(1999) 13, 1721–1726  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu FISH analysis for BCL-1 rearrangements and 12 helps the diagnosis of atypical B cell leukaemias E Matutes1, P Carrara1, L Coignet1, V Brito-Babapulle1, N Villamor1, A Wotherspoon2 and D Catovsky1

Academic Department of 1Haematology and Cytogenetics and 2Histopathology, The Royal Marsden Hospital and Institute of Research, Fulham Road, London SW3 6JJ, UK

We have investigated the diagnostic value of fluorescence in cases of splenic with villous lymphocytes (SLVL) situ hybridisation (FISH) to detect t(11;14) and trisomy 12 in 53 and lymphoplasmacytic lymphomas6–9 have the cases with a B cell leukaemia difficult to classify on clinical and laboratory grounds. These cases were initially diagnosed by t(11;14)(q13;q32) involving the IgH gene locus and the morphology and immunophenotype and in 33 of them, on BCL1/ gene. In addition, few reports have suggested tissue histology, as follows: chronic lymphocytic leukaemia that t(11;14) is present in rare cases of atypical CLL although (CLL), 20, 18 of them with atypical features; B cell prolympho- some of these patients have clinical and/or laboratory features cytic leukaemia (B-PLL), two; mantle-cell lymphoma (MCL), 15; of MCL.6,10–15 splenic lymphoma with villous lymphocytes (SLVL), five; lym- MCL in leukaemic phase may present diagnostic difficulties phoplasmacytic lymphoma, six; , one and, four cases remained unclassifiable. FISH demonstrated BCL-1 with other B-NHL and particularly atypical CLL. The translo- rearrangement in the circulating cells from 15 cases classified cation t(11;14) can be detected by standard cytogenetics, as: MCL (10), atypical CLL (three) and B-PLL (two). A definitive Southern blot, polymerase chain reaction (PCR) and fluor- diagnosis of MCL was made on review of the spleen histology escence in situ hybridisation (FISH).1,3,4,5,8,11,16–19 In addition, in one out of the three atypical CLL with BCL-1 rearrangement. m-RNA or protein expression of the cyclin D1 can be investi- Trisomy 12 was detected in eight cases which included four gated by Northern blot and/or immunohistochemis- atypical CLL, one typical CLL, two MCL and one unspecified B 3,4,8,14,20–22 cell lymphoma by histology and morphology. One of the MCL try. The karyotyping has limitations because of dif- had both trisomy 12 and BCL-1 rearrangement and the other ficulties in obtaining metaphases particularly in chronic was CD5+, CD23+ and had a CLL score of 3, suggesting the lymphoid malignancies. By contrast, FISH allows the study of latter diagnosis. Our findings demonstrate that FISH analysis interphase cells with the possibility of retrospective analysis in is useful to clarify the nature of the disease in patients frozen cytocentrifuge made slides. Furthermore, FISH is more presenting with a B cell leukaemia in which the diagnosis is sensitive than Southern blot to detect BCL-1 rearrangement or difficult by conventional methods. FISH established with cer- 3–5 tainty the diagnosis of MCL by showing BCL-1 rearrangement the t(11;14) and is more specific than cyclin D1 over- in over two-thirds of cases in which this was suspected, includ- expression which may be found in other B cell disorders with- ing blastoid forms, and confirmed the diagnosis of most cases out t(11;14) such as hairy cell leukaemia, marginal zone of atypical CLL. and immunocytomas.20,23 Keywords: FISH; t(11;14); BCL-1; trisomy 12; atypical; B cell We have investigated systematically the diagnostic value of FISH to detect BCL-1 rearrangements in a selected group of B cell leukaemias which were difficult to classify by morphology Introduction and immunological markers. All these cases have also been investigated by FISH for trisomy 12 as this abnormality is The B cell lymphoproliferative disorders are a heterogeneous frequent in atypical CLL.24 group of diseases with distinct clinical and laboratory features. Although chronic lymphocytic leukaemia (CLL) is the most common disease in Western countries, it is apparent that a Materials and methods number of patients, clinically mimicking CLL, correspond to B cell lymphomas (B-NHL) in leukaemic phase. A review of data in our laboratory over a 4-year period (1993–1996) on Patients patients presenting with showed that 48% corresponded to CLL and 30% to B-NHL. Fifty-three patients with lymphocytosis (Ͼ10 × 109/l) have In a number of cases, particularly those evolving with leu- been included. The suspected diagnosis was based on periph- kaemia, there may be difficulties in establishing a precise eral blood morphology and immunological markers.25,26 and diagnosis on the basis of morphology and immunophenotyp- histology, available in 33 cases in whom peripheral nodes ing. Cytogenetic studies may be useful for the differential diag- were present and/or had splenectomy. The initial provisional nosis of the B cell disorders as some of them are associated diagnosis was as follows (Table 1): CLL, which included with distinct karyotypic abnormalities. mostly cases with more than 10% prolymphocytes (CLL/PL) A number of translocations which have in common the and/or atypical CLL, the latter defined by the presence of more breakpoint at 14q32, the locus for the immunoglobulin heavy than 15% cleaved or lymphoplasmacytic cells;24 B-PLL, when chain (IgH) gene have been described in B cell malignancies. the majority of cells were prolymphocytes; MCL characterised Follicular lymphoma carries the t(14;18)(q32;q21) in more by cells with speckled , slightly indented nucleus than 80% of cases whilst 50–70% of mantle-cell lymphoma without prominent nucleolus and occasional cytoplasmic (MCL),1–5 20% of B-prolymphocytic leukemia (B-PLL) and rare vacuolation; follicular lymphoma, characterised by small cells with no visible cytoplasm and smooth dense chromatin, and SLVL in which the cells were small or medium in size and Correspondence: E Matutes; Fax: 44 20 7 351 6420 had prominent cytoplasmic villi. Four cases remained Received 23 April 1999; accepted 20 July 1999 unclassifiable. The two cases with B-PLL were selected on the t(11;14) and trisomy 12 in atypical B cell leukaemias E Matutes et al 1722 Table 1 BCL-1 rearrangement and trisomy 12 (FISH) gated (FITC) streptavidin (Vector Lab, UK), biotinylated goat anti-streptavidin (Vector Lab) and again FITC streptavidin anti- Disease (provisional diagnosis) Number t(11;14) Trisomy bodies. Incubations with mouse anti-digoxigenin followed by of cases 12 tetramethyl-rhodamin isothiocyanate conjugated (TRITC) rab- bit anti-mouse (Sigma, Poole, UK) and TRITC goat anti-rabbit a Atypical CLL 12 2 2 antibodies (Sigma) were used to detect the digoxigenin lab- CLL/PL 6 1a 2 Typical CLL 2 0 1 elled probe. Nuclei were counterstained with DAPl/antifade Mantle-cell lymphoma (MCL) 15 10 2b solution (Vector Lab). B-prolymphocytic leukaemia (B-PLL) 2 2 0 Trisomy 12 was investigated with a chromosome 12 SLVL 5 0 0 centromeric specific probe (Dr Looijenga, Laboratory of Lymphoplasmacytic lymphoma 6 0 0 Experimental Patho-, University Hospital, Rotter- Follicular lymphoma 1 0 0 dam, The Netherlands) using a microwave-based FISH tech- Unspecified B cell lymphoma 4 0 1b nique. Twenty-five nanograms of the probe, previously lab- elled by nick-translation with fluorescein-16-dUTP Atypical CLL, mixture of CLL-like cells and Ͼ15% cleaved and/or (Boehringer) were placed on the slides and these were simul- lymphoplasmacytic cells; CLL/PL, mixture of CLL-like cells and ° more than 10% prolymphocytoid cells and/or immunoblasts. taneously denaturated at 72 C on a hot plate for 2 min. aDetails of these three cases are shown in Table 2. Hybridisation took place in a microwave oven where the bDetails of these three cases are shown in Table 3. slides were irradiated for 5 min at 340 watts. Subsequently the slides were washed once in 0.2 × SSC0.2% Tween 20 for 1 min and mounted with DAPl antifade solution. basis of the translocation t(11;14) previously detected by cyto- The slides were analysed on a Zeiss fluorescent microscope genetics and were used as positive controls. (Axiophot) and FISH images were captured and analysed Immunophenotyping was performed by flow cytometry using a cooled charge coupled device camera (Photometrics) (FACScan) on mononuclear cells as previously described.26 with lPLab (Digital Scientific) software. A minimum of 200 The markers evaluated were: surface immunoglobulin (Smlg), nuclei per sample were analysed. CD2, CD5, CD19, CD22, CD23, CD37, CD79b and FMC7. The normal hybridisation pattern for the BCL-1 rearrange- In selected cases CD10, CD11c, CD25 and CD103 were also ment or t(11;14) was shown by the presence of two pairs of tested. On the basis of the reactivity with CD5, CD23 and juxtaposed signals. The presence of two spots (one green and FMC7 and the intensity of Smlg and membrane CD22 and/or one red) separated by a distance greater than the diameter of a CD79b expression, all cases were given a score as previously single hybridisation spot in combination with two colocalised described.26,27 Each marker has a value of one or zero accord- signals was indicative of the translocation. ing to whether it is typical or atypical for CLL. In most cases of CLL, the scores ranged from 3 to 5 and from 0 to 2 in the other B cell disorders. Results

FISH analysis FISH analysis of t(11;14) and trisomy 12 BCL-1 rearrangement was detected by FISH in 51 to 98% of Peripheral blood isolated mononuclear cells fixed in Carnoy interphase cells from 15 patients. These cases had been pre- solution (methanol–acetic acid 3/1) and spread on slides were liminary classified as: MCL (10), B-PLL (two), CLL/PL (one) and used in all but one case in whom spleen cells were analysed. atypical CLL (two) (Table 1) (Figure 1). Two cell lines, G519 and JVM2, which carry the translocation In normal samples, 94% of interphase nuclei showed two t(11;14)(q13;q32) were used as positive controls and cells pairs of two colocalised signals and 6% showed a pair of split from six normal individuals as negative controls. Three hun- signals (standard deviation: 2.3). The resulting cut-off level dred interphase nuclei were scored in the normal samples. was 10.7%. In the two cell lines, G519 and JVM2, a combi- Two cosmid probes, COS 6.7 and COS H1.5 (Dr E Schuur- nation of one pair of colocalised spots and one pair of dis- ing, Leiden, The Netherlands)28 were used to detect the pres- sociated spots was found in 90% of the interphase nuclei and ence of the t(11;14)(q13;q32). Both are localised on chromo- in all metaphases analysed. some 11 band q13 encompassing a 750 kb region around the Trisomy 12 was detected in 32 to 75% of interphase cells BCL1-MTC. COS 6.7, the most centromeric, is approximately from eight patients (Table 1). In normal controls, 99% of the 400 kb far from the CCND1 gene while COS H1.5 is 250 kb cells had only two signals. The cut-off level was 2%. The cases telomeric from CCND1, covering the area in which most of included: CLL (five) of which one was typical, two atypical the breakpoints have been described in B cell disorders. and two CLL/PL, one unclassifiable B-NHL and two cases pro- FISH was performed according to standard methods. The visionally classified as MCL (see below). One of the latter probes were labelled by nick-translation using biotin 16-dUTP cases had both trisomy 12 and BCL-1 rearrangement. The pat- (COS 6.7) (Boehringer, UK) and digoxigenin 11-dUTP (COS tern of FISH in this case was unusual as 32% of cells were H1.5) (Boehringer). The slides were pretreated with RNAse trisomic for chromosome 12, 36% had four signals and the (100 ␮g/ml) and pepsin (50 mg/ml). Competition hybridisation remaining 32% were disomic. by pre-annealing with 100–200-fold excess of Cot-1DNA was performed before hybridisation on slides previously denatured slide preparations, with a hybridisation mixture containing Clinical and laboratory features of patients with BCL-1 40 ng of each probe. rearrangement Post-hybridisation washes were carried out in 1 × SSC and 0.1 × SSC at 45°C and 60°C, respectively. The biotinylated Mantle-cell lymphoma (10 cases): In 10 cases FISH dem- probe was detected by using fluorescein isothiocyanate conju- onstrated BCL-1 rearrangement and confirmed the suspected t(11;14) and trisomy 12 in atypical B cell leukaemias E Matutes et al 1723 erate and the disease remained stable untreated for 13 years. Recently, this patient underwent splenectomy and the spleen histology was consistent with a diagnosis of MCL. Cells in both cases were CD5+ and CD23− and had low CLL scores (Table 2). The case originally classified as CLL/PL (No. 3) had lym- phadenopathy, splenomegaly and lymphocytosis (Figure 2). The cells were CD5, CD23 and FMC7 negative whilst expressed strong Smlg, weakly CD22 and were CD79b+. Spleen histology showed a diffuse infiltration by pleomorphic lymphoid cells with increased immunoblasts suggesting the diagnosis of CLL/PL or ‘paraimmunoblastic’ transformation of CLL. However on the basis of cyclin D1 expression and the t(11;14) could be finally classified as a ‘blastoid’ transform- ation of MCL

B-PLL: These two cases had typical features of this disease such as marked splenomegaly, no and a raised WBC (80 and 170 × 109/l) with circulating prolympho- cytes with a prominent nucleolus. The cells expressed strong Smlg and were CD5+ and FMC7+ and in one of the two, they were CD23+ (scores 1 and 2).

Clinical and laboratory features of B-NHL with trisomy 12 (Table 3)

Figure 1 The presence of a break in the BCL-1 gene in interphase Two out of the three patients (Nos 4 and 5) were originally cells demonstrated by using two probes which flank the BCL-1 gene and labelled in different colours (red and green). Both signals are colo- classified as MCL and one of them (No. 4) had both trisomy calised when no break is present on 11q13. The Figure shows split 12 and t(11;14) as described earlier. Both these cases signals of a break in the BCL-1 gene between the two probes in a presented with lymphocytosis, organomegaly and one had an case of MCL with t(11;14). aggressive clinical course (Figure 3). Cells were CD5+ and in No. 5, CD23+, with a score of 3. The third patient (No. 6, unspecified B-NHL) presented with generalised lymphadeno- diagnosis of MCL. Seven out of 10 presented with organome- pathy and subsequently developed lymphocytosis and spleno- galy, one of them with bowel involvement and all had bone megaly. Spleen and lymph node histology was consistent with marrow infiltration and raised WBC (15–109 × 109/l) with low-grade lymphoma undergoing transformation. The cells atypical circulating lymphoid cells. The lymphocytes had the were CD5+, CD23− and had a CLL score of 2. In summary, characteristics of the MCL cells in all but one case who had one of the three B-NHL with trisomy 12 corresponded to MCL small cells with condensed chromatin and scanty vacuolated (No. 4), another (No. 5), probably represented atypical CLL cytoplasm. Subsequently, the disease in this patient (CD5+, CD23+) and the third case (No. 6) remained unclassi- underwent transformation with the presence of blastoid forms. fiable B-NHL despite the availability of histology but was not Cells from all these cases had a mature B cell phenotype with a MCL because of the absence of BCL-1 rearrangement while CLL scores ranging from 1–2, except in one case in which the the presence of trisomy 12 supported that it may represented score was 0. CD5 was positive in all but one, whilst all cases an atypical CLL. were CD23 negative. A single patient had both t(11;14) and trisomy 12 and was originally classified on haematological and morphological grounds as MCL in blastic transformation. Discussion He presented with generalised organomegaly, high WBC and had an aggressive clinical course. The circulating lymphocytes This study aimed at clarifying the diagnosis of B cell leu- were large in size, had indented nucleus with reticular kaemia in a group of patients difficult to classify by standard chromatin and scanty cytoplasm and the immunophenotype methodology by investigating the presence of BCL-1 was CD5+, FMC7+, strong CD22 and Smlg, CD79b− and rearrangement/t(11;14)(13;q32) and trisomy 12 by FISH analysis. CD23− (score 1). Our findings demonstrated that 10 out of 15 cases in whom The other five cases in whom MCL was suspected but BCL- a diagnosis of MCL was suspected by morphology and 1 rearrangement or t(11;14) was not detected presented with immunological markers, carried BCL-1 rearrangement. There- organomegaly, lymphocytosis (5 to 48 × 109/l) with CD5+ fore, FISH confirmed that these cases corresponded to MCL lymphocytes and low CLL scores (2 and 3). in leukaemic phase. Such incidence compares with other reported molecular genetic studies in MCL, diagnosed on lymph node histology, in which the frequency of the t(11;14) Atypical CLL and CLL/PL with BCL-1 rearrangement ranges from 50 up to 80%.3–5,17 Whether the remaining cases (Table 2): One out of the two atypical CLL patients (No. 1) in whom a diagnosis of MCL was suspected by lymphocyte presented with marked lymphocytosis, no organomegaly and morphology and phenotype but in which we were unable to had an aggressive clinical course. The other (No. 2) had mod- demonstrate BCL-1 rearrangement, corresponded to MCL with t(11;14) and trisomy 12 in atypical B cell leukaemias E Matutes et al 1724 Table 2 Features of cases diagnosed initially as atypical CLL and CLL/PL with BCL-1 rearrangement

Case Age/Sex Organomegaly WBC ×109/l CLL score CD5 CD23 CD79b Final diagnosis

1 59/M No 300 2 + neg + MCLa 2 55/M splenomegaly 28 1 + neg + MCL 3 50/M nodes and spleen 209 1 neg neg + MCLb

These three cases were disomic for chromosome 12. aNeeds confirmation by histology. bBlastoid form.

absence of such abnormality,3–5 and/or another B cell disease, eg atypical CLL, is uncertain. The clinical features, prominent organomegaly and mild lymphocytosis, and the morphologi- cal and immunophenotypic features of the cells were not those of CLL and/or other lymphoma, such as SLVL. There have been few studies emphasising the value of FISH or fiber FISH to detect t(11;14) in cases of already histologi- cally documented MCL,1,5,18 as well as in atypical CLL.11,13,19 Detection of t(11;14) or BCL-1 rearrangement by FISH seems to be more sensitive than cytogenetics that depends on obtaining metaphases and more accurate than Southern blot and PCR as these may fail to detect BCL-1 rearrangement in approximately 40% of cases due to alternative breakpoints outside the major translocation cluster (MTC) region not detectable by standard probes.3–5 In addition to its sensitivity, FISH is more specific that detecting overexpression of cyclin D1, the gene that is activated by t(11;14) in MCL as this might Figure 2 Circulating cells from a case classified as CLL/PL with the occur through other mechanisms. Thus, cyclin D1 may be t(11;14) (Table 2, case 3). There is a mixture of small lymphocytes upregulated in other B cell diseases, such as hairy cell leu- with clumped chromatin and some large prolymphocytoid cells. kaemia,14,20 marginal zone lymphoma/SLVL,8,9 and even, in an occasional case of MCL5 without evidence of the t(11;14). It is likely that other hitherto unknown mechanisms than t(11;14) lead to the cyclin D1 overexpression in such cases. The presence of the t(11;14) in CLL has been controversial.2 This translocation has been reported with variable frequency (2–12%) in several series.6,10–12,15,29 In one single report up to 50% of CLL were found to have t(11;14),13 but this study did not provide a detailed review of the morphology and/or immunological markers. Thus, it could not be ruled out that cases of MCL had been included. Most of the cases reported had atypical morphology including CLL/PL.6,11,12,15,18,29,30 It should be noted that in the series of Cuneo,11 28% of atypical CLL cases had t(11;14) and trisomy 12 and the majority (78%) of them had del13q14, abnormality, also common but not specific to CLL. This was interpreted as a support of a diag- nosis of CLL. In the present study, three cases initially classi- fied as atypical CLL or CLL/PL had rearrangement of BCL-1 by FISH and none had trisomy 12. One of these cases (No. 2) Figure 3 Circulating cells from a case provisionally classified as MCL without t(11;14) but with trisomy 12 (Table 3, case 5). The could be subsequently reclassified as MCL when spleen his- majority of cells are medium size and have an indented nucleus. tology became available. In another case (No. 3), spleen and lymph node histology was equivocal as to whether they corre-

Table 3 Features of cases diagnosed initially as B-NHL with trisomy 12

Case Age/Sex Organomegaly WBC ×109 CLL score CD5 CD23 CD79b Final diagnosis

4a 68/M nodes and spleen 22 1 + neg neg MCL 5 81/M nodes and spleen 48 3 ++ NT Atypical CLL 6b 62/F nodes 47 2 + neg NT Atypical CLL

aThis case had also BCL-1 rearrangement by FISH analysis. bSpleen histology showed a low-grade lymphoma (undefined) with features of transformation. NT, not tested. t(11;14) and trisomy 12 in atypical B cell leukaemias E Matutes et al 1725 sponded to a blastoid form of MCL or to a CLL/PL, also desig- 4 de Boer CJ, van Krieken JHJM, Schuuring E, Kluin PM. Bcl-1/cyclin nated ‘paraimmunoblastic’ variant of CLL,31 but cyclin D1 D1 in malignant lymphoma. Ann Oncol 1997; 8 (Suppl 2): expression and the presence of BCL-1 rearrangement sup- S109–S117. 5 Vaandrager JW, Schuuring E, Zwikstra E, de Boer CJ, Kleiverda ported a diagnosis of blastoid form of MCL. Recently, the KK, van Krieken JHJM, Kluin-Nelemans HC, van Ommen G-JB, t(11;14) has been reported in two out of four cases of para- Raap AK, Kluin PM. Direct visualization of dispersed 11q13 immunoblastic transformation of CLL/lymphocytic lymphoma chromosomal translocations in mantle cell lymphoma by multi- and it has been speculated that such cases may be intermedi- color DNA fiber fluorescence in situ hybridization. Blood 1996; ate between CLL in transformation and the blastoid form of 88: 1177–1182. MCL.31 It is possible that our case (No. 3) finally classified as 6 Brito-Babapulle V, Ellis V, Matutes E, Oscier D, Khokhar K, MacLennan K, Catovsky D. Translocation t(11;14)(q13;q32) in blastoid MCL with t(11;14) fit within this category. Indeed a chronic lymphoid disorders. Genes Chromos Cancer 1992; 5: case of atypical CLL with t(11;14) undergoing Richter’s trans- 158–165. formation has already been documented.10 In the third case 7 Oscier D, Matutes E, Gardiner A, Glyde S, Mould S, Brito-Baba- (No. 1), the final diagnosis was likely MCL but this required pulle V, Ellis J, Catovsky D. Cytogenetic studies in splenic lym- confirmation by histology. All these three cases had a pheno- phoma with villous lymphocytes. Br J Haematol 1993; 85: 487– type atypical for CLL with scores of 2 or 1. Therefore, these 491. findings indicate that although t(11;14) and/or BCL-1 8 Jadayel D, Matutes E, Dyer MJS, Brito-Babapulle V, Khokhar MT, Oscier DG, Catovsky D. Splenic lymphoma with villous lympho- rearrangement might be a rare feature of atypical CLL and/or cytes: analysis of BCL-1 rearrangements and expression of the CLL/PL, in those cases with both, atypical morphology and cyclin D1 gene. Blood 1994; 83: 3664–3671. immunological markers, the possibility of a MCL needs to be 9 Swerdlow SH, Zukerberg LR, Yang W-I, Harris NL, Williams ME. considered. This is not always taken into account as CLL cases The morphologic spectrum of non-Hodgkin’s lymphomas with with t(11;14) and atypical morphology and immunopheno- BCL1/Cyclin D1 gene rearrangements. Am J Surg Pathol 1996; 20: type have been documented.18 627–640. 10 Cuneo A, Balboni M, Piva N, Rigolin GM, Roberti MG, Mejak C, This study has further confirmed our previous findings that 24 Moretti S, Bigoni R, Balsamo R, Cavazzini PL, Castoldi GL. Atypi- trisomy 12 is mainly found in CLL/PL and atypical CLL. Tri- cal chronic lymphocytic leukemia with the t(11;14)(q13;q32): somy 12 was detected in two cases provisionally classified as karyotype evolution and prolymphocytoid transformation. Br J MCL. One case also had BCL-1 rearrangement and it is poss- Haematol 1995; 90: 409–416. ible that trisomy 12 was a secondary abnormality although 11 Cuneo A, Bigoni R, Negrini M, Bullrich F, Veronese ML, Roberti clonal evolution was not proven. A recent study by Cuneo et MG, Bardi A, Rigolin GM, Cavazzini P, Corce CM, Castoldi G. al32 has shown that up to 28% of MCL have partial or total Cytogenetic and interphase cytogenetic characterization of atypi- cal chronic lymphocytic leukemia carrying BCL1 translocation. trisomy 12 and this feature correlated with a shorter survival. Cancer Res 1997; 57: 1144–1150. The other case (No. 5) originally classified as MCL had a CLL 12 Newman RA, Peterson B, Davey FR, Brabyn C, Collins H, Brunetto + + score of 3, was CD5 , CD23 and may had corresponded to VL, Duggan DB, Weiss RB, Royston I, Millard FE, Miller AA, an atypical CLL. The third case (No. 6) remained undefined Bloomfield CD. Phenotypic markers and BCL-1 gene rearrange- by histology, but trisomy 12 supported the diagnosis of ment in B-cell chronic lymphocytic leukemia: a Cancer and Leu- atypical CLL. kemia Group B study. Blood 1993; 82: 1239–1246. 13 Lishner M, Lalkin A, Klein A, Yarkoni S, Manor Y, Fejgin M, Leytin In summary, our findings demonstrate that FISH analysis V, Ravid M, Amiel A. The BCL-1, BCL-2 and BCL-3 oncogenes investigating the presence of BCL-1 rearrangement and tri- are involved in chronic lymphocytic leukemia. Detection by flu- somy 12 is a useful diagnostic tool to clarify the nature of orescence in situ hybridization. Cancer Genet Cytogenet 1995; those leukaemic B cell disorders which raise diagnostic prob- 85: 118–123. lems due to their atypical morphology, undefined immuno- 14 Bosch F, Jares ES, Campo E, Lopez-Guillermo A, Piris MA, Vil- phenotypic profile and histology. FISH has also the additional lamor N, Tassies D, Jaffe ES, Montserrat E, Rozman C, Cardesa A. advantage of being more sensitive than Southern blot or PCR PRAD-1/Cyclin D1 gene overexpression in chronic lymphopro- liferative disorders: a highly specific marker of mantle-cell lym- to detect the t(11;14) or BCL-1 rearrangement, as well as more phoma. Blood 1994; 84: 2726–2732. specific that cyclin D1 overexpression by Northern blot or 15 Hernandez JM, Meccuci C, Criel A, Meeus P, Michaux L, Van immunohistochemistry, since the latter may be found in a var- Hoof A, Verhoef G, Louwagie A, Schief JM, Michaux JL, Boogaerts iety of B cell conditions other than MCL, with or without M, Van den Berghe H. Cytogenetic analysis of B-cell chronic the t(11;14). lymphoid classified according to morphologic and immunophenotype (FAB) criteria. Leukemia 1995; 9: 2140–2147. 16 Coignet LJA, Schuuring E, Kibbelaar RE, Raap TK, Kleiverda KK, Bertheas MF, Wiegant J, Beverstock G, Kluin PM. 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