Single cell sequencing delineates T-cell clonality and pathogenesis of the parapsoriasis disease group.

BACKGROUND: Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is often underdiagnosed in early stages due to similarities with benign dermatoses such as atopic dermatitis (AD). Furthermore, the delineation from so-called "parapsoriasis en plaque," a disease that can appear either in a small- or large-plaque form, is still controversial. OBJECTIVE: To characterize the parapsoriasis disease spectrum. METHODS: We performed single-cell RNA sequencing of skin biopsies from patients within the parapsoriasis-to-early-stage MF spectrum, stratified for small and large plaques, and compared them to AD, psoriasis and healthy control skin. RESULTS: 6 out of 8 large-plaque lesions harbored either an expanded alpha/beta or gamma/delta T-cell clone with downregulation of CD7 expression, consistent with a diagnosis of early-stage MF. By contrast, 6 out of 7 small-plaque lesions were polyclonal in nature thereby lacking a lymphomatous phenotype, and also revealed a less inflammatory microenvironment than early-stage MF or AD. Of note, polyclonal small- and large-plaque lesions characteristically harbored a population of NPY+ innate lymphoid cells and displayed a stromal signature of complement upregulation and antimicrobial hyperresponsiveness in fibroblasts and sweat gland cells, respectively. These conditions were clearly distinct from AD or psoriasis, which uniquely harbored CD3+CRTH2+ IL13-expressing "Th2A" cells or strong type 17 inflammation, respectively. CONCLUSION: These data position polyclonal small- and large-plaque dermatitis lesions as a separate disease entity, that characteristically harbors a so far undescribed ILC population. We thus propose the new term "polyclonal parapsoriasis en plaque" to this kind of lesions, as they can be clearly differentiated from early and advanced-stage MF, psoriasis and AD on several cellular and molecular levels.

Single-cell RNA sequencing comparison of CD4+, CD8+ and TCR-γδ+ cutaneous T-cell lymphomas reveals subset-specific molecular phenotypes.

BACKGROUND: Malignant clones of primary cutaneous T-cell lymphomas (CTCL) can show a CD4, CD8 or TCR-γδ phenotype, but their individual impact on tumor biology and skin lesion formation remains ill-defined. OBJECTIVES: To perform a comprehensive molecular characterization of CD4+ vs. CD8+ and TCR-γ/δ+ CTCL lesions. METHODS: We performed scRNA-seq of 18 CTCL skin biopsies to compare classic CD4+ advanced-stage mycosis fungoides (MF) with TCR-γ/δ+MF and primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (Berti's lymphoma). RESULTS: Malignant clones of TCR-γ/δ+MF and Berti's lymphoma showed similar clustering patterns distinct from CD4+MF, along with increased expression of cytotoxic markers such as NKG7, CTSW, GZMA, and GZMM. Only advanced-stage CD4+MF clones expressed central memory T-cell markers (SELL, CCR7, LEF1), alongside B1/B2 blood involvement, whereas TCR-γ/δ+MF and Berti's lymphoma harbored a more tissue-resident phenotype (CD69, CXCR4, NR4A1) without detectable cells in the blood. CD4+MF and TCR-γ/δ+MF skin lesions harbored strong type 2 immune activation across myeloid cells, while Berti's lymphoma was more skewed towards type 1 immune responses. Both CD4+MF and TCR-γ/δ+MF lesions showed upregulation of keratinocyte hyperactivation markers such as S100As and KRT16 genes. This increase was entirely absent in Berti's lymphoma, possibly reflecting an aberrant keratinocyte response to invading tumor cells, that could contribute to the formation of the typical ulcero-necrotic lesions within this entity. CONCLUSIONS: Our scRNAseq profiling study reveals specific molecular patterns associated with distinct CTCL subtypes.

Evaluation of mortality, prognostic parameters, and treatment efficacy in mycosis fungoides.

BACKGROUND AND OBJECTIVES: Mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, is characterized by a variable clinical course, presenting either as indolent disease or showing fatal progression due to extracutaneous involvement. Importantly, the lack of prognostic models and predominantly palliative therapy settings hamper patient care. Here, we aimed to define survival rates, disease prediction accuracy, and treatment impact in MF. PATIENTS AND METHODS: Hundred-forty MF patients were assessed retrospectively. Prognosis and disease progression/survival were analyzed using univariate Cox proportional hazards regression model and Kaplan-Meier estimates. RESULTS: Skin tumors were linked to shorter progression-free, overall survival and a 3.48 increased risk for disease progression when compared to erythroderma. The Cutaneous Lymphoma International Prognostic Index identified patients at risk in early-stage disease only. Moreover, expression of Ki-67 >20%, CD30 >10%, CD20+, and CD7- were associated with a significantly worse outcome independent of disease stage. Only single-agent interferon-α and phototherapy combined with interferon-α or retinoids/bexarotene achieved long-term disease control in MF. CONCLUSIONS: Our data support predictive validity of prognostic factors and models in MF and identified further potential parameters associated with poor survival. Prospective studies on prognostic indices across disease stages and treatment modalities are needed to predict and improve survival.

The Reactome Pathway Knowledgebase 2024.

The Reactome Knowledgebase (https://reactome.org), an Elixir and GCBR core biological data resource, provides manually curated molecular details of a broad range of normal and disease-related biological processes. Processes are annotated as an ordered network of molecular transformations in a single consistent data model. Reactome thus functions both as a digital archive of manually curated human biological processes and as a tool for discovering functional relationships in data such as gene expression profiles or somatic mutation catalogs from tumor cells. Here we review progress towards annotation of the entire human proteome, targeted annotation of disease-causing genetic variants of proteins and of small-molecule drugs in a pathway context, and towards supporting explicit annotation of cell- and tissue-specific pathways. Finally, we briefly discuss issues involved in making Reactome more fully interoperable with other related resources such as the Gene Ontology and maintaining the resulting community resource network.

Using the Reactome Database.

Pathway databases provide descriptions of the roles of proteins, nucleic acids, lipids, carbohydrates, and other molecular entities within their biological cellular contexts. Pathway-centric views of these roles may allow for the discovery of unexpected functional relationships in data such as gene expression profiles and somatic mutation catalogues from tumor cells. For this reason, there is a high demand for high-quality pathway databases and their associated tools. The Reactome project (a collaboration between the Ontario Institute for Cancer Research, New York University Langone Health, the European Bioinformatics Institute, and Oregon Health & Science University) is one such pathway database. Reactome collects detailed information on biological pathways and processes in humans from the primary literature. Reactome content is manually curated, expert-authored, and peer-reviewed and spans the gamut from simple intermediate metabolism to signaling pathways and complex cellular events. This information is supplemented with likely orthologous molecular reactions in mouse, rat, zebrafish, worm, and other model organisms. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Browsing a Reactome pathway Basic Protocol 2: Exploring Reactome annotations of disease and drugs Basic Protocol 3: Finding the pathways involving a gene or protein Alternate Protocol 1: Finding the pathways involving a gene or protein using UniProtKB (SwissProt), Ensembl, or Entrez gene identifier Alternate Protocol 2: Using advanced search Basic Protocol 4: Using the Reactome pathway analysis tool to identify statistically overrepresented pathways Basic Protocol 5: Using the Reactome pathway analysis tool to overlay expression data onto Reactome pathway diagrams Basic Protocol 6: Comparing inferred model organism and human pathways using the Species Comparison tool Basic Protocol 7: Comparing tissue-specific expression using the Tissue Distribution tool.

The reactome pathway knowledgebase 2022.

The Reactome Knowledgebase (https://reactome.org), an Elixir core resource, provides manually curated molecular details across a broad range of physiological and pathological biological processes in humans, including both hereditary and acquired disease processes. The processes are annotated as an ordered network of molecular transformations in a single consistent data model. Reactome thus functions both as a digital archive of manually curated human biological processes and as a tool for discovering functional relationships in data such as gene expression profiles or somatic mutation catalogs from tumor cells. Recent curation work has expanded our annotations of normal and disease-associated signaling processes and of the drugs that target them, in particular infections caused by the SARS-CoV-1 and SARS-CoV-2 coronaviruses and the host response to infection. New tools support better simultaneous analysis of high-throughput data from multiple sources and the placement of understudied ('dark') proteins from analyzed datasets in the context of Reactome's manually curated pathways.

Omalizumab-Induced Aspirin Tolerance in Nonsteroidal Anti-Inflammatory Drug-Exacerbated Respiratory Disease Patients Is Independent of Atopic Sensitization.

BACKGROUND: Nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (N-ERD) comprises the triad of chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and intolerance to inhibitors of the cyclooxygenase-1 enzyme. The impact of omalizumab on prevention of aspirin-induced hypersensitivity in N-ERD patients with and without atopic sensitization has not been thoroughly addressed. OBJECTIVE: To investigate the effect of omalizumab treatment on aspirin tolerance in atopic and nonatopic N-ERD patients. METHODS: This single-center, prospective trial evaluated overall omalizumab-induced aspirin tolerability in N-ERD patients by performing aspirin challenge testing before and after 6 months of anti-immunoglobulin E (IgE) therapy. The impact of omalizumab on CRSwNP asthma as well as serum and tissue biomarkers in patients with and without comorbid atopic sensitizations was further analyzed. RESULTS: Out of 33 patients included in the study, 56% developed complete aspirin tolerance and 18% tolerated higher dosages after 24 weeks. Polyp size and disease-specific symptoms (nasal polyp score [NPS] -1.9 ± 0.3, P < .001; Sino-Nasal Outcome Test [SNOT]-20 -16.7 ± 3.7, P < .001; Asthma Control Test [ACT] 3.2 ± 0.7, P < .001) improved in all patients irrespective of atopic sensitization. Effectiveness of omalizumab was accompanied by an increase in mean total serum IgE (307.8 ± 42 kU/L; P < .001) and a decrease in eosinophilic cationic protein (-10.6 ± 6.7 µg/L) and in relative eosinophilia (-2.5 ± 0.7%; P < .01). Whereas there was a significant reduction of tissue IgE (P < .05) in all patients after 4 weeks, the number of local eosinophils decreased only in atopic individuals (P < .05). CONCLUSIONS: Omalizumab induced complete aspirin tolerance in the majority of patients (56%) independent of atopic sensitization and demonstrated clinical efficacy in the treatment of CRSwNP and asthma. Inhibition of IgE can therefore be a promising treatment option in preventing NSAID hypersensitivity reactions in N-ERD patients.

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.

Spatiotemporal Analysis of B Cell- and Antibody Secreting Cell-Subsets in Human Melanoma Reveals Metastasis-, Tumor Stage-, and Age-Associated Dynamics.

Background: The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance. Methods: In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis. Results: In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma. Conclusion: These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

Persistence of mature dendritic cells, TH2A, and Tc2 cells characterize clinically resolved atopic dermatitis under IL-4Rα blockade.

Therapeutic options for autoimmune diseases typically consist of broad and targeted immunosuppressive agents. However, sustained clinical benefit is rarely achieved, as the disease phenotype usually returns after cessation of treatment. To better understand tissue-resident immune memory in human disease, we investigated patients with atopic dermatitis (AD) who underwent short-term or long-term treatment with the IL-4Rα blocker dupilumab. Using multi-omics profiling with single-cell RNA sequencing and multiplex proteomics, we found significant decreases in overall skin immune cell counts and normalization of transcriptomic dysregulation in keratinocytes consistent with clearance of disease. However, we identified specific immune cell populations that persisted for up to a year after clinical remission while being absent from healthy controls. These populations included LAMP3 + CCL22+ mature dendritic cells, CRTH2 + CD161 + T helper ("TH2A") cells, and CRTAM + cytotoxic T cells, which expressed high levels of CCL17 (dendritic cells) and IL13 (T cells). TH2A cells showed a characteristic cytokine receptor constellation with IL17RB, IL1RL1 (ST2), and CRLF2 expression, suggesting that these cells are key responders to the AD-typical epidermal alarmins IL-25, IL-33, and TSLP, respectively. We thus identified disease-linked immune cell populations in resolved AD indicative of a persisting disease memory, facilitating a rapid response system of epidermal-dermal cross-talk between keratinocytes, dendritic cells, and T cells. This observation may help to explain the disease recurrence upon termination of immunosuppressive treatments in AD, and it identifies potential disease memory-linked cell types that may be targeted to achieve a more sustained therapeutic response.

ReactomeGSA - Efficient Multi-Omics Comparative Pathway Analysis.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.

B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma.

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.

The PRIDE database and related tools and resources in 2019: improving support for quantification data.

The PRoteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) is the world's largest data repository of mass spectrometry-based proteomics data, and is one of the founding members of the global ProteomeXchange (PX) consortium. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2016. In the last 3 years, public data sharing through PRIDE (as part of PX) has definitely become the norm in the field. In parallel, data re-use of public proteomics data has increased enormously, with multiple applications. We first describe the new architecture of PRIDE Archive, the archival component of PRIDE. PRIDE Archive and the related data submission framework have been further developed to support the increase in submitted data volumes and additional data types. A new scalable and fault tolerant storage backend, Application Programming Interface and web interface have been implemented, as a part of an ongoing process. Additionally, we emphasize the improved support for quantitative proteomics data through the mzTab format. At last, we outline key statistics on the current data contents and volume of downloads, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas.

Digital image analysis improves precision of PD-L1 scoring in cutaneous melanoma.

AIMS: Immune checkpoint inhibitors have become a successful treatment in metastatic melanoma. The high response rates in a subset of patients suggest that a sensitive companion diagnostic test is required. The predictive value of programmed death ligand 1 (PD-L1) staining in melanoma has been questioned due to inconsistent correlation with clinical outcome. Whether this is due to predictive irrelevance of PD-L1 expression or inaccurate assessment techniques remains unclear. The aim of this study was to develop a standardised digital protocol for the assessment of PD-L1 staining in melanoma and to compare the output data and reproducibility to conventional assessment by expert pathologists. METHODS AND RESULTS: In two cohorts with a total of 69 cutaneous melanomas, a highly significant correlation was found between pathologist-based consensus reading and automated PD-L1 analysis (r = 0.97, P < 0.0001). Digital scoring captured the full diagnostic spectrum of PD-L1 expression at single cell resolution. An average of 150 472 melanoma cells (median 38 668 cells; range = 733-1 078 965) were scored per lesion. Machine learning was used to control for heterogeneity introduced by PD-L1-positive inflammatory cells in the tumour microenvironment. The PD-L1 image analysis protocol showed excellent reproducibility (r = 1.0, P < 0.0001) when carried out on independent workstations and reduced variability in PD-L1 scoring of human observers. When melanomas were grouped by PD-L1 expression status, we found a clear correlation of PD-L1 positivity with CD8-positive T cell infiltration, but not with tumour stage, metastasis or driver mutation status. CONCLUSION: Digital evaluation of PD-L1 reduces scoring variability and may facilitate patient stratification in clinical practice.

Tumor-associated B-cells induce tumor heterogeneity and therapy resistance.

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Greazy: Open-Source Software for Automated Phospholipid Tandem Mass Spectrometry Identification.

Lipid identification from data produced with high-throughput technologies is essential to the elucidation of the roles played by lipids in cellular function and disease. Software tools for identifying lipids from tandem mass (MS/MS) spectra have been developed, but they are often costly or lack the sophistication of their proteomics counterparts. We have developed Greazy, an open source tool for the automated identification of phospholipids from MS/MS spectra, that utilizes methods similar to those developed for proteomics. From user-supplied parameters, Greazy builds a phospholipid search space and associated theoretical MS/MS spectra. Experimental spectra are scored against search space lipids with similar precursor masses using a peak score based on the hypergeometric distribution and an intensity score utilizing the percentage of total ion intensity residing in matching peaks. The LipidLama component filters the results via mixture modeling and density estimation. We assess Greazy's performance against the NIST 2014 metabolomics library, observing high accuracy in a search of multiple lipid classes. We compare Greazy/LipidLama against the commercial lipid identification software LipidSearch and show that the two platforms differ considerably in the sets of identified spectra while showing good agreement on those spectra identified by both. Lastly, we demonstrate the utility of Greazy/LipidLama with different instruments. We searched data from replicates of alveolar type 2 epithelial cells obtained with an Orbitrap and from human serum replicates generated on a quadrupole-time-of-flight (Q-TOF). These findings substantiate the application of proteomics derived methods to the identification of lipids. The software is available from the ProteoWizard repository: http://tiny.cc/bumbershoot-vc12-bin64 .

Tumor-associated B cells in cutaneous primary melanoma and improved clinical outcome.

B cells often infiltrate the microenvironment of human tumors. B cells can both positively and negatively regulate antitumor immune responses. In several human cancers, higher numbers of CD20(+) TAB are associated with a favorable prognosis, whereas in human primary melanomas, this association is contentious. In this study, we determined the association of TAB numbers in cutaneous primary melanoma tissue samples and patients' overall survival. The CD20 immunohistochemistry on archival nonmetastasized and metastasized cutaneous primary melanoma tissues from 2 independent patient cohorts was performed. One cohort was used in class comparison for metastasis, the most important prognostic factor for overall survival, and the other cohort for a subsequent survival analysis. Survival association was further validated with RNA data from a third independent cohort. Whole tissue sections were read automatically via quantitative digital imaging and analysis. Survival data were analyzed by Cox proportional hazard modeling. We discovered that cutaneous primary melanomas without metastasis contain significantly more TAB than primary melanomas that had metastasized. At time of first diagnosis, a higher number of TAB is associated with a significantly better overall survival in patients with cutaneous primary melanomas of >1 mm Breslow depth. Also, higher CD20/CD19 tumor mRNA levels are correlated with a significantly better overall survival. Thus, our data support TAB numbers as a prognostic biomarker in cutaneous primary melanoma patients with a tumor of >1 mm Breslow depth. For a survey in larger studies, whole tissue section analysis seems to be key to accurate assessment of TAB numbers.