Molecular Biology of Gynecologic Cancer

Proto-oncogenes are deoxyribonucleic acid (DNA) sequences found in the genomes of normal and neoplastic tissues that are quite similar to the transforming genes of ribonucleic acid (RNA) tumor viruses. Proto-oncogenes exhibit striking evolutionary conservation, being found in a large number of phylogenetically diverse organisms ranging from Drosophila flies to humans. More than 40 proto-oncogenes have been described, and this list will undoubtedly continue to expand. Protooncogenes encode growth factors, growth factor receptors, and proteins that regulate cell growth and differentiation. Proto-oncogenes are usually grouped according to the primary function or sequence homology of their protein products (Table 1).

TABLE 1. Proto-Oncogene Classification

CSF = colony-stimulating factor; EGF = epidermal growth factor; FGF = fibroblast growth factor;
GTP = guanosine triphosphate; GTPase = guanosine triphosphatase;
PDGF = platelet-derived growth factor

Tumor suppressor genes are a second major group of genes that regulate cell growth. These genes, which include the p53 gene and the retinoblastoma gene, encode DNA-binding proteins that normally act to inhibit cellular proliferation.

Proto-oncogenes and tumor suppressor genes encode proteins that play important roles in every step of the intracellular signal transduction system and permit the cell to respond to external stimuli in a specific, coordinated, and controlled fashion (Fig. 1). The net result of the expression of proto-oncogenes and tumor suppressor genes is the regulation of cell growth and differentiation.

Membrane receptors bind extracellular molecules, such as growth factors and steroid hormones, initiating a cascade of events that culminates in altered gene expression. These receptors are often linked to inner membrane receptors, perhaps the most notable of which are encoded by the ras gene family. The ras genes encode proteins that exhibit guanosine triphosphate (GTP) binding or guanosine triphosphatase (GTPase) activity, or both. These proteins, collectively designated p21, are attached to the inner surface of the cell membrane. These proteins couple membrane receptors to a number of signaling systems, including cyclic adenosine monophosphate and the inositol phospholipid pathway.

Cytoplasmic messenger systems transmit external signals from the cell membrane to the nucleus. This part of the regulatory network involves protein phosphorylation activity and is the target of substances such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, colony-stimulating factor, and insulin-like growth factor (see below).

Numerous proto-oncogenes have been identified that encode nuclear DNA-binding proteins that regulate transcription of other genes. Typically, more than one gene acts in concert with several other genes to trigger a change in proliferation. As an example, increased expression of c-jun and c-fos, followed by an increase in c-myc, is required for the transition from the G0 to the G1 phase of the cell cycle. In contradistinction, the p53 tumor suppressor gene encodes a protein that normally acts to inhibit cellular proliferation. Expression of the p53 gene also increases in cells that have sustained DNA damage so that the damage is not passed to daughter cells before repair.

Data derived from in vitro and in vivo models clearly demonstrate that proto-oncogenes, acting as a subset of a larger group of genes, play crucial roles in the regulation of cellular proliferation and differentiation. Studies of the placenta and endometrium are of particular interest to the gynecologist. In the placenta, the trophoblast cell population changes from one dominated by the “pseudo-malignant” cytotrophoblast to one composed predominantly of the highly differentiated syncytiotrophoblast. This transition is accompanied by discrete changes in the expression of several proto-oncogenes. The expression of c-fms, confined to the placenta and extraembryonic membranes, exhibits a progressive increase in expression during prenatal development in the mouse.¹ In contradistinction, the expression of c-fos is approximately 15-fold greater in cytotrophoblasts than in syncytiotrophoblasts.² The transcription of c-myc appears to be directly associated with proliferative activity of the trophoblast.^{3, 4, 5} Peak c-myc messenger RNA expression occurs at 4 to 6 weeks' estimated gestational age, followed by a rapid decline to barely detectable levels at term. The expression of the Ha-ras proto-oncogene demonstrates little variation with gestational age in placental tissue.

Normal endometrium exhibits cyclic proliferation, differentiation, and exfoliation in response to estrogen and progesterone. These events, which collectively characterize the menstrual cycle, are accompanied by discrete changes in the expression of myc, ras, and fos. Interestingly, the expression of these genes varies not only with the phase of the menstrual cycle, but between the glandular and stromal cell compartments as well.⁶ Cell- and tissue-specific changes in proto-oncogene expression also have been documented in the developing embryo.^{7, 8, 9}

In view of the crucial role that proto-oncogenes and tumor suppressor genes play in normal cell growth and differentiation, it is a logical corollary that the abnormal expression of these genes is causally related to the uncontrolled cellular proliferation that characterizes the neoplastic phenotype. Cellular transformation requires the mutation of several genes via a multistep process. Genetic mutations can result from gene amplification, point mutations, insertional mutagenesis, and deletions or rearrangements, or both (Fig. 2).

Gene amplification, in which the copy number of the gene is increased, is readily detected by southern hybridization analysis. Amplification can result in enhanced gene expression by increasing the amount of template DNA available for transcription. Proto-oncogene amplification is a relatively common event in gynecologic malignancies. In most cases, enhanced oncogene expression accompanies oncogene amplification.

Point mutations, in which the codon sequence is altered, can result in altered gene action by qualitatively altering the gene product. The ras proto-oncogene and the p53 tumor suppressor gene are two of the best-studied genes in gynecologic malignancies that exhibit point mutations. Point mutations of the ras gene occur in approximately 15% of solid tumors. The replacement of a single amino acid in the ras gene product as the result of a base mutation at codon 12, 13, or 61 results in the synthesis of a p21 ras protein with increased transforming ability.¹⁰ The mutated p21 protein exhibits decreased GTPase activity, and the bound GTP is hydrolyzed to guanosine diphosphate more slowly.¹¹ As a consequence, the mutant protein is constitutively activated, and a continuous signal is transduced to the nucleus.

Point mutations of the p53 gene occur at preferential “hot spots” that coincide with the most highly conserved regions of the gene. Loss of normal p53 gene activity removes one of the endogenous “brakes” on cellular proliferation. In addition, point mutations of the p53 gene impair the ability of the cell to repair DNA damage before entry into the S-phase of the cell cycle. As an example, irradiated cells normally exhibit a transient S-phase arrest as p53 gene expression increases and radiation-induced DNA damage is repaired. When a mutated p53 gene is present, the delay at the G1-S checkpoint does not occur, and the DNA damage is passed to the daughter cells. Mutation of the p53 gene is the most frequent genetic alteration found in solid tumors.

Insertional mutagenesis occurs when the regulation of gene expression is altered when DNA sequences that control transcription are deleted, transposed, or placed under the control of a new DNA sequence. Human papillomavirus (HPV), implicated as an important cofactor in the etiology of squamous cell dysplasia and carcinoma, occasionally integrates near the c-myc oncogene during incorporation into the host genome. Juxtaposition of HPV genetic sequences near c-myc has been associated with increased expression of this gene.¹² The HeLa cell line contains integrated HPV18 near the c-myc oncogene, which is also overexpressed. In situ hybridization analysis indicates that multiple copies of this virus also integrate near the c-abl and c-sis oncogenes.¹³

Deletions and rearrangements reflect gross changes in the DNA template that result in the synthesis of a markedly altered protein product.

The transition from normal cervical epithelium to dysplasia to invasive disease is well-defined histologically and provides a useful framework upon which molecular alterations can be evaluated. To date, molecular biologic studies have focused primarily on genetic alterations present in invasive cervical cancer.

Structural alterations of the Ha-ras gene reported to occur in cervical cancer include allelic deletions and point mutations. Riou and associates¹⁴ reported that Ha-ras exhibited allelic loss in the three variable tandem repeats in 36% of heterozygous tumors. Point mutations of Ha-ras, codon 12, were found in 8 of 76 cervical carcinoma specimens, the majority of which were from patients with advanced-stage disease. In addition, 40% of the tumors with codon-12 mutations also exhibited allelic deletions. Other investigators, however, have failed to demonstrate point mutations in a significant proportion of cervical neoplasms.^{15, 16}

The c-erbB gene, which encodes the epidermal growth factor receptor (EGF) receptor, can be overexpressed in cervical neoplasms. Elevated amounts of EGF receptor have been found in severe dysplasia and invasive cervical carcinoma specimens.¹⁷ Kohler and colleagues¹⁸ performed a comprehensive analysis of EGF receptor in cervical carcinoma specimens using a radioligandbinding assay, northern hybridization, and immunohistochemistry. Thirty-four of 42 specimens expressed EGF receptor in high amounts (greater than 10 fmol/mg specific binding) in 42%. Burchuck and co-workers¹⁹ reported that squamous cell carcinomas of the cervix consistently expressed moderate to heavy immunohistochemical staining for EGF receptor.

The c-myc proto-oncogene also has been studied in cervical cancer.^{20, 21,22, 23} Amplification has been reported to occur in 30% to 80% of cases. Of particular interest are the reports by Riou and associates,^{24, 25, 26, 27, 28} who correlated amplification and expression of the c-myc gene with prognosis in early-stage cervical carcinoma. Using northern hybridization and slot blot analysis, they found c-myc overexpression in 33% of 93 cervical carcinoma specimens. They found no apparent relationship between c-myc expression and histology, tumor differentiation, status of the surgical margins, presence or absence of lymphovascular space involvement, or endocervical extension. They considered overexpression of c-myc in lymph node—negative patients, however, to be predictive of subsequent relapse. Three-year survival for patients with normal c-myc expression and negative pelvic lymph nodes was 93%, compared with only 51% for patients with overexpression of the c-myc oncogene and negative lymph nodes. Patients with nodal metastases at the time of radical hysterectomy who had tumors with normal c-myc expression had a 3-year survival of 44%, compared with only 15% for patients with tumors exhibiting overexpression of c-myc.

The expression of c-myc in paraffin-embedded tumor biopsies from patients with advanced-stage disease does not appear to be a prognostic indicator. Symonds and associates²⁹ found that immunohistochemical positivity for c-myc was not associated with time to local recurrence or development of metastases or survival in 55 patients with stage III or stage IV cervix cancer who were treated with neoadjuvant chemotherapy followed by radiation therapy.

The p53 tumor suppressor gene is not commonly mutated in cervical neoplasms. Busby-Earle and colleagues³⁰ studied 57 cases of cervical cancer and found p53 mutations in 2 cases. Helland and co-workers³¹ evaluated 92 cervix neoplasms by PCR and DGGE and found only two examples of p53 mutations. When mutations are present, they are often found in HPV-negative neoplasms. Fujita and associates³² found p53 mutations in 2 of 36 cervical neoplasms.

Immunohistochemical studies of p53 have revealed detectable expression in a variable proportion of cases. Busby-Earle and colleagues reported p53 positivity in 8 of 57 cervical neoplasms, and the pattern of staining was described as sparse.³⁰ Bosari and co-workers, however, reported p53 positivity in 74% of biopsy specimens from invasive squamous cell carcinomas of the cervix.³³ These observations may initially seem inconsistent with the reported rarity of p53 gene mutations in cervical cancer. It is generally presumed that the very short half-life of the normal p53 protein and the small amounts present in normal cells precludes detection by immunohistochemical methods; however, immunohistochemical detection of normal p53 protein has been reported, in colon and lung carcinoma cell lines in the absence of mutations.^{34, 35} Interruption of the degradation of normal p53 protein is the suggested mechanism.³⁶

With respect to dysplastic lesions, Pinion and associates evaluated the amount of Ha-ras p21 protein in cervical biopsy specimens using fluorescent immunohistochemistry with quantitation by automated image analysis.¹⁷ Elevated amounts of the Ha-ras protein product were found in severe dysplasia and invasive carcinoma biopsy specimens as compared with normal cervical tissues. Sagae and colleagues³⁷ also reported that ras p21 protein expression increased with lesion severity.

Berchuck and co-workers reported that the basal keratinocytes of normal cervical epithelium express EGF receptor.¹⁹ They found that keratinocyte differentiation was associated with a decrease in staining intensity of the EGF receptor protein. As one might anticipate, carcinoma in situ lesions, characterized by full-thickness loss of differentiation, exhibited full-thickness expression of EGF receptor.

Investigations that have focused on the oncogenic potential of HPV in cervical neoplasms have provided a framework upon which some of the observations concerning oncogenes and tumor suppressor genes can be unified (Fig. 3). Although there are no data to indicate that HPV by itself is capable of causing either cervical dysplasia or cancer, there is abundant evidence to suggest that it is an important cofactor in the pathogenesis of cervical neoplasms. When found in invasive squamous cell carcinomas of the cervix, HPV16 and HPV18 are integrated into the host genome. On occasion, the HPV may insert near chromosomal fragile sites. Some of these sites are also in close proximity to cellular oncogenes, supporting the hypothesis that insertional mutagenesis may be one mechanism of HPV-related oncogenesis.

Couturier and associates³⁸ demonstrated that HPV sequences were localized to chromosome band 8q24.1, which is also the location of c-myc. In each instance, the c-myc gene was structurally altered or overexpressed. Hori and colleagues³⁹ also reported that HPV16 was integrated in a fragile site near 8q24.1 in a cell line established from a small cell carcinoma of the cervix. Popescu and DiPaolo⁴⁰ have also reported that fragile sites are preferential locations for HPV integration. Apart from insertional mutagenesis, specific HPV protein products appear to exert inhibitory effects on the p53 and retinoblastoma tumor suppressor gene proteins. HPV16 and HPV18 encode proteins that can bind to the gene products of tumor suppressor genes. As a consequence, the normal activity of these tumor suppressor gene products, which is the inhibition of cellular proliferation, is negated. Interaction of HPV16 E7 protein with the retinoblastoma gene product results in inactivation of its function.⁴¹ The E6 protein of HPV16 and HPV18 interacts with the p53 gene product, accelerating its degradation.^{42, 43} The E6-p53 protein interaction is an appealing explanation for the role of HPV in the etiology of cervical dysplasia and neoplasia, although its biologic significance must be clarified.

Although HPV is present in the majority of squamous cell carcinomas of the cervix, there are well-documented, albeit infrequent, instances of HPV-negative cervical neoplasms.^{44, 45} In some of these cases, the p53 protein contains a point mutation that results in a gene product with a prolonged half-life. As a consequence of these mutations, the p53 protein interaction with cellular heat shock proteins is enhanced, and confirmation changes occur that abrogate the p53 genes' product activity as an inhibitor of cellular proliferation. Not all HPV-negative cervical neoplasms, however, contain a mutated p53 gene. In addition, there is no apparent correlation between p53 levels and tumorigenicity. The observation that reintroduction of an intact single copy of chromosome 11 into HeLa cells or SiHa cells reverses their tumorigenicity suggests the presence of an important, but unidentified, tumor suppressor gene that may play a crucial role in the development of cervical cancer.^{46, 47}

Like cervical cancer, endometrial cancer is preceded by precursor lesions that have been histologically characterized. Recent investigations have focused on the molecular genetic changes in both adenocarcinoma and hyperplasia in an effort to determine the genetic mutations that may be causally related to the etiology of this disease.

Point mutations of the ras gene family commonly occur in endometrial adenocarcinoma and atypical adenomatous hyperplasia. The published data are summarized in Table 2.^{48, 49, 50, 51, 52, 53, 54} It appears that ras mutations in endometrial adenocarcinoma most commonly involve the second nucleotide of codon 12 of the Ki-ras gene. Mutations of the Ki-ras gene may signify an “early” event in endometrial carcinogenesis because it is also present in precursor lesions. At this time, abnormal expression and mutations of the ras gene family do not appear to be of prognostic significance. Scambia and co-workers,⁵⁵ however, reported that estrogen receptor—positive neoplasms express higher levels of p21 than estrogen receptor—negative tumors. They observed a similar trend between p21 and the progesterone receptor. Interestingly, they found p21 expression to be unrelated to EGF receptor levels.

TABLE 2. Ki-ras Codon-12 Mutations in Endometrial Neoplasms

Published data concerning p53 mutations in endometrial adenocarcinoma are summarized in Table 3.^{56, 57, 58, 59, 60, 61} Mutations of the p53 tumor suppressor gene have not been described in precursor lesions such as atypical adenomatous hyperplasia, suggesting that this mutation is unlikely to be an early event in the development of endometrial adenocarcinoma.

TABLE 3. p53 Mutations in Endometrial Carcinoma

Amplification of the c-myc oncogene appears to be a relatively common event in endometrial adenocarcinoma, occurring in approximately 30% of cases.⁶² There are no data concerning the frequency of this mutation in precursor lesions.

The c-erbB and HER-2/neu genes commonly exhibit abnormal expression in endometrial adenocarcinoma. In one study of 34 endometrial adenocarcinoma specimens,⁶³ 21 exhibited normal amounts of EGF receptor. In 13 specimens, the amount of EGF receptor was decreased, the greatest reduction occurring in poorly differentiated lesions. In contradistinction, another immunohistochemical study of 69 cases of endometrial carcinoma⁶⁴ demonstrated EGF receptor expression in 49% of the specimens. EGF receptor positivity was correlated with nonendometrioid histologies, metastatic disease at the time of diagnosis, and decreased survival. HER-2/neu overexpression was present in 59% of specimens and was significantly associated with depth of myometrial invasion.

Other studies have also demonstrated a role for HER-2/neu expression as a prognostic indicator. Berchuck and associates⁶⁵ studied 95 endometrial carcinomas using immunohistochemistry and found that overexpression of HER-2/neu correlated with advanced-stage disease and decreased survival. Hetzel and colleagues⁶⁶ reported similar results after evaluating HER-2/neu immunohistochemical expression in 247 endometrial carcinoma specimens. Strong overexpression of HER-2/neu was correlated with decreased progression-free survival and decreased overall survival.

Interestingly, overexpression of the HER-2/neu gene product has been associated with the absence of the estrogen receptor.⁶⁵ This may represent one way in which the neoplasm circumvents its requirement for estrogen-dependent growth.

The c-fms proto-oncogene encodes the colony-stimulating factor (CSF) receptor. Baiocchi and co-workers⁶⁷ reported that five of nine tumor samples expressed M-CSF and that six of nine samples expressed the c-fms transcript. Leiserowitz and associates⁶⁸ demonstrated that the amount of c-fms transcript increases from secretory to proliferative to hyperplastic endometrium. The highest levels were found in adenocarcinoma specimens. It has been proposed that an interaction between M-CSF (the ligand) and c-fms (which encodes the ligand receptor) might contribute to tumor progression and metastatic disease via an autocrine mechanism, in which the tumor stimulates itself to proliferate. In vitro studies have supported this hypothesis.⁶⁹ CSF receptor—positive cell lines were found to exhibit increased invasiveness in the amniotic basement membrane model compared with CSF receptor—negative cell lines after stimulation with CSF.⁶⁹ Overexpression of c-fms has been correlated positively with adverse clinical prognostic indicators.^{67, 68, 70}

Unlike cervical cancer and endometrial cancer, epithelial ovarian cancer does not have an accepted identifiable precursor lesion. In addition, most studies of molecular genetic alterations in ovarian cancer have been performed with tissue from advanced-stage neoplasms. For these reasons, it has been more difficult to differentiate between molecular genetic alterations that are causally related to disease development and alterations that may simply reflect the inherent genetic instability of advanced neoplasms. These difficulties notwithstanding, a number of genetic alterations have been identified in epithelial ovarian cancer.

Amplification of the c-myc gene has been described in approximately one third of advanced-stage epithelial ovarian neoplasms.^{71, 72} Sasano and colleagues⁷³ studied the immunocytochemical localization of c-myc in 18 archival ovarian carcinoma specimens; 14 serous carcinomas were positive for c-myc expression, and 4 of 4 mucinous carcinomas exhibited c-myc expression.

Elevated levels of ras p21 protein have been reported by several investigators.^{74, 75, 76, 77} Amplification of the ras gene has also been reported.^{78, 79, 80, 81} Prognostic roles for ras gene amplification, point mutation, or overexpression have not been identified. In addition, the role of ras gene abnormalities in the initiation and progression of epithelial ovarian cancer remains to be determined.

The HER-2/neu gene is commonly amplified in ovarian cancer. Slamon and co-workers⁸² reported that this gene was amplified in 31 of 120 cases of epithelial ovarian cancer. Following this study, a number of reports have been published concerning the expression of HER-2/neu in epithelial ovarian cancer (Table 4).^{83, 84, 85, 86, 87, 88, 89, 90} It appears that HER-2/neu positivity is more common in well-differentiated lesions, endometrioid cell types, and advanced-stage neoplasms.^{85, 89} The prognostic significance of HER-2/neu overexpression it still being debated; however, the majority of reports suggest little, if any, role.

TABLE 4. HER-2/neu as a Prognostic Indicator in Epithelial Ovarian Neoplasms

The c-erbB proto-oncogene, which encodes EGF receptor, is commonly overexpressed in ovarian carcinoma. Normally, EGF receptor mediates cellular proliferation by binding EGF or transforming growth factor-α (TGF-α).

The c-fms proto-oncogene encodes the receptor for CSF. Baiocchi and associates⁶⁷ found elevated levels of mRNA c-fms transcripts in 78% of 17 stage III ovarian cancers. With the exception of one neoplasm, all of these lesions also expressed M-CSF, which is the ligand for the CSF receptor. Kacinski and colleagues⁹⁰ found that 14 of 14 ovarian cancer specimens expressed fms transcripts and that 7 also expressed M-CSF. It has been suggested that c-fms and M-CSF contribute to autocrine and paracrine induction of neoplastic proliferation and progression in ovarian cancer. Abnormal expression of c-fms has not been demonstrated to be of prognostic significance in multivariate analyses.

Point mutations of the p53 gene have been studied in some detail in ovarian cancer. Kihana and co-workers⁹¹ studied 14 epithelial ovarian cancers and found abnormalities of the p53 gene in 10. Four of these mutations were in exon 7, two were in exon 5, two were in exon 2, one was in exon 8, and one was in exon 6. The point mutations in the p53 gene are typically transitions (purine to purine or pyrimidine to pyrimidine) and suggest mutations resulting from spontaneous errors in DNA synthesis and repair, rather than carcinogen-induced transversions (purine to pyrimidine and vice versa).

Mazars and associates⁹² studied 30 primary ovarian cancer specimens and found that 11 had a mutated allele. Point mutations were restricted to exons 5 and 7. Both the primary lesion and the metastatic lesions from the same patient exhibited the same codon changes, indicating the clonality of epithelial ovarian cancer. Similar results indicating the clonality of epithelial ovarian cancer have been published by other investigators.^{93, 94}

In general, point mutations of the p53 gene result in a protein product with a prolonged half-life, allowing p53 protein to be detected by immunohistochemical methods. Marks and colleagues⁹⁵ examined p53 expression in 107 epithelial ovarian neoplasms, of which 54 exhibited overexpression of p53. There was no correlation between overexpression and stage, grade, or the ability to resect the tumor optimally.

Kohler and co-workers analyzed 53 stage I and II epithelial ovarian neoplasms and reported overexpression in 15.⁹⁶ Stage IC and II neoplasms, poorly differentiated lesions, or neoplasms larger than 10 cm were more likely to overexpress p53 compared with neoplasms without these features, although the differences were not statistically significant. They found that overexpression of p53 was not associated with recurrence or survival. Berchuck and associates⁹⁷ found overexpression of the p53 gene in only 2 of 49 ovarian neoplasms of low malignant potential.

The etiologic role of p53 in ovarian cancer has not been determined, although there is no evidence based on immunohistochemical analyses to suggest that it is an early event. Although p53 is often overexpressed, particularly in advanced-stage and poorly differentiated neoplasms, the true incidence of p53 mutations is not known. Kupryanczyk and colleagues⁹⁸ have clearly demonstrated that immunohistochemical evidence of p53 expression in epithelial ovarian cancer provides an underestimate of p53 mutations compared with the results obtained by molecular genetic analysis of exons 2 to 11. Nonsense mutations, splicing mutations, and deletions might not result in p53 accumulation.

A number of genetic mutations have been described in ovarian cancer, but the specific alterations that are causally involved in the etiology of this disease have not been identified. Presumably, repeated ovulation with disruption of the germinal epithelium, followed by activation of cellular repair mechanisms, provides ample opportunity for somatic gene mutations and deletions to occur.⁹⁹ Recently described in vitro models will promote testing of this hypothesis.¹⁰⁰