Acute leukemias of ambiguous lineage (ALAL) are characterized by ≥ 20% abnormal progenitors without clear differentiation along a single lineage or with commitment to more than 1 lineage. ALAL includes acute undifferentiated leukemia (AUL), lacking definitive lineage markers, and mixed-phenotype acute leukemia (MPAL), which displays features of multiple lineages. Updated diagnostic criteria incorporate immunophenotype and molecular profiling. Due to the genetic heterogeneity of ALAL, there is no standard treatment, though there is growing interest in combining targeted therapies with AML- or ALL-type induction regimens. Retrospective studies support consolidative allogeneic hematopoietic stem cell transplant (HCT), but data remain limited on pre-HCT disease status and outcomes by ALAL subtype.

With IRB approval, we retrospectively analyzed ALAL cases (AUL or MPAL) treated from 2010-2025 at City of Hope. Pathology was reviewed per 2022 WHO/ICC criteria to confirm diagnoses independently. Of 58 patients, 45 underwent HCT (AUL: 12 (27%), MPAL: 33 (73%)). MPAL subtypes included: B/My (n=10), T/My (n=9), B/T (n=2), B/T/My (n=1), BCR::ABL1 (n=7), KMT2Ar (n=2), and ZNF384r (n=2). Median age at diagnosis and HCT was 41 years (range 1-72). Patients were Hispanic (60%), Non-Hispanic White (27%), Asian (7%), Black (4%) and Native Hawaiian (2%). 26 patients underwent blood or marrow HopeSeq Heme Comprehensive testing between July 2018 and February 2025, which includes DNA full exon sequencing of up to 523 genes and RNA fusion detection of up to 165 genes. Recurrent mutations included DNMT3A, RUNX1, WT1, and NF1. Fusions included BCR::ABL1, DDX3X::MLLT10, KMT2Ar, NUP98::NSD1, PICALM::MLLT10, SET::NUP214, TCF3::ZNF384 and IKZF1 oncogenic isoform. Cytogenetics (available for 44/45) showed 26% normal, 29% abnormal, and 49% complex karyotype.

Induction was ALL-type (62%) or AML-type in 38%. Median prior lines of therapy was 2 (range 1-4). Eight patients received HCT2, and one received HCT3. Median time to transplant from diagnosis was 170 days (range 53-3021) and 91% had KPS ≥80. 96% were in CR with < 5% blasts pre-HCT1; 60% were MRD-, 30% MRD+. Among MRD+ patients (n=13), 54% were positive by flow; others were negative by flow but positive by targeted gene-specific qPCR, ClonoSEQ or HopeSeq. Donors included matched related (40%), matched unrelated (29%), mismatched unrelated (11%), haploidentical (16%) and cord blood (4%). Peripheral blood stem cells were used in 80%. Conditioning was myeloablative (60%) and reduced-intensity (38%). Radiation (≥12Gy) was used in 77%, and total marrow/lymphoid irradiation in 6%. Most RIC regimens were fludarabine/melphalan. GVHD prophylaxis included tacrolimus/sirolimus (49%), PTCy-based (29%) and CNI with methotrexate (13%). The median time to neutrophil and platelet engraftment was 15 and 12 days respectively.

Median follow-up was 34.2 months (range 2.6-125.3), and 62.2 months for survivors (n=29). 5-year OS was 64.1% (95% CI 46.8%-77.0%) and PFS 49.8% (95% CI 33.1%-64.3%). The cumulative incidence of relapse was 18.5% at 1 year and 37.7% at 5-years. 5-year NRM was 12.5%. OS did not differ between AUL and MPAL (73.3% vs. 60.2%, p=0.969). MRD+ patients trended toward lower 5-y-OS. Day 100 grade II-IV aGVHD incidence was 40.9% grade II-IV, 8.9% grade III-IV). 5y-cGVHD incidence was 49.5%. 25% had extramedullary disease at relapse post HCT1. Of those, 50% (n=8) underwent HCT2 (all with RIC), with 50% relapsing again; one proceeded to HCT3. Relapsing after HCT may be related to the primitive stem cell origin, (Peretz et al. Nat Commun 2024), typically believed to be drug resistant.

In summary, consolidative allogeneic HCT should be strongly considered for ALAL patients in remission, aligning with prior studies (Munker et al. BBMT 2016; Munker et al. Haematologica 2017; Huang et al. ASCO 2024). Our findings confirm that HCT offers durable long-term disease control with 5-year OS exceeding 60%. Outcomes were comparable between AUL and MPAL, suggesting similar benefit from HCT. Pre-HCT MRD positivity did not impact outcomes post HCT. Additionally, HCT2 was feasible and effective in selected patients, supporting its role as a salvage approach in relapsed ALAL.

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2025
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