Detection of therapeutically relevant and concomitant rare somatic variants in colorectal cancer.

In colorectal cancer (CRC), clinically relevant biomarkers are known for genome-guided therapy that can be detected by both first and next generation methods. The aim of our work was to introduce a robust NGS assay that will be able to detect, in addition to standard predictive single nucleotide-based biomarkers, even rare and concomitant clinically relevant variants. Another aim was to identify truncating mutations in APC and pathogenic variants in TP53 to divide patients into potentially prognostic groups. A multigene panel with hotspots in 50 cancer-critical genes was used. Finally, 86 patients diagnosed with primary or metastatic colorectal cancer were enrolled. In total, there were identified 163 pathogenic variants, among them in the genes most recurrent mutated in CRC such as TP53 (49%), the RAS family genes KRAS and NRAS (47%), APC (43%), and PIK3CA (15%). In 30 samples, two driver mutations were present in one sample, 11 patients were without any mutations covered by this panel. In one patient, a novel variant in BRAF p.D594E was found, not previously seen in CRC, and was concomitant with KRAS p.G12A. In KRAS, a potentially sensitive mutation to anti-EGFR therapy p.A59T was found along with the PIK3CA missense variant p.E545K. It was possible to divide patients into groups based on the occurrence of truncating APC variant alone or concomitant with TP53 or KRAS. Our results demonstrate the potential of small multigene panels that can be used in diagnostics for the detection of rare therapeutically relevant variants. Moreover, the division of patients into groups based on the presence of APC and TP53 mutations enables this panel to be used in retrospective studies on the effectiveness of treatment with anti-EGFR inhibitors.

Acute kidney injury after liver resection in elderly patients.

BACKGROUND: Acute kidney injury (AKI) affects approximately 13% of patients undergoing major abdominal surgery, and is a common and important clinical sign of perioperative injury. The aim of our analysis was to identify risk factors for AKI in elderly patients with no known kidney disease at the time of surgery, and to evaluate their 30-day, 12-month and 5-year survival. METHODS: We performed a retrospective analysis on a group of 785 patients after liver resection to determine the incidence of complications (AKI - according to KDIGO classification, sepsis, cardiovascular and surgical complications). All patients had normal kidney function prior to surgery. We determined risk factors for the development of AKI for two groups of patients, stratified for age: patients younger than 65 years, and patients older than 65 years. RESULTS: The incidence of complications was significantly higher in the group of patients older than 65 years (n = 76) than in younger patients (n = 119) (P = 0.0496). In the group of younger patients, significantly worse 30-day survival was observed for patients who developed AKI (P = 0.0004). We identified the following independent risk factors for AKI: male gender (HR 10,3834; P = 0,0238), histological identification of colorectal carcinoma metastases (HR 2,8651; P = 0,0499), surgery duration longer than 300 min (HR 6,0096; P < 0,0001), blood loss of more than 500 ml (HR 10,5857; P = 0,0012), and the need for more than 500 ml of fresh frozen plasma during surgery ml (HR 2,4878; P < 0,0317). Age was not confirmed to be an independent risk factor for AKI in our study. CONCLUSION: Approaches to treatment should be highly individualized, with assessment of several variables. According to our findings, age should not present a contraindication for the indication of a patient for surgery.

Differential gene expression of immunity and inflammation genes in colorectal cancer using targeted RNA sequencing.

Introduction: Colorectal cancer (CRC) is a heterogeneous disease caused by molecular changes, as driver mutations, gene methylations, etc., and influenced by tumor microenvironment (TME) pervaded with immune cells with both pro- and anti-tumor effects. The studying of interactions between the immune system (IS) and the TME is important for developing effective immunotherapeutic strategies for CRC. In our study, we focused on the analysis of expression profiles of inflammatory and immune-relevant genes to identify aberrant signaling pathways included in carcinogenesis, metastatic potential of tumors, and association of Kirsten rat sarcoma virus (KRAS) gene mutation. Methods: A total of 91 patients were enrolled in the study. Using NGS, differential gene expression analysis of 11 tumor samples and 11 matching non-tumor controls was carried out by applying a targeted RNA panel for inflammation and immunity genes containing 475 target genes. The obtained data were evaluated by the CLC Genomics Workbench and R library. The significantly differentially expressed genes (DEGs) were analyzed in Reactome GSA software, and some selected DEGs were used for real-time PCR validation. Results: After prioritization, the most significant differences in gene expression were shown by the genes TNFRSF4, IRF7, IL6R, NR3CI, EIF2AK2, MIF, CCL5, TNFSF10, CCL20, CXCL11, RIPK2, and BLNK. Validation analyses on 91 samples showed a correlation between RNA-seq data and qPCR for TNFSF10, RIPK2, and BLNK gene expression. The top differently regulated signaling pathways between the studied groups (cancer vs. control, metastatic vs. primary CRC and KRAS positive and negative CRC) belong to immune system, signal transduction, disease, gene expression, DNA repair, and programmed cell death. Conclusion: Analyzed data suggest the changes at more levels of CRC carcinogenesis, including surface receptors of epithelial or immune cells, its signal transduction pathways, programmed cell death modifications, alterations in DNA repair machinery, and cell cycle control leading to uncontrolled proliferation. This study indicates only basic molecular pathways that enabled the formation of metastatic cancer stem cells and may contribute to clarifying the function of the IS in the TME of CRC. A precise identification of signaling pathways responsible for CRC may help in the selection of personalized pharmacological treatment.

Expression of OCT4 isoforms is reduced in primary colorectal cancer.

Introduction: Colorectal cancer (CRC) is one of the most common types of cancer worldwide. The carcinogenesis of CRC is indeed complex, and there are many different mechanisms and pathways that contribute to the development of malignancy and the progression from primary to metastatic tumors. The OCT4A, encoded by the POU5F1 gene, is a transcription factor responsible for the phenotype of stem cells, maintaining pluripotency and regulation of differentiation. The POU5F1 gene is made up of five exons that can create numerous isoforms through alternative promoter or alternative splicing. In addition to OCT4A, other isoforms called OCT4B are also translated into protein; however, their role in cells has been unclear. The aim of our work was to investigate the expression patterns of OCT4 isoforms in primary and metastatic CRC, providing us with useful information about their role in the development and progression of CRC. Methods: Surgical specimens from a total of 78 patients were collected and isolated from primary tumors (n = 47) and metastases (n = 31). The relative gene expression of OCT4 isoforms was investigated using the RT-qPCR method together with the TaqMan probes for particular OCT4 isoforms. Results: Our results suggest significantly downregulated expression of the OCT4A and OCT4Bs isoforms in both primary (p = 0.0002 and p < 0.0001, respectively) and metastatic tumors (p = 0.0006 and p = 0.00051, respectively) when compared with the control samples. We also observed a correlation between reduced expression of all OCT4 isoforms and both primary and left-sided tumors (p = 0.001 and p = 0.030, respectively). On the other hand, the expression of all OCT4 isoforms was significantly upregulated in metastases compared with primary tumors (p < 0.0001). Discussion: Unlike previous reports, we found out that the expression of OCT4A, OCT4Bs, and all OCT4 isoforms was significantly reduced in primary tumors and metastases compared with control samples. On the other hand, we supposed that the expression rate of all OCT4 isoforms may be related to the cancer type and side, as well as to liver metastases. However, further studies are required to investigate the detailed expression patterns and significance of individual OCT4 isoforms in carcinogenesis.

Hypermethylated GRIA4, a potential biomarker for an early non-invasive detection of metastasis of clinically known colorectal cancer.

Introduction: Colorectal cancer (CRC) can develop through several dysregulated molecular pathways, including the serrated pathway, characterized by CpG island methylator (CIMP) phenotype. Although the tumor tissue is a commonly tested material, sample types such as stool or plasma, bring a new, non-invasive approach. Several cancer-related methylated genes have been identified in CRC patients, including gene GRIA4, showing promising diagnostic potential. The aim of our study was to develop a sensitive droplet digital PCR (ddPCR) assay to examine GRIA4 hypermethylation status in CRC patients and evaluate its diagnostic potential in tissue and liquid biopsy samples. Methods: In total, 23 patients participated in this study, 7 patients with primary CRC and 16 patients with liver metastasis of clinically known CRC. We obtained tumor and non-tumor tissues (N=17), blood samples pre- and post-surgery (N=22), and blood of five volunteers without a personal cancer history. We have developed and optimized a ddPCR assay for GRIA4 hypermethylation detection, from tissue and plasma samples. Results: We detected significantly increased GRIA4 methylation in tumor tissues compared to their adjacent non-tumor tissue, p<0.0001. Receiver operating characteristic (ROC) analysis defined cutoff values to separate primary tumors and metastases from non-tumor colon/rectum, specifically 36.85% for primary tumors and 34.81% for metastases. All primary tumors were above this threshold. When comparing the methylation levels of metastatic vs. non-tumor tissue, a smaller increase was observed in liver metastasis versus colon tissue (3.6× gain; p=0.001), then in liver metastasis versus adjacent liver tissue (17.4× gain; p<0.0001). On average, GRIA4 hypermethylation in primary tumor plasma was 2.8-fold higher (p=0.39), and in metastatic plasma, 16.4-fold higher (p=0.0011) compared to healthy individuals. Hypermethylation in metastatic plasma was on average 5.9 times higher (p=0.051) than in primary tumor plasma. After tumor removal surgery, average hypermethylation decrease in plasma was 1.6× for primary (p=0.037) and 4.5× for metastatic patients (p=0.023). Discussion: Based on our data, it can be inferred that GRIA4 serves as a tissue specific biomarker for the colon/rectum tissue, thus is suitable for cancer classification. This biomarker showed the potential to be an attractive target for early non-invasive detection of metastases of clinically known CRC, although additional analysis has to be performed.

Circulating miRNA expression over the course of colorectal cancer treatment.

Colorectal cancer (CRC) is the third-most common cancer type in males and the second-most common cancer type in females, and has the second-highest overall mortality rate worldwide. Approximately 50% of patients in stage I-III develop metastases, mostly localized to the liver. All physiological conditions occurring in the organism are also reflected in the levels of circulating microRNAs (miRNAs/miRs) in patients. miRNAs are a class of small, non-coding, single-stranded RNAs consisting of 18-25 nucleotides, which have important roles in various cellular processes. The aim of the present study was to evaluate a panel of seven circulating miRNAs (miR-106a-5p, miR-210-5p, miR-155-5p, miR-21-5p, miR-103a-3p, miR-191-5p and miR-16-5p) as biomarkers for monitoring patients undergoing adjuvant treatment of CRC. Total RNA was extracted from the plasma of patients with CRC prior to surgery, in the early post-operative period (n=60) and 3 months after surgery (n=14). The levels of the selected circulating miRNAs were measured with the miRCURY LNA miRNA PCR system and fold changes were calculated using the standard ∆∆Cq method. DIANA-miRPath analysis was used to evaluate the role of significantly deregulated miRNAs. The results indicated significant upregulation of miR-155-5p, miR-21-5p and miR-191-5p, and downregulation of miR-16-5p directly after the surgery. In paired follow-up samples, the most significant upregulation was detected for miR-106a-5p and miR-16-5p, and the most significant downregulation was for miR-21-5p. Pathway analysis outlined the role of the differentially expressed miRNAs in cancer development, but the same pathways are also involved in wound healing and regeneration of intestinal epithelium. It may be suggested that these processes should also be considered in studies investigating sensitive and easily detectable circulating biomarkers for recurrence in patients.

Cancer stem cell marker expression and methylation status in patients with colorectal cancer.

The number of individuals diagnosed with colorectal cancer (CRC) has been on an alarming upward trajectory over the past decade. In some countries, this cancer represents one of the most frequently diagnosed types of neoplasia. Therefore, it is an important demand to study the pathology underlying this disease to gain insights into the mechanism of resistance to treatment. Resistance of tumors to chemotherapy and tumor aggressiveness have been associated with a minor population of neoplastic cells, which are considered to be responsible for tumor recurrence. These types of neoplastic cells are known as cancer stem cells, which have been previously reported to serve an important role in pathogenesis of this malignant disease. Slovakia has one of the highest incidence rates of CRC worldwide. In the present study, the aim was to classify the abundance of selected stem cell markers (CD133, CD166 and Lgr5) in CRC tumors using flow cytometry. In addition, the methylation status of selected genomic regions of CRC biomarkers (ADAMTS16, MGMT, PROM1 (CD133), LGR5 and ALCAM) was investigated by pyrosequencing in a cohort of patients from Martin University Hospital, Martin, Slovakia. Samples from both primary tumors and metastatic tumors were tested. Analysis of DNA methylation in the genomic regions of indicated five CRC biomarkers was also performed, which revealed the highest levels of methylation in the A disintegrin and metalloproteinase with thrombospondin motifs 16 and O6-methyguanine-DNA methyl transferase genes, whereas the lowest levels of methylation were found in genes expressing prominin-1, leucine-rich repeat-containing G-protein-coupled receptor 5 and activated leukocyte cell adhesion molecule. Furthermore, tumor tissues from metastases showed significantly higher levels of CD133+ cells compared with that in primary tumors. Higher levels of CD133+ cells correlated with TNM stage and the invasiveness of CRC into the lymphatic system. Although relatively small number of samples was processed, CD133 marker was consider to be important marker in pathology of CRC.

The Difficult Path to Correct Diagnosis of Hepatolithiasis: A Case Report.

Hepatolithiasis is a benign disease, where stones are localized proximal to the confluence of hepatic ducts. The clinical picture may differ depending on whether the stones cause complete, partial, or intermittent biliary obstruction. The course can vary from asymptomatic to fatal, thus, early diagnosis and treatment is critical for a good prognosis. The gold standard in imaging is magnetic resonance cholangiopancreatography (MRCP). However, correct diagnosis can be challenging due to atypical clinical picture and laboratory findings. We present a case where hepatolithiasis was misdiagnosed initially due to incomplete reporting and documentation of MRCP. Choledocholithiasis was diagnosed based on initial MRCP, and endoscopic stone extraction was indicated. However, an unusual post-interventional course and signs of obstructive cholangitis led to an endoscopic re-intervention, which confirmed absence of pathology in extrahepatic biliary ducts. The cholangitis recurrence required intensive antibiotic treatment, and CT examination revealed intrahepatic S3 bile duct dilatation. Thus, a re-evaluation of initial MRCP and repeated MRCP confirmed hepatolithiasis. Further, laparoscopic bisegmentectomy was chosen as the definitive treatment due to the location of the lesion. The patient recovered and remained symptom free upon a 12 month follow up.

Genomic profile and immune contexture in colorectal cancer-relevance for prognosis and immunotherapy.

Colorectal cancer (CRC) is one of the leading cancers in both genders. TNM staging system is still the most commonly used tumor classification and prognostic system. The disadvantage of TNM is that the prognostic information it provides is incomplete, and patients with the same histological tumor stages may differ significantly in the clinical outcome. Therefore, the identification of new prognostic parameters is crucial. The carcinogenic process that gives rise to an individual tumor is unique and tumor microenviroment should be taken into consideration. In CRC, T-cell infiltration is not homogenous, and recent studies are mostly focusing on memory T-cells and CD8 cells in predicting disease-free survival (DFS) and overall survival (OS). It seems that DFS and OS are not only dependent on microsatellite instable or stable status but mostly on the levels of expression of the immune signatures. Also, patients with high infiltration of cytotoxic and memory cells have significantly better outcome. This review consolidates current knowledge and recent research about importance of immune-cell-associated proteins, specific gene profiles of immune cells and immunotherapy in CRC. We also discussed cell-specific signatures in cancer treatment.

Genetic and epigenetic analysis of the beta-2-microglobulin gene in microsatellite instable colorectal cancer.

One of the most common mechanisms of immune evasion in MSI colorectal cancers (CRCs) is loss of HLA class I expression due to mutations in B2M gene which can become a negative predictor for checkpoint blockade therapy. The aim of this study was the determination of prevalence of B2M somatic mutations in MSI CRC patients and relationship between B2M mutations and lymphocytes infiltration and other clinicopathological features as well as detection of methylation changes in B2M promoter region which can be another mechanism of immune escape. In our study, 37 MSI-H and 5 MSI-L patients were selected for screening of B2M mutational and methylation status. The characterization of patients was based on standard histopathological diagnosis and TNM classification; BRAF, KRAS mutations, tumor-infiltrating lymphocytes and peritumoral lymphoid reaction were also determined. MSI analysis was performed using fragment analysis. B2M mutations were identified by Sanger sequencing, and methylation of CpG islands in promoter region was detected by methylation-specific PCR. Heterozygous mutations in the B2M gene were detected in five MSI-H patients (13.5%), while the mutation c.45_48delTTCT was determined in four patients and mutation c.276delC was found in two patients. One of these five patients was compound heterozygote harboring both mutations. Methylation of the promoter region of the B2M gene was observed in one patient with MSI-H colorectal cancer. Detection of genetic and epigenetic changes in B2M gene could be important in personalized therapy for CRC patients as these changes may be one of the mechanisms of secondary resistance of MSI positive tumors to immunotherapy.