Leukemia (2001) 15, 1475–1484 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu BIO-TECHNICAL METHODS SECTION (BTS) BTS Leukemia Detection of translocations affecting the BCL6 locus in B cell non-Hodgkin’s lymphoma by interphase fluorescence in situ hybridization D Sanchez-Izquierdo1, R Siebert2, L Harder2, I Marugan1, A Gozzetti3, HP Price4,SGesk2, JM Hernandez-Rivas5, I Benet1, F Sole´6, T Sonoki4, MM Le Beau3, B Schlegelberger2, MJS Dyer4, J Garcia-Conde1 and JA Martinez-Climent1 1Department of Hematology and Oncology, Hospital Clinico, University of Valencia, Spain; 2Institute of Human Genetics, University Hospital Kiel, Germany; 3Section of Hematology and Oncology, University of Chicago, USA; 4Academic Dept of Haematology and Cytogenetics, Institute of Cancer Research, Sutton, UK; 5Department of Hematology, Hospital Clı´nico, University of Salamanca, Spain; and 6Department of Hematology, Hospital del Mar, Barcelona, Spain Structural alterations in 3q27 affecting the BCL6 locus are potential in their own right including BOB-1, TTF and PIM1. among the most frequent changes in B-NHL. The aim of the Breakpointsat 3q27 are predominantly located in the 5 Ј present study was to establish an interphase-FISH assay for the detection of all diverse BCL6 translocations in B-NHL. Two untranslated region of BCL6, including the promoter and the different approaches were tested, one using a PAC-clone span- first non-coding intron; this region has been termed the major ning the major breakpoint region (MBR) of BCL6 (span-assay), breakpoint region (MBR). In such translocations, the BCL6 and another using two BAC clones flanking the MBR (flank- coding exons are juxtaposed downstream to the promoter assay). Interphase FISH with the span-assay detected the vari- derived from the reciprocal chromosomal partner, resulting in ous BCL6 translocations in seven B-NHL cell lines. The dual- deregulated BCL6 expression.9,10 However, there hasbeen no color flank-assay was evaluated in two laboratories indepen- dently: in normal controls, the cutoff level for false-positive sig- clear link between the presence of BCL6 chromosomal trans- nals was 2.6%, whereas the cutoff level for false-negatives in locationsand deregulated BCL6 expression and it is possible the seven cell lines was 7.5%. To test the feasibility of the FISH that clonal, somatic mutations and deletions within the same strategies, 30 samples from patients with B-NHL with cytog- regulatory region of the BCL6 gene may also contribute to enetic abnormalities of 3q27 were evaluated with both assays. deregulated expression.8,10 In 21 cases, the span-assay indicated a BCL6 rearrangement. Chromosomal translocations involving 3q27 or rearrange- In 18 of the 21 cases, the dual-color flank-assay confirmed the translocation including 12 different partner chromosomal loci. mentsof the BCL6 gene have been identified in about 40% The three false-positive cases detected with the span-assay of diffuse large B-NHL (DLBCL), but they have also been showed trisomy of chromosome 3 by cytogenetic analyses, found at lower frequency in other subtypes of B-NHL.2,7,11,12 and they were correctly classified as non-rearranged with the Whether translocations affecting 3q27 or BCL6 rearrange- flank-assay. In summary, our FISH strategy using two differ- ments are of prognostic value in DLBCL remains controversial. ently labeled flanking BCL6 BAC probes provides a robust, One study reported a favorable prognosis for patients with sensitive, and reproducible method for the detection of com- 11 mon and uncommon abnormalities of BCL6 gene in interphase BCL6 rearrangementsdetected by Southern blot. However, 7,13 nuclei. The routine application of this assay to patients with B- these data have not been confirmed. Resolution of this NHL will allow the assessment of the diagnostic and prognostic point demands an assay that will allow the routine detection significance of BCL6 rearrangements. Leukemia (2001) 15, of all BCL6 translocations. This has been hampered by a num- 1475–1484. ber of different factors. Conventional cytogenetic analysis is Keywords: BCL6 translocations; FISH; B cell lymphoma limited by the location of BCL6 in the terminal portion of chromosome 3q and by the presence of complex three-way translocations within the BCL6 locus.14 Additionally, cyto- Introduction genetically detectable breakpointsin 3q27 might not affect the BCL6 gene. For a regular polymerase chain reaction (PCR) Reciprocal chromosomal translocations involving band 3q27 assay, a complex set of primers for each corresponding are frequent in B cell non-Hodgkin’slymphomas(B-NHL). 1,2 rearrangement would need to be designed to detect all poss- The involved gene in most cases is a zinc finger gene, BCL6, ible rearrangements.8 Currently, the only available techniques and hasbeen found juxtaposednot only to the immunoglob- for the screening of most BCL6 translocations are Southern ulin (IG) genes, but also to a variety of other genes, some of blot analyses and long-distance inverse PCR, but these which have been characterized by molecular cloning.3–8 methods are limited by the necessity for high molecular These genes include not only constitutively expressed genes weight DNA, which isnot alwaysavailable from every patient such as histone H4, but also genes with possible oncogenic and by the technical difficulties associated with these methods.7,8,11–13 Furthermore, the frequent presence of deletionsand mutationswithin the BCL6 MBR may confound Correspondence: JA Martı´nez-Climent, Department of Hematology results of Southern blot analysis. Additionally, the expression and Medical Oncology, Hospital Cli´nico Universitario, University of Valencia, Avda Blasco Iban˜ez, 17, 46010 Valencia, Spain; Fax: 34- of BCL6 protein doesnot correlate with BCL6 gene rearrange- 96-362-2238 ment and therefore immunohistochemical studies cannot be Received 12 January 2001; accepted 9 May 2001 used as a surrogate marker for BCL6 translocations.15 Detection of BCL6 translocations by FISH D Sanchez-Izquierdo et al 1476 Fluorescence in situ hybridization (FISH) is a sensitive Probes used for FISH method for the routine identification of leukemia and lym- phoma-associated chromosomal translocations. Using large Two PAC clonesRMC03P056 and RMC03P061 containing DNA probes such as yeast artificial chromosome (YAC), P1 BCL6 intron 1 were kindly provided by WL Kuo and JW Grey artificial chromosome (PAC) or bacterial artificial chromo- (University of California, San Francisco; http://rmc- some (BAC) spanning common breakpoints, it is possible to www.lbl.gov). BAC clonesRPCI-11 211G3, 528E8 and 690C8 detect most translocations within a specific gene, despite a from the contig HPFCctg 13301 covering the BCL6 locus large number of partner chromosomes.16 However, the sys- (Washington University, http://genome.wustl.edu/) were tematic evaluation of any single color assay is technically dif- obtained from Research Genetics (Huntsville, AL, USA; ficult on patient samples. Thus, for diagnostic purposes, a http://www.resgen.com). more specific approach using two differentially labeled probes Other DNA probes were also used in selected cell lines and from both partner chromosomes leading to the detection of patientsfor the detection of BCL6 translocations to IGH, IGL, the specific fusion signals on interphase nuclei, such as in the and C-MYC genes. For IGH, cosmid c␣1-IGH containing the t(8;14)(q24;q32), t(14;18)(q32;q21) or t(11;14)(q13;q32) trans- C␣1 gene wasused, 17 aswell asPAC 1098L17, kindly pro- locations, is commonly used.17–20 In the case of promiscuous vided by Dr Mariano Rocci (University of Bari, Italy; genes, such as MLL or ETV6, these assays are not of value, http://www.bioserver.biologia.uniba.it), which contains the but translocations can be detected using FISH probes flanking variable region of IGH. For IGL, PAC 1019H10 (also from Dr the MLL or ETV6 breakpoints.21 Few previousstudieshave Rocci) wasselected;thisPAC containsexon 3 of the IGL join- attempted the detection of BCL6 rearrangementsusingFISH. ing segment. By FISH, it was mapped to 22q11 and was fused Two of these reports established assays which were only to C-MYC and BCL2 in several patients and cell lines with applicable to metaphase cells,22,23 whereasanother studyper- t(8;22)(q24;q11) and t(18;22)(q21;q11), respectively (Sanchez formed on interphase cells was limited to the identification of et al, manuscript in preparation). Probes for the C-MYC gene BCL6 translocations involving IGH.24 To date, there hasnot have been described recently.17 DNA from PACs, BACs and been a FISH method available for the routine detection of all cosmids was prepared using the Qiagen (Courtabouef, France) BCL6 translocations. kit system. We report a novel strategy using two differentially labeled flanking BCL6 BAC probes, which provides a robust, sensitive, and reproducible method for the detection of all common and FISH methods uncommon abnormalitiesof BCL6 in B-NHL by interphase FISH. FISH studies were performed on fixed cells from cytogenetic samples. In three patients without available material, FISH was performed on G-banded slides stored at room tempera- ture between 1 and 8 years. DNA from the probes was directly labeled with Spectrum Red (SR) and Spectrum Green (SG) Materials and methods (Vysis, Downers Grove, IL, USA) by nick translation. FISH pro- cedure wasperformed asreported previously. 21 For each experiment, at least 200 interphase nuclei, as well as 20 meta- Cell lines and patients with B-NHL phase