The Effectiveness of Pemetrexed Monotherapy Depending on Polymorphisms in TS and MTHFR Genes as Well as Clinical Factors in Advanced NSCLC Patients

Pathol. Oncol. Res. (2016) 22:49–56 DOI 10.1007/s12253-015-9966-z ORIGINAL ARTICLE The Effectiveness of Pemetrexed Monotherapy Depending on Polymorphisms in TS and MTHFR Genes as Well as Clinical Factors in Advanced NSCLC Patients Tomasz Kucharczyk 1,2 & Paweł Krawczyk 1 & Tomasz Powrózek 1 & Dariusz M. Kowalski 3 & Rodryg Ramlau 4,5 & Ewa Kalinka-Warzocha 6 & Magdalena Knetki-Wróblewska 3 & Kinga Winiarczyk 3 & Maciej Krzakowski 3 & Janusz Milanowski 1,7 Received: 20 November 2014 / Accepted: 3 August 2015 / Published online: 16 August 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract In NSCLC, second-line chemotherapy using pemetrexed or docetaxel has limited efficacy and should be dedicated to selected groups of patients. Pemetrexed is an antifolate compound with the ability to inhibit enzymes (TS, DHFR and GARFT) involved in pyrimidine and purine synthesis. The objective of this study was to evaluate the association between polymorphisms of TS and MHFR genes and clinical outcomes in NSCLC patients treated with pemetrexed monotherapy. DNA was isolated from peripheral blood of 72 non-squamous NSCLC patients treated with pemetrexed. Using PCR and RFLP methods, the variable number of tandem repeats (VNTR), the G > C SNP in these repeats and insertion/deletion polymorphism of TS gene as well as 677C > T SNP in MTHFR gene were analyzed and correlated * Paweł Krawczyk 1 Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin, Poland 2 Postgraduate School of Molecular Medicine, Warsaw Medical University, Żwirki i Wigury 61, 02-091 Warszawa, Poland 3 Department of Lung and Chest Cancer, Oncology Centre-Institute, M. Sklodowska-Curie in Warsaw, W. K. Roentgena 5, 02-781 Warszawa, Poland 4 Greater Poland Center of Pulmonology and Thoracic Surgery of Eugenia and Janusz Zeyland, Poznań, Poland 5 Department of Clinical Oncology, Chair of Cardio-Thoracic Surgery, University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznań, Poland 6 Regional Centre of Oncology in Łódź, Ignacego Paderewskiego 4, 90-993 Łódź, Poland 7 Institute of Agricultural Medicine of Lublin, Kazimierza Jaczewskiego 2, 20-950 Lublin, Poland with disease control rate, progression-free survival and overall survival (OS) of NSCLC patients. Carriers of 2R/3R(G), 3R(C)/3R(G), 3R(G)/3R(G) genotypes showed significantly more frequent early progression than carriers of 2R/2R, 2R/ 3R(C), 3R(C)/3R(C) genotypes of TS gene (p < 0.05). Among carriers of triple 28 bp tandem repeats (3R) in TS gene and C/ C genotype of MTHFR gene a significantly shorter OS was observed (HR = 3.07; p = 0.003). In multivariate analysis, significantly higher risk of death was observed in carriers of both 3R/3R genotype in TS and C/C genotype in 677C > T SNP in MTHFR (HR = 3.85; p < 0.005) as well as in patients with short duration of response to first-line chemotherapy (HR = 2.09; p < 0.005). Results of our study suggested that genetic factors may have a high predictive and prognostic value (even greater than clinical factors) for patients treated with pemetrexed monotherapy. Keywords Non-small cell lung cancer . Pemetrexed monotherapy . TS . MTHFR . Polymorphism Introduction Lung cancer is the most prevalent cause of death due to malignancies worldwide. There is very low percentage of early diagnosed NSCLC patients which results in only 15 % chance of surgical resection [1]. Systemic therapy (chemo- and radiotherapy) is considered to be the main form of lung cancer treatment. Unfortunately, first-line treatment is of low effectiveness and progression is observed in the vast majority of patients. Pemetrexed is one of the novel 3rd generation drugs with the least side effects reported. In the first-line study, cisplatin with pemetrexed proved to be more effective in non- 50 squamous histology of NSCLC and as a result it was registered in only such histology of lung cancer [2]. Second-line treatment with pemetrexed monotherapy is possible in nonsquamous patients with good performance status who progressed after non-pemetrexed first-line chemotherapy, which was proven by a comparative study with docetaxel [3]. Pemetrexed, a multitarget antifolate, is effective in nonsquamous NSCLC and malignant pleural mesothelioma, with the main focus on inhibition of enzymes involved in pyrimidine and purine synthesis such as thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). TS catalyzes transformation of dUMP into dTMP. Decreased levels of dTMP results in inhibition of DNA repair and synthesis, thus cell death [2–4]. Studies have shown that expression levels of pemetrexed target enzymes may alter the effectiveness of the drug [5, 6, 7]. Squamous cell carcinoma was shown to have higher expression of TS than adenocarcinoma, thus being more resistant to pemetrexed treatment [8, 9]. Free circulating folates may regulate pemetrexed targets’ activity. Increased level of methyltetrahydrofolate (5methylTHF) is a result of changes in the activity of 5,10methylenetetrahydrofolate reductase (MTHFR), which results in higher TS activity and thus reduces pemetrexed efficacy [10]. There are three well known polymorphic changes that affect TS mRNA level and TS protein expression: various number of 28-base pair tandem repeats (VNTR) located in the 5′ end untranslated region of TS gene; a single nucleotide polymorphism (SNP) G > C in the second repeat of the 28-bp tandem repeats; and a 6-bp deletion at the 3’ end of the TS gene (1494del6) [11–13]. Polymorphisms in TS gene are known to alter effectiveness of 5-fluorouracil (5-FU) in colorectal cancer [14]. Moreover, MTHFR gene polymorphism 677C > T is causing lower expression of MTHFR and decreased levels of 5-methylTHF in colon and breast cancer cell lines [15]. This retrospective, non-randomized, multicenter study was carried out in order to assess the usefulness of TS and MTHFR gene polymorphisms as predictive markers in NSCLC patients treated with pemetrexed monotherapy. Materials and Methods The whole studied group consisted of 72 NSCLC patients (46 male, 26 female; median age 61) with non-squamous histology, who were treated with pemetrexed monotherapy. Clinical data was collected from all patients. 500 mg/m2 of pemetrexed was administered as an intravenous infusion on day 1 of each 21 day cycle. In order to reduce toxicity, patients received folic acid and B12 vitamin prior to treatment. Kucharczyk T. et al. The response to chemotherapy was assessed according to RECIST criteria. The observation period was from 2008 until February 2014, when progression was observed in 60 patients of whom 45 died. The material for the study were blood samples collected in four oncology centres in Poland. All genetic testing was performed in clinical laboratory in Pneumonology, Oncology and Allergology Department in Lublin. Venous blood (...truncated)