TP53 mutant or wild type, and non-WNT/non-SHH (Groups 3 and 4). WNT-activated medulloblastomas are usually caused by mutations in the CTNNB1 gene (85%–90%), and most remaining cases of CTNNB1 wild type are thought to be caused by germline mutations in APC. So far, the frequencies of CTNNB1 have been reported mainly in North American and European populations. The aim of this study was to report the frequency of CTNNB1 mutations in WNT-activated medulloblastomas in a Latin-Iberian population and correlate with their clinicopathological characteristics.Medulloblastomas are the most common primary malignant brain tumors in children. They are divided into molecular subgroups: WNT-activated, SHH-Activated, CTNNB1 mutation analysis were performed by nCounter and Sanger sequencing, respectively.A total of 266 medulloblastomas from seven different institutions from Brazil (n=211), Portugal (n=38), and Argentina (n=17) were evaluated. Following RNA and DNA isolation from formalin-fixed, paraffin-embedded (FFPE) tumor tissues, the molecular classification and CTNNB1 mutations. CTNNB1 wild-type cases (27%) were more prevalent in female individuals and suggested to be associated with a worse outcome. Among the CTNNB1 wild-type cases, the available analysis of family history revealed two cases with familiar adenomatous polyposis, harboring APC germline variants.WNT-activated medulloblastomas accounted for 15% (40/266) of the series. We observed that 73% of WNT-activated medulloblastomas harbored CTNNB1 mutations in WNT-activated medulloblastomas in our Latin-Iberian cohort compared to frequencies previously described in other populations. Considering that CTNNB1 wild-type cases may exhibit APC germline mutations, our study suggests a higher incidence (~30%) of hereditary WNT-activated medulloblastomas in the Latin-Iberian population.We observed a lower incidence of  TP53 wild type and TP53 mutant, and Groups 3 and 4 are merged in a non-WNT/non-SHH subgroup -activated group accounts for 10% of all medulloblastomas, is commonly observed in children older than 4 years, in an equal proportion of boys and girls, and usually shows no metastasis at diagnosis . This moThe WNT is a conserved pathway that may induce cell proliferation and growth during development via regulation by beta-catenin, which is translocated to the nucleus for binding to transcriptional factors inducing the expression of cyclins and proto-oncogenes . In diffCTNNB1 gene, which encodes beta catenin (CTNNB1 wild type) tumors carry germline APC variants . This study was approved by the institutional review board from Barretos Cancer Hospital (CAAE: 59979816.6.1001.5437). Informed consent was obtained from the patients or familiars before APC germline evaluation.In the present retrospective study, we evaluated 266 FFPE medulloblastoma specimens collected between 2001 and 2022 from naive-treated patients from seven different institutions in Brazil, Argentina, and Portugal: Barretos Cancer Hospital, Brazil (n=119); Federal University of São Paulo UNIFESP (n=20), Brazil; AC Camargo Hospital, Brazil (n=15), Ribeirão Preto Medical School, Brazil (n=37); Child Hospital of Brasília, Brazil (n=20); Italian Hospital of Buenos Aires, Argentina (n=17); and Centro Hospitalar Universitário São João, Portugal (n=38). The frequency of molecular subgroups was previously reported in a subset of cohortCTNNB1 gene analysis, DNA isolation from macrodissected formalin-fixed, paraffin-embedded (FFPE) was performed using the QIAamp DNA Mini Kit , following the manufacturer’s recommendations. For APC gene analysis, DNA was isolated from peripheral blood samples using the QIAamp DNA Blood Mini Kit , following the manufacturer’s instructions. The DNA quantification was performed using the NanoDropVR 2000   . The RNAam, USA) , 14.® FLEX Analysis System available in the Molecular Oncology Research Center of Barretos Cancer Hospital (BCH) using the nCounter® Elements custom panel . The panel comprises three reference genes  and 22 targets for WNT , SHH , Groups 3 , and Group 4 classification  as previously described  mutations were evaluated by NGS (next-generation sequencing) in patients with a family history of colon cancer and/or polyps who have provided consent for germline molecular analysis. Library construction was carried out according to the Barretos Cancer Hospital Hereditary Rare Cancer Solution kit , which includes the genes APC, BRCA2, CEBPA, DICER1, GATA2, SMARCB1, MEN1, NF1, NF2, PALB2, PTCH1, PTEN, RB1, RET, RUNX1, SUFU, TP53, TSC1, TSC2, and VHL, according to the manufacturer’s protocol. Briefly, DNA fragments were generated using an enzymatic fragmentation step. The three subsequent enzymatic steps, end-repair, A-tailing, and ligation to Illumina adapters, were performed in order to produce NGS libraries. A capture-based target enrichment was carried out on the pooled libraries. The quantitation of the final pool of libraries was performed using Qubit dsDNA HS fluorimetric assays . Quality control of fragment size was assessed using DNA ScreenTape analysis . Sequencing was achieved with the final library concentration of 10 pM onto a 600-cycle format V3 flow cell, via Illumina MiSeq platform .® platform .Data analysis was performed in order to detect single nucleotide variants (SNVs), insertions/deletions (indels), and copy number alterations (CNAs). Sequencing FASTQ data were analyzed by the Sophia DDMThe classification of each genomic variant into five different categories, namely, benign (B), likely benign (LB), variant of uncertain significance (VUS), likely pathogenic (LP), and pathogenic (P), were performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines.CTNNB1 mutation, we downloaded the clinical and molecular information on medulloblastomas from The Cancer Genome Atlas (TCGA) consortium at cBioPortal . It included five different whole exome and genome datasets from the Pediatric Cancer Genome Project (PCGP)   , the Sic2, n=37) , Interna2, n=37) , and froThe statistical analysis was performed using the software IBM SPSS statistics version 25. The chi-square or ANOVA tests were applied for qualitative variables, and the Mann–Whitney test was applied for quantitative variables, rejecting the null hypothesis with p<0.05. For survival analyses, the Kaplan–Meier method and the log-rank test were applied.CTNNB1 mutations due to the low quantity and quality of DNA for Sanger sequencing. From the remaining 33 WNT-activated cases , we detected CTNNB1 mutation in 24 (73%) cases, and nine (27%) medulloblastomas were CTNNB1 wild type , Portugal (n=38), and Argentina (n=16). All cases were molecularly classified into the four main medulloblastomas subgroups, namely, WNT activated , SHH activated , Group 3 , and Group 4   populations showed that 29% (n=176) was SHH activated, 39% (n=240) Group 4, 24% (n=148) Group 3, and 8% (n=53) WNT activated. In the WNT-activated subgroup, 87% (n=46) showed hotspot mutation in the CTNNB1, and 13% (n=7) were CTNNB1 wild type  found in five cases, followed by the p. found in three cases, and variants p.(Ser37Tyr), p.(Asp32Tyr), p.(Ser33Cys), and p.(Ser33Phe) were found in two cases each one. The remaining variants were observed only in one case  and p.(Ala43Thr)  populations reveals 47 CTNNB1 variants in 46 WNT-activated medulloblastomas, with all variants being located in the exon 3 of the CTNNB1 gene (The CTNNB1 wild type was significantly higher in Latin-Iberian population (27%) , and no significant associations were observed regarding histology, surgery extension, and metastasis  , and additionally, three patients died due to other reasons , thus were not included in the survival analysis   Figure 3CTNNB1 wild type in our Latin-Iberian population. The analysis of the clinical records available showed the existence of two reports of familial adenomatous polyposis, one from Barretos Cancer Hospital (Brazil) (We found nine WNT-activated medulloblastomas (Brazil) Figure 4APC c.3183_3147delACAAA-p.(Gln1062Ter) pathogenic variant was detected. The proband of this family is a 7-year-old girl diagnosed with medulloblastoma; her brother was also diagnosed with medulloblastoma when he was 6 years old. Their mother had approximately 100 polyps and developed colorectal cancer at the age of 38, fulfilling the criteria of FAP   Figure 4APC germline variant, similarly to the variant detected in the Brazilian family. The patient was a 9.7-year-old girl diagnosed with WNT-activated, CTNNB1-wild type medulloblastoma. At 24 years old, she presented gastrointestinal tumors with hepatic metastasis. Currently, she is 30 years old, and in total remission of the brain tumor.The Portuguese patient harbored a c.3183_3187delACAAA-p.(Gln1062Ter) CTNNB1 gene (~90%) and germline mutations in APC (~10%), which are mutually exclusive , which is higher than those reported in North American and European populations (7%–10%)  . A highe(7%–10%) . Another(7%–10%) . In our (7%–10%) . However(7%–10%) . A compa(7%–10%) . FurtherCTNNB1 mutations in 33 cases and found 73% (24/33) of CTNNB1-mutated cases and 27% (9/33) CTNNB1 wild type. Of note, our frequency of CTNNB1 wild-type WNT-activated medulloblastomas is significantly higher than that reported in North American and European populations, varying from 6.8% . Waszak and colleagues performed whole exome sequencing in 66 WNT-activated medulloblastomas and found somatic CTNNB1 mutations in 89.4% and CTNNB1 wild type in 10.6% of cases8. A recent German study evaluated a large cohort of 191 WNT-activated medulloblastomas and reported 92.2% (176/191) CTNNB1 mutants and 7.8% (15/191) wild-type cases  of WNT-activated medulloblastomas are CTNNB1 wild type.Among the 40 WNT-activated medulloblastomas included in the present study, we successfully evaluated CTNNB1 mutations in different populations needs to be clarified. In the present study, we performed Sanger sequencing of the exon 3 of the CTNNB1 gene, and the studies mentioned above used whole genome or whole exome sequencing , African American, and others   .Disparities in genomic studies due to the under-representation of some populations, such as from South America, were previously demonstrated . ConsequAPC mutations are associated with a 92 times higher risk for developing medulloblastomas than in the general population  had a family history of FAP and associated cancers. Additional malignancies were observed in three patients with APC germline mutations , has been previously detected in families with classic FAP   can be found in the article/This study was approved by the institutional review board from Barretos Cancer Hospital (CAAE: 59979816.6.1001.5437). Informed consent was obtained from the patients or familiars before APC germline evaluation.DM: data collection, experimental procedures, data analysis, and manuscript writing; MB: experimental procedures, results discussion, and manuscript review. AA: oncogenetic analysis, results discussion, manuscript writing, and review. FP: experimental procedures, results discussion, and manuscript review. LL: experimental procedures, manuscript writing, and review. FG: experimental analysis, medical reports review, manuscript writing, and review. AP: experimental procedures, manuscript writing, and review. GT: pathological review of tumor samples from Barreto’s cancer hospital, Brazil, results discussion, manuscript review. IS: pathological review of tumor samples from Barreto’s cancer hospital, Brazil, results discussion, manuscript review. FS: pathological review of tumor, results discussion, and manuscript review. LN: pathological review of tumor, results discussion, and manuscript review. EV: patient’s clinical data collection, and manuscript review. CS: patient’s clinical data collection, and manuscript review. JS: patient’s clinical data collection and manuscript review. SM: patient’s clinical data collection, and manuscript review; ML: Patient’s clinical data collection and manuscript review; GH: patient’s clinical data collection and manuscript review; HG-R: Pathological review of tumor and patient’s clinical data collection, manuscript review. SC: pathological review of the tumor and patient’s clinical data collection and manuscript review. SN: patient’s clinical data collection and manuscript review. MG-d-C: patient’s clinical data colection and manuscript review. JP: pathological review of tumor and manuscript review. FM: patient’s clinical data colection and manuscript review. CJ: medical reports analysis, patients’ treatment protocol review, and manuscript review. BM: medical reports analysis, patients’ treatment protocol review, and manuscript review. RR: supervisor and project coordinator, results discussion, and manuscript writing. All authors contributed to the article and approved the submitted version.


H3-wildtype, and IDH-wildtype, WHO grade 4. Ultimately, we demonstrate that treatment utilizing a radiation-based regimen for GB in a previously radiated tissue can be highly successful despite historical limitations in the management of this disease.Radiation-induced gliomas (RIGs) are an uncommon disease type and a known long-term complication of prior central nervous system radiation exposure, often during childhood. Given the rarity of this malignancy subtype, no clinical trials have explored optimal therapy for these patients, and the literature is primarily limited to reports of patient cases and series. Indeed, the genomic profiles of RIGs have only recently been explored in limited numbers, categorizing these gliomas into a unique subset. Here, we describe two cases of RIG diagnosed as glioblastoma (GB), IDH-wildtype, in adults who had previously received central nervous system radiation for childhood cancers. Both patients demonstrated a surprising complete radiographic response of the postoperative residual disease to front-line therapy, a phenomenon rarely observed in the management of any GB and never previously reported for the radiation-induced subgroup. Both tumors were characterized by next-generation sequencing and chromosomal microarray to identify potential etiologies for this response as well as to further add to the limited literature about the unique molecular profile of RIGs, showing signatures more consistent with diffuse pediatric-type high-grade glioma,  Radiation remains a staple therapeutic option for many pediatric and adult malignancies. Given the mutagenic nature of this therapy, patients receiving ionizing radiation in the management of their disease early in life are at risk for future treatment-related complications, including secondary malignancies , 2. Survde novo gliomas. Indeed, survival data for this disease—which effectively consist of compilations of case reports and retrospective data—have been correspondingly poor with median overall survival (mOS) times of 17 and 9 months for radiation-induced histologic grade 3 and 4 gliomas, respectively (Radiation-induced gliomas (RIGs) have long been anecdotally thought to act more aggressively than other ectively . Despiteectively , 7. For ectively .IDH) mutation and methylguanine-O6-methyltransferase (MGMT) promotor methylation are less likely to be found in RIG, particularly the former, supporting unfavorable outcomes for radiation-induced GB , initially presented to an outside facility with a 3-week history of intractable headaches, relieved by conservative measures, and visual changes described as “seeing fireworks” when his eyes were closed. This improved, but 2 months later, the patient presented to an emergency department for left-sided weakness, fatigue, dizziness, and recurrent headaches with visual changes. An MRI of the head was ultimately completed showing a 5.5 × 5.2 × 4.7 cm posterior parasagittal extra-axial mass with a significant local mass effect and invasion of the superior sagittal sinus and overlying calvarium consistent with a meningioma, presumably radiation-induced. It is noted that there was a 9 × 8 × 8 mm enhancing focus in the left posterior parietal lobe/angular gyrus of unclear etiology but with glioma of consideration. The patient underwent a craniotomy for the resection of the meningioma along with reported embolization at the outside facility; pathology was reported as a grade 2 meningioma.MGMT promotor. The patient sought an opinion at our institution 3 weeks later. An MRI at that time showed substantial enhancing disease regrowth adjacent to the resection cavity, supported by elevated cerebral blood volume and diffusion restriction  regions in 1p, 4q, 5p, 9p, 10q, and 11p  in the setting of traveling to run a marathon. An MRI of the head was completed and demonstrated a 4.5 × 3.4 × 2.9 cm heterogeneously enhancing hemorrhagic mass centered in the left basal ganglia and extending into the left mesial temporal region with significant surrounding T2/FLAIR hyperintense signal, concerning a GB , A2. Thepromotor –D. Targeriations .MGMT promotor methylation, the patient was deemed a poor candidate for standard concurrent TMZ with radiation and underwent hypofractionated intensity-modulated radiotherapy to 40.05 Gy in 15 fractions. Unexpectedly, an MRI of the head 8 weeks after the completion of radiation revealed a marked decrease in residual enhancing tumor and T2/FLAIR signal. The T1 contrast-enhanced tumor further decreased 3 months later and was completely resolved 7 months after the completion of radiation  and/or 1q gain (50%), Chr 13q loss (40%), Chr 14q loss (40%), PDGFRA amplification (36%), CDKN2A loss (29%), BCOR loss (23%), and CDK4 amplification (19%) with findings in the surface tyrosine kinase receptor gene, PDGFRA, and transcriptional co-repressor with influence on apoptosis, BCOR, trending toward and demonstrating significance, respectively. Somatic non-copy number alterations were most often seen in PDGFRA, TP53, MET, and NF1. Deng MY et al. also conducted genetic and expression profiling of 32 RIGs and similarly found that 91% of tumors had methylation profiling consistent with the pedGBM-RTK1 subtype (PDGFRA amplification (53%), CDKN2A/B loss (66%), CDK4 amplification (16%), Chr 1p loss (59%) and/or 1q gain (50%), Chr 13q loss (72%), and Chr 14q loss (45%) as well as Chr 6q loss (56%) and MET amplification (28%). Somatic non-copy number alterations reported included PDGFRA, EGFR, TP53, MET, NTRK2, RAF1, BCOR, and ATRX. TERT promotor and IDH1/2 mutations were distinctly absent along with EGFR amplification and polysomy 7/monosomy 10.To date, the largest studies of high-grade RIGs were published simultaneously in 2021. DeSisto et al. comprehensively evaluated 32 tumors by DNA methylation profiling as well as both DNA and RNA sequencing . Interes subtype . FurtherTP53 (58%), PDGFRA (50%), CDK4 (33%), and CDKN2A/B (33%) in addition to less consistent means of MAP kinase pathway activation  of the WHO Classification of CNS Tumors. Though many RIGs, including both cases here, possess necrosis and/or microvascular proliferation, which in the context of an IDH-wildtype genomic profile is sufficient to establish a diagnosis of GB based on 2021 classification criteria, they actually possess a molecular signature more consistent with diffuse pediatric-type high-grade glioma (PTHGG), 21 of theSpecifically unique to our cases was the marked response to therapy, something not previously reported. Indeed, survival data for RIGs suggest an aggressive disease with poor outcomes , 8, 19. BRCA1  and XPC . While pathogenic alterations in BRCA1 impede the repair of double-stranded DNA breaks as acquired by ionizing radiation, and XPC dysfunction has been shown to increase TMZ-induced DNA damage, neither point mutation correlated with meaningful functional alteration of the resultant protein nor loss of heterozygosity was observed  for the publication of any potentially identifiable images or data included in this article.PG, JH, and HR: acquisition of material. PG, JH, SH, and HR: direct patient management/data implementation. PG, JH, DD, SH, RJ, and HR: analysis of data/information and interpretation of data/information. All authors contributed to the article and approved the submitted version.


Potentilla L. species (Rosaceae) as herbal medicines, a number of species still remain unexplored. Thus, the present study is a continuation of a study evaluating the phytochemical and biological profiles of aqueous acetone extracts from selected Potentilla species. Altogether, 10 aqueous acetone extracts were obtained from the aerial parts of P. aurea (PAU7), P. erecta (PER7), P. hyparctica (PHY7), P. megalantha (PME7), P. nepalensis (PNE7), P. pensylvanica (PPE7), P. pulcherrima (PPU7), P. rigoi (PRI7), and P. thuringiaca (PTH7), leaves of P. fruticosa (PFR7), as well as from the underground parts of P. alba (PAL7r) and P. erecta (PER7r). The phytochemical evaluation consisted of selected colourimetric methods, including total phenolic (TPC), tannin (TTC), proanthocyanidin (TPrC), phenolic acid (TPAC), and flavonoid (TFC) contents, as well as determination of the qualitative secondary metabolite composition by the employment of LC–HRMS (liquid chromatography–high-resolution mass spectrometry) analysis. The biological assessment included an evaluation of the cytotoxicity and antiproliferative properties of the extracts against human colon epithelial cell line CCD841 CoN and human colon adenocarcinoma cell line LS180. The highest TPC, TTC, and TPAC were found in PER7r /g extract and 263.54 mg caffeic acid equivalents (CAE)/g extract, respectively). The highest TPrC was found in PAL7r /g extract), and the highest TFC was found in PHY7 /g extract). The LC–HRMS analysis showed the presence of a total of 198 compounds, including agrimoniin, pedunculagin, astragalin, ellagic acid, and tiliroside. An examination of the anticancer properties revealed the highest decrease in colon cancer cell viability in response to PAL7r (IC50 = 82 µg/mL), while the strongest antiproliferative effect was observed in LS180 treated with PFR7 (IC50 = 50 µg/mL) and PAL7r (IC50 = 52 µg/mL). An LDH (lactate dehydrogenase) assay revealed that most of the extracts were not cytotoxic against colon epithelial cells. At the same time, the tested extracts for the whole range of concentrations damaged the membranes of colon cancer cells. The highest cytotoxicity was observed for PAL7r, which in concentrations from 25 to 250 µg/mL increased LDH levels by 145.7% and 479.0%, respectively. The previously and currently obtained results indicated that some aqueous acetone extracts from Potentilla species have anticancer potential and thus encourage further studies in order to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.Despite the common use of  Potentilla species are widely used in traditional medicine for the treatment of dysentery, diarrhoea, diabetes mellitus, unspecified forms of cancer, and inflammation of the skin [Potentilla species stem from their secondary metabolite composition, which includes a predominant presence of polyphenols, such as hydrolysable and condensed tannins, flavonoids, and phenolic acid, as well as triterpenoids. These substances are associated with antioxidant, anti-inflammatory, and antimicrobial properties [Potentilla species have shown efficacy against various cancer cell lines, e.g., methanol extract from P. discolor inhibited the proliferation and induced the apoptosis of MC3 and YD-15 (human mucoepidermoid carcinoma) [P. recta and P. astracanica decreased viability of HEp-2 (human cervix carcinoma) [P. alba significantly reduced the viability and proliferation of HT-29 (human colon adenocarcinoma) [Potentilla species showed great chemopreventive potential by decreasing the viability and proliferation of LS180 (human colon adenocarcinoma) cells, simultaneously causing substantial damage to their cell membranes while having a significantly weaker impact on normal colon epithelial cell line CCD841 CoN [Potentilla species against human colon cancer cell line LS180 and normal colon epithelial cell line CCD841 CoN. Additionally, identification of the marker metabolites present in extracts using LC–HRMS analysis was conducted to reveal and validate correlations between the qualitative chemical composition of the investigated samples and possible mechanisms of action.Cancer, a non-infectious disease, is one of the most dreadful diagnoses that severely impacts a patient’s life quality. Unfortunately, cancer is a significant and increasing cause of death worldwide. The European Cancer Information System (ECIS) estimated an increase in new cases of cancer in the European Union EU-27) from 2.68 million in 2020 to 3.24 million in 2040, a 21% increase, while the cancer-related death toll is estimated to increase from 1.26 million to 1.66 million cases, a 31.8% increase. Colorectum cancer is the second-most-diagnosed cancer type in EU-27 countries, with over 0.34 million cases in 2020; however, in 2040, it will overtake breast cancer as the most commonly diagnosed cancer type with over 0.43 million cases [7 from 2.the skin ,6. The poperties . NumerouD841 CoN . The prePotentilla species are well-known for their abundance of tannins and flavonoids, which contribute to certain traditional applications aimed at tackling diarrhoea, microbial infections, inflammations of the upper and lower gastrointestinal tract, diabetes mellitus, etc. [Potentilla species were prepared using 70% acetone and were quantitative assessed for the general polyphenolic classes contents using colourimetric methods. The level of phenolic compounds in the extracts from selected Potentilla species are presented in PAL7r and PER7r, were found to contain the highest total phenolic (TPC) and total tannin (TTC) contents /g extract, respectively). On the other hand, among extracts from the aerial parts, PFR7 and PPE7 had the highest TPC and TTC values , while PAU7 and PTH7 revealed the lowest TPC and TTC values . Moreover, PFR7 was found to contain the highest total proanthocyanidin content (TPrC) /g extract), notably higher than that of other herb extracts. According to our previous study and the results herein, extracts from rhizomes, namely, PAL7r and PER7r, had remarkably higher proanthocyanidin contents than their above-ground counterparts  [PAL7r and PER7r had the highest total phenolic acid content (TPAC), followed by PFR7 /g extract, respectively). On the contrary, PAL7r and PER7r had the lowest total flavonoid content (TFC) values, which were significantly lower than those of all other extracts. PHY7 and PPE7 revealed the highest TFC values /g extract, respectively). All the obtained results were significantly higher than the values available in the literature data reported for various extracts from the aerial parts of P. erecta, P. fruticosa, P. nepalensis, P. pensylvanica, and P. thuringiaca [Polyphenols are among the major secondary metabolites that are accountable for the pharmacological activities of plant-based preparations. The major group of polyphenols include flavonoids, phenolic acids, hydrolysable and condensed tannins, lignans, and stilbenes . Potentius, etc. ,12. In oringiaca . Notablyringiaca . MoreovePotentilla species using LC–HRMS (liquid chromatography–high-resolution mass spectrometry) analysis demonstrated the presence of 198 compounds. Among them, three groups of phenolic compounds were dominant in the analysed extracts: tannins, flavonoids, and phenolic acids. Monomeric and dimeric ellagitannins, such as agrimoniin, sanguinis and pedunculagin, are important chemophenetic markers in the Rosaceae family, especially in the Potentilla, Rubus, and Fragaria genera [84, 109, 114, 124, and 128), laevigatin E isomers (37 and 40), agrimoniin (162) and its structural isomer (151), agrimonic acid A or B (102), galloyl-HHDP-glucose , digalloyl-HHDP-glucose (33 and 60) and trigalloyl-HHDP-glucose (131 and 133), galloyl-bis-HHDP-glucose , ellagic acid (135) and its O-pentosides (132 and 163), O-hexosides , and uronic acid  derivatives. The analysis indicated that the one of the most abundant phytochemicals in all the extracts, except PAL7r, was agrimoniin. Agrimoniin has been frequently described as the major phenolic compound in several Potentilla species, such as P. argentea, P. anserina, P. grandiflora P. kleiniana P. norvegica, P. recta, and P. rupestris [135), brevifolincarboxylic acid (46) and its structural isomer (50), and brevifolin (83), were found. Gallotannins were present in a few extracts, which showed the presence of di-, tri-, tetra-, and pentagalloylglucose isomers . However, the analysis revealed the absence of hydrolysable tannins in PAL7r. These findings are in agreement with the previous study, which demonstrated the absence of these metabolites in the aerial parts of P. alba [PAL7r, such as catechin (28), epicatechin (61), and their glucosides , as well as products of their polymerisation, such as A-type procyanidins  and dimeric , trimeric  and tetrameric (56 and 93) B-procyanidins, including procyanidin B1 (25), procyanidin B2 (47), procyanidin B3 (27), procyanidin C1 (94), and procyanidin C2 (34).The identification of the secondary metabolite composition of the aqueous acetone extracts of selected upestris ,21,22. Oupestris . Further92, 119, 161, 166, 184, and 185) as well as isorhamnetin , naringenin (180), kaempferol , quercetin , acacetin (183), and tricin (189 and 195) derivatives. From a chemophenetic perspective, a few of them may be useful as chemical markers of the Potentilla genus, such as both isomers of tiliroside (190), astragalin (kaempferol 3-O-glucoside) (155), isorhamnetin 3-O-glucoside (169), kaempferol 3-O-glucuronide (157), avicularin (quercetin 3-O-arabinoside) (149), hyperoside  (139), isoquercitrin (quercetin 3-O-glucoside) (136), and rutin (quercetin 3-O-rutinoside) (138), which were previously reported to be present in at least one of the Potentilla species investigated to date [1), caffeic acid (29) and its derivatives , coumaric acid , dihydroxybenzoic acid (13), and syringic acid (89) derivatives. The detailed chromatographic data of the analysed samples are shown in Potentilla species may typify their chemical profile as homogeneous.Based on the chromatographic profiles, a number of flavonoids were detected and characterised, including apigenin  and 94.8% (IC50 PFR7 LS180 = 89 µg/mL), respectively. On the contrary, the weakest influence on the metabolic activity of colon cancer cells was noted after treatment with PTH7 and PRI7, which, at a concentration of 250 µg/mL, inhibited cell viability by 58.7% (IC50 PTH7 LS180 = 225 µg/mL) and 57.9% (IC50 PRI7 LS180 = 213 µg/mL), respectively. The strongest reduction (by 36.7%) of the viability of colon epithelial cells was caused by both PME7 and PHY7 , while the weakest effect, as reflected by the IC50 value, was shown by PRI7 (IC50 PRI7 CCD841 CoN = 2402 µg/mL).In the first step, the extract’s influence on both human colon epithelial cell line CCD841 CoN as well as human colon adenocarcinoma cell line LS180 was examined using an MTT assay. Studies were conducted after 48 h of the cells being exposed to either a culture medium (control) or extracts (25–250 µg/mL). As presented in PER7r, PHY7, PME7, and PPE7 was observed at concentrations of 150 and 250 µg/mL, while the beneficial effect of PAL7r, PFR7, and PNE7 was observed at concentrations from 100 to 250 µg/mL. In the case of CCD841 CoN cells, only 3 out of 12 of the investigated extracts inhibited the metabolic activity of colon epithelial cells stronger than 25 µM 5-FU: PAL7r (250 µg/mL); PHY7 (250 µg/mL); PME7 (150 and 250 µg/mL).Used as a positive control for the experiment, 5-fluorouracil (5-FU) at a concentration of 25 µM decreased the metabolic activity of CCD841 CoN and LS180 by 22.2% and 46.2%, respectively. All the investigated extracts at the highest tested concentrations revealed a stronger anticancer effect than 5-FU. Seven of twelve extracts inhibited LS180 cells’ viability better than 5-FU, when used at lower concentrations; the beneficial effect of PAL7r, PER7r, and FFR7 extracts. On several occasions, proanthocyanidins were reported to have a strong influence on colon cancer cell viability. Especially oligomeric proanthocyanidins from grape seeds , which induce the apoptotic cell death of Caco-2  cells manifested by nuclear condensation, caspase-3 and PARP cleavage, and formation of apoptotic bodies [Humulus lupulus L., Cannabaceae) increased the intracellular formation of reactive oxidative species, which was manifested by the augmented accumulation of protein carbonyls and induced cytoskeletal disorganisation of human colon cancer cell line HT-29 [P. argentea, P. grandiflora, P. norvegica, P. recta, and P. rupestris [PAL7r, was shown to have prominent anticancer, antioxidant, and anti-inflammatory activities [Fragaria x ananasa L., Rosaceae) containing agrimoniin as the second-most-abundant phytochemical, in an inflammation-induced colorectal carcinogenesis model, led to downregulating the mRNA expression of the proinflammatory mediators, such as COX-2, iNOS, IL-1β, IL-6, and TNF-α [P. erecta showed the dose-dependent inhibition of UVB-induced or TNF-α stimulated IL-6 and PGE2 production as well as reduced NFκB activation in HaCaT cells (human keratinocytes). Further, a UV erythema study in healthy volunteers revealed that an agrimoniin-enriched fraction significantly inhibited the UVB-induced inflammation process [The obtained results for the MTT assay may be strongly associated with high TPrC, especially in the c bodies . Additione HT-29 . Howeverupestris . This ditivities . It is wtivities . Preclintivities . Phytochnd TNF-α . Moreove process ,38.Potentilla L. extracts was examined in both the normal and cancer cell lines using a BrdU assay . On the contrary, the lowest antiproliferative abilities were revealed by PAU7, which, even at the highest tested concentration, decreased DNA synthesis in LS180 cells by only 14.9% (IC50 PAU7 LS180 = 1495 µg/mL). The antiproliferative effect of the examined extracts was also observed in colon epithelial cells; however, the observed effect was weaker than in cancer cells. The only extract that did not affect divisions of CCD841 CoN was PER7, which was characterised by the highest IC50 value of 3705 µg/mL. On the contrary, the most significant changes in normal cells were observed in response to PAL7r, PFR7, and PER7r, which, at the highest tested concentration, reduced the proliferation of epithelial cells by 36.1%, 38.3%, and 43.9%, respectively . As presented in PAL7r, PFR7, and PER7r, in concentrations from 100 to 250 µg/mL, decreased DNA synthesis more than 5-FU. On the contrary, data collected from colon epithelial cells revealed that most of the investigated extracts  at higher concentrations inhibited DNA synthesis stronger than 25 µM 5-FU, while the antiproliferative effect of PFR7 for the whole range of tested concentrations was higher than the changes induced by analysed cytostatic. However, the presented results correspond with data from our previous study, showing that tested Potentilla species possess similar anticancer potential; moreover, for the PAL7r, PER7r, and PFR7 extracts, the results from a BrdU assay were significantly higher than those for all other tested samples [Vaccinium macrocarpon Aiton, Ericaceae) proanthocyanidin-rich fraction significantly inhibited the viability and proliferation of human oesophageal adenocarcinoma SEG-1 cells. The mechanism involved cell cycle arrest in the G1 phase as well as a significant apoptosis induction [In the next step, the antiproliferative activity of dU assay . All thenduction . Notablynduction . Additionduction . In addinduction .PME7 was, for the whole range of tested concentrations, while PAL7r, PER7, and PHY7, in concentrations from 100 to 250 µg/mL, damaged the membranes of epithelial cells. The indicated extracts at the highest tested concentration increased the LDH level by an average of 11%. Studies conducted on colon cancer cells showed the cytotoxic effect of all the examined extracts for the whole range of tested concentrations. The strongest damage of cancer cell membranes was caused by PAL7r, which in concentrations from 25 to 250 µg/mL increased the LDH level by 145.7% and 479.0% respectively. The weakest cytotoxic effect was noted in colon cancer cells treated with PRI7, PPU7, and PTH7, which, at the highest tested concentration, caused an increase in the LDH level by 245.1%, 254.7%, and 256.0%, respectively. An LDH assay showed that 5-FU in a concentration of 25 µM was not cytotoxic against colon epithelial, while LDH releases were increased in colon cancer cells by 13.4%. All the investigated extracts damaged the colon cancer cell membranes significantly more than 5-FU. For CCD841 CoN cells, significant differences in the LDH concentration between 25 µM 5-FU and the examined extracts were observed in the case of four extracts , for which the cytotoxic impact on colon epithelial cells was reported above. The results of the LDH assay are presumably directly associated with the TTC in the investigated samples. Tannins, due to their specific chemical structure, are known to affect the physical properties of membranes, initiate membrane protein aggregation, increase bilayer adhesion, and regulate cell metabolism [P. anserina and the rhizomes of P. erecta confirmed the formation of urolithins in ex vivo conditions [Extract cytotoxicity was also examined in both normal and cancer colon cell lines using an LDH (lactate dehydrogenase)-based assay . Most oftabolism ,44. The tabolism . NotablyO-glucoside, kaempferol 3-O-glucuronide, and quercetin 3-O-glucuronide were obtained from Extrasynthese . Gallic acid, catechin, and epicatechin were obtained from Carl Roth . Procyanidin B1, procyanidin B2, procyanidin B3, and procyanidin C1 were purchased from Cayman Chemical , while agrimoniin, apigenin, 3-O-caffeoylquinic acid, ellagic acid, astragalin (kaempferol 3-O-glucoside), pedunculagin, avicularin (quercetin 3-O-arabinoside), hyperoside , isoquercitrin (quercetin 3-O-glucoside), and tiliroside (purity > 96%) were previously isolated in the Department of Pharmacognosy of Medical University of Białystok  [The reference phytochemicals, including isorhamnetin 3- Poland) ,49,50,51w:v) solvent ratio to obtain raw extracts. Subsequently. extracts were evaporated to dryness, diluted with water (50 mL). and successively portioned between chloroform (10 × 20 mL). Afterwards. purified extracts were freeze-dried. The list of obtained aqueous acetone extracts from selected Potentilla species detailing plant species, voucher specimen, the parts used and extraction yields are presented in Plants used to obtain material for investigations come from the Medicinal Plant Garden at the Medical University of Białystok  and were collected in June-August 2017–2020. Plants were carefully identified by one of the authors (M.T.), and individual voucher specimens were deposed at the Herbarium of the Department of Pharmacognosy, Medical University of Białystok . Plant material was dried at room temperature in the shade and air temperature and subsequently finely ground with an electric grinder. Accurately weighed 2 g of each powdered dry plant material were separately extracted using an ultrasonic bath  with 70% acetone at a controlled temperature (40 ± 2 °C) for 45 min in a 1:75  . Each ti3) solution . After incubation of the mixture at room temperature for 10 min, the absorbance was measured at 415 nm, and results were expressed as milligrams of rutin equivalents per gram of extract (mg RE/g extract). The determination was repeated at least three times for each sample solution.Total flavonoid content was estimated by the previously described colourimetric method . Each alThe separation and qualitative evaluation of each extract were conducted using a Kinetex XB-C18 column  and Agilent 1260 Infinity LC chromatography system coupled to a photo-diode array (PDA) detector and 6230 time-of-flight (TOF) mass spectrometer . A detailed description of the execution of the above-mentioned assay was presented in the previous study .2 at 37 °C.For the cell culture study, human colon adenocarcinoma cell line LS180 and human colonic epithelial cell line CCD841 CoN were purchased from the European Collection of Cell Cultures  and American Type Culture Collection , respectively. LS180 cells and CCD841 CoN cells were maintained in Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively. Then, 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 g/mL) were added to the cell culture media. Cells were incubated in a humidified atmosphere of 95% air and 5% COExamination of the anticancer potential of extracts was conducted simultaneously on both cancer (LS180) and normal (CCD841 CoN) colon cells. Cells at a density of 5 × 104 cells/mL were plated on 96-well plates. The next day, the cell growth medium was exchanged for fresh medium supplemented with investigated extracts or 25 μM 5-fluorouracil (5-FU). After 48 h of treatment, compound impacts on cell membrane integrity, metabolic activity, and DNA syntexis were examined using LDH, MTT, and BrdU assays, respectively. The description of the execution of indicated tests was previously presented [p < 0.05. The IC50 value (concentration causing proliferation inhibition by 50% compared to control) was calculated according to the method of Litchfield and Wilcoxon [The data were presented as the mean ± standard error of mean (SEM). Statistical analyses were carried out using one-way ANOVA with Tukey’s post hoc test and column statistics. Significance was accepted at Wilcoxon  using GrPotentilla species. The analysis revealed a series of marker metabolites such as agrimoniin, pedunculagin, dimeric and trimeric B-type procyanidins, tiliroside, astragalin (kaempferol 3-O-glucoside), hyperoside  showed cytotoxic effects against normal epithelial cells. Despite the fact that the investigated extracts affected both normal and cancer colon cells, the LS180 cells were more sensitive to tested extracts. Considering the data obtained from all the performed studies, the 2 of the 12 investigated extracts (PFR7 and PER7r) revealed the greatest chemopreventive potential, as manifested by the effective elimination of colon cancer cells, which caused both damage to their cell membranes and inhibition of their proliferation and metabolic activity, with a simultaneous lack of any cytotoxic effect on normal colon epithelial cells and a significantly weaker effect on their metabolism and DNA synthesis compared to cancer cells. The previous [Potentilla species have anticancer potential, however, additional animal and clinical studies, especially including the influence of intestinal flora are required to verify discovered beneficial properties of investigated extracts. Nevertheless, discovered selectivity of the anticancer effects of tested extracts encourages further studies to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.In conclusion, the presented study reports, for the first time, an analysis of the LC–HRMS profile of aqueous acetone extracts from rare previous  and curr


KRAS as a driven mutation followed by SMAD4, CDKN2, and TP53-associated mutations. In clinical aspects, pancreatic cancer presents unspecific clinical symptoms with the absence of screening and early plasmatic biomarker, being that CA19-9 is the unique plasmatic biomarker having specificity and sensitivity limitations. We analyzed the plasmatic exosome proteomic profile of 23 patients with pancreatic cancer and 10 healthy controls by using Nanoscale liquid chromatography coupled to tandem mass spectrometry (NanoLC-MS/MS). The pancreatic cancer patients were subdivided into IPMN and PDAC. Our findings show 33, 34, and 7 differentially expressed proteins when comparing the IPMN vs. control, PDAC-No treatment vs. control, and PDAC-No treatment vs. IPMN groups, highlighting proteins of the complement system and coagulation, such as C3, APOB, and SERPINA. Additionally, PDAC with no treatment showed 11 differentially expressed proteins when compared to Folfirinox neoadjuvant therapy or Gemcitabine adjuvant therapy. So here, we found plasmatic exosome-derived differentially expressed proteins among cancer patients  when comparing with healthy controls, which could represent alternative biomarkers for diagnostic and prognostic evaluation, supporting further scientific and clinical studies on pancreatic cancer.In the oncological area, pancreatic cancer is one of the most lethal diseases, with 5-year survival rising just 10% in high-development countries. This disease is genetically characterized by  A recenA combination of complete resection and systemic multi-agent chemotherapy is the only hope to cure pancreatic cancer. Surgical outcomes have improved due to increased surgical expertise, and more patients are currently considered for resection. However, the overall recurrence rate remains at 70–80%, commonly occurring as distant metastases to the liver, which drove current practice toward neoadjuvant chemotherapy before surgery, regardless of resectability. Current NCCN guidelines recommend Folfirinox or Gemcitabine in combination with nab-paclitaxel as preferred agents. Adjuvant chemotherapy after curative intent surgery in PDAC is evolving with therapies with modified Folfirinox for 24 weeks and Gemcitabine with or without Capecitabine in cases of intolerability to mFolfirinox. Generally, locally advanced or metastatic PDAC patients are considered non-curative and managed with palliative chemotherapy with gemcitabine monotherapy or Folfirinox .KRAS and TP53, have been reported in pancreatic cancer patients [Plasma biomarkers for pancreatic cancer diagnosis and prognosis have been suggested, such as specific exosome and plasma microRNAs that are elevated in pancreatic cancer patients ,16. In apatients ,18. Thispatients ,20,21. Mpatients . Therefopatients ,24. Exospatients ,26. In tEpidemiological data for gender, age, tobacco and alcohol usage, and diabetes for both groups are represented in p-value = 0.0351; df = 14). Regarding the average concentration of exosomes, a significant difference  was observed between the healthy controls (8.83 × 1011 ± 3.5 × 1011 particles/mL) and pancreatic cancer patients (3.70 × 1012 ± 1.8 × 1012 particles/mL). These results were published by Marin et al., 2022 [The average size of exosomes obtained from healthy controls (114.13 ± 13.70 nm) was significantly lower than that of pancreatic cancer patients (117.3 ± 24.68 nm),  and PDAC-S (N = 5) patients, as well as healthy controls (N = 10). Additionally, the proteome of exosomes from PDAC patients treated with neoadjuvant Folfirinox therapy (PDAC-F N = 4), and Gemcitabine-based adjuvant therapy  were analyzed, totalizing 33 patient samples. A high-throughput label-free mass-spectrometry approach was employed to identify and quantify the proteome of the exosomes. Subsequently, statistical and bioinformatics analysis were applied to explore the changes in protein expression across the different sample groups to assess the biological significance of these exosome proteins in the context of tumorigenesis. Initially, 319 different proteins were identified in the exosome samples, with a false discovery rate (FDR) of 1% . However, for a more reliable and representative proteome, only the 135 proteins expressed in at least 70% of the samples in each group were retained for further analysis.In the study’s initial sections, we explored the differential proteome of IPMN and PDAC-S exosomes by comparing their proteomic content with that of healthy control exosomes . Moreover, we analyzed the exosome proteomes between patients with these distinct pancreatic diseases (PDAC-S vs. IPMN). Finally, we investigated the differential proteome of the exosomes from PDAC patients that underwent only surgery and therapy-treated PDAC patients . The identified proteins are presented using their corresponding coding gene symbols.p-value < 0.05, logFC ± 0.58), we identified 33, 34 and seven differentially expressed proteins (DEPs), respectively  revealed that the differential expression profile of identified proteins in IPMN C and PDATo investigate the functions of the identified DEPs in IPMN and PDAC-S samples, as well as the biological processes and pathways that can be impacted by their dysregulated expression, we employed the STRING database (v. 11.5) to predict the main interactions involving each set of DEPs. We also performed enrichment analysis based on the Reactome pathways, GO-MF, and GO-BP collections from MSigDB v. 2023.1 using the IPMN and PDAC-S DEPs separately (FDR < 0.05). The interaction networks exhibited significantly more interactions than expected, indicating that the proteins are biologically connected, at least partially, as a group . This obThe enrichment analysis reveals distinct biological alterations that were uniquely enriched in the IPMN DEPs, PDAC-S DEPs, and some alterations that were common to both diseases .−14) and acute inflammatory response (2.95 × 10−13), involving DEPs such as AGT, ITIH4, F2, and HP; activation of C3 and C5 (FDR = 3.94 × 10−6), with the enriched proteins C3, C5, and CFB; and neutrophil degranulation (FDR = 2.84 × 10−3), involving A1BG, SERPINA1, and SERPINA3 proteins.In the IPMN vs. CT set, the DEPs were especially involved in inflammatory response (FDR = 8.95 × 10−6), involving the apolipoproteins APOA1, APOA2, and APOB; FCGR activation (FDR = 2.61 × 10−5) and CD22 mediated regulation (FDR = 1.68 × 10−5), enriched in immunoglobulins IGKV1-16, IGKV2D-28, IGKV3-15, and IGLV1-40; and wound-healing processes (FDR = 2.44 × 10−8), in which the proteins KNG1, SERPINA1, SERPING1, and VTN were identified.In the PDAC-S vs. CT comparison, the DEPs were involved in the chylomicron assembly and remodeling alterations , hemostasis , innate immune system , and intrinsic pathway of fibrin clot formation . Altogether, C3, F2, SERPING1, CLU, SERPINA1, VTN, and A1BG were identified in these processes.Furthermore, some alterations were enriched in both sets of DEPs (IPMN and PDAC-S), including the complement cascade , coagulation , KRAS signaling , and xenobiotic metabolism hallmarks , while the interferon-gamma response (FDR = 5.52 × 10−3) was the only hallmark enriched for the PDAC-S DEPs. In general, the enrichment analysis based on the cancer modulation corroborates the pathways and GO results and reveals that many of the proteins identified in this study have already been associated with various types of cancer and tumorigenesis processes.Additionally, we also explored the hallmark and cancer module collections of MSigDB v. 2023.1 . Some haThe sets of DEPs identified in this study were analyzed using the NCG (v. 7.1) database to explore their association with cancer-related genes. Among all the DEPs, we identified seven potential or canonical oncogenes and tumor suppressor genes. The comparative analysis of exosome proteomes also aimed to identify proteins whose expression was altered after treatment in patients with PDAC, highlighting potential biomarkers of therapy response and follow-up. This section of the study included those patients who underwent surgery only (PDAC-S) as well as those who received Folfirinox neoadjuvant therapy (PDAC-F) or Gemcitabine adjuvant therapy (PDAC-G). In total, eleven DEPs were identified in the comparisons between these groups, including five immunoglobulins, AGT, C8A, ORM1, F2, SERPINF1, and FCN3 A. FurtheInterestingly, AGT, IGKV2D-28, and C8A presented notable expression patterns B. AGT ex−2), FceRI mediated NF-κB activation (FDR = 1.73 × 10−4), and FceRI mediated MAPK activation (FDR = 6.93 × 10−5).The DEPs set related to the therapies were also identified in biological pathways and processes according to the MSigDB v. 2023.1 database . In thisp-value < 0.05, logFC ± 0.58). The results of this analysis are described in Finally, the PDAC-F and PDAC-G samples were stratified based on treatment duration using a six-month cutoff, considering short (<6) and long (>6) treatment periods, and compared to PDAC-S  is recurrently suggested as a biomarker for different types of carcinoma , but hasThe crosstalk between inflammation and immune alterations is closely associated with tumor predisposition, driving malignant initiation, conversion, and growth, as well as taking part in advanced steps such as invasion and metastasis. Interestingly, we show here that inflammatory and immune system-related alterations, such as activation of C3 and C5, acute inflammatory response, neutrophil degranulation, and complement cascade, stand out as altered processes in IPMN due to exosome protein dysregulations. CFB, C3, and C5, components of the complement system, along with LBP, APOD, and SERPINA1, were some of the DEPs associated with these processes on IPMN.CFB, C3, and C5 are commonly associated with changes in the tumor microenvironment. These proteins are linked to abundant immune infiltrates, immune responses, and senescence, and serve as prognostic markers for various types of cancer ,36,37, iLBP was found to be up-regulated in IPMN compared to healthy controls. LBP is an LPS (lipopolysaccharide)-binding protein that has a positive correlation with PD-L1 (programmed death ligand-1). Yin and collaborators  showed aAltogether, KLKB1, SERPINA1, CFB, C3, and C5 represent DEPs on IPMN that could be further investigated in the context of pancreatic tumorigenesis. These molecular alterations and their associated biological pathways and functions can help to understand the pathological processes that contribute to cancer progression. Moreover, these DEPs could be potential biomarkers for the early detection of IPMN through liquid biopsy methods.PDAC comprises a highly aggressive and lethal form of pancreatic cancer with a notable ability to evade the immune system, creating a tumor microenvironment that promotes immune tolerance and resistance to immune-mediated destruction. This immune evasion, along with other factors, contributes to the aggressive nature of PDAC and its resistance to standard cancer therapies. Our results showed many DEPs related to the immune system and correlated processes. Recently, Huang and collaborators  have comOur proteomic analysis revealed significant differential expression of several proteins in PDAC-S samples compared to healthy controls. Notably, CPN1, IGHV2-26, ITIH3, and CLU were found to be overexpressed, while C4BPB, APOB, CFH, and C1QB showed downregulation in PDAC-S samples.Carboxypeptidases (CPs) form an extensive group of zinc metallopeptidases responsible for various physiological roles by removing C-terminal basic residues from proteins and peptides . CarboxyCPs contribute to the pathogenesis of multiple cancer types . In breaITIH3, another overexpressed DEP in the PDAC-S samples, also acts in the tumor microenvironment contributing to tumor progression. The inter-alpha-trypsin inhibitors (ITI) constitute a family of plasma protease inhibitors that contribute to the stability of the extracellular matrix through covalent binding to hyaluronan . The groClusterin (CLU) is a highly evolutionary conserved glycoprotein that controls crucial physiological processes and several cancer-associated events, including cancer cell proliferation, stemness, survival, metastasis, epithelial-mesenchymal transition (EMT), therapy resistance, and inhibition of programmed cell death, which in turn facilitates cancer growth and recurrence ,56. ThisOur findings revealed the downregulation of C4BP in PDAC-S samples compared to healthy controls. C4b-binding protein (C4BP) is a prominent regulator of the complement system , acting Another component of the complement cascade that was downregulated in PDAC-S is the complement C1q subcomponent subunit (C1QB). The complement cascade, as an integral part of innate immunity, not only acts as a critical mediator in the innate defense against pathogens but also plays a regulatory and anti-inflammatory role in the clearance of immune complexes and dying cells from damaged tissues ,64,65. IThe enrichment analysis of our differentially expressed proteins (DEPs) indicates the potential relevance of innate immune and complement systems, hemostasis, the coagulation cascade, as well as processes associated with IGF regulation, platelet calcium cytosolic levels, and G protein signaling. These findings suggest the involvement of these biological pathways and functions in pancreatic tumorigenesis. In PDAC, the significance of innate immunity has been widely explored due to the presence of intracellular functions for innate immunity proteins within tumor cells . Most ofThe complement system plays a role in the immune surveillance of pathogens and tumor cells. Proteomic studies revealed that extracellular vesicles (EVs) released by metastatic hepatocellular carcinoma (HCC) cells contained a substantial presence of complement proteins . In accoThe hallmarks enriched for our exosome proteomes (IPMN vs. CT and PDAC-S vs. CT) support the main biological pathways and functions discussed earlier, particularly the complement system and related DEPs, such as C3, C4BPB, CFB, CFH, CLU, KLKB1, SERPINA1, and SERPING1. Most of them were also observed in the coagulation hallmark. Interestingly, CFB and SERPINA3, which exhibited higher expression in IPMN and PDAC-S exosomes, have been associated with KRAS signaling ,71. OncoLi and collaborators  performeThe family of apolipoproteins is associated with a variety of cancer types. APOA1 was overexpressed in PDAC-S compared to control, and APOB was downregulated in the plasma of PDAC-S patients. Apolipoprotein A (APOA) seems to be a good indicator of several cancers, such as colon, hepatocellular, and pancreatic cancer; meanwhile, apolipoprotein E (APOE) may have polymorphisms that affect tumor susceptibility. APOC, APOB, and APOD are involved in tumor progression . ApoA1 whttps://clinicaltrials.gov/ct2/show/NCT04862260, accessed on 10 July 2023) in course. This study aims to verify if the addition of a cholesterol shortage on top of Folfirinox treatment in newly diagnosed patients , can lead to a better progression in tumor treatment response. It is expected that a drug-induced cholesterol shortage will slow down or stop the progression of pancreatic adenocarcinomas while increasing the response to chemotherapy .APOB is downregulated in CRC , and its silencing in HCC (hepatocellular carcinoma) resulted in increased cell proliferation rates ,81, indiAnother protein family largely associated with cancer is the complement system proteins. In the context of chemotherapy response, the complement system, together with the tumor microenvironment (TME) plays pivotal roles in cancer progression, and therapy failure . The comSERPINA1, SERPINA3, SERPINC1, SERPINAC1, and SERPINAG1 were upregulated in PDAC-S and IPMN as compared to healthy controls, but not in the direct comparison between IPMN and PDAC-S. The proteins coded by these genes are involved in the complement and coagulation cascade (KEGG Pathways—map04610: complement and coagulation cascade). Wu and collaborators , looked SERPINA3 is an acute-phase protein, so its expression is not a cancer-specific protein and increases in response to inflammation. It acts as an inhibitor of several serine proteases, including pancreatic chymotrypsin and cationic elastase. The expression of SERPINA3 has been previously reported to be increased in tumoral tissues and sera from patients with PDAC, with evidence for correlation with poor survival .SERPINA4 was up-regulated only in IPMN, compared to controls. Some studies showed that SERPINA4 ,92 has aConsidering treatments, AGT was overexpressed in PDAC-S compared to both PDAC g and PDAC-F, while C8A was overexpressed only in PDAC-S compared to PDAC-F, along with ORM1 and SERPINF1. These DEPs may be involved in the drug response but also may reflect the advanced stage of the patient’s receiving chemotherapy. Among hypo-expressed proteins, a member of the immunoglobulin variable chains appears both in PDAC-S versus PDAC-F and PDAC-G.Here, we identified DEP from plasma exosome-derived proteomic profiles of IPMN and pancreatic cancer patients, subdivided into two antagonistic tumor phenotypes, comparing those to healthy control samples. Despite most cases of IPMN being asymptomatic, the molecular changes involved in the IPMN pathology can lead to physiological modifications, creating a favorable environment for the development of PDAC. The early identification of IPMN lesions and comprehension of their associated biological characteristics can be useful to guide patient management and follow-up. Hence, the proteomic alterations observed in the exosomes from IPMN patients can represent potential biomarkers for the detection of IPMN and provide insights into how IPMN influences the tumor microenvironment and neoplastic progression. Moreover, the DEPs identified in PDAC described here could represent biomarkers for diagnostic and prognostic evaluation, which, in association with CA19-9 could potentially improve pancreatic cancer screening and early diagnosis. This report corroborates previous findings and provides additional scientific data on the presence, role, and potential application of exosome proteins to the basic and clinical knowledge of pancreatic cancer. The weakness of this report relies on the reduced number of samples within each pancreatic cancer group, which makes sample heterogeneity a limitation of the findings. However, this report provides data to improve follow-up with specific targets that could be useful as plasma biomarkers for pancreatic cancer. An increased number of samples associated with clinical and treatment data, and especially patient follow-up, should be used to validate such proteins as exosome-derived plasmatic biomarkers in pancreatic cancer.As future perspectives and goals, our group will focus on the validation of the proteins described here in a larger cohort of pancreatic cancer patients. Within this, we highlight a cohort of sequential samples from the same patients in order to perform a temporal evaluation of these proteins. Another future perspective relies on the validation of this finding in an in vivo approach by looking for an exosomal protein biomarker in mouse pancreatic cancer models.g for 10 min, and the plasma was separated and stored at −80 °C.This work was conducted after approval from the Ethics Committee of Fiocruz, Instituto do Cancer do Estado de São Paulo (ICESP) and Hospital do Trabalhador . All sampling and experiments were performed following relevant guidelines, Brazilian regulations, and ethical principles for human research in the Declaration of Helsinki. The project was described to all participants, and a written informed consent and epidemiological questionnaire were obtained from all participants enrolled in the study. A total of 23 pancreatic cancer patients (16 PDAC and 7 IPMN) were recruited from April 2018 to September 2019 with the inclusion criteria as disease confirmation by histopathology and/or surgery at the moment of sample collection. The PDAC patients were subdivided into three groups according to treatment undergone in sample collection, as follows: PDAC-S , PDAC-F , and PDAC g . All pancreatic cancer patients were provided from the Academic Biobank for Research on Cancer at the University of Sao Paulo (USP) (Biobank-USP), located at the Center for Translational Research in Oncology, São Paulo State Cancer Institute , São Paulo, Brazil. The Biobank–USP protocol was approved by the Local Ethics Committee . As a control group, 10 non-cancer participants were recruited from Hospital do Trabalhador, Curitiba PR, Brazil, with the inclusion criteria of non-personal history of any kind of cancer. Demographic and epidemiological data were collected in both groups, while clinical data were collected for pancreatic cancer patients. For each patient, 4 mL of peripheral blood was collected in EDTA tubes, which were immediately centrifuged at 3000× ® Exosome Serum/Plasma Kit (Qiagen cat number ID: 76603), following the manufacturer’s instructions to an initial volume of 0.6 mL plasma. Exosome Tracking Analysis using Nanosight LM-10  was used to determine the concentration and size of exosomes isolated from the blood plasma of pancreatic cancer patients and controls. Exosome samples were diluted in PBS before injection for cell range from 40 to 100 by the reading frame. The videos were set to three runs each of 60 s, the detection threshold was defined as 4, and the camera level was 12. NTA statistical analysis was performed with Minitab Statistical Software 17.0, where samples were submitted to one-way variance analysis (ANOVA), and values were compared with Tukey’s test with a 5% probability level.Nanoparticle isolation was performed with the commercial kit—miRCURYv/v), followed by a standardized In-Gel digestion protocol adapted from [After the isolation of exosomes from plasma, the protein content of 33 samples was quantified by tryptophan fluorescence  then 20 ted from . Brieflyted from . After dThe digested samples (0.5 micrograms) were separated by online nanoscale capillary liquid chromatography and analyzed by nano electrospray tandem mass spectrometry (nanoLC MS/MS) in duplicate injection. The chromatography was performed on an Ultimate 3000 nanoLC (Thermo Fisher Scientific) followed by nanoelectrospray ionization, MS, and MS/MS on an Orbitrap Fusion Lumos (Thermo Fisher Scientific). The chromatographic conditions were as follows: mobile phase A 0.1% formic acid, mobile phase B 0.1% formic acid, and 95% acetonitrile. The flow of 250 nL/min, with a 90 min non-linear  gradient from 5 to 40% B was conducted. The separation was performed on an in-house C18 packed emitter with 15 cm length, 75 μm Internal diameter, packed with 3.0 μm C18 particles (Dr. Maisch—ReproSil-Pur). MS and MS/MS scan parameters were as follows: MS1 acquisition in the Orbitrap analyzer with a resolution of 120,000 m/z window, from 300 to 1500 positive profile mode, with a maximum injection time of 50 ms. MS2 analysis was performed in data-dependent acquisition (DDA) mode of ions with 2–7 charges, 2 s per cycle where the most intense ions were subjected to high energy collisional dissociation (HCD) fragmentation at 30% normalized collision energy, followed by acquisition in the Orbitrap analyzer with a resolution of 15,000 in centroid mode. A dynamic exclusion list of 60 s was applied as well as the internal mass calibration for the MS1 scans. The nESI voltage was 2.3 kV and the ion transfer capillary temperature was 175 °C.Homo sapiens reference database downloaded from Uniprot on 11 January 2021, containing 75,777 entries was used as a database, the reverse database used as a decoy for FDR estimation, 1% FDR for both PSM and protein assignment was accepted, the match between runs and LFQ intensity was enabled. Proteins were only considered for quantification if 2 or more unique peptides were identified for that group. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [2) and normalized by Z-score, with missing values being replaced by values from the normal distribution . The normalization step was performed as recommended by Perseus developers [The spectra identification was performed in MaxQuant version 1.6.17.0  set as fhe PRIDE  partner he PRIDE . The norvelopers .t-tests were applied  to determine the differentially expressed proteins (DEPs) among the comparisons of IPMN vs. CT, PDAC-S vs. CT, IPMN vs. PDAC-S vs. IPMN, PDAC-S vs. PDAC-F, PDAC-S vs. PDAC-G, and PDAC-F vs. PDAC-G. The PDAC-F and PDAC g groups were also subdivided according to the duration of the therapy, using a six-month period as splitting criteria. Additional comparisons (PDAC-S vs. PDAC-F(<6) and PDAC-F(>6); PDAC-S vs. PDAC-G(<6) and PDAC-G(>6)) were performed to assess expression alterations related to therapy duration. Principal component analysis (PCAs) (Perseus) and heatmaps (complexheatmap R package) were used to investigate DEP’s expression patterns across comparisons. The interactive online tool [The samples were grouped according to the following conditions: Controls (CT), IPMN, PDAC-S, PDAC-F, and PDAC-G. Student’s ine tool  was acceThe Molecular Signatures Database   was acceProtein–protein interactions (PPI) were predicted using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING v. 10.5)  with the


Purpose: Prolyl 4-hydroxylase subunit alpha 3 (P4HA3) is implicated in several cancers’ development. However, P4HA3 has not been reported in other cancers, and the exact mechanism of action is currently unknown.Materials and methods: First, the expression profile of P4HA3 was analyzed using a combination of the University of California Santa Cruz (UCSC) database, Cancer Cell Line Encyclopedia (CCLE) database, and Genotype-Tissue Expression (GTEx) database. UniCox and Kaplan-Meier were used to analyze the predictive value of P4HA3. The expression of P4HA3 was analyzed in clinical staging, immune subtypes, and Molecular subtypes. Secondly, the correlation of P4HA3 with immunomodulatory genes, immune checkpoint genes, RNA modification genes, immune cell infiltration, cancer-related functional status, tumor stemness index, DNA mismatch repair (MMR) genes and DNA Methyltransferase was examined. The role of P4HA3 in DNA methylation, copy number variation (CNV), mutational status, tumor mutational burden (TMB), and microsatellite instability (MSI) was also analyzed. In addition, gene set enrichment analysis (GSEA) was used to explore the potential functional mechanisms of P4HA3 in pan-cancer. Finally, P4HA3-related drugs were searched in CellMiner, Genomics of Drug Sensitivity in Cancer (GDSC), and Cancer Therapeutics Response Portal (CTRP) databases.Results: P4HA3 is significantly overexpressed in most cancers and is associated with poor prognosis. P4HA3 is strongly associated with clinical cancer stage, immune subtypes, molecular subtypes, immune regulatory genes, immune checkpoint genes, RNA modifier genes, immune cell infiltration, cancer-related functional status, tumor stemness index, MMR Gene, DNA Methyltransferase, DNA methylation, CNV, mutational status, TMB, and MSI are closely related. Available enrichment analysis revealed that P4HA3 is associated with the epithelial-mesenchymal transition and immune-related pathways. There are currently 20 drugs associated with P4HA3.Conclusion: In human pan-cancer, P4HA3 is associated with poor patient prognosis and multiple immune cells and may be a novel immunotherapeutic target. It may act on tumor progression through the epithelial-mesenchymal transition (EMT) pathway. According to the latest data on cancer from the 2020 statistics, the worldwide cancer burden is expected to increase by 47% by 2040 compared to 2020 . Cancer a-subunits and two ß-subunits. Among them, the catalytic a-subunit has three different isozymes such as P4HA1, P4HA2, and P4HA3 (The extracellular matrix (ECM), the most abundant component of the tumor microenvironment (TME), plays a crucial role in tumor development . Collagend P4HA3 .P4HA3  is a protein-coding gene that belongs to the prolyl 4-hydroxylase family members. A study found that P4HA3 expression is upregulated in patients with renal cell carcinoma (RCC), promoting cancer growth, invasion, and metastasis   interacts with P4HA3 to inhibit pituitary adenoma (PA) growth and metastasis by blocking the PI3K/AKT signaling pathway . P4HA3 is overexpressed in melanoma and promotes tumor proliferation and invasion  . P4HA3 i pathway . In addiinvasion . The aboIn this study, we evaluated the expression of P4HA3 and its relationship with the prognosis of cancer patients. In addition, the relationship between P4HA3 and the tumor immune microenvironment was further analyzed. Our findings provide new insights into the functional role of P4HA3 in human cancer, highlighting the potential mechanisms by which P4HA3 affects the tumor microenvironment and cancer immunotherapy.https://xenabrowser. net/datapages/). The UCSC database includes expression matrices and clinical data of pretreated TCGA tumor patients and normal subjects, including overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI), tumor stage, etc. First, P4HA3 expression data of normal tissues were obtained from the GenotypeTIssup Expression  database. And P4HA3 expression data of tumor cell lines were obtained from the Cancer Cell Line Encyclopedia  database. Second, a uniformly normalized pan-cancer dataset was obtained from the UCSC Xena database to analyze the expression levels of P4HA3 in cancer and the differential expression in cancer and normal samples. Meanwhile, we analyzed the expression of P4HA3 in male and female tumor tissues and the corresponding normal tissues using the R package “gganatogram” (P4HA3 using The Human Protein Atlas (HPA) database (https://www.proteinatlas.org). Many researchers have used this database to analyze the expression levels of target genes, such as Xiong et al., Zhou et al., Liu et al., and Xu et al. used this database to study the expression levels of PIMREG, NAAA, CD2, and TUBA1C respectively, which tentatively proved their analysis results  were downloaded from the UCSC Xena database , C2(IFN-gamma dominant), C3 (inflammatory), C4 (lymphocyte depleted), C5 , and C6 (TGF-b dominant), were obtained in the TISIDB database (https://doi.org/10.1093/bioinformatics/btz210). In addition, the molecular subtype profile of P4HA3 in 13 tumors was also analyzed. We analyzed the association between P4HA3, immune, and molecular subtypes through TISIDB database of pre-processed gene expression data and phenotypic information of TCGA cancers.The expression levels of P4HA3 and immune regulatory genes, immune checkpoint genes, and RNA modification genes were calculated separately using the spearman algorithm.The expression data of 150 marker genes for immune regulation  using various algorithms such as TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, XCELL, EPIC, and TIDE. The TIMER 2.0 database provides a more reliable analysis of target gene and immune cell correlations by using state-of-the-art algorithms to calculate the level of immune infiltration in TCGA tumor data. The results were visualized using the R package “ggplot2” (P4HA3 and the three scores.The correlation between ggplot2” . In addihttps://ngdc.cncb.ac.cn/databasecommons/database/id/6092) as well as 14 cancer-related functional states . The correlation between P4HA3 and 14 cancer-related functional states in each tumor was analyzed and visualized using the R package “ggplot2”. In addition, we obtained DNAss tumor stemness scores calculated from methylation profiles for each cancer from a previous study . Meanwhile, using the TCGA database  we analyzed the relationship between the expression level of P4HA3 and the methylation level of its promoter region and visualized it using the R package “ggplot2”. The correlation between P4HA3 expression and tumor mutational burden (TMB) and microsatellite instability (MSI) in different tumors was analyzed in the TCGA database using Spearman’s test, and the results were visualized by the R package “fmsb”. In addition, we also analyzed the correlation between P4HA3 and five DNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6, PMS2, and EpCAM. and their expression levels with the four methyltransferases DNMAT1, DNMT2, DNMT3A, and DNMT3B. Not only that, but we also analyzed the correlation between P4HA3 expression and somatic cell copy number variation (CNV) in pan-cancer.To understand the mutation characteristics and location of P4HA3, we used the R package “ClusterProfiler” (https://www.gseamsigdb.org/gsea/index.jsp) a hallmark gene set containing 50 critical pathways affecting cancer. The Normalized Enrichment Score (NES) and False Discovery Rate (FDR) of P4HA3 in each path were calculated separately. p-values <0.05 were considered statistically significant.To further understand the biological functions and potential molecular mechanisms of rofiler”  to perfoP4HA3, we analyzed the relationship between P4HA3 expression and drug sensitivity using the CellMiner database (https://ngdc.cncb.ac.cn/databasecommons/database/id/6092). Meanwhile, to further expand our study, we also explored the correlation between P4HA3 expression and drug sensitivity from the Genomics of Drug Sensitivity in Cancer (GDSC) database (https://www.cancerrxgene.org) and Cancer Therapeutics Response Portal (CTRP) database .To understand the potential drugs targeting P4HA3 in normal tissues using the GTEx dataset , cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), kidney renal clear cell carcinoma (KIRC), acute myeloid leukemia (LAML), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and thymoma (THYM) were expressed at higher levels than normal tissues in 16 tumors. In contrast, P4HA3 was expressed at lower levels than normal tissues in eight tumors, including liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), skin cutaneous melanoma (SKCM), thyroid carcinoma (THCA), uterine corpus endometrial carcinoma (UCEC), bladder urothelial carcinoma (BLCA), brain lower grade glioma (LGG), and testicular germ cell tumors (TGCT)  . Finallyand bone . The IHCP4HA3 high expression in BRCA, CESC, COAD, GBM, HNSC, KIRC, kidney renal papillary cell carcinoma (KIRP), LGG, LUSC, OV, READ, STAD, THCA, UCEC, and uveal melanoma (UVM) had a poor prognosis , GBM, COAD, cervical squamous cell carcinoma (CESC), and BLCA , LUAD, LGG, KIRP, KIRC, KICH, GBM, COAD, CESC, BRCA, BLCA, and adrenocortical carcinoma (ACC)  ; P4HA3 wand BLCA ; P4HA3 wand CESC . The surP4HA3 was more highly expressed at higher stages of BLCA, COAD, ESCA, KIRP, READ, STAD, and UVM. In comparison, lower expression in higher stages of ACC, HNSC, KIRC, and LUAD , endothelial cells, and Hematopoietic stem cells in pan-cancer -JQ1, Acrichine, Lomustine, Daunorubicin, 6-Mercaptopurine, and Belinosta1 were significantly and negatively correlated with 16 drugs  play an immunosuppressive role in tumor development and are associated with poor tumor prognosis /PD-L1 resistance by depleting CD8sistance . Becauseotherapy . Thereforognosis . TAMs carognosis . Notablyrognosis . They arrognosis . These rP4HA3 expression with tumor TMB, MSI, MMR Gene, CNA, and mutational status. Among them, elevated P4HA3 expression levels upregulated TMB of UCS, THYM, PRAD, LUAD, LGG, and LAML, as well as MSI of UCEC, PRAD, MESO, COAD, and STAD. In human cancers, P4HA3 expression was significantly associated with five MMR genes, especially in LAML and THCA. We found a P4HA3 gene variation rate of 2.6% in all TCGA tumors, dominated by missense mutations. In addition, epigenetic modifications are one of the main reasons affecting the effectiveness of various tumor immunotherapies, such as ICIs and immune cell therapies   to self-renew and differentiate into mature cells is called stemness . Many st process . Targetin cancer . TGF-β en cancer . TGF-β sg cancer . P4HA3 roma (PA) . Reducedn cancer . Numeroun cancer . This evP4HA3 is expressed at high levels in most cancers, which correlates with the spite of cancers and poor prognosis. We can also assess the effect of tumor immunotherapy, tumor immunosuppression, and drug resistance based on the expression of P4HA3. In addition, P4HA3 can affect the infiltration of tumor immune cells and is likely to promote cancer progression through EMT. Therefore, P4HA3 may serve as a promising biomarker for human cancer diagnosis, treatment, and prognosis and may predict the efficacy of anti-tumor immunotherapy response. This study may provide ideas for future researchers to study the role of P4HA3 in human cancers.Our findings suggest that P4HA3 might affect cancer by participating in some potential signaling pathways, the specific molecular biology of the mechanism of action is not yet clear. These need to be further investigated in depth in the future.Of course, there are some limitations to this study. For example, our study was limited to bioinformatics analysis, which remains to be further validated by basic experiments or clinical trials. In addition, although the present study identified that


Nasopharyngeal carcinoma (NPC) is rare in most parts of the world but endemic in southern Asia. Here, we describe the molecular abnormalities in NPC and point out potential molecular mechanisms for future therapy. This article provides a brief up-to-date review focusing on the molecular pathways of NPC, which may improve our knowledge of this disease, and we also highlight some issues for further research. In brief, some heritable genes are related to NPC; therefore, people with a family history of NPC have an increased risk of this disease. Carcinogenic substances and Epstein–Barr virus (EBV) exposure both contribute to tumorigenesis through the accumulation of multiple genomic changes. In recent years, salted fish intake has decreased the impact on NPC, which implies that changing exposure to carcinogens can modify the risk of NPC. Eradication of cancer-associated viruses potentially eradicates cancer, and EBV vaccines might also prevent this disease in the future. Screening patients by using an EBV antibody is feasible in the high-risk group; plasma EBV DNA measurement could also be conducted for screening, prognosis, and monitoring of this disease. Understanding the molecular mechanisms of NPC can further provide novel information for health promotion, disease screening, and precision cancer treatment. Nasopharyngeal carcinoma (NPC) is rare in most parts of the world but is endemic in southern Asia . AccordiThe association between dietary factors and NPC has been supported by numerous studies, particularly in high-risk populations such as southern China and southeast Asia. The risk factors include frequent consumption of salt-preserved fish, meats, and eggs ,4. The hOther nondietary factors include Epstein–Barr virus infection, tobacco smoke, occupational exposure to fumes, smoke, dust, and chemicals, including formaldehyde. These carcinogenic materials can induce DNA damage, genome instability, and a dysregulated cell cycle that increase the tumorigenesis of NPC. Exposure to carcinogens can also reactivate latent EBV virus ,12. We lEpstein–Barr virus (EBV) is a ubiquitous human g-herpesvirus infecting more than 90% of the adult human population and that establishes life-long latency in epithelial cells and B cells. Its infection is associated with various lymphoid malignancies, including Burkitt lymphoma, Hodgkin lymphoma, B-cell lymphoma, and NK/T-cell lymphoma, as well as two types of epithelial cancer, nasopharyngeal carcinoma (NPC) and subtypes of gastric carcinoma. EBV has two distinct life cycles, namely, lytic (productive) and latent (persistent). After initial replication in nasopharyngeal epithelial cells, EBV passes through the epithelial cells lining the nasopharynx and infects and replicates in naïve B cells. It eventually establishes a benign and stable latency in infected cells in a state of histone-associated episome . PeriodiDuring latency, viral gene expression is limited to a small subset of viral genes that are important for genome maintenance and regulatory functions. There are four types of latency that are defined by the expression profile of late genes. EBV infection in NPC is typically represented by a type 2 latency (default program), in which EBV nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), and LMP2 proteins are produced. In addition, noncoding RNAs, such as EBV-encoded RNAs (EBERs), BamH1 A rightward transcripts (BARTs), and BART miRNAs, are expressed. Expressed viral genes provide necessary replication and survival signals to both EBV and host cells. Latent genes seem to be the key drivers in EBV-associated NPC; however, EBV lytic phase proteins, such as BGLF5 (DNase) and BALF3, promote host genome instability and play important roles in NPC tumorigenesis ,18. TablSimilar to any other type of cancer, early NPC detection is key for a good prognosis. Nasopharyngoscopy is the tool of choice to exam nasopharynx and guide biopsy. Narrow-band imaging (NBI) could be applied for the diagnostic screening and accuracy rate increment of a high-risk population of NPC . The perFor example, an early study showed that measurements of blood EBV DNA could be a predictor of disease recurrence and overall survival in NPC patients . Later, EBV gene products often provide a cell proliferation advantage and anti-death signal. Therefore, targeting the EBV genome or the signaling pathways it stimulates is an attractive way to treat EBV-positive cancers. There are two different approaches used for targeting EBV-positive cancer cells: (1) targeting EBV latent genes and 2) lytic induction therapy. For targeting EBV latent proteins, EBNA1, LMP1, and LMP2 are potential therapeutic targets. Among them, disrupting the function of EBNA1, a protein essential for latent EBV genome replication and maintenance, is gaining the most attention. Strategies include using small molecules or DNA containing the EBNA1 binding sequence or gRNA targeting oriP to interfere with EBNA1 dimerization/oligomerization and DNA binding. The major challenge of these approaches is how to deliver the aforementioned molecules to cancer cells efficiently ,28. More lytic inGenome instability refers to an increased tendency for errors to occur during DNA replication or repair, leading to mutations, deletions, or other alterations in the genome. Genome instability can occur naturally as a result of abnormal cellular processes (endogenous), or it can be induced by exposure to various environmental factors (exogenous), such as radiation, certain chemicals, or viruses. There is evidence to suggest that genome instability plays a role in the development of NPC, and several factors have been implicated in promoting genome instability in NPC. For example, EBV is known to cause DNA damage and inhibit DNA repair mechanisms in infected cells, which can increase the likelihood of mutations and chromosomal abnormalities . In addiRASSF1A) [ADAMTS9), protein tyrosine phosphatase, receptor type G (PTPRG) [PIK3CA and TP53, previously found in NPC, were confirmed in the study. Seven new genes, including BAP1, ERBB2, ERBB3, KRAS, NRAS, KMT2D (MLL2), and TSHZ3, were identified to be significantly associated with NPC [Gene mutations and epigenetic dysregulation are important for NPC tumorigenesis . The losRASSF1A) , ADAM me (PTPRG) , and sev (PTPRG) ,49,50,51 (PTPRG) . NPC hasHLA-A2, HLA-A11, HLA-B46, and HLA-B58. These associations suggest that the immune response plays an important role in the development and progression of NPC. It is worth noting that the association of HLA types with NPC may vary depending on the geographic region and ethnic group. For example, HLA-A2 association with NPC is common in Asians [HLA-A2 in the US have a significantly lower risk than those with other antigens at the A locus [TNFRSF19 on 13q12, MDS1-EVI1 on 3q26, and the CDKN2A-CDKN2B gene cluster on 9p21 [CYP2A13 and NPC susceptibility was found in a Taiwan cohort [CYP2E1 was found to be linked to NPC risk in several studies, including a case–control study conducted in Taiwan [Based on several other GWAS, the major histocompatibility complex (MHC) region on chromosome 6p21, which contains human leukocyte antigen (HLA) genes, was most strongly associated with NPC ,55. HLA n Asians ,57. Cauc on 9p21 . The los on 9p21 ,61. Polyn cohort  and Cantn cohort , polymorn Taiwan ,66,67,68n Taiwan  , cylindromatosis (CYLD), and TNF alpha-induced protein 3 (TNFAIP3), were found in NPC primary tumors in a whole-exome study at a high frequency [NFKBIA regulates NF-κB nuclear translocation. CYLD encodes deubiquitinating enzymes (DUBs), which regulate cell growth and invasiveness by untying K63-linked ubiquitin chains through deubiquitination [TNFAIP3, the gene encoding A20, is associated with a wide panel of inflammatory pathologies [Dysregulation of NF-κB, which is important for angiogenic and tumorigenic regulation, is often observed in cancer. The constitutive activation of NF-κB is associated with the dysregulation of the transcription of genes that encode cytokines, chemokines, adhesion factors, and inhibitors of apoptosis . By EMSArequency ,73. NFKBtination . TNFAIP3hologies . Mutatiohologies .PI3K/AKT pathway regulates a number of cellular processes representing cancer hallmarks, such as cell growth, proliferation, anti-apoptosis, migration, and angiogenesis [PI3K/AKT, either altered expression or mutation of components of the pathway, is found in many types of cancer [PIK3CA (encoding the p110α subunit of PI3K), PTEN (a negative regulator of the pathway), and AKT1/2/3 (encoding AKT isoforms). These mutations result in increased activity of the PI3K/AKT pathway, contributing to the development and progression of NPC. Mutations in PIK3CA promote cell proliferation and migration and inhibit apoptosis in NPC cells [ErbB-PI3K signaling pathway were mutated in 30/128 cases [PIK3CA mutation was one of the most frequently dysregulated oncogenes, activated by hot-spot mutations  and amplification. Mutations in ERBB2, ERBB3, and KRAS were also found. In a separate study [PI3K/AKT pathway activators or regulators  were observed, although less frequently. Together, these findings suggest that ERBB-PI3K pathway activation by genetic alterations exacerbates NPC malignancy [PI3K/AKT pathway can be activated by extracellular signals such as growth factors and cytokines, which bind to their receptors on the cell surface and trigger downstream signaling cascades. LMP1 directly activates PI3K, leading to Akt phosphorylation and activation of several downstream signaling pathways [PI3K/Akt pathway by EBV is thought to contribute to the development of NPC.The ogenesis . The serf cancer ,79. The PC cells . In an N28 cases . PIK3CA te study , mutatiolignancy . In addipathways . The actTP53 and CDKN2A [TP53, encoding the p53 tumor suppressor, regulate the cell cycle. A relatively low incidence of TP53 mutations occurs in NPC compared to the incidence rate (85%) found in head and neck cancers [TP53 mutations are consistently detected more frequently in recurrent and metastatic NPC and are associated with a significant reduction in disease-free and overall survival [A genomic landscape study revealed that the G1/S cell cycle transition was affected in 28% of NPCs, with most of the alterations occurring in  cancers . Howeversurvival ,83. Thissurvival .CDKN2A (p16) and CDKN2B (p15), two negative G1/S regulators, were found in NPC primary tumors [CDKN2A and CDKN2B [CCND1 (cyclin D1) [MYC [Genes controlling the G1/S transition are frequently mutated in NPC. Early driver events in NPC likely involve dysregulation of the cell cycle. Homozygous deletions y tumors . More red CDKN2B . Amplificlin D1) ,85 and MD1) [MYC ,86 loci RASSF1A regulates mitosis and maintains genomic stability by repressing cell cycle-related protein cell division cycle 20 (CDC20) and microtubule proteins. Loss of RASSF1A expression in NPC, either due to 3p21.3 deletion or promoter hypermethylation, is associated with NPC genome instability and tumorigenesis [The tumor suppressor gene igenesis ,87.TGFBR2, TGFBR3, ACVR2A, and SMAD4 [TGF-β/SMAD has a biphasic action in tumorigenesis: a tumor suppressor at the early stage and a tumor promoter at the late stage, suggesting that its function is context dependent. TGF-β signaling regulates G1/S cell cycle transition, apoptosis, senescence, epithelial-to-mesenchymal transition (EMT), metastasis, angiogenesis, and immune responses . Alterednd SMAD4 .TGFBR2, mutations in which attenuated canonical TGF-β-mediated SMAD signaling, was often found in clinical tumor samples when compared with adjacent histological normal epithelial cells and infiltrating lymphocytes [TGFBR2 missense mutations and one contained SMAD4 missense mutation. In addition, patients harboring mutations in TGF-β/SMAD signaling were associated with poor overall survival and poor recurrence-free survival. All these mutations in TGFBR2 and SMAD4 abrogate SMAD-dependent TGF-β signaling, providing evidence that dysregulated TGF-β signaling contributes to exacerbating NPC pathogenesis [Downregulation of phocytes . In anotogenesis . TogetheThe Wnt/β-catenin pathway is involved in the development of embryo and adult tissue hemostasis. It contributes to the development and progression of some solid and hematologic malignancies . A recenCDKN2A and RASSF1, were in these regions. Downregulation of these promoters contributed to NPC tumorigenesis [ARID1A (AT-rich interactive domain-containing protein 1A) was identified to be the most affected gene [ARID1A is a key subunit of the SWI/SNF chromatin remodeling complex, which plays a crucial role in regulating gene expression by altering chromatin structure. Downregulation or loss of ARID1A expression was found to be a common epigenetic alteration in NPC, which may contribute to NPC tumorigenesis by promoting cell proliferation, invasion, and metastasis [ARID1A mutations were present in 17.5% of NPC cases and were associated with poorer overall survival [Epigenetic dysregulation is a hallmark of NPC tumorigenesis. Host genetics, EBV infection, and environmental factors contribute to epigenetic changes, ultimately leading to NPC development. Epigenetic alterations in NPC include DNA methylation, histone modifications, and noncoding RNA dysregulation . In a meigenesis . Pathwayted gene . ARID1A tastasis . One stusurvival .DNMT1, TET1, IDH2), (2) histone modification , and chromatin remodeling  were identified [EBV infection plays a critical role in epigenetic alteration. EBV-driven global-wild epigenetic silencing includes many tumor suppressor genes. Key epigenetic drivers for (1) DNA methylation (Nasopharynx-associated lymphoid tissue (NALT) is different from other lymphoid tissue from organogenesis. NALT plays an important role in mucosa immune response . SecretiThe tumor immune microenvironment (TIME) consists of an immunostimulant TIME and an immunosuppressive TIME. TIME combined with cellular and acellular components. Tumor cells, fibroblasts, endothelial cells, and leukocytes are the main cells of the TIME, while EBV exosomes, cytokines, and chemokines provide mediators between cells and influence tumorigenesis, metastasis, and immune evasion. The microenvironment is heterogeneous and dynamic. The TIME component also serves as a prognostic biomarker and potential target for novel therapy ,99.Since NPC expresses the EBV genome, adoptive T-cell therapy has emerged as a potential therapeutic strategy for NPC. Expression of PDL-1 by cancer cells can inhibit the effector function of adoptively transferred EBV-specific T cells. The combination of EBV-specific adoptive T-cell therapy and programmed cell death-1 blockade therapy has been reported to be more successful .Remarkable progress has been made in the last decade, and the exact molecular mechanisms of NPC have been more clearly defined. Translation of the results of studies still depends on more solid evidence. Research focusing on the genomic, epigenomic, and immune landscapes of NPC is required. Furthermore, immunotherapy might become an important treatment modality in the future.NPC tumorigenesis is a complex process that involves host genetics, viral infection, and environmental factors. It is characterized by a comparatively low mutation rate, extensive hypermethylation, frequent chromosomal abnormalities, and copy number alterations . Primary