CD10 and Das1: a biomarker study using immunohistochemistry to subtype gastric intestinal metaplasia.

BACKGROUND: Intestinal metaplasia (IM) is considered a key pivot point in the Correa model of gastric cancer (GC). It is histologically subtyped into the complete and incomplete subtypes, the latter being associated with a greater risk of progression. However, the clinical utility of IM subtyping remains unclear, partially due to the absence of reliable defining biomarkers. METHODS: Based on gene expression data and existing literature, we selected CD10 and Das1 as candidate biomarkers to distinguish complete and incomplete IM glands in tissues from patients without GC (IM-GC) and patients with GC (IM + GC). Immunohistochemical staining of individually subtyped IM glands was scored after blinding by two researchers using tissue belonging to both IM-GC and IM + GC patients. Whole tissue Das1 staining was further assessed using digital image quantification (cellSens Dimension, Olympus). RESULTS: Across both cohorts CD10 stained the IM brush border and was shown to have a high sensitivity (87.5% and 94.9% in IM-GC and IM + GC patients respectively) and specificity (100.0% and 96.7% respectively) with an overall AUROC of 0.944 for complete IM glands. By contrast Das1 stained mainly goblet cells and the apical membrane of epithelial cells, mostly of incomplete IM glands with a low sensitivity (28.6% and 29.3% in IM-GC and IM + GC patients respectively) but high specificity (98.3% and 85.1% respectively) and an overall AUROC of 0.603 for incomplete IM glands. A combined logistic regression model showed a significant increase in AUROC for detecting complete IM glands (0.955 vs 0.970). Whole tissue digital quantification of Das1 staining showed a significant association with incomplete IM compared to complete IM, both in IM-GC and in IM + GC patients (p = 0.016 and p = 0.009 respectively, Mann-Whitney test and unpaired t test used). Additionally, complete IM in IM + GC patients exhibited significantly more Das1 staining than in IM-GC patients (p = 0.019, Mann-Whitney test). CONCLUSIONS: These findings suggest that CD10 is an outstanding biomarker for complete IM and Das1 may be useful as a secondary biomarker for IM glands at greater risk of progression irrespective of IM subtype. Overall, the clinical use of these biomarkers could lead to improved patient stratification and targeted surveillance.

Molecular and genomic characterisation of a panel of human anal cancer cell lines.

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

SFRP4 drives invasion in gastric cancer and is an early predictor of recurrence.

OBJECTIVE: Gastric cancer patients generally have a poor outcome, particularly those with advanced-stage disease which is defined by the increased invasion of cancer locally and is associated with higher metastatic potential. This study aimed to identify genes that were functional in the most fundamental hallmark of cancer, namely invasion. We then wanted to assess their value as biomarkers of gastric cancer progression and recurrence. DESIGN: Data from a cohort of patients profiled on cDNA expression arrays was interrogated using K-means analysis. This genomic approach classified the data based on patterns of gene expression allowing the identification of the genes most correlated with the invasion of GC. We evaluated the functional role of a key protein from this analysis in invasion and as a biomarker of recurrence after curative resection. RESULTS: Expression of secreted frizzled-related protein 4 (SFRP4) was identified as directly proportional to gastric cancer invasion. This finding was validated in multiple, independent datasets and its functional role in invasion was also confirmed using invasion assays. A change in serum levels of SFRP4 after curative resection, when coupled with AJCC stage, can accurately predict the risk of disease recurrence after curative therapy in an assay we termed PredictR. CONCLUSIONS: This simple ELISA-based assay can help predict recurrence of disease after curative gastric cancer surgery irrespective of adjuvant therapy. The results require further evaluation in a prospective trial but would help in the rational prescription of cancer therapies and surveillance to prevent under or over treatment of patients after curative resection.

High-dimensional analyses reveal a distinct role of T-cell subsets in the immune microenvironment of gastric cancer.

OBJECTIVES: To facilitate disease prognosis and improve precise immunotherapy of gastric cancer (GC) patients, a comprehensive study integrating immune cellular and molecular analyses on tumor tissues and peripheral blood was performed. METHODS: The association of GC patients' outcomes and the immune context of their tumors was explored using multiplex immunohistochemistry (mIHC) and transcriptome profiling. Potential immune dysfunction mechanism/s in the tumors on the systemic level was further examined using mass cytometry (CyTOF) in complementary peripheral blood from selected patients. GC cohorts with mIHC and gene expression profiling data were also used as validation cohorts. RESULTS: Increased CD4+FOXP3+ T-cell density in the GC tumor correlated with prolonged survival. Interestingly, CD4+FOXP3+ T cells had a close interaction with CD8+ T cells rather than tumor cells. High densities of CD4+FOXP3+ T cells and CD8+ T cells (High-High) independently predicted prolonged patient survival. Furthermore, the interferon-gamma (IFN-γ) gene signature and PDL1 expression were up-regulated in this group. Importantly, a subgroup of genomically stable (GS) tumors and tumors with chromosomal instability (CIN) within this High-High group also had excellent survival. The High-High GS/CIN tumors were coupled with increased frequencies of Tbet+CD4+ T cells and central memory CD4+ T cells in the peripheral blood. CONCLUSION: These novel findings identify the combination of CD8+ T cells and FOXP3+CD4+ T cells as a significant prognostic marker for GC patients, which also could potentially be targeted and applied in the combination therapy with immune checkpoint blockades in precision medicine.

Macrophage spatial heterogeneity in gastric cancer defined by multiplex immunohistochemistry.

Tumor-associated macrophages (TAMs), one of the most abundant immune components in gastric cancer (GC), are difficult to characterize due to their heterogeneity. Multiple approaches have been used to elucidate the issue, however, due to the tissue-destructive nature of most of these methods, the spatial distribution of TAMs in situ remains unclear. Here we probe the relationship between tumor context and TAM heterogeneity by multiplex immunohistochemistry of 56 human GC cases. Using distinct expression marker profiles on TAMs, we report seven predominant populations distributed between tumor and non-tumor tissue. TAM population-associated gene signatures reflect their heterogeneity and polarization in situ. Increased density of CD163+ (CD206-) TAMs with concurrent high CD68 expression is associated with upregulated immune-signaling and improved patient survival by univariate, but not multivariate analysis. CD68-only and CD206+ TAMs are correlated with high PDL1 expression.

An orthotopic mouse model of gastric cancer invasion and metastasis.

Gastric cancer is a leading cause of cancer death worldwide, with advanced stage being correlated to the level of tumour invasion and metastasis. Current research is heavily focused on the identification and development of efficacious therapeutics targeting these fundamental hallmarks of cancer, however there are currently no animal models that mimic the invasive phenotypes observed in humans. To address this we have developed an orthotopic mouse model whereby gastric cancer cell lines are tagged with luciferase and injected into the subserosal layer of the stomach. This allows for the monitoring of primary tumour growth and metastasis in real-time as well as quantitation of the degree of tumour invasion through the stomach wall by immunohistochemistry. We have three models based on the degree of invasion and metastasis that are cell line specific: The AGS cells develop into invasive tumours by 4-weeks with no evidence of metastases, MKN45 cells are moderately metastatic with minimal invasion till week 2 and MKN28 cells are highly metastatic and fully invasive by week 1. These models have utility as a tool for testing the efficacy of anti-tumour, anti-invasive and anti-metastatic therapies in the setting of gastric cancer, which currently has poor treatment options.

Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Role of p53 in the progression of gastric cancer.

Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer (GC) but is poorly described in terms of molecular changes. Here, we explored the role of TP53, a commonly mutated gene in GC, to determine if p53 protein expression and/or the presence of somatic mutations in TP53 can be used as a predictive marker for patients at risk of progressing to GC from IM. Immunohistochemistry and high resolution melting were used to determine p53 protein expression and TP53 mutation status respectively in normal gastric mucosa, IM without concurrent GC (IM-GC), IM with concurrent GC (IM+GC) and GC. This comparative study revealed an incremental increase in p53 expression levels with progression of disease from normal mucosa, via an IM intermediate to GC. TP53 mutations however, were not detected in IM but occurred frequently in GC. Further, we identified increased protein expression of Mdm2/x, both powerful regulators of p53, in 100% of the IM+GC cohort with these samples also exhibiting high levels of wild-type p53 protein. Our data suggests that TP53 mutations occur late in gastric carcinogenesis contributing to the final transition to cancer. We also demonstrated involvement of Mdmx in GC.

Influence of natural and synthetic histone deacetylase inhibitors on chromatin.

SIGNIFICANCE: Histone deacetylase inhibitors (HDACIs) have emerged as a new class of anticancer therapeutics. The hydroxamic acid, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™), and the cyclic peptide, depsipeptide (Romidepsin, Istodax™), were approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma in 2006 and 2009, respectively. At least 15 HDACIs are currently undergoing clinical trials either alone or in combination with other therapeutic modalities for the treatment of numerous hematological and solid malignancies. RECENT ADVANCES: The potential utility of HDACIs has been extended to nononcologic applications, including autoimmune disorders, inflammation, diseases of the central nervous system, and malaria. CRITICAL ISSUES: Given the promise of HDACIs, there is growing interest in the potential of dietary compounds that possess HDAC inhibition activity. This review is focused on the identification of and recent findings with HDACIs from dietary, medicinal plant, and microbial sources. We discuss the mechanisms of action and clinical potential of natural HDACIs. FUTURE DIRECTIONS: Apart from identification of further HDACI compounds from dietary sources, further research will be aimed at understanding the effects on gene regulation on lifetime exposure to these compounds. Another important issue that requires clarification.