NOTCH1 Mutations Research Articles

NOTCH1 Signalling: A key pathway for the development of high-risk chronic lymphocytic leukaemia.

NOTCH1 is a cell surface receptor that releases its intracellular domain as transcription factor upon activation. With the advent of next-generation sequencing, the NOTCH1 gene was found recurrently mutated in chronic lymphocytic leukaemia (CLL). Here, virtually all NOTCH1 mutations affect the protein’s PEST-domain and impair inactivation and degradation of the released transcription factor, thus increasing NOTCH1 signalling strength. Besides sequence alterations directly affecting the NOTCH1 gene, multiple other genomic and non-genomic alterations have by now been identified in CLL cells that could promote an abnormally strong NOTCH1 signalling strength. This renders NOTCH1 one of the key signalling pathways in CLL pathophysiology. The frequency of genomic alterations affecting NOTCH1 signalling is rising over the CLL disease course culminating in the observation that besides TP53 loss, 8q gain and CDKN2A/B loss, NOTCH1 mutation is a hallmark genomic alteration associated with transformation of CLL into an aggressive lymphoma (Richter transformation). Both findings associate de-regulated NOTCH1 signalling with the development of high-risk CLL. This narrative review provides data on the role of NOTCH1 mutation for CLL development and progression, discusses the impact of NOTCH1 mutation on treatment response, gives insight into potential modes of NOTCH1 pathway activation and regulation, summarises alterations that have been discussed to contribute to a de-regulation of NOTCH1 signalling in CLL cells and provides a perspective on how to assess NOTCH1 signalling in CLL samples.

EZH2 inhibitor DZNep blocks cell proliferation of GCB-DLBCL cells by upregulating p16

Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Two main subgroups of DLBCL include germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Molecular profiling can further classify DLBCL into four subtypes: MCD (both CD79B and MYD88 L265P), BN2 (NOTCH2 mutation or BCL6 fusion), N1 (NOTCH1 mutation), or EZB (EZH2 mutation or BCL2 fusion). EZH2 inhibitors were recommended for patients with the EZB subtype of DLBCLs; however, little is known about the therapeutic mechanisms. Our results showed that DZNep arrested G1/S phase of GCB-DLBCL cells and inhibited the cell proliferation in vitro through upregulation of p16 by demethylating its promoter. These results suggest that DZNep may have potential as a novel therapeutic agent for DFLBL therapy. This agent may serve as a novel molecular agent to be applied to GCB DLBCL.

Intravascular Large B-Cell Lymphoma Genomic Profile Is Characterized by Alterations in Genes Regulating NF-κB and Immune Checkpoints.

Intravascular large B-cell lymphoma (IVLBCL) is an uncommon lymphoma with an aggressive clinical course characterized by selective growth of tumor cells within the vessels. Its pathogenesis is still uncertain and there is little information on the underlying genomic alterations. In this study, we performed a clinicopathologic and next-generation sequencing analysis of 15 cases of IVLBCL using a custom panel for the detection of alterations in 68 recurrently mutated genes in B-cell lymphomagenesis. Six patients had evidence of hemophagocytic syndrome. Four patients presented concomitantly a solid malignancy. Tumor cells outside the vessels were observed in 7 cases, 2 with an overt diffuse large B-cell cell lymphoma. In 4 samples, tumor cells infiltrated lymphatic vessel in addition to blood capillaries. Programmed death-ligand 1 (PD-L1) was positive in tumor cells in 4 of 11 evaluable samples and in macrophages intermingled with tumor cells in 8. PD-L1 copy number gains were identified in a higher proportion of cases expressing PD-L1 than in negative tumors. The most frequently mutated gene was PIM1 (9/15, 60%), followed by MYD88L265P and CD79B (8/15, 53% each). In 6 cases, MYD88L265P and CD79B mutations were detected concomitantly. We also identified recurrent mutations in IRF4 , TMEM30A , BTG2 , and ETV6 loci (4/15, 27% each) and novel driver mutations in NOTCH2 , CCND3 , and GNA13 , and an IRF4 translocation in 1 case each. The mutational profile was similar in patients with and without evidence of hemophagocytic syndrome and in cases with or without dissemination of tumor cells outside the vessels. Our results confirm the relevance of mutations in B-cell receptor/nuclear factor-κB signaling and immune escape pathways in IVLBCL and identify novel driver alterations. The similar mutational profile in tumors with extravascular dissemination suggests that these cases may also be considered in the spectrum of IVLBCL.

Cdkn2a inactivation promotes malignant transformation of mouse immature thymocytes before the β-selection checkpoint

CDKN2A deletion is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia (T-ALL), occurring across all molecular and immunophenotypic subtypes. CDKN2A encodes two functionally unrelated tumor suppressor proteins, ARF and INK4a, critical regulators of cell cycle and proliferation. Arf has been reported to suppress T-ALL development in post-β-selection thymocytes, but whether CDKN2A acts as a tumor suppressor gene in immature, pre-β-selection thymocytes remains to be elucidated. Resorting to a Rag2-deficient model of T-ALL, driven by the ETV6::JAK2 fusion, we report that Cdkn2a haploinsufficiency at early stages of T-cell development facilitates leukemia development. ETV6::JAK2;Rag2-/-;Cdkn2a+/- T-ALL arose from pre-β-selection thymocytes, since the thymocyte differentiation arrest caused by Rag2 deficiency was maintained in pre-leukemic mice. Furthermore, somatic loss of the Cdkn2a wild-type allele was frequently observed in ETV6::JAK2;Rag2-/-;Cdkn2a+/- T-ALL cells, suggesting a selective advantage for total inactivation of Cdkn2a. Both Cdkn2a-sufficient and Cdkn2a-defective T-ALL presented additional genetic alterations, such as Notch1 mutations and gains of chromosomes 13 and 15. These data indicate that Cdkn2a acts as a gatekeeper for leukemogenesis from the most immature stages of thymocyte development.

AML-283 The Genetic Landscape of NUP98-Rearranged Pediatric Leukemia

Rearrangements involving NUP98 are frequently found in pediatric acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL) and are associated with poor prognosis. Various fusion partners have been identified; however, the genetic basis of how different fusion genes and cooperating mutations contribute to the disease phenotypes and the refractory nature remains to be clarified. To elucidate the transcriptional and genetic background of pediatric NUP98-rearranged acute leukemia. Transcriptomic and genetic analyses were performed on pediatric leukemia with NUP98 rearrangements from multiple clinical studies at St. Jude Children's Research Hospital, by the Italian Association for Pediatric Hematology and Oncology, or by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. No blinding was done in this study. RNA sequencing from 95 patients (median age of 9.0 years) with NUP98-rearranged (NUP98r) leukemia was performed or obtained from previous studies. Eighty-one of these samples were also studied by tumor-normal-paired whole-genome or whole-exome sequencing. We identified 17 unique NUP98 fusion partners from RNAseq data with NSD1 as the most frequent partner (n=44, 46.3%), followed by KDM5A (n=27, 28.4%) and RAP1GDS1 (n=6, 6.3%). We also found rare fusion partners recurrent in this cohort, including LNP1, HOXA13, and HOXD13 (n=2, 2.1%, respectively). Among cooperating mutations, FLT3-ITD and WT1 were frequent in NSD1-AML (57.9% and 52.6%, respectively), and copy number loss of chromosome 13 harboring RB1 was found in 86.3% of NUP98-KDM5A AML with erythromegakaryocytic expression patterns (FAB M6-7). NUP98-RAP1GDS1 fusions are found in both AML (n=3) and T-ALL (n=3) cases and those with T lineage differentiation have concurrent NOTCH1 mutations, indicating that both fusions and cooperating mutations are contributing to the disease phenotypes. We further performed transcriptome analysis of NUP98r AML, integrating various AML subtypes from previous studies. NUP98r AML expressed high MEIS1, PRDM16, and HOXA-B cluster genes similar to AMLs with NPM1 or UBTF mutations, suggesting a molecular basis shared by these subtypes. Genomic and transcriptional profiling of pediatric NUP98-rearranged leukemia identified unique associations among fusions, cooperating mutations, and disease phenotypes.

Investigating a Genetic Link Between Alzheimer’s Disease and CADASIL-Related Cerebral Small Vessel Disease

Monogenic forms of Alzheimer’s disease (AD) have been identified through mutations in genes such as APP, PSEN1, and PSEN2, whilst other genetic markers such as the APOE ε carrier allele status have been shown to increase the likelihood of having the disease. Mutations in these genes are not limited to AD, as APP mutations can also cause an amyloid form of cerebral small vessel disease (CSVD) known as cerebral amyloid angiopathy, whilst PSEN1 and PSEN2 are involved in NOTCH3 signalling, a process known to be dysregulated in the monogenic CSVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The overlap between AD genes and causes of CSVD led to the hypothesis that mutations in other genes within the PANTHER AD–presenilin pathway may be novel causes of CSVD in a cohort of clinically suspicious CADASIL patients without a pathogenic NOTCH3 mutation. To investigate this, whole exome sequencing was performed on 50 suspected CADASIL patients with no NOTCH3 mutations, and a targeted gene analysis was completed on the PANTHER. ERN1 was identified as a novel candidate CSVD gene following predicted pathogenic gene mutation analysis. Rare variant burden testing failed to identify an association with any gene; however, it did show a nominally significant link with ERN1 and TRPC3. This study provides evidence to support a genetic overlap between CSVD and Alzheimer’s disease.

Genotype and Phenotype Differences in CADASIL from an Asian Perspective.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebral small-vessel disease caused by mutations in the NOTCH3 gene. Classical pathogenic mechanisms are associated with cysteine gain or loss, but recent studies suggest that cysteine-sparing mutations might have a potential role as a pathogen. In comparison with CADASIL patients in Western countries, there are several differences in Asian patients: (1) prevalent locus of NOTCH3 mutations (exons 2–6 [particularly exon 4] vs. exon 11), (2) age at symptom onset, (3) prevalence of cerebral microbleeds and hemorrhagic stroke, (4) clinical symptoms, and (5) severity of white matter hyperintensities and typical involvement of the anterior temporal pole in magnetic resonance imaging. Both ethnicity and founder effects contribute to these differences in the clinical NOTCH3 spectrum in different cohorts. More functional investigations from diverse races are needed to clarify unknown but novel variants of NOTCH3 mutations. This review may broaden the spectrum of NOTCH3 variants from an Asian perspective and draw attention to the hidden pathogenic roles of NOTCH3 variants.

Sustained Aurora Kinase B Expression Confers Resistance to PI3K Inhibition in Head and Neck Squamous Cell Carcinoma.

Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Somatic Mutational Landscape in Mexican Patients: CDH1 Mutations and chr20q13.33 Amplifications Are Associated with Diffuse-Type Gastric Adenocarcinoma.

The Hispanic population, compared with other ethnic groups, presents a more aggressive gastric cancer phenotype with higher frequency of diffuse-type gastric adenocarcinoma (GA); this could be related to the mutational landscape of GA in these patients. Using whole-exome sequencing, we sought to present the mutational landscape of GA from 50 Mexican patients who were treated at The Instituto Nacional de Cancerología from 2019 to 2020. We performed a comprehensive statistical analysis to explore the relationship of the genomic variants and clinical data such as tumor histology and presence of signet-ring cell, H. pylori, and EBV. We describe a potentially different mutational landscape between diffuse and intestinal GA in Mexican patients. Patients with intestinal-type GA tended to present a higher frequency of NOTCH1 mutations, copy number gains in cytobands 13.14, 10q23.33, and 12q25.1, and copy number losses in cytobands 7p12, 14q24.2, and 11q13.1; whereas patients with diffuse-type GA tended to present a high frequency of CDH1 mutations and CNV gains in cytobands 20q13.33 and 22q11.21. This is the first description of a mutational landscape of GA in Mexican patients to better understand tumorigenesis in Hispanic patients and lay the groundwork for discovering potential biomarkers and therapeutic targets.

Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, Arbo.

Cell lines represent an essential tool used in preclinical research. Most hematologic malignancies have a wide array of cell lines representing their respective molecular and pathologic spectra. In mantle cell lymphoma (MCL), cell lines become specifically valuable in view of the heterogeneity of this disease. Unfortunately, the number of MCL cell lines that are available for the research community remains small, with only nine cell lines available for purchase through the American Type Culture Collection (ATCC). We have established a novel blastoid MCL cell line, isolated from the malignant pleural effusion of a 69-year-old male with refractory MCL. Arbo was fully characterized with cytogenetics, immunophenotyping, whole exome sequencing and drug sensitivity assays. One of the most notable mutations identified in Arbo (but not in normal tissue) was the missense mutation NOTCH2 R2400*, which has been proposed as a clinically significant mutation in MCL seen in 5% of cases. NOTCH2 R2400* results in a truncated Notch2 protein, leading to a more stable and active protein. Using pharmacologic inhibition of Notch2, we showed a dependence of Arbo on NOTCH2 signaling, as well as a link between CD23 expression on Arbo and NOTCH2 activity. Arbo represents a NOTCH2 mutated model that is useful in MCL as well as other lymphomas with such mutation. We plan to deposit Arbo at the ATCC to be available for the research community.

Inhibition of the Phosphatidylinositol-3 Kinase Pathway Using Bimiralisib in Loss-of-Function NOTCH1-Mutant Head and Neck Cancer.

PI3K/mTOR inhibition leads to apoptosis of NOTCH1-mutant head and neck squamous cell carcinoma (HNSCC) cells. We tested the efficacy of the PI3K/mTOR inhibitor bimiralisib in patients with NOTCH1-mutant HNSCC. Patients with recurrent/metastatic NOTCH1-mutant HNSCC who had progressed during chemotherapy and immunotherapy received bimiralisib until unacceptable toxicity or progression. To assess whether NOTCH1 mutations can be detected in blood, we measured circulating tumor DNA (ctDNA). To assess activated NOTCH1 protein levels, we quantitated cleaved NOTCH1 (cl-NOTCH) by immunohistochemistry. Eight patients were treated, and 6 were evaluable for response. The objective response rate was 17%. For all 8 patients, median progression-free and overall survival was 5 and 7 months, respectively. Bimiralisib was well tolerated, with expected hyperglycemia. Pharmacokinetic values were consistent with published studies. NOTCH1 mutations were detected in 83.3% of ctDNA. Staining for tumor cl-NOTCH1 was negative. The trial closed early due to sponsor insolvency. Although the trial was small, outcomes with bimiralisib were better than the historical standard of care; Results will need to be confirmed in a larger trial. The lack of cl-NOTCH1 was consistent with loss-of-function mutations and validated our mutation function algorithm. The ability to detect NOTCH1 mutations in blood will help future studies. (ClinicalTrials.gov Identifier: NCT03740100).

Role of NOTCH1 mutation as a prognostic factor in chronic lymphocytic leukemia (CLL) in Iraqi CLL patients

Role of NOTCH1 mutation as a prognostic factor in chronic lymphocytic leukemia (CLL) in Iraqi CLL patients

Abstract A36: Building of a large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) repository

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous B-cell malignancy that consists of no fewer than 5 genetic subtypes. Cell lines have been a central model system for DLBCL but fail to fully recapitulate the diversity of genetic alterations that have been observed in large patient cohorts. Approximately 40% of DLBCLs will be refractory to or relapse following first-line R-chemo regimens, with many of these patients destined to receive CD19 CAR T-cells in the third line or later. Due to the relatively recent introduction of CD19 CAR T-cells into standard of care for relapsed/refractory DLBCL, there is a paucity of models in the CAR T relapsed setting. Together, these factors led us to embark upon an effort to build a substantive DLBCL patient-derived xenograft (PDX) repository that encompasses the major DLBCL genetic subtypes and includes CAR T relapsed models. From September 2019 to March 2022, we implanted 231 biopsies with diagnoses of DLBCL (n=120), HGBL (n=17), PMBL (n=2), other high grade B-cell lymphomas (n=87) and other low grade B-cell lymphoma (n=5). Our approaches were modeled from those utilized by the Public Repository of Xenografts (PRoXe: Townsend et al., Cancer Cell 2016), employing implantation of intact core needle biopsies into the renal capsule of NSG mice. Of 206 models that have been monitored for up to 6 months, we have generated PDX tumors from 52 tumors originating from 50 patients (25.2% take rate). The average time from implantation to signs of illness was 8 weeks (2 min, 22 max). The average number of preceding lines of therapy for these models was 3 (0 min, 8 max), with 22 models being generated from biopsies taken subsequent to progression from CD19 CAR T-cells. This included paired biopsies from two patients generated from biopsies taken immediately prior to receiving and following progression from CD19 CAR T-cells. All PDX models were confirmed to be EBV negative by EBER-ISH and subjected to histopathologic and immuno-phenotypical examination to compare their characteristics to an alternative core taken alongside the biopsy used to generate the PDX model. We additionally performed extensive molecular characterization, including RNA, whole-exome (WES), low-pass whole-genome (lpWGS), ATAC and ChIP sequencing for 7 histone post-translational modifications using the Mint-ChIP approach. Epigenetic characterization will be reported at a later date. RNA-seq confirmed both ABC (n=16) and GCB (n=24) subtypes. WES identified cardinal genetic features of all major genetic subtypes of DLBCL, including coexistent MYD88 L265P and CD79B mutations (MCD subtype); EZH2, CREBBP and KMT2D mutations (EZB subtype); NOTCH2 truncating mutations (N2 subtype); SGK1 and TET2 mutations (ST2 subtype); and NOTCH1 mutations (N1 subtype). In summary, we have established and characterized a large LBCL PDX model repository to serve as a resource for mechanistic and pre-clinical studies that will improve our understanding of lymphoma etiology and mechanisms of response/resistance to therapy. Citation Format: Haopeng Yang, Vida Ravanmehr, Jordan C Showell, Estela M Rojas-Neira, Jared M Henderson, Jason R Westin, Michael R Green. Building of a large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) repository [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A36.

Hajdu-Cheney Syndrome: A Novel NOTCH2 Mutation in a Spanish Child in Treatment with Vibrotherapy: A Case Report

A case report of an 11-year-old boy with a de novo variant in NOTCH2 and clinical features characteristic of Hajdu-Cheney syndrome is reported, with acroosteolysis of the distal phalanges of the feet and hands, generalized osteoporosis, musculoskeletal and craniofacial alterations, short stature, bowing of long bones, vertebral anomalies, genu recurvatum, hypertrichosis, joint and skin hyperlaxity, atopic dermatitis, megalocorneas, micrognathia and frequent respiratory infections, among others. Treatment is with bisphosphonates in the framework of bone density improvement and with focal vibration therapy for rehabilitation of the musculoskeletal system and gait improvement. The three generalities of this pathology—phenotypic variability, degenerative character and the presence of generalized osteoporosis and acroosteolysis of the distal phalanges—are seen in this case, whose diagnostic confirmation was made by genetic study.

Induction of a NOTCH3 Lehman syndrome mutation in osteocytes causes osteopenia in male C57BL/6J mice.

Lateral Meningocele or Lehman Syndrome (LMS) is associated with NOTCH3 mutations causing deletions of the PEST domain and a gain-of-NOTCH3 function. We demonstrated that Notch3em1Ecan mice harboring Notch3 mutations analogous to those found in LMS are osteopenic because of enhanced bone resorption. To determine the contribution of specific cell lineages to the phenotype, we created a conditional-by-inversion (Notch3COIN) model termed Notch3em2Ecan in which Cre recombination generates a Notch3INV allele expressing a NOTCH3 mutant lacking the PEST domain. Germ line Notch3COIN inversion caused osteopenia and phenocopied the Notch3em1Ecan mutant, validating the model. To induce the mutation in osteocytes, smooth muscle and endothelial cells, Notch3COIN mice were bred with mice expressing Cre from the Dmp1, Sm22a and Cdh5 promoters, respectively, creating experimental mice harboring Notch3INV alleles in Cre-expressing cells and control littermates harboring Notch3COIN alleles. Notch3COIN inversion in osteocytes led to femoral and vertebral cancellous bone osteopenia, whereas Notch3COIN inversion in mural Sm22a or endothelial Cdh5-expressing cells did not result in a skeletal phenotype. In conclusion, introduction of the LMS mutation in osteocytes but not in vascular cells causes osteopenia and phenocopies Notch3em1Ecan global mutant mice.

Diverse mutations and structural variations contribute to Notch signaling deregulation in paediatric T-cell lymphoblastic lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is an aggressive neoplasm closely related to T-cell acute lymphoblastic leukaemia (T-ALL). Despite their similarities, and contrary to T-ALL, studies on paediatric T-LBL are scarce and, therefore, its molecular landscape has not yet been fully elucidated. Thus, the aims of this study were to characterize the genetic and molecular heterogeneity of paediatric T-LBL and to evaluate novel molecular markers differentiating this entity from T-ALL. Thirty-three paediatric T-LBL patients were analyzed using an integrated approach, including targeted next-generation sequencing, RNA-sequencing transcriptome analysis and copy-number arrays. Copy number and mutational analyses allowed the detection of recurrent homozygous deletions of 9p/CDKN2A (78%), trisomy 20 (19%) and gains of 17q24-q25 (16%), as well as frequent mutations of NOTCH1 (62%), followed by the BCL11B (23%), WT1 (19%) and FBXW7, PHF6 and RPL10 genes (15%, respectively). This genetic profile did not differ from that described in T-ALL in terms of mutation incidence and global genomic complexity level, but unveiled virtually exclusive 17q25 gains and trisomy 20 in T-LBL. Additionally, we identified novel gene fusions in paediatric T-LBL, including NOTCH1-IKZF2, RNGTT-SNAP91 and DDX3X-MLLT10, the last being the only one previously described in T-ALL. Moreover, clinical correlations highlighted the presence of Notch pathway alterations as a factor related to favourable outcome. In summary, the genomic landscape of paediatric T-LBL is similar to that observed in T-ALL, and Notch signaling pathway deregulation remains the cornerstone in its pathogenesis, including not only mutations but fusion genes targeting NOTCH1.

Multi-omics analysis reveals prognostic and therapeutic value of cuproptosis-related lncRNAs in oral squamous cell carcinoma.

Background: Extensive research revealed copper and lncRNA can regulate tumor progression. Additionally, cuproptosis has been proven can cause cell death that may affect the development of tumor. However, there is little research focused on the potential prognostic and therapeutic role of cuproptosis-related lncRNA in OSCC patients. Methods: Data used were for bioinformatics analyses were downloaded from both the TCGA database and GEO database. The R software were used for statistical analysis. Mapping was done using the tool of FigureYa. Results: The signature consist of 7 cuproptosis-related lncRNA was identified through lasso and Cox regression analysis and a nomogram was developed. In addition, we performed genomic analyses including pathway enrichment analysis and mutation analysis between two groups. It was found that OSCC patients were prone to TP53, TTN, FAT1 and NOTCH1 mutations and a difference of mutation analysis between the two groups was significant. Results of TIDE analysis indicating that patients in low risk group were more susceptible to immunotherapy. Accordingly, results of subclass mapping analysis confirmed our findings, which revealed that patients with low riskscore were more likely to respond to immunotherapy. Conclusion: We have successfully identified and validated a novel prognostic signature with a strong independent predictive capacity. And we have found that patients with low riskscore were more susceptible to immunotherapy, especially PD-1 inhibitor therapy.

Prognostic value of baseline genetic features and newly identified TP53 mutations in advanced breast cancer.

Approximately 30% of breast cancer (BC) patients suffer from disease relapse after definitive treatment. Monitoring BC at baseline and disease progression using comprehensive genomic profiling would facilitate the prediction of prognosis. We retrospectively studied 101 BC patients ultimately experiencing relapse and/or metastases. The baseline and circulating tumor DNA‐monitoring cohorts included patients with baseline tumor tissue and serial plasma samples, respectively. Samples were analyzed with targeted next‐generation sequencing of 425 cancer‐relevant genes. Of 35 patients in the baseline cohort, patients with TP53 mutations (P < 0.01), or CTCF/GNAS mutations (P < 0.01) displayed inferior disease‐free survival, and patients harboring TP53 (P = 0.06) or NOTCH1 (P = 0.06) mutations showed relatively poor overall survival (OS), compared to patients with wild‐type counterparts. Of the 59 patients with serial plasma samples, 11 patients who were newly detected with TP53 mutations had worse OS than patients whose TP53 mutational status remained negative (P < 0.01). These results indicate that an inferior prognosis of advanced breast cancer was potentially associated with baseline TP53, CTCF, and NOTCH1 alterations. Newly identified TP53 mutations after relapse and/or metastasis was another potential prognostic biomarker of poor prognosis.

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

Comparison of clinical and neuroimaging features between NOTCH3 mutations and nongenetic spontaneous intracerebral haemorrhage.

The NOTCH3 mutation is a common cause of hereditary cerebral small vessel disease (CSVD) and may be a cause of spontaneous intracerebral haemorrhage (ICH). The aim was to investigate the clinical/imaging features for identifying the NOTCH3-mutation-related ICH. The study was based on a cohort of 749 CSVD patients in Taiwan who received next-generation sequencing of CSVD genes including NOTCH3. Patients with a history of ICH (n=206) were included for analysis. The CSVD neuroimaging markers were compared between the patients with NOTCH3 and those without known genetic mutations. After excluding patients with other causes of ICH (structural lesions, systemic/medication related or amyloid angiopathy) and those without neuroimaging, 45 NOTCH3 mutation patients and 109 nongenetic ICH patients were included. The NOTCH3 mutation patients were more likely to have thalamic haemorrhage, a family history of stroke and more severe CSVD neuroimaging markers. A five-point NOTCH3-ICH score was constructed and consisted of a history of stroke in siblings, thalamic haemorrhage, any deep nuclei lacunae, any hippocampal cerebral microbleed (CMB) and a thalamic CMB &gt;5 (one point for each). A score ≥2 had a sensitivity of 88.9% and a specificity of 64.2% in identifying the NOTCH3 mutation. The NOTCH3 mutation patients had a higher risk of recurrent stroke (9.1 vs. 4.5 per 100 person-years, log-rank p=0.03) during follow-up. The patients with NOTCH3-mutation-related ICH had a higher burden of CMBs in the hippocampus/thalamus and a higher recurrent stroke risk. The NOTCH3-ICH score may assist in identifying genetic causes of ICH.