Academic Department of Haematology and Cytogenetics, Royal Marsden Hospital/Institute of Cancer Research, London/Sutton, UK

Correspondence to: L JA Coignet, at his current address: Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 South First Av, Maywood, IL, 60153, USA; Fax: 708 327 3319

^aCurrent address: Departamento Básico de Medicina, Facultad de Medicina, Hospital de Clinicas, Piso 15, Avda Italia s/n, Montevideo, CP 11600, Uruguay

Deletions of the long arm of chromosomes 11 and 13 are the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders. However, these regions have not been studied so far in B cell prolymphocytic leukemia (B-PLL). We have investigated the incidence of 13q deletions in 18 B-PLL cases by fluorescence in situ hybridization (FISH), using molecular probes for the RB1 and D13S25 loci. Chromosome 11q deletions were evaluated by FISH using the yeast artificial chromosome (YAC) clone 755b11 from the chromosome 11q22.3-q23.1 region, which has been previously shown to be deleted in 20% of cases of chronic lymphocytic leukemia. Chromosome 11q23 deletions were found in 7/18 (39%) cases of B-PLL. Monoallelic loss of RB1, D13S25 and BRCA2 was present in 10/18 (55%), 6/18 (33%) and 3/18 (16%) of the cases, respectively. All the cases with D13S25 and BRCA2 deletion showed RB1 loss. Deletions of 13q14 and 11q23 are frequent chromosome aberrations in B-PLL and, in contrast to CLL, there is a preferential loss of RB1 with respect to the D13S25 locus suggesting that allelic loss of the RB1 gene may play a role in the pathogenesis of B-PLL. Leukemia (2000) 14, 427-430.

B-PLL; FISH; 11q23; 13q14; neoplasia

Introduction

B cell prolymphocytic leukemia (B-PLL) is a distinct leukemia of mature B cells^1,2 characterized by a high white blood cell count with more than 55% of prolymphocytes³ and splenomegaly without significant lymphadenopathy.

The membrane phenotype of the prolymphocyte is quite distinct from that of chronic lymphocytic leukemia (CLL), but has similarities to that of other B cell leukemias. Several chromosome abnormalities have been described, including the t(11;14)(q13;q32), although these are not consistent. Additional abnormalities include trisomy of chromosome 12 and deletions of chromosomes 1, 3 and 6q.⁴ However, little is known about the underlying molecular mechanisms of this rare disease that is frequently associated with resistance to therapy.⁵

Structural abnormalities of the long arm of chromosome 11 (11q) represent a recurrent abnormality in various types of lymphoproliferative disorders.^6,7 Deletions of 11q are the first chromosome abnormalities described in B-CLL, with a common affected region being 11q14-q25.⁸ These abnormalities define a subset of typical CLL patients that show consistent disease progression and reduced survival.⁹ In a recent study, Stilgenbauer et al¹⁰ characterized deletions and translocations involving chromosome bands 11q21-q23. Using a panel of yeast artificial chromosome (YACs) clones organized as a contig, they were able to define a single critical region of 2-3 Mb where all deletions and translocations clustered and that is encompassed by a single YAC clone.

Structural abnormalities of chromosome 13q14 are one of the most frequent cytogenetic changes in lymphoproliferative disorder. It has been observed in 13-25% of the cases in CLL by conventional cytogenetics.¹¹ Molecular analysis and/or interphase cytogenetics have subsequently shown higher proportion of CLL cases with 13q deletions^12,13,14,15 and a recent study on splenic lymphoma with villous lymphocytes (SLVL)¹⁶ demonstrated loss of 13q14 sequences in 50% of the cases investigated.

In the present study, we investigated the frequency of RB1 and D13S25 (13q14) deletions, as well as deletions affecting chromosome band 11q23 as assessed by interphase FISH in a series of 18 B-PLL cases. Where available, conventional cytogenetics was assessed. In addition, we also analyzed the correlation in 11 of our cases with regards to p53 status (ie 17p LOH, P53 mutation and P53 protein).

Materials and methods

Patients

Eighteen cases of B-PLL investigated at our institution between 1990 and 1998 were selected for this study. There were eight females and 10 males, with a median age of 66 years (range, 41-83). Peripheral blood films for morphology analysis and immunologic markers were available in all of the cases studied. Diagnosis was made on the basis of morphologic appearances and detailed immunophenotypic analysis by flow cytometry according to the criteria established by the French-American-British group.¹⁷ All cases were CD19/CD37-positive, CD10 and CD2-negative and expressed strongly surface immunoglobulin, with light chain restriction kappa (13/17) or lambda (4/17), one case was tested only for heavy chain. FMC7 was positive in 13, CD5 in seven and CD23 in five cases.

Peripheral blood samples from five healthy donors were used as normal controls.

Fluorescence in situ hybridization

FISH was performed either on archived material stored in methanol/acetic acid (3:1) or cells were treated with hypotonic solution for 20-40 min at 37°C, re-suspended in methanol/acetic acid (3:1) and stored at -20°C until use.

Probes

For the characterization of chromosome 13q12-14, three probes were used: LSI-RB-1 (containing sequences of the Rb-1 gene) and D13S25 were obtained directly labeled with Spectrum Orange from Vysis (Vysis UK, Richmond, UK). The PAC clone 214k23 (Prof M Stratton, ICR, Sutton, UK) containing BRCA2 was also used. The order of the 13q12-14 probes is as follows: centromere-214k23 (BRCA2)-LSIRB1 - D13S25-telomere. For the study of chromosome 11q, a CEPH Mega-YAC clone, 755b11, encompassing the single critical region of 2-3 Mb where all deletions and translocations clustered¹⁰ was used. A PAC clone specific for the BCR gene on chromosome 22 was used as hybridization quality control (LJAC, unpublished).

FISH

FISH was performed as previously described.¹⁸ The two non-commercial probes were labeled by nick translation with biotin-16-deoxyuridine triphosphate (Boehringer Mannheim, Manheim, Germany). Competition hybridization by pre-annealing with a 50-100-fold excess of Cot-1 DNA was performed before overnight hybridization on slides with previously denatured DNA. Probes were visualized by subsequent incubations with (1) streptavidin-fluorescein isothiocyanate (FITC) (Vector, Peterborough, UK); (2) biotinylated goat anti-streptavidin (Vector); and (3) streptavidin-FITC (Vector). Cells and chromosomes were counterstained with diamidino-phenylindole (DAPI) and embedded in Vectashield (Vector). Digital images were captured using a cooled-coupled device (CCD) camera (Photometrics, Tucson, AZ, USA) mounted on an Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with selective filters for fluorescein, rhodamine and DAPI and controlled by a Quadra 950 MacIntosh computer with SmartCapture software (Vysis). In all cases, a minimum of 200 interphase nuclei was analyzed.

Results

Interphase FISH on five control specimens showed a mean percentage of cells with one hybridization signal in 2.24 ± 0.35, 2.87 ± 0.54, 3.52 ± 0.68, 2.47 ± 0.43 and with three hybridization signals in 0.9 ± 0.41, 1.15 ± 0.26, 1.17 ± 0.37 and 1.24 ± 0.45 for RB1, D13S25, BRCA2 and 755b11 probes, respectively. No hybridization signal was found in 1.02 ± 0.3, 0.98 ± 0.41, 1.43 ± 0.24 and 1.09 ± 0.23 for RB1, D13S25, BRCA2 and 755b11 probes, respectively. Our cut-off levels for monoallelic loss have been arbitrarily set up when 10% of the cells show loss of one hybridization signal for the target and two signals for a control probe for chromosome 22.

Detection of 11q deletion

Interphase FISH revealed 11q hemizygous allelic loss in seven (39%) of 18 B-PLL cases (Figure 1a). The mean percentage of cells with one hybridization signal was 32.6 ± 27.9 (range, 12-97). No case had bi-allelic deletion.

Detection of 13q deletion

Monoallelic deletion of chromosome 13q14 was detected in 10 (55%) of 18 B-PLL. Monoallelic loss of RB-1 was found in 10/18 (55%) (Figure 1b) of the cases whereas hemizygous deletion of D13S25 was found in 6/18 (33%) of cases. The mean percentage of cells with one hybridization signal was 37.5 ± 19.6 (range, 12-70) for RB-1 and 38.8 ± 23.5 (range, 15-85) for D13S25. All the cases with D13S25 deletion (6/18, 33%) had a RB-1 loss. Three (16.6%) of 18 cases had large 13q14 deletion encompassing D13S25, RB-1 and BRCA2. No case showed neither isolated BRCA2 deletion nor bi-allelic deletion for the three 13q genes/marker.

Three (16.6%) of the 18 B-PLL cases showed co-existence of 13q and 11q monoallelic deletion. One had a deletion of the three 13q markers, one showed deletion of D13S25 and RB-1 and one deletion of RB-1 and BRCA2.

Correlation between FISH and P53 status

Previously published P53 results¹⁹ were available in 11 cases. These results involve loss of heterozygosity (LOH), P53 mutation analysis by sequencing and level of expression of the P53 protein, assessed by immunocytochemistry. Eight (72.8%) of the 11 samples showed P53 abnormalities. Out of these eight samples, one (12.5%) did not show any interphase FISH anomaly for 11q and 13q, one sample (12.5%) had a 11q deletion, five (62.5%) had 13q deletion and one (12.5%) both 11q and 13q deletion. Out of the six latter cases, three showed deletion of RB-1 only and three had deletion of both RB-1 and D13S25. Out of the three remaining cases with normal P53 status, two showed a normal pattern and one had an 11q deletion by interphase FISH.

Discussion

Allelic loss of 13q14 and 11q23 have been shown to be recurrent in lymphoproliferative disorders.^{10,12,13,16,20,21} We report here the interphase cytogenetics study of 13q and 11q deletions in a series of 18 B-PLL cases, a rare and aggressive leukemia characterized by a high white cell count and splenomegaly without significant lymphadenopathy. The high incidence of allelic loss at 13q14 and 11q23 suggests the involvement of one or several tumor suppressor genes. The RB1 gene at 13q14.2 is a tumor suppressor gene directly involved in the control of the cell proliferation.^22,23,24 Deletion and/or inactivation of the RB1 gene and its protein product have been postulated to be important in the development or progression of tumor.²⁵ Even though the D13S25 and 11q23 regions have been shown to be recurrently involved in deletion events in lymphoproliferative disorders, the genes implicated remain to be identified and characterized.

In our series of 18 B-PLL cases, chromosome 11q23 deletions were observed in 39% of the cases. Monoallelic loss of RB1 was found in 56% cases and 33% showed hemizygous D13S25 deletions. All the cases with D13S25 deletion showed RB1 loss. Deletion of the BRCA2 containing probe have been observed in 22% of the cases, always associated with RB1 deletion, as seen with D13S25. Three cases showed concurrent 13q14 and 11q23 deletions. The lack of material did not allow us to perform simultaneously 11q and 13q probes hybridization in those cases.

Out of the six cases where conventional cytogenetics was available (data not shown) (Table 1, patient 5), only one case showed 13q14 abnormality. Our interphase FISH results indicate that the breakpoint in this der(20)t(13;20)(q14;p13) translocation was telomeric with regards to D13S25. Two cases showed a t(11;14)(q13;q32) (data not shown) (Table 1, patients 15 and 16). One presented an 11q23 deletion and one a 13q14 deletion.

In our previous study¹⁹ of 11 cases, the overall frequency of p53 mutations in B-PLL was 53% (Table 1), the highest incidence reported within lymphoid neoplasia. Unlike other B cell malignancies, 40% of the detected mutations were either deletion or insertion. Out of these 11 cases, eight (72%) showed p53 abnormalities either by LOH, mutation and/or over-expression. Six of them (75%) had RB1 deletion either alone or associated with D13S25/BRCA2 deletions. These results suggest that RB1 deletions seem to correlate with p53 abnormalities.

We have previously documented a high frequency of allelic imbalance of the RB1 gene and the D13S25 marker in CLL and SLVL.^12,16 In CLL, the D13S25 marker was shown to be deleted in 63% of the cases, with homozygous deletion in 20% of the cases, whereas RB1 was deleted in only 37% of the cases. In contrast, 47% of SLVL cases exhibited monoallelic loss of RB1 and 12% showed hemizygous D13S25 deletion.

In the present series, 56% of the cases had monoallelic loss of RB1 and 33% hemizygous D13S25 deletion. These findings are similar to those observed in SLVL and contrast with CLL where there is a preferential loss of D13S25.

In summary, our data demonstrate that allelic loss at 13q14 and 11q23 are frequent events in B-PLL. These abnormalities are clearly below the detection of conventional cytogenetic analysis, a corresponding chromosomal structural abnormality being seen in one case only. Allelic imbalance at 13q14 show, however, that there is a preferential loss of RB1 with respect to the D13S25 locus as seen in SLVL, but in contrast to CLL. Whether RB1 is the true target of these deletions in B-PLL awaits further studies. Where material is available, inactivation of the second RB1 allele by intragenic deletion, mutation or methylation is currently under investigation.

Acknowledgements

This work was supported by grants from the Kay Kendall Leukemia Fund to LJAC and EM.

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Figure 1  (a) Hybridization of YAC 755b11 (11q) red signal, and chromosome 22 probe green signal on patient 11. One cell shows a normal pattern with two red and green signals whereas two cells show an abnormal pattern with deletion of one red signal indicating an hemizygous deletion at 11q23. (b) Hybridization of PAC 214k23 (13q/BRCA2) green signal, and LSIRB1 probe (13q/RB1) red signal on patient 6. One cell shows two co-localized red and green spots as a normal hybridization pattern whereas the other cell shows a loss of one red signal indicating a hemizygous deletion of RB1 at chromosome band 13q14.

Table 1  Clinical P53 status and FISH data on the 18 B-PLL cases included in our series