Cytoplasmic Light Chain Research Articles

CD69 Expression Predicts Favorable Outcome in Multiple Myeloma Patients Treated with VTD

▪Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (PC). Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification but the clinical and prognostic value of immunophenotyping in MM remains questionable. We have analyzed the prognostic impact of a relatively new marker as CD69. CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous laboratoristic data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. In a previous abstract, we described for the first time in a clinical report, the CD69 expression on pathological PCs of MM patients. Our preliminary data also suggested an intriguing role of CD69 in patients treated with chemotherapy in different stages of disease. In this study, we report a larger setting of 97 patients where we confirmed the expression of CD 69 in 48 of them (49%) (see table I). Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was > 20%.After an induction regimen of treatment with four cycles of VDT (bortezomib, dexametasone, thalidomide), 69 patients were evaluable. 40/69 (65%) of patients obtained at least of a very good partial response or better (Responding pts). In this subgroup of patients 30/45 (66.6%) showed the expression of CD 69. On the contrary only in a little part of partial or less responding patients (NR pts) 9/24 (37.5%) CD69 was detected (see table II) (p=0.02 using a chi squared test and p=0.019 using a Fisher's exact test). Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in >90% of patients. Clinical data were available in all patients and CD69 maintained its association with different response, independently of other prognostic variables.In conclusion CD69 is often expressed in PCM cases, and the expression of this marker is useful to reveal poor prognostic categories and delineate a risk stratification. This molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs.Table IPts CharacteristicsTotalCD69+9748/97SexMale51(52%)Female46(48%)Clinical statusMM non evaluableMM after VTD28/9769/97in VGPR/CR 45/69in PR/SD/PD 24/69Table IIPts treated with VTDResponding ptsNRpts4524CD69+ 39/6930/459/24CD69- 30/6915/4515/24 DisclosuresNo relevant conflicts of interest to declare.

Inconclusive flow cytometric surface light chain results; can cytoplasmic light chains, Bcl-2 expression and PCR clonality analysis improve accuracy of cytological diagnoses in B-cell lymphomas?

BackgroundFlow cytometric immunophenotyping (FCI), is widely used in cytology for distinguishing between B-cell lymphoma (BCL) and reactive lymphocytic proliferations (RLP), mainly by identifying monotypic B-cell populations. Since this cannot always be determined by ratios of surface immunoglobulin light chains (sIg LCs) we wanted to assess if cytoplasmic immunoglobulin (cIg) LCs, Bcl-2 and polymerase chain reaction (PCR) based clonality analysis can improve accuracy of cytological diagnoses of BCL.MethodsOur study included 98 fine needle aspiration biopsies from lymph nodes suspicious for BCL with inconclusive sIg LCs. In all cases PCR clonality analysis was performed in order to determine immunoglobulin heavy chain (IGH) gene and T-cell receptor (TRC) gene rearrangement. In selected cases expression of Bcl-2 and cIg LC were determined by FC.ResultsThirty patients had lymphoma and 68 had reactive lymphocytic proliferations. Three patterns of sIg LCs staining were found: negative, dual positive and difficult to interpret. Percentage of lymphomas was highest in the dual positive group (75 %). Morphology coupled with cIg LCs determination and/or Bcl-2 expression was able to give a correct diagnosis in 83 % of cases. Molecular tests would have been misleading in 15 % of cases because 7/30 BCL were polyclonal and 8/68 RLP were monoclonal.ConclusionsDetermination of cIg LCs, Bcl-2 expression and PCR clonality analysis of B cells improved accuracy of cytological diagnoses in BCL with inconclusive sIg LC. However, clonality determined by PCR was misleading in some cases.

The Reversal of Renal Impairment and its Impact on Survival in Newly Diagnosed Multiple Myeloma Patients

Background: Proliferation rate of plasma cells (PC) is a powerful tool in evaluating all types of monoclonal gammopathies, predicting for progression as well as survival. A flow cytometry (FC)-based method to delineate the cell cycle of clonotypic PCs and determine the proportion in S-phase was developed. This method replaces a labor-intensive slide based methodology, but the prognostic power of this FC approach has not been clearly elucidated. Methods: A total of 1061 patients who had bone marrow (BM) FC PC proliferation analysis at Mayo Clinic, Rochester from May 2012 to December 2013 were studied of who 257 had relapsed disease (RMM). PC proliferation was measured by 8-color FC using antibodies to CD19, CD38, CD138, CD45, and cytoplasmic kappa and lambda Ig light chains, and DAPI DNA staining. The DNA content of the abnormal, clonotypic PCs was specifically analyzed and the %S-phase was determined by measuring the proportion of cells between G0/G1 and G2/M. Patients with inadequate plasma cells for assessment of %SP were designated as 0%. Results: The median follow up from testing for the entire cohort was 13 months (95% CI; 12.7, 13.3); 156 (15%) had died. We specifically examined the clinical features and outcomes based on two cutoffs, %SP 1 and 2. Among relapsed disease, %SP was prognostic for OS using both cutoffs (Table). In univariate analysis, %SP 2, B2microglobulin >5.5, HR FISH (t4; 14, t14;16, t14;20 & del17p) and LDH>221 were all significant, but only %SP 2 was significant in multivariable analysis. Conclusion: The %S-phase of clonal PCs determined by FC is of prognostic value in patients with RMM. This is particularly relevant because it is independent of some of the other known poor prognostic factors such as high risk FISH.

CD69, a New Potential Clinical Marker in Multiple Myeloma

CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. CD69 has not been yet studied in human multiple myeloma, though it has been shown that human chronic lymphocytic lymphoma cells, when induced toward a plasma cell phenotype with tetradecanoyl phorbol acetate (TPA) have increased CD69 expression. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. Therefore, we analyzed the CD69 expression on pathological PCs, from bone marrow samples of 43 patients, by flow cytometry with two aims: to evaluate the real expression of CD69 on pathological PCs and to determine the clinico-pathological significance of this molecule. Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was > 20%. 22 of 43 pts (see table I, group A) were MM resistant/refractory to at least two different chemotherapy regimens (including bortezomib in all patients). 21 patients (table I, group B) were smouldering multiple myeloma (SMM) or MM in at least very good partial response (VGPR) after first line treatment. CD69 was detected on bone marrow PCs in 19 of the 43 patients evaluated (44%). Of the 19 patients with CD69+ (see table II) only 6 (27%) were in the group of refractory/resistant MM, while the majority of these advanced patients, 16/22 (73%), had an absent expression of CD69. On the contrary in the group of SMM/VGPR/CR MM 13 patients (62%) were CD69+ (p=0.04, using a Chi squared test with Yates correction). At the best of our Knowledge this is the first clinical report that confirms CD69 expression on pathological PCs of MM patients. Our preliminary data also suggest an intriguing role of CD69, this molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table IPts CharacteristicsGroup AGroup B2221SexMale8(36%)11(52%)Female14(64%)10 (48%)Clinical statusSMMMM inVGPR/CR9 (43%)12 (57%)Relapsed/refractory22(100%)Number of Previous Therapy (range)3,5 (2-6)1 (0-1)Previous Bor regimenSMM0MM inVGPR/CR12(100%)Relapsed/refractory22(100%)Previous Lena regimenSMM0MM inVGPR/CR0Relapsed/refractory17(77%)Table IIPts ResultsGroup AGroup B2221CD69+ 19/43 (44%)6 (27%)13 (62%)CD69-24/43 (56%)16 (73%)8 (38%) DisclosuresNo relevant conflicts of interest to declare.

Inability of Monoclonal Anti‐light Chain Antibody to Detect Clonal B‐Cells in a Patient with Follicular Lymphoma by Multicolor Flow Cytometry

Our recent publication "Inability of a monoclonal anti-light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry," underlined the importance of choice of antibodies to detect cytoplasmic light chains. Our present study extends this finding for detection of surface immunoglobulin (SIg) light chains on clonal B-cells. Multicolor flow cytometry was used for analyzing bone marrow (BM) from a patient with a CD10-positive follicular lymphoma for infiltrating clonal B-cells. In the BM aspirate, B cells could be identified expressing CD19, CD10, and high levels of CD20. No SIg light chain expression was found on this population of B cells employing monoclonal antibodies. Re-analysis using polyclonal antibodies against SIg light chains, revealed presence of lambda light chains on the CD10positive B-cells. These data illustrate when antibodies against SIg light chains are employed for B-cell clonality assessment, polyclonal antibodies are preferred over monoclonal antibodies.

Inability of a monoclonal anti‐light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry

Multicolor flow cytometry (MFC) is increasingly important for the diagnosis and minimal residual disease (MRD) assessment of patients with plasma cells (PC) dyscrasias, like multiple myeloma. Recently published information shows that immunophenotype of myeloma PC can change over time and normal PC are heterogeneous in the expression of CD19 and CD56. This implies that for a sensitive, reliable detection of MRD clonality assessment by the detection of cytoplasmic kappa and lambda light chains is advisable. Eight-color MFC was used to detect normal and myeloma PC by the expression of CD38 and CD138. Analysis of additional surface antigens like CD45, CD19, CD56, CD27, and the intracellular immunoglobulin light chain distribution were used to differentiate polyclonal from clonal PC. Absence of cytoplasmic light chains expression in a PC subpopulation with an abnormal phenotype suggested the presence of non-secretory plasma cells in the bone marrow (BM) of this patient. This observation however, was contradicted by the presence of free lambda light chains in the patient's serum. After repeating the analysis with polyclonal antibodies against intracellular immunoglobulin light chains instead of monoclonal antibodies, the abnormal PC subpopulation appeared to express lambda light chains. These data illustrate that if clonality assessment of PC is included in disease monitoring, the use of polyclonal over monoclonal antibodies is preferred for the detection of intracellular immunoglobulin light chains.

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.

Complex karyotype and translocation t(4;14) define patients with high-risk newly diagnosed multiple myeloma: results of CMG2002 trial

The prognostic impact of chromosomal abnormalities was evaluated by fluorescence in situ hybridization with cytoplasmic immunoglobulin light chain staining (cIg-FISH) and by classical metaphase cytogenetics in a cohort of 207 patients with newly diagnosed multiple myeloma who were treated with high-dose therapy followed by autologous stem cell transplantation in the CMG2002 clinical trial. The incidence of chromosomal abnormalities detected by FISH was as follows: 52.7% for del(13)(q14), 6.5% for del(17)(p13), 18.6% for t(11;14)(q13;q32), 22.8% for t(4;14)(p16;q32) and 45.7% for gain(1)(q21). Metaphase cytogenetic analysis revealed a complex karyotype in 19.1% and hyperdiploidy in 21.7% of patients. The overall response rate was not influenced by the presence of any studied chromosomal abnormality. Patients with a complex karyotype, those with translocation t(4;14) and those with gain of the 1q21 locus had a shorter time to progression (TTP) and overall survival (OS). Other genomic changes such as translocation t(11;14) and del(13q) had less impact on TTP and OS. In multivariate analysis, complex karyotype, translocation t(4;14) and β2-microglobulin level > 2.5 mg/L were independent prognostic factors associated with poor overall survival. Their unfavorable prognostic impact was even more pronounced if they were present in combination. Patients with t(4;14) present together with a complex karyotype had the worst prognosis, with a median OS of only 13.2 months, whereas patients with a normal karyotype or karyotype with ≤ 2 chromosomal changes had the best outcome, with 3-year OS of 85.9%. In conclusion, complex karyotype, gain of 1q21 region and translocation t(4;14) are major prognostic factors associated with reduced survival of patients with newly diagnosed multiple myeloma treated with autologous stem cell transplantation.

Investigation of 1q21 amplification in patients with multiple myeloma using I-FISH and cIg-FISH

To investigate the prevlance of 1q21 amplification in patients with multiple myeloma (MM) and its correlation with the progression and prognosis of the disease. 1q21 amplification was detected in 48 patients with MM using cytoplasmic light chain immunofluorescence with fluorescence in situ hybridization analysis (cIg-FISH) and interphase fluorescence in situ hybridization (I-FISH) analysis combined with CD138 immunomagnetic cell sorting (MACS). 1q21 amplification (≥ 3 red signals) was detected in 26/48(54.2%) cases by cIg-FISH and 31/48 (64.6%) cases by I-FISH combined with CD138 MACS. There was a good consistency between the two methods (P>0.05). The mortality of patients with 1q21 amplification was significantly higher than those without (P< 0.05). No significant difference was detected in terms of sex, age, Durie-Salmon stage, subgroup and international staging system (ISS) stage between patients with 1q21 amplification and those without (P>0.05). The frequency of 1q21 amplification in MM is high. There was also an association between the amplification and poor prognosis. cIg-FISH is consistent with CD138 MACS combined with I-FISH.

The immunophenotypic stability of plasma cell myeloma by flow cytometry

Flow cytometry (FC) has become increasingly utilized in the diagnosis and monitoring of plasma cell myeloma (PCM), though few studies have evaluated the longitudinal stability of antigen expression. We studied 45 PCM patients by four-color FC for shifts in CD19, CD20, CD38, CD45, CD56, and cytoplasmic light chain expression, between diagnostic/first encounter and positive follow-up analyses. An immunophenotypic (IP) change was defined as gain, loss, or ½ log shift of antigen expression. An IP change was observed in 14/45 (31%) patients, with single IP changes in 9/14, two changes in 2/14, and three changes in 3/14. 3/14 reverted from an aberrant to a normal plasma cell IP, while remaining light chain-restricted. Changes in expression of CD45 occurred in 9/45 (20%), CD19 in 5/45 (11.1%), CD20 in 2/45 (4.4%), and CD56 in 5/45 (11.1%). Approximately 1/3 of PCM cases show IP changes over time, with CD45 the least stable antigen. Recognition of this relative instability is important to avoid narrow targeting of follow-up FC analyses, especially for minimal residual disease monitoring.

Detection of bone marrow involved by nonhematopoietic neoplasms using flow cytometry

Objective To study the value of flow cytometry in identifying metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Methods Twenty-six cell lines representing ten epithelial neoplasms, one lymphoma cell line and one human T cell lymphoblast-like cell line were purchased from American Tissue Culture Collection. From July 2009 to June 2010, five nonhematopoietic neoplasms,fifteen hematopoietic neoplasms and fifteen control patients with complete remession after hematopoietic stem cell transplantation were collected in Beijing Daopei Hospital. Cryopreserved cell lines were thawed and cultured until they entered log phase. After permeabilization, cell lines were analyzed by staining with cytoplasmic CK-FITC antibody using four-color flow cytometer. The percent CK positivity was measured by comparing with negative control. Bone marrow samples were stained with membrane and cytoplasmic antibodies according to our routine methods. Based on lineage markers and blast markers as well as CK expression, the relevant hematopoietic diseases were diagnosed or excluded according to 2008 World Health Organization diagnosis standards. Results All epithelial neoplasm cell lines expressed CK, with average positive percentage 81.1%. All the lymphoid tumor cell lines didn't express CK. Two epithelial neoplasms were CK positive, 100. 0% in thyroid carcinoma and 98. 2% in lung carcinoma, respectively. Hematopoietic tumor and control samples didn't express CK. They expressed relevant hematopoietic markers, such as CD45 as well as lineage markers, or CD138 and cytoplasmic immunoglobulin light chain. Three nonepithelial nonhematopoietic neoplasms didn't express CK. CK positive or negative nonhematopoietic neoplasms didn't express hematopoietic markers such as CD45, HLA-ABC and HLA-DR DP DQ, as well as lineage specific markers. Besides, CK positive might be helpful to suggest epithelial origin. Conclusion Flow cytometry with hematopoietic markers and CK can effectively exclude hematopoietic tumor and identify metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Key words: Neoplasms, glandular and epithelial; Bone marrow neoplasms; Neoplasm invasiveness; Keratins; Flow cytometry

Increased Copy Number of the Interleukin-6 Receptor Gene Is Associated with Adverse Survival in Multiple Myeloma Patients Treated with Autologous Stem Cell Transplantation

Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates plasma cell (PC) growth via the IL-6 receptor (IL-6R). We hypothesized that up-regulation of IL-6R in myeloma cells might confer the growth privilege to myeloma cells over bone marrow (BM) hematopoietic cells. We investigated the frequency and prognostic implication of increased copy number of the IL-6R gene by fluorescence in situ hybridization (FISH) in patients with newly diagnosed multiple myeloma (MM). One hundred two patients with newly diagnosed MM were enrolled. The FISH study for IL-6R was performed using a homemade bacterial artificial chromosome (BAC) probe for IL6R at chromosome 1q21. FISH signals were counted among BM plasma cells sorted by cytoplasmic immunoglobulin light chain staining (cIg FISH). The amplification of IL-6R was detected in 53/102 patients (52.0%). The 5-year overall survival (OS) rate of patients with IL-6R gene amplification was 41.3% versus 44.8% for those with a normal IL-6R (P = .425). In 44 patients treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT), patients with ≥3.1 copy numbers of IL-6R per PC showed adverse 5-year OS compared to those with <2.1 copies of IL-6R gene (44.4% versus 78.0%, P = .024). In multivariate analysis, the increase of IL-6R copy numbers (mean copy/PC ≥3.1) could be considered as an independent prognostic factor for MM patients who underwent ASCT. The gain of the IL-6R gene was frequent in myeloma, showing an association with adverse prognosis in myeloma patients treated with ASCT. These findings suggest the potential role of IL-6R in myeloma cell growth and therapeutic implications of the IL-6R blocker in the future.

Immunophenotypic profile of plasma cell leukemia: A retrospective study in a reference cancer center in India and review of literature

Plasma cell leukemia (PCL) is a rare but aggressive subtype of plasma cell dyscrasia. It is known to present with highly variable morphological features and may mimic with other lymphoid neoplasms. Multicolor flow cytometry (MFC) with availability of newer markers is highly useful in the diagnosis of the plasma cell leukemia. We present an immunophenotypic profile in ten cases of PCL along with their clinical and laboratory findings. We retrospectively studied immunophenotypic profile of 10 cases of plasma cell leukemia (out of 4615 cases of hematolymphoid neoplasms) using five parameter, three color flow cytometric analysis. We also studied their clinical presentation and other laboratory findings. Common clinical features at presentation were weakness, bone pain, anemia, thrombocytopenia and osteolytic lesions. Plasma cell population was identified on strong expression of CD38 and co-expression of CD38 and CD138. CD56 was expressed in 44% cases. CD19 and CD20 were negative in all cases. Surface light chain restriction was seen in 50% cases and in remaining 50% cases revealed cytoplasmic light chain restriction. CD117 was expressed in one out of two cases studied. MFC immunophenotyping is highly useful to differentiate Plasma cell leukemia from other chronic lymphoproliferative disorders with plasmacytoid morphology as well as from non-neoplastic reactive PC and co-expression of CD38 and CD138 is a best combination to identify the plasma cells by MFC.

Intelligent FISH for Myeloma: Enhanced Cytogenetic Aberration Detection with Immunomagnetic Bead Enrichment of Plasma Cells

AbstractAbstract 1918The detection of cytogenetic aberrations in plasma cell neoplasms is definitive for risk stratification. However, metaphase chromosome analysis is limited, due to a low proliferative rate in vitro. Interphase florescent in situ hybridization (FISH) dramatically enhances detection. When detected by FISH, high risk aberrations include complex karyotypes, t(4;14), t(14;16), p53 gene deletions, and hypodiploidy; standard risk aberrations include hyperdiploidy and chromosome 13q deletions detected by FISH only; and low risk aberrations include normal karyotypes, t(6;14), and t(11;14). Despite the utility of interphase FISH in predicting patient outcomes, it is often difficult to detect underlying genetic changes in patients with low levels of clonal plasma cells, such as monoclonal gammopathy of undetermined significance (MGUS) and minimal residual disease (MRD). Plasma cell-targeted FISH can be achieved through 1) simultaneous cytoplasmic light chain staining and FISH, 2) sequential morphologic identification and FISH, 3) immunomagnetic bead depletion of non-plasma cells, or 4) immunomagnetic bead enrichment of plasma cells. Immunomagnetic bead enrichment is superior to other methodologies because it efficiently captures a large number of purified plasma cells for downstream analysis. In this study, we use anti-CD138 coated magnetic beads to enrich bone marrow aspirates containing 1.1% ∼ 5.0% monoclonal plasma cells by flow cytometry, to attain purified samples with approximately 90% plasma cells, for subsequent FISH analysis; we call this procedure Intelligent FISH. We present cytogenetic and morphologic findings on our first 257 cases examined with Intelligent FISH. More than 87% (225/257) of enriched cases exhibit cytogenetic aberrations. In contrast, a matched group of cases analyzed with standard FISH exhibit cytogenetic aberrations in only 31.6% (79/250). These results demonstrate that patients with low levels of plasma cells and normal FISH results may be misclassified as lower risk when they should be considered higher risk, due to lack of sensitivity of conventional FISH techniques. Among our enriched cohort, 22% (57/227) exhibit high risk aberrations, 47% (122/257) exhibit standard risk aberrations, and 30% (78/257) have low risk aberrations. We assess morphologic and immunophenotypic features observed within the different risk groups. These studies show that Intelligent FISH is an efficient and highly valuable technique for accurate risk stratification and detection of minimal disease in patients with plasma cell neoplasms. In particular, it can provide prognostic guidance previously unavailable for the clinical management of MGUS patients. In conclusion, plasma cell enrichment followed by FISH should be incorporated into the standard work-up of MGUS and MRD patients to accurately assess risk, follow-up interval, and treatment options. Disclosures:Keen-Kim:Genoptix Medical Laboratory: Employment. Kaplan:Genoptix Medical Laboratory: Employment. Siva:Genoptix Medical Laboratory: Employment. Wolfe:Genoptix Medical Laboratory: Employment. Nooraie:Genoptix Medical Laboratory: Employment. Chan:Genoptix Medical Laboratory: Employment. Mohrmann:Genoptix Medical Laboratory: Employment.

PH-regulated direct interaction of adenovirus hexon with cytoplasmic dynein intermediate and light intermediate chain subunits

pH-regulated direct interaction of adenovirus hexon with cytoplasmic dynein intermediate and light intermediate chain subunits

IgM Myeloma: Report of Two Cases of a Rare Entity with Unusual Immunophenotype.

Abstract 4901 IntroductionPlasma cell myeloma is characterized by clonal proliferation of plasma cells and serum and/or urine paraprotein, most commonly IgG, IgA or light chain only. IgM, IgD, IgE and non-secretory variants are infrequent. IgM myeloma comprises less than 0.5% of myelomas. These should not be confused with B-cell non-Hodgkin lymphoma showing plasmacytoid differentiation and an IgM paraprotein, particularly lymphoplasmacytic lymphoma. Two cases are reported herein. Case 1A 57 year old female with known MGUS, type II diabetes, rheumatoid arthritis, underwent total thyroidectomy for papillary thyroid carcinoma. Two months later she required excision of a cervical lymph node, identified by radioactive iodine scanning, revealing metastatic papillary thyroid carcinoma. However, during pre-operative evaluation, cytopenias were noted (hemoglobin 7.8 g/dL, platelets 86 × 109L), and renal insufficiency (creatinine 2.1 mg/dL). Subsequent laboratory findings included a serum IgM kappa paraprotein of 6158 mg/dL, normal IgA, decreased IgG of 423 mg/dL, elevated beta-2 microglobulin (7.7 mg/L), elevated serum viscosity (3 cp), mildly decreased serum calcium (8.2 mg/dL). A skeletal survey was negative. Pathologic findings: Peripheral blood showed pancytopenia, marked RBC rouleaux, and occasional atypical plasmacytoid cells (5%). Bone marrow biopsy sections revealed marked hypercellularity with a predominance of plasmacytoid cells. By immunohistochemistry (IHC), these were CD138+, CD117+. MUM1+, weak cytoplasmic kappa+, weak CD79a+, CD45-, CD19-, CD20-, PAX 5-, CD5-, CD10-, CD23-, CD56-, cyclin D1-. Flow cytometry (FC) of peripheral blood also revealed abnormal cells, of comparable phenotype (CD45-, CD19-, CD20-, CD38+, CD138+, CD56-), though CD117 and cytoplasmic light chain expression were not evident. Clinical course: Management included plasma exchange for serum hyperviscosity, pulse steroids and thalidomide. Despite these interventions, her condition worsened, including a progressive decline in renal function, requiring hemodialysis. Eventually she declined further treatment, and expired two months after diagnosis. Case 2An 83 year old male patient presented with fatigue, dyspnea, diffuse pain, paresthesias involving hands and feet of two weeks duration. Past history was remarkable for COPD, lower extremity DVT, and hyperlipidemia. Physical examination revealed no lymphadenopathy or organomegaly. Laboratory studies revealed pancytopenia, IgM kappa paraprotein of 4664 mg/dL, normal serum IgG, diminished IgA, elevated serum creatinin (1.7 mg/dL), normal serum calcium, elevated beta-2 microglobulin (4.9 mg/L) and elevated serum viscosity (3.4 cp). A skeletal survey was negative. Pathologic findings: Peripheral blood revealed pancytopenia with marked RBC rouleaux. Marrow aspirate smears showed abundant abnormal plasma cells, many showing numerous, azurophilic, crystalline cytoplasmic inclusions, Auer rod-like. Bone marrow biopsy was hypercellular and heavily infiltrated by abnormal plasma cells. By IHC, these were CD138+, MUM1+, cyclin D1+, CD56+, cytoplasmic kappa+, CD20+ (sub-set), weak CD117+. FC of marrow aspirate yielded comparable results (CD56 co-expression, bright CD38+ and CD138+, CD19-, CD20-, CD45-). Conventional cytogenetics revealed a normal male karyotype. Interphase FISH revealed t(11; 14), trisomies 11, 13, 14 and 17, with 13q14 and 17p13 deletions. Clinical course: The patient rapidly deteriorated, and expired shortly after hospital admission. ConclusionWe describe two cases of IgM myeloma. The diagnosis in each case was supported by morphology and immunophenotype and, in one case, by interphase FISH. These add to our limited knowledge regarding the biology of this disorder. Recently, it has been suggested that such cases are of relatively uniform immunophenotype, in particular lacking either CD56 or CD117 expression (Feyler S et al, BJH 140, 547-551, 10/2007). Our cases show that the spectrum of immunophenotypic findings in IgM myeloma is broader than has been suggested. In addition, one of the two cases showed Auer-rod like crystalline cytoplasmic inclusions in malignant plasma cells, also a rare finding in this setting. DisclosuresNo relevant conflicts of interest to declare.

Sinonasal extramedullary plasmacytoma

An 81-year-old male presented with a four-month progressive right-sided nasal obstruction and epistaxis due to a polyp. He received antibiotics and intranasal steroid inhalers, without improvement. He had no known history of paranasal sinus disease or allergies. Rhinoscopy revealed an amber polypoid mass with a clear surface, filling the entire nasal cavity. Comprehensive examination was otherwise unremarkable. CT scan demonstrated opacification of the right nasal fossa and the maxillary sinus, without bony erosion. Surgical pathology revealed a polypoid structure lined by attenuated cuboidal epithelium, while its core was massively infiltrated by a relatively monotonous population of smallto medium-sized cells (Fig 1A). The lesional cells illustrated a diffuse membranous staining for CD138 (Fig 1B); further kappa light chain staining highlighted their cytoplasm. Pathologic diagnosis was sinonasal (extramedullary) plasmacytoma. This rare monoclonal gammopathy presents in the head and neck region in 80 percent of the cases, predominantly in the sinonasal cavities. Patients are typically older (median age: 55 yrs), and 75 percent are males. Variable symptoms include nasal obstruction, recurrent rhinosinusitis, and epistaxis. This abnormal accumulation of plasmoblasts is triggered by chemotaxis of immune system cells to the sinonasal mucosa, which leads to an extensive inflammatory reaction and angiogenesis. Presentation can be either an isolated solitary mass or in association with other gammopathies, most commonly multiple myeloma. Differentiation of extramedullary plasmacytoma from other hematological malignancies, such as extranodal marginal zone lymphomas or low-grade lymphomas with plasmacytoid differentiation, is made by immunohistochemistry and flow cytometry. Plasma cell tumors express both CD138 and monoclonal cytoplasmic light chain, either k or l. Treatment includes radiotherapy and/or surgery, with a similar recurrence rate of 12.5 percent. Several authors consider the combination of these modalities as the preferred treatment. The high affinity to the sinonasal tract can be attributed to the rich lymphoid deposits and their role in humoral immunity. The work received the approval of the Institutional Review Board.

1p21 deletions are strongly associated with 1q21 gains and are an independent adverse prognostic factor for the outcome of high-dose chemotherapy in patients with multiple myeloma

Deletions involving chromosome 1p are frequent events in multiple myeloma (MM). As karyotyping and single nucleotide polymorphism-based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we investigated the prevalence and prognostic significance of del(1p21) in 203 MM patients undergoing high-dose therapy and autologous SCT. 1p21 status was also evaluated in 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 41 patients with plasma cell leukemia (PCL). FISH combined with cytoplasmic light chain detection (cIg-FISH) detected hemizygous 1p21 deletions in 18% of the MM, 34% of PCL but none of the MGUS cases. The presence of 1p21 deletions was correlated with 1q21(CKS1B) amplification (P=0.01), and del17p(TP53) (P=0.05) but not with del(13q), t(11;14) or t(4;14). Patients with 1p21 deletions had significantly shorter progression-free survival (PFS; median 14.2 vs 25.4 months, P<0.001) and overall survival (OS; median 39.4 vs 82.3 months, P=0.001) than those without such deletions. In multivariate analysis, del(1p21) was an independent risk factor for PFS (P= 0.003) and OS (P=0.013) after adjusting for del(13q), del(p53), t(4;14) and 1q21 amplifications. Our results indicate that del(1p21) is an independent poor prognostic factor associated with disease progression in MM.

Primary Plasma Cell Leukemia Presented as Progressive Paraplegia: A Case Report

Plasma cell leukemia (PCL) is a rare plasma cell disorder. It is the leukemic variant of multiple myeloma. A 52-year-old man with an atypical presentation of primary plasma cell leukemia is reported. The patient presented with paraparesis which progressively worsened to paraplegia. MRI of the spine showed an extradural mass causing cord compression and multiple bony erosions from soft tissue masses. Peripheral blood film examination and bone marrow aspiration showed numerous plasmablasts. Atypical cells expressed surface and cytoplasmic lambda light chain on immunochemical studies, surface CD45 and CD38. To our knowledge, primary PCL presenting with progressive paraplegia has not been reported in the literature.

ALK-Positive Diffuse Large B-Cell Lymphoma: Report of Four Cases from Peru

BACKGROUND: Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma (ALK-DLBCL) is a rare lymphoma with several differences from ALK-positive anaplastic large cell lymphoma (ALCL). ALK-DLBCL appears as a defined disease entity characterized by the expression of the ALK protein, usually in a granular cytoplasmic fashion. These cells are usually CD20-negative but express plasma cells markers. The most common chromosomal abnormality is a fusion between the ALK and the clathrin gene. Clinically, ALK-DLBCL presents as lymph node-based disease affecting mostly middle-aged men, frequently showing aggressive behavior, a negative prognosis at advanced stage and a strikingly rare bone marrow involvement. Only 44 cases are currently reported in the English and non-English literature.METHODS: This is a retrospective study with data collected on four patients with ALK-DLBCL between 2002 and 2008 in a general hospital from Peru. Immunohistochemical studies were performed to detect expression of B-cell, T-cell, plasma cell markers and oncoviruses (i.e. EBV and HHV8). FISH cytogenetic studies were attempted but were unsuccessful. Clinical characteristics of the cases included age, sex, primary site of involvement, IPI score, Ann Arbor stage, therapy, survival in months and final outcome.RESULTS: The male:female ratio was 1:1; one 13-year-old girl and three adults of 27, 41, and 70 years of age were identified. Two patients had primary extranodal disease (multifocal bone lesions and right nasal fossa involvement) and the remaining two patients had exclusively nodal disease. Stages were I (n=1), II (n=1), III (n=1) and IV (n=1). Two patients had increased LDH levels and three patients reported B symptoms. IPI scores were 0 (n=1), 2 (n=1) and 3 (n=2). All cases exhibited immunoblastic/plasmablastic morphology. By immunohistochemistry the cases showed positive expression of cytoplasmic ALK-1, CD138, monoclonal cytoplasmic light chain, CD45 and EMA. All cases lacked expression of the CD3, CD20 and CD30 antigens. HHV8 and EBV were not expressed. The survival times ranged from 11 to 72 months; the latter was observed in a patient who presented with stage III disease and is still alive without evidence of lymphoma.CONCLUSIONS: ALK-DLBCL is a distinct subtype of B-cell lymphoma, which does not express CD20 and is characterized by marked plasmacytic differentiation. This lymphoma has clinically aggressive behavior and more effective therapies are warranted. However, some cases can have prolonged survival with intensive regimens.Table 1. Clinical characterisitics of the reported casesCaseAgeSexPrimary siteIPIStageTherapySurvival (months)Outcome141FNasal fossa0IARadiotherapy13Alive, free of disease213FCervical lymph node2IIBFrance NHL 96–2002*62Alive, free of disease370MCervical lymph node3IIIBCHOP72Alive, free of disease427MBone3IVBHyperCVAD11Alive, with disease*This regimen is based on induction with vincristine, prednisone, cyclophosphamide, daunorubicin, L-asparaginase and methotrexate, followed by consolidation based on cyclophosphamide, cytarabine, metrotexate, then intensification with vincristine and doxorubicin and manteinance based on metrotexate and mercaptopurine.