Lineage switch (LS), defined as the immunophenotypic transformation of acute leukemia, has emerged as a mechanism of relapse after antigen-targeted immunotherapy, which is associated with dismal outcomes. Through an international collaborative effort, we identified cases of LS after a host of antigen-targeted therapies (eg, CD19, CD22, CD38, and CD7), described how LS was diagnosed, reviewed treatment approaches, and analyzed overall outcomes for this form of postimmunotherapy relapse. Collectively, 75 cases of LS were evaluated, including 53 (70.7%) cases of B-cell acute lymphoblastic leukemia (B-ALL) transforming to acute myeloid leukemia (AML), 17 (22.7%) cases of B-ALL transforming to mixed phenotypic acute leukemia (MPAL)/acute leukemias of ambiguous lineage (ALAL), and 5 (6.7%) cases of rare LS presentation (ie, T-cell ALL to AML). An additional 10 cases with incomplete changes in immunophenotype, referred to as “lineage drift” were also described. With a primary focus on the 70 cases of LS from B-ALL to AML or MPAL/ALAL, LS emerged at a median of 1.5 months (range, 0-36.5) after immunotherapy, with 81.4% presenting with LS within the first 6 months from the most proximal immunotherapy. Although most involved KMT2A rearrangements (n = 45, 64.3%), other rare cytogenetic and/or molecular alterations were uniquely observed. Treatment outcomes were generally poor, with remission rates of <40%. The median overall survival after LS diagnosis was 4.8 months. Outcomes were similarly poor for those with rare immunophenotypes of LS or lineage drift. This global initiative robustly categorizes lineage changes after immunotherapy and, through enhanced understanding, establishes a foundation for improving outcomes of LS.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis related to refractory/relapsing diseases, raising the need for new targeted-therapies. Activating mutations of the IL7-receptor pathway genes (IL7Rp) play a proven leukemia-supportive role in T-ALL. JAK-inhibitors such as ruxolitinib have recently demonstrated preclinical efficacy. However, prediction markers for sensitivity to JAK-inhibitors are still lacking. Herein, we show that IL7R (CD127) expression is more frequent (~70%) than IL7Rp-mutations in T-ALL (~30%). We compared the so-called non-expressers (no IL7R-expression/IL7Rp-mutation), expressers (IL7R-expression without IL7Rp-mutation) and mutants (IL7Rp-mutations). Integrative multi-omics analysis outlined IL7R-deregulation in virtually all T-ALL subtypes, at the epigenetic-level in non-expressers, genetic-level in mutants, and post-transcriptional level in expressers. Ex-vivo data using primary-derived xenografts support that IL7Rp is functional whenever the IL7R is expressed, regardless of the IL7Rp mutational status. Consequently, ruxolitinib impaired T-ALL survival in both expressers and mutants. Interestingly, we show that expressers displayed ectopic IL7R-expression and IL7Rp-addiction conferring a deeper sensitivity to ruxolitinib. Conversely, mutants were more sensitive to venetoclax than expressers. Overall, combination of ruxolitinib and venetoclax resulted in synergistic effects in both groups. We illustrate the clinical relevance of this association by reporting achievement of complete remission in two patients with refractory/relapsed-T-ALL. This provides proof of concept for translation of this strategy into clinics as bridge to transplant. Altogether, IL7R-expression can be used as a biomarker for sensitivity to JAK-inhibition, thereby expanding the fraction of T-ALL patients eligible to ruxolitinib up to nearly ~70% of T-ALL.

Purpose: To assess the impact of PHF6 alterations on clinical outcome and therapeutical actionability in T-cell acute lymphoblastic leukemia (T-ALL). Experimental Design: We described PHF6 alterations in an adult cohort of T-ALL from the French trial Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 and retrospectively analyzed clinical outcomes between PHF6-altered (PHF6ALT) and wild-type patients. We also used EPIC and chromatin immunoprecipitation sequencing data of patient samples to analyze the epigenetic landscape of PHF6ALT T-ALLs. We consecutively evaluated 5-azacitidine efficacy, alone or combined with venetoclax, in PHF6ALT T-ALL. Results: We show that PHF6 alterations account for 47% of cases in our cohort and demonstrate that PHF6ALT T-ALL presented significantly better clinical outcomes. Integrative analysis of DNA methylation and histone marks shows that PHF6ALT are characterized by DNA hypermethylation and H3K27me3 loss at promoters physiologically bivalent in thymocytes. Using patient-derived xenografts, we show that PHF6ALT T-ALL respond to the 5-azacytidine alone. Finally, synergism with the BCL2-inhibitor venetoclax was demonstrated in refractory/relapsing (R/R) PHF6ALT T-ALL using fresh samples. Importantly, we report three cases of R/R PHF6ALT patients who were successfully treated with this combination. Conclusions: Overall, our study supports the use of PHF6 alterations as a biomarker of sensitivity to 5-azacytidine and venetoclax combination in R/R T-ALL.

Given the poor outcome of refractory and relapsing T-cell acute lymphoblastic leukemia (T-ALL), identifying prognostic markers is still challenging. Using single nucleotide polymorphism (SNP) array analysis, we provide a comprehensive analysis of genomic imbalances in a cohort of 317 newly diagnosed patients with T-ALL including 135 children and 182 adults with respect to clinical and biological features and outcomes. SNP array results identified at least 1 somatic genomic imbalance in virtually all patients with T-ALL (∼96%). Del(9)(p21) (∼70%) and UPD(9)p21)/CDKN2A/B (∼28%) were the most frequent genomic imbalances. Unexpectedly del(13)(q14)/RB1/DLEU1 (∼14%) was the second most frequent copy number variant followed by del(6)(q15)/CASP8AP2 (∼11%), del(1)(p33)/SIL-TAL1 (∼11%), del(12)(p13)ETV6/CDKN1B (∼9%), del(18)(p11)/PTPN2 (∼9%), del(1)(p36)/RPL22 (∼9%), and del(17)(q11)/NF1/SUZ12 (∼8%). SNP array also revealed distinct profiles of genomic imbalances according to age, immunophenotype, and oncogenetic subgroups. In particular, adult patients with T-ALL demonstrated a significantly higher incidence of del(1)(p36)/RPL22, and del(13)(q14)/RB1/DLEU1, and lower incidence of del(9)(p21) and UPD(9p21)/CDKN2A/B. We determined a threshold of 15 genomic imbalances to stratify patients into high- and low-risk groups of relapse. Survival analysis also revealed the poor outcome, despite the low number of affected cases, conferred by the presence of chromothripsis (n = 6, ∼2%), del(16)(p13)/CREBBP (n = 15, ∼5%) as well as the newly-identified recurrent gain at 6q27 involving MLLT4 (n = 10, ∼3%). Genomic complexity, del(16)(p13)/CREBBP and gain at 6q27 involving MLLT4, maintained their significance in multivariate analysis for survival outcome. Our study thus demonstrated that whole genome analysis of imbalances provides new insights to refine risk stratification in T-ALL. This trial was registered at www.ClinicalTrials.gov as #NCT00222027 and #NCT00327678, and as #FRALLE 2000T trial.

Methods: We used a patient-specific induced pluripotent stem cell (iPSC) line treated with the mutagenic agent N-ethyl-N-nitrosourea (ENU). Genomic instability was validated using γ-H2AX and micronuclei assays and CGH array for genomic events. Results: An increased number of progenitors (x5-Fold), which proliferated in liquid cultures with a blast cell morphology, was observed in the mutagenized condition as compared to the unmutagenized one. CGH array performed for both conditions in two different time points reveals several cancer genes in the ENU-treated condition, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Transcriptome GEO-dataset GSE4170 allowed us to associate 125 of 249 of the aberrations that we detected in CML-iPSC with the CML progression genes already described during progression from chronic and AP to BC. Among these candidates, eleven of them have been described in CML and related to tyrosine kinase inhibitor resistance and genomic instability. Conclusions: These results demonstrated that we have generated, for the first time to our knowledge, an in vitro genetic instability model, reproducing genomic events described in patients with BC.