Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma.

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

A phase 2 study of lenalidomide, rituximab, cyclophosphamide, and dexamethasone (LR-CD) for untreated low-grade non-Hodgkin lymphoma requiring therapy.

Patients with indolent non-Hodgkin lymphoma (NHL) have multiple treatment options yet there is no consensus as to the best initial therapy. Lenalidomide, an immunomodulatory agent, has single agent activity in relapsed lymphoma. This trial was conducted to assess feasibility, efficacy, and safety of adding lenalidomide to rituximab, cyclophosphamide, and dexamethasone (LR-CD) in untreated indolent NHL patients requiring therapy. This was a single institution phase II trial. Treatment consisted of IV rituximab 375 mg/m2 day 1; oral lenalidomide 20 mg days 1-21; cyclophosphamide 250 mg/m2 days 1, 8, and 15; and dexamethasone 40 mg days 1, 8, 15, and 22 of a 28-day cycle. Treatment continued 2 cycles beyond best response for a maximum of 12 cycles without rituximab maintenance. Thirty-three patients were treated. Median age was 68 (43-83 years). 39% had stage IV disease. Histologic subtypes included 8 follicular lymphoma (FL), 7 marginal zone lymphoma (MZL) (1 splenic, 2 extranodal, and 4 nodal), 15 Waldenström's macroglobulinemia (WM), 1 lymphoplasmacytic lymphoma, 1 small lymphocytic lymphoma, and 1 low-grade B-cell lymphoma with plasmacytic differentiation (unable to be classified better as MZL or LPL). Hematologic toxicity was the most common adverse event. Median time of follow-up was 23.4 months (range 1.8-50.9). The overall response rate was 87.9%, with 30.3% complete response. The median duration of response was 38.7 months. The median progression free survival was 39.7 months, while median overall survival (OS) has not yet been reached. Lenalidomide can be safely added to a simple regimen of rituximab, oral cyclophosphamide, and dexamethasone and is an effective combination as initial therapy for low-grade B-cell NHL.

RNA interference screening identifies lenalidomide sensitizers in multiple myeloma, including RSK2.

To identify molecular targets that modify sensitivity to lenalidomide, we measured proliferation in multiple myeloma (MM) cells transfected with 27 968 small interfering RNAs in the presence of increasing concentrations of drug and identified 63 genes that enhance activity of lenalidomide upon silencing. Ribosomal protein S6 kinase (RPS6KA3 or RSK2) was the most potent sensitizer. Other notable gene targets included 5 RAB family members, 3 potassium channel proteins, and 2 peroxisome family members. Single genes of interest included I-κ-B kinase-α (CHUK), and a phosphorylation dependent transcription factor (CREB1), which associate with RSK2 to regulate several signaling pathways. RSK2 knockdown induced cytotoxicity across a panel of MM cell lines and consistently increased sensitivity to lenalidomide. Accordingly, 3 small molecular inhibitors of RSK2 demonstrated synergy with lenalidomide cytotoxicity in MM cells even in the presence of stromal contact. Both RSK2 knockdown and small molecule inhibition downregulate interferon regulatory factor 4 and MYC, and provides an explanation for the synergy between lenalidomide and RSK2 inhibition. Interestingly, RSK2 inhibition also sensitized MM cells to bortezomib, melphalan, and dexamethasone, but did not downregulate Ikaros or influence lenalidomide-mediated downregulation of tumor necrosis factor-α or increase lenalidomide-induced IL-2 upregulation. In summary, inhibition of RSK2 may prove a broadly useful adjunct to MM therapy.

A comparison of lenalidomide/dexamethasone versus cyclophosphamide/lenalidomide/dexamethasone versus cyclophosphamide/bortezomib/dexamethasone in newly diagnosed multiple myeloma.

Novel agents are considered standard components of induction therapy for newly diagnosed patients with multiple myeloma. We retrospectively compared the results of three consecutive phase 2 clinical trials; RD (lenalidomide/dexamethasone, n=34), CRD (cyclophosphamide/lenalidomide/dexamethasone, n=53) and CyBorD (cyclophosphamide/bortezomib/dexamethasone, n=63) (N=150). Response rates after four cycles of treatment were: ≥near complete response (nCR), 12% vs. 2% vs. 41%, P<0·0001 and very good partial response or better, 35% vs. 30% vs. 65%, P=0·0003, respectively. With all cycles of therapy considered, ≥nCR was 35%, 15% and 41%, P=0·006. However, there is no evidence that one regimen produced superior progression-free survival (PFS) (median: 3·2 vs. 2·3 vs. 2·7years, P=0·11) or overall survival (3-year: 88% vs. 79% vs. 88%, P=0·23). Transplantation did not impact PFS (median: 2·7 vs. 2·3 years, P=0·41) but was associated with improved OS (3-year: 93% vs. 75%, P≤0·001). High genetic risk patients (n=40) had earlier relapse despite lenalidomide or bortezomib (median: 2·1 vs. 2·7years, P=0·45). Grade 3/4 toxicities were least with CyBorD while CRD had most toxicity. In conclusion, CyBorD demonstrated superior responses and less frequent serious toxicity but more neuropathy when compared to RD and CRD. Importantly, 80% of patients treated with modern therapeutic approaches are alive at 4years.

Alkaline phosphatase variation during carfilzomib treatment is associated with best response in multiple myeloma patients.

The ubiquitin-proteasome pathway regulates bone formation through osteoblast differentiation. We analyzed variation alkaline phosphatase (ALP) during carfilzomib treatment. Data from 38 patients enrolled in the PX-171-003 and 29 patients in PX-171-004 studies, for patients with relapsed/refractory myeloma, were analyzed. All patients received 20 mg/m(2) of carfilzomib on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Sixty-seven patients from ALP data were evaluable. In PX-171-003, the ORR (>PR) was 18% and the clinical benefit response (CBR; >MR) was 26%, while in PX-171-004, the ORR was 35.5% overall and 57% in bortezomib-naive patients. ALP increment from baseline was statistically different in patients who achieved ≥ VGPR compared with all others on Days 1 (P = 0.0049) and 8 (P = 0.006) of Cycle 2. In patients achieving a VGPR or better, ALP increased more than 15 units per liter at Cycle 2 Day 1 over baseline. An ALP increase over the same period of time was seen in 26%, 13% and 11% of patients achieving PR, MR, and SD, respectively. This retrospective analysis of patients with relapsed or refractory myeloma treated with single-agent carfilzomib indicates that early elevation in ALP is associated with subsequent myeloma response.

Pharmacokinetic and pharmacodynamic study of two doses of bortezomib in patients with relapsed multiple myeloma.

PURPOSE: Characterize bortezomib pharmacokinetics/pharmacodynamics in relapsed myeloma patients after single and repeat intravenous administration at two doses. METHODS: Forty-two patients were randomized to receive bortezomib 1.0 or 1.3 mg/m(2), days 1, 4, 8, 11, for up to eight 21-day treatment cycles (n = 21, each dose group). Serial blood samples for pharmacokinetic/pharmacodynamic analysis were taken on days 1 and 11, cycles 1 and 3. Observational efficacy and safety data were collected. RESULTS: Twelve patients in each dose group were evaluable for pharmacokinetics/pharmacodynamics. Plasma clearance decreased with repeat dosing (102-112 L/h for first dose; 15-32 L/h following repeat dosing), with associated increases in systemic exposure and terminal half-life. Systemic exposures of bortezomib were similar between dose groups considering the relatively narrow dose range and the observed pharmacokinetic variability, although there was no readily apparent deviation from dose-proportionality. Blood 20S proteasome inhibition profiles were similar between groups with mean maximum inhibition ranging from 70 to 84% and decreasing toward baseline over the dosing interval. Response rate (all 42 patients) was 50%, including 7% complete responses. The safety profile was consistent with the predictable and manageable profile previously established; data suggested milder toxicity in the 1.0 mg/m(2) group. CONCLUSIONS: Bortezomib pharmacokinetics change with repeat dose administration, characterized by a reduction in plasma clearance and associated increase in systemic exposure. Bortezomib is pharmacodynamically active and tolerable at 1.0 and 1.3 mg/m(2) doses, with recovery toward baseline blood proteasome activity over the dosing interval following repeat dose administration, supporting the current clinical dosing regimen.

Lenalidomide plus dexamethasone vs. lenalidomide plus melphalan and prednisone: a retrospective study in newly diagnosed elderly myeloma.

BACKGROUND: The goal of this retrospective study was to compare the efficacy and toxicity of lenalidomide-dexamethasone (len/dex) vs. melphalan-prednisone-lenalidomide (MPR) as upfront therapy for newly diagnosed elderly patients with myeloma. METHODS: Data from 51 patients enrolled in an Italian phase I/II trial and treated with MPR were analyzed and compared with data from 38 patients, seen at the Mayo Clinic, treated with len/dex and enrolled in phase II/III trials. RESULTS: On intention-to-treat analysis, time to progression (median: 24.7 vs. 27.5 months in MPR and len/dex groups, respectively, P = 0.903), progression-free survival (median: 24.7 vs. 27.5 months in MPR and len/dex groups, respectively, P = 0.926), and overall survival (2-yr overall survival: 86.2% in MPR vs. 89.1% in len/dex, P = 0.730) were not significantly different between the two groups. Results were confirmed when the analysis was restricted to MPR and len/dex matched pair mates. Hematologic grade 3-4 toxicities were more common with MPR (neutropenia: 66.7% vs. 21.1%, P < 0.001; thrombocytopenia: 31.4% vs. 2.6%, P < 0.001). Grade 3-4 gastrointestinal events (13.2% vs. 3.9%, P = 0.132), thrombotic events (13.2% vs. 5.9%, P = 0.279), and fatigue (10.5% vs. 3.9%, P = 0.395) were more common with len/dex. CONCLUSIONS: Results show that both MPR and len/dex are efficacious regimens for elderly patients with myeloma. Randomized trials are needed to confirm these results.

IgD multiple myeloma--a clinical profile and outcome with chemotherapy and autologous stem cell transplantation.

IgD myeloma accounts for 2% of myeloma subtypes and has higher incidence of complications and a poorer outcome. The characteristics and outcomes of 25 patients with IgD myeloma (2.6% of all myeloma patients), including 11 patients treated with autologous stem cell transplantation (ASCT) are reported. The presenting features were not unique for myeloma, but it appears that the commonest presenting symptom was bone pain in 20 (80%) patients. Other associated disorders were B-CLL in -1 patient and hairy cell leukemia in another. Three patients had a marked increase in bone marrow reticulin. Three (out of a total of 9) patients had 13q deletion (1 by cytogenetics and 2 by interphase fluorescence in situ hybridisation [FISH]), while 2 patients had a complex karyotype. A total of 11 patients were treated with ASCT, while 14 patients had chemotherapy alone. The response to treatment was: in the ASCT group 2 had a complete response (CR) and 9 had a good partial response (PR), while in the chemotherapy group 6 had a good PR with no CRs. The median overall survival (OS) and progression-free survival (PFS) after autologous peripheral blood stem cell transplantation (APBSCT) has not yet been reached after a median follow up of 4 years. The median PFS after chemotherapy was 1.23 years (95% CI 1.02-1.44). The mean OS transplantation was 5.09 years compared with 2.03 years for those treated with chemotherapy alone. However, this difference was not statistically significant (log rank p=0.09). This small series appears to suggest that outcome for IgD myeloma remains poor with chemotherapy and appears to be superior with ASCT, and larger studies are needed to confirm these findings.

Multiple myeloma involving central nervous system: high frequency of chromosome 17p13.1 (p53) deletions.

Central nervous system (CNS) involvement is an unusual manifestation in multiple myeloma (MM). The molecular basis of CNS myeloma is poorly understood. MM is characterized by translocations involving the immunoglobulin heavy chain (IgH) locus and frequent 13q deletions. Alterations of p53 or c-myc in MM may represent secondary changes associated with disease progression. We investigated nine patients with CNS MM using interphase fluorescence in situ hybridization (FISH) combined with immunofluorescence detection of the cytoplasmic light chain (cIg-FISH) for the presence of above genomic aberrations. Of nine patients studied, eight cases had hemizygous p53 deletion and 4 had 13q deletions. Of the patients with 13q deletions, two had IgH translocations, one involving 4p16.3 (FGFR3), the other involving 16q23 (c-maf). The high incidence of p53 deletions detected by cIg-FISH in CNS myeloma may be a marker for chromosomal instability, and may be associated with metastatic features of myeloma cells.

The t(4;14) is associated with poor prognosis in myeloma patients undergoing autologous stem cell transplant.

The frequency and prognostic relevance of translocations t(11;14) and t(4;14), the most common translocations involving the immunoglobulin heavy chain (IgH) gene in multiple myeloma (MM), were investigated in 128 patients treated with intensive chemotherapy and autologous stem cell transplant. Myeloma cells were identified by cytoplasmic light chain immunofluorescence combined with fluorescence in situ hybridization (cIg-FISH) for detection of translocations t(11;14) and t(4;14). Overall, t(11;14) was detected in 16 of 125 (12.8%) and t(4;14) in 15 of 120 (12.5%) patients. Progression-free and overall survivals were similar for patients with or without t(11;14). However, patients with t(4;14) had significantly shorter progression-free (median 9.9 months vs. 25.8 months; P = 0.0003) and overall survivals (median 18.3 months vs. 48.1 months; P < 0.0001) than patients without t(4;14). The t(4;14) was associated with IgA and t(11;14) with light chain MM. There was no association between the t(11;14) or t(4;14) and other biological parameters including age, gender, haemoglobin, beta-2 microglobulin, C-reactive protein, calcium, creatinine, albumin, or the percentage of bone marrow plasma cells. Multivariate analysis identified t(4;14) as the only adverse prognostic factor for both progression-free survival and overall survival. Our results indicate that the t(4;14) detected by cIg-FISH is associated with a poor prognosis in MM patients receiving intensive chemotherapy and autotransplant.