In situ phenotypic and karyotypic co-detection of aneuploid TCs and TECs in cytological specimens with abnormal cervical screening results

In situ phenotypic and karyotypic co-detection of aneuploid TCs and TECs in cytological specimens with abnormal cervical screening results | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In situ phenotypic and karyotypic co-detection of aneuploid TCs and TECs in cytological specimens with abnormal cervical screening results yanling Wang, Alexander Y Lin, Daisy Dandan Wang, Peter Ping Lin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4324077/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 May, 2025 Read the published version in BMC Cancer → Version 1 posted 10 You are reading this latest preprint version Abstract Background Detection of chromosome aneuploidy is an important method for cervical cancer screening, however, the study of chromosome ploidy in primary cervical tumor cells is limited. A novel immunostaining integrated with fluorescence in situ hybridization (iFISH) strategy phenotypically and karyotypically co-detected the expression of tumor markers and chromosome aneuploidy to investigate the diagnostic values of aneuploid tumor cells (TCs) and tumor endothelial cells (TECs) in all-stage cervical lesion smear specimens. Methods A total of 196 patients were enrolled in this study. Immunofluorescence staining of p16 and Ki67 combined with FISH was applied to quantitatively co-detect and characterize aneuploid CD31 − TCs and CD31 + TECs as well as their subtypes in cervical cytological specimens. The diagnostic values of aneuploid TCs and TECs for high-grade squamous intraepithelial lesions (HSIL+) were investigated by receiver operating characteristic curve analysis. Results The number of total aneuploid CD31 − TCs and their p16/Ki67 + subtypes increased markedly with the severity of cervical lesions, although a similar was not observed on aneuploid CD31 + TECs. To identify HSIL+, the area under the curve (AUC) of tetraploid TCs was the largest (0.739), followed by multiploidy (≥ pentaploid) TCs (0.724) and triploid TCs (0.699). Regarding combined subtypes, the AUC of ≥ tetraploid TCs was 0.745, and their unique diagnosis values were clinically reflected in the vitally high specificity. Conclusion The number of CD31 − TCs was associated with the severity of cervical lesions and aneuploid CD31 − TCs exhibited a remarkable diagnostic specificity for HSIL+. Further studies are required to broaden their other potential clinical utility. cervical cancer aneuploid TC aneuploid TEC iFISH biopsy chromosome 8 Background Cervical cancer is the fourth most common female malignant tumor that severely threatens female health. According to the Global Cancer Statistics 2020, more than 600,000 new cases and more than 340,000 deaths are estimated yearly [ 1 ]. Carcinogenic human papillomavirus (HPV) infection is the most critical condition for cervical cancer. However, HPV infection has a long-term natural history before progressing to cervical cancer. Also, premalignant lesions known as cervical intraepithelial neoplasia (CIN) exhibit varied severities over several years or more than a decade [ 2 ], providing an excellent “window” for the prevention and treatment of cervical cancer. HPV vaccination and cervical screening are the most effective ways of preventing cervical cancer. Given the low rates of vaccination in China [ 3 ], cervical screening remains the first line of prevention for cervical cancer. High-risk HPV (hrHPV) testing is the primary screening method with high sensitivity. A large number of patients have "transient" infections, necessitating the triage of women with hrHPV infection. Although the most frequently used triage strategy, cytological testing has inherent limitations that rely on the training and experience of the pathologist, has strong subjectivity and lacks quality control, which inevitably increases the rate of misdiagnosis [ 4 , 5 ]. Several studies are focused on new biomarkers and cervical screening techniques to maximize the cost-efficiency for patients. Aneuploidy refers to the gain or loss of chromosomes, a common characteristic of malignant tumors, and occurs in 90% of solid tumors [ 6 ]. It is an early event of tumorigenesis and can participate in tumor formation and development, such as affecting cell cycle and genomic stability, producing protein stress response, interfering with cell metabolism, and altering tumor microenvironment [ 7 – 9 ]. A high frequency of aneuploidy is associated with invasion, metastasis, and poor prognosis of various types of tumors. Previous studies have shown that aneuploidy exists in cervical cancer and its precancerous lesions, and hrHPV infection is related to the formation of aneuploidy that increases with the severity of cervical lesions [ 10 , 11 ]. Thus, designing a strategy capable of detecting the aneuploidy in these abnormal cells may have a clinical value in the early diagnosis of cervical lesions, especially high-grade lesions. Immunostaining fluorescence in situ hybridization (iFISH) is a new detection technique developed according to the clinical significance of markers in the diagnosis, classification, prognosis, and treatment guidance of tumors. A simultaneous combination of protein immunofluorescence staining and chromosome karyotype detection isolates and identifies subclasses of aneuploid tumor cells. Some studies have used FISH alone to analyze the chromosomal copy number aberrations of exfoliated cervical cells. The preliminary results proved that the frequency of chromosomal aberrations in exfoliated cervical cells varies with stages of cervical lesions [ 10 , 12 , 13 ]. These studies mainly focused on changes in some chromosome sites, collecting limited data on the chromosomal karyotype. In addition, these studies were incapable of combining tumor markers staining simultaneously. Therefore, there is insufficient evidence on the usage of chromosomal karyotype changes with tumor marker staining in the clinical diagnosis of cervical lesions; several studies are required to quantify the risk stratification of cervical lesions. In the present study, we used iFISH technology to detect aneuploidy in cervical exfoliated cells from patients referred to colposcopy and selected centromere probe 8 (CEP8) to estimate chromosome ploidy, p16, and Ki67 as tumor markers. In addition, CD31 antibody was used to distinguish endothelial cells; tumor-derived endothelial cells (TECs) exhibit aneuploidy, and tumor angiogenesis plays a critical role in tumor progression and metastasis [ 14 , 15 ]. Then, different subtypes of aneuploid tumor cells (TCs) and TECs were screened based on the above characteristics. Next, we assessed the differences in chromosome ploidy and explored the diagnostic accuracy of various subclasses of aneuploid TCs and TECs for detecting high-grade cervical lesions. Materials and Methods Patients and Samples A total of 196 subjects (aged 21–78 years old) treated at the North Campus of Shanghai General Hospital (Shanghai, China) between August 2022 and March 2023 were enrolled in this study. All subjects were referred to colposcopy to detect hrHPV-positive or thin-cytology test (TCT) to detect the abnormalities (according to American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines [ 16 ]). Before colposcopy, cytology samples were collected from the cervical canal and the transformation zone with a cell brush and transferred to the Cell Preservation Solution (Cytelligen, San Diego, CA, USA). The exclusion criteria in this study were as follows: (A) Acute inflammatory period (acute cervicitis), sexual life within 24 h; (B) Menstruation, pregnancy, postpartum within 42 days; (C) History of total hysterectomy, cervical surgery, or cervical physical therapy within 3 months; (D) Received pelvic radiation therapy at any time previously or have a history of other malignancies. Colposcopy and pathological results Macroscopic cervical lesions were directly biopsied under colposcopy conducted by skilled gynecologists. If these lesions were invisible, the biopsies were randomly taken from 3, 6, 9, and 12 points of the cervix. All high-grade squamous intraepithelial lesions (HSIL) patients were hospitalized for standardized treatment. The histopathological results of both colposcopy biopsies and surgical specimens were considered, and the highest pathological index of the cervix was the final diagnosis. All specimens were processed with standard histopathological procedures, and two pathologists evaluated the results in a double-blind manner. The final diagnosis was confirmed by the chief pathologist. The pathological examination results were classified into normal (including cervicitis), low-grade squamous intraepithelial lesions (LSIL, equal to CIN1), HSIL (equal to CIN2-3), and cervical cancer. The final diagnosis results of subjects recruited in this study were as follows: 70 patients with normal cervix, 53 patients with LSIL, 51 patients with HSIL, and 22 patients with cervical cancer. iFISH biopsy of cervical scraping smear specimens iFISH biopsy was applied to co-detect aneuploid CD31 − TCs and CD31 + TECs expressing tumor markers in cervical smear specimens stored in the Cell Preservation Solution (Cytelligen). The specimens were processed according to the manufacturer’s protocol (Cytelligen). Briefly, samples without floccules were clarified by centrifugation at 500 ×g at room temperature for 5 min, followed by transferring the sedimented cells into a 1.5-mL Eppendorf tube containing 800 mL Circulating Rare Cell (CRC) buffer. Samples were thoroughly homogenized using a syringe. The pellet was obtained by centrifugation at 2300 ×g for 3 min and resuspended in 100 mL CRC buffer plus 2 mL of antigen repair buffer for 10 min, followed by incubation at room temperature for 20 min with an antibody cocktail including Alexa Fluor (AF) 488-anti-Ki67, AF 594-anti-p16, and Cy5-anti-CD31 (Cytelligen). Subsequently, the Tissue Fixative (Cytelligen) was dropped on slides and dried overnight. Then, FISH was performed with the centromere probe 8 (CEP 8) SpectrumOrange (Vysis, Abbott Laboratories,Chicago, IL, USA) as described previously [ 17 ]. Briefly, slides were sequentially treated with FR1, FR2, and FR3 and dehydrated in absolute ethyl alcohol. Finally, cells were hybridized with CEP8, washed with FR3 solution, and mounted with the iFISH Full Spectrum Anti-Fade Mounting Medium (Cytelligen). Automated image scanning and analyses The images of aneuploid TCs and TECs on the coated and formatted CTC slides (Cytelligen) were captured and analyzed using an automatic Metafer-iFISH 3D scanning and image analysis system. Five-channel automated X-Y scanning with cross-Z-sectioning of all cells was performed at 1-mm steps of depth (DAPI, CD31, CEP8, p16, and Ki67). Automatic image processing, classification, and statistical analysis were performed based on the expression of tumor markers and chromosome ploidy. Statistical analysis All data were statistically analyzed using IBM SPSS software (version 26.0) and GraphPad Prism software (version 8.0). Differences in continuous variables between groups were compared using the Mann–Whitney U test, and all data were presented as the median ± 95% confidence interval (CI). Non-parametric receiver operating characteristic (ROC) curve analysis was used to compare low-grade (≤ LSIL) to high-grade (≥ HSIL) cervical lesions. Then, a threshold was established for the number of aneuploid TCs and TECs, and the maximum Youden index (sensitivity + specificity − 1) was used to determine the cutoff value. All data in this study were expressed in a ratio of 10,000 percent and logarithms with log10 as the base were displayed in the graph. P < 0.05 was considered statistically significant. Results Identification of aneuploid TCs and TECs Cell images captured by automated image scanning are shown in Fig. 1 . According to previous studies [ 18 , 19 ], we defined the aneuploid TCs as DAPI + /CD31 − /CEP8 > 2 and the aneuploid TECs as DAPI + /CD31 + /CEP8 > 2. Then, aneuploid TC and TEC subtypes, including triploid, tetraploid, and multiploid (≥ pentaploid) TCs, were counted. On the other hand, haploids were not counted due to the small number and no statistical difference in the preliminary experiment. p16 and Ki67 are identified as tumor markers for high-grade cervical lesions in clinical practice [ 5 ]. The expression of p16 and Ki67 on TCs and TECs was observed in the current study. Distribution of aneuploid TCs and TECs in different cervical lesion stages The results of comparing the distribution differences of aneuploid TCs and TECs in different stages of cervical lesions are presented in Fig. 2 as log10 [(total number of aneuploid cells/slide cells)×10000 + 1]. Interestingly, different proportions of aneuploid TCs were detected in the normal cervix infected with hrHPVs, and the proportion of aneuploid TCs increased gradually with the severity of cervical lesions. However, no significant difference was observed between the normal and LSIL groups, but the differences among the other groups were statistically significant (Fig. 2 A); this phenomenon is not obvious in aneuploid TECs (Fig. 2 B). Next, we analyzed the proportion of p16/Ki67(p16 and/or Ki67)-positive aneuploid TCs and TECs, and the images showed that the positive rate of p16/Ki67 increased with the severity of the cervical lesions. Also, the proportion of p16/Ki67-positive aneuploid TCs in cervical cancer was significantly higher than that of HSIL, LSIL, and normal cervix, but this difference was not significant in aneuploid TECs (Fig. 2 C, D). Subclassification of aneuploid TCs and TECs by chromosomal ploidy In order to further understand the distribution and clinical significance of aneuploid TCs and TECs with different chromosome ploidies at all stages of cervical lesions, we divided aneuploid TCs and TECs into 12 subtypes. The results showed that the difference between normal and LSIL groups was not statistically significant in any aneuploid subclass of TCs and TECs. Among all subtypes of aneuploid TCs, the proportion of tetraploid and ≥ pentaploid TCs in patients with HSIL and cervical cancer was significantly higher than that in patients with mild lesions. Although the distribution of triploid TCs was similar, it could not distinguish between LSIL and HSIL (Fig. 3 A). For aneuploid TECs, the altered trend of proportion in each subclass was irregular, and only TECs with ≥ pentaploid could distinguish cervical cancer from HSIL, LSIL, and normal cervix. In the analysis of p16/Ki67-positive aneuploid TCs and TECs, the positive rate of p16/Ki67 in each subclass of aneuploid TCs increased with the severity of cervical lesions (Fig. 3 C). Firstly, the results of the analysis of triploid TCs revealed that the positive rate of p16/Ki67 in the cancer group was much higher than that in the HSIL, LSIL, and normal groups. Compared to tumor cells with triploid, p16/Ki67-positive tetraploid TCs distinguished between patients with HSIL and milder lesions. On the other hand, the proportion of ≥ pentaploid TCs with p16/Ki67-positive could well-distinguish all stages of cervical lesions except between normal and LSIL groups, and the significance was better than that in p16/Ki67-positive triploid and tetraploid TCs. The current data indicated that the higher the ploidy, the greater the degree of chromosomal instability, thus making the cells conducive to the development of malignant tumors. Therefore, the positive rate of tumor markers in aneuploid TCs at different grades of cervical lesions differed markedly. However, this phenomenon did not appear in all subclasses of TECs. As shown in Fig. 3 D, no significant difference was detected in the positive rate of target cells among all groups except the difference in ≥ pentaploid TECs between the HSIL and LSIL groups. Identification of HSIL + by individual and combinations of aneuploid TCs and TECs count In view of the clinical treatment focus on HSIL and above lesions (HSIL+), we divided the pathology into two groups: one was patients with ≤ LSIL and the other was patients with ≥ HSIL. The ROC curve was plotted to evaluate the diagnostic value of each subcategory in predicting HSIL+ (Fig. 4 ). Next, we selected the best cutoff values based on the maximum value of Youden index, and the corresponding sensitivity and specificity are shown in Table 1. The area under the curve (AUC) of tetraploid TCs was the largest (AUC = 0.739, cutoff value: 8.83, sensitivity: 60.3%, specificity: 80.5%), followed by ≥ pentaploid TCs (AUC = 0.724, cutoff value: 1.74, sensitivity: 69.9%, specificity: 69.9%) and triploid TCs (AUC = 0.699, cutoff value: 24.73, sensitivity: 45.2%, specificity: 89.4%). However, the analytical results obtained from aneuploid TCs and TECs with positive p16/Ki67 were unsatisfactory. Their AUC values were small, and the sensitivity of the aneuploid TCs with positive p16/Ki67 was much less than expected, while the specificity was satisfactory. In addition, among all aneuploid TCs, the specificity of ≥ pentaploid TCs with positive p16/Ki67 was the highest (88.6%), followed by pentaploid TCs with positive p16/Ki67 (78.9%) and triploid TCs with positive p16/Ki67 (64.4%). Together, the diagnostic value of a single subclass was mainly reflected in the specificity. Next, we combined some subclasses, and the AUC value of ≥ tetraploid TCs was the largest (AUC = 0.745, cutoff value: 8.74, sensitivity: 71.2%, specificity: 74.8%). The sensitivity and specificity of other combinations are shown in Table 1. Discussion In this study, we evaluated the variation in the number of chromosome 8 aneuploid TCs and TECs at different stages of the cervical lesion. Also, the diagnostic value of aneuploidy subtypes and their combinations for HSIL + was determined. The results showed that the distribution of aneuploid TCs varied with the severity of cervical lesions, indicating that they are optimal indicators of the CIN stage but cannot be found in aneuploid TECs. The subtype analysis of aneuploidy showed that triploid, tetraploid, and ≥ pentaploid TCs could distinguish between HSIL, cervical cancer, and milder lesions and had good clinical value in the diagnosis of HSIL+, especially in the specificity. Compared to subtypes, ≥pentaploid TCs had preferable sensitivity (69.9%), while triploid TCs had preferable specificity (89.4%). However, the sensitivity and specificity of some subcategory combinations were not improved. Previous aneuploidy analysis of cervical cytology was mainly identified by measuring the DNA content of the cell, and the aneuploidy of DNA aided the diagnosis of cervical lesions [ 20 – 22 ]. However, in some cells, although the average molecular weight of DNA is close to that of diploid, the true diploid regions may be missing [ 23 ]. Conversely, aneuploidy karyotype detection is objective. In normal human cells, aneuploidy is rare, except in neuronal and liver cells, most autosomal aneuploidy cells are embryonically lethal [ 7 ]. Strikingly, only tumor cells can maintain or increase proliferation rate when they exhibit aneuploidy. Carcinogenic HPV plays a critical role in inducing cervical cellular aneuploidy, HPV oncoproteins (especially E6 and E7) lead to mitotic defects through mediating cell cycle regulation disorders and affecting centrosome replication and spindle polarity, thus inducing the production of aneuploidy [ 24 – 26 ]. Previous studies have found that the production of tetraploid occurs during early-stage events of cervical cancer that predispose cervical cells to the formation of aneuploidy [ 10 ], our finding was similar when aneuploidy progresses to tetraploidy, the numeral difference of aneuploid TCs between LSIL and HSIL began to make sense(Fig. 3 A). Women with increased tetraploid TCs in cervical exfoliated cells should become our focus of follow-up. Moreover, ≥pentaploidy seems more obvious in cervical cancer, suggesting that the higher level of ploidy was more relevant to the severity of malignancy. Aneuploidy is a form of chromosomal instability, detecting karyotypic alterations provides abundant genetic information on tumor progression which may help us recognize the increased invasiveness and aggressiveness of cervical cancer. In multiple genome sequencing analyses, most of the chromosome arms of cervical cancer cells were gained or lost in different proportions [ 27 , 28 ], additional studies should track karyotype changes during tumor progression and after treatment, in light of aneuploidy karyotype is associated with tumor evolution and drug resistance [ 29 ]. Chromosome 8 abnormalities are closely related to the occurrence and development of various tumors [ 30 , 31 ]. The chromosomal aneuploidy detection method (FISH) using CEP8 has been widely used to evaluate hematological tumors and various solid tumors [ 30 , 31 ]. The abnormality of chromosome 8 has also been confirmed to exist in cervical cancer in previous studies, especially the trisomy of chromosome 8 [ 32 ]. Many cancer-related genes are also located in this chromosome, including c-MYC , CCNE2 , TP53INP1 , and RAD54B [ 28 ], especially c-MYC which exerts a critical role in cell proliferation, differentiation, and apoptosis and is associated with several human tumors [ 33 ]. iFSIH is a new technology introduced for detecting aneuploid cells, mainly used for the detection of circulating tumor cells and circulating tumor endothelial cells [ 30 , 34 ]. To the best of our knowledge, this is the first study wherein the above technique was applied for the detection of aneuploidy in cervical exfoliated cells, confirming the difference in the distribution of chromosome 8 aneuploidy and its subclasses in cervical lesions at all stages. Previous research on chromosomal changes in cervical cytology mainly focused on some chromosomal regions, their sensitivity values for the detection of HSIL + were better. In contrast, our specificity is superior to certain probe sites (such as 3q26&53.3%, 5p15&56.7%, and 20q13&56.7%) [ 12 , 35 ], this may be due to the change of copy number of chromosome arms and bands precedes genome-wide polyploidy. p16 and Ki67 are the most frequently used diagnostic tumor markers for identifying cervical lesions, especially high-grade subtypes. Several studies have shown that the sensitivity of p16/Ki67 double-staining for the diagnosis of HSIL + was significantly higher than that of TCT, although the improvement of specificity was not significant [ 36 ]. In this study, p16/Ki67 was also selected as the object of protein immunofluorescence staining. Interestingly, these results indicated that p16/Ki67 double-staining had a preferable specificity for diagnosing HSIL+; however, the sensitivity was not satisfactory. The differences in these results may be attributed to two aspects. First, previous studies have measured the protein levels of all cells, and false positives could be because p16 is expressed in endometrial tubal metaplasia and cervical endometriosis [ 37 ]. Second, our study only estimated the expression of p16/Ki67 on chromosome 8 in aneuploid tumor cells. Herein, we did not include p16/Ki67-positive diploid tumor cells, which were different from the tumor marker-positive cells counted in other studies. The current study demonstrated some p16/Ki67-positive diploid cells; therefore, we cannot rely solely on markers to identify tumor cells in non-morphological detection due to uncertainty whether these cells are normal or have abnormal chromosome structures. Thus, additional aneuploidy testing may accurately identify tumor cells. Strikingly, E6/E7 play a leading role in the formation of aneuploid cervical cells, perhaps they are more appropriate as phenotypes to be combined with karyotypic detection. Another focus of our study was aneuploid TECs. Tumorigenesis, progression, and metastasis are based on vasculogenesis. Unlike normal blood vessels, tumor vessels contain a majority of cytogenetically abnormal endothelial cells that are characterized by aneuploid chromosomes [ 14 ]. These aneuploid TECs may be derived from tumor cell transdifferentiation and heterotypic cell fusion, thereby exhibiting some characteristics of tumor cells [ 38 ]. Cancerization of stromal cells and transdifferentiation of stem cells may also be involved [ 15 , 39 ]. Positively expressed CD31 is the marker of endothelial cells. Our data showed that the count of CD31 + aneuploid TECs altered dynamically at different stages of cervical lesions and significantly increased in cancerous cells, especially ≥ pentaploid TECs, implying that aneuploid TECs support tumor progression. Moreover, the positive rate of p16/Ki67 aneuploid TECs increased with cervical lesion stages, although no significant difference was noted in each subclassification. Interestingly, these dynamic changes were similar to those reported previously [ 19 ]. The diagnostic role of aneuploid TECs is not remarkable, whereas the presence of aneuploid TECs is crucial for anti-angiogenic therapy, as chromosomal instability may provide a mechanism to alter endothelial cells and render them resistant to drugs [ 29 ]. Some researchers have proposed that aneuploid TEC is more resistant to chemotherapeutic drugs, such as Vincristine and 5-Fluorouracil than normal endothelial cells[ 39 ]. Therefore their potential therapeutic clinical value seems promising for in-depth investigation. Limitations Nevertheless, the present study has some shortcomings. Firstly, the quantity of cells in different specimens varies greatly due to individualized clinical practicing of sample collection for each patient. Secondly, although the number of aneuploid tumors cells in peripheral blood is extremely low in normal subjects as previously reported by others, whether the existence of aneuploid cells in cervix of a large cohort of non-HPV infected healthy subjects remains to be examined. In addition, whether specific subpopulations of aneuploid TCs and TECs and their dynamic changes can predict HPV clearance is yet uncertain in the current study. Thus, additional large cohort clinical studies will be conducted to further optimize and validate aneuploidy and tumor marker-derived biomarkers for maximal benefit of clinical diagnosis of cervical lesions. Conclusions In this study, iFISH, a novel detection technology, was used to detect, characterize and classify aneuploid TCs and TECs in exfoliated cells of cervical lesions at all stages. The current findings indicated that aneuploid TCs and TECs exhibit differences in the quantity, degree, and expression of tumor markers across all stages of cervical lesions. Aneuploid gains were correlated with the severity of cervical lesions. Triploid, tetraploid, and ≥ pentaploid TCs, regardless of p16/Ki67 expression, can distinguish HSIL + with high specificity. Abbreviations HPV human papillomavirus TC tumor cell TEC tumor-derived endothelial cell LSIL low-grade squamous intraepithelial lesion HSIL high-grade squamous intraepithelial lesion FISH fluorescence in situ hybridization CEP8 centromere probe 8 Declarations Ethics approval and consent to participate The study was approved by the Ethic Committee of the Shanghai General Hospital(reference 2021SQ263). All patients received informed consent prior to cervical cytological specimens collection. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. i•FISH ® is the registered trademarks of Cytelligen. Dr. Peter P. Lin is the president at Cytelligen. None of authors owns Cytelligen's stock shares. No additional COI to be disclosed. Funding This research was funded by Shanghai Pujiang Talent Plan(18PJC097) to Y.B.Y, Shanghai Aging and Women and Children’s Health Research Project(2020YJZX0215) to Y.B.Y. Authors’ contributions Y.L.W. contributed conceptualization, validation, formal analysis, investigation, resources, and writing original draft; A.Y.L. and D.D.W. contributed methodology and validation; P.P.L. contributed conceptualization, visualization, writing – original draft, review and editing; X.X.Z contributed data curation, methodology and writing original draft; Y.B.Y contributed review, editing and funding acquisition; Y.P.Z contributed project administration, conceptualization, resources and supervision. All the authors read and approved the final version of manuscript. Acknowledgements Authors thank staffs at Shanghai General Hospital, Cytointelligen (China Medical City, Taizhou, Jiangsu, China) and Cytelligen (San Diego, CA, USA) for providing support. Authors’ information 1 Department of Gynecology and Obstetrics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 Cytelligen, San Diego, CA, USA References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. Moscicki AB, Schiffman M, Burchell A, Albero G, Giuliano AR, Goodman MT, Kjaer SK, Palefsky J. Updating the natural history of human papillomavirus and anogenital cancers. Vaccine. 2012;30(Suppl 5):F24–33. Xia C, Hu S, Xu X, Zhao X, Qiao Y, Broutet N, Canfell K, Hutubessy R, Zhao F. Projections up to 2100 and a budget optimisation strategy towards cervical cancer elimination in China: a modelling study. Lancet Public Health. 2019;4(9):e462–72. Wright TC, Stoler MH, Behrens CM, Sharma A, Sharma K, Apple R. Interlaboratory variation in the performance of liquid-based cytology: Insights from the ATHENA trial. Int J Cancer. 2014;134(8):1835–43. Ebisch RM, Siebers AG, Bosgraaf RP, Massuger LF, Bekkers RL, Melchers WJ. Triage of high-risk HPV positive women in cervical cancer screening. Expert Rev Anticancer Ther. 2016;16(10):1073–85. Weaver BA, Cleveland DW. Does aneuploidy cause cancer? Curr Opin Cell Biol. 2006;18(6):658–67. Tanaka K, Hirota T. Chromosomal instability: A common feature and a therapeutic target of cancer. Biochim et Biophys Acta (BBA) - Reviews Cancer. 2016;1866(1):64–75. Vasudevan A, Schukken KM, Sausville EL, Girish V, Adebambo OA, Sheltzer JM. Aneuploidy as a promoter and suppressor of malignant growth. Nat Rev Cancer. 2021;21(2):89–103. Ben-David U, Amon A. Context is everything: aneuploidy in cancer. Nat Rev Genet. 2020;21(1):44–62. Olaharski AJ, Sotelo R, Solorza-Luna G, Gonsebatt ME, Guzman P, Mohar A, Eastmond DA. Tetraploidy and chromosomal instability are early events during cervical carcinogenesis. Carcinogenesis. 2006;27(2):337–43. Steinbeck RG. Proliferation and DNA Aneuploidy in Mild Dysplasia Imply Early Steps of Cervical Carcinogenesis. Acta Oncol. 2009;36(1):3–12. Luhn P, Houldsworth J, Cahill L, Schiffman M, Castle PE, Zuna RE, Dunn ST, Gold MA, Walker J, Wentzensen N. Chromosomal gains measured in cytology samples from women with abnormal cervical cancer screening results. Gynecol Oncol. 2013;130(3):595–600. Janz KH-H, Castle VPE, Chaudhri N, White N, Wilber K, Morrison LE, Auer G, Burroughs FH, Mark, Sherman E et al. Detection of enomic Amplification of the Human Telomerase Gene (TERC) in Cytologic Specimens as a Genetic Test for the Diagnosis of Cervical Dysplasia. The American journal of pathology 2003, 163(4):1405–1416. Akino T, Hida K, Hida Y, Tsuchiya K, Freedman D, Muraki C, Ohga N, Matsuda K, Akiyama K, Harabayashi T, et al. Cytogenetic Abnormalities of Tumor-Associated Endothelial Cells in Human Malignant Tumors. Am J Pathol. 2009;175(6):2657–67. Hida K, Klagsbrun M. A New Perspective on Tumor Endothelial Cells: Unexpected Chromosome and Centrosome Abnormalities. Cancer Res. 2005;65(7):2507–10. Perkins RB, Guido RS, Castle PE, Chelmow D, Einstein MH, Garcia F, Huh WK, Kim JJ, Moscicki AB, Nayar R, et al. : 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors. J Low Genit Tract Dis. 2020;24(2):102–31. Zhang T, Zhang L, Gao Y, Wang Y, Liu Y, Zhang H, Wang Q, Hu F, Li J, Tan J, et al. Role of aneuploid circulating tumor cells and CD31 + circulating tumor endothelial cells in predicting and monitoring anti-angiogenic therapy efficacy in advanced NSCLC. Mol Oncol. 2021;15(11):2891–909. Cheng H, Wang S, Luan W, Ye X, Dou S, Tang Z, Zhu H, Ping Lin P, Li Y, Cui H, et al. Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer. Chin J Cancer Res. 2021;33(2):256–70. Lei Y, Sun N, Zhang G, Liu C, Lu Z, Huang J, Zhang C, Zang R, Che Y, Mao S et al. Combined detection of aneuploid circulating tumor-derived endothelial cells and circulating tumor cells may improve diagnosis of early stage non‐small‐cell lung cancer. Clin Translational Med 2020, 10(3). Garner D. Clinical application of DNA ploidy to cervical cancer screening: A review. World J Clin Oncol. 2014;5(5):931–65. Costa AF, Ribeiro MGM, Onofre ASC, Miranda Onofre FB. Aneuploidy detection for diagnostic and prognostic use in premalignant and malignant lesions of the uterine cervix: A systematic review. Diagn Cytopathol. 2020;49(2):335–46. Costa AF, Pogere A, Pasinato APBF, Onofre ASC. Onofre FBdM: DNA image cytometry parameters to identify high-grade cervical lesions. Cytopathology. 2022;33(5):563–71. Lauren MF, Merlo Li-san, Wang JW, Pepper, Rabinovitch PS, Maley CC. Polyploidy, aneuploidy and the evolution of cancer. Adv Exp Med Biol. 2010;676:1–13. Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer. 2010;10(8):550–60. Korzeniewski N, Spardy N, Duensing A, Duensing S. Genomic instability and cancer: lessons learned from human papillomaviruses. Cancer Lett. 2011;305(2):113–22. Münger SDK. Centrosomes, Genomic Instability, and Cervical Carcinogenesis. Crit Rev Eukaryot Gene Expr. 2003;13(1):9–23. Ren T, Suo J, Liu S, Wang S, Shu S, Xiang Y, Lang J-H. Using low-coverage whole genome sequencing technique to analyze the chromosomal copy number alterations in the exfoliative cells of cervical cancer. J Gynecologic Oncol 2018, 29(5). Clara Bodelon S, Vinokurova JN, Sampson, Johan A, den Boon JL, Walker MA, Horswill K, Korthauer M, Schiffman ME, Sherman, Rosemary E, Zuna, et al. Chromosomal copy number alterations and HPV integration in cervical precancer and invasive cancer. Carcinogenesis. 2016;37(2):188–96. Vitale I, Manic G, Senovilla L, Kroemer G, Galluzzi L. Karyotypic Aberrations in Oncogenesis and Cancer Therapy. Trends Cancer. 2015;1(2):124–35. Luo S, Ou Y, Zheng T, Jiang H, Wu Y, Zhao J, Zhang Z, You SL. Optimal Strategy for Colorectal Cancer Patients’ Diagnosis Based on Circulating Tumor Cells and Circulating Tumor Endothelial Cells by Subtraction Enrichment and Immunostaining-Fluorescence In Situ Hybridization Combining with CEA and CA19-9. J Oncol. 2021;2021:1–9. Birnbaum D, Adélaïde J, Popovici C, Charafe-Jauffret E, Mozziconacci M-J, Chaffanet M. Chromosome arm 8p and cancer: a fragile hypothesis. Lancet Oncol. 2003;4(10):639–42. Mark HF, Feldman D, Samy M, Sun C, Das S, Mark S, Lathrop J. Assessment of Chromosome 8 Copy Number in Cervical Cancer by Fluorescent in situ Hybridization. Exp Mol Pathol. 1999;66(2):157–62. Shachaf, Catherine M, Felsher DW. Tumor dormancy and MYC inactivation: pushing cancer to the brink of normalcy. Cancer Res. 2005;65(11):4471–4. Lin PP. Integrated EpCAM-independent subtraction enrichment and iFISH strategies to detect and classify disseminated and circulating tumors cells. Clin Translational Med 2015, 4(1). Policht FA, Song M, Sitailo S, O'Hare A, Ashfaq R, Muller CY, Morrison LE, King W, Sokolova IA. Analysis of genetic copy number changes in cervical disease progression. BMC Cancer 2010, 10(1). Chen X, Chen C, Liu L, Dai W, Zhang J, Han C, Zhou S. Evaluation of p16/Ki-67 dual‐stain as triage test for high‐risk HPV ‐positive women: A hospital‐based cross‐sectional study. Cancer Cytopathol. 2022;130(12):955–63. Tringler B, Gup CJ, Singh M, Groshong S, Shroyer AL, Heinz DE, Shroyer KR. Evaluation of p16INK4a and pRb expression in cervical squamous and glandular neoplasia. Hum Pathol. 2004;35(6):689–96. Lin PP. Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis. Cells 2020, 9(6). Hida K, Hida Y, Shindoh M. Understanding tumor endothelial cell abnormalities to develop ideal anti-angiogenic therapies. Cancer Sci. 2008;99(3):459–66. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 May, 2025 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 08 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviews received at journal 24 Jun, 2024 Reviewers agreed at journal 24 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviewers invited by journal 26 May, 2024 Editor invited by journal 03 May, 2024 Submission checks completed at journal 26 Apr, 2024 Editor assigned by journal 26 Apr, 2024 First submitted to journal 25 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4324077","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297476556,"identity":"057db329-21e3-45ad-a013-ef9f20187402","order_by":0,"name":"yanling Wang","email":"","orcid":"","institution":"Shanghai General Hospital","correspondingAuthor":false,"prefix":"","firstName":"yanling","middleName":"","lastName":"Wang","suffix":""},{"id":297476557,"identity":"d18a133f-cdbe-4ec5-898d-0acf994800c6","order_by":1,"name":"Alexander Y Lin","email":"","orcid":"","institution":"Cytelligen","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"Y","lastName":"Lin","suffix":""},{"id":297476558,"identity":"8b3d9c4c-0baf-4b7e-a6b8-d11e22724f76","order_by":2,"name":"Daisy Dandan Wang","email":"","orcid":"","institution":"Cytelligen","correspondingAuthor":false,"prefix":"","firstName":"Daisy","middleName":"Dandan","lastName":"Wang","suffix":""},{"id":297476559,"identity":"9f80be31-3928-47c2-99de-99a36ad0f980","order_by":3,"name":"Peter Ping Lin","email":"","orcid":"","institution":"Cytelligen","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"Ping","lastName":"Lin","suffix":""},{"id":297476560,"identity":"23307f9a-bbae-46fc-9137-3032a41c57a7","order_by":4,"name":"Xuexin Zhou","email":"","orcid":"","institution":"Shanghai General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuexin","middleName":"","lastName":"Zhou","suffix":""},{"id":297476561,"identity":"5f467d70-f727-4c34-bed8-4e4f4ea0c21b","order_by":5,"name":"Yongbin Yang","email":"","orcid":"","institution":"Shanghai General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongbin","middleName":"","lastName":"Yang","suffix":""},{"id":297476563,"identity":"37bf73df-c912-4363-916a-848e6016778d","order_by":6,"name":"Yaping Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDACCRBRUSPHxt58gBQtZ44Z8/EcSyBBC2MLc+I8iRwF4nTIz+4xky5sYEtvY8hhYPhRsY2wFsY5Z8ykZ+6QyW1jOHuAsefMbcJamCVyzKR5z7DltjH2JTAzthGhhQ2spY05nY2Zx4A4LTxQLQlsbMRqkZBIK7aeceaYYRsPW8JBovwiPyN54+2Cihp5+fmPDz74UUGEFiBgkYaxDhClHgiYPxOrchSMglEwCkYoAAC5sDTdVf9d/wAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2024-04-25 12:16:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4324077/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4324077/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-025-14346-y","type":"published","date":"2025-05-26T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83782909,"identity":"2f389d06-0cd5-46e6-9f70-302d9ee39b77","added_by":"auto","created_at":"2025-06-02 16:08:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":702352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4324077/v1/dd451594-d5d8-4baf-8b78-9f5492a30d1b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In situ phenotypic and karyotypic co-detection of aneuploid TCs and TECs in cytological specimens with abnormal cervical screening results","fulltext":[{"header":"Background","content":"\u003cp\u003eCervical cancer is the fourth most common female malignant tumor that severely threatens female health. According to the Global Cancer Statistics 2020, more than 600,000 new cases and more than 340,000 deaths are estimated yearly [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Carcinogenic human papillomavirus (HPV) infection is the most critical condition for cervical cancer. However, HPV infection has a long-term natural history before progressing to cervical cancer. Also, premalignant lesions known as cervical intraepithelial neoplasia (CIN) exhibit varied severities over several years or more than a decade [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], providing an excellent \u0026ldquo;window\u0026rdquo; for the prevention and treatment of cervical cancer. HPV vaccination and cervical screening are the most effective ways of preventing cervical cancer. Given the low rates of vaccination in China [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], cervical screening remains the first line of prevention for cervical cancer. High-risk HPV (hrHPV) testing is the primary screening method with high sensitivity. A large number of patients have \"transient\" infections, necessitating the triage of women with hrHPV infection. Although the most frequently used triage strategy, cytological testing has inherent limitations that rely on the training and experience of the pathologist, has strong subjectivity and lacks quality control, which inevitably increases the rate of misdiagnosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Several studies are focused on new biomarkers and cervical screening techniques to maximize the cost-efficiency for patients.\u003c/p\u003e \u003cp\u003eAneuploidy refers to the gain or loss of chromosomes, a common characteristic of malignant tumors, and occurs in 90% of solid tumors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is an early event of tumorigenesis and can participate in tumor formation and development, such as affecting cell cycle and genomic stability, producing protein stress response, interfering with cell metabolism, and altering tumor microenvironment [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A high frequency of aneuploidy is associated with invasion, metastasis, and poor prognosis of various types of tumors. Previous studies have shown that aneuploidy exists in cervical cancer and its precancerous lesions, and hrHPV infection is related to the formation of aneuploidy that increases with the severity of cervical lesions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, designing a strategy capable of detecting the aneuploidy in these abnormal cells may have a clinical value in the early diagnosis of cervical lesions, especially high-grade lesions.\u003c/p\u003e \u003cp\u003eImmunostaining fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (iFISH) is a new detection technique developed according to the clinical significance of markers in the diagnosis, classification, prognosis, and treatment guidance of tumors. A simultaneous combination of protein immunofluorescence staining and chromosome karyotype detection isolates and identifies subclasses of aneuploid tumor cells. Some studies have used FISH alone to analyze the chromosomal copy number aberrations of exfoliated cervical cells. The preliminary results proved that the frequency of chromosomal aberrations in exfoliated cervical cells varies with stages of cervical lesions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These studies mainly focused on changes in some chromosome sites, collecting limited data on the chromosomal karyotype. In addition, these studies were incapable of combining tumor markers staining simultaneously. Therefore, there is insufficient evidence on the usage of chromosomal karyotype changes with tumor marker staining in the clinical diagnosis of cervical lesions; several studies are required to quantify the risk stratification of cervical lesions.\u003c/p\u003e \u003cp\u003eIn the present study, we used iFISH technology to detect aneuploidy in cervical exfoliated cells from patients referred to colposcopy and selected centromere probe 8 (CEP8) to estimate chromosome ploidy, p16, and Ki67 as tumor markers. In addition, CD31 antibody was used to distinguish endothelial cells; tumor-derived endothelial cells (TECs) exhibit aneuploidy, and tumor angiogenesis plays a critical role in tumor progression and metastasis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Then, different subtypes of aneuploid tumor cells (TCs) and TECs were screened based on the above characteristics. Next, we assessed the differences in chromosome ploidy and explored the diagnostic accuracy of various subclasses of aneuploid TCs and TECs for detecting high-grade cervical lesions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Samples\u003c/h2\u003e \u003cp\u003eA total of 196 subjects (aged 21\u0026ndash;78 years old) treated at the North Campus of Shanghai General Hospital (Shanghai, China) between August 2022 and March 2023 were enrolled in this study. All subjects were referred to colposcopy to detect hrHPV-positive or thin-cytology test (TCT) to detect the abnormalities (according to American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]). Before colposcopy, cytology samples were collected from the cervical canal and the transformation zone with a cell brush and transferred to the Cell Preservation Solution (Cytelligen, San Diego, CA, USA). The exclusion criteria in this study were as follows: (A) Acute inflammatory period (acute cervicitis), sexual life within 24 h; (B) Menstruation, pregnancy, postpartum within 42 days; (C) History of total hysterectomy, cervical surgery, or cervical physical therapy within 3 months; (D) Received pelvic radiation therapy at any time previously or have a history of other malignancies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eColposcopy and pathological results\u003c/h2\u003e \u003cp\u003eMacroscopic cervical lesions were directly biopsied under colposcopy conducted by skilled gynecologists. If these lesions were invisible, the biopsies were randomly taken from 3, 6, 9, and 12 points of the cervix. All high-grade squamous intraepithelial lesions (HSIL) patients were hospitalized for standardized treatment. The histopathological results of both colposcopy biopsies and surgical specimens were considered, and the highest pathological index of the cervix was the final diagnosis. All specimens were processed with standard histopathological procedures, and two pathologists evaluated the results in a double-blind manner. The final diagnosis was confirmed by the chief pathologist. The pathological examination results were classified into normal (including cervicitis), low-grade squamous intraepithelial lesions (LSIL, equal to CIN1), HSIL (equal to CIN2-3), and cervical cancer. The final diagnosis results of subjects recruited in this study were as follows: 70 patients with normal cervix, 53 patients with LSIL, 51 patients with HSIL, and 22 patients with cervical cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eiFISH biopsy of cervical scraping smear specimens\u003c/h2\u003e \u003cp\u003eiFISH biopsy was applied to co-detect aneuploid CD31\u003csup\u003e\u0026minus;\u003c/sup\u003e TCs and CD31\u003csup\u003e+\u003c/sup\u003e TECs expressing tumor markers in cervical smear specimens stored in the Cell Preservation Solution (Cytelligen). The specimens were processed according to the manufacturer\u0026rsquo;s protocol (Cytelligen). Briefly, samples without floccules were clarified by centrifugation at 500 \u0026times;g at room temperature for 5 min, followed by transferring the sedimented cells into a 1.5-mL Eppendorf tube containing 800 mL Circulating Rare Cell (CRC) buffer. Samples were thoroughly homogenized using a syringe. The pellet was obtained by centrifugation at 2300 \u0026times;g for 3 min and resuspended in 100 mL CRC buffer plus 2 mL of antigen repair buffer for 10 min, followed by incubation at room temperature for 20 min with an antibody cocktail including Alexa Fluor (AF) 488-anti-Ki67, AF 594-anti-p16, and Cy5-anti-CD31 (Cytelligen). Subsequently, the Tissue Fixative (Cytelligen) was dropped on slides and dried overnight. Then, FISH was performed with the centromere probe 8 (CEP 8) SpectrumOrange (Vysis, Abbott Laboratories,Chicago, IL, USA) as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, slides were sequentially treated with FR1, FR2, and FR3 and dehydrated in absolute ethyl alcohol. Finally, cells were hybridized with CEP8, washed with FR3 solution, and mounted with the iFISH Full Spectrum Anti-Fade Mounting Medium (Cytelligen).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAutomated image scanning and analyses\u003c/h2\u003e \u003cp\u003eThe images of aneuploid TCs and TECs on the coated and formatted CTC slides (Cytelligen) were captured and analyzed using an automatic Metafer-iFISH 3D scanning and image analysis system. Five-channel automated X-Y scanning with cross-Z-sectioning of all cells was performed at 1-mm steps of depth (DAPI, CD31, CEP8, p16, and Ki67). Automatic image processing, classification, and statistical analysis were performed based on the expression of tumor markers and chromosome ploidy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were statistically analyzed using IBM SPSS software (version 26.0) and GraphPad Prism software (version 8.0). Differences in continuous variables between groups were compared using the Mann\u0026ndash;Whitney U test, and all data were presented as the median\u0026thinsp;\u0026plusmn;\u0026thinsp;95% confidence interval (CI). Non-parametric receiver operating characteristic (ROC) curve analysis was used to compare low-grade (\u0026le;\u0026thinsp;LSIL) to high-grade (\u0026ge;\u0026thinsp;HSIL) cervical lesions. Then, a threshold was established for the number of aneuploid TCs and TECs, and the maximum Youden index (sensitivity\u0026thinsp;+\u0026thinsp;specificity\u0026thinsp;\u0026minus;\u0026thinsp;1) was used to determine the cutoff value. All data in this study were expressed in a ratio of 10,000 percent and logarithms with log10 as the base were displayed in the graph. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of aneuploid TCs and TECs\u003c/h2\u003e \u003cp\u003eCell images captured by automated image scanning are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. According to previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], we defined the aneuploid TCs as DAPI\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e\u0026minus;\u003c/sup\u003e/CEP8\u0026thinsp;\u0026gt;\u0026thinsp;2 and the aneuploid TECs as DAPI\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e/CEP8\u0026thinsp;\u0026gt;\u0026thinsp;2. Then, aneuploid TC and TEC subtypes, including triploid, tetraploid, and multiploid (\u0026ge;\u0026thinsp;pentaploid) TCs, were counted. On the other hand, haploids were not counted due to the small number and no statistical difference in the preliminary experiment. p16 and Ki67 are identified as tumor markers for high-grade cervical lesions in clinical practice [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The expression of p16 and Ki67 on TCs and TECs was observed in the current study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDistribution of aneuploid TCs and TECs in different cervical lesion stages\u003c/h2\u003e \u003cp\u003eThe results of comparing the distribution differences of aneuploid TCs and TECs in different stages of cervical lesions are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e as log10 [(total number of aneuploid cells/slide cells)\u0026times;10000\u0026thinsp;+\u0026thinsp;1]. Interestingly, different proportions of aneuploid TCs were detected in the normal cervix infected with hrHPVs, and the proportion of aneuploid TCs increased gradually with the severity of cervical lesions. However, no significant difference was observed between the normal and LSIL groups, but the differences among the other groups were statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); this phenomenon is not obvious in aneuploid TECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we analyzed the proportion of p16/Ki67(p16 and/or Ki67)-positive aneuploid TCs and TECs, and the images showed that the positive rate of p16/Ki67 increased with the severity of the cervical lesions. Also, the proportion of p16/Ki67-positive aneuploid TCs in cervical cancer was significantly higher than that of HSIL, LSIL, and normal cervix, but this difference was not significant in aneuploid TECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSubclassification of aneuploid TCs and TECs by chromosomal ploidy\u003c/h2\u003e \u003cp\u003eIn order to further understand the distribution and clinical significance of aneuploid TCs and TECs with different chromosome ploidies at all stages of cervical lesions, we divided aneuploid TCs and TECs into 12 subtypes. The results showed that the difference between normal and LSIL groups was not statistically significant in any aneuploid subclass of TCs and TECs. Among all subtypes of aneuploid TCs, the proportion of tetraploid and \u0026ge;\u0026thinsp;pentaploid TCs in patients with HSIL and cervical cancer was significantly higher than that in patients with mild lesions. Although the distribution of triploid TCs was similar, it could not distinguish between LSIL and HSIL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). For aneuploid TECs, the altered trend of proportion in each subclass was irregular, and only TECs with \u0026ge;\u0026thinsp;pentaploid could distinguish cervical cancer from HSIL, LSIL, and normal cervix.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the analysis of p16/Ki67-positive aneuploid TCs and TECs, the positive rate of p16/Ki67 in each subclass of aneuploid TCs increased with the severity of cervical lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Firstly, the results of the analysis of triploid TCs revealed that the positive rate of p16/Ki67 in the cancer group was much higher than that in the HSIL, LSIL, and normal groups. Compared to tumor cells with triploid, p16/Ki67-positive tetraploid TCs distinguished between patients with HSIL and milder lesions. On the other hand, the proportion of \u0026ge;\u0026thinsp;pentaploid TCs with p16/Ki67-positive could well-distinguish all stages of cervical lesions except between normal and LSIL groups, and the significance was better than that in p16/Ki67-positive triploid and tetraploid TCs. The current data indicated that the higher the ploidy, the greater the degree of chromosomal instability, thus making the cells conducive to the development of malignant tumors. Therefore, the positive rate of tumor markers in aneuploid TCs at different grades of cervical lesions differed markedly. However, this phenomenon did not appear in all subclasses of TECs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, no significant difference was detected in the positive rate of target cells among all groups except the difference in \u0026ge;\u0026thinsp;pentaploid TECs between the HSIL and LSIL groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of HSIL\u0026thinsp;+\u0026thinsp;by individual and combinations of aneuploid TCs and TECs count\u003c/h2\u003e \u003cp\u003eIn view of the clinical treatment focus on HSIL and above lesions (HSIL+), we divided the pathology into two groups: one was patients with \u0026le;\u0026thinsp;LSIL and the other was patients with \u0026ge;\u0026thinsp;HSIL. The ROC curve was plotted to evaluate the diagnostic value of each subcategory in predicting HSIL+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Next, we selected the best cutoff values based on the maximum value of Youden index, and the corresponding sensitivity and specificity are shown in Table\u0026nbsp;1. The area under the curve (AUC) of tetraploid TCs was the largest (AUC\u0026thinsp;=\u0026thinsp;0.739, cutoff value: 8.83, sensitivity: 60.3%, specificity: 80.5%), followed by \u0026ge;\u0026thinsp;pentaploid TCs (AUC\u0026thinsp;=\u0026thinsp;0.724, cutoff value: 1.74, sensitivity: 69.9%, specificity: 69.9%) and triploid TCs (AUC\u0026thinsp;=\u0026thinsp;0.699, cutoff value: 24.73, sensitivity: 45.2%, specificity: 89.4%). However, the analytical results obtained from aneuploid TCs and TECs with positive p16/Ki67 were unsatisfactory. Their AUC values were small, and the sensitivity of the aneuploid TCs with positive p16/Ki67 was much less than expected, while the specificity was satisfactory. In addition, among all aneuploid TCs, the specificity of \u0026ge;\u0026thinsp;pentaploid TCs with positive p16/Ki67 was the highest (88.6%), followed by pentaploid TCs with positive p16/Ki67 (78.9%) and triploid TCs with positive p16/Ki67 (64.4%). Together, the diagnostic value of a single subclass was mainly reflected in the specificity. Next, we combined some subclasses, and the AUC value of \u0026ge;\u0026thinsp;tetraploid TCs was the largest (AUC\u0026thinsp;=\u0026thinsp;0.745, cutoff value: 8.74, sensitivity: 71.2%, specificity: 74.8%). The sensitivity and specificity of other combinations are shown in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the variation in the number of chromosome 8 aneuploid TCs and TECs at different stages of the cervical lesion. Also, the diagnostic value of aneuploidy subtypes and their combinations for HSIL\u0026thinsp;+\u0026thinsp;was determined. The results showed that the distribution of aneuploid TCs varied with the severity of cervical lesions, indicating that they are optimal indicators of the CIN stage but cannot be found in aneuploid TECs. The subtype analysis of aneuploidy showed that triploid, tetraploid, and \u0026ge;\u0026thinsp;pentaploid TCs could distinguish between HSIL, cervical cancer, and milder lesions and had good clinical value in the diagnosis of HSIL+, especially in the specificity. Compared to subtypes, \u0026ge;pentaploid TCs had preferable sensitivity (69.9%), while triploid TCs had preferable specificity (89.4%). However, the sensitivity and specificity of some subcategory combinations were not improved.\u003c/p\u003e \u003cp\u003ePrevious aneuploidy analysis of cervical cytology was mainly identified by measuring the DNA content of the cell, and the aneuploidy of DNA aided the diagnosis of cervical lesions [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, in some cells, although the average molecular weight of DNA is close to that of diploid, the true diploid regions may be missing [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Conversely, aneuploidy karyotype detection is objective. In normal human cells, aneuploidy is rare, except in neuronal and liver cells, most autosomal aneuploidy cells are embryonically lethal [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Strikingly, only tumor cells can maintain or increase proliferation rate when they exhibit aneuploidy. Carcinogenic HPV plays a critical role in inducing cervical cellular aneuploidy, HPV oncoproteins (especially E6 and E7) lead to mitotic defects through mediating cell cycle regulation disorders and affecting centrosome replication and spindle polarity, thus inducing the production of aneuploidy [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have found that the production of tetraploid occurs during early-stage events of cervical cancer that predispose cervical cells to the formation of aneuploidy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], our finding was similar when aneuploidy progresses to tetraploidy, the numeral difference of aneuploid TCs between LSIL and HSIL began to make sense(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Women with increased tetraploid TCs in cervical exfoliated cells should become our focus of follow-up. Moreover, \u0026ge;pentaploidy seems more obvious in cervical cancer, suggesting that the higher level of ploidy was more relevant to the severity of malignancy. Aneuploidy is a form of chromosomal instability, detecting karyotypic alterations provides abundant genetic information on tumor progression which may help us recognize the increased invasiveness and aggressiveness of cervical cancer. In multiple genome sequencing analyses, most of the chromosome arms of cervical cancer cells were gained or lost in different proportions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], additional studies should track karyotype changes during tumor progression and after treatment, in light of aneuploidy karyotype is associated with tumor evolution and drug resistance [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChromosome 8 abnormalities are closely related to the occurrence and development of various tumors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The chromosomal aneuploidy detection method (FISH) using CEP8 has been widely used to evaluate hematological tumors and various solid tumors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The abnormality of chromosome 8 has also been confirmed to exist in cervical cancer in previous studies, especially the trisomy of chromosome 8 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Many cancer-related genes are also located in this chromosome, including \u003cem\u003ec-MYC\u003c/em\u003e, \u003cem\u003eCCNE2\u003c/em\u003e, \u003cem\u003eTP53INP1\u003c/em\u003e, and \u003cem\u003eRAD54B\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], especially \u003cem\u003ec-MYC\u003c/em\u003e which exerts a critical role in cell proliferation, differentiation, and apoptosis and is associated with several human tumors [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. iFSIH is a new technology introduced for detecting aneuploid cells, mainly used for the detection of circulating tumor cells and circulating tumor endothelial cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To the best of our knowledge, this is the first study wherein the above technique was applied for the detection of aneuploidy in cervical exfoliated cells, confirming the difference in the distribution of chromosome 8 aneuploidy and its subclasses in cervical lesions at all stages. Previous research on chromosomal changes in cervical cytology mainly focused on some chromosomal regions, their sensitivity values for the detection of HSIL\u0026thinsp;+\u0026thinsp;were better. In contrast, our specificity is superior to certain probe sites (such as 3q26\u0026amp;53.3%, 5p15\u0026amp;56.7%, and 20q13\u0026amp;56.7%) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], this may be due to the change of copy number of chromosome arms and bands precedes genome-wide polyploidy.\u003c/p\u003e \u003cp\u003ep16 and Ki67 are the most frequently used diagnostic tumor markers for identifying cervical lesions, especially high-grade subtypes. Several studies have shown that the sensitivity of p16/Ki67 double-staining for the diagnosis of HSIL\u0026thinsp;+\u0026thinsp;was significantly higher than that of TCT, although the improvement of specificity was not significant [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, p16/Ki67 was also selected as the object of protein immunofluorescence staining. Interestingly, these results indicated that p16/Ki67 double-staining had a preferable specificity for diagnosing HSIL+; however, the sensitivity was not satisfactory. The differences in these results may be attributed to two aspects. First, previous studies have measured the protein levels of all cells, and false positives could be because p16 is expressed in endometrial tubal metaplasia and cervical endometriosis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Second, our study only estimated the expression of p16/Ki67 on chromosome 8 in aneuploid tumor cells. Herein, we did not include p16/Ki67-positive diploid tumor cells, which were different from the tumor marker-positive cells counted in other studies. The current study demonstrated some p16/Ki67-positive diploid cells; therefore, we cannot rely solely on markers to identify tumor cells in non-morphological detection due to uncertainty whether these cells are normal or have abnormal chromosome structures. Thus, additional aneuploidy testing may accurately identify tumor cells. Strikingly, E6/E7 play a leading role in the formation of aneuploid cervical cells, perhaps they are more appropriate as phenotypes to be combined with karyotypic detection.\u003c/p\u003e \u003cp\u003eAnother focus of our study was aneuploid TECs. Tumorigenesis, progression, and metastasis are based on vasculogenesis. Unlike normal blood vessels, tumor vessels contain a majority of cytogenetically abnormal endothelial cells that are characterized by aneuploid chromosomes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These aneuploid TECs may be derived from tumor cell transdifferentiation and heterotypic cell fusion, thereby exhibiting some characteristics of tumor cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Cancerization of stromal cells and transdifferentiation of stem cells may also be involved [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Positively expressed CD31 is the marker of endothelial cells. Our data showed that the count of CD31\u003csup\u003e+\u003c/sup\u003e aneuploid TECs altered dynamically at different stages of cervical lesions and significantly increased in cancerous cells, especially\u0026thinsp;\u0026ge;\u0026thinsp;pentaploid TECs, implying that aneuploid TECs support tumor progression. Moreover, the positive rate of p16/Ki67 aneuploid TECs increased with cervical lesion stages, although no significant difference was noted in each subclassification. Interestingly, these dynamic changes were similar to those reported previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The diagnostic role of aneuploid TECs is not remarkable, whereas the presence of aneuploid TECs is crucial for anti-angiogenic therapy, as chromosomal instability may provide a mechanism to alter endothelial cells and render them resistant to drugs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Some researchers have proposed that aneuploid TEC is more resistant to chemotherapeutic drugs, such as Vincristine and 5-Fluorouracil than normal endothelial cells[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore their potential therapeutic clinical value seems promising for in-depth investigation.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eNevertheless, the present study has some shortcomings. Firstly, the quantity of cells in different specimens varies greatly due to individualized clinical practicing of sample collection for each patient. Secondly, although the number of aneuploid tumors cells in peripheral blood is extremely low in normal subjects as previously reported by others, whether the existence of aneuploid cells in cervix of a large cohort of non-HPV infected healthy subjects remains to be examined. In addition, whether specific subpopulations of aneuploid TCs and TECs and their dynamic changes can predict HPV clearance is yet uncertain in the current study. Thus, additional large cohort clinical studies will be conducted to further optimize and validate aneuploidy and tumor marker-derived biomarkers for maximal benefit of clinical diagnosis of cervical lesions.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, iFISH, a novel detection technology, was used to detect, characterize and classify aneuploid TCs and TECs in exfoliated cells of cervical lesions at all stages. The current findings indicated that aneuploid TCs and TECs exhibit differences in the quantity, degree, and expression of tumor markers across all stages of cervical lesions. Aneuploid gains were correlated with the severity of cervical lesions. Triploid, tetraploid, and \u0026ge;\u0026thinsp;pentaploid TCs, regardless of p16/Ki67 expression, can distinguish HSIL\u0026thinsp;+\u0026thinsp;with high specificity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman papillomavirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor-derived endothelial cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLSIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow-grade squamous intraepithelial lesion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-grade squamous intraepithelial lesion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFISH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCEP8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecentromere probe 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the\u0026nbsp;Ethic Committee of the Shanghai General Hospital(reference 2021SQ263). All patients received informed consent prior to\u0026nbsp;cervical cytological specimens\u0026nbsp;collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;i\u0026bull;FISH\u003csup\u003e\u0026reg;\u003c/sup\u003e is the registered trademarks of Cytelligen.\u0026nbsp;Dr. Peter P. Lin is the president at Cytelligen. None of authors owns Cytelligen\u0026apos;s stock shares. No additional COI to be disclosed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Shanghai Pujiang Talent Plan(18PJC097) to Y.B.Y, Shanghai Aging and Women and Children\u0026rsquo;s Health Research Project(2020YJZX0215) to Y.B.Y.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L.W. contributed conceptualization, validation, formal analysis, investigation, resources, and writing original draft; A.Y.L. and D.D.W. contributed methodology and validation; P.P.L. contributed conceptualization, visualization, writing \u0026ndash; original draft, review and editing; X.X.Z contributed data curation, methodology and writing original draft; Y.B.Y contributed review, editing and funding acquisition; Y.P.Z contributed project administration, conceptualization, resources and supervision. All the authors read and approved the final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank staffs at Shanghai General Hospital, Cytointelligen (China Medical City, Taizhou, Jiangsu, China) and Cytelligen (San Diego, CA, USA) for providing support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Gynecology and Obstetrics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; \u003csup\u003e2\u003c/sup\u003eCytelligen, San Diego, CA, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoscicki AB, Schiffman M, Burchell A, Albero G, Giuliano AR, Goodman MT, Kjaer SK, Palefsky J. Updating the natural history of human papillomavirus and anogenital cancers. Vaccine. 2012;30(Suppl 5):F24\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia C, Hu S, Xu X, Zhao X, Qiao Y, Broutet N, Canfell K, Hutubessy R, Zhao F. Projections up to 2100 and a budget optimisation strategy towards cervical cancer elimination in China: a modelling study. Lancet Public Health. 2019;4(9):e462\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright TC, Stoler MH, Behrens CM, Sharma A, Sharma K, Apple R. Interlaboratory variation in the performance of liquid-based cytology: Insights from the ATHENA trial. Int J Cancer. 2014;134(8):1835\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbisch RM, Siebers AG, Bosgraaf RP, Massuger LF, Bekkers RL, Melchers WJ. Triage of high-risk HPV positive women in cervical cancer screening. Expert Rev Anticancer Ther. 2016;16(10):1073\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeaver BA, Cleveland DW. Does aneuploidy cause cancer? Curr Opin Cell Biol. 2006;18(6):658\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka K, Hirota T. Chromosomal instability: A common feature and a therapeutic target of cancer. Biochim et Biophys Acta (BBA) - Reviews Cancer. 2016;1866(1):64\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasudevan A, Schukken KM, Sausville EL, Girish V, Adebambo OA, Sheltzer JM. Aneuploidy as a promoter and suppressor of malignant growth. Nat Rev Cancer. 2021;21(2):89\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen-David U, Amon A. Context is everything: aneuploidy in cancer. Nat Rev Genet. 2020;21(1):44\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlaharski AJ, Sotelo R, Solorza-Luna G, Gonsebatt ME, Guzman P, Mohar A, Eastmond DA. Tetraploidy and chromosomal instability are early events during cervical carcinogenesis. Carcinogenesis. 2006;27(2):337\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinbeck RG. Proliferation and DNA Aneuploidy in Mild Dysplasia Imply Early Steps of Cervical Carcinogenesis. Acta Oncol. 2009;36(1):3\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuhn P, Houldsworth J, Cahill L, Schiffman M, Castle PE, Zuna RE, Dunn ST, Gold MA, Walker J, Wentzensen N. Chromosomal gains measured in cytology samples from women with abnormal cervical cancer screening results. Gynecol Oncol. 2013;130(3):595\u0026ndash;600.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanz KH-H, Castle VPE, Chaudhri N, White N, Wilber K, Morrison LE, Auer G, Burroughs FH, Mark, Sherman E et al. Detection of enomic Amplification of the Human Telomerase Gene (TERC) in Cytologic Specimens as a Genetic Test for the Diagnosis of Cervical Dysplasia. \u003cem\u003eThe American journal of pathology\u003c/em\u003e 2003, 163(4):1405\u0026ndash;1416.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkino T, Hida K, Hida Y, Tsuchiya K, Freedman D, Muraki C, Ohga N, Matsuda K, Akiyama K, Harabayashi T, et al. Cytogenetic Abnormalities of Tumor-Associated Endothelial Cells in Human Malignant Tumors. Am J Pathol. 2009;175(6):2657\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHida K, Klagsbrun M. A New Perspective on Tumor Endothelial Cells: Unexpected Chromosome and Centrosome Abnormalities. Cancer Res. 2005;65(7):2507\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerkins RB, Guido RS, Castle PE, Chelmow D, Einstein MH, Garcia F, Huh WK, Kim JJ, Moscicki AB, Nayar R, et al. : 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors. J Low Genit Tract Dis. 2020;24(2):102\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Zhang L, Gao Y, Wang Y, Liu Y, Zhang H, Wang Q, Hu F, Li J, Tan J, et al. Role of aneuploid circulating tumor cells and CD31\u0026thinsp;+\u0026thinsp;circulating tumor endothelial cells in predicting and monitoring anti-angiogenic therapy efficacy in advanced NSCLC. Mol Oncol. 2021;15(11):2891\u0026ndash;909.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng H, Wang S, Luan W, Ye X, Dou S, Tang Z, Zhu H, Ping Lin P, Li Y, Cui H, et al. Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer. Chin J Cancer Res. 2021;33(2):256\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei Y, Sun N, Zhang G, Liu C, Lu Z, Huang J, Zhang C, Zang R, Che Y, Mao S et al. Combined detection of aneuploid circulating tumor-derived endothelial cells and circulating tumor cells may improve diagnosis of early stage non‐small‐cell lung cancer. Clin Translational Med 2020, 10(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarner D. Clinical application of DNA ploidy to cervical cancer screening: A review. World J Clin Oncol. 2014;5(5):931\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta AF, Ribeiro MGM, Onofre ASC, Miranda Onofre FB. Aneuploidy detection for diagnostic and prognostic use in premalignant and malignant lesions of the uterine cervix: A systematic review. Diagn Cytopathol. 2020;49(2):335\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta AF, Pogere A, Pasinato APBF, Onofre ASC. Onofre FBdM: DNA image cytometry parameters to identify high-grade cervical lesions. Cytopathology. 2022;33(5):563\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLauren MF, Merlo Li-san, Wang JW, Pepper, Rabinovitch PS, Maley CC. Polyploidy, aneuploidy and the evolution of cancer. Adv Exp Med Biol. 2010;676:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer. 2010;10(8):550\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorzeniewski N, Spardy N, Duensing A, Duensing S. Genomic instability and cancer: lessons learned from human papillomaviruses. Cancer Lett. 2011;305(2):113\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;nger SDK. Centrosomes, Genomic Instability, and Cervical Carcinogenesis. Crit Rev Eukaryot Gene Expr. 2003;13(1):9\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen T, Suo J, Liu S, Wang S, Shu S, Xiang Y, Lang J-H. Using low-coverage whole genome sequencing technique to analyze the chromosomal copy number alterations in the exfoliative cells of cervical cancer. J Gynecologic Oncol 2018, 29(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClara Bodelon S, Vinokurova JN, Sampson, Johan A, den Boon JL, Walker MA, Horswill K, Korthauer M, Schiffman ME, Sherman, Rosemary E, Zuna, et al. Chromosomal copy number alterations and HPV integration in cervical precancer and invasive cancer. Carcinogenesis. 2016;37(2):188\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVitale I, Manic G, Senovilla L, Kroemer G, Galluzzi L. Karyotypic Aberrations in Oncogenesis and Cancer Therapy. Trends Cancer. 2015;1(2):124\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo S, Ou Y, Zheng T, Jiang H, Wu Y, Zhao J, Zhang Z, You SL. Optimal Strategy for Colorectal Cancer Patients\u0026rsquo; Diagnosis Based on Circulating Tumor Cells and Circulating Tumor Endothelial Cells by Subtraction Enrichment and Immunostaining-Fluorescence In Situ Hybridization Combining with CEA and CA19-9. J Oncol. 2021;2021:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirnbaum D, Ad\u0026eacute;la\u0026iuml;de J, Popovici C, Charafe-Jauffret E, Mozziconacci M-J, Chaffanet M. Chromosome arm 8p and cancer: a fragile hypothesis. Lancet Oncol. 2003;4(10):639\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMark HF, Feldman D, Samy M, Sun C, Das S, Mark S, Lathrop J. Assessment of Chromosome 8 Copy Number in Cervical Cancer by Fluorescent in situ Hybridization. Exp Mol Pathol. 1999;66(2):157\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShachaf, Catherine M, Felsher DW. Tumor dormancy and MYC inactivation: pushing cancer to the brink of normalcy. Cancer Res. 2005;65(11):4471\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin PP. Integrated EpCAM-independent subtraction enrichment and iFISH strategies to detect and classify disseminated and circulating tumors cells. Clin Translational Med 2015, 4(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolicht FA, Song M, Sitailo S, O'Hare A, Ashfaq R, Muller CY, Morrison LE, King W, Sokolova IA. Analysis of genetic copy number changes in cervical disease progression. BMC Cancer 2010, 10(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Chen C, Liu L, Dai W, Zhang J, Han C, Zhou S. Evaluation of p16/Ki-67 dual‐stain as triage test for high‐risk HPV ‐positive women: A hospital‐based cross‐sectional study. Cancer Cytopathol. 2022;130(12):955\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTringler B, Gup CJ, Singh M, Groshong S, Shroyer AL, Heinz DE, Shroyer KR. Evaluation of p16INK4a and pRb expression in cervical squamous and glandular neoplasia. Hum Pathol. 2004;35(6):689\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin PP. Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis. Cells 2020, 9(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHida K, Hida Y, Shindoh M. Understanding tumor endothelial cell abnormalities to develop ideal anti-angiogenic therapies. Cancer Sci. 2008;99(3):459\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cervical cancer, aneuploid TC, aneuploid TEC, iFISH biopsy, chromosome 8","lastPublishedDoi":"10.21203/rs.3.rs-4324077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4324077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDetection of chromosome aneuploidy is an important method for cervical cancer screening, however, the study of chromosome ploidy in primary cervical tumor cells is limited. A novel immunostaining integrated with fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (iFISH) strategy phenotypically and karyotypically co-detected the expression of tumor markers and chromosome aneuploidy to investigate the diagnostic values of aneuploid tumor cells (TCs) and tumor endothelial cells (TECs) in all-stage cervical lesion smear specimens.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 196 patients were enrolled in this study. Immunofluorescence staining of p16 and Ki67 combined with FISH was applied to quantitatively co-detect and characterize aneuploid CD31\u003csup\u003e\u0026minus;\u003c/sup\u003e TCs and CD31\u003csup\u003e+\u003c/sup\u003e TECs as well as their subtypes in cervical cytological specimens. The diagnostic values of aneuploid TCs and TECs for high-grade squamous intraepithelial lesions (HSIL+) were investigated by receiver operating characteristic curve analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe number of total aneuploid CD31\u003csup\u003e\u0026minus;\u003c/sup\u003e TCs and their p16/Ki67\u003csup\u003e+\u003c/sup\u003e subtypes increased markedly with the severity of cervical lesions, although a similar was not observed on aneuploid CD31\u003csup\u003e+\u003c/sup\u003e TECs. To identify HSIL+, the area under the curve (AUC) of tetraploid TCs was the largest (0.739), followed by multiploidy (\u0026ge;\u0026thinsp;pentaploid) TCs (0.724) and triploid TCs (0.699). Regarding combined subtypes, the AUC of \u0026ge;\u0026thinsp;tetraploid TCs was 0.745, and their unique diagnosis values were clinically reflected in the vitally high specificity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe number of CD31\u003csup\u003e\u0026minus;\u003c/sup\u003e TCs was associated with the severity of cervical lesions and aneuploid CD31\u003csup\u003e\u0026minus;\u003c/sup\u003e TCs exhibited a remarkable diagnostic specificity for HSIL+. Further studies are required to broaden their other potential clinical utility.\u003c/p\u003e","manuscriptTitle":"In situ phenotypic and karyotypic co-detection of aneuploid TCs and TECs in cytological specimens with abnormal cervical screening results","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 20:08:34","doi":"10.21203/rs.3.rs-4324077/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T11:24:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T05:18:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-24T14:45:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46104262725695235819910737128182431042","date":"2024-06-24T09:59:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322503998048463237661941324647686408342","date":"2024-06-04T14:14:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-26T16:11:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-03T15:33:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-26T05:38:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-26T05:38:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-04-25T12:14:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d37b9d1b-22b7-4f18-a45b-78c9d9aa521c","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:01:48+00:00","versionOfRecord":{"articleIdentity":"rs-4324077","link":"https://doi.org/10.1186/s12885-025-14346-y","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2025-05-26 15:57:35","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2024-05-02 20:08:34","video":"","vorDoi":"10.1186/s12885-025-14346-y","vorDoiUrl":"https://doi.org/10.1186/s12885-025-14346-y","workflowStages":[]},"version":"v1","identity":"rs-4324077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4324077","identity":"rs-4324077","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}