Recent technological advances have resulted in the ability to assess genomic aberrations at both the DNA and RNA levels in a global fashion. This has resulted in a number of new classifications with biological and clinical relevance.
Translocation and cyclin D (TC) classification35
This classification is based on spiked expression of genes deregulated by primary IgH translocations and the universal over-expression of cyclin D genes by either these translocations or another mechanism. The resultant classification identifies eight groups of tumors: those with primary translocations (designated 4p16, 11q13, 6p21, Maf), those that over-expressed CCND1 and CCND2 either alone or in combination (D1, D1&D2, D2), and the rare cases that do not over-express any cyclin D genes (‘none’). Most of the patients with HRD MM fall within the D1 and D1&D2 groups.
The advantage of this classification system is that it focuses on different kinds of mechanisms that dysregulate a Cyclin D gene as an early and unifying event in pathogenesis. The underlying cyclin D deregulation potentially has an important therapeutic implication, as differential targeting of cyclin D may be very useful and add specificity to treatment. Indeed, some potential agents targeting cyclin D2 have been identified in a drug library screen (A. K. Stewart, personal communication).
This classification has great potential for translation into the clinic as it involves the measurement of relatively few markers, and most of the translocations can be detected by FISH. In this regard, cut-offs for each gene/probe used for the TC classification has been derived, and a set of criteria for assigning new cases has been formulated (see Appendix
On the downside, the TC classification does not identify patients with HRD myeloma clearly, with the majority of these patients falling into the D1 and D1&D2 groups. D1&D2 HRD MM appears to have more proliferative disease, but the survival of these patients is not different from those that are D1. In addition, the clinical and biological significance of the D2 group is unclear.
UAMS molecular classification of myeloma
Recently, the group from UAMS (University of Arkansas for Medical Science) derived another MM classification using an unsupervised approach, and identified seven tumor groups characterized by the co-expression of unique gene clusters.42
Interestingly, these clusters also identify tumors with t(4;14), maf translocations, t(11;14) and t(6;14), corresponding to the MS, MF and CD-1 and/or CD-2 groups respectively. In this analysis, t(11;14) and t(6;14) can belong to either the CD-1 or CD-2 group, depending on expression of CD20 and other B-cell-related genes. This is consistent with the finding that t(11;14) and t(6;14) have very similar expression profiles, clinical profiles and outcome. In contrast to the TC classification, the UAMS classification identifies HRD MM as a distinct HY group. However, this may be somewhat misleading, since the HY group, which is about 28% of MM tumors (), includes only about 60% of HRD tumors. The distribution of the remaining HRD tumors among the other six groups has not been clarified, although most are probably in the PR group, defined by increased expression of proliferation-related genes, and the LB group, defined by low bone disease and lower expression of genes associated with bone disease in MM such as FRZB and DKK1.82
The PR, MS and MF groups identify patients with poor prognosis. The PR group, containing patients with t(4;14), t(11;14), and HRD patients, identifies the patients within these categories with more proliferative disease associated with poorer outcome.
Concordance between the translocation and cyclin D (TC) classification and the UAMS (University of Arkansas for Medical Science) molecular classification in non-contaminated samples.
The advantage of the UAMS molecular classification is that it is clinically relevant. It identifies the main genetic subtypes and other clinically relevant subtypes such as the high-risk PR subgroup and the CD20-expressing CD-2 group. It is also interesting that an unsupervised analysis of GEP data essentially identifies the main genetic subtypes of MM, suggesting that the predominant transcriptional heterogeneity seen within MM are driven by these pivotal primary genetic events and/or by progression events such as proliferation in the PR group. One of the deficiencies of this classification is that samples with white-cell and normal plasma-cell contamination were excluded from the classification, as the expression signatures from these contaminating cells may affect assignment to the different groups. Furthermore, classification is based on composite expression of large sets of genes. It is therefore uncertain how this can be applied clinically.
Comparison of TC and UAMS molecular classifications
There is significant overlap between the TC classification and the UAMS molecular classification (). When we compared TC classification of the 414 MM cases assigned a UAMS molecular classification in the original dataset, the MS and MF group correspond to the 4p16 and Maf groups respectively with 100% concordance. The CD-1 and CD-2 groups together correspond to the 11q13 and 6p21 groups (88% concordance). The HY group contains most of the TC D1 and D1&D2 cases (96% concordance). The bulk of the TC D2 cases (67%) fall within the LB group. The PR group contains a mix of patients from the different TC class: 13% 4p16, 4% Maf, 9% 11q13, 21% D1, 19% D1+D2, 28% D2 and 6% None. The PR group is therefore enriched for CCND2-expressing tumors, consistent with the hypothesis that these tumors are more aggressive.
In the original paper, this molecular classification was only applied to non-contaminated samples. We applied the same gene classifier for each molecular subgroup, using prediction analysis for microarrays (PAM analysis), to 145 samples with myeloid contamination in the same UAMS dataset (). The concordance between 4p16 and MS, Maf and MF, D1 or D1&D2 and HY was still excellent. However, in these contaminated samples, the CD-1 and CD-2 groups correspond poorly with 11q13 and 6p21 cases (). It would appear that the TC classification may be more reliable in contaminated samples, especially in identifying the samples with t(6;14) and t(11;14). It is also clear that the distribution of the molecular subtypes by either classification is skewed, with marked under-representation of the tumors with translocations (MF/Maf, MS/4p16, CD-1+CD-2/6p21+11q13) (Tables and ). The reason for this is unclear, but it probably explains the significantly better prognosis of patients from whom the contaminated samples are obtained.42
Concordance between the translocation and cyclin D (TC) classification and the UAMS (University of Arkansas for Medical Science) molecular classification in contaminated samples.
Comparison of concordance within subgroups between contaminated and non-contaminated samples.
Recently, Zhan and colleagues identified an MGUS signature.14
When applied to a large dataset of MM patients, about 30% of MM tumors also express this MGUS signature, which they termed MGUS-L MM. They found that MGUS-L-MM has more benign clinical features and, despite lower complete response to treatment, has better survival. Interestingly, in the two datasets examined, MGUS-L MM is enriched for the CD-1 subtypes and was never seen in the PR subtypes. There were, however, some inconsistencies, as there is enrichment for the HY group in one dataset but not the other.14
It is often difficult to extricate the tumor-cell gene expression signature from contaminating non-malignant cells in the study of minimal plasmacytosis states such as MGUS, even after cell purification. Although the authors try to explain their results in the context of these technical limitations, it is unclear how they define and exclude the effect of contamination. The better survival of patients with MGUS-L MM adds credence to their biological relevance. However, in their survival analysis, the authors have included contaminated samples, and when we re-analyzed the data with these samples excluded, MGUS-L MM no longer has better survival. In a separate analysis of the dataset, we also found that the MGUS-L MM is enriched for cases over-expressing the gene expression signature of normal plasma cells (e.g. polyclonal immunoglobulin genes). In addition, when we applied the MGUS signature to a cohort of MM patients from the Mayo Clinic with high purity of clonal cells (and no signature of normal plasma cells), none of them express the signature (Chng and Bergsagel, unpublished). Therefore, while compelling, the existence of MGUS-L MM as a biological entity is unclear.
Classification by aCGH
When the abnormalities detected by aCGH were used to cluster MM, two groups of patients corresponding to HRD and NHRD MM could be identified. In addition, HRD MM could be further divided into two groups with significantly different event-free survival but not overall survival (when treated with total therapy II). The main differences between these groups are the enrichment of 1q gain, and chromosome 13 loss in the group with poorer prognosis, and the enrichment of chromosome 11 gain in the group with better prognosis. The NHRD MM can also be split into two groups based on chromosome 1, 8 and 16q abnormalities. However, the subgroups of NHRD MM have similar survival.83
These observations require further validation, as the sample size is small and follow-up relatively short. The importance of chromosome 13 deletion in survival of hyperdiploid patients is inconsistent with the results of two large series.43,84
The poor prognostic impact of chromosome 1q gain is well established and is probably mediating most of the difference in survival. The classification of MM by aCGH therefore recapitulates previous FISH findings and does not provide additional prognostic information.