PTEN Mutations Research Articles

Genomic landscape, immune characteristics, and a prognostic mutation signature of advanced cervical cancer in China (162)

Objectives: This study aimed to analyze genomic alteration profiles and immune characteristics of a cohort of Chinese advanced cervical cancer patients to understand why certain patients benefited from molecular targeted therapies, immunotherapy, and their prognostic significance. Methods: PD-L1 expression and clinical-pathologic information from 98 cervical cancer samples were obtained. Cervical cancer data were obtained from The Cancer Genome Atlas (TCGA). Differences in PD-L1 expression and gene mutations between squamous cell carcinoma (SCC) and adenocarcinoma (AC) were analyzed using the Chi-square test or Fisher’s exact test. Differences in gene mutations between our cohort and TCGA cohort were tested by Fisher’s exact test. The relationship between gene mutation and TMB was analyzed by the Mann-Whitney U test. Logistic regression was used to analyze factors influencing TMB-high. Kaplan-Meier survival analyses were applied. Results: Positive PD-L1 expression was significantly higher in cervical SCC than in cervical AC (87% vs 39%, p < 0.001). Frequently mutated genes in cervical cancer included PIK3CA, KMT2D, and KMT2C genes, among others. PIK3CA gene mutation rates were significantly higher in SCC than in AC (p = 0.004). TERT gene mutation rate was significantly higher in our cohort than TCGA cohort (12% vs 1%, p < 0.001). SCC had a significantly higher TMB than AC (p < 0.001). The independent predictors of TMB-high were KMT2C and LRP1B gene mutations (p <0.05). In this study, the median progression-free survival (PFS) was 12.16 months in the PTEN mutant group versus 21.75 months in the wild-type group (p=0.0024). Conclusions: Cervical SCC and AC have different molecular profiling and immune characteristics, suggesting that targeted treatments for SCC and AC patients may improve clinical outcomes. KMT2C and LRP1B gene mutations are independent predictors of TMB-high status in cervical cancer. We have also proposed the prognostic value of PTEN mutation. Objectives: This study aimed to analyze genomic alteration profiles and immune characteristics of a cohort of Chinese advanced cervical cancer patients to understand why certain patients benefited from molecular targeted therapies, immunotherapy, and their prognostic significance. Methods: PD-L1 expression and clinical-pathologic information from 98 cervical cancer samples were obtained. Cervical cancer data were obtained from The Cancer Genome Atlas (TCGA). Differences in PD-L1 expression and gene mutations between squamous cell carcinoma (SCC) and adenocarcinoma (AC) were analyzed using the Chi-square test or Fisher’s exact test. Differences in gene mutations between our cohort and TCGA cohort were tested by Fisher’s exact test. The relationship between gene mutation and TMB was analyzed by the Mann-Whitney U test. Logistic regression was used to analyze factors influencing TMB-high. Kaplan-Meier survival analyses were applied. Results: Positive PD-L1 expression was significantly higher in cervical SCC than in cervical AC (87% vs 39%, p < 0.001). Frequently mutated genes in cervical cancer included PIK3CA, KMT2D, and KMT2C genes, among others. PIK3CA gene mutation rates were significantly higher in SCC than in AC (p = 0.004). TERT gene mutation rate was significantly higher in our cohort than TCGA cohort (12% vs 1%, p < 0.001). SCC had a significantly higher TMB than AC (p < 0.001). The independent predictors of TMB-high were KMT2C and LRP1B gene mutations (p <0.05). In this study, the median progression-free survival (PFS) was 12.16 months in the PTEN mutant group versus 21.75 months in the wild-type group (p=0.0024). Conclusions: Cervical SCC and AC have different molecular profiling and immune characteristics, suggesting that targeted treatments for SCC and AC patients may improve clinical outcomes. KMT2C and LRP1B gene mutations are independent predictors of TMB-high status in cervical cancer. We have also proposed the prognostic value of PTEN mutation.

PTEN Dual Lipid- and Protein-Phosphatase Function in Tumor Progression.

Simple SummaryPhosphatase and tensin homolog deleted on chromosome ten (PTEN) is a multifunctional tumor suppressor with protein- and lipid-phosphatase activities. The inactivation of PTEN is commonly found in all human cancers and is correlated with tumor progression. PTEN-lipid-phosphatase activity has been well documented to dephosphorylate phosphatidylinositol-3, 4, 5-phosphate (PIP3), which hinders cell growth and survival by dampening the PI3K and AKT signaling activity. PTEN-protein-phosphatase activity is less well studied and understood. Recent studies have reported that PTEN-protein-phosphatase activity dephosphorylates the different proteins and acts in various cell functions. We here review the PTEN mutations and protein-phosphatase substrates in tumor progression. We aim to address the gap in our understanding as to how PTEN protein phosphatase contributes to its tumor-suppression functions.PTEN is the second most highly mutated tumor suppressor in cancer, following only p53. The PTEN protein functions as a phosphatase with lipid- and protein-phosphatase activity. PTEN-lipid-phosphatase activity dephosphorylates PIP3 to form PIP2, and it then antagonizes PI3K and blocks the activation of AKT, while its protein-phosphatase activity dephosphorylates different protein substrates and plays various roles in tumorigenesis. Here, we review the PTEN mutations and protein-phosphatase substrates in tumorigenesis and metastasis. Our purpose is to clarify how PTEN protein phosphatase contributes to its tumor-suppressive functions through PI3K-independent activities.

Construction and validation of a glioblastoma prognostic model based on immune-related genes.

BackgroundGlioblastoma multiforme (GBM) is a common malignant brain tumor with high mortality. It is urgently necessary to develop a new treatment because traditional approaches have plateaued.PurposeHere, we identified an immune-related gene (IRG)-based prognostic signature to comprehensively define the prognosis of GBM.MethodsGlioblastoma samples were selected from the Chinese Glioma Genome Atlas (CGGA). We retrieved IRGs from the ImmPort data resource. Univariate Cox regression and LASSO Cox regression analyses were used to develop our predictive model. In addition, we constructed a predictive nomogram integrating the independent predictive factors to determine the one-, two-, and 3-year overall survival (OS) probabilities of individuals with GBM. Additionally, the molecular and immune characteristics and benefits of ICI therapy were analyzed in subgroups defined based on our prognostic model. Finally, the proteins encoded by the selected genes were identified with liquid chromatography-tandem mass spectrometry and western blotting (WB).ResultsSix IRGs were used to construct the predictive model. The GBM patients were categorized into a high-risk group and a low-risk group. High-risk group patients had worse survival than low-risk group patients, and stronger positive associations with multiple tumor-related pathways, such as angiogenesis and hypoxia pathways, were found in the high-risk group. The high-risk group also had a low IDH1 mutation rate, high PTEN mutation rate, low 1p19q co-deletion rate and low MGMT promoter methylation rate. In addition, patients in the high-risk group showed increased immune cell infiltration, more aggressive immune activity, higher expression of immune checkpoint genes, and less benefit from immunotherapy than those in the low-risk group. Finally, the expression levels of TNC and SSTR2 were confirmed to be significantly associated with patient prognosis by protein mass spectrometry and WB.ConclusionHerein, a robust predictive model based on IRGs was developed to predict the OS of GBM patients and to aid future clinical research.

Multiparametric Classification of Non-Muscle Invasive Papillary Urothelial Neoplasms: Combining Morphological, Phenotypical, and Molecular Features for Improved Risk Stratification.

Diagnosis and grading of non-invasive papillary urothelial tumors according to the current WHO classification poses some challenges for pathologists. The diagnostic reproducibility of separating low-grade and high-grade lesions is low, which impacts their clinical management. Whereas papillary urothelial neoplasms with low malignant potential (PUN-LMP) and low-grade papillary non-invasive carcinoma (LG-PUC) are comparable and show frequent local recurrence but rarely metastasize, high-grade papillary non-invasive carcinoma (HG-PUC) has a poor prognosis. The main objective of this work is to develop a multiparametric classification to unambiguously distinguish low-grade and high-grade tumors, considering immunohistochemical stains for p53, FGFR3, CK20, MIB-1, p16, p21 and p-HH3, and pathogenic mutations in TP53, FGFR3, TP53, ERCC2, PIK3CA, PTEN and STAG2. We reviewed and analyzed the clinical and histological data of 45 patients with a consensus diagnosis of PUN-LMP (n = 8), non-invasive LG-PUC (n = 23), and HG-PUC (n = 14). The proliferation index and mitotic count assessed with MIB-1 and P-HH3 staining, respectively correlated with grading and clinical behavior. Targeted sequencing confirmed frequent FGFR3 mutations in non-invasive papillary tumors and identified mutations in TP53 as high-risk. Cluster analysis of the different immunohistochemical and molecular parameters allowed a clear separation in two different clusters: cluster 1 corresponding to PUN-LMP and LG-PUC (low MIB-1 and mitotic count/FGFR3 and STAG2 mutations) and cluster 2, HG-PUC (high MIB-1 and mitosis count/CK20 +++ expression, FGFR3 WT and TP53 mutation). Further analysis is required to validate and analyze the reproducibility of these clusters and their biological and clinical implication.

Indeterminate Thyroid Fine-Needle Aspirations in Pediatrics: Exploring Clinicopathologic Features and Utility of Molecular Profiling

Introduction: The diagnostic utility of molecular profiling for the evaluation of indeterminate pediatric thyroid nodules is unclear. We aimed to assess pediatric cases with indeterminate thyroid fine-needle aspiration (FNA) alongside clinicopathologic features and mutational analysis. Methods: A retrospective review of 126 patients with indeterminate cytology who underwent FNA between January 2010 and December 2021 at the Children’s Hospital of Philadelphia was performed. Indeterminate cases defined by The Bethesda System for Reporting Thyroid Cytopathology (AUS/FLUS or TBSRTC III; FN/SFN or TBSRTC IV; SM or TBSRTC V) were correlated to clinicopathologic and genetic characteristics. Results: Of the 114 surgical cases, 48% were malignant, with the majority of malignant cases diagnosed as follicular variant of papillary thyroid carcinoma (28/55). Risk of malignancy increased with TBSRTC category: 23% for AUS/FLUS, 51% for FN/SFN, and 100% for SM nodules. There were significant differences in surgical approach (p < 0.01), performance of lymph node dissection (p < 0.01), histological diagnosis (p < 0.01), primary tumor focality/laterality (p = 0.04), and lymphatic invasion (p = 0.02) based on TBSRTC classification, with resultant differences in post-surgical risk stratification per American Thyroid Association (ATA) Pediatric Guidelines (p = 0.01). Approximately 89% (49/55) of cases were classified as ATA low risk, and 5 of 6 patients with ATA intermediate- or high-risk disease had SM cytology. Somatic molecular testing was performed in 40% (51/126) of tumors; 77% (27/35) of malignant cases and 38% (6/16) of benign cases harbored driver alteration(s). Of the driver-positive malignant cases, 52% (14/27) were associated with low risk (DICER1, PTEN, RAS, and TSHR mutations), 33% (9/27) were associated with high risk (BRAF mutations and ALK, NTRK, and RET fusions), and 15% (4/27) had unreported risk for invasive disease (APC, BLM, and PPM1D mutations and TG-FGFR1 fusion). Incidence of high-risk drivers increased with TBSRTC category. Approximately 23% (8/35) of patients harboring thyroid malignancy did not have an identifiable driver alteration. Conclusions: Molecular analysis is useful to discriminate benign and malignant thyroid nodules with indeterminate cytology. Patients with driver genetic alteration(s) and indeterminate cytology should consider surgical management secondary to the high incidence (82%; 27/33) of thyroid malignancy in these patients.

Iris and Ciliary Body Melanocytomas Are Defined by Solitary GNAQ Mutation Without Additional Oncogenic Alterations

To analyze the genetic features of melanocytomas and melanomas of the anterior uvea and assess the value of molecular testing for diagnosis and prognostication. Retrospective case-control study. Patients with melanocytoma (n= 16) and melanoma (n= 19) of the anterior uvea. Targeted next-generation sequencing was performed on formalin-fixed, paraffin-embedded tumor tissue from anterior uveal melanocytic tumors and correlated with clinicopathologic features. Presence or absence of accompanying oncogenic alterations beyond GNAQ/GNA11 and their association with histologic features and local recurrence. Hotspot missense mutations in GNAQ/GNA11 were identified in 91% (32/35) of all cases. None of the melanocytomas with or without atypia demonstrated chromosomal imbalances or additional oncogenic variants beyond GNAQ mutation, and none recurred over a median follow-up of 36 months. Additional alterations identified in a subset of melanomas include mutations in BAP1 (n= 3), EIF1AX (n= 4), SRSF2 (n= 1), PTEN (n=1), and EP300 (n= 1); monosomy 3p (n= 6); trisomy 6p (n= 3); trisomy 8q (n= 2); and an ultraviolet mutational signature (n= 5). Local recurrences were limited to melanomas, all of which demonstrated oncogenic alterations in addition to GNAQ/GNA11 (n= 5). A single melanoma harboring GNAQ and BAP1 mutations and monosomy 3 was the only tumor that metastasized. In this study, anterior segment uveal melanocytomas did not display oncogenic alterations beyond GNAQ/GNA11. Therefore, they are genetically similar to uveal nevi rather than uveal melanoma based on their molecular features known from the literature. Molecular testing can be performed on borderline cases to aidrisk stratification and clinical management decisions.

Seborrheic Keratosis With Malignant Transformation (Invasive or Noninvasive Squamous Cell Carcinoma Arising in Seborrheic Keratosis): A Clinicopathologic and Immunohistochemical Study of 11 Cases.

Seborrheic keratosis is a common benign neoplasm composed of basaloid keratinocytes. However, little is known about the malignant transformation of the tumor. Eleven cases of seborrheic keratosis with malignant transformation were analyzed. The 11 patients included 5 male patients and 6 female patients with a median age of 75 years at diagnosis (68-90 years). The tumors arose at various sites from the scalp (n = 3) to the lower leg (n = 2). The median tumor size was 12 (10-32) and 40 (20-75) mm in 7 noninvasive and 4 invasive cases, respectively. One patient exhibited in-transit skin metastasis. Histopathology of the malignant components resembled porocarcinoma or inverted follicular keratosis. Bowenoid and pagetoid spreading was frequently observed. The malignant components expressed cytokeratin 5/6 (100%) and GATA3 (73%), but not cytokeratin 7 (0%), cytokeratin 19 (9%), BerEP4 (0%), c-kit (0%), and NUT (0%). No significant immunoreactivity of YAP1 was observed in any of the cases. Mutant-type immunostaining of p53 and PTEN was observed in 91% and 82% of the cases, respectively. An increase in p16 expression was seen in 6 (86%) of the 7 cases with noninvasive carcinoma, although a loss of p16 immunoexpression was seen in the invasive carcinoma component in 3 (75%) of the 4 cases. This study demonstrated that seborrheic keratosis can undergo malignant transformation, particularly in large-sized lesions in elderly patients. Malignant components mimic porocarcinoma or inverted follicular keratosis. Malignant transformation induced by TP53 and PTEN mutations and tumor invasion by CDKN2A inactivating mutations are suggested in this study.

Targeted molecular profiling of salivary duct carcinoma with rhabdoid features highlights parallels to other apocrine and discohesive neoplasms: which phenotype should drive classification?

Salivary duct carcinoma with rhabdoid features (SDC-RF) is a recently-described salivary gland tumor that bears striking histologic similarity to lobular carcinoma of the breast. While this tumor has an apocrine phenotype that supports classification as a variant of SDC, it infrequently arises in association with conventional SDC. Furthermore, discohesive architecture can be seen in non-apocrine salivary carcinomas, raising the possibility that discohesive growth should define a separate entity. In this study, we aimed to perform comprehensive molecular profiling of SDC-RF to better understand its pathogenesis and classification. We documented the clinicopathologic features of 9 cases of SDC-RF and performed immunostains including AR, GCDFP, and e-cadherin on all cases. We also performed targeted next generation sequencing (NGS) panels on 7 cases that had sufficient tissue available. The SDC-RF represented 8 men and 1 woman with a median age of 67 years (range 63-83 years) and included 6 parotid, 2 buccal, and 1 submandibular primary. All tumors were uniformly composed of discohesive cells with abundant eosinophilic cytoplasm; signet-ring cell features were seen in 2 cases. All tumors were also positive for AR (100%) and GCDFP (100%), and 7 tumors (78%) displayed lost or abnormal e-cadherin. NGS highlighted concomitant PIK3CA and HRAS mutations in 4 tumors, with additional cases harboring TP53, PTEN, and AKT1 mutations. Furthermore, CDH1 alterations were seen in 6 cases, including a novel CDH1::CORO7 fusion. Among 5 patients with follow-up available, 3 (60%) developed local recurrence and widespread distant metastasis and died of disease at a median 20 months (range 10-48 months). Overall, our findings confirm frequent CDH1 mutations and e-cadherin inactivation in SDC-RF, similar to discohesive tumors from other sites. We also highlight an apocrine molecular profile similar to conventional SDC. However, occasional AKT1 mutation and signet-ring features suggest SDC-RF may also be related to mucinous adenocarcinoma. As more salivary tumors with discohesive growth are identified, it may become clearer whether SDC-RF should remain in the SDC family or be recognized as a separate entity.

Mesonephric-Like Adenocarcinoma of Uterine Corpus: A Clinicopathological and Targeted Genomic Profiling Study in a Single Institution

BackgroundMesonephric-like adenocarcinoma (MLA) is a recently characterized, rare, and aggressive neoplasm that mostly arises in the uterine corpus and ovary. MLA shows characteristic pathological features similar to mesonephric adenocarcinoma of the cervix. The origin of MLA is still controversial and recognition of it remains challenging for pathologists. The aim of this study was to enrich the clinicopathological features of MLA in the uterine corpus and explore its molecular alterations by targeted next-generation sequencing (NGS).MethodsFour cases of MLA were identified among a total of 398 endometrial carcinomas diagnosed in our institution between January 2014 and December 2021. Immunohistochemistry and targeted NGS spanning 437 cancer-relevant genes were performed.ResultsThe most common symptom was abnormal vaginal bleeding, and the average age was 68 years. Histologically, the tumors showed a mixture of varied growth patterns including papillary, glandular, tubular, cribriform, solid, and slit-like architectures, which were lined by columnar to cuboidal cells with overlapping vesicular nuclei and sometimes nuclear grooves. Intraluminal eosinophilic colloid-like secretions were focally evident in three of the four cases. Immunohistochemically, the MLAs were positive for GATA3 (4/4), TTF-1 (3/3), luminal CD10 (3/3), calretinin (2/3), and patchy P16 (3/3) and were negative for ER (0/4) and PR (0/4). The expression of P53 was “wild type” (4/4). By targeted NGS, 3/4 (75%), 2/4 (50%), and 1/4 (25%) cases harbored PIK3CA, KRAS, and PTEN mutations, respectively. None of the tumors had mutations in DNA mismatch repair genes, ARID1A/B, POLE, CTNNB1, SMARCA4, or TP53. At the time of diagnosis, three were presented with FIGO IB stage and one with IIIC stage. Two patients received postoperative chemotherapy and radiotherapy and they were alive without evidence of disease at 8 and 56 months follow-up, respectively. One patient developed pulmonary metastasis 13 months after surgery and chemotherapy, and one was dead of the disease 24 months after the operation without adjuvant therapy.ConclusionsMLA is a rare and aggressive malignancy, representing approximately 1% of all endometrial carcinomas. It exhibits mixed architectures associated with distinctive immunophenotype and recurrent KRAS and PIK3CA mutations, supporting classified as of Müllerian origin with mesonephric differentiation.

Identification of BST2 Contributing to the Development of Glioblastoma Based on Bioinformatics Analysis.

Rigorous molecular analysis of the immune cell environment and immune response of human tumors has led to immune checkpoint inhibitors as one of the most promising strategies for the treatment of human cancer. However, in human glioblastoma multiforme (GBM) which develops in part by attracting immune cell types intrinsic to the human brain (microglia), standard immunotherapy has yielded inconsistent results in experimental models and patients. Here, we analyzed publicly available expression datasets to identify molecules possibly associated with immune response originating from or influencing the tumor microenvironment in primary tumor samples. Using three glioma datasets (GSE16011, Rembrandt-glioma and TCGA-glioma), we first analyzed the data to distinguish between GBMs of high and low tumor cell purity, a reflection of the cellular composition of the tumor microenvironment, and second, to identify differentially expressed genes (DEGs) between these two groups using GSEA and other analyses. Tumor purity was negatively correlated with patient prognosis. The interferon gamma-related gene BST2 emerged as a DEG that was highly expressed in GBM and negatively correlated with tumor purity. BST2 high tumors also tended to harbor PTEN mutations (31 vs. 9%, BST2 high versus BST2 low ) while BST2 low tumors more often had sustained TP53 mutations (8 versus 36%, BST2 high versus BST2 low ). Prognosis of patients with BST2 high tumors was also poor relative to patients with BST2 low tumors. Further molecular in silico analysis demonstrated that high expression of BST2 was negatively correlated with CD8+ T cells but positively correlated with macrophages with an M2 phenotype. Further functional analysis demonstrated that BST2 was associated with multiple immune checkpoints and cytokines, and may promote tumorigenesis and progression through interferon gamma, IL6/JAK/STAT3 signaling, IL2/STAT5 signaling and the TNF-α signaling via NF-kB pathway. Finally, a series of experiments confirmed that the expression of BST2 can be significantly increased by IFN induction, and knockdown of BST2 can significantly inhibit the growth and invasion of GBM cells, and may affect the phenotype of tumor-associated macrophages. In conclusion, BST2 may promote the progression of GBM and may be a target for treatment.

PTEN and LKB1 are differentially required in Gli1-expressing mesenchymal cells to suppress gastrointestinal polyposis.

PTEN and LKB1 are intimately associated with gastrointestinal tumorigenesis. Mutations of PTEN or LKB1 lead to Cowden syndrome and Peutz-Jeghers syndrome characterized by development of gastrointestinal polyps. However, the cells of origin of these polyps and underlying mechanism remain unclear. Here, we reveal that PTEN or LKB1 deficiency in Gli1+ gut mesenchymal cells, but not intestinal epithelium, drives polyp formation histologically resembling polyposis in human patients. Mechanistically, although PTEN and LKB1 converge to regulate mTOR/AKT signaling in various tumor contexts, we find that mTOR is essential for PTEN-deletion-induced polyp formation but is largely dispensable for polyposis induced by mesenchymal LKB1 deficiency. Altogether, our studies identify Gli1-expressing mesenchymal cells as a common cell of origin for polyposis associated with PTEN and LKB1 and reveal their engagement of different downstream pathways in gut mesenchyme to suppress gastrointestinal tumorigenesis.

BRCA1, BRCA2, TP53, PIK3CA, PTEN and AKT1 genes mutations in Burkina Faso breast cancer patients: prevalence, spectrum and novel variant.

BRCA1 and BRCA2 are the two most commonly mutated tumor suppressor genes associated with hereditary breast cancer (BC). Also, mutations in TP53, PIK3CA, PTEN and AKT1 were observed at a high frequency in BC with their mutation spectrum exhibiting a subgroup particularity with enormous clinical significance in the prevention, classification and treatment of cancers. Unfortunately, the mutation spectrum of these genes is still unknown in most Sub-Saharan African population. Therefore, using samples from 133 unselected BC patients, we aimed to assess the contribution of these mutations by direct Sanger sequencing. The analysis revealed pathogenic germline variants on BRCA1 exon 11 (c.3331C > T, 0.75%) and BRCA2 exon 11 (c.5635G > T, c.6211delA; 1.5%). Five other pathogenic variants were identified in 61 of the 133 subjects (45.86%), with 39.09% for PIK3CA, 12.78% for TP53. Interestingly, a variant in PIK3CA found in high frequency in our population was different from the one usually found in other populations (c.1634A > C, 38.34%), and four patients carried mutations linked to Cowen Syndrome 5 c.[1634A > C;1658_1659delGTinsC]. A novel variant (c.312G > T) was found in TP53 gene at 12.78%. Overall, mutation carriers were found more in Her2 negative and in patients that underwent surgery and chemotherapy. No pathogenic variant was found in PTEN and AKT1. Our population displayed a high frequency of PIK3CA mutations with an unusual distribution and spectrum as well as a relatively low prevalence of BRCA mutations. Our results provided novel data on an unstudied population and may help in prevention, and the establishment of suitable therapeutic approaches for our population.

Desmoplastic infantile astrocytoma with atypical phenotype, PTEN homozygous deletion and BRAF V600E mutation

Desmoplastic infantile astrocytoma (DIA) is rare, cystic and solid tumor of infants usually found in superficial cerebral hemispheres. Although DIA is usually benign, uncommon cases bearing malignant histological and aggressive clinical features have been described in the literature. We report a newborn patient who was diagnosed with a DIA and died postresection. Pathologic examination revealed that the main part of the tumor had benign features, but the internal region showed areas with a more aggressive appearance, with higher-proliferative cells, anaplastic GFAP positive cells with cellular polymorphism, necrosis foci, vascular hyperplasia with endothelial proliferation and microtrombosis. Genetic study, performed in both regions of the tumor, showed a BRAF V600E mutation and a homozygous deletion in PTEN, without changes in other relevant genes like EGFR, CDKN2A, TP53, NFKBIA, CDK4, MDM2 and PDGFRA. Although PTEN homozygous deletions are described in gliomas, the present case constitutes the first report of a PTEN mutation in a DIA, and this genetic feature may be related to the malignant behavior of a usually benign tumor. These genetic findings may point at the need of further and deeper genetic characterization of DIAs, in order to better understand the biology of this tumor and to obtain new prognostic approaches, a better clinical management and targeted therapies, especially in malignant cases of DIA.

Abstract 5353: PIK3CA and PTEN mutations as drivers of osimertinib resistance in patients with NSCLC

Abstract Osimertinib is a third-generation EGFR-TKI approved for the first-line treatment of EGFR-driven lung adenocarcinomas. High response rates to osimertinib are observed in patients with activating EGFR mutations. However, there is variability in duration of response and patients eventually develop acquired resistance.Preclinical validation of potential acquired resistance mutations identified in relapsed patients treated with osimertinib is a key challenge to develop new strategies to improve outcomes for patients with EGFR mutation positive NSCLC. Tissue and ctDNA NGS analysis from relapsed patients treated first-line with osimertinib in the FLAURA and ORCHARD trials identified PIK3CA gain of function (GOF 11%) and PTEN loss of function (LOF 5%) mutations.To test whether PIK3CA activating mutations can drive resistance to osimertinib we introduced PIK3CA activating variants (PIK3CA-H1047R and PIK3CA-E453K) or knocked-out PTEN in NSCLC lung cancer cell lines harbouring EGFR activating mutations and WT PIK3CA/PTEN using CRISPR/Cas9 technology. Our results show that PIK3CA GOF and PTEN LOF mutations conferred resistance to osimertinib in vitro. Resistance was associated with increased EC50 for osimertinib and diminished apoptotic response in NSCLC PIK3CA GOF and PTEN LOF mutant CRISPR cell lines when compared to parental cells. Protein analysis by western blot also showed an increase in the basal and osimertinib treated levels of pAKT and pS6 in PIK3CA/PTEN CRISPR engineered cell lines, indicating activation of downstream PI3K/AKT and MAPK signalling pathways in the osimertinib-resistant cells. Importantly, our in vitro experiments from multiple cell line models indicated that PIK3CA-mediated resistance to osimertinib could be partially reversed by co-treatment with AKT (capivasertib, AstraZeneca) and PI3K alpha (alpelisib, Novartis) inhibitors; PTEN-mediated resistance could be rescued by co-treatment with capivasertib and PI3K beta (AZD8186, AstraZeneca) inhibitors. We next examined the in vivo efficacy of osimertinib + AKT/PI3K inhibitors therapies in NSCLC xenograft models. Our in vivo analysis show that PIK3CA GOF CRISPR engineered cells displayed diminished response to osimertinib when implanted in mice; importantly, combination with AKT (capivasertib) or PI3K alpha (alpelisib) inhibitors enhanced response on treatment and delayed outgrowth after withdrawal treatment. In addition, we observed that combination treatment with capivasertib induced tumour stasis in a PIK3CA-E454K and two PTENloss PDX models.Altogether our in vitro and in vivo data provide evidence of PIK3CA mutants and PTEN loss-driven mechanisms of resistance to osimertinib and offer possible therapeutic combination strategies for those patients that develop resistance or experience a sub-optimal response to osimertinib through PIK3CA/PTEN mutations Citation Format: Ursula Grazini, Daniel J. O'Neill, Matthew Martin, Chintia Xu, Nicolas Floch, Paul D. Smith, Emanuela Cuomo. PIK3CA and PTEN mutations as drivers of osimertinib resistance in patients with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5353.

Abstract 2557: Slit2 inhibits small cell lung cancer by targeting tumor microenvironment

Abstract Small cell lung cancer (SCLC) is the deadliest subtype of lung cancer with a 5-year survival rate of &amp;lt;5% in extensive-state disease and is designated by US Congress as a “recalcitrant cancer”. Recently approved by FDA, immune checkpoint blockade (ICB) immunotherapy showed marginally improved survival and only in a few patients, suggesting an inert immune tumor microenvironment (TME) and the existence of SCLC subtypes. Therefore, there is a significant need to develop more effective therapies for SCLC. In addition to the nearly universal biallelic inactivation of Rb and Tp53 genes in human SCLC tumors, comprehensive genome-wide screening studies have identified major co-occurring mutations in PTEN, CREBBP, EP300, SLIT2, MLL, COBL, and EPHA7 genes. Among them, SLIT2 showed the highest level of genomic abnormalities defined by a pronounced clustering of mutations and frequent genomic loss. However, the role of Slit2 in SCLC has not been defined yet. By using genetically engineered human SCLC cells overexpressing Slit2 or recombinant Slit2 (rSlit2) protein, we show that Slit2 inhibits SCLC cells proliferation, migration, and invasion properties and tumor sphere formation ability of drug-resistant SCLC cells in vitro. The mechanistic studies have identified inhibition of AKT and β-catenin mediated signaling pathways by Slit2 overexpression. Next, we implanted human SCLC cell lines into the immunocompromised NSG mice subcutaneously followed by treatment with rSlit2 or sterile saline. Analysis of tumor volume over the 4 weeks showed that rSlit2 treatment significantly reduced the growth of SCLC tumors. Further analysis of tumors showed that rSlit2 treated tumors harbor less M2-like CD206+ macrophages. Overall, these results suggest that Slit2 possesses tumor suppressive properties and could be developed as a novel therapeutic agent against SCLC. Citation Format: Dinesh K. Ahirwar, Manish Charan, Swati Misri, Konstantin Shilo, Ramesh Ganju. Slit2 inhibits small cell lung cancer by targeting tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2557.

Abstract 1181: PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy

Abstract Background: The clinical development of farnesyltransferase inhibitors (FTIs) as targeted therapy for HRAS-mutant cancers has demonstrated mixed responses dependent on cancer type. Co-occurring mutations may affect tumor response, supported by previous studies demonstrating that NF1 mutations confer resistance to HRAS inhibition by the FTI tipifarnib in thyroid cancer mouse models. We aimed to determine if PI3K pathway activating mutations altered responses to targeted therapy in HRAS-mutant cancers. Methods: Targeted sequencing data from MSK-IMPACT cohort and DFCI-GENIE (Version 9.0) database was used to investigate co-mutations amongst HRAS-mutant cancers. Fisher’s exact test was used to determine co-altered mutations found predominantly in HRAS-mutant cancers relative to respective KRAS- and NRAS-mutant cancers. ‘RASless’ (KRASlox/HRASKO/NRASKO) mouse embryonic fibroblasts (MEFs) were obtained that in the presence of 600nM tamoxifen (4OHT) resulted in a KRAS knock-out. ‘Rasless’ MEFs were transfected with HRASG13R to create a system for testing sensitivity to FTIs in the presence or absence of WT KRAS, or with concurrent PTEN loss generated by CRISPR-Cas9 technology. Results: A greater proportion of HRAS-mutant cancers had co-altered mutations (48.8%) in genes encoding effectors in the MAPK, PI3K or RTK pathways compared to KRAS- and NRAS-mutant cancers (41.4% and 38.4%, respectively; p&amp;lt;0.05). PTEN mutations were more prevalent in HRAS-mutant NSCLC (21%) compared to KRAS- and NRAS-mutant NSCLC (1% and 2%, respectively; p&amp;lt;0.05). Non-transfected MEFs were sensitized to tipifarnib by introduction of a HRASG13R allele in non-4OHT (IC50: MEF= &amp;gt;3uM, HRASG13R = 324.7nM) and 4OHT (IC50: MEF= &amp;gt;3uM, HRASG13R= 0.62nM; p&amp;lt;0.001) conditions, indicating that WT KRAS confers a relative resistance to the inhibitory effects of the FTI on HRAS. PTEN loss-of-function mutations led to tipifarnib resistance in HRASG13R MEFs in the absence (IC50: &amp;gt;3uM; p&amp;lt;0.001) or presence of 4OHT (IC50: 213.6nM; p&amp;lt;0.001). Combined treatment of HRASG13R/PTEN MEFs with the PIK3CB-specific inhibitor AZD8186 and tipifarnib sensitized cells in non-4OHT (IC50- 100nM:100nM Tipifarnib:AZD8186) and 4OHT (IC50- 100nM:10nM Tipifarnib:AZD8186) conditions. Conclusions: Co-altered mutations of MAPK, PI3K or RTK effectors are found more commonly in HRAS than in KRAS or NRAS-mutant cancers. Co-alteration of PTEN preferentially associated with HRAS-mutations in NSCLC. Deletion of PTEN resulted in resistance to FTI targeted therapy in vitro. Co-altered mutations may predict sensitivity and resistance to FTIs and guide clinical trial design. Citation Format: Aradhya Nigam, Walid K. Chatila, Gnana P. Krishnamoorthy, Alan L. Ho, James A. Fagin, Nikolaus D. Schultz, Brian R. Untch. PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1181.

Abstract 5129: Clinical implication of plasma ctDNA features in HER2-positive gastric cancer treated with combinations of trastuzumab &amp; anti-PD-1 agents

Abstract PURPOSE:Previous studies supported combining PD-1 inhibitors with trastuzumab to treat HER2-positive cancers. Yet proper biomarkers for response/resistance prediction remain unveiled. METHODS: Eligible patients were 18 years of age or older and had histologically confirmed gastric cancer (GC) or gastro-esophageal junction cancer (GEJ). The HER2 status was evaluated with tissue assay, including IHC and FISH. Paired plasma-white blood cell samples were collected prior treatment and assessed with targeted next-generation sequencing (NGS) of circulating tumor DNA (ctDNA). Different therapeutic regimens and ctDNA biomarkers were explored in HER2-positive patients, who received combining therapeutics containing trastuzumab and anti-PD-1 agents. The HER2-positive status was defined as IHC 3+ or IHC 2+/FISH-positive. RESULTS: A total of 49 patients were included. Four patients were excluded due to fail in the quality control for NGS. The HER2 status of tumor tissue and matched plasma ctDNA at baseline display a high concordance of 84.4% (38/45). NGS ctDNA method demonstrated an 84.2% (33/40) sensitivity and a 100% (5/5) specificity to detect HER2 amplification. Among HER2-positive patients, nineteen and eighteen patients were administrated trastuzumab+anti-PD-1±anti-vascular (chemo-free) and trastuzumab+anti-PD-1+chemotherapy (chemo-containing) respectively. Compared to Chemo-free subgroup, the chemo-containing subgroup had numerically higher ORR (61.11% vs 31.58%, p = 0.10) and longer median PFS (8.98 vs 5.43 months, p = 0.54, HR 0.78, 95%CI: 0.35-1.73). Patients with HER2 amplification in ctDNA had higher ORR than those without amplification (56.67% vs 0%, p = 0.009). Besides, an immune-related negative gene set (PTEN mutations, MDM2 amplification and FGF3/4/19 amplification) was also explored. The patients carrying immune-related negative gene alterations achieved decreased ORR (0% vs 54.8%, p = 0.02) and shorter PFS (2.1 vs 6.9 months, p&amp;lt;0.0001, HR 8.21, 95%CI: 2.76-24.48), compared to that of wild type. CONCLUSIONS: The ctDNA display a high consistency with tumor tissue for HER2 status. Compared to chemo-free regimens, chemo-containing regimens trend to demonstrate improved PFS and ORR in HER2-postive GC/GEJ, though no statistically significant. The HER2 amplification and immune-related negative gene alterations in ctDNA might be used as alternative biomarkers for predicting the efficacy of combining therapeutics containing trastuzumab and anti-PD-1 agents. Further studies are warranted to confirm our observation for better management of HER2-positive GC/GEJ. Citation Format: Xiaoyi Chong, Zhengqing Yan, Hua Liu, Lin Cong, Fangli Jiang, Chenhui Xu, Yan Li, Hui Chen, Yanyan Li, Congcong Ji, Shiqing Chen, Xiaochen Zhao, Xiaotian Zhang. Clinical implication of plasma ctDNA features in HER2-positive gastric cancer treated with combinations of trastuzumab &amp; anti-PD-1 agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5129.

Abstract 1504: Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development

Abstract Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole+/P286R;Trp53+/- mice showed accelerated cancer mortality compared to Pole+/P286R;Trp53+/+ mice. Cells from Pole+/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. Somewhat surprisingly, however, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole+/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Further analysis in cells suggests the presence of the mutant polymerase triggers DNA damage in specific contexts. Taken together, our analysis supports the idea that POLE-mediated hypermutagenesis is necessary, but not entirely sufficient, for tumorigenesis. Disabling surveillance of nuclear DNA damage is a likely sufficient factor. Citation Format: Meijuan J. Sun, Vivian S. Park, Wesley D. Frey, Leonard G. Williams, Karl P. Hodel, Juliet D. Strauss, Sydney J. Wellens, James G. Jackson, Zachary F. Pursell. Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1504.

Abstract 3413: Clinical utility of circulating tumor DNA sequencing with a large panel: The experience of Gustave Roussy/National Center for Precision Medicine (PRISM)

Abstract Background: Circulating tumor DNA (ctDNA) sequencing is a promising approach for testing gene alterations and tailoring therapy in cancer patients given, its limited invasiveness, high sensitivity and potential to comprehensively represent tumor heterogeneity. Here, we report the results from a single-center study conducted at Gustave Roussy (Villejuif, France) where we investigated the impact of comprehensive molecular profiling of ctDNA in patients with advanced solid tumors. Methods: Genomic analysis was performed using the Foundation One Liquid CDx Assay (324 genes, tumor mutational burden [TMB], microsatellite instability status). Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB) dedicated to precision medicine, attended by experts in clinical oncology, molecular biology, and clinical genetics. Actionable targets were defined by the MTB according to the existing level of evidence (classified by ESCAT tier), and molecular-based treatment suggestions were proposed where possible. Results: Between December 2020 and November 2021, 1772 patients with metastatic solid tumors underwent molecular profiling. Median time to assay results was 12 days. Results were contributive for 1658 patients (94%). At least one actionable target was detected in 1059 patients (64%) with a total of 1825 theragnostic alterations including : high blood TMB (&amp;gt; 16 mutations/Mb) (N= 243, 13%), alteration of the DNA damage repair response pathway (N=336, 18%), PIK3CA mutations (N=150, 8%), FGFR alterations (N= 67, 4%), MET activations (N=13, 0.7%), ERBB family pathway alterations (N=127, 7%) and PTEN mutations (N=95, 5%). Overall, the MTB recommended a matched therapy for 597 patients (56%) with a total of 819 therapeutic orientations: clinical trials (N= 639, 78%), off label/compassionate use (N=81, 10%), drug with a market authorization (N=51, 6%) and drug within an early access program (N=48, 6%). MTB did not recommend treatment for 462 patients (44%) with targetable molecular alterations for the following reasons: no clinical trial (N=421, 65%), matched treatment was already received (N=169, 26%), worsening of performance status (N= 49, 8%). Conclusions: This large-scale study demonstrates that liquid biopsy with a large NGS ctDNA panel is an efficient approach to match patients to genomically directed clinical trials/targeted therapies. Outcomes of patients treated with matched therapy will be presented at the meeting. Citation Format: Arnaud Bayle, Laila Belcaid, Miha Aldea, Florent Peyraud, Patricia Martin Romano, Félix Blanc-Durand, Rebecca Clodion, Santiago Ponce, Claudio Nicotra, Antoine Hollebecque, Yohann Loriot, Benjamin Besse, Damien Vasseur, Ludovic Lacroix, Etienne Rouleau, Jean-Charles Soria, Fabrice Barlesi, Geoffrey R. Oxnard, Fabrice Andre, Antoine Italiano. Clinical utility of circulating tumor DNA sequencing with a large panel: The experience of Gustave Roussy/National Center for Precision Medicine (PRISM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3413.

Abstract 156: Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter

Abstract Dysregulation of signal transduction pathways is associated with cancer initiation, progression, and recurrence. The PI3K/Akt signaling pathway has been extensively investigated as a therapeutic target due to its mechanistic association with several hallmarks of cancer. Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time. Here we demonstrate the utility of the Incucyte® Kinase Akt Lentivirus Reagent, encoding a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. To demonstrate inhibition of Akt, A549 cells stably expressing the reporter were treated with compounds targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103. Quantification of treatment responses using the Incucyte® Live Cell Analysis System showed concentration-dependent inhibition of Akt activity for all compounds with varying kinetic profiles over 24 hours. To study activation of Akt, HeLa cells were first cultured in the absence of serum to reduce Akt activity. After 4 hours of incubation in serum-free conditions, the cells were treated with either epidermal growth factor (EGF) or recombinant insulin-like growth factor (R3-IGF-1) to activate Akt. An increase in Akt activity was observed for both treatments. While R3-IGF-1-induced activation was sustained over the 12-hour time course, activation by EGF diminished over time. Cell lines with mutations in the tumor suppressor PTEN showed no response to serum starvation or activation with EGF or R3-IGF-1. In addition to monitoring Akt activity over time, the integrated red nuclear restricted protein of the reporter enables concurrent measurements of proliferation. The selective Akt inhibitor MK2206 decreased Akt activity in a similar concentration-dependent manner in both T-47D and MDA-MB-231 cell lines. In contrast, measurements of red object count from the same cells reveal differential effects of MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition of T-47D cell growth but little effect on MDA-MB-231 cells. Overall, these data highlight the utility of the Incucyte® Kinase Akt Lentivirus Reagent to provide valuable kinetic measurements of Akt activity using live cells within a physiologically relevant environment. Citation Format: John N. Rauch, Susan K. Foltin, Libuse Oupicka, Matthew Dilsaver, Grigory S. Filonov, Gillian Lovell, Jasmine Trigg, Cicely L. Schramm. Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 156.