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From www.bloodjournal.org by guest on April 29, 2017. For personal use only. Comparative Genomic Hybridization in Chronic B-Cell Leukemias Shows a High Incidence of Chromosomal Gains and Losses By Martin Bentz, Karin Huck, Stanislas du Manoir, Stefan Joos, Claudius A. Werner, Konstanze Fischer, Hartmut Dohner, and Peter Lichter In chronic B-cell leukemias, fluorescence in situ hybridization revealedchromosomalgainsandlosses not detectedby has greatly improved the ability to detect certain chromobanding analysis.In 8 of these 13 cases, discrepancieswere somal aberrations,as cellsin all phases ofthe cell cycle are furtherinvestigatedusingothermethods, and in allinanalyzed. To obtain a comprehensiveview of chromosomal stances, the CGH findings were confirmed. A limitation of gains and losses, we applied the recently developed techdetecting small deleted regions by CGH was found in one nique of comparative genomic hybridization (CGH) to 28 paexample of 18p.In conclusion, ourdata showthat the results tients with chronic B-cell leukemias. CGH results were comof banding analysesin chronic B-cell leukemias often do not pared with those obtained by chromosome banding analysis reflect the chromosomal changes in the predominant cell and interphase cytogenetics.In 19 of the 28 cases, chromoclone. Thismay be one explanation for the as yet poor corresomal imbalances were detected, including amplified DNA lation between cytogenetic findings and clinical course in sequences in three instances. The most common aberrations this group of neoplasms. included gains of chromosomal material on 8q and12as 0 1995 by The American Societyof Hematology. well aslossesof6q. l l q , 13q. and 17p. In 13cases,CGH C HRONIC B-CELL lymphocytic leukemia (B-CLL) is the most prevalent leukemia inadults and accounts formore than 30% of all leukemiacasesinEuropeand North America. Although chromosomal banding analysis has shown a number of recurrent aberrations in B-CLL, the sole prognosticallyrelevant chromosome abnormalitythat has been identified in multivariate analyses is a complex karyotype associatedwith an inferior outcome.‘ In contrast, in acute leukemias a number of specific chromosomal changes with high prognostic impact have been The difference between these two groups of leukemias may be due to difficulties inidentifying aberrations of the leukemiccell clone in B-CLL.Even when B cell-specific mitogensare used, the leukemic cells may exhibit a low proliferative activity, and in about half of the cases, no chromosomal abnormalities This are is, at part, least in due to mitotic cells arising from nonleukemic T lymphocyte^.^^^ A number of fluorescence in situ hybridization (FISH) studies investigating interphase cells have shown the presence of cell clones carrying chromosomalaberrations in cases where no abnormalities were found by banding analysis: the incidence of both trisomy 12”” and deletions of part of the long arm of chromosome 1 3I4.l5is substantially higher than found by banding techniques. However, interphase FISH depends on the preknowledge of candidate regions and on the availability of suitable DNA probes. Furthermore, only very few From the Deutsches Krebsforschungszentrum, Abt. “Organisation komplexer Genome”, Heidelberg: and the Medizinische Klinik und Poliklinik V, Universirat Heidelberg, Heidelberg, Germany. Submitted November IO, 1994; accepted January 26, 1995. Supported by grants fromthe Fritz Thyssen Stifung and the European Community (GENE-CT 930055). Address reprint requests to Martin Bentz, MD, Deutsches Krebsforschungszentrum, Abt.“Organisation komplexer Genome”, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. The publication costsof this article were defrayedin part b y page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8512-0015$3.00/0 3610 chromosomal regions can be examined in a single experiment. With the recently developed methodof comparative genomic hybridization (CGH),I6 tumor genomes can be rapidly tested for the presenceof chromosomal imbalances (such as partial or complete monosomies and trisomies).“”* Differentially labeled tumor and genomic control DNA are cohybridized to normal metaphase chromosomes under suppression conditions and detected using different fluorochromes. The ratios of the fluorescence intensities generated by tumor and control DNAindicate regions with a normalgenomic content as well as overrepresented and underrepresented sequences within the tumor DNA. We used CGH to identify copy number changes of chromosomal regions in 28 patients with chronic B-cell leukemias. The results were compared with those obtained by Gbanding analysis and interphase cytogenetics. PATIENTS AND METHODS Patient Samples Twenty-eight patients with chronic B-cell leukemias [25 with BCLL and three with B-cell prolymphocytic leukemia (B-PLL): patients 6 , 9, and 28) were examined. In 26 cases, blood specimens were obtained, and in two patients (patients 5 and 1 l), cells from splenectomy samples were obtained. An aliquot of each sample was used for short-term culture and subsequent G-banding analysis. A and genomic DNA was prepared second aliquot was stored -70°C at K digestionandphenol-chloroformextraclaterusingproteinase tion.’” Patients were included in this study depending on the availability of frozen cell material. G-Banding Analysis Mononuclear cells were purified from a Ficoll gradient and cultured at a concentration of1O6 cells per milliliterusing the following mitogens: antihuman immunoglobulin M (IgM; Jackson Immunoresearch Laboratories, West Grove, PA), B-cell growth factor (10%; Cellular Products Inc. Buffalo, NY), and phorbol-l2-myristate-l3acetate (1 to IO ng/mL) or calcium ionophore ( l pmoVL; Calbiochem, La Jolla,CA). Harvesting, slide preparation, and banding were performed as published previou~ly.’~ Between 10 and30 metaphases (median, 20) were analyzed per case. Comparative Genomic Hybridization Hybridization was performed as d e ~ c r i b e d . ” ~Bri ~ ”efly, normal human genomic DNA (control DNA) was labeled with digoxigeninBlood, Vol 85, No 12 (June 15), 1995: pp 3610-3618 From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 361 1 CGH IN CHRONIC B-CELL LEUKEMIAS 11-deoxyuridine triphosphate (dUTP; Boehringer Mannheim, Mannheim, Germany), and tumor DNA was labeled with biotin-16-dUTP (Boehringer Mannheim) by a standard nick translation reaction; DNase I concentration was adjusted to result in an average fragment size of 300 to 1,OOO bp. One microgram of labeled tumor DNA, 1 pg of labeled control DNA, and 70 pg of human Cot I DNA (BRL Life Sciences, Gaithersburg, MD) were cohybridized to slides with metaphase cells prepared from blood of a healthy donor. After hybridization for 2 to 3 days and posthybridization washes, test and control DNA were detected via fluorescein isothiocyanate (FITC) and rhodamine, respectively. Chromosomes were counterstained with 4,6-diamidino-2-phenylindole(DAPI; 200 ng/mL), resulting in a Q banding-like pattern that was used for chromosome identification. Digital Image Analysis Image acquisition, processing, and evaluation were performed as described previo~sly.''.~'Images were acquired using an epifluorescence microscope (Axioplan; Zeiss, Oberkochen, Germany) equipped with a cooled CCD camera (Photometrics, Tucson, AZ). FITC and rhodamine images of the tumor and control DNA were used for quantitative analysis. Ratio profiles of each individual chromosome were calculated using a dedicated software?' For each case, the mean ratio profiles of between 4 and 10 metaphase cells were computed. Thresholds for the identification of imbalances were defined as 0.75 (lower threshold) and1.25 (upper threshold). These are the theoretical values that are expected in a diploid tumor cell population for a monosomy or a trisomy of a certain chromosomal region in 50% of the test cells. Chromosomes or chromosomal regions with fluorescence ratios outside of this interval were considered to be over- or underrepresented, respectively. In studies comparing CGH results with data obtained by other cytogenetic methods, these thresholds were proven to provide robust diagnostic criteria,2I-23 Interphase Cytogenetics The following probes were used for interphase cytogenetics: chromosome-specific plasmid libraries pBS7 and pBS8-; cosmid clones cos-myc72 (8q24)25; a yeast artificial chromosome (YAC) clone mapping to chromososomal band 11q2326;chromosome 12-specific alphoid probe D12Z3 (Oncor Inc, Gaithersburg, MD); phage contig spanning the whole RBI tumor suppressor gene (13q14), provided by Thaddeus Dryja, Cambridge, MAZ7;and four overlapping cosmid clones containing the p53-encoding genomic region (17~13).~* Hybridization was performed on methanoUaceticacidfixed cells as de~cribed.'~ With the exception of one case where only 150 cells were analyzed (hybridization with cos-myc72 in case 6), in all other instances at least 200 cells were evaluated. In case ofthe YAC probe, Ah-polymerase chain reaction (FTR) amplification of the insert was performed as described el~ewhere.~'When interphase analysis was performed with cosmid or phage probes to look for deletions, a further cosmid probe was included as an internal control for hybridization effi~iency.'~ CGH, chromosomal banding analysis, and interphase cytogenetics using probes for chromosomes 12, 13q14, and 17q13 were performed in a blind fashion. RESULTS G-Banding Analysis G-banding analysis was performed in all 28 patients. In five patients, no metaphase cells were found. Thirteen cases exhibited normal karyotypes. In each of the remaining 10 patients with clonal abnormalities, chromosomal imbalances were present. The complete karyotypes are listed in Table l. CGH Analysis In 9 of the 28 patients, no chromosomal gains or losses were found by CGH analysis. In the other 19 patients, the following chromosomal imbalances were identified in more than one case: loss of chromosomal material on 17p (nine cases), 1lq (four cases), 6q (three cases), 13q (three cases), and 18p (two cases); and gains of chromosomal material on 8q (three cases, one of whichwas an amplification), 3q (two cases), 12p (two amplifications at different sites of this chromosome arm in a single patient; Fig 1A through D), trisomy 12 (two cases), 15q (two cases, one of which exhibited an overrepresentation of the whole chromosome 15), and 17q (two cases). The following imbalances of chromosomal regions were detected only once: overrepresentations of 3p, 12q, 18q, and 22q, as well as whole chromosome 19; underrepresentations of 3p, 4q, 7q, 8p, lOq, 15q, and 16p. The complete data for all patients are shown in Table 1. Comparison of CGH and Banding Data Additional Aberrations Found by CGH In 13 of the 28 cases, additional imbalances were detected by CGH (Table 1). These discrepancies can be classified in four different categories. (1) Although normal karyotye was found on banding analysis, clonal abnormalites were found by CGH (six cases: 2, 8, 18, 23, 24, and 27). (2) No metaphase cells were obtained after short-term culture (three cases: 10, 14, and21). (3) By banding analysis, complex karyotype abnormalities were found. The chromosomal origin of some of these aberrations (eg, marker chromosomes) could not be identified by banding analysis in three cases (cases 6, 22, and 28), and in two of these cases, DNA amplifications were detected by CGH. (4) The imbalanced chromosomal region was small and involved in a cryptic translocation (one case, 5). Interphase analysis using specific DNA probes directed against chromosomal regions with discrepant findings was performed in 8 of these 13 cases. The results were as follows. Case 2. In this patient, banding analysis showed a normal karyotype in 15 metaphases. By CGH, a loss of chromosomal material on 1 l q (bands 1lq14 to 1lq25) was detected. Interphase analysis using a YAC probe mapping to chromosomal band 1lq23 showed only one signal in 80.4% of cells (Fig 2F). Case 5. By G-banding, a de1(7)(q32)was found asthe sole chromosomal imbalance. In contrast, CGH analysis showed an overrepresentation of chromosomal material on 8q (bands 8q23 to qter). Furthermore, the terminal deletion on chromosome 7 extended up to chromosomal band 7q31 by CGH analysis. The overrepresentation of the terminal part of chromosome 8 was confirmed by FISH analysis with the cosmid probe cos-myc72 mapping to chromosomal band 8q24: three hybridization signals wereseenin69.4% of interphase cells. Dual color FISH using chromosome-specific DNA plasmid librariesfor chromosomes 7 and 8 resolved both discrepanciesseenin this case:onthefewmetaphasecells From www.bloodjournal.org by guest on April 29, 2017. For personal use only. BENTZ ET AL 3612 . * c 3 .- 0: .B$ gg g 1 6 1 I ._ p -X P B g V g Pmm V5 E E 0 z E S X ; g :o 0 i c P V .- E c F g ;I .iJ -- v) 'E t M g n '9m S r " go pm m n c n c m c 0 Q a 0 8 4 Sd 3E 1 r a nA z ;g F P o + I O = 0 z I 2 U U .- c F I o o o ~ o I jt S E E l 1 0 0 U) m U m c 1 I a 0 'c 3,. m E o 5 : W c .- *I V 8 c z .-c O a zz E! I E i t I I 1 d : U d m + ; l . - + S; S c .-cf S F N c S '+ L c .- d S :g U m E+ o t .gz 1 e 5 1 4 1 0 1 0 0 - m 1 + 5 6U z d F U .-U 0 N I I + g;? 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I SI 3 iz .- e 1 W BC n m v i m 5 P W m $2 C .-c .Ew c 5 ._ v) mQ 0 C U)_m L 2 cm 5 U z 5 E .- mc m o P I L &.S2 I Um N N I .-U F V m 0 U L m m 5 S $ 2 u, +- -a 5 &E?" a - .E E + - c $ c c w w gx N ' U L Q I F A P .E m = c c S I r d e c - 3 5 5s n d - m 5 m c U) C c 0 % ._ $ U a P g 3 .-e 8 D .l W c c c e 1 2 0 ..P c, .- '5 I- g;g a W c P -1" C W 5 I v) c U P+ P vg I o o + E T --f .-- .- U N N g I E " E+" g 3 8a " .- 2 Ep5 U M n 0 Q z.- ._ M ' P i W 'E .- E e- - F C .- c C U m P E D cm at [ I9 t0 E n g? 0 z 0 %t = g c m V 0 z W - I $ 1 = 1 $ 1 I I -g I 1 -6 - r,$ z C a, m - U Fe:: B & ~ mm m '3u m m 5 cmzmcB 'E422 zgs.r m " '" j C. S L sf r .q, c U) B 022 g s g r - m = S 3 P :$g z :=E 235 =g =n F.. z y ; $ 2 2 .-g $ .H?c3B t3E ct3,z4 p3 p " d cI c ,,=g.= .;jegg-= p' z 8""..m,g %$:+g Um ~ . w c a ~"! Q E + Bm m~ 0 = ~ = - ~ ~ E~ { :~ Sg-5 ~ Q fc' cx $ .B $ Q j 5 o m 5 e : ;$ k ;'g g: g p :'ii m4::44444+-5: c zz?i)@ p# $SPOZZ$$$SS $$%$S U z $75 g$;$ $ O S !. g 2 r3 5 . g z g 'ii z z 3 E E m ,E 0 ,E 4 .e g;, 0" 2; g+! f .p$ 3 c - 2 n m m $ g s z 2.;;g,s c .S e zz n . - N m u m W - m mE.=zFzE$Fm2 :: W r i K M R l % % %WL p $ P1 4c * c m m.9 g g 0 B f I - - , C ~ S O ? " = C * V From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 3613 CGH IN CHRONIC B-CELL LEUKEMIAS assessable after hybridization,the additional chromosome 8 material was translocated to the terminal part of the derivative chromosome 7 (Fig 2A). The resulting banding pattern of this derivativechromosome[der(7)t(7;8)(q31;q23)]isindistinguishable from a terminal deletion with breakpoiit the in 7q32. Case 6. In this case, complex karyotype abnormalities were found by banding analysis. In many of the imbalances, the chromosomal origin of the imbalanced material was not identified. By CGH, overrepresentation of the whole chromosome15and material on 12qand 17q, as well as an amplificationmapping to chromosomalband 8q24, were seen.By interphase analysis with the cosmid probe containing the MYC gene, an amplification was detected in 5% of cells as a large, disperse hybridization signal. Inmore than 90%of cells, two focal hybridization signals were seen. A low copy numberamplificationwasalsosuspected by Southern blot analysis (data not shown). Case 8. This patient exhibited anormal karyotyp on banding analysis (21 metaphases analyzed). CGH analysis showed additional material on chromosomes 8 (8q13 to 8qter) and 17q, as well as a loss of material on 16p, 17p, and 18p. Interphase analysis using the cosmid contig spanning the p53 gene on chromosome 1 7 ~ 1 3demonstrated a single hybridization signal in 88.8% of cells. Case 10. In three instances, metaphase preparation was not possible for banding analysis in this patient. CGH analysis showed a deletion on chromosome 13 (bands 13q14 to q21). This wasconfirmed by interphase analysis with the phage contig spanning the RBI gene on chromosome band 13q14: 67% of cells exhibited only one hybridization signal with this probe. Case 21. No metaphase cells were available for banding analysis. By CGH, loss of chromosomal material on l l q and 13q, as well as a trisomy 12 and overrepresentation of chromosomal material on 22q, were found. By interphase cytogenetic analysis, both the trisomy 12 (82% of cells exhibiting three signals) and the deletion on 13q (48.7%of cells showing one signal with the RBI probe) were confirmed. Case 22. In this case, a complex karyotype with a marker chromosome andahomogeneously staining regionwas found by banding analysis. CGH showed a number of imbalances, including two amplification sites (Fig 1). The loss of FISH: chromosomal material on 17p wasconfirmedby 86.9% of cells exhibited one signal after hybridization with the probe spanning the p53 gene. Case 23. G-banding analysis showedanormalkaryotype. By CGH,losses of chromosomal material on 4q(4q22q28), 6q(6q15-q22), and 17p were found. Again, hybridization was performed withthe p53 probe: 90.1% of the nuclei exhibited a single hybridization signal. Case 27. On G-banding, a normal karyotype was found. CGH showed interstitial deletions on 1lq and 13q. The latter was confirmedby FISH using the phage contig spanning the RBI gene; in 8 1.6% of interphase cells, only one hybridization signal was seen. Imbalances Found by Banding Analysis, But Missed by CGH In two cases, imbalances that had been detected by banding analysis were not diagnosed by CGH. Case 12. In this case, an unbalanced whole arm translocation der(l7; 18)(qlO;qlO) resulting in the loss of one copy of 17p and 18p was diagnosed on banding analysis. By CGH, the ratio profile of the short arm of chromosome 18 was shifted towards underrepresentation; however, the threshold for underrepresentation was not reached. By visual inspection, a less intensive FITC fluorescence of 18p was clearly visible. For explanation, see Discussion. Case 20. By banding analysis, metaphases with a normal karyotype and two clones with aberrations were found both had losses of 17p and 18p. In addition, one of these clones (5 of 21 metaphases) had a loss of 3p (whole arm), and the other clone (4 of 21 metaphases) had a loss of 3q (whole arm, two copies of 3p present). By CGH, the profile for chromosome 3was shifted towards underrepresentation without exceeding thethreshold. Interphase analysis with the p53 probe demonstrated that only 55% of cells belonged to one of the aberrant clones. As only part of the aberrant cells had losses of material on 3p or 3q, the percentages of cells carrying the respective imbalances were too low to be detected by CGH. Comparison of CGH With Interphase Cytogenetics In all patients, interphase cytogenetic analysis for the presence of a trisomy 12 as well as for allelic losses of the RBI and TP53 tumor suppressor genes wasperformed. These data have been published p r e v i ~ u s l y . ' ~ ~ ' ~ ~ ' ~ ~ ~ ~ Trisomy 12 In two of the 28 patients, a trisomy 12 had been diagnosed by interphase cytogenetic analysis (Table 1). In both cases, the overrepresentation of the whole chromosome 12was detected by CGH. Loss of the RB1 Gene In 9 of the 28 patients, a loss of one copy of the RB1 tumor suppressor gene had been detected in between 48.7% and 98.1% (median 79%) of interphase cells. By CGH, a loss of chromosomalmaterial involving band 13q14 was detected inonlythree patients (patients 10, 21,and 27). Interestingly, in each of the other six patients with an RBI deletion (patients 2, 7, 17, 23, 24, and 25), the ratio profiles exhibited a shift towards underrepresentation without reaching the diagnostic thresholds. This indicates that deletions involving 13q14 in CLL are often small. Loss of the TP53 Gene Of the 28 patients, 9 had anallelic loss of the TP53 gene in percentages of cells ranging from 24.5% to 90.4% (median, 64.5%; cases 6, 8, 9, 12, 14,20,22,23, and 28). In all cases with the exception of case 14, wherethe deletion was present in only 24.5% of cells, a loss of chromosomal material on 17p was detected by CGH. As shown by banding analysis, in most of these cases, the whole short arm of chromosome 17 was deleted. DISCUSSION CGH has recently been developed and has proven to be a powerful tool for the detection of chromosomal gains and From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 3614 losses in avarietyof tumors.16"X~Z2~'~~""X Using CGH, we identified numerous gains andlosses, as well as three amplification sites, in 28 patients with chronic B-cell leukemias. Many of theimbalances detected with CGHare well known to occur in this typeof leukemia': losses of chromosomal material on 6q, l l q , and 13q as well as gainsof material on chromosome 12. A loss of material on 17p has been found by our group in 17 of 100 patients.2x However, another imbalance, a gain of chromosomal material on 8q, which was detected in 3 of our 28 patients (Fig 2B), had not been described as a frequent structural aberration before. In two of these three cases, the presence of the additional chromosome 8 material was confirmed by other methods. In the third case, no material for further analysis was available. Interestingly, in all cases, the overrepresentation of chromosome 8q was not detected by banding analysis. In twopatients, amplifications ofchromosomal subregions were identified (Figs 1 and 2B). Whereas double minutes or homogeneously staining regions, the cytogenetic hallmarks of gene amplification, have been described in approximately 2% to 3% of acute myeloid leukemias," only a few cases of chronic B-cell leukemias with such abnormalities have been rep~rted.~' In one of our patients (patient 6), the c-myc protooncogene was amplified. This gene is most commonly involved in amplifications of myeloid leukemias:' and amplification of c-myc has been described in one case of BC L L b e f ~ r e . ~In' another patient (patient 22) of our series, two amplificationswere found, both mapping to the short arm of chromosome 12 (12~11-p12 and 12~13; Fig I). Overrepresentation of material on chromosome 12, namely a trisomy 12, is a very common chromosome abnormality in BCLL. Although the chromosomal region 12q13 to q22 has considered been pathogenetically thismost finding suggests that genes on 12pmight also play a significant role in the leukemogenesisof B-cell disorders. On chromosomal band 1 2 ~ 1 3several , candidate genes arelocalized: these include a cyclin gene (cyclin D2), fibroblast growth factor 6, lymphocyte activation gene 3, a G-protein, and the tumor necrosis factor receptor 1," as wellas the recently described ETS-like gene TEL, which is rearranged in some cases of chronic myelomonocytic l e ~ k e m i a . ~Band ' 12~12 is the chromosomal locus of the KRAS protooncogene, which BENT2 ET AL is known to be mutated in some casesof acute lymphoblastic leukemia." Comparison of CGH data and interphase cytogenetic results for trisomy 12and losses of chromosomearm 17p corresponded well. With the exception of one case showing a 17p deletion in only 24.5% of interphase cells, all abnormalities found by interphase analysiswere detected by CGH. The discrepancy found in this case is due to the thresholds used as diagnostic criteria (see above): if the genomes of less than 50% of cells harbor the imbalances, the respective profiles are likely not to reach the thresholds for under- and overrepresentation, respectively (see also case20). Although RBI deletions were found in percentages ranging between 48.7% and 98.1 %, six of nine allelic losses of this chromosomal regiondetected by interphase cytogenetics were missed by CGH analysis. This is most likely due to the small size of the respectivedeletions that may bebeyond the spatial resolution of the CGH analysis. With chromosome preparation techniques and evaluationprocedurescurrentlyused, the size required for the detection of an imbalance may be in the range of the short arm of chromosome 18: a deletion of this region was detected in two of our cases and missed in another case(case 12). Preparation of more elongated chromosomes may increasethespatialresolution. Other strategies to achieve this have been discussed in detail before.*' Comparison of the CGH data with the data obtained by banding analysis showed a high proportion of cases (1 3 of 28) where additional imbalances were found by CGH analysis. Amongthese, there weresix patients that had no chromosome aberrations on banding analysis. Other discrepancies were based on a failure of preparing metaphase cells; complexkaryotypes with markerchromosomes, in whichthe additional or deleted chromosomalregions could be mapped to specific regions of the genome; and one casewith a small overrepresentedregionthat was not detected by banding analysis. In the majority of cases (8 of 13). at least one of the discrepancies was tested by FISH using specific DNA probes, and in each of thesecases,the CGH resultwas confirmed. These dataindicate that in a considerable proportion of CLL cases, additional abnormalities can be found by CGH. In 6 of 13 patients exhibiting a normal karyotype on P Fig 1. Comparative genomic hybridization in CLL. (A) Complete average ratio profile of -sa 22 (seealso B through D). The ratios of FITC to rhodamine fluorescence are plotted along each single chromosome. The mode of the intensity ratios of this case (central line) and the thresholds for overrepresentation (right line) and undarrepresentation (left line) are shown. Underrepresentations ofpart of chromosome 3p as well as an overrepresentation of chromosomalmaterial on 12p are diagnosed.Note the two sharp and 8p. the proximal part of 15q and 17p peaks of the profile on 12p. Underrepresentation of the whole chromosome X is due to the sexes of test (male) and control (female) DNA. The gray shaded areasindicate heterochromatic chromosome regionswith a high content of repetitive sequences that are suppressad bytha Cot l-DNA in the hybridization solution. Consequently, RTC and rhodamine fluorescence intensities are very low in these regions. Minute absolute variations of fluorescence intensities within these regions may result in gross alterations of the ratio profiles. Therefore, they are excluded from evaluation. The same applies to the Y chromosome. (B and C) Normal metaphase spread hybridized with DNA from case 22 detected via FlTC (B, green) and a control DNA detected via rhodamine (C, red). Two very strong band-like hybridization signals are seen on the short arms of chromosomes 12, indicating the presence of amplified DNA sequencesmapping to two different loci on 12p. Weakerstaining X is clearly visible. The corresponding chromosomes are indicated in panel B. (D)FlTC to rhodamine of chromosomes 3p.8p.17p.and in the tumor genome fluorescence intensity ratio image of the same metaphase spreadas in B and C. Chromosomal regions overrepresented are shown in green, whereas underrepresented regions areshown in red. The loss of a chromosome in 100% of cells (X chromosome in this male patient) results in a yellow color in the fluorescence ratio image. (E) Average ratio profiles for chromosomes 17 of the other seven cases exhibiting a deletion of 17p and, in two casas, an overrepresentstion of 17q. From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 3615 CGH IN CHRONICB-CELLLEUKEMIAS A 1 2 3 -.6 .. . 7 8 13 15 14 20 19 9 10 16 21 4 5 11 12 17 18 Bi 22 X E case 6 Case 12 Case 8 Case 20 Case 9 Case 23 Case 26 From www.bloodjournal.org by guest on April 29, 2017. For personal use only. BENTZET AL 3616 B case 8 Case 6 Case 5 a '1 C a 8 Case 14 Case 23 8 Case 4 6 I) D Case 2 11 E Case 27 11 11 11 Case 10 13 13 13 Fig 2. Average ratio profiles ofchromosomes frequently affected by gains or losses. (A, B)In case 5, a cryptic translocationof the overreprechromosome 7 was mapped sented chromosome 8 material to thedeleted chromosome 7 was present. By banding analysis, the breakpoint on more distal than by CGH (see average ratio profile of this case in panel B). A FISH experiment using chromosome-specific DNA libraries for chromosomes 7 (red) and 8 (green), respectively, demonstrates that a part of chromosome 8 is translocated t o the deleted chromosome 7 homolog. Two furthercases showed overrepresentation of chromosome 8 indicated in panel B. (C) Average ratio profiles of cases 4,14, and 23 exhibiting deletions on6q. (D, F) Average ratio profiles of the fourcases with deletions on chromosome l l q . On banding analysis, case 2 had a normal karyotype. Interphase cytogenetic analysis using a YAC probe mapping t o l l q 2 3 was performed. (F) Only one hybridization signal is visible in the cells, indicating a deletion of chromosome l l q . (E) Average ratio profiles of three cases (cases 10,21, and 27) with deletions on 13q. From www.bloodjournal.org by guest on April 29, 2017. For personal use only. CGH IN CHRONIC B-CELL LEUKEMIAS banding analysis, clonal aberrations were found by CGH. In these cases, the analysis of metaphase cells clearly did not reflect the chromosomal changes present in the malignant clone. Such discrepancies between chromosome changes present in the interphase cell fraction but not inthe proliferating cells have been detected in cases of trisomy 12 and deletions of chromosomal band 13q14.9“’In these FISH studies, higher incidences of the respective chromosomal aberrations were found than suspected by banding analysis. In the CGH study presented here, screening for all types of chromosomal imbalances was performed, and almost half of the cases exhibited additional chromosome aberrations that had been missedby banding analysis. This implies that, in cytogenetic studies relying on banding analysis in CLL, possibly relevant karyotype changes are missed in a considerable proportion of cases. In another study, we compared banding and CGH data in 10 cases of myeloid leukemias not showing dis~repancies.~’ In contrast to CLL, in myeloid leukemias, banding data in general reflect the chromosomal changes present in the malignant clone. This may be an explanation for the identification of prognostically relevant chromosomal changes in acute leukemias, whereas such clinically important specific aberrations are still a matter of discussion in CLL. Delineation of frequent chromosomal gains and losses by CGH in CLL may be an important contribution of this new technique to the understanding of this disease. The data obtained by CGH will provide the basis for further analyzing a larger number of cases by FISH using specific DNA probes mapping to the chromosomal regions mostfrequently affected. 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For personal use only. 1995 85: 3610-3618 Comparative genomic hybridization in chronic B-cell leukemias shows a high incidence of chromosomal gains and losses M Bentz, K Huck, S du Manoir, S Joos, CA Werner, K Fischer, H Dohner and P Lichter Updated information and services can be found at: http://www.bloodjournal.org/content/85/12/3610.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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