Polymorphisms in TS, MTHFR and ERCC1 genes as predictive markers in first-line platinum and pemetrexed therapy in NSCLC patients

Pawe Krawczyk 0 1 3 4 5 6 Tomasz Kucharczyk 0 1 3 4 5 6 Dariusz M. Kowalski 0 1 3 4 5 6 Tomasz Powrzek 0 1 3 4 5 6 Rodryg Ramlau 0 1 3 4 5 6 Ewa KalinkaWarzocha 0 1 3 4 5 6 Kinga Winiarczyk 0 1 3 4 5 6 Magdalena KnetkiWrblewska 0 1 3 4 5 6 Kamila WojasKrawczyk 0 1 3 4 5 6 Katarzyna Kaakucka 0 1 3 4 5 6 Wojciech Dyszkiewicz 0 1 3 4 5 6 Maciej Krzakowski 0 1 3 4 5 6 Janusz Milanowski 0 1 3 4 5 6 0 D. M. Kowalski K. Winiarczyk M. Knetki-Wrblewska M. Krzakowski Department of lung and Chest Cancer, Oncology Centre - Institute M. sklodowska-Curie , Warsaw, Poland 1 t . Kucharczyk Postgraduate school of Molecular Medicine, Warsaw Medical University , Warsaw, Poland 2 ) t . Kucharczyk t . Powrzek K. Wojas-Krawczyk K. Kaakucka J. Milanowski Department of Pneumonology, Oncology and allergology, Medical University of lublin , Jaczewskiego 8, 20-954 lublin, Poland 3 J. Milanowski Institute of agricultural Medicine of lublin , lublin, Poland 4 e. Kalinka-Warzocha Regional Centre of Oncology , lodz, Poland 5 R. Ramlau W. Dyszkiewicz Department of thoracic surgery, Poznan University of Medical sciences , Poznan, Poland 6 R. Ramlau W. Dyszkiewicz Wielkopolska Center of Pulmonology and thoracosurgery of eugenia and Janusz Zeyland , Poznan, Poland Purpose We presented retrospective analysis of up to five polymorphisms in TS, MTHFR and ERCC1 genes as molecular predictive markers for homogeneous Caucasian, non-squamous nsClC patients treated with pemetrexed and platinum front-line chemotherapy. Methods the following polymorphisms in Dna isolated from 115 patients were analyzed: various number of 28-bp tandem repeats in 5-UtR region of TS gene, single nucleotide polymorphism (snP) within the second tandem repeat - of TS gene (g>C); 6-bp deletion in 3-UtR region of the TS (1494del6); 677C>t snP in MTHFR; 19007C>t snP in ERCC1. Molecular examinations results were correlated with disease control rate, progression-free survival (PFs) and overall survival. Results Polymorphic tandem repeat sequence (2R, 3R) in the enhancer region of TS gene and g>C snP within the second repeat of 3R allele seem to be important for the effectiveness of platinum and pemetrexed in first-line chemotherapy. the insignificant shortening of PFs in 3R/3R homozygotes as compared to 2R/2R and 2R/3R genotypes were observed, while it was significantly shorter in patients carrying synchronous 3R allele and g nucleotide. the combined analysis of TS VntR and MTHFR 677C>t snP revealed shortening of PFs in synchronous carriers of 3R allele in TS and two C alleles in MTHFR. the strongest factors increased the risk of progression were poor Ps, weight loss, anemia and synchronous presence of 3R allele and g nucleotide in the second repeat of 3R allele in TS. Moreover, lack of application of second-line chemotherapy, weight loss and poor performance status and above-mentioned genotype of TS gene increased risk of early mortality. Conclusion the examined polymorphisms should be accounted as molecular predictor factors for pemetrexedand platinum-based front-line chemotherapy in non-squamous nsClC patients. Introduction lung cancer is the main cause of death among all cancer patients in the world. Over 85 % of such cases are represented by non-small cell lung cancer (nsClC) and only 15 % of those cases have a chance of surgical resection (Dela Cruz et al. 2011). this is why systemic chemotherapy and radiotherapy are the main treatment options in nsClC. standard first-line chemotherapy consisting of platinum compounds and drugs like gemcitabine or vinorelbine, used in nsClC therapy, has proven to be effective, but at a cost of sometimes serious side effects (sculier and Moro-sibilot 2009). therefore, new 3rd generation drugs (pemetrexed) with lower toxicity profiles are being developed and used in combination with platinum compounds in first-line treatment of non-squamous nsClC patients (scagliotti et al. 2008). Platinum compounds are the alkylating agents, which cross-link Dna in several different ways interfering with transcription and cell division. the damaged Dna elicits Dna repair mechanisms or activates apoptosis when repair proves impossible. excision repair cross-complementing group 1 (eRCC1) is an endonuclease, which is involved in Dna repair in nucleotide excision repair mechanism (neR). high expression of eRCC1 and other neR enzymes is associated with a high yield of Dna repair. Increased Dna repair is associated with longer survival of surgically treated patients, but is negative predictive factor for chemotherapy using platinum compounds (Zheng et al. 2007; gazdar 2007). Pemetrexed is a novel cytostatic drug, which has shown efficacy in first and second line of treatment, as well as in maintenance therapy, of non-squamous nsClC and malignant pleural mesothelioma (scagliotti et al. 2008, 2009; al-saleh et al. 2012). Pemetrexed is a multitarget antifolate agent that inhibits enzymes involved in pyrimidine and purine synthesis. Its main target is thymidylate synthase (ts). Inhibition of ts results in decreased amount of thymidyne, which is necessary for Dna repair and synthesis. Pemetrexed also inhibits two other enzymes involved in purine/pyrimidine synthesis pathway: dihydrofolate reductase (DhFR) and glycinamide ribonucleotide formyltransferase (gaRFt). It is also known that the activity of those target enzymes alters the effectiveness of pemetrexed. high expression of ts, DhFR and gaRFt in squamous cell lung cancer and some of lung adenocarcinoma was proven to reduce chemosensitivity of cancer cells to pemetrexed, and pemetrexed was inefficient in patients with this histologic subtype of nsClC (scagliotti et al. 2009; Chattopadhyay et al. 2007; hanauske et al. 2007; Wang et al. 2014; Ceppi et al. 2006). all three enzymes are folate dependent, and hence, the activity of pemetrexed also depends on cell folate level. It has been shown that increased levels of extracellular folates decreased pemetrexed activity in human lung and colon cancer cell lines (Chattopadhyay et al. 2007). enzyme that is indirectly involved in the proper functioning of purine and pyrimidine synthesis pathway is 5,10-methylenetetrahydrofolate reductase (MthFR). It catalyzes an 1 3 irreversible conversion of 5,10-methylenetetrahydrofolate (5,10-methylenethF) to methyltetrahydrofolate (5-methylthF). elevated levels of 5,10-methylenethF causes higher activity of ts and lower effectiveness of antifolate agents in cancers patients (t iseo et al. 2012). thymidylate synthase mRna level is regulated by three different polymorphisms: various number of 28-base-pair (bp) tandem repeats (VntR) in 5 -UtR enhancer region of TS gene (TSER) (Mandola et al. 2003); a single nucleotide polymorphism (snP) g>C in the second repeat of 28-bp repeats; and a 6-bp deletion on the 3 end of the TS gene (1494del6) (Kawakami and Watanabe 2003; Uchida et al. 2004; Mandola et al. 2004; stoehlmacher et al. 2009). VntR and 1494del6 polymorphisms of TS gene are know (...truncated)