Barry H. Smith

Glucose utilization of cerebral gliomas measured by [18F] fluorodeoxyglucose and positron emission tomography

Positron emission tomography was used to measure local cerebral glucose utilization by the 2-[18F]fluoro-2-deoxy-D-glucose technique in 23 patients with cerebral gliomas. All 10 high-grade (III and IV) astrocytomas demonstrated a region of high activity with a glucose consumption of 7.4 ± 3.5 (SD) mg/100 gm per minute. The 13 low-grade (I and II) gliomas had a glucose metabolic rate of 4.0 ± 1.8 mg/100 gm per minute, with no distinctly visible hot spot. Thus, we found a correlation between rate of glycolysis and malignancy in primary cerebral tumors. Cerebral cortical glucose utilization was often depressed in areas adjacent to or neurally connected to the tumor site, and there was focal irregular delta wave EEG activity in these areas.

A Phase I study of intermittent intravenous bromodeoxyuridine (BUdR) with conventional fractionated irradiation.

A Phase I trial of intravenous bromodeoxyuridine (BUdR) and conventional fractionated radiation therapy was performed in 14 patients with glioblastoma multiforme and 7 patients with other poorly radioresponsive tumors. The BUdR was given as a constant intravenous infusion for 12 hr/day for up to 14 days. Thirteen patients received a second 14 day infusion following a 10 to 14 day interruption for bone marrow recovery. Local toxicity (within the radiation field) was minor, with 7 of the 21 patients requiring a brief treatment break for moist skin desquamation. There was no significant CNS toxicity noted clinically nor by autopsy examination. Additionally, no significant enhancement of radiation injury was noted to bowel or liver. However, one patient treated for multiple pulmonary metastases experienced a clinical and radiographic pattern consistent with radiation pneumonitis. Dose-dependent systemic toxicity occurred in bone marrow and skin. Moderate myelosuppression, especially thrombocytopenia, was found following a 14 day cycle of BUdR at and above 650 mg/m2/12 hr infusion. Approximately one-third of patients developed a maculo-papular erythematous rash to the scalp, neck and upper chest. In two patients, the rash became generalized with evidence of epidermolysis on skin biopsy. Pharmacology studies revealed steady-state arterial plasma levels of 2 X 10(-6) M/1 during the 12 hr infusion of 650 to 700 mg/m2. Radiosensitization was measured by a change in the D0 of radiation survival curves of human bone marrow CFUc prior to and following the 14 day infusion in 4 patients. A trend of increasing radiosensitization was noted in most patients as the infusion rate of BUdR was increased from 500 to 870 mg/m2/12 hr. We conclude that the maximum tolerable dose of BUdR is 650 to 700 mg/m2/12 hrs when given as a 2 week intermittent intravenous infusion. Local toxicity is acceptable. The major systemic toxicities are myelosuppression and a maculopapular skin rash.

Depressed cerebellar glucose metabolism in supratentorial tumors

Fifty-four patients with supratentorial tumor and one with brainstem tumor were examined with positron emission tomography (PET) using [18F]fluoro-deoxyglucose (FDG). Twenty-one of these cases had satisfactory studies of the cerebellum. Of these, 12 showed significant metabolic asymmetry between the two cerebellar hemispheres, with the rate of glucose utilization in the hemisphere contralateral to the cerebral tumor being 8-34% lower than on the ipsilateral side, as compared with a right-left asymmetry of only--1.6% +/- 2.1% standard deviation for a group of 5 normal subjects. In these 12 cases the tumor involved the sensorimotor cortex and/or the thalamus with varying degrees of hemiparesis being present. For the remaining 9 patients with no significant cerebellar metabolic asymmetry, the tumor involved regions other than the sensorimotor cortex, and unilateral motor dysfunction was not a prominent clinical feature. The correlation between cerebellar metabolic suppression and unilateral motor dysfunction observed in our cases appears to be due to impairment or interruption of the cortico-thalamo-ponto-olivo-cerebellar circuitry by either the tumor itself or by edema. These results illustrate the ability of FDG-PET scans to detect metabolic changes, not apparent on CT scans, in areas of the brain remote from the primary lesion.

Radiosensitization of hematopoietic precursor cells (CFUc) in glioblastoma patients receiving intermittent intravenous infusions of bromodeoxyuridine (BUdR)

The potential use of bromodeoxyuridine (BUdR) as a radiosensitizer given by an intermittent intravenous route is being studied in a Phase I/II trial at the National Cancer Institute. In order to assess the extent of radiosensitization, we have studied the radiation response of human bone marrow cells CFUc taken from 6 patients prior to and after a 14-day infusion of BUdR. Varying concentrations (1000-1500 mg) of BUdR were infused for 12 hours every 24 hours for up to 14 consecutive days. Cell survival was determined by colony formation of CFUc in soft agar suspension. X ray survival curves were generated over a dose range of 0-300 rad and the slopes of the survival curves (DO) before and after BUdR infusion were compared. Radiation enhancement ratios (ER) (DO pre-BUdR/DO post-BUdR) ranged from 1.0-2.2 and appeared to be BUdR dose dependent. Above 650 mg/m2, the radiation ER was greater than or equal to 1.5. Dose dependent systemic toxicity to bone marrow and skin was also observed with intermittent intravenous infusions of BUdR. From our study, it appears that an intravenous dose of less than 700 mg/m2/12 hours is well tolerated and may result in radiosensitization of CFUc in man.

Response of cultured human brain tumors to nitrosoureas: Correlation with clinical data

An in vitro microcytotoxicity assay was utilized to determine the sensitivity of 58 cultured human malignant gliomas to the chemotherapy agent 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU). Of 58 such tumors, 42 (72%) showed a statistically significant cytotoxic response to BCNU in this assay. For those responding tumor lines, the cytotoxic index ranged from 0.25 to 0.76, with most clustered at the 0.40 level.

Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

Refractory cushing’s disease caused by multinodular ACTH-cell hyperplasia

A patient with pituitary-dependent hypercortisolism, unresponsive to resection of nodules in the anterior lobe, is described. Histochemical stains of the nodules showed multiple, focal, cellular expansions of the fibrovascular stroma. Transitions between normal and expanded adenohypophysial acini were present. Immunoperoxidase stains for ACTH and other pituitary hormones revealed that these multiple foci contained an excess of ACTH-positive cells. Less than 10% of the cells in these foci were negative for ACTH and positive for other hormones. Serial sections showed that these foci of predominantly ACTH-producing acini were not connected. Clinical, morphological, and immunohistochemical data indicated that ACTH-cell hyperplasia caused Cushings disease in this patient. Pathologic study of individual cases should concentrate on determining whether hyperplasia or adenoma exist at the time of surgical exploration of the pituitary gland, since this determination is important to proper treatment. Tentative criteria to recognize ACTH-cell hyperplasia are: 1. Multiple foci of ACTH laden cells. 2. A minor subpopulation of cells of alternate hormone series. 3. Expansion without destruction of acini in the adenohypophysis.

A human ganglioglioma containing paired helical filaments

Neurofibrillary tangles composed of paired helical filaments were found in a human ganglioglioma. This is the first reported occurrence of neurofibrillary tangles in a neoplasm. These tangles were visible light microscopically with hematoxylin-eosin and Bodians stains. They were confirmed as neurofibrillary tangles with Congo red staining under polarized light and with thioflavine S fluorescence. Untrastructurally, the tangles were composed of 10-nm filaments twisted in a helix with 80 nm between constructions. Thus, neoplastic proliferation does not preclude production of paired helical filaments. Cells grew from explants of this tumor, but no paired helical filaments were found in the cells examined. Two other gangliogliomas and normal brain tissue studies by the same procedures did not show paired helical filaments. Gangliogliomas that contain neurofibrillary tangles provide an alternative source of abnormal filaments for analysis.

Flowcytometric and cytogenetic analysis of human cultured cell lines derived from high- and low-grade astrocytomas

SummaryFive human cell lines cultured from high- and low-grade astrocytomas in cerebral hemisphere have been analyzed for DNA and protein distribution by flowcytometric (FCM) and correlated with cytogenetic profiles. Simultaneous calibration with chicken erythrocytes as a co-running standard provided an estimate of chromosomal number of predominate stem cells of each cell line by the ratio of the DNA content of the major peak (G1) to that of chicken erythrocyte (T/E ratio) of FCM. Various lines had different distributions of chromosomal number, ranging from near diploid to tetraploid. Each line had a stem-cell population and chromosomal markers indicative of clonal selection, but no common marker specific to astrocytomas. The histogram of DNA distribution obtained by FCM correlated well with the chromosomal distribution by cytogenetic analysis. In addition, simultaneous measurement of protein and DNA content in multidimensional FCM demonstrated a sigmoid configuration of the profiles, which indicated a gradual increase of protein content associated with an increase of chromosomal number or with progression of cell cycle. To avoid confusion of a bimodal chromosomal distribution with the G2/M phase of the cell cycle, and to determine chromosomal numbers associated with a DNA histogram, simultaneous cytogenetic and FCM study are required. More rapid than cytogenetic analysis, the T/E ratio allows estimation of chromosomal number of the stem-cell population associated with DNA histograms of cultured glioma-derived cell lines.

Membrane and cytoplasmic changes in 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)-sensitive and resistant human malignant glioma-derived cell lines.

Human glioma-derived cell lines previously determined by a microtiter chemotherapy assay to be either ‘sensitive’ or ‘resistant’ to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were treated with BCNU (1–80 µg/ml) and observed using microcinematography, scanning electron microscopy, and transmission electron microscopy. Striking bleb formation and cell retraction were observed to occur in a dose-dependent relationship within minutes in the cells known to be BCNU-sensitive. At 15µg/ ml, 69% of cells showed blebs by 30 min, 87% by 90 min, and 100% by 4 hr. This activity was not seen in BCNU-resistant cells. These morphological changes occur at a time too early to be accounted for by the known BCNU mechanism of DNA alkylation and cross-link formation and suggest that cytoplasmic and/or membrane events may be significant initial events in the cytotoxic actions of BCNU.