J. A. Child

A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.

The association of genetic lesions detected by fluorescence in situ hybridization (FISH) with survival was analyzed in 1069 patients with newly presenting myeloma treated in the Medical Research Council Myeloma IX trial, with the aim of identifying patients associated with the worst prognosis. A comprehensive FISH panel was performed, and the lesions associated with short progression-free survival and overall survival (OS) in multivariate analysis were +1q21, del(17p13) and an adverse immunoglobulin heavy chain gene (IGH) translocation group incorporating t(4;14), t(14;16) and t(14;20). These lesions frequently co-segregated, and there was an association between the accumulation of these adverse FISH lesions and a progressive impairment of survival. This observation was used to define a series of risk groups based on number of adverse lesions. Taking this approach, we defined a favorable risk group by the absence of adverse genetic lesions, an intermediate group with one adverse lesion and a high-risk group defined by the co-segregation of >1 adverse lesion. This genetic grouping was independent of the International Staging System (ISS) and so was integrated with the ISS to identify an ultra-high-risk group defined by ISS II or III and >1 adverse lesion. This group constituted 13.8% of patients and was associated with a median OS of 19.4 months.

Minimal residual disease in myeloma by flow cytometry: independent prediction of survival benefit per log reduction

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.

Long-term Follow-up of MRC Myeloma IX Trial: Survival Outcomes with Bisphosphonate and Thalidomide Treatment

Purpose: Medical Research Council (MRC) Myeloma IX was a phase III trial evaluating bisphosphonate and thalidomide-based therapy for newly diagnosed multiple myeloma. Results were reported previously after a median follow-up of 3.7 years (current controlled trials number: ISRCTN68454111). Survival outcomes were reanalyzed after an extended follow-up (median, 5.9 years). Experimental Design: At first randomization, patients (N = 1,970) were assigned to bisphosphonate (clodronic acid or zoledronic acid) and induction therapies [cyclophosphamide–vincristine–doxorubicin–dexamethasone (CVAD) or cyclophosphamide–thalidomide–dexamethasone (CTD) followed by high-dose therapy plus autologous stem cell transplantation for younger/fitter patients (intensive pathway), and melphalan–prednisone (MP) or attenuated CTD (CTDa) for older/less fit patients (nonintensive pathway)]. At second randomization, patients were assigned to thalidomide maintenance therapy or no maintenance. Interphase FISH (iFISH) was used to analyze cytogenics. Results: Zoledronic acid significantly improved progression-free survival (PFS; HR, 0.89; P = 0.02) and overall survival (OS; HR, 0.86; P = 0.01) compared with clodronic acid. In the intensive pathway, CTD showed noninferior PFS and OS compared with CVAD, with a trend toward improved OS in patients with favorable cytogenics (P = 0.068). In the nonintensive pathway, CTDa significantly improved PFS (HR, 0.81; P = 0.007) compared with MP and there was an emergent survival benefit after 18 to 24 months. Thalidomide maintenance improved PFS (HR, 1.44; P < 0.0001) but not OS (HR, 0.96; P = 0.70), and was associated with shorter OS in patients with adverse cytogenics (P = 0.01). Conclusions: Long-term follow-up is essential to identify clinically meaningful treatment effects in myeloma subgroups based on cytogenetics. Clin Cancer Res; 19(21); 6030–8. ©2013 AACR.

A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma

A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma

Haplotypes in the tumour necrosis factor region and myeloma

This study described the haplotypic structure across a region of chromosome 6 including the tumour necrosis factor (TNF) gene, and investigated its influence on the aetiology of myeloma. A total of 181 myeloma cases from the Medical Research Council Myeloma VII trial and 233 controls from the Leukaemia Research Fund Case Control Study of Adult Acute Leukaemia were included in the analysis. Genotyping by induced heteroduplex generator analysis was carried out for single nucleotide polymorphisms (SNP) located at positions −1031, −863, −857, −308 and −238 of the 5′ promoter region of TNF‐α gene, and 252 in the LT‐α gene; and five microsatellites, TNFa, b, c, d and e. Haplotypes were inferred statistically using the phase algorithm. A limited diversity of haplotypes was observed, with the majority of variation described by 12 frequent haplotypes. Detailed characterization of the haplotype did not provide greater determination of disease risk beyond that described by the TNF‐α−308 SNP. Some evidence was provided for a decreased risk of myeloma associated with the TNF‐α−308 variant allele A, odds ratio, 0·57; 95% confidence interval, 0·38–0·86. The results of this study did not support our starting hypothesis; that high producer haplotypes at the TNF locus are associated with an increased risk of developing myeloma.

In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA‐5 and CD45 expression

The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA‐5 has been reported to identify proliferating ‘precursor’ plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA‐5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45+ and CD45− cells, however, cycling plasma cells were consistently VLA‐5‐. There was close correlation between the expression of VLA‐5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin‐associated molecules CD79a/b (Spearman, n = 10, P < 0·0001). In short‐term culture, cells that were initially VLA‐5‐showed increasing VLA‐5 expression with time. However, simultaneous analysis of the DNA‐binding dye 7‐amino‐actinomycin D demonstrated that this was not as a result of differentiation, as VLA‐5+ plasma cells were all non‐viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA‐5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38+CD138+VLA‐5−. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self‐replenishing population.

A molecular diagnostic approach able to detect the recurrent genetic prognostic factors typical of presenting myeloma

Risk stratification in myeloma requires an accurate assessment of the presence of a range of molecular abnormalities including the differing IGH translocations and the recurrent copy number abnormalities that can impact clinical behavior. Currently, interphase fluorescence in situ hybridization is used to detect these abnormalities. High failure rates, slow turnaround, cost, and labor intensiveness make it difficult and expensive to use in routine clinical practice. Multiplex ligation‐dependent probe amplification (MLPA), a molecular approach based on a multiplex polymerase chain reaction method, offers an alternative for the assessment of copy number changes present in the myeloma genome. Here, we provide evidence showing that MLPA is a powerful tool for the efficient detection of copy number abnormalities and when combined with expression assays, MLPA can detect all of the prognostically relevant molecular events which characterize presenting myeloma. This approach opens the way for a molecular diagnostic strategy that is efficient, high throughput, and cost effective.

The impact of response on bone-directed therapy in patients with multiple myeloma.

Significant benefits for zoledronic acid (ZOL) over clodronate acid (CLO) were seen in the Medical Research Council Myeloma IX randomized trial. ZOL significantly reduced skeletal-related events (SREs), and improved progression-free survival and overall survival (OS), making it the bisphosphonate of choice for newly diagnosed myeloma patients. In this analysis of Myeloma IX data, we have investigated the impact of response on bone disease in 1111 transplant-eligible patients. At posttransplant day 100, complete response (CR) was seen in 48% of patients, very good partial response (VGPR) in 20%, and partial response (PR) in 23%. For patients in VGPR or less, ZOL was superior to CLO in reducing SREs (P = .048), whereas for patients in CR, both agents were equivalent (P = .83). For OS, ZOL was associated with a significant benefit in patients in PR (P = .0091). No difference in OS was seen with patients in CR (P = .91) or VGPR (P = .74). These findings indicate that response category posttransplant may influence the impact of bisphosphonate therapy.

FICTION-TSA analysis of the B-cell compartment in myeloma shows no significant expansion of myeloma precursor cells

Studies utilizing flow cytometry and PCR have shown that the B‐cell compartment in myeloma contains cells which are clonally related to the myelomatous plasma cells. Current data, however, remains inconclusive regarding the extent of this involvement. By combining fluorescent immunophenotyping, tyramine signal amplification and fluorescence in‐situ hybridization (FICTION‐TSA), we have used the presence of numerical chromosomal abnormalities within plasma cells as a clonal marker to examine the CD20+ B‐cell compartment for the presence of aneuploidy. A series of 54 cases of myeloma were screened for the presence of numerical abnormalities of chromosomes 3 and 11. FICTION‐TSA was performed on 13 cases with either trisomy 3 or 11 and on a control group of six cases known to be disomic for the two chromosomes. B‐cell numbers were reduced in the myeloma cases compared to the normal controls (median 1.8% v 3.0%, P = 0.05). In the cases with a chromosomal marker, three signals were seen in a median of 1.88% of CD20+ B cells compared to 2.58% within the control group. Comparison of the two groups using a Wilcoxon‐Mann‐Whitney U test showed no statistical significant difference. Using this data set, it was possible to exclude a 3.03% expansion of clonally related B cells (95% confidence level). We conclude that the B‐cell compartment in myeloma does not represent the major site of clonal expansion, and if clonally related cells are present then the numbers are few.

Plasma fibronectin in myeloproliferative disorders and chronic granulocytic leukaemia

A significant reduction of plasma fibronectin levels was found in polycythaemia vera and myelofibrosis, the lowest levels being found in patients with marked splenomegaly. Plasma fibronectin concentration was normal in essential thrombocythaemia, and only modest reduction was seen in chronic granulocytic leukaemia in either controlled chronic phase or blast cell crisis. In a patient with myelofibrosis, the plasma fibronectin rose from less than 100 mg/l to 177 mg/l after splenectomy. Possible explanations include increased consumption of plasma fibronectin in the expanded mononuclear phagocyte system present in the liver and spleen, reduced hepatic synthesis, and the clearance of circulating immune complexes. Low plasma fibronectin concentrations may increase susceptibility to infection.