TGF α expression in meningioma — tumor progression and therapeutic response

Summary Little 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 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 expression over time, though all material from these procedures was consistent with the diagnosis of histologically benign meningioma.     

Evaluation of response to postradiation eight in one chemotherapy in childhood brain tumors

Ten children, 3 to 15 years of age with high risk primary brain tumors were treated with postradiation eight in one chemotherapy; vincristine, lomustine, procarbazine, hydroxyurea, cisplatin, cytosine arabinoside, cyclophosphamide and methylprednisolone. The tumors comprised of three medulloblastomas, two primitive neuroectodermal tumors, one ependymoblastoma and four anaplastic ependymomas. Treatment involved surgery (two total resection, six subtotal and two biopsy only) followed by conventional radiotherapy (primary tumor: 50–54 Gy, whole brain: 30–45 Gy, and spinal axis: 25–36 Gy). Objective tumor response with radiotherapy was achieved in 7 of 9 patients (78%) (6/8 patients with residual tumor and one patient with complete resection but positive cerebrospinal fluid cytology). Complete response was attained in 4 of 9 patients (44%). Eight in one chemotherapy was initiated four weeks after radiation and repeated at 4 weekly intervals for 5–8 courses. Postradiation eight in one failed to show any additional effect on tumor responses. Median survival was 34 months (range 9–48 months) with five of ten patients alive: four in complete and one in partial remission. All the five survivors were among the patients who had achieved response to initial treatment. This result suggested that degree of response to initial treatment might determine subsequent outcome and thus the choice of modality for initial therapy might be important.     

Interferon effect on glycosaminoglycans in mouse gliomain vitro

Summary The effect of mouse interferon / (MuIFN /) on the production of glycosaminoglycans (GAGs) by mouse glioma G-26in vitro was evaluated. Two GAG species secreted extracellularly by the mouse glioma G-26 were isolated using cellulose acetate electrophoresis. They were identified as hyaluronic acid (HA) and chondroitin sulfate (CS) following enzymatic digestion with enzymes: hyaluronidase and chondroitinase ABC. Further characterization of CS by enzymatic digestion with specific chondroitinases for chondroitin 4-sulfate (CSA) and chondroitin 6-sulfate (CSC), revealed that the isolated CS was neither CSA nor CSC. Therefore, it may be either chondroitin sulfate B (CSB) (dermatan sulfate) or one of the chontroitin sulfate isomers (D-H).The three day incubation of glioma G-26 cells with 8×10-8×104 U/ml of MuIFN / resulted in a dose dependent inhibition of cell proliferation measured by³H-thymidine incorporation and the MTT assay. The significant decrease of the CS (p < 0.008) but not the HA level, (measured densitometrically), was observed following 72 hours (hrs) incubation of G-26 cells with 8 × 10³ U/ml of MuIFN / (IFN treated cells: 0.03 ± 0.007 integrated optical density (IOD); control cells: 0.07 ± 0.01 IOD). The decreased CS production may be the underlying cause of IFN mediated inhibition of glioma cell proliferation.     

Expression of TGFα in Meningiomas

The objective of this study was to examine the expression of transforming growth factor (TGF), a mitogen for many cell types, and its receptor in basic subtypes of meningiomas as well as in meningiomas of varying grade. Formalin-fixed tissues from 26 meningiomas including 15 benign (5 meningothelial, 5 transitional, and 5 fibrous variants), 6 atypical, and 5 malignant examples were immunohistochemically examined for both TGF protein and EGF/TGF receptor protein. In addition, in situ hybridization (ISH) was used to detect TGF mRNA expression. Immunostaining for TGF was strongest in fibrous and atypical meningiomas, followed closely by transitional and malignant tumors. Only weak reactivity was observed in the meningothelial variant. In all but 4 tumors (2 fibrous, 2 atypical), ISH showed TGF mRNA to be present, the signal being stronger in malignant than in conventional or atypical tumors. Lastly, immunostaining for EGF/TGF receptor was positive in all tumors studied. Strong TGF protein expression in meningiomas is commonly associated with fibrous morphology. Although the frequent detection of both TGF protein and its mRNA, as well as of EGF/TGF receptor within tumors of all type and grades, suggests that TGF serves to promote tumor growth, its possible role in tumorigenesis or malignant progression is uncertain.In summary, demonstration of these substances is of no utility in the classification or grading of this common tumor because the differences in their expression among the various meningioma subtypes were not statistically significant.     

Carotenoids,retinol, and vitamin E and risk of proliferative benign breast disease and breast cancer

We investigated the relationship between serum levels of retinol, -carotene, -carotene, lycopene, -tocopherol, and -tocopherol as well as intakes of retinol, carotene, and vitamin E and the risks of breast cancer and proliferative benign breast disease (BBD) in a case-control study of postmenopausal women in the Boston, MA (United States) area. Serum nutrient data were available for 377 women with newly diagnosed stage I or II breast cancer and 173 women with proliferative BBD. Controls were 403 women who were evaluated at the same institutions but did not require a breast biopsy or whose biopsy revealed nonproliferative BBD. We observed no significant associations between serum levels of these micronutrients and risk of proliferative BBD or breast cancer. The risk of breast cancer was decreased among women in the highest quintile of intake of vitamin E from food sources only (odds ratio [OR] for the highest quintile = 0.4,95 percent confidence interval [CI]=0.2–0.9; P, trend across quintiles = 0.02) but less so for total vitamin E intake including supplements (OR=0.7, CI=0.4–1.3; P, trend = 0.07).This project was supported by research grant CA 40429 from the National Cancer Institute. Dr London was supported in part by an Institutional National Research Service Award (CA 09001) from the National Cancer Institute.     

Clearing of cells bearing the bcl-2 [t(14;18)] translocation from blood and marrow of patients treated with rituximab alone or in combination with CHOP chemotherapy

Purpose:Patients who were PCR-positive for B-cellleukemia-lymphoma 2 (bcl-2) gene rearrangement [t(14;18)] wereevaluated for responses to rituximab alone or combined with CHOP. Patients and methods:Patients had relapsed or refractorylow-grade or follicular non-Hodgkins lymphoma (IWF: A–D). Thesingle-agent trial used 375 mg/m² weekly · 4; combinationtherapy included six cycles of CHOP and six 375 mg/m² infusions ofrituximab. Bcl-2analyses of bone marrow (BM) and peripheral blood(PB) samples at base-line and following therapy were performed using a PCRassay. Results:In the single-agent trial, of 70 patients whoseperipheral blood (PB) was bcl-2positive at baseline, 36 becamebcl-2-negative, 13 remained positive, and 21 varied between positive andnegative. The overall response rates (ORRs) were 72%, 31%, and57%, respectively. Twelve of twenty-two patients with repeat bonemarrow (BM) samples were bcl-2-negative three months post-treatment.Of 18 patients in the combination trial, 8 were bcl-2positive in PBand/or BM. All of seven patients positive in PB at baseline and six of sevenpatients positive in BM were negative at the end of therapy; all patientsresponded to treatment (100% ORR). Conclusions:Rituximab, alone or combined with CHOP, eradicatedbcl-2positive cells from PB and BM in over half of the patientstreated and was associated with a high overall clinical response rate. Theimpact on disease-free and overall survival awaits long-term follow up.     

A phase I-II study of 9-cis retinoic acid and interferon-alpha2b in patients with advanced renal-cell carcinoma: an NCIC Clinical Trials Group study.

Although advanced renal-cell carcinoma (RCC) responds poorly to standardtherapies, phase I–II trials have shown activity for combinations ofinterferon-2b (IFN) with a retinoid. Alitretinoin (9-cis RA) isan endogenous retinoid with high binding affinity for both RAR and RXRreceptor families. This phase I–II study enrolled 38 patients with RCCin a dose-escalation study of tolerability, pharmacokinetics (PK), andefficacy of twice daily oral 9-cis RA with subcutaneous IFN. Incontrast to studies with similar doses of daily 9-cis RA, PK studiesfound a consistent reduction in 9-cis RA concentrations of about50% after multiple b.i.d. doses of 30 or 50 mg/m²,independent of cotreatment with IFN. In the phase I portion, toxicitiesincluded systemic symptoms typical of IFN and biochemical abnormalitiespreviously associated with retinoids. Two patients experienced dose-limitingtoxicity at 50 mg/m² b.i.d. of 9-cis RA, thus therecommended phase II dose was 30 mg/m² b.i.d. One of twenty-sixevaluable patients achieved a durable objective partial remission, andrepeated dosing with this regimen was poorly tolerated. This combination ofretinoid and interferon is not recommended for further study in RCC.     

Hereditary persistence of {alpha}-fetoprotein: Case report and review of the literature

Persistently elevated -fetoprotein (AFP) levels of 24 to 30µg/ml (normal <10 µg/ml) were found in a 38-year-old healthyman. Subsequently, AFP was found to be elevated in another five out of 13family members within three generations. The pedigree is consistent with anautosomal dominant inheritance pattern. No discernible disease and nofunctional abnormality appears to be associated with this clinically benigndisorder which has been recorded in the literature on four occasions todate. The reported AFP levels in these other cases ranged from 18 to 198µg/ml.Physiologically, AFP is mainly produced in the liver and the yolk sac ofhuman fetuses more than four weeks old, with peak values of up to 4 mg/ml at12 to 16 weeks of gestation. After birth, AFP levels usually fall, withineight to 12 months, to a very low concentration of <10 µg/ml andpersist at low levels throughout life. However, AFP levels can rise abovenormal in both children and adults in distinct conditions and diseases whichwill be discussed.Hereditary persistence of -fetoprotein (HPAFP) should be consideredin both children and adults with unexplained and persistent elevation of AFPe.g., those screened for hepatocellular carcinoma or diagnosed for germ celltumor. It should also be recognized in AFP screening for neural tube defectsor Down's syndrome during pregnancy. Hereditary persistence of AFP can beeasily confirmed by analyzing AFP levels in family members.     

Manganese superoxide dismutase (MnSOD) polymorphism,alpha-tocopherol supplementation and prostate cancer risk in the alpha-tocopherol,beta-carotene cancer prevention study (Finland)

Objective: Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that plays a key role in protecting the cell from oxidative damage. A polymorphism in the mitochondrial targeting sequence (a valine to alanine substitution), thought to alter transport of the enzyme into mitochondria, has been associated with increased risk for breast cancer with a more pronounced association among women with low intake of dietary antioxidants. We examined the role of MnSOD in the development of prostate cancer in a large, randomized cancer prevention trial of male smokers, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. We hypothesized that MnSOD may be associated with prostate cancer and that long-term antioxidant supplementation (-tocopherol 50 mg/day for five to eight years) could modify the effect on risk. Methods: Logistic regression was used to estimate these associations among 197 cases and 190 controls genotyped and matched for age, intervention group, and clinic. Results: Men homozygous for the MnSOD ala allele had a 70% increase in risk over men homozygous for the val allele (odds ratio, OR = 1.72, 95% confidence interval, CI = 0.96–3.08, p = 0.07). Supplementation with -tocopherol had no impact on the MnSOD–prostate cancer association. Although there was no difference in the association with disease stage, men homozygous for MnSOD ala (compared to MnSOD val/val or val/ala) showed a three-fold risk increase for high-grade tumors (OR = 2.72, 95% CI: 1.15–6.40, p = 0.02). Conclusion: These data suggest an effect of the MnSOD ala/ala genotype on the development of prostate cancer. Our observation of a stronger association with high-grade tumors may have prognostic implications that should also be pursued.     

Endogenous ligands for the epidermal growth factor receptor in human breast tumors do not interfere in an epidermal growth factor receptor radioligand binding assay

Summary Ligands for the epidermal growth factor receptor (EGFR) (e.g. EGF and transforming growth factor alpha, TGF) may be included in membrane preparations from human breast tumors, either complexed or not to EGFR. Assessment of EGFR in human placental membranes (HPM) after preincubation with human EGF (hEGF) or human TGF (hTGF) indicated that the presence of these ligands in a membrane preparation did not affect the apparent number of binding sites, but only resulted in an increased apparent dissociation constant (Kd).To obviate the effect of endogenously bound EGFR ligands in a radioligand binding assay, an acid treatment procedure has been used recently. In the present study we show that acid treatment of mammary tumor membranes does not result in increased EGFR levels but does result in an EGFR enriched membrane preparation by elimination of contaminating cytosol proteins. This however also occurred by washing the membranes with assay buffer at neutral pH.From these experiments we conclude that endogenous ligands, if present in membrane fractions from human breast tumors and occupying EGFR, do not interfere in a multipoint radioligand binding assay.Abbreviations EGF Epidermal Growth Factor - TGF Transforming Growth Factor alpha - EGFR Epidermal Growth Factor Receptor - HAP Hydroxylapatite     

Human Malignant Glioma Therapy Using Anti-αVβ3 Integrin Agents

Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults and is invariably fatal. We have investigated the effect of cyclo-(Arg-Gly-Asp-D-Phe-Val) (cRGDfV) peptide on survival of human malignant glioma cells in vitro and in vivo. Immunofluorescent analyses revealed the presence of _V3 integrin on U-87MG and U-373MG cells, but minimal expression on U-251MG cells. Treatment of U-87MG and U-373MG cells in vitro with cRGDfV (20µg/ml), but not the linear peptide, resulted in the appearance of rounded and loosely attached cells with subsequent cell death. By comparison, neither this cyclic peptide nor its linear homolog had any significant effect on growth and morphology of U-251MG cells. The death of cRGDfV-treated (20µg/ml) glioma cells was blocked by pretreatment (10µM) of cells with DEVD-FMK and LEHD-FMK, inhibitors of caspase-3 and caspase-9, respectively. Moreover, when glioma cells grown as spheroids were treated with cRGDfV (50µg/ml), spheroid formation was markedly reduced. Further, treatment of intracranial U-87MG tumors in scid mice with cyclic peptide significantly (p<0.001) prolonged their survival. These results indicated (i) that cRGDfV induced apoptosis of human glioma cells by binding _V3 integrin expressed on their cell surfaces and (ii) that cRGDfV may be an effective and non-toxic direct anti-tumor therapy for _V3-expressing GBMs.     

Glioblastoma Multiforme in an Asian Population: Evidence for a Distinct Genetic Pathway

In Singapore astrocytic tumours occur in only 25% of patients with primary brain tumours compared to 40–60% in other series. Glioblastoma multiforme arises either de novo as a primary glioblastomas associated with epidermal growth factor receptor (EGFR) and mdm2 over-expression or as a secondary glioblastomas, through malignant progression from low-grade astrocytomas, associated with p53 mutations and PDGFR- over-expression. Using immunohistochemical methods and DNA sequencing, we studied our population of glioblastomas for over-expression of EGFR, mdm2, p53, and PDGFR- as well directly for mutations of the p53 gene. While levels of over-expression of EGFR and mdm2 were consistent with levels expected for primary glioblastomas, levels of p53 and PDGFR- were consistent with levels documented for secondary glioblastomas. Notably 96% of the samples over-expressed p53 as detected with monoclonal antibody pAb 240. Of the 39 samples available for DNA sequencing 18% (7/39) had p53 mutations, including three mutations previously undocumented in glioblastomas. These results provide strong evidence that glioblastomas in Asian patients do not conform to currently accepted models of glioblastoma development, and that clinically defined glioblastomas in these patients show genetic changes consistent with both primary and 'secondary glioblastomas.     

Candidate metastasis-associated genes of the rat 13762NF mammary adenocarcinoma

Summary Differential hybridization was used to isolate genes potentially involved in the process of metastasis. Ten complementary DNAs (cDNAs) that were differentially expressed between a highly metastatic (MTLn3) and a nonmetastatic (MTC.4) line of the rat 13762NF mammary adenocarcinoma were isolated and sequenced. Examination of the EMBL/GenBank database revealed that one of the genes had a high degree of homology (98.8%) to annexin I (also known as calpactin II). Quantitative analysis of Northern blot hybridizations showed that the annexin I-like sequence was expressed 4- to 7-fold higher in MTLn3 than in MTC.4 cells. Steady state mRNA levels were also low in MTLn2, a cell line of low metastatic potential closely related to MTLn3, but were not related to metastatic potential in colon adenocarcinoma or melanoma cells. Two of the cDNAs (designated 8.11 and 10.14) were found to be novel. The expression of 10.14 mRNA (3.2 kb) was 4-fold higher in MTLn3 than in MTC.4 cells. Sequencing of the 10.14 cDNA (2.2 kb) revealed a putative open reading frame of 583 amino acids that was also novel. Expression of 8.11 mRNA (>7 kb) inversely correlated with metastatic potential. Another differentially transcribed gene was highly homologous to ERK2 (extracellular signal related kinase 2), a mitogen-activated protein kinase (MAPK). Northern analysis of ERK2 expression revealed 3-fold higher amounts of a 1.3 kb mRNA in MTLn3 than in MTC.4 cells. Higher levels of ERK2 mRNA were generally seen in the more metastatic human colon but not in melanoma cell lines. We also corroborated the work of Taniguchi (Nucl Acids Res 19:6949, 1991) by independently identifying EF-1 as a putative metastasis-associated gene.Supported by an educational grant from CIBA-GEIGY Corp., at the San Antonio Breast Cancer Symposium, Dec. 8, 1992.     

Ligand-Induced Regulation of ERα and ERβ is Indicative of Human Breast Cancer Cell Proliferation

Two estrogen receptors (ER), ER and ER, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ER protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ER and ER protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ER and ER, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ER in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ER expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ER in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ER expression. Although a change in the ER/ER ratio may play a role in the effect seen in cell proliferation, this study indicates that ER is a poor prognosticator of cell proliferation in breast cancer and that ER is a positive prognosticator of responsiveness to antiestrogen treatment.     

Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells

Verotoxin 1 (VT1) is an E. coli toxin comprising an A subunit with N-glycanase activity, and five smaller B subunits capable of binding to the functional receptor globotriaosylceramide (Gal1-4-Gal1-4-Glcceramide-Gb3). VT is implicated in hemorrhagic colitis and the more serious hemolytic uremic syndrome. VT1 is active against various tumor cell lines in vitro and in vivo. To extend the anti-cancer spectrum of activity of VT to human brain tumors, in the present analysis we studied the effects of VT on the growth of 6 permanent human astrocytoma cell lines. All astrocytoma cell lines analyzed express Gb3 and were sensitive to VT-1 at a dose of 50 ng/ml, but sensitivity was not proportional to the relative Gb3 concentration. VT induced apoptosis in these cells was shown by electron microscopy. Morphological evidence (nuclear shrinkage and chromatin condensation) of apoptosis could be clearly distinguished 1.5 hrs after toxin addition. Ultrastructural preservation of organelles was observed in conjunction with blebbing of the plasma membrane, condensation of chromatin within the nucleus and nuclear shrinkage. Apoptosis was also induced by the recombinant toxin B subunit alone, suggesting that the ligation of Gb3 is the primary induction mechanism. These studies indicate that verotoxin/Gb3 targetting may provide a novel basis for the inhibition of astrocytoma tumour cell growth.