Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma

Abstract

Two oncogenic pathways have been hypothesized for multiple myeloma (MM) and premalignant monoclonal gammopathy of undetermined significance (MGUS) tumors: a nonhyperdiploid pathway associated with a high prevalence of IgH translocations and a hyperdiploid pathway associated with multiple trisomies of 8 chromosomes. Cyclin D1, D2, or D3 expression appears to be increased and/or dysregulated in virtually all MM tumors despite their low proliferative capacity. Translocations can directly dysregulate CCND1 (11q13) or CCND3 (6p21), or MAF (16q23) or MAFB (20q11) transcription factors that target CCND2. Biallelic dysregulation of CCND1 occurs in nearly 40% of tumors, most of which are hyperdiploid. Other tumors express increased CCND2, either with or without a t(4;14) translocation. Using gene expression profiling to identify 5 recurrent translocations, specific trisomies, and expression of cyclin D genes, MM tumors can be divided into 8 TC (translocation/cyclin D) groups (11q13, 6p21, 4p16, maf, D1, D1+D2, D2, and none) that appear to be defined by early, and perhaps initiating, oncogenic events. However, despite subsequent progression events, these groups have differing gene expression profiles and also significant differences in the prevalence of bone disease, frequency at relapse, and progression to extramedullary tumor.

Figure 1.

Identification of TC groups in MM by RNA expression profiling. The samples are 6 peripheral blood-generated plasmablasts and 1 reactive plasmacytosis (PB) (5), 14 bone marrow PCs (PC) from healthy volunteers, samples from newly diagnosed (231) or relapsed (30) MM, and 32 human myeloma cell lines (HMCL). In addition they are all samples from patients with newly diagnosed (231) or relapsed MM (30). Among these 261 samples there are 42 with elevated FGFR3 and/or MMSET (4p16), 19 with elevated MAF or ITGB7 and CX3CR1 as surrogates for MAFB (maf), 7 with elevated CCND3 (6p21), 40 with high CCND1 (11q13), 81 with lower levels of CCND1 (D1), 21 with lower levels of CCND1 and elevated CCND2 (D1+D2), 45 remaining patients with elevated CCND2 (D2), and 6 patients without an elevated cyclin D (none). The relative level of expression of each gene in each sample is shown. For cyclin D1 (CCND1), the expression of the long-form mRNA is in dark green and total mRNA is in light green. The fifth line shows a PI calculated from the log of the median of the normalized values for 12 genes associated with proliferation. The values for PB were extrapolated from a HuFL dataset. The bottom line shows a chromosome 3, 5, 7, 9, 11, 15, 19, 21 index (CI) calculated from the median of the normalized values for genes on a given chromosome. The deviation from 1 for the average of 8 chromosomes (3, 5, 7, 9, 11, 15, 19, and 21) is plotted.

Figure 2.

Biallelic expression of CCND1 alleles in D1 group of MM tumors. Paired genomic DNA and cDNA samples were used to PCR amplify 138-bp exon 4-intron 4 or, respectively, 203-bp exon 4-exon 5 DNA fragments that include an A>G polymorphism at the ultimate exon 4 nucleotide. (A) The resultant fragments were digested with HpaII, which generates 92 + 46-bp fragments or, respectively, 161 + 42-bp fragments when a G is present at the polymorphic site. Paired HpaII-digested samples were subjected to electrophoresis on a 4% Nusieve-0.5% agarose gel. Odd lanes contain genomic fragments; subsequent even lanes contain the corresponding cDNA fragment. Lanes 1-2, homozygous A allele; lanes 3-4, homozygous G allele; lanes 5-22, 9 MM tumor samples. The positions of the 203-bp, 161-bp, 138-bp, and 92-bp fragments are indicated at the left. (B) Paired sequence tracings of genomic and cDNA fragments from representative tumors that have an excess of the A allele (dotted lines), the G allele (solid lines), or equal representation of both alleles. The polymorphic A>G nucleotide and the subsequent G nucleotide are shown. Further details in “Patients, materials, and methods.”

Figure 3.

Trisomy of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 in D1 group. (A) Chromosome localization of genes overexpressed in D1 group and in hyperdiploid ALL. For both the hyperdiploid greater than 50 subgroup of ALL and the D1 group of MM, a list of 618 (ALL) or 545 (MM) genes overexpressed within that group, compared with all other patients with ALL or MM, was generated. From these lists, the ratio of the number of observed to expected genes from each chromosome is plotted. (B) Chromosome index in ALL and MM. For each patient, a chromosome index was calculated for each chromosome using the median level of expression of the genes on that chromosome and plotted with chromosome 1 at the top, down to X at the bottom. No index was calculated for chromosome Y. For the MM samples, this chromosome index was normalized to the median value in BMPCs. A color scale on the left indicates red above 1.3, yellow for 1, and green below 0.7. Patients with MM are grouped as described in Figure 1. The patients with ALL are grouped based on the genetics as described in Yeoh et al (TEL, T, normal, MLL, hyperdiploid, EZA, BCR). See also Supplemental Worksheet, data for Figure 3.

Figure 4.

Shared gene expression profiles for TC groups. (A) Two hundred thirty MM samples were categorized into the 8 TC groups. Five hundred seventy-six genes significantly different between the 8 subtypes (P < .05; Benjamin and Hochberg analysis was used to adjust for the multiple univariate hypothesis tests, with multiple testing correction) were identified. The genes were subjected to hierarchical clustering (Supplemental Worksheet, Table S2). A color code on the bottom identifies the color associated with each TC group. (B) Differences in gene expression between TC groups. A list of 576 genes that differs between the 8 TC groups with a P value less than .05 was generated, and the 13 probe sets corresponding to the genes used to assign the TC class (CCND1, CCND2, CCND3, FGFR3, MMSET, MAF, ITGB7, CX3CR1) were removed. The number of genes from this list that are statistically different (P < .05) in pair-wise comparisons between TC groups is shown, with the intensity of pink shading highlighting the more profound differences. 6 indicates 6p21; 11, 11q13; N, none; 4, 4p16; and M, maf.

Figure 5.

Early oncogenic events in MM. The earliest oncogenic changes, which involve 3 overlapping pathways, probably occur in germinal center B cells and appear to be already present in premalignant MGUS tumors. Two partially overlapping pathways, indicated by IgH translocations (tx) and multiple trisomies, usually generate nonhyperdiploid and hyperdiploid tumors, respectively. A third pathway that leads to monosomy of chromosome 13 or deletion of 13q14 can be present in both types of tumors but occurs with a higher prevalence in nonhyperdiploid tumors, where it occurs in almost all tumors with t(4;14) and t(14;16) but infrequently in tumors with t(11;14). The essentially invariant dysregulation of a cyclin D gene appears to be associated with these early oncogenic changes. The identification of 5 recurrent IgH translocations and the dysregulated expression of a cyclin D gene by expression profiling forms the basis for assignment of MGUS and MM tumors into TC groups as depicted.