Adam Rosenthal

Clinical value of molecular subtyping multiple myeloma using gene expression profiling

Using a data set of 1217 patients with multiple myeloma enrolled in Total Therapies, we have examined the impact of novel therapies on molecular and risk subgroups and the clinical value of molecular classification. Bortezomib significantly improved the progression-free survival (PFS) and overall survival (OS) of the MMSET (MS) subgroup. Thalidomide and bortezomib positively impacted the PFS of low-risk (LoR) cases defined by the GEP70 signature, whereas high-risk (HiR) cases showed no significant changes in outcome. We show that molecular classification is important if response rates are to be used to predict outcomes. The t(11;14)-containing CD-1 and CD-2 subgroups showed clear differences in time to response and cumulative response rates but similar PFS and OS. Furthermore, complete remission was not significantly associated with the outcome of the MAF/MAFB (MF) subgroup or HiR cases. HiR cases were enriched in the MF, MS and proliferation subgroups, but the poor outcome of these groups was not linked to subgroup-specific characteristics such as MAF overexpression per se. It is especially important to define risk status if HiR cases are to be managed appropriately because of their aggressive clinical course, high rates of early relapse and the need to maintain therapeutic pressure on the clone.

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.

Renal insufficiency retains adverse prognostic implications despite renal function improvement following Total Therapy for newly diagnosed multiple myeloma

Renal insufficiency (RI) is a frequent complication of multiple myeloma (MM) with negative consequences for patient survival. The improved clinical outcome with successive Total Therapy (TT) protocols was limited to patients without RI. We therefore performed a retrospective analysis of overall survival, progression-free survival and time to progression (TTP) of patients enrolled in TT2 and TT3 in relationship to RI present at baseline and pre-transplant. Glomerular filtration rate was graded in four renal classes (RCs), RC1–RC4 (RC1 ⩾90 ml/min/1.73 m2, RC2 60–89 ml/min/1.73 m2, RC3 30–59 ml/min/1.73 m2 and RC4 <30 ml/min/1.73 m2). RC1–3 had comparable clinical outcomes while RC4 was deleterious, even after improvement to better RC after transplant. Among the 85% of patients with gene expression profiling defined low-risk MM, Cox regression modeling of baseline and pre-transplant features, which also took into consideration RC improvement and MM complete response (CR), identified the presence of metaphase cytogenetic abnormalities and baseline RC4 as independent variables linked to inferior TTP post-transplant, while MM CR reduced the risk of progression and TTP by more than 60%. Failure to improve clinical outcomes despite RI improvement suggested MM-related causes. Although distinguishing RC4 from RC<4, 46 gene probes bore no apparent relationship to MM biology or survival.

Removing batch effects from purified plasma cell gene expression microarrays with modified ComBat

BackgroundGene expression profiling (GEP) via microarray analysis is a widely used tool for assessing risk and other patient diagnostics in clinical settings. However, non-biological factors such as systematic changes in sample preparation, differences in scanners, and other potential batch effects are often unavoidable in long-term studies and meta-analysis. In order to reduce the impact of batch effects on microarray data, Johnson, Rabinovic, and Li developed ComBat for use when combining batches of gene expression microarray data.We propose a modification to ComBat that centers data to the location and scale of a pre-determined, ‘gold-standard’ batch. This modified ComBat (M-Combat) is designed specifically in the context of meta-analysis and batch effect adjustment for use with predictive models that are validated and fixed on historical data from a ‘gold-standard’ batch.ResultsWe combined data from MIRT across two batches (‘Old’ and ‘New’ Kit sample preparation) as well as external data sets from the HOVON-65/GMMG-HD4 and MRC-IX trials into a combined set, first without transformation and then with both ComBat and M-ComBat transformations. Fixed and validated gene risk signatures developed at MIRT on the Old Kit standard (GEP5, GEP70, and GEP80 risk scores) were compared across these combined data sets.Both ComBat and M-ComBat eliminated all of the differences among probes caused by systematic batch effects (over 98% of all untransformed probes were significantly different by ANOVA with 0.01 q-value threshold reduced to zero significant probes with ComBat and M-ComBat). The agreement in mean and distribution of risk scores, as well as the proportion of high-risk subjects identified, coincided with the ‘gold-standard’ batch more with M-ComBat than with ComBat. The performance of risk scores improved overall using either ComBat or M-Combat; however, using M-ComBat and the original, optimal risk cutoffs allowed for greater ability in our study to identify smaller cohorts of high-risk subjects.ConclusionM-ComBat is a practical modification to an accepted method that offers greater power to control the location and scale of batch-effect adjusted data. M-ComBat allows for historical models to function as intended on future samples despite known, often unavoidable systematic changes to gene expression data.

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.

Identification of novel mutational drivers reveals oncogene dependencies in multiple myeloma.

Understanding the profile of oncogene and tumor suppressor gene mutations with their interactions and impact on the prognosis of multiple myeloma (MM) can improve the definition of disease subsets and identify pathways important in disease pathobiology. Using integrated genomics of 1273 newly diagnosed patients with MM, we identified 63 driver genes, some of which are novel, including IDH1, IDH2, HUWE1, KLHL6, and PTPN11 Oncogene mutations are significantly more clonal than tumor suppressor mutations, indicating they may exert a bigger selective pressure. Patients with more driver gene abnormalities are associated with worse outcomes, as are identified mechanisms of genomic instability. Oncogenic dependencies were identified between mutations in driver genes, common regions of copy number change, and primary translocation and hyperdiploidy events. These dependencies included associations with t(4;14) and mutations in FGFR3, DIS3, and PRKD2; t(11;14) with mutations in CCND1 and IRF4; t(14;16) with mutations in MAF, BRAF, DIS3, and ATM; and hyperdiploidy with gain 11q, mutations in FAM46C, and MYC rearrangements. These associations indicate that the genomic landscape of myeloma is predetermined by the primary events upon which further dependencies are built, giving rise to a nonrandom accumulation of genetic hits. Understanding these dependencies may elucidate potential evolutionary patterns and lead to better treatment regimens.

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.

Treatment to suppression of focal lesions on positron emission tomography-computed tomography is a therapeutic goal in newly diagnosed multiple myeloma

Fluorine-18 fluorodeoxyglucose positron emission tomography with computed tomography attenuation correction (PET-CT) in myeloma can detect and enumerate focal lesions by the quantitative characterization of metabolic activity. The aim of this study was to determine the prognostic significance of the suppression of PET-CT activity at a number of time points post therapy initiation: day 7, post induction, post transplant, and at maintenance therapy. As part of the TT4-6 trial series, 596 patients underwent baseline PET-CT and were evaluated serially during their disease course using peak standardized uptake values above background red marrow signal. We demonstrate that the presence of more than 3 focal lesions at presentation identifies a group of patients with an adverse progression-free survival and overall survival. At day 7 of therapy, patients with complete focal lesion signal suppression revert to the same prognosis as those with no lesions at diagnosis. At later time points, the continued suppression of signal remains prognostically important. We conclude that for newly diagnosed patients with focal lesions, treatment until these lesions are suppressed is an important therapeutic goal as the prognosis of these patients is the same as those without lesions at diagnosis. (clinicaltrials.gov identifiers: 00734877, 02128230, 00869232, 00871013).