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Relationship of human papillomavirus to ploidy in squamous cell carcinomas of the head and neck
Relationship of human papillomavirus to ploidy in squamous cell carcinomas of the head and neck JUAN PABLO RODRIGO, MD, IGNACIO ALVAREZ, MD, JOSÉ ANTONIO MARTÍNEZ, MD, PEDRO SÁNCHEZ LAZO, PhD, SOFÍA RAMOS, PhD, and CARLOS SUÁREZ, MD, León and Oviedo, Spain
To establish the relationship between the presence of human papillomavirus (HPV) gene sequences and the development of genetic abnormalities, 31 squamous cell carcinomas of the head and neck were studied for the presence of HPV types 6b and 16 and the DNA content by flow cytometry. Eighteen (58%) cases were aneuploid. HPV DNA was present in seven (22.5%) tumors. Five of them were positive for the HPV type 6b and two for the HPV type 16. Aneuploidy was correlated with poorly differentiated tumors. No correlation was found between the presence of HPV, DNA content, or tumor differentiation. Consequently, the presence of HPV gene sequences does not seem to be related to a higher incidence of genetic abnormalities in squamous cell carcinomas of the head and neck. (Otolaryngol Head Neck Surg 1999;121:31822.)
T
he association of human papillomavirus (HPV) with dysplasia and carcinoma of the uterine cervix is well known.1-3 In the past few years, a similar relationship has been established between HPV and carcinoma of the upper aerodigestive tract.4-14 There are at least 60 genotypes of HPV, of which HPV types 6 and 11 are associated customarily with laryngeal papillomas, whereas types 6b, 16, and 18 have been related to neoplasias of the same location.4-14 The HPV genome contains eight genes, two of which (E6 and E7 genes) are very similar to genotypes From the Department of Otolaryngology, Hospital de León (Drs. Rodrigo and Alvarez); the Department of Otolaryngology, Hospital Central de Asturias, Oviedo (Drs. Martinez and Suárez); and the Department of Molecular Biology, Universidad de Oviedo (Drs. Lazo and Ramos). Presented at the Annual Meeting of the American Academy of Otolaryngology–Head Neck Surgery, San Francisco, Calif., Sept. 7-10, 1997. Supported by a grant from the Fondo de Investigaciones Sanitarias, Madrid, Spain (grant no. 93/0400). Reprint requests: Juan Pablo Rodrigo, MD, c/o Fernández Ladreda, 32-A 4°B, 33011 Oviedo, Spain. Copyright © 1999 by the American Academy of Otolaryngology– Head and Neck Surgery Foundation, Inc. 0194-5998/99/$8.00 + 0 23/77/90004 318
16 and 18, which are associated with cervical carcinomas.15,16 The cells transfected with the E6 and E7 genes become immortalized, do not differentiate normally, and grow more rapidly than nontransfected cells.17 Furthermore, E6- and E7-transfected cells may become aneuploid.18 HPV E6 and E7 gene products can produce cellular transformation probably because of their capacity to be joined to the products of two other genes that participate in the regulation of cellular proliferation. The HPV E6 gene product binds to the protein codified by the p53 gene, with the result of a more rapid degradation of the same.19 The protein codified by the HPV E7 gene binds to the product of the retinoblastoma gene (Rb), the function of which is necessary for the control of the cellular growth.18 In this way, it is probable that the antiproliferative effects of these two tumor-suppressor genes will be abolished after union to the HPV E6 and E7 gene products. As a result, the cells that contain these genes present an abnormal cellcycle regulation and therefore are subject to genetic mutations. To study the correlation between HPV infection and the presence of alterations in DNA content, we analyzed the integration of the HPV genome by polymerase chain reaction (PCR) and the cellular DNA content by flow cytometry in 31 squamous cell carcinomas of the head and neck. METHODS AND MATERIAL Patients Tumor samples of 31 patients with squamous cell carcinoma of head and neck were obtained from patients undergoing surgical resection of their tumors. Tissue specimens were freshly frozen in liquid nitrogen in the operating room and stored at –70° C until being processed. All included patients had a single primary tumor, and none had undergone treatment before surgery. All but one patient were male, with a mean age of 58 years and a range of 38 to 78 years. The distribution by locations is shown in Table 1, and the distribution by stage, according to the Union Internationale Contre le Cancer (UICC) classification (4th issue), is shown in Table 2. Of 31 cases, 16 (52%) were well differentiated, 10 (32%) were moderately differentiated, and 5 (16%) poorly differentiated.
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Table 1. Distribution of studied cases by site Site
After being mechanically disintegrated in phosphatebuffered saline solution, the samples were filtered by a nylon mesh of 100 µm and thereafter centrifuged. The obtained sediment was digested with trypsin for 10 minutes, filtered by a nylon mesh of 50 µm, and stained with propidium iodide, according to the method of Vindelov et al.20 Samples were analyzed in a flow cytometer (BectonDickinson). As controls of normal DNA content (diploid), human normal lymphocytes were used. The DNA content of the tumor samples was established by comparison with that of the normal controls by use of the DNA index. The DNA index is the ratio between the G0/G1 peaks of the tumor and control DNA histograms (the G0/G1 peak represents the cells that are not synthesizing DNA). By definition the DNA index of the normal controls is 1. The samples were considered to be diploid if the DNA index was found to be 1.0 ± 0.05. If the DNA index was greater than 1.05, we considered it to be hyperdiploid, and if the DNA index was less than 0.95, we considered it to be hypodiploid. The debris and cellular aggregates that could not be filtered were eliminated instrumentally. Detection of HPV-6b and HPV-16 DNA The tissues were minced, homogenized, and digested twice with 100 µg proteinase K (Boehringer Mannheim) per milliliter of digestive buffer (200 mmol/L Tris-HCl [pH 8.0], 25 mmol/L EDTA, 100 mmol/L NaCl, and 0.2% sodium dodecylsulfate) for 8 hours at 37° C. High-molecular-weight DNA was isolated by standard phenol-chloroform extraction, precipitated in ethanol, dissolved in 10 mmol/L Tris and 1 mmol/L EDTA (pH 8.0), and stored at –20° C. DNA concentration was measured by absorbance at 260 nm. After DNA extraction, amplification of HPV-6b and HPV16 E6 and L1 genes was accomplished through PCR, with specific primers for each gene (Table 3). The L1 gene codifies one of the proteins of the viral capsid and is found only in the cells synthesizing viral particles, indicating active infection by the HPV. As positive control, we used the viral DNA cloned in the bacterial plasmid pBR322. The negative control in each case consisted of reaction mixtures of those in which the DNA was omitted. The PCR mixtures contained 0.5 µg of target DNA, 5 µl of ×10 buffer (100 mmol/L Tris-HCl [pH 8.3], 15 mmol/L Cl2Mg, 500 mmol/L ClK, 0.05% Tween 20, 0.05% NP-40, and 0.01% gelatin), 0.2 mmol/L of each dNTP, 1 U of Taq polymerase (Boehringer Mannheim), and 1 µmol/L of each primer in 50 µl total volume, with 50 µl mineral oil overlay. The PCR cycles included 1 minute at each temperature (94° C, 55° C, and 72° C), for a total of 35 cycles, and a final cycle of 72° C for 7 minutes. The PCR products were electrophoresed on 2% agarose gel and visualized with ethidium bromide staining.
319
No. of cases (%)
Oral cavity Oropharynx Glottis Supraglottis Hypopharynx
1 (3%) 12 (39%) 5 (16%) 4 (13%) 9 (29%)
Table 2. Distribution by stage of the studied cases Stage
No. of cases (%)
T stage T1 T2 T3 T4 N stage N0 N1 N2 N3 TNM stage I II III IV
1 (3%) 8 (26%) 10 (32%) 12 (39%) 11 (36%) 6 (19%) 10 (32%) 4 (13%) 1 (3%) 3 (10%) 10 (32%) 17 (55%)
Table 3. Primers used in the PCR Gene
HPV-16/E6 HPV-16/L1 HPV-6b/E6 HPV-6b/L1
Primers
5´-TCAAAAGCCACTGTGTCCTG-3´ 5´-CGTGTTCTTGATGATCTGCA-3´ 5´-CAGGGCCACAATAATGGCAT-3´ 5´-TGCGTCCTAAAGGAAACTGA-3´ 5´-TAAAGGTCCTGTTTCGAGGC-3´ 5´-CGGTTTGTGACACAGGTAGC-3´ 5´-TCTGCTGAAGTAATGGCCTA-3´ 5´-CGTCCCAAAGGATACTGATC-3´
Size (base pairs)
120 451 179 248
Statistic Analysis Statistical analysis was performed by use of χ2 tests with Yates’ correction when appropriate. RESULTS
The clinicopathologic characteristics of the studied patients are shown in Table 4. The HPV E6 gene was detected in seven (22.5%) cases. The HPV L1 gene was not detected in any of these cases. This implies that in all cases the E6 gene was integrated in the genomes of the tumor cells. Five carcinomas contained the HPV-6b DNA, and two the HPV-16 DNA. Figure 1 shows the PCR products in the positive cases.
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Table 4. Clinicopathologic characteristics of the studied cases Case no.
Age (yr)
Tumor site
T stage
N stage
Differentiation
Ploidy
HPV
68 68 43 78 63 46 52 60 70 68 44 67 63 47 53 53 45 71 65 63 56 38 61 63 51 64 59 62 57 53 57
Oropharynx Glottis Hypopharynx Hypopharynx Oropharynx Oropharynx Hypopharynx Hypopharynx Oropharynx Hypopharynx Oropharynx Oropharynx Oropharynx Hypopharynx Hypopharynx Oropharynx Glottis Supraglottis Supraglottis Oropharynx Glottis Oral cavity Supraglottis Oropharynx Oropharynx Supraglottis Glottis Hypopharynx Oropharynx Oropharynx Glottis
4 4 4 4 4 3 2 2 2 4 3 3 3 3 2 3 1 2 2 4 2 4 2 4 4 3 3 3 4 4 3
3 0 2 2 2 2 0 0 2 0 1 1 0 3 1 2 0 3 1 3 0 2 1 2 0 1 0 0 2 2 0
Moderately Poorly Poorly Poorly Moderately Moderately Poorly Well Well Well Moderately Moderately Well Well Well Well Well Moderately Well Well Well Well Moderately Moderately Poorly Well Well Moderately Moderately Well Well
A D A A A A A A A A A A D A D A A D D D D D A A A D D D A D D
N N N N N N N P (6b) N N N P (16) P (16) N N N N N P (6b) N N N N N N N P (6b) P (6b) P (6b) N N
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A, Aneuploid; D, diploid; N, negative; P, positive (type in parentheses).
Table 5. Relationship between histologic grade and ploidy and HPV presence Ploidy
*χ2 †χ2
HPV presence
Differentiation
No. of cases
Diploid
Aneuploid
p Value*
Positive
Well Moderately Poorly
16 10 5
10 (62%) 2 (20%) 1 (20%)
6 (38%) 8 (80%) 4 (80%)
0.04
4 (25%) 3 (33%) 0
Negative
12 (75%) 7 (67%) 5 (100%)
p Value†
0.4
test. test (well-differentiated front to moderately and poorly differentiated cases).
In 13 (42%) cases the tumor had a diploid DNA content, and in 18 (58%) it had an aneuploid DNA content. All the aneuploid cases were hyperdiploid. Of 7 HPVpositive cases, 4 (57%) were diploid and 3 (43%) aneuploid, whereas 9 (37.5%) of the 24 cases that did not show integration of the HPV were diploid and 15 (62.5%) aneuploid. These differences did not reach statistical significance (p = 0.62; Fig. 2). We noted that the well-differentiated carcinomas tended to present a diploid content of DNA, whereas the
moderately or poorly differentiated carcinomas tended to present an aneuploid content. Thus 62% of the welldifferentiated carcinomas were diploid, compared with 20% of the moderate and poorly differentiated; these differences reached statistical significance (p = 0.04; Table 5). On the other hand, there were no statistically significant differences between the histologic differentiation and the presence of the HPV, although none of the positive cases for HPV were poorly differentiated (Table 5).
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Fig 1. Electrophoresis of PCR products from the five cases that were positive to HPV-6b and the two cases that were positive to HPV-16. C+, Positive controls. C–, Negative controls. The size of the fragments, in base pairs (bp), is shown on the left.
Fig 2. Distribution of the studied cases by cellular DNA content and HPV presence.
DISCUSSION
HPV has been found in 10% to 80% of squamous cell carcinomas of the head and neck,4-14 although in the series with greater numbers of patients, the percentage of positive cases is found to be about 20%.6,13 In this work we find a 22.5% integration frequency of the E6 gene for HPV-16 and HPV-6b. On the other hand, the percentage of aneuploid tumors found in our study (58%) is also in agreement with what is previously described, varying from 48% to 76%.21-25 The progressive acquisition of genetic anomalies, including the development of aneuploidy, has been previously described in cells immortalized by HPV types 16 and 18.18,19 Moreover, in carcinomas of the anus, an association between the existence of aneuploidy and the presence of HPV type 16 has been observed.26 Because the products of the HPV E6 and E7 genes bind the proteins codified by tumor-suppressor genes p53 and Rb, respectively, and because these genes act in control of the cell cycle, it is plausible that integration of the HPV
can give cause for genetic instability in the host cell, resulting finally in the development of multiple chromosomal abnormalities. This may be related to the increased proliferative activity observed in HPV-infected cells because cells that are replicating actively are more susceptible to genetic damage than cells at rest.26 However, we have not found a significant relationship between the presence of the HPV types 16 and 6b in tumor cells and the existence of aneuploidy. Thus, of the seven cases that presented HPV E6 gene integration, only three (42%) were aneuploid, which is even greater than the proportion of aneuploid cases (62.5%) among those that were not presenting integration of the HPV. Nevertheless, these results should be interpreted with caution given the small number of cases positive for HPV. In a similar way, in carcinomas with integration, there seems to be a trend of the HPV to present a better histologic differentiation because there is no poorly differentiated case with HPV integration. However, previ-
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ous studies in head and neck carcinomas have not shown a relationship between the degree of histologic differentiation and the integration of the HPV.4-14 Similar results to these have been described in carcinomas of the anus,26 but in carcinomas of the uterine cervix, the presence of the HPV has been associated with a better histologic differentiation.27 These differences suggest that it can vary between the different locations. On the contrary, a significant correlation was found between the ploidy and the degree of histologic differentiation, in such a way that the diploid tumors showed better differentiation than the aneuploid tumors (77% of the well-differentiated tumors were diploid). In conclusion, although the number of positive cases for HPV is too small to establish definitive conclusions, the results of our study suggest that in squamous cell carcinomas of the head and neck the integration of HPV types 16 and 6b is not associated with a greater incidence of aneuploidy. REFERENCES 1. Gupta J, Pilotti S, Rilke F, et al. Association of human papillomavirus type 16 with neoplastic lesions of the vulva and other genital sites by in situ hybridization. Am J Pathol 1987;127:206-15. 2. Thompson CH, Rose BR, Cossart YE. Detection of HPV DNA in archival specimens of cervical cancer using in situ hybridization and the polymerase chain reaction. J Med Virol 1992;36:54-9. 3. Arends MJ, Donaldson YK, Duvall E, et al. HPV in full thickness cervical biopsies: high prevalence of CIN2 and CIN3 detected by a sensitive PCR method. J Pathol 1991;165:301-9. 4. De Villiers EM, Weidauer H, Otto H, et al. Papillomavirus DNA in human tongue carcinomas. Int J Cancer 1985;36:575-8. 5. Lee NK, Ritter D, Gross AE, et al. Head and neck squamous cell carcinomas associated with human papillomaviruses and an increased incidence of cervical pathology. Otolaryngol Head Neck Surg 1988;99:296-301. 6. Hoshikawa T, Nakajima T, Uhara H, et al. Detection of Human papillomavirus DNA in laryngeal squamous cell carcinomas by polymerase chain reaction. Laryngoscope 1990;100:647-50. 7. Bryan RL, Bevan IS, Crocker J, et al. Detection of HPV 6 and 11 in tumours of the upper respiratory tract using the polymerase chain reaction. Clin Otolaryngol 1990;15:177-80. 8. Niedobitek G, Pitteroff S, Herbst H, et al. Detection of human papillomavirus type 16 DNA in carcinomas of the palatine tonsil. J Clin Pathol 1990;43:918-21. 9. Tyan Y-S, Liu S-T, Ong W-R, et al. Detection of Epstein-Barr virus and human papillomavirus in head and neck tumors. J Clin Microbiol 1993;31:53-6. 10. Woods KV, Shillitoe EJ, Spitz MR, et al. Analysis of human
papillomavirus DNA in oral squamous cell carcinomas. J Oral Pathol Med 1993;22:101-8. 11. Shroyer KR, Greer RO. Detection of human papillomavirus DNA by in situ DNA hybridization and polymerase chain reaction in premalignant and malignant oral lesions. Oral Surg Oral Med Oral Pathol 1991;71:708-13. 12. Kasperbauer JL, O’Halloran GL, Espy MJ, et al. Polymerase chain reaction (PCR) identification of human papillomavirus (HPV) DNA in verrucous carcinomas of the larynx. Laryngoscope 1993;103:416-20. 13. Holladay EB, Gerald WL. Viral gene detection in oral neoplasms using the polymerase chain reaction. Am J Clin Pathol 1993; 100:36-40. 14. Tsuchiya H, Tomita Y, Shirasawa H, et al. Detection of human papillomavirus in head and neck tumors with DNA hybridization and immunohistochemical analysis. Oral Surg Oral Med Oral Pathol 1991;71:721-5. 15. Cone RW, Minson AC, Smith MR, et al. Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma. J Med Virol 1992;37:99-107. 16. Stoler MH, Rhodes CR, Whitbeck A, et al. Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Hum Pathol 1992;23:117-28. 17. Woodworth CD, Cheng S, Simpson S, et al. Recombinant retrovirus encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation in human keratinocytes early after infection. Oncogene 1992;7:619-26. 18. Pei XF, Gorman PA, Watt FM. Two strains of human keratinocytes transfected with HPV 16 DNA: comparison with the normal parental cells. Carcinogen 1991;12:277-84. 19. Hurlin PJ, Kaur P, Smith PP, et al. Progression of human papillomavirus type 18–immortalized human keratinocytes to a malignant phenotype. Proc Natl Acad Sci USA 1991;88:570-4. 20. Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983;3:323-7. 21. Burgio DL, Jacobs JR, Maciorowski Z, et al. DNA ploidy of primary and metastatic squamous cell head and neck cancers. Arch Otolaryngol Head Neck Surg 1992;118:185-7. 22. Holm L-E. Cellular DNA amounts of squamous cell carcinomas of the head and neck region in relation to prognosis. Laryngoscope 1982;92:1064-9. 23. Goldsmith MM, Cresson DS, Postma DS, et al. Significance of ploidy in laryngeal cancer. Am J Surg 1986;152:396-402. 24. Guo Y-C, Desanto L, Osetinsky GV. Prognostic implications of nuclear DNA content in head and neck cancer. Otolaryngol Head Neck Surg 1989;100:95-8. 25. El-Naggar AK, López-Varela V, Luna MA, et al. Intratumoral DNA content heterogeneity in laryngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 1992;118:169-73. 26. Noffsinger AE, Hui Y-Z, Suzuk L, et al. The relationship of human papillomavirus to proliferation and ploidy in carcinoma of the anus. Cancer 1995;75:958-67. 27. Wilczynski SP, Bergen S, Walker J, et al. Human papillomaviruses and cervical cancer: analysis of histopathologic features associated with different viral types. Hum Pathol 1988;19: 697-704.
Skull Base and Related Craniofacial Surgery
This interdisciplinary symposium and hands-on cadaver dissection workshop will be held October 27-30, 1999, at the Institute of Anatomy, University of Berne, Berne Switzerland. For further information, contact Prof Dr J. Raveh, Universitätsspital Berne, Fachbereich Schädel-, Kiefer- und Gesichtschirurgie Freiburgstrasse, CH-3010 Berne, Switzerland; phone 41-31-632-3317; fax 41-31-382-0279.
To establish the relationship between the presence of human papillomavirus (HPV) gene sequences and the development of genetic abnormalities, 31 squamous cell carcinomas of the head and neck were studied for the presence of HPV types 6b and 16 and the DNA content by flow cytometry. Eighteen (58%) cases were aneuploid. HPV DNA was present in seven (22.5%) tumors. Five of them were positive for the HPV type 6b and two for the HPV type 16. Aneuploidy was correlated with poorly differentiated tumors. No correlation was found between the presence of HPV, DNA content, or tumor differentiation. Consequently, the presence of HPV gene sequences does not seem to be related to a higher incidence of genetic abnormalities in squamous cell carcinomas of the head and neck. (Otolaryngol Head Neck Surg 1999;121:31822.)
T
he association of human papillomavirus (HPV) with dysplasia and carcinoma of the uterine cervix is well known.1-3 In the past few years, a similar relationship has been established between HPV and carcinoma of the upper aerodigestive tract.4-14 There are at least 60 genotypes of HPV, of which HPV types 6 and 11 are associated customarily with laryngeal papillomas, whereas types 6b, 16, and 18 have been related to neoplasias of the same location.4-14 The HPV genome contains eight genes, two of which (E6 and E7 genes) are very similar to genotypes From the Department of Otolaryngology, Hospital de León (Drs. Rodrigo and Alvarez); the Department of Otolaryngology, Hospital Central de Asturias, Oviedo (Drs. Martinez and Suárez); and the Department of Molecular Biology, Universidad de Oviedo (Drs. Lazo and Ramos). Presented at the Annual Meeting of the American Academy of Otolaryngology–Head Neck Surgery, San Francisco, Calif., Sept. 7-10, 1997. Supported by a grant from the Fondo de Investigaciones Sanitarias, Madrid, Spain (grant no. 93/0400). Reprint requests: Juan Pablo Rodrigo, MD, c/o Fernández Ladreda, 32-A 4°B, 33011 Oviedo, Spain. Copyright © 1999 by the American Academy of Otolaryngology– Head and Neck Surgery Foundation, Inc. 0194-5998/99/$8.00 + 0 23/77/90004 318
16 and 18, which are associated with cervical carcinomas.15,16 The cells transfected with the E6 and E7 genes become immortalized, do not differentiate normally, and grow more rapidly than nontransfected cells.17 Furthermore, E6- and E7-transfected cells may become aneuploid.18 HPV E6 and E7 gene products can produce cellular transformation probably because of their capacity to be joined to the products of two other genes that participate in the regulation of cellular proliferation. The HPV E6 gene product binds to the protein codified by the p53 gene, with the result of a more rapid degradation of the same.19 The protein codified by the HPV E7 gene binds to the product of the retinoblastoma gene (Rb), the function of which is necessary for the control of the cellular growth.18 In this way, it is probable that the antiproliferative effects of these two tumor-suppressor genes will be abolished after union to the HPV E6 and E7 gene products. As a result, the cells that contain these genes present an abnormal cellcycle regulation and therefore are subject to genetic mutations. To study the correlation between HPV infection and the presence of alterations in DNA content, we analyzed the integration of the HPV genome by polymerase chain reaction (PCR) and the cellular DNA content by flow cytometry in 31 squamous cell carcinomas of the head and neck. METHODS AND MATERIAL Patients Tumor samples of 31 patients with squamous cell carcinoma of head and neck were obtained from patients undergoing surgical resection of their tumors. Tissue specimens were freshly frozen in liquid nitrogen in the operating room and stored at –70° C until being processed. All included patients had a single primary tumor, and none had undergone treatment before surgery. All but one patient were male, with a mean age of 58 years and a range of 38 to 78 years. The distribution by locations is shown in Table 1, and the distribution by stage, according to the Union Internationale Contre le Cancer (UICC) classification (4th issue), is shown in Table 2. Of 31 cases, 16 (52%) were well differentiated, 10 (32%) were moderately differentiated, and 5 (16%) poorly differentiated.
Otolaryngology– Head and Neck Surgery Volume 121 Number 3
Analysis of Cellular DNA Content by Flow Cytometry
RODRIGO et al
Table 1. Distribution of studied cases by site Site
After being mechanically disintegrated in phosphatebuffered saline solution, the samples were filtered by a nylon mesh of 100 µm and thereafter centrifuged. The obtained sediment was digested with trypsin for 10 minutes, filtered by a nylon mesh of 50 µm, and stained with propidium iodide, according to the method of Vindelov et al.20 Samples were analyzed in a flow cytometer (BectonDickinson). As controls of normal DNA content (diploid), human normal lymphocytes were used. The DNA content of the tumor samples was established by comparison with that of the normal controls by use of the DNA index. The DNA index is the ratio between the G0/G1 peaks of the tumor and control DNA histograms (the G0/G1 peak represents the cells that are not synthesizing DNA). By definition the DNA index of the normal controls is 1. The samples were considered to be diploid if the DNA index was found to be 1.0 ± 0.05. If the DNA index was greater than 1.05, we considered it to be hyperdiploid, and if the DNA index was less than 0.95, we considered it to be hypodiploid. The debris and cellular aggregates that could not be filtered were eliminated instrumentally. Detection of HPV-6b and HPV-16 DNA The tissues were minced, homogenized, and digested twice with 100 µg proteinase K (Boehringer Mannheim) per milliliter of digestive buffer (200 mmol/L Tris-HCl [pH 8.0], 25 mmol/L EDTA, 100 mmol/L NaCl, and 0.2% sodium dodecylsulfate) for 8 hours at 37° C. High-molecular-weight DNA was isolated by standard phenol-chloroform extraction, precipitated in ethanol, dissolved in 10 mmol/L Tris and 1 mmol/L EDTA (pH 8.0), and stored at –20° C. DNA concentration was measured by absorbance at 260 nm. After DNA extraction, amplification of HPV-6b and HPV16 E6 and L1 genes was accomplished through PCR, with specific primers for each gene (Table 3). The L1 gene codifies one of the proteins of the viral capsid and is found only in the cells synthesizing viral particles, indicating active infection by the HPV. As positive control, we used the viral DNA cloned in the bacterial plasmid pBR322. The negative control in each case consisted of reaction mixtures of those in which the DNA was omitted. The PCR mixtures contained 0.5 µg of target DNA, 5 µl of ×10 buffer (100 mmol/L Tris-HCl [pH 8.3], 15 mmol/L Cl2Mg, 500 mmol/L ClK, 0.05% Tween 20, 0.05% NP-40, and 0.01% gelatin), 0.2 mmol/L of each dNTP, 1 U of Taq polymerase (Boehringer Mannheim), and 1 µmol/L of each primer in 50 µl total volume, with 50 µl mineral oil overlay. The PCR cycles included 1 minute at each temperature (94° C, 55° C, and 72° C), for a total of 35 cycles, and a final cycle of 72° C for 7 minutes. The PCR products were electrophoresed on 2% agarose gel and visualized with ethidium bromide staining.
319
No. of cases (%)
Oral cavity Oropharynx Glottis Supraglottis Hypopharynx
1 (3%) 12 (39%) 5 (16%) 4 (13%) 9 (29%)
Table 2. Distribution by stage of the studied cases Stage
No. of cases (%)
T stage T1 T2 T3 T4 N stage N0 N1 N2 N3 TNM stage I II III IV
1 (3%) 8 (26%) 10 (32%) 12 (39%) 11 (36%) 6 (19%) 10 (32%) 4 (13%) 1 (3%) 3 (10%) 10 (32%) 17 (55%)
Table 3. Primers used in the PCR Gene
HPV-16/E6 HPV-16/L1 HPV-6b/E6 HPV-6b/L1
Primers
5´-TCAAAAGCCACTGTGTCCTG-3´ 5´-CGTGTTCTTGATGATCTGCA-3´ 5´-CAGGGCCACAATAATGGCAT-3´ 5´-TGCGTCCTAAAGGAAACTGA-3´ 5´-TAAAGGTCCTGTTTCGAGGC-3´ 5´-CGGTTTGTGACACAGGTAGC-3´ 5´-TCTGCTGAAGTAATGGCCTA-3´ 5´-CGTCCCAAAGGATACTGATC-3´
Size (base pairs)
120 451 179 248
Statistic Analysis Statistical analysis was performed by use of χ2 tests with Yates’ correction when appropriate. RESULTS
The clinicopathologic characteristics of the studied patients are shown in Table 4. The HPV E6 gene was detected in seven (22.5%) cases. The HPV L1 gene was not detected in any of these cases. This implies that in all cases the E6 gene was integrated in the genomes of the tumor cells. Five carcinomas contained the HPV-6b DNA, and two the HPV-16 DNA. Figure 1 shows the PCR products in the positive cases.
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Table 4. Clinicopathologic characteristics of the studied cases Case no.
Age (yr)
Tumor site
T stage
N stage
Differentiation
Ploidy
HPV
68 68 43 78 63 46 52 60 70 68 44 67 63 47 53 53 45 71 65 63 56 38 61 63 51 64 59 62 57 53 57
Oropharynx Glottis Hypopharynx Hypopharynx Oropharynx Oropharynx Hypopharynx Hypopharynx Oropharynx Hypopharynx Oropharynx Oropharynx Oropharynx Hypopharynx Hypopharynx Oropharynx Glottis Supraglottis Supraglottis Oropharynx Glottis Oral cavity Supraglottis Oropharynx Oropharynx Supraglottis Glottis Hypopharynx Oropharynx Oropharynx Glottis
4 4 4 4 4 3 2 2 2 4 3 3 3 3 2 3 1 2 2 4 2 4 2 4 4 3 3 3 4 4 3
3 0 2 2 2 2 0 0 2 0 1 1 0 3 1 2 0 3 1 3 0 2 1 2 0 1 0 0 2 2 0
Moderately Poorly Poorly Poorly Moderately Moderately Poorly Well Well Well Moderately Moderately Well Well Well Well Well Moderately Well Well Well Well Moderately Moderately Poorly Well Well Moderately Moderately Well Well
A D A A A A A A A A A A D A D A A D D D D D A A A D D D A D D
N N N N N N N P (6b) N N N P (16) P (16) N N N N N P (6b) N N N N N N N P (6b) P (6b) P (6b) N N
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A, Aneuploid; D, diploid; N, negative; P, positive (type in parentheses).
Table 5. Relationship between histologic grade and ploidy and HPV presence Ploidy
*χ2 †χ2
HPV presence
Differentiation
No. of cases
Diploid
Aneuploid
p Value*
Positive
Well Moderately Poorly
16 10 5
10 (62%) 2 (20%) 1 (20%)
6 (38%) 8 (80%) 4 (80%)
0.04
4 (25%) 3 (33%) 0
Negative
12 (75%) 7 (67%) 5 (100%)
p Value†
0.4
test. test (well-differentiated front to moderately and poorly differentiated cases).
In 13 (42%) cases the tumor had a diploid DNA content, and in 18 (58%) it had an aneuploid DNA content. All the aneuploid cases were hyperdiploid. Of 7 HPVpositive cases, 4 (57%) were diploid and 3 (43%) aneuploid, whereas 9 (37.5%) of the 24 cases that did not show integration of the HPV were diploid and 15 (62.5%) aneuploid. These differences did not reach statistical significance (p = 0.62; Fig. 2). We noted that the well-differentiated carcinomas tended to present a diploid content of DNA, whereas the
moderately or poorly differentiated carcinomas tended to present an aneuploid content. Thus 62% of the welldifferentiated carcinomas were diploid, compared with 20% of the moderate and poorly differentiated; these differences reached statistical significance (p = 0.04; Table 5). On the other hand, there were no statistically significant differences between the histologic differentiation and the presence of the HPV, although none of the positive cases for HPV were poorly differentiated (Table 5).
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Fig 1. Electrophoresis of PCR products from the five cases that were positive to HPV-6b and the two cases that were positive to HPV-16. C+, Positive controls. C–, Negative controls. The size of the fragments, in base pairs (bp), is shown on the left.
Fig 2. Distribution of the studied cases by cellular DNA content and HPV presence.
DISCUSSION
HPV has been found in 10% to 80% of squamous cell carcinomas of the head and neck,4-14 although in the series with greater numbers of patients, the percentage of positive cases is found to be about 20%.6,13 In this work we find a 22.5% integration frequency of the E6 gene for HPV-16 and HPV-6b. On the other hand, the percentage of aneuploid tumors found in our study (58%) is also in agreement with what is previously described, varying from 48% to 76%.21-25 The progressive acquisition of genetic anomalies, including the development of aneuploidy, has been previously described in cells immortalized by HPV types 16 and 18.18,19 Moreover, in carcinomas of the anus, an association between the existence of aneuploidy and the presence of HPV type 16 has been observed.26 Because the products of the HPV E6 and E7 genes bind the proteins codified by tumor-suppressor genes p53 and Rb, respectively, and because these genes act in control of the cell cycle, it is plausible that integration of the HPV
can give cause for genetic instability in the host cell, resulting finally in the development of multiple chromosomal abnormalities. This may be related to the increased proliferative activity observed in HPV-infected cells because cells that are replicating actively are more susceptible to genetic damage than cells at rest.26 However, we have not found a significant relationship between the presence of the HPV types 16 and 6b in tumor cells and the existence of aneuploidy. Thus, of the seven cases that presented HPV E6 gene integration, only three (42%) were aneuploid, which is even greater than the proportion of aneuploid cases (62.5%) among those that were not presenting integration of the HPV. Nevertheless, these results should be interpreted with caution given the small number of cases positive for HPV. In a similar way, in carcinomas with integration, there seems to be a trend of the HPV to present a better histologic differentiation because there is no poorly differentiated case with HPV integration. However, previ-
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ous studies in head and neck carcinomas have not shown a relationship between the degree of histologic differentiation and the integration of the HPV.4-14 Similar results to these have been described in carcinomas of the anus,26 but in carcinomas of the uterine cervix, the presence of the HPV has been associated with a better histologic differentiation.27 These differences suggest that it can vary between the different locations. On the contrary, a significant correlation was found between the ploidy and the degree of histologic differentiation, in such a way that the diploid tumors showed better differentiation than the aneuploid tumors (77% of the well-differentiated tumors were diploid). In conclusion, although the number of positive cases for HPV is too small to establish definitive conclusions, the results of our study suggest that in squamous cell carcinomas of the head and neck the integration of HPV types 16 and 6b is not associated with a greater incidence of aneuploidy. REFERENCES 1. Gupta J, Pilotti S, Rilke F, et al. Association of human papillomavirus type 16 with neoplastic lesions of the vulva and other genital sites by in situ hybridization. Am J Pathol 1987;127:206-15. 2. Thompson CH, Rose BR, Cossart YE. Detection of HPV DNA in archival specimens of cervical cancer using in situ hybridization and the polymerase chain reaction. J Med Virol 1992;36:54-9. 3. Arends MJ, Donaldson YK, Duvall E, et al. HPV in full thickness cervical biopsies: high prevalence of CIN2 and CIN3 detected by a sensitive PCR method. J Pathol 1991;165:301-9. 4. De Villiers EM, Weidauer H, Otto H, et al. Papillomavirus DNA in human tongue carcinomas. Int J Cancer 1985;36:575-8. 5. Lee NK, Ritter D, Gross AE, et al. Head and neck squamous cell carcinomas associated with human papillomaviruses and an increased incidence of cervical pathology. Otolaryngol Head Neck Surg 1988;99:296-301. 6. Hoshikawa T, Nakajima T, Uhara H, et al. Detection of Human papillomavirus DNA in laryngeal squamous cell carcinomas by polymerase chain reaction. Laryngoscope 1990;100:647-50. 7. Bryan RL, Bevan IS, Crocker J, et al. Detection of HPV 6 and 11 in tumours of the upper respiratory tract using the polymerase chain reaction. Clin Otolaryngol 1990;15:177-80. 8. Niedobitek G, Pitteroff S, Herbst H, et al. Detection of human papillomavirus type 16 DNA in carcinomas of the palatine tonsil. J Clin Pathol 1990;43:918-21. 9. Tyan Y-S, Liu S-T, Ong W-R, et al. Detection of Epstein-Barr virus and human papillomavirus in head and neck tumors. J Clin Microbiol 1993;31:53-6. 10. Woods KV, Shillitoe EJ, Spitz MR, et al. Analysis of human
papillomavirus DNA in oral squamous cell carcinomas. J Oral Pathol Med 1993;22:101-8. 11. Shroyer KR, Greer RO. Detection of human papillomavirus DNA by in situ DNA hybridization and polymerase chain reaction in premalignant and malignant oral lesions. Oral Surg Oral Med Oral Pathol 1991;71:708-13. 12. Kasperbauer JL, O’Halloran GL, Espy MJ, et al. Polymerase chain reaction (PCR) identification of human papillomavirus (HPV) DNA in verrucous carcinomas of the larynx. Laryngoscope 1993;103:416-20. 13. Holladay EB, Gerald WL. Viral gene detection in oral neoplasms using the polymerase chain reaction. Am J Clin Pathol 1993; 100:36-40. 14. Tsuchiya H, Tomita Y, Shirasawa H, et al. Detection of human papillomavirus in head and neck tumors with DNA hybridization and immunohistochemical analysis. Oral Surg Oral Med Oral Pathol 1991;71:721-5. 15. Cone RW, Minson AC, Smith MR, et al. Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma. J Med Virol 1992;37:99-107. 16. Stoler MH, Rhodes CR, Whitbeck A, et al. Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Hum Pathol 1992;23:117-28. 17. Woodworth CD, Cheng S, Simpson S, et al. Recombinant retrovirus encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation in human keratinocytes early after infection. Oncogene 1992;7:619-26. 18. Pei XF, Gorman PA, Watt FM. Two strains of human keratinocytes transfected with HPV 16 DNA: comparison with the normal parental cells. Carcinogen 1991;12:277-84. 19. Hurlin PJ, Kaur P, Smith PP, et al. Progression of human papillomavirus type 18–immortalized human keratinocytes to a malignant phenotype. Proc Natl Acad Sci USA 1991;88:570-4. 20. Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983;3:323-7. 21. Burgio DL, Jacobs JR, Maciorowski Z, et al. DNA ploidy of primary and metastatic squamous cell head and neck cancers. Arch Otolaryngol Head Neck Surg 1992;118:185-7. 22. Holm L-E. Cellular DNA amounts of squamous cell carcinomas of the head and neck region in relation to prognosis. Laryngoscope 1982;92:1064-9. 23. Goldsmith MM, Cresson DS, Postma DS, et al. Significance of ploidy in laryngeal cancer. Am J Surg 1986;152:396-402. 24. Guo Y-C, Desanto L, Osetinsky GV. Prognostic implications of nuclear DNA content in head and neck cancer. Otolaryngol Head Neck Surg 1989;100:95-8. 25. El-Naggar AK, López-Varela V, Luna MA, et al. Intratumoral DNA content heterogeneity in laryngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 1992;118:169-73. 26. Noffsinger AE, Hui Y-Z, Suzuk L, et al. The relationship of human papillomavirus to proliferation and ploidy in carcinoma of the anus. Cancer 1995;75:958-67. 27. Wilczynski SP, Bergen S, Walker J, et al. Human papillomaviruses and cervical cancer: analysis of histopathologic features associated with different viral types. Hum Pathol 1988;19: 697-704.
Skull Base and Related Craniofacial Surgery
This interdisciplinary symposium and hands-on cadaver dissection workshop will be held October 27-30, 1999, at the Institute of Anatomy, University of Berne, Berne Switzerland. For further information, contact Prof Dr J. Raveh, Universitätsspital Berne, Fachbereich Schädel-, Kiefer- und Gesichtschirurgie Freiburgstrasse, CH-3010 Berne, Switzerland; phone 41-31-632-3317; fax 41-31-382-0279.