Low Minimal Residual Disease Levels Research Articles

Blinatumomab Therapy for Low Level Minimal Residual Disease Detected By Next-Generation Sequencing in Adult B-Cell Acute Lymphoblastic Leukemia

Blinatumomab Therapy for Low Level Minimal Residual Disease Detected By Next-Generation Sequencing in Adult B-Cell Acute Lymphoblastic Leukemia

Blinatumomab Therapy for Low Level Minimal Residual Disease Detected By Next-Generation Sequencing in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Blinatumomab, a bi-specific T-cell engager, has shown clinical benefit in patients with relapsed-refractory and hematologic complete remission with minimal residual disease (MRD)-positive Acute Lymphoblastic Leukemia (ALL). The BLAST trial (Gökbuget N et al., Blood. 2018) demonstrated improved overall survival (OS) and disease-free survival (DFS) in patients with MRD-positive disease (≥0.1%) who achieved MRD response with Blinatumomab. We sought to evaluate the utility of Blinatumomab in low levels of MRD (<0.1%) using clonoSEQ with a sensitivity of (0.0001%). Methods: Adult (18+) B- ALL patients that underwent treatment for B-cell ALL at Norris Comprehensive Cancer Center (NCCC) between 2016 and 2022 who had residual disease tracked using the Next Generation Sequencing (NGS)-based clonoSEQ assay (Adaptive Biotechnologies, Seattle, WA) were included. Patients who received Blinatumomab for MRD positivity were included only if they had a tracked clonoSEQ sequence within one month before starting treatment. Patients who didn't receive Blinatumomab were included if they had an MRD-positive clonoSEQ report. Those who were relapse/refractory when found to be MRD positive were excluded. Cumulative incidence of relapse (CIR) was evaluated using competing risk regression (Fine-Gray method), with death as a competing outcome. At the same time, OS and DFS were analyzed using Cox proportional hazards model. The start time was set to the date of the MRD report, and patients were censored until the last follow-up. Confounder-adjusted KM curves were calculated using direct standardization. Results: 47 patients were included with a median age at diagnosis of 45 years (range: 21-70). Most were Hispanic (N = 33, 70.2%), and the median follow-up time was 12 months. The 3-year OS, DFS, and CIR were 95.4% (95% CI 89.3-100%), 75.1% (95% CI 60.4-93.5%), and 20.3% (95% CI 7.2-38.0%). Twenty-six patients were treated with Blinatumomab (median two cycles, range 1-9), with most (N= 14, 53.8%) having low levels of MRD (<0.1%). Fourteen patients (53.8%) achieved MRD negativity with a median time to MRD negativity of 3.1 months. There were no relapses or death in patients who achieved MRD negativity in the treated group. However, non-responders with Blinatumomab had one death and two relapses. All patients continued Maintenance therapy after receiving Blinatumomab and did not proceed with the transplant. The patients who received Blinatumomab were more likely to be MRD positive on multiparameter flow cytometry (MFC) (60.0% vs. 28.6%, P = 0.042) but had a similar distribution of patient age, treatment type, and receipt of transplant. Additionally, there was a trend toward increased median percent residual cells on clonoSEQ (0.018% vs. 0.003% P =0.244) and flow (0.02% vs 0% P =0.066) in the Blinatumomab treatment group. On univariate analysis, significant predictors of CIR and DFS were flow percent (DFS: HR = 1.54; 95% CI 1.07-2.23; P = 0.019; CIR: HR = 1.83; 95% CI 1.38-2.41; P<0.001) and clonoSEQ residual cell count(DFS: HR = 1.58; 95% CI 1.04-2.41; P = 0.031, CIR: HR = 1.88; 95% CI 1.18-2.97 P=0.008). When controlling for the number of residual cells on close, patients who received Blinatumomab had significantly better CIR(HR = 0.11; 95% CI 0.02-0.69; P = 0.019) and a trend towards improved DFS(HR = 0.21; 95% CI 0.04-1.28; P = 0.092), but no difference in OS. On subgroup analysis of the 31 patients with low levels of MRD (<0.1%), there was no improvement in DFS for patients who were treated with Blinatumomab (HR = 0.41; 95% CI 0.04-3.91; P = 0.44). Of those with low levels of MRD, only two relapsed, and both did not receive Blinatumomab. Conclusions: In B cell ALL, Blinatumomab may offer a CIR benefit to patients with low levels of MRD as detected on clonoSEQ.

Blinatumomab Added to Chemotherapy in Infant Lymphoblastic Leukemia

BackgroundKMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants is an aggressive disease with 3-year event-free survival below 40%. Most relapses occur during treatment, with two thirds occurring within 1 year and 90% within 2 years after diagnosis. Outcomes have not improved in recent decades despite intensification of chemotherapy.MethodsWe studied the safety and efficacy of blinatumomab, a bispecific T-cell engager molecule targeting CD19, in infants with KMT2A-rearranged ALL. Thirty patients younger than 1 year of age with newly diagnosed KMT2A-rearranged ALL were given the chemotherapy used in the Interfant-06 trial with the addition of one postinduction course of blinatumomab (15 μg per square meter of body-surface area per day; 28-day continuous infusion). The primary end point was clinically relevant toxic effects, defined as any toxic effect that was possibly or definitely attributable to blinatumomab and resulted in permanent discontinuation of blinatumomab or death. Minimal residual disease (MRD) was measured by polymerase chain reaction. Data on adverse events were collected. Outcome data were compared with historical control data from the Interfant-06 trial.ResultsThe median follow-up was 26.3 months (range, 3.9 to 48.2). All 30 patients received the full course of blinatumomab. No toxic effects meeting the definition of the primary end point occurred. Ten serious adverse events were reported (fever [4 events], infection [4], hypertension [1], and vomiting [1]). The toxic-effects profile was consistent with that reported in older patients. A total of 28 patients (93%) either were MRD-negative (16 patients) or had low levels of MRD (<5×10−4 [i.e., <5 leukemic cells per 10,000 normal cells], 12 patients) after the blinatumomab infusion. All the patients who continued chemotherapy became MRD-negative during further treatment. Two-year disease-free survival was 81.6% in our study (95% confidence interval [CI], 60.8 to 92.0), as compared with 49.4% (95% CI, 42.5 to 56.0) in the Interfant-06 trial; the corresponding values for overall survival were 93.3% (95% CI, 75.9 to 98.3) and 65.8% (95% CI, 58.9 to 71.8).ConclusionsBlinatumomab added to Interfant-06 chemotherapy appeared to be safe and had a high level of efficacy in infants with newly diagnosed KMT2A-rearranged ALL as compared with historical controls from the Interfant-06 trial. (Funded by the Princess Máxima Center Foundation and others; EudraCT number, 2016-004674-17.)

Inhibition of the PCR by genomic DNA.

qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6. The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer. Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition. Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.

Blinatumomab therapy for B cell acute lymphoblastic leukemia accompanied by persistent or relapsed low-level MRD prior to hematopoietic stem cell transplantation in Chinese children: a case series

BackgroundBlinatumomab could be successfully used to reduce minimal residual disease (MRD) prior to hematopoietic stem cell transplantation (HSCT) in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), but sound evidence is lacking in China.Case presentationThis retrospective study assessed the application of blinatumomab in B-ALL accompanied by persistent or relapsed low-level MRD before HSCT from April 2019 to July 2021. Two cases (Cases 1 and 2) initially achieved remission with MRD < 0.01% upon conventional therapy but had MRD relapse with MRD ≥ 0.01% but < 1% during maintenance treatment. Case 3 had no response to routine treatment, with high MRD (9.88% and 1.23% at days 19 and 46, respectively). Nevertheless, all patients had undetectable MRD. Cases 2 and 3 had undetectable fusion gene following blinatumomab therapy. By bone marrow monitoring (bone marrow morphology, bone marrow MRD and fusion gene) post-HSCT, the patients were persistently negative until May 15, 2022. No patient had serious adverse events before or during blinatumomab treatment.ConclusionsBlinatumomab therapy showed a good performance for three pediatric cases with detectable but low MRD before HSCT in China. However, further prospective studies with large sample sizes are still needed for further clarification.

Long-term Follow-up of CD20-directed Chimeric Antigen Receptor Adoptive T-cell Therapy

Chimeric antigen receptor (CAR) T cell therapy targeting B-cell antigens has led to unprecedented success in inducing complete response (CR) rates in refractory B-cell non-Hodgkin lymphomas (NHLs). We previously reported the results of a pilot trial testing a 3rd generation CAR with 4-1BB and CD28 costimulatory domains targeting CD20 in patients (pts) with relapsed B-cell lymphomas (NCT00621452). 3 pts received cyclophosphamide (CY) 1 g/m2 IV for lymphodepletion 2 days prior to a series of 3 CAR T cell infusions 2 to 5 days apart in escalating doses followed by 2 weeks of low-dose IL-2. Here, we report the long term follow up of 2 of these pts. The third pt, UPN-02, died of progressive mantle cell lymphoma (MCL) and myelodysplastic syndrome at ~26 months (mos) post-treatment. Pt UPN-03, with relapsed MCL, had no evaluable disease after an excisional biopsy following protocol therapy. He relapsed after 2 years with a left flank mass, which was then radiated. He was re-treated with CY lymphodepletion, a dose of 1.8 x 109 CAR T cells/m2, and low-dose IL-2. At 6 weeks post infusion, he developed a small mass at the margin of his previous radiation field suspected to be recurrent MCL. This mass spontaneously regressed 1 mo later. Subsequent CT scans showed a residual soft tissue mass that gradually decreased over time and met criteria for CR by CT criteria. He remained in remission, albeit with detectable minimal residual disease (MRD) by ClonoSEQ assay, for the next 8.4 years until developing a subcutaneous chest mass and axillary adenopathy with biopsy showing recurrent MCL. Pt UPN-04, with relapsed follicular lymphoma (FL), had stable disease by CT at 1 mo but went on to have a partial response at ~3 mos. By 12 mos post-CAR T treatment there was evidence of progressive disease. However, between 2.5 and 3 years post-treatment he experienced resolution of all palpable nodes, and a near-CR was confirmed by CT at 35 mos after treatment. He remained without any clinical signs of progression with low level MRD by ClonoSEQ for the next 4 years. At 7.2 years post-CAR T cell treatment, he was found to have recurrent FL. CAR T cells were detectable for up to 12 mos in UPN-03 and 9 mos in UPN-04 by qPCR assay. B-cell recovery over time in both pts confirmed a lack of functional CAR T cell persistence. Given these findings, along with the durable responses observed in both pts, we hypothesized that these late remissions were caused by endogenous immune responses triggered by CAR T cell therapy. To evaluate this, we performed western blot assays incubating post-infusion serum with autologous tumor cell lysates followed by incubation with an anti-human IgG antibody, which showed new bands at post clinical response timepoints in both pts. ELISPOT assays with PBMCs co-incubated with autologous tumor cells exhibited spikes in IFN-γ secreting cells compared with control cells at timepoints proximal to their clinical responses. These findings suggested the occurrence of both humoral and cellular anti-tumor responses. To better characterize the T-cell response, we performed high-throughput TCR-β sequencing (Adaptive ImmunoSEQ) for UPN-04. We conducted a differential abundance analysis to identify possible tumor-reactive T cells, with the hypothesis that tumor-reactive T cell clones would be increased near the timepoints with clinical responses at ~3 and 35 mos. We identified 12 and 7 sequences, respectively, at these timepoints that were significantly increased compared to baseline (FDR < 0.05) and not present in the CAR-T product; 3 sequences were present at both 3 and 35 mos, and 1 of these was also significantly enriched in a post-infusion tumor biopsy, suggesting possible tumor-specificity. These results support the hypothesis that the late responses were mediated by endogenous T cells rather than CAR T cells. In summary, we report long-term remissions in 2 of 3 pts treated with CD20-targeted CAR T cell therapy, with evidence of endogenous anti-tumor immune responses. Together with previously published reports of epitope spreading after CAR-T for solid tumors, and in mouse models of lymphoma, these results suggest that CAR T cells have the capacity to elicit endogenous anti-tumor immune responses in lymphoma. Our use of CY without fludarabine as lymphodepletion may have facilitated such responses. Future research is warranted to enhance epitope spreading after CAR-T therapy as a bulwark against escape by antigen loss or loss of CAR-T persistence.

Ultrasensitive Circulating Tumor DNA (ctDNA) Dynamics after Autologous CD30.CAR-T Cell Therapy for Relapsed or Refractory (r/r) Classical Hodgkin Lymphoma (CHARIOT Trial)

Introduction Liquid biopsies leveraging circulating tumor DNA (ctDNA) are rapidly emerging as minimally invasive tools to detect, genotype, and monitor diverse tumors. ctDNA dynamics and early responses are well-characterized in non-Hodgkin Lymphomas, especially during frontline immunochemotherapy. However, ctDNA responses and minimal residual disease (MRD) are less well-defined in classical Hodgkin Lymphomas (cHL), especially in the relapsed/refractory setting and after cellular immunotherapy, where significant unmet needs remain. We hypothesized that ultrasensitive ctDNA-based MRD detection by Phased Variant Enrichment & Detection Sequencing (PhasED-Seq; Kurtz et al, 2021 Nat Biotech) can accurately track response and predict progression after investigational therapy of r/r cHL with autologous CD30.CAR-T cells. Method This Phase 2 single arm, multicenter study (NCT04268706) investigates safety and efficacy of CD30.CAR-T cells in cHL patients experiencing progression after at least 3 lines of therapy. In the Pilot part of the study, ctDNA was analyzed as an exploratory biomarker using PhasED-Seq at multiple time-points, including at baseline (pre-treatment), Day 42 post-CD30.CAR-T infusion (D42), and upon progressive disease (PD). Exploratory analyses were performed to assess the predictive role of ctDNA and to compare metabolic tumor response by positron emission tomography (PET) scan with ctDNA-based minimal residual disease (MRD). We profiled 55 serial blood specimens (41 plasma and 14 buffy coat samples) from 14 patients with available samples. Cell-free DNA was profiled by PhasED-Seq in the Foresight Diagnostics CLIA laboratory (Aurora, CO). Baseline plasma and D42 buffy coat specimens were used to genotype each patient's tumor to identify Phased Variants (PVs), and these were used to monitor MRD in subsequent blood specimens. Patients/samples were reported as MRD positive when ctDNA levels exceeded an analytical detection threshold (~1 part per million cfDNA molecules), corresponding to 98% clinical specificity. Result As of 22 July 2022, 17 patients have been screened and 15 patients enrolled in the Pilot segment (median age: 35 years [21-57], 66.7% male). Overall response rate (ORR) after CD30.CAR-T single infusion was 73.3% (n=11), with complete response (CR) rate of 60% (n=9). ORR in 5 patients after the 2nd infusion was 100% (n=5), with CR of 60% (n= 3). ctDNA was successfully genotyped directly from pre-treatment plasma in 12 out of 14 patients where samples were available. Both patients where PV genotyping failed had received bridging cytoreductive therapy before baseline plasma collection. Out of 12 profiled patients, 7 patients achieved CR with median baseline ctDNA concentration of 44.9 hGE/mL. 3 patients had PD with median baseline ctDNA level of 94.0 hGE/mL. One patient with PR and one patient with SD had baseline ctDNA levels of 38.9 hGE/mL and 231.1 hGE/mL, respectively. These results suggest that pre-treatment ctDNA levels could have predictive value on patient response to CAR-T therapy. ctDNA responses largely mirrored radiographic responses. Specifically, among 7 patients achieving radiographic CR, 4 also achieved undetectable ctDNA, and the other 3 had ctDNA responses with low MRD levels (median: 0.2 hGE/mL) at D42. One patient with PR also had low ctDNA concentration at D42 (0.7 hGE/mL). In comparison, 3 patients experiencing PD had relatively higher ctDNA levels (median: 131.4 hGE/mL). Furthermore, ctDNA levels at D42 were significantly correlated with PET Deauville score (p=8.7e-4), and patients with a response had significantly lower ctDNA than those without a response (p=0.004) (Fig B). These results suggest that ctDNA-MRD levels are strongly correlated with metabolic responses determined by PET imaging. Similar results were observed after 2nd infusions of the CAR-T. 3 out of 3 patients achieving CR had low ctDNA concentrations at Day 42 post-2nd infusion (median: 0.2 hGE/mL). Among 2 patients with PR, 1 patient with available sample had 31.9 hGE/mL ctDNA. Conclusion Minimally invasive ctDNA analysis is a viable method to monitor responses, rapidly risk stratify and predict outcomes of patients with r/r cHL treated with CD30.CAR-T therapy. A single infusion of CD30.CAR-T therapy rapidly induces MRD negativity in a significant subset of patients at Day 42. ctDNA as exploratory biomarker will be further evaluated in Pivotal part of the study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BCR::ABL1-Positive Acute Lymphoblastic Leukemia: Different Clinical Behavior and Relevance of Prognostic Features in Typical ALL and in CML-like Disease

Recently we showed that in 20-30% of children diagnosed as BCR::ABL1-positive (Ph+) acute lymphoblastic leukemia (ALL) the BCR::ABL1 fusion is present in a wider clone, involving myeloid cells, non-ALL B-cells and T-cells. This leads to discordant minimal residual disease (MRD) levels assessed by the quantification of two available MRD targets - immunoglobulin (IG)/T-cell receptor (TR) genes rearrangements and BCR::ABL1 fusion. As the cases with multilineage BCR::ABL1 involvement resemble lymphoid blast crisis (LBC) of chronic myeloid leukemia (CML) we named these leukemias "CML-like", as distinct from "typical Ph+ ALL". To reveal biological and clinical differences between the two subtypes, we retrospectively analyzed prognostic relevance of MRD and other features in 147 children with BCR::ABL1-positive ALL treated according to EsPhALL (n = 133) or other (n = 14) protocols. MRD was assessed by DNA-based monitoring of BCR::ABL1 genomic breakpoint and of clonal IG/TR rearrangements. Although overall prognosis of CML-like (n = 48) and typical Ph+ ALL (n = 99) was similar (5-year EFS 60% and 49%; 5-year OS 75% and 73%, respectively), typical Ph+ ALL presented more relapses (12/48 vs. 42/99; p = 0.046) while CML-like patients more often died in the first remission (6/48 vs. 3/99; p = 0.059 for CR1 deaths in total and p = 0.01 for CR1 deaths out of the total deaths). Prognostic role of MRD measured at standard treatment timepoints (TP1 - end of induction IA, day 33; TP2 - end of consolidation IB, week 12) was highly significant in the typical Ph+ ALL (p = 0.0005 for EFS). In contrast, in CML-like patients prognostic impact of MRD was not significant and thus inapplicable for therapy adjustment. Similarly, although diagnostic white blood cell counts were similar in the two subgroups, impact of the hyperleukocytosis ≥ 50 x 109/l on outcome was highly significant in the typical Ph+ ALL (and remained significant in multivariate analyses, being apparent even in patients with fast MRD response), but no prognostic impact was found in CML-like patients. While in the typical Ph+ ALL the NCI risk tended to impact 5-year OS (89% vs. 67%; p = 0.03), there was no impact on prognosis in the CML-like patients. The BCR::ABL1 transcript type (minor/p190 vs. Major/p210), IKZF1 deletion, sex, or the age at diagnosis (< 10 vs. ≥ 10 years) did not show any prognostic relevance. Earlier start of continuous tyrosine kinase inhibitors (TKI) treatment (day 15 vs. day 33) was associated with lower MRD levels in the typical Ph+ ALL but not in CML-like patients. However, earlier TKI start tended to result in better 5-year OS in both subtypes (78% vs. 67%; p = 0.082 and 83% vs. 60%; p = 0.066 for typical Ph+ ALL and CML-like, respectively). To shed more light on the nature of the two subtypes, we now collect additional data (including transcriptome profiles, single cell characteristics, genomic breakpoint features) in order to reveal biological background, cell of origin and further potential differences between the subtypes. Such data could help to find optimal treatment strategies, and also to answer the attractive question whether CML-like disease is in fact a regular CML in LBC or whether there is a wider spectrum of BCR::ABL1-positive leukemias. If these studies lead to a conclusion that CML-like is indeed indistinguishable from LBC-CML, it will be necessary to re-define CML as a disease manifesting - at least in childhood - very often in LBC and with the minor-BCR::ABL1/p190 fusion variant. In conclusion, our data unequivocally confirm that although clinically presenting at diagnosis as one entity, there are two biologically distinct subtypes of BCR::ABL1-positive ALL - typical Ph+ ALL and CML-like disease. The different biology is reflected in treatment response and although the overall survival rates of both subtypes are similar on current protocol, the key reasons of treatment failure are different - toxicity of intensive ALL treatment in CML-like patients and relapses in typical Ph+ ALL. Thus, early distinguishing of the two subtypes is essential to enable optimal treatment approach and therapy adjustments in upcoming trials. For the typical Ph+ ALL, TKI and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, representing one-fourth to one-third of childhood patients diagnosed as BCR::ABL1-positive ALL, no relevant risk factor applicable for therapy tailoring was found so far.

Safety and Anti-Leukemic Activity of CD123-CAR T Cells in Pediatric Patients with AML: Preliminary Results from a Phase 1 Trial

Background The prognosis of pediatric patients with relapsed or refractory (r/r) Acute Myeloid Leukemia (AML) remains dismal. Evaluation of novel therapies such as chimeric antigen receptor (CAR) T cells is therefore urgently needed. CD123 is overexpressed on AML blasts and leukemic stem cells (LSC), making it an attractive target for CAR T-cell directed therapy. Methods and Results We designed a 'bridge to allogeneic hematopoietic cell transplant (HCT)’ Phase 1 study to evaluate the safety and feasibility of treating pediatric patients with r/r AML with CD123-CAR T-cell on four dose levels (DL): DL1: 3x105/kg, DL2: 1x106/kg, DL3: 3x106/kg, DL4: 1x107/kg (NCT04318678). CD123-CAR T-cells were generated from CD4/CD8-selected autologous leukapheresis products using a lentiviral vector, which encoded a CD123-CAR with CD28.z signaling domain and a CD20 safety switch. Patients received lymphodepleting chemotherapy with Fludarabine and Cyclophosphamide followed by a single CAR T cell infusion. To date we have enrolled 12 patients. The median age of treated patients was 17 years (range 12-21years). Except for the first patient who had primary refractory disease, all other patients had relapsed following HCT (n=1-4 previous HCTs). Despite patients being heavily pre-treated, we successfully manufactured products on all patients. The infused products had a predominantly CD4+ effector memory immunophenotype, with a median CD123-CAR+/CD20+ expression of 60.2% (range 46.1-79.4%, N=6). All infused CAR T cell products exhibited potent antitumor activity in vitro when cultured against CD123+ targets (including autologous blasts for 2 patients). We infused 2 patients on DL1 and 3 patients on DL2. Two patients were infused on single patient protocols. All infusions were well tolerated, without any adverse infusion events. Post infusion, we observed isolated fevers that resolved within 24 hours (possible Grade 1 Cytokine Release Syndrome (CRS)). No Grade 2 ≥ CRS or neurotoxicity was seen. Multiplex cytokine analysis showed statistically significant increase of IL15 after lymphodepletion without significant increase in Th1 cytokines or IL6 after T cell infusion. While transient cytopenias were observed, patients did not develop persistent marrow aplasia. No dose limiting toxicities were noted. Disease evaluation was performed at 4-6 weeks following CAR T cell infusion. The 2 patients on DL1 showed no response. In the 3 patients on DL2, we observed: reduction in blast percentage without complete remission (CR) in 1 patient, no response in 1 patient and CR in 1 patient. The patient with CR had isolated extramedullary disease, showing complete resolution of all lesions by week 4 by PET imaging (Figure 1). She recurred 2 months following infusion, coinciding with loss of CAR T-cell detection by qPCR. She received a second infusion off study and again achieved a short-lived CR. One additional patient was infused on dose level 2 off protocol. She achieved morphological CR at day 28 with low level minimal residual disease (0.19%). Correlative studies revealed CD123-CAR T cell expansion in patients on DL2 (Figure 2), but not on DL1. However, peak expansion on DL2 was low in comparison to published reports on CAR T cells for Acute Lymphoblastic Leukemia. Detailed phenotypic analysis revealed that CD123-CAR T cell products were predominantly effector memory (CD45RO+CCR7-) with <2% naïve-like (CD45RO-CCR7+ or CD45RO-CD62L+) T cells. In addition, PD1, TIM3 and CD39 were expressed at a median of 43, 81 and 16% in CD4+ and 3, 72 and 45% in CD8+ T cells. This phenotype could be completely reversed by inhibiting CAR signaling with dasatinib during the manufacturing process, resulting in a CAR T cell product dominated by naïve-like T cells that did not express TIM3, PD1 or CD39, leading to enhanced T-cell effector function. Mechanistic analysis revealed that the observed phenotype could be explained by the transient, increased cell surface expression of CD123 on T cells upon activation during manufacturing, and not by tonic CAR signaling. Conclusions Our initial clinical experience with CD123-CAR T cells for pediatric patients with r/r AML demonstrates feasibility, safety, and evidence of anti-leukemic activity. Upcoming CD123-CAR T cell products will be manufactured in the presence of dasatinib to limit T-cell differentiation and exhaustion as we continue to explore our CD123-CAR T cell therapy approach for pediatric r/r AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Fixed-Duration Immunochemotherapy Approach Combined with Ibrutinib in CLL Produces Deep and Sustained MRD Responses: 5.5-Year Results from the Icll-07 Filo Trial

Introduction In previously untreated, medically fit patients with chronic lymphocytic leukemia (CLL), the availability of targeted agents has led to research interest in achieving deep, durable remissions and improving survival outcomes. The phase II ICLL-07 (NCT02666898) trial by the French Innovative Leukemia Organization (FILO) evaluated a fixed-duration (15-month) immunochemotherapy approach (no more than 4 fludarabine/cyclophosphamide [FC]-obinutuzumab cycles). Primary analysis showed the approach was safe and 62.2% (84/135) of all patients enrolled achieved complete response (CR) with bone marrow (BM) minimal residual disease (MRD) <0.01% (Michallet et al Lancet Haematol 2019;6:e470-9). Follow-up at median 37 months from treatment start showed a persistent MRD benefit beyond end of treatment, high survival rates, and low long-term toxicity (Michallet et al Blood 2021;137:1019-23). Here we report long-term results at median 64 months from treatment start (data cut-off 20 May 2022). Methods Between 27 October 2015 and 16 May 2017, 135 previously untreated, medically fit patients with CLL were enrolled from 27 FILO centers. All received obinutuzumab 1000 mg (8 infusions over 6 cycles) plus ibrutinib (420 mg/day for 9 months). Following evaluation at D1 M9 (N=130 evaluable), those in CR with BM MRD <0.01% then continued only ibrutinib 420 mg/day for 6 additional months (I arm; n=10); otherwise, patients received 4 cycles of FC-obinutuzumab 1000 mg alongside continuing ibrutinib 420 mg/day for 6 additional months (I-FCG arm; n=115). Beyond end of treatment, patients were followed for peripheral blood (PB) MRD every 6 months, response every 3 months, progression-free/overall survival (PFS/OS), and safety. MRD was assessed by 8-color flow cytometry, using a highly sensitive (10-6) technique developed by the FILO group that enables subdivision of classical MRD <0.01% into low-level positive MRD <0.01% and undetectable MRD. Results At D1 M16, undetectable PB MRD rates were 80.0% (8/10) in the I arm and 92.5% (99/107) in the I-FCG arm. Beyond end of treatment, in the I arm the undetectable PB MRD rate decreased rapidly, and by M40 all patients had PB MRD positivity (≥0.01% or low-level positive <0.01%). In the I-FCG arm, the undetectable PB MRD rate was 76.3% (61/80) at M40, and of patients currently investigated at M64, 50/62 (80%) still had undetectable PB MRD (Figure 1). Four relapses occurred beyond end of treatment, including 2 CLL progressions (both Richter's syndrome). Throughout follow-up, no differences in undetectable PB MRD rates were apparent in the I-FCG arm according to the IGHV -mutated versus unmutated status (currently at M64: 20/27 versus 25/35, P=0.26). Overall 4-year PFS and OS rates (95% confidence intervals) were 95.5% (92% to 99 %) and 96.2% (93% to 99.5%), respectively. Of 12 deaths overall, 7 occurred beyond end of treatment, with 5 since last publication due to Richter's syndrome at M50, COVID at M50, lung cancer at M50, myelodysplastic syndrome at M60, and acute myeloid leukemia at M69. Other serious adverse events since last publication were metastatic prostate adenocarcinoma at M50 and suspicion of cerebrovascular accident at M65. Conclusion Long-term results from the ICLL-07 trial in previously untreated, medically fit patients with CLL demonstrated that that our fixed-duration (15-month) immunochemotherapy approach produced deep and sustained undetectable PB MRD responses that were correlated to PFS, without excess toxicity. These long-term MRD results remain the best to date even compared to the new targeted therapy combinations ie ibrutinib +veneteclax or obinutuzumab + venetoclax A randomized trial is needed to compare our approach with a chemotherapy-free strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gimema ALL2418: Interim Analysis of a Phase Iia Study of Feasibility and Effectiveness of Inotuzumab Ozogamicin in Adult Patients with B-Cell Acute Lymphoblastic Leukemia with Positive Minimal Residual Disease before Any Hematopoietic Stem Cell Transplantation

Background: Persistence of minimal residual disease (MRD) is associated with a high rate of treatment failure, relapse, and death in B-cell acute lymphoblastic leukemia (B-ALL). Particularly, patients who remain MRD positive before an allogeneic hematopoietic stem cell transplant (HSCT) have an expected disease-related death rate due to the disease of about 60%. The T cell-engager CD19-directed therapy blinatumomab demonstrated considerable efficacy in this setting, however resistant or unsuitable patients have no alternative therapy. Inotuzumab Ozogamicin (IO) is a CD22-directed antibody-drug conjugate with remarkable efficacy in B-ALL with overt disease. Furthermore, IO does not need a high effective immune system to exert activity. Methods: In this multicenter phase 2 trial (NCT03610438), we are investigating the efficacy of IO in obtaining MRD negativity in two cohorts of 38 Ph+ and 38 Ph- B-ALL patients. IO was administered at the dose of 0.5 mg/sqm on days 1, 8, and 15 of a 28-days cycle. Patients who did not obtain MRD negativity after the 1st IO cycle had the option to receive the 2nd IO cycle maintaining the same dosage. Responding Ph- patients received short-term maintenance with low-dose chemotherapy (alternating vincristine, cyclophosphamide, prednisone, methotrexate, and 6-mercaptopurine), responding PH+ patients received tyrosine kinase inhibitor (TKI) for up to 12 weeks, allowing proper IO washout before HSCT. HSCT ineligible patients entered into low-dose chemotherapy or TKI maintenance for up to 2 years. For the primary endpoint, MRD was centrally assessed at baseline, after course 1, after course 2, and eventually before transplant with high sensitive molecular methods for BCR-ABL1 or V(d)J fusion transcripts. Here, we present the results of an unplanned interim analysis that was generated to evaluate the continuation of the study considering the slow accrual due to the Sars-COV2 pandemic. Results: To date, 51 patients underwent screening procedures, and 43 were considered eligible. We report data from the first 39 patients treated in the study. Nineteen out of 39 patients had Ph- B-ALL and 20/39 Ph+ B-ALL. Eighteen out of 39 (46%) patients were male. The median age was 55 years (22-84), 15/39 patients (38%) were older than 65 years. Demographics were comparable between Ph+ and Ph- patients. Within Ph+, ALL 39% of patients harbored p190 and 61% p210 fusion transcript. Within Ph- B-ALL, 1 patient harbored ALL1/AF4 and 1 patient harbored ALL1/ENL fusion transcript. Most of the patients received 1 (16/39, 42%) or 2 (19/39, 50%) previous lines of therapy; 5 patients (13%) were resistant to blinatumomab, and all of them had Ph- B-ALL. Twelve out of 19 (63%) Ph- B-ALL patients received only 1 IO cycle, 7/19 (37%) received 2 cycles; 5/20(25%) Ph+ B-ALL received only 1 IO cycle, and 15/20 (75%) received 2 cycles. To date, central MRD monitoring is available for 20 patients (12 Ph- and 8 Ph+ B-ALL, ongoing for the remaining cases). Overall, MRD negativity was obtained in 7/20 patients (35%); Particularly, in Ph- B-ALL cohort 5/12 (42%) patients achieved MRD negativity, 4/12 (38%) achieved disease reduction at low level MRD positivity (<10-4); in Ph+ B-ALL cohort, 2/8 (25%) patients achieved MRD negativity. One out of 5 (20%) blinatumomab pre-treated patients obtained MRD negative response. IO was an effective bridge to HSCT for 10/39 patients (26%; in patients <65 years old, 9/24 patients, 37%); Specifically, all 7 patients who obtained MRD negativity after IO, a patient with low-positive MRD and 2 patients with non yet available MRD data were bridged to HSCT. Accordingly, with the caveat of a short median follow-up (2.8 months, range 0.6-20.0 months), overall survival in the study population seems promising (figure 1, median OS not reached). Overall, adverse events collected during the study were limited in number and grading. Also, myelotoxicity was manageable. Out of 39 patients, we documented 1 veno-occlusive disease (2.5%) that occurred within 20 days from the last IO dose and before any HSCT. Conclusions: IO administration demonstrated activity with a good safety profile in the setting of MRD positive B-ALL, including blinatumomab resistant patients. Importantly, IO was capable to bridge 37% of eligible patients to HSCT, augmenting the chance to achieve a cure. The continuation of the study will deliver mature efficacy and survival data for a proper evaluation of this promising strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dissecting MRD Kinetics By Automated Computational Analysis to Improve Outcome Prediction in Mantle Cell Lymphoma: A Bioinformatic Substudy from the Fondazione Italiana Linfomi (FIL) MCL0208 Clinical Trial

Background and Aims. Minimal residual disease (MRD) detection is a validated outcome predictor in mantle cell lymphoma (MCL). Recently, the clinical relevance of repeated MRD monitoring has been described in a prospective clinical trial [Ferrero, Blood 2022]. Nonetheless, complex patterns of MRD kinetics over time and availability of results obtained by heterogeneous tissues (bone marrow, BM, vs peripheral blood, PB) might generate substantial interpretation issues and hamper an easy-to-use application of this predictive biomarker. To overcome the limitation of empirical analysis we tested CONNECTOR, a novel automated computational framework to facilitate the interpretation of MRD kinetics and stratify patients in risk classes based on a solid, algorithm-derived, classification. Patients and methods. ASO RQ-PCR MRD data already generated from BM and PB samples in the FIL MCL0208 trial [Ferrero, Blood 2022], offering first-line high dose chemoimmunotherapy and autologous transplantation (ASCT) to youngers MCL patients, were employed. CONNECTOR (https://qbioturin.github.io/connector/) is a data-driven framework based on Functional Data Analysis for analyzing longitudinal high-dimensional data in a straightforward and revealing way. CONNECTOR allows the aggregation of time-series data through an unsupervised approach in informative clusters. Moreover, we developed a classification approach to predict the risk class for new MRD data. The new MRD curves were allocated to existing risk clusters defined on a training set of MRD data. Results. Without knowledge of any clinical or outcome data, CONNECTOR was run on 117 patients characterized by at least 3 MRD timepoints in BM (median 6, 3-9) and separately on 95 patients with at least 4 MRD timepoints in PB (median 7, 4-9). Starting from BM MRD data, CONNECTOR identified four patient risk Clusters (CrC), based on MRD kinetics, Figure1A. CrC_A comprised 73 patients with an overall quick and stable MRD negativization; CrC_B accounted for 14 patients characterized by later MRD negativization, alternating MRD results, or low-level MRD persistence; the 14 patients in CrC_C were always characterized by early MRD negativization followed by late MRD reappearance; CrC_D was composed of 16 patients with stable MRD positivity or only transient MRD negativization followed by early MRD reappearance. The different trends of MRD kinetics dissected by CONNECTOR fairly predicted patients’ outcomes: median TTP was not reached for CrC_A and B, 36 months for CrC_C and 27 months for CrC_D, respectively (p<0.0001), Figure1B. Similar data were generated when CONNECTOR was run on MRD results from PB samples: CrC allocation of BM vs PB was concordant for 89% of patients and survival analysis on PB results confirmed patients stratification in two groups: favorable MRD kinetics (CrC_A and B, median TTP not reached) vs unfavorable MRD kinetics (CrC_C and D, median TTP 35 months, p<0.0001), as depicted in Figure1B. Finally, we tested the performance of pooling together BM and PB results, to increase the number of evaluable patients (irrespective of eventual missing BM samples). Only the BM point at 12 months after ASCT was considered, together with the PB MRD results of all the available time points. CONNECTOR was used to verify if this mixed MRD pattern was able to correctly reclassify patients in the same CrC. Actually, 89 out of 95 patients (94%) were correctly reclassified (p_mean=0.96, p_sd= 0.07). Discussion. CONNECTOR allowed unsupervised identification of patients clusters with distinct MRD kinetics and highly significant different clinical outcome. Such clusterization proved effective using BM, PB and mixed tissues, as well. Validation of CONNECTOR performance in large, independent prospective trials is currently ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic value of low-level MRD in adult acute lymphoblastic leukemia detected by low- and high-throughput methods

AbstractPersistence of minimal residual disease (MRD) after induction/consolidation therapy in acute lymphoblastic leukemia is the leading cause of relapse. The GMALL 07/2003 study used MRD detection by real-time quantitative polymerase chain reaction of clonal immune gene rearrangements with 1 × 10−4 as discriminating cutoff: levels ≥1 × 10−4 define molecular failure and MRD-negativity with an assay sensitivity of at least 1 × 10−4 defining complete molecular response. The clinical relevance of MRD results not fitting into these categories is unclear and termed “molecular not evaluable” (MolNE) toward MRD-based treatment decisions. Within the GMALL 07/03 study, 1019 consecutive bone marrow samples after first consolidation were evaluated for MRD. Patients with complete molecular response had significantly better outcome (5-year overall survival [OS] = 85% ± 2%, n = 603; 5-year disease-free survival [DFS] = 73% ± 2%, n = 599) compared with patients with molecular failure (5-year OS = 40% ± 3%, n = 238; 5-year DFS = 29% ± 3%, n = 208), with patients with MolNE in between (5-year OS = 66% ± 4%; 5-year DFS = 52% ± 4%, n = 178). Of MolNE samples reanalyzed using next-generation sequencing (NGS), patients with undetectable NGS-MRD (n = 44; 5-year OS = 88% ± 5%, 5-year DFS = 70% ± 7%) had significantly better outcome than those with positive NGS-MRD (n = 42; 5-year OS = 37% ± 8%; 5-year DFS = 33% ± 8%). MolNE MRD results not just are borderline values with questionable relevance but also form an intermediate-risk group, assignment of which can be further improved by NGS.

Preemptive Immunotherapy for Minimal Residual Disease in Patients With t(8;21) Acute Myeloid Leukemia After Allogeneic Hematopoietic Stem Cell Transplantation.

In patients with t(8;21) acute myeloid leukemia (AML), recurrent minimal residual disease (MRD) measured by RUNX1-RUNX1T1 transcript levels can predict relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study aimed to compare the efficacy of preemptive interferon (IFN)-α therapy and donor lymphocyte infusion (DLI) in patients with t(8;21) AML following allo-HSCT. We also evaluated the appropriate method for patients with different levels of RUNX1-RUNX1T1 transcripts. In this retrospective study, consecutive patients who had high-risk t(8;21) AML and received allo-HSCT were enrolled. The inclusion criteria were as follows: (1) age ≤65 years; (2) regained MRD positive following allo-HSCT. MRD positive was defined as the loss of a ≥4.5-log reduction and/or <4.5-log reduction in the RUNX1-RUNX1T1 transcripts, and high-level, intermediate-level, and low-level MRDs were, respectively, defined as <2.5-log, 2.5−3.5-log, and 3.5−4.5-log reductions in the transcripts compared with the pretreatment baseline level. Patients with positive RUNX1-RUNX1T1 could receive preemptive IFN-α therapy or DLI, which was primarily based on donor availability and the intentions of physicians and patients. The patients received recombinant human IFN-α-2b therapy by subcutaneous injection twice a week every 4 weeks. IFN-α therapy was scheduled for six cycles or until the RUNX1-RUNX1T1 transcripts were negative for at least two consecutive tests. The rates of MRD turning negative for patients with low-level, intermediate-level, and high-level RUNX1-RUNX1T1 receiving IFN-α were 87.5%, 58.1%, and 22.2%, respectively; meanwhile, for patients with intermediate-level and high-level RUNX1-RUNX1T1 receiving DLI, the rates were 50.0% and 14.3%, respectively. For patients with low-level and intermediate-level RUNX1-RUNX1T1, the probability of overall survival at 2 years was higher in the IFN-α group than in the DLI group (87.6% vs. 55.6%; p = 0.003). For patients with high levels of RUNX1-RUNX1T1, the probability of overall survival was comparable between the IFN-α and DLI groups (53.3% vs. 83.3%; p = 0.780). Therefore, patients with low-level and intermediate-level RUNX1-RUNX1T1 could benefit more from preemptive IFN-α therapy compared with DLI. Clinical outcomes were comparable between preemptive IFN-α therapy and DLI in patients with high-level RUNX1-RUNX1T1; however, they should be further improved.

Lymphoblasts from Minimal Residual Disease in B-ALL Have a Transcriptional and Chromatin Accessibility Profile Shifted Towards Earlier Hematopoietic Progenitor Biology

Background: Prognosis for children and adults with B-cell acute lymphoblastic leukemia (B-ALL), the most common pediatric malignancy, is heavily dependent on minimal residual disease (MRD) levels at the end of induction. Rates of detectable MRD are dependent on patient age and genomic subgroup. For patients with detectable MRD, the primary therapeutic adjustment is intensification of therapy if possible. Despite the incredible advances in MRD detection and clear associations with prognosis, uncovering the underlying biology of MRD could provide critical insight into mechanisms of therapy resistance and suggest research avenues for precise therapeutic approaches.Methods: We collected samples from paired diagnostic and MRD time points from four patients with B-ALL, with MRD ranging from 0.15-0.84%. Based upon clinical MRD immunophenotype, we performed FACS sorting of lymphoblasts from each sample in order to isolate the rare MRD cells, and performed single-cell RNA sequencing (scRNA-seq) on all four pairs and single-cell ATAC sequencing (scATAC-seq) on two of these pairs. Data were compared across time points and integrated with publicly available datasets studying normal hematopoietic development. Raw scRNA-seq and scATAC-seq data were preprocessed using the Cell Ranger pipeline (10x Genomics, v.3.1.0). Seurat V4 (Hao et al., Cell 2021), Signac (Stuart et al., bioRxiv 2020), and MetaCell (Baran et al., Genome Biology 2019) were used to perform quality control (QC), normalize, identify highly variable features, perform dimension reduction, identify both discretized cell clusters and transient meta cells, and ultimately, visualize using uniform manifold approximation and projection (UMAP).Results: Post scRNA-seq QC, the median number of cells per datasets was 2772, the median number of genes per dataset was 13855 and the median number of UMIs per cell was 6518. Post scATAC-seq QC, the median number of cells per datasets was 1157, the median number of peaks per dataset was 76190 and the median number of peaks per cell was 26642. We compared the diagnosis to MRD time points within each of the four cases. In each case, the transcriptionalprofile of the lymphoblasts changed from diagnosis to MRD timepoints (Fig. 1a), with specific transcriptional clusters enriched in the sorted MRD samples (Fig. 1b). To understand this difference, we compared the time points to publicly available datasets of normal hematopoietic development. First, we compared our data to published bulk RNA-seq data from sorted, well-defined hematopoietic development states (Corces et al., Nature Genetics 2016). We performed principal component analysis (PCA) on this data, and projected our generated scRNA-seq data onto the same principal component (PC) space. While the first PC captures the systematic artifact between the bulk and single-cell data, the second PC captures the developmental pseudo time. In each case, cells from sorted MRD show transcriptional profiles shifterd towards earlier progenitor cells on development pseudo time (Fig. 1c). We performed a similar analysis using scATAC-seq from a paired diagnosis and MRD sample, showing that the chromatin accessibility profile of MRD lymphoblasts reveals the same directional change towards patterns seen in earlier hematopoietic progenitor cells. We repeated a similar data integration on a published scRNA-seq dataset from well annotated normal hematopoietic developmental states (Granja et al. Nature Biotechnology 2019), which again revealed that the transcriptional patterns of MRD lymphoblasts align more closely with common lymphoid progenitors than pre-B cells. (Fig. 1d).Conclusions: Using FACS sorting to isolated rare populations of MRD and subsequent single-cell analyses, we show that therapeutic pressure results in residual disease with an earlier progenitor transcriptional profile, likely determined by altered chromatin accessibility profile. Moving forward, we will explore the drivers of these changing transcriptomic and epigenomic profiles through expanding our number of cases to confirm initial findings, optimizing ATAC sequencing approaches to study the rare cell populations present in low-level MRD, and exploring specific transcription factor motifs driving these differences. Our findings have the potential to inform research into rational therapeutic approaches directed toward the specific biology of low level MRD in B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Disease Kinetics Measured By Circulating Tumor DNA Correlates with Treatment Response after Tafasitamab in Combination with R-CHOP with or without Lenalidomide in First Line Treatment of DLBCL

Background Currently available clinical and biological prognostic factors do not adequately identify first-line DLBCL patients at risk for treatment failure. In DLBCL, circulating tumor DNA (ctDNA) can be utilized as a for molecular disease classification, detect minimal residual disease (MRD) or predict disease progression. We hypothesized that ctDNA detected by the EuroClonality immunoglobulin-based next generation sequencing (IG-NGS) assay correlates with treatment response and outcome.Patients and methods Overall, 451 samples (41 diagnostic fresh frozen paraffin embedded (FFPE) biopsies, 64 peripheral blood mononuclear cells (PBMC) and 346 plasma samples) from patients from a Phase Ib study in de novo DLBCL treated with tafasitamab and R-CHOP or tafasitamab and R-CHOP in combination with Lenalidomide (First-MIND; MOR208C107) were analyzed. DNA from FFPE or diagnostic PBMC was sequenced by using a 2-step PCR approach with the Euroclonality NGS IGH-VJ, IGH-DJ and IGK primer sets (euroclonality.org) (Brüggemann, Leukemia, 2019) with a number of primers adapted for usage in short fragment cfDNA. IG markers were identified at abundance level ≥5% of annotated IG reads and a >1 log higher abundance to the next most frequent clonotype. Disease related clonotypes were traced in plasma during (C2D1 and C4D1 n=34) and at end of treatment (EOT n=32) by a 1-step PCR approach and sequencing of at least 5000 human genome equivalents (hGE) of cell free (cf)DNA. ctDNA levels were determined as the number of specific clonotype molecules per input genome equivalents normalized by a reference standard DNA spiked into each sample as cIT-QC (Knecht, Leukemia, 2019) and reported per plasma volume. Data were analysed by ARResT/Interrogate (Bystry, Bioinformatics, 2017). Patients were reported MRD positive if ≥1 clonal IG rearrangement was detected.Results From 41 patients with available diagnostic FFPE and sufficient DNA quality for marker screening, 34 (89%) had at least 1 detectable clonal marker (IGH-VJ, IGH-DJ or IGK). One clonal IG marker was identified in 20/34 (59%) patients, whereas two or three markers were identified in 9 (27%) and 5 (14%) patients, respectively. Disease specific clonotypes identified in FFPE were recovered in 11/34 PBMC samples (32%) with a median abundance of 0,29% (range 0.006-21.9%) demonstrating a peripheral blood involvement. In 32/33 (97%) pretreatment plasma samples, ctDNA was detected with a median copy number of 172.5/ml plasma. cfDNA and ctDNA levels prior to treatment correlated with IPI and LDH (Fig1). ctDNA dynamics during treatment demonstrated rapid treatment response at C2D1. 23/34 samples were either MRD neg (n=19) or showed a ctDNA reduction ≥ 2 log levels (Fig2). At C4D1 7/34 (20%) samples had low-level detectable MRD and at EOT 29/32 (91%) patients achieved MRD negativity after treatment with tafasitamab and R-CHOP +/- lenalidomide (LOD 2 x 10 -4 for follow-up samples). Early ctDNA dynamics predicted metabolic response when applying a threshold of ctDNA reduction to <1% after the first treatment cycle. 25/28 (89%) patients completing treatment and achieving PET-response could be identified early during treatment by ctDNA assessment.Conclusion Baseline ctDNA levels determined by the EuroClonality IG-NGS assay and absolute cfDNA amounts correlated with pre-treatment risk factors. ctDNA dynamics after the 1 st treatment cycle demonstrated rapid treatment response to tafasitamab + RCHOP +/- lenalidomide and correlated with metabolic response. Assessment of ctDNA dynamics allows early response assessment and might be useful for risk stratification in patients with DLBCL. [Display omitted] DisclosuresKuffer: Morphosys AG: Current Employment. Blair: Morphosys AG: Current Employment.

High Resolution Assessment of Minimal Residual Disease (MRD) By Next-Generation Sequencing (NGS) and High-Sensitivity Flow Cytometry (hsFCM) in the Phase 3 GAIA (CLL13) Trial

High Resolution Assessment of Minimal Residual Disease (MRD) By Next-Generation Sequencing (NGS) and High-Sensitivity Flow Cytometry (hsFCM) in the Phase 3 GAIA (CLL13) Trial

Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001

Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001

Efficacy and toxicity of high-risk therapy of the Dutch Childhood Oncology Group in childhood acute lymphoblastic leukemia.

Children with acute lymphoblastic leukemia (ALL) and high-risk (HR) features have a poor outcome and are treated with HR blocks, often followed by allogenic stem cell transplantation (SCT). This article analyses the outcomes of children treated with HR blocks between 2004 and 2017 according to DCOG ALL10/11 protocols. 1297 patients with newly diagnosed ALL were consecutively enrolled, of which 107 met the HR criteria (no complete remission; minimal residual disease (MRD)>10-3 after consolidation; "MLL-AF4" translocation and in ALL-10 also poor prednisone response). Patients were treated with one induction and consolidation course followed by three HR chemotherapy blocks, after which they received either SCT or further chemotherapy. MRD levels were measured at end of induction, consolidation, and after each HR block. At five years, the event-free survival was 72.8% (95% CI, 64.6-82.0), and the cumulative incidence of relapse was 13.0% (95% CI, 6.3-19.8). Patients with only negative or low-positive MRD levels during HR blocks had a significantly lower five-year cumulative incidence of relapse (CIR) of 2.2% (95% CI, 0-6.6) compared with patients with one or more high-positive MRD levels (CIR 15.4%; 95% CI, 3.9-26.9). During the entire treatment protocol, 11.2% of patients died due to toxicity. The high survival with HR blocks seems favorable compared with other studies. However, the limit of treatment intensification might have been reached as the number of patients dying from leukemia relapse is about equal as the number of patients dying from toxicity. Patients with negative or low MRD levels during HR blocks have lower relapse rates.

Bcl-2 Inhibitor ABT-199 (GDC-0199) Monotherapy Shows Anti-Tumor Activity Including Complete Remissions In High-Risk Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL)

Introduction Novel agents are needed for CLL/SLL patients (pts) R/R to standard therapies. The intrinsic apoptotic pathway is often dysregulated in relapsed CLL/SLL due to a deficiency in pro-apoptotic proteins such as TP53 and overexpression of anti-apoptotic proteins such as Bcl-2. ABT-199 is a selective, potent, orally bioavailable, small molecule Bcl-2 inhibitor that can trigger apoptosis in vitro , even in del(17p) CLL cells, making it a promising agent for the treatment of pts with CLL/SLL. Methods The primary objectives of this phase I, dose-escalation study were to evaluate the safety, pharmacokinetics (PK), determine a maximum tolerated dose, and a recommended phase 2 dose of ABT-199. Secondary objectives were to assess efficacy and to explore biomarkers for response. Pts received a single dose of ABT-199 on Week1 Day -3 or -7 (W1D-3, W1D-7), followed by continuous once-daily dosing from W1D1, until disease progression or unacceptable toxicity. Modifications were made to the dose escalation scheme and tumor lysis syndrome (TLS) prophylaxis and monitoring schedule after TLS was observed in some pts. Evaluations included adverse events (AE; NCI-CTCAE-V4), Cairo-Bishop definitions of laboratory and clinical TLS, PK parameters and response. Results As of July 4, 2013, 56 pts were enrolled in cohorts at doses from 150 to 1200 mg, with a median time on study of 10.0 months (range 0-21.0). 17 (38%) pts had del(17p) and 18 (32%) had fludarabine (F)-refractory disease. 12 of 27 (44%) pts had beta-2 microglobulin levels >3 mg/L and 20 of 24 (83%) had IGVH unmutated status. 22 pts discontinued (D/C) ABT-199: 8 due to AEs, 12 due to progressive disease, 2 for other reasons (1 had a pulmonary embolus requiring Coumadin and 1 went to transplant in response). The most common AEs (All grades, ≥25% pts) were diarrhea (46%), neutropenia (43%), fatigue (34%), upper respiratory tract infection (29%), and cough (25%). Grade (G) 3/4 AEs occurring in ≥4 pts were neutropenia (41%), TLS (11%), thrombocytopenia (10%), hyperglycemia (10%), anemia (7%), and febrile neutropenia (7%). 7 DLTs have occurred to date: 5 TLS (1 G3 laboratory at 50 mg; 1 G4 clinical at 50 mg; 1 G3 laboratory at 100 mg and 2 at 200 mg); 1 G4 neutropenia (600 mg); and sudden death (1200 mg) in the setting of G4 (clinical) TLS. After a single dose of ABT-199 with food, Tmax and T1/2 values were approximately 6 and 17 hrs, respectively, supporting daily dosing. Preliminary efficacy data are summarized in the table below. In pts achieving CR/CRi, minimal residual disease (MRD) was quantified with 4 color flow cytometry using local lab methods (aiming to analyze >200,000 nucleated cells) and interpretation. 8 pts have evaluable results with no detectable MRD in 4 (2 with suboptimal cells analyzed) and low level MRD was found in 4 (0.05, 0.7, 0.75%; quantification pending in 1). Of the pts who had no detectable MRD, 1 pt was F-refractory and del(17p) and 2 pts were F-refractory. Conclusions ABT-199 showed activity in pts with R/R CLL with a response rate of 84% for the study population, including 20% CR/CRi. Pts with high-risk CLL showed similar efficacy with a response rate of 82% in del(17p) and 78% in F-refractory disease. Notably, 3 of the 4 pts who had no detectable MRD and achieved a CR/CRi were high-risk disease pts. This study is continuing enrollment using a revised dosing schedule designed to reduce the identified risk of TLS. A phase 2 monotherapy study in pts with relapsed del(17p) CLL has commenced as well as combination studies with either rituximab or obinutuzumab in pts with relapsed CLL. Disclosures: Seymour Genentech: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Kahl: AbbVie, Inc.: Research Funding; Genentech: Consultancy, Research Funding. Wierda: AbbVie, Inc.: Research Funding; Genentech: Consultancy, Research Funding, Speakers Bureau. Gerecitano: AbbVie: Research Funding; Genentech: Research Funding. Kipps: AbbVie, Inc.: Consultancy, Research Funding. Anderson: AbbVie, Inc.: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employee, which recieves commercial income related to ABT-199, Employee, which recieves commercial income related to ABT-199 Other. Huang: AbbVie, Inc.: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employee, recieves commercial income related to ABT-199, Employee, recieves commercial income related to ABT-199 Other, Employment. Rudersdorf: AbbVie, Inc.: Employment, Stock Other. Gressick: AbbVie, Inc: Employment, Stock Other. Montalvo: AbbVie, Inc.: Employment, Stock Other. Yang: AbbVie, Inc.: Employment, Stock Other. Busman: AbbVie, Inc.: Employment, Stock Other. Dunbar: AbbVie, Inc.: Employment, Stock Other. Cerri: AbbVie, Inc.: Employment, Stock Other. Enschede: AbbVie, Inc.: Employment, Stock Other. Humerickhouse: AbbVie, Inc.: Employment, Stock Other. Roberts: AbbVie, Inc.: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employee, recieves commercial income related to ABT-199, Employee, recieves commercial income related to ABT-199 Other, Employment. View this table: Table: Patient Characteristics and Response Rates (iwCLL 2008 criteria)