Detection of T315I using digital polymerase chain reaction in allogeneic transplant recipients with Ph-positive acute lymphoblastic anemia in the dasatinib era
2020; Elsevier BV; Volume: 81; Linguagem: Inglês
10.1016/j.exphem.2020.01.001
ISSN1873-2399
AutoresYu Akahoshi, Hideki Nakasone, Koji Kawamura, Machiko Kusuda, Shunto Kawamura, Junko Takeshita, Nozomu Yoshino, Yukiko Misaki, Kazuki Yoshimura, Ayumi Gomyo, Aki Tanihara, Masaharu Tamaki, Shun‐ichi Kimura, Shinichi Kako, Yoshinobu Kanda,
Tópico(s)Chronic Lymphocytic Leukemia Research
Resumo•Detection of the T315I mutation at diagnosis and at HSCT using dPCR did not predict relapse after HSCT.•Emergence of the T315I mutation at molecular relapse after HSCT is associated with a higher risk of hematologic relapse.•Detection of the T315I mutation using dPCR is a simple approach and may help physicians switch to appropriate treatments after HSCT in the dasatinib era. Dasatinib, a potent tyrosine kinase inhibitor (TKI), is currently used as first-line treatment for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). However, emergence of the T315I mutation has been found to be a main cause of failure after dasatinib-containing treatments. We assessed the prognostic value of small clones with the T315I mutation at specific time points using the novel technology digital polymerase chain reaction (PCR), which is more sensitive than direct sequencing. This study included 25 consecutive adult patients with Ph+ ALL who underwent allogeneic hematopoietic stem cell transplantation (HSCT) following dasatinib-based chemotherapy at our center. Among six patients who experienced hematologic relapse after HSCT, four harbored the T315I mutation at relapse. However, the detection of small subclones with T315I at either diagnosis or HSCT was not associated with an increased risk of relapse. In contrast, all patients with the T315I mutation at molecular relapse after HSCT (n = 4) eventually had a hematologic relapse, and only two of the 10 patients without the T315I mutation at molecular relapse after HSCT relapsed. In conclusion, the detection of small clones with the T315I mutation at molecular relapse after HSCT, but not before HSCT, could support an early clinical decision to change treatments. Dasatinib, a potent tyrosine kinase inhibitor (TKI), is currently used as first-line treatment for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). However, emergence of the T315I mutation has been found to be a main cause of failure after dasatinib-containing treatments. We assessed the prognostic value of small clones with the T315I mutation at specific time points using the novel technology digital polymerase chain reaction (PCR), which is more sensitive than direct sequencing. This study included 25 consecutive adult patients with Ph+ ALL who underwent allogeneic hematopoietic stem cell transplantation (HSCT) following dasatinib-based chemotherapy at our center. Among six patients who experienced hematologic relapse after HSCT, four harbored the T315I mutation at relapse. However, the detection of small subclones with T315I at either diagnosis or HSCT was not associated with an increased risk of relapse. In contrast, all patients with the T315I mutation at molecular relapse after HSCT (n = 4) eventually had a hematologic relapse, and only two of the 10 patients without the T315I mutation at molecular relapse after HSCT relapsed. In conclusion, the detection of small clones with the T315I mutation at molecular relapse after HSCT, but not before HSCT, could support an early clinical decision to change treatments. Incorporation of tyrosine kinase inhibitor (TKI) has dramatically improved survival outcomes in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) [1Thomas DA Faderl S Cortes J et al.Treatment of Philadelphia chromosome-positive acute lymphocytic leukemia with hyper-CVAD and imatinib mesylate.Blood. 2004; 103: 4396-4407Crossref PubMed Scopus (462) Google Scholar, 2Yanada M Takeuchi J Sugiura I et al.High complete remission rate and promising outcome by combination of imatinib and chemotherapy for newly diagnosed BCR-ABL-positive acute lymphoblastic leukemia: a phase II study by the Japan Adult Leukemia Study Group.J Clin oncol. 2006; 24: 460-466Crossref PubMed Scopus (365) Google Scholar, 3Mizuta S Matsuo K Nishiwaki S et al.Pretransplant administration of imatinib for allo-HSCT in patients with BCR-ABL-positive acute lymphoblastic leukemia.Blood. 2014; 123: 2325-2332Crossref PubMed Scopus (41) Google Scholar]. Allogeneic hematopoietic stem cell transplantation (HSCT) may be avoided in patients who achieve an early molecular response with new-generation TKIs [4Short NJ Jabbour E Sasaki K et al.Impact of complete molecular response on survival in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.Blood. 2016; 128: 504-507Crossref PubMed Scopus (143) Google Scholar,5Jabbour E Short NJ Ravandi F et al.Combination of hyper-CVAD with ponatinib as first-line therapy for patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia: long-term follow-up of a single-centre, phase 2 study.Lancet Haematol. 2018; 5: e618-e627Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. However, given the lack of well-controlled studies, allogeneic HSCT remains the standard strategy and is offered in first complete remission (CR) if feasible. Dasatinib is a second-generation TKI that is 325-fold more potent in vitro than imatinib [6O'Hare T Walters DK Stoffregen EP et al.In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants.Cancer Res. 2005; 65: 4500-4505Crossref PubMed Scopus (918) Google Scholar]. It has recently been used as a first-line treatment in patients with Ph+ ALL [7Rousselot P Coude MM Gokbuget N et al.Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome-positive ALL.Blood. 2016; 128: 774-782Crossref PubMed Scopus (183) Google Scholar,8Ravandi F Othus M O'Brien SM et al.US intergroup study of chemotherapy plus dasatinib and allogeneic stem cell transplant in Philadelphia chromosome positive ALL.Blood Adv. 2016; 1: 250-259Crossref PubMed Google Scholar]. Furthermore, dasatinib exhibits high activity against most imatinib-resistant BCR-ABL1 kinase domain mutations. However, the major cause of relapse is the emergence of clones with the T315I mutation that are resistant to dasatinib [9Soverini S De Benedittis C Papayannidis C et al.Drug resistance and BCR-ABL kinase domain mutations in Philadelphia chromosome-positive acute lymphoblastic leukemia from the imatinib to the second-generation tyrosine kinase inhibitor era: the main changes are in the type of mutations, but not in the frequency of mutation involvement.Cancer. 2014; 120: 1002-1009Crossref PubMed Scopus (107) Google Scholar]. Rousselot et al. [7Rousselot P Coude MM Gokbuget N et al.Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome-positive ALL.Blood. 2016; 128: 774-782Crossref PubMed Scopus (183) Google Scholar] reported that the T315I mutation was detected in 75% of patients who experienced hematologic relapse after dasatinib-based chemotherapy [7Rousselot P Coude MM Gokbuget N et al.Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome-positive ALL.Blood. 2016; 128: 774-782Crossref PubMed Scopus (183) Google Scholar]. Meanwhile, ponatinib, a third-generation TKI, exhibits high activity against Ph+ ALL with BCR-ABL1 kinase domain mutations including T315I. Ponatinib-based chemotherapy might be a promising treatment for Ph+ ALL, but there remains significant concern over the higher incidence of arterial occlusive events [10Cortes JE Kim DW Pinilla-Ibarz J et al.Ponatinib efficacy and safety in Philadelphia chromosome-positive leukemia: final 5-year results of the phase 2 PACE trial.Blood. 2018; 132: 393-404Crossref PubMed Scopus (271) Google Scholar, 11Jabbour E Kantarjian H Ravandi F et al.Combination of hyper-CVAD with ponatinib as first-line therapy for patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia: a single-centre, phase 2 study.Lancet Oncol. 2015; 16: 1547-1555Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 12Jain P Kantarjian H Boddu PC et al.Analysis of cardiovascular and arteriothrombotic adverse events in chronic-phase CML patients after frontline TKIs.Blood Adv. 2019; 3: 851-861Crossref PubMed Scopus (65) Google Scholar]. Although direct sequencing is the standard method for detecting ABL1 kinase domain mutations in clinical practice, it lacks sensitivity to detect minor clones [13Hughes T Deininger M Hochhaus A et al.Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (1006) Google Scholar]. Previous studies indicated that routine TKI prophylaxis before minimal residual disease (MRD) detection after HSCT did not reduce the risk of relapse [14Akahoshi Y Nishiwaki S Mizuta S et al.Tyrosine kinase inhibitor prophylaxis after transplant for Philadelphia chromosome-positive acute lymphoblastic leukemia.Cancer Sci. 2019; 110: 3255-3266Crossref PubMed Scopus (20) Google Scholar,15Pfeifer H Wassmann B Bethge W et al.Randomized comparison of prophylactic and minimal residual disease-triggered imatinib after allogeneic stem cell transplantation for BCR-ABL1-positive acute lymphoblastic leukemia.Leukemia. 2013; 27: 1254-1262Crossref PubMed Scopus (166) Google Scholar]. However, TKI prophylaxis after HSCT for high-risk patients might lead to decrease in relapse. We hypothesized that early detection of small clones with the T315I mutation would predict relapse after HSCT and might help the clinician decide when to change treatments. The aims of this study were to evaluate the presence of the T315I mutation at specific time points using the novel technology of digital polymerase chain reaction (dPCR), which is more sensitive than direct sequencing, and to explore their prognostic value in patients with Ph+ ALL who have received allogeneic HSCT in the dasatinib era. The current study included consecutive adult patients with Ph+ ALL who underwent allogeneic HSCT following dasatinib-based chemotherapy at our center between January 2010 and May 2018. As a policy at our center, allogeneic HSCT was offered to all HSCT-eligible patients. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine or tacrolimus and short-term methotrexate. MRD monitoring was performed at HSCT, at days +30, +90, and +180 and annually after HSCT using bone marrow (BM) samples obtained within 30 days before HSCT and at each time point, respectively. Additional MRD monitoring was performed at the discretion of the attending physician. TKI prophylaxis after HSCT before MRD detection was not given in our center. Written informed consent according to the Declaration of Helsinki was obtained from all patients. All patients during the study period were included in this study. Because the study was retrospective, T315I status did not influence the clinical decision. This study was approved by the institutional review board of Jichi Medical University Saitama Medical Center. MRD was assessed by qualitative and/or real-time quantitative PCR (RQ-PCR) methods after the BM samples were transferred to another clinical laboratory (SRL Co., Japan). Total RNA was extracted from mononuclear cells in BM and transcribed to cDNA. Qualitative PCR was performed by a method detailed previously [16Kawasaki ES Clark SS Coyne MY et al.Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro.Proc Natl Acad Sci USA. 1988; 85: 5698-5702Crossref PubMed Scopus (636) Google Scholar]. RQ-PCR assay was carried out using TaqMan technology (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with a previous report [17Towatari M Yanada M Usui N et al.Combination of intensive chemotherapy and imatinib can rapidly induce high-quality complete remission for a majority of patients with newly diagnosed BCR-ABL-positive acute lymphoblastic leukemia.Blood. 2004; 104: 3507-3512Crossref PubMed Scopus (164) Google Scholar]. For RQ-PCR, the number of transcript copies was normalized relative to glyceraldehyde 3-phosphate dehydrogenase and converted to molecules per microgram of RNA. The detection threshold of RQ-PCR was 50 copies/μg RNA. When RQ-PCR revealed a negative result, qualitative PCR was performed, which was equivalent to a minimal sensitivity of 10–5. Patients who were negative by the qualitative PCR and RQ-PCR methods were considered to be negative for MRD (negative MRD). Molecular relapse after HSCT was defined as the detection of MRD by the qualitative PCR method. We retrospectively analyzed formalin-fixed paraffin-embedded (FFPE) BM aspirate samples at diagnosis, at HSCT, and at the time points of the first molecular and hematologic relapses after HSCT, respectively. FFPE samples were stored in the dark at room temperature (14°C–25°C) for up to 10 years. DNA isolation was performed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. The concentration of nucleic acids used to prepare standard DNA mix was determined with a spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific). The presence of the T315I mutation in the BM samples was determined using the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific) with TaqMan Assay probe/primer sets (C_174580870_10, Thermo Fisher Scientific), including FAM-labeled probe for T315I (5ʹ-FAM-CGGGAGCCCCCGTTCTATATCATCATTGAGTTCATGACCTACGGGAACCTC-MGB-3ʹ) and VIC-labeled probe for wild-type ABL1 (5ʹ-VIC-CGGGAGCCCCCGTTCTATATCATCACTGAGTTCATGACCTACGGGAACCTC-MGB-3ʹ). As the probe/primer sets were designed for the T315I mutation, this assay could not detect other mutations such as the T315A mutation. The genomic DNA extracted from FFPE BM was diluted to 10 to 20 ng/μL before being added to each reaction. Each reaction mix contained a total volume of 15 μL, including 7.5 μL of 2 × QuantStudio 3D Digital PCR Master Mix, 0.75 μL of 10 μmol/L TaqMan Assay (C_174580870_10, Thermo Fisher Scientific), 5.25 μL of nuclease-free water, and 1.5 μL of the diluted genomic DNA. The reaction mix was loaded onto a QuantStudio 3D Digital PCR Chip v2, and the PCR was run on a ProFlex PCR System (Thermo Fisher Scientific) with a cycling protocol as follows: 10 min denaturation at 96°C, 39 cycles of 2 min at 60°C and 30 sec at 98°C, and elongation for 2 min at 60°C. The chips were read on the QuantStudio 3D Digital PCR instrument and analyzed using QuantStudio 3D online Analysis Suite software (Thermo Fisher Scientific). The proportion of the T315I mutation was shown as the ABL1T315I/ABL1 ratio. We further evaluated the sensitivity and the limit of detection for the dPCR assay. HCT116 colorectal carcinoma cells in which the T315I mutation was heterozygously knocked-in (Horizon Discovery, UK) were diluted with nonmutated HCT116 cells (Horizon Discovery) to obtain serial dilutions of 50%, 10%, 1%, 0.1%, 0.05%, 0.01%, and 0% mutated samples. Genomic DNA from these diluted samples was isolated using a QIAamp DNA Mini Kit (Qiagen). In the sensitivity analysis that was replicated three times, this dPCR assay achieved a sensitivity of at least 0.1% (Figure 1). Therefore, when three or more positive partitions corresponding to an ABL1T315I/ABL1 ratio of 0.1% were detected in patients with positive MRD, the sample was defined as true positive for the T315I mutation. The probability of overall survival was estimated with the Kaplan–Meier method with a 95% confidence interval (CI). The cumulative incidences of hematologic relapse, molecular relapse, and non-relapse mortality were calculated using Gray's method. Competing events were death without hematologic relapse for hematologic or molecular relapse, hematologic relapse for non-relapse mortality, and death without GVHD or hematologic relapse for GVHD. All statistical analyses were performed with EZR version 1.37 (Jichi Medical University Saitama Medical Center), which is a graphical user interface for R (R Foundation for Statistical Computing, version 3.2.2) [18Kanda Y Investigation of the freely available easy-to-use software 'EZR' for medical statistics.Bone Marrow Transplant. 2013; 48: 452-458Crossref PubMed Scopus (8859) Google Scholar]. During the study period, 25 patients were included in this study (Table 1). Among these 25 patients, 16 patients received a total body irradiation (TBI)-based myeloablative conditioning regimen with cyclophosphamide (120 mg/kg) and total body irradiation (12 Gy), and one patient not in remission at HSCT additionally received etoposide at 30 mg/kg [19Arai Y Kondo T Shigematsu A et al.Improved prognosis with additional medium-dose VP16 to CY/TBI in allogeneic transplantation for high risk ALL in adults.Am J Hematol. 2018; 93: 47-57Crossref PubMed Scopus (14) Google Scholar]. Eight patients received a reduced-intensity conditioning regimen with fludarabine (125 mg/kg)/melphalan (140 mg/kg) or fludarabine (125 mg/kg)/melphalan (80 mg/kg) and low-dose TBI (4 Gy). The median observation period for survivors was 1,042.5 days (range; 328–3,023 days). Of the 25 patients, three and four patients died of disease progression and treatment-related complications, respectively. The 3-year overall survival rate after HSCT was 70.6% (95% CI: 47.8%–84.9%). The 3-year cumulative incidences of grade II–IV acute GVHD and chronic GVHD were 48.0% (95% CI: 27.2%–66.1%) and 32.0% (95% CI: 14.8%–50.6%), respectively.Table 1Patient characteristics (n =25)Characteristicn (%)Age (y) at HSCT, median (range)50 (19–64)Recipient sex Female13 (52.0) Male12 (48.0)Performance status 015 (60.0) 110 (40.0)Donor source BMT14 (56.0) PBSCT10 (40.0) UCBT1 (4.0)Type of donor Related7 (28.0) Unrelated18 (72.0)HLA compatibility Matched18 (72.0) Mismatched7 (28.0)Conditioning regimen TBI-based MAC17 (72.0) Melphalan-based RIC8 (35.0)GVHD prophylaxis Cyclosporine A-based24 (96.0) Tacrolimus-based1 (4.0)Use of ATG/alemtuzumab No22 (88.0) Yes3 (12.0)Prior transplantation 024 (96.0) 11 (4.0)White blood cell count at diagnosis <30,000/μL12 (48.0) ≥30,000/μL13 (52.0)Breakpoint Minor21 (84.0) Major4 (16.0)Additional cytogenetic abnormalities No20 (80.0) Yes5 (20.0)Disease status at HSCT CR121 (84.0%) CR22 (8.0%) Non-CR2 (8.0%)MRD status at HSCT Positive12 (48.0) Negative13 (52.0)Time (days) from diagnosis to HSCT, median (range)198 (92–911)BMT=bone marrow transplantation; HLA=human leukocyte antigen; MAC=myeloablative conditioning; PBSCT=peripheral blood stem cell transplantation; RIC=reduced-intensity conditioning; UCBT=umbilical cord blood transplantation. Open table in a new tab BMT=bone marrow transplantation; HLA=human leukocyte antigen; MAC=myeloablative conditioning; PBSCT=peripheral blood stem cell transplantation; RIC=reduced-intensity conditioning; UCBT=umbilical cord blood transplantation. Among the 25 patients, 10 BM samples at diagnosis (40.0%) were available. Four of these 10 patients had small clones of the T315I mutation (median ABL1T315I/ABL1 ratio: 0.223%, range: 0.181%–2.115%). The logarithmic transformation of BCR-ABL1 transcripts and white blood cell count (WBC) at diagnosis were not associated with the T315I positivity at diagnosis, respectively (p = 0.762 for BCR-ABL1 transcripts, p = 0.610 for WBC). None of the patients with the T315I mutation at diagnosis developed hematologic relapse after HSCT. Although three patients experienced molecular relapse after HSCT, none of the patients had a positive result for the T315I mutation at molecular relapse. The three patients re-achieved molecular remission after MRD-triggered dasatinib treatment. BM samples at HSCT were available for all 12 patients with positive MRD. Of these patients, six (50.0%) had the T315I mutation at HSCT (median ABL1T315I/ABL1 ratio, 0.247%, range, 0.133 to 20.04%) (Figure 2A). BCR-ABL1 transcripts and WBC count at diagnosis were also not associated with T315I positivity at HSCT, respectively (p = 0.436 for BCR-ABL1 transcripts, p = 1.000 for WBC). Only one patient (16.7%) who had the T315I mutation at HSCT (ABL1T315I/ABL1 ratio: 1.084%) developed hematologic relapse after HSCT at day +77 (ABL1T315I/ABL1 ratio: 12.415%). In contrast, of six patients without T315I mutation at HSCT, two (33.3%) developed hematologic relapse. Overall, 14 (56.0%) and six (24.0%) patients developed molecular and hematologic relapse after HSCT, respectively (Table 2). The cumulative incidences of molecular and hematologic relapse at 3 years were 60.3% (95% CI: 34.8–78.5%) and 25.1% (95% CI: 9.8–43.9%), respectively. The median durations from HSCT to molecular relapse and hematologic relapse were 135.5 days (range, 32–970 days) and 90 days (range: 58–312 days), respectively. The median durations from molecular relapse to hematologic relapse were 22 days (range: 0–244 days). The 3-year cumulative incidences of hematologic relapse, molecular relapse, and nonrelapse mortality were 24.4% (95% CI: 9.6–42.7), 60.3% (95% CI: 34.8–78.5) and 17.3% (95% CI: 3.4–40.2), respectively.Table 2Data for 14 patients who developed molecular relapse after HSCTNo.Age (y)Diagnosis (y)Donor typeConditioningTKI before HSCTMRD at HSCTT315I at HSCTAcute GVHDChronic GVHDTime from HSCT to molecular relapse (d)Treatment after molecular relapseT315I (%) at molecular relapseHematologic relapse (T315I%)Time to hematologic relapse (d)aThe duration from molecular relapse to hematologic relapse.Follow-up duration (d)Current status1502011M-RDCy/TBIIMA/DASA++Grade IIINone62DASAPositive (0.27%)Yes (12.42%)15133Relapse death2412015MM-RDCy/TBIDASA+−Grade INone58ChemotherapyPositive (3.37%)Yes (3.37%)0126Relapse death3452010M-UDCy/TBIIMA/DASA−−Grade INone74DASAPositive (0.30%)Yes (25.55%)29360Relapse death4412017M-UDCy/TBIDASA−−Grade ILimited181DASA→PONA→ChemotherapyPositive (1.53%)Yes (10.36%)101328Under treatment5442012M-UDCy/TBIIMA/DASA++Grade IINone32DASA→PONANegative2,149Still in CR6622012M-UDFlu/TBI10IMA/DASA++Grade IIExtensive90DASANegative187Death (TRM)7602015M-UDFlu/Mel140DASA++Grade IINone229ObservationNegative293Death (TRM)8192010MM-RDFlu/Mel80/TBI4IMA/DASA+−NoneExtensive68DASA→3rd HSCTNegativeYes (0.0%)2442,278Still in CR9602010M-UDFlu/Mel140IMA/DASA+−NoneNone552DASANegative3,023Still in CR10552016M-UDCy/TBIDASA/PONA+−NoneNone43ObservationNegativeYes (0.0%)1596Relapse death11232011MM-RDCy/TBIIMA/DASA+−Grade IIINone292DASANegative2,491Still in CR12312015M-UDCy/TBIDASA+−Grade INone399ObservationNegative1,155Still in CR13392014M-RDCy/TBIIMA/DASA−−Grade ILimited970DASA→PONANegative1,666Still in CR14632015MM-UDFlu/Mel140DASA−−Grade INone289DASANegative1,024Still in CRCR=complete remission; Cy=cyclophosphamide; DASA=dasatinib; Flu=fludarabine; GVHD=graft-versus-host disease; HSCT=hematopoietic stem cell transplantation; IMA=imatinib; Mel=melphalan; MRD=minimal residual disease; M-RD=Matched related donor; M-MRD=mismatched related donor; MM-UD=mismatched unrelated donor; M-UD=matched unrelated donor; PONA=ponatinib; TBI=total body irradiation; TKI=tyrosine kinase inhibitor; TRM=treatment-related mortality.a The duration from molecular relapse to hematologic relapse. Open table in a new tab CR=complete remission; Cy=cyclophosphamide; DASA=dasatinib; Flu=fludarabine; GVHD=graft-versus-host disease; HSCT=hematopoietic stem cell transplantation; IMA=imatinib; Mel=melphalan; MRD=minimal residual disease; M-RD=Matched related donor; M-MRD=mismatched related donor; MM-UD=mismatched unrelated donor; M-UD=matched unrelated donor; PONA=ponatinib; TBI=total body irradiation; TKI=tyrosine kinase inhibitor; TRM=treatment-related mortality. At the time of molecular relapse, the T315I mutation was detected in four patients (28.6%) (median ABL1T315I/ABL1 ratio: 0.915%, range: 0.270%–3.371%) (Figure 2B). All patients with the T315I mutation at molecular relapse developed hematologic relapse, and their disease progressed rapidly (Figure 3) (Table 2, patients 1–4). However, the duration from molecular relapse to hematologic relapse seemed longer in a patient who received ponatinib after molecular relapse (101 days) (Table 2, patient 4). Of the other 10 patients without the T315I mutation at molecular relapse, seven received dasatinib (Table 2). Among these seven patients, dasatinib was switched to ponatinib in two patients because of the continuous detection of MRD, but these patients remained in CR (Table 2, patients 5 and 13). Only two of the 10 patients developed hematologic relapse, but the T315I mutation was not detected at hematologic relapse (Figure 3) (Table 2, patients 8 and 10). At the time of hematologic relapse, four patients (66.7%) had the T315I mutation (median ABL1T315I/ABL1 ratio: 10.34%, range: 3.37%–25.55%) (Table 2). The one patient who had received ponatinib before HSCT because of the T315I mutation detected by direct sequencing developed hematologic relapse without T315I after HSCT (Table 2, patient 10). BCR-ABL1 transcripts and WBC counts at diagnosis were not associated with T315I positivity at hematologic relapse, respectively (p = 0.533 for BCR-ABL1 transcripts; p = 0.400 for WBC). Development of the T315I mutation in the BCR-ABL1 kinase domain is a significant cause of treatment failure for Ph+ ALL in the dasatinib era [9Soverini S De Benedittis C Papayannidis C et al.Drug resistance and BCR-ABL kinase domain mutations in Philadelphia chromosome-positive acute lymphoblastic leukemia from the imatinib to the second-generation tyrosine kinase inhibitor era: the main changes are in the type of mutations, but not in the frequency of mutation involvement.Cancer. 2014; 120: 1002-1009Crossref PubMed Scopus (107) Google Scholar]. In this study, we retrospectively traced small clones with the T315I mutation using dPCR at several specific time points in patients who received allogeneic HSCT following dasatinib-based chemotherapy. Previous studies have suggested that leukemic clones with BCR-ABL1 kinase domain mutations already exist at diagnosis [20Pfeifer H Wassmann B Pavlova A et al.Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL).Blood. 2007; 110: 727-734Crossref PubMed Scopus (182) Google Scholar,21Soverini S Vitale A Poerio A et al.Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis.Haematologica. 2011; 96: 552-557Crossref PubMed Scopus (65) Google Scholar]. They reported that BCR-ABL1 kinase domain mutations at diagnosis, as identified using denaturing high-performance liquid chromatography and direct sequencing, were not associated with dismal outcomes. Meanwhile, a prospective study on dasatinib and low-intensity chemotherapy in elderly patients reported that eight of 10 patients with the T315I mutation at diagnosis by a PCR-based assay relapsed with the T315I mutation [7Rousselot P Coude MM Gokbuget N et al.Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome-positive ALL.Blood. 2016; 128: 774-782Crossref PubMed Scopus (183) Google Scholar]. In contrast to our study, only 10% of the patients in that study received HSCT. The current study suggested that small subclones with T315I at diagnosis and at HSCT were not associated with an increased risk of hematologic relapse after HSCT. Small subclones with T315I might be eradicated by HSCT or multiple treatments other than first- or second-generation TKIs because they did not have any selective advantage under a graft-versus-leukemia effect and intensive chemotherapy including the conditioning regimen. Accordingly, the existence of small subclones with T315I before HSCT might be less useful for predicting outcomes after HSCT. In contrast, all patients who harbored the T315I mutation at the time of molecular relapse after HSCT (n = 4) rapidly developed hematologic relapse, while eight of the 10 patients without T315I at molecular relapse did not. Although the duration from molecular relapse to hematologic relapse in patients with the T315I mutation was quite short (median: 22 days), the administration of ponatinib might prolong hematologic remission. Indeed, in our study, one patient who received post-transplant ponatinib had the longest hematologic remission from molecular to hematologic relapse (101 days). Hence, the current study suggests that detection of the T315I mutation at the time of molecular relapse after HSCT may provide appropriate information for identifying patients who are likely to develop hematologic relapse. However, it remains to be elucidated whether posttransplant ponatinib would completely suppress resistant clones and reduce hematologic relapse in such patients. Interestingly, one patient who had received ponatinib before HSCT had no evidence of the T315I mutation at hematologic relapse, suggesting that treatment failure occurred through some mechanism other than BCR-ABL1 kinase domain mutation. Further studies are required to assess the influence of ponatinib before HSCT. We selected genomic DNA and dPCR to monitor the T315I mutation because FFPE BM could be obtained retrospectively. RNA is less stable than DNA, and the efficiency of cDNA synthesis could vary. Changes in expression of the housekeeping gene could also affect final results. The detection of BCR-ABL1 genomic DNA would provide the accurate proportions of leukemic cells and might be more sensitive than the detection of BCR-ABL1 mRNA [22Pagani IS Dang P Kommers IO et al.BCR-ABL1 genomic DNA PCR response kinetics during first-line imatinib treatment of chronic myeloid leukemia.Haematologica. 2018; 103: 2026-2032Crossref PubMed Scopus (24) Google Scholar]. Meanwhile, fragmentation of FFPE DNA directly influences the success of PCR [23Didelot A Kotsopoulos SK Lupo A et al.Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.Clin Chem. 2013; 59: 815-823Crossref PubMed Scopus (88) Google Scholar]. Because the PCR success of FFPE DNA strongly depends on the size of the amplicon [24Shi SR Cote RJ Wu L et al.DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.J Histochem Cytochem. 2002; 50: 1005-1011Crossref PubMed Scopus (215) Google Scholar,25Gillio-Tos A De Marco L Fiano V et al.Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples.Pathology. 2007; 39: 345-348Abstract Full Text PDF PubMed Scopus (70) Google Scholar], the use of a short amplicon in our study (about 50 bp) could maximize the sensitivity for PCR. Taqman dPCR is a unique method to measure the absolute copy number of target DNA without external standards. Conventional single-nucleotide-polymorphism genotyping using quantitative PCR or direct sequencing, which is the current standard method for detecting ABL1 kinase domain mutations, could not detect these mutations when mutated cells constituted less than 20% of total Ph+ cells [13Hughes T Deininger M Hochhaus A et al.Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (1006) Google Scholar]. 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This is an approximately 200-fold improvement in detection sensitivity compared with conventional direct sequencing. In addition, dPCR enables us to precisely quantify the small population of leukemic cells with the T315I mutation in total DNA without a calibration curve. Although dPCR requires a small amount of input DNA, results are robust within a range of input DNA concentrations. Thus, dPCR using FFPE samples with a wide range of amplicon would reveal a reliable result [23Didelot A Kotsopoulos SK Lupo A et al.Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.Clin Chem. 2013; 59: 815-823Crossref PubMed Scopus (88) Google Scholar]. However, it should be noted that monitoring the emergence of the T315I mutation using genomic DNA did not directly reflect the leukemic cells themselves, although non-leukemic cells were unlikely to acquire the T315I mutation. Therefore, monitoring the T315I mutation by dPCR should be accompanied by conventional methods for detecting MRD by BCR-ABL1 mRNA. In the future, next-generation sequencing technologies will also be available to directly detect and monitor all BCR-ABL1 kinase domain mutations with high sensitivity for clinical practice. However, our inexpensive and simple approach using dPCR might help to guide the decision regarding the choice of treatment. Further studies are warranted to optimize the methods for monitoring T315I mutation and to evaluate pre-emptive strategies with posttransplant ponatinib and other novel agents such as blinatumomab and inotuzumab ozogamicin according to detection of the T315I mutation. The emergence of small clones with the T315I mutation detected by dPCR at molecular relapse after HSCT, not before HSCT, might be useful for predicting hematologic relapse and help physicians change to appropriate treatments after HSCT in the dasatinib era. Detection of the T315I mutation using dPCR combined with the conventional assay for detecting BCR-ABL1 mRNA is a convenient approach that does not require much time or expense. Our results should be confirmed in a large cohort of patients.
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