Background. Despite the improvement of therapeutic regimens, a relevant proportion of multiple myeloma (MM) patients (pts) experience early relapse (ER) [Majithia, Leukemia 2016] and represents an unmet medical need. It is therefore of high clinical interest to identify baseline factors that may predict ER. Aims Methods Data were obtained from 2326 pts enrolled in 8 multi-center clinical trials: NCT01093196, NCT01346787, NCT01857115, NCT01190787, NCT00551928, NCT01091831, NCT01063179 and 2005-004730-41. Here, we included 14 baseline features (fts): age, creatinine, albumin (alb), b2microglobulin (b2m), bone marrow plasma cell (PCbm) were evaluated as continuous variables; free light chain (FLC, λ vs K), M-component subtype (IgA vs others), Revised International Staging System (R-ISS stage II/III vs I), lactate dehydrogenase levels >/≤ upper limit of normal (LDHULN), presence vs absence of chromosomal abnormalities detected by FISH [del17p, t(4;14), t(14;16), t(11;14)], and presence of plasmacytomas as categorical values. Trials were assigned to training and validation set to have a superimposed median (μe) age and follow-up in the two subsets. From the training set, a univariate analysis (UV) on outcome was performed according to both Chi-square and Kruskal tests, as appropriate. Features with p<0.05 were then tested in a multivariate (MV) logistic regression model. Each MV model was based on pts with complete data available. We performed and compared 2 MV models, one including R-ISS and one including fts (LDHULN, alb, b2m and chromosomal abnormalities) defining the R-ISS considered individually. Secondly, each model was tested on the validation set assessing the area under the curve (AUC). From the most accurate model, a score defined low (L), intermediate (I) and high (H) chances of ER. Statistical analysis was performed via R (v.3.5.2). Results ER≤18 models. 10/14 fts were selected based on UV analysis: age, FLC, PCbm, del17p, t(4/14), t(14/16), alb and b2m, LDHULN and R-ISS stage. Pts with complete data were included in the training set (n=923; μe age =66 years [y]; ER=35%) and the validation set (n=313; μe age=67 y; ER=36%), respectively. In the MV incorporating the R-ISS, the R-ISS II/III vs I (OR=1.75, 95% CI:1.26-2.44) and increased PCbm (OR=1.05, 95% CI:1.02-1.08) increased the risk of ER. When the MV analysis was performed including single fts instead of R-ISS, increased PCbm (OR=1.05, 95% CI:1.02-1.08), λ FLC (OR=1.34, 95% CI:1.01-1.79), LDHULN (OR=1.80, 95% CI:1.16-2.81), presence of both del17q (OR=1.58, 95% CI:1.07-2.33) and t(4/14) (OR=2.01, 95% CI:1.36-3.01) increased the probability of ER; increased alb (OR=0.75, 95% CI:0.61-0.94), reduced the risk of ER (table 1). ER≤24 models. 8/14 fts were selected based on UV analysis: FLC, PCbm, del17p, t(4/14), alb and b2m levels, LDHULN and R-ISS stage. Pts with complete data were included in the training set (n=1009; μe age=67 y; ER=45%) and in the validation set (n=352; μe age=67 y, ER=45%). In MV analysis, including R-ISS, both R-ISS (OR=1.88, 95% CI:1.39-2.55) and PCbm (OR=1.04, 95% CI:1.02-1.06) impacted on outcome. When the MV analysis was performed including single fts, λ FLC (OR=1.31, 95% CI:1.01-1.69), PCbm (OR=1.04, 95% CI:1.02-1.07), del17p (OR=1.86, 95% CI:1.30-2.65) and t(4/14) (OR=2.00, 95% CI:1.38-2.88) retained their impact on outcome (table 1). Validation. Each MV model was tested on the validation set. Among the 4 MV models, the ER≤18 incorporating individual fts resulted in the highest AUC (0.66) and was therefore used to build up a prognostic score. Score. The ER score was calculated as 0.047 × PCbm + 0.589 × LDHULN + 0.459 × del17q (IF present) + 0.705 × t(4/14) (IF present) + 0.293 × FLC (IF FLC =λ) - 0.284 × alb. The ER score was calculated as 3 groups of pts with different risks of ER: L (42% of pts; risk of ER18=23%), M (33% of pts; risk of ER18=39%) and H (26% of pts; risk of ER18 =55%). Discussion. This is the first analysis proposing a score that includes standard baseline fts and aims at identifying pts at high risk of ER in the context of novel agent-based therapy. Based on our score, 26% of pts can be defined at high risk. To improve the clinical applicability, the construction of a simplified model with categorized variables is ongoing.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm variant characterized by excessive production of platelets. Since the most common cause of mortality and morbidity in ET patients is thrombosis, the excessive production of platelets may cause thrombotic events. However, little is known about the function of platelets in ET. We report a female patient who presented as asymptomatic, without a remarkable medical history, and ET was diagnosed after an incidental finding of moderate thrombocytosis. Notably, together with thrombocytosis, an abnormal platelet phenotype was found for the presence of a massive, rapid and spontaneous formation of aggregates and platelet hypersensitivity to subthreshold concentrations of aggregating agonists. Bone marrow histopathological examination and genetic analysis with the JAK2 (V617F) gene mutation findings confirmed the initial suspicion of ET. Although the ET patient was placed on aspirin, the persistence of the platelet hyperactivation and hyperaggregability prompted a switch in antiplatelet medication from entero-coated (EC) to plain aspirin. As result, platelet hypersensitivity to agonists and spontaneous aggregation were no longer found. Collectively, our study demonstrates that platelet function analysis could be a reliable predictor of ET and that plain aspirin should be preferred over EC aspirin to attenuate platelet hyperreactivity.

L’emoglobinuria parossistica notturna (EPN) è un disordine ematologico raro caratterizzato da anemia emolitica, trombosi e difetti dell’ematopoiesi. Tale patologia è dovuta a una mutazione somatica del gene PIG-A che comporta la mancata produzione del glicosil-fosfatidil-inositolo (GPI), a cui si ancorano proteine di membrana quali CD55 e CD59 che inibiscono l’attivazione del complemento su diversi tipi cellulari. Dal 2010 il nostro Laboratorio partecipa alla compilazione dell’archivio CLONEPnh. Integrando i dati in esso inseriti con quelli contenuti nell’archivio del nostro Laboratorio è stato valutato il cambiamento negli anni del numero di richieste di ricerca del clone EPN e, in relazione a esso, come è variato il numero dei nuovi cloni individuati. È stata effettuata un’analisi citofluorimetrica a sei colori su sangue periferico, di pazienti afferenti alla Divisione Universitaria di Ematologia dell’Azienda Ospedaliera Città della Salute e della Scienza di Torino, utilizzando il FLAER (tossina batterica inattivata) e i seguenti anticorpi: CD45, CD14, CD33, CD66, CD55, CD59, CD235a. I campioni sono stati acquisiti con citofluorimetro FACSCantoII e analizzati con il software FACS Diva. I dati sono stati inseriti nell’archivio CLONEPnh (www.CLONEPnh.com). Dai dati inseriti nell’archivio CLONEPnh si evince che le richieste di ricerca di EPN sono state 13 nel 2010, 16 nel 2011, 28 nel 2012 e 12 nei primi sei mesi del 2013 e i cloni individuati sono stati, rispettivamente, 1, 0, 1 e 1. Un andamento simile, anche se con un numero decisamente minore di analisi richieste, è stato riscontrato anche negli anni precedenti (dal 2000) analizzando l’archivio del nostro Laboratorio. I nostri dati mostrano un aumento negli anni di analisi effettuate, a cui, però, non è corrisposto un aumento dei cloni individuati. L’aumento delle richieste si può giustificare considerando che la diagnosi di EPN è diventata prevalentemente citofluorimetrica e che le recenti linee guida consigliano, in pazienti con displasie midollari, di ricercare e monitorare cloni di modesta entità mediante la metodica ad alta sensibilità. Tuttavia, per ridurre il costo in termini di tempo e risorse, sarebbero auspicabili l’elaborazione e la validazione di protocolli citofluorimetrici più rapidi con l’utilizzo di un pannello ridotto di reagenti. Paroxysmal nocturnal haemoglobinuria (PNH) is a rare haematological disease characterized by haemolytic anaemia, thrombosis and bone marrow failure. This malignancy arises by somatic mutations of PIG-A gene resulting in a partial or absolute deficiency of cell membrane proteins linked to glycosylphosphatidylinositol (GPI), as CD55 and CD59, involved in the control of complement activation. Since 2010 our laboratory has been participating to the update of the CLONEPnh archive. The data of internal and CLONEPnh archives were merged in order to compare the number of patients positive for PNH clone to the number of samples analyzed. All peripheral blood samples, of patients admitted to the University Division of Haematology of Azienda Ospedaliera Città della Salute e della Scienza di Torino, were stained using FLAER (inactivated bacterial toxin) and a combinations of six monoclonal antibodies: CD45, CD14, CD33, CD66, CD55, CD59, CD235a. The samples were acquired on FACSCanto II flow cytometer and analyzed with FACS Diva software. The data were reported in CLONEPnh archive (www.CLONEPnh.com). The data of CLONEPnh archive show that the analysis carried out were: 13 in 2010, 16 in 2011, 28 in 2012 and 12 in first six months of 2013 and new PNH clones detected were 1, 0, 1 and 1 respectively. The data of our internal archive show a similar trend, with fewer analysis performed, also in previous years (since 2000). Our data show a stable number of PNH clones detected despite an increased number of analysis performed. The essential role of flow cytometry in diagnosis of PNH and the guidelines indication, about search and monitoring of small PNH clones with high sensitivity flow cytometry method, could explain this trend. However, for a better use of economical resources, faster and less expensive flow cytometric protocols must be developed and validated.