- Email: [email protected]
Cytogenetic and fluorescence in situ hybridization characterization of clonal chromosomal aberrations and CCND1 amplification in esophageal carcinomas
Cancer Genetics and Cytogenetics 148 (2004) 21–28
Cytogenetic and fluorescence in situ hybridization characterization of clonal chromosomal aberrations and CCND1 amplification in esophageal carcinomas Yuesheng Jina,b,c, Charlotte Jinc, Simon Lawb, Kent-Man Chub, Hao Zhangb,d, Bodil Strombeckc, Anthony P.W. Yuenb, Yok-Lam Kwonga,* a
Department of Medicine, Queen Mary Hospital, Pokfulam Road, Hong Kong, China Department of Surgery, Queen Mary Hospital, Pokfulam Road, Hong Kong, China c Department of Clinical Genetics, University Hospital, Lund, Sweden d Department of Otolaryngology, Ruijin Hospital, Shanghai Second Medical University, Shanghai, China Received 10 March 2003; received in revised form 28 April 2003; accepted 7 May 2003 b
Abstract
Cytogenetic analyses of four squamous cell carcinomas (SCC) of the esophagus showed complex numerical and structural abnormalities. Chromosomal bands or regions preferentially involved were 11q13, 8q10, 21q10, 3p10~p11, 1p11~q11, 5p11~q11, and 14p11~q11. For the first time, to our knowledge, recurrent aberrations were identified in esophageal SCC, including homogenous staining region (hsr), isochromosomes i(3q) and i(21q), and ring chromosome. Losses of chromosomal material dominated over gains. Recurrent imbalances included under-representation of 4p13~pter, 5q14~qter, 9p22~pter, 10p, 11p13~pter, 12p13~pter, 17p10~pter, 18p11~pter, 21p, and 22p, as well as over-representation of 1q25~qter, 3q, 7q, and 8q. Interestingly, hsr at different chromosomal regions occurred in three of four cases. With the application of fluorescence in situ hybridization (FISH) and multicolor combined binary ratio labeling–FISH with specific DNA probes, it could be shown that in two cases the hsr was derived from chromosome 11 material and that the amplicon included CCND1. Our results, together with previous molecular genetic findings, indicate that CCND1 might be a prime target in 11q13 amplification, and that amplification of this gene might be crucial in the tumorigenesis of esophageal SCC. These observed chromosomal aberrations and imbalances thus provide important information for further molecular genetic investigation of esophageal SCC. 쑖 2004 Elsevier Inc. All rights reserved.
1. Introduction Cancer of the esophagus has a great variation in geographic distribution. Although these neoplasms are uncommon in the United States and many European countries, they are prevalent in China [1]. According to data from the World Health Organization, mortality attributable to esophageal cancer is highest in China, Puerto Rico, and Singapore. The major histologic types of esophageal cancer are squamous cell carcinoma (SCC) and adenocarcinoma. The prognosis of these neoplasms is poor, with a 5-year survival of less than 30% in advanced diseases [2]. The etiology and pathogenesis of this aggressive cancer are poorly understood.
* Corresponding author: Tel.: ⫹852-2-855-4597; fax: ⫹852-2-974-1165. E-mail address: [email protected] (Y.-L. Kwong). 0165-4608/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0165-4608(03)00213-9
In a genome-wide search for molecular alterations underlying tumorigenesis in esophageal SCC, the patterns of aberrations at the chromosomal level might be an essential guide, as has been shown for many other human neoplasms [3]. To our knowledge, however, only eight esophageal SCC with clonal karyotypic changes have been reported to date [4]. Three esophageal SCC had only simple numerical changes, namely ⫺Y and ⫹Y, which presumably did not represent aberration in the tumor parenchyma. The remaining five cases had highly variable chromosome rearrangements. Due to limited chromosomal data and the karyotypic diversity in previously reported tumors, the putative genetic events that might be important in esophageal SCC remain undefined. As part of a systematic study of genomic rearrangements that characterize esophageal SCC, we have detected complex karyotypic aberrations in four tumors, three of which also
22
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
displayed a homogenous staining region (hsr) in different chromosomal locations. To identify the origin of the hsr and target gene(s) in the amplification process, further fluorescence in situ hybridization (FISH) and multicolor combined binary ratio labeling (COBRA)–FISH studies were performed.
2. Materials and methods 2.1. Patients Six patients undergoing esophagectomy/endoscopic biopsy for cancer of the esophagus were studied. None of the patients had been treated with radio- or chemotherapy before the cytogenetic analysis. Samples from two patients did not yield cytogenetic results, whereas the remaining four showed clonal chromosome aberrations. The clinical and histopathologic characteristics of these four patients are summarized in Table 1. 2.2. Karotypic analysis For karyotypic analysis, a piece of the tumor and its marginal tissue was freshly dissected from the surgical specimen and processed for cytogenetic analysis as described [5]. Briefly, the dissected tumor was minced finely with scissors and disaggregated overnight in collagenase (180 U/ mL). The resulting suspension of small cell clumps was washed twice in RPMI-1640 and plated on vitrogen-coated culture flasks. The growth medium was keratinocyte serumfree (KSF) medium (GIBCO 10744-019; Invitrogen Corp., Carlsbad, CA) with 2%–5% fetal calf serum, supplemented with l-glutamine (0.23 mg/mL), penicillin-streptomycin (100 IU/mL and 0.2 mg/mL, respectively), and amphotericine (2.5 µg/mL). The cell growth pattern and morphology were evaluated periodically under inverted microscopy. When tumor cells showed active division (in ~5–10 days), they were harvested by conventional cytogenetic techniques. Harvested cells on slides were incubated overnight at 60⬚C, followed by submersion in 2× standard saline citrate (SSC) at 60⬚C for 3–4 hours before banding with Wright’s stain. The criteria used for clonality and karyotype description were according to the International System for Human Cytogenetic Nomenclature (ISCN 1995) [6]. For an overall assessment of chromosomal aberrations, the findings were also presented as breakpoint and chromosomal imbalance maps. Breakpoints of structural aberrations of each individual tumor were recorded once against the corresponding chromosomal ideograms. For a breakpoint observed more than once in the same clone, or in multiple clones that were related, the breakpoint was still recorded once only. Chromosomal imbalances were recorded in relation to the nearest ploidy. When the same chromosome was involved in both numerical and structural rearrangements that might entail duplication or deletion of that segment, the resultant net imbalance was recorded. Again, the same imbalance found in more than one of the related clones was recorded only once.
2.3. Molecular cytogenetic analysis To delineate the origin of the hsr, COBRA-FISH was performed as described [7], with minor modifications. Whole-chromosome–specific painting probes used for COBRA-FISH were supplied by Cytocell (Adderbury, Banbury, UK). Briefly, slides with metaphase chromosomes were pretreated with RNase A and pepsin according to Wiegant et al. [8]. A probe mix of 8 µL comprising 24 human chromosomes labeled with five distinct fluorochromes by the combination of binary labeling and ratio labeling was applied on the slide. Slides and probes were denatured simultaneously by incubation at 72⬚C on a hot plate. After hybridization for 48–72 hours at 37⬚C in a humid chamber, the slides were washed at 74⬚C in 0.4× SSC for 2 minutes. Chromosomes were counterstained by TNT (0.1 mol/L Tris and 0.15 mol/ L NaCl in 100 mL 0.05% Tween-20) and 4′-6-diamidino2-phenylindole, 2–10 µL and 0.5 mg/mL, respectively, for 10 minutes. The slides were embedded in Citifluor (Ted Pella, Redding, CA) before microscopic evaluation. For analysis, an Axioplan-2 microscope (Zeiss, Oberkochen, Germany) coupled to a cooled charge-coupled device (CCD) camera and a 12-position filter wheel was used. Acquired images were evaluated with the CytoVision ChromoFluor System (Applied Imaging, Newcastle, UK), according to the principles outlined in Szuhai et al. [9]. To determine whether CCND1 was involved in the amplification, a whole-chromosome–specific probe for chromosome 11 (WCP11) and a cosmid containing an insert of CCND1 were used. Cosmid probe preparation was performed according to standard procedures [10]. All DNA probes were labeled with biotin-dUTP or digoxygenin-dUTP (Boehringer Mannheim, Mannheim, Germany) using random hexanucleotides (Amersham, Buckinghamshire, UK). Hybridization and detection were carried out as described by Ho¨glund et al. [11].
3. Results 3.1. Karyotypic analysis Mitotic cells with good quality metaphase chromosomes were obtained in four of six studied cases (Table 1). Karyotypic analysis showed that all four cases had highly complex karyotypes with numerous numerical and structural aberrations. In three cases, hsr at different chromosomal regions were found (Table 1; Figs. 1 and 2). In case 1, hsr was found in chromosomes 4 and 15 and interpreted as der(4) add(4)(p13)hsr(4)(p13) and der(15)add(15)(p11)hsr(15)(p11), respectively. In case 2, an hsr was found in chromosome 11 and the aberration was interpreted as der(11)hsr(11) (q23)add(11)(q23). Case 4 had an hsr that was located at the long arm of chromosome 4, interpreted as der(4)hsr(4) (q31)add(4)(q31). To enable the genomic profile of esophageal SCC to be evaluated more precisely, an esophageal SCC we had reported on previously [12] (Table 1, case 5)
Table 1 Karyotypes based on cytogenetic and multicolor COBRA-FISH analysis in four patients with SCC of the esophagus and a published case for comparison Sex/age
Clinicopathologic features
Tissue
Karyotype
1
M/41
Dysphagia, esophagogastrectomy showed poorly differentiated SCC in distal esophagus, T3N1M0
Tumor
2
M/62
Dysphagia, esophagectomy showed moderately differentiated SCC in distal esophagus, T3N1M0
53~62,XX,⫺Y,⫹del(1)(p22),⫺2,⫹der(3;9)(q10;q10),der(4)t(4;11)(p13;q13)hsr(11)(q13),del(5)(q14),⫹der(7;19)(q10;q10), der(8)t(8;12)(p22;?),⫺9,⫺9,⫺10,der(10;18)(q10;q10),del(11)(p13),⫺11,⫺12,add(14)(p11),der(15)t(11;15)(q13;p11)hsr (2;11)(?;q13)t(2;12)(?;q15),⫹der(16)t(14;16)(q14;q12),⫺17,⫺18,add(18)(p11),⫹der(20)t(8;20)(q13;q13),⫺21,i(21)(q10), der(21;22)(q10;q10),⫺22,⫹r[cp42] 45,X,⫺Y[3]/46,XY[41] 47,X,der(Y)t(Y;17)(q12;q11),⫹2,i(3)(q10),dup(6)(q22q27),⫹7,der(8)t(8;19)(q24;q11),⫹der(8;13)(q10;q10)× 3,der(9;21) (p10;q10),der(11)dup(11)(q14q23)hsr(11)(q13),ider(14)(14qter→q10::q10→q24::9q13→9qter)× 2,⫹i(14)(q10),⫺17, add(22)(q?)[cp60]
3
M/52
4
F/86
Metastatic cervical lymph nodes, SCC of esophagus, surgery not performed Retrosternal pain, esophagectomy showed moderately differentiated SCC in mid-third of esophagus, T4N1M0
Margin Tumor
Margin Tumor
Failure 67,XYY,⫹2,i(3)(q10),⫺4,⫺5,der(6;17)(p10;q10)add(17)(q25),del(6)(q13),⫹7,⫹7,⫹i(8)(q10),⫹9,del(9)(p22)×2,⫺10, add(12)(p13)×1~2,⫺15,⫺17,⫺18,⫺19,⫺21,⫺22[cp49]
Tumor
65~69,X,i(X)(p10)×1~2,Y,del(1)(q11q25),der(1;16)(q10;p10),⫺3,⫺4,der(5;9)(q10;q10),add(7)(q36)×2?4,dic(1;7)(p12;p22), ⫺9,⫺10,⫺10,i(11)(q10),⫺11,⫺12,⫺13,⫺13,⫺14,⫺15,⫺18,⫺18,⫹add(19)(q13),⫹20,⫺21,⫺22,inc[cp19]/100~110, XYY,i(X)(p10)×2,del(1)(q11q25)×2,add(1)(p11)×2,add(3)(p11)×1~2,der(4)add(4)(q31)hsr(4)(q31),⫺5,⫹7,add(7)(q36) ×3~4,⫺8,⫺8,⫺8,⫺9,⫺10,⫺10,⫺10,⫺11,⫺11,i(11)(q10),⫺12,⫺12,⫺13,⫺13,⫺14,⫺14,⫺15,⫺15,⫺16,⫺17,⫺18,⫺18, ⫺18,add(19)(q13),⫺21,⫺21,⫺22,⫺22,inc[cp11] 107~117,Y,XX,⫹der(X;15)(p10;q10),⫹i(X)(p10)×2,add(1)(p11)×2,add(1)(p11)×2,dic(1;7)(p12;p22),⫹del(1)(q11q25)×2, der(2;8)(q10;q10)×2,add(3)(p11)×2,del(3)(q21),⫺4,der(4)add(4)(q31)hsr(q31)×2,add(5)(p11),i(5)(p10),⫹dic(5;13) (q13;p13)×2,⫹7,add(7)(q36)×4,⫺8,⫺9,⫺9,⫺10,⫺10,⫺10,⫺11,⫺11,i(11(q10),⫺12,⫺12,⫺13,add(14)(p11)×3,⫺15,⫹16, ⫺17,⫺18,⫺18,⫺18,⫹add(19)(q13)×2,⫹20,⫺21,?i(21)(q10),⫺22,⫺22,⫹r,inc[cp67] 73~74,XXY,⫹der(1;5)(q10;q10),del(4)(p14),⫹del(4)(q23),⫺5,⫺5,der(5)t(1;5)(q25;p15),der(6)t(6;?;22)(p23;?;q11),⫹der(6) add(6)(p21)del(6)(q23),⫹add(7)(p15),⫺8,⫺8,⫺8,⫹11,der(11)hsr(11)(q13)dup(11)(q14q23)×2,⫺13,idic(13)(p13;p13), ⫺14,der(15)t(9;15)(p13)(p13),⫹16,⫺17,⫺21,⫺22,inc/133~144,idem×2,add(3)(q11),add(3)(p11),der(13)t(1;?;13) (q23;?;p11),inc
Margin
Jin et al. 1995 [12]
Tumor
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
Case no.
Abbreviations: F, female; M, male.
23
24
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
Fig. 1. Representative karyotype from the esophageal SCC of case 1. Arrows indicate hsr.
was also included. A total of 92 breakpoints involving virtually all the chromosomes were identified (Fig. 3). The chromosomal sites rearranged in these esophageal SCC
were nonrandomly distributed. Preferentially involved chromosomal bands or regions were 11q13, 8q10, 21q10, 3p10~p11, 1p11~q11, 5p11~q11, and 14p11~q11. Four
Fig. 2. Representative karyotype from the esophageal SCC of case 2. Arrow indicates the hsr located in chromosome 11.
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
25
Fig. 3. Distribution of the 92 chromosomal breakpoints registered in the five esophageal SCC.
recurrent structural aberrations were identified, including hsr (n = 3), i(3q) (n = 2), i(21q) (n = 2), and ring chromosome (n = 2). Losses of chromosomal material occurred more often than gains (Fig. 4). Imbalances detected in at least three tumors included losses at 4p13~pter, 5q14~qter, 9p22~ pter, 10p, 11p13~pter, 12p13~pter, 17p10~pter, 18p11~pter, 21p, and 22p, and gains at 1q25~qter, 3q, 7q, and 8q. 3.2. Results of COBRA-FISH COBRA-FISH analysis showed that in two cases, the hsr was found to be derived from materials of chromosome 11 (Fig. 5A). In case 1, the hsr on chromosome 15 also contained chromosome 2 material. Further FISH with the use of WCP11 and a CCND1 cosmid showed that CCND1 was amplified in both cases (Fig. 5B). Thus, the derivative chromosomes harboring hsr in case 1 were reinterpreted as der(4)t(4;11)(p13;q13)hsr(11)(q13) and der(15)t(11;15)(q13; p11)hsr(2;11)(?;q13)t(2;12)(?;q15). The derivative chromosome 11 with hsr in case 2 was revised as der(11)dup(11) (q14q23)hsr(11)(q13). 4. Discussion In the present study, we have described in detail the karyotypic aberrations observed in primary culture of esophageal
SCC. To our knowledge, only three previous studies have reported data on primary tumor cell cultures of esophageal SCC. Casalone et al. [13] reported a case with ⫹Y as the sole aberration. Rosenblum-Vos et al. [14] reported six cases, two of which showed ⫺Y. In the remaining four cases, several numerical and/or a few structural aberrations were described. No recurrent aberration was identified. We had previously reported on the latest study, in which a highly complex karyotype including an hsr in chromosomal band 11q13 was detected in a case of esophageal SCC [12]. In contrast to the lack of information on primary tumor cell culture, some karyotypic data were obtained from the cytogenetic study of esophageal carcinoma cell lines. Wang-Peng et al. [15] studied 14 such cell lines and all of them had complex rearranged karyotypes with extensive heterogeneity. Nevertheless, some chromosomal regions seemed to be nonrandomly involved, including 1p11~q11, 1q31, 3p11~p12, 15p11~q11, and 21q11. Aberrations involving 11q, with occasional hsr, had often been mapped to 11q11~q12 in these lines. In general, the karyotypic profile of primary esophageal SCC was similar to that of esophageal SCC cell lines. However, differences in the breakpoint mapping of 11q rearrangements may reflect only the inherent technical difficulty in cytogenetic interpretation. Our observations of complex karyotypes with widespread chromosomal gains and losses support the notion that
26
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
Fig. 4. Distribution of karyotypic imbalances caused by imbalanced structural aberrations or numerical changes in five esophageal SCC. Vertical lines on the left indicate loss of chromosome material, and lines on the right gain of chromosome material.
esophageal SCC develops through a multistage tumorigenesis. Similar to many types of carcinomas, no tumor-specific aberrations could be identified in esophageal SCC. Nevertheless, a nonrandom karyotypic pattern could be delineated. Rearrangements involving chromosomal band 11q13 were a recurrent aberration. Another characteristic feature was the preferential involvement of centromeric and pericentromeric regions, accounting for approximately 45% of the total rearrangements. These resulted in common structural aberrations including isochromosomes, whole-arm translocations, and deletions involving chromosomes 1, 3, 5, 8, and 21. The overall genomic imbalance profile presented herein also allowed the localization of genomic regions frequently involved in esophageal carcinogenesis. The prominent under-representation of genetic materials in chromosomal segments 4p13~pter, 5q14~qter, 9p22~pter, 10p, 11p13~pter, 12p13~pter, 17p10~pter, 18p11~pter, 21p, and 22p, and the over-representation in chromosome segments 1q25~qter, and arms 3q, 7q, and 8q may provide clues for the analysis of gene loci important for esophageal SCC development. Comparative genomic hybridization (CGH) has recently complemented conventional cytogenetics in the characterization of chromosome imbalances in esophageal SCC. Although partly discordant findings have been reported by
different research groups [16–20], these studies in general have revealed widespread imbalances on esophageal SCC, and some genomic regions are nonrandomly involved, which is in line with karyotypic findings. The most common imbalances revealed by CGH were loss of 3p, 4p, 4q, 5q, 9p, and 18q, and gain of 1q, 3q, 7p, 7q, 8q, 20q, and the amplification of 11q13. With some exceptions (e.g., loss of 18q and gain of 20q) these results are in good concordance with the karyotypic imbalances shown by this study. Gene amplification is a common phenomenon observed in many tumor types [21]. High-level amplification of a particular gene or genomic region has been demonstrated in only a few tumors, however, including neuroblastoma, breast cancer, and SCC of the head and neck region. A peculiar cytogenetic feature in esophageal SCC observed in the present study is the frequent occurrence of high-level gene amplification, in the form of hsr, in four out of five esophageal SCC, suggesting that this might be an important mechanism in the formation of esophageal SCC. By the combination of COBRA-FISH and FISH with a CCND1 cosmid probe, it could be shown that in two cases the relevant gene present in multiple copies in hsr was CCND1, which is normally localized at chromosomal band 11q13. CCND1 is a cell cycle–regulating gene that encodes a protein cyclin D1 involved in G1-to-S transition [22]. The amplification of
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
27
Fig. 5. (A) Representative COBRA-FISH karyotype from case 1, showing that the hsr in chromosomes 4 and 15 contain DNA sequences from chromosome 11. (B) Hybridization with a CCND1 probe (red) in esophageal SCC of cases 1 and 2. The chromosomes on the left are normal chromosomes 11; and on the right, derivative chromosomes harboring CCND1 amplification.
CCND1 leads to increased expression of cyclin D1. CCND1 has been extensively studied in a number of epithelial malignancies, particularly carcinomas of the breast and SCC of the head and neck region. In the latter, amplification and overexpression of CCND1 were found in approximately 30% of the primary tumors investigated [23]. Two of the three tumors investigated by FISH in the present study showed amplification of CCND1, implying that this gene might be a prime target in amplification. The pathogenetic significance of the amplification of 11q13 or CCND1 was further accentuated by previous studies that showed molecular evidence of genomic amplification of several loci within
11q13, including the oncogenes INT2, HST1, and CCND1, and overexpression of CCND1 in a subset of esophageal SCC [24–26], as well as the finding of hsr involving chromosome 11q13 in esophageal cancer cell lines [15]. Therefore, this study provides for the first time (to our knowledge) cytogenetic evidence of CCND1 amplification in primary cancer cell cultures. Owing to a lack of suitable specimens, we were not able to confirm if cyclin D1 (product of CCND1) was overexpressed at the protein level in the tumors, which would be another important confirmation of the cytogenetic data observed in this study. Therefore, a crucial point that should be addressed in future studies is to investigate the tumors
28
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
concomitantly by cytogenetic and molecular techniques to define if CCND1 amplification at the cytogenetic level, namely hsr or dmin, is matched with molecular evidence of gene amplification and overexpression. Another question that needs to be addressed when more data become available is whether cytogenetic and molecular evidence of gene amplification is associated with more aggressive tumors histopathologically or clinically, as has been reported in other tumor types such as neuroblastoma and breast cancer [27].
Acknowledgments This work was supported by the Kadoorie Charitable Foundation, research grants from the University of Hong Kong, and grants from the Swedish Cancer Society. Dr. Zhang’s stay at Queen Mary Hospital was sponsored by the Youth Fund from Ruijin Hospital, Shanghai Second Medical University, Shanghai, China.
References [1] International Agency for Research on Cancer. Cancer incidence in five continents. Volume VII. IARC Science Publishers, Lyon, France: 1997, p.1240. [2] Altorki N, Skinner D. Should en bloc esophagectomy be the standard of care for esophageal carcinoma? Ann Surg 2001;234:581–7. [3] Mitelman F, Mertens F, Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997; 15:417–74. [4] Mitelman Database of Chromosome Aberrations in Cancer. Mitelman F, Johansson B, Mertens F, editors. Available at: http://cgap.nci.nih.gov/ Chromosomes/Mitelman. Accessed March 8, 2003. [5] 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. [6] Mitelman F, editor. An international system for human cytogenetic nomenclature. Basel: S. Karger, 1995. [7] Tanke HJ, Wiegant J, van Gijlswijk RP, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J. New strategy for multicolour fluorescence in situ hybridization: COBRA: combined binary ratio labelling. Eur J Hum Genet 1999;7:2–11. [8] Wiegant J, Ried T, Nederlof PM, van der Ploeg M, Tanke HJ, Raap AK. In situ hybridization with fluoresceinated DNA. Nucleic Acids Res 1991;19:3237–41. [9] Szuhai K, Bezrookove V, Wiegant J, Vrolijk J, Dirks RW, Rosenberg C, Raap AK, Tanke HJ. Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH. Genes Chromosomes Cancer 2000;28:92–7. [10] Maniatis T, Fritsch EF, Sambrook J. Molecular cloning. A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.
[11] Ho¨glund M, Johansson B, Pedersen-Bjergaard J, Marynen P, Mitelman F. Molecular characterization of 12p abnormalities in hematologic malignancies: deletion of KIP1, rearrangement of TEL, and amplification of CCND2. Blood 1996;87:324–30. [12] Jin C, Mertens F, Jin Y, Wennerberg J, Heim S, Mitelman F. Complex karyotype with an 11q13 homogeneously staining region in esophageal squamous cell carcinoma. Cancer Genet Cytogenet 1995;82: 175–6. [13] Casalone R, Minelli E, Portensoso P, Giudici A. Clonal duplication of the Y chromosome and fra(X)(q28) in a case of epidermoid carcinoma of the esophagus. Cancer Genet Cytogenet 1990;45:269–71. [14] Rosenblum-Vos LS, Meltzer SJ, Leana-Cox J, Schwartz S. Cytogenetic studies of primary cultures of esophageal squamous cell carcinoma. Cancer Genet Cytogenet 1993;70:127–31. [15] Whang-Peng J, Banks-Schlegel SP, Lee EG. Cytogenetic studies of esophageal carcinoma cell lines. Cancer Genet Cytogenet 1990;45: 101–20. [16] Pack SD, Karkera JD, Zhuang Z, Pak ED, Balan KV, Hwu P, Park WS, Pham T, Ault DO, Glaser M, Liotta L, Detera-Wadleigh SD, Wadleigh RG. Molecular cytogenetic fingerprinting of esophageal squamous cell carcinoma by comparative genomic hybridization reveals a consistent pattern of chromosomal alterations. Genes Chromosomes Cancer 1999;25:160–8. [17] Tada K, Oka M, Hayashi H, Tangoku A, Oga A, Sasaki K. Cytogenetic analysis of esophageal squamous cell carcinoma cell lines by comparative genomic hybridization: relationship of cytogenetic aberrations to in vitro cell growth. Cancer Genet Cytogenet 2000;117:108–12. [18] Mayama T, Fukushige S, Shineha R, Nishihira T, Satomi S, Horii A. Frequent loss of copy munber on the long arm of chromosome 21 in human esophageal squamous cell carcinoma. Int J Oncol 2000;17: 245–52. [19] Yen CC, Chen YJ, Chen JT, Hsia JY, Chen PM, Liu JH, Fan FS, Chiou TJ, Wang WS, Lin CH. Comparative genomic hybridization of esophageal squamous cell carcinoma: correlations between chromosomal aberrations and disease progression/prognosis. Cancer 2001;92: 2769–77. [20] Pimkhaokham A, Shimada Y, Fukuda Y, Kurihara N, Imoto I, Yang ZQ, Imamura M, Nakamura Y, Amagasa T, Inazawa J. Nonrandom chromosomal imbalances in esophageal squamous cell carcinoma cell lines: possible involvement of the ATF3 and CENPF genes in the 1q32 amplicon. Jpn J Cancer Res 2000;91:1126–33. [21] Schwab M. Oncogene amplification in solid tumors. Semin Cancer Biol 1999;9:319–25. [22] Motokura T, Arnold A. Cyclin D and oncogenesis. Current Opin Genet Devel 1993;3:5–10. [23] Schuuring E. The involvement of the chromosome 11q13 region in human malignancies: cyclin D1 and EMS1 are two new candidate oncogenes - a review. Gene 1995;159:83–96. [24] Tsuruta H, Sakamoto H, Onda M, Terada M. Amplification and overexpression of EXP1 and EXP2/cyclin D1 genes in human esophageal carcinomas. Biochem Biophys Res Commun 1993;196:1529–36. [25] Gramlich TL, Fritsch CR, Maurer D, Eberle M, Gansler TS. Differential polymerase chain reaction assay of cyclin D1 gene amplification in esophageal carcinoma. Diagn Mol Pathol 1994;3:255–9. [26] Shinozaki H, Ozawa S, Ando N, Tsuruta H, Terada M, Ueda M, Kitajima M. Cyclin D1 amplification as a new predictive classification for squamous cell carcinoma of the esophagus, adding gene information. Clin Cancer Res 1996;2:1155–61. [27] Schwab M, Amler LC. Amplification of cellular oncogenes: a predictor of clinical outcome in human cancer. Genes Chromosomes Cancer 1990;1:181–93.
Cytogenetic and fluorescence in situ hybridization characterization of clonal chromosomal aberrations and CCND1 amplification in esophageal carcinomas Yuesheng Jina,b,c, Charlotte Jinc, Simon Lawb, Kent-Man Chub, Hao Zhangb,d, Bodil Strombeckc, Anthony P.W. Yuenb, Yok-Lam Kwonga,* a
Department of Medicine, Queen Mary Hospital, Pokfulam Road, Hong Kong, China Department of Surgery, Queen Mary Hospital, Pokfulam Road, Hong Kong, China c Department of Clinical Genetics, University Hospital, Lund, Sweden d Department of Otolaryngology, Ruijin Hospital, Shanghai Second Medical University, Shanghai, China Received 10 March 2003; received in revised form 28 April 2003; accepted 7 May 2003 b
Abstract
Cytogenetic analyses of four squamous cell carcinomas (SCC) of the esophagus showed complex numerical and structural abnormalities. Chromosomal bands or regions preferentially involved were 11q13, 8q10, 21q10, 3p10~p11, 1p11~q11, 5p11~q11, and 14p11~q11. For the first time, to our knowledge, recurrent aberrations were identified in esophageal SCC, including homogenous staining region (hsr), isochromosomes i(3q) and i(21q), and ring chromosome. Losses of chromosomal material dominated over gains. Recurrent imbalances included under-representation of 4p13~pter, 5q14~qter, 9p22~pter, 10p, 11p13~pter, 12p13~pter, 17p10~pter, 18p11~pter, 21p, and 22p, as well as over-representation of 1q25~qter, 3q, 7q, and 8q. Interestingly, hsr at different chromosomal regions occurred in three of four cases. With the application of fluorescence in situ hybridization (FISH) and multicolor combined binary ratio labeling–FISH with specific DNA probes, it could be shown that in two cases the hsr was derived from chromosome 11 material and that the amplicon included CCND1. Our results, together with previous molecular genetic findings, indicate that CCND1 might be a prime target in 11q13 amplification, and that amplification of this gene might be crucial in the tumorigenesis of esophageal SCC. These observed chromosomal aberrations and imbalances thus provide important information for further molecular genetic investigation of esophageal SCC. 쑖 2004 Elsevier Inc. All rights reserved.
1. Introduction Cancer of the esophagus has a great variation in geographic distribution. Although these neoplasms are uncommon in the United States and many European countries, they are prevalent in China [1]. According to data from the World Health Organization, mortality attributable to esophageal cancer is highest in China, Puerto Rico, and Singapore. The major histologic types of esophageal cancer are squamous cell carcinoma (SCC) and adenocarcinoma. The prognosis of these neoplasms is poor, with a 5-year survival of less than 30% in advanced diseases [2]. The etiology and pathogenesis of this aggressive cancer are poorly understood.
* Corresponding author: Tel.: ⫹852-2-855-4597; fax: ⫹852-2-974-1165. E-mail address: [email protected] (Y.-L. Kwong). 0165-4608/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0165-4608(03)00213-9
In a genome-wide search for molecular alterations underlying tumorigenesis in esophageal SCC, the patterns of aberrations at the chromosomal level might be an essential guide, as has been shown for many other human neoplasms [3]. To our knowledge, however, only eight esophageal SCC with clonal karyotypic changes have been reported to date [4]. Three esophageal SCC had only simple numerical changes, namely ⫺Y and ⫹Y, which presumably did not represent aberration in the tumor parenchyma. The remaining five cases had highly variable chromosome rearrangements. Due to limited chromosomal data and the karyotypic diversity in previously reported tumors, the putative genetic events that might be important in esophageal SCC remain undefined. As part of a systematic study of genomic rearrangements that characterize esophageal SCC, we have detected complex karyotypic aberrations in four tumors, three of which also
22
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
displayed a homogenous staining region (hsr) in different chromosomal locations. To identify the origin of the hsr and target gene(s) in the amplification process, further fluorescence in situ hybridization (FISH) and multicolor combined binary ratio labeling (COBRA)–FISH studies were performed.
2. Materials and methods 2.1. Patients Six patients undergoing esophagectomy/endoscopic biopsy for cancer of the esophagus were studied. None of the patients had been treated with radio- or chemotherapy before the cytogenetic analysis. Samples from two patients did not yield cytogenetic results, whereas the remaining four showed clonal chromosome aberrations. The clinical and histopathologic characteristics of these four patients are summarized in Table 1. 2.2. Karotypic analysis For karyotypic analysis, a piece of the tumor and its marginal tissue was freshly dissected from the surgical specimen and processed for cytogenetic analysis as described [5]. Briefly, the dissected tumor was minced finely with scissors and disaggregated overnight in collagenase (180 U/ mL). The resulting suspension of small cell clumps was washed twice in RPMI-1640 and plated on vitrogen-coated culture flasks. The growth medium was keratinocyte serumfree (KSF) medium (GIBCO 10744-019; Invitrogen Corp., Carlsbad, CA) with 2%–5% fetal calf serum, supplemented with l-glutamine (0.23 mg/mL), penicillin-streptomycin (100 IU/mL and 0.2 mg/mL, respectively), and amphotericine (2.5 µg/mL). The cell growth pattern and morphology were evaluated periodically under inverted microscopy. When tumor cells showed active division (in ~5–10 days), they were harvested by conventional cytogenetic techniques. Harvested cells on slides were incubated overnight at 60⬚C, followed by submersion in 2× standard saline citrate (SSC) at 60⬚C for 3–4 hours before banding with Wright’s stain. The criteria used for clonality and karyotype description were according to the International System for Human Cytogenetic Nomenclature (ISCN 1995) [6]. For an overall assessment of chromosomal aberrations, the findings were also presented as breakpoint and chromosomal imbalance maps. Breakpoints of structural aberrations of each individual tumor were recorded once against the corresponding chromosomal ideograms. For a breakpoint observed more than once in the same clone, or in multiple clones that were related, the breakpoint was still recorded once only. Chromosomal imbalances were recorded in relation to the nearest ploidy. When the same chromosome was involved in both numerical and structural rearrangements that might entail duplication or deletion of that segment, the resultant net imbalance was recorded. Again, the same imbalance found in more than one of the related clones was recorded only once.
2.3. Molecular cytogenetic analysis To delineate the origin of the hsr, COBRA-FISH was performed as described [7], with minor modifications. Whole-chromosome–specific painting probes used for COBRA-FISH were supplied by Cytocell (Adderbury, Banbury, UK). Briefly, slides with metaphase chromosomes were pretreated with RNase A and pepsin according to Wiegant et al. [8]. A probe mix of 8 µL comprising 24 human chromosomes labeled with five distinct fluorochromes by the combination of binary labeling and ratio labeling was applied on the slide. Slides and probes were denatured simultaneously by incubation at 72⬚C on a hot plate. After hybridization for 48–72 hours at 37⬚C in a humid chamber, the slides were washed at 74⬚C in 0.4× SSC for 2 minutes. Chromosomes were counterstained by TNT (0.1 mol/L Tris and 0.15 mol/ L NaCl in 100 mL 0.05% Tween-20) and 4′-6-diamidino2-phenylindole, 2–10 µL and 0.5 mg/mL, respectively, for 10 minutes. The slides were embedded in Citifluor (Ted Pella, Redding, CA) before microscopic evaluation. For analysis, an Axioplan-2 microscope (Zeiss, Oberkochen, Germany) coupled to a cooled charge-coupled device (CCD) camera and a 12-position filter wheel was used. Acquired images were evaluated with the CytoVision ChromoFluor System (Applied Imaging, Newcastle, UK), according to the principles outlined in Szuhai et al. [9]. To determine whether CCND1 was involved in the amplification, a whole-chromosome–specific probe for chromosome 11 (WCP11) and a cosmid containing an insert of CCND1 were used. Cosmid probe preparation was performed according to standard procedures [10]. All DNA probes were labeled with biotin-dUTP or digoxygenin-dUTP (Boehringer Mannheim, Mannheim, Germany) using random hexanucleotides (Amersham, Buckinghamshire, UK). Hybridization and detection were carried out as described by Ho¨glund et al. [11].
3. Results 3.1. Karyotypic analysis Mitotic cells with good quality metaphase chromosomes were obtained in four of six studied cases (Table 1). Karyotypic analysis showed that all four cases had highly complex karyotypes with numerous numerical and structural aberrations. In three cases, hsr at different chromosomal regions were found (Table 1; Figs. 1 and 2). In case 1, hsr was found in chromosomes 4 and 15 and interpreted as der(4) add(4)(p13)hsr(4)(p13) and der(15)add(15)(p11)hsr(15)(p11), respectively. In case 2, an hsr was found in chromosome 11 and the aberration was interpreted as der(11)hsr(11) (q23)add(11)(q23). Case 4 had an hsr that was located at the long arm of chromosome 4, interpreted as der(4)hsr(4) (q31)add(4)(q31). To enable the genomic profile of esophageal SCC to be evaluated more precisely, an esophageal SCC we had reported on previously [12] (Table 1, case 5)
Table 1 Karyotypes based on cytogenetic and multicolor COBRA-FISH analysis in four patients with SCC of the esophagus and a published case for comparison Sex/age
Clinicopathologic features
Tissue
Karyotype
1
M/41
Dysphagia, esophagogastrectomy showed poorly differentiated SCC in distal esophagus, T3N1M0
Tumor
2
M/62
Dysphagia, esophagectomy showed moderately differentiated SCC in distal esophagus, T3N1M0
53~62,XX,⫺Y,⫹del(1)(p22),⫺2,⫹der(3;9)(q10;q10),der(4)t(4;11)(p13;q13)hsr(11)(q13),del(5)(q14),⫹der(7;19)(q10;q10), der(8)t(8;12)(p22;?),⫺9,⫺9,⫺10,der(10;18)(q10;q10),del(11)(p13),⫺11,⫺12,add(14)(p11),der(15)t(11;15)(q13;p11)hsr (2;11)(?;q13)t(2;12)(?;q15),⫹der(16)t(14;16)(q14;q12),⫺17,⫺18,add(18)(p11),⫹der(20)t(8;20)(q13;q13),⫺21,i(21)(q10), der(21;22)(q10;q10),⫺22,⫹r[cp42] 45,X,⫺Y[3]/46,XY[41] 47,X,der(Y)t(Y;17)(q12;q11),⫹2,i(3)(q10),dup(6)(q22q27),⫹7,der(8)t(8;19)(q24;q11),⫹der(8;13)(q10;q10)× 3,der(9;21) (p10;q10),der(11)dup(11)(q14q23)hsr(11)(q13),ider(14)(14qter→q10::q10→q24::9q13→9qter)× 2,⫹i(14)(q10),⫺17, add(22)(q?)[cp60]
3
M/52
4
F/86
Metastatic cervical lymph nodes, SCC of esophagus, surgery not performed Retrosternal pain, esophagectomy showed moderately differentiated SCC in mid-third of esophagus, T4N1M0
Margin Tumor
Margin Tumor
Failure 67,XYY,⫹2,i(3)(q10),⫺4,⫺5,der(6;17)(p10;q10)add(17)(q25),del(6)(q13),⫹7,⫹7,⫹i(8)(q10),⫹9,del(9)(p22)×2,⫺10, add(12)(p13)×1~2,⫺15,⫺17,⫺18,⫺19,⫺21,⫺22[cp49]
Tumor
65~69,X,i(X)(p10)×1~2,Y,del(1)(q11q25),der(1;16)(q10;p10),⫺3,⫺4,der(5;9)(q10;q10),add(7)(q36)×2?4,dic(1;7)(p12;p22), ⫺9,⫺10,⫺10,i(11)(q10),⫺11,⫺12,⫺13,⫺13,⫺14,⫺15,⫺18,⫺18,⫹add(19)(q13),⫹20,⫺21,⫺22,inc[cp19]/100~110, XYY,i(X)(p10)×2,del(1)(q11q25)×2,add(1)(p11)×2,add(3)(p11)×1~2,der(4)add(4)(q31)hsr(4)(q31),⫺5,⫹7,add(7)(q36) ×3~4,⫺8,⫺8,⫺8,⫺9,⫺10,⫺10,⫺10,⫺11,⫺11,i(11)(q10),⫺12,⫺12,⫺13,⫺13,⫺14,⫺14,⫺15,⫺15,⫺16,⫺17,⫺18,⫺18, ⫺18,add(19)(q13),⫺21,⫺21,⫺22,⫺22,inc[cp11] 107~117,Y,XX,⫹der(X;15)(p10;q10),⫹i(X)(p10)×2,add(1)(p11)×2,add(1)(p11)×2,dic(1;7)(p12;p22),⫹del(1)(q11q25)×2, der(2;8)(q10;q10)×2,add(3)(p11)×2,del(3)(q21),⫺4,der(4)add(4)(q31)hsr(q31)×2,add(5)(p11),i(5)(p10),⫹dic(5;13) (q13;p13)×2,⫹7,add(7)(q36)×4,⫺8,⫺9,⫺9,⫺10,⫺10,⫺10,⫺11,⫺11,i(11(q10),⫺12,⫺12,⫺13,add(14)(p11)×3,⫺15,⫹16, ⫺17,⫺18,⫺18,⫺18,⫹add(19)(q13)×2,⫹20,⫺21,?i(21)(q10),⫺22,⫺22,⫹r,inc[cp67] 73~74,XXY,⫹der(1;5)(q10;q10),del(4)(p14),⫹del(4)(q23),⫺5,⫺5,der(5)t(1;5)(q25;p15),der(6)t(6;?;22)(p23;?;q11),⫹der(6) add(6)(p21)del(6)(q23),⫹add(7)(p15),⫺8,⫺8,⫺8,⫹11,der(11)hsr(11)(q13)dup(11)(q14q23)×2,⫺13,idic(13)(p13;p13), ⫺14,der(15)t(9;15)(p13)(p13),⫹16,⫺17,⫺21,⫺22,inc/133~144,idem×2,add(3)(q11),add(3)(p11),der(13)t(1;?;13) (q23;?;p11),inc
Margin
Jin et al. 1995 [12]
Tumor
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
Case no.
Abbreviations: F, female; M, male.
23
24
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
Fig. 1. Representative karyotype from the esophageal SCC of case 1. Arrows indicate hsr.
was also included. A total of 92 breakpoints involving virtually all the chromosomes were identified (Fig. 3). The chromosomal sites rearranged in these esophageal SCC
were nonrandomly distributed. Preferentially involved chromosomal bands or regions were 11q13, 8q10, 21q10, 3p10~p11, 1p11~q11, 5p11~q11, and 14p11~q11. Four
Fig. 2. Representative karyotype from the esophageal SCC of case 2. Arrow indicates the hsr located in chromosome 11.
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
25
Fig. 3. Distribution of the 92 chromosomal breakpoints registered in the five esophageal SCC.
recurrent structural aberrations were identified, including hsr (n = 3), i(3q) (n = 2), i(21q) (n = 2), and ring chromosome (n = 2). Losses of chromosomal material occurred more often than gains (Fig. 4). Imbalances detected in at least three tumors included losses at 4p13~pter, 5q14~qter, 9p22~ pter, 10p, 11p13~pter, 12p13~pter, 17p10~pter, 18p11~pter, 21p, and 22p, and gains at 1q25~qter, 3q, 7q, and 8q. 3.2. Results of COBRA-FISH COBRA-FISH analysis showed that in two cases, the hsr was found to be derived from materials of chromosome 11 (Fig. 5A). In case 1, the hsr on chromosome 15 also contained chromosome 2 material. Further FISH with the use of WCP11 and a CCND1 cosmid showed that CCND1 was amplified in both cases (Fig. 5B). Thus, the derivative chromosomes harboring hsr in case 1 were reinterpreted as der(4)t(4;11)(p13;q13)hsr(11)(q13) and der(15)t(11;15)(q13; p11)hsr(2;11)(?;q13)t(2;12)(?;q15). The derivative chromosome 11 with hsr in case 2 was revised as der(11)dup(11) (q14q23)hsr(11)(q13). 4. Discussion In the present study, we have described in detail the karyotypic aberrations observed in primary culture of esophageal
SCC. To our knowledge, only three previous studies have reported data on primary tumor cell cultures of esophageal SCC. Casalone et al. [13] reported a case with ⫹Y as the sole aberration. Rosenblum-Vos et al. [14] reported six cases, two of which showed ⫺Y. In the remaining four cases, several numerical and/or a few structural aberrations were described. No recurrent aberration was identified. We had previously reported on the latest study, in which a highly complex karyotype including an hsr in chromosomal band 11q13 was detected in a case of esophageal SCC [12]. In contrast to the lack of information on primary tumor cell culture, some karyotypic data were obtained from the cytogenetic study of esophageal carcinoma cell lines. Wang-Peng et al. [15] studied 14 such cell lines and all of them had complex rearranged karyotypes with extensive heterogeneity. Nevertheless, some chromosomal regions seemed to be nonrandomly involved, including 1p11~q11, 1q31, 3p11~p12, 15p11~q11, and 21q11. Aberrations involving 11q, with occasional hsr, had often been mapped to 11q11~q12 in these lines. In general, the karyotypic profile of primary esophageal SCC was similar to that of esophageal SCC cell lines. However, differences in the breakpoint mapping of 11q rearrangements may reflect only the inherent technical difficulty in cytogenetic interpretation. Our observations of complex karyotypes with widespread chromosomal gains and losses support the notion that
26
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
Fig. 4. Distribution of karyotypic imbalances caused by imbalanced structural aberrations or numerical changes in five esophageal SCC. Vertical lines on the left indicate loss of chromosome material, and lines on the right gain of chromosome material.
esophageal SCC develops through a multistage tumorigenesis. Similar to many types of carcinomas, no tumor-specific aberrations could be identified in esophageal SCC. Nevertheless, a nonrandom karyotypic pattern could be delineated. Rearrangements involving chromosomal band 11q13 were a recurrent aberration. Another characteristic feature was the preferential involvement of centromeric and pericentromeric regions, accounting for approximately 45% of the total rearrangements. These resulted in common structural aberrations including isochromosomes, whole-arm translocations, and deletions involving chromosomes 1, 3, 5, 8, and 21. The overall genomic imbalance profile presented herein also allowed the localization of genomic regions frequently involved in esophageal carcinogenesis. The prominent under-representation of genetic materials in chromosomal segments 4p13~pter, 5q14~qter, 9p22~pter, 10p, 11p13~pter, 12p13~pter, 17p10~pter, 18p11~pter, 21p, and 22p, and the over-representation in chromosome segments 1q25~qter, and arms 3q, 7q, and 8q may provide clues for the analysis of gene loci important for esophageal SCC development. Comparative genomic hybridization (CGH) has recently complemented conventional cytogenetics in the characterization of chromosome imbalances in esophageal SCC. Although partly discordant findings have been reported by
different research groups [16–20], these studies in general have revealed widespread imbalances on esophageal SCC, and some genomic regions are nonrandomly involved, which is in line with karyotypic findings. The most common imbalances revealed by CGH were loss of 3p, 4p, 4q, 5q, 9p, and 18q, and gain of 1q, 3q, 7p, 7q, 8q, 20q, and the amplification of 11q13. With some exceptions (e.g., loss of 18q and gain of 20q) these results are in good concordance with the karyotypic imbalances shown by this study. Gene amplification is a common phenomenon observed in many tumor types [21]. High-level amplification of a particular gene or genomic region has been demonstrated in only a few tumors, however, including neuroblastoma, breast cancer, and SCC of the head and neck region. A peculiar cytogenetic feature in esophageal SCC observed in the present study is the frequent occurrence of high-level gene amplification, in the form of hsr, in four out of five esophageal SCC, suggesting that this might be an important mechanism in the formation of esophageal SCC. By the combination of COBRA-FISH and FISH with a CCND1 cosmid probe, it could be shown that in two cases the relevant gene present in multiple copies in hsr was CCND1, which is normally localized at chromosomal band 11q13. CCND1 is a cell cycle–regulating gene that encodes a protein cyclin D1 involved in G1-to-S transition [22]. The amplification of
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
27
Fig. 5. (A) Representative COBRA-FISH karyotype from case 1, showing that the hsr in chromosomes 4 and 15 contain DNA sequences from chromosome 11. (B) Hybridization with a CCND1 probe (red) in esophageal SCC of cases 1 and 2. The chromosomes on the left are normal chromosomes 11; and on the right, derivative chromosomes harboring CCND1 amplification.
CCND1 leads to increased expression of cyclin D1. CCND1 has been extensively studied in a number of epithelial malignancies, particularly carcinomas of the breast and SCC of the head and neck region. In the latter, amplification and overexpression of CCND1 were found in approximately 30% of the primary tumors investigated [23]. Two of the three tumors investigated by FISH in the present study showed amplification of CCND1, implying that this gene might be a prime target in amplification. The pathogenetic significance of the amplification of 11q13 or CCND1 was further accentuated by previous studies that showed molecular evidence of genomic amplification of several loci within
11q13, including the oncogenes INT2, HST1, and CCND1, and overexpression of CCND1 in a subset of esophageal SCC [24–26], as well as the finding of hsr involving chromosome 11q13 in esophageal cancer cell lines [15]. Therefore, this study provides for the first time (to our knowledge) cytogenetic evidence of CCND1 amplification in primary cancer cell cultures. Owing to a lack of suitable specimens, we were not able to confirm if cyclin D1 (product of CCND1) was overexpressed at the protein level in the tumors, which would be another important confirmation of the cytogenetic data observed in this study. Therefore, a crucial point that should be addressed in future studies is to investigate the tumors
28
Y. Jin et al. / Cancer Genetics and Cytogenetics 148 (2004) 21–28
concomitantly by cytogenetic and molecular techniques to define if CCND1 amplification at the cytogenetic level, namely hsr or dmin, is matched with molecular evidence of gene amplification and overexpression. Another question that needs to be addressed when more data become available is whether cytogenetic and molecular evidence of gene amplification is associated with more aggressive tumors histopathologically or clinically, as has been reported in other tumor types such as neuroblastoma and breast cancer [27].
Acknowledgments This work was supported by the Kadoorie Charitable Foundation, research grants from the University of Hong Kong, and grants from the Swedish Cancer Society. Dr. Zhang’s stay at Queen Mary Hospital was sponsored by the Youth Fund from Ruijin Hospital, Shanghai Second Medical University, Shanghai, China.
References [1] International Agency for Research on Cancer. Cancer incidence in five continents. Volume VII. IARC Science Publishers, Lyon, France: 1997, p.1240. [2] Altorki N, Skinner D. Should en bloc esophagectomy be the standard of care for esophageal carcinoma? Ann Surg 2001;234:581–7. [3] Mitelman F, Mertens F, Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997; 15:417–74. [4] Mitelman Database of Chromosome Aberrations in Cancer. Mitelman F, Johansson B, Mertens F, editors. Available at: http://cgap.nci.nih.gov/ Chromosomes/Mitelman. Accessed March 8, 2003. [5] 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. [6] Mitelman F, editor. An international system for human cytogenetic nomenclature. Basel: S. Karger, 1995. [7] Tanke HJ, Wiegant J, van Gijlswijk RP, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J. New strategy for multicolour fluorescence in situ hybridization: COBRA: combined binary ratio labelling. Eur J Hum Genet 1999;7:2–11. [8] Wiegant J, Ried T, Nederlof PM, van der Ploeg M, Tanke HJ, Raap AK. In situ hybridization with fluoresceinated DNA. Nucleic Acids Res 1991;19:3237–41. [9] Szuhai K, Bezrookove V, Wiegant J, Vrolijk J, Dirks RW, Rosenberg C, Raap AK, Tanke HJ. Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH. Genes Chromosomes Cancer 2000;28:92–7. [10] Maniatis T, Fritsch EF, Sambrook J. Molecular cloning. A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.
[11] Ho¨glund M, Johansson B, Pedersen-Bjergaard J, Marynen P, Mitelman F. Molecular characterization of 12p abnormalities in hematologic malignancies: deletion of KIP1, rearrangement of TEL, and amplification of CCND2. Blood 1996;87:324–30. [12] Jin C, Mertens F, Jin Y, Wennerberg J, Heim S, Mitelman F. Complex karyotype with an 11q13 homogeneously staining region in esophageal squamous cell carcinoma. Cancer Genet Cytogenet 1995;82: 175–6. [13] Casalone R, Minelli E, Portensoso P, Giudici A. Clonal duplication of the Y chromosome and fra(X)(q28) in a case of epidermoid carcinoma of the esophagus. Cancer Genet Cytogenet 1990;45:269–71. [14] Rosenblum-Vos LS, Meltzer SJ, Leana-Cox J, Schwartz S. Cytogenetic studies of primary cultures of esophageal squamous cell carcinoma. Cancer Genet Cytogenet 1993;70:127–31. [15] Whang-Peng J, Banks-Schlegel SP, Lee EG. Cytogenetic studies of esophageal carcinoma cell lines. Cancer Genet Cytogenet 1990;45: 101–20. [16] Pack SD, Karkera JD, Zhuang Z, Pak ED, Balan KV, Hwu P, Park WS, Pham T, Ault DO, Glaser M, Liotta L, Detera-Wadleigh SD, Wadleigh RG. Molecular cytogenetic fingerprinting of esophageal squamous cell carcinoma by comparative genomic hybridization reveals a consistent pattern of chromosomal alterations. Genes Chromosomes Cancer 1999;25:160–8. [17] Tada K, Oka M, Hayashi H, Tangoku A, Oga A, Sasaki K. Cytogenetic analysis of esophageal squamous cell carcinoma cell lines by comparative genomic hybridization: relationship of cytogenetic aberrations to in vitro cell growth. Cancer Genet Cytogenet 2000;117:108–12. [18] Mayama T, Fukushige S, Shineha R, Nishihira T, Satomi S, Horii A. Frequent loss of copy munber on the long arm of chromosome 21 in human esophageal squamous cell carcinoma. Int J Oncol 2000;17: 245–52. [19] Yen CC, Chen YJ, Chen JT, Hsia JY, Chen PM, Liu JH, Fan FS, Chiou TJ, Wang WS, Lin CH. Comparative genomic hybridization of esophageal squamous cell carcinoma: correlations between chromosomal aberrations and disease progression/prognosis. Cancer 2001;92: 2769–77. [20] Pimkhaokham A, Shimada Y, Fukuda Y, Kurihara N, Imoto I, Yang ZQ, Imamura M, Nakamura Y, Amagasa T, Inazawa J. Nonrandom chromosomal imbalances in esophageal squamous cell carcinoma cell lines: possible involvement of the ATF3 and CENPF genes in the 1q32 amplicon. Jpn J Cancer Res 2000;91:1126–33. [21] Schwab M. Oncogene amplification in solid tumors. Semin Cancer Biol 1999;9:319–25. [22] Motokura T, Arnold A. Cyclin D and oncogenesis. Current Opin Genet Devel 1993;3:5–10. [23] Schuuring E. The involvement of the chromosome 11q13 region in human malignancies: cyclin D1 and EMS1 are two new candidate oncogenes - a review. Gene 1995;159:83–96. [24] Tsuruta H, Sakamoto H, Onda M, Terada M. Amplification and overexpression of EXP1 and EXP2/cyclin D1 genes in human esophageal carcinomas. Biochem Biophys Res Commun 1993;196:1529–36. [25] Gramlich TL, Fritsch CR, Maurer D, Eberle M, Gansler TS. Differential polymerase chain reaction assay of cyclin D1 gene amplification in esophageal carcinoma. Diagn Mol Pathol 1994;3:255–9. [26] Shinozaki H, Ozawa S, Ando N, Tsuruta H, Terada M, Ueda M, Kitajima M. Cyclin D1 amplification as a new predictive classification for squamous cell carcinoma of the esophagus, adding gene information. Clin Cancer Res 1996;2:1155–61. [27] Schwab M, Amler LC. Amplification of cellular oncogenes: a predictor of clinical outcome in human cancer. Genes Chromosomes Cancer 1990;1:181–93.