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  • 1
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 4400-4400
    Abstract: Background Optimization of pre-transplant risk assessment is a crucial issue to improve the allo-HSCT decision making process. Actually 3 major algorithms are in use in clinical practice: the EBMT risk score, the Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) score and - more recently introduced - the refined Disease Risk Index (DRI). DRI was defined to calibrate HSCT outcome across studies and centers. It was developed as a tool to assign patients into risk groups based on disease type and status at the time of transplantation. The aim of the DRI is to provide a robust tool that can be used for prognostication, for the analysis and interpretation of retrospective data, whether conducted in single-center, multicenter, or registry settings, or within the context of the federally mandated center outcome reporting. The DRI can also be used for the stratification of patients entering prospective HCT clinical trials. DRI is not a fixed tool but instead it was conceived to be refined by the transplant community as new information becomes available. Here we are presenting the results of a retrospective study designed to evaluate the 3 aforementioned score in stratification and prognostication of transplant outcome after a haploidentical HSCT (haplo-HSCT). Patients and Methods We included 220 adult patients (pts - 138 male, 82 female) who underwent a haplo-HSCT for hematologic malignancies, between 2006 and 2014 and were reported to our Institutional database. Risk assessment score and outcome analysis included all consecutive pts receiving an haplo-HSCT as 1st allogeneic transplantation. Pts receiving haplo-HSCT as 2nd or 3rd HSCT were excluded from the present analysis. Median age was 49 years (range, 15-77). The cohort included a broad representation of diseases (138/220 acute leukemia, 30 Hodgkin lymphoma); 62 pts were in complete remission at transplant, 158 were presenting active disease. Conditioning regimens mostly rely upon the combination of treosulfan plus fludarabine (201/220) and total body irradiation (range 200 - 400 cGy) was utilized in 52 patients. GVHD prophylaxis consisted mostly of an mTor inhibitor (rapamycin) combined with mycophenolate mofetil. The majority of patients received peripheral blood stem cells from a family haploidentical donor as stem cell source, while only 4 patients received bone marrow transplant. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. Results The median follow-up for survivors was 37 months (r 6-107). The overall survival (OS) at 2-y was 35% and the transplant related mortality at 100-days 23%. The 2y OS according to EBMT / HCT-CI / DRI risk score are reported in table 1.a and figure 1. The evaluation of the HCT-CI impact after DRI stratification was able to show a significant difference in outcome showing better survival for pts with low DRI score and low HCT-CI score as expected (table 1.b). Discussion Refined DRI score and HCT-CI score predict survival after haplo-HSCT. The integrated application of refined DRI and HCT-CI may improve the definition of transplant eligibility for pts candidate to allogeneic HSCT form alternative donors including family haploidentical source. Table 1a. EBMT score 0-3 % pts 4-5 %pts 〉 5 %pts p 51% 17 34% 51 27% 32 0.07 HCT-CI score 0-2 3-4 〉 /= 5 48% 59 36% 31 0% 10 0.0001 DRI score Low-Intermediate High Very-High 61% 32 27% 51 5% 17 0.0001 Table 1b. HCT-CI 0-4 HCT-CI 〉 /=5 p DRILow-Intermediate 64% 0% 0.0001 DRIHigh-Very High 29% 0% Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
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    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 2
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 4488-4488
    Abstract: Abstract 4488 Introduction Definition of leukemia remission requires the lack of disease markers at sub-microscopic level. This is highly important after a potentially curative approach as allogeneic hematopoietic stem cell transplant (HSCT), where early detection of relapse at molecular level may lead to modulation of immunosuppressive therapy or donor lymphocyte infusion (DLI). In AML various approaches has been used to define MRD, but still the majority of AML cases do not have a useful and sensitive MRD marker. Over-expression of Wilms' tumor gene 1 (WT1) in leukemic blasts has been reported in 〉 80% of AML and 〉 40% of MDS. Physiologic hematopoietic stem cell compartments also express WT1, however, a ‘malignant’ WT1 expression can be clearly distinguished based on quantitative detection methods such as semiquantitative RT-PCR. Quantification of WT1 expression levels can detect frequencies of leukemic cells in bone marrow (BM) and peripheral blood (PB) as low as 10-3 and 10-5, respectively. Therefore WT1 expression levels provide a new marker for leukemic blast cells regardless of the type of leukemia especially in the relevant percentage of patients that lacks a specific molecular marker of their disease and thus may be a useful marker MRD and may predict the relapse after allogeneic HSCT. Materials and methods We measured quantitative expression of WT1 at diagnosis, before and after allogeneic transplant monthly for the first six months and then every three months. The quantitative assessment of the WT1 transcript amount was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Results Our study included 19 AML and 6 MDS pts who underwent allogeneic HSCT in our Institute between 12/2007 and 6/2009. Median age at diagnosis was 49 years (range 22-68). Bone marrow samples at diagnosis showed a WT1 median expression level of 5851.66 copies (range 77.98-31203.57). Fifteen pts (60%) had a specific cytogenetic marker that could allow MRD monitoring. At HSCT 17 pts (68%) were in CR, 5 (20%) had a refractory or relapsed disease, while 3 (12%) were transplanted upfront. Six pts (24%) received grafts from a matched sibling donors, 6 (24%) were transplanted from a matched unrelated donor (MUD), 10 (40%) from a familiar haploidentical donor and 3 (12%) received a cord blood unit. Myeloablative conditioning regimen consisted of Treosulfan 42 g/sqm, Fludarabine 150 mg/sqm, ALG 30 mg/kg and Rituximab 500 mg (last two drugs only for alternative donors). After HSCT a rapid decline of WT1 expression levels was observed in all pts that obtained or maintained a condition of CR. Two pts (8%) relapsed and both had an increase in WT1 expression before relapse. In the first relapsed pt, analysis of WT1 showed a dramatically increase between pre transplant level and day +28, while STR showed 100% donor chimerism. Relapse occurred on day +43, still on IST, with 69% of blast at AM evaluation and 40% donor chimerism. This pt was successfully reinduced with chemotherapy followed by allogeneic PBSC infusion without GvHD profilaxys obtaining a rapid reduction of WT1 (43.66). The pt developed GvHD that required a new IST, confirming a strong immune-surveillance of HSCT. The second pt was still on immunosuppressive treatment (IST) at the time of relapse (+136). WT1 in BM on day +30 was 30 cp and then increased gradually to 167 and 190 cp on day +67 and +102, respectively. Cytogenetic analysis and STR chimerism still showed a cCR with 100% donor chimerism. At relapse AM showed 10% blasts, WT1 expression level was 7447 cp with 〉 95% donor chimerism. IST was discontinued and one month later WT1 expression level decreased to 1640 with 〉 95% donor chimerism. In this situation we reasoned that DLI was the best available treatment. The total dose of donor T cell infused was 5 ×105 CD3/Kg. This procedure allowed an immune-mediated leukemia control with reduction of WT1 (1.58), cCR and 100% donor chimerism. Conclusions These data show that WT1 may be useful as a non-specific leukemia marker for monitoring MRD in AML and MDS after allogeneic HSCT and should enable the detection of early relapse allowing intervention at a more favourable stage than at overt relapse. We observed a complete concordance between WT1 expression levels and status of AML before and after allogeneic HSCT. Based on these results cases with increase of WT1 levels after HSCT and without GVHD may be candidate to immune intervention such as discontinuation of immunosuppression and/or DLI. Disclosures: Bonini: MolMed S.p.A.: Consultancy.
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    Publisher: American Society of Hematology
    Publication Date: 2009
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  • 3
    In: American Journal of Hematology, Wiley, Vol. 87, No. 2 ( 2012-02), p. 230-232
    Type of Medium: Online Resource
    ISSN: 0361-8609
    Language: English
    Publisher: Wiley
    Publication Date: 2012
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  • 4
    In: American Journal of Hematology, Wiley, Vol. 86, No. 11 ( 2011-11), p. 914-917
    Type of Medium: Online Resource
    ISSN: 0361-8609
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2011
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  • 5
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2385-2385
    Abstract: Abstract 2385 Background. Allogeneic haemopoietic stem cell transplantation (HSCT) is the best therapeutic option for high-risk hematological disease (HRHD), particularly acute myeloid and lymphoblastic leukemia (AML/ALL). A widely application of HSCT is limited by lack of availability of a suitable donor for every candidate patients (pts). In order to offer a donor to every candidate pts, several centers had developed in the last decade alternative strategy of HSCT, such as umbilical cord blood (UCB) or family haploidentical HSCT (haplo-HSCT). With the aim to treat high-risk leukemia in the ideal appropriate time by allogeneic HSCT, we adopted a policy relying upon HLA typing at entry of every pts diagnosed with HRHD, followed, in absence of a MRD, by the prompt activation of the MUD search. Patients lacking a MRD or a MUD at the appropriate timing are proposed for haplo-HSCT. Methods. Here we report an intention-to-treat analysis of alternative donor HSCT at our Institution for pts diagnosed with high risk AML or ALL. Data were obtained from local institutional database. Results. Between Jan-2004 and July-2010 241 pts (median age 48y, r 15–72) with diagnosis of ALL (60 pts; median age 33y, r 15–64; male 39) or AML (181 pts, median age 51y, r 19–72; male 83) were defined as “high risk status” and received an indication to HSCT according to EBMT (European Group for Blood and Marrow Transplantation) and Northern Italy Leukemia Group (NILG, www.nilg.it) recommendations. In the ALL group 3 pts died before proceed to HSCT, 5 pts are waiting for the identification of a donor. Overall, 52/60 (86%) pts underwent HSCT, the status of disease at transplant was of complete remission (CR) for 27 pts, persistence of disease (PD) for 25. In the AML group 6 pts died before proceed to HSCT, 15 pts are waiting for the identification of a donor. Overall, 160/181 (88%) pts underwent HSCT, the status of disease at transplant was of CR for 97 pts, PD 63. Overall 92 pts activated the research of a MUD, 42 proceed to transplant, 7 received a UCB, 26 received a haplo-HSCT due to absence of a suitable donor in the appropriate time frame or failure to met the criteria to engage a MUD donor. The median time from diagnosis to registry activation was 69 days (r 5–876), the median time from activation to transplant 84 days (r 28–348). In the group of pts in CR at transplant, 37 underwent HSCT from a MRD and 36 from a MUD, 4 pts received a UCB and 47 a haplo-HSCT. Seventy-two pts are alive and in CR at last follow-up, 3/72 after a second transplant from a different donor (haplo-HSCT) and 3/72 after chemotherapy and adoptive immunotherapy to treat hematological relapse. Fifty-two pts died and the causes of death were: relapse (27), infection-related (19), graft-versus-host-disease (GvHD; 5), acute myocardial infarction (1). In the group of pts in PD at transplant, 16 underwent HSCT from a MRD and 6 from a MUD, 3 pts received UCB and 63 a haplo-HSCT. Fifteen pts are alive, 14 in CR, 1 pts under adoptive immunotherapy; 1/14 pts received a second transplant from a different donor (haplo-HSCT) to treat hematological relapse. Seventy-three pts died and the causes of death were: relapse (43), infection (23), GvHD (7). Overall, the median survival is 382 days and the median follow-up for pts alive is 548 days. The 1-year overall survival (OS)/transplant related mortality (TRM)/relapse incidence (RI) are 50,76% 26,96% and 40% respectively. For pts transplanted in CR the 1y OS/TRM/RI are 77,2%, 20,01% and 25% respectively. The outcome analysis per donor source (MRD vs MUD vs haplo-HSCT) is comparable (p=ns). For pts transplanted in PD the 1y OS/TRM/RI are 19,7%, 41% and 67% respectively. The outcome analysis per donor source (MRD vs MUD vs haplo-HSCT) shows a trend of lower RI and TRM in the haplo-SCT vs MRD. Conclusion. The policy adopted provided an allogeneic HSCT in 〉 80% of candidate high-risk acute leukemia patients. No significant differences were registered in outcome for patients transplanted from matched-related, unrelated or family haploidentical donors. Further evaluation and a long-term follow-up will add important evaluation in term of long-term disease control and long-term toxicities. Disclosures: Bonini: MolMed: Consultancy. Bordignon: Molmed: Employment.
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    Publication Date: 2010
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  • 6
    In: British Journal of Haematology, Wiley, Vol. 186, No. 5 ( 2019-09)
    Type of Medium: Online Resource
    ISSN: 0007-1048 , 1365-2141
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    Publisher: Wiley
    Publication Date: 2019
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  • 7
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 5118-5118
    Abstract: Background Targeted therapy with Tyrosine-Kinase-Inhibitors (TKIs) totally modified the course of treatment of Chronic Myeloid Leukemia (CML). The objectives and the needs of treatment have been modified during the last years and the discontinuation of therapy is now a feasible aim. However, a lot of biological data acquired in the last twenty years, showed that degree and mechanisms of Leukemic Stem Cells (LSCs) clearance during TKI treatment are not clearly established as well as the predictive criteria for a stable and prolonged Treatment Free Remission (TFR). The multicentre, prospective, single-arm PhilosoPhi34 study (EudraCT: 2012-005062-34) was designed by the Rete Ematologica Lombarda (REL), to verify the in-vivo activity and time-course of first-line Nilotinib (NIL) therapy on Bone Marrow (BM) CD34+/lin-Ph+ cells clearance. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin- cells at diagnosis and at 12 months (mos) of treatment, for the first 30 evaluable pts, was included. Preliminary GEP data suggested a correlation between different NFKBIA expression at diagnosis and at 12 mos and the achievement of a deeper Molecular Response (MR) (Pungolino et al, AJH 2018). We report here some results of GEP analysis on all enrolled evaluable pts and their possible correlation with clinical data. Methods BM cells were collected and stored at diagnosis and at 12 mos of treatment. CD34+/lin- cells were purified with a Diamond CD34 Isolation Kit Miltenyi (97% of purity). For GEP analysis we used Affymetrix HG-U133 Plus 2.0 microarray and Genechip platform (Affymetrix) and the Affymetrix GeneChip Scanner 3000. Data was pre-processed and normalized using the Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression. P-values were corrected for multiple testing using false discovery rate, for differentially expressed genes confirmation. We chose to analyse different expression of NFKBIA (the inhibitor of NF-kB onco-gene) in order to confirm the preliminary data reported on the first 30 analysed pts. Pts were monitored according to ELN-recommendation. Biological data were correlated with MR at 3, 12 and 36 mos of therapy. We use Fishers test to compare unbalanced group. Results Out of the 87 enrolled pts, 80 completed the first 12 mos of treatment and 78 (1 failure and 77 CCyR) were evaluable for GEP analysis. We observed 2726 genes symbol differentially expressed of which 1868 are coding genes. Among these, JAK-2 showed a down regulation at 12 mos (p: .024). JAK-2 expression ranged from 2.62 to 4.95 at diagnosis and from 1.48 to 5.58 at 12 mos. Only 26/78 pts increased JAK-2 expression that was 〉 4 in 1/26 pts, at diagnosis; 2/26 (7.69%) pts showed a H Sokal. Other 52/78 pts decreased JAK-2 expression that was ≥ 4 in 21/52 pts, at diagnosis; 10/52 (19.23%) pts and 6/21 (28,57%) pts showed a H Sokal. Similarly, when we compared low JAK-2 expression ( 〈 3.5) vs vary high expression (≥ 4) 2/21 vs 6/22 pts had H Sokal (9.52% vs 27.27%; p: .0057). Considering the role of JAK-2 and NFKBIA in cell regulation and survival, we evaluated how the combination of their different expression impact on MR (i.e. NFKBIA increased expression/JAK-2 decreased expression vs NFKBIA decreased expression/JAK-2 increased expression). Data are reported in Table 1 and 2. Conclusion GEP analysis showed a down regulation of JAK-2 expression after 12 mos of first line NIL treatment, in 78 early chronic phase CML pts. Data suggest that high expression of JAK-2, at diagnosis, correlate with H Sokal score. However, H Sokal pts with a JAK-2 down regulation, obtain during treatment similar MR compared to L Sokal pts. Additionally, the study confirms our preliminary observation on 30 pts , concerning the role of NKBIA up - regulation in increasing percentage of earlier and deeper MR . The better condition of NFKBIA and JAK-2 expression (up regulation of NFKBIA and down regulation of JAK-2) is associated with a higher percentage of early MR3 and optimal responses over time, despite the higher number of H Sokal pts in this group. A study with NIL as first line treatment combined with low dose of JAK-2 inhibitor and a natural inhibitor of NF-kB (such as curcuma), during the first year of treatment, to increase the deeper MR rate and the probability of TFR is warrented. Disclosures Rossi: Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Novartis: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.
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    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 8
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 1960-1960
    Abstract: Abstract 1960 BACKGROUND: Human herpesvirus 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Reported clinical manifestations of HHV-6 infection in transplanted patients are skin rash, interstitial pneumonia, bone marrow suppression and encephalitis. Moreover, an increasing number of clinical reports suggest that HHV-6 can facilitate the occurrence of other severe clinical complications of allogeneic HSCT, including Graft-versus-Host Disease (GvHD), ultimately increasining transplant-related mortality. Still, the actual incidence of HHV-6 infection in recipients of HSCT and the causative link between infection and clinical complications remain elusive, mostly due to the small and heterogeneous patient cohorts analyzed to date. METHODS: From January 2009 to July 2011, we retrospectively evaluated 43 consecutive adult patients (median age 51 years) who developed positivity to HHV-6 after allogeneic HSCT for high-risk hematological malignancies. Stem cell donor was for 30 patients family haploidentical, for 5 an HLA identical sibling, and for 8 an unrelated volunteer (1 of which cord blood). The viral load was determined by quantitative PCR in cell-free body fluids such as plasma, bronchoalveolar lavage, cerebrospinal fluid, bone marrow aspirates or in gastrointestinal biopsies. At the time of positivity all patients were receiving acyclovir as viral prophylaxis except 5. Sixteen patients had clinical acute GvHD at time of HHV-6 positivity (grade III-IV in 14), and 33 were profoundly immunosuppressed with variable association of 2–4 immunosuppressive drugs (steroids included). Moreover concomitant CMV positivity was detected in 11 patients, while a severe neutropenia in 12. RESULTS: Median time from allogeneic HSCT to HHV-6 reactivation was 36 days (range: 7–625). In 19 patients HHV-6 was detected in plasma, with a median viral load of 19,454 cp/mL (34-4,524,600); 15 had concomitant fever, 5 skin rash of new onset, 4 impaired liver function, and 5 developed cytopenia subsequently to the infection. In 7 patients HHV-6 was detected in the bone marrow: the median viral load was 163'800 cp/mL (568-1'552'982). In 8 patients, all febrile, HHV-6 was observed in bronchoalveolar lavage samples with a median of 4'149 cp/mL (85–39250). In 16 patients, 10 with documented gut aGvHD, 11 with diarrhoea, HHV-6 was detected in gastrointestinal biopsies with a median of 7'510 cp/mL (120-4'524'600). HHV-6 was found in cerebrospinal fluid in 4 cases (all within 30 days after HSCT); the median viral load was 29'352 cp/mL (4'508-1'552'982); all these patients experienced encephalitis with confusion and anxiety, 2 suffered seizures and 3 showed abnormal findings on brain MRI. Amongst patients with organ localizations of HHV-6 only 28% had concomitant plasma positivity. HHV-6 positivity led to antiviral pharmacological treatment only when associated with clinical manifestations (n=21), using as first choice therapy foscarnet. Amongst the total 43 patients with documented HHV-6 positivity 11 completely solved the clinical event, whereas 19 (44%) died. CONCLUSIONS: HHV-6 infection/reactivation is associated with high morbidity and mortality in patients who undergo allogeneic HSCT. HHV-6 infection typically occurred close to the time of neutrophil engraftment. HSCT from an HLA-mismatched donor and steroid administration were associated with increased risk of active HHV-6 infection. Development of encephalitis was associated with high HHV-6 viral load. The regular monitoring of HHV-6 DNA in allogeneic HSCT recipients, using a real-time PCR assay, may be useful for identifying active HHV-6 infection and for the introduction of a pre-emptive treatment, possibly reducing the incidence of the most severe clinical complications. Disclosures: No relevant conflicts of interest to declare.
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    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 9
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 3080-3080
    Abstract: Background CML is a clonal disorder characterized by the presence of the Philadelphia (Ph) chromosome which encodes for the bcr-abl tyrosine-kinase (TK). Target therapy with the TK inhibitors (TKIs)) has greatly improved its outcome. Treatment with second generation TKIs - e.g. nilotinib (NIL) - results in deeper and faster responses and prevents disease progression. Sustained responses may enable TKI discontinuation. However, even in the event of qPCR negativity, a fraction of patients (pts) experience disease recurrence possibly due to persistence of quiescent leukemic stem cells (LSCs). Degree and mechanisms of LSCs clearance during TKI treatment are not established yet and conflicting results are reported in the literature. Work from the group of Bocchia (Bocchia 2008; Defina 2012) showed reduction of LSCs during long term imatinib (IM) therapy; moreover, in CCyR pts residual LSCs are more rarely detected after NIL compared to IM therapy and, in a small fraction of pts this occurs after very short-term NIL therapy. This data conflicts with in vitro evidence that NIL is not superior to IM in inducing growth suppression in CML LSCs (Konig, 2008). To verify the in vivo activity and time-course of first-line NIL therapy on bone marrow (BM) Ph+ stem cells (CD34+/lin-) clearance, on behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34) was designed. Primary efficacy endpoint was to measure the rate of BM residual CD34+/lin-Ph+ cells in CCyR pts at 6 months of treatment. Methods BM cells were collected and stored at diagnosis and at 3,6 and 12 mos of treatment. CD34+/lin- cells were purified using a Diamond CD34 Isolation Kit Miltenyi (97% of purity). FISH analysis of selected unstimulated CD34+/lin- cells was performed according to standard procedures; considering the low sensitivity of the test, in order to define the test as negative at least 200 nuclei were examined. The A'Hern single stage design was chosen for the present study; considering the CCyR results obtained in the ENESTnd study and the anticipated number of un-evaluable tests, a minimun of 87 pts were required. Results Enrolment of the 87 pts was completed by June 2015. Table 1 summarises pts' characteristics and response to treatment. FISH results are as follows: at 3 mos, 8/65 (12,3%; CI 95%: 2,3%-15,7%) evaluable FISH tested positive (10 negative tests not evaluable); at 6 mos 5/71 (7%; CI 95% :2,3-15,7%) evaluable FISH tested positive (7 negative tests not evaluable); at 12 mos, 0/68 (0%; CI95%:0,0-5,2%) evaluable FISH tested positive (9 negative tests not evaluable). At any time point, Sokal score did not predict for FISH results. However, as outlined in Table 2, H-Sokal score pts are less prevalent among pts who achieve a CCyR, a requirement for FISH analysis. Of the 4 pts who failed the treatments' objectives by 12 mos, 1 was in CCyR with detectable residual CD34+/lin-Ph+ cells at 3 mos; 2 were not in CCyR and with residual CD34+/lin-Ph+ cells at 3mos; 1 was in CCyR and with CD34+/lin-Ph- cells at 3 and 6 mos but with increasing qPCR. Only 1 pt with CD34+/lin-Ph- cells at all time points and with optimal molecular response harboured a NIL-resistant mutation at 26 mos of treatment. None of the 22 pts (including 4 H-Sokal score pts = equal proportion of study cohort) in Molecular Response (MR) 3.0 at 3 mos had a positive FISH at 3 and 6 mos or failed treatment at follow-up. Conclusion. Our final results on the whole cohort of pts confirm our preliminary data on the efficacy of NIL 300 g BID in early clearance of BM LSCs (CD34+/lin-Ph+) in newly diagnosed CP-CML patients tested at 3, 6 and 12 mos of treatment. Moreover, according to our data, fast disease debulking seems crucial for obtaining BM LSCs clearance and it can be speculated that the same mechanism responsible for this early MR 3.0 achievement is also capable of preventing H-Sokal risk pts from failing treatment. Disclosures Orlandi: Ariad: Honoraria; BMS: Honoraria; Novartis: Honoraria.
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    ISSN: 0006-4971 , 1528-0020
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    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 10
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 5122-5122
    Abstract: Introduction Chronic myeloid leukemia (CML) is a stem cell disease characterized by the constitutive activity of the oncoprotein BCR-ABL that activates multiple signal transduction pathways. Tyrosine-kinase inhibitor (TKI) nilotinib successfully inhibits the activation and the proliferative function of BCR-ABL in patients with CP-CML. Despite the success of nilotinib, some patients become refractory suggesting the presence of a population of Philadelphia positive (Ph+) quiescent stem cells escaping the drug activity. Thus, the molecular mechanisms underlying CML remain poorly understood. In this study, we enrolled 87 CP-CML patients (Pungolino et. al. Am J Hematol. 2018). Samples were collected on the behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34), which included 15 centers from Italy. We undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 80 patients at diagnosis vs. the same patients after 12 months of nilotinib to investigate gene expression changes induced by the treatment. Methods We isolated CD34+/lin- cells from BM samples in 87 patients at diagnosis whereas the same cells were also selected from 80/87 patients after 3, 6 and 12 months of nilotinib (Trojani et. al. Cancer Biomark. 2017). BM mononuclear cells (MNCs) as well as BM CD34+/lin- cells of all 80 CML patients were counted at diagnosis and during the treatment with nilotinib (at 3, 6, and 12 months, respectively). Standard FISH tested isolated BM CD34/lin- cells for the 87 patients at diagnosis, and for 80/87 patients after 3, 6 and 12 months of nilotinib treatment, respectively. Therefore, we performed GEP analyses of selected BM CD34+/lin- cells of 80/87 patients at diagnosis vs. the same patients after 12 months of nilotinib treatment. Then, we executed bioinformatic preprocessing and correction for batch effects on raw microarray data. Finally, we conducted differential expression analysis and significantly perturbed genes were subjected to functional clustering. Results We observed a wide variability of the number of BM MNCs as well as the number of the BM CD34+/lin- cells among the 80 CP-CML patients at diagnosis and after 3, 6 and 12 months of nilotinib for each patient (Table 1). Figure 1 showed that the number of the BM CD34+/lin- cells dramatically decreased between the diagnosis and after 3 as well as 6 months of nilotinib. We noticed that the BM CD34+/lin- cells slightly increased between 6 and 12 months of nilotinib which might be caused by the gradual repopulation of the normal CD34+/lin- cells in the bone marrow as FISH results suggested. FISH analysis detected CD34+/lin- Ph+ cells in 87 CP-CML patients at diagnosis. No positive Ph+ nuclei were detected on CD34+/lin- cells of 79/80 patients after 12 months of treatment (to categorize a sample as negative, at least 200 nuclei were examined). All of these 79 patients achieved at least complete cytogenetic response. 1/80 patient relapsed at 12 months. We conducted GEP analyses on 78 subjects because, due to experimental issues, two patients were not considered for differential expression analyses, as the microarray CEL files of the 12 months' samples were corrupted and missed probe intensities for most of the probes. GEP analyses determined 2,959 significantly differently expressed probes between diagnosis and after 12 months of nilotinib treatment. Functional clustering identified some pathways significantly enriched between diagnosis and 12 months of nilotinib. Among these pathways, we found that ABCC4, ABCC5, and ABCD3 genes associated with ATP-binding cassette (ABC) transporters were up regulated at diagnosis. GEP results highlighted that 26 genes belonging to cell cycle, mitosis, DNA damage and repair were over expressed at diagnosis. Moreover, GEP data demonstrated that JAK-STAT signaling pathway was deregulated: JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, SOS1 were over expressed whereas IL22RA1 was under expressed at diagnosis vs. 12 months of nilotinib, respectively. Conclusions In summary, we reported that BM CD34+/lin- cells from CP-CML patients after 12 months of nilotinib were characterized by changes of expression of genes involved in cell cycle checkpoints and mitosis, ABC transporters genes that pump drugs outside form the cells, and JAK-STAT signaling pathway genes responsible for the proliferation, differentiation and cell survival in CML. Disclosures Rossi: Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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