Impending Relapse Research Articles

A Long-Term Safety and Tolerability Study of TV-46000 for Subcutaneous Use in Patients with Schizophrenia: A Phase 3, Randomized, Double-Blinded Clinical Trial.

TV-46000 is a long-acting subcutaneous antipsychotic (LASCA) formulation of risperidone that is approved by the United States Food and Drug Administration for the treatment of schizophrenia in adults. In the phase 3, randomized, double-blind RIsperidone Subcutaneous Extended-release (RISE) study, TV-46000 once monthly (q1m) and once every 2months (q2m) significantly prolonged time to impending relapse compared with placebo [5.0-fold (q1m) and 2.7-fold (q2m)]. This phase 3, randomized, double-blind Safety in Humans of TV-46000 subcutaneous INjection Evaluation (SHINE) study was designed to evaluate the long-term safety, tolerability, and exposure of TV-46000 in schizophrenia. Patients who completed RISE without relapse (rollover) or who were newly recruited (de novo) were eligible for the SHINE study. Patients were initially stabilized on oral risperidone for 12 weeks (completed in RISE for rollover, or in SHINE for de novo). Patients in the de novo cohort and patients who received placebo in RISE were randomized 1:1 in SHINE to receive TV-46000 q1m or q2m for up to 56 weeks. Primary endpoint for SHINE was frequency of reported adverse events (AEs); event rates [ER; events per 100 patient-years (PYs)] were calculated for each AE by patients upon general questioning. Overall, 336patients were randomized in SHINE [TV-46000 q1m, n = 174; TV-46000 q2m, n = 162; of these, de novo, n = 109 and rollover, n = 227 (n = 172 patients were treated and n = 55 received placebo)]. A total of 334 patientswere evaluated for safety [q1m, n = 172 (PY = 97.8); q2m, n = 162 (PY = 104.5)]. Proportions of patients (ER) with ≥ 1 AE and ≥ 1 treatment-related AE were 37% (180.0) and 21% (84.9) for TV-46000 q1m and 46% (157.9) and 20% (70.8) for TV-46000 q2m, respectively. Frequent treatment-related AEs [≥ 3% of patients in either group; proportion of patients (ER)] were injection site pain [q1m, 5% (24.5); q2m, 4% (22.0)] and injection site nodule [q1m, 2% (9.2); q2m, 6%(12.4)]. The proportions of patients with serious AEs was 5% for TV-46000 q1m and 7% for TV-46000 q2m; serious AEs reported for ≥ 2 patients overall were worsening schizophrenia [q1m, n = 1 (< 1%; ER, 1.02); q2m, n = 2 (1%; ER, 1.91)] and hyperglycemia [q1m, n = 1 (< 1%; ER, 1.02); q2m, n = 1 (< 1%; ER, 0.96)]. Ofthreereported deaths, none were related to treatment. Overall, eight patients discontinued treatment because of AEs. Similar or somewhat lower rates of AEs were reported for patients who rolled over from TV-46000 treatment compared with those who had no prior TV-46000 treatment (de novo and placebo rollover). Most AEs related to injection site reactions were mild; no patient had a severe reaction. Results from this long-term safety study add to the favorable safety profiles of TV-46000 q1m and q2m, consistent with other formulations of risperidone and previous studies with TV-46000. ClinicalTrials.gov, NCT03893825; 27 March 2019.

Pre-emptive detection and evolution of relapse in acute myeloid leukemia by flow cytometric measurable residual disease surveillance.

Measurable residual disease (MRD) surveillance in acute myeloid leukemia (AML) may identify patients destined for relapse and thus provide the option of pre-emptive therapy to improve their outcome. Whilst flow cytometric MRD (Flow-MRD) can be applied to high-risk AML/ myelodysplasia patients, its diagnostic performance for detecting impending relapse is unknown. We evaluated this in a cohort comprising 136 true positives (bone marrows preceding relapse by a median of 2.45 months) and 155 true negatives (bone marrows during sustained remission). At an optimal Flow-MRD threshold of 0.040%, clinical sensitivity and specificity for relapse was 74% and 87% respectively (51% and 98% for Flow-MRD ≥ 0.1%) by 'different-from-normal' analysis. Median relapse kinetics were 0.78 log10/month but significantly higher at 0.92 log10/month for FLT3-mutated AML. Computational (unsupervised) Flow-MRD (C-Flow-MRD) generated optimal MRD thresholds of 0.036% and 0.082% with equivalent clinical sensitivity to standard analysis. C-Flow-MRD-identified aberrancies in HLADRlow or CD34+CD38low (LSC-type) subpopulations contributed the greatest clinical accuracy (56% sensitivity, 90% specificity) and notably, by longitudinal profiling expanded rapidly within blasts in > 40% of 86 paired MRD and relapse samples. In conclusion, flow MRD surveillance can detect MRD relapse in high risk AML and its evaluation may be enhanced by computational analysis.

Clonal Dynamics and Relapse Risk Revealed by High-Sensitivity FLT3-Internal Tandem Duplication Detection in Acute Myeloid Leukemia

The ability to detect low-level disease is key to our understanding of clonal heterogeneity in acute myeloid leukemia (AML) and residual disease that elude conventional assays and seed relapse. We developed a high-sensitivity next-generation sequencing (HS-NGS) clinical assay, able to reliably detect low levels (1 × 10−5) of FLT3-ITD, a frequent, therapeutically targetable and prognostically relevant mutation in AML. By applying this assay to 289 longitudinal samples from 62 patients at initial diagnosis and/or clinical follow-up (mean follow-up of 22 months), we reveal the frequent occurrence of FLT3-ITD subclones at diagnosis and demonstrate a significantly decreased relapse risk when FLT3-ITD is cleared after induction or thereafter. We perform pairwise sequencing of diagnosis and relapse samples from 23 patients to uncover more detailed patterns of FLT3-ITD clonal evolution at relapse than is detectable by less-sensitive assays. Finally, we show that rising ITD level during consecutive biopsies is a harbinger of impending relapse. Our findings corroborate the emerging clinical utility of high-sensitivity FLT3-ITD testing and expands our understanding of clonal dynamics in FLT3-ITD–positive AML.

Monitoring ctDNA dynamics in early breast cancer using a novel ultra-sensitive tumor-informed structural variant approach combining whole-genome sequencing and multiplex dPCR.

568 Background: Persistent circulating cell-free tumor DNA (ctDNA) detection during neoadjuvant treatment of early-breast cancer (EBC) indicates high-risk disease. Detection of ctDNA post-resection of EBC (molecular residual disease, MRD) indicates occult metastatic disease and impending disease relapse. For ctDNA to be integrated into EBC management, accessible and scalable diagnostic tools are required. Here, we apply a highly sensitive, personalized tumor-informed approach to ctDNA evaluation predicated on analyses of structural variants (SVs) using a novel digital PCR (dPCR) SV technology. Methods: 170 patients with EBC and eligible for neoadjuvant therapy (NAT) were recruited through the prospective SCAN-B study (NCT02306096) between Dec 2014 and Mar 2019 (25.8% TNBC, 47.1% HR+/HER2- and 24.1% HER2+). Interim results are presented for the first 46 consecutive patients (comprising 567 plasma samples) where minimum QC criteria were met (10% tumor content and 10x sequencing depth). Whole genome sequencing (WGS) was performed on tumor material and personalized multiplex dPCR assays designed tracking up to 8 SVs for use in ctDNA analyses. Plasma samples were collected at baseline, during NAT, pre- and post-surgery and at 6-monthly intervals during follow up. Clinical characteristics and recurrence outcomes were recorded. Results: ctDNA was detected at one or more timepoints prior to surgery in 43/46 (93%) patients across all breast cancer subtypes, at a median variant allele frequency (VAF) of 0.14% (range 0.0002% - 27.6%). ctDNA levels remained detectable at the end of NAT in 24% patients (11/46); 5/11 (45%) ctDNA positive patients experienced disease relapse versus 1/35 (3%) ctDNA negative patients (P=0.002, Fisher’s exact test). At one or more post-operative timepoints, ctDNA was detected in 6/6 (100%) patients who experienced clinical recurrence, with lead times up to 52 months (median 11.8 months, range 3.5 to 52 months). In 40 patients without presentation of clinical recurrence to date, ctDNA was undetectable across 285/287 (99.3%) plasma timepoints collected post-surgery. Post-operative detection of ctDNA associated with poor overall survival (OS) compared to absence of ctDNA (log-rank P&lt;0.0001). Conclusions: In this interim analysis of an ongoing prospective study in patients with EBC receiving NAT, we analyzed plasma for ctDNA using a novel tumor-informed dPCR assay tracking patient-specific SVs. ctDNA detection post-neoadjuvant therapy, prior to surgery associated with high-risk of disease relapse. Postoperative ctDNA detection was observed in 100% patients with clinical recurrence, and associated with poorer OS and long lead-times. Our data demonstrate the feasibility of SVs as an MRD analyte and provide evidence for high levels of clinical sensitivity achievable with this approach in EBC. Clinical trial information: NCT02306096 .

Clonal relapse dynamics in acute myeloid leukemia following allogeneic hematopoietic cell transplantation

AbstractPatients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by using error-corrected next-generation sequencing (NGS) in 74 patients with AML relapsing after alloHCT, evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least 1 MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 month before relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, whereas 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, whereas mutations in signaling genes demonstrated a shorter lead time to relapse. Both DTA (DNMT3A, TET2, and ASXL1) and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS, supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis.

Epileptic spasms relapse is associated with response latency but not conventional attributes of post-treatment EEG.

Relapse of epileptic spasms after initial treatment of infantile epileptic spasms syndrome (IESS) is common. However, past studies of small cohorts have inconsistently linked relapse risk to etiology, treatment modality, and EEG features upon response. Using a large single-center IESS cohort, we set out to quantify the risk of epileptic spasms relapse and identify specific risk factors. We identified all children with epileptic spasms at our center using a clinical EEG database. Using the electronic medical record, we confirmed IESS syndrome classification and ascertained treatment, response, time to relapse, etiology, EEG features, and other demographic factors. Relapse-free survival analysis was carried out using Cox proportional hazards regression. Among 599 children with IESS, 197 specifically responded to hormonal therapy and/or vigabatrin (as opposed to surgery or other second-line treatments). In this study, 41 (21%) subjects exhibited relapse of epileptic spasms within 12 months of response. Longer duration of IESS prior to response (>3 months) was strongly associated with shorter latency to relapse (hazard ratio = 3.11; 95% CI 1.59-6.10; p = 0.001). Relapse was not associated with etiology, developmental status, or any post-treatment EEG feature. This study suggests that long duration of IESS before response is the single largest clinical predictor of relapse risk, and therefore underscores the importance of prompt and successful initial treatment. Further study is needed to evaluate candidate biomarkers of epileptic spasms relapse and identify treatments to mitigate this risk. Relapse of infantile spasms is common after initially successful treatment. With study of a large group of children with infantile spasms, we determined that relapse is linked to long duration of infantile spasms. In contrast, relapse was not associated with the cause of infantile spasms, developmental measures, or EEG features at the time of initial response. Further study is needed to identify tools to predict impending relapse of infantile spasms.

Disease Monitoring in Acute Myeloid Leukemia with CLEC2A qRT-PCR

CLEC2A, C-Type Lectin Domain Family 2 Member A, is a newly discovered AML-restricted antigen. Detailed characteristics, clinical correlations, and therapeutic potential of CLEC2A in AML is presented by Kirkey et. Al. (ASH 2023). CLEC2A is entirely silent in normal hematopoiesis and uniquely expressed in patients with KMT2A rearrangements, providing an ideal therapeutic target in this high-risk subtype of AML. To date, CLEC2A has not been implicated in hematologic malignancies and expression of CLEC2A is entirely absent from normal tissues other than minimal expression in the skin. In addition to its utility as a therapeutic target, CLEC2A provides a unique target for disease monitoring given its lack of expression in normal hematopoietic cells. Here we present a highly sensitive quantitative molecular assay for CLEC2A, establishing its utility for disease monitoring in patients with KMT2A-r AML. In our efforts to evaluate CLEC2A expression and demonstrate the utility of using CLEC2A for disease monitoring in KMT2A-r, we developed a qRT-PCR assay using probes directed at exon 3 of the CLEC2A gene. The assay was optimized with RNA from the OCI-AML2 cells, validated to be CLEC2A-positive by flow cytometry and RNA from Kasumi-1 and K562 cells, both CLEC2A-negative by flow cytometry. We established the sensitivity of the assay through serial dilution of OCI-AML2 in a background of CLEC2A-negative Kasumi-1 or K562 cell RNA with concentrations ranging from 100% to 0.0001% OCI-AML2 RNA. The sensitivity assay demonstrated CLEC2A was detected at a level of 0.001% in both Kasumi-1 and K562. Given the association of CLEC2A with KMT2A-r AML, we evaluated the prevalence of CLEC2A as assessed by qRT-PCR. We screened diagnostic specimens from a large cohort of patients enrolled on recent COG Phase III clinical trials (ages 0-29) with and without KMT2A fusions to establish prevalence of CLEC2A expression. Using our CLEC2A qRT-PCR assay we evaluated diagnostic specimens from 122 patients with (N=76) and without (N=46) KMT2A-r, in addition to RNA from 8 normal bone marrow (NBM) samples, as controls and validated CLEC2A was entirely absent in NBM with median expression 0.13 copies per 1e 4 GUSB, figure 1A. Of evaluable samples, CLEC2A was detected in 65 of 76 (85%) KMT2A-r cases with a median expression of 317.45 copies per 1e 4 GUSB (range 0.03 - 21134.21 copies per 1e 4 GUSB). CLEC2A was detected in 21 of 46 (45%) non-KMT2A-r cases tested with a median expression of 1.37 copies per 1e 4 GUSB (range 0 - 1204.84 copies per 1e 4 GUSB). Further evaluation of KMT2A fusion partner showed that CLEC2A was detected in 100% of MLLT4 (N=6/6), 83.3% in MLLT10 (N=20/24), 72.7% of ELL (N=8/11), and 42.9% in MLLT3 (N=21/49). While CLEC2A expression was seen in both KMT2A-r and non-KMT2A-r AML patients, CLEC2A levels were significantly higher in KMT2A-r patients compared to non-KMT2A-r and NBM samples (p &amp;lt; 0.0001). Since CLEC2A is expressed in AML with no expression in normal hematopoietic cells, we hypothesized that monitoring CLEC2A via qRT-PCR throughout treatment (i.e., at diagnosis, at end of induction [EOI], at relapse) would provide added specificity to traditional flow cytometric disease monitoring. Using paired RNA samples from patients across multiple timepoints, we evaluated the change in CLEC2A expression throughout treatment, figure 1B. Patients with high CLEC2A expression at diagnosis (mean 1670 copies per 1e 4 GUSB, n=39) had decreased expression at EOI (mean 199 copies per 1e 4 GUSB, n=45) indicating treatment response, however high CLEC2A expression was seen at time of relapse (mean 1849 copies per 1e 4 GUSB, n=13) with CLEC2A expression preserved at relapse for all patients. qRT-PCR was able to detect CLEC2A expression in 7 patients at EOI who were in a morphologic CR with 6/7 (86%) patients being MRD-negative, further highlighting the increased sensitivity of CLEC2A qRT-PCR. Here we describe qRT-PCR monitoring of CLEC2A, a novel AML-restricted antigen highly enriched in KMT2A-r AML, provides added specificity and sensitivity to traditional flow cytometric MRD analysis. CLEC2A monitoring via qRT-PCR can be used to assess disease response throughout treatment and evaluate impending relapse as CLEC2A expression is retained at relapse. Additionally, qRT-PCR monitoring of CLEC2A can be incorporated in to pre- and post-HSCT disease evaluations similarly to qRT-PCR analysis of other known high-risk fusions such as NUP98-NSD1 fusions.

Post-CAR-T Minimal Residual Disease (MRD) Monitoring in Mantle Cell Lymphoma Enables Early Relapse Detection

INTRODUCTION: Over 40% of patients with mantle cell lymphoma (MCL) will progress within 1 year after treatment with brexucabtagene autoleucel (Brexu-cel)(Michael Wang et al. ZUMA-2 NEJM 2020). Monitoring minimal residual disease (MRD) of circulating tumor cells (CTC) via next-generation sequencing of the immunoglobulin sequences (IgHTS) with a sensitivity of 1 in million (ClonoSeq, Adaptive Biotechnologies) has previously been shown to identify patients at high risk of MCL relapse (Simone Ferrero et al. Blood 2022). However, there is a limited understanding of MRD monitoring after Brexu-cel. A sub-analysis of 27 patients post-Brexu-cel in ZUMA-2 showed predictive performance of MRD measured at months 1, 3, and 6 in estimating relapse potential. METHODS: Clonotypes were identified from paraffin-embedded tissues and underwent PCR amplification of the IgH, IgH-DJ, and Ig Kappa/lambda regions. CTC MRD assessments were monitored at 1, 3, 6, and 12 months (if applicable) following standard-of-care brexu-cel infusion in all patients with MCL at our center who had at least 3 months of follow-up. PFS and overall survival (OS) were determined by Kaplan-Meier analysis and significance by log-rank test. The comparison between the two groups was conducted using a two-tailed Mann-Whitney test. Significances were calculated using a Fisher exact test between two categorical variables or as otherwise specified. Statistical analysis and plots were generated using Prism 8.4.1 (GraphPad) RESULTS: Between January 2020 and December 2022, all treated patients (n=34) were analyzed. 3 patients were excluded as they were unable to reach day 28 (D28) assessments due to death, progressive disease, or refusal of CAR-T infusion. 5 patients were excluded due to a follow-up of &amp;lt;3 months. The median follow-up time for the 26 included patients was 18.9 months. The median age was 68, 73% were male and 27% were female, 4 median lines of therapy prior, including AutoHSCT (34.6%) or BTKi (92%). The best overall response rate at D28 was 100% with a CR rate of 73% which was similar to ZUMA-2. The median OS was 18.9 months and the median PFS was 15.3 months. We chose to analyze the Day 28 MRD assessment and found 17 of 23 patients (74%) had undetectable MRD and 6 patients had detectable MRD (26%). Due to the relatively small sample size, no significant differences were found between Age, Gender, Pre-treatment LDH, MIPI score, Ki-67, TP53 mutational status, and probability of Day 28 undetectable MRD. Notably, however, 3 of the 6 patients with TP53 mutations had detectable Day 28 MRD. Those patients with detectable MRD compared with those with undetectable MRD had a lower median PFS of 10.74 months vs 17.69 months (p-value= 0.21, Figure 1); although the median OS was similar of 17.69 and 17.84 months, (p-value= 0.75) respectively. 88% of patients (15 out of 17) with undetectable MRD on Day 28 not only maintained this status at 6 months but also showed no radiological disease progression in 93% (14 out of 15) of cases at the time of longest follow-up. A total of 7 patients relapsed during follow-up. MRD predicted 6 impending relapses earlier than PET CT with a median of 294 days (range 15-332 days) prior to progression (Figure 2). CONCLUSION: Evidence from the ZUMA-2 study and our study highlights the significance of early post-CAR-T MRD as an independent prognostic marker for predicting disease recurrence and durable remissions in MCL. Combining MRD measurements with PET-CT at months 1, 3, and 6 after CAR-T therapy can effectively identify high-risk patients for disease progression, offering promising potential to improve outcomes in MCL.

Multiparametric Flow Cytometry-MRD Assay: Lesson from Phase II Trail REL AML 001

Background: Core-binding factor (CBF) AML with RUNX1/RUNX1T1 (AML1/ETO) and CBFB/MYH11 fusion genes is known as a heterogenous group, accounting for about 10% to 15% of AML. In the Phase II Trial REL AML 001 (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345) adult CBF leukemia patients treated with a continuation therapy with Midostaurin were included, and the measurable residual disease (MRD) was performed by molecular MRD assessment (qPCR) and multiparametric flow cytometric-MRD (MCF-MRD) assay. The findings of MCF-MRD assay were compared to qPCR. Methods: 31 patients with AML1/ETO (n=15) and CBFB/MYH11 (n=16) were evaluated between December 2018 to July 2022 with both techniques. A total of 145 determinations (n. AML1/ETO = 56 and n. CBFB/MYH11= 89) were performed. MCF panel included 2 10-color tubes: CD15 FITC/ CD56 PE/ CD34 PerCP-cy5.5/ CD117 PE-cy7/ CD7 APC/ CD13 APC-R700/ CD19 APC H7/ HLADR V450/ CD45 V500C/ CD33 BV630, specific for RUNX1/RUNX1T1 cases and CD64 FITC/ CD2 PE/ CD34 PerCP-cy5.5/ CD117 PE-cy7/ CD200 APC/ CD13 APC-R700/ CD14 APC H7/ HLADR V450/ CD45 V500C/ CD33 BV630, specific for CBFB/MYH11 cases. A mean of 1,733,240 CD45 positive events were acquired, with a mean lower limit of detection (LLOD) of 0.001% (ranging 0.01-0.0003%). In 10 cases (6 RUNX1 and 4 CBFB/MYH11) the molecular and phenotypical features at onset were not available. MCF analyses were performed by InfinicytTM software and reference image tool was used wherever possible. Results: We found a concordance of 76% between MCF cases and RUNX1/RUNX1T1 qPCR, and a concordance of 83% between MCF cases and CBFB/MYH11 qPCR (Tables 1a and 1b). In relapsed cases we observed a dynamic and continuous increase of abnormal MCF events, that was not found in cases without clinical relapse although with positive qPCR (Table 2). The HLADR expression evaluated as the geo mean intensity at presentation and/or at relapse resulted higher in RUNX1/RUNX1T1 patients than in CBFB/MYH11 cases (29845 vs 8979, respectively, p=0.0039 Figure 1). Interestingly, we found in a single case with atypical CBFB/MYH11 fusion gene a high value of HLADR geo mean (44522). The down-regulated expression of HLADR in CBFB/MYH11 blasts may be a useful clue to distinguish AML blasts from the scarcely represented normal CD2 positive myeloid precursors, and from the strongly HLADR positive elements that are found in a regenerating bone marrow background. Conclusion: Although MCF-MRD assay showed lower sensitivity than CBF qPCR, MCF offered interesting informations about the dynamics of the abnormal clone size with time, evaluated as the increasing number of MRD clustering events, which may predict an impending relapse.

Blast Clearance after High Dose-Melphalan Conditioning for Allogeneic Stem Cell Transplantation in Relapsed and Refractory Acute Myeloid Leukemia

Introduction: For patients (pts) with relapsed or refractory acute myeloid leukemia (r/r AML) allogeneic stem cell transplantation (ASCT) has become an established highly effective and potential curative treatment option. Even for pts with active disease, direct ASCT using sequential conditioning regimens has shown promising outcomes. In this retrospective study, we examined prognostic factors for long-term survival in r/r AML pts with active disease with special focus to the prognostic value of blast clearance during condition therapy. Patients and Methods: 186 r/r AML pts treated with sequential conditioning regimens at our center between 2014-2023 were included in our analysis. Patients underwent high-dose melphalan (100 - 140 mg/m 2, day -11 prior ASCT) followed by either fractionated total body irradiation with 8 Gy (TBI, n = 71) or non-TBI regimens (busulfan, n = 101, or treosulfan, n = 14) in combination with fludarabin (120 - 150 mg/m 2) from day -6 before ASCT. TBI-based conditioning was applied almost exclusively in patients aged &amp;lt; 60 years. Graft versus host-disease (GvHD) prophylaxis consisted of calcineurin inhibitors, mycophenolate and anti-T-lymphocyte globulin. In 180 out of 186 patients, blast clearance was assessed by cytologic and flow cytometric blast count via bone marrow (BM) aspirate in median 5 days after Melphalan and prior to continuation of the sequential conditioning therapy. Blast clearance was defined as cytologic BM blast count &amp;lt; 5% and &amp;lt; 0.1% BM cells with leukemia-associated immunophenotype in flow cytometry. Results: Median pts age was 67 years (range 45 - 76 years) in the non-TBI group and 51 years (range 19 - 71 years) in the TBI group, respectively. Median follow up of surviving patients was 46 months. As expected, comorbidities and disease-specific factors varied between these groups. HCTCI scores &amp;gt; 3 points were observed in 14% of TBI pts compared to 32% in non-TBI pts ( p .01) and 45% versus 54% harbored adverse risk genetics according to European Leukemia Net (ELN) 2017 classification ( p .41), respectively. In the non-TBI group, 38% of the pts had a secondary or therapy-related AML compared to 20% of pts in the TBI group ( p .03). HLA-mismatched (&amp;lt;10/10 matched) donor grafts were transplanted in 21% (TBI) vs. 20% (non-TBI) of the pts. 185 of 188 pts received GCS-F mobilized stem cell grafts; 3 pts received a bone marrow graft. Kaplan-Meier estimates for overall survival (OS) with and without blast clearance at 2 years were 69.6% (95% CI 60.1-80.6%) vs. 58% (95% CI 48.8-69.1%, p .33), for event free survival (EFS, event defined as death or overt relapse) 58.9% (95% CI 49-70.8%) vs. 46.1% (95% CI 36.7-58.1%, p .2), respectively. We included the re-occurrence / persistence of defined molecular and cytogenetic markers or decreasing CD34 + bone marrow chimerism (&amp;lt; 95%) after transplant as measures for impending relapse as a modified EFS. The EFS including impending relapse was 49.8% (95% CI 40-62%) vs. 36.4% (27.7-47.8%) after 2 years. A non-significant trend with reduced relapse risk for those patients who achieved blast clearance after Melphalan ( p .1, HR 0.73) was observed. Most patients with impending relapse showed similar survival as patients without evidence of relapse after ASCT. In multivariate cox regression, age, HCTCI scores &amp;gt; 3 points, adverse risk according to ELN 2017 definitions and donor mismatch &amp;lt; 10/10 resulted in adverse prognosis. Especially, patients with mismatched donors displayed highly significant inferior outcomes. Both leukemic burden prior to ASCT as well as blast clearance after melphalan showed no significant correlation with clinical outcome parameters. Conclusion: Sequential conditioning regimens followed by ASCT offer long-term survival for more than 50% of pts with active relapsed or refractory AML. Patients who did not achieve complete blast clearance during conditioning therapy showed a trend towards higher relapse risk after ASCT which did not result in inferior survival. This underscores the importance of close monitoring and early interventions such as accelerated immunosuppression tapering, preemptive donor lymphocyte infusions and targeted therapies in case of impending relapse after ASCT.

Efficacy and safety of TV-46000, a long-acting, subcutaneous, injectable formulation of risperidone, for schizophrenia: a randomised clinical trial in the USA and Bulgaria

TV-46000 is a long-acting, subcutaneous, antipsychotic agent that combines risperidone and an innovative, copolymer-based drug delivery technology in a suspension that was approved in April, 2023 for subcutaneous use. The aim of the phase 3 Risperidone Subcutaneous Extended-release (RISE) study was to evaluate the efficacy of TV‑46000 in schizophrenia. The RISE study consisted of two treatment stages: a 12-week, open-label stabilisation phase with oral risperidone (stage 1), and an open-ended, randomised, double-blind, placebo-controlled, relapse-prevention phase with subcutaneous TV-46000 (stage 2) done at 69 clinical sites across the USA and Bulgaria. Patients diagnosed with schizophrenia more than 1 year before screening by DSM-5 criteria and confirmed at screening by the Structured Clinical Interview for DSM-5 and who had at least one relapse within 24 months before screening were eligible for enrolment. Patients who were outpatients and stabilised in stage 1 continued to stage 2 and were randomly assigned 1:1:1 by a computer-generated randomisation list to receive either subcutaneous TV-46000 once monthly, TV-46000 once every 2 months, or placebo until relapse, early discontinuation, or the study was stopped because the prespecified stopping criterion of at least 90 relapse events was met. The primary endpoint was time to impending relapse of the intention-to-treat patient population in stage 2. This study is registered with ClinicalTrials.gov, number NCT03503318, and is complete. The study enrolled the first patient on June 1, 2018, and the last patient completed on Dec 3, 2020. 1267 patients were screened, 863 enrolled, and 544 (male, n=332 [61%], female, n=212 [39%]; mean [SD] age, 49·3 [10·98] years; Black or African American, n=322 [59%]; White, n=206 [38%]; Asian, n=7 [1%]; Native Hawaiian or other Pacific Islander, n=2 [<1%]; race not reported, n=3 [<1%]; other race, n=4 [<1%]; Hispanic or Latinx, n=117 [22%]) randomly assigned to subcutaneous TV-46000 once monthly (n=183), TV-46000 once every 2 months (n=180), or placebo (n=181). Time to impending relapse was significantly prolonged by 5·0 times with TV-46000 once monthly (hazard ratio, 0·200 [95% CI 0·109-0·367]; p<0·0001) and by 2·7 times with TV-46000 once every 2 months (0·375 [0·227-0·618]; p<0·0001) versus placebo. Most frequently reported treatment-related adverse events (ie, ≥5% of patients in either TV-46000 group) that occurred more often in patients receiving TV-46000 (once monthly or once every 2 months) versus placebo were injection site nodules (7% for TV-46000 once monthly, 7% for TV-46000 once every 2 months, 3% for placebo), weight increased (4%, 6%, 2%, respectively), and extrapyramidal disorder (5%, 3%, 0% respectively). Serious adverse events were reported for eight (4%) patients in the TV-46000 once-monthly group, ten (6%) patients in the TV-46000 once-every-2-months group, and 14 (8%) patients in the placebo group. The safety profile of TV-46000 was consistent with other approved formulations of risperidone. No new safety signals were identified. In patients with schizophrenia, subcutaneous TV-46000 once monthly and once every 2 months significantly delayed impending relapse versus placebo. TV-46000 is an effective long-acting, subcutaneous, antipsychotic agent treatment option in adult patients with schizophrenia, with a favourable benefit-risk profile. Teva Branded Pharmaceutical Products R&D.

Analysis of the Impact of Whole Blood and T-Cell Donor Chimerism after Allogeneic Stem Cell Transplant

Background: Allogeneic stem cell transplant (alloSCT) is one of the few currently available therapies to cure acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Positive outcomes are dependent on engraftment of donor cells to provide hematopoiesis as well as a graft-versus-tumor effect. Measurement of whole blood (WB) and T-cell (TC) chimerism at routine intervals post alloSCT allows for monitoring of engraftment. Development of mixed donor chimerism (MC) may indicate impending graft failure or disease relapse. The ideal frequency for chimerism measurements as well as the role of WB versus TC chimerism remains unclear. In this study, we analyzed the impact of WB versus TC mixed chimerism on survival outcomes in patients with AML and MDS. Furthermore, we studied baseline factors associated with the development of MC. Methods: In this retrospective analysis, adults with AML or MDS who received an alloSCT between January 1, 2016 and February 1, 2022 that had at least 1 donor chimerism measured on Day 30 (D30), Day 60 (D60), Day 90 (D90) at Juravinski Hospital and Cancer Centre (Hamilton, ON, Canada). Baseline demographic and transplant-related characteristics were collected. AML and MDS diagnoses were based on WHO 2016 classification of myeloid neoplasm and acute leukemia. Conditioning intensities were classified according to CIBMTR definitions. A total of 271 transplants were completed during the study period for AML or MDS. 128 were excluded from the analysis (chimerism testing not performed (n=121), second transplants (n=7)). We analyzed a total of 143 patients with median age of 63 years (19 - 76) who were transplanted for AML (n=105) and MDS (n=38). Donors were HLA-matched siblings (MRD, n=26), unrelated donors (MUD (8/8 or 7/8), n=85) or haploidentical related donor (MMRD, n=32). All patients except one received peripheral blood stem cell grafts. For alloSCT, 20 % of patients received myeloablative conditioning (MAC) and 80% received reduced-intensity conditioning (RIC). Complete chimerism (CC) was defined as ≥95%, whereas MC was defined as ≤95%. All chimerism measurements were measured using a PowerPlex® 16 System (Promega) (Hamilton Regional Laboratory Medicine Program, Hamilton, ON). Results: In our cohort, OS at 2 years was 57% and RFS 49%. Of the baseline factors, only disease-risk based on ELN 2022 for RFS with a hazard ratio (HR) of 1.43, 95% CI [0.83, 2.49] for adverse risk and HR 0.53 95% CI [0.23, 1.03] (p=0.03) for favourable risk. WB MC was detected in 22%, 22%, 23% of patients on D30, D60, and D90, respectively. TC MC was detected in 48%, 46%, and 43% of patients on D30, D60, and D90, respectively. WB MC had a strong negative impact on RFS, with a HR of 2.81, 95% CI [1.69, 4.66; p&amp;lt;0.001] on D30, 3.07, 95% CI [1.67, 5.66; p&amp;lt;0.001] on D60 and 4.77, 95% CI [2.47, 9.22; p&amp;lt;0.001] on D90. The influence of TC MC on RFS was also significant at all time points. Of 101 patients who were alive and did not have a relapse prior to D90 and had D30 chimerism and D90 chimerism evaluated, the patients who had a mixed WB and TC chimerism on D90 had a HR of 6.13, 95% CI [2.23, 16.85] and HR 2.73, 95% CI [1.24, 5.98] for RFS, which was statistically significant even after adjusting for known information of D30 WB/TC chimerism status (Table 1). WB MC had a strong negative impact on OS with a HR of 2.67, 95% CI (1.54, 4.64; p=0.001) on D30, 2.81, 95% CI (1.47, 5.38; p=0.002) on D60 and 4.78, 95% CI (2.47, 9.24; p&amp;lt;0.001) on D90. Again, the later time points remained statistically significant, when adjusting for known D30 chimerism status. The influence of TC MC on OS was also significant at all time points, however only a non-significant trend remained at later time points when adjusting for D30 TC chimerism (Table 2). We analyzed baseline factors predictive for D30 chimerism. For WB chimerism, only donor source remained statistically significant (p=0.038) with MMRD having a lower HR for MC 0.39, 95% CI [0.11, 1.41] and MRD having a higher HR for MC 2.33, 95% CI [0.9 - 5.99]. Conclusions: Our data confirm that a mixed chimerism as early as D30 post-alloSCT can have a detrimental impact on outcomes (RFS and OS) in alloSCT. Patients receiving haploidentical alloSCT had a lower risk to develop mixed chimerism in our cohort. These dismal outcomes of patients with mixed chimerism in our cohort provides a strong rationale to study interventions such as novel maintenance strategies or donor-lymphocyte infusions in this patient group.

Specific IgG glycosylation differences precede relapse in PR3-ANCA associated vasculitis patients with and without ANCA rise.

Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares. Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV. Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis (P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P = 0.02). Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre.

Minimal residual disease in patients with diffuse large B-cell lymphoma undergoing autologous stem cell transplantation.

Improved biomarkers are required to guide the optimal use of autologous stem cell transplantation (ASCT) in patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). We hypothesized that minimal residual disease (MRD) identified using immunoglobulin high-throughput sequencing in apheresis stem cell (ASC) samples, post-ASCT peripheral blood mononuclear cell (PBMC), and plasma samples could predict relapse. We studied 159 patients with R/R DLBCL who underwent ASCT, of whom 98 had an ASC sample and 60 had post-ASCT surveillance samples. After a median post-ASCT follow-up of 60 months, the 5-year progression-free survival (PFS) was 48%. MRD was detected in of 23/98 (23%) ASC samples and was associated with very poor PFS (5-year PFS 13% vs 53%, P<.001) and inferior overall survival (52% vs 68%, P= .05). The sensitivity and specificity of ASC MRD positivity for progression and death were 36% and 93%, respectively. Positive ASC MRD remained a significant predictor of PFS in multivariable analysis (hazard ratio [HR], 3.7; P< .001). Post-ASCT surveillance MRD testing of plasma, but not PBMC samples, reliably identified patients with an impending relapse. A positive plasma MRD result was associated with inferior PFS (HR, 3.0; P= .016) in a multivariable analysis. The median lead time from MRD detection to relapse was 62 days (range, 0-518 days). In conclusion, the detection of MRD in ASC samples is associated with a very high risk of relapse, justifying alternative treatment strategies or trials of novel consolidation options in these patients. Furthermore, post-ASCT MRD monitoring may facilitate the evaluation of the early initiation of treatment at molecular relapse. This trial has been registered at www.clinicaltrials.gov as #NCT02362997.

Baseline‐PET derived metrics are the most relevant factors for risk stratification in early‐stage nonbulky HL: preliminary results of the RAFTING trial

Introduction: The efficacy of combined-modality treatment (Tx) with chemotherapy (CT) and radiotherapy (RT) in early-stage Hodgkin Lymphoma (eHL) is offset by long-term morbidity, with a cumulative incidence of 2nd primary malignancy at 40 years of 48.5% (Schaapveld 2015). The RAFTING trial (NCT Id. 04866654) is a phase-2 prospective single-cohort international study to explore the effectiveness of a risk-adapted Tx in non-bulky stage I-IIA eHL, based on the risk of CT failure (TxF) in a single-patient (p.) basis and in a personalized medicine design. Methods: p. are first stratified in 3 classes of TxF risk, depending on (a) modified EORTC criteria (m-EORTC), in which bulky is replaced by a Large Nodal Mass (LNM), defined by a longest ∅ measuring ≥5 cm in CT or PET/CT, (b) Metabolic Tumor Volume (MTV), with a SUVmax threshold method of 41% and cutoff value of 84 ml, and (c) PET-2 result (5-point scale). Tx stratification: Group 1: PET-2 neg. & low MTV p., treated either with 2 (group 1a) or 4 (group 2b) ABVD depending on no or ≥1 m-EORTC criterion presence, addressed, once in CR, to a 3-monthly cell-free tumor DNA (ctDNA) assay (CAPPSeq. Method, Spina 2018); Group 2: group 1 p. with < CR after ABVD or in “limited” relapse (LR), defined by eHL relapse in old and up to 3 new nodal areas, addressed to Involved-Node RT (INRT) and Nivolumab (N), 240 mg. i.v. twice a month for 24 doses; Group 3: PET-2+ and/or high MTV p., treated with the triplet ABVD × 4, INRT, 20 or 30 Gy (A-RxT-N). All PET/CT scans are centrally reviewed by an expert panel. The trial primary endpoint is a 3-Y PFS ≥90% in group 1 p. The secondary endpoints are: (a) Effectiveness of RxT (36 Gy) and N (same schedule of Group 3), in rescue p. with LR (b) effectiveness of the triplet A-RxT-N in high-risk (Group 3) eHL; (c) predictive value of ctDNA in detecting an impending eHL relapse after CT alone. Results: Preliminary results of risk stratification upon enrolment of the first 104/180 (58%) of the pre-planned sample size: in a per-protocol analysis, 88/104 (85%) turned out eligible and 73 stratified for risk. Non-eligibility reasons were higher HL stage, or bulky (10 p.) and early Tx stop, for p./investigator decision: (6p.). Out of 73 p. stratified, 53 (73%) were low-risk (Group 1), and 20 (27%) high risk (Group 3). Out of 53 low-risk p., 16 were in Group 1a: one of them was addressed to salvage Tx with ASCT because of an extended relapse 3 months after CT end. As many as 37 (70%) of low-risk p. belonged to Group 1b, mostly because of the presence of a LNM; two of them had a LR (entering Group 2) and started RxT-N rescue. Twenty p. (27%) had high-risk disease, most (14) for a high MTV, and a minority (6) for a positive PET-2. Updated results will be presented. The research was funded by: Polish Medical Agency ABM Ongoing Trial Keywords: Liquid biopsy, Ongoing Trials, PET-CT No conflicts of interests pertinent to the abstract.

Evaluation of a multimodal pain therapy approach with relapse prophylaxis for back pain (MMS-RFP study): a study protocol for a cluster randomised controlled trial

IntroductionThe need for an interdisciplinary multimodal approach to the treatment of back pain has already been demonstrated by various studies. However, when considering the periods of incapacity to work in...

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) canbe used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Minimal Residual Disease Detection Using Gene Scanning Analysis, Fluorescent Fragment Analysis, and Capillary Electrophoresis for IgH Rearrangement in Adult B-Lineage Acute Lymphoblastic Leukemia: A Cross-Sectional Study.

Minimal residual disease (MRD) is considered the greatest prognostic factor in acute lymphoblastic leukemia (ALL). MRD is a valuable tool for anticipating impending relapse and treatment response assessment. The objective of the present study was to investigate whether the detection of IgH gene rearrangement using polymerase chain reaction (PCR)-based GeneScan analysis could be a complementary method to monitor MRD along with the quantitative realtime PCR (qPCR). In this cross-sectional study, we valued the MRD levels, based on the GeneScanning analysis (GSA), and then compared the data with quantitative real-time polymerase chain reaction at different time points in peripheral blood (PB) samples of adult B-lineage ALL patients (n=35). The specific polymerase chain reaction (PCR) primers for IGH gene FR-1 and fluorescence-labeled J-primer were used and analyzed by capillary gel electrophoresis on a sequencer. The results of this study were compared with the previously reported MRD results obtained by the IGH rearrangements allele-specific oligonucleotide (ASO) -qPCR methods. The total concordance rate was 86.7%, with a P<0.001. MRD results obtained by GSA and ASO-qPCR methods were concordant in all diagnostic samples and samples on the 14th and 28th days of induction therapy. The results of these 2.5 years' follow-ups demonstrated a significant correlation between the two techniques (r=0.892, P<0.001). It seems that the PCR-based GeneScan analysis of IGH gene rearrangement detection may be a valuable molecular technique to distinguish monoclonality from polyclonality. And also, it may be a precise tool to detect the residual leukemic DNA in the PB follow-up samples of patients.

High Molecular Response Rate and Overall Survival with FLT3 Inhibitors As MRD-Guided Salvage Treatment for Molecular Failure in AML

BackgroundFLT3 inhibitors (FLT3i) represent an important new salvage option in patients with relapsed FLT3 mutated AML but response rates are low and survival outcomes remain poor. Many patients with FLT3 AML have co-occurring mutations suitable for sequential molecular monitoring by RT-qPCR, which can be used to identify impending haematological relapse (Heuser, Blood 2021; Ommen, Blood 2010). However, treatment strategies for molecular relapse are not well defined and many patients receive multi-agent chemotherapy or proceed directly to allogeneic stem cell transplant (allo-SCT) despite evidence of poor outcomes (Hourigan, JCO 2019; Dillon, Blood 2020). We aimed to describe response rates and outcomes in patients treated with FLT3 inhibitors for molecular persistence, progression or relapse (collectively 'molecular failure'). Methods In the UK, molecular monitoring for AML is largely centralised, and intervention with FLT3i monotherapy at the time of molecular failure is suggested for patients who had a FLT3 mutation at diagnosis. Consecutive patients undergoing FLT3i salvage were retrospectively identified from 27 participating hospitals. Patients met ELN criteria for molecular failure: persistence >2% after chemotherapy, relapse after previously negative results or progression of ≥1 log10 from low-level positivity. Response was assessed by sequential MRD measurement and was censored at the time of allo-SCT, donor lymphocyte infusion (DLI) or subsequent chemotherapy. Molecular response (MR) was defined as a ≥1 log10 reduction from the pre-treatment result. CRMRD-, overall (OS) and event-free survival (EFS) were defined as per ELN. Molecular event-free survival (mEFS) included molecular progression or relapse, haematological relapse or death. Stored pre and post-treatment samples were retrospectively analysed using a sensitive NGS-based FLT3-MRD assay (Blatte, Leukemia 2019). Results Forty eight patients were identified, aged 5 to 76 years. Thirty-nine (81%) had NPM1 mutations, 4 NUP98::NSD1 fusion, 3 DEK::NUP214, and 1 each CBFB::MYH11 and RUNX1::RUNX1T1. At diagnosis, 44 (92%) had a FLT3-ITD, 6 FLT3-TKD and 2 had both. Molecular failure occurred at a median of 9.6 months (range 0.9 - 31) from AML diagnosis and comprised molecular persistence in 6 (12%), molecular progression in 19 (40%) and molecular relapse in 23 (48%). Patients had received a median of 1 prior line of therapy (range 1-3), which included a FLT3i in 25 (52%) and allo-SCT in 13 (27%). Thirty-two (67%) patients were treated with gilteritinib while 8 (17%) received quizartinib and 8 (17%) sorafenib. Data on treatment toxicity in the first 4 cycles was available for 21 patients. The median number of days of neutropenia and thrombocytopenia and median number of transfusions per cycle was 0. Only 6 of the 21 patients required hospitalisation at any time in the first 4 cycles. Response was assessable in 47 patients, with 30 (64%) achieving MR of whom 19 (40%) had CRMRD-. Six patients (13%) had stable disease and 11 (23%) progressed, including 5 with haematological relapse. Patients treated for molecular relapse had the highest rate of CRMRD- at 55%. CRMRD- was lower in patients with prior FLT3i but the rate of MR was similar, apart from patients currently receiving FLT3i at the time of molecular failure of whom 6 of 13 (46%) had progressive disease. Patients with prior allo-SCT had a high response rate, with 92% achieving MR. Stored pre-treatment samples were available for FLT3 NGS-MRD analysis in 24 patients, with an ITD detected in 20 at a median VAF of 0.04%. The response rate in these patients was 70%. Twenty-four patients received allo-SCT after FLT3i salvage, 18 without requiring additional therapy prior to transplant. Seven patients received DLI. Responses deepened after allo-SCT or DLI, with 77% achieving CRMRD-. At 24 months, OS was 80% (95% confidence interval [CI] 67-96), EFS was 73% (95%CI 59-90) and mEFS was 53% (95%CI 39-72). Late relapses after stopping therapy were noted in 4 patients. Conclusions In contrast with FLT3i monotherapy at the time of frank relapse, treatment at molecular failure is associated with low toxicity, high rates of molecular response and encouraging overall survival, with many patients able to be bridged to allo-SCT or DLI. These results suggest that in patients with an appropriate marker, sequential molecular MRD monitoring should be utilised and the results used to guide pre-emptive therapy Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Utilizing CLL-1 Positive Leukemia Stem Cells As a Marker of Early Treatment Response in Acute Myeloid Leukemia

Background: Current methods to evaluate therapeutic response in Acute Myeloid leukemia (AML) rely primarily on morphological evaluation of leukemia blasts in bone marrow at end of treatment cycles, with complete remission (CR) defined as less than 5% bone marrow blast counts without leukemic phenotype. Measurable residual disease (MRD) has been shown highly prognostic for outcome. MRD by molecular methods is limited to subgroup of patients with defined genetic mutations. MRD detection by Multicolor Flow Cytometry (MFC) is still being standardized. Leukemia stem cells (LSC) has been established as source of treatment resistance and disease recurrence in AML. In this study, we aim to analyze change in LSC during treatment using immunophenotypically defined markers and examine whether information gained from LSC analysis could provide additional insights into disease course and assist management of AML under routine clinical practice setting. Methods: We analyzed flow cytometry data from bone marrow aspirate (BMA) and/or peripheral blood (PB) in patients with AML (excluding acute promyelocytic leukemia) before and after treatment. "Leukemic blasts” were identified by morphology and Leukemia-associated Immunophenotype by MFC. Hematopoietic stem cells (HSCs) gating was based on CD45dim/SSC and CD34+CD38low/- expression. Leukemic stem cells (LSC) fractional change was analyzed using CD371 (CLL-1), CD366 (TIM3), and CD45RA percent of positive over HSCs. A chart review was conducted to determine correlation between LSC fractional change and clinical outcome. Statistical analysis was performed using GraphPad Prism version 9. Results: Fifty AML cases with both pre- and post-treatment data from a single institution in the past 3 years were included in this study. Consistent with published literature, patients with significant reduction in LSC subsets was associated with longer remission, while patients with no significant decrease in the LSC subsets demonstrated no or only a temporary remission. Intriguingly, several patients with drastic reduction in LSC after induction chemotherapy achieved complete remission, despite initial post-treatment bone marrow showing persistence of ">5% leukemia blasts” indicating possibly failed induction therapy. In contrast, in patients with persistently high or increased LSC fraction post-treatment, overt clinical relapse occurred quickly despite "absence” or <1% of leukemia blast differential counts in initial bone marrow. In a series of patients who received allogeneic stem cell transplant (Allo-SCT), rise of LSC fraction post Allo-SCT was associated with early AML relapse (within 6 months), while the decrease of LSC fraction was associated with prolonged remission (Representative patient cases shown in Figure 1). Lastly, among the three LSC-specific markers analyzed, the changes in LSC characterized by CLL-1 expression showed the best correlation with clinical course. In addition, the LSC fraction measured by CLL-1 in the peripheral blood is highly correlated with that of the bone marrow (R2=0.93, P<0.0001). Conclusion: This retrospective study explored the value and applicability of LSC detection in routine clinical practice. Importantly, conventional methods relying on evaluating leukemic blast counts in post-treatment bone marrow can sometimes over- or under- estimate the benefit of certain treatment regimen, leading to inappropriate continuation of the same treatment or switch of a potentially effective treatment regimen. In those groups of patients, the change in LSC fraction provides additional information for early treatment response evaluation, refines outcome prediction, and helps with treatment regimen selection. Trending LSC fractional change identifies impending relapse in post Allo-SCT. LSC fractional change determined by CLL-1 showed the best clinical correlation and should be incorporated into routine evaluation for AML patient in clinical practice, and possibly for those participating in investigational trials. Future studies will focus on collecting more data to validate above findings, determining the thresholds of LSC fractional change which constitute potential treatment response vs failure and testing the feasibility of tracking LSC subsets non-invasively in peripheral blood during treatment to predict therapeutic outcome and potentially guide treatment decisions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal