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Copyright  Blackwell Munksgaard 2002

Eur J Haematol 2002: 69: 309–314 Printed in UK. All rights reserved

EUROPEAN JOURNAL OF HAEMATOLOGY ISSN 0902-4441

Cytogenetic, FISH, and molecular studies in a case of B-cell chronic lymphocytic leukemia with karyotypic evolution Chena C, Cerretini R, Noriega MF, Narbaitz M, Scolnik M, Palacios M, Neme D, Bruno S, Slavutsky I. Cytogenetic, FISH, and molecular studies in a case of B-cell chronic lymphocytic leukemia with karyotypic evolution. Eur J Haematol 2002: 69: 309–314.  Blackwell Munksgaard 2002. Abstract: We report the clinical, cytogenetic, fluorescence in situ hybridization (FISH) and molecular findings in a 54-yr-old male patient diagnosed with B-cell chronic lymphocytic leukemia (B-CLL), who showed progression to a diffuse large B-cell lymphoma (Richter’s syndrome). Genetic studies were performed at diagnosis and during the Richter’s transformation (RT). A clonal karyotype with two dicentric chromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), was found. Both rearrangements were confirmed by FISH. Molecular cytogenetics analysis using p53 probe showed monoallelic loss of this tumor suppressor gene in 43.8% and 77.3% of cells for the first and the second studies, respectively). In both studies, deletions of D13S319 (18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at 13q14 were found. Polymerase chain reaction analysis showed the MBR/JH rearrangement of the bcl-2 gene. FISH studies using LSI bcl-2/ IgH probe allowed quantifying the clonal cell population with this rearrangement (4% and 6.6% of cells at diagnosis and RT, respectively). To our knowledge, this is the first case with a psu dic(12,21) described in B-CLL. The low percentage of cells with the 13q14 deletion and bcl-2/ IgH rearrangement suggests that they were secondary events that resulted from clonal evolution. Our patient had a short survival (9 months) and a clear lack of response to several therapeutic agents, confirming the association of p53 gene deletion and karyotypic evolution with disease progression.

Clonal chromosome abnormalities can be detected in approximately 50% of patients with chronic lymphocytic leukemia (CLL). Different studies found trisomy 12 to be the most frequent chromosome anomaly, followed by structural aberrations of the long arm of chromosomes 13 and 14 (1, 2). However, the introduction of fluorescence in situ hybridization (FISH) studies has demonstrated that the most common chromosome abnormalities in CLL are 13q14 deletions, followed by deletion in 11q22-q23, trisomy 12, monoallelic loss of 17p13, where p53 tumor supressor gene is located, and deletion in 6q21 (2). The most frequent translocation in non-Hodgkin’s lymphoma (NHL) of the B-cell type is t(14,18)(q32.3;q21.3) which is found in follicular,

Christian Chena1, Roxana Cerretini1,5, Mara Fernanda Noriega1, Marina Narbaitz2, Mariano Scolnik3, Mara Fernanda Palacios3, Daniela Neme4, Salvador Bruno4, Irma Slavutsky1 1

Department of Genetics, 2Division of Pathology, Department of Oncological Immunology, 4Division of Oncohematology, Instituto de Investigaciones Hematolgicas `Mariano R. Castex', Academia Nacional de Medicina, Buenos Aires; 5Centro Nacional de Gen,tica M,dica, Buenos Aires, Argentina

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Key words: chronic lymphocytic leukemia; Richter's syndrome; bcl-2 gene; p53 deletion; karyotypic evolution; FISH analysis Correspondence: Lic. Christian Chena, Departamento de Gen,tica, Instituto de Investigaciones Hematolgicas `Mariano R. Castex', Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina Tel: + (5411) 4805–5759/8803 ext 241/291 Fax: + (5411) 4803–9475 e-mail: [email protected] Accepted for publication 31 October 2002

diffuse large-cell lymphomas, and occasionally in other histological subtypes of NHL (3). Molecular studies of this translocation have disclosed a juxtaposition of the bcl-2 gene with the Ig heavy-chain gene locus. On chromosome 18, the breakpoints primarily occur at two loci, the major breakpoint region (MBR) and the minor cluster region (mcr), in approximately 60% and 30% of cases, respectively. On the contrary, cytogenetically detectable t(14,18) has been rarely reported in CLL patients (1, 2). The molecular analysis of this pathology has detected preferentially, although not exclusively, variant (vcr) bcl-2 rearrangements involving the 5¢ flanking region of the bcl-2 gene with the Ig light-chain genes (4). 309

Chena et al. About of 13% of CLL patients evolve into a diffuse large B-cell lymphoma, a process known as Richter’s syndrome (RS). This evolution is usually associated with a rapidly progressive clinical course and poor outcome (5). We report a case of CLL with histologic transformation to RS, refractory to treatment, in which dicentric chromosomes, p53 and 13q14 deletions, and the MBR/JH molecular rearrangement of the bcl-2 gene were found. Case report

A 54-yr-old man was referred to our Institute in October 2000 with generalized non-bulky lymphadenopathies and hepatosplenomegaly. He had no systemic symptoms. The peripheral blood count was: WBC 82 · 109 L)1 (90% lymphocytes, 3% prolymphocytes), Hb 10.4 g dL)1, and platelet count 144 · 109 L)1. LDH was raised to 551 IU L)1 (normal range 140–280 IU L)1) and b2-microglobulin to 10 lg mL)1 (normal value < 3 lg mL)1). A direct Coombs test was positive. In addition BUN was 34 mg dL)1, creatinine 1 mg dL)1, GOT 23 IU L)1, and GPT 19 IU L)1, Total bilirubin was 0.6 mg dL)1. Serum protein electrophoresis showed two oligoclonal bands (0.10 and 0.18 g dL)1). The bone marrow (BM) aspirate showed 70% lymphocytes with typical CLL morphology. The histopathological study of the BM revealed an infiltration of 70% small CD20-positive B-lymphocytes with diffuse pattern. A flow cytometry study showed expression of pan-B antigen (CD19, CD20, CD22) with coexpression of CD5 and CD23. The population was monoclonal as seen by k-chain restriction. The immunophenotypic pattern achieved five points for B-CLL in the scoring system proposed by Matutes et al. (6). A diagnosis of B-CLL, RAI stage III, was made. The patient started with CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone). Right after the second CHOP cycle, an axillary lymphadenophathy that had progressed to 13 · 8 cm in size was found. The histopathological study of a lymph node biopsy showed a pattern of small lymphocytic lymphoma/CLL in transformation to a diffuse large B-cell lymphoma (Richter’s syndrome). The immunohistochemistry, using the Ki67 antigen, disclosed 50% of proliferating cells (Fig. 1). Flow cytometry analysis showed two different sized B-cell populations with identical immunophenotypes, and similar to those found in the initial sample. Subsequently, the patient received fludarabine (25 mg m)2 d)1 · 3 d), cyclophosphamide (1 g m)2d)1), and dexamethasone (40 mg d)1 · 3 d) with further progression of disease to bulky generalized 310

Fig. 1. (a) Section of a lymph node showing large cells with round nuclei and a central nucleolus, prolymphcytes, and a few small lymphocytes (Giemsa stain, 400·). (b) Immunohistochemistry study showing large cells expressing nuclear Ki 67 antigen (clon Mib1, 400·).

lymphadenophathies. He was started on an ESHAP combination chemotherapy (etoposide, cisplatin, cytosine arabinoside). The patient had a short partial remission. However, the disease progressed after the second cycle and the decision of treatment with hyper-CVAD was taken. Following the first phase of this regimen the patient died due to septic shock, 9 months after the diagnosis of CLL . Cytogenetic, FISH, and molecular studies

Cytogenetic studies were performed at diagnosis and during Richter’s transformation (RT). BM cells were cultured in F-10 medium supplemented with 15% fetal calf serum by direct methods and with stimulation of different mitogens (Table 1). G- and C-banding techniques were used. Karyotypic abnormalities were described according to the ISCN (7). For FISH analysis, #17 and #18 whole chromosome painting probes (CAMBIO, Cambridge,

Karyotypic evolution in CLL Table 1. Cytogenetic results on BM cells of a patient with CLL Clinical stage

Culture

Diagnosis

24 h-wM

RT

96 h-PWM 96 h-LPS 72 h-PHA

Karyotype 44, XY, psu-dic(12;21)(q24;q10), dic(17;18)(p11.2;p11.2)[6]/46, XY[16] 44, XY, psu-dic(12;21)(q24;q10), dic(17;18)(p11.2;p11.2)[2]/46, XY[13] 44, XY, psu-dic(12;21)(q24;q10), dic(17;18)(p11.2;p11.2)[7]/46, XY[11] 46, XY[15]

wM, without mitogen; PWM, pokeweed mitogen; LPS, lipopolysaccharide; PHA, phytohemagglutinin; RT, Richter's transformation.

UK), chromosomes #12, #17, and #18 -specific a-satellite DNA probes (VYSIS, Downer Grove, IL), and the locus-specific DNA probes RB-1 for retinoblastoma gene, D13S319 and LSI D13S25 at 13q14 band, p53 at 17p13 (Spectrum Orange), and IgH Spectrum Green/BCL2 Spectrum Orange (VYSIS, Downer Grove, IL) were used. FISH was performed according to manufacturer’s protocols. Hybridization signals were analyzed on the cytogenetic preparations from the patient and controls in 400 interphase nuclei. DNA extraction was performed by conventional means. A nested polymerase chain reaction (PCR) was done in a 50 lL final volume using 1.5 lg of DNA, 0.1 lm oligonucleotide primers, 20 lm of all dNTPs, 2.5 lm Cl2Mg, buffer 1X (10 mm Tris– HCl, 50 mm KCl) and 5 U lL)1 Taq polymerase. The outer and the inner primers described by Gribben et al. (8) for the MBR and the JH consensus region were used. For the first stage, the PCR protocol consisted of 25 cycles that included: denaturation at 94 C for 1 min, annealing at 55 C for 1 min, extension at 72 C for 1 min, and tailing at 72 C for 10 min The second stage of PCR was reamplification of 5 lL of the first PCR product, and consisted of 30 cycles: denaturation at 94 C for 30 s, annealing at 53 C for 1 min, extension at 72 C for 30 s, and tailing at 72 C for 10 min. PCR products were analyzed by electrophoresis in 2% agarose gel with ethidium bromide and visualized under UV light. Positive products give a 180–250 bp specific signal.

Fig. 2. G-banded karyotype of a bone marrow cells showing dic(17;18)(p11.2;p11.2) and psu-dic(12;21)(q24;q10).

Phytohemagglutinin (PHA)-stimulated culture showed only normal metaphases (Table 1). Both rearrangements were confirmed by FISH using painting probes (Fig. 3). The C-banding technique revealed the presence of the inactivated centromere of chromosome 21, indicating the pseudo-dicentric nature of der(12): psu-dic(12,21)(q24;q10). FISH using l-satellite DNA probes of chromosomes 17 and 18 showed a signal of each probe on der(17,18), confirming the dicentric condition of this chromosome: ish dic(17,18)(p11.2;p11.2)(wcp17+wcp18+ D17Z1+D18Z1+). Table 2 shows the molecular cytogenetic analysis. FISH using p53 probe showed monoallelic deletion of this gene, revealing the presence of 43.8% and 77.3% of clonal cells for the first and second studies, respectively. In both studies, deletions of D13S319 and D13S25 loci at the 13q14.3 band with diploid RB-1 were found. Trisomy 12 was not observed.

Results

At diagnosis, cytogenetic analysis showed 6/22 unstimulated BM cells with the karyotype 44, XY, der(12)t(12,21)(q24;q11), der(17,18)(q10;q10) (Fig. 2). Only one cell with 45, XY, der(17,18)(q10;q10) was also observed, suggesting clonal evolution. The second study, performed during RT, showed the same clone in 2/15 and 7/18 metaphases from pokeweed mitogen (PWM)- and lipopolysaccharide (LPS)-stimulated BM cells, respectively.

Fig. 3. Metaphase hybridized with chromosome 12 and 21 painting probes showing normal chromosomes 12 (red) and 21 (green) and psu-dic(12; 21)(q24;q10) (arrow).

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Chena et al. Table 2. FISH analysis in a patient with CLL Clinical stage

FISH (%)

Diagnosis RT

+12 1.0 0.7

RB-1 10.0 6.6

D13S319 18.0 11.7

D13S25 13.2 11.6

p53 43.8 77.3

bcl-2/JH 4.0 6.6

RT, Richter's transformation. Cut-off for positivity (mean of normal control + 3SD) was 2.8%, 12.8%, 10.1%, 7.5%, and 5.4% for + 12 and monoallelic deletions of RB-1, D13S319, D13S25, and p53, respectively. For LSI IgH/BCL2 dual-color probe, the presence of two fusion signals (yellow), one orange, and one green signal indicated the occurrence of the translocation t(14;18).

A nested PCR for the MBR/JH rearrangement of the bcl-2 general showed a positive 200 bp-specific band (Fig. 4). FISH analysis using bcl-2/IgH probe allowed us to quantify the clonal cell population with t(14,18), showing that a small subset of cells had this rearrangement (4% and 6.6% of cells for the first and second studies, respectively) (Table 2) (Fig. 5). Translocation t(14,18) was not detected by conventional cytogenetics.

Fig. 4. Agarosa gel electrophoresis of PCR products showing the MBR/JH rearrangement of the bcl-2 gene. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, patient.

Discussion

In this report we present a B-CLL patient with dicentric chromosomes, monosomy of the p53 gene, a 13q14 deletion, the MBR/JH rearrangement of the bcl-2 gene, and karyotypic evolution, who had a poor clinical course with RT and short survival. As for dic(17,18), only eight B-CLL patients with this anomaly have been reported in the literature: four cases described by Do¨hner et al. (9), three patients referred by Callet-Bauchu et al. (10), and one case recently reported by Espinet et al. (11), four of them as a sole anomaly. Moreover, two reports have described unbalanced whole-arm translocations der(17,18)(q10;q10), as a part of a complex karyotype in which the probable

dicentric condition was not evaluated (12, 13). These findings would suggest this abnormality as a recurrent alteration in B-CLL patients. In the first study of our case, the presence of one cell with dic(17,18) as a sole anomaly would suggest it as the first event leading to CLL that was followed by stepwise clonal evolution in the leukemic cells leading to lymphoma. Thus, an origin of lymphoma from leukemic cells through progressive cytogenetic and molecular cytogenetic changes is proposed. Loss of the short arm of chromosome 17, where the p53 tumor suppressor gene localizes, has been observed in 4% of cytogenetically evaluable CLL patients. However, FISH studies have shown that

Fig. 5. Interphase nuclei hybridized with IgH Spectrum Green/BCL2 Spectrum Orange (VYSIS) probe showing: translocation (two fusion yellow, one red, and one green signal) and no translocation (two green and two red signals).

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Karyotypic evolution in CLL monoallelic deletions of this gene occur in 9–15% of cases (2, 9). Different reports have shown that p53 gene deletions have strong implications in the clinical course of the disease, with significantly shorter median survival, advanced clinical stage, and resistance to chemotherapy. In addition, p53 gene abnormalities occur in about 60% of cases with Richter’s syndrome (2, 9). Our case, with p53 deletion originating in a dic(17,18), showed progression of the disease with a Richter’s transformation and a clear lack of response to several therapeutic agents (alkylating agents, anthracyclin, and purine analogs). In cases reported by Do¨hner et al. (9), FISH and SSCP analysis revealed a biallelic inactivation of the p53 gene. Our case showed a monoallelic loss of p53 as determined by FISH. Because molecular analysis of p53 was not performed, it is unknown whether the remaining allele was also inactivated. Structural aberrations of the long arm of chromosome 12 have been observed in about 5% of CLL cases (1) and in approximately 15% of patients with Richter’s syndrome (14). To our knowledge this is the first case with a psudic(12,21) described in B-CLL. As is known, dicentric chromosomes have been found with different incidences in malignant cells, according to the type of tumor analyzed. The significance of this abnormality depends on whether they contribute to overall gain or loss, or if the translocation results in the formation of a fusion gene (15). In our case, molecular studies showed the MBR/ JH bcl-2 gene rearrangement and FISH analysis demonstrated the presence of this anomaly in a small subpopulation of the leukemic cells, indicating that this finding would be a secondary event. Although the overexpression of bcl-2 gene is a frequent phenomenon in B-CLL cases, the rearrangement of this gene has been observed in a minor subset of patients with this pathology. Among them, only a few CLL patients with the MBR/JH breakpoint have been reported (16–18), vcr rearrangements being the most frequently found (4, 19). Besides, our results agree with those previously reported by Merup et al. (16), who suggested that bcl-2 rearrangements in B-CLL might be a secondary phenomenon that occurs late during disease progression. Finally, our patient showed the 13q14 deletion involving D13S319 and D13S25 loci, with diploid RB-1. Once more, these findings support the idea that a still unknown tumor suppressor gene, named DBM (deleted in B-cell malignancies), is located at 13q14, telomeric to RB-1 (20, 21). In addition, the low percentage of cells with the 13q14 deletion suggests that it was a secondary genetic event that

resulted from karyotypic evolution. Although B-CLL has been considered a genetically stable disease, different studies (22–24) would indicate that clonal evolution is not uncommon in these patients, and would be associated with clinical disease progression. In conclusion, a new case of dic(17;18), with other multiple genetic alterations and clonal evolution, has been presented. The poor clinical course with Richter’s transformation and the short survival observed in this patient support the association of p53 gene deletion and karyotypic evolution with disease progression. Acknowledgements This work was supported by grants from the National Research Council (CONICET), the National Agency of Scientific and Technical Promotion (ANPCyT), Fundacio´n ‘Alberto J Roemmers’, and Fundacio´n Accio´n Oncohematolo´gica.

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