Monitoring of Circulating Tumor Cells by a Combination of Immunomagnetic Enrichment and RT-PCR in Colorectal Cancer Patients Undergoing Surgery.

BACKGROUND: The presence of circulating tumor cells (CTC) has been reported in patients with advanced colorectal cancer. Monitoring CTC (also known as a liquid-biopsy) has recently become the center of interest for low-invasive monitoring of cancer progression and predictive biomarkers testing. Along with high-cost technology and a complex methodology, a straightforward method based on magnetic beads enrichment followed by RT-PCR is set to allow for routine CTC analysis in colorectal cancer patients. OBJECTIVES: The main purpose of this study was to evaluate the possibility of CTC detection in routine monitoring of patients starting before and continuing after surgery. MATERIAL AND METHODS: The investigated group consisted of 30 patients mainly in advanced stages of colorectal cancer. In all patients, CTC detection was performed prior to surgery, in a subset of 14 patients additional sampling was done during and after surgery. In all cases, peripheral blood was processed using AdnaTest ColonCancer kit, which relies on enriching CTCs using EpCAM-functionalized magnetic beads and subsequently identifying tumorspecific CEA, EGFR and GA733-2 mRNA transcripts. RESULTS: Out of all the tested samples, CTC were found in one patient suffering from advanced disease with lung and liver metastases. There, however, the positive finding was confirmed in 3 consecutive samples acquired before, during and shortly after palliative R2 resection. CONCLUSIONS: The presence of CTC may be used to observe post-operative disease development. Due to the overall low CTC detection, further technology development may be necessary before its universal applicability to manage colorectal cancer patients.

HOTAIR long non-coding RNA is a negative prognostic factor not only in primary tumors, but also in the blood of colorectal cancer patients.

Colorectal cancer (CRC) is one of the main causes of death of neoplasia. Demand for predictive and prognostic markers to reverse this trend is increasing. Long non-coding RNA HOTAIR (Homeobox Transcript Antisense Intergenic RNA) overexpression in tumors was previously associated with poor prognosis and higher mortality in different carcinomas. We analyzed HOTAIR expression levels in tumor and blood of incident sporadic CRC patients in relation to their overall survival with the aim to evaluate surrogate prognostic marker for CRC. Tissue donor group consisted of 73 CRC patients sampled for tumor and normal tissue. Blood donor group was represented by 84 CRC patients compared with 40 healthy controls. Patients were characterized for tumor-node-metastasis stage, tumor grade, microsatellite instability and tumor penetration by stromal cells. HOTAIR levels were assessed by real-time quantitative PCR. CRC patients had higher HOTAIR expression in blood than healthy controls (P = 0.0001), whereas there was no difference in HOTAIR levels between tumor and adjacent mucosa of CRC patients. HOTAIR levels positively correlated between blood and tumor (R = 0.43, P = 0.03). High HOTAIR levels in tumors were associated with higher mortality of patients [Cox's proportional hazard, hazard ratio = 4.4, 95% confidence interval: 1.0-19.2, P = 0.046]. The hazard ratio was even higher when blood HOTAIR levels were taken into account (hazard ratio = 5.9, 95% confidence interval: 1.3-26.1, P = 0.019). Upregulated HOTAIR relative expression in primary tumors and in blood of CRC patients is associated with unfavorable prognosis. Our data suggest that HOTAIR blood levels may serve as potential surrogate prognostic marker in sporadic CRC.

Functional, genetic, and epigenetic aspects of base and nucleotide excision repair in colorectal carcinomas.

PURPOSE: DNA repair capacity (DRC) is a determinant not only of cancer development but also of individual response to therapy. Previously, altered base and nucleotide excision repair (BER and NER) have been described in lymphocytes of patients with sporadic colorectal cancer. We, for the first time, evaluate both excision repair capacities in human colon biopsies to study their participation in colorectal tumorigenesis. EXPERIMENTAL DESIGN: Seventy pairs of tumor and adjacent healthy tissues were analyzed for BER- and NER-specific DRC by a comet repair assay. Tissue pairs were further compared for expression levels of a panel of 25 BER and NER genes complemented by their promoter methylation status. RESULTS: We observed a moderate increase of NER-DRC (P = 0.019), but not of BER-DRC in tumors. There was a strong correlation between both tissues for all investigated parameters (P < 0.001). However, 4 NER (CSB, CCNH, XPA, XPD) and 4 BER (NEIL1, APEX1, OGG1, PARP1) genes showed a 1.08- to 1.28-fold change difference in expression in tumors (P < 0.05). Individual gene expression levels did not correlate with overall DRC, and we did not detect any aberrant methylation of the investigated genes. CONCLUSIONS: Our complex analysis showed that tumor cells are not deficient in BER and NER, but rather follow patterns characteristic for each individual and are comparable with adjacent tissue. Alteration of excision repair pathways is not a pronounced event in colorectal carcinogenesis. This study shows the feasibility of DRC evaluation in human solid tissues, representing a complex marker of multigene DNA repair processes.

Utility of cell-free tumour DNA for post-surgical follow-up of colorectal cancer patients.

BACKGROUND: While efficient surgical treatment is the key to prolonged survival of patients with colorectal cancer, post-surgical follow-up is important for the early detection of relapsing disease or of disease progression. Current dispensarization, typically based on imaging CT, PET, MR, is frequently supported by the observation of tumour markers (CEA, CA19-9). Due to their limited sensitivity and selectivity, better tools for monitoring of the disease are desirable. Tumour cell-free DNA (cfDNA) has been recently demonstrated as a new promising molecular marker for observation and early detection of disease progression. PATIENTS AND METHODS: We present results of post-surgical monitoring tumour cfDNA in the cases of seven patients suffering from advanced forms of CRC. We applied a mutation-based approach in which the total cfDNA was screened for a specific somatic mutation present in the primary tumour. We screened a panel of the most frequent somatic mutations covering the genes APC, KRAS, TP53, PIK3CA and BRAF. All patients were tested positive for tumour cfDNA prior to surgery. cfDNA was then evaluated within a week after surgery and subsequently in monthly intervals. RESULTS: We present typical cases of colorectal cancer patients who underwent surgical treatment at different levels of radicality with or without adjuvant chemo/biotherapy. The tumour cfDNA status was found to be always closely correlated with the actual clinical status of the patient. CONCLUSION: The cfDNA appears to be a viable tool for the monitoring of the clinical progression of CRC in patients with cfDNA positivity prior to surgery.

Polymorphisms in miRNA-binding sites of nucleotide excision repair genes and colorectal cancer risk.

Reduced DNA repair capacity and DNA damage accumulation may lead to cancer development. Regulation of and coordination between genes involved in DNA repair pathways is fundamental for maintaining genome stability, and post-transcriptional gene regulation by microRNAs (miRNAs) may therefore be of particular relevance. In this context, the presence of single nucleotide polymorphisms (SNPs) within the 3'untranslated regions of target DNA repair genes could alter the binding with specific miRNAs, modulating gene expression and ultimately affecting cancer susceptibility. In this study, we investigated the role of genetic variations in miRNA-binding sites of nucleotide excision repair (NER) genes in association with colorectal cancer (CRC) risk. From 28 NER genes, we screened among SNPs residing in their 3'untranslated regions and simultaneously located in miRNA-binding sites, with an in silico approach. Through the calculation of different binding free energy according to both alleles of identified SNPs, and with global binding free energies median providing a threshold, we selected nine NER gene variants. We tested those SNPs in 1098 colorectal cancer cases and 1469 healthy controls from the Czech Republic. Rs7356 in RPA2 and rs4596 in GTF2H1 were associated with colorectal cancer risk. After stratification for tumor location, the association of both SNPs was significant only for rectal cancer (rs7356: OR 1.52, 95% CI 1.02-2.26, P = 0.04 and rs4596: OR 0.69, 95% CI 0.50-0.94, P = 0.02; results not adjusted for multiple testing). Variation in miRNA target binding sites in the 3'untranslated region of NER genes may be important for modulating colorectal cancer risk, with a different relevance according to tumor location.

Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls.

Alteration of DNA integrity is a potential cause of cancer and it is assumed that reduced DNA repair capacity and accumulation of DNA damage may represent intermediate markers in carcinogenesis. In this case-control study, DNA damage and nucleotide excision repair capacity (NER-DRC) were assessed in association with sporadic colorectal cancer (CRC). Both parameters were quantified by comet assay in blood cells of 70 untreated incident patients and 70 age-matched healthy controls. mRNA expression and polymorphisms in relevant NER genes were concurrently analyzed. The aim of this study was to characterize incident CRC patients for NER-DRC and to clarify possible relations between investigated variables. Comet assay and mRNA expression analysis showed that CRC patients differ in repair capacity as compared to controls. Patients had a lower NER-DRC and simultaneously they exhibited higher endogenous DNA damage (for both P < 0.001). Accumulation of DNA damage and decreasing NER-DRC behaved as independent modulating parameters strongly associated with CRC. Expression levels of 6 out of 9 studied genes differed between groups (P ≤ 0.001), but none of them was related to DRC or to any of the studied NER polymorphisms. However, in patients only, XPC Ala499Val modulated expression levels of XPC, XPB and XPD gene, whereas XPC Lys939Gln was associated with XPA expression level in controls (for all P < 0.05). This study provides evidence on altered DRC and DNA damage levels in sporadic CRC and proposes the relevance of the NER pathway in this malignancy. Further, alterations in a complex multigene process like DNA repair may be better characterized by functional quantification of repair capacity than by quantification of individual genes transcripts or gene variants alone.

DNA damage and nucleotide excision repair capacity in healthy individuals.

Interindividual differences in DNA repair capacity (DRC) represent an important source of variability in genome integrity and thus influence health risk. In the last decade, DRC measurement has attracted attention as a potential biomarker in cancer prediction. Aim of the present exploratory study was to characterize the variability in DNA damage and DRC on 100 healthy individuals and to identify biological, lifestyle, or genetic factors modulating these parameters. The ultimate goal was to obtain reference data from cancer-free population, which may constitute background for further investigations on cancer patients. The endogenous DNA damage was measured as a level of DNA single-strand breaks and DRC, specific for nucleotide excision repair (NER), was evaluated using modified comet assay, following the challenge of peripheral blood mononuclear cells with benzo[a]pyrene diolepoxide. Additionally, genetic polymorphisms in NER genes (XPA, XPC, XPD, and XPG) were assessed. We have observed a substantial interindividual variability for both examined parameters. DNA damage was significantly affected by gender and alcohol consumption (P = 0.003 and P = 0.012, respectively), whereas DRC was associated with family history of cancer (P = 0.012). The stratification according to common variants in NER genes showed that DNA damage was significantly modulated by the presence of the variant T allele of XPC Ala499Val polymorphism (P = 0.01), while DRC was modulated by the presence of the A allele of XPA G23A polymorphism (P = 0.048). Our results indicate the range of endogenous DNA single-strand breaks and capacity of NER in healthy volunteers as well as the role of potentially relevant confounders. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.

Celiac axis stenosis and lethal liver ischemia after pancreaticoduodenectomy.

Celiac axis stenosis can lead to a fatal hepatic ischemia after pancreaticoduodenectomy unless a simultaneous revascularisation of the celiac circulation is performed. In the present study are reported three cases of celiac axis stenosis, all of which had histologically confirmed periampullary cancer. Case 1: a 50-year-old male with a history of myocardial infarction and liver steatosis; visceral arteriography prior to the surgery demonstrated a celiac axis stenosis. Whipple operation was performed. After removing the specimen, no signs of liver ischemia were found (liver was cholestatic) and pulsation of the hepatic artery was strong. The patient died on the second postoperative day after an abrupt irreversible cardiac arrest. Autopsy proved acute severe hepatic ischemia. Case 2: a 64-year-old female. Preoperative visceral angiography showed significant celiac axis stenosis. As a first step of surgery the root of the celiac trunk was exposed, a fibrotic ring around it was divided. Standard D1 pylorus preserving pancreaticoduodenectomy was performed. Case 3: a 58-year-old female without preoperative angiography, indicated for surgery. After an occlusion test of the gastroduodenal artery the liver became ischemic. Division of the fibrotic ring around celiac axis was performed together with a standard D1 pylorus preserving pancreaticoduodenectomy. No postoperative complications were reported in both case 2 and 3.

Tumor markers in patients with relapse of colorectal carcinoma.

AIM: To evaluate CEA and CA19-9 in a long-term follow-up after radical surgery for colorectal cancer. PATIENTS AND METHODS: A total of 1,090 patients were operated on for colorectal cancer, 716 patients underwent R0 resection, 631 patients were under further surveillance, relapse was diagnosed in 122 patients (20%), 74 patients were indicated for reoperation The resectability of the relapse was 35%. An AxSYM instrument (Abbott) was used for analysis. RESULTS: At the time of relapse both markers were normal in 31% of the patients. When relapse was diagnosed, in patients with normal preoperative levels, CEA and CA19-9 were below cut-off in 48% and 79%, respectively, and in those with primary elevation, they were again elevated in 78% and 64%, respectively. CONCLUSION: The surveillance based only on CEA and/or CA19-9 was cost-effective, but failed to disclose 1/3 of patients suffering from relapse; these markers must be combined with liver and chest imaging methods and colonoscopy.

Multiorgan resections for advanced colorectal cancer.

BACKGROUND: A retrospective review is presented of a single institution's experience with multivisceral resections for locally-advanced colorectal cancer. MATERIALS AND METHODS: Twenty-eight patients, who had undergone RO multiorgan resection, were identified from the database of a total of 1150 patients operated on for colorectal carcinoma in the years 1995-2005 at a single center. There were twelve total pelvic exenterations and 16 patients had undergone en bloc primary tumor resection with adherent organs, such as the spleen, diaphragm, pancreas, stomach, kidney, etc. The patients were followed-up according to a standard protocol. RESULTS: The post-operative mortality was 7%, the average follow-up 21.6 months and the 5-year survival 45%. CONCLUSION: Our results confirmed that, in the case of invasion of colorectal cancer to the adjacent intra-abdominal organs or structures, multiorgan resection 'offers the only chance of potentially-curative treatment.

The role of FDG-PET/CT in the detection of recurrent colorectal cancer.

PURPOSE: The conventional diagnostic techniques used to assess recurrence of colorectal cancer (CRCR) often yield unspecific findings. Integrated FDG-PET/CT seems to offer promise for the differential diagnosis of benign and malignant lesions. The aim of this study was to compare the value of FDG-PET and PET/CT in the detection of CRCR subsequent to colonic resection or rectal amputation. METHODS: The population for this retrospective study comprised 84 patients with suspected CRCR. The sensitivity, specificity and accuracy of PET and PET/CT were calculated for (a) intra-abdominal extrahepatic recurrences, (b) extra-abdominal and/or hepatic recurrences and (c) all recurrences, and tumour marker levels were analysed. RESULTS: The sensitivity, specificity and overall accuracy of PET in detecting intra-abdominal extrahepatic CRCR were 82%, 88% and 86%, respectively, compared with 88%, 94% and 92%, respectively, for PET/CT. The corresponding figures for detection of extra-abdominal and/or hepatic CRCR were 74%, 88% and 85% for PET and 95%, 100% and 99% for PET/CT. Considering the entire population, the sensitivity, specificity and overall accuracy of PET were 80%, 69% and 75%, respectively, compared with 89%, 92% and 90%, respectively, for PET/CT. FDG-PET/CT examination correctly detected 40 out of a total of 45 patients with CRCR. Two of five patients with falsely negative FDG-PET/CT findings had local microscopic recurrences and one had miliary liver metastases. Of 39 patients without CRCR, three showed false positive FDG-PET/CT results. Two of these cases were due to increased accumulation in inflammatory foci in the bowel wall, while one was due to haemorrhaging into the adrenal gland. CONCLUSION: FDG-PET/CT appears to be a very promising method for distinguishing a viable tumour from fibrous changes, thereby avoiding unnecessary laparotomy.

Mesohepatectomy for colorectal liver metastasis. Case report.

A 54-year-old woman was operated for obstructive ileus in 1996. Obstruction was caused by a tumor of the descending colon invading the abdominal wall. Urgent left colectomy with lymphadenectomy was performed. Microscopically six lymphatic nodes were positive. The patient was postoperatively treated with adjuvant chemotherapy. Fifteen months later the patient underwent a resection of central hepatic segments (Couinaud's segment 4,5,8) for metachronous metastasis. At present the patient has no signs of recurrence, she has returned back to her normal life. Despite several unfavorable prognostic factors--obstruction, abdominal wall infiltration, number of positive nodes, short time between primary tumor resection and diagnosis of liver metastasis and centrally located metastasis with satellite lesions, the patient has been surviving for 6 years now.

Routine (18)F-FDG PET preoperative staging of colorectal cancer: comparison with conventional staging and its impact on treatment decision making.

UNLABELLED: The preoperative staging of colorectal cancer (CRC) with (18)F-FDG PET is not as yet generally considered to be evidence based. We have found only 1 study that evaluated (18)F-FDG PET in a nonselected population with proven CRC. Several other studies have concentrated on more advanced disease. The aim of this study was to assess the potential clinical benefit of (18)F-FDG PET in the routine staging of CRC. METHODS: Thirty-eight consecutive patients who had had CRC histologically proven by colonoscopy underwent prospective preoperative staging by plain chest radiography, sonography, CT, and (18)F-FDG PET. Sensitivity, specificity, and accuracy were retrospectively assessed by comparison with the histologic results after surgery (36 patients) or clinical follow-up (2 inoperable cases-both patients died within 1 y of the PET examination). The impact of (18)F-FDG PET on therapeutic decision making was evaluated by comparing medical records before and after (18)F-FDG PET. RESULTS: (18)F-FDG PET correctly detected 95% of primary tumors, whereas CT and sonography correctly detected only 49% and 14%, respectively. Lymph nodes were involved in 7 patients. The sensitivity, specificity, and accuracy of (18)F-FDG PET were 29%, 88%, and 75%, respectively. CT and sonography did not reveal any lymph node involvement. Liver metastases were present in 9 patients. (18)F-FDG PET, CT, and sonography had a sensitivity of 78%, 67%, and 25%, respectively; a specificity of 96%, 100%, and 100%, respectively; and an accuracy of 91%, 91%, and 81%, respectively. (18)F-FDG PET revealed further lesions in 11 patients. Levels of carcinoembryonic antigen and carbohydrate antigen 19-9 tumor markers were elevated in, respectively, only 33% and 8% of cases of proven CRC. (18)F-FDG PET changed the treatment modality for 8% and the range of surgery for 13% of patients. In total, (18)F-FDG PET changed the method of treatment for 16% of patients. CONCLUSION: Plain chest radiography and sonography did not bring any clinical benefits. No correlation was found between the level of tumor markers and the stage of disease. CT is necessary for confirmation of PET findings at extraabdominal sites (PET-guided CT) and for their morphologic specification at abdominal and pelvic sites before an operation. (18)F-FDG PET is the best method for the staging of CRC in all localities, despite the high rate of false-negative PET findings in patients with lymph node involvement. PET should be performed as a first examination after verification of CRC. We propose a PET/CT hybrid system as optimal in the staging of CRC.