Effects of human herpesvirus 6B reactivation on cognitive function in cord blood transplant recipients: a prospective multicenter study.

This prospective multicenter study aimed to determine the effects of human herpesvirus-6B (HHV-6B) reactivation on central nervous system (CNS) function in cord blood transplant (CBT) recipients. Our focus was to track HHV-6B reactivation and evaluate its association with delirium and cognitive function, specifically in the domains of verbal memory, attention/processing speed, and quality of life (QOL). A cohort of 38 patients participated in this study. Of the 37 patients evaluated, seven (18.9%) developed delirium, with six of these cases emerging after HHV-6B reactivation (median lag, 7 days). Evaluation of verbal memory showed that the final trial score for unrelated words at 70 days after transplantation was significantly lower than that before preconditioning (P = 0.004) among patients (n = 15) who experienced higher-level HHV-6B reactivation (median or higher maximum plasma HHV-6 DNA load for participating patients). Patients without higher-level reactivation did not show significant declines in verbal memory scores. QOL was assessed using the 36-item Short-Form Health Survey, and the social functioning score 1 year post-transplantation was significantly lower in patients who experienced higher-level HHV-6B reactivation than in those who did not. Our findings suggest that higher-level HHV-6B reactivation can detrimentally affect certain cognitive functions in CBT recipients.

Impact of Different Fludarabine Doses in the Fludarabine-Based Conditioning Regimen for Unrelated Bone Marrow Transplantation.

The purine analog fludarabine (Flu) plays a central role in reduced-intensity conditioning and myeloablative reduced-toxicity conditioning regimens because of limited nonhematologic toxicities. Few reports assess the impact of different dose of Flu on the clinical outcomes and the Flu doses vary across reports. To compare the effect of Flu dose, the clinical outcomes of patients who received Flu and busulfan (FB; n = 1647) or melphalan (Flu with melphalan (FM); n = 1162) conditioning for unrelated bone marrow transplantation were retrospectively analyzed using Japanese nationwide registry data. In the FB group, high-dose Flu (180 mg/m2; HFB) and low-dose Flu (150/125 mg/m2; LFB) were given to 1334 and 313 patients, respectively. The 3-year overall survival (OS) rates were significantly higher in the HFB group than in the LFB group (49.5% versus 39.2%, P < .001). In the HFB and LFB groups, the cumulative incidences were 30.4% and 36.6% (P = .058) for 3-year relapse and 25.1% and 28.1% (P = .24) for 3-year nonrelapse mortality (NRM), respectively. In the multivariate analysis for OS and relapse, Flu dose was identified as an independent prognostic factor (hazard ratio: 0.83, P = .03; hazard ratio: 0.80, P = .043). In the FM group, high-dose Flu (180 mg/m2; HFM) and low-dose Flu (150/125 mg/m2; LFM) were given to 118 and 1044 patients, respectively. The OS, relapse, and NRM after 3 years did not differ significantly between the HFM and LFM groups (48.3% versus 48.8%, P = .92; 23.7% versus 27.2%, P = .55; 31.9% versus 30.8%, P = .67). These findings suggest that high-dose Flu was associated with favorable outcomes in the FB group but not in the FM group.

Outcomes of Cessation of Nucleos(t)ide Analog Administration on Hepatitis B Virus Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation: A Nationwide Retrospective Study.

Monitoring of hepatitis B virus (HBV)-DNA and HBV-DNA-guided preemptive therapy using nucleos(t)ide analogs (NAs) are recommended to prevent the development of hepatitis due to HBV reactivation after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in recipients with resolved HBV infection. However, little is known about the appropriate duration of NA treatment and the effect of NA cessation on the recurrence of HBV reactivation. This study aimed to clarify the consequences of NA cessation in allo-HSCT recipients with resolved HBV infection who experienced HBV reactivation following transplantation. We retrospectively reviewed the clinical records of recipients with resolved HBV infection (hepatitis B surface antigen [HBsAg]-negative, anti-HBc-positive) before allo-HSCT who had been diagnosed with HBV reactivation (HBsAg-positive and/or HBV-DNA detectable) after allo-HSCT between January 2010 and December 2020. A total of 72 patients from 16 institutions were registered (median age, 60 years; age range, 27 to 73 years; 42 males and 30 females). The day of initial HBV reactivation ranged from day 10 to day 3034 after allo-HSCT (median, 513 days). Anti-HBs were lost in >80% of the patients at the time of HBV reactivation. All 72 patients received preemptive NAs, and no fatal HBV reactivation-related hepatitis was observed. HBV-DNA without hepatitis was continuously detected in 5 patients during the follow-up period. Administration of NAs was discontinued in 24 of 72 patients (33%) by physician decision. Second HBV reactivation occurred in 11 of the 24 patients (46%) in whom administration of NAs was discontinued. The duration of NA treatment did not differ significantly between patients with or without second HBV reactivation. The frequency of further HBV reactivation tended to be lower in patients with an anti-HBs titer of >10 mIU/mL at the time of NA cessation. Multiple reactivations of HBV after NA cessation was common in patients with HBV reactivation who underwent allo-HSCT despite the long duration of NAs. Careful monitoring of HBV-DNA is important even after the discontinuation of NAs in the case with HBV reactivation after allo-HSCT, because multiple reactivations could occur. Active immunization by HB vaccine might be effective for suppressing further HBV reactivation after cessation of NAs.

Outcomes of allogeneic hematopoietic cell transplantation under letermovir prophylaxis for cytomegalovirus infection.

Cytomegalovirus (CMV) infection is a major infectious complication following allogeneic hematopoietic cell transplantation (allo-HCT). Although letermovir (LMV) prophylaxis dramatically reduces the incidence of early clinically significant CMV (csCMV) infection, it remains unclear whether it has a beneficial effect on nonrelapse mortality (NRM) and overall survival (OS). Herein, we evaluated the impact of LMV prophylaxis on posttransplant outcomes using the registry database of the Japanese Society for Transplantation and Cellular Therapy. Adult patients who underwent allo-HCT between 2017 and 2019 were analyzed (n = 6004). LMV prophylaxis was administered to 1640 patients (LMV group) and it significantly reduced the incidence of csCMV infection compared with those not administered LMV prophylaxis (15.4% vs 54.1%; p < 0.01). However, it did not improve the 1-year NRM (hazard ratio [HR], 0.93; p = 0.40) and OS (HR, 0.96; p = 0.49). In the LMV group, 74 patients had breakthrough csCMV infection and showed inferior NRM (HR, 3.44; p < 0.01) and OS (HR, 1.93; p = 0.02) compared with those without infection. After completing LMV prophylaxis, 252 patients had late csCMV infection and showed inferior NRM (HR, 1.83; p < 0.01) and OS (HR, 1.58; p < 0.01). Our findings suggest that managing breakthrough and late csCMV infections is important for improving long-term outcomes.

Outcomes in human T-cell leukemia virus type I carriers after hematopoietic stem cell transplantation for diseases other than adult T cell leukemia/lymphoma: a Japanese national survey.

Background: Human T-cell leukemia virus type I (HTLV-1) is a retrovirus known to cause adult T-cell leukemia/lymphoma (ATL). There are few reports on hematopoietic stem cell transplantation (HSCT) for HTLV-1 carriers with diseases other than ATL. Methods: A total of 25,839 patients (24,399 adults and 1440 children) with pre-transplant HTLV-1 serostatus information recorded in the Japanese National Survey Database who had undergone their first HSCT were analyzed. We investigated the overall survival (OS), transplant-related mortality (TRM), and disease-related mortality (DRM) after HSCT in relation to HTLV-1 serologic status. Findings: Three hundred and forty-eight patients were HTLV-1 antibody carriers. The number of HTLV-1 carriers and noncarriers among adult patients who received allogeneic HSCT (allo-HSCT) or autologous HSCT (auto-HSCT) was 237/15,777 and 95/8920, respectively, and was 16/1424 among pediatric patients who received allo-HSCT. No pediatric HTLV-1 carrier recipients undergoing auto-HSCT were identified. There were no significant differences between HTLV-1 carriers and non-carriers regarding stem cell source, disease risk, or HCT-CI score prior to allo-HSCT. Multivariate analysis of OS (P = 0.020) and TRM (P = 0.017) in adult patients showed that HTLV-1 positive status was a significant prognostic factor. In children, TRM was significantly higher (P = 0.019), but OS was not significantly different. In adult patients who underwent auto-HSCT, HTLV-1 positive status was not a significant prognostic factor. In adult allo-HSCT patients, cytomegalovirus reactivation was significantly more common in HTLV-1 carriers (P = 0.001). Interpretation: HTLV-1 antibody positivity was shown to have a poor prognosis in OS and TRM after allo-HSCT in adult patients and in TRM after allo-HSCT in pediatric patients. Funding: This work was supported in part by the practical research programs of the Japan Agency for Medical Research and Development (AMED) under grant number 17ck0106342h0001.

Risk factors and outcome of Stenotrophomonas maltophilia infection after allogeneic hematopoietic stem cell transplantation: JSTCT, Transplant Complications Working Group.

Stenotrophomonas maltophilia (S. maltophilia) is an aerobic nonfermenting Gram-negative bacillus widely distributed in the environment that has inherent multidrug resistance to beta-lactam and carbapenem antibiotics. S. maltophilia infection (SMI) is known as an important fatal complication following allogeneic hematopoietic stem cell transplantation (HSCT), but its clinical characteristics have not been well clarified. A retrospective study to identify the incidence, risk factors, and outcomes of SMI after allogeneic HSCT was performed using the database of the Japanese nationwide registry, including 29,052 patients who received allogeneic HSCT in Japan between January 2007 and December 2016. A total of 665 patients developed SMI (sepsis/septic shock, 432; pneumonia, 171; other, 62). The cumulative incidence of SMI at 100 days after HSCT was 2.2%. Among risk factors identified for SMI (age ≥ 50 years, male, performance status 2-4, cord blood transplantation [CBT], myeloablative conditioning, Hematopoietic Cell Transplant-Comorbidity Index [HCT-CI] score 1-2, HCT-CI score ≥ 3, and active infectious disease at HSCT), CBT was the strongest risk factor (hazard ratio, 2.89; 95%CI, 1.94-4.32; p < 0.001). The survival rate at day 30 after SMI was 45.7%, and SMI before neutrophil engraftment was significantly associated with poor survival (survival rate 30 days after SMI, 40.1% and 53.8% in patients with SMI before and after engraftment, respectively; p = 0.002). SMI is rare after allogeneic HSCT, but its prognosis is extremely poor. CBT was a strong risk factor for SMI, and its development prior to neutrophil engraftment was associated with poor survival.

Clinical characteristics of late-onset interstitial pneumonia after allogeneic hematopoietic stem cell transplantation.

Non-infectious pulmonary complications after allogeneic hematopoietic stem cell transplantation (HSCT) remain fatal. In particular, information regarding late-onset interstitial lung disease predominantly including organizing pneumonia and interstitial pneumonia (IP) is limited. A retrospective nationwide survey was conducted using data collected from the Japanese transplant outcome registry database from 2005 to 2010. This study focused on patients (n = 73) with IP diagnosed after day 90 post-HSCT. A total of 69 (94.5%) patients were treated with systemic steroids, and 34 (46.6%) experienced improvement. The presence of chronic graft-versus-host disease at the onset of IP was significantly associated with non-improvement of symptoms (odds ratio [OR] 0.35). At the time of last follow-up (median, 1471 days), 26 patients were alive. Of the 47 deaths, 32 (68%) were due to IP. The 3-year overall survival (OS) and non-relapse mortality (NRM) rates were 38.8% and 51.8%, respectively. In the multivariate analysis, the predictive factors for OS were comorbidities at IP onset (hazard ratio [HR]: 2.19) and performance status (PS) score of 2-4 (HR 2.77). Furthermore, cytomegalovirus reactivation requiring early intervention (HR 2.04), PS score of 2-4 (HR 2.63), and comorbidities at IP onset (HR 2.90) were also significantly associated with increased risk of NRM.

Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p = 0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

IMiD/CELMoD-induced growth suppression of adult T-cell leukemia/lymphoma cells via cereblon through downregulation of target proteins and their downstream effectors.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasia associated with human T-cell leukemia virus type 1 (HTLV-1) infection and has an extremely poor prognosis. Lenalidomide (LEN; a second-generation immunomodulatory drug [IMiD]) has been employed as an additional therapeutic option for ATL since 2017, but its mechanism of action has not been fully proven, and recent studies reported emerging concerns about the development of second primary malignancies in patients treated with long-term IMiD therapy. Our purpose in this study was to elucidate the IMiD-mediated anti-ATL mechanisms. Thirteen ATL-related cell lines were divided into LEN-sensitive or LEN-resistant groups. CRBN knockdown (KD) led to a loss of LEN efficacy and IKZF2-KD-induced LEN efficacy in resistant cells. DNA microarray analysis demonstrated distinct transcriptional alteration after LEN treatment between LEN-sensitive and LEN-resistant ATL cell lines. Oral treatment of LEN for ATL cell-transplanted severe combined immunodeficiency (SCID) mice also indicated clear suppressive effects on tumor growth. Finally, a novel cereblon modulator (CELMoD), iberdomide (IBE), exhibited a broader and deeper spectrum of growth suppression to ATL cells with efficient IKZF2 degradation, which was not observed in other IMiD treatments. Based on these findings, our study strongly supports the novel therapeutic advantages of IBE against aggressive and relapsed ATL.

Clinicopathological findings, prognosis, and Epstein-Barr virus infection in rheumatoid arthritis patients with other iatrogenic immunodeficiency-associated T- and NK-cell lymphoproliferative disorders.

BACKGROUND: Other iatrogenic immunodeficiency-associated (OIIA) T- and natural killer (NK)-cell lymphoproliferative disorders (TNK-LPDs) are rare in patients with rheumatoid arthritis (RA). METHODS: We investigated the clinicopathological characteristics, Epstein-Barr virus (EBV) infection, genetic findings, therapeutic response, and prognostic factors in 21 RA patients with OIIA TNK-LPDs and compared these with those of 39 with OIIA B-cell LPDs (B-LPDs) and 22 with non-OIIA B-LPDs. RESULTS: Immunohistologically, 11 patients (52%) showed CD4+ T-LPDs, and 7 had a T follicular helper (TFH) phenotype. The other nine patients (43%) showed CD8+ T-LPDs, and the remaining one (5%) had features of CD3+ CD4- CD8- nasal type TNK-cell lymphoma. CD30+, p53+, and CMYC+ atypical lymphocytes were identified in seven (33%), eight (38%), and five (24%) patients, respectively. In situ hybridisation detected EBV-encoded RNA (EBER) + large atypical lymphocytes in five patients (24%). Nine of 17 patients (53%) showed clonal peaks of TCRγ by polymerase chain reaction. Withdrawal of MTX and biologic drugs was effective in 12 patients (57%), and 8 (38%) received chemotherapies. Two patients with TFH+ or EBV+ CD4+ CD30+ large cell peripheral T-cell lymphoma, one with CD8+ systemic anaplastic large cell lymphoma, and two with systemic EBV+ CD8+ T-cell lymphoma of childhood showed a lethal progressive clinical course within 13 months. Moreover, > 500 U/L LDH, large atypical lymphocytes, expression of CD30, p53, and CMYC, and EBER+ atypical lymphocytes were significantly poor prognostic factors for overall survival (p < 0.05). Median interval from RA onset to OIIA TNK-LPDs was 72 months, which was shorter than 166 months in OIIA B-LPDs (p = 0.003). EBV+ atypical and reactive lymphocytes were frequently found in 15 patients with OIIA TNK-LPDs (71%), in 27 with OIIA B-LPDs (69%), and only in 3 with non-OIIA B-LPDs (14%). CONCLUSIONS: OIIA TNK-LPDs occurred in early phase of RA, compared with OIIA B-LPDs, and occasionally showed a lethal progressive clinical course. Detection of OIIA TNK-LPD patients with poor prognostic factors is necessary. EBV infection in immunosuppressed patients due to persistent RA, MTX, and biologic drugs may play a role in forming the tumour microenvironment and lymphomagenesis of TNK-LPDs.

Impact of Inflammation on Intra-individual Variation in Trough Voriconazole Concentration in Patients with Hematological Malignancies.

The pharmacokinetics of voriconazole shows large intra-individual and inter-individual variability and is affected by various factors. Recently, inflammation has been focused as a significant factor affecting the variability. This study aimed to compare the influence of C-reactive protein (CRP) and other clinical laboratory parameters on intra-individual variability in trough voriconazole concentration and examine the impact of inflammation in patients with hematological malignancies. We conducted a retrospective, single-center, observational cohort study. Forty-two patients with hematological malignancy who received oral voriconazole for prophylaxis against deep mycosis and underwent multiple measurements of trough plasma voriconazole concentration were recruited. Quantitative changes in pharmacological and clinical laboratory parameters (Δ) were calculated as the difference between the current and preceding measurements. Voriconazole concentration/maintenance dose per weight (C/D) was found to correlate positively with CRP level (n = 202, rs = 0.314, p < 0.001). Furthermore, ΔC/D correlated positively with ΔCRP level (n = 160, rs = 0.442, p < 0.001), and ΔCRP showed the highest correlation coefficient among the laboratory parameters. Univariate and multivariate analyses identified ΔCRP (p < 0.001) and Δgamma-glutamyl transpeptidase (γGTP) (p = 0.019) as independent factors associated with ΔC/D. Partial R2 were 0.315 for ΔCRP and 0.024 for ΔγGTP, suggesting markedly greater contribution of ΔCRP to ΔC/D. In conclusion, since clinical laboratory parameters other than CRP had little influence on trough plasma voriconazole concentration, therapeutic drug monitoring and dose adjustment considering fluctuation in CRP level would be important for proper use of voriconazole in patients with hematological malignancies.

RAISING is a high-performance method for identifying random transgene integration sites.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

NPM1-mutation-based measurable residual disease assessment after completion of two courses of post-remission therapy is a valuable clinical predictor of the prognosis of acute myeloid leukemia.

Recent studies have reported that measurable residual disease (MRD) analysis using NPM1 mutations helps determine whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) is indicated in acute myeloid leukemia (AML) patients. However, the optimal timing and cutoff value for measuring MRD using genomic DNA remain undetermined. This study aimed to investigate the optimal timing and cutoff value to ascertain the value of NPM1 mutation in MRD assessment. NPM1-mutated MRD was quantified by real-time polymerase chain reaction of bone marrow samples from 56 patients with NPM1-positive AML who achieved hematological remission. The area under the receiver-operating characteristic curve was greatest when MRD was assessed after two courses of post-remission therapy with a cutoff value of 0.010% (specificity, 68.4%; sensitivity, 87.0%). Patients whose MRD was below the cutoff value throughout the course of treatment had significantly better overall survival and relapse-free survival rates. Of the 33 patients who did not undergo transplantation during the first remission, all of the 11 who were never MRD-negative at any point experienced a relapse. Evaluating MRD with a cutoff value of 0.010% after two courses of post-remission therapy helps predict prognosis and determine the indication for allo-HSCT.

Risk factors and prognosis of non-infectious pulmonary complications after allogeneic hematopoietic stem cell transplantation.

Non-infectious pulmonary complications (NIPCs) following allogeneic hematopoietic stem cell transplantation (HSCT) are relatively rare, but frequently fatal. This study investigated the pre-transplant risk factors for developing NIPCs using Japanese transplant registry database entries from 2001 to 2009. Among 13,573 eligible patients, 535 experienced NIPCs (3.9%). Multivariate analysis identified high recipient age (60 + years: HR 1.85, P = 0.003), HLA mismatch (HR 1.61, P < 0.001), female to male HSCT (HR 1.54, P < 0.001), and unrelated bone marrow transplantation (UR-BMT) (HR 3.88, P < 0.001) as significantly associated with an increased risk of NIPCs. In contrast, a non-total body irradiation (TBI) regimen with reduced intensity conditioning (RIC) were associated with a decreased risk of NIPCs compared with a cyclophosphamide (CY) + TBI regimen (busulfan + CY: HR 0.67, P = 0.009, other non-TBI: HR 0.46, P < 0.001), fludarabine-based RIC (HR 0.52, P < 0.001), and other RIC (HR 0.42, P = 0.003). The mortality rate was significantly worse for patients with NIPCs than those without (HR 1.54, 71 P < 0.001). This large-scale retrospective study suggests that both allo-reactions to donor cells and conditioning regimen toxicity contributed to NIPCs following HSCT.

High-throughput simultaneousquantification offive azole anti-fungal agents and one active metabolite in human plasma using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry.

OBJECTIVES: For patients with hematological malignancy, triazole antifungal agents such as fluconazole (FLCZ), itraconazole (ITCZ), voriconazole (VRCZ), posaconazole (PSCZ) and isavuconazole (ISCZ) are often used for prophylaxis of deep mycosis. Since these azoles exhibit large pharmacokinetic variability, dose adjustment by therapeutic drug monitoring is recommended for some azoles. This study aimed to develop and validate a novel method for simultaneous determination of plasma concentrations of FLCZ, ITCZ, VRCZ, PSCZ, ISCZ and ITCZ-OH, an active metabolite of ITCZ, using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). DESIGN & METHODS: A high-throughput solid-phase extraction method using 96-well MCX µElution Plate was selected as the pretreatment procedure. RESULTS: The calibration curves for FLCZ, ITCZ, ITCZ-OH, VRCZ, PSCZ and ISCZ showed good linearity (back-calculation of calibrators: relative error ≤ 15% [LLOQ: ≤ 20%]) over wide ranges of 100-100000, 20-20000, 40-40000, 20-20000, 5-5000 and 50-50000 ng/mL, respectively. The validation results of all six drugs fulfilled the criteria of the guidance for bioanalytical method validation of the US Food and Drug Administration for within-batch and batch-to-batch precision and accuracy. The extraction recovery rates were good at ≥ 74.9%, and almost no matrix effects were found for all the drugs. The trough (10 h post-dose in 1 patient on PSCZ) drug concentrations in patients with hematologic malignancy who received oral FLCZ, ITCZ, VRCZ or PSCZ were quantified using the method developed. The measurements for all samples were within the ranges of the calibration curves, demonstrating the feasibility of clinical application of the novel method. CONCLUSIONS: We have succeeded in developing a novel high-throughput method using UHPLC-MS/MS for simultaneous quantification of plasma concentrations of FLCZ, ITCZ, ITCZ-OH, VRCZ, PSCZ and ISCZ.

Development of a Sensitive and High-Throughput Assay for Simultaneous Quantification of 5 Tyrosine Kinase Inhibitors and 2 Active Metabolites in Human Plasma Using Ultra-high Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

BACKGROUND: Breakpoint cluster region-Abelson (BCR-ABL) tyrosine kinase inhibitors (TKIs) demonstrate improved therapeutic efficacy in chronic myeloid leukemia (CML). However, drug-drug interactions, nonadherence, and host-related factors may influence plasma concentrations. Therefore, therapeutic drug monitoring may be necessary for patients presenting inadequate treatment responses or adverse events. Herein, the authors aimed to develop a more sensitive and high-throughput method than those previously reported to simultaneously quantify 5 TKIs (imatinib, nilotinib, dasatinib, bosutinib, and ponatinib) and 2 active metabolites (N-desmethyl imatinib and N-desmethyl ponatinib) using ultra-performance liquid chromatography coupled with tandem mass spectrometry. METHODS: Plasma samples were prepared according to a solid-phase extraction protocol using an Oasis MCX µElution plate. The assay fulfilled the requirements of the US Food and Drug Administration for assay validation, with a lower limit of quantification of 0.2 ng/mL for dasatinib, 0.3 ng/mL for N-desmethyl ponatinib, 0.5 ng/mL for N-desmethyl imatinib, bosutinib, and ponatinib, and 2.5 ng/mL for imatinib and nilotinib. RESULTS: Within-batch and batch-to-batch precision at the lower limit of quantification and quality control levels were within 14.3% and 10.9%, respectively. Within-batch and batch-to-batch accuracies ranged from 15.5% to 13.0% and 5.70% to 7.03%, respectively. A positive electrospray ionization mode was used with a run time of 6.0 minutes. The assay applicability was verified by the successful measurement of 78 clinical samples from patients undergoing CML therapy. CONCLUSIONS: The method allows assessment of trough concentrations of TKIs and active metabolites in patients with CML, and hence can be used to assess blood samples in routine clinical settings.