Oncogenic Driver Research Articles

Response to targeted and immune therapy in patients with NSCLC and BRAF non-V600E mutations: A retrospective, multicenter analysis within the national Network Genomic Medicine Lung Cancer (nNGM) in Germany.

8591 Background: Non-small-cell lung cancer (NSCLC) poses a significant global health challenge, with multiple treatment strategies targeting oncogenic drivers. The BRAF V600E mutation, found in 1-3% of NSCLC patients, is effectively targeted by dabrafenib and trametinib, achieving considerable clinical benefits. However, BRAF non-V600E mutations, constituting half of all BRAF mutations, are challenging to treat due to their heterogeneity and lack of information concerning outcome upon targeted and immune therapies. Methods: In order to investigate outcome of patients with BRAF non-V600E mutations upon targeted and (chemo)immune therapy we conducted a retrospective analysis within the national Network Genomic Medicine (nNGM) lung cancer in Germany and. In addition, we evaluated the MEK/ERK activation potential of the BRAF non-V600E mutations from our cohort and their sensitivity to dabrafenib and/or trametinib in vitro. Results: Twenty patients with 11 different BRAF non-V600E mutations were identified. From these patients, 15% achieved partial remission, 40% stable disease, and 40% progressive disease under TKI-therapy. Progression-free survival (PFS) upon targeted therapy ranged from 0.9 to 53.5 months. Notably, patients with MEK/ERK-activating BRAF non-V600E mutations exhibited persistent responses (mPFS 9 months). Specifically, patients with the high-activity mutants p.V600D, p.V600K and p.K601E showed responses to TKI therapy (mPFS 9.2 months). Similar outcome was achieved in patients with activating p.G466V (PFS 4.9 months), p.G469R (PFS 9 months), p.G469V (mPFS 28.2 months) and p.V600_K601delinsE (mPFS 5.6 months) mutations. Interestingly, outcome upon treatment with (chemo)immune therapy for these patients with activating mutations was similar compared to targeted therapy (mPFS 15.0 and 11.7 months). Expectedly, patients with kinase-dead mutants (p.G466R, p.D594E, p.D594G) were not responding to TKIs (mPFS 2.5 months) and had considerably better outcomes under (chemo)immune therapy (mPFS 11.0 and 11 months). Conclusions: Patients with activating BRAF non-V600E mutations can be treated efficiently with BRAF/MEK TKI or (chemo)immune therapy while patients with non-activating mutations do not benefit from targeted therapy but from (chemo)immune therapy. Hence, it is of key importance to apply preclinical activation assays showing good correlations to clinical outcomes in our study prior to applying targeted therapy in patients with unknown BRAF non-V600E mutations. Altogether, our study helps to guide personalized treatment decisions in this patient population.

Prediction of oncogene mutation status in non-small cell lung cancer leveraging radiomics from CT scans: A systematic review and meta-analysis with a special focus on artificial-intelligence-based methods.

e20603 Background: In non-small cell lung cancer (NSCLC), alternative strategies to determine patient oncogene mutation status are essential to overcome some of the drawbacks associated with current methods. We aimed to review the use of radiomics alone or in combination with clinical data and to evaluate the performance of artificial intelligence (AI)-based models on the prediction of oncogene mutation status. Methods: A PRISMA-compliant literature review was conducted. The Medline (via Pubmed), Embase, and Cochrane Library databases were searched for studies published through June 30, 2023, predicting oncogene mutation status in patients with NSCLC using radiomics. Independent meta-analyses evaluating the performance of AI-based models developed with radiomics features or with a combination of radiomics features plus clinical data for the prediction of different oncogenic driver mutations were performed. A meta-regression to analyze the influence of methodological/clinical factors was also conducted. Results: Out of the 615 studies identified, 89 evaluating models for the prediction of epidermal growth factor-1 (EGFR), anaplastic lymphoma kinase (ALK), and Kirsten rat sarcoma virus (KRAS) mutations were included in the systematic review, including a total of 32,084 patients. A total of 38 met the inclusion criteria for the meta-analyses (encompassing 17,066 patients). The AI algorithms' sensitivity/false positive rate (FPR) in predicting EGFR, ALK, and KRAS mutations using radiomics-based models was 0.753 (95% CI 0.721–0.783)/0.346 (95% CI 0.305–0.390), 0.754 (95% CI 0.639–0.841)/ 0.225 (95% CI 0.163–0.302), and 0.744 (95% CI 0.605–0.846)/0.376 (95% CI 0.274–0.491), respectively. A meta-analysis of combined models was only possible for EGFR mutation, revealing a sensitivity/FPR of 0.800 (95% CI 0.767–0.830)/0.335 (95% CI 0.279–0.396). No statistically significant results were obtained in the meta-regression. Conclusions: Radiomics with a specific focus on CT-based models may represent valuable non-invasive tools for the determination of oncogene mutation status in NSCLC. Further investigation is required to analyze whether clinical data might boost their performance.

PD-(L)1 inhibitors plus bevacizumab and chemotherapy as first-line therapy in PD-L1 negative metastatic lung adenocarcinoma: A real-world data.

e20577 Background: Chemotherapies combined with immune checkpoint inhibitors (ICIs) and/or bevacizumab (named IC, BC and IBC respectively) are the preferred first-line options for PD-L1 negative and oncogenic driver wild-type metastatic lung adenocarcinoma. However, the optimal strategy is still undetermined. Methods: This retrospective real-world study enrolled PD-L1 negative metastatic lung adenocarcinoma patients from four cancer centers between January 1, 2018 and June 30, 2022. All the patients received IC, BC, or IBC as the first-line therapies. The efficacy and safety were evaluated. Results: A total of 205 patients were included, with 60, 83, and 62 patients in IC, BC, and IBC groups, respectively. Patients receiving IBC obtained the highest objective response rate (ORR) (43.5%) and disease control rate (DCR) (100%) relative to those receiving IC (40.4%, 84.2%) or BC (40.5%, 96.2%). Compared with the IC (6.74m) or BC (8.28m) group, IBC group improved significantly median progression-free survival (mPFS) (9.53m, P = 0.005). However, the difference in overall survival (OS) were not observed among the three groups. When analyzed based on different clinical and molecular information, we found that male gender, ever smoking, wild-type genes, and adrenal metastasis predicts superior PFS benefit when treated with IBC. In patients with liver metastasis, IBC or BC treatment achieved better PFS compared with IC. No additional adverse reactions were observed in IBC group compared with other two groups. Conclusions: Combined IBC treatment achieved superior DCR and PFS compared with IC or BC treatment in patients with PD-L1 negative metastatic lung adenocarcinoma, while did not increase the adverse events.

Non-clinical evaluation of NT-112, an autologous T cell product engineered to express an HLA-C*08:02-restricted TCR targeting KRAS G12D and resistant to TGF-b inhibition.

e14533 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in oncogenes such as KRAS. Activating mutations in KRAS are among the most prevalent oncogenic driver mutations in human cancers, with KRAS G12D being the most frequent KRAS mutation in colorectal carcinoma and pancreatic cancer. NT-112 is an autologous engineered T-cell product expressing an HLA-C*08:02-restricted TCR that specifically targets the KRAS G12D mutation. NT-112 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-112, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-C*08:02-restricted KRS G12D neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-112 product functionality was evaluated in both in vivo models and in vitro co-culture experiments with a panel of cell lines including those endogenously expressing the KRAS G12D mutation. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. For determination of NT-112 product safety, cross-reactivity and allo-reactivity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-112. Results: High reactivity of the NT-112 TCR against KRAS G12D and HLA-C*08:02-expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against KRAS WT, minimal cross-reactivity in Alanine and Glycine scans, and lack of T-cell activation in the absence of HLA-C*08:02. In vivo, NT-112 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-112 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-112 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant KRAS-targeted cancer immunotherapy.

Beyond Chemoimmunotherapy in Advanced Non-Small Cell Lung Cancer: New Frontiers, New Challenges.

Chemoimmunotherapy is currently the preferred first-line treatment option for the majority of patients with advanced non-small cell lung cancer without driver genetic alterations. Most of these patients, however, will experience disease progression within the first year after treatment initiation and both patients and their physicians will be confronted with the dilemma of the optimal second-line treatment. Identification of molecular targets, such as KRASG12C, BRAFV600X, METexon14, and human epidermal growth factor receptor 2 mutations, and RET rearrangements offer therapeutic opportunities in pretreated patients with corresponding alterations. For those tumors that do not harbor oncogenic drivers, second-line treatment with docetaxel remains the current standard of care despite modest efficacy. Strategies to challenge docetaxel include the combination of immune checkpoint inhibitors (ICIs) with tyrosine inhibitors of multiple kinases or with DNA damage response inhibitors, antibody-drug conjugates, and locoregional treatments for oligoprogressive disease. Next-generation immunotherapy strategies, such as T-cell engagers, immune-mobilizing monoclonal T-cell receptors, chimeric antigen receptor cell therapy, tumor infiltrating lymphocytes, and T-cell receptor cell therapy are being currently investigated in the quest to reverse resistance to ICIs. Importantly, the advent of these new agents heralds a novel spectrum of toxicities that require both the physician's and the patient's education. Herein, we review current and future strategies aiming to outperform docetaxel after chemoimmunotherapy failure, and we provide practical information on how to best communicate to our patients the unique toxicity aspects associated with immunotherapy.

Phase 1 study of BDTX-1535, an oral 4th generation covalent EGFR inhibitor, in patients with recurrent glioblastoma: Preliminary dose escalation results.

2068 Background: Epidermal growth factor receptor ( EGFR) gene is the most frequently altered oncogenic driver in glioblastoma (GBM). In-frame deletion alterations (e.g., EGFRvIII) and missense mutations co-occur in the setting of EGFR gene amplification and are characterized as a hallmark of disease pathogenesis in GBM. BDTX-1535 is an oral, highly potent, brain penetrant, selective, irreversible 4th generation tyrosine kinase inhibitor that targets EGFR alterations in GBM and NSCLC. Preliminary results of the Phase 1 dose escalation study (NCT05256290) of patients with recurrent GBM (rGBM) are presented here. Methods: BDTX-1535-101 is a first-in-human study that enrolled patients with either rGBM harboring EGFR alterations following standard of care or patients with locally advanced or metastatic EGFR mutated NSCLC that progressed on prior EGFR TKIs. Using an adaptive Bayesian optimal interval design in the Phase 1 part, patients were enrolled at increasing dose cohorts and were treated daily for 21-day cycles until treatment discontinuation. The primary objective was to determine the BDTX-1535 recommended Phase 2 dose based on the overall safety, PK, pharmacodynamics, and preliminary antitumor activity. Results: Twenty-seven patients with rGBM were enrolled in the Phase 1 cohort that consisted of 54 patients in total including 27 patients with NSCLC. Patients were treated across seven dose levels (15mg – 400mg QD). The mean age of patients with rGBM was 58.7 years (range 41-85) with 96% of patients with previous temozolomide (TMZ) treatment and a median of 2 lines of prior therapy (range 1-4). The most common all-grade treatment-related AEs across all cohorts were rash (78%), diarrhea (41%), fatigue (15%), stomatitis (11%), decreased appetite (11%), nausea (11%) and paronychia (11%). Gr 3 TRAEs ≥ 10% included rash (19%) reported at 300 or 400 mg QD doses. Plasma exposure of BDTX-1535 increased dose proportionally and had a half-life of ~15h, supporting once daily dosing. The maximum tolerated dose (MTD) is 300 mg QD. Among 19 evaluable patients for response based on RANO criteria, 1 confirmed partial response was observed and 8 patients achieved stable disease. Five patients remained on BDTX-1535 with stable disease for an extended period (>5 months) who previously performed poorly on TMZ with short treatment duration, and 1 patient continues on BDTX-1535 after 16 months of treatment. Conclusions: BDTX-1535 was well-tolerated up to 300mg daily, which is the MTD, and promising preliminary clinical activity was observed in patients with rGBM after relapse on standard of care treatment. Given the inability to reconfirm EGFR status at the time of treatment with BDTX-1535 in this Phase 1 trial, further exploration of BDTX-1535 in a “window of opportunity” study is ongoing (NCT06072586). Clinical trial information: NCT05256290 .

KRAS mutation subtypes responded differently to combination immunotherapy via disturbing myeloid-derived suppressor cells in non-small cell lung cancer.

8635 Background: The combination of immune checkpoint inhibitors (ICIs) and chemotherapy has shown high efficacy in non-small cell lung cancer (NSCLC) patients, but further exploration of the predictive biomarkers is needed. KRAS is the most common driver oncogene in patients with NSCLC. However, the influence of different KRAS mutation subtypes on immunotherapy response remains controversial. We aimed to evaluate the value of KRAS mutation subtypes for predicting response to immunotherapy in NSCLC patients and to further analyze the related tumor immune microenvironment (TiME) phenotypes. Methods: In this retrospective study, clinical data were extracted from the medical records of patients with advanced NSCLC treated with ICIs and whose available molecular analysis included KRAS mutation information between January 2017 and December 2022. NSCLC tissue samples were collected for TiME analysis. Differences in immune cell subsets between different groups were analyzed using the Mann‒Whitney U test. Clinical characteristics, ICI treatment efficacy, progression-free survival (PFS), and overall survival (OS) were analyzed using multivariate Cox models. Results: A total of 1913 eligible KRAS-mutated NSCLC patients were screened, and 137 patients who received systemic immunotherapy were enrolled (108 males vs 29 females), with a median age of 62 (range, 39-84) years. Among them, 126 (92.0%) patients had adenocarcinoma, 99 (72.3%) patients were systemic therapy naïve, and 124 (90.5%) patients received combined immunotherapy. Among the patients with KRAS mutations, 48 (35.0%) had G12C, 20 (14.6%) had G12V, 27 (19.7%) had G12D and 42 (30.7%) had other subtypes. PFS was significantly longer in the G12C subgroup than in the other mutation subgroup (median PFS: 11.3 vs 6.8 months, P = 0.001). The median PFS times in the G12C/G12V subgroup and the other groups were 11.1 months and 6.5 months, respectively ( P= 0.0005). Multivariate analysis of the immunotherapy cohort revealed that G12C/G12V was an independent predictor of PFS ( P = 0.003, hazard ratio (HR): 2.021, 95% CI: 1.265-3.227). Further TiME analysis suggested that the infiltration of myeloid-derived suppressor cells (MDSCs, CD33+CD11b+) in the tumor and stromal areas in the G12C/G12V subgroup was significantly lower than that in the other subgroup (tumor area, P= 0.045; stroma area, P= 0.015). However, no significant difference in PD-L1 expression was found between the G12C/G12V group and the other groups. Conclusions: KRAS G12C and G12V were associated with favorable combined ICI treatment efficacy in patients with NSCLC. Patients with G12C or G12V mutations had longer PFS and lower infiltration of MDSCs. KRAS mutation subtypes are potential predictive biomarkers disturbed by MDSCs for combination therapy in NSCLC patients.

Characterization of diverse targetable ERBB2 alterations in 512,993 patients with solid tumors.

3129 Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) that has demonstrated activity against a range of HER2-expressing and HER2-mutant (mut) solid tumors. We present an extensive landscape of ERBB2 alterations (alt) that may predict sensitivity to HER2-targeted therapies in development with a focus on mutations and cancer types without approved indications. Methods: We queried institutional databases (MSKCC and Foundation Medicine, Inc.) of comprehensive genomic profiling (CGP) performed on solid tumor tissue samples using either MSK-IMPACT (N=83,332; MSK) or FoundationOne/FoundationOne CDx (N=429,661; FMI) and on patient-matched liquid biopsies using FoundationOne Liquid CDx (N=1,922). ctDNA tumor fraction (ctDNA TF) on FoundationOne Liquid CDx was estimated using a composite algorithm. Clinicopathological characteristics and treatment outcomes were annotated for MSK-IMPACT samples. Results: ERBB2 activating alt were detected in 6.6% (n=28,508) and 5.0% (n=4,133) of tumors in the FMI and MSK cohorts, respectively. In FMI tissue samples, ERBB2 alt were most prevalent in gastroesophageal (GEC; 18.1%), bladder (18.0%), salivary gland (14.1%), breast (12.7%), and uterine (12.1%) cancers. The cancer types with the largest number of ERBB2mut tumors, specifically, included NSCLC (n=1,618, 1.9%), breast (n=1,565, 3.5%), colorectal (CRC; n=1,221, 2.3%), bladder (n=855, 8.8%), and GEC (n=660, 3.4%). Across these 5 cancers, ERBB2 mut were most commonly located in the kinase domain (KD; 58%), followed by the extracellular domain (ECD; 28%) and transmembrane domain (TMD; 2%). Rare splice site mut and in-frame deletions resulting in exon 16 loss (Ex16Alt) were also observed (n=76, <0.1%). ERBB2 mut were found to be mutually exclusive with other RTK/MAPK driver oncogenes including: EGFR, KRAS, ALK, MET, BRAF, and RET in NSCLC; FGFR1 in breast cancer; BRAF in CRC; and FGFR3 in bladder cancer. In patient-matched liquid biopsy samples, sensitivity for detecting ERBB2 amplifications (amp; 33.3%, 43/129) was lower than for detecting ERBB2 mut (72.3%, 47/65) across select cancers. However, sensitivity for detection of both alts was improved in liquid samples with elevated ctDNA TF (≥1%), increasing to 52.6% (40/76) for amp and 97.3% (36/37) for mut. In the MSK cohort, 110 of 986 (11.2%) patients with ERBB2 mut non-NSCLC tumors received one or more HER2-targeted therapies, including ADCs. Clinical responses to HER2-targeted therapies were observed in patients harboring KD mut (n=10), ECD mut (n=6), TMD mut (V659E, n=1), and in 1 patient with a V697L mut. Conclusions: CGP detects diverse ERBB2activating alt at a high incidence in solid tumor types beyond NSCLC. These represent patient populations who may benefit from HER2-directed therapies. Trials of novel HER2-targeted agents are necessary to refine our understanding of the biological dependency of HER2 alterations.

Treatment (Tx) inequities in patients (pts) with metastatic non-small cell lung cancer (mNSCLC) harboring actionable driver oncogenes (ADO) in the first-line (1L) setting.

e13673 Background: Targeted treatments (TT) have been approved for pts with mNSCLC harboring ADO in the 1L setting in recent years. This study aimed to understand if subpopulations of mNSCLC pts harboring ADO do not receive equitable access to guideline recommended 1L TT in US clinical settings. Methods: This retrospective study used the Flatiron Health electronic health record-derived, US nationwide, de-identified database. Pts with de novo mNSCLC (stage IVA or IVB) diagnosed between 4/1/2018-6/30/2023 were included if they were ≥18 y, had ≥2 visits after advanced diagnosis (aDx) and ≥90 days follow-up, received any biomarker testing prior to 1L Tx initiation, and harbored ADO (ALK, BRAF, EGFR, MET, NTRK, RET and/or ROS1). Results: Overall 2165 pts met study criteria: mean age was 67 y, 65% female, 11% Medicare insured, 58% commercial insured, 76% treated in the community setting with 11% Medicare and 64% commercial insured (vs 12% and 41% treated in the academic setting with Medicare and commercial insurance), and 45% had smoking history; of non-missing data, 12% had lowest socioeconomic status (SES), 26% with highest SES, 95% had non-squamous NSCLC, and 83% had ECOG performance status ≤1. The median time from aDx to testing result among the 53% non-Latinx (nL) white, 8% nL Black, 7% Latinx, 13% nL Asian, 7% nL other and 13% missing race/ethnicity was 21, 23, 24, 21, 23 and 20 days, respectively; median time from aDx to TT initiation was 38, 38, 43, 36, 47 and 37 days, respectively. Pts significantly more likely to receive 1L TT were (Table): pts of color vs white pts; Medicare vs commercial insurance at community sites; without vs with smoking history. Pts less likely to receive 1L TT were (Table): community vs academic sites; Medicare vs commercial insurance at academic sites. Consistent findings were seen in the 1817 pts with established 1L ADO (ie. ALK, EGFR or ROS1) (Table). Conclusions: When pts with mNSCLC harboring ADO had biomarker testing before Tx initiation, contrary to prior observed racial inequity in NGS testing, there was no racial inequity in receiving 1L TT. However, inequity in receiving 1L TT was observed by insurance type, smoking history and site of care. Interventions are warranted to address these contributors to 1L TT inequities. [Table: see text]

A phase 1 trial evaluating the safety, tolerability, PK, and preliminary efficacy of QTX3034, an oral G12D-preferring multi-KRAS inhibitor, in patients with solid tumors with KRASG12D mutation.

TPS3172 Background: KRAS is one of the most mutated driver oncogenes across solid tumors, and KRAS G12D missense mutations have high prevalence in pancreatic, colorectal (CRC), and endometrial cancers. QTX3034 is a highly-selective, non-covalent, orally bioavailable, multi-KRAS inhibitor with potent activity across several KRAS variants, particularly G12D. QTX3034 binds to GDP-bound forms of mutant and wild-type KRAS with (sub)nanomolar affinities, allosterically inhibiting its conversion to the active state. QTX3034 inhibits KRAS signaling in in vitro studies and displays synergy with EGFR inhibitors. In vivo evaluation of QTX3034 resulted in significant tumor growth inhibition across various tumor models, with tumor regressions observed in 100% of animals in both HPAC pancreatic and GP2D colorectal KRAS G12D xenograft models. QTX3034 exhibits brain penetration in preclinical studies, including an orthotopic model of intracranial efficacy. This clinical trial is now enrolling patients with advanced solid tumors harboring a KRASG12D mutation. Methods: This is a first-in-human, multicenter, open-label phase 1 study in adult patients with advanced solid tumors with KRASG12D mutations, without prior direct KRAS inhibitor treatment (NCT06227377). The primary objectives are to evaluate the safety and tolerability and to determine maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) of QTX3034 alone, and in combination with cetuximab. Secondary objectives are characterization of the PK profile and preliminary antitumor activity. Part 1 consists of two dose-escalation arms: QTX3034 monotherapy (advanced solid tumors) and QTX3034 plus cetuximab (CRC) and will enroll 3-4 subjects per cohort in a Bayesian Logistic Regression Model (BLRM) design. QTX3034 will be administered orally in 21-day cycles until disease progression, intolerable toxicity, or withdrawal from treatment. Exploratory paired biopsies will be collected in Cycle 2 in a group of patients in the dose-escalation phase. Serial ctDNA will be collected in all patients. Following identification of a RP2D, QTX3034 will be evaluated in dose-expansion cohorts of up to 30 patients with various KRASG12D mutated tumor types to determine preliminary activity: pancreatic cancer, CRC, non-small cell lung cancer and endometrial cancer. To further optimize the recommended dose, patients will be randomized between two dose levels in the pancreatic cancer cohort. QTX3034 plus cetuximab will be explored following identification of a recommended combination dose in patients with CRC. Clinical trial information: NCT06227377 .

Multi-region exome sequencing to determine the origin of esophageal spindle-cell squamous cell carcinoma.

4072 Background: Esophageal spindle-cell squamous cell carcinoma (ESS) is a rare biphasic neoplasm composed of carcinomatous and sarcomatous components. However, the genomic origin and gene signature of ESS remain unclear. Methods: A total of 24 patients diagnosed with ESS were enrolled from Shanghai Chest Hospital and underwent either radical resection (23/24) or endoscopic submucosal dissection (1/24). Laser-capture microdissection was employed to isolate cancerous and sarcomatous components from tumor and lymph node metastatic tissues. The cancerous components were subsequently microdissected based on morphology. Whole exome sequencing (WES) was conducted on both tumor tissues and paired white cell samples to analyze genomic profiles. Results: The tumor tissue samples of carcinomatous and sarcomatous components showed high mutational commonality. With the same top high-frequency mutant genes, mutation signatures, and tumor mutation burden, paired samples shared a median of 25.5% mutation sites. Focal gains were found on chromosomes 3q29, 5p15.33, and 11q13.3. Altered genes were mainly enriched in the RTK-RAS signaling pathway. Phylogenetic trees showed a monoclonal origin of ESS. The most frequently mutated oncogene in the trunk was TP53, followed by NFE2L2, KMT2D, and MUC16. It showed a significant association of CDC27, LRP2, APC, and SNAPC4 mutations with poor prognosis in ESS patients. Our data highlight the monoclonal origin of ESS with TP53 as a potent driver oncogene, suggesting new targeted therapies and immunotherapies as treatment options. Conclusions: In conclusion, our results regarding to the mutation characteristics of microdissected samples substantiate the monoclonal origin of both the carcinomatous and sarcomatous components of ESS.

ONECUT2 is a druggable driver of luminal to basal breast cancer plasticity.

Tumor heterogeneity complicates patient treatment and can be due to transitioning of cancer cells across phenotypic cell states. This process is associated with the acquisition of independence from an oncogenic driver, such as the estrogen receptor (ER) in breast cancer (BC), resulting in tumor progression, therapeutic failure and metastatic spread. The transcription factor ONECUT2 (OC2) has been shown to be a master regulator protein of metastatic castration-resistant prostate cancer (mCRPC) tumors that promotes lineage plasticity to a drug-resistant neuroendocrine (NEPC) phenotype. Here, we investigate the role of OC2 in the dynamic conversion between different molecular subtypes in BC. We analyze OC2 expression and clinical significance in BC using public databases and immunohistochemical staining. In vitro, we perform RNA-Seq, RT-qPCR and western-blot after OC2 enforced expression. We also assess cellular effects of OC2 silencing and inhibition with a drug-like small molecule in vitro and in vivo. OC2 is highly expressed in a substantial subset of hormone receptor negative human BC tumors and tamoxifen-resistant models, and is associated with poor clinical outcome, lymph node metastasis and heightened clinical stage. OC2 inhibits ER expression and activity, suppresses a gene expression program associated with luminal differentiation and activates a basal-like state at the gene expression level. We also show that OC2 is required for cell growth and survival in metastatic BC models and that it can be targeted with a small molecule inhibitor providing a novel therapeutic strategy for patients with OC2 active tumors. The transcription factor OC2 is a driver of BC heterogeneity and a potential drug target in distinct cell states within the breast tumors.

Aggressive high-grade NF2 mutant meningiomas downregulate oncogenic YAP signaling via the upregulation of VGLL4 and FAT3/4.

Meningiomas are the most common primary brain tumors in adults. Although generally benign, a subset of meningiomas is of higher grade, shows aggressive growth behavior and recurs even after multiple surgeries. Around half of all meningiomas harbor inactivating mutations in NF2. While benign low-grade NF2 mutant meningiomas exhibit few genetic events in addition to NF2 inactivation, aggressive high-grade NF2 mutant meningiomas frequently harbor a highly aberrant genome. We and others have previously shown that NF2 inactivation leads to YAP1 activation and that YAP1 acts as the pivotal oncogenic driver in benign NF2 mutant meningiomas. Using bulk and single-cell RNA-Seq data from a large cohort of human meningiomas, we show that aggressive NF2 mutant meningiomas harbor decreased levels YAP1 activity compared to their benign counterparts. Decreased expression levels of YAP target genes are significantly associated with an increased risk of recurrence. We then identify the increased expression of the YAP1 competitor VGLL4 as well as the YAP1 upstream regulators FAT3/4 as a potential mechanism for the downregulation of YAP activity in aggressive NF2 mutant meningiomas. High expression of these genes is significantly associated with an increased risk of recurrence. In vitro, overexpression of VGLL4 resulted in the downregulation of YAP activity in benign NF2 mutant meningioma cells, confirming the direct link between VGLL4 expression and decreased levels of YAP activity observed in aggressive NF2 mutant meningiomas. Our results shed new insight on the biology of benign and aggressive NF2 mutant meningiomas and may have important implications for the efficacy of therapies targeting oncogenic YAP1 activity in NF2 mutant meningiomas.

IMPDH Inhibition Decreases TERT Expression and Synergizes the Cytotoxic Effect of Chemotherapeutic Agents in Glioblastoma Cells.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.

Comparison of diagnostic performance between Oncomine Dx target test and AmoyDx panel for detecting actionable mutations in lung cancer

Companion diagnostic (CDx) tests play important roles in identifying oncogenic driver genes and tailoring effective molecularly targeted therapies for lung cancer patients. In Japan, the Oncomine Dx target test (ODxTT) and the AmoyDx pan lung cancer PCR panel (AmoyDx) are prominent CDx tests and only one of these tests is covered by the domestic insurance system. However, these CDx tests cover different target regions and apply different technologies (ODxTT is amplicon-based next-generation sequencing and AmoyDx is multiplex PCR-based assay), which may lead to missing of actionable mutations affecting patient prognosis. Here, we performed a direct comparison analysis of 1059 genetic alterations of eight driver genes from 131 samples and evaluated the concordance between two CDx tests for detecting actionable variants and fusions. When excluding the eight uncovered variants (ODxTT: two variants, AmoyDx: six variants), the overall percent agreement was 97.6% (1026/1051) with 89.0% of overall positive percent agreement (89/100) and 98.5% of overall negative percent agreement (937/951). Of the 25 discordant genetic alterations, two were undetected despite being covered in the AmoyDx (one EGFR variant and one ROS1 fusion). Furthermore, there were potential false positives in the ODxTT (nine MET exon 14 skippings) and in the AmoyDx (five variants, six ROS1 and three RET fusions). These potential false positives in the AmoyDx likely due to non-specific amplification, which was validated by the unique molecular barcoding sequencing. The ODxTT missed two uncovered EGFR rare variants, which was visually confirmed in the raw sequencing data. Our study provides insights into real-world performance of CDx tests for lung cancer and ensures reliability to advance precision medicine.

JUN mediates the senescence associated secretory phenotype and immune cell recruitment to prevent prostate cancer progression

BackgroundProstate cancer develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although activator protein-1 transcription factors such as JUN have been implicated as potential oncogenic drivers, the molecular programs contributing to prostate cancer progression are not fully understood.MethodsWe analyzed JUN expression in clinical prostate cancer samples across different stages and investigated its functional role in a Pten-deficient mouse model. We performed histopathological examinations, transcriptomic analyses and explored the senescence-associated secretory phenotype in the tumor microenvironment.ResultsElevated JUN levels characterized early-stage prostate cancer and predicted improved survival in human and murine samples. Immune-phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of STAT3 activation and IL-1β production. Jun depletion in a Pten-deficient background prevented immune cell attraction which was accompanied by significant reduction of active STAT3 and IL-1β and accelerated prostate tumor growth. Comparative transcriptome profiling of prostate epithelial cells revealed a senescence-associated gene signature, upregulation of pro-inflammatory processes involved in immune cell attraction and of chemokines such as IL-1β, TNF-α, CCL3 and CCL8 in Pten-deficient prostates. Strikingly, JUN depletion reversed both the senescence-associated secretory phenotype and senescence-associated immune cell infiltration but had no impact on cell cycle arrest. As a result, JUN depletion in Pten-deficient prostates interfered with the senescence-associated immune clearance and accelerated tumor growth.ConclusionsOur results suggest that JUN acts as tumor-suppressor and decelerates the progression of prostate cancer by transcriptional regulation of senescence- and inflammation-associated genes. This study opens avenues for novel treatment strategies that could impede disease progression and improve patient outcomes.Graphical

Immunotherapy-relevance of a candidate prognostic score for Acute Myeloid Leukemia

BackgroundAcute Myeloid Leukemia (AML) exhibits a wide array of phenotypic manifestations, progression patterns, and heterogeneous responses to immunotherapies, suggesting involvement of complex immunobiological mechanisms. This investigation aimed to develop an integrated prognostic model for AML by incorporating cancer driver genes, along with clinical and phenotypic characteristics of the disease, and to assess its implications for immunotherapy responsiveness. MethodsCritical oncogenic driver genes linked to survival were identified by screening primary effector and corresponding gene pairs using data from The Cancer Genome Atlas (TCGA), through univariate Cox proportional hazard regression analysis. This was independently verified using dataset GSE37642. Primary effector genes were further refined using LASSO regression. Transcriptomic profiling was quantified using multivariate Cox regression, and the derived prognostic score was subsequently validated. Finally, a multivariate Cox regression model was developed, incorporating the transcriptomic score along with clinical parameters such as age, gender, and French-American-British (FAB) classification subtype. The ‘Accurate Prediction Model of AML Overall Survival Score’ (APMAO) was developed and subsequently validated. Investigations were conducted into functional pathway enrichment, alterations in the gene mutational landscape, and the extent of immune cell infiltration associated with varying APMAO scores. To further investigate the potential of APMAO scores as a predictive biomarker for responsiveness to cancer immunotherapy, we conducted a series of analyses. These included examining the expression profiles of genes related to immune checkpoints, the interferon-gamma signaling pathway, and m6A regulation. Additionally, we explored the relationship between these gene expression patterns and the Tumor Immune Dysfunction and Exclusion (TIDE) dysfunction scores. ResultsThrough the screening of 95 cancer genes associated with survival and 313 interacting gene pairs, seven genes (ACSL6, MAP3K1, CHIC2, HIP1, PTPN6, TFEB, and DAXX) were identified, leading to the derivation of a transcriptional score. Age and the transcriptional score were significant predictors in Cox regression analysis and were integral to the development of the final APMAO model, which exhibited an AUC greater than 0.75 and was successfully validated. Notable differences were observed in the distribution of the transcriptional score, age, cytogenetic risk categories, and French-American-British (FAB) classification between high and low APMAO groups. Samples with high APMAO scores demonstrated significantly higher mutation rates and pathway enrichments in NFKB, TNF, JAK-STAT, and NOTCH signaling. Additionally, variations in immune cell infiltration and immune checkpoint expression, activation of the interferon-γ pathway, and expression of m6A regulators were noted, including a negative correlation between CD160, m6A expression, and APMAO scores. ConclusionThe combined APMAO score integrating transcriptional and clinical parameters demonstrated robust prognostic performance in predicting AML survival outcomes. It was linked to unique phenotypic characteristics, distinctive immune and mutational profiles, and patterns of expression for markers related to immunotherapy sensitivity. These observations suggest the potential for facilitating precision immunotherapy and advocate for its exploration in upcoming clinical trials.

A novel function of STAT3β in suppressing interferon response improves outcome in acute myeloid leukemia

Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3β. While STAT3α is predominantly described as an oncogenic driver, STAT3β has been suggested to act as a tumor suppressor. To elucidate the role of STAT3β in AML, we established a mouse model of STAT3β-deficient, MLL-AF9-driven AML. STAT3β deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3β. Accordingly, STAT3β-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3β expression. Together, our data corroborate the tumor suppressive role of STAT3β in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3β/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3β/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.

Targeting ARNT attenuates chemoresistance through destabilizing p38α-MAPK signaling in glioblastoma

Glioblastoma (GBM) is the most aggressive and lethal brain tumor in adults. This study aimed to investigate the functional significance of aryl hydrocarbon receptor nuclear translocator (ARNT) in the pathogenesis of GBM. Analysis of public datasets revealed ARNT is upregulated in GBM tissues compared to lower grade gliomas or normal brain tissues. Higher ARNT expression correlated with the mesenchymal subtype and poorer survival in GBM patients. Silencing ARNT using lentiviral shRNAs attenuated the proliferative, invasive, and stem-like capabilities of GBM cell lines, while ARNT overexpression enhanced these malignant phenotypes. Single-cell RNA sequencing uncovered that ARNT is highly expressed in a stem-like subpopulation and is involved in regulating glycolysis, hypoxia response, and stress pathways. Mechanistic studies found ARNT activates p38 mitogen-activated protein kinase (MAPK) signaling to promote chemoresistance in GBM cells. Disrupting the ARNT/p38α protein interaction via the ARNT PAS-A domain restored temozolomide sensitivity. Overall, this study demonstrates ARNT functions as an oncogenic driver in GBM pathogenesis and represents a promising therapeutic target.

Trans-activating mutations of the pseudokinase ERBB3

Genetic changes in the ERBB family of receptor tyrosine kinases serve as oncogenic driver events and predictive biomarkers for ERBB inhibitor drugs. ERBB3 is a pseudokinase member of the family that, although lacking a fully active kinase domain, is well known for its potent signaling activity as a heterodimeric complex with ERBB2. Previous studies have identified few transforming ERBB3 mutations while the great majority of the hundreds of different somatic ERBB3 variants observed in different cancer types remain of unknown significance. Here, we describe an unbiased functional genetics screen of the transforming potential of thousands of ERBB3 mutations in parallel. The screen based on a previously described iSCREAM (in vitro screen of activating mutations) platform, and addressing ERBB3 pseudokinase signaling in a context of ERBB3/ERBB2 heterodimers, identified 18 hit mutations. Validation experiments in Ba/F3, NIH 3T3, and MCF10A cell backgrounds demonstrated the presence of both previously known and unknown transforming ERBB3 missense mutations functioning either as single variants or in cis as a pairwise combination. Drug sensitivity assays with trastuzumab, pertuzumab and neratinib indicated actionability of the transforming ERBB3 variants.