Receptor Tyrosine Kinase Pathways Research Articles

Molecular Analysis of Persistent and Recurrent Barrett's Esophagus in the Setting of Endoscopic Therapy.

Early neoplastic progression of Barrett's esophagus (BE) is often treated with endoscopic therapy. Although effective, some patients are refractory to therapy or recur after apparent eradication of the BE. The goal of this study was to determine whether genomic alterations within the treated BE may be associated with persistent or recurrent disease. We performed DNA sequencing on pre-treatment esophageal samples from 45 patients who were successfully treated by endoscopic therapy and did not recur as well as pre-treatment and post-treatment samples from 40 patients who had persistent neoplasia and 21 patients who had recurrent neoplasia. The genomic alterations were compared between groups. The genomic landscape was similar between all groups. Patients with persistent disease were more likely to have pre-treatment alterations involving the receptor tyrosine kinase pathway ( P = 0.01), amplifications of oncogenes ( P = 0.01), and deletions of tumor suppressor genes ( P = 0.02). These associations were no longer significant after adjusting for patient age and BE length. More than half of patients with persistent (52.5%) or recurrent (57.2%) disease showed pre-treatment and post-treatment samples that shared at least 50% of their driver mutations. Pre-treatment samples were genomically similar between those who responded to endoscopic therapy and those who had persistent or recurrent disease, suggesting there is not a strong genomic component to treatment response. Although it was expected to find shared driver mutations in pre-treatment and post-treatment samples in patients with persistent disease, the finding that an equal number of patients with recurrent disease also showed this relation suggests that many recurrences represent undetected minimal residual disease.

Examination of genomic profile of true single circulating tumor cells (sCTC) from hepatocellular carcinoma and its effect on mutational features.

e15039 Background: Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related deaths worldwide. Despite numerous NGS studies unveiling diverse mutational patterns through tissue biopsies, the absence of tissue for biopsy and distinct biomarkers complicates both diagnosis and treatment. The utilization of sCTC genomics holds promise in furnishing clinically relevant and actionable insights to tailor personalized diagnostics and therapy for HCC. We present a comparative analysis of sCTC and paired ctDNA obtained from primary HCC patients for better Tx possibilities. Methods: Genomic profile from 24 sCTCs/CTC clusters (19 sCTCs, 79%; 5 CTC clusters, 21 %) revealed distinct mutational patterns compared to paired ctDNA. Live sCTCs/CTC clusters were isolated from peripheral blood of treatment naïve 12 Hepatobiliary Cancer with advanced and metastatic disease patients using OncoRadar technology. Whole genomes from sCTCs/CTC clusters were amplified, target enriched with hybridization capture using a comprehensive 1080 gene panel-OncoIndx to obtain sequencing libraries, and sequenced on Illumina NextSeq2000 platform in paired end mode (depth 500x). The raw sequence alignment and variant calling was performed using iCare software. Paired ctDNA samples were sequenced the same way, except at the depth of 5500x. Results: Pathogenic (P)/likely pathogenic (LP) variants were detected from all sCTCs, while ctDNA was negative for 40% samples. sCTCs showed at least twice as many P variants compared to paired ctDNA. ATM (50%), PIK3CA (42%), ARID1A /1B (42%), TP53/RB1 (34%), and IDH1 (34%) were among the most frequently mutated genes, while 42% sCTCs showed amplified CCND1/2 variants. In comparison, paired ctDNA had no distinct mutational pattern. Genomic profile of sCTC showed concordance with paired ctDNA for at least one gene mutation when compared at patient level. At pathway level, all sCTCs were mutated for at least one HRR pathway component, while 60% sCTCs had at least one mutation in receptor tyrosine kinase (RTK) pathway (FGFR1, ERBB, ALK, ROS1). sCTC population showed a strong cooccurrence of HRR mutation and epigenetic regulatory pathway mutations (OR =4, CI=0.4-60). At individual mutation level, 30 % concordance was observed in sCTC population, suggesting high mutational heterogeneity. Besides actionable targets, 50% sCTC were enriched with resistance conferring variants such as STK11, NF1, STAT5B, and RB1. Conclusions: The genomic profile of sCTCs in primary HCC pts revealed a complex mutational landscape, offering complementarity to ctDNA for clinical insights. sCTCs exhibited more actionable targets and greater heterogeneity compared to paired ctDNA, making sCTC genomics valuable for pts ineligible for tissue biopsy or lacking clinically relevant information from ctDNA.

DCC-3116 in combination with ripretinib for patients with advanced gastrointestinal stromal tumor: A phase 1/2 study.

TPS11587 Background: Advanced cancers driven by mutations at or downstream of receptor tyrosine kinases (RTKs), including KIT- and PDGFRA-mutant gastrointestinal stromal tumor (GIST), activate autophagy as an escape mechanism in response to RTK pathway inhibitors. ULK1/2 kinases are key regulators that initiate autophagy in response to stress, such as KIT/PDGFRA inhibition in KIT- and PDGFRA-mutant GIST. Therefore, ULK1/2 inhibition represents a potential targeted approach to selectively inhibit autophagy and is an attractive strategy for combination with RTK pathway inhibitors, including ripretinib. Ripretinib is a switch-control KIT/PDGFRA tyrosine kinase inhibitor approved as a ≥fourth-line therapy for advanced GIST. In the INTRIGUE trial, ripretinib demonstrated comparable efficacy and more favorable safety compared with sunitinib in second-line advanced GIST. DCC-3116 is an investigational, selective, potent, switch-control kinase inhibitor of ULK1/2 in development for combination with targeted therapies that activate autophagy, such as ripretinib. In a GIST xenograft model, animals treated with ripretinib demonstrated stable tumor burden while treatment with ripretinib + DCC-3116 resulted in complete regressions. Here, we describe the design of a phase 1/2, multicenter, open-label study evaluating the safety, recommended phase 2 dose (RP2D), and preliminary efficacy of DCC-3116 in combination with ripretinib in advanced GIST (NCT05957367). Methods: This phase 1/2 study includes safety/dose-finding (part 1) and expansion (part 2). In part 1, patients (pts) ≥18 years must have pathologically confirmed GIST with a KIT or PDGFRA mutation and disease progression on or intolerance to ≥1 systemic regimen. Escalating doses of DCC-3116 will be assessed with ripretinib 150 mg once daily. Part 1 will use safety, pharmacokinetics, pharmacodynamics, preliminary efficacy, and a Bayesian optimal interval design to identify the RP2D for part 2 and the maximum tolerated dose. In part 2, second-line pts with confirmed GIST with a KIT exon 11 mutation and disease progression on or intolerance to first-line imatinib will be enrolled. Part 2 will use a two-stage design with non-binding futility and efficacy analyses after stage 1 to explore efficacy of the combination dose selected in part 1. Exclusion criteria for both parts include use of other anticancer treatments, such as ripretinib, and any investigational therapies within 14 days, use of moderate or strong inhibitors or inducers of cytochrome P450 3A4 or P-glycoprotein, symptomatic central nervous system metastases or leptomeningeal disease, and active infections. Primary objectives in part 1 are to identify adverse events and the RP2D. The primary objective of part 2 is to determine the objective response rate per modified Response Evaluation Criteria in Solid Tumors version 1.1. The study is currently enrolling. Clinical trial information: NCT05957367 .

Genomic characterization reveals distinct mutational landscapes and therapeutic implications between different molecular subtypes of triple-negative breast cancer

Triple-negative breast cancer (TNBC) has high heterogeneity, poor prognosis, and limited treatment success. Recently, an immunohistochemistry-based surrogate classification for the “Fudan University Shanghai Cancer Center (FUSCC) subtyping” has been developed and is considered more suitable for clinical application. Seventy-one paraffin-embedded sections of surgically resected TNBC were classified into four molecular subtypes using the IHC-based surrogate classification. Genomic analysis was performed by targeted next-generation sequencing and the specificity of the subtypes was explored by bioinformatics, including survival analysis, multivariate Cox regression, pathway enrichment, Pyclone analysis, mutational signature analysis and PHIAL analysis. AKT1 and BRCA1 mutations were identified as independent prognostic factors in TNBC. TNBC molecular subtypes encompass distinct genomic landscapes that show specific heterogeneities. The luminal androgen receptor (LAR) subtype was associated with mutations in PIK3CA and PI3K pathways, which are potentially sensitive to PI3K pathway inhibitors. The basal-like immune-suppressed (BLIS) subtype was characterized by high genomic instability and the specific possession of signature 19 while patients in the immunomodulatory (IM) subtype belonged to the PD-L1 ≥ 1% subgroup with enrichment in Notch signaling, suggesting a possible benefit of immune checkpoint inhibitors and Notch inhibitors. Moreover, mesenchymal-like (MES) tumors displayed enrichment in the receptor tyrosine kinase (RTK)-RAS pathway and potential sensitivity to RTK pathway inhibitors. The findings suggest potential treatment targets and prognostic factors, indicating the possibility of TNBC stratified therapy in the future.

Abstract PS09-09: Multiomics profiling and molecular classification refine precision treatment strategies for HER2-positive breast cancer

Abstract Background: Anti-HER2 targeted therapy has achieved a series of breakthroughs. However, the current treatment strategy regarding HER2-positive breast cancer remains indiscriminate and lacks specificity, which limits the further improvement of overall treatment response and may lead to overtreatment and extra cost for some patients. Our study aims to reveal the molecular heterogeneity of HER2-positive breast cancer to guide a more precise treatment. Patients and methods: We selected HER2-positive breast cancer patients treated at Fudan University Shanghai Cancer Center between 2013 and 2014 and conducted genomic, transcriptomic, proteomic and metabolomic profiling. We then applied a non-negative matrix factorization algorithm on transcriptomic data to obtain an unsupervised classification. And we further studied the correlation between subtypes and corresponding treatment strategies in multiple cohorts of adjuvant and neoadjuvant therapy. For clinical accessibility, we developed convolutional neural network models through deep learning algorithm based on digital pathology to identify different subtypes. Additionally, we explored novel treatment strategies using the patient-derived organoids (PDOs) models. Results: We established a novel multiomics cohort of 180 HER2- breast cancer patients and classified them into four clinically significant molecular subtypes: (1) A classical HER2-enriched (HER2-CLA, N=51) subtype characterized by strong ERBB2 signaling and remarkable sensitivity to anti-HER2-targeted therapy (pathologic complete response with dual-targeted therapy: 93%). (2) an immunomodulatory (HER2-IM, N=36) subtype characterized by an immune-activated microenvironment and excellent prognosis with current treatment (no relapse in 97% of patients with a median follow-up of 86 months). Tumors of this subtype were therefore candidates for de-escalatory treatment. (3) A luminal-like (HER2-LUM, N=55) subtype distinguished by activated estrogen receptor signaling and (4) a basal/mesenchymal-like (HER2-BM, N=38) subtype enriched in activated receptor tyrosine kinase pathways. HER2-LUM and HER2- BM showed limited benefit from anti-HER2 therapy, and thus, add-on therapies might be needed. The overall area under the curve (AUC) of the convolutional neural network model based on digital pathology for identifying different subtypes is 0.77. In the exploration of novel treatment strategies, we found in the PDO model that the HER2-LUM subtype is more sensitive to a treatment regimen combining standard (chemotherapy and targeted therapy) with subsequent endocrine therapy and CDK4/6 inhibitors compared to other subtypes. Additionally, the HER2-BM subtype demonstrated greater sensitivity to treatment with a combination of EGFR inhibitors, PDGFR inhibitors or VEGFR inhibitors. Conclusion: We uncovered a high degree of molecular heterogeneity in HER2-positive breast cancer and illustrated its impact on treatment response. More precise treatment can be given according to the characteristics of different subtypes, which may achieve good efficacy and simultaneously reduce overtreatment and extra cost. The comprehensive profiling of HER2-positive breast cancers could also serve as an important resource for further exploration. Key Words: HER2-positive breast cancer cohort; molecular classification; targeted therapy; precision treatment; de-escalatory treatment. Citation Format: Yu-Wei Li, Ding Ma, Xiang-Rong Wu, Lei-Jie Dai, Shen Zhao, Yu-Zheng Xu, Xi Jin, Xiao Yi, Ying Wang, Cai-Jin Lin, Yi-Fan Zhou, Tong Fu, Wen-Tao Yang, Ming Li, Hong Lv, Yi-Zhou Jiang, Zhi-Ming Shao. Multiomics profiling and molecular classification refine precision treatment strategies for HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS09-09.

Combined treatment with cetuximab and STA9090 has synergistic anticancer effects on human non-small cell lung cancer.

Cetuximab (CET), a human murine chimeric IgG monoclonal antibody and an inhibitor of epidermal growth factor receptor (EGFR), has been shown to be effective in treating various types of cancer. However, its use is hindered by limitations such as resistance development, variability in patient response, side effects, and challenges in biomarker identification. Therefore, CET is often combined with other targeted therapies or chemotherapies to enhance its effectiveness. In this study, we investigate the anticancer effects and underlying mechanisms of the combination of CET, an EGFR inhibitor, and STA9090, an inhibitor of heat shock protein 90 (Hsp90), in both in vitro and in vivo models of non-small cell lung cancer (NSCLC). The results demonstrate significantly stronger effects on NSCLC cells in response to combination therapy than to treatment with either agent alone, indicating that the combination of CET and STA9090 has potential synergistic effects. Additionally, the combination therapy inhibits tumor growth in a xenograft nude mouse model more effectively than treatment with either agent alone, suggesting improved efficacy when used together. Furthermore, the synergistic effects of the combination therapy are likely due to inactivation of the receptor tyrosine kinase (RTK) pathway, which is overly activated in cancer and contributes to tumor growth, angiogenesis, and metastasis. Consequently, our findings suggest that STA9090 has potent direct antitumor activity and synergizes with CET against NSCLC tumors. It is highly likely that these synergistic effects are mediated through RTK pathway inactivation caused by the combination. Therefore, our findings strongly and consistently support the potential synergistic effect of STA9090, an RTK inhibitor, in combination with EGFR-targeting agents.

Sprouty genes regulate activated fibroblasts in mammary epithelial development and breast cancer

Stromal fibroblasts are a major stem cell niche component essential for organ formation and cancer development. Fibroblast heterogeneity, as revealed by recent advances in single-cell techniques, has raised important questions about the origin, differentiation, and function of fibroblast subtypes. In this study, we show in mammary stromal fibroblasts that loss of the receptor tyrosine kinase (RTK) negative feedback regulators encoded by Spry1, Spry2, and Spry4 causes upregulation of signaling in multiple RTK pathways and increased extracellular matrix remodeling, resulting in accelerated epithelial branching. Single-cell transcriptomic analysis demonstrated that increased production of FGF10 due to Sprouty (Spry) loss results from expansion of a functionally distinct subgroup of fibroblasts with the most potent branching-promoting ability. Compared to their three independent lineage precursors, fibroblasts in this subgroup are “activated,” as they are located immediately adjacent to the epithelium that is actively undergoing branching and invasion. Spry genes are downregulated, and activated fibroblasts are expanded, in all three of the major human breast cancer subtypes. Together, our data highlight the regulation of a functional subtype of mammary fibroblasts by Spry genes and their essential role in epithelial morphogenesis and cancer development.

RAS/Mitogen-Activated Protein Kinase Signaling Pathway in Testicular Germ Cell Tumors.

Germ cell tumors (GCTs) are relatively rare tumors. However, they are the most diagnosed malignancies occurring in the testis among men aged between 15 and 40 years. Despite high aneuploidy and a paucity of somatic mutations, several genomic and transcriptomic assays have identified a few significantly mutated somatic genes, primarily KIT and K-RAS. The receptor Tyrosine Kinase (RTK) pathway and the downstream related Mitogen-Activated Protein Kinase (MAPK) cascades are crucial signal transduction pathways that preside over various cellular processes, including proliferation, differentiation, apoptosis, and responses to stressors. They are well described in solid malignancies, where many of the involved factors are used as prognostic molecular markers or targets for precision therapy. This narrative review focused, in the first part, on PGCs' survival/proliferation and differentiation and on the genetic and epigenetic factors involved in the pathogenesis of testicular germ cell tumors (TGCTs) and, in the second part, on the most recent investigations about the KIT-RAS pathway in TGCTs and in other cancers, highlighting the efforts that are being made to identify targetable markers for precision medicine approaches.

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

Mechanisms of tyrosine kinase inhibitor resistance in renal cell carcinoma.

Renal cell carcinoma (RCC), the most prevalent type of kidney cancer, is a significant cause of cancer morbidity and mortality worldwide. Antiangiogenic tyrosine kinase inhibitors (TKIs), in combination with immune checkpoint inhibitors (ICIs), are among the first-line treatment options for patients with advanced RCC. These therapies target the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase pathway and other kinases crucial to cancer proliferation, survival, and metastasis. TKIs have yielded substantial improvements in progression-free survival (PFS) and overall survival (OS) for patients with advanced RCC. However, nearly all patients eventually progress on these drugs as resistance develops. This review provides an overview of TKI resistance in RCC and explores different mechanisms of resistance, including upregulation of alternative proangiogenic pathways, epithelial-mesenchymal transition (EMT), decreased intracellular drug concentrations due to efflux pumps and lysosomal sequestration, alterations in the tumor microenvironment including bone marrow-derived cells (BMDCs) and tumor-associated fibroblasts (TAFs), and genetic factors such as single nucleotide polymorphisms (SNPs). A comprehensive understanding of these mechanisms opens the door to the development of innovative therapeutic approaches that can effectively overcome TKI resistance, thereby improving outcomes for patients with advanced RCC.

The p110α/ΔNp63α complex mutations in triple-negative breast cancer: Potential targets for transcriptional-based therapies.

Hotspot mutations occurring in the p110α domain of the PIK3CA gene, specifically p110αH1047R/L increase tumor metastasis and cell motility in triple-negative breast cancer (TNBC). These mutations also affect the transcriptional regulation of ΔNp63α, a significant isoform of the p53 protein involved in cancer progression. This study attempts to investigate the transcriptional impact of p110αH1047R/L mutations on the PIK3CA/ΔNp63α complex in TNBC carcinogenesis. We performed site-directed mutagenesis to introduce p110αH1047R/L mutations and evaluated their oncogenic effects on the growth, invasion, migration, and apoptosis of three different TNBC cell lines in vitro. We investigated the impact of these mutations on the p110α/ΔNp63α complex and downstream transcriptional signaling pathways at the gene and protein levels. Additionally, we used bioinformatics techniques such as molecular dynamics simulations and protein-protein docking to gain insight into the stability and structural changes induced by the p110αH1047R/L mutations in the p110α/ΔNp63α complex and downstream signaling pathway. The presence of PIK3CA oncogenic hotspot mutations in the p110α/ΔNp63α complex led to increased scattering of TNBC cells during growth, migration, and invasion. Our in vitro mutagenesis assay showed that the p110αH1047R/L mutations activated the PI3K-Akt-mTOR and tyrosine kinase receptor pathways, resulting in increased cell proliferation, invasion, and apoptosis in TNBC cells. These mutations decreased the repressing effect of ΔNp63α on the p110α kinase domain, leading to the enhancement of downstream signaling pathways of PI3K and tyrosine kinase receptors and oncogenic transformation in TNBC. Additionally, our findings suggest that the physical interaction between the DNA binding domain of ΔNp63α and the kinase domain of p110α may be partially impaired, potentially leading to alterations in the conformation of the p110α/ΔNp63α complex. Our findings suggest that targeting the p110αH1047R/L mutations in TNBC could be a promising strategy for developing transcriptional-based therapies. Restoring the interaction between ΔNp63α and the p110α kinase domain, which is disrupted by these mutations, may provide a new approach to treating TNBC.

Abstract 17144: Pi3 Kinase Activation by Platelet-Derived Purified Exosome Product to Induce Angiogenesis

Introduction: Platelets are heavily implicated in angiogenesis; however, the underlying mechanism remains relatively uncharacterized. The goal of this study was to evaluate the angiogenic potency of a platelet-derived regenerative exosome product (PEP) in an effort to develop a novel treatment for peripheral arterial disease. Here, we evaluated the mechanistic basis of PEP in angiogenesis and it’s use In Vivo in a rodent hind limb ischemia model. Methods/Results: Under Current Good Manufacturing Practices, conditioned medium from apheresis platelet cultures was purified to acquire the exosomes. PEP, enriched in exosome markers CD63, CD9 and Flotillin, induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation In Vitro. Proteomic evaluation of PEP highlighted receptor tyrosine kinase (RTK) pathways and western blot validation demonstrated induction of phospho-MAPK and phospho-AKT following PEP treatment of HUVECs. Systematic evaluation of RTK receptor involvement was performed using small molecule inhibitors and receptor knock-down in HUVECs, ruling out RTK activation as a mode of action. However, small molecule inhibition of PI3 kinase demonstrated elimination of PEP-induced AKT phosphorylation and reduction in PEP-induced angiogenesis In Vitro measured by HUVEC proliferation and migration. In Vivo, evaluation of angiogenesis was performed in a rodent model of hindlimb ischemia treated with PEP embedded in fibrin glue (PEP/TISSEEL). Blood flow was evaluated by SPY angiography on day 0 pre- and post-operation, day 21, and day 28. Animals treated with PEP/TISSEEL showed a significant increase in blood flow compared to sham and TISSEEL treated animals (percent perfusion at 28 days: sham 40.9%, TISSEEL 44.3%, PEP/TISSEEL 100.2%), with significant increase in vascular area on histology (at 28 days vascular area (μm 2 )/muscle area(μm 2 ): healthy control 12.71, sham 4.66, TISSEEL 4.76, PEP/TISSEEL 15.54). Conclusions: Exosome driven activation of PI3 kinase can induce angiogenesis both In Vitro and In Vivo. PEP provides a novel, clinical-grade, off-the-shelf therapy to promote angiogenesis for the treatment of peripheral arterial disease.

Novel Mechanisms of Venetoclax Resistance in Acute Myeloid Leukemia Based on Genomic Rearrangements

Introduction: Venetoclax (VEN), a BCL-2 inhibitor, effectively induces apoptosis in acute myeloid leukemia (AML) cells. Resistance to VEN in the treatment of AML can occur through various mechanisms. Some of the identified resistance mechanisms include: expansion or gain of clones with activation of the receptor tyrosine kinase (RTK) pathway; disruption of TP53 function, a tumor suppressor gene known to play a critical role in cell cycle regulation and apoptosis; selection of AML populations with monocytic differentiation, possibly altering cellular characteristics and response to treatment; overexpression of the MCL1 gene, which encodes for a protein that can counteract the pro-apoptotic effects of venetoclax; switch to lipid metabolism, an adaptive response by leukemia cells that may confer resistance to venetoclax; mutations in BCL-2 and BAX genes, which could affect the activity of these key regulators of apoptosis. While these mechanisms have been extensively studied, the role of rearrangements, often combined with genomic events, has not been fully explored. The INTHEMA protocol (NCT04298892) aimed to identify new VEN resistance mechanisms in patients with relapsed/refractory AML treated with VEN in combination with hypomethylating agents and to provide addressable molecular pathways useful for restoring drug sensitivity or offer novel therapeutic opportunities. Methods: Informed consent was obtained from 21 consecutive patients. Samples were collected from these patients with relapse or refractory disease after VEN + hypomethylating agent treatment. TruSight RNA Pan-Cancer analysis was performed to investigate fusion genes (using capture technology covering 1385 genes), RNA expression, and expressed mutations for each sample. Results: Among the 21 patients, 17 (81%) showed at least one putative mechanism of VEN resistance (as illustrated in the figure). These mechanisms included co-occurring mutations and genomic rearrangements. RTK mutations were found in 14 patients (67%), with specific mutations observed in NRAS, PTPN11, CBL, FLT3, JAK2, cKIT, EPHB6, FGFR and PDGFRb. One patient had a BAX mutation (p.G67R; BH3 domain), three patients exhibited a switch to a monocytic phenotype, and two patients had marker chromosomes. Gene fusions were identified in eight patients (38%): in particular, we discovered a cryptic rearrangement of MECOM (TBL1XR1-MECOM and ETV6_MECOM) in 2 patients, a novel BCL11B fusion ( BCL11B-GSDMC), a CCND2-ETV6 BIRC6-LTBP1, the reciprocal NUP98-NSD1, the canonical CBFB-MYH11 and additional transcripts with less defined roles ( PSPC1-ZMYM2, PER1-DDX5, ELL-SUGP2). Interestingly, most of the genes involved in traslocations, with a potential driver role in therapy resistance ( MECOM, ETV6, CCND2, BCL11B, NUP98) also showed high expression levels in patients harboring the traslocation. Unsupervised clustering of RNA expression data revealed two main patient groups. The first group (24%) demonstrated an “activating-like” signature, characterized by overexpression of CSF3R and IKZF, suggesting a potential target for tyrosine kinase inhibitors. The second group exhibited a “self-renewal like” signature, suggesting a possible benefit from histone deacetylase inhibitors, as previously published. Patients with gene fusions were predominantly clustered within the “self-renewal like” group, particularly in a subgroup (subgroup 4) showing overexpression of HOXA genes. Conclusions: Through deep transcriptomic characterization combined with conventional diagnostics, this analysis uncovered novel mechanisms of VEN resistance while confirming established ones. The distinct gene expression patterns may help tailor targeted therapies, with patients showing the “activating-like” signature potentially benefiting from tyrosine kinase inhibitors and those with the “self-renewal like” signature possibly responding well to histone deacetylase inhibitors. Furthermore, HOXA gene overexpression presents an exciting therapeutic opportunity for selected patients. Further research and clinical validation are needed to translate these findings into actionable clinical strategies.

High-Hyperdiploid Acute Lymphoblastic Leukemia in Children with LZTR1 Germline Variants

Introduction Firstly described in 1967, hyperdiploidy (HD) is the most frequent genetic abnormality in B-cell- precursor acute lymphoblastic leukemia (BCP-ALL) in children, comprising about 25% of all cases. A not yet exactly defined proportion have predisposing pathogenic germline variants in DNA repair pathway genes, chromatin remodeling factors, transcription factors regulating B-cell development (particularly ETV6) or receptor tyrosine kinases pathway genes like RAS/RAF. Among the latter, mutations have mostly been described in PTPN11 and SOS1, but not yet in other components of this central regulatory hub of cellular communication. Germline loss-of-function (LOF) LZTR1 mutations are typically linked to hereditary nerve sheath tumors. However, it remains largely unknown, if other tumor entities are associated with LZTR1 LOF germline mutations, potentially broadening the spectrum of malignancies associated with RASopathies. Aim Our aim is to understand the frequency and the impact of LZTR1 germline variants in HD BCP-ALL of childhood. Material and Methods We analyzed WES data of 283 children with BCP-ALL for the presence of pathogenic variants in the LZTR1 cancer predisposition gene. For a part of the detected variants, we performed further functional analyses to investigate the effect of the alteration on protein function. We applied a Drosophila model that is particularly suited for functional evaluation of Ras pathway activity. Results We identified LZTR1 germline variants in 10/283 (3.5%) patients (Figure 1). Interestingly, in 6 out of 283 (2.1%) children the LZTR1 variants were classified as pathogenic/likely pathogenic (P/LP). The majority of patients (5/6) harboring a P/LP germline LZTR1 variant presented with a HD BCP-ALL. Only two of our patients showed concomitant phenotypic features indicative of an underlying syndromic condition. Patient P1 presented with a HD BCP-ALL at the age of 9 years and mild psychomotor delay. Sequencing revealed a likely pathogenic variant (p.Arg283Trp) that was not yet reported in association with RASopathy syndromes. The second child P8, also diagnosed with a HD BCP-ALL, carried a well-known autosomal dominant mutation described in Noonan syndrome (NS) variant (p.Gly248Arg). The child was characterized by facial dysmorphism, as well as mild developmental delay, although a diagnosis of NS was not established prior to development of the BCP-ALL. The other three patients had no clinical peculiarities other than HD BCP-ALL. We functionally characterized patient derived LZTR1 variants in dLztr1-depleted ISC. Like wild-type hLZTR1 wt, hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg restored control-like ISC lineage production, suggesting that hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg still regulate Ras ubiquitination. In contrast, hLZTR1 p.Tyr535Ter results in even 1.8-fold higher ISC lineage production than dLztr1-RNAi. Beyond ISC production as a readout, we employed a second experimental paradigm directly addressing Ras signalling activity with a translocating modified ERK sensor. In line with Ras activity control of LZTR1, hLZTR1 wt significantly reduced ERK activity, while the putatively LOF variant hLZTR1 p.Tyr535Ter variant significantly increased Ras pathway activation by 1.4-fold over controls. We also noticed that hLZTR1 p.Tyr535Ter induced extensive membrane blebbing and nuclear fragmentation of GFP positive cells likely indicating programmed cell death. Quantification of apoptotic cells revealed an 11.1-fold increase compared to controls, which was not observed for hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg. Most notably, forced expression of hLZTR1 p.Arg283Trp andhLZTR1 p.Tyr535Ter increased mitotic recombination by 4-fold and 3.4-fold, respectively, which was not observed for the hLZTR1 p. Lys761Arg variant. Conclusion In our cohort, approximately 2% of all children with BCP-ALL harboured a P/LP LZTR1 germline variation, not necessarily linked with clinical appearance of Noonan-syndrome-like features, but to the development of a high hyperdiploid karyotype. By applying a Drosophila model, we demonstrated that patient-derived LZTR1 germline variants affect RAS pathway activation, ERK accumulation, cell proliferation, DNA recombination and apoptosis. Figure 1: Depiction of the ten LZTR1 variants detected in an unselected pediatric cohort of 283 BCP-ALL patients

Unidirectional alteration of methylation and hydroxymethylation at the promoters and differential gene expression in oral squamous cell carcinoma.

Background: Oral squamous cell carcinoma (OSCC) is one of the most common types of cancer worldwide. Although overall losses of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been previously observed, a genome-wide, single-base-resolution, and simultaneous mapping of 5mC and 5hmC in OSCC is still unaccomplished. Similarly, the mechanism of how 5mC and 5hmC collectively lead to abnormal gene expression in OSCC is largely unexplored. Using parallel whole-genome bisulfite sequencing (WGBS) and whole-genome oxidative bisulfite sequencing (oxWGBS), we characterized 5mC- and 5hmC-profiles at single-nucleotide resolution in paired primary OSCC samples and their normal adjacent tissues (NATs). We also analyzed the effect of 5mC- and 5hmC-modifications on differential gene expression in OSCC using multi-omics analysis. Results: An overall reduction of both 5mC and 5hmC in various genomic regions have been observed in OSCC samples. At promoter regions, a total of 6,921 differentially methylated regions and 1,024 differentially hydroxymethylated regions were identified in OSCC. Interestingly, compared to bidirectional modification with 5mC and 5hmC, unidirectional modification with 5mC and 5hmC at the promoters is associated with bigger change in the gene expression. Additionally, genes bearing unidirectional modification with 5mC and 5hmC at the promoters are enriched in signaling pathways like cell proliferation, cell differentiation, and receptor tyrosine kinase pathway that are essential for the tumorigenesis. Finally, the grouped expression signature of top 20 genes bearing promoter-unidirectional-modification with 5mC and 5hmC tends to correlate with the clinical outcome of certain subtypes of head and neck squamous cell carcinoma. Conclusion: Using parallel WGBS and oxWGBS analyses, we observed an overall reduction of 5mC- and 5hmC-modifications at various genomic regions in OSCC. Unidirectional modification with 5mC and 5hmC at the promoters is associated with enhanced changes in gene expression in OSCC tissues. Furthermore, such differentially expressed genes bearing unidirectional modifications with 5mC and 5hmC at the promoters might have clinical relevance to the outcome of OSCC.

Molecular classification of hormone receptor-positive HER2-negative breast cancer.

Hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer is the most prevalent type of breast cancer, in which endocrine therapy resistance and distant relapse remain unmet challenges. Accurate molecular classification is urgently required for guiding precision treatment. We established a large-scale multi-omics cohort of 579 patients with HR+/HER2- breast cancer and identified the following four molecular subtypes: canonical luminal, immunogenic, proliferative and receptor tyrosine kinase (RTK)-driven. Tumors of these four subtypes showed distinct biological and clinical features, suggesting subtype-specific therapeutic strategies. The RTK-driven subtype was characterized by the activation of the RTK pathways and associated with poor outcomes. The immunogenic subtype had enriched immune cells and could benefit from immune checkpoint therapy. In addition, we developed convolutional neural network models to discriminate these subtypes based on digital pathology for potential clinical translation. The molecular classification provides insights into molecular heterogeneity and highlights the potential for precision treatment of HR+/HER2- breast cancer.

Laminin 332 expression levels predict clinical outcomes and chemotherapy response in patients with pancreatic adenocarcinoma.

Poor outcomes and chemotherapy resistance for patients with pancreatic adenocarcinoma (PAAD) are a challenge worldwide, and new or improved prognostic biomarkers are urgently required. Individual laminin family members have been established as cancer-associated markers, predicting patient outcomes in many cancer types, including PAAD. Here, we used multiple modalities including RNAseq and gene chip, and genomic and proteomic data to examine the relationships of all laminin genes in PAAD with clinical outcomes. These analyses identified that LAMA3, LAMB3, and LAMC2 expression levels are increased at the mRNA and protein levels in PAAD tumours with evidence of co-regulation. Increased expression of all three genes was associated with decreased promoter methylation status, TP53 mutations, and altered receptor tyrosine kinase (RTK) pathways. Clinically, high LAMA3, LAMB3, and LAMC2 transcript abundance was each related to an advanced histological grade. Moreover, high expression of these genes individually predicted poor patient survival, while a signature of combined high expression of LAMA3, LAMB3, and LAMC2 was a stronger predictor of patient outcomes than each gene alone. Interestingly, cell lines with high expression of LM332 chains were not sensitive to the commonly used PAAD chemotherapy drugs paclitaxel and gemcitabine; however, increased sensitivity was evident for erlotinib, afatinib, gefitinib, and cetuximab epidermal growth factor (EGFR) RTK inhibitors. To explore possible mechanisms, we investigated co-expressed genes, identifying eight hub genes, namely, GJB3, ITGB6, SERPINB5, GPRC5A, PLEK2, TMPRSS4, P2RY2, and TRIM29, which are co-expressed with all three of LAMA3, LAMB3, and LAMC2. Of these, only SERPINB5 provided a stronger predictive value than the laminin-encoding genes. Together, these multiple integrated analyses suggest that the combined expression of LM332 is a useful prognostic biomarker for PAAD and could help patient stratification and therapeutic selection.

Diagnosis and treatment of rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma. The primary tumor is most commonly localized in the head and neck, the urogenital system, or the limbs. Classification by the World Health Organization has distinguished four histopathological RMS subtypes: embryonal, alveolar, pleomorphic, and spindle cell/sclerosing. Differential diagnosis of RMS includes melanoma, malignant neoplasm of peripheral nerve sheaths, liposarcoma, and PEComa. Among typical cytogenetic changes in RMS are chromosomal translocations t(2;13)(q35;q14) and t(1;13) (p36;q14). They lead to the formation of fusion genes that have a prognostic value. In the course of RMS, changes may also be present in signaling pathways, including RAS-PI3K, Wnt/b-catenin, receptor tyrosine kinase pathways, and myogenesis regulation. In 30% of patients at the time of diagnosis of RMS, distant metastases are present, most commonly to lungs, lymph nodes, bones, and bone marrow. Treatment of patients with RMS requires a multidisciplinary approach, and steadily perfected diagnostic techniques contribute to the individualization of therapeutic strategies. Optimal treatment of localized RMS is based on surgery combined with radiotherapy and chemotherapy. If distant metastases are present, the basic therapeutic method is multidrug chemotherapy, most frequently based on vincristine, dactinomycin, ifosfamide/cyclophosphamide, and etoposide. Despite intensive treatment, the 5-year survival index for RMS is not greater than 50%. There are still no unequivocal guidelines concerning the treatment in patients with local or distant recurrences.

Promotion of squamous cell carcinoma tumorigenesis by oncogene-mediated THG-1/TSC22D4 phosphorylation.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-β1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

Mast Cell Involvement in the Pathogenesis of Selected Musculoskeletal Diseases.

In recent years, there has been a noteworthy revival of interest in the function of mast cells (MCs) in the human body. It is now acknowledged that MCs impact a wide array of processes beyond just allergies, leading to a shift in research direction. Unfortunately, some earlier conclusions were drawn from animal models with flawed designs, particularly centered around the receptor tyrosine kinase (Kit) pathway. Consequently, several subsequent findings may have been unreliable. Thus, what is now required is a re-examination of these earlier findings. Nevertheless, the remaining data are fascinating and hold promise for a better comprehension of numerous diseases and the development of more effective therapies. As the field continues to progress, many intriguing issues warrant further investigation and analysis. For instance, exploring the bidirectional action of MCs in rheumatoid arthritis, understanding the extent of MCs' impact on symptoms associated with Ehlers-Danlos syndrome, and unraveling the exact role of the myofibroblast-mast cell-neuropeptides axis in the joint capsule during post-traumatic contractures are all captivating areas for exploration. Hence, in this review, we summarize current knowledge regarding the influence of MCs on the pathogenesis of selected musculoskeletal diseases, including rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, gout, muscle and joint injuries, tendinopathy, heterotopic ossification, and Ehlers-Danlos syndrome. We believe that this review will provide in-depth information that can guide and inspire further research in this area.