Squamocolumnar Junction Cells Research Articles

A Study of Cytokeratin-7 Expression and Clinicopathological Correlation in Dysplasia and Squamous Cell Carcinoma of the Cervix.

Objectives:Cytokeratin (CK) proteins play a vital role in cancer diagnosis, of which,CK-7 is a prominent marker of squamocolumnar junction cells corresponding to the the initiating site of cervical cancer.The current study is aimed to evaluate the expression pattern of CK-7 and to corelate with the clinicopathological features in patients with cervical dysplasia and invasive squamous cell carcinoma. Methodology:The hysterectomy and biopsy specimens from women with cervical dysplasia (n=60) and carcinoma (n=60) were evaluated histopathologically and processed for immunohistochemistry (IHC) staining to assess for CK-7 expression. The relationship between CK-7 expression and tumor characteristics like histological type of cervical intraepithelial neoplasia (CIN), tumor type and grade was evaluated. Data was analyzed using the Chi-square test ,wherein the p value ≤ 0.05 were taken for statistical significance. Results:Positive CK-7 expression was observed in 25 (41.67%) dysplasia and in 34 (56.67%) carcinoma cases. Majority of the cases were CIN III (n=31, 51.67%), large cell non-keratinizing tumor type (n=54, 90%) and moderately differentiated grade of tumor (n=52, 86.67%), out of which 18 (58.1%), 34 (62.96%) and 30 (57.69%) cases were CK-7 positive, respectively. The difference in clinical diagnosis and tumor characteristics over CK-7 expression was significant (p<0.05). The pattern of CK-7 expression in dysplasia and carcinoma cases were diffuse in 23 (38.33%) and 31 (51.67%) respectively and patchy in 2 (3.33%) and 3 (5%) of them, respectively. Conclusion:Significant positive CK-7 expression in cervical dysplasia and carcinoma indicates a good clinical course and its role as a useful predictable marker for cancer progression.

Limitation of cytology and the impacton reduction of cervical cancer

Introduction: Cervical cancer is the third most common malignant tumor in the female population and the fourth cause of death from cancer in women in Brazil. The squamocolumnar junction and the transformation zone concentrate 90% of pre-invasive and invasive cervical lesions. Objective: To evaluate the prevalence of cytology without cells of the squamocolumnar junction and feasibility of active search. Methods: Cross-sectional study at a university hospital between 2017 and 2018. The prevalence of cytology without squamocolumnar junction cells was calculated. A convenience sample was obtained and mean age and relationship with presence of transformation zone cells were calculated. An active search was performed and cytology collected, with estrogen preparation if indicated. Medical records of the other women were analyzed. Results: Squamocolumnar junction cells were not found in 28.84% of samples. Mean age was 53 years, without association with presence of squamocolumnar junction cells (p=0.409). Seventy-six women returned, 36 of which (47.37%) used estrogen. Level 2 or 3 cervical intraepithelial neoplasia, microinvasive carcinoma or cancer was not identified. A total of 134 medical records were analyzed; only 36 women (26.87%) completed screening. Conclusions: The presence of squamocolumnar junction cells indicates quality of cytology; the use of estrogen in postmenopausal women favors its collection. There were difficulties in active search. An immediate repetition of cytology should be considered.

Patterns of Cytokeratin 7 Expression in Cervical Squamous Intraepithelial Lesions

Abstract Introduction/Objective It has been suggested that cervical high-grade squamous intraepithelial lesions (HSIL/CIN2-3) arise from squamocolumnar junction cells that express cytokeratin 7 (CK7). Significant CK7 expression (gradation or full-thickness) has been proposed as a marker of progression of low-grade squamous intraepithelial lesions (LSIL/CIN1) to HSIL and of persistence of HSIL/CIN2. The goal of the study is to survey patterns of CK 7 expression in the different grades of squamous intraepithelial lesions (SILs). Methods 65 cervical specimens (biopsies and excisions) containing 95 lesions of different grades were assessed by immunohistochemical analysis (IHC) for CK7 expression. 26 cases contained more one lesion grade. The diagnosis of HSIL (CIN2-3) was confirmed by p16 IHC. CK7 expression was scored negative, patchy, gradation (i.e., top-down), or full-thickness pattern. In cases with heterogeneous staining, the strongest pattern was used for analysis. Results There was significant variation in patterns within morphologically contiguous lesional foci; staining heterogeneity was noted in 42% of cases. All patterns of expression were encountered in all lesion grades. LSIL/CIN1 (n=47), either alone (n=27) or in combination with HSIL (n=20), often lacked CK7 expression (53%) or were patchy (17%). The frequency of significant (gradation or full-thickness) CK7 expression in LSIL with concomitant HSIL was greater than LSIL occurring alone (40% vs. 22%, respectively). HSIL/CIN3 (n=19) was dominated by full-thickness expression (57%). HSIL/CIN2 (n=29) had a very heterogeneous spectrum of expression with 34% of cases lacking expression. Conclusion CK7 expression is variable across all grades of SILs. LSIL with concomitant HSIL was associated with significant CK7 expression more frequently than LSIL alone. Significant proportion of HSIL, particularly CIN2, lacks CK7 expression. Given this variability, caution is advised regarding the use of CK7 expression as a marker of progression.

Establishment and Molecular Phenotyping of Organoids from the Squamocolumnar Junction Region of the Uterine Cervix.

The metaplastic epithelium of the transformation zone (TZ) including the squamocolumnar junction (SCJ) of the uterine cervix is a prime target of human papilloma virus (HPV) infection and subsequent cancer development. Due to the lack of adequate in vitro models for SCJ, however, investigations into its physiological roles and vulnerability to carcinogenesis have been limited. By using Matrigel-based three-dimensional culture techniques, we propagated organoids derived from the normal SCJ region, along with metaplastic squamous cells in the TZ. Consisting predominantly of squamous cells, organoids basically exhibited a dense structure. However, at least in some organoids, a small but discrete population of mucin-producing endocervix cells co-existed adjacent to the squamous cell population, virtually recapitulating the configuration of SCJ in a TZ background. In addition, transcriptome analysis confirmed a higher expression level of many SCJ marker genes in organoids, compared to that in the immortalized cervical cell lines of non-SCJ origin. Thus, the obtained organoids appear to mimic cervical SCJ cells and, in particular, metaplastic squamous cells from the TZ, likely providing a novel platform in which HPV-driven cervical cancer development could be investigated.

Cytomorphological analysis of cervical cytological smears of women aged over 60 years

ABSTRACT Introduction: Cervicovaginal atrophy is a condition that can affect women after menopause, and cytology is a diagnostic tool useful in such cases. Objective: To evaluate the cytomorphological profile of cervical smears in patients over 60 years old. Methods: Cytopathological examinations of 500 patients over 60 years old were selected consecutively in this cross-sectional, quantitative, retrospective study. Results: Only 114 (22.8%) presented the squamocolumnar junction (SCJ) sampled, and their presence decreased progressively with advancing age (p < 0.001). Most smears (95.6%) were classified as atrophic. Microbiological analysis showed that from the 22 non-atrophic smears, most presented lactobacillus flora. Among the atrophic swabs, the predominant flora was cocci, with 47.2%. Only 4% presented cytological changes: atypical squamous cells of undetermined significance [(ASC-US) - eight cases/40%], atypical squamous cells - cannot exclude high-grade squamous intraepithelial lesion [(ASC-H) - five cases/25%], high-grade squamous intraepithelial lesion [(HSIL) - three/15%], low-grade squamous intraepithelial lesion [(LSIL) - two cases/10%] and adenocarcinoma in situ [(ACI) - two cases/10%]. Among the modified smears, four (20%) presented SCJ cells, and four patients (20%) took hormones (from these, two cases of ASC-H (10%) and two cases ASC-US (10%), showing a relationship between the onset of the lesion and the use of hormones (p < 0.05). Conclusion: The absence of SCJ indicates a diagnostic limitation of sample collection. Although the frequency of lesions has been similar to other studies, and the recommended age range for the examination is between 25 and 60 years, it is important to note that many women older than this range should perform the collection of oncology cytology due to existence of elderly women with risk profile for the disease.

E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant.

Profiling of Discrete Gynecological Cancers Reveals Novel Transcriptional Modules and Common Features Shared by Other Cancer Types and Embryonic Stem Cells.

Studies on individual types of gynecological cancers (GCs), utilizing novel expression technologies, have revealed specific pathogenetic patterns and gene markers for cervical (CC), endometrial (EC) and vulvar cancer (VC). Although the clinical phenotypes of the three types of gynecological cancers are discrete, the fact they originate from a common embryological origin, has led to the hypothesis that they might share common features reflecting regression to early embryogenesis. To address this question, we performed a comprehensive comparative analysis of their profiles. Our data identified both common features (pathways and networks) and novel distinct modules controlling the same deregulated biological processes in all three types. Specifically, four novel transcriptional modules were discovered regulating cell cycle and apoptosis. Integration and comparison of our data with other databases, led to the identification of common features among cancer types, embryonic stem (ES) cells and the newly discovered cell population of squamocolumnar (SC) junction of the cervix, considered to host the early cancer events. Conclusively, these data lead us to propose the presence of common features among gynecological cancers, other types of cancers, ES cells and the pre-malignant SC junction cells, where the novel E2F/NFY and MAX/CEBP modules play an important role for the pathogenesis of gynecological carcinomas.

Microanatomy of the cervical and anorectal squamocolumnar junctions: a proposed model for anatomical differences in HPV-related cancer risk

Human papilloma virus (HPV) infection causes cancers and their precursors (high-grade squamous intraepithelial lesions) near cervical and anal squamocolumnar junctions. Recently described cervical squamocolumnar junction cells are putative residual embryonic cells near the cervical transformation zone. These cells appear multipotential and share an identical immunophenotype (strongly CK7-positive) with over 90% of high-grade squamous intraepithelial lesions and cervical carcinomas. However, because the number of new cervical cancers discovered yearly world wide is 17-fold that of anal cancer, we posed the hypothesis that this difference in cancer risk reflects differences in the transition zones at the two sites. The microanatomy of the normal anal transformation zone (n=37) and topography and immunophenotype of anal squamous neoplasms (n=97) were studied. A discrete anal transition zone was composed of multilayered CK7-positive/p63-negative superficial columnar cells and an uninterrupted layer of CK7-negative/p63-positive basal cells. The CK7-negative/p63-positive basal cells were continuous with—and identical in appearance to—the basal cells of the mature squamous epithelium. This was in contrast to the cervical squamocolumnar junction, which harbored a single-layered CK7-positive/p63-negative squamocolumnar junction cell population. Of the 97 anal intraepithelial neoplasia/squamous cell carcinomas evaluated, only 27% (26/97) appeared to originate near the anal transition zone and only 23% (22/97) were CK7-positive. This study thus reveals two fundamental differences between the anus and the cervix: (1) the anal transition zone does not harbor a single monolayer of residual undifferentiated embryonic cells and (2) the dominant tumor immunophenotype is in keeping with an origin in metaplastic (CK7-negative) squamous rather than squamocolumnar junction (CK7-positive) epithelium. The implication is that, at birth, the embryonic cells in the anal transition zone have already begun to differentiate, presenting a metaplasia that—similar to vaginal and vulvar epithelium—is less prone to HPV-directed carcinogenesis. This in turn underscores the link between cancer risk and a very small and discrete population of vulnerable squamocolumnar junction cells in the cervix.

Carcinogenic HPV infection in the cervical squamo-columnar junction.

Recent studies have suggested the involvement of a unique population of cells at the cervical squamo-columnar junction (SCJ) in the pathogenesis of early (squamous intraepithelial lesion or SIL) and advanced (squamous cell and adeno-carcinomas) cervical neoplasia. However, there is little evidence to date showing that SCJ cells harbour carcinogenic HPV or are instrumental in the initial phases of neoplasia. This study was designed to (1) determine if normal-appearing SCJ cells contained evidence of carcinogenic HPV infection and (2) trace their transition to early SIL. Sections of cervix from high-risk reproductive age women were selected and SCJ cells were analysed by using several techniques which increasingly implicated HPV infection: HPV DNA (genotyping and in situ hybridization)/RNA (PCR), immunostaining for HPV16 E2 (an early marker of HPV infection), p16(ink4), Ki67, and HPV L1 protein. In 22 cases with a history of SIL and no evidence of preneoplastic lesion in the excision specimen, HPV DNA was isolated from eight of ten with visible SCJ cells, six of which were HPV16/18 DNA-positive. In five of these latter cases, the SCJ cells were positive for p16(ink4) and/or HPV E2. Transcriptionally active HPV infection (E6/E7 mRNAs) was also detected in microdissected SCJ cells. Early squamous atypia associated with the SCJ cells demonstrated in addition diffuse p16(ink4) immunoreactivity, elevated proliferative index, and rare L1 antigen positivity. We present for the first time direct evidence that normal-appearing SCJ cells can be infected by carcinogenic HPV. They initially express HPV E2 and their progression to SIL is heralded by an expanding metaplastic progeny with increased proliferation and p16(ink4) expression. Whether certain SCJs are more vulnerable than others to carcinogenic HPV genotypes and what variables determine transition to high-grade SIL remain unresolved, but the common event appears to be a vulnerable cell at the SCJ.

Markers of squamocolumnar junction cells in normal tonsils and oropharyngeal cancer with and without HPV infection.

HPV infection has been identified recently as the causative agent of a subset of squamous cell carcinomas arising in oropharyngeal tonsils. Factors influencing the susceptibility of tonsillar epithelium to HPV-induced oncogenesis are far from being elucidated. A 5-protein signature including cytokeratin (CK)7, anterior gradient (AGR)2, cluster differentiation (CD)63, matrix metalloproteinase (MMP)7, and guanine deaminase (GDA) has recently been found to identify a residual embryonic cell population in the squamocolumnar (SC) junction of the cervix, susceptible to HPV infection, and cancers originating from these cells. The expression of SC junction markers was investigated with immunohistochemistry in normal tonsils and in oropharyngeal carcinomas (OPC) fully characterised for HPV. All markers were constantly expressed in the reticulated epithelial cells of the tonsillar crypts, with variable diffusion and intensity; in OPC, positivity was observed in 36,5%, 29,2%, 39%, 17%, and 25% of cases with respectively AGR2, CK7, GDA, CD63, and MMP7 antibodies. No OPC was positive for all markers; 6 were completely negative. AGR2 and CK7 showed significant association with tumor- and HPV-related parameters. AGR2 expression was associated with tumor origin in the tongue base (p=0.013); CK7 was associated with non-keratinising morphology (p=0.013). p16 tumor cell expression was associated with AGR2 (p=0.021); transcriptionally active HPV infection was associated with AGR2 and CK7 (p=0.024 and 0.043). Expression of SC junction markers in tonsillar crypt cells might be related to the embryological development of tonsillar structures; their partial association with HPV oncogenic infection could help to identify HPV-susceptible cells and related OPC.

A novel blueprint for ‘top down’ differentiation defines the cervical squamocolumnar junction during development, reproductive life, and neoplasia

The cervical squamocolumnar (SC) junction is the site of a recently discovered 'embryonic' cell population that was proposed as the cell of origin for cervical cancer and its precursors. How this population participates in cervical remodelling and neoplasia is unclear. In the present study, we analysed the SC junction immunophenotype during pre- and post-natal human and mouse development and in the adult, processes of metaplastic evolution of the SC junction, microglandular change, and early cervical neoplasia. Early in life, embryonic cervical epithelial cells were seen throughout the cervix and subsequently diminished in number to become concentrated at the SC junction in the adult. In all settings, there was a repetitive scenario in which cuboidal embryonic/SC junction cells gave rise to subjacent metaplastic basal/reserve cells with a switch from the SC junction positive to negative immunophenotype. This downward or basal (rather than upward or apical) evolution from progenitor cell to metaplastic progeny was termed reverse or 'top down' differentiation. A similar pattern was noted in high-grade squamous intraepithelial lesions (HSILs), suggesting that HPV infection of the cuboidal SC junction cells initiated outgrowth of basally-oriented neoplastic progeny. The progressive loss of the embryonic/SC junction markers occurred with 'top down' differentiation during development, remodelling, and early neoplasia. Interestingly, most low-grade SILs were SC junction-negative, implying infection of metaplastic progeny rather than the original SC junction cells. This proposed model of 'top down' differentiation resolves the mystery of how SC junction cells both remodel the cervix and participate in neoplasia and provides for a second population of metaplastic progeny (including basal and reserve cells), the infection of which is paradoxically less likely to produce a biologically aggressive precursor. It also provides new targets in animal models to determine why the SC junction is uniquely susceptible to carcinogenic HPV infection.

A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer

Infection by carcinogenic human papillomaviruses (HPV) results in precancers [cervical intraepithelial neoplasia (CIN)] and cancers near the ectoendocervical squamocolumnar (SC) junction of the cervix. However, the specific cells targeted by HPV have not been identified and the cellular origin of cervical cancer remains elusive. In this study, we uncovered a discrete population of SC junctional cells with unique morphology and gene-expression profile. We also demonstrated that the selected junctional biomarkers were expressed by a high percentage of high-grade CIN and cervical cancers associated with carcinogenic HPVs but rarely in ectocervical/transformation zone CINs or those associated with noncarcinogenic HPVs. That the original SC junction immunophenotype was not regenerated at new SC junctions following excision, not induced by expression of viral oncoproteins in foreskin keratinocytes, and not seen in HPV-related precursors of the vagina, vulva, and penis further support the notion that junctional cells are the source of cervical cancer. Taken together, our findings suggest that carcinogenic HPV-related CINs and cervical cancers are linked to a small, discrete cell population that localizes to the SC junction of the cervix, expresses a unique gene expression signature, and is not regenerated after excision. The findings in this study uncover a potential target for cervical cancer prevention, provide insight into the risk assessment of cervical lesions, and establish a model for elucidating the pathway to cervical cancer following carcinogenic HPV infection.

CD44+ slow‐cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E2 in gastric tumorigenesis

Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow-cycling and, thereby, insensitive to chemo- and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44(+) cells and slow-cycling cells and the underlying mechanisms for the emergence of CD44(+) CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo-columnar junction (SCJ) in normal mouse stomach contains CD44(+) stem cell-like slow-cycling cells and that this characteristic CD44(+) gland was expanded by prostaglandin E2 (PGE(2)) and Wnt signaling in K19-Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19-Wnt1, K19-C2mE and K19-Wnt1/C2mE, revealed that the expansion of CD44(+) SCJ cells is triggered by PGE(2)-mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44(+) gland in gastric tumor of K19-Wnt1/C2mE mouse contains a few BrdU label-retaining quiescent or slow-cycling cells, suggesting that the CD44(+) SCJ cells in normal mouse are candidates for the cell-of-origin of gastric CSCs. These observations suggest that PGE(2)-mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44(+) slow-cycling stem-like cells in SCJ, leading to development of lethal gastric tumors in mice.