Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Hairy Cell Leukemia Is Characterized by Clonal Chromosome Abnormalities Clustered to Specific Regions By Ulla Haglund, Gunnar Juliusson, Birgitta Stellan, and Gosta Gahrton Cytogenetic analysiswas performed on B-cell mitogen-stimulated cells from 36 patients with symptomatic hairy cell leukemia. Evaluable metaphaseswere achievedfrom 30 pa(67%) showed clonal abnormalities. Recurrent tients, and 20 chromosomal aberrations involving chromosomes1,2,5,6, 11, 19, and 20 were found. The abnormalities were mostly deletions andinversions,whereastranslocationsandnumerical abnormalities, except trisomy 5,were rare. Fourteen patients showed multiple clones, which mostly were unrelated and found in different combinations in individual cells. Cells with non-clonal abnormalities identical to those found in clonal changesin other patients were common. Chromosome 5 was involved in clonal aberrationsin 12 of 30 (40%) patients, most commonlyas trisomy 5 (n = 4). or pericentric inversions (n = 6)and interstitial deletions (n = 4) involving band 5q13.Three patients showed two and 1 patient three different clones that involved chromosome5. In addition. 1 patient had a rare constitutional inversion of chromosome 5 with breakpoints at p13.1 and q13.3.Pericentric inversions and interstitial deletions of chromosome 2 occurred clonally in 4 patients (13%) and in single cells of another6 patients. Deletionsofchromosome 1 at band q42 was found in 5 patients, and 1 patient had a translocation between lq42 and a supernumerary chromosome 5. Deletions of 6q and 1 Iq were similar to those commonly found in other lymphoproliferative disorders. Trisomy 5, structural abnormalities 5 involving the pericentromericregionsofchromosomes and 2, and lq42 abnormalities were findings distinguishing the karyotypes in hairy cell leukemia from those of other hematologic malignancies. 0 1994 by The American Society of Hematology. H previously published.' Cells from 18 patients were sampled before treatment, while 13 had been splenectomized and/or had received prior treatment with interferon a (IFNa).Five patients had received multiple regimens, including 2-deoxycoformycin or chlorambucil, before sampling. All patients had active disease, ie, any type and degree of cytopenia with or without symptoms and infectious complications, and all patients were subsequently treated with 2-chlorodeoxyadenosine.'.'' Cell cultures and mitogens. Cells from peripheral blood and BM were cultured in RPM1 1640 supplemented with 10%fetal calf serum (GIBCO, Paisley, Scotland) or 10% human pooled AB serum from regular blood donors, at a cell density of 1 to 2 X 106/mL.In two patients, fibroblasts from skin biopsies were cultured because a constitutional abnormality was suspected. B-cell mitogen-stimulation wasin most cases performed using BSF-MP6, the supematant from a human T-T hybridoma cell line (a generous gift of G. Kjellstrom and A. Rosin, Karolinska Institute, Stockholm, Sweden). MP6 was used at 25% (voYvol) in medium with added heat-killed, formalin-fixed Staphylococcus aureus Cowan strain I (SAC) (1:80 OOO, generously donated by M. Carlsson, Pharmacia AB, Uppsala, Sweden), and human recombinant IL-2 (rIL-2) (10 U/mL, Genzyme, Cambridge, MA).I4 In addition, combinations of lipopolysaccharide (LPS) (100 pg/mL, Sigma, St Louis, MO) and 4P-phorbol 12P-myristate 13a-acetate ( P A ) (2.0 pmol/L, Sigma), or LPS and cytochalasin B (CB) (1.O pg/mL, Aldrich Chemie GmbH, Steinheim, Germany) were used.I5 Pokeweed mitogen (PWM) (5.0 pg/mL, GIBCO, Grand Island, NY) and the supernatant from the Epstein Barr virus-containing B95-8 cell line were occasionally used. AIRY CELL LEUKEMIA (HCL) is a chronic lymphoproliferative disease' constituting 2%of the leukemias. It affects mostly males, with a yearly incidence of six per million. The disease is clinically characterized by anemia, granulocytopenia, thrombocytopenia, monocytopenia, splenomegaly, and impaired immunity. The course might thus be fatal because of an increased risk of opportunistic infe~tions.2'~ Bone marrow (BM) fibrosis is common, and BM morphology is diagnostic. Hairy cells are large differentiated B cells, often exhibiting hair-like protrusions. The hairy cell phenotype is peculiar, with a common expression of the B-cell markers CD19 and CD20, multiple Ig heavy chains: HLA-DR, CD45RA, the monocyte marker CD1 IC, and the interleukin-2 (IL-2) receptor CD25, but occasionally also of other markers, such as CD4, CD5, CD15, or CDlO.' Regarding the karyotype, few investigations on chromosome abnormalities in HCL have been reported. This might in part be explained by the rarity of the disease and the difficulty in obtaining cell material caused by the usually low number of circulating hairy cells and the BM fibrosis. Initially unstimulated cells were studied:.' and a few numerical abnormalities, including trisomy 12, were found. When B-cell mitogens were introduced, more clonal aberrations were observed, most commonly structural abnormalities involving chromosome 14, such as 14q deletions' and 14q+ marker chromosomes." However, no consistent abnormality was linked to HCL. Furthermore, the overall success rate was low, with clonal abnormalities detected in 38 of 153 (25%) studied patients.'' Thus, we were prompted to perform the present investigation, in which several recurrent abnormalities were found. Chromosome abnormalities in regions commonly involved in other hematologic tumors were found, but also those distinguishing HCL from related disorders. PATIENTS AND METHODS Patients. Cytogenetic analyses were performed on cells from 36 patients, 34 men and 2 women, with a mean age of 55 years. The diagnosis was based on typical findings in BMbiopsies and aspirates with cytochemistry, supplemented with flow cytometry using a large panel of antib~dies.~.~.'* Clinical data from 16 of the patients were Blood, Vol 83, No 9 (May l), 1994 pp 2637-2645 From the Division of Clinical Hematology and Oncology, Department of Medicine, Karolinska Institute at Huddinge Hospital, S-l41 86 Huddinge, Sweden. Submitted October 5, 1993; accepted January 3, 1994. Supported by the Swedish Cancer Society, The Swedish Society for Medical Science, The Karolinska Institute's Research Funds, and The Medical Research Council. Address reprint requests to Gunnar Juliusson, MD, Department of Medicine, Huddinge Hospital, S-l41 86 Huddinge, Sweden. The publication costs of this article were defrayed in part by 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 1994 by The American Society of Hematology. 0006-4971/94/8309-0034$3.00/0 2637 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. HAGLUND ET AL 2638 Table 1. Patients With Constitutional, Clonal, and Nonclonal Recurrent Chromosome Abnormalities Patient Age1 Previous Treatment* AbnormalWotal Cells With Constitutional, Clonal, and Nonclonal Recurrent Abnormalities Cell Source1 Mitogenst No. of Metaphases: No. B/ 9.90 TO§ 49 yrs/S, I, C PWM karyotype: Constitutional 211 1 Patients with clonal abnormalities involving chromosome 5 17.92 IK 38 yrs/U BM, B/ MP6 3.91 RH§ 39 yrs/S, I 7.91 GE 50 yrs/S, l BM. B/ LPS, TPA, CB BM/ MP6, LPS TPA, CB 6.92GS 64 yrs/U BM/ MP6 1.90 LHS 51 y r d l 2.92 KK 44 yrs/U B/ LPS, TPA, CB BM. B/ MP6 14/37 5 2 1 2 2 1 416 2 25/47 1 16 1 1 1 3 1.91GP§ 73 yrs/S, l, P, 5.91DK 36 yrsll, dCF 7.90 HEJS 43 yrsll C 23/25 3113 17/43 BM. B, LW LPS, CB, MP6, TPA, PWM 14/36 BM, B/ LPS, CB TPA 2 1/89 BM. B/ LPS. CB "PA 15/29 8 8 1 2 1 2 1 1 1 1 1 1 1 1 2 1 2 3 1 1 1 1 1 1 1 2 1 1 1 1 2 2 1 2 1 l 1 2 2 1 fcontinued on following page) Karyolype 46.XY,inv(5)(p13.lq13.3)~ From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2639 CHROMOSOMEABNORMALITIES IN HCL Table 1. p a t i e m With Constitutional, Clonal, and Nonclonal Recurrent Chromosome Abnormalities (Cont'd) Cells Age1 Patient Source/ Cell Previous Treatment* genst Abnormalities Recurrent Nonclonal andWithClonal, Constitutional, No. of Metaphases: KaryoWpe No. Patients with clonal abnormalities involving chromosome 5 (cont'd) 19.87 KS 74 yrs/U 35.92 LS 69 yrs/U 6.90 JJS 56 yrs/S, I, dCF B/ LPS, CB TPA BM, B/ MP6 BM, B/ LPS, TPA CB, MP6 7114 18/34 3 1 2 4 2 2 20123 1 1 17 1 1 1 Patients with other clonal abnormalities LT 27.92 JEK 52 yrs/U BM, B/ MP6 7.92 AW 64 yrJU BM/ MP6 4.92 SGC 61 yrs/U BM, B/ MP6 LPS, TPA 10.91RB 46 yrs/l BM/ MP6 8/36 8/27 9/25 13/20 9.91 SK 49 yrsll 4.91 NM 40 yrs/l BM/ MP6 BM, B/ TPA LPS, 11/26 12.90 KFJ 73 yrs/U 11.91 58 yrs/U BM/ CB TPA, LPS, BM, B/ MP6 3111 9/33 7118 2 de1(2)(qllq13)or(q13q21) 2 de1(2)(qllq13)or(q13q2l),add(ll)(p?) 1 del(2)(q?q?)11,de1(6)(q25)11 1 inv(2)(pl1-13q11-13)" constitutional karyotype: 45,XY,t(l3;14)(q10;q10)c 2 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 2 1 3 1 1 1 2 2 1 Patients with nonclonal recurrent abnormalities only BA 214 6.91 49 yrdl 21.92 IM B/ 48 vrs/U 3114 CB TPA, LPS, BM, MP6 1 1 1 1 . . Previous treatment: U, untreated; S, splenectomy; I, interferon; dCF, 2'-deoxycoformycin; C, chlorambucil; P, prednisolone. t Evaluated cell sources and cultures: BM, bone marrow; B, blood; LN, lymph node. LPS,CB,TPA,MP6, PWM are mitogens, see text. The bold font indicates cultures in which clonal abnormalities were identified. Unmarked cultures include those with no metaphases. The number of abnormal metaphases includes clonal, nonclonal recurrent, and sporadic aberrations, but not constitutionalabnormalities. § Patients previously reported in ref 3. Nonclonal aberrations that occur as clonal aberrations in other patients. * From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2640 HAGLUND ET AL To obtain elongated chromosomes of normal T cells from patient 9.90, ethidium bromide (10 pg/mL) during the final 2 hours of a phythemagglutinin (PHA)-stimulated lymphocyte culture was used.“ The cells were harvested according to medium color change over a number of days. In general, cells were harvested on days 4 a n d or 5 , with additional harvests in some patients after up to 25 days in culture. Colchicine (Demecolcine, Sigma) to a final concentration of 1.25 or 0.125 p m o m was added 1 hour before harvest. The cells were treated with a hypotonic (75 mmol/L) potassium chloride solution, and fixed in acetic acidmethanol. Slides were, in general, flame dried to improve the spreading of the metaphases. Cytogenetic analysis. Conventional Q- and G-banding techniques were used.”,18 Cytogenetic analysis was performed with a Cytoscan Image analysis system (Applied Imaging, Sunderland, UK). Chromosome abnormalities were given, and clones were defined according to the International System for Human Cytogenetic N o m e n ~ l a t u r e .Two ’ ~ ~ ~cells ~ with identical structural abnormalities, two with identical supernumerary chromosomes, or three with the same missing chromosomes constituted a clone. Single cell abnormalities that appeared as clonal changes in other patients were referred toas “nonclonal recurrent” aberrations. We also regarded nonclonal abnormalities involving recurrent regions, but with breakpoints that did not exactly coincide with those of clonal aberra- tions, as in the case of the pencentric inversions of chromosome 2, as “nonclonal recurrent.” RESULTS In samples from 6 of the 36 patients (17%) no metaphase was found. Twenty male patients of 30 evaluable (67%) showed clonal abnormalities and another two had nonclonal recurrent aberrations (Table 1). Eight patients showed no clonal abnormalities, butonly sporadic chromosome and chromatid aberrations. No evaluable patient lacked chromosomal abnormalities entirely. One patient had a constitutional abnormality that was also found as a clonal change in other patients (see below). Most patients exhibited a large number of different chromosomal abnormalities. The abnormalities were mainly structural, and inversions and deletions werenotably more commonthan translocations. Unstable aberrations, such as dicentrics, rings, and chromatid aberrations also appeared. There were few numerical abnormalities. The chromosomes preferentially involved in clonal abnormalities were Nos. 1, 2, 5, 6, 11, 19, and 20 (Fig 1). Clonal changes involving chromosomes 4, 12, 13, 14, and 15 were 1 2 6 5 bb %-U I I 11 47 ++++++++ 19 20 Fig 1. The locationof clonal and nonclonal recurrent abnormaliiiesin the most frequently affected chromosomes.*A sporadic chromosome aberration includingat least a part of a region involvedin clonal abnormalities, althoughnot with exactly coincident breakpoints. **Constitutional inversion of chromosome 5. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. CHROMOSOME ABNORMALITIES IN HCL 2641 Fig 2. Typical chromosome abnormalities. The abnormal homolog is to the right and the affected regions indicated by arrows on the normal homolog to the left. la) inv(5)(p13.lq13.3)as a constitutional abnormality in patient T6, (b) +inv(5)(pl3ql3)as an acquired abnormality, (c) del(5)(qllql3)or(ql3ql5), (dl del~2l(qllq13~or(q13q21), (e) deI(ll)(q13q21), (f) inv(19)(p13q13). occasionally found. No correlation between the number of clonal abnormalities and the number of metaphases investigated was observed. Cells with clonal abnormalities were frequently seen in multiple cultures, if available (Table l). In our study, MP6 was the single most effective mitogen for hairy cells. No significant impact of previous treatment on the chromosome findings was found, but three of five patients with multiple previous treatment regimens had clonal abnormalities involving chromosome 5 (Table l). Chromosome 5. Thirteen of the 30 patients (43%) with evaluable metaphases showed clonal (n = 12) andor nonclonal recurrent (n = l ) abnormalities of chromosome 5 (Table l). One patient showed three, and three patients two different clones involving chromosome 5. The most commonly occumng abnormalities were inversions (n = 7 including the constitutional inversion, see below) (Fig 2a and b) and interstitial deletions (n = 4) (Fig 2c), all except one involving band 5q13. Supernumerary normal (n = 4) and derivative (n = 4) chromosomes 5 were also common. An additional patient with a del(5)(ql lq13) clone had single cells with trisomy 5 and t(5;13)(q13;q?), respectively, among 89 studied cells. One patient with trisomy 5 had two additional clones with extra chromosome 5 derivatives from a t(1;5) (q42;p15.1) and an inv(5)(p13q13), respectively. Five of the six pericentric inversions included band 5q13 and three of them were found in supernumerary chromosomes (Fig 2b). Sporadic aberrations also involved the 5q13 region: one terminal deletion in 5q13, the t(5; 13)(q13;q?) mentioned above, and one insertion of the segment 5(qlI-l3q22) into a chromosome 8. On the other hand, sporadic translocations involved bands pl, p15, and q35, similar to the sites of clonal translocations (see below). One additional patient had a constitutional pericentric inversion, with the breakpoints p13.1 andq13.3, as defined by the use of high-resolution banding.'" The inversion was present in all metaphases studied from blood and BM during active disease and in complete remission, as well as from skin fibroblasts, whereas no other clonal aberration was seen in the eleven metaphases achieved from BM when involved with HCL. Clonal translocations (n = 3) affected the telomeric parts of either arm, with breakpoints in 5 ~ 1 55q31, , and 5q33-35. Chromosome 2. Clonal andor nonclonal recurrent interstitial deletions (Fig 2d) and inversions involving the pericentric region of chromosome 2 were found in 10 patients (33%). The region common to all these abnormalities was band 2q I 1 (Fig l ) . Because the banding pattern in this region is not very distinct, the exact location of the breakpoints is somewhat uncertain. The true incidence of these aberrations may be higher because of a conservative assessment of the abnormalities. Chromosome I . Terminal deletions of chromosome 1 at band q42 were found as clonal and nonclonal aberrations in three and two patients, respectively. A translocation between lq42 andband p 15.1 of a supernumerary chromosome 5 (see above) was also found. Chromosome 6. Clonal terminal deletions involving band 6q25 occurred in four patients, and as non-clonal abnormalities in another five. One patient exhibited deletions of the long arm with different break points in different cells: q21-23 in one metaphase, q23-25 in two, and q25 in one cell. This patient also had a clonal pericentric inversion comprising most of the chromosome, with breakpoints at p25 and q25. Chromosome 11. Chromosome I 1 was, together with chromosome 5, most frequently involved in clonal abnormalities, such as terminal deletions in band I lq23 and interstitial deletions with the breakpoints q 13q21 or q21q23 (Fig 2e). Pericentric inversions, one clonal and one nonclonal recurrent, involving the break points pl3- 15 and 92 1-23, occurred in two patients. In addition, eight patients showed additional material, the origin of which could not be identified, on the short arm. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. HAGLUND ET AL 2642 Chromosome 19. Clonal pericentric inversions were found in cells from three patients, and in a further two as nonclonal aberrations (Fig 20. One patient exhibited a ring chromosome with undefined breakpoints. Chromosome 20. Clonal terminal deletions of the long arm of chromosome 20, all including the region q13.1 to qter, were found in two patients, and as nonclonal recurrent changes in another two. Numerical abnormalities. Only a few numerical abnormalities were present, except for theadditional chromosomes S (see above). Two patients showed an additional del(l2)(pl1) derivative chromosome, and one patient showed clonally missing chromosomes 7 and 21 together with a marker chromosome. Clonalbreak points. Clonal break points, ie, identical breakpoints involved in different aberrations, clonal or nonclonal, in the same patient, were identified in ten patients. Band Sq13 was involved in different abnormalities in two patients. Also chromosomes 11 and 19 showed terminal and interstitial deletions, inversions, and translocations with coincident break points. One patient showed a deletion in 6q2S and a chromatid break in the same band. Multiple clones. The frequency of cells with clonal abnormalities varied greatly, from 20 of 23 cells (87%) in one patient to zero in others. Fifteen patients had more than one clonal aberration (Fig 3) and 14 of these exhibited distinct multiple clones. Multiple clonal abnormalities often coexisted in different combinations in different metaphases. This is exemplified by one patient with 17 aberrant metaphases of 43 studied; 10 of these contained six different clonal abnormalities in various combinations with other abnormalities, so that no two metaphases had identical karyotype. Constitutional abnormalities. Two patients were found to have constitutional abnormalities, both documented through karyotyping of skin fibroblasts. One patient had an inv(S)(p13.lq13.3), without other clonal changes (see above). The second patient had a Robertsonian translocation between chromosomes 13 and 14, and additionally a clonal pericentric inversion of chromosome 2 in leukemic cells. I 0 1 2 3 4 5 6 Number of abnormalities Fig 3. The number of patients with various numbers of clonal and nonclonal recurrent abnormalities. DISCUSSION Our investigation presented a high frequency of clonal abnormalities in HCL. These were in part abnormalities that are common in different hematologic tumors, and previously described in HCL. However, some of the most frequent abnormalities in our study are rare in other types of leukemias. In contrast to the case in most other hematologic tumors, the structural abnormalities were mostly caused by inversions and deletions, but rarely translocations. Multiple clones and different types of abnormalities involving the same chromosome region in individual patients were other characteristic features. In previously published investigations comprising more than 150 HCL patients, one fourth of the patients that were evaluated, had clonal abnormalities," as compared with two thirds in our study. Recently, fluorescence in situ hybridization techniques, permitting sensitive detection of numerical chromosome abnormalities in interphase cells,have been applied to 24 cases of HCL?' without significant findings. However, no probe for the detection of chromosome 5abnormalities was used. In our study, abnormalities involving chromosomes l , 2, and 5 were the most significant findings. These were typically trisomy 5, structural abnormalities involving band 5q13, pericentric inversions and deletions of chromosome 2, and structural abnormalities of band lq42. Structural abnormalities of chromosome 5 are previously reported in three HCL cases; in two of them band p1 was involved.'"~zzA clonal 5q13.3 breakpoint is reported in one patient with a variant form of HCL as part of a three-way translocation, t(1;S; lO)(p34.3;q13.3;q22.1), with an additional t(S; 14)(q31.1; q l l ) sub-clone." Trisomy 5, pericentric inversions of chromosome S , and other structural abnormalities involving 5q13 are rare in malignant lymphoma and in chronic lymphocytic leukemia (CLL)," the malignancy most closely related to HCL. Of S12 CLL patients with clonal abnormalities included in the database of the International Working Party on Chromosomes in CLL,",24 14 (2.7%) involved chromosome S , mostly as t(1;S) translocations. No single CLL patient had 5p13 abnormalities or pericentric inversions of chromosome S . Trisomy 5 was found in conjunction with other trisomies in 1 CLL patient, and clonal breaks in band 5q13 were found in 3 (0.6%) (G. Juliusson, personal observation, 1993). In similar, int de1(2), inv(2)(pl lq13), and del(l)(q42) are reported in single cases of CLL only." Deletions of Sq, on the other hand, are characteristic features of the myelodysplastic syndromes and secondary myeloid leukemias. However, in these tumors the abnormalities always involve the distal part of the long arm, from q22 to q33," a region encoding several cytokines and cytokine receptors.26 Among numerical abnormalities in HCL, trisomy 3 and 21, and monosomy 7, 10, 12, and 17 have been In our study, trisomy S was the only recurrent numerical abnormality; in addition, one patient showed monosomy 7 and 21. Trisomy 12 is the most common abnormality in CLL,'','5,".27whereas structural abnormalities involving this chromosome are less freq~ent.'~ In HCL, trisomy 12 was the From www.bloodjournal.org by guest on June 18, 2017. For personal use only. CHROMOSOME ABNORMALITIES IN HCL 2643 necessary for tumor formation,@' exemplified by the consisfirst identified chromosome abnormality,6-' but thereafter it tent, progressive accumulation of certain abnormalities in has been found in single patients Structural abnorcolon carcinoma: a RAS gene mutation, del(5q), del(l7p), malities on the short arm of chromosome 12,withbreak and del( 18q):' The recuning chromosomal abnormalities points in 12pl3 and p1 1 were reported by Brito-Babapulle found in this study of HCL may enable the identification of et and +del( 12)(pl1) was also found twice in our study. some important gene loci. Of specific interest was that both these patients with partial The 5q13.3 band is likely to harbor candidate genes intrisomy 12 also had chromosome 5 and 14q abnormalities volved in some transforming event of HCL. This is suggest(patients GP and JJ3), and their HCL showed CLL-like feaed by the frequent occurrence of structural abnormalities tures as regards the phenotype with lack of CD25 expression involving this region,and the rare constitutional inv(5) andor CD5-positivity, high blood lymphoid cell counts, and (p13.lq13.3) abnormality found in our patient TO. The incipoor response. to IFN and deoxyc~formycin.~ dence of constitutional abnormalities is reported to be about Previous studies have indicated recurrent 14q32 transloca0.6% among almost twelve thousand newbornbabies:' Most tions and terminal deletions of chromosome 14 with aberrations were numerical, and balanced structural abnorbreakpoints in the central part of the long arm?*10*22 We malities affecting autosomal chromosomes were found in found two patients with clonal chromosome 14 abnormalities 0.2%;' most commonly as Robertsonian translocations in(above). Such abnormalities are found in all B-cell malignanvolving two of the chromosomes 13, 14, 21, and 22, such cies, and are likely to be unspecific changes in HCL as well as in our patient AW. Constitutional inversions have been as inCLL." Deletions of the long arms of chromosome found in 0.02% of67,000 new born^;^ and in 0.03%of more 6,10*22 and chromosome 1l''*'' in HCL are also characteristic than 3,000 adult patients with hematologic diseases.44None findings in CLL"*" and lymphoma^.^^ Deletions at llq14 of these reported inversions involved chromosome 5, and and of 20q occur in myeloid disorder^.'^ 19q13, a recurrent thus the constitutional inversion of our patient TO is exbreakpoint, is the site of the bcl-3 oncogene?' which was tremely rare. cloned from a rare recurrent but translocation t(14; 19)(q32;q13) in CLL.3' A few other previously reported The RASA gene45 and the dihydrofolate reductase gene (DHFR),& both located to 5q13, might be involved in the clonal abnormalities" were found in single cells from our process leading to chromosome instability andneoplasia. patients, ie, de1(2)(q33-35), de1(9)(q22), and del( 18)(q21). RASA encodes the GAP protein, a GTPase activating protein The nonclonal recurrent abnormalities in our material might that binds to RAS p21 proteins, and is implicated as a receswell indicate clones that were not detected because of a sive oncogene:' Mutant RAS genes may also induce struclimited number of metaphases analyzed. tural chromosome instability.& The RAS genes are involved A characteristic feature of HCL cells was the presence of in the control of cell proliferation, and can be activated in distinct multiple clones with mostly unrelated abnormalities most types of human tumors at varying frequencies:' More that occurred in different combinations in different cells (Tathan 30% of human tumors contain a mutation in one of ble l), without evidence for a karyotypic evolution. In our the RAS genes, N-rus, H-rus, and K - ~ u s , ~located ' at lp13, material, nearly half (14 of 30) of the patients showed unrellp15.5, and 12~12.1,respectively. Another RAS gene, lated and partially related clones. Cytogenetically unrelated clones have been reported in a number of hematologic neoRAB4, is located at 1q42-44." Seventeen of our 30 patients (57%) showed abnormalities, clonal or nonclonal, in lq42, p l a s m ~ . ~They ~ - ~ 'have been estimated to be most common 5q13, the l l p , or 12p regions. Therefore, studies of RAS or in chronic lymphoproliferative disorder^,'^ ie, found in approximately 7% of the cases. The theoryput forward by RASA genes in HCL would be justified. Heim and M i t e l m a ~that ~ ~submicroscopic ~ abnormalities link An inhibition of DHFR,also located to 5q13, could inhibit the different clones together could explain some, but probatheproduction of thymidine monophosphate, and the rebly not all, of the clonal patterns seen in our patients. sulting imbalance of the nucleotide poolin the cell may Specific chromosome regions were also often involved in cause an increase of chromosome abnormalities and aneumore than one kind of aberration. As an example, a segment ploidy:' and also affect the induction of folate-deficient fragof chromosome 1, ie, q12q21, was involved in three different ile sites.52Folate-induced fragile sites coinciding with recuraberrations in the same patient, namely inserted at another rent HCL breakpoints are located at 2q11.2 andq13,l lq13.3 location in chromosome 1 in one metaphase, inserted into and q23.3, and 1 9 ~ 1 3 . Twenty-four '~ of our patients (80%) chromosome 2 at the critical region ql l-l3 in another metahad clonal or nonclonal abnormalities involving at least one phase, and interstitially deleted in a third metaphase.''Jumpof these sites or 5q13. ing translocations" have been described in a few cases of In conclusion, our study has identified certain chromoleukemias and lymphomas, mainly of the B-cell t ~ p e . ~ ~ -some ~ * regions not randomly associated with HCL. Some of The probability of an identical translocation, ie, an abnormalthem seem common to avariety of hematologic tumors, ity with two breakpoints, to occur independently in two difwhereas others, involving bands lq42,2qll, and 5q13, seem ferent cells has been estimated to be 5 X lo-" at the resolucharacteristic for HCL. Molecular studies of genes localized tion of the banding pattern used in the present inve~tigation.~~to these regions and their corresponding proteins might enThese data may point to the presence of a common genetiable the identification of tumorigenic mechanisms. HCL is cally unstable progenitor cell. probably the systemic malignancy with the best treatment Tumorigenesis is considered to be a multistep process. It options, and it would be worthwhile to elucidate genetic has been estimated thatfive to six independent steps are mechanisms that distinguish HCL from other tumors. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2644 HAGLUND ET AL ACKNOWLEDGMENT We are grateful to Professor Lore &h, Academic Hospital, Uppsala, for critical reading of the manuscript, and to Dr Anders RosCn, Linkoping University Hospital, for providing the MP6 mitogen. REFERENCES 1. Bouroncle BA, Wiseman BK, Doan CA: Leukemic reticuloendotheliosis. Blood 13:609, 1958 2. Golomb Hh4, Hadad W: Infectious complications in 127 patients with hairy cell leukemia. Am J Hemato1 16:393, 1984 3. Juliusson G, Liliemark J: Rapid recovery from cytopenia in hairy cell leukemia following treatment with 2-chloro-2’-deoxyadennosine (CdA): Relation to opportunistic infections. Blood 79:888, 1992 4. Melo JV, San Miguel JF, Moss VE, Catovsky D: The membrane phenotype of hairy cell leukemia: A study with monoclonal antibodies. Semin Oncol 11:381, 1984 5. Juliusson G, Lenkei R, Liliemark J: Flow cytometry of blood and bone marrow cells from patients with hairy cell leukemia. Phenotype of hairy cells and lymphocyte subsets following treatment with 2-chlorodeoxyadenosine. Blood (in press) 6. Golomb HM, Lindgren V, Rowley JD: Chromosome abnormalities in patients with hairy cell leukemia. Cancer 41: 1374, 1978 7. Golomb HM, Lindgren V, Rowley JD:Hairy cell leukemia: An analysis of the chromosomes of 26 patients. Virchows Arch B Cell Path01 29:113, 1978 8. Ueshima Y,Alimena G, Rowley JD, Golomb I“: Cytogenetic studies in patients with hairy cell leukemia. Hematol Oncol 1:215, 1983 9. Berger R, Bernheim A, Valensi F, Flandrin G: 14q- in two hairy cell leukemia patients. Cancer Genet Cytogenet 16:91, 1985 10. Brito-Babapulle V, Pittman S , Melo JV, Parreira L, Catovsky D: The 14q+ marker in hairy cell leukemia. A cytogenetic study of 15 cases. Leuk Res 10:131, 1986 11. Juliusson G, Gahrton G: Cytogenetics in CLL and related disorders, in Rozman C (ed): Chronic Lymphocytic Leukaemia and Related Disorders, Baillitre’s Clinical Haematology, v01 6. Philadelphia, PA, Saunders, 1993, p 821 12. Visser L, Shaw A, Slupsky J, Vos H, Poppema SI Monoclonal antibodies reactive with hairy cell leukemia. Blood 74:320, 1989 13. Beutler E: Cladribine (2-chlorodeoxyadenosine). Lancet 340:952, 1992 14. Rosen A, Uggla C, Szigeti R, Kallin B, Lindqvist C, Zeuthen J: A T-helper cell X Molt4 human hybridoma constitutively producing B-cell stimulatory and inhibitory factors. Lymph Res 5:185, 1986 15. Juliusson G, RobbrtK-H, Ost A, Friberg K, Biberfeld P, Nilsson B, Zech L, Gahrton G: Prognostic information from cytogenetic analysis in chronic B-lymphocytic leukemia and leukemic immunocytoma. Blood 65:134, 1985 16. Tomiyasi T, Testa JR: Application of ethidium bromide for high-resolution banding analysis of chromosomes from human malignant cells. Stain Techno1 63:83, 1988 17. Caspersson T, Lomakka G, Zech L The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability. Hereditas 67:89, 1971 18. Zhang S , Qiu J, Gao X: G-banding of human chromosomes. Chin Med J 591210, 1979 19. Harnden DG, Klinger HP, (ed): ISCN 1985: An International System for Human Cytogenetic Nomenclature. Basel, Switzerland, Karger Basel, 1985 20. Mitelman F, (ed): ISCN 1991: Guidelines for Cancer Cytogenetics. Supplement to an International System for Human Cytogenetic Nomenclature. Basel, Switzerland, Karger Basel. 1991 21. Lewis JP, Tanke HJ, Raap AK, Kibbelaar RE, KluinPM, Kluin-Nelemans HC: Hcell leukemia: An interphase cytogenetic study. Leukemia 7:1334, 1993 22. Nishida K, Taniwaki M, Misawa S , Abe T: Nonrandom rearrangement of chromosome 14 at band q32.33 in human lymphoid malignancies with mature B-cell phenotype. Cancer Res 49:1275, 1989 23. Nacheva E, Fischer P, O’Connor S , Bloxham D, Hoggarth C, Marcus R: Complex chromosomal rearrangement in an unusual variant of hairy cell leukemia. Cancer Genet Cytogenet 62: 186. 1992 24. Juliusson G, Gahrton G for the International Working Party on Chromosomes in Chronic Lymphocytic Leukemia (WCCLL): Chromosome abnormalities in B-cell chronic lymphocytic leukemia, in(ed): Cheson BD Chronic Lymphocytic Leukemia, ch 4. New York, NY, Marcel Dekker, 1992, p 83 25. Jacobs A: Genetic abnormalities in myelodysplastic syndrome. Cancer Genet Cytogenet 56:1, 1991 26. Le Beau MM: Cytogenetic and molecular analysis of the deletions of chromosome 5 in myeloid disorders, in The Biology of Hematopoiesis. New York, NY, Wiley-Liss. 1990, p 277 27. Gahrton G, Rob& K-H, Friberg K, Zech L, Bird AG: Nonrandom chromosomal aberrations in chronic lymphocytic leukemia revealedby polyclonal B-cell-mitogen stimulation. Blood 56640, 1980 28. Sadamori A. Sandberg AA: 14q+ and 6q- anomalies In a case with hairy cell leukemia. Cancer Genet Cytogenet 8:89, L983 29. Mitelman F, Kaneko Y,Trent JM: Report of the committee on chromosome changes in neoplasia. Human Gene Mapping 10.5. Cytogenet Cell Genet 55:358, 1990 30. McKeithan TW, Rowley JD, Shows TB, Diaz MO: Cloning of the chromosome translocation breakpoint junction of the t(14; 19) in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 84:9257, 1987 31. Bird ML, Ueshma Y, Rowley JD, Haren JH, Vardiman JW: Chromosome abnormalities in B cell chronic lymphocytic leukemia and their clinical correlations. Leukemia 3:182, 1989 32. Mecucci C, Rege-Cambrin G, Michaux J-L, Tricot G, van den Berghe H: Multiple chromosomally distinct cell populations in myelodysplastic syndromes and their possible significance in the evolution of the disease. Br J Haematol 64:699, 1986 33. Heim S , Mitelman F: Cytogenetically unrelated clones in hematological neoplasms. Leukemia 316, 1989 34. Furuya T, Morgan R, Sandberg AA: Cytogenetic biclonality in malignant disorders. Cancer Genet Cytogenet 62:25, 1992 35. Peterson LC, Lindquist LL, Church S , KayNE: Frequent clonal abnormalities of chromosome band 13q14 in B-cell chronic lymphocytic leukemia: Multiple clones, subclones, and nonclonal alterations in 82 Midwestern patients. Genes Chrom Cancer 4:273, 1992 36. Whang-Peng J, Lee EC, Sieverts H, Magrath IT: Burlutt’s lymphoma in AIDS: Cytogenetic study. Blood 63:818, 1984 37. Shippey CA, Layton M, Secker-Walker LM: Leukemia characterised by multiple subclones with unbalanced translocations involving different telomeric segments: Case report and review of the literature. Genes Chrom Cancer 2:14, 1990 38. Ben-Neriah S , Abramov A, Lerer I, Polliack A, Leizerovilz R, Rabinowitz R, Abeliovich D: “Jumping translocation” in a 17month-old child with mixed-lineage leukemia. Cancer Genet Cytogenet 56:223, 1991 39. Savage JRK: Application of chromosome banding techniques to the study of primary chromosome structural changes. J Med Genet 14:362, 1977 40. Dix D: The role of ageing in cancer incidence: An epidemiological study. J Gerontology: 44:10, 1989 41. Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 61:759, 1990 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. CHROMOSOMEABNORMALITIES IN HCL 42. Jacobs PA, Melville M, Ratcliffe S, KeayAJ, Syme J: A cytogenetic survey of 11680 newborn infants. Ann Hum Genet 37:359, 1974 43. UNSCEAR Report: Ionizing radiation: sources and biological effects. United Nations Scientific Committee on the Effects of Atomic Radiation. New York, NY, United Nations, 1982 44. Benitez J, Valcarcel E, Ramos C, Ayuso C, Cascos AS: Frequency of constitutional chromosome alterations in patients with hematologic neoplasias. Cancer Genet Cytogenet 24:345, 1987 45. Hsieh C-L, Vogel US, Dixon RAF, Francke U: Chromosome localization and cDNA sequence of murine and human genes for ras p21 GTPase activating protein (GAP). Somat Cell Mol Genet 15579, 1989 46. Wasmuth JJ, Bishop DT, Westbrook CA: Report of the committee on the genetic constitution of chromosome 5. Human Gene Mapping 11. Cytogenet Cell Genet 58:261, 1991 2645 47. Spandidos DA, Freshney M, Wilkie NM: Heterogeneity of cell lines derived after transformation of early passage rodent cells by the Ha-rasl human oncogene. Anticancer Res 5:387, 1985 48. Bos JL: The ras gene family and human carcinogenesis. Mutat Res 195:255, 1988 49. Barbacid M: ras genes. Ann Rev Biochem 56:779, 1987 50. Bmns GA, Dracopoli NC: Report of the committee on the genetic constitution of chromosome 1. Human Gene Mapping 11. Cytogenet Cell Genet 58:103, 1991 5 1. Kunz BA: Genetic effects of deoxyribonucleotide pool imbalances. Environ Mutagen 4695, 1982 52. Sutherland GR: Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture. Am J Hum Genet 31:125, 1979 53. Hecht F, Ramesh K, Lockwood DH: A guide to fragile sites on human chromosomes. Cancer Genet Cytogenet 44:37, 1990 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1994 83: 2637-2645 Hairy cell leukemia is characterized by clonal chromosome abnormalities clustered to specific regions U Haglund, G Juliusson, B Stellan and G Gahrton Updated information and services can be found at: http://www.bloodjournal.org/content/83/9/2637.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. Copyright 2011 by The American Society of Hematology; all rights reserved.