Current methods for cervical cancer screening result in an increased number of referrals and unnecessary diagnostic procedures. This study aimed to develop and evaluate a more accurate model for cervical cancer screening.N = 1085), cervical intraepithelial neoplasia , and cervical cancer (N = 551) to predict CIN2+ or CIN3+. A base model using age, cytology, and hrHPV was calculated, and extended versions with additional biomarkers were considered. External validations in two screening cohorts with 3-year follow-up were further conducted .Multiple predictors including age, cytology, high-risk human papillomavirus (hrHPV) DNA/mRNA, E6 oncoprotein, HPV genotyping, and p16/Ki-67 were used for model construction in a cross-sectional population including women with normal cervix  and reduced colposcopy referral rates  compared to hrHPV and cytology co-testing in the cross-sectional population  to predict CIN2+. The AUC further improved when HPV genotyping and/or E6 oncoprotein were included in the base model. External validation in two screening cohorts further demonstrated that our models had better clinical performances than routine screening methods, yielded AUCs of 0.92 (95% CI = 0.91–0.93) and 0.94 (95% CI = 0.91–0.97) to predict CIN2+ and referrals rates of 17.55% (95% CI = 16.24–18.92) and 7.40% (95% CI = 6.50–8.38) in screening cohort I and II, respectively. Similar results were observed for CIN3+ prediction.Compared to routine screening methods, our model using current cervical screening indicators can improve the clinical performance and reduce referral rates.The online version contains supplementary material available at 10.1186/s12916-021-02078-2. Cervical cancer is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women, with an estimated 570,000 new cases and 311,000 deaths in 2018 worldwide [The decision for modern cervical cancer screening programs is often made based on age, cytology, and hrHPV testing results. For example, the USA has different cervical cancer screening guidelines for women in different age groups. For women aged 30 to 65 years, guidelines recommend the use of cytology and hrHPV co-testing due to its high sensitivity. HPV testing has not been recommended in women aged 21–29 without atypical squamous cells of undetermined significance (ASC-US) due to its low specificity . HoweverCervical intraepithelial neoplasia grade 2/3 (CIN2/3) can progress to cervical cancer if left untreated, and therefore identifying women who would benefit from further monitoring and/or treatment is important . HoweverIn recent years, machine learning methods play an important role in selecting an appropriate combination of multiple biomarkers. With the increasing availability of large national databases and computing power, the use of machine learning methods in medical science and health care has been rapidly growing , 13. StuThe aim of this study was to develop models that have better prediction of CIN2+ by using age, cytology, and hrHPV testing, with or without other biomarkers. Models were constructed and evaluated in a cross-sectional population enriched with CIN and cervical cancer. External validation in two screening cohorts was further presented to demonstrate the usefulness of our methods.This study included three populations, one cross-sectional population and two screening cohorts. Women were eligible if they had an intact cervix and no prior history of CIN. Women eligible for the screening cohorts were additionally aged 25 to 65. Women who were pregnant, had a hysterectomy, or received treatment for cervical diseases were excluded. Further details on the study design are provided in Fig. Participants were recruited from five hospitals in China between 2014 and 2015 and included women attending routine cervical cancer screening programs, outpatients referred for colposcopy, and inpatients planning treatment for CIN2+. A questionnaire was used to collect information on demographic factors and obstetrics and gynecology history. Two cervical exfoliated cell samples were collected: one was kept in PreservCyt Solution (Hologic) and aliquoted for cobas HPV (Roche), Aptima HPV (Hologic), Onclarity HPV (BD Diagnostics) testing, p16/Ki-67 dual staining (Roche), and liquid-based cytology (LBC) assessment and the other sample was kept in a Dacron swab for HPV16/18 E6 protein detection (Arbor Vita Corporation). Cervical biopsies were conducted using a protocol as previously described [Both screening cohorts included a baseline phase and a 3-year follow-up phase. Participants in the screening cohort I (SC-I) were recruited from Shanxi Province of China between 2017 and 2020. At baseline, all participants received Aptima HPV, INNO-LiPA HPV genotyping (Innogenetics), and LBC. Aptima HPV positive samples were tested by Aptima HPV16/18/45. Women with HPV16/18/45 positive or abnormal cervical cytology (ASC-US+) were referred for colposcopy and women with HPV16/18/45 results had an additional swab for E6 oncoprotein test collected before colposcopy.Participants in the screening cohort II (SC-II) were recruited from the Inner Mongolia Autonomous Region of China between 2016 and 2019. At baseline, all participants received cobas HPV, INNO-LiPA HPV genotyping, and LBC. Women with HPV16/18 positive or ASC-US+ were referred for colposcopy.For both screening cohorts, women who were HPV positive or had an ASC-US+ cytology continued to annual follow-up visits, and all women regardless the results at baseline came back at the 3rd year for a final visit. At each visit, a LBC specimen was obtained and women with ASC-US+ were referred for colposcopy. Women found to have a diagnosis of CIN2+ at baseline or follow-up exited the study after the colposcopy visit and were referred for treatment.The Onclarity HPV is a PCR assay for the detection of six individual HPV genotypes  and three groups of types . The cobas HPV is another PCR assay for the detection of viral DNA of the 14 hrHPV types, which simultaneously differentiates HPV16 and HPV18. The Aptima HPV is based on the qualitative detection of E6/E7 mRNA of 14 hrHPV types. The Aptima HPV16/18/45 uses the same technology as Aptima HPV for detection of E6/E7 mRNA from HPV16/18/45; the assay differentiates genotype 16 from 18 and 45 but does not differentiate between 18 and 45. INNO-LiPA HPV genotyping assay allows simultaneous and separate detection of 25 different HPV genotypes . All HPV tests were performed at the fully automated system according to the manufacturer’s instructions. The OncoE6 cervical test is an immunochromatographic test for the detection of HPV16/18 E6 oncoprotein. The operation procedures were described previously .Cytology slides were first evaluated by junior cytologists and then diagnosed by senior cytologists. Results were reported using the Bethesda 2014 nomenclature. A second cytology slide was prepared from the residual PreservCyt Solution for p16/Ki-67 dual staining using the CINtecPLUS Cytology kit according to the manufacturer’s instructions for the cross-sectional samples. Technicians were blinded to each other’s findings to minimize bias.Based models of logistic regression and SVM were implemented on the platform of R (Version 3.5.2). Model construction and internal validation were performed in the cross-sectional population, which was randomly split into 70% for a training set and 30% for a testing set.Logistic regression or SVM using age, cytology, and hrHPV as predictors was set as the base model. Among the predictors, age was a continuous covariate; hrHPV testing was dichotomous ; and cytology was a seven-level covariate: negative for intraepithelial lesion or malignancy (NILM), ASC-US, low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells cannot exclude high-grade lesion (ASC-H), atypical glandular cell (AGC), high-grade squamous intraepithelial lesion/adenocarcinoma in situ (HSIL/AIS), and squamous cell carcinoma/adenocarcinoma (SCC/ADC). HSIL and AIS, as well as SCC and ADC, were separately combined because limited cases were available for these levels. CIN2+ or CIN3+, the outcome of interest, was dichotomous. Receiver operating characteristic (ROC) curve (sensitivity and 1-specificity) and the area under the curve (AUC) were used to assess predictive accuracy. Sensitivity, specificity, and colposcopy referral rate were also calculated for current screening methods and models based on the thresholds with the largest Youden Index.α level of 0.05.The base model was extended by substituting hrHPV using different detection methods, i.e., the result of cobas was substituted by Aptima or Onclarity. Additional covariates were also added to the base model, including E6 oncoprotein , p16/Ki-67 , and HPV genotyping . AUCs were compared using the “pROC” package in R. Logistic regression or SVM, which one showed better clinical performance, was chosen for further analysis. Statistical significance was assessed by two-tailed tests with The base model and extended versions with HPV genotyping were applied to both screening cohorts. The extended models with E6 oncoprotein were applied to SC-I only because swab samples were not collected in SC-II. Cytology results diagnosed by junior and senior cytologists were also evaluated in models. Three-year cumulative risks of CIN2+ were estimated by hrHPV and cytology co-testing negative and predicted-negative populations.Table Results for the current screening methods and the proposed models for CIN2+ prediction are presented in Table For the current screening methods, the largest AUCs were obtained by ASC-US+  and HPV mRNA . For co-testing, the AUC was 0.80 (95% CI = 0.79–0.82). The base model showed to have slightly higher AUC compared to current screening methods using either ASC-US+ or hrHPV mRNA. In addition, the base model reduced the number of colposcopy referrals, with a referral rate of 42.76% (95% CI = 38.67–46.92%) compared to 61.62% (95% CI = 59.40–63.80%) by hrHPV and cytology co-testing, 48.25% (95% CI = 45.98–50.53%) by hrHPV mRNA, and 53.16% (95% CI = 50.89–55.41%) by ASC-US+. The referral rates of the base model were further reduced when additional predictors were used . As for SC-II, 28 CIN2+ cases were diagnosed during follow-up, with 11 cases predicted to be positive and 17 cases to be negative at baseline. Women with predicted-negative findings had higher 3-year risks of CIN2+ compared with women with co-test negative .Since the 3-year risk of CIN2+ was higher among women with negative results of the predictive model compared to co-testing, we changed the thresholds from the highest Youden Index to the highest sensitivity for base model prediction. Results for SC-I did not change, whereas SC-II yielded a sensitivity of 100%, specificity of 84.13% (95% CI = 82.88–85.42%), and colposcopy referral rate of 17.23% (95% CI = 15.91–18.61%), which also has a higher specificity and AUC, same sensitivity and lower colposcopy referral rate compared to co-testing at baseline. By using this threshold, 26 out of 28 follow-up CIN2+ cases were predicted to be positive at baseline and 2 cases were negative. Women with predicted-negative findings had slightly lower 3-year risks of CIN2+  compared with 0.09% (95% CI = 0.01–0.32%) of women with co-testing negative.In this study, we developed and evaluated machine learning-based models to predict CIN2+ or CIN3+ for cervical cancer screening. A logistic regression model using hrHPV, cytology, and age was set as the base model due to its superior performances in prediction and colposcopy referral rates reduction. Improved clinical performance of the base model can be gained by incorporating E6 oncoprotein and/or HPV genotyping information. External validation in two screening cohorts further demonstrated that our models had better clinical performances than routine screening methods. The 3-year risks of CIN2+ for the predicted-negative women depended on the thresholds of the model, but the improvement of clinical performance at baseline can be obtained whichever threshold was chosen.Different models were used for cervical cancer screening in previous studies. Karakitsos et al. used the learning vector quantizer neural network classifier on cytological diagnosis, HPV DNA test, E6/E7 HPV mRNA test, and p16 immunostaining to build an algorithm to facilitate the classification of CIN2+. This model improved the AUC (0.916) significantly compared to cytology diagnosis alone (0.866) . A KoreaN = 126). These results showed that additionally testing for the E6 oncoprotein in a limited group of people could yield better screening performance than the base model. In addition, the models recommended fewer women to receive immediate colposcopy compared to HPV and cytology testing alone or co-testing but had the same cost with co-testing in the real-world setting to collect HPV testing and cytology information, hence could reduce unnecessary diagnostic procedures and costs.The clinical performances of our extended models included HPV genotyping and/or E6 oncoprotein showed to be better than the base model in each of the study population assessed, but resulted in a slight increase in cost. HPV genotyping can be a byproduct of HPV testing that has little additional costs but more additional information. E6 oncoprotein is pivotal in initiation and maintenance of oncogenic transformation by HPV  and assoCytology diagnosis is subjective in nature, and its reproducibility and accuracy are affected by the cytologist’s skill . In our Our study demonstrated that machine learning could incorporate multiple screening methods into one algorithm and develop models by the current cervical cancer screening indicators, which has the potential to be a reliable screening method considering its better clinical performance and lower referral rate.Additional File 1:Table S1-S3.Table S1. – Logistic Regression Parameters. Table S2. – SVM Parameters. Table S3. – Clinical performance of current screening methods and models for cross-sectional population and screening cohorts at baseline (CIN3+)


Anal cancer is treated with high dose chemoradiotherapy; but despite this, a minor patient group experience treatment failure. Anal cancer is strongly related to human papilloma virus (HPV) infection and previous smaller studies have shown that fragments of HPV DNA can be detected in blood samples from anal cancer patients. This study measured HPV DNA in 88 patients including both small- and more advanced tumors and detected six different subtypes of HPV. During treatment with chemoradiotherapy, the level of HPV DNA decreased, and three elimination patterns with clinical relevance were identified. Fast elimination correlated to a low risk of failure, slow elimination correlated to risk of failure in the pelvis, and persistent HPV DNA after treatment correlated to a high risk of later distant failure. The results add new information to the increasing interest in research of HPV DNA in HPV related cancers and holds great clinical potential.n = 73), MT (n = 72), EOT (n = 64) and during FU (n = 41). Plasma HPV was detectable in 52 patients and PT pHPV levels correlated to tumor stages. Three elimination patterns were observed during CRT with correlation to outcome: fast responders with no local or distant failures (0/12); slow responders with high risk of local failures (4/20), no distant failures; persistent molecular responders with high risk of distant failures (4/13), but no local failures, p < 0.01. Conclusion: During CRT, pHPV can divide patients with SCCA into three groups with significantly different risk of failure. The use of pHPV can potentially assist in clinical treatment decision.Background and purpose: Circulating tumor DNA (ctDNA) is investigated in various cancers. In squamous cell carcinoma of the anus (SCCA) infection with human papilloma virus (HPV) is found in around 90% of cases and here, plasma HPV (pHPV) can be used as ctDNA. Preliminary data have proved the ability to detect pHPV16 and -18 in SCCA. We have developed a highly sensitive method for measurement of six relevant pHPV subtypes, to investigate the elimination pattern of pHPV during chemo-radiotherapy (CRT) for SCCA and its clinical value. Material and methods: Patients treated at Aarhus University Hospital from 2016–2020 were included. P16 status in the primary biopsy was measured and 82% of patients had P16 positive tumor. Blood samples were collected prior to treatment (PT), mid treatment (MT), end of therapy (EOT), and during follow-up (FU). An in-house multiplex digital droplet PCR method measured pHPV subtypes 16, 18, 31, 33, 51, 58. Results: Samples from 88 patients were drawn PT ( Squamous cell carcinoma of the anus (SCCA) is a rare disease with increasing incidence ,2, and iLocalized SCCA is treated with high-dose chemo-radiotherapy (CRT) leading to an excellent local control of approximately 87 percent . HoweverCirculating free DNA (cfDNA) is a mixture of normal cell DNA fragments and tumor DNA fragments (ctDNA)  in the bConsequently, we have developed a highly sensitive multiplex digital droplet PCR (ddPCR) method detecting the HPV subtypes 16, 18, 31, 33, 51, and 58 in blood samples from a cohort of 88 patients treated with CRT for SCCA.The aims of this study were to evaluate the prognostic value of pHPV in relation to pre-treatment characteristics, asses the clinical value of pHPV elimination patterns, and to determine the landscape of HPV subtypes detected in pHPV in SCCA.Patients with newly diagnosed SCCA at Department of Oncology, Aarhus University Hospital, Denmark, were included in the prospective clinical data and biobank collection study “Plan-A”, Ethical Committee no. 1-10-72-79-16, as described previously .Blood samples were collected prior to CRT (PT) mid through CRT (MT) (+/−7 fractions) and at end of CRT (EOT) and in the follow-up period one to three years after CRT (FU).g for 10 min within two hours and stored at −80 °C until further analysis, according to the standard operating procedures from Bio- and Genome Bank, Denmark [Plasma was centrifuged at 4 °C at 2000  Denmark .A 191 base pair (bp) spike-in DNA  was addeThe spike-in fragment and possible white blood cell (WBC) DNA contamination was measured by multiplex ddPCR. The WBC assay measures B-cell rearranged immunoglobulin genes as previously described . All sam®Taq polymerase  and a multiplex primer-mix targeting the E6 or E7 gene of the relevant HPV subtypes 16, 18, 31, 33, 51, and 58, as well as the EMC7 gene. After pre-amplification the Q5®Taq polymerase was inactivated 10 min at 99 °C. The pre-amplificated material comprised approximately 4000 times the original DNA amount and was afterwards diluted 50 times. Finally, six multiplex ddPCR reactions targeting the EMC7 gene and one of the six HPV subtypes were performed and measured using the Bio-Rad QX200 AutoDG ddPCR system. For further method details see Plasma HPV subtype analysis and quantification were performed on 25 µL purified DNA from the samples and pre-amplified by a 12 cycle PCR reaction, using the Q5P16 immunohistochemistry staining was performed according to standard protocols on paraffin-embedded formalin fixed diagnostic tumor biopsies using an automated system  with the mouse monoclonal antibody Clone E6H4. A cut off level of 70% P16 positivity, was used for classification ,29.p-values below 0.05 were considered significant. Statistical analyses were done using STATA/IC16.0 .Plasma HPV was reported as percentage of total EMC7-65. Wilcoxon–Mann–Whitney test and Kruskal–Wallis equality-of-populations rank test were used for comparison between pHPV and pre-treatment characteristics. Elimination patterns for pHPV are presented on scatterplots of raw data and required a minimum of two measurements per patient during CRT including an EOT measurement, unless pHPV elimination was detected from PT to MT. The association between response patterns and risk of failure was calculated with Fisher’s exact test. Disease free survival (DFS)  and overall survival (OS) (defined as time from inclusion to death of any cause) were presented using the Kaplan–Meier plot. Outcome comparison between patients with PT pHPV level below and above the median pHPV level was analysed using log-rank test and cox regression, presented with hazard ratios (HR). Multivariate analysis was not preformed due to low number of events. n = 58), 60Gy/30 fractions (n = 17) and 54-50Gy/27-25 fractions (n = 12), one patient received 48Gy/30 fractions. Cisplatin based concomitant chemotherapy was administered to 64% of patients. The overall local control rate was 90%, but four patients (4%) did not respond to CRT and five patients (6%) experienced a local recurrence. Four patients (4%) with complete response to CRT experienced subsequent distant failure. The two years DFS and OS were 85%  and 98% , respectively.A total of 88 patients were included. The median follow-up time was 29 months (range 9–53). Pre-treatment characteristics are presented in Blood samples were available from 73 patients PT, 72 at MT and from 64 at EOT. Finally, 41 patients had FU samples drawn. Sample distribution is presented in n = 25). The most frequent HPV subtype identified in the plasma was HPV16 (90%), The overall concordance between P16 and pHPV status was 79%, the sensitivity 82% and specificity 67% for the total cohort, as presented in n = 8) had PT samples with ultra-high pHPV levels, some of which exceeded the amount of cfDNA alleles with a median level of 45.00% (range 15.11–180.90), compared to 0.09% (range 0.00–9.82) for the remaining patients (n = 55). A scatter plot of the sample distribution is illustrated in The mean and median PT pHPV levels in the cohort with pre-amplificated measurements were 9.02% and 0.21%, respectively (range 0.00–180.90). A small group of patients  of these later experienced a local treatment failure.p = 0.81 and p = 0.37 respectively.There was no significant difference between ‘fast responders’, ‘slow responders’, patients with ‘molecular persistent disease’ or patients with pHPV negative status regarding distribution of T- and N classification, n = 29) or 15 +/−7 fractions (n = 12) or 13 +/−7 fractions (n = 4). The median treatment time at MT samples collection was three weeks (15 treatment days (range 10–23)). The Level of the MT pHPV level with relation to number of treatment days is outlined in At time of MT sample patients had received either 16 +/−7 fractions  and experienced a further pHPV increase in the FU sample, 13.96% (95% CI 0.11–86.58), p = 0.31 was detected when comparing high- versus low median PT pHPV levels. The same was observed for DFS with a HR of 4.07 (CI 0.84–19.64) p = 0.08. The number of events was however low. Kaplan–Meier curves are illustrated in A difference in OS with a HR of 2.42 (CI 0.44–13.44) This study describes three clinically relevant elimination patterns of pHPV during and after primary CRT for SCCA and investigates the prognostic value of pHPV prior to treatment as well as methodological and biological aspects of pHPV, when using a multiplex ddPCR targeting the most relevant HPV subtypes.We have developed a multiplex assay to target relevant subtypes for SCCA and demonstrates the presence of the rarer subtypes in pHPV measurement, indicating a clinical relevance of including these in future studies.Our data shows a high sensitivity (82%) and specificity (67%) of pHPV and P16 status when evaluating both high- and low-stage tumors together. In line with previous studies , the senThe lack of primary pHPV detection in the P16 positive cases could be attributed to both methodological and/or biological factors. However, several methodological steps were applied to increase the sensitivity of the assay, including pre-amplifications and volume optimization, resulting in a sensitivity ranging from 0.01–0.001%. Contamination of circulating DNA from normal cells can falsely increase the total cfDNA concentration hampering detection of low frequency ctDNA. Test for contamination with WBC was consequently performed on all samples and only a minority of samples were WBC positive, and no samples were excluded from interpretation. Biological factors influencing pHPV detection includes the tumors ability to shed ctDNA which can be affected by both tumor size and the mitotic rate of the tumor ,35.Interestingly, a subgroup of samples showed ultra-high pHPV levels. This could be due to multiple HPV genome integrations  or as suAnalyses revealed that the pretreatment concentration of pHPV showed a trend towards correlation between stage and level of pHPV, in line with previous observations in other tumor types, and from smaller observations in SCCA ,23. Analn = 18 and n = 21) has, in line with this study, described that lack of complete elimination at EOT is associated with a poorer prognosis [This study provides clinically relevant data with a high sensitivity even for the smallest tumors and describes clinically relevant elimination patterns. Few recent studies have previously presented detection of pHPV in stage I tumors across diagnosis ,24,25,26rognosis ,25. AlthThe number of samples collected during follow-up was limited, but we observed single cases with a significant lead-time between pHPV detected molecular recurrence and relapse detected by imaging , suggesting a strong potential for early detection of local or distant recurrences, as also demonstrated in colorectal cancer . These aWith a highly sensitive multiplex ddPCR method, this study demonstrates the clinical potential of pHPV in support of treatment decisions, treatment evaluation, and potential future tailored treatment for SCCA.


Patients with recurrence of SNIP had a higher proportion of young adults and displayed higher rates of HPV infection, DM, and advanced tumor stage than those without recurrence. HPV infection, young adulthood, DM, and advanced tumor stage could be associated with a high recurrence rate, which suggests that patients with these risk factors could require close follow-up after surgery.Sinonasal inverted papilloma (SNIP) can recur; however, the factors related to tumor recurrence remain unclear. This study aimed to analyze risk factors, including human papillomavirus (HPV) infection, as well as other factors associated with SNIP recurrence. Thirty-two patients who were diagnosed with SNIP and underwent surgery between 2010 and 2019 were enrolled: 24 men and 8 women, with a mean age of 59.2 years. The mean follow-up was 57.3 months. Demographics and information about history of smoking, diabetes mellitus (DM), hypertension, allergic rhinitis, alcohol consumption, tumor stage, surgical approach, and recurrence were reviewed retrospectively. Specimens were investigated using polymerase chain reaction to detect HPV DNA . Seven patients (21.9%) experienced recurrence. HPV DNA was detected in five (15.6%) patients (high-risk subtypes,  Sinonasal inverted papilloma (SNIP) is a benign neoplasm of the nasal cavity and paranasal sinuses and accounts for 0.5–4% of primary nasal tumors . SNIP haThe etiology of SNIP remains unknown. Certain hypotheses have been proposed, but causality has never been established for the suspected factors of smoking, allergy, and human papillomavirus (HPV) infection . SeveralFor more than 30 years, HPV has been suspected to play a major role in the pathophysiology of SNIP. Several studies have reported higher HPV detection rates in recurrent SNIP cases ,6,7, butTherefore, this study aimed to analyze risk factors that are associated with the recurrence of SNIP, containing HPV infection, as well as other risk factors such as age, sex, medical history, smoking, alcohol consumption, tumor stage, and surgical approach.The study population consisted of 32 patients with SNIP who were treated at Kagawa Rosai Hospital between 2010 and 2019. All patients were pathologically diagnosed with SNIP . The meaWe used opt-out to obtain consent for participation in this study.Clinical characteristics were retrieved from patient medical records, which contained information on their medical history, including the presence of non-sinonasal papilloma, diabetes mellitus (DM), hypertension, and allergic rhinitis, as well as information on lifestyle characteristics , occupational and industrial exposures such as welding fumes and organic solvents, primary symptoms, and complications after surgery. From the medical records, lifelong nonsmokers were defined as ‘nonsmokers’, and former smokers and current smokers as ‘smokers’. We also defined lifelong nondrinkers and social drinkers as ‘nondrinkers’, and those who regularly drink several times per week as ‘drinkers’.All patients underwent preoperative computed tomography. The preoperative clinical stages of the initial surgeries were graded according to the Krouse staging system . The tumAll surgeries were performed under general anesthesia using endoscopy, while external approaches, such as the Denker procedure or Caldwell–Luc operation, were added as needed for curative treatment. For complete surgical removal, accurate endoscopic identification and discrimination of the tumor from the surrounding normal mucosa were necessary. The tumor attachment site needed to be completely removed, and after it was removed, the part that was growing into the sinus was also extracted . For theAfter surgical resection, the patients were followed up for at least two years to check for recurrence. Recurrence was defined as the presence of recurrent SNIP after initial curative treatment during the follow-up period. To rule out recurrent disease, patients were followed up after surgery, mainly by endoscopic examination.TM  and primers that detected HPV DNA, as reported previously [HPV DNA was detected using consensus primer-mediated polymerase chain reaction (PCR) assays. Surgically removed tumor lesions were trimmed from formalin-fixed paraffin-embedded (FFPE) blocks of inverted papillomas, followed by deparaffinization, and total DNA was extracted using the QIAamp DNA Micro Kit . A section with a thickness of 3 µm was prepared from the FFPE block, and the location of the lesion was confirmed by hematoxylin and eosin (HE) staining. Then, only the area with a high density of the tumor cells was dissected from the unstained slide, and DNA extraction was performed. PCR was performed using TaKaRa Taqeviously ,14,15. Teviously ,14.We also performed PCR on internal control genes to assess the validity of the HPV DNA amplification results. PCR was performed using the primers for β-actin and GAPDH ,17, and p < 0.05 were accepted as significant, while those with p < 0.1 were considered to indicate a tendency.Differences in baseline characteristics were assessed using the chi-square test or Fisher’s exact test, as appropriate. All statistical analyses were conducted using the statistical software ‘EZR’ (Easy R) . Values The 32 unoperated patients with SNIP included 24 men and 8 women, with a mean age of 59.2 years  at the time of papilloma detection. In all cases, gross total resection was performed. Seven patients (21.9%) experienced a recurrence. Nineteen (59.4%) were smokers and 9 (28.1%) drinkers. The number of patients with non-sinonasal papilloma, allergic rhinitis, DM, or hypertension was 0 (0%), 10 (31.3%), 5 (15.6%), and 11 (34.4%), respectively. There were no patients with occupational and industrial exposures, such as welding fumes or organic solvents. There were 27 patients with nasal obstruction, 4 with epistaxis, and 1 with postnasal drip. The number of tumor attachment sites varied between groups. There were 2 patients (6.3%) with Krouse stage T1, 9 (28.1%) with Krouse stage T2, 21 (65.6%) with Krouse stage T3, and 0 (0%) with Krouse stage T4. An endoscopic approach was performed in 29 patients, and combined approaches  in 3 patients. There were two patients with epistaxis and two with numbness of the cheek as complications. We could control epistaxis in both patients using bipolar cautery. The numbness of the cheek in two patients was resolved a few months after surgery. No major complications were observed. There were 15 patients (46.9%) with enhanced hyperkeratosis or presence of squamous hyperplasia, 0 (0%) with an elevated mitotic index, 0 (0%) with a lack of inflamed polyps, and 0 (0%) with a greater number of aneuploid cells. HPV DNA was detected in 5 (15.6%) of the 32 patients with SNIP. Of these, two were positive for high-risk HPV subtypes, and three were positive for low-risk HPV subtypes.p = 0.025) (p < 0.01). Patients with Krouse stage T3 showed significant associations with recurrence compared to those with Krouse stages T1 or T2 (p = 0.030). Additionally, patients who had HPV high- or low-risk variants showed significant associations with recurrence, compared to those who were negative for HPV variants (p = 0.025).Younger adults (<40 years) showed significant associations with recurrence compared to older adults  . Patientp < 0.01) (Younger patients (<40 years) showed significantly stronger associations with HPV infection (positive for high- or low-risk HPV DNA) than older patients did  .p = 0.0194), and multivariate logistic regression revealed that a younger age at initial diagnosis was the strongest risk factor for neoplasm recurrence [The results of our research on the relationship between clinical or demographic data and recurrence of SNIP showed that younger age <40 years), presence of DM, advanced tumor stage, and HPV DNA were associated with higher recurrence rates. Pähler et al. also reported that patients who presented with recurrent papilloma infection were significantly younger (48.7 years old on average) at the time of initial diagnosis than those (60.2 years old on average) with non-recurrent tumors p = 0.04, and mu0 years, currence ,21. TherPatients with impaired immune responses have a greater tendency to develop HPV-associated disorders ,23,24. Ip = 0.05). Furthermore, Moon et al. reported that the Krouse stage T4 group had more frequent recurrences than the T1, T2, and T3 groups did [Krouse , Han et oups did . This stHPV, an epitheliotropic DNA virus, can infect the epidermis or mucosa in humans. During infection, viral DNA sequences of HPV are incorporated into cellular DNA and play a key role in the promotion of neoplasm growth and malignant transformation of SNIP . The detp = 0.012) [Smoking is considered the most momentous risk factor for the development and recurrence of tumors occurring in the head, neck, and uterine cervix regions ,41. As o= 0.012) . This stA systematic review and meta-analysis indicated that an endoscopic approach was an approving treatment option for SNIP and confirmed a global recommendation that it is the world standard in the treatment of such nose lesions, discovering a lower recurrence rate than that in external approaches. However, the recurrence rate did not show a significant difference between the endoscopic and combined approach groups . Even whDespite being performed in a single institution, this study had limitations. First, the low positivity rate of HPV DNA made it difficult to evaluate the influence of HPV infection on the SNIP recurrence. Only 5 of 32 patients (15.6%) were positive for HPV DNA in the present study. Three recent PCR-based studies detected HPV in 11 of 90 patients (12.2%) , 2 of 19This study examined the risk factors for SNIP recurrence after surgical resection. HPV infection, young adulthood, DM, and advanced tumor stage were associated with a high recurrence rate. These results suggest that patients with these risk factors require information about risk factors and close follow-up after surgery. In the future, a multicenter study with a large sample size will be conducted to establish the relationship between SNIP recurrence and HPV infection, and the relationship between recurrence and other risk factors.


The World Health Organization (WHO) has officially declared a global strategy regarding the Elimination of Cervical Cancer in November 2020, which focuses on preventing cervical cancer through HPV vaccination, screening precancerous lesions and managing and treating invasive cervical cancer . The strAccording to a previous report , 107 (55In this special edition of “Latest National HPV Vaccine Programs and Outcomes in the World”, the status of the global HPV vaccine program and its coverage is demonstrated from various countries. The editors believe that knowing the status of each country may accelerate and support the efforts to meet the goals imposed by WHO and expect that this Special Issue will play a certain role in reaching the goal.First, Dr. Diana Wangeshi Njuguna et al.  show theAnother group show the situation in Japan. In Japan, subsidies from local and national governments for the HPV vaccination program for girls aged 13–16 years commenced in 2010. In 2013, HPV vaccination became a national immunization program in girls aged 12–16 years. However, in June 2013, the Japanese government decided to suspend the governmental recommendation for the HPV vaccine. The rate of HPV vaccination decreased drastically, from 70% to almost zero. This situation has substantially influenced the lower awareness about cervical cancer prevention, even among medical school students . HoweverOther authors present various situations in their respective countries. We need to understand each other’s situations in countries where the HPV vaccine has been successfully disseminated and in countries where it has not been well disseminated, and in this Special Issue, we realize the various steps necessary for increasing of uptake of HPV vaccination. The dissemination of correct knowledge is essential in any country and in any situation. We hope that this special edition of “Latest National HPV Vaccine Programs and Outcomes in the World” will contribute to the improvement of the health of people worldwide.


Fortnite and League of Legends owe their ongoing success in no small part to their massive uptake by streamers, and triple-A releases from major publishers can reliably expect significant attention on streaming platforms. But what about smaller, lower budget games? For independent game developers, the costs and benefits of streaming are less clear. Based on interviews with small commercial indie developers in Toronto and Montréal, this article critically examines different discourses around streaming and commercial indie games, focusing on developer perceptions of the benefits and risks of streaming and its impacts on indie game-making practices, including production, promotion, and community-building. Contrary to persistent popular myths about streaming as the key to ‘discoverability’, commercial indie game development remains a precarious form of cultural work, and indie games collectively attract only a tiny fraction of the overall audience on streaming platforms. There is a high level of uncertainty about the factors that led to a given game’s success, leaving many indie developers ambivalent about leveraging influencer attention and even as they commit significant time and energy trying to doing so.Commercial game makers at all scales of production have increasingly come to incorporate livestreaming into every stage of the game development cycle. Mainstream hits like  Viewers consume billions of hours on a monthly basis, interacting with streamers and fellow spectators via live chat in ways that spill onto social media and ripple outward to shape popular tastes, modes of communication, cultural attitudes, and dominant play styles in game culture . He also determined, based on observing streamers and the affordances of Twitch as a platform, that ‘games with some downtimes, as long as they’re not too long, is great because they have time to engage with their community and talk with people and read the chat’. This is somewhat counterintuitive, since Christopher’s design philosophy and past experience suggest players want a fast-paced game with as little downtime as possible. Tom also touches on this contrast: ‘the streamer demands a certain flow for it to fit inside of a stream. If I’m making a super high stressed action game, that doesn’t work for the streamer as well as it does for the individual player’. That ‘slow time’ allows streamers to more actively engage with their audiences, an essential part of their performance.the big mistake that I didn’t know I was making until I saw it being streamed, which is that I was really trying to get a good difficulty curve from the game, which means sort of introducing things at a steady pace, but not necessarily showing our hand entirely early on. […] it’s only once you get into the second [world] that you start seeing the things that are important, that are not important, that are surprising and that make you sort of realize, “Oh, this game’s a lot deeper than I expected.” But that first world ends up being a really natural stopping spot. So, what I’ve seen is a whole bunch of people who’ve done a single stream of the game where they play for around half an hour to an hour, finish up the first line and then never come back to the game on stream because they feel this shows what the game is about.This poses a dilemma, however, because Curtis feels the game as released is better from a design perspective, even if a more front-loaded structure would be more appealing for streamers and promotional purposes. As Christopher’s example of incorporating downtime also indicates, developers’ instincts about what works for ordinary players must be balanced against what they think will work for content creators, directly informing the design process.Developers are also keenly aware that if streamers are not hooked by a game’s pacing and flow early on, they may not stick with it for long. Curtis feels in retrospect that his most recent game was not structured well for streamers.Twitch’s ‘programmability’ as a platform  extends Several developers talked about plans to build future projects around streaming from the ground up. Holly is hoping to take advantage of the excitement around virtual reality (VR) systems, explaining a concept where ‘the streamer could play it in VR, but the audience could participate in the game itself using the new integration tools’, by voting on what happens in the game, with those interactions incorporated into the VR user interface so the streamer is not ‘cut off’ from the audience. An important factor for Holly is that these integrations are monetizable via viewer donation, with developers getting 20% of the revenue alongside the platform and the streamer, as opposed being left out of the deal as they are in other forms of streaming monetization. She sees this as a pathbreaking idea, since most VR games are not optimized or monetized for streaming, and hopes that the audience-interactive elements will also increase replayability. Curtis has also done experiments with what he calls ‘stream first’ games that are ‘made to be played over Twitch’. With some cultural agency funding, he prototyped ‘a game that was played between the audience and the person streaming’ using the Twitch chat, rather than the official API, and thought it was promising. However, he’s hesitant to turn it into a larger scale project due to the ‘serious money’ required and the lack of well-designed, successful examples of similar games, which he attributes to the fact that some audiences simply want to watch rather than become active participants in the game. Nevertheless, like most developers he has streaming front of mind as he conceptualizes new projects: ‘I’m going to just basically sit down and look at the state of the industry and try to figure out what my plans are next, because it keeps changing’. Indies are navigating a constantly shifting environment, and the language of risk permeates their comments.For Tom, the greatest risk lies in ignoring streamers: ‘A lot of developers would make a game without considering necessarily whether they’re making it for streaming audiences’, waiting until the game is ready to release before contacting streamers with a ‘hope this works out’ approach rather than intentionality. Curtis finds this process ‘super annoying’, since he feels it devalues games designed to be self-contained experiences in favor of ‘endless amounts of content’ and games-as-a-service models. This is exacerbated by what he calls the inscrutable ‘black box of discoverability’ on different platforms, leaving developers mystified about how to find an audience. This skepticism is warranted, according to other developers. Hugh lists off the many ways incorporating streaming-related features can impact a project: ‘additional cost, additional programming time, additional quick fixing, additional [quality assurance]. So you have to be really sure that there’s value in what you’re doing before you commit to spend that money in development’. Christopher is fairly certain there is no value in streaming for his team’s next game, so he’s ‘not going to invest effort and money too much on streaming because these kinds of games almost [never] stream or barely’. Strategic decisions about costs and benefits, imagined audiences, and design ethos, all inflected by platform logics, are now central to commercial indie game development. These strategies are undertaken on the chance – however slim – that streaming can lead to commercial success or notoriety for indie developers.Although the experiences and specific attitudes of our interviewees vary, in the broadest terms indie developers see streaming as a means of promoting their games, alongside marketing, press, social media, public exhibitions, and other forms of promotion. According to developers, the potential value of streaming is highly dependent on the genre of game, and moreover there are many different kinds of streamers, each with different styles of performance and genre preferences, from competitive streamers who often play one game exclusively, to ‘variety streamers’ who rotate games and genres regularly, to ‘niche’ streamers who focus narrowly on a particular genre or subgenre. When the genre of game aligns with the streamers’ particular tastes or play style, Helena says, streamers become ‘very good hype people. If you have a game and you want people to get excited about it and you want to get it to as many people as possible, I feel like streamers are just the connectors’. Developers’ ground-up theories of streaming resonate with Austin Walker’s argument that the affordances of Twitch as a platform encourage a ‘promotional stance’ : 440. WhSome developers see a fairly direct connection between promotion, streaming, and sales. Melvin and Tessa’s accounts of the success of their ‘highly streamable’ competitive party game exemplify the idea of streamers as a form of promotion. Although they did see some spikes in their sales that directly correlated with popular streamers playing the game not long after its release, they place greater emphasis on the fact that they have maintained sales at an unusually steady level for upwards of 3 years, a ‘long tail’ of players discovering the game thanks in part to ongoing streaming and gameplay videos. ‘A lot of them are small, but still people are making content’, which for Melvin and Tessa speaks to the value of fostering paratextual practices as a ‘primary strategy’ for ongoing post-release promotion that they have pursued ‘pretty aggressively’ as they have pushed new content for the game by directly soliciting hundreds of individual streamers. Tessa puts it succinctly: streamers are ‘amplifiers’ and ‘arguably the most effective way that we could possibly have out there to get people’s attention and grow our audience’. Tom ascribes the modest popularity of his own humorous multiplayer game to its ‘replayability’, which he believes encouraged streamers, notably those who played in groups, to ‘keep coming back to it’, correlating with increased sales. Stuart had a similar experience when a high-profile YouTube content creator discovered one of his games several years after release, which he says ‘spiked my sales and then the sales reset, but not to launch, maybe to the year before. It basically, bumped it back a year in terms of the sales, in terms of those numbers. That’s huge’. Stuart directly attributes this ‘reset’ of his game’s long tail to this YouTuber, and he and Christopher both note that the permanent archive of recorded gameplay videos on YouTube may be an even greater asset than livestreamed content since they have more longevity. Several other interviewees drew similar correlations between streaming and long-term success, with the goal of becoming a ‘forever game’ updated over a long period of time for a dedicated audience, as Tom puts it.It’s why some perfume company would pay a model or a celebrity to take a picture with a perfume bottle. It’s like, we want the streamer to play the game because we know that will make the game seem fun to their audience.While certainly this is true in the case of big-name celebrity streamers, smaller or niche streamers can also have a positive impact. Stuart says that his team is deliberately marketing their game to a particular genre niche: ‘our niche streamers are magnitudes smaller. But they are a way more targeted market. I feel like the conversion rate on views to sales would be way higher, like 10 times higher’ because they ‘cater directly to our audience’. In other words, quality is as important as quantity in promotion. Although he is not as convinced of direct sales boosts or measurable return on investment, Hugh contends that ‘we can definitely see that in some cases, our game brought audiences to a Twitch streamer’s channel. And in other cases, the Twitch streamers channel’s audience brought viewers for us for the game’. This leads him to contend that having a game streamed in sufficient numbers can improve discoverability on digital distribution platforms thanks to increased searches and wishlisting. In the same vein, Christopher sees streaming as a useful way to gradually build a player base for in-development games still in beta testing or early access.Helena compares the role of streaming in promotion to celebrity and influencer marketing in other fields:In spite of the opportunities most of our interviewees see in streaming, the strongest theme in our conversations is ambivalence. Indies recognize the inevitability of streaming as a factor in contemporary game development, but frequently express uncertainty about how impactful, reliable, and measurable it really is, and whether actively pursuing it is worth the significant time and effort involved. Although as noted above some developers anecdotally attribute sales or engagement spikes to attention from specific streamers, in many other cases developers report that being featured by streamers with large followings produced no measurable results . Past suDoes that mean that it didn’t have any effect? Does it mean that the effect is going to be felt over the next 12 months as just like a long tail addition to the general visibility of the game? We don’t know.Ultimately he concludes that Twitch is ‘trying to own the channel of communication between the developers and the streamer’, echoing Will Partin’s work on how platforms ‘capture’ previously off-platform monetization strategies (In spite of his game’s popularity with streamers, Melvin also remains ambivalent. At one point, Melvin and Tessa’s studio invested money in the Twitch ‘Bounty Board’ system, which allows developers to make a pot of money available for streamers to claim in exchange for featuring their games. This led to more streamers playing the game, but didn’t have any obvious effect on sales or engagement. ‘What does that mean?’ Melvin wonders, frustrated,rategies . Severalrategies .Considering streaming as just the one thing is kind of saying, all games are the same, all games have the same process […] can we realistically compare a three person VR studio to an 18 person mobile game studio? No, we can’t.Lauren and Carolyn both caution that this makes it difficult to evaluate different indie experiences, since what works well for one game may not work at all for another.All of this raises questions about much of the advice that circulates about streaming for indies. Charlie critiques the popular idea that if you ‘find the right streamer with the right audience […] it’s guaranteed to make all your financial dreams come true as an indie developer’ as a potentially dangerous misconception. Carolyn likewise observes that ‘people think it is an easier thing than it is’ and worries that naivety or overconfidence will lead developers to overemphasize streaming to the detriment other important factors. The concerns discussed above about how amenable different genres are for different kinds of streaming play into this as well. As Lauren puts it,That Dragon, Cancer (more people have played the game for free than have bought it and I find that statistic depressing. […] it all comes back to the nature of our game. Our game is a narrative game that plays like a movie. Once you have seen our game, you don’t really have a reason to play it. And this is the inherent problem with the streaming culture and the game we made. The game we made, it streams well. People enjoy watching it and watching someone play it and it’s a cool experience, but they have no reason to buy it afterwards.The issue was exacerbated by the fact that Holly’s team gave away numerous free promotional copies of the game to streamers, further reducing their overall sales. Anticipating these problems during development, her team considered asking streamers to only play half the game. They decided against it because they didn’t want to sour relationships by coming off as overly controlling, but their fears were borne out.For some indie developers, ambivalence leans toward a wholly negative view of streaming as too risky or even harmful to their games. This reflects a small but significant countercurrent to the generally celebratory discourse around game-based content creators, exemplified by Numinous Games’ charge that YouTube Let’s Play videos hurt sales of their narrative game , Cancer . Of all It feels dangerous to put it into the hands of someone who’s more likely to make fun of it than to appreciate it. […] I know there’s in theory no such thing as bad publicity, but since it’s such a specific and somewhat sensitive game, I just didn’t feel like we should be courting that kind of attention.This again echoes Numinous Games’ concern that That Dragon, Cancer’s deeply personal story of loss would be devalued by content creators, and also resonates with the experiences of queer game developers like Robert Yang, whose games about gay sex and masculinity are frequent targets of gameplay reaction videos and streams that use them as fodder for exaggerated, often profane mockery, and have also been censored by Twitch (Another factor that contributed to Holly’s negative experience was her game’s serious, dark themes. She was worried that streamers – especially those who usually stream more mainstream games – would not take it seriously:y Twitch . Helena,y Twitch .All of this has left Holly fatigued by the overemphasis on streamers in indie game promotion, at least for narrative games: ‘frankly, I’m just disillusioned, I’m like why? Why would I do that? Cool, they’ll play it and no one will buy it’. Several other developers share this skepticism, with Tom even suggesting that a perceived decline in story-oriented games could be related to the rise of streaming, further evidence of a normative effect on game development.Counter-Strike: Global Offensive YouTubers hawking gambling schemes as one example (remediating the games they play – the stream stands as a distinct cultural product (A key factor in all of the different attitudes and perceptions discussed above is the knowledge that streamers are cultural producers in their own right. They may in some cases directly or indirectly promote indie games, but as noted above, cultural intermediation is not their primary function : 51. Thi example , and mor example . If a st product .For some developers, this state of affairs feels unfair or even exploitative. Holly’s negative experience with her game has led her to personally view streamers as profiting off of indies: ‘If they have a large enough audience, they are literally getting money from the audience to be playing a game and or from I guess other ads on Twitch. […] They’re making money off of it’. On the other hand, Hugh understands why some developers feel this way but is critical of the impulse: ‘There is a particular angle that says streamers are parasites, they are producing content off the back of the work that we’re doing. […] the reality is that that’s just not how the world works anymore’. For Hugh, developers need to take streaming as a given of the contemporary industry and make the best of it, rather than treating streamers as competition. Similarly, Lauren argues that streamers and developers alike should approach streaming from a place of collaboration.It is in this potential for platform-mediated collaboration, connection, and community-building that developers see the most direct value in streaming. While the influence of streaming on direct or indirect sales is difficult to pin down, many interviewees point to other, less quantifiable but equally important factors at play, such as community-building and fostering audience engagement. What allows for long tail success like Melvin and Tessa’s is a critical mass of people invested in the game. Cultivating a loyal, participatory community of fan-consumers who feel a personal connection to the creator's work is understood to be essential for contemporary independent cultural production, and social media engagement is a key means of doing so . In CaroTessa, for example, uses streaming as raw material for producing social media posts for her studio: ‘for me it really serves the purpose of creating content that I can use to make the promotion of what’s coming up’. She collects clips of interesting or funny moments from streams, as well as memes, press, and other materials and reworks them into compelling content to share via other channels, a strategy other community managers also employ to generate engagement and build brand recognition. Tessa explicitly ties this to credibility and authenticity, a way of incorporating streamers and viewers into the studio’s community, and she says streamers appreciate this mutually beneficial acknowledgment. The community-building function extends also to shaping that community. Charlie argues that streamed and recorded play not only helps new players grasp the basics of a game, but additionally model normative ways of playing and enjoying it, contributing to emergent community standards more effectively than official developer-produced content or journalistic coverage. Although it was not a major theme in our interviews, some indie developers livestream their own game development work for similar pedagogical reasons . Rather Game developer perspectives on streaming illustrate just how mutable and precarious commercial indie game development continues to be, in spite of the proliferation of streaming-related success stories. The small Canadian developers we spoke with feel the influence of streaming on all aspects of their work and approach its potential risks and benefits ambivalently as they pursue the elusive goal of creative and economic sustainability .In the production process, streaming offers an opportunity for more organic playtesting and tweaking games in response to player experience, but this requires active, ongoing development work. Streaming also has a normative effect on design practices, as developers attempt to conceptualize games that appeal to streamers and viewers, though this may clash with their own design sensibilities. Programmable tools that integrate aspects of the streaming platform directly into games may enhance streamability, but they are often prohibitively costly or labor-intensive for smaller developers. Beyond production, streaming is understood to serve a promotional function, and some developers attribute sales bumps and long-term interest in their games to uptake by streamers. However, the majority of our participants express uncertainty about the value of streaming as a promotional tool, pointing to inconsistent results and frustratingly opaque platforms. For certain kinds of games, the impact of streaming is seen as largely negative, benefitting streamers and the platform more than developers, which has implications for what developers consider commercially feasible. Where developers seem to find streaming more consistently useful is in the less explicitly promotional but no less important community-building aspects of cultural production. Streaming thus becomes one of many venues where developers themselves are compelled to adopt the performative, relational techniques of streamers and other online influencers to cultivate a following for their work.Our findings complicate the optimistic narratives and advice that characterize much of the discourse on streaming and indie games, in which platforms are paradoxically positioned as both the cause of and solution to the problem of discoverability. In fact, the ‘nested precarities’ of the competitive market for indie games, the rapidly changing game industry, and the ambiguous cultural and economic logics of different platforms  are embo


The plastic industry is undergoing drastic changes, due to the customer sustainability perception of plastics, and the eruption of new processes (such 3D printing) and materials (such as renewably sourced resins). To enable a fast transition to high-quality, sustainable plastic applications, a specific methodology could be a key competitive advantage. This novel methodology is focused on improving the objectivity and efficiency of plastic production and the design review process. It is applicable to discrete optimization events in any product lifecycle milestone, from concept design to serial production stages. The methodology includes a natural way to capture plastic-related knowledge and trends, oriented towards building a dynamic “interaction matrix”, with a list of potential optimizations and their positive or negative impacts in a comprehensive set of multi-criteria evaluations. With an innovative approach, the matrix allows the possibility to incorporate a business strategy, which could be different at every lifecycle stage. The business strategy is translated from the common “verbal” definition into a quantitative set of “Target and Restrictions”, making it possible to detect and prioritize the best potential design optimization changes according to the strategy. This methodology helps to model and compare design alternatives, verify impacts in every evaluation criteria, and make robust and objective information-based decisions. The application of the methodology in real cases of plastic material design optimization in the automotive industry has provided remarkable results, accelerating the detection of improvement methods aligned with the strategy and maximizing the improvement in product competitiveness and sustainability. In comparison with the simultaneous application of existing mono-criteria optimization methodologies (such as “Design to Cost” or “Eco Design”) and subjective expert-based reviews, the novel methodology has a reduced workload and risks, confirming its potential for future application and further development in other polymer-based products, such as consumer goods or packaging. Plastic materials have been a revolution for many markets in previous decades, replacing other materials due to their cost competitiveness, design flexibility, light-weight or decoration and finishing properties, widening the access to convenient products and playing an important role in the development of humanity . In the Companies currently producing or designing plastic products must adapt as fast as possible to market requirements and opportunities, reviewing their product and process design to continuously improve and to detect optimization opportunities to improve competitiveness and sustainability. The development and utilization of specific methodologies for this transition could accelerate this adaptation and represent a key competitive advantage for success and sustainability.Due to the intrinsic subjectivity of product design activities, some studies have attempted to understand and evaluate their designs according to attributes , and impRegarding methods for design review and optimization, in some competitive industries, continuous improvement methods are widely applied, such as Lean, Kaizen or Six-Sigma ,30, alsoAs an example, even if applying some existing plastic design methods ,14,29,30The methodology has the objective of plastic product and process design review and optimization at any product lifecycle milestone, from concept to end of life, in order to make it possible to apply in any company and lifecycle stage.It must be capable of capturing the business strategy, which could be different at each milestone, and must be translated into a set of improvement “target” and restrictions.It is focused on the “multi-criteria” evaluation of design alternatives, the detection of optimization strategies according to targets and restrictions, determining changing impacts and making objective and information-based design decisions.The methodology must be easily integrated as an additional “layer” that is compatible with any company size, organization, design strategy or knowledge management system, without required changes, simplifying its implementation and accelerating knowledge capture and application results.The methodology is expected to be applicable to “discrete events”, to simplify the early adoption of the methodology in any product lifecycle phase. It also facilitates the implementation of the methodology, as it does not require any change to the existing methodologies or organizations, in any company size or market setting.The methodology must support knowledge capture as a “Knowledge-Based Engineering” (KBE) tool, helping in “incremental” methodology development and a continuous improvement flow, and ensuring long-term utilization. It must be suitable for the future implementation of learning, artificial intelligence (AI) or data mining procedures.With these main objectives, the methodology development started with a working principle, integration and schema, as shown in The methodology was first developed to be applied in a competitive and exigent market, such as automotive plastic materials. In detail, the first application is the design review and design optimization of the three main vehicle interior and exterior plastic materials, usually representing the majority (around 58%) of the average vehicle plastic weight , . Some other parts include additional processes, such as thermo negative or slush skin formation, skin wrapping, and polyurethane foaming, as well as system assembly, plastic welding, and gluing. The application of the methodology also makes it possible to integrate actors and expertise from different activities—from vehicle design and product engineering, to process engineering and manufacturing processes expertise, as well purchasing and quality support functions. We also considered the application in different lifecycle phases for different vehicles, business strategies, and optimization targets for each event, allowing us to test and validate the methodology in a wide range of representative situations.According to described aim and objectives, the methodology development started from a previously published basic flowchart , which dThe methodology has a set of tools, as shown on the left side of the schema in 1.Optimization team: The first step is to set up the team who will take part in the optimization process. The best strategy for this is defining a multi-disciplinary team representing all company departments and wide knowledge areas. In our application case, the stable collaboration team was 15 people with profiles covering the main company knowledge areas, cumulating near 300 years of experience; as shown in 2.Knowledge “incremental” database: Once the optimization team has been identified, the first step of the methodology development was to request team members to collect related information, and disseminate it to the rest of the team, including design standards, plastic material information, process guidelines, or regulations to be respected. The “incremental” aim of the methodology enabled us to start applications in real cases, even with “partial” knowledge collection, helping to detect lacking areas and develop a comprehensive knowledge base. As an example, in the first application, we detected a lack of benchmark information, as an interesting optimization opportunity; then, an existing benchmark vehicle database  was incl3.Multi-criteria evaluation set: This is usually defined by the company directors, based on existing key performance indicators (KPIs), and is as complete and stable as possible to allow comprehensive and comparable evaluations. In our case, we used the QCDP set of KPIs , which is well known in the automotive and product development industries ,44. HoweThe multi-criteria evaluation set was placed in the columns, as described in the previous paragraph.A list of “potential design optimization” or “changes” was placed in rows, identified by the optimization team members, based on a related knowledge database and expertise, and ordered considering the origin of the opportunity .The interaction matrix is the central tool of the methodology. It concentrates this information in a profitable format, organizing the following information in a matrix format:After the first application in real cases, the team reviewed and approved the set of evaluation criteria, and also identified “reference members” for the evaluation and continuous improvement of each criterion according to their areas of expertise.This interaction matrix was created in a “collaborative” manner by the optimization team, according to the three steps defined in the methodology and shown in Step 1: Each team member could propose changes in their knowledge or expertise area, mainly based on their experience, obtaining qualitative information and considering the methodology as an effective “knowledge capture” system.Step 2: Every change was evaluated based on its positive or negative impact on every criterion of the multi-criteria evaluation set. The author proposing the change and the reference members had to discuss every impacted criterion to reach a base quantitative value or “score” according to an agreed classification, with values from −5 to +5 , according to Arithmetic means of the answered scores, to allow reference members to evaluate their previous scores and agree to set the answer mean as the resultant score for the impact.Detection of dispersions or lack of stability in answer scores based on normal distribution calculation. In this case, disperse values allowed the team to detect the need for the review of the scores and changes to clarify the origin of these dispersions , better explain them to the team, and then re-review the scores to obtain the agreed resultant score for the dispersive values.Step 3: Once the base value for the impacts had been defined, the entire interaction matrix was evaluated by the entire team, using a Delphi or ETE  methodology, and the group “one-voice” opinion output was set. The Delphi method ,46 is weAfter these three steps, and with all these resultant scores, it was possible to build a reviewed interaction matrix, which was the result of the team’s “one voice” and was useful for application in the following stages. It must be highlighted how easily the process could obtain the “one voice” interaction matrix, based on the experts’ knowledge capture and review strategy.The resultant interaction matrix in our application case is  shown in 2.Business strategy definition: Once the interaction matrix had been defined, the next step of the methodology was the tool that allowed the target of the optimization to be fixed according to the company business strategy. This strategy is usually fixed by the company directors and is frequently different at every moment or project milestone. As an example, in the development phase, the key strategy target could be “respect planning and budget”, and in the serial production phase, the target could be “maximizing cost reduction actions, with as less investment as possible”. This strategy is usually fixed by “verbal” qualitative information, and the key is “translating” this verbal information into parametric and quantitative information.The methodology enabled the “translation” of a “verbal” strategy into a set of quantitative “Targets” and “Restrictions” in the evaluation criteria set defined in 2 emissions or waste volume”.Verbal strategy: “During serial production phase, detect cost reduction actions, with no investment, with application delay less than 1 month, and not affecting negatively lifecycle CO→Target: Detect and order changes by favorable impact in the “Production and Manufacturing Cost Reduction” sub-criteria = (CP) ≤ 1→Applicable in serial phase = (DS) ≥ 1;Investment or entry ticket = (CI) ≤ 1;Impact on delivery time/planning = (DL) ≥ −1;2 emissions = (EC) ≥ 0;Impact on lifecycle COImpact on waste volume = (EW) ≥ 0.Restrictions:Translation into quantitative “Target” and “Restrictions” to criteria set:To explain the “translation” process, the next example is based on a real application case, with a relative complex verbal strategy:With the translation of the strategy into a set of target and restrictions, it was possible to define an algorithm to search the “interaction matrix” and detect and classify the changes that met the defined strategy, as shown below, in pseudocode, with comments after the # symbol.→IF: CP ≤ 1; AND → #verify accomplishment of target.IF: DS = 1; AND → #verify accomplishment of restriction 1.IF CI ≤ 0; AND → #verify accomplishment of restriction 2.IF DL ≥ −1; AND → #verify accomplishment of restriction 3.IF EC ≥ 0; AND → #verify accomplishment of restriction 4.IF EW ≥ 0; THEN → #verify accomplishment of restriction 5.SORT: CP (Max→min) → #classify result according to target.Result = [Interaction Matrix: 1→n]; #run through matrix linesIt is possible to apply this kind of algorithm to any mathematic software working with a matrix or also any database or spreadsheet software that can filter and sort information. In the practical application, the lines of the interaction matrix were verified for “target” and “restrictions”. Lines that did not meet these requirements were rejected. As shown in This procedure result was an ordered list of the best “POTENTIAL Optimizations” in the interaction matrix, which respected the business strategy. This allowed profitable work in the following stages: accelerating design improvement and improving work effectiveness in comparison to existing expert subjective optimization searches; and accelerating changes to meet strategy, market, and customer needs.3.Modeling matrix: Once the “POTENTIAL Optimizations” had been detected and listed according to the strategy, the next step was helping the team to verify if these changes were fully applicable to a real case and helping to perform the robust impact modeling and calculation of the “EXPECTED Impacts”, which enabled the team and management to make Go/No Go decisions around changes based on clear and robust information of the impact calculation in all multi-criteria sets, as shown in This tool enabled the addition and modeling of any subjective origin optimization, including modeling as an additional method to capture knowledge and continuously improve the methodology.4.Checks the verified impacts of every Go decision, in order to detect any deviations in previous stages .Captures any subjective origin actions and checks real results in the same way.Collects and captures any new information used during the process: benchmark good practices, regulation changes, knowledge review proposals, etc.Builds an “Improvement Action List” based on this information.Periodically performs a methodology review with the optimization team to review listed actions and, in our case, correct scores of the Delphi validation, as previously described, and update the knowledge base and methodology tools.Continuous improvement tool: The methodology has an “incremental” orientation, which allows it to be applied from the first stages of the knowledge capture process, but it also needs a tool to ensure continuous improvement. This tool performs the following tasks:These tool and actions ensured the periodical review and optimization of the tools of the methodology as a knowledge capture system, with possible development to some automatic detection of deviations and new information, as well as an artificial intelligence application.5.Phase 1: What to do? Kick-off the event, define the team members, extend team collaborators, and optimize the scope. The company directors provide the business strategy for the event, usually in verbal format.Phase 2: How to do it? The team performs the translation into the quantitative “target” and “Restrictions”, applying it to the most updated interaction matrix, and obtaining the list of potential optimizations. The team adds any additional subjective-origin proposal. The resulting optimization list feeds into the modeling matrix for the event.Phase 3: Why do it? The team collects the information and runs calculations until the modeling matrix is complete with detailed calculations of the expected impacts of the optimization list, providing clear and robust information to help directors make decisions regarding the Go-NoGo of every change.Phase 4: How to do it better? The team tracks the application of Go decisions and calculates the verified impacts once changes are applied. The team compares verified impacts with previous phases of potential and expected impacts, and in the case of deviations, develops an improvement action list. If any other deviation or useful information is found during an event, the action list is also evaluated by the team after every optimization event.Methodology application in discrete events: The steps of a typical application in a discrete optimization event, once the main tools of the methodology have been developed, as shown in As mentioned above, the aim of the methodology is to allow application from early stages, and based on knowledge capture, to perform “incremental” development. In our case, one clear opportunity for the development of the interaction matrix was the integration of historical database information. The team detected available databases regarding product modification and problem-solving events from several different companies, plants, and vehicles. These kinds of databases are useful in our case, as a source of historical information to review the interaction matrix, in particular, to review change impact scores in application delays, cost reduction opportunities, and impacts on quality evaluation criteria. All collected databases used formats and codes based on automotive standards, and were relatively easy to compile; then, the statistical software Minitab v17 application was analyzed as follows:Information from cost reduction actions applied in the period 2019–2021 and derived from 27 different plants worldwide. The database has more than 2400 lines, and 1080 modifications directly related to plastic materials were filtered: type classification, as shown in With his information, a statistical analysis was performed to increase the precision of the interaction matrix impact scores.ANOVA analysis of saving as a quantitative variable versus type as an independent/qualitative variable/factor was conducted to obtain information surrounding the potential of each optimization type, based on statistical confidence intervals. The results are shown in ANOVA 1-factor analysis of delay as a quantitative variable versus type as an independent/qualitative variable/factor was performed to obtain information surrounding the application delays of each optimization type. With results, as shown in A database with approximately 4500 lines for problem solving or QRQC  actions was applied in several different vehicles and companies. Then, 694 actions directly related to vehicle plastic materials were filtered. These actions were also classified considering the type of quality issue according to With this information size, statistical analysis was conducted to review the interaction matrix scores for impacts in duality criteria, as described in the following paragraphs.ANOVA analysis of solving delay as a quantitative variable versus defect type as an independent/qualitative variable/factor was performed in order to detect the root causes with the most complex solution and longer solution delay/impact, and the results are shown in All results of the described historical database analysis were incorporated into the methodology applying the continuous improvement tool, sharing the results with the team to update the interaction matrix tool with this information, and applying it to cells highlighted with blue borders, as well as validating it with an additional Delphi boucle, and the resultant cell values are shown by blue lines in the interaction matrix.It is interesting to highlight that it is still possible to improve the methodology with additional analysis of the existing data, or collecting additional databases from different sources, with the application of “Big Data” principles, or any other information source. The “incremental” aim of the methodology and the continuous improvement tool is to help to capture knowledge benefit from this information. The resultant interaction matrix currently in use is shown in The main objective of the methodology development was to improve the design optimization duties, reduce subjectivity, accelerate optimization, and improve the knowledge capture, ensuring the consideration of the company directors’ business strategy and customer and market demands. The methodology was already applied during 2020 and 2021 in four “Discrete Optimization Events” for three different vehicles of the European market; in three different market segments ; and in different vehicle lifecycle milestones, from the design phase to industrialization and serial production phases, as described in As expected, each optimization event had different scopes and strategies, with different teams . This helped to test the methodology in different scenarios, which is the best way to validate it. The four event strategies and results are listed in detail in In order to analyze the effectiveness of these first methodology application events versus the existing expert team’s subjective optimization research in the same events, a comparison of objective evaluation indicators was performed, as shown in These results confirm the effectiveness of the new methodology in comparison to the existing expert team’s subjective optimization research applied in the same period:Higher successful proposal application ratio.More effective change detection member/workload ratio.Accurately estimated impact modeling, and precise information provided to support management decision making, thus reducing risks.Higher verified results and better workload/results ratio, including the workload for methodology development.The higher success ratio of proposals and better methodology performance in comparison to previously existing subjective-based research are probably due to the innovative business strategy translation into targets and restrictions, which makes it much easier to detect most potential optimizations to be studied, and helps to model the alternatives with detailed impacts in the multi-criteria evaluation set, providing robust information to support decision making. In addition, the robustness of the methodology development, based on knowledge capture and historical data sources, is important.The current limitations of the methodology seem to be related to the historical database used, although the massive data checked only partial interaction matrix scores. It is likely that information will still need to be collected in order to increase statistical analysis and information robustness. Another limitation is related to the continuous improvement. Although we started with a limited knowledge database, which could have affected the results of the first applications, the first utilization of the methodology confirmed its potential for lacking knowledge detection and knowledge capture simplicity, rapidly increasing knowledge robustness, but reducing the effectiveness of the first optimization events.The development of the methodology enabled the collection of useful plastic product and process design-related knowledge that was previously varied and based on subjective experience. Knowledge collection was performed in a “natural” way and in a team workload-efficient manner. The construction of the interaction matrix based on this knowledge and the statistical analysis of historical data, reviewed and validated as the expert team’s “one voice”, provided considerable robustness to ensure confident application in real cases, allowing easier understanding and application and reducing subjectivity, representing a powerful tool to collect company knowledge and accelerate transfer and learning.The application of the methodology in real cases in a market as competitive as automotive plastic materials has also provided good results from the early development stages, allowing the team to carry out “incremental” development, detect more optimization changes, model the impacts accurately and provide precise information for decision making. The defined information structure and continuous methodology improvement strategy also enable long-term updates and utilization. It is important to highlight that it has already been used for more than one and a half years, with several successful reviews and optimization/completion loops.Therefore, it is possible to conclude that the developed methodology demonstrated an advantage in comparison to existing subjective optimization research processes. The team members and directors involved in application have provided favorable opinions regarding the effectiveness and effort–results ratio, the robustness of information during the optimization process and the verified results of the application in real cases.In the near future, the methodology will be applied to additional design optimization events to increase its accuracy and effectivity. The application also allows the methodology and utilization experience to be shared with more teams and stakeholders, as a first step for diffusion into potentially interested departments and stakeholders inside the authors’ companies, who have already confirmed their interest in propagating the utilization of the methodology. The authors are also considering the development of training, support materials, and a comprehensive interface, probably as a web-based application that guides and supports users from stand-alone methodology applications into real cases, helping to expand the application into different plastic markets and companies, which maximizes its competitiveness and sustainability impacts. We also expect to further develop the methodology with continuous improvement strategies via the incorporation of additional information databases, related analysis, and periodical knowledge-based reviews to incorporate any new trend, benchmark solution, process, or material.The wide application of this kind of methodology, which is specifically oriented towards accelerating the product and process design optimization of plastic products, enabling plastic product producers to accelerate this adaptation, is the only way to ensure competitiveness, long-term sustainability, and environmental impact reduction in plastic products.