Background In head and neck oncological surgery the goal is to achieve a complete tumor resection with acceptable remaining function and appearance. For oral cavity squamous cell carcinoma (OCSCC) only 15% of the resections are reported as adequate. Since 2013, we have performed intraoperative assessment of resection margins (IOARM) in our institute, based on palpation and visual inspection of the resected specimens by pathologist and surgeon. This has resulted in an improvement of adequate resection margins from 15% to 50%, underlining the importance of IOARM. However, this method is subjective, labor intensive, and logistically challenging. Objective Our aim is to develop an objective method for fast and reliable IOARM based on Raman spectroscopy (RS). Methods RS is a non-destructive objective optical technique that provides information about the molecular composition of tissues. It can discriminate between healthy tissue and tumors. We developed a prototype Raman instrument employing a fiber-optic needle probe. The fiber-optic needle is driven into the OCSCC specimen, from the resection surface towards the tumor. Based on the Raman spectra collected along the insertion path, the location of the tumor border can be determined. From this the resection margin can be determined. Results First tests of the method show that the instrument accurately predicts the achieved resection margins. Per location the measurement and assessment takes 5 seconds. Conclusions This development signifies an important step towards a fast and objective IOARM. The fast measurement time enables an objective inspection of the margins achieved at a large number of locations of the resection surface.

Background Oral cavity squamous cell carcinoma (OCSCC) is the most frequent head and neck cancer. Surgery is the mainstay treatment for patients with OCSCC. In case of bone involvement, the affected bone needs to be resected. Cancer-free bone resection margins (BRMs) are of crucial importance: patients with cancer-free BRMs have a 2 times higher chance of survival. Currently, there is no standard method for intraoperative assessment of BRMs. Bone margin status is only known after tissue decalcification, which takes 1 to 2 weeks. After that time reoperations are highly undesirable, because the surgical defect has healed. Therefore, it is crucial to achieve tumor-free resection surfaces, which requires the possibility of intraoperative assessment of BRMs. Objective The aim of this study was to investigate the potential of Raman spectroscopy (RS) for detection of OCSCC in bone resection surfaces during mandibulectomy. RS is a nondestructive objective optical technique that provides information about the molecular composition of tissues. Methods Raman mapping experiments were performed on fresh mandible resection specimens from patients treated with mandibulectomy for OCSCC. A tumor detection algorithm was created based on water concentration and the high-wavenumber range (2800 cm−1-3050 cm−1) of the Raman spectra. Results Results show that RS can detect OCSCC in bone resection surfaces with a high sensitivity (96%) and specificity (83%; 26 mapping experiments, 22 patients). Conclusions These results form the basis for further development of an RS tool as an objective method for intraoperative assessment of BRMs.

Simple Summary Vulvar squamous cell carcinoma (VSCC) is the most common form of vulvar malignancy, and its incidence has increased in recent years. For better diagnosis and prognostication, and to expand available treatment options, molecular characterization of VSCC is crucial. We sought to identify aberrations in DNA methylation in VSCC, as this has been implicated in the development of several cancers. To this end, we performed genome-wide methylation sequencing on a set of VSCC and normal vulvar tissue using the Infinium MethylationEPIC BeadChip array. We detected 199 genes to be differentially methylated in VSCC compared to normal vulvar tissue. Of these, 194 genes were hyper-methylated, which leads to a loss of function of the genes. As most of these genes are involved in transcription regulator activity, our results suggest that disruption of this process plays an important role in VSCC development. Abstract DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ±0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Background Differentiated oral intraepithelial dysplasia (DOIN) was described by Japanese pathologists in 2007 but is not recognized by the World Health Organization. As in the vulva and penis, differentiated dysplasia in the oral cavity is a diagnostic challenge. Objective We determined reliable histologic criteria for the diagnosis of DOIN and assessed the usefulness of cytokeratins (CK) 13 and 17, combined with proliferation marker KI67. The frequency of DOIN in oral squamous cell carcinoma (OSCC) was also estimated. Methods All OSCC cases from 2014 to 2017 were reviewed for the presence of dysplasia. For differentiated dysplasia, histologic features were studied in detail and diagnostic criteria were established. Interobserver agreement was measured. Immunohistochemistry with CK13 and CK17/Ki67 was performed. Results We noted DOIN in 69% of OSCC cases (143/207). The histologic changes of DOIN were conspicuous in only 27% of cases, whereas in 73% the changes were subtle. Immunohistochemistry with CK13 and CK17 correlated with the histology: loss of CK13 and expression of CK17 in dysplasia, combined with KI67 altered expression. Conclusions DOIN is more frequently associated with oral carcinoma than usual dysplasia. In most cases the histologic changes are subtle. In this study we have attempted to define the histologic criteria. Cytokeratins 13 and 17/KI67 can be useful to support the diagnosis, especially in cases with subtle histologic changes. Recognition of subtle dysplastic changes will lead to progress in knowledge and treatment. We hope that the World Health Organization will recognize DOIN in the future.

OBJECTIVES To define metastatic categories based on their prognostic significance. We hypothesized that oligometastasis in patients with head and neck squamous cell carcinoma (HNSCC) is associated with better post-distant metastasis disease specific survival (post-DM DSS) compared to patients with polymetastasis. Furthermore, the impact on survival of synchronous versus metachronous distant metastasis (DM) occurrence was assessed. MATERIALS AND METHODS Retrospective cohort study in which patients with DM were stratified into three groups: oligometastasis (maximum of 3 metastatic foci in ≤2 anatomic sites), explosive metastasis (≥4 metastatic foci at one anatomic site) and explosive-disseminating metastasis (spread to ≥3 anatomic sites or >3 metastatic foci in 2 anatomic sites). In addition, patients were divided into synchronous versus metachronous DM. RESULTS Between January 1, 2006 and December 31, 2013, a total of 2687 patients with HNSCC were identified, of which 324 patients developed DM. In this group, 115 (35.5%) patients had oligometastasis, 64 (19.8%) patients had explosive metastasis and 145 (44.8%) patients had explosive-disseminating metastasis. Their median post-DM DSS were 4.7 months, 4.1 months and 1.7 months respectively (p < .001). Synchronous DM was associated with more favorable survival rates in univariable and multivariable analyses than metachronous DM with recurrence of the index tumor (6-month post-DM DSS probability of 0.51 vs 0.17, p < .001). CONCLUSION Oligometastasis in HNSCC signifies a better prognosis than a polymetastatic pattern. Metachronous DM occurrence with recurrence of the primary index tumor is associated with an unfavorable prognosis.

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery. The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed. The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

Supposed risk of malignant transformation of salivary gland pleomorphic adenoma (SGPA) is an important reason for aggressive retreatment in recurrent pleomorphic adenoma (RPA). However, although the diagnostic category ‘carcinoma ex‐pleomorphic adenoma’ suggests that malignant transformation of a pleomorphic adenoma is a regular event, this has to date not been shown to occur in sequential lesions of one patient. Here, we show the molecular events in transformation to malignancy of a pleomorphic adenoma of the parotid gland. Detailed molecular analysis revealed an LIFR/PLAG1 translocation characteristic for pleomorphic adenoma and, next to this, a PIK3R1 frameshift mutation and several allelic imbalances. In subsequent malignant recurrences, the same LIFR/PLAG1 translocation, PIK3R1 frameshift mutation, and allelic imbalances were present in addition to TP53 mutations. Thus, this case not only shows malignant transformation of SGPA, but also demonstrates that molecular analysis can be of help in recognising malignancy in the rare instance of RPA.

Objective MET 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. Methods Tumor 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. Results Forty-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). Conclusion MET 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.

Histological diagnosis of differentiated vulvar intraepithelial neoplasia (dVIN), the precursor of human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC), can be challenging, as features of dVIN may mimic those of non-dysplastic dermatoses. To aid the diagnosis, p53-immunohistochemistry (IHC) is commonly used, and mutant expression patterns are used to support a histological diagnosis of dVIN. However, a proportion of dVIN can show wild-type p53-expression, which is characteristic of non-dysplastic dermatoses. Furthermore, recent research has identified a novel precursor of HPV-independent VSCC—the p53-wild-type differentiated exophytic vulvar intraepithelial lesion (de-VIL). Currently, there are no established diagnostic IHC-markers for p53-wild-type dVIN or de-VIL. We evaluated IHC-markers, cytokeratin 17 (CK17), and SRY-box 2 (SOX2), as diagnostic adjuncts for dVIN. For this, IHC-expression of CK17, SOX2, and p53 was studied in dVIN (n = 56), de-VIL (n = 8), and non-dysplastic vulvar tissues (n = 46). For CK17 and SOX2, the percentage of cells showing expression, and the intensity and distribution of expression were recorded. We also performed next generation targeted sequencing (NGTS) on a subset of dVIN (n = 8) and de-VIL (n = 8). With p53-IHC, 74% of dVIN showed mutant patterns and 26% showed wild-type expression. Median percentage of cells expressing CK17 or SOX2 was significantly higher in dVIN (p53-mutant or p53-wild-type) and de-VIL than in non-dysplastic tissues (p < 0.01). Diffuse, moderate-to-strong, full epithelial expression of CK17 or SOX2 was highly specific for dVIN and de-VIL. With NGTS, TP53 mutations were detected in both dVIN and de-VIL. We infer that immunohistochemical markers CK17 and SOX2, when used along with p53, may help support the histological diagnosis of dVIN.