Risk factors for symptomatic venous thromboembolism during therapy for childhood acute lymphoblastic leukemia.

BACKGROUND: Symptomatic venous thromboembolism (VTE) is an unpredictable and life-threatening toxicity, which occurs early in childhood acute lymphoblastic leukemia (ALL) therapy. Approximately 5% of children will experience VTE which is treated with anticoagulation. Asparaginase and corticosteroids are etiologic factors for VTE, however other clinical factors may modify this risk. PROCEDURE: We sought to i) assess published pre-treatment VTE risk factors ii) identify early clinical factors that were associated with VTE and iii) determine whether single nucleotide polymorphisms (SNPs) associated with VTE in non-cancer patients contributed to VTE in children with ALL. We performed a detailed, retrospective analysis of 1021 ALL patients treated between 1998 and 2013. Individual patient records were reviewed to ascertain VTE incidence and document treatment-related clinical variables. RESULTS: The incidence of VTE was 5.1%. Extremes of weight at diagnosis (<5th or >95th centile) was an independent risk factor in multivariable analysis, when added to published risk factors of age ≥10 years and mediastinal mass. When factors during induction/consolidation were considered separately: bacteremia, elevated serum gamma-glutamyl transferase and bilirubin were associated with VTE occurrence. None of the SNPs associated with VTE in non-cancer populations were significantly associated with VTE in our cohort. CONCLUSION: We found two known risk factors (age ≥ 10 years and mediastinal mass) in a large cohort of children treated for ALL and identified other factors associated with VTE such as weight extremes at diagnosis, bacteremia, and abnormal liver function which warrant further study. These VTE risk factors may form the basis of future thromboprophylaxis trials.

Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity.

The frequent loss of heterozygosity of chromosome (Chr) 17 in epithelial ovarian cancer (EOC), particularly high-grade ovarian serous carcinomas (HGOSCs), has been attributed to the disruption of known tumour suppressor genes, such as TP53 (17p13), as well as other genes on this chromosome that alone or in combination have a role in EOC. In a transcriptome analysis of Chr17 genes, we observed significant underexpression of the chemokine CCL2 (17q12) in a small set of HGOSC samples relative to normal ovarian surface epithelial cells and a significant upregulation of CCL2 in the TP53-mutated OV-90 EOC cell line rendered non-tumourigenic as a consequence of genetic manipulation. Here, we report that overexpressing CCL2 in OV-90 resulted in latency of tumour formation at intraperitoneal (i.p.) but not subcutaneous sites in a mouse xenograft model. Overexpressing CCL2 affected cell morphology and exerted modest, but not significant effects on cell viability, colony formation and cell migration. We report significant underexpression of CCL2 by transcriptome analysis (P=0.015) and by immunohistochemistry in 77% of HGOSC samples (n=65). Absent or a very low level of protein expression by immunohistochemistry was also observed in 71% of additional HGOSC samples (n=122). However, CCL2 protein expression did not significantly correlate with overall or disease-free survival. The epithelial cells of normal fallopian tubes, a purported origin of HGOSC, exhibited expression of CCL2 protein by immunohistochemistry. Our results affirm that CCL2 underexpression is a significant feature of HGOSC samples, and that CCL2 overexpression in an EOC cell line model affects tumourigenic potential in the i.p. setting.

Transfer of chromosome 3 fragments suppresses tumorigenicity of an ovarian cancer cell line monoallelic for chromosome 3p.

Multiple chromosome 3p tumor suppressor genes (TSG) have been proposed in the pathogenesis of ovarian cancer based on complex patterns of 3p loss. To attain functional evidence in support of TSGs and identify candidate regions, we applied a chromosome transfer method involving cell fusions of the tumorigenic OV90 human ovarian cancer cell line, monoallelic for 3p and an irradiated mouse cell line containing a human chromosome 3 in order to derive OV90 hybrids containing normal 3p fragments. The resulting hybrids showed complete or incomplete suppression of tumorigenicity in nude mouse xenograft assays, and varied in their ability to form colonies in soft agarose and three-dimensional spheroids in a manner consistent with alteration of their in vivo tumorigenic phenotypes. Expression microarray analysis identified a set of common differentially expressed genes, such as SPARC, DAB2 and VEGF, some of which have been shown implicated in ovarian cancer. Genotyping assays revealed that they harbored normal 3p fragments, some of which overlapped candidate TSG regions (3p25-p26, 3p24 and 3p14-pcen) identified previously in loss of heterozygosity analyses of ovarian cancers. However, only the 3p12-pcen region was acquired in common by all hybrids where expression microarray analysis identified differentially expressed genes. The correlation of 3p12-pcen transfer and tumor suppression with a concerted re-programming of the cellular transcriptome suggest that the putative TSG may have affected key underlying events in ovarian cancer.

Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates.

Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.