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Campeggio, Mimma (2018) Prognostic Role of Minimal Residual Disease before and after Haematopoietic Stem Cell Transplantation in pediatric ALL patients and evaluation of droplet digital PCR applicability in pre-HSCT MRD monitoring. [Tesi di dottorato]

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Abstract (italiano o inglese)

Acute Lymphoblastic Leukemia (ALL) represents the most frequent cancer in childhood. Currently, more than 80% of children with ALL can be cured through intensive and risk-adapted chemotherapy protocols, but unfortunately, the remaining 20% ultimately relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered beneficial for approximately 10% of patients who are at high risk (HR) at frontline therapy according to the AIEOP-BFM protocol criteria, and for the majority of patients after ALL relapse. However, also after HSCT, relapse remains the leading cause of treatment failure in pediatric ALL.
The strongest prognostic factor in childhood ALL is the monitoring of Minimal Residual Disease (MRD). MRD is defined as the persistence, in bone marrow (BM), of leukemic cells not identifiable through cyto-morphological methods. MRD diagnostics has been implemented into major frontline treatment protocols for pediatric ALL, in which it is routinely used to stratify patients into different risk classes: standard risk (SR), medium risk (MR) or high risk (HR) of relapse. The aim of MRD-based stratification is to refine therapy based on risk-class, maximizing cure and minimizing toxicities.
Also for relapsed ALL patients and in patients undergoing HSCT, MRD assessment has been identified as one of the most relevant predictors of prognosis, useful to identify good and poor responders to the therapy. Nevertheless, the clinical significance of MRD in pediatric ALL patients given allogeneic HSCT has not yet been fully validated.
The most widely used approach to detect MRD is represented by real-time quantitative PCR (RQ-PCR), a very sensitive and specific molecular assay. RQ-PCR is based on the patient-specific junctional regions of Immunoglobulin (Ig) and T-cell Receptor (TCR) genes rearrangements, detected on BM aspirates collected at diagnosis (or relapse) of ALL patient.
In the first project of my PhD training (described in Chapter 1) we quantify MRD by RQ-PCR immediately before HSCT, in order to assess its clinical significance and impact on transplant outcome in a large cohort (119) of pediatric ALL patients in first, second or subsequent complete remission (respectively 1CR, 2CR or others CR). In addition, we consecutively analyzed MRD by RQ-PCR in 98/119 and 59/119 ALL patients, respectively during the first (post-HSCT1) and third (post-HSCT3) trimester after HSCT. The aim of these analyses was to address the question of whether MRD evaluation could provide further information to predict the risk of post-transplant leukemia recurrence.
The overall 10-year event-free survival probability (EFSp) for patients with any level of positive MRD pre-HSCT was lower (39% for MRD < 1x10-3 and 18% for MRD ≥ 1x10-3) as compared with negative MRD patients (EFSp = 73%). When patients were analyzed according to the number of CR at HSCT, we observed that different levels of positivity had a different impact on EFSp: low-level MRD positivity had a negative impact only in patients transplanted in second or higher CR; while in first CR, only a high MRD positivity increased the risk of relapse. So pre-transplant MRD assessment confirmed to be a strong predictor of outcome and its effect was consistent throughout the different disease remissions.
We also evaluated the EFSp according to the MRD assessment at post-HSCT1 and post-HSCT3. MRD negativity at early post-transplant was associated with a good EFSp (63%), that was even better when negativity was confirmed also at 3th trimester post-HSCT (pEFS = 84%).
Also the variations of MRD levels over time were important. In particular the change between 1st and 3th trimester allowed to identify 2 categories of patients, with a dramatically different outcome: a group of patients with very poor prognosis (patients with an MRD increasing from post-HSCT1 to post-HSCT3) with an EFSp of only 8%, and a group of patients with very good prognosis (patients with unchanged negative MRD or decreasing to negative MRD and those with unchanged low-positive MRD) with an EFSp ≥ 80%.
Overall, these results confirm that MRD assessment is important both before and after transplant, for early identification of patients with the highest risk of ALL recurrence and with a strong indication to a prompt immunological intervention and to adoption of new drugs.
The second project (described in Chapter 2) was a preliminary study. We focused on a third generation PCR, the droplet digital PCR (ddPCR), that allows for an absolute quantification, with accurate concentration of target DNA. Instead, RQ-PCR allows for a relative quantification, since is based on the comparison with a calibration standard curve made with the diagnostic DNA of patient, for MRD level quantification in follow-up sample. Thus, availability of diagnostic sample can limit RQ-PCR assay.
A broad spectrum of molecular markers has been yet interrogated using ddPCR for diagnostic purposes in various malignancies. Recently, the absolute method was evaluated for MRD quantification in lymphoproliferative disorders of adult, such as lymphomas and ALL; these reports showed a good correlation between quantitative PCR and ddPCR. However, there are still no studies in pediatric ALLs.
In the light of this, we performed ddPCR analyses on BM samples of 65 pediatric ALL transplanted patients with the same primers and probes used for RQ-PCR and in the same reaction conditions. Comparing head-to-head the MRD results obtained with the two molecular approaches, we aimed to investigate the applicability of ddPCR for MRD assessment also in this context. First, we evaluated if positive but not-quantifiable (PNQ) MRD performed by RQ-PCR can be quantified by ddPCR; then we also evaluated the prognostic impact of pre-HSCT MRD levels assessed by ddPCR.
A good level of concordance was found in results of both analyses (Pearson r = 0.98, P < 0.0001) and ddPCR was also able to quantify a various number of sample not-quantifiable by conventional RQ-PCR. Our results suggest that ddPCR has sensitivity, accuracy and reproducibility at least comparable with RQ-PCR.
Statistical analyses have shown no significant differences in prognostic impact on outcome, if patients were stratified according to MRD levels detected by RQ-PCR and ddPCR, since EFSp of PNQ patients was very similar to that of MRD NEG by ddPCR (71% vs 68%, respectively). Despite this, the digital method was able to measure a positive and quantifiable value for 12 ALL patients who relapsed after HSCT, while RQ-PCR technique failed to identify relapse in advance.
These preliminary data confirm that ddPCR may be an accurate and applicable tool for MRD evaluation also in the context of pediatric ALL clinical trials, but highlight the importance of extending the analysis on other retrospectively collected cases, to better define the role of ddPCR for prospective MRD evaluation in pediatric ALLs.

Abstract (seconda lingua)

La Leucemia Linfoblastica Acuta (LLA) rappresenta la patologia tumorale più frequente in età pediatrica. Oltre l’80% dei bambini affetti da LLA viene trattato con successo grazie agli attuali protocolli di chemioterapia intensiva e basata sul rischio di ricaduta, ma sfortunatamente, il restante 20% ricade. Il trapianto di cellule staminali ematopoietiche (TCSE) ha un ruolo fondamentale nella guarigione di circa il 10% dei pazienti definiti ad alto rischio di ricaduta LLA in prima linea e per gran parte dei pazienti recidivati. Sfortunatamente, anche dopo il TCSE, la ricaduta si conferma come principale causa di fallimento terapeutico nelle LLA pediatriche.
Il principale indicatore prognostico nelle LLA infantili è rappresentato dalla Malattia Residua Minima (MRM). La MRM è definita come la persistenza, all’interno del midollo osseo, di cellule leucemiche a livelli non identificabili attraverso esame citomorfologico. La valutazione della MRM è ormai parte integrante dei principali schemi terapeutici di prima linea, in cui viene usata per stratificare i pazienti in diverse classi di rischio di ricaduta (standard, intermedio o alto), con l'obiettivo di adattare la terapia al rischio individuale di ciascun paziente, ottimizzando le cure e riducendo al minimo la tossicità.
Il monitoraggio della MRM è stato identificato come uno dei maggiori fattori predittivi di prognosi, anche per i pazienti ricaduti e per quelli trapiantati, in cui risulta ulteriormente vantaggioso per valutare la risposta alla terapia dei pazienti LLA. Ciononostante, il significato clinico della MRM nei pazienti sottoposti al TCSE non è ancora stato pienamente validato.
L’approccio standard utilizzato per monitorare la MRM è attualmente rappresentato dalla real-time quantitative PCR (RQ-PCR), un saggio molecolare altamente sensibile e specifico, basato sulle regioni giunzionali dei riarrangiamenti dei geni delle immunoglobuline e del recettore dei linfociti T, identificati sugli aspirati midollari della diagnosi (o ricaduta) del paziente LLA.
Nel primo progetto (descritto nel capitolo 1) del mio percorso di dottorato, abbiamo quantificato la MRM mediante PCR quantitativa immediatamente prima del TCSE, per valutare il suo significato clinico e l’impatto sull’outcome in una vasta coorte di pazienti pediatrici affetti da LLA (119), in prima remissione completa (1RC), seconda (2RC) o altre. Abbiamo poi analizzato MRM mediante RQ-PCR in 98/119 e 59/119 pazienti, rispettivamente durante il primo (post-TCSE1) e il terzo (post-TCSE3) trimestre dopo il trapianto. L’obiettivo di queste analisi è stato quello di capire se la valutazione MRM potesse fornire ulteriori informazioni, utili a identificare preventivamente i pazienti con maggior rischio di ricaduta leucemica dopo il trapianto.
Dalle analisi di sopravvivenza in relazione ai livelli di MRM pre-TCSE nei pazienti LLA, qualsiasi livello di positività correla con un outcome sfavorevole (pEFS = 39% per MRM positiva < 1x10-3 e pEFS = 18% per MRM positiva ≥ 1x10-3), rispetto ai pazienti con MRM negativa (pEFS = 73%, P<0.0001). Inoltre, analizzando i pazienti in base al tipo di remissione al TCSE, livelli diversi di positività MRM correlano con un diverso impatto sulla pEFS: bassi livelli di positività MRM indicano infatti, una prognosi sfavorevole solo in pazienti trapiantati in seconda o altre RC, mentre in prima RC solo una positività alta si associa ad un aumentato rischio di ricaduta. Pertanto la MRM pre-TCSE si conferma come importante fattore predittivo di outcome e il suo effetto varia col variare del tipo di remissione al trapianto.
È stata valutata, inoltre, la pEFS dei pazienti in base ai livelli di MRM post-TCSE1 e post-TCSE3; MRM negativa post-TCSE correla significativamente con un outcome favorevole, sia al 1° trimestre (pEFS = 63%), che ancor più se riscontrata al 3° trimestre (pEFS = 84%).
Anche la valutazione prospettica del cambiamento di MRM è risultata significativa. In particolare, valutando la variazione di MRM dal 1° al 3° trimestre post-TCSE, i pazienti con MRM crescente hanno una prognosi sfavorevole (pEFS = 8%), mentre tutti gli altri gruppi correlano con una buona prognosi (pEFS ≥ 80%).
Questi risultati confermano l’importanza del monitoraggio della MRM sia nel periodo precedente che successivo al TCSE, nell’identificare preventivamente pazienti ad alto rischio di ricaduta, possibili beneficiari di interventi immunologici preventivi.
Il secondo progetto trattato (descritto nel capitolo 2) è stato uno studio preliminare, focalizzato su una PCR di terza generazione, la Droplet Digital PCR (ddPCR). Essa consente una quantifica di tipo assoluto, con un’accurata concentrazione del DNA target. La RQ-PCR fornisce, invece, una quantifica di tipo relativo, basata su una curva standard di calibrazione fatta con il DNA della diagnosi del paziente, per la quantificazione dei livelli di MRM di ciascun follow-up. Per cui, la PCR quantitativa può essere limitata dalla disponibilità di materiale diagnostico.
Un ampio spettro di marcatori molecolari è già stato indagato mediante ddPCR per scopi diagnostici in varie patologie tumorali. Studi recenti hanno valutato l’applicabilità della ddPCR nell’ambito delle malattie linfoproliferative dell’adulto, come i linfomi e le LLA, mostrando una buona correlazione dei risultati fra le due metodiche in entrambi gli ambiti. Tuttavia, non sono ancora disponibili lavori che valutino questa correlazione nel campo delle leucemie pediatriche.
Alla luce di questo, abbiamo eseguito analisi ddPCR sugli aspirati midollari di 65 pazienti pediatrici sottoposti a TCSE, utilizzando stessi primer e stesse sonde fluorescenti usati negli esperimenti RQ-PCR, nelle medesime condizioni di reazione. Mettendo a confronto i livelli di MRM emersi coi due approcci molecolari, si è investigata l’applicabilità della metodica assoluta per il monitoraggio della MRM anche in questo contesto. Inizialmente, sono stati valutati campioni risultati, mediante RQ-PCR, positivi ma non quantificabili (PNQ), per verificare se invece si potessero quantificare mediante ddPCR. Successivamente, è stato valutato anche l’impatto prognostico dei livelli MRM pre-TCSE ottenuti tramite ddPCR.
Un buon livello di concordanza è emerso dai risultati ottenuti con entrambe le metodiche (Pearson r = 0.98, P < 0.0001); la ddPCR ha permesso, inoltre, di quantificare numerosi campioni risultati non quantificabili tramite RQ-PCR. I risultati suggeriscono che il metodo assoluto possieda sensibilità, accuratezza e riproducibilità almeno paragonabili alla PCR quantitativa convenzionale.
I pazienti LLA analizzati sono stati stratificati sulla base dei livelli di MRD ottenuti con le due tecniche molecolari, ma nelle analisi di sopravvivenza non sono emerse differenze significative sulla prognosi. Infatti le pEFS dei pazienti con MRM negativa e positiva quantificabile per i due metodi risultano molto simili (rispettivamente 71% e 68%).
Ciononostante, dal presente studio è emerso che col metodo digital sia stato possibile misurare un valore di MRM positivo e quantificabile per almeno 12 pazienti LLA che, in seguito al trapianto, hanno presentato una recidiva; viceversa, la RQ-PCR non era stata in grado di identificare anticipatamente la ricaduta di questi pazienti.
Questi dati preliminari mostrano che la ddPCR possa essere un valido strumento per il monitoraggio della MRM e applicabile anche nel contesto dei trials clinici per pazienti LLA pediatrici. Tuttavia una prosecuzione dello studio ddPCR, con estensione della casistica analizzata, potrebbe essere utile a definire con precisione la significatività delle misurazioni con questa recente metodica.

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Tipo di EPrint:Tesi di dottorato
Relatore:Basso, Giuseppe
Correlatore:Buldini, Barbara
Dottorato (corsi e scuole):Ciclo 30 > Corsi 30 > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE SANITARIA
Data di deposito della tesi:15 Gennaio 2018
Anno di Pubblicazione:15 Gennaio 2018
Parole chiave (italiano / inglese):Malattia Residua Minima / Minimal Residual Disease, Leucemia Linfoblastica Acuta / Acute Lymphoblastic Leukaemia, Trapianto di Cellule Staminali Ematopoietiche / Haematopoietic Stem Cell Transplantation, bambini / children, leucemia / leukaemia, ricaduta / relapse, PCR digitale / droplet digital PCR
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/06 Oncologia medica
Struttura di riferimento:Dipartimenti > Dipartimento di Salute della Donna e del Bambino
Codice ID:10874
Depositato il:26 Ott 2018 09:08
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