Dora W. Hsu

Glycoprotein hormone genes are expressed in clinically nonfunctioning pituitary adenomas.

Approximately 25% of patients with pituitary adenomas have no clinical or biochemical evidence for excess hormone secretion and are classified as having null cell or nonfunctioning adenomas. To characterize the cell type of these tumors, we analyzed pituitary hormone gene expression in clinically nonfunctioning pituitary adenomas using specific oligonucleotide probes for the messenger (m)RNAs encoding growth hormone, prolactin, ACTH, and the glycoprotein hormone subunits, alpha, luteinizing hormone (LH)beta, follicle-stimulating hormone (FSH)beta, and thyroid-stimulating hormone (TSH)beta. Expression of one or more of the anterior pituitary hormone genes was found in 12/14 (86%) of the patients with clinically classified nonfunctioning adenomas. Expression of one or more of the glycoprotein hormone genes (alpha, LH beta, FSH beta, TSH beta) was identified most commonly (79%) with expression of multiple beta-subunit genes in many cases. Expression of alpha-subunit mRNA was found in each of the adenomas from patients expressing one of the beta-subunit mRNAs and in three patients with no detectable beta-subunit mRNA. Although FSH beta and LH beta mRNAs were found with similar frequencies in nonfunctioning adenomas, expression of FSH beta mRNA was generally much more abundant. TSH beta mRNA was detected in only one adenoma. The levels of glycoprotein hormone subunit mRNAs were variable in different adenomas, but the lengths of the mRNAs and transcriptional start sites for the alpha- and beta-subunit genes were the same in the pituitary adenomas and in normal pituitary. Growth hormone and prolactin gene expression were not observed in the nonfunctioning adenomas, but ACTH mRNA was found in a single case. Immunohistochemistry of the adenomas confirmed production of one or more pituitary hormones in 13/14 (93%) nonfunctioning tumors, with a distribution of hormone production similar to that of the hormone mRNAs. These data indicate that pituitary adenomas originating from cells producing glycoprotein hormones are common, but are difficult to recognize clinically because of the absence of characteristic endocrine syndromes and defective hormone biosynthesis and secretion.

Use of MIB-1 (Ki-67) Immunoreactivity in Differentiating Grade II and Grade III Gliomas

The grading of glial tumors has traditionally relied on histological assessment, but the distinction between grade II and grade III gliomas is still a subject of debate. We examined the value of the monoclonal antibody MIB-1 (Ki-67) labeling index (LI) in the differentiation between grade II and grade III gliomas by either the 1993 WHO grading scheme or the St. Anne-Mayo grading scale. The MIB-1 LI in the most densely labeled areas from 80 diffuse cerebral hemispheric gliomas was determined. The tumors included 16 grade II, 31 grade III and 33 grade IV gliomas by the WHO scale. The mean Lis (%) were 0.88 ± 0.29 for grade II, 8.752±1.71 for grade III, and 9.12 ± 1.55 for grade IV gliomas. Analysis of variance indicated a significant difference in mean LIs between grades II and III and grades II and IV (p ≤ 0.0001), but not between grades III and IV. Seven tumors were classified differently by the 2 systems (grade III by WHO, but grade 2 by St. Anne-Mayo), and all had MIB-1 LI over 3%. Univariate analysis showed that MIB-1 LI with a cut-off point at 1.5% was a significant prognostic factor (p ≤ 0.0005). High tumor grade (WHO, p ≤ 0.0002; St. Anne-Mayo, p ≤ 0.0006) and patient age >50 (p ≤ 0.0001) were also significant factors for shorter survival. Using Cox Regression Multivariate Analysis, MIB-1 LI>1.5% was a significant independent predictor of shorter disease survival when paired with tumor grade (p ≤ 0.032), patient age (p ≤ 0.0065), or gender (p ≤ 0.0007). We conclude that the MIB-1 immunoreactivity is useful in distinguishing grade II from grade III gliomas, and maybe more sensitive in assigning aggressive gliomas to grade III than the St. Anne-Mayo grading system.

Prognostic significance of proliferative indices in meningiomas

The prognostic value of tumor proliferative indices in meningiomas was assessed by mitotic counts and by immunocytochemistry using a monoclonal antibody against the proliferating cell nuclear antigen (PCNA) (clone 19A2), a 36-kd nuclear protein involved in DNA synthesis. Sixty-three intracranial meningiomas were classified as benign (26), with atypical features (24) or as malignant (13). The patients included 24 men and 39 women, mean age 54.2 ± 1.7 (mean ± SEM) (range 15–78) at initial presentation. Twenty-four of the 63 primary tumors recurred locally, including 23.1% (6/26) of the benign, 37.5% (9/24) of the atypical, and 69.2% (9/13) of the malignant meningiomas. Among tumors that recurred, 1/9 (11%) of the atypical and 5/9 (55.5%) of the malignant tumors had had macroscopical total excision at the initial surgery. The mean interval to recurrence was 52 ± 11.8 months. The mean progression-free follow-up period for patients without tumor recurrence was 82 ± 8.5 months. Analysis of variance revealed that significant differences existed between tumor grades for both PCNA indices (1.16 ± 0.29% for benign; 14.14 ± 2.07% for atypical and 21.37 ± 5.47% for malignant) and mitotic indices (total counts per ten high power fields) (0.08 ± 0.05 for benign, 4.75 ± 0.91 for atypical and 19.00 ± 4.07 for malignant). Multivariate regression analysis indicated that mitotic index >6 was the single most important factor (p < 0.05) for shorter disease-free interval. Age, sex and total surgical excision were not significant factors. PCNA index was a significant factor in the univariate model, but not in the multivariate model. However, a two-factor-interaction model with PCNA >5% and mitoses >6 was highly significant as a predictor of outcome (p < 0.0001). We conclude that PCNA index may be used as an adjunct with mitotic counts in predicting the clinical course of patients with meningiomas.

Mutant, wild type, or overall p53 expression: freedom from clinical progression in tumours of astrocytic lineage

Abnormalities of the p53 tumor-suppressor gene are found in a significant proportion of astrocytic brain tumours. We studied tumour specimens from 74 patients evaluated over 20 years at the Massachusetts General Hospital, where clinical outcome could be determined and sufficient pathologic material was available for immunostaining. p53 expression studies employed an affinity-purified p53 monoclonal antibody, whose specificity was verified in absorption studies and, in a minority of cases, a second antibody recognising a different epitope of p53. Significant overexpression of p53 protein was found in 48% of the 74 tumours included in this series and high levels of expression were associated with higher mortality from astrocytic tumours (P<0.001, log rank). Multivariate analyses revealed that immunohistochemically detected p53 was an independent marker of shortened progression-free and overall actuarial survival in patients with astrocytic tumours, suggesting that increased expression of p53 plays an important role in the pathobiology of these tumours. In a subset of 36 cases, coding regions of the p53 gene were completely sequenced via SSCP and direct DNA sequencing, revealing that overexpression of p53 protein is not always associated with point mutations in conserved exons of the p53 gene. Finally, we confirmed p53 protein expression in early-passage human glioma cell lines of known p53 mutational status and immunostaining scores. Although grade continues to be the strongest prognostic variable, the use of p53 staining as a prognostic indicator, in contrast to mutational DNA analyses, may be a useful adjunct in identifying patients at higher risk of treatment failure.

TGF α expression in meningioma — tumor progression and therapeutic response

SummaryLittle is known of the molecular genetic mechanisms contributing to meningioma tumor progression. We evaluated a total of 26 clinical cases of meningioma: twenty three patients with meningioma treated at our institution between 1978 and 1990 and three asymptomatic cases found initially at autopsy. In addition, histologically normal meninges obtained at post-mortem examination from 5 cases were evaluated. There were 13 men and 10 women in the patient group with a median age of 48.7 years, treated by surgery and/or irradiation. Median follow-up was 46 months (range 16–152 months). Archival cases and age-matched normal meningeal tissue obtained at autopsy during the same time period were obtained for study. Patients with TGF α scores greater than 3.0 were more likely to fail treatment and had lower overall survival times than those with immunostaining scores of 1 or 2. Three autopsy cases where meningioma had been silent clinically had overall staining scores of 0.75, while 10 samples of normal meninges harvested from 5 cases at autopsy had staining scores of 0. Two patients each underwent 3 surgeries for recurrent tumor, serial specimens showed increased TGF α expression over time, though all material from these procedures was consistent with the diagnosis of histologically benign meningioma.

High PCNA index in meningiomas resistant to radiation therapy

PURPOSE Meningiomas are common intracranial tumors, often well controlled with surgical resection alone. While the efficacy of radiation therapy in improving local control and progression-free survival is well documented, prognostic data substantiate factors that are predictive of poor local control following definitive radiation therapy. PCNA is a DNA polymerase expressed at the highest levels in the S-phase, the most resistant portion of the cell cycle to ionizing radiation in vitro. We investigated the possible correlation between the levels of PCNA expression and the clinical outcome of patients treated with definitive radiation therapy. METHODS AND MATERIALS Archival tissue was collected from 33 cases of meningioma treated at our institution for definitive radiation therapy between 1970 and 1990. Age-matched normal meningeal tissue and asymptomatic meningiomas removed at autopsy served as tissue controls. A standard ABC immumoperoxidase technique employing antibodies to PCNA, PC-10 (Dako, California) was used to stain specimen slides for PCNA. PCNA index was defined as the number of positive nuclei per 10 high-power fields at 400x magnification. Two independent observers scored the slides without prior knowledge of the cases at hand. RESULTS Patients with high PCNA index were less likely to be controlled by therapeutic radiation (p < 0.001, Kaplan-Meier). All patients with a PCNA index greater that 25 failed radiation therapy. Using multivariate analyses, malignant (but not atypical), histology and PCNA index were significant predictors of progression following radiation therapy (p < 0.05, log rank). CONCLUSION PCNA index may be a useful adjunct to more standard histopathologic criteria in the determination of meningioma local control and progression-free survival following therapeutic irradiation. Data on a more expanded population evaluated on a prospective basis will be needed before such criteria are routinely employed in the clinical setting.

Effects of Insulin-induced Hypoglycemia on Cerebrovascular Permeability to Horseradish Peroxidase

The effects of hypoglycemia on cerebrovascular permeability to a protein, horseradish peroxidase (HRP), were studied in mice given 3 or 8 units of crytalline zinc insulin intraperitoneally. HRP (10 mg in 0.1 ml saline) was injected intravenously 15 to 20 minutes prior to sacrifice. Both mildly and severely hypoglycemic groups of mice showed a drastic reduction in the normal transit of HRP across cerebral arterioles. The number of normally-stained vessel segments and of HRP-filled endothelial vesicles decreased in insulin-treated mice. In the brains of severely hypoglycemic mice, however, increased parenchymal HRP accumulation occurred. A ruptured blood vessel was found in the center of one-fourth of the focal exudates examined. Electron microscopic examination revealed thrombin, sometimes extending through the vessel wall, and hemorrhage, yet inter-endothelial tight junctions remained intact. Seizures were associated with severe hypoglycemia in 6 out of 10 mice with serum glucose levels below 40 mg/100 ml following 8 units of insulin, but the number of focal exudates per brain was similar in all 10 mice. We conclude that insulin-induced hypoglycemia is associated with decreased HRP transit across cerebral arterioles, and that severe insulin shock is also accompanied by actual rupture of vessel walls and extravasation of blood and HRP into the parenchyma of the brain.

Functional transplantation of the rat pituitary gland.

OBJECTIVE These studies evaluated the ability of transplanted pituitary cells to restore pituitary function in hypophysectomized rats. METHODS The pituitary glands of neonatal Lewis rats were rapidly removed, enzymatically dispersed, and stereotactically introduced into the third ventricle of hypophysectomized adult male Lewis rats. Four weeks after implantation, plasma levels of anterior pituitary hormones in implanted animals were compared with those of sham-transplanted control animals. RESULTS Plasma levels of prolactin, growth hormone, thyroid-stimulating hormone, and beta-endorphin were below the range of detection in 14 sham-operated animals. In implanted animals, restitution of serum prolactin occurred in 100% of the animals tested, with levels of 2.6 +/- 1.0 ng/ml (mean +/- standard error of the mean; normal, 2-4 ng/ml). Growth hormone was assayable in 71% of the animals, with a mean value of 29 +/- 13 ng/ml over all animals (normal, 1-100 ng/ml); thyroid-stimulating hormone was restored in 68%, with mean resting levels of 79 +/- 13 ng/ml (normal, 100-400 ng/ml); luteinizing hormone levels were found in 53%, with mean levels over all animals of 0.2 +/- 0.1 ng/ml (normal, 0.5-1.0 ng/ml); and beta-endorphin was restored in 45% to high resting levels of 163 +/- 31 pg/ml (normal, 20-30 pg/ml). A challenge with hypothalamic releasing factor and a cold stress test were performed on the animals that had received transplants. Positive hormone responses to both of these tests suggested sensitivity of the pituitary grafts to both endogenous and exogenous sources of stimulation. Histological sections of paraformaldehyde-fixed brains from implanted animals clearly demonstrated survival of clusters of grafted pituitary cells. Positive immunohistochemical staining for adrenocorticotropic hormone and thyroid-stimulating hormone was demonstrated in sections of the grafted tissue. CONCLUSION These data suggest survival of neonatal pituitary transplants in the third ventricle of adult hypophysectomized rats with concomitant restoration of anterior pituitary hormone function.

Spontaneous metastasis, proliferation characteristics and radiation sensitivity of fractionated irradiation recurrent and unirradiated human xenografts☆

PURPOSE Do tumor cells which survive high dose fractionated irradiation exhibit modified metastasis activity, proliferation kinetics, and/or radiation sensitivity? To address this question experimentally, we have studied three recurrent human tumor xenograft systems. METHODS AND MATERIALS Three models were derived from a soft tissue sarcoma (HSTS26T), a colon adenocarcinoma (HCT15), and a glioblastoma (HGL21) which had recurred after 90 Gy, 109 Gy, or 77.4 Gy administered in 30 equal doses, respectively. Their production of spontaneous metastasis and cell proliferation characteristics were studied in early generation xenografts in SCID mice, and were compared to those in their previously unirradiated counterparts. As a control, we have also studied each tumor as a post-surgical recurrence. Specimens from the irradiated recurrent and their unirradiated primary tumors were cultured in vitro and their radiation sensitivity determined by clonogenic assay. RESULTS The three irradiated recurrent tumor systems retained the individual histological features of their unirradiated primary xenografts. A lower metastatic incidence was observed in two of the three irradiated recurrent tumor lines in comparison with their unirradiated control tumors and their surgical recurrent counterparts. No significant differences were found between the irradiated recurrent tumors and their unirradiated counterparts with respect to: volume doubling time, growth time, potential doubling time, mitotic index, PCNA index, and SF2 values. CONCLUSIONS High dose irradiation given in 30 fractions did not increase the metastatic activity in the three human tumor xenograft systems. Furthermore, the fractionated irradiation did not significantly change their proliferation characteristics and cellular radiation sensitivity.

Microvascular Ultrastructure after Perfusion Fixation and Injection of an Electron Dense Medium

Injection of a silicon rubber compound has been used to demonstrate the microvascular architecture of various tissues and organs (Reynolds 1968, Cortel 1969, Beeuwkes 1971, Beeuwkes and Bonventre 1975, Plyley and Groom 1975).