Kyung-Ju Kim

Prognostic implications of tumor-infiltrating FoxP3+ regulatory T cells and CD8+ cytotoxic T cells in microsatellite-unstable gastric cancers

Microsatellite instability (MSI)-high gastric cancers (GC) have better prognosis and higher levels of tumor-infiltrating lymphocytes (TIL) compared with MSI-low or MSI-stable GCs. TILs are part of the adaptive immune response against tumor growth and are associated with improved prognosis in GCs. The aim of this study was to investigate the prognostic significance of CD8+ and FoxP3+ TILs in MSI-high GCs and their relationships with various clinicopathologic characteristics. Intratumoral intraepithelial CD8+ and FoxP3+ TILs were assessed in 99 cases of MSI-high GCs using a computerized image analysis system. TILs were grouped into low- and high-density groups and were analyzed for their relationships with clinicopathologic parameters. A low density was closely associated with a higher TNM stage (P = .040) and invasion depth (P = .044) and more frequent lymphatic and vascular invasion (P = .033 and .015, respectively). GCs with high-density CD8+ or FoxP3+ TILs showed significantly higher overall survival rates than those of GCs with low-density CD8+ or FoxP3+ TILs (P = .017 and .013, respectively, Kaplan-Meier test). In multivariate survival analysis, a high density of FoxP3+ TILs was significantly associated with improved overall survival (P = .027; hazard ratio, 0.269), and the combinatorial status of CD8+ and FoxP3+ TIL density was an independent prognostic factor (P = .003). Our results demonstrate that higher densities of both intratumoral CD8+ and FoxP3+ TILs are associated with good prognosis, suggesting a synergistic activity of these 2 subsets that can be used as an independent prognostic factor in MSI-high GCs.

Prognostic Implication of M2 Macrophages Are Determined by the Proportional Balance of Tumor Associated Macrophages and Tumor Infiltrating Lymphocytes in Microsatellite-Unstable Gastric Carcinoma.

Tumor associated macrophages are major inflammatory cells that play an important role in the tumor microenvironment. In this study, we investigated the prognostic significance of tumor associated macrophages (TAMs) in MSI-high gastric cancers using immunohistochemistry. CD68 and CD163 were used as markers for total infiltrating macrophages and M2-polarized macrophages, respectively. The density of CD68+ or CD163+ TAMs in four different areas (epithelial and stromal compartments of both the tumor center and invasive front) were analyzed in 143 cases of MSI-high advanced gastric cancers using a computerized image analysis system. Gastric cancers were scored as “0” or “1” in each area when the density of CD68+ and CD163+ TAMs was below or above the median value. Low density of CD68+ or CD163+ macrophages in four combined areas was closely associated with more frequent low-grade histology and the intestinal type tumor of the Lauren classification. In survival analysis, the low density of CD163+ TAMs was significantly associated with poor disease-free survival. In multivariate survival analysis, CD163+ TAMs in four combined areas, stromal and epithelial compartments of both tumor center and invasive front were independent prognostic indicator in MSI-high gastric cancers. In addition, the density of CD163+ TAMs correlated with tumor infiltrating lymphocytes (TILs). Our results indicate that the high density of CD163+ TAMs is an independent prognostic marker heralding prolonged disease-free survival and that the prognostic implication of CD163+ TAMs might be determined by the proportional balance of TAMs and TILs in MSI-high gastric cancers.

Adverse prognostic impact of the CpG island methylator phenotype in metastatic colorectal cancer.

Background:The association between the CpG island methylator phenotype (CIMP) and clinical outcomes in metastatic colorectal cancer remains unclear. We investigated the prognostic impact of CIMP in patients with metastatic colorectal cancer treated with systemic chemotherapy.Methods:Eight CIMP-specific promoters (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1, CDKN2A, CRABP1, and MLH1) were examined. The CIMP status was determined by the number of methylated promoters as high (⩾5), low (1–4), and negative (0).Results:A total of 153 patients were included (men/women, 103/50; median age, 61 years; range, 22–80 years). The CIMP status was negative/low/high in 77/ 69/7 patients, respectively. Overall survival (OS) was significantly different among the three CIMP groups, with median values of 35.7, 22.2, and 9.77 months for the negative, low, and high groups, respectively (P<0.001). For patients treated with fluoropyrimidine and oxaliplatin first-line chemotherapy (N=128), OS and progression-free survival (PFS) were significantly different among the three CIMP groups; the median OS was 37.9, 23.8, and 6.77 months for the negative, low, and high groups, respectively (P<0.001), while the median PFS was 9.97, 7.87, and 1.83 months, respectively (P=0.002). Response rates were marginally different among the three CIMP groups (53.4% vs 45.1% vs 16.7%, respectively; P=0.107). For patients treated with fluoropyrimidine and irinotecan second-line chemotherapy (N=86), only OS showed a difference according to the CIMP status, with median values of 20.4, 13.4, and 2.90 months for the negative, low, and high groups, respectively (P<0.001).Conclusions:The CIMP status is a negative prognostic factor for patients with metastatic colorectal cancer treated with chemotherapy.

Expression status of wild-type HSP110 correlates with HSP110 T17 deletion size and patient prognosis in microsatellite-unstable colorectal cancer

It has been recently suggested that the expression levels of mutant HSP110 could be a prognostic marker in colorectal cancer with a high level of microsatellite instability (MSI-H). The aim of our study was to validate the prognostic significance of HSP110 mutation using immunohistochemistry and DNA testing in MSI-H colorectal cancer. Wild-type HSP110 (HSP110wt)-specific immunohistochemistry was performed in 168 MSI-H colorectal cancer tissues, and their expression levels were evaluated using a four-tier scoring system (0/1+/2+/3+). Of these tissues, 167 cases were analyzed for HSP110 T17 deletion. Associations with clinicopathological, molecular and survival parameters were statistically analyzed. The low-level expression of HSP110wt (0/1+) was observed in 40 MSI-H colorectal cancers (24%) and was significantly related to large HSP110 T17 deletions (≥ 4 bp, P<0.001). In survival analysis, patients with low HSP110wt expression (0/1+) showed better disease-free survival compared with those with high expression (2+/3+; P=0.005). This significance in survival difference was maintained in patients with 5-fluorouracil-based chemotherapy-treated tumors (P=0.024) and in those with stage III/IV tumors (P=0.032). Multivariate analysis confirmed the role of HSP110wt expression as an independent prognostic factor (P=0.016, hazard ratio=4.32). In MSI-H colorectal cancer, a low expression of HSP110wt is associated with large HSP110 T17 deletions and better clinical outcome. Immunohistochemistry of HSP110wt can be a simple and valuable tool for the prognostic and therapeutic stratification of patients with MSI-H colorectal cancer.

Differential Features of Microsatellite-Unstable Colorectal Carcinomas Depending on EPCAM Expression Status

Background Recent studies have revealed that a small subset of Lynch syndrome-associated colorectal carcinomas (CRCs) is caused by a germline EPCAM deletion-induced MSH2 epimutation. Based on the finding of this genetic alteration, we investigated the implications of EPCAM expression changes in microsatellite instability-high (MSI-H) CRCs. Methods Expression of EPCAM and DNA mismatch repair proteins was assessed by immunohistochemistry in 168 MSI-H CRCs. Using DNA samples of these tumors, MLH1 promoter methylation status was also determined by methylation-specific real-time polymerase chain reaction method (MethyLight). Results Among 168 MSI-H CRCs, complete loss (CL) and focal loss (FL) of EPCAM expression was observed in two (1.2%) and 22 (13.1%) cases, respectively. Both of the EPCAM-CL cases were found in MSH2-negative tumors without MLH1 promoter methylation. However, only nine of the 22 EPCAM-FL tumors had MSH2 deficiency. Of the 22 EPCAM-FL tumors, 13 showed MLH1 loss, and among them, nine cases were determined to have MLH1 methylation. EPCAM-FL was significantly associated with advanced stage (p=.043), distant metastasis (p=.003), poor differentiation (p=.001), and signet ring cell component (p=.004). Conclusions Loss of EPCAM expression is differentially associated with clinicopathological and molecular features, depending on the completeness of the loss, in MSI-H CRCs.

Differential clinicopathologic features in microsatellite-unstable gastric cancers with and without MLH1 methylation.

Key clinicopathologic features of microsatellite instability-positive (MSI+) gastric cancers (GCs) are that they tend to be located in the antrum and have an intestinal phenotype and an expanding-type growth pattern. They are also associated with a better prognosis. Although MSI occurs mainly as a result of promoter CpG island hypermethylation in the mismatch repair gene MLH1, only a minority of MSI+ GCs develop from genetic mutations of mismatch repair enzymes, including MLH1 and MSH2. Furthermore, it is unknown whether there are differences in the clinicopathologic features of MSI+ GCs with and without MLH1 methylation. The methylation status of 17 genes (including MLH1) was assessed in 102 cases of MSI+ GC to determine whether there was a correlation between the clinicopathologic/molecular features of MSI+ GC and MLH1 methylation status. Compared with MSI+ GCs without MLH1 methylation (n = 22), MSI+ GCs with MLH1 methylation (n = 80) had an older age of onset (66.9 versus 60.9 years, P = .018), were more frequently located in the antrum (86.3% versus 50%, P = .001), exhibited an ulcerofungating gross type of tumor morphology (50.0% versus 9.1 %, P < .001), and had a higher number of unstable microsatellite loci (4.7 versus 3.8, P < .001) and a higher number of methylated genes (11.4 versus 6.2, P < .001). In addition, MLH1-deficient tumors without MLH1 methylation were associated with a better clinical outcome than MLH1-deficient tumors with MLH1 methylation or tumors that retained expression of both MLH1 and MSH2 (P = .002). These findings suggest that MSI+ GCs with and without MLH1 methylation may have different clinicopathologic features. Furthermore, some of the known clinicopathologic features of MSI+ GC, including older age of onset, ulcerofungating gross morphology, and antral location, are not typical of MSI+ GC without MLH1 methylation.

Comparative validation of assessment criteria for Crohn-like lymphoid reaction in colorectal carcinoma

Aims Crohn-like lymphoid reaction (CLR) in colorectal carcinoma (CRC) is associated with a favourable prognosis and microsatellite instability-high (MSI-H) status. However, there is a lack of consensus on optimal criteria for CLR assessment. The aim of this study was to comparatively validate traditional and novel assessment criteria for CLR. Methods CLR status in 212 MSI-H CRCs was assessed independently by two pathologists using three different criteria: (1) traditional semiquantitative criteria (Graham–Appelman criteria), (2) the largest lymphoid aggregate (LA) size-based criteria (Ueno criteria) and (3) LA density-based criteria (Väyrynen–Mäkinen criteria). Results Among the three criteria, the Väyrynen–Mäkinen criteria-based CLR assessment showed the best interobserver agreement (κ value, 0.71; intraclass correlation coefficient, 0.76). Pathologically, intense CLR (grade 2) by Graham–Appelman criteria, active CLR (largest LA size ≥1 mm) by Ueno criteria and high-density CLR (≥0.38 LAs/mm) by Väyrynen–Mäkinen criteria significantly correlated with an early cancer stage (stage I/II). In Kaplan–Meier analysis, both CLR statuses determined by Ueno criteria and Väyrynen–Mäkinen criteria were associated with significant differences in disease-free survival in MSI-H CRC patients (p=0.005 and p=0.001, respectively). In multivariable analysis, both active CLR and high-density CLR proved to be independent favourable prognostic factors in MSI-H CRC (HR, 0.47; 95% CI 0.24 to 0.9 for active CLR and HR, 0.5; 95% CI 0.28 to 0.89 for high-density CLR). Conclusions Our study confirms that the two recently suggested criteria (Ueno criteria and Väyrynen–Mäkinen criteria) for CLR assessment are fairly reproducible methods and can serve as superior prognosticators in CRC.

Gastric-type expression signature in serrated pathway–associated colorectal tumors

Accumulating evidence has indicated that serrated pathway-associated colorectal tumors may be associated with aberrant gastric-type differentiation. Here, we investigated the immunoexpression profiles of gastric-type markers and intestinal-type markers in colorectal tumors, focusing on their relation to serrated pathway-associated tumors. Immunohistochemistry for 7 gastric-type markers (ANXA10, VSIG1, CLDN18, CTSE, TFF2, MUC5AC, and MUC6) and 2 intestinal-type markers (CDX2 and CK20) was performed in 36 normal gastric/colorectal mucosa tissues, 163 colorectal polyps, and 175 microsatellite-unstable colorectal carcinomas (MSI-H CRCs). In normal tissues, all 7 candidate gastric-type markers showed expressional specificity for normal gastric mucosa. Among the colorectal polyps, sessile serrated adenoma/polyps demonstrated the highest positive rate of ANXA10, CLDN18, MUC5AC, and MUC6 expression (87%, 35%, 61%, and 52%, respectively). Microvesicular hyperplastic polyps showed the highest frequencies of ANXA10, VSIG1, and TFF2 positivity (87%, 87%, and 67%, respectively). ANXA10 and MUC6 expression was not detected in all conventional adenomas. In MSI-H CRCs, the expression of ANXA10, TFF2, and MUC5AC was significantly associated with sporadic tumors (P < .001, P = .01, and P < .001, respectively). Moreover, all of the 7 gastric-type markers were significantly related to preferential expression in proximal colon carcinomas among MSI-H CRCs. CDX2 and CK20 expression was retained in all colorectal polyps, whereas there were significantly high frequencies of CDX2 loss (28%) and CK20 loss (29%) in sporadic tumors among MSI-H CRCs. In conclusion, the early gain of gastric differentiation and late loss of intestinal differentiation are immunophenotypic features in the serrated pathway to colorectal carcinoma.

Annexin A10 expression correlates with serrated pathway features in colorectal carcinoma with microsatellite instability

Annexin A10 (ANXA10) has recently been identified as a marker of sessile serrated adenomas/polyps of the colorectum. Although the serrated neoplasia pathway is thought to be involved in the majority of microsatellite instability‐high (MSI‐H) sporadic colorectal carcinomas (CRCs), the clinicopathological implications of ANXA10 expression in CRC are unknown. Here, we evaluated ANXA10 expression status in 168 MSI‐H CRCs by immunohistochemistry. Among 168 MSI‐H CRCs, nuclear staining for ANXA10 in tumor cells revealed 28 cases (17%) with ANXA10‐positive (ANXA10+) tumors. Most of the ANXA10+ tumors were located in the proximal colon (96%, p < 0.001). The ANXA10+ phenotype in MSI‐H CRC was significantly associated with female gender (68%, p = 0.016), CpG island methylator phenotype‐high (CIMP‐H) (68%, p < 0.001), MLH1 promoter hypermethylation (61%, p < 0.001), loss of MLH1 expression (82%, p = 0.019), and wild‐type KRAS status (96%, p = 0.023). Survival analysis revealed no prognostic significance of ANXA10 expression in MSI‐H CRC. In conclusion, ANXA10+ MSI‐H colon carcinomas are characterized by serrated pathway features, including proximal location, female predominance, and high frequencies of CIMP‐H status and MLH1 methylation.

Combined prognostic effect of PD-L1 expression and immunoscore in microsatellite-unstable advanced gastric cancers

BACKGROUND The aim of this study was to evaluate how programmed death-ligand-1 (PD-L1) expression is linked to the immunoscore in the context of the tumor microenvironment and to assess the differential prognostic value of PD-L1 expression according to the immunoscore in 153 patients with microsatellite instability-high (MSI-H) advanced gastric cancer (GC). RESULTS We found that T-PD-L1 (+) and I-PD-L1 (+) were significantly associated with a high immunoscore. The integrated PD-L1 expression of tumor and immune cells was not significantly correlated with the overall survival (OS) of patients. However, a combined survival analysis of PD-L1 expression and immunoscore revealed four distinct subgroups with a statistically significant difference in OS. That is, the PD-L1 (+)/immunoscoreLow group showed the worst and the PD-L1 (+)/immunoscoreHigh group showed the best prognosis. Furthermore, a multivariate analysis revealed that the combined status of PD-L1 expression and immunoscore was an independent and significant prognostic factor for OS in patients with MSI-H GC. MATERIALS AND METHODS The immunoscore was quantified by the number of high-density areas of CD3+ and CD8+ tumor infiltrating lymphocytes both in the tumor regions and compartments (i.e., epithelial and stromal compartments of the tumor center and the invasive front), the scores of which range from I0 to I8. By using immunohistochemistry, the expression of PD-L1 was also analyzed in tumor cells (T-PD-L1) and immune cells (I-PD-L1) using four different cut-off values (1%, 5%, 10% and 50%). CONCLUSIONS Our study revealed that PD-L1 expression is associated with the corresponding immunoscore and that the immunoscore can be a relevant marker for the determination of the prognostic role of PD-L1 expression in MSI-H GCs.Background The aim of this study was to evaluate how programmed death-ligand-1 (PD-L1) expression is linked to the immunoscore in the context of the tumor microenvironment and to assess the differential prognostic value of PD-L1 expression according to the immunoscore in 153 patients with microsatellite instability-high (MSI-H) advanced gastric cancer (GC). Results We found that T-PD-L1 (+) and I-PD-L1 (+) were significantly associated with a high immunoscore. The integrated PD-L1 expression of tumor and immune cells was not significantly correlated with the overall survival (OS) of patients. However, a combined survival analysis of PD-L1 expression and immunoscore revealed four distinct subgroups with a statistically significant difference in OS. That is, the PD-L1 (+)/immunoscoreLow group showed the worst and the PD-L1 (+)/immunoscoreHigh group showed the best prognosis. Furthermore, a multivariate analysis revealed that the combined status of PD-L1 expression and immunoscore was an independent and significant prognostic factor for OS in patients with MSI-H GC. Materials and Methods The immunoscore was quantified by the number of high-density areas of CD3+ and CD8+ tumor infiltrating lymphocytes both in the tumor regions and compartments (i.e., epithelial and stromal compartments of the tumor center and the invasive front), the scores of which range from I0 to I8. By using immunohistochemistry, the expression of PD-L1 was also analyzed in tumor cells (T-PD-L1) and immune cells (I-PD-L1) using four different cut-off values (1%, 5%, 10% and 50%). Conclusions Our study revealed that PD-L1 expression is associated with the corresponding immunoscore and that the immunoscore can be a relevant marker for the determination of the prognostic role of PD-L1 expression in MSI-H GCs.