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Intratumor genomic heterogeneity correlates with histological grade of advanced oral squamous cell carcinoma
Oral Oncology (2006) 42, 740– 744
available at www.sciencedirect.com
journal homepage: http://intl.elsevierhealth.com/journals/oron/
Intratumor genomic heterogeneity correlates with histological grade of advanced oral squamous cell carcinoma q Xinhong Wang a,b,c, Mingwen Fan a,c,*, Xinming Chen b,c, Shuozhi Wang b, Mohd Jamal Alsharif d, Li Wang b,c, Lijiang Liu e, Hao Deng e a
Department of Endodontics, School and Hospital of Stomatology of Wuhan University, China Department of Pathology, School and Hospital of Stomatology of Wuhan University, China c Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology of Wuhan University, Wuhan, Hubei, China d Department of Surgery, School and Hospital of Stomatology of Wuhan University, China e Department of Pathology and Pathophysiology, Medical and Life Science College, Jianghan University, China b
Received 26 October 2005; accepted 24 November 2005
KEYWORDS
Summary To assess the difference in genetic aberration patterns among the invasive tumor front (ITF), center/superficiality and the stroma adjacent to oral squamous cell carcinoma (OSCC), we studied loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosome 9p21 and 17p13 on the three regions by combining laser capture microdissection (LCM) and PCR. We studied 20 OSCC patients with TP53 on chromosome 17p13 and RPS6 on chromosome 9p21. Genomic DNA samples from the ITF, center/superficial and stromal cells adjacent to the tumor were prepared from cryosections using laser-assistant microdissection, then LOH and MI were determined. Cells at the ITF, center/superficiality and stroma showed a high frequency of LOH and MI on chromosomes 17p13 (TP53) and 9p21 (RPS6). Comparison of the patterns of allelic loss and MI encountered at the ITF, center/superficial and stromal cells revealed no concordance. The frequency of RPS6 and TP53 aberration at the epithelial compartment (both ITF and center, 64.7%, 11/17; 70.6%, 12/17) was statistically higher than the stroma (23.5%, 4/17; 43.8%, 7/16) (p < 0.05). Furthermore, for the epithelial compartment, the aberrations proportions of TP53 rose from 60.0% (9/15) to 64.7% (11/17) between the center/superficial part and ITF. Also the rate of RPS6 increased from 29.4% (5/17) to 58.8% (10/17) between
Loss of heterozygosity (LOH); Laser capture microdissection (LCM); The invasive tumor front; Oral; Squamous cell carcinoma; Microsatellite instability (MI)
q
Both the samples of blood and dissected tissue studied in this paper were obtained with the consent of all patients. * Corresponding author. Address: Key Laboratory for Oral biomedical Engineering of Ministry of Education, School and Hospital of Stomatology of Wuhan University, Wuhan, Hubei, China. Tel./fax: +86 27 87647443. E-mail address: [email protected] (M. Fan).
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.11.018
Intratumor genomic heterogeneity correlates with histological grade of advanced OSCC
741
the center/superficial parts and ITF. The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34) (p < 0.05). The current study revealed that intratumor genetic heterogeneity exists in the different histological areas of OSCCs and some particular tumor cell genotypes have correlation with histological patterns. c 2005 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinoma (OSCC) is a common tumor type worldwide. It is well recognized that patients with similar stages of oral cancer may have diverse clinical courses and response to similar treatment. The biological behavior of oral cancer is extremely variable. In the realm of pathology, the most striking features of oral cancer are its heterogeneity and the disparity between biological behavior and morphological classification.1 Heterogeneity has also been observed in the expression of tumor antigen.2–6 At the present, none of the established prognostic factors, solely or in combination, can corporate such various histological criteria.7 Considerable intratumor heterogeneity for genetic alterations has, however, been demonstrated in human prostate carcinoma,8,9 breast cancer,10 ovarian carcinoma11,12 and astrocytoma.13,14 In head and neck HNSCC, intratumor genomic heterogeneity depending on the localization and selection of matched pairs has been tested.15–17 EI-Naggar et al.18 also detected clonal heterogeneity between saliva and matching tumor. In OSCC, little consideration has been given to the individual genetic events occurring in the morphological subpopulations within a single tumor. In this study, areas with different grading of the same tumor were selected on the basis of the histological examination with hematoxylin and eosin of 10 lm frozen sections. Care are taken to the invasive tumor front (ITF) and center/superficial parts, for pathologists have observed for decades that tumor morphology in ITF of the OSCCs frequently shows a lower degree of differentiation and higher grade of dissociation than the center/superficial parts, the ITF represents is the high grade (HG) area and the center/ superficiality is considered as the low grade (LG) area.1,19 We acquired the cells at the (ITF) and the center/superficial part separately using laser capture microdissection. Many studies also showed that LOH in the stroma of patients with tumors is a common event.20–22 In this study, we also examined the LOH of stroma immediately adjacent to corresponding invasive OSCC.
age of 54.6 years (range from 31 to 74 years). Samples included the epithelial–stromal interfaces or the deep invasive margins. The dissected tissues were frozen in liquid nitrogen immediately after surgery and stored at 75 °C until extraction of DNA.
Laser capture microdissection and DNA extraction Serial 10 lm cryosections were stained with H&E to confirm the histopathological diagnosis. Tumor cells at the invasive tumor front (The most progressed 3–6 tumor cell layers or detached tumor cell groups.) at the advancing edge,23 tumor cells of the center and stroma immediately adjacent to the tumor (Fig. 1) were microdissected respectively using a laser microdissection protocol (PALM Company, Germany). Genomic DNA was extracted by proteinase K and salt extraction procedure using Genomic DNA Micro kit (Waston Biotechnologies, Inc., Shanghai, China). Peripherial blood DNA from each patient before the surgery were used as control samples.
Microsatellite analysis Using the PCR, we examined DNA extractions from the microdissected tissues with two polymorphic markers on chromosome 9P21 and 17P13. The makers were RPS6 for 9P21 at the locus of tumor suppressor genes P16 and TP53 for 17P13 at the locus of the P53 tumor suppressor gene, which are known as sites of early genetic changes in oral carcinogenesis. Primer sequences of the two markers were retrieved from the website: www.gdb.org and purchased from Sangon (Sangon Biological Engineering and Technology, Shanghai, China). Each PCR amplification was performed in a 25 ll volume containing standard PCR buffer, 2 lm dNTP, genomic DNA compatible to at least 200 cells, 10 pmol of each primer and 0.25 unite of taq DNA polymerase (Takara, Shuzo Co., Tokyo, Japan).
Materials and methods Patients and specimens Twenty Chinese patients with primary OSCC were treated at the School and Hospital of stomatology of Wuhan University, without any other form of treatment prior to radical surgery, were included in this study. Patients had a mean
Figure 1 Microdissection of cells in the center/superficial part (a), ITF (b) and stroma immediately adjacent to OSCC (c).
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The reactions were conducted for 40 cycles at 95 °C for 30 s, 54–59 °C for 60 s and 72 °C for 60 s followed by a 7 min extension at 72 °C in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, USA) in 500 ll plastic tubes. Amplified PCR products were subjected to electrophoresis in 7% polyacrylamide gels, stained with silver salts. LOH was scored in informative cases when a >50% or more reduction in the ratio of signals from tumor allele. MI was defined as appearance of one or more new allele in tumor DNA as compared with control counterparts. Results were reported as noninformative (NI) when visual inspection could not distinguish two distinct band forms in control DNA after PCR amplification. Each PCR was repeated at least twice, and the same results were obtained.
Statistical analysis Fisher exact test was used to determine the difference in frequencies. All tests were two sided, and p 6 0.05 was considered statistically significant.
Results Table 1 shows the pattern of LOH and MI in each sample. LOH and MI were frequent findings in the cells at the ITF, center/superficiality and stroma adjacent to OSCC. The LOH and MI frequency ranged from 23.5% (4/17) to 64.7% (11/17) of informative cases (Table 2). A comparison of LOH and MI patterns at the ITF and center and stromal cells revealed surprisingly that cells at the three regions showed different patterns of allelic loss for the two loci (Table 1 and Fig. 2). In 10 of 17 informative cases, a heterogenous pattern of LOH of TP53 was detected in the three regions of the same tumor, seven cases showed the same status. Six of 17 informative cases showed different patterns of LOH of TP53 between the ITF and the center/superficial part. Furthermore, in tumor case 11, the ITF lost the lower allele while the center/superficiality lost the upper allele. The result of RPS6 mutations indicated that nine of 18 informative cases showed different genetic alteration in three regions of the same tumor, seven of 18 informative cases showed different patterns between the ITF and center.
Table 1 Results of LOH and MI analysis in ITF, center/ superficial and stroma of 20 patients with OSCC Case no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
TP53
RPS6
I
C
S
I
C
S
} h NI } h NI h } h h s h NI h } } h h h }
} h NI } h NI h } } h h } NI NI h NI } h h }
} } NI h h NI h } } } h } NI } NI s s h } }
MI h } h h NI MI MI } } } NI MI } } h NI h } h
MI } h } } NI MI } } } } NI MI } } h NI } } }
} } h h } } NI MI } } } NI MI } } NI NI } } }
I: the invasive tumor front, C: center/superficial, S: stroma, NI: noninformative, MI: microsallite instability. }: no allelic loss. h: upper allelic loss, s: lower allelic loss.
Table 2
Figure 2 Representative results of allelic imbalance on chromosome 17P13 (TP53) (a) and 9P21 (RPS6) (b) in the ITF, center and stroma cells adjacent to OSCC. N: normal control, I: the invasive tumor front; C: center/superficial; S: the stroma adjacent to OSCC; white arrowheads: loss of either the upper or lower allelic; black arrows: MI, *: NI.
LOH frequency of informative cases in the ITF, center/superficial and stroma in patients
Marker
ITF
Center/superficial
Epithelium
Stroma
TP53 BPS6
11/17(64.7%) 10/17(58.8%)
9/15(60.0%) 5/17(29.4%)
12/17(70.6%) 11/17(64.7%)
7/16(43.8%) 4/17(23.5%)
Intratumor genomic heterogeneity correlates with histological grade of advanced OSCC The ratios of LOH and MI of the three regions were shown in Table 2. They predominated in the epithelial compartments (both ITF and center). LOH and MI overall rates of 70.6% (12/17) and 11/17 (64.7%) were observed on both TP53 and RPS6 in epithelium whereas only rate of 43.8% (7/16) and 23.5% (4/17) were observed on stroma, the difference was statistically significant (p < 0.05). For the epithelial compartment, the aberrations’ proportions of TP53 rose from 60.0% (9/15) to 64.7% (11/17) between the center/superficial part (LG) and ITF (HG). Also the rate of RPS6 increased from 29.4% (5/17) to 58.8% (10/17) between the center/superficial parts (LG) and ITF (HG). The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34) (p < 0.05).
Discussion Histopathological heterogeneity is one of the hallmarks of malignant tumors. The genetic analysis performed in this study not only confirmed the existence of genetic heterogeneity between OSCC and adjacent stroma, but also indicated the existence of intratumor genetic heterogeneity in advanced OSCC. We observed, in some cases studied, different allelic alteration among ITF, center/superficiality and stroma adjacent to OSCC or LOH and MI in one but not the other. In the present study, we showed genetic heterogeneities of both TP53 and RPS6 at the most invasive part (ITF) and center/superficiality of the same tumor. Using methods similar to those used in present study, studies demonstrated a heterogeneous distribution of special mutation in ovarian mucinous tumor,11,12 advanced colorectal adenocarcinoma,24 malignant astrocytomas,13,14 prostate carcinoma8,9 and so on. The intratumor heterogeneity can be explained that within the multiple process of carcinogenesis, clonal populations within the multiple process of carcinogenesis, clonal populations arising within tumors may undergo separate individual genetic changes leading to aggressive growth advantages.9 Differentiation grade is often used as a prognostic marker in OSCC. A correlation between genetic alteration and histological grade of OSCC has little been reported. We compared the genotypes with histological characteristics. We examined the differential genetic compositions of the histological high grade (HG) areas (ITF) and low grade (LG) areas (center/superficiality). The aberrations proportions of TP53 rose from 60.0% to 64.7% between the center/ superficial part (LG) and ITF (HG). Also the rate of RPS6 increased from 29.4% to 58.8% between the center/superficial parts and ITF. The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34). Cheng et al.13 also examined that the incidence of LOH of 9p21 and 10q23-25 were higher slightly in the high grade areas than the low grade areas of the de novo glioblastomas. In the highly invasive OSCC cases, methylation of OSCC cells was detected in the invasive areas, but not in the noninvasive areas.25 The immunohistochemical heterogeneity with respect to p16 and p53 expression has also been reported and both were up-regulated at the invasive front.26–28 The difference between the ITF
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(HG areas) and center/superficiality (LG areas) are tested immunohistochemically in other studies26,29–36 and these studies showed that the ITF consists of the most aggressive cells. Conversely, no correlations were found between tumor cell genotypes and histological patterns, as reported in prostate carcinoma9 and colorectal adenocarcinoma.24 Our observations indicate that some particular tumor cell genotypes have correlation with histological patterns. The higher the grade, the more the genetic alterations as to the two markers studied in this study. We also studied the LOH and MI in the stromal cells adjacent to corresponding tumors. We found that a significant percentage of the stromal cells harbored genetic changes commonly encountered in epithelial parts (both ITF and center/superficiality) of the same tumor. In this present study, of note, both markers demonstrated a statistically higher frequency of LOH and MI in the neoplastic epithelial compartment (70.6% for TP53 and 64.7% for RPS6) compared with the stromal cells adjacent to OSCC (43.8% for TP53 and 23.5% for RPS6). It is similar to the result reported by Kurose et al.22 So the assumption is that the lower frequencies of stromal alterations of TP53 and RPS6 could reflect that alterations of the two markers in the stroma are not dominant and only exert and indirect effect on the adjacent epithelium, or only exert an effect in collaboration with others, to influence the overall process of tumorigenesis.22 On the other hand, we did not encounter a clear concordance between the LOH and MI pattern encountered in the tumor and its adjacent stroma suggesting that the genetic changes taking place in the epithelium and stroma may be occurring independently. Our results is similar to Paterson et al.20 who detected that some of the genetic changes that commonly occur with invasive urothelial carcinoma were frequently found in the adjacent stroma. And they did not found a clear concordance between the LOH pattern encountered in the tumor and its adjacent stroma too. Moinfar et al.21 suggested that several genetic alterations in the stromal cells may precede genotypic changes in the epithelial cells. On the other hand, Kurose et al.22 extrapolated a genetic model of multi-step carcinogenesis for the breast involving the epithelial and stromal compartment and note that genetic alterations occur in the epithelial compartments as the earlier steps followed by LOH in the stromal compartment. Understanding the role of epithelial–stromal interaction in oral carcinogenesis could provide alternate therapeutic approaches to the regulation of cancer growth.21
Acknowledgments We thank Yuan Li and Shichun Xiong for excellent technique assistance.
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744 2. Kerns BJ, Jordan PA, Fareman LL, Berchuck A, Bast Jr RC, Layfield LJ. Determination of proliferation index with MIB-1 in advanced ovatrian cancer using quantitative imagine analysis. Am J Clin Pathol 1994;101(2):192–7. 3. ter Harmsel B, Muden RV. The significance of cell type and tumour growth markers in the prognosis of unscreened cervical cancer patients. Int J Gynecol Cancer 1998;8:336–44. 4. Gavert N, Conacci-Sorrell M, Gast D, et al. A novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers. J Cell Biol 2005;168(4): 633–42. 5. de Vicente JC, Fresno MF, Villalain L, Vega JA, Lopez Arranz JS. Immunoexpression and prognostic significance of TIMP-1 and -2 in oral squamous cell carcinoma. Oral Oncol 2005;41(6): 568–79. 6. Sigalotti L, Fratta E, Coral S, et al. Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5aza-20 -deoxycytidine. Cancer Res 2004;64(24):9167–71. 7. Anneroth G, Batsakis J, Luna M. Review of the literature and a recommended system of malignancy grading in oral squamous cell carcinomas. Scand J Dent Res 1987;95(3):229–49. 8. Alers JC, Krijtenburg PJ, Vissers CJ, Bosman FT, van der Kwast H, van Dekken H. Cytogenetic heterogeneity and histologic tumor growth patterns in prostatic cancer. Cytometry 1995;21(1):84–94. 9. Konishi N, Hiasa Y, Matsuda H, et al. Intratumor cellular heterogeneity and alterations in ras oncogene and p53 tumor suppressor gene in human prostate carcinoma. Am J Pathol 1995;147(4):1112–22. 10. Wild P, Knuechel R, Dietmaier W, Hofstaedter F, Hartmann A. Laser microdissection and microsatellite analyses of breast cancer reveal a high degree of tumor heterogeneity. Pathobiology 2000;68(4–5):180–90. 11. Takeshima Y, Amatya VJ, Daimaru Y, Nakayori F, Nakano T, Inai K. Heterogeneous genetic alterations in ovarian mucinous tumors: application and usefulness of laser capture microdissection. Hum Pathol 2001;32(11):1203–8. 12. Lee JH, Kavanagh JJ, Wildrick DM, Wharton JT, Blick M. Frequent loss of heterozygosity on chromosomes 6q, 11, and 17 in human ovarian carcinomas. Cancer Res 1990;50(9):2724–8. 13. Cheng Y, Ng HK, Ding M, Zhang SF, Pang JC, Lo KW. Molecular analysis of microdissected de novo glioblastomas and paired astrocytic tumors. J Neuropathol Exp Neurol 1999;58(2): 120–8. 14. Shuangshoti S, Navalitloha Y, Kasantikul V, Shuangshoti S, Mutirangura A. Genetic heterogeneity and progression in different areas within high-grade diffuse astrocytoma. Oncol Rep 2000;7(1):113–7. 15. Tremmel SC, Gotte K, Popp S, et al. Intratumoral genomic heterogeneity in advanced head and neck cancer detected by comparative genomic hybridization. Cancer Genet Cytogenet 2003;144(2):165–74. 16. Tabor MP, Brakenhoff RH, van Houten VM, et al. Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications. Clin Cancer Res 2001;7(6):1523–32. 17. Gotte K, Tremmel SC, Popp S, et al. Intratumoral genomic heterogeneity in advanced head and neck cancer detected by comparative genomic hybridization. Adv Otorhinolaryngol 2005;62:38–48. 18. El-Naggar AK, Mao L, Staerkel G, et al. Genetic heterogeneity in saliva from patients with oral squamous carcinomas: implications in molecular diagnosis and screening. J Mol Diagn 2001;3(4):164–70. 19. Bryne M, Boysen M, Alfsen CG, et al. The invasive front of carcinomas. The most important area for tumour prognosis? Anticancer Res 1998;18(6b):4757–64.
X. Wang et al. 20. Paterson RF, Ulbright TM, MacLennan GT, et al. Molecular genetic alterations in the laser-capture-microdissected stroma adjacent to bladder carcinoma. Cancer 2003;98(9):1830–6. 21. Moinfar F, Man YG, Arnould LC, Bratthauer GL, Ratschek M, Tavassoli FA. Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis. Cancer Res 2000;60(9):2562–6. 22. Kurose K, Hoshaw-Woodard S, Adeyinka A, Lemeshow S, Watson C, Eng C. Genetic model of multi-step breast carcinogenesis involving the epithelium and stroma: clues to tumour-microenvironment interactions. Hum Mol Genet 2001;10(18): 1907–13. 23. Piffko J, Bankfalvi A, Ofner D, Rasch D, Joos U, Schmid KW. Standardized demonstration of silver-stained nucleolar organizer regions-associated proteins in archival oral squamous cell carcinomas and adjacent non-neoplastic mucosa. Mod Pathol 1997;10(2):98–104. 24. Baisse B, Bouzourene H, Saraga EP, Bosman FT, Benhattar J. Intratumor genetic heterogeneity in advanced human colorectal adenocarcinoma. Int J Cancer 2001;93(3):346–52. 25. Kudo Y, Kitajima S, Ogawa I, et al. Invasion and metastasis of oral cancer cells require methylation of E-cadherin and/or degradation of membranous beta-catenin. Clin Cancer Res 2004;10(16):5455–63. 26. Piffko J, Bankfalvi A, Tory K, et al. Molecular assessment of p53 abnormalities at the invasive front of oral squamous cell carcinomas. Head Neck 1998;20(1):8–15. 27. Volgareva G, Zavalishina L, Andreeva Y, et al. Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells. BMC Cancer 2004;4:58–68. 28. Svensson S, Nilsson K, Ringberg A, Landberg G. Invade or proliferate? Two contrasting events in malignant behavior governed by p16(INK4a) and an intact Rb pathway illustrated by a model system of basal cell carcinoma. Cancer Res 2003;63(8):1737–42. 29. Piffko J, Bankfalvi A, Ofner D, et al. Prognostic value of histological factors (malignancy grading and AgNOR content) assessed at the invasive tumour front of oral squamous cell carcinoma. Br J Cancer 1997;75(10):1543–6. 30. Noguchi M, Kinjyo H, Kohama GI, Nakamori K. Invasive front in oral squamous cell carcinoma: image and flow cytometric analysis with clinicopathologic correlation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93(6):682–7. 31. Graflund M, Sorbe B, Bryne M, Karlsson M. The prognostic value of a histologic grading system, DNA profile, and MIB-1 expression in early stages of cervical squamous cell carcinomas. Int J Gynecol Cancer 2002;12(2):149–57. 32. Shiina H, Igawa M, Urakami S, Honda S, Shirakawa H, Ishibe T. Immunohistochemical analysis of bcl-2 expression in transitional cell carcinoma of the bladder. J Clin Pathol 1996;49(5):395–9. 33. Bankfalvi A, Krassort M, Vegh A, Felszeghy E, Piffko J. Expression of the E-cadherin/beta-catenin complex and the epidermal growth factor receptor in the clinical evolution and progression of oral squamous cell carcinomas. J Oral Pathol Med 2002;31(8):450–7. 34. Bankfalvi A, Krassort M, Buchwalow IB, Vegh A, Felszeghy E, Piffko J. Gains and losses of adhesion molecules (CD44, Ecadherin, and beta-catenin) during oral carcinogenesis and tumour progression. J Pathol 2002;198(3):343–51. 35. Ondruschka C, Buhtz P, Motsch C, et al. Prognostic value of MMP-2, -9 and TIMP-1,-2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas. Pathol Res Pract 2002;198(8):509–15. 36. Roesch A, Vogt T, Stolz W, Dugas M, Landthaler M, Becker B. Discrimination between gene expression patterns in the invasive margin and the tumour core of malignant. Melanomas Melanoma Res 2003;13(5):503–9.
available at www.sciencedirect.com
journal homepage: http://intl.elsevierhealth.com/journals/oron/
Intratumor genomic heterogeneity correlates with histological grade of advanced oral squamous cell carcinoma q Xinhong Wang a,b,c, Mingwen Fan a,c,*, Xinming Chen b,c, Shuozhi Wang b, Mohd Jamal Alsharif d, Li Wang b,c, Lijiang Liu e, Hao Deng e a
Department of Endodontics, School and Hospital of Stomatology of Wuhan University, China Department of Pathology, School and Hospital of Stomatology of Wuhan University, China c Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology of Wuhan University, Wuhan, Hubei, China d Department of Surgery, School and Hospital of Stomatology of Wuhan University, China e Department of Pathology and Pathophysiology, Medical and Life Science College, Jianghan University, China b
Received 26 October 2005; accepted 24 November 2005
KEYWORDS
Summary To assess the difference in genetic aberration patterns among the invasive tumor front (ITF), center/superficiality and the stroma adjacent to oral squamous cell carcinoma (OSCC), we studied loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosome 9p21 and 17p13 on the three regions by combining laser capture microdissection (LCM) and PCR. We studied 20 OSCC patients with TP53 on chromosome 17p13 and RPS6 on chromosome 9p21. Genomic DNA samples from the ITF, center/superficial and stromal cells adjacent to the tumor were prepared from cryosections using laser-assistant microdissection, then LOH and MI were determined. Cells at the ITF, center/superficiality and stroma showed a high frequency of LOH and MI on chromosomes 17p13 (TP53) and 9p21 (RPS6). Comparison of the patterns of allelic loss and MI encountered at the ITF, center/superficial and stromal cells revealed no concordance. The frequency of RPS6 and TP53 aberration at the epithelial compartment (both ITF and center, 64.7%, 11/17; 70.6%, 12/17) was statistically higher than the stroma (23.5%, 4/17; 43.8%, 7/16) (p < 0.05). Furthermore, for the epithelial compartment, the aberrations proportions of TP53 rose from 60.0% (9/15) to 64.7% (11/17) between the center/superficial part and ITF. Also the rate of RPS6 increased from 29.4% (5/17) to 58.8% (10/17) between
Loss of heterozygosity (LOH); Laser capture microdissection (LCM); The invasive tumor front; Oral; Squamous cell carcinoma; Microsatellite instability (MI)
q
Both the samples of blood and dissected tissue studied in this paper were obtained with the consent of all patients. * Corresponding author. Address: Key Laboratory for Oral biomedical Engineering of Ministry of Education, School and Hospital of Stomatology of Wuhan University, Wuhan, Hubei, China. Tel./fax: +86 27 87647443. E-mail address: [email protected] (M. Fan).
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.11.018
Intratumor genomic heterogeneity correlates with histological grade of advanced OSCC
741
the center/superficial parts and ITF. The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34) (p < 0.05). The current study revealed that intratumor genetic heterogeneity exists in the different histological areas of OSCCs and some particular tumor cell genotypes have correlation with histological patterns. c 2005 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinoma (OSCC) is a common tumor type worldwide. It is well recognized that patients with similar stages of oral cancer may have diverse clinical courses and response to similar treatment. The biological behavior of oral cancer is extremely variable. In the realm of pathology, the most striking features of oral cancer are its heterogeneity and the disparity between biological behavior and morphological classification.1 Heterogeneity has also been observed in the expression of tumor antigen.2–6 At the present, none of the established prognostic factors, solely or in combination, can corporate such various histological criteria.7 Considerable intratumor heterogeneity for genetic alterations has, however, been demonstrated in human prostate carcinoma,8,9 breast cancer,10 ovarian carcinoma11,12 and astrocytoma.13,14 In head and neck HNSCC, intratumor genomic heterogeneity depending on the localization and selection of matched pairs has been tested.15–17 EI-Naggar et al.18 also detected clonal heterogeneity between saliva and matching tumor. In OSCC, little consideration has been given to the individual genetic events occurring in the morphological subpopulations within a single tumor. In this study, areas with different grading of the same tumor were selected on the basis of the histological examination with hematoxylin and eosin of 10 lm frozen sections. Care are taken to the invasive tumor front (ITF) and center/superficial parts, for pathologists have observed for decades that tumor morphology in ITF of the OSCCs frequently shows a lower degree of differentiation and higher grade of dissociation than the center/superficial parts, the ITF represents is the high grade (HG) area and the center/ superficiality is considered as the low grade (LG) area.1,19 We acquired the cells at the (ITF) and the center/superficial part separately using laser capture microdissection. Many studies also showed that LOH in the stroma of patients with tumors is a common event.20–22 In this study, we also examined the LOH of stroma immediately adjacent to corresponding invasive OSCC.
age of 54.6 years (range from 31 to 74 years). Samples included the epithelial–stromal interfaces or the deep invasive margins. The dissected tissues were frozen in liquid nitrogen immediately after surgery and stored at 75 °C until extraction of DNA.
Laser capture microdissection and DNA extraction Serial 10 lm cryosections were stained with H&E to confirm the histopathological diagnosis. Tumor cells at the invasive tumor front (The most progressed 3–6 tumor cell layers or detached tumor cell groups.) at the advancing edge,23 tumor cells of the center and stroma immediately adjacent to the tumor (Fig. 1) were microdissected respectively using a laser microdissection protocol (PALM Company, Germany). Genomic DNA was extracted by proteinase K and salt extraction procedure using Genomic DNA Micro kit (Waston Biotechnologies, Inc., Shanghai, China). Peripherial blood DNA from each patient before the surgery were used as control samples.
Microsatellite analysis Using the PCR, we examined DNA extractions from the microdissected tissues with two polymorphic markers on chromosome 9P21 and 17P13. The makers were RPS6 for 9P21 at the locus of tumor suppressor genes P16 and TP53 for 17P13 at the locus of the P53 tumor suppressor gene, which are known as sites of early genetic changes in oral carcinogenesis. Primer sequences of the two markers were retrieved from the website: www.gdb.org and purchased from Sangon (Sangon Biological Engineering and Technology, Shanghai, China). Each PCR amplification was performed in a 25 ll volume containing standard PCR buffer, 2 lm dNTP, genomic DNA compatible to at least 200 cells, 10 pmol of each primer and 0.25 unite of taq DNA polymerase (Takara, Shuzo Co., Tokyo, Japan).
Materials and methods Patients and specimens Twenty Chinese patients with primary OSCC were treated at the School and Hospital of stomatology of Wuhan University, without any other form of treatment prior to radical surgery, were included in this study. Patients had a mean
Figure 1 Microdissection of cells in the center/superficial part (a), ITF (b) and stroma immediately adjacent to OSCC (c).
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X. Wang et al.
The reactions were conducted for 40 cycles at 95 °C for 30 s, 54–59 °C for 60 s and 72 °C for 60 s followed by a 7 min extension at 72 °C in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, USA) in 500 ll plastic tubes. Amplified PCR products were subjected to electrophoresis in 7% polyacrylamide gels, stained with silver salts. LOH was scored in informative cases when a >50% or more reduction in the ratio of signals from tumor allele. MI was defined as appearance of one or more new allele in tumor DNA as compared with control counterparts. Results were reported as noninformative (NI) when visual inspection could not distinguish two distinct band forms in control DNA after PCR amplification. Each PCR was repeated at least twice, and the same results were obtained.
Statistical analysis Fisher exact test was used to determine the difference in frequencies. All tests were two sided, and p 6 0.05 was considered statistically significant.
Results Table 1 shows the pattern of LOH and MI in each sample. LOH and MI were frequent findings in the cells at the ITF, center/superficiality and stroma adjacent to OSCC. The LOH and MI frequency ranged from 23.5% (4/17) to 64.7% (11/17) of informative cases (Table 2). A comparison of LOH and MI patterns at the ITF and center and stromal cells revealed surprisingly that cells at the three regions showed different patterns of allelic loss for the two loci (Table 1 and Fig. 2). In 10 of 17 informative cases, a heterogenous pattern of LOH of TP53 was detected in the three regions of the same tumor, seven cases showed the same status. Six of 17 informative cases showed different patterns of LOH of TP53 between the ITF and the center/superficial part. Furthermore, in tumor case 11, the ITF lost the lower allele while the center/superficiality lost the upper allele. The result of RPS6 mutations indicated that nine of 18 informative cases showed different genetic alteration in three regions of the same tumor, seven of 18 informative cases showed different patterns between the ITF and center.
Table 1 Results of LOH and MI analysis in ITF, center/ superficial and stroma of 20 patients with OSCC Case no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
TP53
RPS6
I
C
S
I
C
S
} h NI } h NI h } h h s h NI h } } h h h }
} h NI } h NI h } } h h } NI NI h NI } h h }
} } NI h h NI h } } } h } NI } NI s s h } }
MI h } h h NI MI MI } } } NI MI } } h NI h } h
MI } h } } NI MI } } } } NI MI } } h NI } } }
} } h h } } NI MI } } } NI MI } } NI NI } } }
I: the invasive tumor front, C: center/superficial, S: stroma, NI: noninformative, MI: microsallite instability. }: no allelic loss. h: upper allelic loss, s: lower allelic loss.
Table 2
Figure 2 Representative results of allelic imbalance on chromosome 17P13 (TP53) (a) and 9P21 (RPS6) (b) in the ITF, center and stroma cells adjacent to OSCC. N: normal control, I: the invasive tumor front; C: center/superficial; S: the stroma adjacent to OSCC; white arrowheads: loss of either the upper or lower allelic; black arrows: MI, *: NI.
LOH frequency of informative cases in the ITF, center/superficial and stroma in patients
Marker
ITF
Center/superficial
Epithelium
Stroma
TP53 BPS6
11/17(64.7%) 10/17(58.8%)
9/15(60.0%) 5/17(29.4%)
12/17(70.6%) 11/17(64.7%)
7/16(43.8%) 4/17(23.5%)
Intratumor genomic heterogeneity correlates with histological grade of advanced OSCC The ratios of LOH and MI of the three regions were shown in Table 2. They predominated in the epithelial compartments (both ITF and center). LOH and MI overall rates of 70.6% (12/17) and 11/17 (64.7%) were observed on both TP53 and RPS6 in epithelium whereas only rate of 43.8% (7/16) and 23.5% (4/17) were observed on stroma, the difference was statistically significant (p < 0.05). For the epithelial compartment, the aberrations’ proportions of TP53 rose from 60.0% (9/15) to 64.7% (11/17) between the center/superficial part (LG) and ITF (HG). Also the rate of RPS6 increased from 29.4% (5/17) to 58.8% (10/17) between the center/superficial parts (LG) and ITF (HG). The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34) (p < 0.05).
Discussion Histopathological heterogeneity is one of the hallmarks of malignant tumors. The genetic analysis performed in this study not only confirmed the existence of genetic heterogeneity between OSCC and adjacent stroma, but also indicated the existence of intratumor genetic heterogeneity in advanced OSCC. We observed, in some cases studied, different allelic alteration among ITF, center/superficiality and stroma adjacent to OSCC or LOH and MI in one but not the other. In the present study, we showed genetic heterogeneities of both TP53 and RPS6 at the most invasive part (ITF) and center/superficiality of the same tumor. Using methods similar to those used in present study, studies demonstrated a heterogeneous distribution of special mutation in ovarian mucinous tumor,11,12 advanced colorectal adenocarcinoma,24 malignant astrocytomas,13,14 prostate carcinoma8,9 and so on. The intratumor heterogeneity can be explained that within the multiple process of carcinogenesis, clonal populations within the multiple process of carcinogenesis, clonal populations arising within tumors may undergo separate individual genetic changes leading to aggressive growth advantages.9 Differentiation grade is often used as a prognostic marker in OSCC. A correlation between genetic alteration and histological grade of OSCC has little been reported. We compared the genotypes with histological characteristics. We examined the differential genetic compositions of the histological high grade (HG) areas (ITF) and low grade (LG) areas (center/superficiality). The aberrations proportions of TP53 rose from 60.0% to 64.7% between the center/ superficial part (LG) and ITF (HG). Also the rate of RPS6 increased from 29.4% to 58.8% between the center/superficial parts and ITF. The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34). Cheng et al.13 also examined that the incidence of LOH of 9p21 and 10q23-25 were higher slightly in the high grade areas than the low grade areas of the de novo glioblastomas. In the highly invasive OSCC cases, methylation of OSCC cells was detected in the invasive areas, but not in the noninvasive areas.25 The immunohistochemical heterogeneity with respect to p16 and p53 expression has also been reported and both were up-regulated at the invasive front.26–28 The difference between the ITF
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(HG areas) and center/superficiality (LG areas) are tested immunohistochemically in other studies26,29–36 and these studies showed that the ITF consists of the most aggressive cells. Conversely, no correlations were found between tumor cell genotypes and histological patterns, as reported in prostate carcinoma9 and colorectal adenocarcinoma.24 Our observations indicate that some particular tumor cell genotypes have correlation with histological patterns. The higher the grade, the more the genetic alterations as to the two markers studied in this study. We also studied the LOH and MI in the stromal cells adjacent to corresponding tumors. We found that a significant percentage of the stromal cells harbored genetic changes commonly encountered in epithelial parts (both ITF and center/superficiality) of the same tumor. In this present study, of note, both markers demonstrated a statistically higher frequency of LOH and MI in the neoplastic epithelial compartment (70.6% for TP53 and 64.7% for RPS6) compared with the stromal cells adjacent to OSCC (43.8% for TP53 and 23.5% for RPS6). It is similar to the result reported by Kurose et al.22 So the assumption is that the lower frequencies of stromal alterations of TP53 and RPS6 could reflect that alterations of the two markers in the stroma are not dominant and only exert and indirect effect on the adjacent epithelium, or only exert an effect in collaboration with others, to influence the overall process of tumorigenesis.22 On the other hand, we did not encounter a clear concordance between the LOH and MI pattern encountered in the tumor and its adjacent stroma suggesting that the genetic changes taking place in the epithelium and stroma may be occurring independently. Our results is similar to Paterson et al.20 who detected that some of the genetic changes that commonly occur with invasive urothelial carcinoma were frequently found in the adjacent stroma. And they did not found a clear concordance between the LOH pattern encountered in the tumor and its adjacent stroma too. Moinfar et al.21 suggested that several genetic alterations in the stromal cells may precede genotypic changes in the epithelial cells. On the other hand, Kurose et al.22 extrapolated a genetic model of multi-step carcinogenesis for the breast involving the epithelial and stromal compartment and note that genetic alterations occur in the epithelial compartments as the earlier steps followed by LOH in the stromal compartment. Understanding the role of epithelial–stromal interaction in oral carcinogenesis could provide alternate therapeutic approaches to the regulation of cancer growth.21
Acknowledgments We thank Yuan Li and Shichun Xiong for excellent technique assistance.
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