Lay Summary

As at end of May 2020, Italy is one of the country’s most affected by COVID-19 worldwide, with ~230k cases and ~34k deaths attributed to the virus. The Milan area, being one of the epicenters of the Italian epidemic, is experiencing significant pressure on its health system, impacting the lives not only of those directly affected by the disease but also the more vulnerable among the wider patient population, such as pediatric oncology patients. Although data on COVID-19 effects on the young are still sparse, evidence to date suggest that the COVID-19 disease is less severe in children than in adults and the reasons are not yet fully understood. Nevertheless, some groups can still be severely affected and experience serious lung diseases (for instance, children with co-morbidities and simultaneous respiratory infections, or infants without mature immune systems). This project is an experimental research aimed at studying the microbiome of COVID-19 positive  pediatric brain cancer patients and identifying characteristics that can positively or negatively influence the course of brain tumor disease in COVID infected pediatric patients to: 1) define interventions impairing ecological environment favorable for transmission; 2) develop diagnostics and treatment strategies, with specific focus on vulnerable pediatric brain cancer patients.

At time of writing the present proposal, it’s impossible to foresee the course of SARS-CoV-2 pandemic infection; different scenarios stresses that pandemic is likely to last between 18 and 24 months thought  a series of repetitive waves. The present project will provide information to handle pediatric brain tumor patients in the new COVID-19 era.

 

 

Executive Summary

Background

 

Coronavirus disease 2019 or Covid-19 is a recent disease originated in China in late 2019, by now spreads to many countries in the world (Wang, 2020).  The agent of this disease is a betacoronavirus, named SARS-Cov-2 (Lake, 2020). This virus causes a severe acute respiratory syndrome thanks to the binding to angiotensin converting enzyme 2 (ACE-2) receptors present in the alveolar epithelial cells of lung (Magrone, 2020). Interestingly, ACE-2 receptors are also reported to be expressed by gastrointestinal cells facilitating viral entry into the tissue (Zang, 2020). The SARS-Cov-2 RNA can be detected in stool of some patients of Covid-19 for a mean of 11-12 days after the respiratory tract became negative, this means that SARS-Cov-2 might have an impact on the gut microbiota, and in turn, this one might influence the course of this disease (Wu , 2020; Dhar, 2020).

 

Hypothesis

 

The gut microbiota plays a key role in health through its protective, trophic and metabolic actions. It is known that gut microbiota affects the development, behavioral and cognitive functions of the brain (Jenkins, 2016; Diaz, 2011). Taken together, it can be suggested that since the gut microbiota play an important role in immunity, infection caused by  SARS-Cov-2 need to be properly studied in relation to the interaction between the gut microbiota-virus infection and immune response that in turn could modulate the response to radiotherapy in DIPG patients.  Our translational hypothesis is that specific microbiota features can positively or negatively influence the course of COVID infection in pediatric brain cancer patients influencing, in turn, the course of the disease.

 

Goals

 

Provision of uninterrupted and effective pediatric cancer care faces numerous challenges: most childhood cancers are aggressive, necessitating urgent treatment, most children are treated in international collaborative trials and increasingly pediatric tumors are prioritized for treatment with proton therapy, which may entail travelling some distance.

The purpose of the study is to understand characteristics and microbiome profiles of young patients that can positively or negatively influence the course of COVID infection, with focus on pediatric brain cancer patients. Evidence from the study will contribute to the body of research driving practical actions with direct impact on COVID-19 relief for the more vulnerable group of children and young, specifically by:

1) to define the transmission ways and develop diagnostic strategies;

2)  to disclose the ecology in microbiota communities associated to the virus;

3) to understand the microbiota profiles in asymptomatic, pre-symptomatic, and symptomatic residents, at different ages;

4) to define potential diet or pro-biotics interventions impairing ecological environment favorable for transmission.

5) to define ecological environment favorable for transmission within same family members

 

Clinical significance

 

The pandemic is impairing the capacity of the health system to deliver the expected treatment modalities to cancer patients due to shortages of personnel, reduced availability of protective equipment or difficulties in handling with infected children and their caregivers. As in natural disasters, sudden lack in resources may also occur following machine failures, including radiotherapy or particle beam facilities. Limited radiotherapy resources are a major obstacle in improving outcomes in low- and middle-income countries where more than two-thirds of pediatric cancers are diagnosed. Guidelines have to be issued to provide continuous cancer care without increasing the risk of COVID infection and to assess the impact of the infection in the outcome of pediatric brain patients.

 

Experimental design

 

The present proposal is an experimental/translational project to explore the microbiota in COVID-19 positive and control-matched  COVID-19 negative pediatric brain cancer patients.  Adult subjects, such as parents or caregivers will be included to monitor the ways of infection spread. The study will include the following populations to be enrolled:

A) pediatric brain cancer patients having confirmed SARS-Cov-2 positive infection;

B) pediatric brain cancer patients  with SARS-Cov-2 negative status as controls;

C) adult subjects, mainly being children parents or any other caregiver

Covariates (i.e. concomitant diseases, gender, age) will be balanced between the covid-19 positive and negative groups. The comparison between the two groups will provide invaluable information on Covid-19 relief.

 

Methods

 

The project will exploit state-of-the-art in metagenomic profiling which will be able to identify not only bacterial but also viral/fungal genetic material. This data will be analysed in conjunction with detailed clinical data including the treatment and the disease course.

Nasopharyngeal swab and stool specimens will be collected by using dedicated devices. The tubes will contain the DNA/RNA Shield (Zymo), a buffer preserving the genetic integrity and microbiota composition of samples at room temperatures but completely inactivating infectious agents. DNA will be extracted by QIAsymphony DSP Virus / Pathogen Midi kit (Qiagen). DNA will be quantified using a Qubit fluorimeter (ThermoFisher), while quality will be assessed using a 4200 TapeStation (Agilent).

The genome of the SARS-CoV-2 comprises a replicase complex (ORF1ab), Spike gene (S gene), E gene, M gene, N gene, and several unidentified non-structural open reading frames (Khailany, 2020). Since the outbreak of SARS-CoV-2 infection, real-time reverse transcription-polymerase chain reaction (RT-qPCR) assays are the gold standard for clinical diagnosis (Atzrodt, 2020).

Bacterial 16S rRNA genes contain nine hypervariable regions (V1-V9) that will be used as species-specific signature sequences to identify the different microbes present in a sample (Chakravorty, 2007). After DNA extraction, the libraries will be performed with 16S Metagenomics kit (ThermoFisher), which uses bacterial primers containing sequencing adapters and unique sample-specific sequences.

 

Feasibility

 

The feasibility is ensured by the expertise of the team and collaborators involved in the research project.

Dr. Maura Massimino’s research is particularly focused at the study of brain tumors in pediatric age. She has been the PI of two protocols for the diagnosis and treatment of pediatric ependymoma for the Associazione Italiana Ematologia e Oncologia Pediatrica (AIEOP) and she is particularly involved in the activities of the Brain Tumor Subcommittee. Since November 2015 she has been coordinator of the AIEOP Working Group for Childhood Central Nervous System Tumors. She designed and coordinated a study for DIPG patients whose results are, to date, the best published in the field. Together with Dr Biassoni, she is on the Executive Committee of the SIOP DIPG Registry.

The experimental activities will be overseen by Dr, Loris De Cecco, PhD who has scientific and appropriate background, knowledge and skills in the specific field of interest. To manage the multidisciplinarity of the project, a hierarchical organisation scheme will be structured, assigning responsibilities at the different levels of decision and operational intervention.

The team also includes L. Boschetti, PhD, will manage grants, and legal and ethical issues.

The project will be supported by Dr. M.F. Iannò (Biologist) for the experimental activities; Dr. A. Carenzo (Bioinformatician) for data processing and analysis; E. Schiavello, MD - oncologist – that will manage clinical treatments and appropriate sample collection.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest