Affiliations of authors: Laboratory of Epithelial Cancer Biology, Division of Head and Neck Surgery, Department of Surgery (DIK, VBW, AG, IN, LAH, BS), Department of Epidemiology and Biostatistics (LB-P, JS), Department of Pathology (AGH, DC), Memorial Sloan-Kettering Cancer Center, New York, NY; Laboratory of Human Genetics and Hematology, The Rockefeller University, New York (DIK, PG, OL, KP, RD, ADA, BS).
Correspondence to: Bhuvanesh Singh, MD, Laboratory of Epithelial Cancer Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021 (e-mail: singhb{at}mskcc.org)
ABSTRACT
Fanconi anemia is an autosomal recessive disorder characterized by congenital malformations, bone marrow failure, and the development of squamous cell carcinomas (SCCs) and other cancers. Recent clinicopathologic evidence has raised the possibility that an environmental factor such as human papillomavirus (HPV) may be involved in the pathogenesis of SCCs in Fanconi anemia patients. Given the high prevalence of p53 mutations in SCCs among the general population and the lack of p53 mutations in HPV-related carcinogenesis, we evaluated the role of HPV and p53 mutations and polymorphisms in SCC from Fanconi anemia patients. We used polymerase chain reaction (PCR) screening and real-time PCR to detect and quantify HPV DNA in DNA extracted from microdissected SCCs obtained from 24 Fanconi anemia patients (n = 25 SCCs; case subjects) and 50 age-, sex-, and tumor site-matched SCC patients without Fanconi anemia (n = 50 SCCs; control subjects). We PCR-amplified and sequenced exons 49 of the p53 gene from SCC DNA. We detected HPV DNA in 84% of the SCC specimens from the case subjects and in 36% of the SCC specimens from the control subjects (P<.001). The prevalence of p53 mutations in SCCs from the case subjects (0%, 0/25) was statistically significantly lower than that of SCCs from the control subjects (36%, 12/33; P<.001). A greater proportion of patients with Fanconi anemia and SCC were homozygous for Arg72, a p53 polymorphism that may be associated with increased risk for HPV-associated human malignancies, than an ethnically-matched cohort of Fanconi anemia patients without SCC (75% versus 51%; P = .05). These data suggest that Fanconi anemia is associated with increased susceptibility to HPV-induced carcinogenesis.
The high incidence of head and neck and anogenital SCC among Fanconi anemia patients, combined with the high rates of second primary tumors in these patients (4), led us to speculate that an environmental factor may play a role in squamous cell carcinogenesis in these patients. Moreover, the observed distribution of tumors, which involves regions of the body that are at high risk of human papillomavirus (HPV)-associated carcinogenesis, suggests a possible association between SCC and HPV infection in Fanconi anemia patients (812). Because HPV E6 and E7 oncoproteins are expressed in HPV-associated cancers and have been found to inactivate several important tumor suppressor genes including p53 and Rb, respectively, understanding the relationship between HPV and p53 may elucidate the underlying cellular mechanisms responsible for tumor development in the Fanconi anemia population (1316).
We examined the role of HPV infection (as defined by HPV DNA positivity of tumor tissue) in the pathogenesis of SCC among patients with Fanconi anemia by performing a case-control study in which the case subjects were Fanconi anemia patients diagnosed with SCC of the anus, vulva, or head and neck and the control subjects were SCC patients who did not have Fanconi anemia and who were matched to the case subjects in a 2:1 ratio for age, sex, and site of primary tumor. Matching for tobacco and/or alcohol exposure status was not possible because of the small number of appropriate cases in our tissue bank. The cohort of Fanconi anemia patients consisted of 754 diagnosis-confirmed Fanconi anemia patients registered between 1982 and 2001 by the International Fanconi Anemia Registry (IFAR) (2,4). We procured 25 tissue and blood samples from 24 Fanconi anemia patients who had pathologically confirmed SCCs and were treated at one of 24 medical institutions across North America. Tumor tissues (and blood samples, when available) for the control subjects were obtained from the Memorial Sloan-Kettering Cancer Center tumor bank. All tissue specimens were assigned random specimen numbers. To maintain subject anonymity, we collected clinical data independently from the laboratory data and integrated both types of data only after completion of the project. All investigators were blinded to the clinical information until the completion of the study. This study was approved by the Institutional Review Board of The Rockefeller University. We obtained written informed consent from the patients or their next of kin to use clinical data and tissue specimens.
Two experienced pathologists (A. G. Huvos and D. Carlson) reviewed the hematoxylin-eosin-stained sections of the surgical resection specimens to confirm the diagnoses and to identify regions of interest. All specimens were found to contain SCC. All of the vulvar cancers were warty (condylomatous; three cases) or basaloid (usual type; three cases) types, which are associated with HPV infection in the general population, and no cases were of the typical keratinizing type, which in the general population is not associated with HPV (17). Invasive tumor tissue, adjacent non-tumor tissue, and dysplastic tissue (when available) were isolated from the tissue specimens with the use of a laser capture microdissection apparatus (PALM, Bernried, Germany). DNA was extracted from the microdissected tissue (1820) and subjected to HPV DNA detection by polymerase chain reaction (PCR) as previously described (21). HPV DNA positivity was defined as the presence of a 188-base-pair (bp) PCR product on an ethidium bromide-stained agarose gel. The PCR product was purified and sequenced to determine the type of HPV, as previously described (22). We used real-time PCR to confirm HPV DNA detection and to quantify the number of HPV DNA copies per cell, as previously described (22). Only tissue samples that were positive for HPV16 and HPV18 were used for real-time PCR, and tissue samples were considered positive only if they had more than one copy of HPV DNA per 10 cells (22). DNA extracted from microdissected tissue was also analyzed for p53 mutations by PCR amplification of exons 49 of the p53 gene; each amplified exon was sequenced in duplicate using a previously described protocol. (23). To determine p53 codon 72 genotypes, we directly sequenced exon 4 of the DNA extracted from blood samples (DNAeasy; Qiagen, Valencia, CA) of the previously described 24 Fanconi anemia patients with SCC and 72 Fanconi anemia patients without cancer. Because specific ethnic groups vary in the distribution of p53 codon 72 genotypes, the groups of subjects were matched by ethnicity. Statistical analyses were performed using Fishers exact test and the Wilcoxon rank sum test. A two-tailed P value less than or equal to .05 was considered statistically significant for all analyses.
We detected HPV DNA in 21 (84%) of the 25 SCC specimens from the Fanconi anemia case subjects and in 18 (36%) of the 50 SCC specimens from the control subjects (P<.001, Fishers exact test) (Table 1). HPV DNA was not detected in any of the adjacent non-tumor (n = 21) or dysplastic (n = 17) tissues from the Fanconi anemia patients. (We were unable to perform a similar analysis for the control group because of the small size of those tissue specimens and the lack of non-tumor and dysplastic tissue.) A subgroup analysis revealed that 15 (83%) of the 18 head and neck SCCs from the Fanconi anemia case subjects were positive for HPV DNA compared with only 13 (36%) of the 36 head and neck SCCs from the control subjects (P = .002, Fishers exact test). Direct sequencing of PCR products amplified from SCC DNA showed that HPV16 was the most common HPV type detected among the Fanconi anemia-associated SCCs and the control SCCs (19/21 SCCs [90%] and 16/18 SCCs [89%], respectively; P = .55, Fishers exact test). Results of real-time quantitative PCR confirmed the presence of HPV16 or HPV18 in all samples that were positive for HPV DNA by PCR screening with consensus oligonucleotide primers. The median number of HPV DNA copies per genome was 2.6 (range = 0.16249.3 copies/genome) and 3.2 (range = 0.15190.85 copies/genome) in SCCs from Fanconi anemia patients and from the control subjects, respectively (P = .88, Wilcoxon rank sum test; data not shown).
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Storey et al. (25) reported experimental evidence that polymorphic variants of p53 differ in their susceptibility to degradation mediated by HPV-derived E6 oncoprotein. Specifically, they demonstrated that homozygosity for arginine at codon 72 (Arg72) of p53 is associated with an increased susceptibility to E6-induced degradation of p53 in vivo and, in their case series, a sevenfold increased risk for HPV-associated malignancies in humans (26). However, substantial controversy exists about the role of this polymorphism in the susceptibility to HPV-associated carcinogenesis, mainly because large differences in the frequency of Arg72 homozygosity in different populations have made it difficult to define suitable control groups for cancer susceptibility studies (27).
We examined the association between the p53 Arg72 polymorphism and SCC among the cohort of Fanconi anemia patients registered in the IFAR because they are a well-defined population at risk for SCC. We compared the p53 codon 72 genotypes of the 24 Fanconi anemia patients with SCC used in the above analyses with those of 72 Fanconi anemia patients without cancer who were registered in the IFAR and matched to the patients with SCC for ethnicity in a 3:1 ratio. We were unable to amplify exon 4 of the p53 gene of seven control patients, leaving us a total control group of 65 Fanconi anemia patients without SCC. There were no statistically significant differences between the two groups of patients with regard to sex or bone marrow transplant status (25% transplant rate in the case subjects versus 33% transplant rate in the control subjects). A statistically significantly higher proportion of Fanconi anemia patients with SCC was homozygous for the Arg72 polymorphism than Fanconi anemia patients without SCC (75% versus 51%; P = .05, Fishers exact test) (Table 2). Notably, the Fanconi anemia patients with SCC (median age = 30 years; range = 1549 years) were statistically significantly older than the Fanconi anemia patients without SCC (median age = 10.2 years; range = 148 years; P<.001, Wilcoxon rank sum test). To address differences in the at-risk period for cancer development, we compared the genotype frequencies of the 20 non-cancer Fanconi anemia patients older than 15 years with those of the 24 Fanconi anemia patients with SCC (the youngest age for developing Fanconi anemia-associated SCC was 15 years). This subgroup analysis revealed a larger difference in the frequency of p53 Arg72 homozygosity between Fanconi anemia patients with and without SCC (Table 2; P = .015). The odds ratio of SCC associated with Arg72 homozygosity in this subgroup analysis was 5.6 (95% confidence interval = 1.5 to 20.5; P = .01).
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Our results suggest that the high rates of head and neck and anogenital SCCs among Fanconi anemia patients may be associated with the high frequency with which oncogenic HPV DNA is detected in their tumor tissues. Whether this high rate of HPV-associated SCC is caused by an underlying immune dysfunction in Fanconi anemia patients or directly involves the pathway(s) defective in Fanconi anemia is currently not known. Fanconi anemia may be the second inherited syndrome identified, after epidermodysplasia verruciformis, that is associated with an increased susceptibility to HPV-induced carcinogenesis. However, Fanconi anemia-associated SCCs differ from epidermodysplasia verruciformis-associated SCCs in that the former are mucosal and are associated with HPV16, whereas the latter are cutaneous and are associated with HPV5 or HPV8. Moreover, compared with Fanconi anemia patients, epidermodysplasia verruciformis patients appear to have a higher risk for HPV-associated tumorigenesis, which develops in approximately 50% of cases (28). Although mutations in two genes, EVER1 and EVER2, have been identified in epidermodysplasia verruciformis patients by linkage analysis, the precise mechanisms underlying the increased sensitivity to HPV-associated carcinogenesis in these patients are yet to be defined (29).
As with cervical cancer attributable to HPV in the general population, SCC in Fanconi anemia patients is probably also associated with the inactivation of p53 by HPV-associated oncoproteins rather than by direct mutagenesis. In addition, Fanconi anemia patients with homozygosity for Arg72 in p53 have a 5.6-fold increased risk of developing HPV-associated cancers compared with Fanconi anemia patients who do not have Arg72 homozygosity. Thus, Fanconi anemia may represent an excellent model for studying HPV-induced carcinogenesis and associated prevention approaches.
NOTES
Funded in part by Public Health Services grants R37-HL32987 (to A. D. Auerbach) from the National Heart, Lung, and Blood Institute, and RO1-CA82678 (to A. D. Auerbach) and 5T32-CA09685 (to D. I. Kutler) from the National Cancer Institute, and Research Center grants M01-RR00102 and M01-RR06020 (to The Rockefeller University Hospital General Clinical Research Center and The New York Presbyterian Hospital Childrens Clincial Research Center), National Institutes of Health, Department of Health and Human Services; and the 2002 American Society of Clinical Oncology Young Investigator Award (to D. I. Kutler).
B. Singh is a recipient of the George H. A. Clowes, Jr., MD, Memorial Research Career Development Award from the American College of Surgeons.
We thank Nancy Bennett for her excellent editorial assistance.
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Manuscript received April 21, 2003; revised August 27, 2003; accepted September 9, 2003.
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