Combination of flow cytometry and functional imaging for monitoring of residual disease in myeloma.

The iliac crest is the sampling site for minimal residual disease (MRD) monitoring in multiple myeloma (MM). However, the disease distribution is often heterogeneous, and imaging can be used to complement MRD detection at a single site. We have investigated patients in complete remission (CR) during first-line or salvage therapy for whom MRD flow cytometry and the two imaging modalities positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) were performed at the onset of CR. Residual focal lesions (FLs), detectable in 24% of first-line patients, were associated with short progression-free survival (PFS), with DW-MRI detecting disease in more patients. In some patients, FLs were only PET positive, indicating that the two approaches are complementary. Combining MRD and imaging improved prediction of outcome, with double-negative and double-positive features defining groups with excellent and dismal PFS, respectively. FLs were a rare event (12%) in first-line MRD-negative CR patients. In contrast, patients achieving an MRD-negative CR during salvage therapy frequently had FLs (50%). Multi-region sequencing and imaging in an MRD-negative patient showed persistence of spatially separated clones. In conclusion, we show that DW-MRI is a promising tool for monitoring residual disease that complements PET and should be combined with MRD.

Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of "fitter" clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.In multiple myeloma, malignant cells expand within bone marrow. Here, the authors use multi-region sequencing in patient samples to analyse spatial clonal architecture and heterogeneity, providing novel insight into multiple myeloma progression and evolution.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Hyperhaploidy is a novel high-risk cytogenetic subgroup in multiple myeloma.

Hyperhaploid clones (24-34 chromosomes) were identified in 33 patients with multiple myeloma (MM), demonstrating a novel numerical cytogenetic subgroup. Strikingly, all hyperhaploid karyotypes were found to harbor monosomy 17p, the single most important risk stratification lesion in MM. A catastrophic loss of nearly a haploid set of chromosomes results in disomies of chromosomes 3, 5, 7, 9, 11, 15, 18, 19 and 21, the same basic set of odd-numbered chromosomes found in trisomy in hyperdiploid myeloma. All other autosomes are found in monosomy, resulting in additional clinically relevant monosomies of 1p, 6q, 13q and 16q. Hypotriploid subclones (58-68 chromosomes) were also identified in 11 of the 33 patients and represent a duplication of the hyperhaploid clone. Analysis of clones utilizing interphase fluorescence in situ hybridization (iFISH), metaphase FISH and spectral karyotyping identified either monosomy 17 or del17p in all patients. Amplification of 1q21 was identified in eight patients, demonstrating an additional high-risk marker. Importantly, our findings indicate that current iFISH strategies may be uninformative or ambiguous in the detection of these clones, suggesting this patient subgroup maybe underreported. Overall survival for patients with hyperhaploid clones was poor, with a 5-year survival rate of 23.1%. These findings identify a distinct numerical subgroup with cytogenetically defined high-risk disease.

Dose-dense and less dose-intense Total Therapy 5 for gene expression profiling-defined high-risk multiple myeloma.

Multiple myeloma (MM) is a heterogeneous disease with high-risk patients progressing rapidly despite treatment. Various definitions of high-risk MM are used and we reported that gene expression profile (GEP)-defined high risk was a major predictor of relapse. In spite of our best efforts, the majority of GEP70 high-risk patients relapse and we have noted higher relapse rates during drug-free intervals. This prompted us to explore the concept of less intense drug dosing with shorter intervals between courses with the aim of preventing inter-course relapse. Here we report the outcome of the Total Therapy 5 trial, where this concept was tested. This regimen effectively reduced early mortality and relapse but failed to improve progression-free survival and overall survival due to relapse early during maintenance.

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.

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.

Impact of a change in antibacterial prophylaxis on bacteremia and hospitalization rates following outpatient autologous peripheral blood stem cell transplantation for multiple myeloma.

BACKGROUND: Levofloxacin is routinely used for the prevention of invasive bacterial infections during autologous peripheral blood stem cell transplantation (APBSCT). However, increasing rates of bacterial sepsis were noted at our institution among multiple myeloma (MM) patients undergoing outpatient APBSCT with melphalan-based chemotherapy and levofloxacin prophylaxis. We assessed the impact of a change in antibacterial prophylaxis from oral levofloxacin (Period 1) to sequential oral levofloxacin followed by ertapenem (Period 2). METHODS: Electronic medical records were reviewed to identify MM patients who underwent APBSCT in the outpatient clinic between October 2007 and April 2012. RESULTS: Over a 4.5-year period, 165 outpatient APBSCTs were eligible for the analysis. Fewer overall bacteremias occurred during Period 2 as compared with Period 1 (0.5 cases per 100 person-days vs. 2.4 cases per 100 person-days, P<0.001). In addition, fewer patients were hospitalized for neutropenic fever while receiving sequential prophylaxis (45.7% vs. 75.7% of outpatient APBSCT recipients during Periods 2 and 1, respectively; P<0.001). In Kaplan-Meier analysis, receipt of sequential prophylaxis (Period 2) was significantly associated with overall bacteremia-free survival within 30 days after the APBSCT (P<0.001). No significant differences were seen in the number of patients developing Clostridium difficile infection or ertapenem-resistant gram-negative bacteremia between study periods. CONCLUSION: In conclusion, sequential prophylaxis may effectively prevent episodes of bacteremia and hospitalizations in neutropenic MM outpatient APBSCT recipients. Prospective studies that involve larger numbers of MM patients with extended periods of follow-up are ultimately required to define the safety and efficacy of sequential antibacterial prophylaxis.

VTD combination therapy with bortezomib-thalidomide-dexamethasone is highly effective in advanced and refractory multiple myeloma.

Bortezomib (V) was combined with thalidomide (T) and dexamethasone (D) in a phase I/II trial to determine dose-limiting toxicities (DLT's) and clinical activity of the VTD regimen in 85 patients with advanced and refractory myeloma. The starting dose of V was 1.0 mg/m(2) (days 1, 4, 8, 11, every 21 day) with T added from cycle 2 at 50 mg/day, with 50 mg increments per 10 patient cohorts, to a maximum dose of 200 mg. In the absence of DLT's, the same reiteration of T dose increases was applied with a higher dose of V=1.3 mg/m(2). D was added with cycle 4 in the absence of partial response (PR). Ninety-two percent had prior autotransplants, 74% had prior T and 76% abnormal cytogenetics. MTD was reached at V=1.3 mg/m(2) and T=150 mg. Minor response (MR) was recorded in 79%, and 63% achieved PR including 22% who qualified for near-complete remission. At 4 years, 6% remain event-free and 23% alive. Both OS and EFS were significantly longer in the absence of prior T exposure and when at least MR status was attained. The MMSET/FGFR3 molecular subtype was prognostically favorable, a finding since reported for a VTD-incorporating tandem transplant trial (Total Therapy 3) for untreated patients with myeloma (BJH 2008).

Prevention of thalidomide- and lenalidomide-associated thrombosis in myeloma.

The incidence of venous thromboembolism (VTE) is more than 1 per thousand annually in the general population and increases further in cancer patients. The risk of VTE is higher in multiple myeloma (MM) patients who receive thalidomide or lenalidomide, especially in combination with dexamethasone or chemotherapy. Various VTE prophylaxis strategies, such as low-molecular-weight heparin (LMWH), warfarin or aspirin, have been investigated in small, uncontrolled clinical studies. This manuscript summarizes the available evidence and recommends a prophylaxis strategy according to a risk-assessment model. Individual risk factors for thrombosis associated with thalidomide/lenalidomide-based therapy include age, history of VTE, central venous catheter, comorbidities (infections, diabetes, cardiac disease), immobilization, surgery and inherited thrombophilia. Myeloma-related risk factors include diagnosis and hyperviscosity. VTE is very high in patients who receive high-dose dexamethasone, doxorubicin or multiagent chemotherapy in combination with thalidomide or lenalidomide, but not with bortezomib. The panel recommends aspirin for patients with < or = 1 risk factor for VTE. LMWH (equivalent to enoxaparin 40 mg per day) is recommended for those with two or more individual/myeloma-related risk factors. LMWH is also recommended for all patients receiving concurrent high-dose dexamethasone or doxorubicin. Full-dose warfarin targeting a therapeutic INR of 2-3 is an alternative to LMWH, although there are limited data in the literature with this strategy. In the absence of clear data from randomized studies as a foundation for recommendations, many of the following proposed strategies are the results of common sense or derive from the extrapolation of data from many studies not specifically designed to answer these questions. Further investigation is needed to define the best VTE prophylaxis.

Clonal cytogenetic changes and myeloma relapse after reduced intensity conditioning allogeneic transplantation.

To identify a correlation between metaphase cytogenetics and relapse after reduced intensity conditioning (RIC) allotransplant for patients with multiple myeloma, data on 60 patients (median age 52) who received grafts from a sibling (n = 49) or unrelated donor (n = 11) were analyzed. Fifty-three patients (88%) showed chromosomal abnormalities (CA) before the allotransplant, including 42 with abnormalities involving 13q (CA13). Twenty-two patients (41%) relapsed post-allotransplant at a median of 165 days. Of these, 11 patients showed abnormal cytogenetics at the time of post-allotransplant relapse at a median of 167 days. Of 54 patients who developed graft-versus-host disease, relapse occurred in 19 of 48 patients (43%) with CA present before RCI allotransplant, versus 1 of 6 without CA (17%) (P = 0.06). Loss of CA before RIC allotransplant and disease status > PR after RIC allotransplant were significantly associated with a lower risk of post-allotransplant relapse with cytogenetic abnormalities; 5.2 vs 36%, and 18 vs 53%, (both P < 0.05), respectively. The current data suggests that myeloma associated with persistent clonal cytogenetic abnormalities is an entity which most likely escapes the effects of a graft versus myeloma activity, maybe because of acquisition of resistance to immunologic manipulations.

Dialysis-dependent renal failure in patients with myeloma can be reversed by high-dose myeloablative therapy and autotransplant.

To evaluate the role of high-dose melphalan and autologous transplant (AT) in reversing dialysis-dependent renal failure, 59 patients still on dialysis at the time of AT were analyzed. A total of 37 patients had been on dialysis < or =6 months. A 5-year event-free and overall survival rate of all patients after AT was 24 and 36%, respectively. Of 54 patients evaluable for renal function improvement, 13 (24%) became dialysis independent at a median of 4 months after AT (range: 1-16). Dialysis duration < or =6 months prior to first AT and pre-transplant creatinine clearance >10 ml/min were significant for renal function recovery: 12 of 36 (33%) < or =6 months vs one of 18 patients (6%) >6 months on dialysis recovered renal function; 10 of 26 (38%) with >10 ml/min vs three of 28 (11%) with < or =10 ml/min of creatinine clearance (both P<0.05). Quality of response after autotransplant was also significant: 12 of 31 (39%) being greater than partial remission after AT vs one of 21 patients (5%) attaining partial remission or less became independent of dialysis (P<0.05). Our data suggest that significant renal failure can be reversible and AT should be considered early in the disease course.

Use of quantitative ultrasound of the hand phalanges in the diagnosis of two different osteoporotic syndromes: Cushing's syndrome and postmenopausal osteoporosis.

The aim of this study was to assess the ability of the quantitative ultrasound of the hand phalanges to detect different types of osteoporosis resulting from different pathogenetic mechanisms. For this purpose, postmenopausal and glucocorticoid-induced osteoporosis was studied. Thirteen female patients with Cushing's syndrome (CS) resulting from pituitary-dependent bilateral adrenal hyperplasia (10 patients) and from adrenal adenoma (3 patients), and 32 postmenopausal osteoporotic (OP) women, were examined. The two groups of patients were comparable for body mass index (BMI), but CS patients were significantly younger than OP ones (CS 44.5+/-11.6; OP: 73.9+/-3.6). All the patients had femoral neck bone mineral density (BMD) T-score less than -2.0. Cushing patients had a femoral neck BMD similar to that of OP patients (CS: 603+/-66 mg/cm2; OP: 628+/-69 mg/cm2; p=0.19). In contrast, amplitude-dependent speed of sound (AD-SoS) was significantly higher in CS patients than in OP patients (CS: 1997+/-91 m/s; OP: 1707+/-114 m/s; p<0.0001). By adjusting DXA and ultrasound parameters according to age, femoral neck BMD was significantly lower in CS patients and AD-SoS remained significantly higher than in OP patients. These findings indicate that these two different kinds of osteoporosis can be distinguished by ultrasonography and that ultrasound parameters alone cannot be used for evaluating skeletal status in CS patients.

Transplantation as salvage therapy for high-risk patients with myeloma in relapse.

Patients with myeloma relapsing after tandem transplant have a poor survival and treatment options are limited. The role of additional salvage transplant procedures for these patients is unknown. To evaluate the benefit and identify prognostic factors, the outcome of 76 consecutive patients with recurrent myeloma after tandem transplant receiving salvage transplants (ST) was analyzed. Prior to ST, 23 patients (30%) had shown chemosensitive response to preceding salvage chemotherapy: two complete remissions (CR); eight near CRs (nCR: only immunofixation positive); 13 partial remissions (PR >or=75% reduction in M protein). Fifty received an autologous transplant, 22 a sibling-matched allogeneic transplant, and four a matched-unrelated allogeneic transplant. Overall response after ST was 59%: eight CRs (11%); 14 nCRs (18%); 23 PRs (30%). Overall survival (OS) at 2 years was 19%; 2 year event-free survival rate (EFS) 7%. On univariate analysis for survival, only pre-transplant chemosensitive relapse (P < 0.05), serum albumin >3 g/dl (P = 0.001), normal LDH (P = 0.04), and long interval between the second transplant and relapse/progression were significant beneficial factors. In a Cox proportional hazard model, chemosensitive relapse, and albumin >3 g/dl were significant for better OS: hazard ratio (HR) 1.4, 1.7, respectively, while normal LDH, and absence of CA13 were significant for better EFS: HR 1.8, 1.7, respectively. Patients with albumin >3 g/dl who had chemosensitive disease before ST (n = 16) had a median survival of 16 months, compared to 7 months (n = 34) and 2 months (n = 26) for patients with only one (n = 34) or no favorable prognostic factors (n = 28), respectively (P < 0.001). Their survival at 2 years post-ST was 43%, 17% and 11%, respectively. Our study suggests further transplantation should only be considered in the setting of a clinical trial in patients with favorable prognostic factors.

Clinical activity of arsenic trioxide for the treatment of multiple myeloma.

Arsenic has been used since ancient times as a therapeutic agent. However, until recently its use in modern medicine has been restricted to the treatment of a limited number of parasitic infections. In the early 1990s, reports from China described impressive results with arsenic trioxide in patients with de novo, relapsed, and refractory acute promyelocytic leukemia (APL). Other investigators subsequently confirmed these results leading to approval of its use for relapsed or refractory APL in the United States. Investigations of this agent have demonstrated that its efficacy in APL and preclinical tumor models is dependent upon a number of mechanisms, including induction of apoptosis, effects on cellular differentiation, cell cycling, and tumor angiogenesis. Subsequent preclinical studies showed significant activity of arsenic trioxide in multiple myeloma (MM). Based on this, in a phase II trial, we have evaluated the activity of arsenic trioxide in 14 patients with relapsed MM, refractory to conventional salvage therapy. With the dose and schedule used, treatment with arsenic trioxide produced responses in three patients and prolonged stable disease in a fourth patient, with the longest response lasting 6 weeks. Although treatment was reasonably well tolerated, in these patients with extensive prior therapy, 11 developed cytopenia, five associated with infectious complications and three developed deep vein thromboses. The results of this small trial support further investigation of this novel drug for the treatment of patients with relapsed or refractory MM.

Activated protein C resistance in the absence of factor V Leiden mutation is a common finding in multiple myeloma and is associated with an increased risk of thrombotic complications.

Thromboembolism is not uncommon in multiple myeloma (MM) patients on treatment, but its pathogenesis remains poorly understood. We report the results of a prospective randomized trial of 62 newly diagnosed MM patients tested at baseline for hypercoagulability and treated with intensive chemotherapy with or without thalidomide in a randomized fashion. During the induction phase, 12 patients (19%) developed evidence of deep venous thrombosis (DVT), which was significantly more common in the thalidomide arm (36%) than in the control group (3%) (P = 0.001). Fourteen patients (23%) were found to have a baseline-reduced response to activated protein C (APC) in the absence of factor V Leiden mutation. Using a Kaplan-Meier analysis, a significantly higher proportion of patients with APC resistance developed DVT (5/14 versus 7/38; P = 0.04) irrespective of thalidomide administration. The risk of DVT was highest (50%) in patients with APC resistance on thalidomide. None of the patients with normal APC response and not receiving thalidomide developed DVT. In conclusion, in this series, acquired APC resistance was present in almost one-quarter of newly diagnosed myeloma patients and significantly increased the risk of DVT.