PICALM::MLLT10 may indicate a new subgroup of acute leukemias with miscellaneous immunophenotype and poor initial treatment response but showing sensitivity to venetoclax

Abstract The PICALM::MLLT10 fusion gene is a rare but recurrent event in acute leukemia (AL) associated with poor prognosis. It is still confused whether PICALM::MLLT10 can solely correspond to acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) or acute leukemias of ambiguous lineage (ALAL). Here, we reported a series of PICALM::MLLT10 positive AL patients with miscellaneous immunophenotype including T‐ALL, ALAL, AML, and B‐ALL, complex karyotype, half of extramedullary disease (EMD), frequently concomitant PHF6 mutation, and poor initial treatment response to standard chemotherapy aiming to different immunophenotype, but showing sensitivity to combining chemotherapy especially integrated with venetoclax, suggesting this fusion gene may indicate a new subgroup of AL. Eighteen PICALM::MLLT10 positive patients of 533 AL patients (18/533, 3.4%) were identified by RNA sequencing in our center. We found PICALM::MLLT10 positive AL showing miscellaneous immunophenotype, higher expression of leukemic stemness genes and lower expression of biomarkers of venetoclax resistance, more extramedullary involvement, and especially poor response to conventional induction chemotherapy, but may benefit from venetoclax as well as low‐dose Ara‐C, granulocyte colony‐stimulating factor (G‐CSF), and anthracyclines combination chemotherapy. Sequential hematopoietic stem cell transplantation (HSCT) after chemotherapy combined with venetoclax may further improve long‐term survival in AL patients with complete remission (CR) even measurable residual disease (MRD) positive.

Here, we reported a series of PICALM::MLLT10 positive AL patients with unique clinical characteristic and treatment response, suggesting this fusion gene may indicate a new subgroup of AL.

CASE SERIES PRESENTATION
Eighteen PICALM::MLLT10 positive patients of 533 AL patients    2) and CAG or IAG + VEN (Patients 2 and 10, Table 2 and Figure S1G).In addition, two patients including one AML and one ETP-ALL also achieved CR after salvage venetoclax application (Patients 16 and 17, Table 2).In the remaining four patients, one patient who was failure to initial Hyper-CVAD A/B regimen also dramatically achieved CR after the more Hyper-CVAD A regimen combined with venetoclax (Patient 13, Table 2), one patient achieved CR while another still NR with both receiving CLAG (cladribine, cytarabine, G-CSF) regimen (Patients 1 and 4, Table 2), and the B/T MPAL patient still achieve no any remission even after FLT3 inhibitor gilteritinib and blinatumomab application (Patient 7, Table 2).Subsequently 11 patients (nine CR, two with refractory disease) received allogenic hematopoietic stem cell transplantation (allo-HSCT), and all the CR patients survived well after HSCT (follow-up 3-24 months, median 15 months) without relapse, while two NR patients died soon after transplantation because of severe complications (Patients 1 and 7, Table 2).

DISCUSSION
In our patients' series, we have shown that PICALM::MLLT10 positive AL is associated with unique biological characteristic such as miscel- laneous immunophenotype including T-ALL, ALAL, AML, and B-ALL, complex karyotype and frequently concomitant PHF6 mutation.In addition, EMD can be found in half of the total patients.In terms of treatment, this patient group was found to be shown poor initial treatment response to standard chemotherapy but sensitivity to combining chemotherapy especially with venetoclax introduction.We further analyzed the possible treatment mechanism of venetoclax in this patient group and found that BCL-2 itself was highly expressed, indicating that BCL-2 inhibitor should be effective.While MSI2 and HOPX as two known myeloid stemness genes were shown high expression, HOXA9 as target gene directly regulated by PICALM::MLLT10 was also shown high expression, which further indicated that these patients with an earlier leukemic stem cell phenotype can benefit from venetoclax [10].In addition, the lower expression of CD14 and LILRA/B family as biomarkers of venetoclax resistance also verified the efficacy of venetoclax in this subgroup patients [11] (Figure 1C).showed that the cell of origin of leukemia with PICALM::MLLT10 is stem or very early multipotent cell, but not B-cell lineage with a phenotype similar to LSC [12].G-CSF can prime G0/G1 phase LSC into the S phase, which may help the leukemia cells sensitivity to LDAC and anthracyclines [13,14].The above study was also align with our findings.
In addition, venetoclax had been reported eradicating LSCs of AML patients by disrupting the mechanisms of cellular energy metabolism [15].
On the other hand, PHF6 and JAK3 mutations have been confirmed to cooperate and drive T-ALL progression [16], which is consistent with our study showing PHF6 and JAK3 mutations ranking 1 and 2 individually.Furthermore, PICALM::MLLT10 positive AL is clinically characterized by extramedullary involvement [4], also as showing in our study, indicating more aggressive and refractory clinical features.
If extramedullary lesion is persisting, the subsequent allo-HSCT will be delay even though measurable residual disease (MRD) of bone marrow is negative.Moreover, CD7 chimeric antigen receptor (CAR)-T cell [17] or gemtuzumab ozogamicin (GO) may also serve as salvage therapy for patients especially failure of LDAC, G-CSF, and anthracyclines combination chemotherapy as well as venetoclax, but all of these speculations need more clinical data.
Taken together, our data suggested PICALM::MLLT10 positive AL showing miscellaneous immunophenotype, higher expression of leukemic stemness genes and lower expression of biomarkers of venetoclax resistance, more extramedullary involvement, and especially poor response to conventional induction chemotherapy which is alongside with [18], but may benefit from venetoclax as well as LDAC, G-CSF, and anthracyclines combination chemotherapy.Sequential HSCT after chemotherapy combined with venetoclax may further improve long-term survival in AL patients with CR even MRD positive.

( 18 /
533, 3.4%) were identified by RNA sequencing (RNA-seq) in our center from July 2020 to September 2023, including five female and 13 male, with a median age of 32 years (16-50 years).These patients were newly diagnosed AL according to bone marrow morphology and immunology including six ALAL (6/14), seven T-ALL [six early T-cell precursor ALL (ETP-ALL), one cortical T-ALL] (7/27), three AML (3/369), one B-ALL with aberrant expression of myeloid antigen (1/112), and one B/T MPAL (1/11 remission (NR).These NR patients including five ALAL (5/5), four T-ALL (4/6), one AML (1/2), and one B/T MPAL (1/1) subsequently received salvage chemotherapy.It is worth mentioning that five of them (5/11) received combined chemotherapy regimen including LDAC, G-CSF, and anthracyclines such as aclarubicin or idarubicin or homoharringtonine (CAG or IAG or HAG), and two of five patients also further received combination therapy with venetoclax (CAG or IAG + VEN).Finally, all the total five patients dramatically achieved CR (100%, 5/5) after CAG or HAG alone (Patients 3, 8, and 9, Table It has already been reported that the prognosis of PICALM::MLLT10positive T-ALL depends on the stage of leukemia cell maturation arrest.Mature TCRγδ + PICALM::MLLT10 positive T-ALL responds well to standard treatment, whereas TCR-PICALM::MLLT10 positive T-ALL has a strikingly inferior outcome[1], suggesting that an earlier stage of leukemia stem cells (LSCs) is insensitive and resistant to conventional therapy.In our study, these patients showed poor response to initial AML or ALL-like chemotherapy, but especially sensitive to venetoclax F I G U R E 1 PICALM::MLLT10 fusion protein pattern diagram and expression of representative genes including BCL-2, stemness genes, and genes associated with venetoclax (VEN) resistance in 14 PICALM::MLLT10 positive patients.(A) The protein domains of PICALM and MLLT10, fusion gene breakpoints, and their corresponding patient numbers.The numbers inside the circle indicate the number of patients corresponding to that breakpoint fusion pattern.Among them, the PICALM fusion sites were mainly concentrated in exon 17 (n = 6) and exon 19 (n = 8).The MLLT10 fusion site is most common with exon 4 (n = 8).(B) The breakpoints of MLLT10 fusion site, corresponding to exon 4, exon 6, exon 9, and exon 10, respectively.(C) The ordinate is the expression level of the corresponding gene (transcripts per million (TPM) value is quantified).MSI2 and HOPX are two known myeloid stemness genes (high expression in early hematopoietic stem cells).HOXA9 is a target gene known to be regulated by PICALM::MLLT10.The CD14 and LILRA/B gene families are biomarkers (high expression in monocytes) known to be associated with VEN resistance.The results showed higher expression of BCL2 and myeloid stem genes but lower expression of VEN resistance-related biomarkers in PICALM::MLLT10 positive patients.aswell as LDAC, G-CSF, and anthracyclines combination chemotherapy despite the complexity of immunophenotyping and cytogenetics at the first diagnosis.One possible reason may be that PICALM::MLLT10 fusion occur in the early stage of hemopoietic stem cells.Dutta et al.