| Background:Bowen’s disease(BD)is a form of squamous cell carcinoma in situ(SCCis),which usually occurs in fair-skinned elderly and can occur in any part of the skin.The typical clinical manifestations are circular,scaly erythema or flat keratinizing plaques with well-defined boundaries.Although BD is characterized by inert slow growth,it is considered"calm before the storm"and has a 3%to 5%probability of progressing to invasive cutaneous squamous cell carcinoma(cSCC).This type of cSCC was shown to be more aggressive than those that develop from actinic keratoses(AK),with a 20%or higher risk of distant metastasis.In terms of treatment,surgery is limited by large trauma and many contraindications.It has been regarded as a weakly recommended treatment in BD management guidelines and consensus in many countries.Photodynamic therapy(PDT)can effectively treat BD,and it is the preferred treatment for special BD due to its characteristics of minimally invasive,safe,repeatable and induction of anti-tumor immunity.However,due to the limitation of photosensitizer absorption and light source irradiation depth,it is not recommended for the treatment of invasive cSCC at present.BD itself may progress to invasive cSCC.Meanwhile,according to clinical practice and literature reports,PDT is less effective in treating BD than AK,and there is even a reported risk of inducing BD progression to cSCC.As a result,most dermatologists still have concerns when using PDT to treat BD in clinical practice.It seriously affected the clinical application and promotion of PDT.If the efficacy of PDT in the treatment of BD can be confirmed through real-world data,and the key candidate genes that promote the occurrence and progression of BD to cSCC can be explored,it can not only provide a basis for the clinical promotion of PDT treatment of BD,but also be expected to develop reliable molecular identification targets based on differentially expressed genes,and develop reliable combination therapy targets based on key candidate genes.This ultimately provides a guarantee for the accurate diagnosis of BD before PDT treatment,and provides the possibility of expanding PDT treatment cSCC in the future.Unfortunately,there are no clinical reports on the long-term efficacy of PDT in the treatment of BD in Chinese,and the key molecular mechanism of the stepped progression from"normal skin(NS)to BD and then to cSCC"is still unclear.With the development of next generation sequencing(NGS)technology,sequencing accuracy,depth and cost performance are improving,making it possible to reveal gene characteristics at the whole genome or transcriptome level in the field of life science.For more than a decade,NGS has been increasingly active in the field of cancer research and has achieved a wealth of results,especially in tumors that already have targeted drugs,such as trastuzumab(approved for metastatic breast cancer in 1998)and imatinib(approved for chronic myeloid leukemia in 2001).Suggesting that oncology has evolved from the classic use of cytotoxic compounds that target each proliferating cell to targeted therapies that have been found to drive genetic changes in tumourgenesis.This revolutionary change paves the way for personalized medicine,making it possible to optimize precision medicine.In the field of cSCC,NGS technology has also been used worldwide to screen susceptible genes and explore differential expression at the gene level through transcriptome sequencing,in order to find key candidate genes and further clarify the molecular mechanism of disease occurrence and development.However,synchronous sequencing studies on NS,BD and cSCC in Asian population are still blank at present.This study retrospectively analyzed the exact efficacy and existing problems of ALA-PDT in the treatment of BD from our years of clinical practice.Meanwhile,NGS technology was used for transcriptome sequencing of NS,BD and cSCC tissues,in-depth analysis and exploration of key candidate genes promoting the occurrence and progression of BD for cSCC.The aim is to provide a reliable basis for clinical differentiation of BD and cSCC,avoid the risk of clinical application of ALA-PDT in the treatment of BD,provide accurate targets for the development of ALA-PDT sensitizer and cSCC-targeted drugs,and lay a solid foundation for the expansion of ALA-PDT treatment of cSCC and ultimately conquer cSCC.Objectives:This study was carried out in two parts simultaneously.On the one hand,the long-term efficacy and existing problems of ALA-PDT in the treatment of BD were retrospectively analyzed.On the other hand,transcriptome sequencing was performed on 3 pairs of BD and para-cancer normal tissues at the exposed site,3 pairs of BD and para-cancer normal tissues at the non-exposed site,and 6 pairs of cSCC and para-cancer normal tissues at the exposed site based on NGS technology.All this is to achieve the following research objectives:(1)Long-term effective rate was obtained from retrospective analysis data of larger-scale clinical ALA-PDT treatment of BD in the real world,and scientific analysis was conducted on the cause of treatment failure or even further progress to cSCC,providing necessary data support for the clinical application and promotion of PDT treatment of BD.(2)Transcriptome sequencing was used to analyze the differentially expressed genes among NS,BD and cSCC to explore the key molecules and signal pathways in the occurrence and development of disease spectrum,providing more reliable theoretical basis for further mechanism research.(3)Verify the key candidate genes at the cellular level,explore their mode of action and possible mechanism in the process of lineage progression from NS to BD and to cSCC to lay a foundation for the early differential diagnosis of cSCC and the establishment of accurate targets for effective prevention and treatment.Methods:1.Retrospective analysis of long-term efficacy of ALA-PDT in treatment of Bowen’s disease and discussion of existing problems(1)Study design:This was a retrospective observational analysis.Statistical parameters include patients’age,gender,lesion locations,lesion size,duration of lesion,PDT treatment times,anesthesia or not,primary lesion or not,pain values,local fluorescence intensity,total follow-up time,clinical results 3 months after the end of PDT treatment and the last follow-up.(2)Patient selection:BD patients who were histopathologically confirmed and treated with ALA-PDT between February 2010 and June 2020 were enrolled in this study.Excluded patients who lacked comprehensive clinical data,lost follow-up,combined with immunosuppressive diseases,photosensitivity or history of photosensitizer allergy.Lesions that had previously undergone surgical resection or had received other topical treatments within 3 months such as imiquimote,5-fluorouracil,or cryotherapy were also excluded.(3)Treatment protocol:After pretreatment,10%or 20%ALA emulsion was incubated for 3 to 6h,and fluorescence distribution and luminance were observed under Wood lamp for pre-evaluation of drug penetration and absorption.Then,red light irradiation at 635nm was applied with energy density of 100 to 200J/cm~2 and power density of 60 to 150m W/cm~2.Treatment intervals were 2 weeks to 2 months.Follow-up was once at 3,6,and 12 months in the first year after completion of all PDT treatments,and once every 6 months thereafter,with lifetime follow-up recommended(4)Parameter evaluation:Clinical results were divided into complete response(CR),incomplete response(ICR)and recurrence,of which ICR and recurrence were classified as treatment failure(TF).Total clearance rate(TCR)was defined as the percentage of CR lesions without recurrence.Follow-up time was from the end of the last ALA-PDT treatment to the end of the last CR follow-up or switch to another treatment.For CR lesions,the end date of follow-up was the last follow-up without recurrence.(5)Statistical analysis:Single factor variables related to treatment failure were screened by binary Logistic regression analysis,and variables with P<0.1 were selected to establish multiple regression model.P<0.05 was considered statistically significant.2.Transcriptome sequencing revealed the differentially expressed genes of NS,BD and cSCC(1)Sample sampling:12 pairs of tissues confirmed by postoperative histopathology were collected,including 6 cases of cSCC lesions at the exposure site and their paired paracancer normal tissues.Of the 6 BD lesions,3 were located in the exposed site and its adjacent normal tissue,and the other 3 were located in the non-exposed site and its adjacent normal tissue.(2)Library construction and sequencing:Deoxyribonuclease I was used to digest DNA fragments in total RNA of samples,and ribosomal RNA in total RNA was removed by RNase H.Then the purified RNA was randomly segmented,and the broken RNA was used as template to synthesize one-strand c DNA.Two-strand c DNA was synthesized,added base"A"to the 3’end of c DNA.U-tagged second strand template was digested by UDG enzyme for PCR amplification.Agilent 2100 Bioanalyzer was used to detect the fragment size and concentration of the constructed library,and DNB was used for onboard 100bp(PE100)sequencing.(3)RNA-seq data analysis:The quality control of original reads was carried out first,mainly including the removal of reads containing sequencing connectors.Remove reads with N ratio greater than 10%.The number of bases removed with mass value Q≤10 accounted for more than 50%of the low-quality reads in the whole read.Then clean data of r RNA removal were compared to the reference genome,and quantitative analysis of gene expression was performed.Finally,R language was used for data pretreatment,Pearson correlation and hierarchical clustering analysis,and identification of differentially expressed genes(DEGs).(4)Bioinformatics analysis:Functional enrichment analysis of DEGs gene was performed using R language,and gene set enrichment analysis(GSEA)was performed using the entire gene set.Finally,the uniformly up-regulated and down-regulated genes in NS,BD and cSCC were explored.3.The role and the preliminary study on the mechanism of promoting BD progression to cSCC by candidate gene IFI30(1)Expression validation:Transcriptome sequencing results were verified in NS,BD and cSCC tissues by q RT-PCR.Then the relative expression of IFI30 was verified in Ha Cat,A431 and HSC-5 cell lines,respectively.(2)Fluorescence localization:The expression of IFI30 in normal cells or tissues,BD tissues and cSCC cells or tissues can be visually displayed through immunofluorescence detection of cells and tissues,and the expression position of IFI30in cells can be located at the same time.(3)Interference and overexpression:siRNA-IFI30 which interferes with the expression of IFI30 and the recombinant plasmid pc DNA3.1-IFI30 carrying IFI30 were synthesized,respectively.IFI30 gene was knocked down and overexpressed in cSCC cell lines A431 and HSC-5,respectively.The result of downregulation and overexpression were verified by q RT-PCR.(4)Proliferation:The effect of IFI30 on proliferation of cSCC cells was verified by CCK-8 and clonogenesis experiments.(5)Migration:The effect of IFI30 on cSCC cell migration was verified by scratch test;(6)Preliminary study on the mechanism:To explore the possible mechanism of IFI30 gene promoting the occurrence and progression of cSCC through western blot.Results:1.Retrospective analysis of long-term efficacy of ALA-PDT in treatment of Bowen’s disease and discussion of existing problems(1)Statistical data showed that BD mostly occurred in elderly patients,mostly female,mainly distributed in the trunk,followed by the head and face.More than one-third of the lesions were between 1 and 2cm,with an average of 2±1.4cm,and most of them had received 3 or 4 PDT treatments.(2)The median follow-up time of skin lesions was 30 months,and the shortest follow-up time for CR lesions was 18 months.TCR was 89.3%,and recurrence rate was 4.1%.All recurrent cases occurred within 2 years after the end of PDT treatment.Poor treatment response or even occult cSCC was found in TF cases.(3)Pain was the main side effect during ALA-PDT treatment,followed by hyperpigmentation.Other side effects included hypopigmentation,hypertrophic scarring and ulceration.(4)Univariate analysis showed that TF was related to tumor diameter,course of disease,number of ALA-PDT treatments and fluorescence intensity after the first incubation of ALA.Multivariate analysis excluding confounding factors showed that only the number of ALA-PDT treatments and fluorescence intensity after the first incubation of ALA were significantly correlated with TF.2.Transcriptome sequencing revealed the differentially expressed genes of NS,BD and cSCC(1)After gene expression quantification,we obtained a total of 18,485 gene expression quantitative results.The results showed that there was a high correlation coefficient between different individuals(R>0.833).At the same time,unsupervised clustering analysis showed that 12 para-cancer skin samples were one module,6 BD samples were one module,and 6 cSCC samples were one module.Moreover,BD and cSCC have a closer branching relationship.(2)In the difference analysis of cSCC vs NS,a total of 2051 DEGs were identified,of which 925 genes were up-regulated in cSCC and 126 genes were down-regulated in cSCC.In BD vs NS,a total of 1153 DEGs were identified,of which 634 were up-regulated and 519 down-regulated.In cSCC vs BD,a total of 846 DEGs were identified,of which 390 were significantly up-regulated and 456 were significantly down-regulated.(3)GO enrichment analysis showed that DEGs was significantly annotated in epidermal development items,such as epidermal differentiation and development,keratinocyte differentiation,etc.in cSCC vs NS.In addition,immune-related items are annotated,such as cell chemotaxis,T cell activation,leukocyte chemotaxis,etc.In BD vs NS,DEGs mainly involves immune-related items.KEGG functional enrichment analysis showed that cSCC and BD had similar signaling pathways,mainly involving factor-factor receptor interaction,neutrophil extracellular trap formation,cell adhesion molecules and PI3K-Akt signaling pathways.(4)GSEA analysis showed that multiple immune-related signaling pathways were activated in BD and cSCC,while multiple metabolism-related pathways were inhibited in BD and cSCC.The intestinal immune network,cytotoxicity regulated by natural killer cells,Toll-like receptor signaling pathway,chemokine receptor interaction,cytokine receptor interaction,B cell receptor signaling and other immune-related signaling pathways were significantly enhanced during the progression of BD to cSCC.(5)We identified a total of 115 uniformly up-regulated or down-regulated genes in NS,BD and cSCC tissue samples.Among them,37 were significantly up-regulated in NS-BD-CSCC,and 78 were significantly down-regulated in NS-BD-CSCC.3.The role and the preliminary study on the mechanism of promoting BD progression to cSCC by candidate gene IFI30(1)The expression level of IFI30 increased gradually in BD and cSCC tissues,but no significant expression was observed in NS tissues,which was consistent with the transcriptome sequencing results.The expression level of IFI30 in cSCC cell lines A431 and HSC-5 was significantly higher than that in human immortalized keratinocytes Ha Cat.Immunofluorescence in cells and tissues showed that the gene was localized in the cytoplasm.(2)q RT-PCR results showed that si-IFI30 and pc-DNA3.1(+)-IFI30 successfully down-regulated and overexpressed IFI30 in A431 and HSC-5 cells,respectively.(3)The results of CCK-8 proliferation assay and clonogenesis assay showed that IFI30 could significantly promote the proliferation of cSCC cell lines.(4)Scratch test showed that IFI30 could promote the migration of cSCC cell lines.(5)Western blot showed that the expression levels of c-myc,an oncogene involved in cell proliferation,differentiation,metabolism and apoptosis,and cyclin D1,a positive regulator of cell cycle,were significantly decreased in cSCC cell lines after down-regulated IFI30,and vice versa.Conclusions:(1)ALA-PDT is a safe and effective way to treat BD,especially suitable for patients with multiple BD,patients not suitable for surgery,aesthetic sensitive sites or poorly healed sites.Adequate and effective pre-treatment of skin lesions and standard PDT procedures are important factors for effective treatment.Fluorescence intensity after the first ALA incubation is a promising predictor of the final outcome of ALA-PDT therapy for BD.The existence of poor treatment response and occult cSCC requires in-depth exploration from the pathogenesis of BD and the mechanism of the progression from BD to cSCC.(2)At the transcriptome level,BD and cSCC were more similar than NS in terms of overall gene expression and functional enrichment analysis.The gene expression profiles of exposed and unexposed BD sites were very similar.In the process of NS-BD-CSCC,a total of 37 consistent up-regulated genes and 78 consistent down-regulated genes were identified.Multiple immune-related signaling pathways are significantly enhanced during BD progression to cSCC.These signaling pathways and genes will lay a foundation for further exploration the mechanism of BD occurrence and even progressing to cSCC,making it possible to explore new targets for the diagnosis and treatment of BD and cSCC.(3)The expression level of IFI30 gene increased step by step during the progression of NS-BD-cSCC disease spectrum.Its downregulation and overexpression significantly inhibited and promoted the proliferation and migration of cSCC cell lines,respectively.Moreover,it may contribute to the formation and progression of tumors by participating in the cycle,differentiation,metabolism and apoptosis of cancer cells.Further research is urgently needed in the future to lay a foundation for the possible use of IFI30 as an accurate target for effective prevention and treatment of BD and cSCC. |
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