Ludmila Lipska

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. Clin Cancer Res; 18(21); 5878–87. ©2012 AACR.

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.

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.

Circulating Free Tumor DNA in Patient Plasma is a Near-Perfect Marker for Metastatic Spread of Colorectal Cancer: A Study on 165 Patients Undergoing Surgical Treatment

Background: The occurence of circulating epithelial cells and cell-free tumor DNA (cfDNA) in peripheral blood of patients suffering from progressive forms of cancers has first been reported already in 1960s. With the great acceleration of genetic diagnostics by methods of polymerase chain reaction (PCR) the field of non-invasive examination of circulating molecular cancer biomarkers has recently been re-discovered. Aims: To investigate occurence of cfDNA as potential marker of regional and distant metastatic progression of colorectal cancer and to trace the source of cfDNA in patients undergoing surgery treatment at different levels of radicality. Methods: In a group of 165 patients in various stages of the disease tissue samples were initially acquired either as biopsies or resections. Samples were tested for a presence of the most frequent somatic colorectal cancer mutations within KRAS, APC, TP53, BRAF and PIK3CA genes. In addition, multiple plasma samples (n=789) were acquired over a time period covering (i) initial examination, (ii) immediately preceding a surgery (iv) postsurgery and (v) subsequent follow-up. Cell-free tumor DNA was traced in patient plasma by targeting mutations previously detected in tumor tissue. Results: Among patients who were positive for at least one of the detected somatic mutations in tumor tissue (102/165, 62%), cfDNA was present in 39 pre-surgery plasma samples. The frequency of cfDNA was correlated to the disease stage with 0% in Stages 0 and I, 9% in Stage II, 29% in Stage III and 94% in Stage IV. All cfDNA-positive patients (n=21) who underwent radical resection (R0) were free of cfDNA in subsequent testing of samples several days following surgery. All but 4 patients undergoing non-radical surgery, such as partial hepatic resections or paliative surgical treatment, remained cfDNA-positive also after the surgery. In some of the R0 patients, the cfDNA has re-appeared after a period of 18 22 months as a result of disease progression though local nodes or newly discovered metastases with continuous monitoring of the remainder of the group still in progress. Conclusion: Examination of a presence of cfDNA based on scanning plasma samples for presence of specific tumor mutations is a suitable tool for non-invasive monitoring of the disease progression as well as evaluation of surgery outcome. Supported by Czech Ministry of Health grant no. NS9809.

Impact of Postoperative Septic Complications on Recurrence of Colorectal Cancer

Background: About one third of patients racically resected for colorectal cancer develop during follow-up recurrence. Materials and methods: There were 1951 patients operated for colorectal cancer in Surgical Department, Thomayer Hospital Prague, from 1997 to 2015. Radical R0 operation underwent 68% of these patients. Postoperative complications occurred in 457 (34.6%) patients. Impact of postoperative complications on disease free interval was studied in a prospective study. Results: We identified minor complications in 90 patients (6.8%), moderate complications in 28 patients (2.1%), anastomotic leakage in 67 patients (5%) and severe septic complications in 20 patients (1.5%). Another 255 patients (19.3%) had a different, non-inflammatory complications (pulmonary embolism, bowel obstruction, heart failure, etc.). Significantly worse disease-free interval was found in patients with severe septic complications. Conclusion: In our cohort of R0 operated patients, postoperative complication is the second most important prognostic factor following TNM stage of the colorectal cancer. Severe septic complications has an adverse effect on the further course of the disease in terms of relapse. Other potentially septic complications such as anastomotic leakage have no essential impact on recurrence. Therefore, it is necessary to prevent the development of sepsis.

Durable complete response of colorectal cancer metastasis after biochemotherapy

BACKGROUND Resection of the metastatic site is indicated but not always possible in patients with metastatic colorectal cancer (mCRC) who achieve a partial or complete response (CR) to induction systemic treatment. CR after systemic treatment alone is uncommon, and even patients with radiologic CR after induction chemotherapy harbour persistent macroscopic or microscopic residual disease in more than 80% of cases. Occasionally, some metastatic lesions disappear radiologically but others persist after induction systemic treatment. The indication and extent of metastasectomy in these situations is controversial, especially regarding sites with completely regressed metastases. CASE This case report describes a patient with mCRC who achieved a long-term response after biochemotherapy and incomplete metastasectomy. One of the known liver lesions could not be removed due to its disappearance after induction biochemotherapy with FOLFOX and bevacizumab. Further adjuvant chemotherapy using the FOLFOX regimen was administered postoperatively. The patient has been meticulously followed by radiology including repeated positron emission tomography/computed tomography and magnetic resonance scans, clinical examination and tumour markers. No recurrence of cancer has been detected after a follow-up of 5 years. RESULTS AND CONCLUSION CR to systemic treatment is uncommon, but this case report demonstrates that it can be durable in patients with colorectal cancer and liver metastases. This case report indicates that some patients with mCRC can be cured with systemic therapy only, challenging the prevailing paradigm of mCRC therapy.Key words: colorectal cancer - metastasis - chemotherapy - molecular targeted therapy - diagnostic imaging.

Abstract 2406: Validation of a simple low-cost method to monitor ctDNA in patients with solid cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Detection of circulation tumor DNA (ctDNA) in plasma has become a viable option for non-invasive monitoring of patients. Also termed “liquid biopsy” the approach is applicable for pre-diction of response and prediction of resistance to biological therapy (1, 2). Various techniques have been used for ctDNA detection, frequently employing clonal amplification on a digital PCR format (3) with limits of detection (LOD) below 0.01% of mutant alleles. However, these techniques suffer from high complexity, expensive instrumentation, and a considerable cost per sample. We hereby present a simple low-cost alternative that is implementable to routine ctDNA testing. Methods: A panel of PCR amplicons (106 - 174bp) was resolved by denaturing capillary electrophoresis (DCE) revealing minute presence of mutation specific hetero-duplexes. The final panel consisted of clinically relevant oncogenic mutations KRAS, NRAS, BRAF, PIK3CA and EGFR as well as cancer-related mutations in tumor suppressors TP53, APC and CTNNB1. A total of 299 patients was subsequently examined for presence of ctDNA in plasma including 194 with colorectal cancer (CRC), 26 with NSCLC and 79 with pancreatic cancer (PanC). CtDNA status was correlated to TNM stage and tumor markers (CEA and Ca19-9). In a subset of CRC patients (n = 20) the ctDNA was monitored in 2 - 6 month intervals and correlated to the therapy response. Results: The experimental LOD value was in the range between 0.03 - 1% for all tested mutations within the panel. A minimum input amount of DNA was 5 pg (0,005 ng).. The overall rate of ctDNA detection was 32% for CRC (stages I - IV), 31% for NSCLC (stages III - IV) and 27% for PanC (stages II - IV). The highest detection rate, 69%, was observed in Stage IV CRC patients. Comparison with tumor markers (TM) revealed 62% of cases positive for both TM and ctDNA and 13% TM-negative cases with ctDNA positivity. Post-operative absence or persistence of ctDNA was related to the radicality of the surgical treatment and the ctDNA levels were concordant with the response to adjuvant chemotherapy. In several patients a disease progression was signalized based on ctDNA even prior to actual clinical detection by CT imaging. Conclusion: DCE is a simple technique applicable for detection of ctDNA in cancer patients without a need for costly hardware/software equipment. The detection rates are 10 - 15% lower compared to the dedicated dPCR techniques, however, the method requires ca 100x less input DNA, the cost per patient is about 10-fold lower and the turnaround time per test is under 5 hours. Supported by the Czech Ministry of Health Grant 14383. Literature 1. Bettegowda C et al. Sci Transl Med. 2014,6(224):224ra24 2. Douillard JY et al. J Thorac Oncol. 2014, 9(9):1345-1353. 3. Benesova L et al. Anal Biochem 2013,433(2):227-234. Citation Format: Lucie Benesova, Barbora Belsanova, Petra Minarikova, Tereza Halkova, Jiri Pudil, Filip Pazdirek, Milos Pesek, Ondrej Fiala, Jiri Hoch, Miroslav Zavoral, Bohus Bunganic, Miroslav Levy, Ludmila Lipska, Lubos Petruzelka, Miroslav Ryska, Marek Minarik. Validation of a simple low-cost method to monitor ctDNA in patients with solid cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2015-2406

Su1893 Circulating Free Tumor DNA as a Promising Marker for the Prediction of Survival and Monitoring of Remission in Colorectal Cancer After Radical Surgery

PCR assay. Septin9 protein expression in biopsy tissuewas evaluated by immunhistochemistry using a polyclonal antibody for Septin9. Results: mSEPT9 PMR (percent methylation reference) values larger than 1% were detected in 7.7% (1/13) NED, 100% (10/10) adenoma and 100% (16/16) CRC biopsy tissues. In plasma from the same patients, however, mSEPT9 PMR values larger than 0.01% were detected in 10% (2/20) NED, 25% (5/20) adenoma and 75% (15/20) CRC. Immunhistochemical detection of Septin9 protein decreased with increasing disease pathology, with Septin9 expression detected in 100% NED, 50-60% adenoma and 10-20% CRC tissues. Conclusions: mSEPT9 was confirmed as a highly sensitive biomarker for the detection of CRC in blood. While the high level of mSEPT9 in CRC tissue correlated strongly with sensitive detection of these cancers in plasma, the strong correlation was not maintained in tissue and plasma from adenoma patients. Varying degrees of tissue vascularization might underlie this observation, among other explanations. Finally, mSEPT9 in biopsy tissue was inversely correlated with expression of the Septin9 protein.