3Q26 Amplification and Polysomy Of

Human Cancer Biology

3q26 Amplification and Polysomy of Chromosome 3 in Squamous Cell Lesions of the Lung: A Fluorescence In situ Hybridization Study Giuseppe Pelosi,1,4 Barbara Del Curto,1MaurizioTrubia,5 Andrew G. Nicholson,6 Michela Manzotti,1 GiuliaVeronesi,2 Lorenzo Spaggiari,2,4 Patrick Maisonneuve,3 Felice Pasini,7 AlbertoTerzi,8 Antonio Iannucci,9 and GiuseppeViale1,4

Abstract Purpose: An overlapping area of gain at 3q26has been reported in lung squamous cell carcinoma (SCC), but whether this also occurs in preneoplastic/preinvasive squamous cell proliferations and early-stage invasive carcinomas of the lung is still unknown. Experimental Design: We evaluated the prevalence and the clinicopathologic implications of 3q26amplification and polysomy of chromosome 3 in 31preneoplastic/preinvasive squamous cell lesions of the bronchial mucosa and in 139 early-stage invasive pulmonary SCC, both of limited growth within the bronchial wall [early hilar SCC (EHSCC)] and involving the pulmonary parenchyma [parenchyma-infiltrating SCC (PISCC)]. Moreover, mRNA expression of two candidate genes (h-TERC and SKI-like), both mapping to the minimal common amplification region, was also studiedbyquantitative real-time reversetranscription-PCR. Results: 3q26amplification and polysomy of chromosome 3 were confined to malignant samples, with 37% of invasive SCC, and 27% of severe dysplasias/in situ carcinomas showing these chromosomal abnormalities. Amplification (with minimal common amplification region at 3q26.2), polysomy 3, concurrent amplification and polysomy 3, or other changes (monosomy) were found in 25 SCC and 1dysplasia, 24 and 2, 2 and 0, and 1and 0, respectively. Amplification was significantly associated with EHSCC, polysomy 3 with PISCC. 3q26amplification correlated with increased tumor diameter and a history of smoking, whereas polysomy 3 correlated with tumor diameter, pTclass, and p53, p21, and fascin immunoreactivity. No relationship of either 3q26gain or polysomy was found with patients’ survival. Overexpression of h-TERC or SKI-like mRNAwas found in 3q26-amplified or polysomic SCC, with higher levels of h-TERC in the former and of SKI-like in the latter. Conclusions: 3q26amplification and chromosome 3 polysomy may be related to the development of invasive SCC, with differential distribution in tumor subsets, despite substantial histologic uniformity. Both h-TERC and SKI-like may be involved in tumor progression.

A large body of evidence supports the theory that pulmonary hyperplasia, squamous metaplasia and dysplasia, and in situ squamous cell carcinoma (SCC) is not caused by a single SCC (carcinoma in situ [CIS]), occurring over time, either transforming event, but by a steady accrual of subsequent preceding or accompanying invasive tumors (according to the molecular genetic and epigenetic abnormalities, resulting in a so-called sequential theory of morphologic and molecular spectrum of premalignant/preinvasive lesions, i.e., basal cell changes; refs. 1–7). Whether basal cell hyperplasia, squamous metaplasia, and mild squamous dysplasia are common reactive changes or already true neoplastic changes is still unclear, Authors’ Affiliations: Divisions of 1Pathology and Laboratory Medicine, 2Thoracic whereas severe dysplasia and CIS are currently considered high- Surgery, and 3Epidemiology and Biostatistics, European Institute of Oncology, grade preinvasive lesions, with 40% or more individuals 4 5 University of Milan School of Medicine and Cancer Genetic Unit, IFOM developing invasive cancer over time (8, 9). Foundation ^ FIRC Institute of Molecular Oncology Foundation, Milan, Italy; 6Department of Histopatology, Royal Brompton Hospital, London, United In the lung, early-stage invasive SCC [corresponding to Kingdom; 7Division of Medical Oncology, University of Verona, Piazzale Stefani 1; pT1-2N0M0 stage tumor patients according to sixth tumor- and Divisions of 8Thoracic Surgery and 9Pathology, City Major Hospital, Piazzale node-metastasis classification of malignant tumors]may be Stefani 1,Verona, Italy managerially classified as either bronchial wall-limited lesions Received 10/13/06; revised 12/17/06; accepted 12/28/06. (i.e., pT1N0M0), independent of their extension along the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance bronchial tree [the so-called early hilar SCC (EHSCC) according with 18 U.S.C. Section 1734 solely to indicate this fact. to the Japan Lung Cancer Society; refs. 10–24], or parenchyma- Requests for reprints: Giuseppe Pelosi, Divisione di Anatomia Patologica e infiltrating SCC (PISCC; i.e., pT1-2N0M0), independent of Medicina di Laboratorio, Istituto Europeo di Oncologia, Via G. Ripamonti, 435, I- their anatomic site of origin inside the lung, either central or 20141 Milan, Italy. Phone: 39-2-57-48-94-14; Fax: 39-2-57-48-94-17; E-mail: [email protected]. peripheral, but sharing the common feature of parenchymal F 2007 American Association for Cancer Research. invasion. EHSCCs account for up to 2% of all non–small cell doi:10.1158/1078-0432.CCR-06-2483 lung cancer (NSCLC) and up to 10% to 20% of early detected

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Table 1. Clinicopathologic characteristics of 138 SCCs of the lung

Variable All SCC (n = 138) PISCC (n = 105) EHSCC (n = 33) P value (PISCC versus EHSCC) Age (y) V49 5 (3.6%) 4 1 50-59 19 (13.8%) 13 6 60-69 83 (60.1%) 66 17 z70 31 (22.5%) 22 9 0.89 Sex Male 135 (92.8%) 103 32 Female 3 (2.2%) 2 1 0.56 Grading

G1 4 (2.9%) 4 0 G2 91 (65.9%) 66 25 G3 43 (31.2%) 35 8 0.61 Tumor size (cm) <2 31 (22.5%) 11 20 2-4 62 (44.9%) 49 13 >4 45 (32.6%) 45 0 <0.001 pT 1 83 (60.1%) 50 33 2 55 (39.9%) 55 0 <0.001 Performance status Median (range) 90 (70-100) 90 (70-100) 90 (80-100) 0.095 Symptoms Absent 42 (30.4%) 37 5 Local 59 (42.8%) 39 20 Systemic 37 (26.8%) 29 8 0.037 Pack-year Median (range) 60 (0-203) 60 (0-203) 70 (15-232) 0.73 Vital status Alive 86 (62.3%) 60 26 Dead 52 (37.7%) 45 7 0.038 Recurrences No 82 (59.4%) 57 25 Yes 56 (40.6%) 48 8 0.041 Vascular invasion* No 100 (78.1%) 68 32 Yes 28 (21.9%) 27 1 0.0013 Ki-67 index Median (range) 42% (8-77) 41% (13-75) 46.5% (8-77) 0.52

*Vascular invasion was assessed in 128 out of 138 tumors.

lung carcinoma (17, 25–27) and are reportedly associated with with NSCLC. A detailed study dealing with the entire spectrum a longer life expectation in comparison with the other stage I of squamous cell lesions, including both preneoplastic/pre- NSCLC (10, 12–17), although conflicting data have been invasive proliferations and early-stage invasive carcinomas of reported thus far on the issue (17). Therefore, an intriguing the lung, however, is still lacking. question is whether EHSCC represents a specific clinicopath- We used fluorescence in situ hybridization (FISH) analysis to ologic subset of SCC characterized by an inherent, more evaluate the prevalence and the clinicopathologic implications favorable prognosis or rather a more symptomatic tumor of 3q26 region amplification and chromosome 3 polysomy in detectable at an earlier stage. paraffin sections of 170 squamous cell proliferative lesions of Many molecular alterations have been described in the the lung, including 31 preneoplastic/preinvasive squamous cell multistep process of pulmonary SCC development (1–7), but lesions of the bronchial mucosa and 139 early-stage invasive little is known on the role and prevalence of specific cytogenetic SCC with different growth patterns within the bronchial wall alterations (8). Gain at 3q is a common feature of SCC and the pulmonary parenchyma. Moreover, we investigated by (28–31), with an overlapping area of gain at 3q26 having quantitative real-time reverse transcription-PCR (RT-PCR) been reported in SCC at different anatomic sites (32), including assays the expression of two candidate genes mapping the lung (28–30, 33–42), head and neck (43–55), cervix of the 3q26 amplicon, namely, the RNA structural subunit of human uterus (56, 57), and esophagus (58–61). In particular, telomerase (h-TERC or hTR, gene ID 7012; refs. 63, 64) and a Kettunen et al. (42) have recently suggested that multiple cell type–specific mediator of transforming growth factor-h genes located at 3q25fq27 are involved in the tumorigenesis responses (SKI-like or SnoN, gene ID 6498; ref. 65). Our results of pulmonary SCC, and Yokoi et al. (35) have identified h- suggest a different role for 3q26 amplification and chromo- TERC, the RNA component of human telomerase (62), as likely some 3 polysomy in the development of SCC subtypes, with engaged within the 3q26 amplicon in cell lines and patients h-TERC and SKI-like emerging as likely involved genes.

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Materials and Methods invasion to the bronchial wall, without nodal or distant metastases. The clinicopathologic features of 29 of these EHSCC were previously described, and all showed a very good prognosis (14). Relevant data of Patients and tumor samples. A total of 170 consecutive squamous the entire cohort of SCC patients according to tumor type are cell lesions of the lung, including 139 newly diagnosed SCC (136 in summarized in Table 1, including previously unreported data on males and 3 in females) and 31 preneoplastic/preinvasive squamous smoking habit. Inclusion criteria for SCC patients to enter the study cell bronchial lesions from different patients were retrieved from the were pathologic stage I (pT1-2N0M0), radical surgery with extensive files of the Divisions of Pathology of the City Hospital of Verona, the mediastinal lymph node dissection to ensure accurate staging, European Institute of Oncology in Milan, and the Royal Brompton and minimum 30-day postoperative survival, minimum follow-up of Harefield NHS Trust, London. For each case, all paraffin blocks were 5 years, and no (neo)-adjuvant chemoradiotherapy. Accurate follow- retrieved, and the original H&E-stained sections reviewed according to up information was available for all SCC patients, with a mean overall the 2004 WHO classification of lung tumors (1). Formalin-fixed and (OS) and disease-free survival (DFS) of 83 F 53 (median 84) and 77 F paraffin-embedded sections of both SCC and preneoplastic/preinvasive 54 months (median 74), respectively. Fifty-seven patients (41%) squamous cell lesions were used for all experiments. showed recurrent disease, and 53 (38%) died of disease. A total of 33 of the 139 SCC patients met the criteria proposed by the Preneoplastic/preinvasive squamous cell lesions (12 squamous cell Japan Lung Cancer Society for the definition of EHSCC (13, 15), i.e., a metaplasias, 8 squamous cell mild dysplasias, and 11 squamous cell tumor arising in a segmental, lobar, or main bronchus with limited severe dysplasias/in situ SCCs) were obtained from the surgical

Fig. 1. A^D,Representative features of FISH analysis in SCC of the lung showing a normal nucleus with a double signal for both 3q26( green) locus and chromosome 3 centromere (red ;ratio=1;A). Amplified nuclei show a ratio >2 between the two fluorescence signals (B), whereas polysomic nuclei present with more than two specific signals for both probes (C). Concurrent amplification and polysomy is characterized by a simultaneous occurrence of both ratio >2 and more than two specific signals per 3q26and centromere probes ( D).

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Table 2. Prevalence of 3q26 amplification and polysomy 3 in the spectrum of squamous cell lesions of the lung

Type of Types oflesion rearrangement PISCC EHSCC High-grade Low-grade Squamous P value (n = 106) (n = 33) squamous cell squamous cell metaplasia dysplasia/in situ cell dysplasia (n = 12) SCC (n = 11) (n =8) No amplification 70 17 8 8 12 Polysomy 3 21 3 2 0 0 c 3q26 Amplification 14* 13* 1 0 0 0.002 Other Monosomy 1 0 0 0 0

*In each patients’ subgroup, there was a case of concurrent amplification and polysomy. cPresence of rare amplified nuclei only in tumor cells. specimens of other lung cancer patients. These lesions were taken more compared with the various clinicopathologic parameters of the patients’ than 1 cm away from the invasive carcinoma and separated by areas of population including survival, as well as with the immunoreactivity for normal mucosa to avoid any possible pagetoid contamination by several gene products involved in cell cycle control and apoptosis (bcl- invasive SCC cells. All of the patients were preoperatively evaluated 2, p53, p21Waf1, p27/Kip1, cyclin-E, p63, c-kit/CD117), tumor growth with total body computed tomography scan, and tumor stage was (Ki-67 antigen assessment for proliferative fraction, HER-2, epidermal pathologically assessed according to the sixth tumor-node-metastasis growth factor receptor, and microvessel density as a surrogate of classification of malignant tumors. angiogenesis), and tumor cell motility (fascin) by previously detailed Finally, 10 samples of formalin-fixed and paraffin-embedded immunostaining methods (68–71). bronchial tree taken at different levels and pulmonary parenchyma Quantitative real-time RT-PCR. In a few randomly selected from patients with non-neoplastic lung disease (matched for age, sex, amplified, polysomic or negative (neither amplified or polysomic) and smoking habit) constituted the control group for noncarcinoma tumors, two 10-Am-thick sections were cut from paraffin blocks patients. containing at least 90% of tumor cellularity and processed for mRNA Fluorescence in situ hybridization. Two-color FISH assays for 3q26 extraction with Absolutely RNA FFPE Kit (Stratagene, La Jolla, CA) were done on 4-Am-thick paraffin-embedded sections using a Spec- according to the manufacturer’s instructions. The extracted RNA was trumOrange-labeled DNA probe spanning the entire 3q26 chromo- retrotranscribed by standard methods, and an amount of cDNA some region and a conjugated Cy3, SpectrumGreen-labeled, corresponding to 10 ng of the starting RNA was used for centromeric enumeration probe (both from Abbott-Vysis, Downer quantification in an ABI-PRISM 7700 Sequence Detector with SYBR- Grove, IL). For all the other FISH experiments, bacterial artificial GREEN detection (Applied Biosystems, Foster City, CA). Primers used chromosome (BAC) clones specific for 3q25 (RP-11 79M21), 3q26.1 in the study were the following: glyceraldehyde-3-phosphate dehy- (RP-11 297I8), 3q26.2 (RP-11 115B16, RP-11 258G22, RP-11 379K17), drogenase (GAPDH)-F 5¶-GCCTCAAGATCATCAGCAATGC-3¶ and 3q26.3 (RP-11 134M13), and 3q27 (RP-11 125E8), belonging to the GAPDH-R 5¶-CCACGATACCAAAGTTGTCATGG-3¶ as housekeeping 10 Roswell Park Cancer Institute libraries, were selected according to gene; h-TERC-F 5¶-GTGGTGGCCATTTTTTGTCTAAC-3¶ and h-TERC-R 11 esembl genome browser. Clones were validated by FISH analysis on 5¶-TGCTCTAGAATGAACGGTGGAA-3¶;SnoN-F5¶-TCAGCCT- normal fibroblasts to confirm their expected chromosomal localization. GATGCTCCGTGTA-3¶ and SnoN-R 5¶-AAGCCCCAGTGGCAAGTTC-3¶. Esembl database was also queried for known genes, searching in the Thermal conditions were 50jC for 2 min (1 cycle) and 95jC for 10 s A amplified regions. The 4- m-thick paraffin-embedded sections were and 60jC for 15 s (40 cycles). Results for relevant genes were hybridized with the relevant probes labeled by nick translation (66) normalized for GAPDH expression. A SCC sample with no 3q26 using 500 ng of probe labeled with either Fluorolink Cy3-dUTP or amplification or polysomy 3 was used as negative control for both Fluor-X-dCTP (Amersham, Buckinghamshire, United Kingdom). At candidate genes. Triplicate tests were done, and the mean value (F SD) least 100 neoplastic cells were counted for 3q26 (green signals) and was then calculated for each sample. a-satellite sequences of chromosome 3 centromere (red signals). Statistical analysis. Qualitative data were presented as frequencies Amplification of 3q26 was defined as a ratio >2.0 between the relevant and/or percentages and compared using the m2, Fisher exact test, or locus and the chromosome 3 copy number; polysomy by the finding of Wilcoxon-Mann-Whitney test as appropriate. Estimates for OS and DFS more than two specific signals for chromosome 3; chromosome 3 were calculated with the Kaplan-Meier method and compared by the monosomy by a mean of <1.5 signals per cell (67); and concurrent log-rank test. All analyses were carried out using the SAS statistical amplification and polysomy by the presence of three or more copies of software (SAS Institute, Inc., Cary, NC). All P values were based on two- the chromosome 3 centromere per cell with a ratio >2.0 for 3q26. sided testing. Digital images were obtained using a Leica DMRB epifluorescence microscope equipped with a Leica digital camera DC250 (Leica Imaging Systems Ltd, Cambridge, United Kingdom). FITC, Cy3, and 4¶,6- Results diamidino-2-phenylindole fluorescent signals were detected using specific filters. The images were recorded, pseudocolored, and merged Prevalence of 3q26 gain in the spectrum of squamous cell lesions using the QFluoro software (Leica). FISH analysis results were then of the lung and clinicopathologic implications. Representative features of 3q26 amplification and polysomy 3 are depicted in Fig. 1. Overall, 3q26 locus and chromosome 3 abnormalities 10 PieterJ. de Jong at http://bacpac.chori.org/. were detected in 52/139 (37%) locally invasive SCC, including 11 http://www.ensembl.org/index.html 36/106 (34%) parenchyma-infiltrating tumors and 16/33

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(48%) early hilar tumors, and in 3/31 (10%) preneoplastic/ noted between 3q26 amplification or polysomy 3 and both OS preinvasive squamous cell lesions of the bronchial epithelium, and DFS, even after stratifying for tumor type (EHSCC versus all being severe squamous cell dysplasia/CIS (Table 2). Invasive PISCC) or using different cutoff levels. Remarkably, EHSCC tumors showing amplification, polysomy, concurrent amplifi- patients showed a better prognosis than PISCC patients for both cation, and polysomy or monosomy were 27, 24, 2, and 1 case, OS and DFS (Fig. 2A). Marginal differences were also observed in respectively. In preneoplastic/preinvasive squamous cell the same tumor types as stratified for stage 1A (Fig. 2B). lesions, two cases of polysomy and one case exhibiting rare 3q26 minimal common amplification region. FISH analysis amplified nuclei in tumor cells were identified, all occurring in with either BAC probe RP-11 79M21 hybridizing to 3q25 or severe dysplasias/in situ SCC (3/11, 27%). Monosomy was no RP-11 125E8 hybridizing to 3q27 on adjacent paraffin sections longer considered for analysis in our investigation. None of of the 3q26-amplified tumors did not reveal any extra signal for the non-neoplastic control samples showed any such abnor- these loci (Fig. 3). In the 27 amplified invasive SCC (13 EHSCC mality. and 14 PISCC), we found several patterns of gain at 3q26. In All tumors with 3q26 amplification (27 invasive and 1 in situ fact, the use of additional BAC probes targeting different parts carcinoma with rare amplified nuclei) showed a signal ratio per of the 3q26 region indicated that the minimal common region cell up to 4 to 5, with the single exception of one invasive tumor of amplification was centered on 3q26.2 (BAC probes RP-11 with a ratio of 7. Likewise, 16 of the 26 polysomic tumors (24 115B16 and RP-11 379K17), where the smallest amplicon invasive and 2 in situ carcinomas) showed a mean of 3.4 signals identified covers <2 Mb (Fig. 4A and B). per cell (range 3-3.5), with the remaining 10 tumors exhibiting a Expression levels of h-TERC and SKI-like mRNA. The poor mean level of polysomy of 4.7 (range 3-5.5). quality of mRNA in most tumor samples prevented us from Overall, 3q26 gains, either by amplification or polysomy, investigating systematically all tumor samples for the relevant were more prevalent in invasive tumors, either EHSCC or genes. In five tumors (two amplified, two polysomic, and PISCC, than in preneoplastic/preinvasive squamous cell lesions one negative) real-time RT-PCR assessment of h-TERC and (Table 2). Moreover, 3q26 amplification was significantly SKI-like mRNA content was feasible, and the results are associated with EHSCC (13 amplified tumors in 33 EHSCC summarized in Table 4. Although all tumor samples exhibited and 14 amplified tumors in 106 PISCC), whereas polysomy greater amounts of both h-TERC and SKI-like mRNA as was associated with PISCC (21 polysomic tumors in PISCC compared with the negative tumor (showing the same levels of versus 3 polysomic tumors in EHSCC; P = 0.003). h-TERC and SKI-like gene mRNA after normalization with the 3q26 amplification correlated significantly with a tumor housekeeping gene GAPDH), higher levels were found for diameter >2 cm (P = 0.013) and, marginally, with a pack-year mRNA SKI-like than for h-TERC in both amplified and index >15 (P = 0.083). Polysomy 3 correlated significantly with a polysomic tumors. However, h-TERC mRNA expression pre- tumor diameter >2 cm (P = 0.023), pT class (P = 0.019), and p53 vailed in amplified as compared with polysomic tumors, whereas (P = 0.039) and p21 (P = 0.029) immunoreactivity and, the reverse held true for SKI-like. marginally, with strong and diffuse immunoreactivity for fascin (P = 0.109), a marker of cell motility (Table 3). No other Discussion significant correlation was noted with the remaining clinicopathologic and immunohistochemical variables of the patients’ The main findings of our investigation can be summarized as population under evaluation. Moreover, no association was follows: (a) 3q26 amplification and chromosome 3 polysomy

Table 3. Clinicopathologic correlations of 3q26 amplification and polysomy 3 in pulmonary SCCa

Variable Category No gain Polysomy P value* Amplification P value*

Tumor diameter <2 cm 18 2 11 2-4 cm 47 9 6 >4 cm 22 13 0.023 10 0.013 Tumor stage pT1 58 9 16 pT2 29 15 0.019 11 NS Pack-yearc <15 3 2 1 15-60 28 10 11 >60 45 11 NS 5 0.083 p53 expressionb neg 36 5 7 pos 35 17 0.039 14 NS p21 labelling indexx <15% 43 7 10 15% or more 27 15 0.029 7 NS Fascin expressionx <60% 11 0 1 60% or more 59 22 0.109 16 NS

Abbreviation: NS, not significant. *P value according m2 test. cPack-year data were known for 116 patients only. bp53 was assessed in 114/138 tumors; p53-negative tumors were up to 10% of immunoreactive cells, p53-positive tumors showed more than 10% of immunoreactive cells. xMedian value; p21 and fascin immunoreactivity were assessed in 109/138 tumors.

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Fig. 2. A, survival curves according to tumor histology. EHSCC patients show a better OS and DFS as compared with the other stage I PISCC patients. One patient with PISCC (stage 1B, pT2N0M0) showing monosomy for 3q26has not been incorporated in survival analysis. B, marginal differences in survival are present when tumors were stratified for tumor stage 1A(pT1N0M0). likely are late events in the development of SCC of the lung rather than in the squamous cell carcinogenesis of the because they are virtually lacking in preneoplastic/preinvasive bronchial epithelium. The three cases of preneoplastic/prein- lesions; (b) these alterations may be involved in invasiveness vasive squamous cell lesions showing polysomy 3 or rare tumor and progression of some pulmonary SCC, but they do not cells with 3q26 amplification were all in the severe dysplasia/ allow distinction of less aggressive SCC from life-threatening CIS group. Interestingly, Foster et al. (8) have recently shown tumors; (c) 3q26 amplification is more prevalent in EHSCC that 3q26 amplification is a feature of pulmonary squamous than in PISCC, suggesting a different pathogenesis for these CIS able to progress over time to an invasive tumor, thus tumor types; (d) the minimal common amplification region confirming the pivotal role of this change in the transition from corresponds to 3q26.2, and h-TERC and SKI-like may be high-grade preinvasive neoplasia to invasive carcinoma, as also candidate targets for 3q26 gain and polysomy 3. documented in uterine cervix (57, 72) and head and neck (44) The high prevalence of 3q26 gain and polysomy 3 in invasive SCC. The prevalence (1/11, about 10%) of 3q26 amplification pulmonary SCC (as opposed to the lack of these changes in the in severe squamous dysplasia/CIS of the lung, along with its non-neoplastic bronchial epithelium and pulmonary paren- consistent lack in the non-neoplastic bronchial epithelium and chyma and across the spectrum of squamous preneoplastic/ pulmonary parenchyma distant from tumors, are in keeping preinvasive lesions) is consistent with a possible role for these with the results of Singh et al.(44) in head and neck carcinoma, abnormalities in the progression of early-stage infiltrating SCC demonstrating 3q26 amplification in up to 25% of peritumoral

Clin Cancer Res 2007;13(7) April 1, 20 07 200 0 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. 3q26 and Polysomy 3 in Squamous Cell Lesion of the Lung preinvasive lesions and 6% of peritumoral normal mucosa, but in virtually none of the specimens of either preinvasive lesions Table 4. Real-time PCR results in pulmonary SCC or lung normal tissue sampled far from the tumors. The evaluated for mRNA levels prevalence of 3q26 gain is therefore low in preinvasive Case Age Sex Histology Category TERC SKI-like (Sno) squamous cell proliferations in the lung once pagetoid contamination (73, 74) by invasive carcinoma cells is avoided, mean std mean std as we did in the current study. 197 63 M PISCC Amplified 1.9 0.5 3.2 0.6 The relationship between 3q26 amplification, increased 205 69 M PISCC Amplified 2.1 0.2 2.9 0.2 tumor diameter, and cigarette pack-year [as also reported by 111 72 M EHSCC Polysomic 1.6 0.1 6.0 0.1 IEO5 66 M EHSCC Polysomic 1.4 0.1 3.5 0.03 Yan et al. (75)]and between polysomy 3 and indicators of tumor 115 61 M PISCC Negative 1.0 0.1 1.0 0.1 growth (tumor diameter, pT class, and p53 or p21 overexpression) or tumor cell motility (fascin overexpression; Abbreviation: M, male. Table 3) are in keeping with a pivotal role of these abnormalities in driving the tumor progression of some pulmonary SCC subsets. Lack of prognostic implications of 3q26 gains for lung patients’ tumors (pT1-2 N0 M0), which usually show an cancer is at variance with head and neck (44, 48) or cervical (57) inherently more favorable prognosis, as compared with patients SCC, although we have taken into account only early-stage with more advanced disease (76, 77). An intriguing and novel aspect of our investigation is that 3q26 amplification or chromosome 3 polysomy are associated with different types of SCC, either limited to the bronchial wall or infiltrating the pulmonary parenchyma, suggesting a different molecular pathogenesis. These specific molecular profiles and the inherently better prognosis of EHSCC suggest that these neoplasms may represent a peculiar subset of bronchogenic carcinoma (Fig. 2). Therefore, pathologists should be aware of the existence of EHSCC which, albeit rare (14, 17, 25–27), should perhaps be staged as pT1 even when associated with lobar atelectasis of the entire lung or with obstructive pneumonitis in virtue of their better clinical course (ref. 14; Fig. 2). The close association of red (chromosome 3 centromere) and green (3q26 amplicon) signals, the occurrence of discrete areas of immunofluorescence in 3q26-gaining nuclei with signal levels as intense as or even stronger than those detected in nonamplified nuclei, and the lack of extrachromosomal small immunofluorescence signals scattered throughout nuclear area (78) suggest that this chromosomal gain is based on local amplification rather than double minutes. Moreover, increased BAC/centromeric signal ratios were prevalent at 3q26 rather than at either 3q25 or 3q27, excluding additional amplification regions in keeping with the results of Kettunen et al. (42). In addition, we have found that the minimal common amplification region was centered on 3q26.2 (BAC probes RP-11 115B16 and RP-11 379K17), where the smallest amplicon identified covered <2 Mb (Fig. 4A and B). Overall, more than 100 genes are known to map the 3q26 region, many of which are overexpressed in SCC at different anatomic sites (32). In the lung, these include butyrylcholinesterase and glucose transporter 2 (SCL2A2) at 3q26.1-3q26.2 (38, 39), h-TERC at 3q26.2 (35), SKI-like or SnoN (35) at 3q26.2, SCC-related oncogene or defective in cullin neddylation 1, domain containing 1 (DCUN1D1) at 3q26.3 (34, 38), phosphoinositide-3-kinase, catalytic a-polypeptide (PIK3CA) at 3q26.3 (35, 79), ZMAT3 or WIG-1/PAG608 (zinc finger, matrin type 3) at 3q26.3-q27 (37), and ecotropic viral integration site 1 (EVI1) at 3q24-q28 (35). In particular, 3q26.2 is a gene-rich region with several possible candidate oncogenes, and it may be that the amplification of more than one gene product in such a Fig. 3. A and B, FISH analysis on adjacent paraffin sections for 3q26-amplified region contributes to the final malignant invasive phenotype. SCC tumor patients for 3q25 (A)and3q27(B) loci flanking 3q26: the lack h-TERC and SKI-like or SnoN are candidate genes relevant to of additional amplification signals in the form of discrete aggregates of immunofluorescence is proof that amplification pattern is specific for 3q26 pulmonary SCC development (35), and we have tried to chromosomal region. assess whether these genes were overexpressed in carcinomas

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Fig. 4. A and B, FISH analysis on 27 SCC tumor patients using five BAC probes at 3q26region. All values are indicated as the ratio of the average number of BAC signals compared with the average number of centromeric signals for chromosome 3 (BAC/CEN). Green and yellow cells, amplified and nonamplified tumors for each BAC probe, respectively.Tothe right of each row indicating a given patient is reported the estimated genetic distance from pter in megabases. jj, tumors presenting with concurrent amplification and polysomy when tested for the probe spanning the entire 3q26region. with 3q26 amplification or polysomy 3 according to the assessed by comparative genomic hybridization have been tumor type. Despite a limited number of evaluable cases, described in head and neck carcinomas arising from different mRNA levels of both genes were higher in both amplified and anatomic sites (80). polysomic tumors as opposed to negative tumors in keeping In conclusion, our data support the theory that 3q26 with previously reported data (35), SKI-like was preferentially amplification and chromosome 3 polysomy may play a overexpressed in polysomic and h-TERC in amplified carci- different role in the development of different subsets of nomas (Table 4). No relationship was found with common invasive SCC of the lung. Both h-TERC and SKI-like genes confounding factors such as tumor stage, age, sex, and smoke may be involved in this mechanism. index (data not shown). This indicates that different genes may contribute to the development of different types of Acknowledgments invasive SCCs of the lung, despite their similar histologic features, although these results obtained from a few tumor The authors acknowledge the thoracic surgical team at the Royal Brompton Hos- samples are only preliminary. Likewise, genetic differences pital, led by Prof P. Goldstraw, for providing some of the materials used in this study.

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Giuseppe Pelosi, Barbara Del Curto, Maurizio Trubia, et al.

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