Topics:
b-lymphocytes, chronic b-cell leukemias, genes, protein overexpression, translocation (genetics)

Abnormalities of chromosome 7q have rarely been reported in chronic B-cell lymphoproliferative disorders.1  Chromosomal translocation leading to overexpression of the CDK6 gene, which is located in 7q21, has recently been described in splenic marginal zone lymphoma (SMZL).2,3  CDK6 together with CDK4 are serine/threonine kinases that positively regulate the G1 phase progression in association with D-type cyclin. Overexpression of CDK6 is considered to contribute to leukemogenesis through dysregulation of cell proliferation, thereby favoring progression through the G1 phase. It seems likely that CDK6 overexpression is implicated in lymphomas with low proliferative index and, as we show here, may also be involved in other low-grade malignancies such as B-cell chronic lymphocytic leukemia (B-CLL). We have previously described the molecular characterization of a t(7;14)(q21;q32) observed in a case of B-CLL. The t(7;14) involved the immunoglobulin heavy chain (IgH) locus in 14q32 and a transposon-like element located 29 kbp upstream of the CDK6 coding sequence.4  However, no material was available for CDK6 expression analysis in this case. To analyze CDK6 involvement in B-CLL, we have examined the status of the CDK6 gene in bone marrow or blood samples from a further 8 adult patients with B-CLL carrying cytogenetic abnormalities (deletions, duplications, or translocations) involving bands 7q21 to 7q22 (1.5% of our B-CLL series).

B-CLL diagnosis was established according to the World Health Organization Classification of Tumours and was characterized by positivity for CD5 and CD23 expression. All the tumor samples were screened, where possible, by Southern blot, fluorescence in situ hybridization (FISH), reverse-transcriptase real-time quantitative polymerase chain reaction (RT-RQ-PCR), and immunoblotting. In 3 patients, involvement of the CDK6 gene was found by at least 2 different methods. The relevant clinical, biologic, and molecular data for these patients are given in Table 1. FISH and Southern blot analysis showed that an interesting common feature was that in all 3 cases the CDK6 gene was rearranged by reciprocal translocations involving either Igκ, Igλ, or IgH locus and not with only the Igκ light chain locus as has previously been reported in other series.2,3 

Table 1.

Clinical and molecular characteristics of B-CLL patients with the CDK6 rearrangement

Overall survival (y of follow-up)
FISH analysis
SB analysis
Patient
Sex/age at diagnosis, y
Karyotype
CDK6
Ig
1   M/61   Alive (6)   46,XY,t(7;22)(q22;q11), del(13)(q13q14)[12]/46,XY[9]   Split   Igλ split   R  
2   M/79   Alive (3)   46,X,-Y,+12[1]/46,X,-Y,+12, t(7;14)(q21;q32)[16]/46,XY[3]   Split   IgH split   NA  
3
 		 M/61
 		 Died (16)
 		 45,XY,t(2;7)(p11-12;q22),der(8) t(8;17)(p12;p11), -17[5]/47,XY, t(2;7),+5,+12[10]/46,XY[35]
 		 Split
 		 NA
 		 R
 
Overall survival (y of follow-up)
FISH analysis
SB analysis
Patient
Sex/age at diagnosis, y
Karyotype
CDK6
Ig
1   M/61   Alive (6)   46,XY,t(7;22)(q22;q11), del(13)(q13q14)[12]/46,XY[9]   Split   Igλ split   R  
2   M/79   Alive (3)   46,X,-Y,+12[1]/46,X,-Y,+12, t(7;14)(q21;q32)[16]/46,XY[3]   Split   IgH split   NA  
3
 		 M/61
 		 Died (16)
 		 45,XY,t(2;7)(p11-12;q22),der(8) t(8;17)(p12;p11), -17[5]/47,XY, t(2;7),+5,+12[10]/46,XY[35]
 		 Split
 		 NA
 		 R
 

FISH analysis was sequentially performed on the same mitosis with mixed Human BAC Clone GS119p5 and Clone GSI-552A1 (encompassing intron 1, exon 1, and 178 kbp upstream of the coding sequence of the CDK6 gene; Incyte Genomics, San Francisco, CA) and with IgH probes (Vysis, Downers Grove, IL) or Igλ probes (kindly provided by Reiner Siebert). The CDK6 gene was split between chromosomes 7 and 22 in FIO, between chromosomes 7 and 14 in DUP, and between chromosomes 2 and 7 in LAN. Igλ and IgH involvement was further demonstrated in patients 1 and 2, respectively. It may be noticed that the CDK6 GS119p5 probe gave a slight cross-hybridization with 3q chromosome. Southern blot analyses were performed with WS2 probe4  and with CDK6 PCR-amplified cDNA probes: E1 (nucleotides 12 to 298; GenBank accession no. X66365), p123, and p45 (nucleotides 446 to 860 and 61817 to 62434, respectively; GenBank accession no. AC002454). Restriction mapping allowed us to locate the breakpoint in patients 1 and 3 inside the breakpoint cluster region previously described within a 3.6 kbp upstream of the transcriptional start site of CDK6.2  As no mitosis was available for FISH analysis in LAN, Igκ light chain locus involvement was confirmed by Southern blot analysis showing the cohybridization of the CDK6 rearranged band with the Igκ probe (Jκ).

SB indicates Southern blot; R, rearranged; and NA, not available.

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Marked overexpression of CDK6 was observed in all 3 patients with CDK6 rearrangement compared with normal peripheral blood lymphocytes and with B-CLL without CDK6 involvement (Figure 1). This indicates that FISH and CDK6 expression analysis can be successfully used to identify patients with CDK6 involvement. As previously reported,2  CDK6 overexpression can result from aberrant variable-diversity-joining (VDJ) or variable-joining (VJ) recombination, leading to the juxtaposition of the CDK6 gene with the Ig gene enhancer during B-cell differentiation. In addition, our findings demonstrate the involvement of the CDK6 gene in B-CLL, underlying that those abnormalities are not restricted to SMZL.

Figure 1.
Figure 1. Overexpression of CDK6. (A) Quantitative real-time PCR was performed using the LightCycler system with FastStart DNA Master SYBR Green I kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Primers from the CDK6 gene are as follows: forward (5′-AGAAGAAGACTGGCCTAGAG) and reverse (5′-TGGAAGTATGGGTGAGACAGG). Primers from the β-2-microglobulin (β2-MG) gene used to evaluate correcting target molecule amounts are as follows: forward (5′-CCAGCAGAGAATGGAAAGTC) and reverse (5′-GATGCTGCTTACATGTCTCG). Amplification of specific transcripts was confirmed by a melting curve analysis. For PCR calibration, a serial 10-fold dilution series for each gene (ranging from 106 to 10 copies) was amplified and the assay was found to be linear over at least 5 orders of magnitude (CDK6 slope, –3.47; β2-MG slope, –3.43). The absolute copy amount of each sample was obtained by the mean of the following ratio: ([copy number of the CDK6 gene]/[copy number of β2-MG]) × 100. (B) Total cellular proteins were analyzed by Western blotting using anti-CDK6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The same blot was probed for actin to control for equal protein loading. In all samples, tumor cells represented more than 80% of the nucleated cells. RNA and protein were extracted from bone marrow or blood samples from patients with the CDK6 gene rearrangement (FIO, DUP, LAN), from patients with B-CLL without the CDK6 gene rearrangement (B-CLL1 to B-CLL4), and from normal mononuclear peripheral blood cells (C1 to C5).
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Overexpression of CDK6. (A) Quantitative real-time PCR was performed using the LightCycler system with FastStart DNA Master SYBR Green I kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Primers from the CDK6 gene are as follows: forward (5′-AGAAGAAGACTGGCCTAGAG) and reverse (5′-TGGAAGTATGGGTGAGACAGG). Primers from the β-2-microglobulin (β2-MG) gene used to evaluate correcting target molecule amounts are as follows: forward (5′-CCAGCAGAGAATGGAAAGTC) and reverse (5′-GATGCTGCTTACATGTCTCG). Amplification of specific transcripts was confirmed by a melting curve analysis. For PCR calibration, a serial 10-fold dilution series for each gene (ranging from 106 to 10 copies) was amplified and the assay was found to be linear over at least 5 orders of magnitude (CDK6 slope, –3.47; β2-MG slope, –3.43). The absolute copy amount of each sample was obtained by the mean of the following ratio: ([copy number of the CDK6 gene]/[copy number of β2-MG]) × 100. (B) Total cellular proteins were analyzed by Western blotting using anti-CDK6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The same blot was probed for actin to control for equal protein loading. In all samples, tumor cells represented more than 80% of the nucleated cells. RNA and protein were extracted from bone marrow or blood samples from patients with the CDK6 gene rearrangement (FIO, DUP, LAN), from patients with B-CLL without the CDK6 gene rearrangement (B-CLL1 to B-CLL4), and from normal mononuclear peripheral blood cells (C1 to C5).

Figure 1.
Figure 1. Overexpression of CDK6. (A) Quantitative real-time PCR was performed using the LightCycler system with FastStart DNA Master SYBR Green I kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Primers from the CDK6 gene are as follows: forward (5′-AGAAGAAGACTGGCCTAGAG) and reverse (5′-TGGAAGTATGGGTGAGACAGG). Primers from the β-2-microglobulin (β2-MG) gene used to evaluate correcting target molecule amounts are as follows: forward (5′-CCAGCAGAGAATGGAAAGTC) and reverse (5′-GATGCTGCTTACATGTCTCG). Amplification of specific transcripts was confirmed by a melting curve analysis. For PCR calibration, a serial 10-fold dilution series for each gene (ranging from 106 to 10 copies) was amplified and the assay was found to be linear over at least 5 orders of magnitude (CDK6 slope, –3.47; β2-MG slope, –3.43). The absolute copy amount of each sample was obtained by the mean of the following ratio: ([copy number of the CDK6 gene]/[copy number of β2-MG]) × 100. (B) Total cellular proteins were analyzed by Western blotting using anti-CDK6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The same blot was probed for actin to control for equal protein loading. In all samples, tumor cells represented more than 80% of the nucleated cells. RNA and protein were extracted from bone marrow or blood samples from patients with the CDK6 gene rearrangement (FIO, DUP, LAN), from patients with B-CLL without the CDK6 gene rearrangement (B-CLL1 to B-CLL4), and from normal mononuclear peripheral blood cells (C1 to C5).
View largeDownload PPT

Overexpression of CDK6. (A) Quantitative real-time PCR was performed using the LightCycler system with FastStart DNA Master SYBR Green I kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Primers from the CDK6 gene are as follows: forward (5′-AGAAGAAGACTGGCCTAGAG) and reverse (5′-TGGAAGTATGGGTGAGACAGG). Primers from the β-2-microglobulin (β2-MG) gene used to evaluate correcting target molecule amounts are as follows: forward (5′-CCAGCAGAGAATGGAAAGTC) and reverse (5′-GATGCTGCTTACATGTCTCG). Amplification of specific transcripts was confirmed by a melting curve analysis. For PCR calibration, a serial 10-fold dilution series for each gene (ranging from 106 to 10 copies) was amplified and the assay was found to be linear over at least 5 orders of magnitude (CDK6 slope, –3.47; β2-MG slope, –3.43). The absolute copy amount of each sample was obtained by the mean of the following ratio: ([copy number of the CDK6 gene]/[copy number of β2-MG]) × 100. (B) Total cellular proteins were analyzed by Western blotting using anti-CDK6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The same blot was probed for actin to control for equal protein loading. In all samples, tumor cells represented more than 80% of the nucleated cells. RNA and protein were extracted from bone marrow or blood samples from patients with the CDK6 gene rearrangement (FIO, DUP, LAN), from patients with B-CLL without the CDK6 gene rearrangement (B-CLL1 to B-CLL4), and from normal mononuclear peripheral blood cells (C1 to C5).

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Copyright © 2003 by The American Society of Hematology
2003
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