Multicolor fluorescence in situ hybridization characterization of cytogenetically polyclonal hematologic malignancies

Multicolor fluorescence in situ hybridization characterization of cytogenetically polyclonal hematologic malignancies

Cancer Genetics and Cytogenetics 163 (2005) 180–183 Short communication Multicolor fluorescence in situ hybridization characterization of cytogeneti...

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Cancer Genetics and Cytogenetics 163 (2005) 180–183

Short communication

Multicolor fluorescence in situ hybridization characterization of cytogenetically polyclonal hematologic malignancies Josef Davidsson*, Kajsa Paulsson, Bertil Johansson Department of Clinical Genetics, Lund University Hospital, SE – 221 85 Lund, Sweden Received 15 April 2005; received in revised form 30 May 2005; accepted 31 May 2005

Abstract

Several different investigations and methodologies have provided data supporting a monoclonal origin of neoplasia. For example, the vast majority of neoplastic disorders are cytogenetically monoclonal. Occasionally, however, clones with unrelated karyotypic anomalies are found, as, for example, in ~2% of acute myeloid leukemias (AML), myelodysplastic syndromes (MDS), and chronic myeloproliferative disorders (CMD). Whether such a cytogenetic polyclonality represents a polyclonal origin or whether different clones share a submicroscopic primary change, indicating a monoclonal origin, remains to be elucidated. Our objective was to ascertain if cryptic aberrations can be found in cytogenetically polyclonal hematologic malignancies using multicolor fluorescence in situ hybridization (M-FISH). Fourteen AML, MDS, and CMD cases were investigated. In none of these was a cryptic aberration found, common to all subclones, although the karyotypes were revised in two AMLs and one MDS. Thus, all malignancies were still classified as polyclonal after the M-FISH analyses. Based on the present results, we conclude that M-FISH, in general, does not reveal primary cryptic aberrations supporting a monoclonal origin of cytogenetically polyclonal hematologic malignancies. Ó 2005 Elsevier Inc. All rights reserved.

1. Introduction The paradigmatic view in cancer biology is that neoplasia has a monoclonal origin: that is, all tumor cells are derived from a single transformed cell [1]. This theory is based on the fact that several different investigations and methodologies, including cytogenetics, studies of immunoglobulin or T-cell receptor gene rearrangements, and Xinactivation assays, have provided strong evidence for a monoclonal origin of most neoplastic disorders [2–5]. Occasionally, however, cytogenetic investigations reveal clones with different karyotypic anomalies in individual tumors [6–17] and, consequently, this unicellular concept has been questioned. Johansson et al. [16], reviewing cytogenetic polyclonality in hematologic malignancies, reported that 2.6% of acute myeloid leukemias (AMLs), 1.6% of myelodysplastic syndromes (MDSs), and 1.5% of chronic myeloproliferative disorders (CMDs) harbor unrelated clones. The frequencies of cytogenetic polyclonality in treatment-related AML (5.7%) and MDS (2.7%) cases

* Corresponding author. Tel.: 146-46-173398; fax: 146-46-131061. E-mail address: [email protected] (J. Davidsson). 0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.05.013

have been shown to be slightly higher than in de novo disorders [9,15]. Furthermore, some other tumor types, in particular benign hereditary neoplasms and carcinomas of the breast, head and neck region, and pancreas, have been demonstrated to be karyotypically polyclonal in a large proportion of cases [11,13,14]. Still to be answered is whether this cytogenetic polyclonality really indicates a polyclonal origin or whether different clones in fact share a submicroscopic primary change. Our objective was to determine if seemingly unrelated clones harbor a common cryptic aberration, not identified with conventional G-banding analysis. Multicolor fluorescence in situ hybridization (M-FISH) and spectral karyotyping (SKY) analyses have previously been used to identify cryptic translocations in t(9;22)-positive chronic myeloid leukemia [18] and in AMLs with normal karyotypes [19]. In additiondand in this context, notablyd Stark et al. [20] identified, by the use of SKY, a common cryptic aberration in the seemingly unrelated clones in a case of cytogenetically polyclonal AML. Thus, the apparently unrelated clones were in fact a product of clonal evolution [20]. Based on these previous findings, we decided to use M-FISH to screen for cryptic changes in 14 cytogenetically polyclonal hematologic malignancies.

Table 1 Clinical and cytogenetic features of the 14 analyzed cases Dx

Previous Tx [Dx]

1

32/M

AML–NOS

CT [AML]

2 3c 4c 5c 6d

66/M 42/M 33/F 65/F 76/M

AML–NOS AML–M2 AML–M2 AML–M2 AML–M4

d d d d RT [PV]

7 8d 9 10c 11c 12 13 14

49/M 51/F 52/M 74/F 67/F 72/M 78/M 58/M

AML–M5 AML–M5a MDS–NOS MDS–RA MDS–RA MDS–RAEB CMD–NOS CMD–NOS

d RT [BC] d d d d d d

Karyotype

Revised karyotypea

45,XY,217/43~45,idem,add(12)(p11),1mar/46,XY, add(1)(p36),del(9)(p13) 47,X,der(Y)t(Y;3)(q12;q21),18/46,XY,11,der(1;3)(q10;q10) 47,XY,121/47,XY,19 45,X,2X,del(5)(q31)/46,XX,ins(1;?) (p13;?) 45,XX,217,der(18)t(17;18)(q12~21;q23)/46,XX,del(9)(q22) 43~46,XY,add(1)(p36),25,211,211,add(17)(p11), 11~3mar/46,XY,del(20)(q11)

45,XY,217/45,idem,add(10)(q?),1der(?)t(?;10),inc/43~45, idem,t(12;17)(p11;?)/46,XY,t(1;9)(p36;p13) No revision No revision No revision No revision 43~46,XY,der(1)t(1;11)(p36;?),25,der(11)t(11;19)(q2?;?), der(11)t(11;19)t(13;19)(q?;?),der(11)add(11)(p?)t(11;19) t(13;19),der(13)t(11;13)(?;q?),der(17)t(5;17)(p11;p11), add(19),der(19)ins(19;11)t(11;13)/46,XY,del(20)(q11) No revision No revision 46,XY,t(3;21)(q26;q22)/47,idem,113/46,XY,i(17)(q10) No revision No revision No revision No revision No revision

46,XY,der(18)t(11;18)(q13;p11)/47,XY,111 47,XX,18,del(11)(q23)/47,XX,121 46,XY,t(3;21)(q26;q22)/46,XY,i(17)(q10) 46,XX,del(5)(q13q33)/47,idem,18/47,XX,18 46,XX,del(5)(q13q33)/47,XX,18 45,XY,221/45,XY,222 47,XY,19/46,XX,del(7)(q22q32) 46,XY,add(22)(q13)/47,XY,1del(22)(q11)

No. of abnormal metaphases analyzedb 9 (5/2/2) 5 8 13 10 15

(3/2) (5/3) (10/3) (8/2) (11/4)

14 3 15 20 6 3 5 7

(10/4) (2/1) (9/6) (11/6/3) (5/1) (2/1) (4/1) (5/2)

Abbreviations: AML, acute myeloid leukemia; BC, breast cancer; CMD, chronic myeloproliferative disorder; CT, chemotherapy; Dx, diagnosis; F, female; M, male; M2–5a, French–American–British FAB subgroups; MDS, myelodysplastic syndrome; NOS, not otherwise specified; PV, polycythemia vera; RA, refractory anemia; RAEB, refractory anemia with excess of blasts; RT, radiotherapy; Tx, treatment. a Revised based on M-FISH and FISH with WCP probes. b The numbers of analyzed metaphases per clone are given in parentheses. c The original karyotypes of cases 3–5, 10, and 11 have previously been published: Johansson et al., 1999 [16]. d The original karyotypes of cases 6 and 8 have previously been published: Mauritzson et al., 2002 [21].

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Age/sex

Case no.

181

182

J. Davidsson et al. / Cancer Genetics and Cytogenetics 163 (2005) 180–183

2. Materials and methods 2.1. Patients A total of 14 patients with cytogenetically polyclonal hematologic malignancies were included in the study (Table 1); they were selected on the basis of available cells in fixative and a sufficient number of analyzable abnormal metaphases. The study comprised 8 AMLs, 4 MDSs, and 2 CMDs. Nine of the patients were men, five were women; the median age was 58 years (range 32–78). Three of the patients had been treated with radio- or chemotherapy because of a previous neoplastic disorder. The description of karyotypes and criteria for clonality follow the recommendations of ISCN 1995 [22]. 2.2. Fluorescence in situ hybridization For the M-FISH analyses, chromosome painting probes for combined binary ratio labeling (COBRA)–FISH were used, and the COBRA–FISH protocol was in accordance with the one reported by Tanke et al. [23], with minor modifications [18]. Whole chromosome painting (WCP) probes for chromosomes or chromosome arms 1, 5, 9, 10, 11, 12, 13, 15q, 17, 19, and 21q (Vysis, Downers Grove, IL) were used to characterize further abnormalities identified by M-FISH. The WCP analyses were performed as previously described by Barbouti et al. [18]. Fluorescence signals were detected with a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Jena, Germany), and fluorescence images were captured and analyzed using the software of CytoVision Ultra system (Applied Imaging, Newcastle upon Tyne, UK). All metaphases on the slides were analyzed.

3. Results and discussion Between 3 and 20 (median 7) abnormal metaphases could be analyzed in each case (Table 1). The karyotypes were revised in three of the cases as a result of the M-FISH and WCP analyses. Cases 1 and 6, both with initially quite complex karyotypes, were shown to be even more complex, well in agreement with previous studies of cytogenetically complex AML and MDS cases [24]. Furthermore, a novel subclone was identified in case 9 (Table 1). The M-FISH analyses revealed no common cryptic aberration in any of the 14 cytogenetically polyclonal cases. Thus, if cryptic changes truly exist in polyclonal hematologic malignancies, we conclude, based on the present results, that the level of resolution of M-FISH is not sufficient to identify them. It should be stressed that somedor even manydchromosomal aberrations are too small, or otherwise impossible to be visualized by M-FISH and WCP probes (e.g., abnormalities involving subtelomeric regions and paracentric inversions). Furthermore, molecular genetic changes such as point mutations, are, needless to say, not identifiable by these methods. Hence, other investigative approaches are

needed to ascertain the possible existence of submicroscopic chromosomal changes or molecular aberrations in polyclonal malignancies. One possibility to screen for the former changes would be to perform FISH with subtelomeric probes. Telomeres and interstitial telomer-repeat-like sequences play important roles in the formation of chromosomal cryptic aberrations [25]. Furthermore, there are several examples of leukemia-associated translocations, such as t(12;21) (p13;q22) and t(4;14)(p16;q32), which involve subtelomeric regions and which cannot be identified cytogenetically [3]. It is possible to us a 12-color FISH assay with subtelomeric probes in an M-FISH fashion [26] to identify such cryptic changes, as was shown by Brown et al. [27], who used this method to detect the cryptic translocation t(5;11)(q35;p15) in pediatric AMLs with normal karyotypes. However, this method is a cumbersome way to screen large patient materials, because it requires a large number of good quality metaphases. Another possibility could be rainbow cross-species FISH, which generates a specific color-banding pattern for each human chromosome. The ability of this technique to characterize complex intrachromosomal changes has been shown in several hematologic malignancies [28,29]. To our knowledge, however, this method has not been applied to polyclonal cases. Yet another means to screen for submicroscopic changes could be to use arrays of bacterial artificial chromosomes (i.e., BAC arrays). A tiling resolution array of BACs covering the whole human genome has recently been constructed, and this array offers the possibility of detecting a large number of subtle gains and losses of chromosome material, in a single hybridization [30]. A major drawback of this array-based method, however, is that does not identify balanced abnormalities, such as translocations or inversions, unless they harbor concomitant deletionsdchanges that have been shown to be quite common in leukemia-associated translocations [31,32]. Acknowledgments This study was supported by grants from the Swedish Cancer Society. References [1] Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194:23–8. [2] Wainscoat JS, Fey MF. Assessment of clonality in human tumors: a review. Cancer Res 1990;50:1355–60. [3] Mitelman F, Johansson B, Mertens F, editors. Mitelman database of chromosome aberrations in cancer [Internet]. Updated February 2005. Available from http://cgap.nci.nih.gov/Chromosomes/Mitelman. [4] Cazzaniga G, Biondi A. Molecular monitoring of childhood acute lymphoblastic leukemia using antigen receptor gene rearrangements and quantitative polymerase chain reaction technology. Haematologica 2005;90:382–90. [5] van Dijk JP, Heuver LH, van der Reijden BA, Raymakers RA, de Witte T, Jansen JH. A novel, essential control for clonality analysis

J. Davidsson et al. / Cancer Genetics and Cytogenetics 163 (2005) 180–183

[6] [7] [8]

[9]

[10] [11]

[12]

[13] [14]

[15]

[16]

[17]

[18]

[19]

[20]

with human androgen receptor gene polymerase chain reaction. Am J Pathol 2002;161:807–12. Heim S, Mandahl N, Mitelman F. Genetic convergence and divergence in tumor progression. Cancer Res 1988;48:5911–6. Heim S, Mitelman F. Cytogenetically unrelated clones in hematological neoplasms. Leukemia 1989;3:6–8. Kobayashi H, Kaneko Y, Maseki N, Sakurai M. Karyotypically unrelated clones in acute leukemias and myelodysplastic syndromes. Cancer Genet Cytogenet 1990;47:171–8. Johansson B, Mertens F, Heim S, Kristoffersson U, Mitelman F. Cytogenetics of secondary myelodysplasia (sMDS) and acute nonlymphocytic leukaemia (sANLL). Eur J Haematol 1991;47:17–27. Furuya T, Morgan R, Sandberg AA. Cytogenetic biclonality in malignant hematologic disorders. Cancer Genet Cytogenet 1992;62:25–8. Jin Y, Mertens F, Mandahl N, Heim S, Olega˚rd C, Wennerberg J, Bio¨rklund A, Mitelman F. Chromosome abnormalities in eighty-three head and neck squamous cell carcinomas: influence of culture conditions on karyotypic pattern. Cancer Res 1993;53:2140–6. Musilova´ J, Michalova´ K, Zemanova´ Z, Brˇezinova´ J. Multiple unrelated clones in myelodysplastic syndrome and in acute myeloid leukemia. Cancer Genet Cytogenet 1996;88:141–3. Heim S, Teixeira MR, Dietrich CU, Pandis N. Cytogenetic polyclonality in tumors of the breast. Cancer Genet Cytogenet 1997;95:16–9. ˚ , Dawiskiba S, Jin Y, Gorunova L, Ho¨glund M, Andre´n-Sandberg A Mitelman F, Johansson B. Cytogenetic analysis of pancreatic carcinomas: intratumor heterogeneity and nonrandom pattern of chromosome aberrations. Genes Chromosomes Cancer 1998;23:81–99. Pedersen-Bjergaard J, Timshel S, Klarskov Andersen M, Thøger Andersen AS, Philip P. Cytogenetically unrelated clones in therapyrelated myelodysplasia and acute myeloid leukemia: experience from the Copenhagen series updated to 180 consecutive cases. Genes Chromosomes Cancer 1998;23:337–49. Johansson B, Billstro¨m R, Broberg K, Fioretos T, Nilsson P-G, Ahlgren T, Malm C, Samuelsson BO, Mitelman F. Cytogenetic polyclonality in hematologic malignancies. Genes Chromosomes Cancer 1999;24:222–9. Han JY, Kim KH, Kwon HC, Kim JS, Kim HJ, Lee YH. Unrelated clonal chromosome abnormalities in myelodysplastic syndromes and acute myeloid leukemias. Cancer Genet Cytogenet 2002;132: 156–8. Barbouti A, Johansson B, Ho¨glund M, Mauritzson N, Stro¨mbeck B, Nilsson P-G, Tanke HJ, Hagemeijer A, Mitelman F, Fioretos T. Multicolor COBRA-FISH analysis of chronic myeloid leukemia reveals novel cryptic balanced translocations during disease progression. Genes Chromosomes Cancer 2002;35:127–37. Zhang FF, Murata-Collins JL, Gaytan P, Forman SJ, Kopecky KJ, Willman CL, Appelbaum FR, Slovak ML. Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia. Genes Chromosomes Cancer 2000;28: 318–28. Stark B, Jeison M, Gobuzov R, Finkelshtein S, Ash S, Avrahmi G, Cohen IJ, Stein J, Yaniv I, Zaizov R, Bar-Am I. Apparently unrelated

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28] [29]

[30]

[31]

[32]

183

clones shown by spectral karyotyping to represent clonal evolution of cryptic t(10;11)(p13;q23) in a patient with acute monoblastic leukemia. Cancer Genet Cytogenet 2000;120:105–10. Mauritzson N, Albin M, Rylander L, Billstro¨m R, Ahlgren T, Mikoczy Z, Bjo¨rk J, Stro¨mberg U, Nilsson PG, Mitelman F, Hagmar L, Johansson B. Pooled analysis of clinical and cytogenetic features in treatment-related and de novo adult acute myeloid leukemia and myelodysplastic syndromes based on a consecutive series of 761 patients analyzed 1976–1993 and on 5098 unselected cases reported in the literature 1974–2001. Leukemia 2002;16:2366–78. ISCN 1995: an international system for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. Tanke HJ, Wiegant J, van Giljswijk RPM, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vroljik J. New strategy for multi-colour fluroscence in situ hybridisation: COBRA: Combined Binary RAtio labelling. Eur J Hum Genet 1999;7:2–11. Tchinda J, Volpert S, McNeil N, Neumann T, Kennerknecht I, Ried T, Buchner T, Serve H, Berdel WE, Horst J, Hilgenfeld E. Multicolor karyotyping in acute myeloid leukemia. Leuk Lymphoma 2003;44: 1843–53. Obe G, Pfeiffer P, Savage JR, Johannes C, Goedecke W, Jeppesen P, Natarajan AT, Martinez-Lopez W, Folle GA, Drets ME. Chromosomal aberrations: formations, identification and distribution. Mutat Res 2002;504:17–36. Brown J, Saracoglou K, Uhrig S, Speicher MR, Eils R, Kearney L. Subtelomeric chromosome rearrangements are detected using an innovative 12-color FISH assay (M-Tel). Nat Med 2001;7:497–501. Brown J, Jawad M, Twigg SRF, Saracoglu K, Sauerbrey A, Thomas AE, Eils R, Harbott J, Kearney L. A cryptic t(5;11) (q35;p15.5) in 2 children with acute myeloid leukemia with apparently normal karyotypes, identified by a multiplex fluorescence in situ hybridization telomere assay. Blood 2002;99:2526–31. Ried T, Schro¨ck E, Ning Y, Wienberg J. Chromosome painting: a useful art. Hum Mol Genet 1998;7:1619–26. Harrison CJ, Yang F, Butler T, Cheung K-L, O’Brien PC, Hennessy BJ, Prentice HG, Ferguson-Smith M. Cross-species color banding in ten cases of myeloid malignancies with complex karyotypes. Genes Chromosomes Cancer 2001;30:15–24. Ishkanian AS, Malloff CA, Watson SK, deLeeuw RJ, Chi B, Coe BP, Snijders A, Albertson DG, Pinkel D, Marra MA, Ling V, MacAulay C, Lam WA. A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet 2004;36: 299–303. Kolomietz E, Al-Maghrabi J, Brennan S, Karaskova J, Minkin S, Lipton J, Squire A. Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis. Blood 2001;97:3581–8. Godon C, Proffitt J, Dastugue N, Lafage-Pochitaloff M, Mozziconacci MJ, Talmant P, Hackbarth M, Bataille R, AvetLoiseau H. Large deletions 5# to the ETO breakpoint are recurrent events in patients with t(8;21) acute myeloid leukemia. Leukemia 2002;16:1752–4.