Diagnosis Of Waldenstrom's Macroglobulinemia Research Articles

Detection Of MYD88 L265P In Peripheral Blood Of Patients With Waldenström's Macroglobulinemia and IgM Monoclonal Gammopathy Of Undetermined Significance

Waldenström's macroglobulinemia (WM) is characterized by bone marrow (BM) infiltration with lymphoplasmacytic cells and production of an IgM paraprotein. Lymphocytosis is uncommon in WM, and many patients exhibit peripheral B-cell lymphopenia. Recently, by whole genome sequencing, we identified a highly recurrent somatic mutation (L265P) in MYD88. Using real-time allele-specific polymerase chain reaction (AS-PCR), we demonstrated the presence of MYD88 L265P in 93% and 54% of patients with WM and IgM monoclonal gammopathy of undetermined significance (MGUS), respectively (Xu et al, Blood 2013).To clarify the feasibility of using real-time AS-PCR assay to detect MYD88 L265P in peripheral blood, we first examined unselected mononuclear cells from peripheral blood (PB) samples of 88 patients with untreated WM. 81/88 (92%) of these patients were MYD88 L265P positive by bone marrow (BM) examination of CD19 selected B-cells; Of the 81 BM-MYD88 L265P positive patients, 32 (40%) demonstrated MYD88 L265P by AS-PCR in unselected PB mononuclear cells (PBMC) obtained at the time of BM examination. To enhance the ability to detect MYD88 L265P in PB samples, we next used AS-PCR in CD19 selected PBMC obtained from 198 WM patients which included untreated and previously treated patients. Among untreated patients, 106/118 (89.8%) were positive for MYD88 L265P in CD19 selected PB, whereas among previously treated patients, 55/80 (68.8%) were positive for MYD88 L265P (p=0.0002 vs. untreated). Simultaneously analyzed PB and BM samples from 65 untreated WM patients demonstrated positivity for MYD88 L265P in 58/65 (89.2%) and 55/65 (84.6%) of CD19 selected BM and PB samples, respectively. These findings yielded a sensitivity of 94.8%, specificity of 100%, positive and negative predictive values of 100% and 70%, respectively for AS-PCR testing of PB CD19+ cells for MYD88 L265P in untreated WM patients. In addition, we analyzed 12 untreated IgM MGUS patients with paired sampling of CD19 selected PB and BM cells. 6/12 (50%) IgM MGUS patients were positive for MYD88 L265P in BM CD19+ cells, with 5/6 (83%) of these positive patients demonstrating MYD88 L265P by AS-PCR in CD19 selected PB samples.The overall results demonstrate a high concordance of MYD88 L265P status between PB and BM CD19 selected samples, particularly in untreated WM patients. The detection of MYD88 L265P by CD19 selected PB AS-PCR examination provides a convenient and less invasive method to support the diagnosis of WM and IgM MGUS. Disclosures:No relevant conflicts of interest to declare.

Waldenström macroglobulinemia with cryoglobulinemia revealed by amorphous deposits on blood smear

We report the case of an 86-year-old woman who presented with a 2-month history of anemia (9 g/dL) and weight loss. She had a medical history of arthrosis and hyperuricemia. Blood tests showed a normocytic aregenerative anemia with mean corpuscular hemoglobin concentration (MCHM) of 31.6 %, platelets 217 9 10/L, white blood count 3.3 9 10/L with lymphopenia (0.66 9 10/L) associated with elevated CRP (62 mg/L) and hypoproteinemia (58 g/L). TSH and creatinine were normal. Ultrasound and computed tomography revealed a hepatosplenomegaly, and positron emission tomography showed increased uptake in a right inguinal lymph node, biopsy of which revealed no tumor infiltrate. Examination of the blood smear showed amorphous lightly basophilic deposit mimicking white cells or platelet aggregates. The deposits had the same color and appearance as immunoglobulin inclusion in Mott cells, suggesting cryoglobulinemia. Laboratory tests confirmed the presence of cryoglobulin, characterized as an IgM lambda monoclonal protein (3.5 g/L). Due to the lack of evident etiology of this anemia, a bone marrow aspiration was done; smear examination revealed significant infiltration (83 % of the cells) by lymphoplasmocytoid cells which, with the cryoglobulinemia, permitted the diagnosis of Waldenstrom macroglobulinemia. Revelation of lymphoid hemopathy by cryoglobulin precipitates on blood smear remains rare and atypical [1]. The presence of cryoglobulin frequently results in erroneous WBC or platelet counts with pseudoleukocytosis or pseudothrombocytosis [2], whereas in our case

Improved accuracy of discrimination between IgM Multiple Myeloma and Waldenström Macroglobulinaemia by testing for MYD88 L265P mutations

Improved accuracy of discrimination between IgM Multiple Myeloma and Waldenström Macroglobulinaemia by testing for <i><scp>MYD</scp>88</i> L265P mutations

IgM Monoclonal Gammopathy in Gastric MALToma

An 80-year-old male with a 3-year history of IgM kappa paraproteinemia and a presumed diagnosis of Waldenstrom's macroglobulinemia (WM) presented as a new patient to establish care. He had initially presented 3y ears ago with 6 months of intermittent fevers and chills occurring approximately twice monthly. Diagnostic work-up at that point had included positron emission tomography-computed tomography (PET-CT) and MRI, both of which were negative. No esophagogastroduodenoscopy (EGD) was performed as part of the work-up. Blood work revealed an IgM paraproteinemia of 492 mg/dl. He was further evaluated by hematology and rheumatologyphysicians who gavea diagnosis of WM for his paraproteinemia and familial Mediterranean fever (FMF) for his intermittent fevers and chills. The FMF was clinically diagnosed after demonstrating symptom resolutionwith a trial of colchicine. Gene testing was not performed. With the resolution of the fever and chills, management consisted of interval trending of the IgM paraproteinemia. Over a 3-year

Waldenstrom macroglobulinemia presenting with pancreatic mass: a case report and review of literature.

Waldenstrom macroglobulinemia is a rare lymphoplasmacytic lymphoma characterized by a wide range of clinical presentations related to direct tumor infiltration and the production of IgM. Most commonly it presents with cytopenia, hepatosplenomegaly, lymphadenopathy, constitutional symptoms, and hyperviscosity syndrome.We report a case of Waldenstrom macroglobulinemia in an 60-year-old female who initially presented with intermittent abdominal pain. The patient had no peripheral lymphadenopathy. On extensive investigation she was found to have pancreatic mass. The diagnosis of Waldenstrom macroglobulinemia was established after cytomorphology and immunohistochemical analysis of the patient's bone marrow revealed the presence of a lymphoid/lymphoplasmacytoid-like bone marrow infiltrate along with an elevated serum IgM level. The patient responded both clinically and serologically to chemotherapy. This case is unusual because the patient lacked all common clinical features of Waldenstrom macroglobulinemia with exception of anemia.To our knowledge this is the first report of a patient with Waldenstrom macroglobulinemia presenting with a pancreatic mass adding to the spectrum of clinical presentations seen in this disease. This adds to the wide variety of gastrointestinal related clinical presentations of Waldenstrom macroglobulinemia and points to the need for considering Waldenstrom macroglobulinemia along with other lymphoid neoplasms in the differential diagnosis of pancreatic lesions.

Waldenström Macroglobulinemia

The definition of Waldenström macroglobulinemia (WM), originally described in 1944, has been refined substantially over time. The current fourth edition of the World Health Organization of lymphoid neoplasms, in large part, adopted criteria proposed for WM at a consensus conference in 2002. WM is defined as lymphoplasmacytic lymphoma involving the bone marrow associated with a serum immunoglobulin (Ig) M paraprotein of any concentration. Morphologically, WM is composed of a variable mixture of lymphocytes, plasmacytoid lymphocytes, and plasma cells. Immunophenotypically, the neoplastic cells express monotypic IgM and light chain: B lymphocytes express pan-B-cell antigens and surface Ig are usually negative for CD5 and CD10; and plasma cells are typically positive for CD138, CD38, CD45, cytoplasmic Ig, and CD19 (in a substantial subset of cases). The putative cell of origin of WM is a postantigen selected memory B-cell that has undergone somatic hypermutation. The most common cytogenetic abnormality in WM is del(6q), usually in the region 6q23-24.3, present in 40% to 50% of cases. IGH gene translocations are rare and recurrent chromosomal translocations or gene aberrations have not been identified in WM. Here, we provide a historical perspective of WM, review clinical and pathologic aspects of the disease as it is currently defined, and discuss some practical issues in the differential diagnosis of WM that pathologists encounter in the signout of cases.

Temporal and geographic variations of Waldenstrom macroglobulinemia incidence

Waldenstrom macroglobulinemia (WM) is a non-Hodgkin lymphoma (NHL) subtype. Little is known about the incidence and trends for this disease in the United States. Twenty-year data from the Surveillance, Epidemiology, and End Results (SEER) program were used for this study. SEER*Stat was used for data analysis. Of the 95,797 cases of NHL diagnosed between 1988 and 2007 in 9 SEER registries, 1835 (1.9%) were new cases of WM. Median age at diagnosis of WM was 73 years. The overall annual age-adjusted incidence was 0.38 per 100,000 persons per year, which increased with age, ranging from 0.03 in patients aged <50 years to 2.85 in patients aged ≥80 years. The incidence of WM was higher in men (0.54) than in women (0.27; P < .001) and was higher in whites (0.41) than in African Americans (0.18) or other races (0.21; P < .05). The annual percentage change for the whole population was 1.01% (P > .05). The annual percentage change was 1.21% for whites (P < .05) and 0.80% (P > .05) for nonwhites. Significant annual percentage change increases were seen in the group aged 70 to 79 years (1.24%; P < .05) and in 3 geographic registries (P < .001). Although the overall incidence of WM remained steady over time, significant increases in incidence were seen over the past 20 years in whites, in those aged 70 to 79 years, and in 3 geographic registry areas.

Clues to the Pathogenesis of Waldenström Macroglobulinemia and Other Monoclonal IgM Disorders Provided by the Analysis of Immunoglobulin Heavy Chain Gene Rearrangement and Clustering of B-Cell Receptors,

Abstract 3680In B-cell malignancies, biased usage of immunoglobulin heavy chain variable (IGHV) genes and stereotyped clusters of immunoglobulin receptor suggest that a limited set of antigens or superantigens are involved in malignant transformation. In the present study we analyzed the IGH rearrangements of a large series of patients with Waldenström Macroglobulinemia (WM), IgM-monoclonal gammopathies of undetermined significance (IgM-MGUS) and IgM-related disorders (IgM-RD), with the aim to characterize IGH repertoire and to search for clusters of stereotyped receptors.A total of 123 patients with IgM monoclonal gammopathies, including 59 WM, 55 IgM-MGUS and 9 IgM-RD underwent IGHV amplification and direct sequencing. The median age of patients was 63 years (range: 32–86). Diagnosis of WM, IgM-MGUS and IgM-RD was made according to the consensus criteria proposed at the 2nd International Workshop on WM. All IGHV-D-J rearrangements were analyzed using the IMGT databases and the IMGT/V-QUEST tool to identify HCDR3 aminoacid (AA) sequences. HCDR3 sequences were aligned with each other and compared to 28,400 HCDR3 sequences from public databases (EMBL, NCBI, IMGT/LIGM-DB) and from unpublished multi-laboratory databases. Alignments were performed using the multiple sequence alignment software ClustalX (2.0). Criteria for including sequences in a cluster were: >60% aminoacid identity, IGHV gene belonging to the same clan, identical HCDR3 length and same junction residues and/or same IGHD-J genes.A productive monoclonal IGHV-D-J rearrangement was obtained in 99/123 patients, including 55 WM, 37 IgM-MGUS and 7 IgM-RD. IGHV genes were mutated in 94/99 patients (95%). The median somatic hypermutation rate was 93.3% (range 85.5%-97.9%). Four cases showed >98% identity with the germline sequences, whereas one single WM sample showed 100% identity with the germline sequence. When compared with the normal mature B-cell repertoire, a significant over-representation of the IGHV3 family and a significant under-representation of the IGHV1 and IGHV4 families were observed both in WM (87%, 7% and 2% respectively) and in IgM-MGUS (78%, 8% and 8% respectively). IGHV3-23 was the gene most commonly used (29%). Analysis of IGHV3-23 sequences for AA changes in the positions involved in superantigen recognition by IGHV3 subgroup genes showed that the majority of cases had two or more non-permissive AA changes that compromise the capacity to mediate superantigen recognition and binding. The frequency of novel N-glycosylation sites, introduced by the somatic hypermutation process, in this series of patients (12%) was not statistically different from that observed in normal memory B-cells, suggesting that WM, IgM-MGUS and IgM-RD cells do not need to interact with stromal GC environmental elements. The median HCDR3 length was 13 AA (range: 5–29) and was similar in WM, IgM-MGUS and IgM-RD. Intra-WM/IgM-MGUS/IgM-RD search for HCDR3 similarity showed no association that met minimal requirements to define stereotyped receptors. The comparison of WM/IgM-MGUS/IgM-RD sequences with non WM/IgM-MGUS/IgM-RD database showed that WM/IgM-MGUS/IgM-RD sequences are unrelated to known CLL or SMZL subsets. Only one IgM-MGUS occurring in a HCV-positive patient formed a novel subset with other 4 HCV-related lymphoproliferative disorders.The findings of this analysis of WM, IgM-MGUS and IgM-RD IGH sequences indicate that: i) family-usage in the WM, IgM-MGUS and IgM-RD showed a skewed usage compared to normal B-cell repertoire; ii) WM, IgM-MGUS and IgM-RD-specific HCDR3 clusters do not occur to a frequency detectable with currently available databases; iii) WM, IgM-MGUS and IgM-RD sequences are not related to known CLL and SMZL HCDR3 clusters; iv) one IgM-MGUS showed similarities with sequences derived from pathological HCV-related lymphoproliferations, suggesting that, at least in a fraction of cases, HCV may have a pathogenetic role. For the large majority of IgM-related disorders, however, there is little evidence in favor of a B-cell receptor-driven pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Outcome Disparities In Waldenstrom's Macroglobulinemia: A SEER Database Analysis

Abstract 845 Background:Waldenstrom's macroglobulinemia (WM) is a relatively uncommon plasma cell disorder and most of previous literature is limited to small patient series. We have noted ethnic disparities in multiple myeloma, another plasma cell disorder. We undertook a large Surveillance Epidemiology and End Results (SEER) based analysis to describe outcome disparities in different subgroups of WM patients, with a focus on various ethnicities, so that therapeutic resources can be targeted more effectively. Methods:The SEER 17 Registry data (1973–2008) was utilized for patients with confirmed diagnosis of WM. To avoid bias of under representation of different ethnicities, analysis was restricted to patients with a diagnosis date of 1992 or later. Cases that received a diagnosis at death certificate or autopsy, no follow-up records, as well as lacking documentation on age at diagnosis, sex, or race/ethnicity were excluded. Cox proportional hazards models, adjusted for gender, age, race, year of diagnosis, marital status and stratified by SEER registries were used to evaluate associatio n between patient characteristics and survival. All statistical tests were two-sided and utilized the SAS software (v9.2) with a significance level of 0.05. Results:The final analysis included 2,840 WM patients (1,759 males; 62%, 1,081 females; 38%). The studied age-group cohorts included: 18–64 yrs (845; 30%), 65–74 yrs (795; 28%) and >75 yrs (1,200; 42%). Patients were stratified by race/ethnicity: White (2,471; 87%), African-American (AA) (102; 4%), Hispanic (133; 5%), Asian (129; 5%), and Native American (5; 0.1%). Patients were also stratified based on year of diagnosis (before or after 2002) to study the impact of certain novel agents (proteasome inhibitors, IMiDs) on WM treatment. There was a significant difference in the age at diagnosis of WM patients across different ethnicities, with AA the youngest (median 61.5 years) and Whites the oldest (median 73 years) subgroup (p<0.001). (Figure 1) Cause-specific mortality could not be evaluated due to variable documentation of reported causes of death for WM patients in the database. Survival analysis revealed that for all patients, females had better median overall survival (OS) than males (7.3 years vs. 6.2 years, HR 0.834; 95% CI 0.740, 0.939; p=0.003). Among the different age cohorts, patients with age ≥75 years had a significantly worse median OS (4.1 years) than those 65–74 year-old (7.3 years) or 18–64 year-old (10+ years) (p<0.001). Patients diagnosed after 2002 had a significantly better median OS (7.3 years) as compared to patients diagnosed previously (6.1 years) (p=0.002). Hispanics had the worst median OS (5 years) and Whites had the best median OS (6.8 years) across various ethnicities. In a multivariate model using gender, age, marital status, year of diagnosis and race, a significant interaction was noted between race, age and survival, with Hispanics having a significantly worse OS than Whites (HR 1.86; 95% CI 1.269, 2.726; p=0.003) (Figure 2). [Display omitted] [Display omitted] Conclusions:Studies of outcome disparities are important for evaluating disease characteristics and management needs of specific patient populations as well as optimal triaging of healthcare resources. We have performed the largest population-based analysis for WM including various ethnicities in the novel therapeutic agent era. We observed that AA patients had a significantly younger age at WM diagnosis. Older patients had worse OS across all ethnic subgroups and patients diagnosed after 2002 had a better OS suggesting an impact of novel therapeutic agents (e.g., proteasome inhibitors, IMiDs). Hispanic WM patients had the worst OS among all ethnicities. This survival difference was more significant and pronounced in patients >75 years. This suggests that the impact of race on survival is influenced by patient’s age. These results will help in better understanding of various influences on disease biology and clinical behavior. Disclosures:No relevant conflicts of interest to declare.

Identifying Phenotypically and Clinically Distinct Entities within the Spectrum of Marginal Zone Lymphoma and Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia

Abstract 2652The mature B-cell neoplasms marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma (LPL) and Waldenstrom macroglobulinemia (WM) display considerable overlap in clinic presentation, morphology, immunophenotype and genetic features. There is known to be poor reproducibility in the classification of these disorders and often only minimal diagnostic material is available. Multiparameter flow cytometry is a valuable tool for diagnosis and monitoring of B-cell disorders that can be applied to low cellularity samples or when the neoplastic cells represent a minority of total leucocytes. However, even when using large numbers of markers it is not possible to discriminate between MZL, LPL and WM and the aberrant expression of certain markers appears to occur with similar frequency in the different disease categories. The aim of this study was to determine whether there are phenotypically distinct entities within the spectrum of marginal zone and lymphoplasmacytic lymphomas, and to correlate these entities with clinical features.Samples from 146 people with a diagnosis of MZL, LPL or WM (122 bone marrow, 13 peripheral blood, 10 tissue biopsies), 8 with hairy cell leukemia, 10 reactive lymph node biopsies and 10 normal bone marrow samples were analysed using a panel of 39 markers. A minimum of 50,000 cells were acquired and analysed using a BD FACSCanto II with Diva software. Median fluorescence intensities were recorded for neoplastic CD19+ B-cell populations as well as for germinal centre (GC) and non-GC lymph node B-cells, B-progenitors, mature B-cells and plasma cell populations. Unsupervised cluster analysis was performed using dChip software. Clinical features and outcome data were collected and recorded centrally (www.hmrn.org.uk).Cluster analysis identified 5 entities with significant differences in their immunophenotypic profile. Neoplastic cells in all the MZL/LPL/WM groups typically expressed CD31, CD39 and CD49d with lack of CD10, CD23 and weaker expression of LAIR1 (CD305), CXCR5 (CD185) and CD22 compared to normal B-cells. CD5 and CD43 were expressed in a subset of cases but co-expression occurred in <1% of cases. The groups are characterised as follows: (1) clustered with normal mature B-cells, characterised by weaker CD20/CD95 and stronger CXCR5 expression relative to other MZL/LPL/WM cases, shorter survival, anemia and lymphadenopathy, typically requiring therapeutic intervention at diagnosis; (2) clustered with HCL, characterised by CD25 and CD11c expression and a higher degree of extranodal involvement; (3) did not cluster with other B-cell types, characterised by very strong CD79b and surface IgM expression often with anemia requiring supportive care; (4) characterised by strong CD24 expression with relatively good clinical course; and (5) clustered with normal bone marrow plasma cells, characterised by weak CD20, CD24 and IgM expression and also with better clinical features.Group2 year survivalAnemia (Hb <10.5g/dL)Lympho-cytosis (>4 × 10^9/L)Raised B2M (>2.7mg/L)Lymph-adenopathy (≥2 nodes)Extranodal involvementTherapeutic intervention at diagnosis115/24 (63%)10/19 (53%)9/19 (47%)8/10 (80%)12/19 (63%)4/19 (21%)14/19 (74%)228/37 (76%)8/25 (32%)11/25 (44%)6/11 (55%)9/23 (39%)10/23 (43%)9/25 (36%)325/31 (81%)14/24 (58%)5/23 (22%)11/18 (61%)4/19 (21%)5/19 (26%)13/24 (54%)422/27 (81%)5/22 (23%)5/22 (23%)10/17 (59%)5/20 (25%)4/20 (20%)8/22 (36%)522/26 (85%)5/16 (31%)5/16 (31%)6/8 (75%)8/14 (57%)5/14 (36%)5/16 (31%)The results indicate that the current diagnostic categories of MZL and LPL/WM have a great degree of overlap and comprise a number of phenotypically distinct entities which also appear to have different clinical features and outcomes. Enhancing the classification is likely to facilitate diagnosis, monitoring and identification of the underlying features of these B-cell malignancies. Disclosures:Rawstron:BD Biosciences: Patents & Royalties.

Whole-Genome Sequencing Results From 30 Patients with Waldenstrom's Macroglobulinemia

Abstract 434 Introduction:The primary oncogenetic event resulting in malignant transformation in Waldenstrom's Macroglobulinemia (WM) remains to be delineated. We therefore employed whole genome sequencing (WGS) to help identify potential somatic variants in WM. Patients and Methods:Thirty patients meeting consensus criteria for the diagnosis of WM were included for these studies, whose characteristics are depicted in Table 1. CD19-magnetic bead sorting was used for isolation of bone marrow LPC. CD19-depleted PB mononuclear cells were collected as matched normal tissue. For 10 patients, WGS of tumor and matched normal samples was performed, and for 20 additional patients tumor samples alone were completely sequenced. Library construction and WGS was performed by Complete Genomics Inc. Read sequences were aligned to the NCBI Build 37. High confidence somatic variants were identified using cgatools version 1.3. Novel non-synonymous exonic variants for familial and sporadic LPL/WM patients were identified using ANNOVAR using to filter against several large databases including dbSNP version 132, the November 2010 release version of the 1,000 genomes project, and a 46 healthy donor dataset from Complete Genomics, Inc. based on KnownGene annotations. Variants filtered out in this process were checked against the dbSNP132 flagged SNP database for potential clinical significance. Data was further annotated against the Database of Genomic Variants and the Segmental Duplication Database, TargetScan, and transcription factor binding site data from the ENCODE project. When applicable, variants were scored using SIFT, PolyPhen2 and Mutation Taster. Copy number neutral loss of heterozygosity (CNLOH) was identified from the rate of heterozygous variants per 500,000 base pairs, CG content adjusted coverage data, and allele imbalance calculated from the percentage of total reads supporting the less covered allele.Table 1Clinical and laboratory characteristics for 30 WM patients whose lymphoplasmacytic cells were used in WGS studies.MedianRangeAge6241–87Familial WM9 (30%)NAUntreated26 (87%)NABone Marrow Involvement (%)65%5–95%Clonal B–cells(%)196.355.5–100IgM (mg/dL)3,970590–6,580IgMκ/IgMλ monoclonal protein24/6NAIgA (mg/dL)4111–516IgG (mg/dL)519215–3,120Serum B2M (mg/L)3.551.50–12.1Hematocrit (%)31.424.0–41.51Determined by light chain restriction using flow cytometry. Results:Tumor and normal genomes were both sequenced to an average of 66X (range 60–91X) coverage of mapped individual reads. The average gross mapped yield for these genomes was 186.89 (range 171.56–262.03 Gb). Acquired copy number changes were common, and included losses in chromosome 6q (13/30; 43%), gains in chromosome 4 (7/30; 23%), and gains in 6p (3/30; 10%). Large regions of CNLOH were observed in 9/30 (30%) of patients occurring in chromosomes 1, 2, 3, 5, 9, 11, 17, 21, and X. The most frequent somatic variant occurred at position 38182641 in chromosome 3p22.2 in the myeloid differentiation primary response (MYD88) gene, resulting in a non-synonymous change at amino acid position 265 from leucine to proline (L265P) in 26/30 (86.7%) patients. Of these, 4/26 (15%) had a CNLOH covering this position making the variant effectively homozygous. Additional somatic variants occurred in transporter 2, ATP-binding cassette, sub-family B (TAP2) gene in 7/30 (23%) patients; chemokine (C-X-C motif) receptor 4 (CXCR4) gene in 6/30 (20%) patients. Somatic variants were also identified in the coding regions of low density lipoprotein receptor-related protein 1B (LRP1B) gene in 5/30 (17%) patients; mesothelin (MSLN) gene in 4/30 (13%) patients; AT rich interactive domain 1A (ARID1A) gene in 3/30 (10%) patients; histone cluster 1, H1e (HIST1H1E) in 3/30 (10%) patients, and Rap guanine nucleotide exchange factor 3 (RAPGEF3) in 3/30 (10%) patients. Conclusions:The results of this study provide the first reporting of comprehensive WGS efforts in patients with WM, and reveal recurring somatic variants in genes with important regulatory functions including MYD88, TAP2, and CXCR4. Structural and functional validation studies are ongoing and will be updated at the meeting. The results of these studies provide important new insights into the pathogenesis of WM. Disclosures:No relevant conflicts of interest to declare.

Family history of non-hematologic cancers among Waldenstrom macroglobulinemia patients: A preliminary study

BackgroundLittle is known about the epidemiology and etiology of Waldenstrom macroglobulinemia (WM). Despite several studies of the relation between family history and B-cell disorders and WM, family history of non-hematologic cancers has not been systematically investigated. We thus examined associations of family history of breast, colorectal, lung, ovarian, and prostate cancers with WM. MethodsAll probands aged 20–79 years with bone marrow biopsy-confirmed diagnosis of WM between May 1, 1999 and January 1, 2010 at the Bing Center for Waldenstrom Macroglobulinemia were eligible for inclusion in our analysis. We reviewed medical records for eligible probands to determine family history of cancer (defined as a cancer diagnosis for ≥1 first-degree relative(s) of the proband). Using expected values constructed from the United States National Health Interview Survey, we estimated age- and race-standardized rate ratios (RRs) for family history of breast, colorectal, lung, ovarian, and prostate cancers by WM subtype. ResultsFamily history of prostate cancer had the largest overall rate ratio (RR=1.4, 95% confidence limits [CL]: 1.1, 1.7), and among sporadic cases, family history of prostate and breast cancer had the largest rate ratios (prostate: RR=1.3, 95% CL: 1.1, 1.7; breast: RR=1.3, 95% CL: 1.2, 1.6). ConclusionOur study suggests that it may be worthwhile to pursue these associations in a case–control study with uniform selection and data collection for cases and controls, and at least some record-based information on family history.

The Role of Serum Immunoglobulin Free Light Chain in Response and Progression in Waldenstrom Macroglobulinemia

The serum free light chain (sFLC) has been widely used in the assessment of response in patients with multiple myeloma and other plasma cell dyscrasias. However, its use in Waldenstrom macroglobulinemia (WM) has not been previously assessed. We sought to examine the role of sFLC in response and progression of patients with WM. This study was conducted in a cohort of 48 patients with a diagnosis of WM, untreated (n = 20) or relapsed/refractory (n = 28), prospectively treated on a bortezomib and rituximab trial. Involved FLC (iFLC) response occurred in 79% patients versus 60% by M-spike protocol criteria. The median time to response was shorter with iFLC than per protocol (2.1 and 3.7 months; P = 0.05). Progression defined using iFLC also correlated well to progression in the protocol (κ = 0.63). However, the median time to progression (TTP) was more rapid by iFLC than per protocol (13.7 and 18.9 months). We also confirmed that a flare in iFLC in post-rituximab therapy did not correlate with lack of response or shorter TTP. Involved sFLC may be a useful marker of tumor measurement, showing earlier response and progression compared with IgM or M-spike measurements.

Non-random chromosomal deletion clustering at 20q in Waldenström macroglobulinemia

Chromosome change at 20q11–q12, including del(20q), is sometimes reported in plasma cell dyscrasia, but most cases are found during or after chemotherapy. It is therefore still uncertain whether del(20q) is a primary change or therapy-related. We performed cytogenetic studies and fluorescent in situ hybridization (FISH) analysis using 20q12 and 20qter probes to ascertain the possible involvement of 20q in nine patients with Waldenström macroglobulinemia (WM). The FISH study demonstrated deletions of 20q12 and/or 20qter in four of nine patients (44%) with WM at diagnosis, and one of them had the del(20q) chromosome. Moreover, one patient had de novo appearance of the del(20q) chromosome with 20q12 deletion after chemotherapy, although this patient had neither the del(20q) chromosome nor 20q12 deletion at WM diagnosis. Based on the results of this study, we conclude that chromosomal breakage at 20q13 is a non-random genetic change which plays a role in the neoplastic process of WM.

Associated Malignancies in Patients with Waldenström's Macroglobulinemia and Their Kin

We examined the incidence of other malignancies in 924 Waldenström's Macroglobulinemia (WM) patients and their kin. A total of 225 (24.3%) patients had ≥1 additional malignancy, with 63% predating the WM diagnosis. The most common gender-adjusted malignancies were prostate (9.4%), breast (8.0%), non-melanoma skin (7.1%), hematologic (2.8%), melanoma (2.2%), lung (1.4%) and thyroid 1.1%). Among hematologic malignancies, all 13 cases of diffuse large B-cell lymphoma and 4 cases of acute myelogenous leukemia were diagnosed after WM, and were therapy-related. Familial WM subgroup analysis showed a higher incidence of prostate cancer (P=.046) in sporadic WM patients, while patients with familial WM had a higher incidence of lung cancer (P=.0043). An increased incidence of myeloid leukemias (P<.0001) was reported among kin of familial WM patients. These data reveal specific cancer associations with WM, and provide a basis for exploratory studies aimed at delineating a common genetic basis. Additionally, these studies suggest specific cancer clustering based on familial predisposition to WM.

Waldenstrom Macroglobulinemia with CD5+ Expression Presented as Cryoglobulinemic Glomerulonephropathy: A Case Report

Waldenstrom macroglobulinemia (WM) is a B-cell lymphoproliferative disorder associated with bone marrow involvement of lymphoplasmacytic lymphoma (LPL) and an IgM monoclonal gammopathy. Generally B-lymphocytes in LPL do not express CD5 that is important for differential diagnosis of B-cell lymphoproliferative disorders. In WM, various renal diseases and type I cryoglobulinemia are well described separately, but cryoglobulinemic glomerulonephropathy is very rarely reported. A 61-yr-old woman complained of generalized edema, cyanosis of the extremities in cold weather, visual disturbance, and pancytopenia. Bone marrow and renal biopsy showed CD5+ expressing B-cells and cryoglobulinemic glomerulonephropathy. With the diagnosis of WM, she received cyclophosphamide, doxorubicin, vincristine and prednisolone chemotherapy and got complete remission. Here, we report a rare case of WM associated with unusual expression of CD5+ B-lymphocytes and cryoglobulinemic glomerulonephropathy, and emphasize the importance of the clinical features in differentiating CD5+ B-cell lymphoproliferative disorders.

Bendamustine-Based Therapy Is Highly Active In Patients with Relapsed or Refractory Waldenstrom's Macroglobulinemia

AbstractAbstract 4700 Background:Waldenstrom's macroglobulinemia (WM) is an indolent B-cell malignancy characterized by lymphoplasmacytic cell infiltration of the bone marrow and production of an IgM monoclonal protein. Despite advances in treatment, WM remains incurable and novel agents are needed for ongoing disease control. Bendamustine represents an important novel agent for the treatment of B-cell disorders whose activity in WM remains to be clarified. Patients and Methods:We examined the outcome of 30 previously treated patients with the clinicopathological diagnosis of WM who received bendamustine-based therapy. The median prior therapies was 2 (range 1–9), and 16 (53%) patients were refractory to their previous treatment. Baseline characteristics for all patients: Median BM involvement 60%; serum IgM 3,980 mg/dL; Hct 31.0%; serum B2M 3.5 g/L. Treatment consisted of bendamustine administered at 90 mg/m2 IV on days 1, 2 as part of a 4 week cycle, along with rituximab (375 mg/m2 IV) given once on either day 1 or 2 for 24 patients. In the remainder 6 patients, severe rituximab intolerance prevented re-administration of rituximab. In these patients, bendamustine was either administered alone (n=4) or with ofatumumab (1000 mg IV) given on day 1 (n=2) following a test dose of 300 mg IV on day -7 prior to cycle 1 only. Intended therapy consisted of 4–6 cycles of treatment. Plasmapheresis was performed prior to treatment in patients exhibiting symptomatic hyperviscosity, or who had an IgM level >5,000 mg/dL and were to receive monoclonal antibodies in order to prevent a symptomatic IgM flare. Responses were assessed using modified WM consensus criteria, and patients were eligible for response assessment if they completed > 2 cycles of therapy. Results:21 patients completed intended therapy; 9 patients continue on treatment. The median number of treatment cycles for all patients is 4 (range 2–6). Following treatment, median serum IgM levels declined from 3,980 to 1,210 mg/dL (p<0.0001), and hematocrit rose from 31.9% to 34.7% (p=0.005) at best response. The overall and major response rates were 80% and 73%, respectively, with 3 VGPR; 19 PR; 2 MR. 6 patients were non-responders. Responders included those patients receiving bendamustine alone (4 PR), or with ofatumumab (1 PR; 1 MR). With a median follow-up of 5 months, 22/24 responders continue in response. Overall, treatment was well tolerated with grade <2 nausea and diarrhea being the most common toxicities encountered. Three patients developed superficial thrombophlebitis at the site of bendamustine infusion, warranting institution of anticoagulation in 1 patient. Prolonged myelosuppression occurred in 3 patients who received previous nucleoside analogue therapy. One patient previously treated with nucleoside analogues and cyclophosphamide developed MDS, and another patient who received previous cyclophosphamide and bortezomib based therapies transformed to diffuse large B-cell lymphoma following bendamustine-based therapy. Conclusion:Bendamustine-based therapy is active in patients with relapsed or refractory WM and produces high response rates and durable responses both as monotherapy, and in combination with CD20 directed monoclonal antibodies. In patients previously treated with nucleoside analogues, prolonged myelosuppression may occur. Long term toxicities of bendamustine-based therapy remain to be clarified in this patient population. Disclosures:No relevant conflicts of interest to declare.

Clinical and Biological Implications of Hepatitis C Virus Positivity in Waldenstrom's Macroglobulinemia Patients.

Abstract 2934Poster Board II-910 Background:hepatitis C virus (HCV) infection has been associated with increased risk of developing B-cell lymphoprolipherative disorders through chronic immune stimulation. Discordant data have been reported about the prevalence of HCV infection in patients with Waldenström's Macroglobulinemia (WM) and the putative role of HCV in lymphomagenesis of this non-Hodgkin Lymphoma subtype remains controversial. Aim:the current retrospective study was conducted to assess the impact of this infection itself in the clinical and biological features and outcome among WM patients as compared with those with HCV negative WM. Patients and methods:we analyzed 140 WM patients with tested anti-HCV antibody (HCV-Ab). HCV-Ab positivity was detected in 21 cases (15%).Clinical characteristics of patients at diagnosis of WM are reported in the table below.CharacteristicsAll patientsHCV-HCV+PAge y median6363650,495Sex % M/F59/4163/3738/620,052Hb median g/dl12,61311,60,041PLT x 10 9/l median2382502070,028PMN x 10 9/l median3,683,872,650,002IgM mg/dl median2150179625650,843Bone marrow infiltration %:≤30%464460>0,99930-80%323515>0,999≥ 80%222125>0,999LDH U/L median313304,54010,029B2-M ug/mL median2,462,43,650,045Albumine g/dL median4,44,34,40,461Creatinine mg/dL median0,90,90,9>0,999Splenomegaly %2117430,018Lymphadenopathy %1716240,755Neuropathy %119190,248Presence of autoantibodies %1712480,002Cryoglobulins %159480,004 Results:HCV positivity was correlated to lower counts of platelets, neutrophil granulocytes, haemoglobin and with presence of cryoglobulins or autoantibodies and splenomegaly. Interestingly we even found a strong link between the presence of HCV and serum parameters of tumor burden such as beta-2 microglobulin (B2-M) and lactate dehydrogenase (LDH).Furthermore we did not reveal any outcome implications in terms of disease progression needing treatment, time from diagnosis to first therapy, overall survival between HCV- and HCV+ patients.Overall, 88 patients (63%) received treatment for disease progression with schedules including Rituximab in 46 cases (52% of treated patients). Rituximab was administered even in HCV-RNA positive patients associated to Cyclophosphamide and Fludarabine and this did not translate in hepatitis development. HCV-RNA was strictly monitored during immunotherapy and we did not observe any significant flair. Conclusions:in our series of patients HCV infection does not seem to affect clinical outcome of disease. Moreover, in our experience patients with documented infection can receive the same schedule of treatment including intensive chemotherapy even with monoclonal antibodies without development of further toxicity. Disclosures:No relevant conflicts of interest to declare.

Immunophenotypic Analysis Using CD80 and CD86 Staining Improves the Diagnosis of Waldenstrom's Macroglobulinemia by Evidencing a CD80+/CD86- Profile.

Abstract 4389The immunophenotypic characterization is an essential tool in the diagnosis of hematological malignancies but the immunophenotypic profile of Waldenstrom's macroglobulinemia (WM) B cells remains not clearly defined. In order to improve the diagnosis of WM by an immunophenotypic peripheral blood test, we studied within a multicentric protocol the expression of costimulatory molecules CD80 (B7.1) and CD86 (B7.2) on blood B-cells of WM patients diagnosed by monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow (BM) infiltration. Results were compared to those obtained in healthy controls and other chronic B-cell lymphoproliferative disorders (LPD). Immunophenotypic analysis was performed by flow cytometry using six-colour staining. The CD80 and CD86 staining was concomitant with usual analysis of standard antigens (kappa/lambda immunoglobulin light chains, CD20, CD19, CD79b, CD5, CD10, CD23, FMC7, CD38, CD25, CD43, CD11c, CD1c, CD27, IgM, IgD). We first analyzed 65 cases of WM and compared results to 13 healthy control subjects. Among the WM patients, 45 (69%) showed a monotypic kappa or lambda peripheral blood B-cell population with a Matutes score '3. In these patients, CD80 was always positive (>21% of B-cells) and CD86 negative (<18%). The mean responses obtained in WM compared to controls were: CD80 = 44.2±19.5% versus 13±4.6% (p<0.00001), CD86 = 5.4±5.3% versus 4.5±2.1% (p=0.18). Among the 20 patients without monotypic kappa or lambda B-cell population in peripheral blood, 13 showed a CD80+/CD86- profile (CD80 = 33±7.4%, CD86 = 7.6±4.4%). Six of them were studied by molecular aproach (PCR) showing the presence of a clonal B-cell population in all cases. Overall, 90% of WM patients showed a CD80+/CD86- profile in blood. Seven patients without peripheral monotypic kappa or lambda B-cell population evidenced a CD80-/CD86- profile; among them, one was tested by PCR and showed the lack of a clonal B-cell population in blood. We then studied other chronic B-cell LPD with peripheral blood neoplastic involvement by a monotypic kappa/lambda population, including 15 marginal zone lymphoma (MZL), 4 non-Hodgkin lymphomas (NHL) and 28 chronic lymphocytic leukemia (CLL). The mean expression of CD80 was 37±35.6%, 2.5±1.7%, 12.4±20.6%, and of CD86 30.5±38%, 19±27.5%, 15.4±19.3%, respectively. The expression of these antigens in MZL was heterogeneous, but a WM-type CD80+/CD86- profile was never observed in peripheral blood B cells. No NHL and 4 CLL (14%) exhibited a CD80+/CD86- profile. In conclusion, while immunophenotypic analysis usually performed with standard antigens does not allow to define a typical profile of WM, additional CD80 and CD86 staining in blood samples usefully improves the diagnosis of WM in the context of chronic B-cell LPD with peripheral blood involvement having a Matutes score '3. Furthermore, even in the absence of blood involvement detected by classical imunophenotypical aproach, the CD80 and CD86 staining may prove the presence of a peripheral blood monoclonal population, discriminating WM from other B-LPD with plasmatic IgM and lymphoplasmacytic BM infiltration, and diagnosing a WM in 90% of cases. Disclosures:No relevant conflicts of interest to declare.

Keratotic vascular papules over the feet: a case of Waldenström’s macroglobulinaemia-associated cutaneous macroglobulinosis

Waldenström's macroglobulinaemia (WM) is a plasma-cell dyscrasia characterized by the monoclonal proliferation of lymphoplasmacytes. A 48-year-old man presented with a 4-year history of multiple painful, hyperkeratotic deep-seated papules over the pressure areas of both soles. He had a 1-year history of Raynaud's phenomenon, intermittent epistaxis, recurrent vomiting, tingling and numbness, and visual disturbances. Histological examination of a skin biopsy found amyloid-like deposits in the upper and mid dermis involving dermal blood vessels, but apart from periodic-acid-Schiff, various stains gave negative results for amyloid. Direct immunofluorescence was positive for IgM antibody. Hence, a diagnosis of WM with cutaneous macroglobulinosis was made. Immunoelectrophoresis found monoclonal IgM kappa antibody, and bone-marrow examination revealed a lymphoplasmacytoid malignancy. The patient's systemic systems were attributed to hyperviscosity syndrome associated with WM and the cutaneous papules were identified as deposits of excess IgM antibodies. The patient received five cycles of chemotherapy, resulting in nearly complete resolution of the skin lesions and systemic symptoms.