Deletions of CDKN2C in multiple myeloma: biological and clinical implications

Abstract

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.

Figure 1

Deletions of 1p as determined by SNP array. (A) Copy number (CN) is shown in the grey panel to the right of the ideogram, where the line indicates the CN at the point on the chromosome. Homozygous deletion is observed in 1p32.3 in three cases (27, 66 and 71), hemizygous deletion is shown in case 29, and a sample without deletion of 1p32.3 in case 32. (B) The positions of deletions in individual samples are shown as black vertical lines to the right of the ideogram. The green arrow at 1p32.3 indicates the location of CDKN2C and of BAC RP11-278J17. The red arrow at 1p12 indicates the location of BAC RP11-418J17. Cases 27, 66 and 71 had homozygous deletion (blue line) and case 19 had UPD of 1p. (C) FISH of chromosome 1p. An example of normal FISH signal for BACs at 1p32.3 and 1p12 (case 22) (left) and an example of homozygous deletion at 1p32.3 with normal signal at 1p12 (case 27) (right). (D) Deletion of 1p32.3 (CDKN2C) in the model of myeloma pathogenesis. MGUS, monoclonal gammopathy of undetermined significance; SMM, smoldering multiple myeloma; MM, multiple myeloma.

Figure 1

Deletions of 1p as determined by SNP array. (A) Copy number (CN) is shown in the grey panel to the right of the ideogram, where the line indicates the CN at the point on the chromosome. Homozygous deletion is observed in 1p32.3 in three cases (27, 66 and 71), hemizygous deletion is shown in case 29, and a sample without deletion of 1p32.3 in case 32. (B) The positions of deletions in individual samples are shown as black vertical lines to the right of the ideogram. The green arrow at 1p32.3 indicates the location of CDKN2C and of BAC RP11-278J17. The red arrow at 1p12 indicates the location of BAC RP11-418J17. Cases 27, 66 and 71 had homozygous deletion (blue line) and case 19 had UPD of 1p. (C) FISH of chromosome 1p. An example of normal FISH signal for BACs at 1p32.3 and 1p12 (case 22) (left) and an example of homozygous deletion at 1p32.3 with normal signal at 1p12 (case 27) (right). (D) Deletion of 1p32.3 (CDKN2C) in the model of myeloma pathogenesis. MGUS, monoclonal gammopathy of undetermined significance; SMM, smoldering multiple myeloma; MM, multiple myeloma.

Figure 2

Effect of status of CDKN2C on overall survival. Kaplan-Meier estimates of overall survival in cases with alterations of CDKN2C versus cases without alterations of this gene (P = 0.003).

Figure 3

Distribution of the level of expression CDKN2C. Sample expression data are expressed as a percentage of the highest value. The level of expression of CDKN2C was divided into quartiles, 0<2%, 2<4%, 4<10% and 30<100% (y-axis). Cases are grouped as homozygous (hatching), hemizygous (black) and intact CDKN2C (white). Cases with homozygous deletion (A) and those cases with similar expression levels but with hemizygous deletion (B) or intact CDKN2C (C). One case with hemizygous deletion and five cases without alterations of 1p32.3 showed the highest level (30-100%) of expression CDKN2C (D).