Metabolic heterogeneity affects trastuzumab response and survival in HER2-positive advanced gastric cancer.

BACKGROUND: Trastuzumab is the only first-line treatment targeted against the human epidermal growth factor receptor 2 (HER2) approved for patients with HER2-positive advanced gastric cancer. The impact of metabolic heterogeneity on trastuzumab treatment efficacy remains unclear. METHODS: Spatial metabolomics via high mass resolution imaging mass spectrometry was performed in pretherapeutic biopsies of patients with HER2-positive advanced gastric cancer in a prospective multicentre observational study. The mass spectra, representing the metabolic heterogeneity within tumour areas, were grouped by K-means clustering algorithm. Simpson's diversity index was applied to compare the metabolic heterogeneity level of individual patients. RESULTS: Clustering analysis revealed metabolic heterogeneity in HER2-positive gastric cancer patients and uncovered nine tumour subpopulations. High metabolic heterogeneity was shown as a factor indicating sensitivity to trastuzumab (p = 0.008) and favourable prognosis at trend level. Two of the nine tumour subpopulations associated with favourable prognosis and trastuzumab sensitivity, and one subpopulation associated with poor prognosis and trastuzumab resistance. CONCLUSIONS: This work revealed that tumour metabolic heterogeneity associated with prognosis and trastuzumab response based on tissue metabolomics of HER2-positive gastric cancer. Tumour metabolic subpopulations may provide an association with trastuzumab therapy efficacy. CLINICAL TRIAL REGISTRATION: The patient cohort was conducted from a multicentre observational study (VARIANZ;NCT02305043).

Plasma EBV DNA as a prognostic factor in EBV associated gastric cancer: a multicenter, prospective study (EBV PRESAGE study).

Purpose: The Cancer Genome Atlas Research Network identified Epstein-Barr-Virus (EBV)-positive gastric cancer as a distinct molecular subtype. The prevalence is 8-9% and the histological examination shows pronounced lymphocytic infiltration, elevated levels of IFN-γ and consequently overexpression of PD-L1. The role of plasma EBV DNA load as a prognostic factor in patients with this cancer subtype is still to be defined. Methods and analysis: The present multicenter prospective observational study "EBV PRESAGE", involving German and Italian cancer centers, aims to evaluate the prognostic role of plasma EBV DNA in EBV-related gastric cancer (GC). The objective is to study the association between plasma EBV DNA load at different consecutive time points and the patient's prognosis. Every patient with a new diagnosis of gastric cancer (including gastroesophageal junction adenocarcinoma) will be screened for Epstein-Barr encoded small Region (EBER) on tissue biopsies using in situ hybridization (ISH). If EBER ISH is positive, blood analysis for plasma EBV DNA will be conducted. The plasma EBV quantitative analysis will be centralized, and extraction, detection, and quantification of EBV DNA in plasma samples will be performed using real-time PCR. Discussion: We hypothesized that plasma EBV DNA represents a non-invasive tool for monitoring EBV-related GC and might be valuable as a prognostic marker.

Deviating HER2 test results in gastric cancer: analysis from the prospective multicenter VARIANZ study.

PURPOSE: The prospective multicenter VARIANZ study aimed to identify resistance biomarkers for HER2-targeted treatment in advanced gastric and esophago-gastric junction cancer (GC, EGJC). HER2 test deviations were found in 90 (22.3%) of 404 cases (central versus local testing) and were associated with negative impact on survival for trastuzumab-treated patients. Here, we investigated methodological and biological variables that may promote deviating HER2 test results. METHODS: We analyzed HER2 testing procedures and participation in quality assurance programs of 105 participating local pathology laboratories. Furthermore, tumor localization and histological subtypes were compared between patients with centrally confirmed (central HER2 + /local HER2 + , n = 68) and unconfirmed HER2 status (central HER2 -/local HER2 + , n = 68). RESULTS: For central HER2 testing, concordance between in situ hybridization (ISH) and immunohistochemistry (IHC) was 98.3%, with IHC sensitivity of 93.3% (84 IHC + of 90 ISH +), specificity of 99.5% (389 IHC- of 391 ISH-), and a positive diagnosis rate of 97.7%. Central confirmation of the local HER2 IHC scores were seen for the majority of locally HER2- IHC 0/1 (172/178; 96.6%), but less frequently for locally IHC3 + (57/124; 46.0%) cases. Deviation rate was not associated with IHC antibody platform used in the local pathology institute neither with participation in quality-assuring tests. Regarding tumor characteristics, deviating test results were more frequently found in GC vs. EGJC (69.1% vs. 39.7%; p = 0.001) and in Laurén diffuse vs. intestinal subtype (23.5% vs. 5.9%, p = 0.004). CONCLUSION: Tumor localization and histological subtype have an impact on HER2 test deviation rates. Assessment of HER2 remains challenging for GC and EGJC.

Spatial Metabolomics Identifies Distinct Tumor-Specific Subtypes in Gastric Cancer Patients.

PURPOSE: Current systems of gastric cancer molecular classification include genomic, molecular, and morphological features. Gastric cancer classification based on tissue metabolomics remains lacking. This study aimed to define metabolically distinct gastric cancer subtypes and identify their clinicopathological and molecular characteristics. EXPERIMENTAL DESIGN: Spatial metabolomics by high mass resolution imaging mass spectrometry was performed in 362 patients with gastric cancer. K-means clustering was used to define tumor and stroma-related subtypes based on tissue metabolites. The identified subtypes were linked with clinicopathological characteristics, molecular features, and metabolic signatures. Responses to trastuzumab treatment were investigated across the subtypes by introducing an independent patient cohort with HER2-positive gastric cancer from a multicenter observational study. RESULTS: Three tumor- and three stroma-specific subtypes with distinct tissue metabolite patterns were identified. Tumor-specific subtype T1(HER2+MIB+CD3+) positively correlated with HER2, MIB1, DEFA-1, CD3, CD8, FOXP3, but negatively correlated with MMR. Tumor-specific subtype T2(HER2-MIB-CD3-) negatively correlated with HER2, MIB1, CD3, FOXP3, but positively correlated with MMR. Tumor-specific subtype T3(pEGFR+) positively correlated with pEGFR. Patients with tumor subtype T1(HER2+MIB+CD3+) had elevated nucleotide levels, enhanced DNA metabolism, and a better prognosis than T2(HER2-MIB-CD3-) and T3(pEGFR+). An independent validation cohort confirmed that the T1 subtype benefited from trastuzumab therapy. Stroma-specific subtypes had no association with clinicopathological characteristics, however, linked to distinct metabolic pathways and molecular features. CONCLUSIONS: Patient subtypes derived by tissue-based spatial metabolomics are a valuable addition to existing gastric cancer molecular classification systems. Metabolic differences between the subtypes and their associations with molecular features could provide a valuable tool to aid in selecting specific treatment approaches.

Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

HER2 Expression, Test Deviations, and Their Impact on Survival in Metastatic Gastric Cancer: Results From the Prospective Multicenter VARIANZ Study.

PURPOSE: Trastuzumab is the only approved targeted drug for first-line treatment of human epidermal growth factor receptor 2-positive (HER2+) metastatic gastric cancer (mGC). However, not all patients respond and most eventually progress. The multicenter VARIANZ study aimed to investigate the background of response and resistance to trastuzumab in mGC. METHODS: Patients receiving medical treatment for mGC were prospectively recruited in 35 German sites and followed for up to 48 months. HER2 status was assessed centrally by immunohistochemistry and chromogenic in situ hybridization. In addition, HER2 gene expression was assessed using qPCR. RESULTS: Five hundred forty-eight patients were enrolled, and 77 had HER2+ mGC by central assessment (14.1%). A high deviation rate of 22.7% between central and local test results was seen. Patients who received trastuzumab for centrally confirmed HER2+ mGC (central HER2+/local HER2+) lived significantly longer as compared with patients who received trastuzumab for local HER2+ but central HER2- mGC (20.5 months, n = 60 v 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; P < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the HER2 amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a HER2 amplification ratio of ≥ 3.0 were calculated as optimized thresholds for predicting benefit from trastuzumab. CONCLUSION: Significant discrepancies in HER2 assessment of mGC were found in tumor specimens with intermediate HER2 expression. Borderline HER2 positivity and heterogeneity of HER2 expression should be considered as resistance factors for HER2-targeting treatment of mGC. HER2 thresholds should be reconsidered. Detailed reports with quantification of HER2 expression and amplification levels may improve selection of patients for HER2-directed treatment.

Towards Phenotyping of Clinical Trial Eligibility Criteria.

BACKGROUND: Medical plaintext documents contain important facts about patients, but they are rarely available for structured queries. The provision of structured information from natural language texts in addition to the existing structured data can significantly speed up the search for fulfilled inclusion criteria and thus improve the recruitment rate. OBJECTIVES: This work is aimed at supporting clinical trial recruitment with text mining techniques to identify suitable subjects in hospitals. METHOD: Based on the inclusion/exclusion criteria of 5 sample studies and a text corpus consisting of 212 doctor's letters and medical follow-up documentation from a university cancer center, a prototype was developed and technically evaluated using NLP procedures (UIMA) for the extraction of facts from medical free texts. RESULTS: It was found that although the extracted entities are not always correct (precision between 23% and 96%), they provide a decisive indication as to which patient file should be read preferentially. CONCLUSION: The prototype presented here demonstrates the technical feasibility. In order to find available, lucrative phenotypes, an in-depth evaluation is required.

Effects of trastuzumab and afatinib on kinase activity in gastric cancer cell lines.

The molecular mechanism of action of the HER2-targeted antibody trastuzumab is only partially understood, and the direct effects of trastuzumab on the gastric cancer signaling network are unknown. In this study, we compared the molecular effect of trastuzumab and the HER kinase inhibitor afatinib on the receptor tyrosine kinase (RTK) network and the downstream-acting intracellular kinases in gastric cancer cell lines. The molecular effects of trastuzumab and afatinib on the phosphorylation of 49 RTKs and 43 intracellular kinase phosphorylation sites were investigated in three gastric cancer cell lines (NCI-N87, MKN1, and MKN7) using proteome profiling. To evaluate these effects, data were analyzed using mixed models and clustering. Moreover, proliferation assays were performed. Our comprehensive quantitative analysis of kinase activity in gastric cancer cell lines indicates that trastuzumab and afatinib selectively influenced the HER family RTKs. The effects of trastuzumab differed between cell lines, depending on the presence of activated HER2. The effects of trastuzumab monotherapy were not transduced to the intracellular kinase network. Afatinib alone or in combination with trastuzumab influenced HER kinases in all cell lines; that is, the effects of monotherapy and combination therapy were transduced to the intracellular kinase network. These results were confirmed by proliferation analysis. Additionally, the MET-amplified cell line Hs746T was identified as afatinib nonresponder. The dependence of the effect of trastuzumab on the presence of activated HER2 might explain the clinical nonresponse of some patients who are routinely tested for HER2 expression and gene amplification in the clinic but not for HER2 activation. The consistent effects of afatinib on HER RTKs and downstream kinase activation suggest that afatinib might be an effective candidate in the future treatment of patients with gastric cancer irrespective of the presence of activated HER2. However, MET amplification should be taken into account as potential resistance factor.

Regulation of arginase-1 expression in macrophages by a protein kinase A type I and histone deacetylase dependent pathway.

The aim of the current study was to investigate the cAMP-dependent regulation of arginase-1 (ARG1) expression in RAW-macrophages. Basal ARG1 mRNA expression was low and increased upon incubation with the cAMP analogue Br-cAMP. We used selective agonists of protein kinase A type I (PKAI), type II (PKAII) and exchange protein directly activated by cAMP (EPAC) to determine the pathway responsible for ARG1 expression. Activation of PKAI led to a significant up-regulation of ARG1 mRNA expression and arginase enzyme activity. In contrast, neither activation of PKAII nor activation of EPAC affected ARG1 expression. In addition, it has been shown that histone deacetylase (HDAC) activity plays a critical role in cAMP-dependent transcriptional regulation. Incubation with Br-cAMP and the HDAC inhibitor trichostatin A (TSA) led to a concentration-dependent suppression of ARG1 expression. These data indicate that cAMP-dependent activation of ARG1 expression is mediated by PKAI and requires histone deacetylation.

Statins reduce macrophage inflammatory protein-1alpha expression in human activated monocytes.

1. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit a wide variety of anti-atherogenic effects that may be independent of their property to lower plasma cholesterol. 2. In order to systematically investigate these effects at a cellular level, we investigated gene expression in phorbol myristate acetate (PMA)-activated and non-activated human THP-1 monocytes in response to statins using cDNA arrays. 3. Of 588 genes tested, 26 were differentially expressed in the presence of statins. A marked reduction was found for the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). The decrease in MIP-1alpha mRNA expression after incubation with statins was confirmed by quantitative reverse transcription-polymerase chain reaction in THP-1 monocytes and human freshly isolated monocytes. Macrophage inflammatory protein-1alpha protein in THP-1 monocytes was reduced from 377 to 299 and 305 pg/mL by 0.1 micro mol/L simvastatin and 0.01 micro mol/L cerivastatin, respectively. The reduction in MIP-1alpha expression by statins was due, at least in part, to transcriptional inhibition of MIP-1alpha promoter activity. 4. The CC receptor ligand MIP-1alpha is a chemokine that has been implicated in atherosclerotic lesion formation. The present findings suggest that statin-mediated immunomodulation by inhibiting MIP-1alpha could contribute to the beneficial effects of statin therapy independent of lowering plasma cholesterol.

Identification of macrophage arginase I as a new candidate gene of atherosclerosis resistance.

OBJECTIVE: Our laboratory has previously created 2 strains of rabbits with genetically determined high-atherosclerotic response (HAR) and low-atherosclerotic response (LAR). The aim of the present study was to identify new genes of atherosclerosis susceptibility in macrophages from the 2 strains. METHODS AND RESULTS: Suppression subtractive hybridization was used to screen for genes with higher expression in macrophages from LAR rabbits. We identified a cDNA fragment with high homology to human arginase I (AI; 91%) and subsequently cloned the full-length cDNA of the rabbit homologue. Quantitative RT-PCR revealed a significantly higher macrophage AI mRNA expression in LAR rabbits than in HAR rabbits (77428+/-10941 versus 34344+/-4538; P=0.002; copies/10(6) copies beta-actin), which also correlated with a significantly higher arginase enzyme activity. Northern blot analysis led to the identification of a size polymorphism of AI mRNA. This was because of a 413 bp C-repeat insertion in the 3' untranslated region. The shorter transcript variant was predominantly expressed in LAR rabbits and associated with significantly higher AI mRNA expression levels. Transfection experiments indicated decreased mRNA stability of the long AI variant. CONCLUSIONS: High expression of arginase I in macrophages may contribute to atherosclerosis resistance of LAR rabbits, possibly by conferring antiinflammatory effects in the vessel wall.