AKT1 Mutations Research Articles

Several genotypes, one phenotype: PIK3CA/AKT1 mutation-negative hidradenoma papilliferum show genetic lesions in other components of the signalling network

About 60-70% of hidradenoma papilliferum (HP), a benign tumour of the anogenital region, were recently described to harbour mutations in major driver genes of the PI3K/AKT/MAPK-signalling pathways. However, the underlying genetic defects of the non-mutant cases are still unknown. Using a 409 gene panel, we employed targeted next generation sequencing to investigate the mutational landscape in a cohort of seven PI3K/AKT-negative cases and five cases with known hotspot mutations in either PIK3CA or AKT1. In total, we identified 29 mutations in 22 of 409 genes. The four cases with PIK3CA hotspot mutations carried no or only few additional mutations. The AKT1 hotspot mutated case harboured additional mutations in four genes (SYNE1, ADAMTS20, EP400 and CASC5). At least two of these genes are involved in or contribute to the PI3K/AKT-pathway. In the seven non-hotspot mutated cases we observed 18 mutations. Each case carried at least one mutation in a gene contributing to or involved in PI3K/AKT-signalling. Affected genes were PIK3CA (n=1, non-hotspot mutation), PIK3R1 (n=3), SYNE1, AR, IL6ST, PDGFRB, KMT2C, AR, BTK, DST, KAT6A, BRD3, RNF213, USP9X, ADGRB3, MAGI1, and IL7R (each gene mutated once). The identified PIK3CA and PIK3R1 mutations lead to constitutive activated PI3K/AKT-signalling. In conclusion, we demonstrate the genetic basis of HP in all cases. Our data suggest that tumourigenic alterations in the PI3K/AKT-pathway are indispensable in HP and establish a homogenous morphomolecular entity with a functionally converging and selecting tumourigenic mechanism.

High-throughput Exploration of the Network Dependent on AKT1 in Mouse Ovarian Granulosa Cells

The PI3K/AKT signaling pathway is known to regulate a broad range of cellular processes, and it is often altered in several types of cancers. Recently, somatic AKT1 mutations leading to a strong activation of this kinase have been reported in juvenile granulosa cell tumors. However, the molecular role of AKT1 in the supporting cell lineage of the ovary is still poorly understood. To get insights into its function in such cells, we depleted Akt1 in murine primary granulosa cells and assessed the molecular consequences at both the transcript and protein levels. We were able to corroborate the involvement of AKT1 in the regulation of metabolism, apoptosis, cell cycle, or cytoskeleton dynamics in this ovarian cell type. Consistently, we showed in established granulosa cells that depletion of Akt1 provoked altered directional persistent migration and increased its velocity. This study also allowed us to put forward new direct and indirect targets of the kinase. Indeed, a series of proteins involved in intracellular transport and mitochondrial physiology were significantly affected by Akt1 depletion. Using in silico analyses, we also propose a set of kinases and transcription factors that can mediate the action of AKT1 on the deregulated transcripts and proteins. Taken altogether, our results provide a resource of direct and indirect AKT1 targets in granulosa cells and may help understand its roles in this ovarian cell type.

Prognostic implications of a molecular classifier derived from whole-exome sequencing in nasopharyngeal carcinoma.

The aim of this study was to use whole‐exome sequencing to derive a molecular classifier for nasopharyngeal carcinoma (NPC) and evaluate its clinical performance. We performed whole‐exome sequencing on 82 primary NPC tumors from Sun Yat‐sen University Cancer Center (Guangzhou cohort) to obtain somatic single‐nucleotide variants, indels, and copy number variants. A novel molecular classifier was then developed and validated in another NPC cohort (Hong Kong cohort, n = 99). Survival analysis was estimated by the Kaplan‐Meier method and compared using the log‐rank test. Cox proportional hazards model was adopted for univariate and multivariate analyses. We identified three prominent NPC genetic subtypes: RAS/PI3K/AKT (based on RAS, AKT1, and PIK3CA mutations), cell‐cycle (based on CDKN2A/CDKN2B deletions, and CDKN1B and CCND1 amplifications), and unclassified (based on dominant mutations in epigenetic regulators, such as KMT2C/2D, or the Notch signaling pathway, such as NOTCH1/2). These subtypes differed in survival analysis, with good, intermediate, and poor progression‐free survival in the unclassified, cell‐cycle, and RAS/PI3K/AKT subgroups, respectively, among the Guangzhou, Hong Kong, and combined cohorts (n = 82, P = 0.0342; n = 99, P = 0.0372; and n = 181, P = 0.0023; log‐rank test). We have uncovered genetic subtypes of NPC with distinct mutations and/or copy number changes, reflecting discrete paths of NPC tumorigenesis and providing a roadmap for developing new prognostic biomarkers and targeted therapies.

All-Exon TP53 Sequencing and Protein Phenotype Analysis Accurately Predict Clinical Outcome after Surgical Treatment of Head and Neck Squamous Cell Carcinoma.

This study elucidates the clinical impact of surgical treatment of head and neck squamous cell carcinoma (HNSCC) based on a detailed search of all exons of the TP53 gene and p53 protein phenotypic analysis using formalin-fixed paraffin-embedded (FFPE) specimens. Clinically well-annotated FFPE specimens from 317 patients with HNSCC treated by surgery were examined by all-exon TP53 sequencing using a next-generation sequencer and p53 protein phenotype by immunohistochemistry. After excluding human papillomavirus-associated oropharyngeal carcinomas, two risk categories were classified as "p53 adverse function" and "p53 favorable function" based on TP53 mutation status and p53 protein phenotype. Mutation in PIK3CA, AKT, and HRAS was also evaluated by target sequence. Cox proportional hazards regression models were used for statistical analysis of clinical outcomes. Receiver operating characteristic curve analysis was used to determine the optimal surgical margin cutoff for local recurrence. Local control rates were compared between the risk groups using Fisher's exact test. Multivariate analysis identified "p53 adverse function" as an independent poor predictor of overall survival, local control, and distant metastasis-free survival. In oral cavity cancer, the optimal surgical margin cutoff associated with local recurrence was 6mm. In patients with surgical margin > 6mm, the "p53 adverse function" group demonstrated significantly higher local recurrence rate than the "p53 favorable function" group. PIK3CA, AKT, or HRAS mutation did not correlate with improved overall survival. All-exon TP53 sequencing and p53 protein phenotype analysis using FFPE specimens can accurately predict clinical outcomes.

Clinicopathological and molecular differences in colorectal cancer according to location

The incidence, pathogenesis, molecular pathways, and outcomes of colorectal cancer vary depending on the location of the tumor. This study aimed to compare the difference in tumor characteristics and the outcome between right-sided colon cancer and left-sided colorectal cancer (LCRC). A total of 1503 patients with colorectal cancer who underwent surgery at the Taipei Veterans General Hospital between 2000 and 2010 were enrolled in this study. Right-sided colon cancer was defined as cancers in the cecum, ascending colon, and transverse colon, while LCRC was defined as cancers in the splenic flexure colon, descending colon, sigmoid colon, and rectum. The endpoint was overall survival. The mutations were detected via polymerase chain reaction and MASS array. The prognostic value was determined using the log-rank test and the Cox regression analysis. A total of 407 and 1096 cases were classified as right-sided colon cancer and LCRC, respectively. Compared to patients with LCRC, those with right-sided colon cancer had more mucinous type cancer (7.4% vs. 3.5%), poorly differentiated tumor (11.5% vs. 3.6%), and advanced tumor-node-metastasis stage. The risk for peritoneal tumor seeding was higher in the right-sided colon cancer group (12.8% vs. 5.7%). Overall survival was better in LCRC than in right-sided colon cancer ( P=0.036). In our study, right-sided colon cancer had a more advanced tumor stage, a higher risk of peritoneal metastasis, and a poorer outcome than LCRC. Moreover, right-sided colon cancer had more gene mutations in BRAF, KRAS, SMAD4, TGF-β, PIK3CA, PTEN, AKT1, and high microsatellite instability.

Abstract OT1-03-02: CICLADES: Monitoring of ESR1, PIK3CA and AKT1 ctDNA mutations during real-life follow-up of patients with advanced breast cancer treated with endocrine therapy

Abstract Activating ESR1 mutations have recently been reported as a key mechanism leading to Aromatase Inhibitor (AI) resistance. ESR1 mutations occur rarely in primary breast cancers. However, in large retrospective studies, ESR1 mutations occurred in up to 39% of Estrogen-Receptor(ER)-positive metastatic breast cancer resistant to AI. Numerous hotspot mutations have been identified, most of them affecting the ligand-binding domain (LBD) and leading to ligand-independent activation of the ER and to resistance to AI. Phosphatidylinositol 3-kinase (PI3K)/AKT pathway is involved in key Cellular Mechanisms and mutations in PIK3CA and AKT1 are frequently reported in breast cancer. In this study, we propose to use a capture-based Next Generation Sequencing (NGS) assay and to use the barcoding and polishing features in our analysis pipeline. This assay will be able to detect all mutations on AKT1, PIK3CA, ESR1 and other genes on circulating tumor DNA (ctDNA) extracted from blood samples of patients with breast cancer. We consider that this exon-screening strategy is relevant according to the recent knowledge. We plan to prospectively include women with advanced breast cancer about to begin standard-of-care first line endocrine therapy (ET). Patients will be required to have histologically confirmed ER-positive, HER2-negative breast cancer and documented loco-regionally advanced or metastatic disease, not amenable to surgery or radiation with curative intent. Patients with endocrine sensitive disease (no prior ET or relapse more than 12 months after completing adjuvant ET) as well as patients with endocrine resistant disease (relapse while on adjuvant ET or within 12 months of completing adjuvant ET) will be enrolled. ET can be prescribed alone or in combination with a targeted therapy. Nevertheless, we will recruit at least 25% of patients with exclusive ET in the endocrine sensitive group. Peripheral-blood samples, for analysis of ctDNA, will be obtained from participating patients at pre-specified time points: at start of ET to determine the baseline mutational status of ESR1, PIK3CA, AKT1 and other genes included in a panel of genes of interest in solid tumors, and then, at evaluation of response to therapy until disease progression or end of study. Patients will be followed for 36 months or until disease progression. Determination of progression will be done per local investigator. The primary objective is to describe the prevalence of activating ESR1 mutations affecting the LBD, using NGS, from the start of ET to progression or end of study. Secondary objectives include to describe the prevalence of ESR1 mutations affecting other domains, the prevalence of ESR1 mutations in patients with and without endocrine resistance at enrolment and the prevalence of PIK3CA and AKT1 mutations, to demonstrate that ESR1, PIK3CA and AKT1 mutations whatever their times of onset are predictors of progression free survival. As of June 2018, 8 sites were opened to recruitment and 18 pts were included; the target enrollment is 146. The trial is supported by AstraZeneca. Citation Format: Massard V, Uwer L, Salleron J, Deblock M, Kieffer A, Rios M, Gilson P, Lesur A, Harle A, Merlin JL. CICLADES: Monitoring of ESR1, PIK3CA and AKT1 ctDNA mutations during real-life follow-up of patients with advanced breast cancer treated with endocrine therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-03-02.

Abstract P5-11-08: Genetic landscape of prior and resistance to CDK4/6 inhibition in targeted NGS panel analysis of palbociclib or abemaciclib and endocrine versus placebo and endocrine

Abstract Background: CDK4/6 inhibition combined with endocrine therapy is now a standard of care for advanced estrogen receptor (ER) positive breast cancer. Predictive genetic landscape and mechanisms of resistance to CDK4/6 inhibitors have not been described clearly because of limited evidence from clinical samples. We investigated the genetic landscape and mechanisms of resistance to CDK4/6 inhibitors (palbociclib or abemaciclib). Methods: Using driver mutation targeted sequencing, we conducted longitudinal ctDNA analysis in 39 pairs of baseline of primary tumor and end of treatment or monitoring plasma samples. DNA were extracted and the genomic profiles were analyzed based on NGS. Results: In our cohort, the most frequence mutations were PIK3CA (38.5%), TP53(17.9%), CCND1, ZNF703, FGFR1, MAP2K4, RB1, ARID1A, ATRX, DNMT3A. In five paitents harboring RB1 mutations prior to CDK4/6 inhibitor, only one case occured progressing early with 4mos of PFS, and the others were 5.5+, 6.5+, 13.5+, 21+months, it represented different from pre-clinical results that RB1 mutation was associated with resistance to CDK4/6 inhibitor. Interesting, patients with PIK3CA/PTEN/AKT1 mutations (n=15) were likely sensitivity to CDK4/6 inhibitor than those (n=11) with wild type (median PFS: 17 mos vs. 9.5mos, P=0.046). In another cohort of everolimus, new driver mutations emerged in both FGFR1 and ERBB3 at end of everolimus and significantly disappeared in AKT1 mutation. Three patients used palbociclib after progress disease of everolimus, the prognosis were poor with PFS of 1, 6, 9 mos. Evolution of driver gene mutations was uncommon in patients progressing on CDK4/6 inhibitors treatment, but common in patients progressing on everolimus treatment. Conclusions: De novo PIK3CA/PTEN/AKT1 mutations were likely better prognosis biomarkers with the treatment of CDK4/6 inhibitor. RB1 mutation are common and may not contribute to CDK4/6 inhibitor resistance differ from PALOMA3 ctDNA analysis. Our small sample finding indicate it may be a bad stragegy for CDK4/6 inhibitor after progress disease with mTOR inhibitor everolimus. Citation Format: Xu J, Sun T, Wang X. Genetic landscape of prior and resistance to CDK4/6 inhibition in targeted NGS panel analysis of palbociclib or abemaciclib and endocrine versus placebo and endocrine [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-11-08.

Abstract P3-06-04: Clonal evolution of non-malignant proliferative lesions into breast cancers

Abstract [Introduction] Non-malignant proliferative lesions in the breast have been implicated in the development of invasive breast cancer. Previous studies showed that adjacent atypical proliferative lesions and breast cancers shared common genetic alterations, suggesting that these evolved from the same ancestral cell. However, the clonal structure of atypical proliferative lesions and their clonal dynamics during progression to cancer are poorly understood. In this study, we compared genetic profiles (with and without pathogenic germline mutations) of normal mammary ducts, non-malignant proliferative lesions, and cancer tissues from the same patients to illustrate the clonal evolution of cancer from a non-malignant epithelial cell. [Methods] Multiple samples were collected from different proliferative lesions within the cancer-borne breast, including invasive cancers, using micro-dissection from formalin-fixed, paraffin-embedded surgical specimens. Somatic mutations and copy number alterations (CNAs) were then evaluated by whole exome sequencing. [Results] We analyzed a total of 34 samples from 5 premenopausal females carrying estrogen receptor-positive cancers, where the samples were obtained from normal ducts (N = 6), non-atypical (N = 1) and atypical (N = 8) proliferative lesions, and non-invasive (N = 16) and invasive (N = 3) cancers. The number of somatic mutations per sample ranged from 1 to 276 and increased with disease progression, regardless of the germline mutation status. Two cases with bilateral cancers had a pathogenic germline mutation of either BRCA2 or TP53, where no somatic mutations or CNAs were shared by individual proliferative lesions, suggesting multifocal independent cancerous evolutions. By contrast, in the remaining three unilateral cases, no pathogenic germline mutations were detected, but all proliferative lesions, which were separated by a distance of 7-25 mm, shared one or more driver alterations, such as an AKT1 mutation (UID: KU01), concurrent 1q gain and 16q loss (der(1;16)) (UID: KU02), and a GATA3 mutation and der(1;16) (UID: KU03), while harboring private mutations and/or CNAs of their own. The phylogenetic analysis based on the number of shared mutations predicted an early origin of these founder mutations, which frequently predated decades before the onset of cancer. [Conclusions] Our results suggest that early breast cancer development is shaped by the evolution of multiple precancerous clones. These clones are originated from a common ancestor that acquired a founder mutation long before the onset of cancer, followed by branching evolution of multiple clones that acquired additional driver mutations of their own, from which an invasive cancer ultimately develops. In hereditary cases, this process is thought to be substantially promoted multi-focally from within the entire breasts by a germline mutation shared by all mammary cells, frequently resulting in bilateral and/or multifocal breast cancers. Our findings provide unique insight into the early development of breast cancer. Citation Format: Nishimura T, Yoshida K, Kawata Y, Takeuchi Y, Kakiuchi N, Shiozawa Y, Aoki K, Hirata M, Kataoka TR, Sakurai T, Baba S, Shiraishi Y, Chiba K, Takeuchi K, Haga H, Miyano S, Toi M, Ogawa S. Clonal evolution of non-malignant proliferative lesions into breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-04.

Abstract TS2-2: Clinical development of inhibitors of the PI3K pathway

Abstract The activation of the PI3K-AKT-mTOR pathway as a mechanism of resistance to different breast cancer treatments has been well described for years at the preclinical level. This observation, together with the high frequency of alterations of this pathway in patients with breast tumors (especially in the luminal subtype such as PI3KCA mutations, loss of PTEN function or AKT mutations) makes this pathway a highly desirable in inhibiting the growth of these tumor types. The first clinical evidence that inhibition of the PI3K-AKT-mTOR pathway leads to increased progression-free survival in metastatic breast cancer came from the BOLERO-2 trial with the mTORC1-targeting agent everolimus, the only PI3K-AKT-mTOR pathway inhibitor approved to date in breast cancer. Potentially better drugs have subsequently been developed and tested. PI3K inhibitors were designed to potentiate the inhibition of the pathway to optimize treatment efficacy and improve the toxicity profile. The first to be developed were the pan-PI3K inhibitors buparlisib and pictilisib. Results from pivotal clinical studies combining fulvestrant demonstrated increased efficacy but at the cost of increased toxicity that was considered clinically unacceptable. None of them consequently eceived approval for breast cancer treatment. It was hoped that Isoform-specific PI3K inhibitors (alpha or beta-sparing) would improve the toxicity profile and increase efficacy, especially in patients with molecular alterations in the PI3K-AKT-mTOR pathway. We are now witnessing interesting times for these drugs since we have seen the results of the two phase III studies with specific pathway inhibitors, taselisib and alpelisib. During this session we will carefully review the results of both studies in terms of efficacy and toxicity profile as well as those from other phase I-II clinical trials with these and other agents. Representing the main challenges in the future, we still have to determine the best strategy to select those patients who will likely derive greater benefit from these therapies as well as establish the context in which they are most effective, taking into account the mechanisms of resistance that PI3K inhibitors can overcome or those that emerge during treatment with these drugs. Citation Format: Saura Manich C. Clinical development of inhibitors of the PI3K pathway [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr TS2-2.

Abstract P5-11-01: Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD)

Abstract Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER positive breast cancer. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. Based on preclinical and clinical data, SERDs are expected be effective in patients harboring ESR1 mutations. Biomarker analysis was performed on plasma and tumor samples from the Phase I study of GDC-0927 in metastatic breast cancer (Dickler et al, SABCS 2017) with the goal of evaluating activity in both ESR1 mutant and wildtype tumors, and to assess ER pathway modulation. Methods: Hotspot mutations in ESR1, PIK3CA, and AKT1 were analyzed in baseline, on-treatment and end of treatment plasma derived circulating tumor DNA (ctDNA) using the BEAMing assay in patients treated at multiple dose levels of GDC-0927. A subset of samples was analyzed with Foundation Medicine's next generation sequencing ctDNA assay (FACT), which covers genomic alterations in 62 commonly altered genes. Paired pre- and on-treatment biopsies were collected to assess ER pathway modulation. ER, PR, and Ki67 protein levels were analyzed by immunohistochemistry. Gene expression analysis was performed using Illumina's RNA Access library preparation kit followed by paired-end (2x50b, 50M reads) sequencing on the HiSeq. Results: Baseline and on-treatment plasma samples were available for 40 patients. ESR1 and PIK3CA mutations were observed in 52% and 33% of patient baseline samples, respectively (BEAMing method). Mutant allele frequencies (MAF) generally declined in the first on-treatment samples collected for both ESR1 (16 out of 21 samples) and PIK3CA (7 out of 12 samples). The majority of the reductions were greater than 95% relative to baseline. Increases in ESR1 MAFs were observed in later time-points and were not associated with any particular ESR1 mutation. There were six instances for which an ESR1 mutation was detected in an on-treatment sample that was not detected in the baseline sample, three at L536P and one each at D538G, L536H, and S463P, and four out of six with MAFs close to the limit of detection. The FACT assay also detected alterations in CDH1, NF1, PTEN, and TP53 in baseline samples. The relationship between MAF changes and clinical benefit to GDC-0927 will be presented. A predefined, experimentally-derived set of ER target genes were evaluated in pre- and on-treatment tumor biopsy pairs from six patients. Four of the six patients showed evidence of suppression in ER pathway activity, one patient treated at the 1000 mg dose level and three at the 1400 mg dose. The degree of pathway suppression was associated with pre-treatment pathway levels and decreases of ER and Ki67 protein levels. Conclusions: We report here evidence of consistent reduction of ESR1 and PIK3CA ctDNA in patients treated with GDC-0927. ER pathway suppression was observed at both the transcript and protein level confirming pharmacodynamic activity of the SERD. Citation Format: Spoerke JM, Daemen A, Chang C-W, Giltnane J, Metcalfe C, Dickler MN, Bardia A, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez Martin A, Cortes J, Martin M, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Liu L, Li R, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Lackner MR. Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-11-01.

Abstract P2-08-19: Exploratory biomarker analyses of FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) + paclitaxel (PAC) for early triple-negative breast cancer (TNBC)

Abstract Background: The oral AKT inhibitor IPAT is being evaluated in cancers with a high prevalence of PI3K/AKT pathway activation. In the PBO-controlled randomized phase II FAIRLANE trial (NCT02301988), adding IPAT to PAC as neoadjuvant therapy for TNBC led to a numerical increase in pathologic complete response (pCR) in unselected patients (17.1% vs 13.3%), with a greater treatment effect in patients with PIK3CA/AKT1/PTEN-altered tumors (17.9% vs 11.8%). The addition of IPAT also led to an increase in complete response (CR) by MRI (27.6% vs 13.3%) that was enhanced in patients with PIK3CA/AKT1/PTEN-altered tumors (39.3% vs 8.8%) [Oliveira, AACR 2018]. We report an exploratory analysis performed to provide better understanding of potential biomarkers for response. Methods: Pretreatment tumor samples were evaluated for genomic alterations using the FoundationOne® (Foundation Medicine) assay (n=144) and gene expression by RNA-Seq (n=92). Samples were classified into TNBC subtypes based on the method developed by Lehmann and Pietenpol [Lehmann, J Clin Invest 2011]. Tumor-infiltrating lymphocytes (TILs) were quantified using the Salgado method [Salgado, Ann Oncol 2015] (n=135). Results: Of 62 patients (43%) with PIK3CA/AKT1/PTEN-altered tumors, 21 had an activating mutation in PIK3CA or AKT1 and 47 had an alteration in PTEN (6 [3 in each arm] had both PIK3CA mutation and PTEN alteration). Although only 3 patients with PIK3CA/AKT1-mutant tumors achieved a pCR, there was an increased rate of MRI CR with the addition of IPAT to PAC [Table]. In patients with PTEN alterations, both pCR rate and MRI CR rate were increased with IPAT. In patients treated with PBO + PAC, all 4 pCR patients evaluable by RNA-Seq were of the immunomodulatory (IM) subtype. However, in the IPAT + PAC arm, pCRs were also seen in patients with basal-like 1 (BL-1), mesenchymal (M), and mesenchymal stem-like (MSL) subtypes. Consistent with this observation, in the PBO + PAC arm, samples from patients achieving a pCR had significantly higher levels of stromal TILs than those from patients who did not have a pCR, while no difference was observed in the IPAT + PAC arm. Response, n (%)PIK3CA/AKT mutation (n=21)PTEN alteration (n=47) IPAT + PAC (n=11)PBO + PAC (n=10)IPAT + PAC (n=21)PBO + PAC (n=26)pCR1 (9%)2 (20%)4 (19%)3 (12%)CR by MRI5 (45%)1 (10%)8 (38%)2 (8%) Conclusions: This retrospective exploratory biomarker analysis of the phase II FAIRLANE trial of neoadjuvant IPAT for TNBC provides insight into the potential heterogeneity of response and resistance to taxane therapy. The results also hint that response to PAC alone is dependent on baseline immune infiltration and that this dependency might be relieved with the addition of AKT inhibition. Citation Format: Wongchenko MJ, Oliveira M, Saura C, Nuciforo P, Calvo I, Andersen J, Passos Coelho JI, Gil Gil M, Bermejo B, Patt DA, Ciruelos E, Singel SM, Maslyar DJ, Xu N, de la Peña L, Baselga J, Gendreau S, Isakoff SJ. Exploratory biomarker analyses of FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) + paclitaxel (PAC) for early triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-19.

Abstract P4-01-16: Detection of plasma tumor DNA (ptDNA) in patients with hormone receptor-positive HER2-negative (HR+HER2-) early breast cancer (EBC) in clinical remission

Abstract Background: Detection of ptDNA in patients with HR+HER2- EBC in clinical remission may impact recommendations for type and duration of adjuvant endocrine therapy. A sensitive technique to identify tumor mutations in plasma is BEAMing digital PCR. The frequency and timing of detectable mutations in plasma of patients in clinical remission from HR+HER2- EBC are unknown. Methods: We screened a prospective institutional repository for patients that met inclusion criteria. Eligible patients must have been enrolled to the repository between 12/1/2008 (repository start) and 12/31/2016, had HR+HER2- EBC, received follow-up at Johns Hopkins with appointment scheduled between 3/1/2017 and 12/31/2017, completed curative surgery at least 6 months prior to this appointment, been recommended or initiated adjuvant endocrine therapy, and been in clinical remission. Appropriate patients were approached for a current blood sample during their follow-up appointment in 2017. Blood was analyzed using a BEAMing digital PCR platform (Sysmex Inostics OncoBEAM™) for AKT1, PIK3CA, and ESR1 mutations. Results: We identified 67 eligible patients and collected blood from 60. Most patients had relatively low risk disease including 40 patients (67%) with stage I disease, and only 21 patients (35%) received chemotherapy. Patients were evenly divided between receiving tamoxifen or an aromatase inhibitor, and some patients switched from one to the other. The majority of patients (68%) had surgery between 1 and 5 years prior to the current blood draw. Detailed patient characteristics are provided in Table 1. Two out of the 60 patients had detectable ptDNA, both with stage IIA disease. One patient had a mutation in the ESR1 ligand-binding domain P535H 9 months after surgery and while taking adjuvant tamoxifen for 7 months. Sanger sequencing of primary tumor tissue did not reveal this mutation. Another patient had a mutation in PIK3CA exon 9 E542K 9.5 years after surgery and after taking adjuvant tamoxifen for at least 7 years. Amplifying this locus in DNA from primary tumor tissue was unsuccessful; further analysis using droplet digital PCR (ddPCR) is planned. Conclusions: Detection of ptDNA was feasible in a relatively low-risk group of patients with HR+HER2- EBC in clinical remission. Sampling a larger number of patients is needed to gain more understanding of the frequency and timing of detectable ptDNA. Next steps should also focus on determining the natural history of detectable ptDNA in patients with HR+HER2 EBC in clinical remission which may impact adjuvant treatment recommendations. Funding sources: Komen SAC110053, P30 CA06973, Breast Cancer Research Foundation Table 1:Characteristics of included patients N (%)Total patients60Age at diagnosis, median(range)57 (30-77)Female59 (98)Caucasian54 (90)Postmenopausal at diagnosis36 (60)Tumor size <2 cm42 (70)Node negative45 (75)Invasive ductal histology44 (73)Received adjuvant chemotherapy21 (35)Type of adjuvant endocrine therapy Tamoxifen25 (42)Aromatase inhibitor26 (43)Tamoxifen and AI7 (12)None2 (3)Time after surgery 6 months to <1 year6 (10)1 year to <5 years41 (68)5 years to <10 years13 (22) Citation Format: Shah M, Hunter N, Ensminger J, Shinn D, Cole AJ, Quinn HE, Edelstein DL, Wang C, Smith KL, Richardson AL, Cimino-Mathews A, Wolff AC, Cravero K, Park BH, Stearns V. Detection of plasma tumor DNA (ptDNA) in patients with hormone receptor-positive HER2-negative (HR+HER2-) early breast cancer (EBC) in clinical remission [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-16.

Abstract GS1-06: Unraveling lobular breast cancer progression and endocrine resistance mechanisms through genomic and immune characterization of matched primary and metastatic samples

Abstract Background: Invasive lobular breast cancer (ILC) represents the second most common histology of breast cancer, accounts for 10-15% of all invasive cases and generally expresses the estrogen receptor (ER, coded by the ESR1 gene). Little is known about the genomic alterations associated with tumor progression and endocrine resistance in ILC. Here, we therefore molecularly characterized a unique series of matched primary and metastatic ILC. Patients and methods: We retrospectively identified 129 metastatic ER-positive ILC patients from 6 institutions. Following central pathology review and available DNA from the primary tumor (P), the metastasi(e)s (M), as well as from normal tissue, 80 patients (279 samples) were eligible for this study. All but 6 patients (7.5%) received endocrine treatment before metastatic sampling. Low pass whole genome and targeted gene screen (N=20 genes) sequencing was conducted to detect copy number aberrations (CNAs) and mutations associated with ILC metastatic progression respectively. ESR1 mutations were further assessed using droplet digital PCR (ddPCR). Publicly available data from IJB (n=413 ILC Ps), TCGA (n=172 ILC Ps), and MSKCC-IMPACT (n=116 ILC Ms) were used to compare and validate the frequencies of the detected alterations in ILC. Stromal tumor infiltrating lymphocytes (TILs) were assessed by two experienced pathologists. Results: The overall matched CNA comparison revealed a significant positive association between relapse-free survival and the P/M genomic distance defined by the number of CNAs private to P or M (r2= 0.52, p<.001), suggesting that the longer the disease evolution, the more M differs from P. Regarding CNA changes in cancer genes, we observed the acquisition in M of MYC, CCND1, FGFR1/ZNF703, and ZNF217 amplifications in 17, 9, 6 and 6% of the patients, as well as RB1, PTEN, and TP53 deletions in 9, 11, and 6% of the patients, respectively. The matched P/M comparison of the mutations highlighted the acquisition in M of mutations in 11% of the patients for CDH1, in 10% for ESR1, in 8% for ARID1A, in 5% for ERBB2, GATA3, IGF1R, MAP3K1, and PIK3CA, and in 3% for AKT1 and TBX3. Of interest, when comparing the overall CNA frequencies of our Ms to publicly available data from ILC Ps, we observed a higher frequency of CCND1 and MYC amplification, of PTEN, RB1 and TP53 deletions, as well of AKT1, ERBB2, ESR1 and IGF1R mutations in our ILC Ms. Of note, we did not detect any significant difference in CNA or mutational frequency when comparing our ILC Ms to those from MSKCC-IMPAKT. Regarding the immune infiltration, higher TILs in Ps were significantly associated with younger age at diagnosis, high grade tumors, and with mixed non-classic and trabecular histology. A paired analysis revealed no significant difference in TIL levels between P and M. TIL levels in the P or M were not associated with survival. Conclusion: This is to our knowledge the largest metastatic ILC series in which matched P and M samples were interrogated, revealing several genomic alterations, some of which potentially targetable, driving disease progression and endocrine resistance. Citation Format: Desmedt C, Richard F, Majjaj S, Pingitore J, Brown D, Rothé F, Marchio C, Clatot F, Mariani O, Boeckx B, Rouas G, Bertucci F, Galant C, Van den Eynden G, Salgado R, Lambrechts D, Vincent-Salomon A, Piccart M, Pruneri G, Larsimont D, Sotiriou C. Unraveling lobular breast cancer progression and endocrine resistance mechanisms through genomic and immune characterization of matched primary and metastatic samples [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS1-06.

Abstract P2-03-02: Markers of PI3K/Akt, ER & AR pathways and Ki67 expression in relation to tamoxifen outcome in ER+ breast cancer

Abstract Aim: To select tumor biomarkers, included in the AKT pathway, able to predict response to tamoxifen (TAM) in patients with estrogen receptor (ER) positive breast cancer. Patients & Methods: 157 endocrine treatment naïve patients with known ER and HER2 status were treated with TAM: tumour tissue was available either as tissue microarrays (TMA) (n=107) or core biopsies (n=50). After removal of duplicates, ER(-) BCs, & patients without full outcome data 103 patients were available. Ten biomarkers were evaluated by immunohistochemical staining (% positivity and/or H-Score): ER, HER2, Ki67, & phosphorylated forms of MAPK, AKT, ER (Ser118), PRAS40, and IGF1R, as well as pTEN & also androgen receptor (AR) expression. TMA cores were collected from three tumor sites, two central and one peripheral. Tumors were also analysed for AKT1 E17K somatic mutation using BEAMing technology. Patient outcome was assessed by clinical benefit (CB) rate & survival analyses (Time To Progression (TTP) and Time To Death (TTD)). Biomarkers were selected by regression trees to be included in multivariate models for clinical outcomes. False Discovery Rate(FDR) was applied to univariate results. Combinations of pAKT & pPRAS40, Ki67 & ER and ER/AR ratio-were also analysed. Results: Type of Biopsy (Core versus TMA): Generally % positivity was lower in TMAs than cores, for HER2, pER118, pIGF1R and cytoplasmic pAKT and pPRAS40. pAKT, pMAPK and Ki67 were largely unaffected by biopsy type. After FDR testing, significant differences remained for pER118 (p=0.002), pIGF1R (p<0.001), pPRAS40 (p<0.001) and HER2 (P=0.005) positivity levels. Biopsy Location (TMAs): There was no significant difference in % positivity between central & peripheral tumor sites for all biomarkers examined. After FDR correction modest differences were seen for HER2 & pER118 (both p=0.044) only between the two central samples. IHC endpoints versus clinical outcome: Univariate analyses identified only lower levels of pTEN and higher levels of Ki67 (% positivity) as predictive of poor outcome (TTP & TTD) following TAM treatment. In the luminal A (ER+/HER2-) subgroup only Ki67 was predictive of poor outcome (p=0.012). The predictive power of Ki67 for TTP was not improved by combination with pTEN or ER. An inverse ER H-Score association with TTP was confined to the 'luminal A' cohort. AR was not predictive. 71 patients had AR/ER ratio: 61 had ratio <2 (42 had CB & 19 no CB), and 10 had ratio ≥2 (3 CB & 7 no CB) (p<0.02). There was no significant difference in TTP or TTD by AR/ER ratio. Only 2 /146 (<2%) tumors had AKT1 E17K mutations. Conclusions: Biomarker staining cannot be assumed to be the same in TMAs & core biopsies: such data should be combined with caution. Importantly however, there was no evidence of difference in Ki67 between TMA & core samples. There is no evidence to suggest that biomarker staining (including Ki67) at different tumor sites shouldn't be combined, or that ranking of scores differs across such sites. The findings confirmed previous reports that Ki67 & reduced level of PI3K /AKT pathway regulator pTEN can predict resistance to TAM in ER+ BCs. The AKT mutation rate was low. Citation Format: Campbell C, Mathew J, Ellis I, Finlay P, Green A, Bradbury I, Borgquist S, Elebro K, Politz O, Haegebarth A, Gee JM, Robertson JF. Markers of PI3K/Akt, ER & AR pathways and Ki67 expression in relation to tamoxifen outcome in ER+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-02.

Comparative transcriptome analysis reveals K+ transporter gene contributing to salt tolerance in eggplant

BackgroundSoil salinization is one of the most crucial abiotic stresses that limit the growth and production of eggplant. The existing researches in eggplant were mostly focused on salt-induced morphological, biochemical and physiological changes, with only limited works centered on salt-response genes in eggplant at the transcriptomic level.ResultsOur preliminary work found that Zhusiqie (No.118) is salt-tolerant and Hongqie (No.30) is salt-sensitive. Consequently, they were re-named as ST118 and SS30, respectively. ST118 showed less damaged on growth and higher K+/Na+ ratios in leaves than SS30. Comparative-transcriptome analysis was used as a powerful approach to understand the salt-response mechanisms in the leaves and roots of SS30 and ST118. And it revealed that genotype-specific and organ-specific manners exist in eggplant in response to salt stress. Strikingly, the genotype-specific differentially expressed genes (DEGs) in ST118 were considered crucial to its higher salt-tolerance, because the expression patterns of common DEGs in the leaves/roots of the two eggplant genotypes were almost the same. Among them, some transcription factors have been reported to be in response to elevated external salinity, including the members of C2C2-CO-like, WRKY, MYB and NAC family. In addition, the AKT1, KAT1 and SOS1 were up-regulated only in the leaves of ST118. Furthermore, the complementation assays demonstrated that the salt-tolerances of both yeast and Arabidopsis akt1 mutants were enhanced by heterologous expression of SmAKT1.ConclusionThe comparative-transcriptome analysis indicated that the salt-tolerance can be increased by higher transcript level of some genotype-specific genes. This work revealed that eggplants seem to be more inclined to absorb K+ rather than to exclude Na+ under salt stress conditions because seven K+ transporters were significantly up-regulated, while only one Na+ transporter was similarly regulated. Finally, the complementation assays of SmAKT1, which is genotype-specific up-regulated in ST118, suggest that the other TFs and K+ transport genes were worthy of future investigation for their functions in salinity tolerance.

Identification of signaling cascade in the insulin signaling pathway in response to nanopolystyrene particles

The molecular response of animals to nanoplastic particles is still largely unclear. In this study, we employed a modified prolonged exposure system to investigate the molecular response of Caenorhabditis elegans to nanopolystyrene particles. Exposure to nanopolystyrene particles (1 μg/L) significantly decreased expressions of daf-2 encoding an insulin receptor, age-1 encoding a PI3K, and akt-1 encoding an Akt/PKB, and increased expression of daf-16 encoding a FOXO transcriptional factor in insulin signaling pathway. Among these genes, mutation of daf-2, age-1, or akt-1 induced a resistance to toxicity of nanopolystyrene particles, whereas mutation of daf-16 induced a susceptibility to the toxicity of nanopolystyrene particles. RNAi knockdown of daf-16 could further suppress the resistance of daf-2, age-1, or akt-1 mutant to the toxicity of nanopolystyrene particles. The insulin signaling pathway acted in intestinal cells to regulate the toxicity of nanopolystyrene particles. Moreover, sod-3 encoding a manganese superoxide dismutase, mtl-1 encoding a metallothionein, and gpd-2 encoding a glyceraldehyde-3-phosphate dehydrogenase were identified as downstream targeted genes for daf-16 in the regulation of toxicity of nanopolystyrene particles. Therefore, a signaling cascade of DAF-2-AGE-1-AKT-1-DAF-16-SOD-3/MTL-1/GPD-2 was identified in response to nanopolystyrene particles in nematodes. Additionally, this signaling cascade in the insulin signaling pathway may mediate a protective response for nematodes against the adverse effects from nanopolystyrene particles.

Exome Analysis Reveals Genomic Markers Associated with Better Efficacy of Nivolumab in Lung Cancer Patients.

Immune checkpoint inhibitors revolutionized the treatment of non-small cell lung cancer (NSCLC). However, only one-quarter of patients benefit from these new therapies. PD-L1 assessment and tumor mutational burden (TMB) are available tools to optimize use of checkpoint inhibitors but novel tools are needed. Exome sequencing could generate many variables but their role in identifying predictors of response is unknown. We performed somatic and constitutional exome analyses for 77 patients with NSCLC treated with nivolumab. We studied: one-tumor-related characteristics: aneuploidy, CNA clonality, mutational signatures, TMB, mutations in WNT, AKT, MAPK, and DNA repair pathways, and two-immunologic characteristics: number of intratumoral TCR clones, HLA types, and number of neoantigens; and six clinical parameters. A high TMB per Mb, a high number of neoantigens, mutational signatures 1A and 1B, mutations in DNA repair pathways, and a low number of TCR clones are associated with greater PFS. Using a LASSO method, we established an exome-based model with nine exome parameters that could discriminate patients with good or poor PFS (P < 0.0001) and overall survival (P = 0.002). This model shows better ability to predict outcomes compared with a PD-L1 clinical model with or without TMB. It was externally validated on two cohorts of patients with NSCLC treated with pembrolizumab or with nivolumab and ipilimumab as well as in urothelial tumors treated with atezolizumab. Altogether, these data provide a validated biomarker that predicts the efficacy of nivolumab or pembrolizumab in patients with NSCLC. Our biomarker seems to be superior to PD-L1 labeling and TMB models.

AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions.

Aberrant activation of Akt disturbs proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand upstream signaling pathways governing Akt activation. Here, we report that Akt undergoes SETDB1-mediated lysine-methylation to promote its activation, which is antagonized by the Jumonji-family demethylase, KDM4B. Notably, compared with wild-type mice, mice harboring non-methylated mutant Akt1 not only exhibited reduced body size, but also were less prone to carcinogen-induced skin tumors in part due to reduced Akt activation. Mechanistically, Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) interaction with Akt facilitates its interaction with SETDB1 for subsequent Akt methylation, which in turn sustains Akt phosphorylation. Pathologically, genetic alterations including SETDB1 amplification aberrantly promote Akt methylation to facilitate its activation and oncogenic functions. Thus, Akt methylation is an important step synergizing with PI3K signaling to control Akt activation, suggesting that targeting the SETDB1 signaling could be a potential therapeutic strategy for combatting hyperactive Akt-driven cancers.

SETDB1-mediated methylation of Akt promotes its K63-linked ubiquitination and activation leading to tumorigenesis.

The serine/threonine kinase Akt plays a central role in cell proliferation, survival and metabolism and its hyperactivation is linked to cancer progression. Here we report that Akt undergoes K64 methylation by SETDB1, which is crucial for cell membrane recruitment, phosphorylation and activation of Akt upon growth factor stimulation. Furthermore, we reveal an adaptor function of histone demethylase JMJD2A, which recognizes Akt K64 methylation and recruits E3 ligase TRAF6 and Skp2-SCF to the Akt complex, independently of its demethylase activity, thereby initiating K63-linked ubiquitination, cell membrane recruitment and activation of Akt. Notably, cancer associated Akt mutant (E17K) displays enhanced K64 methylation, leading to its hyper-phosphorylation and activation. SETDB1-mediated Akt K64 methylation is upregulated and correlated with Akt hyperactivation in non-small-cell lung carcinoma (NSCLC), promotes tumor development and predicts poor outcome. Collectively, these findings reveal complicated layers of Akt activation regulation coordinated by SETDB1-mediated Akt K64 methylation to drive tumorigenesis.

Genomic Analysis of Posterior Fossa Meningioma Demonstrates Frequent AKT1 E17K Mutations in Foramen Magnum Meningiomas.

Objective Posterior fossa meningiomas are surgically challenging tumors that are associated with high morbidity and mortality. We sought to investigate the anatomical distribution of clinically actionable mutations in posterior fossa meningioma to facilitate identifying patients amenable for systemic targeted therapy trials. Methods Targeted sequencing of clinically targetable AKT1 , SMO , and PIK3CA mutations was performed in 61 posterior fossa meningioma using Illumina NextSeq 500 to a target depth of >500 × . Samples were further interrogated for 53 cancer-relevant RNA fusions by the Archer FusionPlex panel to detect gene rearrangements. Results AKT 1 ( E17K ) mutations were detected in five cases (8.2%), four in the foramen magnum and one in the cerebellopontine angle. In contrast, none of the posterior fossa tumors harbored an SMO ( L412F ) or a PIK3CA ( E545K ) mutation. Notably, the majority of foramen magnum meningiomas (4/7, 57%) harbored an AKT1 mutation. In addition, common clinically targetable gene fusions were not detected in any of the cases. Conclusion A large subset of foramen magnum meningiomas harbor AKT1 E17K mutations and are therefore potentially amenable to targeted medical therapy. Genotyping of foramen magnum meningiomas may enable more therapeutic alternatives and guide their treatment decision process.