Despite rapid development of molecular diagnostics, tumormarker discovery is still challenging. In the past few years the interest of tumor research has gradually turned to proteomics highlighting the observation that secreted proteins are as important as tumor genetics. MS coupled with LC is considered as the „gold standard” quantitative method in tumor protein research. Due to the continuously evolving technical background, growing effort on investigation of HNSCC proteome has also appeared. Initially, studies targeted in vitro cell lines. Analysis of solid tumor tissues opened new ways in tumormarker discovery, as these include the surrounding microenvironment also contributing to the malignant nature of HNSCCs [5]. MS-based protein analysis had been previously possible only on fresh frozen tissue samples with the neccessity of organization steps including planned cryosection and coordination of sample preparation. These drawbacks can be overhelmed by FFPE tissues utilizing deparaffination process [6]. Among existing papers reporting proteomic analysis on FFPE samples, only a few reports address HNSCC [7]. Discovering possible tumor markers can be achieved by finding proteins exclusively expressed by tumor tissue, but protein abundance differences between normal and tumor tissue can also be interpreted as a possible tumor marker. Our aim was to explore protein abundance differences between phenotypically normal and tumor tissue on FFPE laryngeal-hypopharyngeal tumor samples. To our knowledge no LC/MS based proteomical studies exist exclusively investigating LHSCC on FFPE samples.

We found eight and two proteins with significantly higher and lower abundance in LHSCC, respectively, compared to adjacent normal tissue (Table 2).

We foremost found TNC, DYNLL1, COTL1, SPRR2D, SNRNP200, TMED2 and ALDH3A2 abundant in LHSCC by LC/MS. Similar to our findings, one LC/MS study also found NCL levels elevated in LHSCC [8].

We first found DSG1 down-regulated in LHSCC with LC/MS, albeit rather isoform switch among desmogleins seems to be determining in tumor invasivity.

We identified K1C9 down-regulated. One possible explanation for down-regulation is the dedifferentiation. The other probable hypothesis in our opinion is the lack of detectable tryptic peptides of cytokeratins in peritumoral microenvironment due to non-tryptic digestion during invasion.

IPA found no connection between proteins, but we uncovered existing interactions being considered as possible targets for future therapies in LHSCCs (Fig. 1).

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Theoretically, inhibition of TMED2 or its inducer NFE2L2 may suggest a promising tool against HNSCC invasion. NCL can act both as a recruiter and overexpressed protein of EGFR mediated pathways and can facilitate dimerization of EGFR’s cytoplasmic tail. Thus NCL overexpression can be both consequence and initiator of EGFR activation. Therefore interfering NCL can also be promising in EGFR-positive HNSCCs, while down-regulation may serve as a marker of anti-EGFR therapy efficacy.

TNC containing EGF-like repeats may serve as targets against EGFR-positive HNSCCs. Inhibiting EGFR results in TNC down-regulation. Considering the diverse correlations between TNC and EGFR, TNC can serve both a potential target in HNSCC and therapeutic response marker in anti-EGFR treatment.

DYNLL1-related cytoskeletal rearrangement and tumor cell migration can be theoretically inhibited by anti-HGF therapy, as DYNLL1 shows indirect interaction with HGF in HGF/c-MET pathway in HNSCC.

IPA found indirect inhibition of DSG1 by HGF in malignant melanoma highlighting that DSG1 down-regulation contributes to cell-cell adhesion disruption easing invasion. Thus inhibition of HGF can exert anti-tumor effect with maintaining cell-to-cell junctions via stabilizing desmosomes by DSG1 overexpression. Considering that EGFR pathway shares common signals with HGF-mediated routes resulting redundancy and frequently moderate therapeutic response to anti-EGFR treatment, combined inhibition of EGFR and c-MET/HGF pathway is emerging. Interfering redundant pathways (p44/p42 MAPK, PI3K/AKT, STAT) may have the desired anti-cancer effect. Until routinely applied anticancer drug combinations are available simultaneously targeting HGF, EGF and NFE2L2 mediated pathways, inhibition of overexpressed DYNLL, TNC, NCL and TMED2 may exert anti-tumor effect on HNSCC beyond their diagnostic role.

IPA also found unclear interactions. COTL-1 is suggested as tumor-associated protein upregulated on mouse carcinogenesis model. SNRNP200 gene mutation was reported in human cutaneous SCC. IPA found EGFR-ALDH3A2 direct binding with unknown significance. NCL-SNRNP200 direct binding demonstrates NCL’s place in RNA metabolism and identifies SNRNP200 as a nuclear interacting partner.

Contrary to previous MS studies, abundance differences were determined using label-free LC/MS based proteomics exclusively on FFPE LHSCC samples. Feasibility of quantitative LS/MS methods on FFPE samples had been questionable for a long time due to cross-links and formaldehyde induced adducts [9]. Various extraction innovations made protein recovery from FFPE samples as reliable and diagnostically accurate as from fresh-frozen samples [10]. Labeling has several disadvantages compared to label-free technique: protein loss due to each manipulation step, neccessity of prerequisites (e.g. presence of cysteine-containing peptides) and high costs. These disadvantages also can be bypassed with label-free methods.

Our study also has limitations. Interestingly, IPA did not detect SPRR2D and Krt9. This is probably due to the lack of available IPA data. Continuous amplification of stored data can reveal new interactions. The other drawback of our study is the moderate sample number. It should be noted that our primary aim was to design a pilot study for evaluation of protein abundance differences between LHSCC and adjacent healthy tissue.