Targeting refractory/recurrent neuroblastoma and osteosarcoma with anti-CD3×anti-GD2 bispecific antibody armed T cells.

BACKGROUND: The survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits of anti-CD3×anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs). Preclinical studies demonstrated the high cytotoxicity of GD2BATs against GD2+cell lines, leading to the initiation of a phase I/II study in recurrent/refractory patients. METHODS: The 3+3 dose escalation phase I study (NCT02173093) encompassed nine evaluable patients with NB (n=5), osteosarcoma (n=3), and desmoplastic small round cell tumors (n=1). Patients received twice-weekly infusions of GD2BATs at 40, 80, or 160×106 GD2BATs/kg/infusion complemented by daily interleukin-2 (300,000 IU/m2) and twice-weekly granulocyte macrophage colony-stimulating factor (250 µg/m2). The phase II segment focused on patients with NB at the dose 3 level of 160×106 GD2BATs/kg/infusion. RESULTS: Of the 12 patients enrolled, 9 completed therapy in phase I with no dose-limiting toxicities. Mild and manageable cytokine release syndrome occurred in all patients, presenting as grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody-associated pain was minimal. Median overall survival (OS) for phase I and the limited phase II was 18.0 and 31.2 months, respectively, with a combined OS of 21.1 months. A phase I NB patient had a complete bone marrow response with overall stable disease. In phase II, 10 of 12 patients were evaluable: 1 achieved partial response, and 3 showed clinical benefit with prolonged stable disease. Over 50% of evaluable patients exhibited augmented immune responses to GD2+targets post-GD2BATs, as indicated by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines. CONCLUSIONS: This study demonstrated the safety of GD2BATs up to 160×106 cells/kg/infusion. Coupled with evidence of post-treatment endogenous immune responses, our findings support further investigation of GD2BATs in larger phase II clinical trials.

Targeting GD2-positive Refractory/Resistant Neuroblastoma and Osteosarcoma with Anti- CD3 x Anti-GD2 Bispecific Antibody Armed T cells.

Background: Since treatment of neuroblastoma (NB) with anti-GD2 monoclonal antibodies provides a survival benefit in children with minimal residual disease and our preclinical study shows that anti-CD3 x anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs) were highly cytotoxic to GD2+ cell lines, we conducted a phase I/II study in recurrent/refractory patients to establish safety and explore the clinical benefit of GD2BATs. Methods: The 3+3 dose escalation study (NCT02173093) phase I involved 9 evaluable patients with NB (n=5), osteosarcoma (OST) (n=3), and desmoplastic small round cell tumors (DSRCT) (n=1) with twice weekly infusions of GD2BATs at 40, 80, or 160 x 106 GD2BATs/kg/infusion with daily interleukin 2 (300,000 IU/m2) and twice weekly granulocyte-macrophage colony stimulating factor (250 µg/m2). Phase II portion of the trial was conducted in patients with NB at the dose 3 level of 160 x 106 GD2BATs/kg/infusion but failed to enroll the planned number of patients. Results: Nine of 12 patients in the phase I completed therapy. There were no dose limiting toxicities (DLTs). All patients developed mild and manageable cytokine release syndrome (CRS) with grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody associated pain was not significant in this study. The median OS for patients in the Phase I and limited Phase II was 18.0 and 31.2 months, respectively, whereas the combined OS was 21.1 months. There was a complete bone marrow response with overall stable disease in one of the phase I patients with NB. Ten of 12 phase II patients were evaluable for response: 1 had partial response. Three additional patients were deemed to have clinical benefit with prolonged stable disease. More than 50% of evaluable patients showed augmented immune responses to GD2+ targets after GD2BATs as measured by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines. Conclusions: Our study demonstrated safety of up to 160 x 106 cells/kg/infusion of GD2BATs. Combined with evidence for the development of post treatment endogenous immune responses, this data supports further investigation of GD2 BATs in larger Phase II clinical trials.

Oncolytic DNX-2401 virotherapy plus pembrolizumab in recurrent glioblastoma: a phase 1/2 trial.

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Ongoing Response in a Multiply Relapsed Metastatic Posterior Fossa Ependymoma A After Vorinostat and Concomitant Irradiation.

Posterior fossa ependymomas A confer the worst prognosis among all subtypes. They demonstrate distinct epigenetic changes, which can be targeted with epigenetic modifiers like histone deacetylase inhibitors (Vorinostat). We describe a 3-year-old male diagnosed with a posterior fossa ependymoma who had a number of recurrences requiring multimodal therapy. Molecular analysis demonstrated a BCL-6 corepressor mutation, and methylation profiling matched with posterior fossa ependymomas A. He received craniospinal irradiation and focal boost with Vorinostat. Serial imaging after irradiation revealed a progressively decreasing tumor burden with nearly complete resolution of disease at 15 months. Histone deacetylase inhibitors demonstrate promise in treatment of carefully selected cases of ependymoma.

Molecular insights into malignant progression of atypical choroid plexus papilloma.

Choroid plexus tumors are rare pediatric neoplasms ranging from low-grade papillomas to overtly malignant carcinomas. They are commonly associated with Li-Fraumeni syndrome and germline TP53 mutations. Choroid plexus carcinomas associated with Li-Fraumeni syndrome are less responsive to chemotherapy, and there is a need to avoid radiation therapy leading to poorer outcomes and survival. Malignant progression from choroid plexus papillomas to carcinomas is exceedingly rare with only a handful of cases reported, and the molecular mechanisms of this progression remain elusive. We report a case of malignant transformation of choroid plexus papilloma to carcinoma in a 7-yr-old male with a germline TP53 mutation in which we present an analysis of molecular changes that might have led to the progression based on the next-generation genetic sequencing of both the original choroid plexus papilloma and the subsequent choroid plexus carcinoma. Chromosomal aneuploidy was significant in both lesions with mostly gains present in the papilloma and additional significant losses in the carcinoma. The chromosomal loss that occurred, in particular loss of Chromosome 13, resulted in the losses of two critical tumor suppressor genes, RB1 and BRCA2, which might play a possible role in the observed malignant transformation.

Patterns and correlates of preserved humoral immunity to vaccines in children following allogeneic hematopoietic stem cell transplantation.

Data on preservation of vaccine immunity following allogeneic HSCT in children is limited. We investigated vaccine titers and sought correlations with patient characteristics in this study. Twenty-eight cases were retrospectively analyzed. Antibody concentrations against hepatitis A, hepatitis B, 3 poliovirus serotypes, tetanus, diphtheria, measles, mumps, rubella, varicella, and 13 pneumococcus serotypes were measured as part of planned monitoring following HSCT. Protective antibody levels were found for hepatitis A in 79% of the recipients, measles in 54%, all poliovirus serotypes in 50%, tetanus in 50%, rubella in 50%, varicella in 46%, hepatitis B in 46%, mumps in 43%, diphtheria in 29%, and ≥7/13 pneumococcus serotypes in 46%; lowest level observed for diphtheria and highest for hepatitis A prior to starting post-HSCT immunizations. In univariate analysis, patients with non-malignant diseases (P = .03) and without GvHD (P = .04) had more protective titers. A significant positive association was found among vaccine titers against the microorganisms or the serotypes of the same microorganism, which were administered together in the same product, including polio serotypes, diphtheria and tetanus, mumps, measles, and rubella. Higher degrees of sero-positivity are likely to be due to lack of prior chemotherapy in non-malignant disease cases and lesser immunosuppression in patients without GvHD. Monitoring long-term vaccine titers and administering vaccines accordingly could be evaluated for post-HSCT re-immunization practice.

Delayed Granulocyte Colony-Stimulating Factor (G-CSF) Administration after Chemotherapy Reduces Total G-CSF Doses without Affecting Neutrophil Recovery in a Randomized Clinical Study in Children with Solid Tumors.

The use of G-CSF after myelotoxic chemotherapy accelerates neutrophil recovery reducing the risk of febrile neutropenia. Current guidelines recommend initiating G-CSF 24 hours after myelotoxic chemotherapy. However, the optimal timing of post-chemotherapy G-CSF administration has not been elucidated. Our previous work in murine models demonstrated that the reappearance of myeloid progenitors does not occur in bone marrow until 3-4 days after completion of chemotherapy suggesting that delayed G-CSF administration may be equally efficacious compared to current practice. We conducted a prospective, randomized, crossover study to compare the absolute neutrophil count (ANC) recovery after chemotherapy and a delayed G-CSF administration to a standard G-CSF administration schedule with early G-CSF start. A total of 21 children with solid tumors who received 2 identical cycles of myelotoxic chemotherapy were randomized to start receiving G-CSF either 24 hours after completion of chemotherapy or on the day that their ANC dropped below 1,000/mm3. There was no significant difference in the time to neutrophil recovery (ANC > 1,000/mm3 post nadir) between the two G-CSF administration schedules: 16.0 ± 0.5 days in the standard group compared to 16.7 ± 0.4 days in the delayed group (p = 0.36). The total number of G-CSF doses given, however, was significantly less in the delayed group: 6.7 ± 0.6 compared to 10.5 ± 0.6 doses in the standard group (p < 0.0001). Our data show that a delayed administration of post chemotherapy G-CSF resulted in a significant reduction in the number of G-CSF injections without compromising the G-CSF effects on neutrophil recovery.

Pneumocephalus in a Pediatric Patient with Glioma Receiving Trametinib.

The mitogen-activated protein kinase (MAPK) pathway consists of the Ras/Raf/MEK/ERK signaling cascade, and its upregulation plays a major role in the pathogenesis of pediatric astrocytomas and molecular inhibitors of this pathway including trametinib and dabrafenib have been tested in early-phase clinical trials and used by pediatric oncologists in children with BRAF-mutated gliomas. We report a clinical case where a child with progressive BRAF-mutated glioma developed an uncommon and difficult to manage complication - pneumocephalus from intracranial air entry and trapping through dehisced surgical wounds and preexisting skull burr holes. The patient's wound breakdown coincided with skin toxicity from MEK inhibitor therapy. With increasing use of targeted molecular inhibitors in pediatric neuro-oncology, this case illustrates the potentially complicated course of MEK inhibitor therapy in patients with scalp surgical wounds and burr holes that were placed within few weeks from initiation of drug therapy, especially if patients have additional factors that can contribute to poor wound healing such as use of steroids and malnutrition.

Role of Initiating Supportive Care Preceding Veno-occlusive Disease Diagnosis Following Allogeneic Hematopoietic Stem Cell Transplantation in Children.

Severe veno-occlusive disease (VOD) following hematopoietic stem cell transplantation has a high mortality rate. The clinical course of VOD, role of preemptive and aggressive supportive care, and outcomes were investigated in a retrospective study from 2007 to 2014. Defibrotide was not available in all but one case with VOD at our center during the study. Forty-nine allogeneic transplants with intravenous busulfan-based or total body irradiation-based myeloablative conditioning were included. The median after hematopoietic stem cell transplantation day for suspicion of developing VOD (pre-VOD phase) was 6 due to weight gain, hepatomegaly, and/or mild increase in total bilirubin without fulfilling the modified Seattle criteria in 22 cases (45%). Despite fluid restriction, aggressive diuresis, and fresh frozen plasma infusions, 16 patients (33%) developed VOD by +10 days. Five cases (31%) had severe, 9 (56%) moderate, and 2 (13%) mild VOD. Eight cases (50%) required transfer to intensive care. One patient was given defibrotide, which was later discontinued due to concerns of adverse effects. Day +100 survival was 100% with complete resolution of VOD. Preemptive and aggressive supportive care could help achieve favorable outcomes in VOD and may have ameliorated the severity. This approach may be combined with other measures in the prevention/treatment of VOD.

Small Split Doses of CD34+ Peripheral Blood Stem Cells to Support Repeated Cycles of Nonmyeloablative Chemotherapy.

Cumulative myelosuppression is the main limiting factor for administration of repeated cycles of chemotherapy. We present a case series of five pediatric patients with high-risk solid malignancies who received small split peripheral blood stem cells (PBSC) doses of less than 1 × 106/kg CD34+ cells obtained after a single leukapheresis procedure and given after repeated cycles of ICE (ifosfamide, carboplatin, and etoposide) chemotherapy. Mean duration to absolute neutrophil count (ANC) recovery to >1000/mm3 and platelet recovery to >50 × 103/mm3 was 17.1 and 24.3 days. Using split doses of PBSC prevented prolonged neutropenia after repeated cycles of submyeloablative chemotherapy.

Choroid plexus carcinoma in children: the Head Start experience.

BACKGROUND: Choroid plexus carcinoma (CPC) is a rare aggressive intracranial neoplasm with a predilection for young children and a historically poor outcome. Currently, no defined optimal therapeutic strategy exists. The Head Start (HS) regimens have included irradiation-avoiding strategies in young children with malignant brain tumors using high dose chemotherapy to improve survival and minimize neurocognitive sequelae. PROCEDURE: Three sequential HS studies have been conducted from 1991 to 2009. HS treatment strategy has consisted of maximal surgical resection followed by five cycles of intensive induction followed by consolidation myeloablative chemotherapy with autologous hematopoietic stem cell rescue (AuHCR). Irradiation was given following recovery from consolidation based on the patient's age and evidence of residual disease. RESULTS: Twelve children with CPC (median age of 19.5 months) have been treated with HS regimens. Ten patients had >95% resection. Three patients had disseminated disease at diagnosis. Ten patients completed consolidation of whom five are alive, irradiation and disease free at 29, 43, 61, 66 and 89 months from diagnosis. Seven patients experienced tumor recurrence/progression at a median time of 13 months (range 2-43 months). Five patients received irradiation, one for residual disease and four upon progression or recurrence, of whom one is alive at 61 months. The 3- and 5-year progression-free survivals are 58% and 38% and overall survivals 83% and 62% respectively. Late deaths from disease beyond 5 years were also noted. CONCLUSION: Head Start strategies may produce long-term remission in young children with newly diagnosed CPC with avoidance of cranial irradiation.

Hematopoietic cell transplantation and cellular therapeutics in the treatment of childhood malignancies.

Hematopoietic cell transplantation (HCT) represents the most common and effective form of immunotherapy for childhood malignancies. The role of the graft-versus-leukemia effect in allogeneic HCT has been well established in childhood malignancies, but is also associated with short-term and long-term morbidity. HCT may be ineffective in some settings at obtaining control of the malignancy, and as such, cannot be used as a universal cancer immunotherapy. Novel therapies using dendritic cell vaccinations, tumor-infiltrating lymphocytes, and chimeric antigen receptor T cells are being evaluated as potential adjuvants to HCT.

How many sources should be cultured for the diagnosis of a blood stream infection in children with cancer?

UNLABELLED: Blood stream infection (BSI) in a child with cancer is a life-threatening condition while blood cultures (BCs) play a crucial role for the diagnosis. The current practice of obtaining a BC from all sources--peripheral vein, central venous catheter (CVC), all lumens--is controversial and therefore in this study, our aim was to evaluate the utility of obtaining BCs from all these available sources. A retrospective study of BC sets obtained from all newly diagnosed patients with malignancy was conducted. A total number of 633 BC sets from 123 boys and 88 girls (median age: 5 5/12 years) were evaluated during infection episodes from January 1, 2005, to August 31, 2010. Among these cases, 134 were classified as true BSI (21%), 468 as non-BSI, and 31 as false-positive. In 64 cases, the results from peripheral and CVC sources were discordant: 57 catheter positive-peripheral negative and seven catheter negative-peripheral positive. Consequently, seven out of 134 true BSIs (5.2%) would not have been identified if only a BC from a CVC had been obtained. Moreover, if no BCs from all lumens had been obtained we would have missed up to 25% of true-positive BSIs. In the same way, if BCs from all sources had not been drawn we would not have detected up to 52% of true-positive BSIs. CONCLUSION: Obtaining BCs during an infection episode from all sources in a child with cancer is still mandatory.

Predictive factors for blood stream infections in children with cancer.

Blood Stream Infections (BSI) are among the most serious infections in children with cancer and are potentially life threatening. A retrospective study of blood cultures obtained from all newly diagnosed patients--from January 1, 2005 to December 31, 2009--with malignancy was conducted. In this study, our aim was to identify clinical and laboratory variables associated with a BSI in a child with malignancy. Among 1004 separate infection episodes detected in 261 patients, 198 were classified as true BSI (19.7%). Univariate analysis showed that factors such as younger age, race, temperature ≥40°C, presence of chills and hypotension, time interval from the last chemotherapy, treatment for recurrent disease or a history of Stem Cell Transplantation, low hemoglobin, low-Platelets count, and Absolute Neutrophils count less than 4 × 10(9)/L were predictive for a BSI. Patients with a catheter in place and especially if this catheter was tunneled and/or multiple lumen were more likely to have a BSI. Being on antibiotics, the history of a BSI during the previous month and having received a red cell or platelet transfusion during the prior 15 days also increased the likelihood for a BSI. According to a multivariate logistic regression analysis, the factors that remained significant were the younger age, the African American race, the presence of chills or hypotension, the use of tunneled or multiple lumen catheters, the administration of antibiotics during the previous 15 days and a low-PLT count.

Anti-CD3 × anti-GD2 bispecific antibody redirects T-cell cytolytic activity to neuroblastoma targets.

BACKGROUND: The ganglioside GD2 is an attractive target for immunotherapy of neuroectodermal tumors. We tested a unique bispecific antibody anti-CD3 × anti-GD2 (3F8BiAb) for its ability to redirect activated T cells (ATC) to target GD2-positive neuroblastomas. PROCEDURE: ATC were generated from normal human peripheral blood mononuclear cells (PBMC) by stimulating the PBMC with OKT3 and expanding the T cells in the presence of interleukin 2 (IL-2) for 14 days. ATC were armed with 3F8BiAb (100 ng/10(6) cells) or Her2BiAb (50 ng/10(6) cells) prior to use. 3F8 BiAb were tested for its dual-binding specificity to GD2 expressed on cancer cell lines and CD3 expressed on ATC. 3F8BiAb-armed ATC were further tested ex vivo for their cytotoxicity against GD2 positive tumor targets and its ability to induce cytokine response upon binding to targets. RESULTS: GD2 expression in neuroblastoma cells was confirmed by FACS analysis. Specific binding of 3F8BiAb to the tumor targets as well as to ATC was confirmed by FACS analysis. 3F8BiAb-armed ATC exhibited specific killing of GD2 positive neuroblastoma cell lines significantly above unarmed ATC (P < 0.001). GD2BiAb-armed ATC secreted significantly higher levels of Th(1) cytokines and chemokines compared to unarmed ATC (P < 0.001). CONCLUSIONS: These preclinical findings support the potential of a novel immunotherapeutic approach to target T cells to neuroblastoma.

Efficacy of delayed administration of post-chemotherapy granulocyte colony-stimulating factor: evidence from murine studies of bone marrow cell kinetics.

The optimal schedule of post-chemotherapy granulocyte colony-stimulating factor (G-CSF) administration has not been determined. G-CSF is customarily started 24 hours after chemotherapy; however, clinical data demonstrated that delaying G-CSF until 5 days after completion of chemotherapy has not resulted in a longer duration of neutropenia. Here, we examined the optimal timing of post-chemotherapy G-CSF administration in a mouse model, to show that delayed administration does not postpone the appearance of mature granulocytes in the peripheral blood. We also investigated the mechanism of decreased efficacy of the early G-CSF application after chemotherapy by characterizing the changes in bone marrow cellular composition. To our knowledge, we demonstrate for the first time, that early after chemotherapy, the bone marrow is predominantly composed of mature residual granulocytes and very few progenitors and precursors, on which G-CSF would act to generate granulocytes. The point when immature progenitors reappear does not occur in murine bone marrow until 48 hours after a single dose of cyclophosphamide. Our results indicate that the bone marrow cellular composition early after discontinuation of chemotherapy is not optimal for G-CSF action on acceleration of myeloid recovery. Given the high cost of G-CSF prophylaxis, its delayed administration may potentially result in substantial economic benefits.

Delayed introduction of G-CSF after chemotherapy does not affect peripheral blood stem cell yield and engraftment kinetics in children with high-risk malignancies: retrospective study of 45 cases.

We retrospectively compared the effects of two time points of G-CSF (Filgrastim) introduction for PBSC mobilization in 45 children with different malignancies. Seventeen patients received the first G-CSF dose on day 2 or 3 following chemotherapy (group 1). Twenty-eight patients received a "flexible" G-CSF injection schedule when the G-GSF was started at the time of the first platelet count rise during post-chemotherapy recovery phase (group 2). Leukapheresis was performed when WBC recovery reached >2.0 x 10(9)/l or if the peripheral blood CD34(+) cell level was >0.01 x 10(9)/l. A median of 2 (1-4) leukapheresis procedures was performed in both groups to yield a median of 4.2 and 6.1 x 10(6) CD34(+) cells/kg in groups 1 and 2, respectively, which was generally sufficient for auto-transplantation. The proportion of patients with a failure of PBSC collection was similar and G-CSF consumption estimated through the total cycle dose was 2.3 times less in group 2 without increasing infectious risks. The short-term hematological recovery and the early post-transplant course were similar in the two groups. Delayed introduction of G-CSF after chemotherapy allowed PBSC harvest equivalent to that obtained after early G-CSF introduction. This approach could be an interesting alternative in PBSC mobilization but should be assessed by a prospective controlled study.