Xenofon Papanikolaou

Clinical, genomic and imaging predictors of myeloma progression from asymptomatic monoclonal gammopathies (SWOG S0120)

All cases of clinical myeloma (CMM) are preceded by an asymptomatic monoclonal gammopathy (AMG), classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM). We analyzed data from AMG patients (n = 331) enrolled in a prospective, observational clinical trial (S0120). Baseline data from clinical variables, gene expression profiles (GEP) of purified tumor cells, and findings of magnetic resonance imaging (MRI) were correlated with the risk of progression to CMM requiring therapy. GEP of purified tumor cells revealed that all molecular subtypes of CMM are also represented in the AMG phase. An increased risk score (>-0.26) (based on a 70-gene signature, GEP70) was an independent predictor of the risk of progression to CMM. Combination of elevated serum free light chain, M-spike, and GEP70 risk score identified a subset with high risk (67% at 2 years) of progression to CMM requiring therapy. Importantly, absence of these factors in AMM patients predicted low risk similar to MGUS. Detection of multiple (>1) focal lesions by MRI also conferred an increased risk of progression. These data demonstrate that signatures associated with high-risk CMM impact disease risk and support inclusion of genomic analysis in the clinical management of AMGs.

Characterization of the molecular mechanism of the bone-anabolic activity of carfilzomib in multiple myeloma.

Carfilzomib, the next generation of proteasome inhibitor, may increase osteoblast-related markers in patients with multiple myeloma, but the molecular mechanism of its effect on mesenchymal stem cell differentiation to osteoblasts remains unknown. Herein, we demonstrated that carfilzomib significantly promoted mesenchymal stem cell differentiation into osteoblasts. In osteoprogenitor cells and primary mesenchymal stem cells from patients with myeloma, carfilzomib induced increases in alkaline phosphatase activity, matrix mineralization, and calcium deposition via Wnt-independent activation of β-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting analysis and immunofluorescence, we found that carfilzomib induced stabilization of both free and active forms of β-catenin in a time- and dose-dependent manner that was not associated with β-catenin transcriptional regulation. Nuclear translocation of β-catenin protein was associated with TCF transcriptional activity that was independent of the effects of GSK3β-activation and of signaling induced by 19 Wnt ligands, 10 Frizzled receptors, and LRP5/6 co-receptors. Blocking activation of β-catenin/TCF signaling by dominant negative TCF1 or TCF4 attenuated carfilzomib-induced matrix mineralization. Thus, carfilzomib induced osteoblast differentiation via Wnt-independent activation of the β-catenin/TCF pathway. These results provide a novel molecular mechanism critical to understanding the anabolic role of carfilzomib on myeloma-induced bone disease.

Four genes predict high risk of progression from smoldering to symptomatic multiple myeloma (SWOG S0120)

Multiple myeloma is preceded by an asymptomatic phase, comprising monoclonal gammopathy of uncertain significance and smoldering myeloma. Compared to the former, smoldering myeloma has a higher and non-uniform rate of progression to clinical myeloma, reflecting a subset of patients with higher risk. We evaluated the gene expression profile of smoldering myeloma plasma cells among 105 patients enrolled in a prospective observational trial at our institution, with a view to identifying a high-risk signature. Baseline clinical, bone marrow, cytogenetic and radiologic data were evaluated for their potential to predict time to therapy for symptomatic myeloma. A gene signature derived from four genes, at an optimal binary cut-point of 9.28, identified 14 patients (13%) with a 2-year therapy risk of 85.7%. Conversely, a low four-gene score (<9.28) combined with baseline monoclonal protein <3 g/dL and albumin ≥3.5 g/dL identified 61 patients with low-risk smoldering myeloma with a 5.0% chance of progression at 2 years. The top 40 probe sets showed concordance with indices of chromosome instability. These data demonstrate high discriminatory power of a gene-based assay and suggest a role for dysregulation of mitotic checkpoints in the context of genomic instability as a hallmark of high-risk smoldering myeloma.

Metronomic therapy is an effective salvage treatment for heavily pre-treated relapsed/refractory multiple myeloma

Relapsed/refractory multiple myeloma represents a major challenge in multiple myeloma therapy. For patients with relapsed/refractory multiple myeloma, we developed a treatment schema of metronomically scheduled drug therapy. We identified 186 patients who had been treated with metronomic therapy between March 2004 and January 2012 with a median follow up of 24.2 months. Median age was 61 years (range 36–83). Median number of prior therapies was 14 (range 1–51). Median number of completed metronomic therapy cycles was 1 (range 1–5), while 45 of 186 (25%) received 2 or more cycles. Responses included complete remission in 11 of 186 patients (6%), very good partial remission in 12 of 186 (7%), partial remission in 65 of 179 (36%), and minimal response in 29 of 186 (16%), for an overall response rate of 63% (117 of 186). Median overall survival and progression-free survival were 11.2 and 3.6 months, respectively. Hematologic toxicity grading was problematic as 146 of 186 (78%) of patients presented with at least grade 2 thrombocytopenia within 90 days prior to starting metronomic therapy. Grade 4 leukopenia, anemia, and/or thrombocytopenia following metronomic therapy occurred in 108 of 186 (58%), 12 of 186 (6%), and 147 of 186 (79%) patients, respectively. Incidence of grade 3–4 neutropenic fever was 4 of 186 (2%). Most patients (177 of 186, 95%) were treated in an outpatient unit and secondary admissions due to regimen-related toxicity occurred in 37 of 186 (20%). Treatment-related mortality was evident in 2 of 186 (1%). In conclusion, metronomic therapy is an effective late salvage treatment in relapsed/refractory multiple myeloma, with a high overall response rate and a favorable toxicity profile.

Adverse metaphase cytogenetics can be overcome by adding bortezomib and thalidomide to fractionated melphalan transplants

Purpose: To determine whether a reduction in the intensity of Total Therapy (TT) reduces toxicity and maintains efficacy. Experimental Design: A total of 289 patients with gene expression profiling (GEP70)-defined low-risk multiple myeloma were randomized between a standard arm (TT4-S) and a light arm (TT4-L). TT4-L employed one instead of two inductions and consolidations. To compensate for potential loss of efficacy of TT4-L, bortezomib and thalidomide were added to fractionated melphalan 50 mg/m2/d for 4 days. Results: Grade ≥3 toxicities and treatment-related mortalities were not reduced in TT4-L. Complete response (CR) rates were virtually identical (P = 0.2; TT4-S, 59%; TT4-L, 61% at 2 years), although CR duration was superior with TT4-S (P = 0.05; TT4-S, 87%; TT4-L, 81% at 2 years). With a median follow-up of 4.5 years, there was no difference in overall survival (OS) and progression-free survival (PFS). Whereas metaphase cytogenetic abnormalities (CAs) tended to be an adverse feature in TT4-S, as with predecessor TT trials, the reverse applied to TT4-L. Employing historical TT3a as training and TT3b as test set, 51 gene probes (GEP51) significantly differentiated the presence and absence of CA (q < 0.0001), seven of which function in DNA replication, recombination, and repair. Applying the GEP51 model to clinical outcomes, OS and PFS were significantly inferior with GEP51/CA in TT4-S; such a difference was not observed in TT4-L. Conclusions: We identified a prognostic CA-linked GEP51 signature, the adversity of which could be overcome by potentially synergizing anti–multiple myeloma effects of melphalan and bortezomib. These exploratory findings require confirmation in a prospective randomized trial. Clin Cancer Res; 23(11); 2665–72. ©2016 AACR.

Flow cytometry defined cytoplasmic immunoglobulin index is a major prognostic factor for progression of asymptomatic monoclonal gammopathies to multiple myeloma (subset analysis of SWOG S0120).

Multiple myeloma (MM) is a clonal plasma cell (PC) disorder characterized by end organ damage that is in turn characterized by CRAB criteria (calcium and creatinine elevation, anemia and bone lesions).1 It is commonly accepted that nearly all cases of MM are preceded by a clinically benign phase of monoclonal gammopathy of undetermined significance (MGUS) that evolves through a stage of smoldering multiple myeloma (SMM) without end organ damage,2 collectively referred to as asymptomatic monoclonal gammopathies (AMG).3 Although traditionally SMM is considered more prone to MM progression than MGUS, additional variables, such as involved-to-uninvolved free light-chain ratio4 and magnetic resonance imaging-defined focal lesion number and size,5 have been linked to progression to MM and form the basis for the newest International Myeloma Working Group criteria for MM.6 As the treatment of MM has been greatly advanced, emphasis has been placed on identifying patients with AMG at high risk of progression to MM so that, with earlier treatment, end organ damage can be minimized.7 Many new high-risk variables have indeed been identified such as level of circulating plasma cells8 and gene expression profiling (GEP).9, 10 We have previously reported that two-parameter flow cytometry of DNA and cytoplasmic light-chain immunoglobulin (DNA/CIG) is highly predictive of progression-free and overall survival in newly diagnosed MM treated with Total Therapy.11 In the current subset analysis of S0120, we have investigated whether the DNA/CIG assay can also identify patients with AMG at high risk for progression to MM requiring therapy (time to therapy, TTT).12 Of 254 patients enrolled at the University of Arkansas in the observational SWOG S0120 protocol with AMG, 110 had evaluable DNA/CIG information and retained AMG status according to the revised International Myeloma Working Group criteria for MM.6 All patients underwent detailed clinical staging as previously reported.9, 10 DNA/CIG assay was performed on whole bone marrow aspirates along with metaphase cytogenetics and GEP of CD138+ purified PC.13 Imaging studies involved metastatic bone surveys and, in the majority of the cases, magnetic resonance imaging examination of the axial and appendicular skeleton. Details of the DNA/CIG method have been published elsewhere.14, 15 A technical modification of the assay was applied uniformly since August 2006. The assay is based on the two-parameter flow cytometry of cytoplasmic immunoglobulin and DNA of whole bone marrow aspirates. Single-cell suspensions were exposed to anti-light-chain reagents (Dako Kappa and Lambda light chain F(AB)2/FITC conjugated) and then counterstained for DNA with propidium iodide with the addition of RNase. To quantitate the cellular DNA content, DNA index (DI)16 was determined and calculated as the ratio of the mean for each light-chain-positive G0/1 DNA peak divided by the mean of the light-chain-negative diploid G0/1 peak on the X axis. A DI between 0.99 and 1.01 was referred to as diploid, while hyperdiploid implied DI>1.01 and hypodiploid DI<0.99. The excess of kappa- or lambda-positive cells identified the involved or light-chain-restricted (LCR) cell population, the percentage of which was calculated in relation to the total number of gated events. Among the LCR cell population, discrete populations of cells with different DI were identified, which we refer to from here on as DNA stem lines. The involved DNA stem line with the highest percentage was considered dominant. To quantitate the cytoplasmic immunoglobulin content of a light-chain-positive population, the cytoplasmic immunoglobulin index (CIg) was used and calculated from the ratio of the geometric mean of the Y axis (cytoplasmic immunoglobulin fluorescence intensity) for the light-chain-positive G0/1 peak divided by the Y axis geometric mean of the light-chain-negative diploid G0/1 population. The CIg of each distinct DNA stem line was calculated as explained above. Kaplan–Meier methods were used to generate survival distribution graphs, and comparisons were made employing the log-rank test. For continuous variables, the running log-rank method was applied for the calculation of optimal cutoff points. The R2 statistic was used to evaluate the predictive power of different models. Wilcoxon tests were used to compare the medians of continuous measurements between groups. The characteristics of the 110 patients lacking the revised International Myeloma Working Group criteria for MM are portrayed in Supplementary Table 1. The median follow-up time for the 110 patients was 4.8 years. Aneuploidy by DNA/CIG was evident in 64%, all of whom had hyperdiploid stem lines, while additional hypodiploid abnormalities were present in two cases. Low hemoglobin (<10 g/dl) pertained to only 4% (non-plasma cell dyscrasia-related reasons) while creatinine ⩾2 mg/dl was evident in one case due to hypertension-related nephrosclerosis. Metaphase cytogenetic abnormalities (CA) were documented in 16%, a GEP70 score⩾−0.26(ref. 3) pertained to 33% and a recently defined novel GEP4 score⩾9.28(ref. 17) to 12% of patients. We examined the TTT probability of AMG (Table 1). Optimal cutoff points were obtained for all continuous numerical values. We confirm other studies linking older age ⩾65 years, albumin 8.4 The presence of CA, GEP70- and GEP4- high-risk designations was strongly linked to inferior TTT. Among DNA/CIG-derived parameters, CIg 17 were both strongly linked to progression to MM. Other DNA/CIG variables associated with TTT included the presence of aneuploidy and the presence of ⩾2 DNA stem lines (Figure 1). The 26 patients with CIg 17 present in 20 patients conferred a 2-year MM progression rate of 60% versus 9% among the 90 with lower (Figure 1b). Consideration of both DNA/CIG features identified 14 patients displaying two high-risk features with 2-year TTT of 71.4% as opposed to 5.1% in 78 patients with only favorable features, while the presence of one adverse variable present in 18 patients was associated with a 2-year TTT probability of approximately 34% (Figure 1c). Figure 1 Kaplan–Meier plots for the time to progression from AMG to MM requiring therapy according to: CIg, (a) total LCR%, (b) the combination of CIg and total LCR% (c) and the combination of CIg and total LCR% for the SMM population ... Table 1 Cox regression for time to progression to MM In the multivariate model, serum-M⩾3 g/dl, CIg 17% independently conferred adverse outcomes (Table 1). All three parameters combined provided for a high R2 value of 0.861, implying that TTT probability could be accounted for in 86% (Supplementary Table 2). In comparison, the classical criteria of bone marrow plasmacytosis ⩾10% and serum-M⩾3 g/dl had a lower cumulative R2 of 0.632. When only the sub-population of SMM (80 patients; Supplementary Table 3) was considered, DNA/CIG-derived variables retained their statistical significance (Supplementary Table 4). Both LCR>17% and CIg 17% and serum-M⩾3 g/dl; albumin<3.5 g/dl and B2M⩾3.5 mg/l also conferred higher TTT probability for a R2 of 0.862 (Supplementary Tables 4 and 5). The inclusion of GEP variables, available in a subset of 61 patients, identified GEP-4 as a significant variable, dispelling CIg and B2M from the model (R2=0.895; Supplementary Tables 4 and 6). CIg is a measure of plasma cell immunoglobulin production.15 We therefore examined CIg values in patients with MGUS and SMM (both from the S0120 trial), and in newly diagnosed MM patients accrued to Total Therapy 3b.18 Median CIg values declined progressively with the transition from MGUS to SMM and later to MM (10.5 versus 5.6 versus 3.3, P<0.001; Supplementary Figure 1a). To exclude the possibility that the difference in CIg reflects the decreasing percentage of highly secreting normal plasma cells with the evolution of plasma cell dyscrasias,19, 20 the analysis was repeated for strictly aneuploid cases. Again, the evolution from MGUS to SMM to MM was characterized by a progressively lower CIg (16.0 versus 9.1 versus 3.5, P<0.0001; Supplementary Figure 1b). In summary, DNA/CIG offers powerful prognostic information for AMG even in the era of genomic profiling. While LCR% reflects tumor burden, the finding of progressively decreasing CIg with the evolution of plasma cell dyscrasias in this single institution subset analysis of S0120 is novel. It provides evidence that the progression of plasma cell dyscrasias is accompanied by a progressive decline in immunoglobulin production capacity.

The flow cytometry-defined light chain cytoplasmic immunoglobulin index and an associated 12-gene expression signature are independent prognostic factors in multiple myeloma

As part of Total Therapy (TT) 3b, baseline marrow aspirates were subjected to two-color flow cytometry of nuclear DNA content and cytoplasmic immunoglobulin (DNA/CIG) as well as plasma cell gene expression profiling (GEP). DNA/CIG-derived parameters, GEP and standard clinical variables were examined for their effects on overall survival (OS) and progression-free survival (PFS). Among DNA/CIG parameters, the percentage of the light chain-restricted (LCR) cells and their cytoplasmic immunoglobulin index (CIg) were linked to poor outcome. In the absence of GEP data, low CIg <2.8, albumin <3.5 g/dl and age ⩾65 years were significantly associated with inferior OS and PFS. When GEP information was included, low CIg survived the model along with GEP70-defined high risk and low albumin. Low CIg was linked to beta-2-microglobulin >5.5 mg/l, a percentage of LCR cells exceeding 50%, C-reactive protein ⩾8 mg/l and GEP-derived high centrosome index. Further analysis revealed an association of low CIg with 12 gene probes implicated in cell cycle regulation, differentiation and drug transportation from which a risk score was developed in TT3b that held prognostic significance also in TT3a, TT2 and HOVON trials, thus validating its general applicability. Low CIg is a powerful new prognostic variable and has identified potentially drug-able targets.

Reply to “Metronomic chemotherapy beyond misconceptions” - Haematologica 2013;98(11):e145

In their recent comment on our manuscript “Metronomic therapy is an effective salvage treatment for heavily pre-treated relapsed/refractory multiple myelo ma”, Drs. Hatzimichael and Briasoulis generally agree with the findings and results of metronomically scheduled chemotherapy for relapsed refractory patients, a challeng ing group of patients with an unmet medical need. However, the Authors raise some concerns about terminology and definitions, which we would like to clarify. In the reported retrospective study, responses were evaluated on at least two consecutive assessments for every individual case before the start of a new therapy according to the latest IMWG criteria. 1 This allowed us to not only report response rates, but also time to best response. It should be noted that the term “two consecutive assess ments” refers to distinct time points and allows assess ment of response even after a single cycle of therapy. As the Authors correctly point out, the concept of “Metronomic chemotherapy stands in the antipode of MTD (maximum-tolerated dose) chemotherapy and is by concept an angiogenesis targeted cancer therapy”. In fact, the antiangiogenic effect forms the foundation of metro nomic chemotherapy 2 and it is the only one that has been convincingly demonstrated ex vivo . 3-5 For this reason, metronomic chemotherapy is often combined with the anti-VEGF antibody bevacizumab in solid tumors. 6

Clinical Presentation and Gene Expression Profiling of Immunoglobulin M Multiple Myeloma Compared With Other Myeloma Subtypes and Waldenström Macroglobulinemia

Purpose Multiple myeloma (MM) is a clonal bone marrow disease characterized by the neoplastic transformation of differentiated postgerminal B cells. It is a heterogeneous disease both at the genetic level and in terms of clinical outcome. Immunoglobulin M (IgM) MM is a rare subtype of myeloma. Similar to Waldenström macroglobulinemia (WM), patients with MM experience IgM monoclonal gammopathy; however, both diseases are distinct in terms of treatment and clinical behavior. Materials and Methods To shed light on the presentation of IgM MM, its prognosis, and its gene expression profiling, we identified and characterized 21 patients with IgM MM from our database. Results One of these patients presented with a rare IgM monoclonal gammopathy of undetermined significance that progressed to smoldering myeloma. The median survival of the 21 patients was 4.9 years, which was comparable to a matched group of patients with non-IgM MM with similar myeloma prognostic factors (age, gender, albumin, creatinine, anemia, lactate dehydrogenase, β2-microglobulin, cytogenetics abnormalities), but much less than the median survival reported for patients with WM (9 years). We identified a cluster of genes that differ in their expression profile between MM and WM and found that the patients with IgM MM displayed a gene expression profile most similar to patients with non-IgM MM, confirming that IgM MM is a subtype of MM that should be differentiated from WM. Conclusion Because the prognosis of IgM MM and WM differ significantly, an accurate diagnosis is essential. Our gene expression model can assist with the differential diagnosis in controversial cases.

Spectrum of Cerebrovascular Disease in Patients with Multiple Myeloma Undergoing Chemotherapy—Results of a Case Control Study

[This corrects the article DOI: 10.1371/journal.pone.0166627.].