Determinanti genetici di resistenza alla fludarabina nella LLC

Presentazione orale EHA 2013

La resistenza alla fludarabina si verifica in circa il 5-10% dei pazienti con leucemia linfatica cronica (LLC) trattati con questo farmaco. Una dettagliata analisi genomica è stata condotta nei casi di LLC resistenti alla fludarabina, e presentata al recente 18° meeting annuale dell’EHA a Stoccolma, allo scopo di descrivere lo spettro di alterazioni genetiche presenti in questi pazienti e individuare nuovi determinanti molecolari di resistenza alla fludarabina. Accanto a diversi geni mutati ricorrenti, inclusi i già descritti TP53 (presenti nel 27.5% dei campioni prelevati alla diagnosi in 58 pazienti con LLC fludarbina-resistenti), NOTCH1 (24.1%), SF3B1 (17%) and BIRC3 (15.5%), sono stati individuati nuovi geni, fra i quali FAT1 è risultato il più comune (10,3%). FAT1 codifica per una proteina cadherin-like e le sue mutazioni inducono modifiche aminoacidiche nei domini caderinici e intracitoplasmatici della proteina, simili a quelle descritte nei tumori solidi, nei quali l’inattivazione di FAT1 promuove il signaling di Wnt e la crescita tumorale.  La frequenza delle mutazioni di FAT1 fra i casi fludarabina-resistenti era significativamente più alta rispetto a quella osservata nei pazienti non selezionati con LLC alla diagnosi (1,1%), suggerendo un ruolo specifico nella resistenza alla fludarabina.

L’analisi delle Copy Number Alterations (CNA) condotta tramite SNP array su 39 pazienti fludarabina-resistenti ha anche mostrato un maggiore carico di variazioni del numero di copie di DNA nei campioni di pazienti resistenti alla fludarabina (4,8/caso) rispetto a quanto descritto in letteratura nei pazienti con LLC alla diagnosi (~2/caso, Edelmann et al, Blood 2012). Inoltre, il 10% dei casi erano caratterizzati da un numero particolarmente alto di CNA all’interno di un singolo cromosoma, dato consistente con il fenomeno della cromotripsi, una  frammentazione del DNA in molti segmenti riuniti poi in maniera non accurata, nel quale si vengono a creare simultaneamente da poche unità a varie centinaia di mutazioni.

Questi risultati indicano FAT1 come un nuovo gene frequentemente mutato nei casi di LLC resistenti alla fludarabina, con un potenziale ruolo funzionale nella malattia. FAT1 si aggiunge quindi al numero di determinanti genetici attualmente conosciuti di fludarabina-resistenza (NOTCH1, SF3B1, BIRC3, oltre a TP53), complessivamente presenti in circa l’85% dei casi resistenti.

L’abstract completo dello studio presentato all’EHA è riportato in basso:

GENETIC DETERMINANTS OF FLUDARABINE-RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

Monica Messina, Ilaria Del Giudice , Davide Rossi, Sabina Chiaretti, Silvia Rasi, Valeria Spina, Hossein Khiabanian, Antony B. Holmes, Marilisa Marinelli, Giulia Fabbri, Alessandra Rossi, Anna Guarini, Laura Pasqualucci, Raul Rabadan, Riccardo Dalla-Favera, Gianluca Gaidano, Robin Foà.

Division of Hematology, Department of Cellular Biotechnology and Hematology, Sapienza University, Rome, 

Background: The current first line standard of care for fit CLL patients is fludarabine, cyclophosphamide and rituximab, which offers overall response and complete remission rates of 95% and 44%, respectively. Fludarabine-refractoriness (FR), defined as failure to respond or relapse within 6 months of therapy, accounts for 5-10% of treated patients, and continues to represent a challenging clinical problem. The only established player in determining FR is TP53, disrupted in 30-40% of FR-CLL. More recently, next generation sequencing studies led to the identification of new genes (NOTCH1, SF3B1, BIRC3) that are frequently mutated in FR-CLL. However, a comprehensive evaluation of the FR-CLL genome has not been reported.

Aims: We explored the genetic landscape of FR-CLL to provide a comprehensive description of the load and spectrum of alterations that are associated with this condition and to identify novel molecular determinants of FR.

Methods: We integrated Whole-Exome Sequencing (WES, Agilent SureSelect Human All Exon 50Mb, Illumina HiSeq 2000) and Copy Number (CN) analysis (SNP 6.0 arrays, Affymetrix) to study 10 FR-CLL cases sampled prior to the treatment that resulted in refractoriness and their paired germline DNA (discovery panel). Candidate somatic mutations were verified by Sanger sequencing and the entire coding region of recurrently mutated genes (>1 case) was then sequenced in an independent set of 48 FR-CLLs, of which 29 were also subjected to CN analysis. Mutation recurrence was compared versus 174 unselected CLL at diagnosis, to determine preferential association with FR-CLL.

Results: In the 10 discovery cases, we identified 163 non-silent sequence variants (average 16.3/sample, range 9-23), distributed in 151 genes and predominantly represented by missense mutations; additionally, 40 CNAs were found (average 4/sample, range 0-13), accounting for an overall load of genomic alterations of ~20 lesions/sample (range 11-26). The extended analysis of 58 FR-CLLs revealed several recurrently mutated genes – including the previously reported TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (17%) and BIRC3 (15.5%) – but also novel candidates, with the tumor suppressor related gene FAT1, encoding for a cadherin like protein, being the most common (10.3%). FAT1 mutations introduce amino acid changes in the cadherin and intracytoplasmic domains, and are similar to those recently reported in multiple solid tumors, where inactivation of FAT1 promotes Wnt signaling and tumor growth, consistent with a tumorsuppressor role. Importantly, the frequency of FAT1-mutated cases in FR-CLL was significantly higher than that recorded in unselected CLLs at diagnosis (N=2/174, 1.1%, p=0.004), suggesting a specific role in FR. SNP array analysis of 39 patients showed a broader load of CNAs in FR-CLL (4.8/case), than in untreated CLLs described in the literature (~2/case, Edelmann et al, Blood 2012). Furthermore, 10% of cases were characterized by a particularly high number of CNAs within one single chromosome, consistent with the chromotripsis phenomenon.

Summary / Conclusion: FAT1 is a novel commonly mutated gene in FR-CLL (10.3%), which is rarely detected in unselected CLL cases at diagnosis, suggesting a potential functional role in the disease. Patients with FR-CLL present a higher burden of CNAs and chromotripsis compared to untreated CLL described in the literature. The contribution of FAT1 mutation, CNAs and chromotripsis to FR in CLL is under investigation.