Development of the Bruton's tyrosine kinase inhibitor ibrutinib for B cell malignancies.

Ibrutinib is a first-in-class oral covalent inhibitor of Bruton's tyrosine kinase that has demonstrated clinical benefit for many patients with B cell malignancies. Positive results in initial trials led the U.S. Food and Drug Administration to grant ibrutinib three breakthrough therapy designations for mantle cell lymphoma (MCL), del17p chronic lymphocytic leukemia (CLL), and Waldenström's macroglobulinemia (WM). Ibrutinib was approved for these three cancers within 14 months of the original U.S. approval. Additionally, ibrutinib is approved for patient subsets with MCL and/or CLL in >45 other countries. Via a unique mechanism of action, ibrutinib inhibits B cell signaling pathways that regulate the survival, proliferation, adhesion, and homing of cancerous cells. This marks a paradigm shift from the conventional cytotoxic chemotherapy approach to treating B cell malignancies. Ibrutinib continues to be evaluated across a range of B cell malignancies, either as single-agent therapy or in combination with other therapies, and continues to transform the lives of these patients.

Cell signaling-based classifier predicts response to induction therapy in elderly patients with acute myeloid leukemia.

Single-cell network profiling (SCNP) data generated from multi-parametric flow cytometry analysis of bone marrow (BM) and peripheral blood (PB) samples collected from patients >55 years old with non-M3 AML were used to train and validate a diagnostic classifier (DXSCNP) for predicting response to standard induction chemotherapy (complete response [CR] or CR with incomplete hematologic recovery [CRi] versus resistant disease [RD]). SCNP-evaluable patients from four SWOG AML trials were randomized between Training (N = 74 patients with CR, CRi or RD; BM set = 43; PB set = 57) and Validation Analysis Sets (N = 71; BM set = 42, PB set = 53). Cell survival, differentiation, and apoptosis pathway signaling were used as potential inputs for DXSCNP. Five DXSCNP classifiers were developed on the SWOG Training set and tested for prediction accuracy in an independent BM verification sample set (N = 24) from ECOG AML trials to select the final classifier, which was a significant predictor of CR/CRi (area under the receiver operating characteristic curve AUROC = 0.76, p = 0.01). The selected classifier was then validated in the SWOG BM Validation Set (AUROC = 0.72, p = 0.02). Importantly, a classifier developed using only clinical and molecular inputs from the same sample set (DXCLINICAL2) lacked prediction accuracy: AUROC = 0.61 (p = 0.18) in the BM Verification Set and 0.53 (p = 0.38) in the BM Validation Set. Notably, the DXSCNP classifier was still significant in predicting response in the BM Validation Analysis Set after controlling for DXCLINICAL2 (p = 0.03), showing that DXSCNP provides information that is independent from that provided by currently used prognostic markers. Taken together, these data show that the proteomic classifier may provide prognostic information relevant to treatment planning beyond genetic mutations and traditional prognostic factors in elderly AML.

Development and validation of a single-cell network profiling assay-based classifier to predict response to induction therapy in paediatric patients with de novo acute myeloid leukaemia: a report from the Children's Oncology Group.

Single cell network profiling (SCNP) is a multi-parameter flow cytometry technique for simultaneous interrogation of intracellular signalling pathways. Diagnostic paediatric acute myeloid leukaemia (AML) bone marrow samples were used to develop a classifier for response to induction therapy in 53 samples and validated in an independent set of 68 samples. The area under the curve of a receiver operating characteristic curve (AUC(ROC)) was calculated to be 0·85 in the training set and after exclusion of induction deaths, the AUC(ROC) of the classifier was 0·70 (P = 0·02) and 0·67 (P = 0·04) in the validation set when induction deaths (intent to treat) were included. The highest predictive accuracy was noted in the cytogenetic intermediate risk patients (AUC(ROC) 0·88, P = 0·002), a subgroup that lacks prognostic/predictive biomarkers for induction response. Only white blood cell count and cytogenetic risk were associated with response to induction therapy in the validation set. After controlling for these variables, the SCNP classifier score was associated with complete remission (P = 0·017), indicating that the classifier provides information independent of other clinical variables that were jointly associated with response. This is the first validation of an SCNP classifier to predict response to induction chemotherapy. Herein we demonstrate the usefulness of quantitative SCNP under modulated conditions to provide independent information on AML disease biology and induction response.

Functional pathway analysis using SCNP of FLT3 receptor pathway deregulation in AML provides prognostic information independent from mutational status.

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.

Functional pathway analysis in acute myeloid leukemia using single cell network profiling assay: effect of specimen source (bone marrow or peripheral blood) on assay readouts.

BACKGROUND: Single cell network profiling (SCNP) is used to simultaneously measure the effects of modulators on signaling networks at the single cell level. SCNP-based biomarker assays predictive of response to induction therapy and relapse risk in acute myeloid leukemia (AML) patients are being developed. Such assays have typically used bone marrow (BM) as the sample source of blasts. Because circulating peripheral blasts are detectable in ∼65% of AML patients and peripheral blood (PB) sampling is less invasive than BM sampling, this study was performed to assess the effect of sample source on AML blasts signaling as measured in SCNP assay. METHODS: SCNP using multiparametric flow cytometry was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with modulators in 46 pairs of BM mononuclear cells/PB mononuclear cells. The relationship between readouts of modulated intracellular proteins ("nodes") was measured using linear regression, Bland-Altman method, and Lin's concordance correlation coefficient. RESULTS: The majority (156/161) of signaling nodes show strong correlations between paired PB and BM samples independently from the statistical method used. Notable exceptions were two PB samples with almost undetectable levels of circulating blasts compared with paired BM samples. CONCLUSIONS: Our results demonstrate that specimen source (BM or PB) does not significantly affect proteomic signaling in patients with AML and circulating blasts. The ability to use PB as a sample source will facilitate the monitoring of cellular signaling effects following administration of targeted therapies and at time points when BM aspirates are not clinically justifiable.

Functional characterization of FLT3 receptor signaling deregulation in acute myeloid leukemia by single cell network profiling (SCNP).

BACKGROUND: Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative. METHODOLOGY/PRINCIPAL FINDINGS: Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management. CONCLUSIONS/SIGNIFICANCE: These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.

Dynamic single-cell network profiles in acute myelogenous leukemia are associated with patient response to standard induction therapy.

PURPOSE: Complete response to induction chemotherapy is observed in approximately 60% of patients with newly diagnosed non-M3 acute myelogenous leukemia (AML). However, no methods exist to predict with high accuracy at the individual patient level the response to standard AML induction therapy. EXPERIMENTAL DESIGN: We applied single-cell network profiling (SCNP) using flow cytometry, a tool that allows a comprehensive functional assessment of intracellular signaling pathways in heterogeneous tissues, to two training cohorts of AML samples (n = 34 and 88) to predict the likelihood of response to induction chemotherapy. RESULTS: In the first study, univariate analysis identified multiple signaling "nodes" (readouts of modulated intracellular signaling proteins) that correlated with response (i.e., AUC(ROC) > or = 0.66; P < or = 0.05) at a level greater than age. After accounting for age, similar findings were observed in the second study. For patients <60 years old, complete response was associated with the presence of intact apoptotic pathways. In patients > or =60 years old, nonresponse was associated with FLT3 ligand-mediated increase in phosphorylated Akt and phosphorylated extracellular signal-regulated kinase. Results were independent of cytogenetics, FLT3 mutational status, and diagnosis of secondary AML. CONCLUSIONS: These data emphasize the value of performing quantitative SCNP under modulated conditions as a basis for the development of tests highly predictive for response to induction chemotherapy. SCNP provides information distinct from other known prognostic factors such as age, secondary AML, cytogenetics, and molecular alterations and is potentially combinable with the latter to improve clinical decision making. Independent validation studies are warranted.

Palifermin reduces the incidence of oral mucositis in patients with metastatic colorectal cancer treated with fluorouracil-based chemotherapy.

PURPOSE: To characterize the efficacy and safety of palifermin in reducing the incidence of oral mucositis (OM) and diarrhea when administered to patients with metastatic colorectal cancer (CRC) receiving fluorouracil/leucovorin (FU/LV) chemotherapy. PATIENTS AND METHODS: Patients (N = 64) were randomly assigned to receive either placebo or palifermin (40 microg/kg for 3 consecutive days) before each of two consecutive cycles of chemotherapy with FU/LV. The incidence of OM and diarrhea, safety, disease progression, and survival were evaluated. RESULTS: Thirty-six patients received placebo and 28 patients received palifermin. The incidence of WHO grade 2 or higher OM was lower in patients who received palifermin compared with placebo (29% v 61% in cycle 1; 11% v 47% in cycle 2). FU dose reductions in the second chemotherapy cycle were more frequent in the placebo group (31%) than in the palifermin group (14%). Investigators reported lower mucositis scores and patients reported less severe symptoms with palifermin. There were no statistically significant differences in the incidence or severity of diarrhea or in overall survival between the groups. Overall, palifermin was safe and well tolerated. CONCLUSION: Palifermin administered at the indicated dosing regimen (40 microg/kg for 3 consecutive days) before chemotherapy was well tolerated and resulted in a statistically significant and clinically meaningful reduction in the incidence of WHO grade 2 or higher OM in patients with metastatic CRC.

Epratuzumab, a humanized anti-CD22 antibody, in aggressive non-Hodgkin's lymphoma: phase I/II clinical trial results.

PURPOSE: We conducted a single-center, dose-escalation study evaluating the safety, pharmacokinetics, and efficacy of epratuzumab, an anti-CD22 humanized monoclonal antibody, in patients with aggressive non-Hodgkin's lymphoma. EXPERIMENTAL DESIGN: Epratuzumab was administered once weekly for 4 weeks at 120-1000-mg/m2 doses to 56 patients [most (n = 35) with diffuse large B-cell lymphoma]. RESULTS: Patients were heavily pretreated (median, 4 prior therapies), 25% received prior high-dose chemotherapy with stem cell transplant, and 84% had bulky disease (> or =5 cm). Epratuzumab was well tolerated, with no dose-limiting toxicity. Most (95%) infusions were completed within 1 h. The mean serum half-life was 23.9 days. Across all dose levels and histologies, objective responses (ORs) were observed in five patients (10%; 95% confidence interval, 3-21%), including three complete responses. In patients with diffuse large B-cell lymphoma, 15% had ORs. Overall, 11 (20%) patients experienced some tumor mass reduction. Median duration of OR was 26.3 weeks, and median time to progression for responders was 35 weeks. Two responses are ongoing at > or =34 months, including one rituximab-refractory patient. CONCLUSIONS: These data demonstrate that epratuzumab has a good safety profile and exerts antitumor activity in aggressive non-Hodgkin's lymphoma at doses of > or =240 mg/m2, thus warranting further evaluation in this clinical setting.

Temperature-sensitive control of protein activity by conditionally splicing inteins.

Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.

Phase I/II trial of epratuzumab (humanized anti-CD22 antibody) in indolent non-Hodgkin's lymphoma.

PURPOSE: This single-center, dose-escalation study examines the safety, efficacy, and pharmacokinetics of epratuzumab (anti-CD22 humanized monoclonal antibody) in patients with recurrent indolent non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Patients had indolent NHL and recurrent disease after at least one chemotherapy regimen. Epratuzumab was administered intravenously at 120 to 1,000 mg/m2 over 30 to 60 minutes weekly for four treatments. RESULTS: Fifty-five patients received epratuzumab and were assessable for safety; 51 patients were assessable for response. Patients were heavily pretreated (50% had at least four prior regimens) and 49% had bulky disease (> or = 5 cm). Epratuzumab was well tolerated, with no dose-limiting toxicity. Circulating B cells transiently decreased without significant effects on T cells or immunoglobulin levels. More than 95% of infusions were completed in approximately 1 hour. Mean serum half-life was 23 days. Across all dose levels and histologies, nine patients (18%; 95% confidence interval, 8% to 31%) achieved objective response, including three complete responses (CRs). All responses were in patients with follicular NHL: 24% of these patients responded, including 43% in the 360 mg/m2 dose group and 27% in the 480 mg/m2 dose group. No responses were observed in other indolent histologies. Median duration of objective response was 79.3 weeks (range, 11.1 to 143.3 weeks), with median time to progression for responders of 86.6 weeks by Kaplan-Meier estimate. CONCLUSION: Epratuzumab was well tolerated at up to 1,000 mg/m2/wk (for 4 weeks) and had clinical activity. One third of responding patients achieved CR. A 43% objective response rate in follicular NHL patients treated at 360 mg/m2/wk indicates that this dose should be explored in additional studies.

CD22 as a target of passive immunotherapy.

CD22 is a 135-kd B-cell restricted sialoglycoprotein present in the cytoplasm of virtually all B-lineage cells but expressed on the B-cell surface only at mature stages of differentiation. In humans, the vast majority of IgM(+)IgD(+) B cells express cell-surface CD22, while in lymphoid tissues CD22 expression is high in follicular mantle and marginal zone B cells and weak in germinal center B cells. In B-cell malignancies, CD22 expression ranges from 60% to 80% depending on the histological type and on the assays used. The function of the CD22 molecule is uncertain, although recent studies have suggested roles for the molecule both as a component of the B-cell activation complex and as an adhesion molecule. CD22-deficient mice have a reduced number of mature B cells in the bone marrow and circulation; the B cells have a shorter lifespan and enhanced apoptosis, thus indicating a key role of this antigen in B-cell development/survival. After binding with its natural ligand(s) or antibodies, CD22 is rapidly internalized; this provides a potent costimulatory signal in primary B-cell and proapoptotic signals in neoplastic B cells. Preclinically CD22 has been shown to be an effective target for immunotherapy of B-cell malignancies using either "naked" or toxin-labeled or radiolabeled monoclonal antibodies. Clinical trials in patients with non-Hodgkin's lymphoma (NHL) (both indolent and aggressive disease) are now ongoing with a humanized naked anti-CD22 antibody (epratuzumab, Amgen Inc, thousand Oaks, CA and Immunomedics Inc, Morris Plains, NJ) used as single agent or in combination with other monclonal antibodies (ie, rituximab) and/or chemotherapy. Preliminary data from these studies showed these approaches to be effective and well-tolerated.