The profound but frequently transient clinical responses to BRAF
V600
inhibitor (BRAFi) treatment in melanoma emphasize the need for combinatorial therapies. Multiple clinical trials combining BRAFi ...and immunotherapy are under way to further enhance therapeutic responses. However, to which extent BRAF
V600
inhibition may affect melanoma immunogenicity over time remains largely unknown. To support the development of an optimal treatment protocol, we studied the impact of prolonged BRAFi exposure on the recognition of melanoma cells by T cells in different patient models. We demonstrate that autologous CD8
+
tumor-infiltrating lymphocytes (TILs) efficiently recognized short-term (3, 7 days) BRAFi-treated melanoma cells but were less responsive towards long-term (14, 21 days) exposed tumor cells. Those long-term BRAFi-treated melanoma cells showed a non-proliferative dedifferentiated phenotype and were less sensitive to four out of five CD8
+
T cell clones, present in the preexisting TIL repertoire, of which three recognized shared antigens (Tyrosinase, Melan-A and CSPG4) and one being neoantigen-specific. Only a second neoantigen was steadily recognized independent of treatment duration. Notably, in all cases the impaired T cell activation was due to a time-dependent downregulation of their respective target antigens. Moreover, combinatorial treatment of melanoma cells with BRAFi and an inhibitor of its downstream kinase MEK had similar effects on T cell recognition. In summary, MAP kinase inhibitors (MAPKi) strongly alter the tumor antigen expression profile over time, favoring evolution of melanoma variants cross-resistant to both T cells and MAPKi. Our data suggest that simultaneous treatment with MAPKi and immunotherapy could be most effective for tumor elimination.
BackgroundImmune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable ...melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance.MethodsPatient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I-driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models.ResultsRIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs).ConclusionsOur results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.
Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing ...deep penetrating nevus-like morphology. In addition, they have been identified in 1–2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.
Tumor antigen-specific redirection of cytotoxic T cells (CTLs) or natural killer (NK) cells including chimeric antigen receptor (CAR-) and T cell receptor (TCR-) cell therapy is currently being ...evaluated in different tumor entities including melanoma. Expression of melanoma-specific antigen recognized by the respective CAR or TCR directly or presented by HLA molecules is an indispensable prerequisite for this innovative therapy. In this study, we investigated in 168 FFPE tumor specimens of patients with stage I-IV melanoma the protein expression of HER2, TRP2, ABCB5, gp100, p53, and GD2 by immunohistochemistry (IHC). These results were correlated with clinical parameters. Membrane expression of HER2 and GD2 was also investigated in ten melanoma cell lines by flow cytometry for which corresponding tumors were analyzed by IHC. Our results demonstrated that gp100 was the most frequently overexpressed protein (61%), followed by TRP2 (50%), GD2 (38%), p53 (37%), ABCB5 (17%), and HER2 (3%). TRP2 expression was higher in primary tumors compared to metastases (
= 0.005). Accordingly, TRP2 and ABCB5 expression was significantly associated with lower tumor thickness of the primary (
= 0.013 and
= 0.025). There was no association between protein expression levels and survival in advanced melanoma patients. Flow cytometric analysis revealed abundant surface expression of GD2 and HER2 in all melanoma cell lines. The discordant HER2 expression in situ and in vitro suggests a tissue culture associated induction. In summary, our data support the use of gp100 and GD2 as a potential target for developing engineered TCR- or CAR-cell therapies, respectively, against melanoma.
Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes ...encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.
Background
Plasma‐derived tumour‐specific cell‐free nucleic acids are increasingly utilized as a minimally invasive, real‐time biomarker approach in many solid tumours. Circulating tumour DNA of ...melanoma‐specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow‐up of all genotypes, including wild‐type.
Methods
We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N = 175 melanoma, N = 20 normal skin, N = 6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N = 18 melanoma, N = 8 benign nevi). Circulating cell‐free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR.
Results
KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients’ and healthy donors’ plasma (AUC > 86%, p < .0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re‐analysis of single‐cell transcriptomes revealed a pan‐tumour origin of cfRNA, including endothelial, cancer‐associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression‐free survival (PFS) (KPNA2 HR = .54, p = .0362; DTL HR = .60, p = .0349) and overall survival (KPNA2 HR = .52, p = .0237; BACE2 HR = .55, p = .0419; DTYMK HR = .43, p = .0393). Lastly, we found that cfRNA copies significantly increased during therapy in non‐responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p = .0441) and DTYMK (HR = 1.82, p = .018) cfRNA during therapy was predictive of shorter PFS.
Conclusions
In sum, we identified a new panel of cfRNAs for a pan‐tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy‐monitoring tool independent of the melanoma mutational genotype.
Biomarker candidates were identified through unsupervised data mining of melanoma versus benign nevi or healthy skin transcriptomes.
Droplet digital PCR (ddPCR) assays were developed for circulating cell‐free RNA (cfRNA) quantification.
KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy in melanoma.
High baseline cfRNA levels were associated with significantly shorter progression‐free survival.
cfRNA copies significantly increased during therapy in non‐responders regardless of therapy and mutational subtypes.
Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often ...lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi
, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi
, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.
Acral naevi are benign melanocytic tumors occurring at acral sites. Occasionally they can progress to become malignant tumors (melanomas). The genetics of acral naevi have not been assessed in larger ...studies. In our study, a large cohort of 130 acral naevi was screened for gene mutations known to be important in other naevi and melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with clinicopathological parameters. Frequent mutations in genes activating the MAP kinase pathway were identified, including
= 87 (67%)
,
= 24 (18%)
, and one (1%)
mutations.
mutations were almost exclusively V600E (
= 86, 99%) and primarily found in junctional and compound naevi.
mutations were either Q61K or Q61R and frequently identified in dermal naevi. Recurrent non-V600E
,
,
, and
promoter mutations, present in acral melanoma, were not identified. Our study identifies
and
mutations as the primary pathogenic event in acral naevi, however, distributed differently to those in non-acral naevi. The mutational profile of acral naevi is distinct from acral melanoma, which may be of diagnostic value in distinguishing these entities.
The activating receptor NKG2D, expressed on different innate and adaptive cytotoxic lymphocytes, has been demonstrated to play an important role in anti-tumor immunity. Now evidence is provided that ...tumor suppressors control expression of its ligand ULBP2 supporting the role of this receptor-ligand system as an innate barrier against tumor development.
Treatment of patients with advanced metastatic melanoma has for decades been a story of very limited success. This dramatically changed when therapy with anti-PD-1 checkpoint blocking antibodies was ...approved in the USA and Europe in 2014 and 2015, respectively. The therapy exploits the capacity of CD8
T cells to specifically kill tumor cells. Within the tumor microenvironment, CD8
T cell activity is blocked by suppressive signals received via PD-1, an inhibitory co-receptor and so-called checkpoint of T cell activation. PD-1 binds to its ligand PD-L1 on melanoma cells which dampens the T cell's activity. Antibodies blocking inhibitory PD-1/PD-L1 interaction release T cells from suppression. Treatment of late-stage disease melanoma patients with antibodies targeting the PD-1/PD-L1 axis, termed immune checkpoint blocking therapy (ICBT), yields clinical frequently long-lasting responses in 30-40% of cases. Despite this remarkable breakthrough, still the majority of patients resists ICBT or develops resistance after initial therapy response. Administration of anti-PD-1 antibodies in combination with antibodies targeting CTLA-4, another inhibitory immune checkpoint increased clinical responses rate up to 50% but at costs of higher treatment-related toxicities. Thus, strong efforts are now directed toward the understanding of therapy resistance, the identification of biomarkers predicting therapy response, and the development of alternative PD-1-based combination treatment to improve patient outcomes.
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