Faculty Bibliography

There is a significant unmet need in effective therapy for relapsed myeloma patients once they become refractory to bortezomib and lenalidomide. While data from the front line setting suggest bendamustine is superior to melphalan, there is no information defining optimal bendamustine dose in multiply-treated patients. We report a multi-centre randomized two-stage phase 2 trial simultaneously assessing deliverability and activity of two doses of bendamustine (60 mg/m2 vs. 100 mg/m2) days 1 and 8, thalidomide (100 mg) days 1-21 and low dose dexamethasone (20 mg) days 1, 8, 15 and 22 of a 28-d cycle. Ninety-four relapsing patients were treated on trial, with a median three prior treatment lines. A pre-planned interim deliverability and activity assessment led to closure of the 100 mg/m2 arm due to excess cytopenias, and led to amendment of entry criteria for cytopenias. Non-haematological toxicities including thromboembolism and neurotoxicity were infrequent. In the 60 mg/m2 arm, treatment was deliverable in 61.1% subjects and the partial response rate was 46.3% in the study eligible population, with 7.5 months progression-free survival. This study demonstrates bendamustine at 60 mg/m2 twice per month with thalidomide and dexamethasone is deliverable for repeated cycles in heavily pre-treated myeloma patients and has substantial clinical activity.

A sizeable fraction of multiple myeloma (MM) is expected to be explained by heritable factors. Genome-wide association studies (GWAS) have successfully identified a number of common single-nucleotide polymorphisms (SNPs) influencing MM risk. While these SNPs only explain a small proportion of the genetic risk it is unclear how much is left to be detected by other, yet to be identified, common SNPs. Therefore, we applied Genome-Wide Complex Trait Analysis (GCTA) to 2,282 cases and 5,197 controls individuals to estimate the heritability of MM. We estimated that the heritability explained by known common MM risk SNPs identified in GWAS was 2.9% (± 2.4%), whereas the heritability explained by all common SNPs was 15.2% (± 2.8%). Comparing the heritability explained by the common variants with that from family studies, a fraction of the heritability may be explained by other genetic variants, such as rare variants. In summary, our results suggest that known MM SNPs only explain a small proportion of the heritability and more common SNPs remain to be identified.

There is a growing body of evidence supporting the use of epigenetic therapies in the treatment of multiple myeloma. We show the novel HDAC inhibitor CHR-3996 induces apoptosis in myeloma cells at concentrations in the nanomolar range and with apoptosis mediated by p53 and caspase pathways. In addition, HDAC inhibitors are highly synergistic, both in vitro and in vivo, with the aminopeptidase inhibitor tosedostat (CHR-2797). We demonstrate that the basis for this synergy is a consequence of changes in the levels of NFκB regulators BIRC3/cIAP2, A20, CYLD, and IκB, which were markedly affected by the combination. When co-administered the HDAC and aminopeptidase inhibitors caused rapid nuclear translocation of NFκB family members p65 and p52, following activation of both canonical and non-canonical NFκB signalling pathways. The subsequent up-regulation of inhibitors of NFκB activation (most significantly BIRC3/cIAP2) turned off the cytoprotective effects of the NFκB signalling response in a negative feedback loop. These results provide a rationale for combining HDAC and aminopeptidase inhibitors clinically for the treatment of myeloma patients and support the disruption of the NFκB signalling pathway as a therapeutic strategy.

Multiple myeloma is a B-cell malignancy stratified in part by cytogenetic abnormalities, including the high-risk copy number aberrations (CNAs) of +1q21 and 17p(-). To investigate the relationship between 1q21 CNAs and DNA hypomethylation of the 1q12 pericentromeric heterochromatin, we treated in vitro peripheral blood cultures of 5 patients with balanced constitutional rearrangements of 1q12 and 5 controls with the hypomethylating agent 5-azacytidine. Using G-banding, fluorescence in situ hybridization, and spectral karyotyping, we identified structural aberrations and copy number gains of 1q21 in the treated cells similar to those found in patients with cytogenetically defined high-risk disease. Aberrations included 1q12 triradials, amplifications of regions juxtaposed to 1q12, and jumping translocations 1q12. Strikingly, all 5 patients with constitutional 1q12 rearrangements showed amplifications on the derivative chromosomes distal to the inverted or translocated 1q12 region, including MYCN in 1 case. At the same time, no amplification of the 1q21 region was found when the 1q12 region was inverted or absent. These findings provide evidence that the hypomethylation of the 1q12 region can potentially amplify any genomic region juxtaposed to it and mimic CNAs found in the bone marrow of patients with high-risk disease.

AIM/OBJECTIVE:To estimate and compare the extent of myeloma bone disease by skeletal region using whole-body diffusion-weighted imaging (WB-DWI) and skeletal survey (SS) and record interobserver agreement, and to investigate differences in imaging assessments of disease extent and apparent diffusion coefficient (ADC) between patients with pathological high versus low disease burden. MATERIALS AND METHODS/METHODS:Twenty patients with relapsed myeloma underwent WB-DWI and SS. Lesions were scored by number and size for each skeletal region by two independent observers using WB-DWI and SS. Observer scores, ADC, and ADC-defined volume of tumour-infiltrated marrow were compared between patients with high and low disease burden (assessed by serum paraproteins and marrow biopsy). RESULTS:Observer scores were higher on WB-DWI than SS in every region (p<0.05) except the skull, with greater interobserver reliability in rating the whole skeleton (WB-DWI: ICC = 0.74, 95% CI: 0.443-0.886; SS: ICC = 0.44, 95% CI: 0.002-0.730) and individual body regions. WB-DWI scores were not significantly higher in patients with high versus low disease burden (observer 1: mean ± SD: 48.8 ± 7, 38.6 ± 14.5, observer 2: mean ± SD: 37.3 ± 13.5, 30.4 ± 15.5; p = 0.06, p = 0.35). CONCLUSION/CONCLUSIONS:WB-DWI demonstrated more lesions than SS in all regions except the skull with greater interobserver agreement. Sensitivity is not a limiting factor when considering WB-DWI in the management pathway of patients with myeloma.

The unfolded protein response (UPR) remediates endoplasmic reticulum (ER) stress. IRE1, a component of the UPR, senses misfolded protein and cleaves XBP1 mRNA, which is ligated to code for the prosurvival transcription factor. IRE1 also cleaves other mRNAs preceding their degradation, termed regulated IRE1-dependent mRNA decay (RIDD). It has been reported that RIDD may be involved in cell viability under stress and therefore may contribute to cancer cell viability. To investigate RIDD targets that may have functional relevance in cell survival, we identified conserved RIDD targets containing stringent IRE1 RNase target sequences. Using a systematic bioinformatics approach with quantitative-PCR (qPCR) validation, we show that only BLOC1S1 is consistently a RIDD target in all systems tested. Using cancer cell lines, we show that BLOC1S1 is specifically cleaved by IRE1 at guanine 444, but only under conditions of IRE1 hyperactivation. BLOC1S1 cleavage is temporally separate from XBP1 splicing, occurring after depletion of unspliced XBP1. Expression of an uncleavable BLOC1S1 mutant or inhibition of RIDD using an IRE1 RNase inhibitor did not affect cellular recovery from acute ER stress. These data demonstrate that although hyperactivated IRE1 specifically cleaves BLOC1S1, this cleavage event and RIDD as a whole are dispensable for cell viability under acute stress.

Incorporation of novel agents into auto-SCT for patients with multiple myeloma has led to improvement in their outcomes. However, the effects of new drugs, either single or combined, on PBSC mobilization have not been fully evaluated, particularly in phase 3 clinical studies. We analyzed the impact of two novel agent-based induction treatments in patients enrolled in the GIMEMA MMY-3006 study comparing bortezomib, thalidomide and dexamethasone (VTD) versus thalidomide and dexamethasone (TD) in preparation for double auto-SCT. Results showed that a short-term induction therapy with VTD did not adversely affect CD34(+) cell yields as compared with TD (9.75 vs 10.76 × 10(6) CD34(+) cells/kg, P=0.220). For poor mobilizers (<4 × 10(6) CD34(+) cells/kg), 5-year rates of time to progression (TTP), progression-free survival (PFS) and overall survival (OS) were significantly shorter than for successful mobilizers (TTP:17 vs 48%, P<0.0001; PFS: 16 vs 46%, P<0.0001; OS: 50 vs 80%, P<0.0001). These differences were retained across patients randomized to the TD arm; conversely, no differences in outcomes were seen in patients treated with VTD, irrespective of the number of harvested CD34(+) cells. The number of collected PBSCs predicted better outcomes after auto-SCT and VTD overcame the negative impact of a poor stem cell mobilization.

We have sequenced 463 presenting cases of myeloma entered into the UK Myeloma XI study using whole exome sequencing. Here we identify mutations induced as a consequence of misdirected AID in the partner oncogenes of IGH translocations, which are activating and associated with impaired clinical outcome. An APOBEC mutational signature is seen in 3.8% of cases and is linked to the translocation-mediated deregulation of MAF and MAFB, a known poor prognostic factor. Patients with this signature have an increased mutational load and a poor prognosis. Loss of MAF or MAFB expression results in decreased APOBEC3B and APOBEC4 expression, indicating a transcriptional control mechanism. Kataegis, a further mutational pattern associated with APOBEC deregulation, is seen at the sites of the MYC translocation. The APOBEC mutational signature seen in myeloma is, therefore, associated with poor prognosis primary and secondary translocations and the molecular mechanisms involved in generating them.

The detection of minimal residual disease (MRD) in myeloma using a 0.01% threshold (10(-4)) after treatment is an independent predictor of progression-free survival (PFS), but not always of overall survival (OS). However, MRD level is a continuous variable, and the predictive value of the depth of tumor depletion was evaluated in 397 patients treated intensively in the Medical Research Council Myeloma IX study. There was a significant improvement in OS for each log depletion in MRD level (median OS was 1 year for ≥10%, 4 years for 1% to <10%, 5.9 years for 0.1% to <1%, 6.8 years for 0.01% to <0.1%, and more than 7.5 years for <0.01% MRD). MRD level as a continuous variable determined by flow cytometry independently predicts both PFS and OS, with approximately 1 year median OS benefit per log depletion. The trial was registered at www.isrctn.com as #68454111.

Myeloma is characterized by a highly variable clinical outcome. Despite the effectiveness of high-dose therapy, 15% of patients relapse within 1 year. We show that these cases also have a significantly shorter post-relapse survival compared to the others (median 14.9 months vs. 40 months, p = 8.03 × 10(- 14)). There are no effective approaches to define this potentially distinct biological group such that treatment could be altered. In this work a series of uniformly treated patients with myeloma were used to develop a gene expression profiling (GEP)-based signature to identify this high risk clinical behavior. Gene enrichment analyses applied to the top differentially expressed genes showed a significant enrichment of epigenetic regulators as well as "stem cell" myeloma genes. A derived 17-gene signature effectively identifies patients at high risk of early relapse as well as impaired overall survival. Integrative genomic analyses showed that epigenetic mechanisms may play an important role on transcription of these genes.