Primary MyelofibrosisNCT06896344
Role of Mesenchymal Stromal Cell Derived Extracellular Vesicles of Primary Myelofibrosis Patients on CD34+ Cells
An observational study of Primary Myelofibrosis, run by Fondazione IRCCS Policlinico San Matteo di Pavia at 1 site in Italy.
Trial summary
The aim of this study is to demonstrate that, in patients with myelofibrosis (PMF), a chronic neoplastic disease of the bone marrow, some of the cells that form the bone marrow microenvironment (mesenchymal stromal cells, MSCs) can send wrong messages to the CD34+ hematopoietic progenitor cells (HPCs). Read more about this study
- Who is it for?
- Ages 18 years and olderHealthy volunteers can take part
- Conditions the study is for
- Primary Myelofibrosis
- What is being tested?
- In vitro culture
- How many people
- 10 participants
- Where
- 1 siteItaly
- Study dates
- 11 Jan 2024to Aug 2025
- Phase
- Observational
- Are there results?
- Not yet: the study is under way
Key requirements
Who can take part, as listed by the sponsor. Only the study team can confirm whether you are eligible, so take this list to your doctor.
- Ages 18 years and older
- All sexes
- Healthy volunteers accepted
- Adult, Older adult
Study population: PMF (WHO 2016) afferent at the Center for the Study of Myelofibrosis, a national center that receives patients from all the country. Patients will be out of therapy, undergoing bone marrow biopsy for clinical reasons.
Who may be able to join
- Patients with PMF
- JAK2 V617F+ (either homozygous or heterozygous)
- Out of therapy
- At least 18 years old
Who cannot join
- PMF combined to other pathologies
- Previous splenectomy
- Previous bone marrow transplant
Trial locations and contacts
1 site in 1 country. Contact the study team or a site near you to ask about taking part.
Italy1 site, 1 recruiting
- Recruiting
Fondazione IRCCS Policlinico San Matteo di Pavia
Pavia, Pavia, 27100
Margherita Massa, +39 0382502703, m.massa@smatteo.pv.it
About this study
Official title: Microenvironment Cross-talk: Role of Extracellular Vesicles Derived From Mesenchymal Stromal Cells of Patients With Primary Myelofibrosis in Modifying Functional Activities of CD34+ Hematopoietic Progenitor Cells
The aim of this study is to demonstrate that, in patients with myelofibrosis (PMF), a chronic neoplastic disease of the bone marrow, some of the cells that form the bone marrow microenvironment (mesenchymal stromal cells, MSCs) can send wrong messages to the CD34+ hematopoietic progenitor cells (HPCs). CD34+ HPCs in normal conditions reside in the bone marrow until complete maturation; in PMF they leave the bone marrow when they are still progenitor cells and frequently they express the V617F mutation on the JAK2 molecule. In previously published papers, the investigators demonstrated that the MSCs in the bone marrow of patients with PMF are different from those in healthy donors. In this study the investigators want to verify how this difference affects the maturation of CD34+ HPCs of healthy donors and of SET2 cells, a commercially available cell line characterized by the V617F mutation of the JAK2 molecule. In particular, the investigators will verify the effect of MSCs on signals that induce the activation of CD34+ cells, their survival or death, the production of harmful oxidative reagents and their clonal capacity. To obtain these data, the investigators will isolate MSCs from bone marrow blood samples of patients with PMF undergoing bone marrow biopsy for clinical reasons and from healthy subjects, donors of HPCs for transplant. Following a long culture MSCs will be stimulated to release small vescicles containing part of their nuclear and cytoplasmatic molecules. These vescicles will be cultured with CD34+ cells or SET2 cells and their effects will be evaluated. It has been shown that these small vescicles act exactly like MSCs, but they are easier to be used, and could become a very useful acellular drug in the near future.
Read the detailed description
Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by the clonal proliferation of CD34+ progenitor cells. A constitutive activation of the JAK-STAT pathway, due to an acquired gain of function mutation either in JAK2, MPL or CALR genes, plays a relevant pathogenetic role. Moreover, a state of chronic inflammation affects the disease course. Currently, the therapy with the JAK inhibitor Ruxolitinib ameliorates the symptoms of the disease, with no effects on the pathological cell clone; hematopoietic progenitor cell (HPC) transplantation is the only curative option. The investigators previously published that mesenchymal stromal cells (MSCs) from patients with PMF display functional and genetic abnormalities (low proliferative potential, precocious senescence, reduced differentiation potential). Regarding Ruxolitinib, direct effects on MSCs have been described, such as the inhibition of the JAK-STAT signaling, the growth of both healthy donor (HD)- and PMF-MSCs, and the decrease of fibrosis related gene expression. Ruxolitinib has also been reported to reduce MSC secretion of inflammatory cytokines MCP-1 and IL-6, suggesting a role of this drug in modifying the bone marrow (BM) microenvironment. In PMF, as in other hematological malignancies, MSCs participate in the communication between microenvironment and CD34+ cells. Recent studies demonstrated in other pathologies that MSC-derived extracellular vesicles (EVs) and parental MSCs have similar effects. In fact EVs, containing mRNA, microRNA, lipids, and proteins can be incorporated into recipient cells and modify their functions. In normal hematopoiesis, MSC-EV incorporation in CD34+ cells has been shown to induce an activation of the JAK-STAT pathway through increased levels of phospho-STAT5; in addition, a significant decrease in apoptosis and a higher colony-forming unit granulocyte/macrophage clonogenic potential have been reported.
The objectives of this study are:
- to isolate MSCs from BM of PMF patients and HDs. MSCs will be incubated with/out Ruxolitinib. EVs will be obtained following MSC starvation and ultracentrifugations, and stored at -80°C
- to assess EV effects on HD-CD34+ or JAK2V617F+ SET2 cells by evaluation of apoptotic and activation pathways, ROS production and clonogenic capacity. To this regard, confirmatory experiments will be performed with JAK2-mutated CD34+ cells from patients receiving or not Ruxolitinib.
- to perform gene expression profile on CD34+ and SET2 cells after incubation with EVs.
- to investigate EV biocargoes both qRT-PCR and proteomics will be performed on MSC-derived EVs, before and after incubation with Ruxolitinib.
The expected impact will include the identification of modifications in functional activities of CD34+ or SET2 cells following incubation with PMF-MSC-EVs with respect to HD-MSC-EVs, reflecting the effect of an altered microenvironment on HPCs. The investigators also expect the recognition of important targets of severity/progression of the disease by assessing the gene expression profile in CD34+ or SET2 cells that incorporate EVs from different sources.
In addition, the identification of the Ruxolitinib effects on PMF BM microenvironment may be clarified.
This study may allow to act on new targets of disease progression or to support the future use of HD-EVs as an acellular therapy that favours the survival of healthy CD34+ cells or acts against their clonal counterpart.
- Conditions
- Primary Myelofibrosis
- Being tested
- In vitro culture
- Keywords
- Primary Myelofibrosis, CD34, extracellular vesicles
- Medical subject headings
- Primary Myelofibrosis
What participants receive
What participants receive. In a randomised study each person is assigned to one group by chance.
In vitro cultureOther
In vitro culture of bone marrow MNCs to obtain mesenchymal stromal cells
| Study group | Role | What they receive |
|---|---|---|
| Patients with PMF out of therapy undergoing bone marrow biopsy for clinical reasons | Healthy subjects who donate bone marrow hematopoietic progenitor cells Receives: Other: In vitro culture |
What is being measured
The primary outcome is the main question the study is designed to answer.
Effect of bone marrow microenvironment, resembled by EVs, on hematopoietic progenitor cells
The measure of the functional activities of HD-CD34+ cells induced by the bone marrow microenvironment (i.e. MSC derived EVs) of patients with PMF or heathy donors. P-STAT5 molecule as activation index, Bax/Bcl2 ratio as apoptosis indicator, ROS and membrane antigens will be assessed by a unique unit of measure, the mean fluorescence intensity. The change of the different parameters from baseline to the end of the co-culture will be reported.
Measured: Up to 24 weeks
Secondary outcomes (1)
Components of EV biocargoes
Molecular analysis of EV biocargoes derived from either PMF or healthy MSCs
Measured: Through study completion, an average of 1 year
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted26 Mar 2025
- Study start11 Jan 2024
- Primary completionJun 2025 (estimated)
- Study completionAug 2025 (estimated)
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Publications (2)Papers the sponsor has linked to this study.
- Other trials for Primary MyelofibrosisStudies of the same condition, open and under way.
- More trials run by Fondazione IRCCS Policlinico San Matteo di PaviaThe sponsor's other registered studies.
- What the trial phases meanHow a treatment moves from first tests in people to approval.
More details
For doctors and researchers: how the study is designed, who runs it, and its registry IDs.
Study design
- Study type
- Observational
- Observational model
- Case-control
- Time perspective
- Prospective
- Enrolment
- 10 participants (estimated)
Sponsor and investigators
- Lead sponsor
- Fondazione IRCCS Policlinico San Matteo di Pavia
- Sponsor type
- University, hospital or other
- Responsible party
- Principal investigator, Margherita Massa, Fondazione IRCCS Policlinico San Matteo di Pavia
- Investigators
- Margherita Massa, PhD, Principal investigator, IRCCS Policlinico San Matteo Foundation
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT06896344
- Sponsor's protocol ID
- Microenvironment cross-talk
- Other IDs
- 08052022
- Status last verified
- Mar 2025
- First submitted
- 6 Mar 2025
- Last updated
- 2 Apr 2025
- Results posted
- No
- Data monitoring committee
- No
- FDA-regulated drug
- No
- FDA-regulated device
- No
- Individual data sharing
- No
Publications
Zacharaki D, Ghazanfari R, Li H, Lim HC, Scheding S. Effects of JAK1/2 inhibition on bone marrow stromal cells of myeloproliferative neoplasm (MPN) patients and healthy individuals. Eur J Haematol. 2018 Jul;101(1):57-67. doi: 10.1111/ejh.13079. Epub 2018 Apr 30.
PubMed 29645296Preciado S, Muntion S, Corchete LA, Ramos TL, de la Torre AG, Osugui L, Rico A, Espinosa-Lara N, Gastaca I, Diez-Campelo M, Del Canizo C, Sanchez-Guijo F. The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability. Stem Cells. 2019 Oct;37(10):1357-1368. doi: 10.1002/stem.3032. Epub 2019 Jun 11.
PubMed 31184411
Study at a glance
From the registry record, last updated by the sponsor on 2 Apr 2025. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- Microenvironment Cross-talk: Role of Extracellular Vesicles Derived From Mesenchymal Stromal Cells of Patients With Primary Myelofibrosis in Modifying Functional Activities of CD34+ Hematopoietic Progenitor Cells
- ClinicalTrials.gov ID
- NCT06896344
- Acronym / sponsor ID
- Microenvironment cross-talk
- Conditions
- Primary Myelofibrosis
- Keywords
- Primary Myelofibrosis, CD34, extracellular vesicles
- Study type
- Observational
- Recruitment status
- Recruiting
- Enrolment
- 10 participants (estimated)
- Lead sponsor
- Fondazione IRCCS Policlinico San Matteo di PaviaUniversity, hospital or other
- Study start
- 11 Jan 2024 (actual)
- Primary completion
- Jun 2025 (estimated)
- Study completion
- Aug 2025 (estimated)
- First posted
- 26 Mar 2025 (actual)
- Results
- Not posted in the source record
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
The aim of this study is to demonstrate that, in patients with myelofibrosis (PMF), a chronic neoplastic disease of the bone marrow, some of the cells that form the bone marrow microenvironment (mesenchymal stromal cells, MSCs) can send wrong messages to the CD34+ hematopoietic progenitor cells (HPCs). CD34+ HPCs in normal conditions reside in the bone marrow until complete maturation; in PMF they leave the bone marrow when they are still progenitor cells and frequently they express the V617F mutation on the JAK2 molecule. In previously published papers, the investigators demonstrated that the MSCs in the bone marrow of patients with PMF are different from those in healthy donors. In this study the investigators want to verify how this difference affects the maturation of CD34+ HPCs of healthy donors and of SET2 cells, a commercially available cell line characterized by the V617F mutation of the JAK2 molecule. In particular, the investigators will verify the effect of MSCs on signals that induce the activation of CD34+ cells, their survival or death, the production of harmful oxidative reagents and their clonal capacity. To obtain these data, the investigators will isolate MSCs from bone marrow blood samples of patients with PMF undergoing bone marrow biopsy for clinical reasons and from healthy subjects, donors of HPCs for transplant. Following a long culture MSCs will be stimulated to release small vescicles containing part of their nuclear and cytoplasmatic molecules. These vescicles will be cultured with CD34+ cells or SET2 cells and their effects will be evaluated. It has been shown that these small vescicles act exactly like MSCs, but they are easier to be used, and could become a very useful acellular drug in the near future.
Detailed description
Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by the clonal proliferation of CD34+ progenitor cells. A constitutive activation of the JAK-STAT pathway, due to an acquired gain of function mutation either in JAK2, MPL or CALR genes, plays a relevant pathogenetic role. Moreover, a state of chronic inflammation affects the disease course. Currently, the therapy with the JAK inhibitor Ruxolitinib ameliorates the symptoms of the disease, with no effects on the pathological cell clone; hematopoietic progenitor cell (HPC) transplantation is the only curative option. The investigators previously published that mesenchymal stromal cells (MSCs) from patients with PMF display functional and genetic abnormalities (low proliferative potential, precocious senescence, reduced differentiation potential). Regarding Ruxolitinib, direct effects on MSCs have been described, such as the inhibition of the JAK-STAT signaling, the growth of both healthy donor (HD)- and PMF-MSCs, and the decrease of fibrosis related gene expression. Ruxolitinib has also been reported to reduce MSC secretion of inflammatory cytokines MCP-1 and IL-6, suggesting a role of this drug in modifying the bone marrow (BM) microenvironment. In PMF, as in other hematological malignancies, MSCs participate in the communication between microenvironment and CD34+ cells. Recent studies demonstrated in other pathologies that MSC-derived extracellular vesicles (EVs) and parental MSCs have similar effects. In fact EVs, containing mRNA, microRNA, lipids, and proteins can be incorporated into recipient cells and modify their functions. In normal hematopoiesis, MSC-EV incorporation in CD34+ cells has been shown to induce an activation of the JAK-STAT pathway through increased levels of phospho-STAT5; in addition, a significant decrease in apoptosis and a higher colony-forming unit granulocyte/macrophage clonogenic potential have been reported.
The objectives of this study are:
- to isolate MSCs from BM of PMF patients and HDs. MSCs will be incubated with/out Ruxolitinib. EVs will be obtained following MSC starvation and ultracentrifugations, and stored at -80°C
- to assess EV effects on HD-CD34+ or JAK2V617F+ SET2 cells by evaluation of apoptotic and activation pathways, ROS production and clonogenic capacity. To this regard, confirmatory experiments will be performed with JAK2-mutated CD34+ cells from patients receiving or not Ruxolitinib.
- to perform gene expression profile on CD34+ and SET2 cells after incubation with EVs.
- to investigate EV biocargoes both qRT-PCR and proteomics will be performed on MSC-derived EVs, before and after incubation with Ruxolitinib.
The expected impact will include the identification of modifications in functional activities of CD34+ or SET2 cells following incubation with PMF-MSC-EVs with respect to HD-MSC-EVs, reflecting the effect of an altered microenvironment on HPCs. The investigators also expect the recognition of important targets of severity/progression of the disease by assessing the gene expression profile in CD34+ or SET2 cells that incorporate EVs from different sources.
In addition, the identification of the Ruxolitinib effects on PMF BM microenvironment may be clarified.
This study may allow to act on new targets of disease progression or to support the future use of HD-EVs as an acellular therapy that favours the survival of healthy CD34+ cells or acts against their clonal counterpart.
Study design
- Observational model
- Case-control
- Time perspective
- Prospective
- Number of arms
- 1
| Arm | Type | Receives |
|---|---|---|
| Patients with PMF out of therapy undergoing bone marrow biopsy for clinical reasons | – | Other: In vitro cultureHealthy subjects who donate bone marrow hematopoietic progenitor cells |
Who may qualify
- Ages
- 18 years and older
- Sex
- All
- Healthy volunteers
- Accepted
- Age groups
- Adult, Older adult
- Study population
- PMF (WHO 2016) afferent at the Center for the Study of Myelofibrosis, a national center that receives patients from all the country. Patients will be out of therapy, undergoing bone marrow biopsy for clinical reasons.
Key inclusion criteria (4 listed)
- Patients with PMF
- JAK2 V617F+ (either homozygous or heterozygous)
- Out of therapy
- At least 18 years old
Key exclusion criteria (3 listed)
- PMF combined to other pathologies
- Previous splenectomy
- Previous bone marrow transplant
Eligibility is decided by the study team after screening; this overview does not establish that a patient qualifies. Full inclusion and exclusion criteria.
Interventions
| Intervention | Type | Arms |
|---|---|---|
| In vitro cultureIn vitro culture of bone marrow MNCs to obtain mesenchymal stromal cells | Other | Patients with PMF out of therapy undergoing bone marrow biopsy for clinical reasons |
Primary outcome
Effect of bone marrow microenvironment, resembled by EVs, on hematopoietic progenitor cells
Time frame: Up to 24 weeks
The measure of the functional activities of HD-CD34+ cells induced by the bone marrow microenvironment (i.e. MSC derived EVs) of patients with PMF or heathy donors. P-STAT5 molecule as activation index, Bax/Bcl2 ratio as apoptosis indicator, ROS and membrane antigens will be assessed by a unique unit of measure, the mean fluorescence intensity. The change of the different parameters from baseline to the end of the co-culture will be reported.
1 secondary outcome
Components of EV biocargoes
Time frame: Through study completion, an average of 1 year
Results and safety
This study is still under way. Results are not available in the source record yet.
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 2 Apr 2025.
- Background: PubMed 29645296Zacharaki D, Ghazanfari R, Li H, Lim HC, Scheding S. Effects of JAK1/2 inhibition on bone marrow stromal cells of myeloproliferative neoplasm (MPN) patients and healthy individuals. Eur J Haematol. 2018 Jul;101(1):57-67. doi: 10.1111/ejh.13079. Epub 2018 Apr 30.
- Background: PubMed 31184411Preciado S, Muntion S, Corchete LA, Ramos TL, de la Torre AG, Osugui L, Rico A, Espinosa-Lara N, Gastaca I, Diez-Campelo M, Del Canizo C, Sanchez-Guijo F. The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability. Stem Cells. 2019 Oct;37(10):1357-1368. doi: 10.1002/stem.3032. Epub 2019 Jun 11.
- Individual participant dataSharing planned: No
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Referral and contact
AzTrials does not enrol participants or take referrals. Contact the study team or a site listed below; they decide who can take part.
Margherita Massa, PhD principal investigator
IRCCS Policlinico San Matteo Foundation
1 site in 1 country: see every site and its status.
Questions about this trial
What is this clinical trial studying?
An observational study of Primary Myelofibrosis, run by Fondazione IRCCS Policlinico San Matteo di Pavia at 1 site in Italy.
The full registered description is in the About this study section and the official record.
Who may be eligible to take part?
The sponsor lists 4 inclusion and 3 exclusion criteria. Eligibility is decided by the study team after screening, so read the full criteria and contact a listed site.
Is this clinical trial currently recruiting?
The record says: looking for participants now, as of the sponsor's last update on 2 Apr 2025. Status can change between updates, so confirm with the study contact before making plans.
Where is the study taking place?
The record lists 1 site in Italy. Whether a site is taking part right now should be confirmed with that site. See every site.
What are the possible benefits and risks?
They depend on the study and the treatment, and no study can promise a benefit. The study team explains the known risks during informed consent, before anyone joins; the record itself does not list them.
Are results available for this study?
This study is still under way. Results are not available in the source record yet.
How can a doctor refer a patient or contact the research team?
Contact Margherita Massa, PhD (+39 0382502703, m.massa@smatteo.pv.it), or a site near the patient. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
Effect of bone marrow microenvironment, resembled by EVs, on hematopoietic progenitor cells (time frame: Up to 24 weeks). It is what the study was designed to measure, not a finding.
Where can I read the original study information?
On the ClinicalTrials.gov record for NCT06896344, which also lists 2 linked publications.
Does a listing mean the treatment is approved or proven to work?
No. A listing means the study is registered. It does not show that the treatment is safe, works, or is approved by a regulator.
How often is this information updated?
AzTrials copies changed records from ClinicalTrials.gov every night and reloads the whole registry every week. The sponsor last updated this record on 2 Apr 2025.