Conditional survival to assess prognosis in patients with chronic lymphocytic leukemia.

Biomarkers in chronic lymphocytic leukemia (CLL) allow assessment of prognosis. However, the validity of current prognostic biomarkers based on a single assessment point remains unclear for patients who have survived one or more years. Conditional survival (CS) studies that address how prognosis may change over time, especially in prognostic subgroups, are still rare. We performed CS analyses to estimate 5-year survival in 1-year increments, stratified by baseline disease characteristics and known risk factors in two community-based cohorts of CLL patients (Freiburg University Hospital (n = 316) and Augsburg University Hospital (n = 564)) diagnosed between 1984 and 2021. We demonstrate that 5-year CS probability is stable (app. 75%) for the entire CLL patient cohort over 10 years. While age, sex, and stage have no significant impact on CS, patients with high-risk disease features such as non-mutated IGHV, deletion 17p, and high-risk CLL-IPI have a significantly worse prognosis at diagnosis, and 5-year CS steadily decreases with each additional year survived. Our results confirm that CLL patients have a stable survival probability with excess mortality and that the prognosis of high-risk CLL patients declines over time. We infer that CS-based prognostic information is relevant for disease management and counseling of CLL patients.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/µL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

Idelalisib.

Idelalisib (GS-1101, CAL-101, Zydelig®) is an orally bioavailable, small-molecule inhibitor of the delta isoform (p110δ) of the enzyme phosphoinositide 3-kinase (PI3K). In contrast to the other PI3K isoforms, PI3Kδ is expressed selectively in hematopoietic cells. PI3Kδ signaling is active in many B-cell leukemias and lymphomas. By inhibiting the PI3Kδ protein, idelalisib blocks several cellular signaling pathways that maintain B-cell viability. Idelalisib is the first PI3K inhibitor approved by the US Food and Drug Administration (FDA). Treatment with idelalisib is indicated in relapsed/refractory chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and small lymphocytic lymphoma (SLL). This review presents the preclinical and clinical activity of idelalisib with a focus on clinical studies in CLL.

Anti-Angiogenics: Current Situation and Future Perspectives.

Angiogenesis, the process leading to the formation of new blood vessels, is one of the hallmarks of cancer. Extensive studies established that i) vascular endothelial growth factor (VEGF) is a key driver of sprouting angiogenesis, ii) VEGF is overexpressed in most solid cancers, and iii) inhibition of VEGF can suppress tumor growth in animal models. This has led to the development of pharmacological agents for anti-angiogenesis to disrupt the vascular supply and starve the tumor of nutrients and oxygen, primarily through the blockade of VEGF/VEGF receptor signaling. This effort has resulted in 11 anti-VEGF drugs approved for certain advanced cancers, either alone or in combination with chemotherapy and other targeted therapies. However, inhibition of VEGF signaling is not effective in all cancers, and anti-angiogenics have often only limited impact on overall survival of cancer patients. This review focuses on the current status of FDA-approved anti-angiogenic antibodies and tyrosine kinase inhibitors and summarizes the progress and future directions of VEGF-targeted therapy.

Microenvironmental stromal cells abrogate NF-κB inhibitor-induced apoptosis in chronic lymphocytic leukemia.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is known to play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Several NF-κB inhibitors were shown to successfully induce apoptosis of CLL cells in vitro Since the microenvironment is known to be crucial for the survival of CLL cells, herein, we tested whether NF-κB inhibition may still induce apoptosis in these leukemic cells in the presence of protective stromal interaction. We used the specific NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). Microenvironmental support was mimicked by co-culturing CLL cells with bone marrow-derived stromal cell lines (HS-5 and M2-10B4). NF-κB inhibition by DHMEQ in CLL cells could be confirmed in both the monoculture and co-culture setting. In line with previous reports, NF-κB inhibition induced apoptosis in the monoculture setting by activating the intrinsic apoptotic pathway resulting in poly (ADP-ribose) polymerase (PARP)-cleavage; however, it was unable to induce apoptosis in leukemic cells co-cultured with stromal cells. Similarly, small interfering ribonucleic acid (siRNA)-mediated RELA downregulation induced apoptosis of CLL cells cultured alone, but not in the presence of supportive stromal cells. B-cell activating factor (BAFF) was identified as a microenvironmental messenger potentially protecting the leukemic cells from NF-κB inhibition-induced apoptosis. Finally, we show improved sensitivity of stroma-supported CLL cells to NF-κB inhibition when combining the NF-κB inhibitor with the SYK inhibitor R406 or the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, agents known to inhibit the stroma-leukemia crosstalk. We conclude that NF-κB inhibitors are not promising as monotherapies in CLL, but may represent attractive therapeutic partners for ibrutinib and R406.

Drug-perturbation-based stratification of blood cancer.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Spleen Tyrosine Kinase Is Involved in the CD38 Signal Transduction Pathway in Chronic Lymphocytic Leukemia.

The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.

Histopathological regression after neoadjuvant docetaxel, oxaliplatin, fluorouracil, and leucovorin versus epirubicin, cisplatin, and fluorouracil or capecitabine in patients with resectable gastric or gastro-oesophageal junction adenocarcinoma (FLOT4-AIO): results from the phase 2 part of a multicentre, open-label, randomised phase 2/3 trial.

BACKGROUND: Docetaxel-based chemotherapy is effective in metastatic gastric and gastro-oesophageal junction adenocarcinoma, but has not yet been evaluated in the context of resectable patients. Here we report findings from the phase 2 part of the phase 2/3 FLOT4 trial, which compared histopathological regression in patients treated with a docetaxel-based triplet chemotherapy versus an anthracycline-based triplet chemotherapy before surgical resection. METHODS: In this randomised, open-label, phase 2/3 study, eligible participants were recruited from 28 German oncology centres. Patients with resectable gastric or gastro-oesophageal junction cancer who had clinical stage cT2 or higher, nodal positive (cN+) disease, or both were randomly assigned (1:1) to either three preoperative and three postoperative 3-week cycles of intravenous epirubicin 50 mg/m2 on day 1, intravenous cisplatin 60 mg/m2 on day 1, and either fluorouracil 200 mg/m2 as continuous intravenous infusion or capecitabine 1250 mg/m2 orally (two doses of 625 mg/m2 per day) on days 1 to 21 (ECF/ECX group) or four preoperative and four postoperative 2-week cycles of docetaxel 50 mg/m2, intravenous oxaliplatin 85 mg/m2, intravenous leucovorin 200 mg/m2, and fluorouracil 2600 mg/m2 as a 24 h infusion, all on day 1 (FLOT group). Randomisation was done centrally with an interactive web-response system based on a sequence generated with blocks (block size 2) stratified by Eastern Cooperative Oncology Group performance status, location of primary tumour, age, and nodal status. No masking was done. Central assessment of pathological regression was done according to the Becker criteria. The primary endpoint was pathological complete regression (tumour regression grade TRG1a) and was analysed in the modified intention-to-treat population, defined as all patients who were randomly assigned to treatment excluding patients who had surgery but did not provide resection specimens for central evaluation. The study (including the phase 3 part) has completed enrolment, but follow-up is ongoing and this is an interim analysis. The trial is registered with ClinicalTrials.gov, number NCT01216644. FINDINGS: Between Aug 18, 2010, and Aug 10, 2012, 300 patients (152 patients in the ECF/ECX group; 148 patients in the FLOT group) were enrolled into the phase 2 part of the study, 265 of whom (137 in the ECF/ECX group; 128 in the FLOT group) were assessable on a modified intention-to-treat basis. 119 (93%) of 128 patients in the FLOT group and 126 (92%) of 137 patients in the ECF/ECX group were given all planned preoperative cycles of treatment. FLOT was associated with significantly higher proportions of patients achieving pathological complete regression than was ECF/ECX (20 [16%; 95% CI 10-23] of 128 patients vs eight [6%; 3-11] of 137 patients; p=0·02). 44 (40%) of 111 patients in the ECF/ECX group and 30 (25%) of 119 patients in the FLOT group had at least one serious adverse event involving a perioperative medical or surgical complication. The most common non-surgical grade 3-4 adverse events were neutropenia (52 [38%] of 137 patients in the ECF/ECX group vs 67 [52%] of 128 patients in the FLOT group), leucopenia (28 [20%] vs 36 [28%]), nausea (23 [17%] vs 12 [9%]), infection (16 [12%] vs 15 [12%]), fatigue (19 [14%] vs 11 [9%]), and vomiting (13 [10%] vs four [3%]). INTERPRETATION: Perioperative FLOT was active and feasible to administer, and might represent an option for patients with locally advanced, resectable gastric or gastro-eosophageal junction adenocarcinoma. FUNDING: None.

BCR and chemokine responses upon anti-IgM and anti-IgD stimulation in chronic lymphocytic leukaemia.

Dysregulation of B cell receptor (BCR) signalling is a hallmark of chronic lymphocytic leukaemia (CLL) pathology, and targeting BCR pathway kinases has brought great therapeutic advances. Activation of the BCR in lymphoid organs has been associated with CLL cell proliferation and survival, leading to progressive disease. While these responses are mediated predominantly by IgM, the role of IgD is less clear. Seeking to uncover downstream consequences of individual and combined stimulation of the two BCR isotypes, we found an amplification of IgD expression and IgD-mediated calcium signalling by previous stimulation of IgM in CLL. Furthermore, no heterologous downmodulation of the isotypes, as observed in healthy donors, was present. Only marginal downregulation of the expression of various chemokine receptors by α-IgM and α-IgD stimulation was found as compared to normal B cells. Consistently, calcium responses of CLL cells to different chemokines were only weakly affected by preceding BCR activation. In contrast, migration towards the two homeostatic chemokines CXCL12 and CCL21 was differentially regulated by IgM and IgD. While IgM activation reduced migration of CLL cells towards CXCL12, but not CCL21, IgD activation predominantly impacted on CCL21 but not CXCL12-mediated chemotaxis. This indicates that the preference for one chemokine over the other may depend on the functional presence of the two isotypes in CLL. Inhibitors against the kinases Syk, Lyn, and Btk antagonised both BCR- and chemokine-induced calcium signals.

Combination of bendamustine and rituximab as front-line therapy for patients with chronic lymphocytic leukaemia: multicenter, retrospective clinical practice experience with 279 cases outside of controlled clinical trials.

Recently, encouraging results in terms of safety and efficacy have been obtained using bendamustine-rituximab (BR) in untreated chronic lymphocytic leukaemia (CLL) patients enrolled in a phase II study. Here, we report a retrospective international multicenter study of CLL patients treated with BR as front-line therapy. The cohort included 279 patients with progressive CLL from 33 centers (29 Italian, 3 Israeli and 1 German) who received at least 1 cycle of BR as first-line treatment during the 2008-2014 period. The primary objective of this study was to evaluate the efficacy and safety of BR administered as front-line therapy, outside of controlled clinical trials. Median age was 70 years (range, 43-86 years); 62.4% were males and 35.8% had Binet stage C. Forty-two patients (15.2%) were unfit (cumulative illness rating scale [CIRS] score ≥7), and 140 (50.2%) had creatinine clearance ≤70 ml/min. Fluorescent in situ hybridisation analysis, available for 192 cases, showed that 21 (10.9%) had del11q and 18 (9.4%) del17p. The overall response rate (ORR) was 86.4%, with a complete remission rate of 28%. Patients with del17p had an ORR of 66.7%. After median follow-up of 24 months, the 2-year progression-free survival (PFS) was 69.9%; CIRS ≥7, immunoglobulin heavy-chain variable-region (IGHV) unmutated status, del17p and BR dose intensity <80% were independently associated with shorter PFS. Grade III or IV neutropenia, thrombocytopenia, and anaemia were observed in 25.9%, 15.4%, and 15.1% of patients, respectively. Twenty-four patients (8.6%) had severe infections. BR is also an effective and safe regimen for untreated CLL patients, outside of controlled clinical trials.

Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma.

B-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches.

BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia.

Patients with BRAFV600E/K-driven melanoma respond to the BRAF inhibitor vemurafenib due to subsequent deactivation of the proliferative RAS/RAF/MEK/ERK pathway. In BRAF WT cells and those with mutations that activate or result in high levels of the BRAF activator RAS, BRAF inhibition can lead to ERK activation, resulting in tumorigenic transformation. We describe a patient with malignant melanoma who developed chronic lymphocytic leukemia (CLL) in the absence of RAS mutations during vemurafenib treatment. BRAF inhibition promoted patient CLL proliferation in culture and in murine xenografts and activated MEK/ERK in primary CLL cells from additional patients. BRAF inhibitor-driven ERK activity and CLL proliferation required B cell antigen receptor (BCR) activation, as inhibition of the BCR-proximal spleen tyrosine kinase (SYK) reversed ERK hyperactivation and proliferation of CLL cells from multiple patients, while inhibition of the BCR-distal Bruton tyrosine kinase had no effect. Additionally, the RAS-GTP/RAS ratio in primary CLL cells exposed to vemurafenib was reduced upon SYK inhibition. BRAF inhibition increased mortality and CLL expansion in mice harboring CLL xenografts; however, SYK or MEK inhibition prevented CLL proliferation and increased animal survival. Together, these results suggest that BRAF inhibitors promote B cell malignancies in the absence of obvious mutations in RAS or other receptor tyrosine kinases and provide a rationale for combined BRAF/MEK or BRAF/SYK inhibition.

MDSCs: the final frontier of the microenvironment in CLL?

In this issue of Blood, Jitschin et al identify increased numbers of myeloid-derived suppressor cells (MDSCs) in untreated patients with chronic lymphocytic leukemia (CLL) suppressing T cells and inducing regulatory T cells (T(regs)), resulting in impaired immune responses.

Sphingosine-1-phosphate receptors control B-cell migration through signaling components associated with primary immunodeficiencies, chronic lymphocytic leukemia, and multiple sclerosis.

BACKGROUND: Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells. OBJECTIVE: To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor-treated MS, and patients with primary immunodeficiencies. METHODS: S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy. RESULTS: Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, ß-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1. CONCLUSION: Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.

Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T-cells in chronic lymphocytic leukemia.

Antitumor immunity in chronic lymphocytic leukemia (CLL) is hampered by highly dysfunctional T-cells. Although certain T-cell subsets have been reported to be of prognostic significance in this disease, their interplay is complex and it remains incompletely understood which of these subsets significantly drive CLL progression. Here, we determined immunological profiles of 24 circulating T-cell subsets from 79 untreated individuals by multiparametric flow cytometry. This screening cohort included healthy donors, patients with monoclonal B-cell lymphocytosis (MBL), Rai 0 CLL and advanced CLL. We applied multidimensional scaling analysis as rigorous and unbiased statistical tool to globally assess the composition of the circulating T-cell environment and to generate T-cell scores reflecting its integrity. These scores allowed clear distinction between advanced CLL and healthy controls, whereas both MBL and Rai 0 CLL showed intermediate scores mirroring the biological continuum of CLL and its precursor stages. T-cell stimulation and suppression assays as well as longitudinal T-cell profiling showed an increasingly suppressive regulatory function initiating at the MBL stage. Effector function was impaired only after transition to CLL and partially recovered after chemoimmunotherapy. In an independent validation cohort of 52 untreated CLL cases, aberrant T-cell profiles were significantly associated with shorter time to treatment independently of other prognostic parameters. Random forest modeling predicted regulatory T-cell, gamma/delta and NKT-cells, as well as exhaustion of the CD8+ subset as potential drivers of progression. Our data illustrate a pathological T-cell environment in MBL that evolves toward a more and more suppressive and prognostically relevant profile across the disease stages.