Martin H. Dreyling

Codeletion of CDKN2 and MTAP genes in a subset of non-Hodgkin's lymphoma may be associated with histologic transformation from low-grade to diffuse large-cell lymphoma

Identifying the various genetic alterations that contribute to lymphomagenesis is key to our improved understanding of the biological behavior of the disease. Recently, we and others have defined a tumor suppressor region on the short arm of chromosome 9 harboring a cluster of genes, including MTAP, CDKN2A(p16INK4a), and CDKN2B(p15INK4B), which is frequently deleted in a variety of tumor types. To determine whether this region is involved in a particular subset of malignant lymphomas, we have examined 16 cases of diffuse large‐cell lymphoma (DLCL) (including three cases that evolved from low‐grade non‐Hodgkin lymphoma (NHL) (transformed DLCL)), and nine cases of low‐grade NHL that had subpopulations of large cells with a diffuse growth pattern (seven follicular NHL, one chronic lymphocytic leukemia, one mycosis fungoides). Interphase fluorescence in situ hybridization was performed on these samples using a 250‐kb cosmid contig (COSp16), which encompasses MTAP, CDKN2A, and CDKN2B. Six of the 16 DLCLs and one of nine low‐grade NHLs had deletions of COSp16. COSp16 was homozygously deleted in four cases; two cases had hemizygous deletions, and one case had a partial homozygous deletion of the cosmid contig. Three of 13 cases of de novo DLCL, all three transformed DLCLs, and one of nine low‐grade NHL had COSp16 deletions. Although the numbers are small, COSp16 deletion was associated with transformed DLCL in contrast to de novo DLCL (P < 0.04, Fishers exact test) or low‐grade NHL (P < 0.02). The COSp16 deletion was mostly submicroscopic and was not observed in association with any specific recurring cytogenetic abnormalities. These results suggest that targeted deletion of the CDKN2A region occurs in a subset of non‐Hodgkins lymphomas, and may be associated with transformed lymphomas. Genes Chromosomes Cancer 22:72–78, 1998.

CDKN2 gene deletion is not found in chronic lymphoid leukaemias of B- and T-cell origin but is frequent in acute lymphoblastic leukaemia

Summary. Homozygous deletions of the cyclin‐dependent kinase 4 (CDK4) inhibitor gene CDKN2 (pi6, MTS1) have been demonstrated to occur frequently in human cancer cell lines of different origin. However, in most primary tumours the frequencies of CDKN2 deletions are not well defined. We studied primary samples of 100 patients with lymphoid leukaemias [B‐lineage acute lymphoblastic leukaemia (ALL), n = 23; T‐ALL, n= 7; B‐cell chronic lymphocytic (B‐CLL) or prolymphocytic (B‐PLL) leukaemia, =50; T‐CLL/T‐PLL, n= 20] using fluorescence in situ hybridization (FISH) with eight overlapping cosmid clones covering the region on chromosome band 9p21 containing CDKN2. We did not observe any CDKN2 deletions in the 70 patients with chronic lymphoid leukaemias of B‐ or T‐cell origin. Of the 23 patients with B‐lineage ALL, one (4%) exhibited a CDKN2 deletion: in this patient, two clones were detected, one exhibiting a hemizygous and the other a homozygous deletion. On chromosome banding analysis, four patients with B‐lineage ALL had a 9p aberration, whereas all CDKN2 copies were retained. In contrast, six of the seven (86%) patients with T‐ALL exhibited CDKN2 deletions (homozygous, n = 4; hemizygous, n = 2). We conclude that hemizygous or homozygous deletions of the CDKN2 gene occur at high frequency in T‐ALL and at low frequency in B‐lineage ALL, supporting the role of this gene as a tumour suppressor, especially in T‐ALL. However, from our data there is no evidence that CDKN2 is involved in the pathogenesis of chronic lymphoid leukaemias of B‐ or T‐cell origin.

Detection of 9p deletions in leukemia cell lines by interphase fluorescence in situ hybridization with YAC-derived probes

Hemizygous and homozygous deletions of the type I interferon gene cluster (IFN) have been detected in about 20% of acute lymphoblastic leukemias. A putative tumor suppressor gene (TSG) is thought to be located centromeric to the IFN cluster on chromosomal bands 9p21-22. We studied the accuracy of fluorescence in situ hybridization (FISH) for detecting deletions in interphase cells using yeast artificial chromosome (YAC) clones containing all or part of the IFN cluster. FISH probes were generated from YACs (320-1300 kb in size) by a sequence-independent amplification technique (SIA). Fifteen cell lines (nine T-ALL, three B-cell precursor ALL, one B-ALL, one AML, one CML-BC) that had been well characterized by conventional cytogenetic analysis and molecular techniques were analyzed. We were able to detect all numerical changes of the IFN cluster including homozygous and hemizygous deletions accurately and to define subclones of the cell lines. Moreover, in six cell lines we were able to identify subclones. In dilution experiments the detection thresholds for subpopulations with homozygous and hemizygous deletions were determined to be 5% and 7.5%, respectively.

CDKN2 Deletions in Leukemia-Derived Cell Lines: Detection by Fluorescence In Situ Hybridization with Interphase Cells

Hemizygous and homozygous deletions of chromosomal band 9p21 have been detected in various tumor types as well as in more than 20% of acute lymphoblastic leukemia. Recently, the CDKN2 gene (pl6INK4A, MTS I, CDK4I) has been proposed as a candidate tumor suppressor gene because it is frequently deleted in cell lines derived from multiple tumor types. We examined 18 leukemia-derived cell lines (13 T-ALL, 3 B-cell-precursor ALL,1 AML,1 CML-blast crisis) by polymerase chain reaction and Southern blot analysis to further define the frequency and size of 9p deletions in hematological malignancies. Homozygous CDKN2 deletions were detected in 16 cell lines (89%). Interphase fluorescence in situ hybridization (FISH) is a powerful method to detect chromosomal rearrangements, including submicroscopic deletions. Using a cosmid contig of the CDKN2 region, we performed interphase FISH in nine cell lines to study the accuracy of this method in detecting 9p deletions. Interphase FISH determined accurately all deletions of the tumor suppressor region previously detected by Southern blot or PCR. In six cell lines the cosmid contig was completely deleted. In one cell line, the intensity of the hybridization signals was significantly reduced, indicating a partial deletion of the hybridization region. One cell line had a hemizygous deletion which is difficult to detect by molecular techniques. Genomic deletions seem to be the predominant mechanism of CDKN2 inactivation in acute leukemias. Interphase FISH will play an important role in defining the frequency of CDKN2 deletions in primary tumors, because it is able reliably to analyze clinical samples that are contaminated by normal cells.