NOTCH1 Mutations Research Articles

Synergistic immunochemotherapy targeted SAMD4B-APOA2-PD-L1 axis potentiates antitumor immunity in hepatocellular carcinoma

Targeted and immunotherapy combined with interventional therapy can improve the prognosis of advanced cancer patients, and it has become a hot spot to find the new therapeutic schemes, but most of which are not satisfactory. Single-cell RNA sequencing was performed in PDX mouse models with or without TCC therapy. 2-’O-Methylation modification and multiplex immunofluorescence staining were used to explore the function and mechanism of SAMD4B in the immune context of HCC. Here, we propose for the first time a synergistic immunochemotherapy that exerts a potent antitumour effect for patients with advanced hepatocellular carcinoma (HCC) in clinical practice based on three common antitumour drugs and found that HCC patients with new synergistic immunochemotherapy had better three-year overall survival (p = 0.004) and significantly higher survival ratio (increased by 2.3 times) than the control group. We further reveal the immunoregulatory mechanism of synergistic immunochemotherapy through 2’-O-Methylation modification mediated by SAMD4B, a tumour suppressor gene. Mechanistically, SAMD4B, increased by the reduced mutations of upstream genes NOTCH1 and NOTCH2, affected the instability of APOA2 mRNA by 2-’O-Methylation modification of the C-terminus. The decreased APOA2 further attenuated programmed death ligand 1 (PD-L1) level with a direct interaction pattern. The high-SAMD4B tumour tissues contained fewer native CD29+CD8+ T cells, which improved immune microenvironment to achieve the effect of antitumour effect. Overall, we developed a potent synergistic immunochemotherapy strategy that exerts an efficient anti-HCC effect inducing SAMD4B-APOA2-PD-L1 axis to inhibit tumour immune evasion.

NGS-based stratification refines the risk stratification in T-ALL and identifies a very-high-risk subgroup of patients

AbstractWe previously reported a better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 mutations without alterations of K-N-RAS and PTEN genes. Availability of high-throughput next-generation sequencing (NGS) strategies led us to refine the outcome prediction in T-ALL. Targeted whole-exome sequencing of 72 T-ALL–related oncogenes was performed in 198 adults with T-ALLs in first remission from the GRAALL-2003/2005 protocols and 242 pediatric patients with T-ALLs from the FRALLE2000T. This approach enabled the identification of, to our knowledge, the first NGS-based classifier in T-ALL, categorizing low-risk patients as those with N/F, PHF6, or EP300 mutations, excluding N-K-RAS, PI3K pathway (PTEN, PIK3CA, and PIK3R1), TP53, DNMT3A, IDH1/2, and IKZF1 alterations, with a 5-year cumulative incidence of relapse (CIR) estimated at 21%. Conversely, the remaining patients were classified as high risk, exhibiting a 5-year CIR estimated at 47%. We externally validated this stratification in the pediatric cohort. NGS-based classifier was highly prognostic independently of minimal residual disease (MRD) and white blood cell (WBC) counts, in both adult and pediatric cohorts. Integration of the NGS-based classifier into a comprehensive risk-stratification model, including WBC count at diagnosis and MRD at the end of induction, enabled the identification of an adverse-risk subgroup (25%) with a 5-year CIR estimated at 51%, and a favorable-risk group (32%) with a 5-year CIR estimated at 12%. NGS-based stratification combined with WBC and MRD sharpens the prognostic classification in T-ALL and identifies a new subgroup of patients who may benefit from innovative therapeutic approaches. The GRAALL-2003/2005 studies were registered at www.ClinicalTrials.gov as #NCT00222027 and #NCT00327678.

Impact of positive tumor-informed margin and TP53 mutations on surgical outcomes in esophageal squamous cell carcinoma.

e16024 Background: The complete removal of esophageal squamous cell carcinoma (ESCC) with clear margins (R0 resection) is pivotal for patient prognosis. However, it is limited by the potential inaccuracies of conventional histopathology. Next-generation sequencing (NGS) for minimal residual disease (MRD) detection might improve the prediction of postoperative outcomes. Methods: The study cohort included a total of 48 operable ESCC patients, from whom resected esophageal tumors, resection margins, and normal tissues were collected and profiled by a 425-gene panel. Comparisons of mutational features were conducted across various tissue types, and their associations with disease-free survival (DFS) were explored. Results: Mutations were detected in 81.8% (27/33) of normal tissues, 95.8% (46/48) of tumors, and 50.0% (24/48) of resection margins. In general, genetic alterations in resection margins closely resembled those in tumor tissues, but were distinct from normal tissues. Both tumor and margin samples showed higher variant allele frequencies (VAFs) and fewer mutations per patient than normal samples ( p < 0.0001). Moreover, eight patients exhibited identical mutations in paired margin and tumor samples. Notably, one patient out of eight with the same TP53 p.T175H mutation across all sample types exhibited the shortest DFS of only two months. Mutations in TP53 and NOTCH1 were the most common, displaying unique mutational patterns among different sample types. While NOTCH1 mutations were detected more frequently in normal tissues ( p = 0.001), TP53 mutations were enriched in tumors ( p = 0.04). Furthermore, normal tissues exhibited a distinct pattern of mutation sites for TP53 and NOTCH1 compared to those observed in tumor or margin tissues. We characterized tumor-informed margin (TiM) by the presence of identical mutations in both tumor and adjacent margins. The positive TiM status was associated with a shorter DFS, particularly when considering TP53 mutations alone ( p = 0.022) or excluding NOTCH1 mutations ( p = 0.031). Conclusions: A positive TiM status with TP53 mutations is a promising biomarker indicating poor surgical outcomes in ESCC. Mutations within NOTCH1 are common but showed no association with DFS. These findings provide novel insights into the feasibility of genetic profiling of resection margins to facilitate optimizing postoperative therapeutic regimens for ESCC.

Young adults (YA) with non-small cell lung cancer (NSCLC): Snapshot of the oncogenic drivers and immune landscapes.

8538 Background: Approximately 5% of NSCLC occurs in patients 50 years or younger (median age:71) representing distinct clinicopathological features. Reports characterizing genomic alterations are scarce. Using a large real-world (RW) dataset, we characterize oncogenic drivers, and immune landscapes to better understand YA with NSCLC. Methods: 42,326 NSCLC specimens were analyzed using next-generation sequencing of DNA (DNA-592 panel or whole exome) or RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). YA (<=50 years) were categorized into three groups (years): 18-30 (A1, n=61), 31-40 (A2, n=277) & 41-50 (A3, n=1,549); vs >50 (A4, n=40,439). Composition of tumor microenvironment (TME) was estimated from bulk RNA sequencing using QuanTIseq method. RW survival on Osimertinib (Osi-OS) was obtained from insurance claims and calculated from initiation of Osi treatment to last contact and was compared between ages 18-50 and >50 years. Hazard ratio (HR) was calculated using the Cox proportional hazards model. Statistical significance was determined using Chi-square, Fisher’s Exact, Mann-Whitney U and log-rank tests and corrected for multiple comparisons where applicable (q<0.05). Results: Consistent with previous reports, NSCLC in YA was more prevalent in females, non-smokers and was associated with adenocarcinoma histology. Among driver alterations, ALK(IHC+), ROS1, RET and NTRK1 fusion were enriched and KRAS mutations were reduced in YA. Interestingly, while the prevalence of KRASG12D from transition (non-smoker related) and EGFRE746_A750 decreased with age, KRASG12C from transversion increased with age in YA. YA were also associated with improved Osi-OS (HR: 0.79, median Osi-OS=37.4 months vs 32 months in >50 years old, p=0.019). High tumor mutation burden (>=10 mut/Mb), the prevalence of mutations in TP53, KMT2D, NF1, RBM10, NFE2L2 and NOTCH1 increased while GNAS decreased with age. The expression of immune checkpoint genes such as PDCD1LG2(A1/A4: 0.63, A3/A4: 0.89), LAG3(A2/A4: 0.73, A3/A4: 0.89) and IFNG (A1/A4: 0.32, A3/A4: 0.75) were reduced, while M2 macrophage (A2/A4: 1.2) and neutrophil (A2/A4: 1.2) were increased in YA (all q<0.05). Conclusions: We report an increased prevalence of RET and NTRK1 fusions in YA and key differences in distributions of frequent KRAS and EGFR mutations in YA (vs A4) along with decreased expression of immune related genes in the tumor microenvironment. The implications are under active investigation. [Table: see text]

Clinical and molecular characteristics and targeted therapy of pediatric non-infantile fibrosarcoma NTRK rearrangement–related tumors.

10048 Background: Neurotrophic tyrosine receptor kinase (NTRK) rearrangement is rare in tumors and relevant to targeted therapy. Pediatric NTRK rearrangement-related tumors have distinct clinical and molecular features compared to adults, and previous studies on pediatric NTRK rearrangement-related tumors has focused on infantile fibrosarcoma (IFS), the most common subtype. However, the clinical behaviors and molecular profiles of other subtypes of pediatric NTRK rearrangement-related tumors are largely unknown. Methods: In this study, we evaluated the molecular features, treatment response, and prognosis of 28 pediatric patients with non-IFS NTRK rearrangement-related tumors. Next-generation sequencing was performed in 21 patients to identify the NTRK fusion types and their co-occurring alterations, and pathway enrichment analysis was performed to explore the potential mechanisms involving NTRK rearrangement and resistance to tyrosine receptor kinase (TRK) inhibitors. Results: The NTRK fusion partners were diverse and tumor-specific. Several novel NTRK fusions were identified, including SNIP1-NTRK1, AGAP1-NTRK2, CRCT1-NTRK1, ZBTB7B-NTRK1, and SLC6A15-NTRK3. RTK-RAS, NOTCH, TP53, and WNT pathways were top 4 significantly enriched in tested samples. Twelve of 17 patients (70.6%) received TRK inhibitors as salvage treatment achieved complete or partial response. Patients who did not develop DDR, NOTCH, or TP53 pathway-related gene mutations had a high objective response rate of 82%, 82%, and 83%, respectively. Conclusions: Our findings may have important implications for the diagnosis and treatment of non-IFS NTRK fusion-positive tumors in pediatric patients. We also provide potential therapeutic targets for tumors resistant to TRK inhibitors. Clinical trials with TRK inhibitors, either alone or in combination, are required to establish the optimal treatment regimen and sequence in large-cohort pediatric patients with non-IFS NTRK rearrangement-related tumors. Clinical trial information: NCT05076071 .

Increased H19/miR-675 Expression in Adult T-Cell Leukemia Is Associated with a Unique Notch Signature Pathway.

The Notch pathway is a key cancer driver and is important in tumor progression. Early research suggested that Notch activity was highly dependent on the expression of the intracellular cleaved domain of Notch-1 (NICD). However, recent insights into Notch signaling reveal the presence of Notch pathway signatures, which may vary depending on different cancer types and tumor microenvironments. Herein, we perform a comprehensive investigation of the Notch signaling pathway in adult T-cell leukemia (ATL) primary patient samples. Using gene arrays, we demonstrate that the Notch pathway is constitutively activated in ATL patient samples. Furthermore, the activation of Notch in ATL cells remains elevated irrespective of the presence of activating mutations in Notch itself or its repressor, FBXW7, and that ATL cells are dependent upon Notch-1 expression for proliferation and survival. We demonstrate that ATL cells exhibit the expression of pivotal Notch-related genes, including notch-1, hes1, c-myc, H19, and hes4, thereby defining a critical Notch signature associated with ATL disease. Finally, we demonstrate that lncRNA H19 is highly expressed in ATL patient samples and ATL cells and contributes to Notch signaling activation. Collectively, our results shed further light on the Notch pathway in ATL leukemia and reveal new therapeutic approaches to inhibit Notch activation in ATL cells.

Abstract PO2-09-05: Building genomic and transcriptomic data among African American and Black patients with triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive breast cancer associated with poor response and early metastases. It represents a health disparity, with higher incidence and worse outcomes in Black compared to White patients. Further, IBC is enriched for the more aggressive triple-negative receptor subtype, TN-IBC, and this subtype is more prevalent among Black IBC patients. It is critical to identify genetic and molecular features that may guide better treatments for these aggressive cancers across races, and thus imperative to identify and increase racial representation among bioinformatics datasets. We previously performed comprehensive genomic and transcriptomic analyses for TN-IBC patient samples and here examine the differences among self-identified African American or Black (AA/B) and other races among IBC patients. Methods: As previously described, we collected matched blood and baseline tumor samples before treatment from 19 patients with primary TN-IBC. We performed whole exome and RNA sequencing (RNA-Seq) on these samples and compared mutations and differentially expressed genes (DEGs). Results were compared between 3 AA/B patient tissues vs the remaining 16 (Other) cases. Results: Mutations shared by all AA/B patients included amplification of the five most commonly mutated hallmark genes in the complete cohort, ARNT, BCL9, DDR2, FCGR2B and LMNA (all 100% in AA/B patients versus all 50% in the Other cohort). All AA/B patients had a Notch1 mutation (two deletions and one missense mutation, 100% AA/B vs 6% Other (one deletion)). Comparing differentially expressed genes and gene set enrichment analyses of the Broad molecular signatures database (MSigDB) demonstrates the majority of DEGs are downregulated in these AA/B patients. Six of the top 20 downregulated gene sets from the MSigDB C2 group relate to breast cancer subtype and among this TNIBC cohort AA/B tumor expression was downregulated for luminal and normal subtype related genes in multiple gene sets and enriched in the Lien metaplastic related genes. In the MSigDB C5 sets, over half of the top 20 enriched gene ontology sets among AA/B patients related to chromosomes, chromatin, spindle assembly or histone binding, while 16 of the top 20 downregulated enriched sets related to immune function. Multiple KEGG pathways enriched in AA/B cases related to RNA processing and expression and DNA replication and repair. Lastly, hallmark gene sets enriched in AA/B patients were cell cycle related, and targets of Myc, Hedgehog, B-catenin, TGFb, and TNFa via NFKb, while top significantly downregulated sets related to metabolism and immune response. Conclusion: While findings from a small sample size can only be considered hypothesis generating, some patterns emerge from the data among this rare cohort of AA/B TN IBC patients. These tumors appear to be devoid of luminal signals, characterized by cell cycle and chromatin remodeling signals, enriched for signaling typical of stem cell self-renewal, and perhaps more immune-deprived than other TN-IBC. Additional work to build racially diverse IBC transcriptomic data are needed to develop strategies to understand and improve outcomes for these patients. Citation Format: Wencai Ma, Li Zhao, Gayathri Devi, Savitri Krishnamurthy, Larry Coffer, Angela Alexander, Megumi Kai, Xiaoping Wang, Huong Le-Petross, Miral Patel, Bisrat Debeb, Chandra Bartholomeusz, Jianhua Zhang, Xingzhi Song, Andrew Futreal, Naoto Ueno, Rachel Layman, Anthony Lucci, Jing Wang, Wendy Woodward. Building genomic and transcriptomic data among African American and Black patients with triple-negative inflammatory 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 PO2-09-05.

Abstract PO4-24-01: Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and characterized by poor outcomes with a lack of targeted therapies. A comprehensive analysis of the molecular and immune landscape of invasive ductal TNBC can help improve our understanding of TNBC biology and identify novel targets and/or pathways for better management of this disease. Here, we characterized the molecular and immune signature of invasive ductal (ID) TNBC. Methods: 13,036 BC samples (ID TNBC, n=392; ID non-TNBC, n=927) were analyzed by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Real world overall survival (OS) and treatment-associated survival was extracted from insurance claims and calculated from tissue collection or treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: ID TNBC had a higher frequency of TP53, RB1, NF1, PIK3R1, NOTCH1, CREBBP, BRCA1, FANCI, and HRAS mutations (Table 1), NOTCH2 (4.3% vs 0.47%), EGFR (3.4% vs 0%), NFIB (4% vs 0.6%), MYB (3.5% vs 0.5%), CCNE1 (4.4% vs 1.1%) and AKT2 (2.4% vs 0.3%) copy number alterations, and NOTCH2 (1.6% VS 0.2%) fusion (all p < 0.05) compared to invasive ductal non-TNBC. Analysis of inferred immune cell infiltrates showed that ID TNBC had increased infiltration of Tregs (2% vs 1.7%), DC (3.2% vs 2.4%), CD8 T cells (0.5% vs 0.1%), and M1 macrophages (M) (3.4% vs 2.9%), but decreased infiltration of B cells (4.5% vs 6%) and M2M (2.9% vs 4.5%) (all p < 0.05). ID TNBC had increased T cell inflamed score (23 vs -9, p < 0.05), IFNy score (-0.24 vs -0.34, p < 0.05), MHC class I genes (HLA-A, HLA-B, TAP1, TAP2, FC: 1.1-1.5, all p < 0.05), immune checkpoint genes (CD274, PDCD1, CTLA4, PD-L2, FOXP3, IDO, FC: 1.1-2, all p < 0.05) and PD-L1 protein expression (SP142: 49.8% vs. 28.4%; 22c3: 41.7% vs. 16.8%, all p < 0.05). ID TNBC had differential expression of BCL2 family genes (upregulation: BAK1, BID, MCL1, BCL2A1, BCL2L10, FC: 1.1-2.0; downregulation: BAD, BIK, BBC3, BCL2, BCL2L1, BCL2L2, FC: 1.2-2.8, all p < 0.05) compared to invasive ductal non-TNBC. ID TNBC was associated with worse OS compared to ID non-TNBC (mOS: 24.5 vs 54.6 months; HR: 0.5, 95% CI 0.47-0.57; p < 0.00001) but trend towards better survival with pembrolizumab (mOS: 29.4 vs 8.8 month; HR: 0.67, 95% CI 0.28-1.5; p=0.3) treatment. No significant OS difference was noted for by high vs. low pro and anti-apoptotic BCL2 genes except for anti-apoptotic BCL2A1 (higher expression associated with better OS (mOS: 5.7 months, HR: 0.78, 95% CI 0.6-0.99; p=0.047). Conclusion: These data indicate that ID TNBC is associated with distinct mutational profile compared to non-TNBC, has increased T cell inflamed score, IFNy score, MHC class I and immune checkpoint gene expression, and differential immune cell infiltration. Interestingly, TNBC was associated with increased M1 and decreased M2M. There is evidence to suggest that transcriptomically defined M1 high tumors are clinically aggressive and have worse OS in TNBC. BCL2 family genes are not prognostic for TNBC outcomes except BCL2A1, which needs further prospective validation. Table 1: Mutation frequency in invasive ductal TNBC and invasive ductal non-TNBC Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Jose Leone, Priya Jayachandran, Stephanie Graff, Matthew Oberley, George Sledge Jr, Asher Chanan-Khan. Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative 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 PO4-24-01.

Molecular profiling of a bladder cancer with very high tumour mutational burden

The increasing incidence of urothelial bladder cancer is a notable global concern, as evidenced by the epidemiological data in terms of frequency, distribution, as well as mortality rates. Although numerous molecular alterations have been linked to the occurrence and progression of bladder cancer, currently there is a limited knowledge on the molecular signature able of accurately predicting clinical outcomes. In this report, we present a case of a pT3b high-grade infiltrating urothelial carcinoma with areas of squamous differentiation characterized by very high tumor mutational burden (TMB), with up-regulations of immune checkpoints. The high TMB, along with elevated expressions of PD-L1, PD-L2, and PD1, underscores the rationale for developing a personalized immunotherapy focused on the use of immune-checkpoint inhibitors. Additionally, molecular analysis revealed somatic mutations in several other cancer-related genes, including TP53, TP63 and NOTCH3. Mutations of TP53 and TP63 genes provide mechanistic insights on the molecular mechanisms underlying disease development and progression. Notably, the above-mentioned mutations and the elevated hypoxia score make the targeting of p53 and/or hypoxia related pathways a plausible personalized medicine option for this bladder cancer, particularly in combination with immunotherapy. Our data suggest a requirement for molecular profiling in bladder cancer to possibly select appropriate immune-checkpoint therapy.

Preclinical testing of CT1113, a novel USP28 inhibitor, for the treatment of T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive and heterogeneous lymphoid malignancy with poor prognosis in adult patients. Aberrant activation of the NOTCH1 signalling pathway is involved in the pathogenesis of over 60% of T-ALL cases. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase known to regulate the stability of NOTCH1. Here, we report that genetic depletion of USP28 or using CT1113, a potent small molecule targeting USP28, can strongly destabilize NOTCH1 and inhibit the growth of T-ALL cells. Moreover, we show that USP28 also regulates the stability of sterol regulatory element binding protein 1 (SREBP1), which has been reported to mediate increased lipogenesis in tumour cells. As the most critical transcription factor involved in regulating lipogenesis, SREBP1 plays an important role in the metabolism of T-ALL. Therefore, USP28 may be a potential therapeutic target, and CT1113 may be a promising novel drug for T-ALL with or without mutant NOTCH1.

Deciphering age-specific molecular features in cervical cancer and constructing an angio-immune prognostic model.

Cancer incidence is increasingly seen in younger individuals. Molecular distinctions between young and elderly patients at onset are understudied. This study used public databases to explore genomic, transcriptomic, and immune-related features across age groups in cervical cancer. Additionally, it aims to create a prognostic model applicable across diverse age cohorts, enabling precise patient stratification, and personalized therapies. Gene mutations, expression data, and clinicopathological information were obtained from 317 cervical cancer patients. These patients were divided into a young group and an old group based on the median age of onset. The characteristics of differential gene mutation, gene expression, and immune cells analysis were analyzed by R software. Finally, the prognostic model was constructed by univariate Cox, least absolute shrinkage and selection operator, and multivariate Cox regression analyses of angiogenic and immune gene sets. Its validity was further confirmed using an additional 300 cervical squamous cell carcinoma and endocervical adenocarcinoma tissues. Cervical cancer patients at elderly onset age exhibit a significantly higher frequency of NOTCH1 and TP53 driver mutations compared to young patients, along with a notably higher tumor mutational burden. However, there were no significant differences between the 2 groups in terms of genomic instability and age-related mutational signatures. Differential gene expression analysis revealed that the young group significantly upregulated interferon-alpha and gamma responses and exhibited significantly higher activity in multiple metabolic pathways. Immune microenvironment analysis indicated enrichment of dendritic cells and natural killer cells in the young group, while transforming growth factor-β signature was enriched in the elderly group, indicating a higher degree of immune exclusion. A multigene prognostic model based on angiogenesis and T cell immune gene sets showed excellent prognostic performance independent of clinical factors such as age. High-risk groups identified by the model exhibit significant activation of tumor-promoting processes, such as metastasis and angiogenesis. Our study reveals distinct patterns in cancer-driving mechanisms, biological processes, and immune system status between young and elderly patients at onset with cervical cancer. These findings shed light on the age-specific underlying mechanisms of carcinogenesis. Furthermore, an independent molecular prognostic model is constructed to provide valuable references for patient stratification and the development of potential drug targets.

Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.

Mutant IDH inhibitors induce lineage differentiation in IDH-mutant oligodendroglioma

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit theresponse to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

Abstract 1766: CREBBP, NOTCH2 and GNASmutational profile in uterine sarcomas

Abstract Background: Uterine sarcomas (US) present clinical challenges due to their dynamic behavior and limited treatment options. Representing 1-3% of uterine cancer cases, these tumors exhibit rapid growth, high recurrence rates, and resistance to the standard treatments. The present study aimed to validate several Loss-of-function mutations (LOF), obtained by NGS analysis in the CREBBP, NOTCH2, and GNAS sequences, in US through Sanger sequencing. Methods: Twenty-five samples of US were collected between 2000 and 2015 for genomic DNA extraction. An initial genetic screening of the samples by NGS method was followed by Sanger sequencing validation. Bioinformatics analysis through SnapGene confirmed the results. Results: A c.4063G>A mutation was confirmed in uterine leiomyosarcomas (ULMS), in the both strands. Notable variations in the forward strand, such as a C>G substitution at position 38 of the alignment and a degenerate base S at position 113, differentiated ULMS from uterine sarcomas (US). Two ULMSs exhibited the highest substitution rates: one with 18 and another with 6 substitutions. Despite the absence of the c.5527T>C CREBBP mutation, ULMS displayed increased insertions and deletions. Assessing the NOTCH2 gene in US revealed a complex genetic landscape, with a unique SU-specific duplication in the forward strand. The reverse strand maintained high genetic stability. Mutation c.6094C>A was exclusive to one US sample. Notably, the c.7223T>A mutation in NOTCH2 was not identified. Shared mutations between ULMS and US suggest genomic similarities in these regions. In the GNAS analysis, the c.2381A>C mutation was not found. However, in the forward sequence of one US sample, a deletion at the expected substitution site was detected, suggesting a specific genetic alteration. The c.706G>A mutation was consistently present in sample ULMS, indicating genetic stability in this sample. US exhibited a G>C substitution in the reverse sequence, hinting at a unique pattern of genetic variation. These findings underscore the complexity of genetic alterations in these critical gene regions. Conclusion: Our analysis identified specific alterations in ULMS and US. ULMS showed the c.4063G>A CREBBP mutation and unique variations, while US had a specific c.6094C>A NOTCH2 mutation. The absence of the c.2381A>C GNAS mutation in both ULMS and US, along with distinct genetic patterns (c.706G>A in ULMS and G>C substitution in US), underscores significant mutational differences in these critical gene regions. These findings enhance our understanding of uterine sarcoma genetics, prompting further exploration for potential diagnosis, prognosis and therapeutic advancements. Citation Format: Laura Gonzalez dos Anjos, Giovanna Quevedo, Edmund Baracat, Katia C. Carvalho. CREBBP, NOTCH2 and GNASmutational profile in uterine sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1766.

Abstract 7612: Genetic predictors of anti-PD(L)-1 therapy response in hepatocellular carcinoma

Abstract Introduction: Immunotherapy targeting PD-1/PD-L1 shows promise in treating hepatocellular carcinoma (HCC), but standard predictors like PD-L1 expression are unreliable for gauging treatment response. RNA sequencing has identified a BMS 4-gene inflammatory signature that could more accurately forecast clinical outcomes. However, genomic alterations and tumor mutational burden (TMB) haven't consistently predicted anti-PD(L)-1 therapy response in HCC. Therefore, the aim of this research is to identify potential genetic alternation that could serve as predictive marker for anti-PD(L)-1 therapy responses in advanced HCC. Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissues from 38 HCC patients were analyzed using targeted next-generation sequencing with the ACTOncoTM panel before anti-PD(L)-1 therapy. This analysis identified somatic variants across 440 genes and calculated TMB. In silico tools like NetMHC and IEDB predicted neo-peptide-HLA class I binding affinity. Additionally, peripheral blood mononuclear cells provided germline data for paired TMB calculations, with tumor responses assessed by RECIST criteria. Results: Within a cohort of 167 patients with HCC undergoing ICI therapy, 35 (20.9%) responded to treatment, while 132 (79.1%) did not. Available formalin-fixed paraffin-embedded (FFPE) samples from this population included those from 14 responders and 30 non-responders. After conducting quality control measures, which assessed tumor purity, DNA quantity and integrity, as well as sequencing quality, 13 samples from responders and 25 from non-responders qualified for further analysis. Gene alteration analysis identified mutations in TP53 (47%), MUC16 (34%), CTNNB1 (32%), USH2A (16%), ARID1A (13%), NOTCH3 (13%), and ADAMTS16 (11%) of the cases. A TP53 mutation was linked to poor overall survival, whereas mutations in CTNNB1 and the RTK/RAS/RAF pathway did not correlate with clinical outcomes. The median TMB, predicted at 3.2 ± 4.3 mutations per megabase (muts/Mb), was nominally higher in patients who achieved disease control compared to non-responders (4.6 vs. 3.7 muts/Mb, p=0.53). Utilizing paired TMB analysis, those with disease control exhibited a higher TMB trend than non-responders (6.0 vs. 2.2 muts/Mb, p=0.06). Notably, the 13 cases with MUC16 mutations were associated with a significantly higher predicted TMB (10.0 vs. 3.3 muts/Mb, p=0.0015). Conclusions: This study suggests that certain genetic alterations, especially TP53 mutations, may have predictive value for the response to anti-PD(L)-1 therapy in HCC. Moreover, an elevated TMB, particularly when associated with MUC16 mutations, seems to be correlated with improved disease control. Citation Format: San-Chi Chen, Nai-Jung Chiang, MingHuang Chen, Muh-Hwa Yang. Genetic predictors of anti-PD(L)-1 therapy response in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7612.

Abstract 3934: Smoking's influence on divergent paths to lung cancer

Abstract Lung cancer is the leading cause of cancer mortality in the world. Many studies have highlighted how mutagens from cigarette smoke promote specific mutations such as G12 mutations in KRAS, contributing to clonal evolution that leads to lung cancer. Despite the indisputable correlation between smoking and lung cancer, people who have never smoked account for the 7th leading cause of cancer mortality. Given the differences in predominant exposures, we hypothesize that the clonal evolution between people with and without smoking histories are markedly different. Therefore, we analyzed lung samples from people with different cigarette smoking exposure to elucidate smoking’s effect on the somatic mutational landscape in the progression to lung cancer. To observe somatic mutations subjected to different exposures of smoking, we used a rare-mutation detection technique called DuplexSeq to analyze somatic variants from 200+ lung samples. We acquired DNA from cells from bronchoscopy brushings and lung punches of histologically normal tissue from people with and without lung cancer and who currently, formerly, or never smoked cigarettes. Through collaborations, we characterized mutations using bioinformatic pipelines, various databases, and statistical methods. In samples from people who smoke, we observe an enhancement of mutations with higher variant allele frequencies and pervasive positive selection acting on mutations in many cancer-associated genes. From our bronchoscopy brushings, NOTCH1 mutations show the most definitive increase of protein-altering variants, while NF1 and KRAS show a predominance of driver mutation in lung punches. Unexpectedly, people without lung cancer who never smoked had a notable presence of pathogenic mutations in PTEN and PIK3CA. Furthermore, multiple punches from the same individual highlight how expansions of mutations have stronger similarity within an individual than across individuals regardless of smoking status. This association points to the importance of the inherent lung microenvironment in influencing the mutational landscapes in lung cancer progression. Altogether, these results show how smoking increases the oncogenicity of observable mutations in the lung but does not preclude the presence of oncogenic clones, emphasizing the value of precision diagnoses and prevention. Ongoing research will be centered around functional assays to determine the impact of observed mutations on tissue function and longitudinal studies using mutational landscapes as a proxy for lung cancer risk. Citation Format: Edward James Evans, Fabio Marongiu, Emilia Lim, Oriol Pich, Yonghua Zhuang, Dexiang Gao, William Hill, Cian Murphy, Ryan Turner, Moumita Ghosh, York Miller, Mariam Jamal-Hanjani, Charles Swanton, James DeGregori. Smoking's influence on divergent paths to lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3934.

Abstract 2414: Feasibility and clinical utility of blood based NGS in head and neck carcinomas: A single center experience from precision medicine program

Abstract Background: While blood-based next-generation sequencing is increasingly integral to clinical practice across various malignancies, the routine assessment of circulating tumor DNA (ctDNA) in head and neck cancer remains underexplored, with limited supportive data. Methods: This study focused on head and neck cancer patients enrolled in the Gustave Roussy precision medicine program (STING, NCT04932525), undergoing liquid biopsy (LB) with the FoundationOne Liquid CDX panel. The Molecular Tumor Board discussed cases, calculating Tumor Fraction (TF) based on aneuploidy, categorizing it as high or low with a 10% cutoff. Results: The analysis encompassed 239 LBs from 214 patients with relapsed or metastatic head and neck cancers. Of these, 59 LBs were from 57 patients showing stability or positive response (SD/R), while 180 were from 167 patients at diagnosis or disease progression (BL/PD). Notably, 156 (73%) were male, with a median age of 61 years. Within the cohort, 65% of LBs were from patients with squamous cell carcinomas (SCC), 15% adenoid cystic carcinomas (ACC), and 20% other histologies mainly adenocarcinomas. The anatomical distribution included 59 LBs from salivary gland (SG) cancers, 49 oropharynx (OP), 48 oral cavity (OC), and others including larynx, hypopharynx, sinus and nasopharynx. Among LBs performed at BL/PD, 72% were informative, while only 36% were so at SD/R. The proportion of informative biopsies varied across anatomical sites. For SCC, TP53 mutations were most prevalent (71%), followed by TERT and PIK3CA variants, and FGF19 and FGF3 amplifications. ACCs showed NOTCH1 mutations (6/19, 32%) as the most common, followed by TP53 and various other variants. In the entire cohort, clonal hematopoiesis-related variants were identified, with 40% showing at least one of DNMT3A, CHEK2, or ASXL1. Patients with low TF at BL/PD exhibited significantly improved overall survival (21.6 vs. 7.6 months, HR 0.35, 95% CI 0.23 - 0.53, p < 0.0001), consistent across subgroups except for OP.Conclusion: This study demonstrates the feasibility of liquid biopsy in HNSCC and rare head and neck tumors, providing valuable therapeutic and prognostic insights. The findings underscore the potential of liquid biopsy as a valuable tool in clinical decision-making for head and neck cancer patients. Citation Format: Filippo Gustavo Dall'Olio, Damien Vasseur, Lea Loriguet, Francois Regis Ferrand, Antoine Moya-Plana, Yungan Tao, Mariana Iacob, Karim Benihoud, Odile CASIRAGHI, Catherine Brenner, Pierre Busson, Philippe Gorphe, Santiago Ponce, Pierre Blanchard, Etienne Rouleau, Yohann Loriot, Ludovic Lacroix, Antoine Italiano, Ingrid Breuskin, Caroline Even. Feasibility and clinical utility of blood based NGS in head and neck carcinomas: A single center experience from precision medicine program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2414.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce.MethodsIn the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome.ResultsThe median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression.ConclusionOverall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

Molecular characteristics and multivariate survival analysis of 43 patients with locally advanced or metastatic esophageal squamous cell carcinoma.

Esophageal cancer (EC) is an aggressive malignant tumor with poor prognosis and high incidence. It is the sixth leading cause of cancer-related death in the world, and the 5-year overall survival (OS) rate is only 12-20%. The rapid development of next-generation sequencing (NGS) has provided powerful help for the treatment and management of EC patients. Tumor tissue and blood samples of 43 Chinese patients with nonsurgical esophageal squamous cell carcinoma (ESCC) were sequenced using a 425 gene-panel. Genomic profiling was explored and and the Cox proportional hazards model was used to analyze the correlations between gene or signaling pathway alterations and prognosis. In this study, the most common mutated genes were TP53 (90.5%), CCND1 (45.2%), FGF19 (38.1%), NOTCH1 (26.2%), PI3KCA (21.4%) and CDKN2A (19%). Among these mutations, PI3KCA and NOTCH1 showed mutual exclusion to some extent. In the univariate model, mutations in NOTCH1, CBLB and TSC2 genes and tumor mutation burden (TMB) ≥7 were independent biomarkers of OS. NOTCH1 (P=0.007, HR =2.87), CBLB (P=0.011, HR =4.68) and TSC2 (P=0.024, HR =3.7) were significantly associated with poorer OS, and patients with TMB ≥7 had longer OS (P=0.151, HR =0.31). In addition, patients who carried alteration in NOTCH signaling pathway had reduced OS (P=0.014, HR =2.54). NOTCH1, CBLB and TSC2 alterations were found to be potential indicators of poor prognosis in patients with ESCC. TMB was also positively correlated with the OS of ESCC patients, providing valuable insights for their treatment strategies.

Notch signaling genes and CD8+ T-cell dynamics: Their contribution to immune-checkpoint inhibitor therapy in oral squamous cell carcinoma: A retrospective study.

Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T-cell infiltration via PD-L1, which lead to enhanced antitumor immunity and may target for immune-checkpoint inhibitors (ICIs) therapy. This retrospective study analyzed the results of immunohistochemical staining for PD-L1 and CD8+ T-cell infiltration in 10 patients and whole-exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. Four of 10 patients were positive for PD-L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T-cell infiltration via PD-L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.