Molecular oncology in gynecologic cancer: Immunologic Response, Cytokines, Oncogenes, and Tumor Suppressor Genes





Key points





  • The immune system consists of the innate and adaptive immune systems. The innate system is present at birth and consists of natural barriers, natural killer (NK) cells, macrophages, and the complement system. The adaptive immune system adapts to infection and consists of T and B cells.



  • The cellular immune response occurs as a result of T lymphocytes reacting via a surface T-cell receptor (TCR) that processes antigens presented to it by an antigen-presenting cell (APC) in conjunction with human leukocyte (HLA) (major histocompatibility complex; MHC) molecules.



  • T-cell activation can result in activation of helper or inducer (Th) cells, cytotoxic or suppressor T cells , or cytokine production.



  • Th cells recruit macrophages and cytotoxic or suppressor cells.



  • Cytotoxic T cells have the ability to lyse infected cells or signal B cells to produce antibody.



  • Humoral immunity results from antigenic stimulation of a B lymphocyte, which differentiates into a plasma cell and secretes antibody (immunoglobulin).



  • The complement cascade provides a basis for the inflammatory response and can also mediate cytotoxicity.



  • Cytokines (lymphokines) are regulatory substances of the immune system produced as a result of T-cell activation caused by cell damage as a result of a virus or other cells, such as macrophages and monocytes, involved in the immune response.



  • Passive therapy transfers components of the acquired immune system to the recipient with cancer (e.g., monoclonal antibodies directed toward tumor-specific antigens).



  • Active immunotherapy uses a patient’s own immune system for protection against infection (e.g., vaccines).



  • Three types of genes are associated with malignant development—oncogenes, tumor suppressor genes, and DNA mismatch repair genes.



  • Malignant change is seen with point mutations, chromosomal aberration, gene amplification (increase in number of copies), or chromosomal translocation.



  • Ras oncogenes are part of a group of signal transducer oncogenes that relay messages from the membrane to the cell nucleus. Generally they are activated by point mutations.



  • Growth factor genes include C-erb-B2 (Her-2/neu), which can be overexpressed and act as a tumor-specific target for monoclonal antibody therapy; these are especially useful in breast cancer therapy.



  • Nuclear oncogenes include myc and fos and can activate other genes as well as stimulate DNA replication.



  • Angiogenesis is the formation of new blood vessels, allowing tumors to grow.



  • Tumor suppressor genes such as Rb and p53 restrain cell growth. They have two copies and, in general, alteration of both copies leads to a mutant expression, which allows tumorigenesis to occur.



  • BRCA1 and BRCA2 mutations confer a high lifetime risk of breast and/or ovarian cancer. Mutation screening may be appropriate for women with family histories suggesting a hereditary predisposition to breast or ovarian cancer. All epithelial nonmucinous ovarian cancer patients and the majority of breast cancer patients should be offered genetic testing.



  • DNA mismatch repair genes act by recognizing and fixing errors in the DNA helix resulting from incorrect pairings of nucleotides. They prevent the accumulation of genetically damaged material in the cell.



Cancer develops because of the accumulation of successive and multiple molecular lesions that result in an altered cellular phenotype that is self-sufficient in growth signaling; insensitive to antigrowth signals; and capable of tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis , evading apoptosis , deregulating cellular energetics, genome instability, and avoiding immune destruction ( ). These molecular changes can include overexpression , amplification , or mutation of oncogenes ; the failure of tumor suppressor gene function because of a mutation, copy number loss, deletion, or viral infection; and the inappropriate expression of cytokines , growth factors, or cellular receptors. Also, natural or induced immune responses may play a role in the modulation of cancer growth because immune cells such as tumor-associated macrophages may actually cause tumors to grow. Based on a growing understanding of the immune response, biologic pathways, and cancer development, new immunotherapy and targeted therapies for gynecologic malignancies are being developed and are reviewed and summarized in this chapter.


The immunologic response


The immune system has adapted to fight off bacterial or viral infections, but it also plays an important role in the surveillance and control of cancer cell growth. The immune system has two types of responses, innate and adaptive. Innate responses are non–antigen specific, rapid, and do not increase with repetitive exposure to a given antigen . Components of the innate immune system include physical barriers such as epithelial surfaces, macrophages, natural killer (NK) cells, neutrophils, dendritic cells, and components of the complement system. Dendritic cells and macrophages are phagocytic cells that act as antigen-presenting cells (APCs). Macrophages also play an important role in the production of cytokines. Innate immune system forms the initial immune response to invading pathogens and contributes to adaptive immunity, which is composed of T lymphocytes (T cells) and B lymphocytes (B cells) that are involved in cell-mediated immunity and humoral immunity , respectively.


Innate immunity


In contrast to the adaptive immune system, which can recognize a variety of foreign substances, including tumor antigens, the innate immune system can only recognize microbial substances. For the most part, neutrophils, macrophages, NK cells, and dendritic cells are involved in the innate immune response and depend on the recognition of pattern recognition receptors (PRRs), which are encoded in the germline and identically expressed by effector cells. These receptors recognize pathogen-associated molecular patterns (PAMPs), which are expressed by microbes and trigger intracellular signaling cascades that result in inflammation and microbial death. PRRs are expressed constitutively in the host and are not dependent on immunologic memory ( Fig. 27.1 ). Toll-like receptors (TLRs) are PRRs that stimulate type 1 interferon (IFN) production, which has antimicrobial, antiviral, and anticancer activity ( ). TLR agonists are being evaluated for use as vaccine adjuvants in immunotherapy trials for ovarian cancer. NK cells are a subset of the lymphocyte population, can directly kill infected cells, and recognize cells that lack major histocompatibility complex (MHC) class I molecules, such as bacteria ( ). Moretta and colleagues have reported that NK cells are cytotoxic to tumor cells, probably because of a similar lack of MHC class I molecules ( ).




Fig. 27.1


Toll-like receptors (TLRs). TLRs are pattern recognition receptors that recognize microbes, viruses, and cancer cells. TLRs recruit MyD88, which is an adaptor protein that ultimately activates interferon and proinflammatory cytokines. IKK, Inhibitor of nuclear factor kappa B (IκB) kinase; IRAK-4, interleukin-1R–associated kinase-4; IRF-3, interferon regulatory factor-3; IRF-7 , interferon regulatory factor-7; TNF, tumor necrosis factor; TRAF6, TNF receptor–associated factor 6.

(From Takeuchi O, Akira S. Recognition of viruses by innate immunity. Immunol Rev. 2007;220:214-224.)


The complement system plays an important role in the innate immune system and is a complex system consisting of a large group of interacting plasma proteins. Activation by binding to antigen-complexed antibody molecules activates what is termed the classical pathway. In contrast, the alternative pathway is activated by recognition of microbial surface structures in the absence of antibody. Activation of these pathways leads to cleavage of C3 protein into a larger C3b fragment that is deposited on the microbial surface, leading to complement activation of C3a, which serves as a chemoattractant for neutrophils. Complexing of downstream complement proteins C6, C7, C8, and C9 produces a membrane pore in tagged cells that ultimately results in cell lysis. Unfortunately, tumor cells are often resistant to complement-dependent cytotoxicity. The innate immune system is intricately linked to the adaptive immune system by activated macrophages that enhance T-cell activation and complement fragments that can activate B cells and antibody production.


Adaptive immunity


Humoral immunity: B cells and immunoglobulins


In humans, B cells are derived from hematopoietic stem cells and aggregate in the lymph nodes, gastrointestinal tract, or spleen. B lymphocytes synthesize antibodies in response to an activated CD8 + cell or helper T cell (Th2). B lymphocytes then differentiate into plasma cells that secrete large quantities of antibody ( immunoglobulin ) in response to an antigen. Unlike T cells, B cells recognize antigens in an unprocessed state. Each B cell is programmed to secrete a specific type of antibody, and it is estimated that more than 10 7 different antibodies are capable of being produced in response to the presence of foreign antigens ( Fig. 27.2 ).




Fig. 27.2


Overview of specific immune responses. Top row, Humoral immunity. B lymphocytes eliminate microbes by secreting antibodies. Middle and lower rows, Cell-mediated immunity. Helper T lymphocytes activate macrophages or dendritic cells that kill phagocytosed molecules or cytotoxic T lymphocytes that eliminate infected cells.

( APC, Antigen presenting cell; IL, interleukin; TH, helper T cell; TNF-a, tumor necrosis factor alpha. From Chapel H, Haeney M, Misbah S, Snowden N. Essentials of Clinical Immunology. 5th ed. Malden, MA: Blackwell; 2006:35.)


Overall, antibodies have the same basic structure, except for extensive variability in the portion of the structure binding to the specific antigen. Two identical heavy and light chains comprise the basic immunoglobulin (Ig) structure. Each pair is connected by a disulfide bond. Both the heavy and light chains have a variable (V) region at the amino terminus and a constant region (C) at the carboxy terminus. The V region participates in antigen recognition and confers specificity, and the C region enables the antibody to bind to the phagocyte. Five Ig molecules (IgG, IgM, IgA, IgD, and IgE) exist and serve different effector functions. Early in the antibody response, IgM and IgD production occurs and the membrane-bound forms of IgM and IgD bind antigen and activate naïve B cells, leading to B-cell proliferation and clonal selection. IgM is also involved in the activation of the classical pathway of the complement system. Later in the antibody response, the IgG response develops. IgG has a higher specificity for particular antigens and is also responsible for neonatal immunity in the transfer of maternal antibodies across the placenta and gut. Also, IgG causes opsonization of the antigen for phagocytosis by macrophages and neutrophils, as well as activation of the classical pathway of the complement system. NK cells and other leukocytes can bind to IgG- and IgE-coated cells to facilitate antibody-dependent cytotoxicity. IgE mediates hypersensitivity reactions, and IgA is responsible for mucosal immunity.


Cellular immunity: T cells


T cells originate in the bone marrow, differentiate in the thymus, and then circulate in the blood or are harbored in the lymph nodes, spleen, or Peyer patches of the intestine. In contrast to the humoral response, the cellular immune response ( cellular immunity ) depends on direct cell-cell contact. Although antibodies and B-cell receptors may recognize multiple types of antigens, T cells are restricted to peptide antigens and only recognize peptide sequences in the context of membrane-bound host proteins called MHC molecules ( Fig. 27.3 ).




Fig. 27.3


Different routes of antigen presentation. After the antigen is processed into smaller fragments, the major histocompatability complex class (I or II) and these fragments interact with the receptor on the surface of the T cell to activate cytotoxic or helper T cells.

(From Chapel H, Haeney M, Misbah S, Snowden N. Essentials of Clinical Immunology . 5th ed. Malden, MA: Blackwell; 2006:7.)


There are two classes of MHC molecules. Each class presents antigens to different populations of T cells and is responsible for various functions in the cellular immune response. Th cells (which are CD4 + ) respond to antigens bound to class II MHC molecules to secrete cytokines that stimulate the proliferation and differentiation of T cells, other B cells, and macrophages. Class II MHC molecules are expressed primarily by professional APCs, which present phagocytosed and processed extracellular peptides to Th cells.


There are two subsets of Th cells, which differ in their cytokine profiles and elicit different responses. Th1 cells secrete interleukin (IL)–2 and IFN-gamma (IFN-γ) to elicit a cell-mediated inflammatory response. Th2 cells secrete IL-4, IL-5, IL-6, and IL-10 to promote antibody secretion and the humoral response. Although both types are involved in most immune responses, they regulate the magnitude of each through mutual inhibition of cytokine production such that Th2 cell cytokines suppress production of Th1 cell cytokines and vice versa.


Unlike class II MHC molecules, class I MHC molecules are expressed by all nucleated cells in the body and are used to present intracellular peptides for surveillance to circulating cytotoxic T lymphocytes (CTLs). CTLs are also known as CD8 + T cells and directly destroy cells that express foreign antigens that arise after a viral infection or are expressed as a result of tumorigenesis. Therefore CTLs are considered to be primarily responsible for the antitumor immune response. Zhang and colleagues reported that the presence of intratumoral T cells was associated with improved progression-free and overall survival in ovarian cancer patients ( ). This association was confirmed by Sato and colleagues, who documented a survival advantage in patients with a higher CD8 + /CD4 + ratio of intratumoral cells in ovarian cancer patients ( ). Pedersen and associates have successfully treated six platinum-resistant ovarian cancer patients with an infusion of tumor-infiltrating lymphocytes preceded by lymphodepleting chemotherapy and followed by decrescendo IL-2 ( ). Another adoptive immunotherapy strategy is the adoptive transfer of genetically modified T cells, such as chimeric antigen receptor (CAR)–expressing T cells. There are ongoing phase I trials using CAR-T targeting mesothelin (NCT03608618), MUC16 (NCT02498912), and NY-ESO-1 (NCT01567891).


A third class of T cells, regulatory T cells (Tregs), are CD4 + T cells that are present in the peripheral circulation, inhibit immune responses, and prevent autoimmunity. Because most tumor-associated antigens are self-antigens, recognition by immune effector cells is regulated by Tregs through peripheral tolerance. High numbers of Tregs have been found in the peripheral blood of patients with epithelial ovarian cancer, and Tregs preferentially accumulate in the tumor environment, such as ascites and ovarian tumor islets. Curiel and associates have shown that high levels of Tregs are found to be predictive of poor overall survival in a cohort of 70 patients with ovarian cancer ( ). Based on these data, a goal of immunotherapy is to eliminate Tregs with the aim of enhancing innate antitumor immunity, which may be achieved with the use of low-dose cyclophosphamide ( ).


Cytokines


Cytokines are proteins secreted by immune cells that are produced in different phases of the immune response to control its duration and extent. During the activation phase of the immune response, cytokines stimulate growth and differentiation of lymphocytes, whereas in the effector phase of the immune response, they activate other effector cells to help eliminate antigens and microbes. The major classes of cytokines include those that regulate innate immunity, regulate adaptive immunity, and stimulate hematopoiesis.


Cytokines that mediate innate immunity


Interleukins


Interleukins are potent cytokines produced by some leukocytes to affect other leukocytes. IL-1 is released in response to cell damage by macrophages, endothelial cells, and some epithelial cells. Although IL-1 has actions similar to those of tumor necrosis factor (TNF), it lacks the ability to cause septic shock symptoms. Macrophages can secrete a variety of ILs. M1 macrophages secrete IL-12, IL-18, IL-23, IFN-γ, and TNF-alpha (TNF-α) and promote immune responses against tumors and intracellular microbes. IL-12 plays an important role in the transition between cell-mediated immunity and adaptive immunity. IL-12 stimulates NK cells and T cells to produce IFN-γ, which activates macrophages to kill phagocytosed foreign substances; IL-12 also increases cytolytic activity by stimulating CD8 + cells. M2 macrophages produce vascular endothelial growth factor (VEGF), IL-6, IL-10, and prostaglandin E2, all of which have immunosuppressive functions and are found selectively in established tumors. The other ILs stimulate NK and T-cell activation and proliferation and IFN-γ synthesis.


Chemokines


Chemokines are small secreted proteins that are part of the largest known cytokine family. There are approximately 47 peptides and 19 G protein–coupled receptors in humans. Functionally, chemokines released in response to stimuli that cause leukocyte recruitment are considered to be inflammatory, whereas chemokines that cause migration of leukocytes to lymphoid organs are considered to be homeostatic. Chemokines affect tumor establishment in the following ways: determining the extent and type of leukocyte infiltration, promoting angiogenesis, controlling site-specific metastasis, and affecting tumor cell proliferation. Chemokines have been classified into four main subfamilies: CXC, CC, CX3C, and XC. The CXC chemokines (CXCL9, CXCL10, and CXCL11) are induced by IFN-γ and are typical chemoattractants of NK cells ( ; ). In ovarian cancer patients, the expression of CXCR4/CXCL12 correlates with decreased progression-free and overall survival ( ; ; ). Because of the importance of chemokines in gynecologic and other malignancies, CXCR4 inhibitors such as peptide antagonists and neutralizing antibodies have been tested in phase I trials (NCT02179970).


Interferons


Type 1 IFNs, IFN-α and IFN-β, are stimulated by intracellular TLRs and mediate the early innate immune response to viral infections. These cytokines inhibit viral replication, increase expression of class I MHC molecules, and promote a Th1 cell–mediated immune response by promoting T-cell proliferation and NK cell cytolytic activity. IFN-γ, a type II IFN, is principally responsible for macrophage activation and the effector functions of innate and adaptive immune responses.


Cytokines that mediate adaptive immunity


In addition to IFN-γ and transforming growth factor beta (TGF-β), IL-2, IL-4, IL-5, and IL-13 are all involved in the regulation of adaptive immunity. After antigen recognition, the T cells produce IL-2, which causes clonal expansion of activated T cells and additional production of cytokines such as IFN-γ and IL-4. IL-2 stimulates antibody synthesis and B cells by acting as a growth factor. IL-4 not only promotes IgE production from B cells but also stimulates the development of Th2 cells from naïve T cells. IFN-γ is produced by T cells in response to antigen recognition or by NK cells in response to microbes or IL-12. IFN-γ activates the microbicidal function of macrophages, stimulates the expression of class I and II MHC and costimulatory molecules by APCs, promotes the maturation of cells expressing CD4 into Th1 cells, and inhibits the Th2 cell pathway, thereby effectively promoting a cellular immune response. TGF-β inhibits the proliferation of and differentiation of T cells and contributes to immune evasion of tumor cells by inhibiting antitumor host immune responses.


Cytokines that mediate hematopoiesis


Colony-stimulating factors


IL-3 is a multilineage colony-stimulating factor that allows for the differentiation of cells into myeloid progenitor cells, granulocytes, monocytes, and dendritic cells. Granulocyte colony-stimulating factor (G-CSF) is a cytokine produced by macrophages, fibroblasts, and endothelial cells and promotes the mobilization of neutrophils from the bone marrow. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by T cells, macrophages, endothelial cells, and fibroblasts. GM-CSF stimulates the maturation of bone marrow cells into dendritic cells and monocytes. G-CSF and GM-CSF are available pharmacologically and are used in patients undergoing chemotherapy and bone marrow transplantation.


Tumor cell killing and immunotherapy


Immunotherapy has been developed to recognize and destroy tumor cells ( ). Immune modulation, passive therapy, and active therapy are the three major classes of immunotherapy. Immune modulation relies on nonspecific means such as the administration of IL-2, IFNs, checkpoint inhibitors, or bacille Calmette-GueÃÅrin to elicit an immune response. Passive therapy transfers components of the acquired immune system to the cancer patient ( passive immunity ). An example of passive therapy is the use of monoclonal antibodies directed toward tumor-specific antigens . Active therapy uses the woman’s immune system to elicit a response; examples include vaccines composed of peptides, proteins, DNA, or RNA.


Immune modulation has been used in ovarian cancer in the form of adjuvant IFN treatment after surgery and as consolidation therapy after surgery and standard chemotherapy. A phase III trial randomly assigned patients with advanced ovarian cancer to intravenous (IV) cisplatin and cyclophosphamide chemotherapy versus the same regimen with intraperitoneal IFN-γ. Windbichler and colleagues have shown an improvement in progression-free but not overall survival in the IFN arm, with acceptable toxicity ( ). Possible explanations for the improvement in the chemotherapy plus IFN include induction of CTLs, stimulation of NK cells and macrophages, an antiangiogenic effect on tumor vasculature, and the direct inhibition of oncogene expression by high IFN-γ levels in the tumor microenvironment. This study was redone with the standard chemotherapy of carboplatin and paclitaxel with IFN-γ versus chemotherapy alone and showed a survival disadvantage in patients receiving IFN-γ but no difference in progression-free survival (PFS) ( ).


Tumor cells have specific tumor-associated antigens or receptors on their surface that may distinguish them from normal cells. The antigen most often targeted in ovarian cancer is CA-125, a glycoprotein present at elevated levels in the serum of more than 80% of patients with epithelial ovarian cancer. A murine monoclonal antibody (MAb) to CA-125 (oregovomab) was investigated for its therapeutic usefulness as a consolidation treatment in ovarian cancer patients but did not demonstrate an overall survival advantage over control ( ). However, a subset of patients who had evidence of a robust antiantibody immune response in the form of antimurine antibodies had evidence of tumor protection after treatment. Trials of oregovomab, with or without chemotherapy, have also been conducted in patients with recurrent or upfront disease (NCT01616303), and induction of anti–CA-125 T-cell responses correlated with improved survival times ( ). Bevacizumab (Avastin) is a monoclonal antibody directed against VEGF. In two large phase III trials (GOG [Gynecologic Oncology Group] 218 and ICON7 [International Collaborative Group for Ovarian Neoplasm 7]), the incorporation of bevacizumab to primary treatment of ovarian cancer patients produced a clinical benefit ( ; ). In GOG 218 the median PFS was 10.3 months in the control chemotherapy group, 11.2 months in the upfront chemotherapy and bevacizumab and placebo maintenance group, and 14.1 months in the upfront chemotherapy and bevacizumab-throughout group. Additionally, bevacizumab combined with chemotherapy has been approved to be used in recurrent platinum-resistant ovarian cancer because there is a 3.3-month improvement in PFS ( ). Bevacizumab-based therapy is also approved for use in platinum-sensitive ovarian cancer based on an average of 3.4- to 4-month improvement in PFS, as demonstrated in the OCEANS (Ovarian Cancer Study Comparing Efficacy and Safety of Chemotherapy and Anti-Angiogenic Therapy in Platinum-Sensitive Recurrent Disease) and GOG 213 clinical trials ( ; ).


Adoptive T-cell immunotherapy uses the transfer of T cells expanded ex vivo in large numbers because of their ability to kill tumor cells specifically and to proliferate and persist for long periods after transfer. A strong rationale exists for the development of adoptive T-cell therapies in the treatment of ovarian cancer. First, tumor-specific T cells can be found in the peripheral circulation or in tumors in up to 50% of ovarian cancer patients. Second, the fact that intratumoral T cells are associated with improved survival suggests that administering adoptive immunotherapy could produce clinical results ( ). T cells used for adoptive immunotherapy can be derived from peripheral blood lymphocytes (PBLs) or tumor-infiltrating lymphocytes (TILs); however, because TILs are labor intensive and only successful in a subset of patients, investigators are focusing on genetic modification of PBLs to exhibit tumor antigen specificity. T cells can be genetically modified to express either a tumor antigen–specific T-cell receptor (TCR) encoding α and β chains with specificity for tumor-restricted peptide expressed on a given human leukocyte antigen (HLA) molecule or a CAR encoding a transmembrane protein comprising the tumor antigen–binding domain of an Ig linked to one or more T-cell costimulatory molecules ( ).


Another promising immunotherapy in gynecology has been the human papillomavirus (HPV) vaccine for the prevention of vulvar, vaginal, or cervical dysplasia and the corresponding cancers. A study conducted by Munoz and workers has shown that the quadrivalent vaccine against HPV-6, -11, -16, and -18 was up to 100% effective in reducing the risk of HPV-16 and HPV-18–related high-grade cervical, vulvar, and vaginal dysplasias, which may lead to reduction in rates of cervical, vulvar, and vaginal cancers ( ). Listeria monocytogenes, which secretes the antigen HPV-16 E7 and is fused to a nonhemolytic listeriolysin O protein, has been used as a therapeutic vaccine for patients with advanced cervical cancer ( ). HPV vaccine therapy is efficacious and has tremendous implications for the prevention and treatment of gynecologic HPV-related dysplasias and cancers.


Molecular oncology


Cancer development can be sporadic if it is caused by acquired mutations or can be hereditary if caused by inheritance of a mutated gene followed by acquisition of an acquired mutation in the other allele . Genetic alterations occur in three major categories of genes—oncogenes, tumor suppressor genes, and DNA mismatch repair (MMR) genes . Knowledge of how these genes function is a rapidly expanding field and well beyond the scope of this text, but a general overview is provided here.


Oncogenes


An oncogene is a set of genes that when altered are associated with the development of a malignant cell. Functionally, oncogenes are involved in cell proliferation, signal transduction, and transcriptional alteration. Mechanisms of alteration in oncogene function include gene amplification (an increase in the number of copies of the genes in the cell), translocation , or overexpression, which refers to excessive and abnormal protein production. Several classes of oncogenes, such as peptide growth factors, cytoplasmic factors, and nuclear factors, exist. Examples are described in the following section ( Table 27.1 ).



TABLE 27.1

Classes of Genes Involved in Growth Stimulatory Pathways

Adapted from Boyd J, Berchuck A. Oncogenes and tumor suppressor genes. In Hoskins WJ, Young RC, Markman M, et al., eds. Principles and Practice of Gynecologic Oncology. Philadelphia: Lippincott Williams & Wilkins; 2005:93-122.




















































Peptide Growth Factors Corresponding Receptors
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) EGF receptor ( erb -B1), erb -B2 (Her-2/Neu), erb -B3, erb -B4
Heregulin
Vascular endothelial growth factor (VEGF) A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PlGF) 1, PlGF2 VEGFR-1, VEGFR-2, VEGFR-3, neuropilins
Insulin-like growth factors (IGF-I, IGF-II) IGF-I and IGF-II receptors
Platelet-derived growth factor (PDGF) PDGF receptor
Fibroblast growth factor (FGF) FGF receptors
Macrophage colony-stimulating factor (M-CSF) M-CSF receptor (FMS)
Cytoplasmic Factors Examples
Tyrosine kinases Eph family
G proteins K-Ras, H-Ras, N-Ras, RAF
Serine-threonine kinases AKT
Nonreceptor tyrosine kinases Focal adhesion kinase (FAK), src
Nuclear Factors Examples
Transcription factors C-myc, C-jun, C-fos, ARID1A, MYC
Cell cycle progression factors Cyclins, E2F


Peptide growth factors


Epidermal growth factor receptor family


There are four types of Erb B receptors: Erb B1 (commonly known as epidermal growth factor receptor [EGFR], human epidermal growth factor receptor [HER]1), Erb B2 (also known as HER2/neu), Erb B3 (also known as HER3), and Erb B4 (also known as HER4). All Erb B receptors share an extracellular domain that binds ligand, a transmembrane domain, and an intracellular tyrosine kinase domain. In the Erb B pathway, homodimers and heterodimers are formed from the various classes of receptors, resulting in activation of the Ras–Raf–mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)–activated AKT pathway. Although Erb B3 lacks intrinsic kinase activity and Erb B2 has no specific ligand, the formation of heterodimers leads to activation of these classes of receptors.


The Ras-Raf-MAPK pathway is a major downstream target of the Erb B family of receptors and leads to the activation of Ras, causing the activation of MAPKs to regulate transcription of molecules linked to cell proliferation, survival, and transformation ( ). Also, the PI3K-activated AKT pathway serves as another downstream target of the Erb B pathway; it drives tumor progression via increased cell growth, proliferation, survival, and motility. A number of mechanisms such as receptor gene amplification and overexpression, receptor mutations, and autocrine ligand production cause Erb B pathway disruption, leading to tumor formation. EGFR gene mutations have been found in glioblastomas, non–small cell lung cancer, and ovarian cancer. Although, Slamon and colleagues have reported that Her2/neu amplification is found in 20% to 30% of breast cancers and 10% of ovarian cancers, uterine papillary serous cancer have higher Her2/neu expression and HER2 gene amplification than ovarian cancer ( ). Because of the multitude of cancers with genetic alterations or changes in the Erb B family, several potential strategies exist for targeting EGFR, including monoclonal antibodies, low-molecular-weight tyrosine kinase inhibitors (TKIs, many of which are in advanced clinical development), antisense oligonucleotides, and intracellular single-chain Fv fragments of antibodies. A 2018 randomized phase II trial demonstrated an improved progression-free benefit of 4.6 months by adding trastuzumab (a humanized monoclonal antibody targeting Her2/neu) to carboplatin and paclitaxel in patients with advanced or recurrent uterine serous cancer that overexpress Her2/neu ( ).


Angiogenesis and VEGF


Cancer growth requires a sufficient blood supply to extend beyond 1 mm 3 in size. Angiogenesis occurs by sprouting (branching of new blood vessels from preexisting blood vessels) or by nonsprouting (requires the enlargement and splitting of preexisting blood vessels). The tumor vascular environment is characterized by vessels that are irregularly shaped, dilated, tortuous, and disorganized. Angiogenesis is dependent on the relative increase of proangiogenic factors such as VEGF, platelet-derived growth factor (PDGF), and ephrins and their receptors. Also, endothelial cells are genetically stable, unlike tumor cells, thereby potentially increasing the therapeutic value of targeting angiogenesis for cancer therapy ( ).


VEGF is critical to endothelial cell survival, vascular permeability, cell fenestration, and vasodilation. There are seven proteins in this family: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PlGF) 1, and PlGF2. Most human tumors, including those of the lung, thyroid, breast, gastrointestinal, female reproductive tract, and urinary tract, have marked expression of VEGF. There are three VEGF receptors: VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-3 is expressed on the vascular and lymphatic endothelium, unlike the other two receptors, which are expressed on the vascular endothelium. Also, a second class of VEGFRs known as the neuropilins (NRPs) potentiate VEGF-A– and VEGFR-2–mediated actions. Similar to the EGFR family, there are many ligands to receptors in the VEGF family. Ultimately, activation of the VEGF receptors can lead to downstream effects on the MAPK pathway, v-src sarcoma viral oncogene homologue (SRC), PI3K-AKT, focal adhesion kinase (FAK), and Ras-Raf-MAPK superfamily ( ; ; ). Because of the clinical significance of the VEGF pathway in many cancers, anti-VEGF antibodies such as bevacizumab, VEGFR TKIs, and vascular targeting agents have been developed to target this critical pathway ( ; ).


Cediranib is a receptor TKI against VEGFR-1 to -3, platelet-derived growth factor alpha (PDFR-α), and c-kit. In the ICON6 trial, patients with platinum-sensitive recurrent ovarian cancer who received chemotherapy plus cediranib followed by 18 months of cediranib maintenance had a median PFS of 11.0 versus 8.7 months (hazard ratio [HR], 0.57; 95% confidence interval [CI], 0.45 to 0.74) ( ). Cediranib is being combined with the poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitor olaparib in both patients with platinum-sensitive and platinum-resistant ovarian cancer. Liu and colleagues reported an improved PFS of 23.7 versus 5.7 months ( P =.002) and overall survival benefit of 37.8 versus 23.0 months ( P = .047) in patients with platinum-sensitive germline BRCA wild-type/unknown cancer receiving the combination of cediranib and olaparib compared with olaparib ( ).


Ephrin family of ligands and receptors


Tyrosine kinases provide a transfer of a phosphate from adenosine triphosphate (ATP) to tyrosine residues on specific cellular proteins; however, they can also play a role in the development of cancer and tumor progression. Attention has been given to elucidating the role of the ephrin receptor A2 (EphA2) in tumorigenesis and therapeutic targeting. EphA2 belongs to the largest known family of protein tyrosine kinase receptors, the Eph family, and two Eph receptors, A and B, have corresponding ligands. The normal cellular function of EphA2 in epithelial tissue is not completely understood, but in cancer, EphA2 modulates cell growth, survival, migration, and angiogenesis ( ). EphA2 overexpression has been correlated with disease severity and is predictive of a poor outcome in patients with ovarian cancer. Several therapeutic approaches exist for targeting EphA2, including agonist monoclonal antibody, immunotherapy, soluble EphA receptors, and neutral liposomal small interfering RNA (siRNA), some of which are already in clinical trials ( ; ).


Phosphoinositide 3-kinase pathway


The PI3K family is composed of lipid and serine-threonine kinases that control second messengers through phosphorylation. AKT is the predominant downstream target of PI3K and has many targets, including mammalian target of rapamycin (mTOR), signal transducer and activation of transcription (STAT), MAPK, nuclear factor–kappa βÔ¨ and protein kinase C. The activation of the PI3K-AKT pathway controls cell survival with inhibition of apoptosis, cell growth, cell metabolism, RNA translation, and cell proliferation. Also, this pathway has been implicated in chemotherapy resistance ( ).


RAS, another cytoplasmic factor, is a G protein involved in the transmission of growth stimulatory signals from the cell membrane to the nucleus. The RAS family of G proteins is positioned downstream of cell surface receptor tyrosine kinases and upstream of the cytoplasmic cascade of kinases, such as mitogen-activated protein (MAP) kinases. MAP kinases in turn activate nuclear transcription factors such as c-myc, c-jun, and c-fos. It is estimated that about one-third of cancers have point mutations in RAS genes, such as KRAS, HRAS, and NRAS ( ). Because RAS requires the posttranslational modification of the addition of a farnesyl group to the C-terminus to move from the cytoplasm to the inner plasma membrane, inhibitors to farnesylation had been developed but with disappointing results; however, research is ongoing regarding work to use switch II pocket (SII-P) inhibitors to target a pocket in the SW2 region of RAS and inhibitors to target nucleotide exchange ( ).


Tumor suppressor genes


Tumor suppressor genes control cell growth cellular proliferation, and aberrations in tumor suppressor genes can cause malignancy. The retinoblastoma (Rb) gene was the first tumor suppressor gene to be identified and encodes a nuclear protein that regulates G1 phase cell cycle arrest. Knudson and colleagues have proposed the “two-hit” theory to explain the action of tumor suppressor genes. The first hit is the inheritance of the Rb mutated gene and the second hit is the somatic mutation that occurs later and leads to cancer.


In gynecologic malignancies, the most common deregulated tumor suppressor gene is p53, which is located on the short arm of chromosome 17. p53 has key roles as a transcription factor and regulator of the cell cycle and apoptosis. Normal p53 binds to transcriptional regulatory elements in the DNA and acts as a gatekeeper of the genome by responding to DNA damage with the activation of apoptotic effectors such as BAX, FAS, and bcl-2. Missense mutations that change a single amino acid in the encoded protein in exons 5 to 8 are the most common mutations of p53. The resultant mutant proteins can no longer bind to DNA but can bind to and inactivate any normal p53 in a cell. Cells with mutant p53 do not experience cell cycle arrest at the G 1 -S checkpoint before DNA replication and at the G 2 -M checkpoint before mitosis, nor do they undergo apoptosis ( ; ; ).


Tumor suppressor genes such as p53 are found in approximately 10% to 20% of endometrioid cancers, predominantly grade 3, and in many late and sporadic ovarian cancers. In cervical carcinogenesis, the E6 oncoprotein of HPV types 16 and 18 associates with p53 and targets it for degradation, and the E7 oncoprotein of HPV type 16 binds to Rb in infected cells to upregulate proliferation ( ; ). Although p53 mutations are one of the most common mutations in cancer, therapeutic targeting of p53 has met with less than optimal results in several disease sites.


Phosphatase and tensin homologue (PTEN) is the regulatory counterpart of PI3K as a result of dephosphorylating proteins phosphorylated by PI3K. PTEN is a tumor suppressor gene and is found in approximately 20% of endometrial hyperplasia and 50% of endometrioid cancers. Mutations of PTEN occur in exons 3, 4, 5, 7, and 8 targeting the phosphatase domain and regions that control protein stability and localization. Decreased or absent expression of PTEN results in many of the mutations found in endometrioid cancers. Also, epigenetic mechanisms such as promoter hypermethylation and subcellular localization can affect PTEN function in the absence of intragenic mutations.


BRCA1 and BRCA2


Approximately 5% to 10% of breast and 15% of ovarian cancers arise in the setting of a genetic predisposition. Genetic testing for mutations has clinical implications for the patient and patient’s family. The vast majority of these cases are associated with germline mutations in the BRCA1 gene located on chromosome 17q21 and the BRCA2 gene located on chromosome 13q12.3 ( ). Other important pathogenic germline mutations predisposing to ovarian cancer are PALB2, BARD1, BRIP1, RAD51C, and RAD51D. The pattern of inheritance is autosomal dominant, and the prevalence of the mutated gene occurs more often in women of Ashkenazi Jewish descent and in certain French Canadian women. The following women should be tested for a BRCA or other mutations ( ; ; ):




  • A woman with a family history of two or more women with breast cancer or ovarian cancer at any age



  • or



  • A woman with breast cancer before age 50 years, triple negative breast cancer before age 60, two breast cancer primaries, or breast cancer at any age with a family history of other cancers (breast cancer at age younger than 50, ovarian cancer, male breast cancer, pancreatic cancer, or high-grade/metastatic prostate cancer)



  • or



  • A woman at any age with epithelial nonmucinous histology ovarian, fallopian tube, or primary peritoneal cancers



The cumulative risk of developing ovarian cancer by age 70 years is 40% to 50% for BRCA1 mutation and 20% to 25% for BRCA2 mutation carriers, but there is equal breast cancer penetrance in BRCA1 or BRCA2 mutation carriers. The BRCA2 mutation is also associated with male breast cancer and pancreatic, urinary tract, and biliary tract cancers. Unfortunately, a woman with this mutation develops breast or ovarian cancer at a younger age than those who develop sporadic cancers.


BRCA1 and BRCA2 are tumor suppressor genes that encode for large proteins. Similar to other hereditary cancer syndromes, inheritance of a BRCA mutation confers an increased susceptibility to cancer but not an absolute guarantee of developing cancer unless a second inactivation of the allele occurs. Both BRCA1 and BRCA2 encode proteins that are involved in the repair of DNA strand breaks. Most detected mutations are nonsense or frameshift alterations that lead to truncated proteins. BRCA1 and BRCA2 proteins are involved in the pathway mediated by RAD51, which is a protein important in repairing double DNA strand breaks. BRCA1 is also involved in tumor suppression by transcriptional regulation of gene expression, such as being a p53 -independent transactivator of cyclin kinase inhibitor p21. BRCA2 has been identified as a FANCD1 gene, a member of the Fanconi anemia complex. Cells with deficient BRCA1 or BRCA2 are incapable of repairing DNA strand breaks, which leads to genetic instability and tumorigenesis. In BRCA -deficient cells, the defective maintenance of genomic integrity may not only accelerate cancer initiation and progression but also render the cancer more susceptible to therapeutic agents whose cytotoxic potential is mediated through the induction of a specific type of DNA damage that BRCA normally functions to repair. For example, cisplatin and radiation cause DNA interstrand cross-links.


PARPs are a family of multifunctional enzymes that repair DNA single-strand breaks through the repair of base excisions. The inhibition of PARPs leads to the accumulation of DNA double-strand breaks, which are normally repaired by BRCA proteins and thereby provide selectivity of treatment in this BRCA mutation population. Approximately 7% of ovarian cancers will have somatic mutations in BRCA and even more will have a homologous recombination signature, increasing the number of patients who can benefit from PARP inhibitors. Currently there are three commercially available PARP inhibitors: olaparib, rucaparib, and niraparib. PARP inhibitors can be used as first-line maintenance in patients with a germline or somatic BRCA mutation, as second-line maintenance after platinum-sensitive recurrence, and as treatment for patients with germline BRCA mutations ( ). In a large randomized phase III clinical trial (SOLO-1), Moore and colleagues found that patients with ovarian cancer with a germline or somatic BRCA1/2 mutation who had achieved a partial or complete response after initial chemotherapy and had olaparib maintenance had a 70% decrease in the risk of disease progression or death at a median follow-up of 41 months compared with the placebo maintenance group ( ). There are ongoing trials with combinations of PARP inhibitors with antivascular agents and checkpoint inhibitors in gynecologic malignancies.


DNA mismatch repair genes


Hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome, is an autosomal dominant cancer syndrome that predisposes an individual to colorectal, endometrial, gastric, biliary tract, urinary tract, or ovarian cancer. This syndrome is thought to account for all cases of hereditary endometrial cancer and up to 5% of hereditary ovarian cancers. The estimated lifetime risk for endometrial cancer in HNPCC gene carriers is 40% to 60%, corresponding to a relative risk of 13 to 20, whereas that of ovarian cancer is 6% to 20%, corresponding to a relative risk of 4 to 8. Linkage analysis of high-risk families led to the discovery of Lynch syndrome. It was found to be caused by germline mutations in genes responsible for recognizing and fixing errors in the DNA helix, resulting from incorrect pairings of nucleotides during replication or the formation of abnormal loops of DNA. MSH2 (MutS homologue 2) and MLH1 (MutL homologue 1) are the most commonly mutated MMR genes and are located on chromosomes 2p16 and 3p21, respectively. Other MMR genes are MSH6, PMS1, and PMS2, but these occur at a lower frequency. Cells with a defective MMR system exhibit microsatellite instability (MSI). MSI occurs as DNA mismatches cause a shortening or lengthening of repetitive DNA sequences and these mismatches go unchecked. This results in the cancer containing a greater or lesser number of repeats than are present in the normal cells of the individual ( ). A consensus panel of five microsatellite markers (D2S123, D5S346, D17S250, Bat 25, and Bat 26) can be used to identify HNPCC-related cancers compared with sporadic cancers ( ; ; ). In endometrial cancer, MSI can occur from promoter methylation and must be distinguished from MSI caused by an inherited MMR defect.


Taking a family history is the first step in identifying patients with HNPCC. The Bethesda criteria ( Box 27.1 ) seem to be the most sensitive for predicting MMR gene mutations, but the Amsterdam II criteria are more specific. Amsterdam II criteria include the following: colorectal carcinoma and/or endometrial cancer or transitional cell of the ureter or renal pelvis or carcinoma of the small bowel in at least three individuals; one of the patients is a first-degree relative of two other patients; disease occurs in at least two other family members; one of the diagnoses should be made before age 50 years; there is histologic confirmation of the diagnosis; and familial adenomatous polyposis has been excluded. Immunohistochemistry-based universal screening for all colon and endometrial cancer patients will identify 50% more patients with Lynch syndrome than using Amsterdam II or Bethesda criteria.


Aug 8, 2021 | Posted by in GYNECOLOGY | Comments Off on Molecular oncology in gynecologic cancer: Immunologic Response, Cytokines, Oncogenes, and Tumor Suppressor Genes
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