Downregulation of SMOC1 is associated with progression of colorectal traditional serrated adenomas.

BACKGROUND: Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions. METHODS: SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112). RESULTS: SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components. CONCLUSION: Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors.

Serum amyloid A1 recruits neutrophils to the invasive front of T1 colorectal cancers.

BACKGROUND AND AIM: The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs. METHODS: Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n = 49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression. RESULTS: Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils. CONCLUSIONS: Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

Assessment of postoperative common bile duct stones after endoscopic extraction and subsequent cholecystectomy.

BACKGROUND: Common bile duct stones (CBDSs) occasionally cause serious diseases, and endoscopic extraction is the standard procedure for CBDS. To prevent biliary complications, cholecystectomy is recommended for patients who present with gallbladder (GB) stones after endoscopic CBDS extraction. However, CBDS can occasionally recur. To date, the occurrence of CBDS after endoscopic CBDS extraction and subsequent cholecystectomy is not fully understood. Hence, the current study aimed to evaluate the incidence of postoperative CBDSs. METHODS: This retrospective observational study included consecutive patients who underwent postoperative endoscopic retrograde cholangiography after endoscopic CBDS extraction and subsequent cholecystectomy between April 2012 and June 2021 at our institution. After endoscopic CBDS extraction, a biliary plastic stent was inserted to prevent obstructive cholangitis. Endoscopic retrograde cholangiography was performed to evaluate postoperative CBDSs after cholecystectomy until hospital discharge. The outcomes were the incidence of postoperative CBDSs and CBDSs/sludge. Moreover, the predictive factors for postoperative CBDSs were evaluated via univariate and multivariate analyses. RESULTS: Of eligible 204 patients, 52 patients (25.5%) presented with postoperative CBDSs. The incidence rate of CBDS/sludge was 36.8% (n = 75). Based on the univariate analysis, the significant predictive factors for postoperative CBDSs were ≥ 6 CBDSs, presence of cystic duct stones, and ≥ 10 GB stones (P < 0.05). Moreover, male sex and < 60-mm minor axis in GB might be predictive factors (P < 0.10). Based on the multivariate analysis, ≥ 6 CBDSs (odds ratio = 6.65, P < 0.01), presence of cystic duct stones (odds ratio = 4.39, P < 0.01), and ≥ 10 GB stones (odds ratio = 2.55, P = 0.01) were independent predictive factors for postoperative CBDSs. CONCLUSIONS: The incidence of postoperative CBDS was relatively high. Hence, patients with predictive factors for postoperative CBDS must undergo imaging tests or additional endoscopic procedure after cholecystectomy.

Activated macrophages promote invasion by early colorectal cancer via an interleukin 1ß-serum amyloid A1 axis.

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA-sequencing (RNA-seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP-1 cells suggested that interleukin 1ß (IL-1ß) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL-1ß. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1-like/M2-like macrophages at SAA1-positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase-9 in macrophages. Our data suggest that tumor-associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target.

Histopathologic features and fragmentation of polyps with cold snare defect protrusions.

BACKGROUND AND AIMS: Cold snare defect protrusions (CSDPs) include muscularis mucosa (MM) and submucosa tissue. CSDPs are thought to result from fragmentation of the specimen during shallow excision. Our aim in this study was to clarify whether CSDPs are associated with polyp fragmentation. METHODS: We retrospectively analyzed 1026 neoplastic colorectal polyps resected by cold snare polypectomy for which the presence or absence of CSDPs was assessed from the endoscopic image. All prepared specimens were reviewed and assessed for the presence or absence of polyp fragmentation, and the proportion of MM on the stump was measured. In addition, the risk factors for CSDP occurrence were evaluated. RESULTS: CSDPs occurred in 116 of the 1026 polyps (11.3%). Polyp fragmentation was significantly associated with the occurrence of CSDP on univariate analysis (odds ratio [OR], 3.74; P < .001) and multivariate analysis (OR, 3.13; P < .001). The proportion of MM >50% was significantly lower in the CSDP group than in the non-CSDP group (51.5% vs 70.9%, P < .001). CSDPs were significantly associated with a large polyp size (OR, 1.32; P = .007) and a large specimen size (OR, 1.24; P < .001) on multivariate analysis. CONCLUSIONS: The occurrence of CSDP was associated with less MM on the stump and fragmentation of the specimen. Clinically, the presence of CSDP is a good indicator of polyp fragmentation.

Multiloop as a novel traction method in accelerating colorectal endoscopic submucosal dissection.

BACKGROUND AND AIMS: Traction methods have been reported to speed up endoscopic submucosal dissection (ESD). We used the multiloop (M-loop) method as a traction method for colorectal ESD and recorded the submucosal dissection time (SDT) and submucosal dissection speed (SDS). METHODS: From January to August 2018, we used the M-loop method for colorectal ESD procedures and timed the duration and recorded the outcomes. Two experts and eight nonexperts performed the procedures, which were carried out at a tertiary endoscopic center in Japan. RESULTS: A total of 50 patients were treated by colorectal ESD using the M-loop method. The mean SDT was 42.1 ± 4.16 minutes and the mean SDS was 28.0 ± 2.89 mm2/minutes. The mean SDS was 38.9 ± 6.9 mm2/minutes for experts and 25.3 ± 3.1 mm2/minutes for nonexperts. En bloc resection was achieved in 100% of cases. There were 3 adverse events and unfavorable outcomes. CONCLUSIONS: Traction by the M-loop method improved SDS in colorectal ESD. The method can be an effective tool to assist colorectal ESD. Further evaluation of the usefulness of the M-loop method is required in direct comparison with conventional ESD.

Subtypes of the Type II Pit Pattern Reflect Distinct Molecular Subclasses in the Serrated Neoplastic Pathway.

BACKGROUND: Colorectal serrated lesions (SLs) are important premalignant lesions whose clinical and biological features are not fully understood. AIMS: We aimed to establish accurate colonoscopic diagnosis and treatment of SLs through evaluation of associations among the morphological, pathological, and molecular characteristics of SLs. METHODS: A total of 388 premalignant and 18 malignant colorectal lesions were studied. Using magnifying colonoscopy, microsurface structures were assessed based on Kudo's pit pattern classification system, and the Type II pit pattern was subcategorized into classical Type II, Type II-Open (Type II-O) and Type II-Long (Type II-L). BRAF/KRAS mutations and DNA methylation of CpG island methylator phenotype (CIMP) markers (MINT1, - 2, - 12, - 31, p16, and MLH1) were analyzed through pyrosequencing. RESULTS: Type II-O was tightly associated with sessile serrated adenoma/polyps (SSA/Ps) with BRAF mutation and CIMP-high. Most lesions with simple Type II or Type II-L were hyperplastic polyps, while mixtures of Type II or Type II-L plus more advanced pit patterns (III/IV) were characteristic of traditional serrated adenomas (TSAs). Type II-positive TSAs frequently exhibited BRAF mutation and CIMP-low, while Type II-L-positive TSAs were tightly associated with KRAS mutation and CIMP-low. Analysis of lesions containing both premalignant and cancerous components suggested Type II-L-positive TSAs may develop into KRAS-mutated/CIMP-low/microsatellite stable cancers, while Type II-O-positive SSA/Ps develop into BRAF-mutated/CIMP-high/microsatellite unstable cancers. CONCLUSIONS: These results suggest that Type II subtypes reflect distinct molecular subclasses in the serrated neoplasia pathway and that they could be useful hallmarks for identifying SLs at high risk of developing into CRC.

Epigenetic silencing of diacylglycerol kinase gamma in colorectal cancer.

Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.

Endoscopic and molecular characterization of colorectal sessile serrated adenoma/polyps with cytologic dysplasia.

BACKGROUND AND AIMS: Sessile serrated adenoma/polyps (SSA/Ps), which are precursor lesions of colorectal cancer (CRC) with BRAF mutation and the CpG island methylator phenotype (CIMP), develop cytologic dysplasia (CD) during the progression of colorectal tumorigenesis. In the present study we aimed to clarify the endoscopic and molecular signatures of SSA/Ps, with and without CD. METHODS: A series of 208 serrated lesions, including 41 hyperplastic polyps, 90 SSA/Ps, 33 SSA/Ps with CD, and 44 traditional serrated adenomas, were observed and resected using magnifying endoscopy. BRAF and KRAS mutations and methylation of CIMP markers (MINT1, MINT2, MINT12, MINT31, and p16) were analyzed through pyrosequencing. Molecular alterations were then compared with endoscopic and pathologic characteristics. RESULTS: Among SSA/Ps without CD, the Type II-Open pit pattern (Type II-O), BRAF mutation, and CIMP were tightly associated with a proximal colon location. SSA/Ps in the distal colon infrequently exhibited Type II-O and CIMP. By contrast, most SSA/Ps with CD showed Type II-O plus adenomatous pit patterns (Type III or IV), BRAF mutation, and CIMP, irrespective of their locations. CONCLUSIONS: Our results suggest that the Type II-O plus III/IV pit pattern is a common feature of SSA/Ps with CD in both the proximal and distal colon and that this pit pattern is a hallmark of serrated lesions at high risk of developing into CRCs.

Reactivation of hepatitis B virus in patients with undetectable HBsAg undergoing chemotherapy for malignant lymphoma or multiple myeloma.

Despite increasing reports of hepatitis B virus (HBV) reactivation in hematological malignancies, its incidence, and risk factors are still obscure. The aim of this study was to clarify the frequency and risk factors of HBV reactivation in hepatitis B surface antigen (HBsAg) undetectable patients with malignant lymphoma or multiple myeloma, during or after chemotherapy. A total of 109 patients with undetectable HBsAg undergoing chemotherapy for malignant lymphoma or multiple myeloma were enrolled in this study. Anti-hepatitis B surface (anti-HBs) and anti-hepatitis B core (anti-HBc) were checked before treatment, and HBV DNA in sera was quantified monthly during and after chemotherapy. Out of 109 patients, 42 (38.5%) had anti-HBs and 59 (54.1%) had anti-HBc. Among the 59 anti-HBc positive patients, four patients (4/59, 6.8%) showed HBV reactivation during 20.5 median follow-up months. In all four patients with HBV reactivation, peripheral lymphocyte counts before chemotherapy were lower than those without HBV reactivation (P=0.033). HBV reactivation occurred during and after chemotherapy containing rituximab for non-Hodgkin lymphoma. Four patients, who had HBV reactivation, did not develop de novo hepatitis due to HBV reactivation and were able to undergo chemotherapy against malignant lymphoma as scheduled. Monitoring of HBV DNA in sera is useful for the early diagnosis of HBV reactivation, and preemptive therapy is an useful alternative to prevent hepatitis due to HBV reactivation. Patients must be monitored periodically for HBV-DNA levels during and after chemotherapy.

Feasibility of the virtual needle tracking system for percutaneous radiofrequency ablation of hepatocellular carcinoma.

AIM: To evaluate the feasibility of the real-time virtual needle tracking system for percutaneous radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). METHODS: An electromagnetic field created by an ultrasound (US) machine detected the tracking bracket mounted onto the RFA needle. When the needle tip was confirmed to be in the accurate plane extracorporeally, the needle was inserted into the liver using the virtual navigation US system, and RFA was performed. Eight patients with eight liver lesions underwent percutaneous RFA under ultrasound for HCC from October to November 2012 using the real-time electromagnetic virtual needle tracking system (VirtuTRAX). RESULTS: The average size of the tumors was 11.5 mm with one lesion in S4, two in S5, two in S7 and three in S8. Sufficient margins were obtained in a single session in all cases. Using only B-mode, the needle tip was obscured due to the condition of the surrounding liver or subcutaneous fat tissue, but it was identifiable with the use of the virtual needle tracking device in all cases. In one case where the lesion was large, the needle was placed twice deliberately, but the second puncture was made difficult by the ablation artifact of the first puncture. With the tracking device, however, it was possible to perform the second puncture accurately. CONCLUSION: The virtual tracking system is useful in cases where the needle tip is obscured due to surrounding liver conditions or when multiple punctures are necessary due to the ablation artifact's obscuring the needle tip. Freehand puncturing may be possible in the future using this technique with further improvements in the system.

Feasibility of fused imaging for the evaluation of radiofrequency ablative margin for hepatocellular carcinoma.

AIM: To evaluate the feasibility of fusion of conventional imaging modalities to facilitate assessment of ablative margin of radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). METHODS: Two hundred and thirty-nine liver lesions in 109 patients underwent percutaneous RFA under ultrasound for HCC from January 2008 to December 2010. Within these patients, 13 lesions in 12 patients who developed local tumor progression in the follow-up period of at least 8 months were retrospectively reviewed. Imaging obtained before and after RFA was used for creating fused images on a workstation. Ablative margins were assessed using only axial images, and with fused images. RESULTS: The ablative margin was assessed as sufficient in all 13 lesions using side-by-side axial images; however, all lesions were assessed as insufficient with fused imaging evaluation. The reason for the discrepancy of the assessment results were differences in the respiratory dislocation of the liver in the pre- and post-RFA images in eight lesions (61.5%), and rotational displacement of the liver and the torso in five (38.5%). The site of local tumor recurrence relative to the HCC lesion was craniocaudal in 12 lesions, dorsoventral in seven and lateral in seven. In all lesions, the site of local tumor recurrence was congruent with the area of the thinnest ablative margin. CONCLUSION: Assessment of ablative margin with fused imaging revealed insufficiency of ablation previously evaluated as sufficient with conventional axial imaging. Fused imaging evaluation has proved to be an accurate and useful tool for the assessment of RFA margins.

Ratiometric optical temperature sensor using two fluorescent dyes dissolved in an ionic liquid encapsulated by Parylene film.

A temperature sensor that uses temperature-sensitive fluorescent dyes is developed. The droplet sensor has a diameter of 40 µm and uses 1 g/L of Rhodamine B (RhB) and 0.5 g/L of Rhodamine 110 (Rh110), which are fluorescent dyes that are dissolved in an ionic liquid (1-ethyl-3-methylimidazolium ethyl sulfate) to function as temperature indicators. This ionic liquid is encapsulated using vacuum Parylene film deposition (which is known as the Parylene-on-liquid-deposition (PoLD) method). The droplet is sealed by the chemically stable and impermeable Parylene film, which prevents the dye from interacting with the molecules in the solution and keeps the volume and concentration of the fluorescent material fixed. The two fluorescent dyes enable the temperature to be measured ratiometrically such that the droplet sensor can be used in various applications, such as the wireless temperature measurement of microregions. The sensor can measure the temperature of such microregions with an accuracy of 1.9 °C, a precision of 3.7 °C, and a fluorescence intensity change sensitivity of 1.0%/K. The sensor can measure temperatures at different sensor depths in water, ranging from 0 to 850 µm. The droplet sensor is fabricated using microelectromechanical system (MEMS) technology and is highly applicable to lab-on-a-chip devices.

An Integrated Epigenome and Transcriptome Analysis to Clarify the Effect of Epigenetic Inhibitors on GIST.

BACKGROUND/AIM: Epigenetic alterations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). To obtain further insight into the GIST epigenome, we analyzed genome-wide histone modification and DNA methylation in GIST cells. MATERIALS AND METHODS: To reverse epigenetic silencing, GIST-T1 cells were treated with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, and subsequently H3K4me3 levels, the DNA methylome, and the transcriptome were analyzed. RESULTS: Treatment with epigenetic inhibitors not only up-regulated genes with DNA methylation, but also genes related to interferon signaling. ChIP-seq analysis revealed that drug treatment up-regulated H3K4me3 levels in retrotransposons, including endogenous retroviruses (ERV). Finally, utilizing the omics data, we found that hypermethylation of MEG3 is a frequent event and an indicator of poorer prognosis in GIST patients. CONCLUSION: Epigenetic inhibitors may activate interferon signaling via viral mimicry in GIST cells. Moreover, epigenome data could be a useful resource to identify novel GIST-related genes.

[Sarcomatoid carcinoma of the liver: a case report and review of the literature].

A 66-year-old woman presented to our outpatient clinic with abdominal discomfort in April, 2008. Ultrasound revealed a hypo-echoic 10cm mass in the right hepatic lobe but no indication of chronic liver disease, with similar results in her history, on physical exam, and imaging. Serum testing did not identify any systemic disease. The mass was suspected to be intrahepatic cholangiocarcinoma and right hepatic lobectomy was performed. Histologic examination of the specimen revealed numerous spindle cells, and immunostaining confirmed a definitive diagnosis of sarcomatoid carcinoma of the liver. On subsequent review of the case, an abdominal CT performed 2.5 years earlier for unrelated symptoms had shown a 1cm faint low density area in the same location as the mass. The doubling time of this tumor was about 95 days.