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Increased cycling cell numbers and stem cell associated proteins as potential biomarkers for high grade human papillomavirus+ve pre-neoplastic cervical disease.

Canham M, Charsou C, Stewart J, Moncur S, Hoodless L, Bhatia R, Cong D, Cubie H, Busby-Earle C, Williams A, McLoughlin V, Campbell JD, Cuschieri K, Howie S - PLoS ONE (2014)

Bottom Line: Both cytology and histology results were available for most samples with moderate to high grade abnormality.We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3.Samples from women with CIN3 showed increased proliferating cells.

View Article: PubMed Central - PubMed

Affiliation: Human Papillomavirus Group, University of Edinburgh, Edinburgh, United Kingdom; Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
High risk (oncogenic) human papillomavirus (HPV) infection causes cervical cancer. Infections are common but most clear naturally. Persistent infection can progress to cancer. Pre-neoplastic disease (cervical intraepithelial neoplasia/CIN) is classified by histology (CIN1-3) according to severity. Cervical abnormalities are screened for by cytology and/or detection of high risk HPV but both methods are imperfect for prediction of which women need treatment. There is a need to understand the host virus interactions that lead to different disease outcomes and to develop biomarker tests for accurate triage of infected women. As cancer is increasingly presumed to develop from proliferative, tumour initiating, cancer stem cells (CSCs), and as other oncogenic viruses induce stem cell associated gene expression, we evaluated whether presence of mRNA (detected by qRT-PCR) or proteins (detected by flow cytometry and antibody based proteomic microarray) from stem cell associated genes and/or increased cell proliferation (detected by flow cytometry) could be detected in well-characterised, routinely collected cervical samples from high risk HPV+ve women. Both cytology and histology results were available for most samples with moderate to high grade abnormality. We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3. SOX2, TP63 and human gonadotrophin mRNAs were upregulated in high grade disease. Immunohistochemistry showed that SOX2 and TP63 proteins clearly delineated tumour cells in invasive squamous cervical cancer. Samples from women with CIN3 showed increased proliferating cells. We believe that these markers may be of use to develop triage tests for women with high grade cervical abnormality to distinguish those who may progress to cancer from those who may be treated more conservatively.

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Flow cytometric detection of cycling cells in cervical samples.(A) LBC samples stratified by HPV status and histology; samples from women with CIN3 are significantly different from samples with normal cytology and from CIN1 and CIN2, 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus CIN3 group); (B) LBC samples stratified by HPV status and cytology results only; samples with severe dyskaryosis are significantly different from all normal samples 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus severe disease group). * p = <0.05, ** p = <0.01, *** p = <0.001.
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pone-0115379-g004: Flow cytometric detection of cycling cells in cervical samples.(A) LBC samples stratified by HPV status and histology; samples from women with CIN3 are significantly different from samples with normal cytology and from CIN1 and CIN2, 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus CIN3 group); (B) LBC samples stratified by HPV status and cytology results only; samples with severe dyskaryosis are significantly different from all normal samples 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus severe disease group). * p = <0.05, ** p = <0.01, *** p = <0.001.

Mentions: Cell cycle was detected by analysing staining with the DNA chelating dye DRAQ5. Cycling cells were determined to be in the S+G2M phase of the cell cycle as described in Materials and Methods. No aneuploid cells were detected in any samples and cell cycle stages were comparable between samples. Fig. 4 shows that significant increases were seen in cervical samples from women with significant disease compared to normal samples regardless of whether normal samples were HPV-ve or HPV+ve and regardless of whether significant disease was stratified as biopsy proven CIN3 (Fig. 4A) or by cytology determined severe dyskaryosis (Fig. 4B).


Increased cycling cell numbers and stem cell associated proteins as potential biomarkers for high grade human papillomavirus+ve pre-neoplastic cervical disease.

Canham M, Charsou C, Stewart J, Moncur S, Hoodless L, Bhatia R, Cong D, Cubie H, Busby-Earle C, Williams A, McLoughlin V, Campbell JD, Cuschieri K, Howie S - PLoS ONE (2014)

Flow cytometric detection of cycling cells in cervical samples.(A) LBC samples stratified by HPV status and histology; samples from women with CIN3 are significantly different from samples with normal cytology and from CIN1 and CIN2, 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus CIN3 group); (B) LBC samples stratified by HPV status and cytology results only; samples with severe dyskaryosis are significantly different from all normal samples 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus severe disease group). * p = <0.05, ** p = <0.01, *** p = <0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4274002&req=5

pone-0115379-g004: Flow cytometric detection of cycling cells in cervical samples.(A) LBC samples stratified by HPV status and histology; samples from women with CIN3 are significantly different from samples with normal cytology and from CIN1 and CIN2, 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus CIN3 group); (B) LBC samples stratified by HPV status and cytology results only; samples with severe dyskaryosis are significantly different from all normal samples 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus severe disease group). * p = <0.05, ** p = <0.01, *** p = <0.001.
Mentions: Cell cycle was detected by analysing staining with the DNA chelating dye DRAQ5. Cycling cells were determined to be in the S+G2M phase of the cell cycle as described in Materials and Methods. No aneuploid cells were detected in any samples and cell cycle stages were comparable between samples. Fig. 4 shows that significant increases were seen in cervical samples from women with significant disease compared to normal samples regardless of whether normal samples were HPV-ve or HPV+ve and regardless of whether significant disease was stratified as biopsy proven CIN3 (Fig. 4A) or by cytology determined severe dyskaryosis (Fig. 4B).

Bottom Line: Both cytology and histology results were available for most samples with moderate to high grade abnormality.We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3.Samples from women with CIN3 showed increased proliferating cells.

View Article: PubMed Central - PubMed

Affiliation: Human Papillomavirus Group, University of Edinburgh, Edinburgh, United Kingdom; Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
High risk (oncogenic) human papillomavirus (HPV) infection causes cervical cancer. Infections are common but most clear naturally. Persistent infection can progress to cancer. Pre-neoplastic disease (cervical intraepithelial neoplasia/CIN) is classified by histology (CIN1-3) according to severity. Cervical abnormalities are screened for by cytology and/or detection of high risk HPV but both methods are imperfect for prediction of which women need treatment. There is a need to understand the host virus interactions that lead to different disease outcomes and to develop biomarker tests for accurate triage of infected women. As cancer is increasingly presumed to develop from proliferative, tumour initiating, cancer stem cells (CSCs), and as other oncogenic viruses induce stem cell associated gene expression, we evaluated whether presence of mRNA (detected by qRT-PCR) or proteins (detected by flow cytometry and antibody based proteomic microarray) from stem cell associated genes and/or increased cell proliferation (detected by flow cytometry) could be detected in well-characterised, routinely collected cervical samples from high risk HPV+ve women. Both cytology and histology results were available for most samples with moderate to high grade abnormality. We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3. SOX2, TP63 and human gonadotrophin mRNAs were upregulated in high grade disease. Immunohistochemistry showed that SOX2 and TP63 proteins clearly delineated tumour cells in invasive squamous cervical cancer. Samples from women with CIN3 showed increased proliferating cells. We believe that these markers may be of use to develop triage tests for women with high grade cervical abnormality to distinguish those who may progress to cancer from those who may be treated more conservatively.

Show MeSH
Related in: MedlinePlus