Gynecologic Oncology: Clinical Practice and Surgical Atlas, 1st Ed.

Targeted Therapy and Immunotherapy

Robert L. Coleman and Paul J. Sabbatini

COMPONENTS OF THE IMMUNE SYSTEM

While simplistic in its view, the effectors of the immune system are generally divided into those components that support innate immunity and those that support acquired immunity. The classification suggests a dichotomous relationship, but there is necessary and frequent cross-talk between the 2 arms. Innate immunity is active from birth, is the first line of defense against most pathogens, and does not require modification for activity. Acquired immunity largely requires activation of B lymphocytes and T lymphocytes and uses a complex process of activation, modification, expansion, and suppression in response to changing stimuli.

Innate immunity includes physical barriers (skin, mucous membranes), chemical components (hydro-lytic enzymes and complement), and several cellular components. For example, polymorphonuclear lymphocytes are phagocytic cells with lysosomes containing enzymes and generally fight infection. Recent data suggest that neutrophil survival is modulated by T-cell responses, thus illustrating 1 area of cross-communication between the innate and acquired immunity systems. Monocytes are heavily granulated cells that migrate from blood to various tissues and differentiate depending on site. They become Kupffer cells in the liver, microglial cells in the central nervous system, and macrophages in the lung, spleen, and peritoneal surface. Collectively, these differentiated cells (histiocytes and macrophages) become part of the reticuloendothelial system with a major function of phagocytosis of invading entities. The role of the macrophage is much more complex, however, because it produces proinflammatory proteins (cytokines and chemokines) that stimulate the growth of specific immune and inflammatory cells and positions them where needed. They mediate inflammatory responses during wound healing and, after consuming pathogens, can present them to the corresponding T-helper cell. This is another example of communication between traditionally defined innate and acquired immunity effectors. This presentation is done in conjunction with attaching to a major histocompatibility complex (MHC) class II molecule to identify the macrophage as self despite the foreign antigens on its surface. Macrophages can also play a counterproductive role in tumor elimination through the production of molecules that promote tumor growth and angiogenesis (eg, vascular endothelial growth factor [VEGF] and basic fibroblast growth factor [FGF]). Because they promote inflammation, they also release compounds such as tumor necrosis factor (TNF), resulting in nuclear factor-κB activation, which inhibits apoptosis.

Natural killer (NK) cells are granular lymphocytes (10%-15% of total blood lymphocytes) that recognize and destroy tissues that have been altered or stressed, typically by viruses or by malignant transformation. They differ from T lymphocytes in that they do not have antigen-specific receptors. Instead, they have inhibiting receptors that can recognize the MHC class I molecules on normal cells preventing activation. MHC-I expression is absent or aberrant on many virus- and tumor-infected cells. In another example of cooperation between innate and acquired immunity, NK cells participate in the process of antibody-dependent cell-mediated cytotoxicity (ADCC). Immunoglobulin (Ig) G antibodies can bind to infected cells. Fc receptors found on NK cells (also are present on macrophages, neutrophils, and eosinophils) bring the NK cell into contact with the antibody-coated target cell. Upon contact, NK cells release modified cytoplasmic granules containing perforin and granzymes, which promote apoptosis.

The primary effector components of the acquired immune systems are T and B lymphocytes and involve their interaction with antigens. Antigens are any agents that can bind to a component of the immune system, such as binding with an antibody. This is distinct from an immunogen, which also produces an immune response. In general, for an antigen to be immunogenic, it has to be recognized as foreign (ie, tolerance must be broken), has to be large and complex enough (this can sometimes be rectified by using carriers), and for T-cell activation, has to interact with MHC on the antigen-presenting cells. Factors such as dose, schedule, adjuvant therapy, and route of administration have also been shown to have an impact on immunogenicity. A variety of entities may serve as antigens with varying immunogenicity, with common ones including carbohydrates (these can also induce antibody responses without T-cell help), lipids and nucleic acids (both require protein carriers), and proteins.

Biologic Response Modifiers

Cytokines are secreted substances that facilitate a variety of immune response functions. Many are well characterized, and as examples, a few will be discussed here. Classifications are arbitrary because most are pleio-tropic in function. Chemokines are chemoattractant cytokines, which are low molecular weight proteins, that direct the movement of a variety of effector cells to needed locations. Interleukin (IL)-8, for example, which is secreted by macrophages, mobilizes and activates neutrophils and promotes angiogenesis.¹ Colony-stimulating factors cause the differentiation of progenitor cells in the bone marrow, with many produced for clinical use (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, erythropoietin, and thrombopoietin).² Interferons are produced by lymphocytes, macrophages, and dendritic cells and have a wide variety of functions. The interferon-γ produced by T_H1 cells activates NK cells and macrophages and induces the expression of MHC class II on many cell types. Many cytokines also promote inflammation such as IL-1, IL-6, IL-23, and TNF-α.³ Despite diverse functionality, cytokines generally bind and induce polymerization of a cell surface receptor, which activates a signal transduction pathway. It is not well understood how 2 cytokines with diverse functions retain specificity when using the same signal transduction pathway.

PRINCIPLES OF IMMUNOTHERAPY

Humoral Immunity

B lymphocytes (B cells) produce secreted antibodies that can recognize soluble and cell surface molecules in response to an antigen. Each B cell expresses immunoglobulin against a single antigenic determinant, with the immunoglobulin expressed at the cell surface of the B cell. The diversity of specificities in different B cells is generated by rearrangements of the immunoglobulin genes, and new antibody specificities continue to be generated in response to new antigens.⁴ The antigen binds to the B-lymphocyte surface receptor and activates the B cell. B lymphocytes are mobile and migrate to the T-cell rich areas where T-cell help can be provided to promote increased antibody diversity and increased affinity through immunoglobulin gene rearrangements and class switching. Many antigens require costimulation from CD4⁺ helper T cells (T-cell–dependent antigen), but other antigens do not, including nonprotein antigens such as glycolipids (T-cell–independent antigen). Clonal expansion ensues to make multiple copies of the antigen-specific B cell. Differentiation then occurs to form either plasma cells of short duration (antibody-producing cells), memory cells (which are responsible for the amnestic response), or a small population of B cells expressing germline immunoglobulins that have not undergone rearrangement (which are found in the CD5⁺ B-cell population). Antibodies are produced by the plasma cells of 1 of 5 isotopes (IgG, IgA, IgM, IgE, or IgD). The immunoglobulin variable region (called the Fv region) determines antibody specificity and is located in the Fab domain of immunoglobulins. A conformational change in the Fc portion occurs after binding of antibody to antigen, leading to the activation of several effector mechanisms including complement activation. The early antibody response is IgM, but if T-cell help is available, antibody responses mature through immunoglobulin gene rearrangements into the higher affinity IgG classes. These IgG molecules are capable of improved binding to antigen as well as receptors on the bone marrow–derived cells through their Fc domain, thereby expanding effector functions. The response to most nonprotein antigens are IgM class and generally do not mature to IgG responses. IgM is pentameric and has increased avidity to bind multimeric antigens. It then activates complement as its main effector function.⁵ The complement system consists of a variety of blood components with different enzymatic properties, which cause opsonization (coating of pathogens by complement components); recognition by complement receptors on macrophages, monocytes, neutrophils, and dendritic cells; and subsequent activation of these cells, leading to phagocytosis and/or killing. In addition, complement can cause local and direct killing by forming a membrane attack complex that creates holes in membranes of target pathogens and cancer cells, producing complement-dependent cytotoxicity.

IgG antibodies are synthesized following immunoglobulin gene rearrangements, with switches in Fc domains, as the B cell matures in response to T-cell help. IgG antibodies usually have higher affinity than IgM antibodies and can be found in the extracellular space and in the blood. Subclasses of IgG antibodies in humans are especially effective at activating complement and also sensitizing pathogens for killing by NK cells, macrophages, and other cells with complement receptors and immunoglobulin Fc receptors.

The cross-linking of Fc receptors can also lead to activation of the cells and can produce ADCC of tumor cells through the production of cytotoxic molecules such as perforin and granzymes by NK cells. Monoclonal antibodies are commonly used for cancer therapy. Antitumor effects can be in part mediated by antibody binding to critical molecules on the surface of tumor cells, for example by inhibiting tumor cell attachment or growth receptors. However, generally more than 1 mechanism is at work, and Fc receptor–mediated effector mechanisms such as ADCC are also activated.

Cellular Immunity

T lymphocytes arise in the bone marrow and migrate to the thymus, where they differentiate and develop T-cell receptors and CD4 and CD8 coreceptors. The coreceptors bind to the MHC complex (CD4 cells bind to MHC class II molecules, and CD8 cells bind to MHC class I complexes), as shown in Figure 21-1. The MHC binding is required as a first step to ensure the immune attack is directed at the appropriate target and self can be recognized. Antigens must be processed by antigen-presenting cells for recognition (dendritic cells are 1 major example). The combined antigen/MHC molecules are then trafficked to the cell surface for recognition by T-cell receptors, which are encoded by genes of the immunoglobulin family. Just as with antibodies, the diversity of T-cell receptors is generated by rearrangements of these immunoglobulin family genes. Each monoclonal T-cell receptor binds to its appropriate antigen/MHC complex presented on the surface of the antigen-presenting cell. Two signals are required for T-cell activation. The first is the binding of the antigen/MHC complex. The second is a costimulatory signal that comes from the T-cell surface molecule CD28. CD28 engages B7 molecules found on the antigen-presenting cells. Therefore, successful activation of a T cell requires engagement of the T-cell receptor by an appropriately presented antigen/MHC complex in conjunction with CD28 engagement, with B7 as a “safety lock” to be certain activation occurs only in the correct setting.⁶

FIGURE 21-1. Cells expressing major histocompatibility complex (MHC) class II cells interact with CD4⁺ T cells, which produce cytokines. Cells expressing MHC class I cells interact with CD8⁺ T cells, which destroy infected host cells.

Ox40 and 4-1BB are examples of 2 other costimulatory molecules that are upregulated on the surface of activated T cells, which promote survival of T cells and generate T-cell memory responses.⁷ Multiple effector functions are available to T cells once appropriately activated by professional antigen-presenting cells (primarily dendritic cells), including the production of cytokines and cytotoxic molecules, which lead to death of target cells.

The immune response must be suppressed when the threat has resolved. Natural CD4⁺/CD25⁺ T regulatory cells constitute approximately 10% of CD4⁺ cells and participate in this function. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is expressed on these cells. CTLA-4 also binds B7 molecules, but with much higher affinity, thus displacing the required CD28 activation signal. CTLA-4 signaling leads to downregulation of the T-cell response. The manipulation of CTLA-4 expression is being investigated as a novel therapeutic maneuver, with recent data supporting its usefulness in treating patients with melanoma.⁸ Depletion of T regulatory cells may be particularly useful in enhancing the response to tumor vaccines. The multiple opportunities for paired interactions during the T-cell activation process are illustrated in Figure 21-2.

FIGURE 21-2. Selected paired interactions required at the surface of an antigen-presenting cell (APC) and a CD4⁺ T cell that lead to activation and proliferation with cytokine production. MHC, major histocompatibility complex; TCR, T-cell receptor. (Reproduced, with permission, from Dubinett SM, Lee JM, Sharma S, Mule JJ. Chemokines: can effector cells be redirected to the site of the tumor? Cancer J. 2010;16(4):325-335.)

Immunosurveillance

The notion that the immune system may identify and destroy tumors is long-standing. William B. Coley first observed regression in some patients with sarcoma contracting “accidental erysipelas” as early as 1891. Because the rate of spontaneous regression for tumors is rare and patients with functioning immune systems still develop cancer, the confidence in the ability of the immune system to control cancerous growths has varied over the decades. More sophisticated animal models showing the development of carcinoma in animals deficient in various immunologic components, however, allowed the concept of immunosurveillance to grow. Observations noting CD4⁺ and CD8⁺tumor-infiltrating lymphocytes (TILs) in patients with epithelial ovarian cancer further support its role, as illustrated in Figure 21-3. For example, the 5-year overall survival in epithelial ovarian cancer has been related to the presence or absence of TILs (38% vs. 4.5%, ).⁹ A second study also showed improved survival in patients with increased frequencies of intraepithelial CD8⁺ TILs (55 months vs. 26 months; hazard ratio, 0.33; 95% confidence interval [CI], 0.18-0.60; ). In contrast, patients with increased numbers of immune-suppressive CD4⁺CD25HI regulatory T cells have reduced survival.¹⁰ The process is made more complex when we recognize that not only can the immune system protect the host against tumor development, but also some select cancer cells of lower immunogenicity can escape early immunity due to changes in gene expression. This actually leads to an outgrowth of tumors with the capacity to escape recognition, and this process has been termed immunoediting. The immunoediting or “immune sculpting” process, therefore, is responsible for shaping the immunogenicity of the tumors that will eventually form. Considering the effectors of the immune system in the context of both of these processes is important to develop immune-directed therapies with the greatest chance of success.

FIGURE 21-3. Tumor-infiltrating lymphocytes found in epithelial ovarian cancer specimens. (Images contributed by R. Soslow [Memorial Sloan-Kettering Cancer Center, Pathology]).

Escape From Surveillance

An alteration in the function of almost every process discussed herein to facilitate immune activation has been postulated to participate in the ways tumors can evade immune detection. In some cases, antigen presentation is downregulated, or gene deletions or rearrangements may cause reduced expression of the MHC-I complex, thus preventing T-lymphocyte activation. Tumors can also secrete proteins that inhibit T-cell effector action or that promote the development of regulatory T cells that suppress immune function. A recent novel observation showed that certain melanomas can actually remodel their stromal microenvironment so it resembles lymphoid tissue, which recruits regulatory cells to promote tolerance and allow tumor progression.¹¹ Other mechanisms include the down-regulation of intracellular adhesion molecules, changes in molecules responsible for apoptosis signaling, and the development of peripheral tolerance. Based on the increasing number of interacting mechanisms with putative activity that allow immune escape, approaches directed against multiple mechanisms will likely be needed to eradicate immune-tolerant tumor cells.¹²

Immunoprophylaxis

The development of most immunotherapeutic strategies for investigation requires deciding which of the tumor-associated antigens one will target (often multiple). The next step is to select the strategy that will be used (such as dendritic cell vaccination or autologous cell lysates) to generate the proposed effector cells. Some assessment that the effector cells are indeed directed at the target is then prudent. Finally, the clinical merit of the approach must be assessed.

Ongoing advances using newer technologies including serologic analysis of recombinant cDNA expression libraries (SEREX), robust applications of bioinformatics, and seromic profiling techniques have allowed the further characterization of tumor-associated antigens in multiple tumor types.^13,14 There are more than 2000 candidate tumor-associated antigens, and they are generally classified as follows: (1) differentiation antigens, (2) mutational antigens (that are altered forms of proteins), (3) amplification antigens, (4) splice variant antigens, (5) glycolipid antigens, (6) viral antigens, and (7) cancer-testis antigens. Representative examples of each group are provided in Table 21-1.

Table 21-1 Tumor-Associated Antigen Categories With Representative Examples

In addition to selecting the appropriate targets, the next step is to determine the strategy to be used for vaccination. Multiple approaches have been considered, including: a variety of antigens can be given alone or with adjuvant treatment; modified or unmodified tumor cell lysates can be administered (autologous or allogeneic); dendritic cells can be primed with a variety of agents; tumor hybrids with antigen-presenting cells can be made; or DNA alone or in a recombinant fashion can be administered. A variety of cancer vaccine strategies are listed in Table 21-2.

Table 21-2 Cancer Vaccine Strategies

It is important to evaluate the effector cells for the ability to interact with the target. This may be simple and straightforward, or a variety of surrogate approaches may be used to identify whether the biologic end point is being achieved. This may be the most challenging area in immunotherapy because a traditional dose-limiting toxicity finding model does not apply. Radio-immunoassays (enzyme-linked immunosorbent assays [ELISA]) are used to measure antigens, antibodies, or antigen–antibody complexes if appropriate. Antibodies are generally tested for specificity against certain cell surface antigens using fluorescence-activated cell sorting techniques (FACS), or following stimulation with specific antigens in vitro, they can be cultured in the enzyme-linked immunosorbent spot (ELISPOT) assay. Assays for T-cell proliferation and activation are more complex. A cytotoxicity assay (cytotoxic T lymphocytes) measures the ability of cytotoxic T or NK cells to kill radiolabeled target cells (often chromium-51) expressing the appropriate antigen that was initially targeted. The percentage of chromium release can be measured.

Possible effector responses (depending on the strategy selected) are varied and include enhancing phagocytosis, antibody and complement activation, loss of adhesive properties of tumor cells promoted by antibody administration, cytotoxic and helper T lymphocytes, ADCC, activated macrophages, neutrophils, NK/T cells, lymphokine-activated killer cells, and NK cells. More than 1 mechanism is generally at work through direct stimulation or via cross-talk between systems.

CLINICAL PRACTICE IN GYNECOLOGIC ONCOLOGY

Suitability for Immunotherapy

Opportunities to improve the outcome for patients exist by making primary therapy more effective or by exploring the application of “consolidation” or “maintenance” approaches to patients in a complete primary or subsequent remission. One important issue in evaluating immunotherapeutic approaches in ovarian cancer is deciding where in the disease course the novel agent should be evaluated. In general, the minimal disease state is sought, and the remission populations are best suited. The value of treatment in clinical complete remission was first established in acute leukemia, and additional “consolidation” or “maintenance” chemotherapy dramatically improved the outcome for some of these patients. These concepts have not found a place in solid tumor therapy, and the nomenclature remains confusing. Strictly speaking, “consolidation” is best applied to those strategies that are of limited duration, such as a fixed immunization course, and “maintenance” is best used to describe interventions that continue for years (or until progression) such as with trastuzumab. In ovarian cancer, no randomized consolidation study has provided a statistically significant improvement in overall survival, although many attempts have been made. Negative randomized consolidation approaches include both subcutaneous and intraperitoneal interferon-α, high-dose chemotherapy, continued intravenous carboplatin versus whole abdominal radiotherapy (WART), chemotherapy versus observation versus WART, intraperitoneal radioactive phosphorus (phosphorus 32), “non–cross-resistant” chemotherapy in the form of cisplatin and 5-fluorouracil for 3 cycles or topotecan for 4 cycles, the monoclonal antibody oregovomab, which targets CA-125, and the SMART study.^15-17 Consolidation strategies have generally been used in the first remission population; investigational strategies in the second and third remission groups have been rare and all likewise negative to date.¹⁸ Patients with ovarian cancer in remission are ideal candidates for an immunotherapeutic strategy. Recent data highlight the homogeneity of the second and third remission groups who have a progression-free survival (PFS) interval of less than 12 months so that hints of efficacy from a given immunotherapeutic approach could be recognized with a shorter follow-up interval than that required in first remission.

The number of therapeutic strategies under investigation for immunotherapy in patients with ovarian cancer is large. Most trials are pilot studies or phase 1 trials with the goal of assessing safety and immunogenicity. Some have correlated improved outcome with a surrogate such as antibody or T-cell response, and most current trials aim to produce cellular responses. The number of adequately powered randomized trials is few, however, and none has shown definitive efficacy to date. We will consider several examples of immuno-therapeutic approaches that are being evaluated in the clinics, but the list is not exhaustive, and many other approaches have merit.

Antibodies Used as Immunogens

Although some antibodies are administered in the treatment of patients with cancer to convey passive immunity, they may also be used as immunogens and can elicit a complex immune response. Oregovomab (MAb B43.13), which is an IgG1k subclass murine monoclonal antibody that binds with high affinity (1.16 × 10¹⁰/M) to circulating CA-125, has been evaluated. Both cellular and humoral immune responses have been seen with the production of anti-oregovomab antibodies (Ab2), T-helper cells, and cytotoxic T cells in addition to the human anti-mouse antibody (HAMA) response. Nonrandomized clinical studies to date have consistently associated a longer overall survival with immune response. A randomized placebo-controlled trial in patients with stage III or IV epithelial ovarian cancer in first clinical remission receiving oregovomab or placebo showed no benefit using the intent-to-treat population. However, a favorable subgroup of patients (≤2 cm residual at debulking,  U/mL before third cycle, and  U/mL at entry) showed a time to progression advantage favoring vaccination of 24 months versus 10.8 months (hazard ratio, 0.543; 95% CI, 0.287-1.025). This subgroup was appropriately considered to be hypothesis generating, and a follow-up study enrolled 354 patients using the characteristics of this group as eligibility criteria. The median time to progression was 10.3 months (95% CI, 9.7-13.0 months) for the oregovomab group and 12.9 months (95% CI, 10.1-17.4 months) for the placebo group , showing no benefit to oregovomab immunotherapy.¹⁷

Another antibody strategy is immunization with an anti-idiotype vaccine. The hypothesis is that the antigenicity of the immunogen (in this case, the antibody) can be increased by presenting the desired epitope to the now tolerant host in a different molecular environment. The “immune network hypothesis,” which provided the foundation for this approach, was first proposed in the early 1970s and describes an interconnected group of idiotypes expressed by antibodies. The proposed mechanism assumes that immunization with a given antigen will generate the production of antibodies against this antigen (termed Ab1). Ab1 can generate anti-idiotype antibodies against Ab1, classified as Ab2. Some of the anti-idiotypic antibodies (Ab2β) express the internal image of the antigen recognized by the Ab1 antibody and can be used as surrogate antigens. Immunization with Ab2β (the anti-idiotype antibody) can cause the production of anti-anti-idiotype antibodies (classified as Ab3) that recognize the corresponding original antigen identified by Ab1. Ab3 antibodies are also denoted Ab1’ to show that they may differ in their other epitopes compared with Ab1. The relationships of these antibodies to each other are illustrated in Figure 21-4. A previous phase ½ study of abagovomab, the anti-idiotype monoclonal antibody whose epitope mirrors CA-125, suggested that Ab3 production was associated with overall survival. Other studies have shown an increase in interferon-γ expression of CA-125–specific CD8⁺ T cells following immunization, but there has been no specific correlation between the induction of Ab3 and frequencies of CA-125–specific cytotoxic T lymphocytes and T-helper cells. The efficacy of abagovomab in patients in first remission is currently being evaluated in an international phase 3, randomized, double-blind, placebo-controlled study ongoing in approximately 120 study locations (MIMOSA Trial). Outcomes are recurrence-free survival, overall survival, and safety. Preliminary blinded immunogenicity results were reported with 888 patients enrolled onto the study and showed that 68% and 69% of all patients were positive for Ab3 (median values, 62,000 ng/mL and 337,000 ng/mL, respectively), whereas 53% and 63% of patients were positive for HAMA (median values, 510 ng/mL and 644 ng/mL, respectively). Efficacy results will be available in early 2011.¹⁹

FIGURE 21-4. The relationship of antibodies in an anti-idiotypic vaccine strategy. A. The injection of a tumor-associated antigen (TAA) binding antibody (Ab1) leads to an immune response containing antibodies (Ab2) mimicking the structure of the original antigen. B. Vaccination with a TAA-mimicking antibody (Ab2) leads to an immune response directed to the original antigen.

Cancer-Testis Antigen Vaccines

The cancer-testis antigens are a distinct class of differentiation antigens. The family has grown from the original melanoma-associated antigen 1 (MAGE-1) identified in a melanoma cell line to 100 cancer-testis genes or gene families identified in a recent database established by the Ludwig Institute for Cancer Research.²⁰ These antigens share several characteristics, including preferential expression in normal tissues on the testis and expression in tumors of varying histology (including ovarian cancer), and many are members of multigene families that are mostly encoded on chromosome X. Cancer-testis antigen expression has been correlated with clinical and pathologic parameters in a variety of tumors. MAGE-A4 expression shows an inverse correlation between expression and patient survival, for example, in ovarian cancer .

The NY-ESO-1 antigen, initially defined by SEREX in esophageal cancer, is expressed in several tumors, including 40% of epithelial ovarian cancers. NY-ESO-1 MHC class I and II restricted epitopes (recognized by CD8⁺cytotoxic and CD4⁺ helper T cells) have been characterized, including those recognized in conjunction with human leukocyte antigen (HLA)-A2 as well as with other haplotypes. Both NY-ESO-1 peptides and full recombinant protein have been administered to patients on protocols with immunogenicity as the primary end point with various adjuvants. Vaccination has been shown to induce both humoral and T-cell responses.²¹ In a phase 1 trial in patients with epithelial ovarian cancer in first remission immunized with HLA-A*0201–restricted NY-ESO-1b peptide with montanide ISA-51 as the adjuvant,²² treatment was well tolerated. Seven (77%) of 9 patients showed T-cell immunity by tetramer and ELISPOT analyses. Multiple approaches have been used to try and enhance the inherently limited immunogenicity of these peptide vaccinations. Some have included amino acid substitution at the anchor positions of Melan-A/MART-1_26-352L; terminal modification of MART-1_27-35; substitution of cysteine residues for NY-ESO-1; modification of T-cell receptor interacting amino acid residues for carcinoembryonic antigen; and loading of peptides onto autologous dendritic cells. In addition, cytokines and costimulatory molecules have been administered.

Dendritic Cell–Based Vaccines

Dendritic cells are professional antigen-presenting cells. They endocytose, process, and then present tumor antigens to T cells. Many strategies are currently under way to manipulate the dendritic cell for use in immunotherapy. Dendritic cells have been pulsed with tumor-associated peptides or proteins and mRNA-encoded receptors such as folate receptor-α.²³ Other vaccines have been developed by the viral transduction of dendritic cells with tumor-specific genes or through transfection with liposomal DNA or RNA. Another strategy that has been tried to avoid the need to specifically define the effective tumor-associated antigens is to pulse them with tumor lysates or tumor protein extracts. In many cases, preclinical models have suggested protective immunity to subsequent tumor challenge, which supports further interest in investigating the approach.

A specific example includes a study by Czerniecki et al,²⁴ in which advanced breast and ovarian cancer patients were treated with dendritic cells pulsed with HER-2/neu or MUC-1–derived peptides. In 50% of patients, peptide-specific cytotoxic T-cell lymphocytes were generated. Side effects were minimal. Gong and colleagues²⁵ fused human ovarian cells to human dendritic cells and likewise showed the proliferation of autologous T cells, including cytotoxic T-cell activity with lysis of autologous tumor cells by an MHC class I restricted mechanism (ie, demonstrating that the effector cells had the desired activity). Heat shock proteins, which are molecular chaperones that facilitate protein folding, have also been isolated, along with accompanying peptides, and used as immunogens. Heat shock peptide complexes have been shown to interact with dendritic cells via the CD91 receptor. The heat shock proteins are taken up by endocytosis, are cross-presented by MHC-I molecules on the dendritic cells, and result in activation of naïve CD8⁺ cells along with upregulation of costimulatory molecules and the production of cytokines.

Many reported studies have similar immunologic end points, but the clinical interpretation is often difficult from phase 1/2 trials without comparators. Further issues under study include the choice of dendritic cell subtype and maturational status at immunization, antigen loading method, preparation of the cells loaded on the dendritic cells, and route and frequency of administration.

Vaccines Designed to Generate Antibody Responses

Most current vaccines seek to generate cellular responses (often with an accompanying humoral response), but a vaccine is currently in a phase 2 randomized trial in ovarian cancer (Gynecology Oncology Group [GOG] Study 255) that evaluates a vaccine approach primarily designed to augment antibodies. Techniques for the chemical and enzymatic synthesis of carbohydrate and glycopeptide antigens have permitted the development of a variety of synthetic vaccines that depend on antibody production and ADCC as the primary effectors. A variety of options such as different adjuvant therapies, schedules, and methods of conjugation have been tried to enhance immunogenicity. A proposed optimal construct has consisted of an antigen (single or multiple) with the carrier protein keyhole limpet hemocyanin (KLH) and the saponin adjuvant QS-21 (or OPT-821).²⁶ GOG 255 is a randomized trial in patients with second or third complete clinical remission receiving either the adjuvant therapy alone (OPT-821) or a multivalent antigen construct plus OPT-821. The end point is the proportion of patients disease free at 12 months, and accrual is ongoing.

Adoptive Cellular Therapy

Using the adoptive cellular therapy approach, one selects and activates large numbers of lymphocytes and introduces them into a manipulated host environment with a selected target. One way T cells may be modified to recognize tumor-associated antigens is to introduce ex vivo a gene encoding artificial T-cell receptors termed chimeric antigen receptors (CARs) against a specific tumor-associated antigen. The first phase 1 study in patients with epithelial ovarian cancer using gene-modified autologous T cells with reactivity against the ovarian cancer–associated antigen α-folate receptor (FR) has been reported.²⁷ Cohort 1 received T cells with IL-2, and cohort 2 received dual specific T cells followed by allogeneic peripheral blood mononuclear cells. No reduction in tumor burden was seen in any patient. Polymerase chain reaction analysis showed that gene-modified T cells were present in the circulation 2 days after transfer but then declined. An inhibitory factor developed in the serum of 3 of 6 patients tested over the period of treatment that significantly reduced the ability of gene-modified T cells to respond against FR-positive tumor cells. Future studies need to use strategies to increase T-cell persistence. The chimeric receptor approach continues to evolve in specificity against targets expressed in ovarian cancer such as the LeY carbohydrate antigen (expressed on 70% of ovarian cancer cells) or HER-2/neu. Most recently, receptors have been engineered to target the extracellular domain (termed MUC-CD) of MUC16 (CA-125), which is expressed in most ovarian carcinoma.²⁸ In vitro, these CAR-modified, MUC-CD–targeted T cells showed MUC-CD–specific cytolytic activity against ovarian cell lines, and infusion into severe combined immunodeficiency (SCID)-beige mice bearing orthotopic human MUC-CD–positive ovarian carcinoma tumors showed delayed disease progression or eradication. Clinical trials are planned. One necessary challenge to overcome is how to circumvent the multiple mechanisms in the tumor microenvironment that inhibit tumor-targeted T cells. Options under investigation include administering T cells after lymphodepleting chemotherapy, antibody-based blockade of inhibitory ligands, and infusion of proinflammatory cytokines such as IL-12.

Whole Tumor Antigen Vaccines

This strategy seeks to overcome some of the potential problems associated with trying to generate specific immune responses. In the latter case, the response may simply miss the target, it can be limited to only the epitopes provided on the stimulating antigen and actually drive variants of tumor cells that can evade the immune response (immunoediting), or it may be restricted to small numbers of patients of a certain HLA type, as in the case of using HLA-restricted peptides.²² The reason for using whole tumor antigen vaccines is that it allows one to immunize without needing to define the tumor-associated antigens. They can be derived from autologous tumor cells or using an allogeneic strategy. One obvious challenge in using whole tumor antigen vaccines is that the tumor is currently residing in a host where tolerance to the tumor is already present. This tolerance is likely produced in multiple ways, including the production of IL-10 and transforming growth factor (TGF)-β to inhibit T-cell and dendritic cell functions, VEGF to inhibit dendritic cell maturation and differentiation, and soluble Fas ligand, which induces lymphocyte apoptosis. The whole tumor immunogen, therefore, is processed or modified in some way in an attempt to overcome this. Strategies have included using apoptotic whole tumor cells (developed with a lethal dose of irradiation), using necrotic tumor cell lysates (created with repetitive freezing and thawing and often administered as pulsed dendritic cells), and constructing dendritic cell/tumor fusion vaccines.²⁹ The issue of how to increase the immunogenicity of whole tumor vaccines remains a priority. One effective approach has been the use of a replication-deficient herpes simplex virus to infect tumor cells, which are subsequently engulfed and show enhanced ability to both activate NK cells and provide a costimulatory signal for T cells.

TARGETED THERAPIES

The principal tenets of optimal disease management in women with gynecologic malignancies have been the strategic utilization of surgery, cytotoxic chemotherapy, hormonal therapy, and radiotherapy. Although substantial progress has been realized from these practices, disease-specific mortality from gynecologic malignancies still accounts for about 9% of all cancer-related deaths and underscores the need for the development of new therapeutic modalities. Investigation into the mechanisms governing cancer initiation, proliferation, metastases, autophagy, and apoptosis have uncovered a wealth of new opportunities, many of which harbor the potential of reversing the malignant phenotype, selectively inducing cancer cell death, overcoming primary and induced drug resistance, and optimistically improving overall outcomes for patients. Ability to pharmacologically and pharmacodynamically interact with these new “targets” has fostered rapid drug development, some of which is beginning to show merit in the treatment of women with gynecologic malignancy. Because the biology of cancer growth often shares homology across different tumor types, targeted therapies are being investigated where the pathway of aberration is suspected to play an important or dominant role in disease pathogenesis. Although an “Achilles’ heel,” or a solitary activated pathway, is not present in most solid tumors, the opportunity to selectively target key regulatory and survival mechanisms in the tumor microenvironment holds great promise in expanding our therapeutic armamentarium for these women. We review some of these pathways and agents in this section.

Mechanisms of Action

One of the most common events defining the cancer process is dysregulation of protein kinases that govern normal cellular function. In light of this observation, proteins are frequently the targets of anticancer agents. Although there are many ways to affect protein kinase function, including small molecules, monoclonal antibodies, antagomirs, antisense, RNA interference, immuno- and receptor drug conjugates, decoy receptors, allosteric inhibitors, and nanotubes, the intent is to target these aberrancies either restoring normal host function or inducing cell death. The principle challenge is to affect tumor cells without impacting the function of normal host cells. Three relevant mechanisms are important to review.

Interruption of Signal Transduction Pathways

Signal transduction is the process where a ligand, usually lipophobic (eg, a growth factor), meets a receptor or channel on the cell surface and initiates a cascade of events such as kinase activity or dissociation of G-coupled proteins resulting in some cellular response. In contrast, lipophilic ligands (eg, steroids) can penetrate the cell membrane and may affect cellular functions by direct binding to cytoplasmic or nuclear targets. Many of the “small molecules” being developed for cancer therapy involve blocking the tyrosine kinase activity of membrane-bound receptors that are usually influenced by a number of promoting ligands. The prototypical example of a relevant ligand-receptor signal transduction pathway in carcinogenesis is the epidermal growth factor receptor (EGFR) (Figure 21-5). This receptor family is overexpressed and activated in many tumor types, including gynecologic malignancies, and appears to play a key role in disease pathogenesis.³⁰ Binding of the epidermal growth factor (EGF) ligand to the receptor induces tyrosine kinase activity, which leads to receptor dimerization and activation of the pathway driving multiple cellular functions such as cellular proliferation, enhanced cellular motility, resistance to apoptosis, and angiogenesis. Because of the broad spectra of activity, there has been intense interest in developing therapeutics against this pathway. Typically, these targeted agents are classified in 2 broad categories: competitive adenosine triphosphate (ATP)–pocket small-molecule inhibitors and monoclonal antibodies to the receptor’s extracellular domain. The clinical experience of these molecules in gynecologic cancer will be discussed later; however, the crafted directive of these targeted agents is to disrupt ligand/receptor activation in the hopes of blocking the signal transduction pathways leading to cancer cell survival.

FIGURE 21-5. The epidermal growth factor receptor (EGFR) pathway. Ligand binds with the extracellular domain of the EGFR and causes the receptor to dimerize. Upon this, activation of the pathway drives several downstream events including angiogenesis, proliferation, cellular motility, and resistance to apoptosis. Various agents to block this pathway are depicted. See text. LOF, loss of function; VEGF, vascular endothelial growth factor.

Induction of Apoptosis

Normal development and functional physiology are dependent on tight regulation of cellular growth and death. The representation of cancer as “uncontrolled cellular proliferation” attests to the importance dysregulated cellular programmed cell death, or apoptosis, plays in human disease. Phenotypical transformation of normal cell to cancer cell is likely highly influenced by loss of apoptotic function. In addition, resistance to chemotherapy-induced cytotoxicity is frequently the result of cellular escape from apoptotic inducement. Two dominant pathways govern cellular apoptosis: extrinsic, induced via a receptor-ligand interaction (death receptor), and intrinsic, induced via mitochondria-apoptosome signaling (Figure 21-6). The converging points for both pathways are the effector caspases, which are closely regulated by upstream signaling proteins either inducing apoptosis or preventing it. A caspase-independent pathway also exists and appears to be mediated through apoptosis-inducing factor (AIF), which is released from mitochondrial pores under control of Bcl-2 and induces nuclear chromatin clumping. The ultimate declaration of apoptosis is largely the balance of proapoptotic proteins (BAX, BID, BAK, and BAD) and antiapoptotic proteins (Bcl-XI and Bcl-2). Numerous ligands have been identified as substrates for the death receptor including TNF, TNF-related apoptosis-inducing ligand (TRAIL), and Fas. Recently, novel targeted agents harboring agonist activation of this pathway at both the ligand and receptor levels have entered clinical trials.³¹

FIGURE 21-6. The intrinsic and extrinsic pathways of apoptosis. Multiple downstream effectors and cross-talk relationships are depicted and are current targets of drug therapy development.

The intrinsic pathway may be initiated by a number of cellular stressors such as radiation therapy, chemotherapy, hypoxia, infection, and starvation. Mitochondria contribute to the apoptotic process either by increasing permeability, leading to the release of important regulators such as second mitochondrial-derived activator of caspases (SMACs) and nitric oxide, or by developing membrane pores causing the organelle to swell, which can lead to release of cytochrome C. This latter process has been shown to contribute to the formation of the apoptosome, which converts procaspase-9 to its active form, which in turn promotes caspase 3 activation, an effector caspase. Proteins belonging to the BCL-2 family primarily govern the mitochondrial pathway. This is an extensive family that may be divided functionally (pro- or antiapoptotic) or structurally (those that share BCL-2 homology and those expressing only the BH3 domain).³² This latter domain, BH3, is the natural ligand for the BCL-2 family’s protein–protein interaction and has become of great interest in drug therapeutic development.

Finally, p53, the most commonly mutated gene in human malignancy, functions as a transcription factor regulating downstream genes involved in DNA repair, cell cycle arrest, and both the intrinsic and extrinsic apoptotic pathways. p53, when activated, promotes the proapoptotic genes of the BCL-2 family, which inhibit Bcl-2 at the mitochondrial membrane, as well as activate expression of the death receptors, such as DR₅.³¹ In this manner, cross-talk between the intrinsic and extrinsic pathways is extensive. When p53 is dysfunctional, one or both of these pathways may drive carcinogenesis; thus, this serves as rationale to consider combinatorial treatment approaches, such as targeted therapy of the death receptor ligand in combination with cytotoxic chemotherapy.

Stimulation of the Immune Response

As was previously presented, the immune system is a highly complex and interactive network of specialized cells and organs working in conjunction to maintain health. It is of no surprise that attempts at leveraging innate response or inducing a heightened response to cancer cells has been the subject of cancer therapeutic investigation for decades. The slow, albeit measured, clinical progress in this regard is a reflection of the complexity of the system, the evasiveness of cancer cells, and the imperfect models to preclinically study the system. However, the efficiency, selectivity, and sensitivity of the immune response make it one of the most promising avenues of targeted therapy and worthy of the effort.

Key effectors of the immune response include cytokines, such as interferons and interleukins, and antibodies. Contemporary understanding of the interplay between cancer and the immune system suggests that although cancer cells are immunogenic, they do not always elicit a response. This “immunotolerance” is not well understood but may be mediated in part by local anti-inflammatory tumor cytokine production, which may prevent dendritic cells from properly processing tumor cell antigens for a robust immune, anticancer response. Nevertheless, several avenues of investigation have been pursued; the agents used in this regard are called biologic response modifiers (BRMs).

The first BRMs to be created and used in cancer therapy were the interferons. As discussed earlier, this class of compounds has both direct and indirect activity on cancer cells. For example, the interferons can slow cancer cell growth or induce phenotypic transformation into normal cell behavior. Interferons also stimulate NK cells, T cells, and macrophages, which may increase the efficiency of the immune response to effect better anticancer treatment. Several interferon compounds (α, β, and γ,) have been US Food and Drug Administration (FDA) approved for cancer therapy, and many have entered mature clinical investigation, including for gynecologic cancers, albeit with mixed results. For instance, an Austrian phase 3 study randomized 148 women with International Federation of Gynecology and Obstetrics stage IC-IIIC disease to cisplatin/cyclophosphamide with or without subcutaneously administered interferon-γ. PFS at 3 years was significantly improved (17 vs. 48 months; ; Relative Risk (RR), 0.48; 95% CI, 0.28-0.82), and toxicity was considered comparable between the arms. However, a much larger phase 3 study  conducted by the GRACES clinical trial consortium investigating combination paclitaxel/carboplatin with or without interferon-γ-1b in women with advanced-stage ovarian cancer was terminated early due to an interim futility analysis suggesting detrimental effects in the experimental cohort.³³ Clearly, more work in this area is needed.

A second class of cytokines being investigated as cancer therapeutics is the interleukins (IL). These naturally occurring families of compounds have a vast cache of activities in multiple host systems, including lymphoproliferative organs and angiogenesis and immune system effectors, such as lymphocytes and platelets. Currently, IL-2 (aldesleukin), an IL that stimulates growth and differentiation of the T-cell response, is FDA approved for the treatment of meta-static renal cell carcinoma and melanoma. However, in light of the numerous functions ILs drive in the immune and host response to cancer cells, investigators continue to search for key treatment opportunities. For example, it has been known that IL-6, a proinflammatory cytokine that impacts hematopoietic stem cells, is a poor prognostic factor (associated with advanced disease, chemotherapy resistance, early recurrence, and short survival) of several solid tumors, including the gynecologic cancers, and is closely linked to angiogenesis, particularly in ovarian cancer, where high levels are also identified in ascites.³⁴ It also may be an important mediator of the paraneoplastic thrombocytosis phenotype, which is commonly identified in patients with advanced-stage ovarian cancer (Anil Sood, MD, personal communication in 2010). In light of these observations, IL-6–targeted therapies are being developed, including highly selective silencing through short interfering RNA.

MONOCLONAL ANTIBODIES

Antibodies with a specific target created from clones of a unique parent cell are called monoclonal antibodies (MoAbs). Their specificity for a unique target has made them highly desired for cancer therapeutics for which several, such as trastuzumab, bevacizumab, and cetuximab, are already FDA approved. These compounds have been often referred to as the “magic bullets” of disease therapy, a modern realization of the initial hypothesis of selective cytotoxicity forwarded by Paul Ehrlich in 1908.

Development

MoAbs were first developed in murine systems by injecting human cells of interest (eg, cancer cells) and collecting murine plasma containing antihuman murine antibodies. Expansion of these antibodies was initially accomplished in hybridomas created from myeloma cells lacking the ability to secrete antibodies fused with healthy B cells, which were immunized by an antigen of interest. These murine antibodies were largely ineffective as treatment due to poor induction of immune cytotoxicity and the development of HAMAs, which rapidly degraded these agents and, in some cases, led to catastrophic circulatory collapse with repeated exposure. Murine MoAbs are usually represented by the suffix “-omab” (Table 21-3).

Table 21-3 Biologically Targeted Monoclonal Antibody Class and Nomenclature

Humanization

To reduce the immunogenicity associated with murine MoAbs and to enhance their effectiveness, specific modifications were subsequently developed. Three types of MoAbs fall into this “humanization” process: chimeric antibodies, humanized antibodies, and fully human antibodies. All 3 types are used in clinical practice today, including oncology therapeutics. The class of MoAbs can be generally recognized by its nomenclature suffix: chimeric, by “-ximab” (eg, EGFR MoAb, cetuximab); humanized, by “-zumab” (eg, VEGF receptor [VEGFR] MoAb, bevacizumab); and fully human, by “-mumab” (EGFR MoAb, panitumumab). Chimerics are derived by fusing murine variable regions with human constant regions. Humanized MoAbs are created by grafting a small portion (~ 5%) of the immunized murine antibody to a human antibody backbone. The resulting molecule retains specificity for the human target, but due to the latter, largely evades the HAMA response. Although affinity for the primary target may be reduced in the process, several contemporary technologies have been developed to enhance antigen recognition. Fully human MoAbs are produced via transgenic mice, which are genetically engineered to produce human MoAbs following vaccination or by phage display technology. As will be presented later, this class of therapeutic has been central to some of the most important recent cancer treatment advances in gynecologic malignancies.

CLINICAL TARGETS IN GYNECOLOGIC ONCOLOGY

Estrogen Receptor

In light of the central role hormonal steroids play in developmental and adult physiology, it is not surprising that investigation into the role these elements play in proliferative disorders was the basis for some of the earliest forms of cancer therapeutics, particularly in gynecologic malignancies. It is generally well accepted that the bioactivity of estrogen is mediated through either or both estrogen receptors (α and β), which are predominately located in the nucleus. The principal ligand for the estrogen receptor is estrogen, but it may be activated by several growth factors including the epidermal growth factor, insulin, insulin-like growth factor-1, and TGF-α.³⁵ Upon binding of ligand, the estrogen receptor forms either homodimers or heterodimers (in the presence of estrogen receptor-β), which activate transcription of specific genes containing estrogen response elements. In many tissues, this transcription drives proliferation, which if unfettered, can induce aberrations leading to carcinogenesis. This is one of the hypotheses for the development of type I endometrial cancer and may well describe early events in the initiation of ovarian/tubal carcinogenesis and, more recently, metastases. Cells not expressing the estrogen receptor may also respond to estrogen ligand through nongenomic signaling via G-coupled proteins and an associated second messenger. In addition, membrane-bound estrogen receptor-α can activate Akt signaling via interaction with PI3K, particularly in endothelial cells. Conversely, estrogen-independent activation of the estrogen receptor can be induced through the Ras/Raf/ERK pathway in cells that over-express ErbB2. It is interactions such as these that are suspected to contribute to an endocrine-resistant phenotype in breast and ovarian cancers (Figure 21-7). Estrogen receptor-α is predominately expressed in endometrial tissues and stroma, whereas estrogen receptor-β is observed more prevalently in ovarian tissues, although both receptors are expressed there. The function role of these 2 receptors in response to ligand can be complementary or opposing depending on the tissue site. In light of these observations, therapy antagonizing the estrogen receptor/ligand axis has been of interest in many tumors.

FIGURE 21-7. Estrogen receptor (ER) signaling may occur through nuclear or cytoplasmic interactions. Inhibition of apoptosis and aberrant cellular growth can occur via estrogen ligand-independent activation of the ER via Ras/Raf/ERK activation.

The clinical activity of hormonal therapy in endometrial and ovarian cancer is discussed in detail in other chapters; however, it is of interest to comment on the interaction between the estrogen hormone receptor and microRNAs (miRNA). miRNAs are short double-stranded RNA fragments that have near perfect complementarity to a number of genes and are predominately associated with translational repression or degradation.³⁶ Recently, a number of miRNAs have been discovered to reduce estrogen receptor protein translation from estrogen receptor-α, which in turn regulates the expression of controlling miRNAs. The feedback loop is important in tumors where regulatory miRNAs are under suppressive control by estrogen receptor-α overexpression. The relevance of these discoveries will likely be elucidated as therapeutic RNA interference strategies are developed for systemic administration.

Epidermal Growth Factor Receptor

EGFR, like VEGFR, is a tyrosine kinase receptor in the cell membrane. Its ligand, EGF, binds EGFR, which then dimerizes and initiates signal transduction pathways that affect cellular proliferation, motility and invasion, apoptosis, and angiogenesis. EGFR is overexpressed in 60% to 80% of endometrial cancers, 73% of cervical carcinomas, and 68% of vulvar malignancies and is associated with advanced stage and poor prognosis.³⁷ Initial in vivo studies of EGFR inhibitors showed increased chemo- and radiosensitivity of tumors.

Cetuximab is an MoAb against EGFR that has improved survival in patients with head and neck and colorectal carcinoma. This antibody has been tested in combination with carboplatin in patients with EGFR-positive recurrent epithelial ovarian cancer with a response rate of 35% (12% with complete response).³⁸ A trial of cetuximab in combination with carboplatin and paclitaxel in patients with advanced ovarian or peritoneal cancer achieved a complete response of 70%, but 18-month PFS was 38.8% and was not considered a meaningful improvement in outcome over expected activity of carboplatin and paclitaxel alone. Several clinical trials in nearly all gynecologic primaries have now been completed, with both MoAbs and small-molecule tyrosine kinase inhibitors (eg, erlotinib and gefitinib) of this pathway showing modest or limited clinical efficacy. Although overexpression of EGFR would appear to define some aspects of tumor biology, multiple collateral activation pathways below the receptor level are likely contributing to the disconnect between receptor silencing and continued pathway signaling.

Human epidermal growth factor receptor 2 (HER-2) is also a membrane-bound tyrosine kinase receptor in the same family as EGFR. Like EGFR, HER-2 dimerizes upon activation to mediate cell survival, proliferation, and angiogenesis. Approximately 5% to 23% of epithelial ovarian cancers and up to 44% of endometrial cancers overexpress HER-2.³⁹ HER-2 gene amplification has been found to directly correlate with poor clinical outcomes in many malignancies including breast and ovarian cancer. Trastuzumab is a humanized MoAb against HER-2 that has been effective for the treatment of many patients with HER-2–positive breast cancer. In patients with recurrent or progressive epithelial ovarian cancer positive for HER-2 overexpression, 7.3% achieved a clinical response with single-agent trastuzumab, but only 95 of 837 patients screened positive for HER-2, and only 41 patients were eligible for the study. The combination of trastuzumab with paclitaxel and carboplatin for patients with progressive advanced ovarian cancer resulted in a complete response rate of 43%; however, only 7 patients were included in the trial, and only 22 of 321 patients screened showed positive HER-2 gene amplification.⁴⁰ No clinical response has been observed with single-agent trastuzumab in patients with advanced or recurrent endometrial cancer and HER-2 gene amplification.

Pertuzumab is a humanized MoAb that inhibits ErbB2 dimerization with the other ErbB receptors, independent of ErbB2 expression. In a recently completed phase 2 trial of pertuzumab in patients with refractory ovarian cancer, the overall response rate was 4.3%, with 6.8% of patients reporting stable disease at 6 months.⁴¹ A randomized, placebo-controlled, double-blind phase 2 trial investigating pertuzumab in combination with gemcitabine revealed similar rates of PFS between patients treated with or without pertuzumab (3.0 vs. 2.6 months). Interestingly, relative expression of ErbB2 to ErbB3 appeared to indicate clinical benefit by response and PFS criteria. Because ErbB2:ErbB3 heterodimerization is a preferred and potent mitogenic signaling initiator, ErbB3 mRNA appears to reflect efficacy of “on target” pertuzumab binding and may explain the clinical observation. Unfortunately, at this point, it is unclear whether there will be a development plan for the agent in gynecologic cancers.

VEGF-targeted agents appear to have greater activity against cervical cancer than EGF-, EGFR-, and HER-2–blocking agents. A phase 2 trial compared the 2 approaches head to head using pazopanib, a tyro-sine kinase inhibitor that blocks VEGFR and platelet-derived growth factor receptor (PDGFR), versus lapatinib, a tyrosine kinase inhibitor that targets EGFR and HER-2 activity. Pazopanib was superior to lapatinib, with improved PFS and overall survival and minimal toxicity. In a multicenter phase 2 trial of bevacizumab in combination with erlotinib in patients with recurrent ovarian cancer, a response rate of 15% was noted, consistent with the response rate observed with bevacizumab alone.⁴² A randomized phase 2 clinical trial of vandetanib (dual VEGFR/EGFR inhibitor) followed by docetaxel versus vandetanib plus docetaxel is being launched by the Southwest Oncology Group (Trial S0904, ClinicalTrials.gov identifier: NCT00872989).

Despite the apparent lack of activity of EGFR inhibitors in gynecologic cancer, there is rationale for further evaluation of these drugs. Given the high expression of EGFR in gynecologic malignancies and the increased sensitivity of tumors to other cytotoxic therapies when given in combination with EGFR inhibitors, further studies may prove highly beneficial. As illustrated by the discovery that KRAS mutations in colorectal tumors made the tumors resistant to EGFR inhibition, continued strides toward effective onco-logic treatment require a better molecular understanding of carcinogenesis.

Vascular Endothelial Growth Factor

VEGF, also known as vascular permeability factor (VPF), is one of the most well-characterized angiogenesis mediators. VEGF comprises a family of proteins, of which VEGFA (often implied by the term “VEGF”) is the dominant factor in tumor angiogenesis⁴³ (Figure 21-8). There are 3 tyrosine kinase receptors for VEGF, of which VEGFR-2 appears to have the most significant effects on angiogenesis. VEGF is ubiquitous in most human tissue and is upregulated in response to injury or stress. Interaction of VEGFR-2 with its ligand causes homo- or heterodimerization of the receptors, resulting in activation of a cascade of downstream signaling pathways. VEGF activation also results in increased production of nitric oxide and prostaglandin I₂, both vasodilators.⁴⁴ Increased production of VEGF and other growth factors is frequently observed in regions of hypoxia or inflammation and in the presence of activated oncogenes or downregulated tumor suppressor genes. Human papillomavirus (HPV), for example, is the root cause of virtually all cervical cancers. HPV’s E6 protein increases VEGF production by downregulating the tumor suppressor gene p53 and enhancing induction of hypoxia-inducible factor (HIF) 1-α.⁴⁵ Overexpression of VEGF results in increased endothelial cell proliferation, decreased apoptosis, and increased fenestration of endothelial cells. High VEGF expression has been shown to be associated with poor prognosis in most gynecologic malignancies including cervical, endometrial, ovarian, and vulvar cancers.

FIGURE 21-8. The vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. Ligands for the 3 VEGF receptors are depicted above their respective receptor. The neuropilins (NRPs) may act as coreceptors or activators of the pathway and are found on mesenchymal vascular supporting cells called pericytes. PGF, placental growth factor.

Bevacizumab

Bevacizumab is a humanized MoAb against VEGFA that is approved by the FDA for the treatment of metastatic colorectal, non–small-cell lung, renal cell, and breast cancers. Several phase 2 trials of this VEGFA antibody have been performed to assess its activity in gynecologic cancers. Bevacizumab has been most extensively studied in recurrent ovarian cancer patients, where response rates have ranged from 16% to 24% and median overall survival is 10.7 to 17 months when administered either as a single agent or in combination with metronomic cyclophosphamide.^46-48 It has also been shown to have activity in patient with recurrent or persistent endometrial cancer and in patients with progressive or recurrent cervical cancer (Figure 21-9).^49,50

FIGURE 21-9. A-D. Dramatic response to combination paclitaxel and carboplatin with bevacizumab in a patient with widely metastatic recurrent squamous cell carcinoma of the cervix. She had previously been treated with cisplatin-based chemoradiation.

Most studies of bevacizumab in gynecologic cancer have been conducted in patients with recurrent or progressive disease. Following encouraging data in phase 2 studies compared with historical controls, 2 randomized phase 3 studies in untreated advanced ovarian cancer patients have been conducted: GOG 218 (NCT00262847) and ICON-7 (NCT00483782). Each of these trials included an experimental arm with a maintenance treatment phase, which was placebo controlled in GOG 218 and open label in ICON-7. Both trials demonstrated superior clinical activity (hazard for progression) over control and, in the case of GOG 218, over combination paclitaxel, carboplatin, and bevacizumab followed by placebo maintenance. Of interest, the PFS of these “winning” arms is substantively less than that reported by earlier phase 2 data despite a similar proportion of suboptimal stage IIIC patients.

Toxicities associated with bevacizumab in phase 2 trials include hypertension, proteinuria, hemorrhage, neutropenia, venous thromboembolism, pulmonary embolus, congestive heart failure, myocardial infarction, and cerebrovascular ischemia (Table 21-4). Hypertension is the best characterized and most common side effect of the drug. It is thought to be caused by blocking nitric oxide production via inhibiting activation of VEGFR-2 and by endothelial dysfunction in normal tissue. The severity of hypertension is directly correlated with the dose of bevacizumab and the baseline blood pressure of the patient before initiating therapy. The degree of hypertension may also be a biomarker for response to therapy.⁵¹

Table 21-4 High-Grade (Grade 3-5) Toxicities of Biologic Therapies Reported as Related to Investigational Agent Listed According to Agent and Frequency

One of the most alarming potential adverse events associated with bevacizumab is gastrointestinal (GI) perforation and fistula (Figure 21-10). Two phase 2 trials of bevacizumab in treatment of ovarian cancer were stopped early due to a high rate of GI perforation (11% and 15%).^42,47 A retrospective review at Memorial Sloan-Kettering Cancer Center of patients with ovarian carcinoma receiving bevacizumab either in combination or as monotherapy revealed a GI perforation rate of 4% (6 of 160 patients). This is comparable to a compilation of published ovarian cancer trials of bevacizumab that estimates a GI perforation risk of 5.4% (16 of 298 patients). Many of the enrolled patients were heavily pretreated. Some studies have suggested that bowel involvement with ovarian carcinoma, bowel wall thickening, or bowel obstruction on computed tomography imaging; prior radiation therapy; and recent surgery may predispose patients to GI perforation, but strong evidence of association with these factors is still lacking. There are also reports of GI perforations associated with diverticulitis, ulcers, recent anastomosis, or bowel stricture or ischemia.⁵² The etiology of these events is not fully understood but may be related to vascular compromise following VEGF blockade. Although it has yet to be validated in whom bevacizumab administration is without safety concerns, it is prudent to consider these known toxicities relative to benefit and in the context of pre-existing medical infirmity prior to treatment.

FIGURE 21-10. Patient who developed a fistula while on bevacizumab for therapy of recurrent ovarian cancer. The orally administered contrast dye is seen in the anterior abdominal wound.

Other Therapeutics Against VEGF and VEGFR

Sorafenib and sunitinib are 2 tyrosine kinase inhibitors that block the activity of VEGFR, and both are approved by the FDA for targeted cancer therapy in renal cell carcinoma. Sorafenib inhibits several proteins including VEGFR-1, VEGFR-2, VEGFR-3, and PDGFR-α. Clinical investigation of sorafenib in gynecologic malignancies has revealed modest objective tumor responses and significant stable disease. Another multikinase inhibitor that blocks VEGFR and PDGFR, sunitinib, has been found to promote stable disease in women with recurrent ovarian cancer and recurrent or metastatic endometrial or cervix cancer.⁵³ Sorafenib and sunitinib have a similar side effect profile to bevacizumab, with the addition of hand-foot syndrome, which occurs as grade 3 or higher in approximately 13% of recipients (Table 21-4).

In light of the cross-talk pathways that contribute to cancer cell survival, interest has flourished in evaluating combinations of anti-angiogenic agents. An analysis of sorafenib with bevacizumab in patients with ovarian cancer yielded an impressive 43% response rate; however dose reductions of sorafenib were required in 74% of patients due to toxicities.⁵⁴ Eighty-four percent of the ovarian cancer patients in this study experienced grade 1 to 3 hypertension, and grade 1 to 2 hand-foot syndrome occurred in 95%. The toxicities experienced with the drugs in combination were greater than the additive effects of each drug alone. Similar trends of increased response with increased toxicity requiring dose reduction or discontinuation have been observed using bevacizumab with sunitinib or sorafenib in renal cell carcinoma.

Other small-molecule tyrosine kinase inhibitors that target VEGFR include AZD2171, pazopanib, and BIBF-1120. AZD2171 (cediranib) is an oral tyrosine kinase inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α, and c-kit that has been evaluated in phase 2 trials for patients with recurrent epithelial ovarian cancer, fallopian tube carcinoma, or peritoneal cancer. The partial response rate in this population was 10% to 17%, and stable disease was achieved in 13% to 34%.⁵⁵ ICON-6 (NCT00544973) is currently evaluating AZD2171 in a randomized, placebo-controlled, phase 3 trial in patients with recurrent ovarian cancer. Pazopanib is an inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α, PDGFR-β, and c-kit and has been tested in patients with advanced epithelial ovarian, fallopian tube, or primary peritoneal carcinoma. Response rate, as measured by CA-125 decline, was 47%, and 27% of patients had stable disease.⁵⁶ Pazopanib is currently being evaluated as a maintenance therapy in a double-blind, placebo-controlled, phase 3 clinical study in women who have achieved a partial or complete response to primary platinum-based adjuvant chemotherapy (NCT00866697). BIBF-1120, an inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α, PDGFR-,β, and FGF, has been investigated as a single agent in the maintenance setting. Eighty-four patients with best outcome to 1 or 2 previous lines of chemotherapy of either partial or complete response were randomized to either placebo or BIBF-1120. The primary end point was PFS. Overall, patients on placebo had a PFS of 2.8 months compared to 4.8 months in patients treated with BIBF-1120.⁵⁷ These data have prompted a larger phase III trial (NCT01015118) and exploration of chemotherapy combinations as primary therapy for women with ovarian cancer. These agents have similar side effects, the most frequent being hypertension, fatigue, and GI complaints (Table 21-4).

Aflibercept

VEGF Trap, or aflibercept, is a protein containing the VEGF binding regions of VEGFR-1 and VEGFR-2 fused to the Fc region of a human IgG1. This inhibitor resulted in a partial response rate of 11% in women with recurrent platinum-resistant epithelial ovarian carcinoma. Aflibercept was also studied as a single agent in women with refractory ascites. In this trial, the agent was significantly associated with reduced need for paracentesis.⁵⁸ In patients with uterine sarcoma, a phase 2 trial of aflibercept showed that 16% of patients with leiomyosarcoma experienced stable disease for over 6 months, but no response and no stable disease were observed in those with carcinosarcoma.⁵⁹Similar to bevacizumab, aflibercept is also associated with fatigue, hypertension, and GI complaints; a comparison of the 2 is shown in Table 21-5.

Table 21-5 Comparison of Bevacizumab to Aflibercept

PI3K/mTOR/Akt Pathway

The tumor suppressor gene PTEN (phosphate and tensin homolog detected on chromosome 10) is important for normal cellular function. Mutations in PTEN result in decreased apoptosis and are found in up to 83% of endometrioid carcinomas of the uterus. Decreased transcription due to mutation leads to decreased phosphatidylinositol 3-kinase (PI3K) inhibition, increased activity of Akt, and uncontrolled function of mammalian target of rapamycin (mTOR). Elevated activity of mTOR is seen in a vast majority of endometrial cancers as well as approximately 50% of cervical adenocarcinomas and 55% of ovarian carcinomas.⁶⁰ mTOR is a kinase that regulates cell growth and apoptosis.³⁷Temsirolimus, ridaforolimus, and everolimus are mTOR inhibitors that have been tested as single agents in phase 2 studies and found to promote stable disease in 44% of patients with metastatic or recurrent cancer of the endometrium.⁶¹ Side effects of these drugs consist mostly of myelosuppression, hyperlipidemia, hypercholesterolemia, and fatigue. Because aberrations in the PI3K/Akt/mTOR pathway are prolific in gynecologic cancers, drug discovery is keeping pace with several new agents entering the clinical domain (Figure 21-11). These drugs are being studied as single agents and in combination with chemotherapy and hormonal therapy.³⁷

FIGURE 21-11. The PI3K/Akt/mTOR pathway is frequently altered in human malignancy. The multiple effectors of this pathway provide important targets for therapeutic intervention.

Poly(ADP-Ribose) Pathway

There are a total of 17 members of the poly(ADP-ribose) polymerase (PARP) family, of which PARP-1 and PARP-2 orchestrate repair of single-stranded breaks in DNA.⁶² These enzymes bind to DNA at the site of damage and then initiate repair by ribosylation of nearby proteins, leading to base-excision repair at the site of damage and downstream effects on transcription and differentiation (Figure 21-12). Inhibition of PARPs via competitive blockade of the catalytic domain results in accumulation of DNA damage and cell death. BRCA1 and BRCA2 are tumor suppressor genes also important in DNA repair at sites of double-stranded breaks. Homologous recombination at DNA-damaged sites is a high-fidelity method of DNA repair mediated by Rad51 that is dependent on normal BRCA function. Mutations of BRCA genes force the cellular machinery to rely on lower fidelity methods of DNA repair and thus promote genomic instability. The initial studies of PARP inhibitors in BRCA- deficient tumors noted that, although mutations in BRCA increased tumor sensitivity to certain cytotoxic therapies, PARP inhibition causes cell death in this population approximately 3-fold over traditional treatment. By leaving single-stranded breaks unchecked by PARP inhibition, double-stranded DNA breaks are promoted in cells already lacking DNA repair capability, a process known as synthetic lethality. Normal cells with intact BRCA function will be able to repair their double-stranded DNA breaks, making tumor cells more susceptible to this treatment than normal tissue (Figure 21-12). Additionally, PARP inhibition, itself, has been found to suppress expression of BRCA1 and Rad51. Since the discovery of synthetic lethality in 2005, inhibitors of PARP have been studied in BRCA- positive breast cancer and have been found not only to enhance the cytotoxic effects of chemotherapy and radiation, but also to improve outcomes when used as single agents.⁶³

FIGURE 21-12. Poly(ADP-ribose) pathway (PARP). A. The enzyme is important for repair of DNA single-strand breaks. B. In the absence of high-fidelity homologous recombination, such as exists in women whose tumors carry BRCA mutations, tumors engage PARP for continued cell growth and proliferation. This makes them vulnerable to catastrophic damage under the influence of PARP inhibition unless BRCA function returns via reversion mutation.

PARP inhibitors are now being tested in patients with BRCA-positive ovarian cancer. AZD2281 (olaparib) is an oral small-molecule PARP-1 and PARP-2 inhibitor that was tested in 2 phase 1 trials. Among patients with BRCAmutations and ovarian carcinoma treated with olaparib, a response rate of 41% to 53% was noted.⁶⁴ A phase 2 study of AZD2281 in patients with BRCA-positive recurrent ovarian cancer yielded a response rate of 33% at a dose of 400 mg twice a day and 12.5% at a dose of 100 mg twice a day. Side effects of olaparib include GI complaints, fatigue, and myelosuppression. Continued trials of AZD2281 and other PARP inhibitors alone and in combination with chemotherapy are ongoing in patients with BRCA- positive and -negative ovarian and primary peritoneal cancer. Newly developed PARP inhibitors, such as ABT-888, MK4827, and BSI-201, are also currently being tested in gynecologic and nongynecologic tumors.

The activity of PARP inhibitors may not be limited to patients with germline BRCA mutations. Approximately 50% of undifferentiated and high-grade serous ovarian cancers have loss of BRCA1 function.⁶⁵ Many tumors have BRCA-like functional losses such as inactivation of BRCA genes or defects in other genes needed for BRCA-associated DNA repair that yield a clinical outcome similar to cancers with BRCA mutations. There is also increasing evidence that PARP inhibitors enhance the cytotoxic effects of chemotherapy and radiation without regard to BRCA function. These alternative mechanisms of propagating cytotoxic DNA damage may expand the utility of PARP inhibitors to a substantial number of malignancies. PARP inhibitors are currently being tested alone and in combination with chemotherapeutic agents, which may induce a vulnerable tumor homologous recombination phenotype, to evaluate the potential risks and benefits of these drugs among patients with impaired and normal BRCA function.

FUTURE DIRECTIONS

Over the last 5 to 10 years, there has been rapid development and evaluation of molecularly targeted therapies in oncology. The goal of these endeavors is to identify agents against aberrant pathways common among specific tumors that can improve current treatments. Initial phase 2 trials show some promising results, and large phase 3 trials are under way to confirm activity of these agents. There is concern that molecular targeting in treatment of cancer may provide evolutionary pressure to select for tumor cells that are highly resistant to therapy. Targeting multiple pathways of oncogenesis and using molecular inhibitors in combination with other cytotoxic treatments may overcome these selective processes to achieve higher cure rates for patients.

The evolution of investigation of immunotherapy for the treatment of patients with ovarian cancer is following a similar path. Finding targets specific for tumor cells has proven difficult. The range of immune strategies under investigation is wide. They include efforts to increase the effector cell response in specificity, number, and duration, as well as manipulating factors responsible for tolerance and terminating the immune attack. A favorable toxicity profile remains the hallmark of many approaches, and investigating these strategies in an attempt to prolong remission is ongoing. Evolving knowledge regarding the mechanisms of evasion of novel targeted treatments, including those targeting the immune response and other signaling pathways, should lead to better combinations that will hopefully surpass current standard therapy.

REFERENCES

1. Dubinett SM, Lee JM, Sharma S, Mule JJ. Chemokines: can effector cells be redirected to the site of the tumor? Cancer J. 2010;16(4):325-335.

2. Metcalf D. The colony-stimulating factors and cancer. Nat Rev Cancer. 2010;10(6):425-434.

3. Kopf M, Bachmann MF, Marsland BJ. Averting inflammation by targeting the cytokine environment. Nat Rev Drug Discov. 2010;9(9):703-718.

4. Boyd SD, Gaeta BA, Jackson KJ, et al. Individual variation in the germline Ig gene repertoire inferred from variable region gene rearrangements. J Immunol. 2010;184(12):6986-6992.

5. Kojouharova M, Reid K, Gadjeva M. New insights into the molecular mechanisms of classical complement activation. Mol Immunol. 2010;47(13):2154-2160.

6. Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009;27:591-619.

7. Sharma RK, Schabowsky RH, Srivastava AK, et al. 4-1BB ligand as an effective multifunctional immunomodulator and antigen delivery vehicle for the development of therapeutic cancer vaccines. Cancer Res. 2010;70(10):3945-3954.

8. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363(8):711-723.

9. Zhang L, Conejo-Garcia JR, Katsaros D, et al. Intraumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med. 2003;348(3):203-213.

10. Dietl J, Engel JB, Wischhusen J. The role of regulatory T cells in ovarian cancer. Int J Gynecol Cancer. 2007;17(4):764-770.

11. Shields JD, Kourtis IC, Tomei AA, Roberts JM, Swartz MA. Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21. Science. 2010;328(5979):749-752.

12. Yigit R, Massuger LF, Figdor CG, Torensma R. Ovarian cancer creates a suppressive microenvironment to escape immune elimination. Gynecol Oncol. 2010;117(2):366-372.

13. Chatterjee M, Wojciechowski J, Tainsky MA. Discovery of antibody biomarkers using protein microarrays of tumor antigens cloned in high throughput. Methods Mol Biol. 2009;520:21-38.

14. Piura B, Piura E. Autoantibodies to tumor-associated antigens in epithelial ovarian carcinoma. J Oncol. 2009;2009:581939.

15. Alberts DS, Hannigan EV, Liu PY, et al. Randomized trial of adjuvant intraperitoneal alpha-interferon in stage III ovarian cancer patients who have no evidence of disease after primary surgery and chemotherapy: an intergroup study. Gynecol Oncol. 2006;100(1):133-138.

16. Verheijen RH, Massuger LF, Benigno BB, et al. Phase III trial of intraperitoneal therapy with yttrium-90-labeled HMFG1 murine monoclonal antibody in patients with epithelial ovarian cancer after a surgically defined complete remission. J Clin Oncol. 2006;24(4):571-578.

17. Berek J, Taylor P, McGuire W, Smith LM, Schultes B, Nicodemus CF. Oregovomab maintenance monoimmunotherapy does not improve outcomes in advanced ovarian cancer. J Clin Oncol. 2009;27(3):418-425.

18. Sabbatini P. Consolidation therapy in ovarian cancer: a clinical update. Int J Gynecol Cancer. 2009;19(suppl 2):S35-S39.

19. Sabbatini P, Berek J, Casado A, et al. Abagovomab maintenance therapy in patients with epithelial ovarian cancer after complete response post first line chemotherapy: preliminary results of the randomized double blind, placebo controlled multi-center MIMOSA trial. J Clin Oncol. 2010;28(suppl 15). Abstract 5036.

20. Old LJ. Ludwig Institute for Cancer Research. 2007. Available at: http://www.licr.org/. Accessed March 3, 2012.

21. Odunsi K, Qian F, Matsuzaki J, et al. Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer. Proc Natl Acad Sci U S A. 2007;104(31):12837-12842.

22. Diefenbach CS, Gnjatic S, Sabbatini P, et al. Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission. Clin Cancer Res.2008;14(9):2740-2748.

23. Hernando JJ, Park TW, Fischer HP, et al. Vaccination with dendritic cells transfected with mRNA-encoded folate-receptor-alpha for relapsed metastatic ovarian cancer. Lancet Oncol. 2007;8(5): 451-454.

24. Czerniecki BJ, Koski GK, Koldovsky U, et al. Targeting HER-2/neu in early breast cancer development using dendritic cells with staged interleukin-12 burst secretion. Cancer Res. 2007;67(4): 1842-1852.

25. Gong J, Apostolopoulos V, Chen D, et al. J Immunol. 2000; 101(3):316-324.

26. Sabbatini PJ, Ragupathi G, Hood C, et al. Pilot study of a heptavalent vaccine-keyhole limpet hemocyanin conjugate plus QS21 in patients with epithelial ovarian, fallopian tube, or peritoneal cancer. Clin Cancer Res.2007;13(14):4170-4177.

27. Kershaw MH, Westwood JA, Parker LL, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res. 2006;12(20 Pt 1):6106-6115.

28. Chekmasova AA, Brentjens RJ. Adoptive T cell immunotherapy strategies for the treatment of patients with ovarian cancer. Discov Med. 2010;9(44):62-70.

29. Hatfield P, Merrick AE, West E, et al. Optimization of dendritic cell loading with tumor cell lysates for cancer immunotherapy. J Immunother. 2008;31(7):620-632.

30. Rocha-Lima CM, Soares HP, Raez LE, Singal R. EGFR targeting of solid tumors. Cancer Control. 2007;14(3):295-304.

31. Wiezorek J, Holland P, Graves J. Death receptor agonists as a targeted therapy for cancer. Clin Cancer Res. 2010;16(6):1701-1708.

32. Lessene G, Czabotar PE, Colman PM. BCL-2 family antagonists for cancer therapy. Nat Rev Drug Discov. 2008;7(12): 989-1000.

33. Alberts DS, Marth C, Alvarez RD, et al. Randomized phase 3 trial of interferon gamma-1b plus standard carboplatin/paclitaxel versus carboplatin/paclitaxel alone for first-line treatment of advanced ovarian and primary peritoneal carcinomas: results from a prospectively designed analysis of progression-free survival. Gynecol Oncol. 2008;109(2):174-181.

34. Giuntoli RL 2nd, Webb TJ, Zoso A, et al. Ovarian cancer-associated ascites demonstrates altered immune environment: implications for antitumor immunity. Anticancer Res. 2009;29(8): 2875-2884.

35. Keely NO, Meegan MJ. Targeting tumors using estrogen receptor ligand conjugates. Curr Cancer Drug Targets. 2009;9(3): 370-380.

36. Tessel MA, Krett NL, Rosen ST. Steroid receptor and microRNA regulation in cancer. Curr Opin Oncol. 2010;22(6): 592-597.

37. Bansal N, Yendluri V, Wenham RM. The molecular biology of endometrial cancers and the implications for pathogenesis, classification, and targeted therapies. Cancer Control. 2009;16(1):8-13.

38. Secord AA, Blessing JA, Armstrong DK, et al. Phase II trial of cetuximab and carboplatin in relapsed platinum-sensitive ovarian cancer and evaluation of epidermal growth factor receptor expression: a Gynecologic Oncology Group study. Gynecol Oncol. 2008;108(3):493-499.

39. Grushko TA, Filiaci VL, Mundt AJ, Ridderstrale K, Olopade OI, Fleming GF. An exploratory analysis of HER-2 amplification and overexpression in advanced endometrial carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol. 2008; 108(1):3-9.

40. Tuefferd M, Couturier J, Penault-Llorca F, et al. HER2 status in ovarian carcinomas: a multicenter GINECO study of 320 patients. PLoS One. 2007;2(11):e1138.

41. Gordon MS, Matei D, Aghajanian C, et al. Clinical activity of pertuzumab (rhuMAb 2C4), a HER dimerization inhibitor, in advanced ovarian cancer: potential predictive relationship with tumor HER2 activation status. J Clin Oncol. 2006;24(26): 4324-4332.

42. Nimeiri HS, Oza AM, Morgan RJ, et al. Efficacy and safety of bevacizumab plus erlotinib for patients with recurrent ovarian, primary peritoneal, and fallopian tube cancer: a trial of the Chicago, PMH, and California Phase II Consortia. Gynecol Oncol. 2008;110(1):49-55.

43. Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer. 2008;8(8):579-591.

44. Curwen JO, Musgrove HL, Kendrew J, Richmond GH, Ogilvie DJ, Wedge SR. Inhibition of vascular endothelial growth factor-a signaling induces hypertension: examining the effect of cediranib (recentin; AZD2171) treatment on blood pressure in rat and the use of concomitant antihypertensive therapy. Clin Cancer Res. 2008;14(10):3124-3131.

45. Monk BJ, Willmott LJ, Sumner DA. Anti-angiogenesis agents in metastatic or recurrent cervical cancer. Gynecol Oncol. 2010; 116(2):181-186.

46. Burger RA, Sill MW, Monk BJ, Greer BE, Sorosky JI. Phase II trial of bevacizumab in persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer: a Gynecologic Oncology Group study. J Clin Oncol.2007;25:5165-5171.

47. Cannistra SA, Matulonis UA, Penson RT, et al. Phase II study of bevacizumab in patients with platinum-resistant ovarian cancer or peritoneal serous cancer. J Clin Oncol. 2007;25(33):5180-5186.

48. Garcia AA, Hirte H, Fleming G, et al. Phase II clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer: a trial of the California, Chicago, and Princess Margaret Hospital phase II consortia. J Clin Oncol. 2008;26(1):76-82.

49. Aghajanian C, Sill MW, Darcy KM, et al. A phase II evaluation of bevacizumab in the treatment of recurrent or persistent endometrial cancer: a Gynecologic Oncology Group (GOG) study. J Clin Oncol. 2009;27(suppl 15S). Abstract 5531.

50. Monk BJ, Sill MW, Burger RA, Gray HJ, Buekers TE, Roman LD. Phase II trial of bevacizumab in the treatment of persistent or recurrent squamous cell carcinoma of the cervix: a Gynecologic Oncology Group study. J Clin Oncol. 2009;27(7):1069-1074.

51. Scartozzi M, Galizia E, Chiorrini S, et al. Arterial hypertension correlates with clinical outcome in colorectal cancer patients treated with first-line bevacizumab. Ann Oncol. 2009;20(2): 227-230.

52. Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nat Rev Clin Oncol. 2009;6(8):465-477.

53. Biagi J, Oza A, Grimshaw R, et al. A phase II study of sunitinib (SU11248) in patients (pts) with recurrent epithelial ovarian, fallopian tube or primary peritoneal carcinoma: NCIC CTG IND 185. J Clin Oncol. 2008;26(suppl 20S). Abstract 5522.

54. Azad NS, Posadas EM, Kwitkowski VE, et al. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity. J Clin Oncol. 2008;26(22): 3709-3714.

55. Matulonis UA, Berlin S, Ivy P, et al. Cediranib, an oral inhibitor of vascular endothelial growth factor receptor kinases, is an active drug in recurrent epithelial ovarian, fallopian tube, and peritoneal cancer. J Clin Oncol.2009;27(33):5601-5606.

56. Friedlander M, Hancock KC, Rischin D, et al. A phase II, open-label study evaluating pazopanib in patients with recurrent ovarian cancer. Gynecol Oncol. 2010;119(1):32-37.

57. Ledermann J, Rustin G, Hackshaw A, et al. A randomized phase II placebo-controlled trial using maintenance therapy to evaluate the vascular targeting agent BIBF 1120 following treatment of relapsed ovarian cancer (OC). J Clin Oncol. 2009;27(suppl 20S). Abstract 5501.

58. Moroney JW, Sood AK, Coleman RL. Aflibercept in epithelial ovarian carcinoma. Future Oncol. 2009;5(5):591-600.

59. Townsley CA, Hirte H, Hoskins P, et al. A phase II study of aflibercept (VEGF trap) in recurrent or metastatic gynecologic soft-tissue sarcomas: a study of the Princess Margaret Hospital Phase II Consortium. J Clin Oncol.2009;27(suppl 15S). Abstract 5591.

60. Faried LS, Faried A, Kanuma T, et al. Expression of an activated mammalian target of rapamycin in adenocarcinoma of the cervix: a potential biomarker and molecular target therapy. Mol Carcinog. 2008;47(6):446-457.

61. Slomovitz BM, Lu KH, Johnston T, et al. A phase 2 study of the oral mammalian target of rapamycin inhibitor, everolimus, in patients with recurrent endometrial carcinoma. Cancer. 2010; 116(23):5415-5419.

62. Miwa M, Masutani M. PolyADP-ribosylation and cancer. Cancer Sci. 2007;98(10):1528-1535.

63. Drew Y, Plummer R. PARP inhibitors in cancer therapy: two modes of attack on the cancer cell widening the clinical applications. Drug Resist Updat. 2009;12(6):153-156.

64. Fong PC, Boss DS, Yap TA, et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med. 2009;361(2):123-134.

65. Press JZ, De Luca A, Boyd N, et al. Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities. BMC Cancer. 2008;8:17.