Patients In Molecular Remission Research Articles

Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F–positive myeloproliferative neoplasms

AbstractPegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F–positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α–treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F–positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F–positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Long-term outcome in patients with mantle cell lymphoma following high-dose therapy and autologous stem cell transplantation.

Long-term clinical and molecular remissions in patients with mantle cell lymphoma (MCL) after autologous stem cell transplantation (ASCT) have been evaluated in only a few studies. Sixty-five patients with MCL received ASCT (54 first-line ASCT, 10 second-line ASCT, and 1 third-line ASCT). In the case of long-term remission (≥5 years; n = 27), peripheral blood was tested for minimal residual disease (MRD) by t(11;14)- and IGH-PCR at the last follow-up. Ten-year overall survival (OS), progression-free survival (PFS), and freedom from progression (FFP) after first-line ASCT were 64%, 52%, and 59% versus after second-line ASCT 50%, 20%, and 20%, respectively. Five-year OS, PFS, and FFP for the first-line cohort were 79%, 63%, and 69%, respectively. Five-year OS, PFS, and FFP after second-line ASCT were 60%, 30%, and 30%, respectively. Treatment-related mortality (3 months after ASCT) was 1.5%. So far 26 patients developed sustained long-term clinical and molecular complete remissions of up to 19 years following ASCT in first treatment line. Sustained long-term clinical and molecular remissions are achievable following ASCT.

Treatment-free remission after discontinuation of imatinib, dasatinib, and nilotinib in patients with chronic myeloid leukemia

Patients with chronic myeloid leukemia (CML) in the chronic phase receiving tyrosine kinase inhibitor (TKI) therapy are expected to have long-term survival outcomes comparable to those of the general population. Many clinical trials have confirmed that some patients sustain molecular responses without continuing TKI therapy. Treatment-free remission (TFR) is a new goal in treating chronic CML. The safety and outcome of TFR were studied in clinical trials after discontinuing imatinib or the second-generation TKIs dasatinib or nilotinib. TFR was safe in approximately 50% of patients who achieved a deep molecular response to TKI therapy. Patients who relapsed after discontinuing TKI responded immediately to the reintroduction of TKI. The mechanism by which TFR increases the success rate still needs to be understood. The hypothesis that the modulation of immune function and targeting of leukemic stem cells could improve the TFR is under investigation. Despite the remaining questions, the TFR has become a routine consideration for clinicians in the practice of molecular remission in patients with CML.

Circulating tumor DNA (ctDNA) for response assessment in patients with anal cancer treated with definitive chemoradiation.

1 Background: We hypothesized that circulating tumor DNA (ctDNA) clearance could provide an early signal for clinical complete response (cCR) and/ or long-term recurrence compared to standard clinical exam modalities in patients with anal squamous cell carcinoma (ASCC) undergoing definitive chemoradiation (CRT). Methods: Since early 2021 patients with ASCC undergoing CRT at 2 institutions were offered ctDNA monitoring with a commercially available tumor-bespoke multiplex PCR assay. All patients provided written informed consent for ctDNA testing. Patients were clinically restaged, 3-4 months post-CRT, by clinical exam, endoscopy, and/ or MRI and annually with CT chest, abdomen, and pelvis. cCR was defined as no tumor by digital exam, endoscopy and/or MRI. Molecular ctDNA response is described according to cCR, tumor recurrence, and survival. Results: From January 2021 to September 2022, 31 patients with ASCC treated with definitive CRT underwent ctDNA response assessment. The majority (68%) of patients had stage III disease. Patients were treated to a median radiation dose of 54Gy in 27 fractions with combinatorial mitomycin and fluoropyrimidine-based chemotherapy in 94%, and fluoropyrimidine-based chemotherapy alone in 6%. The median follow up was 22 weeks. ctDNA testing was performed in 25 of these patients at baseline, 26 patients during CRT, and 20 patients 30-days post-CRT. At baseline 88% of patients had detectable ctDNA. Patients with stage III, as compared to stage I-II, disease had numerically higher baseline ctDNA levels (26 vs 4 mean tumor molecules per milliliter (MTM/mL), p=0.08). ctDNA levels decreased with treatment (19 vs 0.9 MTM/mL, p=0.05) among the 18 patients with detectable ctDNA and ctDNA tested during CRT, with 50% of patients entering molecular remission. Similarly, ctDNA levels decreased (21 vs 0.2 MTM/mL, p=0.05) among the 16 patients with detectable ctDNA and ctDNA tested post-CRT, with 94% entering molecular remission. All patients in molecular remission were confirmed to have a cCR. Time to molecular ctDNA remission was significantly shorter than time to cCR (median 30 vs 135 days, p <0.01). There were no molecular recurrences among the 16, 14, and 7 patients with ctDNA testing at 2-4 months, 4-8 months, and 8-12 months post-CRT. All patients are alive and without clinical/ radiographic evidence of disease. Conclusions: Surveillance ctDNA monitoring may provide an earlier response assessment for patients with ASCC undergoing CRT compared to standard clinical measures. Longer term follow-up is required to determine if ctDNA response correlates with long term recurrence free survival. Larger trials are needed to assess the clinical utility of integrating molecular ctDNA response in therapeutic response surveillance.

Tvt CAR7: Phase 1 Clinical Trial of Base-Edited "Universal” CAR7 T Cells for Paediatric Relapsed/Refractory T-ALL

Background Genome editing can overcome HLA barriers to generate 'off-the-shelf’ CAR T cell therapies. Despite the success of CAR-T cell therapies in B-cell malignancies, the expression of shared T cell antigens has constrained the development of CAR T cells targeting T-cell malignancies, due to T cell fratricide. Targeted base editing using CRISPR guided cytidine deamination mediates highly precise C→U→T conversion which can directly disrupt gene expression without DNA breaks. This Phase I study will investigate the feasibility and safety of base edited (BE) allogeneic CAR T cells against CD7, disrupted for TCR, CD7 and CD52. The cells are used to secure remission ahead of allogeneic stem cell transplantation (allo-SCT). Investigational medicinal product (IMP) Two allogeneic healthy donor derived BE-CAR7 T cell banks were manufactured from steady state apheresis harvests using a semi-automated process under compliant conditions. Cells were activated with an anti-CD3/CD28 reagent (Transact) and electroporated with codon optimised base editor (coBE) mRNA and three single guide RNAs targeting TRBC, CD52 and CD7. Subsequent lentiviral transduction delivered CAR7 (59-62% efficiency) and magnetic bead processing depleted residual TCRαβ+ T cells (residual 0.1-0.3%). Highly efficient disruption of CD7 and CD52 (98-99%) was confirmed by sequencing. Cells were cryopreserved and QC tested by flow cytometry, ddPCR for copy number (3.1-3.6) and replication competent lentivirus was excluded. Anti-leukemic potency was confirmed in vitro and in human:murine chimera experiments. Trial sponsorship & approvals The TvT CAR7 study (ISRCTN15323014) is an open label, single centre, single arm and non-randomised clinical trial. The trial is sponsored by Great Ormond Street Hospital NHS trust and is supported by MRC, Wellcome Trust and NIHR. Clinical trial authorisation was provided by MHRA and ethical approval by a gene therapy advisory committee (GTAC) ahead of site initiation. Study aims and Objectives The study aims to establish the safety and feasibility of BE-CAR7 to induce molecular remission in children with relapsed/refractory (r/r) CD7-positive T-ALL, ahead of a planned allo-SCT. Assessments include incidence of adverse events, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of BE-CAR7 cells and immune recovery after allo-SCT are monitored. Eligibility, treatment and recruitment The study opened on 14th April 2022 at Great Ormond Street Hospital recruiting children across the UK aged between 6 months and 16 years with r/r CD7+ T-cell malignancies quantifiable in bone marrow (>10-4 by flow or PCR). Exclusions include active uncontrolled infections, pre-existing GvHD or the detection of anti-HLA antibodies against the IMP. Eligible patients receive lymphodepletion with fludarabine (150 mg/sqm), cyclophosphamide (120 mg/kg) and alemtuzumab (1 mg/kg), followed by infusion of 0.2-2.0x106 BE-CAR7 T cells (maximum 5x104/kg TCRαβ T cells). Patients in molecular remission at day +28 proceed to allo-SCT, to remove BE-CAR 7 cells and promote immune reconstitution. The first subject was infused on 4th May 2022. Grade 2 CRS and grade 1 ICANS were observed, but no GvHD. Bone marrow assessment at d+28 revealed morphological remission without count recovery and with PCR-MRD <10-4 and the patient proceeded to reduced intensity allo-SCT as planned. This Phase 1 study aims to treat 10 children in the UK. Conclusion The study has demonstrated the feasibility of manufacturing "off-the-shelf” GMP-compliant base edited CAR7 T cells and is investigating their safety and efficacy against paediatric r/r T-ALL.

Expression Profiles of Human Endogenous Retrovirus in Chronic Myeloid Leukemia at Diagnosis and after TKI Therapy

INTRODUCTION Chronic Myeloid Leukemia (CML) has become a manageable and possibly curable disease due to the use of Tyrosine Kinase inhibitors (TKI). Although the immune system has been shown to be dysfunctional in newly diagnosed CML patients, TKI therapy may restore the function of the immune cells, and thus, the immune system may play a role in the leukemia control. In this complex immune interplay, the modulation and possible role of Human Endogenous Retroviruses (HERVs) expression is still poorly characterized. HERVs represent ~8% of the human genome and have been repeatedly domesticated by the host for major physiological functions, such as the development of placenta and the shaping of complex networks, including the main antiviral pathways of innate immunity. Accordingly, HERVs are still able to both modulate and be influenced by the host immune system and are thus highly investigated in several pathological contexts of immune dysregulation, such as autoimmunity and cancer. The aim of this study was to evaluate the overall HERV transcription profile in CML patients as compared to healthy controls (HC), to explore the regulatory influence of HERVs and their links to CML immune pathogenesis. METHODS The analysis included 10 HC and a total of 23 CML (11 Male, 7 Female, age range 45-84) being either at diagnosis (5 patients) or after TKI treatment (18 patients) for a minimum of 24 months and a molecular response of MR3 or deeper. Total RNA from peripheral blood cells was deep sequenced to obtain transcriptomic profiles, analyzed for the set of HERVs (~3300) and cellular genes (~60700) expression. RESULTS HERV transcriptome is significantly modulated in CML patients, clearly dividing them from HC. In addition, among CML patients, specific signatures of HERV modulation allows to distinguish actively affected patients from the ones that are in molecular remission after TKI treatment. The latter are also very different from HC in terms of HERV expression, possibly due to residual modulation by the disease and/or the prosecution of TKI therapy. Differential expression analyses allowed to identify a total of 575 and 568 deHERVs significantly modulated between CML patients at diagnosis and HC and between CML treated patients in molecular remission (MR3-MR5) and HC (padj: 3.36e-96 to 0.0098 and 8.32e-69 to 0.0099, respectively). In addition, 267 deHERVs were found comparing the same CML patients at diagnosis and during remission (padj: 5.55e-81 to 0.0099). The differential human gene expression revealed about 5800 dysregulated genes (4782 up and 1019 down, padj: 3.96e-260 to 9.5e-30) in CML pts vs HC. The top overexpressed genes were DDX17, VPS13B, PHIP, SMG1, STK10, LRRFIP1 with under-regulation of RNF213, MIR1276, ZRANB2-AS1, AC138028.4 genes, among others. Pairwise comparisons of 5 CML at diagnosis vs samples after treatment with imatinib resulted in a downregulation of ZNF831, EVL, TXK, HAPLN3, SEPTIN1, TNFRSF25 and upregulation of LTF, MPO, ABCA13, BPI, BCL2L15, ERG, MMP8, DEFA4, ANLN, SCN9A genes. CONCLUSIONS The present study provides the first high-throughput snapshot of HERV transcriptome in CML and identify specific sets of HERVs that can distinguish patients at diagnosis, patients in molecular response to TKI therapies and HC. Patient's gene expression profiles revealed that during molecular remission and under continuous treatments is present a disregulated pathway that is different from healthy individuals. Our results can give insights on the role of HERVs in the biology of CML and may add information useful to select the most effective pathways to target to maximize an advantageous immune response and promote a successful TKI discontinuation. Moreover, the following question is open: is there a potential regulatory role of HERVs in the genomic balance of CML patients? Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EICOSANOIDS AS HALLMARKS OF CHRONIC MYELOID LEUKEMIA PROGRESSION AND RESISTANCE TO TKI

Chronic myeloid leukemia (CML) is a Ph-positive myeloproliferative neoplasm giving rise to BCR-ABL1 oncogene. Usually, the disease is diagnosed in the chronic phase (CML-CP) with uncontrolled clonal expansion and high white blood cell count. Without therapy intervention, CML progress to advanced phases (accelerated and blastic phases; CML-AP). The Tyrosine Kinase Inhibitors (TKI) are the first-line therapeutic approach for CML. TKI targets the BCR-ABL tyrosine kinase activity and restrain the disease. The knowledge about the mechanisms involved in disease progression and resistance to TKI, beyond BCR-ABL1 , is limited. The relation among TKI treatment, disease progression and eicosanoids pattern in CML is unknown. Lipid mediators were quantified in healthy individuals (Controls; n = 9) as well as in CML patients in CP (n = 10), CML-AP (n = 13) and CML patients in molecular remission post-TKI therapy (RM; n = 6) using Liquid Chromatography/Mass Spectrometry (LC/MS). Mass spectral data were acquired with negative electrospray ionization. ProteinWizard was used to convert files to be used in bioinformatic subsequent analysis. Mummichog software was used for metabolic pathway enrichment analysis. Heatmaps were generated with the R package gplots and hierarchical clustering was performed with the R package amap with Spearman distance method and ward linkage algorithm. Volcano and bubble plots were obtained with the package ggplot2. We described the metabolomic profiles of CML in different phases and post-TKI therapy. The metabolic patterns were able to distinguish the CML patients in different phases and resistant to TKI. The metabolomic results suggested an association between reprogramming in lipid metabolism and CML progression and resistance to TKI therapy. CML patients showed to be a lower producer of some eicosanoids compared to controls. Interestingly, AP-CML produced less 12-HETE and EPA than CP-CML patients. CML patients in molecular remission produced more EPA in comparison with each other group. Taken together, the results established the eicosanoid profile of CML patients who progress to advanced phases and fail to respond to TKI.

Long-Term Clinical and Molecular Remissions in Patients with Follicular Lymphoma (FL) Following High-Dose Therapy (HDT) and Autologous Stem Cell Transplantation (ASCT)

Background: Long-term clinical and molecular remissions in patients with FL following HDT and ASCT have been evaluated in only a few studies. Results are especially limited for second-line HDT with BEAM (BCNU, etoposide, cytarabine and melphalan). Patients and methods: 73 patients with FL were treated in our institution from 1993 to 2016 (20 1st-line ASCT with total body irradiation (TBI) and cyclophosphamide, 46 2nd-line with BEAM and 8 ≥3rd-line with BEAM). Patients with high-grade transformation before ASCT were excluded. The data were collected retrospectively in 44 and prospectively in 29 patients who participated in two clinical trials: the 1st-line therapy trial of the German Low Grade Lymphoma Study Group (GLSG, principal investigator W. Hiddemann [Lenz G, Blood 2004]) and the salvage therapy LYM1 trial of the EBMT [Pettengell R, JCO 2013]. For the salvage ASCT protocol, most of the patients received 3 - 4 courses of the ESHAP or ICE protocol-since 2001, also usually combined with rituximab. Stem cell apheresis was carried out in the Blood Transfusion Service following these protocols or cyclophosphamide, usually in remission. Patients with partial remission after ASCT received a radiotherapy (RT) with 30-36 Gy in the field of persisting lymphoma, if possible, except participants of the LYM1 trial. Further details are described in an earlier publication of our first 60 patients (Metzner B, Ann Oncol 2013). Response assessment was performed by careful clinical examination (with ultrasound, chest x-ray and partly CT) at regular 3 - 12 monthly time points. In the case of long-term remission (≥ 8 years; n = 26), peripheral blood was tested for minimal residual disease by quantitative t(14;18) RQ-PCR and/or IGH-consensus PCR (n = 25). Results: With a median follow-up of 9.4 years (range 1.2-23.8) 10-year overall survival, progression-free survival and freedom from progression (FFP) after 1st-line ASCT were 80%, 60% and 68%, after 2nd-line ASCT 58%, 37% and 42%, after ≥3rd-line ASCT 50%, 25% and 25%, respectively, with a significant difference for OS, PFS and FFP between 1st-line and ≥3rd-line cohort, for PFS and FFP between 1st- and 2nd-line cohort (p<0.05). The median OS from the initial diagnosis for the 1st-line cohort and the 2nd-line cohort was not yet reached (estimated >23 years); and 9.4 years for the ≥3rd-line cohort; for a subgroup of the 2nd-line cohort with early relapse (time from diagnosis to 1st relapse maximum 24 months, n = 26) not yet reached (estimated >21 years). Ten-year FFP for 2nd-line ASCT and low-risk FLIPI score (n = 16) was 69%, intermediate risk (n = 11) 27% and high risk (n = 18) 29% (p=0.051). No relapses occurred after 8 years following ASCT in 1st or 2nd remission (except one patient 10 years after ASCT). So far, 26 patients developed long-term remissions of at least 8 years (up to 24, median 14 years). 24 of the 25 tested long-term remission patients were MRD-negative at the last follow-up. One patient was MRD-positive (low-level MRD) at the last control (10.6 years after ASCT in 1st line, unchanged as the years before). Five-year non-relapse mortality was 0%. Four of 73 patients developed secondary invasive malignancies in remission after ASCT: in the TBI cohort, 2 of 20 patients; in the BEAM cohort, 2 of 53 patients, (in the 2nd-line ASCT cohort 1 of 45 patients). Conclusion: Sustained long-term clinical and molecular remissions up to > 22 years can be achieved following ASCT in 1st or 2nd treatment line, indicating the potential curative impact of ASCT in FL. [Display omitted] DisclosuresPott:Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Higher Rates of Molecular Response to Peg-IFNa in JAK2V617F Vs. Calr Mutant MPN Patients Are Due to JAK1-Mediated STAT1 Activation and Autoregulation

Introduction: Pegylated interferon alpha (Peg-IFNa) treatment has been described to induce molecular remission in patients (pts) with myeloproliferative neoplasms (MPN), with partial molecular remission (PMR) being achieved in approximately 30-40% of pts. However, it is unclear whether pts with CALR mutations exhibit the same response as pts with JAK2V617F mutations. Also, the precise mechanisms accounting for the responses are still unknown.Methods: We report on hematological and molecular responses, according to ELN guidelines, in 52 MPN pts (JAK2V617F n=26 vs. CALR mutation n=26; ET n=25, prePMF n=8, PMF n=19), treated at Roskilde hospital with Peg-IFNa. The mechanism of IFNa response was assessed in stably transduced 32D murine cell lines and in a cohort of MPN patients within the Czech MPN registry MIND. All pts provided written informed consent, as approved by the local ethics committees. Cell proliferation was assessed by cell counting and/or MTT assay, signaling molecules were quantified using RT-PCR, flow cytometry and/or Western blotting.Results: Pts harboring JAK2V617F mutations were more likely to achieve PMR than those harboring CALR mutations (60% vs. 23%, p<0.05 by Chi square testing), while there was no significant difference in allelic burden before Peg-IFNa treatment. Accordingly, the median allelic burden decrease from baseline in the IFNa responding pts was 66% (range 15-99%) in JAK2V617F- vs. 29% (range 5-82%) in CALR-mutant pts. These differences were present independently of the fractions of ET, prePMF, or PMF pts in the two groups. There was no significant difference in the rates of hematological response. When examining primary cells from the Czech MPN cohort, cells from JAK2V617F positive pts showed higher expression of IFNa response-related genes such as Irf1 (p=0.0350) and Stat1 (trend at p=0.0874). Moreover, IFNa receptor expression was higher in JAK2V617F positive pts (p=0.0471) and the expression levels were associated with hemoglobin (p=0.0139) and hematocrit levels (p=0.0251). This was reflected in 32D cell lines, ectopically expressing both the MPL receptor and either JAK2V617F or CALR del 52. Here, the expression of Irf7, Irf-9, Ifi-27, and Stat2 was significantly higher in JAK2VF vs. CALR mutants (all p<0.001). In addition, JAK2V617F positive cells showed higher STAT1 phosphorylation and Stat1 mRNA expression levels (p=0.0499). In particular, STAT1 showed constitutive phosphorylation in JAK2V617F but not CALR mutant cells, indicating priming of these cells towards an IFNa response. Overexpression of STAT1 but not of a dominant-negative STAT1YF mutant was able to rescue cellular IFNa-induced growth arrest in CALR positive cells to the same level of JAK2V617F positive cells. In contrast, manipulation of STAT2 expression did not alter IFNa signaling. IFNa-induced growth arrest was counteracted by selective JAK1 inhibition (using itacitinib or filgotinib) but not selective JAK2 inhibition (using BMS-911543).Conclusions: MPN pts harboring JAK2V617F mutations show superior Peg-IFNa sensitivity against the malignant clone, as detected by molecular responses, compared to pts harboring CALR mutations. The higher IFNa sensitivity of JAK2V617F mutant cells was reflected in mutant-specific cell lines and is at least in part due to JAK1-mediated increases in STAT1 phosphorylation and STAT1 transcription. These results are of considerable importance for future clinical trials of Peg-IFNa as monotherapy or in combination with JAK inhibitors. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding. Knudsen:Novartis: Other: Payed for ASH2016 registration fee, travel expenses and accommodation. Hasselbalch:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOR Orphan: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.

A drug targeting 5-lipoxygenase enhances the activity of a JAK2 inhibitor in CD34+ bone marrow cells from patients with JAK2V617F-positive polycythemia vera in vitro.

Janus kinase 2 (JAK2) inhibitors, the first targeted treatments for myeloproliferative neoplasms (MPNs), provide substantial benefits, including a marked reduction in splenomegaly and MPN-associated symptoms. However, these drugs rarely induce molecular remission in patients with MPNs. Zileuton, a 5-lipoxygenase (5-LO) inhibitor, has been demonstrated to selectively deplete hematopoietic stem cells (HSCs) expressing a JAK2 point mutation (JAK2V617F) in mouse models of JAK2V617F-induced polycythemia vera (PV). To determine the potential activity of 5-LO inhibitors in combination with JAK inhibitors against human PV HSCs, the present study first analyzed 5-LO expression in CD34+ bone marrow cells from patients with JAK2V617F-positive PV using western blotting and reverse transcription-quantitative PCR, and then examined the effect of zileuton combined with ruxolitinib on colony formation using a colony formation assay. Furthermore, cell cycle and apoptosis in CD34+ cells from patients with PV and healthy volunteers were determined by flow cytometry. In the present study, 5-LO expression was upregulated in CD34+ cells from patients with PV compared with in CD34+ cells from healthy volunteers. Higher levels of leukotriene B4, a product of the 5-LO signaling pathway, were detected in patients with PV compared with in healthy volunteers. Zileuton treatment suppressed the colony formation of CD34+ cells from patients with PV in a dose-dependent manner. Furthermore, zileuton and ruxolitinib exerted their anticancer effects by suppressing hematopoietic colony formation, inducing apoptosis and arresting the cell cycle of human CD34+ cells from patients with PV. The combination of these two drugs exerted a more beneficial effect than either agent alone. Based on these data, zileuton enhanced the antitumor activity of low-dose ruxolitinib in hematopoietic progenitor cells from patients with PV, providing conceptual validation for further clinical applications of combination treatment with ruxolitinib and zileuton for patients with PV.

TKI dose reduction can effectively maintain major molecular remission in patients with chronic myeloid leukaemia.

Targeted therapy for chronic myeloid leukaemia (CML) has allowed for a near-normal patient life-expectancy; however, quality of life and aggravation of existing co-morbidities have posed new treatment challenges. In clinical practice, TKI dose reduction occurs frequently, often on multiple occasions, because of intolerance. We conducted a retrospective 'real-world practice' review of 246 patients receiving lower than standard dose (LD) TKI after the achievement of major molecular response (MR3), because of intolerable adverse events. In 274 of 298 cases of dose reduction (91·9%), MR3 was maintained at median follow-up of 27·3months. One patient progressed to blast crisis while on LD TKI. Two patients developed two new ABL kinase domain mutations (T315I and V299L), of whom one had achieved deep molecular response on an alternative LD TKI at last follow-up. Seventy-six patients eventually discontinued LD TKI and the two-year treatment-free remission (TFR) rate in these patients was 74·1%. The majority of patients with CML in at least MR3 appear to be safely managed with LD TKI, although three of 246 patients had new events (progression and new mutation), indicating that this approach requires vigilance. TKI LD does not prevent the achievement of TFR in this patient population.

Blast MRD CML 1 Trial: Blockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease (MRD) in Chronic Myeloid Leukemia (CML)- a Phase II Study of Adding the Anti-PD-1 Pembrolizumab to Tyrosine Kinase Inhibitors in Patients with Chronic Myeloid Leukemia and Persistently Detectable Minimal Residual Disease: A Trial of the ECOG-ACRIN Cancer Research Group (EA9171)

Blast MRD CML 1 Trial: Blockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease (MRD) in Chronic Myeloid Leukemia (CML)- a Phase II Study of Adding the Anti-PD-1 Pembrolizumab to Tyrosine Kinase Inhibitors in Patients with Chronic Myeloid Leukemia and Persistently Detectable Minimal Residual Disease: A Trial of the ECOG-ACRIN Cancer Research Group (EA9171)

Long-Term Clinical and Molecular Remissions in Patients with Mantle Cell Lymphoma (MCL) Following High-Dose Therapy (HDT) and Autologous Stem Cell Transplantation (ASCT)

Background:Long-term clinical and molecular remissions in patients with MCL following HDT and ASCT have been evaluated in only a few studies. Results are especially limited for remission duration over 5 years. So the curability of this disease remains an open question. Patients and methods:Altogether 61 patients with MCL were treated in our institution with ASCT from 1998 to April 2019 (50 1st-line ASCT, ten 2nd-line ASCT, one 3rd-line ASCT). The data were collected retrospectively in 29 and prospectively in 32 patients who participated in two clinical trials: the 1st-line therapy trials of the German Low Grade Lymphoma Study Group (GLSG, principal investigator W. Hiddemann [Dreyling M, Blood 2005] and the European Mantle Cell Lymphoma Network [Hermine O, Lancet 2016], respectively. The diagnosis was regularly approved by the reference pathology of the GLSG (W.K.). The induction therapy before 1st-line ASCT consisted of 6 courses CHOP (n=11), mostly combined with rituximab and followed by Dexa-BEAM, and 6 alternating courses of R-CHOP and R-DHAP (n=39), respectively. For the salvage treatment patients usually received three to four courses of the DHAP protocol or the ESHAP protocol, since 2001 also combined with rituximab.Stem cell apheresis was carried out in the Blood Transfusion Service following these protocols in remission. High-dose protocols: 1) total body irradiation with cyclophosphamide or melphalan and cytarabin (n=32) or 2) BEAM (n=29). Patients with partial remission after ASCT received a radiotherapy (RT) with 30-36 Gy in the field of persisting lymphoma, if possible (n=4). Since 09/2016 patients received a maintenance therapy with rituximab after ASCT (every 2 months, planned for 3 years, n=14). Further details are described in an earlier publication of our first 36 patients (Metzner B, Ann Hematol 2013). Response assessment was performed by careful clinical examination and by ultrasound, chest x-ray and partly CT at regular 3 - 12 monthly time points. In the case of long-term remission (≥ 5 years; n = 18), peripheral blood was regularly tested twice a year for minimal residual disease (MRD) by quantitative t(11;14) or allel specific IGH RQ-PCR and/or IGH-consensus PCR.Calculations were done using IBM SPSS Statistics Version 25. Data were analysed as of 01 July 2019. Results:With a median follow-up of 5 years (range 0.1-20) 10-year overall survival, progression-free survival (PFS) and freedom from progression (FFP) after 1st-line ASCT were 54%, 46% and 52%, respectively, after 2nd-line ASCT 42%, 20%and 20%, with a significant difference for PFS (p=0.045) and FFP (p=0.014) between 1st-line and 2nd-line cohort.Further prognostic factors (like sex, age, MIPI, bone marrow involvement, remission grade at ASCT: CR vs. PR, type of HDT: TBI vs. BEAM…) seemed to be without relevance (considering the small subgroups). Only one clinical relapse occurred after 5 years following ASCT in 1stor 2ndremission, respectively (one patient 6 years after 1st-line ASCT and another patient 7 years after 2nd-line ASCT and subsequent radiotherapy). So far, 18 patients experienced long-term remissions of at least 5 years (median 9 years, from 5 to 16 years). Fifteen of 17 tested patients were MRD negative at last follow-up, the two MRD positive patients (positive at a low level below the quantitative measuring range) had no clinical signs of relapse at last follow-up. None of these 18 patients had received rituximab maintenance therapy. None showed clinical criteria of "smoldering mantle cell lymphoma" at induction therapy. Treatment-related mortality at 100 days after ASCT was 1.6% (pneumonitis following TBI). One patient developed a secondary invasive malignancy in remission after ASCT (acute myeloid leukemia 4 years following TBI). Conclusion: Sustained long-term clinical and molecular remissions up to 16 years can be achieved following ASCT (without rituximab maintenance therapy), indicating the potential curative impact of 1st-line ASCT in MCL. The 2nd-line ASCT was obviously less effective. Disclosures Dreyling: Celgene: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: scientific advisory board, Research Funding, Speakers Bureau; Bayer: Consultancy, Other: scientific advisory board, Speakers Bureau; Gilead: Consultancy, Other: scientific advisory board, Speakers Bureau; Acerta: Other: scientific advisory board; Novartis: Other: scientific advisory board; Sandoz: Other: scientific advisory board. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P&lt;0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

World Arthritis Day 2018 ‐ Perspectives on Rheumatic musculoskeletal diseases

World Arthritis Day 2018 ‐ Perspectives on Rheumatic musculoskeletal diseases

Tamibarotene maintenance improved relapse-free survival of acute promyelocytic leukemia: a final result of prospective, randomized, JALSG-APL204 study.

Between April 2004 and December 2010, we conducted a prospective randomized controlled study comparing tamibarotene with all-trans retinoic acid (ATRA) in the maintenance therapy of newly diagnosed acute promyelocytic leukemia (APL), and here report the final results of this study with a median follow-up of 7.3 years. Of 344 eligible patients who had received ATRA and chemotherapy, 319 (93%) achieved complete remission (CR). After completion of three courses of consolidation chemotherapy, 269 patients in molecular remission underwent maintenance randomization, 135 to ATRA (45 mg/m2 daily), and 134 to tamibarotene (6 mg/m2 daily) for 14 days every 3 months for 2 years. The primary endpoint was relapse-free survival (RFS). The 7-year RFS was 84% in the ATRA arm and 93% in the tamibarotene arm (p = 0.027, HR = 0.44, 95% CI, 0.21 to 0.93). The difference was prominent in high-risk patients with initial leukocytes ≥ 10.0 × 109/L (62% vs. 89%; p = 0.034). Tamibarotene was significantly superior to ATRA by decreasing relapse in high-risk patients. Overall survival after randomization did not differ (96% vs. 97%; p = 0.520). Secondary hematopoietic disorders developed in nine patients, secondary malignancies in 11, and grade 3 or more late cardiac comorbidities in three. These late complications did not differ between the two arms.

Histomorphological responses after therapy with pegylated interferon \u03b1-2a in patients with essential thrombocythemia (ET) and polycythemia vera (PV)

BackgroundPegylated interferon alfa-2a (PEG-IFN-α-2a) is a potent immunomodulating agent capable of inducing high rate of hematologic and even complete molecular remission in patients with essential thrombocythemia (ET) and polycythemia vera (PV). We recently reported results of a phase 2 trial of PEG-IFN-α-2a in 83 patients with ET and PV after a median follow-up of 83 months. Here we report an analysis of bone marrow (BM) responses in these patients.MethodsAmong 83 patients, 58 (70%, PV 25, ET 31) had evaluable BM samples. BM responses and fibrosis grading were defined according to the International Working Group for Myeloproliferative Neoplasms Research and Treatment, and the European Consensus on grading of BM fibrosis, respectively. BM was assessed prior to enrollment, and every 6–24 months while on therapy in all patients, and after therapy discontinuation in some patients.ResultsThe median age of analyzed 58 patients was 52 years, and 29% were males. After a median follow-up of 84 months, 32 patients are still on study. Hematologic (HR) and molecular responses (MR) were seen in 93 and 69% patients, respectively. Twenty-nine patients (50%) had a BM response, including 13 (22%) with a complete BM response (BM-CR). Moreover, 13 patients (22%) have experienced complete resolution of bone marrow reticulin fibrosis. Patients with BM response had higher duration of HR and MR, and lower discontinuation rate. Furthermore, patients with BM-CR had a higher probability of complete MR. The median duration of BM-CR was 30 months, and 9 patients have maintained their BM-CR (69%), including five who have maintained their response after discontinuation of therapy. Despite this observation, the pattern of HR, MR and BM response, their durability and interrelation was heterogeneous.ConclusionsOur results show the ability of PEG-IFN-α-2a to induce complete BM responses in a subset of ET and PV patients, but its correlation with durable clinically relevant treatment benefit warrants further investigation.Trial registration This study is registered with ClinicalTrials.gov (NCT00452023), and is ongoing but not enrolling new patients.

380 Determination of molecular remission in patients with mycosis fungoides (MF) and Sezary Syndrome (SS) following allogeneic transplant using high throughput sequencing (HTS)

Allogeneic stem cell transplantation (ASCT) is the only potentially curative therapy in pts with advanced stage MF/SS. Currently, we lack predictive markers associated with clinical outcome after ASCT. We utilized HTS of TCRß to evaluate the impact of minimal residual disease (MRD) after ASCT in a prospective clinical trial. Our study included 12 pts with MF and 20 with SS. Median age was 62 (range 20-75). Twenty six pts (81%) had stage IV disease and none achieved complete response from last systemic therapy immediately prior to conditioning. All received non-myeloablative conditioning with total skin electron beam therapy (TSEBT), total lymphoid irradiation (TLI, 8 Gy) and anti-thymocyte globulin (ATG). Pts tolerated ASCT extremely well with 1-year transplant-related mortality of 3%. At 3-months post-transplant, 19 pts achieved complete clinical response and 10 pts had a partial response. Of the 27 pts assessed by the specific and ultra-sensitive (detecting 1 in 150,000 cells) HTS of TCRß, 12 pts (44%) achieved molecular remission in the blood after ASCT. The median time to molecular remission in the blood was 120 days (range 30-730). Of these 12 pts, 11 of them also achieved molecular remission in the skin. Pts who achieved molecular remission in both blood and skin had less chance of disease progression/relapse than those who had MRD, with cumulative incidence of progression/relapse of 13% and 92%, respectively (p=0.0005). Herein, we demonstrate that a non-myeloablative ASCT utilizing our unique TSEBT/TLI/ATG conditioning is safe and effective in treating patients with advanced stage MF/SS. Our findings show that close to half of the pts achieved molecular remission after transplant as determined by HTS of TCRß, which correlated with a very low likelihood of disease progression/relapse. This is the first report of a systematic, rigorous investigation of a clinically meaningful impact of molecular remission in this pt population.

Pegylated interferon alfa-2a in patients with essential thrombocythaemia or polycythaemia vera: a post-hoc, median 83 month follow-up of an open-label, phase 2 trial

BackgroundThe durability of responses and long-term safety of pegylated interferon alpha-2a (PEG-IFN-a-2a) in patients with polycythemia vera and essential thrombocythemia have not been reported. Here, we present long-term efficacy and safety data from a single-center, prospective, phase 2 study, after 7 years of follow-up.MethodsPatients older than 18 years who were diagnosed with essential thrombocythemia or polycythemia vera per 2001 World Health Organization criteria were eligble to enroll.Responses were assessed every 3–6 months: Data were analyzed using descriptive statistics. The rate of leukemia transformation was compared with age- and gender-matched patients who were not treated with PEG-IFN-α-2a.FindingsPEG-IFN-α-2a induced hematologic (80%) and molecular responses (63%) in 83 patients with essential thrombocythemia (n=40) and polycythemia vera (n=43), with median durations of 66 and 53 months, respectively. Thirty-nine percent of hematologic responders and 71% of molecular responders (JAK2V617F+) have maintained some response during follow-up: 48% maintained their best molecular response, including 9 of 10 patients with a complete molecular response. The incidence of major venous-thrombotic events during the study was 1.22/person-year. Overall, 22% of patients discontinued therapy due to treatment-related toxicity. While toxicity rates decreased over time, 5 patients experienced treatment-limiting G3/4 toxicities after 60 months on therapy. Rates of transformation to MF/AML were similar between patients treated with PEG-IFN-a-2a and those from a historical control series.InterpretationPEG-IFN-α-2a can induce durable hematologic and molecular remissions in patients with essential thrombocythemia and polycythemia vera. We suggest a starting dose of 45 mcg/week, and its combination with other drugs should be explored further in clinical trials.

Management of Relapsed Acute Promyelocytic Leukemia Post ATO Upfront Therapy: Open-Labeled Phase II Study Evaluating Role of Proteasome Inhibition

▪Introduction: There is limited data on the best treatment strategy for patients who relapse after upfront arsenic trioxide (ATO) based therapy for acute promyelocytic leukemia (APL). We had recently reported that there was significant micro-environment mediated drug resistance (EM-DR) to ATO which we noted was predominantly mediated by upregulation of the NF-κB pathway. We also noted that this upregulation was more prominent in relapsed APL and that EMDR to ATO could be overcome by the use of proteasome inhibitors (Leukemia 2016 - In Press). Pre-clinical mouse model and preliminary clinical data validated these observations. There has also been a recent concern of mutations in the oncogenic PML-RARA gene following treatment with ATO, with reports suggesting a third of relapsed APL patients having such mutations and that the clinical outcomes in this subset with conventional therapy is poor [NEJM. 2014;370(19): 1864-6].Methods: In this prospective, open-label, non-randomized, phase 2, single center study, we enrolled APL patients in first hematological relapse. Inclusion criteria included presence of t(15; 17) and exposure to ATO as part of up front therapy. Enrolled patients received conventional therapy for relapse, included ATO and ATRA in induction, consolidation. Anthracycline (mitoxantrone) was used only in induction. Additionally, patients received two doses of bortezomib (1.4mg / m2 / dose) in induction and consolidation on days 2 and 5 and one dose in maintenance (10 days/month x 6 months) on day 2. Primary end point was safety (graded according to the NCI Common Terminology Criteria for Adverse Events version 4.0) and the key secondary end points were proportion in complete molecular remission at the end of induction and event free survival (EFS). The study is ongoing and continues to recruit patients. This trial is registered with ClinicalTrials.gov, number NCT01950611.Results: Between Sep 2013 and Mar 2016, 18 patients met eligibility criteria and were enrolled in this study. All 18 patients achieved CHR at a median of 45 days (range: 42-63). Seventeen patients (94.4%) were RT-PCR negative at the end of induction. The median time complete molecular remission was 35 days (range: 21-56 days). None of the patients had any major bleeding or thrombotic events in induction. With the exception of one patient who had Grade IV neurotoxicity which required treatment and recovered after temporarily discontinuing treatment during induction none of the other patients had any Grade III/IV non-hematological toxicity. Other non-hematological events were ≤ Grade II, usually transient and judged to be unrelated to the interventional agent. These included mild headache in 8 (33.3%) and one case (5.5%) each with diarrhea, mild rash, redness of eyes, oral ulcers, vertigo and paresthesia. Auto-SCT was offered to all patients in molecular remission post consolidation. Eight patients (44.4%) underwent an autologous SCT post consolidation while the rest opted to receive maintenance therapy. One patient who opted for maintenance therapy relapsed 6 months after completing maintenance. At an actuarial median follow up of 24 months (range: 4-35) the 3 year KM estimate of EFS was 92.3±7.4% and the cohort had 100% survival at last follow up. This clinical profile and outcome data was compared with a historical cohort that was treated with the same protocol with the exception of bortezomib and the comparative data is summarized in table 1 and figures 1A and 1B.Conclusion: Addition of proteasome inhibitors with ATO is well tolerated. In combination with conventional agents, proteasome inhibitors can potentially improve the clinical outcome of patients with relapsed APL treated with upfront ATO. Our preliminary data also suggests that this combination can obviate the need for an auto-SCT in this setting. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.