Sterile Inflammation and Molecular Aberrations in MDS

Study Overview
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Brief Summary
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The objective of this study is the description of the possible association between genetic mutation/aberration profiles, inflammatory tonus and clinical phenotype based on PROMs and HRQoL. Apart from gaining a better understanding of the causal correlation between genetics, sterile inflammatory processes and QoL (e.g. fatigue) in MDS, this study is supposed to identify potential novel biomarkers and, ultimately, therapeutic targets.

Official Title
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Sterile Inflammation and Molecular Aberrations in Myelodysplastic Syndrome: Determinants of Quality of Life and Vulnerability

Conditions
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Myelodysplastic Syndromes

Intervention / Treatment
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* Diagnostic Test: Next Generation Sequencing
* Diagnostic Test: Tumorimmunological examinations - multiplex assays/quantitative polymerase chain reaction
* Diagnostic Test: flow cytometry
* Diagnostic Test: Metagenomics of stool samples
* Diagnostic Test: Clinical/demographic data
* Other: Elicitation of the HRQoL


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Female and male patients > 18 years MDS, MDS/MPN diagnosis based on current WHO classification. CCUS and CHIP defined by Valent (Valent, Oncotarget, 2018) and by Stauder (Stauder, Blood, 2018) Signed and dated declaration of consent by the patient according to ICH-GCP Guidelines Exclusion Criteria: Any other illness, whether physical or mental, or any laboratory abnormalities which prevent a declaration of consent by the patient Patients with an acute and/or uncontrolled infection, including patients that are afebrile under treatment with antibiotic/antifungal/antiviral prophylactic medication Any pre-existing autoimmune disease requiring a systemic immunosuppression Steroid therapy (>10mg Prednison/day or equivalent), regardless of its necessity up to 4 weeks before inclusion in the study Anamnestic and/or current therapy with hypomethylating agents (HMA) or immunomodulatory imide drugs (IMiDs) Status post allogenic stem cell transplantation Previous or ongoing chemotherapy Pregnancy or breastfeeding period

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| MDS<br>Female and male patients aged 18 years and older MDS, MDS/MPN diagnosis based on current WHO classification. CCUS and CHIP defined by Valent (Valent, Oncotarget, 2018) and by Stauder (Stauder, Blood, 2018) | Diagnostic Test: Next Generation Sequencing<br>* Sequencing of patient samples will be performed in the facilities of ZIMCL. Following DNA extraction, the sequencing of granulocytes as well as of lymphocytes (control) will be carried out. IN addition to whole exome sequencing, a panel from SOPHIA GENETICS (which is also available for routine diagnostics) will be applied including the following MDS specific genes: ASXL1, BRAF, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, HRAS, IDH1, IDH2, KRAS, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, ZRSR. Each patient sample will be bulk sequenced, meaning that relevant mutations are detected down to an allele frequency of 2%.<br>Diagnostic Test: Tumorimmunological examinations - multiplex assays/quantitative polymerase chain reaction<br>* Inflammasome activation is quantified by analyses of inflammasome-associated cytokine patterns. Multiplex assays for the quantification of the inflammasome-specific cytokines will be done from serum as well as from supernatants the stimulated blood cells. Cytokine quantification is carried out with Luminex FlexMap 3D. Serum cytokine levels will be quantitated in parallel. Quantification of RNA expression levels of inflammasome-related gene products will be performed by qPCRs from unstimulated and stimulated (=cryotube) blood cells. The necessary RNA extraction will be performed using a RNA extraction kit.<br>Diagnostic Test: flow cytometry<br>* A detailed evaluation of the individual immune status is being conducted by the analysis of two specialized panels: Panel A provides a broad overview over various immune cell populations, while Panel B identifies T-cell sup-populations.<br>Diagnostic Test: Metagenomics of stool samples<br>* DNA will be extracted from frozen fecal samples applying a bead-beating method using a GNOME DNA Isolation Kit (MP Biomedicals). DNA quality will be assessed using an Agilent 4200 TapeStation (Agilent Technologies). After final precipitation, DNA samples will be re-suspended in TE buffer and stored at -80 °C for further sequencing analysis. To this end, sequencing libraries will be generated using a Nextera XT DNA Sample Prep Kit (Illumina). Library quality will be confirmed using an Agilent 4200 TapeStation. Whole-genome shotgun sequencing of fecal samples will be carried out on a HiSeq2500 platform (Illumina).<br>Diagnostic Test: Clinical/demographic data<br>* Demographic and clinical data include: age, age at initial diagnosis, sex, diagnosis, actual comorbidities, medication at inclusion in study, cytogenetic and molecular profiles and standard laboratory parameters (blood count, differential leukocyte count, biochemistry, iron status, inflammatory markers like CRP, albumin, fibrinogen).<br>Other: Elicitation of the HRQoL<br>* Evaluation of HRQOL, of functional activities and of performance status will be done by the patient and/or the physician using validated scores.<br>|
| control<br>age-matched healthy persons | Diagnostic Test: Next Generation Sequencing<br>* Sequencing of patient samples will be performed in the facilities of ZIMCL. Following DNA extraction, the sequencing of granulocytes as well as of lymphocytes (control) will be carried out. IN addition to whole exome sequencing, a panel from SOPHIA GENETICS (which is also available for routine diagnostics) will be applied including the following MDS specific genes: ASXL1, BRAF, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, HRAS, IDH1, IDH2, KRAS, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, ZRSR. Each patient sample will be bulk sequenced, meaning that relevant mutations are detected down to an allele frequency of 2%.<br>Diagnostic Test: Tumorimmunological examinations - multiplex assays/quantitative polymerase chain reaction<br>* Inflammasome activation is quantified by analyses of inflammasome-associated cytokine patterns. Multiplex assays for the quantification of the inflammasome-specific cytokines will be done from serum as well as from supernatants the stimulated blood cells. Cytokine quantification is carried out with Luminex FlexMap 3D. Serum cytokine levels will be quantitated in parallel. Quantification of RNA expression levels of inflammasome-related gene products will be performed by qPCRs from unstimulated and stimulated (=cryotube) blood cells. The necessary RNA extraction will be performed using a RNA extraction kit.<br>Diagnostic Test: flow cytometry<br>* A detailed evaluation of the individual immune status is being conducted by the analysis of two specialized panels: Panel A provides a broad overview over various immune cell populations, while Panel B identifies T-cell sup-populations.<br>Diagnostic Test: Metagenomics of stool samples<br>* DNA will be extracted from frozen fecal samples applying a bead-beating method using a GNOME DNA Isolation Kit (MP Biomedicals). DNA quality will be assessed using an Agilent 4200 TapeStation (Agilent Technologies). After final precipitation, DNA samples will be re-suspended in TE buffer and stored at -80 °C for further sequencing analysis. To this end, sequencing libraries will be generated using a Nextera XT DNA Sample Prep Kit (Illumina). Library quality will be confirmed using an Agilent 4200 TapeStation. Whole-genome shotgun sequencing of fecal samples will be carried out on a HiSeq2500 platform (Illumina).<br>Diagnostic Test: Clinical/demographic data<br>* Demographic and clinical data include: age, age at initial diagnosis, sex, diagnosis, actual comorbidities, medication at inclusion in study, cytogenetic and molecular profiles and standard laboratory parameters (blood count, differential leukocyte count, biochemistry, iron status, inflammatory markers like CRP, albumin, fibrinogen).<br>Other: Elicitation of the HRQoL<br>* Evaluation of HRQOL, of functional activities and of performance status will be done by the patient and/or the physician using validated scores.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Definition of correlation between molecular aberrations and the sterile inflammatory tonus |  | baseline | 
| Definition how genetic aberrations and associated sterile inflammation impacts on health-related quality of life (HRQoL, e.g. fatigue) and functional activities in patients with MDS, MDS/MPN, or CHIP/CCUS |  | baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Definition of correlation of sterile inflammatory tonus with clinical variables (e.g. progression to secondary acute myeloid leukemia (sAML), complications (e.g. infections), and survival) |  | baseline | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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anemia, health-related quality of life, inflammation