Phase II clinical trial with gemcitabine and paclitaxel sequential monotherapy as first-line treatment for advanced non-small-cell lung cancer (SLCG 01-04)

BACKGROUND: In advanced-stage (IIIB or IV) non-small-cell lung cancer (NSCLC), combination chemotherapy has demonstrated response rates of 20% and a 1-year survival rate of 30%. We conducted a multicentre, open-label, nonrandomised phase II trial to determine the efficacy and tolerability of sequential monotherapy with gemcitabine followed by paclitaxel in chemotherapy-naïve patients with advanced NSCLC. MATERIALS AND METHODS: Between December 2002 and July 2004, the Spanish Lung Cancer Group (SLCG) conducted a study in which 34 patients with advanced (stage IIIB or IV) NSCLC received 1200 mg/m(2) of i.v. gemcitabine on days 1, 8 and 15 of each 28-day cycle for a total of 3 cycles followed by 100 mg/m(2) of weekly i.v. paclitaxel for a maximum of 8 weeks. If objective response or stable disease was achieved, 70 mg/m(2) of weekly i.v. paclitaxel was maintained until disease progression was evident or toxic effects were intolerable. Lung Cancer Symptom Scale (LCSS) analysis was performed. Baseline levels of serum VEGF, EGFR, telomerase reverse transcriptase (hTERT) and K-ras mutations were analysed. The primary endpoint was the objective response rate. RESULTS: The median age of the 34 patients who were enrolled was 67 years (range 46-77), but later 8 patients were excluded; 78.8% were men, 81.8% had performance status 1 and also 81.8% had metastatic disease at diagnosis. The objective response rate was 28% (95% CI, 14.2-47.8); the median overall survival was 7.2 months (95% CI, 2.1-12.3) and the median time to progression (TTP) was 3.1 months (95% CI, 2.5-5.3). Grade 3 or 4 drug-related haematological toxicities were observed in 6 patients. Patients with lower baseline serum VEGF levels had significantly longer survival. CONCLUSIONS: Sequential therapy with gemcitabine followed by paclitaxel was well tolerated with a low proportion of grade 3 or 4 adverse events, the absence of unexpected toxicity and with an improvement in quality of life. Unfortunately, the response rate did not meet the minimally required rate of 20% and the study was prematurely closed. VEGF was identified as a poor prognostic factor for TTP and survival (AU)

Intralesional isotype profiles in human localized cutaneous leishmaniasis lesions.

This immunocytochemical study evaluates the presence of IgG1-4, IgA and IgE immunoglobulins in active lesions of 25 localized cutaneous leishmaniasis patients from three bioclimatic areas (Awa, Afa and Bsha) in Mérida State, Venezuela. All immunoglobulin isotypes except IgE were detected, with variable intensity, in one or more of the epidermal or dermal components of skin lesions. IgG1 and IgG2 were detected significantly more frequently than IgG3, IgG4 and IgA. The ranking of the isotypes according to frequency of detection was the same in all areas: IgG1 = IgG2 > IgG3 = IgG4 = IgA, but considered as whole, all isotypes were detected significantly more frequently in patients from the Awa area than in those from the Bsha area. The predominant expression of isotypes IgG1 and IgG2 suggests a preferential Th1 like immune response. Anti-Leishmania immunoserum stained only parasites and their debris, suggesting that most of the immunostaining was nonspecific.

Vase mini-exon usage by NCAM is not restricted to tumours of neuroectodermal origin.

The neural cell adhesion molecule (NCAM) plays an important role in normal development. Many variants of NCAM are generated through post-transcriptional and post-translational modifications. These variants are tissue-specific and their expression is developmentally regulated. NCAM is also re-expressed in a number of human tumours, including neuroblastoma, rhabdomyosarcoma, Wilms' tumour and Ewing's sarcoma. We have characterized the NCAM variants associated with rhabdomyosarcoma. Polysialylated NCAMs are present in this tumour and, after neuraminidase treatment, they resolve into 2 bands of 140 and 120 kDa. These data were corroborated by Northern-blot analysis where mRNA species of 6.7 and 5.5 kb are detected. These mRNA code for the 140- and 120-kDa NCAM proteins respectively. PCR analysis shows that the previously described VASE mini-exon is also present in NCAM found in rhabdomyosarcoma. The VASE mini-exon, spliced at exon 7-8 junctions, has previously been detected in neural and heart NCAM, as well as in NCAMs found in human small-cell lung carcinoma (SCLC). DNA sequencing confirmed that the VASE mini-exon in rhabdomyosarcoma is identical to that found in neuroblastoma and SCLC.

Thermotherapy v. antimonials: what happens after the healing of cutaneous leishmaniasis lesions?

The long-term effects of meglumine antimoniate (chemotherapy) or exposure to an environmental temperature of 37 degrees C (thermotherapy) on the evolution of Leishmania mexicana infections and on the response to challenge infections six months after treatment were compared in susceptible (BALB/c) and partially resistant (C57BL/6) mice. Thermotherapy was better than chemotherapy in that it healed lesions quicker and prevented relapses in the partially resistant mice during the observation period. However, both treatments appeared equally effective in terms of clinical cure. Neither treatment cleared all parasites from the hosts and both impaired the hosts' immune response to a challenge infection. The results indicate that specific immunity fades with time post-infection and that the persistence of the parasite in a clinically cured host does not maintain protective immunity against challenge infections.

Is leishmaniasis ever cured?

The persistence of parasites in mice cured of Leishmania mexicana infection was investigated by using immunosuppressive drugs and checking for the reappearance of lesions. BALB/c (susceptible) and C57BL/6 (partially resistant) mice infected with 10(4) amastigotes were treated with either thermotherapy or meglumine antimonate and subsequently immunosuppressed with either cyclophosphamide or hydrocortisone. Immunosuppression by either method caused lesions to reappear in both strains of mice regardless of the treatment used to produce clinical cure. In both strains of mice the proportion of animals developing lesions after immunosuppression was greater in the mice cured by the drug. The relevance of these findings to human therapy is discussed.

A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.

Splicing of the VASE exon of neural cell adhesion molecule (NCAM) in human small-cell lung carcinoma (SCLC).

Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.

Expression of the neural cell adhesion molecule (NCAM) on the haemopoietic cell line Nalm-16.

The Nalm-16 cell line was originally described as being of the null acute lymphoblastic leukaemia (ALL) phenotype. Using phenotypic and genotypic markers, we have demonstrated the line carries markers associated with cells of the B lineage. In addition, Nalm-16 binds a series of monoclonal antibodies characterized as predominantly recognising neuroectodermal tissues. Amongst these antibodies, UJ13A, 5.1.H11 and ERIC-1 have been shown to recognise the neural cell adhesion molecule (NCAM). Expression of the NCAM molecule is highly complex, with several isoforms of the protein resulting from the differential splicing of the NCAM mRNA transcript. Western-blot analysis of Nalm-16 cell extracts indicates that cells express the heavily polysialylated form of the molecule found on many embryonic tumours. Neuraminidase digestion indicates that the 140 kD isoform is predominantly expressed on Nalm-16, although the 120 kD isoform is present to a lesser degree. These findings have been confirmed using Northern-blot analysis.

A comparison of the lectin-binding properties of glycoconjugates from a range of Leishmania species.

Constituent glycoconjugates of promastigotes of 14 different Leishmania strains from 6 different species were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently stained with 14 125I-labelled lectins of different specificities. Autoradiography of the gels revealed lectin-specific glycoproteins and other glycoconjugates of known molecular weight. Similarities and differences in antigens and glycoproteins present in the strains are described. The possibility of identification or characterisation of Leishmania species from their electrophoretic behaviour and lectin-binding patterns is unlikely, but these results should be an aid to purification of glycoconjugates from particular strains by lectin-affinity chromatography.

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2.

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.

Monoclonal antibody UJ13A recognizes the neural cell adhesion molecule (NCAM).

Monoclonal antibody (MAb) UJ13A, raised against 16-week-old human foetal brain, recognizes an antigen present on the majority of tissues of neuro-ectodermal origin. The binding profile of this antibody is similar to the known distribution of the neural cell adhesion molecule (NCAM). Although detailed immunohistological and immuno-cytochemical studies with the MAb have been published previously, we demonstrate here, through investigations of selected tissues and cell lines, that the binding profile of an anti-NCAM antiserum is similar to UJ13A. Additional indirect evidence suggesting that UJ13A recognizes NCAM comes from Northern blot analysis of cell lines either binding or not binding UJ13A as determined by indirect immunofluorescence. Only those cell lines known to bind UJ13A express high levels of NCAM mRNA. Western blot analysis of extracts of human brain show that UJ13A recognizes proteins of 180 and 140 kDa in addition to a very weak band of 120 kDa. This data corroborates the suggestion that UJ13A recognizes NCAM as these 3 isoforms of the protein are identified in human brain. Final confirmation of the specificity of UJ13A comes from the study of 3T3 fibroblasts transfected with a cDNA coding for the 125 kDa isoform of human muscle NCAM. UJ13A selectively recognizes these transfectants and binds to a 125 kDa protein isolated from the cells by Western blot analysis. Thus we conclude from these immunological, biochemical and molecular studies that the antigen recognized by the UJ13A MAb is the neural cell adhesion molecule.

Cardiac parasympathetic abnormalities: Cause or consequence of chagas heart disease?

The mechanisms by which Trypanosoma cruzi causes cardiomyopathy are unknown but are the subject o f several hypotheses. In this paper, Diego Davila, Osman Rossell and Jose Donis discuss the aetiology of cardiac failure in Chagas disease and suggest that parasympathetic abnormalities are a consequence of, rather than the cause of, the progressive cardiac enlargement seen in these patients.

Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma.

We describe reagents from 2 workshops which had been identified as recognizing the same or very similar antigens based on their tissue reactivity. Examination of their tissue specificity led us to the conclusion that this was similar to the expression of the neural cell adhesion molecule (NCAM). We also describe the use of a transfection-based assay to show that these reagents do recognize NCAM. 3T3 cells were transfected with a full-length clone of human NCAM. Indirect immunofluorescence studies showed binding of all related antibodies to the transfectants, but not to the control 3T3 cells. In addition, biochemical analysis using certain antibodies in the cluster confirm that they detect NCAM in the transfectants. Our study shows the benefits of using workshops to compare monoclonal antibodies and a molecular approach to define the antigens recognized by such reagents.

The correlation between delayed hypersensitivity, lymphocyte activation and protective immunity in experimental murine leishmaniasis.

The growth of Leishmania major and Leishmania mexicana lesions and the concomitant development of delayed-type hypersensitivity (DTH) to homologous or heterologous soluble antigen was studied in BALB/c and CBA/Ca mice. Although CBA/Ca mice are highly susceptible to L. mexicana, developing non-healing lesions, they are resistant to L. major; while BALB/c mice develop non-healing lesions when infected with either species. The development of resistance was associated with the acquisition of DTH which peaked at 48 h (L. major infected CBA/Ca mice). Non healing lesions were associated with either negative DTH (L. major infected BALB/c mice) or DTH that peaked at 24 h but had significantly subsided by 48 h (L. mexicana infected CBA/Ca and BALB/c mice). The latter response was associated with basophilic infiltration of the skin test site. Pre-irradiating (600 rad) CBA/Ca and BALB/c mice induced resistance against L. mexicana and L. major respectively in conjunction with the appearance of 48 h DTH to the homologous antigen. There was clear dissociation in the skin reactivity produced by the heterologous antigen. Thus L. major-derived antigen failed to produce DTH in L. mexicana infected mice of either strain. L. mexicana-derived antigen on the other hand produced a quicker response and of greater magnitude than the homologous antigen in L. major infected CBA/Ca mice. This correlated well with the strong cross-immunity induced by L. major in these mice to L. mexicana infection.

Reduced hepatic insulin extraction in obesity: relationship with plasma insulin levels.

To elucidate if there are alterations in insulin metabolic clearance in obesity under basal conditions, plasma insulin and C-peptide were measured in 22 obese patients and 8 normal subjects, and the plasma C-peptide to insulin molar ratio was used as an index of hepatic insulin extraction. In obese patients, the C-peptide to insulin molar ratio correlated indirectly with basal plasma insulin levels (r = 0.71; P less than 0.001), being low in the obese patients with higher insulin levels and within the normal range in obese patients in which insulin levels were similar to those of control subjects. It is suggested that hepatic insulin extraction is decreased in obesity, even under basal conditions, but this alteration is only manifested when plasma insulin levels are high.