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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Leukemia. 2014 Mar 12;28(10):2060–2065. doi: 10.1038/leu.2014.98

Quantification of clonal circulating plasma cells in newly diagnosed multiple myeloma: implications for redefining high-risk myeloma

Wilson I Gonsalves 1,2, S Vincent Rajkumar 1,2, Vinay Gupta 1,2, William G Morice 1,2, Michael M Timm 1,2, Preet P Singh 1,2, Angela Dispenzieri 1,2, Francis K Buadi 1,2, Martha Q Lacy 1,2, Prashant Kapoor 1,2, Morie A Gertz 1,2, Shaji K Kumar 1,2
PMCID: PMC4162866  NIHMSID: NIHMS576512  PMID: 24618735

Abstract

The presence of clonal circulating plasma cells (cPCs) is a marker of high-risk disease in all stages of monoclonal gammopathies. However, the prognostic utility of quantitating cPCs using multiparametric flow cytometry in MM patients with current treatments is unknown. There were 157 consecutive patients with newly diagnosed MM seen at the Mayo Clinic, Rochester from 2009–2011 that had their peripheral blood evaluated for cPCs by multi-parameter flow cytometry. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Using a ROC analysis, ≥400 cPCs was considered as the optimal cutoff for defining high-risk disease. The presence of ≥400 cPCs was associated with higher plasma cell proliferation and adverse cytogenetics. The median time-to-next-treatment and OS in patients with ≥400 cPCs (N=37, 24%) was 14 months and 32 months compared with 26 months and not reached for the rest (p<0.001). In a multivariable model, the presence of ≥400 cPCs and older age adversely affected OS. Flow cytometry to quantify cPCs is a valuable test for risk stratifying newly diagnosed MM patients in the era of novel agents. Future studies are needed to determine its role in developing a risk adapted treatment approach.

Keywords: circulating plasma cells, multiple myeloma, survival

INTRODUCTION

Multiple myeloma (MM) is a clonal plasma cell disorder predominantly affecting the elderly, resulting in more than 10,000 deaths annually in the United States.(1) The use of novel therapeutic agents such as immunomodulators and proteasome inhibitors(2), as well as the incorporation of high dose chemotherapy followed by autologous stem cell transplantation (ASCT) in eligible patients(3, 4) has led to significant improvements in the survival of MM patients. However MM remains biologically heterogeneous with significant variability among patients in terms of clinical features, response to therapy and overall survival (OS). Several prognostic variables can help predict this variability in outcomes ranging from clinical-based systems such as the International Staging System (ISS)(5) to more advanced molecular characterizations of the myeloma plasma cells by cytogenetics(6, 7) and gene expression profiling.(8, 9) However with the advancement in technology utilized for laboratory testing and the emergence of new treatments for MM, there has been an evolution in the significance of these previously defined prognostic markers with time.(10, 11)

Prior studies have demonstrated that the presence of clonal circulating plasma cells (cPCs) can identify patients with MM and AL amyloidosis(12, 13) with a shorter survival as well as patients with MGUS and smoldering MM who are at higher risk of progression to MM.(13–15) However, several of these studies utilized a slide-based immunofluorescence assay to detect cPCs, which is a complex and labor intensive process requiring fluorescence microscopy, thus limiting its clinical availability. More importantly, slide based assays have low sensitivity given the relatively small number of cells examined. Conversely, flow cytometry is a readily available tool that can not only detect cPCs rapidly with good correlation with the immunofluorescence-based method(16, 17) but in addition, can also quantify the absolute number of cPCs detected in a more sensitive and reproducible manner.

A previous study from our institution demonstrated the prognostic significance of cPCs in newly diagnosed MM patients during the pre-novel agent era (1998 to 2003), using a non-quantitative flow based method.(13) Given the current routine incorporation of novel agents in the induction treatment of newly diagnosed MM patients and our current knowledge on molecular risk stratification, we set out to evaluate the clinical utility of quantifying cPCs via flow cytometry in newly diagnosed MM patients.

METHODS

Since 2008, the Mayo Medical Laboratories began routinely evaluating the peripheral blood samples of all MM patients for cPCs using flow cytometry rather than the slide-based immunofluorescence assay.(17) We retrospectively evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from October 2009 to November 2011 who had their peripheral blood samples evaluated by flow cytometry prior to beginning therapy. Approval for this study was obtained from the Mayo Clinic IRB in accordance with the federal regulations and the principles of the Declaration of Helsinki.

Six-color multiparametric flow cytometry was performed on peripheral blood mononuclear cells isolated by Ficoll gradient, and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda immunoglobulin light chains. The data was collected using Becton Dickinson FacsCanto II instruments collecting 150,000 events; the flow cytometry data was analyzed using the BDFacs DIVA Software. The gating strategy employed first used the expression of CD38, CD138, and cytoplasmic immunoglobulin light chains to identify all plasma cells in the specimen. The cPCs were then discriminated from polyclonal/normal plasma cells based on differential CD19 and CD45 expression. The cPCs detected were reported as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. The lower limit of cPC detection by this method is 20 cells/150,000 (0.013%). Figure 1 depicts a typical flow cytometry pattern in a patient with kappa-restricted clonal cPC.

Figure 1.

Figure 1

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Shows a typical flow cytometry pattern in a patient with kappa-restricted clonal cPC

The primary end-points of the study were OS and time to next therapy (TTNT). OS was measured from the day of diagnosis to death from any cause, with censoring performed at the date of last contact. TTNT was determined from the day of diagnosis to the day of initiating the next therapy due to a documented relapse or progression of disease, with those alive and relapse free censored at the day of last follow up. Patients who had fluorescent in situ hybridization (FISH) analysis performed on their bone marrow aspirate at diagnosis were categorized as having high risk disease if they had any of the following abnormalities: t(4;14), t(14;16), t(14;20) and del17p. Host and disease variables at diagnosis that were examined for prognostic significance included: age, bone marrow plasma cell percentage, presence of high-risk FISH, ISS stage at diagnosis, plasma cell labeling index (PCLI), serum M spike, urine M spike, hemoglobin, creatinine and LDH. FISH studies and PCLI were performed as previously described.

Statistical analysis was performed using the SAS biostatistical software JMP 9.0.1 (SAS Institute Inc., Cary, NC). Chi-square tests and Fisher exact tests were used to compare differences between the sub-groups of interest. Receiver operating characteristics (ROC) analysis was performed to determine the optimal cut point of cPCs that predicted for worse 2-year mortality. A Kaplan-Meier analysis was used to analyze and create the OS and TTNT curves, and log rank test was used to compare these curves. Finally, a multivariable analysis was performed using the Cox proportions hazards model to assess the influence of various prognostic factors on OS and TTNT.

RESULTS

Peripheral blood PC flow cytometry was performed as part of the initial clinical evaluation of 157 newly diagnosed MM patients from October 2009 to November 2011. Patient and disease characteristics and initial treatments received are described in Table 1. The median age of the patient population was 65 years and 59% were male. Majority of patients (96%) were treated with a novel anti-myeloma agent during their induction therapy with 36% going to an early ASCT. Minority of patients (14%) received their therapy through enrollment on a clinical protocol.

Table 1.

Demographic and clinical characteristics of the 157 newly diagnosed MM patients

Variables All patients (N = 157) Patients with < 400 cPCs (N = 120) Patients with ≥ 400 cPCs (N=37)
Age 65 (39 – 95) 66 (39 – 91) 68 (46–95)
Male (No, %) 93 (59%) 71 (59%) 22 (59%)
Presence of cPCs 85 (54%) 48 (40%) 37 (100%)
No of cPCs/150,000 cells 40 (0 – 46,413) 0 (0–393) 935 (408–46,413)
PCLI 0.8 (0 – 8.6) 0.6 (0 – 4) 1.4 (0 – 8.6)
Bone marrow PC% 45 (5 –100) 40 (5 – 95) 67 (10 – 100)
LDH 160 (3 – 878) 159 (3 – 501) 167 (62 – 878)
Beta-2-microglobulin 3.45 (1.3 – 23.7) 3.1 (1.3 – 23.7) 5.0 (1.4 – 22.4)
Creatinine 1 (0.6 – 5) 1.0 (0.6 – 4.9) 1.0 (0.6 – 5)
Albumin 3.6 (2.1 – 4.5) 3.6 (2.4 – 4.5) 3.5 (2.1 – 4.1)
FISH (available on 125 pts)
 t(4,14) 9 (7%) 4 (4%) 5 (15%)
 t(14,16) 5 (4%) 2 (2%) 3 (9%)
 t(14,20) 1 (1%) 0 (0%) 1 (3%)
 Deletion 17p 17 (18%) 11 (16%) 6 (22%)
 High risk FISH 29 (23%) 16 (18%) 13 (37%)
ISS stage
 -Stage 1 57 (36%) 51 (43%) 6 (16%)
 -Stage 2 62 (40%) 48 (40%) 14 (38%)
 -Stage 3 38 (24%) 21 (17%) 17 (46%)
Initial induction therapy
 Novel agents 150 (96%) 114 (95%) 36 (97%)
  -Thalidomide 12 (8%) 8 (7%) 4 (11%)
  -Lenalidomide 106 (68%) 85 (71%) 21 (57%)
  -Bortezomib 52 (34%) 34 (28%) 18 (49%)
Post-induction ASCT 56 (36%) 46 (38%) 10 (27%)
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Eighty five patients (54%) had cPCs detected by the 6-color flow cytometry with the median number of cPCs in the entire cohort being 40 (range, 0–46,413)/150,000 gated events. The median estimated follow up for the entire cohort was 21 months (95% CI: 17 – 23). The estimated 2-year and 3-year OS for the cohort was 83% and 76% respectively and 25 (16%) patients had died at the time of analysis. Sixty seven patients (43%) had their disease progress during the follow up period for this study requiring second line therapy. Though the median OS was not reached for either group, it was significantly shorter for the patients with any cPCs (N=85; 54%) compared with those lacking detectable cPCs (P=0.019) (Figure 2). The 2-year and 3-year OS for the patients with any cPCs was 76% and 67% compared with 91% and 87% for those with none.

Figure 2.

Figure 2

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Shows the Kaplan-Meier Curve for overall survival (OS) in patients based on the presence of cPCs

Using a ROC analysis, the optimum cutoff predicting for the highest risk of 2-year mortality was around 400 cPCs per 150,000 events that yielded an area under the curve of 0.683 with a sensitivity of 60% and specificity of 75%. Based on this, a cutoff of ≥400 cPCs was used for defining patients with high-risk disease. The patient and disease characteristics of these two groups defined by application of this criterion are shown in Table 1. The median TTNT for patients with ≥400 cPCs (N=37, 24%) was 14 months compared with 26 months for those with < 400 cPCs (N=120, 76%) (p<0.001; Figure 3a). The median OS for those with ≥400 cPCs was 32 months compared with not reached for those with < 400 cPCs (p<0.001; Figure 3b). When characterized by FISH risk status, the presence of ≥400 cPCs predicted for a worse TTNT among standard risk patients than those with < 400 cPCs (P=0.004; Figure 4a). Similarly, the presence of ≥400 cPCs also predicted for a worse OS among standard risk patients than those with < 400 cPCs (P=0.0003; Figure 4b). The 1-year and 2-year OS for standard risk patients by FISH with ≥400 cPCs was 77% and 59% compared to 95% and 90% for those standard risk patients with < 400 cPCs. There was no difference in TTNT (P=0.797) and OS (P=0.876) among high-risk patients by FISH based on the presence or absence of ≥400 cPCs.

Figure 3.

Figure 3

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Shows the Kaplan-Meier Curve for time to next therapy (TTNT) (Panel A) and overall survival (OS) (Panel B) in patients in patients based on the presence of ≥400 cPCs

Figure 4.

Figure 4

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Shows the Kaplan-Meier Curve for time to next therapy (TTNT) (Panel A) and overall survival (OS)(Panel B) in patients with standard risk disease by FISH cytogenetics based on the presence of ≥400 cPCs

Among patients not receiving an ASCT, the median OS for patients with < 400 cPCs (N=74) was not reached in comparison to 32 months for patients with >400 cPCs (N=27) (P = 0.024). Similarly, in patients who received an ASCT, the median OS for patients with < 400 cPCs (N=46) was not reached but was 25 months for the patients with >400 cPCs (N=10) (P <0.001). In patients younger than 65 years of age, though the median OS was not reached in both groups, there were 2 deaths (4%) in the < 400 cPC group (N=54) and 4 deaths (40%) in the >400 cPC group (N=10) (P= 0.003). In patients older than 65 years of age, median OS was not reached in the < 400 cPC group (N=64) but was 32 months in >400 cPC group (N=23) (P=0.021). The clinical characteristics of patients groups stratified by cPC burden were compared: Patients with ≥400 cPCs had higher ISS stage (P = 0.002), creatinine (P = 0.045), PCLI (P <0.001), bone marrow plasma cell % (P <0.001), and high-risk disease by FISH (P = 0.016), compared with those with < 400 cPCs.

The following variables were assessed in a univariate analysis (Table 2) to determine their effects on TTNT and OS: the presence of ≥400 cPCs, age, ISS stage, LDH, bone marrow PC% and FISH risk status. In a univariate model, the presence of ≥400 cPCs predicted for worse TTNT and OS whereas older age, undergoing an ASCT and ISS stage 3 were only associated with worse OS and high-risk FISH was only associated with worse TTNT. In a multivariable analysis (Table 2), only age (P=0.002) and the presence of ≥400 cPCs (P=0.024) retained statistical significance for affecting OS negatively whereas only the presence of ≥400 cPCs (P=0.028) predicted for worse TTNT. Although plasma cell proliferation also serves as a marker of high-risk biology, it was not included in either multivariable as the methodology for measurement transitioned during this time period to a flow based method which was not directly comparable to the antecedent slide-based method.

Table 2.

Univariable and Multivariable analysis of factors predicting worse TTNT and OS

Variable Time to next therapy (TTNT) Overall survival (OS)
Univariable Multivariable Univariable Multivariable
Risk Ratio p Risk Ratio p Risk Ratio p Risk Ratio p
≥400 cPCs 2.20 (1.33–3.53) 0.003 1.85 (1.07–3.11) 0.028 3.89 (1.76–8.64) 0.001 3.16 (1.43–7.08) 0.005
Age 1.21 (1.04–1.39) 0.773 - - 1.07 (1.03–1.11) 0.001 1.06 (1.01–1.10) 0.007
ISS stage 3 1.09 (0.62–1.82) 0.762 - - 2.63 (1.14–5.81) 0.024 2.09 (0.89–4.72) 0.088
LDH 4.57 (0.34–34.4) 0.231 - - 1.07 (0.17–3.72) 0.926 - -
Bone marrow PC% 1.45 (0.62–3.36) 0.392 - - 2.05 (0.47–9.13) 0.342 - -
High risk status by FISH 1.97 (1.10–3.39) 0.024 1.71 (0.94–2.99) 0.078 1.35 (0.48–3.32) 0.544 - -
Post-induction ASCT 0.75 (0.46–1.20) 0.237 0.24 (0.57–0.70) 0.007 0.42 (0.09–1.34) 0.15
Bortezomib-based induction 1.31 (0.79–2.12) 0.286 1.06 (0.41–2.47) 0.891 - -
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DISCUSSION

Several studies have demonstrated the prognostic significance of cPCs across the spectrum of plasma cell disorders such as MGUS(13), smoldering myeloma(14, 15), MM(12, 13) and amyloidosis.(18) Furthermore, cPCs have also predicted for early relapse after ASCT in MM patients(19) and appear to correlate well with response to therapy.(20) However, majority of these studies have utilized a slide-based immunofluorescence assay to detect cPCs which has been a complex and labor intensive process requiring fluorescence microscopy, thus limiting the clinical availability of this test and making it less pragmatic as a prognostic marker. However, the advent of highly sensitive flow cytometry methods, has enabled improved the ease and sensitivity of cPCs detection in the clinical laboratory.

A previous study from our institution demonstrated an independent prognostic value for quantifying CD45 negative cPCs among 50,000 events analyzed per sample by 3-color flow cytometry in patients with newly diagnosed MM from 1998 to 2003.(13) The presence of cPCs detected by flow cytometry was assessed against other well-defined prognostic factors such as albumin, beta-2-microglobulin, age and PCLI.(13) Furthermore, this model appeared to enhance the ISS staging prognostic system in identifying a narrower subset of patients with a particularly poor prognosis.(13) Since 2008, our laboratory has utilized a more advanced 6-color flow cytometer in the routine assessment of peripheral blood samples of MM patients. This allows for rapid quantitative estimation of cPCs from among 150,000 events per sample at a sensitivity of 0.013%, allowing for comparisons both between patient groups as well as for individual patients across different time points. Additionally, availability of multicolor flow cytometers allow for simultaneous assessment of expression of additional antigens such as CD19 and CD45 on the cPCs. This study is the first to evaluate the prognostic value of this more sensitive method of quantifying clonal cPCs in newly diagnosed MM patients who were mostly treated with novel agents while taking into account their current FISH-based risk classification.

This study demonstrates that the number of cPCs detected by flow cytometry continues to remain an independent prognostic factor in patients with newly diagnosed MM treated with novel agents. By utilizing a ROC analysis, we were able to determine that the presence of ≥400 cPCs per 150,000 gated mononuclear events analyzed provided the best possible cutoff for predicting poor outcomes based on 2-year OS. Moreover, this cutoff predicted for a median TTNT of 14 months and an OS of 32 months. The current MM risk stratification system places emphases on the biology of the plasma cells, thus incorporating information provided by molecular cytogenetics or FISH, PCLI and gene expression profiling (the latter two being less widely available) to distinguish between high risk and standard risk patients.(21) However, given the short follow up of 21 months, this was insufficient time for cytogenetics by FISH and ISS stage to be able to indentify patients with poor survival. Thus, this study demonstrates that assessment of the capacity of the clonal plasma cells to mobilize in the peripheral blood, as manifested by the presence of >400 cPCs of all WBCs, is biologically relevant and identifies a subset of patients with a shorter OS and TTNT than expected even prior to traditional prognostic markers such as high risk cytogenetics by FISH and high ISS stage.

The specific pathophysiologic mechanisms and significance of cPCs remain poorly understood. Molecularly, the presence of cPCs in MM patients is believed to reflect a different disease biology due to its association with cytogenetic abnormalities by conventional karyotyping(19) and increased angiogenesis.(22) We observed an association between adverse cytogenetic characteristics by FISH as well as increased proliferation and increased numbers of cPCs. Interactions between MM plasma cells and bone marrow microenvironment are required for proliferation(23) and mediates resistance to treatment.(24) Thus, the presence of cPCs suggests an independence from their bone marrow microenvironment signifying a more self-sustaining and aggressive disease. The mechanisms of independence are not clear, but Paiva et al(25) demonstrated that cPCs have lower expressions of integrins (e.g., CD11a, CD11c, CD29 CD49d, CD49e) and adhesion molecules (CD33 and N-CAM (CD56) creating a lower dependence on the bone marrow microenvironment and higher propensity to circulate in the peripheral blood.

Whole exome sequencing of cPCs and their corresponding bone marrow clonal plasma cells suggest subclonal outgrowth of cPCs from one of the bone marrow plasma cell clones with acquisition of additional mutations over time outside of the bone marrow microenvironment.(Mishima et al. ASH 2013) In contrary, another study suggests fewer cytogenetic alterations in cPCs in comparison to bone marrow clonal plasma cells.(25) Nevertheless, cPCs demonstrate high clonogenic potential supporting a recent disease model that they may represent disseminating neoplastic cells that engraft in various parts of the bone marrow creating metastastic disease.(25, 26) Future longitudinal genomic and immunophenotypic studies of cPCs as well as their corresponding bone marrow plasma cells could shed light on their pathogenesis and role in disease progression.

De novo or primary PCL is a rare and aggressive plasma cell disorder accounting for a very small proportion of newly diagnosed MM patients and is associated with a median survival of 12 months or less based on retrospective series of patients who mostly did not receive novel agent therapy.(27–29) Recent retrospective analyses suggest that the incorporation of novel agent therapies as well as ASCT in the treatment of primary PCL has improved the median survival to a range of 22 to 38 months.(30–32) The original diagnostic criteria of PCL by Noel and Kyle(27) require both more than 20% circulating plasma cells and an absolute count greater than 2 × 109/L plasma cells, although presently, either criteria alone is sufficient for diagnosis.(33) However, both these criteria are dependent upon the ability of the pathologist or hematologist to screen and recognize plasma cells in the peripheral blood smear as well as quantify them in a uniform manner. This precision is not always reproducible across different institutions or medical centers making the diagnosis challenging. This raises an important question on re-defining the diagnostic criteria for PCL by finding an optimal cutoff for cPCs using more sensitive and precise technology such as flow cytometry. This could allow for earlier intervention that could possibly change the natural history of the disease. Future prospective, multi-center studies will be required to achieve this goal.

There are several limitations to our study, the first being its retrospective nature. Secondly, the cutoff of ≥400 cPCs is based on our single institution data and needs to be validated across other institutions. Third, the heterogeneity in induction treatments used limits our ability to assess the predictive value of response to various treatments. Nevertheless, this study suggests that the quantitative estimation of cPCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. This is especially useful given the short follow up time of this study as this parameter appears to be a more powerful early predictor of outcome compared with the current FISH based risk stratification, with its ability to predict for median survival of less than 3 years. Thus, these findings may have implications for revising our definitions of high-risk disease as well as our practice of a risk-adapted approach to initial therapy.

Key point.

The quantitation of clonal circulating plasma cells provides prognostic value in newly diagnosed patients with multiple myeloma

Acknowledgments

This work is supported in part by: Mayo Clinic Hematological Malignancies Program, Paul Calabresi K12 Award (CA96028). Supported in part by grants CA 107476, CA 62242, CA100707, and CA 83724 from the National Cancer Institute, Rockville, MD, USA. Also supported in part by the Jabbs Foundation, Birmingham, United Kingdom and the Henry J. Predolin Foundation, USA.

Footnotes

Contribution: S.K.K. and S.V.R. designed the study, collected and analyzed the data and wrote the manuscript; W.I.G. collected the data and contributed to writing the manuscript; M.A.G., A.D., M.Q.L., W.G.M., M.M.T., P.P.S, V.G., F.K.B., and P.K. contributed to writing and reviewing the manuscript.

Conflict-of-interest disclosure:

S.K.K, W.I.G, M.A.G., M.Q.L., S.V.R., A.D., W.G.M., M.M.T., P.P.S., V.G., F.K.B. and P.K.: These authors declare no competing financial interests.

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