Studio della funzione piastrinica nei pazienti con sindrome mielodisplastica

Background

Myelodysplastic syndromes (MDS) are a heterogenous group of clonai stem celi disorders characterized by trilineage dysplasia resulting in peripheral cytopenias and increased incidence of leukemic transformation¹. Hemorrhagic complications can have serious outcomes in patients with MDS and are among the major causes of death in this population. In a chart review referring to the M. D. Anderson Cancer Center database, 460 patients with MDS died without progression to AML. Bleeding was the only cause of death in 48 (10%) of these patients, while it contributed to death in 90 patients (20%)². Other studies reported that hemorrhagic complications account for about 14%-45% of deaths in MDS patients. Although thrombocytopenia, which is observed in 40-65% of MDS patients, may be a contributing factor³, bleeding complications are observed also in MDS patients with normal platelet count. As a matter of fact, the frequency of fatai bleeding was evenly distributed among ali four lnternational Prognostic Scoring System (IPSS) risk groups⁴, which are defined based on 4 parameters, including the platelet count⁵. Platelet function abnormalities are likely responsible for bleeding events in MDS patients with normal platelet count⁶. Hence, several evidences support the idea that both thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications observed in MDS patients^7,8. Although some abnormalities of platelet function have been documented^9,10, to the best of our knowledge, no published studies evaluated several parameters of platelet function, including platelet aggregation, platelet secretion, the cont~nt of platelet granules, thromboxane A₂ production and the levels of cyclic AMP (cAMP), which is a strong inhibitor of platelet function¹¹, at baseline and after stimulation of adenylyl cyclase with increasing concentrations of prostaglandin E₁ (PGE₁).

Aim of the study

In this study we aim at thoroughly exploring platelet function in patients with MDS or Myeloid Leukemias.

Preliminary data

In 2010 a case report of a woman of 74 years, with no previous bleeding history, was presented¹². She came to the outpatient clinic of Centro Emofilia A Bianchi Bonomi, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico for occurrence of severe mucocutaneous bleedings. Coagulation tests and VWF were norma!. She had mild thrombocytopenia and normocitic anemia, and was later diagnosed with chronic myelomonocytic leukemia. Platelet aggregation and ATP secretion induced by ADP, collagen and U46619 were found to be severely impaired on two separate occasions. CD41/61 and CD42b were normally expressed on her platelets (flow cytometry). The platelet contents of delta-granules (ADP and serotonin) and of alpha-granules .. (fibrinogen), an d se rum thromboxane 82 (TXB2) levels were markedly decreased (the patient was not taking nonsteroidal anti-inflammatory drugs). The concentration of cyclic-AMP in resting platelets was higher than norma( and dramatically increased after exposure of platelets to 1 μmol/L prostaglandin E₁ (PGE₁)¹². Another patient was referred to the Unit at Ospedale San Paolo for severe bleeding manifestations. The patient was diagnosed with MDS and a thorough platelet function evaluation revealed that he had severe abnormalities of platelet function that were comparable to those observed in the previously studied patient with chronic myelomonocytic leukemia.

Methodology

This project will be realized at Ospedale San Paolo, Dipartimento di Scienze della Salute, Università degli Studi di Milano (Milan, ltaly). Approvai by the lnstitutional Review Board of the lnstitution will be requested. The medicai staff of Internai Medicine (Medicina 111) of Ospedale San Paolo will recruit MDS patients, who will sign and informed consent form. An electronic database for recording clinica!, laboratory and experimental data will be created, stored and accessed according to current privacy laws.

Study design
This will be a case control study enrolling about 40 patients with MDS or Myeloid Luekemias and 40 sex-age matched apparently healthy subjects. Ali subjects will be eligible for the study if they meet ali of the following inclusion criteria:

• ≥18 years of age,
• free of drugs known to affect hemostasis for ≥10 days two before blood sampling,
• no drug or alcohol abuse.

Diagnoses of MDS will be done according to the WHO 2008 Consensus Conference criteria. Ongoing medicai treatments will not be modified. Clinica! information including demographics, diagnosis classification, bleeding or thrombosis history, IPSS risk category (score), family history, as well as medication will be collected for each patient.

A venous blood sample (30 ml) will be taken from ali enrolled individuals at the same time in the morning. Laboratory tests of platelet function will be performed in vitro using whole blood, platelet-rich plasma and washed human platelets.

1. Platelet aggregation and secretion induced by collagen, ADP, epinephrine, thrombin and convulxin will be measured by lumiaggregometry.
2. Serum TXB2 will be measured by ELISA.
3. The expression of P-selectin, GPVI, and GPib on the platelet membrane and the binding of PAC-1 (a monoclonal antibody that binds to activated GPIIb-llla) will be measured by flow cytometry.
4. The platelet content of beta-thromboglobulin (contained in the platelet alphagranules), ADP, ATP and serotonin (ali contained in the platelet delta-granules) will be measured by standardized assays.
5. The intraplatelet concentration of cAMP will be measured at baseline and after stimulation with increasing concentrations of PGE 1, using a commercially available ELISA kit.
6. Morphological analysis of platelets will be done by electron microscopy (EM).
7. Attempts will be made to unravel the mechanism(s) by which the platelet cAMP levels are elevated in MDS patients, including the study of Gs protein, adenylate cyclase and phosphodiesterase enzymes.

Statistics

Dichotomous variables will be compared by means of Chi-squared, Fisher’s Exact or the Wilcoxon Rank-sum test, as appropriate. Differences for continuous variables will be tested for statistica! significante by t test, and also multiple regression techniques may be used to adjust analyses for potential confounding factors. Logarithmic transformations will be applied to data with skewed distributions. Correlations between the studied variables and the severity of the bleeding sco re will be evaluated.

Milestone

The first 2 months will be employed to request the lnstitutional Review Board approvai and to set up ali the experimental procedures. During the following 8 months, recruitment of patients and platelet function studies will take piace. The results will then be analyzed and a scientific report will be prepared in the following 2 months.

Facility

The project will be realized at the Laboratory of Hematology and Thrombosis of the Dipartimento di Scienze della Salute of the Università degli Studi di Milano (Milan, ltaly) in collaboration with the Unit of Internai Medicine Clinic (Medicina 111) of the Ospedale San Paolo (Milan, ltaly) under the supervision of professar Marco Cattaneo. The operative units involved can previde facilities, instrumentation and tools necessary to perform ali experiments.

Possible criticai aspects

The only potential criticai aspect could be the difficulty to obtain consensus from our patients to be enrolled in the study.

Relevance and impact of this project

Unravelling the mechanism(s) contributing to the bleeding risk of patients with MDS or Myeloid Leukemias may help improving their management. lt has been shown that 14-45% of deaths in MDS patients are caused by pathological bleeding. The overall incidence of MDS is about 4 cases per 100 000 persons, but it dramatically increases to 40-50 per 100 000 persons older than 70 years. Therefore, its social impact is very relevant and will likely increase in the future.

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References

1. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. British journal of haematology 1976; 33(4): 451-8.
2. Kantarjian H, Giles F, List A, et al. The incidence and impact of thrombocytopenia in myelodysplastic syndromes. Cancer 2007; 109(9): 1705-14
3. Hofmann WK, Ottmann OG, Ganser A, et al. Myelodysplastic syndromes: clinica! features. Seminars in hematology 1996; 33(3): 177-85.
4. Neukirchen J, Blum S, Kuendgen A, et al. Platelet counts and haemorrhagic diathesis in patients with myelodysplastic syndromes. European journal of haematology 2009; 83(5): 477-82.
5. Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012; 120(12): 2454-65.
6. Doli DC, List AF. Myelodysplastic syndromes. The Western journal of medicine 1989; 151(2): 161-7.
7. Maldonado JE, Pierre RV. The platelets in preleukemia and myelomonocytic leukemia. Ultrastructural cytochemistry and cytogenetics. Mayo Clinic proceedings 1975; 50(10): 573-87.
8. Lintula R, Rasi V, lkkala E, et al. Platelet function in preleukaemia. Scandinavian journal of haematology 1981; 26(1): 65-71.
9. Mittelman M, Zeidman A. Platelet function in the myelodysplastic syndromes. lnternational journal of hematology 2000; 71{2): 95-8.
10. Manoharan A, Brighton T, Gemmell R, et al. Platelet dysfunction in myelodysplastic syndromes: a clinicopathological study. lnternational journal of hematology 2002; 76(3): 272-8.
11. Smolenski A. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost 2012; 10(2): 167-76.
12. Lecchi A, Artoni A, Mantovani G, Gelain F, Motta M, Cattaneo M. An acquired hemorrhagic disorder associated with a complex phenotype of platelet dysfunction in a patient with chronic myelomonocytic leukemia. Blood Transfusion 2010; 8 (Suppl. 4). XXI Congresso nazionale SISET 2010.

La sindrome mielodisplastica (MDS) appartiene ad un gruppo eterogeneo di malattie neoplastiche clonali del midollo osseo caratterizzate da displasia trilineare con conseguente citopenia periferica e aumentata incidenza di progressione verso la leucemica acuta¹. Le complicanze emorragiche possono avere gravi conseguenze nei pazienti con MDS e sono tra le principali cause di morte in questa popolazione. In uno studio del M.D. Anderson Cancer Center su 460 pazienti con MDS deceduti senza progressione a leucemia mieloide acuta, il sanguinamento è stata l’unica causa di morte in 48 di questi pazienti (10%), mentre ha contribuito alla morte in 90 pazienti (20%)². Altri studi riportano che le complicanze emorragiche causano circa il 14-45% dei decessi di pazienti con sindromi mielodisplastiche. La trombocitopenia, osservata nel 40-65% dei pazienti con MDS, contribuisce alla patogenesi delle complicanze emorragiche³, tuttavia, si osservano complicanze emorragiche anche in pazienti con MDS che hanno una conta piastrinica normale⁴. Infatti, la frequenza di emorragie fatali è equamente distribuita tra tutti i quattro gruppi di rischio identificati secondo l’International Prognostic Scoring System (IPSS)⁵, che viene definito sulla base di quattro parametri tra cui la conta piastrinica⁶. E, quindi, ipotizzabile che anche le alterazioni della funzione piastrinica siano responsabili delle emorragie nei pazienti con MDS con conta piastrina normale^{7, 8}. Numerose sono, infatti, le evidenze che documentano che non solo la trombocitopenia, ma anche diverse anomalie della funzionalità piastrinica contribuiscano alle complicanze emorragiche osservate nella maggior parte dei pazienti affetti da MDS studiati^{9, 10}. Tuttavia, ad oggi, non ci sono studi pubblicati che valutino contemporaneamente diversi parametri di funzionalità piastrinica, come l’aggregazione, la secrezione, il contenuto di granuli piastrinici, la produzione di trombossano (Tx) A2 e i livelli di AMP ciclico (cAMP) sia in condizioni basali e che dopo la stimolazione. È noto che un aumento di quest’ultimo parametro (cAMP), mai valutato prima in pazienti con MDS, può inibire drasticamente la funzionalità piastrina.