Ruth de Tute

Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia

BACKGROUND A diagnosis of chronic lymphocytic leukemia (CLL) requires a count of over 5000 circulating CLL-phenotype cells per cubic millimeter. Asymptomatic persons with fewer CLL-phenotype cells have monoclonal B-cell lymphocytosis (MBL). The goal of this study was to investigate the relation between MBL and CLL. METHODS We investigated 1520 subjects who were 62 to 80 years of age with a normal blood count and 2228 subjects with lymphocytosis (>4000 lymphocytes per cubic millimeter) for the presence of MBL, using flow cytometry. Monoclonal B cells were further characterized by means of cytogenetic and molecular analyses. A representative cohort of 185 subjects with CLL-phenotype MBL and lymphocytosis were monitored for a median of 6.7 years (range, 0.2 to 11.8). RESULTS Monoclonal CLL-phenotype B cells were detected in 5.1% of subjects (78 of 1520) with a normal blood count and 13.9% (309 of 2228) with lymphocytosis. CLL-phenotype MBL had a frequency of 13q14 deletion and trisomy 12 similar to that of CLL and showed a skewed repertoire of the immunoglobulin heavy variable group (IGHV) genes. Among 185 subjects presenting with lymphocytosis, progressive lymphocytosis occurred in 51 (28%), progressive CLL developed in 28 (15%), and chemotherapy was required in 13 (7%). The absolute B-cell count was the only independent prognostic factor associated with progressive lymphocytosis. During follow-up over a median of 6.7 years, 34% of subjects (62 of 185) died, but only 4 of these deaths were due to CLL. Age above 68 years and hemoglobin level below 12.5 g per deciliter were the only independent prognostic factors for death. CONCLUSIONS The CLL-phenotype cells found in the general population and in subjects with lymphocytosis have features in common with CLL cells. CLL requiring treatment develops in subjects with CLL-phenotype MBL and with lymphocytosis at the rate of 1.1% per year.

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

In multiple myeloma, the use of multiparametric flow cytometry in many laboratories is currently restricted to clinical research studies and the differential diagnosis of unusual cases. This article report the indications of the European Myeloma Network for flow cytometry in patients with monoclonal gammopathies, and the technical recommendations for the analysis of plasma cells. The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Minimal Residual Disease Assessed by Multiparameter Flow Cytometry in Multiple Myeloma: Impact on Outcome in the Medical Research Council Myeloma IX Study

PURPOSE To investigate the prognostic value of minimal residual disease (MRD) assessment in patients with multiple myeloma treated in the MRC (Medical Research Council) Myeloma IX trial. PATIENTS AND METHODS Multiparameter flow cytometry (MFC) was used to assess MRD after induction therapy (n = 378) and at day 100 after autologous stem-cell transplantation (ASCT; n = 397) in intensive-pathway patients and at the end of induction therapy in non-intensive-pathway patients (n = 245). RESULTS In intensive-pathway patients, absence of MRD at day 100 after ASCT was highly predictive of a favorable outcome (PFS, P < .001; OS, P = .0183). This outcome advantage was demonstrable in patients with favorable and adverse cytogenetics (PFS, P = .014 and P < .001, respectively) and in patients achieving immunofixation-negative complete response (CR; PFS, P = .0068). The effect of maintenance thalidomide was assessed, with the shortest PFS demonstrable in those MRD-positive patients who did not receive maintenance and longest in those who were MRD negative and did receive thalidomide (P < .001). Further analysis demonstrated that 28% of MRD-positive patients who received maintenance thalidomide became MRD negative. MRD assessment after induction therapy in the non-intensive-pathway patients did not seem to be predictive of outcome (PFS, P = .1). CONCLUSION MRD assessment by MFC was predictive of overall outcome in patients with myeloma undergoing ASCT. This predictive value was seen in patients achieving conventional CR as well as patients with favorable and adverse cytogenetics. The effects of maintenance strategies can also be evaluated, and our data suggest that maintenance thalidomide can eradicate MRD in some patients.

Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma

In recent years, several studies on large series of multiple myeloma (MM) patients have demonstrated the clinical utility of flow cytometry monitoring of minimal residual disease (flow‐MRD) in bone marrow (BM), for improved assessment of response to therapy and prognostication. However, disturbing levels of variability exist regarding the specific protocols and antibody panels used in individual laboratories. Overall, consensus exists about the utility of combined assessment of CD38 and CD138 for the identification of BM plasma cells (PC); in contrast, more heterogeneous lists of markers are used to further distinguish between normal/reactive PCs and myeloma PCs in the MRD settings. Among the later markers, CD19, CD45, CD27, and CD81, together with CD56, CD117, CD200, and CD307, have emerged as particularly informative; however, no single marker provides enough specificity for clear discrimination between clonal PCs and normal PCs. Accordingly, multivariate analyses of single PCs from large series of normal/reactive vs. myeloma BM samples have shown that combined assessment of CD138 and CD38, together with CD45, CD19, CD56, CD27, CD81, and CD117 would be ideally suited for MRD monitoring in virtually every MM patient. However, the specific antibody clones, fluorochrome conjugates and sources of the individual markers determines its optimal (vs. suboptimal or poor) performance in an eight‐color staining. Assessment of clonality, via additional cytoplasmic immunoglobulin (CyIg) κ vs. CyIgλ evaluation, may contribute to further establish the normal/reactive vs. clonal nature of small suspicious PC populations at high sensitivity levels, provided that enough cells are evaluated.

DCDT2980S, an Anti-CD22-Monomethyl Auristatin E Antibody–Drug Conjugate, Is a Potential Treatment for Non-Hodgkin Lymphoma

Antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL. Mol Cancer Ther; 12(7); 1255–65. ©2013 AACR.

FcRL5 as a Target of Antibody–Drug Conjugates for the Treatment of Multiple Myeloma

Fc receptor-like 5 (FcRL5/FcRH5/IRTA2/CD307) is a surface protein expressed selectively on B cells and plasma cells. We found that FcRL5 was expressed at elevated levels on the surface of plasma cells from the bone marrow of patients diagnosed with multiple myeloma. This prevalence in multiple myeloma and narrow pattern of normal expression indicate that FcRL5 could be a target for antibody-based therapies for multiple myeloma, particularly antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, where limited expression on normal tissues is a key component to their safety. We found that FcRL5 is internalized upon antibody binding, indicating that ADCs to FcRL5 could be effective. Indeed, we found that FcRL5 ADCs were efficacious in vitro and in vivo but the unconjugated antibody was not. The two most effective consisted of our anti-FcRL5 antibody conjugated through cysteines to monomethylauristatin E (MMAE) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (anti-FcRL5-MC-vcPAB-MMAE) or conjugated via lysines to the maytansinoid DM4 through a disulfide linker (anti-FcRL5-SPDB-DM4). These two ADCs were highly effective in vivo in combination with bortezomib or lenalidomide, drugs in use for the treatment of multiple myeloma. These data show that the FcRL5 ADCs described herein show promise as an effective treatment for multiple myeloma. Mol Cancer Ther; 11(10); 2222–32. ©2012 AACR.

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting

Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing.

B‐cell chronic lymphocytic leukaemia cells show specific changes in membrane protein expression during different stages of cell cycle

The proliferating component in chronic lymphocytic leukaemia (CLL) is usually small (<1%) and restricted to a specific micro‐environmental niche. To characterize the proliferating component, CLL cells from bone marrow or lymph nodes of 23 patients were assessed for expression of up to 66 surface antigens in combination with nuclear Ki‐67/MCM6. Ki‐67 expression was associated with step‐wise increases in CD23/CD95/CD86/CD39/CD27 and decreases in CD24/CD69/CXCR4/CXCR5. Ki‐67+ cells showed increased CD38 expression, but with considerable inter‐patient variability: in some cases Ki‐67 expression was only detectable in CD38− CLL cells. The results suggest continuous re‐entry into the cell cycle as no distinct stem cell pool was detectable.

Outcome prediction in plasmacytoma of bone: a risk model utilizing bone marrow flow cytometry and light-chain analysis

The purpose of this study was to use multiparameter flow cytometry to detect occult marrow disease (OMD) in patients with solitary plasmacytoma of bone and assess its value in predicting outcome. Aberrant phenotype plasma cells were demonstrable in 34 of 50 (68%) patients and comprised a median of 0.52% of bone marrow leukocytes. With a median follow-up of 3.7 years, 28 of 50 patients have progressed with a median time to progression (TTP) of 18 months. Progression was documented in 72% of patients with OMD vs 12.5% without (median TTP, 26 months vs not reached; P = .003). Monoclonal urinary light chains (ULC) were similarly predictive of outcome because progression was documented in 91% vs 44% without (median TTP, 16 vs 82 months; P < .001). By using both parameters, it was possible to define patients with an excellent outcome (lacking both OMD and ULC, 7.7% progression) and high-risk patients (OMD and/or ULC, 75% progression; P = .001). Trials of systemic therapy are warranted in high-risk patients.

Minimal residual disease following autologous stem cell transplant in myeloma: impact on outcome is independent of induction regimen

Minimal residual disease (MRD) is a powerful determinant of overall outcome in multiple myeloma (MM). Previous studies have demonstrated that the presence of MRD at the traditional day 100 assessment point following autologous stem cell transplant (ASCT) independently predicts for both progression-free (PFS) and overall survival (OS). This effect on outcome is demonstrable in patients achieving a complete response (CR) and in those with both high-risk and standard-risk cytogenetics.1–4 As a consequence, MRD assessment is currently being considered as a surrogate end point for survival in academic clinical trials and for regulatory drug approval.5,6 Surrogate end points are clearly desirable in MM given the increasing complexity of treatment schedules and continually improving complete response rates and survival, such that trials of up-front therapy require 5–10 years of follow up in order to demonstrate survival differences. Acceptance of MRD as an appropriate end point would also ideally require the demonstration that this effect was independent of the treatment received. We have, therefore, assessed the impact of induction regimen and MRD on outcome in the context of the Medical Research Council (MRC) Myeloma IX trial (ISRCTN68454111). MRC Myeloma IX was a multi-center, randomized phase III trial with protocol and clinical results previously reported.7,8 All patients provided written informed consent. This analysis involves 397 patients randomly assigned to CTD (cyclophosphamide, thalidomide, and dexamethasone; n=189) or CVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone; n=208) for 4–6 cycles, then high-dose melphalan (HDM, 200 mg/m2) and ASCT. Bone marrow (BM) aspirates for MRD assessment were obtained at the end of induction (n=252) and at day 100 post ASCT (n=397). Patients were then randomly assigned to maintenance thalidomide (50–100 mg daily) or no further therapy. Flow cytometry for MRD detection was performed as reported previously. Briefly, we assessed 500,000 cells incubated with 6-color antibody combinations including CD138/CD38/CD45/CD19 with CD56/CD27 in all cases and CD81/CD117 in some cases as required.3 Statistical analyses were landmarked from date of MRD assessment, with a median follow up of 71 months. Fisher’s exact test was used to perform between-group comparisons and survival (OS and PFS) was analyzed using Kaplan-Meier and the log rank tests with a 5% significance level. We have previously reported that the CTD regimen showed superior categorical response rates to CVAD. Overall response rate was 82.5%: CTD versus 71.2% with CVAD (P<0.0001). Similarly, the CR rate was 13.0% versus 8.1% when assessed at the end of induction and 50.0% versus 37.2% post ASCT (P=0.008 and 0.0005, respectively).7 MRD analysis was performed in a subset of 397 patients and a greater proportion of patients became MRD-negative with CTD than with CVAD both at the end of induction (25% vs. 13%; P=0.0039) and post ASCT (71% vs. 54%; P<0.001).3 When outcome is assessed according to MRD status post ASCT, there is a highly significant outcome advantage for those who are MRD negative (median PFS 28.6 vs. 15.9 months, P<0.001; median OS 80.6 vs. 59.0 months, P=0.018).3 When this effect is also assessed according to the induction therapy received, it is clear that the prognostic effect of MRD negativity is identical in those who received CTD and those who received CVAD. For MRD-negative patients, the median PFS was 28.9 months with CTD versus 28.7 months with CVAD (P=0.54) (Figure 1A) while the median OS was 80.6 months versus not reached (P=0.81) (Figure 1B). Similarly, in MRD-positive patients, outcome was identical with each induction regimen, with a median PFS of 14.9 months with CTD versus 15.9 months for CVAD (P=0.96) (Figure 1A) and median OS 58.7 versus 61.9 months (P=0.91) (Figure 1B). This pattern was also demonstrable when the PFS analysis was restricted to patients achieving a conventional immunofixation-negative complete response (Figure 1C). Figure 1. Outcome following autologous stem cell transplant (ASCT) according to day 100 minimal residual disease (MRD) and induction therapy received. The impact of MRD on outcome did not differ according to induction therapy received. For MRD-negative patients ... We and others have previously demonstrated that the outcome of patients is also impacted by MRD at the end of induction as well as following ASCT, such that the outcome is best in those MRD-negative at both time points, and worst in those with detectable disease at both time points.1,3 We have also assessed this according to induction regimen and found no significant differences. In those patients who were MRD-negative at both time points, the median PFS was 44.2 months for those receiving CTD and 40.7 months for those receiving CVAD (P=0.84). Similarly, outcomes were identical in those who were MRD-positive post induction and MRD-negative post ASCT (median PFS 25.0 months, CTD; 24.2 months, CVAD; P=0.56) and in those who were positive at both time points (median PFS 13.3 months, CTD; 14.0 months, CVAD; P=0.79) (Figure 2). Figure 2. Induction regimens do not impact outcome when considered according to MRD assessed both at the end of induction and following ASCT. The outcome of patients who are MRD-negative at both time points (A), MRD-positive post induction and MRD-negative post ... The majority of studies of MRD in myeloma have assessed the value of achieving MRD-negativity, typically with a 10−4 threshold, on outcome. Flow cytometry can also provide a quantitative assessment of residual tumor in those with detectable disease. In a recent analysis, we demonstrated that the level of residual disease is also highly informative, such that an approximate 1-year OS benefit is demonstrable for each log of tumor depletion.4 We have also assessed this effect according to induction regimen and note that a greater proportion of patients receiving CVAD had high levels of MRD ( 0.1%; CVAD n= 68, CTD n=33; P=0.0005) but no difference in PFS was demonstrable across several logs of detectable disease. For those with MRD more than 1% median, PFS was 9.5 months with CTD and 6.5 months with CVAD (P=0.49). A similar pattern was also demonstrable in MRD-positive patients with lower levels of disease (0.1% – <1% median PFS 13.7 months CTD vs. 13.6 months CVAD, P=0.9; 0.01% – <0.1% median PFS 22.1 months CTD vs. 23.6 months CVAD, P=0.84) (Figure 3). Figure 3. Impact of quantitative level of MRD on outcome. The outcome of MRD-positive patients with MRD levels of 0.01% – <0.1% (A), 0.1% – <1% (B) and greater than 1% (C) did not differ with induction regimen (P=0.84, 0.9 and 0.49, ... We would conclude that these data demonstrate that the prognostic impact of MRD is independent of the induction therapy received. The proportion of patients who become MRD-negative (at the 10−4 level) can vary significantly between regimens, but the impact of MRD-negativity is similar regardless of the induction therapy received. It is possible that differences may emerge with more effective induction regimens as levels of disease may then vary over several logs in those patients who are MRD-negative. However, data from those with quantifiable residual disease suggest that outcome is determined by the level of disease rather than the regimen received. This phenomenon has also been clearly demonstrated in B-cell chronic lymphocytic leukemia (CLL) in the German CLL Study Group CLL8 trial.9 In this study, patients were treated with fludarabine-cyclophosphamide (FC) with/without rituximab (R). MRD-negativity was associated with improved PFS and OS, and although a greater proportion of patients became MRD-negative with FCR, there was no difference between the treatment arms when outcome was assessed according to levels of disease. These data provide further evidence to support the role of MRD as a surrogate end point for survival in clinical trials.