Lubos Holubec

Relevance of miR-21 and miR-143 expression in tissue samples of colorectal carcinoma and its liver metastases

MicroRNAs, which are endogenously expressed regulatory noncoding RNAs, have an altered expression in colorectal cancer. The aim of our study was to assess the relationship of miR-21 and miR-143 expression to the prognostic/clinicopathological features of colorectal carcinoma (CRC) and colorectal liver metastases (CLM). The estimation was performed in 46 paired (tumor and control) tissue samples of CRC. Further, we studied 30 tissue samples of CLM. MiR-21 and miR-143 expressions were quantified by using the quantitative reverse transcription polymerase chain reaction method. Relation of miR-21 and miR-143 expression to disease-free interval (DFI) (Wilcoxon; P = 0.0026 and P = 0.0191, respectively) was recorded. There was shorter DFI in patients with a higher expression of miR-21 and, surprisingly, also in patients with a higher expression of miR-143, which is a putative tumor suppressor. There was a higher expression of miR-21 and lower expression of miR-143 in CRC tissue in comparison with adjacent normal colon tissue (P < 0.0001; P < 0.0001, respectively). Similarly, we observed a higher expression of miR-21 and a lower expression of miR-143 in CLM in comparison with normal colon tissue (P < 0.0001; P < 0.0001, respectively). Our results support the hypothesis about oncogenic function of miR-21 and show its relation to DFI. The role of miR-143 in carcinogenesis seems to be more complex.

The role of ABC transporters in progression and clinical outcome of colorectal cancer

Worldwide, colorectal cancer (CRC) is the third most common cancer, with the highest mortality rates occurring in Central Europe. The use of chemotherapy to treat CRC is limited by the inter-individual variability in drug response and the development of cancer cell resistance. ATP-binding cassette (ABC) transporters play a crucial role in the development of resistance by the efflux of anticancer agents outside of cancer cells. The aim of this study was to explore transcript levels of all human ABCs in tumours and non-neoplastic control tissues from CRC patients collected before the first line of treatment by 5-fluorouracil (5-FU)-containing regimen. The prognostic potential of ABCs was evaluated by the correlation of transcript levels with clinical factors. Relations between transcript levels of ABCs in tumours and chemotherapy efficacy were also addressed. The transcript profile of all known human ABCs was assessed using real-time polymerase chain reaction with a relative standard curve. The majority of the studied ABCs were down-regulated or unchanged between tumours and control tissues. ABCA12, ABCA13, ABCB6, ABCC1, ABCC2 and ABCE1 were up-regulated in tumours versus control tissues. Transcript levels of ABCA12, ABCC7 and ABCC8 increased in direction from colon to rectum. Additionally, transcript levels of ABCB9, ABCB11, ABCG5 and ABCG8 followed the reverse significant trend, i.e. a decrease in direction from colon to rectum. The transcript level of ABCC10 in tumours correlated with the grade (P = 0.01). Transcript levels of ABCC6, ABCC11, ABCF1 and ABCF2 were significantly lower in non-responders to palliative chemotherapy in comparison with responders. The disease-free interval of patients treated by adjuvant chemotherapy was significantly shorter in patients with low transcript levels of ABCA7, ABCA13, ABCB4, ABCC11 and ABCD4. In conclusion, ABCC11 may be a promising candidate marker for a validation study on 5-FU therapy outcome.

The role of thymidine kinase in cancer diseases

Thymidine kinase 1 (TK 1-fetal) is a cell cycle-dependent marker that increases dramatically during the S-phase of the cell cycle. In this review, the authors discuss serum levels of thymidine kinase in a variety of neoplasias. Determination of thymidine kinase helps to monitor the follow-up of solid tumours and haematological malignancies as well as indicating the efficacy of adjuvant and palliative chemotherapy. Elevated levels of thymidine kinase must always be interpreted together with a detailed knowledge of the patients condition because nonspecific elevations of serum levels (inflammatory and autoimmune diseases) must be excluded.

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.

Clinical Relevance of the Expression of mRNA of MMP-7, MMP-9, TIMP-1, TIMP-2 and CEA Tissue Samples from Colorectal Liver Metastases

Background: Nowadays we know that survival rates do not differ between repeated and single liver resections for colorectal liver metastases (CLM). To be able to determine patients prone to early recurrence, the use of different markers with a better prognostic value than the routinely employed tumor markers is required. Aim of Study: The aim of our study was to assess mRNA expression of MMP-7, MMP-9, TIMP-1, TIMP-2 and CEA in tissue samples from CLM and their relationship to disease-free interval (DFI) and overall survival (OS). Patients and Methods: The liver tumor biopsies were obtained from 40 patients suffering from CLM treated with radical surgery. mRNA expression levels of CEA, MMPs and TIMPs and a housekeeping gene (GAPDH) were quantified using RT-PCR. Results: The increased expression of CEA, MMP-9 and TIMP-1 in CLM was associated with a short DFI and a high tendency to early CLM recurrence. Statistical analysis confirmed CEA, MMP-9 and TIMP-1 expression as prognostic factors of survival. Conclusion: This study demonstrated the importance of CEA, MMP-9 and TIMP-1 in the prognostication of DFI and OS.

Fibrate treatment and prevalence risk of mild hyperhomocysteinaemia in clinical coronary heart disease patients

Background Several prospective studies reported that fibrates might increase blood total homocysteine (tHcy). In this study we aimed to establish whether the reported fibrate treatment was associated with an increased risk of mild hyperhomocysteinaemia in patients with clinical coronary heart disease, and to establish whether confounding variables may influence this effect. Design A retrospective, case-control analysis. Methods A total of 410 patients, 301 males and 109 females, mean age 59.2 were examined in a Czech sample from the EUROASPIRE II survey. In addition to examinations and measurements, defined by the protocol, we estimated serum total homocysteine (tHcy), folate, B12 vitamin and methylenetetrahydrofolate reductase (MTHFR) genotypes. Results We found significantly higher tHcy concentrations in patients with reported treatment with fibrate (16.6 ± 0.66 μmol/l) compared with no lipid-lowering treatment (13.5 ± 0.64 μmol/l, P< 0.001) or to statin (12.4 ± 0.39 μmol/l, P< 0.001). Concentrations of tHcy ≤15mmol/l (i.e. mild hyperhomocysteinaemia) as a dependent variable were positively associated with age (OR 1.18, P< 0.0003), serum vitamin B12 (OR 0.87, P< 0.003), serum creatinine (OR 1.35, P< 0.0001 and treatment with fibrates (OR 1.30, P< 0.0001), using multiple regression. Using unifactorial or multifactorial analyses, association between fibrate and tHcy is independent from conventional confounders such as age, gender, smoking, folate or B12 concentration, serum creatinine and MTHFR genotypes, however interference of low folate or B12 and fibrate treatment resulted in concentrations of tHcy more than 20 μmol/l. Conclusions Fibrate treatment was associated with a significant increase in prevalence of the risk of mild hyperhomocysteinaemia in coronary patients, independently from conventional confounders.

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment

Sixty percent of patients with colorectal cancer (CRC) are afflicted with distant metastases (liver or lung metastatic process) or a local relapse of malignancy (Bird et al., 2006). The possibilities of surgical and oncological treatment of this disease offer us a large spectrum of treatments including the combination of surgical procedures and consecutive oncological treatments. In the case of radical surgical therapy we can consider the curative access. The main medical problem of CRC is the high rate of recurrences after radically performed surgical therapy. The operability of recurrence is only about 30% in the case of local relapse and 20% in the case of distant metastases (Coleman et al., 2008; Kobayashi et al., 2007). The second dominant problem is the early recurrence of CRC after radical surgical treatment, when the patients undergo a difficult and exhausting procedure with a high risk of perioperative complications without any significant differences in overall survival against modern palliative therapy (Van den Eynde & Hendlisz, 2009). Contemporary clinical and histopathological prognostic factors (staging, grading, etc.) used for the detection of patients with a high risk of relapse and a short overall survival rate and for the indication of adjuvant oncological treatment after radical surgery are not sufficient. Tumor infiltrating lymphocytes (TIL) were described as a good prognostic factor for patients with a high risk of relapse. They are critical indicators of efficient antitumor immunological response. Their number, type and morphology of TIL cells determine resulting tumor prognosis (Atreya & Neurath, 2008; Galon et al., 2006). They could be connected also with the suppression of micrometastatical disease after radical surgery (Gajewski et al., 2006; Pages et al., 2005). We can recognize either the type of immune cells or distinguish their morphological aspects (infiltration of any part of tumor or surrounding of tumor or tributary lymph nodes) (Talmadge et al., 2007).

The Usefulness of Tumor Markers in Patients with Colorectal Carcinoma for the Detection of Local Recurrences and Distant Metastases

AbstractAim: The aim of the study was to evaluate the usefulness of serum tumor marker assessment in patients with colorectal carcinoma for the early detection of local recurrences and distant metastases. Patients and Method: Tumor markers were examined in a group of 517 patients with clinically diagnosed and histologically verified colorectal carcinoma. The following tumor markers were assessed: CEA, CA 19-9, CA 72-4 and thimidinkinase. The statistical evaluation was performed using S.A.S.software (USA). Results: Serum values of all tumor markers were significantly increased in progression of the disease in comparison with those in remission. For detection of local recurrences the highest sensitivities were achieved by CA 19-9 (SN 57%, SP 90%), followed by CEA (SN 48%, SP 90%). For the detection of distant metastases the best seemed to be CEA (SN 63%, SP 90%) followed by CA 72-4 (SN 42%, SP 90%) and CA 19-9 (SN 37%, SP 90%). For the detection of liver metastases in progression the highest sensitivities were shown by CEA (SN 95%, SP 90%) followed by CA 19-9 (SN 72%, SP 90%). For the detection of lung metastases in progression the highest sensitivities were shown by CEA (SN 52%, SP 90%) followed by CA 19-9 (SN 33%, SP 90%). Conclusions: The classification of remission and the prediction of disease progression in patients during follow-up is possible with a high degree of probatility. For the detection of local recurrences CA 19-9 serum values seem to be the best. CEA showed the highest sensitivity for the detection of liver and lung metastases.ZusammenfassungZielsetzung: Ziel dieser Untersuchung war die Bewertung der Nützlichkeit von Serumtumormarkern bei Patienten mit kolorektalen Karzinomen für die frühe Entdeckung von Lokalrezidiven und Fernmetastasen. Patienten und Methode: Wir untersuchten die Tumormarker bei einer Gruppe von 517 Patienten mit klinisch diagnostizierten und histologisch verifizierten kolorektalen Karzinomen. Die folgenden Tumormarker wurden dabei bewertet: CEA, CA 19-9, CA 72-4 und Thimidinkinase. Die statistische Auswertung erfolgte mittels S.A.S.Software. Ergebnisse: Die Serumspiegel aller Tumormarker waren bei fortschreitender Erkrankung signifikant erhöht im Vergleich zu jenen im Remissionsstadium. Zur Entdeckung von Lokalrezidiven wurde die höchste Sensitivität mit dem CA 19-9 (SN 57%, SP 90%) erzielt, gefolgt von CEA (SN 48%, SP 90%). Zum Nachweis von Fernmetastasen schien das CEA die besten Ergebnisse zu bringen (SN 63%, SP 90%), gefolgt von CA 72-4 (SN 42%, SP90%) und CA 19-9 (SN 37%, SP 90%). Bei der Entdeckung von fortgeschrittenen Lebermetastasen zeigte CEA die höchste Sensitivität (SN 52%, SP 90%), gefolgt von CA 19-9 (SN 33%, SP 90%). Schlussfolgerungen: Die Klassifikation der Remission und die Verhersage der Erkrankungsprogression bei Patienten während der Nachuntersuchung ist mit hoher Wahrscheinlichkeit möglich. Zur Entdeckung von Lokalrezidiven zeigen CA 19-9-Serumspiegel die besten Ergebnisse. Das CEA zeigte dagegen bei der Entdeckung von Leber- und Lungenmetastasen die höchste Sensitivität.

IDH1 mutation is associated with lower expression of VEGF but not microvessel formation in glioblastoma multiforme

Introduction Glioblastoma multiforme (GBM) represents the most malignant primary brain tumor characterized by pathological vascularization. Mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) were observed in GBM. We aimed to assess the intra-tumor hypoxia, angiogenesis and microvessel formation in GBM and to find their associations with IDH1 mutation status and patients prognosis. Methods 52 patients with a diagnosis of GBM were included into the study. IDH1 R132H mutation was assessed by RT-PCR from FFPE tumor samples obtained during surgery. The expression of markers of hypoxia (HIF2α), angiogenesis (VEGF), tumor microvascularity (CD31, CD34, vWF, CD105), and proliferation (Ki-67) were assessed immunohistochemically (IHC). IDH1 mutation and IHC markers were correlated with the patient survival. Results 20 from 52 GBM tumor samples comprised IDH1 R132H mutation (38.5%). The majority of mutated tumors were classified as secondary glioblastomas (89.9%). Patients with IDH1 mutated tumors experienced better progression-free survival (P = 0.037) as well as overall survival (P = 0.035) compared with wild type tumors. The significantly lower expression of VEGF was observed in GBM with IDH1 mutation than in wild type tumors (P = 0.01). No such association was found for microvascular markers. The increased expression of newly-formed microvessels (ratio CD105/CD31) in tumor samples was associated with worse patient’s progression-free survival (P = 0.026). Summary No increase in HIF/VEGF-mediated angiogenesis was observed in IDH1-mutated GBM compared with IDH1 wild type tumors. The histological assessment of the portion of newly-formed microvessels in tumor tissue can be used for the prediction of GBM patient’s prognosis.