Philip de Witt Hamer

Background (1) To determine the optimal administration site and dose of indocyanine green (ICG) for blood volume measurement using pulse spectrophotometry, (2) to assess the variation in repeated blood volume measurements for patients after subarachnoid hemorrhage and (3) to evaluate the safety and efficacy of this technique in patients who were treated for an intracranial aneurysm. Methods Four repeated measurements of blood volume (BV) were performed in random order of bolus dose (10 mg or 25 mg ICG) and venous administration site (peripheral or central) in eight patients admitted for treatment of an intracranial aneurysm. Another five patients with subarachnoid hemorrhage underwent three repeated BV measurements with 25 mg ICG at the same administration site to assess the coefficient of variation. Findings The mean ± SD in BV was 4.38 ± 0.88 l (n = 25) and 4.69 ± 1.11 l (n = 26) for 10 mg and 25 mg ICG, respectively. The mean ± SD in BV was 4.59 ± 1.15 l (n = 26) and 4.48 ± 0.86 l (n = 25) for central and peripheral administration, respectively. No significant difference was found. The coefficient of variance of BV measurement with 25 mg of ICG was 7.5% (95% CI: 3–12%). Conclusions There is no significant difference between intravenous administration of either 10 or 25 mg ICG, and this can be injected through either a peripheral or central venous catheter. The 7.5% coefficient of variation in BV measurements determines the detectable differences using ICG pulse spectrophotometry.

The value of chemotherapy in patients with malignant astrocytoma remains controversial. In our laboratories in vitro experiments with organotypic spheroid cultures showed superior effectiveness of anthracyclines. Systemic administration did not provide in therapeutic concentrations so far. Because recent studies on Daunorubicin in liposomes in the treatment of Kaposi sarcoma have shown effectiveness with diminished systemic toxicity, we administered intravenously a single dose of Daunorubicin in liposomes in eight patients at different intervals prior to surgery (12–50 h). In samples taken from tumor, tumor-edge and where possible from adjacent brain, the levels of Daunorubicin and its active metabolite Daunorubicinol were assessed with high performance liquid chromatography. Here we report that high concentrations of Daunorubicin and Daunorubicinol were found in malignant gliomas after systemic administration of liposomal Daunorubicin.

Background Standards for residual tumour measurement after resection of gliomas with no or minimal enhancement have not yet been established. In this study residual volumes on early and late postoperative T2-/FLAIR-weighted MRI are compared. Methods A retrospective cohort included 58 consecutive glioma patients with no or minimal preoperative gadolinium enhancement. Inclusion criteria were first-time resection between 2007 and 2009 with a T2-/FLAIR-based target volume and availability of preoperative, early (<48 h) and late (1–7 months) postoperative MRI. The volumes of non-enhancing T2/FLAIR tissue and diffusion restriction areas were measured. Results Residual tumour volumes were 22% smaller on late postoperative compared with early postoperative T2-weighted MRI and 49% smaller for FLAIR-weighted imaging. Postoperative restricted diffusion volume correlated with the difference between early and late postoperative FLAIR volumes and with the difference between T2 and FLAIR volumes on early postoperative MRI. Conclusion We observed a systematic and substantial overestimation of residual non-enhancing volume on MRI within 48 h of resection compared with months postoperatively, in particular for FLAIR imaging. Resection-induced ischaemia contributes to this overestimation, as may other operative effects. This indicates that early postoperative MRI is less reliable to determine the extent of non-enhancing residual glioma and restricted diffusion volumes are imperative.

Osteopontin (OPN) is a glycophosphoprotein with multiple intracellular and extracellular functions. In vitro, OPN enhances migration of mouse neutrophils and macrophages. In cancer, extracellular OPN facilitates migration of cancer cells via its RGD sequence. The present study was designed to investigate whether osteopontin is responsible for neutrophil and macrophage infiltration in human cancer and in particular in glioblastoma. We found that in vitro mouse neutrophil migration was RGD-dependent. In silico, we found that the OPN gene was one of the 5% most highly expressed genes in 20 out of 35 cancer microarray data sets in comparison with normal tissue in at least 30% of cancer patients. In some types of cancer, such as ovarian cancer, lung cancer and melanoma, the OPN gene was one of those with the highest expression levels in at least 90% of cancer patients. In glioblastoma, the most invasive type of brain tumours/glioma, but not in lower grades of glioma it was one of the 5% highest expressed genes in 90% of patients. In situ, we found increased protein levels of OPN in human glioblastoma versus normal human brain confirming in silico results. OPN protein expression was co-localized with neutrophils and macrophages. In conclusion, OPN in tumours not only induces migration of cancer cells but also of leucocytes.

Cancer spheroids are a valuable model for screening anticancer strategies. However, studies are published using various numbers of spheroids and sections per spheroid. Here, we establish the sample size requirements for valid screening strategies to treat glioma: how many spheroids per experimental group and how many sections per spheroid are required to detect one-third reduction in an endpoint measurement after treatment? From two glioblastoma patients, 32 untreated organotypic spheroids were cultured and sectioned entirely (14–100 sections per spheroid). The viable fraction was determined as endpoint by automated image analysis in sections and used to establish the minimally-detectable difference between a treatment and reference group. Variance was considerable with a coefficient of variance of 21%. The biological variation in viability in sections of spheroids produced 97% of variance when sample size was large. Variance increased when numbers of spheroids but not numbers of sections per spheroid were reduced. A minimum of 12 spheroids per group and one section per spheroid was required for a valid comparison of a treatment group and a control group. When 10 treatment groups and one control group were compared, 16 spheroids per group were required. Thus, the statistical power depended almost entirely on the number of organotypic glioma spheroids and hardly on the number of sections per spheroid. The organotypic glioma spheroid model does not appear to be suitable for high-throughput screening of anticancer strategies, because of the relatively large number of spheroids required. It is the model of choice for low-throughput screening, because this model is far more representative for the parental tumor than any other more efficient glioma model. © 2009 International Society for Advancement of Cytometry

Reproducibility of cryostat section thickness is required for valid quantitative microscopy. This is generally pursued by motorized sectioning using a low but constant speed. The purpose of our study was to compare variation in section thickness between motorized and manual cryostat sectioning. Serial sections were cut from a frozen block of homogenized tissue on different days. Lactate dehydrogenase activity was histochemically detected and calibrated absorbance measurements were taken. The coefficients of variation of measurements was 9.7% for motorized sectioning and 3.3% for manual sectioning. In conclusion, section thickness is similarly reproducible after manual sectioning compared with motorized sectioning, if not better. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc.