Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms.

The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.

Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma.

We have used whole exome sequencing to compare a group of presentation t(4;14) with t(11;14) cases of myeloma to define the mutational landscape. Each case was characterized by a median of 24.5 exonic nonsynonymous single-nucleotide variations, and there was a consistently higher number of mutations in the t(4;14) group, but this number did not reach statistical significance. We show that the transition and transversion rates in the 2 subgroups are similar, suggesting that there was no specific mechanism leading to mutation differentiating the 2 groups. Only 3% of mutations were seen in both groups, and recurrently mutated genes include NRAS, KRAS, BRAF, and DIS3 as well as DNAH5, a member of the axonemal dynein family. The pattern of mutation in each group was distinct, with the t(4;14) group being characterized by deregulation of chromatin organization, actin filament, and microfilament movement. Recurrent RAS pathway mutations identified subclonal heterogeneity at a mutational level in both groups, with mutations being present as either dominant or minor subclones. The presence of subclonal diversity was confirmed at a single-cell level using other tumor-acquired mutations. These results are consistent with a distinct molecular pathogenesis underlying each subgroup and have important impacts on targeted treatment strategies. The Medical Research Council Myeloma IX trial is registered under ISRCTN68454111.

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

XBP1s levels are implicated in the biology and outcome of myeloma mediating different clinical outcomes to thalidomide-based treatments.

Immunoglobulin production by myeloma plasma cells depends on the unfolded protein response for protein production and folding. Recent studies have highlighted the importance of IRE1alpha and X box binding protein 1 (XBP1), key members of this pathway, in normal B-plasma cell development. We have determined the gene expression levels of IRE1alpha, XBP1, XBP1UNSPLICED (XBP1u), and XBP1SPLICED (XBP1s) in a series of patients with myeloma and correlated findings with clinical outcome. We show that IRE1alpha and XBP1 are highly expressed and that patients with low XBP1s/u ratios have a significantly better overall survival. XBP1s is an independent prognostic marker and can be used with beta2 microglobulin and t(4;14) to identify a group of patients with a poor outcome. Furthermore, we show the beneficial therapeutic effects of thalidomide in patients with low XBP1s/u ratios. This study highlights the importance of XBP1 in myeloma and its significance as an independent prognostic marker and as a predictor of thalidomide response.

Homozygous deletion mapping in myeloma samples identifies genes and an expression signature relevant to pathogenesis and outcome.

PURPOSE: Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes. EXPERIMENTAL DESIGN: Using single nucleotide polymorphism-based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome. RESULTS: We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the "cell death" network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets. CONCLUSIONS: We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma.

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

BackgroundThe recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear.Design and MethodsThe expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays.ResultsWe found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples.ConclusionsIn conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.

Streptolysin-O reversible permeabilisation is an effective method to transfect siRNAs into myeloma cells

RNA interference (RNAi) has been shown to be a valuable tool to specifically target gene expression in a number of organisms becoming an indispensable weapon in the arsenal in functional genomics. In this study, we demonstrate that streptolysin-O (SLO) reversible permeabilisation is an efficient method to deliver small interfering RNAs (siRNAs) to hard-to-transfect human myeloma cell lines. We used published, pre-validated siRNAs for ERK2 and non-silencing siRNA control. We transfected siRNAs into human myeloma cell lines using SLO reversible permeabilisation method. Flow cytometry and western blot analysis were performed to assess the effect of SLO on transfection efficiency and ERK2 knockdown. These experiments demonstrate that SLO reversible permeabilisation method is an efficient and easy-to-use method to deliver siRNAs into human myeloma cell lines. Optimised SLO permeabilisation method showed to transfect >80% of JIM-3, H929, RPM18226 and U266 cells, with minimal effect on cell viability (<10%) and cell cycle. Equally important, SLO permeabilisation induced a substantial knockdown of ERK2 at the protein level. These studies demonstrate that reversible SLO permeabilisation can successfully be applied to hard-to-transfect human myeloma cell lines to effectively silence genes. (C) 2008 Published by Elsevier B.V.

A global expression-based analysis of the consequences of the t(4;14) translocation in myeloma

Purpose: Our purpose in this report was to define genes and pathways dysregulated as a consequence of the t(4;14) in myeloma, and to gain insight into the downstream functional effects that may explain the different prognosis of this subgroup.Experimental Design: Fibroblast growth factor receptor 3 (FGFR3) overexpression, the presence of immunoglobulin heavy chain-multiple myeloma SET domain (IgH-MMSET) fusion products and the identification of t(4;14) breakpoints were determined in a series of myeloma cases. Differentially expressed genes were identified between cases with (n = 55) and without (n = 24) a t(4;14) by using global gene expression analysis.Results: Cases with a t(4;14) have a distinct expression pattern compared with other cases of myeloma. A total of 127 genes were identified as being differentially expressed including MMSET and cyclin D2, which have been previously reported as being associated with this translocation. Other important functional classes of genes include cell signaling, apoptosis and related genes, oncogenes, chromatin structure, and DNA repair genes. Interestingly, 25% of myeloma cases lacking evidence of this translocation had up-regulation of the MMSET transcript to the same level as cases with a translocation.Conclusions: t(4;14) cases form a distinct subgroup of myeloma cases with a unique gene signature that may account for their poor prognosis. A number of non-t(4;14) cases also express MMSET consistent with this gene playing a role in myeloma pathogenesis.

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis.

To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-kappaB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM.

Unwrapping microcomputed tomographic images for measuring cortical osteolytic lesions in the 5T2 murine model of myeloma treated by bisphosphonate

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Targeting histone deacetylases for the treatment of immune, endocrine and metabolic disorders

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as histone acetyltransferases or HATs, and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The proinflammatory environment is increasingly being recognised as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential & current development of histone deacetylases for the treatment of diseases for which a proinflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the proinflammatory environment. © 2009 Bentham Science Publishers Ltd.

Toxicity profile of the immunomodulatory thalidomide analogue, lenalidomide : Phase I clinical trial of three dosing schedules in patients with solid malignancies

Thalidomide is an anti-angiogenic agent currently used to treat patients with malignant cachexia or multiple myeloma. Lenalidomide (CC-5013) is an immunomodulatory thalidomide analogue licensed in the United States of America (USA) for the treatment of a subtype of myelodysplastic syndrome. This two-centre, open-label phase I study evaluated dose-limiting toxicities in 55 patients with malignant solid tumours refractory to standard chemotherapies. Lenalidomide capsules were consumed once daily for 12 weeks according to one of the following three schedules: (I) 25 mg daily for the first 7 d, the daily dose increased by 25 mg each week up to a maximum daily dose of 150 mg; (II) 25 mg daily for 21 d followed by a 7-d rest period, the 4-week cycle repeated for 3 cycles; (III) 10 mg daily continuously. Twenty-six patients completed the study period. Two patients experienced a grade 3 hypersensitivity rash. Four patients in cohort I and 4 patients in cohort II suffered grade 3 or 4 neutropaenia. In 2 patients with predisposing medical factors, grade 3 cardiac dysrhythmia was recorded. Grade 1 neurotoxicity was detected in 6 patients. One complete and two partial radiological responses were measured by computed tomography scanning; 8 patients had stable disease after 12 weeks of treatment. Fifteen patients remained on treatment as named patients; 1 with metastatic melanoma remains in clinical remission 3.5 years from trial entry. This study indicates the tolerability and potential clinical efficacy of lenalidomide in patients with advanced solid tumours who have previously received multi-modality treatment. Depending on the extent of myelosuppressive pre-treatment, dose schedules (II) or (III) are advocated for large-scale trials of long-term administration. © 2006 Elsevier Ltd. All rights reserved.

Antibodies against human herpesvirus 8 in black South African patients with cancer

Background Infection with human herpesvirus 8 (HHV-8) has been consistently linked to Kaposi's sarcoma, but its mode of transmission, association with other cancers, and interaction with the human immunodeficiency virus type 1 (HIV-1) are largely unknown. Methods Between January 1992 and December 1997, we interviewed 3591 black patients with cancer in Johannesburg and Soweto, South Africa. Blood was tested for antibodies against HIV-1 and HHV-8 in 3344 of the patients. Antibodies against HHV-8 were detected with an indirect immunofluorescence assay. The intensity of the fluorescent signal correlated well with the titers of antibodies (P<0.001). The relations among the presence of anti–HHV-8 antibodies, sociodemographic and behavioral factors, type of cancer, and the presence or absence of coexistent HIV-1 infection were examined with the use of unconditional logistic-regression models. Results Among the 3293 subjects with cancers other than Kaposi's sarcoma, the standardized seroprevalence of antibodies against HHV-8 was 32 percent, which did not differ significantly from the standardized seroprevalence among black blood donors. Among these 3293 patients, the prevalence of antibodies against HHV-8 increased with increasing age (P<0.001) and an increasing number of sexual partners (P=0.05) and decreased with increasing years of education (P=0.007); it was not strongly associated with HIV-1 infection. Anti–HHV-8 antibodies were more frequent among black than white blood donors (P<0.001). Among the 51 patients with Kaposi's sarcoma, the standardized seroprevalence of antibodies against HHV-8 was 83 percent, significantly higher than the prevalence among those without Kaposi's sarcoma (P<0.001). For 16 other specific types of cancer, including multiple myeloma (108 cases) and prostate cancer (202 cases), the variation in the standardized seroprevalence of antibodies against HHV-8 was not remarkable. At a given intensity of fluorescence of anti–HHV-8 antibodies, Kaposi's sarcoma was more frequent among HIV-1–positive patients than among those who were HIV-1–negative (P<0.001). Conclusions Among black patients with cancer in South Africa, the seroprevalence of anti–HHV-8 antibodies is high and is specifically associated with Kaposi's sarcoma, particularly at high titers.

The spectrum of HIV-1 related cancers in South Africa

Despite the high prevalence of infection by the Human Immunodeficiency Virus (HIV) in South Africa, information on its association with cancer is sparse. Our study was carried out to examine the relationship between HIV and a number of cancer types or sites that are common in South Africa. A total of 4,883 subjects, presenting with a cancer or cardiovascular disease at the 3 tertiary referral hospitals in Johannesburg, were interviewed and had blood tested for HIV. Odds ratios associated with HIV infection were calculated by using unconditional logistic regression models for 16 major cancer types where data was available for 50 or more patients. In the comparison group, the prevalence of HIV infection was 8.3% in males and 9.1% in females. Significant excess risks associated with HIV infection were found for Kaposi's sarcoma (OR=21.9, 95% CI=12.5–38.6), non-Hodgkin lymphoma (OR=5.0, 95%CI=2.7–9.5), vulval cancer (OR=4.8, 95%CI=1.9–12.2) and cervical cancer (OR=1.6, 95%CI=1.1–2.3) but not for any of the other major cancer types examined, including Hodgkin disease, multiple myeloma and lung cancer. In Johannesburg, South Africa, HIV infection was associated with significantly increased risks of Kaposi's sarcoma, non-Hodgkin lymphoma and cancers of the cervix and the vulva. The relative risks for Kaposi's sarcoma and non-Hodgkin lymphoma associated with HIV infection were substantially lower than those found in the West.