Chronic lymphocytic leukemia (CLL) presents a varying incidence of karyotypic abnormalities whose detection is complicated by difficulties in obtaining mitosis for analysis in this type of mature lymphocyte disorder. Since the introduction of molecular cytogenetics (FISH = fluorescent in situ hybridization), applying centromeric probes for chromosome 12 has made it possible to detect a higher percentage of trisomy 12 cases. The objective of the present study was to detect trisomy 12 by FISH (alpha satellite probe) in 13 patients with CLL whose karyotypes by G-banding were either normal or inadequate. Using this method trisomy 12 was detected in three patients in a percentage of positive cells varying from 55.5% to 79%, showing that FISH is a sensitive and highly specific method for trisomy detection and should be routinely performed when the karyotype is normal.A leucemia linfocítica crônica (CLL) apresenta incidência variável de anomalias de cariótipo devido às dificuldades em se obter mitose para análise. Desde a introdução da citogenética molecular (FISH = hibridação in situ por fluorescência) usando sonda centromérica para o cromossomo 12 foi possível detectar uma maior porcentagem de casos com trissomia 12. O objetivo deste trabalho foi de detectar trissomia 12 por FISH (sonda alfa satélite) em 13 pacientes com CLL cujos cariótipos por banda G haviam sido normais ou sem resultado. Três pacientes apresentaram trissomia 12 por este método com uma porcentagem de células trissômicas variando de 55,5 a 79%, demonstrando que a FISH é um método sensível e altamente específico para detecção de trissomia 12.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Disciplina de Hematologia e HemoterapiaUNIFESP, EPM, Disciplina de Hematologia e HemoterapiaSciEL

Chauffaille, Maria de Lourdes Lopes Ferrari

Figueiredo, Maria Stella

Kerbauy, José

Marques, Eliana Azevedo

Oliveira, José Salvador Rodrigues de

Rodrigues, Maria Madalena

Romeo, Maura

Yamamoto, Mihoko

Genetics and Molecular Biology

Chronic lymphocytic leukemia (CLL) presents a varying incidence of karyotypic abnormalities whose detection is complicated by difficulties in obtaining mitosis for analysis in this type of mature lymphocyte disorder. Since the introduction of molecular cytogenetics (FISH = fluorescent in situ hybridization), applying centromeric probes for chromosome 12 has made it possible to detect a higher percentage of trisomy 12 cases. The objective of the present study was to detect trisomy 12 by FISH (alpha satellite probe) in 13 patients with CLL whose karyotypes by G-banding were either normal or inadequate. Using this method trisomy 12 was detected in three patients in a percentage of positive cells varying from 55.5% to 79%, showing that FISH is a sensitive and highly specific method for trisomy detection and should be routinely performed when the karyotype is normal.A leucemia linfocítica crônica (CLL) apresenta incidência variável de anomalias de cariótipo devido às dificuldades em se obter mitose para análise. Desde a introdução da citogenética molecular (FISH = hibridação in situ por fluorescência) usando sonda centromérica para o cromossomo 12 foi possível detectar uma maior porcentagem de casos com trissomia 12. O objetivo deste trabalho foi de detectar trissomia 12 por FISH (sonda alfa satélite) em 13 pacientes com CLL cujos cariótipos por banda G haviam sido normais ou sem resultado. Três pacientes apresentaram trissomia 12 por este método com uma porcentagem de células trissômicas variando de 55,5 a 79%, demonstrando que a FISH é um método sensível e altamente específico para detecção de trissomia 12

Chauffaille, Maria de Lourdes L.F.

Oliveira, Jose Salvador Rodrigues de

Scientific Electronic Library Online - Brazil

Detection of trisomy 12 by fluorescent in situ hybridization (FISH) in chronic lymphocytic leukemia

Maria de Lourdes L.F. Chauffaille

Eliana Azevedo Marques

Jose Salvador Rodrigues de Oliveira

Maria Madalena Rodrigues

Maria Stella Figueiredo

Maura Romeo

Mihoko Yamamoto

José Kerbauy

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Repositório Institucional UNIFESP

531 Detection of trisomy 12 by FISH in CLLGenetics and Molecular Biology, 23, 3, 531-533 (2000)INTRODUCTIONChronic lymphocytic leukemia (CLL) is a malignantlymphoproliferative disorder characterized in 98% of thecases by clonal expansion of B lymphocytes CD 5, CD 19and CD 23 positive.Cytogenetic study is important in prognosis, but maydepend on disease stage (Zwiebel and Cheson, 1998). Themost frequent abnormalities found, totaling 60%, are trisomy12, t(14q), t/del(13q), del(6q) and 11q22-23 (Hallek et al.,1997; Juliusson and Merup, 1998). Patients with chromo-somal abnormalities, particularly when complex, have shortersurvival (Escudier et al., 1993; Xue et al., 1993) while thosewith normal karyotypes respond significantly better to che-motherapy (Han et al., 1988). Karyotype evolution has beenobserved in 20% of the cases and generally in Richter’s trans-formation (Robert et al., 1982; Tsujimoto et al., 1985; Gileset al., 1998).Chromosomal abnormalities are found by conventionalG-banding in only 50% of the patients (Hallek et al., 1997).Some difficulties exist in obtaining mitosis in CLL due tolow mitotic index or because metaphases are not always partof the malignant clone. Since the introduction of molecularcytogenetics (FISH), applying centromeric probes for chro-mosome 12 (alpha satellite) has made possible the detectionof a higher percentage of trisomy 12 cases (Hamblin, 1997).The objective of the present study was to detect tri-somy 12 by FISH in 13 patients whose chromosomal analy-sis by conventional G-banding was either normal or failed.MATERIAL  AND METHODSThirteen CLL patients with normal karyotypes or with-out results due to lack of metaphases were studied. Table Ishows some diagnostic data. The control group was consti-tuted of 3 bone marrow donors with normal karyotypes.The CLL diagnosis resulted from the presence of ab-solute lymphocyte count greater than 5,000/dl, bone mar-row aspiration showing infiltration by lymphoproliferativecells, phenotyping with CD 5, CD 23 and SIg positive cells,and bone marrow biopsy showing massive lymphocyte in-filtration (Brittinger et al., 1997).Study of karyotype was done using peripheral bloodwhose buffy coat was divided and placed in 2 culture flaskswith 8 ml RPMI 1640 medium, pH 7.2, 20% fetal calf se-rum, 1% antibiotics and 100 µl L-glutamin. To the first flaskit was added Pokeweed mitogen (0.1 mg/ml) and, to thesecond, phorbol 12-myristate 13 acetate (TPA, 50 ng/ml)and PHA (0.1 mg/ml) for 72 h. Colcemid was added duringthe last 60 min followed by hypotonic treatment with 0.075mol/l KCl for 20 min and fixation with methanol and aceticacid (3:1) solution repeated 4 times.The slides were banded for trypsin G-banding (GTG);10 metaphases from each culture were analyzed. Chromo-somal abnormalities were described following ISCN (1995).Part of fixed cell (0.5 ml) suspension was separatedfor FISH. This material was centrifuged again and resus-pended; the slides were made by cytocentrifuging (Incibras).After air drying, these slides were washed in 2X SSC for 30min at 37oC and dehydrated in 70, 80 and 95% ethanol for 2min in each. The probe for chromosome 12 alpha satelliteD1273 (Oncor) (1.5 µl) was mixed with 30 µl hybrisol VI(Oncor), both previously heated to 37oC, for 5 min. Theprobe mixture was then denatured at 72oC for 5 min,dropped into the cytospin smear, coverslipped, sealed withrubber cement and incubated for 16 h in a humidified cham-Detection of trisomy 12 by fluorescent in situ hybridization (FISH)in chronic lymphocytic leukemiaMaria de Lourdes L.F. Chauffaille, Eliana Azevedo Marques, Jose Salvador Rodrigues de Oliveira,Maria Madalena Rodrigues, Maria Stella Figueiredo, Maura Romeo, Mihoko Yamamoto and José KerbauyAbstractChronic lymphocytic leukemia (CLL) presents a varying incidence of karyotypic abnormalities whose detection is complicated by diffi-culties in obtaining mitosis for analysis in this type of mature lymphocyte disorder. Since the introduction of molecular cytogenetics (FISH= fluorescent in situ hybridization), applying centromeric probes for chromosome 12 has made it possible to detect a higher percentageof trisomy 12 cases. The objective of the present study was to detect trisomy 12 by FISH (alpha satellite probe) in 13 patients with CLLwhose karyotypes by G-banding were either normal or inadequate. Using this method trisomy 12 was detected in three patients in apercentage of positive cells varying from 55.5% to 79%, showing that FISH is a sensitive and highly specific method for trisomy detectionand should be routinely performed when the karyotype is normal.Disciplina de Hematologia e Hemoterapia da Escola Paulista de Medicina, UNIFESP, Rua Botucatu, 740, 3ο andar, 04023-900 São Paulo, SP, Brasil.Send correspondence to M.L.L.F.C. Fax: +55-11-571-8806. E-mail: chauffaill@hemato.epm.br532 Chauffaille et al.ber at 37oC. Detection and signal amplification were madeby removing coverslip, rinsing the slides in 0.25X SSCfor 5 min at 72oC and phosphate-buffered detergent (PBD)for 2 min at 37oC. Incubation continued with FITC-labeledantidigoxigenin for 5 min at 37oC covered with plastic cov-erslip. They were then rinsed three times in PBD for 2 minand incubated with 20 µl rabbit antisheep antibody for 15min at 37oC. The slides were washed three times in PBD,then incubated with 20 µl FITC antirabbit antibody, for 15min at 37oC, coverslipped. After three PBD washes, 10 µlpropidium iodide with antifade (0.3 µg/ml) was added andthe slides were again coverslipped. They were then analyzedwith a fluorescent microscope (Olympus BMX60) withFITC and rhodamine filters (Chroma Technologies). Theimages were captured through a CCD camera (Cohu) as-sembled to a computer having karyotyping and FISH soft-ware (CytoVision-Applied Imaging). Slides showing morethan 50% cells with fluorescent dots were selected foranalysis. From each slide at least 200 cells were countedand all of them isolated to avoid overlapping signals (Fig-ure 1).RESULTSTable II shows FISH analysis results of the 13 patientsand controls. Each fluorescent dot in a cell represents 12chromosome centromere (Figure 1).Patients 4, 11 and 13 presented trisomy 12 with 55.5,79 and 62% of positive cells, respectively.In the control group the mean of 1.16 cells with threefluorescent dots was observed, and normal range was de-fined as from 0 to 3.24 cells (two standard deviations).DISCUSSIONThe main problem in karyotype study in CLL is the dif-Figure 1 - FISH of case 11. The interphase cells present 3 fluorescent signals.Table I - Patient diagnostic data.Sex Age Date of diagnosis Stage Chemotherapy ∆ts/qt L x 109/dl1 F 46 9/93 B S 1 9.0002 F 60 8/95 A N - 17.0003 F 86 11/93 B S 3 18.9004 M 60 7/94 A N - 17.0005 M 77 5/94 A N - 42.0006 M 70 5/97 A N - 21.0007 F 72 5/98 B S 3 27.0008 F 70 1/92 A S 4 56.0009 M 73 3/98 C S 1 39.00010 F 83 9/99 A N - 12.00011 M 60 2/99 C N - 85.00012 F 83 5/69 C S 2 98.00013 M 68 1/92 C S 2 58.000F = Female; M = male; ∆ts/qt = period of time without chemotherapy in years; L x 109/dl =lymphocytes total count /dl.533 Detection of trisomy 12 by FISH in CLLficulty in obtaining B lymphocyte in mitotic division, evenusing B cell mitogens like PWM or TPA (Hamblin, 1997).Chromosomal abnormalities are described varyingfrom 20 to 70% of CLL cases, occurring more frequentlyin patients with advanced stages of the disease (Zwiebel andCheson, 1998).The FISH method is sensitive and highly specific fortrisomy 12 detection; subsequent to its implementation thenumber of cases reported with this abnormality increasedaround 10%, varying from 12 to 42.6% (Nair et al., 1998).In the present report we detected trisomy 12 in 3 (around23%) out of 13 patients whose karyotypes were normal orfailed.A variation in the percentage of cells with trisomy 12detected by FISH has been reported and some authors havedivided it into two groups: one with small percentage (<10%)and the other with high percentage (>10%). The second groupcorrelates with a more advanced disease stage and, conse-quently, trisomy 12 would relate to an ongoing event (Reed,1998; Nair et al., 1998).A trisomy 12 and atypical morphology correlation wassuggested (nucleous cleavage, lymphoplasmatic character-istics and increased prolymphocyte number) (Matutes etal., 1996) but no correlation with disease stage has beenobserved.Case 4 showed increased CD 20 intensity and surfaceIgM in phenotyping. Great immunological marker variabil-ity has been described in CLL with trisomy 12, though nospecific pattern exists (Döhner et al., 1997).In conclusion, detecting trisomy 12 by FISH was pos-sible in 3 patients (around 23%) with normal or unsuccessfulkaryotype showing that FISH should be used as a routine testin addition to karyotype. More extensive studies are neededto better estimate frequency of this abnormality in patients.RESUMOA leucemia linfocítica crônica (CLL) apresenta incidênciavariável de anomalias de cariótipo devido às dificuldades em seobter mitose para análise. Desde a introdução da citogenética mo-lecular (FISH = hibridação in situ por fluorescência) usando sondacentromérica para o cromossomo 12 foi possível detectar uma maiorporcentagem de casos com trissomia 12. O objetivo deste trabalhofoi de detectar trissomia 12 por FISH (sonda alfa satélite) em 13pacientes com CLL cujos cariótipos por banda G haviam sidonormais ou sem resultado. Três pacientes apresentaram trissomia12 por este método com uma porcentagem de células trissômicasvariando de 55,5 a 79%, demonstrando que a FISH é um métodosensível e altamente específico para detecção de trissomia 12.ACKNOWLEDGMENTSResearch and publication supported by FAPESP.REFERENCESBrittinger, G., Hellriegel, K.P. and Hiddemann, W. (1997). Chronic lympho-cytic leukemia and hairy cell leukemia - diagnosis and treatment: resultsof a consensus meeting of the German CLL-Co-operative Group. Leu-kemia 119 (Suppl. 2): S1-S3.Döhner, H., Stilgenbauer, S., Fischer, K., Bentz, M. and Lichter, P. (1997).Cytogenetic and molecular analysis of B cell chronic lymphocytic leu-kemia: specific chromosome aberrations identify prognostic subgroupsof patients and point to loci of candidate genes. Leukemia 11 (Suppl.2): S19-S24.Escudier, S.M., Leahy, J.M.P., Drach, J.W. et al. (1993). Fluorescent in situhybridization and cytogenetic studies of trisomy 12 in CLL. Blood 81:2702-2707.Giles, F.J., O´Brien, S.M. and Keating, M.J. (1998). Chronic lymphocyticleukemia (Richter´s) transformation. Sem. Oncol. 25: 117-125.Hallek, M., Kuhn-Hallek, I. and Emmerich, B. (1997). Prognostic factors inchronic lymphocytic leukemia. Leukemia 11 (Suppl. 2): S4-S13.Hamblin, T.J. (1997). Trisomy 12 in CLL revised. Leuk. Res. 21: 1025-1026.Han, T., Sadamori, N., Black, A.M.W. et al. (1988). Cytogenetic studies inCLL. Nouv. Rev. Fr. Hematol. 30: 393-395.ISCN (1995). An International System for Human Cytogenetics Nomencla-ture (Mittelman, F., ed.). S. Karger, Basel.Juliusson, G. and Merup, M. (1998). Cytogenetics in chronic lymphocyticleukemia. Sem. Oncol. 25: 19-26.Matutes, E., Oscier, D., Garcia-Marco, D. et al. (1996). Trisomy 12 defines agroup of chronic lymphocytic leukemia with atypical morphology: cor-relation of cytogenetics, clinical and laboratory features in 544 patients.Br. J. Hematol. 92: 382-388.Nair, C.N., Chougule, A., Dhond, S. et al. (1998). Trisomy 12 in chroniclymphocytic leukemia - geographical variation. Leuk. Res. 22: 313-317.Reed, J.C. (1998). Molecular Biology of chronic lymphocytic leukemia. Sem.Oncol. 25: 11-18.Robert, K.H., Gahrton, G., Friberg, K. et al. (1982). Extra chromosome 12 andprognosis in CLL. Scand. J. Haematol. 28: 163-166.Tsujimoto, Y., Jaffe, E., Cossman, J. et al. (1985). Clustering of breakpointson chromosome 11 in human B-cell neoplasms with the t(11;14) chro-mosome translocation. Nature 315: 340-343.Xue,T.H., Marco, J.G., Ellis, J. et al. (1993). Trisomy 12 in CLL detected byFISH. Blood 82: 571-575.Zwiebel, J.A. and Cheson, B. (1998). Chronic lymphocytic leukemia: stagingand prognostic factors. Sem. Oncol. 25: 42-59.(Received February 21, 2000)Table II - Results of FISH analysis.    Case 3 signals 2 signals1 1% 98% 2 2% 91%3 3% 90%4 55.5% 42%5 0 94.3%6 2.5 94%7 0 93.3%8 2.5 94%9 3 91%10 0 98%11 79% 18.6%12 2.3 92%13 62% 36%Control 1 4 (2%) 188 (94%)Control 2 0 189 (94.5%)Control 3 3 (1.5%) 194 (97%)X 1.16 95.16SD 0-3.24 91.96-98.363 signals = Visualization of 3 fluorescent signals incell; 2 signals = visualization of 2 fluorescent sig-nals in cell; X = mean; SD = standard deviation.534 Chauffaille et al.

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Detection of trisomy 12 by fluorescent in situ hybridization (FISH) in chronic lymphocytic leukemia

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