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Adult Precursor B-ALL with BCR/ABL Gene Rearrangements Displays A

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Adult Precursor B-ALL with BCR/ABL Gene Rearrangements Displays A Leukemia (2001) 15, 406–414 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Adult precursor B-ALL with BCR/ABL gene rearrangements displays a unique immunophenotype based on the pattern of CD10, CD34, CD13 and CD38 expression MD Tabernero1, AM Bortoluci1, I Alaejos2,MCLo´pez-Berges2, A Rasillo1, R Garcı´a-Sanz2, M Garcı´a1, JM Sayague´s1, M Gonza´lez2, G Mateo2, JF San Miguel2 and A Orfao1 1Servicio General de Citometrı´a, Departamento de Medicina, and Centro de Investigaciones del Cancer, University of Salamanca; and 2Servicio de Hematologı´a, Hospital Universitario, Salamanca, Spain + The Philadelphia chromosome (Ph ) reflects a balanced stratification and treatment decision making. Unfortunately, a reciprocal translocation between the long arms of chromo- rapid screening method, displaying a high sensitivity and somes 9 and 22 [t(9;22)(q34;q11.2] involving the BCR and ABL + genes. At present, detection of BCR/ABL gene rearrangements specificity, for identification of BCR/ABL precursor-B-ALL is mandatory in precursor-B-ALL patients at diagnosis for cases is not currently available. Although an association prognostic stratification and treatment decision. In spite of the between BCR/ABL gene rearrangements and a common (BII) clinical impact, no screening method, displaying a high sensi- phenotype has long been reported22 it is well-established that tive and specificity, is available for the identification of ␮ + reactivity for CD10 in the absence of cIg and sIg expression – BCR/ABL precursor-B-ALL cases. The aim of the present a common, BII phenotype – is also present in around half of study was to explore the immunophenotypic characteristics of − 22,23 precursor B-ALL cases displaying BCR/ABL gene rearrange- all BCR/ABL precursor-B-ALL cases. Therefore, molecular ments using multiple stainings analyzed by quantitative flow and/or cytogenetic studies directed to the identification of cytometry in order to rapidly (Ͻ1 h) identify unique phenotypes BCR/ABL gene rearrangements should be performed in all pre- associated with this translocation. From the 82 precursor-B- cursor-B-ALL cases, from which only a minor proportion ALL cases included in the study 12 displayed BCR/ABL gene would be positive. rearragements, all corresponding to adult patients, four of The immunophenotypic characteristics of precursor B-ALL which also displayed DNA aneuploidy. Our results show that BCR/ABL+ precursor B-ALL cases constantly displayed a leukemic cells have long been believed to reflect normal hem- homogeneous expression of CD10 and CD34 but low and rela- atopoietic B cell precursors. However, more recent studies tively heterogeneous CD38 expression, together with an have shown that through the simultaneous assessment of sev- aberrant reactivity for CD13. In contrast, this unique phenotype eral antigens almost all precursor-B-ALL cases display pheno- − was only detected in three out of 70 BCR/ABL cases. There- typic aberrations.23–27 Such phenotypic aberrations may be fore, the combined use of staining patterns for CD34, CD38 and associated with specific genetic abnormalities, and it has been CD13 expression within CD10-positive blast cells is highly suggestive of BCR/ABL gene rearrangements in adults with suggested that they could be used to better understand dysreg- precursor B-ALL. Leukemia (2001) 15, 406–414. ulation of protein expression and at the same time they could Keywords: precursor B-ALL; t(9;22)(q34;q11); inmunophenotype; help in identifying cases carrying specific genetic lesions such patterns of expression as t(12;21)28–30 and 11q23 abnormalities.31–33 The aim of the present study was to explore in detail the immunophenotypic characteristics of precursor B-ALL cases Introduction displaying BCR/ABL gene rearrangements in order to rapidly identify unique phenotypes associated with this translocation; The Philadelphia chromosome (Ph+) reflects a balanced patients showing such phenotypic profile should be con- reciprocal translocation between the long arms of chromo- sidered as high priority cases for molecular confirmation of somes 9 and 22 [t(9;22)(q34; q11.2)] involving the BCR and BCR/ABL translocation if the technique is available, or if not ABL genes, respectively.1,2 Three breakpoint cluster regions in categorized as positive in a therapeutic decision-making pro- the BCR gene have been described so far: major(M)- cess. Our results show that the combined use of the patterns minor(m)- and micro (␮)-BCR.3–6 These breakpoints result in of CD34, CD38 and CD13 expression among CD10-positive BCR-ABL fusion proteins that differ in their size and trans- precursor-B ALL adult patients is highly suggestive of BCR/ABL forming potential4,5 the p210, p190 and p230 proteins, gene rearrangements. respectively. While the M-BCR breakpoint is the genetic hall- mark of chronic myeloid leukemia (CML),7,8 Ph+ precursor B- acute lymphoblastic leukemias are usually associated with the m-BCR and to a much lesser extent with M-BCR gene Materials and methods rearrangements.9 Among precursor-B-ALL cases, t(9;22) translocation occurs Patients in around 20–30% of adults and in р5% of children.10–16 From the clinical point of view, Ph+ precursor-B-ALL is asso- ciated with a highly aggressive disease frequently resistant to A total of 82 consecutive patients whose bone marrow (BM) chemotheraphy and with a short survival.14,17–21 Therefore, at samples were referred to the laboratory of the University Hos- present, detection of BCR/ABL gene rearrangements is manda- pital of Salamanca for immunophenotypic and molecular/ tory in precursor-B-ALL patients at diagnosis for prognostic cytogenetic analysis were included in the present study. All patients had an unequivocal diagnosis of ‘de novo’ precursor B-ALL based on morphological, cytochemical and immuno- phenotypic criteria;34 of the patients analyzed, 54 were males Correspondence: A Orfao, Servicio General de Citometrı´a, Laborato- = ± rio de Hematologı´a, Hospital Universitario Salamanca, Paseo de San and 28 were females. In children (n 32) mean age was 4 4 Vicente, 58–182, 37007 Salamanca, Spain; Fax: +34–923–29 46 24 years (median 3 years) while in adults (n = 50) it was 41 ± 19 Received 26 July 2000; accepted 28 November 2000 years (median 36 years). All cases were studied at diagnosis. Immunophenotype of B-ALL with t(9;22)(q34;q11) MD Tabernero et al 407 Immunophenotypic studies in order to be able to specifically gate on leukemic cells. The following information was explored on the leukemic cells for In all cases immunophenotypic studies were performed at each of the antigens analyzed: (1) presence or absence of anti- diagnosis on erythrocyte-lysed whole BM samples upon stain- gen expression; (2) fluorescence intensity, as reflected by the ing with monoclonal antibodies directly conjugated to fluor- mean fluorescence intensity (MFI) expressed in relative linear ochromes. Antigen expression was analyzed on a FACSort fluorescence chanels scaled from 0 to 10 000 (arbitrary units); flow cytometer (Becton Dickinson Biosciences, San Jose, CA, and (3) the pattern of antigen expression (homogeneous vs USA) using triple-stainings for the following combinations of heterogeneous) as reflected by the coefficient of variation (CV) fluorochrome-conjugated monoclonal antibodies (fluorescein of the fluorescence intensity obtained for each fluorochrome- isothiocyanate (FITC), phycoerythrin (PE) and either peridin conjugated monoclonal antibody reagent. From the 82 cases clorophyll protein (PerCP) or the PE/cyanine 5 (Cy5) fluoroch- included in this study 18 displayed a pro-B/BI phenotype, 60 rome tandem) directed against cell surface antigens: corresponded to common BII and four were pre-B/BIII ALL.34 CD4/CD8/CD3, CD7/CD5/CD3, CD19/CD34/CD45, CD10/ CD13/CD19, CD5/CD33/CD20, CD34/CD38/CD19, CD10/ CD20/CD19, CD7/CD2/CD3, CD34/CD22/CD19, Tdt/CD10/ CD19, CD7/CD34/CD38, and kappa/lambda/CD19. In Flow cytometric DNA ploidy studies addition, the expression of MPO, CD79a, IgM and CD3 was also explored at the cytoplasmic level. The analysis of blast cell DNA contents was performed on Briefly, BM samples were obtained and immediately diluted erythrocyte-lysed whole BM samples after specifically staining in phosphate-buffered saline (PBS) containing K EDTA as 3 for both the blast cells and cell DNA. For that purpose the anticoagulant at a proportion of 1/1 (v/v). Afterwards, for sur- Cycloscope LLA reagent kit (IMICO, Madrid, Spain) was used face antigenic stainings, 200 ␮l of PBS-diluted BM samples, strictly following the recommendations of the manufacturer. containing between 0.5 and 1 × 106 nucleated cells were Measurements of DNA cell contents were performed on a placed in each tube and incubated with the appropriate com- FACSCalibur flow cytometer, as previously described.36 bination of monoclonal antibodies for 15 min at room tem- A case was considered to display DNA aneuploidy when perature in the dark. Once this incubation period was fin- G /G -phase blast cells showed a different propidium iodide- ished, 2 ml of FACS lysing solution (Becton Dickinson) diluted 0 1 associated fluorescence intensity as compared to that of nor- 1/10 (v/v) in distilled water were added to each tube and after mal residual G /G BM cells. DNA index was calculated as vigorous vortexing another incubation for 10 min in the dark- 0 1 the ratio between the modal fluorescence intensity of G /G ness (room temperature) was performed. Afterwards, cells 0 1 blast cells and that of the normal G /G residual BM cells. were centrifuged (5 min at 540 g), washed once in 2 ml of 0 1 PBS/tube (5 min at 540 g) and resuspended in 0.5 ml/tube of PBS. For the staining of cytoplasmic antigens (MPO, CD79a and PCR amplification of BCR/ABL transcripts CD3) the Fix & Perm reagent from Caltag Laboratories (San Francisco, CA, USA) was used, strictly following the recommendations of the manufacturer.
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