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Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

The effect of tumor/T-cell interactions on subsequent immune infiltration is undefined. Here, we report that preexposure of melanoma cells to cognate T cells enhanced the chemotaxis of new T cells in vitro. The effect was HLA class I–restricted and IFNγ-dependent, as it was abolished by β2M-knockdown, MHC-blocking antibodies, JAK1 inhibitors, JAK1-silencing and IFNgR1-blocking antibodies. RNA sequencing (RNA-seq) of 73 melanoma metastases showed a significant correlation between the interferon-inducible p150 isoform of adenosine-deaminase-acting-on-RNA-1 (ADAR1) enzyme and immune infiltration. Consistent with this, cocultures of cognate melanoma/T-cell pairs led to IFNγ-dependent induction of ADAR1-p150 in the melanoma cells, as visualized in situ using dynamic cell blocks, in ovo using fertilized chick eggs, and in vitro with Western blots. ADAR1 staining and RNA-seq in patient-derived biopsies following immunotherapy showed a rise in ADAR1-p150 expression concurrently with CD8+ cell infiltration and clinical response. Silencing ADAR1-p150 abolished the IFNγ-driven enhanced T-cell migration, confirming its mechanistic role. Silencing and overexpression of the constitutive isoform of ADAR1, ADAR1-p110, decreased and increased T-cell migration, respectively. Chemokine arrays showed that ADAR1 controls the secretion of multiple chemokines from melanoma cells, probably through microRNA-mediated regulation. Chemokine receptor blockade eliminated the IFNγ-driven T-cell chemotaxis. We propose that the constitutive ADAR1 downregulation observed in melanoma contributes to immune exclusion, whereas antigen-specific T cells induce ADAR1-p150 by releasing IFNγ, which can drive T-cell infiltration.

Comparison between proteome and transcriptome profiling. Related to Figure 1.

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Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

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Supplementary Table from Adenosine-Deaminase-Acting-on-RNA-1 Facilitates T-cell Migration toward Human Melanoma Cells

The effect of tumor/T-cell interactions on subsequent immune infiltration is undefined. Here, we report that preexposure of melanoma cells to cognate T cells enhanced the chemotaxis of new T cells in vitro. The effect was HLA class I–restricted and IFNγ-dependent, as it was abolished by β2M-knockdown, MHC-blocking antibodies, JAK1 inhibitors, JAK1-silencing and IFNgR1-blocking antibodies. RNA sequencing (RNA-seq) of 73 melanoma metastases showed a significant correlation between the interferon-inducible p150 isoform of adenosine-deaminase-acting-on-RNA-1 (ADAR1) enzyme and immune infiltration. Consistent with this, cocultures of cognate melanoma/T-cell pairs led to IFNγ-dependent induction of ADAR1-p150 in the melanoma cells, as visualized in situ using dynamic cell blocks, in ovo using fertilized chick eggs, and in vitro with Western blots. ADAR1 staining and RNA-seq in patient-derived biopsies following immunotherapy showed a rise in ADAR1-p150 expression concurrently with CD8+ cell infiltration and clinical response. Silencing ADAR1-p150 abolished the IFNγ-driven enhanced T-cell migration, confirming its mechanistic role. Silencing and overexpression of the constitutive isoform of ADAR1, ADAR1-p110, decreased and increased T-cell migration, respectively. Chemokine arrays showed that ADAR1 controls the secretion of multiple chemokines from melanoma cells, probably through microRNA-mediated regulation. Chemokine receptor blockade eliminated the IFNγ-driven T-cell chemotaxis. We propose that the constitutive ADAR1 downregulation observed in melanoma contributes to immune exclusion, whereas antigen-specific T cells induce ADAR1-p150 by releasing IFNγ, which can drive T-cell infiltration.

Supplementary Figure from Adenosine-Deaminase-Acting-on-RNA-1 Facilitates T-cell Migration toward Human Melanoma Cells

BackgroundMultiplex immunofluorescence (mIF) can provide invaluable insights into spatial biology and the complexities of the immune tumor microenvironment (iTME). However, existing analysis approaches are both laborious and highly user-dependent. In order to overcome these limitations we developed a novel, end-to-end deep learning (DL) pipeline for rapid and accurate analysis of both tumor-microarray (TMA) and whole slide mIF images.MethodsOur pipeline consists of two DL models: a multi-classifier for classifying multi-channel cell images into 12 different cell types, and a binary classifier for determining the positivity of a given marker in single-channel images. The DL multi-classifier was trained on 7,000 tiles labeled with cell annotations from a publicly available CODEX dataset, consisting of 140 tissue cores from 35 colorectal cancer (CRC) patients. For the binary classifier training, the multi-channel tiles were further split into ∼100,000 single-channel tiles, for which the ground truth was inferred from the known expression of these markers in each cell-type. This DL binary classifier was then utilized to quantify the positivity of various cell state (phenotypic) markers. In addition, the binary classifier was exploited as a cell-typing tool, by predicting the positivity of individual lineage cell markers. The performance of our DL models was evaluated on 1,800 annotations from 14 test tissue cores. The models were further evaluated on a new 6-plex melanoma cohort, stained with PhenoImager®, and were compared to the performance of clustering, manual thresholding or machine learning-based cell-typing methods applied on the same test sets.ResultsOur DL multi-classifier achieved highly accurate results, outperforming all of the tested cell-typing methods, including clustering, manual-thresholding and ML-based approaches, in both CODEX CRC and PhenoImager melanoma cohorts (accuracy of 91% and 87%, respectively), with F1-scores above 80% in the vast majority of cell types. Our DL binary classifier, which was trained solely on the lineage markers of the CRC dataset, also outperformed existing methods, demonstrating excellent F1-scores (>80%) for determining the positivity of unseen phenotypic and lineage markers across the two tumor types and imaging modalities. Notably, as little as 20 annotations were required in order to boost the performance on an unseen dataset to above 85% accuracy and 80% F1-scores. As a result, the DL binary classifier could successfully be used as a cell-typing model, in a manner that is transferable between experimental approaches.ConclusionsWe present a novel state-of-the-art DL-based framework for multiplex imaging analysis, that enables accurate cell typing and phenotypic marker quantification, which is robust across markers, tumor indications, and imaging modalities.

Background: T-cell receptor (TCR) clonality is important for mycosis fungoides (MF) diagnosis. Routine clonality analysis is performed using a polymerase chain reaction (PCR) TCR-γ assay; yet with this method 10–50% of T-cell lymphomas escape detection. TCR-β gene rearrangement is an additional assay. Data about its efficacy is controversial. Objective: Evaluate the role of TCR-β assay in the diagnosis of early MF. Methods: A retrospective study of 61 skin biopsies, 20 from MF patients, 30 from patients suspected to have early MF, and 11 from patients with chronic inflammatory skin disease. Results: Monoclonality was detected in 16/20 (80%) MF cases; 15 (75%) with TCR-β and 12 (60%) with TCR-γ assay. Of the 30 suspected early MF cases, 14 demonstrated monoclonality, all of which with TCR-β and 5 with TCR-γ assay. None of the chronic inflammatory condition samples showed monoclonality. Therefore, TCR-β clonality assay was more sensitive in early MF than TCR-γ (83% vs. 43%, P=0.002). Limitations: A retrospective, relatively small study. Conclusion: TCR-β demonstrated a higher sensitivity rate compared with TCR-γ in early-stage MF. The combined use of the TCR-β and TCR-γ clonality tests can significantly improve the diagnosis rate of early-stage MF.

AIMS An increased prevalence of aggressive histological subtypes, such as micronodular and morpheaform, has been seen, irrespective of the clinical course, in basal cell carcinoma (BCC) following irradiation for tinea capitis. The aim of this study was to assess the histopathological features of BCCs among patients irradiated for tinea capitis and correlate them with the clinical course. METHODS AND RESULTS The medical records and BCC biopsy specimens of individuals who were previously irradiated for tinea capitis were reviewed. Demographic data and clinical characteristics were retrieved. Biopsy specimens were evaluated for histological subtype classification and additional histopathological features. A telephone survey was conducted to assess the clinical behaviour of the tumours. Thirty-one patients (17 male; 14 female) were included. The average age at time of first biopsy was 56 years. The total number of lesions was 185, with 80% of subjects showing multiple lesions. The nodular subtype was the most prevalent, followed by superficial, micronodular and mixed tumours. One-third of the BCCs could be classified as aggressive histologically. Stromal fibroplasia and melanin deposits were common. There was no mortality related to BCC. None of the 17 patients who completed the survey had evidence of local invasiveness or metastases. CONCLUSIONS BCCs following radiation therapy for tinea capitis show unique histological characteristics related to aggressive behaviour. These aggressive features did not reflect the clinical behaviour in the current cohort.

Background T-cell receptor (TCR) clonality may help establish a diagnosis of mycosis fungoides (MF). Routine clonality analysis is performed by using a polymerase chain reaction TCR- gamma assay, yet with this method, 10% to 50% of T-cell lymphomas escape detection. TCR- beta gene rearrangement is an additional assay. Data about its efficacy are controversial. Objective To evaluate the role of TCR-β assay in the diagnosis of early MF. Methods A retrospective study of 61 skin biopsies, 20 from patients with MF, 30 from patients suspected to have early MF, and 11 from patients with chronic inflammatory skin disease. Results Monoclonality was detected in 16 of 20 (80%) MF cases: 15 (75%) with TCR-β and 12 (60%) with TCR-γ assay. Of the 30 suspected cases of early MF, 14 showed monoclonality with TCR-β, and only 5 of 14 showed monoclonality with TCR-γ assay. None of the chronic inflammatory condition samples showed monoclonality. Therefore, TCR-β clonality assay was more sensitive than TCR-γ in early MF (83% vs 43%; P = .002). Limitations This was a retrospective, relatively small study. Conclusion TCR-β showed a higher sensitivity rate compared with TCR-γ in early-stage MF. The combined use of the TCR-β and TCR-γ clonality tests can significantly improve the diagnosis rate of early-stage MF.

Background: Pemphigus vulgaris (PV) is a chronic autoimmune blistering disease. Most patients require long-term therapy with systemic steroids, and a steroid-sparing agent is usually also utilized. Dapsone is a chemotherapeutic agent with anti-inflammatory properties that is used as a steroid-sparing agent in PV. Objective: The aim of the present study was to evaluate the efficacy of dapsone as an adjuvant therapy in patients with PV. Methods: A retrospective analysis of patients' files was performed. All 26 patients included in the study group were treated with dapsone as an adjuvant to systemic steroids for at least 3 consecutive months and were followed up during their dapsone treatment period. Results: After 3 months of treatment with dapsone, 13 patients were in the consolidation phase, 4 patients demonstrated partial remission on minimal therapy, 7 patients demonstrated complete remission on minimal therapy, and 2 patients were defined as treatment failures. The trend of clinical improvement continued after 6 months of treatment and at the study end point. Conclusion: This retrospective case series, one of the largest reported, indicates that dapsone is efficacious and safe for patients with PV in whom it is well tolerated soon after the initiation of treatment.