1,721,390 research outputs found
"Southern blot analysis as ALL-I rearrangements at chromosome 11q23 in acute leucemia"
No full textThe chromosome 11q23 band is a genetic region frequently involved in nonrandom karyotypic abnormalities of acute leukemia. A genomic locus named ALL-1 or MLL, where 11q23 breakpoints are clustered, has been recently cloned and characterized. We have made use of an ALL-1-specific probe in Southern blot experiments to analyze the configuration of this gene in a large series of acute leukemia patients, representative of all different myeloid and lymphoid subtypes. Nine of 145 cases (6.2%) showed abnormal ALL-1 restriction fragments in leukemic DNAs. Of these nine cases, five patients in whom karyotypic data were available displayed chromosome 11q23 aberrations, including t(4;11) (three cases) and t(9;11) (two cases). Immunophenotypic and morphocytochemical characterization of ALL-1-rearranged acute leukemia revealed prevalence of poorly differentiated B lymphoid and/or monoblastic features. Considering the whole series, ALL-1 rearrangements were significantly associated with female sex, higher white blood cell counts at presentation, and very poor clinical outcome. The presence of residual disease was molecularly documented in one case at the time of clinical remission after induction treatment and was followed by early relapse. We conclude that ALL-1 rearrangements are new molecular markers of human leukemia with considerable diagnostic and prognostic relevance
All1 gene at chromosome 11q23 is consistently altered in acute leukemias of early infancy
No full textEarly infancy (< 1 year of age), massive tumor cell burden, and extremely poor prognosis are characteristic features of a particular subset of childhood acute leukemias (AL). In these cases, chromosome aberrations at the 11q23 band are the most frequently reported cytogenetic abnormalities. We have recently cloned a genetic locus named ALL-1, in which DNA breakpoints are clustered in leukemic patients with 11q23 aberrations. Analysis of the ALL-1 genomic configuration in DNA from 15 infants with AL showed specific ALL-1 rearrangements in 12 cases (80%), including 5 with normal karyotypes. These findings indicate that a consistent genetic defect underlies this particular leukemic subse
Retinoic Acid and Arsenic Trioxide sensitize Acute Promyelocytic Leukemia cells to ER stress
No full textPromyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15:17) that results in the expression of the chimeric protein PML-RARα. The fusion of PML, a tumor suppressor that is the major component of the PML-nuclear bodies, with the Retinoic Acid Receptor-α arrests the differentiation program driven by RARα, blocking the leukemic blasts at the promyelocytic stage. Pharmacological doses of Retinoic Acid (RA) are able to remove the block, resume granulocytic differentiation and partially degrade PML-RARα leading to reformation of nuclear bodies. The association of RA with chemotherapy or with arsenic trioxide (ATO), the latter efficiently targeting PML-RARα for degradation, results in high cure rates of acute promyelocytic leukemia (APL). Despite showing a considerably improved safety profile, either RA or ATO are not devoid of toxicity, with the most important and potentially life-threatening one being the so-called retinoic acid differentiation syndrome. We show here that RA-induced differentiation of human APL cell lines and primary blasts dramatically increases their sensitivity to ER stress inducing drugs, like Tunicamycin (Tm), at doses that are not toxic in the absence of RA. Importantly only human progenitors cells derived from APL patients resulted sensitive to the combined treatment with RA and Tm whereas those obtained from healthy donors were not affected. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response, a series of pathways emanating from the ER in case of ER stress, which ensues when higher protein folding activity is required as during differentiation. Although mild, the ER stress induced by RA is sufficient to render differentiating APL cells very sensitive to low doses of Tm. We also show that the UPR pathway downstream of PERK plays a major protective role against ER stress in differentiating cells and, by using a specific PERK inhibitor, we potentiated the toxic effect of the combination of RA and Tm. Moreover we found that low amounts of pharmacologically induced ER stress are also able to strongly increase ATO toxicity even in the absence of RA. Indeed the combination of ATO with Tm efficiently induced apoptosis in RA-sensitive and RA resistant APL cell lines, at doses ineffective in the absence of ER stress. Eventually, we demonstrate that insurgency of oxidative stress, tightly linked with the UPR, is at the basis of the toxicity induced by Tm in combination with RA and/or ATO. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML
Patient-reported outcomes in hematology: Is it time to focus more on them in clinical trials and hematology practice?
No full textIn less than 2 decades, major clinical advances have been made in various areas of hematologic malignancies. Clinicians and patients now frequently face challenging choices regarding various treatments that are often similar in regard to safety or clinical effectiveness; hence, medical decision making has grown in complexity. For example, several novel drugs have been developed as oral agents, introducing an additional challenge in patient management, such as ensuring an optimal adherence to therapy in order to maximize drug effectiveness. This rapidly changing scenario provides a rationale for a more systematic collection of patient-reported outcomes (PRO) in clinical research and routine care. In the former case, PRO may help to better understand overall treatment effectiveness of a new drug being tested. In the latter case, it may aid in making more informed, individualized treatment decisions in daily practice by obtaining more accurate information on the actual symptom burden experienced by the patient. In any case, evaluating PRO requires making several, and often challenging, decisions depending, for example, on the population being studied and the specific setting. Therefore, if PROs are to fulfill their potential of generating clinically meaningful data that robustly inform patient care, special attention should be given to methodological rigor. We outline the value of a more systematic and rigorous implementation of PRO assessment in the current hematology arena, by providing some real world examples of how PRO data have contributed in better understanding the value of new therapies. We also discuss practical considerations in PRO assessment in clinical research
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Impiego del triossido di arsenico nella leucemia acuta promielocitica in recidiva molecolare
Full text linkLa leucemia acuta promielocitica(LAP) è un sottotipo di leucemia mieloide acuta che ha caratteristiche molecolari e cliniche peculiari. Nella maggior parte di casi di LAP,circa il 98%, le cellule leucemiche sono caratterizzate dalla presenza della traslocazione t(15;17), che determina la fusione del gene RARa al gene PML. In conseguenza di questa traslocazione si ha il blocco maturativo delle cellule leucemiche allo stadio di promielocita nella differenziazione granulocitaria. Il trattamento attuale della LAP di solito include una fase di induzione con acido trans retinoico(ATRA) e chemioterapia con antracicline, seguita da una fase di consolidamento che si basa su tre cicli di chemioterapia con antracicline e ATRA e poi una fase di mantenimento con ATRA con o senza basse dosi di chemioterapia per 1-2 anni. Questa strategia terapeutica determina un alto tasso di remissione completa (CR) di circa il 90% e una sopravvivenza globale a 5 anni di circa l’ 80%.Circa il 5%-30% dei pazienti va incontro a recidiva di malattia, principalmente i pazienti con LAP ad alto rischio. Infatti, la recidiva non è comune nei pazienti affetti da forme a rischio basso-intermedio (<10%), mentre il tasso di recidiva nei pazienti ad alto rischio è di circa il 20% (1-2).
La recidiva in sedi extramidollari si verifica approssimativamente in circa il 3% -5% dei pazienti (3). I trattamenti della recidiva di LAP includono l’utilizzo del triossido di arsenico (ATO), dell’anticorpo monoclonale anti-CD33 (gemtuzumab o Mylotarg), e il trapianto di cellule staminali ematopoietiche.
Il triossido di arsenico ha dimostrato efficacia terapeutica anche in pazienti affetti da leucemia acuta promielocitica con malattia avanzata, es. in seconda recidiva dopo più cicli di chemioterapia e /o trapianto autologo o allogenico.
Il meccanismo di azione del triossido di arsenico non è completamente noto.
In vitro, nelle cellule della leucemia acuta promielocitica umana, il triossido di arsenico provoca cambiamenti morfologici e la frammentazione dell’acido desossiribonucleico(DNA) caratteristica dell’apoptosi. Il triossido di arsenico inoltre provoca danno o degradazione della proteina di fusione PML/RARa.
Sono stati presi in esami i tre pazienti trattati presso il nostro Centro. Due pazienti maschi, uno di 46aa ed uno di 16aa ed una paziente femmina di 38aa.
Il paziente di 46aa e la paziente di 38aa erano all’esordio a rischio intermedio, mentre il paziente di 16aa era ad alto rischio. I precedenti trattamenti effettuati sono stati basati in tutti e 3 i casi nel protocollo AIDA.
La fase di malattia era per il paziente di 46aa una prima recidiva molecolare a sette anni dalla 1a RC, per la paziente di 38aa era una prima recidiva molecolare a quattro anni e per il paziente di 16aa una prima recidiva molecolare a 10 mesi.( Tab.1)
Tabella 1. Caratteristiche dei pazienti in studio
Pt Età/S Rischio Terapia di 1 linea Durata 1 RC Tipo di
recidiva
1 46/M Intermedio AIDA 84 m molecolare
+extramid.*
2 38/F Intermedio AIDA2000 48 m molecolare
3 16/M Alto** AIDA2000 10 m molecolare
*Sarcoma mieloide paravertebrale tra D6 e D8.
**WBC 113.000/mmc
Al momento della recidiva, il paziente numero uno ha presentato una localizzazione paravertebrale di sarcoma mieloide come unica sede di recidiva di leucemia promielocitica. Alla risonanza magnetica della colonna effettuata, si è evidenziata la comparsa di una massa solida extramidollare localizzata tra la sesta e l’ottava vertebra. Gli esami ematochimici effettuati comprendenti emocromo e coagulazione completa sono risultati nella norma. Inoltre non sono stati riscontrati promielociti atipici nè all’esame morfologico del sangue periferico nè a livello midollare, confermando pertanto una remissione completa di malattia a livello morfologico, mentre la PCR documentava la presenza di malattia a livello molecolare nel midollo. Pertanto il paziente è stato sottoposto a laminectomia decompressiva, con escissione della massa che all’esame istopatologico mostrava un diffuso infiltrato di elementi monomorfi di taglia media, con nuclei indentati o bilobati, cromatina dispersa,numerosi nucleoli e citoplasma basofilo. Le caratteristiche immunofenotipiche delle cellule neoplastiche erano le seguenti: mieloperossidasi e CD117(c-KIT) fortemente positivi; CD45 debolmente e focalmente espresso, mentre CD34,CD31,CD20;CD79a,CD3,CD56 e CD15 erano negativi. Gli studi di citogenetica sono risultati positivi per la traslocazione t(15;17).
Terapia della recidiva
I tre pazienti in recidiva di LAP sono stati trattati con Triossido di Arsenico ed ATRA in associazione. (Tab.2)
Tabella 2. Terapia della recidiva
Pt Induzione Consolidamento
1 ATO 0.15mg/Kg x 28g
ATRA 45mg/m2/die ATO 5 gg/sett x 4 sett q 4 sett e ATRA q 2 sett
(4 cicli tot)
2 ATO 0.15mg/Kg x 28g
ATRA 45mg/m2/die ATO 5gg/sett x 4 sett q 4 sett e ATRA q 2 sett
( 4 cicli tot)
3 ATO 0.15mg/Kg x 28g
ATRA 25mg/m2/die 1 ciclo+
TMO allogenico
Inoltre i pazienti hanno eseguito terapia intratecale in profilassi con methotrexate e cortisonici. Durante l’intera durata del trattamento nessuno dei pazienti ha presentato complicanze degne di nota.
Risposta alla terapia
I tre pazienti in studio hanno ottenuto una seconda remissione molecolare senza complicanze e sono attualmente rispettivamente in RCm rispettivamente da 13, 22 e 27 mesi. (Tab.3)
Tabella 3. Outcome
Pt Remissione Molecolare Follow-up
(m. in 2RC) Osservazioni
1 Post 1 cons. 13
2 Post 1 cons. 22 Gravidanza a termine dopo
9 mesi dal cons.
3 Post 1 cons. 27
Discussione
Il paziente che ha recidivato dopo sette anni ha presentato un sarcoma mieloide (SM) o sarcoma granulocitico. Il sarcoma mieloide è un raro tumore extramidollare che origina dalle cellule mieloidi immature. Si può verificare in associazione con leucemia mieloide acuta, disordini mieloproliferativi e mielodisplasia. Il SM può presentarsi in diversi modi e colpire qualsiasi organo. Si può verificare in individui in perfetta salute che successivamente sviluppano una leucemia mieloide tipica, o può anche svilupparsi contemporaneamente alla comparsa della leucemia, o dopo la diagnosi od essere una manifestazione della recidiva di malattia dopo il raggiungimento della remissione completa.
Secondo la WHO esistono tre varianti di sarcoma mieloide che si basano sulla predominanza del tipo cellulare e sul loro grado di maturazione:
1) variante mieloblastica
2) variante mieloblasti e promielociti
3) variante promielociti e granulociti neutrofili
Il caso da noi riportato è di un paziente con LAP che dopo sette anni di completa remissione ha presentato un deficit neurologico ed in particolare una paraparesi destra.
La malattia extra midollare alla diagnosi o alla recidiva si sa che si sviluppa nel 3%-8% dei casi di leucemia mieloide acuta, più frequentemente nel tipo mielomonocitica e monoblastica, invece è relativamente rara nella forma promielocitica, ma si è visto un incremento al tempo della recidiva dopo l’avvento del’ATRA.
I siti extramidollari maggiormente coinvolti sono il sistema nervoso centrale e la cute. Sono state fatte due possibili ipotesi sulla relazione tra la malattia extramidollare al momento della recidiva in pazienti affetti da LAP e la terapia con ATRA. La prima suggerisce che il rischio di sviluppare una malattia extramidollare dopo il trattamento con ATRA è dovuto al diretto effetto di questo agente sulle molecole di adesione che determina una maggiore capacità di infiltrazione dei blasti della LAP. La seconda ipotesi e la più accettata è che il verificarsi del sarcoma mieloide alla recidiva è da considerarsi una conseguenza della più lunga sopravvivenza di tali pazienti grazie al successo dei trattamenti.
Da segnalare inoltre il caso della paziente numero due che ha portato a termine una gravidanza e dato alla luce un neonato sano dopo aver ricevuto per la recidiva un trattamento a base di triossido di arsenico (5 cicli).
Riguardo gli effetti collaterali dell’arsenico sul sistema riproduttivo, è noto che elevate esposizioni all’arsenico inorganico determinano nella donna aborti ed infertilità; tuttavia ci sono pochi dati che sono stati ricavati da studi su soggetti esposti ad acqua contenente arsenico o su soggetti che lavorano o vivono vicino a smaltitori. L’interpretazione di questi studi per valutare il reale impatto dell’arsenico sulla fertilità è tuttavia complicata dal fatto che questa popolazione di studio è esposta a molteplici sostanze chimiche (Golub et al,1998). Inoltre, studi su modelli animali hanno mostrato che l’arsenico è teratogeno, determinando malformazioni fetali e difetti di nascita quando somministrato durante la gravidanza. Diversamente dall’arsenico gli effetti della chemioterapia convenzionale sulla fertilità sono stati estesamente studiati e la loro tossicità è stata stabilita ( Nakayaama et al,2008).
La nostra paziente era stata trattata all’esordio della malattia in accordo al protocollo AIDA 2000 con induzione e tre consolidamenti ottenendo la remissione molecolare. A giugno del 2006, quattro anni dopo la fine dell’ultimo consolidamento la paziente aveva presentato la sua prima gravidanza che era però esitata in un aborto spontaneo al 3 mese di gestazione. Subito dopo veniva documentata una recidiva molecolare.
Il trattamento con triossido di arsenico è stato ben tollerato senza gravi tossicità e al termine del consolidamento è stata dimostrata una remissione molecolare completa.
Mentre il danno indotto dalla citotossicità è reversibile in altri tessuti a rapida divisione cellulare, quali il midollo osseo, il tratto gastroenterico e il timo, sembra invece essere progressivo ed irreversibile nell’ovaio, dove il numero di cellule germinali è limitato e non può essere rigenerato.
Prima dell’avvento del triossido di arsenico il trattamento delle recidive di LAP si basava su ulteriore chemioterapia seguita da una fase di intensificazione che consisteva nel trapianto di cellule staminali autologhe o nel trapianto allogenico ( Castagnola et al,1998; Lo Coco etal,1999; Thomas X et al, 2000).
Queste ultime procedure sono associate con un’elevata incidenza di fallimenti ovulatori ed infertilità.
Alla luce della moderna tendenza di utilizzare in prima linea di terapia nella leucemia acuta promielocitica il triossido di arsenico, ci si aspetta che un numero sempre maggiore di pazienti con LAP saranno trattati con ATO in futuro.
Questo permetterà una valutazione su un più esteso numero di pazienti se l’ATO rappresenterà una sicura terapia antileucemica con il rispetto della fertilità umana.
Conclusioni
Benché la nostra casistica sia molto limitata, i casi illustrati confermano la efficacia del triossido di arsenico nella LAP recidivata.
La sommistrazione di ATO precedentemente alla gravidanza non ha determinato sterilità né anomalie fetali.
Lo stesso schema ATO + ATRA è attualmente in fase di sperimentazione clinica in 1 linea. (gruppo GIMEMA
Alterazioni del gene NPM nelle mielodisplasie con delezione 5q-
No full textMyelodysplastic syndromes (MDS) include a heterogeneous group of disease characterized by dysplasia of one or more bone marrow cell lineages, usually with prominent ineffective erythropoiesis and genomic instability leading to anaemia and enhanced risk to transformation to secondary acute myeloid leukemia (AML) . Thus MDS is often diagnosed on the basis of chronic macrocytic anaemia accompanied or not by leukocytopenia and/or thrombocytopenia. The deletion of 5q (5q-) is a frequent clonal chromosomal abnormality in patients with MDS. MDS with 5q- as a sole chromosome alteration is characterized by isolated anaemia, elevated platelet count and a favourable prognosis when compared to other forms of MDS . When the 5q- accompanies additional chromosome defects, it leads to poor-risk karyotypes with dramatically different prognostic features .
NPM1 is a versatile nuclear phosphoprotein that plays multiple roles in ribosome biogenesis and transport, cytoplasmic-nuclear trafficking, centrosome duplication and regulation of p53 .
The NPM1 gene is located in chromosome 5q35 and is involved in a number of human haematopoietic malignancies, such as promyelocytic leukaemia , anaplastic large cell lymphoma , and AML. NPM1 has also been found mutated in approximately 35% of acute myeloid leukaemia cases . Furthermore the 5q region to which NPM1map is deleted in a number of MDS and loss of chromosome 5 is a frequent finding in MDS . In a recent paper, Grisendi and co-workers showed that NPM1 is essential to maintain genomic stability. They demonstrated that NPM1 is haploinsufficient for regulating centrosome duplication as NPM1 heterozygous cells show aberrant centrosome numbers, genomic instability and aneuploidy. We analyzed the presence of NPM1 gene deletion, methylation and mutations in 45 patients affected by MDS and in 5 patients with AML secondary to MDS carrying the 5q- abnormality as sole chromosomal alteration or associated with additional chromosome defects.
Group 1 (17 patients) consisted of patients who present the 5q deletion as sole anomaly as determined by cytogenetics: 10 AR (59%), 1 ARS (6%), 1 AREB (6%), 4 AREB-T (23%) and 1 AML (6%). Group 2 ( 33 patients) consisted of patients who present the 5q deletion associated with additional chromosome defects: 5 AR (15%), 1 ARS (3%), 10 AREB (24%), 13 AREB-T (46%) e 4 AML (12%) The CpG island of the NPM1 gene was unmethylated in all the samples analyzed including patients with isolated 5q- and with complex karyotype. The mutational status of NPM1 exon 12 showed wild type NPM1 in all patient. The FISH analysis of the NPM1 locus revealed deletion of one copy of the gene in 7 cases. Interestingly all the cases with NPM1 deletion are always associated with complex karyotypes and a high-risk disease. May be considered that the aploinsufficienza of the NPM1 gene is not sufficient alone to determine the occurrence of a complex karyotype but may contribute with other genetic mechanisms for its establishment.
With the test of Fisher, has shown a trend of association between the deletion of NPM1 and complex karyotype (p = 0.08); most likely by increasing the number of cases analyzed could be obtained a statistical significance
Immunophenotypic profiling of leukemic stem cells to track FLT3-ITD positive chemo-resistant clones in AML
No full textIntroduction Acute Myeloid Leukemia (AML) is characterized by heterogeneous genetic abnormalities, immunophenotypes and clinical outcomes. Although available treatments induce complete remission (CR) in ~80% of AML patients, some of them will eventually relapse, due to the emergence of resistant clones. Persistence of leukemia stem cells (LSCs) in AML patients achieving CR after chemotherapy, is known to drive disease recurrence and worsen outcome. Therefore, the identification and characterization of LSCs, that generally reside within the CD34+ /CD38- cell fraction and confer resistance to therapy, represents an important challenge. The Internal Tandem Duplication of FLT3 gene (also known as FLT3-ITD mutation), present in approximately 25% of AML patients, has been shown to occur at the LSCs level, and may be a primary event in leukemogenesis. It is also well known that FLT3-ITD mutations represent an independent predictor of poor prognosis in AML, being associated with an increased risk of relapse. According to these data, a recent publication from our group demonstrated that chemo-resistant FLT3-ITD clones, present at AML relapse, were already identifiable at subclonal level at the time of diagnosis. Presence of these subclones actually directly correlates with the detection of CD34/CD123/CD99/CD25 positive cells by multiparameter flow cytometry (MFC). In this line, it has been recently demonstrated that expression of CD99 allows for separation of LSCs from functionally normal hematopoietic stem cells in AML. Based on the preliminary identification of the MFC fingerprint associated with FLT3-ITD mutated cells, our study aimed at better characterizing the phenotypic and genetic profiles of FLT3-ITD positive AML LSC. These cells may sustain resistance mechanisms driving disease relapse, and may be ideal treatment targets. Patients and Methods Following previous results and to better identify patients at risk of relapse, we investigated the FLT3-ITD molecular status at the DNA level in a cohort of 150 patients diagnosed with AML between January 2017 and December 2018. Samples were analysed at at the time of diagnosis, of relapse, and in selected cases during follow-up, using standard procedures. Based on FLT3-ITD V molecular status and samples availability, a sequential gating strategy was carried out in 12 AML samples at diagnosis, to sort the CD34/CD123/CD99/CD25+ (CD38+ and CD38-) LSCs-enriched fraction, the CD34+ stem cell subset (CD123/CD99/CD25-), and T-lymphocytes. Cells were purified using high-speed cell sorting, in collaboration with the IRCCS Santa Lucia Foundation. In order to better characterize FLT3-ITD positive LSCs at the genome level, targeted sequencing was performed on 32 AML samples collected from the 12 AML patients at diagnosis and relapse, using the OncomineTM Myeloid Research Assay panel on the Ion TorrentTM S5 sequencer. This panel contains targeted multibiomarkers to simultaneously detect variants across 40 key genes, 29 driver genes and a broad fusion panel to cover most of the genetic changes associated with AML. Results Enrichment of the FLT3-ITD positive population, characterized by a significantly higher FLT3-ITD mutation load, was observed within the CD34+ compartment of CD123/CD99/CD25+ cells, as compared to MNC (p=0.006). Conversely, the lymphoid/myeloid precursors showed low or absent CD123/CD99/CD25 expression (p=0.002). In one patient with two different FLT3- ITD mutated clones at diagnosis, the LSC population defined by CD34/CD123/CD99/CD25+ expression was homozygous for one ITD mutated clone, likely originated by loss of heterozygosity (LOH) of the mutated allele. Furthemore, based on several studies indicating that LSCs are particularly enriched in the CD34+/CD38- cell fraction, we investigated whether the FLT3-ITD mutation was enriched in the CD34/CD123/CD99/CD25+/CD38- LSCs compartment. In one AML patient, these cells represented the dominant FLT3-ITD mutated population, as compared to the CD34+/CD38+ counterpart. Furtermore, to trace the clonal evolution of LSCs carrying the FLT3-ITD mutation, we sorted different BM-cell population at diagnosis and during follow-up from one AML patient who relapsed 8 months after the initial diagnosis. A progressive increase of the FLT3-ITD allele burden was detected in the CD34/CD123/CD99/CD25+ subset, as compared to the CD34+ cells lacking CD123/CD99/CD25 expression, and to the total MNC population. In this patient, a high FLT3-ITD mutant allele burden in in the sorted LSC was detected already at time of complete morphologic remission, and 2 months VI before haematological relapse. These data confirm that CD34/CD123/CD99/CD25+ LSC may represent the FLT3-ITD reservoir, driver of disease relapse. Finally, based on recent global genome studies carried out by the TCGA network revealing that de novo AML arises from combinations of several recurrent driver gene mutations, we perfomed targeted NGS mutation analysis on the total leukemic cell population (12 pts at diagnosis and 3 pts at relapse) and on several purified leukemia cell fractions in 7 AML patients. We observed a median of 3.5 and 3 mutations per patient at diagnosis and relapse, respectively, with 3 mutations in common in paired diagnostic and relapse samples. In addition to FLT3-ITD mutations, the most frequently mutated genes were NRAS (40% of cases), RUNX1, TET2 (30%), DNMT3A, BCOR, IDH2, NPM1, FLT3-TKD, and KRAS (20%). Mutations in DNMT3A, NPM1, IDH2, RUNX1 and TET2 genes were stable during the disease course. On the contrary, NRAS, FLT3-ITD and EZH2 mutations were lost at the time of relapse in 2 pts, confirming that leukemias may evolve by a process of clonal expansion and/or selection. Comparing the gene mutation load in the diagnostic vs relapse samples, we found an almost costant increase in the variant allele frequency (VAF) for FLT3-ITD, FLT3- TKD, NRAS and RUNX1 (from 0.47%, 2.7%, 3.3%, 3%, and 34% at diagnosis, vs 16%, 9.5%, 39% and 4.2% at relapse). Conversely, in one patient, VAF of DNMT3A and TET2 was similar at diagnosis (41% and 91%) and relapse (49% and 99%), indicating that they may represent early founder mutations, stable during disease course. On the contrary, mutations affecting other myeloid genes such as FLT3 can be gained or lost during disease progression. Aiming at shedding light on the molecular heterogeneity and subclonal structure of AML genetic, we then compared the mutational profiles of MNCs to that of CD34/CD123/CD25/CD99+ highly purified cell population and/or to the CD34+/ CD123/CD25/CD99- counterpart, available for 7 AML patients (N=11 samples). Targeted NGS analysis revealed two different clonal evolution models. In 7 of 11 cases, the same mutation pattern was present both in MNCs and LSC, suggesting that the LSC represent the same genetic background of the bulk leukemic cells. We conclude that in AML, assessment of the genetic profile at diagnosis in a powerful indicator for the presence of somatic mutations, which remain stable VII also at relapse in most cases, with the exception of FLT3-ITD mutations, which may expand at relapse. In this line, muttional analysis of MNC is informative in most cases and is representative of LSC profiles. FLT3-ITD mutate subclones are on the contrary entriched in CD34/CD123/CD99/CD25+/CD38-/+ LSC
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