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Background There are ongoing efforts to reduce CT radiation exposure for the diagnosis of appendicitis. Recent guidelines recommend using clinical scoring systems to triage patients who need imaging examinations. Purpose To determine whether patient triaging with scoring systems can reduce CT use without a loss of diagnostic accuracy in adolescents and young adults suspected of having appendicitis. Materials and Methods This retrospective study used data from a previous multicenter randomized controlled trial conducted between December 2013 and August 2016. Five scoring systems (adult appendicitis, appendicitis inflammatory response, modified Alvarado, Broek, and Christian scores) were used to categorize patients into low-, intermediate-, or high-probability groups. CT use was simulated for only the intermediate-probability group. The primary outcomes were CT reduction rate, sensitivity, and specificity. The CT reduction rate was defined as the proportion of patients in low- and high-probability groups who would not have to undergo CT among all patients. Sensitivity and specificity were calculated in the overall diagnostic pathway using each scoring system and subsequent CT. As a secondary analysis, to maintain the diagnostic accuracy to a level of when CT was used for all patients with suspected appendicitis, new cutoff values for probability group stratification targeting 97.6% sensitivity and 94.9% specificity were applied for each of the scoring systems. Results A total of 2888 patients (mean age ± standard deviation, 28 years ± 9; 1580 women and 1308 men) with suspected appendicitis were evaluated, of whom 1088 had and 1800 did not have appendicitis. The CT reduction rates of the five scoring systems ranged from 55.6% (1606 of 2888 patients) to 71.1% (2053 of 2888), but at the cost of sensitivity (range, 48.7% [530 of 1088] to 81.2% [883 of 1088]) and specificity (range, 79.0% [1422 of 1800] to 97.8% [1761 of 1800]). Targeting 97.6% sensitivity and 94.9% specificity, the CT reduction rates of all five scoring systems were 0% (0 of 2888). Conclusion Using clinical scoring systems in triaging patients for selective CT use led to a considerable loss of diagnostic accuracy. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Mellnick in this issue.

Circulating tumor DNA (ctDNA) may reveal dynamic tumor status during therapy. We conducted serial ctDNA analysis to investigate potential association with clinical outcome in metastatic colorectal cancer (mCRC) patients receiving chemotherapy. Tissue KRAS/NRAS wild-type mCRC patients were enrolled and treated with first-line cetuximab-containing chemotherapy. ctDNA isolated from plasma were analyzed by next generation sequencing (NGS) with 16 targeted gene panel. Among 93 patients, 84 (90.3%) had at least 1 somatic mutation in baseline ctDNA samples (average 2.74). Five patients with KRAS or NRAS hotspot mutation in the ctDNA showed significantly worse progression-free survival (PFS) (p = 0.029). Changes in average variant allele frequency (VAF) in ctDNA showed significant correlation with tumor size change at the time of first response evaluation (p = 0.020) and progressive disease (PD) (p = 0.042). Patients whose average VAF decreased below cutoff (p p = 0.018). At the time of PD, 54 new mutations including KRAS and MAP2K1 emerged in ctDNA. ctDNA sequencing can provide mutation profile that could better reflect tumor mutation status and predict treatment outcome.

Tumor heterogeneity is one of the ongoing huddles in the field of colon cancer therapy. It is evident that there are countless clones which exhibit different phenotypes and therefore, single cell analysis is inevitable. Cancer stem cells (CSCs) are rare cell population within tumor which is known to function in cancer metastasis and recurrence. Although there have been trials to prove intra-tumoral heterogeneity using single cell sequencing, that of CSCs has not been clearly elucidated. Here, we articulate the presence of heterogeneous subclones within CD133 positive cancer stem cells through single cell sequencing. As a proof of principle, we performed phenotype-based high-throughput laser isolation and single cell sequencing (PHLI-seq) of CD133 positive cells in a frozen tumor tissue obtained from a patient with colorectal cancer. The result proved that CD133 positive cells were shown to be heterogeneous both in copy number and mutational profiles. Single cancer stem cell specific mutations such as RNF144A, PAK2, PARP4, ADAM21, HYDIN, KRT38 and CELSR1 could be also detected in liver metastatic tumor of the same patient. Collectively, these data suggest that single cell analysis used to spot subclones with genetic variation within rare population, will lead to new strategies to tackle colon cancer metastasis.

Background Pediatric patients with liver disease require noninvasive monitoring to evaluate the risk of fibrosis progression. This study aimed to identify the significant factors affecting liver stiffness values using two-dimensional shear-wave elastography (2D-SWE), and determine whether liver stiffness can predict the fibrosis stage of various childhood liver diseases. Methods This study included 30 children (22 boys and 8 girls; mean age, 5.1 ± 6.1 years; range, 7 days–17.9 years) who had undergone biochemical evaluation, 2D-SWE examination, histopathologic analysis of fibrosis grade (F0 to F3), assessment of necroinflammatory activity, and steatosis grading between August 2016 and March 2020. The liver stiffness from 2D-SWE was compared between fibrosis stages using Kruskal–Wallis analysis. Factors that significantly affected liver stiffness were evaluated using univariate and multivariate linear regression analyses. The diagnostic performance was determined from the area under the receiver operating curve (AUC) values of 2D-SWE liver stiffness. Results Liver stiffness at the F0-1, F2, and F3 stages were 7.9, 13.2, and 21.7 kPa, respectively (P P P = 0.021, respectively). However, in patients with alanine aminotransferase (ALT) levels below 200 IU/L, the only factor affecting liver stiffness was fibrosis stage (P = 0.030). The liver stiffness value could distinguish significant fibrosis (≥ F2) with an AUC of 0.950 (cutoff value, 11.3 kPa) and severe fibrosis (F3 stage) with an AUC of 0.924 (cutoff value, 18.1 kPa). The 2D-SWE values for differentiating significant fibrosis were 10.5 kPa (≥ F2) and 18.1 kPa (F3) in patients with ALT levels below 200 IU/L. Conclusion The liver stiffness values on 2D-SWE can be affected by both fibrosis and necroinflammatory grade and can provide excellent diagnostic performance in evaluating the fibrosis stage in various pediatric liver diseases. However, clinicians should be mindful of potential confounders, such as necroinflammatory activity or transaminase level, when performing 2D-SWE measurements for liver fibrosis staging.

Background The prognostic potential of Crohn-like lymphoid reaction (CLR) in colorectal carcinoma (CRC) has been investigated through the assessment of different criteria. Methods The prognostic impact of CLR was investigated in 636 CRC patients to compare methods from previously published articles. These methods included CLR measured by number of lymphoid aggregates (LAs) (CLR count), LA size greater than or equal to 1 mm (CLR size), CLR density with a cutoff value of 0.38, and subjective criteria as defined by intense CLR. Results In univariate survival analysis, CLR-positive CRC as defined by the four aforementioned methods was associated with better overall survival (OS) (hazard ratio [HR], 0.463; 95% confidence interval [CI], 0.305 to 0.702; p < .001; HR, 0.656; 95% CI, 0.411 to 1.046; p = .077; HR, 0.363; 95% CI, 0.197 to 0.669; p = .001; and HR, 0.433; 95% CI, 0.271 to 0.690; p < .001, respectively) and disease-free survival (DFS) (HR, 0.411; 95% CI, 0.304 to 0.639; p < .001; HR, 0.528; 95% CI, 0.340 to 0.821; p = .004; HR, 0.382; 95% CI, 0.226 to 0.645, p = .004; and HR, 0.501; 95% CI, 0.339 to 0.741; p < .001, respectively) than CLR-negative CRC, regardless of criteria with the exception of OS for CLR density. In multivariate analysis, two objective criteria (CLR count and CLR density) and one subjective criterion (intense CLR) for defining CLR were considered independent prognostic factors of OS and DFS in CRC patients. Conclusions CLR has similar traits regardless of criteria, but CLR-positivity should be defined by objective criteria for better reproducibility and prognostic value.

Gastric cancer (GC) is a leading cause of cancer morbidity and mortality worldwide. This is due to the heterogeneous features of GC, which consist of a diverse molecular phenotype. Epstein–Barr virus (EBV)-positive GC and microsatellite instability (MSI)-high GC encompass similar epigenetic traits, including high levels of DNA methylation in CpG islands; however, EBV-positive and MSI-high GCs are mutually exclusive. We aimed to elucidate the underlying mechanism of this exclusivity. We knocked out MLH1 in EBV-positive GC cell lines SNU-719 and NCC24 via CRISPR-Cas9, and evaluated the modified cellular properties in vitro and in vivo. The MSI status of each cell line was screened with two marker capillary electrophoresis, and further diagnosed with five marker capillary electrophoresis and parallel sequencing using 21 markers. Initial evaluation showed that cell growth, migration, invasion, and MSI status were not affected by MLH1 silencing. However, with prolonged passage, GC cell lines gradually gained MSI and NCC24 cells were transformed to EBV-positive/MSI-high GC cells after 12 months. Furthermore, MLH1 silencing reduced tumor stemness in SNU-719 and NCC24 regardless of the MSI status in vitro and in vivo. Our findings suggest that EBV-positivity and MSI-high status are mutually exclusive due to the immediate disadvantage in tumor stemness when MLH1 is silenced, whereas the establishment of MSI-high status in EBV-positive GCs required a longer period.

It has been suggested that Fusobacterium nucleatum (Fn) may differentially impact tumor immune responses according to microsatellite instability (MSI) status in colorectal cancers (CRCs). We aimed to reveal the detailed relationship between intratumoral Fn and immune microenvironmental features in MSI-high CRCs. A total of 126 MSI-high CRCs were subjected to analyses for intratumoral Fn DNA load using quantitative PCR and for densities of tumor-infiltrating immune cells, including CD3+ T cells, CD4+ T cells, CD8+ T cells, FoxP3+ T cells, CD68+ macrophages, CD163+ macrophages, and CD177+ neutrophils, at invasive margin (IM) and center of tumor (CT) areas using computational image analysis of immunohistochemistry. Based on the Fn load, the 126 MSI-high CRCs were classified into Fn-high, -low, and -negative subgroups. The Fn-high subset of MSI-high CRCs was significantly correlated with larger tumor size and advanced invasion depth (p = 0.017 and p = 0.034, respectively). Compared with the Fn-low/negative subgroup, Fn-high tumors demonstrated significantly lower density of FoxP3+ cells in both IM and CT areas (p = 0.002 and p = 0.003, respectively). Additionally, Fn-high was significantly associated with elevated CD163+ cell to CD68+ cell ratio in only CT areas of MSI-high CRCs (p = 0.028). In conclusion, the Fn-enriched subset of MSI-high CRCs is characterized by increased tumor growth and invasion and distinct immune microenvironmental features, including decreased FoxP3+ T cells throughout the tumor and increased proportion of M2-polarized macrophages in the tumor center. These findings collectively support that Fn may be linked to pro-tumoral immune responses in MSI-high CRCs.

To evaluate the clinical usefulness of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in children and young adults with Crohn’s disease. From August 2017 to October 2018, 30 patients with Crohn’s disease (21 males and 9 females; mean age 15.1 ± 2.5 years) underwent DCE-MRI with MRI enterography. We assessed the endoscopic finding, pediatric Crohn’s disease activity index (PCDAI), C-reactive protein (CRP) level (mg/dL), Crohn’s disease MR index (CDMI) score, and the perfusion parameters of DCE-MRI (Ktrans, Kep, and Ve) at the ileocecal region between the inactive and active groups based on the histopathologic status. The active Crohn’s disease group showed higher PCDAI, CRP, and CDMI scores than the inactive group (22.2 ± 18.8 vs. 6.3 ± 6.4, p = 0.027; 1.32 ± 1.79 vs. 0.10 ± 0.13, p = 0.005; 7.4 ± 3.9 vs. 4.5 ± 3.0, p = 0.047, respectively). The active Crohn’s disease group showed a higher Ktrans value than the inactive group (0.31 ± 0.12 vs. 0.16 ± 0.46 min−1, p = 0.002). Endoscopic finding; PCDAI, CRP, and CDMI scores; and Ktrans value were significant parameters in the identification of the active Crohn’s disease (p = 0.002, p

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.

Purpose: Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established. Experimental Design: We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method. Results: In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04–0.66), P = 0.011]. Conclusions: TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

Purpose The purpose of this study was to evaluate treatment response to neoadjuvant chemoradiotherapy (CRT) with regard to mucin status in pathology and pretreatment magnetic resonance imaging (MRI) in locally advanced rectal cancer. Materials and Methods Between 2003 and 2011, 306 patients with locally advanced rectal cancer received neoadjuvant CRT followed by surgery, and mucinous adenocarcinoma (MAC) was found in 27 (8.8%). All MAC patients had MRI before and after CRT and mucin proportion at MRI was measured. Therapeutic response was assessed by pathology after total mesorectal excision. To determine the optimal cut-off for mucin proportion in predicting good CRT response (near total or total regression) and negative circumferential resection margin (CRM), the receiver-operating characteristic analysis was performed. Results After neoadjuvant CRT, overall downstaging occurred in 44.4% of MAC and 72.4% of non-MAC (p=0.001), and positive CRM (≤1 mm) was observed more frequently in MAC (p

Background SMAD family member 4 (SMAD4) has gained attention as a promising prognostic factor of colorectal cancer (CRC) as well as a key molecule to understand the tumorigenesis and progression of CRC. Methods We retrospectively analyzed 1,281 CRC cases immunohistochemically for their expression status of SMAD4, and correlated this status with clinicopathologic and molecular features of CRCs. Results A loss of nuclear SMAD4 was significantly associated with frequent lymphovascular and perineural invasion, tumor budding, fewer tumor-infiltrating lymphocytes, higher pT and pN category, and frequent distant metastasis. In contrast, tumors overexpressing SMAD4 showed a significant association with sporadic microsatellite instability. After adjustment for TNM stage, tumor differentiation, adjuvant chemotherapy, and lymphovascular invasion, the loss of SMAD4 was found to be an independent prognostic factor for worse 5-year progression-free survival (hazard ratio [HR], 1.27; 95% confidence interval [CI], 1.01 to 1.60; p=.042) and 7-year cancerspecific survival (HR, 1.45; 95% CI, 1.06 to 1.99; p=.022). Conclusions We confirmed the value of determining the loss of SMAD4 immunohistochemically as an independent prognostic factor for CRC in general. In addition, we identified some histologic and molecular features that might be clues to elucidate the role of SMAD4 in colorectal tumorigenesis and progression.

Background Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR. Methods Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-β, and PI3K). Results Mutation of genes within the WNT, P53, RTK-RAS, TGF-β, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-β pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-β pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p

Background We attempted to elucidate whether p53 expression or TP53 mutation status was associated with cancer-specific survival in adjuvant FOLFOX-treated patients with stage III or high-risk stage II colorectal cancer (CRC). Methods We analysed CRCs (N = 621) for the presence of TP53 alterations and for p53 expression, using targeted resequencing and immunohistochemistry. CRCs were grouped into four subsets according to the p53 expression status, which included p53-no, mild, moderate and strong expression. Results The distributions of CRCs were 19.85, 11.05, 17.7% and 51.5% in the p53-no, mild, moderate and strong expression groups, respectively. Cases in the p53-mild to moderate expression group were associated with a more frequent proximal location, undifferentiated histology, lower N category, extraglandular mucin production, microsatellite instability, CIMP-P1, CK7 expression and decreased CDX2 expression compared with those of cases of the p53-no expression and p53-strong expression groups. According to survival analysis, the p53-mild expression group showed a poor 5-year relapse-free survival (hazard ratio (HR): 2.71, 95% confidence interval (CI) = 1.60–4.60, P

Background This study aimed to investigate the prognostic impact of intratumoral Fusobacterium nucleatum in colorectal cancer (CRC) treated with adjuvant chemotherapy. Methods F. nucleatum DNA was quantitatively measured in a total of 593 CRC tissues retrospectively collected from surgically resected specimens of stage III or high-risk stage II CRC patients who had received curative surgery and subsequent oxaliplatin-based adjuvant chemotherapy (either FOLFOXor CAPOX). Each case was classified into one of the three categories: F. nucleatum–high, –low, or –negative. Results No significant differences in survival were observed between the F.nucleatum–high and –low/negative groups in the 593 CRCs (p = .671). Subgroup analyses according to tumor location demonstrated that disease-free survival was significantly better in F.nucleatum–high than in –low/negative patients with non-sigmoid colon cancer (including cecal, ascending, transverse, and descending colon cancers; n = 219; log-rank p = .026). In multivariate analysis, F. nucleatum was determined to be an independent prognostic factor in non-sigmoid colon cancers (hazard ratio, 0.42; 95% confidence interval, 0.18 to 0.97; p = .043). Furthermore, the favorable prognostic effect of F. nucleatum–high was observed only in a non-microsatellite instability-high (non-MSI-high) subset of non-sigmoid colon cancers (log-rank p = 0.014), but not in a MSI-high subset (log-rank p = 0.844), suggesting that the combined status of tumor location and MSI may be a critical factor for different prognostic impacts of F. nucleatum in CRCs treated with adjuvant chemotherapy. Conclusions Intratumoral F. nucleatum load is a potential prognostic factor in a non-MSI-high/non-sigmoid/non-rectal cancer subset of stage II/III CRCs treated with oxaliplatin-based adjuvant chemotherapy.

BACKGROUND Previous studies on synchronous colorectal carcinoma (SCRC) have reported inconsistent results about its clinicopathologic and molecular features and prognostic significance. METHODS Forty-six patients with multiple advanced tumors (T2 or higher category) who did not receive neoadjuvant chemotherapy and/or radiotherapy and who are not associated with familial adenomatous polyposis were selected and 99 tumors from them were subjected to clinicopathologic and molecular analysis. Ninety-two cases of solitary colorectal carcinoma (CRC) were selected as a control considering the distributions of types of surgeries performed on patients with SCRC and T categories of individual tumors from SCRC. RESULTS SCRC with multiple advanced tumors was significantly associated with more frequent nodal metastasis (p = .003) and distant metastasis (p = .001) than solitary CRC. KRAS mutation, microsatellite instability, and CpG island methylator phenotype statuses were not different between SCRC and solitary CRC groups. In univariate survival analysis, overall and recurrence-free survival were significantly lower in patients with SCRC than in patients with solitary CRC, even after adjusting for the extensiveness of surgical procedure, adjuvant chemotherapy, or staging. Multivariate Cox regression analysis revealed that tumor multiplicity was an independent prognostic factor for overall survival (hazard ratio, 4.618; 95% confidence interval, 2.126 to 10.030; p < .001), but not for recurrence-free survival (p = .151). CONCLUSIONS Findings suggested that multiplicity of advanced T category-tumors might be associated with an increased risk of nodal metastasis and a risk factor for poor survival, which raises a concern about the guideline of American Joint Committee on Cancer's tumor-node-metastasis staging that T staging of an index tumor determines T staging of SCRC.

BackgroundNTRK fusions are emerging tissue-agnostic drug targets in malignancies including colorectal cancers (CRCs), but their detailed landscape in the context of various colorectal carcinogenesis pathways remains to be investigated.MethodsPan-TRK expression was assessed by immunohistochemistry in retrospectively collected colorectal epithelial tumor tissues, including 441 CRCs (133 microsatellite instability-high (MSI-high) and 308 microsatellite stable (MSS)) and 565 premalignant colorectal lesions (300 serrated lesions and 265 conventional adenomas). TRK-positive cases were subjected to next-generation sequencing and/or fluorescence in situ hybridization to confirm NTRK rearrangements.ResultsTRK positivity was not observed in any of the MSS CRCs, conventional adenomas, traditional serrated adenomas, or hyperplastic polyps, whereas TRK positivity was observed in 11 of 58 (19%) sporadic MSI-high CRCs, 4 of 23 (17%) sessile serrated lesions with dysplasia (SSLDs), and 5 of 132 (4%) SSLs. The 11 TRK-positive MSI-high CRCs commonly harbored CpG island methylator phenotype-high (CIMP-high), MLH1 methylation, KRAS/BRAF wild-type, and NTRK1 or NTRK3 fusion (TPM3-NTRK1, TPR-NTRK1, LMNA-NTRK1, SFPQ-NTRK1, ETV6-NTRK3, or EML4-NTRK3). Both NTRK1 or NTRK3 rearrangement and KRAS/BRAF wild-type were detected in all nine TRK-positive SSL(D)s, seven of which demonstrated MSS and/or CIMP-low. TRK overexpression and early dysplastic changes are occasionally co-localized in the crypt base area of SSLs. Age-related occurrence patterns suggest that the progression interval from NTRK-rearranged SSLs to CRCs may be shorter than from BRAF-mutated SSLs to CRCs.ConclusionNTRK-rearranged colorectal tumors develop exclusively through the serrated neoplasia pathway and can be initiated from non-dysplastic SSLs without KRAS/BRAF mutations prior to full occurrence of MSI-high/CIMP-high.

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 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.

Background The introduction of complete mesocolic excision (CME) with central vascular ligation (CVL) for right-sided colon cancer has improved the oncologic outcomes. Recently, we have introduced a modified CME (mCME) procedure that keeps the same principles as the originally described CME but with a more tailored approach. Some retrospective studies have reported the favourable oncologic outcomes of laparoscopic mCME for right-sided colon cancer; however, no prospective multicentre study has yet been conducted. Methods This study is a multi-institutional, prospective, single-arm study evaluating the oncologic outcomes of laparoscopic mCME for adenocarcinoma arising from the right side of the colon. A total of 250 patients will be recruited from five tertiary referral centres in South Korea. The primary outcome of this study is 3-year disease-free survival. Secondary outcome measures include 3-year overall survival, incidence of surgical complications, completeness of mCME, and distribution of metastatic lymph nodes. The quality of laparoscopic mCME will be assessed on the basis of photographs of the surgical specimen and the operation field after the completion of lymph node dissection. Discussion This is a prospective multicentre study to evaluate the oncologic outcomes of laparoscopic mCME for right-sided colon cancer. To the best of our knowledge, this will be the first study to prospectively and objectively assess the quality of laparoscopic mCME. The results will provide more evidence about oncologic outcomes with respect to the quality of laparoscopic mCME in right-sided colon cancer. Trial registration ClinicalTrials.gov ID: NCT03992599 (June 20, 2019). The posted information will be updated as needed to reflect protocol amendments and study progress.

Analyzing cell-free DNA (cfDNA) as a source of circulating tumor DNA is useful for diagnosing or monitoring patients with cancer. However, the concordance between cfDNA within liquid biopsy and genomic DNA (gDNA) within tumor tissue biopsy is still under debate. To evaluate the concordance in a clinical setting, we enrolled 54 patients with metastatic colorectal cancer and analyzed their plasma cfDNA, gDNA from peripheral blood mononuclear cells (PBMC), and gDNA from available matched tumor tissues using ultra-deep sequencing targeting 10 genes (38-kb size) recurrently mutated in colorectal cancer. We first established a highly reliable cut-off value using reference material. The sensitivity of detecting KRAS hotspot mutations in plasma was calculated as 100%, according to digital droplet PCR. We could selectively detect clinically important somatic alterations with a variant allele frequency as low as 0.18%. We next compared somatic mutations of the 10 genes between cfDNA and genomic DNA from tumor tissues and observed an overall 93% concordance rate between the two types of samples. Additionally, the concordance rate of patients with the time interval between liquid biopsy and tumor tissue biopsy within 6 months and no prior exposure to chemotherapy was much higher than those without. The patients with KRAS mutant fragments in plasma had poor prognosis than those without the mutant fragments (33 months vs. 63 months; p

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) is expressed in cells at the base of intestinal crypts, acting as a cellular guide in the maintenance of intestinal crypt architecture. We aimed to investigate the expression profile of EPHB3 in colorectal precancerous lesions and colorectal cancers (CRCs), and assess its prognostic value. EPHB3 expression was higher in CRCs than in normal mucosa and was associated with the intestinal stem cell markers EPHB2, OLFM4, LRIG1, and a proposed cancer stem cell marker, CD44. Enhanced EPHB3 expression significantly declined during the transformation from adenoma to carcinoma and as the tumor invaded into deeper tissue layers. Namely, a substantial reduction of EPHB3 expression was observed in the budding cancer cells at the invasive tumor fronts, which was more extensive than E-cadherin downregulation. In an azoxymethane/dextran sulfate sodium-induced, colitis-associated, CRC model, EPHB3 expression increased along with tumor development. In a large cohort of CRC patients, EPHB3 positivity was observed in 24% of 610 CRCs and was negatively correlated with tumor differentiation, lympho-vascular invasion, and tumor, node, and metastasis stages. EPHB3 was positively associated with microsatellite instability but was associated with neither CpG island methylation, nor with KRAS and BRAF mutations. Notably, EPHB3 positivity was associated with better clinical outcomes, although it was not an independent prognostic marker. Overexpression of EPHB3 in the colon cancer cell line, DLD1, led to decreased cell growth and migration and reduced mitogen-activated protein kinase signaling. Taken together, our data demonstrate the suppressive role of EPHB3 in CRC progression.

Here, we provide an up-to-date review of the histopathology and molecular pathology of serrated colorectal lesions. First, we introduce the updated contents of the 2019 World Health Organization classification for serrated lesions. The sessile serrated lesion (SSL) is a new diagnostic terminology that replaces sessile serrated adenoma and sessile serrated polyp. The diagnostic criteria for SSL were revised to require only one unequivocal distorted serrated crypt, which is sufficient for diagnosis. Unclassified serrated adenomas have been included as a new category of serrated lesions. Second, we review ongoing issues concerning the morphology of serrated lesions. Minor morphologic variants with distinct molecular features were recently defined, including serrated tubulovillous adenoma, mucin-rich variant of traditional serrated adenoma (TSA), and superficially serrated adenoma. In addition to intestinal dysplasia and serrated dysplasia, minimal deviation dysplasia and not otherwise specified dysplasia were newly suggested as dysplasia subtypes of SSLs. Third, we summarize the molecular features of serrated lesions. The critical determinant of CpG island methylation development in SSLs is patient age. Interestingly, there may be ethnic differences in BRAF/KRAS mutation frequencies in SSLs. The molecular pathogenesis of TSAs is divided into KRAS and BRAF mutation pathways. SSLs with MLH1 methylation can progress into favorable prognostic microsatellite instability-positive (MSI+)/CpG island methylator phenotype-positive (CIMP+) carcinomas, whereas MLH1-unmethylated SSLs and BRAF-mutated TSAs can be precursors of poor-prognostic MSI−/CIMP+ carcinomas. Finally, based on our recent data, we propose an algorithm for stratifying risk subgroups of non-dysplastic SSLs.

Background: The prognostic value of immune cells expressing programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) in cancer are controversial, and the potential differential impact of using tissue microarrays and whole tissue sections to assess the positivity of immune cells has not been addressed. Methods: The current study included 30 eligible studies with 7251 patients that evaluated the relationship between tumor-infiltrating lymphocytes expressing PD-1/PD-L1 and overall survival and disease-free survival, or progression-free survival. Subgroup analysis was based on the tissue type of cancer and the type of tissue sampling (tissue microarray or whole tissue section). Results: In the meta-analysis, PD-1-positive and PD-L1-positive tumor-infiltrating lymphocytes had a positive effect on disease-free survival or progression-free survival (hazard ratio [HR] 0.732; 95% confidence interval [CI] 0.565, 0.947; and HR 0.727; 95% CI 0.584, 0.905, respectively). PD-L1-positive tumor-infiltrating lymphocytes had a positive impact on overall survival in studies using tissue microarray (HR 0.586; 95% CI 0.476, 0.721), but had a poor impact when only whole tissue sections were considered (HR 1.558; 95% CI 1.232, 1.969). Lung cancer was associated with good overall survival and disease-free survival (HR 0.639; 95% CI 0.491, 0.831; and HR 0.693; 95% CI 0.538, 0.891, respectively) for PD-1-positive tumor-infiltrating lymphocytes, and colorectal cancer showed favorable disease-free survival (HR 0.471; 95% CI 0.308, 0.722) for PD-L1-positive tumor-infiltrating lymphocytes. Conclusion: Immune cells expressing PD-1 and PD-L1 within tumors are associated with the prognosis. However, the correlation may vary among different tumor types and by the type of tissue sampling used for the assessment.

Aims The precise profile of aberrant expression of thyroid transcription factor-1 (TTF-1) according to antibody clones in colorectal carcinomas (CRCs) has been controversial. Moreover, the detailed clinicopathological and molecular features of CRCs with TTF-1 expression have rarely been investigated. Here, we evaluated the TTF-1 expression status in a large series of CRC cases using three different antibody clones. Methods and Results Immunohistochemistry for TTF-1 using clones 8G7G3/1, SPT24, and SP141 was performed on tumour tissues of 1,319 primary CRCs and 98 corresponding metastatic lesions. Among the 1,319 CRCs, TTF-1 expression was detected in 68 cases using both clones SPT24 and SP141. TTF-1 expression was not detected in any of the cases when clone 8G7G3/1 was used. The 68 CRCs with TTF-1 expression detected using both the SPT24 and SP141 clones were considered TTF-1-positive in this study. TTF-1 positivity was significantly associated with distal tumour location, non-mucinous histology, intact CDX2 expression, and low frequency of KRAS mutations in CRCs. Nearly all TTF-1-positive CRCs showed microsatellite-stable and CpG island methylator phenotype-negative statuses. TTF-1 positivity was also found in all metastatic lesions of the five TTF-1-positive primary CRCs. TTF-1 negativity was maintained in all metastatic lesions of the 93 TTF-1-negative primary CRCs. Conclusions Our study confirmed that the frequency and characteristics of aberrant TTF-1 expression in CRCs vary depending on the antibody clone. Aberrant TTF-1 expression detected by clone SPT24 or SP141 may be encountered preferentially in distally located, conventional pathway-type CRCs. This article is protected by copyright. All rights reserved.

Purpose To evaluate chemoradiotherapy (CRT)-induced changes in the expression levels of programmed death-ligand 1 (PD-L1) and CD8 + tumor-infiltrating lymphocytes (TILs) and prognostic associations in rectal cancer. Methods and Materials We performed a paired analysis using pre-CRT biopsies and the corresponding post-CRT resected tissues of 123 rectal cancer patients undergoing preoperative CRT followed by surgery between 2005 and 2012. Immunohistochemistry of PD-L1 and CD8 was analyzed for the specimens. Results The expression levels of PD-L1 and density of CD8 + TILs increased after CRT ( P + TILs ( P =.020). Patients representing sustained high-to-high PD-L1 expression had poorer overall survival and disease-free interval on univariate Kaplan-Meier analysis ( P =.018 and .029, respectively), with inferior disease-free interval in low-to-low density CD8 + TILs ( P =.010). On multivariate analysis, 2 subgroups with high baseline PD-L1 expression level, the high-to-low and high-to-high alterations, showed worse overall survival (hazard ratio 8.34, 95% confidence interval 1.85-37.53 and hazard ratio 11.03, 95% confidence interval 2.33-52.29, respectively), with the highest mortality risk observed in the high-to-high group. Conclusions This study verified the CRT-induced immunologic shift toward increases in PD-L1 expression and density of CD8 + TILs in rectal cancer patients. The alteration profiles of checkpoint-related molecules identified the patients with poor prognosis, suggesting potential candidates who can benefit from combining CRT and checkpoint inhibitors.

Patients with lysosomal acid lipase (LAL) deficiency and glycogen storage disease (GSD) demonstrated hepatomegaly and dyslipidemia. In our case, a 6-year-old boy presented with hepatosplenomegaly. At 3 years of age, GSD had been diagnosed by liver biopsy at another hospital. He showed elevated serum liver enzymes and dyslipidemia. Liver biopsy revealed diffuse microvesicular fatty changes in hepatocytes, septal fibrosis and foamy macrophages. Ultrastructural examination demonstrated numerous lysosomes that contained lipid material and intracytoplasmic cholesterol clefts. A dried blood spot test revealed markedly decreased activity of LAL. LIPA gene sequencing identified the presence of a novel homozygous mutation (p.Thr177Ile). The patient's elevated liver enzymes and dyslipidemia improved with enzyme replacement therapy. This is the first report of a Korean child with LAL deficiency, and our findings suggest that this condition should be considered in the differential diagnosis of children with hepatosplenomegaly and dyslipidemia.

Summary Background CT radiation is arguably carcinogenic. Results from single-centre studies, mostly retrospective, have advocated lowering the CT radiation dose for the diagnosis of appendicitis. However, adoption of low-dose CT has been slow. We aimed to assess the effectiveness of low-dose CT compared with standard-dose CT in the diagnosis of appendicitis in adolescents and young adults. Methods We did this pragmatic, multicentre, randomi s ed controlled non-inferiority trial at 20 South Korean teaching hospitals with little experience with low-dose CT. Patients aged 15–44 years with suspected appendicitis were randomly assigned (1:1), via computer-generated random assignments (permuted block sizes of two, four, six, and eight) concealed in sequentially numbered envelopes, to receive low-dose CT (2 mSv) or standard-dose CT (≤8 mSv). Randomisation was stratified by site. Group allocation was concealed from patients, outcome assessors, and adverse event adjudicators; care providers, site pathologists, and data collectors were aware of allocation. The primary endpoint was the negative (unnecessary) appendectomy rate among all appendectomies, with a non-interiority margin of 4·5% for low-dose versus standard-dose CT. Primary analy s is was by modified intention to treat, which included all patients who received an appendectomy in the group to which they were assigned. This trial is registered with ClinicalTrials.gov, number NCT01925014. Findings Between Dec 4, 2013, and Aug 18, 2016, we assigned 1535 patients to the low-dose CT group and 1539 patients to the standard-dose CT group. 22 (3·9%) of 559 patients had a negative appendectomy in the low-dose group versus 16 (2·7%) of 601 patients in the standard-dose group (difference 1·3%, 95% CI −0·8 to 3·3; p=0·0022 for the non-inferiority test). We recorded 43 adverse events in 43 (2·8%) of 1535 patients in the low-dose group and 41 adverse events in 40 (2·6%) of 1539 patients in the standard-dose group. One life-threatening adverse event of anaphylaxis caused by an iodinated contrast material occurred in the low-dose group. Interpretation Radiation dose of appendiceal CT for adolescents and young adults can be reduced to 2 mSv without impairing clinical outcomes. In view of the vast number of appendiceal CT examinations done worldwide, use of low-dose CT could prevent a sizeable number of radiation-associated cancers in the future. Funding Korea Health Industry Development Institute, Seoul National University Bundang Hospital, Dasol Life Science, and Bracco Imaging Korea.

Summary Deletion of the HSP110 T 17 mononucleotide repeat has recently been identified as a prognostic marker that is correlated with wild-type HSP110 (HSP110wt) expression in microsatellite instability–high (MSI-H) colorectal cancers. The aim of this study was to assess the correlation between deletion of the HSP110 T 17 repeat and expression of HSP110wt using DNA testing and immunohistochemistry and to determine the prognostic implications of HSP110 T 17 deletion in MSI-H advanced gastric cancers (GCs). The status of HSP110wt expression was evaluated by immunohistochemistry using an HSP110wt-specific antibody in 142 MSI-H advanced GCs. The size of the HSP110 T 17 repeat deletion was analyzed in 96 MSI-H advanced GCs; deletions were divided into small (0-2base pairs) and large deletions (3-5base pairs). Low and high expressions of HSP110wt were detected in 38 (26.8%) and 104 (73.2%) of the 142 cases, respectively. The HSP110 T 17 deletion was observed in 45 (46.9%) of the 96 MSI-H GC samples. Tumors with high expression of HSP110wt showed a tendency to have small or no deletion of HSP110 T 17 . In Kaplan-Meier survival analysis, tumors with a large HSP110 T 17 deletion were associated with favorable overall survival and disease-free survival compared with those with small/no deletion of HSP110 T 17 . However, HSP110 T 17 deletion size was not an independent prognostic factor in multivariate analysis. In summary, deletion of the HSP110 T 17 repeat was frequently observed in MSI-H GCs, and HSP110 T 17 deletion size was inversely correlated with HSP110wt expression status. Large HSP110 T 17 was not a prognostic indicator in MSI-H GCs.

BACKGROUND Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. METHODS Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor β [TGF-β]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. RESULTS The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-β pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. CONCLUSIONS A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society.

Background Tumor microenvironment has recently drawn attention in that it is related with tumor prognosis. Cancer-associated fibroblast also plays a critical role in cancer invasiveness and progression in colorectal cancers. Periostin (POSTN), originally identified to be expressed in osteoblasts and osteoblast-derived cells, is expressed in cancer-associated fibroblasts in several tissue types of cancer. Recent studies suggest an association between stromal overexpression of POSTN and poor prognosis of cancer patients. Methods We analyzed colorectal cancer cases for their expression status of POSTN in tumor stroma using immunohistochemistry and correlated the expression status with clinicopathological and molecular features. Results High level of POSTN expression in tumor stroma was closely associated with tumor location in proximal colon, infiltrative growth pattern, undifferentiated histology, tumor budding, luminal necrosis, and higher TNM stage. High expression status of POSTN in tumor stroma was found to be an independent prognostic parameter implicating poor 5-year cancer-specific survival and 5-year progression-free survival. Conclusions Our findings suggest that POSTN overexpression in tumor stroma of colorectal cancers could be a possible candidate marker for predicting poor prognosis in patients with colorectal cancers.

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.

Background/Aims To evaluate the expression of cellular inhibitor of apoptosis protein 2 (cIAP2) during gastric carcinogenesis after Helicobacter pylori (HP) infection and after HP eradication. Methods We divided non-cancer patients into four groups according to the status of HP infection and atrophic gastritis (AG)/intestinal metaplasia (IM). We compared cIAP2 mRNA expression among these four groups and patients with HP-positive early gastric cancer (EGC) by using real-time polymerase chain reaction (PCR). We evaluated the expression of cIAP2 messenger RNA (mRNA)/protein by using real-time PCR/immunohistochemistry and the degree of apoptosis with a terminal deoxynucleotidyl transferase-mediated nick end labeling assay before and 12 months after endoscopic submucosal dissection (ESD) in HP-positive EGC patients, regardless of whether they had undergone eradication therapy. Results The expression of cIAP2 mRNA was significantly higher in the groups with HP(+), AG/IM(+), and HP-positive EGC than in the control, HP(+), and AG/IM(−) groups (p

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. 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. 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. 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.

CD103 is a transmembrane heterodimer complex that mediates cell adhesion, migration, and lymphocyte homing of cell through interaction with E-cadherin. Recently, CD103+ immune cells in human carcinoma has been investigated as a prognostic factor, however, the correlation between CD103+ immune cells and survival are still elusive. Therefore, a meta-analysis was performed to determine the prognostic value of CD103+ immune cells in solid tumor. Studies relevant to the subject was searched from PubMed, Embase, and Web of Science. Ten studies including 2,824 patients were eligible for the analysis. Tumors positive for CD103+ immune cells were associated with favorable overall survival, disease-free survival, and disease-specific survival. Subgroup analysis revealed that assessing CD103+ immune cells in epithelial and total (both epithelial and stromal) areas or using whole slide section were associated with good prognosis. Furthermore, stromal CD103+ immune cells or CD103+ immune cells evaluated by tissue microarrays were not always significantly prognostic. In conclusion, these results show that CD103+ immune cells are associated with prognosis in solid tumor. However, the region of assessment and selection of material for the evaluation could affect the value of CD103 as a prognostic biomarker.

Purpose The purpose of this study was to reveal the clinicopathological characteristics and prognostic implications associated with fibroblast growth factor receptor 1 (FGFR1) amplification in colorectal cancers (CRCs). Materials and methods We measured the copy number of FGFR1 by droplet digital polymerase chain reaction (ddPCR), and analyzed the FGFR1 expression by immunohistochemistry, in 764 surgically resected CRCs (SNUH2007 dataset, 384 CRCs; SNUH Folfox dataset, 380 CRCs). Results CRCs with ≥ 3.3 copies of the FGFR1 gene were classified as FGFR1 amplified. FGFR1 amplification was found in 10 of the 384 CRCs (2.6%) in the SNUH2007 dataset, and in 28 of the 380 CRCs (7.4%) in the SNUH Folfox dataset. In the SNUH2007 dataset, there was no association between the FGFR1 copy number status and sex, gross appearance, stage, or differentiation. High FGFR1 expression was associated with female sex and KRAS mutation. At the molecular level, FGFR1 amplification was mutually exclusive with BRAF mutation, microsatellite instability, and MLH1 methylation, in both SNUH2007 and SNUH Folfox datasets. Survival analysis revealed that FGFR1 amplification was associated with significantly worse clinical outcome compared with no FGFR1 amplification, in both SNUH2007 and SNUH Folfox datasets. Within the SNUH2007 dataset, CRC patients with high FGFR1 expression had an inferior progression-free survival compared with those with low FGFR1 expression. The FGFR inhibitor, PD173074, repressed the proliferation of a CRC cell line overexpressing FGFR1, but not of cells with FGFR1 amplification. Conclusion FGFR1 amplification measured by ddPCR can be a prognostic indicator of poor clinical outcome in patients with CRCs.

In the present study, we developed a computational method and panel markers to assess microsatellite instability (MSI) using a targeted next-generation sequencing (NGS) platform and compared the performance of our computational method, mSILICO, with that of mSINGS to detect MSI in CRCs. We evaluated 13 CRC cell lines, 84 fresh and 119 formalin-fixed CRC tissues (including 61 MSI-high CRCs and 155 microsatellite-stable CRCs) and tested the classification performance of the two methods on 23, 230, and 3,154 microsatellite markers. For the fresh tissue and cell line samples, mSILICO showed a sensitivity of 100% and a specificity of 100%, regardless of the number of panel markers, whereas for the formalin-fixed tissue samples, mSILICO exhibited a sensitivity of up to 100% and a specificity of up to 100% with three differently sized panels ranging from 23 to 3154. These results were similar to those of mSINGS. With the application of mSILICO, the small panel of 23 markers had a sensitivity of ≥95% and a specificity of 100% in cell lines/fresh tissues and formalin-fixed tissues of CRC. In conclusion, we developed a new computational method and microsatellite marker panels for the determination of MSI that does not require paired normal tissues. A small panel could be integrated into the targeted NGS panel for the concurrent analysis of single nucleotide variations and MSI.

Cancer tissues have characteristic DNA methylation profiles compared with their corresponding normal tissues that can be utilized for cancer diagnosis with liquid biopsy. Using a genome-scale DNA methylation approach, we sought to identify a panel of DNA methylation markers specific for cell-free DNA (cfDNA) from patients with colorectal cancer (CRC). By comparing DNA methylomes between CRC and normal mucosal tissues or blood leukocytes, we identified eight cancer-specific methylated loci (ADGRB1, ANKRD13, FAM123A, GLI3, PCDHG, PPP1R16B, SLIT3, and TMEM90B) and developed a five-marker panel (FAM123A, GLI3, PPP1R16B, SLIT3, and TMEM90B) that detected CRC in liquid biopsies with a high sensitivity and specificity with a droplet digital MethyLight assay. In a set of cfDNA samples from CRC patients (n = 117) and healthy volunteers (n = 60), a panel of five markers on the platform of the droplet digital MethyLight assay detected stages I&ndash, III and stage IV CRCs with sensitivities of 45.9% and 95.7%, respectively, and a specificity of 95.0%. The number of detected markers was correlated with the cancer stage, perineural invasion, lymphatic emboli, and venous invasion. Our five-marker panel with the droplet digital MethyLight assay showed a high sensitivity and specificity for the detection of CRC with cfDNA samples from patients with metastatic CRC.

Context.—Colorectal cancer is a heterogeneous disease entity with 3 molecular carcinogenesis pathways and 2 morphologic multistep pathways. Right-sided colon cancers and left-sided colon and rectal cancers exhibit differences in their incidence rates according to geographic region, age, and sex. A linear tendency toward increasing frequencies of microsatellite instability–high or CpG island methylator phenotype–high cancers in subsites along the bowel from the rectum to the cecum or the ascending colon accounts for the differences in tumor phenotypes associated with these subsites. The molecular subtypes of colorectal cancers exhibit different responses to adjuvant therapy, which might be responsible for differences in subtype-specific survival. Objectives.—To review the clinicopathologic and molecular features of the molecular subtypes of colorectal cancer generated by combined CpG island methylator phenotype and microsatellite statuses, to integrate these features with the most recent findings in the context of the prognostic implications of molecular subtypes, and to emphasize the necessity of developing molecular markers that enable the identification of adenocarcinomas involving the serrated neoplasia pathway. Data Sources.—Based on the authors' own experimental data and a review of the pertinent literature. Conclusions.—Because colorectal cancers arise from 2 different morphologic multistep carcinogenesis pathways with varying contributions from 3 different molecular carcinogenesis pathways, colorectal cancer is a heterogeneous and complex disease. Thus, molecular subtyping of colorectal cancers is an important approach to characterizing their heterogeneity with respect to not only prognosis and therapeutic response but also biology and natural history.

Purpose The aim of our study was to establish a safe and convenient diagnostic method for acute gastrointestinal (GI) graft-versus-host disease (GVHD) in children by determining the sensitivity and negative predictive values of upper and lower endoscopic biopsies for children suspected of GI GVHD. Methods Patients suspected of GI GVHD who received endoscopic evaluation within 100 days after stem cell transplantation and endoscopies between January 2012 and March 2014 in Seoul National University Children's Hospital were included in our study. Results Fifteen patients with a total of 20 endoscopic procedures were included in our study. Sensitivity at the esophagus, stomach, and duodenum were 22.2%, 30.0%, and 80.0%, respectively. Negative predictive values at the esophagus, stomach, and duodenum were 22.2%, 30.0%, and 60.0%, respectively. Overall sensitivity and negative predictive values of upper endoscopic biopsy for GVHD were 77.8% and 50.0%, respectively. Overall sensitivity and negative predictive values of lower endoscopic biopsy for GVHD were 88.9% and 66.7%, respectively. Conclusion We recommend flexible sigmoidoscopy as a safe and accurate diagnostic tool for GVHD, similar to other studies reported previously. However, if there is no evidence of GVHD on sigmoidoscopy with high index of suspicion of GI bleeding, full colonoscopy and upper endoscopy should be considered.

Angiodysplasia (AD) is increasingly being recognized as a major cause of gastrointestinal bleeding. Morphologically flat lesions are common types of AD, whereas the polypoid types are rare. We report a case of multiple polypoid AD in the small bowel causing severe anemia and requiring surgical treatment. A 60-year-old male patient visited our hospital with dyspnea and hematochezia. He had a history of myocardial infarction and was taking both aspirin and clopidogrel. Capsule endoscopy, enteroscopy, computed tomography, and angiography revealed multifocal vascular lesions with a polypoid shape in the jejunum. Surgical resection was performed because endoscopic treatment was considered impossible with the number and the location of lesions. The risk of recurrent bleeding related to the use of antiplatelet agents also contributed to the decision to perform surgery. AD was histologically diagnosed from the surgical specimen. He resumed taking both aspirin and clopidogrel after surgery. He fully recovered and has been doing well during the several months of follow-up.

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.

Giant cell hepatitis with autoimmune hemolytic anemia (AHA) is a rare disease of infancy characterized by the presence of both Coombs-positive hemolytic anemia and progressive liver disease with giant cell transformation of hepatocytes. Here, we report a case involving a seven-month-old male infant who presented with AHA followed by cholestatic hepatitis. The clinical features included jaundice, pallor, and red urine. Physical examination showed generalized icterus and splenomegaly. The laboratory findings suggested warm-type AHA with cholestatic hepatitis. Liver biopsy revealed giant cell transformation of hepatocytes and moderate lobular inflammation. The patient was successfully treated with four doses of rituximab. Early relapse of hemolytic anemia and hepatitis was observed, which prompted the use of an additional salvage dose of rituximab. He is currently in clinical remission.

BACKGROUND A receptor tyrosine kinase for ephrin ligands, EPHB2, is expressed in normal colorectal tissues and colorectal cancers (CRCs). The aim of this study was to investigate EPHB2 expression over CRC progression and determine its prognostic significance in CRC. METHODS To measure EPHB2 mRNA and protein expression, real-time polymerase chain reaction and immunohistochemistry were performed in 32 fresh-frozen and 567 formalin-fixed paraffin-embedded CRC samples, respectively. We further investigated clinicopathological features and overall and recurrence-free survival according to EPHB2 protein expression. RESULTS The EPHB2 level was upregulated in CRC samples compared to non-cancerous tissue in most samples and showed a strong positive correlation with AXIN2. Notably, CD44 had a positive association with both mRNA and protein levels of EPHB2. Immunohistochemical analysis revealed no difference in EPHB2 expression between adenoma and carcinoma areas. Although EPHB2 expression was slightly lower in invasive fronts compared to surface area (p < .05), there was no difference between superficial and metastatic areas. EPHB2 positivity was associated with lymphatic (p < .001) and venous (p = .001) invasion, TNM stage (p < .001), and microsatellite instability (p = .036). Kaplan-Meier analysis demonstrated that CRC patients with EPHB2 positivity showed better clinical outcomes in both overall (p = .049) and recurrence-free survival (p = .015). However, multivariate analysis failed to show that EPHB2 is an independent prognostic marker in CRCs (hazard ratio, 0.692; p = .692). CONCLUSIONS Our results suggest that EPHB2 is overexpressed in a subset of CRCs and is a significant prognostic marker.

There is a close link between Fusobacterium nucleatum and colorectal cancer (CRC) tumorigenesis and chemoresistance. However, the genetic characteristics and clinical significance of CRC related with F. nucleatum remains unclear. This study evaluated the relationship between F. nucleatum, pathway mutation, and patient prognosis. Fusobacterium nucleatum amount in the tumor tissue and adjacent normal tissue were measured by quantitative polymerase chain reaction in adjuvant (N = 128) and metastatic (N = 118) cohorts. Patients were divided into binary (F. nucleatum-high and F. nucleatum-low) according to F. nucleatum amount. Targeted next-generation sequencing of 40 genes included in the 5 critical pathways (WNT, P53, RTK-RAS, PI3 K, and TGF-β) was performed in the adjuvant cohort. Patients with MSI-H and CIMP-H had higher amount of F. nucleatum in tumor tissue. Fusobacterium nucleatum-high patients had higher rates of transition mutation and C to T (G to A) nucleotide change regardless of MSI status. In addition, mutation rate of AMER1 and ATM genes, and TGF-β pathway was higher in F. nucleatum-high patients. Fusobacterium nucleatum-high was associated with poor overall survival (OS) in the palliative cohort (26.4 vs. 30.7 months, p = 0.042). Multivariate analysis revealed F. nucleatum-high as an independent negative prognostic factor for OS [adjusted hazard ratio of 1.69 (95% confidence interval 1.04–2.75), p = 0.034]. However, F. nucleatum amount was not associated with recurrence in the adjuvant cohort. F. nucleatum-high was associated with poor survival in metastatic CRC. In addition, we identified mutational characteristics of colorectal cancer according to F. nucleatum amount.

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 β-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.

// Jung Ho Kim 1, * , Jeong Mo Bae 2, * , Young Seok Song 3 , Nam-Yun Cho 3 , Hye Seung Lee 4 , Gyeong Hoon Kang 2, 3 1 Department of Pathology, SMG-SNU Boramae Medical Center, Seoul, Korea 2 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea 3 Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea 4 Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea * These authors have contributed equally to this work Correspondence to: Gyeong Hoon Kang, e-mail: ghkang@snu.ac.kr Keywords: EPCAM, immunohistochemistry, colorectal cancer, lynch syndrome, prognosis, Pathology Section Received: March 15, 2015 Accepted: September 08, 2015 Published: October 30, 2015 ABSTRACT We aimed to comprehensively investigate the clinicopathologic and molecular implications of altered epithelial cell adhesion molecule (EPCAM) expression in colorectal carcinoma (CRC). EPCAM immunohistochemical expression, EPCAM 3′ end deletion, EPCAM promoter methylation, microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP) were analyzed in large cohorts of human CRCs. Among 218 MSI-high CRCs, complete loss (CL) of EPCAM expression was observed in two cases, both of which displayed MSH2 deficiency and EPCAM 3′ deletion. Thirty-one of the 218 MSI-high CRCs demonstrated the partial loss (PL) of EPCAM expression without EPCAM deletion or methylation and were correlated with CIMP-high and poor disease-free survival. Histologically, foci exhibiting EPCAM loss in EPCAM-PL tumors were dominantly distributed in poorly differentiated tumor components and/or in the invasive tumor front. The implications of EPCAM-PL were further validated in a consecutive series of 726 CRCs. EPCAM-PL ( n = 50; 6.9%) was also associated with CIMP-high and adverse pathologic factors and was confirmed to be an independent poor prognostic factor in CRC (HR, 1.57; 95% CI, 1.04 to 2.39). EPCAM-CL can be used to screen for EPCAM deletion-induced Lynch syndrome-associated CRC, whereas EPCAM-PL can be used as an indicator of tumor aggressiveness and poor prognosis in CRC.

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.

In the current management strategy for colorectal cancer (CRC), fluoropyrimidine-based adjuvant chemotherapy is generally recommended for all patients with stage III CRC and for a subset of patients with high-risk stage II CRC [1]. However, some previous studies have reported that in patients with microsatellite instability–high (MSI-H) CRC, adjuvant chemotherapy based on fluorouracil, which is the most commonly used intravenous fluoropyrimidine agent, had little or no benefit [2]. Although the mechanisms underlying and the factors determining this poor response to fluorouracil-based adjuvant chemotherapy in MSIH CRC patients remain incompletely understood, previous in vitro experiments have revealed that intact DNA mismatch repair function is necessary for fluorouracil to induce apoptotic effects on cancer cells [3,4]. This finding supports the observed resistance of patients with MSI-H CRC to fluorouracil-based adjuvant chemotherapy. MSI-H CRC is characterized by unique pathologic features, including predilections for proximal tumor location, mucinous histology, medullary tumor morphology, signet ring cell tumor component, poor tumor differentiation, tumor-infiltrating lymphocytes, Crohn-like lymphoid reaction and peritumoral lymphoid reaction [5]. Molecularly, MSI-H CRC is caused by DNA mismatch repair deficiency, which is usually due to the inactivation of at least one of the mismatch repair genes, including MLH1, MSH2, MSH6, and PMS2, by germline mutation or acquired promoter hypermethylation [2]. Recent investigations have also determined that germline EPCAM deletion-induced MSH2 epimutation can be one of the causes of Lynch syndromeassociated MSI-H CRC [6,7]. In addition, it is well known that sporadic MSI-H CRC is closely associated with MLH1 methylation, CpG island methylator phenotype and BRAF V600E mutations [2]. Based on the pathologic and molecular heterogeneity of MSI-H CRC, it is strongly expected that there may be pathologic or molecular factors affecting prognostic heterogeneity and differential chemotherapy responses in MSI-H CRC [2]. In this context, our previous investigation revealed that the concurrent loss of caudal type homeobox 2 (CDX2) and cytokeratin 20 (CK20) expression in tumors indicates an aggressive clinical phenotype that is associated with early death or tumor recurrence in patients with MSI-H CRC [8]. Ricciardiello et al. [9] previously reported that in CRC patients, MSI-H status is associated with high expression of thymidylate synthase (TS), the target molecule for fluorouracil. Accordingly, this finding could be a putative underlying mechanism of resistance to fluorouracil chemotherapy in MSI-H CRC patients [9], although this has not been clearly shown [10-12]. Recently, Dorard et al. [13] demonstrated that mutant HSP110 expression and its causal mutation, HSP110 T17 microsatellite deletions, can be prognostic and predictive markers in MSI-H CRC [13]. Furthermore, we have also identified the usefulness of wild-type HSP110 (HSP110wt) immunohistochemistry (IHC) for prognostication in MSI-H CRC [14]. MSI-H CRC is associated with various features, but definitive pathologic or molecular factors that can be used to predict the response to adjuvant chemotherapy in patients with MSI-H CRC have yet to be fully identified. Therefore, we decided to investigate the comprehensive pathologic features that are potentially associated with postchemotherapeutic prognosis in MSI-H CRC patients. Through this intensive analysis, we anticipated identifying the major determining factors for chemotherapy response in MSI-H CRC patients that would be helpful for predicting patient prognosis and establishing treatment strategies in the clinical setting.

The aim of this study is to establish heat shock protein 110 (HSP110) as a prognostic biomarker of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) by considering the intratumoral heterogeneity of HSP110 expression. We performed whole-section immunohistochemistry (IHC) for wild-type HSP110 (HSP110wt) in 164 MSI-H CRCs. The intensity of the HSP110wt expression in tumor cells was semiquantitatively scored (0/1/2/3), and the HSP110wt expression status of each tumor was classified as low or high using the following four scoring criteria: H-score, dominant intensity score, lowest intensity score, and highest intensity score. Among the four criteria, only the dominant intensity score-based dichotomous classification of HSP110wt expression was significantly associated with a difference in disease-free survival (log-rank p = 0.035) in 164 MSI-H CRCs. The HSP110wt-low MSI-H CRCs were significantly correlated with larger deletions in the HSP110 T17 mononucleotide repeat (≥4 bp; p