Chromosomal imbalances in oral squamous cell carcinoma. Examination of 31 cell lines and review of the literature

doi:10.1016/j.oraloncology.2007.05.003

Journal List > NIHPA Author Manuscripts

Oral Oncol. Author manuscript; available in PMC 2009 April 1.

Published in final edited form as:

Oral Oncol. 2008 April; 44(4): 369–382.

Published online 2007 August 2. doi: 10.1016/j.oraloncology.2007.05.003.

PMCID: PMC2362065

NIHMSID: NIHMS45931

Chromosomal imbalances in oral squamous cell carcinoma. Examination of 31 cell lines and review of the literature

Christa Lese Martin,^1,⁵ Shalini C. Reshmi,¹ Thomas Ried,⁶ William Gottberg,⁷ John W. Wilson,^2,⁴ Jaya K. Reddy,³ Poornima Khanna,^1,⁴ Jonas T. Johnson,^3,⁴ Eugene N. Myers,^3,⁴ and Susanne M. Gollin^1,^3,⁴^*

¹ Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania

² Department of Biostatistics and the NSABP Biostatistical Center, University of Pittsburgh, Pittsburgh, Pennsylvania

³ Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsylvania

⁴ University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

⁵ Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia

⁶ National Cancer Institute, NIH, Bethesda, Maryland

⁷ Applied Imaging, Santa Clara, CA

*Corresponding Author Susanne M. Gollin, Ph.D. Department of Human Genetics University of Pittsburgh Graduate School of Public Health 130 DeSoto Street Pittsburgh, PA 15261 Telephone: (412) 624-5390 Fax: (412) 624-3020 Email: sgollin/at/hgen.pitt.edu

The publisher's final edited version of this article is available at Oral Oncol.

Publisher's Disclaimer

Abstract

Classical and molecular cytogenetic analysis, including fluorescence in situ hybridization (FISH) and chromosomal comparative genomic hybridization (CGH), were used to examine genetic changes involved in the development and/or progression of oral squamous cell carcinoma (OSCC). Of 31 OSCC cell lines studied, more than one-third expressed clonal structural abnormalities involving chromosomes 3, 7, 8, 9, and 11. Eleven OSCC cell lines were evaluated using CGH to identify novel genome-wide gains, losses, or amplifications. By CGH, more than half of the cell lines showed loss of 3p, gain of 3q, 8q, and 20q. Further, molecular cytogenetic analyses by FISH of primary tumors showed that the karyotypes of cell lines derived from those tumors correlated with specific gains and losses in the tumors from which they were derived. The most frequent nonrandom aberration identified by both karyotype and CGH analyses was amplification of chromosomal band 11q13 in the form of a homogeneously staining region. Our data suggest that loss of 9p and 11q13 amplification may be of prognostic benefit in the management of OSCC, which is consistent with the literature. The results of this study validate the relationship between these OSCC cell lines and the tumors from which they were derived. The results also emphasize the usefulness of these cell lines as in vitro experimental models and provide important genetic information on these OSCC cell lines that were recently reported in this journal.

Keywords: head and neck cancer, gene amplification, cytogenetics, CGH, biomarker, 11q13

Introduction

In the USA, more than 34,000 new cases of squamous cell carcinoma of the head and neck (SCCHN) are diagnosed annually.¹ If identified early, the prognosis of SCCHN is excellent. However, over the past several decades, SCCHN survival in Caucasians has only improved slightly and in African-Americans, the survival rate has actually decreased.² The overall one, five, and 10 year survival rates in the USA are 84%, 60%, and 48%, respectively.¹ Tobacco and alcohol are proven etiologic factors in SCCHN. They play a role in a process known as field cancerization, whereby all exposed oral epithelial tissue has the opportunity to become neoplastic.³ Due to the field cancerization effect, patients with primary oropharyngeal SCCHN have a 20-fold increased risk of developing a second tumor in the same location.⁴ Although a number of genetic markers have been associated with early diagnosis or prognosis of SCCHN, to the best of our knowledge, none have been implemented in routine clinical laboratory practice, although EGFR gene amplification is being considered, since it is being examined in lung cancer and EGFR inhibitors are available for therapy of amplified tumors.⁵ Further, specific biomarkers for key genes responsible for SCCHN progression, such as EGFR, are essential for the intervention and treatment of SCCHN, but as yet only this gene has been translated to the clinic.

Classical and molecular cytogenetic analyses in conjunction with molecular genetic analyses by our group and others have revealed consistent genetic abnormalities associated with the development and/or progression of SCCHN. This research has revealed the specific genetic changes that take place during head and neck tumorigenesis, such as loss of chromosomal segments 3p, 5q, 7q, 8p, 9p, 11q, and 18q in addition to gains of regions of chromosomes 3q, 5p, 7p, 8q, and 11q.⁶^–¹¹ This study had three goals. First, we karyotyped this series of SCCHN cell lines to identify the genetic alterations in each cell line. Second, we carried out FISH to confirm the relationship between cancer cell lines and the primary tumors from which they were derived. Third, we identified the common abnormalities in SCCHN and correlated our karyotype and CGH findings on the tumors and cell lines with clinical and pathological parameters to identify nonrandom genetic alterations that may serve as prognostic indicators in SCCHN.

Materials and Methods

Tumor samples

After obtaining informed consent, biopsies of 31 oral squamous cell carcinomas (OSCC) were acquired from patients having surgical excision of their tumors at the University of Pittsburgh Medical Center (Table 1). Twenty-three of the tumors were primary OSCC, three patients’ tumors were new primaries (UPCI:SCC026, UPCI:SCC068, and UPCI:SCC099), and five tumors were recurrences (UPCI:SCC002, UPCI:SCC080, UPCI:SCC084, UPCI:SCC090, UPCI:SCC104). Two patients with primary tumors included in this study also had new primaries or recurrences that were also included (UPCI:SCC016/UPCI:SCC026 and UPCI:SCC036/UPCI:SCC104). Five patients received radiation and/or chemotherapy treatment prior to tumor excision (UPCI:SCC002, UPCI:SCC016/UPCI:SCC026, UPCI:SCC078, UPCI:SCC090, UPCI:SCC104).

Table 1

Clinical and histological data on patients/tumors examined in this study.

Upon receipt in the laboratory, the tumor specimen was subdivided into portions for cell culture, DNA extraction, and histopathologic diagnosis. Cell lines were established as described previously.⁷^,¹² Three to four T-25 flasks of tumor cells from each established OSCC cell line were used for DNA extraction for CGH. Prior to DNA extraction, the tumor cells were rinsed in 1X HBSS, pelleted, and frozen at −80° C.

Classical Cytogenetic Analysis

Cytogenetic harvests of the OSCC cell lines were carried out as described previously.¹³ Metaphase spreads were trypsin-Giemsa banded and analyzed. Due to the extent of chromosomal instability in the tumor cell lines, composite karyotypes were not written, since they were very long. Instead, consensus karyotypes (cs), consisting of abnormalities observed in more than 40% of the cells, were prepared from at least eight cells per cell line. Karyotypes were written according to the ISCN (2005)¹⁴ guidelines and images were captured and prepared using an AKS II Automated Karyotyping System (Imagenetics, now Vysis/Abbott Molecular Inc., Des Plaines, IL).

Fluorescence in situ Hybridization (FISH)

To compare chromosome copy numbers between direct harvests of fresh tumor and adjacent oral mucosa cells from OSCC patients and cultured OSCC cells, we used dual-color FISH with alpha-satellite DNA probes for chromosomes 1, 3, 7, and 11, one labeled with biotin and the other labeled with digoxigenin. Alpha-satellite DNA probes for chromosomes 1 (D1Z5), 3 (D3Z1), 7 (D7Z1/D7Z2), and 11 (D11Z1), from Oncor, Inc. (Gaithersburg, MD), were utilized. At least 200 cells from each slide were counted and the number of nuclei with 0, 1, 2, 3, 4, and ≥5 signals was recorded. Results were compared between fresh and cultured tumor cells of the same case to assess whether karyotypic evolution occurred in culture. In addition, comparisons were made between the classical and molecular cytogenetic results to evaluate the reliability of FISH in composing a molecular karyotype from interphase cells.

Comparative Genomic Hybridization (CGH)

Genomic DNA for CGH analysis was isolated from frozen tumor cell lines; DNA from normal male peripheral blood cells served as a control. CGH analysis was completed following the procedure described by du Manoir et al..¹⁵ Slides were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and viewed under an Olympus fluorescence microscope equipped with a filter wheel and attached to a charged-couple device (CCD) camera interfaced with the Cytovision CGH software package (Applied Imaging, Santa Clara, CA). A 100X oil immersion objective was used for image acquisition.

The computer determined the ratio of the test DNA to the control DNA along the length of each chromosome by measuring the fluorescence intensities of the tumor and control DNA. At least five metaphases were captured for each OSCC cell line, resulting in 7 to 20 profiles of each chromosome. By averaging the individual ratio profile for each chromosome from multiple metaphases, the computer generated the overall ratio profile based on previously set guidelines¹⁶ and plotted it beside the corresponding ideogram.

Results

Patient characteristics are shown in Table 1 and consensus karyotypes of the OSCC cell lines in Table 2. OSCC cell lines revealed high frequencies of aneuploidy with multiple chromosome rearrangements; ploidy levels ranged from diploid to pentaploid. Some tumors were comprised of a heterogeneous population of cells, although similar aberrations were observed between subpopulations. Structural rearrangements included unbalanced translocations, Robertsonian translocations, deletions, isochromosomes, and homogeneously staining regions (hsrs). For illustration, a karyotype of UPCI:SCC103 is shown (Figure 1

Table 2

Consensus (cs) karyotypes from 29 OSCC cell lines; aberrations are present in more than 40% of the cells from that population.

Figure 1

Representative karyotype from UPCI:SCC103 passage 10: 63<3n>,-X,-X, -Y,+der(2)t(2;?)(q10;?), −3, −4−5,i(5)(p10),+del(7)(q22), −8, −8,i(8)(q10),+10, +del(10)(q23),+der(11)(pter→q13::hsr::q14→q25::hsr::?), (more ...)

Two patients in this study, UPCI:SCC016 and UPCI:SCC036, each had subsequent tumors that were karyotyped and analyzed. The patient with tumor UPCI:SCC016, a near-triploid tumor, had what was considered clinically a new primary tumor, UPCI:SCC026, which was near-diploid. Aside from amplification of chromosomal band 11q13, comparison of consensus karyotypes did not reveal consistent chromosomal rearrangements. Amplification of 11q13 was observed as an hsr in its native band in UPCI:SCC016 and as an hsr on a derivative chromosome 1 in UPCI:SCC026. The patient with tumor UPCI:SCC036 had what was considered clinically a recurrence, tumor UPCI:SCC104. Although both tumors were near-diploid, no similar chromosomal aberrations were observed. These findings suggest that the development of secondary primary tumors may have occurred independently from the initial tumors in both cases.

Summaries of chromosomal gains and losses by karyotyping and CGH analysis are illustrated in Figures 2

and and3,3

, respectively. Tables 3 and 4 show losses and gains of chromosomal segments observed in at least 20% of 29 OSCC cell lines by karyotype analysis and 11 OSCC cell lines by CGH analysis, respectively. As seen in Table 3, karyotype analysis revealed that nonrandom loss of chromosomal segments occurred more frequently than chromosomal gains in this series of OSCC cell lines. However, CGH analysis showed that gains of genomic material occurred more frequently (Table 4). This observation can be explained by the ability of CGH to characterize gains and losses imparted by marker chromosomes of unknown origin in the consensus karyotypes (Table 2).

Figure 2

Summary of nonrandom chromosomal losses and gains identified by classical cytogenetic analysis of thirty OSCC cell lines. The cell line number corresponding to each line is marked above the lines. Lines to the left or right of the ideograms indicate regions (more ...)

Figure 3

Summary of nonrandom chromosomal losses and gains identified by chromosomal CGH in 11 OSCC cell lines. The cell line number corresponding to each line is marked above the lines. Lines to the left or right of the ideograms indicate regions of loss and (more ...)

Table 3

Chromosomal losses and gains observed most frequently by classical cytogenetic analysis in 29 OSCC cell lines.

Table 4

Chromosomal losses and gains observed most frequently in 11 OSCC cell lines by chromosomal comparative genomic hybridization.^a

Comparison of classical cytogenetic and CGH analyses confirmed many chromosomal alterations. Deletions involving regions of chromosome arms 3p, 4q, 8p and chromosome 18 were found in at least 20% of the cell lines using both methods, as were gains involving chromosome arms 3q, 7p, 9q and chromosomes 19 and 20. Amplification of 11q13 was the most frequently observed abnormality by both karyotype and CGH analyses. However, CGH revealed three novel regions of amplification, 13q21→q22, 14q21→q24, and 19q13.1→qter, each observed in one cell line. In addition, extrachromosomal representations of gene amplification (double minutes) were observed in UPCI:SCC103, and may have resulted from sequence amplification of the 13q21-q22 region.

We compared copy number for chromosomes 1, 3, 7, and 11 between 12 freshly harvested tumors (UPCI:SCC018, 020, 021, 023, 026, 029, 030, 044, 070, 074, 084, 089), corresponding adjacent mucosa cells from three of these (UPCI:SCC029, 030, 044), and the cell lines derived from 11 tumors (UPCI:SCC002, 003, 016, 026, 029, 030, 044, 070, 074, 084, 089). The results from the FISH analysis of chromosomes 1, 3, 7, and 11 in the OSCC are provided in Supplementary Table 1 and Supplementary Figures 1–4. Sixteen tumors were examined for aneuploidy of chromosomes 1 and 3 using centromeric DNA probes. For chromosome 1, 14 (87.5%) tumors were aneuploid, 10 tumors showed gain of chromosome 1, and four tumors showed loss. For chromosome 3, 15 (94%) tumors were aneuploid, 11 tumors showed only copy number gain of chromosome 3, while four tumors showed both loss and gain of chromosome 3. Simultaneous loss and gain of chromosome 3 is best explained by the presence of more than one population of tumor cells identified using FISH, which enables analysis of a larger number of cells than classical cytogenetic analysis. Eight tumors (67%) were aneuploid for chromosome 7, all of which expressed copy number gain. For chromosome 11, 15 tumors were examined for copy number gain or loss. Eleven tumors (73%) were aneuploid for chromosome 11, all of which showed gain of chromosome 11. From these data, it appears that in OSCC, chromosomal gain is a much more frequent event than chromosomal loss. However, many of the chromosomal "gains" most likely represent tumor cell populations that have become tetraploid from aborted cytokinesis in a diploid progenitor population. Therefore, these observations may not represent true aneuploidy, but rather, the evolution from a diploid to a tetraploid tumor. Supplementary Figures 1–4 are histograms that display data collected using FISH with alpha-satellite probes to chromosomes 1, 3, 7, and 11, respectively. Each histogram contains data from those specimens for which fresh and cultured tumor cells were available: UPCI:SCC026, 029, 030, 044, 070, 074, 084, and 089. For comparison, a peripheral blood cell control that was hybridized with the same probe is shown in each graph. Overall, the results illustrated in the histograms demonstrate increased aneuploidies of chromosomes 1, 3, 7, and 11, providing further evidence for their nonrandom involvement in OSCC tumorigenesis. For the most part, the number of signals observed for each chromosome in the direct harvest of the tumor cells corresponded to that of the cultured tumor cells from the same case. Therefore, since corresponding numerical alterations were observed in the directly harvested tumor cells and the cell cultures, these data suggest that little karyotypic evolution has occurred in culture. Clearly, the clone with two copies of the chromosome tested was often reduced in the cell lines, suggesting that our efforts to remove diploid fibroblast-like (stromal) cells from the OSCC cultures was successful, thus validating the successful culturing of OSCC tumor cells. Our results suggest that cultured cells reflect the chromosomal status of the tumors from which they were derived and that analysis of cultured cells is informative of features of the tumors. In addition, the observation of the same number of signals in the direct and cultured tumor cells suggests that clonal selection of the tumor cells has not occurred in vitro.

Discussion

In this study, classical and molecular cytogenetic analyses were used to survey gross genome-wide changes occurring in OSCC cell lines. Our FISH studies of primary tumors showed that the karyotypes of cell lines derived from those tumors correlated with specific gains and losses in the tumors from which they were derived, validating the use of cell lines in OSCC research. Worsham et al.¹⁷ reported similar findings from their study of two head and neck squamous cell carcinomas. Using multi-probe FISH, they compared the ploidy levels observed in cultured tumor cells with those of tumors from which they were derived by examining histologic sections. Cytogenetic aneuploidy was confirmed for the chromosomes examined, demonstrating that the genetic aberrations identified in the tumor cells after a prolonged period in culture were consistent with the genetic aberrations in the original tumors.

Classical cytogenetic analysis was carried out using both short- and long-term cultured tumor cell harvests from 29 OSCC cell lines (Table 2). The majority of breakpoints involved in the chromosomal rearrangements were located in the centromeric regions, consistent with earlier reports of aberrations in head and neck tumors.⁶^,¹⁸

As shown in Table 3, karyotype analysis revealed that nonrandom loss of chromosomal segments occurred more frequently than gains in this series of OSCC cell lines, whereas CGH analysis showed an increased proportion of chromosomal gains rather than losses (Table 4). This observation may be explained by the ability of CGH to further characterize previously unidentified marker chromosomes (Table 2). Overall, analysis of these OSCC lines revealed chromosomal losses at 3p, 4p, 8p, 11q, and l8q and gains at 3q, 5p, 7p/q, 8q, 9q, 11q, 14q, 19q, and 20q.

Regions of chromosome loss

Chromosomal regions lost in OSCC cells most likely harbor tumor suppressor genes that contribute to tumorigenesis. Multiple discrete regions of loss were observed on the short arm of chromosome 3: 3pter→p24, 3pter→p21, 3pter→p14, 3pter→p11 and 3p21→12, consistent with cytogenetic studies of head and neck cancer showing loss of 3p as a key genomic change.⁶^,⁹^,¹⁹ Loss of 3p has been implicated as an early event in SCCHN, as its absence has been shown in dysplastic oral lesions.²⁰^–²² Previously, we too demonstrated alterations or loss of at least one FHIT allele in 22 of 26 OSCC cell lines,²³ consistent with the findings that changes in the FHIT gene and/or protein expression play a key role in the development and/or progression of SCCHN.²⁴^,²⁵ In addition, loss of heterozygosity has been reported for 3pter→p24, 3p21.3, 3p14→cen, 3p21.3→3p13, 3p23→3p21.3, 3p25, 3p24.2→p22, 3p21.1→p12, and 3p14.1→cen.²⁶^–²⁸ Taken together, these studies suggest the presence of at least three tumor suppressor genes located on the short arm of chromosome 3 that may play a role in head and neck tumorigenesis. Several candidate tumor suppressor genes, including HRCA1, RARβand FHIT, reside in regions of chromosome 3p that are most commonly deleted.²⁹^–³¹ Our laboratory has been examining alterations in FANCD2, which codes for a critical DNA damage response protein at 3p26.3, also noted to map to a hotspot of genetic alterations by Weber et al..³²

In this study, loss of chromosome 9p, including breakpoints at 9p21, 9p13, and 9p11, and gain of chromosome 9 from 9q13→qter and 9q32→qter were observed. Deletions of the 9p region have been frequently reported in head and neck cancers by both classical cytogenetic and molecular analyses.⁹ Previous studies identified 9p allelic loss in preinvasive lesions, suggesting that its absence is an early event in SCCHN development.²²^,³³^,³⁴ Band 9p21 is known to contain several tumor suppressor genes, p15/CDKN2B/MTS2, p16/CDKN2A/MTS1, p18/CDKN2C, and p19/CDKN2D, which encode cyclin-dependent kinase inhibitors.³⁵ Alterations in any or all of these genes may lead to uncontrolled cell proliferation through loss of cell cycle checkpoint control, resulting in tumorigenesis.⁹

Interestingly, 95% (19/20) of patients whose tumors contained an intact 9pter-p21 region did not develop a new primary tumor in the followup period, compared to 50% (4/8) of patients with 9p loss, who developed new primary tumors. This observation suggests that deletions in tumor suppressor genes located on chromosome 9p, such as p15/CDKN2B or p16/CDKN2A, may also be important for the development of subsequent SCCHN lesions. This is consistent with the findings of Rosin et al.²² which indicate that 3p and 9p loss in premalignant oral lesions at former cancer sites have a 26.3-fold increase in risk of developing a second oral malignancy compared with lesions that retained these two chromosomal regions.

CGH analysis of our OSCC cells revealed both loss of chromosomal material from 11q22 and amplification of chromosomal band 11q13 in close to 50% of cases examined. Loss of distal 11q will be discussed in the context of 11q13 amplification below.

Nonrandom chromosomal loss of chromosome 18q has frequently been reported in SCCHN.⁸^,¹⁸^,³⁶^–³⁸ In the current study, deletion of the entire long arm of chromosome 18 was observed most frequently by classical cytogenetic analysis, whereas three distinct breakpoints (18q11, 18q12, and 18q22) were identified using CGH. The region 18q21 contains tumor suppressor genes including DCC, SMAD2, SMAD4, SERPINB4, SERPINB5, and SERPINB13.³⁹^–⁴⁴ LOH of the DCC gene has been observed more frequently in patients with decreased SCCHN survival than in those who remain disease-free.⁴⁵ In addition, normal expression of the SERPINB13 gene has been found in oral keratinocytes, oral mucosa, and skin, but exists in lower levels in OSCC, suggesting that its absence may allow SCCHN progression.⁴⁶

Regions of chromosome gain

Many studies have demonstrated gain of chromosomal material on 3q through the formation of an isochromosome 3q, although the majority of extra copies observed in our cell lines resulted from unbalanced translocations. Gains of 3q⁸^,³⁶^,³⁷^,⁴⁷ may suggest the presence of a cancer-related gene, such as the ataxia-telangiectasia and rad3-related gene (ATR) at band 3q22-q24. Our recent studies and those of others have shown that overexpression of ATR could result in tumor initiation and/or progression by promoting chromosomal instability through an aberrant DNA damage response.⁴⁸^–⁵⁰ More telomeric to ATR lies the AIS (TP73L) gene, a human p53 homologue at 3q26-q27, which is present in extra copies, although not amplified technically (≥2.5 copies on a diploid background or ≥5 copies on a tetraploid background), and overexpressed in squamous cell carcinomas.⁵¹^,⁵² AIS/TP73L has been shown to transform rat 1a cells into a malignant phenotype, suggesting that it may also be important for SCCHN tumorigenesis.⁵¹

Gain of chromosome 7p, previously reported by both classical cytogenetic analyses and CGH,⁹ was also observed in this series of OSCC cell lines. Merritt et al.⁵³ first showed amplification and overexpression of the epidermal growth factor gene, mapped to 7p12.3-p12.1, in SCCHN. Subsequent studies by Grandis et al.⁵⁴ have demonstrated that increased protein expression of EGFR and its ligand, TGF-α, are significant predictors for disease recurrence and decreased overall survival, which suggests that EGFR may be a key oncogene for a subset of SCCHN tumors. More recently, it has been shown that the targeted EGFR inhibitor, Erlotinib (Tarceva®) prolonged survival in patients with advanced non-small cell lung cancer who had progressed after standard chemotherapy.⁵

Chromosomal 14q gains in our OSCC cell lines are consistent with the findings in primary tumors from Speicher et al.⁸ and Järvinen et al.,⁵⁵ despite recent findings that showed loss of 14q to be more common.¹⁹ Therefore, the significance of 14q copy number gain in OSCC cell lines remains unclear, although the same genes on 14q that are amplified may be involved in driving the chromosomal gain (see below).

Despite our CGH finding that gain of chromosomal material from 20q was present in more than fifty percent of OSCC cell lines analyzed, 20q amplification has not been detected in primary SCCHN tumors and thus, the prognostic significance in OSCC remains unknown.⁸^,¹⁹ However, amplification of 20q in node-negative breast cancer cells has been shown to result in decreased disease-free patient survival.⁵⁶ Key genes that are gained or amplified on 20q12–13 in breast and/or pancreatic cancer include ZNF217, NCOA3, AIB1, MYBL2, CTSZ, AURKA.⁵⁷^–⁵⁹

Gene amplification

Our CGH analyses revealed three novel regions of amplification, 13q21→q22, 14q21→q24, and 19q13.1→qter, each observed in one cell line. Amplification of 13q21 was reported in one case of OSCC.⁶⁰ The amplicon involving 14q12→q22.1 has been reported in prostate cancer, including overexpression of the TITF1 and MAP4K5 genes.⁶¹ The TITF1 gene codes for thyroid transcription factor 1, a sensitive and specific marker of pulmonary adenocarcinoma, but not extrapulmonary adenocarcinoma. The protein is highly expressed in lung adenocarcinoma, strongly expressed in small cell lung carcinoma and thyroid tumors, although not expressed in SCC of the lung. The MAP4K5 gene codes for mitogen activated protein kinase kinase kinase kinase 5, which maps to 14q11.2-q21 and is a member of the serine/threonine protein kinase family that activates the Jun kinase in mammalian cells. The chromosome 19 amplicon was reported in a comprehensive, correlated study of copy number and gene expression with pathway analysis in laryngeal SCC by Järvinen et al.⁵⁵ and by Nessling et al.⁵⁹ to involve the CCNE1 and AKT2 genes in breast cancer.

CGH analysis of our OSCC cells revealed both loss of chromosomal material from 11q22 and amplification of chromosomal band 11q13 in close to 50% of cases examined. Loss of chromosome material from 11q has been suggested to occur as an intermediate stage in tumor progression, following dysplasia but preceding carcinoma in situ.²⁰ First reported by Jin et al.,⁶² the region distal to 11q13 has been thought to contain at least two tumor suppressor genes implicated in SCCHN progression and a wide variety of other solid tumors.⁶³ Loss of distal 11q was reported in laryngeal SCCs by Järvinen et al..⁵⁵ Haploinsufficiency, as a result of loss of copies of genes involved in the cellular response to DNA double strand breaks including ATM, MRE11, CHEK1, and H2AFX, has been shown to promote chromosomal instability through gene mutation, amplification, and structural chromosome aberrations.⁴⁹^,⁶⁴^–⁶⁶ Our group has shown that OSCC cell lines with distal 11q loss are characterized by a deficient DNA damage response and loss of sensitivity to ionizing radiation.⁵⁰ Further, we have shown in OSCC cell lines that 11q13 amplification usually occurs as a result of breakage-fusion-bridge cycles initiated by a break at the common chromosomal fragile site, FRA11F,¹³^,⁶⁷^,⁶⁸ consistent with other studies in the literature which have demonstrated that gene amplification may result from breakage at chromosomal fragile sites.⁶⁹^,⁷⁰ Common fragile sites are thought to lead to difficulties during DNA replication, and when under replication stress as a result of carcinogen exposure or conditions that occur in precancerous lesions or cancer cells, may lead to replication fork stall or collapse, cell cycle checkpoint induction, and DNA repair.⁷¹ In OSCC, defects in the DNA damage response as a result of genetic loss distal to the 11q13 amplicon and double strand breakage at FRA11F as a result of replication fork collapse may prompt aberrant DNA repair to occur through sister chromatid fusion, leading to chromosomal instability through breakage-fusion-bridge cycles.¹³^,⁶⁷^,⁶⁸ Alternatively, Gibcus et al.⁷² propose that these 11q13 fragile sites are not involved in the breakage necessary for 11q13 amplification, but that the presence of both syntenic transitions and segmental duplications determine the pattern of amplification per the model proposed by Narayanan et al..⁷³

Gene amplification of 11q13 as an hsr has been observed in more than 30–50% of SCCHN tumors⁷^,¹¹^,⁶⁰^,⁶²^,⁷²^,⁷⁴^–⁷⁶ as well as tumors of the aerodigestive tract, bladder, breast, pancreas, lung, ovary, and liver.⁷⁷ The Hittelman group found 11q13 amplification to be an early change in SCCHN,⁷⁸ although more recent studies have shown that gain of the 11q13 region along with loss of 11q14→qter are associated with metastatic lesions of HNSCC,¹⁹ suggesting that 11q13 amplification may be a late event in HNSCC progression. Thus, the timing of this event is controversial. We have observed two copies of the chromosome bearing the 11q13 hsr in many of our near-triploid and near-tetraploid subpopulations, suggesting that the amplification occurred prior to tetraploidization. Chromosomal band 11q13 contains the cyclin D1 gene (CCND1)³⁵ and other genes such as EMS1,⁷⁹^–⁸¹ FGF3 (INT2),⁷^,⁸² FGF4 (HSTF1),⁸³ and the tumor amplified and overexpressed sequence genes 1 and 2 (TAOS1/ORAOV1, TAOS2/TMEM16A).⁸⁴^,⁸⁵ Since the RNA transcript and CCND1 protein have been overexpressed in OSCC cells, it is thought to play an important role in SSCHN progression.⁸⁶ In addition, rapid disease recurrence and poor survival in cases with lymph node involvement have been shown to correlate with cyclin D1 protein overexpression.⁸⁶^–⁸⁹ Our more recent results, confirmed by those of Gibcus et al.⁷² have shown that all but four of 13 genes in the 11q13 amplicon core are overexpressed and this expression appears to be coordinated.⁸⁵ We proposed that 11q13 amplification may be driven by a cassette of genes that provide growth or metastatic advantage to cancer cells. This is supported by the finding that the human 11q13 amplicon core is syntenic to mouse chromosomal band 7F5, which is frequently amplified in chemically-induced murine OSCC. Freier et al.⁹⁰ proposed that coamplification of EMS1 and SHANK2 might have a cooperative effect on OSCC tumor cell motility and invasiveness. Thus, genetic alterations involving chromosome 11 appear to play a key role in OSCC.

Analysis of our data revealed that amplification of 11q13, identified by the presence of an hsr, appeared to be associated with tumor site. Amplification of 11q13 was found to occur more frequently in tumors of the tongue, retromolar trigone, and buccal mucosa. Of the 11 OSCC cell lines we examined by CGH, the most significant finding was the correlation of 11q13 amplification with decreased patient survival. All patients whose tumors lacked 11q13 amplification (6/11) survived, compared to only one of five patients whose tumors expressed 11q13 amplification. This observation further validates the use of 11q13 amplification as a biomarker for patient prognosis, as has previously been reported by several groups.⁸⁸^,⁹¹^–⁹³.

Although previous investigations suggest that CCND1 is the primary cause for OSCC progression in 45% of cases, there are several genes remaining within the 11q13 amplicon that are simultaneously amplified and in some cases, overexpressed at a higher frequency.⁸⁵^,⁸⁶ Due to the identification of genome-wide nonrandom genetic events in OSCC by classical cytogenetic, CGH, and now cDNA microarrays, targeted studies assessing the significance of amplification and overexpression of other genes within the 11q13 amplicon may allow for the elucidation of other key genes associated with HNSCC progression. Further investigation of the biochemical pathways impacted by 11q13 amplification and distal 11q loss are in progress.

In spite of the presence of chromosomal instability, the karyotypes of the cell lines appear to be quite stable over time. Reshmi et al.⁹⁴ analyzed UPCI:SCC040 by G-banding and spectral karyotyping at passage 16 and two ‘clones’ derived by single cell cloning were karyotyped at passage 35. Despite the observation that individual OSCC tumor cells express unique karyotypes and at times unique numerical and structural aberrations, many abnormalities within and among malignant cell populations appear to be clonal and remain constant over time in spite of chromosomal instability resulting from chromosome segregation defects.⁹⁵ This suggests that clonal abnormalities provide a selective growth advantage for high grade tumors, consistent with the suggestion by Albertson et al..⁹⁶ Our cell lines have been examined in our laboratory as late as passage 68 and appear to be remarkably stable. However, any cell line used as a laboratory tool should be tested regularly by DNA fingerprinting and/or karyotyping to be certain that it is the same cell line as expected and has not evolved or more likely, been replaced by HeLa cells or another cell line, including one from another species. Further, cell lines should also be examined at the earliest passage available. Thus, our series of SCCHN cell lines¹² and those of other investigators⁹⁷ may serve as useful laboratory tools for basic and translational research studies. Similar to our recent finding of loss of radiosensitivity associated with distal 11q loss,⁵⁰ or the previous finding of EGFR gene amplification, further investigation of clonal chromosomal abnormalities and genes located therein may lead to useful biomarkers for determining which patients will benefit from targeted therapies and should ultimately assist in the treatment of SCCHN.

Supplementary Material

Supplementary Figure 1. Chromosome 1 copy number determined by FISH in paired fresh and cultured OSCC. The tissue culture was derived from the same dissociated tumor biopsy as the freshly harvested, fixed OSCC cells. DT, directly harvested tumor; CT, cultured tumor; Ctl, lymphocytes used as a FISH control.

Click here to view.^{(1.6M, pdf)}

Supplementary Figure 2. Chromosome 3 copy number determined by FISH in paired fresh and cultured OSCC. The tissue culture was derived from the same dissociated tumor biopsy as the freshly harvested, fixed OSCC cells. DT, directly harvested tumor; CT, cultured tumor; Ctl, lymphocytes used as a FISH control.

Click here to view.^{(1.6M, pdf)}

Supplementary Figure 3. Chromosome 7 copy number determined by FISH in paired fresh and cultured OSCC. The tissue culture was derived from the same dissociated tumor biopsy as the freshly harvested, fixed OSCC cells. DT, directly harvested tumor; CT, cultured tumor; Ctl, lymphocytes used as a FISH control.

Click here to view.^{(1.5M, pdf)}

Supplementary Figure 4. Chromosome 11 copy number determined by FISH in paired fresh and cultured OSCC. The tissue culture was derived from the same dissociated tumor biopsy as the freshly harvested, fixed OSCC cells. DT, directly harvested tumor; CT, cultured tumor; Ctl, lymphocytes used as a FISH control.

Click here to view.^{(1.8M, pdf)}

Click here to view.^{(283K, doc)}

Acknowledgments

This work was supported in part by NIH grants R01DE10513, R01DE14729, and ACS grant EDT-44 to SMG, P60DE13059 to Dr. Eugene N. Myers, R01DE016086 Dr. William S. Saunders, and the University of Pittsburgh Head and Neck Cancer SPORE grant P50 CA097190 to Dr. Jennifer Rubin Grandis, the Mary Hillman Jennings Foundation, and the John R. McCune Charitable Trust. Cytogenetic analyses were carried out in the University of Pittsburgh Cancer Institute Cytogenetics Facility, supported in part by P30CA47904 to Dr. Ronald B. Herberman.

Footnotes

Conflict of interest

None of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias their contribution to this paper.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Jemal A, Siegel R, Ward E, Murray T, Xu J, et al. Cancer Statistics, 2007. CA Cancer J Clin. 2007;57(1):43–66. [PubMed]

2. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, et al. Cancer Statistics, 2004. CA Cancer J Clin. 2004;54(1):8–29. [PubMed]

3. Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. A genetic explanation of Slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res. 2003;63(8):1727–30. [PubMed]

4. Robinson E, Neugut AI, Murray T, Rennart G. A comparison of the clinical characteristics of first and second primary head and neck cancers. A population-based study. Cancer. 1991;68(1):189–92. [PubMed]

5. Tsao M-S, Sakurada A, Cutz J-C, Zhu C-Q, Kamel-Reid S, et al. Erlotinib in lung cancer – Molecular and clinical predictors of outcome. New Engl J Med. 2005;353(2):133–44. [PubMed]

6. Jin Y, Mertens F, Jin C, Åkervall J, Wennerberg J, et al. Nonrandom chromosome abnormalities in short-term cultured primary squamous cell carcinomas of the head and neck. Cancer Res. 1995;55(14):3204–10. [PubMed]

7. Lese CM, Rossie KM, Appel BN, Reddy JK, Johnson JT, et al. Visualization of INT2 and HST1 Amplification in Oral Squamous Cell Carcinomas. Genes Chromosomes Cancer. 1995;12(4):288–95. [PubMed]

8. Speicher MR, Howe C, Crotty P, du Manoir S, Costa J, et al. Comparative genomic hybridization detects novel deletions and amplifications in head and neck squamous cell carcinomas. Cancer Res. 1995;55(5):1010–13. [PubMed]

9. Gollin SM. Chromosomal alterations in squamous cell carcinomas of the head and neck: window to the biology of the disease. Head Neck. 2001;23(3):238–53. [PubMed]

10. Wreesmann VB, Singh B. Chromosomal aberrations in squamous cell carcinomas of the upper aerodigestive tract: biologic insights and clinical opportunities. J Oral Pathol Med. 2005;34(8):449–59. [PubMed]

11. Jin C, Jin Y, Wennerberg J, Annertz K, Enoksson J, et al. Cytogenetic abnormalities in 106 oral squamous cell carcinomas. Cancer Genet Cytogenet. 2006;164(1):44–53. [PubMed]

12. White JS, Weissfeld JL, Ragin CCR, Rossie KM, Martin CL, et al. The influence of clinical and demographic risk factors on the establishment of head and neck squamous cell carcinoma cell lines. Oral Oncol. 2007 In Press. [PubMed]

13. Shuster MI, Han L, Le Beau MM, Davis E, Sawicki M, et al. A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification. Genes Chromosomes Cancer. 2000;28(2):153–63. [PubMed]

14. Shaffer LG, Tommerup N, editors. ISCN. An International System for Human Cytogenetic Nomenclature. S. Karger; Basel: 2005.

15. du Manoir S, Speicher MR, Joos S, Schröck E, Popp S, et al. Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Human Genet. 1993;90(6):590–610. [PubMed]

16. du Manoir S, Schröck E, Bentz M, Speicher MR, Joos S, et al. Quantitative analysis of comparative genomic hybridization. Cytometry. 1995;19(1):27–41. [PubMed]

17. Worsham MJ, Wolman SR, Carey TE, Zarbo RJ, Benninger MS, et al. Chromosomal aberrations identified in culture of squamous carcinomas are confirmed by fluorescence in situ hybridisation. Mol Pathol. 1999;52(1):42–6. [PubMed]

18. Hermsen MAJA, Joenje H, Arwert F, Welters MJP, Braakhuis BJM, et al. Centromeric breakage as a major cause of cytogenetic abnormalities in oral squamous cell carcinoma. Genes Chromosomes Cancer. 1996;15(1):1–9. [PubMed]

19. Bockmühl U, Schluns K, Schmidt S, Matthias S, Petersen I. Chromosomal alterations during metastasis formation of head and neck squamous cell carcinoma. Genes Chromosomes Cancer. 2002;33(1):29–35. [PubMed]

20. Califano J, van der Riet P, Westra W, Nawroz H, Clayman G, et al. Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res. 1996;56(11):2488–92. [PubMed]

21. Roz L, Wu CL, Porter S, Scully C, Speight P, et al. Allelic imbalance on chromosome 3p in oral dysplastic lesions: an early event in oral carcinogenesis. Cancer Res. 1996;56(6):1228–31. [PubMed]

22. Rosin M, Lam WL, Poh C, Le ND, Li RJ, et al. 3p14 and 9p21 loss is a simple tool for predicting second oral malignancy at previously treated oral cancer sites. Cancer Res. 2002;62(22):6447–50. [PubMed]

23. Virgilio L, Shuster M, Gollin SM, Veronese ML, Ohta M, et al. FHIT gene alterations in head and neck squamous cell carcinomas. Proc Natl Acad Sci USA. 1996;93(18):9770–5. [PubMed]

24. Tanimoto K, Hayashi S, Tsuchiya E, Tokuchi Y, Kobayashi Y, et al. Abnormalities of the FHIT gene in human oral carcinogenesis. Brit J Cancer. 2000;82(4):838–43. [PubMed]

25. Gotte K, Hadaczek P, Coy JF, Wirtz HW, Riedel F, et al. Fhit expression is absent or reduced in a subset of primary head and neck cancer. Anticancer Res. 2000;20(2A):1057–60. [PubMed]

26. Maestro R, Gasparotto D, Vukosavljevic T, Barzan L, Sulfaro S, Boiocchi M. Three discrete regions of deletion at 3p in head and neck cancers. Cancer Res. 1993;53(23):5775–9. [PubMed]

27. Wu CL, Sloan P, Read AP, Harris R, Thakker N. Deletion mapping on the short arm of chromosome 3 in squamous cell carcinoma of the oral cavity. Cancer Res. 1994;54(24):6484–8. [PubMed]

28. Ishwad C, Ferrell RE, Rossie KM, Appel BN, Johnson JT, et al. Loss of heterozygosity of the short arm of chromosomes 3 and 9 in oral cancer. Int J Cancer. 1996;69(1):1–4. [PubMed]

29. Boldog FL, Gemmill RM, Wilke CM, Glover TW, Nilsson AS, et al. Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint. Proc Natl Acad Sci USA. 1993;90(18):8509–13. [PubMed]

30. LaForgia S, Lasota J, Latif F, Boghosian-Sell L, Kastury K, et al. Detailed genetic and physical map of the 3p chromosome region surrounding the familial renal cell carcinoma chromosome translocation, t(3;8)(p14.2;q24.1). Cancer Res. 1993;53(13):3118–42. [PubMed]

31. Houle B, Rochett-Egly C, Bradley EWC. Tumor-suppressive effect of the retinoic acid receptor beta in human epidermoid lung cancer cells. Proc Natl Acad Sci USA. 1993;90(3):985–9. [PubMed]

32. Weber F, Xu Y, Zhang L, Patocs A, Shen L, et al. Microenvironmental genomic alterations and clinicopathological behavior in head and neck squamous cell carcinoma. JAMA. 2007;297(2):187–95. [PubMed]

33. van der Riet P, Nawroz H, Hruban RH, Corio R, Tokino K, et al. Frequent loss of chromosome 9p21–22 early in head and neck cancer progression. Cancer Res. 1994;54(5):1156–8. [PubMed]

34. El-Naggar AK, Hurr K, Batsakis JG, Luna MA, Goepfert H, et al. Sequential loss of heterozygosity at microsatellite motifs in preinvasive and invasive head and neck squamous carcinoma. Cancer Res. 1995;55(12):2656–9. [PubMed]

35. Jeannon J-P, Wilson JA. Cyclins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors and their role in head and neck cancer. Clin Otolaryngol. 1998;23(5):420–4. [PubMed]

36. Cowan JM, Beckett MA, Ahmed-Swan S, Weichselbaum RR. Cytogenetic evidence of the multistep origin of head and neck squamous cell carcinomas. J Natl Cancer Inst. 1992;84(10):793–7. [PubMed]

37. Van Dyke DL, Worsham MJ, Benninger MS, Krause CJ, Baker SR, et al. Recurrent cytogenetic abnormalities in squamous cell carcinomas of the head and neck region. Genes Chromosomes Cancer. 1994;9(3):192–206. [PubMed]

38. Takebayashi S, Hickson A, Ogawa T, Jung KY, Mineta H, et al. Loss of chromosome arm 18q with tumor progression in head and neck squamous cancer. Genes Chromosomes Cancer. 2004;41(2):145–54. [PubMed]

39. Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, et al. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science. 1990;247(4938):49–56. [PubMed]

40. Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science. 1994;263(5146):526–9. [PubMed]

41. Schneider SS, Schick C, Fish KE, Miller E, Pena JC, et al. A serine protease inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc Natl Acad Sci USA. 1995;92(8):3147–51. [PubMed]

42. Eppert K, Scherer SW, Ozcelik H, Pirone R, Hoodless P, et al. MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell. 1996;6(4):543–52. [PubMed]

43. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996;271(5247):350–3. [PubMed]

44. Spring P, Nakashima T, Frederick M, Henderson Y, Clayman G. Identification and cDNA cloning of headpin, a novel differentially expressed serpin that maps to chromosome 18q. Biochem Biophys Res Commun. 1999;264(1):299–304. [PubMed]

45. Carey TE, Van Dyke DL, Worsham MJ. Nonrandom chromosome aberrations and clonal populations in head and neck cancer. Anticancer Res. 1993;13(6B):2561–7. [PubMed]

46. Nakashima T, Pak SC, Silverman GA, Spring PM, Frederick MJ, et al. Genomic cloning, mapping, structure and promoter analysis of HEADPIN, a serpin which is down-regulated in head and neck cancer cells. Biochim Biophys Acta. 2000;1492(2–3):441–6. [PubMed]

47. Jin Y, Mertens F. Chromosome abnormalities in oral squamous cell carcinomas. Eur J Cancer B Oral Oncol. 1993;29B(4):257–63. [PubMed]

48. Smith L, Liu SJ, Goodrich L, Jacobson D, Degnin C, et al. Duplication of ATR inhibits MyoD, induces aneuploidy and eliminates radiation-induced G1 arrest. Nat Genet. 1998;19(1):39–46. [PubMed]

49. Gollin SM. Mechanisms leading to chromosomal instability. Semin Cancer Biol. 2005;15(1):33–42. [PubMed]

50. Parikh R, White JS, Huang X, Schoppy DW, Baysal BE, Baskaran R, Bakkenist CJ, Saunders WS, Gollin SM. Loss of distal 11q is associated with DNA repair deficiency and reduced sensitivity to ionizing radiation in head and neck squamous cell carcinoma. Genes Chromosomes Cancer. 2007 In Press.

51. Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA. 2000;97(10):5462–7. [PubMed]

52. Oga A, Kong G, Tae K, Lee Y, Sasaki K. Comparative genomic hybridization analysis reveals 3q gain resulting in genetic alteration in 3q in advanced oral squamous cell carcinoma. Cancer Genet Cytogenet. 2001;127(1):24–9. [PubMed]

53. Merritt WD, Weissler MC, Turk BF, Gilmer TM. Oncogene amplification in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg. 1990;116(12):1394–8. [PubMed]

54. Grandis JR, Melhem MF, Gooding WE, Day R, Holst VA, et al. Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst. 1998;90(11):824–32. [PubMed]

55. Järvinen A-K, Autio R, Saapa-Paananen S, Wolf M, Saarela M, et al. Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarrays. Oncogene. 2006;25(52):6997–7008. [PubMed]

56. Rennstam K, Ahlstedt-Soini M, Baldetorp B, Bendahl PO, Borg A, et al. Patterns of chromosomal imbalances defines subgroups of breast cancer with distinct clinical features and prognosis. A study of 305 tumors by comparative genomic hybridization. Cancer Res. 2003;63(24):8861–8. [PubMed]

57. Collins C, Rommens JM, Kowbel D, Godfrey T, Tanner M, et al. Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma. Proc Natl Acad Sci USA. 1998;95(15):8703–8. [PubMed]

58. Mahlamaki EH, Barlund M, Tanner M, Gorunova L, Hoglund M, et al. Frequent amplification of 8q24, 11q, 17q, and 20q-specific genes in pancreatic cancer. Genes Chromosomes Cancer. 2002;35(4):353–8. [PubMed]

59. Nessling M, Richter K, Schwaenen C, Roerig P, Wrobel G, et al. Candidate genes in breast cancer revealed by microarray-based comparative genomic hybridization of archived tissue. Cancer Res. 2005;65(2):439–47. [PubMed]

60. Huang Q, Yu GP, McCormick SA, Mo J, Datta B, et al. Genetic differences detected by comparative genomic hybridization in head and neck squamous cell carcinomas from different tumor sites: Construction of oncogenetic trees for tumor progression. Genes Chromosomes Cancer. 2002;34(2):224–33. [PubMed]

61. Wolf M, Mousses S, Hautaniemi S, Karhu R, Huusko P, et al. High-resolution analysis of gene copy number alterations in human prostate cancer using CGH on cDNA microarrays: Impact of copy number on gene expression. Neoplasia. 2004;6(3):240–7. [PubMed]

62. Jin Y, Höglund M, Jin C, Martins C, Wennerberg J, et al. FISH characterization of head and neck carcinomas reveals that amplification of band 11q13 is associated with deletion of distal 11q. Genes Chromosomes Cancer. 1998;22(4):312–20. [PubMed]

63. Uzawa N, Yoshida MA, Hosoe S, Oshimura M, Amagasa T, et al. Functional evidence for involvement of multiple putative tumor suppressor genes on the short arm of chromosome 3 in human oral squamous cell carcinogenesis. Cancer Genet Cytogenet. 1998;107(2):125–31. [PubMed]

64. Lobachev KS, Gordenin DA, Resnick MA. The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rerrangements. Cell. 2002;108(2):183–93. [PubMed]

65. Bassing CH, Suh H, Ferguson DO, Chua KF, Manis J, Eckersdorff M, et al. Histone H2AX: A dosage-dependent suppressor of oncogenic translocations and tumors. Cell. 2003;114(3):359–70. [PubMed]

66. Celeste A, Difilippantonio S, Difilippantonio MJ, Fernandez-Capetillo O, Pilch DR, et al. H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell. 2003;114(3):371–83. [PubMed]

67. Reshmi SC, Huang X, Schoppy DW, Black RC, Saunders WS, et al. The relationship between FRA11F and 11q13 gene amplification in oral cancer. Genes Chromosomes Cancer. 2007;46(2):143–54. [PubMed]

68. Reshmi SC, Roychaudhury S, Yu Z, Feingold E, Potter D, et al. Inverted duplication pattern in anaphase bridges confirms the breakage-fusion-bridge (BFB) cycle model for 11q13 amplification. Cytogenet Genome Res. 2007;116(1–2):46–52. [PubMed]

69. Ciullo M, Debily MA, Rozier L, Autiero M, Billault A, et al. Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I. Hum Mol Genet. 2002;11(23):2887–94. [PubMed]

70. Hellman A, Zlotorynski E, Scherer SW, Cheung J, Vincent JB, et al. A role for common fragile site induction in amplification of human oncogenes. Cancer Cell. 2002;1(1):89–97. [PubMed]

71. Glover TW, Arlt MF, Casper AM, Durkin SG. Mechanisms of common fragile site instability. Hum Molec Genet. 2005;14(Spec2):R197–R205. [PubMed]

72. Gibcus JH, Kok K, Menkema L, Hermsen MA, Mastik M, Kluin PM, et al. High-resolution mapping identifies a commonly amplified 11q13.3 region containing multiple genes flanked by segmental duplications. Hum Genet. 2006 doi: 10.1007/s00439-006-0299-6. In Press.

73. Narayanan V, Mieczkowski PA, Kim HP, Petes TD, Lobachev KS. The pattern of gene amplification is determined by the chromosomal location of hairpin-capped breaks. Cell. 2006;125(7):1283–96. [PubMed]

74. Åkervall JA, Michalides RJ, Mineta H, Balm A, Borg A, Dictor MR, et al. Amplification of cyclin D1 in squamous cell carcinoma of the head and neck and the prognostic value of chromosomal abnormalities and cyclin D1 overexpression. Cancer. 1997;79(2):380–9. [PubMed]

75. Snijders AM, Schmidt BL, Fridyland J, Dekker N, Pinkel D, et al. Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma. Oncogene. 2005;24(26):4232–42. [PubMed]

76. Jin C, Jin Y, Gisselsson D, Wennerberg J, Wah TS, et al. Molecular cytogenetic characterization of the 11q13 amplicon in head and neck squamous cell carcinoma. Cytogenet Genome Res. 2006;115(2):99–106. [PubMed]

77. Schraml P, Kononen J, Bubendorf L, Moch H, Bissig H, et al. Tissue microarrays for gene amplification surveys in many different tumor types. Clin Cancer Res. 1999;5(8):1966–75. [PubMed]

78. Izzo JG, Papadimitrakopoulou VA, Li XQ, Ibarguen H, Lee JS, et al. Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification. Oncogene. 1998;17(18):2313–22. [PubMed]

79. Schuuring E, Verhoeven E, Mooi WJ, Michalides RJ. Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas. Oncogene. 1992;7:355–61. [PubMed]

80. Patel AM, Incognito LS, Schechter GL, Wasilenko WJ, Somers KD. Amplification and expression of EMS-1 (cortactin) in head and neck squamous cell carcinoma cell lines. Oncogene. 1996;12(1):31–5. [PubMed]

81. Hui R, Campbell DH, Lee CS, McCaul K, Horsfall DJ, et al. EMS1 amplification can occur independently of CCND1 or INT-2 amplification at 11q13 and may identify different phenotypes in primary breast cancer. Oncogene. 1997;15(13):1617–23. [PubMed]

82. Champeme MH, Bieche I, Hacene K, Lidereau R. Int-2/FGF3 amplification is a better independent predictor of relapse than c-myc and c-erbB-2/neu amplifications in primary human breast cancer. Mod Pathol. 1994;7(9):900–5. [PubMed]

83. Yoshida MC, Wada M, Satoh H, Yoshida T, Sakamoto H, et al. Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer. Proc Natl Acad Sci USA. 1988;85(13):4861–4. [PubMed]

84. Huang X, Gollin SM, Raja S, Godfrey TE. High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells. Proc Natl Acad Sci USA. 2002;99(17):11369–74. [PubMed]

85. Huang X, Godfrey TE, Gooding WE, McCarty KS, Gollin SM. Comprehensive genome and transcriptome analysis of the 11q13 amplicon in human oral cancer and synteny to the 7F5 amplicon in murine oral carcinoma. Genes Chromosomes Cancer. 2006;45(11):1058–69. [PubMed]

86. Jares P, Fernandez PL, Campo E, Nadal A, Bosch F, et al. PRAD1/cyclin D1 gene amplification correlates with messenger RNA overexpression and tumor progression in human laryngeal carcinomas. Cancer Res. 1994;54(17):4813–7. [PubMed]

87. Michalides R, van Veelen M, Hart A, Loftus B, Wientjens E, et al. Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinomas of the head and neck. Cancer Res. 1995;55(5):975–978. [PubMed]

88. Michalides RJ, van Veelen NM, Kristel PM, Hart AA, Loftus BM, et al. Overexpression of cyclin D1 indicates a poor prognosis in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg. 1997;123(5):497–502. [PubMed]

89. Fracchiolla NS, Pruneri G, Pignataro L, Carboni N, Capaccio P, et al. Molecular and immunohistochemical analysis of the bcl-1/cyclin D1 gene in laryngeal squamous cell carcinomas: correlation of protein expression with lymph node metastases and advanced clinical stage. Cancer. 1997;79(6):1114–21. [PubMed]

90. Freier K, Sticht C, Hofele C, Flechtenmacher C, Stange D, et al. Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma. Genes Chromosomes Cancer. 2005;45(2):118–25. [PubMed]

91. Somers KD, Cartwright SL, Schechter GL. Amplification of the int-2 gene in human head and neck squamous cell carcinomas. Oncogene. 1990;5(6):915–20. [PubMed]

92. Åkervall JA, Jin Y, Wennerberg JP, Zatterstrom UK, Kjellen E, et al. Chromosomal abnormalities involving 11q13 are associated with poor prognosis in patients with squamous cell carcinoma of the head and neck. Cancer. 1995;76(5):853–9. [PubMed]

93. Wreesmann VB, Shi W, Thaler HT, Poluri A, Kraus DH, et al. Identification of novel prognosticators of outcome in squamous cell carcinoma of the head and neck. J Clin Oncol. 2004;22(19):3965–72. [PubMed]

94. Reshmi SC, Saunders WS, Kudla DM, Rose Ragin CC, et al. Chromosomal instability and marker chromosome evolution in oral squamous cell carcinoma. Genes Chromosomes & Cancer. 2004;41(1):38–46. [PubMed]

95. Saunders WS, Shuster M, Huang X, Gharaibeh B, Enyenihi AH, et al. Chromosomal instability and cytoskeletal defects in oral cancer cells. Proc Natl Acad Sci USA. 2000;97(1):303–8. [PubMed]

96. Albertson DG, Collins C, McCormick F, Gray JW. Chromosome aberrations in solid tumors. Nat Genet. 2003;34(4):369–76. [PubMed]

97. Lin CJ, Grandis JR, Carey TE, Gollin SM, et al. Head and neck squamous cell carcinoma cell lines: Established models and rationale for selection. Head & Neck. 2007;29(2):163–88. [PubMed]

Formats:

PubMed articles by these authors

PubMed related articles

Recent Activity

Links

Simple NCBI Directory

Getting Started

Resources

Popular

Featured

NCBI Information