Survivin is a novel transcription regulator of KIT and is downregulated by miRNA-494 in gastrointestinal stromal tumors

Gain-of-function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT-independent targets of KIT-regulating mRNAs. We sought to investigate how miR-494 inhibits GIST proliferation and to identify novel target gene. We used microarray-based gene expression analyses to identify pathways and target genes affected by miR-494. The expressional relationship between survivin and miR-494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR-494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR-494, and its expression showed an inverse correlation with miR-494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = -0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR-494-mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR-494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT.ope

Histopathological Variants of Hepatocellular Carcinomas: an Update According to the 5th Edition of the WHO Classification of Digestive System Tumors

Hepatocellular carcinoma (HCC) is heterogeneous in pathogenesis, phenotype and biological behavior. Various histopathological features of HCC had been sporadically described, and with the identification of common molecular alterations of HCC and its genomic landscape over the last decade, morpho-molecular correlation of HCC has become possible. As a result, up to 35% of HCCs can now be classified into histopathological variants, many of which have unique molecular characteristics. This review will provide an introduction to the variously described histopathological variants of HCC in the updated WHO Classification of Digestive System Tumors.ope

LEF1; TFE3; and AR are putative diagnostic markers of solid pseudopapillary neoplasms

The diagnosis of solid pseudopapillary neoplasms (SPNs) is challenging because some SPNs share many similar morphological and immunohistochemical features with other pancreatic neoplasms. In this study, we investigated potential diagnostic markers of SPN. Based on the SPN-specific upregulated genes from a previous DNA microarray and proteome study, we selected six immunohistochemical markers [beta-catenin, androgen receptor (AR), lymphoid enhancer-binding factor 1 (LEF1), transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3), fused in sarcoma (FUS), and WNT inhibitory factor 1 (WIF-1)]. We also evaluated the Ki-67 proliferative index to investigate its associations with prognosis. To validate these markers, we studied 91 SPNs as well as 51 pancreatic ductal carcinomas (PDC) and 48 neuroendocrine tumors (NET) as controls. We found frequent and diffuse nuclear expressions of β-catenin (98.9%), AR (81.3%), LEF1 (93.4%), TFE3 (74.7%), FUS (84.6%), and cytoplasmic expression of WIF-1 (96.7%) in SPNs. In contrast, PDCs and NETs showed no expression. (P < 0.001). When beta-catenin, LEF1, and TFE3 staining were combined, the sensitivity and specificity were 100% and 91.9%, respectively. Four (4.4%) SPNs showed distant metastasis and these tumors were associated with a relatively high Ki-67 proliferative index (≥ 5%; P = 0.013). We identified LEF1, TFE3, and AR as putative diagnostic markers of SPN, auxiliary to β-catenin. Incorporated into an immunohistochemical panel, these markers could be beneficial to distinguish SPN from PDC and NET. In addition, we suggest that the Ki-67 proliferative index can be a predictive marker of metastasis in SPNs.ope

A Case of Merkel Cell Carcinoma of the External Auditory Canal Skin with Annulus and Tympanic Membrane

Merkel cell carcinoma is an extremely rare neuroendocrine neoplasm of the external auditory canal (EAC) skin, which requires wide excision and adjuvant radiation due to a high recurrence rate. In this report, we describe a case of Merkel cell carcinoma arising from the EAC which was successfully treated with endoscopic excision. A 32-years-old female patient with a history of papillary thyroid cancer was diagnosed with an EAC tumor incidentally. There was a erythematous papular lesion on the posterior EAC without any evidence of locoregional metastasis. The patient underwent endoscopic tumor resection under general anesthesia, and the final pathologic report confirmed the diagnosis of Merkel cell carcinoma. There was no clinical or radiographic evidence of recurrence or metastasis of Merkel cell tumor for 41 months after surgical resection alone.ope

Next-generation sequencing with comprehensive bioinformatics analysis facilitates somatic mosaic APC gene mutation detection in patients with familial adenomatous polyposis

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal tumor characterized by numerous adenomatous colonic polyps that often lead to colon cancer. Although most patients with FAP harbored germline mutations in APC gene, it was recently recognized that patients with clinical FAP, but without detectable pathogenic mutations, could be associated with somatic mosaic APC mutation. METHODS: We reanalyzed the nest-generation sequencing (NGS) gene panel testing results of patients who were diagnosed with FAP, but did not have APC mutations, at Yonsei Cancer Prevention Center between July 2016 and March 2018. We tested several variant calling algorithms to identify low level mosaic variants. In one patient with a low frequency APC mutation, NGS analysis was performed together with endoscopic biopsy. Variant calling tools HaplotypeCaller, MuTect2, VarScan2, and Pindel were used. We also used 3'-Modified Oligonucleotides (MEMO)-PCR or conventional PCR for confirmation. RESULTS: Among 28 patients with clinical suspicion of FAP but no detectable pathogenic variants of colonic polyposis associated genes, somatic mosaic pathogenic variants were identified in seven patients. The variant allele frequency ranged from 0.3 to 7.7%. These variants were mostly detected through variant caller MuTect2 and Pindel, and were further confirmed using mutant enrichment with MEMO-PCR. CONCLUSIONS: The NGS with an adequate combination of bioinformatics tools is effective to detect low level somatic variants in a single assay. Because mosaic APC mutations are more frequent than previously thought, the presence of mosaic mutations must be considered when analyzing genetic tests of patients with FAP.ope

Prognosis of stage III colorectal carcinomas with FOLFOX adjuvant chemotherapy can be predicted by molecular subtype

Individualizing adjuvant chemotherapy is important in patients with advanced colorectal cancers (CRCs), and the ability to identify molecular subtypes predictive of good prognosis for stage III CRCs after adjuvant chemotherapy could be highly beneficial. We performed microarray-based gene expression analysis on 101 fresh-frozen primary samples from patients with stage III CRCs treated with FOLFOX adjuvant chemotherapy and 35 matched non-neoplastic mucosal tissues. CRC samples were classified into four molecular subtypes using nonnegative matrix factorization, and for comparison, we also grouped CRC samples using the proposed consensus molecular subtypes (CMSs). Of the 101 cases, 80 were classified into a CMS group, which shows a 79% correlation between the CMS classification and our four molecular subtypes. We found that two of our subtypes showed significantly higher disease-free survival and overall survival than the others. Group 2, in particular, which showed no disease recurrence or death, was characterized by high microsatellite instability (MSI-H, 6/21), abundant mucin production (12/21), and right-sided location (12/21), this group strongly correlated with CMS1 (microsatellite instability immune type). We further identified the molecular characteristics of each group and selected 10 potential biomarker genes from each. When these were compared to the previously reported molecular classifier genes; we found that 31 out of 40 selected genes were matched with those previously reported. Our findings indicate that molecular classification can reveal specific molecular subtypes correlating with clinicopathologic features of CRCs and can have predictive value for the prognosis for stage III CRCs with FOLFOX adjuvant chemotherapy.ope

β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

WNT signaling activation in colorectal cancers (CRCs) occurs through APC inactivation or β-catenin mutations. Both processes promote β-catenin nuclear accumulation, which up-regulates epithelial-to-mesenchymal transition (EMT). We investigated β-catenin localization, transcriptome, and phenotypic differences of HCT116 cells containing a wild-type (HCT116-WT) or mutant β-catenin allele (HCT116-MT), or parental cells with both WT and mutant alleles (HCT116-P). We then analyzed β-catenin expression and associated phenotypes in CRC tissues. Wild-type β-catenin showed membranous localization, whereas mutant showed nuclear localization; both nuclear and non-nuclear localization were observed in HCT116-P. Microarray analysis revealed down-regulation of Claudin-7 and E-cadherin in HCT116-MT vs. HCT116-WT. Claudin-7 was also down-regulated in HCT116-P vs. HCT116-WT without E-cadherin dysregulation. We found that ZEB1 is a critical EMT factor for mutant β-catenin-mediated loss of E-cadherin and Claudin-7 in HCT116-P and HCT116-MT cells. We also demonstrated that E-cadherin binds to both WT and mutant β-catenin, and loss of E-cadherin releases β-catenin from the cell membrane and leads to its degradation. Alteration of Claudin-7, as well as both Claudin-7 and E-cadherin respectively caused tight junction (TJ) impairment in HCT116-P, and dual loss of TJs and adherens junctions (AJs) in HCT116-MT. TJ loss increased cell motility, and subsequent AJ loss further up-regulated that. Immunohistochemistry analysis of 101 CRCs revealed high (14.9%), low (52.5%), and undetectable (32.6%) β-catenin nuclear expression, and high β-catenin nuclear expression was significantly correlated with overall survival of CRC patients (P = 0.009). Our findings suggest that β-catenin activation induces EMT progression by modifying cell-cell junctions, and thereby contributes to CRC aggressiveness.ope

Microsatellite instability test using peptide nucleic acid probe-mediated melting point analysis: a comparison study

BACKGROUND: Analysis of high microsatellite instability (MSI-H) phenotype in colorectal carcinoma (CRC) is important for evaluating prognosis and choosing a proper adjuvant therapy. Although the conventional MSI analysis methods such as polymerase chain reaction (PCR) fragment analysis and immunohistochemistry (IHC) show high specificity and sensitivity, there are substantial barriers to their use. METHODS: In this study, we analyzed the MSI detection performance of three molecular tests and IHC. For the molecular tests, we included a recently developed peptide nucleic acid probe (PNA)-mediated real-time PCR-based method using five quasi-monomorphic mononucleotide repeat markers (PNA method) and two conventional PCR fragment analysis methods using NCI markers (NCI method) or five quasi-monomorphic mononucleotide repeat markers (MNR method). IHC analysis was performed with four mismatch repair proteins. The performance of each method was validated in 166 CRC patient samples, which consisted of 76 MSI-H and 90 microsatellite stable (MSS) CRCs previously diagnosed by NCI method. RESULTS: Of the 166 CRCs, 76 MSI-H and 90 MSS CRCs were determined by PNA method. On the other hand, 75 MSI-H and 91 MSS CRCs were commonly determined by IHC and MNR methods. Based on the originally diagnosed MSI status, PNA showed 100% sensitivity and 100% specificity while IHC and MNR showed 98.68% sensitivity and 100% specificity. When we analyzed the maximum sensitivity of MNR and PNA method, which used the same five markers, PNA method could detect alterations in all five mononucleotide repeat markers in samples containing down to 5% MSI-H DNAs, whereas MNR required at least 20% MSI-H DNAs to achieve the same performance. CONCLUSIONS: Based on these findings, we suggest that PNA method can be used as a practical laboratory test for the diagnosis of MSI.ope

Comprehensive analyses of immunodynamics and immunoreactivity in response to treatment in ALK-positive non-small-cell lung cancer

Background: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). Immune checkpoint inhibitors (ICIs) have not proved efficacy in ALK-positive non-small cell lung cancer so far. In this study, we performed a mouse clinical trial using EML4-ALK transgenic mice model to comprehensively investigate immunomodulatory effects of ALK TKI and to investigate the mechanisms of resistance to ICIs. Methods: EML4-ALK transgenic mice were randomized to three treatment arms (arm A: antiprogrammed death cell protein-1 (PD-1), arm B: ceritinib, arm C: anti-PD-1 and ceritinib), and tumor response was evaluated using MRI. Progression-free survival and overall survival were measured to compare the efficacy. Flow cytometry, multispectral imaging, whole exome sequencing and RNA sequencing were performed from tumors obtained before and after drug resistance. Results: Mouse clinical trial revealed that anti-PD-1 therapy was ineffective, and the efficacy of ceritinib and anti-PD-1 combination was not more effective than ceritinib alone in the first line. Dynamic changes in immune cells and cytokines were observed following each treatment, while changes in T lymphocytes were not prominent. A closer look at the tumor immune microenvironment before and after ceritinib resistance revealed increased regulatory T cells and programmed death-ligand 1 (PD-L1)-expressing cells both in the tumor and the stroma. Despite the increase of PD-L1 expression, these findings were not accompanied by increased effector T cells which mediate antitumor immune responses. Conclusions: ALK-positive tumors progressing on ceritinib is not immunogenic enough to respond to immune checkpoint inhibitors.ope

Association between Fusobacterium nucleatum and patient prognosis in metastatic colon cancer

Recent evidence suggests that Fusobacterium nucleatum (Fn) is associated with the development and progression of colorectal cancer. We aimed to delineate the clinical implications of Fn in metastatic colon cancer. We performed quantitative polymerase chain reaction (qPCR) using DNA samples from synchronous metastatic colon cancer patients with either formalin-fixed paraffin-embedded (FFPE) archival primary site tumor samples or fresh colon tissues. Progression-free survival (PFS)1 and PFS2 were defined as PFS of first- and second-line palliative settings. qPCR for Fn was successfully performed using 112 samples (FFPE, n = 61; fresh tissue, n = 51). Forty-one and 68 patients had right-sided and left-sided colon cancer, respectively. Patients with Fn enriched right-sided colon cancers had shorter PFS1 (9.7 vs. 11.2 months) than the other subgroups (HR 3.54, 95% confidence interval [CI] 1.05-11.99; P = 0.04). Fn positive right-sided colon was also associated with shorter PFS2 (3.7 vs. 6.7 months; HR 2.34, 95% CI 0.69-7.91; P = 0.04). In the univariate analysis, PFS1 was affected by differentiation and Fn positive right-sided colon cancer. The multivariate analysis showed that differentiation (HR 2.68, 95% CI 1.40-5.14, P = 0.01) and Fn positive right-sided colon (HR 0.40, 95% CI 0.18-0.88, P = 0.02) were associated with PFS1. Fn enrichment in right sided colon was not associated with overall survival (OS). Fn enrichment has significantly worse prognosis in terms of PFS1 and PFS2 in patients with right-sided metastatic colon cancers.ope