CD138-positive Plasma Cells Research Articles

High Risk Multiple Myeloma Cases Are Identified In An MMRC Led Study By The SKY92 Gene Signature (MMprofiler)

IntroductionMultiple Myeloma is not a single disease. There is increasing support for risk classification in combination with treatment decision making because of its impact on clinical outcomes. Here we demonstrate additional evidence of the prognostic value of SKY92, an established genetic marker of high risk Multiple Myeloma in a multicenter collection of samples with undisclosed treatments. Materials MethodsA public GEP dataset (MMRC, MMGI portal) contained 114 cases of untreated Multiple Myeloma and was used for SKY92 high risk OS prediction (Kuiper et al. Leukemia 2012). In collaboration with MMRC, OS (with a minimum of at least 2 year follow-up) was collected for 91 of 114 cases for the purpose of this analysis. Briefly, CD138-positive plasma cells had been purified prior to total RNA extraction and subsequent gene expression profiling on Affymetrix U133Plus2.0 GeneChips. The 91 cases represented 9 different clinical sites and their CEL files were normalized as a single batch against a reference cohort of 329 cases after which the SKY92 risk scores were determined as either standard risk or high risk. ResultsSKY92 resulted in 19 high risk (20.9%) versus 72 standard risk (79.1%) cases in the unselected 91 case-cohort. Comparisons with other high risk GEP signatures will be performed. The OS analysis (Figure 1) shows that the HR cases have significantly shorter survival (Hazard Ratio 11, p = 7 x 10-5). Table 1 shows that high risk patients had more elevated B2M (26.3% vs 13.9%), more low albumin (26.3% vs 16.7%) and more high creatinine (26.3% vs 11.0%). There was no difference between high and standard groups in diagnosis dates (not shown). Cause of the 16 (84.2%) deaths among the high risk cases, and 21 (29.1%) deaths among the standard risk cases indicates that high risk contains less disease progression deaths (57.1% vs 31.3%), and more unknown deaths (56.3% vs 23.8%). [Display omitted] [Display omitted] ConclusionsThe SKY92 classifier identified 19 of 91 cases (21%) as high risk, recapitulating the percentage of high risk in previously studied cohorts (Kuiper et al. 2012). Moreover the hazard ratio of 11 when events up to 24 months or 8.18 when all events are considered, emphasizes the unmet medical need of high risk cases identified with SKY92 as 69% of all deaths within 2 years (9/13 death events) were in this category. AcknowledgmentsThis research was performed within the framework of CTMM, the Center for Translational Molecular Medicine, project BioCHIP grant 03O-102. Rafael Fonseca is a Clinical Investigator of the Damon Runyon Cancer Research Fund. This work is supported by grants R01 CA83724, ECOG CA 21115T, Predolin Foundation, Mayo Clinic Cancer Center and the Mayo Foundation. Disclosures:van Beers:Skyline Diagnostics: Employment. Van Vliet:Skyline Diagnostics: Employment. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership. Jagannath:Celgene: Honoraria; Millennium: Honoraria. Jakubowiak:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kumar:Celgene: Clinical Trial Support Other, Membership on an entity's Board of Directors or advisory committees; Cephalon: Clinical Trial Support, Clinical Trial Support Other; Millennium: Clinical Trial Support, Clinical Trial Support Other, Membership on an entity's Board of Directors or advisory committees; Novartis: Clinical Trial Support, Clinical Trial Support Other; Onyx: Clinical Trial Support Other, Membership on an entity's Board of Directors or advisory committees. Lebovic:Celgene: Speakers Bureau; Onyx: Speakers Bureau. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Reece:Onyx: Honoraria; Novartis: Honoraria; Millennium: Research Funding; Merck: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Research Funding. Siegel:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vij:Celgene: Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau. Zimmerman:Celgene: Honoraria; Millennium: Honoraria; Onyx: Honoraria. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

MSH-TP15 Identified By Phage Display Technology Detects a Target Structure For Antibody-Mediated Killing In Multiple Myeloma

Introduction Multiple Myeloma (MM) is a malignant plasma cell disorder with high incidence and mortality. Monoclonal antibodies represent promising new treatment options and are widely applied in cancer therapy, but none have yet been approved for the treatment of MM. Daratumumab and other antibodies have recently shown encouraging results in clinical trials, but there is still a need to identify plasma cell specific target structures suitable for this approach. Methods To identify novel plasma cell surface antigens or epitopes, synthetic human single-chain fragment variable (scFv) phage display libraries were screened by using a newly established subtractive, cellular screening approach. Pre-absorption steps with granulocytes and T lymphocytes were applied with subsequent positive selection for phages that show binding to plasma cell lines INA-6 and JK-6L. The binding patterns of the resulting phage antibodies on various cell types were evaluated by cellular ELISA and flow cytometry. Based on its favorable binding profile, MSH-TP15 was selected and the coding sequence was used to generate a fully human scFv-Fc fusion protein (mini-antibody) for functional assays. Results Screening of phage display libraries by using our novel strategy resulted in a number of phage clones with preferential binding to MM cells. MSH-TP15 was selected based upon its strong binding to five MM cell lines (INA-6, JK-6L, L363, RPMI-8226, U266), while not recognizing a B cell precursor leukemia cell line (SEM), an immature acute myeloid leukemia cell line (KG-1a), and T cell derived acute lymphoblastic leukemia cell lines (CEM, Jurkat). In addition to MM cells, two Burkitt lymphoma cell lines (Daudi, Ramos) were also positive. Importantly, patient-derived CD138-positive malignant plasma cells were recognized while no reactivity was seen on other mononuclear cells of the same patient. No significant binding was seen on peripheral blood T lymphocytes, NK cells, monocytes, and granulocytes from healthy donors. Additional screening on a panel of stably transfected cell lines that express antigens already considered for antibody-based MM therapy (CD38, CD40, CD56, CD70, CD138, CS1, FGFR3, IGF-1R, IL-6R) revealed no reactivity. To subsequently evaluate direct (e.g. growth inhibition) and indirect (e.g. antibody-dependent cell-mediated cytotoxicity; ADCC) antibody effector functions, the coding sequence was used to generate a scFv-Fc fusion protein. This mini-antibody successfully retained its prior established binding properties. The antibody by itself was not capable to induce significant growth inhibition of INA-6, L363, and RPMI-8226 plasma cell lines. In contrast, MSH-TP15 was able to trigger ADCC, an important killing mechanism of therapeutic antibodies, against these myeloma cells with peripheral blood mononuclear cells as effector cells. Conclusions Phage display technology together with a cell-based screening approach led to the generation of an antibody that preferentially binds to MM cells, and is effective in antibody-mediated killing mechanisms. The recognized antigen is distinct from any of the MM antigens currently explored as targets for antibody therapy, and therefore represents a promising target structure suitable for an antibody-based therapeutic approach in myeloma. Disclosures: No relevant conflicts of interest to declare.

Intestinal selenoprotein P in epithelial cells and in plasma cells

The micronutrient selenium and selenium-containing selenoproteins are involved in prevention of inflammation and carcinogenesis in the gut. Selenoprotein P (Sepp1), the plasma selenium transport protein, is secreted primarily from hepatocytes, but Sepp1 mRNA is also abundant in the intestine. By immunofluorescence analysis, we show that Sepp1 levels in epithelial cells of the rat jejunum increase along the crypt-to-villus axis. A different Sepp1 distribution pattern was observed in the rat colon, where the epithelial cells located at the base and at the top of the crypts were similarly positive for Sepp1. In addition, we found pronounced Sepp1 immunoreactivity in CD138-positive plasma cells scattered within the lamina propria of the colon. This hitherto unrecognized presence in terminally differentiated B-cells was corroborated by detection of Sepp1 in plasma cells residing in the rat spleen. Following supplementation with dietary selenium compounds, polarized intestinal epithelial Caco-2 cells secreted Sepp1 into the culture medium across the basolateral membrane. Our data suggest that Sepp1 secreted from epithelial cells may support the intestinal immune system by providing immune cells (including plasma cells) with selenium for the biosynthesis of endogenous selenoproteins.

Increased immunoglobulin G4–positive plasma cells in collagenous sprue

Collagenous sprue is a rare enteropathy whose etiology is unknown, but immune-mediated mechanisms are one of several possibilities. However, the role of immunoglobulin G4 (IgG4)-positive plasma cells has not been studied in collagenous sprue. Endoscopic biopsies from the duodenum with a histologic diagnosis of collagenous sprue (n = 40 from 35 patients), celiac disease (n = 25), peptic duodenitis (n = 15) and normal duodenum (n = 25) were immunohistochemically stained with IgG4 and CD138 antibodies. For each case, the quantities of IgG4- and CD138-positive plasma cells in the lamina propria were estimated by averaging the number in 3 high-power fields (hpf) that showed the highest concentration. Nine of forty collagenous sprue samples showed a mean of 10 or more IgG4 plasma cells per hpf, whereas none of the duodenal control biopsies showed 10 or more IgG4 plasma cells per hpf: celiac disease (P = .01), peptic duodenitis (P = .05), and normal duodenum (P = .01). Our study demonstrates that increased IgG4-positive plasma cells are present in a subset (23%) of collagenous sprue and may play a role in its pathogenesis.

Expression level of microRNA-92a and its clinical significance in multiple myeloma patients

To investigate the relationship between the expression level of microRNA 92a (miR-92a) and del(13q14) and the prognosis of MM patients, and to explore the pathway that miR-92a involved. Bone marrow samples from 53 newly diagnosed MM patients were collected, del(13q14) was analyzed by interphase fluorescence in situ hybridization in sorted CD138 positive plasma cell. The expression of miR-92a in plasma cells was measured by quantitative real-time PCR. The expression of c-jun was detected by Western blot in miR-92a transfected MM cell lines (LP-1, U266 and JJN3). Of the 53 MM patients, del(13q14) was detected in 31 (58.4%) patients. The median levels of miR-92a in MM patients with or without del(13q14) were 27.36±2.61 and 21.87±15.98, respectively (P>0.05). With the median follow-up of 13.5 (0.5-72.5) months, the median duration of progression-free survival of patients with high expression level of miR-92a was significantly shorter than those with low expression level of miR-92a (4.5 months vs 14.0 months, P=0.006). Overexpression of miR-92a in MM cell lines induces time-dependent down-regulation of c-jun. High expression of miR-92a was associated with poor prognosis in MM patients. The expression level of miR-92a was not associated with del(13q14), and the effect of miR-92a on the progress of MM might be involved in c-jun pathway.

Acne inversa (Hidradenitis suppurativa): A review with a focus on pathogenesis and treatment

Acne inversa (AI) is a disabilitating chronic inflammatory disease with major negative impact on quality of life and significant co-morbidities. This is an important link to insights into immune dysfunction, which stimulated therapeutic approaches like tumor necrosis-α inhibitor therapy. This new off-label drug treatment is particularly beneficial when used in combination with wide excision of inflamed skin and subcutaneous tissue. Retinoids have been reported to be helpful in secondary prevention. The standard of therapy in advanced cases is surgery with wide excisions and healing by secondary intention. This treatment results in significant reduction of complaints and achieves satisfactory body contouring.

The novel cytokine interleukin-36α is expressed in psoriatic and rheumatoid arthritis synovium

BackgroundInterleukin (IL)-36α is a recently described member of the IL-1 cytokine family with pro-inflammatory and clearly pathogenic properties in psoriasis.ObjectiveTo determine the IL-36α expression in psoriatic arthritis (PsA) compared to...

Intra-Clonal Heterogeneity Is a Critical Early Event in the Preclinical Stages of Multiple Myeloma and Is Subject to Darwinian Fluctuation throughout the Disease

AbstractAbstract 3941Multiple Myeloma (MM) is a neoplastic proliferation of aberrant plasma cells that has well defined clinical steps including MGUS, SMM, MM and PCL. These clinical stages represent a good model in which to study the multistep acquisition of genetic events underlying cancer progression. The transition of normal plasma cells to myelomatous cells is thought to be the result of the sequential acquisition of “genetic hits” causing the classical hallmarks of cancer. Underlying this transition, the existence and competition between different tumour clones, also termed intra-clonal heterogeneity, has been recently highlighted in a number of human cancers. In this work we have analysed the Darwinian intra-clonal population dynamics of MM in order to better understand disease progression, development of treatment resistance, and the effectiveness of targeted therapies.Next generation sequencing (NGS) is an excellent tool which can identify complex intraclonal relationships in MM allowing the identification of acquired mutations as well as being able to place them within a Darwinian context. This type of model incorporates not only the competition between already existent clones, but also the emergence of new clones during disease progression via the acquisition of novel mutations. Single cell analysis ensures the firm identification of the different clonal populations harbouring common and distinct mutations effectively validating the NGS data.In this work, we have extended our sequence analysis of MM as well as studying paired patient samples at different stages of the disease process including relapses or transitions to later stages of disease. Samples include 6 MGUS patients, 4 high-risk SMM patients that later developed symptomatic MM, 2 MM patients at presentation and relapse, and 1 MM patient that later evolved to PCL. Additional paired patient samples will be added to the final analysis. CD138-positive bone marrow plasma cells were selected to a purity >95%. For SMM-MM paired samples, we used 100 ng of tumour and non-tumour cell DNA to undergo whole genome sequencing, using 120 bp paired-end reads on a GAIIx (Illumina) to a median depth of 32x, with 98% at 1x and 84% at 20x coverage. For the rest of the samples, we used 50 ng of DNA and performed whole exome sequencing using 76 bp paired-end reads on a GAIIx (Illumina) to a median depth of 61x, with 99% at 1x and 85% at 20x exomic coverage. Data were aligned to the human genome (hg19) using Stampy and BWA and acquired SNVs and indels called using GATK. Single cells from a subset of patients were sorted into lysis buffer on 96 well plates using a FACSAria cell sorter. Specific target amplification (STA) was performed before quantitative PCR using genotyping assays specific for the mutations of interest. The STA product was diluted before qPCR interrogation for mutations using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm).The results of this analysis show that: a.The mutational gene spectrum found in MM is consistent with our previous sequencing analysis of t(4;14) and t(11;14) MM cases.b.The mutational landscape and intraclonal diversity seen in paired samples exist at the earliest stages of myeloma including asymptomatic stages such as MGUS or SMM; however, clonal size varies during progression. The number of SNVs in High Rish SMM does not significantly differ from MM with clinical symptoms requiring treatment. Hence, these data suggest that all of the genetic deregulation necessary to give rise to an aggressive clinical state is already present in asymptomatic stages of the disease, but is masked by the existence of a predominant clone with a more benign pattern of behaviour that may compete for access to a putative myeloma niche.c.Different patterns of “clonal tides” are seen at presentation-relapse pairs and following the transition to PCL, consistent with a Darwinian process of tumour evolution. Their high genetic complexity is displayed by the accumulation of multiple mutations in various genes involving diverse biological functions, but that may eventually supply the hallmarks of cancer. These tumour cell populations may be either genetically stable, linearly evolving, or heterogeneous clones with changing predominance. The emergence of new clones, which could be driven by mutations in key genes such as KRAS or BRAF, increases the complexity and effectiveness of targeted therapies. Disclosures:No relevant conflicts of interest to declare.

Immunohistological analysis in diagnosis of plasma cell myeloma based on cytoplasmic kappa/lambda ratio of CD38-positive plasma cells

The accurate determination of cytoplasmic immunoglobulin (cIg) light chain (LC) expression is important to differentiate reactive plasmacytosis from a clonal plasma cell neoplasm such as plasma cell myeloma (PCM). Through retrospective analysis, we studied the cytoplasmic kappa/lambda ratio of CD38-positive plasma cells in the bone marrow from 19 PCM patients and 19 controls. To demonstrate cIg LC expression, the bone marrow was immunostained for IgA, IgG, IgM, kappa, and lambda. The kappa/lambda ratio was defined as the ratio of the kappa-positive cell to the lambda-positive cell in plasma cells. PCM cells were distinguished from normal plasma cells by cut-off levels between 0.59 and 4.0, a sensitivity of 94.7%, and a specificity of 94.7%. The detection of the cytoplasmic kappa/lambda ratio of CD38-positive plasma cells may be a useful tool in the diagnosis of PCM and the correct diagnosis of PCM may be achieved more simply.

Etiology of Nasal Polyps in Cystic Fibrosis: Not a Unimodal Disease

The objective was to determine whether the polyp subtypes observed in cystic fibrosis (CF)-related sinusitis were similar to those observed in non-CF-related sinusitis. Polyp and mucus samples were collected from CF patients who presented for sinus surgery. The polyps underwent histologic and cytochemical evaluation for the presence of lymphocyte cell populations and their respective cytokine markers. The mucus samples were evaluated for DNA content. Of the polyps, 42% had an eosinophilic infiltrate, of which 80% had an additional mixed neutrophilic infiltrate. Of the remaining polyp samples, 42% did not have a granulocytic infiltrate, consistent with non-eosinophilic polyps. All samples had CD138-positive plasma cells. The mucus samples from the patients with CF showed higher extracellular DNA concentrations than did the mucus samples from patients with non-CF sinus disease. Cystic fibrosis-related polyps demonstrated an eosinophil-based dichotomy similar to that of idiopathic non-CF-related polyps. Many also demonstrated neutrophilic infiltrate, indicating that chronic mucus stasis and infection complicate the disease. Agents capable of reducing extracellular DNA may help manage sinusitis in CF patients.

Immunohistological diagnosis of plasma cell myeloma based on cytoplasmic kappa/lambda ratio of CD138-positive plasma cells

For differentiating reactive plasmacytosis from clonal plasma cell neoplasms such as plasma cell myeloma (PCM), it is important to determine the expression of the cytoplasmic light chain accurately. Through retrospective analysis, we studied the cytoplasmic kappa/lambda ratio of CD138-positive plasma cells in bone marrow from 50 patients with PCM and 50 controls by immunohistological analysis. The percentage of cytoplasmic light chain immunoreactive cells out of the total plasma cell population was shown by a novel quantitative image analysis approach using an Aperio ScanScope CS and the Membrane v9 algorithm. PCM cells were distinguished from normal plasma cells by cut-off levels between 0.35 and 5.5, a sensitivity of 100% and a specificity of 98.0%. Detection of the cytoplasmic kappa/lambda ratio of CD138-positive plasma cells could be a useful tool for simple, efficient and accurate diagnosis of PCM.

Abstract 540: Plasma Cell Infiltration and Mucoid Degeneration in the Media of Ascending Aorta in Patients with Coronary Artery Disease

Introduction Atherosclerosis results in inflammatory changes in the aortic intima, but little is known regarding medial changes. The inflammatory diseases of the aorta and large artery display characteristic features of inflammatory cell infiltration in the media, and they are usually diagnosed by medial changes. Aims Atherosclerosis of the ascending aorta coexists with coronary artery disease. The aim of this study was to investigate the atherosclerotic changes in 44 biopsy specimens of media of the ascending aorta associated with coronary artery disease. Methods We compared plasma cells, and matrix metalloproteinase (MMP)-2-, -9- and -12-positive cells immunohistochemically, and we also compared mucoid degeneration and fibrosis determined by special staining using a point-counting method, for groups with a variable number of coronary stenotic (≥75 %) lesions. Results and Conclusion To investigate the appearance of plasma cells, smooth muscle cells, and mucoid degeneration in the media of the atherosclerotic aorta, we examined three cadavers with known atherosclerosis. CD38-positive plasma cells are pleomorphous in the aortic media. Desmin-positive smooth muscle cells appeared fibrous or solitary spindle cells. Mucoid degeneration was shown in all three cadaver cases. In patients with one to three coronary stenotic lesions, plasma cells and mucoid degeneration were low in the aortic media. With four to five lesions, both plasma cells and mucoid degeneration increased significantly compared with those in the group with one to three lesions, and MMP-12-positive cells significantly decreased. In patients with six to nine lesions, the number of plasma cells was significantly lower than in patients with four or five lesions, whereas mucoid degeneration significantly increased. There was no change in fibrosis. In conclusion, both the infiltration of short lived plasma cells and the exudation of proteoglycan are known to be regarded as characteristic features in the autoimmune disease. The delayed stage in our study is suggested to be caused by autoimmune reaction. If the cause of the delayed medial atherosclerosis stage is found, then more effective therapies to suppress its development can be introduced.

Calcific aortic valve stenosis: Immunohistochemical analysis of inflammatory infiltrate

Calcific aortic valve disease is considered a form of atherosclerosis and, like the latter, possibly of inflammatory origin. The aim of our work was to study the pattern of cellular infiltrate in calcific aortic valve stenosis (CAS). Fifteen operatively excised calcified aortic valves were examined by histology and immunohistochemistry (CD20, CD79α, CD3, CD4, CD8, CD68, CD138, CD117, BJK, BJL, IgA, IgD, IgG, IgG4 and IgM). The findings revealed that in CAS, there were chronic inflammatory features with infiltrates comprising lymphocytes, polyclonal plasma cells, histiocytes and mast cells. In T-lymphocytes, CD4 prevailed over CD8. In B-lymphocytes, there was a slight preponderance of CD20 over CD79α. The BJL (lambda)-positive plasma cells prevailed over the BJK (kappa) ones. The CD138-positive plasma cells comprised 24% IgA-, 20% IgD-, 41% IgG- (including 11% of IgG4-) and 15% IgM-positive cells. CAS did not fulfill the criteria of the recently described clinicopathological entity IgG4-related sclerosing systemic disease. The inflammatory process was the same in both subsets of CAS - those with trileaflet (normally formed) valves and those with congenitally bicuspid valves.

Polyclonal IgA Gammopathy Associated with Polyclonal Plasmacytosis in Patients Receiving Lenalidomide Maintenance Therapy

Abstract 5130 INTRODUCTION:Lenalidomide is commonly used in the treatment of multiple myeloma both in the upfront and relapsed settings and appears to also yield promising results as maintenance therapy after autologous stem cell transplantation. The immunomodulatory mechanisms of action of lenalidomide are complex and the side effects of long-term therapy are still not fully known. Here we report a series of patients on lenalidomide maintenance therapy who demonstrated significant elevation of serum polyclonal immunoglobulin A (IgA) levels with associated polyclonal plasmacytosis in the absence of disease progression. METHODS AND RESULTS:We identified 6 patients who, in the course of lenalidomide maintenance therapy, were noted to have asymptomatic elevation of serum IgA levels above normal limits without evidence of monoclonality by immunofixation of serum protein electrophoresis. The original myeloma-associated monoclonal protein was IgG kappa in 5 patients and IgG lambda in 1 patient. Two patients received lenalidomide as part of their induction regimen prior to autologous stem cell transplantation, and transient elevation of IgA level was noted at that time. None of the patients had evidence of IgA gammopathy prior to initiation of lenalidomide. Mean time from initiation of lenalidomide maintenance therapy to elevation of IgA level was 9.8 months (range 2 to 22 months). The mean highest IgA level reached for the 6 patients was 534 mg/dl (range 520 to 1190 mg/dl). Bone marrow aspirate and biopsy were available in 3 patients during the period of IgA elevation. The plasma cell population accounted for 9%, 12% and 20% of the bone marrow nucleated cells on the aspirate. Immunohistochemical staining (HIS) of the bone marrow biopsy showed a moderate increase in the number of CD138 positive plasma cells without light chain restriction. HIS using IgA antibody revealed that the plasma cells were mostly IgA producing. None of the 6 patients had any evidence of progression of their original myeloma-associated paraprotein in the setting of polyclonal IgA elevation. Five patients continue to receive lenalidomide maintenance to date (treatment time length ranging between 17.3 and 43.4 months) with no evidence of serologic or clinical disease progression and with persistently elevated IgA levels. One patient discontinued lenalidomide therapy secondary to development of neuropathy with subsequent normalization of IgA level 21 months later. CONCLUSIONS:Polyclonal IgA gammopathy associated with polyclonal IgA-producing plasmacytosis can occur with lenalidomide maintenance and perhaps lenalidomide treatment. The immunologic basis and implication of this observation is unknown. Awareness of this effect is important especially in view of the associated significant bone marrow plasmacytosis, which may mistakenly be construed as disease relapse or progression in patients who are actually responding to treatment. Disclosures:No relevant conflicts of interest to declare.

Common Fragile Site Gene FRA16D WWOX (WW domain containing oxidoreductase) Is Frequently Altered in Plasma Cells of Multiple Myeloma and MGUS, Possibly Related to Disease Development

Abstract 4646The common fragile sites (CFSs) are regions of profound genomic instability, and hot-spots for deletions and other alterations in cancer cells. We have previously shown that promoter of FHIT is frequently methylated in multiple myeloma (MM) and correlated with worse prognosis. WWOX is located at a CFS region on chromosome 16q23.3, this gene is also a target of alterations in multiple cancers. The objective of current study is to find if this tumor suppressor gene is altered in MM and MGUS. Six myeloma cell lines, bone marrow mononuclear cells (BMMNC) of 165 MM and 25 MGUS patients were subjected to this study after obtaining informed consent. Isolated CD138 positive plasma cells (PC) of 24 MM were examined to see if the alteration occurred really in myeloma cells. Using methylation specific PCR, WWOX promoter methylation was detected in 2 of 6 cell lines, 35% of MM patients and 8% of MGUS patients (p=0.01). The overall survival of the MM patients with methylated WWOX tended to be worse than with unmethylated WWOX (p=0.2). Using nested RT-PCR, aberrant short transcripts of WWOX lacking exons coding SDR domain were detected in 4 cell lines, 68% of MM and 60% of MGUS (p=0.51) In isolated PC, aberrant transcripts of WWOX were detected in 58% of MM and 25% of MGUS (p=0.02). Real time PCR demonstrated higher WWOX expression in isolated PC of MM and MGUS than of BMMNC other than plasma cells (p=0.0001), and those higher WWOX were aberrant type (p=0.001). As aberrant WWOX is known to function as dominant negative for wild type, our results imply that this aberrant WWOX is an oncogene as well as losing tumor suppressor function. Since WWOX is demonstrated to regulate beta-catenin and NF-kB pathway, high frequency of aberrant WWOX expression indicates an important role in myeloma development. Disclosures:No relevant conflicts of interest to declare.

Unicentric castleman's disease located in the lower extremity: a case report

BackgroundCastleman's disease is a rare form of localized lymph node hyperplasia of uncertain etiology. Although the mediastinum is the most common site of involvement, rare cases occurring in lymph node bearing tissue of other localization have been reported, including only a few intramuscular cases. Unicentric and multicentric Castleman's disease are being distinguished, the latter harboring an unfavorable prognosis.Case PresentationHere, we present a case of unicentric Castleman's disease in a 37-year-old woman without associated neoplastic, autoimmune or infectious diseases. The lesion was located in the femoral region of the right lower extremity and surgically resected after radiographic workup and excisional biopsy examinations. The tumor comprised lymphoid tissue with numerous germinal centers with central fibrosis, onion-skinning and rich interfollicular vascularization. CD23-positive follicular dendritic cells were detected in the germinal centers and numerous CD138-positive plasma cells in interfollicular areas. The diagnosis of mixed cellularity type Castleman's disease was established and the patient recovered well.ConclusionsIn conclusion, the differential diagnosis of Castleman's disease should be considered when evaluating a sharply demarcated, hypervascularized lymphatic tumor located in the extremities. However, the developmental etiology of Castleman's disease remains to be further examined.

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.

M protein deposition in the skin: a rare manifestation of Waldenström macroglobulinemia

Waldenstrom macroglobulinemia (WM) is defined by diffuse and nodular bone marrow infiltration of lymphoplasmacytic lymphoma as defined by the World Health Organization (WHO), and by a monoclonal IgM serum paraprotein (macroglobulin) [1]. Clinical symptoms at presentation are frequently unspecific, including fatigue, weakness, and weight loss. Clinical symptoms such as hyperviscosity syndrome, peripheral neuropathy, and glomerular disease are often related to the IgM paraprotein, both in form of circulating IgM and IgM deposition in tissue. The skin constitutes an unusual site of manifestation of WM, both for tumor infiltrates and IgM paraprotein deposition. A 70-year-old male patient was hospitalized in April 2009 with progressive peripheral neuropathy and poor general condition. Further examination revealed small, non-pruritic, soft indolent wart-like papules on both knees. As noted by the patient, the lesions were easily scratched off, bled slightly, leaving scabs and causing additional papules to appear during healing. Laboratory findings included slight anemia with a hemoglobin level of 8.2 mmol/l (normal 8.4–10.9 mmol/l) and a hematocrit reading of 0.379 l/l (normal 0.400–0.530 l/l). The RBC count was 4.22 9 10/l (normal 4.50–5.90 9 10/l) with 3% reticulocytes (normal 6–18%). The thrombocyte count and the differential white blood count were within the normal range. Renal, hepatic and cardial functions were also normal, and there was no splenomegaly or lymphadenopathy. The total protein level was 94.8 g/l (normal 66.0–87.0 g/l), and quantitative immunoglobulin findings were as follows: IgM 23.70 g/l (normal 0.40–2.80 g/l), j-light chains 5.63 g/l (normal 1.50–3.90 g/l), free j-light chains 284.00 mg/l (normal 3.30–19.40 mg/l), free k-light chains 13.90 mg/l (normal 5.71–26.30 mg/l) and a free j-light chains/k-light chains ratio of 20.43 (normal 0.26–1.65). Serum electrophoresis presented an elevated IgM gradient. Serum immunofixation provided a clear precipitation pattern of monoclonal IgM j-type immunoglobulin. The b2 microglobulin level was 1.7 mg/l (normal \2.0 mg/l). Bone marrow microscopy revealed atypical lymphoplasmocytic cells. Immunohistochemistry demonstrated low proliferation and B cell nature of the lymphocytes (25% of all hematopoietic cells) beside a mature CD138 positive plasma cell population (15%) with monotypic restriction for j-light chain and IgM heavy chain (Fig. 1). Given these findings, the diagnosis of Waldenstrom’s macroglobulinemia was established. Skin sections displayed multifocal nodular strongly eosinophilic, PASpositive and Elastica van Gieson, Congo red, and Alcian blue-negative homogeneous deposits in the upper portion of the dermis. Immunohistochemistry presented a faint monotypic staining for IgM and j-light chains, manifesting as multifocal nodular depositions of monoclonal immunoglobulin (Fig. 1). A diagnosis of macroglobulinemia cutis presenting as IgM storage papules was therefore established. To suppress disease activity, the patient received four cycles of Rituximab-CHOP, during which the cutaneous lesions disappeared. B. Oberschmid U. Siebolts (&) A. Beller C. Wickenhauser Institute of Pathology, University of Leipzig, Leipzig, Germany e-mail: udo.siebolts@medizin.uni-leipzig.de

An asymptomatic 61‐year‐old man with BCR‐ABL‐positive bone marrow following autologous transplantation for multiple myeloma

An asymptomatic 61‐year‐old man with <i>BCR‐ABL</i>‐positive bone marrow following autologous transplantation for multiple myeloma

Detection of Minimal Residual Disease of Plasma Cell Myeloma by Five Color Flow Cytometric Immunophenotype Analysis

AbstractAbstract 1903Flow cytometry is widely used to identify a monoclonal proliferation of plasma cells through cytoplasmic immunoglobulin light chain analysis, with a better sensitivity than immunocytochemical staining. Recent studies have demonstrated that neoplastic plasma cells express aberrant surface antigens and immunophenotyping of plasma cells by multiparameter flow cytometry is able to reveal neoplastic plasma cells by their surface antigen profile to a level that is meaningful for the detection of prognostically relevant minimal residual disease. In this study, we compare the sensitivity of detecting abnormal plasma cells by five color flow cytometric immunophenotyping and concurrent cytoplasmic immunoglobulin light chain analysis. Multiparameter analysis was performed with cell surface markers CD45, CD38, CD138, CD19, CD20, CD56, CD117, CD27 and CD28. Plasma cells were identified by bright CD38 and CD138 expression. The bone marrow plasma cells from 8 newly diagnosed or recurrent plasma cell myelomas with 10% or more morphologically identifiable plasma cells were initially analyzed. At least one antigen was found to be abnormally expressed in all 8 cases. CD45, CD19, CD56 and CD117 were most useful in recognizing abnormal plasma cells. Both CD45 and CD19 were negative in all 8 cases. CD56 and CD117 were each positive in 6 cases; at least one of them was positive in all 8 cases; and 4 cases co-expressed both antigens. Thirteen additional cases of plasma cell myeloma in clinical remission with less than 10% plasma cells by bone marrow morphology were studied with antibodies to CD38, CD138, CD45, CD56 and CD117 in a single tube. Eleven cases revealed an abnormal immunophenotype, however, immunoglobulin light chain restriction was detected only in 6 cases. Two cases demonstrated normal phenotype and did not show immunoglobulin light chain restriction. Immunoglobulin light chain restriction was not demonstrated in any cases with less than 0.5% bright CD38 plasma cells. In one case with 9% plasma cells by morphologic examination, the immunoglobulin light chain analysis failed to reveal monoclonal proliferation whereas abnormal expression of both CD56 and CD117 was identified in 50% of the bright CD38 and CD138 positive plasma cells, although flow cytometry only detected a total of 0.5% plasma cells. Abnormal phenotype was detected at a level as low as 0.05% plasma cells by flowcytometry, in cases that less than 1% plasma cells were identified by morphologic examination. Our result suggests that 5 color flow cytometric immunophenotyping is a sensitive and practical way to detect minimal residual disease of plasma cell myeloma in patients under clinical remission. Because rare neoplastic plasma cells may not have abnormal surface antigen profile, and the abnormal phenotype may change after chemotherapy, combination with cytoplasmic immunoglobulin light chain analysis may be necessary to increase the sensitivity and specificity. Disclosures:No relevant conflicts of interest to declare.