Introduction Enhanced diagnostic categorisation improves patient access to information, novel therapies and clinical trials. We previously reported the use of a capture based next-generation sequencing (NGS) panel to improve diagnosis of unclassifiable leukemic indolent B-cell Non-Hodgkin's Lymphomas (LI BNHL) in a prospective multicentre study. We report updated findings including 4-year clinical follow up. Methods 119 patients with LI B-NHL deemed unclassifiable on standard peripheral blood (PB) diagnostics were recruited prospectively from 14 centres around the UK over 4 years. Classic chronic lymphocytic leukaemia (CLL) with Matutes score 4 or 5, CLL-type monoclonal b-cell lymphocytosis (MBL), mantle cell lymphoma (MCL), hairy cell leukaemia (HCL), high grade lymphoma and non-clonal lymphocytosis on standard PB diagnostics were excluded. Sequencing methods using the EuroClonality-NDC assay and analyses for small variants, copy number variants (CNVs) and translocations were described previously (Cross et al. ASH 2020). EuroClonality-NDC confirmed IGH,IGK and/or IGL clonality in all cases. DNA was analysed by 450/850K Illumina methylation array at Ohio State University to provide a DNA methylation-based classifier, as previously described by Yamaguchi et al. Kaplan-Meier method was used to calculate overall survival. Results At 4-year follow up, combining clinical presentation, imaging, morphology, immunophenotyping, somatic mutations and structural variants, 73/119 (61%) of patients could be assigned a diagnostic category with medium to high confidence in most cases. Categories assigned included marginal zone lymphoma (MZL), atypical CLL (aCLL), MCL, lymphoplasmacytic lymphoma, follicular lymphoma, HCL and non-CLL MBL. Majority of cases were categorised as MZL 32/73 (44%) and aCLL 16/73 (22%). The remaining 46/119 (39%) cases remained difficult to categorise even with peripheral blood NGS. Of the MZL cases, 91% were categorised as splenic MZL with 2 nodal MZL and 1 MZL not otherwise specified. 17/32 (53%) of them presented as incidental lymphocytosis and 4/32 (13%) had B symptoms. 22/32 (69%) had documented clinical or radiological evidence of splenomegaly. CD5, ROR1 and CD43 were negative in most cases and CD200 was positive in the majority. NGS detected somatic mutations in 88% of cases. KLF2, TP53 and NOTCH2 were the most common variants. Most common CNVs included 7q-, which was detected in 13/29 (45%) of SMZL cases. Of these 13 cases, 10/13 (77%) had documented splenomegaly. 3 of these cases with 7q- did not have splenomegaly at baseline but developed it during follow up suggesting 7q- could be a predictive marker for development of SMZL. Co-occurrence of trisomy 3 and 18 was found in 3/32 (9%) of cases. t(7;14) CDK6::TRAF associated with SMZL was detected in 1 case. 24/32 (75%) of cases had methylation signatures consistent with SMZL. In the aCLL cohort, 9/16 (56%) presented as incidental lymphocytosis and 3/16 (19%) had B symptoms. 8/16 (50%) and 2/16 (13%) had lymphadenopathy and splenomegaly respectively. Most cases were positive for CD5, ROR1, CD200 and CD81. CD20 expression was moderate to strong in 15/16 (94%) of cases. NGS detected somatic mutations in 71% of cases, with the most common being BIRC3 variant. Most common CNVs include gain of 12 and 13q- in 10/16 (63%) and 4/16 (25%) of cases respectively. t(14;19) IGH::BCL3 involving upstream cluster for BCL3 breakpoint was detected in 2 cases. 13/16 (81%) showed methylation signatures within the HP-CLL or LP-CLL epigenetic clusters. Amongst the whole cohort, 19 cases were lost to follow up and excluded from outcome analysis. 46/100 (46%) of patients needed treatment intervention, including 13/27 (48%) and 10/14(71%) of patients with MZL and aCLL. High grade transformation occurred in 4 patients. 17 patients died with 3 disease-related deaths. 4-year overall survival was 85%, 83% and 92% for the whole cohort, MZL and aCLL respectively. Conclusions Multiparametric integration including NGS data on small variants, copy number changes and structural variants, improves diagnostic categorisation of LI B-NHL on peripheral blood when classic CLL, MCL and HCL have been excluded by immunomorphology and standard FISH testing. This can potentially reduce the need for invasive diagnostic procedures and time to diagnosis while improving access to targeted treatment and clinical trials. A DNA methylation-based classifier is a promising tool for use in these challenging cases.

We analyzed 217 patients with KMT2A-rearranged acute myeloid leukemia (AML) in 2 large sequential randomized trials. Those randomized to FLAG-Ida (fludarabine, cytarabine, granulocyte colony-stimulating factor, idarubucin) had markedly lower rates of relapse than other chemotherapy regimens. Molecular measurable residual disease assessment after cycle 2 was strongly prognostic for relapse and death. The trials were registered at the ISRCTN Registry as AML17 ISRCTN55675535 and AML19 ISRCTN78449203.

Management of R/R ALL remains challenging. While CD19-targeted therapies haveshown compelling clinical activity, existing agents are limited by notable toxicity, inconvenient administration schedules, and limited access. Surovatamig (formerlyAZD0486) is a novel, fully human IgG4 CD19xCD3 bispecific T-cell engager that can address these limitations, with extended half-life enabling dosing every 2 wks (Q2W). Herein, we present updated results from a phase 1 dose-escalation study of surovatamig in R/R B-ALL (SYRUS Part A; NCT06137118), highlighting its promising role in this challenging landscape. Eligible patients (pts) for Part A were aged 16–80 y with R/R CD19+ B-ALL. Surovatamig was administered intravenously in 28-day cycles, starting with triple step-up dosing (SUD, cycle 1 day 1 [C1D1], C1D4, and C1D8). Target dose (TD: 2.4, 7.2, or 15 mg) was administered on C1D15 and then Q2W. The primary objective for Part A was safety and tolerability. Disease assessments were performed by the investigator based on NCCN criteria. Minimal residual disease (MRD) was assessed by local multiparametric flow cytometry (MFC) and central next-generation sequencing (NGS) clonotype assay (eg, ClonoSEQ) if MRD was negative by MFC. As of July 4, 2025, 42 pts were enrolled (TD 2.4 mg [n=13], 7.2 mg [n=12], and 15 mg [n=17]). Median age was 50 y (range 17–77); median prior lines of therapy was 3 (range 2–9). Across doses, 26 pts (62%) had been treated with CD19-targeted treatment (tx) including blinatumomab (43%) and/or CAR-T (40%), and 25 (60%) pts had >50% bone marrow blasts. Overall response rate (ORR; defined as complete remission [CR]/CR with partial or incomplete hematologic recovery) within 3 cycles was 67% (46%, 2.4 mg; 58%, 7.2 mg; 82%, 15 mg). CR rate within 3 cycles was 50% (38%, 2.4 mg; 42%, 7.2 mg; 65%, 15 mg). ORR was 88% (7/8) in pts with extramedullary disease (EMD). Among pts receiving TD of 15 mg, ORR was 88% in pts with prior blinatumomab tx, 73% in pts with prior CAR-T, 86% in double-exposed pts (prior blinatumomab and CAR-T), and 86% in triple-exposed pts (double-exposed + prior inotuzumab ozogamacin). Six of 7 pts with Philadelphia chromosome–positive disease achieved CR within 3 cycles. Among responders, MRD negativity was achieved by MFC in 83% (2.4 mg) and 100% (7.2 mg and 15 mg). By NGS-based assay, MRD negativity with the threshold of 10-4 and 10-6 was achieved in 92% and 60% of evaluable CR pts, respectively. At data cutoff, 14 pts remained on tx; 11 discontinued tx while in CR, of whom 8 proceeded to allogeneic stem cell transplantation and 1 to donor lymphocyte infusion. Among responders (n=28), with a median follow-up of 6.2 mo, median duration of response had not been reached. Only 3 pts experienced relapse, all at 2.4 mg (2 within 3 mo and 1 after 6 mo; 2 were CD19-negative). In these 3 pts who experienced relapse, best MRD status while in CR was as follows: positive by MFC at 10-4, negative by ClonoSeq at 10-4, and negative by ClonoSeq at 10-5. Notably, no relapses have been observed to date in 12 pts who achieved MRD negativity at 10-6. Dose-limiting toxicities were observed in 1 pt (TD 2.4 mg; SUD 0.09/0.27/1.0 mg) with grade (G) 4 thrombocytopenia and G3 ALT/AST elevation and 1 pt (TD 15 mg; SUD 0.27/1.0/2.4 mg) with G4 thrombocytopenia; both resolved and the pts continued planned TD. G3+ related tx-emergent adverse events (AEs) were reported in 9 (21%) pts; 4 fatal infections were reported during C1, none considered related to surovatamig. With optimized SUD (0.09/0.27/1.0 mg, n=23), G2+ CRS events occurred in 9% and no G2+ ICANS events were observed. Following TD administration (n=36), no G3+ CRS events were reported and a G3+ ICANS event was observed in 1 pt (G3, TD 7.2 mg). Plasma cytokine peak levels were highest after D1 dose, which was dose-dependent. Cytokine production was substantially lower after subsequent doses (D4, D8, and D15) across different SUD and TD. Exposure–response analysis (n=34) showed a positive correlation between increased drug exposure (Cavg) and higher ORR. Surovatamig up to a TD of 15 mg is well tolerated in pts with R/R B-ALL. SUD 0.09/0.27/1.0 mg mitigates the incidence and severity of CRS and ICANS. The 15-mg TD showed the highest efficacy, including in pts with prior CD19-targeted therapy and with EMD. These results will guide dose selection for SYRUS Part B (dose optimization) and Part C (efficacy expansion).

CPX-351 is a therapy approved for newly diagnosed (ND) therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC) in patients aged ≥1 year in the US and adult patients in the UK/EU. These approvals were based on the pivotal phase 3 trial of CPX-351 vs 7+3 chemotherapy in adults aged 60-75 years with high-risk/secondary AML showing significantly improved efficacy (median overall survival [OS]: 9.56 vs 5.95 months, one-sided P=0.003; complete remission [CR]/CR with incomplete platelet or neutrophil recovery [CRi]: 47.7% vs 33.3%, two-sided P=0.016) and comparable safety. Two real-world studies, V-RULES (Vyxeos Real-world US Long-term Effectiveness and Safety) and CREST-UK (CPX-351 Real-world Effectiveness and SafeTy Study; NCT05169307) were conducted in the US and UK, respectively, to collect real-world effectiveness and safety data of CPX-351. Here, we present a pooled analyses of these studies in patients aged <60 years to analyze the effectiveness and safety of CPX-351 in this age group. V-RULES and CREST-UK are retrospective, single-arm studies that collected pseudonymized data from the medical records of patients with ND t-AML or AML-MRC who received ≥1 infusion of CPX-351 in routine practice after FDA/EU approval — V-RULES at 10 US centers (October 2017-May 2024) and CREST-UK at 15 UK centers (November 2018-March 2022). The two studies were developed and designed at different times, and, thus, some of the data collection methods differed. Response definitions differed between the two studies (defined as CR/CR with partial hematologic recovery [CRh]/CRi in V-RULES, and CR/CRi in CREST-UK) and were compared side by side in this analysis. Pooled outcomes included Kaplan-Meier (KM)-estimated OS and safety. Overall, 134 patients aged <60 years (V-RULES, N=78; CREST-UK, N=56) received ≥1 induction of CPX-351 (1 cycle, n=114; 2 cycles, n=19; 3 cycles, n=1). Median age at AML diagnosis was 52 years (range: 18-59). The majority of patients (100/111 [90.1%]) had a known ECOG performance status of 0-1. Of patients with available cytogenetic data, 70/132 (53.0%) were classified as adverse risk per Grimwade 2010. Notably, 25/105 patients (23.8%) had mutated TP53 and 35 patients had myelodysplasia-related gene mutations. The median follow-up time was 12.1 months (interquartile range [IQR]: 5.5, 27.5). Among evaluable patients, CR/CRh/CRi was achieved by 49/75 (65.3%) in V-RULES and CR/CRi was achieved by 37/56 (66.1%) in CREST-UK. Among responders, measurable residual disease (MRD) status at best response was assessed in 50 patients (most commonly by flow cytometry [MCF]) and 22/50 (44.0%) patients were MRD-negative. Median OS was 20.0 months (95% confidence interval [CI]: 14.3, 45.4), with a KM-estimated 4-year OS of 37.2% (95% CI: 26.3, 48.1). Early mortality was 6.0% by day 30 and 11.3% by day 60. In total, 61/134 (45.5%) patients underwent hematopoietic cell transplantation (HCT) after CPX-351 treatment. Median OS post-HCT was 47.1 months (95% CI: 34.3, not estimated), with an estimated 4-year OS of 47.2% (95% CI: 26.3, 65.6). In responders, median time to neutrophil (≥500/μL) and platelet (≥50,000/μL) recovery after induction 1 was 35 days (IQR: 30, 41; n=68) and 37 days (IQR: 32, 44; n=64), respectively. Overall, 100/134 (74.6%) patients experienced grade ≥3 treatment-emergent adverse events (TEAEs). Febrile neutropenia (61/134 [45.5%]) and infection (52/134 [38.8%]) were the most common grade ≥3 TEAEs. Out of 134 patients, 47 (35.1%) patients experienced ≥1 serious TEAE. This pooled analysis of the V-RULES and CREST-UK studies provides valuable real-world data on the effectiveness and safety of CPX-351 in adult patients aged <60 years with t-AML or AML-MRC, which is noteworthy as younger adults were not eligible for the CPX-351 phase 3 trial. We observed more favorable response, survival, and HCT outcomes in adults aged <60 years compared with the population of adults aged 60-75 years who were included in the CPX-351 phase 3 trial. Limitations of this study include its retrospective nature with clinician selected treatment in major medical centers. This retrospective real-world pooled analysis of patients treated at 25 US and UK medical centers demonstrates favorable effectiveness and acceptable tolerability of CPX-351 in younger adults with ND t-AML or AML-MRC eligible for intensive chemotherapy.

Favourable outcomes with CPX-351 versus conventional 7 + 3 were demonstrated in the pivotal phase III trial in adults aged 60-75 years with newly diagnosed, highrisk/secondary acute myeloid leukaemia (AML). As a complement to the clinical trial and to address important data gaps, the CPX-351 Real-World Effectiveness and SafeTy (CREST-UK; NCT05169307) study evaluated the use of CPX-351 in routine clinical practice in the UK, in 147 patients with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Best response of complete remission or complete remission with incomplete platelet or neutrophil recovery was achieved by 53% of evaluable patients. Kaplan-Meier median overall survival (OS) was 12.8 months (95% confidence interval 9.2-15.3). Fifty (34%) patients proceeded to haematopoietic cell transplantation (HCT); median OS landmarked from the HCT date was not reached. There were no new safety concerns with CPX-351 identified in CREST-UK. Patients treated with CPX-351 in the outpatient setting spent an average of 24.4, 16.7, 28.2, and 27.7 fewer days on the ward compared with inpatients during first induction, second induction, first consolidation, and second consolidation, respectively. The results from CREST-UK provide valuable insights into the effectiveness, safety, and outpatient delivery of CPX-351 in routine clinical practice in the UK.

KMT2A rearrangements (KMT2Ar) occur in ~5% of adult AML and their outcomes are generally poor, with high rates of early relapse. Menin inhibitors have shown encouraging activity in relapsed/refractory disease, and trials combining these agents with intensive chemotherapy or venetoclax-azacitidine are ongoing. However, the optimal backbone chemotherapy regimen, and the role of MRD during induction, are not well established. We sought to address these issues by examining outcomes of KMT2Ar patients in two large prospective RCTs. Methods NCRI AML17 (2009-2014) and AML19 (2015-2020) enrolled younger adults with newly diagnosed AML. Results from karyotype, FISH, RT-qPCR and RNA sequencing were reviewed to identify all patients with KMT2Ar. MRD was generally assessed by flow cytometry in AML17, while RT-qPCR was used in AML19. Results 218 patients (AML17 n=135, AML19 n=83) were identified with a median age of 41y (range 16 - 68). 8 (4%) had a prior myeloid disorder and 22 (10%) were therapy related. 75 (34%) patients had ELN 2022 intermediate risk disease with t(9;11)/MLLT3::KMT2A and the remaining 66% were adverse risk. 131 patients (60%) had no other cytogenetic abnormalities. Complex cytogenetics (defined here as 3 structural abnormalities in addition to KMT2Ar) were seen in 12 patients (6%) and 7 (3%) had other adverse abnormalities (del5q or -7). FLT3 ITD mutations were present in 9 (4%) and TKD in 15 (7%). Treatment was with DA in 127 (58%), FLAG-Ida in 48 (22%), ADE in 35 (16%) and CPX-351 in 8 (3.7%). 82 (38%) received GO. 196 patients (90%) achieved CR or CRi by the end of the second induction, with 81% in CR/CRi after course 1. Higher rates of refractory disease were seen in patients with a complex karyotype (17%) or other adverse abnormalities (29%). Median follow-up was 4.8y. Day 30 and 60 mortality were 4% and 5% respectively. Median overall survival (OS) was 2.1y (95CI 1.7 - 3.1) and was not significantly different between ELN intermediate (2.7y) and adverse risk (1.9y) disease. On multivariable analyses, higher WCC (HR 1.23, 95CI 1.08-1.41) and complex karyotype (HR 3.46, 95CI 1.72-6.98) were associated with worse survival while FLAG-Ida produced better survival (HR 0.42, 95CI 0.24-0.75). 2-yr OS was 68% (median not reached) for FLAG-Ida compared to 48% (median 1.8y) for other regimens. Of 196 patients achieving remission, 99 (51%) were transplanted in CR1 (60% of ELN adverse and 31% of ELN intermediate patients). CR1 transplant was associated with improved RFS (0.66, 95CI 0.45 - 0.97) and a trend to better OS (HR 0.75, 95CI 0.50 - 1.11), with no heterogeneity based on ELN risk group. 69 patients were included in a direct randomisation of FLAG-Ida (n=42) vs DA (n=27) in AML19. FLAG-Ida was associated with a numerically higher remission after C1 (93% vs 81%, p=0.2) but not after second induction (95% vs 96%). Despite a lower rate of CR1 transplant after FLAG-Ida (40% vs 59%), relapse was significantly reduced (2y CIR 23% vs 61%, p<0.001). Median OS was not reached with FLAG-Ida vs 2.2y for DA, with 2y OS 67% vs 57% (HR 0.54, 95CI 0.26-1.13). No survival difference was seen in the AML17 randomisations of DA vs ADE or DA with 60mg/m2 vs 90mg/m2 of daunorubicin, nor in the AML19 randomisations of GO1 vs GO2 or CPX-351 vs FLAG-Ida. MRD was performed by flow cytometry in 76 and RT-qPCR in 61 patients. 50 patients had a post C2 qPCR result in either PB or BM, with results concordant in all patients with both samples (n=38). 18 patients (36%) remained MRD positive (52% for adverse risk, 14% for intermediate risk). Detectable MRD was strongly associated with relapse (CIR 74% vs 23%; HR 5.58, 95CI 2.4-13.2, p<0.001) and poor OS (2y OS 31% vs 77%, HR 4.42, 95CI 1.9-10.2, p<0.001). Only 3 MRD positive patients remain alive and relapse-free at last follow-up. Flow MRD results were analysed at both post C1 and C2, examining thresholds of either 0.1% or any positivity. The only threshold predictive of outcomes was detection of any positivity post C1, with 60% of patients MRD positive and OS HR 1.88 (95CI 1.00-3.52). Conclusion Here we demonstrate a survival advantage for intensified induction treatment with FLAG-Ida in younger adults with KMT2Ar AML. We also show that molecular MRD after course 2 is a strong predictor of long-term survival. These data are important to inform the design of future trials incorporating menin inhibitors in front-line therapy, and suggest that intensified induction and RT-qPCR MRD should be incorporated into these studies.

We report on a study of next-generation sequencing in 257 patients undergoing investigations for cytopenias. We sequenced bone marrow aspirates using a target enrichment panel comprising 82 genes and used T cells from paired blood as a control. One hundred and sixty patients had idiopathic cytopenias, 81 had myeloid malignancies and 16 had lymphoid malignancies or other diagnoses. Forty-seven of the 160 patients with idiopathic cytopenias had evidence of somatic pathogenic variants consistent with clonal cytopenias. Only 39 genes of the 82 tested were mutated in the 241 patients with either idiopathic cytopenias or myeloid neoplasms. We confirm that T cells can be used as a control to distinguish between germline and somatic variants. The use of paired analysis with a T-cell control significantly reduced the time molecular scientists spent reporting compared to unpaired analysis. We identified somatic variants of uncertain significance (VUS) in a higher proportion (24%) of patients with myeloid malignancies or clonal cytopenias compared to less than 2% of patients with non-clonal cytopenias. This suggests that somatic VUS are indicators of a clonal process. Lastly, we show that blood depleted of lymphocytes can be used in place of bone marrow as a source of material for sequencing.

Gilteritinib is the current standard of care for relapsed or refractory fms related receptor tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia in many countries, however outcomes for patients relapsing after contemporary first-line therapies (intensive chemotherapy with midostaurin, or nonintensive chemotherapy with venetoclax) are uncertain. Moreover, reported data on toxicity and health care resource use is limited. Here, we describe a large real-world cohort of 152 patients receiving single-agent gilteritinib in 38 UK hospitals. Median age was 61 years, and 36% had received >= 2 prior lines of therapy, including a FLT3 inhibitor in 41% and venetoclax in 24%. A median of 4 cycles of gilteritinib were administered, with 56% of patients requiring hospitalization in the first cycle (median, 10 days). Over half of patients required transfusion in each of the first 4 cycles. Complete remission (CR) was achieved in 21%, and CR with incomplete recovery (CRi) in a further 9%. Remission rates were lower for patients with FLT3- tyrosine kinase domain or adverse karyotype. Day-30 and day-60 mortality were 1% and 10.6%, respectively, and median overall survival was 9.5 months. On multivariable analysis, increasing age, KMT2A rearrangement, and complex karyotype were associated with worse survival whereas RUNX1 mutations were associated with improved survival. Twenty patients received gilteritinib as first salvage having progressed after first-line therapy with venetoclax, with CR/CRi achieved in 25% and median survival 4.5 months. Real-world results with gilteritinib mirror those seen in the clinical trials, but outcomes remain suboptimal, with more effective strategies needed.

Favourable outcomes with CPX-351 versus conventional 7 + 3 were demonstrated in the pivotal phase III trial in adults aged 60–75 years with newly diagnosed, highrisk/secondary acute myeloid leukaemia (AML). As a complement to the clinical trial and to address important data gaps, the C PX-351 R eal-World E ffectiveness and S afe T y (CREST-UK; NCT05169307) study evaluated the use of CPX-351 in routine clinical practice in the UK, in 147 patients with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Best response of complete remission or complete remission with incomplete platelet or neutrophil recovery was achieved by 53% of evaluable patients. Kaplan–Meier median overall survival (OS) was 12.8 months (95% confidence interval 9.2–15.3). Fifty (34%) patients proceeded to haematopoietic cell transplantation (HCT); median OS landmarked from the HCT date was not reached. There were no new safety concerns with CPX-351 identified in CREST-UK. Patients treated with CPX-351 in the outpatient setting spent an average of 24.4, 16.7, 28.2, and 27.7 fewer days on the ward compared with inpatients during first induction, second induction, first consolidation, and second consolidation, respectively. The results from CREST-UK provide valuable insights into the effectiveness, safety, and outpatient delivery of CPX-351 in routine clinical practice in the UK.

Molecular failure in NPM1-mutated acute myeloid leukemia (AML) inevitably progresses to frank relapse if untreated. Recently published small case series show that venetoclax combined with low -dose cytarabine or azacitidine can reduce or eliminate measurable residual disease (MRD). Here, we report on an international multicenter cohort of 79 patients treated for molecular failure with venetoclax combinations and report an overall molecular response (>= 1 -log reduction in MRD) in 66 patients (84%) and MRD negativity in 56 (71%). Eighteen of 79 patients (23%) required hospitalization, and no deaths were reported during treatment. Forty-one patients were bridged to allogeneic transplant with no further therapy, and 25 of 41 were MRD negative assessed by reverse transcription quantitative polymerase chain reaction before transplant. Overall survival (OS) for the whole cohort at 2 years was 67%, event -free survival (EFS) was 45%, and in responding patients, there was no difference in survival in those who received a transplant using time -dependent analysis. Presence of FLT3-ITD mutation was associated with a lower response rate (64 vs 91%; P < .01), worse OS (hazard ratio [HR], 2.50; 95% confidence interval [CI], 1.06-5.86;P = .036), and EFS (HR, 1.87; 95% CI, 1.06-3.28; P = .03). Eighteen of 35 patients who did not undergo transplant became MRD negative and stopped treatment after a median of 10 months, with 2 -year molecular relapse free survival of 62% from the end of treatment. Venetoclax-based low intensive chemotherapy is a potentially effective treatment for molecular relapse in NPM1mutated AML, either as a bridge to transplant or as definitive therapy.

Patients with relapsed/refractory acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) have poor outcomes compared with newly diagnosed, treatment-naïve patients. The phase 2, open-label DELPHINUS study evaluated daratumumab (16 mg/kg intravenously) plus backbone chemotherapy in children with relapsed/refractory B-cell ALL (n=7) after ≥2 relapses and children and young adults with T-cell ALL (children, n=24; young adults, n=5) or LL (n=10) after first relapse. The primary endpoint was complete response (CR) in the B-cell ALL (end of Cycle 2) and T-cell ALL (end of Cycle 1) cohorts, after which patients could proceed off study to allogeneic hematopoietic stem cell transplant (HSCT). Seven patients with advanced B-cell ALL received daratumumab with no CRs achieved; this cohort was closed due to futility. For the childhood T-cell ALL, young adult T-cell ALL, and T-cell LL cohorts, the CR (end of Cycle 1) rates were 41.7%, 60.0%, and 30.0%, respectively; overall response rates (any time point) were 83.3% (CR+CR with incomplete count recovery [CRi]), 80.0% (CR+CRi), and 50.0% (CR+partial response); minimal residual disease-negativity (<0.01%) rates were 45.8%, 20.0%, and 50.0%; observed 24-month event-free survival rates were 36.1%, 20.0%, and 20.0%; observed 24-month overall survival rates were 41.3%, 25.0%, and 20.0%; and allogeneic HSCT rates were 75.0%, 60.0%, and 30.0%. No new safety concerns with daratumumab were observed. In conclusion, daratumumab was safely combined with backbone chemotherapy in children and young adults with T-cell ALL/LL and contributed to successful bridging to HSCT. This trial was registered at www.ClinicalTrials.gov as NCT03384654.

Our previous work[1] suggests AML blocks the differentiation of normal haematopoietic stem cells (HSCs) leading to marrow failure. However, many patients do not have uniform pancytopenia where all differentiated blood cells are reduced e.g. some patients have preserved neutrophil counts. We aimed to identify whether the residual mature blood cells at diagnosis of AML are derived from the main AML clone, or from normal HSCs (or pre-leukaemic HSCs) by sequencing highly purified sorted residual neutrophils (CD45+CD13+CD16+) and erythroblasts (CD45-CD71+CD235a+) from 46 fresh AML diagnostic samples using next generation sequencing. T cells and blasts were used as negative and positive controls. Neutrophils had the same mutational profile as the blasts from 25/45 (55%) cases suggesting that neutrophils were derived by differentiation from the AML clone. No mutations in neutrophils from 8/45 (17%) cases consistent with the neutrophils being derived from normal HSCs. Neutrophils had some but not all the mutations seen in the AML clone from 12/45 (27%) cases; in these cases, neutrophils most often bore mutations in ‘early hit’ genes such as DNMT3A or TET2 suggesting they may have derived from pre-leukaemic HSCs. We did not sequence neutrophils in one case due to insufficient yield of DNA. Erythroblast numbers were lower, and we were not able to get sufficient numbers to sequence in all AML. The erythroblasts had the same mutational profile as the blasts from 8 of 17 (47%) sequenced cases and some but not all the mutations seen in the AML clone from 5/17 (29%) cases. Mutations were not seen in erythroblasts from 4/17 (24%) cases. Of 30 cases with preserved neutrophils (>1x109/L) at diagnosis, 20/30 (66%) cases had neutrophils derived from the AML clone, 7/30 (23%) cases had neutrophils derived from pre-leukaemic HSCs and 3/30 (10%) cases had neutrophils derived from normal HSCs. Of the patients with preserved neutrophils, there was a clear association between neutrophil morphology and mutations. Patients with any mutations in the neutrophils (n=27) had neutrophil dysplasia while the 3 cases without mutations in the neutrophils had normal neutrophils morphology. Of the five of AML cases where the haemoglobin was preserved (>100g/l) at diagnosis, two (40%) cases had erythroblasts derived from the AML clone, one (20%) from pre-leukaemic HSCs and two (40%) cases from normal HSCs. In conclusion, where neutrophil numbers are preserved at diagnosis, the neutrophils are derived by differentiation of blasts in two-thirds of patients. In one third of cases the neutrophils derive from normal or pre-leukaemic HSCs suggesting the differentiation block we previously identified in normal HSCs may not be complete in all AMLs. We are testing megakaryocytes using Fluorescence In Situ Hybridization (FISH) to delineate the origin of platelets at diagnosis of AML where the platelets are preserved. This works helps further define the pathology of marrow failure in AML. 1. Miraki-Moud, F., et al., Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation. Proc Natl Acad Sci U S A, 2013. 110(33): p. 13576-81.

British Journal of HaematologyEarly View LETTER TO THE EDITOR Correlation of T-cell receptor constant beta-chain 1 by flow cytometry with molecular T-cell receptor clonality for the investigation of T-cell lymphoproliferation Tania Dexter, Tania Dexter orcid.org/0000-0001-6988-7306 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorTosin Taiwo, Tosin Taiwo The Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorClaire Dearden, Claire Dearden Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorLi Yuan Chan, Li Yuan Chan orcid.org/0000-0003-3737-0905 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorDavid Taussig, David Taussig orcid.org/0000-0002-9621-0146 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorDima El-Sharkawi, Dima El-Sharkawi orcid.org/0000-0002-2752-5814 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorAlan Dunlop, Alan Dunlop The Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorSunil Iyengar, Corresponding Author Sunil Iyengar [email protected] orcid.org/0000-0003-4863-4160 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UK Correspondence Sunil Iyengar, Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Downs Road, Sutton SM2 5PT, UK. Email: [email protected]Search for more papers by this author Tania Dexter, Tania Dexter orcid.org/0000-0001-6988-7306 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorTosin Taiwo, Tosin Taiwo The Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorClaire Dearden, Claire Dearden Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorLi Yuan Chan, Li Yuan Chan orcid.org/0000-0003-3737-0905 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorDavid Taussig, David Taussig orcid.org/0000-0002-9621-0146 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorDima El-Sharkawi, Dima El-Sharkawi orcid.org/0000-0002-2752-5814 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorAlan Dunlop, Alan Dunlop The Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UKSearch for more papers by this authorSunil Iyengar, Corresponding Author Sunil Iyengar [email protected] orcid.org/0000-0003-4863-4160 Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Sutton, UK Correspondence Sunil Iyengar, Department of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, Downs Road, Sutton SM2 5PT, UK. Email: [email protected]Search for more papers by this author First published: 26 February 2024 https://doi.org/10.1111/bjh.19299Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Supporting Information Filename Description bjh19299-sup-0001-DataS1.docxWord 2007 document , 29.4 KB Data S1. bjh19299-sup-0002-supinfo.docxWord 2007 document , 1.5 MB Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Muñoz-García N, Lima M, Villamor N, Morán-Plata FJ, Barrena S, Mateos S, et al. Anti-TRBC1 antibody-based flow cytometric detection of T-cell clonality: standardization of sample preparation and diagnostic implementation. Cancer. 2021; 13:4379. https://doi.org/10.3390/cancers13174379 10.3390/cancers13174379 CASGoogle Scholar 2Shi M, Jevremovic D, Otteson GE, Timm MM, Olteanu H, Horna P. 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Patients with FLT3 -mutated AML have a high relapse rate and suboptimal outcomes. Many have co-mutations suitable for measurable residual disease (MRD) monitoring by RT-qPCR and those destined to relapse can be identified by high or rising levels of MRD, called molecular failure. This provides a window for pre-emptive intervention, but there is little evidence to guide treatment. The use of FLT3 inhibitors (FLT3i) appears attractive but their use has not yet been evaluated. We identified 56 patients treated with FLT3i at molecular failure. The FLT3 mutation was an ITD in 52, TKD in 7 and both in 3. Over half of patients had previously received midostaurin. Molecular failure occurred at a median 9.2 months from diagnosis and was treated with gilteritinib ( n = 38), quizartinib ( n = 7) or sorafenib ( n = 11). 60% achieved a molecular response, with 45% reaching MRD negativity. Haematological toxicity was low, and 22 patients were bridged directly to allogeneic transplant with another 6 to donor lymphocyte infusion. 2-year overall survival was 80% (95%CI 69–93) and molecular event-free survival 56% (95%CI 44–72). High-sensitivity next-generation sequencing for FLT3 -ITD at molecular failure identified patients more likely to benefit. FLT3i monotherapy for molecular failure is a promising strategy which merits evaluation in prospective studies.