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.

Genetic factors influencing the risk of multiple myeloma bone disease.

A major complication of multiple myeloma (MM) is the development of osteolytic lesions, fractures and bone pain. To identify genetic variants influencing the development of MM bone disease (MBD), we analyzed MM patients of European ancestry (totaling 3774), which had been radiologically surveyed for MBD. Each patient had been genotyped for ~6 00 000 single-nucleotide polymorphisms with genotypes for six million common variants imputed using 1000 Genomes Project and UK10K as reference. We identified a locus at 8q24.12 for MBD (rs4407910, OPG/TNFRSF11B, odds ratio=1.38, P=4.09 × 10(-9)) and a promising association at 19q13.43 (rs74676832, odds ratio=1.97, P=9.33 × 10(-7)). Our findings demonstrate that germline variation influences MBD and highlights the importance of RANK/RANKL/OPG pathway in MBD development. These findings will contribute to the development of future strategies for prevention of MBD in the early precancerous phases of MM.

Contribution of Clostridium difficile infection to the development of lower gastrointestinal adverse events during autologous stem cell transplantation.

BACKGROUND: Lower gastrointestinal (GI) adverse events (LGAE) are common afflictions of patients undergoing stem cell transplantation (SCT). Unfortunately, the pathophysiology remains poorly characterized. Emerging data suggest a prominent role of intestinal microbiota; however, contributions of pathogenic gut microbiota such as Clostridium difficile are not well defined. We performed a genome-wide association study (GWAS) to investigate clinical and genetic factors associated with development of LGAE. METHODS: A total of 972 patients undergoing autologous SCT were graded for LGAE based on Common Terminology Criteria for Adverse Events (v 4.0). Germline DNA material was obtained from leukapharesis products and genotyped using Illumina(®) Whole Genome Genotyping Infinium chemistry and HumanOmni1-Quad Bead chips containing over 1.1 million single nucleotide polymorphisms (SNPs) (Illumina, San Diego, California, USA). Statistical models incorporating clinical factors, genetic factors, and a combination of clinical plus genetic factors were utilized to compare patients who developed severe LGAE (grade 2 or above) and others. RESULTS: Among 972 patients, 459 (47.2%) developed severe LGAE. Baseline hemoglobin and hematocrit, estimated glomerular filtration rate, ß2-microglobulin, protocol type, and C. difficile infection (CDI) were associated with severe LGAE on univariate analysis, Genomic comparisons between groups did not reveal any SNPs associated with severe LGAE and neither did incorporation of genetic factors into the clinical model. In addition, 11 candidate SNPs associated with upper GI mucositis were evaluated, alongside clinical factors in a multivariate model. Only CDI was found to be associated with severe LGAE in all models. CONCLUSION: CDI is a prominent factor in the development of LGAE in patients undergoing autologous SCT.

Genetic polymorphisms of EPHX1, Gsk3beta, TNFSF8 and myeloma cell DKK-1 expression linked to bone disease in myeloma.

Bone disease in myeloma occurs as a result of complex interactions between myeloma cells and the bone marrow microenvironment. A custom-built DNA single nucleotide polymorphism (SNP) chip containing 3404 SNPs was used to test genomic DNA from myeloma patients classified by the extent of bone disease. Correlations identified with a Total Therapy 2 (TT2) (Arkansas) data set were validated with Eastern Cooperative Oncology Group (ECOG) and Southwest Oncology Group (SWOG) data sets. Univariate correlates with bone disease included: EPHX1, IGF1R, IL-4 and Gsk3beta. SNP signatures were linked to the number of bone lesions, log(2) DKK-1 myeloma cell expression levels and patient survival. Using stepwise multivariate regression analysis, the following SNPs: EPHX1 (P=0.0026); log(2) DKK-1 expression (P=0.0046); serum lactic dehydrogenase (LDH) (P=0.0074); Gsk3beta (P=0.02) and TNFSF8 (P=0.04) were linked to bone disease. This assessment of genetic polymorphisms identifies SNPs with both potential biological relevance and utility in prognostic models of myeloma bone disease.

Synthesis and analysis of RNA containing 6-trifluoromethylpurine ribonucleoside.

We report the synthesis of a 5'-DMT-2'-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside ((TFM)P) and its use in the site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of (TFM)P-substituted RNA suggest it will be valuable in the study of RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases. Reaction: see text.

Conformational changes that occur during an RNA-editing adenosine deamination reaction.

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.

Analysis of the RNA-editing reaction of ADAR2 with structural and fluorescent analogues of the GluR-B R/G editing site.

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.

Mutagenicity of para-nitroso-dimethyl- and diethyl-anilines.

Low concentrations of para-nitroso-dimethylaniline (NdMA) were mutagenic to Salmonella typhimurium TA100 with optimal effect at 1.5 microM in fluctuation assays, without activating enzymes. The diethyl homologue (NdEA) had little or no mutagenic effect at low concentrations, although the bacteriocidal effects of NdMA and NdEA were similar. At higher bacteriocidal concentrations (approximately LC55-LC80) both NdMA and NdEA were mutagenic. NdMA and some other C-nitroso compounds proved carcinogenic in animal bioassays, and further research is needed to assess the human hazard from exposure to C-nitroso compounds in food, medicines or industry.

Comparative sensitivity of survival-adjusted chi-square and normal statistics for the mutagenesis fluctuation assay.

Three statistics for analysis of microtitre plate mutagenesis fluctuation tests were studied by simulation, and in enzyme-activated assays of dimethylnitrosamine and diethylnitramine. A survival-adjusted chi 2 statistic ('Gsq') was compared with Katz's normally distributed statistic ('Phi'), and with the survival-independent statistic ('Zsq') of Gilbert. When toxicity was either very low or high, the Phi statistic either could not be evaluated over the whole range of possible background mutant frequencies, or sometimes it indicated unusually high levels of statistical significance, even when the other tests were negative. The survival-adjusted Gsq closely followed the Zsq statistic throughout the experimentally useful range of toxicities and mutant background values, with some improvement in sensitivity. Within the range 80 +/- 10% survival approximately, Katz's statistic 'Phi' was the most sensitive. The choice of statistical test could affect the estimate of the minimal effective mutagenic concentration by a factor of 10-100. For screening unknowns, both types of test (Phi and Gsq (or Zsq] may help in detecting suspect pro-mutagens and in designing a confirmatory assay. Bacterial population statistics are needed to assess the value of statistically positive results.

Correlation of human bladder tumor recurrence with changes in clonogenicity of urothelial cells.

Over a 2-year period 340 cystoscopies were performed on 174 patients (126 men and 48 women) followed up for previously treated transitional cell tumor of the bladder or new cases suspected of having bladder tumor. Bladder washings taken at cystoscopy in 66% yielded cells for clonogenic assay. Patients with transitional cell tumors had an average clonogenic index (CI) of 14 (+/- 5) colonies per 10(5) viable urothelial cells, whereas previously tumorous patients with tumor-free bladders had an average CI of 6 +/- 1 colonies (P less than 0.001, Student's t test). Patients with hematuria but no bladder tumor had an average CI of 8 (not significant). In serial examinations with consecutive successful clonogenic assay, 11 of 16 patients remaining tumor-free had constant or falling CI whereas 9 of 12 patients with recurrent tumor had rising CI values (Fisher; P less than 0.025). Changes in the clonogenic index of cells taken from bladder washings paralleled the changes in tumor status at cystoscopy and might have predictive value in this disease.

Mutagenic activation of dialkylnitrosamines by intact urothelial cells.

Intact urothelial cells isolated from bladders of untreated inbred NZO/BIGd or NZC/BIGd mice and NZR/Gd rats activated dimethyl, diethyl, dipropyl, and dibutyl nitrosamines in a liquid culture mutagenesis fluctuation assay using Salmonella typhimurium TA100 as target organism. Rat and mouse urothelial cells were highly effective at 1.5 X 10(5) cells/ml, in O2 gas phase, and no cofactors were required. Relative mutagenic activities were estimated at equitoxic (LD50) concentrations of the 4 nitrosamines. Nitrosamines with odd-carbon chain substitutents were more active than the C-even compounds. Although dibutylnitrosamine is a powerful bladder carcinogen in both rats and mice while the other 3 compounds very rarely cause bladder tumours, the most active promutagens were dipropyl and dimethyl, followed by diethyl and dibutyl nitrosamines. None were active in absence of urothelial cells. Mouse bladder cells were more active than those from NZR rats. There were sex differences in the mice with NZC males and NZO females predominating, but in NZR rats urothelial cells from male and female animals were equally active. These dialkylnitrosamines are widely distributed in the environment, and our results indicate that direct mutagenic activation in the urothelium could be one factor contributing to the incidence of bladder cancer.