Skin cancer in Europe today and challenges for tomorrow.

One in every three cancers diagnosed is a skin cancer. Europe has the global lead in the number of UV-attributable cancer cases with the highest number of melanoma cases worldwide and the second highest number of keratinocyte cancers (KC). Further increases are expected in Europe for the coming decades. Projected increases are highest for KC with increases in incidence around 40% and increases in mortality around 50%, with KC mortality in males approximating melanoma mortality in females. The two main drivers for this skin cancer epidemic are ageing of the population but especially UV exposure. In conclusion, skin cancer represents a major challenge in the cancer field in Europe today and will continue to do so in the coming decades. This calls for a European skin cancer action plan intended to reduce avoidable UV exposure and to prepare the healthcare system to safeguard early diagnosis and treatment of skin cancer.

Skin Cancer Induction by the Antimycotic Drug Voriconazole Is Caused by Impaired DNA Damage Detection Due to Chromatin Compaction.

Phototoxicity and skin cancer are severe adverse effects of the anti-fungal drug voriconazole (VOR). These adverse effects resemble those seen in xeroderma pigmentosum, caused by defective DNA nucleotide excision repair (NER), and we show that VOR decreases NER capacity. We show that VOR treatment does not perturb the expression of NER, or other DNA damage-related genes, but that VOR localizes to heterochromatin, in complexes containing histone acetyltransferase general control of amino-acid synthesis 5-like 2. Impairment of general control of amino-acid synthesis 5-like 2 binding to histone H3 reduced acetylation of H3, restricting damage-dependent chromatin unfolding, thereby reducing NER initiation. Restoration of H3 histone acetylation using histone deacetylase inhibitors, rescued VOR-induced NER repression, thus offering a preventive therapeutic option. These findings underline the importance of DNA damage-dependent chromatin remodeling as an important prerequisite of functional DNA repair.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

European consensus-based interdisciplinary guideline for invasive cutaneous squamous cell carcinoma: Part 2. Treatment-Update 2023.

In order to update recommendations on treatment, supportive care, education, and follow-up of patients with invasive cutaneous squamous cell carcinoma (cSCC), a multidisciplinary panel of experts from the European Association of Dermato-Oncology (EADO), the European Dermatology Forum (EDF), the European Society for Radiotherapy and Oncology (ESTRO), the European Union of Medical Specialists (UEMS), the European Academy of Dermatology and Venereology (EADV), and the European Organisation of Research and Treatment of Cancer (EORTC) was formed. Recommendations were based on an evidence-based literature review, guidelines, and expert consensus. Treatment recommendations are presented for common primary cSCC (low risk, high risk), locally advanced cSCC, regional metastatic cSCC (operable or inoperable), and distant metastatic cSCC. For common primary cSCC, the first-line treatment is surgical excision with postoperative margin assessment or micrographically controlled surgery. Achieving clear surgical margins is the most important treatment consideration for patients with cSCCs amenable to surgery. Regarding adjuvant radiotherapy for patients with high-risk localised cSCC with clear surgical margins, current evidence has not shown significant benefit for those with at least one high-risk factor. Radiotherapy should be considered as the primary treatment for non-surgical candidates/tumours. For cSCC with cytologically or histologically confirmed regional nodal metastasis, lymph node dissection is recommended. For patients with metastatic or locally advanced cSCC who are not candidates for curative surgery or radiotherapy, anti-PD-1 agents are the first-line systemic treatment, with cemiplimab being the first approved systemic agent for advanced cSCC by the Food and Drugs Administration/European Medicines Agency. Second-line systemic treatments for advanced cSCC, include epidermal growth factor receptor inhibitors (cetuximab) combined with chemotherapy or radiotherapy. Multidisciplinary board decisions are mandatory for all patients with advanced cSCC, considering the risks of toxicity, the age and frailty of patients, and co-morbidities, including immunosuppression. Patients should be engaged in informed, shared decision-making on management and be provided with the best supportive care to improve symptom management and quality of life. The frequency of follow-up visits and investigations for subsequent new cSCC depends on underlying risk characteristics.

European consensus-based interdisciplinary guideline for invasive cutaneous squamous cell carcinoma. Part 1: Diagnostics and prevention-Update 2023.

Invasive cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers in white populations, accounting for 20% of all cutaneous malignancies. Overall, cSCC mostly has very good prognosis after treatment, with 5-year cure rates greater than 90%. Despite the overall favourable prognosis and the proportionally rare deaths, cSCC is associated with a high total number of deaths due to its high incidence. A collaboration of multidisciplinary experts from the European Association of Dermato-Oncology (EADO), the European Dermatology Forum (EDF), the European Society for Radiotherapy and Oncology (ESTRO), the European Union of Medical Specialists (UEMS), the European Academy of Dermatology and Venereology (EADV) and the European Organization of Research and Treatment of Cancer (EORTC), was formed to update recommendations on cSCC, based on current literature and expert consensus. Part 1 of the guidelines addresses the updates on classification, epidemiology, diagnosis, risk stratification, staging and prevention in immunocompetent as well as immunosuppressed patients.

Deep learning-based scoring of tumour-infiltrating lymphocytes is prognostic in primary melanoma and predictive to PD-1 checkpoint inhibition in melanoma metastases.

BACKGROUND: Recent advances in digital pathology have enabled accurate and standardised enumeration of tumour-infiltrating lymphocytes (TILs). Here, we aim to evaluate TILs as a percentage electronic TIL score (eTILs) and investigate its prognostic and predictive relevance in cutaneous melanoma. METHODS: We included stage I to IV cutaneous melanoma patients and used hematoxylin-eosin-stained slides for TIL analysis. We assessed eTILs as a continuous and categorical variable using the published cut-off of 16.6% and applied Cox regression models to evaluate associations of eTILs with relapse-free, distant metastasis-free, and overall survival. We compared eTILs of the primaries with matched metastasis. Moreover, we assessed the predictive relevance of eTILs in therapy-naïve metastases according to the first-line therapy. FINDINGS: We analysed 321 primary cutaneous melanomas and 191 metastatic samples. In simple Cox regression, tumour thickness (p < 0.0001), presence of ulceration (p = 0.0001) and eTILs ≤16.6% (p = 0.0012) were found to be significant unfavourable prognostic factors for RFS. In multiple Cox regression, eTILs ≤16.6% (p = 0.0161) remained significant and downgraded the current staging. Lower eTILs in the primary tissue was associated with unfavourable relapse-free (p = 0.0014) and distant metastasis-free survival (p = 0.0056). In multiple Cox regression adjusted for tumour thickness and ulceration, eTILs as continuous remained significant (p = 0.019). When comparing TILs in primary tissue and corresponding metastasis of the same patient, eTILs in metastases was lower than in primary melanomas (p < 0.0001). In therapy-naïve metastases, an eTILs >12.2% was associated with longer progression-free survival (p = 0.037) and melanoma-specific survival (p = 0.0038) in patients treated with anti-PD-1-based immunotherapy. In multiple Cox regression, lactate dehydrogenase (p < 0.0001) and eTILs ≤12.2% (p = 0.0130) were significantly associated with unfavourable melanoma-specific survival. INTERPRETATION: Assessment of TILs is prognostic in primary melanoma samples, and the eTILs complements staging. In therapy-naïve metastases, eTILs ≤12.2% is predictive of unfavourable survival outcomes in patients receiving anti-PD-1-based therapy. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

In vivo imaging of CD8+ T cells in metastatic cancer patients: first clinical experience with simultaneous [89Zr]Zr-Df-IAB22M2C PET/MRI.

Aim/Introduction: Despite the spectacular success of immune checkpoint inhibitor therapy (ICT) in patients with metastatic cancer, only a limited proportion of patients benefit from ICT. CD8+ cytotoxic T cells are important gatekeepers for the therapeutic response to ICT and are able to recognize MHC class I-dependent tumor antigens and destroy tumor cells. The radiolabeled minibody [89Zr]Zr-Df-IAB22M2C has a high affinity for human CD8+ T cells and was successfully tested in a phase I study. Here, we aimed to gain the first clinical PET/MRI experience with the noninvasive assessment of the CD8+ T-cell distribution in cancer patients by in vivo [89Zr]Zr-Df-IAB22M2C with a distinct focus of identifying potential signatures of successful ICT. Material and Methods: We investigated 8 patients with metastasized cancers undergoing ICT. Radiolabeling of Df-IAB22M2C with Zr-89 was performed according to Good Manufacturing Practice. Multiparametric PET/MRI was acquired 24 h after injection of 74.2±17.9 MBq [89Zr]Zr-Df-IAB22M2C. We analyzed [89Zr]Zr-Df-IAB22M2C uptake within the metastases and within primary and secondary lymphatic organs. Results: [89Zr]Zr-Df-IAB22M2C injection was tolerated well without noticeable side effects. The CD8 PET/MRI data acquisitions 24 hours post-administration of [89Zr]Zr-Df-IAB22M2C revealed good image quality with a relatively low background signal due to only low unspecific tissue uptake and marginal blood pool retention. Only two metastatic lesions showed markedly increased tracer uptake in our cohort of patients. Furthermore, we observed high interpatient variability in [89Zr]Zr-Df-IAB22M2C uptake within the primary and secondary lymphoid organs. Four out of five ICT patients exhibited rather high [89Zr]Zr-Df-IAB22M2C uptake in the bone marrow. Two of these four patients as well as two other patients yielded pronounced [89Zr]Zr-Df-IAB22M2C uptake within nonmetastatic lymph nodes. Interestingly, cancer progression in ICT patients was associated with a relatively low [89Zr]Zr-Df-IAB22M2C uptake in the spleen compared to the liver in 4 out of the 6 patients. Lymph nodes with enhanced [89Zr]Zr-Df-IAB22M2C uptake revealed significantly reduced apparent diffusion coefficient (ADC) values in diffusion weighted MRI. Conclusion: Our first clinical experiences revealed the feasibility of [89Zr]Zr-Df-IAB22M2C PET/MRI in assessing potential immune-related changes in metastases and primary and secondary lymphatic organs. According to our results, we hypothesize that alterations in [89Zr]Zr-Df-IAB22M2C uptake in primary and secondary lymphoid organs might be associated with the response to ICT.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

IFN-γ and TNF Induce Senescence and a Distinct Senescence-Associated Secretory Phenotype in Melanoma.

Immune checkpoint blockade (ICB) therapy is a central pillar of melanoma treatment leading to durable response rates. Important mechanisms of action of ICB therapy include disinhibition of CD4+ and CD8+ T cells. Stimulated CD4+ T helper 1 cells secrete the effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF), which induce senescence in tumor cells. Besides being growth-arrested, senescent cells are metabolically active and secrete a large spectrum of factors, which are summarized as senescence-associated secretory phenotype (SASP). This secretome affects the tumor growth. Here, we compared the SASP of cytokine-induced senescent (CIS) cells with the SASP of therapy-induced senescent (TIS) cells. Therefore, we established in vitro models for CIS and TIS in melanoma. The human melanoma cell lines SK-MEL-28 and WM115 were treated with the cytokines IFN-γ and TNF as CIS, the chemotherapeutic agent doxorubicin, and the cell cycle inhibitor palbociclib as TIS. Then, we determined several senescence markers, i.e., growth arrest, p21 expression, and senescence-associated ß-galactosidase (SA-ß-gal) activity. For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, protein arrays, and ELISA. Each treatment initiated a stable growth arrest, enhanced SA-ß-gal activity, and-except palbociclib-increased the expression of p21. mRNA and protein analyses revealed that gene expression and secretion of SASP factors were severalfold stronger in CIS than in TIS. Finally, we showed that treatment with the conditioned media (CM) derived from cytokine- and palbociclib-treated cells induced senescence characteristics in melanoma cells. Thus, we conclude that senescence induction via cytokines may lead to self-sustaining senescence surveillance of melanoma.

A Commotion in the Skin: Developing Melanoma Immunotherapies.

Melanomas are malignant tumors that can partly and very rarely completely regress in response to immune responses. Analyzing the mechanisms underlying this immune-mediated rejection, melanomas became leading in developing general cancer immunotherapy. This resulted in the discovery of tumor-specific neoantigens and mutations autoantigens, now called tumor-associated antigens, and their specific recognition by cytotoxic T lymphocytes. Melanomas were of key importance for the development of adoptive T-cell therapy and active tumor vaccines, namely dendritic cell vaccines. Melanoma therapy with antibodies against CTLA-4 provided the proof of concept that solid cancers can be susceptible to cancer immunotherapy, and melanoma therapy with antibodies against PD-1 resulted in the clinical breakthrough of cancer immunotherapy. Still, about half of patients die from metastatic melanoma. Combining anti‒PD-1 with anti‒CTLA-4 antibodies to increase antitumor immune responses or with targeted therapy improves the overall survival only partially. Recent data revealed a close link between defects in the IFN-γ‒dependent induction of cell cycle control genes and resistance to immunotherapy, which may allow for identifying those patients that respond to immunotherapy and to develop novel therapies, combining cancer immunotherapy with cell cycle inhibitors.

ASC Speck Formation after Inflammasome Activation in Primary Human Keratinocytes.

Chronic UV irradiation results in many changes in the skin, including hyperplasia, changes in dermal structures, and alteration of pigmentation. Exposure to UVB leads to cutaneous damage, which results in inflammation characterized by increased NF-κB activation and the induction of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin- (IL-) 1, or IL-8. IL-1 secretion is the result of inflammasome activation which is besides apoptosis, a result of acute UVB treatment. Inflammasomes are cytosolic protein complexes whose formation results in the activation of proinflammatory caspase-1. Key substrates of caspase-1 are IL-1ß and IL-18, and the cytosolic protein gasdermin D (GSDMD), which is involved in inflammatory cell death. Here, we demonstrate that UVB-induced inflammasome activation leads to the formation of ASC specks. Our findings show that UVB provokes ASC speck formation in human primary keratinocytes prior to cell death, and that specks are, opposed to the perinuclear cytosolic localization in myeloid cells, formed in the nucleus. Additionally, we showed by RNAi that NLRP1 and not NLRP3 is the major inflammasome responsible for UVB sensing in primary human keratinocytes. Formation of ASC specks indicates inflammasome assembly and activation as their formation in hPKs depends on the presence of NLRP1 and partially on NLRP3. Nuclear ASC specks are not specific for NLRP1/NLRP3 inflammasome activation, as the activation of the AIM2 inflammasome by cytosolic DNA results in ASC specks too. These nuclear ASC specks putatively link cell death to inflammasome activation, possibly by binding of IFI16 (gamma-interferon-inducible protein) to ASC. ASC can interact upon UVB sensing via IFI16 with p53, linking cell death to ASC speck formation.

Low-dose total body irradiation facilitates antitumoral Th1 immune responses.

CD4+ T helper cells are capable of mediating long-term antitumoral immune responses. We developed a combined immunotherapy (COMBO) using tumor antigen-specific T helper 1 cells (Tag-Th1), dual PD-L1/LAG-3 immune checkpoint blockade, and a low-dose total body irradiation (TBI) of 2 Gy, that was highly efficient in controlling the tumor burden of non-immunogenic RIP1-Tag2 mice with late-stage endogenous pancreatic islet carcinomas. In this study, we aimed to explore the impact of 2 Gy TBI on the treatment efficacy and the underlying mechanisms to boost CD4+ T cell-based immunotherapies. Methods: Heavily progressed RIP1-Tag2 mice underwent COMBO treatment and their survival was compared to a cohort without 2 Gy TBI. Positron emission tomography/computed tomography (PET/CT) with radiolabeled anti-CD3 monoclonal antibodies and flow cytometry were applied to investigate 2 Gy TBI-induced alterations in the biodistribution of endogenous T cells of healthy C3H mice. Migration and homing properties of Cy5-labeled adoptive Tag-Th1 cells were monitored by optical imaging and flow cytometric analyses in C3H and tumor-bearing RIP1-Tag2 mice. Splenectomy or sham-surgery of late-stage RIP1-Tag2 mice was performed before onset of COMBO treatment to elucidate the impact of the spleen on the therapy response. Results: First, we determined a significant longer survival of RIP1-Tag2 mice and an increased CD4+ T cell tumor infiltrate when 2 Gy TBI was applied in addition to Tag-Th1 cell PD-L1/LAG-3 treatment. In non-tumor-bearing C3H mice, TBI induced a moderate host lymphodepletion and a tumor antigen-independent accumulation of Tag-Th1 cells in lymphoid and non-lymphoid organs. In RIP1-Tag2, we found increased numbers of effector memory-like Tag-Th1 and endogenous CD4+ T cells in the pancreatic tumor tissue after TBI, accompanied by a tumor-specific Th1-driven immune response. Furthermore, the spleen negatively regulated T cell effector function by upregulation PD-1/LAG-3/TIM-3 immune checkpoints, providing a further rationale for this combined treatment approach. Conclusion: Low-dose TBI represents a powerful tool to foster CD4+ T cell-based cancer immunotherapies by favoring Th1-driven antitumoral immunity. As TBI is a clinically approved and well-established technique it might be an ideal addition for adoptive cell therapy with CD4+ T cells in the clinical setting.

Immune checkpoint blockade impairs immunosuppressive mechanisms of regulatory T cells in B-cell lymphoma.

In malignant disease, CD4+Foxp3+ regulatory T cells (Tregs) hamper antitumor immune responses and may provide a target for immunotherapy. Although immune checkpoint blockade (ICB) has become an established therapy for several cancer entities including lymphoma, its mechanisms have not been entirely uncovered. Using endogenously arising λ-MYC-transgenic mouse B-cell lymphomas, which can effectively be suppressed by either Treg ablation or ICB, we investigated which mechanisms are used by Tregs to suppress antitumor responses and how ICB affects these pathways. During tumor development, Tregs up-regulated Foxp3, CD25, CTLA-4 and IL-10, which correlated with enhanced immunosuppressive functions. Thus, in contrast to other tumors, Tregs did not become dysfunctional despite chronic stimulation in the tumor microenvironment and progressive up-regulation of PD-1. Immunosuppression was mediated by direct contacts between Tregs and effector T cells and by IL-10. When λ-MYC mice were treated with ICB antibodies, Tregs revealed a less profound up-regulation of Foxp3, CD25 and IL-10 and a decreased suppressive capacity. This may be due to the shift towards a pro-inflammatory milieu fostered by ICB. In summary, an ICB-induced interference with Treg-dependent immunosuppression may contribute to the success of ICB.

Pretreatment metastatic growth rate determines clinical outcome of advanced melanoma patients treated with anti-PD-1 antibodies: a multicenter cohort study.

BACKGROUND: Checkpoint inhibitors revolutionized the treatment of metastatic melanoma patients. Although tumor burden and lactate dehydrogenase (LDH) are associated with overall survival (OS), the impact of tumor growth kinetics remains elusive and in part contradictory. The aims of this study were to develop a novel simple and rapid method that estimates pretreatment metastatic growth rate (MGR) and to investigate its prognostic impact in melanoma patients treated with antiprogrammed death receptor-1 (PD-1) antibodies. METHODS: MGR was assessed in three independent cohorts of a total of 337 unselected consecutive metastasized stage IIIB-IV melanoma patients (discovery cohort: n=53, confirmation cohort: n=126, independent multicenter validation cohort: n=158). MGR was computed during the pretreatment period before initiation of therapy with anti-PD-1 antibodies nivolumab or pembrolizumab by measuring the increase of the longest diameter of the largest target lesion. Tumor doubling time served as quality control. Kaplan-Meier analysis and univariable as well as multivariable Cox regression were used to examine the prognostic impact of MGR. RESULTS: Pretreatment MGR >3.9 mm/month was associated with impaired OS in the discovery cohort (HR 6.19, 95% CI 2.92 to 13.10, p<0.0001), in the confirmation cohort (HR 3.62, 95% CI 2.19 to 5.98, p<0.0001) and in the independent validation cohort (HR 2.57, 95% CI 1.56 to 4.25, p=0.00023). Prior lines of systemic treatment did not influence the significance of MGR. Importantly, the prognostic impact of MGR was independent of total tumor burden, diameter of the largest metastasis, number of prior lines of systemic treatment, LDH, as well as liver and brain metastasis (discovery and confirmation cohorts: both p<0.0001). Superiority of MGR compared with these variables was confirmed in the independent multicenter validation cohort (HR 2.92, 95% CI 1.62 to 5.26, p=0.00036). CONCLUSIONS: High pretreatment MGR is an independent strong prognostic biomarker associated with unfavorable survival of melanoma patients receiving anti-PD-1 antibodies. Further investigations are warranted to assess the predictive impact of MGR in distinct systemic therapeutic regimens.

Case Report: Combined CDK4/6 and MEK Inhibition in Refractory CDKN2A and NRAS Mutant Melanoma.

There are only limited treatment options for metastatic NRAS mutant melanoma patients with resistance to immune checkpoint inhibitors. Besides activation of the mitogen-activated protein (MAP) kinase pathway, they often have additional disturbances in cell cycle regulation. However, unlike BRAF mutant melanoma, no targeted therapy has yet been approved for NRAS mutant melanoma so far. Here we present a NRAS mutant melanoma patient with response to combined binimetinib and ribociclib therapy following characterization of the molecular defects of the tumor by panel sequencing. Next generation sequencing (708 cancer genes) of a soft tissue metastasis revealed a homozygous deletion of CDKN2A in addition to the previously known NRAS mutation, as well as amplification of CCNE1 and CDK6. Immunohistochemical staining of the altered cell cycle genes confirmed loss of p16, reduced expression of p21 and high expression of CDK6 and cyclin D1. As the patient had been progressive on combined immunotherapy, targeted therapy with combined MEK and CDK4/6 inhibition was initiated as recommended by the molecular tumor board. Response to treatment was monitored with PET/CT and liquid biopsy, serum LDH, and S100. In addition, a patient-derived xenograft (PDX) was used to prove the efficacy of the two drugs in combination. Furthermore, senescence-associated beta-galactosidase staining showed that more cells were senescent under the combination treatment of binimetinib and ribociclib. Our case demonstrates how an individualized, molecular-based therapeutic approach could be found based on next-generation sequencing results. Furthermore our report highlights the fruitful and efficient collaboration of dermatooncologists, human geneticists, molecular pathologists, biochemists, radiologists, and nuclear physicians. Further studies are urgently needed to expand the very limited therapeutic landscape of NRAS mutated melanoma.

Interleukin-10 counteracts T-helper type 1 responses in B-cell lymphoma and is a target for tumor immunotherapy.

To establish strategies for immunotherapy of B-cell lymphoma, it is mandatory to gain deeper insights into the mechanisms of tumor immune escape. In a mouse model of endogenously arising lymphoma, we investigated the impact of IL-10 on the regulation of antitumor responses. Despite progressive functional impairment of NK cells and lack of IFN-γ in the tumor milieu, we found an augmented fraction of T helper type 1 (Th1) cells, which continued to express IFN-γ but also upregulated IL-10 during disease development. Using a lymphoma microenvironment in vitro, we showed that Th1 cells were converted to Foxp3-negative T regulatory type 1 (Tr1) cells, which coexpressed IFN-γ and IL-10 and upregulated PD-1. This differentiation required pre-existing IL-10, which was primarily provided by malignant B cells and dendritic cells. IFN-γ only declined in cells with the uppermost PD-1 levels. Importantly, antibody-mediated IL-10 ablation in vivo improved effector cell functions and significantly suppressed tumor development. While the contribution of IL-10 to cancer immune escape has been controversially discussed in the past, we show that IL-10 suppresses ongoing, potentially protective immune responses in lymphoma and might be a target for immunotherapy.

Therapy of lymphoma by immune checkpoint inhibitors: the role of T cells, NK cells and cytokine-induced tumor senescence.

BACKGROUND: Although antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously growing B-cell lymphoma to analyze the requirements for effective therapy with immune checkpoint inhibitors. METHODS: Growth of spontaneous lymphoma was monitored in mice that received antibodies targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein-4, and the role of different immune cell compartments and cytokines was studied by in vivo depletion experiments. Activation of T and natural killer cells and the induction of tumor senescence were analyzed by flow cytometry. RESULTS: On immune checkpoint blockade, visible lymphomas developed at later time points than in untreated controls, indicating an enhanced tumor control. Importantly, 20% to 30% of mice were even long-term protected and did never develop clinical signs of tumor growth. The therapeutic effect was dependent on cytokine-induced senescence in malignant B cells. The proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were necessary for the survival benefit as well as for senescence induction in the λ-MYC model. Antibody therapy improved T-cell functions such as cytokine production, and long-time survivors were only observed in the presence of T cells. Yet, NK cells also had a pronounced effect on therapy-induced delay of tumor growth. Antibody treatment enhanced numbers, proliferation and IFN-γ expression of NK cells in developing tumors. The therapeutic effect was fully abrogated only after depletion of both, T cells and NK cells, or after ablation of either IFN-γ or TNF. CONCLUSIONS: Tumor cell senescence may explain why patients responding to immune checkpoint blockade frequently show stable growth arrest of tumors rather than complete tumor regression. In the lymphoma model studied, successful therapy required both, tumor-directed T-cell responses and NK cells, which control, at least partly, tumor development through cytokine-induced tumor senescence.

The role of dendritic cells for therapy of B-cell lymphoma with immune checkpoint inhibitors.

Immune checkpoint blocking (ICB) is a promising new tool of cancer treatment. Yet, the underlying therapeutic mechanisms are not fully understood. Here we investigated the role of dendritic cells (DCs) for the therapeutic effect of ICB in a λ-MYC-transgenic mouse model of endogenously arising B-cell lymphoma. The growth of these tumors can be effectively delayed by antibodies against CTLA-4 and PD-1. Tumor-infiltrating DCs from mice having received therapy showed an upregulation of costimulatory molecules as well as an augmented IL-12/IL-10 ratio as compared to untreated controls. Both alterations seemed to be induced by interferon-γ (IFN-γ), which is upregulated in T cells and natural killer cells upon ICB. Furthermore, the enhanced IL-12/IL-10 ratio, which favors Th1-prone antitumor T-cell responses, was a consequence of direct interaction of ICB antibodies with DCs. Importantly, the capability of tumor-infiltrating DCs of stimulating peptide-specific or allogeneic T-cell responses in vitro was improved when DCs were derived from ICB-treated mice. The data indicate that ICB therapy is not only effective by directly activating T cells, but also by triggering a complex network, in which DCs play a pivotal role at the interface between innate and adaptive antitumor responses.

Prognostic role of gamma-glutamyl transferase in metastatic melanoma patients treated with immune checkpoint inhibitors.

BACKGROUND: Hepatic immune-related adverse events (irAE) including elevated liver function tests (transaminases) occur in 1.4-22.3% of melanoma patients receiving immune checkpoint inhibitors (ICPI) and constitute a potentially serious toxicity that is challenging to treat. In contrast to the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST), only little is known about the frequency and impact of gamma-glutamyl transferase (GGT) elevations. METHODS: GGT determined prior to and during therapy of metastatic melanoma patients treated with ICPI were retrospectively assessed in two independent cohorts (PD-1: n = 218, Ipi + Nivo: n = 148). Overall survival (OS) and best objective response were analyzed according to baseline and immune-related GGT (irGGT) elevations during treatment. RESULTS: In multivariate analysis, OS was reduced in patients with elevated baseline GGT (PD-1 group: hazard ratio [HR] 1.76, p = .0073; Ipi + Nivo group: HR 1.77, p = .032). Immune-related GGT elevation was recorded in 17% (PD-1 group) and 38.5% (Ipi + Nivo group). Of these patients, the majority (81 and 68%, respectively) had normal ALT and AST and showed no clinical signs of hepatotoxicity. Patients who experienced irGGT elevation had superior response (PD-1 group: odds ratio [OR] 3.57, p = .00072; Ipi + Nivo group: OR 1.74, p = .12) and OS (PD-1 group: HR 0.37, p = .0016; Ipi + Nivo group: HR 0.33, p = .00050). CONCLUSIONS: The frequency of hepatic irAE is currently underestimated. The addition of the sensitive enzyme GGT to the laboratory panel before and during therapy with ICPI allows to detect two to three times more patients developing hepatic or hepatobiliary toxicity than known so far. Immune-related GGT elevations correlate with response and favorable survival. Precis for use in the Table of Contents The frequency of hepatotoxicity under immune checkpoint blockade is currently underestimated. We suggest the addition of gamma-glutamyl transferase to the laboratory panel in checkpoint inhibitor patients for the detection of hepatobiliary toxicity.