Expression of the soluble vascular endothelial growth factor receptor-1 in cutaneous melanoma: role in tumour progression.

BACKGROUND: Vascular endothelial growth factor (VEGF)-A, placenta growth factor (PlGF) and their corresponding membrane receptors are involved in autocrine and paracrine regulation of melanoma growth and metastasis. Besides the membrane receptors, a soluble form of the VEGF receptor (VEGFR)-1 (sVEGFR-1) has been identified, that behaves both as a decoy receptor, sequestering VEGF-A and PlGF, and as an extracellular matrix (ECM) molecule, promoting endothelial cell adhesion and migration through the interaction with α5ß1 integrin. OBJECTIVES: To analyse whether sVEGFR-1 plays a role during melanoma progression. METHODS: sVEGFR-1 expression was evaluated in a panel of 36 melanoma cell lines and 11 primary human melanocyte cultures by quantitative real-time polymerase chain reaction analysis and in specimens of primary or metastatic melanoma lesions from 23 patients by immunohistochemical analysis. RESULTS: sVEGFR-1 expression was highly upregulated in melanoma cell lines with respect to human melanocytes. Interestingly, cell lines obtained from cutaneous metastases showed a significant reduction of sVEGFR-1 expression, as compared with cell lines derived from primary tumours. These results were confirmed by immunohistochemical analysis of sections from primary skin melanomas and the corresponding cutaneous metastases, suggesting that modulation of sVEGFR-1 expression influences ECM invasion by melanoma cells and metastasis localization. Moreover, we provide evidence that adhesion of melanoma cells to sVEGFR-1 is favoured by the activation of a VEGF-A/VEGFR-2 autocrine loop. CONCLUSIONS: Our data strongly suggest that sVEGFR-1 plays a role in melanoma progression and that low sVEGFR-1/VEGF-A and sVEGFR-1/transmembrane VEGFR-1 ratios might predict a poor outcome in patients with melanoma.

Pre-operative chemotherapy and radiotherapy in breast cancer.

Primary systemic treatment of breast cancer with cytotoxics yields a high response rate and allows conservative surgical procedures in bulky tumours. In order to maximise local control of disease, two innovations were introduced in a pilot study. The first was to identify the good responders after three cycles of chemotherapy and to treat them with three additional cycles. The second was to also give this group of patients a full dose of radiotherapy before surgery with the aim of verifying the rate of pathological complete remissions in view of a possible treatment of breast primary with chemoradiotherapy only. Patients were treated with doxorubicin 60 mg/m2 and cyclophosphamide, 600 mg/m2 both intravenously on day 1, every 21 days for three courses. Partial or complete responders received three more courses followed by radiotherapy (50 Gy plus a 10 Gy boost). The others underwent immediate surgery. A total of 32 patients (median age, 50 years; range 28-69 years); performance status, 0-1; T2 22, T3 8, T4 2) were enrolled and were evaluable for response and side-effects. 9 patients had only three cycles of chemotherapy due to absence of response and 23 patients had six cycles of chemotherapy. Overall, 7 patients had a complete remission, 16 a partial remission and 9 had stable disease, for an overall response rate of 72% (95% confidence interval 53-86%). In the group of patients that completed the programme, two complete pathological remissions were observed and 5 patients had only microfoci of tumour. No toxic death or grade III-IV toxicities were observed. Mild or moderate side-effects included mucositis, nausea/vomiting and leucopenia. In conclusion, our results indicate that the addition of radiotherapy to pre-operative chemotherapy did not significantly enhance the incidence of pathological complete remissions. New primary treatment approaches should be explored in this subset of patients in order to improve outcome.

Biologic and molecular characterization of producer and nonproducer clones from HUT-78 cells infected with a patient HIV isolate.

HUT-78 cells were infected with a reverse transcriptase (RT)-positive supernatant of a culture of peripheral blood lymphocytes (PBL) from an AIDS patient and then cloned. Of these clones, two have been isolated and characterized. Clone D10 is persistently and productively infected with an HIV variant. The clone F12, in spite of the presence of an integrated full-length HIV provirus, does not release virus particles in the medium. D10 and F12 clones substantially differ in terms of protein pattern; that is, D10 is super-imposable to infected HUT-78 cells, whereas F12 exhibits a decreased uncleaved p55 gag precursor and the presence of uncleaved gp160 and of a unique p19, although they do not show qualitative or quantitative differences in viral RNA synthesis. Restriction patterns of F12 proviral DNA do not show major genomic deletions. These results indicate that F12 clone cells carry an HIV genome with minor mutations that probably affect the correct production of viral proteins at a posttranscriptional level. In addition, the F12 clone is resistant to high-multiplicity superinfection with HIV-1 or HIV-2.

Recovery of HIV-related retroviruses from Italian patients with AIDS or AIDS-related complex and from asymptomatic at-risk individuals.

Human retroviruses have been detected in supernatants of cultures of Ficoll-enriched lymphocytes from peripheral blood, lymph nodes and bone marrow of (a) 32 out of 42 patients with Acquired Immunodeficiency Syndrome (AIDS), (b) 34 out of 64 patients with AIDS-related Complex (ARC), (c) 9 out of 18 asymptomatic children born from Human Immunodeficiency Virus (HIV) seropositive mothers, and (d) 9 out of 28 asymptomatic drug abusers or hemophiliacs. Virus detection was monitored by assaying culture supernatants for the presence of Mg++-dependent reverse transcriptase (R.T.) activity. A number of these virus-positive sups were passaged repeatedly in cultures of phytohemagglutinin-stimulated and Interleukin-2 (IL-2) treated fresh lymphocytes from healthy blood donors. Occasionally, multiple samples were obtained at varying time intervals from the same patient and consistently yielded detectable retroviral activity. Several isolates were characterized as closely related if not identical to HIV, HTLV-IIIB strain, since cells from either patients' own lymphocyte cultures or subcultures infected with passaged virus were stained in an indirect immunofluorescent assay with both patients sera and monoclonal antibody against p24 antigen of the HTLV-IIIB strain. Representative isolates, grown on fresh lymphocytes of healthy donors and metabolically labelled with 35S-cysteine, were also analyzed in a radioimmunoprecipitation assay (RIPA) against patients' sera to define their antigenic pattern, which was widely superimposable to that obtained with HTLV-IIIB-infected H9 cells. DNA from lymphocytes infected with 2 representative isolates were Southern-blotted and probed with an insert from a plasmid containing the entire genome of the HTLV-IIIB strain. The hybridization patterns were comparable with those obtained with DNA from H9-infected cells.

[Radiotherapy in the treatment of carcinoma of the major salivary glands]. / La radioterapia nel trattamento dei carcinomi delle ghiandole salivari maggiori.

The authors conducted a retrospective study on 99 patients with malignant tumors of the major salivary glands, who had been treated by radiotherapy between 1976 and 1986: 86 cases of parotid tumors and 13 cases of submandibular gland tumors. Only 28 tumors (28%) were T1-T2, N0-N1. Local control, 3-5 and 8-year survival rates, and complications were considered. Radiation therapy alone was performed on 19 patients (19%), while combined surgical and radiation therapy was performed on 80 patients (80%). The results of radiation therapy alone and combined with surgery are discussed. Loco-regional disease control was obtained in 9 out of 19 patients (47%) by irradiation alone, and by combined irradiation and surgery in 60 out of 80 cases (75%). Distant metastases developed in 23 out of 99 patients (23%). Loco-regional metastases were the most frequent cause of death (20 out of 55 cases: 36%). Severe complications were extremely rare. The results demonstrate not only the advantages of combined treatment but also the value of radiotherapy alone in the treatment of tumors of the major salivary glands.

[Radiotherapy in the treatment of carcinomas of the base of the tongue. Analysis of the literature and personal experience]. / La radioterapia nel trattamento dei carcinomi della base della lingua. Analisi della letteratura ed esperienza personale.

Carcinoma of the base of the tongue is usually treated with radiation therapy. After a review of the literature, the findings are reported of 129 patients with squamous cell carcinoma of the base of the tongue treated in 1979-1983, with a minimum of a 3-year follow-up: 8 T1, 40 T2, 60 T3 and 21 T4 were found; only 48 cases were N0. Advanced stages (stage III and stage IV) were 83% of the total. External photon irradiation (Cobalt-60) was used in all cases; 33 patients underwent chemotherapy or surgery as additional treatment. The overall local control rate was 45.7%. Local control decreased as T stage advanced: T1 62.5%, T2 55%, T3 41.7% and T4 33.3%. The overall regional control rate for lymph nodes clinically palpable was 51.8%. The overall actuarial 5-year survival rate was 29.1%. The 5-year survival rate according to the N-staging varied from 37% for N0-stage to 17.4% for N3-stage. The majority of failures (92.8%) occurred within 2 years since the end of the therapy. Treatment complications, secondary carcinomas and causes of death are also discussed. Radiation therapy has proven to be effective for early-stage carcinoma of the base of the tongue; in more extensive lesions results are poor. Improved results could be obtained by optimal application of radiotherapeutic techniques. Knowledge of the various prognostic factors is essential to administer the therapeutic regimen for a given patient by the characteristics of his particular tumor.

[Carcinoma of the paranasal sinuses. Results with radiotherapy alone or with a radio-surgical combination]. / Carcinomi dei seni paranasali. Risultati del trattamento con radioterapia sola o con associazione radio-chirurgica.

The authors evaluate 117 patients affected by paranasal sinuses carcinoma treated by radiation therapy alone or by post-operative irradiation, from 1976 to 1984. Among these: 4 T1, 21 T2, 48 T3 and 44 T4; 66 cases were squamous cell carcinomas, 23 adenocarcinomas, 16 undifferentiated carcinomas and 12 adenoid cystic carcinomas. The overall 3 and 5 years actuarial survival rates were, respectively, 46% and 27%. The survival rates for stages T1 and T2 were 82 and 48% at 3 and 5 years; for stages T3 and T4 were 36% and 21%. According to the hystological type the survival rate varied from 50% for adenoid cystic carcinomas to 0% for undifferentiated carcinomas at 5 years. The survival rate for the group of patients treated with post-operative irradiation was 37% at 5 years, versus 10% for the group treated by radiotherapy alone. Complications have been minimal. Causes of failure are discussed. In conclusion, the authors consider a multicentric study as necessary to determine the efficacy of various schedules of treatment.

A trial with ribavirin in patients with AIDS or AIDS-related complex.

A therapeutic trial is described which concerns 6 patients with AIDS and 6 patients with ARC who were submitted to a 6 month course of Ribavirin. Ribavirin was administered orally as the following doses: 3.000 mg daily the first week, 2.000 mg daily the second 2 weeks, and 1.000 mg daily up to completion of the 6 months period. No major side-effects were recorded; only a transient anemia was observed in almost all patients at the higher dose; none of them, however, required to be transfused. No improvement was shown by any of the 6 AIDS patients, neither clinically nor according to laboratory test, whereas all 6 patients with ARC experienced a sense of well-being, a clearing of their symptoms and an average weight gain of about 2.5 kg. No significant changes, though, were recorded as for their immune parameters. A remarkable drop of aminotransfereas was also observed in 4 of the patients, who were affected with chronic hepatitis as well. We conclude that additional, if any, Ribavirin trials should be carried out only in ARC patients.

Characterization of the loricrin (LOR) gene as a positional candidate for the PSORS4 psoriasis susceptibility locus.

Psoriasis is a chronic inflammatory disease of the skin with both genetic and environmental risk factors. Non-parametric linkage analyses have mapped many susceptibility loci on different chromosomes. We mapped one of these loci, PSORS4, on human chromosome 1q21. Using the linkage disequilibrium approach, we refined the critical region to a specific genomic interval of about 100 Kb which contains only the loricrin (LOR) gene. Here we report a genetic and functional study of this gene to verify its involvement in psoriasis pathogenesis. We document low expression of LOR in psoriatic skin of patients selected from families in which the disease was segregating with the PSORS4 locus. Re-sequencing of the entire gene in a subset of patients revealed the existence of novel polymorphisms able to influence the protein structure, as shown by molecular modelling studies. However, no evidence for genetic association was detected in a large cohort of Italian nuclear families. This rules out the LOR gene as a candidate for the PSORS4 locus.

Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.