BIRC3 Mutations Research Articles

Normal serum protein electrophoresis and mutated IGHV genes detect very slowly evolving chronic lymphocytic leukemia patients.

More than 35 years after the Binet classification, there is still a need for simple prognostic markers in chronic lymphocytic leukemia (CLL). Here, we studied the treatment‐free survival (TFS) impact of normal serum protein electrophoresis (SPE) at diagnosis. One hundred twelve patients with CLL were analyzed. The main prognostic factors (Binet stage; lymphocytosis; IGHV mutation status; TP53,SF3B1,NOTCH1, and BIRC3 mutations; and cytogenetic abnormalities) were studied. The frequencies of IGHV mutation status, cytogenetic abnormalities, and TP53,SF3B1,NOTCH1, and BIRC3 mutations were not significantly different between normal and abnormal SPE. Normal SPE was associated with Binet stage A, nonprogressive disease for 6 months, lymphocytosis below 30 G/L, and the absence of the IGHV3‐21 gene rearrangement which is associated with poor prognosis. The TFS of patients with normal SPE was significantly longer than that of patients with abnormal SPE (log‐rank test: P = 0.0015, with 51% untreated patients at 5.6 years and a perfect plateau afterward vs. a median TFS at 2.64 years for abnormal SPE with no plateau). Multivariate analysis using two different Cox models and bootstrapping showed that normal SPE was an independent good prognostic marker for either Binet stage, lymphocytosis, or IGHV mutation status. TFS was further increased when both normal SPE and mutated IGHV were present (log‐rank test: P = 0.008, median not reached, plateau at 5.6 years and 66% untreated patients). A comparison with other prognostic markers suggested that normal SPE could reflect slowly advancing CLL disease. Altogether, our results show that a combination of normal SPE and mutated IGHV genes defines a subgroup of patients with CLL who evolve very slowly and who might never need treatment.

Genetic landscape of ultra-stable chronic lymphocytic leukemia patients

BackgroundChronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methodsPatients with absence of disease progression for over 10years (10–34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. ResultsWES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. ConclusionsThe genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

Prognostic Factors in the Era of Targeted Therapies in CLL.

Chronic lymphocytic leukemia is heterogeneous disease characterized by a variable clinical course that is greatly influenced by various patient and disease characteristics. Over the last two decades, advent of new diagnostic methodologies has led to the identification of several factors of prognostic and predictive relevance. Furthermore, recent advances in next-generation sequencing techniques has identified recurrent novel mutations in NOTCH1, SF3B1, BIRC3, and ATM genes whose role as prognostic and predictive markers is currently being investigated. These biologic markers carry new prognostic information and their incorporation into prognostic scoring systems will likely lead to refined multi-parameter risk models. While the prognostic impact of many of the most commonly used markers on clinical outcomes in patients treated with chemo-immunotherapy is well documented, it is important to review their predictive and prognostic role in the era of novel targeted therapies. This article will discuss the currently available information on the clinical relevance of prognostic markers in patients treated with novel targeted therapies.

Novel prognostic molecular factors: a quantum leap in the field of chronic lymphocytic leukemia

Cytogenetic lesions do not completely explain clinical heterogeneity of chronic lymphocytic leukemia (CLL). The 2016 revision of the World Health Organization classification 2008 indicated that molecular lesions of TP53, NOTCH1, SF3B1 and BIRC3 have potential clinical relevance and could be integrated into an updated risk profile. The negative clinical implications of TP53 disruptions are well constituted and patients with these mutations should be considered for novel, small molecule signal transduction inhibitors therapies. Mutations of NOTCH1, SF3B1 and BIRC3 are associated with poor prognosis. Patients with mutated SF3B1 or NOTCH1 genes present shorter time to first treatment compared to unmutated group. NOTCH1 mutations are related to a high risk of Richter's syndrome transformation, especially in case of TP53 disruptions' coexistence. Large studies on MYD88 mutations in CLL have not explained clearly their clinical importance.The aim of this paper is to provide a comprehensive review on novel molecular aberrations identified in CLL.

Mutations of BRAF and BIRC3 Identify a Subgroup of Chronic Lymphocytic Leukemia with Very Poor Prognosis upon FCR Treatment

Mutations of BRAF and BIRC3 Identify a Subgroup of Chronic Lymphocytic Leukemia with Very Poor Prognosis upon FCR Treatment

Distinct molecular genetics of chronic lymphocytic leukemia in Taiwan: clinical and pathogenetic implications.

Differences in chronic lymphocytic leukemia between the Asian and the Western population are widely known. To further clarify these ethnic differences, we profiled the molecular genetics in a cohort of 83 newly diagnosed patients from Taiwan. In detail, we assessed: (i) the usage and the mutational status of the clonotypic immunoglobulin heavy-chain variable region (IgHV) genes, (ii) the presence of VH CDR3 stereotypes, and (iii) TP53, NOTCH1, SF3B1, BIRC3, and MYD88 mutations. The IgHV gene repertoire was biased and distinct from that observed in the West with the most common IgHV genes being IgHV3-23, IgHV3-7, and IgHV3-48. In terms of IgHV gene mutational status, 63.8% of patients carried mutated rearrangements, whereas 22.4% of patients were assigned to stereotyped subsets (6.9% to major subsets and 15.5% to minor ones). The frequencies of NOTCH1, SF3B1, BIRC3 and MYD88 mutations were 9.6%, 7.2%, 1.2%, and 2.4%, respectively; however, the frequency of TP53 mutations was significantly higher (20.5%). Patients with TP53 mutations or del(17p), SF3B1 mutations and unmutated IgHV had a worse outcome compared to the other patients. In conclusion, the differences observed in IgHV properties suggest different pathogenetic factors implicated in the development of chronic lymphocytic leukemia, while the high frequency of TP53 mutations could in part explain the dismal outcome of these patients in Taiwan

Lack of Prognostic Significance of the Conventional and Novel Prognostic Markers in Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases.Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL.Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 and SF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis.Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p<0.0001), and of UM IGHV cases (55% vs 25%, p<0.0001). Analysis of recurrent mutations highlighted a higher prevalence of NOTCH1 mutations (26% vs 8%, p<0.0001) and of BIRC3 mutations (21% vs 1%, p<0.0001) in tris12 vs control group CLL. Conversely, no differences were found in the fraction of cases with TP53 mutations (3% vs 4%, p=0.38) or SF3B1 mutations (7% vs 7%, p=0.89), and in cases expressing ZAP-70 (62% vs 52%, p=0.09). The impact of these features on OS was tested by univariate analysis: in tris12 CLL, only the UM IGHV gene status predicted shorter OS (HR=2.37, p=0.0063), while none of the other characteristics reaching statistical significance as OS predictors (CD49d HR=1.36, p=0.36; CD38 HR=0.42, p=0.052; ZAP-70 HR=3.12, p=0.07; TP53 HR=2.33, p=0.25; NOTCH1 HR=1.40, p=0.22; SF3B1 HR=2.05, p=0.17; BIRC3 HR=1.22, p=0.61). On the other hand, in the control cohort, a significantly higher HR was found for CD49d (HR 3.11, p<0.0001) and CD38 (HR 3.45, p<0.0001) expression, TP53 (HR 2.88, p=0.0026), NOTCH1 (HR 3.57, p<0.0001), and SF3B1 (HR 2.57, p=0.0038) mutations, as well as for the UM IGHV gene status (HR=2.81, p<0.0001), but not for ZAP-70 expression and BIRC3 mutations (HR=1.74 and HR=1.91, p=0.15 and p=0.37, respectively).Conclusions. Mutational status of IGHV genes was the sole prognostic factor able to stratify OS in tris12 CLL. Despite the high frequency of NOTCH1 and BIRC3 mutations, as well as of CD49d and CD38 overexpression, these markers failed to convey a prognostic risk in tris12 CLL. The lack of a significant clinical impact for TP53 and SF3B1 mutations might be partly explained by the low number of mutated cases combined with a relative short follow up in our tris12 cohort. These findings are in keeping with the hypothesis of a different patho-biological mechanism occurring in tris12 CLL, which however remains to be fully elucidated. DisclosuresD'Arena:Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Prognostic Significance of Genomic Alterations in Mantle Cell Lymphoma

BackgroundWhole genome, whole exome, and targeted sequencing have identified various genomic alterations in mantle cell lymphoma (MCL). There remains limited data correlating genomic alterations with clinical outcome in MCL. We describe genomic alterations in MCL using a targeted sequencing platform and correlate these alterations with clinical outcome.MethodsGenomic DNA was isolated from formalin-fixed paraffin-embedded tissue specimens from 55 cases of MCL and screened for clinically relevant mutations by deep-coverage targeted sequencing. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 585 biologically significant cancer-related genes for DNA-Seq. Captured libraries were sequenced to high depth (Illumina HiSeq), averaging >300x. Twenty-eight tumor specimens had corresponding matched normal specimens that facilitated identification of somatic mutations with a high degree of confidence. For the remaining 27 unmatched cases, somatic mutations were inferred according to annotated-based rules. Briefly, non-synonymous variants were considered somatic if they met any of the following criteria: 1) previously reported in the COSMIC database at least 5 times or more, 2) previously reported in published MCL genomic data, or 3) truncating mutations including nonsense mutations, frameshift mutations and splicing mutations that are not at the 3’ end of the genes. The sequencing data were also used for detecting copy number changes including amplifications of established oncogenes or homozygous deletions of known tumor suppressor genes. Overall survival (OS) analyses were performed using Kaplan-Meier and log-rank tests.Results55 cases of newly diagnosed MCL treated at MSKCC from 1999 to 2006 with available specimens were analyzed. Median age of diagnosis was 59 years (range 24-79) with a male predominance (76%). Advanced disease (III/IV) was seen in 93% of cases and an elevated Ki-67 (>=30%) was noted in 41% of patients. Initial treatment included intensive therapy (chemotherapy + ASCT/Allo)(55%), radioimmunotherapy (131I-tositumomab followed by chemotherapy) (20%),bendamustine and an anti-CD20 monoclonal antibody (rituximab or ofatumumab) (11%), single agent anti-CD20 monoclonal antibody (5%), initial observation (7%), and other (2%).The most frequent alterations, above a level of 4%, are shown in Figure 1. The most commonly altered gene was ATM, a tumor suppressor involved in DNA damage repair, and observed in 42% of cases. In addition, known driver mutations in MCL were found, such as TP53 (16%) and CCND1 (11%). Also an amplification of CCND1 was observed in one patient. Alterations in chromatin modifying genes (e.g. KMT2D, MEF2B, WHSC1, SMARCA4) were common, occurring collectively in 38% of the cases. Recurrent alterations in NOTCH1 were identified in 9% of cases. Alterations in BIRC3 (7%) and TRAF2 (4%), key components of the alternative NF-ĸB pathway and mediators of apoptosis, were identified. Homozygous deletion of BIRC3 was also noted in one patient. Homozygous deletion of CDNK2A was noted in 9% of cases. Recurrent promoter mutations were observed in TERT (Telomerase Reverse Transcriptase) gene (13%), which encodes an enzyme involved in cell immortality.With a median follow-up of 7 years, the presence of TP53 mutations was significantly associated with inferior survival compared to TP53-unmutated patients (P<0.001), Figure 2. Notably, homozygous deletion of CDNK2A and mutations in NOTCH1 and KMT2D were not prognostically significant in our series.Conclusion:This analysis further describes the mutational landscape in MCL. Our results are congruent with prior data demonstrating the prognostic relevance of p53 mutations. Several of the genomic alterations identified may have therapeutic significance. For example, TRAF2 and BIRC3 mutations, in the alternative NK-κB pathway, are hypothesized to be associated with ibrutinib resistance. Loss of CDNK2A may confer sensitivity to PRMT5 inhibition. In future,we plan to analyze additional cases of MCL to elucidate the biologic and clinical significance of the observed genetic alterations with the aim of developing mechanism-based, biologically-targeted therapy in MCL. [Display omitted] [Display omitted] DisclosuresKumar:Celgene: Honoraria, Other: Scientific Advisory Board; Celgene: Research Funding; Adaptive Biotechnologies: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding. Zelenetz:Gilead Sciences: Research Funding.

Novel Associations Between Mutations, Prognostic and Clinical Parameters in Untreated Progressive CLL: Data from E1912, a Randomized Phase III Study of the ECOG-ACRIN Cancer Research Group

Sequencing studies have significantly improved our understanding of the CLL genetic landscape and allowed the identification of the first associations between specific mutations and clinical outcome. Hence, mutations in TP53, NOTCH1, SF3B1, BIRC3 and MYD88 are known to be associated with differential prognosis and have been incorporated in CLL risk stratification models. Despite the work performed on these 5 genes, several additional putative tumor-driver genes are recurrently mutated in CLL, but their clinical impact remains unclear. In order to gain further understanding, we performed targeted sequencing in baseline samples of 308 CLL patients (pts) participating in the North American Intergroup study (NAIG) designated as E1912 and led by ECOG. This trial is a randomized Phase III of Ibrutinib (Ibr) and Rituximab (R) vs FCR in previously untreated CLL. Besides the 5 genes aforementioned, we screened another 19 genes recurrently mutated in CLL, and 2 genes (BTK and PLCy2) recently associated with Ibr resistance. The CD5+/CD19+ B lymphocyte population of these pts was flow-sorted. All coding regions were amplified using customized oligos. Libraries were templated and enriched using IonOneTouch2 and IonOneTouch ES systems, respectively. Samples were run on an IonTorrent sequencer using 318TM chips. Variants were called using IonTorrent Somatic VariantCaller. VCF files were annotated using BioR, which provides annotation from NCBI/Ensembl, UCSC, dbSNP, HapMap, 1000Genomes, COSMIC, SIFT, and PolyPhen-2. Somatic variants with a Mapping Quality We found 511 SNVs (442 missense, 45 nonsense and 24 splice sites) and 115 indels. An average coverage of 770X depth was obtained, allowing the detection of minor subclonal mutations ( 1 mutation affecting one or more genes of the pathway. Similar observations were found in mutational hotspots of BIRC3 (30% of mutated cases showed >1 mutation), MED12 (18%), SF3B1 (7%), XPO1 (6%), and NOTCH1 (5%), suggesting the existence of genetic subclones showing convergent evolution in a substantial number of CLL. We found multiple associations between mutations and known prognostic and clinical parameters. These included mutations in NOTCH1 associated with +12 (p=0.005), and expression of ZAP70 (p=0.01), CD38 (p=0.001) and CD49d (p=0.004). Mutant BIRC3 was associated with +12 (p This study, performed on a large cohort of untreated but progressive CLLs, provides novel data about the mutation status of driver genes and their associations with clinical and prognostic parameters. Our data indicate that convergent evolution is a common event in CLL, affecting several driver genes. Furthermore, it reinforces that BTK and PLCy2 mutations are not found in Ibr-naive pts. Finally, this study provides the genetic groundwork for subsequent prospective comparative analyses of the clinical outcome for CLL cohorts treated with Ibr-R vs FCR in this phase 3 NAIG trial. Disclosures O9Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Fonseca:Novartis: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Novartis: Consultancy; Janssen: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Sanofi: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Bayer: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms. Shanafelt:Genentech: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Whole Exome Sequencing from Nine Independent Sites of Extraosseous Disease in a Single Patient with Relapsed Multiple Myeloma Show That Extramedullary Disease Arise through a Combination of Branched and Parallel Evolution

Introduction: Multiple myeloma (MM) is the second most common hematologic malignancy. Recent sequencing studies show evidence of massive genetic heterogeneity reflected in multiple parallel subclones already at diagnosis. Different subclones respond differently to given therapy. The role of treatment-driven subclonal skewing and intrinsic acquired mutations is poorly understood in the relapse setting. At relapse, the distribution of subclones throughout the bone marrow and at extramedullary sites is currently unknown. Methods: We performed a research autopsy on a single individual with IgG kappa MM who survived with multiple metastatic sites of disease for 10 years after diagnosis. Genomic DNA was extracted from histologically confirmed snap frozen normal tissue and nine independent sites of extraosseous disease. Whole exome sequencing (150X) was performed by the MSK Genomics Core. Data were filtered to remove germline polymorphisms and enrich for high quality somatic variants. Phylogenetics and subclonal analyses were performed using Treeomics and SCHISM.Results: The average on target coverage was 177X with 73% of bases covered a minimum of 100x. A total of 6348 somatic variants were initially identified. After filtering for high quality somatic variants, 1330 remained with 220 of these variants common to all MM samples and an average of 628 variants per sample. Annotation of high quality variants revealed potentially deleterious somatic mutations in NRAS, FAM46C, DYNC1, CREBBP, ATM, BIRC3, MGA, PPP6C and SMARCA4. Phylogenetics analyses (shown below) indicated the metastases arose through a combination of branched and parallel evolution, with the ATM mutation arising in one branch (one metastasis) that was distinct from a second clonal population containing the NRAS, FAM46C, BIRC3, CREBBP, DYNC1 and PPP6C mutations that were present in all eight other metastases. The MGA and SMARCA4 mutations further identified two additional subclones arising in from the NRAS/FAM46C mutant common ancestral clone. Clonality analyses independently supported this hierarchy by identifying NRAS and FAM46C as early clonal events and MGA and SMARCA4 as late events in the progression of this patient's disease. It also suggested a degree of subclonal mixing within each metastatic site.Conclusions: Using whole exome sequencing from nine independent sites of extraosseous disease in a single MM patient with relapse 10 years after initial diagnosis, we show that extramedullary disease arise through a combination of branched and parallel evolution. Two additional patients have also undergone research autopsy and results will be presented at the meeting. [Display omitted] DisclosuresLandau:Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx/Amgen: Research Funding; Spectrum Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Hassoun:Binding Site: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Takeda: Consultancy, Research Funding. Korde:Medscape: Honoraria. Landgren:Medscape Myeloma Program: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding; BMS: Honoraria; Merck: Honoraria; Celgene: Honoraria, Research Funding.

Targeting Ikkα in CLL: Inhibition of Non-Canonical NF-κb Signaling Decreases Survival and Proliferation of CD40L-Stimulated Primary CLL Cells

The stress-inducible transcription factor, NF-κB, plays an important in role in B-cell malignancies, regulating expression of a plethora of genes including those that drive proliferation, apoptosis and survival. Non-canonical (’alternative’) NF-κB signalling, resulting in processing of the p100 subunit to p52 and dimerization with RelB, is less well-studied than the canonical pathway. Previous studies on NF-κB signalling, particularly in CLL, showed that the RelA (canonical) subunit is highly expressed and associated with poor outcome. In multiple myeloma and lymphoma, mutations in genes that alter NF-κB activity (NOTCH1, BIRC3, MYD88, TRAF2/3) can promote cell survival, by increasing activation of the CD40 receptor or NIK (NF-κB-inducing kinase), resulting in constitutive non-canonical NF-κB signalling. More recently, a comprehensive study showed that recurrent mutations in TRAF3, BIRC3 and NFKB2, all of which regulate non-canonical NF-κB signalling, also occur in CLL (Puente XS et al, Nature 2015). Taken together, these data suggest that non-canonical NF-κB signalling is important in CLL and that this pathway represents a novel therapeutic target. We hypothesised that receptor-activated proliferation in CLL cells is reliant on non-canonical NF-κB-regulated gene transcription, and that targeting this pathway would decrease CLL cell survival. Since the IKKα kinase phosphorylates p100, which in turn leads to active p52/RelB dimers in the nucleus, it is a key driver of the non-canonical signalling pathway. We used cell line models and patient-derived CLL cells to evaluate a series of selective, first-in-class IKKα inhibitors.Initially, we assessed 20 novel IKKα inhibitors in MEC-1 (CLL-like, BIRC2/3 and TP53 deleted) and Maver-1 (mantle cell lymphoma, TRAF3 deleted) cell lines, as these are representative of B-cell malignancies with constitutive activation of non-canonical NF-κB. Compounds S and U were found to be potent (cell-free IC50 values < 50nM) and selective (e.g. >50-fold over IKKβ). In growth inhibition studies, compound S inhibited cell proliferation with GI50 values of 4.3 μM (MEC-1) and 2.7 μM (Maver-1) whereas compound U gave GI50 values of 0.47 μM (MEC-1) and 0.5 μM (Maver-1). We then examined selected cases from our CLL cohort. Western blotting of nuclear extracts showed that expression of NF-κB subunits in patient-derived CLL cells was heterogeneous: RelA was constitutively expressed in most cases, and although expression of the non-canonical subunits, p52 and RelB, varied, high levels of p52 were associated with high RelB expression, indicating that non-canonical signalling is active in CLL. The panel of 20 IKKα inhibitors were also tested in viability and apoptosis assays in patient-derived CLL cells, revealing LC50 values ranging from 0.5 μM (Compound U) - 5μM (48 hr exposure). Decreasing cell survival correlated with increased caspase 3/7 activation. To model the tumour microenvironment, we used CD40L-expressing fibroblasts in co-culture with primary CLL cells to stimulate NF-κB signalling and CLL cell proliferation. CD40L stimulation led to increased levels of nuclear RelB and p52 levels after 2-8 hrs co-culture, concomitant with activation of IKKα, as demonstrated by rapid phosphorylation of p100 subunit. Compound S was evaluated in CD40L-stimulated CLL cells where it was shown to inhibit p100 processing and decrease levels of nuclear RelB and p52. Compound S significantly reduced CD40L-stimulated proliferation of CLL cells following 7 day treatment (1μM), in a similar fashion to the clinically-used PI3Kδ inhibitor, Idelalisib, and was also effective at reducing proliferation of TP53-mutated patient-derived CLL cells. Compound S had little or no effect on accumulation of nuclear RelA (p65), indicating selectivity of these inhibitors for IKKα over IKKβ and suggesting that targeting receptor-stimulated IKKα in B cells may avoid ’global’ toxicity anticipated by use of IKKβ-targeting therapies.Future studies will examine the gene expression profile following IKKα inhibition in CLL, and also interrogate the underlying genotypes among patients that may drive NF-κB signaling. These data provide proof of concept that targeting non-canonical NF-κB signaling is a valid therapeutic strategy in CLL. DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease (MRD) Status in FCR-Treated CLL Patients at the End of Treatment Influences Progression Free Survival (PFS), Results of the Ctrial-IE (ICORG) 07-01/ CLL Ireland Study, with Mutational Analysis Providing Additional Insight

Fludarabine, cyclophosphamide and rituximab (FCR) therapy results in a complete remission (CR) rate of 72% and a median progression free survival (PFS) of 80 months in non-del (17P) CLL1. Achieving an MRD negative (MRD-ve) CR after completing therapy is an early surrogate marker for overall survival (OS) and PFS2. Specific genetic CLL subtypes determined by fluorescent in-situ hybridisation (FISH) analysis, immunoglobulin mutation IgVH, NOTCH1 and SF3B1 status determine response to chemotherapy3,4.We completed a multi-centre prospective study between 2008 and 2012, with a median follow up of 62.6 months using MRD status to determine length of therapy. Patients who achieved an MRD-veCR after 4 courses of FCR received no further therapy and the remaining patients completed 6 cycles of FCR. MRD status was tracked 6 monthly in patients who became MRD-veuntil MRD was detected. The genetic subtype was also analysed but did not influence treatment.Fifty-two patients {35M;17F, median age 61years (range 37-73)} were enrolled. Forty-six patients completed the MRD assessment after 4 cycles. Eleven patients discontinued assigned FCR therapy for the following reasons: prolonged cytopenia (4); non-compliance (1); autoimmune haemolytic anaemia (2); renal impairment (1); pleural effusion (1); not recorded (2).Eighteen (34.6%) patients achieved an MRD-veCR after 4 cycles and a further11 after 6cycles resulting in 29/52 (55.8%) MRD-veCRs in total. The median PFS was 72.3 months (95% Confidence Interval 61.3-84.1 months) and the median OS has not been reached. Patients who attained an MRD-veversusMRD+vestatus had a prolonged PFS (81.1 vs 46.2 months, p<0.0002). No difference in PFS was observed between patients reaching an MRD-veCR after 4 versus 6 cycles (median PFS81.1 vs 84.1 months,p=0.29).FISH results were available for 48 patients; del(13q) in 16/48 (33%), del(11q) in 15/48 (31%) no abnormality in 12/48 (25%), trisomy 12 in 4 (8%) and other abnormality in 1 patient. The IgVH status was unfavourable in 34/52 (65%), SF3B1 mutations were detected in 5/51 (9.8%) and NOTCH1 mutations in 10/52(19.2%) patients respectively, comparable to published studies of first-line treatment in CLL3,4.The median PFS for patients with good risk IgVH was not reached. Del(11q) did not impact on PFS (median PFS 66.5 vs 78.9 months, p=0.7301). SF3B1 and NOTCH1 mutated patients had a shortened PFS (median PFS 38.4 vs 71.1 months, p=0.038 and median PFS 62.4 vs 82.2 months p=0.0302, respectively).In conclusion abbreviated FCR therapy is effective for patients achieving MRD-veremission after 4 cycles. SF3B1 and NOTCH1 mutated patients had a short PFS and may benefit from alternative first-line treatment. This finding emphasizes the role mutational profiling will play in optimising and personalising therapy in CLL in the future.Reference:Tam C, O'Brien S, WierdaW, et al. “Long-term results of the fludarabine, cyclophosphamide and rituximab regimen as initial therapy of chronic lymphocytic leukemia” Blood 2008 Aug 15;112(4):975-80Böttcher S, Ritgen M, Fischer K, et al. "Minimal Residual Disease Quantification is an Independent Predictor of Progression-Free and Overall Survival in Chronic Lymphocytic Leukemia: A Multivariate Analysis From the Randomized GCLLSG CLL8 Trial" J Clin Onc 2012 Mar 20; 30(9):980-8.StilgenbauerS,SchnaiterA,PaschkaP, et al. "Gene mutations and treatment outcome in chronic lymphocyticleukemia: results from the CLL8 trial" Blood 2014 May 22;123(21):3247-54Chiaretti S, Marinelli M, Del Giudice I, et al."NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukaemia undergoing first-line treatment: correlation with biological parameters and response to treatment"LeukLymphoma 2014 Dec; 55(12):2785-92 [Display omitted] [Display omitted] [Display omitted] DisclosuresCrotty:BMS, Takeda, Novartis, Janssen, Roche: Honoraria. O'Dwyer:Glycomimetics: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

NF-κB activation in chronic lymphocytic leukemia: A point of convergence of external triggers and intrinsic lesions

The nuclear factor-κB (NF-κB) pathway is constitutively activated in chronic lymphocytic leukemia (CLL) patients, and hence plays a major role in disease development and evolution. In contrast to many other mature B-cell lymphomas, only a few recurrently mutated genes involved in canonical or non-canonical NF-κB activation have been identified in CLL (i.e. BIRC3, MYD88 and NFKBIE mutations) and often at a low frequency. On the other hand, CLL B cells seem 'addicted' to the tumor microenvironment for their survival and proliferation, which is primarily mediated by interaction through a number of cell surface receptors, e.g. the B-cell receptor (BcR), Toll-like receptors and CD40, that in turn activate downstream NF-κB. The importance of cell-extrinsic triggering for CLL pathophysiology was recently also highlighted by the clinical efficacy of novel drugs targeting microenvironmental interactions through the inhibition of BcR signaling. In other words, CLL can be considered a prototype disease for studying the intricate interplay between external triggers and intrinsic aberrations and their combined impact on disease evolution. In this review, we will discuss the current understanding of mechanisms underlying NF-κB deregulation in CLL, including micro-environmental, genetic and epigenetic events, and summarize data generated in murine models resembling human CLL. Finally, we will also discuss different strategies undertaken to intervene with the NF-κB pathway and its upstream mediators.

Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors.

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s).

Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia

AbstractGenomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients.

Clinical Impact of Clonal and Subclonal TP53, SF3B1, BIRC3, and ATM Mutations in Chronic Lymphocytic Leukemia

Abstract Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

Kinetics of CLL Subclonal Architecture: Spontaneous Disease Progression or Treatment-Induced Selection?

Recent advances in whole genome assessment have highlighted the presence of marked intraclonal heterogeneity in chronic lymphocytic leukaemia (CLL). It has been suggested that this heterogeneity plays an important role in disease progression and that treatment may facilitate the outgrowth of CLL subclones with a high proliferative advantage. Recent years have also seen the development of a number of novel therapeutic approaches for CLL, including a range of targeted treatments as well as immunochemotherapy. Consequently, there is a need to assess the effects that various treatments have on the subclonal architecture of CLL during disease progression as any changes could potentially influence future clinical decisions.In this study a cohort of 133 CLL tumours was analysed for the mutational status of 6 genes recurrently mutated in CLL; ATM, TP53, SF3B1, MYD88, BIRC3, and NOTCH1; by deep targeted sequencing with an allele depth of 3000-5000 reads. Mutation(s) of at least one gene was detected in 74 of these patients. Prior to treatment, mutations with allelic frequencies (AF) both greater than and less than 50% were identified for all genes. However, the dominant mutation of ATM, TP53, BIRC3 or NOTCH1, presented with an AF greater than 50% in the majority of cases, whereas SF3B1 and MYD88 mutations typically exhibited an AF less than 50%. Generally, there was frequent coincidence of ATM mutations with SF3B1 or BIRC3 mutations; NOTCH1 mutations often occurred concurrently with either ATM, BIRC3 or SF3B1. Occasionally, TP53 mutations were found concommitantly with SF3B1 mutations and less frequently with BIRC3 mutations, whereas sole MYD88 or TP53 mutations characterised another two subsets of patients. Additionally, BIRC3, SF3B1 and MyD88 mutations were mutually exclusive in this cohort, as were ATM and TP53 mutations, with the exception of 2 cases upon relapse. Time to first treatment (TTFT) and overall survival (OS) were shorter for patients with one or more mutated gene than those without a mutation in any of these six genes. Furthermore, the presence of mutations either in multiple genes or multiple mutations within a gene conferred a more inferior TTFT.To determine the impact of therapy upon the mutational status, 31 paired pre-treatment and relapse samples were assessed. The response to treatment was heterogeneous with AFs of mutations of all the genes both increasing and decreasing in various patients irrespective of the treatment regimen. The most frequent response observed in this cohort of patients was the appearance of novel, sometimes multiple mutations of TP53 which were undetected in pre-treatment samples. Similarly, we observed the emergence of one or more novel ATM and NOTCH1 mutations and less frequently BIRC3 and SF3B1 upon relapse in a subset of patients. Of note, changes in mutational status did not appear to be influenced by the type of treatment, chemotherapy (n=19) vs chemoimmunotherapy (n=12).Patient follow-up cannot determine whether changes in subclonal architecture are treatment-induced or a consequence of natural disease progression, thus we monitored the CLL subclonal architecture of 5 representative CLL cases during xenotransplantation by multiplexed-FISH analysis. In the absence of treatment, 3 of the 5 CLL xenografts faithfully recapitulated the subclonal architecture of the patient samples. Intriguingly however, in the remaining 2 untreated xenografts the subclonal architecture significantly deviated (P<0.0001) from that observed in the patients, one of which displayed a striking similarity with the subclonal selection detected in the patient following chlorambucil treatment. This suggested that the change in CLL subclonal architecture post-treatment could be a reflection of natural CLL progression.In summary, we conclude that subclonal architecture in CLL is complex, dynamic and impacts upon overall prognosis. Subclonal changes are not always a consequence of therapeutic intervention but may be caused by natural disease progression. Further studies, including in vivo modelling, are necessary to ascertain when subclonal selection in CLL is treatment-induced from that caused by natural disease progression. DisclosuresNo relevant conflicts of interest to declare.

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.

Oncogenic activity of BIRC2 and BIRC3 mutants independent of nuclear factor-κB-activating potential.

BIRC2 and BIRC3 are closely related members of the inhibitor of apoptosis (IAP) family of proteins and play pivotal roles in regulation of nuclear factor-κB (NF-κB) signaling and apoptosis. Copy number loss for and somatic mutation of BIRC2 and BIRC3 have been frequently detected in lymphoid malignancies, with such genetic alterations being thought to contribute to carcinogenesis through activation of the noncanonical NF-κB signaling pathway. Here we show that BIRC2 and BIRC3 mutations are also present in a wide range of epithelial tumors and that most such nonsense or frameshift mutations confer direct transforming potential. This oncogenic function of BIRC2/3 mutants is largely independent of their ability to activate NF-κB signaling. Rather, all of the transforming mutants lack an intact RING finger domain, with loss of ubiquitin ligase activity being essential for transformation irrespective of NF-κB regulation. The serine-threonine kinase NIK was found to be an important, but not exclusive, mediator of BIRC2/3-driven carcinogenesis, although this function was independent of NF-κB activation. Our data thus suggest that, in addition to the BIRC2/3–NIK–NF-κB signaling pathway, BIRC2/3–NIK signaling targets effectors other than NF-κB and thereby contributes directly to carcinogenesis. Identification of these effectors may provide a basis for the development of targeted agents for the treatment of lymphoid malignancies and other cancers with BIRC2/3 alterations.

Small Subclones Harboring NOTCH1, SF3B1 or BIRC3 Mutations Are Clinically Irrelevant in Chronic Lymphocytic Leukemia

Introduction. Ultra-deep next generation sequencing (NGS) allows sensitive detection of mutations and estimation of their clonal abundance in tumor cell populations. TP53 mutations identified by ultra-deep NGS significantly impact on survival of chronic lymphocytic leukemia (CLL) patients, independent of their representation in the tumor clone and even if restricted to a small fraction of leukemic cells.Purpose. Here we aim at assessing the frequency, prognostic impact and evolution during disease course of NOTCH1, SF3B1 and BIRC3 mutations identified by ultra-deep NGS in newly diagnosed CLL.Methods. The study was based on a consecutive series of 304 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; del13q/+12/del11q/del17p: 51/21/8/9%; median follow-up: 8.1 years). By ultra-deep NGS, TP53 mutations (exons 4-8) occurred in 15% of CLL with a median clonal abundance of 18% (range 0.2-95%). NOTCH1 (hg19 g.chr9:139390596-139390829), SF3B1 (exons 14, 15) and BIRC3 (exons 6, 9) mutations were screened on peripheral blood samples by amplicon-based ultra-deep NGS (454 Life Sciences) (average depth: 2437). A bioinformatic algorithm was applied to call non-silent variants out of background NGS noise. Variant calling by the algorithm was validated by Sanger sequencing or, if the variant was below the sensitivity threshold of Sanger sequencing, by both duplicate ultra-deep NGS and allele specific PCR (AS-PCR). Variant allele frequency (VAF) was corrected for tumor representation.Results. Ultra-deep NGS identified 46 NOTCH1 mutations (median VAF 24%; range: 1.4-64%) in 14% (43/304) CLL, 43 SF3B1 mutations (median VAF 16%; range: 0.5-48%) in 11% (35/304) CLL and 37 BIRC3 mutations (median VAF 5.6%; range: 0.2-47%) in 8% (26/304) CLL. In cases harboring more than one mutation (NOTCH1=3; SF3B1=2; BIRC3=5), the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. Out of the variants identified by ultra-deep NGS, a significant fraction of NOTCH1 (n=14; median VAF: 3.9%; range: 1.4-9%), SF3B1 (n=25; median VAF: 4.3%; range: 0.5-17%) and BIRC3 (n=30; median VAF: 1.4%; range: 0.2-6%) mutations were missed by Sanger sequencing because their VAF was below the sensitivity threshold of the assay (~10%). The molecular spectrum of these small subclonal mutations was highly consistent with that of mutations that were visible by Sanger sequencing. The overall survival of cases harboring solely small subclonal mutations of NOTCH1, SF3B1 and BIRC3 was similar to that of wild type cases and longer than that of cases with clonal lesions (Fig. 1). Outcome-driven statistical approaches (maxstat and recursive partitioning) were applied to identify the optimal cut-off of the VAF in order to test whether the clonal representation of mutations correlated with CLL survival. Application of these approaches to TP53 mutations failed in identifying a clear cut-off, suggesting that TP53 variants are relevant to the outcome of CLL independent of their VAF. Conversely, in the case of NOTCH1, SF3B1 and BIRC3 mutations, these approaches consistently identified 25%, 35% and 1%, respectively, as the best VAF cut-off values for outcome prediction (Fig. 1), thus suggesting that small subclones harboring NOTCH1, SF3B1 or BIRC3 mutations are clinically irrelevant. By multivariate analysis, NOTCH1 mutations >25% of VAF (HR: 1.8; p=.038), SF3B1 mutations >35% of VAF (HR: 2.9; p=.005) and BIRC3 mutations >1% of VAF (HR: 1.8; p=.044) were significantly associated with an increase in the hazard of death, after adjusting for TP53 lesions (HR: 2.9; p<.001), del11q (HR: 2.2; p=.007), +12 (HR: 1.3, p=.174) and del13q (HR: 0.8; p=.512). Among cases harboring small mutated subclones, longitudinal deep NGS of sequential samples documented the outgrowth of the variants to a clonal level in 1/5 cases for NOTCH1, in 5/12 for SF3B1 and in 3/10 for BIRC3. While selection of NOTCH1 and BIRC3 variants did not associate neither with disease feature nor with treatment, the outgrowth of subclonal SF3B1 mutations was restricted to cases showing unmutated IGHV genes (p=.015).Conclusions. Small mutated subclones of the NOTCH1, SF3B1 and BIRC3 genes appear to be clinically irrelevant. Mutations of NOTCH1 and SF3B1 require a substantial clonal representation to be harmful. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.