Fiona M. Ross

Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders

Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

Prognostic Subgroups in B-Cell Chronic Lymphocytic Leukemia Defined by Specific Chromosomal Abnormalities

BACKGROUND AND METHODS Specific chromosomal abnormalities have been shown to affect the overall survival of patients with acute leukemia, but the possibility that specific chromosomal defects may influence the course of B-cell chronic lymphocytic leukemia (CLL) is controversial. We assessed this possibility as follows: blood mononuclear cells from 433 patients with B-cell CLL in five European centers were cultured with B-cell mitogens, and banded metaphases were studied. RESULTS Three hundred ninety-one patients could be evaluated cytogenetically, and 218 had clonal chromosomal changes. The most common abnormalities were trisomy 12 (n = 67) and structural abnormalities of chromosome 13 (n = 51; most involving the site of the retinoblastoma gene) and of chromosome 14 (n = 41). Patients with a normal karyotype had a median overall survival of more than 15 years, in contrast to 7.7 years for patients with clonal changes. Patients with single abnormalities (n = 113) did better than those with complex karyotypes (P less than 0.001). Patients with abnormalities involving chromosome 14q had poorer survival than those with aberrations of chromosome 13q (P less than 0.05). Among patients with single abnormalities, those with trisomy 12 alone had poorer survival than patients with single aberrations of chromosome 13q (P = 0.01); the latter had the same survival as those with a normal karyotype. A high percentage of cells in metaphase with chromosomal abnormalities, indicating highly proliferative leukemic cells, was associated with poor survival (P less than 0.001). Cox proportional-hazards analysis identified age, sex, the percentage of cells in metaphase with chromosomal abnormalities, and the clinical stage of the disease (Binet classification system) as independent prognostic variables. CONCLUSIONS Chromosomal analysis provides prognostic information about overall survival in addition to that supplied by clinical data in patients with B-cell CLL.

The role of maintenance thalidomide therapy in multiple myeloma: MRC Myeloma IX results and meta-analysis

Thalidomide maintenance has the potential to modulate residual multiple myeloma (MM) after an initial response. This trial compared the effect of thalidomide maintenance and no maintenance on progression-free survival (PFS) and overall survival (OS) in MM patients. After intensive or nonintensive induction therapy, 820 newly diagnosed MM patients were randomized to open-label thalidomide maintenance until progression, or no maintenance. Interphase FISH (iFISH) analysis was performed at study entry. Median PFS was significantly longer with thalidomide maintenance (log-rank P < .001). Median OS was similar between regimens (log-rank P = .40). Patients with favorable iFISH showed improved PFS (P = .004) and a trend toward a late survival benefit. Patients with adverse iFISH receiving thalidomide showed no significant PFS benefit and worse OS (P = .009). Effective relapse therapy enhanced survival after progression, translating into a significant OS benefit. Meta-analysis of this and other studies show a significant late OS benefit (P < .001, 7-year difference hazard ratio = 12.3; 95% confidence interval, 5.5-19.0). Thalidomide maintenance significantly improves PFS and can be associated with improved OS. iFISH testing is important in assessing the clinical impact of maintenance therapy. Overview analysis demonstrated that thalidomide maintenance was associated with a significant late OS benefit. This trial was registered at www.isrctn.org as #ISRCTN68454111.

Minimal Residual Disease Assessed by Multiparameter Flow Cytometry in Multiple Myeloma: Impact on Outcome in the Medical Research Council Myeloma IX Study

PURPOSE To investigate the prognostic value of minimal residual disease (MRD) assessment in patients with multiple myeloma treated in the MRC (Medical Research Council) Myeloma IX trial. PATIENTS AND METHODS Multiparameter flow cytometry (MFC) was used to assess MRD after induction therapy (n = 378) and at day 100 after autologous stem-cell transplantation (ASCT; n = 397) in intensive-pathway patients and at the end of induction therapy in non-intensive-pathway patients (n = 245). RESULTS In intensive-pathway patients, absence of MRD at day 100 after ASCT was highly predictive of a favorable outcome (PFS, P < .001; OS, P = .0183). This outcome advantage was demonstrable in patients with favorable and adverse cytogenetics (PFS, P = .014 and P < .001, respectively) and in patients achieving immunofixation-negative complete response (CR; PFS, P = .0068). The effect of maintenance thalidomide was assessed, with the shortest PFS demonstrable in those MRD-positive patients who did not receive maintenance and longest in those who were MRD negative and did receive thalidomide (P < .001). Further analysis demonstrated that 28% of MRD-positive patients who received maintenance thalidomide became MRD negative. MRD assessment after induction therapy in the non-intensive-pathway patients did not seem to be predictive of outcome (PFS, P = .1). CONCLUSION MRD assessment by MFC was predictive of overall outcome in patients with myeloma undergoing ASCT. This predictive value was seen in patients achieving conventional CR as well as patients with favorable and adverse cytogenetics. The effects of maintenance strategies can also be evaluated, and our data suggest that maintenance thalidomide can eradicate MRD in some patients.

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.

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.

Cyclophosphamide, thalidomide, and dexamethasone (CTD) as initial therapy for patients with multiple myeloma unsuitable for autologous transplantation

As part of the randomized MRC Myeloma IX trial, we compared an attenuated regimen of cyclophosphamide, thalidomide, and dexamethasone (CTDa; n = 426) with melphalan and prednisolone (MP; n = 423) in patients with newly diagnosed multiple myeloma ineligible for autologous stem-cell transplantation. The primary endpoints were overall response rate, progression-free survival, and overall survival (OS). The overall response rate was significantly higher with CTDa than MP (63.8% vs 32.6%; P < .0001), primarily because of increases in the rate of complete responses (13.1% vs 2.4%) and very good partial responses (16.9% vs 1.7%). Progression-free survival and OS were similar between groups. In this population, OS correlated with the depth of response (P < .0001) and favorable interphase fluorescence in situ hybridization profile (P < .001). CTDa was associated with higher rates of thromboembolic events, constipation, infection, and neuropathy than MP. In elderly patients with newly diagnosed multiple myeloma (median age, 73 years), CTDa produced higher response rates than MP but was not associated with improved survival outcomes. We highlight the importance of cytogenetic profiling at diagnosis and effective management of adverse events. This trial was registered at International Standard Randomized Controlled Trials Number as #68454111.

Aberrant global methylation patterns affect the molecular pathogenesis and prognosis of multiple myeloma

We used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal gammopathy of undetermined significance [MGUS], presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell-cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.

Deletion of chromosome 13 detected by conventional cytogenetics is a critical prognostic factor in myeloma

In myeloma, the prognostic impact of different strategies used to detect chromosome 13 deletion (Δ13) remains controversial. To address this, we compared conventional cytogenetics and interphase fluorescence in situ hybridization (iFISH) in a large multicenter study (n=794). The ability to obtain abnormal metaphases was associated with a poor prognosis, which was worse if Δ13, p53 deletion or t(4;14) was present, but only Δ13 remained significant on multivariate analysis. Patients with Δ13, by either cytogenetics or iFISH, had a poor prognosis. However, when cases with Δ13 detectable by both cytogenetics and iFISH were separated from those detected by iFISH only, the poor prognosis of iFISH-detectable Δ13 disappeared; their outcome matched that of patients with no detectable Δ13 (P=0.115). Addition of ploidy status to iFISH-Δ13 did not affect the prognostic value of the test. Indeed both cytogenetics and iFISH Δ13 divided both hyperdiploidy and nonhyperdiploidy into two groups with similar prognoses, indicating that the poor prognosis of ploidy is entirely due to its association with Δ13. We conclude that Δ13 detected by metaphase analysis is a critical prognostic factor in myeloma. Absence of Δ13, even in those patients yielding only normal or no metaphases, is associated with a relatively good prognosis.

Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders

The European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or break-apart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.