La leucemia linfatica cronica (LLC) è la leucemia più diffusa nei Paesi occidentali. Il decorso clinico della LLC può spaziare da una condizione indolente, a progressione lenta, ad una forma più aggressiva che può portare a una morte precoce. È importante, per identificare i pazienti che necessitano di immediato trattamento clinico, la continua ricerca di nuovi e più accurati marcatori prognostici. Tra questi, il cariotipo complesso (CK) (definito dalla presenza di più di 3 anomalie nello stesso clone) è emerso come un indipendente marcatore prognostico negativo. Il CK include una varietà di anomalie citogenetiche, da quelle numeriche a quelle strutturali. In questo studio è stata eseguita l’analisi citogenetica su 90 pazienti con la LLC e il cariotipo complesso che non sono mai stati trattati farmacologicamente. È stato valutato l’impatto prognostico di ogni singola anomalia citogenetica in relazione alla sopravvivenza globale (O.S) e il tempo al primo trattamento (TTFT). Tra tutte le anomalie, le traslocazioni sbilanciate hanno mostrato di avere un indipendente impatto prognostico, con un peggioramento dell’OS e un più breve TTFT. È stata eseguita anche l’analisi dei profili di espressione genica su 23 dei 90 pazienti, 11 con traslocazioni sbilanciate e 12 senza. La presenza di traslocazioni sbilanciate ha identificato una categoria di pazienti con diverse caratteristiche geniche. Tra i geni differentemente espressi, l’attenzione è stata focalizzata su SLAMF1, un gene coinvolto nei processi di attivazione dei linfociti B, apoptosi e controllo del ciclo cellulare. L’espressione di SLAMF1 è stata analizzata in tutti gli esordi di LLC (349), conservati a Ferrara tra il 2009 e il 2018, attraverso l’uso della droplet digital PCR, usando la β-actina come gene di controllo. I pazienti sono stati divisi in tre diversi gruppi in base all’espressione di SLAMF1. Un livello di espressione superiore a 6,24 è stato considerato come alto, mentre i pazienti con livello inferiore a 2,8 sono stati inseriti nel gruppo con espressione considerata bassa. I restanti pazienti sono stati posizionati nel gruppo a espressione intermedia. Questa divisione è risultata inversamente sovrapponibile a una divisione dei pazienti basata sul loro rischio prognostico. Quelli con una bassa espressione di SLAMF1 sono risultati avere un TTFT più breve e un peggiore OS. Nella patogenesi della LLC i miRNA giocano un ruolo importante. La delezione dei miR-15a e miR-16-1, coinvolti nel pathway di Bcl2, può causare l’insorgenza della LLC. È ipotizzabile che i miRNAs possano essere la causa della down-regolazione di SLAMF1 nei pazienti con LLC e traslocazioni sbilanciate. Per capire meglio il ruolo che questi miRNAs svolgono nel regolare l’espressione di SLAMF1, è stato eseguito un saggio di luciferasi analizzando i miRNA normalmente over-espressi in casi di LLC, sospettati di essere i responsabili della down-regolazione di SLAMF1, ovvero i miR-17/92, miR132 e miR-148a. I risultati della luciferasi non hanno mostrato differenze nell’espressione di SLAMF1 a seguito dell’azione di questi miRNA. Questo indica che i miRNA non sono responsabili per la down-regolazione di SLAMF1 e che questo meccanismo rimane tutt’ora sconosciuto. Capire i meccanismi molecolari che regolano lo sviluppo della malattia permetterebbe di identificare nuovi target terapeutici. Il CK è un marcatore prognostico negativo, ma la presenza di specifiche anomalie citogenetiche, le traslocazioni sbilanciate, possono modificare il suo valore prognostico. L’espressione di SLAMF1 sulla superficie dei linfociti B può rappresentare un nuovo marcatore prognostico veloce e facile da misurare. Queste scoperte possono avere una grande rilevanza clinica per stratificare i pazienti in categorie di rischio, permettendo di selezionare il trattamento clinico più adatto.

Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia in western countries. CLL’s clinical course can range from an indolent condition, with a slow progression, to an aggressive form, which can lead to an early death. The discovery of new and more accurate prognostic markers is crucial for identifying patients in immediate need of treatment. Complex Karyotype (CK) (≥ 3 chromosome aberrations in the same clone) has emerged as an independent negative prognostic marker. CK includes a variety of cytogenetic aberrations, from numerical to structural abnormalities. In this study, it was performed a cytogenetic analysis on 90 treatment-naïve CLL patients with CK. It was evaluated the prognostic impact of each cytogenetic abnormality for the Overall Survival (OS) and Time To First Treatment (TTFT). Among all the abnormalities, unbalance translocations had displayed an independent prognostic impact, with a worse OS and TTFT. It was performed a gene expression profile analysis on 23 of the 90 patients, representative of the entire cohort, 11 with unbalance translocation and 12 without. It emerged that the presence of unbalanced translocation identified a subset of patients with distinct molecular features. Among the differentially expressed genes, the attention was focused on SLAMF1, a gene involved in lymphocyte activation, apoptosis, and cell cycle control. It was decided to analyze the expression of SLAMF1 in all CLL onset (349) stored in the Ferrara laboratory from 2009 to 2018. Through a droplet digital PCR, it was studied the expression of SLAMF1. The housekeeping gene, β-actin, was used as a standard control. The patients were divided into three different groups based on SLAMF1 expression. Patients with an expression above 6,24 belonged in the high-expression group. A value below 2,8 was considered low, while the rest of the patients were classified as an intermediate group. This partition inversely overlaps with the division of patients based on their prognostic risks. Patients with lower expression of SLAMF1 had a shorter TTFT. and O.S. In CLL miRNAs play an important role in the pathogenesis of the disease. The deletion of the miR-15a and miR-16-1, involved in the Bcl2 pathway, causes the development of CLL. It could be possible that miRNAs are responsible for the downregulation of SLAMF1 in CLL patients. To clarify the role played by these miRNAs in the regulation of SLAMF1 expression it was performed a luciferase assay. The miRNAs selected as possibly responsible for SLAMF1 regulation were the ones up-regulated in CLL: miR-17/92, miR132 and miR-148a. The results of the luciferase assay show no difference in SLAMF1 expression based on the actions of miRNAs. This indicates that miRNAs are not responsible for the dysregulation of SLAMF1. The mechanisms remain unknown. Due to the clinical and molecular heterogeneity of CLL, a better understanding of the molecular pathway that regulates the development of CLL is important for highlighting new possible treatment targets. Although CK is a negative prognostic marker, in this study, it was proven that specific cytogenetic anomalies could worsen the CK prognostic significance. Also, the expression of SLAMF1, easy to measure on the surface of B lymphocytes, could represent a new prognostic marker. These findings will be helpful to separate the patients into risk categories and to select the best treatment.

CLINICAL AND BIOLOGICAL MEANING OF GENOMIC COMPLEXITY IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND COMPLEX KARYOTYPE (CK).

MELANDRI, AURORA
2021

Abstract

Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia in western countries. CLL’s clinical course can range from an indolent condition, with a slow progression, to an aggressive form, which can lead to an early death. The discovery of new and more accurate prognostic markers is crucial for identifying patients in immediate need of treatment. Complex Karyotype (CK) (≥ 3 chromosome aberrations in the same clone) has emerged as an independent negative prognostic marker. CK includes a variety of cytogenetic aberrations, from numerical to structural abnormalities. In this study, it was performed a cytogenetic analysis on 90 treatment-naïve CLL patients with CK. It was evaluated the prognostic impact of each cytogenetic abnormality for the Overall Survival (OS) and Time To First Treatment (TTFT). Among all the abnormalities, unbalance translocations had displayed an independent prognostic impact, with a worse OS and TTFT. It was performed a gene expression profile analysis on 23 of the 90 patients, representative of the entire cohort, 11 with unbalance translocation and 12 without. It emerged that the presence of unbalanced translocation identified a subset of patients with distinct molecular features. Among the differentially expressed genes, the attention was focused on SLAMF1, a gene involved in lymphocyte activation, apoptosis, and cell cycle control. It was decided to analyze the expression of SLAMF1 in all CLL onset (349) stored in the Ferrara laboratory from 2009 to 2018. Through a droplet digital PCR, it was studied the expression of SLAMF1. The housekeeping gene, β-actin, was used as a standard control. The patients were divided into three different groups based on SLAMF1 expression. Patients with an expression above 6,24 belonged in the high-expression group. A value below 2,8 was considered low, while the rest of the patients were classified as an intermediate group. This partition inversely overlaps with the division of patients based on their prognostic risks. Patients with lower expression of SLAMF1 had a shorter TTFT. and O.S. In CLL miRNAs play an important role in the pathogenesis of the disease. The deletion of the miR-15a and miR-16-1, involved in the Bcl2 pathway, causes the development of CLL. It could be possible that miRNAs are responsible for the downregulation of SLAMF1 in CLL patients. To clarify the role played by these miRNAs in the regulation of SLAMF1 expression it was performed a luciferase assay. The miRNAs selected as possibly responsible for SLAMF1 regulation were the ones up-regulated in CLL: miR-17/92, miR132 and miR-148a. The results of the luciferase assay show no difference in SLAMF1 expression based on the actions of miRNAs. This indicates that miRNAs are not responsible for the dysregulation of SLAMF1. The mechanisms remain unknown. Due to the clinical and molecular heterogeneity of CLL, a better understanding of the molecular pathway that regulates the development of CLL is important for highlighting new possible treatment targets. Although CK is a negative prognostic marker, in this study, it was proven that specific cytogenetic anomalies could worsen the CK prognostic significance. Also, the expression of SLAMF1, easy to measure on the surface of B lymphocytes, could represent a new prognostic marker. These findings will be helpful to separate the patients into risk categories and to select the best treatment.
CUNEO, Antonio
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2488207
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