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Two human melanoma lines were transduced by a retroviral vector with the gene of the human interleukin-2 (IL-2) and characterized for their immunological properties in comparison with the parental lines. Transduction resulted in the production of biologically active IL-2 in the average amounts of 2,282 and 2,336 pg/ml per 105 cells per 24 hr over 3 and 2 months by the Mel4932/IL-2 and the MelB6/IL-2 lines, respectively. Melanoma-transduced cells lost their tumorigenicity in nude mice. No major changes in the phenotype were observed in IL-2 gene-transduced lines. In fact, more than 90% of cells expressed class I and II(DR) HLA, adhesion molecules, integrins, and melanoma-associated antigens. Irradiation with 100–400 Gy, while inhibiting tumor cell growth in vitro, allowed the release of IL-2 by the transduced cells for at least 5 weeks. The two melanoma lines also maintained susceptibility to lysis by lymphokine-activated killer (LAK) cells and by a HLA-A2-restricted melanoma-specific cytotoxic T ...

Sister chromatid exchange (SCE) analysis was carried out on peripheral blood lymphocytes of 20 familial malignant melanoma (FMM) and 39 sporadic malignant melanoma (SMM) untreated patients, belonging to 10 and 39 families, respectively. The study was extended to 39 unaffected close relatives of FMM patients, to 187 unaffected close relatives of SMM patients, and to 20 unaffected unrelated individuals (control group), all examined under the same conditions. The mean SCE rates/cell were significantly higher in MM families than in the control group, and in melanoma patients than in their close relatives. The mean SCE levels of FMM and SMM patients, (8.4 +/- 0.8 and 8.0 +/- 0.3, respectively) were similar, and so were the distributions of individuals in classes of increasing SCE values (with a modal value at 7-8 SCEs/cell). The mean SCE levels of close relatives of FMM and SMM patients were also similar (5.4 +/- 0.2 and 5.4 +/- 0.1, respectively, with a modal value at 4-5 SCEs/cell), and slightly higher than in the control group (4.7 +/- 0.2 SCEs/cell). More than 7 SCEs/cell were observed in the majority (41 of 59) of FMM or SMM patients, in a smaller fraction (25 of 227) unaffected relatives, and in none of 20 unrelated unaffected individuals. These observations favor the hypothesis that higher SCE levels may be an expression of constitutional lesions predisposing to this neoplastic disease.

In order to study the immunogenetics of myasthenia gravis (MG), we analysed the TCR and HLA-class II genes from Italian and Californian myasthenic patients. We investigated polymorphisms of the TCR using the full length cDNA probes pGA5 and the pT10 for the alpha and beta chains, respectively. The 6.3 kb and 2.0 kb polymorphic markers, revealed by the PssI enzyme and the alpha chain probe, were shown to be significantly associated with MG. Italian MG patients were HLA typed, and allele frequencies showed a significant association of DR3 and DQw2 with MG. The relative risk calculated for DR3 was 7.4. T-cell proliferative responses to peptides of the AchR alpha chain were also studied and no associations with TCR RFLP analysis or HLA-class II typing were observed.

We conducted a randomized trial comparing expectant management versus immunotherapy with paternal leukocytes to improve obstetric outcome in women with unexplained recurrent abortion. Eligible for the study were women with unexplained recurrent abortion (three or more miscarriages and no live birth), negative findings of immunological screening and no inhibition of the mixed lymphocyte culture. These women were seen for the first time between October 1988 and March 1991 in a network of obstetric departments in Northern Italy. Subjects positive for HLA DR3 or with a partner positive for hepatitis virus B antigen were not eligible. A total of 44 women entered the study. Patients were randomly allocated to immunotherapy (22 women) or expectant management (22 women). Women allocated to immunotherapy were given 200 x 10(6) purified paternal lymphocytes before pregnancy. Median follow-up was 24 months (range 10-39) in the immunotherapy group and 25 months (range 11-38) in the expectant management group. Out of the 22 women randomized to immunotherapy, 16 became pregnant and the corresponding value was 14 in the expectant management group. Spontaneous abortion occurred in six out of the 16 pregnancies observed in the treated women. Among the 14 pregnancies observed in the expectant management group, two aborted and one late fetal death occurred. The cumulative proportions of women who became pregnant over 4 years were 37 and 45% in the immunotherapy and expectant management groups respectively; this difference was not significant. No adverse effect was observed in treated women.

We investigated the utility of serum S100 determined by means of immunoradiometric assay in a cohort of 438 patients affected by cutaneous melanoma (126 untreated and 312 previously treated). Using 0.2 microg/l cut-off value, determined in 134 healthy blood donors, the sensitivity was 4.2% in stage I patients (4/94), 5.3% in stage II patients (1/19), and 38.5% in stage III patients (5/13). Even though the sensitivity increased progressively from stage I to stage II and III, these differences were not statistically significant. The prognostic significance of S100 evaluation at diagnosis was investigated in terms of survival but no statistical correlation between S100 basal levels and survival was found. In the 312 previously treated patients serum S100 levels were correlated to disease extent, high levels of the marker were observed in 42.8% (9/21) of patients with local recurrence, in 32% (16/50) of patients with lymph node and/or in-transit metastases, in 77.3% (17/22) of patients with distant metastases, and in patients with NED, the specificity of the marker was 96.8% (212/219). The difference between these groups were statistically significant. In conclusion, S100 protein was abnormally high in patients with metastatic malignant melanoma. Serial S100 measurements in a follow-up study are necessary to test the importance of the protein in the management of patients with metastatic malignant melanoma.

We analysed the effect of HLA loci on the secondary sex ratio, and investigated whether allele sharing between parents and between mother and child, or child homozygosity, affected the viability of male embryos, which are generally less resistant to unfavourable conditions during pregnancy. The sharing conditions at the B and DR loci showed significantly differing effects: HLA-B seemed to favour female births, while, in pregnancies subsequent to the first, HLA-DR seemed to favour male births. Both HLA-B and DR loci seemed to work through immunological mechanisms.

We have evaluated the frequency of HLA class I and II antigens in 205 Italian patients with hepatocellular carcinoma (HCC) and 749 blood donors (controls). Moreover, we have looked for correlations between HLA antigen frequencies and HBV and/or HCV infections in HCC patients. We found great differences in HLA antigen frequencies considering only two groups: HCC patients and controls. The polymorphism is smaller when we consider the different groups of HCC patients in regard to the previous viral infections (HBV and/or HCV). The most interesting finding is the higher frequency of Cw7, B8 and DR3 in almost all groups of HCC patients. It is well known, that the HLA A1, Cw7, B8, DR3 antigen haplotype is associated with a rapid decline of CD4 cells, and HLA B8, DR3 positive subjects may display some changes in immune parameters and are prone to develop several immunological diseases. Thus HCC might be the result of a lower sensitivity (genetically given) to mitogenic stimuli of HBV and HCV.

The survival of stage I melanoma patients was evaluated and compared with the detectable expression of HLA antigens. Of 904 patients who were surgically treated, 219 were HLA typed on peripheral blood lymphocytes. Four consecutive HLA typings were considered necessary. Median follow-up was 8 years. Two main groups of patients were considered: (a) patients with consistent detectable expression of antigens; and (b) patients with inconsistent detectable expression of antigens. Patients with consistent HLA antigens detection had an 8-year survival rate of 87.7% compared with 49.2% of patients with an inconsistent rate (P 10−7). Multivariate analysis of survival of the 182 HLA-typed patients who survived at least 24 months from surgery showed that two of the criteria had an independent impact on survival: tumour thickness (P 0.02) and HLA typing (P 2 × 10−5). Inconsistent detection of HLA antigens on peripheral blood lymphocytes during the first 24 months after surgery is an indicator of poor prognosis in stage I melanoma patients.

From May 1994 to February 1995 five HLA-A2 patients affected with melanoma metastases were treated with active immunization with IL4 transfected, irradiated, HLA-A2 + allogeneic melanoma cells. They were injected with 50 × 10 6 cells at day 1, 13, 26, 55. Two patients received 4 vaccinations, 1 patient 3 vaccinations and 2 patients (actually not evaluables) were injected 2 times. According to the WHO toxicity grading 2 patients presented grade 2 local toxicity and 3 patients had grade 1. No systemic toxicity was recorded. No evidence of clinical response was obtained. At the present, 4 patients are alive with disease and 1 patient is alive without disease after surgical excision of a cutaneous metastasis.

Sister chromatid exchange (SCE) was analyzed in stimulated lymphocytes and skin fibroblasts in members of three families with cutaneous malignant melanoma (CMM). Two of these families were characterized by familial CMM; the other family had one patient affected by CMM and two others with other cutaneous melanocytic lesions. All the patients had undergone surgery but no chemotherapy. Higher and differing SCE rates were found in lymphocytes and in fibroblasts of all patients. A wide range of SCE distribution was found in patients with high SCE rate. A few healthy close relatives also showed relatively high SCE rates and wide range distributions. These subjects may be regarded as a subset of family members at high risk for developing cancer. The variability of SCE rates and distribution may reflect genetic heterogeneity of CMM.

Two groups of malignant melanoma (MM) patients were considered: the group of 140 patients previously published and a new one of 58, both typed for HLA-A, -B and -C antigens at the Istituto Nazionale Tumori of Milan. The control group consisted of healthy blood donors not related to the patients. Only the HLA-Bw35 antigen frequency was significantly decreased in both groups of patients. To investigate the HLA-A and -B blanks, 62 MM patients and 90 healthy controls, who showed non-homozygotic blanks when they were firstly HLA typed, volunteered to repeat the HLA typging three or more times in a 2-year period. A significant increase in both HLA-A and -B blanks in the patients as compared to controls was noticed (p = 3 × 10−4 and 3 × 10−5, respectively). In the future, attempts should be made to correlate the HLA antigen and blank frequencies with the evolution of the disease and, also, to verify the hypothesis that the HLA-Bw35 antigen may be an associated resistance factor against the tumor.

Sister chromatid exchange (SCE) and the proliferative pattern of phytohemagglutinin-stimulated lymphocytes were examined in 36 nonfamilial cutaneous malignant melanoma (CMM) patients. One close relative of each of 27 CMM patients was also examined. All the patients had undergone surgical treatment for the neoplasm, but had received no chemotherapy or radiotherapy. The SCE rates were found to be higher and more variable in a significant fraction of CMM patients, and in relatively fewer unaffected relatives, which is in contrast to findings in unrelated subjects taken as controls. Also, variable and higher proportions of cells in metaphase of the first cell cycle (Ml), after 72-hr culture in the presence of bromodeoxyuridine, were more often found among the CMM patients than in the controls; however, no effect of clinical progression of the neoplastic disease on SCE rates or on the lymphoproliferative pattern was observed. The present study indicates heterogeneity among subjects who develop CMM and suggests that the peculiarities of SCE rates and of the lymphoproliferative patterns observed in some of the CMM patients and in a few of their close relatives may be connected with the mechanism of onset of the neoplasm.

Ninety insulin-dependent diabetic children were HLA-typed in order to elucidate the role played by HLA complex-linked genes in the pathogenesis of diabetes melHtus of childhood. HLA-Aw30 and HLA-Bw35 were significantly increased and decreased, respectively, in the diabetic group as compared with controls. In relation to age at onset of diabetes, HLA-B8 was significantly increased in the 0-5-year group. By dividing the patients according to the season at onset of the disease, only HLA-Aw30 in the October-January group reached the level of significance (P=0.05).

One hundred and forty melanoma patients, divided in 2 groups, i.e., patients with clinical evidence of melanoma and patients with no clinical evidence of melanoma, were typed for HLA-A, -B and -C antigens and compared with 340 to 905 (according to each HLA antigen examined) healthy adult blood donors. Overall, a highly significant increase in HLA-B40 antigen (p = 5.6 × 10−7) and a decrease in HLA-BW35 (p = 1.6 × 10−4) was observed. No relevant difference was found between the 2 groups. Moreover, an unexpected excess of HLA blanks was observed at both A and B loci in the first group (p = 1.3 × 10−2 and p = 3.3 × 10−6, respectively) and only at the B locus in the second (p = 1.2 × 10−2), when the patients were compared to 288 healthy individuals HLA typed at the same time and with the same HLA antisera as the patients. The increase in HLA blanks in melanoma patients deserves further investigation to ascertain whether it may be due to the tumor not yet surgically removed or may be referred to technical pitfalls.

Six families with familial malignant melanoma (FMM) were HLA-A, -B, and -C typed to ascertain whether or not FMM would segregate with the HLA complex. The HLA-B12 antigen was present in five of the six families. In three families the HLA-FMM linkage could be analyzed: linkage was possible in two but not in the third. These findings suggested that two types of FMM may exist: a more frequent type (five cases) that apparently segregates with the HLA complex and another (one case) that does not segregate with the HLA complex. Moreover, a factor included in the HLA region or another factor linked to it may have played an important role, in five of the six families, in FMM pathogenesis, whereas in the sixth family its role may have been performed by some other factor. These findings are consistent with the hypothesis of two complementary factors (one of which was included in the HLA complex) for the promotion of FMM with dominant inheritance and high penetrance.

The relationship between sister chromatid exchange (SCE) and DNA damage or carcinogenesis has been the subject of numerous investigations (1–6). Altered frequencies of SCEs have been reported in cancer patients (7–13), but their meaning with regard to cancer development has not yet been elucidated.

There is some evidence that genes at loci on the lower end of chromosome 14, encoding for the immunoglobulin heavy chains allotypes (Gm), may influence susceptibility to human tumors. We examined the Gm and Km (IgK light chain) allotype distribution in a sample of 41 patients with familial malignant melanoma and in 79 healthy relatives. An increased frequency of the haplotype carrying the Gm (2) allotype, namely Gm (1, 2, 17;..; 21), seemed to be peculiar to patients, since it was almost twice as frequent in them than in the healthy population and four times as frequent with respect to the healthy relatives. Our findings are in keeping with previous suggestions that in Caucasian melanoma patients genes of the immunoglobulin heavy chain constant region, or Gm-linked genes, may enhance susceptibility to malignant melanoma.

One hundred and twenty-four subjects belonging to 25 families, 51 with familial malignant melanoma (FMM), and 186 subjects belonging to 41 families, 41 with sporadic malignant melanoma, were typed for the HLA A, B, C and DR loci of the HLA system. There was the same statistically significant difference in the frequency of the haplotype A9, B35, Cw4 between each group of patients and the respective healthy relatives (p = 0.01, p = 0.01 and p = 4 × 10−3, respectively). Moreover, the higher frequency of the haplotype A9, B35, Cw4 in the healthy members of the FMM families (42.46 %) compared with the healthy members of the SMM families (23.44 %) indicates that in the latter group other individuals are at risk for the disease. Furthermore, the different frequency of haplotypes B5, DR5 and B5, Cw1 suggests that differences exist between the two groups of healthy relatives. These observations confirm that the HLA region is involved in the etiology of malignant melanoma.