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Detection of Circulating Lung Cancer Cells with Strong Thymidylate Synthase Reactivity in the Peripheral Blood of a Patient with Pulmonary Adenocarcinoma Treated with Pemetrexed

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... Furthermore, a study by Zucali et al. demonstrated that TS (Thymidylate Synthase) is overexpressed in MPM tissues and is a strong predictor of responsiveness of MPM patients to Pemetrexed/Carboplatin (119). It is thus possible that detection of TS in circulating MPM cells or as circulating cell-free RNA might prove to be an interesting predictive biomarker (120,121). ...
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Malignant pleural mesothelioma (MPM) is an aggressive tumor linked to asbestos exposure. Although the risk factors for MPM are well-known, the majority of MPM patients are diagnosed at an advanced stage and have a very poor prognosis. Circulating biomarkers for early diagnosis remain to be identified, and the current standard for MPM diagnosis relies on pleural biopsies. Robust non-invasive tests for the screening of asbestos-exposed subjects are therefore an important unmet clinical need. This review provides a critical summary of recent liquid biopsy-based studies aimed at discovering novel blood-based circulating biomarkers for the early diagnosis and prognostic stratification of MPM patients.
... Das et al. measured ERCC1 expression by immunohistochemical (IHC) analysis of CTCs from 17 patients with advanced NSCLC treated with platinumbased chemotherapy and found similar results: low ERCC1 expression on CTCs correlated with longer PFS [110]. Similarly, thymidylate synthase expression (associated with pemetrexed sensitivity in tumor samples) on CTCs has the same predictive effect [111]. Concerning immune checkpoints inhibitor (ICI) treatment, PDL1 expression (evaluated by IHC analysis) on tumor samples seems to correlate with the tumor response. ...
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Molecular characterization of tumor cells is a key step in the diagnosis and optimal treatment of lung cancer. However, analysis of tumor samples, often corresponding to small biopsies, can be difficult and does not accurately reflect tumor heterogeneity. Recent studies have shown that isolation of circulating tumor cells (CTCs) is feasible in non-small cell lung cancer patients, even at early disease stages. The amount of CTCs corresponds to the metastatic potential of the tumor and to patient prognosis. Moreover, molecular analyses, even at the single-cell level, can be performed on CTCs. This review describes the technologies currently available for detecting and capturing CTCs, the potential for downstream molecular diagnostics, and the clinical applications of CTCs isolated from lung cancer patients as screening, prognostic, and predictive tools. Main limitations of CTCs are also discussed.
... In a small study, CTCs were used to detect ERCC1 expression and patients with detectable levels had a significantly shorter PFS compared to patients with undetectable ERCC1 levels [71]. Case reports demonstrating CTCs staining positive for thymidylate synthase (the target for pemetrexed (Eli Lilly, USA)) [72] raise the prospect of this as a potential predictive biomarker. Using CTCs to test for therapeutically relevant biomarkers was reviewed recently by Becker et al. [73]. ...
... Nonetheless-as discussed before-identifying genes that are associated with an aggravated outcome is an important method to form a candidate oncogene pool that is available for further work, such as in vitro studies or biomarker guided therapy trials [4]. Further studies are warranted and currently employed by our group to validate these genes including tactics to identify these genes in circulating tumor cells [23,24]. ...
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Background Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC. Methods 47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha). Results EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01). Conclusions EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.
... 20 In this study, samples were tested using IHC, which could explain the different results. 20,48,49 The advantage of IHC is the higher level of evidence (mRNA versus translationated protein) and the analysis of tumor cells only. Nevertheless, the standard IHC has disadvantages in comparison with qPCR analysis. ...
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Background: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. Methods: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. Results: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. Conclusions: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.
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Lung cancer is the leading cause of cancer-related death worldwide with a 5-year survival rate of less than 15%, despite significant advances in both diagnostic and therapeutic approaches. Combined genomic and transcriptomic sequencing studies have identified numerous genetic driver mutations that are responsible for the development of lung cancer. In addition, molecular profiling studies identify gene products and their mutations which predict tumour responses to targeted therapies such as protein tyrosine kinase inhibitors and also can offer explanation for drug resistance mechanisms. The profiling of circulating micro-RNAs has also provided an ability to discriminate patients in terms of prognosis/diagnosis and high-throughput DNA sequencing strategies are beginning to elucidate cell signalling pathway mutations associated with oncogenesis, including potential stem cell associated pathways, offering the promise that future therapies may target this sub-population, preventing disease relapse post treatment and improving patient survival. This review provides an assessment of molecular profiling within lung cancer concerning molecular mechanisms, treatment options and disease-progression. Current areas of development within lung cancer profiling are discussed (i.e. profiling of circulating tumour cells) and future challenges for lung cancer treatment addressed such as detection of micro-metastases and cancer stem cells. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Thymidylate synthase (TYMS) is an important enzyme for 5-fluorouracil (5-FU) metabolism in metastatic colorectal cancer (mCRC) patients. The search for this enzyme in circulating tumor cells (CTCs) can be a powerful tool to follow-up cancer patients. mCRC patients were enrolled before the beginning of 5-FU-based chemotherapy. The blood was filtered on Isolation by Size of Epithelial Tumor Cells (ISET), and the analysis of TYMS expression in CTCs was made by immunocytochemistry. Additionally, we verified TYMS staining in primary tumor and metastasis from the same patients. There were included 54 mCRC patients and 47 of them received 5-FU-based chemotherapy. The median CTCs number was 2/mL. We were not able to analyze immunocytochemistry in 13 samples (9 patients with absence of CTCs and 4 samples due to technical reasons). Therefore, TYMS expression on CTCs was analyzed in 34 samples and was found positive in 9 (26.5%). Six of these patients had tumor progression after treatment with 5-FU. We found an association between CTC TYMS staining and disease progression (DP), although without statistical significance (P= 0.07). TYMS staining in primary tumors and metastases tissue did not have any correlation with disease progression (P= 0.67 and P= 0.42 respectively). Patients who had CTC count above the median (2 CTCs/mL) showed more TYMS expression (P= 0.02) corroborating with worse prognosis. Our results searching for TYMS staining in CTCs, primary tumors and metastases suggest that the analysis of TYMS can be useful tool as a 5-FU resistance predictor biomarker if analyzed in CTCs from mCRC patients. This article is protected by copyright. All rights reserved. © 2015 UICC.
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This study is to evaluate whether thymidylate synthase (TS) or thyroid transcription factor 1 (TTF1) protein expression can predict clinical outcomes for pemetrexed-based chemotherapy in patients with nonsquamous non-small cell lung cancer (NSCLC). Two hundred eighty-five consecutive patients with nonsquamous NSCLC treated with pemetrexed-based chemotherapy were immunohistochemically analyzed for the expressions of TS and TTF1. TS and TTF1 expression were successfully analyzed in 193 and 284 cases, respectively. Tumors with TS-negativity or TTF1-positivity were more frequent in patients who were female, younger, had adenocarcinoma, or had never smoked. Higher response rates for pemetrexed-based chemotherapy were associated with TS-negativity (33.7% versus 14.1%, p = 0.002) and TTF1-positivity (28.1% versus 9.8%, p < 0.001). In univariate analysis, progression-free survival for pemetrexed-based chemotherapy was significantly longer in groups with adenocarcinoma (2.9 versus 1.4 months, p = 0.001), TS-negativity (4.1 versus 2.0 months, p = 0.001), and TTF1-positivity (3.8 versus 1.3 months, p < 0.001). In multivariate analysis, TS-negativity (hazard ratio [HR] = 0.70; 95% confidence interval [CI], 0.51-0.97) and TTF1-positivity (HR = 0.51; 95% CI, 0.35-0.73) were associated with longer progression-free survival. Patients with TTF1-positive tumors also had significantly longer overall survival times than patients with TTF1-negative tumors (25.4 versus 14.2 months, HR = 0.55; 95% CI, 0.39-0.77). Low TS or high TTF1 protein expression was significantly associated with better clinical outcomes in nonsquamous NSCLC patients who were treated with pemetrexed-based chemotherapy. The predictive role of TS or TTF1 expression should be further validated in a prospective randomized study.
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Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are target enzymes of inhibition by pemetrexed, an antifolate for treatment of advanced non-small-cell lung cancer (NSCLC). This study is to evaluate the association of TS and DHFR expressions and the treatment efficacy of pemetrexed in NSCLC patients. From January 2006 to October 2008, patients with advanced NSCLC treated with pemetrexed after prior chemotherapy were included. The TS and DHFR expressions in tumor tissues were examined by immunohistochemistry and evaluated by a semiquantitative histologic score (H-score). The H-score was derived from the degrees of intensity of tumor cells multiplied by the percentage of positive neoplastic cells. The medical records were reviewed and analyzed with respect to patients' characteristics, histology types, treatment responses and survivals. Among 268 NSCLC patients treated with pemetrexed, 49 had tumor specimens available for TS and DHFR evaluation. The TS expression was positively correlated with DHFR expression (r(2)=0.11, p=0.02). Patients with low TS (≤150) expression had a longer median progression-free survival (PFS) than those with high TS (>150) expression (4.8 vs. 3.4 months; p=0.01). Patients with low DHFR expression (≤120) also had a longer median PFS than patients with high DHFR expression (>120), which was not statistically significant (5.8 vs. 3.6 months; p=0.33). In patients with adenocarcinoma, the low TS patient group also had a longer median PFS and a longer median overall survival (OS) as compared with patients with high TS expression (PFS, 4.8 vs. 3.8 months, p=0.03; OS, 21.4 vs. 10.0 months, p=0.03). Nevertheless, the association of DHFR expression level and median PFS as well as OS were not statistically significant. TS expression, rather than DHFR, may be an important predictive factor for treatment efficacy of pemetrexed in NSCLC patients.
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Increasing the sensitivity and specificity of detecting circulating carcinoma cells of patients with hepatocellular carcinoma (HCC) is very important for monitoring recurrence. To establish a novel method of detecting circulating carcinoma cells. For method development, 3 sets of controls using HCC cell line HepG2 cells were used. (A): Serial dilutions of HepG2 cells were directly used to extract total RNA for nested reverse transcription-polymerase chain reaction (RT-PCR). (B): Five milliliter of healthy blood was spiked with a serial dilution of HepG2 cells and was used for Ficoll density gradient centrifugation to recover cells. The cells were used to extract total RNA for RT-PCR. (C): After cell recovery with the same procedure as B, the cells were sorted sequentially by CD45 and Ber-EP4 immunomagnetic beads and used for RNA extraction and RT-PCR. For clinical samples, 44 patients with HCC and 7 healthy subjects were included. The alpha-fetoprotein mRNA was amplified using nested RT-PCR technique. The spiking experiments using HepG2 cells showed that 10 cells in 5 mL blood could be detected by method C and an excellent dose-response to the number of spiked cells. Whereas, method B lacked any dose-response and would yield high false-positive rates. In clinical samples, the improved method led to a positive detection rate of 52.9%, 76.9%, and 92.9% in Child-Plug class A, B, and C, respectively. There was significant difference between class A and class C (P<0.05). The total positive detection rate was 72.7%. Combining negative and positive immunomagnetic beads with RT-PCR technique may improve the sensitivity and specificity of detecting circulating HCC cells.
Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer
  • G V Scagliotti
  • P Parikh
  • Von Pawel
Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol. 2008; 26:3543-3551. [PubMed: 18506025]