Figure 37-1 Intra-abdominal spread of ovarian cancer. Ovarian cancers metastasize through lymphatics to lymph nodes at the level of the renal hilus, through blood vessels to the liver and other organs, but most frequently over the surface of the parietal and visceral peritoneum from pelvis to diaphragm. Blast Jr RC, Mills GB. Molecular pathogenesis of epithelial ovarian cancer. In: Mendelsohn J, Howley P, Israel M, Gray JW, Thompson CB, eds. The Molecular Basis of Cancer, 3rd ed. Philadelphia, Pa: Saunders-Elsevier; 2008:441-454.
Given the location of the ovaries within the pelvic cavity and the difficulty in assessing abnormalities on routine gynecologic examination, the disease is diagnosed only after it has metastasized in approximately 80% of cases. Ovarian cancer is often described as a “silent killer,” but the disease is generally symptomatic, even at early stages in 89% of cases. 8 Symptoms are not, however, specific and are generally attributed to benign gastrointestinal, genitourinary, musculoskeletal, or gynecologic conditions. Detection of a pelvic mass by physical examination or transvaginal sonography generally prompts exploratory surgery to remove the primary tumor and as much of the metastatic disease as possible—so-called cytoreductive surgery. Chemotherapy is generally given for 18 weeks thereafter using a combination of cytotoxic drugs including a taxane (paclitaxel or docetaxel) and a platinum derivative administered intravenously or directly into the peritoneal cavity.
In the 20% of patients with disease that is still localized to the ovaries (stage I), the prognosis is excellent, with up to 90% survival at 5 years using currently available surgery and chemotherapy. As the disease spreads to the other pelvic organs (stage II), to the peritoneal cavity and retroperitoneum (stage III), or to the hepatic parenchyma, pleural cavity, or lymph nodes outside the abdomen (stage IV), the prognosis becomes progressively worse, with a 5-year survival of less than 10% in the last group. Overall, 5-year survival rates have improved significantly (P < .05) from 37% in the 1970s to 45% in the 2000s, 2 related in large part to improvements in cytoreductive surgery and combination chemotherapy with carboplatin and paclitaxel. Over the past decade, however, median 5-year survival has not improved for patients with newly diagnosed advanced-stage ovarian cancer treated on clinical protocols of the Gynecologic Oncology Group. Moreover, long-term survival for women with advanced disease has not improved dramatically over the past three decades, and 70% of patients eventually succumb to the disease.
Figure 37-2 Intraperitoneal metastases from epithelial ovarian cancer studding the peritoneal surface.
Cellular and Molecular Characteristics of Ovarian Cancer Cells
Heterogeneity of Ovarian Cancers
As in many other malignancies, epithelial ovarian cancer is a clonal disease that arises from a single cell in more than 90% of cases. 9 Despite a clonal origin, epithelial ovarian cancers exhibit marked heterogeneity at a molecular, cellular, and clinical level.
Cell Proliferation
Among cancers from different patients with invasive cancer, the fraction of cycling cells can vary from 1% to 79% with a mean of 9% to 34% in different series. 10 Cyclin D1, cyclin E, and CDK2 are upregulated in a minority of cancers with their DNA copy numbers or protein levels correlating inversely with survival. Conversely, the p16, p21, and p27 CDK inhibitors are downregulated or mislocalized in a fraction of cancers, associated with a poorer outcome.
Figure 37-3 Different histotypes of epithelial ovarian cancer.
Histotypes
Ovarian cancers exhibit distinct histotypes—serous, endometrioid, clear cell, mucinous—that resemble epithelial components of normal fallopian tube, endometrium, vagina, endocervix, or intestine (Figure 37-3 ). Histotypes differ with regard to risk factors, genetic abnormalities, expression of tumor markers, and response to chemotherapy. 10 Each histotype exhibits a distinctive pattern of gene expression judged by array analysis, real-time reverse transcriptase–polymerase chain reaction (RT-PCR), and immunohistochemistry. 11 Molecular alterations in ovarian cancers of different histotypes correlate with changes in the normal tissues that they resemble morphologically. The HOX family of homeobox genes plays an important role in determining the histotype of ovarian cancers. 12 During normal development, HOXA9 is solely expressed in the primordia of the fallopian tubes, HOXA10 in the developing uterus, HOXA11 in the lower uterine segment and cervix, and HOXA13 in the future upper vagina. Expression of these HOX genes is retained in adult tissues but is not observed in ovarian surface epithelia. Expression of HOXA9, HOXA10, and HOXA11 is recapitulated in serous, endometrioid, and mucinous epithelial ovarian cancers, and enforced alterations in expression alter cellular histotype, indicating a causal role. 12
Low and High Grade (Type I and II)
Low- and high-grade ovarian cancers differ not only in histologic differentiation, but also in pathogenesis, genotype, rate of growth, prognosis, and response to therapy, permitting separation into Type I (low-grade) and Type II (high-grade) lesions. 13–15 Type I cancers include low-grade serous, mucinous, endometrioid, and clear cell histotypes and are often diagnosed in early stage (I or II), grow slowly, and resist conventional chemotherapy. Low-grade tumors frequently express estrogen receptors and may respond to tamoxifen or aromatase inhibitors. The more prevalent Type II cancers include high-grade serous, endometrioid, or undifferentiated histotypes, present at late stage (III or IV), grow aggressively, and respond to conventional chemotherapy, but only occasionally to endocrine therapy. Thus, the distinction between Type I and Type II ovarian cancers can inform choice of treatment. 15,16
Whereas Type II serous cancers appear to arise de novo from the walls of ovarian cysts or the surfaces of the ovary or fallopian tube, Type I low-grade serous cancers can grow from noninvasive serous “borderline” tumors of low malignant potential in 60% of cases. High-grade Type II ovarian cancers respond to primary chemotherapy with carboplatin and paclitaxel in approximately 70% of cases. Low-grade serous tumors are resistant, but not refractory, to primary platinum-based therapy. Recurrent low-grade serous ovarian cancer has a very low rate of response. 17 Low-grade mucinous and clear cell histotypes respond to conventional chemotherapy in only 26% and 15% of cases, respectively. 2,17
Low-grade serous cancers exhibit a relatively normal karyotype with wild-type TP53 and BRCA1/2, but exhibit frequent mutations in KRAS genes in 19% to 54% of cases. Low-grade serous cancers express the insulin-like growth factor receptor, and the majority overexpress the IGF-1 growth factor, providing a potential target for therapy. Frequent mutations of KRAS are found in mucinous cancers and in adjacent borderline tumors, consistent with the mutated gene driving malignant progression. A similar pattern of gene expression has been observed in clear cell and low-grade endometrioid carcinomas, consistent with a common cell of origin. 18 Similar mutations have been found in both histotypes. Inactivating mutations of ARID1A, a chromatin remodeling gene, have been reported in 49% of ovarian clear cell carcinomas and 30% of endometrioid ovarian cancers. 19,20 Mutations of PPP2R1A, the regulatory subunit of a serine-threonine phosphatase required for chromosome segregation, have been found in 7% of clear cell ovarian cancers. 19 Phosphatidylinositol-3-kinase (PI3K) signaling is activated in low-grade endometrioid cancers through inactivating mutations and epigenetic silencing of PTEN and activating mutations of PIK3CA. In nonepithelial granulosa cell tumors, recurrent single base mutations (402C-G) of FOXL2, a transcription factor implicated in granulosa cell differentiation, have been found in 97% of cases. 21
Thus, low-grade Type I cancers appear to be driven by mutations that activate Ras/MAP and PI3K signaling in the context of a relatively normal karyotype with wild-type TP53 and BRCA1/2. By contrast, Type II high-grade serous cancers exhibit numerous copy number abnormalities with frequent amplifications and deletions, but with mutations in a very limited number of genes including TP53 and BRCA1/2. The Cancer Genome Atlas Research Network (TCGA) analyzed copy number abnormalities in 489 high-grade serous ovarian cancers, detecting amplification of more than 30 growth stimulatory genes. 22 Amplification and overexpression of genes in the PI3K family occur in more than 50% of Type II cancers, activating the PI3K pathway and conferring “PI3Kness.” 23 In the absence of germline abnormalities of BRCA1/2, homologous DNA repair can be compromised by somatic mutations of BRCA1 and BRCA2 within the cancer alone, BRCA2 can be silenced, and upstream mutations can downregulate BRCA function combined with mutations in other genes potentially involved in homologous recombination, producing “BRCAness” in up to 50% of patients with Type II cancers. 24 DNA sequencing of exons from 316 cancers detected mutations of TP53 in 96%, most of which appeared to be inactivating. 22 Of the 26,000 genes, only a few were mutated in 2% to 4% of cases, including NF1, Rb1, BRCA1, BRCA2, and CDK12. Unlike Type I cancers, fewer than 1% of Type II cancers had mutations of BRAF, PI3KCA, KRAS, or NRAS. Despite the low prevalence of Rb1 mutations, dysfunction of the Rb pathway was found in 67% of high-grade serous cancers. As in the case of epithelial cancers at other sites, both TP53 and Rb were inactivated in two thirds of Type II ovarian cancers. As indicated earlier, low-grade tumors have a high frequency of mutations in PIK3CA, KRAS, ARID1A, and other putative oncogenes and tumor suppressor genes, whereas high-grade ovarian cancers are characterized by mutations in TP53 and BRCA1/2 and marked alterations in DNA copy number. These distinct molecular characteristics indicate that interconversion from Type I to Type II cancers is either a very rare event or does not occur, and thus Type I and Type II tumors likely represent independent diseases. Embracing this concept and performing independent clinical trials and tailored therapy for Type I and Type II tumors will be necessary to improve patient outcomes.
Other Prognostic Subtypes Based on Gene Expression
The pattern of gene expression has been used to identify prognostic subgroups. The Australian Ovarian Cancer Study Group profiled 285 serous and endometrioid tumors to identify six molecular subtypes. 25 Two subtypes were associated with low malignant potential serous tumors and low-grade endometrioid ovarian cancers, whereas the other four transcriptional profiling based subtypes included high-grade serous and endometrioid histotypes including a “mesenchymal” subtype. The immunoreactive subtype expressed T-cell chemokine ligands CXCL11 and CXCL10 and the receptor CXCR3, consistent with a higher level of infiltration by leukocytes. Importantly, lymphocytic tumor infiltration has been associated with an improved outcome. Cases in the proliferative cluster exhibited high expression of the HMGA2 and SOX11 transcription factors, low expression of MUC1 and MUC16 mucins, and high expression of proliferation markers such as MCM2 and PCNA. Differentiated cases have high expression of MUC16, MUC1, and the secretory fallopian tube marker SLPI. Mesenchymal cancers were associated with high expression of HOX genes and markers for stromal components including myofibroblasts (FAP) and microvascular pericytes (ANGPTL2 and ANGPTL1). These subtypes were not associated with changes in overall survival, although a prognostic signature was developed that included 193 genes. Subsequent analyses developed a prognostic “Classification of Ovarian Cancer” (CLOVAR) using gene expression that distinguished groups with markedly different median survival (23 vs. 46 months) and resistance to platinum therapy (63% vs. 23%). 26 Although these classifications can help to stratify future trials, profiles with higher positive and negative predictive value for response to conventional and novel agents will be required in order to affect clinical management.
Figure 37-4 Histopathologic features of K-Ras transformed, immortalized human ovarian surface epithelial cells. With permission from Liu, J, Yang G, Thompson-Lanza JA, et al. A genetically defined model for human ovarian cancer. Cancer Res 2004; 64:1655-1663. PMID: 14996724.
Immortalization
Telomerase activity is increased in 80% to 90% of ovarian cancers. Substantially greater telomerase activity is found in ovarian cancers than in borderline lesions or normal ovaries. High-grade serous ovarian cancers demonstrate high degrees of genomic instability and copy number abnormalities, possibly related to bridge-fusion breakage that occurs at telomeric crisis, before the upregulation of telomerase. Immortalization of human ovarian epithelial cells can be achieved by the introduction of human telomerase reverse transcriptase (hTERT) after disruption of the TP53 and/or Rb pathways using SV40T/t antigen or siRNA against Rb. 27 Cells immortalized with SV40 T/t and hTERT can be transformed with mutant Ras, producing cancers resembling low-grade human ovarian cancers that grow as nodules on the peritoneum and exhibit serous papillary histology (Figure 37-4 ). 2 Other combinations of gene aberrations can generate tumorigenic lines from normal ovarian or fallopian tube epithelium, providing a potential approach to explore the roles of specific genomic aberrations in ovarian oncogenesis.
Genomic Abnormalities in Sporadic Ovarian Cancers
A number of genetic and epigenetic abnormalities have been detected in DNA from sporadic ovarian cancers, including amplification, mutation, hypomethylation, chromatin modification, deletion, loss of heterozygosity, and promoter methylation (Table 37-1 ). 2,14,22 High-grade Type II ovarian cancers are genetically unstable both in terms of DNA copy number and in exhibiting a moderate mutation rate (Figure 37-5 ). For unknown reasons, areas of genomic aberration are associated with changes in RNA splicing. The CDK12 gene has been implicated in splicing and is mutated and probably inactivated in 3% of high-grade serous cancers. Aberrant expression of different splicing factors has been observed in ovarian cancer, and altered splicing may affect not only adhesion (CD44), but also the activity of putative oncogenes (EVI-1b and AML-1), as well as the metabolism of xenobiotics (CYP1A1) or of multidrug resistance (SPF45). 2 Processing of miRNAs is also impaired in a fraction of ovarian cancers, with decreased levels of Dicer and Drosha in more than half of ovarian cancers. 28 Low Dicer expression was significantly associated with advanced tumor stage (P = .007) and poor survival (P = .02), whereas low Drosha expression was associated with suboptimal surgical cytoreduction (P = .02). Gene silencing with shRNA, but not siRNA, may be impaired in cells with low Dicer expression. Mutations of Dicer and Drosha are rare in high-grade serous cancer, but mutations of Dicer have been detected in 29% of nonepithelial ovarian cancers. 29 Abnormalities in miRNAs have been found in many different types of cancers related to gain or loss of DNA copies, methylation, or mutation. 30 In ovarian cancers, copy number abnormalities have been found in 37% of 283 loci known to contain miRNAs. 2 A number of miRNAs may act as tumor suppressors and are downregulated in high-grade serous ovarian cancers, including Let-7a/b/d/f and miRs -15/16, -22, -31, -34a/b/c, -125b, -127-3p, -140, -145, -152, -155, -181a, -199a, and -382. 22,30 Conversely, several miRNAs are upregulated and can promote ovarian oncogenesis or chemoresistance, including miRs -15a/16, -20A, -23a/b, -30a/b/c, -92, -93 and -106a, -135b, -141, -200a/b/c, -244, -299-5p, -302d, and -373. Upregulated miRNAs are being evaluated as biomarkers.
Figure 37-5 Copy number abnormalities in cancers from different sites. GBM, Glioblastoma multiforme. With permission from Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 2011;474:609-615; and Douglas Levine.
Copy number abnormalities (CNAs) are particularly common in epithelial ovarian cancers. Candidate oncogenes and tumor suppressor genes have been mapped to most but not all of the abnormal sites. Amplification of 1q22 includes the RAB25 oncogene. Among the genes encoded on the 3q26 amplicon, protein kinase C iota (PKCiota), SnoN, MDS1-ecotropic viral integration site-1 (mecom/EVI1), and the p110-α catalytic subunit of phosphoinositide-3-kinase (PIK3CA) are overexpressed in a fraction of ovarian cancers and are associated with a poor prognosis. 2 PKCiota protein is required for the establishment and maintenance of epithelial cell polarity. Levels of aberrant PKCiota are markedly increased and/or mislocalized in the majority of serous ovarian cancers and are associated with increased cyclin E protein expression and proliferation. 2 Cyclin E1 amplification and protein levels correlate with a worsened outcome in ovarian cancer. The FGF-1 peptide growth factor encoded by a gene in the amplicon at 5q31 can stimulate cancer growth, stromal growth, and angiogenesis. An amplicon at 8q24 contains c-myc, which is amplified in up to 40% of ovarian cancers, inducing factors required for proliferation and activating telomerase. Another amplicon on chromosome 19q contains the p85 β subunit of PI3K as well as AKT2, a target of PI3 kinase. Another major amplicon at 20q13.2 contains the BTAK/Aurora kinase gene that upregulates c-Myc and activates telomerase.
Loss of tumor suppressor function has been observed in ovarian cancers (Table 37-2 ). 2,14 In some cases, inactivating mutations have been associated with loss of heterozygosity (LOH) (BRCA1, BRCA2, TP53, PTEN), but in others epigenetic changes alone (RASSF1A, DLEC1) or in combination with LOH (ARHI, BRCA1, LOT-1, PEG3, WWOX) have silenced suppressor function. As described earlier, somatic mutation of TP53 is observed in nearly all high-grade type II ovarian cancers. Because of the dominant negative activity of some mutant TP53 protein, TP53 function can be lost with a single genetic event. The pattern of transitions, transversions, and deletions within mutated TP53 genes resembles the pattern of mutations in factor IX deficiency (hemophilia B) in the germline that is thought to be related to spontaneous deamination during DNA replication. 5 If spontaneous mutation during proliferation is a critical mechanism driving carcinogenesis, genetic events requiring only a “single hit” may be favored.
Table 37-3
Oncogenes Associated with Epithelial Ovarian Cancer 14,22
Additional targetable genes with low or high gain of copy number in >20% of high-grade serous ovarian cancers include AKT1, AKT3, CDK2, IL8RB, EPCAM, ERBB3, FGFR2, HDAC4, HSP90AB1, HSP90B1, IGF1, IGFR1, LPAR3, MAP3K6, MAPK15, MAPKAPK2, MAPKAPK5, MECOM, MSTN, MTOR, NCAM1, NOS1, NOS3, PIK3CD, POLB, POLE, RHEB, RICTOR, PPS6KC1, RAPTOR, SKI1, STAT1, STAT4, TERT, TGFB1, TGFB2, TGFBR3, TNFRSF9, VEGFA. 22
Function of imprinted growth regulatory genes can also be lost in a single genetic or epigenetic event. Approximately 70 human genes are imprinted with only one allele expressed at conception, during embryonic development, and in each normal adult cell. Silencing of the maternal or paternal allele is inherited epigenetically. Among the candidate tumor suppressor genes whose function is downregulated in ovarian cancer (see Table 37-2), at least four are imprinted: ARHI (DIRAS3), LOT-1, NDN, and PEG3. ARHI encodes a 26-kDa GTPase that has 50% to 60% homology to Ras and Rap and that is downregulated in more than 60% of ovarian cancers, associated with decreased time to progression. 31 Expression can be downregulated by multiple mechanisms including LOH, methylation and silencing of the functional allele, transcriptional regulation with E2F1 and E2F4, and shortened mRNA half-life. 32 Re-expression of ARHI at physiologic levels inhibits clonogenic growth and motility of ovarian cancer cells that lack its expression, inhibiting STAT3 translocation, downregulating cyclin D1 and inducing p21WAF1/CIP1 and p27KIP1, producing G1 arrest. ARHI reexpression decreases levels of HIF1-α and inhibits signaling through Ras/MAP and PI3K/TSC2/mammalian target of rapamycin (mTOR), inducing autophagy and tumor dormancy. 33 Among the other imprinted tumor suppressor genes, LOT-1 is a zinc finger protein that presumably acts as a transcription factor 2 and PEG3 is required for TP53-induced apoptosis. 2 Consequently, imprinted genes may regulate the proliferation, motility, and survival of ovarian epithelial cells through multiple mechanisms.
Activation of oncogenes occurs through amplification, overexpression, or mutation in ovarian cancers (Table 37-3 ). 2,14 The two most common aberrations, mutations of members of the PI3K/AKT pathway and of the Ras/RAF pathway, are discussed in the following sections. The FOXM1 and NOTCH pathways are also aberrant in high-grade serous ovarian cancers, albeit at a lower frequency. Abnormalities of receptor and nonreceptor kinases have also been documented.
In contrast to breast cancer, where HER-2 is amplified and overexpressed in 20% to 30% of cases, HER-2 overexpression was found in only 11% of 837 epithelial ovarian cancers in a Gynecologic Oncology Group trial in which trastuzumab produced objective responses in only 7% of tumors with HER-2 overexpression. 2 Unlike lung cancer, the tyrosine kinase domain of EGFR is rarely mutated in ovarian cancer, but the receptor can be amplified in up to 20% of cases. A characteristic constitutively activating deletion in the extracellular domain of EGFR (EGFRvIII), first demonstrated in glioblastomas, has also been detected in a small fraction of ovarian cancers. 2 Constitutive activation of EGFR has, however, not been detected in ovarian cancer cell lines. Inhibition of the EGFR with erlotinib and gefitinib produced only modest clinical activity in ovarian cancer, where objective regression has been observed in 4% to 6% of cases.
Cyclin E maps to an amplicon that is found in a significant minority of ovarian cancers. Knockdown of neighboring genes has shown that CCNE1 is a driver required for clonogenicity that is associated with chemoresistance. 2 Proteomic studies using reverse phase protein analysis have shown that overexpression of CCNE1 protein marks a distinct cluster of cases with poor prognosis.
BTAK/Aurora A kinase, a serine-threonine kinase required for chromosome segregation and centrosome function, is amplified in 10% to 25% and activated in 48% of ovarian cancers. 2 BTAK/Aurora A kinase regulates telomerase activity. Forced expression of Aurora A induces centrosome amplification, cell cycle progression, and chemoresistance to cisplatin and paclitaxel mediated through AKT in a TP53-dependent manner. Chemical inhibition of BTAK/Aurora A kinase downregulates NF-κB, Bcl-XL, and Bcl-2 and enhances sensitivity to chemotherapy. Another cytoplasmic serine-threonine kinase, salt-induced kinase 2 (SIK2), localizes to centrosomes and phosphorylates c-NAP1, permitting centrosome splitting. 34 SIK2 is overexpressed in 30% of ovarian cancers associated with a worse prognosis. Downregulation of SIK2 enhances sensitivity to paclitaxel.
RAB25, a member of the RAB family of small G-proteins implicated in apical vesicle trafficking and polarity, is amplified and overexpressed in approximately half of ovarian cancers and is associated with a poor prognosis. Forced expression of RAB25 protein markedly increased anchorage-dependent and anchorage-independent proliferation; prevented apoptosis and anoikis, including that induced by chemotherapy; and enhanced growth in xenografts. 2 Inhibition of apoptosis was associated with a decrease in the expression of the pro-apoptotic molecules BAK and BAX, as well as activation of the PI3K/AKT pathway. RAB25 is sufficient to increase cellular metabolism and render cells resistant to acute metabolic stress, demonstrating a link between polarity, vesicle trafficking, and cellular metabolism.
In addition to the kinases, growth factors, and signal transducing proteins, members of the family of eukaryotic initiation factors (eIFs) have been implicated in ovarian oncogenesis. The initiation factor eIF-5A2 maps to a region on 3q26 that is amplified in ovarian cancers. Overexpression of eIF-5A2 is associated with advanced-stage ovarian cancer. 2 Forced expression of eIF-5A2 stimulates and antisense inhibits ovarian cancer growth and tumorigenicity.
Several transcription factors are also overexpressed in ovarian cancers. The c-Myc gene is amplified in up to 40% of ovarian cancers, and protein levels are increased in approximately one third of cases, including clear cell and endometrioid histotypes. 2 The c-Myc protein induces E2F1, E2F2, E2F3, and telomerase while blocking TP53-mediated transcription of p21. Despite the probable importance of c-Myc amplification in oncogenesis, no apparent association with survival has been found. A number of approaches to target cells with c-Myc aberrations are beginning to emerge. The association of cMyc with metabolism and particularly in the role of glutamine in cancer biology provides a potential avenue to therapy. The Hippo pathway translational co-activator YAP has also been implicated as a possible oncogene in ovarian cancer that can drive progression and induce resistance to chemotherapy. 35
Aberrant Signaling
Several tyrosine kinase growth factor receptors can be activated in epithelial ovarian cancers, including EGFR, HER-2/ HER-3, M-CSFR, IGFRI, FGFR4, and PDGFR. Binding of relevant ligands to one or more of these receptors can activate signaling through Ras/MAP, PI3K, JNK, JAK/STAT, PKC, and PLCγ or PLC-x with a concomitant alteration in calcium fluxes. Among these several signaling pathways, the Ras/MAP and PI3K pathways appear to be particularly important in regulating the growth, survival, metastatic potential, and drug resistance of ovarian cancer cells. Although the RAS/MAPK and PI3K pathways exhibit few mutations in high-grade serous ovarian cancers, the pathways are frequently activated. The PI3K pathway may be activated by changes in copy number of multiple pathway components as well as by loss of PTEN and INPP4B. Indeed, in the TCGA dataset, the RB1 pathway was dysregulated in 67% of high-grade serous cancers and the PI3K pathway in 45%. 22 The NOTCH signaling pathway was dysregulated in 22%. Stimulation of FGFR4 by FGF-1 activates mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and the WNT signaling pathways. 36
In many tumor types, K- or H-Ras is activated by mutations at codons 12 or 61, permitting constitutive binding of GTP rather than GDP to the Ras protein. Activated Ras can signal through the MAPK, PI3K, or Ral pathways, affecting proliferation, survival, motility, invasion, and drug resistance. As noted earlier, the Ras gene is mutated in approximately half of Type I low-grade serous cancers, but rarely in Type II high-grade cancers. Nevertheless, Ras activity as assessed by GTP binding is increased in many Type II high-grade cancers.
The PI3K pathway regulates survival, proliferation, motility, angiogenesis, glucose metabolism, and drug resistance. Activation of the PI3K pathway is observed in 50% of ovarian cancers through multiple mechanisms including amplification or activating mutation of the PI3K p110 α, activating mutation of the PI3K regulatory subunit p85, inactivating mutation of the PTEN phosphatase and amplification of AKT2 (see Table 37-3). 2 The p110 subunit of PI3K is amplified in 9% to 22% of ovarian cancers, overexpressed in 32%, and mutated in 9% to 12%, predominantly in low grade. 2 The pattern of PIK3CA mutation is histotype dependent, being common in endometrioid and clear cell tumors, but rare in serous cancers. The AKT serine-threonine kinase is generally activated physiologically by the products of PI3K. However, in 12% to 27% of ovarian cancers, the AKT2 kinase gene is amplified and is associated with an increase in AKT kinase activity. 2 The p70S6 kinase is downstream of both PI3K and AKT kinase in the signaling cascade. The p70S6 kinase may have a critical role in modulating drug resistance in that rapamycin, an inhibitor of p70S6 kinase, can potentiate sensitivity of some ovarian cancer cell lines to cisplatin-induced apoptosis. 2 Inhibition of activated PI3K decreases cell growth, induces apoptosis, and potentiates paclitaxel chemotherapy. The PI3K pathway is being explored as a therapeutic target in many cancer lineages, including ovarian cancer.
Normal ovarian surface epithelium expresses small amounts of macrophage colony stimulating factor (M-CSF, CSF-1), but little if any of fms, the tyrosine kinase receptor for this ligand. At least 70% of ovarian cancers express and secrete substantially greater amounts of M-CSF, and approximately 50% of cancers express the fms receptor. 2 Interaction of M-CSF with fms stimulates invasiveness and upregulates urokinase-like plasminogen activator (uPA). Expression of uPA correlates with tumorigenicity of ovarian cancer cell lines in xenograft models. 2
Among the nonreceptor tyrosine kinases, the Src tyrosine kinase can be physiologically activated in ovarian cancer cell lines, but the Src gene is rarely amplified or mutated in surgical specimens. 2 Stat3 is phosphorylated by the Janus kinase (JAK) and Src kinase. Phosphorylated Stat3 forms dimers that can be translocated to the nucleus, binding DNA and inducing transcription of genes required for proliferation (cyclin D, c-Myc, c-Fos), survival (Bcl-2, Bcl-XL, XIAP, survivin), and angiogenesis (VEGF). Stat3 can be activated on the intracellular domains of peptide growth factor receptors (EGFR) and cytokine receptors (IL-6R) or by direct interaction with either Src or Abl. Activated pStat3 has been detected in 86% of 322 ovarian cancers. 2 Nuclear localization of pStat3 has been observed in 71% of cases, associated with decreased overall survival. Autocrine and paracrine stimulation with IL-6 activates Stat3, increasing both proliferation and motility. Inhibition of JAK inhibits IL-6–stimulated chemotaxis toward serum and haptotaxis toward fibronectin. 2 Knockdown of Stat3 with siRNA inhibits motility and prevents translocation of Stat to focal adhesions. The imprinted tumor suppressor gene ARHI inhibits proliferation and motility, binds to Stat3, and sequesters it in the cytoplasm, preventing translocation to the nucleus and to focal adhesions. 2 In addition, ARHI can prevent binding of Stat3 to DNA Stat Response Elements in promoter regions.
The endothelin A peptides (ET-1, ET-2, and ET-3) are potent mitogens for several human tumors. ET-1 and its ETA receptor (ETAR) are overexpressed in primary and metastatic ovarian cancers, providing the potential for autocrine stimulation. 2 Interaction with ET-1 also transactivates EGFR, stimulates proliferation, blocks apoptosis, activates integrin-like kinase (ILK), upregulates matrix metalloproteinases (MMPs), and increases vascular endothelial growth factor (VEGF) expression, enhancing angiogenesis.
Lysophosphatidic acid (LPA) is produced constitutively by mesothelial cells and some ovarian cancer cells and accumulates at high levels in the ascites of nearly all ovarian cancer patients. 37 LPA can be detected in the plasma of most patients at all stages of disease. The LPA-2 and LPA-3 receptors are markedly upregulated in ovarian cancers. 2 Interacting with these receptors, LPA stimulates calcium influx, proliferation, motility, chemotaxis, invasion, and resistance to chemotherapeutic agents, signaling through the RAS/MAP and PI3K pathways. LPA is a potent inducer of VEGF, IL8, IL6, and gro, implicating LPA in the accumulation of ascites, neovascularization, and metastasis. LPA can cross activate the EGFR receptor family and other receptor tyrosine kinases, potentially contributing to the activity of these receptors in ovarian cancer. Both the enzyme producing LPA, autotaxin, and the enzymes degrading LPA (LPPs) are aberrant in ovarian cancer. Drugs targeting LPA production and action are potent inhibitors of metastasis.
All three TGF-β isoforms are expressed by normal ovarian surface epithelial cells and regularly inhibit their proliferation, maintaining autocrine growth inhibition. 2 Loss of expression of TGF-β or loss of responsiveness to the growth inhibitory factor is detectable in a fraction of ovarian cancers. Moreover, TGF-β can stimulate the motility and invasiveness of transformed cells. Although mutation in Smad4 is observed in a fraction of ovarian cancers, both TGF-βRI and TGF-βRII receptors are generally intact, as is Smad signaling. 2 Loss of growth inhibition and increased invasiveness may relate to EVI-1 overexpression that is observed in 43% of ovarian cancers 2 and is thought to inhibit transcription of TGF-β–responsive genes. SnoN and AML1, which bind to and regulate Smads, are also aberrant in ovarian cancer.
Müllerian inhibition substance (MIS) bears homology to TGF-β, binds to a receptor with similar structure and function, and is produced by the Sertoli cells of the testis and granulosa cells of the ovary. 2 During embryonic development of gonadal structures, MIS induces atrophy of the Müllerian duct in male mammals. MIS inhibits growth of human epithelial ovarian cancer cells in culture and in xenografts. Binding of MIS to MISII G-protein–coupled receptors upregulates p16, produces G1 cell cycle arrest through an Rb-independent mechanism, and induces apoptosis. Some 56% of human ovarian cancers express MISII receptors, and clonogenic growth can be inhibited with MIS in more than 80% of cancers bearing receptors. MIS enhanced the anticancer activity of suboptimal doses of chemotherapeutic agents against human and murine ovarian cancer cell lines in culture and as xenografts. 2
Stem Cells
Both normal and malignant tissues are thought to contain small subpopulations of stem cells with unlimited replicative potential that can be passed serially from mouse to mouse in vivo or as spheroids in cell culture. In cancers, tumor-initiating cells that have stem cell–like characteristics are generally resistant to chemotherapy and radiotherapy. The phenotype of human ovarian cancer stem cells remains to be fully defined. Increased tumor-initiating potential has been associated with CD133+ALDH1+ and CD44+CD117+ subpopulations in ovarian cancer cell lines and clinical specimens. 38 CD133 has been associated with stem cells from normal and malignant tissues arising at multiple sites. Aldehyde dehydrogenase mediates resistance to certain drugs and toxins. CD44 is the hyaluronate receptor required for adhesion, and CD117 is the transmembrane tyrosine kinase growth factor receptor, c-Kit. CD24 can be associated with both subpopulations and marks ovarian cancer cells with tumor-initiating capacity and chemoresistance. CD24+ ovarian cancer cells express a number of stem cell biomarkers including Nestin, Beta-catenin, Bmi-1, Oct3/4, Notch1, and Notch4. Recent studies suggest that normal ovarian stem cells occupy a niche in the hilus of the mouse ovary at the junction between the ovarian surface epithelium, the peritoneal mesothelium, and the tubal (oviductal) epithelium. The cells cycle slowly and express a number of stem cell markers including ALDH1, LGR5, LEF1, CD133, and CK6B. Spheroids can be passaged serially in culture, and cells can be transformed by inactivating TP53 and Rb1. 39
Animal Models
Among the animal models, spontaneous ovarian cancers occur in approximately 40% of egg-laying hens 4 years of age. 40 Some 46% of ovarian cancers have mutations of TP53, and the majority express MUC16. Chickens have been used to test different hormonal strategies for preventing epithelial ovarian cancer.
Murine ovarian epithelial cells have been engineered to express different combinations of oncogenes and tumor suppressors. 40 When target cells were derived from transgenic mice that lacked TP53 expression, the addition of any two of three oncogenes—c-Myc, activated K-Ras, and activated AKT—were sufficient to induce high-grade cancer in ovarian surface epithelial cells. In mice that were deficient for both TP53 and BRCA1, Myc overexpression is sufficient to induce cancers. In a serous murine ovarian cancer model, disruption of either Rb or TP53 produced relatively few ovarian cancers, whereas disruption of both Rb and TP53 produced a greater number of high-grade epithelial ovarian cancers, albeit with long mean latency (227 days). High-grade ovarian epithelial cancers were induced in a third model when SV40 T antigen, which blocks both TP53 and Rb, was driven by the Müllerian hormone type 2 receptor (Ambr2) promoter. A model for low-grade ovarian cancer has been generated in mice in which the PTEN gene was disrupted and an oncogenic form of KRasG12D was expressed selectively in ovarian surface epithelial cells, using mice in which Cre recombinase was driven by the Amhr2 promoter. Finally, high-grade serous cancers of the fallopian tube have developed in in mice where PTEN and Dicer were conditionally depleted using the Amhr2-Cre mouse strain. Consequently, ovarian cancers have been associated with loss of TP53, Rb, PTEN, and Dicer function, as well as activation of Ras, AKT, and Myc. To date, however, murine models have not succeeded in mimicking the abnormal DNA copy numbers observed in human ovarian cancers, possibly related to the telomere length in murine cells and relative resistance to telomeric crisis.
Interaction of Ovarian Cancer Cells with the Microenvironment
Loss of Adhesion, Epithelial-Mesenchymal Transition, Invasion, and Metastasis
Several molecular alterations contribute to the distinctive pattern of metastasis observed in ovarian cancer. For those cancers that arise from the surface of the ovary or fimbriae of the fallopian tube, cancer cells must dissociate from the basement membrane and then survive anoikis within the peritoneal cavity. Whereas normal ovarian surface epithelial cells bind to both laminin and to collagen in the basement membrane, loss of adhesion to laminin occurs following malignant transformation. 2 In the normal ovary, the α6β4 integrin laminin receptor is detected over the entire basal surface of epithelial cells at points of contact with the basement membrane, whereas solid ovarian cancers exhibit only focal expression of this integrin. Ascites tumor cells have markedly decreased expression of α6 and β4 , consistent with the possibility that downregulation of integrin expression releases tumor cells from the basement membrane. Transformed cells can dissociate individually or as multicellular aggregates or spheroids that are carried passively by peritoneal fluid to sites of metastasis in the peritoneum and omentum. Interaction of integrins with other cancer cells in aggregates can regulate activation of FAK, ILK, PI3K, and AKT. Signaling through these pathways can determine whether cancer cells undergo apoptosis or anoikis on dissociation from the substratum. In addition to changes in integrin expression, alterations occur in the cadherins that regulate adhesion between epithelial cells. Normal surface epithelial cells of the ovary and fallopian tube have low levels of E-cadherin and may depend on N-cadherin to maintain association between epithelial cells. 41,42 In contrast to cancers that arise from many other organs, E-cadherin levels can actually increase during malignant transformation as ovarian cancers differentiate into multiple histotypes, but only low levels of E-cadherin are found in poorly differentiated ascites cells where N- and P-cadherin are often upregulated.
Epithelial-to-mesenchymal transition (EMT) occurs during metastasis in many carcinomas, including those that arise in the ovary. Increased expression of the transcription factors Slug and Snail are associated with loss of intercellular adhesion, as well as specific repression of adherens junction components (E-cadherin and β-catenin), tight junction components (occludin and ZO-1), desmosomal junction components (Dsg2), and neutrophil gelatinase-associated lipocalin (NGAL). 2 However, as noted earlier, the histologic differentiation of ovarian cancer cells potentially driven by homeobox genes can override the effects of EMT and result in increased E-cadherin levels. N-cadherin and vimentin are increased and Rac1, Rho A, and cdc42 GTPases are activated, as ovarian cancer cells assume a spindle shape and become more motile. EMT of ovarian cancer cells can be driven by endothelin A, EGF, LPA, Rab25, bone morphogenic protein-4 (BMP4), hypoxia, and 17β-estradiol. EMT can facilitate invasion of basement membrane and local stroma for those epithelial ovarian cancers that arise in cysts.
To implant on the peritoneal surface, ovarian cancer cells must attach to mesothelial cells through β1 integrins, CD44, and MUC16. β1 integrins on the cancer cell surface bind to VCAM-1, fibronectin, laminin, and type IV collagen on mesothelial cells and to type I and III collagens on the underlying basement membrane. 2,43 Antibodies that block anti-β1 integrin or matrix proteins can prevent adherence of some, but not all, ovarian cancer cell lines to mesothelial monolayers.
Mesothelial cells also express hyaluronic acid, to which the CD44 hyaluronate receptor can bind. Whereas normal ovarian cells express the canonical CD44S receptor, more than 70% of ovarian cancers exhibit a diverse mixture of CD44 splice variants. 2 Anti-CD44 antibodies can partially block adhesion of ovarian cancer cells to peritoneal mesothelial cells and can reduce the frequency of peritoneal metastases. 2 Ezrin, part of the submembrane linking complex that connects CD44 to the cytoskeleton, is strongly expressed in 49% of ovarian cancers and is associated with reduced overall survival. 2 Knockdown of ezrin with siRNA inhibits invasiveness. Interestingly, ascites tumor cells have decreased CD44 expression.
MUC16 on the surface of ovarian cancer cells can bind to mesothelin, a glycosylphosphatidylinositol-anchored glycoprotein expressed on the surface of mesothelial cells. MUC16, expressed by 80% of ovarian cancers, is a high-molecular-weight (more than 1 million Da), highly glycosylated mucin with a cytoplasmic tail. The extracellular domain of MUC16 contains at least 40 repeating subunits of 154 amino acids. 44 MUC16 peptides dock with mesothelin, and binding of ovarian cancer cells to mesothelial cells can be blocked with anti-mesothelin antibodies. 2 Interaction appears to depend on interaction with N-glycans associated with MUC16. Knockdown of MUC16 decreases the invasiveness of ovarian cancers. 2
Shed MUC 16 (CA125) has provided a serum biomarker for monitoring the course of ovarian cancer during treatment. 45 Increases or decreases in CA125 have correlated with disease course in more than 80% of patients with elevated serum levels of the marker. CA125 has been used to distinguish malignant from benign pelvic masses, identify persistent disease, and detect disease recurrence. 46 Although individual values of CA125 or annual transvaginal sonography (TVS) have not been sufficiently sensitive or specific for early detection of ovarian cancer, 47 sequential two-step strategies have demonstrated greater specificity where rising CA125 identifies a small fraction of patients who would benefit from TVS. 48 Greater sensitivity can be attained with multiple biomarkers. 49
Degradation of basement membrane at the site of local invasion and in metastases requires upregulation of type 1-MMP, MMP-2, MMP-9, uPA, and kallikrein activity, resembling changes during ovulation. The ability of tumor cells to migrate and to invade matrigel membranes is increased by VEGF, EGF, heregulin, TGF-β, BMP4, hepatocyte growth factor, M-CSF, TNF-α, heregulin, and LPA. Signaling through Ras/MAP, PI3K/AKT/p70S6K, Src and Stat has been implicated in migration and invasion. Recent work points to the importance of fibronectin-mediated activation of α5β1 integrin, which signals through c-Met, Src, and FAK to stimulate invasion and metastasis. 50 Effector proteases, including MMP2, MMP7, MMP9, IGFBP2, uPA, and the kallikreins, have all been associated with ovarian cancer cells in culture and in pathologic specimens. MMP-2 and MMP-9 are associated with very early dysplastic lesions where basement membrane is breaking down. 2 Ovarian stromal cells are an important source for several of the proteases, including MMP-9, which has been associated with infiltrating monocytes.
The human kallikreins (hKs) include some 15 different serine proteases with a high degree of homology that map to a cluster on chromosome 19q13.4. 2 Twelve are transcriptionally upregulated in ovarian cancer. In aggregate, the kallikreins degrade multiple matrix components including fibronectin, vitronectin, laminin, and collagen I, II, III, and IV. Transfection of hK4, hK5, hK6, and hK7 does not affect proliferation, but increases invasiveness in vitro and formation of peritoneal metastasis in nu/nu mice. Kallikrein activity is inhibited physiologically by serpins and antithrombin-3. Several kallikreins are being evaluated as biomarkers for detection or prognostication in ovarian cancer where hK5, hK6, hK7, hK8, hK10, and hK11 are upregulated and hK14 downregulated in tissue and in serum. Elevated hK5, hK6, hK7, and hK15 have been associated with a poor prognosis and elevated hK8 and hK9 with a good prognosis.
Binding of cancer-associated integrins to extracellular matrix can stimulate chemotaxis and invasion. Adhesion of ovarian cancer cells to collagen and clustering of collagen binding integrins activates integrin-mediated signaling via SRC kinases to induce expression of EGR1, resulting in transcriptional activation of the MT1-MMP promoter and subsequent MT1-MMP–catalyzed collagen invasion. 2 Laminin, fibronectin, and collagen can all enhance chemotactic activity associated with activation of Ras/MAP, whereas enhanced invasion is observed only with laminin and collagen. The α3, α6 , and β1 integrin-mediated signaling through Ras/MAP, Erk, and AKT have been implicated in chemotaxis and invasion.
Stress hormones—including epinephrine, norepinephrine, and cortisol—have been shown to upregulate MMP-2 and MMP-9 in ovarian cancer cells, enhancing invasion and angiogenesis. Chronic behavioral stress produced higher levels of tissue catecholamines, greater tumor burden, and more invasive growth in an orthotopic murine model of ovarian cancer. 51 β2 adrenergic receptor-driven cyclic AMP (cAMP)-protein kinase A (PKA) and Src signaling increase migration, invasion, and vascularization, enhancing tumor growth and increasing expression of VEGF, MMP-2, and MMP-9. 52 Moreover, among ovarian cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality.
Ovarian cancer metastases grow efficiently within the peritoneal cavity, but not as well at other sites. 43 In the past, peritoneovenous shunts were used to palliate intractable ascites, resulting in the systemic infusion of large numbers of ovarian cancer cells. At postmortem, the majority of ovarian cancer patients did not develop widely disseminated macroscopic hematogenous metastases, consistent with the possibility that the “soil” is indeed as important as the “seed.” 53 Predilection for the omentum may be explained by the ability of adipocytes to secrete adipokines including IL-8 that promote chemotaxis and invasion of ovarian cancer cells, as well as their ability to provide fatty acids as an energy source for rapid cancer growth. 54
Angiogenesis
Angiogenesis is an important component of metastatic potential. In primary ovarian cancers, microvessel density has correlated directly with the propensity to metastasize and inversely with disease-free survival. 2 VEGF, PDGF, acidic FGF, basic FGF, angiopoietin 1 and 2, IL-6, and IL-8 can all contribute to angiogenesis in different ovarian cancers. 2,55–57 Most ovarian cancers express VEGF, which stimulates proliferation of endothelial cells and serves as a survival factor both for endothelial cells and for ovarian cancer cells that express VEGFR family members. Treatment with the anti-VEGF antibody bevacizumab has produced an objective response rate of 16% in patients with recurrent ovarian cancer and has stabilized disease for 5.5 months in 50%. 56 In randomized trials in first line, the addition of bevacizumab to conventional chemotherapy has increased progression-free survival by 2.7 to 3.8 months (P = .004, P < .001). 58,59 Robust predictive biomarkers for response to bevacizumab are still being sought. Platelet-derived growth factor (PDGF) has been detected in areas of increased blood flow in ovarian cancer. 2 Pericytes—cells that cover endothelial cells and stabilize vessels—express PDGFR and secrete VEGF, creating a paracrine loop with vascular endothelial cells that secrete PDGF and express VEGFRs. Consequently, both receptors and both cell types might be targeted for more effective antivascular therapy.
Other targets may prove useful for antivascular therapy. 57 FAK is overexpressed in more than two thirds of human ovarian cancers associated with shorter survival. In addition to enhancing migration, invasion, and metastasis of cancer cells, FAK activation increases VEGF transcription, angiogenic cytokine production, and pericyte migration. Chemical inhibition or siRNA knockdown of FAK in xenograft models has slowed cancer growth, inhibited angiogenesis, and enhanced taxane sensitivity. Dll4, one of the Delta-like ligands for NOTCH, is overexpressed in 72% of ovarian cancers and is an independent predictor of poor survival. Inhibiting Dll4 inhibits tumor growth by inducing nonproductive angiogenesis with increased vascular density and decreased perfusion of tumors. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with bevacizumab alone in animal models. The availability of gamma secretase inhibitors and anti-Dll4 antibodies should facilitate translation to the clinic. Zeste homolog 2 (EZH2) is a polycomb protein that has been detected in tumor-associated endothelial cells in a fraction of ovarian cancers with a poor prognosis. VEGF from cancer cells induces EZH2, which methylates and silences vasohibin1 (VASH1), a potent anti-angiogenic factor. 60 Silencing EZH2 in tumor-associated endothelial cells with siRNA inhibited angiogenesis and reduced xenograft growth by reactivating VASH1. Finally, EphA2 is a transmembrane tyrosine kinase receptor that is overexpressed in 76% of ovarian cancers with later stage and higher grade. EphA2 receptor activation is required for VEGF-mediated endothelial cell migration and has been associated with vasculogenic mimicry by cancer cells. Microvessel density is increased and MMp-2 and MMP-9 increased in clinical samples that overexpress EphA2. Agonistic monoclonal antibodies, receptor-TRAPs, and siRNA have all proven effective in preclinical models.
Immunologic and Inflammatory Factors
Cytokines and Chemokines
Ovarian cancers can express up to 1000 times more TNF-α than normal ovarian epithelial cells. Some 80% of ovarian cancers express TNF-α, regulated translationally and transcriptionally through NF-κB. Ovarian cancer cells can also express both TNFRI and TNFRII, receptors that permit both autocrine and paracrine stimulation. Exogenous TNF-α or IL-1α enhances the expression of endogenous TNF-α and increases levels of IL-1α, IL-6, CCL2, CXCL8, and M-CSF. 61 TNF-α can exert contrasting effects on different ovarian cancers by inhibiting, failing to effect, or stimulating tumor cell proliferation. In 10% to 25% of tumor cells taken directly from patients, TNF-α can stimulate clonogenic growth. Knockdown of endogenous TNF-α has inhibited the growth and dissemination of ovarian cancer xenografts. 2 Clinical trials have been undertaken with infliximab, which blocks TNF-α in ovarian cancer patients. Interaction of TNF, CXCL12, and IL6 in an autocrine cytokine network can influence angiogenesis, myeloid cell infiltration, and NOTCH signaling.
Interleukin-6 (IL-6) also plays an important role in mediating paraneoplastic thrombocytosis. Thrombocytosis is associated with a poor prognosis and elevated plasma levels of thrombopoietin and IL-6. 62 Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti–IL-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing IL-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. Treatment with an anti-platelet antibody significantly reduced tumor growth and angiogenesis in tumor-bearing mice.
Immunosuppression
Immunodeficiency has been documented in patients with ovarian cancer. Advanced disease has been associated with defects in delayed cutaneous hypersensitivity and in the humoral immune response. Before treatment, T-cell numbers and subsets in peripheral blood have been comparable to controls, but functional defects of B cells have been detected. 2 Following primary chemotherapy, T-cell function is also compromised. T cells isolated from ascites fluid or tumor tissue exhibit decreased expression of the TCR-zeta chain, and downregulation of the TCR-zeta chain can be produced ex vivo by co-culture of T cells with macrophages or soluble tumor-derived factors. 2 The presence of CD4+CD25+FOXP3+ regulatory T cells suppresses specific T-cell–mediated immunity in tumor masses but not in stroma and has been associated with decreased survival. 2 Plasmacytoid dendritic cells favor the induction of tolerance. 63 Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of Treg cells. Ovarian cancers can also produce TGF-β and several immunosuppressive factors including IL-10, VEGF, fibronectin, and mucins. 2
Humoral Immune Response
Despite an immunosuppressive environment, antibodies against tumor-associated antigens can be found in the blood of ovarian cancer patients. Correlation of antibodies against MUC1 with favorable risk factors has raised the interesting hypothesis that immunity to mucins might suppress the development of ovarian cancers, although there may be many different reasons for the development of anti-MUC1 antibodies. 2 Antibodies against mesothelin, TP53, and HER2 have been found in ovarian cancer patients and are being explored as potential biomarkers for early diagnosis and monitoring.
Cellular Immune Response
T cells from ovarian cancer patients can kill autologous tumor cells following in vitro activation. Cytotoxic T cells can be generated by incubating peripheral blood lymphocytes with dendritic cells that have been pulsed with extracts of autologous, but not allogeneic, ovarian cancers. 2 Cytotoxic T cells bear Fas ligand and induce apoptosis in cells that express Fas, which includes most ovarian cancers. Restricted expression of T-cell receptor V-β subtypes has been observed in tumor-associated lymphocytes, consistent with antigen-driven expansion of specific clones. Aberrantly glycosylated mucins, including MUC1, are expressed by most ovarian cancers, and T cells reactive with MUC1 and MUC16 molecules have been obtained from ovarian cancer patients. TP53 is mutated in approximately 70% of all ovarian cancers and in virtually all high-grade serous ovarian cancers, and T cells reactive with TP53 can be detected in some 50%, but are also found in a similar fraction of controls with benign disease. 2 T cells reactive with HER-2 epitopes have been isolated from the ascites fluid of ovarian cancer patients. Other antigens have been recognized by T cells from ovarian cancer patients, including folate receptor-α, NY-ESO-1, MAGE, Sp17, survivin, and telomerase.
T-cell antigen epitopes are recognized in the context of specific major histocompatibility complex (MHC) determinants. Normal ovarian surface epithelial cells express class I but not class II MHC components. In ovarian cancers, class I determinants are expressed in approximately 80%, and class II determinants are expressed in 40%. 2 The level of class I MHC expression in epithelial ovarian cancer cells has correlated with the degree of T-cell infiltration in vivo and the ability to expand T cells in vitro in the presence of low levels of IL-2. Low class I MHC expression is a poor prognostic factor in aneuploid ovarian cancers. Antibodies reactive with autologous tumor cells have also been identified.
Ascites contains widely varying fractions of lymphocytes, macrophages, mesothelial cells, and cancer cells. In one study, an average of 51% CD8 T cells, 10% CD4 T cells, and 27% CD14 macrophages were encountered. 2 A variety of chemokines—CCL-2, 3, 4, 5, 8, and 22—and their receptors—CCR1, 2a, 2b, 3, 4, 5, and 8—were detected, and a direct correlation was found between CCL5 and the CD3 T-cell infiltration. Chemokine CXCL12 and its unique receptor CXCR4 have been implicated in metastasis of several different cancers. CXCL12 was found in 91% of ovarian cancers and CXCR4 in 59%. 2 Expression of CXCR4 was associated with decreased disease-free and overall survival. CXCL12 and VEGF are both induced in ovarian cancer cells by hypoxia and synergize to induce tumor vessels. 2 CXCL12 also attracts plasmacytoid dendritic cells into ascites that further enhance angiogenesis by secreting IL-8 and TNF-α. 2
Among the cells that infiltrate solid ovarian cancers, T cells are most prevalent, B cells are rare, and macrophages vary in number. In addition to the specific cytotoxic effects of T cells, the interferons produced by activated T cells can inhibit tumor growth, inhibit IL-8 secretion, block angiogenesis, upregulate MHC, and augment mucin expression. Intratumoral T cells have been found in 55% of ovarian cancers and are associated with a 5-year survival rate of 38%, compared with 4.5% for patients whose tumors lack T-cell infiltrates. 2 Both CD4 and CD8 cells can be found at tumor sites. The presence of CD8 T cells and a high CD8/CD4 ratio has correlated with the most favorable prognosis, related to the adverse effect of CD4+CD25+FOXP3+ regulatory T cells within the CD4+ population. 2 Ovarian cancer cells can secrete M-CSF and MCP1 that exert potent chemotactic activity for macrophages. Cytokines and factors released from activated macrophages can stimulate (IL-1, IL-6, TNF) or inhibit (nitric oxide, TNF) tumor growth. Tumor-associated macrophages have impaired phagocytic activity and effector function for ADCC. Cytotoxic NK cells have been detected in ascites fluid and in solid ovarian cancers. Despite these many potential immune effector mechanisms, most ovarian cancers grow progressively.
Based on the immunobiology of ovarian cancer, a number of strategies have been evaluated for the treatment of the disease. 64 Cancer vaccines have included idiotype anti-MUC16 (CA125), 65 MUC1, CEA, and NY-ESO-1. An alternative approach has used autologous ovarian cancer extracts, viral oncolysates or primed dendritic cells with or without depletion of TREGS using metronomic daily cyclophosphamide. Adoptive immunotherapy has been performed using tumor infiltrating lymphocytes (TILs) expanded ex vivo with IL-2. In an early nonrandomized trial, consolidation of primary therapy with TILs and IL-2 improved progression-free and overall survival in a small group of ovarian cancer patients. Substantial effort has been directed in the laboratory toward developing methods for optimal expansion and antigen-specific stimulation of TILs, as well as to introducing T-cell receptors or chimeric antigen receptors (CARs) into T cells. Monoclonal antibodies have been used to regulate T-cell activity. Ipilimumab, an anti-CTLA4 that inactivates a T-cell checkpoint, has produced anecdotal responses in ovarian cancer patients.
Conclusion
Despite major progress over the past decade, many critical questions remain to be answered if we are to develop therapeutic approaches that will optimally benefit patients. An understanding of the pathogenesis of epithelial ovarian cancer should permit earlier detection and more effective, potentially less toxic, therapy. We will need to embrace the concept that ovarian cancer consists of multiple independent diseases with two major subgroups of Type I and Type II, with the corollary that clinical trials and translational studies must be performed independently on each disease type. Biomarkers in addition to CA125 and imaging techniques with higher resolution than TVS must be identified to improve the ability to detect small volumes of disease in the ovary or fallopian tube for both early diagnosis and patient monitoring. Autoantibodies against overexpressed wild-type proteins and mutant TP53 provide attractive biomarker candidates, and SQUID imaging with targeted magnetic nanoparticles poses a potentially transformative technology. Low-grade Type I cancers are driven by Ras mutations, PI3K activation, and paracrine IGF signaling, in the context of wild-type TP53 and expression of ER and PR. Combinations of drugs that inhibit MEK, the PI3K pathway, and IGFR need to be tested in Type I ovarian cancers. Hormonal therapy, particularly in the context of targeted therapy, should be explored, especially considering the recent outcomes of trials of hormonal manipulation and mTOR targeting in breast cancers. High-grade Type II ovarian cancers are driven by copy number abnormalities that affect PI3K, NOTCH, and other pathways with loss of TP53 and/or BRCA1/2 function. Dual inhibition of PI3Kness and BRCAness should be evaluated in Type II ovarian cancers based on synergistic activity seen animal models. A variety of drugs targeting the PI3K pathway are in trials in ovarian cancer, and PAPR inhibitors that act as synthetic lethal with defects in BRCA1/2 or homologous recombination have shown activity in selected patients—providing an opportunity for combination trials, which indeed have just been initiated. Development of strategies that target mutant TP53 or act as a “synthetic lethal” with mutant TP53 constitutes a significant knowledge gap that needs to be filled. A number of compounds that can bind and potentially normalize function of TP53 with specific hotspot mutations have been developed and are entering clinical trials. Relevant genetically engineered murine models are needed that are driven by copy number abnormalities. Robust biomarkers for stem cell–like cells must be identified and strategies devised to eliminate dormant cancer cells. Predictive biomarkers are also needed to identify patients most likely to benefit from bevacizumab. Several other angiogenic targets must be evaluated, including FAK, Dll4, EZH2, and EphA2. Finally, therapy with vaccines, adoptive therapy, and checkpoint and other immunoregulatory antibodies must be combined strategically with targeted agents.
Acknowledgments
These studies were supported in part by a grant from the National Cancer Institute R01 CA135354, by the M.D. Anderson SPORE in Ovarian Cancer NCI P50 CA83639, the Shared Resources of the M.D. Anderson CCSG NCI P30 CA16672, the Ovarian Cancer Research Fund, the National Foundation for Cancer Research, philanthropic support from the Zarrow Foundation and Stuart and Gaye Lynn Zarrow, Golfers Against Cancer, the Kaye Yow Foundation, and the Mossy Family Foundation.
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