Investigating the Microbiome and Volatile Organic Compound Profile of Children With Neuroblastoma

Investigating the Microbiome and Volatile Organic Compound Profile of Children With Neuroblastoma - a Pilot Study

Background: Malignant tumors may lead to a catabolic state with loss of muscle and adipose tissue. The full picture of catabolism is termed cachexia and is associated with significant morbidity and mortality of cancer patients. Although the full picture is rarely observed up to 50% of children with cancer suffer from significant malnourishment. Additionally to tumor-induced catabolism, side-effects of chemotherapy may be problematic for the patients. In this regard up to 60% of children suffer from gastrointestinal mucositis presenting with nausea, vomiting, diarrhea or constipation and abdominal pain. In the worst case, mucositis may lead to bacterial translocation with life-threatening inflammatory response. Clinically this may require a reduction of the dosage or the number of chemotherapy cycles resulting in reduced effectivity. Up to now the therapy of mucositis is only symptomatic. Recent research of the applicant has shown a significant reduction of Lactobacilli in mice with neuroblastoma (a malignant childhood tumor). The dysbiosis was associated with catabolism, increased gut permeability and inflammation. Astonishingly, chemotherapy alone also leads to a significant reduction of Lactobacilli compared to sham mice, which may be linked to the development of mucositis clinically. Overall, the intestinal microbiome seems to play an essential role in the development of tumor-associated catabolism and chemotherapy-induced mucositis. Aim: The aim of this project is to determine if the changes in the intestinal microbiome observed in mice can also be seen in children with neuroblastoma. Methods: One part of the study will include 10 children with neuroblastoma (inclusion after verification of the diagnosis) and 10 healthy controls. The fecal microbiome will be determined by 16S-ribosomal deoxyribonucleic acid (rDNA) pyrosequencing. Volatile organic compounds in the breath will be sampled and measured by Gas Chromatography/Mass Spectroscopy. A basic science human work package will address the question if there are differences. In the second part serial investigations in children with neuroblastoma will assess whether or not these patients show alterations of the intestinal microbiome under chemotherapy.

Neuroblastoma group: Children with neuroblastoma enrolled after verification of diagnosis. Sampling before initiation of chemotherapy and under chemotherapy. Control group: healthy children without gastro-intestinal or pulmonary disease recruited from paediatric surgery.

10 children with neuroblastoma. Inclusion after verification of diagnosis and informed consent. Sampling of fecal microbiome (Initial microbiome, microbiome under chemotherapy, final microbiome), fecal volatile organic compounds (initial fecal volatile organic compounds, fecal volatile organic compounds under chemotherapy and final fecal volatile organic compounds) and breath organic volatile compounds (initial breath organic compounds, breath volatile organic compounds under chemotherapy and final breath volatile organic compounds). Samples will be taken after verifying diagnosis before initiation of chemotherapy, 1 week after completion of each cycle and 3 weeks after the end of chemotherapy.

10 children without gastro-intestinal or pulmonary disease as age and sex matched controls to the neuroblastoma group. Patients will be recruited from paediatric surgery. Inclusion after informed consent. Sampling of fecal microbiome (initial fecal microbiome), fecal volatile organic compounds (initial fecal volatile organic compounds) and breath organic volatile compounds (initial breath volatile organic compounds). Samples will be taken as age and sex matched controls for the neuroblastoma group. Sampling will be done once after obtaining informed consent.

Stool sampling for fecal microbiome analysis by 16S rDNA pyrosequencing. Neuroblastoma group and Control group.

Volatile organic compound analysis of stool samples by gas chromatography/mass spectroscopy Neuroblastoma group and Control group.

Breath sampling for organic compound analysis by gas chromatography/mass spectroscopy Neuroblastoma group and Control group.

Stool sampling under chemotherapy of children in neuroblastoma group (1 sample 1 week after completion of each chemotherapy cycle). Chemotherapy according to Société Internationale d´Onclogie Pediatrique Neuroblastoma Group (SIOPEN) guidelines

Stool sampling under chemotherapy of children in neuroblastoma group (1 sample 1 week after completion of each chemotherapy cycle). Chemotherapy according to SIOPEN guidelines. Neuroblastoma group

Breath sampling under chemotherapy of children in neuroblastoma group (1 sample 1 week after completion of each chemotherapy cycle). Chemotherapy according to SIOPEN guidelines. Neuroblastoma group

Difference of alpha and beta diversity, relative abundance of fecal bacteria at different levels (phylum, class, order, family and genus levels) between neuroblastoma and control group

Neuroblastoma group: within 48h after diagnosis, before initiation of chemotherapy. Control group: within 24h after obtaining informed consent.

Change of alpha and beta diversity, relative abundance of fecal bacteria at different levels (phylum, class, order, family and genus levels) under chemotherapy in the neuroblastoma group

Within 48h after diagnosis, before initiation of chemotherapy; 1 week after each chemotherapy cycle and 3 weeks after the end of chemotherapy.

Body weight (in kg) and height (in m) will be determined to calculate the body mass index (BMI in kg/m^2).

Neuroblastoma group: within 48h after diagnosis. Control group: within 24h after obtaining informed consent.

Body weight (in kg) and height (in m) will be determined to calculate the Body mass index (BMI in kg/m^2).

Within 48h after diagnosis, before initiation of chemotherapy; 7 days after completion of each chemotherapy cycle and 3 weeks after the end of chemotherapy.

Assessment of the mucositis score according to the WHO criteria (WHO handbook for reporting results of cancer Treatment; WHO Offset publication no 48) The score contains 5 subitems which are evaluated separately. At the end a total score is derived by adding the results of all items. Subitem 1: oral mucosa; range 0 (best) to 4 (worst) Subitem 2: nausea and vomiting; range from 0 (best) to 4 (worst) Subitem 3: diarrhea; range from 0 (best) to 4 (worst) Subitem 4: constipation; range from 0 (best) to 4 (worst) Subitem 5: abdominal pain; range from 0 (best) to 4 (worst)

Within 48h after diagnosis, before initiation of chemotherapy; 7 days after completion of each chemotherapy cycle and 3 weeks after the end of chemotherapy.

Neuroblastoma group: within 48h after diagnosis. Control group: within 24h after obtaining informed consent.

Neuroblastoma group: within 48h after diagnosis. Control group: within 24h after obtaining informed consent.

Within 48h after diagnosis, before initiation of chemotherapy; 1 week after each chemotherapy cycle and 3 weeks after the end of chemotherapy.

Within 48h after diagnosis, before initiation of chemotherapy; 1 week after each chemotherapy cycle and 3 weeks after the end of chemotherapy.

Inclusion Criteria: Age 2-8 years Neuroblastoma group: verified neuroblastoma Control group: absence of pulmonary or gastro-intestinal disease Written parental informed consent obtained Exclusion Criteria: Active gastro-intestinal or pulmonary disease Antibiotic or probiotic treatment within 3 weeks before sampling Negative parental informed consent