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Introduction

Pain is a feared and common complication in patients with cancer (1,2), which increases in prevalence and intensity with disease progression (3,4), and influences multiple aspects of quality of life (5–8). Pharmacogenetic, pharmacokinetic and pharmacodynamic variations among individuals result in a wide variety of responses to pain sensation and analgesics; therefore, investigations of biomarkers for opioid treatment have been performed to improve the efficacy of morphine treatment (9). According to a review published in 2015 (10), numerous studies have proposed that the catechol-O-methyltransferase (COMT) 472G→A (rs4680, p.Val158Met) genotype may be a predictive biomarker for the response to morphine treatment. In these studies, patients with the G/G genotype received the highest dose of morphine (11,12), while those with the A/A genotype received the lowest morphine dose (13). However, the majority of these studies had a small sample size, were retrospective, or were targeting non-cancer pain (14). A large sample study has indicated that none of the 112 single nucleotide polymorphisms (SNPs) in 25 candidate genes demonstrated significant associations with opioid dose (15); however, the quantity of concomitant analgesics [non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen] and the method for dose determination of opioids remain unclear. Furthermore, the mean Brief Pain Index (16) following treatment was relatively high (15).

An exploratory study was planned, which included 100 patients with opioid-treatment-naïve cancer, in whom the use of NSAIDs and acetaminophen, as well as the method of dose determination of morphine were kept constant. The investigation was divided into a pilot study of 50 cases, and a replication study (the current study) of 50 more cases. Expression levels of functional genetic variants as predictive biomarkers of the response to morphine were examined. The pilot study indicated that COMT 472G→A may be a predictive biomarker (17). The aim of the current prospective study was to replicate these findings in an independent cohort of 50 opioid-treatment-naïve patients exhibiting various types of cancer.

Materials and methods

Patients and samples

This prospective study was conducted from April 2011 to March 2012 at the Kindai University Faculty of Medicine (Osaka, Japan). A total of 50 patients with opioid-treatment naïve and histologically confirmed malignant neoplasms who were scheduled to undergo opioid treatment were evaluated in the study. These patients were recruited and selected by non-probabilistic convenience sampling by their physician from the outpatients and inpatients service of the Department of Medical Oncology (Kindai University Faculty of Medicine). All patients met the following inclusion criteria: Aged ≥18 years, a verified cancer diagnosis, cancer pain suitable for morphine treatment (excluding patients with neuropathic pain, predominant incidental pain, glomerular filtration rate <30 ml/min, and history of opioid/drug abuse or alcoholism from a self-reported questionnaire), daily use of NSAIDs and able to provide written informed consent. The required dose of morphine (day 1) was evaluated in 50 patients, and was assessed in 48 patients, (2 were excluded who could not continuously receive morphine due to adverse effects or mortality; Fig. 1).

Figure 1. Schema of the study design. NSAIDs, non-steroidal anti-inflammatory drugs; NRS, numerical rating scale; SNPs, single nucleotide polymorphisms; COMT, catechol-O-methyltransferase.

Clinical features, including age, sex, Eastern Cooperative Oncology Group performance status (PS) (18), and types of primary malignant neoplasm were recorded. Morphine was administered using the standard method, including titration [NCCN Guidelines (19), Adult Cancer Pain], performed by specialized palliative care doctors. Immediate release (IR) morphine (5 mg) was administered orally, and efficacy and adverse effects were reassessed at 60 min. When the pain score was not decreased, the same dose of IR morphine (5 mg) was re-administered. Dose titration was performed to decrease pain by ≥33% on the numerical rating scale (NRS) (20) pain scale at day 1 (pre-treatment), as well as to reduce the NRS to a score of ≤3.

Criteria for discontinuation of dose titration were appearance of adverse effects graded ≥3 (Common Terminology Criteria for Adverse Events, v4.0) (21), or an attending physician determining titration to be difficult to continue, even if the adverse effect was graded ≤2. Controlled release (CR) morphine, six times the dose of IR morphine, was administered orally once per day. If pain was insufficiently controlled by 10 mg IR morphine in total, 60 mg CR morphine was administered at night on day 1. The attending physician could reduce the dose of CR morphine based on the general condition of the patient. Morphine administration was discontinued following a side effect of grade ≥3. Standardized information on potential benefits and adverse effects was provided to the patients. The study was approved by the Regional Committee for Medical Research Ethics, Kindai University Faculty of Medicine. Informed consent was obtained from all participants in the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Pain score

The pain score was assessed using a 0–10 NRS (0, no pain to 10, worst imaginable pain) on day 1 (pre-treatment), day 1 (post-treatment) and day 8 (one week after treatment) by asking patients the following question: “How intense was your average pain for the past 24 h?”

Genotyping

SNPs of COMT 472G→A (rs4680; p.Val158Met) modulate the genetic response to opioid medications (22). In an exploratory study of 50 cases, a functional genotype analysis of opioid receptor µ 1 118A→G (rs1799971; p.Asn40Asp) and COMT 472G→A (rs4680; p.Val158Met) was performed to establish the predictive biomarkers for the treatment outcome of morphine, based on the plasma level of morphine and the required dose according to genotype on days 1, 2 and 8. On day 1, blood was sampled for genotyping, and the pain score was recorded on days 1 (before and after treatment), 2 and 8. The required dose of morphine on day 1 was significantly higher in patients with the COMT G/G genotype, compared with those with A/A and A/G genotypes in the previous pilot study (P=0.03) (16). Therefore, in the current prospective study of 50 independent cases, COMT alone was the main focus.

Genomic DNA was isolated from blood samples using a QIAamp(r) DNA Mini kit (cat. no. 51304; Qiagen GmbH, Hilden, Germany) and was subjected to a genotype analysis using Taqman® SNP Genotyping Assay for COMT rs4680 (cat. no.4362691; Thermo Fisher Scientific Inc., Waltham, MA, USA) and a StepOnePlus™ Real-Time PCR Systems (cat no. 376600; Thermo Fisher Scientific Inc.).

Statistical analysis

All data are expressed as means ± standard deviation. Differences in the effects of morphine between gene polymorphism groups were evaluated using a Mann-Whitney U test. Two-sided P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS software (v19.0; IBM SPSS, Armonk, NY, USA).

Results

Patient characteristics

The characteristics of the 50 patients (25 men and 25 women) are presented in Table I. The median age was 63 years (age, 36–80 years; 62±9.17). The primary tumors were as follows: Lung cancer, n=19 (38%); breast cancer, n=9 (18%); colorectal cancer, n=5 (10%); head and neck cancer, n=4 (8%); gastric cancer, n=3 (6%); unknown primary cause, n=3 (6%); pancreatic cancer, n=2 (4%); gallbladder cancer, n=2 (4%); and others, n=3 (6%). More than 80% of the subjects exhibited metastasis and a PS of 0 to 2. The median required doses of morphine were 31.2 mg (20–60 mg) on day 1 and 32.6 mg (20–60 mg) on day 8. The pain NRS before and after treatment did not differ between the G/G and non-G/G groups (P=0.177). A dose of 60 mg morphine was required in 4 cases of lung cancer and 1 case of unknown primary cancer. The causes of pain were lymph node metastases and bone metastases in 4 cases each, and pleural seeding in 1 case. Features of 5 high-dose morphine-requiring cases, defined as requiring 2 or more cycles of 5 mg IR morphine, are presented in Table II.

Table I. Clinical characteristics of the patients (n=50).

Table I.

Clinical characteristics of the patients (n=50).

Characteristics	Patients (n)
Age, years
<65	27
≥65	23
Sex
Male	25
Female	25
Performance status
0–2	40
3–4	10
Genotype: COMT 472G→A (rs4680; p.Val158Met)
G/G	29
A/G	19
A/A	2
Tumor type
Lung	19
Breast	9
Colorectal	5
Head and neck	4
Gastric	3
Unknown primary	3
Gallbladder	2
Pancreas	2
Others	3
Required dose of morphine on day 1 (mg)
20	2
30	43
60	5
Required dose of morphine on day 8 (mg)
As required	8
20	3
30	30
60	6
90	1
N.E.	2
Day 1 (pre-treatment) pain NRS, mean (SD)
All patients	6.88 (2.43)
G/G	7.07 (2.25)
Non-G/G (A/A and A/G)	6.62 (2.69)
Day 1 (post-treatment) pain NRS, mean (SD)
All patients	2.46 (1.46)
G/G	2.38 (1.45)
Non-G/G (A/A and A/G)	2.57 (1.50)
Day 8 pain NRS, mean (SD)
All patients	3.60 (2.73)
G/G	3.17 (2.63)
Non-G/G (A/A and A/G)	4.19 (2.80)

[i] COMT, catechol-O-methyltransferase; N.E, not evaluated; NRS, numerical rating scale; SD, standard deviation.

Table II. Characteristics of patients with high-dose morphine-requiring cases.

Table II.

Characteristics of patients with high-dose morphine-requiring cases.

Patient	Age (y)	Sex	PS	COMT genotype	Tumor type	Pain NRS (before administration)
1	63	M	1	G/G	Lung	8
2	59	M	1	G/G	Lung	7
3	70	M	2	G/G	Lung	10
4	64	F	1	G/G	Unknown primary	8
5	60	F	2	G/G	Lung	8

[i] PS, performance status; COMT, catechol-O-methyltransferase; NRS, numerical rating scale.

Pain titration

Morphine at the required dose was administered to patients as a controlled-release agent. On day 1, dose titration was performed to reach NRS ≤3 and pain control ≥33%. Titration was successful in 76% of all cases, but unsuccessful in 60% of G/G genotype cases.

Genotypes of COMT and treatment outcomes of morphine

A functional genotype analysis of COMT 472G→A (rs4680; p.Val158Met) was performed to establish the predictive biomarkers for the outcome of morphine treatment (Fig. 2). The outcome based on the required morphine dose on days 1 and 8 was examined according to the genotype. The required dose of morphine (mg) on day 1 in GG, AG, and AA were 35.2±11.5, 29.5±2.3, and 25.0±7.1, respectively, and was significantly higher for patients with the COMT G/G genotype, compared with those with non-G/G (A/A and A/G) genotypes (P=0.013).

Figure 2. Genotypes were evaluated for COMT 472G→A (rs4680; p.Val158Met). The treatment outcome of morphine was examined based on the required dose (day 1). Comparisons are for G/G vs. A/A and A/G of COMT. The P-value was calculated using a Mann-Whitney U test. COMT, catechol-O-methyltransferase.

Discussion

To the best of our knowledge, the present study is the first to demonstrate the reproducibility of differences between required doses of morphine among individuals, due to genetic polymorphisms, in an independent cohort of opioid-treatment-naïve patients exhibiting various types of cancer. The most important finding is that patients with the COMT G/G genotype require a higher dose of morphine (day 1), compared with patients with A/A and A/G genotypes.

The morphine effect varies due to genetic differences; therefore, genetic polymorphisms are considered to be predictive markers. Various clinical studies have demonstrated that the required dose of morphine is higher in subjects with a COMT G/G genotype, compared with other genotypes, in cancer and non-cancer pain (11–14). The current findings are consistent with these results. A large sample study has indicated that none of 112 SNPs in the 25 candidate genes showed significant associations with opioid dose (15); however, this study lacked clarity regarding the doses of concomitant analgesics, the dose determination of opioids, and the high average total dose of morphine of 90 mg/day. Despite this dose, residual pain was relatively high (pain NRS in the past 24 h, 3.20±2.10 subsequent to analgesic use and no data for pain NRS prior to analgesic use) (15). This suggests a high proportion of refractory pain. By contrast, in the current study, the dose of concomitant analgesics was controlled (all patients received daily regular doses of NSAIDs and acetaminophen), the method of dose titration was consistent, and pain was relatively well-controlled (pain NRS following treatment, 2.46±1.46). Patients who report a higher NRS for baseline pain generally exhibit a larger change in raw pain intensity (23). The patients in the present study had relatively strong pain (pain NRS in the past 24 h, 6.88±2.43) that was relatively well-controlled, and this may have made it easier to identify a difference due to genetic polymorphism.

Notably, the required dose of morphine on day 1 was significantly higher in patients with the COMT G/G genotype. The enzyme activity of COMT is defined as high in G/G, intermediate in G/A and low in A/A. Impairment of the COMT enzyme suppresses the production of enkephalin, which causes subsequent opioid receptor expression upregulation (10). In the A/A genotype, the function of the µ receptor is weak and patients tend to be sensitive to pain. However, the density of µ receptors is proposed to be increased in this genotype, which may be the reason for the low quantity of morphine required (24,25).

The nature of the pain NRS questionnaire may also be relevant. As patients with the COMT A/A genotype are more sensitive to pain, the reported NRS may be higher than that for the quantity of morphine required (26). A meta-analysis by Lee et al (27) revealed an association between fibromyalgia and the COMT A/A genotype.

The present study has various limitations. First, it is not possible to rule out that differences in patients’ background between the COMT G/G genotype and COMT non-G/G genotype groups may be responsible for the higher required dose of morphine in the G/G group. Although there was no significant difference in age between the G/G genotype and non-G/G genotype groups (P=0.658), the PS tended to be poor in the G/G genotype group (P=0.053). However, there were no patients with poor PS (3 and 4) in the high-dose morphine group. The COMT G/G genotype group has a marginally higher NRS score (prior to treatment) than the non-G/G genotype group. Although there was no significant difference in the NRS score before treatment between these groups (P=0.177), NRS scores tended to be higher in high-dose morphine-requiring cases compared with other cases. Thus, patient backgrounds require evaluation in future studies. In addition, the patients were included in this study based on the decision of attending physicians in a single hospital; consequently, the results may not be generalizable to other centers. Furthermore, disease duration, comorbidity, use of drugs other than opioids, NSAIDs, acetaminophen, and psychosocial factors, including education level and occupation, were not examined. Finally, the sample size is not considered to be large enough for a genetic study, which increases the rate of false positives. Therefore, the current results should be treated with caution until the present study is repeated using a larger population sample.

In conclusion, within these limitations, these results indicate that the COMT genotype influences the outcome of morphine treatment; therefore, it may be useful as a predictive biomarker for morphine treatment. The frequency of the GG allele has been found to be around 20% in previous studies (11,12,25), but close to 50% in a study targeting Japanese patients (17). This demonstrates ethnic differences in allele frequencies. This observation supports that it is clinically meaningful not to choose morphine as the first opioid in G/G patients with cancer pain, as higher doses may be required. However, these findings are from a prospective observational study, and an interventional study is required for further evaluation.

Acknowledgements

The present study was supported by the Third-Term Comprehensive 10-Year Strategy for Cancer Control and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare (grant no. H22-037). The authors would like to thank Mrs. Tomoko Kitayama, Mrs. Erina Hatashita, Mrs. Kiyoko Kuwata, Mrs. Haruka Yamaguchi, Mr. Hiromasa Wadaguri and Mrs. Akiko Mizumoto for their technical assistance.

References