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. 3A). 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.