A reduction in the magnitude of corneal astigmatism of ≥1 D was observed in 52.8% of eyes. No significant changes were observed in corneal endothelial density (p = 0.317). Significant changes were found in the anterior vertical coma component (p = 0.002) as well as in the spherical aberration of the posterior corneal surface (p = 0.004). Only two relevant complications were described: one corneal microperforation with penetration of the ring segment into the anterior chamber , and a case of ring extrusion . ICRS implantation in children keratoconus eyes allows a reduction of corneal astigmatism, irregularity, and aberrations, leading to a significant visual improvement.The short-term safety and efficacy of intracorneal ring segment (ICRS) implantation in keratoconus eyes of children are investigated in this study. A retrospective interventional case series study including a total of 33 keratoconus eyes (age 8 to 17 years) that had undergone ICRS  implantation was conducted. Information about visual, refractive, pachymetric, corneal topographic and aberrometric, and corneal endothelial changes during a 3-month follow-up were extracted and analysed. A significant improvement was observed in logMAR corrected distance visual acuity ( Keratoconus is a degenerative corneal disease that is commonly manifested during puberty, with the possibility of progressing until the third or fourth decade of life . This coAlthough keratoplasty is a potential solution in the most advanced keratoconus cases, both penetrating and deep lamellar keratoplasty are not exempt from complications such as rejection of the donor button, vascularization and infectious diseases . In chilRetrospective interventional case series including a total of 33 keratoconus eyes (21 men and 12 women) that had undergone ICRS implantation using the femtosecond laser technology for the creation of the tunnels. All cases included were treated and followed at Clinica de Ojos Larco Vision , with all surgeries performed in the period from May 2015 to July 2018. Inclusion criteria were keratoconus diagnosis according to the standard criteria that consider the presence of the following signs: anterior corneal topographic asymmetric bowtie pattern, KISA ≥ 100, and one or more biomicroscopic keratoconus signs, such as Fleischer ring, significant corneal thinning, Vogt striae, conical protrusion on the cornea at the apex or anterior stromal scar [A complete preoperative ophthalmological examination was performed in all cases including measurement of uncorrected (UDVA) and corrected distance visual acuity (CDVA), autorefraction , cycloplegic and manifest refraction, slit lamp evaluation of the anterior segment, fundus evaluation, pupillometry, specular microscopy for corneal endothelial cell density analysis  and corneal topographic, pachymetric and aberrometric evaluation with the Scheimpflug imaging-based system Pentacam . Postoperative examinations were performed the day after surgery, at 1 week, 1 month, and 3 months after surgery. In the last postoperative visit, a complete eye examination identical to the preoperative exam was performed. The importance of not rubbing the eyes and maintaining the anti-allergic treatment was remarked to all patients and their relatives, with the additional recommendation of attending to eye controls every 3 months.All patients received indications and anti-allergic treatment prior to surgery and it was maintained indefinitely.All surgical procedures were performed by the same experienced surgeon (PL) under topical anaesthesia . The femtosecond laser platform FS 200  was used to create the corneal incision and tunnels. The tunnels were created at 70% of corneal depth ensuring that at least 100 μm of corneal tissue was present under the tunnel. Keraring segments  were implanted in all cases, with a selection of the number of segments to implant as well as their diameter (SI5 or SI6), arc length and thickness according to the manufacturer’s nomogram and the scotopic pupil size measured. https://www.imim.cat/ofertadeserveis/software-public/granmo/. Assuming a paired test in a same group for detecting significant differences in CDVA, the statistical power was 84.0% for the detection of a minimum difference of −0.16 logMAR (minimum change in CDVA observed at 3 months postoperatively), considering an alpha error of 0.05, the sample of 33 eyes, and the standard deviation of differences in CDVA at 3 months after surgery (0.31 logMAR).The statistical power associated to the sample size selected was calculated using the online calculator, p-value of less than 0.05 was considered as statistically significant.Statistical analyses were performed with a commercially available software package . Normality of data samples was evaluated by means of the Kolmogorov-Smirnov test. When parametric analysis was possible, the Student t test for paired data was used for comparisons between preoperative and postoperative visits, whereas the Wilcoxon ranked sum test was applied to assess the significance of such differences when parametric analysis was not possible. The Pearson or Spearman correlation coefficient was used to assess the level of correlation between preoperative corneal astigmatism and the clinical outcomes obtained depending if the condition of normality could be assumed or not. For all statistical tests, a A total of 33 eyes of 33 patients with mean age of 14.9 years  were enrolled. The sample was comprised of 21 males (63.6%) and 12 females (36.4%). A total of 15 right (41.7%) and 21 left eyes (58.3%) were included. The model of ring segments SI5 was implanted in a total of 16 eyes (44.4%), whereas a total of 20 eyes (55.6%) were implanted with the SI6 ring segments. In all cases except in 12 eyes, two ring segments were implanted , with the following distribution in terms of arc length: 90° in 11 eyes , 120° in 18 eyes , 150° in 15 eyes , 160° in 9 eyes , 210° in 4 eyes , and 340° in 3 eyes . Concerning ring segment thickness, the distribution of cases was as follows: 150 μm in 3 eyes , 200 μm in 14 eyes , 250 μm in 12 eyes , and 300 μm in 31 eyes .p ≥ 0.066). However, a significant improvement was also observed in logMAR CDVA (p = 0.005). Concerning keratometric readings, a statistically significant reduction was observed in K1 (p = 0.001), K2 (p < 0.001), and KM (p < 0.001). Consequently, a significant reduction was observed in the magnitude of corneal astigmatism (p < 0.001), with 11.1% (4/36) and 69.2% (18/26) of eyes with magnitude of anterior corneal astigmatism of 3 D or less before and 3 months after surgery, respectively (p < 0.001) (p = 0.647).ectively . A total< 0.001) . Concernp = 0.002) as well as the spherical aberration of the posterior corneal surface (p = 0.004). No significant changes were observed in the remaining aberrometric components (p ≥ 0.200). A poor although significant correlation was found between preoperative coma RMS and the change induced in this parameter with surgery . No correlation was found between the preoperative magnitude of spherical aberration and the change induced in this parameter with surgery .p = 0.317), with mean preoperative and 3-month postoperative values of 2795.08 cell/mm2  and 2624.50 cell/mm2 , respectively.Concerning safety and complications, only two relevant complications were described, one corneal microperforation with penetration of the ring segment into the anterior chamber , and a case of ring extrusion  . No signIn the last two decades, advances in keratoconus management have been focused on avoiding corneal transplantation . Althougp = 0.001). Alfonso et al. [Concerning the refractive outcomes, a reduction in magnitude was observed in sphere, cylinder and SE in most of patients, but not reaching statistical significance in the overall sample. This is consistent with the variability of ICRS outcomes reported in the peer-reviewed literature according to the corneal topographic phenotypic profile and the level of severity of the disease ,25,26. Ro et al.  reportedo et al.  also repThe change induced at 3 months postoperatively in the magnitude of anterior corneal astigmatism was strongly correlated with the preoperative magnitude of anterior corneal astigmatism. This correlation was negative which indicated that less reduction of corneal astigmatism was achieved in those cases with lower levels of baseline corneal toricity. This suggests that no more undercorrection was present in cases of high levels of corneal astigmatism. However, despite the greater astigmatic corrective effect achieved in very toric corneas, the impact of potential misalignments of the meridian of correction is higher, as demonstrated in previous series . In any The analysis of changes in corneal aberrations in the current series revealed that statistically significant changes were found in the vertical coma component of the anterior corneal surface as well as in the spherical aberration of the posterior corneal surface. There was a trend to a reduction of primary coma RMS that did not reach statistical significance, as has been reported in other studies evaluating the corneal aberrometric outcomes after KeraRing implantation . The maiFinally, the safety of the procedure was confirmed with the gain of lines of CDVA and the presence of only two relevant complications, a corneal microperforation with penetration of the ring segment into the anterior chamber and a ring extrusion. These complications have been also reported in series with adult patients . In our As limitations of the study, it should be noted that no validated questionnaires were used to assess and characterize changes in symptomatology. This should be investigated in future series evaluating the impact of ICRS implantation in children with keratoconus. Likewise, the results of this technique in a longer follow-up period should be reported to confirm the long-term safety of the procedure. Despite these limitations, this article adds new information in terms of tomographic and corneal aberrometric changes to the currently available peer-reviewed literature ,14,33 abIn conclusion, the implantation of ICRS in children is a safe procedure, which is not exempt from complications like the two reported cases, related to manipulation of the eyes, despite having been warned not to do it and that they were solved without problems. The case of the migration of the ICRS to the anterior chamber had a previous history of micro perforation 3 months before the implantation of the ICRS. Possibly, the combination with CXL is necessary in most cases to avoid keratoconus progression, but more studies are still needed to confirm this and to evaluate the safety and efficacy of this combination. Abreu et al.  reported


Investigation of protein expression in cancer cells is an important part of the diagnostic process. Increasing knowledge about expression of different proteins has been exploited for prognostic assessments and in some cases also for selection of treatment. The p53 protein has proven important in development of various cancers, and the expression of this protein and its signaling pathway is therefore of interest when examining cancer patient samples. Here, we present mass cytometry as a tool for detection of p53 expression. Mass cytometry allows for measurement of up to 50 parameters per sample with single cell resolution, and we aim to demonstrate its potential for p53-focused research.TP53 mutational status. Results: The p53 protein can be detected in cell lines and in primary samples by mass cytometry. By combining antibodies for p53-related signaling proteins with a surface marker panel, we show that mass cytometry can be used to decipher the single cell p53 signaling pathway in heterogeneous patient samples. Conclusion: Single cell profiling by mass cytometry allows the investigation of the p53 functionality through examination of relevant downstream signaling proteins in normal and malignant cells. Our work illustrates a novel approach for single cell profiling of p53.Purpose: The p53 protein and its post-translational modifications are distinctly expressed in various normal cell types and malignant cells and are usually detected by immunohistochemistry and flow cytometry in contemporary diagnostics. Here, we describe an approach for simultaneous multiparameter detection of p53, its post-translational modifications and p53 pathway-related signaling proteins in single cells using mass cytometry. Method: We conjugated p53-specific antibodies to metal tags for detection by mass cytometry, allowing the detection of proteins and their post-translational modifications in single cells. We provide an overview of the antibody validation process using relevant biological controls, including cell lines treated in vitro with a stimulus (irradiation) known to induce changes in the expression level of p53. Finally, we present the potential of the method through investigation of primary samples from leukemia patients with distinct  TP53 is mutated, the expression level of the (mutant) p53 protein is elevated. This is largely due to the absence of activation of a p53 downstream response, including transactivation of Mdm2, which leads to stabilization and accumulation of the mutated p53 protein  (REA529), p53and Ki67 a. To furm AML017 b. By perTP53 mutated patient. We investigated the force directed layout by visualizing the cells originating solely from patient AML017  was characterized by lower p53 expression. By assessing the expression of the p53 downstream target p21(CIP1/WAF1) and the proliferation marker Ki67, we observed a seemingly inverse correlation. Indeed, the Ki67 expression was high, while p21(CIP1/WAF1) expression was low. The reversed was observed in the p53 low population . Ki67 was also expressed in this population.Individual analysis of the different patients using viSNE can be found in In this study, we demonstrate the use of mass cytometry to examine p53 expression in cell lines and patient samples of leukemia. Investigation by mass cytometry demands for single cell suspension. Hematological malignancies are therefore particularly suitable, as the samples naturally are in single cell suspension. Thus, peripheral blood and bone marrow do not require extensive processing by mechanical and/or enzymatical degradation as solid tissues demand for. Peripheral blood and bone marrow samples can be fixed shortly after sampling using commercially available reagents  or proteomic stabilizer (Smart Tube Inc.). This greatly reduces the risk of altering intracellular signaling profiles prior to fixation. Furthermore, due to the possibility to simultaneously perform broad phenotyping and detect expression of multiple intracellular proteins, mass cytometry allows for assessment of protein expression in specific cellular subsets in primary samples. However, limitations of the technology do exist, and these are thoroughly addressed in Spitzer et al. . In shorTP53 mutations (specific amino acid sequences in mutated TP53) could enable mass cytometry to distinguish different mutations. Alternative ways of detecting specific mutations could be to use RNA probes toward specific mutations; a strategy similar to proximity ligation assays for RNA (PLAYR) or metal in situ hybridization (MISH) [In contrast to immunoblot analysis of p53 proteins, single cell detection by mass cytometry does not allow discrimination of molecular weight of the p53 species detected. This is especially a challenge in regard to the detection of p53 isoforms. To our knowledge, no isoform-specific antibodies developed have currently been validated to show compatibility with detection of non-denatured proteins by flow- or mass cytometry. Development of such specific antibodies could allow the assessment of biology and functionality of the p53 isoforms. Future development of antibodies with improved specificity could ease the identification of endogenous p53 isoforms, and as mass cytometry allows for high-dimensional data collection, thorough assessment of the individual isoforms’ properties in a natural system could be achieved. As the method’s span of detection relies solely on appropriate probes , development of specific monoclonal antibodies directed toward specific n (MISH) ,35.Detection of p53 using antibodies is limited by the antibody specificity, sensitivity, and affinity to p53 in the particular sample preparation. The p53 protein is encoded by a gene that may be the origin to more than 13 pre-mRNA splicing isoforms . FurtherTP53 mutations affect the p53 expression level by using different cell lines for validation experiments. As these cell lines express p53 in varying degrees, we show the importance of titrating antibodies on relevant samples. HL60 cells do not express full-length p53 due to deletion in both alleles [TP53 mutation, which results in high protein expression [Careful use of technical and biological validation is essential for establishing mass cytometry detection, e.g., by a standardized set of cell lines. Similarly, post-translational modifications may be validated by in vitro stimulated cell lines. Here, we demonstrate how  alleles , while Npression . An antipression ,42. Of npression . In thisIn general, mass cytometry is regarded as a less sensitive platform than flow cytometry and low-abundant targets may therefore theoretically be more difficult to detect by mass cytometry. However, the low background signal combined with almost negligible signal spillover in mass cytometry partly compensates for this . The detResting healthy cells express only low levels of p53, and thus, to properly detect p53 by mass cytometry, we argue that careful selection of isotope conjugate during antibody panel design is of importance. This was also evident from our observations when conjugating the same p53 antibodies to two different metal isotopes. Therefore, it is important that the antibodies in a panel are tested and titrated on that exact instrument. We encourage users of mass cytometry to carefully assess the signal generated by the different metal channels, and if feasible, conjugate aliquots of the same antibody to different isotopes for comparison of signal intensity. In our case, the two 153Eu antibody conjugates consistently generated stronger signals than the two 175Lu conjugates in all cell samples expressing low levels of p53 protein. In the cell lines expressing high levels of p53 protein, the signal differences were visually indistinguishable.We previously compared two-dimensional gel immunoblot with flow cytometry analysis of intracellular proteins in AML . ImmunobTP53 mutations, and that these mutated subpopulations could indicate higher risk of relapse [TP53 mutations in clinical diagnostics is sequencing, while loss of TP53 can be detected by fluorescent in situ hybridization (FISH). Although TP53 is mutated, the protein can still be translated. The resulting protein product may deviate from normal conformation and thus not retain normal protein function. Downstream targets of p53 might therefore not be affected by p53 activation. Our investigation of patient AML017 exemplifies such an event, where p53 is expressed at high levels in the majority of the malignant population, although the downstream target p21(CIP1/WAF1) remains low. We hypothesize that this pattern is due to the protein product of mutated TP53 in this patient, which is unable to induce p21(CIP1/WAF1) transcription as a consequence of loss-of-function. However, we also observed a second small population, corresponding to approximately 1% of the cells in this patient, where lower levels of p53 were detected. Here, the expression pattern of p21(CIP1/WAF1) and Ki67 was reversed; a pattern indicative of normal p53 function. This could be due to expression of wild type p53 in this small subpopulation of malignant cells. An alternative explanation could be p53-independent activation of p21(CIP1/WAF1) [TP53 wild type patient, we observed the expected expression pattern of p53 and p21(CIP1/WAF1). Yet, despite high p21(CIP1/WAF1) expression, the malignant cell population also exhibited high Ki67 expression, indicative of high mitotic activity. An explanation for this inconsistency could be that this patient harbors other driver mutations causing excessive mitotic activity. Numerous other mutations and aberrant signaling pathways in AML have been described to be associated with non-mutational p53 dysfunction [An increasing number of reports have shown that hematological malignancies may have a minute cell population carrying  relapse ,49. We hP1/WAF1) . Unfortufunction . Howevermut (Y5) antibody unexpectedly yielded signal in both the TP53 wild type and the TP53 mutated cell lines and patient samples. This suggests that the Y5 antibody binds to wild type p53, and illustrates the importance of careful validation of the antibodies. However, a recent report demonstrated the presence of p53 protein with “pseudo-mutant” conformation in pre-leukemic hematopoietic stem/progenitor cells and in leukemic blast cells in TP53 wild type patients [In our experiments, the p53patients . This waThe work presented here is focused on the application of mass cytometry in assessment of hematological malignancies. Although we argue that peripheral blood and bone marrow are especially suitable samples for this application, the method is by no means limited to samples of such origin. Several publications presenting mass cytometry analyses of single cell samples originating from solid tissues have been published ,54,55. WIn summary, mass cytometry has a great potential for p53-centric investigation of primary patient samples. It allows for thorough investigation of surface markers, enabling broad immunophenotyping combined with intracellular signaling proteins, far exceeding current routine methods such as flow cytometry. As precision medicine is emerging and the span of patients eligible for this treatment approach is expanding, increased knowledge of the disease state in every individual patient is of importance and can potentially provide clinical information relevant for future treatment choices. In this work, we demonstrated how currently available mass cytometry technology can be applied to investigate the p53 network in primary clinical samples. Development of p53 isoform-specific antibodies as well as modification- and conformation-specific antibodies compatible with the mass cytometry platform would add increasing value to experiments focusing on p53 and its functions in different malignancies.This study has been performed in accordance with the Declaration of Helsinki and in agreement with approved local protocols. The study was approved by the regional ethics committee; Regionale komiteer for medisinsk og helsefaglig forskningsetikk (REK) Vest, University of Bergen, Bergen, Norway (Project REK2012/2245). Before study initiation, all subjects provided written informed consent.w/w), 20.35% formaldehyde (w/w) and 15.65% diethylene glycol (w/w)) according to manufacturer’s protocol. The samples were stored at −80 °C in NaCl 0.9%. Bone marrow from the HCL patients was aspirated by standard medical procedure into syringes containing heparin, and further sample processing was performed as described for peripheral blood.Blood samples were collected by phlebotomy of a peripheral vein or drawn from a Hickman central venous catheter into EDTA vacutainers . Red blood cells were lysed, and white blood cells were fixed using BD lyse/fix Buffer 5X . OV-90 and Raji cells were purchased from American Type Culture Collection . All cell lines were cultured in RMPI-1640 medium  supplemented with 10% heat inactivated Fetal Bovin Serum , 1% L-arginine (Sigma-Aldrich) and 1% Penicillin-streptomycin (Sigma-Aldrich), in incubators at 37 °C and 5% COmut (Y5) were purchased from Cell Signaling Technology , Miltenyi Biotec  and Abcam , respectively. These antibodies were ordered as custom formulation suspended in BSA- and azide free buffer. This is important for the mass cytometry technology, as carrier BSA will compete with the antibody for binding to the metal tags during conjugation. The antibodies were conjugated to the metal tags according to Fluidigm’s MaxPar Antibody Labeling Kit protocol . In the conjugation process, the antibodies are covalently bound to a polymer. The polymer includes a chelating group which forms a non-covalent non-reversible interaction with the metal.The details of the mass cytometry antibody panel are described in The p53-specific antibodies were titrated and validated using appropriate cell line and primary cell controls, as described in the results section. Antibody titrations were performed by five 1:1 serial dilution of the antibodies, starting at 1:50 dilution. Five equal barcoded cell aliquots were stained with the resultant titration series. During titration, all channels susceptible for potential spillover were kept empty, in order to evaluate the actual signal spillover the concentrations tested.k-nearest neighbor density estimation to form cell clusters, using a range of different k-values. The optimal k-value (number of clusters) was found using the “elbow point/switch point”. To increase biological interpretability, similar clusters were manually merged. We visualized the data by a force directed layout, which separates the clusters according to relative similarity. Unsupervised clustering using x-shift and graphical representation of the data was performed using the VorteX stand-alone software, downloaded from nolanlab/vortex GitHub.All samples included in the validation and titration were barcoded as described in Fluidigm’s MaxPar Cell ID protocol (PRD 023 V1). After barcoding, the cells were stained with extracellular and intracellular antibodies according to the Fluidigm Phospho Protein staining protocol (PN 400278 A4), with some minor adjustments. This includes the addition of two 20 min incubation steps with 200IU heparin  immediately preceding the addition of the extracellular and intracellular antibody cocktails. The final heparin concentration during antibody incubation was 100IU. This was done to block unspecific antibody binding by eosinophils, as previously described . In addiTP53 gene, and the post-translational modification by acetylation at Lysine 382 can be detected by mass cytometry using antibody probes. We further demonstrate the potential of the technology by analyzing patient samples, including assessment of p53 expression in specific cellular subsets. Mass cytometry allows detection of multiple parameters simultaneously, enabling mapping of the p53 network. Thus, these results demonstrate that mass cytometry is a promising tool, which could reveal novel insight into p53 biology in multiple malignancies. Future development of p53 isoform-specific antibodies compatible with this technology could have the potential to further advance the field of p53 research by mass cytometry.In this work, we demonstrate that the p53 protein, the non-functional protein of a mutated


Interactions between DNA and DNA-binding proteins play an important role in many essential cellular processes. A key function of the DNA-binding protein p53 is to search for and bind to target sites incorporated in genomic DNA, which triggers transcriptional regulation. How do p53 molecules achieve “rapid” and “accurate” target search in living cells? The search dynamics of p53 were expected to include 3D diffusion in solution, 1D diffusion along DNA, and intersegmental transfer between two different DNA strands. Single-molecule fluorescence microscopy enabled the tracking of p53 molecules on DNA and the characterization of these dynamics quantitatively. Recent intensive single-molecule studies of p53 succeeded in revealing each of these search dynamics. Here, we review these studies and discuss the target search mechanisms of p53. Genome editing method has been used to investigate and engineer gene functions in life science. The genome encompasses DNA sequences that encode genes, and gene editing is the genetic engineering of a specific DNA sequence, including insertion, deletion, modification, and replacement. The main player in genome editing is a type of protein that can bind to DNA, known as DNA-binding proteins. DNA-binding proteins include enzymes, which can cut DNA or ligate two DNA molecules, and transcription factors, which can activate or deactivate gene expression. These proteins are classified into DNA sequence-specific and nonspecific binders.6 and 109 bp of DNA, respectively. In contrast, the DNA sequence that is bound by the proteins is just typically 5–30 bp. For example, assuming that a single protein searches for the target coded in 109 bp of genome by repeatedly associating and dissociating with random sites in the genome (3D diffusion), the search time is estimated to be much longer than the physiological time which is estimated from several minutes to a half of an hour in cells. However, the natural proteins complete the target search exactly within the physiological search time. This gap is called the “target search problem.” How do DNA-binding proteins solve this problem and function on the target?The sequence-specific DNA-binding proteins have a common mechanism to search for and bind to their target DNA sites encoded in a genome. Successful target binding by transcription factors can trigger the regulation of the cellular functions, whereas the failure of the target search and binding is known to cause various diseases including cancers. In addition, the target binding is delicately regulated depending on genome functioning. Accordingly, the target search and binding by DNA-binding protein are one of the indispensable reactions in life. The bacterial and human genomes comprise approximately 10To solve this problem, three target search dynamics are currently proposed: 3D diffusion; 1D diffusion, in which a protein moves along DNA; and intersegmental transfer, in which a protein transfers between two different DNA strands without dissociating ,3. TheorThe transcription factor p53 can induce multiple tumor suppression functions, such as cell cycle arrest, DNA repair, and apoptosis. Furthermore, p53 is known as the guardian of the genome, and it contains an N-terminal (NT) domain, core domain, linker, tetramerization (Tet) domain, and C-terminal (CT) domain . The corTo solve the target search problem, it is necessary to differentiate and characterize each search dynamic. Single-molecule fluorescence microscopy can track a single molecule moving along DNA and/or transferring between two DNA molecules ,22,23,24Several elegant methods for producing DNA arrays were developed in order to acquire data on many molecules simultaneously ,27,28. AIn 2008, Tafvizi et al. succeeded in observing the movement of p53 tetramer along a non-target DNA sequence using single-molecule fluorescence microscopy . A thermTafvizi et al. further revealed that 1D diffusion of p53 along the non-target DNA sequence does not depend on a monovalent salt concentration; this suggested “1D sliding,” in which p53 moves along DNA while maintaining contact with the DNA ,29. BaseWe clarified that p53 possesses two 1D diffusion modes with different diffusion coefficients by altering the structure of p53 on DNA ,36 Figu. The fasIf p53 reaches the target DNA sequence using 1D diffusion, it will recognize and bind to the target. Considering the fast target search by DNA-binding proteins, one might predict a high efficiency of target recognition and binding . ContrarIn the cell, many DNA-binding proteins bind to and cover DNA, which could block the 1D diffusion of p53 along DNA and hinder its target search. In the nucleus, genomic DNA is stored at high concentrations. In these conditions, it may be possible that p53 possesses a special mechanism to bypass the obstacles on DNA by transferring from one DNA strand to another a.8 M−1s−1). This suggests that even if p53 encounters DNA-bound obstacles in the cell, it can bypass these via ultrafast transfer. In contrast, p53 does not transfer from the target DNA sequence. Accordingly, once p53 binds to the target DNA sequence, it is not repelled from the target DNA by nearby DNA in the cell.We conducted a kinetic ensemble measurement to monitor the transfer process of p53 from fluorescent to non-fluorescent DNA . FluoresTo directly examine p53 transfer between DNA segments, we prepared crisscrossing DNA in the flow cell using the modified DNA garden method and tested whether p53 moves between DNA segments at the crisscross points  Figure b. The crConsidering the in vitro data described above, p53 is presumed to solve the target search problem by utilizing 3D diffusion, 1D diffusion along DNA, and intersegmental transfer between two DNAs in the cell. In 3D search, p53 can associate to non-target DNA near the diffusion limit . The assConsidering the biological functions of p53, one may wonder why the target search by p53 is not optimized in terms of facilitation. Under various cellular stresses such as DNA damage and tumorigenesis of the cell, p53 turns the function “ON” and induces DNA repair and apoptosis. In contrast, p53 should keep the function “OFF” under normal conditions. It would be dangerous if p53 were to accidentally bind to the target DNA sequences and induce apoptosis in normal conditions. To prevent this malfunction, p53 may keep target recognition low under normal conditions at the expense of target search speed in stress conditions.To regulate multiple functional pathways as well as the discrete ON/OFF switch, p53 has three different strategies. One strategy is to increase the p53 copy number under stressed conditions . The incThe target search dynamics of p53 in vivo have been investigated by single-molecule fluorescence imaging. For example, Mazza et al. succeeded in tracking p53 molecules one by one in live cells using HILO illumination . They shTarget search is not limited to DNA-binding proteins, such as p53. Various processes can be considered as target search problems, including biomolecules searching for binding partner molecules or animals seeking food. Interestingly, a common mechanism known as intermittent search is widely used in biology . IntermiSingle-molecule fluorescence microscopy has clarified that p53 solves the target search problem by utilizing 3D diffusion, 1D diffusion along DNA, and intersegmental transfer between two DNAs. As predicted by theoretical studies, target search by p53 can be facilitated by combining these three strategies. In particular, considering the ultrafast association to DNA, 1D diffusion along DNA, and ultrafast intersegmental transfer, target searching by p53 is somehow optimized toward facilitation. In contrast, the low target recognition by p53 decelerates target searching. Furthermore, p53 may prevent malfunction under normal conditions at the expense of the target search speed in stress conditions. Accordingly, the target search strategy of p53 may be well designed to satisfy biological requirements.Further single-molecule investigations could deepen the understanding of target search by p53. In the cell, histone proteins bind to and condense DNA, which may affect target search by p53 . Single-Since many DNA-binding proteins exist in the cell and have different structures and functions, it is interesting how such DNA-binding proteins solve the target search problem. For example, if certain DNA-binding proteins differ in structure and function, their target search mechanisms may each differ as well ,104,105.


TP53 genomic locus is a target of mutational events in at least half of cancers. Despite several decades of study, a full consensus on the relevance of the acquisition of p53 gain-of-function missense mutants has not been reached. Depending on cancer type, type of mutations and other unidentified factors, the relevance for tumour development and progression of the oncogenic signalling directed by p53 mutants might significantly vary, leading to inconsistent observations that have fuelled a long and fierce debate in the field. Here, we discuss how interaction with the microenvironment and stressors might dictate the gain-of-function effects exerted by individual mutants. We report evidence from the most recent literature in support of the context dependency of p53 mutant biology. This perspective article aims to raise a discussion in the field on the relevance that context might have on p53 gain-of-function mutants, assessing whether this should generally be considered a cell non-autonomous process.The  Mutant p53 GOF effects have been shown to vary in different settings, potentially depending on cancer types, experimental models and conditions.Reciprocal interaction between microenvironment and mutant p53 GOF effects exists.Similarly to wt p53, mutant p53 proteins can contribute to transduction of extrinsic signalling.What is the effective contribution of mutant p53 GOF effects to the cancer progression?How relevant is the microenvironmental context in determining mutant p53 GOF effects?Can microenvironmental factors be targeted to abolish mutant p53 GOF pro-oncogenic functions?5. Originally identified as a major executor of the response to DNA damage, with a more recent revision, p53 is considered a molecular hub for the interactions between stressors  and cellular biological responses12  theory in p53 mutation26. The shift of p53 from the wild-type status to the mutant protein therefore appears to turn p53 into an oncogene. This basic, consistent and generally accepted consideration has, however, not always found solid support in the experimental data. Clear GOF phenotypes have been shown for many hot-spot mutations, such as p53 R175H and R273H. Introduction of p53 R175H and R273H into p53-null cells promotes growth in in vitro soft-agar assays and in injected nude mice. In contrast, genetically engineered mouse models carrying mouse homologous mutations (p53 R172H and R270H) did not show any alteration in survival compared with p53-null mice.An additional critical aspect of the mutations in p53 is the frequently observed protein products. Eighty percent of p53 mutations are missense, leading to generation of neomorphic proteins36. However, a complementary approach based on functional and transcriptional analyses of CRISPR-Cas9-generated isogenic human leukaemia cell lines suggested a selective advantage associated with a dominant negative effect of p53 mutants but no evidence of any GOF. This study also reported no evidence of GOF effects on the clinical outcome of patients with myeloid malignancies37. Whether this is different from solid tumours, where a pro-invasion and pro-metastatic programme might benefit from mutant p53 GOF, remains to be determined. However, formal evidence for the existence of p53 mutant GOF effects could be found in the dependency displayed by cancers on sustained expression of p53 mutant proteins38. Ablation of mutant p53 in allotransplanted, xenotransplanted and autochthonous mouse cancer models impairs tumour growth, promoting cell death and tumour regression or stagnation42. While these results allow pharmacological targeting of the mechanisms leading to p53 mutant stabilisation as an anticancer approach, at the basic molecular level, they formally demonstrate that expression of p53 mutant proteins is not equal to the loss of the functional wild-type version. However, a general consensus has not yet been achieved on the relevance of the contribution of p53 GOF effects to tumorigenesis.Puzzling results have also been shown in myeloid malignancies. GOF p53 R172H was seen to accelerate complex-karyotype acute myeloid leukaemia in mouse models by promoting cell fate plasticity through the pluripotency factor FOXH143. This is reflected in an influence of extrinsic signalling on p53 mutant behaviour and on the ability of the p53 mutant to modulate the response to extrinsic factors. In light of the central role of functional (wt) p53 in the response to cellular stress, a fundamental conserved implication of the mutant proteins in the interaction with microenvironment is not surprising  transcriptionally promotes the expression of Hsps, which in turn interact with p53 mutants, inhibit the E3 ubiquitin ligase activity of MDM2 and CHIP and induce the stability of p53 mutant proteins. The mechanisms leading to the stabilisation of p53 mutants are therefore influenced by extrinsic stressors able to trigger the activation of HSF1. Similarly, mechanical stimuli from the tumour tissues have also been shown to result in p53 mutant stabilisation. Rho-A is a sensor of extracellular matrix (ECM) stiffness. In response to stiff a ECM, Rho-A activation and actin-dependent mechanotransduction induce activation of Hsp90 via the mechanosensitive HDAC6 deacetylase, thus leading to stabilisation of mutant proteins50. Similar to HSF1, Rho-A is also frequently hyperactive in tumour tissues. Notably, Rho-A is regulated by mutant p53 activity itself. Activation of Rho-A is influenced by GEF-H1 and RhoGDI, which are influenced by GOF p53 mutants, feeding a forward loop that results in further stabilisation of the p53 mutant. Disruption of this loop with zoledronic acid (ZA) and geranylgeranyl transferase inhibitors (GGTIs), which inhibit the geranylgeranylation of Rho-A required for its activation on the plasma membrane, impairs p53 mutant stability51  determines the expression of fundamental components of the basal lamina, such as laminin-γ2 and collagen type VIIa1, in hypoxic tumours56. Consistently, vessel density correlates with the expression of p53 missense mutations in human breast cancers57.GOF p53 mutants can also facilitate a pro-tumorigenic microenvironment by promoting the secretion of IL-8 and GRO-α by cancer cells. Similar to the p53 mutant/HIF-1 molecular model, p53 mutants appear to hijack the E2F2 transcriptional factor. The p53 mutant/E2F2 complex controls the promoter of inhibitor of DNA binding 4 (ID4), which binds and stabilise mRNAs of IL-8 and GRO-α. Conditioned media from p53 mutant cells promote the in vitro proliferation of endothelial cells, and p53 mutants favour angiogenesis in vivo in xenotransplanted models58. Cooperation between mutant p53 and Sp1 facilitates transcriptional control of ENTPD5, and this appears to mediate p53 mutant-dependent growth, architectural tissue remodelling, migration, invasion and lung colonisation as assessed in tail vein-implantation mouse models58.N-glycosylation of cellular surface proteins, such as receptors and integrins, is also altered by p53 status. Ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) is a mutant p53 target gene involved in promoting the folding of N-glycosylated membrane proteins in the endoplasmic reticulumEvidence exists that p53 mutants can influence the microenvironment. Simplification tends to indicate how “mutants” direct the composition of the extracellular milieu; however, it is reasonable to speculate that different mutations will have different impacts on this process. However, the large variety of mutations makes the resolution of this issue very challenging Fig. .Fig. 1p560, Rb62 or Bcl-267. The p53 mutant can dictate instructions to the microenvironment, which in turn are influenced by additional extrinsic factors that direct signals on the cancer cells that are differentially integrated based on the p53 mutational status (Fig. In this perspective, we suggest that p53 GOF is a non-cell-autonomous phenomenon, which would make this gene very peculiar if compared with other oncogenes/oncosuppressors, such as KRas51. The limited ability of in vitro and xenotransplanted models to recapitulate the natural cancer microenvironment might represent a particular concern in the interpretation of the data on p53 GOF. The addiction that cancer cells develop to p53 mutations is a specific relevant aspect in this context. While strong support for the existence and relevance of p53 GOF comes from evidence that in vivo tumours are dependent on p53 expression, no evidence exists of the reproducibility of this effect in vitro. This questions not only the work done to dissect the mechanisms underlying p53 GOF but also the utility of in vitro models for developing therapeutic approaches to target p53 mutant stability.It is remarkable that a significant fraction of work on p53 GOF is performed in in vitro systems or in xenotransplanted models. Compelling evidence indicates that plastic dishes provide altered extracellular matrix interactions to cancer cells, influencing the stability of p53 mutants and consequentially providing artificial responses68.It appears optimistic to envisage general major mechanisms responsible for p53 GOF, but it is possibly also very optimistic to envisage the existence of p53 mutant of similar mechanisms in different contexts. This raises complexity in the study of p53 GOF, but even more, it presents challenges in identifying strategies to target this gene with general approaches in cancer patients. While a massive effort in drug development against p53 mutant tumours has been invested in the two past decades, the outcome is very soon expected to be delivered. This should also help in understanding how feasible the approach is and direct efforts for future investments


Forty years of research has proven beyond any doubt that p53 is a key regulator of many aspects of cellular physiology. It is best known for its tumor suppressor function, but it is also a regulator of processes important for maintenance of homeostasis and stress response. Its activity is generally antiproliferative and when the cell is damaged beyond repair or intensely stressed the p53 protein contributes to apoptosis. Given its key role in preventing cancer it is no wonder that it is the most frequently mutated gene in human cancer. Surprisingly, a subset of missense mutations occurring in p53 (gain-of-function) cause it to lose its suppressor activity and acquire new functionalities that turn the tumor suppressor protein into an oncoprotein. A solid body of evidence exists demonstrating increased malignancy of cancers with mutated p53 in all aspects considered “hallmarks of cancer”. In this review, we summarize current findings concerning the cellular processes altered by gain-of-function mutations in p53 and their influence on cancer invasiveness and metastasis. We also present the variety of molecular mechanisms regulating these processes, including microRNA, direct transcriptional regulation, protein–protein interactions, and more. One of the results of the changes in demographic structure of many societies is an increasing prevalence of cancer. It is an extremely diverse disease, with many types of cancers originating in various organs. With progress in molecular diagnostics, cancers that previously have been thought of as a single disease are now known to have molecular subtypes that are predictive of their disease course and outcome. Being diverse as they are, most—if not all—cancers possess several features defined by Hanahan and Weinberg in 2000 as hallmarks of cancer ,2. TheseTP53 gene encodes p53 protein that, in its wild type form, acts as a tumor suppressor. In an active state, it is a homotetramer that binds to its specific target sites and modifies gene expression. Activity of p53 is crucial for control of cell cycle progression, apoptosis, cellular senescence, DNA repair, and genome maintenance. The crucial role of p53 in preventing cancer formation is underlined by the fact that, based on tens of thousands of records in International Agency for Research on Cancer (http://p53.iarc.fr) [The iarc.fr) , over 50iarc.fr) ,7,8; thiiarc.fr) ; this moiarc.fr) ,11,12,13Generation of p53 NULL mice in the early 1990s demonstrated in vivo that it is indeed a major tumor suppressor ,15. The Trp53-/R248Q and Trp53R248Q/R248Q genotypes when compared with their Trp53−/− counterparts [Trp53R248W/R248W mice presented more complex tumor spectrum than the Trp53−/− mice, but no significant change in survival [Trp53+/R172H genotype exhibited marked increase in frequency of metastasis when compared with their Trp53+/− counterparts, but no decrease in overall survival was noted [Trp53+/R172H genotype exhibited marked increase in frequency of metastasis when compared with their Trp53+/− counterparts [Trp53+/R172P mice [Phenotypic variations of GOF p53 depend on the residue that is mutated as well as the amino acid that replaces the wild type variant. Although pooled patients with germline p53 missense mutations (Li-Fraumeni syndrome) do not develop the tumors earlier than the patients with NULL genotypes, early onset (7 years difference) is observed when the population of patients bearing R248Q (but not R248W) mutation is compared to NULL population. In addition, the distribution of primary tumor sites and the tumor spectrum in the large IARC cohort differs between patients bearing NULL and several “hot spot” mutated alleles ,27. Simiterparts , whereassurvival . In a muas noted . In anotas noted , the micterparts . Interes72P mice . In a bi72P mice . In an i72P mice . Similar72P mice ,29. Thes72P mice . AlthougFor local invasion or distant metastasis to happen, tumor cells that are already immortalized still have to break several barriers. First, the cells have to acquire mobility and capability of extracellular matrix degradation. This, in conjunction with sensitivity to certain chemokines, enables directional migration through extracellular matrix. At this stage the tumor is capable of local invasion. If the cells can penetrate lymphatic or blood vessel wall, then they can travel to the local lymph node or a remote location, respectively. To survive in a blood or lymphatic vessel, the cells need to be resistant to anoikis (lose their anchorage dependence) and also evade immune cells. The organs targeted by metastases are non-random and that points to the fact that cancer cells exhibit a certain degree of tropism. That tropism results from sensitivity to certain chemokines and ability to adhere to the endothelium at specific locations. Such phenotypes are often connected to changes in expression of chemokine receptors and integrins. It is important to understand that a similar p53-induced change in phenotype may be caused by a number of molecular mechanisms and by a direct or indirect activity of mutated p53. These mechanisms include changes in miRNA, lncRNA, and interactions of p53 with transcription factors or chromatin. Conversely, a certain molecular mechanism may be involved in modification of various phenotypes.CD44, SERPINE1 (PAI-1) and CCND1 (Cyclin D1) [In the process of spontaneous acquisition of metastatic properties, cancer cells often undergo epithelial–mesenchymal transition (EMT). This is a broad phenotypic change that allows the cells to gain mesenchymal properties such as ability to migrate or detach from surrounding cells. Although the changes in cells are dramatic, the whole process is controlled by a relatively small number of transcription factors such as SNAI1 (Snail), SNAI2 (Slug), TWIST1, and ZEB1/2 ,32,33. Tclin D1) . A more clin D1) , wherebyclin D1) . Indeed,clin D1) . An examclin D1) . Nearly clin D1) . A mechaclin D1) . In thisclin D1) .Following the reports that wild type and mutated p53 variants regulate several aspects of cancer initiation and progression through miRNA ,43 furthTrp53-/R172H cells derived from murine pancreatic ductal adenocarcinoma [Mutations in p53 may lead to phenotypic changes that do not present full EMT profile or that are of different nature, yet still increase metastatic potential. These include sequestration and inactivation of p63 and p73 that has been observed in mouse models  and demoarcinoma , a depenarcinoma . In a siarcinoma . A novelarcinoma .Another, relatively less known class of RNA molecules is long non-coding RNA (lncRNA). These molecules may serve as scaffolds for assembly of protein complexes. It has been recently discovered that two lncRNAs—lnc273-31 and lnc273-34—were upregulated in colorectal cancer cell culture model following the expression of p53-R273H. They are required for increased cell migration, proliferation and chemoresistance in vitro. In a murine xenotransplant model these lncRNAs dramatically accelerate tumorigenesis. Their expression is also significantly elevated in colorectal cancer patient tissues that bear R273H mutation in p53 .Changes in cellular physiology induced by GOF mutations in p53 that manifest as clinically observed increase in metastasis may also concern changes in cytokine production and cytokine sensitivity as well as integrins and cytoskeleton organization. Although some of them are similar to what is happening during EMT, they may also occur as isolated phenotypes. It was reported that cancer cells expressing any of several mutated p53 variants  released exosomes enriched in miR-1246 that were taken up by tumor-associated macrophages. This reprogrammed the macrophages into anti-inflammatory immunosuppression mode with increased release of TGF-β, which in turn supported tumor growth . A case A link between inflammation and cancer has been established long ago and indeed inflammation is considered one of the “hallmarks of cancer” ,2. The tTrp53-/R172H mice elevated expression of PLAC8 (onzin) was found when compared to Trp53−/− counterparts; this resulted in an increase in metastatic traits in osteosarcoma cells, both in vivo and in vitro. The molecular mechanism involved increased expression of CXCL5 that, in an autocrine manner, enhanced ERK1/2 phosphorylation. Observed phenotypes were reproduced by addition of exogenous CXCL5 and they could be abolished by specific inhibition of ERK1/2. However, some phenotypic changes resulting from p53-R172H expression were not prevented by Cxcl5 gene silencing, indicating that other mechanisms were also involved.A tumor may be likened to a wound that never heals, thus inducing chronic inflammation. There are multiple ways in which tumor cells adapted to sustain and benefit from an ongoing inflammatory process. Due to the limited scope of the article the effects of loss of p53 activity will not be elaborated upon, but rather some examples of GOF p53 mutation-induced changes in the development of inflammation and their consequences for tumor invasion and metastasis will be presented. The key cellular regulator of inflammation is NF-κB and it was demonstrated in a number of in vitro and in vivo models that mutated p53 prolongs its activation in response to TNF-α and thus promotes chronic inflammation that eventually leads to tumorigenic transformation . In glioTrp53−/− or Trp53-/R270H background it was shown that while the Trp53−/− animals rarely developed invasive tumor phenotype, it was commonly found in Trp53-/R270H mice. The authors conclude that this might have been caused by an observed increase in chromatin accessibility resulting in upregulation of approximately 350 genes, many of which could be classified as proinflammatory [Several mechanisms related to mutated p53-induced changes in epigenetic gene expression regulation were proposed. In a transgenic mouse colon cancer model carrying deletions in Apc in a ammatory . In a reammatory . A mechaammatory . The incammatory . An indiammatory . Interesammatory . Table 2The best characterized metabolic shift in cancer cells is known as Warburg effect . In brieAfter forty years of scientific efforts directed at dissecting the role of p53 in tumor development and evolution an enormous amount of observations was made. An initial view that p53 was an oncoprotein was abandoned in favor of its tumor suppressor role. Several years later it turned out that under certain circumstances p53 may indeed become an oncoprotein. If one looks at the tumor development as a process of evolution and adaptation to hostile environment—patient organism—one cannot miss the high prevalence of certain mutations in p53. These overrepresented gain-of-function mutations were demonstrated to provide the tumor with numerous advantages in its struggle for survival and expansion. However, that also means that tumors rely on and are addicted to mutated p53 which potentially presents clinically actionable opportunities. This brief review presented a number of molecular mechanisms utilized by mutated p53 to exert its activities: epigenetic regulation, direct and indirect transcriptional control, miRNA-mediated control, and mRNA stability modification. Some of these act in a cell-autonomous manner and some in autocrine or paracrine manner. Certainly there are effects and modes of action that are unknown yet. However, due to the high prevalence of these mutations in cancer patients, every effort directed at identifying the molecular mechanisms of clinically relevant phenotypes may result in identification of actionable therapeutic targets.


Due to Here we have organized this Research Topic titled “Role of p53 in cell metabolism, ferroptosis, and stemness” and wish to collect manuscripts about the emerging roles of p53 in mediating cell metabolism, ferroptosis, and stemness, which link p53 with diverse diseases, particularly tumors . We haveReinisch et al. found that under acute fasting, p53 in BAT was activated to induce the expression of miR-92a-1-5p. They also demonstrated a fructose transporter Slc2a5 to be a repressive target of miR-92a-1-5p. The downregulation of Slc2a5 reduced the import of fructose, glycolysis, and finally thermogenesis. This study revealed a novel p53/miR-92a-1-5p/Slc2a5 axis in fasting-regulated thermogenesis.MicroRNA is a type of short non-coding RNA with diverse functions by targeting different genes . p53 is Lei et al. in their comprehensive review focused on the role of p53 in the brain. They firstly introduced a basic background of p53 and its regulation. They then discussed how p53 regulates various diseases in the brain, including glioma, cerebral stroke, and neurodegenerative diseases.Zhou et al. wrote a short review about ferroptosis and lymphoma, highlighting that targeting the p53-mediated ferroptotic pathway may be a promising method to treat lymphoma. pan and Wang contributed a review about p53 and ferroptosis in osteosarcoma. In this review, they classified ferroptosis into typical and atypical types. In both types of ferroptosis, p53 has a vital role. Ferroptosis also contributes to diseases other than cancer, such as in the regulation of diabetes. In the review by Du et al., authors depicted the major mechanism of ferroptosis and its effect in diabetes. Notably, they pointed out the crucial role of p53-regulated ferroptosis in the pathology of diabetes. In an interesting study from Hu et al., authors investigated the function of a bioactive peptide G1dP3 in MH7A cells. They showed that G1dP3 significantly suppressed the viability of MH7A cells by inducing ferroptosis. Mechanistically, they demonstrated that G1dP3 could activate p53 expression, resulting in the suppression of the SLC7A11/GPX4 axis.Ferroptosis is a newly discovered cell death type, which is caused by the iron dependent peroxidation of cell membranes . p53 hasLiu et al. presented an informative review about how p53 mutation or deletion remolds the tumor immune microenvironment. There are at least three ways that mutant p53 or loss of p53 contributes to an immunosuppressive status: 1) induce immunosuppressive cytokines and downregulate proinflammatory factors; 2) augment the expression of immunosuppressive ligands; and 3) facilitate immunosuppressive cells differentiation. Correspondingly, targeting mutant p53 would be a reasonable choice to restore the tumor immune microenvironment. Corazzari and Collavin wrote an attractive review to summarize the ferroptosis-regulatory activity of both wild type and mutant p53. They proposed that the regulated ferroptosis could be a stress response to diverse stimuli in and out of the tumor cells. K120 is a critical site for p53 function. Monti et al. analyzed the transactivation activity of the p53 K120R mutant. The K120R mutation abrogates the acetylation at this site, preventing p53 from promoting apoptosis. However, this mutant could still retain some activity to modulate cellular metabolism. Mutant p53 also influences the efficacy of tumor treatment. Li et al. reported a pulmonary sarcomatoid carcinoma case with p53 T125K mutation. Fortunately, treatment of Tislelizumab in combination with Anlotinib enabled this patient to go into complete remission. We hope that this therapeutic regimen may benefit other similar patients.p53 functions as a checkpoint molecule to suppress tumorigenesis. To overcome this, p53 is often mutated or deleted in tumor cells. Mutant p53 may have different functions compared to wild type p53 . Liu et Wei et al. discussed the crosstalk between p63 and STAT3 in the regulation of cancer stemness.Other members of the p53 family, such as p63 and p73, also have fundamental roles in tumor . Signal To summarize, this Research Topic of papers present novel information about some unconventional functions of p53 (and p63). Elucidating the mechanisms of p53 that underlie its versatile roles will not only benefit the basic research about p53 and tumor biology, but also open promising doors for developing therapeutics targeting p53 and related pathways for the treatment of cancer and other diseases. We hope this Research Topic can bring new knowledge and ideas to those who are interested in the function of p53.


These findings reveal unique aspects of the amount of various cannabis forms smoked/vaped in relation to gender and provides preliminary evidence of cannabis consumption behaviors in relation to changing social and cultural contexts.Limited research examines changes in quantities of various forms of smoked/vaped cannabis among regular consumers, including emerging adults  in Canada. This information is particularly relevant in the current context of emerging cannabis behaviors among EAs related to political amendments , vaping-related lung illnesses (EVALI), and unprecedented pandemics (COVID-19). This study investigated the impact of legalizing recreational cannabis use in Canada, the EVALI epidemic, and the COVID-19 pandemic on the quantity of smoked/vaped forms of cannabis in relation to gender differences. EAs retrospectively self-reported the quantity of herb, hash, concentrates, joint size, and the number of joints and vaping cartridges in relation to three consecutive developments: pre-legalization, post-legalization; pre-EVALI, post-EVALI, pre-COVID-19, and during COVID-19. The quantity of herb use significantly increased among heavy users, and vaping quantity significantly increased among light users. Overall, an increasing incremental trend was observed in the average quantity of cannabis forms used over time. Males consumed higher quantities of all cannabis forms than females. More males than females reported using concentrates ( In Canada, cannabis consumption rates are characterized by marked differences in relation to age and sex ,2. For eContrary to its widespread permissive use and its presumptions as a ‘benign’ substance among EAs ,6, cannaThe complexity of measuring cannabis-related effects and risks are further complicated by sex-specific neurobiological mechanisms, gender-specific consumption differences, and motives for use. Nascent evidence suggests a higher sensitivity to cannabis among females, and their vulnerability based on the accelerated progression to problematic use , althougEmerging trends of cannabis consumption behaviors and diversified profiles associated with recreational cannabis legalization in Canada since 17 October 2018 warrants research to examine the quantity of cannabis used, as it may vary across products and methods of use ,15. The p < 0.001). This increase was more prominent among students aged 21 and above , rising from 40.4% to 60.9% (p < 0.001). Similarly, according to recent nationwide data from Canada, there was an increase in cannabis use between 2018 and 2019, specifically among individuals aged 25 and older (from 13.1% to 15.5%) and males (from 17.5% to 20.3%) .n = 11), did not smoke or vape cannabis in the last 12 months (n = 13) , or did not consent (n = 7).Participation was limited to the following inclusion criteria: reported smoking and/or vaping cannabis in the last 12 months, currently living in Canada at the time of the study, and 18 to 29 years old. The survey was restricted to the English language, and consent was required to participate. Respondents that met the inclusion criteria and consented were then directed to the survey questions, which was designed to take 20 to 25 min to complete. No compensation was provided. All responses were anonymous. Convenience sampling was used to recruit participants 18 to 29 years old through social media platforms (Facebook and Twitter) across Canada and from Ontario Tech University. Recruitment and data collection occurred from 4 May 2020 to 31 August 2020. Respondents were identified as cannabis users if they reported smoking and/or vaping cannabis in the last 12 months. Results of 343 individuals were reviewed, of which 312 were eligible. Respondents were excluded if they were not 18–29 years old  Prior to Legalization (approximately October 2018); (2) since legalization; (3) prior to EVALI (approximately November 2019); (4) since EVALI; (5) prior to imposed COVID-19 preventative measures (approximately March 2020); (6) since imposed COVID-19 preventative measures.International Cannabis Policy Study (ICPS) . Reference images were provided to quantify 1 g of hash and concentrate (derived from ).For vaping cannabis oil/vape juice, participants were asked: “What size vaping cartridge do you ‘primarily’ use?”, and “How many vape cartridge(s) or refill(s) did you use in a month?”. Vaping quantity was determined based on the number of vape cartridges/refills used in a usual month . Numeric assumptions were inputted for options “less than” or “more than” the amount indicated. Measuring vaping quantity by the number of cartridges over a longer timeframe than a day is found to be effective in measuring consumption. Therefore, the consumption of vaping was examined by the average of vaping cartridges used in a month. Evidence shows most respondents prefer to report the number of cartridges or tank refills (51%), and fewer users finish 1 cartridge/tank in a day . Data foParticipants reported the cumulative lifetime days of smoking or vaping, using the following questions: ‘How many days in your lifetime have you smoked cannabis?’; ‘How many days in your lifetime have you vaped cannabis?’. A binary variable was created to differentiate an occasional user  from an experienced user  based on lifetime days of use. Occasional users included smoking or vaping: 1 to 2; 3 to 10; or 11 to 99 days in their life. Experienced users included those that reported 100 to 999 or more than 1000 days of lifetime smoking or vaping . FrequenDue to the limited outbreak and awareness of EVALI in Canada, participants were asked, “Prior to taking this survey did you know about EVALI?”. The options were dichotomized (“yes” or “no”). Demographic characteristics included sex  and postal region. Participants were asked, “what was your sex at birth?”, and provided the first three digits of their postal code . Postal codes were grouped by province.p < 0.05) using the estimation method for Rouder’s mixed design. The Bayes factor test yields a BF quantifying how well the alternative hypothesis (H1) predicts the empirical data relative to the null hypothesis (H0) [Data were analyzed using SPSS v 27 (IBM SPSS Statistics 27 ©). Chi-square tests were used to analyze differences in relation to the form of cannabis used most of the time by sex . A one-wsis (H0) ,59. The sis (H0) . AnalyseThe average quantity of cannabis use per month for each form was calculated by multiplying it by the frequency of smoking/vaping categories to reflect the number of days used per month  in relation to each time period. Smoking frequency for each time period was multiplied by the herb quantity, joint size and joint quantity reported. Vaping frequency for each time period was multiplied by the number of cartridge refills in order to estimate the average quantity of vaping cartridges used in one month.The six time periods were divided into four time periods by calculating the average quantity of use between the periods after October 2018, and before November 2019 to create one category (October 2018 to November 2019) given that the quantity of herb, joints and vaping remained consistent in each period. In addition, the average quantity between the two periods—since EVALI (November 2019) and before COVID-19 (March 2020)—were calculated to show the quantity of use from November 2019 to March 2020.2 (1) = 5.02, p < 0.05). Hash was indicated as the least primary form used by males (3%) and females (5%) . More than three quarters of participants (76%) indicated a postal region from Ontario; few participants were from other Canadian provinces and territories (24%). The most popular form of cannabis used as the primary form was herb for males and females (91%), followed by cannabis vaping oil/vape juice, which was endorsed by 15% of males and 15% of females. More males (15%) than females (7%) reported using cannabis concentrates as the primary form (ꭓles (5%) . Only 24The majority of the sample comprised highly experienced (exclusive) cannabis smokers, with 63% of males and 54% of females reporting 100 or more lifetime days of smoking. The mean (SD) age of smoking cannabis onset was 17 (±2) years, while the age of vaping onset was higher at 21 (±3) years (only 2% of users had exclusively vaped cannabis in the last 12 months).BF (14) = 4.496), p < 0.001, showing strong evidence for an effect on the quantity of herb used over time in the sample population (Bf > 3)  = 0.939, p = 0.41), but a statistically significant difference for females  = 5.585, p = 0.004), showing that herb quantity almost doubled from 0.67 g to 1.2 g from pre-legalization to since the COVID-19 pandemic. Post hoc comparisons revealed that a significant increase was evident from before and after legalization for females (p = 0.005), in which herb quantity increased from an average of 0.67 g to at least 1 g personally used on a typical day.(Bf > 3) . AnalyseBF (14) = 65.776, p < 0.001) (1) showing an effect on joint quantity over time was indicated (Bf > 3). Analyses separated by sex showed that there was a significant increase in joint quantity for males  = 3.05, p = 0.049 and for females  = 4.902, p = 0.006). The average number of joints smoked on a typical day by females was highest during the pandemic period (M = 0.86). In relation to joint size, the overall results indicate no evidence for an effect on joint size in relation to the events (BF (14) = 0.903, p < 0.001). Although analyses stratified by sex showed no difference for males  = 1.83, p = 0.17), a borderline significant difference was found for females  = 2.67, p = 0.049).Joint quantity and joint size significantly differed over time (< 0.001) . Strong BF (14) = 0.195 p < 0.001)  = 2.84, p = 0.40) or females  = 2.49, p = 0.11). Changes in hash quantity also indicate moderate evidence for no effect across the time intervals (BF (14) = 0.066, p < 0.001). Due to the small subsample of participants using hash, we were unable to stratify based on sex.In addition, results indicate no difference in concentrate quantity in relation to the events (< 0.001) . AnalyseBF  = 0.954 p < 0.001), although the Bf < 3  = 2.922, p = 0.048). Although there was no significant difference for males  = 1.296, p = 0.29), an average increase is seen before and after legalization and since the COVID-19 pandemic, but a decrease from before and after EVALI  = 9.25, p < 0.001) for the total sample and for females specifically  = 7.86, p < 0.001). The quantity of use calculated based on the average joint size and number of joints also significantly increased for the total sample  = 6.25, p = 0.003) and for females  = 4.06, p = 0.022). F  = 9.03, p < 0.001). Joint quantity also increased significantly among heavy users  = 9.48, p < 0.001). There was an increase over time in relation to vaping for both heavy and light users; however, this association was only significant among light users F  = 5.89, p = 0.005.This study contributes to the complex and evolving landscape of cannabis consumption by examining changes associated with various quantity measures of cannabis forms consumed through smoking and vaping before and after political amendments , public health concerns (EVALI) and during unprecedented natural societal health threats (COVID-19). The findings from this study provide insight into possible changes in cannabis consumption among EAs as a result of public health events. The current legislation on recreational cannabis consumption puts EAs in a social environment that allows for easy accessibility and experimentation and makes them vulnerable to health risks. Recent evidence has shown an increase in the prevalence of cannabis users since legalization . The curFor a clearer estimation of quantity, the quantity measures of the various cannabis forms in this study can be compared to a recent Canadian study that established an algorithm for measuring the quantity of various cannabis forms in standard joint size equivalents . CallaghOverall, the primary findings of this study suggest that cannabis quantity may change over time and in relation to specific events. This is indicated by incremental increases in the average quantity of cannabis forms including dried herb, concentrates, number of joints, and vaping cartridges, from before legalization of cannabis in Canada (October 2018), to after the declaration of the COVID-19 pandemic (March 2020). Specifically, an increasing trend was found over time in relation to the quantity of dried herb, which was the most common form used in this study. It is also interesting to note that in some cases, the quantity of use is lower at the start of each new event relative to the end of the previous one. Although joint size and the number of joints smoked on a typical day increased after legalization, an average decrease was observed from the time frame following legalization and before EVALI was declared an epidemic. However, while joint size decreased, the number of joints per day increased from before to after the declaration of the pandemic for females. These inconsistent patterns which show an average increase in relation to specific events followed by a decline suggest that cannabis consumption behaviors may be associated with changing social and cultural contexts, events, and life trajectory (negative events) among EAs. Additional longitudinal research is needed to further explore the varying patterns of cannabis use over time.Although THC-containing vaping products have been found to play a key role in the propagation of the EVALI epidemic ,61, our In line with the literature, cannabis vaping may lead to an increase in quantity over time, as vaping becomes a consistent and repetitive behavior, and leads to more puffs being taken during each session, thus limiting the time that one vaping cartridge might last . ConsequThe increase in quantity of cannabis used after legalization found in this study, may have been a result of developed interest for the effects and engagement in the liberty of cannabis laws that allowed accessibility to diverse strains and forms . FollowiDuring the COVID-19 pandemic, studies focused on the quantity of joints used in a day, suggesting boredom, stress, and loneliness as motives of increased substance use ,71. SpecIt is important to note that although research indicates an increase in the prevalence of females using cannabis, more research is required to delineate the effects of specific forms of cannabis used in relation to sex-specific mechanisms . For exaThis study sheds light on cannabis-use trends in relation to single-item indicators by examining specific measures in relation to the quantity of various cannabis forms where limited research exists. These findings reveal unique aspects of the amount consumed of various cannabis forms in relation to sex and may aid in determining adverse or harm-reduction measures of consumption. Continued efforts to examine quantity can have important implications for the prevention and treatment of cannabis-related problems. In addition, to our knowledge, this is the first study to examine how quantity measures of multiple cannabis forms may change in relation to legalization, EVALI, and COVID-19 by sex-specific analyses in Canada. At the same time, this paper contributes to establishing standardized criteria for measuring cannabis, which would promote uniformity in quantifying its consumption, thereby aiding the development of research and clinical assessment standards. This, in turn, would enable clinicians to better identify individuals who are at a higher risk of acute and/or chronic issues resulting from cannabis use by gaining insights into how consumption patterns affect health outcomes. Additionally, considering gender-specific factors is important, as significant epidemiological and clinical distinctions have been observed between males and females in relation to cannabis use.Despite these strengths, there are limitations to consider. First, asking participants to retrospectively report cannabis consumption behaviors in relation to specific measurements of quantity may have introduced recall bias, and the events of legalization, EVALI and COVID-19 were primed in the survey as trigger points for recall during data collection. Second, our study recruited individuals that were 18 years old who were minors during the time of cannabis becoming legalized in Canada (approximately 16 years old); therefore, they may have experienced different changes in cannabis use than respondents of age. In addition, the sample size was relatively small and largely from Ontario.Due to the cross-sectional exploratory design of this study, we cannot make any causal inferences. Therefore, it is unclear if the events had a direct effect on the quantity of consumption. Similarly, any changes in relation to cannabis quantity consumed post-legalization of recreational cannabis use in Canada (17 October 2018) may be attributed to legalization and not necessarily reflect the subsequent public heath events, including the outbreak of EVALI and the COVID-19 pandemic. Differences in length between the time periods is also a limitation, given that the mean consumption in one period may not reflect exactly the same process and has the same meaning in other periods. Nonetheless, there seems to be a slow adoption of incremental increases in consumption post-legalization, which was followed by successive events that are relevant to cannabis use and may have influenced users to adapt their consumption. In addition, experimental use is prone to influence the natural progression of increased consumption, as users may transition to more frequent and daily use over time . FurtherMoreover, infrequent, or varying use, and sharing—which is common among cannabis users—can limit the reported capacity ,54. It iIn addition, more people reported using the herb in its dried form and in the form of joints than any other form. This may have limited the power to examine significant changes in quantity for other forms in this study, including, hash and concentrates. Due to power considerations, we were not able to stratify by sex for changes in relation to hash quantity. Finally, examining vaping quantity is difficult as there are a diverse range of vaping devices, forms of cannabis used in vaping devices, as well as the size of cartridges and tanks used .Given the substantial changes  in relation to cannabis policy, corresponding health risks associated with cannabis products and its derivatives, and emerging respiratory illnesses (EVALI and COVID-19) that make cannabis users that smoke or vape particularly vulnerable, examining changes in consumption can provide timely evidence of health-risk behaviors. Greater access to cannabis may increase the quantity of use over time and subsequently build tolerance, particularly for heavy users. Therefore, the quantity of consumption may be critical in addressing the progression of cannabis use disorder, as well as the relationship between measures of consumption and physiological health risks. Sex-specific differences in cannabis smoking and vaping behaviors underscore the need for gender- and sex-focused research as sub-groups may respond differently to factors that may influence cannabis use. Further research is needed to examine the predictors of changes in cannabis consumption behaviors and how these patterns may be influenced by the complex interactions of biological and psychosocial factors.


For malignant transformation, the expression of p62 in the nucleus (p = 0.03) was significantly lower in malignant transformation cases, whereas the expression of p62 in the cytoplasm (p = 0.03) and the aggregation expression (p < 0.01) were significantly higher. Our results suggest that the function of p62 is altered by its subcellular localization. In addition, defects in selective autophagy occur in cases of malignant transformation, suggesting that p62 is a potential biomarker of the risk of malignant transformation of OPMDs.To determine the intracellular behavior of p62, a marker of selective autophagy, in oral potentially malignant disorders (OPMDs). This retrospective study includes 70 patients who underwent biopsy or surgical resection and were definitively diagnosed with OPMDs. Immunohistochemical staining for p62, XPO1, p53, and ki67 was performed on all samples and positive cell occupancy was calculated. We statistically investigated the correlation between protein expression in OPMDs and the association between malignant transformation, clinicopathological characteristics, and occupancy. ki67 expression was negatively correlated with p62 expression in the nucleus ( Oral potentially malignant disorders (OPMDs) are a newly defined concept by the World Health Organization (WHO), which defined them in 2017 as “clinical conditions that carry risk of cancer development in the oral cavity, regardless of whether they are clinically definite precursor lesion or normal mucosa” [Anil et al. reported the presence and extent of epithelial dysplasia as predictors of the development of oral leukoplakia in cancer . AlthougTreatment options for OPMDs include follow-up, removal of disease triggers such as inappropriate restorations and sharp edges of teeth, medication, and surgical resection ; howeverSelective autophagy is a mechanism that selectively degrades bacteria, specific organelles such as mitochondria, and aggregates of polyubiquitinated proteins . DegradaRecently, autophagy has been linked to various diseases, including Alzheimer’s disease, Parkinson’s disease, and cardiovascular disease . It has Yoshida et al. reported the expression of p62 in oral leukoplakia; however, to the best of our knowledge, there are no studies on the expression of p62 in OPMDs . Lin et Of the 104 patients who visited the Department of Oral and Maxillofacial Surgery at the University of Tsukuba Hospital between 2014 and 2021, underwent biopsy or surgical resection, and received a definitive diagnosis of OPMDs, 70 patients provided their informed consent to participate in the study. The specimens retrieved from the patients were formalin-fixed, and paraffin-embedded blocks were prepared for pathological examination. For patients who underwent both biopsy and surgical resection, the specimen obtained during surgical resection was used based on its condition. Clinical and clinicopathological data were obtained from medical records. All the specimens were diagnosed by at least two pathologists. Regarding malignant transformation of OPMDs, since OPMDs have the propensity to recur after surgical resection and is similar to OSCC, the following definition of local recurrence of OSCC was used as a reference: a local recurrence is defined as less than 2 cm away from or occurs within 3 years of the primary tumor and a second primary tumor as more than 2 cm from or occurs more than 3 years after the primary tumor in OSCC [Clinicopathological characteristics including age, sex, drinking habits, smoking habits, location, disorder, presence of epithelial dysplasia, and development of cancer in the 70 cases included in the study are shown in This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the University of Tsukuba Hospital .All tissue sections had been fixed in formalin, paraffin-embedded, and sectioned to a thickness of 4 μm. Human liver cancer tissue sections were used as positive controls for p62, p53, and ki67, and human endometrioid adenocarcinoma tissue sections were used for XPO1 as specified in the protocol. Staining without primary antibodies was used as the negative control.After deparaffinization and rinsing, samples were pretreated with a high pH system antigen activation solution  in a microwave oven at 95 °C for 20 min. Endogenous peroxidase activity was removed by treatment with a mixture of 0.3% hydrogen peroxide in 100% methanol for 10 min. After thorough rinsing, the samples were incubated with primary antibodies against p62 , XPO1 , p53 , and ki67  for 60 min at room temperature, and ki67  primary antibodies at room temperature for 60 min. The samples were washed and treated with a secondary antibody (Histofine Simple Stain MAX-PO(MULTI), Nichirei Bioscience, Tokyo, Japan) for 30 min at room temperature and treated with 3,3′-diaminobenzidine tetrahydrochloride  for 5 min to detect antigen–antibody binding. Finally, hematoxylin was used to counterstain the nuclei.The expression of p62, XPO1, p53, and ki67 in the mucosal epithelium of the stained sections was evaluated. Although both strong and weak expressions were observed, we judged the cells as either positive or negative. A staining intensity equivalent to that of the positive control was considered positive. Images of representative strong and weakly expressing cases of each marker are shown in All immunostained markers were assessed using an optical microscope . Regions showing representative staining in sections were selected at a low-magnification field of view (5×) and evaluated at a medium-magnification field of view (20× and 40×). Three oral surgeons  determined the number of all epithelial cells and the number of positive cells for each marker at random. The positive cell occupancy rate for each was calculated, considering each receiver operating characteristic.Staining for p62 was evaluated separately for nucleus, cytoplasm, and aggregation. In accordance with our previous research, we defined a cell positive for p62 aggregation staining as one with at least one dot of accumulation in the cytoplasm in 20× and 40× field of view . RepreseThe correlation of protein expression in all OPMDs cases was evaluated using Spearman’s correlation test and is presented as a scatter plot. In addition, univariate analysis was performed using Fisher’s exact test with clinical characteristics and the Mann–Whitney U test for the association with each protein expression rate in cases of malignant transformation of OPMDs.p values less than 0.05 were considered statistically significant.All SPSS software ver. 28  was used for statistical analysis.p < 0.01). The expression of p62 in the cytoplasm positively correlated with ki67 expression (p < 0.01) and XPO1 expression (p < 0.01). XPO1 expression positively correlated with ki67 expression (p = 0.03) and p53 expression (p = 0.03). The correlations for proteins other than these five were not significantly different.The expression levels of p62, XPO1, p53, and ki67 in OPMDs, which were correlated using the Spearman’s correlation test, are shown in p = 0.03), p62 in cytoplasm (p = 0.03), and aggregation (p < 0.03) were associated with malignant transformation. Actually, the expression of p62 in the nucleus was significantly lower in the malignant transformation cases, whereas p62 expression in the cytoplasm and aggregation was significantly higher. In contrast, the expression levels of XPO1, p53, and ki67 were not associated with malignant transformation.The results of univariate analysis of the association between the malignant transformation of OPMDs and the clinical characteristics or the expression rate of each protein are shown in This study yields two major findings. First, in OPMDs, nuclear p62 expression was negatively correlated with cell proliferation, whereas cytoplasmic expression was positively correlated with cell proliferation. In addition, p62 cytoplasmic expression correlated with XPO1 expression, suggesting that p62 may be transported to the cytoplasm by XPO1. In the cases of OPMDs malignant transformation cases, the p62 expression was altered in the subcellular localization from the nucleus to the cytoplasm, and the p62 aggregation expression was also significantly higher, suggested that selective autophagy is abnormal.Selective autophagy plays a role in maintaining cellular homeostasis but is known to play a dual role in malignant tumors, depending on the type, stage, and genetic context of the cancer. In the early stages of tumorigenesis, it prevents chronic tissue damage by maintaining the genomic stability and by preventing the accumulation of carcinogenic mutations and inhibits the accumulation of cancer inducers. This enabled it to function as a cancer suppressor; however, in the late stages of tumorigenesis, it strengthens cancer cell survival and stress resistance by preventing tumor cell damage and satisfying high metabolic cravings. This eventually promotes tumorigenesis and leads to the accumulation of therapeutic resistance, which contributes to the progression and metastasis of malignant tumors . In othep62 is an adaptor protein for selective autophagy, which is resolved by autophagy along with its target proteins; therefore, the accumulation of p62 is thought to signify autophagy impairment . AlthougOriginally, p62 was a shuttle protein that continuously and rapidly moved between the nucleus and cytoplasm, and this movement was regulated by the phosphorylation and aggregation of p62 . Variouski67 is a protein expressed in all cell cycles except quiescence (G0 phase) and is used as a cell proliferation marker . ki67 isXPO1 binds to the nuclear export signal (NES) of its target protein, forms a complex, and is transported from the nucleus to the cytoplasm via the nuclear membrane . XPO1 paIn addition to autophagy, p62 is involved in the Keap1-Nrf2 pathway . p62 hasp53 acts as a tumor suppressor by inducing genes involved in cell cycle arrest, apoptosis, senescence, and repairing DNA . p53 is Several studies have reported changes in p62 subcellular localization in normal tissues and malignant tumors. Lin et al. reported no difference in p62 nuclear expression but significantly increased the p62 cytoplasmic expression in OSCC compared to normal oral mucosa. The combination of the high p62 cytoplasmic expression and the low p62 nuclear expression in OSCC is also associated with a lower overall survival and disease-specific survival . KitamurThis study has several limitations. First, in this study, 6 cases (8.6%) were malignantly converted, which is similar to the malignant conversion rate reported previously . HoweverIn this study, we found that the properties of p62 in OPMDs with respect to cell proliferative capacity were reversed between the nuclear and cytoplasmic expression. In addition, in cases of malignant transformation of OPMDs, the subcellular localization of p62 changes from nuclear to cytoplasmic, and p62 aggregation increases, suggesting that selective autophagy is impaired. This indicates that changes in the subcellular localization of p62 may be a potential biomarker for assessing the risk of malignant transformation in OPMDs.