Gregory J. Ahmann

Initial genome sequencing and analysis of multiple myeloma

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.

Prognostic value of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma

A specific role for increased level of expression of CKS1B, as a consequence of chromosome 1q21 copy number gain, has been postulated as both pathogenic, as well as a powerful clinical prognostic factor in multiple myeloma (MM). The purpose of this study is to determine the clinical associations and prognostic impact of copy number gain at chromosome 1q21 (with a bacteria artificial chromosome clone containing CKS1B) and CKS1B gene level of expression in MM. We studied the chromosome region 1q21 for copy number change in a cohort of myeloma patients treated by high-dose therapy with stem-cell rescue (HDT) (n=159). A separate cohort of patients, treated by HDT was studied for CKS1B messenger RNA expression by gene expression profiling (n=67). 1q21 gain was then correlated with clinical parameters and survival. Gain of 1q21 copy number was detected in about a third of MM and was associated with more proliferative disease and poor-risk cytogenetic categories such as t(4;14), and chromosome 13 deletion. Both 1q21 gain and increase gene expression level were significantly associated with reduced survival. However, neither is an independent prognostic marker in MM on multivariate Cox proportional hazard analysis.

A novel three-color, clone-specific fluorescence in situ hybridization procedure for monoclonal gammopathies

We have developed a three-color cytoplasmic immunoglobulin (cIg) and fluorescence in situ hybridization (FISH) technique to detect plasma cell chromosomal aneuploidy in patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and amyloidosis (AL). Immunofluorescent-labeled antibodies to detect light chain expression and six directly labeled alpha-satellite chromosome specific enumeration probes (CEP) were used simultaneously to detect aneuploidy of the plasma cells. The six probes were specific for chromosomes 7, 9, 11, 15, 18, and X. The technique was tested in 12 consecutive patient samples (5 MM, 2 MGUS, 3 SMM, and 2 AL). Based on the alpha-satellite signals, we found trisomic clones for CEP 7 (4 of 12), CEP 11 (4 of 12), CEP X (1 of 12), CEP 9 (8 of 12), CEP 15 (7 of 12), and CEP 18 (5 of 12). Trisomic clones of at least one of the six chromosomes were present in 9 of 12 patients. We believe that this technique efficiently identifies monotypic plasma cells and permits simultaneous analysis of numeric chromosome anomalies by FISH in emerging neoplastic cells. We are in the process of applying this technique to a series of about 100 newly diagnosed monoclonal gammopathy patients.

Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations

Chromosomal hyperdiploidy is the defining genetic signature in 40–50% of myeloma (MM) patients. We characterize hyperdiploid-MM (H-MM) in terms of its clinical and prognostic features in a cohort of 220 H-MM patients entered into clinical trials. Hyperdiploid-myeloma is associated with male sex, kappa immunoglobulin subtype, symptomatic bone disease and better survival compared to nonhyperdiploid-MM (median overall survival 48 vs 35 months, log-rank P=0.023), despite similar response to treatment. Among 108 H-MM cases with FISH studies for common genetic abnormalities, survival is negatively affected by the existence of immunoglobulin heavy chain (IgH) translocations, especially those involving unknown partners, while the presence of chromosome 13 deletion by FISH did not significantly affect survival (median overall survival 50 vs 47 months, log-rank P=0.47). Hyperdiploid-myeloma is therefore a unique genetic subtype of MM associated with improved outcome with distinct clinical features. The existence of IgH translocations but not chromosome 13 deletion by FISH negatively impacts survival and may allow further risk stratification of this population of MM patients.

Plasma interleukin-6 and C-reactive protein levels in reactive versus clonal thrombocytosis

PURPOSE Evaluate the discriminatory value of plasma interleukin-6 or C-reactive protein levels in clonal thrombocytosis compared with those in reactive thrombocytosis. PATIENTS AND METHODS A comparative analysis of quantitatively measured laboratory values in a prospectively studied group of consecutive patients. The setting was a tertiary referral center consisting of two hospitals and an outpatient clinic. Plasma interleukin-6 and C-reactive protein levels were measured in 91 consecutive patients with thrombocytosis (platelet count > or = 600 x 10(9)/L). The cause of thrombocytosis was determined by reviewing the medical histories and follow-up data without knowledge of the corresponding laboratory values. Sixty-four patients had reactive thrombocytosis, 20 had clonal thrombocytosis, and 7 had clonal thrombocytosis plus reactive thrombocytosis. Plasma interleukin-6 was measured by an enzyme-linked immunosorbent assay, and C-reactive protein was measured with rate immunonephelometry. RESULTS Interleukin-6 levels were undetectable in all the patients with clonal thrombocytosis, whereas they were increased in 60% of the patients with reactive thrombocytosis or clonal thrombocytosis plus reactive thrombocytosis. There was a correlation between interleukin-6 and C-reactive protein levels (r = .6), and the median and range values of both levels differed significantly between the clonal thrombocytosis group and the other two groups (P < 0.0001). In 81% of the patients with reactive thrombocytosis, levels of either interleukin-6 or C-reactive protein were elevated. There was no correlation between interleukin-6 and C-reactive protein levels and the platelet count. CONCLUSIONS An elevated interleukin-6 level is rare in uncomplicated clonal thrombocytosis and suggests reactive thrombocytosis. However, an isolated normal value has little discriminatory value. Measurement of C-reactive protein level may be used as a less expensive surrogate for measurement of interleukin-6. Repeatedly low levels of both interleukin-6 and C-reactive protein are most consistent with clonal thrombocytosis.

Detection of myeloma cells in the peripheral blood by flow cytometry.

Bone marrow plasma cells (PC) from patients with multiple myeloma (MM) express monoclonal cytoplasmic immunoglobulin (clg) light chain, strongly express CD38, and usually lack or dimly express CD45. The detection of malignant plasma cells in the peripheral blood (PB) by immunofluorescence microscopy (IM) distinguishes patients with active MM from those with stable disease. The aim of this study was to learn whether two-color (CD38 and CD45) flow cytometry (FC) on whole blood specimens (WBFC) and three-color FC (CD38, CD45, and anti-kappa or lambda clg) on mononuclear cells could identify circulating PC as well as the standard, more labor intensive IM technique. Split-samples of PB from 73 patients with plasma cell proliferative disorders were examined by both techniques. WBFC detected CD38+ CD45- cells in 94% (33/35) of patients with circulating monoclonal PC detected by IM and three-color FC detected monoclonal CD38+ CD45- cells in 77% (27/35) of these cases. The absolute number of monoclonal PC detected by IM was compared to the FC methods and the Spearman rank correlations were 0.77 with WBFC and 0.80 with three-color FC. This study indicates that WBFC, using antibodies to CD38 and CD45, offers a practical and reliable method to detect and quantify circulating malignant PC in patients with MM.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Deletions of 17p13.1 and 13q14 are uncommon in Waldenström macroglobulinemia clonal cells and mostly seen at the time of disease progression

Waldenström macroglobulinemia (WM) is a plasma cell dyscrasia characterized by a monoclonal IgM paraproteinemia. Deletions of 17p13.1 and 13q14 are associated with tumor progression and worsened outcome in multiple myeloma (MM), and we thus investigated WM patients for their presence. Patients (n = 40) were required to have a > or = 1.5 g/dl serum IgM paraproteinemia and a monoclonal lymphoplasmacytic infiltrate. We used interphase fluorescence in situ hybridization (FISH) with probes that localized to 17p13.1(LSI p53/CEP 17) and 13q14 (D13S319 and LSI 13 Rb). Of 40 successfully studied patients for 17p13.1(p53) deletions, 6 were abnormal, consistent with hemizygous deletion (15%). Of 37 cases successfully studied for the 13q14 deletions, 6 were also abnormal with one pair of signals deleted (16%). Patients with deletions were more likely to be later in the course of the disease. No obvious clinical associations were noted with the exception that patients with 17p13.1(p53) deletions had a higher percent involvement of clonal cells in the bone marrow. Deletions of these two regions are uncommon in WM, being more common in the late stages of the disease, thus unlikely playing a role in primary disease pathogenesis.

Biologic and genetic characterization of the novel amyloidogenic lambda light chain–secreting human cell lines, ALMC-1 and ALMC-2

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge, no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach, we established the genetic relationship between the cell lines and the primary patient cells, and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patients disease. Importantly, we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity, the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover, these cell lines will provide an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology.

Chromosomal abnormalities in systemic amyloidosis

Primary systemic amyloidosis (AL) is a plasma cell disorder characterized by deposition of monoclonal light chains in different organ systems. Although multiple and complex numerical chromosomal abnormalities have been described in patients with multiple myeloma, it is currently unknown whether such changes occur in systemic amyloidosis.