Genetic analysis and allele-specific expression of SMAD7 3'UTR variants in human colorectal cancer reveal a novel somatic variant exhibiting allelic imbalance.

BACKGROUND: Considering the impact of SMAD7 deregulation in colorectal cancer (CRC) progression and the significance of single nucleotide variant (SNV)-mediated disruptions of microRNA (miRNA)-dependent regulation for cancer susceptibility, our study aimed to analyze genetic variation in the SMAD7 3' untranslated region ( 3'UTR) in CRC, measure differences in allelic mRNA expression, and evaluate its interference with miRNA-mediated post-transcriptional regulation. PATIENTS AND METHODS: This study included 80 patients with different CRC stages and six human colon cancer cell lines of various histological origins. SMAD7 3'UTR was analyzed by direct sequencing, followed by the relative quantification of differential allelic expression of detected variants by allele-specific qRT-PCR. In silico tools were employed for predictions of regulatory consequences of detected variants. RESULTS: A total of four different SNVs in one cell line and nine patients were found, among which were a novel somatic point variant and three already known germline variants (rs16950113, rs1050799536, and rs1043778717). All evaluated SNVs exhibited variable extents of allelic imbalance in expression. In silico analysis predicted significant effects of SNVs on miRNA binding efficiency, with each SNV disrupting existing and creating new target sites for one or more miRNAs. CONCLUSION: Imbalance observed in the expression of SNV alleles altering miRNA binding suggests that all investigated SNVs are potential contributing factors impacting SMAD7 expression regulation in CRC that further studies should investigate.

Pilot proteomic study of locally advanced rectal cancer before and after neoadjuvant chemoradiotherapy indicates high metabolic activity in non-responders' tumor tissue.

PURPOSE: In the search for candidate predictive biomarkers to evaluate response to neoadjuvant chemoradiotherapy (nCRT) in rectal cancer, only a few studies report proteomic profiles of tumor tissue before and after nCRT. The aim of our study was to determine differentially expressed proteins between responders and non-responders before and after the therapy in order to identify candidate molecules for prediction and follow-up of response to nCRT. EXPERIMENTAL DESIGN: The study has included tissue sections of rectal tumor and non-tumor mucosa from five responders and five non-responders taken before and after nCRT from patients with locally advanced rectal cancer. Extracted proteins were analyzed by LC-MS/MS analysis followed by a set of bioinformatics analyses. RESULT: Proteomics analysis provided a mean of approximately 1050 protein identifications per sample. A comparison of proteomic profiles between responders and non-responders has identified 18 differentially expressed proteins. Pathway analysis demonstrated high metabolic activity in non-responders' tumors before nCRT, indicating the presence of intrinsic chemoradioresistance in these subjects. Two proteins associated with poor prognosis in colorectal cancer, ADAM10 and CAD, were identified as candidate predictive biomarkers as they were present in non-responders only. CONCLUSIONS AND CLINICAL RELEVANCE: Shortlisted proteins from our study should be further validated as candidate biomarkers for response to routinely applied nCRT protocols.

Application of nanofat for treatment of traumatic faecal incontinence after sphincteroplasty - A pilot study.

AIM: The aim of this study was to investigate whether the application of nanofat containing stem cells improves continence in women who had previously undergone anal sphincteroplasty with unsatisfactory long-term outcomes. METHOD: This prospective pilot study included nine women with various degrees of anal incontinence who had previously undergone anal sphincteroplasty due to obstetric trauma. In all patients, the Wexner Incontinence Score (WS) and Faecal Incontinence Quality of Life Score (FIQLS), as well as anal manometry and endoanal ultrasound measurements, were performed before the procedure and during follow-up. In all patients, liposuction was performed and 50 ml of raw lipoaspirate was obtained and processed using a NanoFat Kit device. Approximately 20 ml of the mechanically emulsified and filtrated fat was obtained and the anal sphincter complex was infiltrated with it. Patient follow-up was conducted in person or via telephone 6 and 12 months after the procedure. RESULTS: The squeeze pressure was significantly increased 6 months after the procedure (p = 0.01). The external anal sphincter measured at the 12 o'clock position was significantly thicker (p = 0.04). A significant decrease in the WS was observed both 6 and 12 months after the procedure compared with baseline values (p < 0.05 for both). CONCLUSION: This study is the first to show that the application of nanofat as an injectable product improves continence in patients with unsatisfactory results after sphincteroplasty, suggesting it to be a promising and effective therapeutic tool. The procedure is safe and can be easily performed as an ambulatory procedure.

Nucleic acid-based markers of response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer.

The progress that has been made in the treatment of rectal cancer has mostly resulted from multimodality strategy approach that combines surgery, chemotherapy and radiotherapy. In locally advanced rectal cancer (LARC), surgery remains the primary treatment, while neoadjuvant chemoradiotherapy (nCRT) is used to downsize or downstage the tumor before surgical resection. Highly variable response to nCRT observed in LARC patients raises the need for biomarkers to enable prediction and evaluation of treatment response in a more efficient and timely manner than currently available tools. The search for predictive biomarkers continues beyond blood proteins, which have failed in subsequent validation studies. This review presents nucleic acids-based markers and their predictive potential in LARC patients. Most of the candidate biomarkers come from relatively small single-institution studies. The only candidate biomarker that emerged as relevant in more than a single study was elevated levels of Fusobacterium nucleatum nucleic acids in tumor tissue. Considering that this marker is easily accessible through non-invasive analysis of faecal samples, its predictive potential is worth further validation. The other candidate nucleic acid-based biomarkers require more consistent studies on larger cohorts before they can be considered for use in clinical setting.

Prognostic potential of circulating miR-93-5p in patients with colorectal cancer liver metastases.

This study aimed to examine the expression pattern of tumoral and circulating miR-93-5p in patients with colorectal cancer (CRC) liver metastasis (CRLM) and to explore its predictive and prognostic potential. CRLM tissue, surrounding non-tumor liver tissue, and serum were obtained from 35 patients with CRLM. The expression pattern of tissue and circulating miR-93-5p in patients with CRLM was determined using quantitative polymerase chain reaction, using miR-16-5p for normalization. Sample-based cut-off values for CRLM and serum miR-93-5p expression were calculated using Receiver Operating Characteristic curve analysis to stratify the patients into high and low miR-93-5p expression groups which were that compared with patients' clinicopathological data, therapy response, one-year disease-free survival, and disease recurrence. Relative miR-93-5p expression was higher in CRLM in comparison to the non-metastatic liver tissue (p<0.001). CRLM miR-93-5p expression showed moderate negative correlation with carcinoembryonic antigen levels (r=-0.406; p=0.016). There were no differences in high-/low-miR-93-5p expression and therapy responders vs. non-responders, which was confirmed in vitro using metastatic and normal colonic cells SW620 and HCEC-1CT, respectively. No difference was observed in one-year recurrence-free survival in patients with high vs. low miR-93-5p expression in CRLM or serum. However, high miR-93-5p serum levels were significantly associated with early disease recurrence (p=0.035). In conclusion, miR-93-5p serum levels could be potentially used as a prognostic factor for early disease recurrence in CRLM patients.

SMAD4-201 transcript as a putative biomarker in colorectal cancer.

BACKGROUND: Transcripts with alternative 5'-untranslated regions (UTRs) result from the activity of alternative promoters and they can determine gene expression by influencing its stability and translational efficiency, thus executing complex regulation of developmental, physiological and pathological processes. Transcriptional regulation of human SMAD4, a key tumor suppressor deregulated in most gastrointestinal cancers, entails four alternative promoters. These promoters and alternative transcripts they generate remain unexplored as contributors to the SMAD4 deregulation in cancer. The aim of this study was to investigate the relative abundance of the transcript SMAD4-201 in colorectal cell lines and tissues in order to establish if its fluctuations may be associated with colorectal cancer (CRC). METHODS: Relative abundance of SMAD4-201 in total SMAD4 mRNA was analyzed using quantitative PCR in a set of permanent human colon cell lines and tumor and corresponding healthy tissue samples from patients with CRC. RESULTS: The relative abundance of SMAD4-201 in analyzed cell lines varied between 16 and 47%. A similar relative abundance of SMAD4-201 transcript was found in the majority of analyzed human tumor tissue samples, and it was averagely 20% lower in non-malignant in comparison to malignant tissue samples (p = 0.001). Transcript SMAD4-202 was not detectable in any of the analyzed samples, so the observed fluctuations in the composition of SMAD4 transcripts can be attributed to transcripts other than SMAD4-201 and SMAD4-202. CONCLUSION: The expression profile of SMAD4-201 in human tumor and non-tumor tissue samples may indicate the translational potential of this molecule in CRC, but further research is needed to clarify its usability as a potential biomarker for early diagnosis.

The role of haematological parameters in predicting the response to radical chemoradiotherapy in patients with anal squamous cell cancer.

BACKGROUND: Historically, the treatment of choice for anal cancer had been abdominoperineal resection (APR). Radical radiotherapy with concurrent 5-fluorouracil plus mitomycin C chemotherapy was later established as standard therapy, although with a failure rate of 20-30%. The aim of this study was to evaluate the outcomes after radical chemoradiotherapy (CRT), prognostic and predictive factors and patterns of failure. PATIENTS AND METHODS: This study included 47 patients treated with radical CRT for patohistologicaly confirmed anal squamous cell carcinoma. Analysed haematological parameters included: neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and haemoglobin level. The final logistic regression model included treatment break period. Tumour response was assessed at 24 weeks from CRT completion. Follow-up was performed every 3 months during the first two years, and every 6 months thereafter. RESULTS: A complete clinical response (CR) was detected in 30 patients (63.8%). Patients who did not achieve a 6-months CR and those who had a CR after 6 months but then relapsed were referred to surgical treatment. With combined CRT and surgical salvage treatment the CR rate was 80.9%. Patients with CR after 6 months had significantly longer disease-free survival (DFS), progression-free survival (PFS), and overall survival (OS). A significant effect on the 6-month response was confirmed for PLR (p = 0.03). CONCLUSIONS: Important prognostic factors associated with CR were baseline haemoglobin level and period of treatment interruptions. Potential haematological prognostic factors could be PLR and NLR, which can be routinely determined by low-cost and minimally invasive methods.

SMAD7 and SMAD4 expression in colorectal cancer progression and therapy response.

Inhibitory SMAD7 and common mediator SMAD4 play crucial roles in SMAD-dependent TGF-ß signaling that is often disrupted in colorectal cancer (CRC). This study aimed to profile the expression of SMAD7 and SMAD4 in primary and metastatic CRC and to evaluate their significance in disease progression and therapy response. The expression of SMAD7 and SMAD4 genes was analyzed by quantitative real-time PCR in tissues from 35 primary and metastatic CRC patients and in vitro in 7 human cell lines originating from colon tissue. Expression levels of SMAD7 and SMAD4, as well as their ratio, were determined and their association with tumor characteristics and response to therapy were evaluated. SMAD4 level was significantly lower in tumors compared to non-tumor tissues in both primary (p = 0.001) and metastatic (p = 0.001) CRC patients, while tumor expression of SMAD7 was significantly lower from non-tumor tissue only in metastatic patients (p = 0.017). SMAD7/SMAD4 ratio was elevated in CRC primary tumor tissues and cell lines compared to corresponding non-tumor tissues and cell line, respectively (p = 0.003). SMAD7 expression was significantly elevated in primary tumor tissues obtained from responders to neoadjuvant chemoradiotherapy (nCRT) compared to non-responders (p = 0.014). Alterations of expression and ratio of SMAD7 and SMAD4 in CRC cell lines, primary rectal cancer, and liver metastasis emphasize the importance of these genes in different stages of disease progression. Differential expression of SMAD7 in responders versus non-responders to nCRT should be further investigated for its potential predictive value.

Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients.

Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups-patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be interpreted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted personalized CRC patient management.

Prognostic Significance of CDH1, FN1 and VIM for Early Recurrence in Patients with Colorectal Liver Metastasis After Liver Resection.

PURPOSE: There are limited data on expression of epithelial-mesenchymal transition (EMT) markers in patients with colorectal liver metastases (CRLM). The study aim was to evaluate the expression and prognostic significance of E-cadherin (CDH1), fibronectin (FN1) and vimentin (VIM) in patients with CRLM after curative-intent liver resection. PATIENTS AND METHODS: Thirty patients with CRLM managed by curative-intent liver resection were included in this prospective pilot study. Blood samples, colorectal liver metastases and surrounding non-tumor liver tissue were collected. Expression of CDH1, FN1 and VIM was analyzed by quantitative real-time polymerase chain reaction. Expression in CRLM and non-tumor liver tissue was compared, while expression in serum was correlated with CRLM expression. One-year recurrence-free survival was compared between patients with low and high CDH1, FN1 and VIM expression. RESULTS: The expression of CDH1 was similar in CRLM and non-tumor liver tissues, while FN1 and VIM expression was significantly lower in metastatic tissue (P=0.003 and pP<0.001, respectively). Serum expression of CDH1 and VIM was detected in 66.7% and 93.3% of patients, respectively, while FN1 was not detected in any of the patients. The correlation of CDH1 and VIM expression between CRLM and serum was not statistically significant. Decreased CDH1 expression in CRLM and decreased VIM expression in serum were associated with early recurrence after surgical treatment of CRLM. CONCLUSION: Lower expression of CDH1 in CRLM and lower serum expression of VIM were found to be associated with early recurrence after liver resection for CRLM.

Perineural invasion as a prognostic factor in patients with stage I-III rectal cancer - 5-year follow up.

BACKGROUND: Rectal cancer (RC) is one of the most common diagnosed cancers, and one of the major causes of cancer-related death nowadays. Majority of the current guidelines rely on TNM classification regarding therapy regiments, however recent studies suggest that additional histopathological findings could affect the disease course. AIM: To determine whether perineural invasion alone or in combination with lymphovascular invasion have an effect on 5-years overall survival (OS) of RC patients. METHODS: A prospective study included newly diagnosed stage I-III RC patients treated and followed at the Digestive Surgery Clinic, Clinical Center of Serbia, between the years of 2014-2016. All patients had their diagnosis histologically confirmed in accordance with both TMN and Dukes classification. In addition, the patient's demographics, surgical details, postoperative pathological details, differentiation degree and their correlation with OS was investigated. RESULTS: Of 245 included patients with stage I-III RC, lymphovascular invasion (LVI) was identified in 92 patients (38%), whereas perineural invasion (PNI) was present in 46 patients (19%). Using Kaplan-Meier analysis for overall survival rate, we have found that both LVI and PNI were associated with lower survival rates (P < 0.01). Moreover when Cox multiple regression model was used, LVI, PNI, older age, male gender were predictors of poor prognosis (HR = 5.49; 95%CI: 2.889-10.429; P < 0.05). CONCLUSION: LVI and PNI were significant factors predicting worse prognosis in early and intermediate RC patients, hence more aggressive therapy should be reserved for these patients after curative resection.

Altered organization of collagen fibers in the uninvolved human colon mucosa 10 cm and 20 cm away from the malignant tumor.

Remodelling of collagen fibers has been described during every phase of cancer genesis and progression. Changes in morphology and organization of collagen fibers contribute to the formation of microenvironment that favors cancer progression and development of metastasis. However, there are only few data about remodelling of collagen fibers in healthy looking mucosa distant from the cancer. Using SHG imaging, electron microscopy and specialized softwares (CT-FIRE, CurveAlign and FiberFit), we objectively visualized and quantified changes in morphology and organization of collagen fibers and investigated possible causes of collagen remodelling (change in syntheses, degradation and collagen cross-linking) in the colon mucosa 10 cm and 20 cm away from the cancer in comparison with healthy mucosa. We showed that in the lamina propria this far from the colon cancer, there were changes in collagen architecture (width, straightness, alignment of collagen fibers and collagen molecules inside fibers), increased representation of myofibroblasts and increase expression of collagen-remodelling enzymes (LOX and MMP2). Thus, the changes in organization of collagen fibers, which were already described in the cancer microenvironment, also exist in the mucosa far from the cancer, but smaller in magnitude.

Combined Diagnostic Efficacy of Neutrophil-to-Lymphocyte Ratio (NLR), Platelet-to-Lymphocyte Ratio (PLR), and Mean Platelet Volume (MPV) as Biomarkers of Systemic Inflammation in the Diagnosis of Colorectal Cancer.

BACKGROUND: Systemic inflammation in colorectal cancer (CRC) may be reflected by neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and mean platelet volume (MPV). This study was designed to investigate the efficiency of preoperative NLR, PLR, and MVP as a tool for the assessment of tumor characteristics in newly diagnosed patients with CRC. PATIENTS AND METHODS: For 300 patients and 300 healthy volunteers, complete blood counts with automated differential counts were performed. The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count; PLR was calculated by dividing the absolute platelet count by the absolute lymphocyte count. The diagnostic performance of NLR, PLR, and MVP was estimated by ROC curve. RESULTS: ROC curve analysis showed high diagnostic efficacy of NLR and PLR in CRC patients with cut-off values of 2.15 (AUC = 0.790, 95% CI 0.736-0.884, Se = 74.1%, and Sp = 73%) and 123 (AUC = 0.846, 95% CI 0.801-0.891, Se = 73.5%, and Sp = 80%) compared to healthy controls, respectively. The diagnostic efficacy of three combined markers was superior compared with individual markers (AUC = 0.904, 95% CI 0.812-0.989, Se = 96%, and Sp = 70%). CONCLUSION: NRL, PLR, and MPV may be useful markers in diagnostic and early recognition of different stages of CRC; additionally combined all together have stronger diagnostic efficacy.

Deletion and Single Nucleotide Polymorphisms in Common Glutathione-S Transferases Contribute to Colorectal Cancer Development.

Glutathione-S transferases (GSTs) are xenobiotic-conjugation enzymes involved in the detoxification process of heterocyclic aromatic amines and polycyclic aromatic hydrocarbons, widely recognized risk factors of colorectal cancer (CRC) development. Polymorphism in GSTs often leads to alteration or complete lack of enzyme activity, which might have an effect on CRC carcinogenesis. Aim of this study was to investigate GST gene variants as risk factors in patients with CRC. A total of 523 CRC patients administered for surgical resection and 400 matched controls were included. Deletion polymorphism of GSTs M1 and T1 was investigated by polymerase chain reaction. Single nucleotide polymorphism of GST A1 and P1 was investigated by restriction fragment length polymorphism method. The association between GST genotype and risk of CRC development was found in carriers of GSTT1-null and GSTP1-variant genotypes individually (p = 0.050 and p = 0.016, respectively). Furthermore, statistically significant association was found when combination of GSTP1-variant genotype with any of other three common GST genotypes was analyzed with respect to CRC susceptibility. Additionally, patients with combined GSTM1-null/GSTT1-null/GSTA1 low-activity/GSTP1-variant genotype showed 2.71-fold increased risk of developing CRC (p = 0.037). This study supports hypothesis that GST polymorphisms might have an important role in the process of the CRC development. Additionally, GSTM1-null/ GSTT1-null/ GSTA1 low-activity/ GSTP1-variant genotype could be combination of GST genotypes whose carriers are more prone to CRC development.

Polymorphism RAD51 172G > T in Serbian patients with colorectal cancer.

PURPOSE: The RAD51 gene plays an important role in homologous strand exchange in DNA repair. Two common single nucleotide polymorphisms in this gene, 135G>C and 172G>T, were associated with altered gene transcription. While 135G>C was already linked to breast and colorectal cancers in certain populations, 172G>T is far less investigated, although sporadic studies showed it could be a prognostic factor for some cancer lesions. The purpose of this study was to investigate RAD51 172G>T polymorphism in Serbian population, its association with colorectal carcinoma, as well as correlation with disease characteristics and response to neoadjuvant chemoradiotherapy. METHODS: The 172G>T polymorphism was evaluated by PCR-RFLP method in blood samples of 209 colorectal cancer patients and 43 healthy subjects who served as controls. The distribution of genotypes was also analyzed in respect to several tumor characteristics in cases where histopathological data were available. RESULTS: A significant association between the RAD51 172G>T polymoprhism and desmoplastic reaction of colorectal cancer was demonstrated. The 172G allele was found to be significantly more frequent in patients with more intensive desmoplastic response of the tumor tissue. CONCLUSIONS: The results of our study suggest that the 172T allele of RAD51 may be a favorable prognostic factor in Serbian patients with colorectal cancer, although larger prospective studies are required to confirm this finding.

Single Atomic Layer Ferroelectric on Silicon.

A single atomic layer of ZrO2 exhibits ferroelectric switching behavior when grown with an atomically abrupt interface on silicon. Hysteresis in capacitance-voltage measurements of a ZrO2 gate stack demonstrate that a reversible polarization of the ZrO2 interface structure couples to the carriers in the silicon. First-principles computations confirm the existence of multiple stable polarization states and the energy shift in the semiconductor electron states that result from switching between these states. This monolayer ferroelectric represents a new class of materials for achieving devices that transcend conventional complementary metal oxide semiconductor (CMOS) technology. Significantly, a single atomic layer ferroelectric allows for more aggressively scaled devices than bulk ferroelectrics, which currently need to be thicker than 5-10 nm to exhibit significant hysteretic behavior (Park, et al. Adv. Mater. 2015, 27, 1811).

Nitric oxide synthesis modulation - a possible diagnostic and therapeutic target in colorectal cancer.

PURPOSE: Considering the contradictory literature data about the role of nitric oxide (NO) in colon carcinogenesis, the purpose of this study was to examine the changes of L-arginine metabolites in colon cancer and surrounding tissue as possible molecular markers of tumor behavior after surgery and the possibility of NO synthesis modulation in new individualized therapeutic strategies. METHODS: The study encompassed 50 patients who underwent surgery for colorectal cancer (CRC). The three tissue specimens were taken by surgery (tumor, adjacent and healthy tissue) and the concentrations of NO2+NO3, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were determined in the tissue specimens. RESULTS: The results proved higher NO2+NO3 concentrations in adjacent tissue compared to the tumor, implicating high angiogenic potential of the tumor-surrounding tissue, which could have clinical importance in the assessment of the probability of tumor local recurrence and metastasis. Increased ADMA concentrations in tumor tissue associated with low NO levels, could lead to new therapeutic strategies directed to the use of inhibitors of NO synthesis as ideal candidates for molecular therapy of CRC. ADMA concentration in adjacent tissue was an independent predictor of distant metastasis. CONCLUSIONS: The obtained results suggest that determination of the examined biomarkers in CRC and adjacent tissue samples could give useful information about tumor proliferative and angiogenic potential, which in turn could enable individualization of therapy and the choice of proper adjuvant therapy in patients with CRC.

Cell-free DNA as biomarker and source for mutation detection in primary colorectal cancer.

PURPOSE: To analyze if cell-free (cf)DNA levels and the presence of KRAS and BRAF mutations in serum could be used as diagnostic biomarkers in patients with primary colorectal cancer (CRC). METHODS: This study included 92 individuals who were operated due to primary CRC (N=52;study group) and to hemorrhoids (N=40;control group). Serum cfDNA levels were measured with real-time PCR (RT-PCR) using PicoGreen dsDNA quantitation reagent. Colorectal tissue and related blood and serum samples taken at the time of surgery were subjected to DNA extraction and analysis of KRAS and BRAF mutations based on multiplex SNaPshot assay and DNA sequencing. RESULTS: The average cfDNA concentration was lower in patients of the study group (20±7 ng/µL) in comparison to controls (34±9 ng/µL) and this difference was statistically significant (p<0.001). The SNaPshot analysis detected KRAS c35 mutations in colorectal tumor tissue in 14 cases, but the presence of the mutation was not confirmed in cfDNA extracted from blood samples of these patients. CONCLUSIONS: The level of serum cfDNA in CRC is decreased in comparison to patients with hemorrhoids, which questions the usefulness of cfDNA as cancer biomarker. Also, cfDNA does not appear to be suitable as a source for mutation detection in this disease.