Increased Plasma Ghrelin Level in Lung Cancer Cachexia¹ Free

Cancer cachexia, which is a catabolic state characterized by weight loss and muscle wasting, occurs in some patients with advanced lung cancer (1, 2) and is a strong independent risk factor for mortality in such patients (3). Furthermore, chemotherapy may cause anorexia because of its toxicity (4). Thus, impaired energy balance is one of the most important problems in patients with lung cancer.

Ghrelin is a novel GH³ -releasing peptide, isolated from the stomach, which is identified as an endogenous ligand for growth hormone secretagogues receptor (5). Recently, ghrelin has been shown to cause a positive energy balance by decreasing fat utilization through GH-independent mechanisms (6). In addition, both intracerebroventricular and peripheral administration of ghrelin have been shown to elicit potent, long-lasting stimulation of food intake via activation of neuropeptide Y neurons in the hypothalamic arcuate nucleus (7, 8, 9). These findings raise the possibility that ghrelin plays an important role in the regulation of metabolic balance. However, little information is available regarding the pathophysiology of ghrelin in cachexia associated with lung cancer. In addition, the relationship between plasma ghrelin and anorexia induced by chemotherapy remains unknown.

Thus, the purposes of this study were: (a) to investigate whether plasma ghrelin level is associated with cachexia and disease severity in patients with lung cancer; and (b) to examine whether plasma ghrelin level is influenced by anorexia after chemotherapy.

Forty-three patients with cytologically or histologically established lung cancer at Hiroshima University Hospital between January and July 2001 were included in this study. Patients with one or more of the following conditions were excluded: (a) recent antineoplastic therapy such as chemotherapy, radiation therapy, or surgery; (b) the presence of active infection or gastric ulcer; (c) other primary cachectic states such as thyroid disease or severe liver disease; and (d) chronic renal impairment (serum creatinine level ≥ 1.5 mg/dl). None of the patients was receiving parenteral or tube feeding or had undergone gasterectomy. ECOG performance status was used as an index of the patients’ general condition. Food intake was measured using a self-assessment numerical rating scale ranging from 0 to 10, where 0 indicates absolutely no appetite, and 10 indicates an extremely good appetite. The study included 21 healthy control subjects (17 men and 4 women, mean age = 63, range = 33–78 years). Healthy control subjects were healthy hospital personnel without recent body weight loss or gain, and they had no acute or chronic disease and no regular medication. All patients gave informed and written consent, and the study was approved by the local committee and conducted in accordance with good clinical practice guidelines.

Patients were divided into two groups by the presence or absence of cachexia. Cachexia was defined clinically as documented nonintentional dry weight loss of >5 kg (all >10% of their previous normal weight) at least in ≤3 months (10). All cachectic patients also complained of weight loss. Consequently, 21 of 43 patients with lung cancer were defined as having cancer cachexia.

Blood samples were taken from the antecubital vein between 7 and 8 a.m. after an overnight fast. The blood was immediately transferred into a chilled glass tube containing disodium EDTA (1 mg/ml) and aprotinin (500 units/ml) and centrifuged immediately at 4°C. Before RIA, plasma samples were extracted using Sep-Pak C18 cartridges (Waters, Milford, MA; Ref. 11). RIA for plasma ghrelin was performed as described previously (11). Briefly, a polyclonal antibody was raised against the COOH-terminal fragment [13–28] of rat ghrelin in a rabbit. A maleimide-activated mariculture keyhole limpet hemocyanin-[Cys 0]-ghrelin [13–28] conjugate was used for immunization. The RIA incubation mixture consisted of 100 μl of standard ghrelin or unknown sample, normal rabbit serum, and 200 μl of antiserum at a dilution of 1:10,000. After 12-h incubation at 4°C, 100 μl of ¹²⁵I-labeled ligand (15,000 cpm) were added to the mixture. After 36-h incubation at 4°C, 100 μl of goat antirabbit IgG antiserum were added. Free and bound tracers were separated by centrifugation at 3,000 rpm for 30 min after incubation for 24 h at 4°C. Pellet radioactivity was quantified using a gamma counter. This assay for plasma ghrelin was performed in one batch run and in a blinded fashion. The antiserum exhibited 100% cross-reactivity with rat and human ghrelin [13–28]. Intraobserver variability of ghrelin measurement was <6%, and its interobserver variability was <9%.

We examined the time course of plasma ghrelin level in 17 patients with inoperable clinical stage III or IV who underwent single agent chemotherapy or combination therapy. Patients undergoing chemotherapy were divided into two groups by the presence or absence of decreased food intake. Anorexia including decreased food intake was evaluated by the toxicity grading criteria of the National Cancer Institute-Common Toxicity Criteria. Decreased food intake was defined as toxicity at least grade 2 of the grading criteria and was observed in 10 of the 17 patients on day 8 after chemotherapy. Thus, blood sampling for ghrelin measurement was performed in patients with decreased food intake (n = 10) and those without (n = 7) at baseline and on days 8 and 21 after the start of the first chemotherapy course. The patients were required to have measurable disease, ECOG performance status of 0–2, adequate bone marrow, and adequate renal and liver function. Changes in body weight after chemotherapy were determined with the following formula: % changes in body weight = (body weight on days 8 or 21 − baseline body weight)/baseline body weight × 100.

There was no significant difference in age, sex, body mass index, tumor stage, histology, or ECOG performance status between patients with and without decreased food intake. Chemotherapy regimens were decided by the attending physicians ad libitum (Table 1). Platinum-containing chemotherapy was more frequently used in patients with decreased food intake (n = 8) than in those without (n = 2).

The original accrual goal of this study was 40 patients. This sample size was based on 80% power so one could detect a 30% elevation in plasma ghrelin level of patients with cachexia. All data were expressed as mean ± SE unless otherwise indicated. Comparisons of parameters between the two groups were made by Fisher’s exact test or unpaired Student’s t test. Comparisons of parameters among the three groups were made using one-way ANOVA, followed by Newman-Keuls test. Comparisons of the time course of parameters between the two groups were made by two-way ANOVA for repeated measures, followed by Newman-Keuls test. 95% CIs for the differences between the two groups were calculated. Multiple regression analysis was applied to determine independent relations of clinical parameters with plasma ghrelin level. P < 0.05 was considered statistically significant.

The clinical characteristics of patients with and without cachexia and control subjects are summarized in Table 2. There was no significant difference in age or sex among the three groups. Neither tumor stage nor histology differed between patients with and without cachexia. Body mass index was significantly lower in cachectic patients than in noncachectic patients and in control subjects. ECOG performance status, a score of severity of general condition, was significantly higher in cachectic patients with lung cancer than in noncachectic patients. Numerical rating scale, a marker for food intake, was significantly lower in cachectic patients. Serum albumin level was significantly lower, and LDH was significantly higher in cachectic patients than in noncachectic patients and control subjects. There was no significant difference among the three groups in serum total cholesterol, triglyceride, fasting glucose, or creatinine.

Plasma ghrelin level did not significantly differ between all patients with lung cancer and control subjects (157 ± 10 versus 132 ± 8 fmol/ml, P = 0.1). However, plasma ghrelin level was significantly higher in cachectic patients (180 ± 17 fmol/ml) than in noncachectic patients (135 ± 10 fmol/ml) and control subjects (Fig. 1). In addition, plasma ghrelin level tended to increase with the severity of ECOG performance status (Fig. 2). In particular, plasma ghrelin level was significantly higher in patients both with ECOG performance status 2 and 3 than in controls. Multiple regression analysis (R² = 0.39; P = 0.013) also demonstrated cachexia and ECOG performance status were independently correlated with plasma ghrelin level in patients with lung cancer (Table 3). There were no significant correlations between plasma ghrelin level and biochemical markers, such as plasma albumin, total cholesterol, fasting glucose, and creatinine.

Decreased food intake was observed in 10 of the 17 patients after chemotherapy. There was no significant difference in baseline clinical characteristics between patients with and without decreased food intake (Table 4). In addition, baseline plasma ghrelin level did not significantly differ between the two groups (Fig. 3). Interestingly, plasma ghrelin level significantly increased in patients with decreased food intake after chemotherapy (from 136 ± 11 fmol/ml to 170 ± 16 fmol/ml on day 8, 179 ± 20 fmol/ml on day 21 after start of chemotherapy), whereas the ghrelin level did not significantly change in patients without decreased food intake (Fig. 3). The differences between the two groups were 7 fmol/ml (95% CI, 0–13 fmol/ml) at baseline, 58 fmol/ml (95% CI, 50–65 fmol/ml) on day 8, and 61 fmol/ml (95% CI, 54–68 fmol/ml) on day 21. Numerical rating scale, body weight, and serum albumin level significantly decreased in patients with decreased food intake after chemotherapy, whereas these parameters did not significantly change in those without. No significant differences in serum sodium, neutrophil count, or serum creatinine between the two groups were seen after chemotherapy (Table 4). There was no significant difference in response rate for chemotherapy based on the WHO criteria (12) between patients with and without decreased food intake (P = 0.15).

In the present study, we demonstrated that: (a) baseline plasma ghrelin level was significantly higher in cachectic patients with lung cancer than in noncachectic patients and control subjects; and (b) follow-up plasma ghrelin level increased in the presence of anorexia after chemotherapy.

Severe weight and appetite loss associated with cachexia is an important problem in several types of cancer, including lung cancer (1, 2). In the present study, a marked decrease in body mass index, appetite loss, and low serum albumin level was observed in cachectic patients with lung cancer. These results suggest the presence of impaired energy balance in cachectic patients with lung cancer. In addition, both ECOG performance status and serum LDH level were significantly higher in cachectic patients with lung cancer than in noncachectic patients. These results suggest that a cachectic condition is associated with the severity of lung cancer. Interestingly, plasma ghrelin level was significantly higher in cachectic patients with lung cancer than in noncachectic patients and control subjects. Multiple regression analysis demonstrated that cachexia and ECOG performance status were independently correlated with plasma ghrelin level. Recently, peripheral administration of ghrelin has been reported to induce a positive energy balance and weight gain by decreasing fat utilization and increasing carbohydrate utilization, through a GH-independent mechanism (6). These results raise the possibility that increased plasma ghrelin may represent a compensatory mechanism under conditions of anabolic/catabolic imbalance in cachectic patients with lung cancer. In the present study, plasma ghrelin level increased in proportion to ECOG performance status, which is associated with the severity and mortality of patients with lung cancer (13). Taken together, measurement of plasma ghrelin may be a simple, noninvasive, and relatively inexpensive method to assess the development of cachexia and disease severity in patients with lung cancer.

A recent animal study has demonstrated that endogenous production of ghrelin is enhanced by fasting (14). Therefore, in the present study, ghrelin measurement was repeated after chemotherapy to examine whether plasma ghrelin level is influenced by anorexia. After chemotherapy, numerical rating scale, body weight, and serum albumin level were significantly decreased in patients with anorexia, reflecting deterioration of nutrition. Expectedly, plasma ghrelin level was significantly increased in patients with decreased food intake on days 8 and 21 after the start of chemotherapy, although the ghrelin level did not significantly change in patients without decreased food intake. These results suggest that anorexia may influence plasma ghrelin level in patients with lung cancer. Ghrelin has been shown to elicit potent, long-lasting stimulation of food intake via activation of neuropeptide Y neurons in the hypothalamic arcuate nucleus (7, 8, 9). Thus, ghrelin synthesis may be up-regulated as a compensatory mechanism by chemotherapy-induced anorexia. It should be noted that plasma ghrelin level appears to be easily influenced by short-term appetite loss. This result is consistent with earlier findings that ghrelin is rapidly influenced by fasting and refeeding (6). Appetite loss is known to be one of the most important adverse effects of chemotherapy (15). Thus, measurement of plasma ghrelin may serve as a marker for the severity of chemotherapy-induced toxicity.

A number of studies has examined the effects of medical treatment on cancer anorexia (10, 16). Nevertheless, some cases are refractory to those treatments. Ghrelin may have beneficial effects on energy metabolism not only through GH-dependent mechanisms but also through GH-independent mechanisms. Thus, it would be interesting to investigate whether supplementation of ghrelin would attenuate the development of cachexia in the most severe forms of lung cancer.

This study included some study limitations: (a) this study is a cross-sectional observational study, and the patient numbers are small; additional investigation in a large scale study would be needed; (b) platinum-containing chemotherapy was more frequently used in patients with decreased food intake than in those without; nonetheless, platinum does not have direct biological effects on the stomach which produces ghrelin; and (c) renal dysfunction induced by platinum was not observed in the present study; thus, elevated plasma level of ghrelin is unlikely to be attributable to administration of platinum.

In conclusion, baseline plasma ghrelin level was elevated in cachectic patients with lung cancer, and follow-up ghrelin level increased in patients with anorexia after chemotherapy. Considering the positive energy effects induced by ghrelin, increased ghrelin may represent a compensatory mechanism under catabolic–anabolic imbalance in cachectic patients with lung cancer.

We thank Drs. Keiichi Kondo, Yoshinori Haruta, Tetsuya Oguri, and Kazunori Fujitaka for referral of patients and helpful advice.