Enterococcus faecalis from clinical mastitis of cattle and determine their antimicrobial resistance and virulence determinants to evaluate their potential public health significance. A total of 105 composite milk samples  were analyzed. E. faecalis were isolated by culturing on enterococcal selective media and identified by PCR and sequencing. Antimicrobial resistance phenotype was elucidated by the disc diffusion method, and MIC was determined by broth microdilution method according to CLSI guidelines. Detection of antimicrobial resistance and virulence genes was done by PCR. E. faecalis were isolated from 11.25% (9/80) of the clinical mastitis and 4% (1/25) of the apparently healthy cattle milk samples. The disc diffusion test revealed 40% isolates as resistant to tetracycline and azithromycin, respectively. Among them, 20% (2/10) of isolates showed resistance to both tetracycline and azithromycin. Tetracycline-resistant isolates showed MIC ranging from ≥64 to >128 μg/ml and carried tetracycline-resistant genes tetK, tetL, and tetM in 25%, 25%, and 50% of the resistant isolates, respectively. On the other hand, all the isolates were sensitive to amoxicillin, ampicillin, bacitracin, chloramphenicol, gentamicin, penicillin, and vancomycin. In addition, the isolates carried at least one of the nine virulence genes screened with pil having the highest frequency, followed by fsrB, fsrC, ace, sprE, gelE, and agg genes. Positive correlations were evident between ace, fsrC, gelE, and sprE genes that are associated with the attachment and biofilm formation in E. faecalis. E. faecalis isolated in this study carried antibiotic resistance and virulence determinants which explain their competence to be potential human pathogens.This study was designed to identify  Bovine mastitis is one of the most costly production diseases affecting dairy industries globally . In BangEnterococcus faecalis is considered as the major environmental mastitis-causing pathogen, and the occurrence of this pathogen was reported as 18% in mastitic cow's milk [E. faecalis was reported in animal-originated food including meat, milk, and their products and linked to human diseases such as urinary tract infection [E. faecalis transmission to humans preferably through raw milk [Mastitis is caused by a diverse group of organisms that originate from the environment or are transmitted from an infected udder, termed environmental and contagious mastitis, respectively . Controlw's milk . Besidesw's milk . E. faecnfection –10. Theiraw milk .E. faecalis are intrinsically susceptible to carbapenems, vancomycin, tetracycline, and fluoroquinolones; moreover, acquired resistance to the antibiotics through transposons or plasmids has been reported [E. faecalis might be difficult to treat with most commercial antimicrobials.Enterococci are well known for having a high level of resistance against a wide variety of antimicrobial substances, developed by both intrinsic and acquired mechanisms. They are intrinsically resistant to virtually all cephalosporins, aminoglycosides, clindamycin, and trimethoprim-sulfamethoxazole . E. faecreported . Due to E. faecalis. Many researchers have reported several virulence factors in E. faecalis that might be involved in the severity of diseases in humans and animals [agg, ace) and cytolysis and dissemination of E. faecalis into the host  [E. faecalis has biofilm-forming machinery  that allows the bacterium to adhere to biotic and abiotic surfaces and confers additional antimicrobial resistance [E. faecalis to humans, and eventually affect the choice of drug [In addition to antimicrobial resistance, virulence factors contribute to the pathogenesis of  animals . Studiesnd sprE) . Furthersistance . The mulsistance . As ente of drug .E. faecalis isolated from bovine clinical mastitis. Therefore, we aimed to conduct the present study to determine the antimicrobial resistance and virulence determinants in E. faecalis isolated from clinical mastitis of cattle having potential public health significance.To the best of our knowledge, no data yet exist from Bangladesh on the evaluation of antimicrobial resistance and virulence patterns in The present study was conducted in Dhaka , Mymensingh , and Sirajganj  districts of Bangladesh between January 2019 and June 2021. A total of 105 milk samples were collected comprising 80 from cattle with clinical mastitis and 25 apparently healthy cattle. Cow with clinical mastitis was identified by the residential farm veterinarian based on gross changes in the udder  and/or milk (flakes and/or clots). For the identification of apparently healthy cattle, the California Mastitis Test was used . Cow's mμl of the enriched sample was spread onto modified Edwards medium (MEM)  and incubated aerobically overnight at 37°C. Black color colonies characteristic of Enterococcus spp. obtained on MEM were screened for bacterial morphology by Gram's staining. At least three colonies showing characteristics of Enterococcus spp. were purified by subsequent streaking onto MEM. Crude genomic DNA was extracted from the purified colonies by boiling method and subjected to PCR targeting ddl gene [E. faecalis using the primers provided in Supplementary Table μl volume with 10 μl 2X GoTaq® G2 Green Master Mix , 10 pmol of each primer  with an initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 54°C for 30 sec, extension at 72°C for 1 min, and a final extension step at 72°C for 5 min. Representative isolates which were positive for the ddl gene were further confirmed by sequencing of 16S rRNA using the primers 8F and 1492R [Enrichment of milk samples was performed in Luria Bertani (LB) broth as described earlier . 100 μl ddl gene  for the nd 1492R   were interpreted following the guidelines of the Clinical and Laboratory Standards Institute [β-lactams (amoxicillin 10 μg (AMX), ampicillin 10 μg (AMP), and penicillin G 10 μg (P)), aminoglycosides (gentamicin 30 μg (GEN)), glycopeptides (vancomycin 30 μg (VA)), macrolides (azithromycin 15 μg (AZM)), phenicols (chloramphenicol 30 μg (C)), polypeptides (bacitracin 10 units (B)), and tetracyclines (tetracycline 30 μg (TE)). E. coli strain ATCC25922 was used as the control bacterial strain, and kanamycin (30 μg) was used as the control antibiotic in each experiment. Each experiment was performed three times to confirm the reproducibility of the results. Isolates resistant to three or more antimicrobial classes were considered multidrug resistant (MDR) [Disc diffusion method  was perfnstitute . A totalnt (MDR) .μg/ml and from 0.5 to 128 μg/ml, respectively. Plates were interpreted according to CLSI guidelines, and MIC breakpoint of each sample against their respective antibiotic was recorded where growth was significantly reduced, ignoring tiny buttons or light or faint turbidity [E. coli ATCC 25922 was included in each trial as the quality control strain. Isolates exhibiting MIC > 500 μg/ml gentamicin are considered as high-level gentamicin resistance (HLGR), otherwise considered as wild or low-level resistance to gentamicin [All the isolates were subjected to broth microdilution, to determine the minimum inhibitory concentration (MIC) against gentamicin  and tetracycline  following CLSI guidelines . Test plurbidity . Each teE. faecalis isolates showing antimicrobial resistance were screened for the presence of antimicrobial-resistant genes by PCR. Genes conferring resistance to tetracycline  were screened using the primers enlisted in Supplementary Table (aac(6′)−Ie − ″)aph − Ib, aph(2″) − Ic, aph(2″) −Id, aph(3″) −IIIa, aacC2 and aacC4) and vancomycin  were screened considering current trends of gentamicin and vancomycin resistance in Enterococcus spp. [μl with 20 μl 2x GoTaq® G2 Green Master Mix , 20 pmol of each primer  with an initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing for 1 min at different temperatures according to the primers used  and submitted to GenBank.16S rRNA gene of the representative Data obtained from this study were incorporated into Excel 2013  and then transferred to statistical tools for further analysis.E. faecalis. A p value less than 0.05 was considered statistically significant in all tests.By the Statistical Package for the Social Sciences (SPSS) software , a Fisher's exact test was conducted to find the possible variations between the occurrences of E. faecalis, to evaluate the associations between resistance genes of E. faecalis isolates, and finally to determine the interrelation between different virulence genes of E. faecalis isolates. A statistically significant p value was less than 0.05. The bivariate analysis was performed in SPSS software (version 25).Pearson correlation coefficient was enumerated by a bivariate analysis to assess the correlations between antimicrobials resistant to isolated E. faecalis following culture and PCR targeting the ddl gene  was higher than milk samples originating from apparently healthy cattle ; however, the difference was not statistically significant (p = 0.281)  of milk samples were positive for ddl gene , Table 1ddl gene . The seqE. faecalis isolates were found sensitive to the entire nine antibiotics tested except tetracycline and azithromycin. Forty percent (40%) of the isolates were found sensitive to tetracycline and azithromycin, respectively , 25% (1/4), and 50% (2/4), respectively (aph(3″) − IIIa, aacC2, and aacC4 were detected in 12.5% (1/8), 12.5% (1/8), and 37.5% (3/8) of the isolates, respectively; and one (1) of the isolates carried vanB gene. Besides, none of the antibiotic-resistant gene was detected in four (4) isolates examined. In bivariate analysis, high positive significant correlations were observed in between aac2 and tetM (p = 0.035), and aph(3″) − IIIa and tetL gene (p = 0.001)  .E. faecalis isolates were found positive for at least one virulence gene, where 80% (8/10) of the isolates harbored four or more virulence genes. Occurrence of pil gene (100%) was higher than virulence genes fsrB (80%), fsrC (60%), ace (60%), sprE (30%), gelE (20%), and agg (10%) , and gelE and sprE (p = 0.01) virulence genes of E. faecalis. On the other hand, a negative moderate significant correlation was observed between agg and fsrB (p = 0.035) virulence genes of E. faecalis (All the gg (10%) . Howeverfaecalis .E. faecalis are the most common enterococci species in clinical mastitis; however, there is no study yet in Bangladesh in detecting antimicrobial resistance and virulence factors of this organism from clinical bovine mastitis. From this perspective, we undertook the present study to identify E. faecalis with their antimicrobial resistance, corresponding resistance genes, and virulence factors from cattle showing mastitis.Antimicrobial resistance is a global public health concern. Indiscriminate or irrational use of medically important antimicrobials in animal production is claimed as a major driver of antimicrobial resistance transfer to humans . In animE. faecalis in milk samples of cattle having mastitis was 11.25% which is higher than previously reported as 0.2% in Germany and the United States [E. faecalis in clinical mastitis was reported in Belgium (20%) and South Korea (86.5%) [E. faecalis in milk samples represents a threat to human health as they can be transmitted to humans via the consumption of contaminated milk or milk products [In this study, the occurrence rate of d States , 33. How (86.5%) , 35. TheE. faecalis is quite alarming since it could lead to treatment failure and potential life-threatening diseases in humans if proper antimicrobials are not selected. Interestingly, the isolates described in this study were sensitive to seven (7) other antibiotics tested including penicillin and gentamicin. Gentamicin is the most widely used aminoglycosides against enterococci; however, due to the emergence of HLGR, gentamicin monotherapy becomes ineffective in such cases. In case of low-level resistance, gentamicin or other aminoglycosides are suggested in combination therapy with cell wall inhibitors like penicillin and glycopeptides against Enterococcus [E. faecalis in the study areas. However, antibiotic sensitivity testing is warranted before prescribing any antibiotics to control mastitis as a diverse group of microorganisms are involved in this disease pathogenesis.Tetracycline is one of the most commonly used antimicrobials in animal production for disease control and growth promotion. Widespread uses of this antimicrobial lead to the emergence of tetracycline-resistant bacteria . In addirococcus , 38. BasE. faecalis isolates for nine (9) different tetracycline-resistant genes where the isolates carried either of the three tetracycline-resistant genes tetK or tetL or tetM, indicating the diversity of the tetracycline-resistant genes in the study area. Detection of tetK, tetL, and tetM genes in E. faecalis isolated from subclinical or clinical bovine mastitis has been reported earlier [E. faecalis isolated from mastitis in Bangladesh needs further investigation with more samples and isolates.Tetracycline resistance is conferred by diverse tetracycline-resistant genes located on horizontally transferable elements. At least thirty different tetracycline-resistant genes have been described so far , 39. In  earlier , 41. HowaacC2 and aacC4) in some of the E. faecalis. These genes are known to confer resistance to gentamicin [E. faecalis pathogenicity. In addition, one of the E. faecalis isolates carried vanB gene despite phenotypic vancomycin sensitivity indicating that the gene is dormant or nonfunctional in this strain. The presence of aacC2, aacC4, and vanB genes might not be associated with antibiotic resistance in E. faecalis isolated in this study but poses a threat of transferring these resistance determinants to other enterobacteria having clinical significance in human infections.Antibiotic resistance is conferred by a diverse mechanism including the presence of respective genes or bacterial metabolism , 43. Howntamicin , 46. HowE. faecalis is dependent on its establishment, adherence, invasiveness, and ability to overcome the host defense system and biofilm formation—an important attribute of the bacteria that facilitates its persistence in adverse environmental conditions [E. faecalis isolates were positive for virulence gene pili and six for ace genes. The pili and ace are two important virulence factors whose products are associated with adhesion and colonization in the host [E. faecalis isolates also carried virulence genes fsrB, fsrC, sprE, and gelE, whose products are linked to biofilm formation and its strength in E. faecalis [ace and fsr genes, as well as the sprE and gelE genes, indicates that the isolated E. faecalis are strong biofilm former, as evidenced by previous studies [E. faecalis isolates have the characteristics of a potential human pathogen. In addition, these virulence properties might be linked to E. faecalis persistence in the udder environment. However, to ascertain these possibilities, phenotypic expression of the virulence properties is suggested, which could not be performed due to a lack of our laboratory facilities and funding.Pathogenesis of nditions . In thisthe host . In addifaecalis . Further studies . It is cE. faecalis from bovine clinical mastitis. The occurrence of these resistant isolates in milk samples from mastitis as well as apparently healthy cattle is of public health concern. E. faecalis isolated in this study might be strong biofilm formers. Their biofilm-associated virulence determinants and antimicrobial resistance could be the reason for their persistence in the udder environment and resistance to antimicrobial therapy. Their virulence properties and resistance to medically important antimicrobials indicate their potential to induce human or animal diseases that might be difficult to treat if proper antimicrobials are not selected. Thus, an antibiotic sensitivity test is suggested before prescribing any antimicrobials for mastitis.For the first time in Bangladesh, in this study, we have detected antimicrobial resistance and virulence determinants of


Eating disorders (EDs) emerge most often in adolescent girls. While the basic neural and psychiatric mechanisms of ED development remain unknown, their incidence has increased with sociocultural modernization. To determine what aspects of modern culture are related to EDs, we examined the relationship between predisposition to EDs and interest in several modern cultural factors among young female university students in Bangladesh.A cross-sectional study was conducted in a population of 196 female students aged 18–29 years in a university in Bangladesh. Their predisposition to EDs was examined using the Eating Attitudes Test-26 (EAT-26), and their interest in modern culture was evaluated by grading interest in fashionable clothes, Indian TV shows/serials, luxury food and social network activities, and by examining involvement in cultural activities such as playing, dancing, parties and singing. The relationship between predisposition to EDs and interest in modern culture was then examined. The relation between EAT-26 score and body mass index (BMI) was also examined.The EAT-26 revealed that 37% of the participants were at risk of developing an ED. Correlation analyses showed that a high EAT-26 score was correlated with a high interest in fashionable clothes and Indian TV shows/serials, but not with a high interest in luxury food or social network activities, or involvement in cultural activities, such as playing, dancing, parties and singing. Further, EAT-26 questions in the dieting category and oral control category, but not the bulimia and food preoccupation category, were correlated with interest in cultural factors. EAT-26 score showed no significant correlation with BMI.The results of this study indicate that the risk of EDs is related to an interest in several facets of modern culture. Monitoring the cultural interests of adolescent females, who are continually exposed to modern culture and highly susceptible to EDs, will contribute to the prevention of EDs.The online version contains supplementary material available at 10.1186/s12905-023-02186-6. Eating disorders (EDs) are characterized by abnormal eating behaviors, which typically emerge in females during or prior to adolescence , 2. PatiThe incidence of EDs has increased continually over the past several decades worldwide including Asia, in parallel with the modernization of society that is promoted by industrialization and urbanization –8. IndusThe risk of EDs has been evaluated using the Eating Attitudes Test-26 (EAT-26) questionnaire, which consists of 26 questions related to dieting, bulimia and preoccupation with food, and oral control . This teParticipants’ interest in luxurious food and fine dining was chosen as a representative aspect of modernized food culture. Interest in fashionable clothes was examined because image of beauty and body image disturbances are considered to underlie EDs –20. InteA total of 208 women were initially recruited from the three campuses of Dhaka International University . Exclusion criteria were the age over 30 (n = 5) and incomplete answers to the questionnaire (n = 7). As a result, 196 participants aged 18–29 years (mean ± standard deviation [SD] range: 22.2 ± 1.9 years) were subjected to analysis. The sample size was determined using G*Power 3.1 software based on a power of 80%, significance level of 0.05, and a medium effect size of 0.25, to detect differences among four groups with different levels of interest in cultural factors, which gave a required total sample size of 180.Orientation sessions for the study were conducted by teachers and other faculty members regarding the purpose, procedure, maintenance of confidentiality and anonymity, and limited usage of the data for research. After the explanation, all participants provided informed consent orally. Almost all of the potential subjects agreed to participate in the study.The survey was conducted between March and July 2019. After informed consent had been obtained, the EAT-26 survey was administered by providing written questionnaire forms. The validity and reliability of EAT-26 in Bangladesh had been shown previously , 16. StuAll the questionnaires were provided in the university classrooms. Several tens of participants in a classroom were delivered with written questionnaire forms and filled in answers, supervised by teachers or faculty staffs who gave instructions and responded to technical questions from participants. After collecting the questionnaire forms with answers, incomplete forms having blank columns were excluded from analysis.The EAT-26 was administered according to standard methods . The que2) [BMI was calculated by dividing the weight in kilograms by the square of height in meters (kg/m2) . BMI was2)  as follor). The relationships of EAT-26 total and subscale scores with interest in modern culture were analyzed by Spearman’s rank evaluation test and a one-way analysis of variance (ANOVA) with the Tukey–Kramer post hoc test. The relationship between ED risk groups (“no risk” and “at risk” groups) and the level of interest in modern culture was assessed by the chi-square test. A multivariate logistic regression analysis was further conducted to adjust for possible interrelations among interest in different cultural factors. The relationships between the scores for each question on the EAT-26 and interest in modern culture were analyzed by Spearman’s rank evaluation test and the Kruskal–Wallis test. In all analyses, P < 0.05 was taken to indicate statistical significance. Logistic regression analysis was performed using JMP software . All other analyses were performed using IBM SPSS Statistics software . All graphs were constructed using GraphPad Prism software .The association between BMI and EAT-26 score was determined by calculating Pearson’s correlation coefficient  were in the age range 18–23 years. Figure r = 0.076, P = 0.29)  were analyzed as representative aspects of modern culture. The numbers of participants and percentages of total participants with strong, moderate, little, and no interest in fashionable clothes were 56 (28.6%), 58 (29.6%), 62 (31.6%), and 20 (10.2%), respectively; those for luxury food/fine dining were 48 (24.5%), 58 (29.6%), 64 (32.7%), and 26 (13.3%), respectively; those for Indian TV shows were 10 (5.1%), 21 (10.7%), 52 (26.5%), and 113 (57.7%), respectively; and those for Facebook were 38 (19.4%), 72 (36.7%), 66 (33.7%), and 20 (10.2%), respectively. The numbers of participants who had taken part in playing, dancing, partying, and singing were 36 (18.4%), 34 (17.3%), 19 (9.7%), and 48 (24.5%), respectively; 68 (34.7%) selected “other” activities.Next, we examined whether interest in the above aspects of modern culture were correlated with EAT-26 scores   and Indian TV shows (P = 0.0016) , Facebook (P = 0.55), and involvement in cultural activities (P = 0.78) was not significantly correlated with EAT-26 score  Fig. A and C. 16) Fig. A. Partic16) Fig. C. IntereP = 0.032; chi-square test) (Table n = 121) and compared with the group of participants not involved in these activities (n = 75). The results showed a significant association for interest in Indian TV (P = 0.030) with an odds ratio of 2.48  Table . There wTaken together, these analyses showed that young female students with higher levels of interest in fashionable clothes and those with a stronger interest in Indian TV tended to have higher EAT-26 scores and a higher ED risk.r = 0.22, P = 0.002; Spearman’s rank evaluation test), while there were no significant correlations for other combinations , bulimia and food preoccupation category (6 questions), and oral control category (7 questions)  . The difference in dieting subscale scores was not statistically significant but had a relatively small P-value  . The difference in the bulimia and food preoccupation subscale scores was not statistically significant   Fig. C. MultipVA) Fig. A. AlthouVA) Fig. B.P = 0.012, one-way ANOVA)  and between the strong and no interest groups (P = 0.011). The difference in oral control subscale scores was not statistically significant, although the P-value was relatively small  . The difference in the bulimia and food preoccupation subscale scores was not statistically significant   Fig. D. MultipVA) Fig. F, and muOverall, the analyses indicate that interest in fashionable clothes and Indian TV shows tended to be correlated with the dieting and oral control EAT-26 subscale scores, where interest in fashionable clothes showed a stronger correlation with the oral control subscale score and interest in Indian TV shows a stronger correlation with the dieting subscale score.We finally examined which questions on the EAT-26 questionnaire were correlated with interest in fashionable clothes and Indian TV shows Table . In SpeaThen, group differences were examined with the Kruskal–Wallis test Table . Among tThese analyses for each question indicate that interest in fashionable clothes and Indian TV shows is positively correlated with particular questions in the dieting and oral control categories of the EAT-26.The present study shows that a high EAT-26 score was correlated with a high interest in fashionable clothes and Indian TV shows/serials, but not with a high interest in luxury food or social network activities, or involvement in cultural activities, such as playing, dancing, partying, and singing. Specific questions in the EAT-26 dieting and oral control categories, but not the bulimia and food preoccupation category, were correlated with interest in these modern cultural factors. These results indicate that the risk of EDs is related to an interest in several aspects of modern culture.The incidence of EDs has increased with industrialization and urbanization worldwide, including Asia –8. ConsiOur study shows a correlation between interest in fashionable clothes and ED risk. Adolescent females experience physical and identity-related changes, and acquire a greater awareness both of their own bodies and those of others . This grOur study also revealed a correlation between ED risk and interest in Indian TV shows/serials. TV programs, especially those delivered from India to Bangladesh, convey images of the beauty and body size ideals created by the fashion and beauty industries. Images associated with beauty ideals may contribute to body dissatisfaction, defined as the discrepancy between one’s actual and ideal body shape . In factInterest in fashionable clothes and Indian TV shows were significantly correlated with the dieting and oral control EAT-26 subscale scores in the present study. This is consistent with the notion that body image disturbance and ED development are closely linked –20. FashAn analysis of the scores for each question on the EAT-26 also showed correlations between interest in fashionable clothes/Indian TV shows and dieting/oral control questions. Interestingly, the questions showing significant correlations were different between interest in fashionable clothes and Indian TV shows (see Table Social media platforms, including Facebook, Twitter, Instagram, and YouTube, allow the viewing and sharing of user-generated content. The rapid growth of social media has been shown to be associated with a number of psychiatric disorders, including EDs , 29. A cIn contrast to interest in fashionable clothes and Indian TV shows, our study did not show a relationship between interest in luxury food/fine dining and ED risk. While abnormal food intake is the central symptom of EDs, food avoidance and binge eating/purging are considered to be manifestations of a body image disturbance, rather than causes of EDs , 20. EDsLifestyles differ among populations even in the same country. Although a positive correlation between EAT-26 total score and BMI was reported in a previous study in Bangladesh , our stuThis study shows that young female university students in Bangladesh with a strong interest in fashionable clothes and Indian TV shows as aspects of modern culture tended to have a greater ED risk, and that these interests were correlated with the dieting and oral control EAT-26 subscale scores, but not with the bulimia and food preoccupation subscale score. These results are consistent with the suggestion that the development of EDs is related to increased body awareness and body image disturbances. Monitoring the cultural interests of adolescent females, who are continually exposed to modern culture and highly susceptible to EDs, may contribute to the prevention of EDs.Additional file 1. Relationships between interest in modern culture and scores for each EAT-26 question.


The traditional Chinese medicine (TCM) formula 01 for nasopharyngeal carcinoma (NPC01) is used in the management of head and neck cancers (HNCs), but whether NPC01 has an impact on the HR-QOL of patients with HNCs is unknown.This was a retrospective study of patients with HNCs who were treated between January 2019 and January 2020 at the Head & Neck Cancer Center of Tianjin Medical University Cancer Institute & Hospital. The patients were grouped according to whether they received NPC01 or not (controls). All patients routinely completed the EORTC QLQ-C30 and QLQ-H&N35 modules before and after 3 months of systemic treatment. Health economics were collected.P = 0.004). All other characteristics were comparable between the two groups . In EORTC QLQ-C30, physical functioning (P = 0.03), fatigue (P = 0.04), pain (P = 0.02), appetite loss (P = 0.02), and constipation (P = 0.01) scores were improved more in the NPC01 group than in controls. In EORTC H&N35, the scores for pain (P = 0.02), swallowing (P = 0.01), and dry mouth (P = 0.02) were better in the NPC01 group than in controls. The NPC01 cost was 38.94 RMB/patient compared with 12.81 RMB/patient for regular follow-up, but considering insurance coverage, the financial burden was not higher.The patients who received NPC01 were older than the controls (48.6 ± 11.4 vs. 43.4 ± 8.8 years, The results suggest that NPC01 improves HNCs-related symptoms and HR-QOL. Head and neck cancer (HNC) is a heterogeneous collection of malignancies arising in the mucosal linings of the upper aerodigestive tract including the oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx –3. HNC mTaiping Huimin He Ji Ju Fang (an ancient book of pharmacology of TCM formulae) of the Song Dynasty (AD 960–1279) in China. Liang-Ge-San has been used for centuries in China for the treatment of fever, constipation, pharyngitis, and rhinitis [The traditional Chinese medicine (TCM) formula 01 for nasopharyngeal carcinoma (NPC01) has shown some efficacy in improving the symptoms and signs of HNC . NPC01 irhinitis . Some corhinitis , 15, 16.rhinitis , 17, 18.rhinitis , 17. NevThis study retrospectively analyzed HNC patients treated in the Head & Neck Cancer Center of Tianjin Cancer Hospital from January 2019 to January 2020. This study was approved by the ethics committee of the Tianjin Medical University Cancer Institute & Hospital, Tianjin Medical University (Reference Number: 201902157). The need for informed consent was waived by the committee. The inclusion criteria were: 1) pathologically confirmed HNC and 2) regular follow-up after completing comprehensive treatment including surgery, radiotherapy, and chemotherapy . The excHNC patients treated with NPC01 were included into the treatment group, while those patients who did not receive NPC01 treatment were included into the control group.The ingredients of NPC01 are shown in the Table All patients routinely received HR-QOL measurement before and after 3 months of systemic treatment. The European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-H&N35 are commonly used to measure the quality of life in HNC patients –21. To aThe surveys were conducted by the staff of the Integrated traditional Chinese and Western Medicine Department. Trained psychologists and physicians interviewed patients face-to-face. The questionnaires were administered by a medical doctor. Patient screening was conducted in the outpatient department. The outpatient department had relevant publicity on recruiting research subjects, and patients were informed of the research contents during picking up medicine or waiting in the outpatient department. Each participant was informed of the objectives of this study both in oral and written form. Informed consent from participants was obtained. The interviewers allowed participants to complete the self-assessment questionnaires independently. Participants were encouraged to seek consultation and ask questions as needed.P < 0.05 was considered statistically significant.All data were processed using SPSS 16.0 . Qualitative data were presented as numbers and percentages and analyzed using the chi-square test or Fisher’s exact test. Continuous data were found to be normally distributed and analyzed using the t-test. A value of P = 0.004). All other characteristics were comparable between the two groups of patients .Of the 390 HNC patients screened, 71 patients treated with NPC01 for 3 months and 63 patients who received regular treatments were finally included in this study . Table P = 0.03), fatigue (P = 0.04), pain (P = 0.02), appetite loss (P = 0.02), and constipation (P = 0.01) scales in the QLQ-C30 questionnaire were significantly improved in the NPC01 group. The scores of pain (P = 0.02), swallowing (P = 0.01), and dry mouth (P = 0.02) scales in the H&N35 questionnaire were also significantly improved in the NPC01 group. After 3 months, the scores of NPC01-treated patients in most scales were increased, but there was no statistically significant difference. Meanwhile, the scores of 5 scales  in the NPC01 group were lower than those in the control group, but it was not statistically significant.Significant differences were observed in 5 of the 15 scales in the QLQ-C30 and 3 of the 13 scales in the H&N35 between the two groups (There was no deaths or serious adverse events. Treatment-limiting adverse events were reported in all participants, including headache, dizziness, limb numbness, abdominal distention, stomachache, and diarrhea after drug administration Table , and allTCM NPC01 has been extensively applied for the management of HNC , 17, 18,This study aimed to investigate the effect of NPC01 on the HR-QOL outcomes in HNC patients. NPC01 is usually given after surgery, radiotherapy, and chemotherapy as an adjuvant therapy  to managThe functional and esthetic organs of HNC patients are inevitably sacrificed following tumor resection. Whether surgery, radiation, chemotherapy, or any combination of them can bring a host of potential complications or adverse results after treatment. Hospital support services for HNC survivors remain inadequate . CurrentAlthough NPC01 has been applied in our cancer center for many years, the formal scientific evaluation of NPC01 is still a brand new field. As the first step in developing a clinical application program of NPC01 for cancer patients, this study is conducted to explore the prospect and experience of NPC01. We also realize that the use of NPC01 is a self-help process with profound cultural deposits, which is related to the traditional Chinese philosophy of life.HNC risk is strongly associated with low socioeconomic status . Even thThis study also has some limitations. Firstly, this study uses a retrospective cohort in a single institution. Secondly, the sample size of this study is small. Additional clinical trials are still required to evaluate the benefits of NPC01 . In addiIn conclusion, NPC01 can improve HR-QOL results in HNC patients. Further research on NPC01 is warranted to determine its exact effectiveness and safety. The findings of this study enrich the relevant knowledge of NPC01 in the treatment of HNC and provide useful information for future clinical research in this field.In conclusion, NPC01 can improve HR-QOL in patients with HNC compared with controls with regular follow-up. NPC01 is an effective,economical and safe treatment.


Posterior wall acetabular fractures remain one of the most difficult fracture injuries to treat. Accurate assessment of fracture characteristics and appropriate preoperative surgical strategies are essential for excellent reduction. This paper evaluates the feasibility and effectiveness of a one-stop computerized virtual planning system for the surgical management of posterior wall acetabular fractures.52 cases of posterior wall acetabular fractures treated surgically were selected in our department between January 2015 and December 2020 for retrospective analysis. 52 cases were classified into group A (25 patients) and group B (27 patients) according to whether computerized virtual planning procedures were performed preoperatively. In group A, virtual surgical simulation was conducted using a one-stop computerized planning system preoperatively. In group B, traditional surgery was employed. Reduction quality, surgical time, blood loss, hip function, complications, and instrumentation time were compared between the two groups.The actual surgery for all patients in group A was essentially the same as the virtual surgery before the operation. Compared to group B, patients in group A had markedly shorter surgical time (−43 min), shorter instrumentation time (−20 min), and less intraoperative blood loss (−130 ml). However, no significant statistical difference was observed in reduction quality and hip function. The complication rate was slightly lower in group A (4/25) than in group B (7/27), without a significant difference.The one-stop computerized virtual planning system is a highly effective, user-friendly and educational tool for allowing the cost-efficient surgical simulation of posterior wall acetabular fractures and providing a more individualized therapeutic schedule. The one-stop computerized planning system is feasible to treat posterior wall acetabular fractures, which is an effective method than the conventional treatment of posterior wall acetabular fractures.Trial registration: retrospective registration. Posterior wall fractures are the most common type of acetabular fractures, approximately 1/4–1/3 of all acetabular fractures, which have worse results than most other patterns even in the hands of experienced surgeons . LetournCurrently, three-dimensional computed tomography (3D-CT) has been widely used to identify fracture morphology and spatial relationship among bone blocks , 10. WhiModern image processing and computer technology make it technically and financially easier for orthopedic surgeons to use virtual planning software in clinical practice , 16, 17.Herein, a one-stop computerized virtual planning system is proposed to enable a series of consecutive preoperative procedures including automatic segmentation, virtual fracture reduction, and internal fixation simulation via a personal computer by surgeons. This study evaluates the feasibility and effectiveness of the one-stop planning system for treating posterior wall acetabular fractures. Moreover, a retrospective comparison is conducted on the results of patients who suffer from posterior wall acetabular fractures with and without a virtual surgical planning system.The Department of Orthopedics in the General Hospital of Central Theater Command performed a retrospective case-control study between January 2015 and December 2020. The Institutional Research Ethics Committee approved the study, and all participants provided written informed consent.The inclusion criteria include: (i) isolated posterior acetabular wall fractures; (ii) age equal to or greater than 18 years. The exclusion criteria include (i) inability to undergo surgery within 3 weeks of injury; (ii) pathologic or open posterior wall acetabular fractures; (iii) history of hip injuries; (iv) along with pelvic or femoral head fractures; (v) patients with incomplete radiographic data and a follow-up time of fewer than 12 months.52 cases with posterior wall acetabular fractures between January 2015 and December 2020 were selected for analysis. Patients were classified into groups A and B based on whether to perform the surgery by the new method of a one-stop computerized planning system or the traditional method, respectively. The new method consisted of 3D virtual fracture model reconstruction, reduction and implantation simulation. There were 25 patients in group A and 27 patients treated by the conventional technique in group B. Patient characteristics are summarized in Table In group A, thin-slice CT axial images of the pelvis were output to the DICOM file and input to the computerized virtual system  on a personal computer.The one-stop preoperative planning process on software was as follows: Firstly, the appropriate threshold for bone was measured by default and a virtual 3D model of the pelvis was reconstructed. Then fracture fragments were separated using a 3D automatic-segmentation function. The approximate extent of each part was approximately marked using the device cursor, followed by a software algorithm precisely and automatically defining the fragment border when adjacent fragments were defined as separated parts. Different colors were then assigned to each fracture fragment to make it easier for surgeons to identify different fragments Fig. . FollowiAll patients were placed laterally on a radiolucent table and all operations were conducted under general anesthesia by two experienced trauma surgeons. In both groups, the Kocher–Langenbeck (K–L) approach was used to expose the posterior wall.In group A, aided by detailed preoperative planning, key anatomical landmarks were precisely exposed with less invasiveness and appropriate sequential procedures for fracture reduction were performed. After adequate fracture reduction, miniplates were placed in the pre-determined position, and then fixation was enhanced by the patient-specific reconstruction plates to improve its stability and prevent postoperative fixation loss Fig. . The numIn group B, the fracture nature was learned by surgeons to make the surgical plan using plain radiographs and 3D-CT scans conventionally. The same surgical procedure was used in this group of patients. The miniplates and reconstruction plates were placed empirically without the assistance of one-stop planning system. The reconstruction plates were all contoured intraoperatively, and the time required for implantation was recorded. Fracture reduction and positioning of miniplates and reconstruction plates were confirmed using intraoperative fluoroscopy in both groups.The same management was conducted in both groups. Prophylactic antibiotic (cefazolin sodium) was routinely administered and continued for 48 h postoperatively. The drainage amount was recorded continuously and the drainage tube was removed at the 48th hour postoperatively. All the patients were treated with low molecular weight heparin (LMWH) as anticoagulation therapy. Routine chemoprophylaxis for heterotopic ossification was not used. 3D-CT of the pelvis and standard pelvic plain films (Judet and AP views) were obtained within one-week post-operation and rehabilitation training was guided by rehabilitation doctors for all patients after surgery. All patients were followed up at 1, 2, 3, 6, and 12 months after surgery and annually thereafter. During the follow-up, postoperative complications, clinical function, and fracture healing were recorded.Fracture reduction quality, complications, blood loss, instrumentation time, and surgical time were assessed. Instrumentation time refers to the time needed for plate fixation, including fixing plate configuration adjustments, drilling screw holes, measuring screw lengths, and locking plates. Based on the criteria of Matta, the quality of posterior wall fracture reduction was expressed as poor (> 3 mm), imperfect (2–3 mm), and anatomic (0–1 mm) . During n) frequencies. Chi-square and Wilcoxon rank-sum tests were used for Categorical variables as appropriate. P value < 0.05 was defined as statistical significance.SPSS software version 20.0 and student t-test were used for continuous variables. Continuous variables with a normal distribution were expressed as the mean ± standard deviation. Categorical variables were expressed as relative (%) and absolute (p > 0.05). The mean follow-up periods were 28.24 ± 8.13 and 29.56 ± 7.63 months in groups A and B (p = 0.550). No difference was found in the fracture healing time between the two groups .Demographic data, duration of surgery, preoperative sciatic nerve damage, hip dislocation, concomitant injury, and fracture side are presented in Table p = 0.002; 138.64 ± 42.37 vs. 182.26 ± 47.99 min, p = 0.001). It was also found that the mean instrumentation time of group A was markedly shorter compared to group B . According to the Matta scoring system, in group A, anatomic, imperfect, and poor grades were observed in 22 (88.0%), two (8.0%), and one (4.0%) cases, respectively. In group B, anatomic, imperfect, and poor grades were observed in 21 (77.8%), four (14.8%) and two (7.4%) cases, respectively. The quality of fracture reduction was similar between both groups (P = 0.337)  , fair in one (4.0%), good in seven 28.0%), and excellent in 16 (64.0%). In group B, the function results at the final follow-up were graded as poor in one case (3.7%), fair in two (7.4%), good in nine (33.3%), and excellent in 15 (55.6%). No significant statistical difference was found in the hip function outcomes between both groups  of group A and three (11.1%) of group B. In group A, no patient developed avascular necrosis of the femoral head after the operation. In group B, one (3.7%) patient developed avascular necrosis of the femoral head and was treated with total hip arthroplasty one year postoperatively. Based on the Brooker classification, five patients developed the class I heterotopic ossification (HO) with no clinical symptoms (two in group A and three in group B). No perioperative complications including incision infection, iatrogenic sciatic nerve injury, and deep vein thrombosis occurred in either group. None of the patients had plate failure, screw loosening, miniplate displacement, or intra-articular screw placement at the follow-up.The acetabular fracture is caused by severe intra-articular trauma, frequently from high-velocity injury , 3. The During the past decade, virtual preoperative planning of orthopedic surgery based on a virtual reality model has become increasingly common with the development of computer technology and image processing , 19, 27.This study is unique in case and control groups where patients with similar fracture types were included, who were surgically treated by the same Kocher–Langenbeck approach. Thus, the interference factors could be reduced as much as possible with a higher level of reliability. This study aims to compare the outcomes of the posterior wall acetabular fractures treated by the one-stop preoperative planning system and the traditional method. Compared to the conventional surgical method in group B, both surgical time and blood loss in group A were significantly reduced by the computerized virtual planning system. The decreased surgical time was mainly due to preoperative surgical simulation in a 3D virtual model. Psychological studies have suggested that cognitive preparation and mental readiness are necessary for successful procedures, especially for beginners . Given tThere are also some limitations in the present study. First, the process of repositioning fragments is manual, which is still not precise and fast enough, especially for fractures with severe comminution. Further study may better address this problem by using the mirrored image from the opposite acetabulum, which may be more efficient. Second, the shape of the virtual contoured plate is too ideal to bend the real plate because the destruction of the screw hole should be avoided when bending. Therefore, the virtual contoured plate may not match each brand of the reconstruction plates. Third, the present study is a retrospective design with a relatively small sample size. Further prospective randomized controlled trials with a large sample size may ascertain the role of this one-stop computerized virtual planning system in the surgical treatment of posterior wall acetabular fractures.The one-stop computerized virtual planning system is an efficient, user-friendly and educational tool, which allows cost-efficient surgical simulation of posterior wall acetabular fractures and provides a more individualized treatment plan. With the assistance of a one-stop planning system, shorter surgical and instrumentation time, and less blood loss were observed during the surgery of posterior wall acetabular fractures. Consequently, the one-stop planning system is feasible to treat posterior wall acetabular fractures, which is an effective method than the conventional treatment of posterior wall acetabular fractures.


There is still uncertainty on whether ionizing radiation from CT scans can increase the risks of cancer. This study aimed to identify the association of cumulative ionizing radiation from CT scans with pertaining cancer risks in adults.Five databases were searched from their inception to November 15, 2020. Observational studies reporting cancer risks from CT scans in adults were included. The main outcome included quantified cancer risks as cancer case numbers in exposed/unexposed adult participants with unified converted measures to odds ratio (OR) for relative risk, hazard ratio. Global background radiation (2.4 mSv per year) was used as control for lifetime attribution risk (LAR), with the same period from incubation after exposure until survival to 100 years.25 studies were included with a sum of 111,649,943 participants , comprising 2,049,943 actual participants from 6 studies with an average follow-up period as 30.1 years ; 109,600,000 participants from 19 studies using LAR. The cancer risks for adults following CT scans were inordinately increased . Moreover, cancer risks elevated with increase of radiation dose , and multiple CT scan sites . The risk of solid malignancy was higher than leukemia. Notably, there were no significant differences for age, gender, country, continent, study quality and studying time phrases.Based on 111.6 million adult participants from 3 continents , this meta-analysis identifies an inordinately increase in cancer risks from CT scans for adults. Moreover, the cancer risks were positively correlated with radiation dose and CT sites. The meta-analysis highlights the awareness of potential cancer risks of CT scans as well as more reasonable methodology to quantify cancer risks in terms of life expectancy as 100 years for LAR.CRD42019133487.The online version contains supplementary material available at 10.1186/s12885-022-10310-2. As an auxiliary diagnostic imaging modality, CT scans play an important role in the diagnosis of various complicated diseases . As new Substantial follow-up works are now underway to determine whether patients experiencing ionizing radiation from CT scans can significantly increase the risk of cancer. Indeed, many lines of evidence have shown that ionizing radiation from CT scans is associated with cancer/tumor risks –12. WherHowever, researchers questioned the validity of the earlier indirect estimates based on uncertain risk projections for radiation , whereasThis systematic review and meta-analysis was successfully registered in the International Prospective Register of Systematic reviews (PROSPERO) on 28 April 2019, with the Registration number CRD42019133487. The initial enrollment was to study the relationship between CT screening and cancer risk in children and adolescents. We revised the registry for adult CT scans and cancer risks due to a published study by another group during our studying process ..Observational studies, cohort studies, and case-control studies were included in this systematic review and meta-analysis, while studies assessed review articles, proceedings, case series and case reports were excluded. No restrictions to the language of publication were applied to select primary studies. We added extra studies by additional hand search of the reference lists of related articles.Patients meeting the following requirements were included in this study: 1) Patients were older than 18 years for their first CT scan, 2) Patients did not have any malignant diseases (such as precancerous symptoms or early symptoms of the cancer) prior to their first CT scan, 3) Patients with follow-up of more than 1 year after their first scan. 4) Patients underwent at least one CT scan. Those with the following conditions were excluded: 1) Patients with incomplete demographic data or data errors, 2) Patients lost follow-up for a variety of factors.Included patients received one or more electronically archived CT scans. The absorbed dose from a CT scan mainly depends on factors including age, sex, examination site, year of scan , and maCancer risks, including each part of the human body with at least 1 year lag period after first exposure.The potential associations between adult CT scan exposure radiation doses and cancer risks, including lifetime attribute risk (LAR).The study included two outcome measures.A thorough search was conducted in the following global databases, PubMed, Medline, EMBASE, Web of Science, Springer Link, Cochrane Library, from inception through Nov 15th 2020 with terms including “Adult”, “computerized tomography, X ray”, and “Cancer”. Search strategies were designed by an experienced librarian and revised by another librarian according to the PRESS (Peer Review of Electronic Search Strategies) checklist . Three rThe ROBs of each study were assessed independently by two authors according to Newcastle-Ottawa Scale ..e/Ru for RR (Relative Risk), Re (1- Ru)/[Ru (1- Re)] for OR , and (Or/Oe)/(Cr/Ce) for HR  , Ι2 = 94.18; combined, OR, 5.89 , Ι2 = 99.61%; Fig. P = 0.00; Table The cancer risks for adults following CT scans were inordinately increased . Cancer risks of exposed men and women were not significantly different  for adults in terms of age .There was no evidence of publication bias  to explore the impact of scanning year and countries on cancer risks. Scanning year, countries, and continents of studies did not alter cancer risks (P = 0.00) . In details, the cancer risks were lowest for radiation dose of less than 15 mSv , highest for dose greater than 55 mSv , intermediate for dose of 15 to 55 mSv , with significant differences between triple groups  . Moreover, the relationship between cancer risk and radiation dose was more significant in the < 45 years group (P = 0.00), while there was no significant difference in 45–65 years groups .Dose-response analysis showed that the cancer risks significantly increased linearly with radiation dose of CT scans . Heterogeneity was present in all analyses . Meta-regression (Table S5) identified data type, CT sites, and radiation dose contributing to heterogeneity for cancer risk, with no factors substantially reducing heterogeneity between studies.The meta-analysis presents novel and complete evidence for the common and important issue concerned by clinicians, patients and the public, i.e., exposed CT scans and pertinent cancer risks for adults in their remaining lives. Based on 2,049,943 actual and 109,600,000 LAR/LBR participants from 9 countries, cancer risks for adults undergoing CT scans were successfully identified. Importantly, studies using LAR have been neglected in previous synthesized reports; partly due to the paucity of an appropriate control/unexposed population in quantify OR for these studies.Indeed, selecting a control group (LBR) for LAR studies was challenging for such meta-analysis with global included studies. Participants unexposed to CT scans receive cosmic radiation (background radiation) in the earth, with various radiation dose range in different regions . When baSo far, no studies have quantified the cancer risk of low-dose radiation. One of the most important reasons is insufficient sample size. According to the evaluation of a total of 1000 mSv for a sample of 1000 people, the sample size needed to evaluate 100 mSv would be 100,000, 10 mSv would be 10 million . In thisAmple evidence from Japanese atomic bomb survivors indicates that children are more susceptible than adults to the deleterious effects of ionizing radiation . ChildreIn 2007, the International Commission on Radiological Protection (ICPR) published a guide to the basic principles of appropriate radiation Protection based on radiation exposure. Since then, almost all international standards and national regulations dealing with radiation protection have been based on the recommendations of the Committee. Consequently, 2007 should be a milestone for CT scan in terms of dose reduction. Even in this study, no significant difference was found between radiation and cancer risk compared with studies published before or after 2007. What is clear is that researchers will adjust their protective strategies to a higher level in the plethora of exposure situations, as ICRP itself states.Firstly, There are various confounders for cancer incidence , includiBased on 111.6 million adult participants from 3 continents , this meta-analysis identifies an inordinately increase in cancer risks from CT scans for adults . Moreover, the cancer risks were positively correlated with radiation dose and CT sites. The meta-analysis highlights the awareness of potential cancer risks of CT scans as well as more reasonable methodology to quantify cancer risks in terms of life expectancy as 100 years for LAR.Additional file 1 Table S1. Search strategy and resultsAdditional file 2 Table S2. Contacting authors for additional informationAdditional file 3 Table S3A. Newcastle-Ottawa Quality Assessment Scale – Case–control studies. (.docx). Table S3B. Newcastle-Ottawa Quality Assessment Scale – Cohort studiesAdditional file 4 Table S4. Ionizing radiation and cancer risks sensitivity analysisAdditional file 5 Table S5. Meta regression analysisAdditional file 6 Fig. S1. Bubble plot depicting the relationship between “dose” as radiation to CT scans and “response” as cancer risksAdditional file 7 Fig. S2. Forest plot of cancer risk at different doses from CT scansAdditional file 8 Fig. S3. Forest plot of cancer risks at radiation doses from CT radiation exposure in age groups Additional file 9 Video. The association of cumulative ionizing radiation from CT scans with pertinent cancer risks in adults. (.mp4)


This study aimed to evaluate the impact of anastomotic leakage (AL) on oncological outcomes after restorative rectal cancer surgery.Patients who underwent anterior resection for rectal adenocarcinoma between January 2011 and December 2017 were retrospectively reviewed. Data were collected from three colorectal surgery centers. Patients with grade B and C leaks according to the International Study Group of Rectal Cancer classification were identified and compared with the control group. Estimated recurrence and survival rates were compared using the log-rank method and Cox regression analysis.p < 0.001). However, distant recurrence rates were similar  between the groups. Kaplan-Meier curves showed that patients with AL had a reduced 5-years local recurrence-free survival . AL  and node involvement  were significantly associated with LR in multivariate analysis. AL was significantly associated with DFS only at univariate analysis , with a difference between 5-year DFS of patients with and without AL . Only male gender, pT3-4 stage, and node involvement were identified as independent prognostic factors for reduced DFS in the multivariate Cox regression analysis.A total of 367 patients were included in the study, with a mean follow-up of 59.21 months. AL occurred in 64 patients (17.4%). Fifteen patients with AL (23.5%) developed local recurrence (LR) compared to 17 (4.8%) in the control group (In this cohort of patients, AL was associated with a significant risk of LR after rectal cancer surgery. Total mesorectal excision (TME), described by Heald , 2, decrHowever, radical surgery is associated with a high risk of perioperative complications, permanent stoma, and functional impairment . AlthougThis study aimed to investigate the impact of AL on the recurrence and survival of patients undergoing sphincter-preserving surgery for rectal cancer.We conducted a retrospective review to identify all patients who underwent restorative anterior rectal resection for adenocarcinoma at three different colorectal surgery centers from January 2011 to December 2017.We included patients with histologically proven primary rectal tumors located within 15 cm of the anal verge who underwent surgery using an open or minimally invasive approach. Patients who did not meet the inclusion criteria, such as those treated in emergency settings or for palliative purposes, those who underwent rectal surgery for benign pathologies, those who had no restorative surgery , those who underwent trans-anal TME or local excision with trans-anal endoscopic microsurgery (TEM) and patients lost to follow-up, were excluded.The disease in all patients was staged using pelvic MRI, and chest and abdominal CT scans. Neoadjuvant chemoradiotherapy was performed for locally advanced mid–low rectal tumors (T3-4 and/or N+) followed by TME. For upper third rectal cancer, a mesorectal excision was performed by resecting from at least 5 cm below the distal margin of the tumor.Baseline patient characteristics and cancer-related and operative data were collected from each participating center and successively merged in a comprehensive anonymized database.Anastomotic leak was defined and classified according to the International Study Group of Rectal Cancer criteria . Grade AOncological outcomes included disease-free survival (DFS), local recurrence (LR), and distant recurrence (DR), defined as the presence of a histopathologically proven or high radiological suspicion of tumor in the pelvis and outside the pelvis, respectively. Patients were followed-up every 3–6 months for the first 2 years after surgery and then every 6 months for a total of 5 years. CT scans of the thorax, abdomen, and pelvis; serum markers; and colonoscopy were performed according to the guidelines . Lost toχ2 test with or without Yates correction was performed. Differences between means were compared using the Mann-Whitney U test and Student’s t-test. Univariate and multivariate forward stepwise logistic regression model (minimum AIC) were performed considering local recurrence as binary dependent variable. Survival time data of local recurrence and DFS were estimated using the Kaplan-Meier method and differences were analyzed using the log-rank Mantel-Cox test. Multivariate analysis of DFS was then performed using Cox logistic regression model. Only variables with p value <0.2 at univariate analysis were entered in multivariate models. The results were reported as Odds Ratio (OR) or Hazard Ratio (HR) with 95% confidence intervals, when appropriated. Statistical significance was set at p < 0.05.Statistical analysis was performed using SPSS version 26 software  for Windows and StataCorp (2019) Stata Statistical Software Release 16 . First, data normality was tested using the Shapiro-Wilk or Kolmogorov-Smirnov tests. Data and counts for dichotomous variables were presented as frequencies. Continuous data were presented as mean ± one standard deviation (SD) or as median and interquartile (25%–75%) or minimum–maximum range. To compare differences in frequencies, Fisher's exact test or the In total, 419 patients underwent restorative rectal cancer surgery between January 2011 and December 2017. The study population included 367 patients because 52 patients (12.4%) were lost to follow up.p = 0.007), and the pathological T stage was more advanced (The baseline patient characteristics and perioperative details are described in advanced .p < 0.001). However, DR rates were similar between the groups .The oncological outcomes are shown in p < 0.001) and node involvement  were significantly associated with LR in multivariate analysis. . Kaplan-Meier curves showed that patients with AL had a reduced 5-year LRFS  (The median follow-up was 60.18 (37.10–77.18) months in the AL group and 58.12 (38.72–77.22) months in the control group (< 0.001) .p = 0.037) but this was not confirmed at multivariate analysis (p = 0.04) when compared to that of the control group  and decreased cancer-specific survival  in patients with AL. Additionally, AL did not increase DR , as we demonstrated in the present study. The association of AL with LR after rectal resection was also confirmed in the most recent similar systematic reviews (p = 0.005) and a decrease in DFS  at the 5-year follow-up were found in patients with AL. Similar to our results, AL was not a significant predictor of DR .Despite the evidence suggested by the reports of these influential institutions, our results are consistent with those of a recent meta-analysis involving 11,353 patients . Only st reviews , 19 and  reviews , 20. Fin reviews , where ain vitro and may therefore affect tumor recurrence to the peritoneum in vivo (The mechanism by which AL increases LR after rectal cancer surgery remains unclear. Postoperative sepsis may induce an inflammatory response. Some data suggest that the systemic inflammatory response participates in the progression of metastatic disease in patients with colorectal cancer . The rel in vivo . Further in vivo . Therefo in vivo , 27.Otherwise, AL might lead to local implantation of viable cancer cells at the anastomotic site at the time of surgery , 29. FinOn the other hand, risk factors for AL such as male gender, obesity, previous radiotherapy and T stage are well established , 31. We Although we found an AL rate of 17.4%, which is slightly higher than the 9–11% published elsewhere , 33, 34,This study has some limitations. The data were retrospectively collected, which has the risk of patient selection bias. We only included patients with grade B and C leaks because routine postoperative imaging was not performed to detect asymptomatic leaks. Furthermore, the diverting stoma was performed according to the surgeon's preference and no details regarding adjuvant chemotherapy or other phatological features were provided.Anastomotic leakage contributes to adverse oncologic outcomes such as LR after restorative rectal cancer surgery. Therefore, prevention to minimize the risk is essential, and careful surveillance and tailored oncologic assessments should be considered.


Efforts have been made to improve the risk stratification model for patients with diffuse large B-cell lymphoma (DLBCL). This study aimed to evaluate the disease prognosis using machine learning models with iterated cross validation (CV) method. A total of 122 patients with pathologically confirmed DLBCL and receiving rituximab-containing chemotherapy were enrolled. Contributions of clinical, laboratory, and metabolic imaging parameters from fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) scans to the prognosis were evaluated using five regression models, namely logistic regression, random forest, support vector classifier (SVC), deep neural network (DNN), and fuzzy neural network models. Binary classification predictions for 3-year progression free survival (PFS) and 3-year overall survival (OS) were conducted. The 10-iterated fivefold CV with shuffling process was conducted to predict the capability of learning machines. The median PFS and OS were 41.0 and 43.6 months, respectively. Two indicators were found to be independent predictors for prognosis: international prognostic index and total metabolic tumor volume (MTVsum) from FDG PET/CT. For PFS, SVC and DNN (both with accuracy 71%) have the best predictive results, of which outperformed other algorithms. For OS, the DNN has the best predictive result (accuracy 76%). Using clinical and metabolic parameters as input variables, the machine learning methods with iterated CV method add the predictive values for PFS and OS evaluation in DLBCL patients. The age-standardized rate  for male and female in Taiwan was only 3.44 and 2.54, respectively1. The causes of DLBCL are not well understood. The DLBCL can arise in any part of the body and is often an aggressive type of malignancy. Although it usually arises from the normal B lymphocytes, it can also represent a malignant transformation from other types of B-cell lymphoma. Typically, the first sign of this disease is the rapidly growing tissue or mass sometimes associated with the B symptoms . The diagnosis of DLBCL is made pathologically of the biopsied tissue. There are several subtypes of DLBCL identified which differ in their clinical presentations, symptoms, aggressiveness, morphology, immunophenotypes, gene expression, and the prognosis2. Because of its aggressiveness with complex outcomes due to the heterogeneous entity, complete pretreatment evaluation including prognostic classification is important for the disease management.Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma and accounts for approximately one-third of non-Hodgkin’s lymphoma3.In the past 20 years, the international prognostic index (IPI) has been one of the most useful tools to evaluate the prognosis in patients with aggressive NHL including DLBCL. In the evaluation of IPI, one point is assigned for each of the following risk factors: (1) age over 60 years, (2) Stage III or IV disease, (3) elevated level of serum lactate dehydrogenase (LDH), (4) Eastern Cooperative Oncology Group (ECOG) performance status of 2–4, and (5) more than 1 extra-nodal site involvement of disease . The 5-year overall survival (OS) for low risk (IPI: 0–1), low-intermediate risk (IPI: 2), high-intermediate risk (IPI: 3), and high risk (IPI: 4–5) is 73%, 51%, 43%, and 26%, respectively4. However, patients with the same IPI score may still suffer from different outcomes either due to early relapse or refractory disease. Great efforts have been made to improve the evaluation models for risk stratification8, and more reliable prognostic predictors are pressingly needed to differentiate patients who are more likely to have poorer outcome9. Further information gained from different imaging modalities to build a more reliable prediction model for clinical practice is helpful and valuable.Since the addition of rituximab  into the first-line chemotherapy  (R-CHOP), the 5-year survival rate of DLBCL has been improved12. It has been reported in the literature that there are a variety of quantitative parameters deriving from image which have potential utility to predict prognosis or treatment outcome. The standardized uptake value (SUV) is the most commonly used parameter in FDG PET/CT and has been proved to be a significant prognostic predictor in DLBCL14. The percentage change in maximal SUV (SUVmax) between initial and delayed phase images is defined as the retention index (RI) which is found to have significant prognostic potential to predict overall survival (OS) in patient with DLBCL15. Beyond SUV and RI, the total metabolic tumor volume (MTVsum) has also been shown to be a predictor for survival outcome in many previous studies19. It was also reported that an elevated MTVsum, independent from IPI, is a predictor for shorter progression-free survival (PFS) and OS in patients with DLBCL20.As stated above, pretreatment staging is crucial. The fluorine-18 fluorodeoxyglucose (FDG), a glucose analog, can be used to measure the degree of glucose utilization. The uptake degree of FDG detected by positron emission tomography/computed tomography (PET/CT) represents the tissue metabolism on the whole-body and functional images. The FDG PET/CT has been widely used in pretreatment staging of disease and assessment of treatment response for patients diagnosed with DLBCL23.Learning is the process of behavior improvement over time via discovering new information. If the referred process is achieved by machine rather than human brain, it is called machine learning. The experience acquiring from the existing examples helps to find the optimal solution for coming problems in the machine learning process. Due to rapid accumulation of larger and larger raw data, the traditional methods cannot handle well and the big data concept emergences over time with the development of information technologies. Machine learning in which algorithms were used by the computers with a certain order when performing operations is a subset of artificial intelligence. Based on the training data, machine learning algorithms can search for optimal connection weights of a prespecified neural network model in order to make decisions and predictions. Some machine learning models, in which sophisticated mechanism such as high-order and non-linear interactions between predictors and the responses were used, have shown ability to improve overall clinical prediction in various conditions29. However, there are few reports, in which using modern machine learning models and incorporating FDG PET/CT metabolic imaging parameters, to perform the outcome prediction in DLBCL.Nonetheless, due to the heterogenous entity of DLBCL, the prognostic parameters of DLBCL, the total effects of these parameters, and individual weight of each parameter remain to be an issue deserving further research. Previous studies have reported that machine learning algorithms, using either molecular profiling data or combined clinical and genetic data, helped to do disease classification, diagnosis, and prognosis predictionTherefore, this study aims to develop the logistic and neural network models to predict DLBCL clinical outcome based on PFS and OS. Both clinical and metabolic parameters from FDG PET/CT scans were used as predictors. The performance of the models is also compared.This is a retrospective study in which medical records of malignant lymphoma patients diagnosed at the Kaohsiung Medical University Hospital were reviewed. The Institutional Review Board of Kaohsiung Medical University Hospital approved the reviewing process of the clinical data (KMUHIRB-E(I)-20180275). Patient consent was waived because all the clinical data were retrospectively collected via the medical chart reviewing, and the waiver for subject informed consent was also approved by the Institutional Review Board of Kaohsiung Medical University Hospital. The inclusion criteria were recent diagnosis and histologically proven DLBCL. The patients had to be 18 years old or older without known previous or concurrent malignant disease. All patients underwent a complete pre-treatment work-up, including whole-body FDG PET/CT scan, bone marrow biopsy, clinical history, physical examination, and standard laboratory tests. All methods were carried out in accordance with relevant guidelines and regulations. The definite diagnosis was made by experienced pathologists based on the World Health Organization classification of lymphoid neoplasms in 2016. Only the “diffuse large B-cell lymphoma, NOS” type were enrolled. Patients’ clinical stage patient was determined with the Ann Arbor staging system and was achieved by the multi-disciplinary consensus by experts in various professional fields including radiology, nuclear medicine, pathology, and hematology.All patients received 4–6 cycles of rituximab-containing chemotherapy, that is, rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP), as an initial therapy. Involved field radiation therapy was administered for patients with initial clinical bulky disease or residual tumor presentation after completion of the chemotherapy. Patients experiencing refractory or relapsed disease were treated with salvage chemotherapy or received autologous stem cell transplantation (ASCT) with high-dose chemotherapy if clinically indicated. The therapeutic regimens and plans followed the National Comprehensive Cancer Network (NCCN) guidelines for B-cell lymphoma according to the year when patient was diagnosed, and the detail was decided with consensus in the multi-disciplinarily combined conference of lymphoma in accordance with the patient’s clinical condition. The PFS was defined as the time from diagnosis to disease relapse, progression or death related to lymphoma. The OS was defined as the time from diagnosis to death from any cause.We collected available information  during the pre-treatment workup as the input variables for the machine learning models. The output is a binary label regarding patient clinical outcome at the 3-year timepoint. That is, the output for PFS indicates whether the patient relapse or progresses after 3 years from the date of diagnosis; and for OS, whether the patient remained alive after 3 years from the date of diagnosis. This is a binary classification problem.In this study, we used the following machine learning techniques to create prediction models: (1) logistic regression (LR), (2) random forest (RF), (3) support vector classifier (SVC), (4) deep neural network (DNN), and (5) fuzzy neural network (FNN).30. So far, however, it has been rarely used in the field of cancer prediction and prognosis. Here, scikit-learn package (0.23.2) was carried to conduct the random forest model and the SVC with regularization parameter C = 1.0 and radial basis function kernel. Fourth, DNN model is a learning machine composed of multiple processing layers including many intermediate hidden layers. Each hidden unit in the hidden layer composes of the non-linear activation functions that are transformed from linear combination of predictors31. In the current study, four-layer feedforward model with adaptive moment estimation optimizer, rectified linear unit (Relu) activation function for hidden layers, and sigmoid activation function for output layer conducting with Keras package (2.3.1) was used. The model of DNN with architecture of 2-6-4-1 was structured and a total of 51 parameters were developed. Lastly, an FNN is intrinsically a fuzzy system represented as a neural network. Suppose the fuzzy rules in the rule base are given in the form as shown below, where x is the input, y is the output, and A, B are fuzzy sets:If Then First, we fitted an LR model using scikit-learn package (version 0.23.2) for Python (3.7.9). After processing, the prediction outcomes as well as the confusion matrix were computed. Second, the RF model, which is an ensemble of decision trees with bootstrapped training samples for tree induction, was adopted. In this study, the parameter setting for number of trees was 100. Third, SVC is a part of the support vector machine (SVM), which is one of the most robust prediction models. The SVC algorithm formally creates a hyperplane in which the data can be separated into two classes with the maximal soft margin. The SVC has been used in a broad range of classification and pattern recognition problems ranging from speech, text recognition, protein function prediction, and handwriting analysisIn this fuzzy system, we specifically use the singleton fuzzifier, product inference engine, center-average defuzzifier, and Gaussian membership functions for the fuzzy sets. Then the fuzzy system can be configured as a neural network, called an FNN. We implemented the FNN using Tensorflow (1.15.4) as a neural network layer with a total of 64 parameters. The source code for defining the FNN layer is provided in the supplementary file.For better and reliable estimation of the predicting capability of various learning machines, we used 10-iterated fivefold cross validation (CV) with shuffling process. Fivefold CV was conducted and the whole process was then repeated 10 times, resulting in 50 testing results. Then the mean and standard deviation on the 50 testing errors was calculated to evaluate the predicting power of each model. According to the 50 testing results, the confusion matrix results were subsequently computed. The F1 values were also calculated to compare the prediction ability.https://www.medcalc.org; 2021) was used to perform Kaplan–Meier survival analysis, Cox proportional hazard model, and area under the curve (AUC) index for receiver operator characteristic (ROC). A p < 0.05 was considered statistically significant.MedCalc Statistical Software version 20.014  female and 65 (66.6%) males, were included in the study, with the mean age 61.3 ± 17.0 years , hemoglobin (Hb), and platelet count], liver function test , albumin, creatinine, β2-microglobulin, and LDH. The metabolic parameters from FDG PET/CT imaging were also obtained. The mean value of maximal SUV of tumor (SUVt) and maximal SUV of biopsied site (SUVb) was 16.12 ± 8.25 and 14.36 ± 8.77 respectively. The mean values of MTVsum and total lesion glycolysis (TLG) were 664.48 ± 968.67 cmThe survival analysis revealed the clinical outcome shows in Fig. 3 . The Kaplan–Meier survival analysis revealed patient with MTVsum ≥ 165.40 had a significantly poor PFS  revealed to be significantly correlated with the patients’ PFS. The multivariate analysis further disclosed that IPI and MTVsum were independent prognostic factors for PFS Table . The cutFor subsequent machine learning applications, we used IPI and MTVsum as the input predictors. The IPI is the international prognostic index clinically used for lymphoma, which is an integer from 0 to 5. The MTVsum is the total metabolic tumor volumes of whole-body lesions, which is the summation of whole-body tumor volumes when they showed abnormal FDG uptake. The MTVsum is a positive real number. The output is a binary label regarding patient clinical outcome  at the 3-year timepoint.The dataset is partitioned into training set and testing set. We use the training set to train the model. Then, we use the testing set to evaluate the performance of the model. The evaluation is based on commonly used statistical indicators given below:For fivefold CV, the whole dataset is randomly divided into 5 disjoint sets. In each iteration of the fivefold CV, one set is specified as the testing data and the rest are training set. After the training process using training data, the pre-specified metric or performance index  is computed on the testing data. This training/testing process is performed for 5 times producing 5 testing results. The whole process is repeated 10 times constituting the so called 10-iterated fivefold CV and 50 testing results of the statistical indicators are obtained. Finally, we calculate the means and standard deviations of the 50 testing results serving as the performance of the model.p = 0.0425; DNN vs. LR, p = 0.0140) and FNN  models. No statistical significance toward RF was noted (p = 0.4131). The results of the algorithms used are summarized in Table Using IPI and MTVsum as input predictors, the machine learning algorithms used namely LR, RF, SVC, DNN and FNN, had an accuracy of 0.66 ± 0.10, 0.69 ± 0.14, 0.71 ± 0.14, 0.71 ± 0.10, and 0.67 ± 0.08 respectively. The specificity was 0.74 ± 0.04, 0.76 ± 0.12, 0.84 ± 0.12, 0.77 ± 0.11, and 0.79 ± 0.14 respectively. The AUC index for ROC of these predictive models were 0.66 ± 0.10, 0.70 ± 0.13, 0.71 ± 0.13, 0.69 ± 0.09, 0.66 ± 0.10, respectively. Among these five models, SVC (0.71 ± 0.14) and DNN (0.71 ± 0.1) had the highest prediction accuracy and are statistically significant in comparison with the LR . The Kaplan–Meier survival analysis revealed patient with MTVsum ≥ 190.19 had a significantly poor OS  were significantly correlated with the patients’ OS. Further multivariate analysis disclosed that IPI and MTVsum were independent prognostic factors for OS Table . The cutUsing IPI and MTVsum as input predictors for the machine learning algorithms, the predictive accuracy showed 0.73 ± 0.05 for LR, 0.72 ± 0.12 for RF, 0.75 ± 0.13 for SVC, 0.76 ± 0.10 for DNN, and 0.75 ± 0.09 for FNN respectively 46. It is believed that machine learning methods become an alternative means for handling complex and large data sets and for model generation47.As a subspecialty of artificial intelligence, machine learning denotes the development of algorithms obtaining parameters and models optimally representing the available data. There are two parts in the learning process, (1) estimating the unknown parameters from a known data set in a system, and (2) predicting new outputs of the system using the weights of these parameters. Nowadays, the amount of information to be interpreted and examined when predicting the malignant disease prognosis has been increasing rapidly. The evidence-based medicine is based on randomized controlled trials in which large patient populations and clinical data is handled. In the future, the clinical trials can be better conducted with machine learning approaches, and thus new findings can be obtained using the available data more easily. The machine learning-based clinical decision support systems, in recent years, have been emerging in dealing with certain clinical situations29. Another paper used the hybrid machine learning approach, more specifically, both clinical and genomic date to create a single classifier to predict patient outcomes in DLBCL27. Ando et al.25 conducted the FNN model and 4 genes transcriptional profiling data to predict lymphoma survival, yielding an accuracy of 73.4% in comparison with the 68.5% accuracy using the Cox model and 17 genes. Another article by Ando et al.26 reported the FNN model as a powerful tool for extracting significant biological markers affecting prognosis in DLBCL. Biccler et al.48 collected patients from the Swedish and Danish cohorts and developed a new prognostic model based on machine learning approaches which outperformed known prognostic predictors for patients with DLBCL.The literature review disclosed some studies evaluating patient survival in DLBCL using machine learning models. Shipp et al. reported the successful prediction of patient OS via supervised machine learning algorithms dealing with oligonucleotide microarray gene-expression data49 established a tumor microenvironment (TME) related prognostic signature for DLBCL patients. When combining with IPI components, it is a promising prognostic model not only to help clarifying immune responses in the DLBCL microenvironment but also to indicate new clinical applications for immune therapy and individualized therapy in patients with DLBCL. In the current study, we incorporated both clinical  and the metabolic image parameter from FDG PET/CT scans  as the prognostic predictors. Additionally, both 3-year PFS and OS evaluations for 122 patients with DLBCL were performed using 5 different learning machines. For PFS, SVC and DNN had the highest survival estimation with a prediction accuracy of 71%. For OS, the DNN model has the highest accuracy for the prediction estimation (76%), and the FNN and SVC models have accuracy of 75%. The results are similar with that reported by Ando et al.25.In the similar study published in 2021, Pan et al.Although the current study was dealing with a relatively small patient population, we tried to develop 5 machine learning models with iterated CV method to predict clinical outcomes in patients with DLBCL. The machine learning methods may add the values for the survival prediction. It allowed clinicians to pay more attention to the follow-up and therapeutic strategies if high-risk patients were early identified. In the future, we hope that the modern predictive modeling approaches can be applied rather than barely using clinically available dichotomized variables or risk scores to predict patient survival. Further studies dealing with prospective design, larger patient population, and more specific histological subtypes based on different molecular or genetic presentations, may be conducted. Moreover, the prognostic modeling can be grounded on a combination of clinical, pathological, molecular, and metabolic imaging information.In the current study using machine learning algorithms with iterated CV method in patients with DLBCL, the best results for PFS were obtained with SVC and DNN techniques which outperform LR, RF, and FNN methods. For OS evaluation, the best results were obtained with DNN technique outperforming FNN, SVC, LR, and RF methods.Supplementary Information 1.Supplementary Information 2.


All patients were diagnosed and/or treated for melanoma at the Veneto Oncological Institute and the University Hospital of Padua  in 1998–2014. Patients were categorized in exposed or unexposed groups according to their home address and the geographical classification of municipalities affected by PFAS contamination as provided by Veneto Government in 2018. Presence of mitoses was found in 70.5% of exposed patients and 58.7% of unexposed patients (p = 0.005). Median follow-up was 90 months (IQR 59–136). 5-year overall survival was 83.7% in exposed patients and 88.0% in unexposed patients (p = 0.20); 5-year disease-specific survival was 88.0% in exposed patients and 90.9% in unexposed patients (p = 0.50); 5-year disease-free survival was 83.8% in exposed patients and 87.3% in unexposed patients (p = 0.20). Adjusting for imbalanced characteristics at baseline (presence of mitoses), survival was not statistically different between exposed and unexposed patients . Although the magnitude of PFAS exposure was not quantifiable, our findings suggested that exposure to PFAS was associated with higher level of mitosis in melanoma patients, but this did not translate into a survival difference. Further studies are required to investigate this relationship and all effects of PFAS on prognosis.Per- and polyfluoroalkyl substances (PFAS) are endocrine disrupting chemicals which could be associated with cancer development, such as kidney and testicular cancers, pancreatic and hepatocellular carcinoma and thyroid tumor. Available scientific literature offers no information on the role of PFAS in melanoma development/progression. Since 1965, a massive environmental contamination by PFAS has occurred in northeastern Italy. This study compared histopathology and prognosis between melanoma patients exposed ( The incidence of melanoma is continuously increasing in both adult and pediatric populations around the world, with a faster pace compared to other malignancy , 2. The This was a retrospective cohort study on PFAS exposure in patients who were diagnosed and /or treated for primary melanoma at Veneto Institute of Oncology and at the University Hospital of Padua between 1998 and 2014.All patients who were diagnosed and/or treated for primary melanoma in 1998–2014 at Veneto Institute of Oncology and at the University Hospital of Padua  were considered for inclusion in this study. The two participating hospitals are level III referral centers located in the Veneto Region . Most patients are referred for diagnosis and/or first-line treatment, while some patients are referred for disease progression after being treated in local level II centers. Main inclusion criteria were age ≥ 15 years and living in the Veneto Region. In 2018, the regional administration produced a document (no. 691 of 21 May 2018) where regional areas were classified according to PFAS contamination . In our All melanoma diagnoses were histologically confirmed according to the fourth edition of the World Health Organization classification of skin tumors and the staging was updated to the 8th edition of the Union for International Cancer Control (UICC) TNM Classification of Malignant Tumours , 14. TheAll data were extracted from a local database. Data collection included demographics , tumor characteristics  and follow-up information. Follow-up data were extracted from scheduled visits. Overall survival (OS) was calculated from date of diagnosis to date of death or last visit. Disease-specific survival (DSS) was calculated from date of diagnosis to date of disease-related death, or date of last visit/disease-unrelated death. Disease-free survival (DFS) was calculated from date of diagnosis to date of recurrence, or date of last visit/death. Recurrence could include local recurrence, regional skin/in-transit metastases, regional lymph node metastases and/or distant metastases.Continuous data were summarized as median and interquartile range (IQR). Comparisons between exposed and unexposed cohorts were performed using the Chi Square test or Fisher’s exact test , and Mann–Whitney test (continuous data). A logistic regression model was estimated to assess the relationship between PFAS exposure and presence of mitoses, adjusting for age and sex. Survival estimates were calculated using the Kaplan–Meier method and compared between exposed and unexposed cohorts using log-rank test  and Cox regression models with unbalanced baseline characteristics as additional independent variables . Effect sizes were reported as odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI). No adjustment for multiple testing was applied given the exploratory purpose of the study. All tests were two-sided and a p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using R 4.1  .The study was approved by the Ethics Committee of the Veneto Institute of Oncology (CESC-IOV) on 20 January 2020 . The study was conducted according to Helsinki Declaration principles, and all patients gave their consent to have their anonymized data used for scientific purposes.p = 0.005; Tablep = 0.005). No other statistically significant differences were found in the two cohorts  and 488 melanoma patients living outside the PFAS areas (“unexposed cohort). All patients were Caucasian and aged > 15 years. Demographics and tumor characteristics are reported in Table p = 0.20); 5-year DSS was 88.0% in exposed patients and 90.9% in unexposed patients (p = 0.50); 5-year DFS was 83.8% in exposed patients and 87.3% in unexposed patients (p = 0.20) , while higher number of mitoses per mm2 was associated with impaired OS , DSS  and DFS .At a median follow-up of 90 months (IQR 59–136), overall 135 patients died (84 from the disease and 51 due to other causes) and 542 were alive, while the information was not available in five patients who were lost to follow-up. 5-year OS was 83.7% in exposed patients and 88.0% in unexposed patients (In the last decade, the contamination of water by PFAS has gained increasing interest in medical research. Previous studies showed an association between PFAS exposure and testicular Leydig cell adenomas, pancreatic and hepatocellular carcinoma or thyroid tumor in the rats , 17, 18,