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In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 4092-4092

Abstract: Background. Waldenstrom macroglobulinemia (WM) is a B-cell malignancy characterized by bone marrow (BM) infiltration of clonal lymphoplasmacytic cells, which produce a monoclonal immunoglobulin M. MYD88L265P mutation may be considered as a founder event because of it high frequency in WM. WM cells may acquire additional genetic hits that may potentially promote disease progression: CXCR4 or CD79B mutations, copy number variation,…TP53 is a tumor suppressor gene that functions as regulator influencing cellular responses to DNA damage. Little is known regarding TP53 alteration in WM. Our aim was to screen TP53 mutation in a large cohort of WM at diagnosis to analyze the genomic landscape of WM using targeted next generation sequencing (NGS) and genome wide single nucleotide polymorphism array (SNPa) and to identify clinical and biological characteristics. Method. BM samples of 125 WM (mean age: 67 years) were analyzed at diagnosis. Tumoral DNA was extracted following CD19 B cell selection. TP53 mutations were analyzed by targeted NGS to scan the coding exons of TP53. MYD88L265P, CD79A, CD79B, and CXCR4mutations were analyzed by sanger sequencing and/or NGS. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 62 cases. CN-LOH (copy neutral- loss of heterozygosity) and CNA (copy number aberration) were mapped using console 3.02 software (Affymetrix). Flow cytometry was performed to assess P53 and p21 expression after nutlin3a exposition to characterize functional mutant of TP53. Viability and cell growth of treated cells were determined using the MTS assay. Results. We have identified TP53 mutations using NGS in 7.3 % of WM (6 non-sense, 3 frameshift mutations located in the DNA binding domain) (TP53mut WM). The mutation load of TP53 varied from 13% to 98.9% (mean: 62.0%) using the variant allele frequency in NGS. We next examined the effects of nutlin-3a which is an mdm2 inhibitor on WM patients CD19+ cells genotyped for TP53 mutation. Nutlin-3a increased the expression of p53 and p21 in TP53Wild WM patients using flow cytometry (n=6). In contrast, in TP53MutWM cells, no significant variation of p53, p 21 and viability using MTS assay was observed suggesting the presence of functional mutation of TP53. The minimal deleted region of 17p in 17p deleted (TP53Del) samples was mapped using SNP array and contained 79 genes, among which was systematically comprised the loss of TP53. A high correlation between TP53 mutation and deletion 17p (p 〈 106) was observed. One case of CN-LOH was observed at TP53 locus (1,6% of cases). Overall, we have identified alteration of TP53 locus including mutation, deletion and copy neutral loss of heterozigosity in 11, 2% of WM. Using SNP array, we found a relationship between deletion 17p, alteration of TP53 locus including mutation, UPD or del17p (TP53Alt) and TP53Mutand a greater frequency of genomic aberrations in WM compared toTP53wild (p=0.01, p=0.024 and p=0.06 respectively). A higher frequency of WM patients with more than 3 CNA identified by SNPa was observed in TP35Mut group (p=0.03) and del17p group (p 〈 0.00001). No association was observed between TP53Mut and CXCR4 and MYD88mutations. We thoughtto identify clinical and biological characteristics of WM according to TP53Mutand/or Del17pfeatures. With a median follow-up of 5 years, 33 (26%) patients had died. 69% of cases were treated. Front line therapy included rituximab-based regimens in 76%, alkylating agent in 78%, fludarabine in 6%. The WM with TP53alteration, irrespective of TP53Mut or del17p, displayed features of adverse prognosis in regards to higher serum levels of b2m (89% versus 40%, p=0.012), and also greater IPSSWM score 2 and 3 (50% versus 30%, and 43% versus 30%, p=0.041, respectively). Importantly, the presence of TP53alteration, irrespective of TP5Mut or del17p, was associated to poor outcome in overall survival in our series, TP53alteration (p=0.003), del17p (p=0.002), and TP53Mut(p=0.015). TP53 alteration prognostic value was independent of CXCR4 or MYD88L265Pmutations. Conclusion: A low frequency of TP53 mutation was observed in WM at diagnosis. We identified a genomic signature associated to their presence. In addition, a pejorative prognostic value of TP53 mutation was observed in WM highlighted the need of new therapeutic in this sub group of WM. Disclosures Leleu: TEVA: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.4092.4092

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: British Journal of Haematology, Wiley, Vol. 191, No. 3 ( 2020-11), p. 506-509

Type of Medium: Online Resource

ISSN: 0007-1048 , 1365-2141

URL: Issue

URL: Article

DOI: 10.1111/bjh.v191.3

DOI: 10.1111/bjh.17008

Language: English

Publisher: Wiley

Publication Date: 2020

detail.hit.zdb_id: 1475751-5

In: Blood, American Society of Hematology, Vol. 136, No. Supplement 1 ( 2020-11-5), p. 29-30

Abstract: Introduction: Although survival dependence on Bcl2 is a well-known aspect of the pathophysiology of chronic lymphocytic leukemia (CLL), the mechanisms of Bcl-2 dysregulation are incompletely understood. Recurrent translocations involving BCL2 and immunoglobulin genes, including t(14;18)(q32;q21) and variants such as t(2;18) or t(18;22), are classically observed in follicular lymphoma or germinal center diffuse large B-cell lymphoma (GC DLBCL), but are uncommon ( & lt;5%) in CLL and usually associated with an indolent clinical course. Here, we characterize the mutational landscape and the functional Bcl-2 family dependencies of BH3 proteins in BCL-2-rearranged (BCL2-R) CLL. We used a functional approach known as BH3 profiling which measures the proximity of a cell to the threshold of apoptosis ("priming") and identifies which anti-apoptotic proteins a cell depends on for survival. Methods: Clinically annotated primary samples from BCL2-R CLL patients identified by karyotype were obtained from the French Innovative Leukemia Organization network and Dana-Farber Cancer Institute. Primary samples from CLL without BCL2 rearrangement were used as a control (ctrl CLL). Next generation sequencing (NGS) was performed using a custom-designed panel of 29 genes, including among others: BIRC3, NOTCH1, FBXW7, MLL2, RAS pathway, SF3B1 and TP53. The mean coverage obtained was 2000X (limit of detection (LOD): 1%). Digital droplet PCR (ddPCR) was used to quantify NOTCH1 c.7544_7545delCT (LOD: 0.025%). Protein expression (Bcl2, Mcl1, Bim) was assessed by Western blot. Baseline BH3 profiling was performed as per Ryan et al., Bio Chem 2016. To mimic the lymph node microenvironment, viability assays were performed in co-culture with the stromal cell line NK.tert. Viability was assessed by AnnexinV/Hoechst staining. Ex vivo drug treatments included: BCL2i (inhibitor): venetoclax; MCL-1i: AZD5991, S63845 and BCLXLi: A133. Statistical analyses were by unpaired and paired t-test with a two-tailed nominal p ≤ 0.05 considered as significant. Results: In our cohort of 110 patients, the median age was 70 years old, and 79% were male. BCL2-R were t(14;18) in 77.2%, t(18;22) in 16.3% and t(2;18) in 6.3% of patients. The translocation involving BCL2 gene was isolated in 23.6% of cases, and was associated with trisomy 12 in 45.4% of patients. The most frequently mutated genes in this cohort were in the NOTCH pathway (NOTCH1 mutation: 43.6 %, mostly subclonal (mean of variant allelic frequency: 6.1%) and FBXW7: 4.5%)) and RAS pathway (KRAS, NRAS, BRAF: 9.1%). BCL2 mutations were observed in 22.8% of the 57 examined cases. No mutation previously described in venetoclax resistant CLL, such as F104L or G101V variant, were observed. Furthermore, MLL2 mutations were observed in 14.5% cases and were significantly associated with complex karyotype (p=0.01) and trisomy 12 (p=0.04). Others mutated genes were: BIRC3 (5.4%), TP53 (3.6%), SF3B1 (1%) and MYD88 L265P(1%). No mutations in EZH2, CREBBP or EP300 were found. In 15 CLL representative samples from each group (BCL2-R and ctrl), Bcl2 protein expression was significantly higher in BCL2-R CLL (ratio Bcl2/actin 0.94 vs 0.74, p=0.009) as was expression of the pro-apoptotic protein Bim (ratio Bim/actin: 2.059 vs 1.524, p=0.007). BH3 profiling demonstrated that BCL2-R CLL and ctrl CLL samples (n=23 in each group) had comparable overall priming (cyto-C release 66.1% vs 63.3%, ns) and Bcl-2 dependence (cyto-C release 75.4% vs 76.3%, ns). Both also had low dependence on Bcl-xL (cyto-C release 8.2% vs 8.8%, ns). In contrast, Mcl-1 dependence was found to be significantly lower in BCL2-R CLL (cyto-C release 15.6% vs 37.4%, p & lt;0.0001). Consistent with our BH3 profiling results, the activity of venetoclax and the Bcl-xLi (A133) did not differ significantly between the 2 groups (n=15). In contrast, both Mcl-1i were less active in the BCL2-R group: average viabilities after 24h treatment with AZD5991 were 76.4% vs 56.3% (p=0.006) and with S63845 77.3% vs 62.9% (p=0.02) in the BCL2-R vs ctrl group, respectively. Conclusion: The genomic landscape of BCL2-R CLL is characterized by a high frequency of trisomy 12, subclonal NOTCH and RAS pathway mutations, as well as BCL2 and MLL2 mutations. Protein expression, BH3 profiling and viability assays data are consistent with nearly exclusive dependence on Bcl-2. Our data suggest that Bcl-2 inhibition should be favored over Mcl-1 inhibition in BCL2-R CLL. Disclosures Herbaux: Roche: Consultancy, Honoraria, Research Funding. Laribi:abbvie: Honoraria, Research Funding; amgen: Research Funding; novartis: Honoraria, Research Funding; takeda: Research Funding. Ysebaert:Roche: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Morel:Janssen: Honoraria. Guieze:abbvie: Honoraria, Other: advisory board, travel funds; janssen cilag: Honoraria, Other: advisory board, travel funds; roche: Other: travle funds; gilead: Honoraria, Other: travel funds; astrazanecka: Honoraria, Other: advisory board. Brown:Sun: Research Funding; Acerta: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other; Invectys: Membership on an entity's Board of Directors or advisory committees, Other: DSMC; Gilead: Consultancy, Research Funding; BeiGene: Consultancy; Catapult: Consultancy; Dynamo Therapeutics: Consultancy; Eli Lilly and Company: Consultancy; Juno/Celgene: Consultancy; Kite: Consultancy; MEI Pharma: Consultancy; Nextcea: Consultancy; Novartis: Consultancy; Octapharma: Consultancy; Pfizer: Consultancy; Rigel Pharmaceuticals: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Loxo: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Janssen: Honoraria; AbbVie: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2020-139210

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Clinical Cancer Research, American Association for Cancer Research (AACR), Vol. 23, No. 20 ( 2017-10-15), p. 6325-6335

Abstract: Purpose: TP53 is a tumor-suppressor gene that functions as a regulator influencing cellular responses to DNA damage, and TP53 alterations are associated with pejorative outcome in most B-lymphoid disorders. Little is known regarding TP53 alteration in Waldenstrom's macroglobulinemia (WM). Experimental Design: Here, we have explored the incidence of TP53 alteration using Sanger sequencing and ultradeep-targeted sequencing in 125 WM and 10 immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS), along with the clinical features and the associated genomic landscape using single-nucleotide polymorphism array and mutational landscape in an integrative study. Results: Overall, we have identified alteration of TP53 locus including mutation, deletion, and copy-neutral LOH in 11.2% of WM. TP53 mutation was acquired in 7.3% of patients with WM at diagnosis, being absent in IgM MGUS, and was highly correlated to deletion 17p. No correlation with CXCR4 mutations was observed. Patients with TP53 alteration had a greater number of genomic abnormalities. Importantly, WM with TP53 alteration had a significantly shorter overall survival, particularly in symptomatic WM, and independently of the international prognostic scoring system for Waldenstrom macroglobulinemia (IPSSWM) score. Specific treatment for WM with TP53 may have to be studied. Nutlin-3a–targeted p53 signaling induced cytotoxicity preclinically, along with new compounds such as ibrutinib, PrimaMet, or CP31398 that bypass p53 pathway in WM, paving the path for future treatment-tailored options. Conclusions: Our results highlight the clinical significance of detection of TP53 alteration in WM to determine the prognosis of WM and guide the treatment choice. Clin Cancer Res; 23(20); 6325–35. ©2017 AACR.

Type of Medium: Online Resource

ISSN: 1078-0432 , 1557-3265

URL: Article

DOI: 10.1158/1078-0432.CCR-17-0007

Language: English

Publisher: American Association for Cancer Research (AACR)

Publication Date: 2017

detail.hit.zdb_id: 1225457-5

detail.hit.zdb_id: 2036787-9

In: Clinical Cancer Research, American Association for Cancer Research (AACR), Vol. 22, No. 6 ( 2016-03-15), p. 1480-1488

Abstract: Purpose: Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4mut) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia. Experimental Design: We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study. Results: We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4mut Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q. Conclusions: Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4mut, characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia. Clin Cancer Res; 22(6); 1480–8. ©2015 AACR.

Type of Medium: Online Resource

ISSN: 1078-0432 , 1557-3265

URL: Article

DOI: 10.1158/1078-0432.CCR-15-0646

Language: English

Publisher: American Association for Cancer Research (AACR)

Publication Date: 2016

detail.hit.zdb_id: 1225457-5

detail.hit.zdb_id: 2036787-9