Mutations In NOTCH1 Gene Research Articles

Squamoid Eccrine Ductal Carcinoma Displays Ultraviolet Mutations and Intermediate Gene Expression Relative to Squamous Cell Carcinoma, Microcystic Adnexal Carcinoma, and Porocarcinoma

Squamoid eccrine ductal carcinoma is a rare infiltrative tumor with morphologic features intermediate between squamous cell carcinoma (SCC) and sweat gland carcinomas such as microcystic adnexal carcinoma. Although currently classified as a sweat gland carcinoma, it has been debated whether squamoid eccrine ductal carcinoma is better classified as a variant of SCC. Furthermore, therapeutic options for patients with advanced disease are lacking. Here, we describe clinicopathologic features of a cohort of 15 squamoid eccrine ductal carcinomas from 14 unique patients, with next-generation sequencing DNA profiling for 12 cases. UV signature mutations were the dominant signature in the majority of cases. TP53 mutations were the most highly recurrent specific gene alteration, followed by mutations in NOTCH genes. Recurrent mutations in driver oncogenes were not identified. By unsupervised comparison of global transcriptome profiles in squamoid eccrine ductal carcinoma (n = 7) to SCC (n = 10), porocarcinoma (n = 4), and microcystic adnexal carcinoma (n = 4), squamoid eccrine ductal carcinomas displayed an intermediate phenotype between SCC and sweat gland tumors. Squamoid eccrine ductal carcinoma displayed significantly higher expression of 364 genes (including certain eccrine markers) and significantly lower expression of 525 genes compared with other groups. Our findings support the classification of squamoid eccrine ductal carcinoma as a carcinoma with intermediate features between SCC and sweat gland carcinoma.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [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 518.

Role of Mutation of NOTCH1 gene in development of oral squamous cell carcinoma: a narrative review.

Cancer of the oral cavity has numerous types and, among all, oral squamous cell carcinoma represents >90% of all cancers of the oral area. Oral squamous cell carcinoma arises from the squamous lining of the oral cavity. Across the globe, most commonly it develops in the regions of tongue followed by floor of the mouth, and lower lip. Neurogenic locus notch homolog protein 1 gene has its association with oral squamous cell carcinoma and is known to be associated with both oncogenic and tumour suppressor roles. The current narrative review comprised literature published from 2013 to 2023. It was searched on Google Scholar, PubMed and Google databases. Globally, neurogenic locus notch homolog protein 1 mutations are associated with the development of oral squamous cell carcinoma. Most of the mutations are linked to ligand bind epidermal growth factor-like repeat region of extracellular domain of neurogenic locus notch homolog protein 1. Once activated, the pathway is involved in tumour progression and metastasis. The Asians compared to Caucasians are more affected by neurogenic locus notch homolog protein 1 mutations.

IMMUNOPHENOTYPE OF LEUKEMIC CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH NOTCH1 AND SF3B1 GENE MUTATIONS.

The typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis, but the expression of some antigens varies and has prognostic value. There are data that reduced CD20 expression is associated with NOTCH1 and SF3B1 gene mutations. To determine a high-risk group of CLL patients for prediction of unfavorable NOTCH1 and SF3B1 gene mutations based on immunophenotyping of leukemic cells. Flow cytometric and molecular-genetic analysis (mutations of NOTCH1, SF3B1, and TP53 genes using the polymerase chain reaction followed by direct sequencing) was performed in a group of 86 previously untreated CLL patients. The immunophenotype of leukemic cells of all examined patients met the criteria of CLL diagnosis. NOTCH1 gene mutations were found in 21 patients (24.4%), and SF3B1 gene mutations - in 7 patients (8.1%). There were no TP53 gene mutations among the examined patients. A decreased number of CD20+CD5+ cells and a downward trend in the relative index of mean fluorescence intensity (iMFI) of CD20+ cells were found in patients with NOTCH1 and SF3B1 gene mutations. Based on the iMFI level (higher and/or lower than 3.0) and the number of CD20+CD5+ cells among all B-cells (higher and/or lower than 50%), we distinguished CLL cases with low and relatively high levels of CD20 antigen expression. Using ROC analysis and the parameter of low CD20 antigen expression, we could predict the presence of NOTCH1 and SF3B1 gene mutations in 73.3 ± 0.06% of patients (p = 0.001). The risk of NOTCH1 and SF3B1 gene mutations in cases with low CD20 antigen expression was 6.96 (95% CI = 2.53-19.18; p = 0.0001). The revealed regularities were statistically significant for patients in whom the diagnosis was established in all Binet - Rai stages except A0-AI. Our data confirmed a reduced CD20 expression in CLL patients with NOTCH1 and SF3B1 mutations. In addition, an approach was proposed to identify high-risk CLL patients for prediction of such mutations: previously untreated CLL patients at advanced Binet - Rai stages (BII, CIII, CIV) with a reduced number of double-positive CD20+CD5+ cells in peripheral blood and/or low iMFI of CD20+ cells.

RISK FACTORS AND EARLY SIGNS OF CRITICAL CONDITIONS IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA ADMITTED TO THE INTENSIVE CARE UNIT

Relevance: Acute lymphoblastic leukemia (ALL) is the most common cancer among children, accounting for nearly a quarter of all childhood cancers.
 The study aimed to determine the risk factors and signs of critical conditions in children with acute lymphoblastic leukemia admitted to an intensive care unit (ICU).
 Methods: The approach used was a systematic review. Data was collected from sources published in 2019-2023. Four cohort studies, four retrospective analyses, two literature reviews, one case-control study, and one case study were included in this systematic review.
 Results: The prognosis in pediatric ALL depends on the initial number of blast cells in the peripheral blood. Patients with B-cell precursor acute lymphoblastic leukemia (BCP ALL) and low blast cell numbers survived better than patients with T-cell acute lymphoblastic leukemia (T-ALL) and low cell count. IL1B and NLRP1 genetic polymorphisms enhanced ALL risk and reduced infectious comorbidity. However, these gene polymorphisms must be confirmed in juvenile leukemia. KRAS, FLT3, NRAS, PTPN11, KMT2D, PTEN, and NOTCH1 gene mutations affected pediatric ALL patient features and treatment results. These mutations demonstrate the relevance of genetic profiling in risk classification and tailored management. Gene variations and availability of effective medication contributed. Pediatric BCP-ALL patients with the PAX5P80R mutation had worse 5-year overall survival, higher white blood cell counts, male preponderance, and more genetic abnormalities. Pediatric BCP-ALL focused on genetic analysis and risk stratification. Children of African American and European American ancestry showed varied incidence, recurrence, and outcome rates for ALL. African American children exhibited lower incidence but greater recurrence rates and poorer prognosis than European American children.
 Conclusion: Risk factors for these patients’ admission to ICU include comorbidities, infectious diseases, hypoxia, and hemodynamic instability, as well as age and baseline white blood cell count at diagnosis.

Genetic Lesions in Russian CLL Patients with the Most Common Stereotyped Antigen Receptors.

Chronic lymphocytic leukemia (CLL) is one of the most common B-cell malignancies in Western countries. IGHV mutational status is the most important prognostic factor for this disease. CLL is characterized by an extreme narrowing of the IGHV genes repertoire and the existence of subgroups of quasi-identical stereotyped antigenic receptors (SAR). Some of these subgroups have already been identified as independent prognostic factors for CLL. Here, we report the frequencies of TP53, NOTCH1, and SF3B1 gene mutations and chromosomal aberrations assessed by NGS and FISH in 152 CLL patients with the most common SAR in Russia. We noted these lesions to be much more common in patients with certain SAR than average in CLL. The profile of these aberrations differs between the subgroups of SAR, despite the similarity of their structure. For most of these subgroups mutations prevailed in a single gene, except for CLL#5 with all three genes affected by mutations. It should be noted that our data concerning the mutation frequency in some SAR groups differ from that obtained previously, which could be due to the population differences between patient cohorts. The research in this area should be important for better understanding the pathogenesis of CLL and therapy optimization.

Detection of High-Frequency Mutation Genes in HIV-Positive and HIV-Negative Lymphomas

Objective Compare HIV-positive lymphoma with HIV-negative lymphoma mutation genes differences, screen specific mutations genes in HIV-positive lymphoma, analyze high frequency mutation genes in HIV-positive lymphoma patients, and explore the effect of HIV infection on lymphoma gene mutation and disease development. It can provide a theoretical basis for the clincal diagnosis and targeted therapy HIV-positive lymphoma. Methods A total of 7 HIV-positive lymphoma patients and 13 HIV-negative lymphoma patients who were treated in Chongqing University Cancer Hospital from September 2020 to September 2021 were enrolled. Tumor tissue samples were detected by second-generation sequencing technology, and gene mutations in HIV-positive lymphoma patients were analyzed by gene heat map, comparing the gene mutation differences between HIV-positive and HIV-negative lymphoma patients. Results Compared with HIV-negative lymphoma patients, more and more complex gene mutation sites were detected in HIV-positive lymphoma patients. Single nucleotide mutation of MYC gene was more common in HIV-positive lymphoma patients, while deletion of KMT2D gene was more common in HIV-negative lymphoma patients. And TP53 gene is highly mutated in both HIV-positive and HIV-negative lymphoma patients. However, mutations of TET, CCND3, TNFRSF14, ARID1A, TNFAIP3, NFKBIE, CXCR4, STAT3, NOTCH1 and other gene mutation with low mutation abundency occurred only in HIV-positive lymphoma patients. NOTCH2, PRDM1, EZH2, CARD11, EP300, MEF2B, CREBBP and other gene mutations occurred only in HIV-negative lymphoma patients. Conclusion HIV-positive lymphoma patients are more prone to gene mutations, and the gene mutations are more complex and diverse. MYC gene mutations and TP53 gene mutations play an important role in the occurrence and development of HIV-positive lymphoma, while the occurrence and development of HIV-negative lymphoma are more related to KMT2D gene mutations and TP53 gene mutations.

Characteristics and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia patients

Objective: To explore the characteristics, clinical features and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia (T-ALL) patients. Methods: The clinical data of 61 T-ALL patients who underwent second-generation gene sequencing in Henan Provincial People's Hospital from March 2016 to March 2021 were retrospectively analyzed. There were 46 males and 15 females, with a median age [M (Q1, Q3)] of 18 (11, 30) years. The relationship between NOTCH1/FBXW7 gene mutation characteristics, clinical and laboratory parameters and their impact on event free survival (EFS) and overall survival (OS) were analyzed. Results: NOTCH1 gene mutations were found in 34 cases (55.7%, 34/61), including 22 cases of heterodimer domain (HD) mutations (64.7%), 7 cases of proline/glutamate/serine/threonine (PEST) mutations (20.6%), and 5 cases of both HD and PEST mutations (14.7%). FBXW7 gene mutations were detected in 9 cases (14.8%, 9/61), of which 5 cases had both NOTCH1 and FBXW7 gene mutations. Twenty-three (37.7%, 23/61) cases were wild type. The median white blood cell count of patients in NOTCH1/FBXW7 gene mutations group and wild-type group was 76.4×109/L (8.3×109/L, 149.2×109/L), 54.1×109/L (5.3×109/L, 156.6×109/L), respectively. Moreover, the hemoglobin was (89.1±27.1) g/L and (99.5±23.1) g/L, respectively, and the median proportion of bone marrow primordial cells was 84.5% (69.0%, 91.3%) and 60.0%(35.0%, 80.0%), respectively. The gene expression rate of SIL-TAL1, Hox11 and Hox11L2 was 7.9% (3/38) vs 17.4% (4/23), 18.4% (7/38) vs 4.3% (1/23), 5.3% (2/38) vs 13.0% (3/23), respectively (all P>0.05). However, the median platelet level in the NOTCH1/FBXW7 gene mutations group was 60.5×109/L (36.8×109/L, 100.3×109/L), which was lower than that in the wild-type group [116.0×109/L (63.0×109/L, 178.0×109/L)] (P=0.018). The median number of gene mutations in the group with NOTCH1/FBXW7 gene mutations group was 2.5 (1.8, 4.0), which was more than that in the group without NOTCH1/FBXW7 gene mutations group [0 (0, 1.0)] (P<0.001). The median EFS and OS of adult NOTCH1/FBXW7 gene mutations group were 28.0 (95%CI: 7.3-48.7) months and 30.0 (95%CI: 8.9-51.1) months, respectively, which were better than those of adult wild-type group [4.5 (95%CI: 0-11.6) months and 9.0 (95%CI: 0-19.1) months] (P=0.008 and 0.014).The median EFS and OS of children NOTCH1/FBXW7 gene mutations group were 12.0 (95%CI: 10.4-13.6) months and 19.0 (95%CI: 13.6-24.4) months, respectively, and those of wild-type group were 10.0 (95%CI: 8.9-11.1) months and 21.0 (95%CI: 0-51.4) months, respectively (P=0.673 and 0.434). Conclusions: The mutation rate of NOTCH1/FBXW7 gene is higher in T-ALL patients. Patients with NOTCH1/FBXW7 gene mutations group have lower platelet count and better EFS and OS. NOTCH1/FBXW7 gene mutation may be used as a hierarchical basis for individualized treatment of adult T-ALL patients.

PB1871: PROGNOSTIC SIGNIFICANCE OF THE NOTCH1 GENE MUTATION AND OTHER LABORATORY FACTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background: An important stage in the management of patients with CLL is the prediction of the course of the disease in order to assess the need for an earlier onset of therapy and the use of alternative therapy on an individual basis, depending on the detectable prognostic markers. Aims: To determine the interrelationship of clinical-laboratory (as well as molecular-genetic) prognostic markers with various courses of CLL. Methods: We have examined 107 patients diagnosed with CLL in SI «RRC RM&HE» from 2017 to 2020. This cohort was divided into 2 groups: 1st group (49 patients) without signs of progression of CLL, and the 2nd group (58 patients) with clinical signs of disease progression after 3 years of observation. The median age in the 1st group was 61 years, in the 2nd - 62 years. The material for the study was venous blood. To determine the immunophenotype of tumor cells we used the flow cytofluorimeter FacsCanto II. Detection of β2-mg, TK and TPA was performed using the chemiluminescent analyzer. To determine the mutations of the NOTCH1 gene (34th exon), SSCP-PCR was used, followed by direct sequencing of DNA samples with conformational polymorphism. We used nonparametric statistics methods calculated in the Statistica 10.0. Statistically significant differences were considered for p<0.05. Results: Patients in group 1 differed from patients in group 2 in levels of β2-mg-1.8 and 3.5 μg/L (p<0.001) and LDH-220 and 326 U/L (p<0.002). TK levels (p<0.001) and TPA (p<0.011) were also significantly higher in group 2 (7.7 and 49.8 IU/L) compared with patients in group 1 (4.2 and 34.3 IU/L). According to immunophenotyping data, statistically significant differences were obtained on the Zap70 marker: in the 1st group the median was 10.1%, in the 2nd group - 21.6%. As a result of molecular genetic analysis, mutations in the NOTCH1 gene were identified in 14 patients. It was found that patients without progression at the time of inclusion in the study in the presence of mutations in the NOTCH1 gene had a statistically significantly shorter progression-free survival (mean 33.4 months (95% CI [22.0-44.8]) compared with patients without mutations (59.2 months (95% CI [54.1–64.3]) (p=0.018), which indicates the association of NOTCH1 mutations with a poor prognosis for disease progression in patients with CLL. Summary/Conclusion: It was found that markers such as LDH, β2-mg, TK, TPA, ZAP70 and NOTCH1 gene mutation have certain prognostic significance, which was confirmed by comparing their levels in patients with and without CLL progression after 3 years of observation. We can use NOTCH1 gene mutation as an additional factor in predicting the course of CLL. Knowledge of reliable prognostic markers may allow to initiate in-time treatment or to adjust therapy for patients with high-risk CLL with a probable increase in their life span.

Characteristics of Notch signaling pathway and its correlation with immune microenvironment in SCLC

ObjectivesTo explore the characteristics of Notch pathway in small cell lung cancer (SCLC), clarify the expression of delta-like protein 3 (DLL3) in SCLC patients undergoing surgical treatment, and explore the correlation between DLL3 expression and clinicopathological features, prognosis, and immune microenvironment. MethodsA total of 134 patients who received surgical treatment and were diagnosed as SCLC by postoperative histopathology were enrolled. All exon gene sequencing was performed in tumor tissues and adjacent normal tissues of 28 patients to clarify the genomic characteristics of SCLC. The expressions of DLL3 and programmed cell death 1 ligand 1 (PDL1) were detected by immunohistochemistry (IHC) in 101 postoperative pathological specimens. ResultsNotch receptor mutations are common, but the overall mutation rate of Notch ligand family is not high and mutually exclusive. The mutation status of Notch and Notch1 gene were not related to the prognosis. Notch1 gene mutation was significantly negatively correlated with the expression of PD-L1. DLL3 was highly expressed in SCLC and had no significant correlation with prognosis. There was a positive correlation between DLL3 expression and PD-L1 expression. Patients with positive DLL3 expression and no NOTCH 1 gene mutation had higher PD-L1 expression. SCLC patients with such characteristics may be more likely to benefit from immunotherapy. ConclusionThis study preliminarily explored the genomic characteristics and immune microenvironment of surgical resection of SCLC in China, and found the correlation between Notch gene mutation, DLL3 expression and PD-L1 expression, which provided a new idea for selecting appropriate treatment strategies for SCLC in the future.

Analyzing The Mutations Of Notch1 And Sf3b1 Genes In Cases With CLL Detected Isolated 13q Deletion

Chronic lymphocytic leukemia (CLL) is known as type of leukemia originating from clonal mature B lymphocytes and has genetic heterogeneity. Many studies have been done to clarify the genome of CLL. In these studies, del(13q) is reported as the most common chromosomal aberration. Although this anomaly is associated with good prognosis when isolated, patients have clinical heterogeneity. In addition, in many gene mutations including TP53, NOTCH1 and SF3B1 gene mutations, poor prognosis has been identified. The prognostic significance of these genes has begun to be demonstrated. According to these data, we aimed to determine the mutation rate of NOTCH1 and SF3B1 genes and to investigate the prognostic effects (disease stages, TTFT and OS) in 43 cases with isolated del(13q) by using FISH method. We investigated the most common mutations of NOTCH1 and SF3B1 gene in CLL by using Sanger sequencing method. While frameshift 7541_7542delCT mutation was detected in the NOTCH1 gene in 1 out of 42 CLL cases with clinical heterogeneity, mutation in the SF3B1 gene couldn’t be detected. As a result of our study, it was observed that NOTCH1 and SF3B1 gene mutations weren’t associated with isolated del(13q) which is compatible with the literature data. Our study is the first study that evaluates NOTCH1 and SF3B1 gene mutations with prognostic parameters of isolated del(13q) with CLL patients in the Turkish population. As a result; It was concluded that there may be different reasons responsible for the clinical heterogeneity of isolated del(13q) cases and further studies are needed to reveal these reasons

NOTCH1 mutations as prognostic marker in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity with poor prognosis. The dysregulation of Notch signaling pathway has been implicated in the OSCC tumorigenesis. However, the clinical implication of NOTCH1 mutation status in OSCC remains unelucidated. We extracted the NOTCH1 gene mutations from a whole exome sequencing dataset of 168 frozen OSCC tumor specimens and validated these NOTCH1 gene mutations by Sanger sequencing. We also assessed these NOTCH1 gene mutations and its pathological significance in our OSCC tumor tissues using immunohistochemistry. Univariate and multivariate analyses were also used to determine whether the association between NOTCH1 mutation status and prognostic factors was independent of other parameters. In this study, we have identified 44 (26.19 %) NOTCH1 gene mutations from a whole-exome sequencing of 168 OSCC formalin-fixed, paraffin-embedded (FFPE) tissue specimen. These mutations distributed in different NOTCH1 function domains, including the EGF-like repeats region, negative regulatory region, and Ankyrin repeats region. The immunohistochemical staining analysis revealed that NOTCH1 expression was increased in oral cancer tissues. In addition, of the 43 OSCC tumors with NOTCH1 mutations, we observed that the majority were negative for NOTCH1 intracellular domain 1 (NICD1) staining (76.74 %), and 10 tumors were positive for NICD1 staining (23.26 %). In conclusion, our study suggested that NOTCH1 expression is associated with the progression of OSCC. We also demonstrated that presence of a mutated NOTCH1 gene will help prognostic stratification in OSCC when combined with other clinicopathologic parameters.

Genetic Lesions in Russian CLL Patients with the Most Common Stereotyped Antigen Receptors

Background: The features of the IGHV gene repertoire in CLL have been widely discussed in the last 20 years. In approximately 30% of cases of CLL, highly homologous "stereotyped" antigen receptors (SARs) are expressed, representing quasi identical amino acid sequences. Associations with age, disease severity, and cytogenetic aberrations have already been reported for certain SARs. It should be noted that the predictive value of certain SARs could be higher than that of IGHV mutation status. Several SAR subgroups have already been identified as independent prognostic factors in CLL (CLL#1, CLL#2, CLL#8 demonstrate an extremely aggressive course of the disease, CLL#4 - indolent). It was also shown that SAR subgroups may associate with distinct profiles of genetic lesions. Aim: To study the frequency of cytogenetic aberrations and mutations in the TP53, NOTCH1, and SF3B1 genes in CLL patients with the most common SARs in Russia. Methods: The study included 62 patients with CLL diagnosed and followed up from 2012 to 2020. Sequencing of the IGHV gene and SAR assignment were done according to the ERIC recommendations. NOTCH1 gene mutations (exon 34) were assessed by fragment analysis or by NGS using primers described by Campregher et al. TP53 gene mutations (exons 2-11) were determined using NGS according to Pavlova et al. SF3B1 gene mutations (exons 14-16) were studied in 26 patients by NGS. 51 patients underwent a FISH study for the presence of 17p13/TP53 deletions; 29 - additionally for 11q23/ATM and 13q14 deletions (Abbott, USA). Results: CLL#1, CLL#6, and CLL#3 are the most common SAR subsets in Russian CLL patients. Here we report data for 30 patients with CLL#1, 17 with CLL#6, and 15 with CLL#3. In CLL#1 group, 10 (33.3%) patients had mutations in the TP53 gene. In 7 cases, the mutation burden exceeded 35%, in three patients it was less than 10%. In 7 (23.3%) patients, mutations in the NOTCH1 gene were detected, in 5 - c.7544_7545delCT, in 1 - c.7558_7561delCTTC and in 1 - p.Q2444X. Simultaneous mutations in the NOTCH1 and TP53 genes were found in two patients. Deletion 17p13was found in 8 patients from 25 studied (32%). In 7 patients TP53 mutation and del17p13 were observed simultaneously. Deletion 11q23 was found in 6 out of 15 patients (40%). No cases with the simultaneous occurrence of del11q23 and del17p13 or mutations in the TP53 gene were observed. SF3B1 gene mutations in this group were not investigated. In the CLL#6 group, 6 (35.3%) patients had mutations in the TP53 gene (in two of them two clones with different TP53 mutations were observed). In one case, the mutation was less than 10%. In 4 (23.5%) patients NOTCH1 gene mutations (all c.7544_7545delCT) were detected, in two cases the mutation burden was less than 5%. Simultaneous NOTCH1 and TP53 gene mutations were found in two patients. Deletion 17p13 was found in 4 patients from 14 studied (28.5%). In 3 patients TP53 mutations and del17p13 were observed simultaneously. Deletion 11q23 was found in 1 patient out of 10. No SF3B1 gene mutations were found in this group (in 11 patients tested). In CLL#3 group, only 2 (13%) patients had mutations in the TP53 gene, with one having 2 different clones and the other having 4 (this patient also had del17p13, the only one in this group). Also, only 2 (13%) patients had c.7544_7545delCT deletion in the NOTCH1 gene. SF3B1 gene mutations were found in 8 (53.3%) patients. Conclusions: Our data show that in CLL#1, CLL#6, and CLL#3 subsets genetic lesions are much more common than in CLL patients in general. This is in part consistent with earlier European studies. However, in our sample, TP53 mutations and del17p13 are much more frequent in CLL#1 and CLL#6 groups. This may be due to a more sensitive approach (NGS) for mutation detection we used. Furthermore, our cohort included patients relapsed after the FCR treatment. The discrepancy in the detection rate of del11q23 in the CLL#1 subset can be explained by the small sample. Also, population differences in the development of the disease cannot be ruled out. Further studies of genetic lesions associated with certain SAR subgroups may improve diagnostics and therapy of CLL and impact the understanding of disease pathogenesis. Disclosures No relevant conflicts of interest to declare.

Overcoming NOTCH1-Driven Chemoresistance in T-Cell Acute Lymphoblastic Leukemia Via Metabolic Intervention with Oxphos Inhibitor

Overcoming NOTCH1-Driven Chemoresistance in T-Cell Acute Lymphoblastic Leukemia Via Metabolic Intervention with Oxphos Inhibitor

Association between tumor mutation profile and clinical outcomes among Hispanic Latina women with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 15%-20% of all breast cancer types. It is more common among African American (AA) and Hispanic-Latina (HL) women. The biology of TNBC in HL women has been poorly characterized, but some data suggest that the molecular drivers of breast cancer might differ. There are no clinical tools to aid medical oncologists with decisions regarding appropriate individualized therapy, and no way to predict long-term outcomes. The aim of this study was to characterize individual patient gene mutation profiles and to identify the relationship with clinical outcomes. We collected formalin-fixed paraffin-embedded tumors (FFPE) from women with TNBC. We analyzed the gene mutation profiles of the collected tumors and compared the results with individual patient's clinical histories and outcomes. Of 25 patients with TNBC, 24 (96%) identified as HL. Twenty-one (84%) had stage III-IV disease. The most commonly mutated genes were TP53, NOTCH1, NOTCH2, NOTCH3, AKT, MEP3K, PIK3CA, and EGFR. Compared with other international cancer databases, our study demonstrated statistically significant higher frequencies of these genes among HL women. Additionally, a worse clinical course was observed among patients whose tumors had mutations in NOTCH genes and PIK3CA. This study is the first to identify the most common genetic alterations among HL women with TNBC. Our data strongly support the notion that molecular drivers of breast cancer could differ in HL women compared with other ethnic backgrounds. Therefore, a deeper understanding of the biological mechanisms behind NOTCH gene and PIK3CA mutations may lead to a new treatment approach.

CLL-263: Genetic Lesions Associated with CLL#1 Subset Patients in Russia

Background About 30% of cases of CLL express highly homologous “stereotype” antigenic receptors (SARs) which can be assigned to different subsets. Several SARs have already been identified as independent prognostic factors for CLL (CLL#1, CLL#2, CLL#8 are associated with extremely aggressive course of the disease, CLL#4 - indolent). It is suggested that different SARs may also associate with the occurrence of certain genetic lesions. Aim To evaluate the frequency of cytogenetic aberrations and TP53, NOTCH1 gene mutations in Russian patients of CLL#1 subset, the most common CLL subset in Russia. Methods We tested 20 CLL patients with SAR CLL#1 for NOTCH1 (34 exon) and TP53 (2-11 exons) gene mutations by fragment analysis and NGS sequencing. FISH study for the presence of 17p13/TP53, 11q23/ATM, 13q14 deletions and 12 trisomy also was done. Results TP53 gene mutations were detected in 5 patients (25%); all mutations were described as pathogenic. Also, in 5 (25%) patients, NOTCH1 gene mutations were detected. Simultaneous mutations in the NOTCH1 and TP53 genes were detected in two patients (10%). Deletion 17p13 was found in 4 patients (27%) from 15 evaluated. Three patients simultaneously had a mutation in the TP53 and del17p13 gene; in all these cases, the mutated TP53 allele burden was higher than 40%. In 5 of 11 patients, 11q23 deletion was found (45%). There were no cases with the simultaneous occurrence of del11q23 and del17p13 or mutations in the TP53 gene. In one patient, both del11q23 and a mutation in the NOTCH1 were found. One patient showed a 13q14 biallelic deletion (simultaneously with del17p13 and mut TP53). Trisomy of the 12th chromosome was not detected. Conclusion Our data show that in Russian patients with CLL#1 subset, genetic lesions are much more common than in patients with CLL in general. That is mostly consistent with earlier European studies. Further studies of genetic lesions in CLL#1 and other subsets are required for better understanding of CLL pathogenesis and improving diagnostics and therapy.

NOTCH-1 Gene Mutations Influence Survival in Acute Myeloid Leukemia Patients.

Background:Although NOTCH-1 gene mutations were reported to contributes to leukemogenesis in lymphocytic leukemias, its role in acute myeloid leukemia (AML) remains unclear. Therefor; this study was designed to determine the prevalence and clinical impact of NOTCH-1 mutations in AML patients. Materials and Methods:In the current study, NOTCH-1 gene mutations were identified in Bone Marrow samples obtained from fifty primary AML patients before start of therapy using Sanger sequencing. Results: NOTCH-1 gene mutations were detected in 6 out of 50 AML cases (12%). The three mutations were (two mutations C7318A in the Pest domain exon 34); (another 2 in the Pest domain Del 7,344, ins C7349, G7356A and the last ones in the HD-N exon-26 (Del A4609). The clinical findings in the mutant AML (mu AML) patients did not significantly different as compared to the un mutated (unmut) AML patients. There is significant association between CD7 aberrant expression and NOTCH-1 mutations. The complete remission was significantly higher in unmut AML cases as compared to mut AML ones (P=0.024). Multivariate (Age; Gender; Bone Marrow Blast cells; NOTCH-1 mutations) Cox regression analysis revealed that NOTCH-1 mutation is an independent risk factor for AML overall survival (P<0.001). The OS in unmut AML group (21.2 months) was significantly longer as compared to mut AML one (1.2 months) (P<0.001). Conclusion:Our data indicate that NOTCH-1 gene mutations were detected in 12% of AML patients. These mutations displayed bad clinical outcome on AML patients. Therapeutic targeting of NOTCH-1 could be a potentially effective approach to combat master oncogenic drivers in AML.

Dysregulation of Notch signaling in cardiac mesenchymal cells of patients with tetralogy of Fallot.

Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients. Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated. The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients. The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.

Myeloid/Lymphoid Neoplasm With FGFR1 Rearrangement Accompanying RUNX1 and NOTCH1 Gene Mutations

Background: Myeloid/Lymphoid Neoplasm with FGFR1 Rearrangement is a rare kind of hematological malignant disease.Case presentation: A 37-year-old male patient experienced three distinct disease stages from myeloproliferative neoplasm (MPN), T-cell lymphoblastic lymphoma (T-LBL) to a much more complexed phage of a mixed phenotype acute leukemia (MPAL). Both genetic and genomic alternations were detected including chromosomal abnormality and genic mutations.Result: Karyotyping and fluorescence in situ hybridization (FISH) analysis of either bone marrow or lymph node sample confirmed the presence of the FGFR1 rearrangement. Amplifications of RUNX1, ERG, and U2AF1 genes were identified by next generation sequencing. Furthermore, a frame-shift variant of F330fs*>149 in the RUNX1 gene and a missense mutation of R2263Q in NOTCH1 were also detected.Conclusion: The FGFR1 rearrangement functions as a trigging oncogenic event. Then other genetic events such as RUNX1 and/or NOTCH1 alternations further lead to progression of disease with trilineage blasts assignment.

MS05.03 Notch Signalling

The Notch signaling pathway is a highly conserved pathway that has a vital role in embryonic development and post-embryonic functions such as hematopoiesis, neural cell development and angiogenesis (1). The signaling pathway is activated by the interaction of 4 Notch receptors and their interactions with 5 different ligands. Signaling is characterized by juxtacrine interaction between ligands and receptors on neighboring cells or within the same cell. Alterations in this pathway have been detected in several tumors both as tumor promoter and tumor suppressor. Notch signaling does appear to have relevance in non-small cell lung cancers (NSCLC) (2). In approximately 30% of NSCLCs loss of Numb, a negative regulator of Notch1, leads to increased Notch activity and about 10% of NSCLCs have gain of function mutations in Notch1. A meta-analysis demonstrated that higher expression of Notch1 was correlated with more advanced tumors (3). In addition, higher expressions of both Notch1 and Notch3 were associated with poor prognosis (4,5). In pre-clinical studies inhibition of Notch3 signaling has reduced growth of lung cancers. In addition, it has been demonstrated that there is cross talk between Notch3 and EGFR pathways and inhibition of both pathways can induce expression of anti-apoptotic protein BIM. Finally, Notch signaling may have a role in induction of the epithelial to mesenchymal phenotype (EMT) in cancers (6). EMT is known to be associated with resistance to both cytotoxic agents and targeted agents and inhibition of Notch signaling can not only reverse EMT but also can enhance the anti-tumor activity of cytotoxic agents such as cisplatin and targeted agents such as erlotinib. The EMT phenotype is frequently observed in a sub-population of cancer cells with self-renewal capacity or cancer stem cells. Notch signaling may be crucial to survival of cancer stem cells and persistence of this population may contribute to resistance to therapeutic agents. In squamous cell lung cancers Notch signaling may have tumor suppressive properties (7). Loss of function mutations in Notch family of genes, predominantly in Notch receptors, are frequently identified in several squamous cell cancers including squamous cell cancer of the lung. Similarly, loss of function mutations in Notch genes, particularly Notch1 have been identified in small cell lung cancer (SCLC). Expression of Notch receptor in a mouse model of SCLC reduced tumor burden, suggesting its tumor suppressive properties. The expression of DLL3, one of the Notch ligands is induced in SCLC by a key transcription factor ASCL1.DLL3 is shown to downregulate Notch signaling in SCLCs and enhance the carcinogenic phenotype. All the above data suggest that Notch signaling is highly contextual. In some tumors this pathway may have tumor suppressive properties but in others tumor promoting properties. Defining the role of this pathway in tumor types may guide development of therapeutic strategies targeting the Notch signaling pathway. Bigas A, Espinosa L. The multiple usages of Notch signaling in development, cell differentiation and cancer. Curr Opin Cell Biol 2018;55:1-7. Westhoff B, Colaluca IN, D’Ario G, et al. Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci 2009;106:22293-8. Yuan X, Wu H, Xu H, et al. Meta-analysis reveals the correlation of Notch signaling with non-small cell lung cancer progression and prognosis. Sci Rep 2015;5:10338. Donnem T, Andersen S, Al-shibili K, et al. Prognostic impact of Notch ligands and receptors in nonsmall cell lung cancer: coexpression of Notch-1 and vascular endothelial growth factor-A predicts poor survival. Cancer 2010:116:5674-85. Hassan WA, Yoshida R, Kudoh S, et al. Evaluation of role of Notch3 signaling pathway in human lung cancer cells. J Cancer Res Clin Oncol 2016;142:981-93. Yuan X, Wu H, Han N, et al. Notch signaling and EMT in non-small cell lung cancer: biological significance and therapeutic application. J Hematol Oncol 2014;7:87. Nowell CS, Radtke F. Notch as a tumor suppressor. Nat Rev Cancer 2017;17:145-159. squamous, Tumor suppressor, NOTCH