Recurrent B-cell Research Articles

Vivovec™ Surface-Engineered Lentiviral Particles Mediate In Vivo CAR T Generation with Potent and Highly Durable Activity in Non-Human Primates and Tumor-Bearing Humanized Mice

Introduction: Ex-vivo generated autologous CAR T cell therapy has transformed the treatment of hematologic malignancies, driving deep and durable responses in patients refractory to conventional therapies. However, multiple challenges including complex manufacturing, high cost, and toxic pre-conditioning regimens limits access to these therapies. To overcome these challenges, a novel lentiviral vector platform, VivoVec™, is being developed. The VivoVec™ platform comprises lentiviral particles surface-engineered with a multi-domain fusion (MDF) protein assembled from T cell activating and costimulatory ligands along with a CAR transgene payload, designed to provide an off-the-shelf solution for generation of CAR T cells in vivo. Here we have evaluated the performance of VivoVec™ particles in multiple preclinical models. Following a single VivoVec™ particle injection, CAR T cells are efficiently generated in vivo and expand in response to cognate antigen, eradicate target antigen expressing cells, and form CAR T cell memory populations, in the absence of lymphodepleting chemotherapy or exogenous supportive cytokines. Methods: Mice (NSG MHCI/II knockout): Mice were engrafted with CD19+ Nalm6 tumors and resting human PBMCs, followed by intraperitoneal administration of varying doses of VivoVec™ carrying an aCD19 CAR payload. Absolute CAR T-cell numbers in peripheral blood were quantified by flow cytometry, and Nalm6 tumor burden was assessed by bioluminescence imaging. Non-Human Primates ( Macaca nemestrina): VivoVec™ surface engineered with NHP-specific surrogate MDF and carrying a human/NHP cross-reactive aCD20 CAR payload. VivoVec™ was administered via intranodal injection to 3 animals at doses of 0.77e8/kg TU in 1 animal and 2.50 e8/kg TU in 2 animals. aCD20 CAR T expansion and B-cell depletion were continuously evaluated by flow cytometry approximately weekly over ~4 months. Body weight, body temperature, neurological exams, serum chemistry panels, and complete blood counts were assessed weekly for the duration of the study. Results: VivoVec™ cultured with resting PBMCs selectively bind T cells and some NK cells in an MDF surface engineering-dependent manner, resulting in T cell activation, transduction, and CAR expression. The resultant CAR T cells demonstrate polyfunctional activity through antigen-specific tumor cell killing of Nalm6 tumor cells, cytokine secretion, and proliferation in response to serial antigen exposure. VivoVec™ administration led to dose-dependent generation of CAR T cells in Nalm6-tumor bearing humanized NSG MHCI/II knockout mice. In vivo generated CAR T cells expanded and mediated potent and durable anti-Nalm6 tumor activity. Non-Human Primates: In the two animals receiving 2.50e8 TU/kg of particles, aCD20 CAR T cells were detected in peripheral blood by flow cytometry with an early peak expansion by day 10 (representing 25.9% or 61.3% of total T cells, respectively). In the first animal, B-cell aplasia was observed from day 7 to day 42, when a small recovering B-cell population was detectable. This was closely followed by a secondary CAR T-cell expansion peaking at day 51: the CAR T cell population represented 43.2% of T cells present in the blood and resulted in recurrent B-cell aplasia sustained through day 70. Persistent B cell aplasia in the second animal receiving the 2.50e8 TU/kg dose began at day 7 and continues to be monitored. VivoVec™ was well-tolerated with no evidence of toxicity associated within, or shortly after the period of administration. Mild CRS was observed in association with both early and late CAR T cell expansions. Conclusions: VivoVec™ with MDF surface engineering enables potent and specific generation of functional CAR T cells in vivo in multiple preclinical models, including immune competent NHPs. Importantly, the generation and expansion of CAR T cells occurs in the absence of lymphodepleting chemotherapy, or supportive exogenous cytokines, thus the MDF engineering could impart properties consistent with a natural T cell effector response followed by formation of functional memory T cells. VivoVec™ represents a novel therapeutic platform in oncology that has the potential to overcome many of the challenges associated with the current ex vivo CAR T cell approaches. Additional data from ongoing NHP studies will be provided at the time of presentation.

Chimeric Antigen Receptor T-Cell Therapy of Neurolymphomatosis Monitored by FDG PET/CT.

18 F-FDG PET/CT was performed to evaluate possible recurrent B-cell lymphoblastic lymphoma in a 34-year-old man. The images showed multiple foci of increased activity in the nerve root and peripheral nerve. A biopsy confirmed the diagnosis of neurolymphomatosis. After receiving chemotherapy, PET/CT showed progressive disease. The patient subsequently received the CD-19 chimeric antigen receptor T-cell therapy. A follow-up PET/CT acquired 30 days after chimeric antigen receptor T-cell therapy revealed no abnormal FDG activity.

67. Recurrent high grade B-cell lymphoma masquerading as a dentigerous cyst

B-cell lymphoma arises primarily within the lymph nodes but can present at extra-nodal sites. We present an unusual case of recurrent B-cell lymphoma presenting as a suspected dentigerous cyst in the mandible.

Top advances of the year: Pediatric oncology.

Accelerated discovery and collaborative research continue to highlight the remarkable progress that has been made in the diagnosis and treatment of pediatric cancers. This manuscript highlights important discoveries on how precision oncology is being incorporated into the diagnosis and treatment of childhood cancer at the national level to identify promising new therapies using a tumor-agnostic approach. In addition, we have highlighted three articles that incorporate genomics and cell-free DNA to better classify, monitor and incorporate risk-based therapies for children with medulloblastoma. Finally, we highlighted the important role of monclonal antiobody therapy in the treatment of recurrent B-cell leukemia and newly diagnosed high-risk neuroblastoma.

Efficacy and safety of chimeric antigen receptor T-cells treatment in central nervous system lymphoma: a PRISMA-compliant single-arm meta-analysis.

Chimeric antigen receptor (CAR) T cells are used to treat refractory and recurrent B-cell lymphoma. When administered intravenously, CAR T cells can be detected in cerebrospinal fluid, and thus represent a promising method for the treatment of central nervous system lymphoma (CNSL). This meta-analysis aimed to clarify the effectiveness and safety of CAR T-cell therapy in the treatment of CNSL. Studies involving patients with CNSL who received CAR T-cell therapy that reported overall response (OR), complete response (CR), and partial response (PR) were included. A random-effects or fixed-effects model with double arcsine transformation was used for the pooled analysis and 95% confidence intervals (CI) were determined for all outcomes. Eight studies, comprising 63 patients, were identified and were included in the meta-analysis. The pooled OR and CR rates after treatment with CAR T cells were 69% (95% CI, 56-81%) and 51% (95% CI, 37-64%), respectively. The pooled rate of progressive disease after remission was 38% (95% CI, 21-55%). The pooled rate for neurotoxicity grade 3 or above was 12% (95% CI, 3-24%, I2 = 0.00%, p = 0.53). No treatment-related deaths were reported. CAR T-cell therapy is a promising option for the treatment of CNSL owing to a high short-term remission rate and controllable side effects. However, the high recurrence rate after remission must be addressed. Long-term follow-up data with large sample sizes are also needed to better assess the effectiveness and safety of CAR T-cell therapy. This meta-analysis was registered in the international prospective register of systematic reviews (PROSPERO) (CRD42022301332).

Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy

AbstractThe discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell–omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Formation of necrotizing vasculitis in the patient with diffuse B-cell large cell lymphoma on immune checkpoint inhibitor (PD-1) therapy

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have significantly improved the prognosis and clinical outcomes for patients with malignancies in many types of cancer. Nivolumab is a human monoclonal antibody that blocks the interaction between PD-1 and PD-L1/2, thereby preventing inhibition of T-cells, which contributes to the antitumor response. The use of ICT can lead to autoimmune aggression and the development of adverse events. Most of these are eliminated on their own after ICT therapy and steroid treatment have been discontinued.
 CLINICAL CASE DESCRIPTION: A 73-year-old patient with recurrent large B-cell lymphoma, on the background of R-CHOP, R-CHOP-mini, R-GDOx therapy incomplete clinical effect and frequent relapses have been registered. On immunohistochemistry, PD-L1 was positive in 90 % of the tumor cells. A decision has been made to administer the checkpoint inhibitor (PD-1) nivolumab, which developed a necrotizing vasculitis after three injections.
 CONCLUSION: On temporary withdrawal of the drug and administration of prednisolone at a dose of 1 mg/kg/day, the skin has recovered completely. When the PD-1 inhibitor was reapplied, there was no recurrence of autoimmune aggression. The treatment has been continued in the full way.

A Mathematical Description of the Bone Marrow Dynamics during CAR T-Cell Therapy in B-Cell Childhood Acute Lymphoblastic Leukemia.

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients’ experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T-cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells, and CAR T-cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T-cells, and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T-cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load, and leukemia burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model.

Application of pixantrone in a III line of treatment as a bridge therapy to allotransplantation of stem cells

Diffuse large B-cell lymphomas are the most common group among all lymphomas. Despite the effectiveness of the I line therapy in 10–40% of patients, the possibility of relapse should be taken into account. The anthracycline antibiotics used in the II line therapy carry a high risk of cardiac complications. In patients with refractory or recurrent disease, emergency treatment is used, aiming for the transplantation of hematopoietic cells. If the II line therapy fails, the prognosis is bad. A new chance for patients with refractory or recurrent B-cell lymphoma is pixantrone dimaleate, a drug structurally related to anthracyclines with a significantly lower cardiotoxic potential. The paper presents a case of a 65-year-old patient with relapsing DLBCL lymphoma, after two treatment lines: R-CHOP and R-DHAP, and after autologous transplantation of stem cells, in which the use of pixantrone in the treatment of recurrence allowed to achieve remission and prepare the patient for the allo-HSCT procedure.

Anakinra in Preventing Severe Chimeric Antigen Receptor T-Cell Related Encephalopathy Syndrome in Patients With Recurrent or Refractory Large B-cell Lymphoma

Anakinra in Preventing Severe Chimeric Antigen Receptor T-Cell Related Encephalopathy Syndrome in Patients With Recurrent or Refractory Large B-cell Lymphoma

Inotuzumab Ozogamicin and Chemotherapy in Treating Patients With Recurrent or Refractory B-cell Acute Lymphoblastic Leukemia

Inotuzumab Ozogamicin and Chemotherapy in Treating Patients With Recurrent or Refractory B-cell Acute Lymphoblastic Leukemia

Clinical analysis of donor-derived CD19 chimeric antigen receptor T lymphocytes for the treatment of recu-rrent acute B-cell leukemia after allogeneic hematopoietic stem cell transplantation

Objective To discuss the cure effect and side effects of donor anti-CD19 chimeric antigen receptor T lymphocytes(CD19 CAR-T)for treating recurrent acute B-cell leukemia after allogeneic hematopoietic stem-cell transplantation(Allo-HSCT), and to analyze the influencing factors for this therapy. Methods The clinical data of 5 acute B-cell leukemia patients were analyzed retrospectively who relapsed after Allo-HSCT and received donor CD19 CAR-T therapy at Beijing Children′s Hospital from July 2015 to October 2017.Disease status before infusion, conditioning regimen, reinfusion cell dose, and side-effect of CAR-T infusion, changes in the related immunological indicators, and follow-up treatment results were investigated. Results One patient had no effect, other patients got remission or minimal residual disease(MRD) negative within 4 weeks after CAR-T infusion, and the middle time was 14 days.Peripheral CAR-T peak happened 2 weeks after CAR-T infusion.By the last follow, 2 patients died of leukemia, 3 patients were alive, and 1 case of them lived with tumor after CD19 negative relapse, others lived with disease-free condition.Cytokine release syndrome(CRS) was the most common side effect, happening in 1 to 2 weeks after infusion, 1 patient had neurologic toxicities, and 2 patients had suspicious graft-versus-host disease. Conclusions Donor CD19 CAR-T therapy has a good short-term effect for relapsed B-cell leukemia patients after Allo-HSCT, but long-term effect requires further observation; CRS is the most common side-effect.Off-target and cell exhaustion are the main reasons for defeat. Key words: Donor chimeric antigen receptor T cell; CD19; Relapse acute leukemia; Allogeneic hematopoietic stem cell transplantation

Possibilities of therapy recurrent b-cell chronic lymphocytic leukemia associated with autoimmune complications: data from literature and personal observation

The description of the clinical observation of the successful therapy with ibrutinib recurrent B-cell chronic lymphocytic leukemia associated with autoimmune complications is given. The reasons for occurrence the autoimmune complications of CLL, their effect on the disease prognosis as well as the tactics of conducting similar patients have been considered.

FIRST CLINICAL RESULTS OF ADCT‐402, A NOVEL PYRROLOBENZODIAZEPINE‐BASED ANTIBODY DRUG CONJUGATE (ADC), IN RELAPSED/REFRACTORY B‐CELL LINEAGE NHL

Introduction: CD19 expression has been demonstrated in many types of NHL, including FL and DLBCL. Pyrrolobenzodiazepine (PBD) is a DNA cross-linking agent. ADCT-402, a humanized antibody directed against human CD19 conjugated to a PBD dimer toxin, has demonstrated potent pre-clinical anti-tumor activity against CD19-expressing B-cell malignancies. Interim results of the first in-human clinical study of ADCT-402 in the difficult-to-treat relapsed/refractory NHL setting are reported here. Methods: Patients (pts) with recurrent B-cell NHL who had failed or were intolerant to established therapies, or had no other treatment options available, were assigned IV infusions of ADCT-402 every 3 weeks (1 cycle) according to a 3 + 3 dose-escalation study design. No intra-patient dose escalation was allowed. Safety, tolerability, and efficacy were assessed. Results: As of 3 Feb 2017, 37 pts (23 male, 14 female; median age: 69 years [range 24–85]; median number of previous therapies: 3 [range 1–10]) have been recruited. Diagnoses included DLBCL (n = 22), MCL (n = 7), FL (n = 6) and other B-cell NHL (n = 2). Pts received ADCT-402 doses ranging from 15 to 200 μg/kg (median cycles: 3 [range 1–11]). Treatment-emergent adverse events (TEAEs) included fatigue (21 [56.8%] pts), peripheral edema (10 [27.0%] pts), and nausea (9 [24.3%] pts). Grade ≥ 3 TEAEs included decreased neutrophil count (4 [10.8%] pts), increased γ-glutamyltransferase (3 [8.1%] pts), neutropenia (3 [8.1%] pts), and thrombocytopenia (2 [5.4%] pts). TEAEs in 4 (10.8%) pts led to ADCT-402 withdrawal (increased γ-glutamyltransferase; increased blood alkaline phosphatase and γ-glutamyltransferase; fatigue and edema; abdominal pain and myalgia). No DLTs were reported and the MTD was not reached. 15/34 (44.1%) evaluable pts achieved a CR or PR (overall response [OR]), including 9/22 (40.9%) pts with DLBCL. 2/15 pts progressed by data cut-off. One 30 μg/kg pt continues in CR for >24wks. Table 1 shows the best OR by dose. 10/17 (58.8%) pts in the ≥120 μg/kg cohort achieved OR, including 7/12 (58.3%) with DLBCL. Figure 1 shows nodal regression. Preliminary PK and anti-drug antibody data will be reported at the meeting. Conclusions: ADCT-402 demonstrates encouraging single-agent anti-tumor activity and manageable toxicity in recurrent B-cell lineage NHL. At doses >120 μg/kg, the OR in DLBCL is 58%. Further evaluation in specific NHL subtypes is warranted. A dose expansion in DLBCL is planned. http://clinicaltrials.gov/show/NCT02669017 * Best visit response based on the 2014 Lugano Classification Criteria Key: CR, complete response; OR, overall response (CR+PR); PD, progressive disease; PR, partial response; SD, stable disease Keywords: CD19; diffuse large B-cell lymphoma (DLBCL); non-Hodgkin lymphoma (NHL).

Genomic analysis of adult B-ALL identifies potential markers of shorter survival

B lymphoblastic leukemia (B-ALL) in adults has a higher risk of relapse and lower long-term survival than pediatric B-ALL, but data regarding genetic prognostic biomarkers are much more limited for adult patients. We identified 70 adult B-ALL patients from three institutions and performed genome-wide analysis via single nucleotide polymorphism (SNP) arrays on DNA isolated from their initial diagnostic sample and, when available, relapse bone marrow specimens to identify recurring copy number alterations (CNA). As B-cell developmental genes play a crucial role in this leukemia, we assessed such for recurrent deletions in diagnostic and relapse samples. We confirmed previous findings that the most prevalent deletions of these genes occur in CDKN2A, IKZF1, and PAX5, with several others at lower frequencies. Of the 16 samples having paired diagnostic and relapse samples, 5 showed new deletions in these recurrent B-cell related genes and 8 showed abolishment. Deletion of EBF1 heralded a significant negative prognostic impact on relapse free survival in univariate and multivariate analyses. The combination of both a CDKN2A/B deletion and an IKZF1 alteration (26% of cases) also showed a trend toward predicting worse overall survival compared to having only one or neither of these deletions. These findings add to the understanding of genomic influences on this comparably understudied disease cohort that upon further validation may help identify patients who would benefit from upfront treatment intensification.

Clonal relationships in recurrent B-cell lymphomas.

Immunoglobulin (Ig) gene rearrangements remain largely unmodified during the clonal expansion of neoplastic cells. We investigated the clonal relationships between lymphoma components at diagnosis and at relapse by analyzing Ig gene rearrangements. A BIOMED-2 multiplex polymerase chain reaction (PCR) assay was performed in 27 patients using formalin-fixed paraffin embedded tissues, with subsequent cloning and sequencing of the amplified Ig genes in 17 patients. All 27 cases of primary and corresponding relapsed tumors showed monoclonal rearrangements of the Ig genes by BIOMED-2 PCR. Whereas IgVH or IgVK fragment lengths were identical in 8/27 pairs (30%), fragment lengths differed in 19/27 pairs (70%). In 17 cases analyzed by sequencing, an identical VDJ gene rearrangement was confirmed in 4/4 pairs (100%) with the same fragment lengths and in 10/13 pairs (77%) with different fragment lengths. Four of 17 primary lymphomas had multiple VDJ rearrangements, and three of them showed an unrelated relapse. Unrelated relapse was observed in 1/8 mantle cell lymphomas, 1/5 diffuse large B-cell lymphomas, and a large B cell lymphoma developed in a patient with a small lymphocytic lymphoma. Unrelated relapses developed after a longer disease-free interval and tended to show poorer outcome compared with related relapse. In summary, relapse of a lymphoma from an unrelated clone is uncommon, but can occur in B-cell lymphomas. Clonal relationships should be determined by sequencing of the Ig genes, and not just by comparing the PCR product size.

Abstract B19: Population pharmacokinetic-pharmacodynamic (PKPD) modeling of ibrutinib in patients with B-cell malignancies.

Abstract Ibrutinib (IBRU) is an oral Bruton's tyrosine kinase (BTK) inhibitor, approved by US FDA for the treatment of chronic lymphocytic leukemia (CLL/SLL) and mantle cell lymphoma (MCL) patients having received at least one prior therapy. A nonlinear mixed-effects population model was developed to describe the PK of IBRU in patients with B-Cell malignancies and to establish the effect of pathophysiological covariates on its PK behavior. The relationship between PK and BTK engagement in peripheral blood mononuclear cells (PBMC) was also explored. IBRU PK data (3477 observations in 245 patients) were available in patients with MCL, CLL/SLL and recurrent B-cell malignancies at dose levels from 1.25 to 12.5 mg/kg and at fixed doses from 420 to 840 mg once daily. An additional phase 2 study in 119 patients with MCL (772 observations) treated at 560 mg once daily was used to validate the PK model. BTK occupancy was assessed (694 observations in 127 patients) in PBMCs using a fluorescent affinity probe. Various models were tested on the data using the first-order conditional estimation method as implemented in NONMEM version 7.1. A 2-compartment linear model with sequential zero-first order absorption and first order elimination was able to accommodate available PK data, including those of the validation dataset (prediction errors <15%). PK was dose- and time- independent. IBRU was rapidly absorbed, extensively distributed (volume of distribution at steady-state ~ 10,000 L) and cleared (apparent oral clearance ~1000 L/h). Relative bioavailability in the fasting state was about one third lower compared to the fed condition used in the clinical trials. No significant effect of other pathophysiological covariates on the PK was found (including sex, age or indication) except for body weight and coadministration of antacids, which had a marginal effect on the volume of distribution and duration of absorption, respectively. Analysis of PK-BTK engagement suggested that IBRU is a potent inhibitor of the BTK activity and that its interaction with BTK is rapid and durable. Citation Format: Italo Poggesi, Maria Luisa Sardu, Eleonora Marostica, Juthamas Sukbuntherng, Betty Y. Chang, Jan de Jong, Xavier Woot de Trixhe, An Vermeulen, Giuseppe De Nicolao, Susan Mary O'Brien, John C Byrd, Ranjana H Advani, Danelle Frances James, William Deraedt, Darrin Beaupre, Michael Wang. Population pharmacokinetic-pharmacodynamic (PKPD) modeling of ibrutinib in patients with B-cell malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B19.

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia.

BackgroundWe present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL.ResultsWe used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (‘other’ subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5.ConclusionsOur findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0039-z) contains supplementary material, which is available to authorized users.

Abstract 4634: Population pharmacokinetic model of ibrutinib, a Bruton's tyrosine kinase inhibitor, for the treatment of B-cell malignancies

Abstract Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are B-cell malignancies with initial high response rate to chemoimmunotherapy, but are largely considered incurable. Ibrutinib (PCI-32765), an oral Bruton's tyrosine kinase inhibitor recently approved to treat MCL, is under development for other B-cell malignancies. We developed a population pharmacokinetic (PK) model for describing data collected to date in clinical trials with ibrutinib. Ibrutinib plasma data were available from 3 clinical trials: 1) a phase 1 dose-escalation study in recurrent B-cell malignancies (doses: 1.25-12.5 mg/kg and 560 mg fixed); 2) a phase 1b/2 dose-finding study in CLL (doses: 420 and 840 mg); 3) an open-label, phase 2, fixed-dose study in MCL (dose: 560 mg). Overall, ≈3477 observations were collected in 245 patients following single and repeated daily dosing on different treatment days. A 2-compartment model with sequential zero to first order absorption and first order elimination was implemented. Analyses were performed with a log-transform-both-sides approach. Additive and exponential models were used to describe residual and inter-individual variability, respectively. The first-order conditional estimation method was implemented using NONMEM v 7.1. A linear model constructed with data collected following single and repeated doses of ibrutinib at different dose levels demonstrated that the PK was dose and time independent. Ibrutinib was rapidly absorbed and was characterized by a high oral plasma clearance (≈1000 L/h with between-subject variability of 21.9%; this, for a dose of 560 mg, would lead to an average steady state concentration of ≈22 ng/mL, ie, ≈50 nM) and a high apparent volume of distribution at steady state (≈10,000 L). Though both values are confounded by absolute bioavailability, these values suggest that ibrutinib clearance and volume are high. The half-lives of distribution and terminal phases were estimated to be <1 h and ≈14 h, respectively. Administration of ibrutinib in fasting state was characterized by a 67% relative bioavailability compared to both meal conditions used in the clinical trials and administration after a high-fat meal. The PK parameters were not found to be significantly different between studies and clinical indications. Body weight and coadministration of antacids had a marginal effect on volume of distribution and duration of absorption processes, respectively. No significant effect of covariates such as age, gender, patient pretreatment, or clinical chemistry data was found. Population parameters and inter-individual variability were estimated with good precision. The model provided satisfactory goodness of fit, individual fittings, and visual predictive checks. In conclusion, the proposed population PK model was able to accommodate the plasma concentration-time profiles of ibrutinib across various trials. Citation Format: Eleonora Marostica, Juthamas Sukbuntherng, David Loury, Jan De Jong, Xavier Woot de Trixhie, An Vermeulen, Giuseppe de Nicolao, Susan O'Brien, John C. Byrd, Ranjana Advani, Jesse McGreivy, Italo Poggesi. Population pharmacokinetic model of ibrutinib, a Bruton's tyrosine kinase inhibitor, for the treatment of B-cell malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4634. doi:10.1158/1538-7445.AM2014-4634

CXCL12 Mediates Immunosuppression in the Lymphoma Microenvironment after Allogeneic Transplantation of Hematopoietic Cells

Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen-presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)-based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12-dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT.