Background: TRIM37 (Tripartite Motif Containing 37) is an E3 ubiquitin ligase for histone H2A and inhibits transcription in several genes. However, it is not known whether it plays a role in gastric cancer (GC). In this study, we tested whether TRIM37 acts as a cancer-promoting factor by being overexpressed in GC.Methods: We analyzed GC cell lines and 124 primary tumors, which were curatively resected in our hospital between 2001 and 2003.Results: Overexpression of the TRIM37 protein was detected in almost all GC cell lines and GC samples (76 out of 124 cases) and was significantly correlated with lymphatic and venous invasion, advanced T-Stage, N-Stage, histology and high recurrence rate. Patients with TRIM37 overexpressing tumors had a worse survival rate than those with non-expressing tumors (P=0.0057). Moreover, TRIM37 positivity was identified as an independent factor predicting worse outcomes . The apoptotic cell analysis showed that the knockdown of TRIM37 increased apoptosis in comparison with the control. In TRIM37 overexpressing GC cells, knockdown of TRIM37 suppressed the migration and invasion.Conclusions: TRIM37 plays a crucial role in tumor malignant potential through its overexpression and highlight its usefulness as a prognostic factor and potential therapeutic target in GC. MET and ERBB2; mutations in TP53, APC, and E-cadherinK-rasp16β-catenin; and inactivation of the mismatch-repair gene hMLH1, which is associated with microsatellite instability Gastric cancer (GC) is one of the leading causes of death from cancer worldwide Tripartite motif-containing 37 (TRIM37) has been identified as an oncogenic H2A ubiquitin ligase. In addition, TRIM37 is located at the critical region of 17q23, in which genomic high-copy amplification has been reported to contribute to the progression of several types of cancer, including prostate cancer TRIM37 expression in TRIM37-overexpressing GC cells suppressed cell proliferation, migration, invasion, and chemoresistance to cisplatin (CDDP). Our results provided evidence that TRIM37 could be an important molecular marker for determining the malignant properties of tumors and could be a promising therapeutic target in patients with GC.In this study, we wished to investigate the effects of TRIM37 overexpression and activation in GC. Consequently, we demonstrated that TRIM37 was frequently overexpressed in GC cell lines and primary GCs, and the overexpression of TRIM37 was identified as an independent factor associated with a poor prognosis. We also demonstrated that knockdown of 2. Paraffin-embedded primary GC tissue samples were collected from 124 consecutive GC patients who had undergone curative gastrectomy at the Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine  between 2001 and 2003. Paraffin blocks are stored at room temperature in the dark and we stained the sliced specimens within two weeks. Relevant clinical and survival data were obtained for all patients. All experimental methods were carried out in accordance with relevant guidelines and regulations. Written informed consent was obtained from all patients to use their tissue specimens for research purposes, and the study was approved by the institutional review boards of Kyoto Prefectural University of Medicine. (ERB-C-67) None of the patients underwent endoscopic mucosal resection, palliative resection, preoperative chemotherapy, or radiotherapy, and none of them had synchronous or metachronous multiple cancers in other organs. Disease clinical and pathological stages were defined in accordance with the International Union against Cancer tumor-lymph node-metastases (TNM) classification A total of seven GC cell lines  and a fibroblast cell line (WI-38) were used in this study. All cell lines were purchased from RIKEN BioResource Center Cell Bank , where the cell lines were authenticated by short tandem repeat (STR) profiling prior to distribution. HGC27 cells were cultured in Dulbecco's Minimum Essential Medium (DMEM). All other cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium . All media were supplemented with 100 mL/L fetal bovine serum (FBS) . All cell lines were cultured in a humidified incubator at 37°C with 5% COTRIM37; Applied Biosystems) according to the manufacturer's instructions. The results of gene expression were calculated as a ratio between TRIM37 and an internal reference gene  that provided a normalization factor for the amount of RNA isolated from a specimen. This assay was performed in triplicate for each sample.Total RNA was extracted from cell lines using an RNeasy Mini Kit . The reverse transcription reaction was performed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). The total RNA of normal organs was purchased from Takara Bio Inc., Shiga, Japan . The abundance of mRNA was measured by qPCR using a StepOnePlus PCR system (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software v2.0 (Applied Biosystems), using TaqMan Gene Expression Assays  were purchased from Sigma Aldrich , anti-ACTB antibodies were purchased from Santa Cruz Biotechnology , and anti-ZEB1 antibodies and anti-E-cadherin antibodies were purchased from Cell Signaling Technology (USA). Cells were lysed and their proteins were extracted using M-PER® Mammalian Protein Extraction Reagent .TRIM37  or Luciferase (Luc) 5'-CGUACGCGGAAUACUUCGA-3'  was transfected into cells (10 nmol/L) using Lipofectamine RNAiMAX  according to the manufacturer's instructions. The knockdown of a target gene was confirmed by western blotting.For loss-of-function analysis by the knockdown of endogenous gene expression, each of the siRNAs targeting For measurements of cell growth, the number of viable cells at various time points post-transfection was assessed by a colorimetric water-soluble tetrazolium salt assay . The cell cycle position was evaluated 72 h post-transfection by fluorescence-activated cell sorting (FACS), as described elsewhere At 72 h post-transfection, siRNA-transfected cells were harvested and stained with fluorescein isothiocyanate-conjugated annexin V and phosphatidylinositol using an Annexin V Kit . A Becton Dickinson Accuri™ C6 flow cytometer was used to analyze the proportion of apoptotic cells.4 cells; MKN7, 5 × 104 cells per well) and incubated overnight under normal culture conditions. The cells were then incubated with CDDP (8 μM) and PTX (0.1 μM). At 48 h after the addition of the anticancer drug, apoptotic cell analysis was performed as mentioned above.To assess the chemoresistance of GC cell lines to CDDP and paclitaxel (PTX), NUGC4 (wild-type TP53) and MKN7 (mutant TP53) that had been transfected with siRNA-TRIM37 and its control were plated onto 6-well plates  were seeded into the upper chamber with serum-free medium. Complete growth-medium was added to the lower well of each chamber. The transfectants were incubated for 22 h and then migrated or invasive cells on the lower surface of the filters were fixed and stained with Diff-Quik stain . The stained cell nuclei were counted directly in triplicate, as described elsewhere Transwell migration and invasion assays were carried out in 24-well modified Transwell Boyden chambers . The upper surface of 6.4-mm-diameter filters with 8-µm pores was precoated with (invasion assay) or without (migration assay) Matrigel (BD Transduction). The siRNA transfectants  for 30 min at room temperature, the sections were further incubated at room temperature for 1 h with anti-TRIM37 antibodies (1:1000). PBS was used for all dilutions and washings. Bound primary antibodies were detected using the EnVision™+ Horseradish Peroxidase System . HRP labeling was visualized using color development with diaminobenzidine tetrahydrochloride. Slides were counterstained with Mayer's hematoxylin.Primary tumor samples were fixed with 10% formaldehyde in phosphate buffered saline (PBS) and embedded in paraffin. Paraffin blocks are stored at room temperature in the dark and we stained the sliced specimens within two weeks. A horseradish peroxidase (HRP) staining method was used. Briefly, following deparaffinization, antigen retrieval was performed by heating the samples in 10 mmol/L citrate buffer (pH 9.0) at 95°C for 60 min. Endogenous peroxidases were quenched by incubating the sections in 3% HFor the scoring of TRIM37 expression, the intensity was evaluated for each case . The expression of TRIM37 was graded as high expression (intensity score ≥ 2 of tumor cells showing immunopositivity), or low expression (intensity score ≤ 1 of tumor cells showing immunopositivity) under high-powered (×200) microscopy P < 0.05 level.Clinicopathological categorical variables pertaining to the corresponding patients were analyzed for significance using the Chi-square test or Fisher's exact test. Differences in non-categorical variables among subgroups were tested using the non-parametric Mann-Whitney U-test. For the analysis of survival, Kaplan-Meier survival curves were constructed for groups based on univariate predictors, and differences between the groups were tested with the log-rank test. Univariate and multivariate survival analyses were performed using the likelihood ratio test of the stratified Cox proportional hazards model. Differences were assessed with a two-sided test and were considered significant at the TRIM37 was overexpressed in GC cell lines compared with cells from healthy organs . TRIM37 mRNA overexpression was observed in GC cell lines and the testis compared with healthy organs and the fibroblast cell line WI-38, suggesting that this gene is a cancer-testis antigen . We examined the status of a TP53 mutation in GC cell lines by western blotting. The status of these TP53 mutations in various cell lines was positively associated with those in the database . Note that, among TP53-mutated GC cell lines, KATO-III and HGC27 cells have a p53 gene deletion and a frameshift mutation, respectively.Quantitative RT-PCR analysis was performed to test whether  antigen  A. TRIM3Materials and methods section. In primary cases, TRIM37 expression was negative in the non-tumorous gastric mucosal cell population . Kaplan-Meier survival estimates showed that TRIM37 immunoreactivity in tumor cells was significantly associated with overall survival according to the extent of each intensity score . In the scores of intensities, the TRIM37 high-expression group, with a score ≥ 2 for tumor cells showing immunopositivity, showed significantly poorer overall survival  and relapse-free survival  compared with the low-expression group .Because TRIM37 was overexpressed in almost all GC cell lines, it was hypothesized that TRIM37 would also be highly expressed in GC tissues and would be associated with carcinogenesis and malignant outcomes. We examined the prognostic and clinicpathological significance of TRIM37 expression in primary tumor samples of GC, based on the immunohistochemical staining pattern of this protein. TRIM37 was observed mainly in the cytoplasm of cancer cells. We classified 124 GC samples into either positive or negative groups according to the intensity of TRIM37 staining among tumor cells, as described in the Table 1. Protein expression of TRIM37 was significantly associated with being male, histopathological differentiation, venous invasion, lymphatic invasion, advanced pT and pN stage, and recurrence rate. A Cox proportional hazards regression analysis (Table 2) identified TRIM37 immunoreactivity in tumor cells as an independent factor that predicted a worse overall survival rate , along with advanced pathological stage. Following gastrectomy, TRIM37 protein expression was significantly associated with recurrence (P =0.028), in particular lymphatic metastasis (P = 0.042) (Table 3).To test the hypothesis that TRIM37 protein abundance contributes to malignant features in GC, we assessed the expression of TRIM37 in primary GC tissues by immunohistochemistry. The relationship between the expression of TRIM37 and clinicopathological characteristics is summarized in TRIM37 as an oncogene whose overexpression may be associated with gastric carcinogenesis, we first performed a cell-proliferation assay using siRNAs specific to TRIM37 to investigate whether knockdown of TRIM37 would suppress the proliferation of GC cells that overexpress TRIM37. In the p53 wild-type cell line, NUGC4, and the p53 mutant cell line, MKN7, expression of the TRIM37 protein was efficiently knocked-down by the introduction of a TRIM37-specific siRNA (siRNA-TRIM37), and compared with a luciferase-specific siRNA (siRNA-Luc) as a negative control . The proliferation of these cell lines was particularly suppressed following the knockdown of endogenous TRIM37 expression .To gain insights into the potential role of TP53 wild-type NUGC4 cells with siRNA-TRIM37 resulted in an accumulation of cells in G2/M phase compared with their transfection with control siRNA, while transfection of TP53 mutant MKN7 with siRNA-TRIM37 resulted in an accumulation of cells in the sub-G1 phase compared with transfection with control siRNA .To investigate the mechanisms by which the knockdown of TRIM37 suppressed cell proliferation, we performed a cell cycle analysis and an apoptosis assay. FACS analysis demonstrated that transfection of TP53 wild-type NUGC4 and TP53 mutant MKN7 with siRNA-TRIM37 increased early apoptosis (annexin V-positive/PI-negative) and late apoptosis (annexin V/PI-double positive), respectively, at 72 h post-transfection compared with transfection with control siRNA . These findings suggest that the knockdown of TRIM37 overexpression induces cell apoptosis.Apoptotic cell analysis showed that transfection of Table 1, protein expression of TRIM37 was significantly associated with both venous and lymphatic invasion in clinical samples. To confirm the relationship between TRIM37 and cell migration and invasion in vitro, transwell migration and invasion assays were performed. We examined the ability of TP53 wild-type NUGC4 and TP53 mutant MKN7 cells transfected with siRNA-TRIM37 to move through pores under different conditions. An uncoated membrane was used for the migration assays, whereas a Matrigel-coated membrane was used for the invasion assays. As Figures  show, the number of siRNA-TRIM37-transfected NUGC4 and MKN7 cells that migrated into the lower chamber was significantly lower compared with siRNA-control-transfected cells under both conditions. These results suggest that the overexpression of TRIM37 may enhance the ability of GC cells to migrate and invade in both the p53 wild-type and the p53 mutant cell lines.As shown in TRIM37 suppressed the production of ZEB1 and induced the production of E-cadherin in both TP53 wild-type and TP53 mutant cells . These results suggest that overexpression of TRIM37 induces cell migration and invasion via the loss of E-cadherin. Figure  shows a hypothetical model of the overexpression of TRIM37 in GC cells.The most important hallmark of EMT is the loss of E-cadherin, which is mediated by ZEB1. TP53 wild-type NUGC4 and TP53 mutant MKN7 with siRNA-TRIM37 increased early apoptosis, at 72 h post-transfection, compared with transfection with control siRNA. However, when treated with PTX, chemosensitivity was not related to the status of TRIM37 expression in either the TP53 wild-type NUGC4 or TP53 mutant MKN7 .Recently, it has been reported that high levels of expression of TRIM37 are associated with chemoresistance to CDDP in esophageal cancer and osteosarcoma In this study, we demonstrated that TRIM37 is frequently overexpressed in primary GC cells and GC cell lines, and that this overexpression is significantly associated with malignant features of tumors and poor outcomes. Furthermore, the knockdown of TRIM37 inhibits cell proliferation, migration, invasion, and chemoresistance to CDDP. These findings suggest that TRIM37 plays a crucial role in tumor malignant potential through its overexpression and highlight its usefulness as a prognostic factor and potential therapeutic target in GC.Recent studies identified that the mono-ubiquitination of histones is one of the most representative epigenetic markers and is intimately involved with Polycomb protein-mediated transcriptional gene-silencing. Polycomb complexes have crucial functions in cellular differentiation and development in vitro analyses, knockdown of TRIM37 overexpression induced G2/M cell-cycle arrest in wild-type TP53 cells and G0/G1 cell-cycle arrest in mutant TP53 cells. Balestra et al. demonstrated that TRIM37 acts to prevent centriolere duplication in HeLa cells TP53 cells. Indeed, as shown in our immunohistochemical analysis, the overexpression of TRIM37 is related to the presence of lymphatic and venous invasion. Moreover, patients with GC that showed high levels of TRIM37 expression GC had worse prognostic outcomes and a higher incidence of lymphatic recurrence (Table 3). Chen et al. also reported that TRIM37 was significantly associated with lymph node metastasis in vitro findings supported the in vivo results of our immunohistochemical analysis and strongly suggested that TRIM37 plays a pivotal role in the malignant potential of GC.Chen et al. reported that TRIM37 promote cell invasion and metastasis in GC. In addition, our study revealed that TRIM37 promote cell proliferation and CDDP resistance Recent studies regarding chemotherapy identified that the downregulation of TRIM37 in esophageal cancer and osteosarcoma cells presented CDDP-induced cell apoptosis In conclusion, we clearly demonstrated the frequent overexpression of the TRIM37 protein and its prognostic value in patients with GC. Although studies involving larger cohorts will be necessary to validate these findings before moving on to explore their application to clinical settings, our results provide evidence that TRIM37 could be a crucial molecular marker for determining the malignant properties of GC cells and also that it could be a target for molecular therapy in patients with GC.Supplementary figure S1.Click here for additional data file.


R2 = 0.75, Rho = 0.83, p < 0.0001) than traditional SDMT. Mathematical adjustment for motoric disability of the dominant hand, measured by another smartphone test, compensates for the disadvantage of touch-based test. Averaging granular home measurements of the digital biomarker also increases accuracy of identifying true neurological decline.As the burden of neurodegenerative diseases increases, time-limited clinic encounters do not allow quantification of complex neurological functions. Patient-collected digital biomarkers may remedy this, if they provide reliable information. However, psychometric properties of digital tools remain largely un-assessed. We developed a smartphone adaptation of the cognitive test, the Symbol-Digit Modalities Test (SDMT) by randomizing the test’s symbol-number codes and testing sequences. The smartphone SDMT showed comparable psychometric properties in 154 multiple sclerosis (MS) patients and 39 healthy volunteers (HV). E.g., smartphone SDMT achieved slightly higher correlations with cognitive subscores of neurological examinations and with brain injury measured by MRI ( Most countries already experience a shortage of neurologists. By 2025 in the United States, the average demand for neurologists is expected to eclipse the supply by 20% or moreCurrent solutions of shifting care for neurological patients to primary care providers, while simultaneously decreasing the length of individual patient-encounters for neurologists, lead to delays or mistakes in diagnoses and suboptimal patient outcomes. Indeed, a comprehensive neurological examination takes 40–60 min to perform and years to master. Consequently, such a demanding exam is rarely performed in contemporary clinical practice, depriving patients of reliable measurements of their disease progression necessary for optimal therapeutic decisions.3. NeuFun is designed to recreate the essential domains of neurological examination, allow patients to do testing from home, and have the results forwarded to their clinicians—a capacity essential for telemedicine, e.g., in the times of a global pandemic. Provided that each test in the NeuFun validates its psychometric properties against the gold standard, its use may speed up the identification and referrals of neurological patients from primary care providers to neurologists, help neurologists to focus their examination on affected neurological domains, and reliably track neurological disability during the disease course.An alternative solution is to develop a surrogate of neurological examination which is accessible, reliable, and sensitive to changes in disease course. To meet this need, we have created a collection of smartphone tests, the Neurological Functional Test Suite (NeuFun), administered in a patient-autonomous manner3. Here, we have tested psychometric properties of the modified Symbol-Digit Modalities Test (SDMT). The SDMT is a cognitive processing speed test traditionally administered by neuropsychologists4. Participants are given a key of nine symbol-digit pairs along with a sequence of symbols. They are then asked to use the key to match as many symbols in the sequence to their corresponding numbers as possible within 90 s. In the written format, participants write the matching numbers on the paper. In the verbal format, participants say the number and test administrators write in participants’ responses. This operator-dependency makes SDMT prohibitively expensive to administer in routine neurology practice. Whereas neuropsychologists administer written followed by oral versions, MS investigators use predominantly oral SDMT to minimize influence of the hand disability on test performance5. The common SDMT administration uses a single form, copyrighted by Western Psychological Services, raising a possibility of code memorization on repeated testing, as demonstrated in the oral SDMT for both healthy volunteers (HV) and MS patients7.Previous studies have optimized and validated tests that measured motoric and cerebellar functionsTo prevent memorization, we introduce several adaptations of the smartphone SDMT. For instance, the symbol-digit key pairing changes randomly with each trial. While this key is displayed on the top of the screen during test duration, participants are shown each symbol  one at a time. This prevents inputting the numbers out of sequence in unsupervised testing. To input their responses, participants touch the corresponding number on a virtual number keypad  SDMT to the Paced Auditory Serial Addition Test (PASAT-3), a gold standard that is part of the MS functional composite (MSFC), used in clinical trials. In the PASAT-3, participants are told a sequence of numbers at intervals of 3 s. Participants are asked to add the most recently stated number in the sequence to the prior number and state the result before the next number in the sequence is announced.We developed both touch-based and oral versions of smartphone SDMT, and each subject could choose which version they wanted to use. Very few have chosen the oral version of the test, and during the training phase, we noticed that the oral version generated errors when the word-recognition technology failed to correctly identify responses in patients with hypophonia or dysarthria. None of the tested subjects were so disabled that they could not use the touch-based smartphone SDMT. Therefore, we focused on this version and developed mathematical adjustment for motoric dysfunction to compensate for the shortcoming of the touch-based test.p < 0.0001, CI: [13–23 points difference],  Memorization of the symbol-digit code: Because most of our patients have experienced traditional SDMT before, they may have memorized the symbol-digit code. (2) The traditional SDMT copyrighted by Western Psychological Services facilitates performance as the first 26 items on the scoring sheet originate only from the first 6 symbols (out of 9) in the symbol-digit key10 10 Fig. .oral SDMT5, the test-retest reliability was assessed by correlation coefficients, which ranged from 0.76 to 0.97. Therefore, we assessed test-retest reliability of written and smartphone SDMT in our MS#1 cohort when SDMT was measured six months apart. This revealed Rho = 0.95 , R2 = 0.90, p < 0.0001 for written and Rho = 0.87 , R2 = 0.80, p < 0.0001 for smartphone SDMT. We conclude, that despite randomization, test-retest reliability of smartphone SDMT is comparable to the published oral SDMT data and slightly lower than the non-randomized written SDMT.In the MS Outcome Assessment Consortium (MS-OAC) analysis of written SDMT may reflect unwanted learning effect, effectively representing a “noise” in test measurement. To formally test this hypothesis, we compared the construct and predictive validity of smartphone and written SDMT. We included an alternative cognitive test, PASAT-3, which is included in MSFC as a gold-standard . In contrast, both SDMTs exhibited significant correlations with dominant hand (DH) cerebellar functions , vision , and eye movements . As would be expected from the lesser motoric demand of touching a screen versus writing numbers, the smartphone SDMT had slightly weaker correlation with DH cerebellar functions than written SDMT. On the other hand, smartphone SDMT correlated stronger with vision and eye movements, indicating a more demanding hand-eye coordination.NeurEx also identified contributions of remaining neurological functions to cognitive test performance. As expected for an oral test not dependent on eye or motoric functions, PASAT-3 did not correlate with any other NeurEx subsystem, except with the brainstem/lower cranial nerve subscore that includes hearing and articulation functions  and brain atrophy (BPFr)12; therefore, we assessed analogous correlations.Criterion validity of the traditional While PASAT-3 did not correlate significantly with these MRI measures, smartphone SDMT had slightly stronger correlation coefficients in comparison to traditional written SDMT: Rho 0.46 versus 0.43 for BPFr and Rho −0.45 versus −0.43 for T2LL  regression. In the EN models, the BPFr and T2LL were predictor variables, while any NeurEx factor which showed strong evidence of correlation with SDMT results (including age) were included as covariates. Because complex models are susceptible to overfitting , their validity must be tested in an independent cohort. Therefore, we randomly divided our MS cohort to training (2/3) and validation (1/3) subcohorts  than the written SDMT  can replace the neurologists scores in EN models. When we replaced the NeurEx scores of DH cerebellar and upper motoric strength with tapping results in the smartphone SDMT EN model, the model validated only slightly worse than the model that included clinicians’ data  analysis showed that in all the longitudinal data, including the “learning” period, between-patient data clustering accounted for 87% of the total variance in the smartphone SDMT score 11.Next, we plotted the intra-individual differences between two traditional written SDMT tests collected six months apart Fig. , Table 2ero Fig. , we usedp = 1.0; ∆ NeurEx comparison + 5.2 versus +7.5: p = 0.4). As evidenced by positive numbers, in contrast to EDSS, both CombiWISE and NeurEx measured progression of clinical disability, which was equal in patients whose SDMT scores declined by at least four points and patients whose scores did not.Thirty MS patients (26.8%) fulfilled four-point SDMT decline criterion, but we observed no statistically significant differences in either CombiWISE or NeurEx disability progression between MS patients who fell below and above this threshold  change = 13-point decline. Only three MS subjects (2.7%) declined in SDMT performance beyond this threshold during six months follow-up. This cohort is too small to obtain reliable statistics, but the median CombiWISE and NeurEx changes were higher in this small group in comparison to the remaining 109 subjects with MS .We conclude that both written and smartphone SDMT have similar variance, requiring a difference of 13 to 14 SDMT points to identify decline that exceeds test-retest fluctuation in SDMT performance when comparing only two SDMT tests within the same subject.Next, we hypothesized that granular collection of data may lower this threshold of true deterioration when comparing period averages. The Gaussian distributions of test-retest data suggests random distribution of noise, that could be limited by averaging multiple weekly tests within individual patients, akin to “repeated measures”. Thus, within the granular testing cohort, we compared the variance of single adjacent tests, with an average of two, three, or four adjacent tests Fig. . We obseWe conclude that when a subject performs SDMT only twice, the decline of more than 14 points reliably exceeds natural variability in the test performance and should trigger a search for structural cause, such as MS exacerbation of progression of cognitive deficit. Lower level of SDMT performance decline should trigger repeated testing. In this regard, granular collection offers advantage over episodic collection of SDMT data, as averaging multiple test repetitions lowers the threshold for identifying true decline in test performance in individual subjects.1. Because this situation is unlikely to improve, it is imperative to find alternative reliable measurements of neurological disability. The digital health revolution spurred development of “medical” apps. However, a recent conference on digital health, Digital Health: From Science to Application  concluded that technical development of these apps is easy in comparison to validating their value for intended use, in accordance with FDA guidance17. Consequently, most medical apps are released without assessment of their psychometric properties18.Neurological examination is a powerful tool to identify, localize, and grade neurological deficits. However, economic pressures limit access to this essential tool worldwide19.To assure broad use of NeuFun, we focused our developmental efforts on tests that can be self-administered on smartphones instead of tablets due to the broad availability of the former, including in developing countries. This would keep the cost of the test low and allow telemedicine with home administration of the test. However, the adaptation of paper-based tests to much smaller smartphone screens requires greater involvement of visual system/hand-eye coordination Figs. , 4. This5, we followed this framework to validate smartphone SDMT. Despite randomization, smartphone SDMT has comparable discriminative, psychometric, construct, predictive, and criterion validity to published parameters of oral SDMT. In addition, previous publications on oral SDMT often excluded subjects with hearing/speaking disabilities and with comorbidities such as alcoholism and depression to limit noise in SDMT performance14. This over-estimates the psychometric validity of oral SDMT compared to routine practice. We did not exclude any MS patients based on disability or comorbidities. We also blinded divergent assessments , limiting bias in assessing psychometric characteristics of both SDMT tests.As the published MS-OAC developed an excellent framework for validating the oral SDMT5) and validates the ability of smartphone SDMT to reliably measure MS-related brain injury.The touch-based smartphone SDMT adaptation has predicted limitations in that motoric disability of the performing hand affected SDMT performance. However, as illustrated, careful mapping of contributing neurological functions permits mathematical adjustment for measured disabilities as covariates to isolate the tested neurological domain. Using this methodology, the smartphone SDMT results, when adjusted for visual disability and DH dexterity, explained a stunning 75% of the variance in the EN linear regression model against a composite of brain atrophy and T2LL in the independent validation cohort. This remarkable criterion validity compares favorably to oral SDMT in MS  decline in test performance. Indeed, our use of non-linear regressions yielded an identical result  previously reported in natalizumab-treated MS patientsThus, our paper demonstrates that, in principle, SDMT is adaptable to self-administered digital tests that may be broadly utilized in neurology practices and guide therapeutic decisions. This brings an associated challenge to correctly interpret the test results. In this regard we acknowledge that our HV data are still limited and need to be expanded to provide reliable normative data across all age-groups. The other problem we addressed is the threshold of decline in SDMT performance that exceeds natural variation and therefore may represent actionable change due to e.g., MS relapse or disease progression. We demonstrated that this threshold is quite large, representing over 14-point decline when the subject performs the test only twice, which can be lowered to 7-point decline if the person performs the test four times and averages the test performance. Because these data-driven, intra-individual thresholds exceed currently accepted four-point decline in SDMT performance identified on a group level as clinically meaningful, we want to clarify the reasons for this apparent discrepancy.14. These MS patients had a battery of neurocognitive tests twice, on average 2-4 years apart. From ten cognitive tests administered, only the change in SDMT differentiated the two MS groups with statistical significance: the subjects whose vocational status declined showed decline on SDMT by 3.0 +/− 8.0 points, while patients with a stable vocational status showed SDMT improvement of 1.3 +/− 6.6 points. We note that SD of intra-individual SDMT changes measured in this highly pre-selected MS cohort still greatly exceeds four-point threshold. Considering the accepted cut-off of 2 SD to differentiate true biomarker change from random test variation, even this cohort generates a threshold of 13–16 SDMT points decline on the individual level. The papers that corroborate the four-points SDMT decline threshold on a group level did not measure test-retest variance on an individual level15 and did not measure the predictive power of four-point SDMT decline on vocational status of MS patients in an independent, prospective MS cohort.The paper that suggested four-point decline in SDMT as “clinically meaningful” studied retrospectively two groups of MS patients: those who experienced decrease in employment status, and those who did not5). Finally, we determined only the threshold of decline in the intra-individual performance that exceeds natural variation; we had too few MS patients who exceeded this threshold during the follow-up period to determine the “clinical significance” of such decline.Thus, the apparent discrepancy of the conclusions between this and previously published papers resides in the way this “clinically-meaningful” threshold was applied; the previous studies applied this threshold on a group level, whereas we sought to determine the threshold that can alert the subjects and their clinician to the actionable intra-individual decline in test performance. Indeed, four-point SDMT decline on a group level should be deemed clinically-meaningful, as longitudinal MS studies lasting less than three years did not measure any decline in SDMT performance , and the results are stored in a secured online database under alphanumeric code that lacks personal identifiable information (PII). The smartphone SDMT uses the Android operating system (Android 9 and up), with graphics optimized for only two types of smartphones used for all subjects (Google Pixel XL/2XL) in colorblind-friendly visualizations. To assure equivalence of the test between the smartphones, no vector-based rescaling was used. Instead, the size and location of the graphics is predefined and displaced equivalently on different phones. The SDMT symbols are Unicode characters.This study was approved by and carried out in accordance with the recommendations of the Central Institutional Review Board of the National Institutes of Health (NIH). All subjects gave a written or digital informed consent in accordance with the Declaration of Helsinki.Healthy volunteers (HV) and MS participants enrolled in the protocol: Comprehensive Multimodal Analysis of Neuroimmunological Diseases in the Central Nervous System .#1. HV who underwent all study procedures . The HV inclusion criteria are: (1) At least 18 years old; (2) Normal vital signs; (3) Able to give informed consent; (4) Able to undergo all research procedures. HV exclusion criteria were: (1) Systemic inflammatory disorder, or inflammatory or non-inflammatory neurological diseases; (2) Previous or current history of alcohol and substance abuse; (3) Contraindications to associated procedures.Two HV cohorts were recruited: HV group (#2) uses the NeuFun remotely to mimic a real-world situation (n = 27). To mimic the population that would provide normative data via the App marketplace, this sub-study collects no personally identifiable information (PII) and poses no inclusion/exclusion criteria. Subjects are self-declared to be healthy and sign digital informed consent that tested their comprehension of the testing instructions and associated risks/benefits directly via smartphone. They then input their age (in years), gender, and take the test under assigned alphanumeric code. Because no systemic differences were identified between HV cohorts #1 and #2 (not shown), the two HV cohorts were merged for the analyses.Because this number of HV is understandably limited, we recruited a second HV cohort solely to obtain normative NeuFun data: Smartphone app 22.Inclusion criteria for patients in the NCT00794352 protocol are: (1) Clinical syndrome consistent with immune-mediated CNS disorder; and/or (2) Neuroimaging evidence of inflammatory and/or demyelinating CNS disease; (3) Age ≥12 years; (4) Adults that are able to give informed consent, or minors with a parent or legal guardian able to consent, with child willing to assent; (5) Able to undergo all research procedures. The exclusion criteria included: (1) Medical condition that would make participation impossible or risky; (2) Medical contraindications for MRI; (3) Unwilling to consent for collection of biological samples. The diagnosis of MS was based on 2010 and (after 2017) 2017 McDonald’s MS diagnostic criteria#1. MS patients tested only in the NIH clinic, on average every 6–12 months (n = 154). These patients had neurological examinations and a brain MRI within 1–48 h of the NeuFun. Clinician documented neurological examinations in NeurEx, an iPad-based app that allows fast and intuitive documentation of neurological examination, including its spatial information, by touching human body diagrams. NeurEx then automatically calculates traditional disability scales, including Expanded Disability Status Scale (EDSS)11. #2. MS sub-cohort with granular NeuFun collection at home (n = 15). These MS patients expressed interest, were given a smartphone pre-loaded with NeuFun, and were asked to test at least once per week around the same time of day. Subjects who completed at least 20 sittings from MS#2 and HV#2 cohorts comprise the longitudinal/granular data. The first test sitting from subjects in all cohorts comprised the cross-sectional data.Paralleling the two HV cohorts, we also have two cohorts of MS patients: Participant demographics are summarized in Tables 23. Locally anonymized and encrypted T1 magnetization-prepared rapid gradient-echo (MPRAGE), or fast spoiled gradient-echo (FSPGR) images and T2 weighted three-dimensional fluid attenuation inversion recovery (3D FLAIR) DICOM files were analyzed by an automated segmentation algorithm LesionTOADS24, implemented into a cloud service for medical image processing by QMENTA (www.qmenta.com). The sequences are anterior commissure-posterior commissure aligned, co-registered and skull stripped. The T1 image is additionally bias-field corrected. The segmentation is performed by combining a topological and statistical atlas resulting in computed volumes for each segmented tissue in mm3. The segmentation maps were quality checked (M.V.) with errors corrected by re-running the algorithm, or uncorrectable scans were excluded.The details of the MRI sequences were previously publishedAll subjects received a sequential alphanumeric identification code. NeuFun, written SDMT, and PASAT-3 were collected by investigators blinded to neurological examination and MRI results. Clinicians performing neurological examinations were blinded to MRI and NeuFun results. MRI analyses were performed by investigators blinded to clinical examination and NeuFun outcomes. All MS patients and HVs with available data in the research database were included, irrespective of level of disability or comorbidities.Time is an essential determinant in the utility of a digital test: because NeuFun is designed to recreate the neurological examination, it consists of many tests. If their administration takes too long, subjects will not complete the suite. Because we could not find published rationale for the selection of 90 s for the standard SDMT, we determined the shortest SDMT duration necessary to provide reliable information using test-retest reliability data from individuals who completed two 90-s trials in the same sitting. Spearman correlation coefficients of the number of correct answers were generated for every 5 s of the duration of the two trials . Expectedly, these correlations increased with the duration of the test, but eventually plateaued around Rho = 0.9 Fig. . Using np < 0.001). This is clinically irrelevant compared to between participant’s variance that ranges from 0.2 to 0.9 correct answers per second  between 75 s and 90 s tests identified miniscule difference of 0.003 points/s  a Bland–Altman plot assessed test-retest bias  we increased the outlier score to the closest limit of agreement on the Bland–Altman plot Fig. .26  and Wilcoxon rank-sum test (unpaired data). Correlation are assessed using Spearman correlation coefficients, and associations that control for confounding variables are conducted using elastic net (EN) regression ; captures the degree in which the regression line follows a 1:1 trajectory, with CCC = 1 representing 100% agreement. An intraclass correlation coefficient  for all analyses are reported as 95%.glmnet R package), the cross-sectional data was randomly divided into training (2/3) and validation (1/3) cohorts stratified for age and SDMT scores (Supplementary Fig. In the EN regression (7.There is a preprint version of this paperFurther information on research design is available in the Supplementary InformationReporting Summary


KAI1 (CD82) is a metastasis suppressor gene known to be down-regulated in carcinomas of breast, prostate and many other organs. The mechanism of KAI1 down-regulation is complex and not well understood. Here, we investigate the role of 8 SNPs (not previously studied) in KAI1 gene that could influence its expression in tumor tissue samples of breast cancer patients from the Eastern province of Saudi Arabia. KAI1 gene were selected from the NCBI website (dbSNP) and were then filtered for those SNPs causing stop codon mutations (rs139889503 and rs150533529) or nonsynonymous mutation in the 5’-UTR . SNPs genotyping was performed using TaqMan SNP Genotyping Assay and the results were correlated with KAI1 protein expression profile by immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) samples of breast cancer and control none-neoplastic tissues. Single nucleotide polymorphisms (SNPs) in KAI1 expression by IHC was observed in all none-neoplastic breast tissue samples and only in 35% out of the 59 breast cancer tissue samples. None of the samples was homozygous for the stop codon allele A in the SNP rs139889503 or allele T in the SNP rs150533529. The SNPs in the 5-UTR, rs11541048, rs115500759, and rs182579675, were only present in the homozygous state for the G and C alleles respectively in both cancer and control samples. The other SNPs in the 5’-UTR  had no significant difference in the allele distribution between KAI1 expressing or none-expressing tissue samples. KAI1 expression.Our findings showed no significant effect of the studied SNPs on down-regulation of  KAI1 (CD82) is a well-known metastasis suppressor trans-membrane glycoprotein. KAI1 prevents metastasis by inhibiting cancer cells mobility and infiltration . To achieve its function, KAI1 protein interacts with other cell membrane proteins such as adhesion proteins, growth factor receptor (GFR), and signaling pathways . Different types of cancers showed KAI1 loss of expression especially those in advanced stages . The mechanism behind down-regulation of KAI1 expression during cancer development is complex, mediated by transcription control, alternative splicing, and even post-translational remodeling . Only a few studies investigated gene mutation, loss of heterozygosity (LOH), or promoter hypermethylation as a cause of KAI1 down-regulation . One of the early studies revealed that neither mutation (on 10 prostatic cancer samples) nor allelic loss (on 34 prostatic cancer samples and 12 metastatic lymph nodes) is the cause of KAI1 gene down-regulation . A similar observation was made in another study . Further studies also revealed that epigenetic changes such as methylation of promoter region is not essential to KAI1 loss of expression . A later report also showed that loss of KAI1 expression is not related to mutation, LOH, epigenetic regulation of the promoter, or p53 mutation . On the other hand, a study on 52 ovarian carcinomas found a missense alteration at codon 241 (ATC to GTC), which causes valine to isoleucine substitution in the peptide sequence but it occurs in normal tissues in addition to tumor cells . One more study discovered a significant correlation between a SNP on exons 3 (-29166 C>T) and the development of colorectal cancer of high grade and advanced stage .Breast cancer is a common cancer in the Eastern Province of Saudi Arabia with a high mortality rate which is mostly associated with its dissemination. Mechanism of cancer dissemination, in general, is mediated by complex effects of promoting genes and suppressor genes . KAI1 expression, which could be if proved, a possible mechanism of KAI1 loss of expression in breast cancers.To our knowledge, after a thorough literature review, no previous study was done on the relationship between the SNPs (selected in our study) and the expression level of KAI1. We aim here to investigate the possibility of a significant association between these selected SNPs, and the Sample collectionFifty-nine formalin-fixed paraffin-embedded (FFPE) tissue samples of breast cancer in addition to 20 FFPE none-neoplastic breast tissue samples (controls) were collected from the archive of the pathology department at King Fahd Hospital of the University (KFHU). All hematoxylin and eosin slides from tumor and control samples have been examined and verified by a breast pathologist. This study received an ethical approval from the Institutional Review Board (IRB) at Imam Abdulrahman Bin Faisal University (IRB number: IRB–2015-01-095).Immunohistochemistry study (IHC)KAI1 expression profile of cancer and control samples in the study, has been retrieved from our previous study  which has been performed on FFPE tumor and control samples’ paraffin sections (3-4μm thick), on Ventana Ultraview Autostainer using Ventana DAB detection universal kit  following the standard protocol with further optimization, using as primary antibody . The antibody expression level of each sample was classified into: partial, diffuse or no expression.Immunohistochemical DNA extractionDNA was extracted from two 10-micrometer tissue sections using the FFPE DNA purification kit  according to the manufacturer’s instructions. The tissue sections were first deparaffinized using 350ul of Qiagen deparaffinization solution for three minutes  followed by 1ml of Xylene for 2 minutes. DNA quality and concentration were assessed using NanoDrop2000 .SNP selectionKAI1 gene for SNP using the NCBI website, 3594 SNP were identified in homosapiens KAI1 gene. All intronic and synonymous SNPs were excluded. The remaining SNPs were selected if they cause stop codon or if they were nonsynonymous and were located in the 5’-untranslated region. The following SNPs were predicted to cause stop or nonsense mutation: rs139889503, rs150533529, rs11541048, rs77359459, rs115500759, rs182579675, rs200238062, rs372733853.After screening the SNP genotypingGenotyping of the KAI1 SNPs was performed by TaqMan assay . The following SNPs were available in the company’s database as validated assays; rs139889503, rs150533529, rs11541048, rs372733853. The other SNPs were ordered as an assay on demand. A twenty-microliter reaction volume containing specific primers and probes for each SNP separately was run on the 7500 Fast real-time PCR thermocycler  according to the manufacturer’s instructions. The software ABI 7500 was used for the endpoint allele calling.Data analysisKAI1 expression by IHC. Significant correlation was considered at P < 0.050.Data were collected in an Excel sheet and be prepared for statistical analysis by IBM SPSS software v.24. Pearson’s correlation coefficient (2-tailed) has been performed to measure the strength of the association between each of the mentioned SNPs and the level of The expression of KAI1 by IHCKAI1 by immunohistochemistry from breast cancer tissue and none-neoplastic breast tissues using anti-CD82 TS82B . Out of 59 tumor samples included in the current study, 35% (n=22/59) showed expression of KAI1 protein . The remaining 65% (n=37/59) showed loss of KAI1 expression. All the 20 control samples showed expression of KAI1.In a previous study, we investigated the expression level of Genotyping of the selected SNPsKAI1. Over three thousand SNPs were identified in the homosapiens KAI1 gene. We have then excluded all SNPs that are intronic or produce synonymous mutations. The remaining SNPs were further refined to SNPs which produce a stop codon (rs139889503 and rs150533529) or if they were nonsynonymous mutations and were in the 5’-untranslated region  in the second SNP rs150533529. However, 4 cases, including two normal controls, were heterozygous for this SNP  of the cases. We searched the dbSNP database for SNPs in the gene coding region for d region . None ofthis SNP . The norKAI1 expression and identified six non-synonymous SNPs . Therefore, its expression is usually downregulated in many tumors particularly in advance stages. Understanding the mechanism of KAI1 expression status in breast cancer tissues. We investigated the effect of SNPs that may produce stop codon and lead to truncation of the protein on KAI1 expression. None of the samples was homozygous for the allele that produces stop mutation. However, four cases, including two normal controls, were heterozygous for these SNPs. The normal tissue showed expression of KAI1 in the two cases. While in the tumor samples, KAI1 was expressed in 50% (n=1/2) of the cases. This indicates that the heterozygous state of these two SNPs does not affect KAI1 expression.Our findings showed that most of tumor and control samples in the study showed almost the same SNP profile with no significant association with KAI1 gene on its expression. The 5’-UTR is an untranslated sequence in an mRNA directly upstream of the start codon, which plays an important role in the regulation of protein expression by influencing the translation of its transcript. Similarly, no effect was observed for the non-synonymous SNPs in the 5’-untranslated region (5’-UTR) of the KAI1 on the risk of breast cancer when there is lymph nodes involvement . Therefore, there could be no one SNPs that by itself affects the KAI1 expression but rather a combined effect of multiple SNPs.These SNPs were not previously studied, and no data was available about them in the literature other than the databases. A study investigating SNP in multiple metastasis inhibitor genes including KAI1 did not find a significant association between particular SNP and risk of breast cancer but rather found an effect of multiple variants in several genes including KAI1 gene could be a less frequent event . A study on patients with head and neck cancer concluded that germline mutation in The small sample size could be a limitation in our study. Therefore, further studies are required to understand KAI1 down-regulation in tumor tissue which could be not only at the germline level but also at the transcriptional or translational level (epigenetic regulation) .KAI1 expression in breast cancer, which again makes mutations a less likely mechanism of KAI1 loss of expression. In conclusion, our findings did not reveal significant association between the studied SNPs and the


Human papillomavirus (HPV) infections account for over 600,000 new cancer cases every year . HPV is Methodological advances in microbiome sequencing and analysis have enabled these recent sweeping advances in knowledge. In particular, 16S ribosomal RNA (rRNA) amplicon sequencing is frequently used. This cost-effective method sequences specific hypervariable regions of the 16S rRNA gene and clusters the identified bacteria into operational taxonomic units (OTUs) that can quantify diversity metrics and relative abundances, as well as provide genus-level identification [These deficiencies could be overcome with whole-genome shotgun (WGS) sequencing. WGS deciphers broad regions of entire microbial genomes with significantly more sequencing depth but at a greater cost and use of bioinformatics resources than 16S rRNA analysis . DespiteHPV cancers may be uniquely affected by the microbiome since these solid tumors arise in the mucosa of the orogenital tract, which each have unique and diverse microbiomes. Insights into the potential influence of the microbiome on viral persistence, immune response, host-mucosal environment, and cancer treatments for HPV-related cancers are just beginning to emerge. In this review, we will discuss how the microbiome may play a pivotal role in the formation of HPV-driven cancers.Lactobacillus depletion, anaerobic bacteria predominance, and Lactobacillus iners dominance. These CSTs were significantly associated with preinvasive disease, increased disease severity, and disease invasiveness. Additionally, increased vaginal microbe diversity and richness were associated with increased rates of HPV infection and persistence, as well as higher CIN severity [Sneathia was significantly enriched in CIN samples in multiple studies, and its presence was associated with changes in immune mediators [The role of the microbiome in HPV-driven diseases has been extensively studied in cervical intraepithelial neoplasia (CIN). severity –13. Sneaediators ,14. But,Lactobacillus species have been better studied and appear to serve primarily protective roles with some exceptions. L. crispatus dominance has been strongly associated with a healthy vaginal microbiome and is responsible for producing high quantities of lactic acid and secretion of protective proteins throughout mucosal microenvironment [L. iners is the most commonly reported Lactobacillus-dominated CST in CIN patients. L. iners produces low amounts of lactic acid with no reported host-protective peptide production. L. iners CSTs are also the most likely to transition to CSTs characteristic of CIN patients, possibly because of its ability to adapt to a variety of pH environments and its distinct lack of bacteriocin synthesis genes, all of which allow abnormal cervicovaginal bacteria to thrive [ironment . Converso thrive ,16.Lactobacilli abundance, increased predominance of abnormal anaerobic bacteria, and increased diversity [Lactobacilli resulting in abnormally high vaginal pH (>4.5) can cause bacterial overgrowth and decreases in protective flora [An association between bacterial vaginosis (BV) and CIN has long been suggested . CIN andiversity . Studiesve flora . This dive flora . Additiove flora . Women wve flora . Furtherve flora . BV incive flora . All of HPV persistence is necessary but insufficient for the formation of ICC, and it is now believed the local microbiota may play a role in tumorigenesis. Lactobacillus spp., increases in overall bacterial diversity and richness, and an increased predominance of Fusobacterium spp. As with CIN, the ICC microbial profile resembles that of BV. The transition of CIN to invasive disease does not seem to result in major vaginal microbiome shifts, as similar states of dysbiosis facilitate the persistence and progression of HPV-driven disease. Although observed in both cervical disease states, Fusobacterium predominance was more commonly observed in ICC patients. Fusobacterium was found to be associated with increased levels of IL-4 and transforming growth factor (TGF)-β1 mRNA, suggesting a role in immunosuppression within the ICC microenvironment [Lactobacillus spp. This decline in the predominating protective bacteria results in increased vaginal pH levels, weakened pathogenic defenses, and damaged mucosal barriers [16S rRNA studies assessing the cervicovaginal microbiome account for the majority of ICC studies. ICC patients exhibit decreases in abundance of ironment . Overallbarriers ,17,25. Ebarriers . TogetheGemella and Leuconostoc, while Haemophilus correlated with HPV infection [Despite numerous studies assessing the microbiome in oral cavity cancers (OCCs) and OPCs, most of these studies do not specify HPV positivity or instead focus on non-HPV–related oral cancers. The section labeled HNSCC (Head Neck Squamous Cell Carcinoma) in nfection ,28. 16S nfection ,28. ThisLactobacillus spp. were found to be significantly associated with HPV-positive OPC patient saliva samples [Lactobacillus spp. using high-resolution 16S rRNA analysis. A subset of OPC patient samples were enriched with commensal species from the vaginal flora, including L. gasseri/johnsonii and L. vaginalis, not seen in control groups nor saliva from the Human Microbiome Project [Lactobacillus in the oral microbiome of these samples is not well understood, as Lactobacillus is often protective in vaginal and oral contexts [Lactobacillus during oral HPV disease.Interestingly,  samples ,29. In aBeyond cervical cancers and OPCs, research characterizing the microbiome of other HPV-driven cancers is relatively nonexistent. Characterization of the anal cancer microbiome would be particularly interesting because of direct interactions with the gut microbiota, but studies have yet to be published, likely because of disease rarity.The identification of bacterial composition changes in HPV-associated cervical cancers and OPCs has provided valuable insight into potential target populations of interest and possible biomarkers of disease and disease-state.Lactobacillus presence, increased microbial diversity, and increased predominance of atypical, anaerobic bacteria. These distinct features of BV are involved in the overall weakening of the immune and mucosal defense response against invading pathogens and pathogen clearance through a variety of mechanisms, including the release of mucin-degrading enzymes, disrupted pH maintenance, cytokine modulation, and chronic inflammation. In comparison, research characterizing the microbiome of HPV-driven oral and oropharyngeal cancers is limited. But still, microbiome profiles of OCC and OPC patients exhibit significantly different microbiota compositions when compared to healthy controls. Additionally, these cancers exhibit a significant decrease in microbial diversity, which will likely benefit future studies because the pool of potentially pathogenic bacteria is much more limited. Specific genera found within the oral microbiome of HPV+ OPC and OCC patients like Gemella, Leuconostoc, and, unexpectedly, commensal vagina Lactobacillus spp. provide an interesting starting point for future investigations.CIN and ICC patient 16S rRNA bacterial profiles have largely been established across multiple studies, and they exhibit shared characteristics pathognomonic to BV, including decreased The microbiome field regarding HPV-driven cancers is quickly emerging with the majority of research dedicated to characterizing bacterial profiles, but much is still unknown. Outside of HPV-driven cancers, microbiome research on viruses, fungi, and other microorganisms of the human flora and their relationships to disease are gaining interest ,31. BactWhile all HPV-driven cancer microbiomes require additional taxonomic characterization, the field would benefit immensely from mechanistic studies that describe the relationship of microbiota and the immune response to HPV infection, clearance, and carcinogenesis. More sensitive sequencing techniques and metabolomics could provide insight into specific microorganisms and associated gene expression and metabolite changes. Combining these data with rigorous immune analyses such as antigen stimulation, immune profiling, and computational analysis of sequence homology could provide critical molecular clues in understanding the microbiome’s role in tumor immunity activation and anergy. Continued understanding of the pathogenic microbiota through clinical investigation and validation in preclinical models could help rapidly shed light on this evolving field.


The objective of this study was to determine the result reproducibility and performance of the BD Onclarity human papillomavirus (HPV) assay (Onclarity) on the BD Viper LT platform using both contrived and clinical specimens. Reproducibility was assessed in BD SurePath liquid-based cytology (LBC) medium (SurePath) using contrived panels  or clinical specimens . In addition, specimens from 3,879 individuals from the Onclarity trial were aliquoted prior to or following cytology processing and tested for HPV. TC) scores for HPV ranging from −0.07 to 0.31. Positivity rates were close between the Cervex-Brush and Cytobrush/spatula for all age groups tested. Onclarity results are reproducible and reliable, regardless of sample collection before or after cytology aliquoting. Onclarity performs well regardless of the method of specimen collection (Cervex-Brush or Cytobrush/spatula) for cervical cancer screening.The objective of this study was to determine the result reproducibility and performance of the BD Onclarity human papillomavirus (HPV) assay (Onclarity) on the BD Viper LT platform using both contrived and clinical specimens. Reproducibility was assessed in BD SurePath liquid-based cytology (LBC) medium (SurePath) using contrived panels  or clinical specimens . In addition, specimens from 3,879 individuals from the Onclarity trial were aliquoted prior to or following cytology processing and tested for HPV. Finally, specimens were collected using either the Cervex-Brush or Cytobrush (or Cytobrush/spatula) for comparison of HPV results. Contrived specimens showed >95% concordance with the expected results, and pooled clinical specimens had standard deviations and coefficients of variation ranging from 0.87 to 1.86 and 2.9% to 5.6%, respectively. For precytology and postcytology aliquot analyses, specimens showed >98.0% overall agreement and mean differences in cycle threshold ( Over 95% of cervical cancer cases are caused by persistent infection with 13 to 14 human papillomavirus (HPV) genotypes 14, which in vitro diagnostics (IVD) marked for the detection of 14 high-risk HPV genotypes (14–The BD Onclarity HPV assay (“Onclarity”)  is clinically validated through U.S.- and non-U.S.-based studies for the detection of 14 high-risk HPV genotypes –13. Oncl–types 14–. In addiThe Onclarity assay is performed on samples obtained from liquid-based cytology (LBC) specimens collected using a Cervex-Brush or Cytobrush (or Cytobrush/spatula) device. However, the order of aliquoting for the Onclarity assay can vary based on the respective screening strategy employed by each laboratory. For example, sites utilizing HPV primary screening with cytology triage will perform HPV testing from an initial LBC aliquot (precytology) and use the remaining vial/specimen for cytology. Conversely, sites employing a screening program with primary cytology testing and HPV triage testing will perform cytology testing first, followed by HPV testing from the specimen after cytology processing . Therefore, it is important to establish that the performance of the HPV assay is unaffected by the order in which the aliquot is taken.Contrived specimens and pooled clinical specimens were utilized to test reproducibility within Onclarity assay runs and between Onclarity assay runs, study sites, operators, reagent lots, and days of operation. In addition, data from pre- and postcytology aliquot specimens were analyzed to determine whether the Onclarity assay performance is impacted by the order in which the sample is aliquoted . Finally, from BD SurePath (“SurePath”)  vials obtained during the Onclarity trial, Onclarity assay results were compared in specimens obtained using two different collection devices in order to determine whether performance results are affected based on the method of sampling for endocervical specimens.Women ≥21 years of age (women >65 years of age were included if they met U.S. Preventive Services Task Force (USPSTF) screening recommendations) were invited to join the Onclarity trial between 2013 and 2015. Initially, 33,858 subjects (across 31 collection sites) were enrolled; the trial population, criteria for inclusion/exclusion, and procedures involving LBC collection, cytology testing, colposcopy/biopsy procedures, and histology examination/diagnosis were described previously . By cytoC5 [specimens called positive approximately 5% and negative 95% of the time]), low-positive specimens , and moderate-positive specimens (3× C95 [specimens approximately three times above the C95 level and expected to be positive 100% of the time]). These determinations were made based on the assay cycle threshold (TC) values relative to the clinical cutoff point (C95) associated with the assay. Pooled clinical specimens positive for HPV16, -18, -45, -31, -33/58, or -52 were diluted  to a detection level close to the C95 . Negative panel members were created by pooling high-risk-HPV-negative clinical specimens. All panel members were stored at −20°C prior to Onclarity assay testing. Standard deviations and coefficients of variability for PCR mean cycle times within a run, between runs, between operators, between sites, between reagent lots, and between days  were all factors used as outcome measures of reproducibility.For reproducibility testing, contrived panel members were prepared using SiHa, HeLa, and MS751 transformed cell lines that express HPV16, -18, and -45, respectively. Aliquots from each cell panel preparation were added to an HPV-negative SurePath clinical matrix to yield high-negative specimens  or a Cytobrush Plus GT and a Pap Perfect plastic spatula (“Cytobrush/spatula”)  and stored/transported in SurePath LBC specimen vials. Clinical specimens were processed (as described below) and utilized for HPV testing via the Onclarity assay on the BD Viper LT system (“Viper LT”) .For precytology and postcytology specimens, central laboratory personnel vortexed the SurePath LBC specimen and manually aliquoted 0.5 ml of the specimen into an HPV LBC diluent tube . Aliquoting from SurePath LBC specimen vials to diluent tubes was performed in the same order as the specimen vials were received. Following the removal of the 0.5-ml precytology aliquot, 8.0 ml of the specimen was removed from the SurePath LBC vial, and a cytology slide was processed  according to the manufacturer’s instructions. A final 0.5-ml aliquot from residual fluid in the SurePath LBC vial was manually transferred into a second HPV LBC diluent tube . Thus, pre- and postcytology aliquot diluent tubes were obtained from the same SurePath LBC specimen vials; both diluent tubes were sent to one of four laboratories that ran Viper LT testing . There was a minimal delay for the postcytology aliquot specimens while cytology slides were prepared; this was within the validated room-temperature storage time. Overall, 3,879 SurePath vials were utilized in the study to provide pre- and postcytology aliquot pairs for HPV testing and analysis.The details for HPV testing with the Onclarity assay on the Viper LT system using LBC specimens were described previously , 17. BriTC scores (between 34.2 and 38.3 for HPV16 and between 29.6 and 34.2 for the other 13 genotypes) were required to ensure that genotypes were being detected in proximity to the clinical cutoff. The limit of detection around the clinical cutoffs for HPV16  is around 1,500 viral genome copies/ml of undiluted SurePath medium; for the other 13 genotypes , it ranges from 3,000 to 10,000 viral genome copies/ml. Additional information regarding this issue is available in the product’s information-for-use document  daily, for 9 days. Three different reagent lots were utilized: one lot per 3 days of testing. Panel members were randomized, and technical staff were blind to genotypes in each panel member. A total of 162 results (54 per testing site) were expected for each panel member. Percent agreement  analyses were performed for high-negative, low-positive, and moderate-positive contrived specimens. The acceptance criterion for HPV assay performance during testing of panel members was predetermined: for low-positive specimens, it was 94%, and for moderate-positive specimens, it was 98% (Table S1). For clinical specimen analysis, specific mean document .TC scores, including mean differences between the two, were calculated, and statistical comparison was performed using a paired t test. Linear regression was performed for high-risk HPV genotype detection between pre- and postcytology aliquot specimens.For pre- versus postcytology aliquot comparison, positive, negative, and overall agreements were determined using the precytology aliquot result to define positive and negative. Mean  pre- and postcytology aliquot TC scores, HPV TC scores, and high-risk HPV positivity rates were analyzed in three intended-use populations  and different age groups . The mean HBB TC score was calculated by averaging each specimen’s three internal control TC score results. The HPV TC score was calculated by selecting the strongest TC score from nine channels, excluding subjects without an HPV TC score result. The HBB and HPV TC scores were compared using a two-sample t test. The P value that was determined using the Satterthwaite approximation for degrees of freedom was reported. To test whether HPV positivity rates were different between the two collection devices, Fisher’s two-sided exact test was performed.Data for comparison of collection devices were generated at four testing sites in the United States from a precytology aliquot. HBB TC score met the acceptance criteria; the overall standard deviations and percent coefficients of variation ranged from 0.87 to 1.86 and 2.9% to 5.6%, respectively, with the greatest variation being observed within replicates on the same instrument run  by Onclarity , HPV18 (HeLa), and HPV45 (MS751) to spike an HPV-negative clinical specimen matrix at prespecified low- and moderate-positive concentrations. As shown in ment run . HPV-negnclarity .TC score results are plotted in x axis and those from the postcytology aliquot on the y axis. Although there was a slight difference in the distribution of TC scores between pre- and postcytology aliquot groups, the results corresponding to precytology aliquots and postcytology aliquots were linear and represented a one-to-one correlation.Individual Onclarity results from precytology aliquot versus postcytology aliquot specimens were compared. The total numbers of individual results were as follows: 77 for HPV16, 34 for HPV18, 65 for HPV31, 59 for HPV33/58, 42 for HPV45, 72 for HPV51, 80 for HPV52, 195 for HPV35/39/66, and 149 for HPV56/59/66. Individual Onclarity assay TC scores were determined for Onclarity results from specimens that were positive for any high-risk HPV genotype (n = 773) or the individual genotype HPV16 (n = 77), HPV18 (n = 34), or HPV45 (n = 42)  being no greater than 0.31 cycle from zero. Statistical analyses revealed no significant difference between the mean TC scores from pre- and postcytology aliquot specimens for any of the genotype categories.Mean (n = 42) . Pre- ann = 989 for Cervex-Brush and n = 964 for Cytobrush); NILM, ≥30 years of age ; and primary screening, ≥25 years of age . To compare Onclarity performances for each collection device, the TC values were averaged for all samples with a signal (TC < 40) on the Viper LT system  (Table S4). No significant difference was observed between the Cervex-Brush and the Cytobrush (or Cytobrush/spatula) across the three screening populations or by age group  . The HPV positivity rates  for specimens collected with the Cervex-Brush and Cytobrush (or Cytobrush/spatula) devices were not significantly different across all three screening populations. In addition, positivity rates with both collection devices tended to decrease with increasing age in the primary screening population (Onclarity performances were compared following collection with either the Cervex-Brush or the Cytobrush (or Cytobrush/spatula) in three screening populations: ASCUS, ≥21 years of age  investigated here . Onclarity met reproducibility criteria for contrived specimens containing individual genotypes 16, 18, and 45 and for pooled clinical specimens positive for either HPV16, -18, -45, -31, -33/58, or -52. The overall agreement of the results from the Onclarity assay using precytology aliquot and postcytology aliquot samples for each of the subject populations was above 98%, with the lower bound of the 95% confidence interval being ≥93%. Finally, HPV positivity rates and HPV mean TC value for HPV16 (38.3) is approximately 4 cycles higher than that for the other eight Onclarity results (34.2), which may explain the low reproducibility of results for the HPV16 high-negative specimens relative to the other genotype results. However, the Onclarity assay has been clinically validated for HPV16 detection, and previous results for HPV16 from screening populations have demonstrated good specificity and positive predictive values for the detection of the individual HPV16 genotype  and one lot (lot 2). It is not clear that these two instances represent a true depiction of Onclarity assay performance for differentiating HPV16-negative specimens from HPV16-positive specimens around the cutoff. The clinical cutoff genotype , 19–24. otype 19–. Similarotype 19–. In thisThe Onclarity assay is an FDA-approved and CE-marked HPV test for which clinical validation has previously been established 11–, 20–32. 20–11–22–The Onclarity assay is the only HPV assay approved for extended genotyping  in the primary screening population (≥25 years of age). Importantly, the Onclarity assay provides coverage of the three HPV genotypes  that represent approximately 77% of invasive cervical cancer . Wright TC scores of individual results for HPV16, -18, -45, -31, -33/58, and -52 from HPV-positive pooled clinical specimens also demonstrate the reproducibility of the Onclarity assay. In addition, 100% of HPV-negative specimens  were associated with TC values above the cutoff for a positive result (38.3 on the HPV16 channel and 34.2 for channels relative to the other eight HPV channels). This reproducibility is important as countries in North America, Europe, Australia, and Asia continue to consider extended and full genotyping as a triage approach to improve risk detection for high-grade cervical disease during HPV primary screening. Publications from both the Onclarity clinical trial and Kaiser Permanente Northern California have previously demonstrated the potential benefit of extended genotyping to identify either those with NILM cytology as being at high-enough risk for a referral for colposcopy  or those with ASCUS/low-grade squamous intraepithelial lesion (LSIL) as being of low-enough risk to return for follow-up as opposed to a referral for immediate colposcopy   , 22, 32.r HPV56) .a priori represents early or receding infections; thus, enrichment is likely occurring in this cytology group for HPV-positive results that are close to the clinical cutoff of the assay. Qualitative HPV assays show more variability at low infection levels.As many HPV assays are nucleic acid amplification based, precytology aliquot specimens are typically preferred for HPV testing prior to LBC processing. However, current cervical cancer screening recommendations, both inside and outside the United States, vary based on the age of the screening population . Approaches to cervical cancer screening also vary from country to country. In Europe, for example, approximately 55% of countries utilize cytology as the primary screening modality, with 45% of countries utilizing some combination of cytology and HPV testing . DependiHere, the collection device had no overall impact on the HPV result. In addition, there was no observed effect of the collection device type across age groups, and therefore, either collection device should be effective for different screening populations . The squamocolumnar junction (SCJ) is an anatomical area in which cellular transformation occurs at a high rate and is a common region in which abnormal cells develop. With age, the cervical transformation zone  recedes into the cervical canal , which rTC < 40) and signal-positive (TC ≤ 38.3) TC values is smaller (the cutoff is closer than the limit of detection) than that for the other eight HPV results: signal negative, 34.2 < TC < 40; signal positive, TC ≤ 34.2. Finally, histological outcomes were not used here to determine whether Onclarity assay results involving clinical specimens corresponded to performance compared to histological outcomes. However, the objective of this study was to determine the analytical performance of the Onclarity assay using clinical and contrived specimens, irrespective of the ability of the Onclarity assay to detect cancer or precancer. The clinical performance of the Onclarity assay, compared to histology as a reference, has been described extensively elsewhere . Finally, as discussed above, regarding the HPV16 high-negative results, bias could have affected the accuracy of our results for HPV16 compared to the other eight HPV results as the difference in the HPV signal-negative (38.3 < lsewhere , 20, 23.Overall, the results here characterize the impact of preanalytical activities on Onclarity assay reproducibility and provide evidence for the potential flexibility of the Onclarity assay within different workflows during cervical cancer screening. This includes sample collection devices, aliquoting order, and other laboratory workflow practices. Regardless of each of these factors, the results obtained with the Onclarity assay on the Viper-LT system were robust and reproducible.


To investigate distributions of cervical lesions and factors associated with the severity of the cervical lesions in high-risk HPV (hr-HPV) positive women with atypical squamous cells of undetermined significance (ASC-US) cytology.Clinical information of 250,000 women who underwent HPV and cytological test was collected from January 2012 to January 2019. The association between the severity of the cervical lesions and hr-HPV genotypes, hr-HPV viral load, and ages, were analyzed in hr-HPV-positive/ASC-US women.2 = 35.03, p < 0.0001). Compared with the 51–60-year-old group (21.65%), the women in ≤ 30 (40.52%), 31–40 (39.67%), and 41–50 (34.22%) year-old groups had significantly higher risk of HSIL+. The women in ≤ 51–60 (2.68%) and > 60 (3.41%) year-old groups were at increased risk for cervical cancer, compared with the ≤ 30-year-old group (0.61%).3459 hr-HPV-positive/ASC-US women were enrolled in this study. Overall, 43.51% of women with ASC-US had normal histological results, 34.35% had high-grade squamous intraepithelial lesion (HSIL), and 1.30% had cervical cancer. The rate of HSIL or worse (HSIL+) in women with single HPV16 infection (63.09%) was the highest, followed by HPV33 (57.50%), HPV51 (36.11%), HPV58 (36.11%), HPV52 (28.28%), HPV18 (26.37%), HPV66 (19.35%), HPV39 (18.92%), HPV53 (15.00%), and HPV56 (8.51%). Detection rate of HSIL+ in low, intermediate and high viral-load groups were 15.87% (n = 30), 34.91% (n = 74) and 40.68% (n = 214) (Cochran-Armitage Trend test χASC-US women with HPV 16/18/33/51/52/58 single infection and multiple infections, as well as high HPV viral loads, have high risk of HSIL+. Cervical cancer is the third most common cancer among women worldwide and is the second most common to breast cancer in Asia . ApproxiThinprep cytologic test (TCT) is a commonly used method for cytology test . HoweverThere are three common HPV tests in China. The Cobas 4800 HPV test performs DNA extraction, Polymerase Chain Reaction amplification, and real time detection by an automated sample preparation, to detect 14 hr-HPV genotypes. The HPV GenoArray test simultaneous identification of 21 individual HPV genotypes and efficient detection of single or multiple HPV infection, which preforms DNA amplification and HybriBio’s proprietary flow through hybridization technique. Hybrid capture 2 test (HC2) is one of the most frequently applied test to detect the presence of any 13 hr-HPV types, which can provide a quantitation of the viral DNA load.This study aimed to investigate the distribution of hr-HPV genotypes, the association between the severity of the cervical lesions and hr-HPV viral loads, and age-stratified prevalence of high-grade squamous intraepithelial lesion (HSIL) and cervical cancer, by analyzing of clinical information of HPV-positive/ASC-US women in China, which would help us find an appropriate triage of women with ASC-US cytology in the future.All procedures in studies involving human participants were performed in accordance with ethical standards and approved by the Ethics Committee of Shandong University [2018 (054)].And the requirement for written informed consent was waived by the Ethics Committee of Shandong University.This retrospective study included women who accepted TCT test, HPV test and colposcopy at Qilu Hospital of Shandong University from January 2012 to January 2019.Inclusion criteria: (1) women with ASC-US cytology and hr-HPV infections; (2) women accepted colposcopic examination and underwent cervical biopsy under colposcopic guidance; (3) women with complete cervical cervix.Exclusion criteria: (1) women with history of treatments to cervical lesions, such as cervical surgery, laser, freezing and medication, et al.; (2) women with malignant tumors such as ovarian cancer and endometrial cancer; (3) women with autoimmune diseases or receiving immunotherapy; (4) women with pregnancy; (5) women who had received the HPV vaccine; (7) women with smears of insufficient quality .No vaginal washing, intravaginal medication or physical therapy for 3 days before the samples were collected. No sexual behavior within 24 h and women were in non-menstrual period when the samples were collected. Specimen collection, specimen preparations, and results of test are performed, according to the instructions of the manufacturers, respectively.ThinPrep  preparations was used and cytological diagnosis was performed by experienced cytopathologists, according to the Bethesda system 2011 .HC2 : semi-quantitative detection of 13 hr-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Positive by the ratio relative-light-unit/cut-off (RLU/CO) was > 1.0 .the Cobas 4800 System : Qualitative detection of HPV 16, HPV 18 and 12 hr-HPV .the HPV GenoArray test : qualitative detection of 15 kinds of hr-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68; 6 kinds of low risk HPV (lr- HPV) types: 6, 11, 42, 43, 44 and 81 (CP8304).Women could choose one of three HPV tests:All women, enrolled in this study, underwent visual inspection with acetic acid and insulin inspection with Lugo's iodine (VILI) and colposcopy. If any of suspicious lesion were found among the above-mentioned examinations, multi-point biopsy was taken directed by colposcopy at the suspected lesion; otherwise, a random four-quadrant cervical biopsy was obtained at the squamocolumnar junction. Women who were not satisfied with colposcopy should underwent Endocervical Curettage, if necessary.The histologic diagnosis was based on the consensus diagnosis of two experienced gynecologic pathologists. All gynecologic pathologists were blinded to results of TCT, HPV test and colposcopic examination. The results of biopsy reports include: Normal; low-grade squamous intraepithelial lesion (LSIL) (including CIN 1), high-grade squamous intraepithelial lesion (HSIL) (including CIN 2–3), and cervical cancer. HSIL or worse (HSIL+) includes HSIL and cervical cancer. The gold standard for this study was the histologic diagnosis.This work was supported by the foundation of National Natural Science Foundation of China and Science, Grant/Award Number: 81874105; National Clinical Research Center for Gynecological Oncology, Grant/Award Number: 2015BAI13B05; National Natural Science Foundation of China and Science, Grant/Award Number: 81372809; and National Key Research & Development Program of China, Grant/Award Number: 2016YFC1302903. The funding sources acknowledged the design of the study but did not influence the process of our study, including data collection, analysis, and interpretation and the reporting of results.p values < 0.05.Statistical analyses were performed using the SAS 9.4  for windows. The categorical variables were expressed by a percentage (%). Cochran-Armitage Trend test was used to analyze the detection rate of HSIL+ in different viral load groups. The binary Logistic regression model was used to analyze the detection rate of HSIL+ and cervical cancer in different age groups. Results were considered statistically significant at Clinical information of 250,000 women who underwent HPV and TCT test at Qilu Hospital of Shandong University from January 2012 to January 2019 was retrospectively collected and analyzed , 1,043 women had single HPV infection, and 554 women had multiple HPV infections. 1245 women were diagnosed with hr-HPV positive by HC2 test. The remaining hr-HPV positive 241 women who lost their results of HPV test, were classified as ANY group in this study. The average age of women was 39.86 ± 9.94 years (range: 25–80 years). The median number of pregnancies was 3 (range: 0–14) and the median number of births was 1 (range: 0–8).This study divided the age into 5 age groups ≤ 30 , 31–40 , 41–50 , 51–60 , and > 60 .The common hr-HPV genotype was shown in Table The HSIL+ detection rates were different in different hr-HPV genotypes, and the rates of HSIL+ ranged from 8.51 to 63.09% in ASC-US women with single hr-HPV infection. The HSIL+ detection rate of women with single HPV16 infection (63.09%) was the highest, followed by HPV33 (57.50%), HPV51 (36.11%), HPV58 (36.11%), HPV52 (28.28%), HPV18 (26.37%), HPV66 (19.35%), HPV39 (18.92%), HPV53 (15.00%), and HPV56 (8.51%). And, other hr-HPV genotypes  were grouped into the group of "others" due to the small number of people included in women with the single hr-HPV infection. The HSIL+ detection rate in the "others" group was 30.09%  of the women in this study were diagnosed with cervical cancer, including 24 women with HPV16 infections (including 19 women with HPV16 single infection and 5 women with HPV16 multiple infections), 3 women with HPV18 single infection, 1 woman with HPV33 single infection, 1 woman with 12 hr-HPV infection (from Cobas 4800 HPV test), and 16 women with unidentified hr-HPV genotypes infections .2 = 35.03, p < 0.0001).1245 women tested positive for hr-HPV by HC2 test. 42.93% (n = 398) of women with ASC-US had normal histological results, 22.76% (n = 211) had LSIL, 33.23% (n = 308) had HSIL, and 1.08% (n = 308) had cancer. 34.30% (n = 318) of women were diagnosed with HSIL+. According to the viral load of hr-HPV, women were divided into 3 groups: low viral load group , medium viral load group , and high viral load group . The pathological distribution of these three viral-load groups was shown in Table Age-stratified prevalence of HSIL+ and cervical cancer in hr-HPV-positive/ASC-US women is shown in Fig. The cervical cancer detection rates were 0.61%, 0.95%, 1.38%, 2.68% and 3.41%, respectively, in group of women aged ≤ 30 years, 31–40 years, 41–50 years, 51–60 years and > 60 years. When regarding the rate of cervical cancer of women in ≤ 30-year-old group who had the lowest rate of cervical cancer as the compared baseline (OR as 1.00), the women in ≤ 51–60  and > 60  year-old groups were at increased risk for cervical cancer. There was no statistically significant increase in the rates detection of cervical cancer in the group of 31–40 years by 55.74%  and the group of 41–50 years by 126.23% , compared with the ≤ 30-year-old group  in cervical screenings of this center, which was suggested the center strictly controlled the frequency of occurrence of ASC-US and reduced the phenomenon of ascus as the “Garbage Dump”, according previous research . Some stIn this cross-sectional study, the rates of HSIL+ ranged from 8.51% of HPV 56 to 63.09% of HPV16 in ASC-US women with single hr-HPV infection. The HSIL+ detection rates in women with HPV multiple infections was more than 39.77%. Previous research reported HPV-positive/ ASC-US women would have similar the 2-year cumulative risk of HSIL+ and be clinically equivalent with women with LSIL Pap results. We look forward to relevant longitudinal researches on relationship between HPV genotypes and risk of cervical lesions.2 = 35.03, p < 0.0001) in this study.Systematic review , 21 and Due to hormone levels, the use of intrauterine device, more sexual behavior and inflammation in younger women, the cells of those with benign morphological changes mixed with cells of true precancerous lesions, and it may interfere doctors’ diagnosis.HPV prevalence rate among younger women are higher, and the rate of HPV prevalence is highly age-related and decreases with age . AccordiThe previous research found that CIN1 and a subset CIN2 lesions are clinical manifestations of the result of productive hr-HPV infectio , which cOlder women could clear newly HPV infections (including HPV16 infections) as quickly as younger women . So, theLower estrogen levels, thinning of the epithelium, decreasing lactobacilli and some genital tract infections combined diabetes etc., create favorable conditions for HPV infection in older women, especially for > 60 years old women.Another subset of CIN2 lesions and CIN3 lesions are clinical manifestations of the result of transforming hr-HPV infection, which is characterized by a dysregulated expression of E6 and E7 viral oncogenes . It woulWhat’s more, persistent hr-HPV infection is a risk factor for cervical cancer and the risk of persistence increases with the increasing age of the woman. Therefore, older ASC-US women have a higher cervical cancer detection rate.The highest detection rate of HSIL+ (40.52%) was in the age group of women aged ≤ 30 years, and the lowest detection rate of HSIL+ (21.65%) was in the 51–60-year-old group in this study. A large longitudinal cohort study lasting 7 years within the Guanacaste population showed older women had a similar or slightly decreased risk of HSIL+ and especially CIN 3 compared with younger women . In addiHowever, this study showed there was no statistically significant increase in the rate detection of HSIL in the group of > 60 years by 36.49% (29.55% vs 21.65%), compared with the 51–60 years group, which may be related to the relatively limited sample we studied.A 2013 Cochrane Meta-analysis studied triage ASC-US by repeat cytology versus HPV testing and found that similar pooled CIN2+ and CIN3+ specificities that likely will translate to similar overtreatment rates . So, oneWhat’s more, this study found that women infected with HPV52 and HPV58 were also more common, except HPV16 and HPV18, in China. Therefore, it seems a wise choice to get a vaccine containing multiple hr-HPV types. Currently, there are two-valent HPV vaccines (HPV 16/18), four-valent HPV vaccines (HPV 6/11/16/18), and nine-valent HPV vaccines (HPV 6/11/16/18/31/33/45/52/58). Therefore, women should choose nine-valent HPV vaccines in China, if conditions permit.Combined with knowledge about the biology of CIN and results of this study, here are a few suggestions to triage women with ASC-US cytology. Firstly, we should ensure the quality of TCT test and strictly control the occurrence frequency of ASC-US. It has been suggested that good management requires that the frequency of ASC be maintained at < 5% of all cervical screenings , and ASCASC-US women with HPV 16/18/33/51/52/58 single infection and multiple infections, as well as high HPV viral loads, have high risk of HSIL+. In addition, the rate of HSIL+ was higher and the rate of cervical cancer was lower in younger hr-HPV-positive/ ASC-US women, in contrast with older women.This study was a retrospective single-center study with the possibility of incomplete data collection and limited data sources. What’s more, women enrolled in this study were tested by there HPV tests and different HPV tests varied in sensitivity and specificity. Relatively large prospective studies were expected to confirm the findings of this study in the future.