Induction of IgG antibodies directed to a M(r) 31,000 melanoma antigen in patients immunized with vaccinia virus melanoma oncolysates.

Pre- and postimmunization sera from eight tumor-free melanoma patients undergoing vaccinia melanoma oncolysate (VMO) therapy were used to investigate the humoral response to antigens from infected and uninfected melanoma cells and from vaccinia virus. Immunodetection on Western blots showed that all patients, in addition to reacting to several other proteins, developed IgG antibodies to a M(r) 31,000 protein antigen within 1 month of immunization. This M(r) 31,000 antigen is expressed both on VMO and on melanoma metastases in situ, disappears in primary cultures of these metastases, and is absent in extracts from vaccinia virus, from human melanoma cell lines, and from normal melanocytes, suggesting that this M(r) 31,000 protein is reexpressed following vaccinia virus infection of human melanoma cells. Periodate treatment of the blotted antigens abolished reactivity of patients' postimmunization sera with the M(r) 31,000 antigen, thus showing that this antigen is a glycoprotein and that the relevant epitope is likely to reside on its carbohydrate moiety. These anti-M(r) 31,000 IgG antibodies were absent in the sera of VMO-treated patients before immunization, absent in the serum of a normal donor hyperimmunized with vaccinia virus, and absent in normal human sera. In addition, these anti-M(r) 31,000 antibodies appeared 1 week after the first VMO injection, remained stable during the treatment, and decreased when the treatment was stopped. Such antibodies can also be demonstrated in sera of melanoma patients bearing metastases but disappeared following resection of their metastases. Thus, in melanoma patients, immunization with VMO induces an antibody response directed against a M(r) 31,000 glycoprotein likely to represent a new melanoma antigen. Further identification of this antigen could be of utmost interest for the further development of melanoma vaccines.

[Response in patients with melanoma to immunization using melanoma oncolysates of vaccine virus]. / Réponses de malades atteints de mélanome à l'immunisation par oncolysats de mélanomes au virus de la vaccine.

Thirty-two patients with high risk melanoma (either primary melanoma of the limbs or trunk, or recurrent melanoma) and clinically disease-free following appropriate surgical treatment were immunized with a vaccinia virus oncolysate made from a pool of 4 human melanoma cell lines. Injections were given id weekly for 3 months, and then bi-monthly for a further 21 months or until relapse. Treated patients have been under study for 11-72 months, and 15 of them for more than 36 months. Twelve patients received a full 24-month treatment: 3 relapsed and 10 are alive (9 of them disease-free) with a survival of 34-72 months. One patient is still under treatment. Nineteen patients relapsed during treatment: among the 13 patients that relapsed early during the course of treatment, 9 patients died after a survival of 5-30 months and 4 are alive with a survival of 30-59 months; among the 6 patients that later relapsed, 2 patients died after a survival of 21 and 29 months and 4 are alive with a survival of 16-69 months. An analysis of the patients' disease-free survival and overall survival was made using the actuarial method, and limited to 5 years: the disease-free survival curve shows a 35% plateau reached after 40 months, and the survival curve shows a 60% plateau reached after 30 months. The patients' responses to the immunization antigens expressed by the oncolysate were studied. Lymphocytes from immunized patients do respond in vitro to the stimulation by oncolysate in the presence of low amounts of IL-2, and this response is greater than that of normal individuals. IgG antibody production to gangliosides with N-glycolyl neuraminic acid is of prognostic significance, the increase in IgG anti-ganglioside antibody in patients after 3 and 6 months of treatment being linked to the absence of relapse in these patients. Finally, preliminary results show, in several patients under treatment, the appearance of antibodies directed against a 31 kD protein of the oncolysate not detectable in the vaccinia virus or in melanoma cell lysates. Such results are in accordance with previously reported ones from similar studies conducted by other investigators and tend to indicate the efficacy of vaccinia virus oncolysate immunization in the treatment of high risk melanoma.

Positive relationship of clinical and serologic responses to vaccinia melanoma oncolysate.

In this phase Ia/Ib trial, vaccinia melanoma oncolysate (VMO) is a virus-augmented melanoma cell membrane vaccine that has been shown to be safe and to stimulate the production of antimelanoma antibodies in high-risk melanoma patients treated in a surgical adjuvant setting. One patient with stage I and 38 patients with stage II melanoma were entered in the study between December 1984 and October 1985, with a mean follow-up of approximately 17 months. Each patient received a smallpox booster injection followed one week later by the first of 13 weekly intradermal injections of 2.0 mg of VMO. At the end of 13 weeks, injections were given every other week for 12 months or until recurrence. Clinical results show that 25 of the 39 patients had no evidence of disease as of December 1986. Moreover and more importantly, statistical comparison of patients in this study with 39 matched controls shows a significant increase in disease-free survival for the patients treated with VMO. Serum obtained prior to treatment and at three-month intervals during treatment was tested in a Staphylococcus protein A rosette assay for reactivity with melanoma cell lines. All pretreatment samples (39/39) were negative, and 64% became positive by 12 months after appropriate dosage escalations. Moreover, enzyme-linked immunosorbent assay showed a positive correlation between anti-melanoma IgG antibody titer and disease-free survival.

Serological evaluation of melanoma patients in a phase I/II trial of vaccinia melanoma oncolysate (VMO) immunotherapy.

Vaccinia melanoma oncolysates (VMO) were tested in a Southeastern Cancer Study Group (SECSG)-sponsored phase I/II multiinstitutional trial. Forty-eight patients with stage I or II disease were placed on study at six different dose levels of VMO and two different dose schedules, immediate or delayed. Patients' sera, obtained before treatment and every 3 months following initiation of treatment, were tested for antimelanoma antibodies using a Staphylococcus protein A (SpA) assay. Pretreatment sera were negative in 46 of 47 patients, and only two of 19 patients on delayed treatment developed reactivity by 6 months. However, 13 of 23 on immediate treatment developed reactivity, including eight of eight at the higher doses (1.5 and 2.0 mg). Neither anti-HLA antibody tested by a standard microcytotoxicity assay nor circulating immune complexes measured by both Clq and conglutinin binding assays were produced as a result of the immunization. The demonstration of immunogenicity of VMO at the 2 mg dose and immediate schedule supported the rationale for the use of this dose and schedule for the ongoing second phase Ia/Ib trial and for the future phase III randomized prospective study.

A Southeastern Cancer Study Group phase I/II trial with vaccinia melanoma oncolysates.

Vaccinia melanoma oncolysates (VMO) were tested in a Southeastern Cancer Study Group (SECSG) Phase I/II trial. Forty-eight patients with high-risk Stage I or pathologic Stage II disease were placed on study at six different dose levels and two different treatment regimens. Patients were monitored for toxicity to the VMO after each injection. Patients' sera were tested for anti-human melanoma reactivity with the Staphylococcus Protein A (SpA) assay. Toxicity was minimal at all doses tested. In only 2 of 19 patients on delayed treatment did reactivity develop in the SpA assay by 6 months. However, 13 of 23 patients on immediate treatment showed reactivity, including 8 of 8 at the two highest doses. Since the VMO appears to be safe at all of the doses tested, and because of the immunogenicity of the VMO at the higher doses as demonstrated by the SpA assay, the 2-mg dose level, for immediate treatment, was chosen for use in future trials.

A preliminary trial of vaccinia oncolysates in the treatment of recurrent melanoma with serologic responses to the treatment.

A preliminary trial was designed as a toxicity/feasibility study using a fixed dose of vaccinia melanoma oncolysates (VMO) to treat recurrent stage II and stage III (skin, subcutaneous, and nodal metastases only) melanoma. There were no adverse consequences of the therapy, and 4 of the 12 patients treated seemed to have responded to the treatment by the criteria of the study. Sera from the six patients with the longest survival showed immunoreactivity to human melanoma lines in a Staphylococcus protein A assay (SpA) after 3 months of therapy. While the specificity of this immunoreactivity remains to be determined, the discovery of posttreatment serologic activity in a SpA assay permits investigation of the degree of VMO immunostimulation at different dose levels of the biologic. This assay may provide the means to quantitate optimal biologic dose for future melanoma oncolysate trials. The Southeastern Cancer Study Group is now conducting a phase I/II trial with these vaccinia melanoma oncolysates using the SpA assay to monitor this trial.

[Structural and numerical chromosome abnormalities in human malignant melanoma cell lines (author's transl)]. / Anomalies chromosomiques numériques et structurales dans 36 lignées de mélanome malin humain.

Characteristics and cytogenetic markers of 36 human malignant cell lines were studied. Structural abnormalities of chromosomes 1, 2, 3, 6, 7, 9, 11, 12 and 22 were the most frequent. Relative polysomy for chromosome 3, 7, 16, 19, 20, 22 and monosomy for chromosome 9 were observed. Each cell line contained structural and/or numerical abnormalities of chromosome 7, but structural abnormalities of this chromosome were only observed in cell lines derived from metastases; while only polysomy of chromosome 7 was observed in cell lines derived from primaries or local recurrences.

[Value of ultrastructural morphology in the preparation of oncolysates of human malignant melanoma]. / Intérêt de la morphologie ultrastructurale dans la préparation des oncolysats de mélanome malin humain.

The culture of human cutaneous malignant melanomas is an important stage in the preparation of oncolysates for therapeutic purposes. The authors recall the definition of oncolysate: lysis of the malignant melanocyte by vaccinia virus liberates masked antigens which when reinjected into the patient accelerate or restimulate the production of antibodies. Culture is used to increase the number of malignant melanocytes, i. e. antigenic material, which is an essential precaution in tumours of small size. Production of oncolysate requires the exclusive use of the malignant melanocyte, i. e. the importance of morphological identification during the "in vitro" phase. The optical criteria defined by Fedoroff are inadequate and a source of error. Ultrastructural studies render this identification more valid, insofar as the normal and pathological markers which are the melanosomes are better known. Ultrastructural studies may be used to differentiate the malignant melanocyte from the ordinary macrophage in media which are black, and light cultures to distinguish fibroblastic growth, with no antigenic value, from the malignant melanocyte without pigment (achromic), recognisable by its premelanosome.