Methotrexate is a cornerstone in the treatment of childhood leukemia. When given in high-dose as part of the initial phase of treatment, gastrointestinal toxicity is a known problem. However when children reach maintenance treatment with low-dose methotrexate this is not described as a significant challenge. Children with juvenile idiopathic arthritis are another patient group receiving low-dose methotrexate. Among these patients gastrointestinal intolerance is such a significant problem that treatment may be ceased. The aim of this project is to create a greater understanding of gastrointestinal intolerance associated to low-dose methotrexate treatment by investigating the differences between these two patient groups, investigating genetic and psychological factors.

Background:

Childhood leukemia (Acute Lymphoblastic Leukemia, ALL) is the most frequent malignancy in the pediatric population (1). Survival rates have reached above 85% due to advances in chemotherapy. High-dose methotrexate (MTX) is a mainstay in the treatment of ALL (2-4). Gastrointestinal toxicity is a well known challenge associated to high-dose MTX treatment (5). However this is not described as a significant problem among these children when they reach maintenance treatment and receive low-dose MTX treatment. Children with Juvenile Idiopathic Arthritis (JIA) also receive low-dose MTX treatment. Among these patients MTX induced gastrointestinal intolerance is a significant problem. Studies have shown that more than half of the JIA patients have problems tolerating MTX because of nausea which may lead to cessation of treatment (6,7). It is largely unknown why MTX causes nausea but both pharmacogenetic and psychological factors are thought to play a role (8).

JIA is the most common chronic pediatric rheumatic disease. Without treatment the disease causes significant short- and long-term disability and quality of life impairment. Prognosis has greatly improved within recent years as a result of substantial progress in disease management and MTX is a mainstay in the treatment of JIA (9-13).

The aim of this project is to create a greater understanding of MTX induced gastrointestinal intolerance among children with leukemia and children with JIA. The project will focus on nausea and investigate the level of nausea in the two patient groups and compare if a difference does exist. The investigators will further investigate pharmacogenetic and psychological factors and their relation to the patients' level of nausea.

Pharmacogenetic background:

MTX is absorbed from the gastrointestinal tract and transported to the bloodstream by transporter proteins in the enterocytes. Elimination of MTX is primarily renal but a percentage of MTX passes through enterohepatic circulation first (14,15). Studies on the use of high-dose MTX treatment to children with ALL suggest that fast hepatic clearance of MTX increases the degree of gastrointestinal toxicity. It has further been shown that patients with mutations (Single Nucleotide Peptides, SNPs) in the gene encoding the hepatic transporter protein SLCO1B1, responsible for transporting MTX into the liver, have less gastrointestinal toxicity to high-dose MTX (16). The transporter protein encoded by SLC19A1 is placed both in the liver and the enterocytes (15).

Studies on adult patients diagnosed with either psoriasis or rheumatoid arthritis and treated with low-dose MTX, and studies on ALL patients in high-dose MTX show that SLC19A1 is associated to the concentration of MTX in plasma and erythrocytes as well as MTX-related toxicity (1,17-20). Transporter proteins that transport MTX out of the liver and into the bile ducts are encoded by ABCC2 and ABCB1(15). Studies on adult patients with rheumatoid arthritis in low-dose MTX, adults with Chronic Myeloid Leukemia and studies on ALL patients in high-dose MTX have shown that both genes are pharmacokinetically important and ABCC2 has further been shown associated to MTX-related gastrointestinal toxicity (5,21-24). Previous studies on patients with rheumatoid arthritis, childhood leukemia and JIA have shown that the frequency of clinically relevant SNPs in genes encoding MTX-related transporter proteins are: SLCO1B1 (70.3%), ABCB1 (20.6%), ABCC2 (56.8%), SLC19A1 (31%) (5,16,17,19,21,25-28).The investigators therefore believe it conceivable to find the selected genetic polymorphisms for MTX-related transporter proteins in the participating study population.

Pharmocogenetic Aims:

Investigate if MTX-related gastrointestinal intolerance is associated to the enterohepatic circulation. Specifically, to determine if SNPs in genes encoding MTX-related liver transporter proteins are associated to the level of drug induced gastrointestinal side effects.

Pharmacogenetic Hypotheses:

Patients with SNPs in genes encoding MTX-related transporter proteins in the liver have a lower level of enterohepatic circulation of MTX and thus have a lower level of nausea than other patients.

Psychological Background:

Studies on adult cancer patients have shown that psychologically based nausea occurring after treatment with chemotherapy is a possible explanation for the nausea which is out of proportion with the medicine's emetogenic potential. It is also a possible explanation for the great variation in the level of cancer patients' nausea after the same type of chemotherapy (29,30). Studies on children in chemotherapy for different cancers have shown that psychological factors, such as coping strategies and anxiety, affect the degree of chemotherapy associated nausea and vomiting (31-33). The investigators want to investigate if this is also the case for children with leukemia in maintenance treatment with low-dose MTX as well as JIA patients in low-dose MTX-treatment.

Psychological aims:

To investigate if the nausea-coping strategies and anxiety level of the study population are associated to the degree of MTX-induced gastrointestinal intolerance. And investigate if there is a difference between the two patient groups.

Psychological hypotheses:

Insufficient psychological coping strategies and high level of anxiety may cause psychologically based nausea. Thus patients with a high level of anxiety and insufficient coping strategies will have a higher degree of nausea to MTX treatment than other patients.

Perspectives This project will contribute to a greater understanding of MTX-induced intolerance in children with leukemia and JIA. This will also be beneficial for all other children who receive low-dose MTX treatment. The project will optimize the maintenance treatment of childhood leukemia and the treatment of JIA by using patient SNP genotypes to determine who can tolerate MTX and who cannot. Additionally, identify patients where psychological intervention may diminish MTX-induced nausea.

Materials and methods

The study population comprises:

1. Children diagnosed with ALL in maintenance treatment
2. Children diagnosed with JIA according to ILAR criteria

For inclusion/exclusion criteria see under "Eligibility Criteria". For each patient a list of disease specific characteristics will be noted. The level of MTX-induced intolerance is determined by an electronic "nausea-diary" for four weeks, containing a "faces of nausea scale" based on a "faces of pain scale" (34). The gastrointestinal side effects will further be evaluated by a Methotrexate-Intolerance-Severity-Score questionnaire (MISS) (35).

Pharmacogenetic methods:

Blood samples on each patient will be used to determine the prevalence of SNPs in genes encoding MTX-related transporter proteins (genes including: SLCO1B1, SLC19A1, ABCC2, ABCB1). The genetic analysis will be performed at the Institute of Biomedicine, Aarhus University. Genotyping are carried out using the Sequenom MassARRAY Genotyping system (Sequenom, San Diego, CA). In brief multiplex PCR are performed in 5 μl reactions containing 10 ng of genomic DNA, 1.25 x PCR buffer, 0.5 mM dNTP, 100nM of each primer and 0.5U Taq polymerase using standard cycling conditions. The PCR products are then treated with SAP and the probe extension reaction (iPLEX) carried according to the iPLEX standard protocol (Sequenom). The desalted samples are analyzed using a Bruker matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer (Sequenom) and the genotypes determined using the MassARRAY Typer 3.4 software (Sequenom). The blood sample will also be used to measure the concentration of MTX-polyglutamates in the erythrocytes (Ery-MTX-PG). The measurement of Ery-MTX-PG will be performed by HPLC-technique at the children's oncology lab, Rigshospitalet, Copenhagen, Denmark.

2) Psychological methods: The patients' coping strategies will be investigated using a nausea-coping-questionnaire developed from a pain-coping questionnaire, which has specifically been developed for children and previously been translated to Danish, validated and used in earlier studies. It consists of eight subscales of main coping strategies: Information seeking, problem solving, seeking social support, positive self-statements, behavioral distraction, cognitive distraction, externalizing and internalizing/catastrophizing (36,37,39,40). The patients' level of anxiety will be assessed by "Beck Youth Inventories" (BYI), which previously have been translated into Danish and validated. The BYI is brief and easy to use. It consists of 20 sentences that investigate fear, worrying and physiological symptoms associated with anxiety (38).

Statistics Power calculation: This is based on the primary target measure (the degree of nausea) quantified by the "faces of nausea scale" in the nausea diary. The scale has the end points: "no nausea" and "extreme nausea", in numbers 0 to 5. Results from an earlier study using the faces of pain scale show that mean scores were 0.74 (SD=0.70) and 0.91(SD=0.86) (36). From a clinical assessment the investigators expect the mean nausea score for the group of JIA patients to be 2.5 and 1 for the ALL patients. The standard deviation is expected to be similar to the study above (SD=0.9). If the level of significance (alpha) is 0.05, the power is 80% and the ratio between the group sizes is 2, then the calculated size of the study population will be 10 patients in the JIA group and 5 in the ALL group. If the mean nausea score only is 2 for the group of JIA patients, but still 1 for the group of ALL patients, and the standard deviation is greater than expected, SD=2. But alpha still is 0.05, the power is 80%, and the ratio between the group sizes is still 2. Then the calculated size of the study population will be 96 patients in the JIA group and 48 in the ALL group.

Research Plan

The research group consists of:

From Department of Pediatrics, Aarhus University Hospital Skejby:

Main applicant: Nini Kyvsgaard Brix Nørgaard, PhD-student (enrolled at Aarhus University), M.D.

Main supervisor: Troels Herlin, Professor, DMSc; Co-supervisor: Torben Stamm Mikkelsen, MD, PhD.

From Institute of Biomedicine, Aarhus University:

Co-supervisor: Thomas Juhl Corydon, Associate Professor, PhD, Deputy Head of Institute for Biomedicine, Aarhus University.

From Institute for Psychology, Aarhus University:

Co-Supervisor: Mikael Thastum, Professor, PhD, Head of Clinical Psychology, Aarhus University.

The PhD-student will perform and coordinate the daily execution of the project with guidance from the group of supervisors. The first and second year of the project will be dedicated to the collection of patient data and blood samples. Firstly, the inclusion of the JIA patients will commence. Then the inclusion of the ALL patients. The third year will be dedicated to analysis of gene polymorphisms and MTX levels, data analysis and compilation of manuscripts for publication. The research group has extensive research experience and clinical expertise within this area.

Feasibility The laboratory facilities to take the blood samples and carry out the initial handling of them are available. The final analysis will be performed as specified under 'pharmacogenetic methods'. The investigators have access to software to perform the questionnaires electronically, as well as access to statistical support.

Inclusion Criteria:

• children diagnosed with JIA according to ILAR criteria, whom are followed at the departments of pediatrics at Aarhus University Hospital Skejby and Odense University Hospital.
• and children diagnosed with ALL, whom are followed at the departments of pediatrics at Aarhus University Hospital Skejby, Aalborg University Hospital, Odense University Hospital and Rigshospitalet.
• aged 9 years and above
• currently treated with low-dose MTX for at least six weeks

Exclusion Criteria:

• Children with cognitive difficulties will be excluded
• Children without ability to speak Danish will be excluded.

see under project - and group description