Proteomics characterization of HPV-independent Vulvar Cancer Tumour Progression and Metastasis by Mass Spectrometry

Vulvar cancer is the fourth most common gynecological cancer around the world, constituting 5% of all gynecological cancers and 0.6% of total cancer cases in 2016. As a relatively rare cancer type, the average vulvar cancer incidence rate is three in 100 000 women each year. But, Australian Indigenous showed significantly higher incidence rate than the global level. Previous etiological studies have identified two distinctive oncogenetic origins of vulvar cancer based on the presence of HPV infection or not. In general, over 60% of patients, especially those diagnosed at the postmenopause age or with a history of precancerous lesions of differentiated VIN (dVIN), are classified as the high metastatic risk HPV-independent type vulvar cancer. Currently, most scientific efforts of vulvar cancer research are given to the low-risk HPV-dependent type, whereas protein studies focused on the HPV-independent type are quite limited. The protein biomarker is an important diagnostic tool in cancer early detections and metastasis predictions. However, there is no protein biomarker targeting on HPV-independent vulvar cancer available in the clinical practice yet. Tumour biomarkers, such as TP53 and CA125, do not have enough sensitivity and specificity in vulvar cancer detection. Therefore, new protein biomarker candidates are needed for current vulvar cancer diagnosis and prognosis. Mass spectrometry (MS) is a high-throughput protein analysis and biomarker discovery technique in modern cancer studies. Recent developments of cut-edge MALDI mass spectrometry imaging (MALDI-MSI) and nano- LC-ESI-MS label-free quantification (LFQ) provide new opportunities to reveal differential abundances and spatial distributions of proteins from vulvar cancer tissue samples. In this thesis, protein markers for HPV-independent vulvar cancer carcinogenesis and metastasis were identified from the MS based tissue characterization and validated by Immunohistochemistry (IHC). Firstly, the intermedia filament protein, Cytokeratin 5 (CK5), was identified as a tissuespecific biomarker for HPV-independent vulvar cancer in the MALDI-MSI analysis of 12 representative patient samples, which has been published in the manuscripts. Other potential tissue markers,β-Actin (ACTB) as an example, were also detected with differential abundances among benign epithelium, differentiated vulvar intraepithelial neoplasia (dVIN), vulvar squamous cell carcinoma (VSCC) and metastatic lymph nodes (LN). These proteins could be used as tissue markers to distinguish VSCC from dVIN and other vulvar tissues. The following results of this thesis focused on proteins with potential discriminative abilities between lymph node metastasis (LNM) positive and LNM negative HPV-independent vulvar cancer patients. Two vulvar cancer VSCC tissue microarrays (TMAs) built from 28 patient samples (16 LNM positive and 12 LNM negative) were analyzed using the MALDI-MSI data. Peptide peaks of N-myc downstream regulated 1 (NDRG1) and Annexin 8 (ANXA8) showed higher discriminative powers in the Receiver Operating Characteristic Curve analysis with Area Under Curve scores of 0.641 and 0.625 respectively. Further Canonical Corresponding Analysis and Linear Discrimination Analysis (CCA-LDA) classification based on log-transformed peptide peaks achieved the best 28 out of 28 corrections in determining LNM status of TMA patients. Proteins with differential abundance were further validated by the LFQ and IHC staining, which could be used as molecular references complement to the histopathological classification system. Lastly, four Indigenous HPV-independent vulvar cancer patients were studied in Chapter 5 to compare differential protein abundances between Indigenous and non-Indigenous patients in LFQ experiment. As the result, the Periostin (POSTN) demonstrated significantly lower abundance in Indigenous vulvar cancers as compared to other non-Indigenous cancers. Besides, NDRG1 and Ras-related protein 1A (RAP1A) were identified able to determine Indigenous VSCCs with and without LNM. Spatial distributions of those proteins of interest were revealed by the MALDI-MSI and IHC staining on consecutive tissue sections. In addition, the Reactome Pathway Analysis suggested neutrophil degranulation and metabolism alternations could be key biological processes involved with the prevalence of vulvar cancer in Indigenous communities. Discovery proteins associated with vulvar cancer progression is important for vulvar cancer diagnosis and prognosis while understanding differential protein abundance in Indigenous patients will help to improve vulvar cancer treatments. This study proved MALDI-MSI and nano-LC-ESIMS/ MS LFQ are ideal methods for vulvar cancer protein identification and quantification. Protein identified in this study will be promising vulvar cancer tissue markers in the future practice.

Author dc:creator

• Zhang, Chao

Advisors dc:contributor.advisor

• Hoffmann, Peter
• Oehler, Martin K.

• Vulvar cancer
• mass spectrometry
• MALDI-MSI
• proteomics

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/2440/120298

OAI identifier oai:identifier

oai:digital.library.adelaide.edu.au:2440/120298