Landscape of NOTCH1 mutations and co-occurring biomarker alterations in chronic lymphocytic leukemia.

NOTCH1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and has emerged as a marker of poor prognosis. In addition to coding NOTCH1 mutations involving exon 34, non-coding NOTCH1 mutations involving the 3' UTR have been described in a limited number of chronic lymphocytic leukemia (CLL) patients and were associated with adverse outcomes. In this study, 1574 CLL patients were assessed using targeted sequencing with a 29 gene panel and the results were correlated with prognostic characteristics. NOTCH1 mutations were detected in 252 (16%) patients, including both coding (220/252, 14%), non-coding (24/252, 1.5%) and a mixture of coding and non-coding (8/252, 0.5%) NOTCH1 mutations. NOTCH1 mutations were more commonly seen in patients with unmutated IGHV, ZAP70 positivity and CD38 positivity. Mixed NOTCH1 mutations were also more commonly seen in patients with unmutated IGHV and ZAP70. There was no association between mixed NOTCH1 mutations and CD38 expression in this cohort. The most common cytogenetic alteration detected in patients with coding and mixed NOTCH1 mutations was trisomy 12, whereas del13q was the most common cytogenetic alteration detected in patients with non-coding NOTCH1 mutation. The most common gene mutations co-occurring with coding NOTCH1 mutations were: TP53 (23.2%), SF3B1 (16.4%) and SPEN (10%). The most common gene mutations co-occurring with non-coding NOTCH1 mutations were: SF3B1 11(34.4%), ATM 4(12.5%) and TP53 4(12.5%). CLL patients with clonal coding and non-coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 (4.3 vs 10.0 years and 0.9 vs 10.0 years respectively, p < 0.05). Similarly, CLL patients with subclonal coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 (5.6 vs 10.0 years, p < 0.05). CLL patients with subclonal non-coding NOTCH1 mutations also had a shorter time-to-first treatment than patients with wild type NOTCH1 mutations, however, the difference was not significant (5.1 vs 10.0 years, p = 0.15). These data confirm that both coding and non-coding NOTCH1 mutations carry adverse prognostic impact and need to be included in sequencing assays performed for the prognostic workup of CLL patients.

From the archives of MD Anderson Cancer Center: Untreated leukemic non-nodal mantle cell lymphoma with relapse as pleomorphic variant mantle cell lymphoma 21 years later.

Leukemic, non-nodal mantle cell lymphoma (MCL) is a distinct, rare, indolent variant of mantle cell lymphoma, but can relapse aggressively. It can present with lymphocytosis with chronic lymphocytic leukemia (CLL)-like morphologic and immunophenotypic features as was initially considered in the index case. However, at time of splenectomy, two years later cyclin D1 overexpression was shown and the disease was realized to be leukemic non-nodal MCL. The patient was followed for 21 years, without therapy, before he developed clinically aggressive MCL with lymphadenopathy. Lymph node biopsy showed MCL, pleomorphic variant. We review the literature and discuss the features of leukemic non-nodal MCL as well as the potential pitfalls in diagnosis. Furthermore, we are not aware of another cases reported with a 21 year interval from initial diagnosis of leukemic non-nodal MCL to aggressive MCL.

Clinico-pathologic characteristics and outcomes of the World Health Organization (WHO) provisional entity de novo acute myeloid leukemia with mutated RUNX1.

We studied the characteristics of the provisional category de novo acute myeloid leukemia (AML) with mutated RUNX1 (AML-RUNX1mut) proposed by the World Health Organization (WHO). Until now, most published studies have combined de novo and secondary AML-RUNX1mut. We compared the clinicopathologic characteristics and outcomes of WHO-defined de novo AML-RUNX1mut with de novo AML without RUNX1 alterations (AML-RUNX1wt). We performed sequential NGS to assess RUNX1 mutation stability over disease course. We identified 46 de novo AML-RUNX1mut patients [32 (70%) men, 14 (30%) women; median age, 66.5 years] with 54 RUNX1 mutations [median VAF, 32% (2-97%)]. Point mutations clustered within the runt-homology-domain and frame-shift mutations within the transactivation domain. Compared with AML-RUNX1wt, AML-RUNX1mut showed male predominance (p = 0.02), higher frequency of SRSF2 (p = 0.02), and ASXL1 (p = 0.0004) mutations and normal karyotype (p = 0.01), and absent NPM1 mutations (p = 0.0002). De novo AML-RUNX1mut showed no significant difference in overall survival (OS) compared with AML-RUNX1wt (median: 26 vs. 32 months) (p = 0.71). AML-RUNX1mut with clonal RUNX1 mutation (≥20% VAF) had shorter OS than subclonal <20% VAF (23 months vs. undefined; p = 0.04). However, the difference was not significant when compared with AML-RUNX1wt (23 vs. 32 months; p = 0.23). No significant OS difference was noted between de novo AML-RUNX1mut and AML-NOS-RUNX1wt. By sequential multigene mutation profiling, RUNX1 mutation disappeared at relapse in one of ten patients. Overall, the findings support separate categorization of this entity. However, there is no significant outcome difference compared with AML-RUNX1wt.

Data on MECOM rearrangement-driven chromosomal aberrations in myeloid malignancies.

Data in this article presents the results of conventional cytogenetics and fluorescence in situ hybridization (FISH) analyses in 129 patients with confirmed MECOM rearrangement (https://doi.org/10.1016/j.cancergen.2019.03.002) [1]. Generally, the MECOM rearrangement has arisen through translocation, inversion, and insertion and/or unknown mechanism. In addition to the typical chromosomal aberrations, inv(3)(q21q26.2) and t(3; 3)(q21; q26.6) [2-4], over 50% of cases presented here exhibit a wide spectrum of MECOM rearrangement-driven, atypical chromosomal aberrations, including inv(3) with breakpoint other than 3q21; t(1; 3); t(2; 3); t(3; 6); t(3; 8); t(3; 12); t(3; 17); t(3; 21) as well as an insertion of 3q26.2 into different chromosomes. These cases are thoroughly characterized by karyotyping, interphase-, metaphase-, map-back FISH and whole chromosomal painting (WCP) analyses.

Association of gene mutations with time-to-first treatment in 384 treatment-naive chronic lymphocytic leukaemia patients.

This study correlated somatic mutation results and known prognostic factors with time-to-first treatment (TTFT) in 384 treatment-naïve (TN) chronic lymphocytic leukaemia (CLL) patients to help determine disease-specific drivers of early untreated CLL. CLL DNA from either peripheral blood or bone marrow underwent next generation targeted sequencing with a 29-gene panel. Gene mutation data and concurrent clinical characteristics, such as Rai/Binet stage, fluorescence in situ hybridisation (FISH), ZAP70/CD38, karyotype and IGHV mutation, status were analysed in univariable and multivariable analyses to identify associations with TTFT. TTFT was defined as time from diagnosis to initial treatment. In univariable analyses, mutated ATM (P < 0·001), NOTCH1 (P < 0·001) and SF3B1 (P = 0·002) as well as unmutated IGHV (P < 0·001), del(11q) (P < 0·001) and trisomy 12 (P < 0·001) by hierarchal FISH and advanced Rai (P = 0·05) and Binet (P < 0·001) stages were associated with shorter TTFT. Importantly, del(17p), mutated TP53 and complex karyotype were not associated with shorter TTFT. In a reduced multivariable analysis, mutated ATM (P < 0·001) and unmutated IGHV status (P < 0·001) remained significant, showing their importance in early leukaemogenesis. High-risk prognostic markers such as del(17p), mutated TP53 and complex karyotype, were not correlated with TTFT, suggesting that these abnormalities have limited roles in early disease progression but are more important in relapsed CLL.

Early T precursor acute lymphoblastic leukaemia/lymphoma shows differential immunophenotypic characteristics including frequent CD33 expression and in vitro response to targeted CD33 therapy.

The differential immunophenotypic characteristics of early T precursor (ETP) acute lymphoblastic leukaemia/lymphoma (ALL) remain incompletely characterized. The study group (n = 142) included 106 (74·7%) men and 36 (25·3%) women with a median age of 34·9 years (range, 2-79) at diagnosis. Patients were subtyped by flow cytometry immunophenotyping as follows: 33 (23·2%) ETP; 32 (22·5%) early non-ETP; 60 (42·2%) thymic; and 17 (12·1%) mature. Excepting definitional markers, there was a significant differential expression of the markers CD2, CD10, CD33 and TdT between ETP-ALL and non-ETP-ALL. Positive CD33 expression (≥20% of leukaemic blasts) was detected in 21/33 (63%) ETP-ALL compared with 17/95 (17·9%) non-ETP-ALL (P < 0·001). Notably, targeted anti-CD33 therapy with IMGN779 resulted in significant growth inhibition and increased apoptosis in ETP-ALL cells in vitro. An 11-marker T-ALL immunophenotype score discriminated reliably between ETP and non-ETP ALL. Longitudinal analysis of ETP-ALL cases in this study demonstrated that the immunophenotype may be occasionally dynamic but is largely stable over the disease course. In summary, identification of ETP-ALL might be enhanced by using an 11-marker T-ALL immunophenotype score. CD33 expression is frequent in ETP-ALL, and in vitro data suggest that exploring anti-CD33 therapy in ETP-ALL is warranted.

Deciphering the complexities of MECOM rearrangement-driven chromosomal aberrations.

MECOM rearrangement is associated with rapid disease progression and poor prognosis in myeloid neoplasms. Previous studies were often based on 3q26.2 abnormalities without confirmation of MECOM status. The frequency of MECOM rearrangement and attribution of various chromosomal aberrations remain poorly characterized. This study presented 129 cases with confirmed MECOM rearrangement by karyotyping and multiple FISH methodologies. MECOM rearrangement arose through translocation (49.6%, n = 64), inversion (40.3%, n = 52), insertion (5.4%, n = 7) or unknown mechanism(s) (4.7%, n = 6). The classic inv(3)(q21q26.2) was dominant (n = 50) in inversion-driven MECOM rearrangement; and 3 of them also had double inv(3). For translocation-driven MECOM rearrangement, t(3;21) was most common (n = 15), followed by t(2;3) (n = 13), t(3;12) (n = 10), t(3;3) (n = 9), t(3;8) (n = 6), t(3;6) and t(3;17) (n = 4 each), t(1;3) and t(3;?) (n = 1 each). Cases with t(3;3)-, t(3;12)-, and insertion-driven MECOM rearrangement were prone to exhibit a complex karyotype, while cases with t(2;3)-, t(3;21)- and insertion-driven MECOM rearrangement were prone to have an "unbalanced" MECOM FISH signal pattern, likely caused by uncommon breakpoint(s) within the target of 5'MECOM probe. Therefore, atypical chromosomal aberrations and/or mechanisms are involved in MECOM rearrangement. Confirmation/exclusion of MECOM rearrangement is necessary in all cases with a 3q26.2 abnormality. (Word count: 190).

DDX41 mutations in myeloid neoplasms are associated with male gender, TP53 mutations and high-risk disease.

Myeloid neoplasms with germline DDX41 mutations have been incorporated into the 2017 WHO classification. Limited studies describing the clinicopathologic features and mutation profile are available. We searched for myeloid neoplasms with a DDX41 gene mutation tested by an 81-gene next-generation sequencing panel over a 7-month period. We identified 34 patients with myeloid neoplasms with DDX41 abnormalities; 26 (76%) men and 8 women (24%) [median age, 70 years], 20 acute myeloid leukemia (AML), 10 myelodysplastic syndrome (MDS), 1 chronic myelomonocytic leukemia (CMML) and 3 myeloproliferative neoplasms (MPN). Fifty-nine DDX41 variants were detected: 27 (46%) appeared somatic and 32 (54%) were presumably germline mutations. The majority of presumed germline mutations were upstream of the Helicase 2 domain (93%) and involved loss of the start codon (30%). The majority of somatic mutations were within the Helicase 2 domain (78%), with the missense mutation p.R525H being most common (67%). There was a significant difference in the location of germline or somatic mutations (P < .0001). Concomitant mutations were detected involving 19 genes, but only TP53 (n = 11, 32%), ASXL1 (n = 8, 24%), and JAK2 (n = 4, 12%) were recurrent. Twenty (59%) patients showed diploid cytogenetics. Twenty-three (68%) patients presented with AML or MDS-EB-2, suggesting an association with high-grade myeloid neoplasm. Patients with myeloid neoplasms carrying DDX41 mutations show male predominance (3:1), higher age at presentation, association with TP53 mutations, and association with high-grade myeloid neoplasms in our cohort at a referral cancer center setting. These findings support the recognition of myeloid neoplasms with DDX41 mutation as unique, need for germline confirmation, and further assessment of family members.

Routine sequencing in CLL has prognostic implications and provides new insight into pathogenesis and targeted treatments.

Chronic lymphocytic leukaemia (CLL) is a genetically heterogeneous disease characterised by genomic alterations and gene mutations that may portend worse survival or resistance to treatments. A total of 680 blood or bone marrow samples underwent targeted sequencing of 29 genes previously identified as being mutated in CLL, which were correlated to known prognostic clinical characteristics. Overall, 400 (59%) patients were treatment-naïve (TN) and 280 (41%) were relapsed/refractory (R/R). Most patients (70%) had ≥1 mutation, with TP53 (22%), SF3B1 (18%), NOTCH1 (13%) and ATM (13%) being the most commonly mutated genes. A higher proportion of R/R patients had mutations in SF3B1 (P = 0·01) and TP53 (P < 0·001). Patients with mutated IGHV CLL more often had mutations in KLHL6 (P = 0·001) and MYD88 (P < 0·001). Pairwise associations showed mutational co-occurrences in the TN group including SF3B1/ATM [false discovery rate (FDR) < 0·05] and NOTCH1/POT1 (FDR < 0·01). Recurrent mutations resulting in premature truncation prior to the ubiquitination domains of NOTCH1 in its PEST domain and BIRC3 in its RING domain can produce proteins that constitutively activate CLL. Frequent missense mutations, such as K700E in SF3B1 and E571K in XPO1, have unknown function but are most likely to be activating mutations. Future directions include using these mutations to identify pathways for therapeutic targeting and rational drug design.