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Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-β1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-β1, only a few activate the cytokine via cell type–specific mechanisms. The cellular source of the active TGF-β1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-β1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-β1 activation by GARP-expressing cells. GARP:TGF-β1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-β1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-β1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

Unlimited replicative potential is one of the hallmarks of cancer cells. In melanoma, hTERT(telomerase reverse transcriptase) is frequently overexpressed because of activating mutations in its promoter, suggesting that telomerase is necessary for melanoma development. We observed, however, that a subset of melanoma metastases and derived cell lines had no telomere maintenance mechanism. Early passages of the latter displayed long telomeres that progressively shortened and fused before cell death. We propose that, during melanoma formation, oncogenic mutations occur in precursor melanocytes with long telomeres, providing cells with sufficient replicative potential, thereby bypassing the need to re-activate telomerase. Our data further support the emerging idea that long telomeres promote melanoma formation. These observations are important when considering anticancer therapies targeting telomerase.

Melanoma is not only one of the most immunogenic cancers but also one of the most effective cancers at subverting host immunity. The role of T lymphocytes in tumor immunity has been extensively studied in melanoma, whereas less is known about the importance of B lymphocytes. The effects of plasma cells (PCs), in particular, are still obscure. The aim of this study was to characterize pathological features and clinical outcome of primary cutaneous melanomas associated with PCs. Moreover, we investigated the origins of the melanoma-associated PCs. Finally, we studied the outcome of patients with primary melanomas with PCs. We reviewed 710 melanomas to correlate the presence of PCs with histological prognostic markers. Immunohistochemistry for CD138 and heavy and light chains was performed in primary melanomas (PM) and in loco-regional lymph nodes (LN), both metastatic and not metastatic. In three PM and nine LN with frozen material, VDJ-rearrangement was analyzed by Gene Scan Analysis. Survival analysis was performed on a group of 85 primary melanomas >2 mm in thickness. Forty-one cases (3.7%) showed clusters/sheets of PCs. PC-rich melanomas occurred at an older age and were thicker, more often ulcerated and more mitotically active (P<0.05). PCs were polyclonal and often expressed IgA in addition to IgG. In LN, clusters/sheets of IgA+ PCs were found both in the sinuses and subcapsular areas. Analysis of VDJ-rearrangements showed the IgA to be oligoclonal. Melanomas with clusters/sheets of PCs had a significantly worse survival compared with melanomas without PCs while, interestingly, melanomas with sparse PCs were associated with a better clinical outcome (P=0.002). In conclusion, melanomas with sheets/clusters of PCs are associated with worse prognosis. IgG and IgA are the isotypes predominantly produced by these PCs. IgA oligoclonality suggests an antigen-driven response that facilitates melanoma progression by a hitherto unknown mechanism.

Melanoma is not only one of the most immunogenic cancers but also one of the most effective cancers at subverting host immunity. The role of T lymphocytes in tumor immunity has been extensively studied in melanoma, whereas less is known about the importance of B lymphocytes. The effects of plasma cells (PCs), in particular, are still obscure. The aim of this study was to characterize pathological features and clinical outcome of primary cutaneous melanomas associated with PCs. Moreover, we investigated the origins of the melanoma-associated PCs. Finally, we studied the outcome of patients with primary melanomas with PCs. We reviewed 710 melanomas to correlate the presence of PCs with histological prognostic markers. Immunohistochemistry for CD138 and heavy and light chains was performed in primary melanomas (PM) and in loco-regional lymph nodes (LN), both metastatic and not metastatic. In three PM and nine LN with frozen material, VDJ-rearrangement was analyzed by Gene Scan Analysis. Survival analysis was performed on a group of 85 primary melanomas >2 mm in thickness. Forty-one cases (3.7%) showed clusters/sheets of PCs. PC-rich melanomas occurred at an older age and were thicker, more often ulcerated and more mitotically active (P<0.05). PCs were polyclonal and often expressed IgA in addition to IgG. In LN, clusters/sheets of IgA+ PCs were found both in the sinuses and subcapsular areas. Analysis of VDJ-rearrangements showed the IgA to be oligoclonal. Melanomas with clusters/sheets of PCs had a significantly worse survival compared with melanomas without PCs while, interestingly, melanomas with sparse PCs were associated with a better clinical outcome (P=0.002). In conclusion, melanomas with sheets/clusters of PCs are associated with worse prognosis. IgG and IgA are the isotypes predominantly produced by these PCs. IgA oligoclonality suggests an antigen-driven response that facilitates melanoma progression by a hitherto unknown mechanism.

Genome hypomethylation is a common epigenetic alteration in human tumors, where it often leads to aberrant activation of a group of germline-specific genes, commonly referred to as “cancer-germline” genes. The cellular functions and tumor promoting potential of these genes remain, however, largely uncertain. Here, we report identification of a novel cancer-germline transcript (CT-GABRA3) displaying DNA hypomethylation-dependent activation in various tumors, including melanoma and lung carcinoma. Importantly, CT-GABRA3harbors a microRNA (miR-105), which has recently been identified as a promoter of cancer metastasis by its ability to weaken vascular endothelial barriers following exosomal secretion. CT-GABRA3also carries a microRNA (miR-767) with predicted target sites in TET1and TET3, two members of the ten-eleven-translocation family of tumor suppressor genes, which are involved in the conversion of 5-methylcytosines to 5-hydroxymethylcytosines (5hmC) in DNA. Decreased TET activity is a hallmark of cancer; here, we provide evidence that aberrant activation of miR-767 contributes to this phenomenon. We demonstrate that miR-767 represses TET1/3mRNA and protein expression and regulates genomic 5hmC levels. Additionally, we show that high CT-GABRA3transcription correlates with reduced TET1mRNA levels in vivo in lung tumors. Together, our study identified a cancer-germline gene that produces microRNAs with oncogenic potential. Moreover, our data indicate that DNA hypomethylation in tumors can contribute to reduced 5hmC levels via activation of a TET-targeting microRNA.

Melanoma patients have high frequencies of T cells directed against antigens of their tumor. The frequency of these antitumor T cells in the blood is usually well above that of the anti-vaccine T cells observed after vaccination with tumor antigens. In a patient vaccinated with a MAGE-3 antigen presented by HLA-A1, we measured the frequencies of anti-vaccine and antitumor T cells in several metastases to evaluate their respective potential contribution to tumor rejection. The frequency of anti–MAGE-3.A1 T cells was 1.5 × 10−5 of CD8 T cells in an invaded lymph node, sixfold higher than in the blood. An antitumor cytotoxic T lymphocyte (CTL) recognizing a MAGE-C2 antigen showed a much higher enrichment with a frequency of ∼10%, 1,000 times higher than its blood frequency. Several other antitumor T clonotypes had frequencies >1%. Similar findings were made on a regressing cutaneous metastasis. Thus, antitumor T cells were ∼10,000 times more frequent than anti-vaccine T cells inside metastases, representing the majority of T cells present there. This suggests that the anti-vaccine CTLs are not the effectors that kill the bulk of the tumor cells, but that their interaction with the tumor generates conditions enabling the stimulation of large numbers of antitumor CTLs that proceed to destroy the tumor cells. Naive T cells appear to be stimulated in the course of this process as new antitumor clonotypes arise after vaccination.

The identification of antigens recognised on human tumours by autologous T-lymphocytes has opened the way for vaccination strategies involving defined tumour antigens. These vaccinations are therapeutic, i.e. they involve patients with detectable disease. Tumour regressions have been observed in a minority of melanoma patients in Phase I/II trials. Some of these regressions have been complete and long lasting. Improving the efficacy of therapeutic vaccines will critically depend on their capacity to trigger a robust immune response, on the development of appropriate methods to monitor these antitumour immune responses to vaccination and on a better understanding of the mechanisms used by tumours to escape immune attack. Finally, the initiation of large randomised Phase III trials will determine the impact of these vaccines on melanoma treatment.

Genes of the MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells, which do not carry HLA molecules.Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-A family was tested with reverse transcriptase-polymerase chain reaction in lymphoma samples. Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12, and 1 lymphoepithelioid T-cell lymphoma expressed geneMAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4. In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease. In the positive samples, the Reed-Sternberg cells were strongly stained whereas the surrounding cells were not. These results indicate that Hodgkin disease may be a target for specific immunotherapy involving MAGE-A4 antigens.

Genes of the MAGE-Afamily are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells, which do not carry HLA molecules.Therefore, peptides encoded by MAGE-Agenes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-Afamily was tested with reverse transcriptase-polymerase chain reaction in lymphoma samples. Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12, and 1 lymphoepithelioid T-cell lymphoma expressed geneMAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4.In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease. In the positive samples, the Reed-Sternberg cells were strongly stained whereas the surrounding cells were not. These results indicate that Hodgkin disease may be a target for specific immunotherapy involving MAGE-A4 antigens.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-β1, a pro-fibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-β1, only a few activate the cytokine via cell type–specific mechanisms. The cellular source of the active TGF-β1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-β1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-β1 activation by GARP-expressing cells. GARP:TGF-β1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garpin either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-β1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-β1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

Genes of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.

Genes of the MAGE, BAGE, GAGE,and LAGE-1/NY-ESO-1families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO1) is a physiological immunoregulatory mechanism often hijacked by tumors. Our recent extensive study of IDO1 protein expression in human tissues showed expression in mature dendritic cells and in pulmonary and placental endothelial cells. IDO1 was also expressed in 56% of tumors, either by tumoral, stromal, or endothelial cells. These results and reagent will guide the clinical development of IDO1 inhibitors for cancer therapy.

Metastatic melanoma lesions sometimes host ectopic lymphoid structures exhibiting ongoing adaptive immune responses. Here, we discuss how this finding fits in our current view of antitumor immunity.