Platelet-derived endothelial cell growth factor/thymidine phosphorylase immunohistochemical expression in lymphoid tissue and lymphoid malignancies.

The catabolic enzyme thymidine phosphorylase (TP) plays a crucial role in nucleic acid metabolism by regulating the availability of thymidine. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor that was recently shown to be TP. The angiogenic properties of PD-ECGF/TP are attributable to a reduction of thymidine levels that results in a promotion of endothelial cell proliferation. Early studies showed a higher concentration of TP in macrophages than in parenchymal cells and in neoplastic than in nonneoplastic tissues. We examined the immunohistochemical expression of PD-ECGF/TP in reactive lymphoid tissues (lymph node and tonsil), as well as in a series of 20 cases of Hodgkin's disease and 31 cases of non-Hodgkin's lymphomas. Macrophages, sinus lining cells, and cells with dendritic morphology, of both follicular dendritic and interdigitating reticular cell type, presented a prominent nuclear and cytoplasmic positivity in reactive lymphoid tissue and in malignant lymphomas. Small lymphocytes and the neoplastic population were always negative, whereas endothelial staining was variable and showed no correlation to the type or grade of the lymphomas. In Hodgkin's disease (with the exception of the nodular lymphocyte predominance type) and some cases of high-grade non-Hodgkin's lymphomas, the positive dendritic cells formed a dense meshwork closely surrounding the neoplastic population. Our results suggest that the reported upregulation of PD-ECGF/TP activity in lymphoid malignancies is attributable to the nonneoplastic population, especially to cells of dendritic morphology.

TAL-1 protein expression in vascular lesions.

The distribution of TAL-1 protein, an important vascular promoter in mice, has been examined immunohistochemically in a range of human vascular lesions and normal tissues. Formalin-fixed, paraffin-embedded vascular lesions including granulation tissue, haemangiomas, Kaposi's sarcomas, spindle cell haemangioendotheliomas, and angiosarcomas, were examined using a monoclonal antibody to recombinant TAL-1. Endothelial cells in all lesions gave positive immunostaining of variable intensity. Granulation tissue and spindle cell areas of the vascular tumours gave the strongest staining (nuclear and cytoplasmic). The better-differentiated endothelial cells within the tumours and resident well-formed vessels were less positive and some cells were in fact negative. The malignant endothelial cells in angiosarcomas showed less intense positive staining than KS cells. This study has shown TAL-1 protein expression in a range of reactive, benign, and malignant vascular lesions. Protein expression appears to be stronger in the spindle cell areas, perhaps reflecting greater expression in less-differentiated endothelial cells.

Relationship of elevated tumour thymidine phosphorylase in node-positive breast carcinomas to the effects of adjuvant CMF.

BACKGROUND: Thymidine phosphorylase (TP) catalyses the reversible phosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate. High expression of TP in cell lines potentiates the effects of the cytotoxic drugs 5-fluorouracil and methotrexate, both of which are used in the cyclophosphamide, 5-fluorouracil and methotrexate (CMF) treatment regimen of breast cancer. PATIENTS AND METHODS: We therefore examined the expression of this enzyme in 328 invasive breast carcinomas using immunohistochemistry and assessed whether the expression of this enzyme by the tumour predicts patients response to CMF in node-positive patients. RESULTS: Whereas no significant difference in either relapse-free survival (RFS) (P = 0.2) or overall survival (OS) (P = 0.07) was observed between TP-negative and -positive tumours in non-treated patients, there was a significant increase in both RFS (P = 0.02) and OS (P = 0.02) in patients treated with CMF in TP-positive compared with TP-negative tumours. A multivariate analysis of the 134 node-positive patients demonstrated that in ductal carcinomas, TP was an independent variable for OS. CONCLUSIONS: This pilot study suggests that patients with TP-positive tumours have a significant survival benefit when treated with CMF and supports the hypothesis that TP enhances tumour sensitivity to the anti-metabolites 5-fluorouracil and methotrexate.

Platelet-derived endothelial cell growth factor (thymidine phosphorylase) expression in lung cancer.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an enzyme with angiogenic and cell motility properties. Moreover, it is involved in the transformation of fluoropyrimidines into active cytotoxic metabolites. In the present study, the expression of PD-ECGF in normal lung and lung cancer was immunohistochemically evaluated using the P-GF.44C monoclonal antibody. Alveolar and tumoural macrophages were invariably stained and were used as an internal control for assessment of the staining. Alveolar epithelium was always negative, whilst bronchiolar epithelium showed occasional positive reactivity. Normal lung and tumour endothelium was occasionally positive. Positive staining in more than 50 per cent of cells was observed in 23/71 squamous carcinomas (32 per cent), 16/38 (42 per cent) adenocarcinomas, and 2/6 (33 per cent) adenosquamous carcinomas. Differentiated areas and areas of squamous metaplasia were more strongly positive than other tumour areas. All 22 small cell carcinomas and one carcinoid tumour were negative. The present study provides a baseline for future studies in non-small cell lung cancer to correlate PD-ECGF expression with tumour vascularization, prognosis, and response to chemotherapy.

The distribution and expression of the two isoforms of DNA topoisomerase II in normal and neoplastic human tissues.

In mammalian cells, there are two isoforms of DNA topoisomerase II, designated alpha (170-kDa form) and beta (180-kDa form). Previous studies using cell lines have shown that the topoisomerase IIalpha and beta isoforms are differentially regulated during the cell cycle and in response to changes in growth state. Moreover, both isoforms can act as targets for a range of anti-tumour drugs. Here, we have analysed the normal tissue distribution in humans of topoisomerase IIalpha and beta using isoform-specific antibodies. In addition, we have studied expression of these isoforms in 69 primary tumour biopsies, representative either of tumours that are responsive to topoisomerase II-targeting drugs (breast, lung, lymphoma and seminoma) or of those that show de novo drug resistance (colon). Topoisomerase IIalpha was expressed exclusively in the proliferating compartments of all normal tissues, and was detectable in both the cell nucleus and cytoplasm. In biologically aggressive or rapidly proliferating tumours (e.g. high-grade lymphomas and seminomas), there was a high level of topoisomerase IIalpha, although expression was still detectable in colon tumours, indicating that expression of this isoform is not sufficient to explain the intrinsic drug resistance of colon tumours. Topoisomerase IIbeta was expressed ubiquitously in vivo and was localized in both the nucleoli and the nucleoplasm. This isoform was present in quiescent cell populations, but was expressed at a generally higher level in all tumours and proliferating cells than in normal quiescent tissues. We conclude that topoisomerase IIalpha is a strict proliferation marker in normal and neoplastic cells in vivo, but that topoisomerase IIbeta has a much more general cell and tissue distribution than has topoisomerase IIalpha. The apparent up-regulation of topoisomerase IIbeta in neoplastic cells has implications for the response of patients to anti-tumour therapies that include topoisomerase II-targeting drugs.

Platelet-derived endothelial cell growth factor expression correlates with tumour angiogenesis and prognosis in non-small-cell lung cancer.

Angiogenesis is a recently described prognostic factor in non-small-cell lung cancer. Platelet-derived endothelial cell growth factor (PD-ECGF), shown to be the enzyme thymidine phosphorylase (TP), induces angiogenesis in vitro and in vivo. High intracellular levels of the enzyme are associated with increased chemosensitivity to pyrimidine antimetabolites. PD-ECGF/TP expression was evaluated immunohistochemically in surgically resected specimens from 107 patients with operable non-small-cell lung cancer using the P-GF,44C monoclonal antibody. High expression of PD-ECGF/TP was found in 25% of cases and was associated with high vascular grade (P = 0.01). Fourteen of 32 (44%) high vascular grade tumours showed a positive reactivity for PD-ECGF/TP vs 13/75 (17%) of low/medium vascular grade. Positive expression was observed more frequently in T2-staged cases than in T1 (P = 0.04). While overall survival was not affected (P = 0.09), subset analysis revealed that node-negative patients with positive PD-ECGF/TP expression had a worse prognosis (P = 0.04). The results suggest that PD-ECGF/TP may be an important molecule involved in angiogenesis in non-small-cell lung cancer. Up-regulation of the enzyme defines a more aggressive tumour phenotype in patients with node-negative disease. Assessment of vascular grade and PD-ECGF/TP expression should be taken into account in the design of randomized trials assessing the role of adjuvant chemotherapy in non-small-cell lung cancer.

Thymidine phosphorylase expression in Kaposi sarcoma.

AIMS: To examine the immunohistochemical distribution of thymidine phosphorylase (TP) in all clinicopathological subtypes of Kaposi sarcoma. METHODS: Thirty two biopsy specimens of Kaposi sarcoma (29 patients) were studied. Six of these patients represented classic, six endemic, eight HIV associated, seven post-immunosuppression/transplant related, and two unclassified variants of Kaposi sarcoma. The average age was 49 years (range 22-83 years) and the male: female ratio 24:5. Four samples of angiosarcoma and one of spindle cell haemangio-endothelioma were stained in parallel. All specimens were fixed in formalin, embedded in paraffin wax and processed routinely. Immunohistochemistry was carried out using an antibody directed against CD31 (JC70) and the monoclonal antibody P-GF.44C against TP. RESULTS: All biopsy specimens showed immunoexpression for TP. The spindle cell component stained more strongly than newly formed endothelium lined vessels and normal, resident vessels at a distance from the lesions. CONCLUSIONS: The strong immunoexpression of TP suggests up-regulation of TP and a role for TP in angiogensis in Kaposi sarcoma. The mechanism for the up-regulation of TP remains unknown, but viral infections may trigger it. The differential staining of the various cell components of Kaposi sarcoma also suggest that TP either plays a role in the differentiation and maturation of Kaposi sarcoma or is a reflection of such changes.

The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase is up-regulated in breast cancer epithelium and endothelium.

Tumour angiogenesis is a complex multistep process regulated by a number of angiogenic factors. One such factor, platelet-derived endothelial cell growth factor has recently been shown to be thymidine phosphorylase (TP). TP catalyses the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine. Although known to be generally elevated in tumours, the expression of this enzyme in breast carcinomas is unknown. Therefore, we used ribonuclease protection assays and immunohistochemistry to examine the expression of TP in 240 primary breast carcinomas. Nuclear and/or cytoplasmic TP expression was observed in the neoplastic tumour epithelium in 53% of tumours. Immunoreactivity was also often present in the stromal, inflammatory and endothelial cell elements. Although endothelial cell staining was usually focal, immunoreactivity was observed in 61% of tumours and was prominent at the tumour periphery, an area where tumour angiogenesis is most active. Tumour cell TP expression was significantly inversely correlated with grade (P = 0.05) and size (P = 0.003) but no association was observed with other tumour variables. These findings suggest that TP is important for remodelling the existing vasculature early in tumour development, consistent with its chemotactic non-mitogenic properties, and that additional angiogenic factors are more important for other angiogenic processes like endothelial cell proliferation. Relapse-free survival was higher in node-positive patients with elevated TP (P = 0.05) but not in other patient groups. This might be due to the potentiation of chemotherapeutic agents like methotrexate by TP. Therefore, this enzyme might be a prediction marker for response to chemotherapy.

Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.

Polycystic kidney disease 1 (PKD1) is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease. We have studied PKD1 mRNA, with an RNase protection assay, and found widespread expression in adult tissue, with high levels in brain and moderate signal in kidney. Expression of the PKD1 protein, polycystin, was assessed in kidney using monoclonal antibodies to a recombinant protein containing the C terminus of the molecule. In fetal and adult kidney, staining is restricted to epithelial cells. Expression in the developing nephron is most prominent in mature tubules, with lesser staining in Bowman's capsule and the proximal ureteric bud. In the nephrogenic zone, detectable signal was observed in comma- and S-shaped bodies as well as the distal branches of the ureteric bud. By contrast, uninduced mesenchyme and glomerular tufts showed no staining. In later fetal (>20 weeks) and adult kidney, strong staining persists in cortical tubules with moderate staining detected in the loops of Henle and collecting ducts. These results suggest that polycystin's major role is in the maintenance of renal epithelial differentiation and organization from early fetal life. Interestingly, polycystin expression, monitored at the mRNA level and by immunohistochemistry, appears higher in cystic epithelia, indicating that the disease does not result from complete loss of the protein.

The distribution of the deleted in colon cancer (DCC) protein in human tissues.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins and is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is known to occur, resulting in different isoforms of the protein. Using five antibodies against the DCC gene product (three monoclonal antibodies raised in our laboratory, one commercially available antibody, and a rabbit polyclonal antibody), we have demonstrated by immunostaining a DCC protein isoform in reticuloendothelial cells in human thymus, tonsil, and lymph node. This can be distinguished from another isoform described in normal colonic epithelium, because this latter is not demonstrable with the antibodies we have used. It could not be detected in normal colonic epithelium, polyps or colorectal carcinomas. This restrictive distribution suggests that not all DCC gene products are important in colonic cancer.

Immunohistochemistry in apparently normal bone marrow trephine specimens from patients with nodal follicular lymphoma.

AIM: To establish the role of immunohistochemistry (using a limited panel of antibodies) in detecting minimal involvement by follicular lymphoma in routinely processed bone marrow trephine specimens, which show no obvious morphological (light microscopic) evidence of lymphoma; to determine whether bcl-2 immunostaining in bone marrow distinguishes between benign and malignant infiltrates in a patient with nodal follicular lymphoma. METHODS: Twenty seven consecutively selected paraffin wax embedded, formalin fixed bone marrow trephine specimens were stained with the following antibodies: anti-bcl-2, anti-CD79a, anti-CD3, and kappa and lambda light chains, using the Streptavidin biotin complex technique. RESULTS: Five of the 27 cases, which showed no evidence of involvement by follicular lymphoma on routine stains, showed monotypic B cells on immunohistochemistry. Two of the cases were diffuse, while the remaining three showed mini-aggregates around bony trabeculae. In all five cases the lymphomatous infiltrates were strongly bcl-2 positive. Reactive B lymphoid nodules did not show the same degree of bcl-2 positivity, and negative cells could be discerned within the reactive nodules. CONCLUSIONS: There is merit in studying so-called negative bone marrows immunohistochemically in order to detect minimal involvement by follicular lymphoma. A limited panel of antibodies including anti-bcl-2, anti-CD79a and anti-CD3 is usually adequate to accomplish this. Strongly bcl-2 positive lymphoid aggregates in the bone marrow of patients with nodal follicular lymphoma are indicative of lymphoma.