PI3K Mutations Research Articles

Activated PI3K-delta syndrome in an Egyptian pediatric cohort with primary immune deficiency

BackgroundActivated Phospho-Inositide 3 (PI3) Kinases Delta syndrome (APDS) can underlie primary immune deficiency. The prevalence and phenotypic characterization of these patients are not well described in Egypt. ObjectivesTo describe patients with APDS in hospitalized children with recurrent respiratory tract infections with suspected primary immune deficiency. Methods79 patients were included in the study. E1021K and E525K mutations of PI3K δ chain gene were screened by Sanger sequencing technique. Resultsone patient was heterozygous to E1021K mutation; a female child was diagnosed clinically as Combined Immune Deficiency with CD4 and B lymphopenia and markedly deficient IgG and increased IgM. The E525K mutation was not detected in our cohort. ConclusionsScreening for APDS in patients with recurrent respiratory tract infections with undefined antibody deficiency or combined immune deficiency with or without bronchiectasis is required. These patients need great attention to benefit from the available treatment. Further studies on the Egyptian population are recommended to increase the knowledge about the prevalence and phenotypic characterization of this disease in Egypt.

Co-targeting EGFR and mTOR with gefitinib and everolimus in triple-negative breast cancer cells

Triple-negative breast cancers (TNBC) are unlikely to respond to hormonal therapies and anti-HER2-targeted therapies. TNBCs overexpress EGFR and exhibit constitutive activation of the PI3K/AKT/mTOR signalling pathway. We hypothesized that simultaneously blocking EGFR and mTOR could be a potential therapeutic strategy for the treatment of TNBC. We examined the antitumour activity of the mTOR inhibitor everolimus combined with the EGFR tyrosine kinase inhibitor gefitinib in TNBC cell with or without activating mutations in the PI3K/AKT/mTOR signalling pathway. We demonstrated that everolimus and gefitinib induced synergistic growth inhibition in the PI3K and PTEN-mutant CAL-51 cell line but not in the PTEN-null HCC-1937 cell line. The antiproliferative effect was associated with synergistic inhibition of mTOR and P70S6K phosphorylation, as well as a significant reduction in 4E-BP1 activation in the CAL-51 cell line. We also showed that combination therapy significantly inhibited cell cycle progression and increased apoptosis in this cell line. Gene and protein expression analysis revealed significant downregulation of cell cycle regulators after exposure to combined treatment. Collectively, these results suggested that dual inhibition of mTOR and EGFR may be an effective treatment for TNBC with activating mutations of PI3K.

Long-term outcomes and clinicogenomic correlates in recurrent, metastatic adenoid cystic carcinoma.

Little is known about the long-term impact of local and systemic therapies for recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC), or the clinical significance of molecular alterations. We identified 72 R/M cases among 123 ACC patients from our institution. We report long-term outcomes, predictors of recurrence and survival, and the impact of sequential cancer-directed therapy among R/M patients. We integrate genomic data for 36 sequenced ACC patients. Median overall survival (OS) from initial diagnosis was 35.1 ys (95%CI: 25.8-37.3) for R/M ACC patients. 10-y OS among R/M patients was 84.7%, worse for patients with extra-pulmonary metastatic disease (p=0.02). Only initial disease stage predicted recurrence (OR 1.69, p=0.03). Longer time to first R/M treatment predicted improved survival (p<0.01); those treated≤3years from their R/M diagnosis had poor outcomes (p=0.01). R/M patients who received systemic therapy vs. active surveillance had similar survival (p=0.35). Molecular findings predicted outcomes: 10-y OS: 100% MYB, 53.3% PI3K, 32.1% NOTCH1 and others, p=0.03. PI3K mutations predicted a longer disease-free interval (p=0.04). Underlying disease biology remains the strongest predictor of outcomes in R/M ACC. Shorter time to R/M therapy predicts poor outcomes. Molecular alterations are prognostic, and PI3K mutations identify an intermediate-risk ACC subgroup.

Abstract P3-11-01: An updated systematic review and meta-analysis of the safety and efficacy of phosphoinositide 3-kinase inhibitors (PI3Ki) in advanced breast cancer

Abstract Background In advanced breast cancer (ABC), the addition of pan-PI3Ki to the standard of care (SOC) treatment improves tumor response rate and progression free survival (PFS). Yet, further development of these drugs has been limited by their safety profile. Instead, selective PI3Ki were assessed and the Food and Drug Administration has recently approved the use of a selective PI3Ki based on a phase 3 randomized trial. Therefore, we updated our systematic review and meta-analysis to evaluate whether there is a difference in the efficacy and/or safety of pan versus selective PI3Ki in ABC. Methods The electronic databases Ovid PubMed, Cochrane Central Register of Controlled Trials and Embase, were searched for relevant publications reporting randomized controlled trials between January 2000 and June 2019. Pooled hazard ratios (HR) for progression-free survival (PFS), and pooled odds ratios (OR) for objective response rate (ORR), disease control rate (DCR) and toxicity were computed and weighted using Mantel-Haenszel method and generic inverse variance. Subgroup analyses compared patients with and without PI3K pathway activation. Results Seven studies comprising 3,597 patients were included (5 studies with pan-PI3Ki and 2 studies with selective PI3Ki). Pan and selective PI3Ki decreased the risk of progression by 21% and 32% compared to SOC: HR: 0.79, 95%CI 0.0.71-0.88 and 0.68, 95%CI 0.57-0.81 respectively, p for test for subgroup differences: 0.16. An improvement in ORR was more pronounced with the use of selective versus pan PI3Ki: OR 2.67, 95%CI 1.78-4.00 and OR 1.31, 95%CI 1.01-1.71 respectively, p for test for subgroup differences: 0.004 and DCR was only improved with selective PI3Ki: OR 1.85 95%CI 1.37-2.51. A statistically significant increase in the toxicity of any grade and of grade 3 and higher was observed with pan and selective PI3Ki: 1) for any grade toxicity: OR 5.51, 95%CI 3.05-9.96, and OR 3.77, 95%CI 2.16-6.58 respectively, p for test for subgroup differences 0.16; 2) for grade 3 or higher toxicity: OR 1.91, 95%CI 1.76-2.08 and OR 2.41, 95%CI 2.07-2.82 respectively; p for test for subgroup difference 0.01. In subgroup analyses, the PFS improvement was more pronounced in the PI3K mutant subgroup: HR 0.72, 95%CI 0.63-81 for the PI3K mutant subgroup and HR 0.83, 95%CI 0.73-0.96 for the PI3K wild-type subgroup, p for test for subgroup differences 0.11. Conclusion While selective PI3Ki have a better effect on ORR, DCR and PFS in patients with ABC compared to pan PI3Ki; their toxicity profile is still clinically substantial. Rigorous safety monitoring and early medical intervention are needed to reduce treatment discontinuation, optimize efficacy and render these drugs more clinically usable. Citation Format: Jacques Raphael, Syed Hussaini, Kathleen I Pritchard, Prakesh Shah, Phillip Blanchette, Ricardo Fernandes, Danielle Desautels. An updated systematic review and meta-analysis of the safety and efficacy of phosphoinositide 3-kinase inhibitors (PI3Ki) in advanced 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 P3-11-01.

Preclinical drug testing for clinical trial planning of novel combinative therapy with PI3K and MAPK inhibitors in colorectal cancer(CRC).

179 Background: Combinative inhibitors with multiple actions to target downstream effectors are good strategy to reduce resistance occurrence. This is a preclinical study to evaluate efficacy of Palbociclib (P; CDK4/6 inhibitor) in combination with either Gedatolisib (G; P13K/mTOR dual inhibitors) or PD0325901 (PD; MEK 1/2 inhibitor) in CRC cell models. Methods: Five CRC cell lines with hot-spots mutations in PI3K and MAPK pathways genes are used. Cell growth assay is used to evaluate antiproliferative response to P, G and PD alone and in combination. RPPA is used to clarify the role actions of combinative drugs on resistance development and new druggable proteins. Paired t-test is used. Results: All cell lines except LS1034 (IC50=7.2µM) were sensitive to G. LS411N and SNUC4 were sensitive to P whereas Caco2, DLD1 and LS1034 were resistant (IC50&gt;15µM). All cells lines were sensitive to PD. When tested in combination, P+G has synergistic response in all cells lines, except for LS411N (CI=0.9; p=0.02). P+G is highly synergistic in LS1034 with KRAS mutation (CI=0.1; p=0.11). P+G is also synergistic in DLD1 with co-occurring PIK3CA and KRAS mutations (CI=0.6; p=0.04). P+PD is synergistic in all cells lines except for LS411N. In SNUC4 with PIK3CA mutation, both drug combinations have equal synergism (CI=0.5; p&lt;0.05). RPPA analysis is in progress and will be included in final abstract. Conclusions: This offers good rationale for further clinical development of P+G as beneficial therapy in treatment-resistant CRC patients. P+G offers better alternative to P+PD since MEK inhibitor resistance had been associated with tumours harbouring KRAS with(out) PIK3CA mutations. BRAFV600E and ERBB2 mutations are useful negative predictors in this cohort. [Table: see text]

Comparison of efficacy of aspirin plus epirubicin, oxaliplatin, and capecitabine versus epirubicin, oxaliplatin, and capecitabine alone in patients with locally advanced and metastatic gastric cancer: A randomized clinical trial.

384 Background: Aspirin was long known to prevent cancer, the last decade revealed its therapeutic role via varied mechanisms like inhibition of platelet activation, COX and PI3K pathway. Since PI3K/AKT/mTOR is one of the pathways activated in gastric cancer and Giampieri et al (2016) showed improved response rates, PFS and OS with addition of aspirin to capecitabine in heavily pretreated metastatic colorectal cancer,a cancer in which efficacy of aspirin is related to presence of PI3K mutations,we aimed to compare the efficacy of aspirin added to a standard regime EOX with EOX alone in locally advanced and metastatic gastric cancer. Methods: All patients with advanced gastric cancer coming to JIPMER,Department of Medical oncology between march 2017 to may 2019 were screened for eligibility in the trial.Those eligible were randomly assigned to standard EOX or standard EOX plus 150 mg of daily aspirin.Tumor measurements were performed at baseline,then after 3-4 cycles (interim response) and the response to treatment was assessed by the radiologist who was blinded to treatment arms according to RECIST1.1 criteria.Toxicity profiles were recorded as per CTCAE v 4.03.In per protocol analysis,response rates, PFS(progression free survival) and OS (overall analysis) were analysed for patients who received ≥3 cycles and had an evaluable interim response. Results: 95 patients were randomised.In per protocol analysis, 70 patients were included. The results are shown in table. Conclusions: No statistically significant difference was seen with respect to response rates, PFS, OS and toxicity, although there was a higher ORR (overall response rate=complete response,CR +partial response, PR) and OS seen in EOX plus aspirin arm. Clinical trial information: CTRI/2017/11/010651. JIPMER,Puducherry,India [Table: see text]

International Retrospective Study of Allogeneic Hematopoietic Cell Transplantation (HCT) for Activated Phosphoinositide 3-Kinase Delta (PI3K) Syndrome

Mutations resulting in increased PI3K signaling confer increased risk of B cell lymphoma, recurrent infections, poor viral control, and autoimmunity. Allogeneic HCT is curative, but the optimal approach to HCT for these patients is still evolving as experience grows. Herein, we present the clinical outcomes of 27 patients transplanted for activating PI3K mutations. Required approvals were obtained by contributing centers. Nineteen <i>PIK3CD</i> patients and 8 <i>PIK3R1</i> patients received 22 and 10 HCTs respectively, at median age 12 years (range 2-66) at time of first HCT. Significant pre-HCT comorbidities were common, Fig 1. Conditioning platforms varied in intensity; most included serotherapy (84%). With median survivor follow up of 26.3 months from first HCT (range 2.4-71.8), overall survival was 85% (3 infectious deaths and 1 due to organ toxicity); graft failure (GF)-free survival (GFFS) was estimated at 80% at 1 year but declined to 68% by 2 years, Fig 2. Neutrophil engraftment occurred at median 15.5 days (range 11-33) with autologous recovery in 1 patient at day +14 and another deceased prior to engraftment. Four engrafted patients are alive and well despite mixed donor chimerism (<95%). Nine (33%) patients required 24 total subsequent unplanned donor cell infusions (DCI) for mixed chimerism (n=15), poor graft function (n=2), infection (n=1), GF (n=3), malignancy relapse (n=1), or to improve immune reconstitution (n=2). Grade 2-4 acute graft versus host disease (GVHD) occurred in 27% patients, with Grade 3-4 acute GVHD and limited self-resolved chronic GVHD in 1 (4%). Organ toxicities and infectious complications are summarized in Fig 3; CMV infection requiring treatment occurred in 11 (41%) patients with disease in 3 (11%). Serotherapy use, conditioning intensity, and degree of donor human leukocyte antigen (HLA) match were not significantly associated with cumulative incidence of GF or subsequent unplanned DCI (with death as a competing risk), while mTOR inhibitor use post-HCT was associated with subsequent unplanned DCI for graft augmentation or GF (p=0.006). An mTOR inhibitor was used within the year following 12 HCTs, for primary GVHD prophylaxis (n=7) or to treat disease manifestations or GVHD; loss of chimerism and/or need for subsequent DCI occurred in 9 of these. Patients with activating PI3K mutations are at overall significant risk of GF or need for subsequent DCI. While we found no association with GF or DCI for discrete elements such as serotherapy use, donor source, or conditioning intensity, small numbers preclude conclusions regarding the optimal HCT approach, and more HCTs with longer follow up are needed to refine these preliminary findings. Post-HCT mTOR inhibitor exposure was associated with need for subsequent unplanned DCI, perhaps by providing a survival advantage to host lymphocytes, and may thus be detrimental in these patients during the early post-HCT timeframe.

Translational Highlights in Breast and Ovarian Cancer 2019 - Immunotherapy, DNA Repair, PI3K Inhibition and CDK4/6 Therapy.

In the near future, important translational questions of clinical relevance will be adressed by studies currently in progress. On the one hand, the role of PD-L1 expression must be further understood, after it was found to be relevant in the use of atezolizumab in first-line therapy of patients with metastatic triple-negative breast cancer (TNBC). No association between efficacy and PD-L1 expression was found in a neoadjuvant study that included pembrolizumab in TNBC. The pathological complete response rate (pCR) was higher in both patient groups with and without PD-L1 expression when pembrolizumab was added to chemotherapy. Another future question is the identification of further patient groups in which efficacy of PARP inhibitors is seen, which are licensed for the pBRCA1/2 germline mutation. These include, for example, patients with mutations in other genes, which are involved in homologous recombination, or patients with tumours that show an abnormality in global tests of homologous recombination deficiencies (HRD tests). The question of whether a PARP inhibitor can be given and with which chemotherapy combination partners is currently being investigated in both breast and ovarian cancer. While the data on improved overall survival are being consolidated for the CDK4/6 inhibitors, knowledge of molecular changes during the therapy and during progression on the therapy is growing. Both the accumulation of PI3K mutations and also PTEN changes might play a part in planning subsequent therapies. This review article summarises these recent developments in breast cancer and in part also in ovarian cancer.

The Clinical and Functional Relevance of the Transforming AKT E17K Mutation in T-ALL

INTRODUCTION. The importance of IL-7R signaling has been thoroughly studied in the context of both normal T-cell development and leukemogenesis. Upon IL-7 induced heterodimerization of the IL-7Rα and the γc chain, downstream signaling routes regulate survival and proliferation of differentiating T-cells. JAK-STAT, MAPK-ERK and PI3K-AKT pathways are aberrantly activated in T-cell acute lymphoblastic leukemia (T-ALL) patients by recurrent gain-of-function mutations of the IL-7R signaling pathway. IL-7R signaling mutations are a result of mutations in IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS, PI3K or AKT that are found in nearly 35% of pediatric T-ALL patients and are associated with steroid resistance and inferior event-free survival. Here we focus on downstream AKT activation in relation to cellular transformation and steroid resistance. We previously demonstrated that AKTE17K transforms IL3-dependent BaF3 cells in contrast to wild type AKT, while wild type AKT drives steroid resistance in SUP-T1 T-ALL cells in contrast to mutant AKTE17K. AIM. Our aim was to identify functional differences between AKT and AKTE17K molecules in respect to cellular transformation and steroid resistance. We assessed activation level, cellular localization and downstream signaling events that may explain these functional differences. RESULTS. For this study, we used doxycycline-inducible SUP-T1 cell lines that overexpress wild type AKT or mutant AKTE17K molecules. Using immunohistochemistry, we revealed abundant cytoplasmic localization of wild type AKT but predominant plasma membrane localization of AKTE17K and extends previous findings by others. Furthermore, we found evidence for phosphorylation of wild type AKT on serine-473 (S473) and threonine-308 (T308) residues in both cytoplasmic and nuclear fractions, indicating that part of induced AKT becomes activated. In contrast, expression of AKTE17K was accompanied with abundant phosphorylation of T308 while having relatively low levels of S473 phosphorylation. The T308/S473 phosphorylation ratio was 5 times higher for AKTE17K compared to wild type AKT. These results are in line with current views on AKT activation that S473 phosphorylation facilitates the opening of the AKT pleckstrin homology domain and membrane docking, after which AKT is phosphorylated (activated) on the T308 residue by PDK1. The constitutive plasma membrane localization and open conformation of AKTE17K as a result of its E17K pleckstrin homology domain mutation leaves it independent of S473 phosphorylation and results in enhanced T308 phosphorylation by PDK1. Consequently, AKTE17K mutant cells may exhibit reduced cellular sensitivity to allosteric AKT inhibitors but not to type I ATP-competitive inhibitors. Indeed, SUP-T1 cells expressing wild type AKT or mutant AKTE17K are equally sensitivity to the ATP-competitive AZD5363 inhibitor, but AKTE17K cells display reduced sensitivity to the MK2206 allosteric inhibitor compared to AKT wild type cells. In an attempt to explain enhanced steroid resistance by expression of wild type AKT but not mutant AKTE17K, we found no differences in AKT-mediated S134 phosphorylation of the NR3C1 steroid receptor or impaired nuclear import of NR3C1 following steroid treatment (Piovan et al., Cancer Cell 2013). This indicates that AKT induces steroid resistance by other mechanisms in our SUP-T1 cell system. CONCLUSIONS. In contrast to wild type AKT overexpression, we demonstrated that AKTE17K has (i) transforming activity, (ii) does not yield cellular resistance to steroid treatment, (iii) is predominantly localized at the plasma membrane, (iv) has high T308 over S473 phosphorylation and (v) is less sensitive to the MK2206 allosteric AKT-inhibitor. These observed differences are likely caused by the constitutively open AKTE17K conformation. Since no differences in NR3C1 phosphorylation or nuclear shuttling were observed in our model, we hypothesize other mechanisms underlie the provoked steroid resistance by AKT wild type overexpression. To identify these novel mechanisms, we are currently performing an unbiased phospho-protein array to identify differentially phosphorylated AKT substrates. Disclosures No relevant conflicts of interest to declare.

TMOD-25. GLIOBLASTOMA ORGANOIDS: A MODEL SYSTEM FOR PATIENT-SPECIFIC THERAPEUTIC TESTING

Abstract Glioblastoma treatment options remain limited due to its aggressive and invasive nature. It is increasingly appreciated that molecular heterogeneity between tumors and within tumors likely contributes to the lack of therapeutic advances. To maintain the inherent heterogeneity of glioblastoma, we employed a novel method to rapidly culture glioblastoma organoids (GBOs) directly from neurosurgical resection. GBOs are routinely generated around two weeks following initial resection. Comprehensive histologic and sequencing analyses demonstrated similarity to primary tumors. Leveraging clinical molecular and sequencing data, selected GBOs were treated with radiation/temozolamide and targeted inhibitor therapies. The effect on proliferation was measured by the percentage of KI67+ cells and gene set enrichment (GSEA) analysis was performed to compare the pre-treated expression signature amongst responsive and non-responsive tumors. Treatment of organoids with radiation/temozolamide led to a decrease in the percentage of KI67+ cells in four of eight patient-derived organoid lines with some evidence of correlative radiographic response Gene sets associated with radiation response and TNF signaling were enriched in radiation/temozolamide sensitive GBOs. GBO response to EGFR inhibition via gefitinib treatment was specific to EGFR altered tumors, whose expression also enriched for EGF signaling pathway expression. Two GBOs had downstream NF1 mutations that responded to the MEK inhibitor trametinib. On GSEA, gene expression of NF1 mutated GBOs enriched for RAS signaling. One GBO line was found to have a PI3K mutation and responded dramatically to mTOR inhibition via everolimus. Dichotomous efficacy of MEK and mTOR inhibition was also noted by tumor-specific changes in GBO diameter following treatment. This novel culturing method of GBOs maintains intertumoral and intratumoral heterogeneity and allows for therapeutic testing within two weeks of neurosurgical resection. As clinical sequencing because increasingly prevalent, GBOs may become a valuable tool to functionally test mutation-specific treatment strategies in a patient-specific manner within a clinically relevant time frame.

1561P - Incidence of T790M in NSCLC patients progressed to gefitinib, erlotinib, and afatinib: A study on circulating tumour DNA

BackgroundInsights into the mechanism of resistance to first generation EGFR-TKIs may provide important information for further patient management, including the choice of second-line treatments. T790M is a gatekeeper mutation of the ATP binding pocket of the EGFR kinase domain and is the most common mechanism of resistance to first-generation EGFR-TKIs. Owing to its biologic relevance in the response of NSCLC to the selective pressure of treatments, the present study investigated in circulating cell-free DNA (cfDNA) if the occurrence of T790M at progression differed among gefitinib, afatinib, and erlotinib. MethodsThis study included patients with NSCLC bearing EGFR activating mutation, and given gefitinib (G), erlotinib (E) or afatinib (A) as first-line treatment. Plasma samples for the analysis of cfDNA were taken at disease progression (PD) and analyzed by a ddPCR using the ddPCR EGFR Mutation Assay. In selected cases, a rebiopsy was performed to confirm the absence of the T790M in negative plasma. ResultsA total of 83 patients were enrolled; 42 patients received G/E and 41 received A. Patients’ characteristics were comparable across the two groups. Median time to progression (TTP) was 14.4 in G/E vs 10.2 months in A group (p=0.09). Forty-seven out of 83 patients (56.6%) were positive for the T790M in plasma. There was a higher incidence of the T790M in patients who progressed to G/E than in patients treated with A: 33 (79%) vs 14 (34%), respectively (p=0.0001). To confirm the absence of the T790M, a rebiopsy was feasible in 7 patients of the G/E group and in 23 of the A group. The analysis of the cytological sample confirmed the absence of the T790M, and PI3K mutation was found in both groups in 1 patient (2%). Three patients (7%) had MET amplification in the A group. Afatinib dosage was reduced in 15 patients to 30mg; T790M was not correlated with the dose reduction, being detectable in 6 patients who needed the reduction and in 8 who received the full standard dose (p=0.54). ConclusionsIn conclusion, even though gefitinib, erlotinib, and afatinib belong to the same class of EGFR-TKIs, differences in the appearance of resistance mutation are demonstrated in the present study and this finding may have implications in the choice of 2nd-line treatment. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

729P - Genomic profiling in Chinese biliary tract cancer patients with PI3K/AKT/mTOR pathway and RAS gene mutations

BackgroundBiliary tract cancers (BTCs) are a group of relatively rare invasive carcinomas including gallbladder carcinoma (GBC), intrahepatic (ICC), hilar (HCCA) and extrahepatic (ECC) cholangiocarcinoma. Clinical trials of PI3K/AKT/mTOR inhibitors have revealed PI3K and PTEN mutations as effective biomarkers. This study aimed to analyze the gene mutations involved in this pathway and other gene mutations in Chinese BTC patients and to analyze the profiling characteristics of each subtype. MethodsA total of 661 (125 ECC, 159 GBC, 47 HCCA, and 330 ICC) BTC patients were included in this study. FFPE and matching blood samples from these patients were collected and sequenced using next-generation sequencing targeting 450 cancer genes. Genomic alterations including single nucleotide variants, insertions, and deletions, copy number variations and fusions were assessed. ResultsA total of 17.9% (118/661) of patients were identified harboring PI3K/AKT/mTOR pathway mutations. In preclinical data, RAS gene (KRAS/HRAS/NRAS) mutations might lead to reduced sensitivity to mTOR inhibitors. There was 11.8% (39/330) in ICC, 7.2% (9/125) in ECC, 12.8% (6/47) in HCCA and 25.2% (40/159) in GBC detected with PI3K pathway mutations and RAS wildtype, where the GBC proportion was statistically significantly higher than any other subtypes. A total of 3.6% (24/661) of patients presented co-occurrence of RAS and PI3K pathway mutations, but this was not found in HCCA. The percentages of co-occurrence in ICC, ECC and GBC were 3.6% (12/330), 4.0% (5/125) and 4.4% (7/159), respectively. The top 3 mutated genes in the PI3K/AKT/mTOR pathway were PIK3CA at 45.8% (54/118), PTEN at 22% (26/118) and TSC2 at 9.3% (11/118). PIK3CA mutations and amplification were discovered in 52 patients and 2 patients, respectively, including 23 ICC and 23 GBC patients, the most frequent being E542K (12/52) and52) mutations./52) mutations. ConclusionsIn Chinese BTC patients, the incidence (3.6%) of RAS and PI3K pathway mutation’s co-occurrence was generally low. Among BTC subtypes, GBC patients had the highest mutation proportion (25.2%) involved in the PI3K/AKT/mTOR pathway with wildtype RAS. Legal entity responsible for the studyDan Liu. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

A Patient-Derived Glioblastoma Organoid Model Maintains Intertumoral and Intratumoral Heterogeneity for Therapeutic Testing

Abstract INTRODUCTION Glioblastoma remains invariably lethal due to its aggressive and invasive nature. It has been increasingly appreciated that molecular heterogeneity between tumors and within tumors likely contributes to the lack of efficacy of numerous clinical trials. METHODS In order to maintain the inherent heterogeneity of glioblastoma, we employed a novel method to rapidly culture glioblastoma organoids (named GBOs) directly from neurosurgical resection. Cultures were generated from minced glioblastoma tissue in defined media free of serum, exogenous EGF/fibroblast growth factor (FGF), and matrigel so as to minimize selection bias. Over 30 GBO lines have been generated to date. Comprehensive histologic and sequencing analyses were performed to assess similarity to primary tumors. Leveraging clinical molecular and sequencing data, selected GBOs were treated with radiation/temozolamide, EGFR inhibition (gefitinib), MEK inhibition (trametinib), and mTOR inhibition (everolimus) for 7 d and analyzed by percent KI67+ cells following treatment. Gene set enrichment and gene ontology analysis was performed from pretreated sequencing data. RESULTS Rounded GBOs form within 2 wk and maintain high similarity to the primary tumor based upon histology as well as by sequencing analysis. Treatment with radiation/temozolamide led to a decrease in the proportion of KI67+ cells in 3 of 8 tumors with some evidence of correlative clinical radiographic response. GBO response to gefitinib treatment was specific to EGFR altered tumors and enriched for EGF related gene sets. Two GBOs had downstream NF1 mutated tumors that responded to MEK inhibition with gene set enrichment for RAS signaling. Despite resistance to other experimental treatments, 1 GBO line was found to have a PI3K mutation and responded significantly to downstream mTOR inhibition. CONCLUSION This novel culturing method of GBOs maintains both intertumoral and intratumoral heterogeneity. As clinical sequencing because increasingly prevalent, GBOs may become a valuable tool for functionally testing mutation-specific treatment strategies in a patient-specific and clinically relevant time frame.

Outcome Analysis Among Meningioma Genomic Subgroups Identifies Decreased Recurrence Free Survival in PI3K Activated Tumors

Abstract INTRODUCTION Previous studies have established relationships between meningioma molecular features and clinical outcome, including deoxyribonucleic acid (DNA)-methylation patterns, TERT promoter mutations, and various chromosomal copy number changes. These relationships stratify patients according to risk of recurrence and progression, thereby guiding difficult postoperative decisions regarding adjuvant therapies and frequency of follow-up. However, the associations of somatic driver mutations with prognosis is relatively less explored, and may yield actionable insights regarding meningioma pathogenesis and patient management. METHODS Available outcome-related data for over 450 meningiomas was collected from retrospective chart review, including extent-of-resection, postoperative therapy, radiological recurrence, long-term clinical events, and Karnofsky performance score. All samples underwent targeted and/or whole-exome sequencing followed by independent classification based on (i) driver mutation and (ii) COSMIC mutational signature. Statistical relationships were investigated between genomic and patient outcome variables using Fisher's exact tests, Kaplan-Meier curves, and Cox proportional hazards modeling. RESULTS We found that KLF4 meningiomas were more likely to illicit long-term symptoms in patients, while POLR2A tumors exhibited the highest average number of years until recurrence. Significant relationships were not identified between mutational signature and outcome. At 5 yr, we observed divergence in recurrence-free survival (RFS) between PI3K activated (AKT1 or PIK3CA mutant) and non-PI3K grade 1 meningiomas. Multivariable Cox proportional hazards analysis confirmed PI3K mutants as an independent significant predictor of RFS from grade, gender, Ki-67, and other established features. CONCLUSION Patients with KLF4-mutant meningiomas, which typically occur in the skull base and with elevated edema, may experience increased incidence of long-term symptoms. Meningiomas harboring activating PI3K mutations exhibit decreased progression free survival, suggesting they could benefit from closer radiologic monitoring or adjuvant therapies. Our results further validate the utility of genomic profiling in meningioma patients, and suggest the need for multimodal molecular integration for optimal prognostic stratification.

Abstract 2132: Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma

Abstract Introduction: Sinonasal ITAC is a rare tumor commonly related to occupational wood or leather dust exposure. Clinical management has improved,however, recurrences are frequent, and the overall 5-year survival rateremains poor (60%). Therefore, there is a clear need for new therapeutic options. The aim of this study was to identify genetic alterations as specific targets for personalized treatment. Experimental procedures: Next Generation Sequencing using SureSelect QXT (Ilumina) was performed on a panel of 120 genes against which drugs have been approved by the FDA. Tumor and germline DNA of 25 patients was sequenced, for one case also the recurrence and metastasis. Pools were sequenced in a MiSeq system with a minumum coverage of 500X. To select somatic and pathogenic variants, the raw sequencing data were manually curated as follows: all silent mutations and variants with allele frequency &amp;gt;5% were discarded, only variants with tumor frequency &amp;gt;10 % of readings were considered; by comparing with the germline sample, only variants that appeared exclusively in the tumor sample were selected, as well as homozygous changes in the tumor that were heterozygous in the germline. Results: Five tumors (20%) did not show any somatic alteration in the 120 genes tested.In the remaining 20 tumors, mutations were found in 33 genes. The most relevant were involved in the EGFR-PI3K-AKT-MTOR, the MAPK-ERK and the Wnt signaling pathways, indicating an important role in ITAC tumorigenesis. The most frequently affected genes were tyrosin-kinase receptors (44%), especially the ERBB family (20% of cases with mutations) and FGFR1 (20% of tumors with gene copy number amplifications). Thirty-six percent and 28% of tumors showed alterations in the MAPK-ERK and PI3K-AKT-MTOR cascades respectively. PI3K mutations were particulary frequent (7 different variants in 5 tumors.) With regard to the Wnt pathway, both APC and CTNBB1 genes appeared mutated in 3 cases. In one patient, the primary tumor, recurrence and lymph node metastasis were investigated. All somatic alterations observed in the primary tumor (mutations in ATM, BRC2, KMT2B and amplifications of MET, ATM and FGF1R) remained present in both recurrence and metastasis, excluding the possibility of a second primary tumor. Additional mutations in the recurrence (NTRK1, TSC2 and PIK3R2) were also seen in the metastasis, while no alterations occurred in the metastasis exclusively. Confirmatory studies are underway correlating the most relevant sequencing results with gene and protein expression data of the affected pathways. Conclusions: By sequencing 120 clinically actionable genes in ITAC, several alterations were found that could be used as targets for new, personalized therapies. Examples include Dovitinib against FGFR1 amplification, antibody and small molecule inhibitors of ERBB, BRAF and MTOR, and tankyrase inhibitors targeting the Wnt pathway. Citation Format: Cristina Riobello, Virginia N. Cabal, Paula Sánchez Fernández, Rocío García-Marín, Blanca Vivanco, Fernando López, José L Llorente, Mario Hermsen. Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2132.

Abstract 353: A novel molecule with profound tumor killing activity

Abstract LY3343544: A novel MET antibody drug conjugate that shows profound pre-clinical in vivo anti-tumor activities, irrespective of MET pathway dependence MET is over-expressed in many types of human tumors. Due to the heterogeneity of human tumors, MET antibodies or small molecule inhibitors have benefited only small subsets of patients with tumors driven by signaling through the c-Met pathway. The patient selection strategies to identify those tumors with MET activation dependence are helpful in predicting sensitivity to many of these inhibitors. It was reported previously that Lilly’s MET antibody, emibetuzumab, showed clinical activity in selective NSCLC patients with high MET IHC staining (90% to 100% 3+ positive) when it was combined with erlotinib in Phase II clinical Trials. In searching for a better treatment for patients carrying the MET overexpression tumors regardless other co-existing mutations, we developed LY3343544, a novel antibody drug conjugate (ADC) molecule that consists of emibetuzumab conjugated with the potent microtubule inhibitor MMAE using a unique lysine conjugation approach. Upon binding to MET, LY3343544 is internalized via receptor-mediated endocytosis. LY3343544 maintains the similar binding and internalization activities to the cell surface MET as compared to emibetuzumab in the competitive cell binding assay and the internalization assay. We reported here that LY3343544 showed profound anti-tumor activity in a preclinical mouse models, and overcome intrinsic resistance mechanisms including KRAS, BRAF, PI3K and TP53 mutations. LY3343544 kills tumor cells expressing a wide range of MET levels on the cell surface and is capable of killing a variety of MET-overexpressing tumor cells including pancreatic, cholanglocarcinoma, colorectal, NSCLC, gastric, head and neck tumor cells in vitro. In contrast, LY3343544 does not kill human normal endothelial cells and normal epithelial cells, no activity on human peripheral blood mononuclear cells with or without activation as well as in cell-based assays. Moreover, LY3343544 is more stable in rodent PK studies than typical inter chain Cys VC-MMAE conjugates and showed tumor regressions in colorectal, NSCLC, gastric and pancreatic mouse xenograft models. Furthermore, LY3343544 shows profound tumor regression in &amp;gt;50% of PDAC PDX models (n=40): 20% complete response (CR); 22.5% partial response (PR); and 17.5% stable disease (SD); overall disease control rate (DCR) is 60%. In addition, LY3343544 shows tumor growth inhibition in cholangiocarcinoma PDX model that is resistant to emibetuzumab. In summary, LY3343544 is highly potent in killing a variety of tumor cells in cell-based killing assays. It demonstrated good stability in vivo and profound anti-tumor efficacy in multiple mouse xenograft models and patient-derived xenograft models thus is a promising agent to treat many types of cancers. Citation Format: Ling Liu, Aaron D. Wrobleski, Yin Yin, Wei Zeng, Xianming Chen, David J. Stokell, Sheng-bin Peng, Amita Datta-Mannan, Gregory P. Donoho, Philip W. Iversen, Philip Hipskind, Yiqing Feng. A novel molecule with profound tumor killing activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 353.

PF354 HOW TELOMERE LENGTH SCREENING CAN BE HELPFUL FOR THE DIAGNOSIS OF CYTOPENIC PATIENTS?

Background: Telomere-related disorders are a clinically and genetically heterogeneous group of diseases characterized by premature telomere shortening and proliferative failure of a variety of tissues.The main organs subject to failure of function and fatty or fibrotic replacement are the bone marrow, the lung, and the liver. All 3 may be affected in an individual or a pedigree, with variable severity.Screening for telomeropathies is of particular importance in cytopenic young patients as the disorders encompass a wide spectrum and clinical presentation can therefore be subtle. Aims: To evaluate lymphocyte and granulocyte telomere length (TL) in cytopenic patients and its correlation withphenotypical and hematological features. Methods: TL in lymphocytes and granulocytes was measured using flow fluorescence in situ hybridization (flow FISH) and compared to percentiles (p) of reference ranges. All patients underwent bone marrow evaluation and for some patients genetic tests were performed (NGS or Sanger sequencing). Furthermore, we recorded for all patients somatic and hematological features (see table). Somatic abnormalities and grade of cytopenia were scored according to scores published in other genetic marrow failure syndromes (Fanconi Anemia, from Svahn et al. Am J Hematol 2016). Dyskeratosis congenita (DC) phenotype was considered as established when one of the typical symptoms/signs was present (skin,nails, leuko/erythrtoplakia, lung, liver fibrosis). Results: During the timespan from april 2015 to February 2019 we studied 36 cytopenic patients, M/F 1.25, median age 12.5 years (range 1–51 years). Based on the evaluation of telomere lenght, we divided our patients into 3 groups - Group 1: no telomere lenght abnormalities: 25 patients - Group 2: telomere lenght between 1st and 5thp in at least one of cells subsets analyzed (lymphocytes or granulocytes): 3 patients - Group 3: telomere lenght <1st p in at least one of cells subsets analyzed (lymphocytes or granulocytes): 8 patients Patients hematological and phenotypic features are summarized in table 1 Patients with phenotypic abnormalities were 20/36, including 4 with established DC features. Patients with cytopenia of central origin were 23/36, 17 with Bone Marrow Failure, 5 with Myelodysplasia and 1 with Juvenile Myelomonocytic Leukemia. In 20 patients we performed genetic tests (NGS or Sanger sequencing) and we identified in 14/20 patients a pathogenic mutation (3 Fanconi Anemia mutations, 1 Blackfan Diamond Anemia, 1 LIG4 mutation, 2 TACI mutations, 1 PI3K mutation, 1 NFKB mutation, 1 GATA mutation, 1Ohdo syndrome, 3 DC mutations −1 DKC1, 1 TERT,1 TERC-). In patients with TL <5°p (group 2 and 3) we found that established DC phenotype was more frequent (p = 0.0011).According to the data available in the literature, all patients with genetically confirmed DC had a telomere <1st centile. Furthermore, the following combinations: phenotypic abnormalities+cytopenia and cytopenia+ established DC phenotype (with or without genetic confirmation) had excellent concordance with telomere shortening (Cohen's kappa coefficient >0.81).Summary/Conclusion: Our findings suggest that in patients with cytopenia, telomere shortening is more likely to be detected in those who have established DC phenotype and/ or other somatic abnormalities. Based on this it might be advisable to test telomere length in these categories in order to detect telomere disease subject that might be otherwise missed.

The spatiotemporal evolution of early-stage non-small-cell lung cancer.

8539 Background: Lung cancer is a genetically heterogeneous disease. The genomic basis of tumorigenesis and cancer cell spread, as well as intratumor heterogeneity (ITH) and subclonal evolutionary patterns might correlate with patients’ clinical outcomes. In this prospective study, we aimed to investigate such associations through comprehensive spatiotemporal genomic profiling in early-stage non-small cell lung cancers (NSCLCs). Methods: We performed deep targeted sequencing (GeneseeqPrime, 425 genes) of 503 primary tumor regions and 141 metastatic lymph node tumors from surgery and 378 longitudinal plasma biopsies (pre- and post-operation) across 128 Stage I-III NSCLC patients. ITH and phylogenetic tree for each patient were analyzed and correlated with clinical outcomes. Longitudinal and phylogenetic ctDNA analyses were further performed. Results: Spatial ITH varied among patients and was associated with clinical phenotypes. Geographical stratification of clonal structure, with localized confinement of subclones, was linked with slower tumor progression. In contrast, early expansion of subclones to multiregions was associated with rapid tumor growth and lymph node metastases. EGFR and TP53 mutations were nearly always clonal, whereas subclonal mutations in PI3K, WNT and TGF-beta pathway that occurred later in evolution were found in more than 50% of the patients. By tracking these phylogenetic events, we identified five evolutionary subtypes with distinct clinical outcomes, including a rare subtype characterized by independent origin of multiple EGFR driver mutations. ctDNA profiling could capture the spatial ITH to a certain extent with additional unique signatures. Further longitudinal and phylogenetic ctDNA analyses indicated early detection of relapse and adjuvant chemotherapy resistance. Conclusions: ITH is a key factor associated with clinical outcomes of early-stage NSCLC patients, which show diverse evolutionary subtypes underpinning the disease progression such as lymph metastasis and relapse. ctDNA sequencing can be used to capture spatial ITH, predict recurrence and track drug resistance.

Predictors of survival in chemorefractory colorectal liver metastases treated with Y90 radioembolization.

e15044 Background: The aim of the study was to evaluate molecular signatures and timing of yttrium-90 (Y90) as prognostic factors of survival following Y90 radioembolization (RE) in patients with chemorefractory colorectal liver metastases (CRLM). Methods: In a retrospective single-institution study, patients with unresectable, chemorefractory CRLM who had undergone genomic analysis prior to Y90 RE from 2013 to 2018 were identified. Timing of Y90 (following failure of first line vs following multiple lines of chemotherapy) and oncogenic mutation status (MAPK and PI3K pathways) were evaluated as predictors of survival length following Y-90. Survival was measured from first Y90 using Kaplan-Meier estimation and predictors of outcome were identified using log-rank analysis. Results: Overall, 58 patients with chemorefractory CRLM who underwent genomic analysis prior to Y-90 were identified. Of these, 26 (44.8%) had MAPK mutations, and 2 (3.5%) had PI3K mutations. Median survival after first Y-90 RE was 12.9 months. There was significantly prolonged overall survival (OS) in patients receiving Y90 immediately following failure of the first line of chemotherapy (FOLFOX ± bevacizumab) versus patients who underwent multiple lines of chemotherapy prior to Y90 (median OS of 16.9 vs 11.4 months for others; p = 0.023). When stratifying the cohort based on the timing of Y90, patients with WT-MAPK (n = 8) had prolonged OS if they underwent Y-90 following failure of the first line of chemotherapy (median OS of 22.3 vs 12.9 mo; p = 0.019). Patients with WT-PI3K (n = 19) had prolonged OS if they underwent Y90 following failure of the first line of chemotherapy (median OS of 16.9 vs 12 mo; p = 0.037). When analyzing the entire cohort non-stratified by timing of Y90, genetic mutation was not found to be a predictor of prolonged OS. Conclusions: Y90 RE after failure of first line of chemotherapy in CRLM appears to confer prolonged survival specifically for WT-MAPK and WT-PI3K oncogenes. Conversely, the prognosis of more favorable genetic status appears to be negated if Y90 RE is performed after failure of second or third line chemotherapies.

DIPG-03. TARGETING PI3K USING THE BLOOD BRAIN BARRIER PENETRABLE INHIBITOR, GDC-0084, FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating, incurable childhood brain cancer. 80% of patients harbour a genetic lesion to Histone-H3 with a substitution of lysine 27 for a methionine (K27M) (H3F3A/H3.3 and HIST1H3B/H3.1) which results in the loss of methylation, interfering with chromatin function, activating oncogenic transcription. Unfortunately, H3 mutations are not directly targetable. RTK-RAS-PI3Kpathway alterations occur in more than half of these patients (69%), and may represent a more promising therapeutic target. However, PI3K inhibitors have been notoriously ineffective due to their inability to cross the blood-brain-barrier (BBB). Targeting downstream components of the PI3K-AKT-mTOR signalling axis is a promising paradigm for PI3K mutant patients. This project aims to overcome the limitations of targeting downstream effectors of the PI3K pathway by utilising a novel PIK3CA specific, BBB permeable inhibitor, GDC-0084, currently in clinical trials for Glioblastoma (GBM) and DIPG. METHODS: Utilising DIPG patient derived (PDX) cells harboringH3K27M mutations (n=6), and +/- PI3Kmutations, we examined the in-vitroefficacy of GDC-0084 on cell proliferation compared to Rapamycin. Phosphoproteomic profiling is currently underway investigating the role GDC-0084 has on inhibition of downstream oncogenic signalling pathways. Assessment of efficacy in a PDX in-vivo model is currently in progress. RESULTS: GDC-0084 significantly reduced the growth of all DIPG cell lines regardless of whether they harboredPI3K mutations (p=&amp;lt;0.001), and were significantly more cytotoxic than the mTORC1 inhibitor Rapamycin (p=&amp;lt;0.01). DIPG cell lines were also more sensitive to PI3K inhibition by GDC-0084 compared to GBM cell lines (n=4, p=&amp;lt;0.001) highlighting the therapeutic potential of GDC-0084 for the treatment of DIPG. Combining GDC-0084 and Vandetanib in DIPG revealed synergistic effects. CONCLUSIONS: While there are currently no therapeutics effective against the transcriptional programs elicited by H3 mutations, these studies re-ignite the potential for PI3K inhibition as a therapeutic target for DIPG.