Advanced cutaneous squamous cell carcinoma (cSCC) encompasses unresectable and metastatic disease. Although immune checkpoint inhibition has been approved for this entity recently, a considerable proportion of cases is associated with significant morbidity and mortality. Clinical, histopathological, and radiological criteria are used for current diagnostics, classification, and therapeutic decision-making. The identification of complex molecular biomarkers to accurately stratify patients is a not yet accomplished requirement to further shift current diagnostics and care to a personalized precision medicine. This article highlights new insights into the mutational profile of cSCC, summarizes current diagnostic and therapeutic standards, and discusses emerging diagnostic approaches with emphasis on liquid biopsy and tumor tissue-based analyses. Cumulative UV-exposure of the skin remains a key factor for the development of invasive cSCC, whilst changing demographics towards aging populations contributes to a consistently rising - though hardly subsumable - incidence of disease worldwide. The relevance of invasive cSCC is denoted by a range of twice the incidence of melanoma in European Caucasians of up to 10 times in fair-skinned populations in Australia2. Recent findings also suggest a sex-biased susceptibility of cSCC highlighting a certain protective role of female immunity3.Cutaneous squamous cell carcinoma (cSCC) is the second most frequent skin tumor arising from malignant progression of keratinocytesUltraviolet radiation (UVR) is a central factor of carcinogenesis. It results in a high tumor mutational burden, which may challenge advances in precision medicine in advanced cSCC. In the last years, many studies aimed at the characterization of the mutational landscape of cSCC. They provided insight into the mutanome of advanced cSCC populations, which may help to point out new therapeutic targets and resistance mechanisms. Both tissue- and liquid biopsy-based approaches are under investigation for biomarker identification. A thorough understanding of cSCC mutagenesis and its mutational landscape will be helpful in the development and application of these techniques in advanced cSCC.In this perspective, we therefore first summarize current knowledge on cSCC mutagenesis and mutational landscape presumably relevant for precision medicine approaches. We then discuss current diagnostics and treatment of advanced cSCC with perspectives of molecular biological tissue- and liquid biopsy-based techniques towards precision medicine.4. Besides a direct mutagenic effect, UVR may contribute to tumor development and progression via enhancement of multiple other processes such as local inflammation and immunosuppression5.Chronic cumulative exposure to UVR, predominantly UVA- and UVB-light, constitutes the most important risk factor for cSCC. The UV-mediated induction of photoproducts like cyclobutane pyrimidine dimers (CPDs) or pyrimidine-6,4-pyrimidone dimers (6-4PPs), among other alterations, may result in typical UV fingerprint mutations, if not repaired by nucleotide excision repair (NER)6. What we do know is that sun-exposed skin already is composed of thousands of mutants within every cell and a high clonal heterogeneity, in 18–32% exhibiting clones with about a handful driver mutations known for cSCC, but phenotypically not showing malignant transformation7.Understanding of the genomic correlates of a clinical multi-step evolvement of invasive cSCC from sun-exposed skin via “precancerous” lesions like actinic keratoses (AK) or Bowen’s disease (BD) and in situ cSCC (cSCCis) has been subject of many studies Table . Despite8. This has been reasoned both due to a temporally increased cumulative exposure to UVR, but also by the finding, that NER capacity significantly reduces with age, resulting in an increasingly inefficient DNA repair machinery9.CSCC and precursor lesions are mostly seen in elderly patients, aged, sun-exposed skin, which has been demonstrated to be an extended mosaic of multiple clones, predisposed to acquire further, potentially transforming events11. Current data suggest a similar tumor mutational burden (TMB) of cSCC in immunocompromised versus immunocompetent patients12. However, there is evidence of distinct alterations, such as a higher frequency in HRAS mutations and a mutational signature related with exposure to azathioprine treatment14. This indicates that advanced cSCC arising upon immunosuppression may have an altered mutational landscape compared to advanced cSCC arising predominantly from UVR. The molecular biological identification of driver or druggable mutations and the development of a tissue- or liquid biopsy-based assay for its stratification would be a desirable advancement, especially in a setting of SOTR, in which targeted alternatives to immune system promoting immunotherapy could help circumvent difficult medical situations.Besides UVR, many other factors contributing to the carcinogenesis of cSCC have been recognized. Discussion of all factors would be beyond the scope of this perspective. However, an important factor to mention here is immunosuppression: Immunosuppressed patients, especially solid organ transplant recipients (SOTR), are a considerable group with a 100-fold increased risk of developing aggressive cSCC15. At this point, we do have considerable insight into the mutanome of cSCC and precursor lesions. We even have an idea of temporal significance of respective mutations. Unfortunately, we do not know in detail yet, which events become vital for transformation into invasive cSCC, aggressive disease or metastatic progression, after all.As UVR leads to a high mutational burden, cSCC is characterized by a diverse mutational landscape. Tissue- and liquid biopsy-based diagnostic approaches may depend on a thorough understanding of driving mutational events. In this perspective, we therefore conceived an up-to-date overview of selected reports on the somatic mutational landscape of cSCC and their contribution to the field Table . A recen6. Therefore, one central question is, which mutation is the result of genetic drift or selection respectively. Largescale studies shedding light on temporospatial heterogeneity are therefore needed. This could be realized with upcoming technologies like single cell sequencing or spatial transcriptomics. With these methods only recently a tumor-specific keratinocyte (TSK) population localized to the leading edges of cSCC could be identified, which, in conjunction with basal and adjacent stromal and immune cells, exhibited invasive and immunosuppressive features16. Further transcriptomic, proteomic and metabolomic studies recently highlighted important correlations for cSCC and precursor lesions19. Particularly noteworthy in context on advanced cSCC, Shapanis et al. reported a significant correlation of poor outcome with increased annexin A5 (ANXA5) and dolichyl-diphosphooligosaccharide-protein glycosyltransferase noncatalytic subunit (DDOST) expression in a retrospective study of patients, which exhibited metastatic disease up to 5 years after primary surgery of cSCC20. They developed a prediction model of ANXA5 and DDOST showing higher sensitivity and specificity than cSCC clinical staging systems for estimating likelihood of metastases in cSCC20.As an example, TP53 and NOTCH1 are among the most frequently mutated genes in cSCC, but these mutations do not seem to drive clonal growth beyond a certain size without additional genetic, epigenetic or environmental contribution21. Based on a detected ERBB3 mutation, lapatinib was added to nivolumab resulting in stabilization of disease21. However, if frequent NOTCH1 loss-of-function mutation would be targeted, Martincorena et al. realistically stated that we might successfully treat 60% of cSCC, but with a considerable collateral damage to physiologically normal skin7.Another perspective is the identification of targetable events in the mutational landscape of cSCC. Jones et al. described a patient who experienced disease progression while on nivolumab treatmentTogether, there is strong evidence that, for a deeper understanding of inter- and intrapersonal moieties in cSCC and subsequently the advancement of diagnostics and therapeutic decisions, identification of composite biomarkers rather than single biomarkers could picture the sophisticated interplay between tumor genetics, tumor mutational environment (TME), and other host factors.22. However, metastatic disease does not necessarily need to be clinically apparent as a large tumor.Cutaneous manifestations of advanced cSCC are primarily investigated clinically Fig. . Importa23. At the presence of risk factors or clinical suspicion, lymph node sonography is performed for evaluation of locoregional metastases24. Further radiological assessment via computed tomography (CT) and cranial magnetic resonance imaging (cMRI) is sought in cases of suspected metastasis or suspicious findings in sonography , targeted therapy (cetuximab) or immunotherapy (interferon) have been explored. Unfortunately, overall results were not encouraging, leaving advanced cSCC patients a rather dismal prognosis. However, PD-1 checkpoint inhibitor cemiplimab has been demonstrated a milestone in advanced cSCC treatment just recently: In open-label nonrandomized Phase-II pivotal EMPOWER-cSCC-1 trial, cemiplimab induced response rates of 47%, which led to FDA approval in September 2018 and EMA approval in June 201926. In June 2020, pembrolizumab was approved for treatment of advanced cSCC by FDA, based on KEYNOTE-629 trial, underlining the rapidly increasing role of immuno-oncology in the therapeutic spectrum of cSCC27.Although the vast majority of cSCC are successfully eradicated by surgical complete excision with excellent prognosis, aggressive cSCC subgroups are associated with a higher likelihood of recurrence, metastasis or even deathIn general, current diagnostic standard of care in cSCC is already considerably efficient and easy to perform in comparison to tumor entities of internal organs, for example. However, in terms of advanced cSCC and especially prediction upon systemic treatment, this might not be the fact yet: although cemiplimab became a first-line gold standard treatment in advanced cSCC early after approval, up to 50% of the patient collective might not profit long-term. This is presumably a result of a precision medicine diagnostic gap of current standard of care diagnostics. In contrast to other entities like for example melanoma, there are only few reliable prognostic or predictive biomarkers. In the following, we will therefore discuss both the current status and possible future development and challenges of selected tumor tissue- and liquid biopsy-based diagnostic molecular biological techniques in advanced cSCC.29. Liquid biopsy may also provide a platform of real-time monitoring of tumor heterogeneity and residual tumor load and holds the potential for development of personalized therapeutic regimens31.There are potential advantages to blood-based liquid biopsy over tissue-based techniques to monitor tumor development during immunotherapeutic treatment. Blood can be easily drawn serially in a minimally invasive and reproducible manner, paving the way for optimal cancer surveillance28. Up to date, few reports on proteomic plasma profiling (PPP) in cSCC have been presented33. However, besides an often limited stability, proteins are frequently not specific for cancer28. The combination of circulating tumor DNA (ctDNA) and PPP to form a composite biomarker may provide the advantage of increased specificity and sensitivity. Cohen et al. reported on CancerSEEK, a blood-based test, in which both ctDNA and PPP are combined and have been evaluated on eight surgically resectable cancer entities34. CancerSEEK was demonstrated to identify patients even with low tumor burden or at early stage of disease with sensitivities of ~70–98%34. Unfortunately, cSCC was not included in this test. Part of this reason may be the frequency of cSCC in the population and proportionally fewer rapidly advancing cases as compared to breast cancer or lung cancer. Further, circulating proteins used in the CancerSEEK panel were previously found to be specific for certain entities. No specific circulating protein has been identified for advanced cSCC so far, possibly making combination for ctDNA and PPP difficult in this case. This may in turn be the consequence of cSCC not yet being in focus of the latter studies, as described above. Thus, further reports on PPP and ctDNA analysis in advanced cSCC are required. In the authors’ opinion, PPP in combination with ctDNA or circulating tumor cell (CTC) analysis might exhibit potential for future development of a composite biomarker panel in advanced cSCC.Circulating proteins may be easily isolated from blood with the advantages of high sensitivity and easy standardization35. Dysregulation of miRNA expression is involved in most cancer hallmarks such as regulation of cell apoptosis, invasion, proliferation, or migration36. For cSCC, a plethora of differentially regulated miRNAs with important roles in formation and progression of disease have been identified. These have been thoroughly reviewed recently38. MiRNAs may be detected in peripheral blood and other body fluids40. Extracellular vesicles may contain considerable miRNA amounts41. Their stability in blood makes them potential candidates for valuable biomarkers43. In cervical SCC different miRNA patterns have been identified predictive biomarkers for lymph node metastasis in early-stage disease44. In another cohort of 79 cSCC patients, prognostic sub-groups based on miRNA-203 and miRNA-205 expression patterns could be defined45. However, a more profound knowledge on miRNA signatures as biomarkers for monitoring therapeutic responses upon immunotherapy of advanced cSCC will be important for translation into clinic.MicroRNAs (miRNAs) are endogenous, small non-coding RNAs, which may control basal cell biological processes via post-transcriptional regulation of gene expression47.Besides investigations on the utility of miRNAs as a supplemental biomarker in advanced cSCC, both topical and systemic miRNA targeting strategies such as introduction of defective miRNA or blocking of miRNA overexpression  have been presented and are subject to futher investigations in cSCC49. It is easy and well-established isolation as well as detection of also rare mutations delineate ctDNA a promising candidate not only for prognostication of advanced CSCC, but also monitoring of disease as well as the detection of minimal residual disease28. Depending on the status of disease and tumor burden, sensitivity may be limited. However, multiple assays like digital droplet PCR, modified next generation sequencing (NGS), standard NGS or sanger sequencing are available with high differences in sensitivity, but also considerable differences in cost and turn-around time for each sample to be analyzed30.The mutant fraction of cell-free DNA representing tumor cell-derived DNA circulating in blood is termed ctDNA. It is released by apoptotic and/or necrotic tumor cells and mostly studied in plasma, but can also be isolated from other body fluids, such as saliva53. Latest results in this tumor entity revealed a correlation of ctDNA quantity and tumor burden, the prediction of lymph node metastasis and overall survival by a copy number instability score (CNI), making ctDNA a promising candidate biomarker for molecular diagnostics54. A so far unique demonstration of detection of HPV ctDNA in oral HNSCC clearly associated pre- and post-treatment ctDNA levels from blood and saliva with treatment success or failure and identified disease recurrence early after treatment58. Moreover, Cabel et al. provided a first insight in this regard into anal SCC by demonstrating detectability of HPV ctDNA and post-treatment association with poor outcome55. Thus, future ctDNA based investigations on cSCC, especially in previously HPV-associated locations, should be intiated.As for cSCC, ctDNA analysis holds a potential which has not been exploited so far. In contrast, for head and neck squamous cell carcinoma (HNSCC), ctDNA could be detected in several studies demonstrating feasibility alreadyAs mentioned above and in contrast to HNSCC, for most cSCCs a high TMB due to cumulative UV-exposure is characteristic. Additionally, only few actionable driver mutations are known, resulting in the current challenge of ctDNA detection against the background of intratumoral mutational heterogeneity. Compared to HNSCC, molecular data on cSCC is rather rare in publicly available databases. Therefore, defining ctDNA libraries and ctDNA panels for routine diagnostics will be crucial for success of ctDNA analysis in cSCC. Besides these current pressing action points in cSCC there are several unanswered key questions such as interpretation of ctDNA detectability and origin in the considerable cohort of patients with multiple cSCC or field cancerization respectively Box . DiffereBox 1 Projected challenges in use of liquid biopsy in advanced cSCCTMB and multiple candidate driver mutations characterize cSCC. Which alterations can be diagnostically addressed? Preparation of libraries and panels of ctDNA will presumably prove more suitable than single probes.- A high clonal heterogeneity, e.g. upon treatment. Sensitivity of ctDNA and CTC is a common issue and has to be evaluated in the latter regard.- Against the background of multiple frequent mutations, detection of seldom mutations might be of relevance to monitor Field cancerization or the co-occurence of multiple cSCC sites might confound the picture of the lesion of interest. Could tumor-derived material such as ctDNA be released into the blood stream both by the primary tumor of interest leading to advanced disease and secondary tumors? Other epithelial cancers have been successfully identified by liquid biopsy at an early stage previously. A realistic underlying mechanism could therefore hypothetically be that detectable ctDNA was measurable already in cases of localized invasive cSCC. In that case, a subsequent challenge could be traceability of liquid biopsy signals back to the ”causative“ primary tumor leading to advanced disease. One possible attempt could be mutational profiling of the respective tumor tissues combined with ctDNA panel analysis. It could be speculated that this approach might be too costly and laborious for later implementation into routine clinical diagnostics of advanced cSCC cases.- Detectability of CTCs by label dependent or independent techniques in advanced cSCC? Using label dependent techniques, for example, feasibility of CTC detection will rely on cellular markers on CTC. As cSCC is from epithelial origin, established techniques using epithelial markers could be a potential approach.- 59. For cutaneous HNSCC, a pilot study including ten patients with regional metastatic disease successfully identified CTCs in 80% (up to 44 cells/9 ml blood)60.CTCs are intact viable tumor cells released by primary tumors or metastatic tissue into the blood at low concentrations. They have been used in clinical studies for real-time monitoring of tumor evolution under immunotherapy in tumor entities like melanoma61. Although a meta-analysis of pre-treatment PD-L1 CTC expression in HNSCC did not reveal significant associations with progression-free survival, Strati et al. reported that increased PD-L1 expression on CTCs in locally advanced HNSCC was an independent prognostic marker of decreased overall survival and progression-free survival after radio-chemotherapy65. Currently, a couple of studies are further investigating immune-marker expression on CTCs and their correlation with tumor tissue markers or blood-based markers in HNSCC67. However, cSCC remains to be investigated in regard of CTC characterization  treatment in tumor entities like melanoma69. The current perspective is that a high TMB may result in a high neoantigen load, which may lead to increased T-cell activity, thus enhancing anti-tumor response70. Based on the assumption of a correlation between TMB and overall response rate, cSCC has therefore been previously projected to exhibit among the highest response rates to anti PD-1 treatment71. However, advanced cSCC is comparatively rare and therefore might be often not included in large studies on TMB or represented by a fairly small sample size. Additionally, recent findings indicate that the effect of a high TMB depends on the treatment context, respectively on prior ICI treatment and type of ICI regimen72. Larger studies on the prognostic and predictive value of TMB regarding ICI treatment in advanced cSCC should be striven for. Moreover, several limiting barriers for TMB to be adopted as a biomarker into clinical practice yet have to be overcome73. Of these, a lack of harmonization of applied methods to investigate TMB across studies, adequate methods to convert TMB estimates across different panels and missing of robust predictive cut-offs are considered the central limitations73.TMB is a biomarker measuring total counts of somatic mutations per megabase of the investigated tumor genome75. However, other studies did not find correlation of PD-L1 expression with prognostic features in cSCC76. This illustrates the current limitations for clinical use of PD-L1 as an exclusive biomarker in advanced cSCC, although it might be determined a valuable marker in a composite panel. Further studies with larger cohorts are needed to understand and confirm whether testing for PD-L1 aids in cSCC prognostication or prediction upon ICI treatment. Heterogeneity in findings may be reasoned by a considerable methodological and definitional variability among conducted studies77.The expression of PD-L1 on tumor cells has been associated with poor prognosis due to promotion of an immunosuppressive TME in many cancer entities. However, an inverse correlation has been reported between PD-L1 expression and poor prognosis in long-term follow-up analyses. For cSCC, PD-L1 expression has been associated with an increased risk of metastatic disease and the presence of high-risk pathological findings78. Cancer is increasingly defined as a complex interplay between the tumor and the host’s immune system78. In 2006, Galon et al.79 demonstrated in situ-analysis of infiltrating adaptive immune cells to be a valuable prognostic tool in colorectal cancer patients. They introduced “Immunoscore” as a method assessing type, density and location of in situ-T-cell infiltrates within colorectal tumor tissue and discovered a prognostic superiority to mere classification of patients regarding the extent of tumoral spread79. The concept of this immune texture is based on the dependency of tumor immune microenvironment interplaying with the efficacy of an immunotherapeutic treatment. Based on an individual’s immune cell signature, treatment outcome is sought to be predicted. Immunoscore data on cSCC has not been published so far. The authors see an applicability of this technique for future cSCC prediction to immunotherapeutic response critically: cSCC inherently exhibits a high mutational load. As described above, this is considered a favorable feature in terms of immunotherapeutic addressability. However, it may also reflect tumor heterogeneity taking reports on multiple clones even within one primary tumor into consideration7. Furthermore, this tumor-based technique would demand combination with another approach allowing for identification of the primary cSCC site relevant for advanced disease at a clinical coincidence of other cSCC tumor sites.The extent of tumoral spread serves as a standard parameter for classification of patients into stages. This categorization of patients revealed heterogeneous prognostic populations within staged subgroupsIn the field of histology and histopathology, chemical analysis of tumor tissue, mostly from FFPE samples, is considered diagnostical standard to accurately define a cancer entity and tumor subtypes. In order to more precisely define tumor subgroups on an individual basis, gene expression profiling is an increasingly used technique to molecularly characterize tumor tissue. For example, gene expression of tumor tissue may be quantified by DNA microarray or high-throughput real time-PCR (RT-PCR) with RNA from FFPE samples. Differentially expressed genes may be correlated with clinical impact on parameters like therapeutic responsiveness or response prediction. Such analyses allow for composition of gene expression profiles (GEP) or definition of signatures that may be further evaluated as clinical biomarkers in prospective trials.80 identified potential candidate genes and susceptibility loci of cSCC compared to non-cSCC controls via GEP analysis. This and other recent reports demonstrate the potential of GEP for defining cSCC biomarkers and understanding molecular pathogenesis of cSCC17.Ioannidis et al.81. It should be noted that there is not a clear recommendation for the optimal timing of testing yet82. As GEP testing is now available in cSCC, an expert panel has recently underlined the concern of early incorporation into clinical routine until definitive results of prospective trials become available82. It is therefore currently recommended for use as an additional data point instead of a surrogate for standard of care diagnostics or treatment82.Recently, a promising 40-gene expression profile test  to predict metastatic risk in localized high-risk cSCC has been presented that could complement current staging systems for patients with high-risk cSCCThe majority of cSCC cases are considered localized disease and current standard-of-care screening for localized cSCC and antecedent lesions is well-established, easy to perform and cost-efficient. Of note, a considerable cohort of inoperable and metastatic cases can be deducted from a hardly subsumable yet rising incidence of cSCC cases worldwide. In contrast to localized disease, current diagnostics of advanced cSCC does not reflect the clinical precisional medicine need in the era of immunotherapeutic treatment. Recent studies addressing molecular biology of advanced cSCC have set the basis for a broader knowledge on the mutational landscape of this tumor entity. Despite recent scientific advances, it remains difficult to identify significant and actionable driver mutations of advanced disease. Reliable biomarkers for prognostication, monitoring and characterization of disease are a desired, not yet clinically realized need in advanced cSCC. It will be important to implement state-of-the art techniques into routine diagnostics of advanced cSCC to address current challenges like detection of disease or monitoring treatment response in order to further personalized medicine. To achieve this goal, diagnostics should not only focus on tissue-based analysis. Liquid biomarker analysis of CTC, ctDNA, miRNA, or PPP in combination with other techniques hold a, partly unexploited, potential in advanced cSCC and should become a central part of future investigations. The development of novel biomarkers is most likely to supplement the established diagnostic standard rather than substituting it Fig. .


Picobirnaviruses (PBVs) are small, double stranded RNA viruses with an ability to infect a myriad of hosts and possessing a high degree of genetic diversity. PBVs are currently classified into two genogroups based upon classification of a 200 nt sequence of RdRp. We demonstrate here that this phylogenetic marker is saturated, affected by homoplasy, and has high phylogenetic noise, resulting in 34% unsolved topologies. By contrast, full-length RdRp sequences provide reliable topologies that allow ancestralism of members to be correctly inferred. MAFFT alignment and maximum likelihood trees were established as the optimal methods to determine phylogenetic relationships, providing complete resolution of PBV RdRp and capsid taxa, each into three monophyletic groupings. Pairwise distance calculations revealed these lineages represent three species. For RdRp, the application of cutoffs determined by theoretical taxonomic distributions indicates that there are five genotypes in species 1, eight genotypes in species 2, and three genotypes in species 3. Capsids were also divided into three species, but sequences did not segregate into statistically supported subdivisions, indicating that diversity is lower than RdRp. We thus propose the adoption of a new nomenclature to indicate the species of each segment . Picobirnaviruses (PBVs) are small, double stranded RNA viruses with two genome segments encoding a capsid and RNA-dependent RNA polymerase (RdRp). PBVs are typically isolated from fecal samples and associated with diarrhea in immunocompromised individuals or as coinfections of gastrointestinal viruses and bacteria . SheddinThe PBV genus has been divided into two genogroups (G-I and G-II) on the basis of genome segment 2 (RdRp) phylogeny. There has been an attempt to further classify species by virtue of the infected host , but given the ability to readily jump from one species to the next, this is clearly inaccurate ,12. LikeRecent advances in technologies, including phylogenetic methods and associated computational capacity, along with the ability to readily obtain full genomes via metagenomic next generation sequencing (mNGS), can lead to improved classification of groups. Here we continue efforts to establish a consistent phylogenetic methodology to provide a more accurate evolutionary analysis and better understanding of PBV phylogenetic diversity.n = 2542 sequences) containing all Picobirnavirus (PBV) genus capsid and RdRp coding regions were downloaded from the GenBank database (http://www.ncbi.nlm.nih.gov/) accessed on 1 February 2021. Unique sequences were obtained by filtration according to De la Cruz et al. [Two sequence datasets  with the option E-INS-I to decrease the penalty in the gaps. To determine the level of agreement among the different methodologies with an estimated ratio, the accuracy of the alignments was assessed by comparing the measurement of precision determined by the modeler score to the measure of recall determined by the sum of pairs score (SP-score) algorithms implemented in FastSP v. 1.6.0 [Nucleotides and amino acid sequences of PBV capsid and RdRp were aligned using the three most common multiple sequence alignment (MSA) methodologies: CLUSTAL W in the BioEdit Sequence Alignment Editor , multiplv. 1.6.0 .The impact of the phylogenetic inference on picobirnavirus topology reconstruction was evaluated by two tree-building methodologies: (i): distance-building, e.g., neighbor-joining (NJ)  and (ii)To perform multiple hypothesis testing on phylogenetic data, topologies were evaluated by several conventional testing procedures including the Kishino and Hasegawa test (KH) , the ShiPairwise sequence comparison (PASC)  and the Using both ML trees obtained for each PBV genomic segment (coding sequences for RdRp and capsid), a face-to-face phylogenetic tree visualization was computed with an R-script using functions in the R ‘ggtree’ package  and tidyA starting point in the evaluation of the genetic relationships established by different viral strains is to determine the reliability of the tree topology and the accuracy of the different markers used to yield the inferred tree structure. We started by evaluating three major aspects including saturation, homoplasy, and phylogenetic noise in the most common phylogenetic marker in RdRp used to resolve PBV phylogeny. First, an assessment of saturation of substitution was performed by plotting the absolute number of transitions and transversions versus genetic distance, comparing the commonly used 200-RdRp marker A. For thHomoplasy leads to incomprehensible evolutionary history by overestimating genetic similarity . A largeNext, we evaluated phylogenetic noise using maximum likelihood mapping. The phylogenetic quartet is the fundamental unit of the tree graph for which levels exceeding 30% equate to noise. For the RdRp-200 marker, we observed a large percentage of dots (taxa) grouping in the vertices of the triangles such that 34.4% of topologies remained unsolved  C. With >Multiple sequence alignment (MSA) is a fundamental first step in phylogenetic estimations and as such, alignment errors can lead to significant bias during topology reconstruction ,47. The Phylogenetic tree methods were then evaluated with the MAFFT alignment deploying the same battery of statistical tests . ML yielCurrent PBV nomenclature refers to only two genogroups based upon RdRp. Refseq strains widely recognized as GG-I and GG-II reside within PBV-R1 and PBV-R2 lineages defined here A. PBV-R3Using the threshold values determined by PASC analysis B, subdivPASC analysis was then applied to 422 capsid sequences following ML tree reconstruction A. As witOur rigorous analysis demonstrates the need to standardize phylogenetic methodologies. Starting with the choice of genetic markers, we show clearly that the information contained within the 200 nt region of RdRp was insufficient to reliably distinguish groups. Transversions outnumbered transitions, mutations saturated the length of the sequences, and more than 1/3 of taxa remained unresolved. Trees derived from full-length sequences remedied all these shortcomings and in turn provided new biological insights into the evolution of PBV. The proper combination of the alignment algorithm (MAFFT) and phylogenetic tree reconstruction (maximum likelihood) also avoided bias and achieved greater resolution of taxa. Adopting a uniform set of analysis criteria is imperative so that different research groups are evaluating picobirnavirus sequences in the same manner.PBV has not been isolated in culture, nor are there serotypes, etc., to distinguish strains. The International Committee on the Taxonomy of Viruses (ICTV) has endorsed, among other phylogenetic and biological criteria, the use of genome-wide nucleotide or amino acid sequence identity thresholds for the classification of novel virus isolates.. Complete RdRp sequences segregated into three lineages, which based upon pairwise genetic distances, we determined are unique species of PBV. The idea that PBVs genogroups I and II diverged into different genera has been suggested in recent studies ,4,16, hoFull-length capsids were also classified into three species, however PASC analysis indicates there were not statistically supported subdivisions therein, only clades in PBV-C1. The lack of observed genetic diversity in capsid may be a reflection of incomplete or biased reporting. Indeed, the vast majority of capsid sequences in GenBank are from Marmota himalayana, whereas for RdRp there is a greater balance of vertebrate entries A,B. An aWe urge the adoption of the phylogenetic classification scheme and nomenclature set forth here. As with Influenza A virus  and more recently suggested for infectious bursal disease virus (IBDV), a Birnavirus member , we suggest picobirnaviruses ought to be named according to their capsid and RdRp species . As questions persist regarding whether PBVs are bacterial or vertebrate viruses, a separate debate continues as to whether they are disease-causing, strictly opportunistic infections, or simply a bystander ,58. It dWe established that the previously accepted classification of PBV with a 200 nt region of RdRp needs to be replaced by an analysis of full-length sequences using MAFFT alignments and maximum likelihood phylogenetic tree reconstruction. RdRp and capsid segments were each found to have diverged into three species, independent of host and with an ability to freely reassort. Agreed upon methodologies, taxonomic assignments, and nomenclature will further the field’s understanding of PBV evolutionary dynamics and whether emerging strains have the potential to cause disease.


Ectopic pancreas is basically a benign disease and is not always necessary to be removed. However, all types of neoplasms occurring in the normal pancreas such as ductal adenocarcinomas and intraductal papillary mucinous neoplasms (IPMNs) may develop even within ectopic pancreas. We recently encountered an extremely rare case of ectopic pancreas in the gastric antrum associated with IPMN possessing a GNAS mutation.GNAS at exon 8 (Arg201Cys).A 71-year-old Japanese woman complained of epigastric pain. Computed tomography and upper gastrointestinal endoscopy showed an intramural cystic mass in the antrum of the stomach. Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) biopsy did not give a definitive diagnosis, and the patient underwent resection of the lesion. Histology of the resected specimen showed that the gastric intramural lesion was ectopic pancreas. Moreover, the lesion contained dilated duct components with tubulo-villous epithelial proliferation consistent with pancreatic IPMN. Since the covering epithelial cells had highly atypical nuclei, the lesion was diagnosed as IPMN with high grade dysplasia. Immunohistochemistry showed that the IPMN component showed to be MUC2-, MUC5AC-, and CDX2-positive but MUC1- and MUC6-negative. Mutational analyses using genomic DNA revealed that the IPMN component had a mutation of GNAS mutation. Although there were 17 cases of ectopic pancreas with IPMN including 6 cases of gastric ones reported in the English literature, this is the first case of ectopic pancreas with IPMN which was proved to have GNAS mutation. Intimate preoperative examinations including imaging analyses and EUS-FNA biopsy/cytology are recommended to decide whether the lesion has to be resected or not even if they are not effective for getting the right diagnosis.We finally diagnosed this case as gastric ectopic pancreas accompanied by intestinal type IPMN with high grade dysplasia possessing  Ectopic pancreas, also known as pancreatic heterotopia or aberrant pancreas, is defined as pancreatic tissue that presents in organs other than the pancreas. Its predilection site is the stomach, duodenum, and Meckel’s diverticulum of the ileum, and it often affects submucosal, proper muscular and/or subserosal layers of them , 2. EctoIn the pancreas, many different types of neoplasms can occur. Most of them originate from pancreatic exocrine tissue especially duct component. Ductal adenocarcinomas are the most common tumor type of ductal origin with solid characterization, and intraductal papillary mucinous neoplasms (IPMNs) are a less common tumor type of ductal origin with cystic change. Although IPMNs generally proliferate within the pancreatic duct, they could have intraductal malignant lesion with rather high incidence and even might invade outside the ductal structure as the result of tumor progression. Thus, they can be divided into three categories: intraductal papillary mucinous adenoma (IPMA), IPMN with high-grade dysplasia , and invasive IPMC.On the other hand, IPMNs can be classified into four subtypes by histology and immunohistochemistry (IHC) for mucin core proteins (MUCs)/CDX2: gastric, intestinal, pancreatobiliary, and oncocytic types , 4. GastK-ras, GNAS, p16, TP53, CTNNB1, etc [K-ras mutation could be found in both pancreatic duct adenocarcinomas and IPMNs, but GNAS mutation at exon 8 (Arg201Cys or Arg201His) is specifically detected in IPMNs [Pancreatic neoplasms such as pancreatic duct adenocarcinomas, IPMNs and solid pseudopapillary neoplasms might have gene mutations of GNAS mutation. We report here the case with a review of the literature.Ectopic pancreas is basically a benign disease and does not always require resection. However, all types of neoplasms occurring in the normal pancreas such as ductal adenocarcinomas and IPMNs may develop even within ectopic pancreas –21. TherA 71-year-old Japanese woman complaining of epigastric pain came to Hyogo College of Medicine Hospital. Computed tomography showed an intramural cystic tumor having a maximum diameter of 70 mm in the antrum of the stomach  was observed in the gastric antrum Fig. a. Cut suGNAS mutation at codon 201 in exon 8 (Arg201Cys) . Thus, the lesion was finally diagnosed as gastric ectopic pancreas accompanied by intestinal type IPMN with high grade dysplasia (non-invasive adenocarcinoma) possessing GNAS mutation (Arg201Cys). The lesion was considered to be completely resected.Moreover, the lesion contained dilated duct components with tubulo-villous epithelial proliferation Fig. c at the ys) Fig.  but not Ectopic pancreas is generally a benign disease, but there is a possibility that neoplasms occurring in the normal pancreas such as ductal adenocarcinomas and IPMNs may develop even within the ectopic pancreas –22. We ePathological changes other than neoplasms such as cyst formation and inflammation/abscess which can occur in the normal pancreas could also happen in ectopic pancreas. The lesions might cause symptoms such as abdominal pain, epigastric discomfort, nausea, vomiting, and bleeding , althougPancreatic IPMN exhibits papillary epithelial proliferation and mucus production, and is classified into four subtypes  according to mucus traits and histological features , 4. InteGNAS mutation at exon 8 (Arg201Cys or Arg201His) among pancreatic neoplasms [GNAS mutation but none of pancreatic duct adenocarcinomas have such a mutation [K-ras mutation, while approximately half of pancreatic IPMNs have the mutation [GNAS and K-ras mutations, only GNAS mutation, only K-ras mutation, or neither of them. Each subtype  of pancreatic IPMNs appears to have GNAS and/or K-ras mutations at similar frequency [GNAS mutation but not K-ras mutation. This is the first reported case of ectopic pancreas with IPMN component which is proved to have GNAS mutation. Since all of intestinal type IPMNs have GNAS mutations and since invasive cancer associated with IPMNs appears to be much more frequent in intestinal type IPMN, detection of GNAS mutation in the specimen from intramural gastric lesion by preoperative EUS-FNA biopsy/cytology could become an indicator suggesting that the tumor should be excised.Pancreatic IPMNs specifically have eoplasms , 6. Apprmutation . On the mutation . Thus, arequency . In the Imaging tests could suspect ectopic pancreas of the stomach, but preoperative accurate diagnosis of the lesion by them is usually difficult. In the case of gastric ectopic pancreas with IPMN, the situation may be similar. Moreover, preoperative EUS-FNA biopsy/cytology and incisional biopsy cannot always obtain appropriate material for pathological diagnosis like in our case. Thus, definitive diagnosis of gastric ectopic pancreas without IPMN or with IPMN is often made by the histological examination of the resected tissue. Many gastric GISTs have solid appearance by imaging, but some GISTs might show cystic change especially in those with platelet-derived growth factor receptor alpha gene mutation. Indeed, preoperative clinical diagnosis of our case was GIST with cystic change, and the final diagnosis of ectopic pancreas with IPMN was made by the resected specimen. Although both ectopic pancreas with IPMN and GIST with cystic change have to be resected because of possibility with malignant characteristics, systematic lymph node dissection might not be required in the case of GISTs because they usually could not metastasize to lymph node. In any case, we have to consider that gastric intramural lesion with cystic change might be ectopic pancreas with IPMN component.GNAS mutation. This is the first case of ectopic pancreas with IPMN which was proved to have GNAS mutation. Intimate preoperative examinations including imaging analyses and EUS-FNA biopsy/cytology are recommended to decide whether the lesion should be resected or not even if they are not effective for getting the accurate diagnosis.We experienced an extremely rare case of gastric ectopic pancreas accompanied by intestinal type IPMN with high grade dysplasia possessing


Solitary fibrous tumors (SFTs) are rare soft tissue neoplasms that can impact the central nervous system (CNS). SFTs comprise <1% of all primary CNS tumors. Here, we describe a rare case of intradural, extramedullary SFT arising within the thoracic spine that was treated with surgical resection. Histological features were evaluated and revealed a highly cellular tumor with positive expression of BCL2, CD34, CD99, and STAT6 proteins that are consistent with a diagnosis of SFT. We discuss the use of surgical intervention for long-term disease control of spinal SFT and evaluate the role of postoperative radiation therapy in management strategies. Lastly, we review the literature reports of intradural, extramedullary SFTs in the thoracic spine. The importance of molecular characterization by histopathology to properly determine diagnosis and prognosis is emphasized. STAT6 and NAB2 genes. Diagnosis is typically made based on the nuclear localization of STAT6 that results from this fusion event and is a surrogate marker used in place of whole-genome sequencing [A meningeal solitary fibrous tumor (SFT) and hemangiopericytoma were previously described as separate entities having common genetic and molecular properties . These rquencing . The addquencing ,2. Origiquencing . UnfortuThe current best treatment practices for SFT consist of safe, gross total resection (GTR) regardless of the site of origin. Although adjuvant radiotherapy (RT) has been proposed to reduce recurrence risk, survival outcomes remain mixed -6. AccurHere, we present a rare case of intradural, extramedullary SFT (grade III) arising within the thoracic spine. We discuss the clinical features and review in detail the relevant histopathology. We also provide a review of the reported literature cases of intradural, extramedullary SFTs identified within the thoracic spine.An 81-year-old woman with a past medical history of ulcerative colitis, hypothyroidism, and hypertension presented with lower back pain progressing to paraparesis and decreased sensation in the lower extremities over the course of eight months. At presentation, she had already lost the ability to ambulate and had difficulty controlling bowel movements for over two weeks. Magnetic resonance imaging (MRI) of the thoracic spine demonstrated severe spinal cord compression due to a contrast-enhancing, intradural, extramedullary lesion at the T11-12 level measuring 3.1 cm in the greatest dimension  [Here, we present a rare case of a primary intradural, extramedullary WHO Grade III SFT arising within the thoracic spine in an 81-year-old woman. While SFT arising within the spine has been described previously, intradural extramedullary tumors remain rare with only 46 cases in the thoracic spine as identified in 28 separate retrospective studies (Table wannoma) . Immunoswannoma) ,10. It rwannoma) . We did SFT and hemangiopericytoma were previously distinguished as separate diagnoses with similar molecular profiles, but the term hemangiopericytoma has been retired. All CNS tumors of this kind are now classified as SFTs . In the Spinal SFT can be classified into four anatomic locations for surgical planning, similar to other spinal tumors. This includes vertebral (osseous), paravertebral, spinal canal, and mixed location. For lesions within the spinal canal, three subtypes exist that are extradural, intradural extramedullary, and intradural, intramedullary. Spinal SFT typically presents with focal deficits due to mass effect at the corresponding vertebral level. In the case presented here, progressive tumor growth and compression of the spinal cord at T11 resulted in paraparesis and decreased sensation bilaterally in the lower limbs over an eight-month period.SFT is often misdiagnosed radiographically given the similarities to other spinal lesions on imaging and lack of radiographic pathognomonic findings. Common misdiagnoses include meningioma, schwannoma, hemangioma, metastases, and primary bone malignancies. Therefore, a definitive diagnosis is typically made by pathology after resection of the mass.CNS SFT is graded based on the mitotic index. Five mitoses per 10 high power fields (hpf) represents the threshold between WHO Grade II and Grade III disease [The question of whether spinal cord SFT is more benign with respect to intracranial counterparts remains unknown. In this case, we observed relatively benign tumor behavior despite common genetic precursor variants with previously reported aggressive SFT CNS neoplasms . One hypNAB2-STAT6 fusion events using genome sequencing with a focus on exon and intron breakpoints. Variation in exon fusions has been linked to recurrence rates in conventional SFTs versus previously described hemangiopericytomas that exhibit more aggressive phenotypes [Given the rare occurrence of SFT spinal neoplasms, no prospective, randomized control trials are available. Our approach and outcome support the current standard of care for SFT that includes safe maximal resection. In general, we support aggressive resection for optimal treatment, but acknowledge that this must be evaluated on a case-by-case basis. Progression-free survival and overall survival remain unknown. For this 81-year-old patient, a residual tumor was left in the foramen since definitive diagnosis at the time of operation remained unclear on frozen section analysis. Since definitive diagnosis was not known at this time, frozen section analysis clearly indicating more aggressive cellular phenotypes may have indicated more aggressive resection. This elderly patient was already severely functionally affected prior to surgical resection. Therefore, it was reasonable to offer a smaller but effective resection in the face of the unknown diagnosis. In a younger patient, it may have been indicated to unroof the foramen and commit to a small segment fusion in the case that the frozen section exhibited evidence of high-grade aggressive disease. The current consensus indicates that stereotactic radiosurgery can be useful to prevent disease progression and reduce recurrence, thereby lengthening progression-free survival time . Howeverenotypes .We reviewed the literature for cases of intradural, extramedullary SFT in the thoracic spine and provided this in Table NAB2-STAT6 gene fusion, a characteristic feature of SFT. Our findings support the maximum possible GTR as an appropriate surgical intervention in accordance with the current standard of treatment, although adjuvant radiotherapy can be considered after STR to hamper disease progression. High-risk features including age ≥55 years, high mitotic index, and the presence of tumor necrosis warrant close follow-up. By reviewing reported cases of intradural, extramedullary SFT in the thoracic spine, we identify a distinct lack of metastatic disease in this subtype that may inform future management strategies. However, recurrence remains common. Future studies centered around mechanisms driving faster SFT recurrence in the spine versus the more aggressive intracranial tumors will be essential to understanding the disease process to optimize treatment. A comparative analysis of vascular characteristics of spinal versus intracranial SFTs may provide insights into pathogenesis and prognosis. Such studies should include a detailed investigation into different NAB2-STAT6 fusion events using whole-genome sequencing since different exon fusion sites have been linked to recurrence rates and aggressive behavior in conventional SFTs.SFT neoplasms of the CNS and spinal column are rarely reported in the literature. Because of low incidence rates, it is a challenge to construct prospective studies to elucidate the molecular nature and optimal management of spinal SFT. It is imperative to determine the effectiveness of adjuvant RT and chemotherapy to maximize survival rates, therapeutic outcomes, and reproducibility. This is particularly important as factors portending a more aggressive phenotype remain unknown. In this report, we describe a rare case of intradural, extramedullary SFT of the thoracic spine, the second reported case in a female ≥80 years. Tumor cells expressed STAT6 re-localized to the nucleus that is a sensitive and specific surrogate biomarker for


Human oncogenic viruses account for at least 12% of total cancer cases worldwide. Epstein–Barr virus (EBV) is the first identified human oncogenic virus and it alone causes ~200,000 cancer cases and ~1.8% of total cancer-related death annually. Over the past 40 years, increasing lines of evidence have supported a causal link between EBV infection and a subgroup of lung cancers (LCs). In this article, we review the current understanding of the EBV-LC association and the etiological role of EBV in lung carcinogenesis. We also discuss the clinical impact of the knowledge gained from previous research, challenges, and future directions in this field. Given the high clinical relevance of EBV-LC association, there is an urgent need for further investigation on this topic. Lung cancers (LCs) are the number one killer among cancers in the U.S. and estimated to cause more than 131,000 deaths in 2021 [LC is a complex and multifactorial malignant disease with a currently unclear etiology. It is believed that genetic, epigenetic, and environmental factors all can contribute to lung cancer development. Among those known risk factors, smoking is a definitive oncogenic factor for LC development. Nevertheless, the incidence of LCs is only slowly declining even after the dramatic reduction of smoking through public health awareness campaigns. To date, only a small portion (10–20%) of smokers develop LCs . With a Epstein–Barr virus (EBV) is also known as human herpesvirus 4 and belongs to the gamma-herpesvirus subfamily, in the same family with Kaposi’s sarcoma-associated herpesvirus (KSHV). EBV is the first human oncogenic virus discovered in 1964, which currently accounts for ~200,000 annual cancer cases and ~1.8% of all cancer-related deaths globally ,11. EBV Like other herpesviruses, EBV employs two distinct stages to complete its life cycle: latency and lytic cycle (or lytic reactivation). After an initial, usually asymptomatic infection, EBV will establish a life-long persistent infection in the host. During latency, the EBV genomes remain as episomes and replicate in the S-phase. The replicated episomes are then partitioned into the daughter cells during cell division. In order to avoid host immune surveillance, only a limited repertoire of viral genes are expressed. Notably, abundant viral non-coding RNAs with low antigenicity are also synthesized to facilitate the viral replication. In response to certain physiological or pathological stimuli, the latent genome can be reactivated, characterized by a series of highly ordered events. The viral immediate-early gene products, such as Zta and Rta, will function as a molecular switch to turn on the entire lytic cascade and induce the expression of ~100 viral early and late genes. Ultimately, this leads to the production of new infectious virions ,17.The infectious virions can be transmitted through body fluids such as blood and urine, but they are mainly spread through saliva exchange. Like other human herpesviruses, except varicella-zoster virus (VZV), vaccine-based blockade of EBV infection is still in the development stage and not yet an option. It is estimated that ~90% of the global adult population are currently carrying EBV . To dateAccumulating evidence has shown that both viral latency and lytic cycle are required for EBV pathogenesis. There are approximately 100 open reading frames encoded by the EBV genome. Among them, some latent genes such as EBV-encoded nuclear antigen 1 (EBNA1) , EBV-encThe human lung is not a sterile anatomic site and it is regularly in contact with the external environment . A diverA possible association of EBV with LCs was first supported by the finding that some LC patients had a significantly higher level of anti-EBV IgA in the sera compared to the healthy control individuals . Later oIn addition to the uncommon LELC subtype, emerging evidence has shown that EBV is also present in the tumor cells of conventional NSCLC subtypes including lung squamous-cell carcinomas (LUSC) and lung adenocarcinomas (LUAD) ,45,46,47Here, we summarized the studies reporting the status and potential roles of EBV in lung carcinogenesis. Studies were identified by searching the PubMed database and only articles (including abstracts) published in English were reviewed. The latest literature search was performed in March 2021. Having carefully evaluated the extracted studies, we selected 42 relevant studies and the EBV status in LCs was summarized in Having reviewed the data, we found that the association of EBV and LCs shows significant differences based on tumor histology types and geographical sites where the studies were carried out. The reported incidence rates vary from 0 to 100% . EBV posThe variation of incidence appears also affected by the sample sizes and methodologies used for EBV infection. Notably, the majority of the studies used traditional viral screening methods such as polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC). Due to the inherent limitations of these traditional screening methods , the accuracy of the incidence data was questionable. We reasoned that an ideal detection approach should cover the full sequence of LC cells and search for any present EBV genetic materials.To date, the next-generation sequencing (NGS) technology has been successfully utilized to discover and interrogate various oncogenic pathogens. NGS uses an unbiased method to globally assess all the exogenous microorganisms within a tumor sample with high sensitivity and specificity. Several research teams, including ours, have successfully utilized NGS  to interrogate exogenous pathogens associated with human cancers ,83,84,85In addition to the traditional methods, we utilized our unbiased RNA-seq based informatics approach to comprehensively interrogate the involvement of EBV in LCs in our recent work . RNA-seqThe low EBV infection rate (~0.6%) observed in our study indicates that EBV is unlikely to play a significant role in the development of LCs in the western countries, but it may contribute to the development of a subset of LC cases . In areaEBV exhibits various latency programs in the infected lymphoid and epithelial cells . The typThe high level of BART is consistent with a true EBV latency since BART is more highly expressed in the infected epithelial cells than in B cells. Although previous studies have been unable to detect protein from endogenous BART  ,89, the Several novel alternative splicing events of LMP2A have been detected in various EBV-associated cancers ,92,93. IIn some EBV-associated cancers such as NPC and GC, EBV is known to alter the tumor immune microenvironment to facilitate the carcinogenesis ,81. In aIndeed, elevated levels of multiple immune checkpoint molecules, such as indoleamine 2,4-dioxygenase (IDO), programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte-activation gene 3 (LAG3), B and T lymphocyte attenuator (BTLA), and v-domain Ig suppressor of T cell activation (VISTA) were also detected in EBV(+) LCs . FurtherAlong this line, we also detected a high level of BNLF2a gene expression in the absence of significant expression of other viral lytic genes in the EBV(+) LCs . BNLF2a Unlike other EBV-associated cancers such as NPC and GC, the EBV-mediated alteration of cellular signaling pathways in lungs is poorly characterized. Having examined the cellular transcriptome of EBV(+) LCs, we found the activation of several EBV-associated oncogenic pathways and inhibition of multiple tumor suppressors . It inclSo far, the mutational landscape has only been explored in the LELC subtype of EBV(+) LCs. Emerging evidence has shown that major driver mutations for EBV-negative (EBV(−)) LCs such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma (KRAS), mesenchymal epithelial transition factor (MET), anaplastic lymphoma kinase (ALK), and c-ros oncogene 1 (ROS1) are rarely observed in the EBV(+) LCs ,107,108.Recently, Hong and colleagues utilized whole exome sequencing, targeted deep sequencing and single-nucleotide polymorphism array to survey the genomic landscapes of 91 EBV(+) LC samples . They foMeanwhile, Chau et al. conducted a whole genome sequencing analysis of 57 EBV(+) LCs with LELC subtypes . They foIn another study, Chen and colleagues did whole genome sequencing analyses of eight EBV(+) LC samples with LELC subtype . The somIn general, tumor viruses are necessary, but not sufficient for their associated cancer development. In accordance with this information, we found that primary lung epithelial cells cannot be immortalized by EBV infection alone (Lin unpublished data). Thus, in the context of EBV-associated LCs, we postulated that EBV infection only contributes to a portion of the oncogenic events. Additional genetic and epigenetic mutations and co-factors such as chronic inflammation, immunosuppression, and environmental mutagens are also required for this multistep oncogenic process. We now favor a disease model in which the impact of EBV infection on the lung carcinogenesis might be a consequence of the aberrant establishment of viral latency in lung epithelial cells that have already undergone premalignant changes . In our Understanding of the EBV-lung epithelial cell interaction depends on appropriate in vitro and in vivo experimental models. A lung cancer cell line carrying natively infected EBV should be an ideal in vitro system to explore the complex interplay between EBV and the host cells. To our knowledge, such cell systems have not yet been reported. We thus utilized our NGS pipeline to analyze the RNA-seq data sets of 182 lung cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) cohort , includiOne remaining puzzle is how EBV gains access to the lung epithelium when the EBV life cycle is believed to occur in oral epithelial cells and B cells. Although we cannot rule out the possibility that lung epithelium may serve as a normal reservoir for EBV infection, another possible explanation is that some inflammation events preceding lung cancer may help the EBV infection. Since lung tissue is not a sterile environment, the constant interaction between pulmonary microbiota and lung tissue may elicit inflammation that subsequently attracts lymphocytes and certain lytic-inducing epithelium-derived extracellular vesicles ,113 to fIn contrast to the well-established route for EBV entry into B cells, it is still largely unclear how EBV enters epithelial cells. We reasoned that EBV may enter the lung epithelial cells through multiple mechanisms: (1) cell-to-cell mechanism through which EBV can enter lung epithelial cells by cell membrane contact or the formation of cell-in-cell structure of EBV-infected B cells and uninfected epithelial cells ; (2) CelThe observation that EBV(+) LCs are mostly seen in Asian populations and that the disease is more prevalent in NPC endemic regions raises the possibility that some viral genomic variations may contribute to the unusual geographic distribution of EBV(+) LCs. As a DNA virus, EBV has long been thought to have a highly conserved genome. However, increasing lines of evidence have shown that EBV genome polymorphism may contribute to its oncogenicity. For example, Feng and colleagues reported that a particular single nucleotide polymorphism in the EBV genome (SNP G155391A) leading to an RPMS1 variant is strongly associated with NPC but not other EBV-related malignancies . It willThe low survival rate of LCs partially results from the fact that LCs are largely diagnosed in their late/advanced stages when surgical treatment is no longer an option. However, since EBV itself can serve as a great biomarker, routine application of EBV serology or nucleic acid testing could offer early diagnosis of EBV(+) LCs during the surgically treatable stages of this disease. Additionally, the EBV testing could also be useful in monitoring treatment and providing prognostic information. Further, since EBV(+) LCs usually do not carry the EGFR mutation, which makes conventional tyrosine kinase inhibitor (TKI)-based therapy ineffective. Thus, stratification of cancer patients with EBV(+) LCs will help choose more efficient therapeutic regimen. For example, since EBV(+) LCs usually express higher levels of PD-L1 as well as other immune checkpoint molecules, the new immune checkpoint inhibitors may help improve the overall survival of EBV(+) LCs by preventing immune evasion of the EBV-infected tumor cells. In the future, EBV(+) LC patients may also benefit from next-generation drugs targeting the EBV gene products that exclusively expressed in the cancer cells.Together, better understanding of EBV-associated lung carcinogenesis will provide important mechanistic basis for the development of personalized cancer therapy and diagnostics that will ultimately benefit many cancer patients.