Current Targeted Therapies in HER2-Positive Gastric Adenocarcinoma

Abstract

Gastric cancer is one of the most common types of cancer in the world, usually diagnosed at an advanced stage. Despite the advances in specific anticancer agents' development, the survival rates remain modest, even in early stages. In 15%–20% of cases, the human epidermal growth factor receptor 2 (HER2) overexpression was identified. We conducted a general review to summarize the progress that has been made in the targeted treatment of HER2-positive esogastric junction or gastric adenocarcinoma. According to our findings, trastuzumab is the only validated anti-HER2 agent in locally advanced or metastatic disease and its adjuvant effectiveness is assessed in a RTOG phase III study. In a previously treated advanced disease, the maytansine derivate TDM 1 failed to be approved as a second-line regimen, and the tyrosine kinase inhibitor, lapatinib, shows modest results. The antiangiogenics have not been analyzed in specific populations and targeting the mesenchymal–epithelial transition factor (MET) receptor, overexpressed in up to 46% of the advanced disease, seems encouraging. Regarding the checkpoint inhibitors, based on KEYNOTE 059 multilevel ongoing trial, stratified according to the HER2 and programmed death-ligand (PD-L) 1 status, pembrolizumab was approved for third-line treatment of gastric or gastroesophageal junction adenocarcinoma.

Introduction

Gastric cancer (GC) is the fourth most frequent type of cancer in the world and, despite the decrease in specific mortality, it remains the second most common cause of death. The survival rates are modest, with a 5-year overall survival (OS) from 57% to 79% in stage I, and about 4% in stage IV of the disease. The main cause appears to be the advanced GC at diagnosis, and in the low-income countries the prevalence of stages III–IV reaches up to 92%.^1

–4

In metastatic disease, the response rate of the various first-line chemotherapy regimens ranges from 20% to 40%, which allows a median survival of around 9 months compared with 5 months for patients treated with best supportive care.^5

–8

Gastric adenocarcinoma represents ∼95% of the malignant tumors of the stomach and shows an overexpression of human epidermal growth factor receptor 2 (HER2) in about 20% of the cases.^9

–12

HER2 was described for the first time, 25 years ago in relation with breast tumors. It is a tyrosine-kinase (TK) cellular growth receptor, with no known ligand so far. It becomes active by homo- and heterodimerization with receptors belonging to the same family of human epidermal growth factors, of which HER2-HER3 plays an important role in the carcinogenesis process, mostly through the phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway activation.^13

–17

In gastric adenocarcinoma, its expression was more frequently reported in the intestinal (53%) than in the diffuse (8%) or mixed histological subtype.^18
–20

The HER2 overexpression was described as an independent negative prognostic factor in breast cancer; in gastric localization, it was found to correlate with other pathological features of poor prognosis like serosal infiltration, lymphatic invasion, and high grade or large tumor size (>5 cm).^21

–24

Trastuzumab, the first anti-HER2 monoclonal antibody, developed in 1990, interferes with receptor functionality by several intricate mechanisms: dimerization's block, receptor's internalization, and degradation, promoting the activity of tyrosine kinase–ubiquitin ligase c-Cbl, cyclin-dependent kinase inhibitor p27 accumulation, cell cycle arrest in G1 phase, PI3K/Akt pathway inhibition, and antibody-dependent cellular cytotoxicity.^25
–27 Trastuzumab became successfully integrated into the standard chemotherapy regimen either in a perioperative or metastatic setting in HER2+ breast cancer. Added to docetaxel in the first line advanced disease, led to a 6.4% improvement in a 4-year disease-free survival (DFS) (78.6% vs. 72.2%, p < 0.0001), with a HR = 0.85 (95% CI: 0.70–1.04, p = 0.11).^28,29

Trastuzumab has demonstrated its efficacy in GC too. The ToGA (Trastuzumab for GC) trial, added to the standard chemotherapy treatment, permitted a significant OS improvement, in the metastatic HER2+ patients.³⁰

Faced with a much lower life expectancy and given the progress obtained in the HER2+ breast cancer counterpart, we proceed to a general review regarding the advancements in the HER2+ GC immunophenotype treatment. We included the gastroesophageal junction (GEJ) adenocarcinoma as well, due to the similarities in the tumor growth and patterns of spread.^31

–34

Anti-HER2 Standard Treatment

HER2 overexpression is considered in GC, because of the incomplete predominantly basolateral receptor membrane distribution and higher intratumoral heterogeneity compared with breast cancer, when membranous staining in immunohistochemistry (IHC) is either high, moderate, or low, but positive in the in situ hybridization technics (fluorescent, chromogenic or silver-FISH/CISH/SISH), the test HER2 gene amplification, is at least 10% of the cells in the surgical specimen. As regards biopsy, the stain must concern, at least five cohesive cells. Trastuzumab is, up until now, the only target drug ever approved for HER2+ GC.³⁵

ToGA, a phase III, open-label, international, randomized controlled trial, in 584 included patients that overexpressed the HER2 protein, validated trastuzumab in the first line with fluoropyrimidines and cisplatin, given every 3 weeks (q3w) for six cycles. 5-Fluorouracil (5-FU) 800 mg/m² continuous intravenous infusion on days 1–5 or capecitabine 1000 mg/m² orally, twice daily for 14 days followed by a 1-week rest, were administered, in association with cisplatin 80 mg/m² on day 1 and trastuzumab at a dose of 8 mg/kg (loading dose) on day 1 of the first cycle, followed by 6 mg/kg q3w, intravenous infusions. The median OS improvement was about 13.8 months versus 11.1 months (HR = 0.74, CI: 0.60–0.91, p = 0.0046), 6.7 months versus 5.5 months (HR = 0.71, CI: 0.59–0.85, p = 0.0002) the median progression-free survival (PFS) and the objective response rate (ORR) was significantly higher than in the standard treatment arm (47% vs. 35%, p = 0.0017).

The adverse event's occurrence was similar in both arms, excepting grade 3–4 diarrhea (9% vs. 4%).³⁰

A high expression of the HER2 protein was defined as IHC 2+ and FISH + or IHC 3+, and a low one as IHC 0–1 and FISH +. One cost-effective analysis of the Asian ToGA population found that trastuzumab administration in the IHC 2+ subgroup is debatable: HR for death was 0.78 (95% CI: 0.55–1.10) versus 0.66 (95% CI: 0.50–0.87) in the IHC 3+ patients.³⁶

Based on the hypothesis, following preclinical findings in the HER2+ metastatic breast cancer that the high burden metastatic tissue might have a higher clearance of trastuzumab, higher doses have been assessed, wishing for improvement upon ToGA outcomes. In the phase IIIb HELOISE study, 10 mg/kg every 3 weeks Trastuzumab administration along with cisplatin/capecitabine chemotherapy was not found either more toxic or more efficacious in the patients bearing gastric or GEJ adenocarcinoma.^37

–40

There are several other regimens that have been assessed for efficacy along with the targeted agents in the metastatic setting.

Faced with the requirements of hydration, sometimes difficult to apply in frail patients, and to the gastrointestinal and renal toxicity of cisplatin, based on the REAL2 trial showing oxaliplatin–cisplatin equivalence in advanced GC, a multicenter, phase II study evaluated trastuzumab added to the oxaliplatin–capecitabine first-line regimen in HER2+ advanced disease. The partial response has been recorded in 33 of 46 enrolled patients and for an average follow-up of 28.6 months, the median PFS was 9.2 months (95% CI: 6.5–11.6) and the median OS 19.5 months (95% CI: 15.5–26.0).^41,42

In 47 patients previously exposed to chemotherapy, weekly trastuzumab–paclitaxel association led to a PFS of 5.09 months (95% CI: 3.79–6.49), an OS of 16.8 months (95% CI: 13.5–18.6), and a 37.0% ORR (95% CI: 23–52).⁴³

Trastuzumab in the Perioperative Setting

Knowing the advantage brought by trastuzumab in the breast cancer neoadjuvant treatment, its association to capecitabine–oxaliplatin regimen in the resectable HER2+ GC was evaluated in the NEOHX phase II study, the patients being assigned to receive three courses of chemotherapy before and after surgery, followed by trastuzumab administration until 12 months completion. For the 24.1 months, the median follow-up, in 36 patients' DFS rates at 18 and 24 months were 71% and 60%, respectively.⁴⁴ The undefined benefits of trastuzumab in this setting is also being evaluated in the INNOVATION phase II study that randomizes patients with stage Ib-III GC to receive either neoadjuvant chemotherapy alone (cisplatin and 5-FU/capecitabine) or in association with trastuzumab and pertuzumab.⁴⁵

There are ongoing studies assessing the safety of trastuzumab concomitance with irradiation or along with other fluoropyrimidine agents such as S-1 an oral tegafur-containing product already used in advanced GC, in the localized disease (Table 1).^46

–49

Table 1.

Trastuzumab Testing Studies

Study/first author/year	Sponsor/promoter	Setting	Phase	No patients	Status	ORR%	DFS/RFS	OS (months)
ToGA/Bang/2010 ³⁰	Hoffmann-La Roche	LA/M HER2+ (IHC3+ or FISH+) GEJ/GC	III	584	Completed	47	PFS = 6.7	13.8
NEOHX/Rivera/2015⁴⁴	Hoffmann-La Roche	LA resectable HER2+ GEJ/GC	II	36	Completed	NA	NA	NA
INNOVATION⁴⁵	EORTC	LA resectable HER2+ GEJ/GC	II	NA	Recruiting	NA	NA	NA
TOXAG Study⁴⁶	Hoffmann-La Roche	Stages IB-IIIB resected HER2+ (IHC3+ or FISH+) GEJ/GC	II	35	Ongoing, not recruiting	NA	NA	NA
RTOG 1010⁴⁷	NCI	Stages IB-IIIB HER2+ GEJ or EC	III	591	Ongoing, not recruiting	NA	NA	NA
JCOG1301/Kataoka/2015⁴⁹	National Cancer Center Research and Development Fund	LA HER2+ (IHC3+ or FISH+) GEJ/GC	II	130	Recruiting	NA	NA	NA

DFS, disease-free survival; EC, esophageal cancer; EORTC, European Organization for Research and Treatment of Cancer; FISH, fluorescence in situ hybridization; GC, gastric cancer; GEJ, gastroesophageal junction; HER2, human epidermal growth factor 2; IHC, immunohistochemistry; LA, locally advanced; M, metastatic; NA, not available; No, number (pts number); NCI, National Cancer Institute; ORR, objective response rate; OS, overall survival; PFS, progression-free survival.

The Pathways and Mechanisms of Resistance to Trastuzumab

One-third of HER2+ GC patients receiving upfront trastuzumab will experience early disease progression.^50,51

Several criteria have been proposed to define the different types of trastuzumab resistance to guide the second-line targeted molecules' choice in the HER2+ breast cancer phenotype. Thus, the radiological progression within 3 months or recurrence in 12 months since the last adjuvant administration defines the primary resistance while the refractoriness is considered in the case of progression after two or more lines of trastuzumab-containing regimens.^52
–54

Resistance may be either intrinsic (pre-existing resistance), or drug-induced (acquired resistance).

The intrinsic resistance seems to be related mostly to the structural alterations of the receptor or of the intracellular downstream proteins, altering the signaling pathway.⁵⁵ In the acquired resistance, several mechanisms are described, such as mucin synthesis at the receptor level, interfering with antibody fixation, truncated form of HER2 receptor, heterodimerization with the members of the same family for example, HER1, HER3, crosstalk with other tyrosine kinase membrane receptors for example, IGFR, cMET, and aberrant activation of the internal signalization pathway (MAPK and PI3K/AKT).^56

–60

There are new researches regarding anti-HER2 mechanisms of resistance in GC.

A recent preclinical study revealed that metastasis associated in the colon cancer 1 (MACC1) gene, a regulator of MET/AKT signaling pathway, might contribute to trastuzumab resistance, facilitating tumor progression by promoting the Warburg effect, a phenomenon describing that for their energy requirements many cancer cells use glycolysis preferentially and not mitochondrial oxidative phosphorylation.^61,62 Using the established trastuzumab-resistant GC cell lines, this in vitro study evaluated the impact of trastuzumab combined with oxamate on the tumor growth and metabolism in a xenograft model of HER2+ GC cells. The results showed that MACC1, already known as a poor prognosis factor in GC, was upregulated in the trastuzumab-resistant cell lines, its downregulation reversed this resistance, whereas its overexpression augmented the Warburg effect and triggered the PI3K/AKT pathway, leading to the MACC1–PI3K/AKT–Warburg effect the pathway description in trastuzumab resistance of the HER2+ GC cells.⁶³

The acquired resistance to trastuzumab in the GC experimental models was also explored by analyzing the trastuzumab-resistant subclones, derived from the HER2-overexpressing GC cells, each cell line with a distinct resistance mechanism and with a different diffusion pattern. The subclones (NCI-N87, AKG, and KKP cells), obtained after 12 months exposure to escalating doses of trastuzumab, presented a lower growth rate than the parental cell lines. The HER2 and HER3 were highly expressed in the NCI-N87 cells, but HER3 was also found in KKP and AKG, positivity evaluation made by IHC. HER4 was mostly expressed in the AKG cells.

Starting from breast cancer, where White et al. showed that the overexpression of IQGAP1, a scaffold protein found in all human tissues, is associated to trastuzumab-induced resistance by decreasing the HER2 expression and HER2-stimulated activation of the PI3K/AKT pathway, correlated with its presence in aggressive forms of GC, and the same pattern was explored for HER2+ GC.^64
–66 IQGAP1 managed to obtain trastuzumab sensitivity restoration in the NCI-N87 subclones once its expression was silenced. Moreover, the AKG and KKP cells showing IQGAP1 expression were identified as being involved in the acquired resistance.⁶⁷

Another described mechanism of resistance to Trastuzumab in GC was the miR-223-FBXW7 pathway, after knowing that F-box and WD repeat domain-containing 7 (FBXW7) is one of the main causes of carcinogenesis, cancer development, and drug resistance, being associated with poor prognosis in gastrointestinal cancers.^68

–72 Following the establishment of the trastuzumab-resistant GC cells 23 microRNAs (miRs), involved in multiple pathways as cell proliferation, apoptosis, or drug resistance, were upregulated in the resistant cells compared with the parental cell lines. miR-223 was described as its overexpression lowers the FBXW7 expression and also the GC cells sensitivity to trastuzumab, while its inhibition is reversing this effect.^73,74 Furthermore, miR-223 overexpression in suppressing considerably trastuzumab-induced apoptosis.⁷⁵

The overexpression of IQGAP1 or the MACC1–PI3K/AKT–Warburg effect and the miR-223-FBXW7 pathways are only a few of the possible resistance trastuzumab mechanisms, that should be fully understood and further looked into, focusing on the need for research on the signaling pathways or drug targets.

Several pharmacological strategies to circumvent trastuzumab resistance have been identified and are under development in gastric or GEJ adenocarcinoma.

Armamentarium Against Resistance

Trastuzumab beyond progression

Extrapolating the existing data in HER2+ breast cancer, effective strategies for the trastuzumab-resistant disease have also been studied in GC.

One of the strategies found to be effective against the acquired resistance is continuing trastuzumab administration beyond disease progression, by changing the cytotoxic or the molecule that it has been associated with.^76,77

A conjugated antibody–drug composed of trastuzumab and a microtubule inhibitor DM1, a maytansine derivative (TDM1), showed efficacy in tumors considered resistant to Trastuzumab. The phase III EMILIA trial results showed a significant improvement in the median PFS (9.6 months vs. 6.4 months, HR 0.65, 95% CI: 0.55–0.77, p < 0.001) and the median OS, 31 months versus 25 months (HR 0.68, CI: 0.55–0.85, p < 0.001) for TDM1 versus lapatinib plus capecitabine in the pretreated HER2+ breast cancer, resulting in its approval as the second-line treatment of HER2+ breast cancer.^78,79

Encouraged by these findings, the phase II/III GATSBY trial was designed to assess the efficacy in the advanced HER2+ GC patients who progressed after the first-line treatment. The patients were randomized into three arms: TDM1 3.6 mg/kg q3w, TDM1 2.4 mg/kg qw, or taxane-containing chemotherapy: docetaxel 75 mg/m2 q3w, or weekly administration of paclitaxel 80 mg/m². The results show no OS benefit over taxanes, failing to be approved as second-line regimen in HER2+ unresectable GC.⁸⁰

HERBIS-5, an open-label, multicentric, phase II study assessed the efficacy and safety of irinotecan (CPT-11) 150 mg/m², q2w added to a standard dose of trastuzumab in Japanese patients with advanced HER2+ chemorefractory disease. The median PFS and OS were 3.7 and 7.5 months, respectively, showing a promising activity of this association.⁸¹

The phase II study assessing MM-111, a novel bispecific antibody active in preclinical model that blocks HER3 activation in the HER2+ tumors, along with paclitaxel and trastuzumab in Japanese population, were prematurely closed by lack of efficiency in terms of PFS and OS.⁸²

Tyrosine kinase inhibitors

An alternative antireceptor strategy is represented by the intracellular small-molecule ATP inhibitors of the tyrosine kinase domain.

Lapatinib was one of the first tyrosine kinase inhibitor (TKI) developed that showed activity in cells with acquired resistance to trastuzumab, reversible inhibitor of HER2 tyrosine kinase activity in addition to that of its coreceptor EGFR. In HER2+ breast cancer, it represents a valid second-line option for women who progressed after anthracycline, taxane, and trastuzumab-containing regimen in metastatic setting, being approved in combination with capecitabine, another indication being in postmenopausal women, not currently intended for chemotherapy, with hormone receptor-positive breast cancer, in association with an aromatase inhibitor.^83
–85

In gastroesophageal adenocarcinoma, lapatinib in monotherapy showed a modest activity. In a phase II SWOG study, which enrolled 47 chemo-naive patients with advanced disease, 9% had partial remission and 23% stable disease, results explained by the nonspecific population analysis, as HER2 and EGFR statuses were not among the enrollment criteria.⁸⁶

In the TRiO-013 LOGiC trial, lapatinib was assessed along with capecitabine and oxaliplatin, in patients with previously untreated HER2+ advanced GEJ. The results showed that this regimen did not increase OS, but a slight improvement has been reported in a preplanned exploratory subgroup analysis of Asian patients [16.5 months (95% CI: 13.3–20.2) in the lapatinib arm vs. 10.9 months (95% CI: 9.0–14.9) in the placebo arm] and those younger than 60 years of age [12.9 months (95% CI: 11.1–16.0) in the lapatinib arm vs. 9 months (95% CI: 7.8–11.3) in the placebo arm]. No correlation between the HER2 status and survival was observed.⁸⁷

Antiangiogenic Therapies in HER2+ GC: Getting Closer

The vascular endothelial growth factor (VEGF) overexpression has been reported in almost half of the gastric adenocarcinoma patients, more frequently in the intestinal type, reaching up to 76% according to Song et al. analysis, where the majority of the population was stage III–IV disease, tumor size larger than 5 cm, and poorly differentiated adenocarcinoma histology.^88

–92 A prospective biomarker analysis showed that higher levels of VEGF-A correlate with low survival rates.^93
–95

Inadequate tissue oxygenation induces hypoxia-inducible factor 1 alpha (HIF1α) release, a key active angiogenic factor that stimulates VEGF secretion.^96,97 The HER2 overexpression was found to upregulate VEGF-A synthesis.⁹⁸

VEGF-Trap is a decoy receptor fusion protein of second and third immunoglobulin (Ig) domain of VEGFR1 and VEGFR2, respectively, fused to the Fc of human IgG1.⁹⁹

The therapeutic efficacy of trastuzumab-VEGF-Trap association was tested in HER2 GC xenografts expressing NCI-N87^Luc+, the cells constructed from the human GC cell line NCI-N87. The two-agent combination leads to an inhibition of proliferation and an increase of apoptosis in the tumor cells as well as to a reduction of the tumor vascular density, compared with either molecule separately. The exposure to trastuzumab resulted in a decrease of Akt-Erk phosphorylation and VEGF expression in the NCI-N87^Luc+ cells, implying their downregulation by target lesion.^98,100,101

Bevacizumab failed to prove a survival benefit in patients with advanced GC, in the phase III AVAGAST study (12 months vs. 10 months, HR = 0.87, CI: 0.73–1.03, p = 0.1002) or in the similarly designed for Chinese patients, AVATAR trial (10.5 months for the chemotherapy and Bevacizumab arm vs. 11.4 months in the chemotherapy and the placebo arm, HR = 1.11, 95% CI: 0.79–1.56, p = 0.56). There was no preplanned stratification or a post hoc subgroup analysis considering the HER2+ population in any of the studies.^102,103

The combination of capecitabine, oxaliplatin, trastuzumab, and bevacizumab was evaluated in a multicenter phase II study enrolling 35 metastatic esophagogastric cancer patients with high expression of HER2 protein treated by not more than one previous chemotherapy line. This regimen was well tolerated and showed an enhancing effect between bevacizumab and trastuzumab with a median OS of 26.92 months (95% CI: 11.31–36.36), median PFS of 13.93 months (95% CI: 8.5–22.33), and a 76.5% RR (95% CI: 0.59–0.88).¹⁰⁴

Ramucirumab is a monoclonal antibody directed against VEGFR-2, which is approved as second line for metastatic GC, both in monotherapy and chemotherapy associated, showing improved results on OS, PFS, and disease control. Associated to trastuzumab and capecitabine/cisplatin, ramucirumab is currently investigated in a phase II study enrolling patients with metastatic HER2+ GEJ and GC that have received no prior chemotherapy for stage IV disease.¹⁰⁵

Researching Second Line and Beyond

EGFR pointing

The transmembrane TK receptor EGFR is expressed in 60% of GC cases and it was found to mainly correlate with advanced stages and poor prognosis.^106

–109

Afatinib, irreversible EGFR, HER2, and HER4 TKI receptors, displayed an antitumoral activity in vivo in a study whose main objective was to evaluate the sensibility of PET⁹⁰ Zirconium–Trastuzumab in HER2+ GC assessment, and in monitoring pharmacodynamic effects of this drug.¹¹⁰ Currently, it was evaluated for clinical benefit in a phase II trial (NCT01522768) in HER2+ trastuzumab-refractory esophagogastric cancer in heavily treated patients. The results showed that 19 patients have responded to treatment, 2 had a partial response, and 6 stable disease. The common side effects included rash, diarrhea, nausea/vomiting, and fatigue.¹¹¹

Dacomitinib, irreversible pan-HER inhibitor, evaluated in a phase II trial that enrolled 27 previously treated HER2+ GC patients, of whom seven previously exposed to an anti HER2 agent, reported a median OS of 7.1 months and a 4 months PFS of 22.2%. Two partial responses and 9 stable diseases were obtained, the response rate was 7.4%, and the disease control rate was 40.7%, of whom almost half of the patients displayed a tumor volume shrinkage by up to 50%. The frequent toxicities were rash, diarrhea, and fatigue, most of which were grade 1 or 2 (Table 2).^112,113

Table 2.

Ongoing Studies of anti-HER2 Target Agents

Study/first author/year	Sponsor/promoter	Setting	Phase	No patients/status	Compound	ORR%	DFS/RFS (months)	OS (months)
S0413/Iqbal/2011⁸⁶	NCI	LA/M GC, ADK FIRST LINE T unexposed	II	47 Completed	Lapatinib	NA	TTF = 1.9	4.8
TRIO-013/LOGiC/Hecht/2015⁸⁷	Novartis	LA HER2+ GC/OC FIRST LINE T unexposed	III	545 Ongoing	Lapatinib	53	PFS = 6.0	12.2
NCT01402401⁹³	Novartis Pharmaceuticals	LA HER2+ GEJ/GC, beyond FL, T exposed	II	21 Completed	AUY922 Trastuzumab	NA	NA	NA
NCT01522768¹¹¹	Memorial Sloan Kettering Cancer Center	LA/M HER2 EGC beyond FL, T Refractory	II	40 Recruiting patients	Afatinib (BIBW 2992)	NA	NA	NA
NCT01152853/Oh/2016¹¹³	Seoul National University Hospital	R/LA/M HER2 GEJ/GC beyond FL, T exposed	II	27 Completed	Dacomitinib PF-00299804	7.4	PFS = 2.1	7.1

ADK, adenocarcinoma; DFS, disease-free survival; EGC, esophagogastric cancer; FL, first line; GC, gastric cancer; GEJ, gastroesophageal junction; HER2, human epidermal growth factor 2; LA, locally advanced; M, metastatic; NA, not available; No, number (pts number); ORR, objective response rate; OS, overall survival; PFS, progression-free survival; R, recurrent; T, trastuzumab.

Against mTOR system

The serine/threonine kinase mTOR that integrates multiple signals from the hormones and growth factors, playing a main role in the control of hyperplastic cell survival or apoptosis, nucleates two different multiprotein complexes called mTOR complex 1 (mTORC1) and 2 (mTORC2). The inhibition of mTOR appears to play an important part in optimizing the effects of HER2 inhibitors.^{114

–122}

The first described anti-mTOR agent was everolimus, an oral molecule approved for the treatment of renal carcinoma, breast cancer, and progressive neuroendocrine tumors of pancreatic origin.^123
–125

In a phase I trial, everolimus associated to mitomycin C (MMC) was assessed in previously treated metastatic GEJ or GC patients. The 16 enrolled patients have been tested for HER2 status, PTEN, PIK3CA, AKT1, CTNNB1, and E-cadherin1 expression and mutations were divided into three arms to receive 5, 7.5, or 10 mg/day of everolimus along with MMC, 5 mg/m² q3w. Most of the adverse events were grade 3, especially leukopenia and neutropenia. Three (18.75%) patients had a partial response, and four (25%) had stable disease. The antitumoral activity, according to RECIST 1.1 criteria, was highest in the 10 mg/day arm with no associations between the response to therapy and the HER2 status.¹²⁶

Targeting MET

MET, a TK receptor protein activated by hepatocyte growth factor and the scatter factor, is overexpressed in up to 46% of the advanced GC and MET gene amplification, which has been reported in up to 10%–20% of GC, being associated with a more aggressive disease.^{127

–132}

Synergistic effects with lapatinib in the HER2+ GC cell lines overexpressing MET were shown by foretinib, an oral molecule, multi TKI for MET and VEGFR. These data suggest that the associated therapy of a MET inhibitor and a targeted anti-HER agent should be tested in a clinical setting on the HER2+ patients who progressed on trastuzumab or lapatinib.^133,134

Other ways against resistance

Heat shock protein 90 (HSP90) is a chaperone protein overexpressed in the cancer cells that enables the activation of many oncoproteins. A reduction of the ErbB2 protein levels was observed in HER2+ breast cancer and also the GC cell lines after HSP90 blockade, leading to growth inhibition and downstream signaling by PI3K and ErBB3.¹³⁵

The preclinical data have demonstrated the NVP-AUY922 activity, a novel HSP90 inhibitor, against GC cells with synergistic effect when associated with chemotherapy, such as 5-FU and oxaliplatin. A multicentric single-arm phase II trial is evaluating the efficacy and safety of AUY922 in association with trastuzumab, in patients with advanced HER2+ GC after failure of prior trastuzumab-based therapy.^136,137

Immunotherapy in HER2+ GC

Inside the GC, it has recently identified four biomolecular distinct subtypes according to the Cancer Genome Atlas (TCGA), providing a starting point for clinical trials testing new targeted therapies: Epstein–Barr virus (EBV)-positive tumors, microsatellite unstable (MSI) tumors, tumors with chromosomal instability, and genomically stable tumors.¹³⁸

According to this classification, the EBV and the MSI subgroups seem to benefit the most from the immunotherapy, since EBV-positive tumors are characterized by extreme DNA hypermethylation, PIK3CA mutations and JAK2, programmed death-ligand (PD-L) 1, and PD-L2 amplifications and the MSI subgroup is characterized by MLH1 hypermethylation, associated with a higher mutation rate that includes TP53, KRAS, PIK3CA, ERBB3, PTEN, HLA-B, B2M, or NF1.¹³⁹

No HER2 positivity in the EBV+ cases was found in a study of 465 gastroesophageal adenocarcinoma patients that underwent primary resection. Additionally, the study shows inferior rates of EBV positivity in GEJ adenocarcinoma compared with GC.¹⁴⁰

Immune checkpoint pathways

After the success of immunotherapy for malignant melanoma, leading to Food and Drug Administration (FDA) approval of several molecules, including nivolumab (fully human antibody against PD-1) also used in renal, lung, head and neck, and urinary bladder cancer, ipilimumab (fully human antibody against CTLA-4) or pembrolizumab (fully human antibody against PD-1) available for head, neck, and lung cancer, ongoing studies are evaluating immune checkpoint pathways in GC, too.^141
–143

KEYNOTE-012 is a phase Ib study that enrolled 39 PD-L1+ patients with recurrent or metastatic GC or GEJ adenocarcinoma. The patients were assigned to receive pembrolizumab 10 mg/kg q2w for up to 24 months or until complete response, disease progression, or unacceptable toxicity. The results of the study showed a 22% overall response rate, a 24% PFS rate, and a 69% OS rate at 6 months, with a tolerable toxicity.^144,145

Based on these promising results and the fact that PD-L1 is a predictive biomarker for pembrolizumab in lung cancer, a phase II study, KEYNOTE-059, was developed to further evaluate pembrolizumab in GC. In the multilevel study design, a stratification was preplanned according to the HER2 and PD-L1 status, in one arm to be included patients with any PD-L1 status and any HER2 status, pretreated by at least two lines of chemotherapy with or without trastuzumab, another arm with HER2−, any PD-L1 status chemo-naive patients, and in a third-arm patients HER2− and PD-L1+, in the first line too.¹⁴⁶ The recent results show an ORR of 60% (68.8% in PD-L1+ and 37.5% in PD-L1−) in the HER2−, any PD-L1 cohort, and an ORR of 11.2% (15.5% in PD-L1+ and 5.5% in PD-L1−) in any HER2 or PD-L1 status cohort.^147,148 Currently, pembrolizumab is FDA approved for patients with recurrent locally advanced or metastatic gastric or GEJ adenocarcinoma whose tumors express PD-L1, who have received at least two lines of chemotherapy with fluoropyrimidine and platinum-based antineoplastic drugs, and, if appropriate, HER2/neu-targeted therapy.¹⁴⁹

In HER2+ GC, several of these therapies have been and are still being tested in phase II and III trials (Table 3).^{150

–158} Recently, pembrolizumab received FDA-accelerated approval for first tissue/site agnostic indication, for adult and pediatric patients with unresectable or metastatic MSI-H or mismatch repair deficient (dMMR) solid tumors that have progressed following prior treatment or with MSI-H or dMMR colorectal cancer that has progressed following therapy consisting of oxaliplatin, irinotecan, and fluoropyrimidine.^159,160

Table 3.

Current Clinical Trials Assessing the Role of Immunotherapy in Gastric Cancer HER2+

Phase	Population	Compound	Target	Associated treatment	Status	Trial ID
I	M GC/BC HER2 IHC 2+, FISH–SL	HER2-Peptid-Vakzine Imiquimod Sargramostim,	ECD, TM HER2/neu p185neu	Cyclophosphamide	Recruiting	NCT02276300¹⁵⁰
I	LA/M BC/GC/EC HER2+ Beyond SL	IL 12	Anti-Her2/neu Ab, amino terminus H chain	Trastuzumab	Completed	NCT00004074¹⁵¹
I	R BC, GC HER2+ SL	IL 12	Anti-Her2/neu Ab, amino terminus H chain	Trastuzumab paclitaxel	Completed	NCT00028535¹⁵²
I	M, EC/GC/BC HER2+ Beyond SL	CHP-HER2 CHP-NY-ESO-1	Truncated HER2, 146 amino acid residues NH₂ terminus (146HER2) CD4 T-cell	OK-432	Completed	NCT00291473¹⁵³
I	R/M, GC/EG HER2+ SL	AVX901	ECD, TM HER2/neu	—	Ongoing, not recruiting	NCT01526473¹⁵⁴
I/II	M BC/GC HER2+ SL T exposed	NK cells infusion	MCF-7 Her2/neu	Trastuzumab IL 2	Recruiting	NCT02030561¹⁵⁵
Ib/II	LA/M HER2+ GEJ/GC Beyond SL T exposed	Margetuximab pembrolizumab	FcγRIIIA NK (CD16A allelic expression) Anti PD-1	—	Recruiting	NCT02689284¹⁵⁶
II	R/M GEJ/GC HER2+/- FL, previous Pl-FU-T SL	Pembrolizumab	PD-1	Cisplatin 5-FU capecitabine	Ongoing, not recruiting	NCT02335411¹⁴⁶
II	LA/M or unresectable HER2+/- EC, GEJ or GC, HER2+/- SL Pl-FU-T in HER2+ pts	MEDI4736	PD-L1	Capecitabine trastuzumab	Recruiting	NCT02678182¹⁵⁷
III	LA/M HER2+/- GC or GEJ SL Pl-FU-T in HER2+ pts	Pembrolizumab	PD-1	Paclitaxel	Ongoing, not recruiting	NCT02370498¹⁵⁸

Ab, antibody; BC, breast cancer; EC, esophageal cancer; ECD, extracellular domain; FISH, fluorescence in situ hybridization; FL, first line; 5-FU, 5-fluorouracil; GC, gastric cancer; GEJ, gastroesophageal junction; HER2, human epidermal growth factor 2; IHC, immunohistochemistry; IL-12, interleukin-12; LA, locally advanced; M, metastatic; NK, natural killer; Pl-FU-T, platinum-fluoropyrimidine therapy; R, recurrent; SL, second line; TM, transmembrane domain.

Correlated with the TCGA classification data, this opens a new horizon for the MSI-H gastric tumors that may derive a particular benefit.

Further impact over the HER2+/HER2− patients must be clarified.

Conclusions

HER2 overexpressing or amplified GC is a poor prognostic disease, even in early stages.

Despite the availability of trastuzumab as upfront target treatment for advanced or metastatic stages, the improvements in survival remain modest.

In HER2+ breast cancer, significant progress has been obtained by HER2 multilevel targeting strategy, even from the front line.

Starting with laboratory identification, there are differences between breast and GC and HER2 expression, diversity might be due to the various antibody membranous distribution in the cancer cells or intratumoral heterogeneity.¹⁶¹

Also, the lessons should be learned and extrapolated in GC mainly with regard to the resistance mechanism. Pharmacological methods of overcoming the HER2-targeted treatment resistance have to be developed specifically for the gastric and EG HER2+ phenotype.

Trastuzumab is the only validated anti-HER2+agent in locally advanced or metastatic disease, showing an improvement in both OS and PFS and its perioperative effectiveness is currently under evaluation. TDM1 did not manage to show the same benefit in advanced HER2+ GC as it showed in HER2+ breast cancer. A slight improvement was displayed by lapatinib in a post hoc analysis in Asian and younger patients.

Even though VEGF overexpression is frequently found in GC adenocarcinomas, Bevacizumab failed to show any survival benefit in phase III trials, but better results appeared once trastuzumab is associated and the appropriate patient selection is made. Studies are needed to further analyze antiangiogenics in specific HER2 population for potentially active molecularly targeted drugs. Therefore, optimal postprogression therapy for HER2+ metastatic GC remains unknown.

Targeting MET receptor, associated with anti-HER2 TKI, seems promising.

The checkpoint inhibitors that caused a paradigm shift in melanoma treatment can be assessed in HER2+ GC too and early clinical data are encouraging. The best outcome in the MSI-H subgroup of GC patients is awaited due to the early KEYNOTE-059 results and also because of the accelerated approval of pembrolizumab based on molecular markers instead of the conventional approach.

Biomarkers are urgently needed to be explored to select patients, more likely to respond to a particular target agent, and also accurate work-up diagnosis for HER2 detection is mandatory to obtain the best results provided by the anti-HER2 therapy.

The heterogeneity expressed by HER2+ GC, challenges in the irreproducible data from breast cancer and the faulty capacity of understanding oncogenic pathways put together with inappropriate studies, design stratification are some of the pitfalls that are in the way of achieving superior treatment responsiveness.

The treatment of HER2+ GC remains a challenge, in consequence, molecular and clinical research should represent a priority in this field, and maybe running post hoc analysis to propose a stratified phase 3 studies may represent an appropriate approach.

Footnotes

Disclosure Statement

There are no existing financial conflicts.

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