Rebecca K.M. Protheroe

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.

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.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material. Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis. Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor. Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Timing of acquisition of deletion 13 in plasma cell dyscrasias is dependent on genetic context

Monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) are characterized by an expansion of monoclonal plasma cells and can progress to symptomatic multiple myeloma. This study assessed the incidence and the association of monosomy 13 with IgH translocations, ploidy status and deletions of 16q23 and TP53 in a large series of MGUS and SMM patients. Background Multiple myeloma, monoclonal gammopathy of undetermined significance and smoldering multiple myeloma harbor common chromosomal abnormalities but the prevalence and relative association of aberrations in these diagnostic groups remains controversial. We investigated these aspects in a large series of patients. Design and Methods Chromosome 13 deletion (Δ13), deletion of TP53, ploidy status and immunoglobulin heavy chain (IgH) translocations were evaluated by fluorescence in situ hybridization in patients with monoclonal gammopathy of undetermined significance (n=189), smoldering multiple myeloma (n=127) and multiple myeloma (n=400). Results Overall, Δ13 (25%, 34% and 47%), 16q23 deletions (6%, 8% and 21%) and 17p13 deletions (3%, 1% and 10%) were less frequent in patients with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in those with multiple myeloma. When distinct genetic groups were considered, no differences in the prevalence of Δ13 were found with t(4;14)(p16;q32) and t(14;16)(q32;q23) among the three diagnostic groups; in contrast Δ13 was rarer in t(11;14)(q13;q32) in patients with monoclonal gammopathy (1/28) and smoldering myeloma (2/13) than in those with multiple myeloma (40%). Similar results were seen for the few t(6;14)(p21;q32) cases: 0/3 patients with monoclonal gammopathy or smoldering myeloma had the Δ13, whereas 4/6 (67%) patients with multiple myeloma and this translocation also had the deletion. In multiple myeloma patients with both an IgH translocation and Δ13, the proportions of cells affected by the two abnormalities were similar, as was the case for t(4;14) and t(14;16) monoclonal gammopathy patients positive for Δ13. In contrast, in monoclonal gammopathy patients with t(14;20)(q32;q11), the translocation was present in almost all cells, while the Δ13 was present in only a sub-population. Conclusions These results indicate that the presence and time of occurrence of Δ13 depends on the presence of specific concurrent abnormalities. The observation that Δ13 was extremely rare in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma with translocations directly involving cyclin D genes (CCND1 and CCND3) suggest a possible role of Δ13 in the progression of the disease specifically in these genetic sub-groups. (clinicaltrials.gov identifier: ISRCTN 68454111; UKCRN ID 1176).

Age has a profound effect on the incidence and significance of chromosome abnormalities in myeloma

A simple high throughput micro-fluorescence in situ hybridisation technique (FISH) was used to detect chromosome 13 deletions (Δ13), immunoglobulin heavy chain (IgH) rearrangements, t(11;14)(q13;q32), t(4;14)(p16;q32), t(14;16)(q23;q32), p53 loss, and numerical changes of chromosomes 3, 6, 7, 9, 10, 11 and 17 in 228 cases of multiple myeloma (MM), including 33 asymptomatic/smouldering MM (SMM). The patients were not part of a clinical trial and were from 30 different hospitals. In all, 98.4% of cases were abnormal, with 43% having IgH rearrangements and 42% Δ13. The low incidence of IgH rearrangements was due to a decrease in this finding with age (P=0.001) and the relatively high proportion of elderly patients in our study population (41% >70 years old). The incidence of specific IgH translocations was t(4;14) 11%, t(11;14) 16% and t(14;16) 3%. Univariate statistical testing showed Δ13 (P=0.002), and t(14;16) (P=0.005) to be associated with shorter survival. This effect was exaggerated for patients aged 70 years or under but no effect on survival was seen for those over 70 years. In younger patients t(4;14) (P=0.044) and p53 deletion (P<0.001) were also significant poor prognostic indicators. Multivariate analysis showed Δ13 and t(14;16) to be independent prognostic variables when considered with age and clinical parameters.

THE T(14,20) IS A POOR PROGNOSTIC FACTOR IN MYELOMA BUT IS ASSOCIATED WITH LONG-TERM STABLE DISEASE IN MONOCLONAL GAMMOPATHIES OF UNDETERMINED SIGNIFICANCE

A large series of plasma cell dyscrasias (n=2207) was examined for translocations which deregulate the MAF genes, t(14;20)(q32;q12) and t(14;16)(q32;q23), and their disease behavior was compared to a group characterized by the t(4;14)(p16;q32) where CCND2 is also up-regulated. The t(14;20) showed low prevalence in myeloma (27/1830, 1.5%) and smoldering myeloma (1/148, <1%) with a higher incidence in MGUS (9/193, 5% P=0.005). Strong associations with del(13) (76%), non-hyperdiploidy (83%) and gain of 1q (58%) were seen but no association with an IgA M-protein or absence of bone disease was noted. All three translocations were associated with poor outcome in myeloma, but strikingly all t(14;20) MGUS/smoldering myeloma cases (n=10) had stable, low level disease. In contrast, the 10 t(14;16) and 25 t(4;14) MGUS/smoldering myeloma cases were associated with both evolving and non-evolving disease. None of the associated genetic abnormalities helped to predict for progression from MGUS or smoldering myeloma. (Clinicaltrials.gov identifier: ISRCTN 68454111; UKCRN ID 1176)

Frequent upregulation of MYC in plasma cell leukemia.

Plasma cell leukemia (PCL) is a rare form of monoclonal gammopathy, which can originate de novo or evolve from multiple myeloma (MM) as a terminal leukemic phase. Previous cytogenetic studies of PCL have reported the presence of complex karyotypes with involvement of multiple unidentified chromosomal regions. We report here the analysis of 12 PCL (10 primary and two secondary) by metaphase and FISH analysis combined with oligonucleotide array data (244 k, Agilent). Interphase‐FISH results were compared with those from a series of 861 newly diagnosed patients with MM. Cytogenetic analysis was successful on 11 patients, all of whom showed clonal chromosomal abnormalities. Compared with MM, t(11;14)(q13;q32) (42% versus 15%; P = 0.027) and t(14;16)(q32;q23) (25% versus 4%; P = 0.010) were more frequent in PCL, although neither the specific partner chromosome involved in the IgH translocation nor the ploidy status predicted for survival. Chromosomes 1, 8, 13, and 16 showed the highest number of copy number alterations with 8q24 being the chromosomal region most frequently involved. In eight of 12 patients we found abnormalities (translocations, one amplification, small deletions, and duplications) that directly targeted or were very close to MYC. Only four of these changes were detected by routine FISH analysis using commercial probes with the others exclusively detected by arrays. Quantitative reverse transcription polymerase chain reaction demonstrated that these different abnormalities were associated with increased levels of MYC mRNA. We conclude that MYC dysregulation by complex mechanisms is one of the major molecular events in the oncogenesis of PCL.

Loss of 1p and rearrangement of MYC are associated with progression of smouldering myeloma to myeloma: sequential analysis of a single case

This case report suggests that loss of 1p and rearrangement of MYC are associated with progression of smoldering myeloma to to multiple myeloma. We report serial genetic studies on a young female patient initially diagnosed with asymptomatic smouldering myeloma who progressed to symptomatic myeloma 4.5 years after presentation. An unbalanced translocation, der(14)t(4;14)(p16;q32), was initially found in all plasma cells plus deletions of other chromosomal regions as detected by array-based comparative genomic hybridization. Deletion of chromosome 13 was observed in a minor population of plasma cells (<20%) for the first two years, increasing to 100% of plasma cells by the time of multiple myeloma diagnosis. Loss of 1p and a rearrangement of MYC were first observed in a small population of plasma cells one year prior to the clinical diagnosis of multiple myeloma, but these subclones increased rapidly in size to become the major population suggesting that they were directly involved in the transformation process. This case report provides a unique insight into the mechanisms of disease progression from smouldering multiple myeloma to multiple myeloma.

Deletion of 16q identified by FISH is an independent adverse prognostic marker in multiple myeloma

Chromosomal translocations lead to oncogene activation in a significant number of haematological malignancies. Those involving the immunoglobulin heavy chain locus, IGH, at chromosome band 14q32 are frequently observed in B-cell malignant proliferation. A small number have been described in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). However, their biological and clinical significance is currently unknown. Detailed fluorescence in situ hybridisation (FISH) and molecular studies were carried out on a series of BCP-ALL patients with chromosomal abnormalities involving 14q32. Novel and recurrent translocations affecting different chromosomes were highlighted. Refined FISH mapping identified putative IGH partner genes at, or flanking, the translocation breakpoints. Four translocations: two previously reported, t(14;19)(q32;q13), t(8;14)(q11;q32), and two novel, t(14;14)(q11;q32)/ inv(14)(q11q32) and t(14;20)(q32;q13), were identified. Molecular analyses showed that four different members of the CAATT enhancer binding protein (CEBP) gene family were involved: CEBPA (19q13, n59), CEBPD (8q11, n58), CEBPE (14q11, n53) and CEBPB (20q13, n52). One patient with a t(14;19)(q32;q13) was observed to involve the fifth family member CEBPG (19q13, n51). Breakpoints were located within the 30 untranslated region (UTR) of CEBPA and either 30 UTR or 50 of CEBPE, whereas breakpoints in 8q11 were B30 kb centromeric of CEBPD. Where material was available, over-expression of target genes was shown by quantitative real-time PCR. Overall, this study has demonstrated for the first time the involvement of five members of the same gene family in a single subtype of haematological disease. It has indicated that transcriptional upregulation of CEBP gene family members, by juxtaposition to IGH, is important in BCP-ALL: a mechanism in complete contrast to that involving CEPBA in acute myeloid leukaemia.