Higher Complete Remission Rate Research Articles

CD58 Could be As a Leukemic Marker in Relapsed/Refractory B-ALL Patients after Multiline Therapies

It has been verified that CD58, a member of the immunoglobulin superfamily, is expressed in B-cell lymphoblastic leukemia (B-ALL) at a higher level and is a marker of minimal residual disease in B-ALL. A study reported that CD58 was expressed in 99.4% of B-ALL cases at diagnosis. Nevertheless, little is known regarding CD58 expression in relapsed/refractory (r/r) B-ALL patients having received multiline therapies especially chimeric antigen receptor (CAR) T-cells. Although CD19-specific CAR-T therapy resulted in high complete remission (CR) rates, some patients relapsed after CAR-T and part of them showed CD19 loss, under this circumstance, additional B-cell antigen specifically expressed on the B-ALL cells could help us to identify the leukemia cells. In this study, we investigated the CD58 expression in r/r B-ALL patients before CAR-T, and the influence of CAR-T cells on CD58 antigen of lymphoblasts in patients failed or relapsed after CAR-T. CD58 on leukemic cells of r/r B-ALL patients has been routinely detected by flow cytometry in our center. Data of r/r B-ALL patients were collected from May 2019 through May 31, 2022. Samples were detected on an 8-color BD FACSCanto II flow cytometer, and analyzed by FACS Diva software from BD or Kaluza software from Beckman Coulter. Antigen full expression, partial expression and no expression were defined as >80%, 20%-80% and <20% of gated cells displaying the antigen of interest, respectively. A total of 274 r/r B-ALL patients planned to be administered CD19 CAR-T cells were assayed CD58 antigen before CAR-T, all of them had received multiline chemotherapies, 61 (22.3%) underwent allogeneic hematopoietic cell transplant (allo-HCT) and 6 cases had a history of antibody (CD20, CD19 or CD22) treatment. The median age was 12 years (range, 1-75 years), including 97 adults and 177 children younger than 18 years. Among all 274 patients, 228 (83.2%, 228/274) showed CD58 expression, consisting of 180 (78.9%, 180/228) cases with full expression and 48 (21.1%, 48/228) with partial expression; whereas 46 (16.8%, 46/274) patients were CD58-negative, demonstrating that CD58 was expressed on the majority (83.3%) of r/r B-ALL patients after treated by multiple intensive chemo drugs and even allo-HCT. Then, we observed the influence of CAR-T cells on the expression of CD58 antigen of lymphoblasts. We followed-up 57 patients who had CD58 expression before CD19 CAR-T but failed or relapsed after CAR-T. Of them, 45.6% (26/57) cases received the further treatment as a consolidation following CD19 CAR-T (CD22 CAR-T, 14; allo-HCT, 12). Among 57 patients, 10 were CD58-negative while 47 cases still showed CD58-positive, the frequency of CD58 expression was 82.5% (47/57) after treated by CAR-T cells and allo-HCT. When we excluded the 26 patients who received following CD22 CAR-T or allo-HCT, and only considered the 31 patients exposed to one-time CD19 CAR-T cells, the CD58 expression rate was 87.1% (27/31). It was reported that CD58 loss in tumor cells induced resistance to CAR-T therapy in cell lines and mouse models. We therefore analyzed the influence of CD58 expression on the treatment response of CAR-T cells. Among 274 r/r B-ALL patients received CD19 CAR-T, 6 were unable to evaluate the treatment response due to death or hospital discharge within 15 days after cell infusion, the remaining 268 patients had evaluation data including 223 cases with CD58 expression (full or partial expression) and 45 without CD58 expression. After CD19 CAR-T therapy, the CR (including MRD+CR) rate in patients with CD58 expression was 91.9% (205/223) while it was 95.6% (43/45) in patients without CD58 expression, indicating that CD58 negativity was irrelevant to CAR-T treatment failure in r/r B-ALL. In conclusion, CD58 antigen was expressed in the majority (83.2%) of r/r B-ALL cells without exposing to CAR-T treatment, and still expressed on the lymphoblasts of most patients (>80%) failed or relapsed after CAR-T, therefore, it could be as a leukemic marker for identifying tumor cells in r/r B-ALL patients who have received multiline therapies including allo-HCT and CAR-T. Additionally, CD58-negative patients achieved a similar high CR rate as CD58-positive patients did after CD19 CAR-T.

A Phase Ib Study of the Safety and Efficacy of Venetoclax When Combined with Escalating Doses of Omacetaxine in Patients with Relapsed/Refractory Acute Myeloid Leukemia Failing Treatment with Venetoclax-Containing Regimens (VEN-OM)

Background: Venetoclax (VEN) combined with hypomethylating agents (HMA) are increasingly used as first line (1L) treatment in AML patients (pt) intolerant of intensive chemotherapy, due to the high complete remission (CR) rate (66.4%), tolerance of therapy and a median duration CR of 17.5 months. However, >60% of pts discontinued VEN/HMA (n=286) during the VIALE-A trial, mostly due to disease progression (DiNardo et al.N Engl J Med2020; 383:617-29). Outcomes for such pts receiving 2L and 3L therapies for relapsed/refractory (R/R) AML are poor and may be due to development of resistance to BCL-2 inhibition through mechanisms including the upregulation of the anti-apoptotic proteins BCL-XL or MCL1. In vitro studies demonstrate that VEN-resistant leukemia stem cells (LSC) are re-sensitized to BCL-2 inhibition when MCL-1 is also inhibited (Ramsey et al. Cancer Discov 2018; 8:1566-81). Novel MCL-1 inhibitors are thus in phase 1 clinical trials in R/R AML (e.g. NCT02675452), however there have been concerns for cardiotoxicity. Metabolic studies (Stevens et al. Nat Commun 2018: 9, 3694) indicate that the CD123+ malignant stem cell compartment of pts with high-grade MDS rely on upregulation of the protein synthesis machinery and mitochondrial oxidative phosphorylation (OxPhos). Omacetaxine (OM), a protein translation inhibitor both inhibits OxPhos and down-regulates (other) proteins with a short half-life such as MCL1. Treatment of a xenograft AML model with a combination of OM and VEN indicates in vivo synergistic effects in impairing mitochondrial respiration, ATP production, BCL-2 inhibition and MCL-1 downregulation (Fig 1A). We hypothesize this combination will prove effective in pts with R/R AML including those resistant to VEN-containing regimens. A similar drug combination is being studied by Dinardo et al. in R/R AML and MDS pts with RUNX1 mutations. Methods: VEN-OM is a single arm, open-label, phase Ib dose-escalation study in R/R AML pts (18-75y). The trial utilizes an innovative Bayesian Optimal Interval (BOIN) adaptive design (Yuan et al. Clin Cancer Res 2016)to determine the maximum tolerated dose (MTD), with a target dose-limiting toxicity (DLT) rate of 2%, and 4 pre-specified dose levels of OM, involving 24-30 pts. Major eligibility: ECOG 0-2, adequate organ function, R/R AML having progressed on ≥ 1L of therapy, which must have included a VEN-containing regimen. Exclusion criteria: Prior use of OM, peripheral blast count > 25 × 109/L, unresolved ≥ grade 2 clinically significant nonhematologic toxicities from prior anticancer therapy, unresolved ≥ grade 2 disseminated intravascular coagulation (DIC), other active malignancy within 1 year before study entry, or active infections. Induction Phase: Beginning with the first pt treated at the lowest dose of OM (0.625 mg/m2 q12h), OM dose escalation and de-escalation will involve comparison of the observed DLT rate at the current dose with a pair of fixed, pre-specified boundary rates. After a VEN ramp-up phase (days 1-3), the VEN dose is fixed at 400mg daily on days 4-28. OM doses are given days 1-7. OM dose will be escalated for each subsequent pt until the first DLT occurs, whereupon the current dose cohort is expanded to 3. In simulations of these conditions with a target DLT rate of 20%, the percentage correct selection of the MTD is between 60% with enrollment of 20 pts and 70% with 24 pts. Consolidation Phase: OM dose will be as per induction dose for days 1-7 of each cycle. VEN dose is 400mg days 1-21. Regimen comprises 3 cycles: Induction cycle x 1 and Consolidation cycles x 2 (Fig 1B). Primary endpoint is MTD of the combination of VEN and OM. Secondary endpoints include overall response rate after 3 cycles, characterization of adverse effects, and estimates of EFS and OS at 12 months. Exploratory objectives include measurement of validated markers of early renal injury including urine TIMP-2 x IGFBP7 and KIM-1 to identify pts susceptible to OM-induced nephrotoxicity and potential correlation with OM efficacy. Our previous experience with OM added to 7+3 ("AML-02", ASH 2022 abstract submitted) suggests that quantification of BM leukemia blast protein synthesis rates (Walker et al. Clin Cancer Res 2021, 27:819-30) and 'BH3 profiling' at presentation predicts outcomes, and these correlative studies will also be performed. The study recently opened, with the first patient completing induction without complications. Trial registered as (NCT04926285). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax (VEN) Plus Intensive Chemotherapy (IC) with FLAG-IDA in Patients (Pts) with Newly Diagnosed (ND) and Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Despite the high complete remission (CR) rates achieved with IC, relapse rates remain high underscoring the need for novel combination regimens capable of inducing deeper remissions via eradicating measurable residual disease (MRD). Recently, several studies have shown promising clinical activity of VEN when combined with different IC regimens in pts with ND AML with composite CR rates (CRc) above 90% and MRD-ve rates exceeding 80% (Wang 2022; Kadia 2021). FLAG-IDA + VEN has also shown significant clinical activity in pts with ND AML with a CRc rate of 89% and MRD-ve remission rate of 93% (DiNardo 2022). Methods: We conducted a single-center retrospective study to assess the clinical activity of FLAG-IDA + VEN in pts with AML. During induction, VEN was initially given for 14 days which was later on reduced to 7 days due to encouraging results by Kadia et al. Standard cytogenetic (CN) analysis was performed and molecular data was obtained using a 40-gene NGS platform. Responses were assessed using the modified International Working Group response criteria and included CR, CR with incomplete count recovery (CRi), morphologic leukemia-free state (MLFS), and partial remission (PR). Time-to-event analyses were estimated using the Kaplan-Meier method and compared using the log rank test. Results: From March 2020 to May 2022, 24 pts with AML (17 ND and 7 R/R) were treated using FLAG-IDA + VEN (Table 1). Median age was 48 years (range, 21-68) with male predominance (67%). Among the 17 pts with ND AML (8 de novo, 5 AML-MRC, 2 treated-secondary, 2 therapy-related), 3 had extramedullary disease (EMD). The most common ELN CN risk groups were complex (N=5), KMT2A-rearranged (N=4), other adverse (N=3), and diploid (N=3). Eight pts (47%) harbored a RAS mutation. Median time to initiation of therapy was 5 days (range, 2-14). Twelve pts (71%) [KMT2A-rearranged, N=4; complex CN, N=3; diploid, N=3; other adverse, N=1; other intermediate, N=1] achieved CR/CRi including 1 pt with EMD. Median time to ANC (≥ 500) and platelet (≥ 50,000) recovery were 29 days (range, 19- 42) and 25 days (range, 17- 42), respectively. Median number of consolidation cycles given were 1.5 (range, 1- 5). MRD status was available in 6 out of 12 pts and all 6 pts achieved MRD negativity using flow cytometry (<10-3). Median follow-up among ND AML pts was 4.3 months (mths) (range, 0.7- 13.2). Among the 12 responders: (1) 3 pts received allo-SCT [KMT2A-rearranged, N=2; (-7), N=1] after 7, 4, and 3 mths, respectively, and remain alive in CR 10+, 7.5+, and 3+ mths, respectively, after initiation of therapy, (2) 6 pts with ELN favorable (N=3), intermediate (N=2), and adverse (N=1) risk AML remain in CR/CRi for 6+, 4+, 1+, 3+, 2+, and 2+ mths, respectively, (3) 3 pts with ELN adverse risk AML (including 1 pt with de novo MLLr AML and EMD) achieved CR and relapsed after 0.7, 1.7, and 7.6 mths, respectively. One pt died due to progressive disease (PD) 4.7 mths after relapse and the remaining 2 pts are receiving salvage therapy and alive 4.3+ and 5.2+ mths after relapse, respectively. All nonresponders (N=5) had ELN adverse risk AML including 2 pts with EMD and 2 pts with treated secondary AML. All 5 nonresponders died due to PD including 3 pts who had no response to HMA+VEN based salvage therapy. Among the 7 pts with R/R AML, 6 were in salvage 1 and 1 pt received prior allo-SCT. Responses were observed in all pts: 4 CR, 1 CRi, 1 MLFS, and 1 PR. Median time to count recovery in R/R AML pts was 33 days (range, 24-78). Median follow-up in R/R AML pts was 6.2 mths (range, 1.5- 15.6). Of the 5 pts who achieved CR/CRi, 4 were bridged to allo-SCT and 1 pt died in CR after 3 cycles of consolidation due to sepsis. Of the 4 SCT recipients: (1) 2 pts relapsed 2 mths after allo-SCT and died 4 and 6.5 mths, respectively, after allo-SCT; (2) 2 pts (KMT2A-rearranged and NPM1mut) remain alive in CR for 8+ and 14+ mths, respectively. Two pts with complex CN achieved MLFS and PR after 1 cycle of therapy and died due to PD and fungal pneumonia after 5.3 and 1.5 mths of starting therapy, respectively. Median overall survival (OS) for pts with ND AML and R/R AML were 13.2 mths (95% CI: Not reached) and 6.2 mths (95% CI: 5-7), respectively (p=0.62; Figure 1). Among pts with ND AML, median OS was not reached among responders and was 2.6 mths in non-responders (p=0.002). Conclusion: FLAG-IDA + VEN is a feasible and active regimen in AML. Longer follow-up and larger multicenter studies are needed to confirm the efficacy of this regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone Marrow Fibrosis Is Associated with Non-Response to CD19 CAR-T Therapy

Background: Despite the use of CD19-directed CAR-T cell therapy (CAR19) for treatment of relapsed/refractory (R/R) B-acute lymphoblastic leukemia (B-ALL), we cannot predict patient response before infusion. While CAR19 therapies show high complete remission (CR) rates, 15% of patients do not respond (NR), while others eventually relapse (REL). The B-ALL tumor microenvironment may impact efficacy of CAR T cell therapy. We evaluated whether pre-infusion bone marrow biopsy (BMB) parameters predict response to CAR-T cell therapy. Methods: Patients treated with CAR19 for R/R B-ALL from 2012-2021 at the Children's Hospital of Philadelphia were identified. BMBs from pre-CAR and 28 days post-CAR timepoints were evaluated. RNA sequencing-based gene expression profiling was performed (EdgeSeq Whole Transcriptome Panel, HTG Diagnostics). Differential expression (DE) was assessed by DESeq2 in HTG Reveal software. Pathway analysis was done using Metascape. BMBs were evaluated with reticulin special stain and CD3 immunostaining and quantified by expert scoring and digital image analysis. Modified Bauermeister scale ≥ 2 was considered severe fibrosis. Sustained Responders (SR) were defined as patients who did not relapse after achieving CAR19-mediated CR. 63 cases of primary and relapsed B-ALL were stained with CD3 and reticulin to determine baseline variation in fibrosis and CD3 infiltration before any immunotherapy. Results: DE and pathway enrichment analysis of NR and SR pre-CAR infusion samples showed significant NR up-regulation of genes in Skeletal System (p < 0.001) and Connective Tissue Development (p < 0.001), Epithelial to Mesenchymal Transition (p < 0.001), and ECM Structural Constituent (p < 0.001) pathways. Cytokine signaling was not significantly different between groups. Several fibrosis-associated genes, TGFB3 (5.67x, p = 0.009), PRELP (19.26x, p = 0.001), EGR1 (4.85x, p = 0.005), and DUSP1 (4.51x, p = 0.009), were among the ten most significantly upregulated genes. NRs also demonstrated the highest levels of pre-CAR fibrosis. 76% of NRs had a fibrosis score ≥ 2 compared to 56.7% for CRs and 46.7% for REL, and a higher average fibrosis score (2.23), compared to CR (1.46), SR (1.59), and REL (1.47) groups (p = 0.00776, p = 0.03, p = 0.00999, respectively; Mann-Whitney U-Test, two-tailed). CD3 staining was inversely correlated to reticulin fibrosis. Among non-immunotherapy treated patients, 70.3% of primary and 55.6% of relapsed cases had severe fibrosis, while 51.9% of primary and 77.8% of relapsed cases lacked CD3 infiltration. Fibrosis was higher in hyperdiploid, hypodiploid, iAMP21, PAX5-rearranged, and complex genetic subtypes of B-ALL, while CD3 infiltration was lower in PAX5-rearranged, MLL, TCF3-PBX1 and relapsed subtypes of B-ALL. Conclusions We describe for the first time the association between bone marrow fibrosis and response to CAR19 therapy in B-ALL. ECM and Connective Tissue Development pathway expression in NRs exceeds that of both their treatment-responsive peers, as well as expected physiological expression of bone growth/remodeling. Moreover, severe fibrosis and diminished lymphocytic infiltrates were noted in high-risk genetic subtypes of B-ALL even before administration of any immunotherapy. As such, up-regulated fibrosis-associated genes such as PRELP, EGR1, DUSP1, and TGFB3 could be targeted to improve response to CAR19 therapy. Additionally, reticulin staining is a widely available assay that could be used to assess fibrosis in bone marrow biopsies and serve as a useful heuristic tool in predicting the likelihood of therapy success. Ultimately, evaluation of pre-CAR infusion fibrosis levels in conjunction with other molecular and morphological markers may allow for more informed decision making, targeted therapeutic plan development, and patient outcome optimization in R/R B-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax Plus Decitabine for Young Adults with Newly Diagnosed ELN Adverse-Risk Acute Myeloid Leukemia: Updated Results of a Phase 2 Trial

Background: The young adults with newly diagnosed (ND) ELN adverse-risk acute myeloid leukemia (AML) fit for intensive chemotherapy (IC) have low complete remission (CR) rate and dismal outcomes compared with AML in favorable- and intermediate-risk categories. Venetoclax (VEN) combination with hypomethylating agents is a new standard of care for older and unfit AML patients (pts) and has been demonstrated to improve composite complete remission and overall survival (OS). We previously reported that VEN plus decitabine (DEC) as frontline treatment for young pts with ND ELN adverse-risk AML and demonstrated promising efficacy and safety at median follow-up of 2.7months in a phase 2 trial (Chen et al, ASH 2021). Here we present an updated analysis of efficacy and safety results in the ongoing study. Methods: This multicenter, single-arm, phase 2 trial (NCT04752527) enrolled young pts (aged between 18 and 59) with ND AML, who met the adverse risk group according to 2017 ELN risk stratification screened by fast next-generation sequencing (NGS), multiplex RT-PCR and fluorescence in situ hybridization (FISH) within 72 hours, and were eligible for standard induction chemotherapy. The study design was previously reported (Chen et al, ASH 2021). The primary outcome was composite complete remission (CRc: CR plus CR with incomplete blood count recovery [CRi] and morphologic leukemia free state [MLFS]). Secondary outcomes were OS, event-free survival (EFS) and safety. The data cut-off date was July 31, 2022. Results: Between February 12, 2021 and July 31, 2022, 286 patients aged between 18 and 59 with ND AML underwent screening. 78 pts were classified as ELN adverse-risk category by fast NGS (within 72 hours) together with multiplex RT-PCR and FISH and 42 pts were enrolled, comprising 26 males and 16 females. Median age was 39 years (range, 19-58) (Table 1). As of the cut-off date, 40 pts completed the first cycle of VEN+DEC and 37 pts completed two courses. In cycle 1, 31 (77.5% [95% CI, 61-89]) pts achieved CRc, 8 (20% [95% CI, 10-36]) pts had partial remission and 1 (2.5% [95% CI, 0-15]) had no response. 24 (77.4% [95% CI, 58-90]) of 31 pts achieving CRc had measurable residual disease (MRD) negativity assessed by multiparameter flow cytometry (MFC) with a sensitivity of 10-3-10-4(0.1%-0.01%). Pts with RUNX1 and FLT3-ITD mutations had higher CRc rates of 83.3% and 80%, respectively. Pts harboring TP53 and ASXL1 mutations achieved encouraging CRc rates of 62.5% and 75%, respectively. The CRc rates in pts with 11q23/KMT2A rearrangements and complex karyotype were 66.7% and 88.9%, respectively. The CRc and MRD negativity were achieved in 91.9% (34/37, 95% CI, 77-98) and 82.3% (28/34, 95% CI, 65-93) of the pts after the second course of treatment. 31 (73.8%) of 42 pts received allogeneic hematopoietic stem cell transplantation (HSCT), among them 26 patients achieved complete remission before HSCT, 3 received HSCT after morphologic relapse and 2 with salvaged HSCT. At median follow-up of 10.2 months (range, 0.8-17), the median OS and EFS were not reached (Figure1a, b). Estimated 12-months OS was 92.9% (95% CI, 79-98) and EFS was 71.4% (95% CI, 55-84). Among enrolled pts achieving CRc in cycle 1, median time to blood cell count recovery for absolute neutrophil count (ANC) ≥1000 cells per μL was 30 days (range, 16-NR), and for platelet count ≥50 000 platelets per μL was 20 days (range, 0-NR). Among 40 pts evaluable for safety analysis, the most common non-hematological adverse events were infections with grade 3 or worse neutropenia in 18 (45%) pts and febrile neutropenia in 11 (27.5%) pts. Notable serious adverse events were pneumonia in 5 (12.5%) pts and sepsis in 1 (2.5%). Mortality rates of 30-day and 60-day were 0%. Conclusions: VEN plus DEC was an effective, lower-intensity regimen that was well tolerated for young adults with ND ELN adverse-risk AML, producing high rates of CR and encouraging EFS and OS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interpreting Immune Reconstitution and Clinical Outcome By Single-Cell Transcriptomic Analysis in T-ALL Patients Undergoing Donor-Derived CD7 CAR-T Immunotherapy

Background Twenty-five percent of T-ALL patients experience relapse after allo-HSCT, which indicates a dismal prognosis and represents a significant unmet clinical need. CD7 is continuously expressed on T-ALL cell surface at higher levels than that on normal T cells and is therefore considered as a viable CAR T therapeutic target. However, the impact of CD7-positive normal T and NK cell clearance on the development, function, and homeostasis of immune system remains to be addressed. Methods We designed an open label, phase I clinical trial enrolled patients with a pathologically confirmed CD7-positive T-ALL/LBL (ChiCTR2200058969). After a 6-day manufacturing, the patients received donor-derived CD7 CAR T cells at the target dose of 1×105/kg. To minimize CD7 CAR T-cell mediated fratricide, we used IntraBlock technology to prevent CD7 expression on CAR T cells surface. Single cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) were adopted to profile immune statues changes before and after CD7 CAR T-cell treatment. Results Seven eligible patients were enrolled between November 2020 and December 2021, and all were followed up until May 1, 2022. Three patients were diagnosed with R/R T-ALL and 4 with R/R T-LBL at relapse. All patients had a history of allo-HSCT prior to enrollment. Patients were heavily pretreated with a median of 4 (3-8) lines of therapies before enrollment. The rate of CRS and ICANS was 85.7% and 14.3%, respectively. Only one patient had a grade 3 CRS and a serious grade 4 ICANS, resolved by dexamethasone and tocilizumab. Only two patients (28.6%) developed GVHD. Hematologic CAR T cells related adverse events (AE) included leukopenia, thrombocytopenia and neutropenia were observed in 6 patients (85.7%). Infections occurred in 5 patients (71.4%). On day 28, the patients who had bone marrow involvement achieved minimal residual disease (MRD) negative complete remission (CR, n=1) or CR with incomplete hematologic recovery (CRi, n=2); The patients who had CNS leukemia achieved CR on day 28. Five patients with their EMD encountered CR at day 90. The optimal ORR and CR were 100% and 100%, respectively. The CR/CRi rate was 85.7% on month 3. The median followed-up time was 4 months (range 3-16 months). Two patients died of serious infection and one patient died of brain hemorrhage. Four patients were alive until the cut-off time. Additionally, CD7-positive T-cells in the peripheral blood (including normal and abnormal T cells) were eliminated in seven patients on day 11 after CD7 CAR T infusion. However, CD7-negative T cells dramatically increased in all patients. In addition, the CD4/CD8 ratio decreased, driven by the increase in the proportion of CD8-positive T cells. The absolute numbers of T/B/NK cells (excluding CAR+ and leukemia T cells) showed a transient decline, and recovered by a low cells number at day 21. PBMC samples from four patients (two alive and two died of infection) were tested by scRNA-seq. The profiling of immune cells demonstrated that the severe infection after treatment is mainly caused by the loss of monocytes. This kind of monocytes loss is related to the decline of high developmental potential subset of monocytes before treatment. Moreover, the reconstructed T cell populations may have normal or even enhanced immune functions than pre-treatment T cells in all samples, which is supported by the higher expression levels of TCRs, T cell activation pathway, and T cell function pathway in post-treatment samples. The main character of the reconstructed T cell populations is its similarity with T cells in autoimmune diseases. This includes the expansion of GZMH+ T cells, decline of CD4+ naïve T cells, and activation of autoimmune disease related pathways. We also observed consistently upregulation of S100A8 and S100A9 in leukocyte lineage as a signature of the patients with relapse lymphoma. Conclusion Among 7 patients with r/r T-ALL enrolled in this trial, donor-derived CD7 CAR T cells exhibited efficient expansion and achieved a high complete remission rate with manageable safety profile. Data from scRNA-seq of PBMCs indicated a fulfilled T lymphocytes immune function by CD7-negativeT cells. The major challenge on prevent post-treatment infection may be the protection from loss of monocytes. Key words CD7 CAR-T immunotherapy; Immune Reconstitution; Single-cell Transcriptomic Analysis; T-ALL

Comparative Efficacy and Safety of Biological Agents in the Treatment of Lupus Nephritis: A Network Meta-Analysis

Background: To date, no studies have described randomized controlled trials (RCTs) evaluating the effectiveness and safety of various biological agents used in induction therapy for lupus nephritis. Objectives: We designed this study to assess the relative efficacy and safety of some of these biological agents in patients with lupus nephritis. Method: We collected data from RCTs that examined the efficacy and safety of any biological agents for lupus nephritis and then used these data to complete a Bayesian network meta-analysis to combine the direct and indirect evidence from these studies. Results: We identified nine RCTs evaluating rituximab, abatacept, belimumab, anifrolumab, obinutuzumab, ocrelizumab, and low-dose interleukin-2 (IL-2) across 1,480 patients. Low-dose IL-2, obinutuzumab, rituximab, and belimumab achieved complete remission in a significant proportion of respondents when compared with that in the control. Ranking probability based on the surface under the cumulative ranking curve (SUCRA) indicated that low-dose IL-2 had the highest probability of achieving complete remission, followed by obinutuzumab, rituximab, belimumab, anifrolumab, abatacept, ocrelizumab, and the control. The risk of serious adverse events (SAE) tended to be lower for low-dose IL-2, rituximab, belimumab, and obinutuzumab than for the control. SUCRA-based ranking indicated that IL-2 had the highest probability of being safe, followed by rituximab, belimumab, obinutuzumab, control, anifrolumab, abatacept, and ocrelizumab. Conclusions: Low-dose IL-2 was the most effective induction treatment for patients with lupus nephritis and had the lowest potential for SAE. Higher complete remission rates and a more favorable safety profile suggest that low-dose IL-2, obinutuzumab, rituximab, and belimumab may be superior to the current control as treatments for lupus nephritis.

Rituximab in Subepidermal Blistering Diseases

Background: Although rituximab is known to be effective in the treatment of pemphigus, its role in subepidermal autoimmune blistering diseases is unclear and currently limited to off-label use. Summary: This is a meta-analysis of case reports, case series, and retrospective studies on the effectiveness and safety of rituximab in bullous pemphigoid, mucous membrane pemphigoid, ocular pemphigoid, and epidermolysis bullosa acquisita. We compared remission and relapse rates in patients who received rituximab with those who only received conventional medical therapy. Comparisons were also made among disease subgroups. Key Message: The present analysis suggests that patients with subepidermal autoimmune blistering diseases treated with rituximab achieve a higher rate of complete remission and encounter their first relapse after a longer time interval. However, time to remission and total relapse rates were similar between groups. Adverse events and mortality rates were no more common in patients who received rituximab. This analysis was limited by the absence of randomized controlled trials and the observation that rituximab was used as a late rescue therapy in most reports. In conclusion, rituximab may be effective in subepidermal blistering disease, but randomized controlled studies are required for the validation of current observational data.

Megestrol acetate dispersible tablets with a 5-HT3 receptor antagonist and dexamethasone vs. 5-HT3 receptor antagonist plus dexamethasone, can better control chemotherapy-induced nausea and vomiting: a randomized controlled study.

A reasonable and effective control of chemotherapy-induced nausea and vomiting (CINV) plays an important role in the comprehensive treatment of cancer. Megestrol belongs to the 17α-hydroxyprogesterone derivative and is a highly effective synthetic progesterone. Recorded in the instructions may improve appetite and cachexia in patients with advanced tumors. In recent years, clinical practice and small sample studies have shown that megestrol combined with chemotherapy can improve CINV. This randomized controlled trial aimed to evaluate the clinical efficacy and safety of megestrol acetate combined with a 5-Hydroxytryptamine (5-HT3) receptor antagonist and dexamethasone in patients with CINV. Patients with malignant tumors who were treated with cisplatin-containing chemotherapy in our hospital from September 2018 to December 2019 were enrolled. A total of 120 patients were selected and randomly assigned to receive either megestrol acetate dispersible tablets with a 5-HT3 receptor antagonist and dexamethasone (megestrol group) or a 5-HT3 receptor antagonist plus dexamethasone (control group). Megestrol acetate dispersible tablets: 160 mg orally every morning from the day of chemotherapy until it lasts for ten days. Abstract IV of the quality-of-life scale for cancer patients in China was used to assess the quality of life (QOL) of the participants. All adverse reactions during chemotherapy were assessed according to the CTCAE 4.03 evaluation standard issued by the National Cancer Institute and divided into five grades according to severity. For the control of nausea, the rates of complete prevention were significantly higher in the megestrol group than in the control patients during the delayed [53.3% (31/60) vs. 30.0% (18/60), P=0.012] and overall [40.0% (24/60) vs. 15.0% (9/60), P=0.002] observation periods. Moreover, the megestrol combination treatment group also achieved markedly higher rates of complete remission of vomiting than the control group during the delayed observation period [76.7% (46/60) vs. 51.7% (31/60), P=0.001], achieving an overall higher proportion of remission during the study period [68.3% (41/60) vs. 46.6% (28/60), P=0.0016]. The triple antiemetic protocol using megestrol acetate with a 5-HT3 receptor antagonist plus dexamethasone can improve CINV symptoms caused by highly emetogenic chemotherapy (HEC) with cisplatin, with an excellent control effect and few adverse reactions, especially for delayed CINV. Chinese Clinical Trial Registry ChiCTR1800017953.

ALL-424 Updated Results from the Phase II Study of Blinatumomab in Combination With Ponatinib in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Blinatumomab and ponatinib both induce high rates of complete molecular remission (CMR) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). Their combination may generate durable responses and decrease the reliance on allogeneic stem cell transplantation (allo-SCT). To evaluate the combination of blinatumomab and ponatinib in patients with Ph-positive ALL. Patients with newly-diagnosed (ND) or relapsed/refractory (R/R) Ph-positive ALL were treated on this phase II trial. Blinatumomab was given for up to 5 cycles at standard doses, and ponatinib was started at 30mg daily during cycle 1, then lowered to 15mg daily upon achieving CMR. Among responders, ponatinib was maintained for a minimum of 5 years. Patients received 12 doses of prophylactic intrathecal chemotherapy. The primary endpoint was the CMR rate in the ND cohort, and the overall response rate (ORR; CR and CRi) in the R/R cohort. Forty-nine patients were treated: 35 ND (median age, 57 years; range, 22-83 years) and 14 R/R (median age, 38 years; range, 24-61 years). Among R/R patients, 43% were in Salvage ≥2. One early death within 4 weeks was observed among ND patients due to intracranial hemorrhage, none among R/R patients. The ORR was 96% in the ND cohort and 92% in the R/R cohort, with respective CMR rates of 85% and 79%. Only one patient with ND disease underwent allo-SCT because of persistently detectable BCR::ABL1 transcript levels; 6 patients (46%) with R/R disease underwent subsequent allo-SCT. After a median follow-up of 11 months (range, 1-46 months), no relapses were observed in the ND cohort with similar 2-year OS and EFS rates of 93%. In the R/R cohort, the estimated 2-year OS and EFS rates were 61% and 42%, respectively. This combination showed a favorable safety profile, with most adverse events of grade 1-2. One patient discontinued ponatinib due to toxicity. The chemotherapy-free combination of blinatumomab and ponatinib demonstrated robust clinical activity in Ph-positive ALL, with high rates of CMR and durable remissions. This strategy will potentially obviate the need for chemotherapy and allo-SCT in many patients, particularly in the frontline setting.

Autologous Anti-CD19 CAR T-Cells immunotherapy in relapsed/refractory acute lymphoblastic leukemia patients. A systematic review and meta-analysis

Introduction: Acute Lymphoblastic Leukemia (ALL) is the most prevalent malignancy in children; however, when the neoplasm becomes refractory/relapses (R/R) the cure possibilities are practically null. Objectives: To analyze the Anti-CD19 Chimeric Antigen Receptors (CAR) T-Cells immunotherapy efficacy in the treatment of R/R ALL, providing evidence about the efficacy and safety of the therapy for the analyzed group. Methods: The study consisted of a systematic review and meta-analysis based on the analysis of indexed articles. The searches were carried out with the terms: “acute lymphoblastic leukemia”, “CAR T”, and “CD19-specific chimeric antigen receptor”. Results: Only 18 of the 94 articles obtained initially met the inclusion criteria and were selected for review, totaling 637 patients. Thus, it was observed in the responses that approximately 81% of the patients achieved a Complete Response; 7% did not respond; the neoplasm relapsed in 17% of the cases; and 6.1% of the patients died. The main side effects found were Cytokine Release Syndrome (CRS), Severe Cytokine Release Syndrome, and Neurotoxicity, present in 36.3%, 29%, and 24% of patients, respectively. Conclusion: Anti-CD19 CAR T-Cells immunotherapy is an effective therapy, capable of producing high rates of complete remission in R/R ALL treatment.

Histopathological and Clinical Features of Methotrexate-Associated Lymphoproliferative Disorders and Post-Transplant Lymphoproliferative Disorders

Objective: To study histopathological and clinical features of methotrexate-associated lymphoproliferative disorders (MTX-LPD) and post-transplant lymphoproliferative disorders (PTLD).Material and Methods: A retrospective study on 30 cases of MTX-LPD and 2 cases of PTLD from 2006 to 2021.Results: By histopathology, the MTX-LPD group had 21 cases of lymphoma (MTX-Lymphoma) and 9 cases of reactive changes (MTX-Reactive). The PTLD group included diffuse large B-cell lymphoma and polymorphic PTLD (1 case each). The distinctive findings in MTX-Lymphoma and PTLD were association with Epstein-Barr virus (EBV) (8/12 cases, 66.7%) and CD30 positivity (13/18 cases, 72.2%). The MTX-LPD group had median MTX dosage of 10 mg/week, median MTX cumulative dosage of 2,613.75 mg, and median duration of MTX usage of 2,186 days. The 14 MTX-LPD and PTLD patients has median duration to response after the varied interventions of 47 days and the time to the first complete remission (CR) of 126 days. The MTX-Reactive patients had a significantly higher absolute lymphocyte count, younger median age, fewer B symptoms, higher rate of single site involvement, less extranodal involvement, shorter duration to response, less time to enter CR, and higher CR rate than the MTXLymphoma patients (p &lt; 0.05).Conclusion: Histopathology in MTX-LPD and PTLD patients can vary from reactive changes to lymphoma. EBV study and CD30 immunostaining help identify MTX-LPD and PTLD. History of MTX usage and other causes of immunodeficiency should be considered before diagnosing lymphoma. MTX discontinuation or reduction of immunosuppressant dosage are recommended before administrating combined chemotherapeutic agents in unresponsive cases.

Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1–7. However, CAR-T cell therapy currently has several limitations8–12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR–Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

Treatment for relapsed acute promyelocytic leukemia.

The advent of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the outcomes of acute promyelocytic leukemia (APL); nevertheless, a small fraction of patients still experience relapse. Due to the infrequency of APL relapse coupled with the rapid change in the therapeutic landscape, there are limited available data regarding the treatment of relapsed APL. In this situation, however, ATO-based therapy has been shown to result in high rates of hematological and molecular complete remission (CR). Autologous hematopoietic cell transplantation (HCT) is considered the postremission therapy of choice when patients achieve molecular CR, whereas recent studies have suggested that molecular CR is not prerequisite for the success of autologous HCT. Allogeneic HCT can be reserved for selected patients, i.e., those who cannot achieve CR and those who relapse after autologous HCT, because of high toxicities and the expectation of highly favorable outcomes with autologous HCT during CR. For patients who are ineligible for HCT, prolonged administration of ATRA + ATO would be a viable option. To further refine the therapy for patients with relapsed APL, it is imperative to aggregate clinical data of patients who relapse after the ATRA + ATO frontline therapy within the framework of national and international collaboration.

Safety and efficacy of mosunetuzumab, a bispecific antibody, in patients with relapsed or refractory follicular lymphoma: a single-arm, multicentre, phase 2 study

Mosunetuzumab is a CD20 × CD3 T-cell-engaging bispecific monoclonal antibody that redirects T cells to eliminate malignant B cells. In a phase 1 study, mosunetuzumab was well tolerated and active in patients with relapsed or refractory B-cell lymphoma. We, therefore, aimed to evaluate the safety and anti-tumour activity of fixed-duration mosunetuzumab in patients with relapsed or refractory follicular lymphoma who had received two or more previous therapies. We conducted a single-arm, multicentre, phase 2 study at 49 centres in seven countries (Australia, Canada, Germany, South Korea, Spain, UK, and USA). All patients were aged 18 years or older with histologically confirmed follicular lymphoma (grade 1-3a) and an Eastern Cooperative Oncology Group performance status of 0-1. Patients had disease that was relapsed or refractory to two or more previous lines of treatment, including an anti-CD20 therapy and an alkylating agent. Intravenous mosunetuzumab was administered in 21-day cycles with cycle 1 step-up dosing: 1 mg on cycle 1 day 1, 2 mg on cycle 1 day 8, 60 mg on cycle 1 day 15 and cycle 2 day 1, and 30 mg on day 1 of cycle 3 and onwards. Patients with a complete response by investigator assessment using the International Harmonisation Project criteria completed treatment after cycle 8, whereas patients with a partial response or stable disease continued treatment for up to 17 cycles. The primary endpoint was independent review committee-assessed complete response rate (as best response) in all enrolled patients; the primary efficacy analysis compared the observed IRC-assessed complete response rate with a 14% historical control complete response rate in a similar patient population receiving the pan class I PI3K inhibitor copanlisib. Safety was assessed in all enrolled patients. This study is registered with ClinicalTrials.gov, number NCT02500407, and is ongoing. Between May 2, 2019, and Sept 25, 2020, we enrolled 90 patients. As of the data cutoff date (Aug 27, 2021), the median follow-up was 18·3 months (IQR 13·8-23·3). According to independent review committee assessment, a complete response was recorded in 54 patients (60·0% [95% CI 49·1-70·2]). The observed complete response rate was significantly higher than the historical control complete response rate with copanlisib of 14% (p<0·0001), thereby meeting the primary study endpoint. Cytokine release syndrome was the most common adverse event (40 [44%] of 90 patients) and was predominantly grade 1 (23 [26%] of 90) and grade 2 (15 [17%]), and primarily confined to cycle 1. The most common grade 3-4 adverse events were neutropenia or neutrophil count decreased (24 [27%] of 90 patients), hypophosphataemia (15 [17%]), hyperglycaemia (seven [8%]), and anaemia (seven [8%]). Serious adverse events occurred in 42 (47%) of 90 patients. No treatment-related grade 5 (ie, fatal) adverse event occurred. Fixed-duration mosunetuzumab has a favourable safety profile and induces high rates of complete remissions, allowing potential administration as an outpatient regimen, in patients with relapsed or refractory follicular lymphoma and two or more previous therapies. F Hoffmann-La Roche and Genentech.

Mosunetuzumab, a bispecific antibody, in patients with relapsed or refractory follicular lymphoma.

Follicular lymphoma has a chronic clinical course with an overall survival of 80% at 10 years, justifying its classification as an indolent B-cell lymphoma. 1 Batlevi CL Sha F Alperovich A et al. Follicular lymphoma in the modern era: survival, treatment outcomes, and identification of high-risk subgroups. Blood Cancer J. 2020; 10: 74 Google Scholar Unfortunately, follicular lymphoma is a heterogenous disease, and about one in five patients relapses within 2 years after the start of initial treatment. 2 Casulo C Byrtek M Dawson KL et al. Early relapse of follicular lymphoma after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone defines patients at high risk for death: an analysis from the National LymphoCare study. J Clin Oncol. 2015; 33: 2516-2522 Google Scholar Furthermore, retrospective data have shown that progression-free survival and overall survival substantially decrease in the relapsed setting, reflecting the clinical experience that patients with follicular lymphoma become more treatment-resistant in later lines of therapy. 1 Batlevi CL Sha F Alperovich A et al. Follicular lymphoma in the modern era: survival, treatment outcomes, and identification of high-risk subgroups. Blood Cancer J. 2020; 10: 74 Google Scholar Thus, despite the generally indolent nature of follicular lymphoma, a great unmet medical need exists for efficient treatments for patients with relapsed or refractory follicular lymphoma, and the therapeutic armamentarium for later lines of treatment is particularly scarce. Safety and efficacy of mosunetuzumab, a bispecific antibody, in patients with relapsed or refractory follicular lymphoma: a single-arm, multicentre, phase 2 studyFixed-duration mosunetuzumab has a favourable safety profile and induces high rates of complete remissions, allowing potential administration as an outpatient regimen, in patients with relapsed or refractory follicular lymphoma and two or more previous therapies. Full-Text PDF

Application of High-Efficiency Cell Expansion and High-Throughput Drug Sensitivity Screening for Leukemia Treatment.

This study is to assess the clinical value of in vitro primary cell high-efficiency expansion and high-throughput drug sensitivity screening (HEHDS) system in leukemia, and we evaluated a cohort of 121 patients with acute myeloid leukemia (AML) and 27 patients with acute lymphoblastic leukemia (ALL) using HEHDS. Bone marrow aspirates were collected from patients with leukemia. Purified leukemic cancer cells were obtained, cultured, and screened with a panel of 247 FDA-approved compounds by HEHDS technology. Ninety-six patients received HEHDS-guided therapy while 52 patients who were subjected to physician directed therapy served as controls. ALL patients who received treatment guided by HEHDS showed higher rate of complete remission (CR) than that of patients in the non-HEHDS group (90.91% vs. 56.25%). Similarly, AML patients received HEHDS-guided therapy were found to have greater CR rate, when compared with patients who received physician-directed therapy (45.88% vs. 25%). There was a significantly higher rate of CR in HEHDS-guided therapy group compared to the non-HEHDS group. The application of HEHDS could be beneficial for leukemia treatment.

Efficacy and safety of leflunomide combined with corticosteroids for the treatment of IgA nephropathy: a Meta-analysis of randomized controlled trials

Objective We performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the efficacy and safety of leflunomide combined with corticosteroids, compared with corticosteroids alone, for IgA nephropathy. Materials and methods Studies were retrieved by searching of PubMed, Embase, Cochrane’s Library, China National Knowledge Infrastructure (CNKI), and Wanfang databases on 11 October 2021. A random-effect model incorporating the heterogeneity was used to pool the results. The efficacy outcomes included the complete remission rate of proteinuria, overall response rate (the combined rates of patients with complete and partial remission of proteinuria), changes of urine protein excretion (UPE), serum creatinine (SCr), and estimated glomerular infiltrating rate (eGFR). Results Nineteen studies were included. Patients receiving the combined therapy had a higher complete remission rate (relative risk [RR]: 1.29, 95% CI: 1.08–1.55, p = 0.006; I 2 = 0%) and overall response rate (RR: 1.18, 95% CI: 1.10–1.26, p < 0.001, I 2 = 0%) compared to patients who received CS alone. Besides, combined therapy was associated with significantly reduced levels of UPE (mean difference [MD]: −0.30 g/24h, 95% CI: −0.43 to −0.16, p < 0.001; I 2 = 34%) and SCr (MD: −7.55 mmol/L, 95% CI: −11.06 to −4.04, p < 0.001; I 2 = 34%), and increased level of eGFR (MD: 6.51 mL/min/1.73 m2, 95% CI: 4.06–8.97, p < 0.001; I 2 = 0%). The incidence of adverse events was not significantly different. Conclusions Combined treatment with leflunomide and corticosteroids was more effective than corticosteroids alone for patients with IgA nephropathy.

P342: BASELINE GENE EXPRESSION ANALYSIS OF RELAPSED ACUTE B-LYMPHOBLASTIC LEUKEMIA PATIENTS TREATED WITH INOTUZUMAB OZOGAMICIN

Background: Despite introduction of novel agents, relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) carries dismal outcome. Inotuzumab ozogamicin (InO) is a humanized anti-CD22 monoclonal antibody conjugated to calicheamicin approved for R/R B-ALL able to obtain high CR rates but of short duration. Mechanisms underlaying InO resistance are largely unknown. Aims: To characterize the baseline differentially expressed genes in a series of R/R B-ALL patients in relation with patient response to InO in order to individuate potential pathways involved in resistance. Methods: Gene expression profile of 18 R/R B-ALL patient samples was analyzed with RNA-seq, before InO exposure. All patients received at least 1 InO course. Patient population was divided in poor/non-responders (NR) and responders (R), based on InO response. R were defined as patients with duration of response (DoR) ≥3 months after bone marrow (BM) complete remission (CR) achievement. NR were defined either refractory or with a DoR <3 months after CR achievement. The list of significant (p<0.05) differentially expressed genes (DEGs) in NR, as compared to R whose absolute fold change (FC) was ≥2, was analyzed. P values were corrected with the Benjamini-Hochberg algorithm (false discovery rate; FDR). Gene expression results were analyzed with QIAGEN Ingenuity Pathway Analysis (IPA). Results: Eighteen R/R BM samples of CD22-positive B-ALL were analyzed with RNA-seq. Eight patients were defined as NR and 10 patients as R. Patient disease characteristics in NR and R groups were homogeneous for age, sex, number of Philadelphia (Ph)-positive ALL patients, duration of 1st remission, number of previous therapy lines including hematopoietic stem cell transplantation (HSCT) and Blinatumomab. At the time of InO therapy all patients had morphological BM relapse and all were CD22-positive. Median CD22 expression percentage on leukemic blasts was 100% (range 100-100) in NR patients and 100% (range 70-100) in R patients (p=0.177). Two patients in R group had a CD22 expression blast percentage of 70% and 76%. Median CD22-fluorescent intensity (CD22-FI) in NR as compared to R was 75.38 (IQR 59.58, 89.51) and 136.51 (IQR 114.38, 151.57) respectively, with significantly lower values in NR group (p = 0.04). Overall, 370 genes were differentially expressed (p<0.05) in NR, as compared to R patients, of which 32 were significantly differentially expressed (corrected p < 0.05, FC ≥2). Thirty-one were down- and 1 was up-regulated in NR vs R. DEGs were involved in basophil differentiation, carbon dioxide transport, erythrocyte and myeloid cell development, heme metabolic and porphyrin-containing compound biosynthetic processes, cation and cellular ion homeostasis. Both IPA upstream regulator and regulator effect analysis identified the serine/threonine homeodomain-interacting protein kinase-2 (HIPK2) as predicted downregulated. The inhibition of HIPK2 was predicted to be the causal upstream condition of the under-expression of six DEGs from the set (FECH, ANK1, SCL4A1, EPOR, GATA1, HBZ) with activation Z score of -2.449 and p value of overlap = 1.02E-09. No difference in terms of HIPK2 expression was appreciated in the two groups, suggesting downregulation at post-transcriptional level. Image:Summary/Conclusion: A unique pattern of gene expression signature based on HIPK2 downregulation was identified in poor responders to InO, providing potentially important insights in mechanisms of resistance. HIPK2 downregulation needs to be further validated. CT and CP contributed equally

Single-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy

AbstractVenetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.