RNA expression of 6 genes from metastatic mucosal gastric cancer serves as the global prognostic marker for gastric cancer with functional validation.

BACKGROUND: Molecular analysis of advanced tumors can increase tumor heterogeneity and selection bias. We developed a robust prognostic signature for gastric cancer by comparing RNA expression between very rare early gastric cancers invading only mucosal layer (mEGCs) with lymph node metastasis (Npos) and those without metastasis (Nneg). METHODS: Out of 1003 mEGCs, all Npos were matched to Nneg using propensity scores. Machine learning approach comparing Npos and Nneg was used to develop prognostic signature. The function and robustness of prognostic signature was validated using cell lines and external datasets. RESULTS: Extensive machine learning with cross-validation identified the prognostic classifier consisting of four overexpressed genes (HDAC5, NPM1, DTX3, and PPP3R1) and two downregulated genes (MED12 and TP53), and enabled us to develop the risk score predicting poor prognosis. Cell lines engineered to high-risk score showed increased invasion, migration, and resistance to 5-FU and Oxaliplatin but maintained sensitivity to an HDAC inhibitor. Mouse models after tail vein injection of cell lines with high-risk score revealed increased metastasis. In three external cohorts, our risk score was identified as the independent prognostic factor for overall and recurrence-free survival. CONCLUSION: The risk score from the 6-gene classifier can successfully predict the prognosis of gastric cancer.

Aberrant Methylation of Somatostatin Receptor 2 Gene Is Initiated in Aged Gastric Mucosa Infected with Helicobacter pylori and Consequential Gene Silencing Is Associated with Establishment of Inflammatory Microenvironment In Vitro Study.

The loss-of-function variants are thought to be associated with inflammation in the stomach. We here aimed to evaluate the extent and role of methylation at the SSTR2 promoter in inflammation and gastric tumor formation. A whole-genome bisulfite sequencing analysis revealed that the SSTR2 promoter was significantly hypermethylated in gastric tumors, dysplasia, and intestinal metaplasia compared to non-tumor tissues from patients with gastric cancer. Using public data, we confirmed SSTR2 promoter methylation in primary gastric tumors and intestinal metaplasia, and even aged gastric mucosae infected with Helicobacter pylori, suggesting that aberrant methylation is initiated in normal gastric mucosa. The loss-of-function of SSTR2 in SNU638 cell-induced cell proliferation in vitro, while stable transfection of SSTR2 in AGS and MKN74 cells inhibited cell proliferation and tumorigenesis in vitro and in vivo. As revealed by a comparison of target genes differentially expressed in these cells with hallmark molecular signatures, inflammation-related pathways were distinctly induced in SSTR2-KO SNU638 cell. By contrast, inflammation-related pathways were inhibited in AGS and MKN74 cells ectopically expressing SSTR2. Collectively, we propose that SSTR2 silencing upon promoter methylation is initiated in aged gastric mucosae infected with H. pylori and promotes the establishment of an inflammatory microenvironment via the intrinsic pathway. These findings provide novel insights into the initiation of gastric carcinogenesis.

Dysplastic Stem Cell Plasticity Functions as a Driving Force for Neoplastic Transformation of Precancerous Gastric Mucosa.

BACKGROUND & AIMS: Dysplasia carries a high risk of cancer development; however, the cellular mechanisms for dysplasia evolution to cancer are obscure. We have previously identified 2 putative dysplastic stem cell (DSC) populations, CD44v6neg/CD133+/CD166+ (double positive [DP]) and CD44v6+/CD133+/CD166+ (triple positive [TP]), which may contribute to cellular heterogeneity of gastric dysplasia. Here, we investigated functional roles and cell plasticity of noncancerous Trop2+/CD133+/CD166+ DSCs initially developed in the transition from precancerous metaplasia to dysplasia in the stomach. METHODS: Dysplastic organoids established from active Kras-induced mouse stomachs were used for transcriptome analysis, in vitro differentiation, and in vivo tumorigenicity assessments of DSCs. Cell heterogeneity and genetic alterations during clonal evolution of DSCs were examined by next-generation sequencing. Tissue microarrays were used to identify DSCs in human dysplasia. We additionally evaluated the effect of casein kinase 1 alpha (CK1α) regulation on the DSC activities using both mouse and human dysplastic organoids. RESULTS: We identified a high similarity of molecular profiles between DP- and TP-DSCs, but more dynamic activities of DP-DSCs in differentiation and survival for maintaining dysplastic cell lineages through Wnt ligand-independent CK1α/ß-catenin signaling. Xenograft studies demonstrated that the DP-DSCs clonally evolve toward multiple types of gastric adenocarcinomas and promote cancer cell heterogeneity by acquiring additional genetic mutations and recruiting the tumor microenvironment. Last, growth and survival of both mouse and human dysplastic organoids were controlled by targeting CK1α. CONCLUSIONS: These findings indicate that the DSCs are de novo gastric cancer-initiating cells responsible for neoplastic transformation and a promising target for intervention in early induction of gastric cancer.

Comparison of East-Asia and West-Europe cohorts explains disparities in survival outcomes and highlights predictive biomarkers of early gastric cancer aggressiveness.

Surgical resection with lymphadenectomy and perioperative chemotherapy is the universal mainstay for curative treatment of gastric cancer (GC) patients with locoregional disease. However, GC survival remains asymmetric in West- and East-world regions. We hypothesize that this asymmetry derives from differential clinical management. Therefore, we collected chemo-naïve GC patients from Portugal and South Korea to explore specific immunophenotypic profiles related to disease aggressiveness and clinicopathological factors potentially explaining associated overall survival (OS) differences. Clinicopathological and survival data were collected from chemo-naïve surgical cohorts from Portugal (West-Europe cohort [WE-C]; n = 170) and South Korea (East-Asia cohort [EA-C]; n = 367) and correlated with immunohistochemical expression profiles of E-cadherin and CD44v6 obtained from consecutive tissue microarrays sections. Survival analysis revealed a subset of 12.4% of WE-C patients, whose tumors concomitantly express E-cadherin_abnormal and CD44v6_very high, displaying extremely poor OS, even at TNM stages I and II. These WE-C stage-I and -II patients tumors were particularly aggressive compared to all others, invading deeper into the gastric wall (P = .032) and more often permeating the vasculature (P = .018) and nerves (P = .009). A similar immunophenotypic profile was found in 11.9% of EA-C patients, but unrelated to survival. Tumours, from stage-I and -II EA-C patients, that display both biomarkers, also permeated more lymphatic vessels (P = .003), promoting lymph node (LN) metastasis (P = .019), being diagnosed on average 8 years earlier and submitted to more extensive LN dissection than WE-C. Concomitant E-cadherin_abnormal/CD44v6_very-high expression predicts aggressiveness and poor survival of stage-I and -II GC submitted to conservative lymphadenectomy.

Mapping of the perigastric lymphatic network using indocyanine green fluorescence imaging and tissue marking dye in clinically advanced gastric cancer.

BACKGROUND: Using indocyanine green (ICG) fluorescence imaging and tissue marking dyes (TMDs), perigastric lymphatic mapping and their pathological correlation were examined to see whether ICG staining covers all metastatic lymph nodes (LNs) in advanced gastric cancer (AGC). METHODS: Patients with AGC who underwent open distal or total gastrectomy were enrolled. ICG was serially injected intraoperatively into the subserosa along the greater and lesser curvatures. Stomach specimens were examined under a near-infrared camera. ICG-stained LNs were named, excised, and tattooed with different colored TMDs to retrace the exact location after pathological examinations. RESULTS: A total of 687 LNs and 69 LN stations were examined from 11 patients. The map of the perigastric lymphatic network showing the topography of ICG-stained and ICG-unstained LNs, including metastatic information, was successfully reconstructed. The average number of ICG-stained and ICG-unstained LNs were 23.6 ±â€¯12.3 (37.8%) and 38.8 ±â€¯17.1 (62.2%), respectively. LN metastases were present in 28 LN stations of 8 patients. Of 8 cases with LN metastases, 40% (11.1-75% per case) of metastatic LNs were stained by ICG. Of 28 metastatic LN stations, 21 (75.0%) were covered by ICG, and actual metastatic LNs were stained in 16 LN stations (57.1%). In 4/8 cases (50%), all metastatic LN stations showed ICG signals. CONCLUSIONS: ICG fluorescence imaging and TMD are useful tools for visualizing the perigastric lymphatic network and retracing the exact location of ICG-stained LNs in AGC. However, ICG imaging is still not recommended for selective LN dissection in AGC because of the limited staining of perigastric LNs.

Comprehensive Molecular Characterization of Adenocarcinoma of the Gastroesophageal Junction Between Esophageal and Gastric Adenocarcinomas.

OBJECTIVE: To investigate the molecular characteristics of AGEJ compared with EAC and gastric adenocarcinoma. SUMMARY OF BACKGROUND DATA: Classification of AGEJ based on differential molecular characteristics between EAC and gastric adenocarcinoma has been long-standing controversy but rarely conducted due to anatomical ambiguity and epidemiologic difference. METHODS: The molecular classification model with Bayesian compound covariate predictor was developed based on differential mRNA expression of EAC (N = 78) and GCFB (N = 102) from the Cancer Genome Atlas (TCGA) cohort. AGEJ/cardia (N = 48) in TCGA cohort and AGEJ/upper third GC (N = 46 pairs) in Seoul National University cohort were classified into the EAC-like or GCFB-like groups whose genomic, transcriptomic, and proteomic characteristics were compared. RESULTS: AGEJ in both cohorts was similarly classified as EAC-like (31.2%) or GCFB-like (68.8%) based on the 400-gene classifier. The GCFB-like group showed significantly activated phosphoinositide 3-kinase-AKT signaling with decreased expression of ERBB2. The EAC-like group presented significantly different alternative splicing including the skipped exon of RPS24, a significantly higher copy number amplification including ERBB2 amplification, and increased protein expression of ERBB2 and EGFR compared with GCFB-like group. High-throughput 3D drug test using independent cell lines revealed that the EAC-like group showed a significantly better response to lapatinib than the GCFB-like group (P = 0.015). CONCLUSIONS: AGEJ was the combined entity of the EAC-like and GCFB-like groups with consistently different molecular characteristics in both Seoul National University and TCGA cohorts. The EAC-like group with a high Bayesian compound covariate predictor score could be effectively targeted by dual inhibition of ERBB2 and EGFR.

CD44v6 High Membranous Expression Is a Predictive Marker of Therapy Response in Gastric Cancer Patients.

In gastric cancer (GC), biomarkers that define prognosis and predict treatment response remain scarce. We hypothesized that the extent of CD44v6 membranous tumor expression could predict prognosis and therapy response in GC patients. Two GC surgical cohorts, from Portugal and South Korea (n = 964), were characterized for the extension of CD44v6 membranous immuno-expression, clinicopathological features, patient survival, and therapy response. The value of CD44v6 expression in predicting response to treatment and its impact on prognosis was determined. High CD44v6 expression was associated with invasive features (perineural invasion and depth of invasion) in both cohorts and with worse survival in the Portuguese GC cohort (HR 1.461; 95% confidence interval 1.002-2.131). Patients with high CD44v6 tumor expression benefited from conventional chemotherapy in addition to surgery (p < 0.05), particularly those with heterogeneous CD44v6-positive and -negative populations (CD44v6_3+) (p < 0.007 and p < 0.009). Our study is the first to identify CD44v6 high membranous expression as a potential predictive marker of response to conventional treatment, but it does not clarify CD44v6 prognostic value in GC. Importantly, our data support selection of GC patients with high CD44v6-expressing tumors for conventional chemotherapy in addition to surgery. These findings will allow better stratification of GC patients for treatment, potentially improving their overall survival.

Predictive biomarkers for 5-fluorouracil and oxaliplatin-based chemotherapy in gastric cancers via profiling of patient-derived xenografts.

Gastric cancer (GC) is commonly treated by chemotherapy using 5-fluorouracil (5-FU) derivatives and platinum combination, but predictive biomarker remains lacking. We develop patient-derived xenografts (PDXs) from 31 GC patients and treat with a combination of 5-FU and oxaliplatin, to determine biomarkers associated with responsiveness. When the PDXs are defined as either responders or non-responders according to tumor volume change after treatment, the responsiveness of PDXs is significantly consistent with the respective clinical outcomes of the patients. An integrative genomic and transcriptomic analysis of PDXs reveals that pathways associated with cell-to-cell and cell-to-extracellular matrix interactions enriched among the non-responders in both cancer cells and the tumor microenvironment (TME). We develop a 30-gene prediction model to determine the responsiveness to 5-FU and oxaliplatin-based chemotherapy and confirm the significant poor survival outcomes among cases classified as non-responder-like in three independent GC cohorts. Our study may inform clinical decision-making when designing treatment strategies.

Assessment of copy number in protooncogenes are predictive of poor survival in advanced gastric cancer.

The copy number (CN) gain of protooncogenes is a frequent finding in gastric carcinoma (GC), but its prognostic implication remains elusive. The study aimed to characterize the clinicopathological features, including prognosis, of GCs with copy number gains in multiple protooncogenes. Three hundred thirty-three patients with advanced GC were analyzed for their gene ratios in EGFR, GATA6, IGF2, and SETDB1 using droplet dPCR (ddPCR) for an accurate assessment of CN changes in target genes. The number of GC patients with 3 or more genes with CN gain was 16 (4.8%). Compared with the GCs with 2 or less genes with CN gain, the GCs with 3 or more CN gains displayed more frequent venous invasion, a lower density of tumor-infiltrating lymphocytes, and lower methylation levels of L1 or SAT-alpha. Microsatellite instability-high tumors or Epstein-Barr virus-positive tumors were not found in the GCs with 3 or more genes with CN gain. Patients of this groups also showed the worst clinical outcomes for both overall survival and recurrence-free survival, which was persistent in the multivariate survival analyses. Our findings suggest that the ddPCR-based detection of multiple CN gain of protooncogenes might help to identify a subset of patients with poor prognosis.

Programmed Death Ligand 1-Expressing Classical Dendritic Cells MitigateHelicobacter-Induced Gastritis.

BACKGROUND & AIMS: Helicobacter pylori has been reported to modulate local immune responses to colonize persistently in gastric mucosa. Although the induced expression of programmed cell death ligand 1 (PD-L1) has been suggested as an immune modulatory mechanism for persistent infection of H pylori, the main immune cells expressing PD-L1 and their functions in Helicobacter-induced gastritis still remain to be elucidated. METHODS: The blockades of PD-L1 with antibody or PD-L1-deficient bone marrow transplantation were performed in Helicobacter-infected mice. The main immune cells expressing PD-L1 in Helicobacter-infected stomach were determined by flow cytometry and immunofluorescence staining. Helicobacter felis or H pylori-infected dendritic cell (DC)-deficient mouse models including Flt3-/-, Zbtb46-diphtheria toxin receptor, and BDCA2-diphtheria toxin receptor mice were analyzed for pathologic changes and colonization levels. Finally, the location of PD-L1-expressing DCs and the correlation with H pylori infection were analyzed in human gastric tissues using multiplexed immunohistochemistry. RESULTS: Genetic or antibody-mediated blockade of PD-L1 aggravated Helicobacter-induced gastritis with mucosal metaplasia. Gastric classical DCs expressed considerably higher levels of PD-L1 than other immune cells and co-localized with T cells in gastritis lesions from Helicobacter-infected mice and human beings. H felis- or H pylori-infected Flt3-/- or classical DC-depleted mice showed aggravated gastritis with severe T-cell and neutrophil accumulation with low bacterial loads compared with that in control mice. Finally, PD-L1-expressing DCs were co-localized with T cells and showed a positive correlation with H pylori infection in human subjects. CONCLUSIONS: The PD-1/PD-L1 pathway may be responsible for the immune modulatory function of gastric DCs that protects the gastric mucosa from Helicobacter-induced inflammation, but allows persistent Helicobacter colonization.

Can endoscopic ultrasonography (EUS) improve the accuracy of clinical T staging by computed tomography (CT) for gastric cancer?

INTRODUCTION: With the introduction of new therapeutic options for gastric cancer treatment, more precise preoperative staging of gastric cancer is needed. The purpose of this study was to evaluate the role of endoscopic ultrasonography (EUS) for improving the accuracy of clinical T staging by computed tomography (CT) for gastric cancer. MATERIALS AND METHODS: A total of 2636 patients underwent stomach protocol CT (S-CT) and EUS, followed by gastrectomy for primary gastric adenocarcinoma between September 2012 and February 2018 at Seoul National University Hospital. The results of preoperative S-CT and EUS were compared to the postoperative pathologic staging. RESULTS: The overall accuracy of S-CT and EUS for T staging were 69.4% and 70.4%, respectively. When T staging was divided into T1-2 and T3-4 for clinically advanced gastric cancer (AGC), the positive predictive value for T3-4 using S-CT, EUS, and a combination of both modalities was 73.8%, 79.3%, and 85.6%, respectively. In 114 cases of indeterminate lesions between cT1 and cT2 by S-CT, EUS had a better prediction rate than the final decision based on endoscopy or the agreement between the two experts (Match rate: EUS vs. final decision, 69.3% vs. 58.8%). CONCLUSION: EUS can be a complementary diagnostic tool to clinical T staging of gastric cancer by CT for selecting T3-4 lesion.

RUNX3 methylation drives hypoxia-induced cell proliferation and antiapoptosis in early tumorigenesis.

Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFß and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.

Immunoscore is a strong predictor of survival in the prognosis of stage II/III gastric cancer patients following 5-FU-based adjuvant chemotherapy.

The prognostic impact of Immunoscore (IS) in gastric cancer (GC) patients treated with adjuvant chemotherapy remains unelucidated. We evaluated the CD3 + , CD8 + , and Foxp3 + T-lymphocyte densities in tumor centers and invasive margin regions of 389 patients with surgically resected stage II/III GC who received 5-FU-based adjuvant chemotherapy and investigated the impact of IS on survival. In univariate analysis, high CD3 + , CD8 + , and Foxp3 + T-lymphocyte densities in the invasive margin were correlated with better prognosis (all P < 0.05). Patients with high IS had significantly longer disease-free survival (DFS; P < 0.001) and overall survival (OS; P < 0.001). In multivariate analysis, IS demonstrated a powerful prognostic impact on patient outcome [DFS, hazard ratio (HR) = 0.465; 95% confidence interval (CI), 0.306-0.707, P < 0.001; OS, HR = 0.478; 95% CI, 0.308-0.743, P = 0.001]. Additionally, although all EBV-positive cases had high IS, IS was similar in both microsatellite instability (MSI)-high and microsatellite stable (MSS)/MSI-low groups (83.3% and 80.5%, respectively). Subgroup analysis according to MSI status revealed that high IS patients had significant DFS and OS benefits in both MSS/MSI-low (DFS, HR = 0.527, 95% CI, 0.341-0.816, P = 0.004; OS, HR = 0.528, 95% CI, 0.334-0.837, P = 0.007) and MSI-high (DFS, HR = 0.166, 95% CI, 0.033-0.826, P = 0.028; OS, HR = 0.177, 95% CI, 0.036-0.883, P = 0.035) groups. Thus, the assessment of immune cell infiltration based on IS may provide a strong indicator of survival in stage II/III GC patients with curative resection following 5-FU-based adjuvant chemotherapy.

Comprehensive genetic features of gastric mixed adenoneuroendocrine carcinomas and pure neuroendocrine carcinomas.

We aimed to determine the pathogenesis of gastric mixed adenoneuroendocrine carcinoma (MANEC) and pure neuroendocrine carcinoma (NEC), which is largely unknown. Targeted DNA sequencing was performed on 34 tumor samples from 21 patients - 13 adenocarcinoma (ADC)/NEC components from MANECs and eight pure NECs - and 21 matched non-neoplastic gastric tissues. Mutational profiles of MANECs/NECs were compared with those of other tumors using public databases. The majority (64.1%; 59/92) of mutations in MANEC were shared by both ADC and NEC components. TP53 was the most commonly mutated gene in MANEC (69.2%, 9/13) and pure NEC (87.5%, 8/9). All TP53 mutations in MANEC were pathogenic mutations and were shared by both ADC and NEC components. A subset of TP53WT MANECs had a microsatellite-unstable phenotype or amplifications in various oncogenes including ERBB2 and NMYC, and the only TP53WT pure NEC harbored MYC amplification. Compared to NEC in other organs, NECs arising from the stomach had unique features including less frequent RB1 mutations. Differentially altered genes of MANEC ADC components were significantly associated with receptor tyrosine kinase signaling pathways, while differentially altered genes of MANEC NEC components were significantly associated with the NOTCH signaling pathway. Our data provide evidence suggesting a possible clonal origin of ADC and NEC components of MANEC, and we found that gastric MANECs and pure NECs are distinct entities with unique mutational profiles and underlying protein networks. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expression of human leukocyte antigen class I and ß2-microglobulin in colorectal cancer and its prognostic impact.

Downregulation of human leukocyte antigen (HLA) class I has been postulated to be a mechanism of adaptive immune escape in various tumors, especially microsatellite instability-high (MSI-H) colorectal cancer (CRC). In this study, we aimed to investigate HLA class I and ß2-microglobulin (ß2M) expression in MSI-H and microsatellite-stable (MSS) CRCs and determine its prognostic impact. The representative areas from the tumor center (TC) and tumor periphery (TP) from 300 CRCs, including 161 MSI-H and 139 MSS cases, were selected to construct a tissue microarray. Immunohistochemistry (IHC) for HLA A/B/C, ß2M, CD3, and CD8 was performed. Reduced HLA A/B/C expression was detected in 113 (70.2%) MSI-H and 54 (38.8%) MSS cases, while reduced ß2M expression was observed in 69 (42.9%) MSI-H and 17 (12.2%) MSS cases. Although reduced ß2M expression was associated with higher pathological tumor (pT) stage in MSI-H CRC with borderline significance, no association was found between HLA A/B/C and ß2M expression and survival. Interestingly, reduced HLA A/B/C expression in MSS was associated with higher stage, and reduced HLA A/B/C and ß2M expression was an independent prognostic factor in multivariate analysis. In conclusion, reduced HLA A/B/C and ß2M expression was frequently observed in immunotherapy-naive MSI-H CRC, suggesting the possibility of primary resistance to immune checkpoint inhibitor. Interestingly, downregulation of HLA A/B/C and ß2M was associated with poor prognosis in MSS cancers. Overall, IHC for HLA A/B/C and ß2M might be a feasible predictive or prognostic tool in CRC.

SMOC2, an intestinal stem cell marker, is an independent prognostic marker associated with better survival in colorectal cancers.

We aimed to investigate the expression profile of SPARC-related modular calcium-binding protein 2 (SMOC2) during colorectal cancer (CRC) progression and assess its prognostic impact in CRC patients. In our study, we showed that SMOC2 transcript level was higher in CRC samples than in normal mucosa (P = 0.017); this level was not associated with candidate cancer stem cell markers (CD44, CD166, CD133, and CD24) or intestinal stem cell markers (LGR5, ASCL2, and EPHB2) except for OLFM4 (P = 0.04). Immunohistochemical analysis showed that SMOC2-positive cells were confined to the crypt bases in the normal intestinal mucosa, hyperplastic polyps, and sessile serrated adenomas, whereas traditional serrated adenomas and conventional adenomas exhibited focal or diffuse distribution patterns. In total, 28% of 591 CRCs were positive for SMOC2, but SMOC2 positivity had negative correlations with lymphatic invasion (P = 0.002), venous invasion (P = 0.002), and tumor stage (P < 0.001). However, a positive association with nuclear ß-catenin expression was seen. Furthermore, while upregulated SMOC2 expression was maintained during the adenoma-carcinoma transition, it decreased in cancer cells at the invasive front but did not decline further during lymph node metastasis. SMOC2 positivity showed no correlations with molecular abnormalities, including microsatellite instability, CpG island methylator phenotype, and mutations of KRAS and BRAF. In addition, we showed comprehensively that SMOC2 positivity is an independent prognostic marker for better clinical outcomes in a large cohort of CRC patients (P = 0.006). In vitro studies also demonstrated that induced SMOC2 expression in DLD1 cells exerts a suppressive role in tumor growth as well as in migration, colony, and sphere formation abilities. Taken together, our results suggest SMOC2 as a candidate tumor suppressor in CRC progression.

Prognostic Impact of Frozen Section Investigation and Extent of Proximal Safety Margin in Gastric Cancer Resection.

BACKGROUND AND AIMS: Guidelines propose different extents of macroscopic proximal margin for gastric cancer and frozen margin investigation in selected cases, but data is lacking. This study was to evaluate the necessary extent of macroscopic proximal margin, accuracy of frozen margin investigation, and prognostic impact of tumor-free proximal margin length in pT2-pT4 gastric cancer. STUDY DESIGN: Proximal and distal frozen margins were routinely investigated intraoperatively in all pT2-pT4 gastric cancers resected between 2011 and 2017. Macroscopic and microscopic proximal margin lengths were correlated. For R0-resections, survival analysis was performed for distal gastrectomy (DG) with microscopic proximal margin length ≤3 cm versus >3 cm. RESULTS: Overall, 1484 patients were included. Microscopic proximal margin lengths were macroscopically more often misestimated in diffuse histology (P = 0.0004), but extent of underestimation in centimeter was similar to intestinal and mixed/undetermined type (P = 0.134). Fifteen cases (1.0%) resulted in R1-resection, 10 at distal, and 5 at proximal margin but none with macroscopic proximal margin ≥3 cm and negative frozen section. Overall agreement of frozen margin and final pathology was 2951/2968 (99.4%). Proximal margin length in DG did not correlate with survival or recurrence in R0-resected patients. DISCUSSION: Diffuse histology is at higher risk for underestimation of proximal margin length, but extent of underestimation is similar in other Laurén subtypes. If ≥3 cm macroscopic proximal margin length is applied with intraoperative frozen margin confirmation, R1-resection can be avoided. CONCLUSION: In pT2-T4a gastric cancer, proximal margin of ≥3 cm plus frozen margin confirmation provides high oncological safety. In DG patients with R0-resection, proximal margin length does not correlate with survival or recurrence.

Increased HOXC6 mRNA expression is a novel biomarker of gastric cancer.

In this study, we aimed to investigate the molecular biomarkers that are pivotal for the development and progression of gastric cancer (GC). We analyzed clinical specimens using RNA sequencing to identify the target genes. We found that the expression of HOXC6 mRNA was upregulated with the progression of cancer, which was validated by quantitative real time PCR and RNA in-situ hybridization. To compare the protein expression of HOXC6, we evaluated GC and normal gastric tissue samples using western blot analysis and immunohistochemistry. We detected significantly higher levels of HOXC6 in the GC tissues than in the normal controls at both mRNA and protein levels. The expression levels of HOXC6 mRNA in patients with advanced gastric cancer (AGC) were significantly higher than those in patients with early gastric cancer (EGC). Kaplan-Meier curves showed that high expression of HOXC6 mRNA is significantly associated with poor clinical prognosis. Our findings suggest that HOXC6 mRNA may be a novel biomarker and can be potentially valuable in predicting the prognosis of GC patients. Especially, HOXC6 mRNA in-situ hybridization may be a diagnostic tool for predicting prognosis of individual GC patients.

Prediction of TP53 mutations by p53 immunohistochemistry and their prognostic significance in gastric cancer.

BACKGROUND: Recently, molecular classifications of gastric cancer (GC) have been proposed that include TP53 mutations and their functional activity. We aimed to demonstrate the correlation between p53 immunohistochemistry (IHC) and TP53 mutations as well as their clinicopathological significance in GC. METHODS: Deep targeted sequencing was performed using surgical or biopsy specimens from 120 patients with GC. IHC for p53 was performed and interpreted as strong, weak, or negative expression. In 18 cases (15.0%) with discrepant TP53 mutation and p53 IHC results, p53 IHC was repeated. RESULTS: Strong expression of p53 was associated with TP53 missense mutations, negative expression with other types of mutations, and weak expression with wild-type TP53 (p<.001). The sensitivity for each category was 90.9%, 79.0%, and 80.9%, and the specificity was 95.4%, 88.1%, and 92.3%, respectively. The TNM stage at initial diagnosis exhibited a significant correlation with both TP53 mutation type (p=.004) and p53 expression status (p=.029). The Kaplan-Meier survival analysis for 109 stage II and III GC cases showed that patients with TP53 missense mutations had worse overall survival than those in the wild-type and other mutation groups (p=.028). Strong expression of p53 was also associated with worse overall survival in comparison to negative and weak expression (p=.035). CONCLUSIONS: Results of IHC of the p53 protein may be used as a simple surrogate marker of TP53 mutations. However, negative expression of p53 and other types of mutations of TP53 should be carefully interpreted because of its lower sensitivity and different prognostic implications.

Author Correction: Evaluation of molecular subtypes and clonal selection during establishment of patient-derived tumor xenografts from gastric adenocarcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.