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Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1–3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity.

The prognostic impact of human papillomavirus (HPV) in oropharyngeal cancer is generally acknowledged, and HPV‐status is assessed routinely in clinical practice. Paradoxically, while the oral cavity seems the predilection site for productive HPV‐infections, figures on HPV‐attribution in oral cavity squamous cell carcinoma (OCSCC) differ widely, and prognostic impact is uncertain. Major obstacles are the lack of reproducible assays to detect HPV in nonoropharyngeal cancers, the relatively small cohorts studied and consequently the shortfall of convincing data. In our study, we used a validated, nucleic acid‐based workflow to assess HPV‐prevalence in a consecutive cohort of 1016 OCSCCs, and investigated its prognostic impact. In parallel, we analyzed p16‐immunohistochemistry (p16‐IHC) as surrogate marker for transforming HPV‐infection and independent prognosticator. All OCSCC‐patients diagnosed between 2008 and 2014 at two Dutch university medical centers were included (N = 1069). Formalin‐fixed, paraffin‐embedded (FFPE)‐samples of 1016 OCSCCs could be retrieved. Punch biopsies were taken from the tumor area in the FFPE‐blocks and tested for HPV. P16‐IHC was performed on 580 OCSCCs, including all HPV‐positive tumors. From 940 samples (92.5%), nucleic acids were of sufficient quality for HPV‐testing. In total, 21 (2.2%) OCSCCs were HPV DNA‐positive. All HPV DNA‐positive tumors were E6 mRNA‐positive and considered as true HPV‐positive. There was no difference in survival between HPV‐positive and HPV‐negative OCSCCs. In total, 46 of 580 (7.9%) OCSCCs were p16‐immunopositive, including all HPV‐positive tumors. Survival was comparable in p16‐positive and p16‐negative OCSCCs. To conclude, HPV‐prevalence is very low in OCSCC and neither HPV‐status nor p16‐status affects outcome. Based on these data, determining HPV‐status in OCSCC seems irrelevant for clinical management., What's new? While human papillomavirus (HPV)‐infection is prognostic for oropharyngeal squamous cell carcinoma (OCSCC), its role and prognostic impact in OCSCC remain uncertain. In the present study, nucleic acid‐based HPV‐testing of a cohort of 1016 tumor samples collected from OCSCC patients diagnosed between 2008 and 2014 reveals very low HPV‐prevalence in OCSCC. In addition, no difference was found in survival between HPV‐positive and HPV‐negative tumors. The findings indicate that HPV is uncommon in OCSCC and, when present, has no impact on survival, providing new and important insight into the ongoing debate regarding the role of HPV in OCSCC.

Simple Summary Head and neck cancer is the sixth most common cancer type worldwide, comprising tumors of the upper aero/digestive tract. Approximately 50% of these cancers originate in the oral cavity. Depending on disease stage, oral cancer patients are treated with single-modality surgery, or in combination with radiotherapy with or without chemotherapy. Despite advances in these modalities, the 5-year survival rate is merely 50%. Therefore, implementation of targeted therapies, directed against signaling molecules, has gained attention. One potential target is the MET protein, which can be present on the surface of cancer cells, orchestrating aggressive behavior. As cancer cells can shed the extracellular part of MET from their surface, it is important to identify for MET positive patients whether they possess the entire and/or only the intracellular part of the receptor to assess whether targeted therapies directed against the extracellular, intracellular, or both parts of MET need to be implemented. The receptor tyrosine kinase MET has gained attention as a therapeutic target. Although MET immunoreactivity is associated with progressive disease, use of targeted therapies has not yet led to major survival benefits. A possible explanation is the lack of companion diagnostics (CDx) that account for proteolytic processing. During presenilin-regulated intramembrane proteolysis, MET's ectodomain is shed into the extracellular space, which is followed by gamma-secretase-mediated cleavage of the residual membranous C-terminal fragment. The resulting intracellular fragment is degraded by the proteasome, leading to downregulation of MET signaling. Conversely, a membrane-bound MET fragment lacking the ectodomain (MET-EC-) can confer malignant potential. Use of C- and N-terminal MET monoclonal antibodies (moAbs) has illustrated that MET-EC- occurs in transmembranous C-terminal MET-positive oral squamous cell carcinoma (OSCC). Here, we propose that ectodomain shedding, resulting from G-protein-coupled receptor transactivation of epidermal growth factor receptor signaling, and/or overexpression of ADAM10/17 and/or MET, stabilizes and possibly activates MET-EC- in OSCC. As MET-EC- is associated with poor prognosis in OSCC, it potentially has impact on the use of targeted therapies. Therefore, MET-EC- should be incorporated in the design of CDx to improve patient stratification and ultimately prolong survival. Hence, MET-EC- requires further investigation seen its oncogenic and predictive properties.

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/β-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and β-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.

ObjectiveMET positivity is independently associated with survival in oral squamous cell carcinoma (OSCC). Since MET is a known orchestrator of invasive tumor growth, we investigated its association with LNM in early oral tongue squamous cell carcinoma (OTSCC). As it is recommended by the NCCN to use tumor depth of invasion (DOI) in making decisions on elective neck dissection (END), the results obtained for MET positivity were aligned with those for DOI > 4 mm. The cutoff value used in our institution.MethodsTumor samples from patients who underwent primary tumor resection and neck dissection between 1995 and 2013, were collected from the archives of the Leiden and Erasmus University Medical Center. Immunohistochemistry with D1C2 was performed to identify MET negative (< 10% uniform positivity) and MET positive (≥ 10% uniform positivity) cancers. ROC curve analysis and the Chi-squared test were used to investigate the association of MET positivity with LNM (pN+ and occult). Binary logistic regression was used to investigate the association of MET positivity with LNM.ResultsForty-five (44.1%) of the 102 cancers were MET positive. Ninety were cN0 of which 20 were pN+ (occult metastasis). The remaining 12 cancers were cN+, of which 10 were proven pN+ and 2 were pN0. MET positivity was associated with LNM with a positive predictive value (PPV) of 44.4% and a negative predictive value (NPV) of 82.5% for pN+. For the occult group, the PPV was 36.8% and the NPV was 88.5%. Regression analysis showed that MET positivity is associated with pN+ and occult LNM (p-value < 0.05).ConclusionMET positivity is significantly associated with LNM in early OTSCC, outperforming DOI. The added value of MET positivity could be in the preoperative setting when END is being considered during the initial surgery. For cases with DOI ≤ 4 mm, MET positivity could aid in the clinical decision whether regular follow-up, watchful waiting, or END is more appropriate. Realizing that these preliminary results need to be independently validated in a larger patient cohort, we believe that MET positivity could be of added value in the decision making on END in early OTSCC.

ObjectiveThe depth of invasion (DOI) is considered an independent risk factor for occult lymph node metastasis in oral cavity squamous cell carcinoma (OCSCC). It is used to decide whether an elective neck dissection (END) is indicated in the case of a clinically negative neck for early stage carcinoma (pT1/pT2). However, there is no consensus on the cut-off value of the DOI for performing an END. The aim of this study was to determine a cut-off value for clinical decision making on END, by assessing the association of the DOI and the risk of occult lymph node metastasis in early OCSCC.MethodsA retrospective cohort study was conducted at the Erasmus MC, University Medical Centre Rotterdam, The Netherlands. Patients surgically treated for pT1/pT2 OCSCC between 2006 and 2012 were included. For all cases, the DOI was measured according to the 8th edition of the American Joint Committee on Cancer guideline. Patient characteristics, tumor characteristics (pTN, differentiation grade, perineural invasion, and lymphovascular invasion), treatment modality (END or watchful waiting), and 5-year follow-up (local recurrence, regional recurrence, and distant metastasis) were obtained from patient files.ResultsA total of 222 patients were included, 117 pT1 and 105 pT2. Occult lymph node metastasis was found in 39 of the 166 patients who received END. Univariate logistic regression analysis showed DOI to be a significant predictor for occult lymph node metastasis (odds ratio (OR) = 1.3 per mm DOI; 95% CI: 1.1–1.5, p = 0.001). At a DOI of 4.3 mm the risk of occult lymph node metastasis was >20% (all subsites combined).ConclusionThe DOI is a significant predictor for occult lymph node metastasis in early stage oral carcinoma. A NPV of 81% was found at a DOI cut-off value of 4 mm. Therefore, an END should be performed if the DOI is >4 mm.