British Journal oJ Haematobgg, 1995. 91. 865-870

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

MARTIN SCHRODER,' ULRIKE MATHIEU,4 MARTIN H. DREYLING,2 STEFAN K. BOHLANDER,2 ANNE H A G E M E I ~ E R ,

BERNA H. BEVERLOO, OLUFUNMILAYO I. o L O P A D E , 2 STEPHAN S T I L G E N B A U E R , ' KONSTANZE FISCHER, ' MARTIN B E N T Z , PETER L I C H T E R ~ AND HARTMUT DOHNER' 'Medizinische Klinik und Poliklinik V, Universitut Heidelberg, Heidelberg, Germany; 2Section of HernatologylOncology, Department of Medicine, University of Chicago, Chicago, Illinois, U.S.A.; 3Department of Cell Biology and Genetics, Erasmus Universiteit, Rotterdam, The Netherlands; 4Abteilung Organisation komplexer Genome, Deutsches Krebsforschungszentrum, Heidelberg, Germany

Received 8 May 1995; accepted for publication 25 July 1995

Summary. Homozygous deletions of the cyclin-dependent kinase 4 (CDK4) inhibitor gene CDKN2 (p16, MTS1) have been demonstrated to occur frequently in human cancer cell lines of different origin. However, in most primary tumours the kequencies of CDKN2 deletions are not well defined. We studied primary samples of 100 patients with lymphoid leukaemias [B-lineage acute lymphoblastic leukaemia (ALL), n = 2 3 : T-ALL, n = 7; B-cell chronic lymphocytic (B-CLL) or prolymphocytic (B-PLL) leukaemia, n = 50; T-CLLIT-PLL, n = 201 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 (homozy- gous, n = 4; hemizygous, n = 2). We conclude that hemi- zygous 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.

Keywords: CDKN2 (p16; MTSl), fluorescence in situ hybridization, chronic Iymphocytic leukaemia, prolympho- cytic leukaemia, acute lymphoblastic leukaemia.

Links between cell-cycle control and malignant transforma- tion have been determined over the past few years. Moving cells through the cell cycle, the cyclin-dependent kinases (CDKs), together with their cofactors. the cyclins, play an important role in the regulation of eukaryotic cell prolifera- tion (for review see Hartwell & Kastan. 1994). At a cell cycle checkpoint, cyclin D1/CDK4 triggers G1/S transition via phosphorylation of the Rb protein, pRb. Subsequently, phosphorylated pRb can no longer bind and repress the transcription factor E2F which mediates the transcription of several genes involved in the control of cell proliferation.

Correspondence: Dr Hartmut Dohner, Medizinische Klinik und Poliklinik V, Universitat Heidelberg, Hospitalstr. 3, 691 15 Heidel- berg, Germany.

CDK4 is either part of a quaternary complex containing a protein of M, 16K named p l6 , or associates separately with p16 in a binary complex. Studying the catalytic activity of cyclin Dl/CDK4 to phosphorylate pRb in vitro, p16 was identified as a specific inhibitor of CDK4 (Serrano et al, 1993). Due to this regulatory role in the cell cycle, it was suggested that p16 had tumour suppressor function. In multiple human cancers, chromosome band 9p21 is involved in deletions (Diaz et al, 1988: Olopade et al, 1992). Screening a large series of melanoma cell lines with sequence-tagged sites (STSs) for deletions of 9 ~ 2 1 , a small consensus region of < 40 kb was localized (Weaver-Feld- haus et al. 1994). By DNA sequencing, a region that was identical with the previously described gene (Serrano et al, 1993) encoding the CDK4 inhibitor p16 was identified

0 1995 Blackwell Science Ltd 865

866 Martin Schroder et a1 (Kamb et al, 1994). Originally named multiple tumour suppressor 1 (MTSl), the official symbol for this gene is now CDKN2 (cyclin-dependent kinase inhibitor 2). Analysis of 290 cell lines from 12 different types of tumours with CDKN2-specific STSs revealed a high frequency of homo- zygous deletions of CDKN2 (46%) (Kamb et al, 1994). Of melanoma cell l i e s with hemizygous CDKN2 deletions, 41% had a mutant second allele of the gene. However, the frequencies of CDKN2 abnormalities in primary tumour samples are not well defined. Some groups demonstrated frequent involvement of the gene (Caldas et al, 1994; Mori et al, 1994), whereas others observed only few cases of CDKN2 mutations (Cairns et al, 1994; Okamoto et al, 1994; Spruck et al, 1994). This controversy remains unresolved. Fluores- cence in situ hybridization (FISH) has been shown to be a very sensitive method for the detection of gene deletions in leukaemias (Rowley et al, 1990; Stilgenbauer et al, 1993; Dohner et al, 1994, 1995). Compared to Southern blot analysis or PCR amplification, FISH enables the identifica- tion of a gene deletion on the single cell level and also the distinguishing between hemizygous and homozygous dele- tions. Moreover, the detection of a gene deletion in a subpopulation of cells is possible. To determine the frequency of CDKN2 deletions in haematological malignancies, we studied primary samples of 100 patients with acute and chronic lymphoid leukaemias with a cosmid contig probe encompassing the tumour suppressor region on 9p2 1.

PATIENTS AND METHODS

Patient samples. Five probands and 100 patients with acute and chronic lymphoid leukaemias were analysed. The leukaemias were classified as follows: pre-preB acute lymphoblastic leukemia (ALL) (CD19+. CDlO-), n = 7: common ALL (CD19+, CDlO'), n = 14; pre-B ALL (CDlY', cytoplasmic Ig'), n = 2; the ages of the patients with &lineage ALL ranged from 3 months to 71 years (median 36 years); of the 7 T-ALL, five (nos. 24, 25. 27, 28 and 29) were cortical T-ALL (cytoplasmic CD3+, CDl'), one (no. 30) was a mature T-ALL (surface CD3', TCRyG'), and one (no. 26) had a heterogenous phenotype expressing both cytoplasmic and surface CD3: ages 5-57 years (median 31 years); B-cell chronic lymphocytic leukaemia (B-CLL), n = 48, ages 39-81 years (median 60 years): B-cell prolymphocytic leukaemia (B-PLL), n = 2, ages 65 and 62 years: T-CLL, n = 5, ages 53-71 years (median 62 years): T- PLL, n = 15, ages 45-79 years (median 62 years). For FISH studies, mononuclear cells were obtained from blood ( n = 69), bone marrow (n = 9), lymph node ( n = 5) or splenic tissue (n = 2). In the childhood ALL ( n = 15), total nuclear cells from bone marrow were used. In the ALL cases the blast counts in the specimens ranged from 45% to 98% (median 71.5%). Cells were exposed to hypotonic treatment ( 0 . 0 7 5 ~ KCl, 16min at 37°C) and fixed with methanol/ acetic acid. Chromosome banding analysis was performed according to standard methods.

Fluorescence in situ hgbridization. For FISH experiments a pool of eight overlapping cosmid clones ('COS pl6 ' ) covering approximately 250kb of the CDKN2 region on 9p21 were

used (Dreyling et al, 1995). This cosmid contig identified all CDKN2 deletions that had been detected by molecular genetic methods in series of 10 cell l i e s (data not shown). As a positive control for the hybridization efficiency, the yeast artificial chromosome (YAC) clone 9 77E10 (1470 kb: CEPH YAC-library), mapping to 6q2 7, was used. In one case the YAC clone 253F8 (450kb: Fujita et al, 1992). mapping to 9q13 was used to identify a derivative chromosome 9.

The DNA was amplified and labelled using the recently described PCR technique of sequence-independent amplifica- tion (Bohlander et al, 1994) with incorporation of biotin-16- dUTP or digoxigenin-1 1-dUTP (Boehringer Mannheim. Germany). FISH was performed as described (Stilgenbauer et al, 1993). To prevent false-positive results caused by inefficient hybridization or chromatin loss, analysis was only performed on slides with high hybridization efficiency, indicated by two 9 77E10 signals in > 90% of the nuclei. Fluorescence signals were enumerated in 200 interphase nuclei using single band-pass filters. Dualcolour images were taken using a cooled charge coupled device (CCD) camera (Photometrics Ltd, Tucson, U S A . ) . To define the cut- off level for the diagnosis of a CDKN2 deletion, hybridization experiments were performed on blood samples from five probands using the same combination of DNA-probes.

RESULTS

Controls Hybridization of COS p16 and YAC 977E10 (as an internal control) to metaphase cells of five probands resulted in strong hybridization signals on the respective target regions (COS p16 on chromosome band 9p21, YAC 977310 on band 6q27). By analogy to previous FISH studies (Stilgenbauer et al, 1993; Dohner et al, 1995), the cut-off level for the diagnosis of a CDKN2 deletion was defined by the mean + 3 standard deviations (SD) of the frequency of interphase nuclei exhibiting only one COS p16 signal (mean 3.9%; SD 2.9%; cut-off level 12.6%).

B-lineage and T-ALL Of the 23 patients with B-lineage ALL, one patient (no. 21) exhibited a CDKN2 deletion: two clones were detected, one exhibited a hemizygous (53.6% nuclei with one COS p l6 signal), and the other a homozygous deletion (25.8'26 nuclei with no COS p16 signal). Data from chromosome banding analysis are available for 20/23 patients with B-lineage ALL. Patient 21 with the CDKN2 deletion had no 9p aberration. Cytogenetic changes of 9p were seen in four B-lineage ALL patients but they did not result in loss of FISH signal for CDKN2. Patient 1 4 had a balanced t(9;11)(p21;q23) and patient 18 had a translocation of 9p on a derivative chromosome 12 der(l2). A de1(9)(p21) was seen as additional chromosome in patient 15 with a hyperdiploid karyotype and also in a tetraploid clone of patient 20. The vast majority of cells exhibited three and four COS p16 fluorescent signals, respectively, indicating that there was no gene deletion in the hyperdiploid and tetraploid clone.

In contrast, six of the seven (86%) patients with T-ALL exhibited a CDKN2 deletion: four patients had a

0 1995 Blackwell Science Ltd, British Journal of Haematology 91: 865-870

CDKN2 Gene Deletion in Lymphoid Leukaemia 8 6 7 Table 1. Data on hemi- and homozygous CDKN2 deletion in 30 patients with B- or T-lineage ALL as detected by fluorescence in situ hybridization with the COS p l b probe.

COS p16 signals

None One Patient Age Immunophenotype (%) Banding analysis: 9p aberration

1 2 3 4 5 6 7 8 9

10 11 12 13 14 1 sa 16 17 18 19

21 22 23 24 25 26 27 28 29 30

2Ob

22Y 19Y 2 8 ~ 58Y 29Y 58Y 44Y 2 7~ 7 1 ~ 5 5Y 5 7Y 22Y

3m 4Y 2Y 2Y

1OY

2Y 3Y

28Y 2Y

29Y 35Y

5Y 12Y 5Y 5 7Y

1 3 ~

1 4 ~

2 9 ~

Pre-pre-B ALL Pre-pre-B ALL Pre-pre-B ALL Pre-pre-B ALL Pre-pre-B ALL Pre-pre-B ALL Pre-pre-B ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Common ALL Pre-B ALL Pre-B ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL

3.5 2.5 0.5 2.5 1.5 2.5 0.5 1.5 2.5 1.5 1.0 1.0 1.7 0.0 0.5 0.0 1.2 2.5 0.4 3.4

2 5.8 0.5 1.6 0.5

74- 5 96.0 87.3 9 5.0 4.5 0 4

1.0 1.0 2.0 3.5 3.0 1.5 3.5 2.5 2.5 4.0 2.5 2.5 8.7 7.1 0.5 2.0 7 4 2.8 5.0 2.5

53.6 3 .0 5.0 2.5 1.0 0.9 0.4 1.8

63.2 844

No 9p aberration No 9p aberration No metaphases No 9p aberration No 9p aberration No Yp aberration No 9p aberration No metaphases ND ND No 9p aberration ND No 9p aberration

+ del(9) (p2lorp22) in a hyperdiploid clone No 9p aberration No 9p aberration der (9) t (9:4;13) (pl l;q31-?:ql3),der (12)t(9:12) (pl1:pl l ) No 9p aberration del(9) (p21) x2 in a tetraploid clone No 9p aberration No 9p aberration No 9p aberration ND ND Monosomy 9 No 9p aberration No 9p aberration de1W ( ~ 2 1 ) dic(9;12) (p12;p12)

t(9;l l) (p22: q23)

Bold =percentages of nuclei exceeding the cut-off level (12.6%): y = years: m =months: ND = not done; apercentage of nuclei with 2 and 3 COS p16 signals: 21.5% and 76.1%: bpercentage of nuclei with 3 and 4 COS p16 signals of 200 nuclei with four 9?'7B10 signals: 1.5% and 94.5?/0.

homozygous, and two patients had a hemizygous CDKNZ deletion. Chromosome banding analysis was performed in five of these seven cases. In two patients, cytogenetic loss of 9p correlated with hemizygous deletion of CDKN2, patient 29 with de1(9)(p21) and patient 30 with a dicentric chromosome, dic(9;12)(p12;p12). In the three other cases (nos. 26.27 and 28). homozygous deletion of CDKN2 did not correspond to visible cytogenetic changes of chromosome 9p using banding techniques. The percentages of interphase nuclei with one COS p16 signal (cases with hemizygous CDKNZ deletion) or no COS p16 signal (cases with homozygous CDKN2 deletion) are summarized in Table I. Examples of in situ hybridization images are given in Fig 1.

B-CLLIB-PLL and T-CLL/T-PLL We did not observe any hemi- or homozygous CDKN2 deletion in 50 patients with B-CLL/B-PLL and 20 patients

Table 11. Frequency of CDKN2 dektions in 100 patients with lymphoid leukaemias detected by fluorescence in situ hybridization (FISH) with the COS p16 probe.

CDKN2 deletion

Patient group Homozygous Hemizygous

B-CLL/PLL (n = 50) 0/50 0/50 T-CLL/PLL (n = 20) 0120 0.20 B-lineage ALL (n = 23) 1*/23 1*/23 T-ALL (n = 7) 4/7 (571%) 217 (28*h0h)

*One patient (no. 21) exhibited two clones, one with a hemizygous and the other with a homozygous CDKN2 deletion.

8 1995 Blackwell Science Ltd, British loitrnal of Huematology 91: 865-870

868 Martin Schroder et a1

Fig 1. Analysis of the CDKN2 gene by fluorescence in situ hybridization (FISH). (a) Metaphase cell and two interphase nuclei of patient 3 0 with T- cell acute lymphoblastic leukaemia (T-ALL) and a hemizygous CDKN2 gene deletion. Note the two control fluorescent signals (977E10: Cy-3) but only one COS p16 signal (FITC) in the interphase nuclei and in the metaphase cell. The two FITC signals represent the two chromatids of one CDKN2 allele (arrowhead). (b) Interphase nuclei of patient 2 7 with T-ALL and a homozygous CDKNZ gene deletion. In one interphase nucleus both CDKN2 alleles are retained (detected with FITC). Four nuclei exhibit loss of both CDKN2 alleles: only the two control fluorescent signals (977310; Cy-3) are detected.

with T-CLL/T-PLL (Table 11). The percentage of interphase nuclei with two COS p16 signals ranged between 89.0% and 98.5% (mean 94.1%; SD 2.2%). 25/70 patients were analysed by chromosome banding: 20 of these patients had clonal chromosome abnormalities. In one patient with T-PLL an unbalanced translocation involving band 9pl3 was identified. However, two COS p16 signals were detected in 96.7% of interphase nuclei. Dual-colour hybridization to metaphase preparations with COS p16 and YAC 253F8 mapping to 9q13 showed that the second CDKN2 allele was retained on the derivative chromosome 9. Thus, the break- point on 9p was most likely telomeric to CDKN2. Table I1 summarizes the frequencies of CDKN2 deletions in the 100 patients with lymphoid leukaemias.

DISCUSSION

After the initial reports on frequent losses or mutations of the putative tumour suppressor gene CDKN2 in human cancer cell lines (Kamb et al, 1994; Nobori et al. 1994) a controversy arose whether these changes were involved in the development of the cancers or whether they arose during the establishment of the cell l i e s . Alternatively, cells bearing a CDKN2 deletion could have a selective growth advantage in culture. In primary tumour samples CDKN2 mutations were detected in oesophageal squamous cell carcinoma (52%; Mori et al, 1994), pancreatic adenocarci- noma (38%; Caldas et al, 1994) and non-small cell lung cancer (30%; Hayashi et al, 1994). In contrast, Cairns et a1

a 1995 Blackwell Science Ltd, British Journal of Haematology 91: 865-870

CDKN2 Gene Deletion in Lymphoid Leukaemia 869 (1994) identified CDKN2 mutations in only two of 75 primary tumour samples of different origin that exhibited loss of hekrozygocity (LOH) at 9p21. Okamoto et al(1994) found only one CDKN2 mutation in 2 5 primary tumour samples from oesophageal (n = 12), lung (n = 7) and liver (n = 6) carcinomas.

At present, little data is available on CDKN2 abnormalities in haematological malignancies. We analysed primary tumour samples from 100 patients with acute and chronic lymphoid leukaemias by FISH.

B-lineage and T-ALL We demonstrate that CDKN2 deletions occur at high frequency in T-ALL and at low frequency in B-lineage ALL; 617 (86%) patients with T-ALL and 1/23 (4%) patients with B-lineage ALL had either a homozygous or a hemizygous CDKN2 deletion. The data are difficult to compare with recently published series because of the different techniques applied. Given the low frequency of intragenic CDKN2 mutations in ALL (Quesnel et al, 1995), CDKN2 inactivation seems to occur primarily via a deletion in this disease. However, not all cases exhibit homozygous CDKN2 deletion. Since even the presence of only one allele may be insufficient for a normal phenotype, the identification of hemizygous CDKN2 deletions is important. In contrast to quantitative Southern blot analysis, FISH enables the unambiguous diagnosis of hemizygous gene deletions. In our study, 2/30 patients with ALL exhibited a hemizygous CDKN2 deletion. In addition, in one case (patient 21) we identified two clones: one exhibited a hemizygous and the other a homozygous CDKN2 deletion indicating clonal evolution in a subpopula- tion of the leukaemic cells. By Southern blot analysis, Hebert et al(1994) found homozygous CDKN2 deletions in 20124 (83%) patients with T-lineage ALL and in 2/31 (7%) patients with B-lineage ALL. In an additional six cases, possible hemizygous deletions were detected. In contrast, Quesnel et al (1995) reported homozygous CDKN2 deletions in only 3/ 12 (2 5%) T-lineage ALL and in 6/39 (1 5%) B-lineage ALL as detected by Southern blot analysis. They concluded that homozygous CDKN2 deletion may not be associated with a specific immunophenotype in ALL. Regarding the Blineage ALL. differences in the frequencies of CDKN2 deletions may reflect differences in patient selection. In our series the majority of patients had a hyperdiploid karyotype, whereas Quesnel et a2 (1995) had a majority of patients with pseudodiploid karyotypes. Interestingly, in another study, homozygous CDKN2 deletions were detected in 11/53 (20%) &lineage ALL but in none of 11 T-lineage ALL by Southern blot analysis (Stranks et al, 1995).

In our study a discrepancy in the data obtained from chromosome banding analysis was noted in seven cases. In four cases homo- or hemizygous CDKN2 deletions were detected by FISH, whereas no 9p aberration was found by banding analysis. The discrepancies indicate that in these cases the deletions occurred at the submicroscopic level. Interestingly, in three Rlineage ALL cases banding analysis revealed a deletion or unbalanced translocation of 9p, whereas there was no CDKN2 deletion by -FISH. The most likely explanation is that the breakpoints on 9p21 are distal

to the COS p16 target sequences or that 9p material is not lost but masked in a complex karyotype. Also the 250 kb genomic DNA probe may miss small deletions at the CDKN2 locus. Moreover, another locus on 9p different from CDKN2 may be important in the tumorigenesis of some cases of ALL. Indeed, there is evidence suggesting another candidate tumour suppressor gene in band 9p21 (Cairns et aI, 1994; Stranks et al, 1995). In a recent study Chaganti et al(1995) demonstrated the absence of CDKN2 deletions in diffuse large-cell lymphomas with LOH for various loci at 9p21 and suggested the presence of one or more tumour suppressor genes different from CDKN2 in this region.

B-CLL/B-PLL and T-CLLIT-PLL No CDKN2 deletion was identified in the 70 cases with chronic lymphoid leukaemias of B- or T-cell origin. This was consistent with the data obtained from chromosome banding in our patients where no 9p aberrations were described except in one patient with a T-PLL. In this case an unbalanced translocation involving 9p was found. However, both CDKN2 copies were retained on FISH analysis. By analogy to the three ALL cases described above, another locus on 9p may be involved in this case of T-PLL. The absence of CDKN2 deletions in these types of leukaemia is in concordance with recent reports (Quesnel et aI, 1995; Stranks et al, 1995). Alternative mechanisms of cell-cycle dysregulation, such as loss of Rb-l (Stilgenbauer et al, 1993; Dohner et aI, 1994) or loss of TP53 (Dohner et al, 1995), seem to be important in the pathogenesis of chronic lymphoid leukaemias.

ACKNOWLEDGMENTS

We gratefully acknowledge Dr Patricia Bray-Ward and Dr David C. Ward for providing us with YAC clone 977E10, and Dr Michel Koenig for providing us with YAC 253F8.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Do 436/3-2), the Tumorzentrum Heidelberg/Mannheim (Forschungsschwerpunkt 1/1. l), the European Community (GENECT 930055). and the J. S. McDonnell Foundation.

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