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. Author manuscript; available in PMC: 2016 Jan 27.
Published in final edited form as: Cancer Ther. 2009;7:49–52.

Analysis of bone marrow plasma cells in patients with solitary bone plasmacytoma

Archana Bhaskar 1, Ritu Gupta 1,*, Atul Sharma 2, Lalit Kumar 2, Paresh Jain 1,§
PMCID: PMC4729369  NIHMSID: NIHMS752342  PMID: 26834513

Summary

Local radiotherapy is the treatment of choice for solitary bone plasmacytoma (SBP) and the role of adjuvant systemic chemotherapy in preventing progression to multiple myeloma (MM) is controversial. The purpose of this study was to examine the presence of systemic disease in the form of neoplastic plasma cells (PC) in bone marrow of patients with SBP. Flow cytometric immunophenotyping of PC was carried out on bone marrow aspirate of 7 patients using monoclonal antibodies: CD19 FITC, CD45 FITC, CD20 FITC, CD52 PE, CD117 PE, CD56 PE, CD38 PerCP-Cy5.5, CD138 APC, anti-kappa (κ) FITC and anti-lambda (λ) PE. The neoplastic as well as normal PC were identified in bone marrow aspirate of all the patients at the time of diagnosis; the neoplastic PC ranged from 0.1%to 0.7% of all BM cells and 33.5% to 89.7% of total BMPC. The κ:λ ratio was normal in all the samples ranging from 0.5% to 1.6%. The present work shows the presence of systemic disease in the form of neoplastic PC in bone marrow of patients with SBP. Prospective studies would be required to study if the levels of neoplastic PC in the bone marrow may help us identify patients who are likely to progress to overt MM and benefit from systemic chemotherapy.

Keywords: Bone marrow plasma cells, solitary bone plasmacytoma, multiple myeloma, neoplastic plasma cells, flow cytometry

I. Introduction

Plasmacytoma are clonal proliferation of plasma cells (PC) that are identical to multiple myeloma (MM) but have a single bone lesion - solitary bone plasmacytoma (SBP); or less commonly, a soft tissue mass-extramedullary plasmacytoma. Solitary bone plasmacytoma make up about 5% of PC neoplasms and the common sites are in marrow areas with active hematopoiesis (Soutar et al, 2004). The diagnosis of SBP requires presence of a localized mass of clonal PC, absence of other lytic lesion on skeletal survey, no evidence of plasmacytosis in the bone marrow and absence of anemia, hypercalcaemia, or renal involvement. These tumors are usually treated with localized radiotherapy but the outcome varies and as many as 75% of patients develop MM with a median time to progression of 2-4 years (Soutar et al, 2004). Monoclonal paraproteinemia is seen in 24-72% of patients with SBP and persistence of M-protein for more than one year after radiotherapy is the only independent adverse prognostic factor predicting progression to MM (Dimopoulos et al, 2000; Wilder et al, 2002). In patients with non-secretory SBP at presentation, other parameters which can predict the likelihood of progression to MM need to be established.

Monoclonal gammopathy of undetermined significance (MGUS) is another hematological condition where about 16% patients have been reported to develop MM with a median follow-up period of 10 years (Kyle, 1993). Using aberrant immunophenotype to identify neoplastic PC, it has been demonstrated that the bone marrow of patients with MGUS contain normal as well as neoplastic PC and predominance of neoplastic PC in the bone marrow plasma cell compartment at the time of diagnosis correlates with a higher risk of progression to MM (Perez-Persona et al, 2007). The immunophenotyping of bone marrow plasma cells in SBP has not been investigated so far and since a significant number of these patients progress to MM, we hypothesized that the bone marrow of patients with SBP might contain neoplastic PC at the time of diagnosis, which may correlate with risk of progression to MM.

II. Materials and Methods

A. Patients

A total of 7 patients who fulfilled the criteria of SBP are included in this study.

Bone marrow aspirates was collected in ethylene diamine tetraacetic acid from all the patients after obtaining informed consent as per the guidelines of the ethical committee of the institute. Serum and urine protein electrophoresis was performed on all the patients as part of routine diagnostic work-up.

B. Flow cytometric immunophenotyping

Immunophenotyping studies were carried out on the bone marrow aspirates using the pre-titrated volumes of the following monoclonal antibodies: CD19 FITC, CD45 FITC, CD20 FITC, CD52 PE, CD117 PE, CD56 PE, CD38 PerCP-Cy5.5, CD138 APC (BD Biosciences, San Jose, CA, USA) Kappa (κ) FITC and Lambda (λ) PE (Serotec). Staining was done using standard whole blood lysis technique. Briefly, for tube assessing only surface antigens an aliquot of BM aspirate containing 2 × 106 cells was processed using 2 ml of ammonium chloride for 10 minutes at room temperature followed by washes in phosphate buffer saline containing sodium azide and resuspended in paraformaldehyde. To study cytoplasmic κ and λ light chains expression, fixation and permeabilization prior to staining was carried out as per manufacturer’s recommendation (Serotec). Acquisition was done on a flow cytometer (BD FACS Calibur or BD FACS Canto) equipped with facility for at least 4-color immunophenotyping and 105 events were acquired in each tube.

C. Flow cytometric data analysis

Analyses were carried out using FCS Express V3 (De Novo Software). Plasma cells were identified based on dual expression of CD38 and CD138 and expression of other antigens was analyzed on this gated population of PC. A tube with cells labeled only with gating reagents was included as negative control with every sample. Normal PC were defined as CD19+CD56− (Harada et al, 1993). A PC was defined as neoplastic when it displayed at least two aberrant antigens i.e. CD19−, CD56+, CD45−, CD52+, CD117+, CD20+. The κ:λ ratio was defined as abnormal if < 0.5 or > 3 (Morice et al, 2007).

III. Results

Plasma cells were easily identified based on dual expression of CD38 and CD138 in the bone marrow aspirates and varied from 0.17% to 1.05%. The neoplastic as well as normal PC were seen in the bone marrow aspirates of all the cases at the time of diagnosis and ranged from 0.10% to 0.70% and 0.06% to 0.49% of all the bone marrow cells respectively (Figure 1). Neoplastic PC comprised 33.5% to 89.7% (median: 60.8%) of the total bone marrow plasma cells. Similar to other studies, the aberrant immunophenotype rather than the light chain restriction pattern was useful in identification of low number of neoplastic plasma cells in the bone marrow as the κ:λ ratio was normal in all the samples (range: 0.5% to 1.6%). Protein electrophoresis studies on serum and urine revealed an M-band in five cases at the time of diagnosis and in two patients subsequent to therapy (Table 1). During a median follow-up period of 7 months, two of the patients progressed to MM (Table 1).

Figure 1.

Figure 1

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Flow cytometric dot-plots showing immunoprofile of bone marrow plasma cells in a patient with solitary bone plasmacytoma. Arrows indicate neoplastic plasma cells.

Table 1.

Profile of seven patients with solitary bone plasmacytoma

Age(y)/S
ex		 Site NPC/all
BMPC (%)		 k/λ ratio SPE Progression
to MM
At diagnosis At follow-up
(months)
53/F Femur 60.8 1.3 P P (18) Yes
37/M Tibia 33.5 1 P N (18) No
58/M Femur 49.7 1.5 N N (7) No
38/M L5 vertebra 69.8 1.6 P N (7) No
64/M Rib 56.6 1.5 N N (6) No
35/F D6 vertebra 70.6 0.8 P N (5) No
50/M Humerus 89.7 0.5 P P (4) Yes
Open in a new tab

BMPC indicates bone marrow plasma cells; SPE, serum protein electrophoresis; NPC, neoplastic plasma cells; MM, multiple myeloma; k, kappa; λ, lambda; P, positive; N, negative

IV. Discussion

In patients with SBP, the persistence of monoclonal protein for more than one year after therapy is the only independent adverse prognostic factor predicting progression to myeloma and complete disappearance of the monoclonal protein after therapy is associated with lower risk of progression to myeloma (Wilder et al, 2002). Definitive local radiotherapy is the treatment of choice for SBP as the disease is believed to be localized and the response to radiotherapy is measured by documenting the reduction in the levels of monoclonal protein, which is a slow process. There is no suitable parameter which can predict the likelihood of progression to myeloma upfront at the time of diagnosis itself in patients with this plasma cell proliferative disorder.

The role of adjuvant chemotherapy in preventing progression to MM is controversial. Some reports have suggested that adjuvant chemotherapy may delay progression to MM while others consider it to be non-beneficial (Mayr et al, 1990; Holland et al, 1992; Shih et al, 1995; Aviles et al, 1996; Tsang et al, 2001). The SBP as per their defining criteria do not exhibit bone marrow plasmacytosis and unlike MGUS, the existence of neoplastic PC in the bone marrow of patients with SBP is largely unknown. Our study demonstrates, for the first time, the existence of systemic disease albeit of low load, in the form of neoplastic PC in the bone marrow of patient with SBP at the time of their initial diagnosis The rapid progression to myeloma in the second case in this study was associated with relatively high numbers of neoplastic PC in the bone marrow.

Small number of cases studied and the relatively short duration of follow-up preclude a definitive opinion on value of these findings at this time. However, given the ease of flow cytometric quantitative detection and enumeration of neoplastic PC in the bone marrow, studies with larger sample size and longer follow-up may reveal potential benefits on evaluation of the effect of radiotherapy alone or along with adjuvant chemotherapy on the systemic load of neoplastic PC in patients of SBP. Further, it might be of interest to ascertain if a certain level of neoplastic PC in the bone marrow of patients with SBP correlates with risk of progression to overt MM and help in identifying patients who may benefit from systemic chemotherapy.

Acknowledgments

We gratefully acknowledge support of Professor Subroto Sinha and Dr. Neera Nath, Department of Biochemistry, who made available the flow cytometric laboratory facility to carry out this work.

This study was supported by research funding from Department of Science & technology, Government of India to RG under the SERC-Fast track scheme. Grant number: SR/FT/L-29/2005.

Abbreviations

κ

kappa

λ

lambda

MGUS

monoclonal gammopathy of undetermined significance

MM

multiple myeloma

PC

plasma cell

SBP

solitary bone plasmacytoma

Biography

graphic file with name nihms-752342-b0002.gif

Ritu Gupta

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