Harnessing autophagy to overcome mitogen-activated protein kinase kinase inhibitor-induced resistance in metastatic melanoma.

BACKGROUND: Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogen-activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance. OBJECTIVES: To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy. METHODS: Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib-induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib-resistant melanoma cell survival and invasion, respectively. RESULTS: CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF-mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271-expressing subpopulations. Trametinib-induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271-expressing melanoma subpopulations with RNA interference and small-molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9-tetrahydrocannabinol and Vps34 - reduced survival of MEKi-resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi-resistant cells in an in vivo zebrafish xenograft. CONCLUSIONS: These results highlight a novel mechanism of MEKi-induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug-resistant melanoma cells to the cytotoxic effects of MEKi.

Kinesin family member 20A is a novel melanoma-associated antigen.

It has been shown recently that immunotherapy for advanced melanoma is effective. However, in order to improve the efficacy of immunotherapy, the identification of more specific melanoma-associated antigens is urgently needed. Kinesin family member 20A (KIF20A) has been reported to be a promising immunotherapeutic target for pancreatic cancer. To investigate the expression of KIF20A in melanoma, we performed quantitative reverse transcript (RT)-PCR and western blotting analyses of melanoma cell lines. We also investigated primary melanomas and naevus tissues with immunohistochemistry and real-time RT-PCR. KIF20A expression was detected in 59% of melanomas and 12% of naevi by immunohisto-chemistry, and 64% of melanomas and 60% of naevi by real-time RT-PCR. The primary melanomas that were positive for KIF20A showed a significantly greater thickness than those that were negative, and patients with KIF20A+ melanoma tended to develop recurrence earlier. These results suggest that immunotherapy with KIF20A may be a novel treatment option for advanced melanoma.

Premalignant quiescent melanocytic nevi do not express the MHC class I chain-related protein A.

The MHC class I chain-related protein A (MICA) is an inducible molecule almost not expressed by normal cells but strongly up-regulated in tumor cells. MICA-expressing cells are recognized by natural killer (NK) cells, CD8+ abTCR and gdTCR T lymphocytes through the NKG2D receptor. Engagement of NKG2D by MICA triggers IFN-g secretion and cytotoxicity against malignant cells. Although most solid tumors express MICA and this molecule is a target during immune surveillance against tumors, it has been observed that high grade tumors from different histotypes express low amounts of cell surface MICA due to a metalloprotease-induced shedding. Also, melanomas develop after a complex process of neotransformation of normal melanocytes. However, the expression of MICA in premalignant stages (primary human quiescent melanocytic nevi) remains unknown. Here, we assessed expression of MICA by flow cytometry using cell suspensions from 15 primary nevi isolated from 11 patients. When collected material was abundant, cell lysates were prepared and MICA expression was also analyzed by Western blot. We observed that MICA was undetectable in the 15 primary nevi (intradermic, junction, mixed, lentigo and congenital samples) as well as in normal skin, benign lesions (seborrheic keratosis), premalignant lesions (actinic keratosis) and benign basocellular cancer. Conversely, a primary recently diagnosed melanoma showed intense cell surface MICA. We conclude that the onset of MICA expression is a tightly regulated process that occurs after melanocytes trespass the stage of malignant transformation. Thus, analysis of MICA expression in tissue sections of skin samples may constitute a useful marker to differentiate between benign and malignant nevi.

In situ analysis of antigens on malignant and benign cells of the melanocyte lineage. Differential expression of two surface molecules, gp75 and p89.

Monoclonal antibodies (mAb) were selected for differential binding to sections of freshly frozen biopsy material of human malignant melanomas and their precursor lesions, the melanocytic nevi. Both melanomas and normal nevi expressed human Ia-like antigens, transferrin receptor and the transferrin-related molecule p97. In contrast, only 1 nevus of 21 tested expressed both glycoprotein gp75, defined by mAb 15.75, and protein p89, defined by mAb P3.58, whereas 12 of 15 melanomas tested expressed both antigens. mAb P3.58 reacted with one additional melanoma and one nevus. The expression of these two molecules therefore appears to be correlated with the appearance of the malignant phenotype of melanocytes.

[Reactivity of five different antibodies with benign and malignant melanocytic lesions]. / A különbözo antitestek reaktivitása benignus és malignus melanocitás daganatokban.

At the histological examination of an increasing number of melanocytic tumors there is a need to use various immunohistochemical methods. Currently, we are supplied by several antibodies working well on formalin-fixed, paraffin-embedded samples. We have tested five antibodies (S-100, HMB-45, Melan-A, MITF, PNL-2) on 34 benign and 34 malignant melanocytic tumors. We examined the specificity and sensitivity in the junctional and dermal component separately, with special consideration to features disturbing the evaluation (regression, halo-like inflammation, etc.). We have concluded that the histological diagnosis of melanocytic tumors is based on the detailed examination of traditional HE slides and the immunohistochemical methods only confirm or weaken our opinion.

Immunohistological characterisation of tumour infiltrating lymphocytes in melanocytic skin lesions.

BACKGROUND: Although the presence of tumour infiltrating lymphocytes (TIL) is a constant feature in melanomas, their immunophenotypic characterisation is still incomplete. We hypothesise that the transition from normal skin to benign naevi (BN) to melanocytic dysplastic naevi (MDN) to radial growth phase cutaneous malignant melanoma (RGP-CMM) to vertical growth phase cutaneous malignant melanoma (VGP-CMM) is associated with alterations in TIL. This study attempted to test this hypothesis and to characterise TIL in the melanocytic skin lesions. METHODS: In total, 74 lesions (12 BN, 12 MDN, 13 RGP-CMM, 26 VGP-CMM, and 11 metastatic melanomas) were examined using immunoperoxidase staining methods and antibodies targeting leukocyte common antigen (LCA+), T (CD3+) and B (CD20+) lymphocytes, and resting cytotoxic T cells (TIA-1+). RESULTS: Histologically, the transitions from normal skin to BN to MDN to RGP-CMM to VGP-CMM was associated with a gradual increase in the numbers of TIL (total, parenchymal, stromal, perivascular, and epidermal TIL, as well as TIL at the base of the lesions). The numbers of TIL were higher at the stroma than at the parenchyma. Similarly, immunostaining revealed that these transitions were associated with a gradual increase in the staining values (staining intensity, percentage of positive cells, and immunoreactivity score) for LCA+, CD20+, CD3+, and TIA-1+cells. The number of CD3+ cells was higher than that of CD20+ cells. All these differences between the normal skin and the lesional ones reached statistical significance (p<0.01). The majority of CD3+ cells were TIA-1+ T cells with cytotoxic potential. Compared with primary melanomas, there was a decrease in TIL in metastatic melanomas. CONCLUSIONS: The gradual increase in TIL during melanoma tumorigenesis may reflect increased antigenicity of the tumour cells. Although both humoral and cell mediated immunity are involved in melanomagenesis, the latter seems to have the major role. The immune profile of MDN suggests their intermediacy between BN and CMM.

Immunophenotypic markers to differentiate between benign and malignant melanocytic lesions.

BACKGROUND/AIMS: The authors investigated the expression of S100A1, S100A6, S100B, MelanA, and CEA in conjunctival naevi, primary acquired melanosis (PAM), conjunctival melanoma, and uveal melanoma in order to assess their potential usefulness in the pathological differential diagnosis of these entities. METHODS: Paraffin embedded sections of 18 conjunctival naevi, 14 PAM, 16 conjunctival melanomas, and 20 uveal melanomas were immunostained for S100A1, S100A6, S100B, MelanA, and CEA, and expression was scored semiquantitatively. RESULTS: Expression of S100A1 differed significantly between conjunctival naevi and conjunctival melanoma, with percentages of positive cells of 30.6% and 71.4%, respectively. Conjunctival melanomas had high average scores for S100A1 and S100B (71.4%, 62.9%, respectively), while uveal melanomas also had high S100A1 but low S100B scores (88.5%, 18.5%, respectively). MelanA was highly variable; naevi and uveal melanoma had higher average scores than conjunctival melanoma. CEA was hardly detectable in all four groups. CONCLUSION: S100A1 seems to be a possible candidate to differentiate conjunctival naevi from conjunctival melanoma. S100B seems to differentiate between uveal melanoma and conjunctival melanoma. However, the study size was small and therefore the data have to be confirmed by others.

Inhibition of leukocyte migration by tumor-associated antigens in soluble extracts of human malignant melanoma.

Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected in response to tumor-associated antigens of human melanoma. The antigens were 3 M KCl-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61%) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCl extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCl extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-1 disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results indicate that the direct LMI assay may be used to measure cell immune reactivity against melanoma-associated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.

Phenotyping of lesions of melanocyte origin with monoclonal antibodies to melanoma-associated antigens and to HLA antigens.

The antigenic profiles of a large number of surgically removed human benign and malignant lesions of melanocyte origin have been analyzed with the use of monoclonal antibodies (MoAb) against la antigens, against the HLA-A,B,C-beta 2-microglobulin molecular complex, against a cytoplasmic melanoma-associated antigen (MAA), and against membrane-bound MAA. Membrane-bound MAA include a high-molecular-weight MAA (HMW-MAA), a 115K MAA, and a 100K MAA. Appearance of the HMW-MAA and of the cytoplasmic MAA, as well as cytoplasmic distribution or loss of HLA-A,B,C antigens, occurs in benign lesions. Additional appearance of Ia antigens is associated with malignant transformation of melanocytes. The antigenic profile defined by the battery of MoAb used displays differences among benign lesions of different histogenesis, between benign and malignant lesions, and among malignant lesions with different histopathologic properties. These results suggest that phenotyping of surgically removed lesions with anti-MAA and anti-HLA MoAb may contribute to the understanding of the steps involved in tumor progression of melanocytes and may aid in the diagnosis of lesions with unusual histopathologic features.

Ultrastructural evidence for destruction in the halo nevus.

Nine halo nevi in various stages of regression were examined by electron microscopy for fine structural evidence of an immunological mechanism of tumor cell destruction and halo formation. Early regressing lesions (Stage I) showed nevus cells associated with infiltrating lymphocytes, monocytes, and plasma cells, but without nevus cell destruction. In later lesions (Stages II and III), vacuolar cytolysis was commonly observed in nevus cells. In Stage III lesions, portions of nevus cells are found within macrophages. The electron microscopic findings of lymphocyte, monocyte, and plasma cell infiltration of the tumor followed by vacuolar cytolysis support the concept of an immune reaction in regressing halo nevi.

Mouse monoclonal antibody (FKH1) detecting human melanoma-associated antigens.

A mouse monoclonal antibody, FKH1, was produced to detect cytoplasmic melanoma-associated antigen. FKH1 was raised using cultured human melanoma cell line KHm-6 as an immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against cell lines and normal and neoplastic tissues. Positive reactions were seen against 5 human melanoma cell lines. It stained cytoplasm of melanoma cells in a diffuse and granular pattern in indirect immunofluorescence. Immunoelectron microscopy showed diffuse distribution of immunoreactant in the cytoplasm of KHm-1 cells excluding melanosomes and other organelles. Reactivity against frozen and alcohol-fixed, paraffin-embedded melanocytic tumors was also tested with IIF or indirect or avidin biotinylated horseradish peroxidase complex immunoperoxidase techniques. All cases of frozen sections from benign and malignant melanocytic tumors showed positive staining with FKH1. In fixed tissues, however, reactivity was 11 of 14 (79%) in malignant melanoma and 28 of 42 (67%) in other melanocytic tumors. FKH1 did not react against normal melanocytes and nonmelanocytic tumors except APUDoma and 2 glioblastoma cell lines. It failed to stain the B-16 mouse melanoma cell line, neuroblastoma cell line, breast carcinoma cell line, and T-cell lymphoma cell line. Normal human peripheral nerves were nonreactive with FKH1. In immunoelectroblot study, FKH1 bound with proteins having molecular weight of 71,000 and 55,000 extracted from KHm-6 cells. It was suggested that FKH1 is a useful monoclonal antibody in diagnostic study of human malignant melanoma specimens.

Immunoperoxidase localization of lymphocyte subsets in the host response to melanoma and nevi.

Using an avidin:biotin immunoperoxidase system with monoclonal antibodies to lymphocyte subsets, we have investigated the host response to malignant melanoma and melanocytic nevi in frozen sections. Eight primary melanomas, eight metastases, three dysplastic nevi, and two dermal nevi were studied with antibodies T11, T4, T8, and B1. Sections were read in a semiquantitative manner by two observers. Virtually all lymphocytes in these lesions were T-cells (T11 positive). In all primary melanomas, in the majority of metastases, and in all dysplastic nevi, both T4- and T8-positive cells were present. In two of eight metastases, tumor cells stained with T4, and in one case, melanoma cells stained with B1 antibody. The host response to melanoma involves primarily T-cells and includes both the helper:inducer (T4) and suppressor:cytotoxic (T8) subsets.

Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas.

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma cells.

Immunohistochemical analysis of malignant melanomas and nevocellular nevi with monoclonal antibodies to distinct monomorphic determinants of HLA antigens.

Using an indirect immunoperoxidase technique, 20 nevocellular nevi, 5 dysplastic nevi, 14 primary cutaneous melanomas, and 24 metastatic melanomas were tested with a panel of monoclonal antibodies to monomorphic determinants of Class I (HLA-A,B,C) and Class II (la-like) major histocompatibility complex antigens. Class I HLA and beta 2-microglobulins were not detected on the majority of nevus cells but were expressed by 3 of 5 dysplastic nevi, by the majority of tumor cells in 12 of 14 primary cutaneous melanomas, and in 13 of 24 metastases. The different expression of Class I HLA and beta 2-microglobulins in primary and metastatic lesions suggests that loss of these antigens may be associated with progression of malignancy. Class II HLA were not detected in common nevi but were locally present in 1 of 5 dysplastic nevi, 7 of 14 cases of primary cutaneous melanoma, and all 24 cases of metastatic lesions tested. These findings suggest that increase in Class II HLA expression may be associated with progression of malignancy. The staining patterns obtained with monoclonal antibodies to distinct determinants of Class I HLA and Class II HLA were superimposable within each type of antigen. Therefore, the discrepancies in the literature about the expression of histocompatibility antigens by lesions of melanocytic origin are not likely to reflect the different specificity of the antibodies used by the various investigators.

Cell division cycle-associated protein 1 as a new melanoma-associated antigen.

Immune checkpoint inhibitors have increased the median survival of melanoma patients. To improve their effects, antigen-specific therapies utilizing melanoma-associated antigens should be developed. Cell division cycle-associated protein 1 (CDCA1), which has a specific function at the kinetochores for stabilizing microtubule attachment, is overexpressed in various cancers. CDCA1, which is a member of cancer-testis antigens, does not show detectable expression levels in normal tissues. Quantitative reverse transcription polymerase chain reaction and immunoblotting analyses revealed that CDCA1 was expressed in all of the tested melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. An immunohistochemical analysis and a Cox proportional hazards model showed that CDCA1 could be a prognostic marker in malignant melanoma (MM) patients. CDCA1-specific siRNA inhibited the cell proliferation of SKMEL2 and WM115 cells, but did not reduce the migration or invasion activity. These results suggest that CDCA1 may be a new therapeutic target of melanoma.

Eruptive nevi of the palms and soles.

The sudden eruption of atypical and benign melanocytic nevi has been associated with a number of disease states and primary skin conditions. Most case reports and series of patients have linked eruptive nevi with blistering skin disease or immunosuppression. Subsets of patients in the immunosuppressed category have remarkably increased numbers of nevi on the palms and soles. We describe a case of multiple eruptive nevi of the palms and soles in association with immunosuppression, and the potential underlying mechanisms promoting such nevogenesis are explored. Although both the absolute number of nevi and presence of dysplastic nevi have been correlated with an increased relative risk of melanoma, actual risk of melanoma in patients with eruptive nevi is unknown.

PD-L1 expression in melanocytic lesions does not correlate with the BRAF V600E mutation.

PD-L1 expression in melanoma correlates with response to PD-1 pathway-blocking antibodies. Aberrant tumor-cell PD-L1 expression may be oncogene driven and/or induced by IFNγ. Melanomas express PD-L1 in association with tumor-infiltrating lymphocytes (TIL), but the potential contribution of the BRAF V600E mutation (BRAFmut) to induced PD-L1 expression has not been determined. Fifty-two archival melanocytic lesions were assessed for PD-L1 expression, TIL infiltration, and BRAFmut simultaneously. IFNγ-induced PD-L1 expression in cultured melanomas was assessed in parallel according to BRAF status. Melanocyte PD-L1 expression was observed in 40% of specimens, and BRAFmut was observed in 42% of specimens, but no significant concordance was found between these variables. Almost all melanocytes displaying PD-L1 expression were observed to be adjacent to TILs, irrespective of BRAF status. TIL(-) lesions were not more likely to be associated with BRAFmut, when compared with TIL(+) lesions. Baseline expression of PD-L1 by melanoma cell lines was virtually nil, regardless of BRAFmut status, and the intensity of IFN-induced PD-L1 expression in melanoma cell lines likewise did not correlate with BRAF mutational status. PD-L1 expression in melanocytic lesions does not correlate with the BRAFmut. Thus, distinct populations of melanoma patients will likely benefit from BRAF inhibitors versus PD-1 pathway blockade.

Beta 2 microglobulin expression in normal melanocytes, nevocellular nevi, and malignant melanomas.

Expression of beta 2 microglobulin (beta 2M), a light chain of class 1 HLA antigen, was studied in normal melanocytes and in benign and malignant melanocytic tumors by use of immunohistochemical methods. By immunoelectron microscopy, normal melanocytes were shown to express beta 2M on the cell surface. In lentigo maligna melanomas and acral lentiginous melanomas, the mean percentages of beta 2M-positive tumor cells were significantly lower in thick (greater than 1.50 mm) primary lesions and metastases than in thin (less than or equal to 1.50 mm) primary lesions. The evidence suggests that melanocyte-derived melanoma clones with a low grade of malignancy preserve class 1 HLA expression, and that the clones with a high grade of malignancy tend to lose the antigen expression. Nevus cells in common nevi have little or no expression of beta 2M. In halo nevi, however, beta 2M were detected on nevus cells in the lesions associated with inflammatory infiltration. Immunohistochemical analyses of the cellular composition of the inflammatory cells in halo nevi demonstrated the presence of cytotoxic T cells together with helper/inducer T cells, Langerhans cells, and macrophages. It appears that nevus cells of halo nevi are destroyed by cytotoxic T cells and that class 1 HLA antigens expressed on nevus cells play an important role in the target cell recognition and lysis by specific cytotoxic T cells.