Composite scoring system and optimal tumor budding cut-off number for estimating lymph node metastasis in submucosal colorectal cancer.

BACKGROUND: Tumor budding is associated with lymph node (LN) metastasis in submucosal colorectal cancer (CRC). However, the rate of LN metastasis associated with the number of tumor buds is unknown. Here, we determined the optimal tumor budding cut-off number and developed a composite scoring system (CSS) for estimating LN metastasis of submucosal CRC. METHODS: In total, 395 patients with histologically confirmed T1N0-2M0 CRC were evaluated. The clinicopathological characteristics were subjected to univariate and multivariate analyses. The Akaike information criterion (AIC) values of the multivariate models were evaluated to identify the optimal cut-off number. A CSS for LN metastasis was developed using independent risk factors. RESULTS: The prevalence of LN metastasis was 13.2%. Histological differentiation, lymphatic or venous invasion, and tumor budding were associated with LN metastasis in univariate analyses. In multivariate models adjusted for histological differentiation and lymphatic or venous invasion, the AIC value was lowest for five tumor buds. Unfavorable differentiation (odds ratio [OR], 8.16; 95% confidence interval [CI], 1.80-36.89), lymphatic or venous invasion (OR, 5.91; 95% CI, 2.91-11.97), and five or more tumor buds (OR, 3.01; 95% CI, 1.21-7.69) were independent risk factors. In a CSS using these three risk factors, the rates of LN metastasis were 5.6%, 15.5%, 31.0%, and 52.4% for total composite scores of 0, 1, 2, and ≥ 3, respectively. CONCLUSIONS: For the estimation of LN metastasis in submucosal CRC, the optimal tumor budding cut-off number was five. Our CSS can be utilized to estimate LN metastasis.

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.

Hyalinizing Trabecular Tumor of the Thyroid Gland, a Diagnostic Challenge in Fine-Needle Aspiration Cytology: Case Report.

Hyalinizing trabecular tumor (HTT) is a rare thyroid tumor with low to minimal malignant potential. HTT is often misinterpreted as other thyroid tumors, including papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC), on fine-needle aspiration (FNA) cytology, because of its overlapping cytologic features, such as nuclear grooves and intranulcear pseudoinclusions. Although cytopathologists cannot definitely conclude HTT by FNA cytology, suspicion of HTT is necessary to avoid misdiagnosing HTT as PTC or MTC and to avoid unnecessary aggressive treatment. Here, we report a case of HTT with novel cytologic features in CellPrep liquid based cytology that was diagnosed as suspicious for papillary carcinoma by FNA and finally diagnosed as HTT in the surgical specimen.

Merkel Cell Carcinoma Metastatic to Pleural Fluid: A Case Report.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine carcinoma of the skin that shows locoregional or distant metastasis. Metastasis of MCC to body cavity effusion is extremely rare; only three cases have been reported so far. Metastatic MCC in effusion cytology shows small blue round cells with fine stippled chromatin like other small blue round cell tumors such as small cell lung carcinoma or lymphoma. The diagnosis of metastatic MCC can grant patients good chances at recently advanced therapeutic options. Here, we present a case of metastatic MCC to pleural effusion with characteristic single file-like pattern.

CpG Island Methylator Phenotype-High Colorectal Cancers and Their Prognostic Implications and Relationships with the Serrated Neoplasia Pathway.

The concept of a CpG island methylator phenotype (CIMP) was first introduced by Toyota and Issa to describe a subset of colorectal cancers (CRCs) with concurrent hypermethylation of multiple CpG island loci. The concept of CIMP as a molecular carcinogenesis mechanism was consolidated by the identification of the serrated neoplasia pathway, in which CIMP participates in the initiation and progression of serrated adenomas. Distinct clinicopathological and molecular features of CIMP-high (CIMP-H) CRCs have been characterized, including proximal colon location, older age of onset, female preponderance, and frequent associations of high-level microsatellite instability and BRAF mutations. CIMP-H CRCs arise in sessile or traditional serrated adenomas and thus tend to display the morphological characteristics of serrated adenomas, including epithelial serration, vesicular nuclei, and abundant cytoplasm. Both the frequent association of CIMP and poor prognosis and different responses of CRCs to adjuvant therapy depending on CIMP status indicate clinical implications. In this review, we present an overview of the literature documenting the relevant findings of CIMP-H CRCs and their relationships with the serrated neoplasia pathway.

Associations and prognostic implications of Eastern Cooperative Oncology Group performance status and tumoral LINE-1 methylation status in stage III colon cancer patients.

BACKGROUND: Low methylation status of LINE-1 in tumors is associated with poor survival in patients with colon cancer. Eastern Cooperative Oncology Group performance status (ECOG-PS) is a method to assess the functional status of a patient. We retrospectively evaluated the relationship between ECOG-PS and LINE-1 methylation in colorectal cancers (CRCs) and their prognostic impact in CRC or colon cancer patients receiving adjuvant 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX). RESULTS: LINE-1 methylation and microsatellite instability were analyzed in stage III or high-risk stage II CRCs (n = 336). LINE-1 methylation levels were correlated with clinicopathological features, including PS and recurrence-free survival (RFS). The association between the tumoral LINE-1 methylation level and PS was observed (OR = 2.56, P < 0.001). Differences in LINE-1 methylation levels in cancer tissue between the PS 0 and 1 groups were significant in patients older than 60 years (P = 0.001), the overweight body mass index group (P = 0.005), and the stage III disease group (P = 0.008). Prognostic significances of LINE-1 methylation status or combined PS and LINE-1 methylation statuses were identified in stage III colon cancers, not in stage III and high-risk stage II CRCs. Low LINE-1 methylation status was closely associated with a shorter RFS time. The difference between PS(0)/LINE-1(high) and PS(≥1)/LINE-1(low) was significant, which suggests that colon cancer patients with concurrent PS(≥1)/LINE-1 (low) have a higher recurrence rate. CONCLUSIONS: PS was associated with LINE-1 methylation in CRC tissue. LINE-1 methylation was associated with RFS in stage III colon cancer patients who were treated with adjuvant FOLFOX chemotherapy. Combined PS and LINE-1 methylation status might serve as a useful predictor of cancer recurrence.

Are clinicopathological features of colorectal cancers with methylation in half of CpG island methylator phenotype panel markers different from those of CpG island methylator phenotype-high colorectal cancers?

CpG island methylator phenotype (CIMP)-high (CIMP-H) colorectal cancer (CRC) is defined when a tumor shows methylation at greater than or equal to 60% of CIMP panel markers. Although CRCs with methylation at 50% of panel markers are classified as CIMP-low/CIMP-0 tumors, little is known regarding the clinicopathological and molecular features of CRCs with methylation at 4/8 panel markers (4/8 methylated markers) and whether they are akin to CIMP-H or CIMP-low/CIMP-0 CRCs in terms of their clinicopathological or molecular features. A total of 1164 cases of surgically resected CRC were analyzed for their methylation status in 8 CIMP panel markers, and the frequencies of various clinicopathological and molecular features were compared between CRCs with 0/8, 1/8 to 3/8, 4/8, and 5/8 to 8/8 methylated markers. CRCs with 4/8 methylated markers were closer to CRCs with 5/8 to 8/8 methylated markers in terms of sex distribution, mucin production, serration, nodal metastasis, CK7 expression, CK20 loss, and CDX2 loss frequencies and overall survival rate. CRCs with methylation at 4/8 markers were closer to CRCs with 1/8 to 3/8 methylated markers in terms of less frequent right colon location and poor differentiation. CRCs with 4/8 methylated markers showed the shortest overall survival time compared with CRCs with 0/8, 1/8 to 3/8, 4/8, or 5/8 to 8/8 methylated markers. In terms of clinicopathological and molecular features, CRCs with 4/8 methylated markers appeared to be closer to CIMP-H than to CIMP-low/CIMP-0 and would thus be better classified as CIMP-H if the CRCs require classification into either CIMP-H or CIMP-low/CIMP-0.

Distribution of intestinal stem cell markers in colorectal precancerous lesions.

AIMS: Intestinal stem cell (ISC) markers such as LGR5, ASCL2, EPHB2 and OLFM4, and their clinical implications have been studied extensively in colorectal cancers (CRCs). However, little is known about their expression in precancerous lesions of CRCs. Here, we investigated the expression and distribution of ISC markers in serrated polyps and conventional adenomas. METHODS AND RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that all ISC markers were up-regulated significantly in conventional adenomas with low-grade dysplasia (CALGs) compared with other lesions. RNA in-situ hybridization confirmed that CALGs exhibited strong and diffuse expression of all ISC markers, which indicate a stem cell-like phenotype. However, normal colonic mucosa, hyperplastic polyps and sessile serrated adenomas harboured LGR5(+) cells that were confined to the crypt base and demonstrated an organized expression of ISC markers. Notably, in traditional serrated adenomas, expression of LGR5 and ASCL2 was localized to the ectopic crypts as in the normal crypts, but expression of EPHB2 and OLFM4 was distributed in a diffuse manner, which is suggestive of a progenitor-like features. CONCLUSIONS: The expression and distribution profile of ISC markers possibly provides insights into the organization of stem and progenitor-like cells in each type of precancerous lesion of CRC.

Annexin A10 expression in colorectal cancers with emphasis on the serrated neoplasia pathway.

AIM: To validate the utility of Annexin A10 as a surrogate marker of the serrated neoplasia pathway in invasive colorectal cancers (CRCs). METHODS: A total of 1133 primary CRC patients who underwent surgical resection at Seoul National University Hospital between January 2004 and December 2007 were enrolled. Expression of Annexin A10 was evaluated by immunohistochemistry using tissue microarray and paired to our findings on clinicopathologic and molecular characteristics of each individual. CpG island methylator phenotype was determined by MethyLight assay and microsatellite instability was determined by high performance liquid chromatography. KRAS and BRAF mutation status was evaluated by direct sequencing and allele-specific PCR. Univariate and stage-specific survival analyses were performed to reveal the prognostic value of Annexin A10 expression. RESULTS: Annexin A10 expression was observed in 66 (5.8%) of the 1133 patients. Annexin A10 expression was more commonly found in females and was associated with proximal location, ulcerative gross type, advanced T category, N category and TNM stage. CRCs with Annexin A10 expression showed an absence of luminal necrosis, luminal serration and mucin production. CRCs with Annexin A10 expression were associated with CpG island methylator phenotype, microsatellite instability and BRAF mutation. In survival analysis, Annexin A10 expression was associated with poor overall survival and progression-free survival, especially in stage IV CRCs. CONCLUSION: Annexin A10 expression is associated with poor clinical behavior and can be used a supportive surrogate marker of the serrated neoplasia pathway in invasive CRCs.

Nuclear maspin expression correlates with the CpG island methylator phenotype and tumor aggressiveness in colorectal cancer.

It has been suggested that nuclear expression of maspin (mammary serine protease inhibitor; also known as SERPINB5) in colorectal cancer (CRC) is associated with proximal colonic tumor location, mucinous and poorly differentiated histology, microsatellite instability-high (MSI-H), and poor prognosis. Based on these findings, there may be a potential association between nuclear maspin expression and the CpG island methylator phenotype (CIMP) in CRC, but no study has elucidated this issue. Here, we evaluated maspin protein expression status by immunohistochemistry in 216 MSI-H CRCs. CIMP status was also determined by methylation-specific quantitative PCR method (MethyLight) using eight CIMP markers (MLH1, NEUROG1, CRABP1, CACNA1G, CDKN2A (p16), IGF2, SOCS1, and RUNX3) in 216 MSI-H CRCs. Associations between maspin expression status and various pathological, molecular, and survival data were statistically analyzed. Among the 216 MSI-H CRCs, 111 (51%) cases presented nuclear maspin-positive tumors. Nuclear maspin-positive MSI-H CRCs were significantly associated with proximal tumor location (P = 0.003), tumor budding (P < 0.001), lymphovascular invasion (P = 0.001), perineural invasion (P = 0.008), absence of peritumoral lymphoid reaction (P = 0.045), lymph node metastasis (P = 0.003), distant metastasis (P = 0.005), advanced AJCC/UICC stage (stage III/IV) (P = 0.001), and CIMP-high (CIMP-H) status (P < 0.001). Patients with nuclear maspin-positive tumors showed worse disease-free survival than patients with nuclear maspin-negative tumors (log-rank P = 0.025). In conclusion, nuclear maspin expression is molecularly associated with CIMP-H rather than MSI-H, and clinicopathologically correlates with tumor aggressiveness in CRC.

Transglutaminase 2 expression and its prognostic significance in clear cell renal cell carcinoma.

BACKGROUND: A few recent studies have demonstrated a possible role of transglutaminase 2 (TG2) in tumorigenesis or progression of renal cell carcinoma (RCC). The aim of this study was to examine TG2 expression and its clinicopathologic significance in a large number of human clear cell RCCs (CCRCCs). METHODS: We analyzed 638 CCRCC patients who underwent partial or radical nephrectomy between 1995 and 2005. The expression of TG2 was determined by immunohistochemistry and categorized into four groups, according to staining intensity: negative (0), mild (1+), moderate (2+), and strong (3+). RESULTS: TG2 staining intensity was negative in 8.5% of CCRCC (n=54), 1+ in 32.6% (n=208), 2+ in 50.5% (n=322), and 3+ in 8.5% (n=54). Strong TG2 expression was correlated with high Fuhrman nuclear grade (p=.011), high T category (p=.049), metastasis (p=.043) and male sex (p<.001) but not with N category.The survival analysis showed a significant association between strong TG2 expression and worse overall and cancer-specific survival (p=.027 and p=.010, respectively). On multivariate analysis, strong TG2 expression was a marginally significant prognostic indicator for Fuhrman nuclear grade and TNM staging (p=.054). CONCLUSIONS: Our study is the first to demonstrate the clinicopathologic significance of TG2 expression in a large number of human CCRCC samples. Strong TG2 expression was associated with high nuclear grade and poor prognosis.

Diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration cytology of pancreatic lesions.

BACKGROUND: Endoscopic ultrasound-guided fine needle aspiration cytology (EUS-FNAC) is currently the most commonly used procedure for obtaining cytologic specimens of the pancreas. It is accurate, minimally invasive, safe and cost-effective. However, there is discrepancy between cytological and surgical diagnoses. This study was aimed at evaluating the diagnostic accuracy of EUS-FNAC of the pancreas. METHODS: We performed a retrospective review of 191 cases of pancreatic lesions initially diagnosed by EUS-FNAC with subsequent histological diagnosis between 2010 and 2012 in the Department of Pathology, Seoul National University Hospital. Cytologic and surgical diagnoses were categorized into five groups: negative, benign, atypical, malignant, and insufficient for diagnosis. Subsequently, 167 cases with satisfactory yield in both surgical and cytology specimens were statistically analyzed to determine correlations with diagnosis. RESULTS: In comparison to surgical diagnoses, cytologic diagnoses were true-positive in 103 cases (61.7%), true-negative in 28 cases (16.8%), false-positive in 9 cases (5.4%), and false-negative in 27 cases (16.1%). The diagnostic accuracy was 78.4%, sensitivity was 79.2%, and specificity was 75.7%. The positive predictive value was 92.0%, and negative predictive value was 50.9%. CONCLUSIONS: EUS-FNAC has high accuracy, sensitivity, specificity and positive predictive value. Overcoming the limitations of EUS-FNAC will make it a useful and reliable diagnostic tool for accurate evaluation of pancreatic lesions.

Prognostic significance of promoter CpG island hypermethylation and repetitive DNA hypomethylation in stage I lung adenocarcinoma.

In carcinogenesis of peripheral pulmonary carcinomas, multiple genetic and epigenetic alterations are involved. In this study, we quantified methylation levels of repetitive DNA elements (L1 and Alu) and six CpG island methylator phenotype (CIMP)-panel markers in various lesions representing steps in the development of lung adenocarcinoma (ADC), including atypical adenomatous hyperplasia, adenocarcinoma in situ, and invasive ADC. We then assessed methylation levels in an independent set of stage I ADCs (n = 100) and correlated methylation status with clinicopathological findings and clinical outcome. The pattern of changes in the methylation levels of L1 and Alu was different during progression of the lesion along the process of multistep carcinogenesis. A methylation level of >52.4 % of L1 and of >19.7 % of Alu in stage I ADC was associated with shorter cancer-specific survival in univariate but not in multivariate analysis. A tumor to normal lung tissue methylation ratio of >0.693 of L1 was an independent parameter heralding poor prognosis for stage I ADC patients. Methylation of CIMP-related genes was found in ADC. Stage I ADC cases without methylation of any of the six markers had a significantly shorter cancer-specific survival than ADC with methylation of one or more markers. The combination of tumor to normal L1 methylation ratio > 0.693 and absence of methylation of CIMP markers correlated independently with shorter cancer-specific survival. In conclusion, our findings suggest that Alu hypomethylation is an early and L1 hypomethylation a later event during multistep pulmonary carcinogenesis. The prognostic significance of the combination of methylation status of L1 and CIMP markers must be validated in large-scale studies of pulmonary ADC.

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.

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.

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.

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.

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.

Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer.

Several previous studies have demonstrated that the CDX2-negative (CDX2) and/or CK20-negative (CK20) phenotypes of colorectal cancers (CRCs) might be associated with high levels of microsatellite instability (MSI-H). The aim of this study was to investigate the clinicopathologic and molecular features of MSI-H CRCs with different CDX2/CK20 expression statuses. The CDX2 and CK20 expression statuses were immunohistochemically evaluated in 109 MSI-H CRC tissue samples, and the correlations of these statuses with clinicopathologic, molecular, and survival data were statistically analyzed. Of the 109 MSI-H CRCs, 15 were CDX2 (13.8%), and 19 were CK20 (17.4%). The simultaneous loss of CDX2 and CK20 expression (CDX2/CK20) was observed in 9 cases (8.3%). CDX2 loss was correlated with lymph node metastasis, poor differentiation, MLH1 loss, the mutation of BRAF, and CpG island methylator phenotype-high (CIMP-H) status. Right-sided tumor location, nodal metastasis, poor differentiation, and CIMP-H status were significant characteristics of CK20 tumors. The CDX2/CK20 phenotype was associated with older age (above 56 y), higher stage (stage III or IV), deep invasion (pT3 or pT4), lymph node metastasis (pN1 or pN2), poor differentiation (nonmedullary/non-signet ring cell type), the mutation of BRAF, and CIMP-H status among MSI-H CRCs. Patients with CDX2/CK20 tumors exhibited worse overall and disease-free survival compared with the patients with CDX2 and/or CK20 tumors (P<0.001). In the multivariate analysis for disease-free survival, the CDX2/CK20 phenotype was an independent prognostic factor for MSI-H CRC (P=0.030, hazard ratio=3.288). The CDX2/CK20 phenotype defines a distinct subgroup of MSI-H CRCs with poor differentiation, CIMP-H status, and unfavorable prognosis.

Clinical outcomes of patients with microsatellite-unstable colorectal carcinomas depend on L1 methylation level.

BACKGROUND: The prognostic significance of microsatellite instability (MSI) in colorectal cancers (CRCs) has been addressed in many studies since the initial description of better survival rates in MSI-positive (MSI+) tumors than in MSI-negative (MSI-) tumors. Recent studies have demonstrated that a higher degree of hypomethylation of long interspersed nuclear element-1 (L1) is related to poor prognosis of CRCs and that a wide variation of L1 methylation levels exist within MSI+ CRCs. Our aim was to identify whether L1 and Alu methylation status could predict clinical outcomes within MSI+ CRCs. METHODS: We analyzed 207 MSI+ CRCs for their methylation levels in L1 and Alu repetitive DNA elements using pyrosequencing and correlated them with clinicopathological information including survival data. RESULTS: Univariate survival analysis showed that low Alu methylation status (<18.60%) and low L1 methylation status (<53.00%) were significantly associated with shorter overall survival time (log-rank test, P = 0.009 and P < 0.001, respectively). Multivariate analysis using nine parameters (Alu methylation status, L1 methylation status, patient's age, disease stage [tumor, node, metastasis staging system], differentiation, Crohn-like lymphoid reaction, KRAS/BRAF mutation status, CpG island methylator phenotype [CIMP] status, and peritumoral lymphocytic infiltration), which were significantly prognostic in MSI+ CRCs, revealed that low L1 methylation status was an independent prognostic factor of MSI+ CRCs (P = 0.009), whereas low Alu methylation status was not. CONCLUSIONS: Clinical outcomes of MSI+ CRCs depend on L1 methylation status, suggesting that lower L1 methylation status serves as a significant prognostic parameter of adverse prognosis in MSI+ CRCs.