Role of the anatomic site in the association of HLA class I antigen expression level in metastases with clinical response to ipilimumab therapy in patients with melanoma.

BACKGROUND: The clinical response to immune checkpoint inhibitors (ICIs) in only part of the treated patients, in conjunction with the potentially serious side effects associated with this type of therapy, has emphasized the need to identify biomarkers to select patients who may benefit from ICI treatment. The aim of our study was to test human leukocyte antigen (HLA) class I and II expression in melanoma metastases as potential biomarkers of response to ipilimumab and survival in patients with metastatic melanoma, since these molecules play a crucial role in the interactions of malignant cells with host's immune system. MATERIALS AND METHODS: HLA class I and II antigen expression level in pretreatment surgical tissue samples (50 lymph node and 35 cutaneous or subcutaneous metastases) from 30 patients was analyzed by immunohistochemical staining with monoclonal antibodies. Expression levels were correlated to intratumoral density of lymphocytes expressing cluster of differentiation (CD)8, CD45RO, CD4, forkhead box P3 (FOXP3) and/or programmed cell death protein 1 (PD-1), to clinical response to treatment, and to patients' survival. RESULTS: HLA class I antigen expression level in lymph node metastases, but not in cutaneous or subcutaneous metastases was significantly correlated to density of CD8+ and CD45RO+ T cells and of lymphocytes expressing PD-1, as well as to clinical response and to patients' survival. CONCLUSIONS: Our results corroborate the role of HLA class I expression level (alone or in combination with T-cell density values) as a predictive biomarker of response to ipilimumab in patients with melanoma. In addition, our results show that this association is influenced by the anatomic site of the metastasis used to measure the HLA class I antigen expression level.

Correlation with lymphocyte infiltration, but lack of prognostic significance of MECA-79-positive high endothelial venules in primary malignant melanoma.

High endothelial venules (HEVs) are specialized vessels in lymphoid organs, supporting lymphocyte trafficking from the blood. As the presence of these vessels was described recently in tumors, it was proposed that they could facilitate the development of antitumor immune response, resulting in improved prognosis. The aim of our study was to analyze the correlation of the density of HEVs with that of the different immune cell types as well as with the clinicopathologic parameters and the disease outcomes in patients with cutaneous melanoma. Primary melanoma samples of 118 patients were analyzed retrospectively by immunohistochemical labeling and quantitation of vessels stained with the MECA-79 antibody, as well as a panel of eight different immune cell types (CD8 and CD45RO T cells, lymphocytes expressing the CD25, CD134, or CD137 activation markers, FOXP3 regulatory T cells, CD20 B cells, and DC-LAMP mature dendritic cells). Correlations of MECA-79 vessel density with that of the immune cells, as well as with clinicopathologic parameters and disease outcomes were evaluated. We showed that the number of MECA-79 vessels correlates strongly with the peritumoral density of B and T lymphocytes. Moreover, higher HEV numbers were detected in tumors hosting tertiary lymphoid structures as well as in those of axial location compared with the ones in the extremity and in men compared with women, whereas no association was found with patient age, tumor thickness, histologic type or ulceration, or with the survival of melanoma patients. The density of MECA-79 HEVs in primary melanomas shows a correlation with B and T-lymphocyte density and differences according to the presence of tertiary lymphoid structures, tumor site, and the sex of the patient. However, it has no prognostic value.

Tumor-infiltrating immune cells as potential biomarkers predicting response to treatment and survival in patients with metastatic melanoma receiving ipilimumab therapy.

Monoclonal antibodies targeting immune checkpoints are gaining ground in the treatment of melanoma and other cancers, and considerable effort is made to identify biomarkers predicting the efficacy of these therapies. Our retrospective study was performed on surgical tissue samples (52 lymph nodes and 34 cutaneous/subcutaneous metastases) from 30 patients with metastatic melanoma treated with ipilimumab. Using a panel of 11 antibodies against different immune cell types, intratumoral immune cell densities were determined and evaluated in relation to response to ipilimumab treatment and disease outcome. For most markers studied, median immune cell densities were at least two times higher in lymph node metastases compared to skin/subcutaneous ones; therefore, the prognostic and predictive associations of immune cell infiltration were evaluated separately in the two groups of metastases as well as in all samples as a whole. Higher prevalence of several immune cell types was seen in lymph node metastases of the responders compared to non-responders, particularly FOXP3+ cells and CD8+ T lymphocytes. In subcutaneous or cutaneous metastases, on the other hand, significant difference could be observed only in the case of CD16 and CD68. Associations of labeled cell densities with survival were also found for most cell types studied in nodal metastases, and for CD16+ and CD68+ cells in skin/s.c. metastatic cases. Our results corroborate the previous findings suggesting an association between an immunologically active tumor microenvironment and response to ipilimumab treatment, and propose new potential biomarkers for predicting treatment efficacy and disease outcome.

Ectopic lymphoid structures in primary cutaneous melanoma.

Ectopic lymphoid structures have been described in several tumor types including metastatic lesions, but not primary tumors, of patients with melanoma. Here we present evidence of B-cell follicles in primary cutaneous melanomas, being present in 39 of 147 cases (27 %). B-cell clusters were associated with T lymphocytes, most of which belonging to CD45RO(+) memory T cells. A network of CD21(+) follicular dendritic cells was demonstrated in 8 of 22 cases studied (36 %). MECA-79(+) HEV-like venules were observed in the neighborhood of the follicles in the majority of cases, however, their presence was not confined to tumors hosting ectopic lymphoid structures. The appearance of B-cell aggregates did not show association with the outcome of the disease, although a trend for their higher prevalence was observed in thicker tumors. Our results show that neogenesis of lymphoid structures does occur in primary melanomas, albeit with lower frequency compared to that reported in metastases.

Immune cell profile of sentinel lymph nodes in patients with malignant melanoma - FOXP3+ cell density in cases with positive sentinel node status is associated with unfavorable clinical outcome.

BACKGROUND: Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions. METHODS: In this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients. Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome. RESULTS: Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes. Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied. In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients. The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease. CONCLUSIONS: Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells. The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance. These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients. On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients.

Sex-dependent liver colonization of human melanoma in SCID mice--role of host defense mechanisms.

The possibility that endocrine factors may influence the clinical course of malignant melanoma is suggested by the superior survival data of women. In preclinical models we observed a higher rate of colony formation by human melanoma cells in male compared to female SCID mice, but only in the case of the liver and not in other organs. The gender difference could be seen at an early phase of colony formation. On the other hand, in our human melanoma cell lines we failed to detect steroid receptor protein expression, and treatment with sex hormones did not considerably influence their in vitro behavior. Investigating the possible contribution of host cells to the observed gender difference, we performed in vivo blocking experiments applying pretreatment of the animals with Kupffer cell inhibitor gadolinium chloride and the NK cell inhibitor anti-asialo GM1 antibody. While Kupffer cell blockade enhanced melanoma liver colonization equally in the two sexes, a more prominent increase was observed in female than in male mice in the case of NK cell inhibition. Further supporting the importance of NK cells in the lower liver colonization efficiency of melanoma cells in females, gender difference in colony formation was lost in NSG mice lacking NK activity. Although in humans no organ selectivity of gender difference in melanoma progression has been observed according to data in the literature, our results possibly indicate a contribution of natural host defense mechanisms to gender difference in survival of patients with melanoma or other tumor types as well.

Prognostic impact of B-cell density in cutaneous melanoma.

Studies on the prognostic importance of tumor-infiltrating lymphocytes have mainly focused on T cells, while little is known about the role of tumor-infiltrating B lymphocytes. We investigated the prevalence of CD20(+) B cells by immunohistochemistry in primary melanoma samples of 106 patients and analyzed in relation to clinicopathological parameters and patients' survival. The majority of samples contained a significant amount of B lymphocytes, predominantly dispersed in the stroma surrounding tumor deposits (mean peritumoral and intratumoral densities: 178.7 ± 156.1 vs. 4.9 ± 6.9 cells/mm², respectively). B cells organized in follicle-like aggregates were also observed in 26% of the samples. B-cell density correlated with that of activated (CD25(+) or OX40(+)) T lymphocytes. Infiltration by CD20(+) lymphocytes did not correlate with tumor thickness, while the presence of B-cell aggregates was observed more frequently in thick melanomas. On the other hand, B-cell infiltration was more pronounced in nonmetastatic or lymph node metastatic tumors, compared to visceral metastatic ones. Accordingly, high number of these cells provided significant survival advantage (P = 0.0391 and P = 0.0136 for intra- and peritumoral infiltration, respectively). Furthermore, combination of peritumoral B-cell density with the number of activated T lymphocytes identified patient subgroups with different disease outcome, which was most favorable in the case of high density, while very poor in the case of low density of both cell types. Multivariate survival analysis identified tumor thickness and CD20(+)/OX40(+) cell density combination as significant independent prognostic factors. Taken together, our results show correlation between low number of CD20(+) B lymphocytes and melanoma progression, indicating a possible role of tumor-infiltrating B cells in antitumoral immune response. It was also reflected in better outcome of the disease since the density of B lymphocytes alone as well as in combination with that of activated T cells proved of prognostic importance in patients with malignant melanoma.

FOXP3+ cell density in primary tumor has no prognostic impact in patients with cutaneous malignant melanoma.

Regulatory T cells (Tregs) have been implicated as inhibitors of antitumor immune reactions. However, data on the relevance of their prevalence at tumor sites in influencing disease outcome are controversial. The aim of our study was to investigate the role in tumor progression and the prognostic impact of the density of lymphocytes expressing FOXP3, a transcription factor expressed predominantly by CD4(+)CD25(+) Tregs, in primary cutaneous melanoma. We examined the infiltration of FOXP3(+) cells by immunohistochemistry in tumor samples from 97 patients and evaluated in relation to patient and tumor parameters. The degree of infiltration by FOXP3(+) cells did not show correlation with the thickness of melanomas. Moreover, no associations were found with metastasis formation during the 5-year follow-up period, patient survival, or any other clinicopathologic parameters studied. These results suggest that the presence of FOXP3(+) lymphocytes in primary tumors is not of prognostic importance in human cutaneous melanoma.

Density of DC-LAMP(+) mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor.

As the most potent antigen presenting cells, dendritic cells (DCs) play key roles in the immune response against tumors. Their density in the tumor tissue has been associated with prognosis in patients with various cancers. However, few studies have been aimed at the presence and maturation state of DCs in cutaneous melanoma, with regard to their potential clinical correlates. In this study, the density of DCs expressing CD1a and the maturation marker DC-LAMP was determined by immunohistochemistry in primary tumor samples from 82 patients with cutaneous malignant melanoma. Intratumoral and peritumoral cell densities were analyzed in relation to tumor thickness and the subsequent development of metastases, as well as to patients' survival. CD1a(+) DCs were found both infiltrating melanoma cell nests and in the surrounding stroma, while DC-LAMP(+) mature DCs were generally confined to the peritumoral areas, associated with lymphocytic infiltrates. DC density values significantly correlated with the number of activated (CD25(+) or OX40(+)) T lymphocytes (p < 0.001). The degree of infiltration by CD1a(+) and DC-LAMP(+) DCs showed strong inverse correlation with the thickness of melanomas (p < 0.001). High peritumoral density of mature DCs was associated with significantly longer survival (p = 0.0195), while density of CD1a(+) cells had a prognostic impact of borderline significance (p = 0.0610). Moreover, combination of high peritumoral CD1a(+) or DC-LAMP(+) cell density with high number of CD25(+) or OX40(+) lymphocytes identified patient subgroups with more favorable survival compared to other subgroups. A multivariate survival analysis involving DC and activated T-cell densities alone and in combinations, as well as traditional prognostic factors, identified high DC-LAMP(+) cell/high OX40(+) cell density and Breslow index as independent predictors of good prognosis. These results suggest that the presence of CD1a(+) DCs primarily depends on the thickness of melanomas, without direct relationship with the patients' survival. On the other hand, the density of mature DCs, especially in association with that of activated T cells, proved of prognostic importance, suggesting that these parameters could be considered as signs of a functional immune response associated with better outcome of the disease.