Although the incidence of anal cancer in the general population is low, at 0.9 cases per 100,0001 compared with cervical cancer (at 8 cases per 100,000), men who have sex with men (MSM) are at increased risk to develop anal cancer. Among HIV-negative MSM, the incidence of anal cancer is estimated to be as high as 35 per 100,000,2 an incidence that is several times higher than the current rate of cervical cancer in the general population of women in the United States. The current rate of anal cancer among HIV-positive MSM is estimated to be at least double that of HIV-negative MSM.3 These alarming numbers may increase further, because highly active antiretroviral therapy (HAART) is increasing survival in the HIV-positive population and, consequently, increases the likelihood that unscreened and untreated precursor lesions may progress to cancer.
Anal intraepithelial neoplasia (AIN) is histologically and biologically similar to cervical intraepithelial neoplasia (CIN) and is the likely precursor lesion to anal carcinoma.4 One cross-sectional study showed that AIN is common among HIV-negative MSM (7%) and is even more common among HIV-positive MSM (36%).5 In recent years, several studies have been published indicating that AIN is also commonly found in HIV-positive women (up to 26%).6-8 The likely anal cancer precursor, high-grade AIN (AIN 2 and AIN 3), is also referred to as a high-grade squamous intraepithelial lesion (HSIL). Low-grade AIN (AIN 1 and condyloma) is also referred to as a low-grade squamous intraepithelial lesion (LSIL) and is not considered to be a cancer precursor.
Like cervical cancer, anal cancer is strongly associated with human papilloma virus (HPV) infection, and risk factors for the 2 cancers are similar. HPV DNA has been identified in more than 99% of cervical cancers9 and in most anal cancers.10,11 More than 100 different types of HPV have been described to date, and mucosal HPVs that infect the anogenital tract generally are classified as low-risk or high-risk HPV. Whereas the low-risk HPVs usually are associated with benign lesions such as warts, high-risk HPVs are found in invasive cancers.
In vitro studies using cervical cell lines indicate that the oncogenic properties of high-risk HPV types can be attributed mainly to 2 viral proteins: E6 and E7. These proteins disrupt DNA repair and cell cycle control through interaction with the tumor suppressors p53 and RB respectively.12,13 It is also well established that expression of the HPV E6 and E7 genes induces genomic instability in cell culture,14-16 and chromosomal copy number imbalance has been described previously for cervical and anal cancer.17-21 Furthermore, HPV DNA is integrated into the host cell genome at increasing frequency with the progression of CIN to cervical cancer.22 Establishing the role that HPV plays in the initiation of genomic instability is an important goal for our understanding the pathogenesis of the disease.
An increasing number of technologies recently have been developed to allow large-scale analysis of genomic alterations in cancer tissues. Whereas lymphoid-derived tumors commonly harbor simple chromosomal translocation events leading to fusion proteins with oncogenic properties, solid tumors usually present with complex chromosomal rearrangements leading to multiple DNA copy number imbalances.23 Comparative genomic hybridization (CGH) has been particularly useful in genome-wide identification of large regions of DNA copy number abnormalities (CNAs).24 The utility of this metaphase chromosome-based methodology has been hampered by its low resolution (5-15 megabases [Mb]), however, resulting in an inability to detect small regions of CNAs. In recent years, microarray-based comparative genome hybridization (aCGH) has been developed.25,26 This technique represents a promising new tool to conduct high-resolution genome-wide analysis of CNAs, allowing better detection of small regions of CNAs and providing a better chance to identify genes involved in tumorigenesis.
We previously performed chromosomal CGH and reported CNAs in AIN tissues of various grades.17 In the present study, we characterized CNAs in AIN tissues using aCGH with human inserts in bacterial artificial chromosomes (BACs) as spotted targets. These BACs were selected to be regionally distributed across the human genome, with an average spacing of 1.4 Mb, to better delineate the extent of copy number changes in AIN grades I, II, and III. The commonly occurring gain in chromosome 3q was of particular interest because it represents a recurrent change in multiple types of carcinomas.27,28 It has been reported to represent an important event in the transition from severe dysplasia to invasive carcinoma of the cervix18,20,29 and is the most common alteration in HPV-positive vulvar tumors.30,31 We also performed quantitative real-time polymerase chain reaction (PCR) analysis (quantitative microsatellite analysis [QuMA])32 to delineate an amplicon found on chromosome arm 3q better. Finally, to determine the relation between CNAs and HPV DNA integration, we performed HPV restriction site (RS) PCR analysis on these same samples.
MATERIALS AND METHODS
This study was conducted with the approval of the Committee on Human Research of the University of California, San Francisco. AIN biopsy samples were obtained as described previously.17 A total of 46 frozen biopsies from 44 men were collected, embedded in optimum cutting temperature (OCT)™ (Tissue-Tek; Sakura-Fivetek, Inc., Torrance, CA), and immediately frozen and stored in liquid nitrogen until use. Histopathologic findings were interpreted by a single pathologist on all samples using 2 hematoxylin-eosin (HE)-stained 8-μm sections. To study common changes in DNA copy number, we performed aCGH on an additional 5 AIN samples that previously were characterized by chromosomal CGH17 and known to carry genomic abnormalities. To avoid bias, only these samples were considered when studying common copy number changes and were excluded from all other comparative analyses.
Isolation and Purification of DNA
Fresh-frozen samples were sliced on a cryostat into 20-μm sections, washed once in phosphate-buffered saline (PBS), resuspended in 600 μL of lysis solution (500 μL of Nuclei Lysis Solution from the Wizard Genomic DNA purification kit [Promega, Madison, WI] plus 100 μL of 0.5-M EDTA, pH 8.0), and homogenized at 5000 to 8000 rpm for 2 minutes on ice with a Tissue Tearor homogenizer (Fisher Scientific, Los Angeles, CA). These homogenates were purified as described by Pinkel et al.26
Human Papillomavirus Genotyping
HPV genotyping was performed on DNA extracted from each lesion by PCR using MY09/MY11 primer pairs followed by dot blot hybridization, as described previously.4 The integrity of the cellular DNA in each sample was tested for PCR amplification using specific primers for human β-globin.
HPV16 integration events were evaluated using an RS-PCR method as described by Thorland et al33 with the following modifications. RS-PCR was performed using 4 restriction site oligonucleotides (RSOs) designed to recognize specific restriction sites (BamH1, EcoR1, Sau3a, and Taq1) in a multiplex PCR format as originally described by Weber et al34 using 25 to 50 ng of template DNA. For each sample, 4 PCRs were set up. Each PCR contained a mixture of all 4 RSOs and 1 of 4 HPV16-specific primers. The 4 HPV16-specific primers used were: HPV16-768-24D, HPV16-1545-26D, HPV16-2386-25D, and HPV16-2929-24D.33,35 An aliquot of each reaction was submitted to a second round of multiplex RS-PCR using HPV16 nested primers. The entire amplification reaction was then loaded onto a 2% agarose gel for electrophoresis. Bands were excised from the gel, and the PCR products were extracted using the Qiagen gel extraction kit (Qiagen, Valencia, CA). The PCR product was sequenced at the University of California, San Francisco (UCSF) Genome Core using the appropriate sequencing primers.33 To test our capacity to retrieve integration sites using this method, we analyzed DNA extracted from the cervical cell line SiHa, which is known to harbor a single HPV16 integration site at 13q22.24
Microarray Based Comparative Genomic Hybridization
Genomic DNA was random-primed in a 25-μL reaction by combining 10 to 300 ng of genomic DNA and a random primer solution from the BioPrime DNA Labeling System (InVitrogen, Carlsbad, CA). The mixture was heated at 100°C for 10 minutes, placed on ice for 5 minutes, and combined with deoxynucleoside triphosphate (dNTP) solution (400 μM each of deoxyadenosine triphosphate [dATP], deoxyguanosine triphosphate [dGTP], and deoxycytidine triphosphate [dCTP] and 250 μM of deoxythymidine triphosphate (dTTP) in 10 mM of Tris-HCl, pH 7.6), 80 μM of Cy3-deoxyuridine triphosphate (dUTP) or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), plus 40 U of Klenow fragment from a BioPrime DNA labeling System (InVitrogen). This solution was incubated at 37°C overnight and purified using a Sephadex microspin-G50 column (Amersham Biosciences).
Random-primed genomic DNA (approximately 10 μg) for the test (Cy3) and the reference (Cy5) was pooled, mixed with approximately 500 μg of COT-1 DNA (InVitrogen), and ethanol precipitated. Hybridization to BAC arrays consisting of 2464 BAC clones, each printed in triplicate on chromium slides and kindly provided by Drs. D. Albertson and D. Pinkel (UCSF Comprehensive Cancer Center), was performed as described in published reports.25,36,37
Image Acquisition and Analysis
Microarray images were acquired on a custom-built charge-coupled device (CCD) camera imager to obtain 16-bit 1024 × 1024 pixel 4′-6-diamidine-2-phenylindole (DAPI), Cy3, and Cy5 images.25 Image processing was performed using SPOT software38 to segment the array spots and to calculate the log2 ratios of the total integrated Cy3 and Cy5 intensities of each spot after subtraction of local background intensity. Using a second custom program called SPROC, spots were automatically filtered to remove spots with fewer than 15 pixels, spots with low Cy3 and Cy5 correlation (<0.7), or spots with a low value of the combined Cy3 and Cy5 intensities (<150). The genomic locations of all BAC clones were positioned and ordered on the University of California, Santa Cruz (UCSC) genome assembly database of July 2003 (hg16) (http://www.genome.UCSC.edu).
Each BAC clone was spotted in triplicate, and clones with only 1 valid spot or clones with a large standard deviation (SD) of the log2 ratio (>0.15) among the duplicate and/or triplicate spots were excluded from analysis. To further test for BAC clone quality, we performed 4 hybridizations of Cy3-labeled normal female reference DNA versus the same DNA with Cy-5 label, and we eliminated any clones that gave data in fewer than 70% of the hybridizations. In addition, we calculated the mean log2 ratio and the SD of the log2 ratio for each clone over these 42 hybridizations. Clones that showed a mean log2 ratio >0.16 or a SD of the log2 ratio >0.13 were eliminated from analysis. Any clone that showed an individual-clone CNA in more than 10% of these arrays was eliminated. Finally, a number of clones in these arrays were mapped to the Y chromosome or were unmapped. These clones were eliminated from analysis. This filtering resulted in elimination of 236 clones from the original 2464.
To define a regional CNA, we determined a threshold log2 ratio. Results for each hybridization were used to calculate the SD of the log2 ratio, excluding clones on the X chromosome and regions that showed obvious CNAs. A gain or loss (CNA) was defined when a clone showed a log2 ratio >2.5 SDs for that hybridization or when a clone showed a log2 ratio >1.5 SDs and it was mapped adjacent to a clone with log2 ratio >2.5 SDs.
Samples and Human Papillomavirus Genotype
Forty (91%) of 44 participants were HIV-positive. Their median CD4 count was 439 cells/mm3, and the range was 19 to 852 cells/mm3. Forty-two anal biopsy samples were obtained from individual patients, and 2 patients contributed 2 specimens. Anal biopsy samples were examined microscopically and classified as follows: 5 normal, 2 condyloma, 7 AIN-I, 10 AIN-II, 17 AIN-III, 3 unknown grade, and 2 with predominantly colonic tissue. Table 1 provides a summary of the HPV genotyping and histologic grading of these specimens. HPV DNA was found in 37 (95%) of 39 AIN samples. HPV16 was the most common HPV type and was detected in 17 (45%) of 38 of the samples positive for HPV DNA, including 3 samples showing multiple HPV types. Multiple HPV types were detected in 5 samples.
Human Papillomavirus Integration
Using RS-PCR analysis on cases, as expected, a single sequence corresponding to the published integration site for SiHa cells was identified (data not shown). The specificity of the RS-PCR assay for HPV16 was tested on 2 samples with HPV18, and these samples yielded no PCR product. All HPV16-containing AIN samples produced a PCR product corresponding to a normal HPV sequence. A summary of the integration results is shown in Table 2. Coexisting within the same lesion, integrated HPV16 was identified in 4 samples, with 1 sample showing 2 integration sites. Using the cellular flanking sequence retrieved from sequencing, we were able to map 4 integration sites precisely using the UCSC genome assembly of July 2003. The short flanking sequence retrieved from the fifth integration site did not match any sequence from the genome assembly and, consequently, could not be mapped. Interestingly, the viral-cellular junction in this sample contained a 9-base pair (bp) direct repeat.
In 6 of the cases, we found HPV16 with large deletions (2888-6631 bp) that disrupted the E1 or E2 open reading frame. One case showed 3 such rearrangements, and 2 other cases showed integrated and deleted HPV16. In 3 samples, the rearranged viral genome joined the plus strand of the E1 or E2 gene to the minus strand of the L1 gene open reading frame. For 1 deletion, we observed a fusion of the E1 gene plus strand to the corresponding strand of the L1 gene (nuc. 6702), and for the other 6 deletions, we observed a fusion of the E1 or E2 plus strand to the plus strand of the long control region of HPV between nucleotides 7719 and 7899 (GenBank: NC_001526).
Frequency of Genomic Instability
Regional CNAs were identified and characterized by aCGH in 8 newly analyzed samples (Fig. 1). Although sample A147 seems to be largely normal, a small but well-defined region of DNA loss was present in chromosome 4q. A summary of the grading of samples with regional CNAs is shown in Table 3. There were no regional CNAs in any of the 31 other newly analyzed samples. AIN of higher grade severity was more likely to show genomic aberrations than AIN of lower grades. More than one third of AIN-III samples (35%) showed CNAs by aCGH compared with 20% of AIN-II and 0% of the AIN-I samples, although this difference was not statistically significant (P > 0.2). Interestingly, we found a strong correlation between the status of the HPV genome and the presence of CNAs. Whereas all 6 CNA-positive samples with HPV16 showed integrated and/or rearranged HPV, only 2 of 10 CNA-negative samples showed rearranged HPV and none showed integration. This correlation was significant with an odds ratio of 44, and a 95% confidence interval of 2.0 to 218.0 (P = 0.007, Fisher exact test).
The frequency of CNAs across the genome was evaluated on the complete set of 13 cases that showed any genetic abnormalities (Fig. 2). This set consisted of 8 cases identified in this study and 5 cases previously characterized by chromosomal CGH and reanalyzed here using aCGH. In samples showing CNAs, recurrent gains were observed on chromosomes 1 (54%), 3q (77%), 8p (38%), and 20q (38%). The gain on chromosome arm 8p showed a sharp raise in frequency between BAC clones RP11-112O8 and RP11-262B15 (8p: nuc.4.8-9.9 Mb), and the gain in 20q11 occurred between clones RP11-134I8 and RP11-138A15 (20q: nuc.31.4-36.7 Mb). Microarray CGH analysis also showed several regions with high frequencies of copy number loss. The most common regions of loss were located on chromosomes 2q (30%), 6q (30%), 7q (58%), 11q (49%), and 15q (30%). The single BAC clone in 6q that showed a high frequency of loss also seemed to be commonly lost in our control versus control hybridizations. Thus, we believe it is an unstable clone and that this result most likely was an artifact.
Whereas most of the 3q gains in our study involved the entire arm, 3 cases were informative in mapping the centromeric proximal origin of this CNA to a genome position between chr3:125 Mb and chr3:127 Mb using the UCSC assembly database of July 2003 (hg16). The distal portions of all 3q amplicons were not informative, however, and the CNA appeared to extend all the way to the 3q telomere.
To validate the region of amplification on chromosome 3q, we performed QuMA on DNA from 5 of the AIN samples that showed 3q amplification. The results (data not shown) confirmed the proximal origin of the amplicon and the absence of termination of the 3q amplicon short of the telomere.
aCGH also showed the amount of gain or loss at each of the CNAs (Fig. 3). The common CNAs in chromosomes 1, 8, and 20 were not highly amplified (1 extra copy), whereas the region on chromosome 3q was more highly amplified (1.5 extra copies). Chromosome arm 4q showed a highly amplified region (4 extra copies), although this CNA occurred in only 1 high-grade AIN sample. This region was immediately followed by a loss at 4q21 that was identified in a second sample as well. Chromosome 17q25 was present in high copy number (3 extra copies) in 1 sample. The common regions of loss in chromosome arms 2q and 11q showed a single copy loss. Whereas the average copy number at 2q22 was similar for all clones in that region, there was an increase in the frequency of chromosome loss between clones RP11-119N3 and RP11-364H22 (2q: nuc. 137.9-151.9 Mb).
Our study uses the recently developed method of aCGH to reveal regional DNA CNAs in AIN lesions that are thought to be predecessors of anal cancer. Some of the most commonly occurring CNAs were on chromosome arms 1q (gain), 3q (gain), 7q (loss), 8p (gain), 11q (loss), 15q (loss), and 20p (gain). Other studies have shown a number of similar genomic alterations in cervical lesions, including CIN and invasive cervix cancer. Umayahara et al39 showed gains in chromosome arms 1q and 3q and losses in chromosome arm 11q in CINs and cervical cancer, and Kirchhoff et al20 showed gains in 1q, 3q, 8p, and 20q as well as losses in 7q for CINs. Whereas these previous studies of cervical cancer showed several other CNAs (the most common being +1p, −2q, −4p&q, −5p, and −6p), the high degree of coincidence between the reported alterations and our results for AIN likely reflect common disease pathways between these 2 HPV-related diseases.
Our data suggest that a close link exists between HPV integration and/or rearrangement and the presence of DNA CNAs. This strongly suggests that in AIN, HPV integration and/or rearrangement leads to genomic instability. Our results are supported by a recent study on CIN and microinvasive cervical cancer that showed that integration of HPV16/18 into host genome correlated with increased occurrence of numeric aberrations of chromosomes as an indicator of genetic instability.40 Sequence modification, including direct and inverted repeats, orphan nucleotides, and stretches of purines and pyrimidines, frequently have been observed at viral-cellular junctions in cervical cancer.35,41,42 Two of the 5 integrations sites identified in our study carried such modifications.
To define the region of amplification in 3q, we used aCGH analyses and QuMA to define the centromeric proximal terminus as chr3:125 to 127 Mb. This amplicon region commonly extended to the 3q terminus at chr3:199 Mb. Chromosomal CGH studies that have been performed on CINs and invasive cervical cancers showed a similar result, with essentially all amplicons extending to 3qter.18,39,43 The detection of a 3q gain in more than 70% of AIN samples with CNAs suggests that this copy number gain is an early event in the tumorigenesis process of AIN. This result is in contrast to a previous finding in cervical cancer that 3q gain was associated with a transition to malignant phenotype29 but is in agreement with a later report that 3q gain was commonly detected (6 of 13 cases) in moderate cervical dysplasia.20 The report of Kirchhoff et al20 stated that higher sensitivity of their CGH was the best explanation for this later finding. Because aCGH has higher sensitivity than chromosomal CGH,23 our results are consistent with the Kirchhoff explanation.
Because we seldom found variations in the amount of CNA within this 75-Mb region of amplification on chromosome arm 3q, we infer that there are several oncogenes dispersed along the region and that multiple genes are activated to progress from low-grade AIN to high-grade AIN. In the region from 3q21 to 3qter, 30 genes have been reported to serve as proto-oncogenes and/or were shown to be increased in various cancers. TP73L, located at 3q28, generates a p53-like product lacking the transactivation domain and can serve as dominant negative for expression of p53, thus acting as an oncogene. H-RYK (3q22.2), an atypical member of the receptor tyrosine kinase family that has been shown to activate the mitogen-activated protein kinase (MAPK) pathway,44 is overexpressed in ovarian tumors.45 Another is LAMP3 (3q27.1), a lysosomal-associated transmembrane protein that serves as a ligand for the E-selectin molecules present on endothelial cells, perhaps serving to promote or facilitate the metastatic process of tumors.
In the 3q region, there also are 3 genes that all are associated with the AKT pathway; MRAS (a RAS-related protein [3q22.3]) as well as PIK3CB (3q22.3) and PIK3CA (3q26.32), the 2 subunits of phosphoinositide-3-kinase. The AKT pathway signals control of apoptosis through BCLX, the caspase cascade and/or nuclear factor (NF)-κB control. In addition, the AKT pathway recently was shown to activate human telomerase reverse transcriptase (hTERT) to induce telomerase activity.46
Most of the samples in this study, including samples with high-grade AIN, were from HIV-positive study participants. This is consistent with earlier observations that HIV-positive MSM are likelier to have AIN and to progress to high-grade AIN than HIV-negative MSM.47,48 Our data do not allow us to determine the role of HIV infection in HPV genomic rearrangement or integration or in the genomic instability that leads to CNAs. Given the high proportion of HIV-positive men and women who develop anal high-grade AIN, however, it is likely that these are occurring more commonly and perhaps at a faster rate among HIV-positive men and women. It is possible that HPV genomic rearrangement, integration, and CNAs play a role in the increased risk of anal cancer associated with HIV infection.
In summary, we have shown that approximately 30% of high-grade AIN cases harbor DNA CNAs and that the presence of these alterations is highly correlated with integration and/or rearrangement of HPV. We also identified at high resolution a number of chromosomal regions that are frequently gained or lost in AIN. The striking similarities between anal and cervical neoplasia in histologic changes, HPV infections, and genomic alterations suggest a common tumorigenesis pathway. Further characterization at the molecular level and identification of deregulated genes are critical to our understanding and management of these diseases. Given the increased risk of anal cancer in HIV-positive men and women and their increased AIN lesion size compared with that in HIV-negative individuals, studies to determine the prognostic significance of these changes should be valuable to determine those cases or regions of AIN most in need of treatment to prevent anal cancer.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
anal intraepithelial neoplasia; anal cancer; array comparative genomic hybridization; human papillomavirus; quantitative microsatellite analysis