HER2 expression beyond breast cancer: therapeutic implications for gynecologic malignancies.

Several studies have sought to evaluate potential biomarkers useful for predicting drug responsiveness and disease course. In breast cancer HER2 overexpression has been associated with both resistance to chemotherapeutic agents and also is predictive of a favorable response to other agents ( 73 , 74 ). In a recent study of metastatic breast cancer patients treated with trastuzumab and paclitaxel, Jung and colleagues found that high HER2 amplification index and high class III β-tubulin expression were predictive of a good response to therapy ( 74 ). The utility of molecular markers including HER2, Ki67, ER and PR were evaluated in 72 patients with advanced ovarian cancer. None of these markers were associated with progression free survival, however Ki67 nuclear expression (a marker of proliferation) was associated with a higher rate of complete response (p=0.05). HER2 overexpression was only 5% in this study and as such was not found to have a predictive or prognostic role in this study ( 75 ). Some larger studies in ovarian cancer patients have shown however the potential predictive role of HER2. The status and role of HER2 and class III β-tubulin was also evaluated in metastatic extramammary Paget's disease ( 76 ). In one study 63% of extramammary Paget's disease tumors overexpressed HER2 and positive class III β-tubulin reactivity was observed in 22% of these tumors. Class III β-tubulin expression is associated with taxane resistance. The authors suggested that HER2-targeted immunotherapy combined with taxane derivatives is a therapeutic option for metastatic extramammary Paget's disease based on high HER2 and low class III β-tubulin expression results. Both HER2 and class III β-tubulin may be appropriate biomarkers for guiding treatment of this disease ( 76 ).

Exome-wide analyses have recently greatly contributed to a better understanding of the biology of human neoplasms through the identification of mutations and copy number variations in genes crucial for the development of human tumors. More importantly, these studies have paved the way for a more rational drug design and the development/implementation of novel therapies specifically targeted against molecular aberrations present in a variety of human tumors. The transmembrane epidermal growth factor type II receptor (i.e., HER2), represents the prototype of a stable molecular abnormality endowed with well-characterized functional consequences that is detectable in several of the most common human solid tumors including but not limited to breast, ovarian, endometrial, colon, non-small cell lung cancer, prostate and cervical cancer ( 1 - 4 ). Importantly, HER2 overexpression has been shown to correlate with a worse survival in both node-positive and node-negative breast cancer patients and to be of prognostic and potential therapeutic value in other solid tumor types including multiple gynecologic malignancies ( 5 , 6 ). The location of HER2 on the cell surface has contributed to its appeal as an immunotherapy target. Trastuzumab (human monoclonal anti-HER2 antibody) has provided a distinct therapeutic advantage in not only breast cancer but in other tumor types, for example HER2 positive advanced gastric or oesophagogastric junction adenocarcinoma. As such Trastuzumab has received United States Food and Drug Administration (FDA) approval for the treatment of HER2 overexpressing breast and metastatic gastric cancer. The role of trastuzumab in gynecologic malignancies is still being explored with a Phase II trial underway in advanced stage uterine serous endometrial cancer.

The human epidermal growth factor type II receptor HER2 (c-erbB2) gene product is a transmembrane receptor protein that includes a cysteine-rich extracellular ligand-binding domain, a hydrophobic membrane spanning region, and an intracellular tyrosine kinase domain. With no direct ligand identified to date, HER2 functions as a preferred partner for heterodimerization with other members of the epidermal growth factor receptor family ( i.e ., HER1 or ErbB1, HER3 or ErbB3, and HER4 or ErbB4) and thus plays an important role in coordinating the complex ErbB signaling network that is responsible for regulating cell growth and differentiation ( 7 ). In normal cells, there are two copies of the gene per cell. In the presence however of HER2 amplification there may be as many as 100 c-erbB2 genes per tumor cell ( 8 - 10 ). This gene amplification results in overexpression of HER2 at both the mRNA and protein levels. The over-expression of HER2 at such a high level results in the tyrosine kinases becoming constitutively activated. This is thought to be the result of crowding of adjacent HER2 receptors in the cell membrane ( 8 - 11 ). The transactivation of the tyrosine kinase part of the receptor subsequently activates gene transcription factors and other cell cycle regulatory molecules including downstream protein pathways such as Ras/Raf/mitogen-activated protein kinase (Ras/Raf/MAPK) and phosphatidylinositol-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathways ( 12 ). Alterations of cell cycle regulatory molecules may be critical for the formation and maintenance of the transformed phenotype resulting from HER2 gene amplification and protein overexpression ( 13 ). On the other hand, blockade of HER2 function may result in the reversal or prevention of these effects on cell cycle regulators.

HER2 protein expression has a potential key role as both a prognostic marker and as therapeutic option in gynecologic tumors. The elucidation of genetic mutations which may modify tumor response to targeted agents and the correlation with HER2 expression is expected to provide important information for future cancer treatment. The advent of pharmaco-genomics and targeted therapy has provided the possibility for tailored tumor treatment and with molecular profiling of gynecologic tumor types comes the added potential for discovering novel and/or improved therapeutic strategies aimed at a particular gene product. Enhanced understanding of tumor heterogeneity will further facilitate a multifaceted approach to cancer treatment from the point of diagnosis with the hope of achieving a durable response.

Currently, HER2 expression status is routinely determined by immunohistochemistry (IHC), followed with additional fluorescent in situ hybridization (FISH) assays to verify equivocal IHC results. FISH identifies the number of HER2 gene copies in conjunction with the number of chromosome 17 centromere (CEP17) copies. It is a very sensitive and specific method which utilizes formalin-fixed, paraffin-embedded tissue (FFPE). FISH can also be applied to cell blocks or cytologic specimens. Generally, FISH scoring is considered more objective and quantitative than IHC scoring, however reproducibility is dependent on the thickness of tissue sections and the interpretation as well as recognition of the invasive component. FISH is not only more expensive than IHC but also is more time consuming requiring appropriate trained personal and the use of a fluorescence microscope ( 14 ). Chromogenic in situ hybridization (CISH) is another cost-effective and valid nucleic acid assay now being used which allows for the detection of the HER2 oncogene amplification using conventional enzymatic reactions and is applicable to FFPE tissues to which IHC was previously performed ( 13 ). Silver-enhanced in situ hybridization (SISH) like chromogenic in situ hybridization is a new bright-field technique that has been introduced for determining HER2 status. Both CISH and SISH combine features of IHC and fluorescent in situ hybridization. SISH is quicker to perform than FISH and has few requirements. SISH is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology and reduces error rates ( 14 ) ( 15 ). SISH is scored similarly to CISH with 5 or fewer copies of the HER2 gene per nucleus in more than 50% of tumor cells defining no amplification and high amplification defined as more than 10 copies or large clusters or a mixture of multiple dots and large clusters of HER2 present in more than 50% of cancer cells ( 14 ). SISH has been shown to have high concordance rate with FISH in previous studies ( 15 ) ( 16 ). Multiple studies have found the lack of a perfect correlation between gene amplification quantification and HER2 protein expression ( 11 ). Importantly, breast cancer studies have noted that HER2 protein product overexpression can also be found in the absence of gene amplification ( 17 ). Similar discrepancies between FISH and IHC results for HER2 have been noted in endometrial cancer. This underscores the need to consistently validate protein expression in c-erbB2 amplified tumors as protein expression may be discordant due to post-transcriptional modifications in mammalian cells ( 18 ). In the past some of the variation in HER2 overexpression and gene amplification has at least in part resulted from differences in the testing methods, interpretation, and scoring criteria used. HER2 intensity determination by IHC previously was by FDA scoring criteria. The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) in 2007 published recommendations for optimal HER2 testing performance. This new testing algorithm relies on accurate and reproducible assay performance. Also included in the recommendations were methods to reduce assay variation such as specimen handling. The panel also suggested an algorithm for defining positive, negative and equivocal test results for both HER2 protein expression and gene amplification ( 19 ). The (ASCO/CAP) IHC score of protein expression is on a scale of 0 to 3+ and has generally been adopted widely. A score of 0-1+ represents a negative result and is assigned if there is no staining or weak, incomplete membrane staining in any proportion of tumor cells. An equivocal IHC results consists of specimens scored as 2+ and is described as complete membrane staining that is either non-uniform or weak in intensity but with obvious circumferential distribution in at least 10% cells or very rarely tumors that show complete membranes staining of 30% or fewer tumor cells. IHC positive result is 3+ cell surface expression defined as uniform intense membrane staining of >30% of invasive tumor cells ( 19 ). HER2 protein overexpression has been found in several tumor types including breast, esophageal, lung, cervical, endometrial and ovarian cancer. Characterization of this increased expression has been exploited mostly in patients with breast cancer where it has a prognostic, predictive and therapeutic target value. Continued interest in the expression of this plasmalemmal glycoprotein in other malignancies has resulted in several papers describing the possible mechanism of HER tumorgenesis. The investigation of HER2 expression in gynecologic malignancies has produced strong evidence that overexpression is associated with increased tumor aggression. The mechanism of tumor aggressiveness includes resistance to immunologic mediators such as tumor necrosis factor (TNF-α), activated macrophages and lymphocyte-activated killer cells ( 20 ). HER2 overexpressed tumors have been found to have increased amounts of factors governing tumor invasiveness and angiogenesis. In breast, cervical, endometrial and ovarian cancer, there have been several studies linking the amplification of the c-erbB2 gene with chemo-resistance and overall poor survival ( 21 - 24 ). Also in breast cancer, discordant HER2 expression has been found between primary and metastatic sites. Interestingly, studies in gastric and epithelial ovarian cancer have shown that there is instead a corresponding level of HER2 expression in primary and metastatic sites ( 25 - 27 ).

Although multiple mechanisms of action have been attributed to anti-HER2 monoclonal antibodies including inhibition of tumor proliferation and/or promotion of cell cycle arrest (secondary to decrease HER2 receptor dimerization) and induction of apoptosis, strong experimental evidence suggests that engagement of Fc receptors on effector cells (i.e., mainly natural killer (NK) cells) represents the dominant component of the in vivo activity of these antibodies against tumors. Humanized monoclonal antibodies (mAb) against HER2 are known to induce tumor lysis through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and consistent with this view, the efficacy of these mAb in mice defective for Fc has been previously demonstrated to be only about 30% of the activity shown by the intact antibody when it is able to engage the Fc receptors on NK cells ( 77 ). This theory is further supported by experimental studies comparing the in vitro and in vivo activities of IgG and F(ab’) 2 fragments in a mixture of three monoclonal anti-HER2 antibodies. In these reports, the in vitro anti-proliferative and pro-apoptotic effects of IgG and F(ab’) 2 were similar but only the IgGs had significant antitumor activity in vivo. The importance of the Fc portion of trastuzumab for effective in vivo activity is supported by studies demonstrating that even a mixture of three anti-HER2 monoclonal antibodies which were highly effective at inducing cell death in vitro still required Fc-mediated effector function for optimal functioning in vivo ( 78 ). Clinical results have also shown that there is a improved response to trastuzumab in patients with a particular Fc polymorphism resulting in a higher NK affinity to IgG1, again lending support that ADCC inclusive of its mediators is critical for the in vivo efficacy of trastuzumab ( 79 , 80 ). Moreover other clinical data has demonstrated that patients responding to neoadjuvant trastuzumab showed a four-fold increase in antibody-dependent lytic activity from isolated peripheral blood mononuclear cells in contrast to patients without a response ( 81 ) . As mentioned above, trastuzumab (Herceptin®, Genentech, CA, USA) is a humanized monoclonal IgG1 antibody that works both through recruitment of NK cells and initiation of ADCC as well as abrogation of downstream effectors ( 77 , 81 , 82 ). It is FDA-approved as an adjunct to cyclophosphamide, paclitaxel and doxorubicin in the treatment of early-stage HER2 positive, node-positive breast cancer and as a single agent for adjuvant treatment of early-stage, HER2 positive, high-risk ER/PR-negative breast cancers following multi-modality anthracycline-based therapy ( 83 ). Based on sound biologic plausibility, there is also considerable interest in applications for HER2 overexpressing gynecologic malignancies. As assessed by standard 51 chromium release assays, trastuzumab results in ADCC in the range of 25–60% against USC that overexpress HER2 ( 84 ). The therapeutic potential of anti-HER2 immunotherapy in vitro against HER2 expressing carcinosarcomas of the female genital tract has recently been investigated ( 67 ). Our research group described HER2 gene and protein expression in primary carcinosarcoma cell lines and also for the first time demonstrated in vitro trastuzumab-mediated ADCC against uterine and ovarian carcinosarcoma cell lines. Although only a minority of carcinosarcoma demonstrated HER2 overexpression, few would argue against the addition of trastuzumab to the armamentarium of agents for treating HER2 positive carcinosarcoma patients in view of the high rate of recurrence and the generally poor responses to chemotherapy ( 67 ). Several case reports exist in the literature of trastuzumab clinical activity in heavily pretreated advanced stage endometrial carcinoma patients with HER2 overexpression by IHC. The histological subtypes reported on were uterine serous and high-grade endometrioid tumors. The responses varied from complete response to stable disease with the longest period of stable disease being 11 months ( 85 - 87 ). Despite encouraging case reports ( 85 - 87 ), when evaluated as a single-agent, trastuzumab 4 mg/kg in week 1 then 2 mg/kg weekly until disease progression in stage III/IV or in recurrent endometrial cancers at the Phase II level in GOG-181B failed to demonstrate significant activity ( 52 ). Since combination therapy with trastuzumab and chemotherapy is generally more effective than single agent trastuzumab in HER2 positive breast and gastric cancer, a multi-institutional Phase II trial is underway to investigate whether the addition of trastuzumab to paclitaxel and carboplatin chemotherapy improves progression free survival (PFS) when compared to paclitaxel and carboplatin alone in stages III/IV and recurrent USC patients overexpressing HER2 at 3+ level by IHC or positive by FISH ( 88 ). Pertuzumab (Omnitarg®, Genentech, South San Francisco, CA, USA) is a humanized IgG1 mAb HER heterodimerization inhibitor that binds domain II of the erbB2 receptor. Compared to trastuzumab, pertuzumab inhibits a broader array of downstream signal transduction pathways through abrogation of lateral signal transduction ( 89 - 93 ). El-Sawhi and colleagues ( 84 ) investigated the sensitivity of USC cell lines to heterologous peripheral blood lymphocytes (PBLs) in the presence of pertuzumab (2.5 μg/mL), trastuzumab (2.5 μg/ml), and the combination of both in cell lines expressing high levels of HER2. Significant cytotoxicity was achieved with pertuzumab (mean ± SD: 61 ± 25.6%; range, 32.5-67.3%; p=0.0001) and trastuzumab (mean ± SD: 56.3 ± 14.2%; range, 32.5-77.1%; p=0.0001) compared with control, (i.e., PBLs alone [mean ± SD: 3.2±5.6%; range, 0-19%] or PBLs and rituximab (anti-CD20) [mean ± SD: 2.5 ± 3.8%; range, 0-13%]). Combination of the two antibodies significantly increased ADCC in low HER2 expressing USC cell lines ( 84 ). Trastuzumab emtansine (T-DM1, Genentech/Roche) is a novel antibody-drug conjugate that combines trastuzumab with targeted delivery of the antimicrotubule agent DM1. DM1 belongs to the maytansine class of chemotherapeutic agents. On average 3-4 molecules of DM1 bind to each trastuzumab molecule. TDM-1 has demonstrated robust clinical activity in heavily-pretreated patients with trastuzumab-refractory HER2 positive breast cancer with a 43.6% objective response rate and median PFS of 9.6 months ( 94 ). The common side effects reported with this new drug are fatigue, thrombocytopenia and epistaxis. Similarly promising anti-tumor activity has been noted in HER2 positive gastric tumors with resistance to trastuzumab ( 95 ). However no clinical trials have yet been conducted in gynecologic cancer. Clinical trials exploring T-DM1 therapy in HER2 positive advanced/recurrent and/or refractory USC are warranted. Faulty immunosurveillance represents an important contributor in cancer development and progression. Even though immune responses such as those mediated by natural killer cells are thought to play an important role to keep tumor growth in check, eventually these mechanisms become overwhelmed and immunologic escape occurs, at which time disease becomes apparent ( 96 - 98 ). The cytokine interleukin (IL)-2 controls the growth and differentiation of a number of lymphocyte subsets, notably CD8+ cytotoxic T cells (T c ), NK cells, and CD4+ helper T regulatory cells (T regs ). High-dose recombinant IL-2 (aldesleukin, Proleukin®, Novartis, Switzerland) was approved by the Federal Drug Administration (FDA) as early as 1998 for treatment in metastatic melanoma and renal cell carcinoma ( 99 ). IL-2 therapy is plagued by significant pulmonary and hepatic toxicities and may lead to parallel expansion of T regs able to blunt antitumor immune responses, thereby limiting the beneficial effects of expanding tumor-reactive CD8+ T c ( 100 , 101 ). Augmenting targeted immunotherapy in particular natural killer cell function with IL-2 or with agonist monoclonal antibody for costimulatory receptors on NK cells is a promising strategy ( 102 ). El-Sawhi and colleagues demonstrated enhanced cytotoxicity when pertuzumab was combined with low doses of IL-2 (mean ± SD: 46.9 ± 11%; range, 22.4-74.2%) versus pertuzumab alone (mean ± SD: 41.5±12.8%; range, 15.8-71.9%) (p=0.04) in preclinical experiments. Similarly, pertuzumab in combination with IL-2 induced significantly higher cytotoxicity when compared with PBLs treated with IL-2 (p= 0.03) or PBLs treated with IL-2 in the presence of rituximab (p=0.03). In contrast, no significant increase in cytotoxicity was detected after 5 hours of IL-2 treatment in the absence of pertuzumab or in the presence of rituximab ( 84 ). One mechanism by which tumors escape immune surveillance is through antigen evolution and upregulation of membrane complement regulatory proteins (mCRPs) that may hinder complement-dependent pathways ( 103 ). The mCRPs CD46 (membrane cofactor protein), CD55 (decay-accelerating factor) and CD59 (protectin) have been shown to be upregulated in colorectal, cervical, prostate and renal cell carcinomas ( 104 - 106 ). mCRPs upregulation lead to inactivation of C4b/C3b, dissociation of C3/C5-convertases and prevention of membrane-attack complex assembly ( 107 ). Recently, our research group has shown that USC overexpress CD46, 55 and 59 relative to normal endometrial cells; knockdown via siRNAs of CD55 and CD59 but not CD46 significantly sensitized USC to CDC (from 6.8 to 11%) and ADCC (from 48 to >60%) ( 108 ). Inhibition of mCRPs on type II endometrial cancers harboring c-erbB2 gene amplification may prove to be a useful strategy to improve the response of these aggressive tumors to trastuzumab-mediated CDC and ADCC ( 108 ). Blocking mTOR complex-1 induces upregulation of HER2 expression mediated by mTOR complex-2 in preclinical models ( 109 ). Clinical benefits were seen in patients with trastuzumab-resistant HER2 positive breast cancer treated with everolimus (mTOR inhibitor) in phase I/II trials ( 110 , 111 ). Thus, evaluation of mTOR inhibitors in combination with HER2 targeted therapy should be investigated in gynecologic cancer. Dual tyrosine kinase inhibitors (TKI) such as lapatinib have shown efficacy in HER2 positive trastuzumab-resistant breast cancers, esophageal and gastric adenocarcinomas. Lapatinib's additive or synergistic effects with chemotherapy have also been demonstrated in these tumor types ( 109 , 112 ). Lapatinib is a reversible dual inhibitor of both HER2 and EGFR and in preclinical models has shown effectiveness in restoring trastuzumab sensitivity ( 113 ). Accumulation of the truncated form of HER2, p95-HER2, which lacks the trastuzumab binding site but is able to maintain tyrosine kinase activity is one mechanism of trastuzumab resistance. Lapatinib is able to inhibit p95-HER2 phosphorylation and as a result reduce growth of HER2 driven malignancies ( 114 ). Pan-HER TKIs which inhibit epidermal growth factor family receptors and their downstream pathways have also proved beneficial in solid tumor clinical trials ( 115 ). Also new in therapeutic approaches for solid tumors is the targeting of heat shock protein 90 (hsp90) molecular chaperone. Hsp90 is the overseer for several oncogenic proteins including HER2, AKT and others involved in signal transduction and cell cycle regulation ( 116 , 117 ). Hsp90 inhibitors have shown encouraging clinical activity in HER2 positive metastatic breast cancer. Considering that most solid tumors, including gynecologic tumors, are complex and have several genetic abnormalities, a single targeted agent is unlikely to be effective over a long period of time and combination therapy is crucial. Hsp90 inhibitors are thus currently under evaluation in early phase clinical trials as single agent therapy or in combination with trastuzumab ( 118 , 119 ). ( Table II ) Radioimmunotherapy has been explored in the management of colon, metastatic renal and prostate cancer with encouraging results ( 120 - 122 ). Preclinical studies have recently shown the safe applicability of radioimmunotherapy in gynecologic cancer and other tumor types ( 123 , 124 ). A phase I trial evaluating lead-212 ( 212 Pb)-trastuzumab in patients with HER2 positive ovarian, pancreatic, colon, gastric, endometrial or breast cancer patients documented to have peritoneal studding or positive washings (intraperitoneal disease) is ongoing. This represents a potential treatment approach for patients with metastatic disease utilizing this lead isotope with a short path length specifically targeted to malignant cells by the trastuzumab antibody ( 125 ). While trastuzumab is an effective immunotherapeutic agent against a variety of tumors overexpressing HER2, it potentially has limitations of eventual drug resistance and risk of cardiotoxicity, especially in patients on previous anthracycline based regimens. Therefore interest naturally surfaced overtime in anti-HER2 vaccines as experience with trastuzumab grew. The use of a vaccine that induces or stimulates a preexisting anti-HER2 immune response has several advantages including fewer injections for patients but the most important being the possibility of establishing a memory immune response capable of preventing disease recurrence. Consistent with this view, several clinical trials are underway in patients with stage II, III or stage IV breast, ovary, colon or non-small cell lung cancers with HER2 expression. A phase I/II randomized trial of HER2 peptide-loaded DC vaccination with or without cyclophosphamide for consolidation therapy of advanced ovarian cancer was reported in 2011. This study revealed that HER2 peptide-loaded DC vaccination elicited modest immune response. Of 11 patients receiving the vaccine, 2 recurred during vaccination, 3 patients recurred at 6, 17 and 26 months and 6 patients had no evidence of disease at 36 months. This study also showed that a single intravenous dose of cyclophosphamide (300mg/m 2 ) has no effect on the number of circulating T regs despite previous evidence reported of cyclophosphamide enhancing tumor immunotherapy by eliminating T regs ( 126 - 128 ). Immunological tolerance against HER2 is a significant obstacle to effective vaccination against this oncoprotein ( 129 ). Generally toxicities reported with the HER2 vaccine have been commonly local reactions of erythema, induration, pruritus and inflammation. The most frequent systemic toxicities were fatigue, headache, arthralgias, myalgias, chills, bone and back pain. No additive cardiotoxicity or autoimmunity has been confirmed as a result of this vaccine therapy.