Background:The use of β-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether β-blockers could be used in combination with chemotherapy for the treatment of neuroblastoma.Methods:Seven β-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma.Results:Three β-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. β-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, β-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, β-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01).Conclusion:β-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.

BackgroundAngiosarcomas are rare malignant tumors of vascular origin that represent a genuine therapeutic challenge. Recently, the combination of metronomic chemotherapy and drug repositioning has been proposed as an attractive alternative for cancer patients living in developing countries.MethodsIn vitro experiments with transformed endothelial cells were used to identify synergistic interactions between anti-hypertensive drug propranolol and chemotherapeutics. This led to the design of a pilot treatment protocol combining oral propranolol and metronomic chemotherapy. Seven consecutive patients with advanced/metastatic/recurrent angiosarcoma were treated with this combination for up to 12 months, followed by propranolol-containing maintenance therapy.FindingsGene expression analysis showed expression of ADRB1 and ADRB2 adrenergic receptor genes in transformed endothelial cells and in angiosarcoma tumors. Propranolol strongly synergized with the microtubule-targeting agent vinblastine in vitro, but only displayed additivity or slight antagonism with paclitaxel and doxorubicin. A combination treatment using bi-daily propranolol (40 mg) and weekly metronomic vinblastine (6 mg/m2) and methotrexate (35 mg/m2) was designed and used in 7 patients with advanced angiosarcoma. Treatment was well tolerated and resulted in 100% response rate, including 1 complete response and 3 very good partial responses, based on RECIST criteria. Median progression-free and overall survival was 11 months (range 5–24) and 16 months (range 10–30), respectively.InterpretationOur results provide a strong rationale for the combination of β-blockers and vinblastine-based metronomic chemotherapy for the treatment of advanced angiosarcoma. Furthermore, our study highlights the potential of drug repositioning in combination with metronomic chemotherapy in low- and middle-income country setting.FundingThis study was funded by institutional and philanthropic grants.

We performed a Phase I clinical trial of donor derived CD19-specific chimeric antigen receptor T-cells (CAR T-cells) for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene capacity limitations of traditional viral vectors, CAR T-cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, one patient developed a gradually enlarging retroperitoneal tumor due to a CAR expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection of a second CAR T-cell tumor in thoracic para-aortic lymph nodes in an asymptomatic patient. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell derived lymphoma progressed and one patient died. We describe the first two cases of malignant lymphoma derived from CAR gene modified T-cells. Although CAR T-cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. The trial was registered at www.anzctr.org.au as ACTRN12617001579381.

CD19-specific chimeric antigen receptor (CAR19) T-cells effectively induce remission of B-cell malignancy, but the cost and complexity of production using viral vectors is a factor limiting widespread application. Furthermore, the small cargo capacity of viral vectors may hamper future development of more heavily engineered CAR T-cells. We demonstrated the feasibility of generating CAR19 T-cells from HLA-matched donors of sibling allogeneic hematopoietic stem cell transplant (HSCT) patients via a simple and inexpensive method using the high-capacity piggyBac transposon. A cohort of 10 patients with relapsed or refractory B-cell acute lymphoblastic leukemia or aggressive lymphoma following HSCT were the first human subjects to receive piggyBac-generated CAR19 T-cells. Treatment with intra-patient escalating doses of CAR19 T-cells was effective, with all 9 evaluable patients achieving complete remission. At a median follow-up of 18.0 months, 5 patients remained in complete remission of B-cell malignancy. One patient died of viral sepsis. Four patients developed cytokine release syndrome of maximum grade 2, and no neurotoxicity or new graft-versus-host disease occurred. However, two patients developed malignant CAR19 T-cell tumors, one of whom was successfully treated; one patient died of the secondary tumor. The piggyBac system represents a feasible alternative to viral vectors for the generation of effective CAR19 T-cells, but its oncogenic potential in the context of the described production process will need to be addressed before any further clinical use is possible. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Glucocorticoids are important regulators of bone cell differentiation and mesenchymal lineage commitment. Using a cell-specific approach of osteoblast-targeted transgenic disruption of intracellular glucocorticoid signaling, we discovered a novel molecular pathway by which glucocorticoids, mainly through the mature osteoblast, regulate the cellular mechanisms that govern cranial skeleton development. Embryonic and neonatal transgenic mice revealed a distinct phenotype characterized by hypoplasia and osteopenia of the cranial skeleton; disorganized frontal, parietal and interparietal bones; increased suture patency; ectopic differentiation of cartilage in the sagittal suture; and disturbed postnatal removal of parietal cartilage. Concurrently, expression of Mmp14, an enzyme essential for calvarial cartilage removal, was markedly reduced in parietal bone and cartilage of transgenic animals. Expression of Wnt9a and Wnt10b was significantly reduced in osteoblasts with disrupted glucocorticoid signaling, and accumulation of β-catenin, the upstream regulator of Mmp14 expression, was decreased in osteoblasts, chondrocytes and mesenchymal progenitors of transgenic mice. Supracalvarial injection of Wnt3a protein rescued the transgenic cranial phenotype. These results define novel roles for glucocorticoids in skeletal development and delineate how osteoblasts -under steroid hormone control -orchestrate the intricate process of intramembranous bone formation by directing mesenchymal cell commitment towards osteoblastic differentiation while simultaneously initiating and controlling cartilage dissolution in the postnatal mouse.

No abstract

The anti-angiogenic activity of chemotherapy is both dose- and schedule-dependent. While conventional maximum tolerated dose (MTD) chemotherapy exerts only mild and reversible anti-angiogenic effects, low-dose metronomic (LDM) chemotherapy was developed to specifically target tumour angiogenesis. However, the long-term effects of either MTD or LDM chemotherapy on vascular endothelial cells have never been investigated. Here, we demonstrated that repeated exposure to MTD and LDM chemotherapy differentially impact on the angiogenic potential and chemosensitivity of immortalized endothelial cells. Repeated MTD vinblastine treatment of vascular endothelial cells led to an increased proliferation rate and resistance to paclitaxel. In contrast, repeated LDM treatment with vinblastine or etoposide impaired the angiogenic potential of endothelial cells and increased their chemosensitivity. This effect was associated with a significant decrease in βII- and βIII-tubulin expression. Functional analysis using siRNA showed that silencing the expression of βIII-tubulin in endothelial cells significantly decreased their capacity to form vascular structures and increased their sensitivity to the anti-angiogenic and vascular-disrupting effects of chemotherapy, whereas silencing βII-tubulin expression had no effect. Collectively our results show that LDM chemotherapy impairs the angiogenic potential of endothelial cells while increasing their chemosensitivity—an effect at least in part mediated by the down-regulation of βIII-tubulin expression. Furthermore, our study suggests that βIII-tubulin represents an attractive therapeutic target to increase the anti-angiogenic effects of chemotherapy and overall anti-tumour efficacy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-012-9321-x) contains supplementary material, which is available to authorized users.

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300 ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.