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Progranulin (PGRN) has been characterized as an autocrine growth and survival factor and is known to stimulate tumorigenesis and proliferation of several types of cancer cell. However, little is known about the prognostic role of PGRN in colorectal cancer (CRC). A retrospective analysis was performed for patients with colorectal cancer who underwent curative resection between May 2013 and June 2015. PGRN expression in tumor cells was semi-quantitatively categorized (no expression, 0; weak/focal, 1+; moderate/focal or diffuse, 2+; strong/diffuse, 3+), and high expression was considered for tumors graded ≥2+ staining intensity. A total of 109 patients (28 stage I, 32 stage II, and 49 stage III) were analyzed. Thirty-eight patients (35%) had tumors with high PGRN expression, and there was a trend of elevated pre-operative CEA and CA19-9 levels in patients with high PGRN-expressing tumors compared to those with low PGRN-expressing tumors (CEA, 49% vs. 21%; CA19-9, 21% vs. 7%). The 3-year recurrence-free survival (3Y-RFS) and overall survival rates were 83.7% (95% CI, 76.8-90.6) and 96.0% (95% CI, 92.3-99.7), respectively. Patients with high PGRN-expressing tumors had a worse rate of 3Y-RFS (66.8%) compared to those with low PGRN-expressing tumors (92.4%; p = 0.010). Multivariate analysis showed that high PGRN expression, age (>66 years), stage (III), and perineural invasion (+) were independent prognostic factors associated with poor RFS after adjusting for confounding factors including sex, MSI, tumor location, KRAS, and lympho-vascular invasion. PGRN overexpression was significantly associated with poor RFS in patients with CRC who have undergone curative resection.

Background: With the recent development of molecular tests for various biomarkers, it has become even more important to prepare adequate tissue samples. However, little is known about how the effect of cold ischemia time or formalin fixation time can affect KRAS mutation detection in colorectal cancer., Methods: This study included the results of KRAS mutation tests for colorectal cancer in 401 specimens. We investigated clinicopathologic factors that may affect DNA quality of formalin-fixed, paraffin-embedded (FFPE) tissue including specimen type, cold ischemia time, and formalin fixation time and assessed the detection rate of the KRAS mutation in samples with varying DNA quality., Results: Sample DNA quality for KRAS mutation test was better in biopsy specimens, which showed markedly shorter cold ischemia time and shorter formalin fixation time compared to resection specimens. A cold ischemia time of one hour or less was associated with better sample DNA quality. But the formalin fixation time was not a significant factor when it fell within the range performed in routine pathology diagnosis. When prolonged formalin fixation was tested, we confirmed that the specimen DNA quality gradually got worse from one month to three months., Conclusions: The biopsy specimens showed better sample DNA quality for KRAS mutation test compared to resection specimens. In a routine diagnostic pathology setting, the cold ischemia time was an important factor affecting DNA quality and the formalin fixation had a wide time range for optimal DNA quality., (Copyright © 2019. Published by Elsevier GmbH.)

Background: More than half of the patients selected based on KRAS mutation status fail to respond to the treatment with cetuximab in metastatic colorectal cancer (mCRC). We designed a study to identify additional biomarkers that could act as indicators for cetuximab treatment in mCRC., Methods: We investigated 58 tumor samples from wild type KRAS CRC patients treated with cetuximab plus irinotecan (CI). We conducted the genotyping for mutations in either BRAF or PIK3CA and profiled comprehensively the expression of 522 kinase genes., Results: BRAF mutation was detected in 5.1 % (3/58) of patients. All 50 patients showed wild type PIK3CA. Gene expression patterns that categorized patients with or without the disease control to CI were compared by supervised classification analysis. PSKH1, TLK2 and PHKG2 were overexpressed significantly in patients with the disease control to IC. The higher expression value of PSKH1 (r = 0.462, p < 0.001) and TLK2 (r = 0.361, p = 0.005) had the significant correlation to prolonged PFS., Conclusion: The result of this work demonstrated that expression nature of kinase genes such as PSKH1, TLK2 and PHKG2 may be informative to predict the efficacy of CI in wild type KRAS CRC. Mutations in either BRAF or PIK3CA were rare subsets in wild type KRAS CRC.

Purpose: Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies., Experimental Designs: Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial., Results: Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22-21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib., Conclusions: ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors.

Background/aim: CD133 is an important, but not exclusive, biomarker of colorectal cancer (CRC) stem cells., Materials and Methods: In order to identify other CRC stem cell-specific genes, we performed a comparative expression profiling of CD133(+) and CD133(-) cell populations in primary and metastatic tumors from four patients with CRC. CD133(+) and CD133(-) CRC cells were isolated using MagSweeper and used for whole-transcriptome analysis with RNA-Seq., Results: We found that in CD133(+) cells, 17 genes (RNASE2, PRB2, IL4, MGC27382, CLEC4C, SALL3, GIMAP1, ISG15, LOC728875, ZIK1, ICAM2, CCDC7, CDYL2, LRRC2, ZEB1, OSTF1 and CCDC144B) were significantly up-regulated compared to CD133(-) CRC cells. Among them, IL4 has been known as an inducer of survivin implicated in the survival and proliferation of cancer cells. However, the prognostic value of survivin in CRC is controversial. We evaluated survivin expression in formalin-fixed paraffin-embedded tumor samples of 188 patients with CRC by immunohistochemistry. Survivin over-expression was detected in 85 patients (45.2%) and was significantly associated with primary tumor sites (p=0.028), lymph node metastasis (p=0.029) and advanced III/IV CRC stages (AJCC 7; p=0.001). Furthermore, survivin up-regulation correlated with reduced disease-free survival (DFS; p=0.021) and overall survival (OS; p<0.000) and was proved to be an independent prognostic factor for both DFS and OS in multivariate analysis., Conclusion: Our data suggest that CD133(+) CRC stem cells have a distinct expression pattern and that survivin, up-regulated by differentially expressed IL-4, is a candidate biomarker for the prediction of recurrence and survival in CRC., (Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.)

Purpose: Preclinical data has demonstrated the potential of simvastatin to overcome cetuximab resistance in KRAS mutant CRC patients. Therefore, we designed a study using simvastatin/cetuximab/irinotecan for KRAS mutant CRC patients who are refractory to irinotecan and oxaliplatin-based chemotherapy., Patients and Methods: In this phase II study, patients received 500 mg/m(2) cetuximab, 150-180 mg/m(2) (day 1), and 80 mg simvastatin (once daily, days 1-14, every 2 weeks). The primary endpoint was the objective response rate (ORR). Secondary endpoints were progression-free survival (PFS), overall survival (OS), the disease control rate (DCR), and safety. We also analyzed the relationship between the RAS gene expression signature score and treatment response to simvastatin/cetuximab/irinotecan., Results: Fifty-two KRAS mutant CRC patients were enrolled. The ORR (complete response [CR], 0; partial response [PR], 1) was 1.9 % (95 % confidence interval [CI], -1.8-5.6). The DCR (CR, 0; PR, 1; stable disease, 33) was 65.4 % (95 % CI, 52.5-78.3). The median PFS and OS from the time of study drug administration were 7·6 months (95 % CI, 4.4-10.8) and 12.8 months (95 % CI, 9.5-16.2), respectively. The most common grade 3/4 adverse events were anemia (28.8 %), neutropenia (13.5 %), and diarrhea (7.7 %). The RAS signature score was significantly correlated with the maximal change in target lesions from baseline (r = 0.57, P = 0.014)., Conclusion: The simvastatin/cetuximab/irinotecan regimen showed promising efficacy and safety in KRAS mutant CRC patients who failed irinotecan and oxaliplatin-based chemotherapy. The RAS signature may be a novel predictor of treatment response to cetuximab-combined chemotherapy in CRC patients.

Purpose: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare group of tumors with a wide spectrum of clinical behavior. However, there are no known clinically relevant biomarkers to predict metastasis., Experimental Design: To investigate differential gene expression signatures of metastatic vs non-metastatic NETs, we studied cell cycle regulatory genes in 19 metastatic and 22 non-metastatic colorectal NETs by PCR arrays. Immunohistochemistry (IHC) and quantitative real-time RT-PCR were performed to verify the results and another set of 38 GEP-NETs were further studied for validation., Results: We first delineated six candidate genes for metastasis including ATM, CCND2, RBL2, CDKN3, CCNB1, and GTSE1. ATM was negatively correlated with metastatic NETs (p<0.001) with more than 2-fold change compared to non-metastatic NETs. Overexpression of ATM protein by IHC was strongly correlated with high ATM mRNA levels and low Ki-67 labeling index. Patients with ATM-negativity by IHC showed significantly decreased overall survival than patients with ATM-positivity (median OS, metastatic vs non-metastatic NETs; 2.7 years vs not reached; p = 0.003) and 85.7% of metastatic NETs were ATM-negative. In another validation set of GEP-NETs, decreased mRNA of ATM gene was associated with metastasis and remained significant (p = 0.023)., Conclusions: ATM down-regulation was strongly associated with metastatic NETs when compared with non-metastatic NETs and ATM may be a potential predictive marker for metastasis as well as a novel target in metastatic GEP-NETs.

Mucin occasionally accumulates intracellularly in colorectal mucinous adenocarcinomas, resulting in signet-ring cell morphology. In the current practice of pathology, there is no definitive rule on how to report a minor component of signet-ring cells in colorectal mucinous adenocarcinomas. We hypothesized that the absence of signet-ring cell component might have a favorable effect on survival of mucinous adenocarcinoma patients. To assess the biological characteristics of colorectal mucinous adenocarcinoma, we analyzed its clinicopathological features, microsatellite instability status, and survival outcomes and compared them with those of colorectal signet-ring cell carcinoma. A total of 266 consecutive colorectal mucinous adenocarcinoma patients and 65 signet-ring cell carcinoma patients were included. Mucinous adenocarcinomas, by comparison with signet-ring cell carcinomas, were characterized by development at an older age, less frequent vascular invasion and lymph node metastasis, and lower TNM stage at presentation. A total of 21 (22%) of 95 mucinous adenocarcinomas and 12 (19%) of 63 signet-ring cell carcinomas were high-frequency microsatellite instability. Patients with mucinous adenocarcinoma had significantly better survival than those with signet-ring cell carcinoma (P<0.0001) or than those with signet-ring cell carcinoma showing >50% extracellular mucin by volume (P<0.0001). In univariate analysis, absence of signet-ring cell component (P=0.0197), absence of vascular invasion, decreased invasion depth, no lymph node metastasis, and lower TNM stage had a favorable effect on survival of mucinous adenocarcinoma patients. Absence of vascular invasion, no lymph node metastasis, and lower TNM stage had a favorable effect on survival of signet-ring cell carcinoma patients. Multivariate analysis showed that higher TNM stage and T stage 4 were independent predictors of poor outcome in patients with mucinous adenocarcinoma. Our observations strongly suggest that pathologists should report the percentage of signet-ring cell component in colorectal mucinous adenocarcinomas and mucinous adenocarcinoma has different biologic behavior compared with signet-ring cell carcinoma.