Faculty Bibliography

The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis

Radiotherapy has remained the mainstay of treatment for children with intrinsic, diffuse pontine tumors in spite of over 20 years of clinical trials attempting to validate the additional role of chemotherapy. Conventional phase II clinical trials conducted in patients with recurrent or progressive brainstem gliomas using single chemotherapy agents such as cyclophosphamide, carboplatin, cisplatin, etoposide and thiotepa or combinations of chemotherapy agents have produced low response rates in the range of 15-20%. Preradiotherapy chemotherapy phase II trials in newly diagnosed patients have yielded similar results with a therapeutic window as long as 4 months. Preliminary data from protocols employing high-dose chemotherapy with stem cell support have also met with disappointment. The one published phase III trial conducted by the Children's Cancer Study Group comparing radiotherapy with radiotherapy plus chemotherapy (CCNU, vincristine and prednisone) failed to establish a benefit to multimodality therapy. Current studies include the use of radiosensitizing chemotherapy (carboplatin, estramustine and cisplatin) in newly diagnosed patients and more intensive neoadjuvant chemotherapy trials. Major advances in our management await insights into molecular vulnerability of high-grade gliomas

PURPOSE: To determine in a prospective randomized trial the effect on survival, progression-free survival, and patterns of relapse of a decrease in the neuraxis radiation dose from 3,600 cGy in 20 fractions to 2,340 cGy in 13 fractions in patients with newly diagnosed medulloblastoma between 3 and 21 years of age with low T stage (T1, T2 and T3A), minimal postoperative residual tumor, and no evidence of dissemination (M0). METHODS AND MATERIALS: Between June 1986 and November 1990, the Children's Cancer Group and the Pediatric Oncology Group randomized 126 patients in a two-arm study comparing the two different doses of neuraxis irradiation. In both arms, the posterior fossa received 5,400 cGy in 30 fractions. All patients were staged with myelography, postoperative lumbar cerebrospinal fluid cytology, and postoperative contrast-enhanced cranial computerized tomography to ensure no evidence of dissemination and no more than 1.5 cm3 residual tumor volume. Overall survival, progression-free survival, and patterns of recurrence were carefully monitored. Prospective endocrine and psychometric studies were performed to determine the benefit of decreasing the neuraxis radiation dose. RESULTS: Following an interim analysis at a median time on study of 16 months, the study was closed, since a statistically significant increase was observed in the number of all relapses as well as isolated neuraxis relapses in patients randomized to the lower dose of neuraxis radiation. CONCLUSIONS: In patients with newly diagnosed medulloblastoma considered to have a good prognosis on the basis of low T stage, minimal residual tumor after at least subtotal resection, and no evidence of dissemination after thorough evaluation, there is an increased risk of early relapse associated with lowering the dose of neuraxis radiation from 3,600 cGy in 20 fractions to 2,340 cGy in 13 fractions.

A new analytical affinity chromatography method was developed for measuring the free [Zn2+] concentration in bovine milk. The column was generated by immobilizing avidin and attaching biotinylated metallothionein (MT) on controlled-pore glass beads. Zinc bound to the MT column at physiological free [Zn2+] concentration and was dissociated again in an elution buffer of pH 2. The distributions of extrinsically added 65Zn and native zinc in different fractions of milk were virtually identical, validating the use of extrinsic labeling in studies of the free [Zn2+] concentration in milk. Extrinsically labeled whey fractions were mixed with standard solutions whose free [Zn2+] concentrations were calculated by computer model. 65Zn retained by the column provided an indication of free [Zn2+] concentration in the mixture, and by interpolation, in the original milk. The free [Zn2+] concentration measured by the affinity chromatography method in the milk of a group of six cows was 90.4 +/- 29.7 pM. This value is similar to estimates of free [Zn2+] concentrations in other biological fluids by entirely different methods. Measurement of free [Zn2+] may be helpful in understanding the physiology and biochemistry of zinc metabolism.

BACKGROUND: Insulin attenuates serotonin-induced Ca2+ influx, the intracellular Ca2+ transient, and contraction of cultured vascular smooth muscle cells from dog femoral artery. These studies were designed to test whether insulin-induced glucose transport was an early event leading to the inhibitory effects of insulin on Ca2+ influx, intracellular Ca2+ concentration, and contraction in these cells. METHODS AND RESULTS: Insulin 1 nmol/L stimulated the 30-minute uptake of [3H]2-deoxyglucose in these cells via a phloridzin-inhibitable mechanism. Contraction of individual cells was measured by photomicroscopy, intracellular Ca2+ concentration was monitored by measuring fura 2 fluorescence by use of Ca(2+)-sensitive excitation wavelengths, and Ca2+ influx was estimated by the rate of Mn2+ quenching of intracellular fura 2 fluorescence when excited at a Ca(2+)-insensitive wave-length. In the presence of 5 mmol/L glucose, preincubation of cells for 30 minutes with 1 nmol/L insulin inhibited 10(-5) mol/L serotonin-induced contraction of individual cells by 62% (P < .01) and decreased the serotonin-stimulated component of Mn2+ influx by 78% (P < .05). Removing glucose from the preincubation medium or adding 1 mmol/L phloridzin completely eliminated these effects of insulin. Insulin lowered the serotonin-induced intracellular Ca2+ peak by 37% (P < .05), and phloridzin blocked this effect of insulin. When glucose uptake was increased to the insulin-stimulated level by preincubation of the cells for 30 minutes with 25 mmol/L glucose in the absence of insulin, serotonin failed to stimulate Mn2+ influx, the serotonin-induced Ca2+ peak was decreased by 46% (P < .05), serotonin-induced contraction was inhibited by 60% (P < .01), and addition of insulin did not further inhibit contraction. CONCLUSIONS: Since the effects of insulin on serotonin-stimulated Ca2+ transport, intracellular Ca2+ concentration, and contraction were dependent on glucose transport and were duplicated when glucose transport was stimulated by high extracellular glucose concentration rather than insulin per se, it is concluded that insulin-stimulated glucose transport is an early event that leads to decreased Ca2+ influx and contraction in vascular smooth muscle.

PURPOSE: To determine clinical characteristics and response to treatment for children with supratentorial primitive neuroectodermal tumors (S-PNETs). PATIENTS AND METHODS: After surgery and staging, 55 patients aged 1.5 to 19.3 years with S-PNETs were randomized to receive craniospinal radiotherapy (RT) followed by eight cycles of 1-(2-chloro-ethyl)-3-cyclohexylnitrosourea (CCNU), vincristine (VCR), and prednisone (standard treatment) or two cycles of 8-in-1 chemotherapy followed by RT and then eight additional cycles of 8-in-1. RESULTS: Three-year Kaplan-Meier estimates (estimate +/- SE) of survival and progression-free survival (PFS) rates for patients with confirmed diagnoses of S-PNET were 57% +/- 8% and 45% +/- 8%, respectively; survival and PFS rates for children with PNETs located in the pineal region were 73% +/- 12% and 61% +/- 13%, respectively, and were significantly different from the other S-PNETs (P < .03). The 8-in-1 arm had greater toxicity than the standard-treatment arm. Distributions of PFS between the two treatment groups were not significantly different (P > .5). Other univariate prognostic factors that influenced PFS included metastasis (M) stage (P < .03: M0 50% +/- 9% v M1-4 0%) and age (P < .02: 1.5 to 2 years 25% +/- 13% v > or = 3 years 53% +/- 9%). CONCLUSION: In this first randomized treatment trial for S-PNETs in children, no significant differences were detected between the two treatment groups. M0 and pineal site of involvement were independent predictors of a better outcome. However, survival was better than previously reported.

A dialysis method was developed for measuring free Zn2+ concentration in plasma and milk for determination of the electrochemical gradient for Zn2+ across the mammary gland. This method used the zinc content of casein after dialysis as the metal ion indicator because zinc in the calcium phosphate-citrate complex inside casein micelles is dependent on the free Zn2+ concentration of an associated aqueous phase. Zinc, calcium and magnesium distribution in milk confirmed the high zinc binding by bovine casein. Zinc in the casein, dialyzed against standards or unknowns, was measured by atomic absorption spectrophotometry. Average free Zn2+ concentrations measured by the dialysis method in plasma and milk of five cows were 0.141 and 0.331 nmol/L, respectively. The equilibrium potential of Zn2+ ions across the mammary epithelium, calculated from free Zn2+ concentrations in blood and milk measured by the dialysis method, was -11.4 mV, consistent with the mammary electrical potentials noted in previous studies. Therefore, no electrochemical gradient for zinc between the two compartments was apparent, and it is not necessary to invoke an active transport mechanism in the mammary gland to explain the higher zinc concentration in milk than in plasma of most species.

PURPOSE: To examine the effects of lomustine (CCNU), a commonly used nitrosourea, and craniospinal radiation therapy on the subsequent development of restrictive lung disease (RLD) following treatment for malignant brain tumors. PATIENTS AND METHODS: Pulmonary function testing with measurement of lung volume, spirometry, and diffusion capacity was performed in 28 patients who had received CCNU and/or radiation therapy as treatment for a malignant brain tumor. The median age at the time of treatment was 11.4 years (range, 3.9 to 36.7) and radiation therapy was completed 6 months to 11.6 years (median, 2.6 years) before testing. Patients were divided into four groups based on prior therapy. Group 1 received involved-field irradiation and a CCNU-containing chemotherapy regimen (n = 7); group 2, craniospinal irradiation with a boost to the primary tumor site and a CCNU-containing chemotherapy regimen (n = 6); group 3, craniospinal irradiation with a boost to the primary tumor site and a non-CCNU-containing chemotherapy regimen (n = 7); and group 4, craniospinal irradiation with a boost to the primary tumor site without chemotherapy (n = 8). RESULTS: Fourteen patients (50%) had findings consistent with RLD. One of seven patients (14.3%) who received CCNU without spinal irradiation had RLD, whereas 13 of 21 (61.9%) who received spinal irradiation with or without CCNU had RLD (P = .038), including four of eight patients treated with craniospinal irradiation alone. Logistic regression analysis showed that only spinal irradiation was a significant predictor for RLD. Patients who received spinal irradiation were 4.3 times more likely to have RLD than those who did not receive spinal irradiation. CONCLUSION: Spinal irradiation may be a risk factor for the development of RLD