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

Blood ◽  
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.

scholarly journals Primary vitreoretinal lymphomas display a remarkably restricted immunoglobulin gene repertoire

2020 ◽  
Vol 4 (7) ◽  
pp. 1357-1366
Author(s):  
Nabila Belhouachi ◽  
Aliki Xochelli ◽  
Myriam Boudjoghra ◽  
Claude Lesty ◽  
Nathalie Cassoux ◽  
...  
Keyword(s):  
B Cell ◽  
Central Nervous System Lymphoma ◽  
B Cell Lymphoma ◽  
Gene Repertoire ◽  
Immunoglobulin Gene ◽  
Heavy Chains ◽  
Vitreoretinal Lymphoma ◽  
Myd88 L265p Mutation ◽  
Myd88 L265p ◽  
Antigen Selection

Abstract Primary vitreoretinal lymphoma (PVRL) is a high-grade lymphoma affecting the vitreous and/or the retina. The vast majority of cases are histopathologically classified as diffuse large B-cell lymphoma (DLBCL) and considered a subtype of primary central nervous system lymphoma (PCNSL). To obtain more insight into the ontogenetic relationship between PVRL and PCNSL, we adopted an immunogenetic perspective and explored the respective immunoglobulin gene repertoire profiles from 55 PVRL cases and 48 PCNSL cases. In addition, considering that both entities are predominantly related to activated B-cell (ABC) DLBCL, we compared their repertoire with that of publicly available 262 immunoglobulin heavy variable domain gene rearrangement sequences from systemic ABC-type DLBCLs. PVRL displayed a strikingly biased repertoire, with the IGHV4-34 gene being used in 63.6% of cases, which was significantly higher than in PCNSL (34.7%) or in DLBCL (30.2%). Further repertoire bias was evident by (1) restricted associations of IGHV4-34 expressing heavy chains, with κ light chains utilizing the IGKV3-20/IGKJ1 gene pair, including 5 cases with quasi-identical sequences, and (2) the presence of a subset of stereotyped IGHV3-7 rearrangements. All PVRL IGHV sequences were highly mutated, with evidence of antigen selection and ongoing mutations. Finally, half of PVRL and PCNSL cases carried the MYD88 L265P mutation, which was present in all 4 PVRL cases with stereotyped IGHV3-7 rearrangements. In conclusion, the massive bias in the immunoglobulin gene repertoire of PVRL delineates it from PCNSL and points to antigen selection as a major driving force in their development.

MYD88 L265P mutation promoted malignant B cell resistance against T cell-mediated cytotoxicity via upregulating the IL-10/STAT3 cascade

2018 ◽  
Vol 64 ◽  
pp. 394-400 ◽  
Author(s):  
Huiying Qiu ◽  
Shenglan Gong ◽  
Lili Xu ◽  
Hui Cheng ◽  
Lei Gao ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Resistance ◽  
Myd88 L265p Mutation ◽  
Myd88 L265p

MYD88 L265P Mutation in Waldenstrom's Macroglogulinemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1307-1307 ◽  
Author(s):  
Stephanie Poulain ◽  
Christophe Roumier ◽  
Audrey Decambron ◽  
Aline Renneville ◽  
Charles Herbaux ◽  
...  
Keyword(s):  
B Cell ◽  
High Frequency ◽  
Marginal Zone ◽  
B Cell Lymphoma ◽  
Marginal Zone Lymphoma ◽  
Chromosome 3 ◽  
Homozygous Mutation ◽  
Mutation Status ◽  
Myd88 L265p Mutation ◽  
Myd88 L265p

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.

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 ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2667-2667 ◽  
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.

Genomic Comparison Of Clonal B-Cells In Waldenstrom Macroglobulinemia (WM) Versus IgM MGUS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 400-400
Author(s):  
Bruno Paiva ◽  
Luis A Corchete ◽  
Norma Gutierrez ◽  
María-Belén Vidriales ◽  
Irene Aires-Mejia ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Copy Number ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Significant Proportion ◽  
Molecular Profile ◽  
Healthy Donors ◽  
Myd88 L265p

Abstract It is hypothesized that similarly to multiple myeloma, also in WM there may be a continuum between IgM MGUS, smoldering (SWM) and symptomatic WM, rather than these entities being considered as separate. The very low frequency of MYD88 L265P initially reported in IgM MGUS suggested that this could be implicated in disease transformation. However, using sensitive ASO-PCR a significant proportion of patients of patients with IgM MGUS already harbors the MYD88 mutation. Thus, the molecular mechanisms driving the malignant transformation of WM remain largely unknown. Here, we used high-sensitive 8-color multidimensional flow cytometry (MFC) to detect and sort the specific B-cell clone in BM samples (N=31) from a total of 22 newly-diagnosed WM patients (8 symptomatic, 14 SWM) as well as 9 patients with IgM MGUS. The later 9 cases had negative BM biopsy, but light-chain restricted clonal B-cells (typically CD22low, CD25+, sIgM+, LAIR1-) were identified by MFC (median 1.74%, range 0.2%-7.04%). MYD88 L265P was detected on FACS-sorted (purity ≥97%) clonal B-cells from 9/9, 13/14 and 7/8 IgM MGUS, SWM and WM patients, respectively. We first compared the genomic profile of clonal B-cells through high density Cytoscan750K array. Overall, IgM MGUS, SWM and WM patients showed a median of 2, 1.5, and 3 copy number abnormalities (CNA)/case, respectively [defined by >25 consecutive imbalanced markers/segment, >100Kb genomic size and <50% overlapping variants with patient-paired control DNA (n=6), or unpaired DNA from BM normal B-cells from 20 healthy donors]. Whole chromosomal imbalances were detected in IgM MGUS (+18), SWM (+3, +12) and WM patients (+4, +12, +18, +19). Gain and deletion of chromosomal arms was also detected in the 3 disease stages: 3q+, 6q-, 8p-, 13q-, 17p-, 18q+ in IgM MGUS; 11q- in SWM; and 6q-, 17p-, 18q+, 22q- in WM. Thus, genomic imbalances typically observed in WM (3q+,6q- or 18q+) were already detectable in clonal B-cells from IgM MGUS patients. Trisomy 4 was not present, nor CNA at 4q33-34 (previously ascribed with increased susceptibility for IgM MGUS and WM). One minimal amplified region at 8q11.23 was noted in 6 of the 31 patients (19%). Median number of copy-number-neutral loss of heterozygosity (CNN-LOH) was also similar between IgM MGUS, SWM and WM (median of 3, 2, and 3 CNN-LOH/case, respectively). Of note, two IgM MGUS patients showed CNN-LOH in minimal deleted regions often detected in the aggressive forms of the disease such as 6q16.1and 6q25.3. In accordance to the genomic profiles, preliminary analysis of gene expression profiles (GEP; HumanGene 1.0ST) between FACS-sorted clonal B-cells from IgM MGUS, SWM and WM patients showed virtually no deregulated genes (SAM Excel add-in with a FDR q-value<10-5). Consequently, we grouped patients together (n=14) and compared them to normal BM B-cells from healthy donors. Moreover, taking into consideration the aberrant phenotypes of the Waldenström’s clone, a specific comparison was made between the GEP of clonal B-cells vs CD22+/CD25- normal B-cells (n=6) as well as the small subset of normal BM B-cells that display the typical CD22low/CD25+ WM phenotype (n=4). Clonal B-cells showed de-regulation of 776 genes (92 down- and 684 up-regulated) as compared to CD22+/CD25- normal B-cells. By enrichment analysis (Ingenuity Pathways), top upstream regulators such as IFNg, the B-cell receptor (BCR) complex, and the synovial apoptosis inhibitor 1 (SYVN1) were activated in clonal B-cells, while the IL1 receptor antagonist (IL1RN) was inhibited. Well-known genes such as PRDM1, CD27, IL2Rα (CD25) or TRAF3 were also up-regulated in clonal B-cells. Noteworthy, up to 27 genes over-expressed by clonal B-cells were already up-regulated in normal BM CD22low/CD25+ B-cells vs CD22+/CD25- normal B-cells. Accordingly, when compared to the CD22low/CD25+ normal B-cell counterpart, GEP of clonal B-cells was far less deregulated (185 genes being infra-expressed). In fact, genes such as IL1R2, TLR4, TNFRSF1A, IGF1R, FCER1G or TNFSF13B (target molecules of the NFKB and IL-6 pathways) were down-regulated in the WM clone vs CD22low/CD25+ normal B-cells. In summary, our results show that clonal B-cells from IgM MGUS patients already show a molecular profile that overlaps with that of WM, and suggest that the Waldenström’s clone may arise from normal CD22low/CD25+ BM B-cells. Disclosures: No relevant conflicts of interest to declare.

PREVALENCE OF MYD88 L265P MUTATION IN HISTOLOGICALLY PROVEN, DIFFUSE LARGE B-CELL VITREORETINAL LYMPHOMA

Retina ◽  
2016 ◽  
Vol 36 (3) ◽  
pp. 624-628 ◽  
Author(s):  
Harish Raja ◽  
Diva R. Salomão ◽  
David S. Viswanatha ◽  
Jose S. Pulido
Keyword(s):  
B Cell ◽  
Vitreoretinal Lymphoma ◽  
Myd88 L265p Mutation ◽  
Myd88 L265p ◽  
Large B Cell

scholarly journals Prognostic value of MYD88 L265P mutation in diffuse large B cell lymphoma via droplet digital PCR

2020 ◽  
Vol 22 (2) ◽  
pp. 1243-1256 ◽  
Author(s):  
Jing Niu ◽  
Zhiping Ma ◽  
Aijiang Nuerlan ◽  
Sijing Li ◽  
Wenli Cui ◽  
...  
Keyword(s):  
B Cell ◽  
Prognostic Value ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Large B Cell Lymphoma ◽  
Myd88 L265p Mutation ◽  
Myd88 L265p ◽  
Large B Cell

scholarly journals Distinctive Genomic Alterations in Testicular Diffuse Large B Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3655-3655
Author(s):  
Connie Batlevi ◽  
Franck Rapaport ◽  
Lu Wang ◽  
Andrew M. Intlekofer ◽  
Amanda R. Copeland ◽  
...  
Keyword(s):  
B Cell ◽  
Copy Number ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Snp Array ◽  
B Cell Lymphoma ◽  
Immune Recognition ◽  
Variant Allele Frequency ◽  
Large B Cell Lymphoma ◽  
Myd88 L265p

Abstract Background: Primary testicular diffuse large B cell lymphoma (DLBCL) is an uncommon malignancy portending a poor prognosis with increased risk of central nervous system disease. Phenotypically, most primary testicular lymphomas have a non-germinal center B-cell like (non-GCB) origin. To identify the genetic characteristics of testicular DLBCL, we evaluated DNA copy number and mutational profiling using SNP array and a next generation targeted sequencing platform. Methods: Twelve cases of testicular DLBCL with patient consent for tissue specimens and sufficient tumor tissue were retrospectively identified. Cell of origin was determined by Hans immunohistochemistry (IHC) model. We performed a custom, targeted deep-sequencing assay of 585 cancer genes (HemePACT) on matched tumor and normal pairs. Barcoded pools were sequenced on Illumina HiSeq 2500 to 500-1000x coverage per sample Sequencing was compared to a matched normal tissue control (N=10) if available or alternatively a pooled normal tissue control. We excluded all mutations either present at a high variant allele frequency in the matching germline samples, present in two databases of inherited variants (DBSNP and 1000 genomes) or present in one databases of inherited variants and absent from COSMIC. We evaluated copy number and allelic imbalance with an Affymetrix OncoScan SNP-array. IHC was performed for select genes. Results were compared to a panel of non-testicular DLBCL previously described (N=78). Results: The median age of the patients was 55.1 years (range 21.9-77.9). Patients had clinical stage IE (50%) and IV (50%) disease. All samples were sequenced from pre-treatment biopsies. Eleven of 12 patients were initially treated with R-CHOP chemotherapy, intrathecal methotrexate and radiation. Treatment history for one patient was unknown. We identified 124 mutations in 12 cases of testicular DLBCL. The most common mutation was MYD88 occurring in 10/12 patients (83%) with 6 mutations in non-GCB and 2 mutations in GCB (Fig 1A). The MYD88 L265P allele was most frequent and occurred in 9/12 patients (75%). The median MYD88 L265P variant allele frequency was 0.36 (range 0.07-0.51) with normal copy number status at that loci. In contrast, MYD88 mutations were less frequent in DLBCL without testicular involvement, 12/37 (32%) non-GCB and 3/41 (7%) GCB DLBCL, p<0.05 by Fisher's t-test (Fig 1B). Furthermore, mutations in CD79B were significantly more common in testicular DLBCL (5/12, 42%) versus non-testicular DLBCL (7/78, 9%). Concurrent mutations affecting the BCR receptor pathway was noted in 10/12 patients (83%): CD79B (5/12, 42%), TNFAIP3 (1/12, 8%), CARD11 (1/12, 8%) (Fig 1A). Frequency of TNFAIP3, CARD11 was not statistically significant between testicular versus non-testicular DLBCL. Other commonly mutated pathways include epigenetics (10/12, 83%) and immune recognition (6/12, 50%). Deregulation of immune recognition was noted by HLA-A (3/12, 25%) and beta-2-microglobulin (2/12, 17%) mutations as well as loss of HLA locus in 8/10 samples (80%) (Fig 1C). IHC revealed 6/10 (60%) cases with no MHC Class I expression of the tumor cells. MHC Class I negative cases also lacked B2M expression (3/6) or displayed mislocalization of B2M to the cytosol (3/6). PD-L1 was expressed by lymphoma cells as assayed by IHC in 1/10 (10%) cases but no amplification or mutation was identified. No copy gains of the 9p24.1, PD-1, PD-L1, PD-L2 locus were identified via HemePACT or SNP array. Although mutations in CDKN2A/B were not identified in HemePACT, SNP array confirmed loss of CDKN2A/B at the 9p21 loci in 3/10 cases (30%). Conclusion: Targeted genomic sequencing and SNP array analysis have identified a distinctive genetic pattern with alterations of MYD88, BCR pathway mutations, and immune recognition deficiency in testicular DLBCL compared to non-testicular DLBCL. These findings may have implications in guiding the design of future treatment strategies for testicular DLBCL. Disclosures Younes: Novartis: Research Funding; Janssen: Honoraria; Johnson and Johnson: Research Funding; Takeda Millenium: Honoraria; Seattle Genetics: Honoraria, Research Funding; Sanofi-Aventis: Honoraria; Bayer: Honoraria; Bristol Meyer Squibb: Honoraria; Curis: Research Funding; Celgene: Honoraria; Incyte: Honoraria.

scholarly journals Clinical Significance of MYD88 Mutation in Patients with Diffuse Large Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3881-3881 ◽  
Author(s):  
Ritsuko Seki ◽  
Takashi Okamura ◽  
Koteda Satoko ◽  
Kuniki Kawaguchi ◽  
Kei Noumura ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
B Cell ◽  
Clinical Significance ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Probe System ◽  
Allele Specific ◽  
Myd88 L265p Mutation ◽  
Myd88 L265p

Abstract Mutation of the MYD88 has recently been identified in activated B cell like diffuse large B cell lymphoma (DLBCL) and enhanced cell proliferation systems such as JAK-STAT and NF-kB signaling pathways. However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive and an automatic method utilizing guanine-quenching probes (QP) to detect mutation and investigated the relationship between MYD88 L265P mutation and clinical significance. We amplify a DNA fragment including the mutation to intend for by PCR and associate it with Q-probe with complementary sequence, using the temperature that Q-probe dissociates varying according to a conformity degree of the complementarity sequence. We judge it by detecting the fluorescence to be provided by dissociation. Results were obtained from 1ul of DNA solution(10ng) within 90 min by the method. Detected mutations were identical between QP method and allele-specific PCR (AS-PCR).Eighty-nine patients with a diagnosis of de novo DLBCL made between 1999 and 2014, and treated with CHOP or R-CHOP therapy. We retrospectively analyzed the outcome of 89 patients (age range; 21-88 and 59% were female). The median follow-up time was 4.4 y. Survival analyses were performed using the Kaplan-Meier method. None of the patients had a known history of human immunodeficiency virus infection. MYD88 L265P mutation was both assessed by Q-probe system that can detect low levels of mutant DNA and allele-specific TaqMan polymerase chain reaction assay. We performed the direct sequence method using 3130 Applied Biosystem Genetic Analyzer as antithesis. The cell-of-origin was determined based on immunohistochemical (IHC) stains for CD10, BCL-6 and MUM-1 by Hans' algorithm. MYD88 L265Pmutation was detected in 25.8% (23/89) in various tissues of DLBCL. MYD88 mutations occurred more frequently in males (P<0.05), cases without B symptoms (P<0.05). MYD88 mutation was infrequent in DLBCL arising in lymph nodes (10.6%), but more frequently found in extranodal sites such as testes (83%, 5/6), nasal (75%,9/12), central nervous system (50%,2/4), and leg (100%,1/1). In agreement with recent studies, we found no mutated cases among gastric cases. As somatic mutations in MYD88 was reported to be the most frequent alterations found in non-GCB type, we further analyzed GCB or non-GCB type by IHC. MYD88 mutations were predominantly observed in the non-GCB type (74%, 17/23), compared with 26%, 6/23 in GCB type. Overall survival (OS) for 3 years were 84.2% and 70.2% in patients with wild-type MYD88 and in MYD88 mutation group (P=0.366), respectively. Progression-free survival (PFS) for 3 years, 76.9% and 64.3% in patients with wild type and in mutated group (P=0.156), respectively. However, all four cases with CNS relapse had this mutation, 2 originated from testis, and remained 2 from lymph nodes. Our results confirm the remarkable site-specific occurrence of MYD88 mutation. In addition, Q-probe system for detection of MYD88 mutation was very useful because of its sensitivity and in the case who obtained only a small amount of biopsy specimen. MYD88 L265Ppromotes survival of malignant lymphoid cells through several mechanisms. Further large scale study should be necessary for more understanding of biological and clinical significance of DLBCL patients with MYD88 mutation. Disclosures No relevant conflicts of interest to declare.

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