The Comprehensive Cancer Centre, Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, People's Republic of China
The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, 321 Zhongshan Rd, Nanjing 210008, People's Republic of China
Different chemotherapeutic agents currently available are effective only in certain subsets of patients. Predictive biomarkers will be helpful in choosing those agents and can improve the clinical efficiency by a more personalized chemotherapeutic approach. Raltitrexed is a novel water-soluble quinazoline folate analogue and can improve the efficiency of gastric cancer treatment, but its predictive biomarker remains unclear. The aim of our study was to investigate the role of plasma and tumor thymidylate synthase (TS) mRNA levels as predictive biomarkers for raltitrexed in gastric cancer. In total, 125 freshly removed gastric tumor specimens and corresponding blood samples before surgery were collected. Raltitrexed sensitivity was determined by histoculture drug response assay procedures. TS mRNA levels in tumor and plasma were determined by quantitative reverse transcription polymerase chain reaction. Plasma TS mRNA level in cancer patients was significantly higher than in healthy subjects (p = 0.009) and was significantly correlated with TS mRNA level in tumor tissues (r = 0.665, p < 0.001). Tumor and plasma TS mRNA expression levels were significantly lower in raltitrexed-sensitive group than in resistant group (p = 0.007 and 0.013, respectively). The sensitivity and accuracy of raltitrexed sensitivity prediction based on plasma TS mRNA levels were 82 and 60%, respectively, whereas the prediction based on tumor TS mRNA reached 70% sensitivity and 68% accuracy. These results indicate that TS mRNA level in plasma can mirror tumor TS mRNA level, and both of them can be used to predict raltitrexed sensitivity in gastric cancer.
Gastric cancer remains one of the leading causes of cancer death worldwide.1, 2 The median survival for advanced (stages III–IV) gastric cancer patients is <1 year.2 Up to now, there is no “gold standard” chemotherapy for gastric cancer. Although some chemotherapeutic agents (including 5-FU, docetaxel, oxaliplatin, capecitabine and irinotecan) have been recommended for the treatments of advanced gastric cancer, with any combination of those agents, the response rate is only approximately 30–50%.3, 4 In the last 5 years, with the advance of pharmacogenomic technology, tailor chemotherapy based on the specific genetic profile of individual patients has come of age.5, 6 In an attempt to improve the clinical efficiency, it is important and necessary to identify biomarkers capable of discriminating the patients who are likely to respond to certain chemotherapeutic agents.3, 7–11
Raltitrexed, a new kind of water-soluble quinazoline folate analogue, acts as a direct and specific thymidylate synthase (TS) inhibitor. Preclinical data have suggested that raltitrexed could improve therapeutic efficacy for a variety of advanced solid tumors, including gastric cancer, and a considerable number of corresponding phase I/II clinical studies are undergoing.12 Although TS is a major target of raltitrexed, the value of its expression level in predicting raltitrexed sensitivity of gastric cancer is unclear. A recent study reported that increased TS protein expression was related to resistance to raltitrexed in colon cell lines.13 Downmodulation of TS represented enhancement of raltitrexed sensitivity in colon cells.14 Thus, we hypothesized that TS mRNA level may serve as a predictive biomarker for raltitrexed in gastric cancer.
Great efforts have also been made in recent years to identify noninvasive or minimally invasive tumor biomarkers for sensitivity prediction, especially for stage IV patients, whose biopsies are not always easy to obtain or enough for gene detection. Genomic, epigenomic and transcriptomic levels can vary at metastatic and primary tumors from the same patients.15 Tumor-derived RNA species successfully detected in plasma and other body fluids have potential for use in disease and treatment assessment.16–21 Our earlier studies have demonstrated that cell-free mRNAs in malignant effusions have value in diagnoses and sensitivity prediction.22, 23 It has been reported that circulating mRNAs in the blood are surprisingly stable with the way of packaging themselves into exosomes (such as microparticles, microvesicles or multivesicles) to protect themselves from degradation.24 Therefore, mRNA can be isolated from plasma and be reversely transcribed and amplified by PCR. Although TS mRNA levels in colon25 and breast cancer21 patients have been reported to be associated with advanced stages, and it also has been reported as a possible marker of clinical outcome in breast cancer,21 information on plasma TS mRNA in raltitrexed sensitivity prediction is not available and the relationship of TS mRNA levels between plasma and corresponding tumor remains to be known.
In our study, we investigated the role of plasma and tumor TS mRNA as a predictive biomarker for raltitrexed sensitivity in gastric cancer. Our goals were the following: (i) to establish an accurate and convenient method to determine the cell-free TS mRNA contents in plasma using very small amounts of plasma samples; (ii) to collect 125 gastric tumor specimens for raltitrexed sensitivity assessment and TS mRNA detection; at the same time, to collect corresponding blood samples before surgery from the same 125 patients for plasma TS mRNA detection; (iii) to analyze the relationship between plasma and tumor TS mRNA expression levels and between TS mRNA expression and raltitrexed sensitivity.
Our data suggested that there were significant correlations between plasma and tumor TS mRNA levels and between TS mRNA expression levels and raltitrexed sensitivity, indicating that TS mRNA level in plasma could mirror tumor mRNA level, and that both plasma and tumor TS mRNA could be used to predict raltitrexed sensitivity in gastric cancer.
Material and Methods
All specimens and relevant clinical data were obtained from the Department of Oncology and General Surgery of Drum Tower Hospital, Nanjing, China during the period from August 2010 to September 2011. The specimens include 125 freshly removed gastric tumors, blood samples from the 125 corresponding patients before surgical operation and 30 age-matched healthy subjects. Twenty-two paired blood samples before and 1 month after operation (chemotherapy naive) were also collected for gene detection and analysis. The fresh gastric tumors and blood samples were kept at 4°C and sent to the laboratory within 15 min of collection. Each tumor tissue was divided into two parts: (i) one part was kept in 4°C Hanks' balanced salt solution with 1% penicillin/streptomycin and its chemosensitivity was detected in vitro by histoculture drug–response assay (HDRA)26, 27; (ii) the remaining part was left in formalin and made into formalin-fixed paraffin-embedded (FFPE) tumor blocks for pathological observation and gene detection. Gloves were worn during the whole process of treatment. Diagnosis of patients with gastric tumor was confirmed by histopathology. Clinical and histopathological data, including sex, histology, tumor site, stage, histological grade and lymph node metastasis were all collected. Clinical characteristics of the patients are summarized in Table 1. Informed consent was obtained from all patients and the protocols for our study were approved by the Human Research Protective Committee of Drum Tower Hospital.
Table 1. Patient characteristics
Histoculture drug response assay
HDRA procedures were performed as reported by Furukawa and colleagues with slight modification.26, 27 Briefly, the fresh tumor tissues were obtained and sent to the laboratory within 4°C Hanks' balanced salt solution with 1% penicillin/streptomycin. Specimens were washed with Hanks' balanced salt solution twice and minced into small pieces to approximately 10 mg, which were then placed on prepared collagen surfaces in 24-well microplates. There were eight parallel culture wells for raltitrexed testing and eight parallel culture wells for control. Plates were incubated for 7 days at 37°C in the presence of drugs dissolved with RPMI 1,640 medium containing 20% fetal calf serum and left in a humidified atmosphere containing 95% air–5% CO2. Concentration of drugs was 10 μg/mL for raltitrexed according to its peak plasma concentration in patients. After histoculture, 100 μL of Hank's balanced salt solution containing 0.1 mg/mL type I collagenase (Sigma, Shanghai, China) and 100 μL of 3-(4,5-dimethyl-2-thiazotyl)-2,5-diphenyl-2H-tetrazolium bromide solution and dissolved in 5 mg/mL phosphate buffer solution were added to each culture well and incubated for another 16 hr. After extraction with dimethyl sulfoxide, absorbance of the solution in each well was read at 540 nm. Absorbance per gram of cultured tumor tissue was calculated from the mean absorbance of tissue from eight parallel culture wells, and the tumor tissue weight was determined before culture. The inhibition rate was calculated by using the following formula: Inhibition rate (%) = (1 − T/C) × 100, where T is the mean absorbance of treated tumor/weight and C is the mean absorbance of control tumor/weight. The HDRA was regarded as valuable when the mean absorbance of extracted formazan at 540 nm of the control tumor was 15 or more per gram. When the inhibition rate of the drug was a negative value, it was regarded as zero, which meant absolutely no chemosensitivity. Based on statistical optimization analyses,28 tumors were defined as sensitive to raltitrexed if the inhibition rate was ≥33.63% and as resistant if the inhibition rate was <33.63%.
mRNA expression level detection
Total RNA extraction from plasma
Two milliliter of blood was collected in EDTA tubes. It was immediately centrifuged at 3,000g for 10 min at 4°C, followed by recentrifugation at 12,000g for 10 min at 4°C. Every 750 μL of plasma was carefully separated and was mixed with 2,250 μL TRIzol LS reagent (Invitrogen, Carlsbad, CA) thoroughly, and stored at −80°C until use. Each plasma-TRIzol LS mixture was thawed and mixed with 600 μL of chloroform. The RNA lysate was separated into three phases by centrifugation at 14,000g for 15 min at 4°C. The upper layer with RNA inside was transferred to new RNase-free tubes and further purified with a column named RNA pure link mini kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions and dissolved in 35 μL of RNase-free water followed by treatment with DNase I (Ambion, Carlsbad, CA).
Total RNA extraction from FFPE tumor tissues
The remaining part of each fresh gastric tumor specimen was left in formalin immediately after surgery. In all, 24 hr later, they were made into FFPE tumor blocks for pathological observation and gene detection. Seven 7-μm sections were prepared from primary tumor blocks that contained at least 80% tumor cells. After hematoxylin–eosin staining, the cancerous parts were microdissected and transferred into a microcentrifuge tube. RNA was isolated in accordance with a proprietary procedure (European patent number EP1945764-B1). Briefly, paraffin was removed by xylene, and microdissected cancerous parts were lysed in a proteinase K-containing buffer at 60°C for 16 hr. RNA was purified by phenol and chloroform extractions followed by precipitation with isopropanol in the presence of sodium acetate at −20°C. The RNA pellet was washed in 70% ethanol and resuspended in 53 μL of RNase-free water followed by treatment with DNase I (Ambion, Carlsbad, CA).
Quantitative reverse transcription polymerase chain reaction assessment of gene expression
M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA) was used to generate cDNA for quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect the expression of β-actin (used as endogenous control) and TS. Each batch of reaction included a positive control from commercial human lung and liver RNA (Stratagene, La Jolla, CA) as calibrators and negative controls without RNA and reverse transcriptase. Total RNA of 1 μg was used for each RT reaction. Template cDNA was amplified with specific primers and probes for β-actin and TS using Taqman Universal Master Mix (Applied Biosystems, Foster City, CA). The Assay IDs for the primers and probes were as follows (Applied Biosystems, Foster City, CA): Hs99999903_m1 (β-actin), Hs00426586_m1 (TS). The qRT-PCR was performed to quantify gene expression using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR conditions were 50°C for 2 min, 95°C for 15 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Relative gene expression quantifications were calculated according to the comparative Ct method. Final results were determined by the formula3, 29 mRNA expression level = 2−ΔΔCt and were analyzed with the Stratagene analysis software.
The Mann–Whitney U test and the Kruskal–Wallis test were used to test the associations between TS mRNA levels and clinical characteristics and between raltitrexed sensitivity and clinical characteristics. The Mann–Whitney U test was used to compare raltitrexed-sensitive and raltitrexed-resistant groups according to TS mRNA levels in plasma and tumor tissues. The Spearman's rank correlation test was used to correlate the TS mRNA levels between plasma and paired tumor tissues. Wilcoxon matched pairs tests was used to compare plasma mRNA levels between cancer patients and age-matched healthy subjects, and plasma TS mRNA levels between before and after operation. To compare the value of TS mRNA levels in plasma and tumors as predictive biomarkers, receiver operating characteristic (ROC) curves were plotted and compared to area under the curve (AUC). A p-value of <0.05 was considered statistically significant. Statistical analysis was performed using the SPSS, version 16.0.
The inhibition rate of raltitrexed in freshly removed gastric tumor specimens
Among the 125 studied patients, plasma and tumor TS mRNA levels and in vitro chemosensitivity were successfully assessed in 109 individuals, of whom 9 were Stage I, 26 were Stage II, 72 were Stage III and 2 were Stage IV. All the patients' characteristics are summarized in Table 1. The mean inhibition rate of raltitrexed in freshly removed gastric tumor specimens was 48% (range, 2.50–88.21%). There was no significant association between the clinical characteristics and the inhibition rates of raltitrexed (Table 1).
TS mRNA levels in plasma and tumor tissues
In the total 109 samples, with the same calibrators, the TS mRNA levels in tumor tissues were around eightfold higher than those in plasma. The mean mRNA level in tumor tissues was 9.43 (range, 0.13–46.55) and the mean mRNA level in plasma was 1.20 (range, 0.02–7.25). In addition, there was no significant association between clinical characteristics and plasma or tumor TS mRNA expression levels (Table 1). However, TS mRNA levels in both tumor tissues and plasma were slightly higher in stage IV (Fig. 1).
Plasma TS mRNA levels in cancer patients were fourfold higher than in healthy people (cancer patients: 1.20; healthy subjects: 0.31; p = 0.009), and were positively related with tumor TS mRNA levels (r = 0.665, p < 0.001, Fig. 2). However, there was no correlation between plasma TS mRNA levels and tumor volume or between plasma TS mRNA levels and LDH (data not shown).
The correlation between TS mRNA expression levels and the sensitivity to raltitrexed
In the 109 samples, tumor and plasma TS mRNA levels were lower in the raltitrexed-sensitive group than in the raltitrexed-resistant group (tumor: 7.80 vs. 12.59, p = 0.007, Fig. 3a; plasma: 0.91 vs. 1.62, p = 0.013, Fig. 3b; Mann–Whitney U test). ROC curves were made for raltitrexed sensitivity prediction using tumor and plasma TS mRNA levels, respectively (Fig. 4). The AUCs were 0.671 (95% CI: 0.561–0.780, p = 0.006) in tumor and 0.674 (95% CI: 0.557–0.790, p = 0.005) in plasma. With the optimized cutoff value of 6.79, the sensitivity and accuracy of raltitrexed sensitivity prediction based on tumor TS mRNA levels were 70 and 68%, respectively. With the optimized cutoff value of 0.56, plasma TS mRNA-based prediction reached 82% sensitivity and 60% accuracy.
TS mRNA levels in plasma before and after operation
TS mRNA levels in 22 paired blood samples before and 1 month after operation (chemotherapy naive) were compared. TS mRNA levels decreased significantly in 20 out of 22 gastric cancer patients 1 month after operation (p = 0.021, Wilcoxon matched pairs test, Fig. 5).
Studies with the TS inhibitor have reported that high TS mRNA and/or protein expression is suitable as a prognostic factor for poor response to 5-FU-based chemotherapy.30–35 Also, in vitro study has reported that pemetrexed sensitivity of freshly removed surgical specimens and the TS mRNA expression levels were significantly associated.7 A clinical study of 49 patients in nonsmall cell lung cancer also showed that the patient group with lower TS mRNA levels had a longer median progression free survival and a longer median overall survival as compared to patients with high TS expression when treated with pemetrexed.8 In addition, in vitro study mentioned that increased TS protein expression was found in raltitrexed-resistant colon cell lines13 and that downmodulation of TS protein could enhance the effects of raltitrexed sensitivity in resistant tumor cells.14 All those indicate that TS may have a key role in determining the sensitivity of TS inhibitors like 5-FU, pemetrexed and raltitrexed. However, till now, no large-scale clinical samples included study was available concerning the association of TS mRNA expression and sensitivity to raltitrexed in gastric cancer.
In recent years, the detection of cell-free mRNA in the plasma and serum of human subjects has opened up new investigational opportunities. Several candidate genes (β-catenin,36 hTERT,19, 20 CK1917, 18 mRNA, etc.) have been proven to provide valuable information for early diagnosis, tumor staging and disease monitoring. Some genes such as cyclin D1 and TS mRNA have been reported having correlation with survival in breast cancer.21 However, only very few studies have investigated their value for chemosensitivity prediction in gastric cancer. In our study, there was a significant correlation between plasma TS mRNA levels and raltitrexed sensitivity. Also, we concluded that the plasma TS mRNA measurement can be an attractive means for raltitrexed sensitivity prediction. Plasma TS mRNA-based prediction reached 82% sensitivity and 60% accuracy, whereas tumor TS mRNA-based prediction reached 70% sensitivity and 68% accuracy. It seems that TS plasma mRNA level is more sensitive but less specific. This may be because molecular alterations in circulation precede malignant pathological changes in tumors, but they are more likely to be influenced by other factors in blood.15 Nevertheless, these results form a rational basis for the design of clinical trials to evaluate clinical value of plasma TS mRNA in raltitrexed sensitivity prediction, and that will be especially helpful for patients without sufficient tumor tissues for gene detection when chemotherapeutic agents needed to be chosen.
In our study, there was a significant difference in plasma TS mRNA levels between normal control and cancer patients. This result, in agreement with the previous reports,21 indicates that the increase of plasma TS mRNA levels in gastric cancer patients may have a tumor origin. In addition, the decrease of TS mRNA levels in most blood samples after tumor removal could also explain that cell-free mRNAs were tumor related. Notably, in 2 out of the 22 follow-up patients who had palliative gastrectomy and had more than 17 lymph node metastasis, the TS mRNA levels did not decrease but slightly increased after the operation. This may be owing to the fact that cancer cells might still exist in those patients and keep releasing mRNA into the blood. Moreover, in our study, TS mRNA levels in plasma were significantly associated with the levels in tumor tissues, which is in agreement with a similar conclusion reported earlier,19 and indicted that cell-free mRNA could mirror the tumor information.
Studies have reported that TS would be a poor prognostic marker, and that TS mRNA in patients was associated with advanced stages in colon cancer and breast cancer.21, 25 In our study, there was no correlation between plasma TS mRNA levels and stages although TS mRNA levels were slightly higher in stage IV. This could be owing either to inadequate numbers of stage IV patients included in our study to reflect objective results, or to the heterogeneity of cancer.
The chemosensitivity testing method we adopted here is HDRA, which has been demonstrated by a variety of studies as a useful predictor of response to chemotherapy at different cancerous sites, mainly gastrointestinal cancer.26 The collagen sponge-gel-supported histoculture conserves the original phenotypic characteristics and potential cellular interactions of tumor cells,37 and highly mimics the growth of the tumors in vivo.38 Good correlations between efficacy rate for an individual agent using HDRA in vitro assay and clinical response rate to each agent have been reported previously in various cancer.26, 39–43 Therefore, HDRA has been recognized as a convenient and useful predictor for response to chemotherapy. Nonetheless, we should note that our study may have some technical limitations. The tissue specimens tested might not be representative of the behavior of the patient's whole tumors because of the cancer heterozygote, and there may unavoidably exist an imperfect relationship between tumor response and survival because of treatment associated adverse events.44 To overcome this problem, we designed eight parallel culture wells for raltitrexed sensitivity testing and eight parallel culture wells for control from different parts of one patient's tumor sample. Moreover, more adequately designed clinical trials will be carried out for further confirmation.
Taken together, our study is the first to demonstrate the role of TS mRNA levels in plasma and tumors as potential predictive biomarkers for raltitrexed sensitivity in gastric cancer. Plasma TS mRNA will be especially meaningful for patients without sufficient tumor tissue for gene expression analysis. For example, gastric cancer patients with low levels of plasma TS mRNA may benefit from raltitrexed-based chemotherapy. Large-scale retrospective and prospective studies will be needed to further investigate the role of plasma and tumor TS mRNA levels as predictive biomarkers for raltitrexed in gastric cancer.