Potential Predictors of Sensitivity to Pemetrexed as First-line Chemotherapy for Patients with Advanced Non-Squamous NSCLCs

According to WHO statistics, the incidence and fatality rate of lung cancer increase year by year. And in China, more than 75% of patients with non-small cell lung cancer (NSCLC) present with locally advanced (stage IIIB) or metastatic (stage IV) disease at diagnosis (Zhou et al., 2011). For patients in this setting, platinum-based chemotherapy is recommended as first-line treatment according to current guideline (Pfister et al., 2003). Some studys showed non-inferior efficacy and better tolerability for PEM plus cisplatin than for cisplatin plus other chemotherapy agents eg.:gemcitabine or docetaxel especially for patients with adenocarcinoma (Reck et al., 2009; Scagliotti et al., 2009; Klein et al., 2010). PEM is an antifolate that inhibits multiple enzymes involved in purine and pyrimidine synthesis. The mechanism of action consists of the inhibition of three key enzymes in the folate metabolic pathway, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT) (Giovannetti et al., 2005). This mechanism leads to depletion of fully


Introduction
According to WHO statistics, the incidence and fatality rate of lung cancer increase year by year.And in China, more than 75% of patients with non-small cell lung cancer (NSCLC) present with locally advanced (stage IIIB) or metastatic (stage IV) disease at diagnosis (Zhou et al., 2011).For patients in this setting, platinum-based chemotherapy is recommended as first-line treatment according to current guideline (Pfister et al., 2003).Some studys showed non-inferior efficacy and better tolerability for PEM plus cisplatin than for cisplatin plus other chemotherapy agents eg.:gemcitabine or docetaxel especially for patients with adenocarcinoma (Reck et al., 2009;Scagliotti et al., 2009;Klein et al., 2010).PEM is an antifolate that inhibits multiple enzymes involved in purine and pyrimidine synthesis.The mechanism of action consists of the inhibition of three key enzymes in the folate metabolic pathway, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT) (Giovannetti et al., 2005).This mechanism leads to depletion of fully reduced folate, ultimately resulting in disruption of nucleotide synthesis for both pyrimidines and purines.Pemetrexed, once in the cell, is an excellent substrate for polylpolyglutamate synthetase, leading to extensive intracellular polyglutamate derivates that are more potent inhibitors of the described enzymes.Polyglutamated pemetrexed is retained intracellularly longer than the parent compound, resulting in more prolonged cytotoxic effects (E.Esteban et al., 2009).It is reported that resistance to PEM is correlated with high pre-treatment TS, GARFT ,and DHFR expression in NSCLC cell (U.Eismann et al., 2006).TS expression is regarded as the most meaningful predictor for sensitivity or resistance to PEM in fresh tumor tissue.But for patients with advanced NSCLC, no enough tumor tissue is available for the detection of TS, GARFT, and DHFR expression.And limited by the condition of experiment in different hospitals and regions, the test result cannot be in full compliance with the actual situation.Thus at present, PEM is given to patients with advanced NSCLC, regardless of whether TS, GARFT ,and DHFR is over-expressed.In an attempt to indentify predictors of clinical indictors for sensitivity of PEM and design a model for the judgment of PEM response, we conduct this study to identify factors that could indentify patients who could mostly benefit from PEM therapy.

Patients
Patients were required to be pathologically/ cytologically diagnosed with advanced non-squamous NSCLC in Jiangsu Cancer Hospital & Research Institute from June 2006 to February 2013, without history of chemotherapy; to sign an informed consent before treatment; to have a score of karnofsky performance status ≥ 70; to be 25 to 75 years of age.Other eligibility criteria included: adequate hematological (white blood cell count > 3.0×10 9 and platelet count > 150×10 9 ), liver (bilirubin and transaminases < 1.5 times the upper normal limit) and renal function (creatinine leval < 1.5 times the upper normal limit); patients were excluded from this study if they failed to complete two cycles of chemotherapy, or with any serious medical or psychiatric condition, or other malignancies.Pregnant or lactating women are also excluded from this study.
We retrospectively reviewed the records, pathological reports, and imaging studies of eligible patients and then compared the characteristics of responders with those of none-responders.These groups of patients were analyzed for significance of sex, age, smoking history, pathological differentiation, platinum combined, hemoglobin and sites of metastasis.

Treatment
PEM was given at a dose of 500 mg/m 2 with a platinum that is recommended by NCCN.All the patient received two cycles of therapy and 21 days as a cycle.

Radiologic Evaluation
For all patients, we reviewed the imaging studies performed at the initiation of therapy.Follow-up imaging was performed after two cycles of chemotherapy.All imaging was reviewed by one thoracic radiologist.Nonresponders were identified by clinical reports for hospital radiologic studies.We classified the therapeutic results as complete or partial remission and stable or progressive disease according to the response evaluation criteria for solid tumors (Therasse et al., 2000).

Statistical Methods
We investigated the associations between sensitivity to pemetrexed and patients characteristics.Potential prognostic factors explored included sex, age, smoking history, the level of pathological differentiation, platinum combined, hemoglobin and sits of metastasis.The primary objective was to identify the factors associated with response to PEM.The associations between response and the covariates were assessed using univariate logistic regression analysis.All variables in the univariate analysis were then included in multivariable logistic regression model with pemetrexed response as the dependent variable.

Results
One hundred and nine patients with advanced NSCLC who had no history of chemotherapy accepted PEM with a platinum as first line chemotherapy for two cycles.The overall observed response rate was 83%.univariate logistic regression analysis.Seventy percent of patients were older than 65 and 68% well differentiated adenocarcinoma.Twenty percent of patients had liver metastases.We found that age and platinum were significant in predicting the sensitivity of PEM (p<0.05).Then all the factors were tested in a multivariable logistic regression analysis.The result showed in Table 2.No significant difference in response rate to PEM was found in those potential predictors

Discussion
Cisplatin-based chemotherapy for patients with advanced NSCLC results in a small but statistically significant improvement in survival, as compared with supportive care.Combination of a platinum plus a new agent continues to be the standard of care (Marino et al., 1994).
PEM is an effective and well tolerated chemotherapeutic agent.Based on previous studies, it is considered that PEM is proper for patients who were pathologically diagnosed with lung adenocarcinoma (Rodrigues-Pereira et al., 2011).However, we think it is important to clarify patients who could mostly benefit from this treatment.PEM is recommended for patients with advanced NSCLC and adenocarcinoma, regardless of whether TS, GARFT, and DHFR is over-expressed.Considering the general characteristics of patients with advanced NSCLC and the pharmacokinetics of PEM, we intend to clarify predictors of response for this regimen and we hypothesis that sex, age, differentiation level, etc. could be possible predictors.Age at diagnosis influences survival, and women with squamous or anaplastic tumors have a better prognosis than men with the same pathological types (Rossing et al., 1982).PEM inhibits the folate dependent enzymes (although all the patients were given the standard vitamin supplementation ).It may also interfere the metabolism of hemoglobin.And hemoglobin, age and sex may reflect the general condition of a patient, so we included sex, age, tumor differentiation in logistic regression model.It was reported that cisplatin-based chemotherapy is slightly superior to carboplatin-based chemotherapy in terms of response rate (Ardizzoni et al., 2007), suggesting that platinum could be a factor influencing the response of PEM.In addition, cigarette smoking is reported to change induction of hepatic microsomal enzymes in man and animals (Jusko, 1978), indicating that smoking history and cancer metastasis especially liver metastasis could associated with metabolism of chemotherapeutic agents.Therefore, impact of platinum, smoking habit and live metastasis was also estimated.
In this study, we found that age and platinum had significant impact on the response of PEM therapy by univariate analysis.However, all the factors imposed no significantly different influence on response rate of PEM therapy according to multivariable analysis.Our study provided a research direction.Considering the sample size of this study is not large enough to detect minor difference of these variables regarding the influence on the response, a further study containing sufficient number of cases is needed to re-confirm this result.

Table 1 . Univariate Analysis of Features Associated with Sensitivity to Pemetrexed
Table 1 lists patient demographics and the results of potential prognostic factors for PEM sensitivity using