Re-envisioning risk biomarkers in acute myeloid leukemia (AML) at single-cell level

Project Information

reMARK-AML

Grant agreement ID: 101123346

DOI

10.3030/101123346

Project closed

EC signature date 17 July 2023

Start date 1 August 2023

End date 31 January 2025

Funded under

• European Research Council (ERC)

Total cost

No data

EU contribution

€ 150 000,00

Coordinated by

UNIVERSITA DEGLI STUDI DI PERUGIA
Italy

Periodic Reporting for period 1 - reMARK-AML (Re-envisioning risk biomarkers in acute myeloid leukemia (AML) at single-cell level)

Reporting period: 2023-08-01 to 2025-01-31

Summary of the context and overall objectives of the project

Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancies that despite recent progresses remains an unmet medical need. Genetic markers are used to define different disease categories and prognostic groups. One of the most frequent is mutations in the Fms-like tyrosine kinase 3 (FLT3) gene, particularly internal tandem duplication (ITD). FLT3-ITD represents an important adverse risk factor, and although targetable with specific approved inhibitors that are routinely used worldwide, it mostly requires allogeneic hematopoietic stem cell transplant to give patients prospective of cure. FLT3-ITD represents specifically the ‘tip of the balance’ for the prognostic definition of a particular leukemia that is the AML with nucleophosmin (NPM1) gene mutation, the most frequent AML, accounting for about one-third of cases. Indeed, only when in absence of FLT3-ITD (FLT3-ITDneg), patients with NPM1 mutations are regarded as favorable outcome with standard chemotherapy alone. Yet a significant proportion - up to about 40% - of these patients will relapse, and notably, about 20-30% undergo relapse with emergence of mutations, including FLT3-ITD, originally not detected at diagnosis by standard approaches.
We previously found that single FLT3-ITDpos cells at either diagnosis or during treatment can be clinically relevant as they acquire drug resistance, may emerge after therapy and initiate relapse in patients otherwise regarded as FLT3-ITDneg and thus at favorable risk at presentation. The solution would be to intercept these cells earlier and adapt targeted therapy in order to prevent relapse. In our PoC study, we validated our hypothesis in patients and provided data that revealed the value of single FLT3-ITDpos cells as novel biomarker to re-define prognosis, and possibly refine treatment strategy, in the specific setting of NPM1-mutated AML.
Biomarkers are specific characteristics of a disease that can be measured and evaluated in an objective manner. FLT3-ITD has the characteristics of diagnostic, prognostic ad predictive biomarker. Therefore, studies on FLT3-ITD as biomarker are complex because of the different implications, but at the same time may have a great impact. The aim of this project was to develop a reliable assay that can be translated into the clinics, and address clinical issues since it requires validation in clinical studies involving patients. The final idea is that every new case of AML with mutated NPM1 and FLT3 wild-type according to standard-of-care would need to be tested with the novel approach to be assigned to the correct risk category and adapt targeted therapy. The gain would be to bring new knowledge for AML management. The impact would be tremendous at different levels, leading to
1) refinement in definition of prognostic risk categories in AML;
2) change in the diagnostic flow-chart and guideline for AML;
3) adoption of the concept of adaptive therapy in AML based on the demonstrated predictive value of the new biomarker.
This project is ground breaking as it could change prognostic risk parameters and the paradigm of treatment in AML by shifting the concept of biomarker and therapeutic target to the single-cell level, and precision therapy to adaptive treatment strategy.
Patients will highly benefit from the results of this project, since their assignment to the correct risk category will allow them to have access to targeted therapy and start, since the beginning, the correct therapeutic path preventing relapses. Industries would benefit from the development and commercialization of the newly developed diagnostic tool as well as from the extended use of the specific drug.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Single-cell targeted DNA sequencing (scDNAseq) is a novel technique, which allows to deeply characterize cells and related mutations, and is currently the only technological approach able to detect within the disease bulk very small cellular subpopulations carrying the target mutation. We adopted the Tapestri platform from Mission Bio, and set up a first strategic technological approach for our study. Indeed, the final goal of the project is the development of a prototype fit-for-purpose diagnostic assay for early detection of cells carrying the FLT3-ITD mutation, and the design of innovative adaptive therapeutic approaches following their interception. Thirty out of the planned 42 patients have been recruited. Bone marrow samples were collected at diagnosis, post-treatment and at relapse. On the same samples, diagnostic analyses have been performed according to standard of care (SoC). Samples were appropriately cryopreserved for experimental preparation. All the patient data were collected in a dedicated database. scDNAseq was performed on bone marrow cells, libraries prepared following manufactures instruction by Tapestri instrument. To optimize activities, we used a high quality, cost-effective sequencing service. Analysis on patients samples was performed using the Mosaic Mission Bio proprietary bioinformatics analysis platforms. The raw data will be uploaded in the publicly available Gene Expression Omnibus repository (GEO).
In our preliminary findings, we were able to intercept rare cells carrying FLT3-ITD mutation in a diagnosis sample of a patient otherwise regarded as negative for FLT3-ITD. We were able to find these cells also in another patient who was regarded as negative for FLT3-ITD at diagnosis and who relapsed with positive cells. In a third patient, also regarded as negative for FLT3-ITD, we observed that the leukemic blasts injected in immunocompromised mice, gave rise to a very aggressive disease. By sequencing, we found that these cells were positive for FLT3-ITD. scDNAseq on the diagnosis sample revealed the presence of few FLT3-ITD positive cells at diagnosis as well. Finally, we found FLT3-ITD positive cells in the diagnosis sample of a patient who was in remission but came back to our hospital with molecular relapse. The patient is now under surveillance and the cells are on sequencing to check for the presence of FLT3-ITD. Despite the accrual of a number of patients lower than planned, our proof-of-concept data were considered already clinically relevant and a sufficieet to foster us to proceeding to the phase of developing - in collaboration with an industrial partner - an easy-to-apply novel diagnostic tool with deep sensitivity to be able to detect single FLT3-ITD cells. The study and observation of patients will proceed as described and possibly further extended.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our PoC data support the development of a new faster and accurate diagnostic tool for AML with profound implications for healthcare and society. Indeed, it fosters personalized medicine since advanced diagnostics can help tailored treatments based on individual genetic or biomarker profiles.
The research team has taken contacts with the industrial partners involved in the research and experimental development activities and will foster these relationships and establish new ones for the clinical application of the results of the project.
It will be necessary further research and funding to promote this development by interdisciplinary collaboration and partnerships between biotech and healthcare fields, design new clinical trials and strengthen real-world testing to validate efficacy and reliability of our study