Objective The present study aims to establish a risk profile for high-grade cervical lesions and cervical cancer (CIN2 + ) in women undergoing colposcopy at the Hospital do Câncer de Barretos, through the analysis of Human Papillomavirus (HPV) infection, cervical cytology, and patient's age.Methods Retrospective cross-sectional study based on a computerized database of women aged ≥ 18 years old who underwent colposcopy at the Prevention Department of the Hospital do Câncer de Barretos from 2017 to 2019.Results A total of 3,411 women were included, 58.0% were positive for high-risk-HPV test, with a higher prevalence of CIN2+ for HPV16 (30.3%) and other HPV (45.0%). Cytological findings that suggest invasive cervical cancer (squamous cells or adenocarcinoma), regardless of the status of HPV test, showed 100% diagnosis of CIN2 + , while atypias that suggest high-grade lesions, HSIL and ASC-H, positive for HPV test, showed in 86 and 55.2%, respectively, diagnosis of CIN2 + . ASC-H cytological results among women aged > 40 years old and negative HPV were mainly associated with benign findings. We observed that ≤ CIN1 has a higher prevalence among older women with negative HPV, while for high-grade lesions there is an increase among young women HPV16- and/or 18-positive. In cancer diagnosis, we observed a predominance of HPV 16/18 regardless of the age group.Conclusion The highest risks of precursor lesions and cervical cancer were found among women with positive HPV 16/18 tests and severe cytological atypia in population screening tests. In addition, cytological findings of ASC-H HPV negative in women > 40 years old usually represent benign findings in histological investigation. Cervical cancer is one of the most frequent types of cancer among women worldwide, being the fourth in incidence and mortality, with an estimated 604,127 new cases in 2020.Persistent infection by human papillomavirus (HPV) is the leading cause of cervical cancer, with 14 types considered oncogenic (hrHPV). Types 16 and 18 together are responsible for ∼ 70% of squamous cell carcinomas, followed by types 31, 33, 45, 52, and 58.The early detection of precursor lesions through population screening programs and the expansion of HPV vaccination coverage can minimize the impact of cervical cancer.The initiatives to implement molecular tests in population screening are justified because there is evidence that the presence of hrHPV infection, even among women with normal cytology, increases the risk of CIN2+ and may contribute to bringing the diagnosis forward by more than a decade depending on the case.The association of viral type with the age of the infected woman may also represent an increased risk factor for precursor lesions. Positivity for HPV16 in CIN2+ cases showed an increased risk among English women aged 24 to 44 years old, and for the group aged from 45 to 64 years old, there was a greater association with viral types other than 16/18.Considering the cytological findings, the association of moderate to severe atypia and hrHPV infection among patients referred for colposcopy represents a high risk for histological diagnosis of high-grade and invasive lesions.Therefore, cytological results, HPV testing, and age have demonstrated their potential in the stratification of risk for precursor lesions and cervical cancer.Women ≥ 18 years old who underwent colposcopy at the Cancer Prevention Unit of the HCB from 2017 to 2019 were included. Patients undergoing colposcopy were previously screened by cervical cytology, and HPV tests were performed as a cotest on the same sample of screening cytology or collected during the colposcopy examination. Inconclusive results or findings related to other sites  were disregarded.This was a retrospective cross-sectional study based on a computerized database of the Department of Cancer Prevention of the HCB.The cervical cancer screening program includes conventional cervical smear and liquid based cytology . Samples were sent to the Pathology laboratory for preparation of slides according to the manufacturer's instructions using BD PrepMate and BD PrepStain.For the detection of HPV DNA, the Cobas HPV Test X480TM  was used. This test detects 14 types of HPV, with HPV 16 and 18 alone and HPV-others encompassing the oncogenic types in a grouping: HPV-31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.Women are referred for colposcopy due to abnormal cervical screening at the HCB. During colposcopy, biopsy is performed when the gynecologist identifies a lesion in the examination. Biopsy was not performed when squamocolumnar junction (SCJ) was visualized and no lesion was identified by the gynecologist. Finally, when SCJ was not visualized and no lesion was identified by the gynecologist, an endocervical curettage (ECC) was performed.The gold standard considered was the combination of colposcopic examination and anatomopathological results for cases undergoing biopsies or excisional therapeutic procedures (excision of the transformation zone of the cervix [TZE]). The final histological diagnosis was established according to the most severe result obtained.p-value was < 0.05. A confidence interval (CI) of 95% was considered. IBM SPSS Statistics for Windows version 27  was used for data storage and statistical analysis.Analyses of comparisons of proportions were performed using the chi-squared test, considering that the differences between the groups were statistically significant when theThe present study was conducted in accordance with the guidelines for good clinical practice and was approved by the Ethics Committee of the HCB under CAAE number 50357321.9.0000.5437.A total of 3,411 women aged from 18 to 93 years old (mean 42.5 years old) who underwent colposcopy between March 2017 and December 2019 were included; 27.4% of the participants were tested for HPV at the time of screening (associated with cytology), and 72.6% were tested during colposcopy. The positivity rate for HPV was 58.0% , with 802 positive for HPV16/18 (40.5%) and 1,177 positive for other HPV types (59.5%). HPV16 alone was detected in 19.6% of the cases. Among the women positive for types 16/18, there was a higher prevalence (38.4%) of CIN3. Among the women positive for other viral types than 16/18, 52.8% had no intraepithelial lesions or cancer. In the group of women who tested negative for HPV, 86% had benign findings on colposcopy associated with pathological findings. Our findings show the evidence of false positives in DNA-HPV results since we find ∼ 5% of CIN2+ findings in HPV-negative cases, which could be explained by the possibility of a few cases of cervical cancer not being caused by HPV .Regarding cytology, the presence of atypical squamous cells of undetermined significance, which cannot rule out a high-grade lesion (ASC-H), was the most frequent finding (40.8%) and the cytological classification that most commonly pushes the patient to the complementary examination of colposcopy. The mean ages of the women diagnosed with high-grade intraepithelial lesions were 36.4 years old and, for cases of cervical cancer, 43.8 years old .Considering the diagnoses of CIN2/3 and cervical cancer, the HPV positivity rates were 90.8% and 94.5%, respectively. Types 16 and 18 were detected in 50.8% of the women with CIN2/3 and in 81.7% of the patients diagnosed with cancer. HSIL and ASCH were the types of cytological atypia most frequently associated with the diagnosis of CIN2+ .For the analysis of HPV positivity in the CIN2+ and CIN3+ populations, there was greater positivity in the group of other HPVs  and HPV16 alone . The most frequent combination was observed in the combined HPV16 and other HPV groups, namely, 14.4% for CIN2+ and 14.0% for CIN3+ .Positivity for HPV 16 and 18 was associated with a higher prevalence of diagnosis of CIN2, CIN3 and cancer , while other HPV types and negative HPV showed higher prevalence of diagnosis of ≤ CIN1 . Regarding cytology, the diagnoses of benign and low-grade intraepithelial lesions were more frequent among women with cytology showing ASCUS, LSIL or NILM. CIN2/3 was more frequent among women with AIS, IA, SCC, HSIL (invasive) or HSIL cytology .Patients classified as ASCH were the most frequent finding in cytology . In totThere was a higher prevalence of infection by type 16/18 among younger women diagnosed with CIN2/3 and the involvement of the other viral types increased proportionally with increasing age. Among women diagnosed with cancer, a higher incidence of infection by type 16/18 was also found in all age groups, but there was a tendency for other viral types to increase beginning at 35 years old .The present study aimed to stratify the risk of precursor lesions and cervical cancer among women who were attended at a reference center for colposcopy, considering age, the cytological test result, and HPV genotype.The possibility of molecular tests associated with colposcopic and cytological evaluation increases the accuracy of diagnosis, and the high negative predictive value of HPV tests confers a low risk of relevant tissue damage among women who test negative. In our study, 41.19% of the women were negative for hrHPV, of which 86% were negative for intraepithelial lesions and cervical cancer. Other studies have demonstrated the association between colposcopic and histological findings and HPV infection status.In the qualitative evaluation of the composition of viral types among women who tested positive, it was observed that the majority (59.5%) was infected with non-16/18 types. However, there is a clear trend toward benign findings and low-grade intraepithelial lesions in this group of women, whereas type 16/18 increases the risk of CIN3 + . The ESTAMPA study conducted in Latin American countries also found an association between HPV16/18 positivity and the severity of tissue lesions .Cytological atypia classified as ASCH was the most frequent, and in 40.8% of the cases, the need for colposcopic examination was justified. Among the women who received this cytological report, 50% tested positive for HPV, and a trend of CIN2/3 diagnoses was observed for the group of younger women. Other studies demonstrated the association of ASCH with precursor lesions and cancer according to HPV status.There is a clear association of ASCH results with factors of atrophy of the cervical epithelium among postmenopausal women, and when associated with negative HPV > 40 years old, they usually represent benign findings in histological investigation.Women with SCC and AI cytology, regardless of the HPV test status, had a 100% diagnosis of CIN2 +/CIN3 + , which reflects the high specificity of cytology directly associated with the degree of atypia. When associated with positive HPV, the cytology HSIL and ASCH presented a diagnosis of CIN2+ in 86 and 55.2% of patients, respectively. In the absence of HPV infection, CIN2+ detection decreases to < 50% for these same cytological results. These results agree with those of another study that reported a specificity of 86.6% in the diagnosis of CIN2+ among women with HSIL and positive HPV16/18.In view of the risks of developing precursor lesions and cervical cancer, the detection of HPV has shown great benefits, especially due to the increased sensitivity in detecting CIN3 + .Our study did not aim to evaluate the primary screening of cervical lesions by molecular tests but focused on the importance of stratifying the risk of women previously screened by cytological tests, considering age and the results of the investigation for HPV by genotyping. The results demonstrate the importance of additional molecular tests, especially among women with cytological tests classified as inconclusive changes and atypia of undetermined significance.In addition, the possibility of partial HPV genotyping may indicate the need for clinical procedures and personalized follow-up protocols for each patient with individual risk stratification. Although it was not the focus of the present study, the HPV vaccinated populations may also be an additional benefit with HPV genotyping for those viral types that are not protected by the vaccine.The results described here reinforce the importance of types 16/18 in the carcinogenesis of cervical cancer and add evidence regarding the participation of other viral types in a large proportion of women diagnosed with precursor lesions.The highest risks of precursor lesions and cervical cancer were found among women with positive HPV 16/18 tests and severe cytological atypia in population screening tests. Patients with cytology classified as ASCH may have a risk of CIN2+ stratified by the HPV test, and the number of positive tests will be higher among younger women < 40 years old.


This study aimed to investigate the differences in long-term oncological outcomes between high-risk human papillomavirus (HR-HPV) negative and HR-HPV positive early-stage cervical cancers.We retrospectively analysed 2061 cases of early-stage cervical cancer from the Chinese cervical cancer clinical diagnosis and treatment database. Kaplan-Meier curves were used to describe the survival outcomes of different HR-HPV infections. Cox proportional hazard regression model was used to analyze and determine independent risk factors.K-M analysis revealed no significant difference in 5-year OS between HR-HPV negative and HR-HPV positive groups . A significant difference was observed in 5-year DFS between the HR-HPV negative and HR-HPV positive groups . Cox proportional hazard regression model indicated that HR-HPV infection (negative vs. positive) was an independent factor influencing 5-year DFS after early cervical cancer surgery . HR-HPV infection (negative vs positive) was not an independent factor influencing 5-year OS after early cervical cancer surgery (OS: P=0.813). After 1:1 PSM pairing, there was no significant difference in 5-year OS and DFS between HR-HPV negative group and HR-HPV positive group . Cox multivariate analysis indicated that HR-HPV infection was not an independent factor influencing 5-year OS and DFS after early cervical cancer surgery .The tumour results of HR-HPV negative group and HR-HPV positive group were similar, after eliminating the differences in known variables that affect the oncological outcomes of cervical cancer. The treatment plan of HR-HPV positive cervical cancer is suitable for HR-HPV negative cervical cancer. Cervical cancer is the fourth most common malignant tumor that threatens women’s health worldwide. According to data from the International Agency for Research on Cancer, it is estimated that there will be approximately 604,000 new cases and 342,000 deaths due to cervical cancer globally in 2020. In low-income developing countries and regions, the number of new cases and deaths due to cervical cancer ranks second among female malignant tumor . Notablyhttp://apps.who.int/trialsearch/).This study was a multicentre, retrospective, observational study, a cervical cancer specialized disease database (n=63926) that covers consecutive patients with cervical cancer in 37 hospitals in mainland China treated since January 2004. The Southern Hospital Ethics Committee of Southern Medical University reviewed the establishment of the cervical cancer database (Ethics No. NFEC-2017-135). The identifier of the clinical trial is CHiCTR180017778  or magnetic resonance imaging (MRI), and a few patients are detected by positron emission tomography-CT. The oncological outcomes were estimated according to the recorded information, and the last day of the return visit or telephone follow-up was defined as the last follow-up. In this database, the final International Federation of Gynecology and Obstetrics (FIGO) stage was corrected by tumor size according to the FIGO 2018 staging system. Tumor size was determined by final pathological records.Entry conditions and grouping were as follows (1): Chinese female, age ≥ 18 years; (2) FIGO stage included IA1 (lymphatic vascular space infiltration (LVSI)-positive) - IIA2 stage (including unknown sub-stages of IA (LVSI-positive), IB, IIA); (3) histological type was squamous cell carcinoma, adenocarcinoma, and adeno-squamous cell carcinoma; (4) no preoperative adjuvant therapy was administered; (5) surgical approach was laparotomy; (6) operation method: IA1 (LVSI-positive), IA (LVSI-positive), and IA2 patients underwent QM-B type surgery, while the remaining patients underwent QM-C type surgery; (7) survival outcomes were available; (8) Availability of HR-HPV status. The exclusion criteria were as follows: (1) accidental discovery of cervical cancer, pregnancy complicated by cervical cancer, stump cancer, and other types of malignant tumors concurrently; (2) patients who did not meet the inclusion criteria.The staging rules for cervical cancer in FIGO 2018 are based on the combination of clinical imaging and pathological diagnosis results. The following four points should be noted for staging: 1. Two or more senior physicians should conduct a joint physical examination to clarify the clinical staging. When conditions permit, it is best to perform pelvic examination under anesthesia. 2. When there are differences in stages, the earlier stage shall prevail. 3. Allow imaging and pathological examination results to be used for staging. 4. The diagnosis of minimally invasive carcinoma must be made by an experienced pathologist based on cervical conization specimens.In this study, all patients were tested for HPV by in-house polymerase chain reaction (PCR). For cervical cancer patients with negative HR-HPV in the first screening, the second sampling and testing were conducted by the same method. Patients who tested negative twice were classified as HR-HPV negative patients.The 5-year DFS was defined as the date from the operation to the date of death due to cervical cancer or recurrence of cervical cancer. OS was defined as the date from the operation to the date of death from any cause. Patients with no evidence of recurrence or death were defined by the date of the last follow-up date or the last outpatient visit.Continuous variables are summarized by means ± standard deviation, while count variables are summarized by frequency and percentage. The comparison between the mean values of continuous variables is conducted using independent sample t-tests, and the comparison between the rates of counting data groups adopts χ 2 Test, rank variable adopts nonparametric rank sum test. The t-test and the χ 2 Test were used to analyze the clinical pathological characteristics and differences between the HR-HPV negative group and the HR-HPV positive group in early cervical cancer populations. The statistical software used was Statistical Product and Service Solutions 23.0 . The P-value <0.05 was considered statistically significant.Kaplan-Meier curves were used to describe the survival outcomes of different HR-HPV infections. Cox proportional hazard regression model was used to analyze and determine independent risk factors, and estimate the hazard ratio (HR) and 95% confidence interval (CI) of the impact of HR-HPV infection on the 5-year OS and DFS rates. In the Cox proportional risk regression models, we included clinical variables regarded as known factors affecting the oncological outcomes of cervical cancer .In the propensity score matching (PSM) analysis, patients in the HR-HPV negative group were matched to patients in the HR-HPV positive group based on propensity score to reduce bias. Then, a new group of patients was constructed with different HR- HR-HPV infection but similar other clinicopathological features. The propensity score of each patient to receive HR-HPV negative patients was calculated by logistic regression model, which included clinical variables of known factors affecting the oncological outcomes of cervical cancer . This propensity score was used for one-to-one matching cases with the nearest neighbor matching with variance of 0.02.A total of 2,061 cases met the enrolment criteria. The detailed data-filtering process is presented in Comparison of oncological outcomes between HR-HPV negative and HR-HPV positive surgical cases of early cervical cancer 2061 cases of cervical cancer in IA1~IIA2 stage met the initial inclusion criteria, including 153 cases in HR-HPV negative group and 1908 cases in HR-HPV negative group Table 1.The survival analysis revealed no significant difference in 5-year OS  between the HR-HPV-negative and HR-HPV positive groups, but there was a significant difference between the HR-HPV negative group and the HR-HPV positive group in the 5-year DFS   was not an independent factor influencing 5-year postoperative death due to early cervical cancer (OS: P = 0.813) Table 2.Comparison of oncological outcomes between HR-HPV-negative and HR-HPV positive surgical cases of early cervical cancer after further enrolment and matching.Meet the initial inclusion criteria and strictly follow the histological type, LVSI, postoperative adjuvant therapy 1:1 matching. The matching tolerance is 0, including 153 cases each in the HR-HPV positive and HR-HPV negative group Table 3.The survival analysis showed that there was no statistically significant difference between the HR-HPV negative and the HR-HPV positive group in the 5-year OS , and there was no statistically significant difference between the HR-HPV negative and the HR-HPV positive group in the 5-year DFS  Table 4,In this study, our previous study showed that HR-HPV infection (negative vs. positive) is an independent influencing factor for recurrence/death of early cervical cancer 5 years after surgery. However, after PSM matching to eliminate relevant confounders, we found that HPV infection was not an independent influencer of recurrence/death after early cancer surgery.This study was based on the real conditions in some parts of Chinese Mainland. in order to explore the impact of HR-HPV infection on the oncological outcome of early cervical cancer after laparotomy. The subjects were patients with stage IA1~IIA1 cervical cancer treated by laparotomy. This study was a multicenter study based on the real-world study, covering a large database of 63926 cases in 37 hospitals of different regions, levels and categories in China. It can reflect the real research situation of oncological outcomes of IA1~IIA1 cervical cancer patients with different HR-HPV infection in China after laparotomy.At present, studies have confirmed that cervical cancer is caused by HR-HPV infection. persistent infection with HR-HPV  can cause cancer of the cervix . HPV plaHowever, several recent studies have shown that HR-HPV infection has a paradoxical impact on the prognosis of cervical cancer. Liana et al. believe that HPV-negative cervical cancer patients were significantly more likely to have adverse outcomes than HPV 16/18-positive patients  . Ping LiThere are still some reports suggesting that there is no significant correlation between HPV infection and tumor prognosis. A recent systematic study found that there was no statistically significant association between HPV16 and/or HPV18 positive and overall survival or disease-free survival of cervical cancer . In a stWith the further study of cervical cancer, the relationship between HPV infection and prognosis has been changing. HPV infection is a decisive factor in the occurrence of cervical cancer, but in actual clinical work, a small number of cervical cancer patients have negative HPV detection. HPV negative squamous cervical carcinoma is very rare. HPV positivity of among adenosquamous cancers (ADS) may be up to 86%, the prevalence of HPV among adenocarcinoma (ADC) varies between the subtypes  , 23. TheIn order to avoid the influence of different pathological tissue types, LVSI, and postoperative adjuvant therapy on the tumor outcome of cervical cancer patients as much as possible, this study strictly controls them to eliminate the influence of the above differences on the tumor outcome of cervical cancer patients. However, there are still limitations of HPV detection in clinical practice. Sampling errors may be the primary cause of false negative HPV testing. For example, low cellularity (due to cancer necrosis and/or inflammation), influence of blood or lubricants, cell fixation or cell lysis may lead to classification errors. It is reported that the use of formalin fixed and paraffin embedded samples had an impact on DNA preservation and subsequent HPV-DNA test results, leading to the high prevalence of HPV negative tumors . The lowIn addition, many other factors may also be influencing factors that have no significant correlation between HPV infection and oncological outcome of IA1~IIA1 cervical cancer patients after abdominal surgery. The sample size is not large enough, the definition of HPV infection status  is different, the treatment plans received by cervical cancer patients are different, the statistical definition of survival rate is different, and there are many relative confounding factors in the actual clinical treatment process.Real-world research has garnered increasing attention in recent times, as exemplified by the “Basic Considerations for Real-World Evidence Supporting Drug Research and Development” issued by Chinese State Drug Administration in May 2019. Although treatments were not standardized, this report represents the status of cervical cancer diagnosis and treatment in China. Moreover, this study adopted PSM to eliminate baseline heterogeneity between groups. Crucially, this study more realistically reflected the treatment status and oncological outcomes of Chinese patients with HPV-negative and HPV positive IA1–IIA2 cervical cancer, providing evidence that may not be available from randomized controlled trials.This study has several limitations that stem from the retrospective nature of data collection. Although patients were matched based on perioperative factors to minimize bias, unknown confounding factors not captured in the dataset may have created residual bias in the results. Further, this study only focused on the analysis of survival outcomes of treatment groups with different HPV conditions after laparotomy for cervical cancer, and did not analyze the impact of specific conditions on the oncological outcome in postoperative radiotherapy, chemo-therapy and follow-up treatment. We look forward to a multicenter prospective study with a larger sample and a longer follow-up time.In conclusion, HPV-negative cervical precancerous lesions are not common in clinical practice, and their clinical characteristics and prognosis are not more favorable than those of HPV positive lesions. This study explored the impact of HPV infection on oncological outcomes of early cervical cancer by assessing patients with stage IA1-IIA2 cervical cancer undergoing surgery in parts of mainland China, encompassing 37 different regions, grades, and categories in China. This multicenter study based on real-world research contributes to previous gaps in the literature, as we provide novel insight into oncological outcomes after treatment for HPV-negative and HPV positive stage IA1–IIA2 cervical cancer in China. We aim to conduct further research in this area in order to provide a theoretical basis and novel ideas for individualized and differentiated treatment of different types of cervical lesions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by The Southern Hospital Ethics Committee of Southern Medical University (Ethics No. NFEC-2017-135). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.MH: Writing – review & editing. XS: Writing – original draft, Writing – review & editing. PaL: Writing – original draft. HZ: Writing – original draft. LS: Writing – original draft. WW: Writing – original draft. SJ: Writing – original draft. HW: Writing – original draft. PiL: Writing – review & editing. CC: Writing – review & editing.


Garcinia. Unlike α-mangostin, to date, there have only been a few studies on the biological activity and derivatization of β-mangostin. In this study, two novel glycosylated derivatives of β-mangostin were successfully synthesized via a one-pot enzymatic reaction. These derivatives were characterized as β-mangostin 6-O-β-d-glucopyranoside and β-mangostin 6-O-β-d-2-deoxyglucopyranoside by TOF ESI/MS and 1H and 13C NMR analyses. Beta-mangostin showed cytotoxicity against KB, MCF7, A549 and HepG2 cancer cell lines, with IC50 values ranging from 15.42 to 21.13 µM. The acetylcholinesterase and α-glucosidase inhibitory activities of β-mangostin were determined with IC50 values of 2.17 and 27.61 µM, respectively. A strong anti-microbial activity of β-mangostin against Gram-positive strains  was observed, with IC50 values of 0.16, 0.18 and 1.24 µg ml−1, respectively. Beta-mangostin showed weaker activity against Gram-negative strains  as well as Candida albicans fungus, with IC50 and MIC values greater than the tested concentration (greater than 32 µg ml−1). The new derivatives of β-mangostin showed weaker activities than those of β-mangostin, demonstrating the important role of the hydroxyl group at C-6 of β-mangostin in its bioactivity.Beta-mangostin is a xanthone commonly found in the genus  Unlike α-mangostin, so far, there have been few studies on the biological activity of β-mangostin and its new derivatization. There are a few reports on the anti-cancer activity of β-mangostin, such as growth inhibition in MCF-7 breast cancer cells 1H NMR spectra of compounds (1) and (2) showed an anomeric proton signal at δ 4.62 , confirming the presence of a sugar residue (δ 7.15 and 7.18 ppm for compounds (1) and (2), respectively) were shifted to those reported in the literature for β-mangostin [δ 2.27 and 1.35 ppm (d-2-deoxyglucopyranoside (β-Man2DG) and compound (2) was β-mangostin 6-O-β-d-glucopyranoside (β-ManGlc).The  residue . The posangostin . The sug1.35 ppm  20]. Th. Th1H NM. 3.2. 3.2.150 values ranging from 15.42 to 21.13 μM  and β-ManGlc (2), was investigated using an MTT assay ,12. Beta21.13 μM . Meanwhi50 value of 18.04 μM for 72 h treatment was similar to that reported by Syam et al. [50 of 16.5 μM for 48 h and 26 μM for 24 h treatment [et al. [50 = 81.9 μM) (The cytotoxic activity of β-mangostin on the MCF-7 cell line with an IC81.9 μM) . Accordi50 value varies widely. In HeLa cervical cancer cells, the IC50 value was 27.2 µM [50 values of 4.8, 5.8 and 10.3 µM for C6, U251 and T89G cells, respectively [The cytotoxic activity of β-mangostin has also been reported in other cell lines, and its IC 27.2 µM . On glioectively . Severalectively .et al. [d-2-deoxyglucopyranoside) and Man-6DG (α-mangostin 6-O-β-d-2-deoxyglucopyranoside), suppressed the growth and migration of hepatocellular carcinoma cell lines  [in vitro by downregulating hypoxia-inducible factor-1 and endothelial vascular growth factor [In this study, the cytotoxic activity of the β-mangostin glycosyl derivatives was lower than that of β-mangostin. However, further research on the mechanisms of apoptosis, metastasis and related signalling pathways is required to clarify the role of these new derivatives. It has been previously reported that β-mangostin did not cause cytotoxicity or cell cycle suppression in hepatocellular carcinoma cell lines  ,24 or Heet al. , two deo, HepG2) . In addih factor .50 values ranging from 15.42 to 21.13 μM, while α-mangostin exhibited IC50 values with broader range, from 0.24 μM to 81.9 μM depending on the cell line  of α-mangostin had similar toxicity to α-mangostin [Alpha-mangostin and β-mangostin exhibited different toxicities in the five tested cell lines. Beta-mangostin exhibited a fairly stable effect with ICell line . In termangostin , whereas. 3.2.2AChE plays the main role in hydrolysis of acetylcholine (90%) along with butyrylcholinesterase (BuChE) to regulate cholinergic neurotransmission –29. AChEet al. [The results in et al. .Table 3et al. [H-xanthen-9-one) while compound (5) -1,3,6-trihydroxy-8-isopentyl-7-methoxy-9H-xanthen-9-one) was the most potent inhibitor of BuChE with IC50 values of 5.26 μM and 7.55 μM, respectively [et al. [50 = 2.403 µM) in comparison with BuChE (IC50 = 31.221 µM) [According to Chi et al. , a serieectively . Similar [et al.  showed t.221 µM) .50 value of 2.17 µM) in comparison to synthesized derivatives. Similar natural xanthone compounds including α-mangostin and γ-mangostin had strong inhibitory activity against AChE with IC50 values of 2.14 µM and 1.31 µM, respectively [Currently, four drugs are used for treatment of Alzheimer's disease: donepezil, rivastigmine, galantamine and the glutamate antagonist memantine. Galantamine, an alkaloid extracted from the Amaryllidaceae family, is a naturally occurring AChE inhibitor. The advantage of natural AChE inhibitor substances is that in addition to anti-AChE activity, there are other beneficial activities, such as antioxidant activity –35. Seveectively . In addiectively –41.. 3.2.3d-glucose and reduced blood sugar levels. Therefore, one strategy for the treatment of diabetes is inhibition of α-glucosidase.Alpha-glucosidase is an important enzyme that affects the blood glucose levels. These enzyme inhibitors slow the release of 50 = 27.62 µM) when compared with the positive control acarbose (IC50 = 208.62 µM), with an inhibitory potency ratio of 7.55. All glycoside derivatives seemed to lose this activity, with IC50 values higher than the tested concentrations (greater than 124 µM for β-Man2DG and greater than 50 µM for β-ManGlc). This result is similar to that of α-mangostin, with α-glucosidase inhibitory activity at an IC50 of 31.1 µM, while synthetic compounds  lost this activity [From the results in activity .Table 450 values of the investigated substances, such as synthetic hydroxylxanthones [et al. [50 = 1.5 μM) > α-mangostin (IC50 = 5.0 μM) > β-mangostin (IC50 = 14.4 μM) [The structural relationship and α-glucosidase inhibitory activity were also determined by analysing the ICanthones . The resanthones . Similar [et al. , showing14.4 μM) . This ma. 3.2.4B. subtilis and L. fermentum with the same MIC values of 0.5 μg ml−1 and IC50 values of 0.16 μg ml−1 and 0.18 μg ml−1, respectively. For the remaining strains, β-mangostin and its derivatives (1) and (2) exhibited weak inhibitory activity, with MIC values higher than the tested concentrations.The results in S. aureus  and Enterococcus , β-mangostin showed no antibacterial activity (MIC > 200 μg ml−1), whereas α- and γ-mangostin exhibited the highest activities among the eight xanthones studied (MICs ranging from 3.13 to 6.25 μg ml−1) [In a previous report on the antibacterial activity of xanthone against μg ml−1) . Based oμg ml−1) . Therefo. 4d-2-deoxyglucopyranoside (1) and β-mangostin 6-O-β-d-glucopyranoside (2), were synthesized by enzymatic methods. Although the biological activities of these derivatives have been reduced compared to those of β-mangostin, further research is required to clarify the role of the functional groups of these derivatives in the mechanisms of anti-cancer, antibacterial, antidiabetic and anti-Alzheimer effects.The natural compound β-mangostin has potential activities associated with cancer cell toxicity, inhibition of AChE and α-glucosidase, and Gram-positive antibacterial activity. Two new derivatives of β-mangostin, β-mangostin 6-O-β-


Several studies showed that human papillomavirus (HPV) affects male fertility, but its impact on female fertility and in vitro fertilization (IVF) outcome is not yet clear.Objective of this observational, prospective, cohort study was to evaluate the prevalence of HPV infection in women candidate to IVF, and the effects of HPV infection on the kinetic of embryonic development and on IVF outcome. A total number of 457 women candidate to IVF were submitted to HR-HPV test; among them, 326 underwent their first IVF cycle and were included in the analysis on IVF results.8.9% of women candidate to IVF were HPV-positive, HPV16 being the most prevalent genotype. Among the infertility causes, endometriosis was significantly more frequent in HPV-positive than in negative women . Granulosa and endometrial cells resulted HPV-positive in 61% and 48% of the women having HPV-positive cervical swab, respectively. Comparing HPV-positive and negative women at their first IVF cycle, no significant difference was observed in the responsiveness to controlled ovarian stimulation (COS) in terms of number and maturity of retrieved oocytes, and of fertilization rate. The mean morphological embryo score was comparable in the two groups; embryos of HPV-positive women showed a quicker development in the early stages, with a significantly shorter interval between the appearance of pronuclei and their fusion. In the following days, embryo kinetic was comparable in the two groups until the early blastocyst stage, when embryos of HPV-positive women became significantly slower than those of HPV-negative women. Overall, these differences did not affect live birth rate/started cycle, that was comparable in HPV-positive and negative women .(a) the prevalence of HPV infection in women candidate to IVF is similar to that observed in the general female population of the same age range; (b) HPV infection migrates along the female genital apparatus, involving also the endometrium and the ovary, and perhaps participates in the genesis of pelvic endometriosis; (c) HPV slightly affects the developmental kinetic of in vitro-produced embryos, but does not exert an effect on live birth rate. Human papillomavirus (HPV) infection represents a quite frequent sexually transmitted disease, as half of the sexually active males and females may be affected during lifetime . The maiHPV is a DNA-virus with specific tropism for human epithelial tissue. There are almost 200 HPV different genotypes, classified as low risk (LR-HPV) or high risk (HR-HPV) on the basis of their ability to induce neoplastic transformation of the infected cells , 5. AlthRecent studies suggest that HPV may affect fertility and perhaps also the efficacy of in-vitro fertilization (IVF) –11. In tThe effect(s) of HPV on female fertility are unclear. It was reported that HPV-related cervical cytology abnormalities are more common in infertile women than in the general population . FurtherThe present study was designed as an observational prospective cohort study, and was aimed at evaluating both the prevalence of HPV in women undergoing IVF, and the impact of HPV infection on embryo morphokinetic and IVF outcome.A total number of 457 women with couple’s infertility, aged 30–43 years, in the waiting list of IVF program at the Physiopathology of Reproduction and IVF Unit of S. Anna Hospital (University of Turin), were submitted to a cervical swab for HR-HPV test. Among them, 326 underwent their first IVF treatment in the study time period (January – October 2021) and were included in the analysis of IVF outcome. For those who resulted positive for HPV in the cervical swab, HPV genotyping and cervical cytology (PAP test) were performed. Moreover, in case of cervical positivity, the presence of HPV-DNA was also tested in endometrial cells (collected during a mock embryo-transfer before starting IVF procedure) and in granulosa cells .The study was carried out in accordance to the Declaration of Helsinki and was authorized by the local Ethical Committee (Ref. number 0000055). Signed informed consent was obtained from all patients.Cervical sampling was accomplished approximately one month before starting IVF procedure using a specific sampling set , gently scraping cells from the cervical canal and from the internal and external portion of the cervix. Cervical samples were stored in a 10 ml transport medium , and labelled with an ID number.Briefly, cervical samples were processed by QIAsymphony®DSP , an automatic system based on magnetic particle technology, for the isolation of human cervical cells from the preserving solution. Subsequently, isolated cervical cells were tested by the Hybrid Capture 2 assay High-Risk HPV DNA test  by using the Rapid Capture System®  according to the manufacturer’s instructions. HC2 test is a nucleic acid hybridization assay with signal amplification for qualitative detection of 14 HPV types in cervical cells . HC2 results were expressed as the ratio between the specimen’s light emission (relative light units [RLU]) and the mean of three concurrently tested positive controls (CO). Samples were considered positive when the ratio (RLU/CO) was ≥ 1  , and 2 µTo test HPV presence, a 150 bp L1 region was amplified with GP5+/GP6 + consensus primers, allowing detection of a broad range of HPV types , 34. ConSamples testing positive for GP5+/GP6 + PCR were genotyped by using INNO-LiPA® HPV Genotyping Extra II Amplification test . This assay can identify 32 different HPV genotypes at the same time using reverse hybridization, after a preliminary step of amplification. PCR reactions were carried out in thermocycler (GeneAmp PCR System 9700) using SPF10 biotinylated consensus primers.The biotinylated amplicons were denatured and hybridized with specific oligonucleotide probes fixed on membrane strips. After reverse hybridization, they were incubated with BCIP/NBT chromogen at 20–25° C on a shaker yielding a purple precipitate, and the results were visually interpreted .Controlled Ovarian Stimulation (COS) with gonadotropins was accomplished according to standardized protocols. Follicular growth was monitored by serial transvaginal ultrasound (TV-US) and measurement of circulating estradiol (E2) every second day from day 7. When at least two follicles reached 18 mm mean diameter, with appropriate E2 levels, a single subcutaneous injection of 10.000 IU hCG  was administered to trigger ovulation.Transvaginal US-guided oocyte pick-up (OPU) was performed 35–37 h later under local anesthesia . Follicular fluid was aspirated and immediately observed under stereomicroscope. Cumulus-oocytes complexes (COCs) were washed in buffered medium  and incubated in controlled atmosphere  using appropriate culture medium .The same day of OPU, semen samples were examined to assess sperm concentration, motility and morphology according to the World Health Organization guidelines ; then they were prepared by density gradient centrifugation in order to select normally motile and morphologically normal spermatozoa. A few hours after OPU, oocytes were fertilized by conventional IVF or ICSI (Intra-Cytoplasmic Sperm Injection) according to semen parameters.Normal fertilization was assessed 16–18 h later (Day 1) by evaluating the presence of two pronuclei (2PN) and the extrusion of the second polar body.Embryos were cultured in pre-equilibrated cleavage medium  overlain with mineral oil up to day 3 of development; at this stage, a change of medium was performed and a new one  was kept until the blastocyst stage (day 5–6). Embryo morphological evaluation was first performed on day 2 using the Integrated Morphology Cleavage Score (IMCS) , and theEmbryo transfer (ET) in uterus was performed on day 5 using the soft catheter Sydney Guardia  under transvaginal US guidance, as previously reported by our group .The primary endpoint for power calculation was the prevalence of HPV infection among infertile women candidate to IVF. Accordingly, the power calculation was made as follows: considering the available epidemiologic data, we hypothesized that HPV infection could be 10% more frequent among women undergoing IVF than in the general population, and 450 subjects would have allowed to reach a statistical power of 80% with 0.05 alpha error.2 test , and the results were expressed by mean and standard deviation or by percentage. All statistical tests were for unpaired data and two-sided; a p-value < 0.05 was considered significant.Analyses were performed subdividing women undergoing IVF according to the presence or absence of HPV infection in the cervical swab. The data normal distribution was assessed using Kolmogorov-Smirnov test. Comparison between HPV-positive and -negative women was performed using the Student t-test (continuous variables) or the χAmong the 457 recruited women, 41 (8.9%) resulted HPV-positive at the cervical swab. Their mean age was 36.5 ± 3.7 years, and HPV prevalence was higher in younger women and decreased linearly with age, similarly to what observed in the general population of the same age range (not shown). Among infertility causes, endometriosis was the only to be significantly more frequent in HR-HPV-positive than in negative women .In case of HPV-positivity, a Pap Test was performed to evaluate the presence of cytological lesions: 80% of patients resulted negative for intraepithelial lesions or malignancy (NILM), 16.6% and 3.3% had LSIL or HSIL, respectively. After genotyping, HPV16 was the most prevalent genotype (17.8%), followed by HPV18 and 59 (8.21%), 45 and 66 (6.85%), 31, 33, 51 and 52 (4.11%). According to what suggested by experts of the Low Genital Tract Infections Unit of our Institution, patients with HSIL were temporarily excluded from IVF program, whereas patients with LSIL were admitted.Interestingly enough, granulosa and endometrial cells resulted positive for HPV in 61% (25/41) and 48% (20/41) of women who were HPV-positive at the cervical swab, respectively. Of note, endometrial biopsy was performed using a modified Vabra catheter having an outer part in order to avoid any contamination from the cervix.Basal clinical characteristics and IVF outcome of the 326 patients who underwent their first IVF in the time period of the study are summarized in Tables The ovarian responsiveness to COS was comparable in the two groups, leading to similar oocyte yield; even egg competence appeared similar, with comparable fertilization rate, cleavage rate, and embryo availability Table . The embOverall, 845 embryos were analyzed using the Time-Lapse System; 446 reached the blastocyst stage, among which 202 were obtained from HPV-positive and 244 from negative women Table . In the The IVF outcome of patients with or without HPV infection in granulosa cells was comparable for all clinical parameters, including the rate of mature oocytes , that of fertilized oocytes , and the embryo morphological score . Also the morphokinetic analysis of embryos belonging to patients with granulosa-positive or negative cells was not significantly different, as well as the LBR/SC (not shown).Differently, HPV-positivity of endometrial cells resulted in a much lower LBR/SC (14.7%) than that observed in women with HPV-negative endometrium (42.8%), but the difference was not statistically significant, likely due to the limited number of observations.The role of HPV in human infertility was previously investigated in a few studies. A higher incidence of HPV infection was demonstrated in the semen of infertile men –42, and Differently, the effect(s) of HPV on the woman’s reproductive potential was never clearly defined, although some studies suggested that HPV might negatively affect woman’s fertility. It was reported, in fact, that HPV-related cervical cytology abnormalities were more common in infertile women than in the general population , and a cIn the present study, we observed a group of 457 women candidate to IVF for various indications: all underwent HPV testing on a cervical swab, and the prevalence of HPV infection was 8.9%, in line with the general female population of the same age range in our geographical area. A higher incidence of HPV infection was observed at younger age, and even this was common to both infertile women and the general female population. Different causes of infertility were similarly distributed in couples with HPV-positive or negative cervical swab, with the exception of endometriosis, that was significantly more frequent among HPV-positive women. As hypothesized by others , our molAs for the efficacy of IVF, we evaluated only first IVF cycles ending with blastocyst transfer in order to compare homogeneous groups of HPV-positive and negative women, excluding other factors potentially able to influence IVF outcome. Overall, our observations do not suggest any negative effect of female HPV infection on the key steps of IVF procedure: in fact, HPV-positive women had a similar responsiveness to COS, similar oocyte maturation rate, fertilization rate and embryo morphological quality than their negative controls. Finally, the clinical pregnancy rate and the miscarriage rate were comparable , leading to a similar LBR/started cycle.Using the time-lapse technology to detect any possible, even subtle, effect of HPV on embryonic development, we observed only small differences, not leading to a significantly different clinical outcome. Indeed embryos of HPV-positive women showed a quicker development in the early stages, but later on, in the late stages of growth to blastocyst, they became slower than those of HPV-negative women, in agreement to what observed in the mouse by Henneberg et al. , who repIn patients with HPV in granulosa cells, the virus did not seem to affect follicular development, oocyte maturation and competence. Differently, the presence of HPV in the endometrium was apparently associated with a lower embryo implantation rate, but this observation was made on such a small number of cases, that it definitely needs to be eventually confirmed by novel studies.In conclusion, the present study (a) confirms that the prevalence of HPV infection in women candidate to IVF is comparable to the general female population of the same age range, (b) shows that HPV can move along the female genital apparatus and could be somehow involved in the genesis of pelvic endometriosis, and (c) shows that HPV slightly affects the developmental kinetic of in vitro-produced embryos, without significantly affecting the main IVF outcomes, including the live birth rate per started cycle.


We then evaluated HPV+ HNSCC, which lacks premalignant tissue, and uncovered its previously unknown progression, identifying early drivers. We converted relative timing estimates of driver mutations and HPV integration to years before diagnosis based on a clock-like mutational signature. We associated the timing of transitions to aneuploidy with increased intratumor genetic heterogeneity and shorter overall survival. Our approach can establish previously unknown early genetic progression of cancers with unobtainable premalignant tissue, supporting development of experimental models and methods for early detection, interception and prognostication.Analysis of premalignant tissue has identified the typical order of somatic events leading to invasive tumors in several cancer types. For other cancers, premalignant tissue is unobtainable, leaving genetic progression unknown. Here, we demonstrate how to infer progression from exome sequencing of primary tumors. Our computational method, PhylogicNDT, recapitulated the previous experimentally determined genetic progression of human papillomavirus-negative (HPV Getz and colleagues demonstrate a computational approach to estimate the timing of genomic drivers in early tumor progression from cancer types that do not have premalignant disease, using HPV-negative and HPV-positive head and neck squamous cell carcinoma as examples. That order can provide clues to etiology and cell-intrinsic mechanisms in tumorigenesis, informing studies of how normal tissue becomes a tumor, and can also provide ways to detect and treat early disease stages and identify early clonal events as promising targets for therapy of later invasive tumors3.Deciphering the temporal order of genetic lesions throughout the steps of cancer progression has long been a goal of cancer research13. However, there are many cancer types with poorly defined, undetectable or difficult-to-biopsy premalignant lesions22 whose genetic progression thus remains speculative.For cancers with well-defined pathologic progression from normal tissue, for example, adenoma through carcinoma in situ to invasive carcinoma, analysis of lesions along that trajectory has provided corresponding genetic progression models24. We recently developed PhylogicNDT, an integrated suite of tools, to reconstruct the clonal architecture and relative timings of genetic events using a coherent probabilistic framework26 and have successfully applied it in several contexts29.Here, we demonstrate how to infer the typical order of genetic events in a cancer type from exome sequencing of primary tumor samples taken long after cancer initiation. The genome of an invasive tumor includes information about its initial genetic progression, which is recorded in patterns of somatic mutations and copy number changes as cells evolve into an invasive cloneWe asked whether more readily available whole-exome sequencing (WES) of primary tumors could similarly provide information on genetic progression.8 determined the genetic progression of classic HNSCC, typically associated with tobacco and alcohol use, from loss-of-heterozygosity (LOH) analysis along the pathologic progression from normal tissue to initial lesion, dysplasia and carcinoma in situ to invasive carcinoma , which provides a unique opportunity to validate and extend such progression models. A quarter of a century ago, Califano et al.oma Fig. . That mo38 abbreviated HPV+ HNSCC) has remained unknown39. Premalignant lesions have not been identified reliably for HPV+ HNSCC40. The early genetic events in classic HPV– HNSCC that disrupt the CDKN2A and TP53 loci are infrequent in HPV+ HNSCC, as the E7 and E6 viral protein products provide corresponding tumorigenic functions42. Whether HPV+ HNSCCs converge thereafter to the same genetic progression as HPV– HNSCC is unclear. Computational reconstruction provides a unique opportunity to determine the genetic progression of HPV+ HNSCC and explore whether the well-known differences between HPV+ and HPV– HNSCC in response to therapy35 are associated with their different genetic paths to invasive cancer.In contrast to classic HNSCC, the genetic progression of the increasingly prevalent and clinically important HNSCC associated with human papillomavirus  identify the genetic progression of HPV+ HNSCC. We started with HNSCC from The Cancer Genome Atlas (TCGA) project43 and collected samples from 43 additional oropharyngeal HNSCCs to increase the representation of HPV+ cases .We thus applied PhylogicNDT to WES data from primary HNSCC to (1) demonstrate the reconstruction of genetic progression in HPV– HNSCC, extending the number and details of timed events, and describe the previously unknown order of genetic progression of HPV+ HNSCC. We document the timing of different types of aneuploidy and their strong association with intratumor heterogeneity and survival. Additionally, we converted the relative timing estimates of main drivers and HPV integration to years before diagnosis based on a clock-like mutational signature.Here, we report the validation of our computational approach in HPVThis report provides a framework for studying genetic progression of cancer types for which premalignant tissue is unobtainable. This framework enables uncovering mechanisms of cancer initiation and early development in types of cancer for which the rarity, inaccessibility, or lack of premalignant tissue previously made their genetic progressions undecipherable.TP53 (red) leading to a R248Q protein alteration was seen in both final copies of arm 17p, consistent with the mutation preceding the duplication.The data in Fig. 26. Examples of inferred orders of events in two HPV– tumors are shown in Fig. π, with π = 0 and π = 1 reflecting the times of the first and last clonal events, respectively26.Relative timing of all genetic events within each tumor is estimated across the genome by applying these principles in a probabilistic computational frameworkπ value distributions) of selected driver events across a cohort of tumors to obtain a posterior distribution for the typical relative timings of the events. The median relative timing (mRT) for each genetic event provides a point estimate for ordering events into a genetic progression model.Next, PhylogicNDT combines the within-tumor timing , progressing to dysplasia with −17p (which includes TP53) and −3p. The transition to carcinoma in situ is associated with −13q, −11q and −14q, and, finally, progression to invasive cancer is associated with −6p, −8 and −4q. Even the partial orderings of within-tumor timing estimates were generally consistent with the Califano model . Indeed, the early loss of arm 8p, not evaluated by Califano et at., agreed with its frequent loss in early oral cavity dysplasia31.Computational timing analysis, based solely on primary tumor specimens, recapitulated and substantially expanded the empirical progression model of Califano et al. Fig. . NotablyCDKN2A tumor suppressor that is also inactivated by frequent (23.5%) and early (mRT = 0.27) point mutations or homozygous deletions . Nearly all HPV– HNSCC  had at least one such disruption of CDKN2A.The first and most prevalent event was –9p , the location of the TP53 was also frequent and early via mutation  or −17p , with at least one such event in 369 HPV– tumors (87.6%). Our timing of TP53 and CDKN2A single-nucleotide variations (SNVs) agreed with the long-appreciated presence of TP53 (ref. 30) and CDKN2A (ref. 32) mutations in dysplastic tissue of the head and neck. The third of the earliest genetic events identified by Califano et al., −3p, occurred in 339 tumors (80.5%); its mRT of 0.12 was similar to that of TP53 disruptions.Disruption of PIK3CA locus, was later (mRT = 0.51), suggesting a role in progression rather than initiation. Other arm-level gains and losses covered a wide range of relative timings.We further identified prevalent and early −9q  and −8p  events. By contrast, the frequent +3q event (55.6%), containing the 34.Based on the Califano et al. model, we mapped relative timings to pathologic progressions. Precursor lesions become dysplastic near an mRT of ~0.25, between timings of −17p (0.18) and −13q (0.33). The transition to carcinoma in situ is near 0.7 (mRT of −14q and −4q) and that to invasive carcinoma at or after ~0.75 . Our timing of +3q (mRT = 0.51) and +8q (mRT = 0.57) before this estimate of the transition to carcinoma in situ agrees with findings  of these gains in dysplastic tissueFBXW7, NOTCH1, CASP8, FAT1, NSD1, HRAS, EP300, CREBBP, KMT2D, PIK3CA, NFE2L2, HLA-A, SPEN and KMT2C. mRT values of events in the first nine genes were less than 0.69, supporting early roles in tumorigenesis before carcinoma in situ. Although individually of low prevalence (each <25%), a mutation in at least one of these first nine genes occurred in over 60% of HPV– HNSCCs (260/421). Mutations in PIK3CA, NFE2L2, HLA-A, SPEN and KMT2C were at later mRT values, presumably late in pathologic progression.We further established relative timing of SNVs in several lower-prevalence driver genes (in order of increasing mRT): – HNSCCs. These whole-genome events were late in progression  and 0.80 (WGD); Fig. Finally, whole-genome events, leading to whole-genome copy number profiles that are predominantly triploid (WGT) or whole-genome doubling of both alleles (WGD) resulting in tetraploid samples, occurred in nearly half (47.7%) of HPV– HNSCC, we turned to HPV+ HNSCC, where pathologic progression has not been identified44. These tumors typically arise in crypts of tonsils and related lymphoid tissues at breaks of the basement membrane46 that allow viral access to infect basal epithelial cells and ready entry of transformed cells into the lymphatics . Median tumor purity was 0.48 (range of 0.12 to 0.98), and median ploidy was 2.14  for each HPV+ tumor via PhylogicNDT SinglePatientTiming . Arm-level events −13q and +8q were also both highly prevalent (>40%) and early , suggesting frequent roles in early HPVTRAF3, ZNF750 and NOTCH1 had mRT values of 0.55 or earlier, while others had mRT values of 0.75 or greater. Except for PIK3CA (mRT = 0.83), none of the 13 potential driver genes were mutated in more than 15% of HPV+ tumors, although nearly three-quarters of tumors  had a mutation in at least 1 potential driver gene. Only 10 of 101 HPV+ HNSCCs had genome-wide events leading to WGT or WGD.Of 13 low-prevalence potential driver genes, mutations only in + HNSCC, the HPV E7 and E6 viral gene products inactivate the functions of CDKN2A and TP53, whereas in HPV– HNSCC, those functions are lost via somatic events42. We sought to determine other similarities and differences in the progression of these two major HNSCC subtypes.In HPV+ and HPV– HNSCC. Even beyond expected differences associated with CDKN2A and TP53, 20 genetic events were significantly more prevalent in HPV– HNSCC, including SNVs in FAT1, NOTCH1, CASP8, HRAS, the −3p highly prevalent in HPV– HNSCC and 15 other arm-level somatic copy number alterations . Notably, whole-genome events leading to WGT or WGD were nearly five times more prevalent in HPV–  than in HPV+ tumors . However, 14 events were found at higher prevalence in HPV+ than in HPV– HNSCC .First, we compared the frequencies of genomic events in HPV+ and HPV– tumors, with APOBEC (SBS2/SBS13) and aging signatures (SBS1/SBS5) enriched in HPV+ tumors and the smoking signature (SBS4) enriched in HPV– tumors.Comparing mutational signatures showed that the prevalence of all signatures differed between HPV+ and HPV– cohorts  and 12 more at q < 0.2.Next, to compare the timing of events, we examined 42 events present in at least three cases in each of the HPVrts Fig. . We combmutation . We dete– progression, −9p, −17p and mutation of TP53 (CDKN2A or TP53 inactivation), were earlier than in HPV+ tumors, as expected from the different mechanisms for inactivating the function of these tumor suppressor genes. Lower-prevalence mutations in CREBBP, NOTCH1, FBXW7, FAT1 and NFE2L2 were also earlier in HPV– tumors.Early high-prevalence genetic events in HPV+ progression, +3q and −11q, were significantly earlier in HPV+ than in HPV– progression, supporting their roles as early HPV+ driver events. WGD in HPV+ HNSCC was also found to occur significantly earlier than in HPV– tumors, despite the much lower prevalence of WGD in HPV+ tumors. Other arm-level events earlier in HPV+ progression were gains +1q, +17q, +6p, +20p, +20q and +8q and loss of 6q.The early and high-prevalence genetic events in HPV– HNSCC, prevalence of some genetic events differed markedly among the major anatomic subdivisions oral cavity, oropharynx and larynx. NSD1 mutations were preferentially laryngeal, while almost all CASP8 mutations were in oral cavity tumors (Supplementary Table – HNSCC subsets with mutations in NSD1 (n = 54) or CASP8 (n = 50), comparing their timings against other HPV– tumors , which showed no significant subsite preference  activity in NSD1-mutated tumors . Nearly two-thirds of driver events (46 of 65) had differential prevalence  and mutations in HRAS (36% versus 2.7%), FAT1 (52% versus 20.8%), EPHA2 (18% versus 2.4%), EP300 (18% versus 4.6%) and NOTCH1 (38% versus16.2%). TP53 (50% versus 82.2%) and NSD1 (4% versus 14%) mutations and losses −17p (18% versus 59.3%), −3p (30% versus 87.3%) and −11q (8% versus 46.4%) were significantly less prevalent. CASP8-mutated tumors thus are evidently driven more by mutations in multiple driver genes than by chromosome arm gains or losses. Single-individual timing diagrams of CASP8-mutated cases show early CASP8, TP53 and CDKN2A mutations  and lower prevalence of +17p (19.0% versus 31.0%) and +8p  score as a measure of genetic heterogeneity, defined as the median-normalized width of the distribution of mutant allele fractions , reflecting the genomic regions with copy numbers different from the modal copy number level, provided an overall measure of aneuploidy. Noticeably, this measure was near linearly related to the MATH score  single-tumor data  were available for 28 oropharyngeal tumors newly collected for this study. Although the mutations and subclonal composition could differ substantially between paired samples  in HPV+ tumors. We found that these rates were also similar among tumor anatomical sites  mutations  as a function of participant age at diagnosis .Real-time timing estimates of WGT and WGD support a model with an abrupt transition from the WGD state to WGT  fits the data well Fig. . About 3The relationship between FGA and the timing of the WGD/WGT event  occur around 30–40 years before diagnosis, while HPV+ early events (+3q and +9q) occur around 20–30 years before diagnosis  and analyzed multiplicities and CCFs  tumors are typically diagnosed many years after key driver events, which can happen at different ages , high FGA showed an association with overall survival similar to that of high MATH. High- versus low-score hazard ratios ) were FGA  and MATH . Aneuploid tumors or tumors with whole-genome events leading to WGD or WGT had weaker associations with overall survival . Replacing MATH with FGA or WGD/WGT . Aneuploidy class analyzed similarly was just above the significance threshold . Applying the Akaike information criterion (AIC), MATH  had the strongest association with survival, although genomic instability measures had similar AIC scores .To evaluate these associations in more individuals, including those with HPVWGT Fig.  in an ot+ HNSCC.Early genetic progression and aneuploidy development in tumors thus can be modeled computationally using WES data from primary tumors, providing findings about processes strongly associated with survival. Timing analysis is possible even when premalignant tissue is unobtainable, as in HPV+ HNSCC fills a major gap in knowledge about the progression of genetic events in this increasingly prevalent and clinically significant disease37, a cancer whose lack of premalignant tissue frustrated prior attempts at timing40. Analysis of HPV– HNSCC verified that our computational approach could recapitulate the progression in classic HPV– disease54 and extend the progression model to additional events in significant subsets of HPV– disease. These results on cohorts of primary HNSCC specimens suggest that this approach could be applied to other types of cancer whose early genetic progression is unknown because premalignant tissue is difficult or impossible to obtain.Our analysis of HPV– HNSCC to a total of 61 SNV or copy number alteration events, of which 17 had >40% prevalence. Our timing estimates agreed well with the Califano et al. model8 and with more recent studies that refined and extended it34. Comparing predicted mRT values , with over 60% of HPV– HNSCCs having a mutation in at least one of these genes. Of those, we found anatomic and genetic differences among tumors, with CASP8 mutations almost solely in the oral cavity and NSD1 predominant in laryngeal tumors. By contrast, tumors with NOTCH1 mutations, long suspected to play a role in HPV– HNSCC, showed no anatomic preference or frequency or timing differences from other HPV– HNSCC. Genes with mutations at later mRT  are more likely to be important for progressing to later stages of the disease.Additionally, we identified genes with early mutations that likely play roles in HPV+ HNSCC, gain of chromosome arm 3q and loss of arm 11q are both earlier and more frequent than in HPV– HNSCC, indicating important early roles during HPV+ progression. We estimate that genomic integration of HPV viral DNA occurs early in tumor development, consistent with HPV being the main contributor to cancer initiation.In HPVPIK3CA locus, whose activation by copy number gain or mutation occurs often in HNSCC56; 74% of HPV+ and 62% of HPV– HNSCC showed genetic alterations related to the PIK3CA locus. Gain of 3q was found at an earlier mRT in HPV+ tumors (0.05) than in HPV– tumors . As HPV E6 leads to inactivation of the p53 protein product42, and TP53 mutations occur in only 3% of HPV+ HNSCC, loss of additional genes on 17p presumably are important. Rather than the very early loss seen in HPV– tumors, 17p loss occurs at intermediate stages of HPV+ disease progression (mRT of 0.66), suggesting that those genes are involved in later stages of tumor development.Similarly surprising in HPV– disease and 20–30 or more years in HPV+ tumors, with HPV integration also early in the development. WGD-to-WGT conversion seems to follow an abrupt transition model, with a specific transition probability per year.Using a clock-like mutational signature to convert relative event timing estimates to years before diagnosis, we identified that early founding events can occur as many as 30–40 years before tumor diagnosis in HPV63. Identification of WGD timing before invasion might be used to identify HPV– HNSCC at higher risk of progression. Other early drivers can be the basis of additional therapeutic trials, including in the preventive setting for high-risk individuals.This work helps clarify the nature of outcome-associated genomic disruption measures. Almost all tumors with a whole-genome event leading to WGT or WGD had high MATH values. Similar to high MATH, high FGA was also associated with shorter overall survival in HNSCC when therapies and standard clinical and pathologic characteristics were taken into account. Measures of genomic disruption can be used to classify individuals into high- and low-risk groups for monitoring disease with strategies such as minimal residual disease assays22, including multiple rare tumor types, could be of great clinical importance. Many such cancers have uncertain etiology, with effective treatment avenues less well defined than in more prevalent tumors. Establishment of progression trajectories, early drivers and similarity of progression to other cancer types can lead to development of improved disease models, better treatment strategies and clinical management and more accurate prediction of disease course.Applying our approach to other cancers whose premalignant tissue is seldom or never obtainableThis research complied with all relevant ethical regulations. Human research on individuals at Massachusetts Eye and Ear (MEE) was approved by the MEE Human Studies Committee under protocol HSC 11-024H, with informed consent obtained from participants. Participants were not compensated.43 and 45 individuals from MEE with oropharyngeal tumors whose WES data met quality control criteria (see below). TCGA clinical data were as in previous work51. Clinical data from MEE corresponding to TCGA data fields were extracted from participant records. Median time to death for 218 participants was 13.7 months (IQR of 8.4 to 26.5 months); follow-up time for the 313 last known to be alive was approximately twice as long . HPV status of TCGA tumors was assessed by RNA sequencing43; for MEE tumors, clinical HPV annotations were used. Clinical data are provided as Source Data for Fig. We analyzed data from 486 individuals with head and neck cancer in TCGA43. Tumor portions from MEE participants were frozen at liquid nitrogen temperature; participant-matched frozen blood provided control DNA for assessing tumor-specific mutations. Twenty-eight of the MEE tumor samples had two subsamples taken for separate processing.TCGA tumor and control tissue or blood samples were as previously reported25. Analysis proceeded from paired aligned bam files, which were inputted into a standard WES somatic variant-calling pipeline, including MuTect for calling somatic SNVs64, Strelka for calling small insertions and deletions65, deTiN for estimating tumor-in-normal contamination66, ContEst for estimating cross-participant contamination67, AllelicCapSeg for calling allelic copy number variants68 and ABSOLUTE for estimating tumor purity, ploidy, CCFs and absolute allelic copy number68. Artifactual variants were filtered out using a token panel of normals filter, a blat filter and an oxoG filter. For TCGA tumors in which the WES data did not yield sufficiently high-quality copy number data from AllelicCapSeg (361 tumors), we used HapSeg on single-nucleotide polymorphism (SNP) arrays as a substitute step. MEE tumors underwent a similar variant-calling pipeline as the TCGA tumors, but we did not substitute any AllelicCapSeg results with HapSeg on SNP arrays.Tumors from TCGA had undergone WES or WGS, as described– and HPV+ subsets using SignatureAnalyzer70 on the somatic SNV calls resulting from the variant-calling pipeline.We performed SBS signature analysis separately on the HPV26.We used PhylogicNDT to estimate relative event timing within individual tumors (SinglePatientTiming) and to combine timing information among tumors (LeagueModel). This analysis suite has been described in detail elsewhere68 to determine purity and ploidy, allele-specific SNV multiplicity estimates and purity-corrected copy number variation values from WES or WGS reads were used to set a within-tumor partial ordering of events. For example, on a chromosomal region that has been doubled, a tumor-specific mutation present at two copies is timed before the doubling, while a mutation present at only one copy is timed after the doubling. Tumor-specific genetic events are mapped on a π scale (as a probability distribution) from 0 to 1 (refs. 26), representing the first and last clonal genetic events. Estimated distributions are corrected for power to detect based on coverage profiles. The proportion of clonal events per megabase occurring before each genetic event in a tumor is defined as the π score for that event. Clonal events that cannot be timed are assigned a uniform π distribution, and the last clonal and all subclonal events are assigned π scores of 1, represented as a delta function δ(π − 1). Uncertainties arising from sequencing are incorporated into a distribution of π values for each event within the tumor. Thus within-tumor timing uses information from all tumor-specific SNV and copy number variation events.After ABSOLUTE analysisπ-score distributions for each genetic event shared among a number of tumors into a consensus genetic relative timing progression. This consensus relative timing is generated by repeated sampling from the single-tumor π distributions of the shared events among multiple subsets of tumors. On this relative timing scale, a value of 0 represents the earliest shared event, and 1 is the last clonal shared event. The result is a distribution of relative timing scores for each shared event among the tumors based on comparison of within-tumor π scores among shared events. This was repeated for 200 iterations via resampling at an average of 63% without replacement, and the union of the relative timing score traces for each iteration was used as the final timing score distribution.PhylogicNDT LeagueModel uses a Bayesian Monte Carlo Markov chain (MCMC) approach to combine single-tumor + and HPV– HNSCC or between subsets of HPV– tumors, we performed a similar MCMC approach to the LeagueModel algorithm, but for each resampling iteration, we calculated the difference in the relative timing scores between subsets. Point estimates for timing differences were calculated as the median of the final trace, and one-way P values were calculated as the proportion of trace samples that was greater than 0. These were converted to two-way P values (P2-way = 1 − 2|0.5 − P1-way|) and then to q values using multiple hypothesis correction.To compare consensus genetic progressions between HPVπ-score distribution calculated by using only CpG>T mutations with PhylogicNDT SinglePatientTiming. The resulting distribution for a specific event was then combined across participants to produce a cohort-level distribution for the typical time of the event in terms of years before diagnosis. Timing of HPV integration events was performed by aligning structural variation breakpoints and spanning reads at 53 integration sites across 11 individuals with available WGS data and performing a similar timing analysis as for mutations . Because APOBEC mutations are often clustered near HPV integration sites, we excluded APOBEC mutations for the timing estimate. Often several integration sites were identified per participant, and sites on copy number regions with the earliest estimate of real time were used as representative for the participant.To estimate the real-time timing of somatic events, we used a robust linear regression model by removing the top and bottom 5% of slope outliers to fit the rate of clonal CpG>T  mutations  corrected for copy number and multiplicity as a function of the age at diagnosis  or doubling (WGD) was based on the corresponding ploidy group (triploid or tetraploid).The FGA absent a whole-genome event was the fraction of the genome deviating by more than 0.2 from a value of one copy for either allele. With a whole-genome event, FGA was calculated as the fraction of genome deviating by more than 0.2 from a value of two copies for either allele.No statistical method was used to predetermine sample size. As there were no groups defined by experimental manipulations, no blinding was performed. Random resampling is inherent in PhylogicNDT, as described above.P values within each HNSCC subset. Survival analysis used the survival71 (version 3.3-1) and rms72 (version 6.3-0) packages under version 4.2.0 of R73, extending a previously described model for HNSCC survival51. Cox survival models were stratified by anatomic site to satisfy the proportional hazards assumption, and model calibration was checked by bootstrap resampling.Event prevalence comparisons, associations among measures of intratumor heterogeneity and other routine analyses were performed with standard statistical routines in Python or R. Frequentist tests (except for inherently one-sided chi-square statistics) were two sided. The Benjamini–Hochberg false discovery rate correction was applied to event prevalence comparison Further information on research design is available in the Reporting SummarySupplementary Tables 1–7+ and HPV– HNSCC. a, Number and percentage of cases having each of 66 genomic events for 421 HPV– and 101 HPV+ HNSCCs. Fisher’s exact test two-sided P values are reported for comparison between HPV– and HPV+ tumors. Benjamini–Hochberg false discovery rate-corrected q values are also shown. b, List of differentially prevalent events at q < 0.1. c, List of events not differentially prevalent (q < 0.1). d, Differentially prevalent events in b favored in HPV– HNSCC. e, Differentially prevalent events in b favored in HPV+ HNSCC. Supplementary Table 2. Clonal event prevalence comparison between HPV+ and HPV– HNSCC, like Supplementary Table 1 but restricted to clonal events. Supplementary Table 3. Event prevalence by anatomic subsite for HPV– HNSCC. Breakdown by anatomic subsite of number and percentage of cases having specified genomic events: larynx, 114 tumors; oral cavity, 274 tumors; oropharynx, 33 tumors. Fisher’s exact test P values and Benjamini–Hochberg q values are as in Supplementary Table 1. Supplementary Table 4. Event prevalence comparison between NSD1-mutated and other HPV– HNSCC, like Supplementary Table 1a–c but comparing 54 NSD1-mutated HPV– HNSCC against 367 other HPV– HNSCC. Supplementary Table 5. Event prevalence comparison between CASP8-mutated and other HPV– HNSCC, like Supplementary Table 1a–c but comparing 50 CASP8-mutated HPV– HNSCCs against 371 other HPV– HNSCCs. Supplementary Table 6. Event prevalence comparison between NOTCH1-mutated and other HPV– HNSCCs, like Supplementary Table 1a–c but comparing 70 NOTCH1-mutated HPV– HNSCCs against 342 other HPV– HNSCCs. Supplementary Table 7. Real-time timing estimates for whole-genome copy-number events.Supplementary Table 1. Event prevalence comparison between HPV