Bone marrow failure Bone marrow failure: A condition that occurs when the bone marrow stops making enough healthy blood cells. The most common of these rare diseases are aplastic anemia, myelodysplastic syndromes (MDS) and paroxysmal nocturnal hemoglobinuria (PNH). Bone marrow failure can be acquired (begin any time in life) or can be… syndromes are not only serious and debilitating diseases but they can also progress toward leukemia. Identifying the specific factors contributing into this malignant transition is extremely important. Our group focus in past few years has been the role of the immune system in bone marrow failure and we showed the importance of specific subtypes of immune cells in both pathogenesis and malignant transformation of these diseases. The AAMDSIF research grant will allow us to focus on differences in immune changes in aplastic anemia aplastic anemia: (ay-PLASS-tik uh-NEE_mee-uh) A rare and serious condition in which the bone marrow fails to make enough blood cells - red blood cells, white blood cells, and platelets. The term aplastic is a Greek word meaning not to form. Anemia is a condition that happens when red blood cell count is low. Most… and paroxysmal nocturnal hemoglobinuria paroxysmal nocturnal hemoglobinuria: (par-uk-SIZ-muhl nok-TURN-uhl hee-muh-gloe-buh-NYOOR-ee-uh) A rare and serious blood disease that causes red blood cells to break apart. Paroxysmal means sudden and irregular. Nocturnal means at night. Hemoglobinuria means hemoglobin in the urine. Hemoglobin is the red part of red blood cells. A… (PNH) and will enable us to utilize the state of the art technologies (such as multidimensional mass cytometry and high throughput DNA sequencing) to further understand the role of immune system in these diseases as well as identifying new important biological markers for diagnosis and classification of aplastic anemia and PNH.
The aim of this grant is to identify the specific immune dysregulation in paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA)/PNH, and also correlate the interplay between the PNH cells and specific T-cell subsets called NKT-cells in a sequential longitudinal study. We will also identify ways that different therapies modulate the immune response and utilise it for the therapeutics of marrow failures. Cutting edge technologies such as mass-cytometry (CyTOF) and high precision DNA sequencing (deep sequencing) are being used to address these questions.
Progress so far Patient recruitment:
We have identified 89 patients with PNH through the King’s College London (KCL) tissue bank which are consented to participate in research projects (10 patients were recruited since last year). Among these patients, we have access to peripheral blood mononuclear cells (PBMCs) of 26 patients at time of diagnosis and following therapy with C5 inhibitor (Eculizumab). PBMCs from these patients are stored as viable cells and being used for this study. We have also recruited 16 AA patients with a small to moderate PNH clone clone: To make copies. Bone marrow stem cells clone themselves all the time. The cloned stem cells eventually become mature blood cells that leave the bone marrow and enter the bloodstream. (with no cases of hemolytic PNH) and 8 age matched healthy volunteers (HDs) as control groups thus far.
Identifying the specific immune signature(s) for PNH and AA/PNH
The first aim of this research was to identify a specific signature which identifies AA/PNH patients and could potentially predict their response to immunosuppressive therapy immunosuppressive therapy: Immunosuppressive drug therapy lowers your body's immune response. This prevents your immune system from attacking your bone marrow, allowing bone marrow stem cells to grow, which raises blood counts. For older patients with acquired aplastic anemia, immunosuppressive drug therapy is the… (IST). We have used CyTOF technology and multiple bioinformatics packages to identify potential immune signature(s), which predicts response to immunosuppression in AA/PNH. This enabled us to distinguish two distinct subpopulations of human T-cells (known as T regulatory cells (Tregs)), in AA and HDs and to demonstrate clear differences in AA that predicted response to treatment. The results from this study are under revision for publication and the AAMDSIF has been acknowledged in this work. Up to now, blood samples from 5 PNH patients have been stained and analysed pre and post Eculizumab Eculizumab: Eculizumab (Soliris ®) is given as an IV into a vein at the doctor’s office or at a special center. The procedure usually takes about 35 minutes. You will probably get an IV once a week for the first 4 weeks. Starting in the 5th week, you will get a slightly higher dose of Soliris every 2 weeks. … by the same method (10 samples) and the preliminary data suggest that type of Treg cells could also predict response to therapy in PNH. We have successfully optimized the CyTOF panel for identification of another important immune cells in PNH (CD8+ T-cell). We were able to identify the classic CD8+ T-cell subpopulations as well as novel clusters of cells in PNH patients. We will continue to analyse more samples by CyTOF to confirm the persistence and biological relevance of the identified clusters in PNH, AA/PNH.
Plans for the second year
We would like to continue this work by accomplishing the following tasks:
1. Finalise the CyTOF run for all collected samples from PNH patients pre and post Eculizumab and finalise the data analysis.
2. Once the CyTOF data analysis has been completed, and the set of markers which accurately define the identified cells in PNH, these markers will be used to isolate these cells and perform the additional molecular studies as outlined in our application.
The ultimate goal of the second part is to identify NKT-cells within the expanded population of cells and see whether these cells can distinguish PNH from AA/PNH and how they would affect the response to Eculizumab.