Thymic tumors are a group of rare mediastinal malignancies that include three different histological subtypes with completely different clinical behavior: the thymic carcinomas, the thymomas, and the rarest thymic neuroendocrine tumors. Nowadays, few therapeutic options are available for relapsed and refractory thymic tumors after a first-line platinum-based chemotherapy. In the last years, the deepening of knowledge on thymus’ biological characterization has opened possibilities for new treatment options. Several clinical trials have been conducted, the majority with disappointing results mainly due to inaccurate patient selection, but recently some encouraging results have been presented. In this review, we summarize the molecular alterations observed in thymic tumors, underlying the great biological differences among the different histology, and the promising targeted therapies for the future. Primary Thymic Epithelial Tumors (TETs) are rare mediastinal tumors arising from thymic epithelial cells, with a reported annual incidence ranging from 1.3 to 3.2 per million . TCs dis18F-FDG-PET for the best planning of the treatment is available, showing a correlation between histological grade and 18F-FDG uptake [18F-FDG uptake and glucose transporter 1 (GLUT1), hypoxia-inducible factor-1 α (HIF-1α), vascular endothelial growth factor (VEGF), microvessel density (MVD), and p53 immunohistochemical (IHC) expression was observed [Clinical management in TETs is mainly driven by disease stage, with the Masaoka-Koga staging system currently routinely adopted for its optimal correlation with the prognosis ,8,9. CT G uptake ,11. Moreobserved .A radical surgical resection represents the best treatment strategy for early-stage disease. On the contrary, the treatment of locally advanced or oligometastatic disease can be challenging and requires a multidisciplinary approach, including surgery, chemotherapy, and radiation therapy . SystemiIn the last decade, the wide implementation of high throughput technologies in solid tumors has allowed the identification of a broad spectrum of molecular aberrations and altered signaling pathways in TETs, leading to the definition of distinct molecular profiles in TMs and TCs. The identification of specific aberrations in TETs has paved the way for novel therapeutic targeted strategies investigated in phase 1/2 studies . HoweverIn this review, we will summarize the genomic background of thymic tumors and the emerging molecular classification, with a significant focus on the biologic rationale explaining the possible use of targeted agents in this heterogeneous group of rare thoracic cancers . We willThe different histological subtypes of TETs harbor specific molecular alterations, as revealed by the comprehensive genomic analysis performed within The Cancer Genome Atlas (TCGA) project . Indeed,The genomic mutational profile of TETs is characterized by enrichment of C > T mutations within CpG islands, a mutational signature associated with aging and in agreement with the median age of onset . general transcription factor II-I (GTF2I), HRAS, TP53, and NRAS. Clonality analyses revealed that the mutations in all four genes probably occurred at the onset or in the very early stages of tumor development. None of the four most frequently mutated genes in TETs is amenable for targeted inhibition to date [Whole-Exome Sequencing (WES) on 117 samples of TETs and paired normal tissue has identified four recurrently mutated genes:  to date . TP53, CYLD, cyclin-dependent kinase inhibitor 2A (CDKN2A), BRCA1 associated protein 1 (BAP1), and polybromo 1 (PBRM1) [KIT, discoidin domain receptor tyrosine kinase 2 (DDR2), platelet-derived growth factor receptor alpha (PDGFRA), ROS1, insulin-like growth factor 1 receptor (IGF1R) [KIT and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) the most frequently altered genes. Other targets included PDGFRA, fibroblast growth factor receptor 3 (FGFR3), protein patched homolog 1 (PTCH1), F-box and WD repeat domain containing 7 (FBXW7), breast cancer type 2 susceptibility protein (BRCA2), isocitrate dehydrogenase 1 (IDH1), human epidermal growth factor receptor 2 (ERBB2), and ERBB3 [Overall, TCs have been found to carry a higher number of mutations than TMs with recurrent mutations of known cancer-related genes, including  (PBRM1) . Sequenc (IGF1R) . DNA seqnd ERBB3 .Regarding the tumor mutational burden (TMB), TETs are characterized by the lowest TMB among adult cancers. Comparing the TMB between TCs and TMs, a significant increase of TMB in TC samples could be observed . In the forkhead box C1 (FOXC1) is codified [medium-sized neurofilament (NEFM) and ryanodine receptor type III (RYR3), presenting a sequence similarity with autoimmune targets [Chromosomal copy number alterations (CNAs) have been described with high frequency in TETs and are usually associated with B2 and B3 TMs and with TCs . Chromoscodified , is assocodified . Further targets .MLH1 by promoter hypermethylation, O-6-methylguanine-DNA methyltransferase (MGMT) methylation and loss of its protein expression as well as methylation of the promoter region of CDKN2 have been frequently reported in TETs [Epigenetic alterations, such as aberrant DNA methylations, have been frequently observed and they also correlate with histological type and clinical stage. Silencing of tumor suppressor genes as  in TETs . Non-cod in TETs .KIT mutations are found in less than 10% of cases, with a wide spectrum of mutations not always sensitive to KIT inhibitors [c-KIT is often overexpressed in TCs (79–88%), whereas hibitors ,24. A sihibitors  with fewhibitors . Activathibitors . IGF-1R hibitors . The exphibitors ,28. As fhibitors . TETs cahibitors ,31. Addihibitors . Proteomhibitors .According to genomic analyses, TETs were classified into distinct molecular subtypes, with a good correlation with histological classification and prognosis . GTF2I and RAS wild-type tumors, frequently associated with MG. Subtype 2 is mainly composed of TCs and tumors typically present chromosome 16q loss. Subtype 3 (AB-like) includes essentially type AB TMs and tumors generally are GTF2I mutated and RAS wild-type. Finally, subtype 4 (A-like) contains a mix of type A and AB TMs and is characterized by GTF2I and RAS mutated tumors [GTF2I mutant group is enriched in type A and AB TMs; the T-cell signaling gene profile group is composed principally by type B1, B2, and AB TMs; the chromosomally stable group includes mainly type B2 TMs; the chromosomally unstable group is principally represented by TCs and type B2 and B3 TMs. Both chromosomally stable and unstable clusters are enriched in MG cases. Interestingly, the T-cell signaling gene profile subgroup is enriched for genes related to costimulatory and coinhibitory T-cell signaling, implying an abundance of PD1-expressing CD8+ T cells, that may respond to immunotherapy [Radovich et al. described 4 clusters of TETs based on a multi-omic unbiased clustering, which integrated mutation, CNA, mRNA, and miRNA expression, DNA methylation, and protein expression data. Subtype 1 (B-like) is principally represented by type B TMs and is characterized by d tumors . Other aotherapy . These wotherapy ,34.Based on molecular characterization of TETs, many targeted therapy clinical trials have been led .GTF2I, genes of the PI3K/AKT/mTOR pathway, genes of the RAS family, and others. Other genomic alterations shared with TC are discussed in the respective section.As previously reported, there are recurrent mutations in some genes in TM that also define distinct subgroups of TETs. Those comprise GTF2I is a gene located in the long arm of chromosome 7 at position 11.23 (7q11.23) [7q11.23) . It enco7q11.23) ,37. Seve7q11.23) ,39. TFII7q11.23) .GTF2I and gene fusions have been observed, respectively, in TETs and soft tissue angiofibromas (GTF2I/NCOA2 fusion), acute promyelocytic leukemias (GTF2I/RARA fusion), and pilocytic astrocytoma (GTF2I/BRAF fusion) [Mutations of  fusion) ,38.GTF2I mutation in TETs always occurs at the same codon and seems to be pathognomonic, clonal, and oncogenic. Indeed, the L424H mutation has been observed only in TETs, while other tumors rarely present a mutation in GTF2I and always in different codons. Moreover, clonality analyses showed that this mutation is clonal, suggesting a very early onset in tumor development [GTF2I L424H mutation has a high prevalence in TETs (39–43.4%), especially in type A (82–100%) and AB (70–100%) TMs, while it is less frequent in more aggressive subtypes [GTF2I mutation prevalence is associated with disease stage, since it is more frequent in early (57%) than advanced (19%) stages. Notably, TM patients with GTF2I mutant tumors lived longer than those with GTF2I wild-type tumors  [The elopment . The GTFsubtypes ,19,41. F vs 88%) ,42.GTF2I L424H mutation observed in TETs affects a specific amino acid sequence, which is a non-canonical destruction box, involved in TFII-I proteasomal degradation. In presence of DNA damage, TFII-I is ubiquitinated and degraded by the proteasome complex. The L424H missense mutation determines a RILLAKE-to-RILHAKE alteration in the destruction box sequence that hampers TFII-I recognition for degradation. As a result, TFII-I turnover is reduced with consequent upregulation of downstream pathways, such as those involved in cell proliferation, cell morphogenesis, receptor tyrosine kinase signaling, retinoic acid receptors, neuronal processes, and the WNT and SHH signaling pathways [GTF2I in TETs [The typical pathways ,35,37. Opathways ,19,35,38 in TETs .GTF2I with its correlated pathways could be a potentially interesting targeted treatment. At the moment, no targeted therapies have been developed for patients carrying this mutation.Considering the high prevalence of the mutation and the oncogenic role in TETs, The PI3K/AKT/mTOR signaling pathway is essential for the regulation of many cellular processes, such as proliferation, survival, metabolism, and angiogenesis, and is deregulated in many cancer types .PI3K, AKT, TSC, and mTOR, have been observed in both TMs and TCs [The PI3K/AKT/mTOR pathway activation plays a crucial role in TETs growth and can thus be exploited by drugs targeting mTOR or other inhibitors acting on the same pathway ,45. Muta and TCs ,45,46. MTP53, kelch-like ECH-associated protein 1 (KEAP1) and CDKN2A, were observed in 27% of patients, without association with time to treatment failure (TTF) [A phase 2 single-arm study of everolimus, an mTOR inhibitor, enrolled patients with TMs (N = 32) and TCs (N = 18) after at least one previous platinum-based chemotherapy . The sture (TTF) .PI3K inhibitors have been investigated in preclinical studies and showed potential activity in TETs . A singlThe insulin-like growth factor (IGF) pathway regulates several biological processes, such as metabolism and cell growth. IGF I and II bind IGF-R1, which is a heterotetrameric transmembrane glycoprotein with an intracellular tyrosine kinase domain. IGF-R1 is encoded by a gene located in the long arm of chromosome 15 at position 26.3 (15q26.3) and is expressed ubiquitously, including in immune cells. The binding of IGF to IGF-R1 is modulated by the IGF binding proteins (IGFBPs 1-6) and leads to the activation of IGF-and of two major pathways: the insulin receptor substrate (IRS)/PI3K/AKT/mTOR pathway, with mainly metabolic effects, and the SHC/RAS/MAPK pathway, with mainly mitogenic effects ,48.In the thymus, IGF-1 has been shown to increase the thymic epithelial cell population and affect thymocyte development and chemokine expression . IncreasAll histological subtypes of TETs have some degree of IGF-1R expression, especially aggressive subtypes and those at advanced disease stage ,51. FurtCixutumumab, a monoclonal antibody that binds IGF-1R and promotes its degradation, has been studied in a phase 2 trial in 49 pre-treated patients with TET (12 TCs and 37 TMs) . In the RAS is mutated in 10–30% of all human cancers [KRAS is mutated in 85% cases, while NRAS (12%) and HRAS (3%) are less common. The majority of RAS mutations occur at codons 12, 13, or 61 and determines constitutive activation of RAS [RAS proteins are a family of kinases with intrinsic guanosine-triphosphatase activity that mediate and integrate signal transduction from a multitude of cellular signals to their principal effectors that are RAF kinases and the PI3K/AKT/mTOR pathway. In humans, there are 4 isoforms of RAS, encoded by 3 genes: a gene located in the short arm of chromosome 12 at position 12.1 (12p12.1) encodes for 2 different splice variants of KRAS (KRAS4a and KRAS4b); a gene located in the short arm of chromosome 1 at position 13.2 encodes for NRAS (1p13.2); a gene located in the short arm of chromosome 11 at position 15.5 encodes for HRAS (11p15.5) . RAS is  cancers : KRAS isn of RAS . RAS mutations are more frequent in TCs than in TMs (18.5% vs 10%) [HRAS mutation is more frequent in A/AB TMs, while NRAS is more frequent in TCs [RAS mutations in TETs usually occur at known gain-of-function codons  and are associated with worse prognosis [RAS proteins are frequently mutated also in TETs 7–18.5%), especially HRAS and NRAS .5%, espe,54. Overt in TCs . RAS mutrognosis ,54. Recerognosis ,57,58. ASSTRs are expressed in TETs, thus the efficacy of octreotide, a somatostatin analog, with and without prednisone has been investigated by three phase 2 studies ,31,59. TMembers of the SRC family are tyrosine kinases involved in the transduction of signals for embryonal development and cellular growth. In the thymus, SRC is involved in thymic epithelial cell maturation. SRC role in the development of many types of cancer is well established so that SRC inhibitors have been developed . SaracatEGFR mutations are rare [EGFR has a central role in the regulation of epithelial tissue development and homeostasis and its deregulation is observed and successfully targeted in many cancer types ,63,64. Tare rare ,54,65. Rare rare ,67,68, bare rare .KIT, CYLD, and angiogenesis-related genes, that are also offer targets for specific treatments. Moreover, TCs show alterations in tumor suppressor genes  and in epigenetic regulators. Other genomic alterations shared with TM are discussed in the respective section.As extensively showed, TC has a different biologic and mutational landscape compared to TMs within TETs. The most relevant and characterizing alterations occur in KIT is a gene located in the long arm of chromosome 4 at position 12 (4q12) and encodes for a type III receptor tyrosine kinase, c-KIT (CD117), involved in many cellular processes. Binding with its ligand, the stem cell factor (SCF), triggers c-KIT dimerization and autophosphorylation of the tyrosine residues, protein kinase activation, and downstream activation of many signal transduction pathways including MAPK, PI3K/AKT/mTOR, PLCγ/DAG/IP3, JAK/STAT, and SRC pathway, with consequent stimulation of cell survival, proliferation, motility/invasion and angiogenesis [The proto-oncogene ogenesis ,71. Mutaogenesis ,75,76,77KIT mutations are found in less than 10% of cases. On the other hand, c-KIT overexpression is rare in TMs (2–4%), and no known mutation, except for a KIT deletion in a patient with AB thymoma reported on TCGA PanCancer Atlas, have been reported [KIT reported in TCs that show different drug susceptibility are V560del at exon 11, H697Y at exon 14, L576P at exon 11, Y553N at exon 11, D820E at exon 17, V559G at exon 11, 577–579del at exon 11, and K642E at exon 13 [Among TETs, TCs frequently present c-KIT overexpression 46–79%), whereas reported ,78,79,80%, wherea exon 13 ,82,83,84 exon 13 ,81,83,85 exon 13 ; D820E mKIT mutation [KIT mutations sensitive to imatinib [KIT sensitive mutations should be designed in order to assess the real activity of imatinib. However, the overall rarity of TETs and KIT mutations in these tumors makes a prospective trial unfeasible.Two phase 2 trials investigated the activity of imatinib in pre-treated patients with TETs. Neither study met its primary endpoint (ORR), but patients were not selected by the presence of mutation ,87. Indeimatinib ,83,85. FSunitinib, a multikinase inhibitor of VEGFR, c-KIT, and PDGFR among others, was studied in a phase 2 trial with promising results . The stuKIT mutations [KIT mutation (D820E at exon 17) [Sorafenib, another multikinase inhibitor of RAF, VEGFR, c-KIT, PDGFR, and other kinases , showed utations ,82,84,90cylindromatosis deubiquitinase (CYLD) gene is located in the long arm of chromosome 16 at position 12.1 (16q12.1) [The 16q12.1) . It enco16q12.1) . Ubiquit16q12.1) . CYLD pr16q12.1) ,93. CYLD is also involved in thymus development, in particular in differentiation and maturation of thymic medullary epithelial cells (mTECs): in fact, CYLD is a positive regulator of T-cell receptor signaling during the double-positive to single-positive transition of thymocytes, and also controls the nuclear entry of Bcl-3. Moreover, CYLD regulates the AIRE  expression in mTECs that is crucial for T-cell development . Because CYLD has a central role in inflammation, cell death, cell cycle progression, cell migration, DNA damage, and WNT signaling, CYLD loss-of-function is associated with the deregulation of NFκ-B, JNK, c-MYC, and AKT and consequent tumor development ,95, suchCYLD mutation in TETs is more frequent than in other tumors, especially in TCs with a prevalence >10%. CYLD-deficient TET cells, in presence of INFγ, upregulate PD-L1 via AKT-mediated increased STAT1 expression and increased activity of the STAT-IRF1 axis. CYLD loss also determines an increase in IRF1 in an INFγ-independent way by an increase in the basal Lys63-linked ubiquitination and consequent AKT activation. Activated AKT phosphorylates GSK3β and prevents it from phosphorylating IRF1, leading to missed IRF1 ubiquitination. The final effect is an increase in IRF1 half-life and activity [CYLD mutation was identified in 5 of the 36 tumors in which targeted exome sequencing was conducted, and it was associated with high PD-L1 expression. A post-hoc analysis showed a non-significant trend between CYLD mutation and longer PFS and OS [activity ,98. Overactivity . Of noteactivity ,99. CYLDCYLD mutation or loss might serve as a potential biomarker of response to ICIs to better patient selection.These findings suggest that Angiogenesis is one of the hallmark of cancer and is sustained by the production of several growth factors, such as VEGF, PDGF, transforming growth factor beta (TGFβ), and angiopoietins. Nevertheless, tumor vessels are usually immature and defective, with consequent induction of hypoxia, decreased immune cell infiltration, increased risk of tumor dissemination, and reduced efficacy of drugs and radiotherapy ,101.Remarkably, the expression of vascular growth factors and their receptor has been observed in TETs, with a correlation between high levels and aggressive histology types . RecentlAs angiogenesis has a central role in cancer development and progression, many drugs targeting this process have been developed and are currently available and approved for different cancer types ,105. BevIn respect to new antiangiogenic drugs, a recent case report described the efficacy of anlotinib, a multi-target tyrosine kinase inhibitor (TKI) that targets VEGFR, FGFR, PDGFR, and c-KIT, in a patient with refractory TC who achieved a SD with a PFS of 23 months . HoweverBAP1 (8%), SET domain containing 2 (SETD2) (6%), additional sex combs like 1 (ASXL1) (4%), DNA methyltransferase 3 alpha (DNMT3A) (4%), ten-eleven translocation 2 (TET2) (4%), Wilms tumor 1 (WT1) (4%) and SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) (3%). The prevalence of these mutations is higher when considering only TCs, being 13% for BAP1 mutations, 9% for SETD2 mutations, 6% for DNMT3A mutations, and 4% each for ASXL1, SMARCA4, TET2, and WT1 mutations, respectively [Epigenetic processes regulate the transcriptional status of genes, chromosomal domains or entire chromosomes through chromatin remodeling, histone modification, DNA methylation/demethylation and interaction with ncRNAs, inducing a phenotype change without modifying the underlying DNA sequence . TETs, aectively .MGMT is a frequently aberrantly methylated gene in TETs. This occurred far more commonly in TCs than TMs (74% vs 29%), with a significant association with loss of gene expression [MGMT promoter is correlated with a higher sensitivity to alkylating agents in different cancer types, including gliomas, lymphomas, and pancreatic NETs [Methylation of DNA is the most studied epigenetic mechanism and is obtained by the addition of a methyl group to a cytosine residue in the context of CpG dinucleotides. CpG dinucleotides are present diffusively in the whole genome but are aggregated in CpG-rich regions, referred to as CpG islands. Methylation is catalyzed by different DNA methyl-transferases (DNMTs): DNMT1, DNMT3A, and DNMT3B. Methylation of CpG islands impedes interaction of DNA regulating regions with transcription factors or promotes recruitment of inhibitory proteins with the final effect of transcription silencing. On the other hand, demethylation, controlled by TET methylcytosine dioxygenases (TET1-3), eventually promotes transcription and has a fundamental role during embryogenesis ,110,111.pression . Methylatic NETs ,115,116,Histones  are basic proteins with positive charge that pack DNA into repeating nucleosomal units and condensing them into chromatin. Histones are subjected to post-translational modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation, that modify the DNA-histone and histone-histone interactions, and regulate transcription by modulating access to chromatin by DNA translation machinery . An unbaPTEN, while miR-145-5p is a negative regulator of EGFR expression [ncRNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), have a fundamental role in transcriptional regulation: miRNAs bind and inhibit mRNAs; lncRNAs interact with transcriptional regulation proteins, influencing chromatin structure and regulating mRNAs expression; circRNAs regulate mRNA splicing; all those ncRNAs have also a “sponge effect”, that allow a reciprocal regulation . In cancpression . Deregulpression . The theBAP1 is the most frequently mutated epigenetic regulatory gene in TCs. This tumor suppressor gene is located in the short arm of chromosome 3 at position 21.1 (3p21.1) and encodes for a deubiquitinating enzyme. Germline heterozygous BAP1 mutations are responsible for the BAP1-cancer syndrome, an autosomal dominant condition characterized by high susceptibility to developing cancers, particularly uveal melanoma, malignant mesothelioma, cutaneous melanomas, renal cell carcinoma, and cholangiocarcinoma [BAP1 and other genes encoding epigenetic regulators may sensitize tumor cells to histone methyltransferase enhancer of zeste homolog 2 (EZH2) inhibitors, such as tazemetostat, which are currently entering early phases clinical trials but have not yet been investigated in TETs [arcinoma . BAP1 dearcinoma ,125. Sinarcinoma . Moreove in TETs .TP53 gene is located in the short arm of chromosome 17 at position 13.1 (17p13.1) and encodes for the tumor-suppressor protein p53 [TP53 is the most frequent mutated gene in human cancer since about 50% of tumors harbor a mutation in this gene. Mutations of TP53 determine a loss-of-function in the onco-suppressive activity of the protein, but also a gain-of-function in oncogenic properties of the mutant p53 [tein p53 , which ctein p53 . TP53 istant p53 ,129.In healthy thymic epithelial cells, p53 is a key regulator of mTEC differentiation, through the RANK-NFκ-B pathway, and controls the expression of the tissue-restricted antigens. A p53 deficiency determines an aberrant thymopoiesis and an altered T-cell peripheral homeostasis with consequent abnormal immunological tolerance .TP53 is one of the most frequently mutated genes also in TETs, especially in TCs, in which mutations in TP53 were reported in 18.5–26% of cases and are associated with worse outcomes with respect to TP53 wild-type tumors [e tumors ,132,133.TP53 are also associated with resistance to chemotherapy because of the involvement of multidrug resistance gene 1 (MDR1/ABCB1) [Mutations in 1/ABCB1) . Many mo1/ABCB1) ,134. Dru1/ABCB1) ,134. To Cell-cycle transition through the four phases G1, S (DNA synthesis), G2, and M (mitosis) is strictly regulated by the cyclin-dependent kinases (CDKs) and upstream signaling pathways, such as mitogen, hormone, or growth factor stimulation. CDK3/cyclin C complex regulates the entry into cell cycle from G0 (quiescence). In the early phase G1, Cyclin D activates CDK4 and CDK6, which phosphorylate the tumor suppressor Retinoblastoma (RB) protein. Phosphorylated RB releases the E2F transcription factors, resulting in gene expression required for transition into the S phase. The CDK2/cyclin E complex completes the transition from G1 to S phase. The progression through phase S is controlled by the CDK2/cyclin A complex, while phase G2 is regulated by the complex CDK1/cyclin A, and CDK1/cyclin B complex completes the mitosis process. The cyclin kinase inhibitors (CKIs), such as p16, p21, and p27, negatively regulate the cell-cycle progression ,136.CDKN2A/B, hyper-methylation of their promoter, and loss of expression of p16, p21, and p27 [CDKN2A (9p21) lead to p16 decrease and CDK4/6 hyper-activation and are associated with a worse prognosis in TCs [Dysregulations in CDKs, cyclins, and CKIs are frequent in human cancers, leading to abnormal cell proliferation. Cell-cycle aberrations have been also described in TETs, with alterations mainly in the CDK/RB pathway ,137. The and p27 . FurtherInhibitors of the CDKs have been studied in many human cancers, and are currently approved for hormone-sensitive breast cancer treatment ,140.The role of milciclib, an oral inhibitor of CDKs, tropomyosin receptor kinase A (TRKA), and SRC family kinases, was investigated in two phase 2 studies . The CDKExportin 1 (XPO1) gene is located in the short arm of chromosome 2 at position 15 (2p15) and encodes for XPO1, a nuclear exporter of proteins and RNAs [and RNAs . The excand RNAs . TMs andand RNAs . EGFR and MYC [Selinexor is a selective inhibitor of XPO1 that promotes its proteasomal degradation and consequently forces the nuclear localization and functional activation of tumor-suppressor proteins, prevents the oncoprotein mRNA translation, and causes cell cycle arrest and apoptosis in malignant hematologic and solid tumor cells ,146,147. and MYC . In TET cells, selinexor determined nuclear accumulation of the tumor-suppressor proteins FOXO3a, p53, and p27. Additionally, selinexor determined cell-cycle arrest through the shuttling of many proteins that regulate cell-cycle progression and apoptosis due to the induction of the pro-apoptotic proteins BIM and BAX. Of note, GTF2I is another target of XPO1 . A phase 1 trial evaluated the safety and efficacy of selinexor in 189 patients with advanced solid tumors. Remarkably, 4 TET patients were included, one had a PR, and three patients had SD, making selinexor a potential drug of interest for the future of TET treatment .Thymic neuroendocrine tumors (tNETs) are rare primary thymic neoplasms characterized by neuroendocrine differentiation, accounting for 2% of all neuroendocrine tumors, and about 5% of all thymic malignancies . tNET arThe 25% of tNETs arise in patients affected by multiple endocrine neoplasia type 1 (MEN1), a genetic disorder that predisposes to developing different kinds of neuroendocrine tumors ,154. AppBecause tNET are rare tumors, data to guide optimal treatment are limited and came from small retrospective trials and case series. Surgery is still the only curative-intent treatment and a complete resection represents the most significantly favorable prognostic factor for survival. Adjuvant radiation therapy (RT) plays a role in subtotally resected or locally advanced unresectable nonmetastatic disease. The evidence supporting the benefit of adjuvant RT is limited and, although it is associated with improved local control, there is no evidence of a survival benefit ,158. ForTP53 (18.5%), followed by KIT (7.4%) and PDGFRA (5.6%), which are commonly altered also in TETs [Even if the knowledge of the genetic variability of TMs and TCs has been deepened in recent years, still today, little is known about tNETs molecular characteristics. Sakane et al. have sequenced by NGS with a panel including 50 common cancer-related genes 54 patients with thymic neoplasia, including 48 TCs and 6 tNETs. The authors reported no significant differences in mutation frequency between TC and tNETs. The 3 most frequently mutated genes were  in TETs . CurrentAs there is no standard treatment for patients with advanced TETs after PD to platinum-based chemotherapy, several strategies, including targeted molecules, are being explored in different therapeutic settings .As surgery has a prominent role in the therapeutic strategy and outcome of thymic malignancies, neoadjuvant treatment with the aim of reducing tumor size and improving surgical outcomes might translate into better overall survival. With this aim, the preoperatory association of cetuximab and neoadjuvant chemotherapy (CAP regimen) in patients with resectable clinical Masaoka stage II-IVA TM or TC is under investigation in a phase 2 trial (NCT01025089).In addition, many clinical trials are investigating the potential role of targeted therapies as single-agent therapies or in combination with other types of systemic treatment . Indeed, the effectiveness of targeted agents in refractory/relapsed TETs, could be enhanced by adopting combination strategies, hence representing a promising approach.The multi-target TKI regorafenib has different targets involved in tumor angiogenesis and cell proliferation . A single-arm phase 2 trial (RESOUND) explores the activity of regorafenib in patients with different metastatic solid tumors refractory to available standard treatment, including TM (type B2–B3) and TC (NCT02307500).Similarly, a phase 2 trial investigates the activity of sunitinib in patients with type B3 TM or TC who have received at least one prior platinum-containing chemotherapy regimen .To date, several trials are ongoing to better define the role of multi-targeted TKI in TETs when associated with other systemic treatments .The RELEVENT study is an open-label phase 2 study of the combination of ramucirumab, carboplatin, and paclitaxel that will evaluate activity and safety in the first-line setting for relapsed or metastatic TETs of any histological type (NCT03921671). Of note, this study will evaluate the mutational status of a subset of genes, polymorphisms, and selected miRNA expression .A phase 2 trial is assessing the activity of pembrolizumab, an anti-PD 1 monoclonal antibody, and sunitinib in participants with TC, not amenable to curative treatment (NCT03463460). Similarly, a multicentric, open-label, single-arm phase 2 study (PECATI study) will evaluate the efficacy and safety of the pembrolizumab-lenvatinib combination in pretreated immunotherapy-naïve patients with TC (NCT04710628).Another phase 1/2 study will evaluate the safety and preliminary activity of the oral VEGFR/PDGFR kinase inhibitor vorolanib (CM082) combined with the anti-PD-1 nivolumab in patients with thoracic malignancies, including TC (NCT03583086).In conclusion, the combination of TKI and immunotherapy may represent a promising strategy, although TKI induced cardiotoxicity risk could overlap with immune-mediated cardiological toxicity risk . The toxNucleocytoplasmic transport is often altered in TETs . An ongoDeregulation of TGF-β signaling pathway is observed across tumor types, as a consequence of increased expression of TGF- β or mutations/deletions of other axis components  . MultiplMesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas. Anetumab ravtansine (BAY 94-9343) is an antibody-drug conjugate directed against mesothelin expressing cancer cells and able to induce a bystander effect on neighboring mesothelin-negative tumor cells, displaying encouraging preliminary antitumor activity in heavily pretreated patients . A PhaseThymic neoplasia are rare malignancies with limited therapeutic options. Recent advances in the understanding of TET biology fostered by the wide access to new technologies, such as NGS, allowed to identify features underpinning molecular differences between TM and TC, some of which also served as potential targets for specific treatments. Despite the dramatic clinical and biological diversity, many clinical trials had enrolled patients with either histology due to the rarity of TETs. A short-term goal for TETs investigation should be tailoring clinical trials to histology and molecular subtypes. Recently, promising results from targeted therapies and immunotherapy have been reported, but safety and appropriate response biomarkers identification are still open questions. Although several studies have been led and targets have been identified, no targeted treatment is currently approved for TET patients in Europe.


Simulation results show that, under oblique incidence with TE polarization, ED-SLRs with extremely narrow linewidth down to 12 nm and high quality factor up to 409 can be excited in the mid-infrared regime. By varying the incidence angle, the ED-SLR can be tuned over an extremely large spectral region covering almost the entire mid-infrared regime. We further numerically show that, by changing the GST crystalline fraction, the ED-SLR can be actively tuned, leading to nonvolatile, reconfigurable, and narrowband filtering, all-optical multilevel modulation, or all-optical switching with high performance. We expect that this work will advance the engineering of Mie SLRs and will find intriguing applications in optical telecommunication, networks, and microsystems.Mie surface lattice resonances (SLRs) supported by periodic all-dielectric nanoparticles emerge from the radiative coupling of localized Mie resonances in individual nanoparticles through Rayleigh anomaly diffraction. To date, it remains challenging to achieve narrow bandwidth and active tuning simultaneously. In this work, we report extremely narrow and actively tunable electric dipole SLRs (ED-SLRs) in Ge Surface lattice resonances (SLRs), sometimes referred to as collective resonances or diffractively coupled localized resonances, were initially observed in periodically arranged plasmonic nanoparticles, owing to the coherent interference between the localized surface plasmon resonance (LSPR) and the Rayleigh anomaly (RA) diffracted light ,2. BecauQ is intrinsically linked to the ratio of energy stored to the energy lost by an oscillator, and can be estimated as evaluated as the ratio of the resonance wavelength to the linewidth. Castellanos et al. xSbyTez, T) [2Te3 , Sb2S3, d Sb2Se3 ,20, haved Sb2Se3 ,21,22,23d Sb2Se3 , tunabled Sb2Se3 ,27,28,29d Sb2Se3 ,31,32,33d Sb2Se3 ,35,36 wed Sb2Se3 ,38. Based Sb2Se3 ,42,43,44d Sb2Se3  were als2Se2Te5 nanorod array under oblique incidence with TE polarization. We will show that, by increasing the incidence angle from 0° to 70°, the resonance wavelength of the ED-SLR can be tuned over a very broad spectral region, which spans from 3.171 μm to 4.908 μm and covers almost the entire mid-infrared regime (3–5 μm). Meanwhile, the linewidth decreases dramatically to 12 nm, and the corresponding quality factor increases by more than ten times, from 38 to 409. We will also show that, by changing the GST crystalline fraction, which can be done using nanosecond [In this work, we provide a solution to this problem by launching ED-SLRs in a Genosecond  or femtonosecond  optical x- and y-directions, diameter The operation principle of the metasurface is designed as follows. When GST is in the amorphous state, the ED-SLR can be excited for the TE-polarization, leading to extremely narrow bandpass filtering in reflection and correspondingly bandstop filtering in transmission, as illustrated by The designed metasurface can be fabricated using the state-of-the-art nanofabrication processes following . A GST fThe total reflectance and transmittance spectra, ollowing ,47,48. Iken from , are theFor the TE polarization, In sults in   and  , these Therefore, we have shown that the operation wavelength of the high-performance dynamically tunable optical filtering, modulation or switching can be conveniently tuned by varying the incidence angle.2S3 and Sb2Se3 suffer from much lower loss [In conclusion, we have numerically demonstrated extremely narrow and actively tunable ED-SLRs in GST metasurfaces. Simulation results have shown that the extremely narrow ED-SLR can be excited close to the wer loss ,20. We t


PD-L1, LAG3 and T-bet were associated (Cramér’s V=0.3) with MSI/dMMR tumors and with a shorter overall survival , whereas high levels of expression of EOMES, TRAF1, PD-L1, FCRL4, BTLA and SIGLEC6 were associated with a shorter overall survival , independently from the molecular subtype of CRC. Expression levels of PD-L1, PD-1, LAG3, EOMES, T-bet, and TIGIT were significantly correlated with each other and associated with genes coding for +CD4 and +CD3+CD8 T cell markers and +CD94+EOMES+T-bet+NKp46 cell markers, , which identify a subset of group 1 innate lymphoid cells, namely conventional (c)NK cells. Expression of TRAF1 and BTLA co-occurred with both T cell markers, CD3γ, CD3δ, CD3ε, CD4, and B cell markers, CD19, CD20 and CD79a . Expression of TGFβ1 was associated only with CD4+ and +CD3ε+CD8 T cell markers . Expression of PD-L2 and IDO1 was associated  only with cNK cell markers, whereas expression of FCRL4, SIGLEC2 and SIGLEC6 was associated  with +CD20+CD79a+CD19 B cell markers. Morphometric examination of immunostained CRC tissue sections, obtained from a validation cohort of 53 CRC patients, substantiated the biostatistical findings, showing that the highest percentage of immune exhaustion gene expressing cells were found in tumors from short-term survivors and that functional exhaustion is not confined to T lymphocytes, but also involves B cells, and cNK cells. This concept was strengthened by CYBERSORTx analysis, which revealed the expression of additional immune exhaustion genes, in particular FOXP1, SIRT1, BATF, NR4A1 and TOX, by subpopulations of T, B and NK cells. This study provides novel insight into the immune exhaustion landscape of CRC and emphasizes the need for a customized multi-targeted therapeutic approach to overcome resistance to current immunotherapy.Colorectal cancer (CRC) is one of the most common cancer worldwide, with a growing impact on public health and clinical management. Immunotherapy has shown promise in the treatment of advanced cancers, but needs to be improved for CRC, since only a limited fraction of patients is eligible for treatment, and most of them develop resistance due to progressive immune exhaustion. Here, we identify the transcriptional, molecular, and cellular traits of the immune exhaustion associated with CRC and determine their relationships with the patient’s clinic-pathological profile. Bioinformatic analyses of RNA-sequencing data of 594 CRCs from TCGA PanCancer collection, revealed that, in the wide range of immune exhaustion genes, those coding for  Colorectal cancer (CRC) ranks third for incidence, after breast and lung cancers, and second for mortality, after lung cancer . MortaliAdvances in patient-tailored therapy, which is based on the mutational, transcriptomic, and proteomic profiles of individual CRC , and on In this study, using bioinformatics, we analyzed the transcriptional profile, and concomitant immune cell context, of the immune exhaustion in CRCs of the TCGA PanCancer collection, and its relationship with patients’ clinical-pathological characteristics and follow-ups. A validation cohort of CRC patients, which includes short- and long-term survivors, provided tumor samples to localize and quantify, by immunohistochemistry and computerized morphometric analysis, immune exhaustion gene expression at protein level.Our findings reveal that high levels of expression of specific immune exhaustion genes occurs in MSI molecular subtype, however, independently from the molecular subtype of CRC, high expression levels of a range of immune exhaustion genes regulating different immune cell subsets, are significantly associated with worst prognosis. Biostatistical co-occurrence and morphological co-localization of the expression of immune exhaustion genes and markers of T and B lymphocytes, and of conventional (c) NK cells, strongly suggest that, regardless of the molecular subtype of tumor, CRC-associated immune exhaustion is not only a “T cell matter”. CIBERSORTx analysis corroborates this concept revealing that the expression of relevant immune exhaustion-related genes, detected in the different T lymphocyte subpopulations, also involves B and NK cell subpopulations.Profiling of functional immune exhaustion in individual CRC can lead to the development of a successful patient tailored therapy to counteract metastasis and disease recurrence.Colorectal Adenocarcinoma TCGA PanCancer collection”, which includes 594 CRC cases . The expression of the genes listed in the For bioinformatic analyses, RNA sequencing (seq) data  of tumor samples from the “RC cases Table 1,ρ) was used to exclude correlations between gene expression and patients’ age, sex and TNM staging.For each CRC sample, the Z-score of the expression level for each gene of interest was calculated and compared to the mean of all the samples in the study. Samples with a Z-score ≥ 2 were considered high-expressing, whereas samples with a Z-score < 2 were considered low-expressing. Survival curves were constructed (with CRC cases for which both gene expression and follow-up data were available) using the Kaplan-Meier method and survival differences were analyzed by the Log-rank test, whereas the association between gene expression and CRC molecular subtypes was assessed using Cramér’s V test. Gene co-occurrence analysis was performed by odds ratio (OR) calculation, while Spearman’s correlation coefficient -normalized RNA-seq data of the “BERSORTx , a compuBERSORTx , and batα level of 0.05, using Stata, version 13 .All statistical tests were evaluated at an To validate bioinformatic data at the protein level, we collected colon tissue samples and clinicopathological profiles of patients  who undeThe study was reviewed and approved by the Ethical Committee of the “G. d’Annunzio” University and Local Health Authority of Chieti, Italy. The study was performed, after written informed consent from patients, in accordance with the principles outlined in the Declaration of Helsinki.CRC samples, obtained from the validation cohort, based on standard sampling protocols , were fi2 field, on single or double immunostained sections, with Qwin image analysis software (version 2.7) . The Qwin image analyses ensures the following highly reproducible steps: 1) image acquisition; 2) conversion of RGB image (true colors) to binary image (black and white); 3) filtering to remove noise; 4) counting of immunostained cells or measurement of positively stained area. Six to eight high-power fields, randomly chosen, were evaluated for each section and two sections per sample were analyzed. Results were expressed as mean percentage ± standard deviation (SD) of positive cells/number of total cells per field. Immunostained sections were examined by two pathologists in a blind fashion, with very good agreement . Representative images of immunostained sections for exhaustion markers were captured in the stroma, between tumor glands, or in lymphoid aggregates, because in these areas their expression was more evident, regardless of the number of lymphoid-like structures in tumor samples from short versus long-term survivors, which has been the subject of previous investigations .For the bioinformatics, statistical analyses have been described above. For morphometric studies, between-group differences were assessed by Student’s inhibitory receptors, immunosuppressive mediators, and transcription factors, as listed in the Functional and exhausted immune cells display distinct transcriptional programs , 20. To MLH1, MSH2, MSH6 or PMS2)  with MSI/dMMR tumors, which were diagnosed in 63/594 (10.61%) of CRC patients  . This deotherapy . Bioinfopatients Table 5.PD-1, LAG3 and T-bet in CRC samples, was related with patients’ clinical outcome, Kaplan Meier survival analysis was performed for patients with CRC, of all stages and molecular subtype. As shown in PD-1 (PD-1high) had an OS of 29.43 months, versus 91.21 months of patients bearing tumors with a low level of expression (PD-1low) .To assess whether the expression of high, or T-bethigh was 34.68 and 39.82 months, respectively, versus 91.63 and 91.45 months of patients with LAG3low, or T-betlow tumors . Odds ratio calculation revealed that the expression of LAG3 was strongly associated with that of PD-1 . In agreement with this finding, high levels of co-expression of these genes were also associated with a shorter OS  , which were distinguished, by outcome, as short-term  and long-term  survivors Table 6.PD-1, LAG3 and T-bet was detected in lymphoid aggregates and scattered among the neoplastic glands, or at intra-epithelial sites, whereas LAG3 was also occasionally expressed by clusters of colonic cancer cells (p<0.001) in CRC samples from short-term survivors, than in tumor samples from long-term survivors, as reported in the Expression of p<0.0001) of double positive cells than tumors from long-term survivors . Notably, co-expression of high levels of T-bet and EOMES led to a further reduction in the OS , than patients bearing PD-L1low or TRAF1low CRCs  than in CRC samples from long-term survivors  higher in short-term survivors than in long-term survivors. Moreover, immunostaining for FCRL4 and double staining for both CD20 and FCRL4 performed on CRC samples from short-term survivors, also showed mean percentages of single or double positive cells significantly (p<0.0001) higher than that found in CRC samples from long-term survivors  correlated with each other and were associated with the expression of genes coding for CD4, CD8a, and CD3γ, CD3δ and CD3ε chains, with an OR >1.5; p<0.05, which suggests that expression of these immune exhaustion genes primarily involves +CD8 and +CD3+CD4 T cells. Furthermore, expression of TRAF1 co-occurred with those of PD-1, PD-L1, EOMES, T-bet, TIGIT, BTLA, CD3γ, CD3δ, CD3ε, CD4 and CD8a, with an OR >2, p<0.05.Expressions of BTLA co-occurred with the expression of PD-1, PD-L1, EOMES, T-bet, TIGIT, TRAF1 CD3γ, CD3δ, CD3ε and CD4 , whereas TGFβ1 co-occurred with PD-1, PD-L1, LAG3, CD3γ, CD8a, and CD4 .+ cells, in the lymphoid aggregates, or scattered in the stroma and CRC epithelia, substantiated the biostatistical findings  cells, but also involved both naïve and memory B cells, and that expression of SIRT1 and NR4A1 was extended to naïve B cells, whereas expression of BATF was also found in resting NK cells.BTLA, FCRL4 and SIGLEC6, that were found to be associated with a worst outcome , which also dampens BCR signaling , which was confirmed by immunohistochemistry  CRC tissue sections, from short-term survivors of the validation cohort, revealed a distinct expression of TIGIT, IDO1 and PD-L2 among the neoplastic glands, mostly in the areas infiltrated by EOMESTaken together, immunohistochemical and morphometric analyses substantiated the biostatistical findings, which suggested that CRC-associated immune exhaustion, demonstrated by high levels of a wide range of immune checkpoint molecules, affects T and B lymphocytes, and cNK cells, and is associated with a worse clinical outcome, independently of the disease stage and molecular subtype.Despite the survival benefit of early detection through increased screening, CRC remains a leading cause of cancer related death , which iPD-L1, LAG3 and T-bet in the context of MSI tumors -associated factors (TRAF) protein family, which plays important roles in the immune system as key intracellular signaling molecules in TNFR, Toll-like receptor (TLR), cytokine and antigen receptor signaling pathways . TRAF1 eBoth PD-1, which binds to PD-L1 and PD-L2, and LAG3, which binds to MHC class II molecules, can be found on T and B lymphocytes, macrophages, dendritic cells (DCs), and NK cells –59. In C+ T cells , a member of the tumor necrosis factor receptor superfamily (TNFR-SF) . The inh T cells . BTLA bl T cells –67. Alth T cells . Further T cells .In vivo, inhibition of TGFβ1 signaling in CD8+ T cells, through expression of a dominant-negative receptor, improved the function of exhausted T cells . Expression of BATF involves CD8+ cells .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Cutaneous sweat gland tumors form an extremely diverse and heterogeneous group of neoplasms that show histological differentiation to the sweat apparatus. Due to their rarity, wide diagnostic range, and significant morphological overlap between entities, their accurate diagnosis remains challenging for pathologists. Until recently, little was known about the molecular pathogenesis of adnexal tumors. Recent findings have revealed a wide range of gene fusions and other oncogenic factors that can be used for diagnostic purposes and, for some, can be detected by immunohistochemistry. Among other organs containing exocrine glands, such as salivary glands, breasts, and bronchi, most of these biomarkers have been reported in homologous neoplasms that share morphological features with their cutaneous counterparts. This review aims to describe these recent molecular and immunohistochemical biomarkers in the field of sweat gland tumors.NFIB, MYB, and MYBL1 fusion), cutaneous mixed tumor (PLAG1 fusion), cylindroma and spiradenoma and their carcinomas thereof (NF-κB activation through CYLD inactivation or ALKP1 hotspot mutation), hidradenoma and hidradenocarcinoma (MAML2 fusion), myoepithelioma (EWSR1 and FUS fusion), poroma and porocarcinoma , secretory carcinoma , tubular adenoma and syringo-cystadenoma papilliferum (HRAS and BRAF activating mutations). Sweat gland tumors for which there are no known molecular abnormalities will also be briefly discussed, as well as potential future developments.Cutaneous sweat gland tumors are a subset of adnexal neoplasms that derive or differentiate into the sweat apparatus. Their great diversity, rarity, and complex terminology make their pathological diagnosis challenging. Recent findings have revealed a wide spectrum of oncogenic drivers, several of which are of diagnostic interest for pathologists. Most of these molecular alterations are represented by gene fusions, which are shared with other homologous neoplasms occurring in organs containing exocrine glands, such as salivary and breast glands, which show similarities to the sweat apparatus. This review aims to provide a synthesis of the most recent immunohistochemical and molecular markers used for the diagnosis of sweat gland tumors and to highlight their relationship with similar tumors in other organs. It will cover adenoid cystic carcinoma ( Benign and malignant cutaneous adnexal tumors are rare . They spAn update of the nosology of tumors of cutaneous appendages has been proposed by the fourth edition of the WHO classification of cutaneous neoplasms . At the MYB gene, skin cylindromas represent a distinct tumor entity with frequent CYLD mutation. Furthermore, some of these biomarkers are related to a whole group of adnexal neoplasms since they are detected in benign and malignant cases, while some evidence suggests a multistep progression from a benign precursor with a specific molecular background to their malignant counterpart. Histopathological classifications attempted to separate the wide variety of adnexal neoplasms based on their differentiation to specific parts of the skin appendages, including apocrine, eccrine, follicular, sebaceous, and multilineage differentiation. The cell of origin remains the subject of debate and investigation for many neoplasms. The difficulty in determining the cell of origin is related in part to the biology of many fusion-driven sweat gland tumors. Indeed, chimeric fusion proteins confer a new, often aberrant, histological phenotype that lacks a physiological cellular counterpart to correlate with for classification purposes. Hopefully, most of these gene fusions are highly specific in each morphological context, being mostly restricted to a single subtype of tumor.Most skin carcinomas, especially basal and squamous cell carcinomas, exhibit an ultraviolet mutational signature and a high tumor mutational burden, which are considered key oncogenic events in their initiation and progression ,4. On thThis review aims to provide a synthesis of the most recent immunohistochemical  and moleNFIB and MYB fusion), mixed tumor (PLAG1 fusion), cylindroma and spiradenoma (NF-κB activation through CYLD inactivation or ALKP1 hotspot mutation), hidradenoma (CRTC1/3 and MAML2 fusion), myoepithelioma (EWSR1 and FUS fusion), poroma (YAP1 and NUTM1 fusion), secretory carcinoma (ETV6 and NTRK3 fusion), tubular adenoma and syringocystadenoma papilliferum (HRAS and BRAF activating mutations). For each entity, data regarding the respective homologous extracutaneous tumors will be mentioned.We will review, in alphabetical order, adenoid cystic carcinoma  and MYBL1::NFIB (20–23%) [MYB rearrangement. Extracutaneous cases lacking these recurrent alterations have been reported with alternative fusion partners (ACTN1) and amplification of MYB [MYB), and MYB proto-oncogene like 1 (MYBL1) are genes mapped on chromosomes 6 and 8, respectively. MYB and MYBL1 rearrangements and amplification cause cell proliferation [NSR, MET, EGFR, IGF1R, and IGF2 (89). The NOTCH pathway genes are commonly altered in advanced adenoid cystic carcinoma and can be targeted by specific therapies [Molecular biology: Fusions of MYB or MYBL1 with NFIB are detected in 40–97% of adenoid cystic carcinomas with very high specificity ,16,19,2020–23%) ,22,23,24% ,22,23,n of MYB . MYB oveferation  through herapies .MYB::NFIB fusions have been reported in tumors diagnosed as wild-type CYLD cutaneous cylindroma in a single report [MYB fusion in bona fide cutaneous cylindroma is highly questionable.Discordant or controversial data: Contrary to the high specificity reported by numerous studies ,16,19,20e report , but thie report . Since cGeneral features: Cutaneous mixed tumor .Related terminology: The homologous neoplasm in the salivary gland, breast, and lung is called pleomorphic adenoma.Immunohistochemistry: Like adenoid cystic carcinoma, a cutaneous mixed tumor shows epithelial and myoepithelial differentiation but also a stromal component. The panel of antibodies to highlight its multiple cell populations includes SOX10, p63, PS100, calponin, SMA, EMA, c-KIT, CEA, and various cytokeratins ,13,14. FCTNNB1, LIFR, and CHCHD7. This explains why pleomorphic adenomas of the salivary gland demonstrate a wide range of PLAG1 and HMGA2 fusions: CTNNB1::PLAG1, LIFR::PLAG1, CHCHD7::PLAG1, TCEA1::PLAG1, HMGA2::FHIT, HMGA2::NFIB, and HMGA2::WIF1. Although NDRG1::PLAG1 and TRPS1::PLAG1 fusions have been described in cutaneous cases [HMGA2 fusion has been reported in cutaneous mixed tumors. More studies are required to define more precisely the genetic landscape of cutaneous mixed tumors and, in particular, their relationship with myoepithelioma when their myoepithelial component is predominant (cf. myoepithelioma).Molecular biology: Recurrent rearrangements of PLAG1 are detected in 33% of cutaneous mixed tumors by FISH , which ius cases , anotherus cases . To dateDiscordant or controversial data: A PHF1::TFE3 fusion transcript was detected in one case morphologically diagnosed as malignant chondroid syringoma . HoweverCYLD or ALPK1 mutations. Spiradenoma differs from cylindroma by its nodular architecture and lymphocytic infiltrate. On rare occasions, both neoplasms show a morphological and phenotypical overlap with adenoid cystic carcinoma. Most cases occur sporadically; however, some cases arise in the context of a predisposition syndrome known as Brooke–Spiegler syndrome. Multiple cylindromas, spiradenomas, trichoepitheliomas, and basal cell salivary gland tumors occur in this autosomic dominant disorder linked to CYLD mutations.General features: Cylindroma and spiradenoma  are relaRelated terminology: Homologous neoplasms in the salivary gland and breast are called basal cell adenoma (membranous variant of) and adenocarcinoma. Confusingly, the term ‘cylindroma’ has historically been used in the salivary gland to describe adenoid cystic carcinoma.Immunohistochemistry: Cylindroma and spiradenoma staining patterns are similar to ACC ,13,14: SALPK1 and CYLD [CYLD mutations affect coding and splicing, resulting in inactivation of CYLD [CYLD mutations compared to approximately a third of cases of spiradenoma [DNMT3A have also been described in cylindroma [ALPK1 mutations involve predominantly a hotspot in the alpha-kinase domain (p.V1092A) and are detected in 43% and 28% of spiradenomas and spiradenocarcinomas, respectively. Consistent with a model of progression and transformation from precursor spiradenoma, loss-of-function TP53 mutations are restricted to spiradenocarcinoma [Molecular biology: Spiradenoma and cylindroma are related to oncogenic activation of the NF-κB pathway, triggered by mutually exclusive mutations of and CYLD . A wide  of CYLD . Most caradenoma . Disruptlindroma . ALPK1 mCYLD-defective cylindroma [CYLD-proficient dermal cylindroma demonstrated MYB fusions [CYLD-defective cylindromas, however, demonstrate upregulation and overexpression of MYB and its target genes [CYLD mutations have recently been described in a subset of high-risk HPV-positive head and neck squamous cell and anal carcinomas, which show histological features close to cylindroma and have been designated as CYLD-mutant cylindroma-like basaloid carcinoma [Discordant or controversial data: MYB fusions are highly specific and are considered the hallmark of ACC ,16,19,20lindroma . A singl fusions , which w fusions   is located on chromosome 19 and activates cAMP-responsive element binding protein (CREB) signaling. Mastermind-like 2 (MAML2) is located on chromosome 11 and is associated with the Notch signaling pathway. EGFR overexpression has been found in the whole spectrum of cutaneous hiradenoma, namely hidradenocarcinoma, atypical hidradenoma, and hidradenoma [EGFR in cutaneous cases lacked to demonstrate a clear correlation between the protein expression and the amplification of EGFR. Nonetheless, the EGFR signaling could still represent the mechanism of action of the CRTC1::MAML2 fusion protein since the CRTC1 portion of the chimeric protein acts on AREG, which is a ligand of EGFR. In addition, some cases of salivary mucoepidermoid carcinoma that lack CRTC1::MAML2 fusion rather demonstrate amplification and overexpression of EGFR and higher grade [MAML2 gene fusion in hidradenoma and hidradenocarcinoma requires more studies.Molecular biology: Half to 75% of the cases of hidradenoma have  fusions . Similar fusions ,52,53. Hfication ,55. CREBradenoma . Howeverer grade . In thiser grade . ConsideEWSR1::POU5F1 fusion has been described in tumors diagnosed as hidradenoma in a single report [cf. myoepithelioma).Discordant or controversial data: e report  but has e report . A subsee report   predominantly affects the sun-exposed area of the head and neck and has a high recurrence rate, slow growth, and low metastatic potential. MAC is an asymmetric and deeply infiltrative tumor that can present three components arranged in a vertical gradient, some of which may be lacking: small keratinizing cysts on the surface, solid aggregates in the center of the lesion, and, finally, deep tubular structures. As the histology of MAC in the superficial part of the skin resembles the histology of syringoma and basal cell carcinoma, an accurate diagnosis of MAC can be difficult on superficial biopsy. No specific molecular or immunohistochemical hallmark has yet been discovered.MYH9 mutations [Related terminology: Microcystic adnexal carcinoma is specific to the skin and lacks a homologous neoplasm in other organs. Solid carcinoma, reported in a series of 14 cases, may represent a variant of microcystic adnexal carcinoma . A low-gutations .Immunohistochemistry: Immunostains aim to differentiate between MAC, desmoplastic trichoepithelioma, basal cell carcinoma, and squamous cell carcinoma. In this setting, BerEP4, p63, and Ki67 can be used. BerEP4 shows negative or very infrequent staining in MAC, including the solid variant, while strong BerEP4 staining suggests basal cell carcinoma ,66,67,68Molecular biology: Analysis of mutation burden, UV signature, mutational status of 400 cancer-relevant genes, and copy number variations showed few recurrent somatic mutations and, in most cases, a lack of an ultraviolet signature, a counterintuitive finding regarding the preferential occurrence of MAC in chronically sun-exposed skin [TP53 mutations and frame-preserving indel mutations located in the kinase domain of JAK1, copy number alterations with gain of JAK2, MAF, MAFB, JUN, or FGFR1, and loss of CDKN2A. These data were consistent with another study in which sequencing indicated a mutation of TP53 with loss of CDKN2A and CDKN2B in metastatic MAC [sed skin . The samatic MAC . Anotheratic MAC . ConsideEWSR1 or FUS [EWSR1::PBX3 fusion. Malignant cases are extremely rare.General features: Myoepithelioma  is a rar1 or FUS . Unlike Related terminology: The term myoepithelioma is used regardless of the organ considered. Depending on the classification, myoepithelial tumors are considered mesenchymal.Immunohistochemistry: There is no specific immunostain for myoepithelial differentiation. Myoepitheliomas generally exhibit the expression of cytokeratins, S100 70–80%), SOX10 (30–82%), calponin (86%), EMA (65%), GFAP (45%), SMA (35%), desmin (15%), and p63 (15%) ,78,79. S%, SOX10 EWSR1 belongs to the TET family of proteins, a highly conserved group of multifunctional, RNA-binding proteins that includes fused in sarcoma (FUS). EWSR1 and FUS are related in both structure and function, are expressed in most cell and tissue types, and serve a multifunctional purpose and predominantly reside in the nucleus. Fusion proteins involving EWSR1 and FUS act as aberrant transcription factors . While EWSR1 gene fusions are lacking in cutaneous mixed tumors, many recurrent gene fusions involving the EWSR1 and FUS genes have been reported in cutaneous and soft tissue myoepithelioma [EWSR1::PBX3 fusions are more frequent in syncytial myoepithelioma compared to other cutaneous myoepitheliomas [EWSR1::POU5F1 and EWSR1::PBX3 are the two most frequent fusions [FUS::KLF17, EWSR1::PBX1, EWSR1::ZNF444, EWSR1::KLF15, FUS::POU5F1, EWSR1::KLF17, EWSR1::FUS, EWSR1::ATF1, and EWSR1::VGLL1 are less frequent [POU5F1-rearranged myoepitheliomas show a distinctive clear cell appearance that can be mistaken for hidradenoma, and PBX1-rearranged myoepitheliomas show sclerosis [SMARCB1 (INI1) is observed in a subset of malignant cases but has not been reported on the skin [Molecular biology: thelioma , with a heliomas ,81,82. I fusions , while Ffrequent ,86,87,88clerosis . Loss ofthe skin ,90,91.Discordant or controversial data: EWSR1::POU5F1 translocation is frequent in a subset of myoepithelial tumors with frequent extensive clear cell morphology that can be mistaken for hidradenoma [EWSR1::POU5F1 had been reported in tumors diagnosed as hidradenoma in a single report [radenoma . In thise report  but was e report   and NUT (clone C52B1) ,95,96: l1 fusion . NUT immarcinoma ,99. A hiarcinoma ,101,102.arcinoma ,104, andarcinoma . AE1/AE3arcinoma . GATA3 [arcinoma , SOX10 [arcinoma , and GCDarcinoma  are usuaMolecular biology: Recurrent YAP1 rearrangements have been detected in 88% and 63% of cases of poroma and porocarcinoma, respectively [YAP1::MAML2 fusion in porocarcinoma [YAP1::NUTM1 and WWTR1::NUTM1. Consistent with immunohistochemical data, a single study reported that YAP1::NUTM1 fusion was more common in benign and malignant poroid hidradenoma than in poroma and porocarcinoma [BRD3/4::NUTM1 fusion has been described in exceptional cases of primary or metastatic cutaneous adnexal carcinoma [CDKN2A, EGFR, ERBB2, FGFR3, HRAS, KRAS, NRAS, PIK3CA, TP53, and RB1 [BRCA2, and deleterious alterations in multiple homologous recombination repair pathway components. Although gene fusions in this study were not tested to confirm diagnosis, these data encourage investigation of targeting the p53 axis, PARP inhibition, and immunotherapy in the treatment of porocarcinoma [ectively , with suarcinoma  and in parcinoma . Yes1 asarcinoma . Other farcinoma . BRD3/4:arcinoma ,99. All arcinoma . Porocar and RB1 ,114,115.arcinoma . General features: Cutaneous secretory carcinoma  and ‘mammary-type secretory carcinoma of the skin’.Immunohistochemistry: NTRK fusions can be detected by immunohistochemistry using a panTRK antibody (clone EPR 17341) with various performances and staining patterns, with a sensitivity up to 100% for secretory carcinoma [ETV6, TRK immunohistochemistry is expected to stain the nucleus, while the cytoplasm and/or membrane are stained when alternative fusion partners are involved [arcinoma ,118,119.involved . Other pinvolved ,122. Molecular biology: Independent of the organ considered, most cases of secretory carcinoma are associated with ETV6::NTRK3 fusion [NTRK) family of genes includes NTRK1, NTRK2, and NTRK3, which regulate cell survival and differentiation. NTRK gene fusions have been shown to be actionable genomic events with the demonstration of highly efficacious outcomes with the use of TRK kinase inhibitors, such as larotrectinib and entrectinib [NFIX::PKN1 fusion has also been described [ETV6::NTRK3 fusion is not specific and is detected in infantile fibrosarcoma, cellular mesoblastic nephroma, melanocytic Spitz nevus, pediatric astrocytoma, a subset of thyroid papillary carcinoma, leukemia, NTRK-rearranged mesenchymal spindle cell tumors, and inflammatory myofibroblastic tumors [3 fusion ,124. Therectinib . An alteescribed . Owing tescribed . The ETVc tumors ,144,145.BRAF, KRAS, and HRAS. Both tubular adenoma and syringocystadenoma papilliferum can develop sporadically or more frequently on the genetic background of a cutaneous mosaic RASopathy, such as nevus sebaceus of Jadassohn [General features: Tubular adenoma and syringocystadenoma papilliferum SCAP,  are relaadassohn ,148.Related terminology: The salivary and bronchus homologous neoplasm is called sialadenoma papilliferum.BRAF p.V600E mutation, with excellent sensitivity and specificity with the anti-BRAF p.V600E antibody (clone VE1) [Immunohistochemistry: Immunostains can be used successfully to detect cases with one VE1) . EpithelMolecular biology: BRAF p.V600E mutations are detected in 50–64% of syringocystadenomas papilliferum and 66% of tubular adenomas, respectively. Tumors that arise in nevus sebaceus almost constantly harbor HRAS or KRAS mutations [BRAF, HRAS, and KRAS increase cell growth, differentiation, and survival. In sporadic tumors, HRAS p.G13R mutations are found in 7–26% of cases, while KRAS p.G12D mutations are less frequent [BRAF mutation in both the glandular and contiguous hyperplastic squamous epithelium [BRAF p.V600E mutation in most cases, while reports of RAS mutations are currently lacking [ARID2, CDKN2A, ERBB4, FAT1, FGF10, FGFR1, GNA11, KDM5C, MAGI2, NFKBIA, NOTCH1, PIK3CA, RAC1, RICTOR, SLIT2, TERT, TP53 mutations. More studies are needed to discover the molecular landscape of the malignant counterpart of SCAP. A case of digital papillary adenocarcinoma with BRAF p.V600E has been published but could represent a malignant form of tubular adenoma [utations . Activatfrequent ,152. DNAfrequent . Verrucoithelium ,154,155. lacking ,157. No  adenoma . In thisGeneral features: Endocrine mucin-producing sweat gland carcinoma EMPSGC,  is a lowRelated terminology: The related homologous neoplasm is called solid papillary adenocarcinoma of the breast. Endocrine mucin-producing sweat gland carcinoma may represent a precursor of mucinous sweat gland carcinoma (neuroendocrine type) ,160. LowImmunohistochemistry: Endocrine mucin-producing sweat gland carcinoma (EMPSGC) expresses INSM1, synaptophysin (90%), chromogranin (71%), ER (98%), PR (96%), and AR ,162. OthBRD4, PPP4R2, and RTEL1), regulation of transcription/post transcriptional processing , and tumor suppressors  [Molecular biology: Identified mutations affect genes involved in DNA damage response/repair (d LATS2) . Other sd LATS2) ,168,169.MYB, MYBL1, PLAG1, HMGA2, NTRK3, and a BRAF mutation in the appropriate morphological context is diagnostic of adenoid cystic carcinoma, cutaneous mixed tumor, secretory carcinoma, syringocystadenoma papilliferum, and tubular adenoma. On the other hand, the demonstration of fusion of YAP1, NUTM1, MAML2, EWSR1, FUS, and mutation of CYLD and ALPK1 points to the poroid, hidradenomatous, myoepithelial, cylindromatous, and spiradenomatous categories of tumors, and these alterations do not predict prognosis or malignancy, which is based on other characteristics, such as morphology and proliferation. Other alterations are used for diagnostic and theranostic purposes, such as the BRAF p.V600E mutation and the fusion of NTRK3.Although some of these biomarkers are directly linked to a specific diagnosis, other markers are associated with a whole spectrum. In fact, demonstrating a rearrangement of Some cases still lack any detectable molecular or immunohistochemical hallmark, and there is not a single answer to this question: variation in the sensitivity of the molecular techniques used, unreported gene fusion, alternative molecular mechanisms, and misinterpretation of pathological findings may contribute to the variation in prevalence of observed anomalies. The exact prevalence of these molecular anomalies is still unknown given the lack of data and large studies that include bona fide cases.Numerous other sweat gland tumors and related carcinomas still lack extensive molecular or immunohistochemical data, notably histiocytoid carcinoma, papillary digital adenocarcinoma, polymorphous sweat gland carcinoma, squamoid eccrine ductal carcinoma, mucinous carcinoma, apocrine carcinoma, and adnexal adenocarcinomas without other specification (NOS). This lack of data is the consequence of the combination of their rarity and diversity, which prevents large-scale studies from focusing on their molecular background. Networks of pathologists with common and robust diagnostic criteria, collaborative work, and collections of these rare neoplasms are of utmost importance to improve their diagnosis and allow proper future research ,170,171.The molecular landscape of cutaneous adnexal tumors has changed dramatically in the past few years. The addition of robust biomarkers in an integrated molecular–pathological approach is more likely to positively impact the reproducibility of diagnoses. Due to their great variety, rarity, complex terminology, and overlapping microscopic and phenotypic features, the pathological diagnosis of rare cutaneous neoplasms is challenging. However, these novel molecular and immunohistochemical hallmarks are expected to improve overall diagnostic accuracy due to their high specificity when integrated into an appropriate clinical and pathological context. Ultimately, since the entities will be systematically validated with a specific hallmark, standardized diagnoses will pave the way for future studies investigating the prognosis and therapeutic management of these rare cutaneous tumors.