Cytogenetic and molecular genetic characterization of the ‘high hyperdiploid’ B‐cell precursor acute lymphoblastic leukaemia cell line MHH‐CALL‐2 reveals a near‐haploid origin

Abstract

British Journal of HaematologyVolume 154, Issue 2 p. 275-277 correspondenceFree Access Cytogenetic and molecular genetic characterization of the ‘high hyperdiploid’ B-cell precursor acute lymphoblastic leukaemia cell line MHH-CALL-2 reveals a near-haploid origin Hanan E. Aburawi, Hanan E. Aburawi Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this authorAndrea Biloglav, Andrea Biloglav Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this authorBertil Johansson, Bertil Johansson Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this authorKajsa Paulsson, Kajsa Paulsson Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this author Hanan E. Aburawi, Hanan E. Aburawi Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this authorAndrea Biloglav, Andrea Biloglav Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this authorBertil Johansson, Bertil Johansson Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this authorKajsa Paulsson, Kajsa Paulsson Department of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, Lund, Sweden.E-mail: kajsa.paulsson@med.lu.seSearch for more papers by this author First published: 26 April 2011 https://doi.org/10.1111/j.1365-2141.2011.08601.xCitations: 5AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Childhood acute lymphoblastic leukaemia (ALL) may be divided into subgroups with different prognostic and clinical features based on the presence of specific acquired genetic aberrations. Cases with 51–67 chromosomes constitute the high hyperdiploid subgroup, characterized by non-random gains of chromosomes X, 4, 6, 10, 14, 17, 18, and 21 (Paulsson & Johansson, 2009). Additional genetic aberrations, such as microdeletions and point mutations, are sometimes present, but how they contribute to the leukemogenic process remains largely unknown. Functional in vitro studies of high hyperdiploid ALL are hampered by a lack of such cell lines, due to high hyperdiploid cells being exceedingly difficult to culture. In fact, to the best of our knowledge, only one cell line, MHH-CALL-2, is available from this ALL subtype. The aim of the present study was to perform genetic studies of MHH-CALL-2 to enable future use of this cell line as a model system for in vitro studies of high hyperdiploid childhood ALL. The MHH-CALL-2 cell line was established in 1993 from a peripheral blood sample obtained from a Caucasian 15-year-old female diagnosed with B-cell precursor ALL (Tomeczkowski et al, 1995). The karyotype has variably been described as 52,XX,+X,+10,+18,+18,+21,+21 (Tomeczkowski et al, 1995) and 52,XX,+8,+10,+18,+18,+21,+21 (The DSMZ database; http://www.dsmz.de/), i.e., with ambiguity regarding trisomy X/8. We performed G-banding analysis according to standard methods, showing a modal number of 51, with trisomies X and 18 and tetrasomy 21, and addition of a large marker chromosome. Multicolour-fluorescence in situ hybridization (M-FISH) using combined binary ratio labelling probes (Raap & Tanke, 2006) confirmed these gains and showed that the marker was a derivative chromosome 18 resulting from an unbalanced t(15;18) (Fig 1A, B); the latter was confirmed with whole chromosome painting probes (Applied Spectral Imaging, Neckarhausen, Germany). Because the translocation was unbalanced, single nucleotide polymorphism (SNP) array analysis (see below) enabled mapping of the breakpoints to 15q13.1 and 18q22.1 (Fig 1C). Taken together, the cell line MHH-CALL-2 was found to have the karyotype 51,XX,+X,+18,+der(18)t(15;18)(q13.1;q22.1),+21,+21. Neither +8 nor +10 were seen, in contrast to previous reports. Interphase FISH using a centromeric probe for chromosome 10 and the AML1/ETO (RUNX1/ RUNX1T1) probe set for chromosomes 8/21 (Abbott Molecular Inc., Des Plaines, IL, USA) confirmed disomy 8 and 10. Figure 1Open in figure viewerPowerPoint Results of cytogenetic, fluorescence in situ hybridization (FISH), and SNP array analyses of MHH-CALL-2. (A, B) G-banding and multicolour-FISH showing the karyotype 51,XX,+X,+18,+der(18)t(15;18)(q13.1;q22.1),+21,+21. The der(18)t(15;18) is indicated by an arrow in panel A. (C) SNP array results. Top panels show the logR ratios, where copy number changes are shown as ratios above (gain) or below (loss) zero. Lower panels show the B allele frequency (BAF) values, indicating genotypes. Homozygous SNP reporters have values of 0 or 1; heterozygous SNP reporters have values of c. 0·5. The top chromosome is chromosome 18. Four copies are present from 18pter-q22.1, visible as increased logR ratios and retained heterozygosity (‘2:2’ tetrasomy). 18q22.1-qter is present in three copies, visible as increased logR ratios and skewed BAF values of c. 0·33 and c. 0·67. The lower chromosome is chromosome 9, which is a uniparental isodisomy, visible as logR ratios of c. 0 and complete loss of heterozygosity. A small homozygous deletion, including CDKN2A, is seen in 9p21, visible as decreased logR ratios and random BAF values due to the low signal intensities. SNP array analysis was done utilizing the Illumina 1M-quad bead Infinium BD BeadChip platform (Illumina, San Diego, CA, USA) as previously described (Paulsson et al, 2010). Copy number variants were excluded based on comparison with the Database of Genomic Variants (http://projects.tcag.ca/variation/). The analysis showed trisomy X and tetrasomies 18 [including one extra normal copy and the +der(18)t(15;18)] and 21, with doubling of both homologues (‘2:2’ tetrasomy). It also revealed what we would like to denote total ‘UPIDity’, i.e., all disomic chromosomes were uniparental isodisomies (UPIDs), detectable as complete loss of heterozygosity without concurrent copy number loss. Importantly, we have previously shown that only a minority of the disomies in high hyperdiploid ALL are UPIDs (Paulsson et al, 2010). Instead, universal UPIDity is suggestive of a near-haploid (23–29 chromosomes) or low hypodiploid (30–39 chromosomes) origin (Onodera et al, 1992; Paulsson & Johansson, 2009). Near-haploidy/low hypodiploidy occurs in <1% of childhood ALL (Harrison et al, 2004). More than half of such cases harbour a second clone with a chromosomal content in the high hyperdiploid range, where the original leukaemic cell has doubled all of its chromosomes (Harrison et al, 2004). In some patients, the original clone is no longer present and only the doubled clone, with a high hyperdiploid modal number, is seen. However these cases are generally considered to belong to the near-haploid/hypodiploid subgroup and are not ‘true’ high hyperdiploid ALL (Paulsson & Johansson, 2009). Thus, our findings suggest that MHH-CALL-2 should not be regarded as a high hyperdiploid cell line in spite of its chromosome number, but rather as a near-haploid. For patients, this distinction is important to make, as near-haploid childhood ALL has a dismal prognosis, with event-free survival rates of only c. 30% 3 years after diagnosis, in contrast to 75–80% for high hyperdiploid cases (Moorman et al, 2003; Harrison et al, 2004; Forestier et al, 2008). Reverse transcriptase-polymerase chain reaction was performed according to standard methods for ETV6/RUNX1 [t(12;21)(p13;q22)], BCR/ABL1 p190 and p210 [t(9;22)(q34;q11)], and TCF3/PBX1 [t(1;19)(q23;p13)] and Southern blot analysis was done using a probe for MLL (11q23). No abnormalities were detected, in line with the fact that these aberrations are categorized as ALL subgroups distinct from high hyperdiploidy. Furthermore, mutation analyses of FLT3, NRAS, KRAS, and PTPN11, performed as previously described (Paulsson et al, 2008), were all negative. The SNP array analysis showed that the breakpoints of the der(18)t(15;18) were at 27 636 979 bp (NCBI 36.1) in chromosome 15 and at 60 525 373 bp in chromosome 18; neither breakpoint was in a gene. The SNP array analysis also revealed a small homozygous deletion of the region 21 813 952–22 078 260 bp in 9p21 (Fig 1C), including CDKN2A, in line with the results from Mullighan et al (2007) showing that this gene is almost always lost in cases with <46 chromosomes. Taken together, our data suggest that the MHH-CALL-2 arose by a multistep process, with (i) initial near-haploidy with 26 chromosomes followed by (ii) CDKN2A deletion, (iii) doubling of the chromosomal content to 52 chromosomes, (iv) formation of a der(18)t(15;18) and (v) loss of an X chromosome. At least the first three steps must have occurred before establishment of the cell line, as the original leukaemia had 52 chromosomes. Considering the previously reported G-banding results, which deviate from ours by addition of a chromosome and no der(18) t(15;18), one may speculate that loss of one of four X chromosomes and acquisition of der(18)t(15;18) occurred after MHH-CALL-2 was established; i.e., they were not present in the patient. To summarize, the present study clearly shows that the MHH-CALL-2 cell line is in fact derived from a near-haploid childhood ALL, not a high hyperdiploid leukaemia as previously surmized. It is thus unsuitable as a model system for functional studies of the latter leukaemic subtype. Unfortunately, this also means that there is no high hyperdiploid ALL cell line available, hampering investigations of this large group of childhood ALL. Acknowledgements The SNP array experiments were performed by Sciblu Genomics at Lund University, Lund, Sweden. 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Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume154, Issue2July 2011Pages 275-277 FiguresReferencesRelatedInformation