Federica Cattina

Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.

CDKN2A/B Alterations Impair Prognosis in Adult BCR-ABL1–Positive Acute Lymphoblastic Leukemia Patients

Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1–positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis–relapse samples were further available and analyzed for 19 (19%) cases. Results: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014). Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1–positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413–23. ©2011 AACR.

Prospective Phase II Study on 5-Days Azacitidine for Treatment of Symptomatic and/or Erythropoietin Unresponsive Patients with Low/INT-1–Risk Myelodysplastic Syndromes

Purpose: This phase II prospective study aimed to evaluate the efficacy and safety of 5-days azacytidine (5d-AZA) in patients with low-risk myelodysplastic syndromes (MDS). Second, single-nucleotide polymorphism (SNP) genetic profile and phosphoinositide-phospholipase C (PI-PLC) β1 levels were studied to evaluate possible biologic markers able to predict the hematologic response. Experimental Design: The study tested a lower intensity schedule of azacytidine. The treatment plan consisted of 75 mg/sqm/d subcutaneous administered for 5 days every 28 days, for a total of 8 cycles. Results: Thirty-two patients were enrolled in the study. The overall response rate was 47% (15 of 32) on intention-to-treat and 58% (15 of 26) for patients completing the treatment program. In this latter group, 5 (19%) achieved complete remission (CR) and 10 (38%) had hematologic improvement, according to the International Working Group (IWG) criteria. Three patients have maintained their hematologic improvement after 37, 34, and 33 months without other treatments. Moreover, 21 and 2 of 26 cases completing 8 cycles were transfusion-dependent for red blood cells and platelets at baseline, respectively. Of these, 7 (33%) and 2 (100%) became transfusion-independent at the end of the treatment program, respectively. Grade 3–4 neutropenia occurred in 28% of patients and 4 patients died early due to infections or hemorrhage. SNP results were not significantly correlated to the clinical outcome, whereas PI-PLCβ1 level anticipated either positive or negative clinical responses. Conclusions: 5d-AZA is safe and effective in a proportion of patients with low-risk MDS. PI-PLCβ1 gene expression is a reliable and dynamic marker of response that can be useful to optimize azacytidine therapy. Clin Cancer Res; 19(12); 3297–308. ©2013 AACR.

Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positive acute lymphoblastic leukemia in complete response

Monitoring of minimal residual disease (MRD) by quantification of BCR-ABL1 transcript levels has become a main part of the management of patients with BCR-ABL1-positive acute lymphoblastic leukemia (ALL) in treatment with tyrosine kinase inhibitors (TKIs). The failure to achieve molecular negativity shortly after starting TKI has been demonstrated to be predictive of relapse, suggesting that an accurate measurement of low BCR-ABL1 levels may have a role in preventing hematological relapse. Despite the big efforts made by many European laboratories within the European Study Group, at the time of writing a standardized procedure to quantify and express results is still missing for BCR-ABL1-positive ALL. In this study, in order to detect with high sensitivity low levels of BCR-ABL1 transcripts, we used a new technology and a new molecular approach based on microfluidic digital polymerase chain reaction (dPCR) using Taqman chemistry and we compared obtained results with those generated by the conventional method based on reverse transcriptase PCR reaction (RQ-PCR) for BCR-ABL1 and total ABL1, with TaqMan chemistry and with Applied Biosystems instrument. We demonstrated the dPCR is high-sensitive (able to detect a single copy of BCR-ABL1) and reliable (results are comparable to those obtained by BCR-ABL1 quantification with conventional technology), allowing an accurate monitoring of BCR-ABL1-positive ALL patients in complete remission.

EXTRACORPOREAL PHOTOPHERESIS (ECP) FOR TREATEMENT OF ACUTE AND CHRONIC GRAFT VERSUS HOST DISEASE: AN ITALIAN MULTICENTRIC RETROSPECTIVE ANALYSIS ON 94 PATIENTS ON BEHALF OF THE GRUPPO ITALIANO TRAPIANTO DI MIDOLLO OSSEO (GITMO)

Background Extracorporeal photopheresis (ECP) is considered a valid second-line treatment for acute and chronic graft versus host disease (GVHD). Methods Ninety-four patients with acute GVHD (aGVHD) (n = 45) and chronic GVHD (cGVHD) (n = 49), retrospectively recruited in 6 Italian centers, were submitted to ECP for second-line treatment. At the time of ECP, 22 (49%) and 23 (51%) of 45 patients with aGHVD were nonresponsive and in partial remission (PR) after steroids, respectively, and all the 49 patients with cGVHD were steroid refractory. Results Forty-one (91%) of 45 patients with aGVHD achieved complete remission (CR) after ECP. Fifteen (33%) of 45 patients developed cGVHD. The CR rate in patients who started ECP being nonresponsive and in PR after steroid was 86% and 96%, respectively. After a median follow-up of 20 months (range, 2-72), 15 (33%) of 45 patients developed cGHVD and 16 (35%) of 45 patients died, in 3 cases for aGVHD. A trend for a better survival was seen among patients who started ECP in PR after steroid (80% vs 50% at 2 years; P = 0.07). Overall, 22 (45%) of 49 patients and 17 (35%) of 49 patients with steroid refractory cGHVD achieved CR and PR after ECP, respectively. After a median follow-up of 27 months, 44 (90%) of 49 patients are alive, 21 of whom (48%) are on steroid. Conclusions Extracorporeal photopheresis is confirmed as an effective second-line treatment in both aGVHD and cGVHD, because it can induce a response in more than 80% of the patients and a long-term survival in at least 50% of the cases.

Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph−) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph− ALL.

Profiling of drug-metabolizing enzymes/transporters in CD33+ acute myeloid leukemia patients treated with Gemtuzumab-Ozogamicin and Fludarabine, Cytarabine and Idarubicin

Genetic heterogeneity in drug-metabolizing enzyme/transporter (DMET) genes affects specific drug-related cancer phenotypes. To investigate the relationships between genetic variation and response to treatment in acute myeloid leukemia (AML), we genotyped 1931 variants on DMET genes in 94 CD33-positive AML patients enrolled in a phase III multicenter clinical trial combining Gemtuzumab-Ozogamicin (GO) with Fludarabine–Cytarabine–Idarubicin (FLAI) regimen, with the DMET Plus platform. Two ADH1A variants showed statistically significant differences (odds ratio (OR)=5.68, P=0.0006; OR=5.35, P=0.0009) in allele frequencies between patients in complete/partial remission and patients without response, two substitutions on CYP2E1 (OR=0.13, P=0.001; OR=0.09, P=0.003) and one on SLCO1B1 (OR=4.68, P=0.002) were found to differently influence liver toxicity, and two nucleotide changes on SULTB1 and SLC22A12 genes correlated with response to GO (OR=0.24, P=0.0009; OR=2.75, P=0.0029). Genetic variants were thus found for the first time to be potentially associated with differential response and toxicity in AML patients treated with a combination of GO–FLAI regimen.

Postremission sequential monitoring of minimal residual disease by WT1 Q-PCR and multiparametric flow cytometry assessment predicts relapse and may help to address risk-adapted therapy in acute myeloid leukemia patients

Risk stratification in acute myeloid leukemia (AML) patients using prognostic parameters at diagnosis is effective, but may be significantly improved by the use of on treatment parameters which better define the actual sensitivity to therapy in the single patient. Minimal residual disease (MRD) monitoring has been demonstrated crucial for the identification of AML patients at high risk of relapse, but the best method and timing of MRD detection are still discussed. Thus, we retrospectively analyzed 104 newly diagnosed AML patients, consecutively treated and monitored by quantitative polymerase chain reactions (Q‐PCR) on WT1 and by multiparametric flow cytometry (MFC) on leukemia‐associated immunophenotypes (LAIPs) at baseline, after induction, after 1st consolidation and after 1st intensification. By multivariate analysis, the factors independently associated with adverse relapse‐free survival (RFS) were: bone marrow (BM)‐WT1 ≥ 121/104 ABL copies (P = 0.02) and LAIP ≥ 0.2% (P = 0.0001) (after 1st consolidation) (RFS at the median follow up of 12.5 months: 51% vs. 82% [P < 0.0001] and 57% vs. 81%, respectively [P = 0.0003]) and PB‐WT1 ≥ 16/104 ABL copies (P = 0.0001) (after 1st intensification) (RFS 43% vs. 95% [P < 0.0001]) Our data confirm the benefits of sequential MRD monitoring with both Q‐PCR and MFC. If confirmed by further prospective trials, they may significantly improve the possibility of a risk‐adapted, postinduction therapy of AML.

Peripheral Blood WT1 Expression Predicts Relapse in AML Patients Undergoing Allogeneic Stem Cell Transplantation

To evaluate if WT1 expression may predict relapse after allo-SCT, we analyzed WT1 levels on peripheral blood (PB) and bone marrow (BM) before and after allo-SCT in 24 AML patients with WT1 overexpression at diagnosis. Five copies of WT1/ABL × 104 from PB were identified as the threshold value that correlated with relapse after allo-SCT. The same correlation was not identified when WT1 expression was assessed from bone marrow (BM). Eight out of 11 (73%) patients with a pre-allo-SCT PB-WT1 ≥ 5 and 4/13 (31%) patients with a pre-allo-SCT PB-WT1 < 5 relapsed, respectively (P = 0.04). The incidence of relapse was higher in patients with PB-WT1 ≥ 5 measured after allo-SCT, at the 3rd (56% versus 38%; P = 0.43) and at the 6th month (71% versus 20%; P = 0.03). Patients with pretransplant PB-WT1 < 5 had significantly better 2-year OS and LFS than patients with a PB-WT1 ≥ 5 (81% versus 0% and 63% versus 20%) (P = 0.02). Our data suggest the usefulness of WT1 monitoring from PB to predict the relapse in allotransplanted AML patients and to modulate the intensity of conditioning and/or the posttransplant immunosuppression in an attempt to reduce the posttransplant relapse risk.

Treatment of Chronic Myeloid Leukemia Elderly Patients in the Tyrosine Kinase Inhibitor Era

The prevalence of chronic myeloid leukemia (CML) is expected to double in the next 15 years. The introduction of imatinib significantly changed the prognosis of CML, challenging the concept of a fatal disease. Nowdays, imatinib, nilotinib and dasatinib are registered for first-line treatment of CML patients in chronic phase (CP). Considering elderly patients, the most extensively studied TKI is imatinib, that induces a rate of cytogenetic and molecular responses comparable between the younger and the elderly patients. Once a CCgR with imatinib is achieved, the probability to be alive and disease free at 8 years is more than 80%. These results confirm that imatinib has to be considered the first-line treatment for the elderly and that the CCgR is the guide parameter for treatment modulation and the most solid marker of long term outcome. Nevertheless, older patients tolerate imatinib worse in comparison to the younger, and this causes a higher rate of therapy discontinuation and less adherence to chronic treatment. Thus, the toxic profile of each TKI is one of the most important factors driving the choice of the best drug. Another important factor is the potency of the TKI. Since nilotinib and dasatinib are more potent than imatinib in inducing cytogenetic and molecular responses, they could be preferred for increasing the proportion of patients who can achieve deeper molecular responses, allowing treatment discontinuation. This approach is intriguing, but it is still experimental. Another therapeutic strategy could be the identification of the minimal effective dose of TKI in order to maintain the CCgR, but also this approach is under clinical investigation.