PTEN Mutations Research Articles

Clinical description of two cases of Cowden syndrome and the implication regarding thyroid cancer.

Thyroid cancer is one of the most common manifestations of Cowden syndrome, yet the syndrome is rare. The incidence of Cowden syndrome is 1 in 200,000. The diagnosis can be made clinically when patients present with a combination of symptoms such as mucocutaneous lesions with a strong personal or family history of thyroid, breast, endometrial, and colorectal cancer. A high index of suspicion is required to provide a clinical diagnosis utilizing major and minor criteria. Once a clinical diagnosis is made, genetic testing for a PTEN mutation, a tumor suppressor gene, is recommended. Cancer surveillance should be performed for those with positive genetic testing as well as those with negative genetic testing who still meet clinical diagnostic criteria. We present two cases of Cowden syndrome: one case involving an increasing number of thyroid nodules in a patient with known Cowden syndrome and another patient with a strong family history of cancer, personal history of follicular thyroid cancer, and numerous colonic polyps on screening colonoscopy. These cases demonstrate how early diagnosis of Cowden syndrome can help detect early cancer in both the patient and affected relatives. Diagnosing Cowden syndrome helps pre-risk stratification for early cancer screening. The diagnosis of Cowden syndrome can be made with a combination of major and minor criteria: any two major criteria with or without a minor criterion; one major and one minor criterion; or three minor criteria. Patients who meet the diagnostic criteria for Cowden syndrome should undergo genetic screening.

Investigating the effects of PTEN mutations on cGAS-STING pathway in glioblastoma tumours

BackgroundPTEN is a tumour suppressor gene and well-known for being frequently mutated in several cancer types. Loss of immunogenicity can also be attributed to PTEN loss, because of its role in establishing the tumour microenvironment. Therefore, this study aimed to represent the link between PTEN and cGAS-STING activity, a key mediator of inflammation, in tumour samples of glioblastoma patients.MethodsTumour samples of 36 glioblastoma patients were collected. After DNA isolation, all coding regions of PTEN were sequenced and analysed. PTEN expression status was also evaluated by qRT-PCR, western blot, and immunohistochemical methods. Interferon-stimulated gene expressions, cGAMP activity, CD8 infiltration, and Granzyme B expression levels were determined especially for the evaluation of cGAS-STING activity and immunogenicity.ResultsMutant PTEN patients had significantly lower PTEN expression, both at mRNA and protein levels. Decreased STING, IRF3, NF-KB1, and RELA mRNA expressions were also found in patients with mutant PTEN. Immunohistochemistry staining of PTEN displayed expressional loss in 38.1% of the patients. Besides, patients with PTEN loss had considerably lower amounts of IFNB and IFIT2 mRNA expressions. Furthermore, CD8 infiltration, cGAMP, and Granzyme B levels were reduced in the PTEN loss group.ConclusionThis study reveals the immunosuppressive effects of PTEN loss in glioblastoma tumours via the cGAS-STING pathway. Therefore, determining the PTEN status in tumours is of great importance, like in situations when considering the treatment of glioblastoma patients with immunotherapeutic agents.

TGF-β and BMP signaling are associated with the transformation of glioblastoma to gliosarcoma and then osteosarcoma.

Gliosarcoma, an isocitrate dehydrogenase wildtype (IDH-WT) variant of glioblastoma, is defined by clonal biphasic differentiation into gliomatous and sarcomatous components. While the transformation from a glioblastoma to gliosarcoma is uncommon, the subsequent transformation to osteosarcoma is rare but may provide additional insights into the biology of these typically distinct cancers. We observed a patient initially diagnosed with glioblastoma, that differentiated into gliosarcoma at recurrence, and further evolved to osteosarcoma at the second relapse. Our objective was to characterize the molecular mechanisms of tumor progression associated with this phenotypic transformation. Tumor samples were collected at all 3 stages of disease and RNA sequencing was performed to capture their transcriptomic profiles. Sequential clonal evolution was confirmed by the maintenance of an identical PTEN mutation throughout the tumor differentiation using the TSO500 gene panel. Publicly available datasets and the Nanostring nCounter technology were used to validate the results. The glioblastoma tumor from this patient possessed mixed features of all 3 TCGA-defined transcriptomic subtypes of an IDH-WT glioblastoma and a proportion of osteosarcoma signatures were upregulated in the original tumor. Analysis showed that enhanced transforming growth factor-β (TGF-β) and bone morphogenic protein signaling was associated with tumor transformation. Regulatory network analysis revealed that TGF-β family signaling committed the lineage tumor to osteogenesis by stimulating the expression of runt-related transcription factor 2 (RUNX2), a master regulator of bone formation. This unusual clinical case provided an opportunity to explore the modulators of longitudinal sarcomatous transformation, potentially uncovering markers indicating predisposition to this change and identification of novel therapeutic targets.

“De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

Glioblastoma is a clinically and molecularly heterogeneous disease, and new predictive biomarkers are needed to identify those patients most likely to respond to specific treatments. Through prospective genomic profiling of 459 consecutive primary treatment-naïve IDH-wildtype glioblastomas in adults, we identified a unique subgroup (2%, 9/459) defined by somatic hypermutation and DNA replication repair deficiency due to biallelic inactivation of a canonical mismatch repair gene. The deleterious mutations in mismatch repair genes were often present in the germline in the heterozygous state with somatic inactivation of the remaining allele, consistent with glioblastomas arising due to underlying Lynch syndrome. A subset of tumors had accompanying proofreading domain mutations in the DNA polymerase POLE and resultant “ultrahypermutation”. The median age at diagnosis was 50 years (range 27–78), compared with 63 years for the other 450 patients with conventional glioblastoma (p < 0.01). All tumors had histologic features of the giant cell variant of glioblastoma. They lacked EGFR amplification, lacked combined trisomy of chromosome 7 plus monosomy of chromosome 10, and only rarely had TERT promoter mutation or CDKN2A homozygous deletion, which are hallmarks of conventional IDH-wildtype glioblastoma. Instead, they harbored frequent inactivating mutations in TP53, NF1, PTEN, ATRX, and SETD2 and recurrent activating mutations in PDGFRA. DNA methylation profiling revealed they did not align with known reference adult glioblastoma methylation classes, but instead had unique globally hypomethylated epigenomes and mostly classified as “Diffuse pediatric-type high grade glioma, RTK1 subtype, subclass A”. Five patients were treated with immune checkpoint blockade, four of whom survived greater than 3 years. The median overall survival was 36.8 months, compared to 15.5 months for the other 450 patients (p < 0.001). We conclude that “De novo replication repair deficient glioblastoma, IDH-wildtype” represents a biologically distinct subtype in the adult population that may benefit from prospective identification and treatment with immune checkpoint blockade.

Abstract A061: The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination

Abstract Background: Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive and rare malignancies accounting for 10% of all tissue sarcomas. Around 50% develop in patients with neurofibromatosis type 1 carrying mutations in the NF1 gene and the other 50% occur sporadically. NF1 is a tumor suppressor gene and a negative regulator of RAS signaling pathway. However, mutation of the NF1 gene is not sufficient to drive MPNST and a series of further mutational events must accumulate in genes other than NF1, such as in CDKN2A and PRC2 complex components. Mutations in p53 or PTEN and/or mutations or copy number variations in oncogenes, such as EGFR or c-Met can also be detected in MPNST. Herein we explore the effects of LSD1 inhibition with iadademstat as a single agent in a panel of MPNST cell lines, both NF1-related and sporadic, and in combination with inhibitors of the MEK-ERK and PI3K/AKT signaling pathways. Materials and Methods: The cell viability in 12 MPNST cell lines, both commercial and derived from patients’ primary tumors, was assessed after 6 days of treatment with iadademstat as a single agent. Synergisms between iadademstat and the MEK inhibitor selumetinib or the PI3K inhibitor copanlisib were also evaluated after 6 days of treatment in three selected responsive cell lines and analyzed with Calcusyn and Combenefit software. Additionally, the effects of iadademstat in RAS pathways (pAKT and pERK levels) were also evaluated in these cell lines. Results: Iadademstat displayed sub-nanomolar activity as a single agent in 8 out of 12 cell lines tested, both NF1-related and sporadic, with different degrees of viability reduction. This was further confirmed with two additional LSD1 inhibitors, with iadademstat being the most potent inhibitor tested. Highly-responsive cell lines showed a decrease in pAKT levels while medium- and low-responsive cell lines displayed no significant differences. pERK levels were not affected. Moreover, the combination of iadademstat and selumetinib or copanlisib displayed strong synergy in cell viability assays in both commercial and patient-derived iadademstat-sensitive cell lines. Conclusions: The LSD1 inhibitor iadademstat is efficacious as single agent in nearly 70% of the MPNST cell lines tested, both NF1-related and sporadic and this efficacy increases synergistically in combination with selumetinib or copanlisib. Therefore, LSD1 inhibitors alone or in combination emerge as a potential treatment for MPNST, an indication with high unmet needs with no approved targeted therapies available. Citation Format: Natalia Sacilotto, Cristina Mascaró, Edgar Creus, Juana Fernández, Conxi Lázaro, Ana Limón, Robert Soliva, Jordi Xaus. The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A061.

Abstract B017: Clinical utility of next generation sequencing in advanced colorectal cancer: The earlier the better

Abstract Background. Since next generation sequencing (NGS) was approved in solid tumor in Korea, it has been usually performed when patients were progressed on standard chemotherapy in advanced colorectal cancer (CRC). Recently, new targeted agents have been developed rapidly, so the NGS is recommended as early as possible. In this study, we analyzed NGS results and investigated clinical benefit in advanced CRC. Patient and method. Between October 2018 to December 2022, NGS data in 85 advanced CRC patients in St. Vincent’s Hospital, Suwon, Korea were retrospectively analyzed. Results. Seven patients had metachronous, and others had synchronous metastasis. The median age was 58 (34-84), and 59 (69.4%) were male. Fourteen (16.5%) were right-side, 40 (47.1%) were left-side colon, and 30 (35.3%) were rectum. The median time from diagnosis of advanced CRC to NGS was 13 (0-81) months, and median time to death after NGS was 8 (0.3-43.0) months. NGS platform were ‘Oncomine comprehensive assay Plus’ in 71 (83.5%), and the others were various platforms in K-master project. NGS analyses revealed new therapeutic target in 17 (20%): high tumor mutational burden (n = 2), KRAS G12C (n = 4), KRAS (n = 1) or BRAF (n = 2) mutation not routinely screened at initial, BRAF class II mutation (n = 2), Her2/3 mutation (n = 2), and NTRK fusion (n = 1). Among those, two patients with KRAS G12C mutation enrolled in clinical trial of KRAS G12 inhibitor, and one patient with NTRK fusion received NTRK inhibitor (3.5% of total). Among RAS wild type population (n=30), we identified hyperselected patients (n = 18, [60%], absence of either KRAS, NRAS, PTEN, and extracellular domain EGFR mutations, HER2 and MET amplifications, and ALK, RET or NTRK1 fusions by PARADIGM trial), which showed longer survival in cetuximab-treated population (p&amp;lt;0.001). Specific mutation including KRAS, NRAS, BRAF, TP53, or APC did not impact on prognosis. High TMB (≥10Mut/Mb) were associated with longer survival (p = 0.020). For patients with oligometastasis, hyperselected patients (p = 0.062) showed good prognosis. Conclusion. The NGS results provided additional predictive or prognostic information in advanced CRC patients. Application of NGS as early as possible might help to make a clinical decision. Citation Format: Ho Jung An, Hyunho Kim, Hyung Soon Park, Hyeon-Min Cho, Bong-Hyeon Kye, Ri Na Yoo, Ji-Yeon Moon, Sung Hwan Kim, Jonghoon Lee, Hyo Chun Lee. Clinical utility of next generation sequencing in advanced colorectal cancer: The earlier the better [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B017.

The identification of a PTEN-associated gene signature for the prediction of prognosis and planning of therapeutic strategy in endometrial cancer.

Endometrial cancer (EC) is one of the most common malignancies among women. To improve the prognosis and treatment of EC, finding out a phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-associated prognostic signature would be beneficial. EC clinical data, genetic mutation data, and transcriptome data were downloaded from The Cancer Genome Atlas (TCGA) database. To clarify the specific PTEN-associated signature, cox regression analyses were performed. The clinical value of the selected signature on the overall survival (OS) and the secretoglobin family 2A member 1 (SCGB2A1)-independent analysis, immune and functional analysis were investigated respectively. Five hundred and fourteen EC samples were screened and PTEN mutation occupied 57%. Enrichment analysis indicated that mutant-type PTEN was enriched for pathways related to the upregulated human T-cell leukemia virus-1 (HTLV-1) infection and estrogen signaling pathway. SCGB2A1 was identified by cox regression analysis. Immune analysis exhibited significant immune infiltration with higher expression of T cells, B cells, and macrophage groups. Immune-checkpoint transcripts CD274 molecule (CD274), and cytotoxic T-lymphocyte associated protein 4 (CTLA4), hepatitis A virus cellular receptor 2 (HAVCR2), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PDCD1), PDCD1 ligand 2 (PDCD1LG2), T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), and sialic acid binding immunoglobulin like lectin 15 (SIGLEC15) were discovered statistically different. In addition, the low-SCGB2A1 group had worse OS than the high-SCGB2A1 group. SCGB2A1 showed significant area under the curve (AUC) values in a time-dependent receiver operating characteristic (ROC) analysis. Prevalence of microsatellite instability (MSI) was detected and SCGB2A1 showed a negative correlation with EC. Immune checkpoint blockade (ICB) response indicated a worse immune response in the low-SCGB2A1 group. The distribution of one-class linear regression (OCLR) scores reflected the negative correlation between messenger RNA expression-based stemness index (mRNAsi) and prognostic gene expression. Furthermore, several SCGB2A1-related signaling pathways in EC were identified. SCGB2A1 is a prognostic immunometabolic signature for patients with EC, which may help improve the prognosis and therapeutic effect.

Abstract C020: First-in-human phase 1/1b trial of the first-in-class bi-steric mTORC1-selective inhibitor RMC-5552 in patients with advanced solid tumors

Abstract Activation of the mTOR pathway plays a role in the oncogenesis and progression of many cancer types, and is frequently co-altered in RAS-driven tumors. First-generation mTORC1-selective inhibitors were limited by incomplete inhibition of 4EBP1 phosphorylation, while second-generation pan-mTOR inhibitors had a low therapeutic index due to mTORC2-mediated toxicity such as hyperglycemia. RMC-5552 is a first-in-class third-generation bi-steric mTOR inhibitor that is mTORC1-selective via its FKBP12-binding moiety and potently inhibits phosphorylation of the 4EBP1 tumor suppressor via a linked ATP-competitive moiety, addressing these key limitations of prior mTOR inhibitors in preclinical models. RMC-5552 is being investigated in a Phase I/Ib study (NCT04774952). Eligible patients (pts) were ≥18 yrs old with ECOG 0-1 and had advanced solid tumors that had failed or were ineligible for standard-of-care treatments. Pts received RMC-5552 as a weekly infusion. To address mucositis, a common toxicity in pts treated with mTOR inhibitors, pts prophylactically used dexamethasone mouthwash either alone or with tacrolimus mouthwash (TM), which binds to the RMC-5552 presenter protein FKBP12. Objectives included assessment of safety/tolerability (CTCAEv5), defining the maximum tolerated and recommended Phase 2 doses, pharmacokinetics, and preliminary efficacy (RECIST v1.1). As of June 12, 2023, 47 pts have been evaluated at 6 dose levels from 1.6 mg to 12 mg. The most common (&amp;gt; 20%) treatment-related adverse events (TRAE, any grade) were stomatitis/mucositis (45%), fatigue (40%), nausea (38%), decreased appetite (32%), and vomiting (23%). Treatment-related events of hyperglycemia occurred in 4% of pts (both grade 2) and were not dose limiting. The most common grade 3 TRAE at dose levels ≥6 mg was mucositis/stomatitis, which was dose-dependent and limited tolerability in the absence of TM. Between 8 and 12 mg, the rate of treatment related stomatitis/mucositis was 65% (15% grade 3) in pts treated without TM (N = 20) versus 30% (no grade 3) in pts treated with TM (N = 10). TM prophylaxis enabled dose escalation beyond dose levels previously considered intolerable due to mucositis (10-12 mg; escalation ongoing), and unbound plasma exposures at 8-12 mg were within the range that induced significant anti-tumor activity in preclinical models. The disease control rate was 67% among efficacy evaluable pts (N = 39). One pt with a salivary gland tumor harboring a PTEN mutation had a confirmed partial response (PR) with 78% reduction and 16 months duration of response. In addition, molecular responses of circulating tumor DNA were observed in pts with mTOR pathway variants. Updated efficacy results will be presented. These early data indicate that RMC-5552 monotherapy is tolerated at doses predicted to be efficacious, and that selective inhibition of mTORC1 alleviates mTORC2-mediated toxicity. Dose escalation is ongoing, and mucositis prophylaxis has enabled further dose intensification for future combination studies with RAS(ON) inhibitors. Citation Format: Alison M Schram, Abdul Rafeh Naqash, Eric B Haura, Jonathan W Riess, Susanna V Ulahannan, Sai-Hong I Ou, Anna Capasso, Pamela N Munster, Michael L Cheng, W Clay Gustafson, Bojena Bitman, Sumit Kar, Zhican Wang, Lin Tao, Justin G Meyerowitz, Howard A Burris. First-in-human phase 1/1b trial of the first-in-class bi-steric mTORC1-selective inhibitor RMC-5552 in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C020.

Germline PTEN genotype-dependent phenotypic divergence during the early neural developmental process of forebrain organoids.

PTEN germline mutations account for ~0.2-1% of all autism spectrum disorder (ASD) cases, as well as ~17% of ASD patients with macrocephaly, making it one of the top ASD-associated risk genes. Individuals with germline PTEN mutations receive the molecular diagnosis of PTEN Hamartoma Tumor Syndrome (PHTS), an inherited cancer predisposition syndrome, about 20-23% of whom are diagnosed with ASD. We generated forebrain organoid cultures from gene-edited isogenic human induced pluripotent stem cells (hiPSCs) harboring a PTENG132D (ASD) or PTENM134R (cancer) mutant allele to model how these mutations interrupt neurodevelopmental processes. Here, we show that the PTENG132D allele disrupts early neuroectoderm formation during the first several days of organoid generation, andresults in deficient electrophysiology. While organoids generated from PTENM134R hiPSCs remained morphologically similar to wild-type organoids during this early stage in development, we observed disrupted neuronal differentiation, radial glia positioning, and cortical layering in both PTEN-mutant organoids at the later stage of 72+ days of development. Perifosine, an AKT inhibitor, reduced over-activated AKT and partially corrected the abnormalities in cellular organization observed in PTENG132D organoids. Single cell RNAseq analyses on early-stage organoids revealed that genes related to neural cell fate were decreased in PTENG132D mutant organoids, and AKT inhibition was capable of upregulating gene signatures related to neuronal cell fate and CNS maturation pathways. These findings demonstrate that different PTEN missense mutations can have a profound impact on neurodevelopment at diverse stages which in turn may predispose PHTS individuals to ASD. Further study will shed light on ways to mitigate pathological impact of PTEN mutants on neurodevelopment by stage-specific manipulation of downstream PTEN signaling components.

Source, co-occurrence, and prognostic value of PTEN mutations or loss in colorectal cancer

Somatic PTEN mutations are common and have driver function in some cancer types. However, in colorectal cancers (CRCs), somatic PTEN-inactivating mutations occur at a low frequency (~8–9%), and whether these mutations are actively selected and promote tumor aggressiveness has been controversial. Analysis of genomic data from ~53,000 CRCs indicates that hotspot mutation patterns in PTEN partially reflect DNA-dependent selection pressures, but also suggests a strong selection pressure based on protein function. In microsatellite stable (MSS) tumors, PTEN alterations co-occur with mutations activating BRAF or PI3K, or with TP53 deletions, but not in CRC with microsatellite instability (MSI). Unexpectedly, PTEN deletions are associated with poor survival in MSS CRC, whereas PTEN mutations are associated with improved survival in MSI CRC. These and other data suggest use of PTEN as a prognostic marker is valid in CRC, but such use must consider driver mutation landscape, tumor subtype, and category of PTEN alteration.

Prostate cancer patient stratification by molecular signatures in the Veterans Precision Oncology Data Commons.

Veterans are at an increased risk for prostate cancer, a disease with extraordinary clinical and molecular heterogeneity, compared with the general population. However, little is known about the underlying molecular heterogeneity within the veteran population and its impact on patient management and treatment. Using clinical and targeted tumor sequencing data from the National Veterans Affairs health system, we conducted a retrospective cohort study on 45 patients with advanced prostate cancer in the Veterans Precision Oncology Data Commons (VPODC), most of whom were metastatic castration-resistant. We characterized the mutational burden in this cohort and conducted unsupervised clustering analysis to stratify patients by molecular alterations. Veterans with prostate cancer exhibited a mutational landscape broadly similar to prior studies, including KMT2A and NOTCH1 mutations associated with neuroendocrine prostate cancer phenotype, previously reported to be enriched in veterans. We also identified several potential novel mutations in PTEN, MSH6, VHL, SMO, and ABL1 Hierarchical clustering analysis revealed two subgroups containing therapeutically targetable molecular features with novel mutational signatures distinct from those reported in the Catalogue of Somatic Mutations in Cancer database. The clustering approach presented in this study can potentially be used to clinically stratify patients based on their distinct mutational profiles and identify actionable somatic mutations for precision oncology.

Prognostic and predictive biomarkers for anti-EGFR monoclonal antibody therapy in RAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis

BackgroundRAS mutations affect prognosis in patients with metastatic colorectal cancer (mCRC) and have been identified as strong negative predictive markers for anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy, but many tumors containing wild-type RAS genes still do not respond to these therapies. Some additional biomarkers may have prognostic or predictive roles, but conclusions remain controversial.MethodsWe performed a meta-analysis and systematic review of randomized controlled trials comparing anti-EGFR mAb therapy with alternative therapy that investigated the prognostic and predictive impact of additional biomarkers in RAS wild-type (wt) mCRC patients. Hazard ratios (HRs) and 95% confidence intervals (CIs) for progression-free survival (PFS) and overall survival (OS) and odds ratios (ORs) for objective response rate (ORR) were calculated. The prognostic value of biomarkers was investigated by separately pooling HR and OR for different treatment groups in an individual study. The predictive value was assessed by pooling study interactions between treatment effects and biomarker subgroups.ResultsThirty publications reporting on eighteen trials were selected, including a total of 13,507 patients. In prognostic analysis, BRAF mutations were associated with poorer PFS [HRs = 3.76 (2.47–5.73) and 2.69 (1.82–3.98)] and OS [HRs = 2.66 (1.95–3.65) and 2.45 (1.55–3.88)] in both the experimental and control arms; low miR-31-3p expression appeared to have longer PFS and OS. In terms of predictive effect, a lack of response to anti-EGFR therapy was observed in patients with BRAF mutant tumors (Pinteraction < 0.01 for PFS). Patients with tumors with any mutation in the KRAS/NRAS/BRAF/PIK3CA gene also showed similar results compared with all wild-type tumors (Pinteraction for PFS, OS, and ORR were < 0.01, < 0.01 and 0.01, respectively). While low miR-31-3p expression could predict PFS (Pinteraction = 0.01) and OS (Pinteraction = 0.04) benefit. The prognostic and predictive value regarding PIK3CA mutations, PTEN mutations or deletions, EGFR, EREG/AREG, HER2, HER3, and HER4 expression remains uncertain.ConclusionsIn RAS wt mCRC patients receiving EGFR-targeted therapy, BRAF mutation is a powerful prognostic and therapy-predictive biomarker, with no effect found for PIK3CA mutation, PTEN mutation or deletion, but the combined biomarker KRAS/NRAS/BRAF/PIK3CA mutations predict resistance to anti-EGFR therapy. Low miR-31-3p expression may have positive prognostic and therapy predictive effects. Evidence on the prognostic and predictive roles of EGFR and its ligands, and HER2/3/4 is insufficient.

Multi-omic profiling reveals discrepant immunogenic properties and a unique tumor microenvironment among melanoma brain metastases

Melanoma brain metastases (MBM) are clinically challenging to treat and exhibit variable responses to immune checkpoint therapies. Prior research suggests that MBM exhibit poor tumor immune responses and are enriched in oxidative phosphorylation. Here, we report results from a multi-omic analysis of a large, real-world melanoma cohort. MBM exhibited lower interferon-gamma (IFNγ) scores and T cell-inflamed scores compared to primary cutaneous melanoma (PCM) or extracranial metastases (ECM), which was independent of tumor mutational burden. Among MBM, there were fewer computationally inferred immune cell infiltrates, which correlated with lower TNF and IL12B mRNA levels. Ingenuity pathway analysis (IPA) revealed suppression of inflammatory responses and dendritic cell maturation pathways. MBM also demonstrated a higher frequency of pathogenic PTEN mutations and angiogenic signaling. Oxidative phosphorylation (OXPHOS) was enriched in MBM and negatively correlated with NK cell and B cell-associated transcriptomic signatures. Modulating metabolic or angiogenic pathways in MBM may improve responses to immunotherapy in this difficult-to-treat patient subset.

PATH-31. RECURRENT COPY NUMBER CHANGES IN GLIOBLASTOMA, IDH-WILDTYPE: ARE OTHER +7/-10 PATTERNS CLINICALLY RELEVANT?

Abstract Glioblastoma (GBM), IDH-wildtype (WT) is currently defined as an IDH-WT and H3-WT diffuse astrocytic glioma with molecular diagnostic features including whole +7/−10, TERT promoter (TERTp) mutation, and EGFR amplification and/or morphological diagnostic features such as necrosis and microvascular proliferation. To further understand the diversity of copy number signatures of GBM, IDHWT and evaluate the diagnostic utility of other recurrent copy number changes, we analyzed paired chromosomal microarray and next-generation sequencing mutational profiling data from 418 adult IDH-WT and H3-WT tumors with GBM morphological diagnostic features. The most common molecular features were TERTp mutations (85%), CDKN2A/B heterozygous/homozygous deletion (78%/66%), +7/-10 (63%), EGFR amplification (41%), +19 (36%), PTEN mutations (35%), partial -13 (33%), partial -14 (33%), +20 (32%), TP53 mutations (32%), and partial -22 (29%). At least one molecular diagnostic biomarker was present in 92% (n=385) of cases, and 29% (n=121) of cases had all three. The +7/-10 biomarker was the most frequent chromosomes 7/10 copy number change (68%, n=265). Other +7/-10 patterns were seen in 30% (n=125) of cases and included isolated +7 or -10, combinations of +7 and -10 with partial +7, partial -10 or 10 copy neutral loss of heterozygosity (cnLOH). Overall, 93% (390/418) of cases had whole and other chromosome 7/10 copy number changes. In comparison to tumors with +7/-10, tumors with other +7/-10 patterns were significantly enriched for partial 13 loss (Bonferroni adjusted p=0.04) but were not associated with significant difference in patient overall survival (p=0.30, 158/418 had available survival data). Our findings reiterate and expand the spectrum of copy number changes associated with GBM, IDH-WT. The similar overall survival rates between cases with the +7/-10 biomarker and those with other +7/-10 patterns suggest that other +7/-10 patterns may be functionally equivalent to +7/-10 and diagnostically relevant as a molecular diagnostic biomarker for this tumor type.

BIOM-31. TARGETED DNA SEQUENCING OF RECURRENT GLIOBLASTOMA IDENTIFIES MOLECULAR FACTORS ASSOCIATED WITH CLINICAL OUTCOMES

Abstract PURPOSE The goal of our project was to define the molecular features of recurrent glioblastoma (rGBM) and their prognostic significance. METHODS We retrospectively reviewed 121 consecutive glioblastoma patients who underwent re-resection at a single institution from 2016-2021. Primary treatment in all patients included maximal safe resection followed by radiotherapy with concurrent and adjuvant temozolomide (TMZ). Capture-based targeted DNA sequencing for point mutations and copy number analysis was performed on all re-resection samples. Overall (OS) and progression-free survival (PFS) from re-resection were analyzed with the Kaplan-Meier method and Cox regression analyses. RESULTS Median age at first recurrence was 57 years (range, 27-78) and follow up from re-resection was 11 months. Salvage therapy comprised re-irradiation (n = 52, 43%), temozolomide (TMZ) (n = 39, 32%), lomustine (n = 47, 39%), and/or bevacizumab (n = 22, 18%). Median OS and PFS were 12.1 and 4.4 months, respectively. Median tumor mutation burden (TMB) was 4 mut/Mb (range, 1-650), average unstable microsatellite sites (MSI) were 1.16% (range, 0-20), and MGMT was methylated in 71 (59%) patients. Longer OS was found after postoperative TMZ (19.1 vs. 9.1), KPS &amp;gt; 70 (13.6 vs. 11.1), and/or re-irradiation (12.6 vs. 8.4) (p&amp;lt; .05). On multivariate analysis, TMZ (HR 0.2, 0.079-0.48), re-irradiation (HR 0.27, 0.12-0.58), and mutations in PDGFRA (HR 0.24, 0.067-0.83) or PTEN (HR 2.3, 1.1-4.8) genes were associated with longer OS after re-resection (p&amp;lt; .05). Multivariate predictors of re-irradiation response included MSI &amp;gt;1.16 (HR 0.25, 0.084-0.75), and mutations in PDGFRA (HR 0.04, 0.005-0.33), TERT (HR 0.16, 0.033-0.78), or PIK3R1 (HR 0.18, 0.041-0.8) genes. In the 46 matched primary/recurrent tumors, 40 (87%%) had a change in a mutation and 9 (20%) demonstrated hypermutation, of which 4 (44%) had mismatch repair deficiencies. CONCLUSIONS Our study outlines the molecular factors associated with survival and response to re-irradiation in rGBM as well as, the molecular differences between primary/recurrent samples.

Outcomes of fertility preservation treatments in patients with endometrial cancer with different molecular classifications based on an NGS panel.

The molecular classification of endometrial cancer has previously been shown to be associated with clinical outcomes. However, there are insufficient data to support the routine use of molecular classification for the treatment of patients seeking fertility preservation. Here, we retrospectively investigated 90 patients received fertility-sparing treatment. We used a next generation sequencing (NGS) panel to classify these patients into four subtypes. All patients received hormonal therapy combined with hysteroscopy. Therapeutic effects were evaluated by hysteroscopy every three months during the treatment. Patients with POLE mutations had the highest disease progression rate (50.0%, P=0.013), while the microsatellite instability-high (MSI-H) group had the highest recurrence rate (50.0%, P=0.042). PIK3CA mutation (hazard ratio (HR): 0.61; 95% confidence interval (CI): 0.37-0.99; P=0.046), overweight (HR: 0.56; 95% CI: 0.32-0.96; P=0.033) and obesity (HR: 0.44; 95% CI: 0.20-0.95; P=0.036) were associated with a significantly lower cumulative complete response (CR) rate. The combination of gonadotropin-releasing hormone analogues (GnRH-a) and letrozole (HR: 3.43; 95% CI: 1.81-6.52; P< 0.001) was associated with a significantly higher cumulative CR rate. KRAS mutation was significantly associated with disease progression (P=0.002). In wild-type TP53 patients, PTEN and PIK3CA mutations significantly prolonged the duration of treatment to achieve CR (log rank P=0.034; P=0.018). The implementation of molecular classification for EC patients undergoing fertility-sparing treatment is promising and can facilitate the selection of appropriate medical regimes to achieve better outcomes in patients with EC who require fertility preservation treatment.

Cancer and Autism: How PTEN Mutations Degrade Function at the Membrane and Isoform Expression in the Human Brain

Mutations causing loss of PTEN lipid phosphatase activity can promote cancer, benign tumors (PHTS), and neurodevelopmental disorders (NDDs). Exactly how they preferentially trigger distinct phenotypic outcomes has been puzzling. Here, we demonstrate that PTEN mutations differentially allosterically bias P loop dynamics and its connection to the catalytic site, affecting catalytic activity. NDD-related mutations are likely to sample conformations of the functional wild-type state, while sampled conformations for the strong, cancer-related driver mutation hotspots favor catalysis-primed conformations, suggesting that NDD mutations are likely to be weaker, and our large-scale simulations show why. Prenatal PTEN isoform expression data suggest exons 5 and 7, which harbor NDD mutations, as cancer-risk carriers. Since cancer requires more than a single mutation, our conformational and genomic analysis helps discover how same protein mutations can foster different clinical manifestations, articulates a role for co-occurring background latent driver mutations, and uncovers relationships of splicing isoform expression to life expectancy.

Identification of cancer driver mutations in liquid-based cytology samples for the screening of endometrial diseases

BackgroundEndometrial cancer (EC) is one of the leading causes of cancer death among women and early detection is crucial for its successful treatment. We previously showed that cytological examination combined with genetic analysis using liquid-based cytology (LBC) samples improved the diagnostic sensitivity of EC.MethodsAmong 218 individuals who underwent endometrial screening by LBC, 208 samples were analysed by cancer-panel sequencing. Excluding 13 samples with low sequence coverage, we further analysed 195 samples.ResultsAmong the 195 women analysed, 39 and 15 were eventually shown to suffer from malignant endometrial and non-endometrial neoplasms, respectively. Driver mutation(s) were found in nine of the 15 cytology-negative patients with endometrial malignancy, corroborating that combination of cytological and genetic analyses should increase the sensitivity of the diagnosis of malignant endometrial neoplasms. Importantly, driver alterations were found in 32 of 125 women without malignant or premalignant diseases, which raises caution for the interpretation of genetic alterations detected in the endometrial samples. Comparison between the driver mutations in the 32 subjects and those in the 29 endometrial malignancies unveiled that the number of mutations and mutations in PTEN, CTNNB1, and TP53 may be applied for the assessment and prediction of development of EC.ConclusionsThe genetic analysis combined with liquid-based cytology is useful to improve the diagnostic sensitivity of endometrial neoplasms. However, caution must be taken when cancer-associated mutation(s) are detected in the genetic analysis. Further investigation may clarify the risk of malignant endometrial neoplasms in women with driver mutation(s).

Mutational Landscape and In-Silico Analysis of TP53, PIK3CA, and PTEN in Patients with Breast Cancer from Khyber Pakhtunkhwa.

Herein, we report the mutational spectrum of three breast cancer candidate genes (TP53, PIK3CA, and PTEN) using WES for identifying potential biomarkers. The WES data were thoroughly analyzed using SAMtools for variant calling and identification of the mutations. Various bioinformatic tools (SIFT, PolyPhen-2, Mutation Taster, ISPRED-SEQ, SAAFEQ-SEQ, ConSurf, PROCHECK etc.) were used to determine the pathogenicity and nature of the SNVs. Selected interaction site (IS) mutations were visualized in PyMOL after building 3D structures in Swiss-Model. Ramachandran plots were generated by using the PROCHECK server. The selected IS mutations were subjected to molecular dynamic simulation (MDS) studies using Gromacs 4.5. STRING and GeneMANIA were used for the prediction of gene-gene interactions and pathways. Our results revealed that the luminal A molecular subtype of the breast cancer was most common, whereas a high percentage of was Her2 negatives. Moreover, the somatic mutations were more common as compared to the germline mutations in TP53, PIK3CA, and PTEN. 20% of the identified mutations are reported for the first time from Khyber Pakhtunkhwa. In the enrolled cohort, 23 mutations were nonsynonymous SNVs. The frequency of mutations was the highest in PIK3CA, followed by TP53 and PTEN. A total of 13 mutations were found to be highly pathogenic. Four novel mutations were identified on PIK3CA and one each on PTEN and TP53. SAAFEQ-SEQ predicted the destabilizing effect for all mutations. ISPRED-SEQ predicted 9 IS mutations (6 on TP53 and 3 on PIK3CA), whereas no IS mutation was predicted on PTEN. The TP53 IS mutations were TP53R43H, TP53Y73X, TP53K93Q, TP53K93R, TP53D149E, and TP53Q199X; whereas for PIK3CA, the IS mutations were PIK3CAL156V, PIK3CAM610K, and PIK3CAH1047R. Analysis from the ConSurf Web server revealed five SNVs with a highly conserved status (conservation score 9) across TP53 and PTEN. TP53P33R was found predominant in the grade 3 tumors, whereas PTENp.C65S was distributed on ER+, ER-, PR+, PR-, Her2+, and Her2- patients. TP53p.P33R mutation was found to be recurring in the 14/19 (73.6%) patients and, therefore, can be considered as a potential biomarker. Finally, these mutations were studied in the context of their potential association with different hormonal and social factors.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced &amp;gt; 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.