Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment.

Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours.

Microdialysis assessment of methamphetamine-induced changes in extracellular neurotensin in the striatum and nucleus accumbens.

Stimulants of abuse such as cocaine and methamphetamine (METH) have dramatic effects on tissue neurotensin (NT) levels in the striatum and nucleus accumbens. Presumably these effects are due to the ability of such drugs to increase dopamine transmission. Because changes in dopamine activity appear to influence NT systems, we examined the effects of increasing doses of METH on extracellular NT levels in the medial striatum and nucleus accumbens using in vivo microdialysis in conscious rats. At the lowest dose tested (0.5 mg/kg), METH almost doubled the extracellular concentration of NT in both regions. When the dose of METH was increased to 5.0 mg/kg, extracellular NT concentration was elevated, but only to approximately 150% of control. At the highest dose examined (15.0 mg/kg), extracellular NT was not altered compared to pretreatment control levels. The role of DA D-1 and D-2 receptors in mediating these effects was determined by combining specific antagonists with the low dose of METH. The D-1 antagonist SCH 23390 blocked the METH-induced increase in extracellular NT levels in the striatum, but not in the nucleus accumbens. Pretreatment with the D-2 antagonist, eticlopride, blocked the increase in extracellular NT in both regions. Changes in striatal NT extracellular levels after a single METH injection were compared to the alterations in tissue NT levels following multiple administrations of the same doses of METH. Tissue levels were significantly elevated with 5 or 15 mg/kg METH in the medial, but not the lateral, striatum. There was not a clear correlation observed between the METH effects on striatal NT tissue levels and extracellular NT concentration.

Maintenance chlorambucil after CVP in the management of advanced stage, low-grade histologic type non-Hodgkin's lymphoma. A randomized prospective study with an assessment of prognostic factors.

One hundred sixty-two patients with Stages III and IV non-Hodgkin's lymphoma of low-grade histologic type were treated with combination chemotherapy using cyclophosphamide, vincristine, and prednisolone (CVP) followed by radiotherapy to sites of previous bulk disease. The patients were randomized to receive either follow-up alone or "maintenance" chemotherapy with 2 years of intermittent chlorambucil. A complete remission was obtained in 56% of patients and the median survival was 64 months (median follow-up, 74 months). Multivariate analysis revealed stage (P less than 0.0001) and Karnofsky performance status (P = 0.021) to predict complete response (CR) and the achievement of a CR (P less than 0.0001), female sex (P = 0.008), the absence of bulk disease (P = 0.038) and low serum alkaline phosphatase (P = 0.002) to predict prolonged survival. The median relapse-free survival (RFS) of the complete responders was 41 months. A prolonged RFS was predicted by low stage (P = 0.014), low serum lactic dehydrogenase (LDH) (P = 0.045) levels, and by the administration of maintenance chlorambucil (P = 0.045). A prolonged survival of the complete responders was predicted by a low number of nodal sites of involvement with lymphoma at presentation (P = 0.022) and lack of liver involvement (P = 0.011). The administration of oral maintenance therapy with chlorambucil for a full 2 years was only possible in 38% of patients, mainly because of progression of disease and the induction of thrombocytopaenia, but despite this it prolonged the median RFS by 38 months and its use could be considered when future studies are being designed.