DNA Copy Number Changes in Cervical Adenocarcinoma¹

Cancer of the uterine cervix is the leading cause of cancer death among women worldwide. The estimated number of new cervical cancer cases per year is 500,000 of which 79% occur in the developing countries (1).

In the United States, the age-adjusted incidence rates per 100,000 for all invasive cervical cancers decreased by 36.9% over 24 years (1973–1977 versus 1993–1996). Similarly, the age-adjusted incidence rates for SCCs³ declined by 41.9%. In contrast, the age-adjusted incidence rates for ACs increased by 29.1%. The proportion of AC increased by 107.4% relative to all cervical cancers, by 95.2% relative to SCC, and by 49.3% in the population of women at risk (2).

The etiology of the cervical AC is less clear. Initially, an association between continuous use of oral contraceptives and the increased incidence of cervical AC had been suggested. However, other investigators found no convincing evidence for a direct link of oral contraceptive use to cervical neoplasia (3). The HPV is considered to be the most important cofactor in the development of cervical SCCs. Cervical ACs might also be related to HPV. Thus, a Scandinavian study found HPV in 71% of the 131 analyzed cervical ACs. HPV 18 was the most prevalent (52%), followed by HPV 16 (33%) and other types (15%). The prevalence of HPV infection was age-dependent. In women <40 years, HPV was present in 89%, whereas in women ≥60 years, HPV was observed in only 43% of the cases. The HPV-positive ACs were represented by an age distribution similar to that of cervical squamous carcinomas with a maximum age in the 40–49 age group. HPV-negative ACs occurred mostly in elderly women (4, 5).

Previous LOH (6), FISH, and CGH analyses identified common and contrasting genetic alterations in cervical carcinoma (Table 1). Overall, the data presented in Table 1 shows similar percentages of alterations throughout the selected chromosomal regions. However, within a region the amount of reported alterations differs. Heselmeyer et al. (7) showed that gains of chromosome 3q sequences could be the most consistent chromosomal aberration during the transition from high-grade cervical intraepithelial neoplasia to early invasive cervical carcinoma. Furthermore, they suggested (8) that gains on chromosomes 1q, 3q, and 5p could be particularly frequent in advanced-stage cervical carcinomas (clinical stage IIB-IV). We analyzed recently 62 cases of SCC of clinical stage Fédération Internationale des Gynaecologistes et Obstetristes IB, and demonstrated a correlation between the number of genomic alterations by CGH and poor prognosis. Chromosome 18q losses were associated significantly with poor disease outcome (9). In SCC, the most frequent aberrations were DNA sequence losses on chromosomes 4q (53%), 3p (52%), and 13q (45%).

There are only few molecular studies of cervical ACs. In cervical ACs (10), frequent LOH has been demonstrated for chromosome 4p (4p15.3). LOH on 4p was significantly more frequent in AC (82%) compared with SCC. It has been concluded that different patterns of DNA copy number changes between SCC and AC may represent gene regions targeted by different gene-environment interactions in these tumor subtypes. Yang et al. (11) presented recently a CGH study in 20 cases of cervical AC. They found that DNA copy number gains were more common than losses. The most consistent region of chromosomal gain was mapped to chromosome arm 3q and was found in 70% of the cases. Other recurrent amplifications of genetic material were detected on 17q (45%), 1p (30%), 1q (25%), and 11q (20%). DNA losses were seldomly observed, occurring primarily in under-represented regions of chromosome arms 4q, 13q, and 18q. Copy number changes on 17q are of particular interest, because important genes are located in the proximity of 17q11–21. These genes are HER-2/neu, BRCA1, and nm23. Alterations of these genes have been detected in human ACs of the breast (12, 13) and ovary (12), in which an association between genomic alterations and poor prognosis of the disease has been reported (14). However, there are few reports on genomic alterations of these genes in invasive cervical cancer (15, 16, 17, 18, 19).

Here, we investigated patterns of copy number changes in cervical ACs by CGH to analyze the genotype-phenotype correlation and to investigate their potential role for disease progression. In addition, we compared this new data with our results published recently of a CGH analysis in SCC (9).

Twenty-two radically operated cervical ACs with clinical stage IB from the years 1986–1995 were identified from the archives of the Institute for Pathology in Basel. Tumors were staged according to the fifth edition of the Union International Contre le Cancer (20) and the American Joint Committee on Cancer Tumor-Node-Metastasis System (21). Tumor volume, depth of invasion, histological grade, and presence of vascular invasion were assessed in all of the tumors as additional prognostic parameters. Five of 22 patients with cervical ACs had regional lymph node metastases. Clinical follow-up data were available for all of the patients. Disease-specific survival was defined as the time between primary treatment and death of patients as a result of the tumor.

Specimens of cervical ACs were trimmed to enrich for tumor cells by excising tumor tissue from the paraffin block. The excised tumor tissue was re-embedded in a paraffin block. The first and the last sections were stained with H&E to verify the presence of at least 75% tumor cells in the sample. Genomic DNA was isolated by proteinase K extraction according to standard protocols.

CGH analysis was performed as described (9, 22). Briefly, 20 μm-thick sections were deparaffinized and suspended in DNA extraction buffer containing 0.5 mg/ml proteinase K. Additional proteinase K was added 24 and 48 h later, for a total incubation time of 72 h. Two μg of tumor DNA were nick translated by using a commercial kit (BioNick kit; Life Technologies, Inc., Gaithersburg, MD). SpectrumGreen Direct-Labeled Total Human Genomic dUTPs (Vysis, Inc., Downers Grove, IL) was used for the direct labeling of tumor DNA. SpectrumRed-labeled normal reference DNA (Vysis) was used for cohybridization.

The hybridization mixture consisted of 200 ng of SpectrumGreen-labeled tumor DNA, 200 ng of SpectrumRed-labeled normal reference DNA, and 20 μg of human Cot-1 DNA (Life Technologies, Inc.) dissolved in 10 μl of hybridization buffer [50% formamide, 10% dextran sulfate, and 2 × SSC (pH 7.0)]. Hybridization was performed over 3 days at 37°C to normal metaphase spreads (Vysis, Inc.). Posthybridization washes were performed as described previously. Digital images were collected from six to seven metaphases using a Photometrics cooled CCD camera (Microimager 1400; Xillix Technologies, Vancouver, British Columbia, Canada) and a Sun workstation. The Vysis software program was used to calculate average green:red ratio profiles for each chromosome. At least four observations per autosome and at least two observations per sex chromosome were included in each analysis according to previous recommendations (23).

CGH experiments included a tumor cell line (Spectrum Green-labeled MPE-600 DNA; Vysis) with known aberrations (positive control) and a hybridization of two differentially labeled sex-mismatched normal DNAs to each other (negative control). Metaphases were used only if the color ratio of sex-mismatched normal DNAs was ≤0.66 on the X chromosome. The thresholds used for the definition of DNA sequence copy number gains and losses were based on the results of CGH analyses of formalin-fixed normal tissues. Gains of DNA sequences were defined as chromosomal regions where both the mean green:red fluorescence ratio and its SD were >1.20, whereas losses were defined as regions where both the mean and its SD were <0.80. Over-representations were considered amplifications when the fluorescence ratio values in a subregion of a chromosome arm exceeded 1.5. In negative control hybridizations, the mean green:red ratio occasionally exceeded the fixed 1.2 cutoff level at the after chromosomal regions: 1p32-pter, 16p, 19, and 22. Therefore, these known G-C-rich regions were excluded from all of the analyses.

HER-2/neu protein expression of the tumors was analyzed using the HercepTest kit (DAKO Diagnostics, Glostrup, Denmark) according to the manufacturer’s instructions. Briefly, the deparaffinized tissue sections were first incubated in a 95°C water bath for 40 min to induce epitope retrieval and then at room temperature for 30 min with the prediluted primary antibody to HER-2/neu. The bound primary antibody was visualized by use of the dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat antirabbit immunoglobins as provided by the manufacturer. We included the positive and negative control cell lines supplied with the HercepTest kit in each IHC assay to ensure the validity of the staining. Stained slides were analyzed by light microscopy by use of a ×10 objective. We scored immunohistochemical staining according to the manufacturer’s instructions. Tissue samples were classified as positive if they had a score of 2+ or 3+ and as negative if they had a score of 0 or 1+.

For the detection and typing of HPV DNA of HPV types 6/11, 16/18, or 31/33/51 in the specimens we used an in situ DNA hybridization procedure (ENZO PathoGene DNA Probe Assay; ENZO Diagnostics, Farmingdale, NY). Specific hybridization between the HPV DNA probes and DNA in the specimen was determined by the detection of biotin. As a chromogen we used 3-amino-9-ethylcarbazole mixed with hydrogen peroxide in acetate buffer. After counterstaining with hemalum and mounting with Crystal/Mount (Biømeda, Foster City, CA), the stained cells were observed by light microscopy.

Results are given as mean values and SD. Relationships between categorical features and counts were evaluated by the nonparametric method of the Mann-Whitney U test. Contingency table analysis was used to analyze the correlations among carcinomas with and without regional lymph node metastasis, and to analyze for frequency differences of genomic aberrations between AC and SCC. Survival was defined as the time between primary treatment and death with distant metastasis. Patients that survived were censored at the time of last follow-up. Survival analysis was performed using the Kaplan-Meier method with a log-rank test. The median values of the numbers of DNA aberrations, DNA sequence losses, and gains were used as cutoff points to define patients with high and low numbers of corresponding aberrations. A Cox proportional hazards analysis was used to test for independent prognostic information. Statistical analyses were performed by use of the StatView 4.5 Software program (Abacus Concepts, 1995).

All of the cervical ACs were HPV positive. Of 22 cervical ACs, 17 were positive for DNA of HPV 16/18, and 5 were positive for DNA of HPV 31/33/51. Thus, all of the tumor samples of both histological types of invasive cervical cancer were high-risk HPV positive.

Genomic aberrations were detected by CGH in all of the tumors. There was a median number of 6 aberrations per tumor (range: 0–12), 5 losses (range: 0–10), and 1 gain (range: 0–3). Losses were observed most frequently on chromosomes Xq (50%), Xp (36%), 18q (36%), 4q (36%), 9p (32%), 13q (27%), 5q (27%), and 3p (23%). Gains frequently involved chromosome 17q (54%) and 20q (23%). No high-level DNA amplifications were detected (Table 2). The total number of genomic aberrations was similar in patients with (7.0 ± 4.6) and without (5.1 ± 2.9) regional lymph node metastasis.

Survival data were available for all of the patients. There was a mean follow-up of 53.5 ± 38.9 months (median: 42 months). Clinical outcome of patients with regional lymph node metastasis (survival time: 20.4 ± 9.9; median: 17 months) was significantly worse than of nodal negative patients (P < 0.02). Therefore, survival analysis to study the association of genetic changes with clinical outcome was restricted to patients without regional lymph node metastasis, the mean follow-up time for them being 63.2 ± 38.9 months (median: 60 months).

To compare both histological types of cervical cancer, we used the data of our recently published CGH analysis of 62 Fédération Internationale des Gynaecologistes et Obstetristes stage IB cervical SCCs (9). These tumors were studied in the same laboratory with the same CGH protocol and the same evaluation criteria. The mean follow-up time was similar in both tumor sets. The 5-year survival was 70% in SCC and 64% in AC. There was no significant difference in the observed frequency of the total number of genomic aberrations between ACs and SCCs (Table 2). No differences were observed between the subgroups of tumors with and without regional lymph node metastasis. All of the specific aberrations were analyzed for frequency differences between AC and SCC. Copy number gains on chromosome 17q were more frequent in cervical ACs than in SCCs (54% versus 27%; P < 0.008). On the other hand, losses on 4p (P < 0.03), 3p (P < 0.08), and 13q (P < 0.08) were more common in SCCs than in ACs (Table 3). All of the samples of both invasive cervical ACs and SCCs were HPV positive.

The analysis of the association between genomic alterations and survival showed that DNA sequence losses on chromosome 18q are significantly associated with poor overall survival (Fig. 1) in node-negative ACs. There was no difference in the prognosis of tumors with a high number of aberrations (≥6 per tumor) compared with tumors with a low number of aberrations. Cox proportional hazard analysis in all of the cervical ACs with the parameters depth of invasion, histological grade, vascular invasion, tumor volume, loss of 11p, loss of 18q, gain of 17q, DNA losses, and gains indicated that regional lymph node status, depth of invasion, vascular invasion, and loss of 18q were independent prognostic variables (Table 4).

We found DNA copy number gains frequently on chromosome 17q. Therefore, we included Her-2 expression analysis in our investigation. Among the 22 tumors HER-2/neu positivity (score 2+) was detected in only 2 tumors (9%; Fig. 2), and one of them also showed DNA sequence gains on chromosome 17q.

In this study, we analyzed genomic aberrations in cervical ACs. Neither the number of DNA sequence losses nor the number of DNA sequence gains per tumor were related to disease outcome. However, DNA sequence losses on chromosome 18q were associated with a poor prognosis. Losses on 18q also were an important prognostic parameter in our CGH study on SCC. Three candidate tumor suppressor genes DCC (24), DPC4 (25), and MADR2 (26) have been identified on the long arm of chromosome 18. Two of these genes, DPC4 and MADR2, are of particular interest, because they are important mediators in the transforming growth factor β pathway. For the DCC gene, Klingelhutz et al. (27) showed that the progression of HPV-transformed epithelial cells to tumorigenic cells was accompanied by LOH affecting the DCC gene in a way that resulted in loss of DCC expression. Tumorigenicity was suppressed when a DCC expression vector was transfected back into these cells, thus concluding that the DCC gene suppresses the malignant phenotype of transformed epithelial cells. Therefore, sequence losses on 18q might contribute to a more aggressive tumor cell phenotype in cervical SCC as well as in cervical AC.

The distribution pattern of genomic aberrations in cervical ACs was different from the genomic pattern in SCCs. There were significantly more copy number losses on chromosome 4p in SCCs and significantly more copy number gains on chromosome 17q in ACs. This might suggest different pathways in malignant cell transformation in different histological types of cervical cancer.

In this study, >50% of patients with cervical AC had chromosome 17q copy number gains. There were no high level amplifications on chromosome 17q. The HER-2/neu (ErbB2) proto-oncogene, the metastasis-suppressor gene nm23, and the BRCA1 gene are located on chromosome 17q. HER-2/neu protein overexpression has been associated with poor prognosis in a variety of tumors (28), but its significance in cervical cancer of different histological types still remains unclear. Only 9% of the cervical ACs showed an HER-2/neu protein over-expression. This is consistent with previous studies, showing rare HER-2/neu expression in cervical ACs (16, 17). These findings suggest that amplification of HER-2/neu is rare in cervical ACs and that low level chromosome 17q copy number gains are not associated with HER-2/neu overexpression.

nm23 has been identified as a metastasis-suppressor gene also located on 17q. Its expression was found to be inversely correlated with metastasis and poor prognosis in breast cancer (29), colorectal cancer (30), and ovarian carcinomas (31). In cervical AC but not in SCC, the reduced expression of the nm23 protein was related to unfavorable disease outcome (19).

The BRCA1 locus may interact with HER-2/neu, nm23, and possibly other loci in a certain way during malignant progression (32). To our knowledge, there is no report on the significance of BRCA1 alterations in both histological types of invasive cervical cancer.

In summary, our data highlight chromosomal regions that may harbor important genes for both histological types of cervical cancer. DNA sequence losses of chromosome 18q may be relevant for progression in squamous cell as well as ACs. The higher frequency of chromosomal copy number gains on chromosome 17q and the different frequency distribution of losses in AC compared with SCC indicate a diverging genomic pattern and may be indicative for multiple genetic pathways in cervical tumorigenesis.

We thank Elisabeth Schultheiss, Carole Egenter, and the staff of the Institute for Pathology, University of Basel, for their excellent technical support.