched) and 5 samples of cutaneous diffuse large B cell lymphoma, not otherwise specified (DLBCL-NOS) (1 matched and 4 unmatched). This is the largest whole exome cohort of cutaneous DLBCL to date. We applied algorithms routinely used by our lab to identify putative cancer promoting mutations and to compare the genetic landscape with DLBCL, both nodal and extranodal.Broadly, the landscape of mutations in DLBCL-LT is similar to that of activated B-cell like (ABC) DLBCL and most analogous to primary central nervous system lymphoma (PCNSL), a type of extranodal ABC DLBCL. Similar to PCNSL, we found frequent disease-defining oncogenic gene mutations in MYD88 (85%), CD79B (38%), and TBL1XR1 (38%), and loss of function mutations in tumor suppressor genes KMT2D (15%), CDKN1B (15%), and CD58 (8%). We observed frequent mutations in PIM1 (54%), OSBPL10 (31%), BTG2 (31%), and IGLL5 (15%), which are either known or proposed targets of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation (aSHM) also seen in PCNSL. Importantly and similarly to PCNSL, we found alterations at the PDL-1 and PDL-2 locus, but instead of copy number gains, we discovered frequent PD-L1 and PD-L2 translocations (40%) that may translate into overexpression of PD-L1 .This spectrum of mutations differed significantly compared to our DLBCL-NOS cohort, which did not have the above oncogenic mutations and lacked any PD-L1 or PD-L2 chromosomal alterations. Finally, we report novel oncogenic mutations in DLBCL-LT including MYD88 p.S243N, CD79B p.L199P, CCND3 p.T283P and p.P284S, BRAF p.V600E, and CARD11 p.R113Q and novel loss of function mutations in KMT2D, CDKN1B, CD58, MSH2, PIK3R1, MSH2, and BMF .Collectively, we identified recurrent mutations in multiple key cancer-related biological pathways, including epigenetic modulators (TBL1XR1, HIST1H1E, and KMT2D), NF-κB signaling pathway (MYD88, CD79B, and CARD11), MAPK signaling pathway (BRAF), PI3K signaling pathway (PIK3R1), JAK/STAT signaling pathway (PIM1), cell cycle regulators (CCND3, CDKN1B and BTG2), and immune escape (CD58). All of the DLBCL-LT samples in our cohort contained one or more gene mutations that can be targeted by agents that are currently FDA-approved or under clinical trials, such as BRAF inhibitors, JAK/STAT inhibitors, PIM inhibitors, PI3K inhibitors, NF-kB inhibitors, MYD88 inhibitors, and PD-1/PDL-1 inhibitors.Altogether, our data identified targetable putative driver gene mutations in DLBCL-LT. These mutational signatures bear broad similarities to PCNSL. Because of the relative rarity of this disease, we hope these efforts will allow for its inclusion, when relevant, in trials involving genotypically similar DLBCL. DisclosuresNo relevant conflicts of interest to declare.

The Role of Human Papilloma Virus in Dictating Outcomes in Head and Neck Squamous Cell Carcinoma.

The Human Papilloma Virus (HPV) is an oncogenic virus which is associated with the development of head and neck squamous cell carcinoma (HNSCC), predominantly within the oropharynx. Approximately 25% of oropharyngeal squamous cell carcinoma (OPSCC) cases worldwide are attributable to HPV infection, with an estimated 65% in the United States. Transmission is via exposure during sexual contact, with distinctive anatomical features of the tonsils providing this organ with a predilection for infection by HPV. No premalignant lesion is identifiable on clinical examination, thus no comparative histological features to denote the stages of carcinogenesis for HPV driven HNSCC are identifiable. This is in contrast to HPV-driven cervical carcinoma, making screening a challenge for the head and neck region. However, HPV proffers a favorable prognosis in the head and neck region, with better overall survival rates in contrast to its HPV negative counterparts. This has resulted in extensive research into de-intensifying therapies aiming to minimize the morbidity induced by standard concurrent chemo-radiotherapy without compromising efficacy. Despite the favorable prognosis, cases of recurrence and/or metastasis of HPV positive HNSCC do occur, and are linked with poor outcomes. HPV 16 is the most frequent genotype identified in HNSCC, yet there is limited research to date studying the impact of other HPV genotype with respect to overall survival. A similar situation pertains to genetic aberrations associated in those with HPV positive HNSCC who recur, with only four published studies to date. Somatic mutations in TSC2, BRIP1, NBN, TACC3, NFE2l2, STK11, HRAS, PIK3R1, TP63, and FAT1 have been identified in recurrent HPV positive OPSCC. Finding alternative therapeutic strategies for this young cohort may depend on upfront identification of HPV genotypes and mutations which are linked with worse outcomes, thus ensuring appropriate stratification of treatment regimens.

In silico study of gene-gene interaction of PIK3R1and PIGF deleterious SNPs in correlation to the preeclampsia risk and its haplotype effect on antihypertensive treatment

Introduction: The PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1) and PlGF (Placenta growth factor)genes share some common pathways with eNOS,( Endothelial nitric oxide synthase) that plays a vital role in angiogenesis of blood vessels and regulating endothelial function. The PIK3R1 gene encodes the receptor for the p85α regulatory subunit of the phosphoinositide-3-kinase (PI3K), that involved in endothelial cell migration. The PlGF gene encodes for the placenta growth factor, a homolog of vascular endothelial growth factor (VEGFA) that is involved in angiogenesis. PIK3R1 and/or PlGF mutations may cause dysregulation of eNOS contributing to endothelial dysfunction in preeclampsia. This study aims to analyze the effect of mutation of the PIK3R1 and PlGF genes on the structure and function of PIK3R1 and PGF protein that may have an important role in pathogeneses of preeclampsia. Methodology: The data on human PIK3R1and PGF genes were retrieved from dbSNP/NCBI. Ten prediction algorithms; SIFT, PROVEAN, Polyphen, SNAP2, SNPs&GO, PANTHER PhD-SNP, I-Mutant, Mutpred, and Hope were used to analyzing the effect of nsSNPs on functions and structure of the PGF and PIK3R1 protein. STRING and KEGG databases were used for PGF and PIK3R1 protein-protein interaction. Results and Discussion: As per the dbSNP database, the humanPIK3R1gene contained 365 missense mutations. A total 3nsSNPs (T239M, S229W, E47K) and 2 nsSNPs (H125Y, V59G) were predicted to have the most damaging effects on the structure and function of PIK3R1 and PGF respectively. STRING and KEGG revealed that PIK3R1and PGF had strong interactions with proteins involved in the VEGF signaling pathway and PI3K-Akt signaling pathway. PIK3R1 is confirmed to be linked to important diseases like preeclampsia and can affect its treatment response. Conclusion: Gene-gene interaction is an important factor in preeclampsia treatment, effect of mutation of the PIK3R1 and PlGF genes on the structure and function of PIK3R1 and PGF protein may have an important role in pathogeneses of preeclampsia. Also, these are linked to its treatment effect. This document gives formatting instructions for authors preparing papers for publication in the journal. Authors are encouraged to prepare manuscripts directly using this template. This template demonstrates the format requirements for the Journal.

Binimetinib activity in PIK3R1-mutant patient-derived xenografts (PDX) implanted into immunodeficient mice.

e13062 Background: The PIK3R1 gene is genetically altered in ̃3% of breast cancers in the Western and ̃17% in the Eastern worlds. We recently reported that breast cancer cells lacking protein expression of PIK3R1 had elevated levels of activated MEK, sensitizing them to the MEK inhibitor trametinib. To better understand whether MEK inhibition is a therapeutic option for breast cancer patients with mutated PIK3R1, we tested the newer generation MEK inhibitor, binimetinib, in a PDX mouse model. Methods: Two PDx models (table) were tested in athymic nude-foxn1nu (immune-compromised) mice. Both patients had had metastatic, grade 3 breast cancer and expressed moderate RNA levels of PIK3R1. Results: Body weights of treatment arms were unchanged throughout the experiment. The TNBC PDx study was halted early at 13 days because tumors reached the predefined maximum size of 1500 mm2. The binimetinib treated mice experienced slowed tumor growth (522.8 +/- 172.8%) compared to the vehicle control (720.7 +/- 229.1%), (p=0.0693). In the HR+ PDx model, the combination of binimetinib plus tamoxifen resulted in a statistically significant decrease (371.5 +/- 108.1%) in tumor volume compared to tamoxifen alone (525.0 +/- 154.1%; p = 0.0302). Conclusions: Our previous and current results suggest that mutation in PIK3R1 sensitizes cells to MEK inhibitors. Binimetinib inhibited growth in both models. This inhibition was of borderline significance in the TNBC model and was statistically significant in the ER+, HER2- model. To the best of our knowledge, these results provide the first evidence of binimetinib efficacy in PIK3R1-mutant, HR+, HER2- PDX immunodeficient mice. Plans for a phase II trial in patients with PIK3R1-mutant, HR+, HER2- breast cancer are underway.[Table: see text]

Genomic profiles of BRAF inhibitor resistance mechanisms in metastatic colorectal cancer.

e15527 Background: BRAF mutations are present in 5-10% of metastatic colorectal cancers (mCRCs). Patients with mCRC whose tumors harbor BRAF V600 mutation generally respond poorly to standard chemotherapy. Treatment with BRAF inhibitors has been shown to improve outcomes, whereas the resistance develops through undefined mechanisms. Therefore, it is imperative to accurately comprehend the genomic profiling of resistance mechanisms to BRAF inhibitors in mCRC for exploring its clinical treatment strategies. Methods: We analyzed next-generation sequencing results in 520 cancer-associated genes for 22 patients who had BRAF V600E mutant mCRC in order to characterize genomic predictors of treatment outcome and track the acquired resistance (AR) mechanisms during the vemurafenib/dabrafenib/encorafenib treatment in combination with cetuximab +/- trametinib. The median age of the patients was 61 years old, 54.5% were male, 54.5% had right-sided mCRC, and 81.8% received one or more prior chemotherapy lines. Tissue and/or plasma samples were collected at baseline (prior to anti-BRAF treatment) and upon disease progression (PD). Objective tumor responses were radiologically assessed every 6 weeks according to RECIST v1. 1. Results: By Feb 2021, treatment had been discontinued in 15 (63.2%) of the patients due to disease progression, while the other 7 cases were still under treatment. The median PFS (mPFS) for all patients was 4.5 months. The overall response rate was 32%, and the disease control rate was 86%. In addition to BRAF V600E, the most common concomitant mutations were identified in TP53 (20/22), RNF43 (8/22), LRP18 (7/22), APC (7/22) and PIK3CA (5/22). Patients with baseline alterations in RNF43 (n = 8; p = 0.0466), or RECQL4 (n = 4; p = 0.0406) had significantly longer PFS than their respective wild type counterparts. In contrast, baseline alterations in PPP2R2A (n = 3; p = 0.0131), RUNX1T1 (n = 3; p = 0.0147), and receptor tyrosine kinase (RTK) signaling pathway genes (n = 6; p = 0.0057) were each significantly associated with shorter PFS. Among the 15 patients whose disease progressed, 9 were identified with newly developed AR mutations in PD tumor or plasma samples. MET amplification was the most common AR mechanism (n = 4) followed by BRAF amplification, KRAS, NRAS mutations (n = 3 each), and MAP2K1, PIK3R1 mutations (n = 1 each). Q61 substitution was the most dominant form for both KRAS and NRAS AR mutations (83%, 5/6). In terms of signaling pathway, MAPK (67%, 6/9) and RTK (44%, 4/9) pathway alterations were most frequently observed. Conclusions: Our study illustrated the landscape of genetic alterations in patients with BRAF V600E mutant mCRC upon BRAF inhibitor treatment, which could be critical to predict responses to BRAF inhibitors and guide personalized clinical decision-making after disease progression.

Efficacy of everolimus-containing chemotherapy in HER2 negative metastatic breast cancer patients with PI3K/AKT/mTOR mutations: A retrospective study.

e13069 Background: The PI3K/AKT/mTOR pathway is frequently altered in breast cancer. Everolimus is a selective inhibitor of mammalian target of Rapamycin (mTOR).The benefit of everolimus in metastatic breast cancer patients with PI3K/AKT/mTOR mutations remains unclear, especially in TNBC patients. Our study aimed to investigate the efficacy of the everolimus-containing chemotherapy in metastatic breast cancer patients with PI3K/AKT/mTOR mutations. Methods: 13 cases of HER2- metastatic breast cancer with PI3K/AKT/mTOR mutations treated with everolimus-containing chemotherapy were analyzed. The genetic profile in PI3K/AKT/mTOR pathway was studied. Results: Overall, 4 cases were hormone receptor (HR) positive and 9 were TNBC. 7 patients only had PIK3CA mutations, 1 patient had only PIK3R1 mutations, and 5 patients had other mutations in the pathway in addition to PIK3CA, including PTEN, PIK3CB, CTNNB1, FGFR1 and TSC2. 5 cases were treated as first or second line chemotherapy, and 8 were as third line or above. The ORR (CR+PR) was 38.5% (5/13), and DCR (CR+PR+SD) was 84.6% (11/13), including 5 cases of PR, 6 cases of SD, and 2 cases of PD. The median PFS was 5.8 months (95% CI 3.7-9.7), and the median OS was 14.3 months (95% CI 6.47-null). However, the ORR of TNBC patients was higher than that of HR-positive, which were 44.4% (4/9) and 25% (1/4), respectively ( p = 0.070). The mPFS was also longer than that of HR-positive with 5.8 months versus 1.7 months, as well as the mOS with 14.3 versus 3.8 months. We also found that the patients carrying other mutations besides PIK3CA in the PI3K/AKT/mTOR pathway had a longer PFS compared to the patients without other mutations, which was 9.7 months and 4.3 months respectively ( p = 0.272). And the DCR of the two groups were 100% and 75% respectively ( p = 0.451). Conclusions: We found that everolimus-containing chemotherapy was effective in the treatment of HER2- metastatic breast cancer with PI3K/AKT/mTOR pathway activation. TNBC patients had a trend of longer mPFS than HR+ patients. And the patients carrying other mutations besides PIK3CA in the PI3K/AKT/mTOR pathway have a trend of longer PFS and higher DCR compared to the patients without other mutations. However, trails with larger samples are needed for further verification.

Selected Articles from This Issue

Angiosarcomas (AS) are rare, aggressive tumors arising from vascular tissues, and harbor extensive genomic events such as driver gene mutations and chromosomal translocations. However, recurrent fusion products and specific genomic events that promote disease progression have thus far proven elusive. In this study, Kim and colleagues employ RNA sequencing data from human angiosarcomas and canine hemangiosarcomas—which closely mirror human AS—and identify 25 novel fusion products among the human and canine samples. Though the fusions were themselves non-recurrent, they co-occured alongside recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS, suggesting that genomic instability induced by these mutations may play a role in the genomic complexity of the disease. Further, the novel fusions were uniformly associated with increased angiogenic signaling, indicating that diverse genomic events in AS likely converge on the activation of a specific transcriptional program. Taken together, the data nominate a host of new molecular targets for investigation and clarify a unifying role for diverse genomic events in the pathogenesis of AS.Extracellular vesicles (EV) have emerged as a key mediator of intercellular signaling and have been linked to the pathogenesis of cancer. EV are loaded with different “cargo” depending on their source, and the characterization of these cargoes is important to understanding and contextualizing their role. Here, Ikeda and colleagues use proteomics to quantitatively probe the differences between EV from cancerous versus normal colon. Employing an ex vivo method for harvesting tissue-exudative EV (Te-EV) from patient samples, the authors find that the amino acid transporter CAT1 is enriched in Te-EV from colorectal cancer (CRC). CAT1-positive EV were found at higher abundance in the plasma of CRC patients and were shown to mediate CRC pathogenesis: by modulating the metabolism of arginine and nitric oxide in endothelial cells, CAT1-positive EV from CRC tissues promoted vascular cell proliferation and tubule formation. These observations identify a key mechanism underlying angiogenesis in CRC and nominate CAT1-positive EV as a potential plasma biomarker for CRC development and progression.Oncogene signaling is a potent driver of tumorigenesis and progression, but cells inherently resist oncogenic transformation via feedback loops leading to cellular senescence. Understanding how oncogene-induced senescence (OIS) is subverted by tumors is critical to the development of new methods to clinically manage cancers driven by BRAF- and RAS-activating mutations. Here, Garnett and colleagues employ a genetic screen to identify mediators of OIS in the context of BRAF activation. The authors found that Amylin, a 37-amino acid peptide that promotes glycolysis, is a key molecular determinant of the response to oncogene signaling. Amylin-ablated cells underwent a series of metabolomic and epigenetic changes that circumvented the onset of senescence in response to BRAF signaling. However, exogenous supplementation of Amylin to the cell growth medium restored the ability of cells to enter a senescent state. These data identify Amylin as a key control node governing the response to oncogene signaling in cancer cells, and suggest that Amylin treatment may hold clinical benefit for patients with oncogene-driven cancers.Acute myeloid leukemia (AML) is a genetically diverse cancer with over ten different molecular subtypes characterized by specific genomic events. EGFR pathway signaling has also been described in AML cells, but most AML cells paradoxically do not express any EGFR family members on the cell surface. In this study, Kam and colleagues address this discrepancy and identify intracellular expression of truncated ERBB2 isoforms in AML cells and myelodysplastic syndrome, an AML precursor lesion. These ERBB2 isoforms—which are also known to be pathogenic in breast cancer—mediated active signaling via classical EGFR-related pathways and were sensitive to inhibition with ERBB2 tyrosine kinase inhibitors. While ERBB2 inhibition was effective as a single agent, combination of the ERBB2 inhibitor lapatinib with the BCL-2 inhibitor venetoclax resulted in marked synergism at all tested doses. These observations nominate ERBB2 inhibitors for further investigation to complement the growing arsenal of therapeutic options for AML patients.

PI3K activation promotes resistance to eribulin in HER2-negative breast cancer.

Eribulin is a microtubule-targeting agent approved for the treatment of advanced or metastatic breast cancer (BC) previously treated with anthracycline- and taxane-based regimens. PIK3CA mutation is associated with worse response to chemotherapy in oestrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic BC. We aimed to evaluate the role of phosphoinositide 3-kinase (PI3K)/AKT pathway mutations in eribulin resistance. Resistance to eribulin was evaluated in HER2- BC cell lines and patient-derived tumour xenografts, and correlated with a mutation in the PI3K/AKT pathway. Eleven out of 23 HER2- BC xenografts treated with eribulin exhibited disease progression. No correlation with ER status was detected. Among the resistant models, 64% carried mutations in PIK3CA, PIK3R1 or AKT1, but only 17% among the sensitive xenografts (P = 0.036). We observed that eribulin treatment induced AKT phosphorylation in vitro and in patient tumours. In agreement, the addition of PI3K inhibitors reversed primary and acquired resistance to eribulin in xenograft models, regardless of the genetic alterations in PI3K/AKT pathway or ER status. Mechanistically, PI3K blockade reduced p21 levels likely enabling apoptosis, thus sensitising to eribulin treatment. PI3K pathway activation induces primary resistance or early adaptation to eribulin, supporting the combination of PI3K inhibitors and eribulin for the treatment of HER2- BC patients.

Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy. IMPLICATIONS: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

Adult-type granulosa cell tumor of the ovary: a FOXL2-centric disease.

Adult‐type granulosa cell tumors (aGCTs) account for 90% of malignant ovarian sex cord‐stromal tumors and 2–5% of all ovarian cancers. These tumors are usually diagnosed at an early stage and are treated with surgery. However, one‐third of patients relapse between 4 and 8 years after initial diagnosis, and there are currently no effective treatments other than surgery for these relapsed patients. As the majority of aGCTs (>95%) harbor a somatic mutation in FOXL2 (c.C402G; p.C134W), the aim of this study was to identify genetic mutations besides FOXL2 C402G in aGCTs that could explain the clinical diversity of this disease. Whole‐genome sequencing of 10 aGCTs and their matched normal blood was performed to identify somatic mutations. From this analysis, a custom amplicon‐based panel was designed to sequence 39 genes of interest in a validation cohort of 83 aGCTs collected internationally. KMT2D inactivating mutations were present in 10 of 93 aGCTs (10.8%), and the frequency of these mutations was similar between primary and recurrent aGCTs. Inactivating mutations, including a splice site mutation in candidate tumor suppressor WNK2 and nonsense mutations in PIK3R1 and NLRC5, were identified at a low frequency in our cohort. Missense mutations were identified in cell cycle‐related genes TP53, CDKN2D, and CDK1. From these data, we conclude that aGCTs are comparatively a homogeneous group of tumors that arise from a limited set of genetic events and are characterized by the FOXL2 C402G mutation. Secondary mutations occur in a subset of patients but do not explain the diverse clinical behavior of this disease. As the FOXL2 C402G mutation remains the main driver of this disease, progress in the development of therapeutics for aGCT would likely come from understanding the functional consequences of the FOXL2 C402G mutation.