One-step molecular detection of the MYD88 L265P mutation by unlabeled probe genotyping analysis

Abstract Abstract 1307 Background. Mutation of MYD88 gene has recently been identified in activated B-cell like diffuse B-cell lymphoma, and enhanced JAK STAT and NF-kB signalling pathways. Whole exome sequencing study in Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. Although the genetic background is not fully deciphered in WM, the role of NF-kB and JAK STAT pathways has been demonstrated in WM; which underlying mechanisms of deregulation remain to be elucidated. We aimed to analyze MYD88 mutation in exon 5 and to characterize the clinical significance of this genetic alteration in 67 WM. Method. 67 patients (42 males, 25 females) diagnosed with WM were included in this study, along with 9 patients with chronic lymphocytic leukemia (CLL), 4 multiple myeloma (MM) and 9 marginal zone lymphoma (MZL) were also studied. Patients were untreated at time of BM collection and gave informed consent prior to research sampling. Clinical features, immunophenotypic markers using flow cytometry (Matutes score panel, CD38, CD138, CD27, CD80), conventional cytogenetic, FISH and SNP array data (n = 46) were analysed. B cells from bone marrow and T cells from blood were isolated respectively using B cell isolation kit and Pan T isolation kit (Myltenyi Biotech). For DNA sequencing of exon 5 of MYD88, the exon 5 of MYD88 gene was amplified from genomic DNA by PCR. The purified PCR products were directly sequenced in both directions using BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, CA, USA) and analyzed on the Applied Biosystems 3130xl Genetic Analyzer. Data were analyzed with SeqScape software version 2.5 (Applied Biosystems). Results. MYD88 L265P mutation (MYDmut) was observed in 79% of patients, including homozygous mutation in two patients (3%). MYD88 mutation was not identified in T lymphocytes isolated from 4 WM patients that confirmed MYD88 mutation was acquired in the tumoral cells. We haven't observed any other mutation on exon 5. We then sought for other mechanisms of MYD88 gene alteration, such as copy number alteration (CNA) and copy neutral –loss of heterozygosity (CN-LOH) also considered as an acquired UPD (uniparental disomy) at MYD88 locus. We found an UPD at MYD88 locus in solely one patient (2%), and haven't identified any deletion at 3p22. On the contrary, we observed a gain on chromosome 3 at 3p22 locus (including MYD88 gene) in 7/57 (12%) patients. Taking together, we identified alteration of the MYD88 locus in 85% of patients with WM, by either gain-of-function mutation (79%) or CNA (12%). Interestingly, we found gain on chromosome 3 more frequently in the MYDwildgroup than in the MYDmutgroup (p=0.02). Twenty one percent of the patients with WM had no mutation of MYD (MYDwild), and were characterized with a female predominance, a splenomegaly, gain of chromosome 3 and CD27 expression. We did not observed difference in terms of survival according to the MYD88 mutation status. MYD88 mutation was not related to deletion 6q, gain of 4, deletion 11q, deletion 17p, deletion 13q14 in our study. Interestingly, deletion 7q, a frequent cytogenetic aberration in marginal zone lymphoma, was rare in our series (4/57; 7%) and was independent of MYD88 mutation status (2 in the MYDwild and 2 in the MYDmut) (p=ns). No MYD88 L265P mutation was observed in CLL and MM. In MZL, 1/9 patient without M monoclonal component had a MYDL265p mutation. Conclusion. These results confirm a high frequency of MYD88 L265P mutation in WM that may become a useful biomarker for diagnostic in WM and may help better understand the physiopathogeny of WM. Disclosures: No relevant conflicts of interest to declare.

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Prevalence and Clinical Significance of the MYD88 (L265P) Somatic Mutation in Patients with Waldenström Macroglobulinemia, IgM-Monoclonal Gammopathy of Undetermined Significance or Other Mature B-Cell Neoplasms.

Blood ◽

10.1182/blood.v120.21.2667.2667 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2667-2667 ◽

Cited By ~ 1

Author(s):

Marzia Varettoni ◽

Luca Arcaini ◽

Silvia Zibellini ◽

Emanuela Boveri ◽

Sara Rattotti ◽

...

Keyword(s):

B Cell ◽

Marginal Zone ◽

Lymphoproliferative Disorders ◽

Marginal Zone Lymphoma ◽

Splenic Marginal Zone Lymphoma ◽

Specific Pcr ◽

Allele Specific ◽

Myd88 L265p Mutation ◽

Myd88 L265p ◽

Monoclonal Component

Abstract Abstract 2667 Waldenström Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by bone marrow infiltration by lymphoplasmacytic lymphoma associated with a monoclonal component of IgM type in the serum. WM is often preceded by an IgM monoclonal gammopathy of undetermined significance (IgM-MGUS). The cumulative probability of progression of IgM-MGUS to WM or to other lymphoproliferative disorders is approximately 1.5% per year. Other mature B-cell neoplasms such as splenic marginal zone lymphoma (SMZL) and B-cell chronic lymphoproliferative disorders (B-CLPD) can carry an IgM monoclonal component and should therefore be considered in differential diagnosis with WM. In a study based on parallel sequencing of the whole genome of lymphoplasmacytic cells and paired normal tissue from WM patients, Treon et al (Blood. 2011;118:Abstract 300) have identified a highly recurrent somatic mutation with oncogenic activity in the myeloid differentiation primary response (MYD88) gene, leading to a change from leucine to proline at position 265 of the aminoacid sequence [MYD88 (L265P)]. Targeted Sanger resequencing showed MYD88 (L265P) in 90% of WM patients, but only in a minority of patients with IgM-MGUS or other mature B-cell neoplasms such as SMZL. We developed an allele-specific PCR for the MYD88 (L265P) mutation, and studied 58 patients with WM, 77 with IgM-MGUS, 84 with splenic marginal zone lymphoma (SMZL) and 52 with B-cell chronic lymphoproliferative disorders (B-CLPD). DNA was obtained from bone marrow cells (n=204) and peripheral blood (n=67). The aims of this study were: i) to assess the prevalence of the mutation in WM, IgM-MGUS, SMZL, and B-CLPD; ii) to analyze the relationship between MYD88 (L265P) mutation and clinical phenotype; iii) to evaluate the impact of the mutation on the risk of progression from IgM-MGUS WM or other lymphoproliferative disorders. The MYD88 (L265P) mutation was detected in 58/58 (100%) patients with WM, either asymptomatic (n=39) or symptomatic (n=18), and in 36/77 (47%) patients with IgM-MGUS. In addition, it was detected in 5/84 (6%) patients with SMZL and in 3/52 (6%) with B-CLPD; of these MYD88 (L265P)-positive subjects, 4 SMZL and 2 B-CLPD patients carried a serum IgM monoclonal component, while the remaining B-CLPD patient carried a double (IgM and IgG) monoclonal component. Compared with IgM-MGUS patients with wild-type MYD88, those carrying MYD88 (L265P) had significantly higher levels of IgM (P<.0001), lower levels of IgG (P=.04) and IgA (P=.04), and higher incidence of Bence-Jones proteinuria at diagnosis (P=.002). During the follow-up, 9 patients with IgM-MGUS progressed to WM (7 cases) or to marginal zone lymphoma (2 cases). Using a case-control approach, the risk of evolution of patients with MYD88 (L265P) was significantly higher as compared to that of patients with wild-type MYD88 sequence (OR 4.7, 95% confidence interval 0.8–48.7, P=.047). In conclusion, the findings of this study indicate that: i) the allele-specific PCR we developed is able to detect the MYD88 (L265P) mutation in all patients with WM and in nearly half the patients with IgM-MGUS, and therefore represents a useful diagnostic tool; ii) MYD88 (L265P) is an uncommon molecular lesion in SMZL and in B-CLPD, but is associated with an IgM monoclonal component in the few positive patients, suggesting that some cases of B-CLPD might be included in the spectrum of WM; iii) in IgM-MGUS, the mutation is associated with greater disease burden and higher risk of disease progression, and therefore represents a useful prognostic marker. Disclosures: No relevant conflicts of interest to declare.

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Examination of Genetic Aberrations in Chlamydophila Psittaci negative MALT Lymphomas of the Ocular Adnexa

Blood ◽

10.1182/blood.v120.21.1569.1569 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1569-1569

Author(s):

Daxing Zhu ◽

Offiong F Ikpatt ◽

Sander R Dubovy ◽

Yasodha Natkunam ◽

Jennifer R Chapman-Fredricks ◽

...

Keyword(s):

B Cell ◽

Copy Number ◽

Chromosomal Alterations ◽

Dual Color ◽

Pathway Activation ◽

Myd88 L265p Mutation ◽

Igh Rearrangement ◽

Myd88 L265p ◽

A20 Gene ◽

Antigen Selection

Abstract Abstract 1569 Ocular adnexal mucosa associated lymphoid tissue lymphomas (OAMALTL) are the most common lymphomas of the eye. The etiology and pathogenesis of ocular adnexa MALT lymphomas (OAMALTL) are still unknown and the association with Chlamydophila psittaci (C. psittaci) has been shown in only some geographic regions. Only few small studies specifically examined for the presence of t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH, t(14;18)(q32;q21)/IGH-MALT1, t(3;14)(p13;q32)/FOXP1-IGH translocations or for TNFAIP3 (A20) mutations/deletions, which may contribute to the activation of canonical nuclear factor (NF)-kB pathway in OAMALTL. Herein we sought to comprehensively examine the frequency of these translocations as well as CARD11 and MYD88 (L265P) mutations, in addition to A20 mutations /deletions in a large cohort of C. psittaci negative OAMALTL. A total of 47 OAMALTL originating in the orbit (22), conjunctiva (19) and lacrimal gland (6) were used for analysis. Dual color fusion probes for t(14;18)(q32;q21) IGH-MALT1, t(11;18)(q21;q21) BIRC3-MALT1 and dual color break apart probes for MALT1, BCL6, and CEP18 (chromosome 18 centromere) were used for FISH analysis in all the analyzed tumors. Dual color break apart probe for IGH was used for selected tumors. Extracted DNA was used for PCR amplification and sequencing of the coiled-coil domain of CARD11, exon 5 of MYD88, in which the L265P mutation was previously reported in diffuse large B-cell and MALT lymphomas, and all the coding exons of the A20 gene. A20 gene copy number variation was analyzed by TaqMan Copy Number Assay (Applied Biosystems). DNA extracted from peripheral blood lymphocytes of 3 healthy volunteers and OCI-LY8 and Jeko-1 cell lines served as calibrator and positive controls for A20 deletion. CARD11 mutations were not found in all the analyzed tumors. The MYD88 L265P mutation was detected in 3 (6.4%) tumors. A total of 9 A20 mutations were identified in 7 (14.9%) tumors. One tumor harbored 3 distinct mutations (F149C; 1bp deletion in exon 3 and 2bp deletion in exon 5). Among the 9 detected A20 mutations, the majority (89%) would produce truncated proteins due to out-frame insertion/deletion (7), while one of these deletions was located in a known splicing site. Only 2 missense mutations were observed, including one in a tumor in which 2 concomitant deletions were also present (described above). A20 heterozygous deletions were detected in 7 (14.9%) tumors. There was no association between A20 mutations and heterozygous deletion in any of the analyzed tumors. None of the tumors harbored a concomitant A20 mutation/deletion and MYD88 L265P mutation. A total of 5 tumors harbored chromosomal alterations: additional copy of BCL6, most probably due to trisomy 3, in 2 tumors, an additional copy of IGH in 1, extra copies of both IGH and MALT1 in 1, and extra copies of IGH, MALT1, BIRC3 together with IGH rearrangement to unidentified partner in 1. t(14;18)(q32;q21) IGH-MALT1 and t(11;18)(q21;q21) BIRC3-MALT1 were not detected in any of the analyzed tumors. A20 mutations were detected in a tumor with extra signals of both IGH and MALT1 genes as well as in one of the tumors with an additional copy of BCL6. The tumor with complex chromosomal aberrations including the IGH rearrangement to an unidentified partner also harbored A20 deletion. 26 of the analyzed tumors previously were shown to exhibit evidence of antigen selection based on the analysis of IGHV mutation pattern (PLoS One. 2011;6(12):e29114). There was no association between antigen selection and A20 mutations/deletions, MYD88 (L265P) mutation and chromosomal alterations. Overall, our data suggest that translocations characteristic for MALT lymphomas are rarely observed in OAMALTL. The MYD88 (L265P) mutation is also uncommon, while most of the A20 mutations/deletions affect only one allele and thus most probably do not lead to NF-kB activation. This raises a question on what are the mechanisms for canonical NF-kB signaling pathway activation in OAMALTL and if it is indeed activated in these tumors. It is possible that NF-kB signaling pathway activation maybe due to B cell receptor signaling, as may be reflected by association with presence of antigen selection that is observed in major fraction of these tumors. Immunohistochemical studies addressing these questions are underway and will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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MYD88 Negative Waldenstrom Macroglobulinemia Has Distinct Clinical & Biological Features As Compared To Its MYD88 Mutant Counterpart

Blood ◽

10.1182/blood.v122.21.4299.4299 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4299-4299

Author(s):

Nikhil V Patkar ◽

Prashant Deshpande ◽

Russel Mascarenhas ◽

PG Subramanian ◽

Prashant Tembhare ◽

...

Keyword(s):

Bone Marrow ◽

Partial Response ◽

Conflicts Of Interest ◽

International Prognostic Index ◽

Post Treatment ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Abstract Introduction Waldenstrom Macroglobulinemia (WM) harbors a mutation in MYD88 gene (MYD88L265P) with frequencies varying from 67% to 91%. Although of diagnostic use its clinical significance in terms of prognosis and treatment response is unclear. We retrospectively analyzed WM for MYD88 L265P mutation, immunogenetic profile (presence of somatic hypermutations and biased gene usage) & correlated these with standard clinical variables including prognosis and patient outcome. Patients & Methods 32 cases WM (diagnosed as per WHO 2008/2001 criteria) were retrospectively accrued from 2007-2013. Genomic DNA extracted from bone marrow aspirate smears was subjected to an allele specific oligonucleotide PCR to detect the MYD88L265P mutation using fluorescently labeled primers followed by capillary electrophoresis. Immunogenetics was assessed in 29 patients. Clonal FR1/FR2 regions of the VH gene were amplified & sequenced. Sequence data was compared to the closest germline sequences on NCBI & IMGT databases. Laboratory variables (Hb, WBC, platelet, M Protein concentration, S. IgM, b2M level, S. Globulin, LDH, %of lymphoplasmacytic lymphocytes) were evaluated at baseline along with the International Prognostic Index (ISSWM). Response evaluation was done as per VIth International Consensus guidelines after treatment as well as at last follow up. 2-tailed Student's t-Test & Chi squared test were applied for statistical analysis. Results Median age was 60 years (range: 46-77), male predominant (87.5%).Majority of patients had cytopenia (90.6%) of one or more blood lineages. Median IWSSM was 3 (n=26). The median follow up was 21.5 months (range: 1 week to 82 months). Majority of patients were treated with cyclophosphamide/vincristine/prednisone ± rituximab (55.1%), followed in others by bendamustine/rituximab(13.8%) or fludarabine/cyclophosphamide/rituximab,(13.8%) or cyclophosphamide/thalidomide/dexamethasone (10.3%). MYD88 L265P mutation was found in 84.3% (27/32) of patients. The immunogenetic results here pertain only to samples with productive IGHV gene rearrangement [22/29 (∼76%) cases]. 96% of cases revealed somatic hypermutations. 59% of cases showed a biased use for the VH3 gene followed by VH4 (22.7%) and VH1 (18.18%). The commonest gene used was IGHV3-7 (27.3%) followed by IGHV1-18 (18.2%). Clinical features separating MYD88 negative from MYD88 mutated WM are seen in Table 1. MYD88 negative WM presented with lower number of infiltrating tumor cells in the bone marrow (p=0.05), older age (p=0.02) and had a lower IWSSM score at presentation (p=0.03) as compared to mutated WM. Majority of the MYD88 negative group were in VGPR,(very good partial response) or CR (complete response) (75%:VGPR/CR) post treatment as compared to MYD88 mutated patients [21%: VGPR/CR, 31.6%: PR (partial response): 26.3%, SD (stable disease):15.8%, PD (progressive disease):6.3%]. At the last follow up 44.4% of MYD88 mutated WM had PD where as no patient in MYD88 WT had changed their initial post treatment status. Two patients with MYD88 mutation died due to disease related complications. Conclusion Our data indicates that WM is a biologically heterogeneous subset dichotomized by MYD88 mutations. WM patients with MYD88 mutations present at younger age with high tumor burden in the bone marrow, high risk of progression and poor therapeutic response. Although limited in number, MYD88 negative WM patients were not associated with PD as compared to the mutated group. Overall MYD88mutation may be considered as an adverse prognostic factor in WM. Disclosures: No relevant conflicts of interest to declare.

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