Mutations In NOTCH1 Gene Research Articles

Assessment of the Progression Risk of Chronic Lymphocytic Leukemia

Objective: to identify the interconnection of laboratory parameters with different courses of chronic lymphocytic leukemia (CLL) and to develop a comprehensive model for the assessment of the risk of the disease progression. Material and methods. The study included 127 patients with CLL whose laboratory parameters were evaluated (general and biochemical blood tests, β2-microglobulin, thymidinekinase, tissue polypeptide antigen (TPA), immunophenotypic markers, and also NOTCH1 gene mutations). Results. For the prediction of the course of the disease the most informative were such markers as β2-microglobulin, thymidinekinase, ZAP-70, CD38, and TPA. Based on the obtained data, a model of the risk assessment for CLL progression with high sensitivity and specificity was developed. The progressive-free survival (PFS) was evaluated in two groups of the patients of different risk (low and high) assigned to them according to the prognostic model. In the patients from the low-risk group PFS was determined to be 60 months, and in the high-risk group it was equal to 29.4 months. And it was found out that the patients without progression at the time of inclusion in the study with the presence of mutations of the NOTCH1 gene had a shorter PFS in comparison with the patients without mutations, which may indicate a link between the mutations of the NOTCH1 gene and the unfavorable prognosis for the disease progression. Conclusion . The integrated application of the prognostic factors in the form of a CLL progression risk assessment model allows to stratify CLL patients into high and low risk groups and to predict the probability and progression rate at the time of the diagnosis and during the treatment.

PROSPERO: A study to determine the utility of focused genomic profiling to guide selection of drug therapy in salivary gland cancer.

6086 Background: For most patients with recurrent or metastatic salivary gland cancer (RM-SGC), there are no standard therapies. Many patients undergo genomic profiling to guide selection of targeted therapy. The MSK-IMPACT study applied a 468 gene next generation sequencing (NGS) panel, identifying actionable mutations in 34/114 patients (30%) with RM-SGC. Minimising cost will facilitate application within publically funded healthcare systems. We therefore sought to determine the utility of genomic profiling using a focused 24 gene targeted NGS panel to identify actionable mutations in RM-SGC with a sub-group analysis in adenoid cystic carcinoma (ACC) and non-ACC sub-types. Methods: From January 2017 to 2018, 125 patients with RM-SGC provided informed consent to an ethically approved study. Clinical and demographic characteristics were collected. DNA was extracted from FFPE samples and analysed using Qiagen GeneRead DNAseq Targeted Panel V2 in the Manchester Centre for Genomic Medicine Diagnostic Laboratory, an NHS clinically accredited lab. A custom bioinformatic pipeline was validated to detect single nucleotide variants and indels ( < 40bp) to 5% mutant allele frequency. Alterations were categorised following American College of Medical Genetics guidelines and Association for Molecular Pathology tiering. Results: DNA from 101 tumours (69 major, 32 minor salivary gland) was sequenced with 95% coverage at > 350x read depth over the target enrichment. 65 patients had adenoid cystic carcinoma (ACC) and 36 had non-ACC SGC. Median age was 55 years (range 18-80). 43 actionable alterations were identified in 33 patients within the following genes: TP53 (21%), PIK3CA (8%), ERBB2 (6%), PTEN (3%), BRAF (2%), EGFR (T790M) (1%), and AKT1 (1%). Targeted therapy was selected based on genomic findings in 12% of these patients. In ACC patients, actionable alterations were seen in 25% compared with 55% of non-ACC patients (9 adenocarcinoma, 5 salivary duct carcinoma, 3 carcinoma ex pleomorphic adenoma, 2 mucoepidermoid carcinoma and 1 myoepithelial carcinoma). Conclusions: This study identified actionable alterations in 33% of SGC patients using focused genomic profiling, demonstrating comparable utility to larger research panels. This focussed panel is being expanded to include emerging biomarkers such as NOTCH gene mutations, with NOTCH inhibitors currently in trials in ACC. Greater access to basket studies incorporating therapies matched to genomic alterations will maximise the clinical utility of this approach.

Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy.

Bicuspid aortic valve disease is common and is associated with ascending aortic aneurysms. Vascular smooth muscle cell (VSMC) apoptosis is characteristic of the ascending aorta of bicuspid patients, and NOTCH1 gene mutations have also been linked to the disease. NOTCH signalling is a fundamental cell signalling pathway, which dictates cell fate decisions including apoptosis. Our objective was to elucidate the role of NOTCH signalling in VSMC apoptosis and differentiation in bicuspid aortopathy. Ascending aortic biopsies were obtained from 19 bicuspid and 12 tricuspid aortic valve patients and were sub-classified into 4 groups according to the maximum ascending aortic diameter (aneurysmal ≥45 mm). Apoptotic VSMCs were counted by light microscopy using a TUNEL assay. Gene expression of key regulators of NOTCH signalling (NOTCH1 and HES1), apoptosis (BAX and BCL-2) and VSMC differentiation (MYH11, CNN1 and MYH10) were quantified using quantitative real-time PCR. Primary VSMCs were cultured from 2 tricuspid aortic valve and 2 bicuspid aortic valve patients, NOTCH signalling was inhibited with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, and the gene expression was again quantified. The apoptotic cell count was significantly higher in bicuspid aortic valve patients (3.2 cells/50000 μm2 vs 1.1 cells/50000 μm2; P = 0.033). There was a trend towards lower apoptotic cell count in the aneurysmal versus non-aneurysmal tricuspid and bicuspid groups and an increased ratio of proapoptotic gene expression, which was not statistically significant. This was associated with a 2.8-fold increase in contractile gene expression (P = 0.026) and a 2.0-fold increase in NOTCH signalling gene expression in bicuspid versus tricuspid aortic valve patients (P = 0.022). NOTCH inhibition in cultured VSMCs induced a similar pattern of increased proapoptotic and procontractile gene expressions. This preliminary study suggests that NOTCH activation in the non-aneurysmal bicuspid aortas may underlie aortopathy by influencing VSMC apoptosis and differentiation. NOTCH signalling manipulation may provide a therapeutic target for preventing aneurysms in bicuspid patients. Further studies with larger sample sizes are needed to substantiate the present findings.

Analysis of LPL gene expression in patients with chronic lymphocytic leukemia

The IGHV mutational status is one of the most important markers for chronic lymphocytic leukemia(CLL) prognostication. Lipoprotein lipase(LPL) gene expression was found to correlate with IGHV status and was suggested as its surrogate marker. Recent data reported that LPL expression might be influenced by pivotal signalling pathways in CLL. This study aimed to assess LPL gene expression in relation to key immunogenetic and molecular markers of CLL, including IGHV mutational status, B-cell receptor (BCR) stereotypy, TP53, NOTCH1, and SF3B1gene mutations. Materials andMethods: Expression of LPL mRNA was measured in peripheral blood mononuclear cells of 73CLL patients by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). IGHV, NOTCH1, TP53, and SF3B1 gene mutation analysis was performed by PCR amplification and direct sequencing. 44of 73 (60%) CLL cases were categorized as LPL-positive based on the cut-off value established by ROC (receiver operating characteristic) curve analysis. LPL expression was significantly associated with IGHV mutation status (r=0.684; p < 0.0001) and tended to correlate with presence ofNOTCH1 gene mutations (p = 0.113). BCR stereotyped cases showed higher LPL expression values in comparison to unstereotyped cases in the LPL-positive group of patients (p=0.041). LPL expression was associated with a shorter overall survival in the entire СLL group (median 107vs 143, p=0.048) as well as in Binet A patients, albeit with borderline significance (median 139vs not reached, p=0.086). LPL expression was found to be closely correlated with IGHV gene mutational status and overall survival, proving LPL as prognostic marker in CLL. Our results also indicate a possible relationship between aberrant expression of LPL and BCR- and NOTCH1-dependent signalling pathways.

ARRB1-Induced NOTCH1 Degradation Is Inhibited By Mir-223 in T-ALL

Leukemia is the most common malignant tumor in children under 15 years old, which is divided into several subtypes, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL) and chronic myeloid leukemia (CML), based on the disease phases and effected cells. Each subtype has its specific molecular feature and key regulation factors. In our previous studies, we reported that β-arrestin1 (ARRB1), the pivotal scaffold protein to transduce various cellular signals, could bind with EZH2 to increase Bcr/Abl H4 acetylation level and thus promote CML progression (Brit J Cancer 2014, 111(3): 568-76). ARRB1 could enhance DNMT1 activity and PTEN methylation, decrease PTEN expression and promote self-renew of B-ALL leukemia initiating cells (LICs) (Cancer Lett 2015, 357(1): 170-8.). ARRB1 could increase P300 to bind with SP1 to hTERT promoter, and thus increase hTERT transcription/expression, telomerase activity, telomere length and cell senescence in B-ALL LICs (Cell Death Diff 2017, 8(4): e2756.). However, little is known in the T-ALL, which about 70% have the mutations of NOTCH1 gene. Here, we unveil ARRB1 could curb the progression of T-ALL cells in vitro and in vivo, while the expression of ARRB1 was suppressed by the aberrant increased miR-223. Mechanistically, ARRB1 could recruit DTX1, the E3 ubiquitin ligase, to promote the ubiquitination and degradation of NOTCH1 protein in T-ALL. Furthermore, Overexpression of ARRB1-derived miR-223 sponge BUTR was incompatible with cell proliferation and induces apoptosis in T-ALL cells. Collectively, our results for the first time revealed that ARRB1 acted as a tumor suppressor by promoting NOTCH1 degradation in T-ALL cells where miR-223 effectively antagonized ARRB1 functions. This provides that miR-223 may serve as a valid drug target for developing novel and efficacious T-ALL therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of Notch Signaling in Acute Myeloid Leukemia (AML)

Background: Notch signaling is a developmental pathway involved in normal and malignant hematopoiesis. Mutations in Notch genes lead to T-ALL and are associated with poor prognosis in B-CLL. However its role in AML is still under investigation, with controversial results. In addition, although the pathway consists of 4 receptors and 5 ligands, many studies are mostly based on single receptor or ligand.Aims: This study was conducted to determine if the expression level of each Notch receptor and ligand are associated with known prognostic factors and patient's survival.Methods: AML primary cells were collected from 79 AML patients after informed consent. The follow-up was for 36 months. Flow cytometry analysis was used to study the expression of each Notch receptor and ligand. Prognostics or risk factors considered were sex, white blood counts, FAB and cytogenetics. The Mann-Whitney test was used for analyzing differences in expression levels between two groups. Spearman test was used for correlation between Notch expression levels and prognostics factors. Differences in overall (OS) and progression free (PFS) survival were established through Gehan-Breslow-Wilcoxon test.Results: Consistently with previous findings, we found expression of Notch1, Notch2, Notch3, Notch4, Jagged1, Jagged2 and DLL3 in AML samples. There were no differences in expression levels between male and female. For each receptor or ligand, higher expression levels were found in more immature samples (M0, M1, and M2). Notch3, Notch4 and Jagged2 were enriched in adverse cytogenenetics risk groups compared to favorable cytogenetics risk patients. Then, for each receptor or ligand, patients were divided into two groups; patients with higher expression levels and patients with lower expression levels. Analyzing OS and PFS, we found that patients with lower expression levels of Notch4, Jagged2 and DLL3 displayed a longer survival compared to patients with higher expression levels.Conclusion: Given its oncogenic role in T-ALL and other malignancies Notch1 has been the more studied receptors in hematological malignancies. However, we have previously demonstrated that Notch4, DLL3 and Jagged2 are all involved in survival of AML and B-ALL cells. Studying the prognostic value of all Notch receptors and ligands, the current research clearly shows that higher levels of Notch4 and Jagged2 are found in poor cytogenetics risk groups and are associated with a shorter patient's survival. These demonstrations suggest that expression levels of Notch receptors and ligands in AML patient's samples at diagnostic could stand as prognostic marker for clinical care. DisclosuresNo relevant conflicts of interest to declare.

Study on Immunophenotypes and Gene of Acute Lymphoblastic Leukemia

To retrospectively analyze the immunophenotyping, fusion gene and gene mutation of 30 acute lymphoblastic leukemia (ALL) cases and to investigate the relationship between the analysis results and the clinical therapeutic effect and prognosis. Thirty All phtients were collected from the First Hospital of Harbin, Institute of Hematology and Oncology Department of Pediatrics from August 2015 to June 2016. According to the classification of FAB standard, 27 cases were B system ALL, 3 cases were T system ALL. All patients were diagnosed by bone marrow cell morphology, immunophenotype, cytogenetics and molecular biology detetions, the differentiation antigens on membrane surface and in cytoplasm of ALL cells, and 43 kinds of fusion gene qualitative screening（BCR-ABL， AML1-ETO, PML-RARα and so on） were qualitative screened and ALL gene mutations（IKZF1, TP53, PAX5, JAK1, JAK2, CRLF2, PHF6, NOTCH1, FBXW7, PTEN）were detected by next generation sequencing(NGS). (1) Among 30 ALL patients, the incidence of B-ALL(90.00%) was higher than that of T-ALL(10.00%). (2) 27 cases of B-ALL expressed CD19, CD22, CD10, CD34 and so on. CD19 and CD22 were the most diagnostic antigens of B-ALL. (3) 3 cases of T-ALL mainly expressed cCD3, CD7, CD10, cTDT and so on; cCD3 and CD7 were the most diagnostic antigens of T-ALL. (4) The quantitative screening of 30 cases of ALL 43 fusion genes found BCR-ABL,TEL-AML1 and E2A-PBX1, MLL-AF6, MLL-AF4, and SIL-TAL1 fusion gene was positive in 1 case each; NGS detection of gane mutations associated with ALL showed that: 3 cases of B-ALL found that TP53 mutation occured 3 casas of B-ALL, TET2 I1762V mutations in 1 cases, 3 patients (2 cases of T-ALL, 1 cases of B-ALL) showed NOTCH1 gene mutation. After a cycle of treatment, the efficacy of adult B-ALL treatment (28.57%) was significantly lower than that of child B-ALL (95.00%), and the survival rate of child B-ALL was significantly better than that of adult B-ALL until July 10, 2017, and the differences were significant. The immunophenotype technology of leukemia and molecular biology has an important guiding role in the diagnosis of leukemia, selection of treatment plan and evaluation of curative effect, and it is the complement of bone marrow cell morphology diagnosis.

Induced pluripotent stem cells with NOTCH1 gene mutation show impaired differentiation into smooth muscle and endothelial cells: Implications for bicuspid aortic valve-related aortopathy

ObjectiveThe NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. MethodsThe iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1−/− (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1−/− and wild type cells. ResultsThe expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1−/−NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1−/− NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1−/−CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1−/−ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. ConclusionsNOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve.

Abstract 49: Synergistic antileukemic therapies in NOTCH1-induced T-ALL

Abstract Over 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL) harbor activating mutations in Notch1 gene, making this receptor a promising therapeutic target for T-ALL. Υ-secretase inhibitors (GSIs), which block a proteolytic cleavage required for NOTCH1 activation, have been clinically explored for the treatment of T-ALL. However, lack of primary responses and relapses have prevented further development of GSIs in the clinic, highlighting the need to identify novel therapies synergistic with NOTCH1 inhibition for the treatment of highly aggressive NOTCH1-mutant T-ALL. To identify new antileukemic drugs for T-ALL we designed an expression-based drug screen utilizing drug profiles contained in the Connectivity Map (cMAP) to search for positive connections with gene signatures driven by NOTCH1 inhibition in this disease. This analyses uncovered 17 compound candidates for synergistic activity with NOTCH1 inhibition, including drugs with known mechanism of action: inhibitors of the mTOR/PI3K/AKT pathway and histone deacetylase inhibitors; as well as compounds with unrelated mechanisms, pyrvinium pamoate (antihelmintic) and withaferin A (antiangiogenic). Pair-wise gene set enrichment analyses on the expression profile driven by each of these drugs in T-ALL cells revealed compounds with transcriptional programs partially overlapping those driven by PI3K/AKT inhibitors, and drugs with distinctive expression profiles, such as withaferin A, suggestive of a unique mode of action. We next characterized the activity of each of the cMAP hits alone and in combination with the GSI DBZ in vitro using a panel of 5 NOTCH1-mutant T-ALL cell lines (CUTLL1, DND41, HPBALL, RPMI and CCRF-CEM). In this analysis, 5 drugs scored as highly active (IC50&amp;lt;0.5) and synergistic (combination index, CI &amp;lt;0.4) in combination with the GSI DBZ: withaferin A, parthenolide, rapamycin, wortmannin, and vorinostat. In addition, 3 out of the 5 drug candidates displayed proapoptotic activity by flow cytometry in cells treated with each of the drugs alone and in combination with DBZ. Among them, withaferin A stood up as the most cytotoxic GSI-synergistic drug candidate. In addition, evaluation of withaferin A activity as single agent and in combination with DBZ in genetic mouse models and primary-derived xenografts of NOTCH1-mutant T-ALL in vivo demonstrated strong antileukemic effects for the combination of GSI and withaferin A. Functional annotation and DeMAND algorithm analyses on the expression profile of withaferin A revealed downregulation of genes and pathways involved in protein translation and identified 21 components of the ribosome and the translation machinery as potential effectors of the antitumoral activity of the drug in T-ALL, including two subunits of the eIF2A translation complex (eIF2S1 and eIF2S2) which controls translation initiation via phosphorylation of the eIF2S1 subunit under conditions of cellular stress. Polysome profiling, nascent protein, and Western blot analyses of cells treated with withaferin A confirmed an inhibitory effect of the drug in protein translation mediated by eIF2S1 phosphorylation. Consistently, expression of a non-phosphorylable mutant form of eIF2S1 in leukemia cell lines rescued the effects of withaferin A in vitro. Our results illustrate the power of our expression-based and in vivo validation approach toward the identification of antitumor drug combinations for the treatment of human cancer. Citation Format: Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Yue Qin, Daniel Herranz, Mukesh Bansal, Tiziana Girardi, Elisabeth Paietta, Martin S. Tallman, Jacob M. Rowe, Kim De Keersmaecker, Andrea Califano, Adolfo Ferrando. Synergistic antileukemic therapies in NOTCH1-induced T-ALL [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 49.

Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer

The Notch signaling pathway mediates cell fate decisions1,2 and is tumor suppressive or oncogenic depending on the context2,3. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine (NE) fate4–6. In small cell lung cancer (SCLC), an aggressive NE lung cancer7, loss-of-function NOTCH mutations and the inhibitory effects of ectopic Notch activation indicate that Notch signaling is tumor suppressive8,9. Here, we show that Notch signaling can be both tumor suppressive and pro-tumorigenic in SCLC. Endogenous activation of the Notch pathway results in a NE to non-NE fate switch in 10-50% of tumor cells in a mouse model of SCLC and in human tumors. This switch is mediated in part by Rest/Nrsf, a transcriptional repressor that inhibits NE gene expression. Non-NE Notch-active SCLC cells are slow growing, consistent with a tumor suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to NE tumor cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumor growth and delays relapse. Thus, SCLC tumors generate their own microenvironment via activation of Notch signaling in a subset of tumor cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select SCLC patients.

Prognostic Value of Recurrent Molecular Genetics and Epigenetics Abnormity in T Lymphoblastic Lymphoma / Leukemia -Review

T lymphoblastic lymphoma / leukemia is a strong invasive and has a high incidence of various molecular genetic abnormalities. The NOTCH1 / FBXW7 mutation is one of the most common mutations, and related with good prognosis in T-LBL / ALL. PTEN mutation, a poor prognostic factor, could be overcome by NOTCH1 mutations in pediatric patients to some extent. Patients with MLL gene abnormality and loss of heterozygosity 6q have worse prognosis than those with normal karyotype. The incidence of MLL gene abnormality, RUNX1 mutation and DNMT3A mutation in early precursor T-lymphoblastic leukemia was higher than that of other mature subtypes, which could be used as risk stratification factors. Epigenetic abnormalities play an important role in the malignant transformation of stem cells. Epigenetics treatment such as demethylation therapy may improve the prognosis of high-risk patients. In this article, the NOTCH1,FBXW7,PTEN and RAS gene mutations and prognosis, the molecular and cellular abnormalities and prognosis, as well as epigenetic abnormality and prognosis are reviewed.

NOTCH1 Mutations in Aortic Stenosis: Association with Osteoprotegerin/RANK/RANKL.

Background. The NOTCH pathway is known to be important in the pathogenesis of calcific aortic valve disease, possibly through regulators of osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK), and its ligand (RANKL) system. The purpose of the present study was to search for possible associations between NOTCH1 gene mutations and circulating levels of OPG and soluble RANKL (sRANKL) in patients with aortic stenosis (AS). Methods. The study was performed on 61 patients with AS including 31 with bicuspid and 30 with tricuspid aortic valves. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Serum OPG and sRANKL levels were assessed. Results. In total, 6 genetic variants of the NOTCH1 gene including two new mutations were identified in the study group. In an age- and arterial hypertension-adjusted multivariable regression analysis, the serum OPG levels and the OPG/sRANKL ratio were correlated with NOTCH1 missense variants. All studied missense variants in NOTCH1 gene were found in Ca(2+)-binding EGF motif of the NOTCH extracellular domain bound to Delta-like 4. Conclusion. Our results suggest that the OPG/RANKL/RANK system might be directly influenced by genetic variants of NOTCH1 in aortic valve calcification.

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the “watch and wait” interval and after standard chemotherapy.

Expression of circulating miRNAs associated with lymphocyte differentiation and activation in CLL\u2014another piece in the puzzle

Expression of microRNAs is altered in cancer. Circulating miRNA level assessed in body fluids commonly reflects their expression in tumor cells. In leukemias, however, both leukemic and nonleukemic cells compose circulating miRNA expression profile of peripheral blood. The latter contribution to extracellular miRNA pool may result in specific microenvironmental signaling, which promotes proliferation and survival. In our study, we used qT-PCR to assay peripheral blood serum of 22 chronic lymphocytic leukemia (CLL) patients for the expression of 84 miRNAs associated with activation and differentiation of B and T lymphocytes. Results were analyzed regarding the most important prognostic factors. We have found that the general expression of examined miRNAs in CLL patients was lower as compared to healthy volunteers. Only miR-34a-5p, miR31-5p, miR-155-5p, miR-150-5p, miR-15a-3p, and miR-29a-3p were expressed on a higher level. Alterations of expression observed in CLL patients involved miRNAs associated both with B and T lymphocyte differentiation and activation. The most important discriminating factors for all functional miRNA groups were trisomy 12, CD38 expression, B2M level, WBC, and NOTCH1 gene mutation. Correlation of expression of miRNAs related to T lymphocytes with prognostic factors proves their supportive function in a leukemic microenvironment. Further studies utilizing a larger test group of patients may warrant the identification of circulating miRNAs that are key players in intercellular interactions and should be considered in the design of microenvironment-targeted therapies.

Does Notch play a tumor suppressor role across diverse squamous cell carcinomas?

The role of Notch pathway in tumorigenesis is highly variable. It can be tumor suppressive or pro‐oncogenic, typically depending on the cellular context. Squamous cell carcinoma (SCC) is a cancer of the squamous cell, which can occur in diverse human tissues. SCCs are one of the most frequent human malignancies for which the pathologic mechanisms remain elusive. Recent genomic analysis of diverse SCCs identified marked levels of mutations in NOTCH1, implicating Notch signaling pathways in the pathogenesis of SCCs. In this review, evidences highlighting NOTCH's role in different types of SCCs are summarized. Moreover, based on accumulating structural information of the NOTCH receptor, the functional consequences of NOTCH1 gene mutations identified from diverse SCCs are analyzed, emphasizing loss of function of Notch in these cancers. Finally, we discuss the convergent view on an intriguing possibility that Notch may function as tumor suppressor in SCCs across different tissues. These mechanistic insights into Notch signaling pathways will help to guide the research of SCCs and development of therapeutic strategies for these cancers.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia.

CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

CD49d Prevails over the Novel Recurrent Mutations As Independent Prognosticator of Overall Survival in Chronic Lymphocytic Leukemia

Background. CD49d, the alpha-chain of the integrin heterodimer VLA-4, has been identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p (17p-) chromosome involving TP53 (Bulian et al, JCO, 2014). In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized.Aim. To test the relevance of CD49d in molecular subgroups of CLL defined by NOTCH1, SF3B1 and BIRC3 gene mutations.Methods. The study was based on a cohort of 778 unselected CLL (422 untreated and 356 treated cases) all characterized for CD49d expression (CD49dhigh, ≥30% positive cells by flow cytometry in 229 cases), IGHV mutational status (unmutated, UM, in 262 cases), karyotype abnormalities according to the hierarchical stratification (13q- in 308 cases, +12 in 103 cases, 11q- in 64 cases), tested at diagnosis, along with mutations/deletions (hereinafter, disruption) of TP53 (disrupted in 84 cases, including 58 cases with 17p-), BIRC3 (disrupted in 59 cases), and mutationsof NOTCH1 (mutated in 81 cases), SF3B1 (mutated in 54 cases)tested at diagnosis in 65% of cases, in all cases before therapy. Chromosome abnormalities by FISH were defined by using a 5% cut-off; IGHV, TP53, BIRC3, NOTCH1 and SF3B1 mutations were investigated by Sanger sequencing. Median follow-up of patients was 80 months with 173 deaths.Results. i) association with CD49d: a CD49dhigh phenotype associated with age ≥65 years (p=0.0001), Rai stage I or higher (p>0.0001), UM IGHV status (p>0.0001), absence of 13q- (p=0.0001), presence of +12 (p<0.0001), presence of NOTCH1 mutations (p<0.0001). ii) CD49d prognostic impact: in univariate analysis, the following covariates had a significant impact on OS: age ≥65 years (hazard ratio/confidence interval (HR/CI)= 3.98/2.77-5.73; p<0.0001), Rai stage I or higher (HR/CI=1.81/1.33-2.46; p=0.0002), high CD49d expression (HR/CI=2.62/1.94-3.54; p<0.0001), UM IGHV status (HR/CI=3.03/2.24-4.10; p<0.0001), presence of 13q- (HR/CI=0.52/0.37-0.71; p=0.0001), +12 (HR/CI=1.59/1.08-2.33; p=0.0183), 11q- (HR/CI=2.16/1.42-3.30; p=0.0004), NOTCH1 mutations (HR/CI=2.66/1.82-3.88; p<0.0001), SF3B1 mutations (HR/CI=1.99/1.24-3.20; p=0.0048), BIRC3 disruption (HR/CI=2.41/1.58-3.68; p<0.0001), TP53 disruption (HR/CI=3.23/2.28-4.57; p<0.0001). In a multivariate analysis model including all these variables, high CD49d expression (HR/CI=1.76/1.28-2.42; p=0.0006), UM IGHV status (HR/CI=2.21/1.58-3.09; p<0.0001), presence of 11q- (HR/CI=2.11/1.35-3.31; p=0.0011), TP53 disruption (HR/CI=2.93/2.04-4.22; p<0.0001), and NOTCH1 mutations (HR/CI=1.76/1.16-2.67; p=0.0082) emerged as independent prognosticators, along with age ≥65 years (HR/CI=3.73/2.58-5.39; p<0.0001). iii) CD49d prognostic impact using the integrated hierarchical mutational/cytogenetic model: by integrating gene mutations and cytogenetic aberrations according to Rossi et al (Blood, 2013), a multivariate analysis model was applied with the following covariates: age, CD49d, IGHV status, TP53/BIRC3 disruption, NOTCH1 mutations/SF3B1 mutations/11q-, normal karyotype/+12, 13q-. Again, CD49dhigh phenotype (HR/CI=1.88/1.38-2.54; p=0.0001), UM IGHV status (HR/CI=2.16/1.54-3.01; p<0.0001), presence of NOTCH1 mutations/SF3B1 mutations/11q- (HR/CI=1.62/1.11-2.38; p=0.0139), and of TP53/BIRC3 disruption (HR/CI=2.67/1.92-3.70; p<0.0001) retained their independent prognostic impact, along with age ≥65 years (HR/CI=3.58/2.28-5.17; p<0.0001). Notably, CD49dhigh CLL patients experienced shorter OS than the CD49dlow cases in the context of each mutational/cytogenetic group (TP53/BIRC3 disruption, p=0.0064; NOTCH1 mutations/SF3B1 mutations/11q-, p=0.05; normal karyotype/+12, p=0.0153; 13q-, p=0.0126; see Figure).Conclusion. CD49d was confirmed as independent negative OS prognosticator in CLL also when the novel recurrent alterations of NOTCH1, BIRC3, and SF3B1 genes were considered. In this setting, TP53 disruption and NOTCH1 mutations remained independent OS prognosticators, allegedly for their physiopathological roles in CLL cell immuno-chemoresistance. [Display omitted] DisclosuresGaidano:Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.

Genomic Profiling Identifies Recurrent Genetic Alterations in SCLC

Abstract SCLC is characterized by oncogenic TP73 rearrangements and inactivating mutations in NOTCH genes.

Concerning consequences of blocking Notch signaling in satellite muscle stem cells.

Concerning consequences of blocking Notch signaling in satellite muscle stem cells.

Prognostic Impact of NOTCH1 Hotspot Mutation in TP53-Mutated Patients with Chronic Lymphocytic Leukemia

IntroductionA presence of activating mutations in NOTCH1 gene has been recently associated with reduced survival and chemo-immunotherapy resistance in chronic lymphocytic leukemia (CLL). However, a prognostic significance of the NOTCH1 mutations with respect to TP53mutation status has not been fully explained yet.MethodsAn examined cohort included 409 patients with CLL enriched for high risk cases; in 121 patients consecutive samples were investigated. To determine the TP53 mutation status, a functional analysis of separated alleles in yeast (FASAY, exons 4-10) combined with direct sequencing was performed; the ambiguous cases were retested using an ultra-deep next generation sequencing (MiSeq platform; Illumina). The presence of NOTCH1 hotspot mutation (c.7544_7545delCT) was analyzed using direct sequencing complemented by allele-specific PCR in the selected samples. In several patients harboring concurrent NOTCH1 and TP53 mutations, single separated cancer cells were examined using multiplex PCR followed by direct sequencing. A correlation between mutation presence and patient overall survival, time to first treatment and other molecular and cytogenetic prognostic markers was assessed using Log-rank (Mantel-cox) test and Fisher’s exact test, respectively.ResultsThe NOTCH1 and TP53 mutations were detected in 16% (65/409) and 27% (110/409) of the examined patients, respectively; a coexistence of these mutations in the same blood samples was observed in 11% (19/175) of the mutated patients. The detected increased mutation frequency attributes to more unfavorable profile of the analyzed cohort; in the TP53-mutated patients missense substitutions predominated (75% of TP53 mutations).As expected, a significantly reduced overall survival in comparison to the wild-type cases (147 months) was observed in the NOTCH1-mutated (115 months; P = 0.0018), TP53-mutated (79 months; P < 0.0001) and NOTCH1-TP53-mutated patients (101 months; P = 0.0282). Since both NOTCH1 and TP53 mutations were strongly associated with an unmutated IGHV gene status (P < 0.0001 and P = 0.0007), we reanalyzed the IGHV-unmutated patients only and interestingly, the impact of simultaneous NOTCH1 and TP53 mutation presence on patient survival was missed in this case (P = 0.1478).On the other hand, in the NOTCH1 and/or TP53-mutated patients significantly reduced time to first treatment was identified as compared to the wild-type cases (41 months vs. 25 months in NOTCH1-mutated, P = 0.0075; 17 months in TP53-mutated, P < 0.0001; and 18 months in NOTCH1-TP53-mutated patients, P = 0.0003). The similar results were observed also in the subgroup of the IGHV-unmutated patients, with the exception of patients carrying sole NOTCH1 mutation (P = 0.2969). Moreover, in the NOTCH1-TP53-mutated patients an increased frequency of del(17p)(13.1) was found in comparison to the TP53-mutated patients only (72% vs. 56%); this cytogenetic defect was not detected in the patients with sole NOTCH1 mutation.Our results might indicate, that NOTCH1 mutation could preferentially co-selected with particular, less prognostic negative type of TP53 defects. Notably, in our cohort the NOTCH1 mutation predominated in the patients harboring truncating TP53 mutations localized in a C-terminal part of the TP53 gene behind the DNA-binding domain (P = 0.0128). Moreover, in one of the NOTCH1-TP53-mutated patients the analysis of separated cancer cells revealed a simultaneous presence of NOTCH1 mutation and TP53 in-frame deletion in the same CLL cell. In contrast, in the other examined NOTCH1-TP53-mutated patient the concurrent NOTCH1 mutation and TP53 missense substitution (with presumed negative impact on patient prognosis) were found in different CLL cells.ConclusionsThe parallel presence of NOTCH1 hotspot mutation might be detected in a significant proportion of TP53-mutated patients and it seems to be associated with less prognostic unfavorable TP53 mutations. Nevertheless, these preliminary data should be further confirmed in a large cohort of patients.This study was supported by projects VaVPI MSMT CR CZ.1.05/1.1.00/02.0068 of CEITEC, IGA MZ CR NT13493-4/2012, NT13519-4/2012 and CZ.1.07/2.3.00/30.0009. DisclosuresBrychtova:Roche: Travel grants Other. Doubek:Roche: Travel grants Other.