Correspondence 1445 The paternal 9 and the maternal are preferentially rearranged in chronic myeloid leukaemia

Leukemia (2004) 18, 1445–1448. doi:10.1038/sj.leu.2403404 results also showed that the normal 9 and 22 Published online 10 June 2004 were always of maternal and paternal origin, respectively, a finding that was totally consistent with the parental origin of the TO THE EDITOR rearranged chromosomes. However, as the 9 þ and 22 þ hybrids (unlike the 9q þ and Ph þ cell hybrids) can also be In the majority of patients with chronic myeloid leukaemia (CML), derived from normal cells where t(9;22) is not present, the a reciprocal chromosome translocation t(9;22)(q34.1;q11.2) origi- parental haplotypes of the normal and 22 might nates two derived products known as the Philadelphia (Ph) have not been fully reciprocal to the haplotypes of the chromosome and 9q þ following rearrangement at definite BCR 1 rearranged chromosomes. The fact that this did not happen and ABL regions. While breakpoints are well characterised can be explained, regardless of the high tumour mass of the at the cytogenetic and molecular levels, the parental origin of 2–7 patients herein studied, by the selective advantage, in vitro,of the rearranged chromosomes is controversial. To clarify cell hybrids derived from tumour cells with respect to those this issue, we analysed cell hybrids segregating the derived derived from normal cells. Usually, only the first three visible chromosomes and the normal, nonrearranged, chromosomes 9 colonies to appear in a plate are cloned, while only one colony and 22. Cell hybrid panels were created with bone marrow per plate was used for this analysis. Certainly, fast growing cell aspirates from four CML patients following cell fusion with lines are more likely to form visible colonies, a reason why they recipient, Hprt-deficient, rodent cell lines (RAG and AKO-15). are favourably selected by this procedure. The four panels derived from the patients were PCR screened for A simple probability estimate indicates that a translocation amplifying variable markers located in regions across 9q34.1 involving the paternal chromosome 9 and the maternal and 22q11.2 breakpoints (Tables 1 and 2). This allowed us to chromosome 22 represents one-fourth of all potential 9;22 separate hybrid cell lines into different classes per panel: 9 þ translocations and that our findings, in four patients, could have (cells containing chromosome 9 and lacking 9q þ , 22 and Ph), only occurred at random with P ¼ 1/256 ¼ 0.0039. This was 9q þ (containing the 9q þ chromosome and lacking 9, 22 and concordant with previous findings in 15 Ph þ patients, whose Ph), 22 þ (containing chromosome 22 and lacking 9, 9q þ and paternal and maternal chromosomes 9 and 22 were identified Ph) and Ph þ (containing the Ph chromosome and lacking 9, by cytogenetic markers,2 but differed from other reports using 9q þ and 22). All Ph þ lines, when screened by multiplex RT- 5,6 3,4,7 PCR,8 detected BCR-ABL transcripts, subsequently confirmed by cytogenetic markers and others using molecular markers cDNA sequencing, demonstrating the presence of a transcrip- in which the parental origin of the rearranged chromosomes was tionally active BCR-ABL . Another cell hybrid class, 9 þ / not coincident with our findings. It has also been proposed, 22 þ , containing both 9q and 22q regions across breakpoints based on theoretical probability estimates, that the participation and shown to be BCR-ABL þ by PCR assays, was also identified of chromosomes 9 and 22 in t(9;22), regardless of the parental and two of these cell lines were used as control in each panel. origin of each chromosome 9 or 22 homologue, is statistically These cell lines must contain, at least, the normal chromosomes more likely to occur between chromosomes of different parental 9 9 and 22 in addition to the Ph chromosome or the 9q þ origin as a strict consequence of chance. This proposition chromosome and the Ph chromosome. Altogether, 53 hybrid implies that similar proportions of t(9;22) would occur by cell lines, derived from four patients, were analysed (Table 3). rearranging the paternal chromosome 9 and the maternal In order to demonstrate the parental origin of each chromo- chromosome 22 on one side, and the maternal chromosome 9 some in the hybrid cell lines derived from CML patients, we and the paternal chromosome 22 on the other. A close screened eight variable regions of chromosome 9 and seven examination of previous reports in which the parental origin of variable regions of chromosome 22 and compared our results both rearranged chromosomes was determined in CML patients with similar assays in DNA extracts from at least one of the showed that most translocations occurred between chromo- patients’ parents and from 9 þ /22 þ hybrid cells. Supplemen- somes of different parental origin, but the number of cases was tary Tables 1–8 show the chromosome haplotypes found in the very small and, in one t(9;22), both paternal chromosomes were different cell hybrid classes in all panels. A summary of our rearranged.5 Moreover, these few cases reported to date and our results is presented in Table 4 for each of the four hybrid cell own data do not indicate that the two putative types of t(9;22) classes/per panel/per marker in which the parental origin with chromosomes of different parental origin might be equally (maternal or paternal) of the alleles is indicated. Of the 15 likely because there seems to be a higher incidence of variable markers, 14 proved to be informative in 489 of 535 PCR translocations involving the paternal chromosome 9 and the assays, indicating that the paternal chromosome 9 and the maternal chromosome 22 than those resulting from a parental maternal chromosome 22 are preferentially involved in the reciprocation. characteristic t(9;22)(q34.1;q11.2) rearrangement of CML. These These analyses, however, must take into consideration that the data might not be strictly comparable due to the different approaches used for determining the parental origin of chromo- Correspondence: Dr HN Seua´nez, Genetics Division, Instituto somes involved in t(9;22). Our results were provided by ˆ ´ Nacional de Cancer, Rua Andre Cavalcanti, 37, 4th floor, 20231- screening regions that are closely placed, both proximally and 050 Rio de Janeiro, RJ, Brazil; Fax: þ 55 21 3233 1423; E-mail: [email protected] distally, to cytogenetic breakpoints. This makes it highly unlikely Received 15 December 2003; accepted 20 April 2004; Published that our data could be biased by somatic recombination online 10 June 2004 between breakpoint regions and these markers as it might occur

Leukemia Correspondence 1446 Table 1 Amplimers used for the amplification of chromosome 9 markers

Amplimers Amplimer Cytogenetic Size of amplified Type of repeat Annealing Maximum reference allocation of fragment (bp) temperature (1C) heterozygosity amplified marker

Mfd135CA* GDB: 180704 9q31.1 Dinucleotide 53 0.8380 Mfd135GT 116–150 C3B2-1* 9q31.3 105–137 Dinucleotide 52 0.8840 C3B2-2 GDB: 185718 Mfd 178CA* GDB: 180718 9q31.3 Dinucleotide 51 0.8030 Mfd178GT 187–203 Mfd94CA* 9q33.1 135–159 Dinucleotide 55 0.8050 Mfd94GT GDB: 180558 Mfd77CA* GDB: 180555 9q33.2 Dinucleotide 55 0.5530 Mfd77GT 89–97 GSN.PCR1.1* 9q33.2 111–147 Dinucleotide 53 0.7610 GSN.PCR1.2 GDB: 178525 1627-1* GDB: 185720 9q33.3 Dinucleotide 55 0.8100 1627-2 136–154 DBH.PCR2.1* 9q34.2 235–280 Dinucleotide 65 0.7238 DBH.PCR2.2 GDB: 196477

*Sense primer 50 labelled with 6-FAM. bp ¼ base pairs; GDB ¼ Database (www.gdb.org).

Table 2 Amplimers used for the amplification of chromosome 22 markers

Amplimers Amplimer Cytogenetic Size of amplified Type of repeat Annealing Maximum reference allocation of fragment (bp) temperature (1C) heterozygosity amplified marker

F8VWFP.PCR4.1** GDB: 277263 22q11.1 Tetranucleotide 53 F8VWFP.PCR4.2 329–349 0.6100 TOP1P2.PCR1.1** 113–155 Dinucleotide 50 0.9200 TOP1P2.PCR1.2 GDB: 188759 22q11.2 CYP2D8P.PCR2.1** GDB: 180365 22q12.3 Dinucleotide 58 CYP2D8P.PCR2.2 108–130 0.8000 MB-1F** 217–219 Dinucleotide 53 0.4712 MB-1R GDB: 270294 22q12.3 IL2RB.PCR1.1** GDB: 188757 22q13.1 Dinucleotide 51 125–135 0.9100 IL2RB.PCR1.2 TG-01** GDB: 180405 22q13.1 Dinucleotide 56 0.9100 TG-02B 149–163 CYP2D8P.PCR1.1** GDB: 179878 22q13.3 Dinucleotide 51 CYP2D8P.PCR1.2 98–116 0.8000

**Sense primer 50 labelled with NED. bp ¼ base pairs; GDB ¼ Genome Database (www.gdb.org). TOP1P2.PCR1.1/1.2 amplify a marker that is distally located (bp 23 485 119–3 485 250) with respect to BCR (bp 21 847 702–21 982 691) according to http://www.ensembl.org/Multi/blastview.

between breakpoint regions and the very distant cytogenetic the M-BCR4 and ABL polymorphic alleles.7 Moreover, our own markers previously used for identifying the parental origin of data were not affected by any selection bias with respect to the chromosomes 9 and 22.2,5,6 Moreover, as our molecular establishment of hybrid cell panels because cell fusion experi- markers were also allocated outside the BCR and ABL , ments were successful with all patients that this was attempted. our selection criterion was unrelated to the presence of The fact that the paternal chromosome 9 and the maternal polymorphisms in these loci. This excluded the possibility that chromosome 22 are preferentially involved in t(9;22) was our results could be biased by this variability as it might have initially associated to imprinting,2 although monoallelic expres- been the case of previous studies in which patient selection sion of BCR and ABL was clearly ruled out.10,11 Thus, the relied on the presence of polymorphic CGG repeats at the 50- mechanisms responsible for the distinctive parental participation unstranslated BCR region,3 endonuclease cleavage sites inside of chromosomes 9 and 22 in t(9;22) remains to be elucidated.

Leukemia Correspondence 1447 Table 3 Hybrid cell classes and the number of hybrid cell lines per panel

Cell hybrid PCR amplifications Number of cell lines class With chromosome 9 amplimers With chromosome 22 amplimers Panel 1 Panel 2 Panel 3 Panel 4 Total

Mfd135CA/GT DBH.PCR2.1/2.2 F8VWFP.PCR4.1/4.2 MB-1F/1R

9+ + + ÀÀ412310 22+ ÀÀ + + 436215 9q+ + ÀÀ+ 242412 Ph+ À ++À 12238 9+/22+ + + + + 2 2228 Total 13 12 14 14 53 + ¼ Presence of amplified products; À¼absence of amplified products.

Table 4 Parental origin of chromosomes 9, 22, 9q+ and Ph

Panel Cell N Chromosome 9 markers Chromosome 22 markers hybrid class Proximal to 9q34.1 Distal Proximal Distal to 22q11.2

Mfd135 C3B2 Mfd178 Mfd94 Mfd77 GSN 1627 DBH F8VW TOP CYP2 MB IL2 TG CYP1

1 9+ 04 M M M M M M M M NEG NT NT NEG NT NT NT 2 9+ 01 M M M M M M M M NEG NT NT NEG NT NT NT 3 9+ 02 M M M M M M M M NEG NT NT NEG NT NT NT 4 9+ 03 M M M M M M M M NEG NT NT NEG NT NT NT

1 22+ 04 NEG NT NT NT NT NT NT NEG P P P NI P P P 2 22+ 03 NEG NT NT NT NT NT NT NEG P P P NI NI NI P 3 22+ 06 NEG NT NT NT NT NT NT NEG P P P NI P P P 4 22+ 02 NEG NT NT NT NT NT NT NEG NI P P NI P P P

1 9q+ 02 P P P P P P P NEG NEG M M NI M M M 2 9q+ 04 P P P P P P P NEG NEG M M NI NI NI M 3 9q+ 02 P P P P P P P NEG NEG M M NI M M M 4 9q+ 04 P P P P P P P NEG NEG M M NI M M M

1 Ph+ 01 NEG NEG NEG NEG NEG NEG NEG P M NEG NEG NEG NEG NEG NEG 2 Ph+ 02 NEG NEG NEG NEG NEG NEG NEG P M NEG NEG NEG NEG NEG NEG 3 Ph+ 02 NEG NEG NEG NEG NEG NEG NEG P M NEG NEG NEG NEG NEG NEG 4 Ph+ 03 NEG NEG NEG NEG NEG NEG NEG P NI NEG NEG NEG NEG NEG NEG Total 45 M ¼ maternal allele; P ¼ paternal allele; N ¼ number of hybrid cells tested; NI ¼ noninformative; NT ¼ not tested; NEG ¼ absence of amplified products.

Acknowledgements Supplementary Information

This work was supported by Instituto Nacional de Caˆncer (INCA) Supplementary information is available on the Leukemia and Fundac¸a˜o Ary Frauzino (Rio de Janeiro, Brazil). We are website (http://www.nature.com/leu/). grateful to Drs Arthur Moellman and Claudete Klumb, to other members of the staff of the Haematology Service-INCA and the Bone Marrow Transplantation Centre-INCA for samples and References clinical data. We are grateful to Claudio Vieira da Silva for technical support and to Amersham Biosciences for lending the 1 Barnes DJ, Melo JV. Cytogenetic and molecular genetic aspects of chronic myeloid leukemia. Acta Haematol 2002; 108: 180–202. MegaBACE Genetic profiler software. This work was approved by 2 Haas OA, Argyriou-Tirita A, Lion T. Parental origin of chromosomes the local Committee of Medical Ethics in accordance with the involved in the translocation 9;22. Nature 1992; 359: 414–416. guidelines of the Helsinki declaration. 3 Riggins GJ, Sherman SL, Phillips CN, Stock W, Westbrook CA, Warren ST. CGG-repeat polymorphism of the BCR gene rules out 1 R Olicio 1Genetics Division, Instituto Nacional de Caˆncer, predisposing alleles leading to the . MB Rivero1 Rio de Janeiro, Brazil; and Genes Chromosomes Cancer 1994; 9: 141–144. HN Seua´nez1,2 2Department of Genetics, Universidade Federal 4 Litz CE, Copenhaver CM. Paternal origin of the major breakpoint cluster do Rio de Janeiro, Rio de Janeiro, Brazil region in chronic myeloid leukemia. Blood 1994; 83: 3445–3448.

Leukemia Correspondence 1448 5 Maserati E, Seghezzi L, Pasquali F, Locatelli F. Parental origin of 8 Cross NC, Melo JV, Feng L, Goldman JM. An optimized multi- chromosomes 9 and 22 involved in the Ph chromosome transloca- plex polymerase chain reaction (PCR) for detection of BCR-ABL tion in chronic myelocytic leukemia. Cancer Genet Cytogenet fusion mRNAs in haematological disorders. Leukemia 1994; 8: 1998; 107: 151–152. 186–189. 6 Nakamura H, Itoyama T, Niikawa N, Sadamori N, Tomonaga M. No 9 Jankovic´ GM, Cˇ olovic´ MD, Bogdanovic´ AD, Cˇ olovic´ NR, Jankovic´ parental origin bias for the rearranged chromosomes in myeloid SJ. Parental reciprocation in t(9;22) vs genomic imprinting. leukemias associated with t(9;22), t(8;21) and t(15;17). Leukemia Leukemia 1996; 10: 1402. Res 1998; 22: 793–796. 10 Fioretos T, Heisterkamp N, Groffen J. No evidence for genomic 7 Melo JV, Xiu-Hua Y, Diamond J, Goldman JM. Balanced parental imprinting of the human BCR gene. Blood 1994; 83: 3441–4344. contribution to the ABL component of the BCR-ABL gene in chronic 11 Melo JV, Xiu-Hua Y, Diamond J, Goldman JM. Lack of imprinting myeloid leukemia. Leukemia 1995; 9: 734–739. of the ABL gene. Nat Genet 1994; 8: 318–319.

Biochemical pathway of antigen processing by HLA class II molecules in B cell lymphomas

Leukemia (2004) 18, 1448–1450. doi:10.1038/sj.leu.2403384 peptide-loaded HLA-DR molecules at the cell surface in various Published online 20 May 2004 types of B cell lymphomas and compared them with normal B lymphocytes from spleen and peripheral blood. Lymph node TO THE EDITOR biopsies were obtained from 16 patients with four diffuse B cells (DLCL), four mantle cells (MCL), four follicular (FL), and Non-Hodgkin’s B cell lymphomas are characterised by the four lymphocytic (LL) lymphomas. Diagnosis was established expression of both human leukocyte antigen (HLA) class I and according to the WHO classification. B lymphocytes, except for class II molecules. As HLA class II molecules are constitutively peripheral blood (positive purification), were purified by expressed only in professional antigen-presenting cells (APCs), B negative immunomagnetic selection (final population 498% lymphomas are considered as professional APCs. Contrary to the CD19 þ cells). A total of 16 lymphomas expressed HLA-DR majority of tumour cells that usually express only HLA class I molecules at their surface (Table 1). Different populations of molecules, B cell lymphomas can also be considered as an HLA-DR complexes were exposed at the cell surface of B original model for cellular immunotherapy involving the CD4T lymphocytes. These include peptide-loaded HLA-DR complexes lymphocyte component in direct contact with tumour cells. We and HLA-DR/CLIP complexes. Strikingly, four MCLs and three and others previously reported that CD4T lymphocytes may play DLCLs presented no or few HLA-DR/CLIP complexes at their a direct role by cell–cell contact and could not only directly surface when compared to other lymphomas and normal B target tumour cells expressing HLA class II molecules (review by Wang1) but also by differentiation or cell cycle progression. Moreover, modifications of the second messengers in the signalling pathway via class II HLA-DR molecules were Table 1 Purified B lymphocytes were analysed by flow cytometry previously described in B lymphomas.2 This could be of interest in the field of cellular immunotherapy where APCs and Lymphoma DR DR-CLIP Ratio (%) DR-CLIP/DR particularly dendritic cells are used to generate specific antitumour CD4 and CD8T lymphocytes. DLCL 1 750 87 11.6 HLA class II molecules biosynthesis begins in the endoplas- DLCL 2 283 4 1.5 mic reticulum. They are subsequently targeted at compartments DLCL 3 312 13 4.2 intermediately between late endosomes and lysosomes called DLCL 4 1560 10 0.6 MCL 1 2797 12 0.4 MIIC (for MHC class II compartment) where antigen loading can MCL 2 698 0 0 take place, before targeting to the cell surface (review by MCL 3 1067 1 0.1 3 Alfonso and Karlsson ). During this intracellular pathway, the MCL 4 1235 5 0.4 Invariant chain (Ii) blocks the peptide-binding groove of HLA- LL 1 1685 37 2.2 DR molecules and is sequentially degraded until the generation LL 2 1780 66 3.7 of a peptide called CLIP (Class II associated Invariant chain LL 3 488 15 3.1 LL 4 212 10 4.7 peptide). The loading of antigenic peptides instead of CLIP is FL 1 1055 9 0.8 then catalysed by HLA DM in the MIIC, which is in turn FL 2 712 43 6 modulated by HLA DO (selectively expressed in B lymphocytes FL 3 1374 34 2.5 and a specific negative modulator of HLA DM outside the MIIC). FL 4 1086 23 2.1 In this study we explored the key components of the Control 1 899 34 3.8 biochemical pathway leading to the expression of antigenic Control 2 1312 22 1.7 Control 3 1892 40 2.1 Control 4 1190 75 6.3 Correspondence: Dr F Garban, De´partement de cance´rologie et Mean fluorescence intensity for total HLA-DR (mAb L243) and HLA d’he´matologie, CHU de Grenoble, BP 217, 38 043 Grenoble Cedex 9, DR/CLIP (mAb CerCLIP) is presented for each sample and were France; Fax: þ 33 4 76 76 56 61; obtained by subtracting the value of the isotype control to the E-mail: [email protected] sample value. The ratio of HLA-DR/CLIP vs total surface HLA-DR was 3N. Magniez and C. Roucard have equally contributed to this work. calculated. Controls 1 and 2 are from spleen, controls 3 and 4 are from Received 31 July 2003; accepted 12 February 2004; Published online peripheral blood. DLCL, diffuse large cell lymphomas; LL, lymphocytic 20 May 2004 lymphomas; FL, follicular lymphomas; MCL, mantle cell lymphomas.

Leukemia