Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA. METHODS: Using a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies. RESULTS: PEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility. CONCLUSIONS: The authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

Herceptin.

The biology of the human epidermal growth factor (EGF) receptor-2 (HER2) has been reviewed numerous times and provides an excellent example for developing a targeted cancer therapeutic. Herceptin, the FDA-approved therapeutic monoclonal antibody against HER2, has been used to treat over 150,000 women with breast cancer. However, the developmental history of Herceptin, the key events within the program that created pivotal decision points, and the reasons why decisions were made to pursue the monoclonal antibody approach have never been adequately described. The history of Herceptin is reviewed in a way which allows the experience to be shared for the purposes of understanding the drug discovery and development process. It is the objective of this review to describe the pivotal events and explain why critical decisions were made that resulted in the first therapeutic to successfully target tyrosine kinases in cancer. New approaches and future prospects for therapeutics targeting the HER family are also discussed.

A recombinant human enzyme for enhanced interstitial transport of therapeutics.

Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

A novel approach to thymidylate synthase as a target for cancer chemotherapy.

Tumor cell resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious problem often associated with increased intracellular TS. Clinically, another problem that arises from the use of TS inhibitors is toxicity, which develops, in part, because normal cells may be adversely affected by doses of inhibitor that do not impact tumor cells. To circumvent this problem, we have devised a new strategy called enzyme-catalyzed therapeutic activation (ECTA), which takes advantage of overexpressed TS to enzymatically generate cytotoxic moieties preferentially in tumor cells. We show herein that tumor cells expressing elevated levels of TS are preferentially sensitive to NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine. We find support for the proposed mechanism of NB1011 in the following results: 1) positive relationship between TS protein level and sensitivity to NB1011 in engineered HT1080 tumor cells, designed to express defined levels of TS protein; 2) NB1011 activity is enhanced on tumor cells which express endogenous elevated TS; 3) cytotoxicity of NB1011 is blocked by raltitrexed (Tomudex); 4) NB1011 selection of TS-overexpressing MCF7TDX tumor cells results in recovery of cell populations and clones with diminished TS levels (and restored sensitivity to raltitrexed). A preliminary comparison of TS mRNA levels in multiple normal tissues versus colon tumor samples suggests that selective tumor cytotoxicity of NB1011 may be possible in the clinical setting. Because NB1011 cytotoxicity is dependent upon activation by TS, its proposed mechanism of action is distinct from current TS-targeted drugs, which require inhibition of TS to be effective.

Enzyme-catalyzed therapeutic agent (ECTA) design: activation of the antitumor ECTA compound NB1011 by thymidylate synthase.

The in vivo administration of enzyme-inhibiting drugs for cancer and infectious disease often results in overexpression of the targeted enzyme. We have developed an enzyme-catalyzed therapeutic agent (ECTA) approach in which an enzyme overexpressed within the resistant cells is recruited as an intracellular catalyst for converting a relatively non-toxic substrate to a toxic product. We have investigated the potential of the ECTA approach to circumvent the thymidylate synthase (TS) overexpression-based resistance of tumor cells to conventional fluoropyrimidine [i.e. 5-fluorouracil (5-FU)] cancer chemotherapy. (E)-5-(2-Bromovinyl)-2'-deoxy-5'-uridyl phenyl L-methoxyalaninylphosphoramidate (NB1011) is a pronucleotide analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), an antiviral agent known to be a substrate for TS when in the 5'-monophosphorylated form. NB1011 was synthesized and found to be at least 10-fold more cytotoxic to 5-FU-resistant, TS-overexpressing colorectal tumor cells than to normal cells. This finding demonstrates that the ECTA approach to the design of novel chemotherapeutics results in compounds that are selectively cytotoxic to tumor cell lines that overexpress the target enzyme, TS, and therefore may be useful in the treatment of fluoropyrimidine-resistant cancer.

p53 gene therapy in a rat model of hepatocellular carcinoma: intra-arterial delivery of a recombinant adenovirus.

p53 tumor suppressor gene therapy has been proposed for cancers characterized by inactivation of p53 function, and successful therapy will require efficient strategies for gene delivery. To maximize transgene expression in tumors, a clinical strategy has been proposed to treat neoplasms in the liver via hepatic artery administration of a recombinant adenovirus encoding wild-type p53 (rAd-p53). We have developed a syngeneic rat model using a p53mut hepatocellular carcinoma cell line (McA-RH7777) that results in multifocal liver tumor nodules to provide experimental support for this strategy. Treatment of McA-RH7777 cells with rAd-p53 in vitro resulted in efficient transgene expression, growth suppression, and apoptosis. Intrahepatic artery dosing with rAd-p53 or an adenovirus encoding beta-galactosidase (rAd-betagal) increased transgene expression in tumor tissue and decreased systemic exposure when compared with i.v. dosing. Daily hepatic artery dosing of rAd-p53 suppressed tumor growth when compared with untreated rats or animals treated with rAd-betagal. These data demonstrate the potential for arterial gene delivery to tumors using recombinant adenoviruses, and support continued investigation of rAd-p53 gene therapy for liver malignancies.

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells.

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.

p53-mediated accumulation of hypophosphorylated pRb after the G1 restriction point fails to halt cell cycle progression.

This study analyses whether the inability of p53 to induce G1 arrest after the restriction point relates to an inability to modulate pRb phosphorylation. Transient p53 overexpression in normal human diploid fibroblasts and p53-deficient cancer cells led to increased levels of the cyclin-dependent kinase inhibitor p21 cip1/Waf1/Sdi1 and an accumulation of hypophosphorylated pRb in cells growing asynchronously and in cells synchronized in late G1 or M. Similarly, gamma-irradiation of asynchronous, late-G1, or S phase fibroblasts led to an increase in hypophosphorylated pRb. Experiments with fibroblasts expressing the HPV16 E6 protein indicated that accumulation of hypophosphorylated pRb required functional p53. Progression into and through S phase was not altered by the presence of hypophosphorylated pRb in late G1, consistent with the failure of p53 to mediate G1 arrest in cells that are past the restriction point. These data indicate that accumulation of hypophosphorylated pRb has significantly different effects on cell cycle progression in early G1 versus late G1 or S phase.

Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo.

Alterations in the p53 tumor-suppressor gene occur in 35-60% of human glioblastomas, and re-introduction of p53 can suppress neoplastic growth. To evaluate the potential for p53 gene therapy of glioblastoma, we have analyzed the response of human glioblastoma cell lines in vitro and in vivo to experimental therapy with replication-deficient recombinant adenoviruses encoding wild-type p53 (rAd-p53). Western blot analyses showed high-level expression of p53 protein after treatment with rAd-p53, and transgene expression was dependent on promoter strength. A p53-specific dose-dependent inhibition of in vitro cellular proliferation was observed in 5 of 6 cell lines, and growth inhibition corresponded to adenovirus-mediated gene transfer and expression. p53-specific cell death was quantitated by release of the lactate dehydrogenase enzyme. Fragmentation of DNA into nucleosomal oligomers and the occurrence of a hypodiploid cell population detected by flow cytometry provided evidence for apoptosis. Studies in nude mice demonstrated that ex vivo infection with rAd-p53 suppressed the tumorigenic potential of human glioblastoma cells. Furthermore, direct injection of rAd-p53 into established s.c. xenografts inhibited tumor growth. Our observations suggest that re-introduction of wild-type p53 may have potential clinical utility for gene therapy of glioblastoma.

Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors.

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.

186Re-labeled antibodies to p185HER2 as HER2-targeted radioimmunopharmaceutical agents: comparison of physical and biological characteristics with 125I and 131I-labeled counterparts.

Overexpression of the HER2/neu protooncogene has been shown to correlate with poor clinical prognosis. A murine monoclonal antibody (4D5) directed against the extracellular domain (ECD) of p185HER2 has been shown to inhibit in vitro and in vivo growth of carcinomas overexpressing HER2 and has been humanized (rhuMAb HER2). The objective of the study was the identification of an agent which might be useful for in vitro studies, tumor imaging and/or radioimmunotherapy by linking beta-emitting radionuclides to these HER2-targeted antibodies. Murine 4D5 and humanized rhuMAb HER2 were radiolabeled with 125I, 131I or 186Re. Physical characteristics (TCA precipitability, SDS-PAGE, size exclusion chromatography), binding affinities to the HER2 ECD (in an ELISA and on SK-BR-3 cells) and antiproliferative activities of the radiolabeled antibodies were determined. Although 131I-4D5 and 131I-rhuMAb HER2 usually retained > 85% ECD binding, they exhibited increased aggregation and fragment content, drastically reduced antiproliferative activities and poor stability upon storage at 4 degrees C. For these antibody preparations, conservation of binding did not necessarily correlate with preservation of bioactivity indicating the importance of bioactivity determinations in radiolabeled antibody studies. Conversely, 4D5 and rhuMAb HER2 labeled with 125I or 186Re maintained physical properties, ECD binding, antiproliferative activities and were stable upon storage at 4 degrees C for at least 8 days. The superior retention of physical and biological characteristics of 186Re-labeled 4D5 and rhuMAb HER2 compared with their 131I-labeled counterparts suggests the potential for their use as radioimaging and radioimmunotherapeutic agents in the treatment of HER2 overexpressing tumors.

Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein.

Human malignancies are often characterized by mutations of the p53 tumor suppressor gene. In a large proportion of cases, the mutation results in production of an altered protein that can bind and inactivate the wild-type gene product. This "dominant-negative" activity of mutant p53 molecules may limit the utility of p53 gene therapy of cancer. Using replication-deficient recombinant adenoviruses (rAd-p53) as a p53 gene delivery system, we evaluated the effects of p53 reintroduction on a series of 45 human cell lines containing wild-type, mutated, or no p53 protein. Results indicate a p53-specific, dose-dependent, and promoter-specific growth inhibition of a majority of p53-altered cell lines that correlates with the degree of adenovirus transgene expression. Similar effects were not observed on cells containing wild-type p53. rAd-p53 inhibited the growth of cells expressing various mutant p53 proteins including those characterized as "dominant negative mutants", and the antiproliferative effects were not abrogated by high levels of endogenous mutated p53 protein. In vivo, rAd-p53 also suppressed tumor growth and increased survival of nude mice bearing tumors that express mutant p53. These results support a role for p53 gene therapy of cancer, including malignancies harboring mutations in this tumor suppressor gene.

Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography.

We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications.

Recombinant human retinoblastoma protein inhibits cancer cell growth.

Aberrant expression of the tumor suppressor gene RB1 is associated with a variety of solid tumors and hematopoietic neoplasms. Certain cancer cell lines in which the protein encoded by RB1 (p110RB) is absent have been reported to show decreased growth rate, clonogenicity, or tumorigenicity following insertion of a transcriptionally active RB1 gene. We asked whether these RB-deficient cells could be growth inhibited by direct exposure to purified p110RB. We report a decrease in uptake of tritiated thymidine by 5637 bladder carcinoma cells (RB-negative) when purified recombinant p110RB is added to culture media. Internalization of the protein by cells and translocation to the nucleus are demonstrated by immunohistochemistry, FACS, and detection of radiolabeled protein in subcellular fractions. Next, we chose a well-described leukemia cell culture model to investigate the potential effect of recombinant p110RB in clinical disease. We observed dose-related decreases in cell number of colony formation in vitro in 8 of 20 acute myelogenous leukemia samples, 7 of which did show endogenous p110RB detectable by immunohistochemistry. Histological appearance following exposure to p110RB shows cytoplasmic vacuolization and nuclear lobulation of degenerating cells. We conclude that purified p110RB added to culture media is internalized by cells, translocated to the nucleus, and exerts a growth-inhibitory effect on certain cancer cell types.

Inhibition of tumor cell proliferation in vitro and in vivo by exogenous p110RB, the retinoblastoma tumor suppressor protein.

Reconstitution of retinoblastoma gene (RB) deficient tumor cells with RB generally leads to growth suppression in vitro and/or reduced tumorigenicity in nude mice. An alternate approach to gene replacement is the delivery of the RB gene product (p110RB) into cells lacking its expression. In this report we demonstrate that exogenously added p110RB is taken up by and localized to the nucleus of cultured cells and has growth suppression properties similar to endogenous RB. RB-negative (RBneg) tumor cells are preferentially growth inhibited while most RB-positive (RBpos) tumor cells and normal cells are much less sensitive. We have extended these studies to relevant nude mouse xenograft models for human lung cancer. Local or systemic administration of p110RB inhibits tumor growth in treated animals. These results represent the first use of a tumor suppressor protein as a potential cancer therapeutic.

Retinoblastoma protein monoclonal antibodies with novel characteristics.

We have developed a family of monoclonal antibodies directed against the retinoblastoma gene product (p110RB). One of these monoclonal antibodies, 3C8, binds p110RB near the C-terminal end of the protein (aa886-aa905). It was characterized by immunoblotting, ELISA, fluorescence-activated flow cytometry and immunohistostaining. It was shown to be useful for the detection of p110RB in formalin-fixed and paraffin-embedded tissue sections. Because 3C8 binds outside of regions shown to be involved in p110RB interactions with other cellular proteins, it may be an especially useful reagent for the reliable detection of p110RB in tumor cells, and for the isolation by affinity chromatography of p110RB complexes with other cellular proteins.

Inhibition of human lung cancer cell line growth by an anti-p185HER2 antibody.

p185HER2, the product of the c-erbB-2 or HER2 gene, is a membrane-bound tyrosine kinase that has structural similarity to the epidermal growth factor receptor. Functionally, interaction of HER2 with its ligand or p185HER2 antibodies affects the growth and differentiation of HER2-expressing breast cancer cell lines. As p185HER2 is also expressed in human lung cancers and human lung cancer cell lines, we hypothesized that these cell lines would also respond to p185HER2 antibodies. To test this hypothesis, we cultured human non-small cell lung cancer cell lines in the presence of a p185HER2 monoclonal antibody called 4D5. 4D5 inhibited the growth of p185HER2-expressing cell lines in a dose-dependent fashion. In addition, BEAS.2B, a p185HER2-nonexpressing bronchial epithelial cell line, was transfected with the HER2 cDNA, resulting in high-level p185HER2 expression, and growth of BEAS.HER2 was now inhibited by 4D5 exposure. Mechanistically, 4D5 appeared to have a weak agonist effect on the tyrosine kinase function of p185HER2, as exposure of p185HER2-expressing cell lines to 4D5 resulted in increased p185HER2 phosphorylation. Furthermore, inhibition of tyrosine kinase function with Genistein reversed the 4D5-induced growth inhibition. Therefore, 4D5 can regulate the growth of p185HER2-expressing lung cancer cell lines through agonist effects on p185HER2.

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies.

The HER2 protooncogene encodes a receptor tyrosine kinase, p185HER2. The overexpression of p185HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 kappa antibody), was also tested in soft-agar growth assays for activity against p185HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a "humanized" 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185HER2.

c-erbB-2 oncoprotein in screen-detected breast carcinoma: an immunohistological study.

This study was aimed at comparing screen-detected and symptomatic breast carcinomas with regard to the prevalence of positive immunostaining for c-erb-B-2 oncoprotein. Paraffin sections of 81 breast carcinomas detected through the National Screening Programme were examined for the over-expression of c-erbB-2 oncoprotein using the avidin-biotin immunoperoxidase technique and 2 different specific antibodies: 21N and 4D5. Twenty-one other carcinomas detected in symptomatic patients were similarly examined for comparison. The screen-detected lesions were of 2 types: palpable and impalpable which required needle localization prior to surgical removal. Of the 81 screen-detected tumours, only 8 were c-erbB-2 positive, compared with 5 out of 21 non-screen-detected tumours. When only invasive carcinomas were considered, 5 out of 63 screen-detected cases were positive, compared with 5 out of 15 symptomatic cases. Four out of the 5 screen-detected positive invasive carcinomas were clinically palpable. Our results show an obvious trend towards a lower prevalence of c-erbB-2 oncoprotein over-expression in screen-detected carcinomas. As this over-expression is thought to be associated with increased tumour aggressiveness, the findings suggest a low prevalence of aggressive tumours in screen-detected lesions, particularly impalpable invasive carcinomas, identified so far.

Loss in expression of the retinoblastoma gene product in human gliomas is associated with advanced disease.

The retinoblastoma susceptibility gene (RB), the prototype of the class of tumor suppressor genes, is inactivated in a number of human malignancies. We investigated a possible role of RB in human brain tumors. Immunoprecipitation revealed frequent loss of RB protein expression in glioma cell lines (8/24), which was accompanied by lack of RB encoded transcripts. Among seventeen primary brain tumors studied by Western blotting, loss of Rb protein expression was observed in WHO grade 3 and 4 gliomas (3/10). However, none of the low grade gliomas and the other primary brain tumors investigated lacked RB protein expression. These data suggest a role for RB in glial malignancy, and loss of Rb expression appears to be associated with glial tumor progression.