Abstract
Aim: To evaluate whether expression of L-type amino acid transporter 1 (LAT1) in pretreatment rectal cancer biopsies is predictive of tumour response to neoadjuvant hyperthermo-chemoradiotherapy (HCRT). Patients and Methods: Forty-four patients with rectal adenocarcinoma who received neoadjuvant HCRT were investigated. LAT1 expression was immunohistochemically evaluated using pretreatment biopsies. The operation was performed after 2-3 months following HCRT and each resected specimen was graded by the histological criteria of the Japanese Classification of Colorectal Carcinoma. Results: A positive LAT1 expression was recognized in 50.0% (22/44) of patients. Resected specimens were divided into 2 groups according to the histological grading criteria: good response (n=29) and poor response (n=15). LAT1-negative tumours had an 81.8% probability of good response and 18.2% probability of poor response. LAT1 expression showed marginally significant association with response to HCRT (p=0.05). Conclusion: LAT1 may be a useful predictive marker of response to HCRT in rectal cancer.
Recently, preoperative chemoradiotherapy for locally advanced rectal cancer (LARC) has been increasingly used as a neoadjuvant treatment (1-6). The pathologic downstaging after neoadjuvant chemoradiotherapy is correlated with improved survival, decreased local recurrence, and a higher rate of sphincter-preserving surgeries (7-9). Heat is cytotoxic for cells in hypoxic, poor perfusion and low pH environments that are specifically found in tumour tissues. It has been clearly shown that hyperthermia in combination with radiotherapy increases the cytotoxic effects (10). Therefore, in this study local hyperthermia was added to chemoradiotherapy (HCRT) in order to enhance of the local control.
Prospective identification of patients who have a higher likelihood of responding to preoperative neoadjuvant therapy could be important in deceasing treatment morbidity. In addition, patients who are unlikely to respond could be offered alternative approaches to therapy. Although recent studies have evaluated the potential of genetic biomarkers to predict the outcome in LARC treated with neoadjuvant chemoradiotherapy, there is no effective method of predicting which patients will respond to neoadjuvant therapy (11).
Amino acid transporters are essential for the growth and proliferation of normal and transformed cells (12, 13). Among various types of amino acid transports, system L is a Na+-independent, large and neutral amino acid transport agent (12, 14). L-Type amino acid transporter 1 (LAT1) transports large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine and histidine (15, 16). Previous studies showed that LAT1 is highly expressed in proliferating tissues including tumour cell lines and primary human tumours and played an essential role in the growth of tumour and the survival of patients (16-18). Kaira et al. described that the positive incidence of LAT1 expression was approximately 30% in colon cancer (19). Moreover, LAT1 expression was higher in the metastatic lesions of colon cancer than in the primary lesions. However, it is unclear whether LAT1 expression could predict treatment response in human neoplasms.
The aim of the present study was to determine whether the expression of LAT1 in pretreatment rectal cancer biopsies is predictive of tumour response to HCRT.
Patients and Methods
Patients. Between 2003 and 2006, 44 patients with proven rectal adenocarcinoma who received HCRT followed by surgery were investigated. For diagnostic work-up, all patients underwent staging for distal metastases with computed tomography of the abdomen and thorax. T stage was determined by magnetic resonance imaging, especially T2-weighted imaging. The extent and location of the tumour was classified according to the Japanese Classification of Colorectal Carcinoma (20). Patient characteristics are shown in Table I. The median follow-up was 44.7 months (range: 3.5-77.0 months) for all patients and that for the 35 patients who were still alive at the last follow-up was 50.0 months (range: 29.7-77.0 months).
Treatments. The treatment protocol for LARC has been described in previous reports (21). Radiotherapy was delivered by 10-MV X-rays through an anteroposterior or three- or four-field box technique. The clinical target volume encompassed the primary tumour and the entire mesorectal tissue. The total radiation dose was 40-50 Gy, with daily fractions of 2 Gy on 5 consecutive days per week. Chemotherapy consisted of 5-FU (250 mg/m2 per day) and leucovorin (25 mg/m2 per day) administered by continuous infusion at night for 5 days a week in the 1st, 3rd and 5th weeks of radiation. Hyperthermia was performed for 2-5 sessions once a week with an 8 MHz radiofrequency capacitive heating equipment (Thermotron-RF 8; Yamamoto Vinita Co. Ltd., Japan). The operation was performed after 2-3 months following HCRT.
LAT1 expression analysis. LAT1 expression was evaluated using pretreatment biopsies by immunohistochemical staining with an affinity-purified polyclonal rabbit anti-human LAT1 antibody (16). An oligopeptide corresponding to amino acid residues 497 to 507 of human LAT1 (CQKLMQVVPQET) was synthesised. The NH2-terminal cysteine residue was introduced for conjugation with keyhole limpet hemocyanin. Antipeptide antibody was produced as described elsewhere (22). For immunohistochemical analysis, antiserum was affinity purified as previously described (22).
Patient characteristics.
The detailed protocol for LAT1 immunostaining has been published previously (19). Immunohistochemical staining was performed on paraffin sections using a polymer peroxidase method (Envision+ horseradish peroxidase; Dako Cytomation, Denmark). Briefly, deparaffinised, rehydrated sections were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. To expose antigens, sections were microwaved in 10 mmol/l sodium citrate buffer (pH 6.0) for 5 min and cooled for 30 min. After rinsing in 0.05 mol/l TBS containing 0.1% Tween 20, the sections were incubated with affinity-purified anti-LAT1 antibody (1.2 mg/ml; 1:3,200) overnight at 4°C. Thereafter, they were incubated with Envision+ rabbit peroxidase (DAKO, Carpinteria, CA) for 30 min. The peroxidase reaction was performed using 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.05 mol/l Tris-HCl buffer (pH 7.4). Finally, nuclear counterstaining was done with Mayer's haematoxylin. For negative control, the incubation step with the primary antibody was omitted. The specificity of immunoreactions using the anti-LAT1 antibody was established in previous studies (18, 23).
LAT1 expression was considered positive only if distinct membrane staining was present. Staining intensity was scored as follows: 1, <10% of tumour area stained; 2, 10% to 25% stained; 3, 26% to 50% stained; and 4, >51% stained. The tumours in which stained tumour cells made up more than 10 % of the tumour were graded as positive. According to this scoring protocol, two investigators among from the author team, who had no prior knowledge of the clinical data, independently graded the staining intensity in all cases. To test the intraobserver variability, each section was reassessed by the same investigator after the first assessment had been completed. The time interval between the first and second assessments was at least 4 weeks. The interobserver variability was also determined by comparing the values of the first measurements of two investigators.
Histopathological response for HCRT. Each resected specimen was examined for histological changes after HCRT according to the histological criteria of the Japanese Classification of Colorectal Carcinoma (20). Grades were assigned according to the amount of necrosis, degeneration and/or lytic change of the tumour in the estimated total amount of the lesion (Table II). Grading of histopathological response was performed by a pathologist blinded to the immunohistochemistry results.
Histological criteria for the assessment of response to neoadjuvant therapy.
Statistical analysis. The association between LAT1 expression and histological response for HCRT was analyzed using the Fisher's exact test. Statistical analysis was performed using StatView J-5.0 Japanese version (HULINKS, Inc. Japan).
Results
LAT1 expression. LAT1 immunostaining was detected in carcinoma cells in tumour tissues and was localised predominantly on their plasma membrane. All positive cells revealed strong membranous LAT1 immunostaining. A positive LAT1 expression was recognised in 50.0% (22/44) of tissues.
Correlation between LAT1 expression and pathological evaluation. The numbers of resected specimens given grades 1a, 1b, 2 and 3 were 5, 10, 20 and 9, respectively. Patients were divided into two groups; those with grade 2 and 3 were considered to have a good response to HCRT, while those with grade 1a and 1b were considered to have a poor response to HCRT. LAT1-negative tumours had an 81.8% probability of good response and 18.2% probability of poor response, while LAT1-positive tumours had a 50.0% probability of good response and 50.0% probability of poor response. LAT1 expression showed marginally significant association with response to HCRT (p=0.05) (Table III).
Discussion
Recently, neoadjuvant chemoradiotherapy has been increasingly used for LARC, and approximately 45% of LARC patients respond to neoadjuvant chemoradiotherapy in terms of downstaging by at least one T stage (24). However, there is no useful molecular biomarker to predict the outcome after neoadjuvant chemoradiotherapy. Thymidylate synthase and epidermal growth factor receptor (EGFR) polymorphisms, along with the quantitative assessment of EGFR and p21, have been shown potential to predict response to treatment in rectal cancer (11). However, it is unclear whether LAT1 expression could predict response and outcome after treatment in patients with rectal cancer. In the present study, the patients with LAT1-positive expression tended to have a poor response for HRCT. The up-regulation of LAT1 has been reported in gliomas, oesophageal carcinomas, non-small cell lung cancer (NSCLC), urothelial carcinomas of the upper urinary tract and prostate cancer (25-30). However, there is no description of the relationship between treatment response and LAT1 expression in these human neoplasms. Further studies should investigate whether LAT1 expression is a biomarker of resistance to treatment in human neoplasms.
Relationship between LAT1 expression and pathological evaluation.
LAT1 expression has been associated with the Ki-67 labelling index, indicating an up-regulation of metabolic activity (31). The tumours with LAT1 expression have a tendency for rapid proliferation. In addition, highly proliferative activity can lead to hypoxic components in tumour tissue because of insufficient blood supply. Hoskin et al. showed that there is a correlation between hypoxia markers (glucose transporter-1 protein and carbonic anhydrase IX) and Ki-67 in bladder cancer (31).
The present study had some limitations, which are discussed briefly. The use of biopsies rather than whole tumours to assess LAT1 should be interpreted with caution. Biopsy samples are only parts of the tumour and are not representative of the whole tumour tissue. Another limitation of the study was due to the fact that it was performed retrospectively on a small number of patients. Further investigations with an increased number of cases are required.
- Received April 22, 2010.
- Revision received September 3, 2010.
- Accepted September 10, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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