Quantification of Marrow Plasmacytosis in HIV Patients

Abstract Introduction: A reactive, polyclonal increase in marrow plasma cells in pts with HIV is relatively common, reported to occur in 25% of cases in one series (1) and recently noted to be "usually seen" (2). However, quantification of the degree of marrow plasmacytosis has not been well described. Methods: Pts with a diagnosis of HIV and who had a bone marrow aspirate and biopsy performed at our institution over a 10 year period (2/2004-9/2014) were reviewed. Pts with a diagnosis of myeloma were excluded. A 500 cell count was performed on each marrow aspirate and plasma cell percentage determined. Results: 65 pts were identified; 9 were excluded due to dry taps, so there were 56 evaluable marrow aspirates. Reasons for performing the aspirate and biopsy included: lymphoma staging (NHL in 11/65, HL in 4/65); abnormal SPEP (2/65); abnormal MRI (1/65); FUO (14/65), Kaposi's sarcoma/fevers (3/65), pancytopenia (15/65), thrombocytopenia (6/65), neutropenia (2/65) and anemia (10/65). The average marrow plasma cell percentage was 4.6%, with a range from 0- 21% (median 4%). Only 7/56 (12.5%) had >10% plasma cells. Of these 7 cases, only one had an identifiable cause (multicentric Castleman's) for the plasmacytosis. The 6 others had no neoplastic or infectious etiology, except for HIV itself (including one with 21% plasma cells). All had polyclonal plasmacytosis. Conclusions: In this small series of HIV marrows the degree of plasmacytosis was generally mild (average of 4.6%, which is barely above the upper limit of normal range of 3.5%). However, there were a minority of cases which exhibited significant plasmacytosis (as high as 21%) without any cause other than HIV. This suggests that, while significant marrow polyclonal plasmacytosis is usually not seen in HIV, it may occur as a result of HIV itself without any other identifiable cause. 1) Karchen DS and Frost AR. The bone marrow in HIV-related disease: Morphology and clinical correlation. Am J Clin Path. 1991; 95(1): 63-71. 2) Leibman HA and Tulpule A. "Hematologic manifestations of HIV/AIDS". Chapter 159. Hematology: Basic Principles and Practice, 6th edition. 2013. Disclosures No relevant conflicts of interest to declare.

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Should CD138 Immunohistochemistry be Standard Recommended Practice for Bone Marrow Evaluation of Plasma Cell Neoplasms?

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa161.241 ◽

2020 ◽

Vol 154 (Supplement_1) ◽

pp. S110-S110

Author(s):

A Vijayanarayanan ◽

K Inamdar ◽

M Menon ◽

P Kuriakose

Keyword(s):

Bone Marrow ◽

Plasma Cell ◽

Linear Correlation ◽

Plasma Cells ◽

Pearson Correlation ◽

Protein Electrophoresis ◽

Pearson Correlation Coefficient ◽

Cell Percentage ◽

Significant Linear Correlation ◽

Plasma Cell Neoplasms

Abstract Introduction/Objective Myeloma diagnosis by a pathologist requires 10% plasma cells (PC) or a biopsy proven plasmacytoma in addition to myeloma defining events. PC% > 60% is a biomarker of malignancy under this definition. WHO allows for assesment of plasma cell percentage either by aspirate count or by CD138 immunohistochemistry (IHC). There is lack of consensus on aspirate smear adequacy for PC% estimation. Uneven distribution of plasma cells, hemodilution and/or patchy infiltration can lead to gross underestimation. We compared PC% by aspirate count and CD138 IHC and established corelation with serum protein electrophoresis (SPEP) values. Methods 67 myeloma cases were included after excluding cases with suboptimal or inadequate aspirate smears. Two hematopathologists evaluated the diagnostic marrow (therapy naive) for PC% by aspirate count and CD138 IHC on biopsy/clot section. Corresponding SPEP and Free light chain (FLC) values were obtained. Correlation coefficent was calculated using Pearson correlation coefficient (GraphPad Prism). Results The Ig subtypes included IgG (41/67) and IgA (17/67). 12 cases had available FLC values. Both average and median PC% by CD138 IHC was considerably higher (50%, 52%) compared to aspirate count (29%, 21%). However, PC% by aspirate smear count and CD138 IHC demonstrated a significant linear correlation (r=0.71, p60% by CD138 (and not by aspirate count). Conclusion CD138 IHC based PC% is consistently higher, nevertheless, statistically significant linear corelation is observed between aspirate count PC% and CD138 IHC. A significant linear correlation is observed between CD138 IHC and SPEP (IgG and IgA), however, no such correlation is observed with aspirate count. More cases were diagnosed as myeloma (11%) and higher propotion of cases (35%) had biomarker of malignancy i.e. PC% >60% by CD138 IHC. Based on these findings, we propose estimation of PC% by CD138 immunostain be a recommended standard practice for better clinicopathologic and biologic correlation.

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Do patients with MGUS require a bone marrow biopsy?

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.8117 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 8117-8117

Author(s):

J. Singh ◽

A. K. Malani ◽

C. H. Huang ◽

M. Hashmi ◽

S. C. Mathur ◽

...

Keyword(s):

Bone Marrow ◽

Regression Analysis ◽

Bone Marrow Biopsy ◽

Plasma Cell ◽

Linear Correlation ◽

Plasma Cells ◽

Bone Marrow Biopsies ◽

Cell Percentage ◽

Serum Igg ◽

Immunoglobulin Levels

8117 Monoclonal gammopathy of undetermined significance (MGUS) increase in prevalence with age and it is associated with risk of progression to plasma cell disorder. According to ASH guidelines, patients (pts) should have a complete blood count (CBC), creatinine, calcium, and a complete bone survey and periodic follow up. There has been no clear-cut guideline regarding the role of bone marrow biopsy in these patients. There is suggestion in the literature that bone marrow aspiration and biopsy is indicated if the M protein is 1.5 g/dL. Hypothesis We hypothesize that the increase in serum immunoglobulin is correlated with an increase in plasma cell in the bone marrow biopsy. Methods: We performed a retrospective chart review of 327 MGUS veteran patients seen from 2002 to 2005. Diagnostic criteria for MGUS were defined as <3 g/dL serum monoclonal protein, <10 % plasma cells in the bone marrow and absence of radiographic or laboratory abnormality related to the plasma cell proliferative process. Patients with smoldering myeloma were excluded. Bone marrow biopsies were available on 97/327 patients. Bone marrow biopsy with plasma cell percentage, serum protein electrophoresis (SPEP) and immunofixation (SFE), and immunoglobulin levels of these patients were retrieved and statistical analysis was performed by using Pearson correlation coefficient and linear regression analysis to detect the correlation between plasma cell percentage and immunoglobulin levels. Results: Of the 97 patients whom the bone marrow biopsy was available, 66 patients had IgG, 15 had IgA and 16 had IgM monoclonal paraprotein. There was linear correlation between serum IgG and IgA levels with the percentage of plasma cells in the bone marrow. (p< 0.001 and < 0.02 respectively. By regression analysis, using a cut off value of 10% plasma cells in the bone marrow, the predicted level of IgG and IgA immunoglobulin was 2124 mg /dl and 1564 mg/dl respectively. There was no correlation between IgM immunoglobulin and plasma cell percentage in the marrow. Conclusion: There is a linear correlation between serum IgG and IgA immunoglobulin with plasma cell percentage in the bone marrow. Bone marrow biopsy with plasma cell percentage of 10% or higher may be predicted in patients with MGUS with IgG or IGA above 2g/dl and 1.5g/dl respectively. No significant financial relationships to disclose.

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Significance of Bone Marrow Plasma Cell Percentage in Patients with Monoclonal Gammopathy of Unknown Significance Developing Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.5688.5688 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5688-5688

Author(s):

Mona L Vekaria ◽

Bharat Rao ◽

Philip Kuriakose

Keyword(s):

Risk Factors ◽

Bone Marrow ◽

Bone Marrow Biopsy ◽

Plasma Cell ◽

Monoclonal Gammopathy ◽

Plasma Cells ◽

Time To Progression ◽

Bone Marrow Biopsies ◽

Monoclonal Protein ◽

Cell Percentage

Abstract Introduction: Monoclonal gammopathies are characterized by the detection of a monoclonal immunoglobulin in the serum or urine and underlying proliferation of a plasma cell/B lymphoid clone. (1) Patients with monoclonal gammopathy of undetermined significance (MGUS) have a clonal plasma cell population in the marrow (<10%) and secrete a monoclonal protein in the serum (<3g/dL) and/or urine. However, they lack clinical features of overt Multiple Myeloma (MM) (lytic bone lesions, anemia, renal impairment and hypercalcemia). In a study from the Mayo Clinic, 59 of 241 patients with MGUS (24%) developed MM over a period of 22 years. (2) The interval from recognition of monoclonal protein to diagnosis of MM ranged from 2-29 years, indicating that patients with MGUS need to be followed indefinitely. Many risk factors have been looked at to identify those with MGUS who are at the highest risk to progress into MM. We hypothesize that a higher number of plasma cells would correlate with a greater risk of progression to MM and sought to find out if this could be documented by arbitrarily dividing patients between < or ≥5% plasma cells seen on initial bone marrow biopsy. Methods: We retrospectively reviewed patients diagnosed with MGUS at Henry Ford Hospital between 1999-2013 who underwent a bone marrow biopsy for documenting plasma cell percentage. In addition to this, we also recorded serum hemoglobin, calcium, creatinine, monoclonal protein type and amount, serum free light chains, beta-2 microglobulin and urine for monoclonal protein at the time of diagnosis of MGUS as well as last completed values. For patients that had skeletal surveys we noted if lytic lesions were present at diagnosis, as well as cytogenetics and karyotype evaluations on bone marrow biopsy samples, if completed. Results: 120 patients with bone marrow biopsies were reviewed. Out of this 17 patients were noted from initial bone marrow biopsy to have ≥10% plasma cells. The remaining 103 patients were categorized as having MGUS. While we were not able to complete full statistical analyses, we did note that 14 of these 103 (13.6%) patients went on to develop overt MM. Further evaluation of these patients revealed that 8 of 14 (57%) had bone marrow biopsies showing ≥5% plasma cells. Interestingly the average time to progression into MM in this subgroup was 1,879 days whereas in the 6 of 14 (43%) with bone marrow biopsy showing <5% plasma cells had average time to progression into MM of 1,965 days. Abnormal cytogenetics and karyotypes of the bone marrow biopsy were also seen in 37.5% of the subgroup of patients with ≥5% plasma cells whereas it was only seen in 16.7% of the subgroup of patients with <5% plasma cells. With statistical data analyses we hope to prove significance in the above collected data as well as make further correlations in regards to risk factors in patients with MGUS. Conclusion: While we have not been able to complete full statistical analyses of the collected data yet, basic review of the above patients with MGUS and ≥5% plasma cells in the bone marrow biopsy showed a trend to develop MM faster by an average of 86 days than those that had <5% plasma cells. These same patients also were more likely to have abnormal cytogenetics and karyotypes of their bone marrow biopsies. There is a need for further investigations to be done in patients with MGUS and higher risk features. It is important that hematologists be able to recognize a high risk MGUS patient as this would lead to closer monitoring and consideration for earlier aggressive treatment to potentially delay progression into overt MM. Disclosures No relevant conflicts of interest to declare.

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Bone Marrow Biopsy May Be Required During the Initial Evaluation of Individuals with Asymptomatic Monoclonal Gammopathy

Blood ◽

10.1182/blood.v118.21.5067.5067 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5067-5067

Author(s):

Meletios Athanasios Dimopoulos ◽

Evangelos Terpos ◽

Maria Gkotzamanidou ◽

Evangelos Eleutherakis-Papaiakovou ◽

Magdalini Migkou ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsy ◽

Plasma Cell ◽

Light Chain ◽

Monoclonal Gammopathy ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Incidental Finding ◽

M Protein ◽

Monoclonal Protein

Abstract Abstract 5067 The incidental finding of a monoclonal gammopathy during workup for various conditions or in the context of a routine check-up is increasingly common. Several “patients” are then referred for diagnostic evaluation of their monoclonal gammopathy and additional workup is needed. It has been proposed that a bone marrow (BM) aspirate and biopsy is indicated when the monoclonal protein (M-protein) is ≥1.5 g/dL, when abnormalities are noted in the complete blood cell count, serum creatinine level, serum calcium level, or radiographic bone survey, in individuals with non-IgG monoclonal gammopathy and in those with an abnormal serum free light chain (FLC) ratio. The aim of this study was to identify factors that could aid in the evaluation of individuals presenting with asymptomatic monoclonal gammopathy and in whom invasive diagnostic testing with a bone marrow biopsy is considered. Thus, we analyzed our database and identified patients who were referred to the Department of Clinical Therapeutics of the University of Athens, Greece, for evaluation of asymptomatic monoclonal gammopathy and in whom a BM trephine biopsy, a serum and urine protein electrophoresis (SPEP) with immunofixation and quantitative immunoglobulins were performed. SPEPs were scanned and M-protein was measured using imaging analysis software. Patients with a monoclonal M-protein ≥ 3 g/dl (30 g/L), i.e. those diagnosed with asymptomatic/smoldering myeloma (SMM) or Waldenstrom's macorglobulinemia based on the standard criteria, were not included in the analysis. Clonality of BM plasma cells or lymphoplasmacytes was assessed by immunohistochemistry. Patients who eventually were diagnosed with plasma cell related conditions (i.e. amyloidosis, peripheral neuropathy, dermatoses, etc.) were also excluded from the analysis. Our analysis included 161 patients: 53% were females, median age was 64 year (range 33–89 years), 53% had a monoclonal IgG protein, 15.5% had a monoclonal IgA protein, 24% a monoclonal IgM protein and 2.5% had only a monoclonal light chain, while 4% had a biclonal protein. In 64% of patients the monoclonal light chain was kappa and in 37% was lambda. The median serum M-protein was 0.948 g/dl (range 0.1–2.99 g/dl); 52% of patients had an M-protein of <1 g/dl and 79% of <2 g/dl. Immunoparesis of at least one of the uninvolved immunoglobulins was present in 38% of cases and of both of the uninvolved immunoglobulins in 6%. Median BM infiltration by monoclonal plasma cells or lymphoplasmacytes was 15%. In 66.5% of individuals there was a BM infiltration of ≥10% by monoclonal plasma cells or lymphoplasmacytes, while in 10% of the studied cases the BM infiltration was ≥50%. A significant correlation of the size of M-protein and of the infiltration of the BM was found (R=0.592, p<0.001). However, 27% of patients with M-protein <0.5 g/dl had ≥10% clonal plasma cells or lymphoplasmacytes in their BM biopsies. The respective rates were 46% for those with M-protein <1 g/dl, 54% for those with M-protein 1.5 g/dl and 58% for those with M-protein <2 g/dl. Ninety per cent of those who had immunoparesis of at least one of the uninvolved immunoglobulins had ≥10% clonal plasma cells or lymphoplasmacytes. A BM infiltration of ≥10% was more frequent in individuals with a monoclonal IgG or IgA protein (72% and 80%, respectively) vs. 45% of those with a monoclonal IgM protein (p=0.015). Light chain isotype, age and gender were not predictive of the degree of BM plasma cell infiltration. In multivariate analysis, immunoparesis of at least one of the uninvolved immunoglobulins (OR: 6.45, 95% CI: 2.32–18, p<0.001), an IgG or IgA monoclonal protein (OR: 2.67, 95% CI: 1.1–6.4, p=0.028) and an M-protein of ≥1 g/dl (OR: 5.4, 95% CI: 2.23–13) were independently associated with the presence of ≥10% of clonal infiltration in BM biopsy. By combining the above risk factors we found that in those who had all three, 97% had ≥10% clonal cells in the BM biopsy, while in those with 0–1 of the above factors the probability to find ≥10% clonal cells was 43%. These findings indicate that even patients with low risk for BM infiltration by clonal plasma cells, may be diagnosed as SMM when a BM biopsy is performed. In conclusion, our data on a large number of individuals with asymptomatic monoclonal gammopathy who underwent a BM biopsy may indicate that the latter exam may provide useful information and could be included in the standard initial workup of these individuals. Disclosures: No relevant conflicts of interest to declare.

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Appearance of Monoclonal Plasma Cell Diseases in Whole Body MRI in 544 Patients and Correlation with Parameters of Disease Activity

Blood ◽

10.1182/blood.v120.21.4966.4966 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4966-4966

Author(s):

Jens Hillengass ◽

Kerstin Kilk ◽

Karin Listl ◽

Thomas Hielscher ◽

Kai Neben ◽

...

Keyword(s):

Bone Marrow ◽

Disease Activity ◽

Plasma Cell ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Whole Body ◽

Focal Lesions ◽

Diffuse Infiltration ◽

Whole Body Mri ◽

Cell Percentage

Abstract Abstract 4966 Focal lesions (FL) and diffuse tumor cell infiltration detected by whole body MRI (WB-MRI) have been demonstrated to be of prognostic significance for predicting progression free and overall survival in patients with monoclonal plasma cell diseases. In this trial we have assessed 544 unselected and untreated patients: 138 with monoclonal gammopathy of undetermined significance (MGUS), 157 with smoldering myeloma (SMM), and 249 with multiple myeloma (MM). WB-MRI was performed on two identical 1. 5 Tesla MRI-scanners with body array curls. Assessment of FL was done by two experienced radiologists blinded to the diagnosis of the patients in consensus. We found focal lesions in 23. 9%, 34. 4%, and 80. 3% of patients with MGUS, SMM and MM, respectively, and a diffuse infiltration was detected in 38. 4%, 45. 9%, 71. 5% of the corresponding patients. The differences between MGUS, SMM and MM patients were statistically significant (p<0. 0001). The presence of FL as well as the presence of a diffuse infiltration was correlated with an increased plasma cell percentage in bone marrow (p<0. 0001) and monoclonal protein concentration (p=0. 001). Further categorization of the diffuse infiltration patterns in WB-MRI into minimal, moderate, severe and “salt&pepper” patterns, was able to identify a significant correlation between both M-protein and plasma cell percentage in bone marrow as well as age. In MGUS and SMM patients, FL were more often detected in patients with a diffuse background, while in MM patients, FL were present at the same rate across the diffuse infiltration subgroups. In summary bone marrow infiltration in WB-MRI is significantly different between stages of plasma cell disease and correlates with established markers of disease activity. Disclosures: No relevant conflicts of interest to declare.

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Observations On Eosinophil Ifiltrates in The Bone Marrow Of Patients With Multiple Myeloma. Clinicopathological Correlates

Blood ◽

10.1182/blood.v122.21.5338.5338 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5338-5338

Author(s):

Finella MC Brito-Babapulle ◽

Tanya Cranfield ◽

Robert B Corser ◽

Helen Dignum ◽

Christopher James ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Osteolytic Lesion ◽

Inverse Correlation ◽

Eosinophil Infiltration ◽

Bone Marrow Biopsies ◽

Lytic Lesions

Abstract Mouse eosinophils have been shown in 2011 to be required for the maintenance of long lasting plasma cells in the bone marrow and in maintaining the bone marrow plasma cell microenvironment. Human eosinophils have been shown by Wong et al to support multiple myeloma cell proliferation via a mechanism independent of IL6. We looked at bone marrow biopsies taken from patients who had a paraprotein and in whom a diagnosis of multiple myeloma was suspected. These samples were taken solely for the purposes of diagnosisng multiple myeloma and were retrospectively reviewed from the point of view of degree of eosinophil infiltration and its correlation with tumour load, bone lytic lesions, plasma cell morphology, whether blastic, crystalline inclusions, Mott cells, flame cells and or lymphoplasmacytoid. There were no cases of IGD or E myeloma or osteosclerotic myeloma.Nonsecretory myeloma and cases of light chain myeloma with or without amyloid were included in the series. Biopsies were not performed from osteolytic lesion unless biopsy was necessary to make a diagnosis of myeloma. Myeloma was diagnosed when plasma cell infiltrate was greater than 10% on bone marrow aspirate with a paraprotein and or lytic lesions. Eosinophil infiltration did not correlate with any of the tumour clinicopathological markers but showed an inverse correlation with degree of plasmacytosis. Eosinophils were hardly ever found in marrow aspirates that had over 70% plasma cells. They were usually found in trephine sections of bone marrow in areas where there was Grade I/II fibrosis and were often found in close proximity to focal areas of plasma cell infiltration. Whether eosinophils play a role in preventing or maintaining malignant plasma cell recurrence is currently being studied. Disclosures: No relevant conflicts of interest to declare.

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Comparison Of Hevylite™ Assay and Plasma Cell Immunophenotyping For Response Evaluation and Residual Disease Characterisation In IgA Myeloma

Blood ◽

10.1182/blood.v122.21.5319.5319 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5319-5319

Author(s):

Daniela Lakomy ◽

Stephanie Lemaire-Ewing ◽

Cedric Rossi ◽

Jessica Borgeot ◽

Jean-Noël Bastie ◽

...

Keyword(s):

Bone Marrow ◽

Binding Site ◽

Plasma Cell ◽

Light Chain ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Response To Treatment ◽

Response Evaluation ◽

Bone Marrow Plasma

Abstract Introduction The evaluation of multiple myeloma response to treatment as defined by international guidelines is currently based on morphologic examination of bone marrow plasma cells, serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and serum free light chain assay. For several years new tools are available as bone marrow plasma cell immunophenotyping and the HevyliteTM assay. HevyliteTM IgA assay provides an automated evaluation of serum heavy/light chain ratio (HLC) of the involved and uninvolved immunoglobulin (Ig) (i.e. IgAΚ/IgAλ). This is particularly interesting in IgA myeloma where the use of SPEP is limited due to a frequent comigration of monoclonal IgA with other proteins. We therefore compared the IgA quantification by Hevylite™ assay and the bone marrow plasma cell immunophenotyping for response evaluation and residual disease characterisation in IgA myeloma. Methods Hevylite™ assay, SPEP, IFE were performed in eleven IgA myeloma patients at different times: after induction chemotherapy, after the consolidation phase and after autologous stem-cell transplantation (ASCT). In the same time, minimal residual disease (MRD) assessment was performed on bone marrrow by multiparameter flow cytometry (MFC). Hevylite™ assay was performed on a Binding Site SPAplus analyser (Hevylite, Binding Site, Birmingham, UK) following the manufacturer recommendations. SPE and IFE were realized on Sebia Hydrasys analyser (Sebia, Evry, France) and results were read by two experienced biologists. Results 1. We found a perfect agreement between the IFE and immunophenotyping results at each time of evaluation, for positive results as for negative results. 2. The SPEP was contributive only in two patients and in these cases it was less sensitive than IFE. In the other patients, the monoclonal IgA migrated in beta region and/or as multiple bands, making the quantitative estimation difficult. 3. In all patients, when MRD by MFC was undetectable and IFE was negative, the HLC ratio was normal. 4. In 3 patients, HLC ratio was consistent with the IFE and MRD by MFC at each time of evaluation. Nevertheless, in 8 patients out of 11, while HLC ratio became normal, MRD by MFC and IFE were still positive. In all cases, the normalization of HLC ratio was followed, at the next step of evaluation, by the normalization of MFC and IFE. 5. In 5 patients, the normalization of HLC ratio occurred before ASCT, while IFE and MRD by MFC were still positive. Nevertheless, after ASCT, IFE and MRD by MFC became also negative, in accordance with the HLC ratio (Table 1). Conclusions During the evaluation of response to treatment of IgA myeloma, we observed a normalization of HLC ratio (Hevylite™ IgA assay) preceding the normalization of MRD by MFC and IFE. This could be explained by the fact that IFE and immunophenotyping provide very sensitive information but only on the monoclonal component. HLC ratio reflects the balance between the monoclonal and polyclonal Igs of involved and uninvolved isotype. A normalization of HLC ratio can be interpreted as an increasing polyclonal Ig proportion parallel with a decreasing monoclonal Ig proportion and may reflect the reconstitution of polyclonal plasma cells. If confirmed by other studies and long term follow-up, HLC ratio could be a non-invasive predictive marker of a good response in IgA myeloma. Disclosures: No relevant conflicts of interest to declare.

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Single Cell RNA-Seq Reveals Clonal Consistency and Differential Malignancy in Extramedullary Plasmacytoma of Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.4808.4808 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4808-4808

Author(s):

Shuang Geng ◽

Jing Wang ◽

Mingyi Chen ◽

Wenming Wang ◽

Yuhong Pang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Single Cell ◽

Plasma Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Extramedullary Plasmacytoma ◽

Rna Seq ◽

Malignant Plasma Cell

Abstract Extramedullary Plasmacytoma (EMP) is a minor yet devastating metastatic form of Multiple Myeloma (MM), shortening patients' survival from 10 years to 6 months on average. Genetic cause of EMP in MM is yet to be defined. Transcriptome difference between EMP+ patients and EMP- patients is studied here on single cell level by RNA Sequencing (RNA-Seq). We sorted CD38+CD138+ malignant plasma cells from bone marrow and peripheral blood samples by flow cytometry, then picked up single malignant plasma cell and performed single cell RNA-Seq with SmartSeq2 protocol followed by Tn5-based library preparation from bone marrow, peripheral blood and extramedullary tissue of EMP patients. From the single cell RNA-Seq results, in bone marrow we found differential gene expression between EMP+ and EMP- samples, such as CTAG2, STMN1 and RRM2. By comparing circulating malignant plasma cells in PBMC and malignant plasma cell from the sample EMP+ patient, we observed metastatic clone in blood with the same VDJ immunoglobulin heavy chain as in bone marrow. Several genes' expression of these metastatic cells are down-regulated than in bone marrow, such as PAGE2, GTSF1, DICER1. These genes may correlate with egress capability of MM cells into peripheral to become circulating plasma cells (cPCs), and EMP eventually. Disclosures No relevant conflicts of interest to declare.

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CD81: A Novel, Specific and Highly Sensitive Marker in Flow Cytometric Diagnosis of Plasma Cell Dyscrasia

Blood ◽

10.1182/blood.v118.21.2880.2880 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2880-2880

Author(s):

Prashant Ramesh Tembhare ◽

Constance Yuan ◽

Neha Korde ◽

Irina Maric ◽

Katherine Calvo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Antigen Expression ◽

Plasma Cell Dyscrasia ◽

Fluorescent Intensity ◽

Reliable Marker ◽

Sensitive Marker

Abstract Abstract 2880 Background: The percent abnormal plasma cells (aPC) as determined by flow cytometry (FC) has been shown to be an independent risk factor for progression from myeloma precursor disease (monoclonal gammopathy of uncertain significance, MGUS; smoldering multiple myeloma, SMM) to multiple myeloma (MM). However, differentiation of aPCs from normal PCs (nPCs) in these patients is challenging. MM cell lines are know to underexpress the tetraspanin proteins (e.g. CD81, CD82) in comparison to nPCs. Although CD81, a nonglycosylated tetraspanin, is robustly expressed on the surface of nPCs, little information is available regarding its expression in the aPCs of MM, SMM and MGUS. In this study we evaluate the expression of CD81 in conjunction with CD19, CD45 and CD56 in bone marrow aPCs and nPCs from patients with MM, SMM and MGUS. Methods: Bone marrow aspirates from 41 patients (9 MGUS, 22 SMM, 7 MM, 3 non-neoplastic with clinical suspicion of MGUS) were analyzed with 8-color multiparametric FC using a panel of antibodies (CD138, CD38, CD19, CD20, CD27, CD28, CD45, CD56, CD81, CD13, CD14, CD16, CD3, CD34 and intracellular kappa & lambda light chains). The pattern of surface antigen and intracellular light chain expression was utilized to determine the percent aPC (defined as monoclonal with aberrant antigen expression) and percent nPC (defined as polyclonal with normal antigen expression). In all cases the pattern of antigen expression was evaluated in the aPCs; additionally, in cases with greater than 5% nPCs (19/41 patients: 8 MGUS, 8 SMM and 3 non-neoplastic) the pattern of antigen expression was evaluated in the nPCs. The ability to detect clonal aPC by evaluation of FC pattern of antigen expression was determined and compared for CD19, CD45, CD56 and CD81. We also examined the sensitivity and specificity of the CD19 and CD81 combination verses the conventional combination of CD19, CD56 and CD45 (Perez-Persona et al, Blood 2007) for the detection of clonal aPC. Results: CD81 was strongly expressed by nPC (average mean fluorescent intensity (MFI): 11500, standard deviation (SD): 5061, range: 5347–21657) in contrast to aPC with abnormally weak expression (average MFI: 1487, SD: 887, range: 647–4311). CD81 was a highly reliable marker for the detection of clonal PC; with 90% sensitivity and 100% specificity. It was the most specific and second most sensitive marker in our study (Table 1). CD81 was equally sensitive in detection of aPCs in MGUS, SMM and MM. Evaluation of the combined pattern of expression of CD19 and CD81 resulted in 100% sensitivity and 100% specificity for detection of aPC, which is greater than the conventional combination of CD19, CD56 and CD45, yielding 100% sensitivity but 90% specificity, for diagnostic evaluation of aPC. Conclusions: CD81 is a highly reliable marker in the detection of abnormal plasma cells in MM, SMM and MGUS. The combined approach of CD19 and CD81 is superior to other conventional marker combinations (i.e. CD19, CD45, and CD56) in terms of detection of clonal plasma cells and may replace their use in the clinical evaluation of bone marrow aspirates for plasma cell processes. Furthermore, it should help widening the applicability of minimal residual disease testing in MM. Disclosures: No relevant conflicts of interest to declare.

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Constitutively High Expression of CD137L on Primary MM Cells Could be Inhibited By Chemotherapy but Re-Emerged after Desease Relapse

Blood ◽

10.1182/blood.v124.21.5198.5198 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5198-5198

Author(s):

Chengcheng Fu ◽

Hui Liu ◽

Ling Ma ◽

Shuang Yan ◽

Juan Wang ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Lines ◽

Plasma Cell ◽

Plasma Cells ◽

De Novo ◽

Conflicts Of Interest ◽

B Cell Lymphoma ◽

High Expression ◽

Normal Plasma

Abstract Objective: Multiple myeloma (MM) is a kind of malignant monoclonal plasma cell disorder. CD137L molecules are important member of TNFsuperfamily. Under physiological conditions, CD137L express on the surface of various active APCs and participate in keeping the balance of immune system. Under pathological conditions, CD137L could express on the surface of various tumor cells. For example, in hematologic malignancies, about 35% of AML with FAB type M4 or M5, and most CLL, B-cell lymphoma cells express CD137L. CD137L molecules play an important role in the maturation process of B cells. Active CD137L can promote proliferation, differentiation and immune response of B cells. But when B cells transform to plasma cells, the majority of B antigens will be lost, such as CD19, CD20, HLA-DR. CD137L are also lost in the process of transformation. Our previous studies showed that there is no expression of CD137L and CD137 molecular on normal plasma cells. But CD137L were highly expressed on MM cell lines U-266, KMS-11, My-5, LP-1 and 8266. CD137L signaling promoted U266 cell proliferation remarkably, which could be blocked by Bortizomib. CD137L signaling also pushed MM cells in the G1 phase to enter the S phase. To understand the expression of CD137L and CD137 on MM primary cells，We examined the bone marrow specimens of MM patients, then analyzed the relationship between the expression of CD137L and the clinical characteristics of patients. Methods: Flow cytometry was used to detect the CD137L and CD137 expression. Markers of normal plasma cell were CD45+CD38+CD138+CD19+CD56-, Markers of abnormal plasma cell were CD45-/CD45lowCD38++CD138+CD19-CD56+. bone marrow specimens from 127 cases of MM patients treated in the First Affiliated Hospital of Soochow University from 2012 to 2013 and the normal control from 10 volunteers were collected with the consent of patients and volunteers. 45 patients were newly diagnosed, 72 patients were checked after treatment, 10 patients were relapsed and refractory; 68 patients were males, 59 patients were females; The median age was 58(36-73); 3 pts were D-S staging I, 14 pts II, 59 pts III; 25 pts were ISS staging I, 65 pts II, 35 pts III. Results: Normal plasma cells didn’t express CD137L and CD137 molecular .CD137L molecular but not CD137 were expressed on primary MM cells in all 45 de novo patients. The median level was 46.7 (3.6–96.7)%, significantly higher than that on normal plasma cells (P <0.05). A retrospective analysis had been made to find no correlation between CD137L expression and patients’ age, gender, type, DS stage, ISS stage , LDH, creatinine, serum calcium, AKP, M protein and extramedullary plasmacytoma. The CD137L expression of MM cells from 12 de novo patients treated with PAD were decreased gradually with the increase of treatment courses and improvement of efficacy. The expression of CD137L on MM cells in 79 patients grouped in CR (n = 12), PR (n = 51), recurrent (n = 16) were 4.5 (0–18)%，10 (−056)% (P<0.05), 30.5 (5.3–95)% (P = 0.00) respectively. Conclusions: CD137L molecular without CD137 were constitutively highly expressed on MM cell lines and primary MM cells, but hardly on normal plasma cells. There was no relationship between the expression of CD137L with patients’ clinical and biological characteristics. But our data showed that constitutively high expression of CD137L molecular on primary MM cells could be inhibited by chemotherapy but re-emerged after desease relapse. Disclosures No relevant conflicts of interest to declare.

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