Transition from Squamous Cell Carcinoma to Adenocarcinoma in Adenosquamous Carcinoma of the Lung
2000; Elsevier BV; Volume: 156; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64999-1
ISSN1525-2191
AutoresHironobu Kanazawa, Masahito Ebina, Nozomu Ino-oka, Minoru Shimizukawa, Toru Takahashi, Shigefumi Fujimura, Tadashi Imai, Toshihiro Nukiwa,
Tópico(s)Cancer-related Molecular Pathways
ResumoThe heterogeneity of tumor cells is frequently observed in lung cancer, but the clonality of these cells has not yet been established. The distinct components of 12 lung adenosquamous carcinomas were compared by genetic alterations of p53 and K-ras, chromosomal abnormalities at 9p21 and 9q31–32, and immunohistochemical reactions. The immunoreactivity of p53 was consistent in both adenocarcinomatous and squamous cell carcinomatous components as well as in the transitional areas, retaining the morphological characteristics of the distinct components. The same p53 mutation was found in both components of each tumor with p53 overexpression. No K-ras mutations were detected in any of the tumors examined. Three of the four tumors with chromosomal abnormalities detected, one at 9p21 and two at 9q31–32, had coincident abnormalities between the distinct components, whereas one tumor deleted homozygously at 9p21 (D9S259) in the adenocarcinomatous component with loss of heterozygosity in the other component. The expression of squamous cell carcinoma-related antigen in adenocarcinomatous components was significantly higher than that of lung adenocarcinomas (57 ± 5.8% vs. 1.0 ± 0.5%, P < 0.0001), whereas Mucin 1 expression is less in these components (9.0 ± 4.9% vs. 55 ± 8.2%, P = 0.003). These results suggest monoclonal transition from squamous cell carcinoma to adenocarcinoma in lung adenosquamous carcinoma. The heterogeneity of tumor cells is frequently observed in lung cancer, but the clonality of these cells has not yet been established. The distinct components of 12 lung adenosquamous carcinomas were compared by genetic alterations of p53 and K-ras, chromosomal abnormalities at 9p21 and 9q31–32, and immunohistochemical reactions. The immunoreactivity of p53 was consistent in both adenocarcinomatous and squamous cell carcinomatous components as well as in the transitional areas, retaining the morphological characteristics of the distinct components. The same p53 mutation was found in both components of each tumor with p53 overexpression. No K-ras mutations were detected in any of the tumors examined. Three of the four tumors with chromosomal abnormalities detected, one at 9p21 and two at 9q31–32, had coincident abnormalities between the distinct components, whereas one tumor deleted homozygously at 9p21 (D9S259) in the adenocarcinomatous component with loss of heterozygosity in the other component. The expression of squamous cell carcinoma-related antigen in adenocarcinomatous components was significantly higher than that of lung adenocarcinomas (57 ± 5.8% vs. 1.0 ± 0.5%, P < 0.0001), whereas Mucin 1 expression is less in these components (9.0 ± 4.9% vs. 55 ± 8.2%, P = 0.003). These results suggest monoclonal transition from squamous cell carcinoma to adenocarcinoma in lung adenosquamous carcinoma. Although tumor heterogeneity is a fundamental feature of carcinogenesis and cancer progression,1Mabry M Nelkin B Falco JP Barr LF Baylin SB Transitions between lung cancer phenotypes-implications for tumor progression.Cancer Cells. 1991; 3: 53-58PubMed Google Scholar, 2Kern SE Clonality: more than just a tumor-progression model.J Natl Cancer Inst. 1993; 85: 1020-1021Crossref PubMed Scopus (29) Google Scholar, 3Godfrey M Molecular heterogeneity: a clinical dilemma; clinical heterogeneity: a molecular dilemma.Am J Hum Genet. 1993; 53: 22-25PubMed Google Scholar the mechanism by which it occurs remains largely unknown.4Heppner GH Tumor heterogeneity.Cancer Res. 1984; 44: 2259-2265PubMed Google Scholar, 5Williams GT Wynford-Thomas D How many clonality be assessed in human tumours?.Histopathology. 1994; 24: 287-292Crossref PubMed Scopus (13) Google Scholar The heterogeneity of tumor cells is frequently observed in lung cancer.1Mabry M Nelkin B Falco JP Barr LF Baylin SB Transitions between lung cancer phenotypes-implications for tumor progression.Cancer Cells. 1991; 3: 53-58PubMed Google Scholar, 6Ashley DJ Davies HD Cancer of the lung: histology and biological behavior.Cancer. 1967; 20: 165-174Crossref PubMed Scopus (9) Google Scholar, 7Roggli VL Vollmer RT Greenberg SD McGavran MH Spjut HJ Yesner R Lung cancer heterogeneity: a blinded and randomized study of 100 consecutive cases.Hum Pathol. 1985; 16: 569-579Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 8Dunnill MS Gatter KC Cellular heterogeneity in lung cancer.Histopathology. 1986; 10: 461-475Crossref PubMed Scopus (68) Google Scholar The main pattern consists of a regional distribution of individual cells, similar to their immediate neighbors, whereas more distant cells tend to be similar to their immediate neighbors.6Ashley DJ Davies HD Cancer of the lung: histology and biological behavior.Cancer. 1967; 20: 165-174Crossref PubMed Scopus (9) Google Scholar, 7Roggli VL Vollmer RT Greenberg SD McGavran MH Spjut HJ Yesner R Lung cancer heterogeneity: a blinded and randomized study of 100 consecutive cases.Hum Pathol. 1985; 16: 569-579Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 8Dunnill MS Gatter KC Cellular heterogeneity in lung cancer.Histopathology. 1986; 10: 461-475Crossref PubMed Scopus (68) Google Scholar, 9Coons SW Johnson PC Shapiro JR Cytogenetic and flow cytometry DNA analysis of regional heterogeneity in a low grade human glioma.Cancer Res. 1995; 55: 1569-1577PubMed Google Scholar According to fundamental concepts concerning the carcinogenesis of lung cancer, field cancerization, and the multistep carcinogenic process, the pathogenesis of these heterogeneous tumor cells would be explained by various carcinogenic stages involving monoclonal or polyclonal expansion.5Williams GT Wynford-Thomas D How many clonality be assessed in human tumours?.Histopathology. 1994; 24: 287-292Crossref PubMed Scopus (13) Google Scholar, 10Lee SJ Mao L Hong WK Biology of preneoplastic lesions.in: Roth JA Cox JD Hong WK Lung Cancer. 2nd ed. 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Armed Forces Institute of Pathology, Washington, DC1995: 279-286Google Scholar According to the most recent World Health Organization classification of lung tumors, a definitive diagnosis of lung adenosquamous carcinoma requires a minimum of 10% of each component in the whole tumor.15Travis WD Colby TV Corrin B Shimosato Y Brambilla E Histological Typing of Lung and Pleural Tumors. 3rd ed. Springer-Verlag, Berlin1999Crossref Google Scholar Although morphological characterizations of both components of adenosquamous carcinoma have been made, no genetic approach to the clonality of adenosquamous carcinoma has yet been attempted. In this study, we performed topographic genotyping and immunohistochemical analysis on 12 adenosquamous carcinoma tumors with a definitive diagnosis, free from either anti-cancer chemotherapy or irradiation therapy before sampling, that would cause genetic changes to the cancer cells. As topographic genotyping, comparative DNA sequences of p53, K-ras, and chromosomal analysis at 9p21 and 9q31–32 were performed on the distinct components in each tumor. p53 mutations occur in approximately one-half of non-small-cell carcinomas at various points within hot spots,16Hollstein M Sidransky D Vogelstein B Harris C p53 mutations in human cancer.Science. 1991; 253: 49-53Crossref PubMed Scopus (7783) Google Scholar supposed as a good marker for detecting the clonality of cancer cells.17Sidransky D Mikkelsen T Schwechheimer K Rosenblum ML Cavanee W Vogelstein B Clonal expansion of p53 mutant cells is associated with brain tumour progression.Nature. 1992; 355: 846-847Crossref PubMed Scopus (662) Google Scholar, 18Holst VA Finkelstein S Colby TV Myers JL Yousem S P53, and K-ras mutational genotyping in pulmonary carcinoma, spindle cell carcinoma, and pulmonary blastoma: implications for histogenesis.Am J Surg Pathol. 1997; 21: 801-811Crossref PubMed Scopus (78) Google Scholar K-ras mutations have been reported in 27 to 46% of adenocarcinomas of the lung, and less or not at all in squamous cell carcinoma.19Rodenhuis S Slebos RJC Boot AJM Everts SG Mooi WJ Wagenaar SS van Bodegom PC Bos JL Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung.Cancer Res. 1998; 48: 5738-5741Google Scholar, 20Suzuki Y Orita M Shiraishi M Hayashi K Sekiya T Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products.Oncogene. 1990; 5: 1037-1043PubMed Google Scholar, 21Mills NE Fishman CL Rom WN Dubin N Jacobson DR Increased prevalence of K-ras oncogene mutaions in lung adenocarcinoma.Cancer Res. 1995; 55: 1444-1447PubMed Google Scholar 9p2122Wiest JS Franklin WA Otstot JT Forbey K Varella-Garcia M Rao K Drabkin H Gemmill R Ahrent S Sidransky D Saccomanno G Fountain JW Anderson MW Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene.Cancer Res. 1997; 57: 1-6PubMed Google Scholar, 23Lukeis R Irving L Garson M Hasthorpe S Cytogenetics of non-small cell lung cancer: analysis of consistent non-random abnormalities.Genes Chromosomes Cancer. 1990; 2: 116-124Crossref PubMed Scopus (136) Google Scholar and 9q31–3224Miura K Suzuki K Tokino T Isomura M Inazawa J Matsuno S Nakamura Y Detailed deletion mapping in squamous cell carcinomas of the esophagus narrows a region containing a putative tumor suppressor gene to about 200 kilobases on distal chromosome 9q.Cancer Res. 1996; 56: 1629-1634PubMed Google Scholar are frequent abnormal loci in squamous cell carcinoma, but less in adenocarcinoma of the lung. 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The degrees of immunoreactivity were quantified according to the distribution of immunoreactive tumor cells among whole tumor cells according to our previous study.36Ebina M Steiberg SM Mulshine JL Linnoila RI Relationship of p53 overexpression and up-regulation of proliferating cell nuclear antigen with the clinical course of non-small cell lung cancer.Cancer Res. 1994; 54: 2496-2503PubMed Google Scholar The main purposes of this study were to clarify the clonality of adenosquamous carcinoma of the lung as a typical model of heterogeneous tumors and to elucidate the process of carcinogenesis and cancer progression of these tumors. Such information may suggest successful therapies for heterogeneous tumors.1Mabry M Nelkin B Falco JP Barr LF Baylin SB Transitions between lung cancer phenotypes-implications for tumor progression.Cancer Cells. 1991; 3: 53-58PubMed Google Scholar, 2Kern SE Clonality: more than just a tumor-progression model.J Natl Cancer Inst. 1993; 85: 1020-1021Crossref PubMed Scopus (29) Google Scholar, 3Godfrey M Molecular heterogeneity: a clinical dilemma; clinical heterogeneity: a molecular dilemma.Am J Hum Genet. 1993; 53: 22-25PubMed Google Scholar The tumors of 12 patients with adenosquamous carcinoma of the lung were obtained at the Hospital of the Institute of Development, Aging and Cancer, Tohoku University (cases 1–8), and the Central Hospital of Aomori Prefecture (cases 9–12). Eight of these tumors (cases 1–4 and 9–12) were resected by surgical operation; the others (cases 5–8. were autopsied (Table 1). Two patients (cases 10 and 11) are without smoking habit; the rest are current or former smokers (Table 1) with the range of Brinkman index for cigarette smoking from 690 to 1400 (958 ± 104, mean ± SEM). Informed consent for genetic analysis of these tumors was obtained from each patient or their families. None of these patients was treated by anti-cancer chemotherapy or irradiation before the tumors were sampled. Metastatic lesions were available in three cases (cases 3, 5, and 8), and were examined by microscopic observation. A pathological diagnosis of adenosquamous carcinoma was based on the WHO Histological Typing of Lung Tumors15Travis WD Colby TV Corrin B Shimosato Y Brambilla E Histological Typing of Lung and Pleural Tumors. 3rd ed. Springer-Verlag, Berlin1999Crossref Google Scholar and checked by at least three independent pathologists. Three to five sampled specimens of each tumor were formalin-fixed and paraffin-embedded, and 10 serial sections 3 μm thick were cut from each block. The first sections were stained with hematoxylin-eosin (H&E), the second sections for periodic acid-Schiff (PAS)-alcian blue staining, and the remaining eight sections were used for immunohistochemical study. Three additional sections 8 μm thick were cut and used for microdissection of the two distinct components of each tumor to extract DNA as described below. Ten primary tumors of lung adenocarcinoma and ten primary tumors of lung squamous cell carcinoma, surgically resected in our hospital, were also used in our pilot study to get control reactions by immunohistochemistry, especially for non-commercially available antibodies against human squamous cell carcinoma-related antigen (F2H7C) and against human MUC1 (MUSEII).Table 1Characteristics of Patients and Adenosquamous Carcinomas Examined, and p53 Status in Each ComponentCase no.Age (y)/genderSmokingStageSamplingPredominant p53 componentIHC AD/SQp53 mutation AD/SQ172 /MformerIoperatedSQ (80%)N /NWT/WT265 /MformerIIoperatedSQ (80%)N /NWT/WT368 /McurrentIIIAoperatedSQ (70%)N /NWT/WT468 /FcurrentIIIAoperatedAD (80%)P /P167Gln to Arg/167Gln to Arg589 /MformerIVautopsiedAD (60%)P /P297His to Tyr/297His to Tyr668 /McurrentIVautopsiedSQ (60%)N /NWT/WT778 /McurrentIVautopsiedSQ (80%)P /P210Asn to Asp/210Asn to Asp874 /McurrentIVautopsiedAD (70%)N /NWT/WT968 /McurrentIIoperatedSQ (60%)P /PND1063 /FnoIoperatedAD (60%)P /PND1160 /FnoIoperatedSQ (80%)N /NWT/WT1265 /McurrentIIIAoperatedSQ (80%)N /NWT/WTM, male; F, female; AD, adenocarcinomatous component; SQ, squamous carcinomatous component; IHC, immunohistochemistry; P, positive; N, negative; WT, wild-type; ND, not determined;167Gln to Arg, mutation from glutamine to arginine at codon 167; 297His to Tyr, mutation from histidine to tyrosine at codon 297; 210Asn to Asp, mutation from asparagine to asparatic acid at codon 210; cdns, codons. Open table in a new tab M, male; F, female; AD, adenocarcinomatous component; SQ, squamous carcinomatous component; IHC, immunohistochemistry; P, positive; N, negative; WT, wild-type; ND, not determined;167Gln to Arg, mutation from glutamine to arginine at codon 167; 297His to Tyr, mutation from histidine to tyrosine at codon 297; 210Asn to Asp, mutation from asparagine to asparatic acid at codon 210; cdns, codons. The two different foci in each tumor, determined by subsequent H&E-stained sections, were scraped off individually from three deparaffinized thick sections under microscopic observation. To avoid the contamination of each sample from different foci in one section, the tissue sections were dried up before sampling, and only the foci to be scraped off were wetted using microcapillary tips. The tissue fragments were collected into microtubes and digested as previously described.40Teneriello MG Ebina M Linnoila RI Henry M Nash JD Park RC Birrer MJ p53 and Ki-ras gene mutations in epithelial ovarian neoplasms.Cancer Res. 1993; 53: 3103-3108PubMed Google Scholar For the analysis of loss of heterozygosity at 9p21 and 9q31–32, additional DNA samples were extracted from tumor-free lung tissues, which were available in cases 1 to 8 and used for control DNA for each tumor. For detecting p53 mutations within exons 5 to 8, the isolated DNA samples from the two different components of each tumor were amplified using primer sets as described previously.41Hall KL Teneriello MG Taylor RR Lemon S Ebina M Linnoila RI Norris JH Park RC Birrer MJ Analysis of Ki-ras, p53, and MDM2 genes in uterine leiomyomas and leiomyosarcomas.Gynecol Oncol. 1997; 65: 330-335Abstract Full Text PDF PubMed Scopus (71) Google Scholar The amplified polymerase chain reaction (PCR) products were cloned into a pCRII vector (Invitrogen Corp., Carlsbad, CA) and purified through Qiagen Mini Columns (Qiagen Inc., Chatsworth, CA). The mixtures of 20 clones of each DNA sample were sequenced by a dsDNA cycle sequencing system (Gibco BRL, Rockville, MD) using primers end-labeled with [γ-32P] ATP (Amersham Corp., Arlington, IL. according to the manufacturers' manuals. Mutations were accepted when the genetic alterations were detected in the independent PCR products of the same DNA samples. The isolated DNA samples from the two different two components in each tumor were also analyzed for K-ras mutations at codons 12, 13, and 61 using PCR-restriction fragment length polymorphisms (RFLP. analysis, and direct sequencing. As for codon 12, PCR-primer introduced restriction with enrichment for mutant alleles (PIREMA) was used as described previously.21Mills NE Fishman CL Rom WN Dubin N Jacobson DR Increased prevalence of K-ras oncogene mutaions in lung adenocarcinoma.Cancer Res. 1995; 55: 1444-1447PubMed Google Scholar Briefly, the PCR products (192 bp) were digested with BstNI (TOYOBO, Osaka, Japan), and the digested products were amplified again in the same protocol with the first PCR. As positive controls of the K-ras mutations at these sites, the extracted DNA samples of the cell lines NCI-H358 (TGT) and NCI-H727 (GTT)42Mitsudomi T Viallet J Mulshine JL Linnoila RI Minna JD Gazdar AF Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines.Oncogene. 1991; 6: 1353-1362PubMed Google Scholar were used, in addition to human genomic DNA (Clontech Laboratories, Inc., Palo Alto, CA) as a normal control (GGT). Direct sequencing of DNA samples was also performed for detecting other possible mutations at codon 12, 13, and 61 using a double-strand DNA cycle sequencing system (Gibco BRL. with the primers end-labeled with [γ-32P]ATP (Amersham Corp.) according to a previous study.40Teneriello MG Ebina M Linnoila RI Henry M Nash JD Park RC Birrer MJ p53 and Ki-ras gene mutations in epithelial ovarian neoplasms.Cancer Res. 1993; 53: 3103-3108PubMed Google Scholar Three different DNA samples from each tumor were used for microsatellite analysis, collected independently from the adenocarcinomatous components, squamous carcinomatous components, and tumor-free sites. Microsatellite analysis was omitted in cases 9 to 12, which lacked tumor-free materials. Six microsatellite markers were used in this study to distinguish the chromosomal regions at 9p21 (D9S265, D9S126, D9S259) and 9q31–32 (KM9.1, D9S177) using primer sequences reported before.22Wiest JS Franklin WA Otstot JT Forbey K Varella-Garcia M Rao K Drabkin H Gemmill R Ahrent S Sidransky D Saccomanno G Fountain JW Anderson MW Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene.Cancer Res. 1997; 57: 1-6PubMed Google Scholar, 24Miura K Suzuki K Tokino T Isomura M Inazawa J Matsuno S Nakamura Y Detailed deletion mapping in squamous cell carcinomas of the esophagus narrows a region containing a putative tumor suppressor gene to about 200 kilobases on distal chromosome 9q.Cancer Res. 1996; 56: 1629-1634PubMed Google Scholar The primers were end-labeled with 0.2 μCi each of [γ-32P] ATP and 0.07 U of T4 polynucleotide kinase (Gibco BRL). PCR amplifications were performed in 10 μl reaction volumes, including 50 ng of genomic DNA, 0.6 pg of labeled primer, 50 μmol/L of each dNTP (Takara), and 0.5 U of Taq DNA polymerase (Takara). After initial denaturation at 94°C for 3 minutes, 40 cycles of PCR (30 seconds at 94°C, 60 seconds at 50°C, and 60 seconds for 72°C) were performed, with final extension for 10 minutes at 72°C. PCR products were mixed with loading buffer, heat denatured, and electrophoresed in 8% polyacrylamide-urea-formamide gel. The intensity of the radioactivity of the signal was measured using Bio-Imaging Analyzer system (Fuji Film Co., Minami-Ashigara, Japan), and loss of heterozygosity was defined as a reduction in the intensity of one allele in the tumor DNA by at least 50% as compared with the corresponding normal DNA.22Wiest JS Franklin WA Otstot JT Forbey K Varella-Garcia M Rao K Drabkin H Gemmill R Ahrent S Sidransky D Saccomanno G Fountain JW Anderson MW Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene.Cancer Res. 1997; 57: 1-6PubMed Google Scholar, 24Miura K Suzuki K Tokino T Isomura M Inazawa J Matsuno S Nakamura Y Detailed deletion mapping in squamous cell carcinomas of the esophagus narrows a region containing a putative tumor suppressor gene to about 200 kilobases on distal chromosome 9q.Cancer Res. 1996; 56: 1629-1634PubMed Google Scholar When both alleles were deleted, the results were confirmed at least twice. The mouse monoclonal antibody antiserums used in this study were as follows: anti-human CEA (ZC23, Histofine, Tokyo), anti-human CA19-9 (1116 NS 19-9, CIS bio International, Tokyo), anti-human SCC (F2H7C, kindly provided by Dr. Kato, Yamaguchi University School of Medicine, Yamaguchi, Japan), anti-human VEGF (Santa Cruz Biotechnology, Santa Cruz, CA), anti-human MUC1 (MUSEII, kindly provided by Dr. Imai, Sapporo University, Sapporo, Japan), anti-human p53 (DO-7, Dako Corporation, Carpinteria, CA), anti-human PCNA (PC-10, Dako), and anti-human p21Waf1/Cip1 (Ab-1, Oncogene Science). The optimal dilution of each immunohistochemical reaction was determined by pilot studies as follows: antibodies against CEA and CA19-9 were diluted according to the manufacturer's recommendations, anti-SCC at 1:100, anti-VEGF at 1:500, anti-MUC1 at 1:50, anti-p53 at 1:20, anti-PCNA at 1:20, and anti-p21Waf1/Cip1 at 1:10. Primary incubation was performed overnight at 4°C with these primary antibodies, and staining was performed using the avidin-biotinylated peroxidase complex technique using Vectastain kits (Vector Laboratories, Burlingame, CA) according to our previous study.36Ebina M Steiberg SM Mulshine JL Linnoila RI Relationship of p53 overexpression and up-regulation of proliferating cell nuclear antigen with the clinical course of non-small cell lung cancer.Cancer Res. 1994; 54: 2496-2503PubMed Google Scholar For the reactions with the antibodies against p53 and Waf1, microwave pretreatment was performed using 0.01 mol/L sodium citrate buffer at pH 6.0. To quantify the immunoreactive tumor cells, five random sites each per one adenocarcinomatous and one squamous carcinomatous component of the same tumor were color-photographed and observed at 1000× as the final magnification using parallel sampling lines (one nucleus once). The degrees of immunoreactivity of each component were determined by percentage of the positive tumor cells among total tumor cells, positive and negative (approximately 300), according to the quantification methods.36Ebina M Steiberg SM Mulshine JL Linnoila RI Relationship of p53 overexpression and up-regulation of proliferating cell nuclear antigen with the clinical course of non-small cell lung cancer.
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