Neuropathological assessment of the olfactory bulb and tract in individuals with COVID-19.

The majority of patients with Parkinson disease (PD) experience a loss in their sense of smell and accumulate insoluble α-synuclein aggregates in their olfactory bulbs (OB). Subjects affected by a SARS-CoV-2-linked illness (COVID-19) also frequently experience hyposmia. We previously postulated that microglial activation as well as α-synuclein and tau misprocessing can occur during host responses following microbial encounters. Using semiquantitative measurements of immunohistochemical signals, we examined OB and olfactory tract specimens collected serially at autopsies between 2020 and 2023. Deceased subjects comprised 50 adults, which included COVID19 + patients (n = 22), individuals with Lewy body disease (e.g., PD; dementia with Lewy bodies (n = 6)), Alzheimer disease (AD; n = 3), and other neurodegenerative disorders (e.g., progressive supranuclear palsy (n = 2); multisystem atrophy (n = 1)). Further, we included neurologically healthy controls (n = 9), and added subjects with an inflammation-rich brain disorder as neurological controls (NCO; n = 7). When probing for microglial and histiocytic reactivity in the anterior olfactory nuclei (AON) by anti-CD68 immunostaining, scores were consistently elevated in NCO and AD cases. In contrast, microglial signals on average were not significantly altered in COVID19 + patients relative to healthy controls, although anti-CD68 reactivity in their OB and tracts declined with progression in age. Mild-to-moderate increases in phospho-α-synuclein and phospho-tau signals were detected in the AON of tauopathy- and synucleinopathy-afflicted brains, respectively, consistent with mixed pathology, as described by others. Lastly, when both sides were available for comparison in our case series, we saw no asymmetry in the degree of pathology of the left versus right OB and tracts. We concluded from our autopsy series that after a fatal course of COVID-19, microscopic changes in the rostral, intracranial portion of the olfactory circuitry -when present- reflected neurodegenerative processes seen elsewhere in the brain. In general, microglial reactivity correlated best with the degree of Alzheimer's-linked tauopathy and declined with progression of age in COVID19 + patients.

Autoimmunity Against Surfactant Protein B Is Associated with Pneumonitis During Checkpoint Blockade.

RATIONALE: Immune checkpoint inhibitor-related pneumonitis is a serious autoimmune event affecting up to 20% of patients with non-small cell lung cancer, yet the factors underpinning its development in some patients and not others are poorly understood. OBJECTIVES: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis. METHODS: The study cohort consisted of non-small cell lung cancer patients who gave blood samples before and during immune checkpoint inhibitor treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with non-small cell lung cancer and patients with melanoma. MEASUREMENTS AND MAIN RESULTS: Across both cohorts, patients who developed pneumonitis had higher pre-treatment levels of immunoglobulin G autoantibodies targeting surfactant protein-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4+ interferon-gamma-positive surfactant protein B-specific T cells, and expanding T cell clonotypes recognizing this protein, accompanied by a pro-inflammatory serum proteomic profile. CONCLUSIONS: Our data suggest that the co-occurrence of surfactant protein-B-specific immunoglobulin G autoantibodies and CD4+ T cells is associated with the development of pneumonitis during ICI therapy. Pre-treatment levels of these antibodies may represent a potential biomarker for elevated risk of developing pneumonitis and on-treatment levels may provide a diagnostic aid.

Characterization of PRAME immunohistochemistry reveals lower expression in pediatric melanoma compared to adult melanoma.

Pediatric melanomas are rare tumors that have clinical and histological differences from adult melanomas. In adult melanoma, the immunohistochemical marker PRAME is increasingly employed as a diagnostic adjunct. PRAME is also under investigation as a target structure for next-generation immunotherapies including T-cell engagers. Little is known about the characteristics of PRAME expression in pediatric melanoma. In this retrospective study, samples from 25 pediatric melanomas were compared with control groups of melanomas in young adults (18-30 years; n = 32), adult melanoma (>30 years, n = 30), and benign melanocytic nevi in children (0-18 years; n = 30) with regard to the immunohistochemical expression of PRAME (diffuse PRAME expression >75%/absolute expression). Pediatric melanomas show lower diffuse PRAME expression (4%) and lower absolute PRAME expression (25%) compared to young adult melanomas (15.6%/46.8%) and adult melanomas (50%/70%). A significant age-dependent expression could be observed. An analysis of event-free survival shows no prognostic role for PRAME in pediatric melanoma and young adult melanoma, but a significant association with diffuse PRAME expression in adulthood. The age dependency of PRAME expression poses a potential pitfall in the diagnostic application of melanocytic tumors in young patients and may limit therapeutic options within this age group. The immunohistochemical expression of the tumor-associated antigen PRAME is an increasingly important diagnostic marker for melanocytic tumors and is gaining attention as a possible immunotherapeutic target in melanoma. As the available data primarily stem from adult melanoma, and given the clinical and histological distinctions in pediatric melanomas, our understanding of PRAME expression in this specific patient group remains limited. The age-dependent low PRAME expression shown here constrains the use of this marker in pediatric melanoma and may also limit the use of immunotherapeutic strategies against PRAME in young patients.

Isolated melanoma cells in sentinel lymph node in stage IIIA melanoma correlate with a favorable prognosis similar to stage IB.

BACKGROUND: The American Joint Committee on Cancer 8th edition (AJCC v8) defines sentinel lymph nodes (SLN) containing any tumor cells as positive SLN. Consequently, even thin melanomas with isolated tumor cells (ic) in SLN are classified as stage IIIA, making them candidates for adjuvant therapy. OBJECTIVES AND ENDPOINTS: We aimed to evaluate survival outcomes of melanoma stage IIIA (ic) and compare them with stage IIIA with lymph node (LN) metastases > 0.1 mm. Primary endpoints were relapse-free survival (RFS) and distant metastases-free survival (DMFS). Secondary endpoint was melanoma specific survival (MSS). RESULTS: The discovery cohort from the Department of Dermatology, University Hospital Tuebingen, included 237 patients; confirmation cohort included 143 patients from the DeCOG trial. The Tuebingen cohort included 95 patients with stage IIIA (ic) and 142 patients with stage IIIA. The DeCOG trial included 39 patients with stage IIIA (ic) and 104 patients with stage IIIA. In the Tuebingen cohort, 10-year RFS rates for stage IIIA (ic) and IIIA were 84% (95% CI 75-94) and 49% (95% CI 39-59), respectively (p < 0.001). 10-year DMFS rates for stage IIIA (ic) and IIIA were 89% (95% CI 81-97) and 56% (95% CI 45-67), respectively; (p < 0.001). In the DeCOG cohort, 10-year RFS for stage IIIA (ic) and stage IIIA were 88% (95% CI 78-99) and 35% (95% CI 7-62), respectively; (p = 0.009). 10-year DMFS for stage IIIA (ic) and IIIA was 88% (95% CI 77-99) and 60% (95% CI 39-80), respectively (p = 0.061). CONCLUSION: Stage IIIA (ic) melanoma exhibits a prognosis similar to stage IB. Recommendation of adjuvant therapy in Stage IIIA (ic) warrants thorough discussion.

Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy.

BACKGROUND: MEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib. METHODS: The effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi. RESULTS: Diethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib. CONCLUSIONS: Disulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

Long-term survival of stage IV melanoma patients: evaluation on 640 melanoma patients entering stage IV between 2014 and 2017.

PURPOSE: Since the introduction of immune checkpoint inhibitors (ICI) and targeted therapies (TT), survival rates of metastatic melanoma patients have increased significantly and complete remissions are no longer rarities. Consequently, there is an increasing number of long-term survivors who have not yet been comprehensively characterized. METHODS: We included melanoma patients who entered stage IV between 2014 and 2017 and survived at least 5 years after entering stage IV. Descriptive statistics were performed to characterize the applied systemic therapies, response rates and to report which of these patients are still alive today. RESULTS: 640 patients entered stage IV at the University Hospital Tuebingen. Of these, 207 patients (32%) were still alive at least 5 years after entering stage IV. Details of applied therapies and response rates were available in 176 patients (85%). About 90% of patients (n = 159) were still alive at the time of analysis. Median survival since first stage IV diagnosis was 6.0 years (range 5-9 years). An impressive majority of patients (n = 146, 83%) were no longer receiving systemic therapy at the time of evaluation. Complete remission under first line systemic therapy was seen in 36% of the patients. CONCLUSION: This dataset comprises the largest available cohort of long-term surviving stage IV melanoma patients. Since 90% of patients in our cohort are still alive today, we expect an increasing number of long-term survivors in the future. Our data indicate the need for specific follow-up programs addressing the needs of long-term survivors.

A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Histopathologic regression in patients with primary cutaneous melanoma undergoing sentinel lymph node biopsy is associated with favorable survival and, after metastasis, with improved progression-free survival on immune checkpoint inhibitor therapy: A single-institutional cohort study.

BACKGROUND: Histopathologic regression of cutaneous melanoma is considered a favorable prognostic factor, but its significance in clinical practice remains controversial. OBJECTIVE: To investigate the prognostic importance of regression in patients with primary cutaneous melanoma undergoing sentinel lymph node (SLN) biopsy and to assess its significance in patients progressing to an unresectable stage requiring systemic therapy. METHODS: We retrospectively reviewed patients with newly diagnosed melanoma undergoing SLN biopsy between 2010 and 2015 and available information on histopathologic regression (n = 1179). Survival data and associations of clinical variables with SLN status were assessed. RESULTS: Patients with regressive melanoma showed favorable relapse-free (hazard ratio [HR], 0.52; P = .00013), distant metastasis-free (HR, 0.56; P = .0020), and melanoma-specific survival (HR, 0.35; P = .00053). Regression was associated with negative SLN (odds ratio, 0.48; P = .0077). In patients who progressed to an unresectable stage, regression was associated with favorable progression-free survival under immune checkpoint inhibition (HR, 0.43; P = .031) but not under targeted therapy (HR, 1.14; P = .73) or chemotherapy (HR, 3.65; P = .0095). LIMITATIONS: Retrospective, single-institutional design. CONCLUSIONS: Regression of cutaneous melanoma is associated with improved prognosis in patients eligible for SLN biopsy as well as in patients with unresectable disease receiving systemic therapy with immune checkpoint inhibitors.

Baseline metastatic growth rate is an independent prognostic marker in patients with advanced BRAF V600 mutated melanoma receiving targeted therapy.

BACKGROUND: Targeted therapy (TT) of BRAF V600 mutated unresectable melanoma with inhibitors of the MAPK pathway achieves response rates of up to 76%, but most patients develop secondary resistance. Albeit TT is strikingly efficacious during the first days of treatment, even in advanced cases, long-term survival is highly unlikely, especially in patients with unfavorable baseline characteristics like elevated lactate dehydrogenase (LDH). In patients treated with anti-PD-1 immune checkpoint inhibitors, elevated baseline metastatic growth rate (MGR) was the most important prognostic factor. Here, we aimed at investigating the prognostic impact of MGR in patients with unresectable melanoma receiving TT. METHODS: Clinical records of 242 patients with at least one measurable target lesion (TL) receiving TT at seven skin cancer centers were reviewed. Baseline MGR was determined measuring the largest TL at baseline and at one earlier timepoint. RESULTS: Overall survival (OS) and progression-free survival (PFS) were significantly impaired in patients with an MGR > 3.9 mm/month (median OS: 11.4 vs. 35.5 months, P < 0.0001; median PFS: 4.8 vs. 9.2 months, P < 0.0001). Multivariable analysis of OS and PFS revealed that the prognostic impact of elevated MGR was independent of LDH, presence of brain and liver metastases, tumor burden, and line of treatment. The prognostic significance of elevated MGR was highest in patients with normal LDH. CONCLUSIONS: Baseline MGR is an important independent prognostic marker for OS and PFS in melanoma patients treated with TT. Its implementation in clinical routine is easy and could facilitate the prognostic stratification.

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.

Outcome and treatment-related adverse events of combined immune checkpoint inhibition with flipped dosing in a real-world cohort of 79 patients with metastasized melanoma.

Introduction: Combined immune checkpoint inhibition (ICI) with ipilimumab and nivolumab is a widely used treatment regimen for metastatic melanoma with non-resectable metastases. Nevertheless, the standard dose of ipilimumab 3 mg/kg bw and nivolumab 1 mg/kg bw is associated with a high rate of treatment-related adverse events (trAEs) (59% grade 3-4). In the CheckMate 511 study, it could be shown that flipped dosing with ipilimumab 1 mg/kg bw and nivolumab 3 mg/kg bw resulted in a significant reduction of trAE. Methods: We have also used this regimen in the clinical setting and report the trAE, progression-free survival, and overall survival for 79 patients with metastatic melanoma who started combined ICI in the flipped dosing between March 2019 and April 2020. Results: in total, 40 patients started first-line, 50% of whom had an elevated lactate dehydrogenase level at baseline. The disease control rate of these patients was 50%. The 2-year overall survival rate 67%. Moreover, 33% of the patients suffered grade 3 or 4 treatment related adverse events. Discussion: The results of our study correspond very well to the results of the CheckMate 511 study (2-year OS: 65%, grade 3-4 immune-related side effects: 35%). Combined ICI with ipilimumab 1 mg/kg bw and nivolumab 3 mg/kg bw seems to be an equally effective but better-tolerated therapy regimen for metastasized melanoma patients, also in a real-world cohort.

Autoreactive T cells targeting type II pneumocyte antigens in COVID-19 convalescent patients.

BACKGROUND: The role of autoreactive T cells on the course of Coronavirus disease-19 (COVID-19) remains elusive. Type II pneumocytes represent the main target cells of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autoimmune responses against antigens highly expressed in type II pneumocytes may influence the severity of COVID-19 disease. OBJECTIVE: The aim of this study was to investigate autoreactive T cell responses against self-antigens highly expressed in type II pneumocytes in the blood of COVID-19 patients with severe and non-severe disease. METHODS: We collected blood samples of COVID-19 patients with varying degrees of disease severity and of pre-pandemic controls. T cell stimulation assays with peptide pools of type II pneumocyte antigens were performed in two independent cohorts to analyze the autoimmune T cell responses in patients with non-severe and severe COVID-19 disease. Target cell lysis assays were performed with lung cancer cell lines to determine the extent of cell killing by type II PAA-specific T cells. RESULTS: We identified autoreactive T cell responses against four recently described self-antigens highly expressed in type II pneumocytes, known as surfactant protein A, surfactant protein B, surfactant protein C and napsin A, in the blood of COVID-19 patients. These antigens were termed type II pneumocyte-associated antigens (type II PAAs). We found that patients with non-severe COVID-19 disease showed a significantly higher frequency of type II PAA-specific autoreactive T cells in the blood when compared to severely ill patients. The presence of high frequencies of type II PAA-specific T cells in the blood of non-severe COVID-19 patients was independent of their age. We also found that napsin A-specific T cells from convalescent COVID-19 patients could kill lung cancer cells, demonstrating the functional and cytotoxic role of these T cells. CONCLUSIONS: Our data suggest that autoreactive type II PAA-specific T cells have a protective role in SARS-CoV-2 infections and the presence of high frequencies of these autoreactive T cells indicates effective viral control in COVID-19 patients. Type II-PAA-specific T cells may therefore promote the killing of infected type II pneumocytes and viral clearance.

Autoimmunity in immune checkpoint inhibitor-induced immune-related adverse events: A focus on autoimmune skin toxicity and pneumonitis.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, their use is frequently associated with immune-related adverse events (irAEs) potentially affecting any organ. The mechanisms mediating such irAEs remain poorly understood and biomarkers to predict the development of irAEs are lacking. Growing evidence shows the importance of self-antigens in mediating irAEs during ICI therapy, in particular the well-described melanocyte differentiation antigens in melanoma patients. This review will focus on two novel classes of self-antigens involved in mediating autoimmune skin toxicity and pneumonitis in non-small cell lung cancer patients treated with immunotherapy. T cells specific for these self-antigens are thought to not only mediate irAEs but are thought to simultaneously mediate anti-tumor responses and are therefore associated with both autoimmune toxicity and response to ICI therapy. We further discuss emerging cellular and proteomic immune signatures of irAEs that may serve as biomarkers to help predict which patients are at higher risk of developing these irAEs. The determination of new tumor antigens involved in ICI therapy and the identification of related biomarkers brings us a step forward in the mechanistic understanding of ICIs and will help to monitor patients at higher risk of developing irAEs. Lastly, we discuss the current challenges in collecting research samples for the study of ICI-related mechanisms and in distinguishing between immune signatures of response and those of irAEs.

Prognosis in stage II melanoma of the head and neck depends on the histological subtype.

BACKGROUND AND OBJECTIVES: The melanoma guideline is mainly based on the AJCC stage. There is no difference according to histological subtypes such as superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM) or nodular malignant melanoma (NM). We aimed to evaluate whether patients with LMM have a different clinical course from patients with SSM/NM. This is particularly important as adjuvant anti-PD-1 therapy is approved for stage IIB and IIC melanoma. PATIENTS AND METHODS: Data were extracted from the Central Registry of Malignant Melanoma. Only patients with LMM, SSM, and NM of the head and neck with primary diagnosis between 01/01/2000 and 12/31/2019 were included. Progression-free survival (PFS), melanoma-specific survival (MSS), and pattern of metastases were analyzed for the LMM group compared to SSM/NM. RESULTS: The LMM cohort (n = 902) had significantly better MSS than the SSM/NM cohort (n = 604). There was no difference in PFS. The 5-year MSS of the stage II LMM cohort was 88.5% (95% CI 81.4-95.6) compared to 79.7% (95% CI 72.8-86.6) in the stage II SSM/NM cohort. CONCLUSION: It does not appear appropriate to use adjuvant therapy in stage II LMM patients to the same extent as in patients with SSM/NM.

Interleukin 17 signaling supports clinical benefit of dual CTLA-4 and PD-1 checkpoint inhibition in melanoma.

Recent studies suggest that BRAFV600-mutated melanomas in particular respond to dual anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibition (ICI). Here we identified an over-representation of interleukin (IL)-17-type 17 helper T (TH17) gene expression signatures (GES) in BRAFV600-mutated tumors. Moreover, high baseline IL-17 GES consistently predicted clinical responses in dual-ICI-treated patient cohorts but not in mono anti-CTLA-4 or anti-PD-1 ICI cohorts. High IL-17 GES corresponded to tumor infiltration with T cells and neutrophils. Accordingly, high neutrophil infiltration correlated with clinical response specifically to dual ICI, and tumor-associated neutrophils also showed strong IL-17-TH17 pathway activity and T cell activation capacity. Both the blockade of IL-17A and the depletion of neutrophils impaired dual-ICI response and decreased T cell activation. Finally, high IL-17A levels in the blood of patients with melanoma indicated a higher global TH17 cytokine profile preceding clinical response to dual ICI but not to anti-PD-1 monotherapy, suggesting a future role as a biomarker for patient stratification.

Histopathologic abundance of pigmentation correlates with disease-specific survival in malignant melanoma but is not independent of current AJCC pT stage.

The increasing number of melanoma patients makes it necessary to develop best possible strategies for prognosis assessment in order to recommend appropriate therapy and follow-up. The prognostic significance of tumor cell pigmentation has not been fully elucidated. Hematoxylin and eosin (H&E)-stained sections of 775 melanomas diagnosed between 2012 and 2015 were independently assessed for melanin pigment abundance by two investigators, and the impact on melanoma-specific survival was calculated. Unpigmented melanomas (n = 99) had a melanoma-specific survival of 67.7%, melanomas with moderate pigmentation (n = 384) had a melanoma-specific survival of 85.9%, and strongly pigmented melanomas (n = 292) had a melanoma-specific survival of 91.4% (p < .001). In an analysis of melanoma-specific survival adjusted for pT stage and pigmentation, we found a nonsignificant impact of pigmentation abundance with a hazard ratio of 1.277 (p = .74). The study presented here provides evidence in a German cohort that patients with pigmented melanomas have a more favorable prognosis than those diagnosed with nonpigmented melanomas. Moreover, the abundance of pigmentation already seems to provide a first prognostic estimate. However, it does not appear to provide significant additional value for prognostic assessment according to the AJCC 2017 pT classification.

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.