Circulating tumor DNA tracking in patients with pancreatic cancer using next-generation sequencing.

BACKGROUND: Pancreatic cancer remains one of the most devastating malignancies due to the absence of techniques for early diagnosis and the lack of target therapeutic options for advanced disease. Next Generation Sequencing (NGS) generates high throughput and valuable genetic information when evaluating circulating tumor DNA (ctDNA); however clinical utility of liquid biopsy in pancreatic cancer has not been demonstrated yet. The aim of this study was to evaluate whether results from a Next Generation Sequencing panel on plasma samples from pancreatic cancer patients could have a clinical significance. METHODS: From December 2016 to January 2020, plasma samples from 27 patients with pancreatic ductal adenocarcinoma at two different tertiary Spanish Hospitals underwent ctDNA testing using a commercial NGS panel of 65 genes. Clinical data were available for these patients. VarsSome Clinical software was used to analyse NGS data and establish pathogenicity. RESULTS: Evaluable NGS results were obtained in 18 out of the 27 plasma samples. Somatic pathogenic mutations were found mainly in KRAS, BRCA2, FLT3 and HNF1A, genes. Pathogenic mutations were detected in 50% of plasma samples from patient diagnosed at stages III-IV samples. FLT3 mutations were observed in 22.22% of samples which constitute a novel result in the field. CONCLUSIONS: Liquid biopsy using NGS is a valuable tool but still not sensitive or specific enough to provide clinical utility in pancreatic cancer patients.

CA19-9 capability as predictor of pancreatic cancer resectability in a Spanish cohort.

CA19-9 serum has been suggested as a marker of unresectability but different cut-off levels have been published. A cut-off of 500 U/ml is currently considered in an international consensus as biological criteria of borderline resectable pancreatic adenocarcinoma. To evaluate whether serum CA19-9 threshold of 500 U/ml could be adequate predictor of resectability in pancreatic adenocarcinoma. Multicenter, observational, prospective study performed in Spain including 203 patients diagnosed with pancreatic adenocarcinoma. 43 (21.2%) cases were resectable and 160 (78.8%) unresectable. Among the 176 preoperative CA19-9 available values, 98 (58.3%) were ≤ 500 U/ml and 73 (42.7%) > 500 U/ml. Resectability rate in those patients with CA19-9 ≤ 500 U/ml was 60% while it was found to be 18% when CA19-9 > 500 U/ml. Statistical model to predict resectability based on CA19-9 provide an AUC of 0.6618 (95% CI 0.53-0.83) when only CA19-9 values > 500 U/ml are studied. Serum levels of CA19-9 higher than 500 U/ml are indicative of unresectable disease, however reduced sensitivity and specificity lead to a limited clinical applicability for resectability.

Influence of Dopaminergic System Genetic Variation and Lifestyle Factors on Excessive Alcohol Consumption.

AIMS: To examine the role of genetic and environmental factors in the pathogenesis of alcohol dependence in a Spanish cohort of women and men. METHODS: We analyzed the relationship between 56 genetic variants in 7 genes associated with the dopaminergic reward pathway and excessive alcohol consumption. The study sample (N = 1533, of which 746 were women) consisted of 653 heavy consumers and 880 very low consumers from the Spanish subcohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Single nucleotide polymorphisms (SNPs) were genotyped using a customized array. Lifestyle variables were also examined to assess associations between genetic and environmental factors. RESULTS: No statistically significant differences were found between cases and controls for the allele frequencies in five genes: TH, SLC18A2, DRD1, DRD3 and COMT. Conversely, some alleles of the 12 SNPs from the DRD2 locus and the 5 from the MAOA locus showed significant associations with excessive alcohol consumption. Namely, rs10891556 (DRD2) proved to be the only SNP positively correlated with excessive alcohol consumption in both sexes. DRD2 rs1800497 and rs877138 were significantly associated in men, whereas DRD2 rs17601612 and rs4936271 and MAOA rs5906898 were associated with excessive alcohol consumption in women. A correspondence analysis provided an overall lifestyle profile of excessive drinkers, who were predominantly men who smoked, had large intakes of meat, small intakes of fruit and vegetables, whose jobs did not require high education levels and who engaged in little physical activity. CONCLUSIONS: It has shown the influence of dopaminergic pathway in the genetics of alcohol dependence with differences between men and women and providing a lifestyle profile of excessive drinkers.

Molecular characterization of oral squamous cell carcinoma using targeted next-generation sequencing.

OBJECTIVES: Many genetic factors play an important role in the development of oral squamous cell carcinoma. The aim of this study was to assess the mutational profile in oral squamous cell carcinoma using formalin-fixed, paraffin-embedded tumors from a Taiwanese population by performing targeted sequencing of 26 cancer-associated genes that are frequently mutated in solid tumors. METHODS: Next-generation sequencing was performed in 50 formalin-fixed, paraffin-embedded tumor specimens obtained from patients with oral squamous cell carcinoma. Genetic alterations in the 26 cancer-associated genes were detected using a deep sequencing (>1000X) approach. RESULTS: TP53, PIK3CA, MET, APC, CDH1, and FBXW7 were most frequently mutated genes. Most remarkably, TP53 mutations and PIK3CA mutations, which accounted for 68% and 18% of tumors, respectively, were more prevalent in a Taiwanese population. Other genes including MET (4%), APC (4%), CDH1 (2%), and FBXW7 (2%) were identified in our population. CONCLUSIONS: In summary, our study shows the feasibility of performing targeted sequencing using formalin-fixed, paraffin-embedded samples. Additionally, this study also reports the mutational landscape of oral squamous cell carcinoma in the Taiwanese population. We believe that this study will shed new light on fundamental aspects in understanding the molecular pathogenesis of oral squamous cell carcinoma and may aid in the development of new targeted therapies.

Loss of the transcription factor GLI1 identifies a signaling network in the tumor microenvironment mediating KRAS oncogene-induced transformation.

Although the biological role of KRAS is clearly established in carcinogenesis, the molecular mechanisms underlying this phenomenon are not completely understood. In this study, we provide evidence of a novel signaling network regulated by the transcription factor GLI1 mediating KRAS-induced carcinogenesis. Using pancreatic cancer (a disease with high prevalence of KRAS mutations) as a model, we show that loss of GLI1 blocks the progression of KRAS-induced pancreatic preneoplastic lesions in mice with pancreas-specific Cre-activated oncogenic mutant kras. Mice lacking GLI1 develop only low-grade lesions at low frequency, and in most cases, the pancreata are histologically normal. Further characterization of the phenotype showed a decrease in the activation of STAT3 in pancreatic preneoplastic lesions; STAT3 is a transcription factor required for the development of premalignant lesions and their progression into pancreatic cancer. Analysis of the mechanisms revealed a key role for GLI1 in maintaining the levels of activated STAT3 through the modulation of IL-6 signaling. GLI1 binds to the IL-6 mouse promoter and regulates the activity and expression of this cytokine. This newly identified GLI1/IL-6 axis is active in fibroblasts, a known source of IL-6 in the tumor microenvironment. Sonic hedgehog induces GLI1 binding to the IL-6 promoter and increases IL-6 expression in fibroblasts in a paracrine manner. Finally, we demonstrate that mutant KRAS initiates this cascade by inducing the expression of Sonic hedgehog in cancer cells. Collectively, these results define a novel role for GLI1 in carcinogenesis acting as a downstream effector of oncogenic KRAS in the tumor microenvironment.

Changes in the levels of thioredoxin and indoleamine-2,3-dioxygenase activity in plasma of patients with colorectal cancer treated with chemotherapy.

Increased oxidative stress and indoleamine-2,3-dioxygenase (IDO) activity have been reported in cancer, but their relationship with chemotherapy remains unknown. The aim of the present study was to examine wether the chemotherapy treatments used in colorectal cancer had an additional effect on oxidative stress and on IDO activity. Plasma samples were collected from 27 colorectal cancer patients on cytostatic treatment, 27 with cytostatic drugs plus monoclonal antibodies (cytostatic-Mabs) and 15 non-treated patients. All patients with colorectal cancer had high plasma malondialdehyde (MDA), thioredoxin (Trx) levels, and elevated IDO activity in plasma (IDOp) and in dendritic cells (IDOc). This study shows that treatment with cytostatics have an effect on oxidative stress by increasing MDA levels and by decreasing Trx levels and IDO activity. However, treatment with cytostatic-Mabs showed no effect on MDA levels but decreased Trx levels, and the IDO activity showed values similar to the healthy group. Significant correlations between plasma IDO activity and the levels of Trx (r = 0.2062, p < 0.05) and MDA (r = 0.2873, p < 0.005) were observed. Furthermore, our study suggests that IDO activity measured as kynurenine levels could be used as a marker of the response to the chemotherapy treatments, although further studies are necessary.

Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models.

BACKGROUND: Statins may have therapeutic effects on hepatocarcinoma (HCC). This type of disorder is the most common malignant primary tumour in the liver. Our objective was to determine whether pravastatin had a therapeutic effect in vitro and in vivo models. METHOD: We design in vitro and in vivo model. In vitro we used PLC and determine cell proliferation. In vivo, we used and animal model to determined, PCNA and MAT1A expression and transaminases levels. RESULTS: We found that pravastatin decreases cell proliferation in vitro (cell proliferation in pravastatin group was 82%, in sorafenib group 51% and in combined group 40%) and in vivo (in pravastatin group 80%, in sorafenib group 76.4% and in combined group 72.72%). The MAT1A levels, was significantly higher in Pravastatin group (D 62%, P 94%, S 71%, P + S 91%). The transaminases levels, decreased significantly in Pravastatin group (GOT and GPT levels D 619.5 U/L; 271 U/L) (P 117.5 U/L; 43.5 U/L) (S 147 U/L; 59 U/L) (P + S 142 U/L; 59 U/L). CONCLUSION: The combination of pravastatin + sorafenib were more effective than Sorafenib alone.

No evidence for PML-RARa bcr1 fusion gene in colorectal cancer.

Colorectal cancer is the third most prevalent cancer and a leading cause of cancer death. Metastatic colorectal cancer patients are treated with anti-EGFR monoclonal antibodies in combination with chemotherapy; however, the efficiency is only 10-20% of such patients. An increasing amount of data has demonstrated that response to anti-EGFR therapies is confined to patients with KRAS and BRAF wild type tumors but still some of these patients are non responders to this treatment. The presence of oncogenic deregulation of different members of EGFR downstream signaling or crosstalk molecules could predict the lack of response in these patients. In this study, 40 wild type KRAS and BRAF colorectal tumors were analyzed to elucidate whether PML-RARa bcr1 fusion gene may play a role in colorectal carcinogenesis. Specifically we want to determine if this fusion could be responsible for the inability to respond to anti-EGFR monoclonal antibodies. Here, for the first time it is reported, that PML-RARa bcr1 fusion is not responsible for colorectal tumor development and also, this translocation is not predicting the lack of efficacy of anti-EGFR therapies in wild type KRAS and BRAF colorectal cancer patients. These results also suggest that PML-RARa is unlikely to be a promising target for adjuvant therapy in colorectal cancer patients.

KRAS assay selection: sensitivity and accuracy in clinical application.

KRAS mutation status is routinely tested before the administration of chemotherapeutic agents that target EGFR such as cetuximab and panitumumab. Various commercial assays to analyze KRAS mutational status are currently certified for in vitro diagnostic use. However, the differences in the operational characteristics of the different assays have not been investigated. Since different assays could be different in specificity, sensitivity and precision, studies to clarify the best option for an adequate determination of KRAS mutation status are necessary. Until a gold standard for testing KRAS mutation status is established, the treating physician must interpret the results of any given assay keeping this limitation in mind.

Effect of neoadjuvant chemotherapy in hepatic steatosis.

UNLABELLED: Chemotherapy drugs often produce side effects in the liver. In recent years, there has been speculation about the ability to produce hepatic steatosis in patients treated with 5-fluorouracil and oxaliplatin. This prospective study examines whether these drugs can produce steatosis in patients with neoadjuvant treatment who were operated on for liver tumors. PURPOSE: Our objective was to assess the effect of neoadjuvant chemotherapy (NAC) on the development of hepatic steatosis in the healthy liver. PATIENTS AND METHODS: This was a prospective study based on 32 patients divided into two groups. The presence of steatosis was assessed using a histological score (Kleiner classification) and a biochemical method (Folch method) for patients from both groups. RESULTS: A total of 14 patients (44%) had hepatic steatosis and half of these were in each group. The steatosis was moderate to severe (grades 2-3) in 4 patients (13%), 2 in each group. The mean levels of triglycerides in the liver were 33.38 and 29.94 mg/g in group I and group II, respectively, with the difference not being statistically significant. CONCLUSIONS: Almost half of the patients treated with NAC for liver neoplasia developed steatosis. Nevertheless, NAC does not seem to increase the risk of hepatic steatosis.

KRAS, BRAF, EGFR and HER2 gene status in a Spanish population of colorectal cancer.

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical-pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.

PIK3CA mutations in KRAS and BRAF wild type colorectal cancer patients. A study of Spanish population.

The objective of the work was to study PIK3CA mutations in wild type KRAS and BRAF colorectal cancer. Clinicopathological data and paraffin-embedded specimens were collected on 73 patients who underwent colorectal resections at General Yagüe Hospital in Burgos. KRAS, BRAF and PIK3CA status were analyzed by real-time PCR in all patients. PIK3CA mutations were present in 8.22% of wild type KRAS and BRAF colorectal cancers. The most frequent mutation is E545K/D in exon 9 which represents 83.3% of all mutations. By contrast, we did not found any tumour harbouring H1047R mutation in exon 20. Among the patients who undergo a curative resection of colorectal cancer, PIK3CA mutation is present in an important percentage of KRAS and BRAF wild type tumours. PIK3CA mutation may be considered as it could be a hypothetic reason to be not responder to anti-EGFR antibodies.

TAp73 is one of the genes responsible for the lack of response to chemotherapy depending on B-Raf mutational status.

BACKGROUND: Although there have been many studies on the p73 gene, some of its functions still remain unclear. There is little research on the relationship between p73 gene transcription and its protein expression and the response to certain drugs such as oxaliplatin and cetuximab, which are drugs currently used in colorectal cancer.The purpose of this study was to evaluate the impact of TAp73 expression on oxaliplatin and cetuximab-based chemotherapy in colorectal cancer cell lines with different K-Ras and B-Raf mutational status. METHODS: TAp73 was analyzed in three colorectal tumor cell lines HT-29, SW-480 and Caco-2. mRNA TAp73 was determined using Real time PCR; TAp73 protein by immunoblotting and cell viability was analyzed by the MTT method. RESULTS: We found that mRNA and TAp73 protein were decreased in cells treated with oxaliplatin (in monotherapy or combined with cetuximab) when B-Raf is mutated. This was statistically significant and was also associated with higher cell viability after the treatment. CONCLUSIONS: Here, for the first time we report, that there is a signaling loop between B-Raf activation and p73 function.Low expression of TAp73 in colorectal cancer cell lines with mutated B-Raf may be involved in the lack of response to oxaliplatin in monotherapy or combined with cetuximab.

Validation of miR-1228-3p as Housekeeping for MicroRNA Analysis in Liquid Biopsies from Colorectal Cancer Patients.

BACKGROUND: Circulating microRNA (miRNA) analysis is a growing research field. However, it usually requires an endogenous control or housekeeping (HK) in order to normalize expression of specific miRNAs throughout different samples. Unfortunately, no adequate HK for circulating miRNA analysis is still known in the colorectal cancer (CRC) context whereas several have been suggested. Hence, our aims were to validate the previously suggested miR-1228-3p as HK for CRC studies, to compare its suitability with the widely used miR-16-5p, and to evaluate the influence of hemolysis on both miRNAs. METHODS: We analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) the expression of miR-1228-3p, miR-16-5p and the spike-in cel-miR-39 in a set of 297 plasmas (92 CRC, 101 advanced adenomas -AA-, and 100 controls) and 213 serum samples (59 CRC, 74 AA and 80 controls). We also analyzed both miRNAs depending on the hemolysis degree in 7 plasmas and 31 serums. RESULTS: Levels of miR-1228-3p and miR-16-5p did not show significant differences between groups although miR-16-5p exhibited more variability in plasma and serum samples. Importantly, the combination of cel-miR-39 and miR-1228-3p was the most stable one. Moreover, we observed that miR-16-5p was significantly influenced by hemolysis in contrast with miR-1228-3p that exhibited no correlation with this confounding factor in both biofluids. CONCLUSION: MiR-1228-3p has been validated as an adequate endogenous control for circulating miRNA analysis in CRC and AA liquid biopsies.

Plasma MicroRNA Signature Validation for Early Detection of Colorectal Cancer.

OBJECTIVES: Specific microRNA (miRNA) signatures in biological fluids can facilitate earlier detection of the tumors being then minimally invasive diagnostic biomarkers. Circulating miRNAs have also emerged as promising diagnostic biomarkers for colorectal cancer (CRC) screening. In this study, we investigated the performance of a specific signature of miRNA in plasma samples to design a robust predictive model that can distinguish healthy individuals from those with CRC or advanced adenomas (AA) diseases. METHODS: Case control study of 297 patients from 8 Spanish centers including 100 healthy individuals, 101 diagnosed with AA, and 96 CRC cases. Quantitative real-time reverse transcription was used to quantify a signature of miRNA (miRNA19a, miRNA19b, miRNA15b, miRNA29a, miRNA335, and miRNA18a) in plasma samples. Binary classifiers (Support Vector Machine [SVM] linear, SVM radial, and SVM polynomial) were built for the best predictive model. RESULTS: Area under receiving operating characteristic curve of 0.92 (95% confidence interval 0.871-0.962) was obtained retrieving a model with a sensitivity of 0.85 and specificity of 0.90, positive predictive value of 0.94, and negative predictive value of 0.76 when advanced neoplasms (CRC and AA) were compared with healthy individuals. CONCLUSIONS: We identified and validated a signature of 6 miRNAs (miRNA19a, miRNA19b, miRNA15b, miRNA29a, miRNA335, and miRNA18a) as predictors that can differentiate significantly patients with CRC and AA from those who are healthy. However, large-scale validation studies in asymptomatic screening participants should be conducted.

HIV Epidemiology in Uganda: survey based on age, gender, number of sexual partners and frequency of testing.

BACKGROUND: Human Immunodeficiency Virus (HIV) is a major cause of morbidity and mortality in the world. When compared to the developed countries where HIV prevalence is on the decline, sub-Saharan Africa has experienced either a rise or stagnation in rates. OBJECTIVES: The aim of this study was to test and educate the community in the villages of Masajja and Kibiri of Wakiso district in Uganda for HIV and safe sex practices. METHODS: A sociodemographic survey was also performed to obtain data for gender, age, number of sexual partners during the previous year, frequency of testing and if ever tested positive for other sexually transmitted diseases (STDs). RESULTS: While 7 of the tested individuals were positive for HIV, 77 reported that they had once tested positive for other STDs. Of the 7 HIV positive individuals, 4 were females and 3 males. Over half of the tested individuals reported only one sexual partner in past 12 months and more than a quarter were sexually active with more than one partner. Majority of our population also reported getting HIV tested every 6 months or less. CONCLUSION: Robust implementation of methods such as education and frequent testing can lower Uganda's prevalence of HIV even further.

Pancreatic Cancer in Lynch Syndrome Patients.

Although colorectal cancer (CRC) is the most common cancer type in Lynch syndrome (LS) families, patients have also increased lifetime risk of other types of tumors. The accumulated risk of pancreatic cancer (PC) in LS patients is around 3.7% and developed tumors often present a characteristically medullary appearance with prominent lymphocytic infiltration. LS patients are considered in high risk for PC development as they present 8.6-fold increase compared with the general population. Here we review PC cases reported in LS patients and current management guidelines. Literature data show that LS is clearly associated with PC and recent publications also demonstrated a connection with pancreatic neoplasic precursor lesions such as intraductal papillary mucinous neoplasms (IPMN) in these patients. While screening techniques are well established for CRC detection, clear strategies are not yet uniform for PC. Magnetic resonance imaging (MRI) and/or endoscopic ultrasound every 1-2 years in MMR mutation carriers with PC in a first or second-degree relative is recommended. Better pancreatic cancer detection strategies should be urgently defined due to the importance of early diagnosis in this disease.

Antimicrobial susceptibility testing before first-line treatment for Helicobacter pylori infection in patients with dual or triple antibiotic resistance.

AIM: To evaluate the efficacy of antimicrobial susceptibility-guided therapy before first-line treatment for infection in patients with dual or triple antibiotic resistance. METHODS: A total of 1034 patients infected by Helicobacter pylori (H. pylori) during 2013-2014 were tested for antimicrobial susceptibility. 157 of 1034 (15%) patients showed resistance to two (127/1034; 12%) and to three (30/1034; 3%) antibiotics. Sixty-eight patients with dual H. pylori-resistance (clarithromycin, metronidazole or levofloxacin) were treated for 10 d with triple therapies: OAL (omeprazole 20 mg b.i.d., amoxicillin 1 g b.i.d., and levofloxacin 500 mg b.i.d.) 43 cases, OAM (omeprazole 20 mg b.i.d., amoxicillin 1 g b.i.d., and metronidazole 500 mg b.i.d.) 12 cases and OAC (omeprazole 20 mg b.id., amoxicillin 1 g b.i.d., and clarithromycin 500 mg b.i.d.) 13 cases based on the antimicrobial susceptibility testing. Twelve patients showed triple H. pylori-resistance (clarithromycin, metronidazole and levofloxacin) and received for 10 d triple therapy with OAR (omeprazole 20 mg b.id., amoxicillin 1 g b.i.d., and rifabutin 150 mg b.i.d.). Eradication was confirmed by 13C-urea breath test. Adverse effects and compliance were assessed by a questionnaire. RESULTS: Intention-to-treat eradication rates were: OAL (97.6%), OAM (91.6%), OAC (92.3%) and OAR (58.3%). Cure rate was significantly higher in naïve patients treated with OAR-10 compared to patients who had two or three previous treatment failures (83% vs 33%). Adverse events rates for OAL, OAM, OAC and OAR were 22%, 25%, 23% and 17%, respectively, all of them mild-moderate. CONCLUSION: Antimicrobial susceptibility-guided triple therapies during 10 d for first-line treatment leads to an eradication rate superior to 90% in patients with dual antibiotic H. pylori resistance.

Glypican-1 in exosomes as biomarker for early detection of pancreatic cancer.

On June 24, 2015 Nature published an article entitle "Glypican-1 identifies cancer exosomes and detects early pancreatic cancer'', which demonstrates that exosomes positives for the proteoglycan glypican-1 (GPC1) are expressed in serum of patients with pancreatic cancer since very early stages but not in benign pancreatic disease. Additionally, these GPC1(+) circulating exosomes correlate with tumor burden and could be used as prognostic biomarker in pre and post-surgical patients. The study is pioneer since GPC1 biomarker in exosomes offers better sensitivity and specificity than any other under evaluation or used in clinical practice. However, methodology for exosomes isolation still remains at investigational phase. Further studies are need to translate this technology to a practicable clinical method. Further research is also required to validate this biomarker in larger prospective cohort including more cases of premalignant lesions and then replicated results could possible guide changes in clinical practice.