PIK3R1 Mutations Research Articles

Mutational landscape of primary and recurrent Ewing sarcoma

IntroductionEwing sarcoma (ES) is a highly aggressive malignancy of bone and soft tissues characterized by the presence of a genetic fusion involving the EWSR1 gene. More than one-third of patients develop distant metastases, which are associated with unfavorable prognosis. Knowledge about the disease’s genetic landscape may help foster progress in using targeted therapies in the treatment of ES.Aim of the studyThe objective is to assess the mutational landscape of ES in pretreatment samples, tumor samples after neoadjuvant chemotherapy, and in metastatic/recurrent tumors in children and adultsMaterial and methodsDNA from 39 formalin-fixed paraffin-embedded tumor samples of 22 patients (17 adults, 5 children) were analyzed by targeted next generation sequencing (NGS) using the Oncomine Comprehensive Assay v3gene panel. Additional functional analyses were performed between patient subgroups.ResultsAll samples were characterized by low tumor mutation burden (< 10 mut/Mb). The most commonly mutated genes were PIK3R1 (59%) and POLE (50%). The most widely detected variants in biopsy samples were PIK3R1 T369I (50%), FGFR1 E159K, and TP53 at codon 72 (both in 27.3%). Additionally, the ATR,BRCA1, RAD50,ATM,CHEK1, and NBN genes showed a significantly higher number of mutations in ES. Mutations in PIK3R1 were significantly more frequent in adults, while mutations in the pathways responsible for cell cycle control, DNA repair, and transcriptional regulation were more frequent in children.ConclusionsBesides EWSR1 fusion, ES is characterized by numerous point mutations that are potential targets for precision medicine. There is high genomic heterogeneity that may explain differences in outcomes between patient subgroups.

Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma

SummaryBackground Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.

DIPG-51. ACVR1 MUTATIONS PROMOTE TUMOR GROWTH IN MODELS OF DIFFUSE INTRINSIC PONTINE GLIOMA

Mutations in the gene encoding activin A receptor type 1 (ACVR1) are found in approximately 25% of diffuse intrinsic pontine gliomas (DIPGs), a pediatric glioma with 2-year survival rate of less than 10%. ACVR1mutations frequently coincide with activating PIK3CA or PIK3R1 mutations, indicating a potential cooperative effect of BMP and PI3K signaling in gliomagenesis. We used genetically engineered mice with inducible knock-in of Acvr1R206H or Pik3caE545K alleles, such that cre-mediated recombination resulted in expression of the gain of function mutated genes from their endogenous promoters at physiological levels. Cre-mediated deletion in GFAP-CreER;Pik3caE545K/+;p53cKO mice (Pik3ca;p53) mediated Trp53 deletion and expression of Pik3caE545K in glial progenitors, and spontaneously induced high-grade glioma (HGG) in mice with complete penetrance. Heterozygous knock-in of the Acvr1R206H allele accelerated tumorigenesis and impaired survival in Pik3ca;p53 mice (Acvr1;Pik3ca;p53). Transcriptomic analysis of Acvr1;Pik3ca;p53 tumors compared to Pik3ca;p53 littermate controls, as in patient-derived tumors, revealed broad molecular signatures associated with cell fate commitment and chromosome maintenance. Pharmacologic inhibition of ACVR1 was sufficient to impair growth in human patient-derived DIPG cell lines. Together, our studies show that ACVR1 activation promotes tumor growth in spontaneous mouse HGG and patient-derived DIPG cells, suggesting that ACVR1 inhibition may produce a clinically significant therapeutic effect in DIPG.

PATH-22. COMPREHENSIVE ANALYSIS OF DIVERSE LOW-GRADE NEUROEPITHELIAL TUMORS WITH FGFR1 ALTERATIONS REVEALS A DISTINCT MOLECULAR SIGNATURE OF ROSETTE-FORMING GLIONEURONAL TUMOR

Abstract The FGFR1 gene encoding fibroblast growth factor receptor 1 has emerged as a frequently altered oncogene in the pathogenesis of multiple low-grade neuroepithelial tumor (LGNET) subtypes including pilocytic astrocytoma (PA), dysembryoplastic neuroepithelial tumor (DNT), rosette-forming glioneuronal tumor (RGNT), and extraventricular neurocytoma (EVN). These activating FGFR1 alterations in LGNET can include tandem duplication of the exons encoding the intracellular tyrosine kinase domain, in-frame gene fusions most often with TACC1 as the partner, or hotspot missense mutations within the tyrosine kinase domain (either p.N546 or p.K656). However, the specificity of these different FGFR1 events for the various LGNET subtypes and accompanying genetic alterations are not well defined, nor are the histopathologic features of pilocytic astrocytomas with FGFR1 alterations versus those harboring the more common BRAF mutations or fusions. Here we performed comprehensive genomic and epigenomic characterization on a diverse cohort of 30 LGNET with FGFR1 alterations. We identified that RGNT harbors a distinct epigenetic signature compared to other LGNET with FGFR1 alterations, and is uniquely characterized by FGFR1 kinase domain hotspot missense mutations in combination with either PIK3CA or PIK3R1 mutation, often with accompanying NF1 or PTPN11 mutation. In contrast, EVN harbors its own distinct epigenetic signature and is characterized by FGFR1-TACC1 fusion as the solitary pathogenic alteration. Additionally, DNT and PA are characterized by either kinase domain tandem duplication or hotspot missense mutations, occasionally with accompanying NF1 or PTPN11 mutation, but lacking the accompanying PIK3CA or PIK3R1 mutation that characterizes RGNT. The glial component of LGNET with FGFR1 alterations typically has a predominantly oligodendroglial morphology, and many of the pilocytic astrocytomas with FGFR1 alterations lack the biphasic pattern, piloid processes, and Rosenthal fibers that characterize pilocytic astrocytomas with BRAF mutation or fusion. Together, this analysis refines the classification and histopathologic spectrum of LGNET with FGFR1 alterations.

TMOD-14. CREATION OF A GENETICALLY ENGINEERED MOUSE MODEL OF ANAPLASTIC ASTROCYTOMA DRIVEN BY THE IDH1-R132H ONCOGENE

Abstract Despite the high prevalence of IDH1-R132H mutations in lower grade gliomas, the ability to study this mutation in vivo has been hampered by a lack of faithful mouse models. Therefore, we used a CRISPR/Cas9- and AAV-based strategy to create a genetically engineered mouse model (GEMM) of astrocytoma driven by IDH1-R132H that recreates the genetic landscape of human IDH1 mutant astrocytoma. IDH1 mutations in astrocytomas often co-occur with mutations in TP53, ATRX, and either PIK3R1 or PIK3CA. Using human astrocytes immortalized via expression of telomerase (which phenocopies ATRX loss) and HPV E6 and E7 oncoproteins (which phenocopy p53 and pRb loss, respectively), we found that PIK3R1 and IDH1 oncogenes cooperate to promote anchorage-independent cell growth in vitro and orthotopic brain tumor formation in vivo. These data identified a combination of clinically relevant mutations that we hypothesized could be leveraged to cause spontaneous astrocytoma formation in mice. To simultaneously engineer Idh1, Pik3ca, Tp53, and Atrx mutations in mouse brain tissue, we intracranially injected adeno-associated virus (AAV) expressing Cre recombinase and sgRNAs targeting murine Atrx and Tp53 genes into four mouse strains with the following conditional alleles: 1) LSL-Cas9; 2) LSL-Cas9; LSL-Pik3caH1047R, 3) LSL-Cas9; LSL-Idh1R132H, and 4) LSL-Cas9; LSL-Idh1R132H; LSL-Pik3caH1047R. Grade III anaplastic astrocytomas preferentially formed 9-14 months after injecting the mice carrying both the Idh1 and Pik3ca conditional alleles. These astrocytomas harbored all intended mutations, expressed astrocytoma lineage markers, and displayed elevated (R)-2-hydroxyglutarate, the oncometabolite produced by mutant Idh1. To create an additional model with shorter tumor latency, we transplanted glioma stem-like cells derived from our GEMM into recipient mice to produce Idh1 mutant astrocytoma allografts. These allografts provide a tractable platform for preclinical therapeutic studies. Taken together, our findings show that IDH1 and PI3K oncoproteins cooperate to promote gliomagenesis and unveil new genetically faithful mouse models of mutant IDH1-driven astrocytoma.

A novel PIK3R1 mutation of SHORT syndrome in a Chinese female with diffuse thyroid disease: a case report and review of literature

BackgroundSHORT syndrome is a rare genetic disease named with the acronyms of short stature, hyper-extensibility of joints, ocular depression, Rieger anomaly and teething delay. It is inherited in an autosomal dominant manner confirmed by the identification of heterozygous mutations in PIK3R1. This study hereby presents a 15-year-old female with intrauterine growth restriction, short stature, teething delay, characteristic facial gestalts who was identified a novel de novo nonsense mutation in PIK3R1.Case presentationThe proband was admitted to our department due to irregular menstrual cycle and hirsutism with short stature, who had a history of intrauterine growth restriction and presented with short stature, teething delay, characteristic facial gestalts, hirsutism, and thyroid disease. Whole-exome sequencing and Sanger sequencing revealed c.1960C > T, a novel de novo nonsense mutation, leading to the termination of protein translation (p. Gln654*).ConclusionsThis is the first case report of SHORT syndrome complicated with thyroid disease in China, identifying a novel de novo heterozygous nonsense mutation in PIK3R1 gene (p. Gln654*). The phenotypes are mildly different from other cases previously described in the literature, in which our patient presents with lipoatrophy, facial feature, and first reported thyroid disease. Thyroid disease may be a new clinical symptom of patients with SHORT syndrome.

PIK3R1 MUTATION: A RARE GENETIC CAUSE OF BRONCHIECTASIS

PIK3R1 MUTATION: A RARE GENETIC CAUSE OF BRONCHIECTASIS

Abstract B18: Single-arm, open-label, phase II study of LY3023414 for the treatment of recurrent or persistent endometrial cancer

Abstract Background: Activating mutations of the PI3K/mTOR pathway are highly prevalent in endometrial cancers (EC), but monotherapy targets of this pathway have shown modest activity and are associated with poor tolerance. LY3023414 is a selective inhibitor of class I PI3K isoforms, mTOR, and DNA-PK. Building on phase I experience of LY3023414, we sought to evaluate the efficacy and safety of LY3023414 in PI3K pathway activated advanced EC. Methods: Eligible patients (pts) had PI3K pathway activation defined as (1) loss of PTEN function including whole or partial gene deletion, frame shift mutations, or non-sense mutations (PTEN missense mutations excluded) or (2) activating mutation in PIK3CA, AKT1, PIK3R1, PIK3R2, or mTOR. Up to 4 prior lines of therapy were allowed. Each cycle was 21 days. Efficacy assessments were performed every 2 cycles and treatment-related adverse events (AEs) were assessed per CTCAE v. 4.03. The primary objective was best overall response rate by RECIST 1.1. Results: Of the 28 pts enrolled, 25 (89%) had 1-2 lines of therapy and 3 pts (11%) had 3-4 prior lines of therapy. LY3023414 was dosed at the RP2D of 200 mg orally twice daily. Three pts were excluded from efficacy assessment due to toxicity, noncompliance or early patient withdrawal. The clinical benefit rate (CBR), defined as the percentage of pts with complete response (CR) + partial response (PR) + stable disease (SD) ≥12 weeks, was 56% (14/25). Five patients achieved PR; 9 pts achieved SD at best response. Three of the 5 PRs were sustained from 44-52 weeks; 1 pt with PR is still on therapy &amp;gt; 84 weeks. The median PFS and median OS were 2.53 (CI 1.18-2.99) months and 9.24 (CI 5-NR) months, respectively. Twenty-one pts underwent further genomic sequencing; 16 pts (76%) had a PIK3CA mutation, 6 pts (29%) had a PIK3R1 and 10 pts (48%) had a PTEN mutation. There were no significant co-mutations identified and a correlation of molecular alterations with response was not identified. Across all cycles, the most common grade 3 LY3023414-related adverse events (AEs) were lymphopenia (n=10), hypokalemia (n=5), hypophosphatemia (n=4), hyponatremia (n=3), and anemia (n=3). There were 3 LY3023414-related grade 4 AEs: allergic reaction (n=1), CPK rise (n=1) and hyponatremia (n=1). There were no LY3023414-related G5 AEs. Conclusions: In PI3K pathway-altered, heavily pretreated advanced EC, LY3023414 demonstrated a manageable safety profile and promising single-agent efficacy. Enrollment is now complete and updated response data will be presented. Citation Format: Maria M. Rubinstein, David Hyman, Imogen Caird, Roisin O'Cearbhaill, Rachel Grisham, William Tew, Martee Hensley, Paul Sabbatini, Carol Aghajanian, Alexia Iasonos, Helen Won, Duygu Selcuklu, Catherine Zimel, Vicky Makker. Single-arm, open-label, phase II study of LY3023414 for the treatment of recurrent or persistent endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B18.

Prevalence of Phosphatidylinositol-3-Kinase (PI3K) Pathway Alterations and Co-alteration of Other Molecular Markers in Breast Cancer.

Background: PI3K/AKT signaling pathway is activated in breast cancer and associated with cell survival. We explored the prevalence of PI3K pathway alterations and co-expression with other markers in breast cancer subtypes.Methods: Samples of non-matched primary and metastatic breast cancer submitted to a CLIA-certified genomics laboratory were molecularly profiled to identify pathogenic or presumed pathogenic mutations in the PIK3CA-AKT1-PTEN pathway using next generation sequencing. Cases with loss of PTEN by IHC were also included. The frequency of co-alterations was examined, including DNA damage response pathways and markers of response to immuno-oncology agents.Results: Of 4,895 tumors profiled, 3,558 (72.7%) had at least one alteration in the PIK3CA-AKT1-PTEN pathway: 1,472 (30.1%) harbored a PIK3CA mutation, 174 (3.6%) an AKT1 mutation, 2,682 (54.8%) had PTEN alterations (PTEN mutation in 7.0% and/or PTEN loss by IHC in 51.4% of cases), 81 (1.7%) harbored a PIK3R1 mutation, and 4 (0.08%) a PIK3R2 mutation. Most of the cohort consisted of metastatic sites (n = 2974, 60.8%), with PIK3CA mutation frequency increased in metastatic (32.1%) compared to primary sites (26.9%), p < 0.001. Other PIK3CA mutations were identified in 388 (7.9%) specimens, classified as “off-label,” as they were not included in the FDA-approved companion test for PIK3CA mutations. Notable co-alterations included increased PD-L1 expression and high tumor mutational burden in PIK3CA-AKT1-PTEN mutated cohorts. Novel concurrent mutations were identified including CDH1 mutations.Conclusions: Findings from this cohort support further exploration of the clinical benefit of PI3K inhibitors for “off-label” PIK3CA mutations and combination strategies with potential clinical benefit for patients with breast cancer.

Comprehensive analysis of diverse low-grade neuroepithelial tumors with FGFR1 alterations reveals a distinct molecular signature of rosette-forming glioneuronal tumor

The FGFR1 gene encoding fibroblast growth factor receptor 1 has emerged as a frequently altered oncogene in the pathogenesis of multiple low-grade neuroepithelial tumor (LGNET) subtypes including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor (DNT), rosette-forming glioneuronal tumor (RGNT), and extraventricular neurocytoma (EVN). These activating FGFR1 alterations in LGNET can include tandem duplication of the exons encoding the intracellular tyrosine kinase domain, in-frame gene fusions most often with TACC1 as the partner, or hotspot missense mutations within the tyrosine kinase domain (either at p.N546 or p.K656). However, the specificity of these different FGFR1 events for the various LGNET subtypes and accompanying genetic alterations are not well defined. Here we performed comprehensive genomic and epigenomic characterization on a diverse cohort of 30 LGNET with FGFR1 alterations. We identified that RGNT harbors a distinct epigenetic signature compared to other LGNET with FGFR1 alterations, and is uniquely characterized by FGFR1 kinase domain hotspot missense mutations in combination with either PIK3CA or PIK3R1 mutation, often with accompanying NF1 or PTPN11 mutation. In contrast, EVN harbors its own distinct epigenetic signature and is characterized by FGFR1-TACC1 fusion as the solitary pathogenic alteration. Additionally, DNT and pilocytic astrocytoma are characterized by either kinase domain tandem duplication or hotspot missense mutations, occasionally with accompanying NF1 or PTPN11 mutation, but lacking the accompanying PIK3CA or PIK3R1 mutation that characterizes RGNT. The glial component of LGNET with FGFR1 alterations typically has a predominantly oligodendroglial morphology, and many of the pilocytic astrocytomas with FGFR1 alterations lack the biphasic pattern, piloid processes, and Rosenthal fibers that characterize pilocytic astrocytomas with BRAF mutation or fusion. Together, this analysis improves the classification and histopathologic stratification of LGNET with FGFR1 alterations.

Abstract 2508: Characteristics and clinical correlations of PI3K/AKT/mTOR pathway genetic variations in Chinese endometrial carcinoma

Abstract Background: Next-generation sequencing (NGS) and the resulting target-based precision therapy has shown promising efficacy in many tumors. The PI3K/AKT/mTOR pathway is the most common abnormal pathway in endometrial carcinoma. Here we report the genetic characteristics of the PI3K/AKT/mTOR pathway in Chinese endometrial cancer and explored their clinical application to support further precision therapy strategy studies. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor samples and matched peripheral blood samples of 76 Chinese endometrial adenocarcinoma patients were collected for YuansuTM NGS-panel assay (OrigiMed, Shanghai). Pathological diagnosis was confirmed by independent pathologists. Genomic alterations (GAs) included single base substitution, short and long insertions/deletion (Indel), copy number variation, gene fusion, and rearrangement. Tumor mutational burden (TMB) and microsatellite instability (MSI) were assessed. FIGO stage, age, gender, and differentiation degree were collected for clinical application analysis. Results: GAs in the PI3K/AKT/mTOR pathway were found in 93.4% (71/76) of patients and were mostly substitution/Indels (75.0%) and truncations (24.5%). The most common GAs were PTEN (72.4%), PIK3CA (51.3%), PIK3R1 (32.9%), FBXW7 (9.2%), and AKT1 (7.9%). Hotspot mutation sites were p.R130X (17.9%) of PTEN and p.H1047X (21.2%) and p.R88Q (15.4%) of PIK3CA. The median TMB was 7.55 Muts/Mb, but patients with PTEN, PIK3CA, and PIK3R1 mutations had a significantly higher TMB (PTEN: 20.9 vs 2.5 Muts/Mb, respectively, p &amp;lt; 0.001; PIK3CA: 23.2 vs 5 Muts/Mb, respectively, p = 0.003; PIK3R1: 25.5 vs 6.2 Muts/Mb, respectively, p = 0.037). MSI-high was found in 9.2% of patients. PIK3R1 mutation patients were younger compared to wild type patients (51 vs 56 years old, respectively, p = 0.024), whereas FBXW7 mutation patients were older (63 vs 54 years old, respectively, p = 0.040). The rates of PTEN mutations and truncation variations were lower in FIGO IV stage (33.3% and 41.7%, p = 0.023 and p = 0.002, respectively) and higher in highly differentiated tissues (92% and 92%, p = 0.009 and p = 0.035, respectively), indicating that these GAs were associated with low grade phenotypes. A 56-year-old female with metastatic endometrial cancer, who could not tolerate a needle biopsy and standard treatment protocols, had ctDNA testing. A PTEN deletion was found and she received mTOR inhibitor everolimus treatment and achieved a partial response which lasted for more than 12 months. Conclusions: Our study revealed gene variation characteristics of the PI3K/AKT/mTOR pathway in Chinese endometrial adenocarcinoma patients and the clinical correlations of these variations. PTEN mutations and truncation variations were associated with low grade phenotypes. This study was helpful for optimizing the usage of mTOR inhibitor everolimus. Citation Format: Fengxia Xue, Yingmei Wang, Haifang Wang, Jun Zhang, Li Meng, Honglin Guo, Junping Shi. Characteristics and clinical correlations of PI3K/AKT/mTOR pathway genetic variations in Chinese endometrial carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2508.

Abstract 675: First-in-class approved phosphoinositide-3-kinase inhibitor Alpelesib (BYL 719): Examination of disease-specific activity in human tumor 3 dimensional primary culture organoids

Abstract Phosphoinositide 3-kinases (PI3Ks) are hetero-dimeric lipid kinases that are frequently dysregulated in cancer. Class I PI3K activity is upregulated in many breast cancers via mutations in PIK3CA, PIK3R1, PTEN and AKT1 making PI3K inhibition attractive as a therapeutic target. BYL719, an oral α-specific PI3K inhibitor that selectively inhibits p110α was granted FDA approval for estrogen receptor (ER) + breast cancer following the successful completion of the SOLAR-1 trial (Andre, F. et al NEJM, 2019). To explore the clinical utility of BYL 719 in human cancer, we applied the Ex Vivo Analysis of Programmed Cell Death (EVA/PCD) in surgical specimens obtained from 388 individual patients. Methods: EVA/PCD technique uses enzymatic and mechanical disaggregation followed by density centrifugation to isolate human tumor 3-D organoids of desired size. Morphologic and metabolic measures of cell death provide lethal concentration 50% (LC50), interpolated from 5-point dose response curves and then compared by modified Z-score to provide disease-specific activity. BYL 719 correlations with other classes of drugs are performed by Pearson Moment with significance by two tailed T-test. Results: By rank order the highest BYL719 activity was found in Uterine&amp;gt;Breast&amp;gt;Colon&amp;gt;Gastric&amp;gt;Ovary&amp;gt; Pancreas. The lowest BYL719 activity was found for Melanoma&amp;lt;Heme&amp;lt;GBM&amp;lt;Neuroendocrine&amp;lt;Sarcoma&amp;lt;NSCLC. Drug correlations were significant (Bonferroni-correction) for Idelalisib, Pictilisib, BEZ 235 and Everolimus as well as Gefitinib, Lapatinib, Selumetinib and Palbociclib. Among cytotoxic agents tested BYL719 activity only correlated with CDDP and 5FU. Conclusions: Whilst BYL 719 has been FDA approved for ER (+) breast cancer, our results suggest a strong rationale for its use in uterine cancer. As uterine cancer is associated with mutations in PTEN and PI3K, the BYL719 activity identified by EVA/PCD is highly consistent with the known molecular profiles. In an analysis of 87 breast cancer specimens, by subgroup (ER, PR, HER2, BRCA, etc.) only previously untreated patients revealed significantly higher BYL 719 activity. Based upon mechanism of action, correlations with PIK3 and PTEN inhibitors are expected, but those with EGFR, HER2 and CK 4/6 inhibitors may offer insights into potential synergies currently being evaluated by EVA/PCD. Discussion: BYL719's approval marks the introduction of a new drug class into clinical therapy. With a growing number of small molecule signal transduction inhibitors, the capacity to target PIK3 offers the opportunity to forge new drug combinations. The EVA/PCD platform's capacity to examine disease-specific activity and drug synergy offers the opportunity to streamline drug development, accelerate the introduction of new classes of agents and explore novel drug combinations. Citation Format: Robert Alan Nagourney, Adam J. Nagourney, Peter Tran, Paula Bernard, Federico Francisco, Steven S. Evans. First-in-class approved phosphoinositide-3-kinase inhibitor Alpelesib (BYL 719): Examination of disease-specific activity in human tumor 3 dimensional primary culture organoids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 675.

Next-generation sequencing analysis of endometrial screening liquid-based cytology specimens: a comparative study to tissue specimens

BackgroundLiquid-based cytology (LBC) is now a widely used method for cytologic screening and cancer diagnosis. Since the cells are fixed with alcohol-based fixatives, and the specimens are stored in a liquid condition, LBC specimens are suitable for genetic analyses.MethodsHere, we established a small cancer gene panel, including 60 genes and 17 microsatellite markers for next-generation sequencing, and applied to residual LBC specimens obtained by endometrial cancer screening to compare with corresponding formalin-fixed paraffin-embedded (FFPE) tissues.ResultsA total of 49 FFPE and LBC specimens (n = 24) were analyzed, revealing characteristic mutations for endometrial cancer, including PTEN, CTNNB1, PIK3CA, and PIK3R1 mutations. Eight cases had higher scores for both tumor mutation burden (TMB) and microsatellite instability (MSI), which agree with defective mismatch repair (MMR) protein expression. Paired endometrial LBC, and biopsied and/or resected FFPE tissues from 7 cases, presented almost identical mutations, TMB, and MSI profiles in all cases.ConclusionThese findings demonstrate that our ad hoc cancer gene panel enabled the detection of therapeutically actionable gene mutations in endometrial LBC and FFPE specimens. Endometrial cancer LBC specimens offer an alternative and affordable source of molecular testing materials.

206-OR: Generation of Induced Pluripotent Stem Cells Derived from a Patient with PIK3R1 Mutation and Analysis of Defects in Insulin Action in Hepatocytes Differentiated from These Cells

Background: A protocol of differentiation form induced pluripotent stem cells (iPSCs) to hepatocytes has been previously reported, and such iPSCs-derived hepatocytes possessed morphological and biochemical characteristics similar to authentic hepatocytes. It is however unknown whether iPSCs-derived hepatocytes normally respond to metabolic regulatory hormones including insulin. Mutations in the gene for p85α subunit of PI3K (PIK3R1), an essential element of insulin action, give rise to severe insulin resistance in human. Such cases are very rare, and only ∼30 families with PIK3R1 mutations have been reported as of today. Methods and Results: We established a protocol of differentiation from iPSCs to hepatocytes that respond to insulin. In hepatocytes differentiated from iPSCs derived from healthy subject with this protocol, phosphorylation of AKT and the expression of the lipogenic genes FASN as well as SREBF1c were induced by insulin. Furthermore, the expression of the gluconeogenic gene G6PC was upregulated by a combination of cAMP and dexamethasone, which was markedly impaired by the addition of insulin. We then generated iPSCs from a severe insulin resistant diabetic patient with the PIK3R1 c.1945C&amp;gt;T mutation, a hot-spot mutation in this gene. Insulin-induced gene expression along with phosphorylation of AKT was impaired in hepatocytes differentiated from the patient-derived iPSCs. We also repaired the mutation of PIK3R1 in the patient-derived iPSCs by the CRISPR/Cas9 genome editing. Insulin responsiveness was recovered in hepatocytes differentiated from the resultant iPSCs in which the mutation was repaired. Conclusion: We established a differentiation protocol of hepatocytes in which insulin’s metabolic actions can be evaluated. The usefulness of this protocol was validated with the use of iPSCs derived from a severe insulin-resistant patient with PIK3R1 mutation. Disclosure T. Hamaguchi: None. Y. Hirota: Other Relationship; Self; Sanofi. T. Takeuchi: None. M. Koyanagi-Aoi: None. T. Aoi: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH &amp; Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Clinical efficacy and molecular effects of lenvatinib (Len) and letrozole (Let) in hormone receptor-positive (HR+) metastatic breast cancer (MBC).

1019 Background: Preclinical studies show cross talk between RET and estrogen receptor, with at least additive treatment (Tx) effect of Len, a RET inhibitor, with Let. Our previous work concluded a recommended phase 2 dose (RP2D) of Len 14mg daily and Let 2.5mg daily (Lim, ASCO 2019). We present efficacy data from dose escalation and expansion cohorts. Methods: Safety, tolerability and efficacy data of MBC patients in both dose escalation (Len dose level 1 [DL]:20mg, DL -1:16mg and DL -2:14mg) and expansion (Len 14mg) cohort of this phase Ib/II study of combination Len+Let study was analysed. Patients were treated with single-agent Len for 2 weeks, followed by Len+Let until disease progression (PD). Serial tumor biopsies at baseline, after Len alone, 4 weeks post Len+Let, and upon PD, were sequenced for 440 genes with the ACTOnco+ platform. Results: A total of 33 pts (DL1 6pts, DL-1 6pts, DL-2 + expansion 21pts) with median 5 lines of prior Tx (range 0-11) were enrolled; 87.9%, 75.8%, and 75.8% had prior endocrine therapy (ET), ET+CDK4/6 inhibitor (i), and chemotherapy (CT) respectively. Objective response rate (ORR), disease control rate (DCR) ≥6 months (m), median duration of response (DOR), and percentage progression-free (PPF) at 12m were 33.3%, 45.5%, 11.5m (range 6.3-22.4), and 27.2% respectively. Among patients who previously progressed on CDK4/6i (n = 25), ORR, DCR ≥6m, median DOR, and PPF at 12m were 24.0%, 40.0%, 13.7m (range 6.3-18.2), and 12.0% respectively. Of note, 3/25 (12%) patients had durable response to Len+Let lasting ≥12m, despite having only modest PFS on ET+CDK4/6i (3, 7, and 12 months respectively). Most frequent all-grade toxicities (tox) were HTN (n = 15, G3:15), hypothyroidism (n = 20, G3:0) and fatigue (n = 13, G3:2), with no G4/5 tox. No new toxicity signals were observed compared to dose escalation phase. Pre-treatment tumor molecular profiling showed responders to be more likely to harbor NEFH, USH2A and PTCH1 mutations, while non-responders were more likely to carry PIK3R1, APC and PALB2 mutations. Sequencing of serial biopsies showed downregulation of BRD4, PTCH1, KIT, NTRK1 and CREBBP after Len treatment. Conclusions: Len+Let showed significant anti-tumor activity with meaningful duration of response, even in pts who failed prior CT or ET+CDK4/6i. The results support further investigation in randomized studies. Tumor profiling identified mutations associated with response and insights on molecular effects of lenvatinib. Clinical trial information: NCT02562118 .

Truncation of Pik3r1 causes severe insulin resistance uncoupled from obesity and dyslipidaemia by increased energy expenditure

ObjectiveInsulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance. MethodsThe human pathogenic Pik3r1 Y657∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically. ResultsLike humans with SHORT syndrome, Pik3r1WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia. ConclusionsPik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted.

Clinical, Immunological, and Functional Characterization of Six Patients with Very High IgM Levels.

Very high IgM levels represent the hallmark of hyper IgM (HIGM) syndromes, a group of primary immunodeficiencies (PIDs) characterized by susceptibility to infections and malignancies. Other PIDs not fulfilling the diagnostic criteria for HIGM syndromes can also be characterized by high IgM levels and susceptibility to malignancies. The aim of this study is to characterize clinical phenotype, immune impairment, and pathogenic mechanism in six patients with very high IgM levels in whom classical HIGM syndromes were ruled out. The immunological analysis included extended B-cell immunophenotyping, evaluation of class switch recombination and somatic hypermutation, and next generation sequencing (NGS). Recurrent or severe infections and chronic lung changes at the diagnosis were reported in five out of six and two out of six patients, respectively. Five out of six patients showed signs of lymphoproliferation and four patients developed malignancies. Four patients showed impaired B-cell homeostasis. Class switch recombination was functional in vivo in all patients. NGS revealed, in one case, a pathogenic mutation in PIK3R1. In a second case, the ITPKB gene, implicated in B- and T-cell development, survival, and activity was identified as a potential candidate gene. Independent of the genetic basis, very high IgM levels represent a risk factor for the development of recurrent infections leading to chronic lung changes, lymphoproliferation, and high risk of malignancies.

Classification of gastric cancer by EBV status combined with molecular profiling predicts patient prognosis.

PurposeTo identify how Epstein‐Barr virus (EBV) status combined with molecular profiling predicts the prognosis of gastric cancer patients and their associated clinical actionable biomarkers.Experimental DesignA next‐generation sequencing assay targeting 295 cancer‐related genes was performed in 73 EBV‐associated gastric cancer (EBVaGC) and 75 EBV‐negative gastric cancer (EBVnGC) specimens and these results were compared with overall survival (OS).Results PIK3CA, ARID1A, SMAD4, and PIK3R1 mutated significantly more frequently in EBVaGC compared with their corresponding mutation rate in EBVnGC. As the most frequently mutated gene in EBVnGC (62.7%), TP53 also displayed a mutation rate of 15.1% in EBVaGC. PIK3R1 was revealed as a novel mutated gene (11.0%) associated almost exclusively with EBVaGC. PIK3CA, SMAD4, PIK3R1, and BCOR were revealed to be unique driver genes in EBVaGC. ARID1A displayed a significantly large proportion of inactivated variants in EBVaGC. A notable finding was that integrating the EBV status with tumor mutation burden (TMB) and large genomic instability (LGI) categorized the tumors into four distinct molecular subtypes and optimally predicted patient prognosis. The corresponding median OSs for the EBV+/TMB‐high, EBV+/TMB‐low, EBV‐/LGI‐, and EBV‐/LGI+ subtypes were 96.2, 75.3, 44.4, and 20.2 months, respectively. The different subtypes were significantly segregated according to distinct mutational profiles and pathways.ConclusionsNovel mutations in PIK3R1 and TP53 genes, driver genes such as PIK3CA, SMAD4, PIK3R1, BCOR, and ARID1A, and distinguished genomic profiles from EBVnGC were identified in EBVaGC tumors. The classification of gastric cancer by EBV, TMB, and LGI could be a good prognostic indicator, and provides distinguishing, targetable markers for treatment.

Abstract P4-09-04: Prevalence of phosphatidylinositol-3-kinase (PI3K) pathway alterations and co-alteration of other markers in breast cancer

Abstract Background: PI3K/AKT signaling pathway is often activated in breast cancer (BC), through mutations in PIK3CA or AKT1, and alterations in PTEN, thus promoting cell survival. PI3K and AKT inhibitors have been shown to have significant activity against tumor progression and to overcome resistance in BC. We aim to explore the prevalence of PI3K pathway alterations and co-expression with other markers in different BC subtypes. This approach could allow identification of novel drug combinations that have the potential to elicit synergistic growth inhibition and be further explored in the clinical trial setting. Methods: Molecular profiles of 4895 female and male BC cases submitted to Caris Life Sciences were reviewed to identify pathogenic or presumed pathogenic mutations in PIK3CA, AKT1, PTEN, PIK3R1 and PIK3R2 using 592-gene next generation sequencing (NGS; average read depth 500 ×). Cases with loss of PTEN by IHC were also included. Co-mutation frequency with PIK3CA-AKT1-PTEN alterations were examined, including homologous recombination deficiency (HRD) genes and DNA damage response (DDR) pathways, markers of response to immune-oncology (IO) agents and RAS signaling pathway. Cases were classified in BC subtypes according to the ASCO-CAP guidelines. Results: The median age was 58 (range: 17-90). Gender frequency and primary/ metastatic site frequency were similar across BC subtypes. 3558 (72.7%) cases had at least one alteration in the PIK3CA-AKT1-PTEN pathway: 1472 (30.1%) cases harbored a PIK3CA mutation, 174 (3.6%) harbored an AKT1 mutation, and 2682 (54.8%) had PTEN alterations (PTEN mutation in 344, 7.0% and/or PTEN loss by IHC in 2516, 51.4% of cases). The most common hotspot mutations in PIK3CA were in the kinase domain (H1047R in 567 cases, 38.5% of all PIK3CA alterations) and in the helical domain (E545K in 304 cases, 20.7% of all PIK3CA alterations). Uncommon activating PIK3CA mutations were seen in 8.4% of breast tumors. The single hotspot mutation, E17K, was the most common AKT1 mutation (n=164, 94.3% of all AKT1 mutations). 81 (1.7%) tumors harbored a PIK3R1 mutation, with 66 unique alterations identified (4 pathogenic, 55 presumed pathogenic), and 4 (0.08%) cases harbored a PIK3R2 mutation (G373R mutation). With respect to the different BC subtypes, PIK3CA was the most frequent alteration in HER2 positive BC (present in 96.2% of mutated cases). Within HER2 negative subtypes, PTEN was most frequently altered and PTEN mutation or PTEN loss by IHC was present in 79.3% of mutated cases. Triple negative breast cancer (TNBC) was the subtype with the lowest frequency of PIK3CA mutations (18.0% in TNBC vs. 37% in other subtypes). The frequency of selected co-mutations with PIK3CA-AKT1-PTEN alterations is illustrated in table 1. Notable co-alterations in this cohort include increased PD-L1 expression and high tumor mutational burden (TMB). There were no significant increases in the frequency of mutations in DDR pathway mutations (not shown) in the PIK3CA-AKT1-PTEN altered cohort. Conclusions: We observed a high prevalence of expected hotspot mutations in PIK3CA and AKT1 across BC subtypes. There was a significant increase in PD-L1 expression in tumor cells, and high TMB in PIK3CA-AKT1-PTEN mutated cohorts. A similar association was seen with RAS signaling pathways. Further development of drugs that affect the PIK3CA-AKT1-PTEN pathway in all BC subtypes and combination with drugs that target the immune system may be of interest. Table 1. Selected co-alterations based on statistical significancePathwayGene/ ProteinAll subtypes (%)HR+ HER2+ (%)HR- HER2+ (%)HR+HER2- (%)TNBC (%)MTWTMTWTMTWTMTWTMTWTHomologous recombinationBRCA13.02.71.00.60.00.80.8*1.8*6.16.0BRCA24.15.41.02.53.62.34.1*7.9*4.43.0PALB20.7*1.3*0.00.00.00.00.5*2.5*0.90.3Possible predictors of IO benefitPD-149.850.038.552.266.766.737.541.766.058.1PD-L1 (SP142)6.9*4.2*3.30.78.14.93.33.011.68.1(Tumor Cells)PD-L1 (SP142)29.226.650.018.825.027.312.916.742.140.4(Immune Cells)MSI0.70.60.00.01.20.80.80.40.71.2TMB-High (≥ 10 mut/Mb)22.9*18.8*36.0*17.2*29.825.921.2*16.6*23.621.3Chromatin remodelingARID1A12.4*18.6*19.423.87.118.917.121.55.89.6ARID20.70.80.01.90.00.70.80.60.60.9RAS-RAF-MEK-ERK HRAS0.7*0.1*0.00.00.00.00.20.11.40.3KRAS2.0*1.1*1.00.00.00.01.91.22.22.0NRAS0.20.00.00.00.00.00.20.00.30.0BRAF0.50.11.00.01.20.00.60.30.40.0OthersTP5360.9*53.1*66.360.885.586.738.4*25.6*84.786.4CDH110.3*6.1*9.1*2.5*4.82.215.1*9.6*4.9*2.3*NF16.2*2.1*9.5*0.8*2.73.65.8*1.8*6.7*2.8*RB15.5*2.6*2.21.33.63.03.9*1.7*7.85.0ERBB22.3*3.4*3.03.13.56.73.23.51.0*2.3*Notes: MT = at least 1 pathogenic mutation in PIK3CA, AKT1, or PTEN or PTEN loss by IHC. WT = no pathogenic mutations in PIK3CA, AKT1, and PTEN.*statistically significant difference between MT and WT Citation Format: Katia Khoury, Antoinette Tan, Andrew Elliott, Joanne Xiu, Zoran Gatalica, Arielle L Heeke, Claudine Isaacs, Paula R Pohlmann, Lee S Schwartzberg, Michael Simon, Michael W Korn, Sandra M Swain, Filipa Lynce. Prevalence of phosphatidylinositol-3-kinase (PI3K) pathway alterations and co-alteration of other markers in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-04.

S4-KL-1 UPDATE OF WHO2016 CLASSIFICATION OF ADULT DIFFUSE GLIOMAS

The World Health Organization (WHO) central nervous system (CNS) tumor classification has represented the primary source of diagnosis and grading criteria of brain tumors. Nonetheless, recent advances of studies on their molecular alterations require more rapid update of recommendations for clinical practice. To accomplish this, cIMPACT-NOW (the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy) was established in 2016 and has published four updates. For adult gliomas, update 1 clarified the use of the term NOS (Not Otherwise Specified) and proposed a new term NEC (Not Elsewhere Classified); update 2 revised clarifications regarding diffuse astrocytoma/anaplastic astrocytoma, IDH-mutant; update 3 proposed molecular criteria for an IDH-wildtype diffuse or anaplastic astrocytic glioma without histological features of glioblastoma, which would behave similarly to a grade IV glioblastoma. Nonetheless, no consensus on pathologic or molecular markers that could be incorporated into a more clinically relevant grading scheme for IDH-mutant gliomas has been reached. The molecular alterations previously studied using relatively large cohorts include CDKN2A/B homozygous deletion, CDK4 amplification, RB1 mutation/homozygous deletion, PIK3CA or PIK3R1 mutations, PDGFRA amplification, NMYC amplification, global hypomethylation, genomic instability and chromosome 14 loss. The proliferative activity, based on the mitotic count or Ki67 indices, and other morphologic features typical of a high grade that might stratify the risk better than the current criteria have also been evaluated. Despite the discordance among the results of previous studies, CDKN2A/2B homozygous deletions have been shown prognostic significance in high-grade IDH-mutant astrocytomas and microvascular proliferation stratifies IDH-mutant gliomas lacking a CDKN2A homozygous deletion, suggesting that the integration of molecular information and traditional histological findings is still essential for achieving maximum risk stratification of adult cases of IDH-mutant diffuse gliomas. The grading scheme for adult IDH-mutant as well as wild-type gliomas should therefore be revised in the next WHO update.