Increasing evidence suggests that circulating cell-free DNA (cfDNA) testing might allow for monitoring the response to anti-EGFR monoclonal antibodies in patients with metastatic colorectal carcinoma (mCRC). However, few data are available in treatment-naïve patients. We tested cfDNA samples obtained from mCRC patients enrolled in a phase III trial of the anti-EGFR monoclonal antibody cetuximab plus chemotherapy as first-line treatment. Analysis of serial plasma samples revealed a complex dynamic of RAS/BRAF mutations in response to treatment, with transitory peaks of these mutations that were not associated with resistance to therapy. Overall, our findings suggest that early appearance of RAS/BRAF mutations in the plasma of patients receiving first-line anti-EGFR agents in combination with chemotherapy should not be considered as marker of resistance.n = 37), at 8 weeks of treatment (n = 32), progressive disease  and 3 months after PD (n = 21). cfDNA testing was performed using the Idylla™ ctKRAS and ctNRAS-BRAF tests and the Oncomine Pan-Cancer Cell-Free Assay. Analysis of basal samples revealed RAS/BRAF mutations in 6/37 cases. A transient RAS positivity not associated with PD was observed at 8 weeks in five cases that showed no mutations at baseline and PD. The frequency of mutant cases increased at PD (33.3%) and decreased again at 3 months after PD (9.5%). The median progression-free survival (mPFS) of patients RAS/BRAF mutant at PD was 7.13 months versus 7.71 months in wild-type patients (p = 0.3892). These data confirm that the occurrence of RAS/BRAF mutations in mCRC patients receiving anti-EGFR agents is relatively frequent. However, the cfDNA dynamics of RAS mutations in patients treated with anti-EGFR agents plus polychemotherapy are complex and might not be directly associated with resistance to treatment.Analysis of plasma-derived cell-free DNA (cfDNA) might allow for the early identification of resistance in metastatic colorectal carcinoma (mCRC) patients receiving anti-EGFR monoclonal antibodies. We tested plasma samples from the Erbitux Metastatic Colorectal Cancer Strategy (ERMES) phase III trial of FOLFIRI+Cetuximab in first-line treatment of RAS/BRAF wild-type mCRC. Samples were collected at baseline ( The epidermal growth factor receptor (EGFR) signaling plays a relevant role in the pathogenesis and progression of colorectal carcinoma (CRC) ,2. In thIn addition to primary resistance mechanisms, the activity of anti-EGFR monoclonal antibodies in CRC is limited by the development of molecular alterations producing acquired resistance ,13,14,15Importantly, the mechanisms of acquired resistance to EGFR monoclonal antibodies in mCRC patients were discovered though testing the cell-free DNA (cfDNA) isolated from plasma. The analysis of cfDNA has several advantages over tissue testing, including the possibility to repeat the test over the time, thus monitoring the molecular evolution of the disease.Studies have shown that the appearance of RAS mutations in cfDNA anticipates clinical progression of the disease . These dThe availability of plasma samples from patients enrolled in the ERMES trial offers a unique opportunity to study the dynamics of RAS/BRAF mutations in the cfDNA under the pressure of both polychemotherapy and anti-EGFR antibodies, and to evaluate the prognostic and predictive role of cfDNA testing in RAS/BRAF wild-type patients receiving first-line anti-EGFR-based therapy. In particular, we evaluated whether early cfDNA testing might allow for identifying patients with RAS/BRAF mutations, leading to resistance to anti-EGFR agents, who thus might benefit from a different therapeutic strategy.n = 37), 8 weeks of treatment (n = 32), PD (n = 36) and at 3 months after PD (n = 21). Overall, 10 patients were randomized in arm A and 27 in arm B.The ERMES study is a phase III randomized trial of FOLFIRI+Cetuximab until progressive disease  compared to 8 cycles of FOLFIRI+Cetuximab followed by Cetuximab alone until PD (arm B) in first-line treatment of KRAS/NRAS/BRAF wild-type mCRC patients. Plasma samples were collected from 37 mCRC patients enrolled in ERMES at baseline  as assessed by local pathology laboratories.g using a refrigerated centrifuge and, without disturbing the bottom red blood cell layer, the supernatant from the top layer of the tube was transferred into the new collection tube. Another centrifugation was performed for 10 min at 3000× g in order to remove the platelets. The supernatant was transferred in criovials and stored at −80 °C. The plasma samples were shipped at the centralized laboratory in dry ice.The plasma samples were isolated from 10.0 mL of whole blood in EDTA Vacutainer tubes. After the collection, the blood samples were immediately processed. Cells were removed by centrifugation for 10 min at 1600× Plasma samples were analyzed using the fully automated Idylla™ ctKRAS and Idylla™ ctNRAS-BRAF mutation test . For each sample, 1 mL of plasma was loaded into the Idylla™ cartridge. The Idylla™ ctKRAS test covers 21 KRAS mutations in exons 2, 3 and 4; the ctNRAS-BRAF test covers 18 mutations in exons 2, 3 and 4 of NRAS gene and 5 mutations in BRAF codon 600.The circulating total nucleic acids (cTNA) were extracted from 4 mL of plasma samples using MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit  and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). An amount of 2–20 ng of cfTNA was used to prepare libraries. Targeted libraries were performed using the Oncomine Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), following the manufacturer’s recommendations. The Oncomine Pan-Cancer Cell-Free Assay assesses genetic alterations in 52 driver genes and includes the following: hotspot genes and short indels in AKT1, ALK, AR, ARAF, BRAF, CHEK2, CTNNB1, DDR2,EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, RAF1, RET, ROS1, SF3B1, SMAD4 and SMO; gene fusions in ALK, BRAF, ERG, ETV1, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK3, RET and ROS1; MET exon 14 skipping; copy number variations of CCND1, CCND2, CCND3, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, MET and MYC; and coverage of tumor suppressor genes APC, FBXW7, PTEN and TP53. The final concentration of each library was determined by Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific). Barcoded libraries were diluted to 100 pM, pooled in equal volume aliquots and then loaded on to the Ion Chef™ Instrument (Thermo Fisher Scientific) for emulsion PCR, enrichment and loading onto the Ion S5 540 chip. The sequencing runs were performed on the Ion S5 XL System (Thermo Fisher Scientific). The data were analyzed by Ion Torrent Suite Software v.5.12 and using Ion Reporter Software v5.14.Tissue samples were analyzed with the Oncomine Solid Tumor DNA kit (Thermo Fisher Scientific). The panel analyzes hotspot and targeted regions of the following 22 genes implicated in colon and lung cancers: ALK, EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, MET, DDR2, KRAS, PIK3CA, BRAF, AKT1, PTEN, NRAS, MAP2K1, STK11, NOTCH1, CTNNB1, SMAD4, FBXW7 and TP53. Libraries were prepared starting from 10 ng of genomic DNA (measured using the Qubit fluorometer in combination with the Qubit dsDNA HS Assay Kit) according to the manufacturer’s instructions. Then, 100 pM of each equalized library was multiplexed and clonally amplified on Ion sphere particles (ISPs) by emulsion PCR performed on the Ion One Touch 2 instrument with the Ion PGM template OT2 200 kit (Thermo Fisher Scientific). The template ISPs were enriched, loaded on an Ion 316 chip and sequenced on a PGM sequencer with the Ion PGM™ sequencing 200 kit v2 according to the manufacturer’s instructions. The data were analyzed by Ion Torrent Suite Software v.5.12 and using Ion Reporter Software v5.14.n = 37), at 8 weeks of treatment (n = 32), at progressive disease  and at 3 months after PD (n = 21). The analysis performed with the Idylla™ ctKRAS/NRAS/BRAF assay was successful for all but one sample at 3 months after PD for whom the KRAS test was invalid  enrolled in the ERMES trial using the Idylla platform. In particular, we studiedd available plasma samples at baseline . Cases 01-0019 and 29-001 were positive at baseline and at every available subsequent time point . Four caThe peculiar dynamics of these cases with a wave of transient KRAS mutations is shown in The fraction of KRAS/NRAS/BRAF cfDNA-positive cases significantly increased at PD, with 14 mutations detected in 12/36 patients (33.3%). In particular, the Idylla™ test identified six KRAS, three NRAS and five BRAF variants, with two cases carrying either a KRAS or NRAS mutation and a BRAF V600E variant . An addiWe had a significant dropout of cases at 3 months after PD. In fact, only 21 patients had available plasma samples at PD. Such dropout is probably due to the fact that patients with disease progression left the study to receive a second line of therapy in the context of clinical practice. In particular, all patients received a second-line regimen not containing an anti-EGFR agent.Only 2 of the 21 cases with available samples at 3 months after PD (9.5%) showed either a KRAS or a KRAS and NRAS variants . Such reWe next assessed the correlation between the presence of KRAS/NRAS/BRAF mutations in the cfDNA and the outcome of patients who received first-line chemotherapy plus cetuximab. The presence of KRAS/NRAS/BRAF mutations at baseline, at 8 weeks, at PD or at any of these points did not correlate with response to therapy (data not shown). However, the dynamics of RAS mutations did correlate with patients’ outcomes on treatment. We had paired 8-week samples available for six cases with RAS or BRAF mutations at baseline. In 4/6 cases, we observed a partial response to therapy that was associated with plasma samples becoming negative at week 8. The only baseline KRAS-positive patient with persistent KRAS mutation at week 8 did not respond to therapy but had a stabilization of the disease. The BRAF-positive case had a PD with a very short PFS of 1.38 months, while showing an increase of the BRAF MAF in the cfDNA .Interestingly, the four cases with transient RAS mutation increase at week 8 all had PR as the best response, while case 01-0017 with a RAS-positive sample at week 8 and BRAF positivity at PD had CR as the best response.p = 0.3892; HR 1.346, CI 95% 0.6844 to 2.647)  of patients with KRAS/NRAS/BRAF mutations at PD was 7.13 months versus 7.71 months in patients with wild-type plasma samples (o 2.647) . Similaro 2.647) .Testing plasma-derived cfDNA has several advantages over tissue testing, namely being able to better recapitulate the heterogeneity of advanced cancers and allowing for the monitoring of the molecular evolution of the disease . In partSeveral studies demonstrated a good concordance between tissue and cfDNA testing of RAS mutations in patients with mCRC ,31,32,33Although RAS/BRAF mutations are well-defined mechanisms of resistance to anti-EGFR agents ,41,42,43We also observed a transient increase in the levels of RAS mutations at week 8 in five patients who became RAS negative at PD. A transient increase of RAS-positive clones has been previously described in mCRC patients receiving anti-EGFR monoclonal antibodies, who still became RAS positive at PD . It mustThe rate of RAS/BRAF-positive cases at PD following treatment with an anti-EGFR monoclonal antibody ranged between 32% and 96% in previous studies, a significant difference that was most likely due to the limited number of patients and the different technologies used for testing ,22,23,24This study has several limitations due to the low number of patients included in our analysis. However, our preliminary findings suggest a complex dynamic of RAS/BRAF mutations in mCRC patients treated with first-line anti-EGFR monoclonal antibodies in combination with polychemotherapy. Additional studies are needed to clear whether cfDNA monitoring might guide therapeutic decisions in mCRC patients.


Background: To date, useful diagnostic applications of p16 IHC have been documented in gynecological pathology both for HPV-related and non-HPV-related lesions. In the present article, we reported our experience with the novel anti-p16 INK4a antibody (clone BC42), whose expression was tested across all different gynecologic neoplasms; we also compared it to the traditional E6H4 clone. Moreover, we discussed and explored all the diagnostic applications of p16 IHC in gynecologic pathology. Methods: Consultation cases covering a 5-year period (2016–2020) regarding gynecological neoplastic and non-neoplastic lesions in which immunohistochemistry for p16, clone E6H4 was originally performed, were retrospectively retrieved from the files of our institution. Immunohistochemical staining for p16ink4a (BC42)  and p16ink4a (E6H4)  was performed by using the Ventana automated immunostainer . The immunostaining pattern was defined as negative, focal/patchy, or diffuse. Results: A total of 196 cases, represented by 36 high-grade SIL/CIN3 of the uterine cervix, 30 cervical adenocarcinomas, 22 cervical squamous cell carcinoma, 70 endometrial carcinomas, 25 high grade serous ovarian carcinomas, 6 uterine adenomatoid tumors, and 10 uterine leiomyosarcomas were included in this study. Results showed concordant staining quality of both clones on all tested neoplastic tissues. Conclusions: The novel anti-p16 antibody (BC42 clone) appeared as an alternative to the current E6H4 for use in gynecological neoplasms, offering similar levels of positivity and equally reliable staining results. Normal vulvar squamous epithelium and squamous cell hyperplasia lack p16 immunostaining, while some authors reported a focal and heterogeneous expression in lichen sclerosus .Regarding vulvar intraepithelial neoplasia (VIN), two main pathogenetic types are recognized: (1) Classic VIN, accounting for approximately 90% of cases, (2) differentiated (simplex) VIN, accounting for the remaining 10%. While classic VIN is related to HPV infection, differentiated VIN is not linked to HPV and is commonly associated with lichen sclerosus . TherefoMoreover, the staining pattern of p16 is helpful in the distinction of the two main categories of classic VIN: Low-grade SIL (VIN 1) and High-grade SIL VIN 2/3). In fact, a continuous strong nuclear or nuclear and cytoplasmic staining of the basal cell layer, which extends upward involving at least one-third of the epithelial thickness (block staining), is highly suggestive of precancerous high-grade SIL (VIN 2/3). On the other hand, p16 staining in VIN1 is usually limited to the basal cell layer . In fact,7,10,11.Invasive vulvar neoplasms follow the same p16 staining pattern of their precursor lesions; in fact, HPV-related invasive squamous cell carcinomas are p16-positive, while non-HPV-related invasive tumors are mainly p16-negative ,5. An exp16 expression in the squamous cervical epithelium is encountered in dysplastic, HPV related lesions: Low grade squamous intraepithelial lesion  and high grade squamous intraepithelial lesion ; on the other hand, p16 is usually absent in normal squamous epithelium as well as in inflammatory conditions and in the metaplastic squamous epithelium ,13,14,15L-SIL lesions display a nuclear and cytoplasmic positivity for p16 limited to the lower third of squamous epithelium; however, different staining patterns have been reported according to HPV type of infection: Intermediate and high-risk HPV-related L-SIL usually display strong and continuous p16 expression while low risk HPV-related L-SIL exhibits a less specific weak or focal p16 staining ,14,15,16The most useful p16 application in cervical pathology is in the diagnosis of H-SIL. In fact, intense continuous p16 staining within the upper two thirds of the squamous epithelium is highly suggestive of the presence of a high-grade dysplastic lesion, although a small number of H-SILs can show no staining by p16 immunohistochemistry ,5,18. P1Endocervical glandular preneoplastic and neoplastic lesions related to HPV infection exhibit p16 positivity, which is not encountered in normal endocervical glands ,22. Diffuse p16 positivity is nearly always encountered in endocervical adenocarcinoma in situ (AIS), (given its association with high-risk HPV) and is of great diagnostic utility in the differential diagnosis with benign glandular lesions such as microglandular hyperplasia (MGH), tuboendometrial metaplasia (TEM), and endometriosis ,22,23. MAnother diagnostic field in which p16 IHC plays a key role is in the distinction between endocervical and endometrial adenocarcinoma since the precise site of origin  has important prognostic and therapeutic implications ,27,28,29However, to better clarify the endometrial versus endocervical origin of neoplasms, p16 must be integrated with broader immunohistochemical panels, which include estrogen and progesterone receptors as well as vimentin, monoclonal carcinoembryonic antigen, and mismatch repair proteins ,29,30,31Finally, p16 has been recently proposed as a diagnostic marker to differentiate atypical polypoid adenomyoma (APA) from myoinvasive endometrioid carcinoma; indeed, p16 often shows diffuse positivity in the fibromyomatous stroma of APA and negativity or focal positivity in the stroma of myoinvasive carcinoma ,33,34.Despite the fact that p16 has limited diagnostic utility in mesenchymal tumors, interesting data have been published regarding its expression in uterine smooth muscle tumors. Several studies reported significant differences in p16 staining intensity and frequency between leiomyomas, STUMPs, and leiomyosarcomas. In fact, diffuse and strong staining for p16 is usually encountered in leiomyosarcomas and less frequently in STUMPS, while most leiomyomas are reported to be p16 negative ,35,36,37Uterine tumors resembling ovarian sex cord tumors (UTROSCT) represent rare mesenchymal tumors with sex cord differentiation where sporadic reports have highlighted p16 positive staining ,39. Finap16 IHC has limited diagnostic applications in ovarian neoplasms. In general, p16 is negative or weekly positive in most ovarian tumors, including benign and borderline serous tumors, endometrioid, mucinous, clear cell tumors, as well as sex cord and germ cell neoplasms ,42,43,44The only primitive ovarian tumor exhibiting diffuse and strong p16 expression is high-grade serous ovarian carcinoma. This finding contrasts with the low and weak staining encountered in low grade, borderline, and serous neoplasms ,43,44,45We have compared the new anti-p16 INK4a antibody  to clone E6H4, in terms of staining results on various neoplastic tissues of the female genital tract in order to assess its diagnostic reliability in daily diagnostic practice.All consultation cases covering a 5-year period (2016–2020) regarding gynecological neoplastic and non-neoplastic lesions in which immunohistochemistry for p16, clone E6H4, was originally performed, were retrospectively retrieved from the files of our institution.Pathology reports, hematoxylin-and-eosin (H&E), and immunohistochemical stained slides were reviewed by 2 expert pathologists , in order to confirm the initial histological diagnoses. Sections (~4 μm) of formalin-fixed paraffin-embedded tissues were deparaffinized, rehydrated through a series of alcohol/water solutions, followed by blocking of endogenous peroxidases with a 3% hydrogen peroxide solution. Tissues were subjected to heat-induced antigen retrieval using a modified citrate buffer in a pressure cooker  and were heated to 110 °C for 15 min. Both p16 antibodies  were applied to target tissues for 60 min. Detection of the p16 antibody was accomplished using a MACH 4 Universal HRP-Polymer detection system . In a final detection step, 3,3′-diaminobenzidine (DAB) was applied for visualization. Slides were briefly counterstained in a modified Mayer’s hematoxylin. Immunohistochemical staining was performed by using the Ventana automated immunostainer . Diaminobenzidine was used as the chromogen, and sections were counterstained in Harris’ hematoxylin. Positive controls were included in each immunostaining run. These comprised a cervical squamous carcinoma with diffuse, strong positivity with p16. p16 immunostaining pattern was defined as negative, focal/patchy, or diffuse. A focal pattern consisted of patchy and discontinuous staining with mainly cytoplasmic localization and minor nuclear staining. A diffuse pattern was defined as diffuse and strong nuclear and cytoplasmic staining.A total of 196 cases, represented by 36 high-grade SIL/CIN2-3 of the uterine cervix, 30 cervical adenocarcinomas, 22 cervical squamous cell carcinoma, 70 endometrial carcinomas (40 endometrioid and 30 serous), 25 high grade serous ovarian carcinomas, 6 uterine adenomatoid tumors, and 10 uterine leiomyosarcomas were included in this study.Results showed concordant staining quality of both clones on all neoplastic tissues tested. All tested cases and comparative immunohistochemical results are reported in Strong and diffuse staining pattern with both BC42 and E6H4 clones was observed in 35/36 cervical high-grade SIL cases ; in detaStrong and diffuse p16 staining was also encountered in all usual-type endocervical adenocarcinomas (30 cases) and cervical squamous cell carcinomas (22 cases). Regarding p16 expression in endometrial tumors, focal/patchy pattern of p16 staining, was detected with both clones in 32/40 (80%) tested in endometrial endometrioid carcinomas ; the remOvarian high-grade serous carcinomas also showed identical staining results with both clones. In detail, strong and diffuse p16 positivity with BC42 and E6H4 clones was observed in 21/25 and 20/25 cases, respectively, with the remaining cases showing a “patchy” pattern of staining .In recent years, p16 has been extensively investigated as a diagnostic aid in various scenarios in gynecologic pathology. Like with all markers, in each of these scenarios, p16 is neither 100% specific nor 100% sensitive for a given lesion. In the gyneco-pathological scenario, there are several areas where p16 is of undoubted value, usually in combination with other markers. These include: (i) The distinction of uVIN from dVIN in vulvar site; (ii) the identification of small high-grade CIN lesions, in particular at cauterized cervical resection margins; (iii) the separation of high grade CIN from benign mimics; (iv) the distinction between low grade and high grade CIN; (v) the distinction of cervical AIS from benign mimics; (vi) the identification of cervical small cell carcinoma; (vii) its potential prognostic role as predictor of HSIL outcome; (viii) the distinction of an endometrial and an endocervical adenocarcinoma; (ix) the differential diagnosis between uterine endometrioid adenocarcinomas and serous carcinoma (in particular in presence of glandular pattern); (x) the confirmation of a metastatic cervical adenocarcinoma in the ovary.It is likely that as the full range of p16 immunoreactivity is further investigated, additional useful applications will be discovered. Specific areas worthy of future investigation include the value of p16 in the categorization of problematic uterine smooth muscle neoplasms, the differential expression of p16 in low-grade and high-grade ovarian serous carcinomas, and the role of p16 as a diagnostic marker of APA. Different tested monoclonal clones are commercially available, but data comparing protocols of use are lacking ,49. In rRecently, a novel anti-p16 antibody has emerged as an alternative to the current E6H4 for use in histopathological diagnosis. It has been obtained by immunizing Balb/C mice with a recombinant human p16 protein . The Anti-p16 INK4a antibody [clone BC42] has been already tested on various normal (30 types) and neoplastic tissues (12 types), revealing equivalent exceptional staining quality, high levels of sensitivity and specificity compared to the current used clone E6H4, in particular in: Cervical adenocarcinoma (22 cases); cervical intraepithelial neoplasia (CIN) (24 cases); cervical squamous carcinoma (16 cases); head and neck cancer (12 cases); endometrial carcinoma (48 cases) and ovarian cancer (12 cases) .Our study represents the first one testing staining quality and applicability of the new clone BC42 on a larger number of gynecological cases (196 cases), also including uterine leiomyosarcomas and adenomatoid tumors. Results showed high staining quality on all neoplastic tissues tested, similarly to the current used clone E6H4.In particular, a strong and diffuse staining pattern with both BC42 and E6H4 clones was observed in 97.22% of H-SIL, in 100% of usual type cervical adenocarcinomas, cervical squamous cell carcinomas, and uterine adenomatoid tumors, and, respectively, in 84% vs. 80% ovarian high-grade serous carcinomas.-Pre-analytical variables: Specimen collection and handling, tissue fixation , and protocol of processing-Analytical variables: Choice of immunohistochemistry protocol, reagent variability, antigen retrieval technique, and technician training/expertise-Post-analytical variables: Evaluation of positive/negative controls, morphological correlations, diagnostic and prognostic significance, correlation with other data, interpretation, and reporting of results, and experience/expertise of the pathologistWe must be aware that p16 staining, as well as any type of immunohistochemical staining, can be affected by:The goal consists in ensuring reproducibility, obtaining high quality stained sections with minimal inter-observer variability in diagnostic report and in promoting inter-laboratory standardization, which are all synonyms of quality assurance, defined by the College of American Pathologists (CAP) as a ‘‘process of assuring that all pathology services involved in the delivery of patient care have been accomplished in a manner appropriate to maintain excellence’’ . We retaP16 IHC is of crucial diagnostic importance in several fields of gynecopathology, and its area of application is further increasing.The new anti-p16 antibody (BC42 clone) is emerging as an alternative to the current E6H4 for use in cancer pathological diagnosis, offering similar levels of positivity and equally reliable staining results.We have to keep in mind that to achieve the excellence in a pathological diagnosis, concerted actions with regard to laboratory standardization, quality assurance, coupled with progressive automation of all procedures and technical/medical skills are needed for the proper utility of p16 IHC in daily practice.


Diffuse large B-cell lymphoma (DLBCL) is the commonest form of lymphoid malignancy, with a prevalence of about 40% worldwide. Its classification encompasses a common form, also termed as “not otherwise specified” (NOS), and a series of variants, which are rare and at least in part related to viral agents. Over the last two decades, DLBCL-NOS, which accounts for more than 80% of the neoplasms included in the DLBCL chapter, has been the object of an increasing number of molecular studies which have led to the identification of prognostic/predictive factors that are increasingly entering daily practice. In this review, the main achievements obtained by gene expression profiling (with respect to both neoplastic cells and the microenvironment) and next-generation sequencing will be discussed and compared. Only the amalgamation of molecular attributes will lead to the achievement of the long-term goal of using tailored therapies and possibly chemotherapy-free protocols capable of curing most  patients with minimal or no toxic effects. Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoid malignancy, with a prevalence of about 40% worldwide . It consIn the Revised Fourth Edition of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, DLBCL is subdivided into morphologic variants, molecular subtypes, and distinct disease entities  1]. Nev. Nev1]. Based on a recent survey of 3550 DLBCL patients who mostly underwent R-CHOP with curative intent, the 5-year overall survival and cumulative incidence of relapsed/refractory disease corresponds to 65.3% and 23.1% of cases, respectively [In recent years, DLBCL-NOS has been the object of the extensive application of high-throughput technologies, which has led to the identification of prognostic/predictive factors that are increasingly entering daily practice.MYC, BCL2, and/or BCL6 (HGBCL-D/TH) by FISH, as underlined by Sehn and Salles in their review on DLBCL published in the New England Journal of Medicine on 4 March 2021 [Although DLBCL-NOS is the main focus of this review, the borders between DLBCL-NOS and high-grade B-cell lymphoma (HGBCL)  will alsrch 2021   Alizadeh and coworkers first reported that DLBCLs could be divided into two main subtypes with a gene signature related to the germinal center B-cell (GCB) and activated B-lymphocytes from the peripheral blood (ABC), respectively . Such a The main limitation of conventional GEP was the need for fresh or frozen (FF) samples, which were available for a small minority of patients followed up at reference centers. Therefore, many attempts were made to find surrogates for GEP through the search for immunohistochemical markers ,16,17,18In 2014, a new approach was proposed based on targeted digital GEPFF and was successfully applied to mRNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples (Lymph2Cx) . In partThe COO determination provided less significant prognostic information when applied to cases enrolled in some trials ,27,28. TThe main limitation of targeted GEP applied to routine biopsies is the need for platforms which are not available in all pathology laboratories, unlike immunohistochemistry. This problem, as well as the test costs and need for basic bioinformatic skills, can be overcome by a hub-and-spoke organization, which is also required for the application of the array of molecular techniques at the basis of precision medicine (see below).MYC, BCL2, and/or BCL6 [FISH analyses have shown that B-cell lymphomas, regarded as DLBCLs-NOS based on morphology and phenotype, could carry double or triple rearrangements of /or BCL6 . These c/or BCL6 ,30. Acco/or BCL6 . However/or BCL6 . Most im/or BCL6 , these c/or BCL6 ,32.MYC/BCL2 DH and non-DH GCB-DLCBLs. With their respective signatures, these investigators were, as expected, able to identify most DH lymphomas, as well as many non-DH lymphomas which were actually found in about half of the identified patients and showed a poorer response to standard chemoimmunotherapy. These findings suggest that many of the patients harbored genetic or even epigenetic alterations that produced similar gene expression changes in the tumor cells, as recognized by the respective signatures. This does not come as a surprise. In fact, activation of an oncogene or oncogenic pathway can be produced by multiple mechanisms besides MYC/BCL2 DH, for example MYC upregulation through translocation and gene amplification. Apart from structural alterations, MYC expression or activity can be enhanced through transcriptional and posttranscriptional events.In 2018, investigators from two groups used GEP signatures to identify high-risk patients with DLBCL in FFPE series. Sha et al.  used a BMYC, BCL-2, and NFKBIA . The high-risk (MBN Sig-high) subgroup, characterized by higher expression levels of MYC and BCL-2 and a lower expression of NFKBIA, could be used to identify a significant fraction of ABC DLBCLs and the vast majority of double-hit cases, allowing for further risk stratification within the GCB/U subset. These results were validated in three independent series including Sha’s cohort based on the REMoDL-B trial [MYC, BCL-2, and NFKBIA can refine the prognostic stratification in DLBCL.Derenzini et al.  used a tignaling ), in 186-B trial ,36, a ph-B trial , an explFinally, Mottok et al.  developeBy means of a gene profiling analysis of nearly 500 FF DLBCL samples, in 2008 Lenz et al. first demonstrated that the expression of peculiar gene sets, namely “Stromal-1” and “Stromal-2” signatures, respectively correlated with good- and poor-outcome subgroups of R-CHOP-treated patients independently of COO . AlthougTo overcome this limitation, in 2018 Ciavarella and co-workers  generatep-value = 0.001), PFS , and OS . Multivariate analyses adjusted for the IPI factors showed the signature was independent of COO, MYC rearrangements, and double-expressor (DE) status. LAMIS-high and simultaneous DE status characterized a patient subgroup with dismal prognosis and greater probability of early relapse.In a parallel work, Staiger et al.  proposedBeyond the prognostic value of TME, only a few studies have provided comprehensive biological insights into the putative link between B-cell genomics and functional patterns of immune and stromal components while suggesting new rationales for future therapeutic approaches.Tripodo et al.  reportedEZH2 mutations, lower T cell infiltration, and poorer outcome [Intriguing research integrating transcriptomic, genetic, and immunophenotypic data of 347 DLBCLs demonstrated that MHC loss, particularly in GC-derived tumors originating from the centroblast-rich DZ, is associated with a strong enrichment of Finally, very recent work by Kotlov et al.  providedBy all means, in-depth TME analysis still represents an approach that can significantly improve the prognostication of DLBCL and even further tune the identification of different risk groups within the same COO category, predicting the response to targeted therapies.Over the last few years, several proposals for a genetic classification of DLBCL have been published. Hereunder, the main contributions will be summarized and discussed based on the technical approach used.EZH2, SGK1, GNA13, SOCS1, STAT6, and TNFRSF14, which were mutated in GCB tumors, and ETV6, MYD88, PIM1 and TBL1XR1, which were mutated in ABC tumors. Interestingly, MLL2 mutations were associated with those of MYC, while TP53 mutations occurred in a mutually exclusive fashion with KLHL6. CRISPR screening revealed that knockout of EBF1, IRF4, CARD11, MYD88, and IKBKB was selectively lethal in ABC DLBCL cell lines, as was knockout of ZBTB7A, XPO1, TGFBR2, and PTPN6 in the GCB lines. On prognostic grounds, MYC mutations were strongly associated with poorer survival, as were mutations in CD79B and ZFAT. Mutations in NF1 and SGK1 were associated with more favorable survival. Furthermore, in ABC DLBCLs, genetic alterations in KLHL14, BTG1, PAX5, and CDKN2A were associated with significantly poorer survival, while those in CREBBP were associated with favorable outcomes. In the GCB-DLBCL group, genetic alterations in NFKBIA and NCOR1 were associated with poorer prognosis, while alterations in EZH2, MYD88, and ARID5B were all associated with a significantly better prognosis. The authors developed a multivariate supervised learning approach for defining the association of survival with combinations of genetic markers (150 genetic driver genes) and gene expression markers . This led to the proposal of a three-subgroup molecular risk model that was found to outperform all existing predictors . However, the recent application of this model to 499 DLBCLs by Bolen et al. [In 2017, Reddy and coworkers  reportedTwo studies published in 2018 proposed a molecular subclassification of DLBCLs that had potential prognostic and therapeutic implications ,52. TheyChapuy et al.  describeBCL6, and mutations of genes involved in the NOTCH2 and NF-kB pathways . Besides the multiple genetic lesions of genes involved in immune escape , these C1 cases carried MYD88 mutations which were non-L265P, unlike what was observed in the cases included in C5. Notably, C1 cases had a rather favorable course and revealed potential therapeutic targets related to NOTCH2 and BCL6 signaling and immune evasion mechanisms. C5 tumors, which behaved more aggressively than the C1 ones, showed mutations of L265PMYD88, CD79B, PIM1, TBL1XR1, GRHPR, and BTG1, SV of 18q, and activation of the NF-kB pathway. In addition, they carried ongoing mutations, being at least in part under the effect of AID. Potential targets for C5 cases corresponded to BCR/TLR signaling and BCL2.Most cases included in clusters (Cs) 1 and 5 were classified as ABC. However, they showed important differences on molecular and prognostic grounds. C1 cases were thought to derive from marginal-zone B-cells, as they showed a stable mutational pattern, structural variants (SVs) of BCL2 mutations with concordant SVs. They also exhibited frequent mutations in chromatin modifiers, KMT2D, CREBBP, and EZH2, and increased transcriptional abundance of EZH2 targets by gene set enrichment analisys (GSEA). These tumors also had alterations in the B-cell transcription factors MEF2B and IRF8, and indirect modifiers of BCR and PI3K signaling (TNFSF14(HVEM), HCNV1, and GNA13). In addition, C3 tumors had two alternative mechanisms of inactivating PTEN: focal 10q23.31/PTEN loss and predominantly truncating PTEN mutations, events that play a role in the process of lymphomagenesis. C4 DLBCLs were characterized by mutations in four linker and four core histone genes, multiple immune evasion molecules , BCR/PI3K signaling intermediates , NF-kB modifiers , and RAS/JAK/STAT pathway members (BRAF and STAT3). Comparison of the C3 and C4 genetic signatures further revealed that these GCB-DLBCLs utilized distinct mechanisms to perturb common pathways such as PI3K signaling. In contrast to C3 DLBCLs, C4 tumors rarely exhibited PTEN alterations but harbored more frequent RHOA mutations. In addition, C4 DLBCLs rarely exhibited BCL2 alterations and had higher mutational density. The distinct genetic features of C3 and C4 GCB-DLBCLs led Chapuy et al. to suggest specific targeted therapies including inhibition of BCL2, PI3K, and the epigenetic modifiers EZH2 and CREBBP in C3 GCB tumors, and JAK/STAT and BRAF/MEK1 blockade in C4 GCB-DLBCLs. Last but not least, C3 cases had a far worse prognosis.Cs 3 and 4 were significantly enriched in GCB cases but were characterized by different genetic lesions and responses to chemoimmunotherapy. The majority of DLBCLs in C3 harbored TP53 by mutations and 17p copy loss. In addition, they often exhibited copy loss of 9p21.13/CDKN2A and 13q14.2/RB1, perturbing chromosomal stability and cell cycle. C2 tumors also had significantly more driver somatic copy number alterations (SCNAs) and a higher proportion of genome doubling events. This cluster included both GCB- and ABC-DLBCLs, as did prior DLBCL cohorts with TP53 mutations in targeted analyses [13q31.31/miR-17-92 copy gain and 1q42.12 copy loss, were also more common in these DLBCLs, which were characterized by a rather unfavorable prognosis.C2 DLBCLs harbored frequent biallelic inactivation of A further cluster, termed 0, was also detected, which apparently lacked significant genetic alterations. However, as the C0 group consisted almost exclusively of T-cell rich/histiocyte-rich B-cell lymphomas, the obtained results might have been largely influenced by the small number of neoplastic cells.BCL2 and MYC alterations. Tumors with cooccurring BCL2 and MYC SVs were significantly more frequent in C3 DLBCLs.The authors further evaluated Importantly, the coordinate genetic signatures reported by Chapuy et al. predicted outcomes independent of IPI which could suggest new combination treatment strategies and, more broadly, provide a roadmap for actionable DLBCL classification .L265PMYD88 and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Interestingly, Schmitz and co-workers enriched their series with unclassified DLBCLs. The latter turned out to frequently carry mutations affecting SPEN and NOTH2 as well as BCL6 fusions. ABC cases were enriched in L265PMYD88 and CD79B or NOTCH1 mutations, with the two conditions being mutually exclusive. GCB tumors showed the co-occurrence of EZH2 mutations and BCL2 translocations. The MCD and N1 subtypes were dominated by ABC cases, while EZB included mostly GCB tumors, and BN2 had contributions from all GEP subgroups. Overall, about 45% of the samples were classified into the genetically pure subtypes of DLBCL.By their integrated approach, Schmitz et al.  identifiMYD88L265P or CD79B aberrations (mutation or amplification), with 42% bearing both abnormalities. The MCD subtype showed a frequent gain or amplification of SPIB, encoding a transcription factor that, with IRF4, defines the ABC phenotype and promotes plasmacytic differentiation. Known tumor suppressors in MCD include CDKN2A, ETV6, BTG1, and BTG2, and putative tumor suppressors include TOX, SETD1B, FOXC1, TBL1XR1, and KLHL14. The tumor suppressor TP53 was mutated significantly less often in MCD as compared to other subtypes. Immune editing appeared prominent in MCD genomes, with 76% acquiring a mutation or deletion of HLA-A, HLA-B, or HLA-C and 30% acquiring truncating mutations targeting CD58.The MCD subtype displayed 82% of cases carrying NOTCH2 mutation or amplification, SPEN mutation, or mutation in DTX1, a NOTCH target gene. BCL6 fusion, the other BN2 hallmark, occurred in 73% of cases. BCL6 fusions were enriched in cases with NOTCH2, SPEN, or DTX1 lesions to a significantly greater extent in BN2 than in non-BN2 cases. Genetic aberrations (mutations or amplifications) targeting regulators of the NF-kB pathway were a prominent feature of BN2. These more often affected TNFAIP3, PRKCB, and BCL10. Other likely gain-of-function events included mutations targeting cyclin D3 and CXCR5, whereas inactivating lesions targeting the immune regulator CD70 suggested immune escape.BN2 was dominated by NOTCH pathway aberrations, with 73% acquiring a NOTCH1 mutations and aberrations targeting transcriptional regulators of B-cell differentiation , which may contribute to its plasmacytic phenotype. TNFAIP3 mutations in N1 could reinforce this phenotype by fostering NF-KB-induced IRF4 expression.N1 was characterized by BCL2 translocation, EZH2 mutation, and REL amplification, as well as inactivation of the tumor suppressors TNFRSF14, CREBBP, EP300, and KMT2D. The germinal-center homing pathway involving S1PR2 and GNA1314 was disrupted in 38% of EZB cases. JAK-STAT signaling was promoted in about half cases by a STAT6 mutation or amplification or by a mutation or deletion targeting SOCS1. PI3K target of rapamycin signaling turned out to be activated in 23% of cases by MTOR mutations or the amplification of MIR17HG. Immune editing was of interest in EZB genomes since 39% acquired lesions in the major histocompatibility complex class II pathway genes CIITA and HLA-DMA.EZB was enriched for most of the genetic events previously ascribed to GCB-DLBCL, including CD79B and MYD88L265P mutations.The four subtypes differed significantly in PFS and OS, with the BN2 and EZB subtypes having much more favorable outcomes than the MCD and N1 subtypes. The predicted 5-year OS rates for the MCD, N1, BN2, and EZB subtypes were 26%, 36%, 65%, and 68%, respectively. Within ABC DLBCL, patients with MCD had significantly inferior survival as compared with those with BN2, and patients with either MCD or N1 had significantly inferior survival as compared with patients with ABC tumors that were not genetically classified. Within GCB-DLBCL, there was a trend toward inferior OS among patients with EZB as compared with those with other GCB tumors. The COO subgroups and genetic subtypes independently contributed to survival in a multivariate analysis. Conversely, the IPI score did not vary significantly among the genetic subtypes, but the latter significantly added to IPI. A trend toward increased extranodal involvement  was a feature of MCD, which reflected the frequent BCL2 translocations than in other EZB tumors. MCD tumors also had high BCL2 mRNA expression as compared with other cases, a finding due to mechanisms other than translocation or amplification.On therapeutic grounds, constitutive BCR signaling activation was most frequent in MCD and least frequent in EZB, but genetic alterations involving the BCR cascade occurred in all genetics subtypes, suggesting that constitutive BCR signaling is a pervasive aspect of DLBCL pathogenesis. BN2 was notably enriched for BCR–NF-kB and IKK regulator aberrations. In addition to NF-kB, survival of DLBCL cells turned out to be promoted by antiapoptotic BCL2 family members, which were targeted by genomic amplification or translocation in 17.4% of cases. As expected, BCL2 mRNA levels were significantly higher in EZB tumors with BCL2, NOTCH2, and MYD88 recapitulated the above-mentioned studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. The results obtained by Lacy et al. are summarized in Lacy et al.  applied TMEM30A loss-of-function mutations which were associated with a favorable outcome and were uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, accounting for the improved treatment outcome. Furthermore, they found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, Ennishi et al. showed that TMEM30A loss-of-function increased B-cell signaling following antigen stimulation—a mechanism conferring selective advantage during B-cell lymphoma development. These findings suggested intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.Ennishi et al.  performeBCL2/BCL6 rearrangement data. Schmitz’s cohort was used as training set, while those from Chapuy et al. and Ennishi et al. were used for validation. Wright et al. developed a model that is summarized in Finally, Wright et al.  developeThe paper of Lacy et al.  was accoThe heterogeneity of genetic lesions is much greater than was thought until a couple of years ago. This has been highlighted by liquid biopsy (LB) ,60,61. BThe continuous development of sequencing and bioinformatic techniques will allow us to achieve the long searched-for goal of using customized therapies based on the molecular characteristics of each individual tumor. Some approaches do appear to be more easily and cheaply applicable to daily life. Nevertheless, the more comprehensive the bioinformatic approach is, the higher the likelihood of overcoming today’s standard chemoimmunotherapy and designing chemotherapy-free protocols capable of curing most  patients, with minimal or no toxic effects.


However, the destruction of the ozone layer in the past few decades has increased the risk of skin aging and wrinkling caused by excessive exposure to ultraviolet (UV) radiation, which may also promote the risk of skin cancer development. The promotion of public health recommendations to avoid sunlight exposure would reduce the risk of skin cancer, but it would also enhance the risk of vitamin D3 insufficiency/deficiency, which may cause disease development and progression. In addition, the ongoing global COVID-19 pandemic may further reduce sunlight exposure due to stay-at-home policies, resulting in difficulty in active and healthy aging. In this review article, we performed a literature search in PubMed and provided an overview of basic and clinical data regarding the impact of sunlight exposure and vitamin D3 on public health. We also discuss the potential mechanisms and clinical value of phototherapy with a full-spectrum light  as an alternative to sunlight exposure, which may contribute to combating COVID-19 and promoting active and healthy aging in current aged/superaged societies.Most humans depend on sunlight exposure to satisfy their requirements for vitamin D Notes of Nursing: What it is and What it is Not, Florence Nightingale (1820–1910), the founder of modern nursing, also mentioned that light is one of five essential points for the health of houses, in addition to pure air, pure water, drainage and cleanliness  (n = 518)”, “sun exposure and public health [found by citation matching] (n = 8)”, “vitamin D biosynthesis and aging (ageing) [Filter applied: Review] (n = 551)”, “sunlight and COVID-19 (n = 301)”, “COVID-19 and UV and latitude (n = 6)”, “UV radiation and COVID-19 (n = 221)”, “phototherapy and COVID-19 (n = 111)”, “founder of phototherapy and dermatology (n = 9)”, “phototherapy and psychiatry (Filter applied: Review) (n = 162)”, “phototherapy and sleep disorder (n = 677)”, “phototherapy and Alzheimer’s disease and dementia (Filter applied: Review) (n = 38)”, “ COVID-19 and risk factors and obesity and diabetes (n = 645)”, “COVID-19 and vitamin D deficiency (n = 369)”, “COVID-19 and angiotensin and ACE2 (n = 4708)”, “COVID-19 and angiotensin and ACE2 and vitamin D (n = 51)”, “COVID-19 and adipokines (n = 38)”, “COVID-19 and alarmins (n = 36)”, “angiotensin and alarmins (n = 14)”, “COVID-19 and oxidative stress and antioxidants (n = 221)”, “COVID-19 and gut dysbiosis (n = 124)”, “COVID-19 and fecal  microbiota transplantation (n = 37)”, “sunlight and gut dysbiosis (n = 4)”, “visible light and opsins (Filter applied: Review) (n = 556)”, “visible light and opsins and lung (n = 14)”, “visible light and opsins and adipose tissues (n = 6)”, or “opsins and cancer (n = 176)”. We then integrate our previous and present data and discuss the potential mechanisms and clinical value of phototherapy with full-spectrum light  as an alternative to sunlight exposure for contributing to active and healthy aging, notably in the era of COVID-19.In this review article, we summarize the impact of sunlight exposure and vitamin D3 insufficiency/deficiency [The first published application of phototherapy was conducted by Niels Ryberg Finsen (1860–1904), who developed a carbon arc lamp for the treatment of skin tuberculosis (lupus vulgaris), and his clinical contribution in dermatology was awarded the Nobel Prize in Physiology or Medicine in 1903 . The mecficiency . Well-deficiency ,32,33.3  in nonalcoholic steatohepatitis (NASH), and phototherapy with full-spectrum light  12 h/day for 6 weeks could elevate vitamin D3 levels, resulting in the amelioration of NASH progression in rats [3 and lipid transfer/metabolic proteins, such as apolipoprotein E (apoE) and adiponectin, by phototherapy with full-spectrum light [3 in the skin [3 [3 metabolism partly through the induction of CYP27B1 for active vitamin D3 generation. In support of our observations, a recent review article mentioned the fundamental role of red and near-infrared light in improved health status induced by sunlight exposure [3 biosynthesis should be further explored using full-spectrum light with UV cut-off filters. In addition to the clinical impact of phototherapy in terms of the elevated vitamin D3 in NASH patients [3 and healthy microbiota composition. Interestingly, accumulating evidence suggests the impact of microbiota on circadian rhythms and human health [3 insufficiency/deficiency, circadian rhythm disruption and gut dysbiosis.In our previous study, we demonstrated lower expression of vitamin D in rats . In thisum light . Howeverum light ) are faithe skin , and a lthe skin . Interes skin [3  in humanexposure . The imppatients , we and patients  peritonipatients , and foopatients . Althougn health . These e3 insufficiency/deficiency and COVID-19, suggesting the therapeutic potential of vitamin D3 supplementation for the prevention and treatment of SARS-CoV-2 infection [3 supplementation could modulate unbalanced RAS and ACE2 downregulation, resulting in induction of the ACE2/Ang-(1–7)/Mas receptor (MasR) axis for protection against ALI/ARDS [There are many risk factors associated with the severity and mortality of COVID-19, including aging, overweight-obesity, hypertension, diabetes, and lung, cardiovascular, and kidney diseases . In addinfection ,46,47,48nfection ,50,51,52nfection , blockadnfection ,55 , suggesting the involvement of RAS in obesity, gut dysbiosis, COVID-19, and ALI/ARDS [In addition, adipose tissues are one of the largest endocrine organs and a source of proinflammatory mediators and adipokines, which may create chronic low-grade inflammatory preconditioning ,59. TherALI/ARDS .Recent studies have pointed to the significance of early detection of danger signals for the classification of COVID-19 patients as being at high risk of mortality. Alarmins are possible danger signals associated with COVID-19 and comorbidities , and S103 and the reduction in risk factors. However, a recent randomized clinical trial with a single oral dose of vitamin D3  did not reduce the hospital length of stay in patients with moderate to severe COVID-19 [3 (5000 IU) for two weeks reduced the time to recovery for symptoms such as cough and gustatory sensory loss among mild to moderate COVID-19 patients with suboptimal vitamin D3 status [3 supplementation who achieved serum 25(OH)D3 levels ≥30 ng/mL had a lower risk of SARS-CoV-2 infection, severity, and mortality than unsupplemented controls [3 supplementation may be effective for the prevention of SARS-CoV-2 infection and the treatment of symptoms, but deciding the dose and duration of vitamin D3 supplementation must be an important point for achieving better COVID-19 outcomes. Most importantly, misuse of vitamin D3 supplementation may rarely cause vitamin D intoxication, leading to hypercalcemia and serious kidney, heart, and neurological problems [Based on the current understanding of risk factors, prognostic factors, and mechanisms of action of SARS-CoV-2, one of the promising strategies for the prevention of infection and recovery from severe illness may be the maintenance of optimal levels of vitamin DCOVID-19 . FurtherCOVID-19 . On the 3 status . Anothercontrols . These cproblems ,83. On tproblems .3 without the risk of vitamin D intoxication because it would be expected to generate sufficient vitamin D3 under the appropriate exposure regimen (12 h/day for 6–9 weeks) [3, and multiple standard-dose exposures  over a period of time would be required. Second, phototherapy with full-spectrum light would ameliorate the adipose tissue dysfunction, which causes insulin resistance, proinflammatory cytokine release, and altered adipokine production [Firmicutes/Bacteroidetes ratio [Firmicutes/Bacteroidetes ratio was reported in COVID-19 patients and was reduced in the recovery state [Lachnospiraceae_NK4A136_group (phylum Firmicutes) as a food allergy-associated bacteria [Lachnospiraceae in the lung microbiota of patients with ARDS [Clostridium difficile infection patients with COVID-19 [Parabacteroides goldsteinii (phylum Bacteroidetes) was identified as a beneficial bacterial species enriched by phototherapy with full-spectrum light [Parabacteroides goldsteinii plays an important role in the anti-obesity effect of polysaccharides isolated from Hirsutella sinensis [Parabacteroides goldsteinii [Parabacteroides goldsteinii. Finally, our preliminary data suggest the impact of phototherapy with full-spectrum light on altered expression of ACE2 and DPP4 (CD26), receptors for SARS-CoV-2 entry in inflamed colon tissues  ,41; i.e.oduction . Reducedoduction . Our preoduction . A recenoduction . Third, oduction . A similoduction ,72 and ioduction ,88, resury state . In our ith ARDS . InteresCOVID-19 ,92. In asinensis , a tradisinensis . Recentldsteinii , suggest tissues . Due to 3 biosynthesis, alters adipokine production (elevated adiponectin), modulates microbiota composition , and reduces many risk factors  associated with COVID-19 and severe illness  [3 biosynthesis may be different in each individual, and seasonal changes in sunlight exposure may also affect it. We also need to consider the adverse effects  caused by excessive UV exposure. In addition, many people, such as night-shift workers, elderly people with difficulty walking, and people who are hospitalized, may be limited to receiving enough benefits from sunlight. In current aged/superaged societies and an ongoing global pandemic of COVID-19, we need to consider ways to enhance our immunity to maintain suitable health. Notably, the stay-at-home policy may reduce the time for outdoor activities, resulting in vitamin D3 insufficiency/deficiency [3 supplementation, phototherapy with full-spectrum light is an alternative approach to stimulate vitamin D3 biosynthesis with minimal risk of skin damage and adjust circadian rhythms, which are quite important for biological, physiological, and immunological activities in all living organisms. To achieve healthy aging, the age-dependent decline in vitamin D3 biosynthesis should be actively adjusted by vitamin D3 supplementation and phototherapy with full-spectrum light, which may reduce the risk of various diseases, such as osteoporosis, metabolic syndromes, allergies, infectious disorders, mental/neurological disorders and cancers . Although further small and large cohort studies are necessary, phototherapy with full-spectrum light could be a reasonable approach with a low potential risk of adverse events, and we highly recommend changing the indoor light environment to full-spectrum light at homes and public spaces, such as schools, working places, clinics, and hospitals as well as nursing homes. The cost of full-spectrum light  may be a critical issue, but there are many beneficial impacts of phototherapy with full-spectrum light, including the generation of sufficient vitamin D3 and the maintenance of healthy gut microbiota composition as well as suitable circadian rhythm. All of these are indispensable for achieving active and healthy aging in current aged/superaged societies.Sunlight exposure and appropriate exercise may be the best ways to maintain healthy conditions. However, vitamin Dmelanin) ,99. Therficiency  and an ificiency . In addi cancers a. In thepatients b. Althou3 insufficiency/deficiency, and it may lead us to achieve active and healthy aging in the era of COVID-19.In conclusion, we propose phototherapy with full-spectrum light as one of the potential strategies to prevent disease progression associated with vitamin DOPN1SW , OPN2, OPN3, and OPN4 genes are widely found and differentially expressed in human brain areas and potentially regulate the circadian photoentrainment of the central biological clock [Recently, the impact of blue light  on SARS-CoV-2 inactivation was reported . Furtheral clock . The impal clock ,115,116,al clock . Taken tFurther investigations, including the screening of optimal conditions of phototherapy, such as light strength, effective wavelength, and the duration of exposure, should be considered in future preclinical and clinical trials.


Erythropoietin (EPO) acts on multiple tissues through its receptor EPOR, a member of a cytokine class I receptor superfamily with pleiotropic effects. The interaction of EPO and EPOR triggers the activation of several signaling pathways that induce erythropoiesis, including JAK2/STAT5, PI3K/AKT, and MAPK. The canonical EPOR/JAK2/STAT5 pathway is a known regulator of differentiation, proliferation, and cell survival of erythroid progenitors. In addition, its role in the protection of other cells, including cancer cells, is under intense investigation. The involvement of EPOR/JAK2/STAT5 in other processes such as mRNA splicing, cytoskeleton reorganization, and cell metabolism has been recently described. The transcriptomics, proteomics, and epigenetic studies reviewed in this article provide a detailed understanding of EPO signalization. Advances in this area of research may be useful for improving the efficacy of EPO therapy in hematologic disorders, as well as in cancer treatment. In classical erythropoiesis, erythropoietin (EPO) binds to an EPO receptor (EPOR) homo-dimer, activating EPO/EPOR signal transduction. EPO has two non-identical binding sites on EPORs: a high-affinity G151 (nano-molar) on the first and a low-affinity R103 (micro-molar) on the second receptor. The main signal cascades activated by EPO are JAK2/STAT5 cascade, phosphatidylinositol 3-kinase (PI3K) cascade, RAS/MAP kinase cascade, and protein kinase C (PKC) cascade [Janus kinase 2 (JAK2) is a non-receptor tyrosine kinase that represents a pivotal role in signal transduction via its association with EPOR. There are diverse models of EPO/EPOR signaling. One supposes that EPO binding stimulates dimerization of the EPOR, which is important for signalization . Investi−/− mice exhibit reduced tyrosine phosphorylation of EPOR and decreased STAT5 activity. The importance of FYN in erythropoiesis is also supported by the blunted responsiveness of Fyn−/− mice to stress erythropoiesis [Signal transducer and activator of transcription 5 (STAT5) refer to two proteins that share 94% structural homology and are transcribed from separate genes, STAT5A and STAT5B. However, they are both essential in erythroid differentiation  and is popoiesis . Anotheropoiesis . Besidesopoiesis . Recently, 121 new signals and/or EPO/EPOR/JAK2 target proteins, together with their EPO-modulated domains and phosphosites , were identified. These included actin assemblage modifiers with adaptors DLG-1, DLG-3, WAS, WASL, and CD2AP, and erythroid cytoskeletal targets such as spectrin A, spectrin B, adducin 2, and glycophorin C. Metabolic regulators such as aldolase A, pyruvate dehydrogenase α1, and thioredoxin-interacting protein were also found as an EPO target set . FurtherEPO-dependent STAT5 target genes involved in erythropoiesis are well-known. These are the following: Bcl-XL, Bcl2l1, Pim1, Cish, Socs3, Podocalyxin, Maltr3, Chac1, Ccrn4l, Socs2, Tnfr-sf13c, Lrp8, Cmtm6, Gdf3, Oncostatin-m, Rpl12, Lyl1, Gas5, Pim3, Bim, Trb3, Serpina-3G, Irs2, Suv420h2, Gypc Cdc25a, Btg3, p27-kip1, Trb2, Klf3, Cyclin-G2, Cyclin-D2, CyclinB1-IP-1, Spi2A, and others ,25. SomeMany new targets have emerged in EPOR/JAK2/STAT5 signaling in erythroid cells. Singh et al.  found 16The occupation of STAT-binding sites seems to be cell-type- and developmental-stage-specific. That makes the signaling pathways, epigenetic, and transcriptional changes downstream of EPOR unique in different kinds of cells. For example, in the well-designed study, novel factors such as REST, an epigenetic modifier central to neural differentiation and plasticity, and NFR1, a key regulator of antioxidant response and mitochondrial biogenesis, were found in fetal neural progenitor cells. EPO treatment of these cells also altered the epigenetic landscape, identifying 1150 differentially bound histone H3 lysine 4 dimethylated regions, which are known as active and poised enhancers and transcription start sites . The mosIn fact, evidence is emerging that specific chromatin remodeling is required for STAT binding to a subset of loci, and epigenetic regulation is a crucial and dynamic part of their gene regulation activity. Multiple studies have shown that STAT5 can also function as a transcriptional repressor by recruiting demethylating or deacetylating epigenetic modifiers to specific gene loci . An expen = 24), PI3K (n = 45), and FOXO (n = 25). The epigenetic landscape near genes involved in regulating the actin cytoskeleton (n = 31), glycosylation (n = 153), and transport (n = 221) was also altered within the first hour of exposure, suggesting that cytoskeletal reorganization may be regulated in part at the epigenetic level. To uncover how EPO modulates the erythroid epigenome, an epigenetic profiling study was performed using an ex vivo murine cell system that underwent synchronous erythroid maturation in response to EPO stimulation . AuthorsMutational analysis of the eight EPOR tyrosine residues indicated that three of them, Y343, Y460, and Y464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity. Moreover, phosphorylation at these three residues was necessary for full activation of STAT5, which is a critical downstream mediator of JAK2 V617F-induced oncogenic signaling . Jeong eAlthough EPO stimulation of cultured EPOR-expressing breast cancer cells did not result in increased proliferation, over activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT were observed . EPO-indNon-small cell lung carcinoma (NSCLC) cell line H838 expresses functional EPORs and their treatment with EPO-reduced cisplatin-induced apoptosis . AnotherSimilarly, the co-existence of JAK2 and phosphorylated-JAK2, and STAT5 and phosphorylated STAT5, which are all involved in the mitogenic signaling of EPO, has been found frequently in the cells of malignant tumors of female reproductive organs, stomach, choriocarcinoma, melanoma, and capillary endothelial cells. Most of the phosphorylated JAK2 or phosphorylated STAT5-positive cells, however, had disappeared after the injection of monoclonal antibodies against EPO or a soluble form of EPOR . In HUVETo date, epoetin alpha, beta, and darbepoetin alpha have been approved for anemia treatment in cancer patients. Although epoetins act on the same EPOR, differences in pharmacodynamics and pharmacokinetics through variations in glycolylation degree are expected. However, though both epoetins alpha and beta demonstrated comparable efficacy and safety profiles, epoetin beta achieved targeted hematocrit levels with lower doses  and keptN642H, was observed in many forms of peripheral T-cell leukemia/lymphomas [N642H, under the hematopoietic vav-promoter, developed an aggressive CD8+ T-cell leukemia with organ infiltrations by CD8+, CD4+, and γδ T-cells [N642H was confirmed by a transplantation model derived from the transgenic mouse in NKT [Under physiological conditions, STAT5 expression is regulated by prolactin, growth hormone, insulin growth factor, estrogen, and progesterone signaling pathways, and plays a significant role in a normal mammary cell differentiation, proliferation, and survival . Watowicymphomas ,60,61 anymphomas  and neutymphomas . Moreove T-cells . The once in NKT  and γδ Te in NKT  mentioneBone marrow-derived endothelial cells from monoclonal gammopathy (MGEC) of undetermined significance and multiple myeloma endothelial cells (MMEC) were assessed for the possibility that EPO might play a role in MGECs- and MMECs-mediated angiogenesis. Lamanuzzi et al.  showed tSimilar signal transduction, including the STAT5 pathway, is mediated via EPOR-βcR, although EPOR splice variants  and EPORProtection mediated by EPO/EPOR/JAK2 results in pancreatic β cell proliferation and survival, increased angiogenesis, and reduced inflammation in the islets , as wellSTAT5 is also required for lipid degradation and its deficiency supports adiposity and impairs lipid mobilization in the adipocytes of mice ,81,82. IThe upregulation of STAT5 and its downstream gene products, Bcl-xL and XIAP, demonstrated significant protective effects on astrocytes (during 24–48 h anoxia) and A9 dopaminergic neurons of the substantia nigra . Both efIn rats with experimental epilepsy, EPO protected myocardial cells from apoptosis via the JAK2/STAT5 pathway, and the inhibition of JAK2/STAT5 signalization by AG490 reduced the antiapoptotic effects of EPO . On the Recently, several new target genes of the EPO/EPOR/STAT5 signaling cascade have been discovered, especially in erythroid cells. These genes and their protein products are mainly involved in the processes of epigenetic regulation, mRNA splicing, molecular signaling, EPOR turnover, and negative regulation of STAT5 signaling. However, they can also be implicated in the regulation of cell metabolism and cytoskeletal reorganization. Transcriptomic analyses and mapping of EPO/EPOR target genes are also necessary for tumor and normal cells, on which only signaling and the effect of EPO/EPOR have been described so far, e.g., protective, antiapoptotic, trophic, and other. New EPO/EPOR target genes in cancer cells could serve as markers for various means of monitoring both disease activity and cell responses to various therapies, including the administration of rHuEPO to tumor patients.We reviewed most recent scientific papers and the data from the biomedical literature found in PubMed. Studies were obtained using the following keywords: “STAT5” or “erythropoietin” or “erythropoietin receptor” and “erythropoiesis” or “cancer cell” or “normal cell” or “signaling”.


The USA has a high burden of childhood asthma. Previous studies have observed associations between higher blood lead levels and greater hypersensitivity in children. The objective of the present study was to estimate the association between blood lead concentrations during early childhood and an asthma diagnosis between 48 and 72 months of age amongst a cohort with well-characterized blood lead concentrations. Blood lead concentrations were measured at 6, 12, 18, 24, 36, and 48 months of age in 222 children. The presence of an asthma diagnosis between 48 and 72 months was assessed using a questionnaire which asked parents or guardians whether they had been told by a physician, in the past 12 months, that their child had asthma. Crude and adjusted risk ratios (RR) of an asthma diagnosis were estimated for several parameterizations of blood lead exposure including lifetime average (6 to 48 months) and infancy average (6 to 24 months) concentrations. After adjustment for child sex, birthweight, daycare attendance, maternal race, education, parity, breastfeeding, income, and household smoking, age-specific or composite measures of blood lead were not associated with asthma diagnosis by 72 months of age in this cohort. H) cells favoring the production of TH2 and associated cytokines  had asthma?” Children were considered to have physician-diagnosed asthma if their parent/guardian reported a diagnosis at any one of these three time points.Venous blood samples were collected when children were 6, 12, 18, 24, 36, 48, 60, and 72 months of age , 21. Allr = 0.21 (p < 0.05) between 6 and 24 months and r = 0.89 (p < 0.05) at 60 and 72 months, supporting the use of imputation.Average and peak blood lead concentrations were calculated from serial blood lead measures, similar to previous analyses in this cohort , 24. LifAt each visit, parents/guardians completed a questionnaire concerning the child’s health history, parental health, socioeconomic status, demographic information, and the home environment. Covariates of interest included data collected from the child’s birth record such as sex, birthweight (grams), and maternal parity. Maternal race (white/non-white) and whether the child was ever breastfed were collected at the child’s 6-month visit. The average adjusted income variable used in the analysis incorporated household income as well other sources of income  reported at the 54- through 72-month visits. This variable was calculated by averaging household income reported at 54, 60, 66, and 72 months, adding any average other income reported from that same time period, and subtracting out the average rent paid during that same period. Data collected at the child’s 72-month follow-up included maternal education , more than high school), total number of cigarettes smoked per day in the household, and any child daycare attendance in the past 12 months.Statistical models included blood lead concentration as a continuous variable or as a three-level ordinal classification variable with categories ≤ 5.0 μg/dL, 5.0–9.9 μg/dL, and ≥ 10.0 μg/dL. These groupings were chosen based on recent Centers for Disease Control and Prevention (CDC) reference levels, clinical utility, and as having significance for informing public health policy , 27. Forp values for linear trend were considered statistically significant if p < 0.05. All data management and analyses were performed using the SAS software system .The risk ratio for asthma and respective 95% confidence interval (CI) were estimated using log-binomial regression for each parameterization of lead. In total, 10 primary models were fit: five unadjusted, and five adjusted, examining (1) concurrent 48-month blood lead levels, (2) peak 48-month blood lead levels, (3) lifetime average at 48-month blood lead levels, (4) peak 24-month blood lead levels, and (5) infancy average at 24-month blood lead levels. As a secondary analysis, continuous parametrizations of age-specific lead measurements at 6, 12, 18, 24, 36, 48, 60, and 72 months of age were examined. Risk ratios and Among the 222 children with complete data, 70 31%) had a concurrent blood level at 48 months < 5 μg/dL, 99 (45%) had a level between 5 and 9.9 μg/dL, and 53 (24%) had a level ≥ 10 μg/dL (Table % had a cFifty-two children (23%) had been told by a doctor that they had asthma in the last 12 months during at least one of the three visits at 48, 60, or 72 months  . Our stur = 0.22, p = 0.0004) between higher postnatal blood lead levels and IgE concentrations, after adjusting for age [Previous epidemiological studies have observed higher IgE concentrations among children with higher blood lead levels, with mean blood lead levels in the range of 1–2 μg/dL , 14, 33. for age . An addi for age , 33.n = 2782) observed a moderate Kappa of 0.60 agreement, and a high specificity of correctly identifying a physician asthma diagnosis of 95.9% [Limitations of this study include the assessment of asthma via caregiver report of a physician’s diagnosis. Some misclassification may have been introduced if parents or guardians did not recall whether their child had been diagnosed. A study comparing parent-reported asthma diagnoses with health claims  and peak blood lead concentrations as determinants of asthma risk. In addition, the prospective nature of the cohort provided data on important early life confounders such as maternal parity and child birthweight, which might not be readily available in a cross-sectional design. Furthermore, we collected sociodemographic data at multiple time points which permitted construction of variables—such as average adjusted household income—that considered multiple time periods and sources of income, which served to reduce residual confounding, which could otherwise distort the lead-asthma association.In conclusion, we did not observe an association between child blood lead levels and physician diagnosed asthma by age 72 months in this cohort. Although prenatal lead exposure has been linked with childhood IgE levels, the direct effect of prenatal lead exposure on asthma is not known.


Conjunctival melanoma (CM) accounts for 5% of all ocular melanomas and arises from malignantly transformed melanocytes in the conjunctival epithelium. Current therapies using surgical excision in combination with chemo- or cryotherapy still have high rates for recurrences and metastatic disease. Lately, novel signal transduction-targeted and immune checkpoint inhibitors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein-1 (PD-1) receptor inhibitors, BRAF- or MEK-inhibitors for systemic treatment of melanoma have improved the outcome even for unresectable cutaneous melanoma, improving patient survival dramatically. The use of these therapies is now also recommended for CM; however, the immunological background of CM is barely known, underlining the need for research to better understand the immunological basics when treating CM patients with immunomodulatory therapies. Immune checkpoint inhibitors activate tumor defense by interrupting inhibitory interactions between tumor cells and T lymphocytes at the so-called checkpoints. The tumor cells exploit these inhibitory targets on T-cells that are usually used by dendritic cells (DCs). DCs are antigen-presenting cells at the forefront of immune response induction. They contribute to immune tolerance and immune defense but in the case of tumor development, immune tolerance is often prevalent. Enhancing the immune response via DCs, interfering with the lymphatic pathways during immune cell migration and tumor development and specifically targeting tumor cells is a major therapeutic opportunity for many tumor entities including CM. This review summarizes the current knowledge on the function of lymphatic vessels in tumor growth and immune cell transport and continues to compare DC subsets in CM with related melanomas, such as cutaneous melanoma and mucosal melanoma. GNAQ or GNA11, [Melanomas vary in their behavior, genetics, and response to therapies. It is, therefore, important to look at melanoma subtypes in a differentiated way. Currently, we are seeing significant therapeutic success for cutaneous melanoma with molecular inhibitors and immune checkpoint inhibitors. Success in ocular melanoma is limited because of the lack of therapeutic standards and clinical trials for conjunctival melanoma (CM) and, in general, too little is known about the immunology of ocular tumors and the tumor microenvironment. We, therefore, may miss ocular features if drugs tested only for cutaneous melanomas are uncritically applied to ocular melanoma patients. It is important not to consider or treat all melanomas the same. For example, another completely different ocular melanoma subtype is uveal melanoma. It originates from non-epithelial melanocytes and differs significantly compared to dermal, mucosal and conjunctival melanoma in terms of mutations, with mostly primary mutations located in r GNA11, , which ar GNA11, , comparer GNA11, .The overall mortality rate, e.g., for CM, is still high and is approximately 30% . TherefoThis review summarizes the similarities and differences of CM  and Phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway have been shown [NRAS, BRAF, KIT, or the tumor suppressor gene neurofibromin 1 (NF1) lead to an increase in signal transduction from the cell membrane to the nucleus, which in turn activates differentiation, growth and survival [NRAS, a small GTPase, are found in approximately 20% of CM and in up to 30% of cutaneous and other mucosal melanomas [NRAS activates BRAF a component of the MAPK pathway and PI3-K of the PI3-K/AKT/mTOR pathway [BRAF occur in 14–50% of CM cases. The most frequent BRAF mutation is the V600E mutation (80–90%), [BRAF activates the mitogen-activated protein kinase (MEK) protein by phosphorylation, which leads to activation of the extracellular-signal regulated kinases (ERK) protein, leading to cell growth [In CM, mutations in the mitogen-activated protein kinase , ,66,67. BBRAFV600E mutations are typically found in cutaneous melanoma areas without sun damage and develop from nevi [BRAF are found in sun exposed skin melanoma [BRAF mutations in cutaneous melanoma (32–60%) [BRAF mutations are found in the sun-exposed epibulbar conjunctiva with a more than twofold frequency [rom nevi ,69, whilmelanoma . Compare(32–60%) ,72,73,74(32–60%) ,77,78,79requency .NF1 dysfunction occurs in 30% of CM, in 8–18% in mucosal melanoma and in 12–25% in cutaneous melanoma [NF1 is, as mentioned above, a tumor suppressor protein [An melanoma ,80. NF1  protein .TERT) are found in 32–43% of CM [TERT mutations are mainly found in UV exposed areas and correlate with a shorter metastatic free survival [TERT mutations have been seen in 85% of metastatic cutaneous melanoma tissues, and 33% of primary cutaneous melanomas [Mutations in the promoter of the telomerase reverse transcriptase , a member of the PI3K/AKT/mTOR pathway, have been detected in 0–11% of CM cases [KIT mutations are very rare in cutaneous melanoma (0–2%) and occur more often in mucosal melanoma (4–21% of cases) [KIT leads to ligand-independent auto-phosphorylation of intracellular tyrosine residues, resulting in downstream activation of PI3K [KIT is crucial for the normal development of melanocytes acting as an important mitogen [Mutations in the receptor tyrosine kinase KIT CD1, a membeCM cases ,86. KIT f cases) ,21. The  of PI3K . KIT is  mitogen .The total mortality of CM is around 30% . The tumThe preferred therapy for local CM is surgical excision combined with brachy-, cryo-, chemo- or immunotherapy ,95 revi.BRAF V600E or V600K mutation are offered a BRAF and MEK inhibitor, as this prolongs the recurrence-free survival, according to the melanoma guideline [BRAF mutations or advanced disease, although there are no studies on this. Novel treatment options are summarized in A wide range of new therapeutic approaches have already made it into clinical trials. Already today, patients with dermal melanoma from AJCC 2017 stage IIIA-D are offered an anti-PD1 therapy, or from stage III A–D with a uideline ,98,99. SBRAF, NRAS, NF1, and KIT genes as mentioned above. In case reports, BRAF inhibitors in combination with or without MEK inhibitors have shown good therapeutic effects, up to complete response, with acceptable side effects [One possibility for new therapy is the treatment with targeted molecular inhibitors. The MAPK and PI3K/AKT/mTOR pathways provide molecular targets ,65. Both effects ,106.BRAF and NRAS mutants, reviewed in [Cao et al. showed that inhibition of both BRAF and MEK act synergistically, at least in three CM cell lines  and was more effective than BRAF inhibition alone . Similariewed in .KIT mutations were rarely detected in CM  or programmed cell death protein-1 (PD-1)  and immuThe CTLA-4 inhibitor ipilimumab is an approved therapy option for metastatic cutaneous melanoma . In a caFurthermore, in case of insufficient response different immune checkpoint inhibitors can be combined (like a PD-1 inhibitor together with a CTLA-4 inhibitor), however, at the cost of increased side effects including diarrhea, fatigue, pruritus or nausea ,146,147.Initial clinical studies showed positive effects of therapeutic inhibition of ERK1/2, the PI3K-AKT-mTOR pathway and CDK4/6 in cutaneous melanoma . In the Although initial studies have yielded promising results, only case reports of immune checkpoint inhibitors in CM are currently available and no larger, controlled trials have yet been conducted.DC vaccination is another hot topic in oncology and several studies have been performed to understand the mechanisms behind this approach and clinical trials were performed or are ongoing, summarized in , althougDCs are able to initiate adaptive immune responses  and actiDC vaccination entails the adoptive transfer of autologous, activated, or antigen-loaded DCs into cancer patients. The DCs are isolated or generated in vitro and manipulated before reinfusion . This haThe overall patient response rate using DC vaccination is up to 15% ,159, whiLymphatic vessels play a central role in the maintenance of tissue fluid balance, dietary lipid absorption and the trafficking of immune cells . In healFrom capillaries, the lymph is channeled via pre-collectors into collecting lymphatic vessels, which carry it to the regional lymph nodes and after exiting through efferent lymphatics via the left and right lymphatic duct to the venous circulation.Elegant cannulation studies, which analyzed the composition of lymph by tracing immune cells, labeled with photo-convertible fluorescent proteins, identified T-cells as major cargo of afferent lymphatics followed by antigen presenting DCs and few B-cells ,166. PerDevelopmentally, already the earliest lymphatic progenitors express the transcription factor PROX1 and appear at the dorsal roof of the cardinal vein . PROX1 iFollowing postnatal maturation, the lymphatic vessel system acquires a quiescent state. LEC quiescence may also be revoked by lymphangiogenic, physiologic, but mainly pathologic stimuli, including tumor development, but also wound healing, obesity, or inflammatory scenarios like myocardial infarction, inflammatory bowel disease, or corneal transplantation ,177,178.The conjunctiva contains lymphatic vessels, which lately have been shown ex vivo using OCT signaling in porcine eyes  or in viRegarding CM, the presence of lymphatic vessels has been demonstrated in a CM mouse model , as wellThe interaction of DCs and tumor draining lymphatic vessels but also tumor-induced changes in lymph nodes have been studied poorly in CM. Tumor draining lymph nodes often present an altered immunologic microenvironment compared to non-tumor-associated lymph nodes. An increased frequency of regulatory T-cells in tumor-draining lymph nodes correlates with a worse prognosis for cutaneous melanoma patients. . This isDCs are professional antigen presenting cells, which capture, process and present antigens to T-cells  after re1 and cDC2.The main function of conventional DCs (cDCs) is antigen presentation . Their p1 express toll-like receptor 2, 3, 4, 9, 11, 12 and 13  and recognize pathogen associated molecular patterns (PAMPs) [1 are the main producers of interleukin 12 (IL-12), interferon α (IFN-α) and IFN-λ and also express IL-6 and tumor necrosis factor α (TNF-α) when they are activated by PRRs [1 in mice recognize exogenous soluble and cell-associated antigens which they present to CD8+ T-cells on MHCI [In mice, lymphoid cDC (PAMPs) ,206. cDC by PRRs ,207. cDC on MHCI ,209. The on MHCI ,211,212.1 are also specialized to take up material from dead tumor cells and transport it to tumor-draining lymph nodes where they present it to anti-tumor CD8+ T-cells [In the tumor context, cDC T-cells ,214.1 cells is low, e.g., in tg(Grm1)EPv mice, the amount of cDC1 positive cells in skin melanomas was less than 1% of the CD45+ cells, compared to more than 2% cDC2 and about 8% Langerhans cells [1 still had important anti-tumoral effects. Intratumoral cDC1 can attract T-cells [−/− mice, cDC1 are missing and this leads to a failure to reject immunogenic tumors and a worse response to immune checkpoint therapies [Although in skin melanoma, the amount of cDCns cells , cDC1 st T-cells , stimula T-cells  and secr T-cells . In Batfherapies ,217,219.1 express TLR1, 3, 6, 8 and 10 [1 are the main producers of type 3 interferon and cross-present antigens to cytotoxic T-cells [1 are able to stimulate allogeneic or autologous T-helper cells [Human cDC8 and 10 ,220,221. T-cells . In conter cells .2 in mice present pathogens on MHCII molecules [2 in the spleen detect pathogens within the cytoplasm and express TLR 1, 2, 4, 5, 6, 7, 8, and 9 [2 express proinflammatory cytokines like TNF-α, IL-6 and low levels of IL-10 and play an important role in chemo attraction [Lymphoid cDColecules ,224. cDC8, and 9 ,206. Indtraction ,225.2 can be subdivided in ESAMhi (80% of cDC2), which are more efficient in Th2 polarization and in ESAMlo [2, which prime Th1 cells more efficiently [cDCn ESAMlo ,227 cDC2iciently ,227.2 are among others CD11chi SIRPα+ and can be subdivided into CD5hi and CD5lo cDC2 [hi DCs showed better migration to the lymph node and induce more potently T-cell proliferation, compared to CD5lo cDC2 [Human cDC5lo cDC2 ,228. CD55lo cDC2 , indicat2 decreased in the skin melanoma compared to the tumor free tissue over time, whereas no difference was seen for Langerhans cells (LCs) and cDC1 [2 are important to induce a protective antitumor CD4+ T-cell immune response and may act as a target of T-reg suppression [In the peripheral blood of melanoma patients, no significant difference was seen regarding disease stage and CD11b+ DCs . In a trand cDC1 . Binnewipression .CD103++ CD11b- mDCs, CD103- CD11b+ mDCs and CD103++ CD11b+  [1 and are found mainly in connective tissues [Tissue migratory or non-lymphoid DCs in mice can be subdivided into three main subsets: nal DCs) . Further tissues . The int tissues . CD103-  tissues . Tissue- tissues ,200,233.In mouse skin melanoma models, CD103+ CD11b+ cells have been shown to be the only antigen presenting cells, transporting tumor antigen to the lymph nodes and subsequently priming CD8+ T-cells, although they represent only a minor population of DCs infiltrating the tumor . They weLCs are found in the epidermal layer of the skin, in the epithelium of the cornea, buccal, gingival and genital mucosae and lead to regulatory T-cell proliferation in a steady state, but limit the activation in an inflamed state ,235.+ T-cells, to Th17 and Th22 cells [Human LCs are among others LANGERIN+ . In huma22 cells ,237,238.22 cells .pDCs are the major producers of type 1 interferons ,204 and + T-helper cells and cytotoxic T-cells to lead to an anti-viral immune response [pDCs in mice are CD317+ CD209++ Ly6c++ ,202. TLRresponse .Human pDCs, like murine pDCs, respond to viral infections and produce type 1 interferon in response to TLR7 and TLR9 and type 3 interferons in response to TLR9 ,244. The2 in melanoma patients receiving DC vaccination [Imiquimod has been approved for local treatment and promotes pDCs and induces cytotoxicity [Others showed that human pDCs are more efficient in attracting cytotoxic CD8+ T-cells than cDCcination . Interfetoxicity .+ and CD8+ T-cells due to the secretion of IL-1, IL-12, IL-23 and TNF-α [moDCs originate from monocytes and appear in inflammation, cancer, or infection ,237,247.nd TNF-α .2 [In general, little is known about DC subsets in the conjunctiva per se and in CM. However, a few DC subsets have been described in the healthy conjunctiva: Ohbayashi et al. used confocal microscopy for detecting DCs in the conjunctiva and found a small number of conventional DCs but almost no plasmacytoid DCs (pDCs) . Langerh2  and with2 . Khandew2  via CCR72 .DCs play also an important role in the development of autoimmune diseases of the ocular surface like dry eye disease. It was shown that Thrombospondin-1 (TSP-1) is expressed on DCs of the ocular surface .Additionally, in infectious diseases, DCs play a critical role by presenting pathogenic antigens to effector T-cells. For the conjunctiva, it was recently shown that goblet cells, which provide the mucin layer of the tear film, are in close contact with conjunctival DC. Contreras-Ruiz et al. could show that, on one hand, goblet cells control the activation status of conjunctival DC by expression of TGF-β2 and TSP-1 inducing a tolerogenic phenotype . On the In pterygium, a benign conjunctival tumor that centripetally invades the clear cornea, laser scanning confocal microscopy revealed in patients that in both the clear cornea, as well as in the pterygium, the amount of DCs was significantly increased . An AustIn malignant CM, Wolf et al. recently characterized the tumor microenvironment of CM in more detail. Next to natural killer T-cells (NKT), B-cells and mast cells, they found an enrichment of pDC and activated DCs in CM compared to healthy conjunctiva . For pDC+ MHCII+ DCs have been identified in the tumor microenvironment: cDC1, cDC2 and moDCs [In the field of skin melanoma, more is known about the DC subtypes. Here three major subsets of CD11cnd moDCs ,263,264.In several studies of human melanomas, a low number of intratumoral DCs have been associated with a worse prognosis ,266.Wculek et al. reviewed that tumor-associated DCs, for example, in ovarian cancer, cutaneous melanoma, gastric cancer and lung carcinoma have a reduced function in differentiation, activation and stimulation of immune response ,268,269.CM is a very rare tumor with a high rate of mortality and local recurrence. Despite some similarities with cutaneous melanoma, CM is a mucosal melanoma and only very little is known about immunological responses or DC subsets in CM. Promising new therapeutic options are targeted molecular inhibitors, immune checkpoint inhibitors and DC-immunotherapy. Although more and more knowledge about DCs and their subtypes is becoming known and single studies have shown effects of different DC subtypes in several diseases of the ocular surface, unfortunately very little is known about the subtypes in CM so far. Whether individual subtypes are important and similar to other cancer types, should be shown by future studies to exploit possible therapeutic effects.Therefore, much remains to be learned about the composition and function of DCs and their subsets in CM to establish new therapy options.