Global miRNA profiling reveals key molecules that contribute to different chronic lymphocytic leukemia incidences in Asian and Western populations.

It has been known for decades that the incidence of chronic lymphocytic leukemia (CLL) is significantly lower in Asia than in Western countries, but the reason responsible for this difference still remains a major knowledge gap. Using GeneChip® miRNA array to analyze the global microRNA expression in B lymphocytes from Asian and Western CLL patients and healthy individuals, we have identified microRNA with CLL-promoting or suppressive functions that are differentially expressed in Asian and Western individuals. In particular, miR-4485 is upregulated in CLL patients of both ethnic groups, and its expression is significantly lower in Asian healthy individuals. Genetic silencing of miR-4485 in CLL cells suppresses leukemia cell growth, whereas ectopic expression of miR-4485 promotes cell proliferation. Mechanistically, miR-4485 exerts its CLL-promoting activity by inhibiting the expression of TGR5 and activating the ERK1/2 pathway. In contrast, miR-138, miR-181a, miR- 181c, miR-181d, and miR-363 with tumor-suppressive function are highly expressed in Asian healthy individuals. Our study suggests that differential expression of several important microRNA with pro- or anti-CLL functions in Asian and Western B lymphocytes likely contributes to the difference in CLL incidence between the two ethnic groups, and that miR-4485 and its downstream molecule TGR5 could be potential therapeutic targets.

Sustained remissions in CLL after frontline FCR treatment with very-long-term follow-up.

Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) achieves durable remissions, with flattening of the progression-free survival (PFS) curve in patients with mutated immunoglobulin heavy chain variable gene (IGHV-M). We updated long-term follow-up results from the original 300-patient FCR study initiated at MD Anderson in 1999. The current median follow-up is 19.0 years. With this extended follow-up, the median PFS for patients with IGHV-M was 14.6 years vs 4.2 years for patients with unmutated IGHV (IGHV-UM). Disease progression beyond 10 years was uncommon. In total, 16 of 94 (17%) patients in remission at 10 years subsequently progressed with the additional follow-up compared with the patients in our prior report in 2015. Only 4 of 45 patients (9%) with IGHV-M progressed beyond 10 years. Excluding Richter transformation, 96 of 300 patients (32%) developed 106 other malignancies, with 19 of 300 (6.3%) developing therapy-related myeloid neoplasms (tMNs), which were fatal in 16 of 19 (84%). No pretreatment patient characteristics predicted the risk of tMNs. In summary, FCR remains an option for patients with IGHV-M chronic lymphocytic leukemia (CLL), with a significant fraction achieving functional cure of CLL. A risk-benefit assessment is warranted when counseling patients, balancing potential functional cure with the risk of late relapses and serious secondary malignancies.

Venetoclax consolidation in high-risk CLL treated with ibrutinib for ≥1 year achieves a high rate of undetectable MRD.

Patients receiving ibrutinib for CLL rarely achieve undetectable measurable residual disease (U-MRD), necessitating indefinite therapy, with cumulative risks of treatment discontinuation due to progression or adverse events. This study added venetoclax to ibrutinib for up to 2 years, in patients who had received ibrutinib for ≥12 months (mo) and had ≥1 high risk feature (TP53 mutation and/or deletion, ATM deletion, complex karyotype or persistently elevated ß2-microglobulin). The primary endpoint was U-MRD with 10-4 sensitivity (U-MRD4) in bone marrow (BM) at 12mo. Forty-five patients were treated. On intention-to-treat analysis, 23/42 (55%) patients improved their response to CR (2 pts were in MRD + CR at venetoclax initiation). U-MRD4 at 12mo was 57%. 32/45 (71%) had U-MRD at the completion of venetoclax: 22/32 stopped ibrutinib; 10 continued ibrutinib. At a median of 41 months from venetoclax initiation, 5/45 patients have progressed; none have died from CLL or Richter Transformation. In 32 patients with BM U-MRD4, peripheral blood (PB) MRD4 was analyzed every 6 months; 10/32 have had PB MRD re-emergence at a median of 13 months post-venetoclax. In summary, the addition of venetoclax in patients treated with ≥12mo of ibrutinib achieved high rate of BM U-MRD4 and may achieve durable treatment-free remission.

A phase 1 study to evaluate the safety, pharmacology, and feasibility of continuous infusion nelarabine in patients with relapsed and/or refractory lymphoid malignancies.

BACKGROUND: Nelarabine is a purine nucleoside analogue prodrug approved for the treatment of relapsed and refractory T-cell acute lymphoblastic leukemia (R/R T-ALL) and lymphoblastic lymphoma (T-LBL). Although effective in R/R T-ALL, significant neurotoxicity is dose-limiting and such neurotoxicity associated with nucleoside analogues can be related to dosing schedule. METHODS: The authors conducted a phase 1 study to evaluate the pharmacokinetics and toxicity of nelarabine administered as a continuous infusion (CI) for 5 days (120 hours), rather than the standard, short-infusion approach. RESULTS: Twenty-nine patients with R/R T-ALL/LBL or T-cell prolymphocytic leukemia (T-PLL) were treated, with escalating doses of nelarabine from 100 to 800 mg/m2 /day × 5 days. The median age of the patients was 39 years (range, 14-77 years). The overall response rate was 31%, including 27% complete remission (CR) or CR with incomplete platelet recovery (CRp). Peripheral neuropathy was observed in 34% of patients, including four ≥grade 3 events related to nelarabine. Notably, there was no nelarabine-related central neurotoxicity on study. The maximum tolerated dose was not reached. Pharmacokinetic data suggested no relationship between dose of nelarabine and accumulation of active intracellular ara-GTP metabolite. Higher intracellular ara-GTP concentrations were statistically associated with a favorable clinical response. CONCLUSION: Preliminary evaluation of continuous infusion schedule of nelarabine suggests that the safety profile is acceptable for this patient population, with clinical activity observed even at low doses and could broaden the use of nelarabine both as single agent and in combinations by potentially mitigating the risk of central nervous system toxicities.

A phase two study of high dose blinatumomab in Richter's syndrome.

Richter's Syndrome (RS) is an aggressive transformation of CLL, usually clonally-related diffuse large B-cell lymphoma (DLBCL), characterized by frequent TP53 mutations, intrinsic chemoresistance and poor survival. TP53-independent treatments are needed. We conducted a single center, phase 2, investigator-initiated study of high dose blinatumomab (maximum 112 mcg/d after initial, weekly dose escalation), NCT03121534, given for an 8-week induction and 4-week consolidation cycle. Responses were assessed by Lugano 2014 criteria. Serial multi-parameter flow cytometry from blood was performed to identify patient-specific biomarkers for response. Nine patients were treated. Patients had received a median of 4 and 2 prior therapies for CLL and RS, respectively. Five of 9 had del(17p) and 100% had complex karyotype. Four patients had reduction in nodal disease, including one durable complete response lasting >1 y. Treatment was well tolerated, with no grade >3 cytokine release syndrome and 1 case of grade 3, reversible neurotoxicity. Immunophenotyping demonstrated the majority of patients expressed multiple immune checkpoints, especially PD1, TIM3 and TIGIT. The patient who achieved CR had the lowest levels of immune checkpoint expression. Simultaneous targeting with immune checkpoint blockade, especially PD1 inhibition, which has already demonstrated single-agent efficacy in RS, could achieve synergistic killing and enhance outcomes.

STAT3 Activates the Pentraxin 3 Gene in Chronic Lymphocytic Leukemia Cells.

Pentraxin-related protein 3 (PTX3), commonly produced by myeloid and endothelial cells, is a humoral pattern recognition protein of the innate immune system. Because PTX3 plasma levels of patients with chronic lymphocytic leukemia (CLL) are high and most circulating cells in patients with CLL are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from seven of seven patients with CLL produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes coexpressing CD19 and CD5, and confocal microscopy showed that PTX3 is present in the cytoplasm of CLL cells. Because STAT3 is constitutively activated in CLL cells, and because we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 Abs precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an EMSA, we established and used a dual-reporter luciferase assay to confirm that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 enhanced apoptosis of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

Cyclin-dependent kinase inhibitor fadraciclib (CYC065) depletes anti-apoptotic protein and synergizes with venetoclax in primary chronic lymphocytic leukemia cells.

Fadraciclib (CYC065) is a second-generation aminopurine CDK2/9 inhibitor with increased potency and selectivity toward CDK2 and CDK9 compared to seliciclib (R-roscovitine). In chronic lymphocytic leukemia (CLL), a disease that depends on the over-expression of anti-apoptotic proteins for its survival, inhibition of CDK9 by fadraciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription in vitro; these actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and induced apoptosis. While the simulated bone marrow and lymph node microenvironments induced Mcl-1 expression and protected CLL cells from apoptosis, these conditions did not prolong the turnover rate of Mcl-1, and fadraciclib efficiently abrogated the protective effect. Further, fadraciclib was synergistic with the Bcl-2 antagonist venetoclax, inducing more profound CLL cell death, especially in samples with 17p deletion. While fadraciclib, venetoclax, and the combination each had distinct kinetics of cell death induction, their activities were reversible, as no additional cell death was induced upon removal of the drugs. The best combination effects were achieved when both drugs were maintained together. Altogether, this study provides a rationale for the clinical development of fadraciclib in CLL, either alone or in combination with a Bcl-2 antagonist.

STAT3 induces the expression of GLI1 in chronic lymphocytic leukemia cells.

The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1 activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1 and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1 as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.

The multi-kinase inhibitor TG02 induces apoptosis and blocks B-cell receptor signaling in chronic lymphocytic leukemia through dual mechanisms of action.

The constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

CXCL13 plasma levels function as a biomarker for disease activity in patients with chronic lymphocytic leukemia.

The chemoattractant CXCL13 organizes the cellular architecture of B-cell follicles and germinal centers. During adaptive immune responses, CXCL13 plasma concentrations transiently increase and function as a biomarker for normal germinal center activity. Chronic lymphocytic leukemia (CLL) cells express high levels of CXCR5, the receptor for CXCL13, and proliferate in pseudofollicles within secondary lymphoid organs (SLO). Given the morphologic and functional similarities between normal and CLL B-cell expansion in SLO, we hypothesized that CXCL13 plasma concentrations would correlate with CLL disease activity and progression. We analyzed CXCL13 plasma concentrations in 400 CLL patients and correlated the findings with other prognostic markers, time to treatment (TTT), CCL3 and CCL4 plasma concentrations, and in vivo CLL cell proliferation. We found that CXCL13 plasma concentrations were higher in CLL patients with active and advanced stage disease, resulting in a significantly shorter TTT. Accordingly, high CXCL13 levels correlated with other markers of disease activity and CCL3 levels. Higher CLL cell birth rates in vivo also associated with higher CXCL13 plasma concentrations. Interestingly, elevated CXCL13 plasma levels normalized during ibrutinib therapy, and increased in ibrutinib resistance patients. Collectively, these studies emphasize the importance of CXCL13 in crosstalk between CLL cells and the SLO microenvironment.

AMG-176, an Mcl-1 Antagonist, Shows Preclinical Efficacy in Chronic Lymphocytic Leukemia.

PURPOSE: Survival of CLL cells due to the presence of Bcl-2 and Mcl-1 has been established. Direct inhibition of Bcl-2 by venetoclax and indirect targeting of Mcl-1 with transcription inhibitors have been successful approaches for CLL. AMG-176 is a selective and direct antagonist of Mcl-1, which has shown efficacy in several hematologic malignancies; however, its effect on CLL is elusive. We evaluated biological and molecular effects of AMG-176 in primary CLL cells. EXPERIMENTAL DESIGN: Using samples from patients (n = 74) with CLL, we tested effects of AMG-176 on CLL and normal hematopoietic cell death and compared importance of CLL prognostic factors on this biological activity. We evaluated CLL cell apoptosis in the presence of stromal cells and identified cell death pathway including stabilization of Mcl-1 protein. Finally, we tested a couplet of AMG-176 and venetoclax in CLL lymphocytes. RESULTS: AMG-176 incubations resulted in time- and dose-dependent CLL cell death. At 100 and 300 nmol/L, there was 30% and 45% cell death at 24 hours. These concentrations did not result in significant cell death in normal hematopoietic cells. Presence of stroma did not affect AMG-176-induced CLL cell death. IGHV unmutated status, high ß2M and Mcl-1 protein levels resulted in slightly lower cell death. Mcl-1, but not Bcl-2 protein levels, in CLL cells increased with AMG-176. Low concentrations of venetoclax (1-30 nmol/L) were additive or synergistic with AMG-176. CONCLUSIONS: AMG-176 is active in inducing CLL cell death while sparing normal blood cells. Combination with low-dose venetoclax was additive or synergistic.

Ofatumumab is safe and effective as front-line treatment in older patients with chronic lymphocytic leukemia and severe co-morbidities, including other malignancies.

BACKGROUND: The majority of patients with chronic lymphocytic leukemia (CLL) are older and have multiple comorbidities, including other cancer diagnoses. These patients are routinely excluded from participation in clinical trials. OBJECTIVE: In this phase II study, we determined the activity and toxicity of ofatumumab, a fully human anti-CD20 monoclonal antibody, in older patients with CLL, poor performance status and comorbidities. METHODS: Treatment-naïve patients with CLL aged ≥65 years with an ECOG performance status of 2-3 or Charlson comorbidity index ≥2 were eligible. Ofatumumab was administered intravenously weekly for the first month, then monthly for a total of 12 months. RESULTS: Thirty-four patients were enrolled. Median age was 73 years, and 29% had another cancer diagnosis. Among 32 patients evaluable for response, the overall response rate was 72%. We observed complete responses in 19% of patients and partial responses in 53%. The median progression-free survival duration was 21 months, and the estimated proportion of patients alive at 36 months was 87%. All 34 patients were evaluable for toxicity. Treatment was well tolerated, with infusion-related reactions being the most common treatment-related adverse event. Only one patient had a grade 3 infection. Additional grade 3 adverse events that may have been related to ofatumumab were diarrhea, nausea/vomiting, hyperglycemia, pulmonary embolism, and hypersensitivity reaction, each in one patient. No grade 4 adverse events were observed. CONCLUSION: Single-agent ofatumumab is a well-tolerated and effective therapeutic approach for treatment-naïve older patients with CLL; it can be safely administered to patients with significant comorbidities and other cancer diagnoses.

The BET inhibitor GS-5829 targets chronic lymphocytic leukemia cells and their supportive microenvironment.

Despite major improvements in treatment outcome with novel targeted therapies, such as the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, chronic lymphocytic leukemia (CLL) remains incurable in the majority of patients. Activation of PI3K, NF-κB, and/or MYC has been linked to residual disease and/or resistance in ibrutinib-treated patients. These pathways can be targeted by inhibitors of bromodomain and extra-terminal (BET) proteins. Here we report about the preclinical activity of GS-5829, a novel BET inhibitor, in CLL. GS-5829 inhibited CLL cell proliferation and induced leukemia cell apoptosis through deregulation of key signaling pathways, such as BLK, AKT, ERK1/2, and MYC. IκBα modulation indicates that GS-5829 also inhibited NF-κB signaling. GS-5829-induced apoptosis resulted from an imbalance between positive (BIM) and negative regulators (BCL-XL) of the intrinsic apoptosis pathway. The antileukemia activity of GS-5829 increased synergistically in combinations with B-cell receptor signaling inhibitors, the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib, and the SYK inhibitor entospletinib. In cocultures that mimic the lymph node microenvironment, GS-5829 inhibited signaling pathways within nurselike cells and their growth, indicating that BET inhibitors also can target the supportive CLL microenvironment. Collectively, these data provide a rationale for the clinical evaluation of BET inhibitors in CLL.

STAT3-Induced Wnt5a Provides Chronic Lymphocytic Leukemia Cells with Survival Advantage.

The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.

Time-to-progression after front-line fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy for chronic lymphocytic leukaemia: a retrospective, multicohort study.

BACKGROUND: Fludarabine, cyclophosphamide, and rituximab (FCR) has become a gold-standard chemoimmunotherapy regimen for patients with chronic lymphocytic leukaemia. However, the question remains of how to treat treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia. We therefore aimed to develop and validate a gene expression signature to identify which of these patients are likely to achieve durable remissions with FCR chemoimmunotherapy. METHODS: We did a retrospective cohort study in two cohorts of treatment-naive patients (aged ≥18 years) with chronic lymphocytic leukaemia. The discovery and training cohort consisted of peripheral blood samples collected from patients treated at the University of Texas MD Anderson Cancer Center (Houston, TX, USA), who fulfilled the diagnostic criteria of the International Workshop on Chronic Lymphocytic Leukemia, had received at least three cycles of FCR chemoimmunotherapy, and had been treated between Oct 10, 2000, and Oct 26, 2006 (ie, the MDACC cohort). We did transcriptional profiling on samples obtained from the MDACC cohort to identify genes associated with time to progression. We did univariate Cox proportional hazards analyses and used significant genes to cluster IGHV-unmutated samples into two groups (intermediate prognosis and unfavourable prognosis). After using cross-validation to assess robustness, we applied the Lasso method to standardise the gene expression values to find a minimum gene signature. We validated this signature in an external cohort of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia enrolled on the CLL8 trial of the German Chronic Lymphocytic Leukaemia Study Group who were treated between July 21, 2003, and April 4, 2006 (ie, the CLL8 cohort). FINDINGS: The MDACC cohort consisted of 101 patients and the CLL8 cohort consisted of 109 patients. Using the MDACC cohort, we identified and developed a 17-gene expression signature that distinguished IGHV-unmutated patients who were likely to achieve a long-term remission following front-line FCR chemoimmunotherapy from those who might benefit from alternative front-line regimens (hazard ratio 3·83, 95% CI 1·94-7·59; p<0·0001). We validated this gene signature in the CLL8 cohort; patients with an unfavourable prognosis versus those with an intermediate prognosis had a cause-specific hazard ratio of 1·90 (95% CI 1·18-3·06; p=0·008). Median time to progression was 39 months (IQR 22-69) for those with an unfavourable prognosis compared with 59 months (28-84) for those with an intermediate prognosis. INTERPRETATION: We have developed a robust, reproducible 17-gene signature that identifies a subset of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia who might substantially benefit from treatment with FCR chemoimmunotherapy. We recommend testing the value of this gene signature in a prospective study that compares FCR treatment with newer alternative therapies as part of a randomised clinical trial. FUNDING: Chronic Lymphocytic Leukaemia Global Research Foundation and the National Institutes of Health/National Cancer Institute.

Minimal residual disease undetectable by next-generation sequencing predicts improved outcome in CLL after chemoimmunotherapy.

Patients with chronic lymphocytic leukemia (CLL) who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD) status after first-line fludarabine, cyclophosphamide, and rituximab (FCR) have prolonged progression-free survival (PFS), when assessed by an assay with sensitivity 10-4 (MRD4). Despite reaching U-MRD4, many patients, especially those with unmutated IGHV, subsequently relapse, suggesting residual disease <10-4 threshold and the need for more sensitive MRD evaluation. MRD evaluation by next-generation sequencing (NGS) has a sensitivity of 10-6 (MRD6). To better assess the depth of remission following first-line FCR treatment, we used NGS (Adaptive Biotechnologies Corporation) to assess MRD in 62 patients, all of whom had BM U-MRD by multicolor flow cytometry (sensitivity 10-4) at end-of-FCR treatment. Samples from these patients included 57 BM samples, 29 peripheral blood mononuclear cell (PBMC) samples, and 32 plasma samples. Only 27.4% of the 62 patients had U-MRD by NGS. Rate of U-MRD by NGS was lowest in BM (25%), compared with PBMC (55%) or plasma (75%). No patient with U-MRD by NGS in BM or PBMC was MRD+ in plasma. Patients with mutated IGHV were more likely to have U-MRD by NGS at the end of treatment (EOT; 41% vs 13%, P = .02) than those with unmutated IGHV. Median follow-up was 81.6 months. Patients with U-MRD at EOT had superior PFS vs MRD+ patients, regardless of sample type assessed (BM, P = .02, median not reached [NR] vs 67 months; PBMC, P = .02, median NR vs 74 months). More sensitive MRD6 testing increases prognostic discrimination over MRD4 testing.

Trabectedin Reveals a Strategy of Immunomodulation in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasia characterized by protumor immune dysregulation involving nonmalignant cells of the microenvironment, including T lymphocytes and tumor-associated myeloid cells. Although therapeutic agents have improved treatment options for CLL, many patients still fail to respond. Some patients also show immunosuppression. We have investigated trabectedin, a marine-derived compound with cytotoxic activity on macrophages in solid tumors. Here, we demonstrate that trabectedin induces apoptosis of human primary leukemic cells and also selected myeloid and lymphoid immunosuppressive cells, mainly through the TRAIL/TNF pathway. Trabectedin modulates transcription and translation of IL6, CCL2, and IFNα in myeloid cells and FOXP3 in regulatory T cells. Human memory CD8+ T cells downregulate PD-1 and, along with monocytes, exert in vivo antitumor function. In xenograft and immunocompetent CLL mouse models, trabectedin has antileukemic effects and antitumor impact on the myeloid and lymphoid cells compartment. It depletes myeloid-derived suppressor cells and tumor-associated macrophages and increases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1+ CLL cells, PD-L1+ monocytes/macrophages, and PD-1+ T cells. Thus, trabectedin behaves as an immunomodulatory drug with potentially attractive therapeutic value in the subversion of the protumor microenvironment and in overcoming chemoimmune resistance.