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.

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.

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.

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.

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.

Differential prognostic impact of CD8+ T cells based on human leucocyte antigen I and PD-L1 expression in microsatellite-unstable gastric cancer.

BACKGROUND: The aim of the study was to determine the human leucocyte antigen class-I (HLA-I), programmed death-ligand 1 (PD-L1) expression and tumour-infiltrating lymphocytes (TILs) of microsatellite instability-high gastric cancer. METHODS: The HLA-I expression type was determined by immunohistochemistry of HLA-A, HLA-B, HLA-C and ß2-microglobulin in the centre of the tumour (CT) and in the invasive margin (IM) of samples from 293 patients (total loss vs. preserved type). PD-L1 expression and TIL density was examined immunohistochemically. HLA-I genotyping was also performed. RESULTS: The expression loss of the HLA-I molecules was significantly associated with low TIL density. According to survival analyses, the HLA-I expression type and PD-L1 positivity were not independent prognostic factors. The TIL density had no prognostic implication when survival analysis was performed for the whole patient group; however, high CD8+ TIL infiltration was significantly associated with good prognosis in only HLA-I-preserved-type/PD-L1-positive group (p = 0.034). The homozygosity of the HLA-I allele was more frequently observed in the total loss type group. CONCLUSIONS: We confirmed differential prognostic implication of CD8+ TILs according to the HLA-I and PD-L1 expression. Determination of the HLA-I expression could be helpful to select patients who would benefit from anti-PD-1/PD-L1 therapy.

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.

Alterations in the Rho pathway contribute to Epstein-Barr virus-induced lymphomagenesis in immunosuppressed environments.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas (EBV+-DLBLs) tend to occur in immunocompromised patients, such as the elderly or those undergoing solid organ transplantation. The pathogenesis and genomic characteristics of EBV+-DLBLs are largely unknown because of the limited availability of human samples and lack of experimental animal models. We observed the development of 25 human EBV+-DLBLs during the engraftment of gastric adenocarcinomas into immunodeficient mice. An integrated genomic analysis of the human-derived EBV+-DLBLs revealed enrichment of mutations in Rho pathway genes, including RHPN2, and Rho pathway transcriptomic activation. Targeting the Rho pathway using a Rho-associated protein kinase (ROCK) inhibitor, fasudil, markedly decreased tumor growth in EBV+-DLBL patient-derived xenograft (PDX) models. Thus, alterations in the Rho pathway appear to contribute to EBV-induced lymphomagenesis in immunosuppressed environments.

Combination of L1 methylation and tumor-infiltrating lymphocytes as prognostic marker in advanced gastric cancer.

BACKGROUND: High density of tumor-infiltrating lymphocyte (TIL) is known to be associated with prolonged survival time, whereas tumoral-L1 hypomethylation has been associated with shortened survival time in patients with gastric cancer (GC). Since L1-methylation level is high in lymphocytes, higher density of TIL could lead to higher measurement of L1-methylation level in cancer tissues which contain cancer cells as well as non-neoplastic cells, including TIL. Putative interaction of TIL in the relationship between L1-methylation level and survival led us to explore combinatory statuses of tumoral-L1-methylation level and TIL density as a prognostic marker in GC. METHODS: TIL and tumoral-L1-methylation level were measured in advanced GC samples (n = 491), using CD3 immunohistochemistry and pyrosequencing-methylation analysis, respectively. TIL density was measured in tumor center and invasive front areas. RESULTS: TIL density correlated with tumoral-L1-methylation level but the relationship was weak. Combinatory statuses of L1-methylation level and CD3 TIL density were found to be statistically significant in survival analysis. Multivariate analysis revealed that the relationship between combinatory statuses and survival was independent. Prognostic value of the combinatory statuses at invasive front was significant in an independent set. CONCLUSIONS: Our findings indicate that tumoral-L1-methylation level is correlated with TIL density and that combinatory statuses might help to find a subset of GCs with worse clinical outcome in GCs with low-L1-methylation status or a subset of GCs with better clinical outcome in GCs with high-L1-methylation status.

Identification of a molecular signature of prognostic subtypes in diffuse-type gastric cancer.

BACKGROUND: Although recent advances in high-throughput technology have provided many insights into gastric cancer (GC), few reliable biomarkers for diffuse-type GC have been identified. Here, we aim to identify a prognostic and predictive signature of diffuse-type GC heterogeneity. METHODS: We analyzed RNA-seq-based transcriptome data to identify a molecular signature in 150 gastric tissue samples including 107 diffuse-type GCs. The predictive value of the signature was verified using other diffuse-type GC samples in three independent cohorts (n = 466). Log-rank and Cox regression analyses were used to estimate the association between the signature and prognosis. The signature was also characterized by somatic variant analyses and tissue microarray analysis between diffuse-type GC subtypes. RESULTS: Transcriptomic profiling of RNA-seq data identified a signature which revealed distinct subtypes of diffuse-type GC: the intestinal-like (INT) and core diffuse-type (COD) subtypes. The signature showed high predictability and independent clinical utility in diffuse-type GC prognosis in other patient cohorts (HR 2.058, 95% CI 1.53-2.77, P = 1.76 × 10-6). Integrative mutational and gene expression analyses demonstrated that the COD subtype was responsive to chemotherapy, whereas the INT subtype was responsive to immunotherapy with an immune checkpoint inhibitor (ICI). Tissue microarray analysis showed the practical utility of IGF1 and NXPE2 for predicting diffuse-type GC heterogeneity. CONCLUSIONS: We present a molecular signature that can identify diffuse-type GC patients who display different clinical behaviors as well as responses to chemotherapy or ICI treatment.

microRNA-30a arbitrates intestinal-type early gastric carcinogenesis by directly targeting ITGA2.

BACKGROUND: Spasmolytic polypeptide-expressing metaplasia (SPEM) is considered a precursor lesion of intestinal metaplasia and intestinal-type gastric cancer (GC), but little is known about microRNA alterations during metaplasia and GC developments. Here, we investigate miR-30a expression in gastric lesions and identify its novel target gene which is associated with the intestinal-type GC. METHODS: We conducted in situ hybridization and qRT-PCR to determine miR-30a expression in gastric tissues. miR-30a functions were determined through induction or inhibition of miR-30a in GC cell lines. A gene microarray was utilized to confirm miR-30a target genes in GC, and siRNA-mediated target gene suppression and immunostaining were performed. The Cancer Genome Atlas data were utilized to validate gene expressions. RESULTS: We found down-regulation of miR-30a during chief cell transdifferentiation into SPEM. MiR-30a level was also reduced in the early stage of GC, and its level was maintained in advanced GC. We identified a novel target gene of miR-30a and ITGA2, and our results showed that either ectopic expression of miR-30a or ITGA2 knockdown suppressed GC cell proliferation, migration, and tumorigenesis. Levels of ITGA2 inversely correlated with levels of miR-30a in human intestinal-type GC. CONCLUSION: We found down-regulation of miR-30a in preneoplastic lesions and its tumor-suppressive functions by targeting ITGA2 in GC. The level of ITGA2, which functions as an oncogene, was up-regulated in human GC. The results of this study suggest that coordination of the miR-30a-ITGA2 axis may serve as an important mechanism in the development of gastric precancerous lesions and intestinal-type GC.

Somatic mutational profiles of stage II and III gastric cancer according to tumor microenvironment immune type.

We aimed to determine somatic mutational profiles of stage II/III gastric cancers (GCs) according to their tumor microenvironment immune types (TMITs), which classify cancer based on co-assessment of PD-L1 expression and CD8+ tumor infiltrating lymphocytes. Eighty patients with stage II/III GC were classified as follows: TMIT I (PD-L1+ /CD8High ), TMIT II (PD-L1- /CD8Low ), TMIT III (PD-L1+ /CD8Low ), and TMIT IV (PD-L1- /CD8High ). Deep targeted sequencing using a panel of 170 cancer-related genes was performed on an Illumina HiSeq-2500 system. Most frequently mutated genes included GNAQ (41.3%), TP53 (38.8%), CREBBP (35.0%), and MAP3K1 (35.0%). PIK3CA mutations were observed more frequently in TMIT I (45.8%) and III (66.7%), than in II (12.0%) and IV (8.0%). Other genes with enriched mutations within TMIT I included ATM (33.3%), BRCA2 (33.3%), MAP3K4 (29.2%), and FLT4 (25.0%). FGFR3, MAP3K1, and RUNX1 mutations were more frequently found in TMIT II. TMIT III had a unique somatic mutation profile harboring enriched mutations of histone modifiers including CREBBP and KMT2A, and we found FGFR2 amplification exclusively within TMIT IV. Fuzzy clustering analysis based on somatic mutation frequencies identified a hypermutated group (cluster 1) and a hypomutated group (cluster 2). Cluster 1 had significant associations with TMIT I, EBV+ GCs, and MSI-H GCs (P = .023, .014, and .004), and had better overall survival (P = .057) than Cluster 2. TMIT I, EBV+ , and MSI-H GCs were estimated to have greater tumor mutational burden (P = .023, .003, and .015). By analyzing somatic mutation profiles according to TMIT classification, we identified TMIT-specific genetic alterations that provide clues for biological linkage between GC genetics and microenvironment.

Microsatellite Instability and Programmed Cell Death-Ligand 1 Expression in Stage II/III Gastric Cancer: Post Hoc Analysis of the CLASSIC Randomized Controlled study.

OBJECTIVE: We investigated microsatellite instability (MSI) status and programed cell death ligand 1 (PD-L1) expression as predictors of prognosis and responsiveness to chemotherapy for stage II/III gastric cancer. BACKGROUND: The clinical implications of MSI status and PD-L1 expression in gastric cancer have not been well-elucidated. METHODS: Tumor specimens and clinical information were collected from patients enrolled in the CLASSIC trial-a randomized controlled study of capecitabine plus oxaliplatin-based adjuvant chemotherapy. Five quasi-monomorphic mononucleotide markers were used to assess tumor MSI status. PD-L1 expressions of tumor and stromal immune cells were evaluated using immunohistochemistry. RESULTS: Of 592 patients, 40 (6.8%) had MSI-high (MSI-H) tumors. Among 582 patients available for immunohistochemistry evaluation, PD-L1 was positive in tumor cells (tPD-L1) of 16 patients (2.7%) and stromal immune cells (sPD-L1) of 165 patients (28.4%). Multivariable analysis of disease-free survival (DFS) showed that MSI-H and sPD-L1-positivity were independent prognostic factors [hazard ratio 0.301 (0.123-0.736), 0.714 (0.514-0.991); P = 0.008, 0.044), as were receiving chemotherapy, age, tumor grade, and TNM stage. Although adjuvant chemotherapy improved DFS in the microsatellite-stable (MSS) group (5-year DFS: 66.8% vs 54.1%; P = 0.002); no benefit was observed in the MSI-H group (5-year DFS: 83.9% vs 85.7%; P = 0.931). In the MSS group, sPD-L1-negative patients, but not sPD-L1-positive patients, had significant survival benefit from adjuvant chemotherapy compared with surgery only (5-year DFS: 66.1% vs 50.7%; P = 0.001). CONCLUSION: MSI status and PD-L1 expression are clinically actionable biomarkers for stratifying patients and predicting benefit from adjuvant chemotherapy after D2 gastrectomy for stage II/III gastric cancer.

Development and Validation of an Easy-to-Implement, Practical Algorithm for the Identification of Molecular Subtypes of Gastric Cancer: Prognostic and Therapeutic Implications.

BACKGROUND: Gastric cancer (GC) is a heterogeneous disease, and substantial efforts have been made to develop a molecular biology-based classification system for GC. Analysis of the genomic signature is not always feasible, and thus, we aimed to (i) develop and validate a practical immunohistochemistry (IHC)- and polymerase chain reaction (PCR)-based molecular classification of GC and (ii) to assess HER2 status according to this classification. MATERIALS AND METHODS: A total of 894 consecutive patients with GC from two individual cohorts (training, n = 507; validation, n = 387) were classified using Epstein-Barr virus (EBV) in situ hybridization, microsatellite instability (MSI) testing, and IHC for E-cadherin and p53. RESULTS: We were able to classify patients into five groups in the training cohort: group 1 (MSI+), group 2 (EBV-, MSI-, non-epithelial-mesenchymal transition [non-EMT]-like, p53-), group 3 (EBV+), group 4 (EBV-, MSI-, non-EMT-like, p53+), and group 5 (EBV-, MSI-, EMT-like). In the training cohort, each group showed different overall survival (OS) after gastrectomy (p < .001); group 1 had the best prognosis, and group 5 showed the worst survival outcome. The significant impact of the classification system on OS was also verified in the validation cohort (p = .004). HER2 positivity was observed in 6.5% of total population, and most of HER2-positive cases (93.1%) were included in groups 2 and 4. CONCLUSION: We developed and validated a modified IHC- and PCR-based molecular classification system in GC, which showed significant impact on survival, irrespective of stage or other clinical variables. We also found close association between HER2 status and non-EMT phenotype in our classification system. IMPLICATIONS FOR PRACTICE: Molecular classification of gastric cancer suggested by previous studies mostly relies on extensive genomic data analysis, which is not always available in daily practice. The authors developed a simplified immunohistochemistry- and polymerase chain reaction-based molecular classification of gastric cancer and proved the prognostic significance of this classification, as well as the close association between HER2 status and certain groups of the classification, in the largest consecutive cohort of gastric cancer. Results of this study suggest that this scheme is a cost-effective, easy-to-implement, and feasible way of classifying gastric cancer in daily clinical practice, also serving as a practical tool for aiding therapeutic decisions and predicting prognosis.

A First-in-Human Phase I Study of GC1118, a Novel Anti-Epidermal Growth Factor Receptor Antibody, in Patients with Advanced Solid Tumors.

LESSONS LEARNED: GC1118 is a novel fully human anti-epidermal growth factor receptor (EGFR) antibody with unique binding epitopes and different ligand-binding inhibitory activity compared with cetuximab or panitumumab.GC1118 showed promising antitumor activity, especially in patients with colorectal cancer resistant to prior EGFR antibody. Skin toxicities were more common and diarrhea was less frequent compared with other anti-EGFR antibodies. BACKGROUND: GC1118 is a novel monoclonal antibody targeting epidermal growth factor receptor (EGFR) with more potent ligand inhibition than cetuximab or panitumumab. We conducted a first-in-human, phase I study of GC118 in patients with refractory solid tumors. METHODS: In the dose escalation part, GC1118 was administered on days 1, 8, 15, and 22, followed by a 2-week rest, during which dose-limiting toxicities (DLTs) were evaluated. In the expansion part, patients were enrolled into three cohorts (Cohort 1 [C1], patients with colorectal cancer [CRC] without prior anti-EGFR treatment; Cohort 2 [C2], patients with CRC with tumors resistant to anti-EGFR therapy; Cohort 3 [C3], EGFR-overexpressing gastric cancer). RESULTS: In the dose escalation part, 24 patients were treated at five dose levels: 0.3, 1.0, 3.0, 4.0, and 5.0 mg/kg. In the 5.0 mg/kg cohort, two patients experienced DLTs (skin toxicities). The maximum-tolerated dose (MTD) was 4.0 mg/kg. Common adverse events were skin toxicities. In the expansion part, 39 patients were enrolled. In Cohort 1, stable disease (SD) was observed in 58%; in Cohort 2, partial response (PR) 17% and SD 8%; in Cohort 3, PR 8% and SD 17%. CONCLUSION: GC1118 showed promising antitumor activity and was well tolerated. Infrequent diarrhea compared with other anti-EGFR antibodies might be advantageous for further development.

Clinicopathologic significance of human leukocyte antigen class I expression in patients with stage II and III gastric cancer.

Human leukocyte antigen class I (HLA I) molecules composed of alpha (heavy) chain, including HLA-A, -B, or -C encoded by HLA genes, and beta-2-microglobulin (ß2M) are membrane proteins on all nucleated cells that display peptide antigens for recognition by CD8-positive cytotoxic T cells. Here, we examined the clinicopathologic signification of HLA I expression in patients with gastric cancer (GC). Immunohistochemistry was performed to detect HLA A/B/C, ß2M, CD8, p53, and programmed death-ligand 1 (PD-L1) in the center and invasive margin of the tumor in 395 stage II and III GCs using tissue array method. Additionally, Epstein-Barr virus (EBV) infection and microsatellite instability (MSI) status were investigated. Negative expression of HLA A/B/C and ß2M was observed in 258 (65.3%) and 235 (59.5%) of 395 stage II and III GCs, respectively. Negative HLA I expression was significantly associated with aggressive clinicopathologic features. Furthermore, negative expression of HLA A/B/C and ß2M was inversely correlated with CD8-positive cytotoxic T cell infiltration, EBV-positivity, and PD-L1 expression (all p < 0.001). Patients with HLA A/B/C-negative GC had worse overall survival (OS) (p = 0.019) and combined analysis with both HLA A/B/C and ß2M expression status significantly predicted OS in univariate (p = 0.004) and multivariate survival analysis (p = 0.016). Negative expression of HLA A/B/C and ß2M was frequently observed in stage II and III GCs, particularly with the aggressive clinicopathologic features, and correlated with an unfavorable prognosis and host immune response status. These findings contribute to further development of immunotherapy.

Expression Profile of LGR5 and Its Prognostic Significance in Colorectal Cancer Progression.

We investigated the expression profile of leucine-rich, repeat-containing, G-protein-coupled receptor 5 (LGR5) during colorectal cancer (CRC) progression and determined the prognostic impact of LGR5 in a large cohort of CRC samples. LGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 68% of 788 CRCs and was positively correlated with older age, moderately to well-differentiated cells, and nuclear ß-catenin expression. Enhanced LGR5 expression remained persistent during the adenoma-carcinoma transition, but markedly declined in the budding cancer cells at the invasive fronts, which was not due to altered wingless-type mouse mammary tumor virus integration site family (Wnt) or epithelial-mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS or BRAF mutation. Notably, LGR5 positivity was an independent prognostic marker for better clinical outcomes in CRC patients. LGR5 overexpression attenuated tumor growth by decreasing ERK phosphorylation along with decreased colony formation and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony-forming and migration capacities in LoVo cells. Taken together, our data suggest a suppressive role of LGR5 in CRC progression.

Combinatory low methylation statuses of SAT-α and L1 are associated with shortened survival time in patients with advanced gastric cancer.

BACKGROUND: L1 and SAT-α are repetitive DNA elements that undergo demethylation in association with cancerization. Unlike L1 hypomethaylation, nothing is known regarding the prognostic implication of SAT-α hypomethylation alongside L1 hypomethaylaton in gastric cancers. METHODS: Formalin-fixed paraffin-embedded samples from 492 cases of advanced gastric cancer were analyzed to determine their L1 and SAT-α methylation status using pyrosequencing methylation assay. RESULTS: L1 and SAT-α methylation statuses were correlated with clinicopathological parameters, including survival. L1 or SAT-α methylation levels were lower in gastric cancers with venous invasion or nodal metastasis than those without. L1 methylation was lower in gastric cancers with lymphatic emboli than in those with no lymphatic emboli, but was higher in gastric cancers with perineural invasion than in those with no perineural invasion. Multivariate survival analysis revealed that both tumoral L1 and SAT-α hypomethylations were found to correlate independently with OS (HR = 1.477; 95% CI 1.079-2.021 and HR = 1.394; 95% CI 1.011-1.922, respectively) and RFS (HR = 1.477; 95% CI 1.090-2.001 and HR = 1.516; 95% CI 1.106-2.078, respectively). Combined L1 and SAT-α hypomethylation turned out to correlate independently with OS (HR = 2.003; 95% CI 1.268-3.164) and RFS (HR = 2.226; 95% CI 1.411-3.510). CONCLUSION: Not only tumoral L1 hypomethylation, but also tumoral SAT-α hypomethylation was found to be independent prognostic parameters in patients with advanced gastric cancer. SAT-α methylation status can be used to further divide gastric cancers with L1 hypomethylation into subsets of gastric cancers with better and worse prognosis.