Host chemokine signature as a biomarker for the detection of pre-cancerous cervical lesions

Background The ability to distinguish which hrHPV infections predispose to significant disease is ever more pressing as a result of the increasing move to hrHPV testing for primary cervical screening. A risk-stratifier or “triage” of infection should ideally be objective and suitable for automation given the scale of screening. Results CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL12 emerged as the strongest, candidate biomarkers to detect underlying disease [cervical intraepithelial neoplasia grade 2 or worse (CIN2+)]. For CIN2+, CCL2 had the highest area under the curve (AUC) of 0.722 with a specificity of 82%. A combined biomarker panel of six chemokines CCL2, CCL3, CCL4, CXCL1, CXCL8, and CXCL12 provides a sensitivity of 71% and specificity of 67%. Conclusion The present work demonstrates that the levels of five chemokine-proteins are indicative of underlying disease. We demonstrate technical feasibility and promising clinical performance of a chemokine-based biomarker panel, equivalent to that of other triage options. Further assessment in longitudinal series is now warranted. Methods A panel of 31 chemokines were investigated for expression in routinely taken archived and prospective cervical liquid based cytology (LBC) samples using Human Chemokine Proteomic Array kit. Nine chemokines were further validated using Procartaplex assay on the Luminex platform

Development of an in-house ELISA to detect anti-HPV16-L1 antibodies in serum and dried blood spots

Measuring anti-HPV antibody levels is important for surveillance of the immunological response to both natural infection and vaccination. Here, an ELISA test for measurement of HPV-16L1 antibodies was developed and validated in sera and dried blood spots. An in-house ELISA was developed for measuring anti-HPV-16L1 IgA and IgG levels. The assay was standardized against WHO international standard serum and validated on serum, dried blood spots and cervical liquid based cytology samples from women attending colposcopy clinics in Scotland. Antibody avidity index was also measured in serum samples. The average HPV 16-L1 specific IgG and IgA levels measured in sera, in women attending a routine colposcopy service were 7.3 units/ml and 8.1 units/ml respectively. Significant correlations between serum and dried blood spot eluates for both IgG and IgA were observed indicating that the latter serve as a credible proxy for antibody levels. Average IgG Avidity Index was 35% (95% CI 25%-45%) suggesting previous, historical challenge with natural infection. This ELISA has potential for use in epidemiological and field studies of antibody prevalence and if coupled with avidity measurement may be of use in individual case monitoring of vaccine responses and failures

Increased Cycling Cell Numbers and Stem Cell Associated Proteins as Potential Biomarkers for High Grade Human Papillomavirus+ve Pre-Neoplastic Cervical Disease

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

Evaluation of HarmoniaHPV test for detection of clinically significant human papillomavirus infection using the VALGENT framework

Aim: The VALidation of HPV GENotyping Tests (VALGENT) is a framework for comparison and validation of HPV tests with genotyping capabilities. In this study, the clinical performance of a single tube HPV test -HarmoniaHPV- was assessed in SurePathTM samples and compared to a clinically validated reference test, the GP5+/6+ Enzyme ImmunoAssay (GP5+/6 + EIA). Methods: HarmoniaHPV test is a real-time, PCR based, limited genotyping HPV test which detects 14 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 with HPV16, and HPV 18 reported individually. Clinical performance was assessed using 998 unselected, cervical screening samples enriched with 297 cytologically abnormal specimens (100 atypical squamous cells of unspecified significance, 100 low-grade squamous intraepithelial lesions, 97 high-grade squamous intraepithelial lesions). Cases were defined as women diagnosed with histologically confirmed cervical intraepithelial neoplasia 2 or more (>CIN2, N = 122). Results: Using the manufacturer recommended (un-adjusted) cut-offs, HarmoniaHPV had non-inferior sensitivity for detection of > CIN2 but showed inferior specificity. A cut-off optimisation exercise was therefore carried out and optimised cut-offs for each individual channel rendered a sensitivity and specificity of HarmoniaHPV that was non-inferior to GP5+/6 + EIA. Analytically, the test showed excellent intra- and inter-laboratory reproducibility, which improved further with the use of the optimised cut-offs. Conclusion: HarmoniaHPV when operated with optimised cut-offs fulfils the international clinical criteria for use in cervical cancer screening on SurePath samples. The optimised cut-offs warrant additional testing and independent validation

Proteomic array detection of stem cell associated proteins in pooled aliquots from 9 HR-HPV+ve cervical samples from women with CIN3 (A) and 9 HPV –ve samples with normal cytology (B); (C & D) map and key for array spots; (E) average pixel density ranked by ratio of pooled CIN3:normal samples for each protein on the array; (F) sox2, TP63 and HCG mRNAs are upregulated in HPV+ve cervical samples with severe dyskaryosis compared to HPV–ve samples with normal morphology (* = p<0.05, ** p<0.01, Mann Whitney test).

<p>Proteomic array detection of stem cell associated proteins in pooled aliquots from 9 HR-HPV+ve cervical samples from women with CIN3 (A) and 9 HPV –ve samples with normal cytology (B); (C & D) map and key for array spots; (E) average pixel density ranked by ratio of pooled CIN3:normal samples for each protein on the array; (F) sox2, TP63 and HCG mRNAs are upregulated in HPV+ve cervical samples with severe dyskaryosis compared to HPV–ve samples with normal morphology (* = p<0.05, ** p<0.01, Mann Whitney test).</p

TP63 and SOX2 staining in cervical biopsies.

<p>Immunohistochemical staining of cervical biopsies. Bars  =  50µm.(A) normal cervix no primary control; (B) normal cervix stained with anti-SOX2; (C) Squamous cell cervical carcinoma no primary control; (D) representative TP63, and (E) representative SOX2, staining of tumour cells. For both TP63 and SOX2 staining was seen in the nucleus of positive cells (examples indicated by solid arrows); negative cells were a minority of tumour cells (examples indicated by unfilled arrows). (F) Image analysis results of % nuclear +ve tumour cells in biopsies. Parallel sections from 11 cases were stained with SOX2 and TP63. Tumour cells were evaluated for their nuclear expression of the transcription factors. There was no significant difference between the data for SOX2 and TP63 (Wilcoxon signed rank test).</p

Flow cytometric detection of cycling cells in cervical samples.

<p>(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.</p

Samples with increased TRA-1-60+ve cells from patients with CIN3 have enhanced expression of stem cell proteins.

<p>A) Flow cytometric analysis of samples used for proteomic microarray. Dotted lines show that the three samples with a significant increase in TRA-1-60 +ve cells have similar levels of cycling cells to the other three CIN3 samples. *  = p<.05. B) Image-J pixel density analysis of stem cell proteomic microarray. SOX2, TP63 and HCG are further increased in samples with increased TRA-1-60+ve cells. C) Inverted image of the scan of the array from TRA1-60+ve samples; D) Inverted image of the scan of the array from TRA1-60 –ve samples.</p

Disease status, age range and cytology results for cervical LBC samples obtained from the Scottish national HPV Archive.

<p>Disease status, age range and cytology results for cervical LBC samples obtained from the Scottish national HPV Archive.</p