Abstract
Aim: Increased serum or urinary concentrations of neopterin are predictive of poor prognosis in patients with tumors across a spectrum of primary locations. Less information is available about the significance of changes of urinary neopterin concentrations during therapy. The aim of the present study was to examine the association between urinary neopterin and toxicity of radiotherapy. Patients and Methods: We analyzed changes of urinary neopterin and toxicity of therapy in 12 patients with head and neck carcinoma during external-beam radiation. Urinary neopterin was determined daily by high-performance liquid chromatography. Results: In addition to a trend for increased neopterin concentrations during radiation therapy, a significant association between changes of neopterin and toxicity and vice versa was observed with a rise of neopterin predicting a later manifestation of toxicity as well as manifestion of toxicity predicting a later rise of neopterin. Conclusion: Urinary neopterin is predictive of toxicity in patients with head and neck carcinoma. An association between toxicity and subsequent rise of urinary neopterin concentrations was also observed.
The term head and neck carcinoma is based on topographical location and encompasses neoplasms of different morphologies and biological behaviors. Similarly to other tumor types, the treatment of head and neck carcinomas is based on a multimodality therapeutic strategy. While patients with early disease are treated with a single modality (surgery or radiation), patients with more advanced tumors may be cured only with a multimodal approach that encompasses a combination of surgery and radiation, chemoradiation (external-beam radiation combined with platinum-based systemic chemotherapy) or radiation with targeted biological therapy (cetuximab) (1, 2). The combination of external-beam radiation with systemic therapy (chemotherapy or targeted therapy) increases efficacy at the price of significantly higher toxicity. Therefore, not only delivery of external beam radiation, brachytherapy or chemotherapy, but also supportive care, including administration of anti-emetics, adequate hydration, nutritional intervention or local care, are essential in the management of patients with head and neck cancer.
Administration of anticancer therapy induces an inflammatory response. Changes of physiology associated with systemic inflammatory response, including metabolism of trace elements and vitamins, may play an important role in the toxicity of combined-modality treatment. The presence of systemic inflammatory or immune response may be assessed by measuring circulating cytokine concentrations. However marked fluctuations of systemic cytokine concentrations and the need for repeated office visits and venepuncture represent major obstacles complicating the use of serial cytokine determinations for the longitudinal study of inflammatory phenomena.
Neopterin is a pteridine produced from guanosine triphosphate (GTP) by activated macrophages. The activity of GTP cyclohydrolase I, the enzyme catalyzing the production of neopterin, is induced by interferon-γ (IFN-γ). IFN-γ is produced by T-lymphocytes and natural killer cells, and serum concentrations of IFN-γ reflect the systemic immune response. The production of IFN-γ is also enhanced by pro-inflammatory cytokines, such as interleukin-1 and interleukin-6, and the pro-inflammatory cytokines are known to enhance and, to a lesser degree, induce neopterin production. Thus, neopterin concentrations reflect both systemic immune and inflammatory responses. Neopterin may be measured in serum or in urine, and neopterin concentrations in both have been validated as indicators of systemic immune and inflammatory responses in a wide range of disorders, including malignant tumors (3-7). The determination of neopterin in serial urine samples circumvents the need for repeated venepuncture. Moreover, neopterin is stable in refrigerated samples for up to two weeks, and samples for repeated assessment may thus be collected by the patient and stored between regular office visits. Urinary neopterin concentrations are relatively stable over time in patients with cancer in the absence of complications (8).
Little is known about changes in urinary neopterin during radiotherapy. Recently, we reported on the negative prognostic significance of increased urinary neopterin concentrations in patients with head and neck carcinoma (9). In the present study, we evaluated daily urinary neopterin measurements in a subgroup of this cohort during external-beam radiation in relation to the toxicity of treatment.
Patients and Methods
Twelve patients, eight males and four females, mean age of 55±12 years, with histologically-verified head and neck carcinoma were included in the study. All patients were treated with external-beam radiation (70 Gy in 35 fractions). Systemic chemotherapy (cisplatin, 40 mg/m2 weekly) was administered concomitantly with external-beam radiation to seven patients, and to two patients, external-beam radiation was administered in combination with cetuximab (loading dose 400 mg/m2 followed by 250 mg/m2 weekly). Intensity-modulated radiotherapy was administered to 6 patients, and a 3-dimensional technique was used for six patients. The primary tumor was localized in the larynx in three patients, in the oropharynx in five patients, in the nasopharynx in one patient and in the oral cavity in two patients. One patient had cervical lymphadenopathy of unknown primary. One patient had stage II, one patient had stage III, seven patients had stage IVA, and two patients had stage IVB disease. Stage could not be determined in one patient.
Before the start of therapy percutaneous endoscopic gastrostomy and tracheostomy were performed in 11 and 2 patients, respectively. The investigations were approved by the Institutional Ethical Committee of the Hospital Bulovka (registration number 23.4.2007/2171/EK-Z), and all patients signed an informed consent. Results from this cohort describing the changes of serum retinol and α-tocopherol and correlation of retinol and α-tocopherol with urinary neopterin (determined once weekly) have already been published (9).
Early urine samples were collected before the start of external-beam radiation and systemic chemotherapy and then daily during the course of treatment, and stored at −20°C until analysis. Urinary neopterin and creatinine were determined using high performance liquid chromatography system Prominence LC20 (Shimadzu, Kyoto, Japan) by a modification of a method described in detail earlier (6, 9), and the neopterin concentrations were expressed as neopterin/creatinine ratio (mmol/mol creatinine).
Toxicities that were evaluated daily during the therapy included nausea, vomiting, mucositis, xerostomia, pain, and eye, ear, skin, laryngeal, pharyngeal and upper gastrointestinal toxicity. The toxicity was rated using the Common Terminology Criteria for Adverse Events version 3.0 (http://ctep.cancer.gov). The performance status was also assessed daily.
For the comparison between pre-treatment neopterin concentrations and concentrations during the therapy, the Wilcoxon signed-rank test was used. To examine the association between the urinary neopterin/creatinine ratio as an independent variable and toxicity as response variable, a logistic regression model was used. The aim was to identify the lag (in days) of toxicity associated with rising neopterin concentration. In this case, organ toxicity has a polynomial distribution (grades 0-4), with grade 0 as baseline. Because of correlation between the subsequent daily measurements in each patient, SAS GLIMMIX procedure was used for fitting longitudinal mixed-effect logistic regression models that included a random intercept to induce a compound symmetry covariance structure for repeated measurements on individual patients. To examine the association between toxicity as an independent (categorical) variable and urinary neopterin/creatinine ratio as a response variable, a generalized mixed model was applied (using SAS GLIMMIX procedure). The analyses were performed using the SAS software (version 9.2; SAS Institute, Cary, NC, USA). The decision on statistical significance was based on a value of p=0.05.
Results
In general, a trend for increasing urinary neopterin concentrations was evident during radiotherapy, but when mean neopterin concentrations were compared to mean pre-treatment concentrations, this trend reached statistical significance only late throughout the course of treatment (during weeks 6 and 7; Table I).
A significant association was observed between changes of neopterin concentration and changes in toxicity. The rise of urinary neopterin concentration significantly (p<0.01) preceded the change of upper gastrointestinal toxicity which occurred with a delay (lag) of six days, and nausea, with a lag of eight days (Table II). A less pronounced effect (p<0.05) depending on neopterin rise was observed for nausea, with a lag of 7, 10, 13 and 14 days, xerostomia with no lag (lag=0 days) and upper gastrointestinal toxicity with a lag of 8 and 23 days (Table II).
When the urinary neopterin/creatinine ratio was evaluated as a response variable with log-normal distribution, highly significant (p<0.01) effects of changes of nausea, mucositis and performance status on urinary neopterin concentrations were observed (Table III). A less pronounced effect (p<0.05) on urinary neopterin concentrations was observed for nausea, vomiting, mucositis, skin toxicity, xerostomia, laryngeal toxicity, pharyngeal toxicity, upper gastrointestinal tract toxicity, pain and performance status (Table III).
Discussion
In the present report, we expand the findings from a previous study of a decrease of serum retinol and a late increase of urinary neopterin in patients with head and neck carcinoma treated with external-beam radiation (9), with an analysis of the association between daily urinary neopterin measurements and daily symptom scores. Despite fluctuations, a general trend of increasing urinary neopterin concentrations was evident during therapy of external-beam radiation, but statistically significant elevations of neopterin concentrations were observed only during the last two weeks of therapy. Evidently, the changes of urinary neopterin concentrations were predictive of toxicity associated with the therapy. Increased neopterin concentrations preceded increased upper gastrointestinal toxicity by about one week from the onset of neopterin increase. While the association between the rise of urinary neopterin and increased gastrointestinal toxicity may be explained by the effect of inflammatory activation on the manifestations of toxicity, the pathogenesis of the response of nausea may be more complex and may involve rebound phenomena that are reflected in the reduction of nausea intensity after a certain delay. Urinary neopterin concentration was also weakly, associated with xerostomia. On the other hand, urinary neopterin was significantly increased following the changes of the intensity of nausea, vomiting, mucositis, skin toxicity, xerostomia, laryngeal toxicity, pharyngeal toxicity, upper gastrointestinal tract toxicity and performance status. The changes of urinary neopterin that resulted from changes of toxicity were delayed by lag times ranging from 5 to 24 days. The strongest effect on neopterin concentrations was observed for nausea, mucositis and performance status. These data indicate an effect of these toxicities and the changes of performance status on immune activation that is reflected in urinary neopterin concentration. The pathogenesis of changes of neopterin concentrations resulting from the changes of severity of nausea, mucositis and laryngeal toxicity may also involve rebound phenomena as these changes were in both directions, with an increase of neopterin concentration followed by a later significant concentration decrease. These observations are in line with earlier reports associating urinary neopterin with an increase of parameters of intestinal permeability that reflect gastrointestinal toxicity or complications of chemotherapy in general (10, 11). Present data indicate that the interaction between systemic immune and inflammatory responses and manifestations of toxicity goes in both directions. Not only inflammatory responses induce the manifestations of toxicity, but also the toxicity results in subsequent significant changes of concentrations of neopterin, a biomarker of systemic immune and inflammatory responses.
Enhanced neopterin production resulting in increased serum or urinary concentrations has been amply documented in patients with different primary tumors (6, 11-13), but less is known about neopterin in patients with head and neck carcinoma. In a study of 23 patients with untreated squamous cell carcinoma of the oral cavity and in 12 patients with recurrent disease reported by Murr et al. (14), increased urinary neopterin was observed in 19 patients, including nine (75%) with recurrent disease, and increased urinary neopterin was associated with poor prognosis in both the univariate and multivariate analyses. In an earlier report on the present cohort that evaluated neopterin only once weekly, significantly increased neopterin concentrations were evident only at visits six and seven weeks after the start of treatment, and increased pre-treatment neopterin concentrations in an expanded cohort predicted poor survival (9). In patients with advanced cancer, increased urinary neopterin concentrations have been demonstrated to be associated with other laboratory parameters indicating lowered lymphocyte function (5, 15). Similarly to the statistically significant increase of urinary neopterin concentrations demonstrated in the present series, despite the limited size of the cohort, an increase of neopterin production has been observed after systemic administration of different cytokines (12), chemotherapy (16, 17), and external-beam radiation (18).
Daily neopterin measurements were performed in organ transplant recipients, and a rise in urinary neopterin was an early indicator of acute complications in this population (19). Similarly, daily monitoring of urinary neopterin was performed in patients with cancer, and an increase of neopterin concentration preceded the emergence of complications, while a decrease was associated with tumor control (8). Nevertheless, data documenting utilization of daily neopterin measurements in correlation with treatment complications in patients with cancer are still very limited. To the best of our knowledge, the present study is the first to report an association between changes of urinary neopterin and toxicity of external-beam radiation.
Clinical trials of new drugs or combination focus increasingly on issues associated with tolerance and effect of therapy on the quality of life. Many aspects of toxicity are reported by the patients and, therefore, influenced by subjective factors. An objective laboratory test that would support patient-reported outcome is needed. Determination of urinary neopterin is of advantage in this setting, since repeated venepuncture is avoided; urine samples may be taken at home, without the need for daily office visits, stored and delivered at planned hospital visits. This approach could be useful in future clinical trials, and illustrates the potential use of laboratory methods in the management of patients with cancer (20).
In conclusion, urinary neopterin concentrations increased during external-beam radiation therapy in patients with head and neck carcinoma. The present data indicate that inflammatory responses have an effect on subsequent manifestations of toxicity and they further show an effect of toxicity on neopterin, as a parameter of systemic immune and inflammatory responses. Daily measurement of neopterin concentrations may be useful in the assessment of the condition of the patient and the effect of therapeutic interventions.
Acknowledgements
This study was supported by the research project Biomedreg CZ.1.05/2.1.00/01.0030 and the project of Palacký University LF_2013_010.
- Received June 12, 2013.
- Revision received July 18, 2013.
- Accepted July 19, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved