The neurovascular unit in diffuse intrinsic pontine gliomas

Aims: Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a median overall survival of eleven months. Lack of chemotherapy efficacy may be related to an intact blood-brain barrier (BBB). In this study we aim to investigate the neurovascular unit (NVU) in DIPG patients. Methods: DIPG biopsy (n = 4) and autopsy samples (n = 6) and age-matched healthy pons samples (n = 20) were immunohistochemically investigated for plasma protein extravasation, and the expression of tight junction proteins claudin-5 and zonula occludens-1 (ZO-1), basement membrane component laminin, pericyte marker PDGFR-β, and efflux transporters P-gp and BCRP. The mean vascular density and diameter were also assessed. Results: DIPGs show a heterogeneity in cell morphology and evidence of BBB leakage. Both in tumor biopsy and autopsy samples, expression of claudin-5, ZO-1, laminin, PDGFR-β and P-gp was reduced compared to healthy pontine tissues. In DIPG autopsy samples, vascular density was lower compared to healthy pons. The density of small vessels (<10 µm) was significantly lower (P<0.001), whereas the density of large vessels (≥10 µm) did not differ between groups (P = 0.404). The median vascular diameter was not significantly different: 6.21 µm in DIPG autopsy samples (range 2.25-94.85 µm), and 6.26 µm in controls (range 1.17-264.77 µm). Conclusion: Our study demonstrates evidence of structural changes in the NVU in DIPG patients, both in biopsy and autopsy samples, as well as a reduced vascular density in end-stage disease. Adding such a biological perspective may help to better direct future treatment choices for DIPG patients

Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay.

The methyl-thiazol-tetrazolium (MTT) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > or = 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukemia (ALL) samples with > or = 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > or = 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with < 70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > or = 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > or = 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunoamagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay

PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation

Poly ADP-ribose polymerase (PARP) is a protein involved in single strand break repair. Recently, PARP inhibitors have shown considerable promise in the treatment of several cancers, both in monotherapy and in combination with cytotoxic agents. Synthetic lethal action of PARP inhibitors has been observed in tumors with mutations in double strand break repair pathways. In addition, PARP inhibition potentially enhances sensitivity of tumor cells to DNA damaging agents, including radiotherapy. Aim of this study is to determine the radiosensitizing properties of the PARP inhibitor Olaparib in childhood medulloblastoma, ependymoma and high grade glioma (HGG). Increased PARP1 expression was observed in medulloblastoma, ependymoma and HGG, as compared to non-neoplastic brain tissue. Pediatric high grade glioma, medulloblastoma and ependymoma gene expression profiling revealed that high PARP1 expression is associated with poor prognosis. Cell growth inhibition assays with Olaparib resulted in differential sensitivity, with IC50 values ranging from 1.4 to 8.4 μM, irrespective of tumor type and PARP1 protein expression. Sensitization to radiation was observed in medulloblastoma, ependymoma and HGG cell lines with subcytotoxic concentrations of Olaparib, which coincided with persistence of double strand breaks. Combining PARP inhibitors with radiotherapy in clinical studies in childhood high grade brain tumors may improve therapeutic outcome

Clinical and self-reported markers of reproductive function in female survivors of childhood Hodgkin lymphoma

Purpose: To evaluate the impact of treatment for Hodgkin lymphoma (HL) on clinical reproductive markers and pregnancy outcomes.Methods: This study was embedded within the DCOG LATER-VEVO study; a Dutch, multicenter, retrospective cohort study between 2004 and 2014. Serum anti-Müllerian hormone (AMH), follicle stimulating hormone (FSH), inhibin B, antral follicle count (AFC), and self-reported (first) pregnancy outcomes were evaluated in female childhood HL survivors and controls.Results: 84 HL survivors and 798 controls were included, aged 29.6 and 32.7 years old at time of assessment. Median age at HL diagnosis was 13.4 years. Cyclophosphamide equivalent dose (CED-score) exceeded 6000 mg/m2 in 56 women and 14 survivors received pelvic irradiation.All clinical markers were significantly deteriorated in survivors (odds-ratio for low AMH (&lt; p10) 10.1 [95% CI 4.9; 20.6]; low AFC (&lt; p10) 4.6 [95% CI 2.1; 9.9]; elevated FSH (&gt; 10 IU/l) 15.3 [95% CI 5.7; 41.1], low Inhibin B (&lt; 20 ng/l) 3.6 [95% CI 1.7; 7.7], p &lt; 0.001). Pregnancy outcomes were comparable between survivors and controls (± 80% live birth, ± 20% miscarriage). However, survivors were significantly younger at first pregnancy (27.0 years vs 29.0 years, P = 0.04). Adjusted odds-ratio for time to pregnancy &gt; 12 months was 2.5 [95% CI 1.1; 5.6] in survivors, p = 0.031. Adverse outcomes were specifically present after treatment with procarbazine and higher CED-score.Conclusion: HL survivors appear to have an impaired ovarian reserve. However, chance to achieve pregnancy seems reassuring at a young age. Additional follow-up studies are needed to assess fertile life span and reproductive potential of HL survivors, in particular for current HL treatments that are hypothesized to be less gonadotoxic.</p

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor

AbstractVirotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer

Possibilities for tailored and targeted therapy in paediatric acute myeloid leukaemia

The clinical outcome of acute myeloid leukaemia (AML) in children has improved considerably using intensive chemotherapy and/or stem cell transplantation. This leads to cure in 50-70% of patients, and also results in significant morbidity and mortality. Hence, we need other ways to improve the cure rate. This review discusses possibilities for tailored therapy, reviewing in vitro cellular drug sensitivity data. The results provide suggestions regarding the adaptation of clinical protocols in certain AML subgroups, although further clinical studies will show whether this is effective. Secondly, we review type 1 genetic abnormalities (such as receptor tyrosine kinase mutations) that result in enhanced survival and proliferation of leukaemic cells, which can be detected in approximately 50% of paediatric AML samples, and are non-randomly associated with French-American-British type and cytogenetic subgroups. FLT3 internal tandem duplication is associated with poor clinical outcome, and may be used for risk-group stratification. The first results with small molecule inhibitors in adult AML do not suggest their use in children as yet. International collaboration is needed to further improve outcome by developing treatment protocols for subgroups of paediatric AML

Proteasome inhibition as novel treatment strategy in leukaemia

Following its success in multiple myeloma (MM), proteasome inhibition has become a topic of interest as novel treatment strategy of cancer. By simultaneously affecting multiple pathways in the cancer cell, such as deregulation of the programmed degradation of many cellular proteins, proteasome inhibition causes rapid apoptosis of these cells. Both in rapidly proliferating leukaemic cell lines and in primary leukaemic cells isolated from patients, proteasome inhibition results in antileukaemic activity. The normal counterparts of these cells are much more resistant to proteasome inhibitors (PI), thereby resulting in a favourable therapeutic index. Importantly, while leukaemic stem cells are sensitive to proteasome inhibition, normal haematopoietic stem cells are still viable after drug exposure. Nowadays, many PIs are being identified; bortezomib is the most well known since obtaining Food and Drug Administration approval for clinical use in MM. This review summarises the biological and clinical aspects of proteasome inhibition and discusses the potential role of these inhibitors in the treatment of leukaemia

Cellular drug resistance in childhood leukemia

The response to chemotherapy is determined essentially by two factors: first, pharmacokinetic factors, determining which concentration of drug reaches the malignant cells, and second, cellular drug resistance of these cells, determining how many of them will be killed by that concentration of drug. The study of cellular drug resistance has been stimulated by the development of short-term 'total cell kill' assays, such as the MTT assay, for use on patient samples. The drug resistance profiles differed markedly between ALL and ANLL, between immunophenotypic and karyotypic subgroups within ALL, and between initial and relapsed ALL. The results of the MTT assay showed a significant relation between the antileukemic activity of prednisolone in vitro and the clinical response to systemic monotherapy with that drug. At multivariate analysis including several well-known prognostic factors (WBC, age, immunophenotype) only the in vitro resistance to prednisolone, dexamethasone, l-asparaginase, and daunorubicin was significantly related to clinical outcome. At multiple regression analysis, combination of the results for prednisolone, l-asparaginase, and vincristine made it possible to distinguish between three patient groups with increasing levels of drug resistance and markedly different probabilities of 2-year disease-free survival: 100%, 83%, and 60%. These results show that in vitro drug resistance testing can give a correct prediction of prognosis, superior to that of currently used prognostic factors. Stratification of prognostic groups based on the results of drug resistance testing is feasible and should be introduced into new clinical trials. Many questions now remaining could be answered within carefully designed preclinical and clinical studies