Trastuzumab For Gastric Cancer Research Articles

State of the art and upcoming trends in HER2-directed therapies in gastrointestinal malignancies.

This review critically evaluates the evolution and current status of human epidermal growth factor receptor 2 (HER2)-directed therapies in upper gastrointestinal (GI) malignancies, a timely and relevant inquiry given the dynamic shifts in therapeutic strategies over the past decade. Initial enthusiasm following the Trastuzumab for Gastric Cancer (ToGA) study's demonstration of trastuzumab's efficacy, however, encountered hurdles due to subsequent trials showing limited progress, underscoring the necessity for a reevaluation of therapeutic approaches and the exploration of novel agents. The review highlights significant breakthroughs in the form of immune checkpoint inhibitors and innovative therapeutic technologies, which have redefined treatment paradigms and shown promising efficacy in HER2-positive cases. Emerging treatments such as trastuzumab deruxtecan (T-DXd), zanidatamab and evorpacept further illustrate the ongoing efforts to leverage unique mechanisms of action for improved HER2-positive antitumor activity. The advancements in HER2-directed therapies underscore a pivotal era in the management of upper GI malignancies. These developments not only reflect the profound impact of integrating novel therapeutic combinations but also highlight the critical role of ongoing research in overcoming resistance mechanisms and tailoring treatment to individual disease profiles.

Liquid biopsy based HER2 amplification status in gastric cancer patients indicates clinical response

Gastric carcinomas are among the most common cancers in Germany, with approximately 18,000 new cases per year.About 10 years ago, based on results of the Trastuzumab for gastric cancer (ToGA) trial, the addition of the monoclonal antibody trastuzumab to a platinum-fluoropyrimidine chemotherapy backbone became the standard-of-care 1st-line therapy for human epidermal growth factor receptor 2 (HER2)-positive gastric cancers. Only patients with primary HER2 gene amplification benefit from this therapy. Thus, accurate HER2 gene amplification detection is predictive and critical for therapy selection. As a gold standard the HER2 status is currently determined in tumor tissue specimens using immune histochemistry and fluorescent in situ hybridisation. However, HER2 amplification is detectable in only about 20 % of gastric carcinomas. The recent approval of an antibody-drug conjugate Trastuzumab deruxtecan (T-DXd) and the establishment of a new subgroup of HER2-low tumors due to the bystander effect associated with T-DXd increases the relevance of precise HER2 diagnostics.Aim of this analysis was to determine the HER2 amplification status from circulating DNA fragments in blood using a HER2 Copy Number Variation assay to establish a minimal invasive approach.For the present study, a digital droplet PCR-based method was validated relative to established tissue-based methods. Furthermore and most importantly, the changes of HER2 status during therapy were investigated in seven patients indicating that the changes of HER2 status and number of HER2 copies detected in blood can reflect on therapy efficiency and uncover treatment resistance.

Immunoexpression of PTEN, HER2/neu, and Ki-67 in endoscopic gastric carcinoma biopsies; their correlation with histological subtypes and one-year survival.

Gastric carcinoma is a major cause of cancer-related morbidity and mortality worldwide. Gastric neoplasms arise from genetic and epigenetic changes in various genes. Present study evaluates the immunoexpression of PTEN, HER2/neu, and Ki-67 in endoscopic gastric carcinoma biopsies and correlates the expression of these proteins with clinicopathological features. Adequate endoscopic biopsies of 27 cases of gastric carcinoma were evaluated for World Health Organization (WHO) and Lauren's classification subtypes along with HER2/neu, PTEN, and Ki-67 immunoexpression. HER2/neu immunostaining was scored as proposed in the Trastuzumab for gastric cancer (ToGA) trial while PTEN staining and downregulation were assessed using an immunoreactive score. The cut-off for Ki-67 expression was taken as 90th percentile of the values in adjacent non-neoplastic tissue. All statistical analysis was done at 5% level of significance with SPSS v22 statistical software. Tubular adenocarcinoma was the commonest WHO histological subtype and 56% of cases were of intestinal type as per Lauren's classification. 55.6% of cases showed a complete loss of PTEN expression in neoplastic tissue. 17 of the 19 cases with adjacent non-neoplastic tissue showed PTEN downregulation in neoplastic tissue. 81.5% of cases had a high Ki-67 index and HER2/neu overexpression was noted in 36% of cases. All the four cases who died had high Ki-67 proliferation indices; 3 patients had loss of PTEN expression and HER2/neu overexpression. We conclude that these immunomarkers can play important role in the behavior of gastric carcinomas and can be targeted for new therapies.

Immunohistochemical expression of FOXP3 in gastric carcinoma; its relation to Ki-67 proliferation marker, HER2/neu expression, and other clinicopathological parameters

ABSTRACT Gastric cancer is common cancer in the world. Contradictory results regarding FOXP3 expression in gastric carcinoma were detected and the role of Ki-67 in prognosis is not completely understood. Furthermore, due to increasing use of anti-HER2 drug trastuzumab for gastric cancer, assessment of HER2 expression becomes important. This study tried to assess the FOXP3 expression in gastric carcinoma and to study the relation between its expression and Ki-67 and HER2/neu expression and relation between their expression and other clinicopathological variables. This retrospective study was carried out on 60 gastric adenocarcinoma cases. Tissue microarrays and immunohistochemical staining for FOXP3, Ki-67 and HER2/neu were done and then assessed and scored. HER2/neu expression showed significant relation to Lauren histological type and lymph node status. High Ki-67 index was related significantly to patients’ age, lympho-vascular tumor emboli, peri-neural invasion, tumor grade, lymph node status, and cancer stage. There was significant relation between high FOXP3 expression and patients’ age, lympho-vascular tumor emboli, peri-neural invasion, tumor grade, lymph node status, and cancer stage. Direct positive significant relationships between HER2/neu, Ki-67, and FOXP3 expression were noticed. Finally, high FOXP3 expression, positive HER2/neu, and high Ki-67 nuclear proliferation index may be an indication of the aggressiveness of gastric carcinoma.

HER2 Splice Site Mutation c.1899-1G>A as the Potential Acquired Resistance to Trastuzumab in a Patient with HER2-Positive Gastric Adenocarcinoma.

The addition of trastuzumab to chemotherapy regimen is the standard of care for human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer; however, most patients eventually acquire trastuzumab resistance. Although some resistance mechanisms to trastuzumab-based regimens have been proposed, further understanding is required for developing therapeutic strategies to overcome the resistance. In the present work, we attempted to determine the possible resistance mechanism to trastuzumab in a patient with HER2-positive stage IV gastric adenocarcinoma. In this study, we first report the nucleotide change c.1899-1G>A at the intron 15 acceptor splice site promoting exon 16 deletion of HER2 as the potential mechanism of trastuzumab resistance in HER2-positive gastric adenocarcinoma. KEY POINTS: The combination of trastuzumab with chemotherapy is considered to be the standard therapy for HER2-positive advanced gastric cancer (GC), but most of the patients eventually acquire trastuzumab resistance. The mechanisms of resistance to trastuzumab in GC are poorly characterized. To the best of the authors' knowledge, this study is the first to implicate HER2 c.1899-1G>A, which results in exon 16 skpping, as the acquired resistance mechanism to trastuzumab in HER2-positive gastric adenocarcinoma. This work provides insights into the potential molecular mechanism of trastuzumab resistance, which is crucial in developing effective therapeutic strategies for HER2-positive GC patients refractory to trastuzumab.

Effectiveness of Trastuzumab in Routine Clinical Practice: A Population-based Study of Patients with HER-2-positive Oesophageal, Gastroesophageal and Gastric Cancer

AimsIn the pivotal Trastuzumab for Gastric Cancer (ToGA) trial, trastuzumab improved median survival in patients with advanced HER-2-positive gastric and gastroesophageal cancer from 11.1 to 13.8 months; however, its effectiveness in routine clinical practice has not been evaluated. Our objective was to evaluate the uptake and outcomes of trastuzumab in a population-based cohort of patients with oesophageal, gastroesophageal and gastric cancer in Ontario, Canada. Materials and methodsThe Ontario Cancer Registry and linked treatment records were used to identify all patients with oesophageal, gastroesophageal and gastric cancer treated with trastuzumab during 2012–2017. Outcomes were analysed from the time of first trastuzumab cycle and included a primary outcome (survival) and secondary outcomes (uptake, delivery, 30-day hospital admission and 30-day mortality). Trends over the study period and survival were evaluated. ResultsIn total, 476 patients with oesophageal, gastroesophageal and gastric cancer received trastuzumab during the study period. The mean age was 62 years, 78% (370/476) were male, and 65% (312/476) had gastric cancer. The annual number of patients receiving trastuzumab increased over the study period (53 in 2012 and 101 in 2017). The median number of cycles of trastuzumab delivered was six. Thirty-day hospital admission and mortality rates were 17% and 4%, respectively. The median overall survival was 282 days (9.3 months). ConclusionsThe median survival of patients treated with trastuzumab for advanced oesophageal, gastroesophageal and gastric cancer in routine practice is substantially less than that observed in the pivotal clinical trial. Studies of comparative effectiveness using real-world data offer insight into outcomes achieved in routine practice.

Trastuzumab Specific Epitope Evaluation as a Predictive and Prognostic Biomarker in Gastric Cancer Patients

While human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) antibodies bind to the intracellular domain, trastuzumab binds to the extracellular epitope of HER2 receptor: target of drug action. We aimed to evaluate clinical significance of the new IHC method assessing the target of trastuzumab in gastric cancer (GC) patients and compare with conventional methods. Sixty-nine trastuzumab-treated GC patients were enrolled from two different cohorts. Additionally, we enrolled 528 consecutive GC patients to evaluate prognostic implications of HER2 test methods. HER2 status was assessed by trastuzumab IHC, HER2 IHC (4B5), and HER2 silver in situ hybridization (SISH). HER2 IHC showed 3+ in 48/69 trastuzumab-treated patients (69.6%), however, trastuzumab IHC showed 3+ in 25 (36.2%). Patients with trastuzumab IHC ≥2+ had significantly better progression-free survival (PFS) and overall survival (OS) than their counterpart (p = 0.014). In univariate analysis, trastuzumab IHC ≥2+ and HER2 IHC 3+ were only significant predictive factors for OS in trastuzumab-treated patients. Of the 528 consecutive GCs, patients with trastuzumab IHC ≥2+ had shorter disease-free survival (DFS) and OS (p = 0.008 and 0.031, respectively), while conventional methods failed to reveal any significant survival differences. HER2 assessment by trastuzumab IHC was different from conventional HER2 test results. Trastuzumab IHC was suggested to be a significant predictive factor for trastuzumab responsiveness and prognostic factor for consecutive GCs.

HER-Family Ligands Promote Acquired Resistance to Trastuzumab in Gastric Cancer.

Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line-based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer.

Trastuzumab induces PUMA-dependent apoptosis and inhibits tumor growth in gastric cancer.

Gastric cancer (GC) is one of the most prevalent cancers worldwide. Trastuzumab has been approved for the treatment of metastatic GC, gastroesophageal junction cancer, and breast cancer. However, the mechanisms involved in trastuzumab‐induced GC cell apoptosis remain largely unknown. In this study, we investigated the underlying mechanisms of trastuzumab‐mediated suppression of GC cell growth both in vitro and in vivo. We found that trastuzumab treatment induces p53 upregulated modulator of apoptosis (PUMA) expression in GC cells, through the NF‐κB pathway following AKT inhibition and glycogen synthase kinase 3β (GSK3β) activation. We also observed that PUMA was necessary for trastuzumab‐induced apoptosis in GC cells. Moreover, PUMA deficiency suppressed apoptosis and the antitumor effect of trastuzumab in xenograft models. Finally, computerized tomography (CT) and immunohistochemistry results showed that patients with increased activation of PUMA were more sensitive to trastuzumab treatment than those with low PUMA expression. These results indicate that trastuzumab induces PUMA‐dependent apoptosis and inhibits tumor growth in GC, suggesting that PUMA plays a critical role in mediating the antitumor effects of trastuzumab in GC. PUMA induction may be used as a marker of trastuzumab sensitivity.

HER2-targeted gold nanoparticles potentially overcome resistance to trastuzumab in gastric cancer

An issue of concern is that no current HER2-targeted therapeutic agent is effective against Trastuzumab (Tmab)-resistant gastric cancer. Gold nanoparticles (AuNPs) are promising drug carriers with unique characteristics of a large surface area available for attachment of materials such as antibodies. Here, we created HER2-targeted AuNPs (T-AuNPs) and examined their therapeutic efficacy and cytotoxic mechanisms using HER2-postive Tmab-resistant (MKN7) or Tmab-sensitive (NCI-N87) gastric cancer cell lines. In vitro, T-AuNPs showed stronger cytotoxic effects than controls against MKN7 and NCI-N87 cells although Tmab had no effect on MKN7 cells. Autophagy played an important role in T-AuNP cytotoxic mechanisms, which was considered to be driven by internalization of T-AuNPs. Finally, T-AuNPs displayed potent antitumor effects against NCI-N87 and MKN7 subcutaneous tumors in in vivo mouse models. In conclusion, HER2-targeted AuNPs with conjugated Tmab is a promising strategy for the development of novel therapeutic agents to overcome Tmab resistance in gastric cancer.

Clinicopathological factors associated with HER2-positive gastric cancer: A meta-analysis.

Background:The treatment of patients with advanced gastric cancer remains a most challenging task in the clinical practice. Recently, targeted therapies have significantly impacted the treatment strategy for many common malignancies. The use of trastuzumab, a monoclonal antibody against human epidermal growth factor receptor 2 (HER2; also known as ERBB2), plus chemotherapy proved to improve median overall survival in patients with advanced gastric cancer, compared with chemotherapy alone in Trastuzumab for Gastric Cancer (ToGA) trial. However, the prognostic value of HER2 status in gastric cancer remains controversial. Therefore, the aim of this study was to investigate the clinical pathology significance of HER2 overexpression in resectable gastric cancer for selecting the right patients with gastric cancer who may benefit from trastuzumab treatment.Methods:Publications reported the clinicopathological factors associated with HER2 status in gastric cancer from 2012 to 2017 were collected. The literature databases, such as “Cochrane Library”, “Sciencedirect”, “Springer”, “PubMed”, “Embase”, were extensively searched to retrieve the clinical studies of HER2 expression in gastric cancer. The major outcomes measures were odds ratios (ORs) and their 95% CIs. Statistical analysis was carried out by Revman software 5.3. The Newcastle–Ottawa scale was used to assess the quality of evidence.Result:Fifteen studies met our inclusion criteria. This study demonstrated that the pooled OR for HER2 positivity was associated with being male (OR: 1.42; 95% CI: 1.23–1.64), well/moderately differentiated tumor (OR: 2.76; 95% CI: 1.72–4.45), and for intestinal-type tumor (OR: 0.31; 95% CI: 0.25–0.38). However, it had no correlation with depth of tumor (P = .07), venous invasion (P = .82), and lymphovascular invasion (P = .24).Conclusion:HER2-positive expression was associated with male gender, intestinal type, and well/moderate cell differentiation. We recommend that those gastric cancer patients who may benefit from trastuzumab treatment should be subjected to targeted therapies. However, detecting HER2 status may contribute to the target therapy for gastric carcinoma using trastuzumab. This would be strengthened by further studies incorporating comorbidity data, and outcomes from centralized programs.

Central validation of HER2 status to determine heterogeneity of marker expression in HER2-positive gastric cancer (GC).

12 Background: 10-20% of GC overexpress HER2, a membrane-bound receptor tyrosine kinase (RTK) which belongs to the epidermal growth factor receptor (EGFR) family. Drugs directed against HER2 have shown mixed success in the treatment of advanced GC. While trastuzumab, a monoclonal antibody addressing HER2 has been approved for 1st-line treatment of stage IV HER2+ GC, trastuzumab-emtansine failed to improve outcomes in 2nd-line and lapatinib, a small molecular RTK inhibitor of HER2 and EGFR was not effective in 1st- and 2nd-line. Until now, primary and secondary resistance against HER2-directed treatment of GC is not well understood. The VARIANZ study aims to assess mechanisms influencing efficacy of trastuzumab in HER2+ GC. Methods: In this multicenter study, patients who receive medical treatment for advanced GC are recruited in 31 sites. The HER2 status is verified centrally by two dedicated GI pathologists using immunohistochemistry (IHC, DCS, HI608C0I) and chromogenic-in-situ hybridization (CISH, Zytomed Systems, C-3022-40). Results: From May 2014 to August 2016, we have enrolled 316 patients in this ongoing project (72% male, median age 64 years). At present, 281 samples were fully characterized for the HER2 status. According to criteria from the Trastuzumab for Gastric Cancer (ToGA) study, 53 of 281 samples were characterized HER2+ by central testing. In 38 samples that were diagnosed as HER2+ by local pathologists the HER2 status could not be verified centrally. 7 HER2- probes in local testing were characterized as HER2+ by central testing. The overall deviation rate between local and central testing is 27%. HER2 gene amplification in HER2+ tumors with deviating local report (mean HER2/CEP17: 2.8 ± 0.9, range between 1.9 and 4.5) is lower compared to HER2+ tumors and confirmed local report (mean HER2/CEP17: 5.5 ± 2.6; range between 2.2 and 11.0; p = 0.014). Conclusions: HER2-expression in GC is heterogeneous and still not easy to assess. Variability between local and central HER2 assessment is significant. Robust biomarkers predicting response or resistance to HER2 and other target therapies are needed. Clinical trial information: NCT02305043.

Quantitative proteomic analysis of HER2 expression in the selection of gastric cancer patients for trastuzumab treatment

BackgroundA wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies.Patients and methodsGC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics.ResultsQuantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy.ConclusionsProteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods.

571 - Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer

The HER2 receptor is overexpressed in 7-34% of gastric cancer (GC) tumors, being a potential therapeutic target. Trastuzumab, an anti-HER2 monoclonal antibody, in combination with platinum therapy was approved in 2010 as first-line treatment for advanced GC tumors overexpressing HER2. Despite the clinical benefit of trastuzumab, development of acquired resistance to treatment limits the efficacy of trastuzumab. To study the mechanisms of trastuzumab acquired resistance we generated, upon chronic exposure to trastuzumab, a trastuzumab-resistant cell line derived from the human HER2-positive GC cell NCI-N87, hereafter named NTR2.4. The resistant phenotype of NTR2.4 cell line was associated with an increased basal activation of SRC, MAPK/ERK and PI3K/mTOR HER2 downstream pathways that persisted upon exposure to trastuzumab. Trastuzumab treatment induced a compensatory upregulation of HER3 and an increased expression of the HER family ligands EGF, AREG, HB-EGF, and NRG1 in NTR2.4 compared with the trastuzumab-sensitive NCI-N87 cell line. Ligand stimulation induced resistance to trastuzumab in NCI-N87 cells. These results were replicated in another model of trastuzumab-resistance, OTR6 cell line, suggesting an important role of HER receptor plasticity and HER family ligand in trastuzumab efficacy and resistance. Analysis of clinical samples from HER2-positive GC patients treated with trastuzumab showed higher EGF concentration in serum samples and increased EGF mRNA expression in tumor biopsies at trastuzumab progression compared with samples previous to trastuzumab treatment. Treatment with Pan-HER, a novel antibody mixture against EGFR, HER2, and HER3, was effective in both trastuzumab-sensitive and trastuzumab-resistant cell lines and also in the presence of ligands. Altogether, our results suggested that Pan-HER would be a potential candidate to overcome trastuzumab resistance in advanced GC patients.

Recent Strategies for Treating Stage IV Gastric Cancer: Roles of Palliative Gastrectomy, Chemotherapy, and Radiotherapy.

Recently, many strategies have been reported for the effective treatment of gastric cancer. However, the strategy for treating stage IV gastric cancer remains controversial. Conducting a prospective phase III study in stage IV cancer patients is difficult because of heterogeneous performance status, age, and degree of cancer metastasis or extension. Due to poor prognosis, the variance in physical status, and severe symptoms, it is important to determine the optimal strategy for treating each individual stage IV patient. In the past decade, many reports have addressed topics related to stage IV gastric cancer: the 7th Union for International Cancer Control (UICC) TNM staging system has altered its stage IV classification; new chemotherapy regimens have been developed through the randomized ECF for advanced and locally advanced esophagogastric cancer (REAL)-II, S-1 plus cisplatin versus S-1 in RCT in the treatment for stomach cancer (SPIRITS), trastuzumab for gastric cancer (ToGA), ramucirumab monotherapy for previously-treated advanced gastric or gastro-oesophageal junction adenocarcinoma (REGARD), and ramucirumab plus paclitaxel versus placebo plus paclitaxel in patients with previously-treated advanced gastric or gastro-oesophageal junction adenocarcinoma (RAINBOW) trials; and the survival efficacy of palliative gastrectomy has been denied by the reductive gastrectomy for advanced tumor in three Asian countries (REGATTA) trial. Current strategies for treating stage IV patients can be roughly divided into the following five categories: palliative gastrectomy, chemotherapy, radiotherapy, gastric stent, or bypass. In this article, we review recent publications and guidelines along with above categories in the light of individual symptoms and prognosis.

Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.

HER2-positive advanced gastric cancer patients frequently develop resistance to trastuzumab through mechanisms still poorly understood. In breast cancer, other members of the HER-family are known to be involved in trastuzumab-resistance, as is overexpression of the scaffold protein IQGAP1. In the present work, we investigated acquired resistance to trastuzumab in gastric cancer experimental models. Trastuzumab-resistant (HR) subclones derived from 3 HER2-overexpressing gastric cancer cells were generated and characterized for alterations in HER2-signaling mechanisms by next-generation sequencing, immunohistochemical, western blot and qRT-PCR techniques, and molecular modeling analysis. All subclones showed a reduced growth rate with respect to parental cell lines but each had a different resistance mechanism. In NCI N87 HR cells, characterized by a marked increase in HER2-signaling pathways with respect to the parental cell line, trastuzumab sensitivity was restored when IQGAP1 expression was silenced. AKG HR subclone showed higher HER3 protein expression than the parental line. High nuclear HER4 levels were observed in KKP HR cells. In conclusion, our study revealed that high IQGAP1 expression leads to resistance to trastuzumab in gastric cancer. Furthermore, 2 new mutations of the HER2 gene that may be involved in acquired resistance were identified in AKG HR and KKP HR subclones.

Molecular mechanism of secondary-resistant for trastuzumab in gastric cancer.

58 Background: The ToGA trial demonstrated the significant efficacy of trastuzumab combined with chemotherapy in patients with HER2-positive GC. Although trastuzumab has become a key drug in cancer treatment, the resistance of breast cancer to trastuzumab is a major problem in clinical practice. The aim of this study was to identify molecular mechanism that regulates the sensitivity of HER2-positive GC cells to trastuzumab. Methods: (i) Establishment of trastuzumab-resistant GC cell: We cultured the HER2-positive cell line in the presence of trastuzumab (ii) microRNA array: We examined the miR expression profile in the parent and resistant cells using miR array analysis. (iii) Identification of novel miR-gene pathway: We focused on miR-223-FBXW7 pathway based on miRarray analysis. Results: (i) We established resistant cell without significant changes in HER2 or p-HER2 in the presence of trastuzumab continuously for 6 months. (IC 50: 0.17 vs. 988.8μg/ml) (ii) We identified 23 miRs up-regulated in the resistant cells as compared with the parental cells. Of these identified miR, 40% miRs are located on 19q13.42. (iii) the miR-223/FBXW7 pathway regulates the sensitivity of a HER2-positive gastric cancer cell line to trastuzumab through the modulation of apoptosis. Conclusions: This study suggested that the miR-223-FBXW7 pathway can be a crucial clue to the mechanism of resistance to trastuzumab in GC, leading to the development of individualized treatment in clinical practice.

A Retrospective Analysis for Patients with HER2-Positive Gastric Cancer Who Were Treated with Trastuzumab-Based Chemotherapy: In the Perspectives of Ethnicity and Histology.

PurposeWhile the Trastuzumab for Gastric Cancer (ToGA) trial demonstrated the efficacy and safety of trastuzumab-based chemotherapy in HER2-positive metastatic gastric cancer, the overall survival (OS) benefit was not found in Asian and diffuse-type cancer patients. The aim of the study is to investigate predictive markers for trastuzumab-based chemotherapy.Materials and MethodsData of patients with HER2-positive gastric cancer treated with trastuzumab-based chemotherapy were analyzed retrospectively.ResultsA total of 168 Asian patients were included. The median age was 60 years (range, 27 to 85 years) and the male:female ratio was 118 (70.2%):50 (29.8%). Fourteen (8.3%), 63 (37.5%), 75 (44.6%), and 11 (6.5%) patients had well, moderately, poorly-differentiated tubular adenocarcinoma and signet ring cell carcinoma, respectively. With 14 complete responses and 73 partial responses, the response rate was 50.6%. The median progression-free survival (PFS) was 10.2 months (95% confidence interval [CI], 8.7 to 11.7), and the median OS was 18.5 months (95% CI, 16.4 to 50.6). Next, we investigated the effect of poorly-differentiated histology (PDH, poorly-differentiated tubular adenocarcinoma+signet ring cell carcinoma) on clinical outcomes. The median PFS (8.9 months vs. 11.5 months, p=0.16) was slightly inferior in PDH patients, and the median OS was significantly shorter in PDH patients (14.6 months vs. 19.0 months, p=0.025).ConclusionWhile subset analysis of the ToGA trial demonstrated that trastuzumab-based chemotherapy may not be beneficial for Asians and patients with PDH, our data may suggest that even in Asian patients and patients with PDH, trastuzumab-based chemotherapy could be associated with improved clinical outcomes in patients with HER2-positive gastric cancer.

Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.

In the present study, we demonstrate that prolonged treatment by trastuzumab induced resistance of NCI-N87 gastric cancer cells to trastuzumab. The resistant cells possessed typical characteristics of epithelial to mesenchymal transition (EMT)/cancer stem cells and acquired more invasive and metastatic potentials both in vitro and in vivo. Long term treatment with trastuzumab dramatically inhibited the phosphorylation of Akt, but triggered the activation of STAT3. The level of IL-6 was remarkably increased, implicating that the release of IL-6 that drives the STAT3 activation initiates the survival signaling transition. Furthermore, the Notch activities were significantly enhanced in the resistant cells, companied by upregulation of the Notch ligand Jagged-1 and the Notch responsive genes Hey1 and Hey2. Inhibiting the endogenous Notch pathway reduced the IL-6 expression and restored the sensitivities of the resistant cells to trastuzumab. Blocking of the STAT3 signaling abrogated IL-6-induced Jagged-1 expression, effectively inhibited the growth of the trastuzumab resistant cells, and enhanced the anti-tumor activities of trastuzumab in the resistant cells. These findings implicate that the IL-6/STAT3/Jagged-1/Notch axis may be a useful target and that combination of the Notch or STAT3 inhibitors with trastuzumab may prevent or delay clinical resistance and improve the efficacy of trastuzumab in gastric cancer.

Abstract 1444: Sensitivity to trastuzumab for gastric cancer is regulated by miR-223/FBXW7 pathway

Abstract Background: A recent large-scale phase III study (the ToGA trial) demonstrated the significant efficacy of trastuzumab in addition to chemotherapy in patients with HER2-positive gastric cancer (GC). Although trastuzumab has become a key drug in recent cancer treatment, resistance to trastuzumab for breast cancer is a major problem in clinical practice. However, it is unclear whether similar mechanisms of trastuzumab resistance act in GC. F-box and WD repeat domain-containing 7 (FBXW7) is the substrate recognition components of an evolutionarily conserved SCF (complex of SKP1, CUL1, and F-box protein)-type ubiquitin ligase complexe, which has been well characterized and shown to have important roles in regulating the stability of multiple oncoprotein substrates, including cyclin E, c-Myc, Notch, c-Jun, mTOR, and MCL1. Therefore, FBXW7 is recognized to be one of the major causes of drug resistance. The aim of the current study is to clarify the novel microRNA (miR)-FBXW7 pathway regulating resistance to trastuzumab. Method: Using 5 GC cell lines, we examined the following items in vitro. 1. We examined the status of FBXW7 expression by quantitative real-time reverse transcription-PCR (qRT-PCR). 2. We established the trastuzumab resistance GC cell line with continuous trastuzumab administration. Parent GC cell line and resistance GC cell line are subjected to miR PCR array. 3. We focused on miR223-FBXW7 pathway. We examined the status of HER2, miR223, FBXW7, and its target gene MCL1 expression by up or down regulation of miR-223 in GC cell lines. 4. We investigated whether the miR-223-FBXW7 pathway can affect the resistance of trastuzumab. Result: FBXW7 and miR-223 expression showed inverse correlation. Moreover, miR-223 expression was increased and FBXW7 expression was decreased whereas MCL1 expression, downstream targets of FBXW7, was increased while no significant change was observed both in HER2 and p-HER2 in GC trastuzumab resistance cells. Overexpression of miR-223 decreased FBXW7 expression persisting HER2 and p-HER2 expression and can induce the resistance of trastuzumab. The suppression of miR-223 increased FBXW7 expression persisting HER2 and p-HER2 expression and can simultaneously restore the sensitivity of trastuzumab.Conclusion: Our current study revealed for the first time that regulated the sensitivity of HER2-positive GC cell line to trastuzumab. These findings suggest that this pathway may be crucial to the mechanism of resistance to trastuzumab in GC, which may lead to the development of individualized treatment in clinical practice. Citation Format: Kojiro Eto, Masaaki Iwatsuki, Takatsugu Ishimoto, Hideo Baba. Sensitivity to trastuzumab for gastric cancer is regulated by miR-223/FBXW7 pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1444. doi:10.1158/1538-7445.AM2014-1444