Current knowledge on the molecular biology of VSCC is scarce. In recent years, novel treatments that could exploit the virology of HPV and/or enhance the host immune response have been proposed, still there is a paucity of clinical trials in VSCC [7]. Moreover, individualization of the currently available treatment modalities is hampered by the lack of reliable prognostic factors approved for clinical practice. The FIGO staging was shown not to be fully correlated with VSCC patients’ prognosis [17]. Even the tumor-free pathological resection margin distance of < 8 mm, long considered as a significant risk factor of vulvar recurrence, has lately been challenged as being associated with local recurrence [18]. Patients with multiple positive LNs benefit from adjuvant radiation therapy following surgery, yet in patients with a single positive LN the survival benefit still remains debatable [19–21]. Likewise, the role of adjuvant chemoradiotherapy in patients with LN involvement, as well as of neoadjuvant treatment modalities in advanced VSCC cases are not well defined.
The experimental path we applied, from global proteomic analysis to the targeted and quantitative PRM analysis of specific proteins, is the progress towards understanding the mechanisms underlying VSCC progression. A similar approach was previously confirmed by Sandberg et al. [22] while detecting protein alterations related to HPV infection in a set of VSCC tumors. An increasing evidence suggests that HPV-harboring VSCC tumors are less aggressive, though some ambiguities remain regarding the prognostic value of HPV status because of the different HPV testing methods applied, and the low numbers of cases examined in some studies [23–25]. In our study, we analyzed a cohort of early-stage VSCC patients with a long-term follow-up after surgery, which enabled stratification of cases according to treatment outcomes. Tumor proteome pathway fingerprinting with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes was performed to investigate the phenotypic changes driving VSCC tumor growth and promoting its aggressive behavior. We identified the inflammatory response as the most prevalent constituent of the top pathways revealed to be altered based on enrichment or depletion of DEPs in tumors of VSCC patients who progressed, vs those of patients who were disease-free over a long follow-up time. This notion, based on VSCC tumor proteomics data combining cancerous and micro-environmental cellular alterations, seems to be particularly justified given no immune-related pathways relevant to DEPs differentiated VSCC and normal vulvar tissues.
The immune infiltrate of VSCC and its precursor lesions by innate and adaptive immune cells, recently reviewed by Abdulrahman et al. [26], point towards immunotherapeutic options to augment the current treatment modalities. The results of our “modelling” of VSCC tumorigenesis at the level of regulatory pathways and sets of molecules, suggest that the innate immune response is a key attribute accompanying vulvar cancer progression. Local chronic inflammation and systemic inflammatory reactions that are implicated in cancer development include the activation of platelets and the coagulation system [27]. Local inflammatory reactions may result from a disturbed balance between the host and its commensal microbes and may promote cancer formation [28]. Neutrophil elastase (ELANE), PRTN3 and cathepsin G (CTSG) are the main neutrophil serine proteases (NSPs) released by activated neutrophils at the sites of inflammation, and are mediators of innate immune responses to microbial threats [13, 29]. In response to bacterial infection, in otherwise healthy individuals, NSPs connect innate immunity and coagulation by promoting local thrombus formation and microvascular occlusion, to isolate the pathogens and prevent the systemic spread of invading microorganisms [30, 31]. PRTN3, which – along with CTSG and ELANE – is a potent platelet agonist, induces platelet activation [32, 33].
In our study, the Complement and coagulation cascades and Staphylococcus aureus infection KEGG pathways were the two pathways most significantly differentiating d-fVSCC and progVSCC samples. Noteworthy, these findings refer to the causes of VSCC progression (not the effects of disease progression) as just the primary tumors were examined in the discovery phase of the study. Commensal S. aureus colonization may occur on human skin and in the nose, and up to 40% of adults are reported to be carriers [34]. Bacteria and fungi residing in the female genital tract cause common and recurrent vulvovaginal microflora disturbances leading to i.a. vulvovaginal inflammation. In otherwise healthy women who contract genital tract infections, Staphylococcus aureus is an infrequent but important pathogen [35, 36]. S. aureus DNA was demonstrated to be strongly associated with squamous cell carcinoma of the skin [34]. Olejek et al. [37] found 90% of VSCC, as assessed prior to surgery, to contain pathogenic bacterial flora, including S. aureus species in 16% (7/43) of the analyzed cases. It must be noted, however, that the Staphylococcus aureus infection KEGG pathway may not be species-specific. However, there are no data on the vulvovaginal microbiota make-up in cancer patients to support the concept of promoting VSCC progression by specific microbes. Several microorganisms of the vulvovaginal region that cause bacterial vaginosis and trichomoniasis had previously been associated also with cervical cancer [38, 39]. Vaginal dysbiosis is suggested to be a risk factor for HPV infection and persistence. Moreover, there is a trend toward an increased risk of cervical cancer progression (though not reaching statistical significance) in patients with vaginal dysbiosis [38]. Our preliminary PCR genotyping findings do not support an association between the presence of bacterial DNA in VSCC tumors neither with hrHPV infection nor VSCC progression. These results require verification with more throughput (covering more microbial species) and a quantitative method, preferably based on vulvar swabs rather than vulvar tissue samples. Yet, our data clearly confirm the presence of pathogens, including S. aureus, within vulvar tumors.
Tumor-educated platelets (TEPs) induce the local and systemic responses to tumors growth and thus a TEP-based liquid biopsy was proposed for cancer diagnostics [40]. In VSCC, the preoperative NLR and PLR values were shown to be associated with the most important prognostic factor, the nodal status [16]. As NLR and PLR counts did not differ between d-fVSCC and progVSCC patients in our study, the results of GO analysis, i.e. identification of platelet degranulation GO term in d-fVSCC vs progVSCC global proteomic analysis, as well as elevation of serum PRTN3 concentration during VSCC progression and metastasis formation, suggest neutrophil and platelet activation during the disease course, rather than their increased numbers.
In our study, a global proteomic approach revealed a multitude of DEPs in d-fVSCC versus progVSCC tumors. The panel of proteins that passed the discovery phase based on their statistically significant differential expression, was verified by PRM analysis. The results of verification with targeted proteomics, by accurately quantitating protein levels in tumors samples, provided a list of reliable candidate biomarkers having “progression-classifier” properties, and these proteins need to be validated in future studies. In the consecutive steps of global data verification and narrowing-down of the list of DEPs, in order to determine the histopathological context and cell localization of the two proteins, we performed IHC analysis of VSCC tumors with anti-HMGA2 and anti-PRTN3 antibodies.
HMGA2 belongs to the family of high-mobility group proteins, the second most abundant chromatin proteins after histones [41]. HMGA proteins are indirect transcription regulators that alter chromatin structure and affect, either positively or negatively, the transcription of a variety of target genes including inducible genes related to the immune system functions [42, 43]. HMGA2 is highly expressed during embryogenesis and rarely in adult tissues [44]. Observed in many solid tumors types, HMGA2 overexpression correlates with poor survival [45]. In tumors of epithelial origin, HMGA2 was shown to be essential for tumor progression and metastasis by inducing the epithelial-mesenchymal transition (EMT) via interaction with the TGFβ signaling pathway [46]. HMGA2 overexpression, caused by the translocations at the 12q15 region, was first identified in benign mesenchymal tumors [47]. HMGA2 overexpression can also result from down-regulation of let-7 microRNAs [10]. In our study, we have observed decreasing levels of circulating let-7c in plasma samples of VIN and VSCC patients. Recently, HMGA2 was demonstrated by RT-PCR and IHC to be expressed in VSCC tumors but not in the normal vulva tissue [48]. In our study, HMGA2 tissue abundance was associated with the aggressive phenotype of VSCC. HMGA2 is known as a nuclear protein, however, in our VSCC sample set HMGA2 staining was also clearly cytoplasmic. The possibility of the presence of the HMGA2 protein at more than one, just nuclear, cellular compartment was indicated previously, in the approach focused on identifying low-molecular-mass proteins, revealing HMGA2 to be secreted from oral cavity squamous cell carcinoma cell lines [49].
The second verified candidate protein biomarker, PRTN3 (myeloblastin) belongs to a family of NSPs which digest matrix components in inflammation and, independently of their proteolytic activity, possess microbicidal properties [11]. PRTN3 contributes to neutrophil transendothelial migration via interaction with CD177 [50]. The PRTN3 protein, derived from mature neutrophils, is detectable in the healthy donors’ circulation [51]. The presence of anti-neutrophil cytoplasmic antibodies (ANCAs), predominantly directed against the neutrophil serine proteinase (PR)-3, but also against myeloperoxidase (MPO), is characteristic for granulomatosis with polyangiitis and vasculitis (GPA) [52]. GPA belongs to ANCA-associated vasculitis (AAV), a group of vasculitides characterized by neutrophil-rich inflammation of small vessels and the presence of circulating ANCAs. Interestingly, AAV - including GPA - has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse [53]. Bacterial infections lead to neutrophil activation, which triggers NETosis, and indeed in AAV patients NETs were shown to be present in skin lesions, kidney glomeruli and in thrombi [54]. NETs consist of i.a. pro-inflammatory proteins, such as high-mobility group box 1 (HMGB1) that was shown to be elevated in AVV patients’ sera [55]. In the pathogenesis of GPA, besides neutrophils, the key effector cells (https://clinicaltrials.gov/ct2/show/NCT01862068), also activated platelets are proposed to play an important role [56]. NETs can activate the alternative complement pathway and contribute to thrombus formation while in turn platelets induce NETosis with HMGB1 expression on the platelets mediating the process [54].
In our study, PRTN3 was quantitated along with ANCA in serum samples of VIN and VSCC patients with an intention to identify a secreted protein that might prove to become a circulating biomarker of VSCC progression. Indeed, circulating PRTN3 and ANCA, were found to be elevated in VSCC patients as compared to healthy female donors. This observation seems to be related to neutrophil or platelet activation rather than a change in their numbers. Both local and systemic abundance of PRTN3 found in VSCC patients, suggests similarities in the mechanisms underlying VSCC and GPA development. These analogies might include a key role of bacterial infection in these conditions.
PRTN3 has been proposed as a therapeutic target for example in chronic obstructive pulmonary disease (COPD), an inflammatory condition associated with neutrophilic inflammation [57]. Sivelestat, a selective ELANE and PRTN3 inhibitor (https://pharos.nih.gov/idg/ligands/sivelestat), was analyzed in the four clinical trials on lung injury and respiratory failure (ClinicalTrials.gov). Sivelestat was shown to improve the mortality rates of patients with sepsis associated with acute respiratory distress syndrome and disseminated intravascular coagulation [58]. ELANE and PRTN3 along with CTSG are NSPs stored in neutrophil granules and externalized during neutrophil activation at inflammatory sites [29]. In pathological conditions, these NSPs may interact with the complement pathway by cleaving C5a receptor (C5aR/CD88) expressed on neutrophils thereby inactivating its C5a-induced signaling ability, and causing inefficient bacterial clearance [31]. ELANE activity is associated with poor outcomes in numerous malignancies, and a recent study of Lerman et al. [59] points towards ELANE as a biomarker and therapeutic target in prostate cancer. ELANE, PRTN3 and CTSG play a regulatory role in noninfectious inflammatory diseases and should be of interest as potential therapeutic targets in cancer management. Recently, Kam et al. [60] reported an inhibition of HMGB1 with sivelestat to be able to promote myelodysplastic syndrome (MDS) cell death and alter innate immune responses via suppression of NFkB pathways. As HMGA2, another high-mobility group protein, also promotes the release of pro-inflammatory cytokines through regulation of the NFκB pathway [61], one might hypothesize that sivelestat could also block HMGA2, providing an additive benefit in VSCC therapy.