We have observed that the differential activation of genes involved in acute inflammatory processes () between regressing and progressing melanoma lesions obtained after immunotherapy are dramatic. Among 30,000 studied genes in regressing metastases we mostly detected upregulation of the genes related to the immune rejection with a great enrichment for transcripts located in Chromosome 6, where antigen presentation and many inflammation related genes are located. We propose that these differences are due to efficient tumor recognition and elimination of regressing metastases by the activated immune system “triggered” by HLA Class I upregulation. However, the systemic immunotherapy cannot induce the regression of all lesions. Because of the major differences in HLA expression, we propose that progressing lesions are not recognized by immune cells due to irreversible alterations in HLA Class I.
The transcriptional pattern expressed by regressing metastases was quite similar, independently of the type of immunotherapy used. This suggests that the mechanisms leading to tumor rejection converge in a unique pathway (). In this context, the expression pattern of regressing lesions was also similar to that observed after imiquimod or IL-2 administration in basal cell carcinoma,20,21
graft versus host disease, autoimmunity or acute infection resulting in clearance of pathogen.23
This has been postulated as the existence of an immunologic constant of rejection.24,25
The molecular mechanisms used to eliminate all of them converge in a final pathway consisting of the expression of antigen presentation, ISGs and IEFs. The expression pattern that we have found is ultimately an interferon Type II signature most probably mediated by a release of interferon-gamma by tumor infiltrating T cells, even when the melanoma patient was treated with interferon alpha2b.
A common pathway of T cell mediated rejection is showed in different pathological entities.
Immunohistochemistry staining of tumors showed the correlation between tumor infiltration and the expression of genomic transcripts implicated in tumor rejection (ISGs and IEFs). Several studies have described the presence of tumor-antigen specific cytotoxic T lymphocytes in the tumor microenvironment.23
Nevertheless, these cells are unable to eliminate tumor cells.23
In this study, immune cells potentially induced or activated by the immunotherapy can recognize and eliminate lesions that previously were low immunogenic and invisible for the immune system. However, this immunomodulation occurred only in a subset of metastases, while another group of lesions continued to grow in spite of systemic activation of immune surveillance mechanisms. Therefore, intratumoral factors are most likely to be responsible for the failure of the immune response within the progressing metastases. Genome transcriptional analysis indicates that antigen presentation is the most critical pathway to determine tumor regression. Previous studies12,13
and microdissected tumor from both patients confirm that specific HLA-A, B and C high expression is associated with immune infiltrate and tumor regression, meanwhile a low or negative HLA Class I transcription correlate with tumor progression (). Interestingly, we did not find differences in markers corresponding to regulatory T cells (Tregs) or myeloid derived suppressor cells (MDSC) when both types of metastases were compared.
Correlation of HLA class I surface expression with tumor response after immunotherapy
Our observations suggest that immunotherapy promotes a modification of tumor microenvironment, leading to a release of immune stimulating factors by immune infiltrating cells. This immune stimulation will lead to upregulation of HLA expression in tumor cells with reversible alterations of HLA Class I expression (“Soft” lesions) and, subsequently, these tumor cells will be recognized and destroyed by the antigen-specific T cells. These T cells, as they recognize tumor cells, produce more proinflammatory factors such as IFN-γ, IL-2, TNF-α and GM-CSF.26
This, in turn, triggers a positive and self perpetuating feedback between tumor and immune cells until tumor rejection occurs. In contrast, if cancer cells bear irreversible defects in HLA Class I genes (“Hard” lesions), antigen presentation remains defective after immunotherapy impairing the amplification of the immune response in situ
and promoting their escape from immune recognition.26
Our prediction is that “soft” and “hard” HLA Class I tumor lesions will coexist during the natural history of tumor development. However after immunotherapy tumor cells with “soft” lesion will upregulate antigen presentation and can be rejected. In contrast, those tumor cells bearing “hard” and irreversible genetic defects will prevail and progress to kill the host.27
This clearing would lead to the selection of a tumor variant with additional alteration of HLA Class I expression.28,29
To the best of our knowledge, this is the first comparison of the transcriptional profile of melanoma metastases with different responses after immunotherapy in the same patient, showing the “molecular image” of tumor rejection. Our results strongly suggest that the genetic makeup of individual tumor cells is a major factor determining immune responsiveness. In particular the capacity to modulate tumor HLA Class I expression.