Stephanie Poulain

MYD88 L265P mutation in Waldenstrom macroglobulinemia

Mutation of the MYD88 gene has recently been identified in activated B-cell-like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and nuclear factor κB (NF-κB) signaling pathways. A whole exome-sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-κB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM.

MYD88 L265P mutation contributes to the diagnosis of Bing Neel syndrome

Bing‐Neel syndrome (BNS), a rare neurological syndrome associated with Waldenström macroglobulinaemia (WM), is a direct involvement of the central nervous system by lymphoplasmacytoid cells characterized with an adverse prognostic. The MYD88 L265P mutation has been identified in the vast majority of patients with WM. The diagnosis of BNS is often challenging because of the variety of clinical presentations associated with difficult histological techniques. We hypothesized that identification of MYD88 L265P mutation in the cerebrospinal fluid (CSF) would contribute to the diagnosis of BNS in addition to imaging, flow cytometry and cytology. We identified MYD88 L265P mutation in the CSF and the bone marrow of all cases of BNS using quantitative polymerase chain reaction qPCR and Sanger sequencing. Copy neutral loss of heterozygosity including MYD88 was observed in one case. No mutation of CXCR4, CD79A and CD79B was observed in parallel. We further showed that monitoring the quantitative expression of MYD88 L265P mutation might be a useful molecular tool to monitor response to chemotherapy using qPCR. In conclusion, identification of MYD88 L265P mutation might be a new molecular‐based biomarker tool to add to the diagnostic and monitoring armamentarium for BNS.

Balancing Risk Versus Benefit in the Treatment of Waldenström's Macroglobulinemia Patients with Nucleoside Analogue-Based Therapy

Nucleoside Analogues (NA) are considered as appropriate agents in the treatment of Waldenströms Macroglobulinemia (WM). There are sporadic reports on increased incidence of transformation to high grade non-Hodgkin lymphoma (transformation to NHL) and development of therapy related-myelodysplasia/acute leukemia (t-MDS/AML) among WM patients treated with NA. Several studies have been conducted in Europe and in the United States to retrospectively examine the incidence of such events in WM patients. The incidences of transformation to NHL and t-MDS/AML ranged from 4.7% to 8%, and from 1.4% to 8.9%, respectively, and demonstrated an increased incidence of these late events among WM patients treated with NA. The effect of these secondary malignancies needs to be better evaluated in prospective studies, especially in young patients. These NA treatment-associated risks should not by themselves be used to justify avoidance of NA therapy for WM patients but should be used in considering risk versus benefit for a particular patient given the expanding options of therapy for WM patients.

Genome wide SNP array identified multiple mechanisms of genetic changes in Waldenstrom macroglobulinemia

SNP array (SNPa) was developed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) without copy number changes, CN‐LOH. We aimed to identify novel genomic aberrations using SNPa in 31 WM with paired samples. Methylation status and mutation were analyzed on target genes. A total of 61 genetic aberrations were observed, 58 CNA (33 gains, 25 losses) in 58% of patients and CN‐LOH in 6% of patients. The CNA were widely distributed throughout the genome, including 12 recurrent regions and identified new cryptic clonal chromosomal lesions that were mapped. Gene set expression analysis demonstrated a relationship between either deletion 6q or gain of chromosome 4 and alteration of gene expression profiling. We then studied methylation status and sought for mutations in altered regions on target genes. We observed methylation of DLEU7 on chromosome 13 in all patients (n = 12) with WM, and mutations of CD79B/CD79A genes (17q region), a key component of the BCR pathway, in 15% of cases. Most importantly, higher frequency of ≥3 CNA was observed in symptomatic WM. In conclusion, this study expands the view of the genomic complexity of WM, especially in symptomatic WM, including a potentially new mechanism of gene dysfunction, acquired uniparental disomy/CN‐LOH. Finally, we have identified new potential target genes in WM, such as DLEU7 and CD79A/B. Am. J. Heamtol. 88:948–954, 2013.

Expression of the multidrug resistance‐associated protein in myelodysplastic syndromes

In the myelodysplastic syndromes (MDS), P‐glycoprotein (P‐gp) expression is clinically associated with drug resistance, whereas the clinical significance of multidrug resistance‐associated protein (MRP1) is uncertain. Bone marrow from 56 patients with MDS, including six with refractory anaemia (RA)/RA with ringed sideroblasts (RARS), 23 cases of RA with excess blasts/in transformation (RAEB/T), four patients with chronic myelomonocytic leukaemia (CMML) and 23 cases of MDS having progressed to acute myeloid leukaemia (MDS‐AML), were studied. MRP1 expression was investigated by immunocytochemistry (ICC) and by flow cytometry using MRPm6 monoclonal antibody. The efflux test using calcein‐AM (CAM) ± probenecid to evaluate MRP1 activity was performed in ten of the 56 patients. Twenty‐eight of the 56 cases (50%) expressed MRP1. MRP1 expression was more frequent in MDS‐AML than in MDS (70% vs. 36%). The efflux test using CAM was positive in three out of the ten patients tested. The results were in agreement with expression of MRP1 in six cases, and were discordant in four cases (1 MRP‐/CAM+, 3 MRP+/CAM–). No correlation was observed between MRP1 expression and P‐gp, lung resistance‐associated protein (LRP) or CD34 expression, although there was a trend for more frequent MRP1 expression in P‐gp‐positive cases in MDS‐AML (P = 0·08). Ten of the 26 patients treated with intensive chemotherapy achieved complete remission including six out of 16 MRP1+ and four out of ten MRP1– cases (P = NS). In conclusion, MRP1 expression was correlated with disease stage in MDS in our study. As for P‐gp, discordant expression/function of MRP1 could be found in some cases, suggesting the existence of non‐functional transport proteins in MDS. MRP1 expression did not seem to be a prognostic factor in MDS in our experience.

Novel M-Component Based Biomarkers in Waldenström's Macroglobulinemia

Waldenstroms macroglobulinemia (WM) is an indolent B-cell lymphoma of the lymphoplasmacytic type accompanied by a serum IgM component. However, conventional IgM quantification lacks sensitivity, does not precisely reflect tumor burden of WM, and, although being the main marker for monitoring response to treatment, may not be accurate. New serum M-component based biomarkers were developed for routine practice in recent years, such as the Freelite® test and more recently the Hevylite test®. Studies have shown that Freelite was a prognostic marker for time to treatment in WM that helps monitoring disease response or progression. Hevylite measures IgMkappa and IgMlambda, separately, and might provide true quantitative measurement of the IgM M-spike. Although current data are preliminary, Hevylite® might replace the current technique to measure IgM M-spike in the years to come. We summarize herein studies conducted to delineate the role of these tests in WM.

Recommendations for the diagnosis and initial evaluation of patients with Waldenström Macroglobulinaemia: A Task Force from the 8th International Workshop on Waldenström Macroglobulinaemia

The diagnosis of Waldenström macroglobulinaemia (WM) can be challenging given the variety of signs and symptoms patients can present. Furthermore, once the diagnosis of WM is established, the initial evaluation should be thorough as well as appropriately directed. During the 8th International Workshop for WM in London, United Kingdom, a multi‐institutional task force was formed to develop consensus recommendations for the diagnosis and initial evaluation of patients with WM. In this document, we present the results of the deliberations that took place to address these issues. We provide recommendations for history‐taking and physical examination, laboratory studies, bone marrow aspiration and biopsy analysis and imaging studies. We also provide guidance on the initial evaluation of special situations, such as anaemia, hyperviscosity, neuropathy, Bing‐Neel syndrome and amyloidosis. We hope these recommendations serve as a practical guidance to clinicians taking care of patients with a suspected or an established diagnosis of WM.

High-Throughput Genomic Analysis in Waldenström's Macroglobulinemia

Single-nucleotide polymorphism array (SNPa) and array-based comparative genomic hybridization (aCGH) are among the most sensitive genomic high-throughput screening techniques used in the exploration of genetic abnormalities in Waldenströms macroglobulinemia (WM). SNP and aCGH allow the identification of copy number abnormalities (CNA) at the kilobase level thus identifying cryptic genetic abnormalities unseen by lower-resolution approaches such as conventional cytogenetic or fluorescence in situ hybridization (FISH). CNA were identified in nearly 80% of cases by aCGH that delineated in addition minimal altered regions. At gene level, remarkable findings affecting genes involved in the regulation of the NF-kB signaling pathways were identified, such as biallelic inactivation of TNFAIP3 and TRAF3. SNPa also allowed characterization of copy neutral losses such as uniparental disomies (UPD), which is an important and frequent mechanism of gene alteration in cancer cells. Herein, we summarize the current knowledge of WM genomic basis using these high-throughput techniques.

Update on therapeutic options in Waldenstrom macroglobulinemia

Waldenström macroglobulinemia (WM) is a B‐cell disorder characterized primarily by bone marrow infiltration with lymphoplasmacytic cells (LPCs), along with demonstration of an IgM monoclonal gammopathy in the blood. WM remains incurable, with 5–6 yr median overall survival for patients with symptomatic WM. The main therapeutic options include alkylating agents, nucleoside analogues, and rituximab, either in monotherapy or in combination. Studies involving combination chemotherapy are ongoing, and preliminary results are encouraging. However, there are several limitations to these approaches. The complete response rate is low and the treatment free survival are short in many patients, no specific agent or regimen has been shown to be superior to another, and no treatment has been specifically approved for WM. As such, novel therapeutic agents are needed for the treatment of WM. In ongoing efforts, we and others have sought to exploit advances made in the understanding of the biology of WM so as to develop new targeted therapeutics for this malignancy. These efforts have led to the development of proteasome inhibitors, of them bortezomib, several Akt/mTor inhibitors, such as perifosine and Rad001, and immunomodulatory agents such as thalidomide and lenalidomide. Many agents and monoclonal antibodies are currently being tested in clinical trials and seem promising. This report provides an update of the current preclinical studies and clinical efforts for the development of novel agents in the treatment of WM.

Expression of Lung Resistance Protein and Correlation with Other Drug Resistance Proteins and Outcome in Myelodysplastic Syndromes

The major vault lung resistance protein LRP is a cytoplasmic protein involved in drug resistance, especially in acute myeloid leukemia. We looked for LRP overexpression, using immunocytochemistry with LRP 56 monoclonal antibody, on marrow slides from 41 cases of myelodysplastic syndromes (MDS). LRP overexpression (LRP+) was defined by expression of LRP 56 in at least 20% of marrow blasts. LRP overexpression was seen in 19 (46%) cases. Concordant results between LRP overexpression and P-glycoprotein (PGP) expression were seen in 66% of the cases (p = 0.03), and discordant results (LRP+ and PGP-, or LRP- and PGP+) in 33% of the cases. No correlation was seen between LRP overexpression and FAB type, karyotype, CD34, p53 expression and bcl2 overexpression in blasts. Furthermore, in the 18 cases treated with anthracycline-AraC intensive chemotherapy and the 7 cases treated with low dose AraC, the response rate was not significantly different in LRP+ and LRP- patients. Survival was also similar in LRP+ and LRP- patients. In conclusion, LRP overexpression is probably more frequent in MDS than in de novo AML and, as in AML, is only partially correlated with PGP expression. In our experience, however, LRP was not a prognostic factor for response to chemotherapy and survival in MDS.