B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in IntroductionChronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia with an immunophenotype expressing the T-cell marker CD5 together with CD19, CD20, CD23, and dim-surface immunoglobulin. 1 Although immunophenotypically similar to the normal B1 lymphocytes, CLL cells have a distinct mRNA gene expression profile that most approximates a postgerminal memory B cell. 2 For many years CLL has been viewed as a nonproliferating leukemia based on the nonproliferating blood compartment; however, as with normal B cells, it has come to be recognized that CLL cell proliferation probably occurs in sites where microenvironmental stimulation occurs such as the lymph nodes and spleen. In such sites, proliferation centers are observed with a high proportion of dividing CLL cells expressing survivin that are often surrounded by either T cells or accessory stromal cells capable of providing cytokine costimulation. 3,4 Studies administering heavy water allow accurate measurement of all body compartments of CLL and assess the birth rate of CLL tumor cells in vivo. 5 These studies have demonstrated a broad range of proliferation of CLL cells that varies based on disease state and also immunoglobulin heavy chain variable (IVGH) mutational status. 5,6 In particular, a higher tumor birth rate is noted in CLL patients with IVGH unmutated disease and ZAP-70 expression. Multiple studies have documented evidence of enhanced B-cell receptor (BCR) signaling in patients with IVGH unmutated disease or those with increased ZAP-70 expression. [7][8][9] Thus, accessory cytokines, cell-cell contact in the microenvironment, and also BCR-signaling coupled to B-cell proliferation appear sentinel to CLL progression and pathogenesis.While understanding of CLL biology has improved dramatically, until very recently integration of these findings to treatment interventions has been lacking. Specifically, treatment has included alkylators, nucleoside analogs, and their combination where small advances in improved response and progression-free survival (PFS) have been noted. 10,11 However, these therapies have had very little impact on overall survival of CLL. The addition of the chimeric CD20 antibody, rituximab, perhaps represents the most significant advance in CLL therapy. Rituximab single agent activity 12 and phase 2 studies combining it with fludarabine (FR) or fludarabine and cyclophosphamide (FCR) have demonstrated improved overall survival (OS) over historical controls. 13,14 A randomized trial of FCR versus fludarabine or cyclophosphamide alone 15 demonstrated significant improvement in response; PFS and OS. While the presumptive mechanism of rituximab in CLL has been assumed to be immunologic (reviewed in Jaglowski and Byrd 16 ), a recent study demonstrated a direct effect on BCR-signaling in both normal and malignant B cells via perturbation of membrane rafts by CD20 anti...

Importance The Bruton’s Tyrosine Kinase inhibitor ibrutinib is effective in patients with chronic lymphocytic leukemia (CLL). Reasons for discontinuation from this drug and outcomes following discontinuation have not been evaluated outside of clinical trials with relatively short follow-up. Objective To determine features associated with discontinuation of ibrutinib and outcomes. Design 308 patients participating in four sequential trials of ibrutinib were included. These trials accrued patients included in this analysis from May 2010 until April 2014, and data were locked in June 2014. Setting The Ohio State University Comprehensive Cancer Center Participants Patients with CLL enrolled on 4 sequential clinical trials. Main Outcome Measure Patients were evaluated for time to discontinuation, reasons for discontinuation, and survival following discontinuation. For patients who discontinued due to progression, targeted deep sequencing was performed in samples at baseline and relapse. Results With a median follow-up of 20 months, 232 patients remain on therapy, 31 have discontinued because of progression, and 45 have discontinued for other reasons. Disease progression includes Richter’s transformation or progressive CLL. Richter’s appeared to occur early and CLL progressions later (cumulative incidence at 12 months: 4.5% (95% CI: 2.0% to 7.0%) and 0.3% (95% CI: 0% to 1.0%), respectively). Median survival following Richter’s transformation was 3.5 months (95% CI: 0.3–6.0), and 17.6 months (95% CI: 4.7-not reached) following CLL progression. Sequencing on peripheral blood from 8 patients with Richter’s transformation revealed 2 with mutations in BTK, and a lymph node sample showed no mutations in BTK or PLCγ2. Deep sequencing on 11 patients with CLL progression revealed BTK or PLCγ2 mutations in all. These mutations were not identified pre-treatment in any patient. Conclusions and Relevance This single institution experience with ibrutinib confirms it to be an effective therapy and identifies, for the first time, baseline factors associated with ibrutinib discontinuation. Outcomes data show poor prognosis after discontinuation, especially for those patients with Richter’s transformation. Finally, sequencing data confirm initial reports associating mutations in BTK and PLCγ2 with progression and clearly show that CLL progressions are associated with these mutations, while Richter’s transformation is likely not.

IntroductionChronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the United States, with approximately 15 000 new cases and approximately 4500 deaths per year. 1 CLL is characterized by a B1 monoclonal lymphocyte immunophenotype with expression of the surface antigens CD19, CD5, CD20, CD23, and dim surface immunoglobulin G. The cell of origin of CLL is uncertain, but a gene expression pattern most similar to a mature memory B cell has been hypothesized. 2 In addition, CLL cells display disrupted apoptosis that is caused by both primary tumor features and codependent stromal elements. 3 Although many patients are asymptomatic at diagnosis, CLL is a progressive disease that in most patients eventually will require treatment. Once they become symptomatic, patients have a relatively short overall survival, ranging from 18 months to 6 years, with a 22.5% 10-year survival expectation. 4 Common treatments for CLL include alkylating chemotherapeutic drugs (such as chlorambucil and cyclophosphamide), purine analogs (such as fludarabine), and rituximab (used in combination with fludarabine, fludarabine and cyclophosphamide, or pentostatin and cyclophosphamide). Newer studies with either single-agent bendamustine or alemtuzumab have been shown to have improved response and progression-free survival over alkylator-based therapy. However, no current treatment option results in curative therapy, and all patients eventually relapse. This provides strong justification for developing additional types of therapies for CLL. Of particular interest are therapies that target signal transduction pathways essential to CLL cell survival mechanisms that are known to be aberrantly activated.One such pathway is the phosphoinositide 3-kinase (PI3K) pathway. The PI3K pathway is acknowledged as a key component of cell survival in many cancers, including CLL. It is activated by receptors, or the small guanosine triphosphatase Ras, and is made up of various classes of PI3K isoforms. 5 There are 3 classes of PI3K isoforms; however, only the class I isoforms phosphorylate inositol lipids to form second messenger phosphoinositides. Specifically, class I PI3K enzymes convert PtdIns(3,4)P 2 into PtdIns(3,4,5)P 3 , in the cell membrane that recruit, via binding to the amino-terminal pleckstrin homology domain, downstream signaling proteins such as Tec kinases, phosphatidylinositol-dependent kinase, Akt, integrin-linked kinase, and Rac guanine exchange factor. Class I isoforms are made up of 2 subsets (IA and IB). Class IA encompasses p110␣, p110␤, and p110␦ (catalytic domains), bound by p85, p50, or p55 (regulatory domains). Class IB is made up solely of the p110␥ (catalytic domain) bound by the regulatory domain p101. The p110␣ and p110␤ isoforms are ubiquitously expressed, and knock-out mice for both are embryonic lethal. 6 It is thought that this widespread functionality of PI3K signaling is at An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed ...

Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and MethodsPatients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. ResultsWith a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. ConclusionRelapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.

BACKGROUND.Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS.Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8 + T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4 + T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025.

In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110␦ (PI3K-␦) pathway. Inhibition of IntroductionLenalidomide is an immune modulatory agent currently approved for marketing in multiple myeloma and myelodysplasia. Lenalidomide is also clinically active in lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia (CLL). 1,2 The potential mechanisms of action of lenalidomide in these different diseases are multiple. 3 Application of lenalidomide in CLL has been associated with development of antitumor antibodies and reversal of hypogammaglobulinemia 4 but can also induce a disease-specific side effect of tumor flare and cytokine release. 3,5 The downstream manifestations of cytokine release, including increased serum basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), have previously been associated with promoting CLL survival 6-11 and also with decreased response to lenalidomide in CLL. 2 Understanding the mechanisms of these responses to lenalidomide is relevant to the development of this agent in CLL and to the logical design of future combination studies.We have recently demonstrated that lenalidomide can increase surface expression of CD154 on CLL cells while promoting normal B cells to produce immunoglobulin G (IgG) and IgM. 4 After clinical treatment with lenalidomide, we were also able to demonstrate a similar phenotype in CLL cells in patients receiving CD154 gene therapy, 12 including up-regulation of DR5 and BID. In one patient with pretreatment evidence of residual normal B cells, lenalidomide induced the development of antibodies, including generation of antitumor-directed ROR-1 antibodies. 4 The mechanism by which this occurred was shown in vitro to involve upstream activation of the phosphatidylinositol 3-kinase (PI3K) pathway. 4 Immune activation of CLL cells with up-regulation of costimulatory molecules, such as CD40, CD80, and CD86, 4,5,13 may also be responsible for lenalidomide-induced tumor flare. Given that CLL cell activation could have both favorable immunemodulating effects and detrimental clinical effects from tumor flare and also production of antiapoptotic cytokines, such as bFGF and VEGF, we sought to characterize whether a specific PI3K isoform was responsible for CLL activation by lenalidomide. This work is highly relevant given the observed preclinical 14 and clinical activity 15 of the PI3K p110␦ (PI3K-␦) inhibitor, CAL-101, in the treatment of CLL, the development of other compounds targeting B-cell receptor signaling, 16,17 and the potential interest in administering these newer drugs with lenalidomide as therapy for CLL. Methods Cell culture and treatment reagentsWritten, informed consent...

Key Points• Pretreatment near-tetraploidy is associated with advanced Rai stage, deletion of 17p, and complex karyotype.• Pretreatment near-tetraploidy is an independent risk factor for ibrutinib discontinuation via Richter transformation.Ibrutinib is a highly effective targeted therapy for chronic lymphocytic leukemia (CLL).However, ibrutinib must be discontinued in a subset of patients due to progressive CLL or transformation to aggressive lymphoma (Richter transformation). Transformation occurs early in the course of therapy and has an extremely poor prognosis. Thus, identification of prognostic markers associated with transformation is of utmost importance. Neartetraploidy (4 copies of most chromosomes within a cell) has been reported in various lymphomas, but its incidence and significance in CLL has not been described. Using fluorescence in situ hybridization, we detected near-tetraploidy in 9 of 297 patients with CLL prior to beginning ibrutinib treatment on 1 of 4 clinical trials (3.0%; 95% confidence interval [CI], 1.4%-5.7%). Near-tetraploidy was associated with aggressive disease characteristics: Rai stage 3/4 (P 5 .03), deletion 17p (P 5 .03), and complex karyotype (P 5 .01). Neartetraploidy was also associated with ibrutinib discontinuation due to Richter transformation (P , .0001), but not due to progressive CLL (P 5 .41). Of the 9 patients with near-tetraploidy, 6 had Richter transformation with diffuse large B-cell lymphoma. In a multivariable model, near-tetraploidy (hazard ratio [HR], 8.66; 95% CI,; P , .0001) and complex karyotype (HR, 4.77; 95% CI,; P 5 .01) were independent risk factors for discontinuing ibrutinib due to transformation. Our results suggest that near-tetraploidy is a potential prognostic marker for Richter transformation to assess in patients going on ibrutinib. IntroductionIbrutinib is a first-in-class oral covalent inhibitor of Bruton tyrosine kinase (BTK) 1 approved to treat chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and Waldenstrom macroglobulinemia. Ibrutinib has rapidly changed the landscape of CLL treatment, with high response rates and prolonged remission durations in both relapsed/refractory CLL and previously untreated patients.2-5 Despite these strides, a subset of patients relapse on ibrutinib. Patients relapse primarily with progressive CLL or Richter transformation, an aggressive transformation into lymphoma, predominantly diffuse large B-cell lymphoma.6 Most patients progressing with CLL acquire mutations in either the C481 ibrutinib-binding pocket of BTK or downstream activating mutations in PLCG2 that bypass the need for signaling through BTK. progress via disease transformation. 6 Identifying prognostic markers associated with Richter transformation is critical at this time as an increasing number of patients have begun to receive ibrutinib and those whose CLL transforms have very aggressive disease and poor prognosis. 6,9 Complex karyotype ($3 unrelated chromosomal abnormalities) has been associated with ibrutinib discontinuation due to progr...

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123− lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a “bridge-to-transplant” before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.