AKT1 Mutations Research Articles

172TiP - The K-BASKET trial: A prospective phase II biomarker-driven multiple basket trial in Korean solid cancer patients

BackgroundPrevious studies reveal that key oncogenic pathways are shared across various human cancer types, and molecularly targeted therapies show clinical efficacy, influenced by predictive biomarkers rather than by tumor histologic types. These results raised the concept of biomarker-driven therapy that enables personalized cancer treatment regardless of tumor location and origin. We hereby introduce the designs and goals of the K-BASKET trial (Korea-biomarker-driven multi-arm drug-screening, knowledge and evidence-generating targeted trial), enabling to match the right drug to right target like NCI-MATCH trial. Trial designThis is a proof-of-concept phase II multiple basket trial to test the clinical significance of matched targeted therapies with comprehensive biomarker profiling. The patients with refractory metastatic or unresectable cancer of various origins will be screened for molecular biomarkers by next-generation sequencing, immunohistochemical staining, and in situ hybridization. Those who have cancer with druggable biomarkers will be treated with matched targeted agents. Three treatment arms for the second step are the followings; 1) Patients with MET amplification or exon 14 skipping MET mutation will be assigned to the TAS-115 arm, a novel multikinase inhibitor. 2) Patients with activating PIK3CA or AKT mutations will be assigned to the TAS-117 arm, a novel selective AKT inhibitor. 3) The remaining patients will be screened for PD-L1/MSI/EBV status and those with positive PD-L1/MSI/EBV results or PolD/PolE mutations will be treated with PD-L1 monoclonal antibody. The clinical efficacy and safety of the targeted therapy, as well as the feasibility of molecular profiling and drug matching, will be tested. This study is particularly relevant because: 1) it will identify new indications and predictive biomarkers of targeted therapies in common cancer types in Asian; 2) it is one of the earliest basket trials in tumor immunotherapy and molecular profiling. We anticipate that this trial will provide valuable information on biomarker-driven therapy in clinical practices of personalized treatment. Legal entity responsible for the studyYonsei University. FundingNational R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (HA16C0018). DisclosureAll authors have declared no conflicts of interest.

625P - Clinical relevance of circulating tumour (ct)DNA genotyping for first-line cetuximab-based treatment monitoring in metastatic colorectal cancer (mCRC): A prospective multicentric study

BackgroundGenotyping of ctDNA characterizes the molecular profile and monitors tumor molecular dynamics, but the clinical applicability of next generation sequencing (NGS) DNA sequencing has not been assessed in a large prospective cohort of mCRC patients (pts). MethodsmCRC pts with RAS wt tumors treated in first line with chemotherapy (CT) + cetuximab in 16 Spanish hospitals were included. Plasma samples were collected baseline (BL) and every 2 cycles until progression. ctDNA was isolated and sequenced with NGS platform Oncomine Colon cfDNA Assay which identifies hotspot mutations (mut) in AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53 and APC. RAS ctDNA mut were also assessed by dPCR (BEAMing) at BL. Detection of any mut in plasma was considered as ctDNA+. Mut in BRAF/RAS/MAP2K1/PIK3CA were considered as anti-EGFR resistance. Results101 pts were enrolled (65.3% male; median age 64.5y; 77.6% left colon). RAS mut were detected in 11 pts BL by BEAMing. After quality assessment, 84 samples were sequenced by NGS BL and at least one mut was detected in 65 samples (77.3%; median 1, range 0-5) and 10 RAS mut were detected. Plasma samples from 47 pts were also analyzed after 4 cycles of treatment (C4) and mut were detected in 30 pts (63.8%; median 1, range 0-3). Median mutant allele fraction for paired samples was 24.9 at BL and 0.56 at C4 (p=0.0045). Treatment clinical benefit (CB: PR, CR and SD≥16weeks) was observed in 72 out of 85 pts. BL ctDNA+ or C4 ctDNA change were not associated with CB (p=0.722). Assessment of RAS BL mut was not associated with CB (p=0.243 by NGS and p=0.712 by BEAMing), while the absence of RAS mut at C4 predicted treatment CB (93.2% vs. 6.8% RAS mut p=0.02). Median PFS for all pts was 10.13 month. ctDNA+ at BL or C4 were not correlated with PFS (HR 0.8; p=0.73). No differences in PFS were found in ctDNA RAS mut vs RAS wt BL (BEAMing+ HR=1.2 p=0.79 and NGS+ HR=2.6 p=0.052). Among all anti-EGFR resistant mut, only ctDNA RAS mut were detected at C4 (N=4). Persistence or emergence of RAS mut at C4 were strongly associated with PFS (12.9m vs. 4.2. HR 8.8 p=0.0003). ConclusionsEarly RAS ctDNA dynamics predicts benefit to first line CT+cetuximab in mCRC pts. Legal entity responsible for the studyGrupo de Tratamiento de los Tumores Digestivos (TTD), Spain. FundingMerck-Serono. DisclosureJ. Vidal Barrull: Honoraria (self), Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Amgen; Honoraria (self): Sysmex-Inostics. E. Elez Fernández: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck-Serono; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Sanofi. F. Losa: Honoraria (self): Amgen; Honoraria (self): Merck-Serono. R. Salazar: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Guardant-Helth; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Ferrer; Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Novartis; Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy: MSD. J. Tabernero: Honoraria (self), Advisory / Consultancy: Array Biopharma; Honoraria (self), Advisory / Consultancy: Merck-Serono; Honoraria (self), Advisory / Consultancy: Molecular Partners; Honoraria (self), Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (institution), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: Foundation Medicine; Honoraria (self), Advisory / Consultancy: Halio DX SAS. E. Aranda Aguilar: Honoraria (self): Celgene; Honoraria (self): Merck-Serono; Honoraria (self): Roche; Honoraria (self): Sanofi. B. Bellosillo: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Biocartis; Advisory / Consultancy: Novartis; Advisory / Consultancy: Thermofisher. C. Montagut Viladot: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck-Serono; Advisory / Consultancy: Sysmex-Inostics; Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy: Biocartis; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Guardant-Helth; Advisory / Consultancy: Symphogen. All other authors have declared no conflicts of interest.

Pathological and molecular characteristics of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer characterized by the presence of many dermal tumor emboli in the papillary and reticular dermis of the skin overlying the breast. IBC patients, compared to other breast cancer patients, have more frequently metastatic axillary lymph nodes. IBC is often high grade, negative for hormone receptors and presents with amplification of the HER2 gene. Invasive IBC is frequently of ductal phenotype, even if a specific histological distinction for these lesions has not been described. The pathogenesis and evolution of IBC are strongly dependent upon tumor microenvironment, characterized by several macrophages/monocytes and lymphocytes. The tumor and microenvironment cells are well molecularly characterized, showing the main contributor of inflammatory pathways in tumor biology of IBC. In addition, several molecular alterations are described in this tumor, such as mutations of ERBB2, KRAS, BRAF, EGFR, PIK3CA, PTEN, AKT1, and AKT3 genes that could suggest a therapeutic stratification of IBC patients with the combination of different biological target therapies.

1957P - Comparative molecular analyses between microsatellite stable BRAFV600E mutant colorectal cancers and BRAFV600E mutant melanomas

BackgroundSeveral studies in BRAFV600E colorectal cancers (CRC) have failed to replicate the efficacy of BRAF inhibitors in treatment of BRAFV600E melanomas (Mel), suggesting the existence of different resistance mechanisms as well as molecular and biological differences between the 2 tumor types. Herein, we examine these differences. MethodsTumor samples submitted to Caris Life Sciences for NGS DNA sequencing (592 gene panel) and RNA sequencing (54 gene panel) between the years 2015 and 2019 were retrospectively studied. Only microsatellite stable (MSS) tumors with either BRAFV600E mutation or wild type (WT) BRAF status were included in this initial analysis. Chi-square tests determined differences. ResultsA total of 8409 tumor samples were analyzed: CRC (N=7453: BRAFV600E=373; WT=7080) and Mel samples (N=956: BRAFV600E=274; WT=682). The most frequently mutated genes in BRAFV600E CRC were TP53 (81%), APC (26%), and SMAD4 (22.4%), compared to CDKN2A (22%), PTEN (13%), and TP53 (13%) in BRAFV600E Mel. Molecular alterations that are significantly more frequent in BRAFV600E CRC vs BRAFV600E Mel but not significantly different between BRAF WT CRC vs BRAF WT Mel included RSPO3 fusions (CRC=22.6% vs Mel=0%, P=0.027); RNF43 deletions (2.0% vs 0%, p=0.019); and mutations in RNF43 (18.8% vs 0%, P<0.001), AKT1 (3.5% vs 0.4%, p=0.008), and MTOR (2.4% vs 0.4%, p=0.037). RSPO3 fusions and RNF43 mutations/deletions are mutually exclusive. Significantly more frequent alterations in BRAFV600E Mel compared to BRAFV600E CRC (but not in either WT) included deletions in PTEN (7% vs 3%, p=0.005) and mutations in PTEN (12.9% vs 5.4%), MITF (1.9% vs 0%), STK11 (1.5% vs 0%), ERCC2 (1.1% vs 0%), FANCC (1.2% vs 0%), and CDK4 (1.5% vs 0%) (p<0.05 for all). ConclusionsBRAFV600E CRC carry molecular alterations that are distinct from BRAFV600E Mel. RSPO3 fusions and RNF43 alterations are prevalent in BRAFV600E CRC tumors in a mutually exclusive manner, suggesting that Wnt pathway activation via genetic alterations in upstream Wnt pathway regulators may play an important role in BRAFV600E CRC tumorigenesis and resistance to BRAF inhibitors. Drug combinations that target both the MAPK and Wnt pathways may warrant further investigation. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureM.E. Salem: Travel / Accommodation / Expenses: Caris Life Sciences. J. Xiu: Full / Part-time employment: Caris Life Sciences. A. Grothey: Travel / Accommodation / Expenses: Caris Life Sciences. R.M. Goldberg: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. Z. Gatalica: Full / Part-time employment: Caris Life Sciences. R. Feldman: Full / Part-time employment: Caris Life Sciences. M. Saul: Full / Part-time employment: Caris Life Sciences. W..M. Korn: Full / Part-time employment: Caris Life Sciences. A.F. Shields: Research grant / Funding (institution), Travel / Accommodation / Expenses: Caris Life Sciences. J.L. Marshall: Advisory / Consultancy: Caris Life Sciences. H.J. Lenz: Travel / Accommodation / Expenses: Caris Life Sciences. All other authors have declared no conflicts of interest.

105P - Prospective testing of circulating tumour DNA in metastatic breast cancer facilitates clinical trial enrollment and precision oncology

BackgroundThe increasing availability of targeted agents for treatment of metastatic breast cancer (mBC) necessitates accurate and timely molecular characterisation of disease. As a minimally invasive test, circulating tumour DNA (ctDNA) is well positioned to overcome many of the limitations associated with traditional tumor biopsies. Here, we established a program to assess the feasibility of routine prospective ctDNA testing for the clinical management of mBC patients. MethodsDetection of somatic mutations from patient plasma was performed using a multiplexed droplet digital PCR (ddPCR) approach to identify hotspot mutations in PIK3CA, ESR1, ERBB2 and AKT1. In parallel, a subset of samples were also analysed via next generation sequencing (targeted amplicon (TA) sequencing and low-coverage whole-genome sequencing). Results were discussed at a multidisciplinary breast cancer meeting prior to therapy selection. Results234 mBC patients were enrolled on this study, with a median age at diagnosis of 54 years (28-80) and a median of 2 lines of prior therapy. The average turnaround time for ctDNA testing using ddPCR was 9 days (1-49). Using ddPCR, 80/234 (34.2%) patients had ≥1 mutation identified, with 52/234 (22.2%) patients having an alteration in PIK3CA, 35/234 (15.0%) in ESR1, 9/234 (3.8%) in AKT1 and 2/234 (0.9%) in ERBB2. TA sequencing performed in the first 159 patients, identified actionable mutations (classified using the OncoKB database) in 63 patients (39.6%) and showed that a mean variant allele fraction of>5% was significantly associated with inferior overall survival (Hazard ratio: 1.8; 95% Confidence interval: 1.1-3.1; p<0.02). Of 97/234 patients where an actionable alteration was identified, the result influenced clinical management in 41 (42.3%), including 18 who were enrolled in a clinical trial. In one patient initially diagnosed with ER+/HER2- disease, a HER2 gene amplification was identified through ctDNA analysis leading to the initiation of HER2-targeted treatment and a near complete metabolic response to treatment. ConclusionsProspective ctDNA testing of mBC patients is a practical and feasible approach to guide clinical trial enrolment and patient management. Legal entity responsible for the studyThe authors. FundingNational Health and Medical Research Council Australia. DisclosureS.Q. Wong: Travel / Accommodation / Expenses: Bio-Rad Laboratories. S. Dawson: Research grant / Funding (self): Genentech. All other authors have declared no conflicts of interest.

Sulfated polysaccharide of Sepiella maindroni ink targets Akt and overcomes resistance to the FGFR inhibitor AZD4547 in bladder cancer.

Rapid appearance of resistance to fibroblast growth factor receptor (FGFR) inhibitors hampers targeted regimens in bladder cancer. In the present study, we evaluated whether SIP-SII, a sulphated derivative of the polysaccharide in Sepiella maindroni (spineless cuttlefish) ink used in traditional Chinese medicine, could attenuate resistance to FGFR inhibition in bladder cancer cells. In vitro assays indicated that SIP-SII reduced cell viability and migration, restricted cell cycle progression, and increased apoptosis in parallel with decreased AKT phosphorylation and downregulation of CDK4, MMP2, and Bcl-2 in RT112 and JMSU1 cells. Synergistic effects on cell viability were observed when SIP-SII was combined with the small-molecule FGFR inhibitor AZD4547. Specific Akt targeting by SIP-SII was suggested by the fact that neither Akt knockdown nor the selective PI3K inhibitor BKM120 enhanced the inhibitory effects of SIP-II, while expression of a constitutively active Akt mutant rescued SIP-SII effects. Furthermore, subcutaneous transplantation of RT112 xenografts confirmed the superiority and tolerability of combined SIP-SII and AZD4547 administration over monotherapy regimens. The present study thus provides pre-clinical evidence of the ability of SIP-SII to improve FGFR-targeted therapies for bladder cancer by inhibiting Akt.

AKT1 internal tandem duplications and point mutations are the genetic hallmarks of sclerosing pneumocytoma.

Sclerosing pneumocytoma is a unique benign neoplasm of the lungs. The molecular alterations in sclerosing pneumocytoma are not well understood. In a previous whole-exome sequencing study, recurrent AKT1 point mutation was observed in about half of the cases of sclerosing pneumocytoma. However, in the remaining half, cancer-related mutations have still not been identified. In this study, we first analyzed the raw sequence data from the previous whole-exome sequencing study (PRJNA297066 cohort). Using Genomon-ITDetector, a special software for detection of internal tandem duplications, we identified recurrent internal tandem duplications in the AKT1 gene in 22 of the 44 tumor samples (50%). All the cases positive for AKT1 internal tandem duplications lacked AKT1 point mutations. Next, we performed targeted next-generation sequencing in an independent cohort of sclerosing pneumocytoma from our hospital (VGH-TPE cohort), and again identified recurrent AKT1 internal tandem duplications in 20 of the 40 (50%) tumor samples analyzed. The internal tandem duplications resulted in duplications of 7 to 16 amino acids in a narrow region of the Pleckstrin homology domain of the AKT1 protein. This region contains the interaction interface between the Pleckstrin homology and kinase domains, which is known to play a critical role in the activation of the AKT1 protein. Moreover, we found that AKT1 internal tandem duplications were mutually exclusive of other forms of AKT1 mutations, including point mutations and short indels. Taking all forms of AKT1 mutations together, we detected AKT1 mutations in almost all the sclerosing pneumocytomas in our study (PRJNA297066 cohort: 41 out of 44 cases, 93%; VGH-TPE cohort: 40 out of 40 cases, 100%). Our results suggest that AKT1 mutation is the genetic hallmark of sclerosing pneumocytoma. These results would help in better understanding of the pathogenesis of sclerosing pneumocytoma.

OS8.2 Risk stratification in chordoid meningioma depends on mutation status and epithelial differentiation

Abstract BACKGROUND Meningiomas are heterogenous tumors and their pathologic classification in the WHO Classification of Tumors of the CNS comprises 13 recognized histologic variants. Chordoid meningioma is a rare and more aggressive histologic variant of meningioma. Due to paucity of cases, the molecular profiling and patient risk stratification were not addressed in this variant. MATERIAL AND METHODS We performed an integrated clinical, NHERF1 immunohistochemical (IHC) and next generation sequencing (NGS) analysis of WHO grade II chordoid meningiomas from 30 patients (35 cases), representing the largest study to date. The NGS was performed by using a custom Agilent-generated 295-gene library containing genes with recurrent mutations in adult and pediatric, primary and metastatic brain cancer. Five of these cases cases were confirmed by performing NGS at Tempus on the xT panel containing 596 genes, and an additional 10 atypical (non-chordoid) meningioma, WHO grade II, cases were sequenced at Tempus, for comparison. RESULTS The patients with chordoid meningioma have demographic characteristics different from the general population: younger age of onset (45 vs 65), skull base (SB) location for the female patients (6:1), and increased recurrence rate (25% at 5 years). NHERF1 microlumen extent directly correlated with skull base location (p=0.0002), and segregated tumors into three cell differentiation categories -fibroblastic (13.3%), epithelial/poorly-differentiated (26.7%) and epithelial/well-differentiated (60%). NGS identified NF2 and TRAF7, as the most common mutated genes, correlating with the location and epithelial differentiation of the tumors. Mutations in AKT1, KLF4 and MN1 were found in isolated well-differentiated skull base tumors. Mutations in genes not previously involved in meningioma pathogenesis were also found. CONCLUSION The integrated tumor analysis stratified the patients into: (1) HIGH RECURRENCE RISK , characterized by tumors harboring NF2 mutations and low epithelial differentiation and (2) LOW RECURRENCE RISK , characterized by tumors exhibiting either epithelial differentiation, skull base location and TRAF7/AKT1/KLF4 mutations, or fibroblastic differentiation, non-skull base location and other mutations. This is the first study of the integrated mutational landscape of chordoid meningioma with important implications for patient risk stratification and management.

AKT1E17K Activates Focal Adhesion Kinase and Promotes Melanoma Brain Metastasis.

Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1E17K had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1E17K displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase (FAK). AKT1E17K expression in melanoma cells increased invasion and this was reduced by pharmacologic inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets. IMPLICATIONS: This study suggests that AKT1E17K promotes melanoma brain metastasis through activation of FAK and provides a rationale for the therapeutic targeting of AKT and/or FAK to reduce melanoma metastasis.

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.

The role of skeletal muscle Akt in the regulation of muscle mass and glucose homeostasis

ObjectiveSkeletal muscle insulin signaling is a major determinant of muscle growth and glucose homeostasis. Protein kinase B/Akt plays a prominent role in mediating many of the metabolic effects of insulin. Mice and humans harboring systemic loss-of-function mutations in Akt2, the most abundant Akt isoform in metabolic tissues, are glucose intolerant and insulin resistant. Since the skeletal muscle accounts for a significant amount of postprandial glucose disposal, a popular hypothesis in the diabetes field suggests that a reduction in Akt, specifically in skeletal muscle, leads to systemic glucose intolerance and insulin resistance. Despite this common belief, the specific role of skeletal muscle Akt in muscle growth and insulin sensitivity remains undefined. MethodsWe generated multiple mouse models of skeletal muscle Akt deficiency to evaluate the role of muscle Akt signaling in vivo. The effects of these genetic perturbations on muscle mass, glucose homeostasis and insulin sensitivity were assessed using both in vivo and ex vivo assays. ResultsSurprisingly, mice lacking Akt2 alone in skeletal muscle displayed normal skeletal muscle insulin signaling, glucose tolerance, and insulin sensitivity despite a dramatic reduction in phosphorylated Akt. In contrast, deletion of both Akt isoforms (M-AktDKO) prevented downstream signaling and resulted in muscle atrophy. Despite the absence of Akt signaling, in vivo and ex vivo insulin-stimulated glucose uptake were normal in M-AktDKO mice. Similar effects on insulin sensitivity were observed in mice with prolonged deletion (4 weeks) of both skeletal muscle Akt isoforms selectively in adulthood. Conversely, short term deletion (2 weeks) of skeletal muscle specific Akt in adult muscles impaired insulin tolerance paralleling the effect observed by acute pharmacological inhibition of Akt in vitro. Mechanistically, chronic ablation of Akt induced mitochondrial dysfunction and activation of AMPK, which was required for insulin-stimulated glucose uptake in the absence of Akt. ConclusionsTogether, these data indicate that chronic reduction in Akt activity alone in skeletal muscle is not sufficient to induce insulin resistance or prevent glucose uptake in all conditions. Therefore, since insulin-stimulated glucose disposal in skeletal muscle is markedly impaired in insulin-resistant states, we hypothesize that alterations in signaling molecules in addition to skeletal muscle Akt are necessary to perturb glucose tolerance and insulin sensitivity in vivo.

VE-Cadherin Is Required for Lymphatic Valve Formation and Maintenance

SUMMARYThe lymphatic vasculature requires intraluminal valves to maintain forward lymph flow. Lymphatic valves form and are constantly maintained by oscillatory fluid flow throughout life, yet the earliest steps of how lymphatic endothelial cells are able to respond to fluid shear stress remain unknown. Here, we show that the adherens junction protein VE-cadherin is required for the upregulation of valve-specific transcription factors. Conditional deletion of VE-cadherin in vivo prevented valve formation in the embryo and caused postnatal regression of nearly all lymphatic valves in multiple tissues. Since VE-cadherin is known to signal through β-catenin and the VEGFR/AKT pathway, each pathway was probed. Expression of a constitutively active β-catenin mutant or direct pharmacologic activation of AKT in vivo significantly rescued valve regression in the VE-cadherin-deficient lymphatic vessels. In conclusion, VE-cad-herin-dependent signaling is required for lymphatic valve formation and maintenance and therapies to augment downstream pathways hold potential to treat lymphedema in patients.

Vascular malformations syndromes: an update.

To provide an update of vascular malformation syndromes by reviewing the most recent articles on the topic and following the new International Society for the Study of Vascular Anomalies (ISSVA) 2018 classification. This review discusses the main features and diagnostic approaches of the vascular malformation syndromes, the new genetic findings and the new therapeutic strategies developed in recent months. Some vascular malformations can be associated with other anomalies, such as tissue overgrowth. PIK3CA-related overgrowth spectrum (PROS) is a group of rare genetic disorders with asymmetric overgrowth caused by somatic mosaic mutations in PI3K-AKT-mTOR pathway that encompass a heterogeneous group of rare disorder that are associated with the appearance of overgrowth. CLOVES syndrome and Klippel-Trénaunay syndrome are PROS disease. Proteus syndrome is an overgrowth syndrome caused by a somatic activating mutation in AKT1. CLOVES, Klippel-Trénaunay and Proteus syndromes are associated with high risk of thrombosis and pulmonary embolism. Hereditary hemorrhagic telangiectasia is an autosomic dominant disorder characterized by the presence of arteriovenous malformations. New therapeutic strategies with bevacizumab and thalidomide have been employed with promising results.

Intestine-specific activity of insulin signaling pathway in response to microgravity stress in Caenorhabditis elegans

Insulin signaling pathway is conserved and important during the control of biological processes in organisms. In this study, using the simulated microgravity system in Caenorhabditis elegans, we examined the response of insulin signaling pathway to simulated microgravity. After simulated microgravity treatment for 24-h, we observed the significant decrease in expressions of daf-2 encoding an insulin receptor and two genes (age-1 and akt-1) encoding kinase proteins, and increase in expression of daf-16 encoding a FOXO transcriptional factor. Simulated microgravity also induced an obvious translocation and accumulation of DAF-16::GFP signals in the nucleus. Mutation of daf-2, age-1, or akt-1 caused the resistance to toxicity of simulated microgravity, and daf-16 mutation caused the susceptibility to toxicity of simulated microgravity, indicating the protective response of insulin signaling to simulated microgravity. RNA interference (RNAi) knockdown of daf-16 could suppress the resistance of daf-2, age-1, or akt-1 mutant nematodes to toxicity of simulated microgravity, implying the involvement of signaling cascade of DAF-2-AGE-1-AKT-1-DAF-16 in regulating the response to simulated microgravity. Tissue-specific activity analysis demonstrated that the insulin signaling pathway only acted in the intestine to regulate the response to simulated microgravity. Moreover, in the intestine, insulin signaling functioned synergistically with p38 mitogen-activated protein kinase (MAPK) signaling to regulate the response to simulated microgravity. Therefore, our results suggest the crucial role of intestinal insulin signaling pathway in response to simulate microgravity in nematodes.

Molecular profiling and molecular classification of endometrioid ovarian carcinomas

ObjectiveEndometrioid ovarian carcinomas (EOCs) comprise 5–10% of all ovarian cancers and commonly co-occur with synchronous endometrioid endometrial cancer (EEC). We sought to examine the molecular characteristics of pure EOCs in patients without concomitant EEC. MethodsEOCs and matched normal samples were subjected to massively parallel sequencing targeting 341–468 cancer-related genes (n = 8) or whole-genome sequencing (n = 28). Mutational frequencies of EOCs were compared to those of high-grade serous ovarian cancers (HGSOCs; n = 224) and EECs (n = 186) from The Cancer Genome Atlas, and synchronous EOCs (n = 23). ResultsEOCs were heterogeneous, frequently harboring KRAS, PIK3CA, PTEN, CTNNB1, ARID1A and TP53 mutations. EOCs were distinct from HGSOCs at the mutational level, less frequently harboring TP53 but more frequently displaying KRAS, PIK3CA, PIK3R1, PTEN and CTNNB1 mutations. Compared to synchronous EOCs and pure EECs, pure EOCs less frequently harbored PTEN, PIK3R1 and ARID1A mutations. Akin to EECs, EOCs could be stratified into the four molecular subtypes: 3% POLE (ultramutated), 19% MSI (hypermutated), 17% copy-number high (serous-like) and 61% copy-number low (endometrioid). In addition to microsatellite instability, a subset of EOCs harbored potentially targetable mutations, including AKT1 and ERBB2 hotspot mutations. EOCs of MSI (hypermutated) subtype uniformly displayed a good outcome. ConclusionsEOCs are heterogeneous at the genomic level and harbor targetable genetic alterations. Despite the similarities in the repertoire of somatic mutations between pure EOCs, synchronous EOCs and EECs, the frequencies of mutations affecting known driver genes differ. Further studies are required to define the impact of the molecular subtypes on the outcome and treatment of EOC patients.

Combined Microsatellite Instability and Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST) Might Be a More Promising Immune Biomarker in Colorectal Cancer.

The form of microsatellite instability (MSI) affecting tetranucleotide repeats known as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has emerged as a new potential biomarker in multiple cancers. In colorectal cancer (CRC), the correlation between EMAST and MSI mutations remain inconclusive. We evaluated 1,505 patients with CRC using five EMAST markers (D20S82, D20S85, D8S321, D9S242, and MYCL1) and the Bethesda panel of MSI markers. Most commonly, mutations involved in CRCs were identified by MassArray Assay, and DNA repair genes were analyzed by next-generation sequencing. Clinical characteristics and prognostic relevance were correlated with EMAST and MSI. Tumors that were EMAST positive and MSI high (MSI-H) were detected in 159 (10.6%) and 154 (10.2%) of 1,505 patients with CRC. Patients were divided into four groups according to EMAST and MSI status (EMAST-positive and MSI-H, EMAST-positive and microsatellite-stable [MSS], EMAST-negative and MSI-H, and EMAST-negative and MSS). The EMAST-positive and MSI-H group was associated with female predominance, higher prevalence of proximal colon tumors, early stage tumors, poorly differentiated tumors, mucinous histology, and higher incidence of mutations in PI3KCA, BRAF, TGFBR, PTEN, and AKT1 compared with other groups. Furthermore, compared with only EMAST-positive tumors or only MSI-H tumors, tumors that were both EMAST-positive and MSI-H had a higher frequency of MLH1, MSH3, MSH6, PMS2, and EXO1 gene mutations. Finally, the presence of EMAST-positive and MSI-H tumors was a good prognostic indicator in CRC. High mutations in several DNA repair genes in EMAST-positive and MSI-H tumors suggest that this subtype of CRC might be more suitable for treatment with immune therapy. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a unique molecular subtype of colorectal cancer (CRC). The current study demonstrated that the EMAST-positive and MSI-high (MSI-H) group was associated with female predominance, higher prevalence of proximal colon tumors, early stage tumors, poorly differentiated tumors, mucinous histology, and higher incidence of mutations in PI3KCA, BRAF, TGFBR, PTEN, and AKT1 compared with other groups. Most importantly, high mutations in DNA repair genes and MSI-related genes in EMAST-positive and MSI-H tumors suggest that this subtype of CRC might be more suitable for treatment with immune therapy compared with MSI-H tumors alone.

Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation

BackgroundThe PI3K/AKT pathway is activated through PIK3CA or AKT1 mutations and PTEN loss in breast cancer. We conducted a phase II trial with an allosteric AKT inhibitor MK-2206 in patients with advanced breast cancer who had tumors with PIK3CA/AKT1 mutations and/or PTEN loss/mutation.MethodsThe primary endpoint was objective response rate (ORR). Secondary endpoints were 6-month progression-free survival (6 m PFS), predictive and pharmacodynamic markers, safety, and tolerability. Patients had pre-treatment and on-treatment biopsies as well as collection of peripheral blood mononuclear cells (PBMC) and platelet-rich plasma (PRP). Next-generation sequencing, immunohistochemistry, and reverse phase protein arrays (RPPA) were performed.ResultsTwenty-seven patients received MK-2206. Eighteen patients were enrolled into the PIK3CA/AKT1 mutation arm (cohort A): 13 had PIK3CA mutations, four had AKT1 mutations, and one had a PIK3CA mutation as well as PTEN loss. ORR and 6 m PFS were both 5.6% (1/18), with one patient with HR+ breast cancer and a PIK3CA E542K mutation experiencing a partial response (on treatment for 36 weeks). Nine patients were enrolled on the PTEN loss/mutation arm (cohort B). ORR was 0% and 6 m PFS was 11% (1/9), observed in a patient with triple-negative breast cancer and PTEN loss. The study was stopped early due to futility. The most common adverse events were fatigue (48%) and rash (44%). On pre-treatment biopsy, PIK3CA and AKT1 mutation status was concordant with archival tissue testing. However, two patients with PTEN loss based on archival testing had PTEN expression on the pre-treatment biopsy. MK-2206 treatment was associated with a significant decline in pAKT S473 and pAKT T308 and PI3K activation score in PBMC and PRPs, but not in tumor biopsies. By IHC, there was no significant decrease in median pAKT S473 or Ki-67 staining, but a drop was observed in both responders.ConclusionsMK-2206 monotherapy had limited clinical activity in advanced breast cancer patients selected for PIK3CA/AKT1 or PTEN mutations or PTEN loss. This may, in part, be due to inadequate target inhibition at tolerable doses in heavily pre-treated patients with pathway activation, as well as tumor heterogeneity and evolution in markers such as PTEN conferring challenges in patient selection.Trial registrationClinicalTrials.gov, NCT01277757. Registered 13 January 2011.

Abstract 925: PDX models of ER+ endocrine-resistant metastatic breast cancer identify Polo-like kinase 1 (PLK1) as a therapeutic target

Abstract Background: Metastatic estrogen receptor positive (ER+) breast cancers (BC) are genetically heterogeneous and a significant proportion of patients display intrinsic or acquired resistance to endocrine treatments, mTOR and CDK4/6 inhibitors. The objective of this study was to identify new therapies in clinically relevant PDX models of metastatic ER+ BC progressing on endocrine treatment and/or palbociclib. Methods: PDX models were generated by engraftment of spinal bone metastases from 120 BC patients. PDXs were molecularly characterized using SNP-arrays, targeted exome sequencing and global gene expression (GEX) analysis. The anti-tumor efficacy of the PLK1 inhibitor volasertib was evaluated in vivo compared to fulvestrant and palbociclib in two PDX models. PLK1 abundance was analyzed by immunohistochemistry in PDXs and compared with the original primary tumor and bone metastasis from each patient. PLK1 expression measured by RT-PCR was assessed in a cohort of 441 BC patients with 15-year follow-up. GEX analysis of PLK1was assessed in patients treated with neoadjuvant anastrazole. Mechanistic studies were performed in a panel of endocrine resistant BC cell lines. Results: Ten PDX have been established, 8 were ER+ and 2 triple-negative (TN). Genomic alterations included mutations in AKT1, PI3KCA, BRCA2, GATA3, NF1, and amplifications of FGFR1, CCND1 and CCNE2. Comparative pathway analysis of bone metastases derived PDX and patients' primary tumors showed enrichment for pathways associated with mitotic nuclear division, chromatid segregation and G2/M transition. PLK1 was the top commonly up-regulated gene within these pathways. Treatment of an endocrine-resistant ER+ PDX, harboring amplification of CCND1, CCNE2, FGFR1 and high expression of CDK1, showed only partial response to palbociclib and resistance to the FGFR inhibitor, AZD4547. However, PLK1 inhibitor volasertib induced rapid tumor shrinkage and complete response within 5 weeks. Importantly, volasertib was also highly effective when tested as second line therapy in palbociclib pre-treated xenografts. In-vitro inhibition of PLK1 by siRNA or volasertib inhibited tumor proliferation without affecting the expression of ER-regulated genes, suggesting an ER-independent function of PLK1 in regulating cell proliferation. Assessment of the clinical validity of PLK1 expression, revealed a strong association with poor metastases free survival (p&amp;lt;0.0001) in ER+ BC, but not in HER2+ or TNBC. Finally, in a cohort of patients treated with neoadjuvant anastrozole, on-treatment gene expression of PLK1 was significantly (p&amp;lt;0.0001) associated with poor response. Conclusion: We show that inhibition of PLK1 is a new potential treatment strategy for metastatic ER+ BC. Additional experiments are ongoing in PDX and cell lines to investigate G2/M phase dependence of ER+ BC and to identify predictive biomarkers. Citation Format: Elodie Montaudon, Joanna Nikitorowicz-Buniak, Laura Sourd, Rania El Botty, Ahmed Dahmani, Léa Huguet, Ludivine Morriset, Pierre Painsec, Fariba Nemati, Sophie Vacher, Sophie Chateau-Joubert, Sunil Pancholi, Camilla Rega, Ricardo Ribas, André Nicolas, Didier Meseure, Anne Salomon, Zakia Tariq, Keltouma Driouch, Florence Coussy, Guillaume Dutertre, Paul Cottu, Ivan Bièche, Lesley-Ann Martin, Elisabetta Marangoni. PDX models of ER+ endocrine-resistant metastatic breast cancer identify Polo-like kinase 1 (PLK1) as a therapeutic target [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 925.

Abstract 2934: AKT mutant allele-specific activation dictates pharmacologic sensitivities

Abstract The objective of this study is to explore the biochemical characteristics and therapeutic sensitivity profiles of novel, non-E17K, less frequent AKT1 mutations, and thereby expand the biomarker of sensitivity to AKT inhibition in molecularly defined cancer patients. AKT is a critical signaling node that translates phosphoinositide 3-kinase (PI3K) pathway stimulation into diverse cellular effects. Gain of function AKT1 mutations arise in diverse human cancers, of which E17K is the most common. Presence of AKT1 E17K mutation renders these tumors susceptible to AKT inhibition. Nevertheless, the long tail of potentially activating mutations in AKT is largely uncharacterized, thereby limiting our ability to act clinically in prospectively sequenced advanced cancer patients. We performed a population-scale candidate driver mutation discovery analysis in AKT1, AKT2 and AKT3 in a cohort of 41,075 retrospectively and prospectively sequenced primary and metastatic cancers, and explored both their functional and biological impact as well as their therapeutic sensitivity. Our results demonstrated that some, but not all of the identified AKT missense mutations activated PI3K signaling in a growth factor-independent manner, and sensitized tumor cells to diverse AKT inhibitors. By contrast, we discovered a different class of small in-frame paralogous AKT duplication mutants that induced distinctive structural changes, leading to a far greater degree of membrane affinity, AKT activation, pathway dependence, and hyper-sensitivity to ATP-competitive AKT inhibitors, while conferring resistance to allosteric AKT inhibitors. Leveraging a co-clinical trial framework, we are now enrolling patients on the basis of these mutations in a basket study involving AKT alterations. One such case was that of a castration-resistant metastatic prostate cancer patient who harbored AKT2 duplication mutant, and subsequently responded to AKT inhibition. Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacological sensitivities to AKT inhibition. Citation Format: Tripti Shrestha Bhattarai, Tambudzai Shamu, Swati Patel, Alexander Gorelick, Matthew T. Chang, Elena I. Gavrila, JianJong Gao, Mark T. Donoghue, Paul S. Gao, Tara Soumerai, Wassim Abida, Lilian M. Smyth, David M. Hyman, David B. Solit, Barry S. Taylor. AKT mutant allele-specific activation dictates pharmacologic sensitivities [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 2934.

Abstract 2515: Comprehensive analysis of the histologically distinct components of an adenosquamous carcinoma of the lung

Abstract Background: Adenosquamous carcinoma (ASC) is a rare subtype of non-small-cell lung cancer, consisting of an adenocarcinoma (AC) and a squamous cell carcinoma (SCC) component. Little is known about the molecular makeup of the two components in ASC. In this study, we applied a comprehensive analysis including whole exome sequencing (WES) and RNA sequencing to understand the evolutionary relationship between the AC and SCC components of an ASC of the lung. Methods:FFPE tumor tissue of three ASC patients was used in this study. Samples were stained with FastRed to visualize the nuclei. AC and SCC components were scratched separately from histological slides for DNA and RNA extraction. WES analysis was performed to assess the mutational profile and copy number alterations (CNAs). The transcriptome was investigated in each of the components using RNA sequencing. Results:WES analysis of the AC and SCC components of one patient revealed a common trunk of 118 mutations, including driver mutations EGFR exon 19 deletion and TP53 p.192*, indicating a common clonal origin of the components and the presence of bona fide drivers in the common ancestor. AC shows more private mutations than SCC (234 vs 134). Potential drivers are private to AC included TP53 and AKT1 mutations, while TLX1 and TRBV5-1 mutations are restricted to SCC. AC and SCC have highly similar CNA profiles, suggesting that all CNAs are early evolutionary events of the ASC. Mutational signature of the truncal mutations is similar to that of the mutations private to AC, while mutations private to SCC showed a distinct pattern enriched in T&amp;gt;G, suggesting that SCC may have derived from an AC ancestor cell. Transcriptomic profiling shows that genes related to oxidative phosphorylation, fatty acid metabolism and peroxisome pathways are highly expressed in AC, in contrast to genes related to epithelial-mesenchymal-transition, hedgehog signaling and IL6-JAK-STAT3-signaling pathways in SCC. Conclusion:Macroscopic dissection and separate analysis of the histologically distinct components of an ASC of the lung demonstrated the clonal relatedness of the AC and SCC. The diverse phenotype of the components is associated with distinct genetic profiles and mutational signatures. Analysis of additional ASCs of the lung may reveal genetic and/or transcriptomic underpinnings of the phenotypes. Citation Format: Arthur Krause, Maria R. De Filippo, Thomas Lorber, Spasenija Savic, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. Comprehensive analysis of the histologically distinct components of an adenosquamous carcinoma of the lung [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 2515.