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Cytogenetic, FISH, and molecular studies in a case of B-cell chronic lymphocytic leukemia with karyotypic evolution

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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
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  Cytogenetic, FISH, and molecular studiesin a case of B-cell chronic lymphocyticleukemia with karyotypic evolution Clonal chromosome abnormalities can be detectedin approximately 50% of patients with chroniclymphocytic leukemia (CLL). Different studiesfound trisomy 12 to be the most frequent chromo-some anomaly, followed by structural aberrationsof the long arm of chromosomes 13 and 14 (1, 2).However, the introduction of fluorescence  in situ hybridization (FISH) studies has demonstrated thatthe most common chromosome abnormalities inCLL are 13q14 deletions, followed by deletion in11q22-q23, trisomy 12, monoallelic loss of 17p13,where p53 tumor supressor gene is located, anddeletion in 6q21 (2).The most frequent translocation in non-Hodg-kin’s lymphoma (NHL) of the B-cell type ist(14,18)(q32.3;q21.3) which is found in follicular,diffuse large-cell lymphomas, and occasionally inother histological subtypes of NHL (3). Mole-cular studies of this translocation have discloseda juxtaposition of the bcl-2 gene with the Igheavy-chain gene locus. On chromosome 18, thebreakpoints primarily occur at two loci, themajor breakpoint region (MBR) and the minorcluster region (mcr), in approximately 60% and30% of cases, respectively. On the contrary,cytogenetically detectable t(14,18) has been rarelyreported in CLL patients (1, 2). The molecularanalysis of this pathology has detected preferen-tially, although not exclusively, variant (vcr)bcl-2 rearrangements involving the 5 ¢  flankingregion of the bcl-2 gene with the Ig light-chaingenes (4). Chena C, Cerretini R, Noriega MF, Narbaitz M, Scolnik M, PalaciosM, Neme D, Bruno S, Slavutsky I. Cytogenetic, FISH, and molecularstudies in a case of B-cell chronic lymphocytic leukemia with karyotypicevolution.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 patientdiagnosed with B-cell chronic lymphocytic leukemia (B-CLL), whoshowed progression to a diffuse large B-cell lymphoma (Richter’s syn-drome). Genetic studies were performed at diagnosis and during theRichter’s transformation (RT). A clonal karyotype with two dicentricchromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), wasfound. Both rearrangements were confirmed by FISH. Molecularcytogenetics analysis using p53 probe showed monoallelic loss of thistumor suppressor gene in 43.8% and 77.3% of cells for the first and thesecond studies, respectively). In both studies, deletions of D13S319(18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at13q14 were found. Polymerase chain reaction analysis showed theMBR/J H  rearrangement of the bcl-2 gene. FISH studies using LSI bcl-2/IgH probe allowed quantifying the clonal cell population with thisrearrangement (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 inB-CLL. The low percentage of cells with the 13q14 deletion and bcl-2/IgH rearrangement suggests that they were secondary events thatresulted 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 evolu-tion with disease progression. Christian Chena 1 , RoxanaCerretini 1,5 , Mara FernandaNoriega 1 , Marina Narbaitz 2 ,Mariano Scolnik 3 , Mara FernandaPalacios 3 , Daniela Neme 4 , SalvadorBruno 4 , Irma Slavutsky 1 1 Department of Genetics,  2 Division of Pathology, 3 Department of Oncological Immunology,  4 Division ofOncohematology, Instituto de InvestigacionesHematolgicas `Mariano R. Castex', Academia Nacionalde Medicina, Buenos Aires;  5 Centro Nacional deGentica Mdica, Buenos Aires, ArgentinaKey words: chronic lymphocytic leukemia; Richter'ssyndrome; bcl-2 gene; p53 deletion; karyotypicevolution; FISH analysisCorrespondence: Lic. Christian Chena, Departamento deGentica, Instituto de Investigaciones Hematolgicas`Mariano R. Castex', Academia Nacional de Medicina,Pacheco de Melo 3081, 1425 Buenos Aires, ArgentinaTel: +(5411) 4805–5759/8803 ext 241/291Fax: + (5411) 4803–9475e-mail: cpchena@hematologia.anm.edu.arAccepted for publication 31 October 2002 Eur J Haematol 2002: 69: 309–314Printed in UK. All rights reserved Copyright    Blackwell Munksgaard 2002 EUROPEANJOURNAL OF HAEMATOLOGY ISSN 0902-4441 309  About of 13% of CLL patients evolve into adiffuse large B-cell lymphoma, a process known asRichter’s syndrome (RS). This evolution is usuallyassociated with a rapidly progressive clinical courseand poor outcome (5). We report a case of CLLwith histologic transformation to RS, refractory totreatment, in which dicentric chromosomes, p53and 13q14 deletions, and the MBR/J H  molecularrearrangement of the bcl-2 gene were found. Case report A 54-yr-old man was referred to our Institute inOctober 2000 with generalized non-bulky lymph-adenopathies and hepatosplenomegaly. He had nosystemic symptoms. The peripheral blood countwas: WBC 82  ·  10 9 L ) 1 (90% lymphocytes, 3%prolymphocytes), Hb 10.4 g dL ) 1 , and plateletcount 144  ·  10 9 L ) 1 . LDH was raised to 551 IUL ) 1 (normal range 140–280 IU L ) 1 ) and  b 2 -micro-globulin to 10  l g mL ) 1 (normal value <3  l gmL ) 1 ). A direct Coombs test was positive. Inaddition BUN was 34 mg dL ) 1 , creatinine 1 mgdL ) 1 , GOT 23 IU L ) 1 , and GPT 19 IU L ) 1 , Totalbilirubin was 0.6 mg dL ) 1 . Serum protein electro-phoresis showed two oligoclonal bands (0.10 and0.18 g dL ) 1 ).The bone marrow (BM) aspirate showed 70%lymphocytes with typical CLL morphology. Thehistopathological study of the BM revealed aninfiltration of 70% small CD20-positive B-lympho-cytes with diffuse pattern. A flow cytometry studyshowed expression of pan-B antigen (CD19,CD20, CD22) with coexpression of CD5 andCD23. The population was monoclonal as seen by k -chain restriction. The immunophenotypic patternachieved five points for B-CLL in the scoringsystem proposed by Matutes  et al  . (6). A diagnosisof B-CLL, RAI stage III, was made. The patientstarted with CHOP chemotherapy (cyclophospha-mide, doxorubicin, vincristine, and prednisone).Right after the second CHOP cycle, an axillarylymphadenophathy that had progressed to13  ·  8 cm in size was found. The histopathologicalstudy of a lymph node biopsy showed a pattern of small lymphocytic lymphoma/CLL in transforma-tion to a diffuse large B-cell lymphoma (Richter’ssyndrome). The immunohistochemistry, using theKi67 antigen, disclosed 50% of proliferating cells(Fig. 1). Flow cytometry analysis showed twodifferent sized B-cell populations with identicalimmunophenotypes, and similar to those found inthe initial sample.Subsequently, the patient received fludarabine(25 mg m ) 2 d ) 1 ·  3 d),cyclophosphamide(1 g m ) 2 d ) 1 ),and dexamethasone (40 mg d ) 1 ·  3 d) with fur-ther progression of disease to bulky generalizedlymphadenophathies. He was started on an ESHAPcombination chemotherapy (etoposide, cisplatin,cytosine arabinoside). The patient had a shortpartial remission. However, the disease progressedafter the second cycle and the decision of treatmentwith hyper-CVAD was taken. Following the firstphase of this regimen the patient died due to septicshock, 9 months after the diagnosis of CLL . Cytogenetic, FISH, and molecular studies Cytogenetic studies were performed at diagnosisand during Richter’s transformation (RT). BMcells were cultured in F-10 medium supplementedwith 15% fetal calf serum by direct methods andwith stimulation of different mitogens (Table 1).G- and C-banding techniques were used. Karyo-typic abnormalities were described according to theISCN (7).For FISH analysis, #17 and #18 whole chromo-some painting probes (CAMBIO, Cambridge, Fig. 1.  (a) Section of a lymph node showing large cells withround nuclei and a central nucleolus, prolymphcytes, and afew small lymphocytes (Giemsa stain, 400 · ). (b) Immuno-histochemistry study showing large cells expressing nuclearKi 67 antigen (clon Mib1, 400 · ). Chena  et al. 310  UK), chromosomes #12, #17, and #18 -specific a -satellite DNA probes (VYSIS, Downer Grove,IL), and the locus-specific DNA probes RB-1 forretinoblastoma gene, D13S319 and LSI D13S25 at13q14 band, p53 at 17p13 (Spectrum Orange), andIgH Spectrum Green/BCL2 Spectrum Orange(VYSIS, Downer Grove, IL) were used. FISHwas performed according to manufacturer’s proto-cols. Hybridization signals were analyzed on thecytogenetic preparations from the patient andcontrols in 400 interphase nuclei.DNA extraction was performed by conventionalmeans. A nested polymerase chain reaction (PCR)was done in a 50  l L final volume using 1.5  l g of DNA, 0.1  l m  oligonucleotide primers, 20  l m  of alldNTPs, 2.5  l m  Cl 2 Mg, buffer 1X (10 m m  Tris– HCl, 50 m m  KCl) and 5 U  l L ) 1 Taq polymerase.The outer and the inner primers described byGribben  et al  . (8) for the MBR and the J H consensus region were used. For the first stage,the PCR protocol consisted of 25 cycles thatincluded: denaturation at 94   C for 1 min, anneal-ing at 55   C for 1 min, extension at 72   C for1 min, and tailing at 72   C for 10 min The secondstage of PCR was reamplification of 5  l L of thefirst PCR product, and consisted of 30 cycles:denaturation at 94   C for 30 s, annealing at 53   Cfor 1 min, extension at 72   C for 30 s, and tailing at72   C for 10 min. PCR products were analyzed byelectrophoresis in 2% agarose gel with ethidiumbromide and visualized under UV light. Positiveproducts give a 180–250 bp specific signal. Results At diagnosis, cytogenetic analysis showed 6/22unstimulated 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, showedthe same clone in 2/15 and 7/18 metaphasesfrom pokeweed mitogen (PWM)- and lipopolysac-charide (LPS)-stimulated BM cells, respectively.Phytohemagglutinin (PHA)-stimulated cultureshowed only normal metaphases (Table 1). Bothrearrangements were confirmed by FISH usingpainting probes (Fig. 3). The C-banding techniquerevealed the presence of the inactivated centromereof chromosome 21, indicating the pseudo-dicentricnature of der(12): psu-dic(12,21)(q24;q10). FISHusing  l -satellite DNA probes of chromosomes 17and 18 showed a signal of each probe on der(17,18),confirming the dicentric condition of this chromo-some: ish dic(17,18)(p11.2;p11.2)(wcp17+wcp18+D17Z1+D18Z1+).Table 2 shows the molecular cytogenetic analy-sis. FISH using p53 probe showed monoallelicdeletion of this gene, revealing the presence of 43.8% and 77.3% of clonal cells for the first andsecond studies, respectively. In both studies, dele-tions of D13S319 and D13S25 loci at the 13q14.3band with diploid RB-1 were found. Trisomy 12was not observed. Table 1. Cytogenetic results on BM cells of a patient with CLLClinical stage Culture KaryotypeDiagnosis 24 h-wM 44, XY, psu-dic(12;21)(q24;q10),dic(17;18)(p11.2;p11.2)[6]/46, XY[16]RT 96 h-PWM 44, XY, psu-dic(12;21)(q24;q10),dic(17;18)(p11.2;p11.2)[2]/46, XY[13]96 h-LPS 44, XY, psu-dic(12;21)(q24;q10),dic(17;18)(p11.2;p11.2)[7]/46, XY[11]72 h-PHA 46, XY[15]wM, without mitogen; PWM, pokeweed mitogen; LPS, lipopolysaccharide; PHA,phytohemagglutinin; RT, Richter's transformation. Fig. 3.  Metaphase hybridized with chromosome 12 and 21painting probes showing normal chromosomes 12 (red) and21 (green) and psu-dic(12; 21)(q24;q10) (arrow). Fig. 2.  G-banded karyotype of a bone marrow cells show-ing dic(17;18)(p11.2;p11.2) and psu-dic(12;21)(q24;q10). Karyotypic evolution in CLL 311  A nested PCR for the MBR/J H  rearrangement of the bcl-2 general showed a positive 200 bp-specificband (Fig. 4). FISH analysis using bcl-2/IgH probeallowed us to quantify the clonal cell populationwith t(14,18), showing that a small subset of cellshad this rearrangement (4% and 6.6% of cells forthe first and second studies, respectively) (Table 2)(Fig. 5). Translocation t(14,18) was not detected byconventional cytogenetics. Discussion In this report we present a B-CLL patient withdicentric chromosomes, monosomy of the p53gene, a 13q14 deletion, the MBR/J H  rearrangementof the bcl-2 gene, and karyotypic evolution, whohad a poor clinical course with RT and shortsurvival. As for dic(17,18), only eight B-CLLpatients with this anomaly have been reported inthe 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 unbalancedwhole-arm translocations der(17,18)(q10;q10), as apart of a complex karyotype in which the probabledicentric condition was not evaluated (12, 13).These findings would suggest this abnormality as arecurrent alteration in B-CLL patients. In the firststudy of our case, the presence of one cell withdic(17,18) as a sole anomaly would suggest it as thefirst event leading to CLL that was followed bystepwise clonal evolution in the leukemic cellsleading to lymphoma. Thus, an srcin of lymphomafrom leukemic cells through progressive cytogeneticand molecular cytogenetic changes is proposed.Loss of the short arm of chromosome 17, wherethe p53 tumor suppressor gene localizes, has beenobserved in 4% of cytogenetically evaluable CLLpatients. However, FISH studies have shown that Fig. 4.  Agarosa gel electrophoresis of PCR productsshowing the MBR/J H  rearrangement of the bcl-2 gene. Lane1, molecular weight marker; lane 2, positive control; lane 3,negative control; lane 4, patient. Fig. 5.  Interphase nuclei hybridized with IgH Spectrum Green/BCL2 Spectrum Orange (VYSIS) probe showing: translo-cation (two fusion yellow, one red, and one green signal) and no translocation (two green and two red signals). Table 2. FISH analysis in a patient with CLLClinical stage FISH (%)+12 RB-1 D13S319 D13S25 p53 bcl-2/J H Diagnosis 1.0 10.0 18.0 13.2 43.8 4.0RT 0.7 6.6 11.7 11.6 77.3 6.6RT, 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, andp53, respectively. For LSI IgH/BCL2 dual-color probe, the presence of two fusionsignals (yellow), one orange, and one green signal indicated the occurrence of thetranslocation t(14;18). Chena  et al. 312  monoallelic deletions of this gene occur in 9–15%of cases (2, 9). Different reports have shown thatp53 gene deletions have strong implications in theclinical course of the disease, with significantlyshorter median survival, advanced clinical stage,and resistance to chemotherapy. In addition, p53gene abnormalities occur in about 60% of caseswith Richter’s syndrome (2, 9). Our case, with p53deletion originating in a dic(17,18), showed pro-gression of the disease with a Richter’s transfor-mation and a clear lack of response to severaltherapeutic agents (alkylating agents, anthracyclin,and purine analogs). In cases reported by Do ¨hner et al  . (9), FISH and SSCP analysis revealed abiallelic inactivation of the p53 gene. Our caseshowed a monoallelic loss of p53 as determined byFISH. Because molecular analysis of p53 was notperformed, it is unknown whether the remainingallele 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 ourknowledge this is the first case with a psu-dic(12,21) described in B-CLL. As is known,dicentric chromosomes have been found withdifferent incidences in malignant cells, accordingto the type of tumor analyzed. The significance of this abnormality depends on whether they con-tribute to overall gain or loss, or if the translo-cation results in the formation of a fusion gene(15).In our case, molecular studies showed the MBR/J H  bcl-2 gene rearrangement and FISH analysisdemonstrated the presence of this anomaly in asmall subpopulation of the leukemic cells, indica-ting that this finding would be a secondary event.Although the overexpression of bcl-2 gene is afrequent phenomenon in B-CLL cases, the rear-rangement of this gene has been observed in aminor subset of patients with this pathology.Among them, only a few CLL patients with theMBR/J H  breakpoint have been reported (16–18),vcr rearrangements being the most frequentlyfound (4, 19). Besides, our results agree with thosepreviously reported by Merup  et al  . (16), whosuggested that bcl-2 rearrangements in B-CLLmight be a secondary phenomenon that occurs lateduring disease progression.Finally, our patient showed the 13q14 deletioninvolving D13S319 and D13S25 loci, with diploidRB-1. Once more, these findings support the ideathat a still unknown tumor suppressor gene, namedDBM (deleted in B-cell malignancies), is located at13q14, telomeric to RB-1 (20, 21). In addition, thelow percentage of cells with the 13q14 deletionsuggests that it was a secondary genetic event thatresulted from karyotypic evolution. AlthoughB-CLL has been considered a genetically stabledisease, different studies (22–24) would indicatethat clonal evolution is not uncommon in thesepatients, and would be associated with clinicaldisease progression.In conclusion, a new case of dic(17;18), withother multiple genetic alterations and clonal evolu-tion, has been presented. The poor clinical coursewith Richter’s transformation and the short survi-val observed in this patient support the associationof p53 gene deletion and karyotypic evolution withdisease progression. Acknowledgements This work was supported by grants from the National ResearchCouncil (CONICET), the National Agency of Scientific andTechnical Promotion (ANPCyT), Fundacio ´n ‘AlbertoJ Roemmers’, and Fundacio ´n Accio ´n Oncohematolo ´gica. References 1. J uliusson  G, O scier  DG, F itchett  M,  et al.  Prognosticsubgroups in B-cell chronic lymphocyte leukemia definedby specific chromosomal abnormalities. N Engl J Med1990; 323 :720–724.2. D o¨hner  H, S tilgenbauer  S, B enner  A,  et al.  Genomicaberrations and survival in chronic lymphocytic leukemia.N Engl J Med 2000; 343 :1910–1916.3.  Mittelman F ,  Johansson B , M ertens  F, eds. MitelmanDatabase of Chromosome Aberration in Cancer. 2002http://cgap.nci.nih.gov/Chromosomes/Mitelman.4. 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