Heparan sulphate epitope-expression is associated with the inflammatory response in metastatic malignant melanoma.

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope-specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3(+) cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3(+) cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3(+) cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3(+) cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3(+) cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.

On the effect of biochemotherapy in metastatic malignant melanoma: an immunopathological evaluation.

Although immunotherapy and biochemotherapy have shown promise, producing a subset of durable responses, for the majority of patients with metastatic melanoma the prognosis is still poor. Therefore there is a great need for predictive tests to identify patients with a high probability of responding. Furthermore, there is also a need for a better understanding of the mechanisms of action during treatment in order to be able to monitor the relevant antitumour reactivity during treatment and to optimize the efficacy of future immunotherapy and biochemotherapy. In the present study histopathological regression criteria were used to study the efficacy of biochemotherapy. Thirty-two patients with metastatic malignant melanoma (18 with regional disease and 14 with systemic disease) were treated with biochemotherapy (cisplatin 30 mg/m2 intravenously on days 1-3, dacarbazine 250 mg/m2 intravenously on days 1-3 and interferon-alpha2b 10 million IU subcutaneously 3 days a week, every 28 days). Pre-treatment fine needle aspirates were obtained from metastases to analyse the number of tumour-infiltrating CD4+ lymphocytes. Therapeutic efficacy was evaluated in metastases resected after treatment using histopathological criteria of tumour regression. Comparisons were also made with metastases from 17 untreated patients, all with regional disease. Regressive changes of 25% or more (of the section area) were found in two of the 17 untreated patients with regional disease compared with 13 of the 18 patients with regional disease and 10 of the 14 patients with systemic disease after biochemotherapy. Fifty per cent of the patients with regional disease showed a high degree of regressive changes (75-100% of the section area) after biochemotherapy. These results demonstrate the occurrence of an antitumour reactivity in the majority of patients. Patients with extensive regressive changes in 75-100% of the analysed biopsies were also found to have a longer overall survival (P = 0.019). In patients with regional disease there was a close correlation between a larger number of CD4+ lymphocytes pre-treatment and a higher degree of regressive changes post-treatment (P < 0.05). Thus, immunohistochemical analysis of tumour biopsies shortly after treatment seems to be a good surrogate endpoint. This technique also allows detailed analysis of antitumour reactivity and escape mechanisms.

Bcl-2 expression in metastatic malignant melanoma. Importance for the therapeutic efficacy of biochemotherapy.

For the majority of patients with metastatic malignant melanoma the prognosis is poor. Immunotherapy and biochemotherapy have shown promise with a subset of durable responses, but there is still a great need for a better understanding of the mechanisms of action during treatment to optimize future treatment schedules. In the present study Bcl-2 expression was studied in biopsies from ten patients with metastatic malignant melanoma (five with regional disease and five with systemic disease) treated with biochemotherapy (cisplatinum 30 mg/m2 days 1-3, DTIC 250 mg/m2 days 1-3 i.v. and Interferon-alpha2b 10 MIU s.c. 3 days a week, on a 28-day cycle). The expression of Bcl-2 by the tumour cells was separately recorded in areas of histopathological regressive changes and in areas of unaffected tumour growth. Comparisons were made with biopsies from 14 untreated patients. In 10 of 10 treated patients a high expression of Bcl-2 by the tumour cells was found in areas of unaffected tumour growth. In contrast, only in 5 of 13 untreated patients was a high expression of Bcl-2 by the tumour cells found in these areas ( P=0.008). A significant difference was also found in the expression of Bcl-2 by the tumour cells between areas of unaffected tumour growth and areas of histopathological regressive changes ( P=0.03). The significantly higher expression of Bcl-2 by the tumour cells in areas of unaffected tumour growth in treated patients compared to untreated patients indicates that clones with a high expression of Bcl-2 may be present after therapy, preventing apoptosis and eventually in many patients resulting in progressive disease. Supporting this concept, a difference was also found between the expression of Bcl-2 in areas of unaffected tumour growth, i.e. in areas of treatment failure, and the expression in areas of histopathological regressive changes. Thus immunohistochemical analysis of tumour biopsies shortly after therapy seems to be a good surrogate endpoint that allows a detailed analysis of Bcl-2 expression. The high expression of Bcl-2 shown in unaffected tumour areas after therapy suggests the need for additional treatment, e.g. Bcl-2 antisense therapy.

Biochemotherapy of metastatic malignant melanoma. On down-regulation of CD28.

Immunotherapy and combination treatments such as biochemotherapy have shown promise, with higher response rates and a subset of durable responses; however, as the majority of responses are still of short duration, they do not provide any survival benefit. There is therefore a great need to better understand the mechanisms whereby tumours escape immune surveillance. The present study examines the expression of CD28 in patients with untreated and treated melanoma metastases. Twenty-eight patients with metastatic malignant melanoma were treated by biochemotherapy (cisplatinum 30 mg/m(2) days 1-3, DTIC 250 mg/m(2) days 1-3 i.v., and IFN-alpha2b 10 million IU s.c. three days a week for 28 days treatment cycle). Tumours were resected post-biochemotherapy and analysed for the expression of CD28 in CD4(+) and CD8(+) lymphocytes in areas where histopathological regressive changes had occurred, and close to tumour cells in areas of unaffected tumour growth using a double-staining technique. A high percentage of the lymphocytes in areas with regressive changes were found to be CD4(+)CD28(-). In contrast, the vast majority of CD4(+) lymphocytes migrating close to the tumour cells were found to be CD28(+) (P<0.001). A similar difference in the expression of CD28 was also found for the CD8(+) subset (P=0.004). A difference in down-regulation of the expression of CD28 was found between CD4(+) and CD8(+) lymphocytes both in the areas of regressive changes and in the unaffected tumour areas. The present study demonstrates that extensive down-regulation of the co-stimulatory factor CD28 is found in metastases following biochemotherapy. These results indicate that parameters of importance for the immune function have already undergone modification after one or two treatment cycles and that this down-regulation occurs in particular in areas with regressive tumour changes.