Escherich, Carolin S.

Abstract Background: Older adults with newly diagnosed ALL treated with conventional chemotherapy have poor survival due to high rates of toxic death and disease relapse. The Alliance A041703 trial (NCT0373981) tested chemotherapy-free treatment with the anti-CD22 antibody-drug conjugate inotuzumab ozogamicin (InO) followed by the anti-CD19-CD3 bifunctional T-cell engager blinatumomab (blina) without maintenance in older adults to reduce toxicity and increase efficacy of therapy for this patient (pt) population. We previously reported with 22-month (mo) median follow-up that the study met its primary endpoint with a 1-year (yr) event-free survival (EFS) of 75% (90% confidence interval [CI] 63-89% lower bound exceeding null rate of 10%; JCO 2023 41, 7006). Here we report secondary outcomes with 45-mo median follow-up: 3-yr EFS, 3-yr overall survival (OS), response, and relapse characteristics. Methods: Eligible pts were ≥60 yrs old with newly diagnosed, Ph-negative, CD22-positive (≥20% of blasts) B-cell ALL without planned allogeneic HCT (alloHCT), liver disease, or CNS involvement at enrollment. Induction IA was InO 0.8 mg/m2 day (d) 1, 0.5 mg/m2 d8, d15 IV on a 21d cycle. Pts with adequate cytoreduction (marrow blasts reduced ≥50% or marrow cellularity ≤20%) after IA went to IB if in CR/CRi (InO 0.5 mg/m2 d1, d8, d15 IV; 28d cycle) or IC if no CR/CRi (InO 0.8 mg/m2 on d1 and 0.5 mg/m2 on d8 and d15 IV; 28d cycle). Those without cytoreduction to IA started course II blina. Pts without events in IA/B/C received course II blina continuous IV (9 mcg/d x 7d then 28 mcg/d x 21d; 14d break; 28 mcg/d x28d). Pts with CR/CRi to InO received 2 more 28d cycles of blina; others received 3 more. CNS prophylaxis was methotrexate 15 mg intrathecal x 8 doses. EFS was time from therapy start to failure to achieve CR/CRh/CRi by the end of course II, progression, relapse, or death, whichever occurred first, censored at last follow up. OS was time from registration to death from any cause censored at the last known alive date. Results: The median age of the 33 treated pts was 71 yrs (range 60-84). Eight pts had prior therapy for alternate malignancy in CR for >2 yrs prior to enrollment (6 multiple myeloma, 1 non-Hodgkin lymphoma, 1 breast cancer). Median CD22 expression was 92% (range 21-100%) and 49% had NCCN poor-risk genetics. Five pts underwent alloHCT in first CR/CRi and 2 pts received POMP maintenance after early discontinuation of blina (1 toxicity, 1 physician choice). The CR/CRi rate after InO was 85% (1 early death/refractory, 2 refractory, 2 undetermined due to low marrow cellularity on remission assessment marrow) with CR/CRi rate of 97% by completion of course II blina. With 45 mo median follow-up, the estimated 3-yr EFS was 48% (95% CI 33-70%) with a median EFS of 31 mo (95% CI 17.4-not reached [NR]) and no events after 31 mo. Sixteen events occurred: 12 relapses, 1early death, and 3 deaths in remission (1 encephalopathy, 1 relapse of multiple myeloma, 1 pulmonary complications after alloHCT). No isolated CNS relapse has been reported. Among relapses, 10 had immunophenotyping. Four (40%) were CD19-negative, one of which was also CD22-negative. Among CD19-negative relapses, genetics were hypodiploid (2), Pax5alt (1), and ZNF384-like (1, also CD22-negative). TP53 mutation or loss was observed in 5/12 (42%) relapses. Thirteen pts died. The estimated 3-yr OS was 59% (95% CI 44-79%) with median OS NR (95% CI 32-NR) with no deaths after 36 mo. In univariate analysis, EFS and OS were not impacted by age, gender, genetic risk (NCCN), prior therapy for alternate malignancy, or alloHCT in first remission. EFS was better with higher CD22 expression at diagnosis (≥90% vs <90%, 3-yr EFS 60% vs 25%, P=0.044). EFS and OS were worse for detectable minimal residual disease (MRD) at any timepoint by ASO-PCR. Undetectable MRD after course IA (1 cycle InO induction) was associated with EFS with a 3-yr EFS of 52% vs 0% for detectable MRD (median NR vs 8 mo, hazard ratio [HR] 0.07, P=0.004) and a 3-yr OS of 100% vs 0% for detectable MRD (median NR vs 20 mo, HR 0.00, P=0.0014). Conclusions: Longer follow-up of chemotherapy-free treatment with InO and blina for older pts with newly diagnosed, Ph-negative, CD22-positive, B-cell ALL reveals durable remissions and survival. The A041703 regimen is a compelling option for first-line treatment of this pt population. Support: U10CA180821, U10CA18088; https://acknowledgments.alliancefound.org.

PURPOSE Older patients with ALL receiving conventional chemotherapy have poor survival due to toxic death and relapse. We hypothesized that a chemotherapy-free, targeted regimen using the anti-CD22 antibody-calicheamicin conjugate inotuzumab ozogamicin followed by the bispecific anti-CD19/CD3 T-cell engager blinatumomab would reduce toxic death and yield high rates of prolonged remission and survival. METHODS Eligible patients were age 60 years and older with untreated, Philadelphia chromosome (Ph)–negative, CD22-positive, B-cell ALL. Patients received up to two cycles of inotuzumab ozogamicin followed by four or five cycles of blinatumomab with intrathecal methotrexate CNS prophylaxis. The primary end point was 1-year event-free survival (EFS). RESULTS The 33 eligible patients had a median age of 71 years (range, 60-84) and a median CD22 expression of 92% (range, 21%-100%). Eight (24%) had previous chemotherapy or radiation for other cancers, six for multiple myeloma. The composite complete remission rate was 85% after two cycles of inotuzumab ozogamicin and 97% by the end of two cycles of blinatumomab. At a median follow-up of 30 months, the 1-year EFS and overall survival were 75% (95% CI, 61 to 92) and 85% (95% CI, 73 to 98), respectively. EFS was shorter with lower CD22 expression or detectable measurable residual disease at any time point. CONCLUSION Inotuzumab ozogamicin then blinatumomab without maintenance chemotherapy in older patients with untreated, Ph-negative, CD22-positive, B-cell ALL yields a high remission rate and excellent EFS. Given the lack of standard, safe, and effective therapies in this population, the regimen should be considered a standard treatment option.

Abstract Inotuzumab ozogamicin (InO) is an antibody-calicheamicin conjugate with high efficacy in lymphoid malignancies. It targets the B-cell surface protein CD22, which is expressed in most B-cell acute lymphoblastic leukemia (B-ALL) cases, albeit with variable intensity. However, factors governing CD22 expression and thus leukemia sensitivity to InO remain incompletely understood. Using multiomic characterization of 196 human B-ALL samples, coupled with ex vivo InO sensitivity profiling, we showed that early leukemia differentiation arrest at the pre–pro-B stage is associated with resistance to InO. Screening of 1639 transcription factor genes identified early B-cell factor 1 (EBF1) as a key regulator of CD22 expression (false discovery rate of 7.1 × 10−4). When comparing the assay for transposase-accessible chromatin with sequencing profiling results of the most InO-sensitive and -resistant cases (50% lethal concentration <10th vs >90th percentile, n = 18), the binding motif for EBF1 was strikingly enriched in regions with differential open chromatin status (P = 8 × 10−174). CRISPR interference targeting EBF1 binding sites at the CD22 locus led to an ∼50-fold reduction in cell surface CD22 expression and, consequently, an ∼22-fold increase in InO resistance in ALL cell lines. Interestingly, within BCR::ABL1 ALL, we observed intrasubtype heterogeneity linked to EBF1 transcriptional downregulation (P = 1.1 × 10−15) and/or somatic alteration (P = .004), which led to reduced CD22 expression (P = 8.3 × 10−11) and ex vivo and in vivo resistance to InO. Collectively, these findings point to the direct impact of EBF1 on CD22 expression during B-cell development, which, in turn, contributes to interpatient variability in InO response, even within the same subtype of B-ALL.

Abstract Inotuzumab ozogamicin (InO) is an antibody-calicheamicin conjugate with striking efficacy in B-cell acute lymphoblastic leukemia (B-ALL). However, there is wide interpatient variability in treatment response, and the genetic basis of this variation remains largely unknown. Using a genome-wide CRISPR screen, we discovered that the loss of DNA nucleotidylexotransferase (DNTT) is a primary driver of InO resistance. Mechanistically, the downregulation of DNTT attenuated InO–induced DNA damage response, cell cycle arrest, and mitochondrial apoptotic priming, thereby ultimately leading to leukemia resistance to InO. Ex vivo leukemia InO sensitivity was highly associated with DNTT expression in ALL blasts with substantial intraleukemia heterogeneity as revealed by single-cell RNA sequencing. Among patients with B-ALL enrolled in the Children's Oncology Group trial AALL1621, we observed consistent DNTT downregulation in residual blasts following InO treatment. The selection of DNTT-low blasts by InO therapy was also recapitulated in vivo using patient-derived xenograft models. Collectively, our data indicate that DNTT is a key regulator of calicheamicin response in leukemia and thus a potential biomarker for individualizing InO therapy in B-ALL.

Background: Inotuzumab Ozogamicin (InO) is an antibody-cytotoxin conjugate with high activity in B-cell acute lymphoblastic leukemia (B-ALL). InO targets the B-cell surface protein CD22, which is expressed in most B-ALL cases, albeit with great variability in expression levels. Emerging evidence suggest that this variation is associated with the stage of leukemia differentiation arrest [PMID: 21348573]. However, the exact relationship between CD22 expression and leukemia sensitivity to InO, particularly in the context of leukemia developmental state, remains unclear. Furthermore, our understanding of the molecular mechanisms regulating CD22 transcription in B-ALL is still incomplete. Methods: To comprehensively investigate the genomic basis of variability in leukemia CD22 expression and thus B-ALL sensitivity to InO, we performed a multi-omic characterization of 196 primary human B-ALL samples, using whole transcriptome sequencing (RNAseq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), coupled with ex vivo profiling of InO sensitivity. Additionally, genomic editing studies were performed in Nalm6 and REH B-ALL cell lines, and patient derived xenograft (PDX) models were used for in vitro and in vivo validations. Results: Applying digital cytometry to leukemia bulk RNAseq data to infer the B-ALL developmental state, we found that blast cells with early Pre-pro-B-like signature had low levels of CD22 expression and were resistant to InO. As B-cell differentiation is controlled by stage-specific transcription factors (TFs), we performed a targeted CRISPR library screen of 1,639 TF genes and identified EBF1 as a key activator of CD22 expression (FDR=7.1x10-4). In primary B-ALL samples, EBF1 expression was significantly correlated with CD22 expression (R=0.69, P<0.0001) and leukemia response to InO (measured as LC50, R=-0.42, P<0.0001). In addition, TF footprint analysis of leukemia ATAC-seq data identified EBF1 as the most differentially activated TF comparing InO-sensitive and -resistant B-ALL cases (P=8x10-174). Blocking the EBF1 binding sites within the CD22 promoter by CRISPR interference resulted in a 50-fold and 49-fold decrease in cell surface CD22 expression, and a 22-fold and 25.7-fold increase in InO LC50 for Nalm6 and REH, respectively, confirming the direct impact of EBF1 on CD22 transcription and InO sensitivity. Because BCR::ABL1-positive ALL showed the greatest variability in InO sensitivity among all B-ALL subtypes, we further investigate the role of the EBF1/CD22 axis on InO sensitivity within this subtype. Unsupervised clustering of gene expression profiles revealed two distinct groups, namely BCR::ABL1 ALL with multilineage (BCR::ABL1-M) or lymphoid only (BCR::ABL1-L) involvement [PMID: 38153913]. Compared to BCR::ABL1-L, BCR::ABL1-M displayed markedly reduced CD22 expression (P=8.3×10-11) and resistance to InO (P=1.9×10-4), likely due to the lower expression and frequent somatic alteration of EBF1. Finally, we established PDX models for representative cases of BCR::ABL1-M and BCR::ABL1-L. Strikingly, InO treatment did not affect leukemia progression in the BCR::ABL1-M PDX model, whereas the BCR::ABL1-L PDX model achieved complete remission and has remained leukemia free to date. Conclusion: Collectively, these data demonstrate the direct impact of EBF1 on CD22 expression during B-cell development, and explain the heterogeneity of InO sensitivity, even within the same molecular B-ALL subtype.