Comparison of circulating DNA in malignant neoplasia from diverse locations: Investigating a diagnostic role.

CONTEXT: Circulating free DNA (cfDNA) analysis has emerged as novel noninvasive diagnostic biomarker in several solid tumors. Raised levels have been reported in several malignancies and may correlate with clinicopathological and treatment response. The current study was designed to assess the diagnostics of cfDNA in different tumor types of malignancies correlating with tumor (T), nodes (N), and metastases (M) stage. DESIGN: Serum samples were collected from treatment naïve cases with histologically diagnosed tumors including 23 brain tumors, 48 breasts, 50 gallbladder carcinoma (GBC), 13 lungs, 68 oral squamous cell carcinoma (OSCC), and 25 normal controls. CfDNA was quantified with real-time polymerase chain reaction (PCR), Invasive ductal carcinoma (IDC) using beta-globin gene amplification. Cut off values for diagnostics were calculated using receiver operating curve analysis. RESULTS: Contrary to other cfDNA studies where it was postulated that cfDNA would not cross the blood-brain barrier and reach the systemic circulation, we found detectable cfDNA in glioma with median (Q1-Q3) of 349.22 ng/ml (19.87-1276.58). Median cfDNA concentration in breast, gallbladder, lung, oral and normal controls was 328.72 (128.38-624.44), 778.50 (589.88-1864.35), 348.73 (194.67-483.61), 386.27 (47.88-959.67), and 74.12 (49.66-120.00), respectively. Grades I and II glioma had significantly lower levels compared to Grades III and IV (P = 0.0001). Significant difference in median cfDNA values in IDC and GBC was observed with increasing tumor grades, stage, T stage, nodal stage and metastasis and with stage of OSCC cases. CONCLUSION: CfDNA levels showed good diagnostic discrimination in glioma, GBC, breast, lung carcinoma, and OSCC. Significant increase in titers was evident with increase in cancer stage from I to IV in breast, GBC and OSCC.

Plasma-derived candidate biomarkers for detection of gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Reliable and affordable biomarker-based assays with high sensitivity and specificity for the detection of this cancer are a clinical need. With the aim of studying the potential of the plasma-derived extracellular vesicles (EVs), we carried out quantitative proteomic analysis of the EV proteins, using three types of controls and various stages of the disease, which led to the identification of 86 proteins with altered abundance. These include 29 proteins unique to early stage, 44 unique to the advanced stage and 13 proteins being common to both the stages. Many proteins are functionally relevant to the tumor condition or have been also known to be differentially expressed in GBC tissues. Several of them are also present in the plasma in free state. Clinical verification of three tumor-associated proteins with elevated levels in comparison to all the three control types-5'-nucleotidase isoform 2 (NT5E), aminopeptidase N (ANPEP) and neprilysin (MME) was carried out using individual plasma samples from early or advanced stage GBC. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC. These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC.

The clinical significance of preoperative serum fibrinogen levels and platelet counts in patients with gallbladder carcinoma.

BACKGROUND: Gallbladder carcinoma (GBC) was the most common malignancy of biliary tract. Patients with malignancies frequently present with activated coagulation pathways, which might potentially related to tumor progression and prognosis. The purpose of the study was to investigate the clinical significance of preoperative serum fibrinogen levels and platelet counts in GBC patients. METHODS: The preoperative fasting serum fibrinogen levels and platelet counts of 58 patients with GBC were measured by AUV2700 automatic biochemical analyzer, as well as 60 patients with cholesterol polyps and 60 healthy volunteers. Kaplan-Meier survival analysis was applied to show the correction between fibrinogen levels and outcome after surgery. RESULTS: The fibrinogen levels of patients with GBC were significantly higher than healthy gallbladder and cholesterol polyp of gallbladder (p < 0.001 and p < 0.001, respectively). In GBC, fibrinogen levels were associated with tumor depth (p = 0.001), lymph node metastasis (p = 0.002), distant metastasis (p < 0.001) and Tumor Node Metastasis (TNM) stage (p < 0.001). The levels in TNM stage IV disease were significantly higher than stage III or stage I + II disease (p = 0.048 and p < 0.001, respectively), and in TNM stage III disease were significantly higher than stage I + II disease (p = 0.002). Furthermore, the overall survival was better in low fibrinogen level group than in high fibrinogen level group (p < 0.001). However, thrombocytosis was not significantly associated with overall survivals (p > 0.05) in multivariate analysis. CONCLUSIONS: The preoperative serum fibrinogen levels and platelet counts might be reliable biomarkers for the occurance of disease, tumor depth, lymph node metastasis, distant metastasis and advanced TNM stage in patients with GBC. The serum fibrinogen levels might be a prognostic factor to predict outcome for GBC patients suffering from surgery treatment. Anticoagulation therapy might be considered to control cancer progression in future studies.

Exploration of differentially-expressed exosomal mRNAs, lncRNAs and circRNAs from serum samples of gallbladder cancer and xantho-granulomatous cholecystitis patients.

Gallbladder cancer (GBC) is the most common biliary tract malignancy worldwide. Although a growing number of studies have explored the mechanism of GBC, thus far, few molecules have been discovered that can be utilized as specific biomarkers for the early diagnosis and therapeutic treatment of GBC. Recent studies have shown that exosomes not only participate in the progression of tumors, but also carry specific information that can define multiple cancer types. The present study investigated the expression profiles of coding (or messenger) ribonucleic acids (mRNAs) and non-coding RNAs (ncRNAs, including long non-coding RNAs [lncRNAs] and circular RNAs [circRNAs]) in plasma-derived exosomes from GBC patients. Using high-throughput RNA sequencing and subsequent bioinformatic analysis, a number of differentially expressed (DE) mRNAs, lncRNAs, and circRNAs were identified in GBC exosomes, compared to their expressions in xantho-granulomatous cholecystitis (XGC) exosomes. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) analyses were then conducted to investigate the potential functions of these DE RNAs. Furthermore, the interaction networks and competing endogenous RNA networks of these DE RNAs and their target genes were investigated, revealing a complex regulatory network among mRNAs and ncRNAs. In summary, this study demonstrates the diagnostic value of plasma-derived exosomes in GBC and provides a new perspective on the mechanism of GBC.

Diagnostic Value of Inflammatory Factors in Patients with Gallbladder Cancer, Dysplasia, and Cholecystitis.

BACKGROUND: Involving pre-sampled patients with cholecystitis, dysplasia, and adenocarcinoma, the present study aimed to compare the neutrophil/lymphocyte (NLR), monocyte/lymphocyte (MLR), platelet/lymphocyte (PLR) ratios, and plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW) values and to determine their prognostic importance. METHODS: The present study involved 187 cholecystectomy specimens that were diagnosed as cholecystitis, dysplasia, and adenocarcinoma. Preoperative neutrophil, monocyte, lymphocyte, and platelet counts, NLR, MLR, and PLR ratios, and PCT, MPV, and PDW levels of the same patient groups were retrospectively recorded. RESULTS: In the present study, the cut-off values for dysplasia of NLR, PLR, and MLR were found as 1.61, 81.45, and .19, whereas those for cancer of NLR, PLR, and MLR were 2.65, 182.69, and .35, respectively. The NLR, PLR, and MLR values of the chronic cholecystitis and chronic calculous cholecystitis groups were statistically significantly lower than those of the chronic active calculous cholecystitis group (P < .01). The NLR and MLR values of the non-cancer and non-dysplasia groups were statistically lower than those of the cancer and dysplasia groups (P < .05). CONCLUSION: According to the results of the present study, using additional imaging methods, acute-phase cholecystitis can be distinguished using preoperative neutrophil and monocyte counts, and NLR, PLR, and MLR cut-off values can be used to distinguish dysplasia, which is the antecedent of gallbladder cancer. It is thought that this might provide patients with an advantage in terms of early treatment and survival.

Extracellular vesicle-shuttled miRNAs as a diagnostic and prognostic biomarker and their potential roles in gallbladder cancer patients.

Circulating microRNAs (miRNAs) in serum extracellular vesicles (EVs) are a promising biomarker in cancer. We aimed to elucidate the serum EVs miRNA biomarkers to identify patients with gallbladder cancer (GBC) and to clarify their potential roles. One hundred nineteen serum EVs from GBC and non-GBC individuals were isolated by pure-EVs-yieldable size-exclusion chromatography, and then were analyzed using a comprehensive miRNAs array and RT-qPCR-based validation. The functional roles of the identified miRNAs were also investigated using GBC cell lines. Serum EVs miR-1246 and miR-451a were significantly upregulated and downregulated, respectively in GBC patients (P = 0.005 and P = 0.001), in line with their expression levels in cancer tissue according to an in silico analysis. The combination of CEA and CA19-9 with miR-1246 showed the highest diagnostic power (AUC, 0.816; Sensitivity, 72.0%; Specificity, 90.8%), and miR-1246 was an independent prognostic marker of GBC (Hazard ratio, 3.05; P = 0.017) according to a Cox proportional hazards model. In vitro, miR-1246 promoted cell proliferation and invasion, while miR-451a inhibited cell proliferation and induced apoptosis with the targeting of MIF, PSMB8 and CDKN2D. Taken together, miR-1246 in serum EVs has potential application as a diagnostic and prognostic marker and miR-451a may be a novel therapeutic target in GBC.

The expression of growth differentiation factor 15 in gallbladder carcinoma.

PURPOSE: This study initially explored the expression of GDF-15 in gallbladder carcinoma and its clinical significance, and analyzed the correlation between the expression of GDF-15 and the clinicopathological features as well as the prognosis of patients with gallbladder carcinoma. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of GDF-15 in the serum of 42 patients with gallbladder cancer. The control group included 24 patients with cholecystitis and 20 healthy volunteers. The immunohistochemical method (IHC) was used to detect the expression of GDF-15 in 42 cases of gallbladder tumor tissue and 35 cases of adjacent non-tumor gallbladder tissue specimens. RESULTS: The results of ELISA showed that the concentration of GDF-15 in serum was considerably higher in gallbladder cancer patients than that in gallbladder benign lesions and healthy volunteers (p=0.006, p<0.001). In the group of patients with gallbladder cancer, the consistence of GDF-15 in patients with lymph node metastasis was significantly higher than that of patients without lymph node metastasis (p<0.001). Immunohistochemical staining showed that the expression of GDF-15 in gallbladder carcinoma was markedly higher than that in non-tumor gallbladder tissues (p=0.003), and the high expression of GDF-15 was significantly correlated with the differentiation grade of gallbladder carcinoma and tumor TNM stage (p=0.005, p=0.002). CONCLUSION: GDF-15 is related to the occurrence and development of gallbladder cancer. GDF-15 in serum can be used as a potential marker for the diagnosis of gallbladder cancer and can be used to predict the lymph node metastasis of gallbladder cancer.

Roles of Salmonella typhi and Salmonella paratyphi in Gallbladder Cancer Development.

BACKGROUND: Typhoid (Salmonella typhi and paratyphi) carriers and gall bladder cancer (GBC) are endemic in northern India. Results of previous studies about association of typhoid carriers with GBC are inconsistent. We studied antibodies against Salmonella typhi and paratyphi in serum samples of patients with GBC. METHODS: We performed modified Widal test for antibodies against Salmonella typhi (Vi and O) and Salmonella paratyphi (AO and BO) antigens in patients with GBC (n=100), xanthogranulomatous cholecystitis (XGC, n=24), chronic cholecystitis (CC, n=200) and healthy controls (HC, n=200). RESULTS: Serum antibodies against Salmonella were more frequently positive in GBC (22%) and XGC (29%), particularly in males in age ≥50 years (GBC: 47% and XGC: 50%) vs. HC (0) (p <0.01). Vi antibody was more common in GBC (13%, OR:9.8) and XGC (8%, OR:5.9) than HC (2%). O antibody was more common in GBC (8%, OR: 8.6) and XGC (8%, OR: 9.0) than HC (1%). O antibody was also more common in males with GBC (12%) than CC (1%) and HC (1%) (P=0.02 and p <0.001, respectively). AO (6%) and BO (4%) antibodies were detected in GBC, particularly in males, than HC (0), (p <0.01). Salmonella antibodies were more frequent in GBC with GS than those without GS (50% vs. 20%, OR=3.94, P=0.01). CONCLUSIONS: Salmonella carrier state was more common in GBC and XGC, particularly in elderly males than HC. The Vi antibody was more common in GBC and XGC than HC. Salmonella infection was more common in GBC with GS than those without GS.

Inflammatory profiles in Chilean Mapuche and non-Mapuche women with gallstones at risk of developing gallbladder cancer.

Chile has high incidence rates of gallbladder cancer globally, particularly among Amerindian women, who also have a high prevalence of gallstones. We examined differences in inflammatory biomarkers between Mapuche and non-Mapuche women from the Chile Biliary Longitudinal Study, a cohort of women with ultrasound-detected gallstones. We randomly selected 200 Mapuche women frequency matched to non-Mapuche women on age and statin use Inflammatory biomarkers were analyzed using a multiplex assay and linear regression to assess associations of a priori markers (CCL20, CXCL10, IL-6, and IL-8) with ethnicity. Novel biomarkers were analyzed using exploratory factor analysis (EFA) and sufficient dimension reduction (SDR) to identify correlated marker groups, followed by linear regression to examine their association with ethnicity. The mean values of IL-8 were higher in Mapuche than non-Mapuche women (P = 0.04), while CCL20, CXCL10, and IL-6 did not differ significantly by ethnicity. EFA revealed two marker groups associated with ethnicity (P = 0.03 and P < 0.001). SDR analysis confirmed correlation between the biomarkers and ethnicity. We found higher IL-8 levels among Mapuche than non-Mapuche women. Novel inflammatory biomarkers were correlated with ethnicity and should be studied further for their role in gallbladder disease. These findings may elucidate underlying ethnic disparities in gallstones and carcinogenesis among Amerindians.

Serum CA 19-9 and CA 125 as a diagnostic marker in carcinoma of gallbladder.

BACKGROUND: Gall bladder carcinoma is endemic in North India along the Ganges belt. Most of the cases usually present in late stage when prognosis is poor. That mandates a necessity for proper screening in these areas for gall bladder lesions. Tumor markers CA 19-9 and CA 125 have been studied in various GI cancers and may also help in the screening, diagnosis and evaluation of gall bladder carcinoma. Aims: To assess serum CA19-9 and serum CA125 in patients with gall bladder lesions and find out a cut off value for diagnosis of carcinoma gallbladder. METHODS AND MATERIAL: Study included 118 cases, with female: male ratio of 4:1.Out of it, 91 (77 %) cases were benign and 27 (23 %) were malignant. Patients' sera was collected and analyzed for CA19-9 and CA 125 by CMIA method. RESULTS: The Mean (SD) value of CA19-9 for benign and malignant cases was found to be 12.86 (17.54) and 625.35(186.52) U/ml. For CA 125 it was found to be 17.98(13.69) and 239.63(73.72) U/ml respectively. The difference was statistically significant (P< 0.001). When Mean - 2SD value of malignant lesions were taken as cut off a value of CA 19-9 and CA 125 were found be 252.31 U/ml & 92.19U/ml respectively, found to be significant to suggest /diagnose a case of carcinoma gall bladder along with clinicoradiological findings. Taking these value as cut off Sensitivity & Specificity for CA 19-9 and CA 125 in detecting malignant cases were found to be 100% & 98.90% and 100% & 94.50% respectively. CONCLUSIONS: It is concluded that both serum CA 19-9 and serum CA 125 may act as a good adjunct for diagnosis of cases of carcinoma gallbladder along with imaging studies. However, changes in CA19-9 are more significant than CA 125.

Incidence and Preoperative Predictor Factors of Gallbladder Cancer Before Laparoscopic Cholecystectomy: a Systematic Review.

INTRODUCTION: Many cases of gallbladder cancer (GBC) were made incidentally after cholecystectomy for presumed benign disease. The aim of this review is to assess the preoperative predictor factors of gallbladder cancer. METHODS: This systematic review was conducted according to PRISMA guidelines when it was applicable. We conducted bibliographic researches on October 2nd, 2019, in the following sources: The National Library of Medicine through PubMed, Cochrane database, and Google scholar. We have assessed the univariate and multivariate analysis outcomes. RESULTS: We included ten studies. Incidence of incidental GBC was 0.36%. Seven studies reported age as a significant predictive factor of iGBC. Comorbidities were the second significant predictor. One study found that iGBC group was more likely to have elevated TB, DB, PAL, and ALT. Another study reported a significantly higher rate of TB, PAL, and AST. One study concluded that elevated CA19-9 combined with CEA or CA-125 was significantly more frequent in the group with iGBC. Polyps, porcelain GB, GB wall thickness, and CBD dilation were reported to be associated with iGBC. iGBC group were more likely to have solitary and larger GS and gallbladder wall thickening, essentially focal. CONCLUSION: Incidence of iGBC was 0.365% varying between 0.19 and 1.6% of laparoscopic cholecystectomy and about 50% of GBC cases. This highlights the deficiency of preoperative diagnostic features. Despite the efforts made, the rate of this condition is still high, underlining the need of new radiological technologies.

The C-reactive protein to albumin ratio is an excellent prognostic predictor for gallbladder cancer.

A number of inflammation indicators based on C-reactive protein (CRP) and albumin have been widely used to predict the prognosis in several types of tumors, but their functions in gallbladder cancer (GBC) have rarely been explored. The aim of our study is to evaluate and compare the prognostic values of the C-reactive protein to albumin ratio (CAR), Glasgow prognostic score (GPS), modified Glasgow prognostic score (mGPS) and high-sensitivity modified Glasgow prognostic score (HS-mGPS) in patients with GBC. 144 GBC patients who received curative surgery in our hospital from January 2010 to May 2017 were enrolled in this research. The Kaplan-Meier analysis showed that the median OS of the patients in the high CAR group was significantly shorter than the patients in the low group (p < 0.001), and higher scores of GPS, mGPS and HS-mGPS were also associated with decreased OS, respectively. However, according to the Receiver Operating Characteristic (ROC) curve, the CAR was superior to the other prognostic scores in determining the prognosis for the GBC patients. In the multivariate analysis, CAR was verified as an independent risk factor for poor prognosis, together with tumor differentiation, T stage and postoperative complications. All in all, compared to the other three CRP-albumin-related prognostic predictors, CRA is a better indicator in predicting poor long-term outcomes in GBC patients after radical surgery.

Immunoproteomics approach revealed elevated autoantibody levels against ANXA1 in early stage gallbladder carcinoma.

BACKGROUND: Early diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of 'tumor-associated antigens (TAAs) that elicit humoral response' in early stage GBC patients. METHODS: Total protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins. RESULTS: Image analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed 'high' expression levels in 72% of GBC cases whereas all the controls showed 'low' expression levels. CONCLUSIONS: The study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

Overexpression of microRNA-205-5p exerts suppressive effects on stem cell drug resistance in gallbladder cancer by down-regulating PRKCE.

Some microRNAs (miRs or miRNAs) have been reported to function as tumor suppressors in gallbladder cancer (GBC). However, the specific effect of miR-205-5p on GBC remains unclear. The objective of the present study was to unravel the effects of miR-205-5p on the drug resistance in GBC. For this purpose, the expression of miR-205-5p and protein kinase C ϵ (PRKCE) was quantified in the peripheral blood sample harvested from GBC patients and healthy volunteers. Then the relationship between miR-205-5p and PRKCE was validated. After isolating the GBC stem cells, ectopic expression and depletion experiments were conducted to analyze the effect of miR-205-5p and PRKCE on cell proliferation, drug resistance, apoptosis, and colony formation rate as well as the expression of apoptotic factors (Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved caspase 3). Finally, the mouse xenograft model of GBC was established to verify the function of miR-205-5p in vivo. Intriguingly, our results manifested that miR-205-5p was down-regulated, while PRKCE was up-regulated in peripheral blood samples and stem cells of patients with GBC. Moreover, miR-205-5p targeted PRKCE and negatively regulated its expression. The overexpression of miR-205-5p or silencing of PRKCE inhibited the drug resistance, proliferation, and colony formation rate while promoting apoptosis of GBC stem cells. Additionally, the overexpression of miR-205-5p attenuated drug resistance to gemcitabine but promoted the gemcitabine-induced cell apoptosis by inhibiting the PRKCE in vivo. Overall, an intimate correlation between miR-205-5p and PRKCE is a key determinant of drug resistance of GBC stem cells, thus, suggesting a novel miR-205-5p-based clinical intervention target for GBC patients.

The Clinical Role of Preoperative Serum CA19-9 and Carcinoembryonic Antigen (CEA) Levels in Evaluating the Resectability of Advanced Gallbladder Cancer.

BACKGROUND The present study was designed to study the ability of preoperative serum concentrations of the tumor-associated biomarkers carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9) and adjusted CA19-9 to assess the resectability of advanced gallbladder cancer (GBC). MATERIAL AND METHODS This retrospective study included patients with potentially resectable stage II-IV (AJCC 8th) GBC examined at our institution between January 2012 and December 2016. Receiver operating characteristic (ROC) curve analysis was used to determine the predictive value and optimal cut-off point of tumor-associated biomarkers for curative resection. RESULTS Pathological examination of the 309 patients included in this study found that 169 (54.7%) underwent R0 (curative) resection, whereas 121 (39.2%) underwent R1/2 (non-curative) resection, and 19 (6.1%) were unresectable. The mean serum concentrations of CEA, CA19-9 and adjusted CA19-9 were significantly lower in patients who underwent R0 resection than in the other groups. ROC curve analysis showed that adjusted CA19-9 concentration was better able to predict resectability (area under the curve, 0.774; 95% confidence interval, 0.722-0.826; P<0.001) than total bilirubin, CEA, and CA19-9 concentrations. The optimal cut-off for adjusted CA19-9 concentration was 47.63 U/mL, which had a sensitivity of 69.82%, a specificity of 75%, a positive predictive value of 77.12% and a negative predictive value of 67.31%. CONCLUSIONS Adjusted CA19-9 concentration is an easily calculated parameter superior to CA19-9 and CEA concentrations in predicting the resectability of advanced gallbladder cancer.

Raised CA19-9 and CEA have prognostic relevance in gallbladder carcinoma.

BACKGROUND: Role of tumor markers in gall bladder carcinoma (GBC) is not well established. We evaluated the prognostic value of carbohydrate antigen 19-9 (CA19-9) and carcinoma embryonic antigen (CEA) in patients with GBC. METHODS: Of the 225 patients of GBC enrolled,176 patients were included in the study (excluded 49 patients with jaundice). Patients were divided into 3 groups; resectable n = 92, unresectable n = 17, metastatic n = 67. The clinico-pathological characteristics, tumor markers and survival data were analysed. The cutoff values of CA19-9 & CEA for predicting metastases were computed using receiver operating characteristic curve. Kaplan Meir survival and Cox regression analysis were done for factors predicting survival and recurrence. RESULTS: The median value of Ca19-9 was significantly higher in metastatic group [resectable: 21.3, unresectable: 53.9 and metastatic: 79; p < 0.001] but not for CEA [3.5, 7.8 and 5 ng/ml (p = 0.20)]. A cutoff value of 72 IU/ml for CA19-9, 5 ng/ml for CEA had a sensitivity and specificity of 52 and 80%, 51 and 72% respectively for detection of metastatic disease. Median, 3-year & 5-year survival were significantly lower in patients with CEA > 4 (p = 0.041), Ca19.9 > 37 (p = 0.019), T3/T4 (p = 0.001), node positive (p = 0.001) and presence of perineural invasion (p = 0.001). However, on multivariate analysis, only Ca19.9 > 37 predicted recurrence (p = 0.002, HR 5.8). CONCLUSIONS: Raised CA19.9 and CEA predict metastatic disease in patients with GBC without jaundice with a high specificity and may help in prognostication of the patient. CA19-9 was better than CEA in prediction of tumor burden and in predicting recurrence.

Exosomal piRNA profiling revealed unique circulating piRNA signatures of cholangiocarcinoma and gallbladder carcinoma.

Cholangiocarcinoma (CCA) and gallbladder carcinoma (GBC) are biliary tract cancers with poor five-year survival and high recurrence rates. Both CCA and GBC patients suffer from lack of circulating diagnostic biomarkers at the early stage. Extracellular vesicles, especially exosomes, have been emerged as promising diagnostic sources for cancers due to easy and quick accessibility. Hence, identification of exosomal biomarkers provides a novel strategy for CCA and GBC diagnosis. Here, five CCA patients and four GBC patients were enrolled for exosomal small RNA sequencing. Our data showed that exosomal piwi-interacting RNA (piRNA) populations were altered in the plasma of CCA and GBC patients. In comparison to healthy individuals, 694 and 323 piRNAs were upregulated in CCA and GBC, respectively, while 36 and 191 piRNAs were downregulated. Interestingly, sequencing results predicted that piR-2660989, piR-10506469, piR-20548188, piR-10822895, piR-hsa-23209, and piR-18044111 were upregulated in both CCA and GBC plasma. Importantly, we further included blood samples from 50 health individuals, 40 CCA patients, and 25 GBC patients and found that piR-10506469 were significantly increased in the exosomes of plasma from both CCA and GBC patients. Moreover, we analyzed the expression levels of differentially expressed exosomal piRNAs in the plasma of CCA and GBC patient before and after surgeries and found that piR-10506469 and piR-20548188 were significantly decreased in patients underwent surgeries. Taken together, our data revealed that exosomal piRNAs those are differentially expressed in CCA and GBC plasma may serve as potential biomarkers for the diagnosis of CCA and GBC.

Epigenetic Biomarkers in Gallbladder Cancer.

Gallbladder cancer (GBC) is associated with various nongenetic and genetic factors. Lack of specific and sensitive diagnostic markers has significantly impacted the mortality of this disease. Here we discuss the recent discovery of epigenetic changes that show great promise as diagnostic biomarkers as well as potential therapeutic targets for GBC.

Surgical Indication for Advanced Gallbladder Cancer Considering the Optimal Preoperative Carbohydrate Antigen 19-9 Cutoff Value.

BACKGROUND: Selecting patients who will benefit from resection among those with advanced gallbladder cancer (GBCa) having poor prognostic factors is difficult. METHODS: One hundred twenty-one patients who underwent resection for stage II-IV GBCa and 19 unresected patients (unresectable group) were enrolled. The clinical impact of carbohydrate antigen 19-9 (CA19-9) and advanced surgical procedures for GBCa was evaluated. RESULTS: The optimal CA19-9 cutoff value (based on the greatest difference in overall survival) was 250 U/mL. CA19-9 ≥250 U/mL was found to be an independent prognostic factor. Patients with CA19-9 <250 U/mL who developed jaundice (median survival time [MST], 49.1 months) or who required major hepatectomy (MST, 21.5 months) or pancreatoduodenectomy (PD; MST, 50.3 months) had a better prognosis than those with CA19-9 ≥250 U/mL who developed jaundice (MST, 16.1 months; p = 0.061) or who required major hepatectomy (MST, 9.2 months; p = 0.066) or PD (MST, 8.6 months; p = 0.025); their prognosis was comparable to that of the unresectable group (jaundice: p = 0.145, major hepatectomy: p = 0.292, PD: p = 0.756). CONCLUSIONS: Even if GBCa patients develop jaundice or require major hepatectomy, or combined PD, resection can be considered for those with CA19-9 <250 U/mL. However, surgical indication should be carefully determined in patients with CA19-9 ≥250 U/mL.

Baseline derived neutrophil-to-lymphocyte ratio as a prognostic biomarker for non-colorectal gastrointestinal cancer patients treated with immune checkpoint blockade.

BACKGROUND: Biomarkers in non-colorectal gastrointestinal (GI) cancer patients receiving immune checkpoint blockades (ICBs) are still limited. METHODS: Data were prospectively collected from a discovery cohort (n = 53) and a validation cohort (n = 107) in patients with non-colorectal GI cancer receiving ICB, as well as a chemotherapy-only cohort (n = 171). System inflammatory markers and derived neutrophil-to-lymphocyte ratio (dNLR) were determined as biomarkers by univariate and multivariate analyses. RESULTS: A higher level of dNLR (cutoff = 3) was associated with shorter overall survival (OS) in discovery and validation cohorts. In pooled cohort, disease control rate (DCR) (28% vs. 48.1%) was associated with dNLR (p = .017). In univariate analysis, original tumor site, tumor histopathology, number of metastases, and dNLR were correlated with OS. In multivariate analysis, higher dNLR level was correlated with reduced OS (10.43 months vs. 4.20 months, p < .001). In chemotherapy-only cohort, dNLR was also correlated with DCR and OS. CONCLUSION: Higher dNLR level was correlated with worse outcomes, suggesting that dNLR may help risk-group stratification and assist disease management strategies as a prognostic biomarker for non-colorectal GI patients receiving ICB.