Myelodysplasia-Related Mutations in Adults and End-of-Induction Measurable Residual Disease Are Associated with Inferior Outcome in Patients with Acute Leukemia of Ambiguous Lineage
Authors: Chatterjee Gaurav , Mawalankar Gojiri , Kusurkar Saurabh , Madan Pratishtha , Paulose Elwin , Joshi Swapnali , Bhanshe Prasanna , Chaudhary Shruti , Ghogale Sitaram , Deshpande Nilesh , Shetty Dhanlaxmi Lalit , Jindal Nishant , Mirgh Sumeet , Shetty Alok , Chichra Akanksha , Sengar Manju , Gokarn Anant , Punatar Sachin , Nayak Lingaraj , Roy Moulik Nirmalya , Srinivasan Shyam , Dhamne Chetan , Jain Hasmukh , Bagal Bhausaheb , Narula Gaurav , Banavali Shripad , Khattry Navin , Rajpal Sweta , Tembhare Prashant , Subramanian Papagudi Ganesan , Gujral Sumeet , Patkar Nikhil 
Published: December 2024

Purpose
Recent updates on hematolymphoid neoplasm have included myelodysplasia-related (MR) mutations (SRSF2, ZRSR2, SF3B1, ASXL1, BCOR, EZH2, U2AF1, STAG2) as class-defining for acute myeloid leukemia (AML-MR). The outcome of patients with acute leukemia of ambiguous lineage (ALAL) and MR mutations remain unknown. Here, we document the genomic spectrum of a large cohort of ALAL and analyse the clinical impact of MR mutations.
Patients and Methods
A total of 116 patients with ALAL for which samples were received for molecular testing at Tata Memorial Centre over 6 years (2018-2023) were included in this study. The diagnosis of ALAL was established using a 5-tube, 10-13 color flowcytometry assay as per WHO criteria. Diagnostic samples were sequenced using a targeted, 137-gene capture-based acute leukemia panel. RNA-sequencing to identify chimeric gene fusions were performed in 59.5% (69/116) of patients. Measurable residual disease (MRD) was evaluated at end-of-induction (EOI) timepoint using 10-16 color flowcytometry assays. EOI-MRD ≥0.01% was considered positive. Overall survival (OS) and event free survival (EFS) were used as the end points. Clinical impact of variables was analysed using log-rank test and Cox proportional hazards regression model.
Results
The cohort included 53 children (median age 10 (1-17) years) and 63 adults (median age 33 (19-73) years). The distribution of the cohort according to WHO diagnosis were T/Myeloid MPAL (55, 47.4%), B/Myeloid MPAL (17, 14.7%), MPAL with KMT2A-rearrangement (11, 9.5%), ALAL NOS (11, 9.5%), MPAL with BCR::ABL1 (10, 8.6%), B/T MPAL (10, 8.6%), and B/T/Myeloid MPAL (2, 1.7%). Most commonly mutated genes included RAS-pathway mutations (total 38, 32.8%: NRAS (23, 19.8%), KRAS (7, 6%), NF1 (7, 6%), and PTPN11 (5, 4.3%)), MR mutations (total 28, 24.1%: U2AF1 (10, 8.6%), EZH2 (7, 6%), BCOR (3, 2.6%), ASXL1 (3, 2.6%), SF3B1 (2, 1.7%), SRSF2 (2, 1.7%), ZRSR2 (1, 0.9%), and STAG2 (1, 0.9%)), FLT3 (22, 19%), WT1 (16, 13.8%), RUNX1 (14, 12.1%), NOTCH1 (10, 8.6%), CEBPA (9, 7.8%), PHF6 (9, 7.8%), and TP53 (7, 6%). Other than BCR::ABL1 and KMT2A rearrangements, only fusions recurrent in &gt;1 cases were NUP98::NSD1 and PICALM::MLLT10 (2 each). Most common structural alteration was -7/del7(q) (7, 6%). Among the most recurrent genes, only WT1 (14/16) and CEBPA (8/9) showed preferential distribution among T/Myeloid MPALs (P=0.02). The MR mutations were uniformly distributed across the immunophenotypic subtypes (11/55 T/Myeloid, 4/17 B/Myeloid, 5/10 B/T) and age groups.
Outcome data were available for 88 patients, with a median follow-up of 25.5 months. Median OS and EFS of the entire cohort were 39.8 months and 27.8 months, respectively. Out of the 88 patients, 74 were treated with ALL-directed regimen and 14 patients received AML-directed therapy. No significant impact of age (P=0.75), sex (P=0.64), TLC (P=0.14), mutation burden (P=0.95) or WHO/immunophenotypic subgroups (P=0.69) was observed on OS or EFS. Presence of MR mutations were associated with significantly inferior OS (median OS 16.7 vs 55.1 months, HR = 2.02, 95% CI = 1.0-4.2, P=0.038) and showed a trend towards inferior EFS (median EFS 14.7 vs 40.1 months, HR = 1.9, 95% CI = 0.95-3.8, P=0.063). 19 out of 20 patients with MR mutations were treated with ALL-directed therapy. In particular, MR mutations in adults were predictive of inferior outcomes (median OS 21 vs 55.1 months, HR 2.5 , 95% CI = 1.0-6.0, P=0.038; median EFS 21 vs 28.4 months, HR 2.5, 95% CI = 1.1-6.5, P=0.031). CEBPA mutations demonstrated a trend towards superior outcome (median OS not reached vs 30.8 months, HR 0.2, 95% CI = 0.027-1.5, P=0.078; median EFS 56.8 vs 27.7 months, HR 0.34, 95% CI = 0.081-1.4, P=0.13). No other mutations showed impact on OS/EFS. EOI-MRD was positive in 63.9% (46/72) patients. EOI-MRD positivity predicted inferior outcome (median OS 30.1 vs 70.3 months, HR 3.6, 95% CI = 1.3-9.7, P=0.0065; median EFS 21 vs 56.8 months, HR 2.8, 95% CI = 1.2-6.3, P=0.011).
Conclusion
We provide genomic landscape of mutations in ALAL and show that MR mutations in adults and EOI-MRD positivity predict inferior outcome in these patients. These data may provide rationale for AML-directed therapy in adults with immunophenotype of ALAL demonstrating MR mutations, and support the inclusion of MR mutations as class-defining for AML in recent WHO/ICC classifications.