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Purpose: Current immunomagnetic enrichment method can only detect general epithelial antigens of circulating tumor cells (CTC). Further characterization of the CTCs to provide specific information on the tumor type is not possible. We attempted to overcome this drawback by developing the methodology for using a gastrointestinal-specific anti-cytokeratin (CK) 20 antibody to detect CTCs in colorectal cancer patients' blood.Experimental Design: The protocol was validated using a colorectal cancer SW480 cell line. The clinical significance of findings in colorectal cancer was investigated by detecting CK20-positive CTCs (pCTC) in patients with colorectal cancer, other common cancers, colorectal adenoma, benign colorectal diseases, and normal subjects. Moreover, the malignant nature of CK20 pCTCs was examined by comparing chromosome 17 aberration patterns with those from the corresponding primary tumors.Results: The assay successfully showed CK20-positive SW480 cells. When applied in patient samples, the detection rates were 62% (132 colorectal cancer patients; median number = 11 CTCs), 0% (120 patients with other common cancers), 6% (50 colorectal adenoma patients), 0% (120 patients with benign colorectal diseases), and 0% (40 normal subjects). Furthermore, statistical analysis showed that CK20 pCTC numbers were associated with tumor-node-metastasis stage and lymph node status. Using the median CK20 pCTC numbers as the cutoff points, stratified groups of colorectal cancer patients had significant differences in their recurrence, metastasis, and survival. Finally, chromosome 17 aneusomy in 90% of colorectal cancer patients with CK20 pCTCs matched with those from the primary tumors.Conclusions: Detection of CK20 pCTCs using the new protocol could generate clinically important information for colorectal cancer patients.

Supplementary Figure 1. Validation of the lncRNA microarray data using qRT-PCR. Supplementary Figure 2. Expression levels of DZnep-induced lncRNAs in a panel of HCC cell lines and non-tumor cell lines. Supplementary Figure 3. Expression levels of DZnep-induced lncRNAs in paired human HCC tissues and adjacent non-tumor tissues. Supplementary Figure 4. High specificity of ChIP assay in measuring true interaction between EZH2 and the promoters of EZH2 targeted genes. Supplementary Figure 5. Inhibition of TCAM1P-004 and RP11-598D14.1 with siRNAs promoted cell proliferation. Supplementary Figure 6. Knockdown of TCAM1P-004 and RP11-598D14.1 with shRNAs inhibited cell apoptosis, promoted colony formation ability and transformation ability, and stimulated tumor growth in xenograft mice model. Supplementary Figure 7. Overexpression of TCAM1P-004 and RP11-598D14.1 repressed cell colony formation and transformation abilities, and tumor growth in xenograft mice model. Supplementary Figure 8. Overexpression of TCAM1P-004 or RP11-598D14.1 promoted apoptosis in subcutaneous tumors derived from Hep3B cells. Supplementary Figure 9. Inhibition of TCAM1P-004 or RP11-598D14.1 could induce tumorigenesis of non-tumor MIHA cells. Supplementary Figure 10. KEGG pathway enrichment analysis of TCAM1P-004 and RP11-598D14.1 regulated genes. Supplementary Figure 11. qRT-PCR validated signaling pathways related to TCAM1P-004 and RP11-598D14.1. Supplementary Figure 12. Expression of DDIT and PFKFB4 in HCC cell lines and HCC tumor samples. Supplementary Figure 13. Expression of PFKFB4 in HCC tumor samples. Supplementary Figure 14. Alignment analysis between lncRNA TCAM1P-004 and RP11-598D14.1. Supplementary Figure 15. EZH1 showed no significant role in HCC.

Long noncoding RNAs (lncRNA) play critical roles in the development of cancer, including hepatocellular carcinoma (HCC). However, the mechanisms underlying their deregulation remain largely unexplored. In this study, we report that two lncRNAs frequently downregulated in HCC function as tumor suppressors and are epigenetically silenced by histone methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11-598D14.1 were inhibited by EZH-mediated trimethylation of H3K27me3 at their promoters. Downregulation of TCAM1P-004 and RP11-598D14.1 was frequently observed in HCC tumors compared with adjacent normal tissues. Both lncRNAs inhibited cell growth, cell survival, and transformation in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demonstrated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These lncRNA–protein interactions were critical in regulating p53, MAPK, and HIF1α pathways that promoted cell proliferation in HCC. Overexpression of EZH2 was critical in repressing TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/RP11-598D14.1–regulated pathways were prevalent in human HCC. Aberrant suppression of TCAM1P-004 and RP11-598D14.1 led to loss of their tumor-suppressive effects by disrupting the interaction with IGF2BP1, HIST1H1C, and STAU1, which in turn promoted HCC development and progression. Collectively, these findings demonstrate the role of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor growth and suggest that EZH2 may serve as a therapeutic target in HCC.Significance:EZH2-mediated loss of lncRNAs TCAM1P-004 and RP11-598D14.1 hinders the formation of tumor suppressor lncRNA–protein complexes and subsequently promotes HCC growth.

Supplementary Tables S1-S3 from Clinical Significance of Cytokeratin 20-Positive Circulating Tumor Cells Detected by a Refined Immunomagnetic Enrichment Assay in Colorectal Cancer Patients

Background & Aims\ud Metabolic syndrome can lead to the clinical manifestation of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) as part of the natural history of NAFLD. Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations.\ud Methods\ud Here, tumor-normal pairs from 100 subjects diagnosed with NAFLD-HCC were subject to next generation sequencings. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD to NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of underlying CTNNB1 S45P driver mutation. Syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mut and TNFRSF19 in reshaping the tumor microenvironment.\ud Results\ud Mutational process operative in NAFLD-liver inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. ChIP-seq integrated with transcriptome and immune profiling showed for the first time a transcriptional axis of CTNNB1mut/TNFRSF19/repressed senescence-associated secretory phenotype-like (SASP-like) cytokines (including IL6 and CXCL8). This phenomenon could be reverted by Wnt-modulator ICG001.\ud Conclusions\ud The unique mutational processes in NAFLD-liver and NAFLD-HCC alludes to a “field effect”. Whereby, distinct aberrations and shift in driver events reveal a gain-of-function role of CTNNB1 mutations in immune exclusion via TNFRSF19 inhibition of SASP-like features.\ud LAY SUMMARY\ud The increasing prevalence of metabolic syndrome in adult populations poised NAFLD-induced HCC to be the major type of liver cancer of the 21st century. We showed a strong “field effect” in NAFLD-liver from mutational signatures detected and a mechanistic path of activated β-catenin in reshaping the tumor-immune microenvironment.

Serologic and molecular surveillance of serum collected from 152 suspected scrub typhus patients in Myanmar revealed Orientia tsutsugamushi of genotypic heterogeneity. In addition, potential co-infection with severe fever with thrombocytopenia syndrome virus was observed in 5 (3.3%) patients. Both scrub typhus and severe fever with thrombocytopenia syndrome are endemic in Myanmar.

Background/Aims: ATP-binding cassette (ABC) transporters have been shown to regulate tumor initiating cells in various cancer types and specific genotype associated with cancer risk. ABCF1 was on priority among the ABC transporters because the gene located at 6p21, the chromosome region associated with a number of HCC risk genetic loci including the HLA genes. Its family member ABCF2 was also examined as their expression levels were highly correlated and associated with HCC recurrence. Subjects and Methods: A total of 300 HCC and 300 healthy blood samples (99.3% and 94.7% Chinese respectively) were prospectively collected with informed consent. All patients had been diagnosed with primary HCC and underwent partial hepatectomy. Clinical information including sex, age, tumor stage and survival outcomes were collected prospectively. Genomic DNA were extracted from blood samples and SNPs were examined by TaqMan genotyping assay, Sequenom MassARRAY platform and direct sequencing. Results: Sixteen SNPs and three INDELs were examined in ABCF1 and ABCF2 in the germline DNA of 300 HCCs and 300 healthy individuals. Among the 19 loci investigated, ABCF1 rs1264440 (916-99A>G) and rs4148252 (*1delA), ABCF2 rs3823589 (-43+219C>A) and rs75100208 (368-94C>A) were significantly associated with HCC risk (OR: 1.424, 95%CI: 1.03-1.97, P=0.032; OR: 1.446, 95%CI: 1.04-2.00, P=0.026; OR: 1.635, 95%CI: 1.09-2.46, P=0.018; OR: 1.479, 95%CI: 1.09-2.18, P=0.049, respectively). Patients with any two of the four variant genotype, compared with those with all wild type, revealed elevated HCC risks (OR: 1.657, 95%CI: 1.16-2.37, P=0.006). Importantly, patients with all four variant genotypes demonstrated augmented HCC risk (OR: 2.295, 95%CI: 1.27-4.15, P=0.006). Conclusions: ABCF1 genetic variants rs1264440, rs4148252 and ABCF2 genetic variants rs3823589, rs75100208 were associated with significantly increased HCC risk. Augmented cancer risk was observed among patients possessed all four variant genotypes in ABCF1 and ABCF2. Further mechanistic studies are warranted to comprehend the underlying mechanisms. Citation Format: Philip Chun Yeung, Kelvin Kwok Chai Ng, Tan To Cheung, Charing Ching-Ning Chong, Paul Bo San Lai, Siu Tim Cheung. ABCF1 and ABCF2 genetic variants in association with hepatocellular carcinoma (HCC) risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1436.

Postoperative pancreatic fistula (POPF) after pancreaticoduodenectomy (PD) remains a dreadful complication. Duct-to-mucosa pancreaticojejunostomy (DTMPJ) is a commonly performed anastomosis after PD. This study aims to evaluate whether there is a size limit of pancreatic duct below which POPF rate increases significantly after DTMPJ.A retrospective study was performed from a database with prospectively collected data on consecutive patients undergoing DTMPJ.Between the years 2003 and 2019, a total of 288 patients with DTMPJ were recruited. POPF occurred in 56.3% of the patients, of which 43.8% were biochemical leak, 8.7% were grade B, and 1.4% were grade C. Overall operative morbidity was 51.4%, of which 19.1% were major complications. Five patients (1.7%) died within 90 days of operation. Patients with grade B/C POPF had significantly soft pancreas (DTMPJ can be safely performed for pancreatic duct1 mm without significantly increased POPF risk.

Background The aim of the study was to evaluate the current opinion and clinical decision-making process of international pancreatologists, and to systematically identify key study questions regarding the diagnosis and treatment of chronic pancreatitis (CP) for future research. Methods An online survey, including questions regarding the diagnosis and treatment of CP and several controversial clinical case vignettes, was send by e-mail to members of various international pancreatic associations: IHPBA, APA, EPC, ESGE and DPSG. Results A total of 288 pancreatologists, 56% surgeons and 44% gastroenterologists, from at least 47 countries, participated in the survey. About half (48%) of the specialists used a classification tool for the diagnosis of CP, including the Mayo Clinic (28%), Mannheim (25%), or Buchler (25%) tools. Overall, CT was the preferred imaging modality for evaluation of an enlarged pancreatic head (59%), pseudocyst (55%), calcifications (75%), and peripancreatic fat infiltration (68%). MRI was preferred for assessment of main pancreatic duct (MPD) abnormalities (60%). Total pancreatectomy with auto-islet transplantation was the preferred treatment in patients with parenchymal calcifications without MPD abnormalities and in patients with refractory pain despite maximal medical, endoscopic, and surgical treatment. In patients with an enlarged pancreatic head, 58% preferred initial surgery (PPPD) versus 42% initial endoscopy. In patients with a dilated MPD and intraductal stones 56% preferred initial endoscopic ± ESWL treatment and 29% preferred initial surgical treatment. Conclusion Worldwide, clinical decision-making in CP is largely based on local expertise, beliefs and disbeliefs. Further development of evidence-based guidelines based on well designed (randomized) studies is strongly encouraged.

There is a growing concern upon the finding of many drug induced Cushing’s syndrome because of inadvertent use of glucocorticoids (GC) either prescribed or as alternative medicine in Myanmar. These patients are presenting with diversity of clinical problems ranging from hypertension, diabetes to acute adrenal crisis due to hypothalamic-pituitary-adrenal (HPA) axis suppression and secondary adrenal insufficiency (AI). The present study aimed to assess the delta cortisol (the degree of cortisol increments) during the first short Synacthen test (SST) as a factor predictive of adrenal function recovery in patients with drug induced Cushing’ syndrome with secondary AI and to determine the proportion of patients who recovered from AI within six-month follow-up. This was a hospital based prospective analytical study that enrolled a total of 52 patients with drug induced Cushing’s syndrome with secondary AI from January 2018 to June 2019. Secondary AI is defined by morning basal serum cortisol ≤ 400 nmol/L with Synacthen stimulated peak cortisol level ≤ 550 nmol/L and serum ACTH < 60 pg/ml. The follow-up SSTs were performed at three-month and six-month after first SST, and the patients with morning basal serum cortisol > 400 nmol/L (or) Synacthen stimulated peak serum cortisol > 550 nmol/L during follow-up SSTs are defined as recovered adrenal function group. In this study, a total of 52 patients were treated with modified regimen of physiological dose of prednisolone with tapering schedule or stress dose GC based on the basal serum cortisol levels up to six months. Among them, nearly half (n=25 / 48.1%) of the patients with drug induced Cushing’s syndrome with secondary AI achieved normal adrenal function within six-month follow-up. It was found that mean values for delta cortisol were not statistically significant between recovered and non-recovered groups, 118.6 nmol/L (SD 72.3) and 97.2 nmol/L (SD 64.2) respectively. The delta cortisol during the first SST could not predict strongly (AUC - 0.6, 95% CI - 0.44 to 0.76, P = 0.2) the recovery of adrenal function in patients with drug induced Cushing’s syndrome with secondary AI and it was inconsistent with previous studies. The older age of the patients, oral route of administration of drugs causing Cushing’s syndrome and comorbid hypertension were found to be more significant in the non-recovered group. In conclusion, the present study did not support the evidence that the delta cortisol during the first SST could predict adrenal function recovery in patients with drug induced Cushing’ syndrome with secondary AI. Reference: (1) Baek et al., 2016; Recovery of Adrenal Function in Patients with Glucocorticoids Induced Secondary AI. Endocrinol Metab.31, pp. 153–160. (2) Pofi et al., 2018; The Short Synacthen Test Can Be Used to Predict Recovery of HPA Axis Function. J Clin Endocrinol Metab.103(8), pp. 3050–3059.

Hepatocellular carcinoma (HCC) is a major global health problem and its treatment outcomes are limited by therapeutic resistance. Cancer stem cells are the roots of tumor growth, recurrence, metastasis and treatment resistance. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor well known to promote carcinogenesis in a number of tumors. The present study aims to investigate the role of STAT3 in HCC. Total STAT3 levels were examined by qRT-PCR in patients with primary HCC who underwent curative partial hepatectomy. Elevated STAT3 levels were significantly associated with poor recurrence-free survival and overall survival of HCC patients (log-rank test, P = 0.019 and 0.008, respectively). Immunohistochemistry (IHC) was performed to determine the activation status of STAT3 in HCC. Phosphorylated (p)-Y705 and p-S727 staining was detected in HCC tissues, albeit with varying patterns of expression. To investigate the efficacy of STAT3-targeted therapeutics in HCC, napabucasin, a cancer stemness inhibitor targeting STAT3-driven gene transcription, and the specific small interfering RNA (siRNA) were examined in HCC cell lines Hep3B, HepG2 and Huh7. Both napabucasin and siRNA prominently downregulated RNA and protein levels of total STAT3 and p-STAT3 of HCC cells. Napabucasin reduced viability, induced death, hindered migration, impaired colony and spheroid formation efficiency of HCC cells, as well as lowered expression levels of the cancer stem cell markers granulin-epithelin precursor (GEP) and CD133 in whole genome RNA sequencing analysis. Notably, napabucasin combined with 5-fluorouracil (5-FU) synergistically inhibited cell proliferation and induced apoptosis, suggesting its potential in sensitizing HCC cells to chemotherapeutic agents. In addition, napabucasin diminished the colony and spheroid formation abilities of chemo-resistant HCC cells (acquired resistance to 5-FU with over 10-fold increase) to a similar extent as their parental counterparts, indicating its potency in targeting chemo-resistant HCC cells via cancer stemness inhibition. Furthermore, napabucasin also suppressed HCC tumor growth in vivo, accompanied with reduced total STAT3 and p-STAT3 levels and proliferation (Ki-67). Overall, the present study highlighted the prognostic significance of STAT3 in HCC, and its functional role in the control of chemo-resistance and cancer stemness in HCC through targeted therapeutics. Citation Format: Carol Lee, Elaine Yee-Ling OR, Charing Ching-Ning Chong, Tan To Cheung, Iris Ming-Jing Xu, Linda Wing-Chi Ng, Marcus Hung-Lam Law, Peter Tin-Chung Li, Stephen Lam Chan, Anthony Wing-Hung Chan, Philip Chun Yeung, Kelvin Kwok-Chai Ng, Paul Bo-San Lai, Siu Tim Cheung. STAT3 regulates chemo-resistance and cancer stemness in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4151.

The effect of statins in preventing hepatocellular carcinoma (HCC) recurrence after curative liver resection has been controversial. Some retrospective studies identified perioperative use of statins was associated with significantly lower risk of HCC recurrence and improve recurrence-free survival (RFS) after liver resection, among patients with or without chronic hepatitis viral infection in multivariate survival analysis. However, a recent single-center study enrolled 430 cases in Taiwan revealed that the protective effect of statins was only significant in univariate analysis but not significant in multivariate analyses. To evaluate the effect of statins on the risk of HCC recurrence after curative liver resections, we performed a systematic review and meta-analysis on this topic. A systematic search of Medline, Embase, and Web of Science was conducted through December 2019. Studies were included if they evaluated perioperative exposure to statins, reported the recurrence of HCC after curative resection, and reported hazard ratios (HR) of multivariable analysis by Cox proportional hazards model. Summary HR estimates with 95% confidence intervals (CI) were calculated using the random-effects model. The analysis included 5 studies reporting the recurrence of HCC among 8,586 patients after liver resection, in which 411 of them received perioperative statins treatments. A meta-analysis of the studies showed a significant (39%) reduction in the risk of HCC recurrence among patients who took statins (adjusted HR, 0.61; 95% CI, 0.49-0.76), with no or little heterogeneity among studies (χ2=5.58, df=4, I2=28%). In conclusion, perioperative statins use may improve recurrence-free survival and decrease the risk of HCC recurrence after curative liver resection. Further prospective studies and larger randomized controlled trials (RCTs) are warranted to validate the conclusion. Statin usersNon-statin usersAdjusted Hazard RatioStudyTotalTotalWeightIV, Random, 95%CIYear of publicationWu 2012175439437.6%0.68 (0.53-0.87)2012Lee 2016132207831.2%0.66 (0.49-0.90)2016Kawaguchi 2017317037.0%0.34 (0.12-0.75)2017Nishio 20184360014.5%0.42 (0.25-0.71)2018Young 2019304009.8%0.79 (0.41-1.52)2019Total (95% CI)4118175100.0%0.61 (0.49-0.76)Heterogeneity: Tau2=0.02; Chi2=5.58, df=4 (p=0.23); I2=28%Test for overall effect: Z=4.43 (p Citation Format: Chun Philip Yeung, Siu Tim Cheung, Kwok Chai Kelvin Ng, Bo San Paul Lai, Ching Ning Charing Chong. Statin use associated with reduced risk of hepatocellular carcinoma recurrence following liver resection: A systematic review and meta-analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2345.