Considerable interlaboratory variation in PD-L1 positivity for head and neck squamous cell carcinoma in the Netherlands- A nationwide evaluation study.

AIMS: Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) are eligible for first-line immune checkpoint inhibition if their tumour is positive for programmed death ligand 1 (PD-L1) determined by the combined positive score (CPS). This nationwide study, using real-world data, investigated the developing PD-L1 testing landscape in the first 3 years after introduction of the test in HNSCC and examined interlaboratory variation in PD-L1 positivity rates. METHODS: Pathology reports of HNSCC patients mentioning PD-L1 were extracted from the Dutch Pathology Registry (Palga). Tumour and PD-L1 testing characteristics were analysed per year and interlaboratory variation in PD-L1 positivity rates was assessed using funnel plots with 95% confidence limits around the overall mean. RESULTS: A total of 817 PD-L1 tests were reported in 702 patients among 19 laboratories; 85.2% of the tests on histological material were stated to be positive. The national PD-L1 positivity rate differed significantly per year during the study period (79.7-89.9%). The use of the recommended 22C3 antibody increased from 59.9 to 74.3%. A total of 673 PD-L1 tests on histological material from 12 laboratories were analysed to investigate interlaboratory variation. Four (33%) deviated significantly from the national mean of PD-L1-positive cases using CPS ≥ 1 cut-off, while two (17%) deviated significantly for CPS ≥ 20 cut-off. CONCLUSION: In the first 3 years of PD-L1 assessment in HNSCC, the testing landscape became more uniform. However, interlaboratory variation in PD-L1 positivity rates between Dutch laboratories was substantial. This implies that there is a need for further test standardisation to reduce this variation.

Using Intratumor Heterogeneity of Immunohistochemistry Biomarkers to Classify Laryngeal and Hypopharyngeal Tumors Based on Histologic Features.

Haralick texture features are used to quantify the spatial distribution of signal intensities within an image. In this study, the heterogeneity of proliferation (Ki-67 expression) and immune cells (CD45 expression) within tumors was quantified and used to classify histologic characteristics of larynx and hypopharynx carcinomas. Of 21 laryngectomy specimens, 74 whole-mount tumor slides were scored on histologic characteristics. Ki-67 and CD45 immunohistochemistry was performed, and all sections were digitized. The tumor area was annotated in QuPath. Haralick features independent of the diaminobenzidine intensity were extracted from the isolated diaminobenzidine signal to quantify intratumor heterogeneity. Haralick features from both Ki-67 and CD45 were used as input for a principal component analysis. A linear support vector machine was fitted to the first 4 principal components for classification and validated with a leave-one-patient-out cross-validation method. Significant differences in individual Haralick features were found between cohesive and noncohesive tumors for CD45 (angular second motion: P =.03, inverse difference moment: P =.009, and entropy: P =.02) and between the larynx and hypopharynx tumors for both CD45 (angular second motion: P =.03, inverse difference moment: P =.007, and entropy: P =.005) and Ki-67 (correlation: P =.003). Therefore, these features were used for classification. The linear classifier resulted in a classification accuracy of 85% for site of origin and 81% for growth pattern. A leave-one-patient-out cross-validation resulted in an error rate of 0.27 and 0.35 for both classifiers, respectively. In conclusion, we show a method to quantify intratumor heterogeneity of immunohistochemistry biomarkers using Haralick features. This study also shows the feasibility of using these features to classify tumors by histologic characteristics. The classifiers created in this study are a proof of concept because more data are needed to create robust classifiers, but the method shows potential for automated tumor classification.

Association of histological features with laryngeal squamous cell carcinoma recurrences: a population-based study of 1502 patients in the Netherlands.

BACKGROUND: Recurrences remain an important problem in laryngeal squamous cell carcinoma. Little has been described about histological characteristics of the primary laryngeal tumor that may be associated with recurrences. Identifying risk factors for recurrences might help in adapting treatment or follow-up. Using real-life population-based data, we aimed to identify histological features of the primary tumor associated with recurrences and overall survival. MATERIAL AND METHODS: Demographic, clinical and treatment information on all first primary invasive laryngeal tumors diagnosed in 2010-2014 (N = 3705) were extracted from the population-based nationwide Netherlands cancer registry (NCR) and linked to PALGA, the nationwide Dutch pathology registry, to obtain data on histological factors and recurrences. For a random 1502 patients histological information i.e., keratinization, perineural invasion (PNI+), vascular invasion (VI+), growth pattern, degree of differentiation, extracapsular spread (ECS+), cartilage- and bone invasion and extralaryngeal extension, was manually extracted from narrative pathology reports and analyzed for locoregional recurrence and overall survival using cox regression analysis. RESULTS: In total, 299 patients developed a locoregional recurrence and 555 patients died. Keratinization (HR = 0.96 (95%CI: 0.68-1.34) p = 0.79), two or three adverse characteristics (PNI+, VI+, non-cohesive growth) (HR = 1.38 (95% CI: 0.63-3.01) p = 0.42), and ECS+ (HR = 1.38 (95% CI: 0.48-4.02) p = 0.55) were not associated to recurrence. For death, also no significant association was found. CONCLUSION: In this population-based real-life dataset on laryngeal carcinoma in the Netherlands, histological factors were not associated with locoregional recurrences or overall survival, but future studies should investigate the role of these features in treatment decisions.

Clear Cell Neoplasms of Salivary Glands: A Diagnostic Challenge.

This review focuses on the heterogenous group of clear cell neoplasms of salivary glands and attempts to identify major differential diagnostic features. Within the head and neck region, clear cells are found most commonly in salivary gland tumors, but may also be seen in tumors of squamous or odontogenic epithelial origin, primary or metastatic carcinomas, benign or malignant melanocytic lesions, or benign or malignant mesenchymal tumors. Clear cells occur fairly commonly among a wide variety of salivary gland neoplasms, but mostly they constitute only a minor component of the tumor cell population. Clear cells represent a major diagnostic feature in two salivary gland neoplasms, epithelial-myoepithelial carcinoma and hyalinizing clear cell carcinoma. In addition, salivary gland neoplasms composed predominantly of clear cells could also include clear cell variants of other salivary neoplasms, such as mucoepidermoid carcinoma and myoepithelial carcinoma, but their tumor type-specific histologic features may only be available in limited nonclear cell areas of the tumor. Diagnosing predominantly clear cell salivary gland tumors is difficult because the immunoprofiles and morphologic features may overlap and the same tumor entity may also have a wide range of other histologic presentations. Many salivary gland tumors are characterized by tumor type-specific genomic alterations, particularly gene fusions of the ETV6 gene in secretory carcinoma, the MYB and MYBL1 genes in adenoid cystic carcinoma, the MAML2 gene in mucoepidermoid carcinoma, the EWSR1 gene in hyalinizing clear cell carcinoma, and others. Thus, along with conventional histopathologic examination and immunoprofiling, molecular and genetic tests may be important in the diagnosis of salivary gland clear cell tumors by demonstrating genetic alterations specific to them.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Comparison of three PD-L1 immunohistochemical assays in head and neck squamous cell carcinoma (HNSCC).

Expression of programmed cell death-ligand 1 (PD-L1) is being used as predictive biomarker for immunotherapy in head and neck squamous cell carcinoma (HNSCC). Several antibodies are available for PD-L1 testing and multiple staining and scoring methods are used. This study aimed to compare the performance of two PD-L1 standardized assays (SP263 and 22C3 pharmDx) and one laboratory-developed test (LDT) (22C3) in HNSCC using the tumor proportion score (TPS) and the combined positive score (CPS). Pretreatment biopsies from 147 HNSCC patients were collected in a tissue-microarray (TMA). Serial sections of the TMA were immunohistochemically stained for PD-L1 expression using 22C3 pharmDx on the Dako Link 48 platform, SP263 on the Ventana Benchmark Ultra platform, and 22C3 as an LDT on the Ventana Benchmark Ultra. Stained slides were assessed for TPS and CPS. Cutoffs of ≥1% and ≥50% for TPS and ≥1 and ≥20 for CPS were used. Concordance between the different staining assays was moderate to poor for TPS (intraclass correlation coefficient (ICC) 0.46) as well as for CPS (ICC 0.34). When stratifying patients by clinically relevant cutoffs, considerable differences between the assays were observed: concordance was poor for both TPS and CPS. Generally, SP263 stained a higher percentage of cells than the other assays, especially when using the CPS. Moderate concordance was shown between three different PD-L1 immunohistochemical assays and considerable differences in PD-L1 positivity were observed when using clinically relevant cutoffs. This should be taken into account when using PD-L1 expression to guide clinical practice.

False-negative programmed death-ligand 1 immunostaining in ethanol-fixed endobronchial ultrasound-guided transbronchial needle aspiration specimens of non-small-cell lung cancer patients.

AIMS: Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation. METHODS AND RESULTS: FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results. CONCLUSION: Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT.

Emerging Entities and New Diagnostic Markers for Head and Neck Soft Tissue and Bone Tumors.

Bone and soft tissue tumors of the head and neck are relatively uncommon tumors that often represent a diagnostic challenge because of the wide range of entities that must be considered in the differential diagnosis. Over the past few years, classification of bone and soft tissue tumors has evolved primarily because of substantial contributions from molecular genetics, with the identification of new markers that are increasingly used to complement histopathologic findings in the routine diagnostic workup. This review focuses on the recently described mesenchymal tumors that preferentially involve the head and neck region, with a focus on the most relevant novel immunohistochemical and molecular findings, including gene fusions and mutations, that can help in the diagnosis and in the assessment of clinical behavior.

High-grade Transformation/Dedifferentiation in Salivary Gland Carcinomas: Occurrence Across Subtypes and Clinical Significance.

High-grade transformation (HGT) or dedifferentiation has been described in a variety of salivary gland carcinomas, including acinic cell carcinoma, secretory carcinoma, adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, polymorphous adenocarcinoma, low-grade mucoepidermoid carcinoma, and hyalinizing clear cell carcinoma. High-grade (HG) transformed tumors are composed of a conventional low-grade component characterized by specific microscopic and immunohistochemical features for the given entity, intermingled with or juxtaposed to areas of HG morphology. This is usually either poorly differentiated adenocarcinoma, carcinoma not otherwise specified, or undifferentiated carcinoma, in which the original line of differentiation is lost. The HG component is composed of solid nests of anaplastic cells with large vesicular pleomorphic nuclei, prominent nucleoli, and abundant cytoplasm. Frequent mitoses and extensive necrosis may be present. The Ki-67 labeling index is consistently higher in the HG component. The molecular genetic mechanisms responsible for HGT of salivary gland carcinomas are largely unknown, though p53 inactivation and human epidermal growth factor receptor 2 overexpression and/or gene amplification have been demonstrated in the HG component in a few examples, the frequency varies for each histologic type. Salivary gland carcinomas with HGT are more aggressive than conventional carcinomas, with a higher local recurrence rate and a poorer prognosis. They have a high propensity for cervical lymph node metastasis suggesting a need for a wider resection and neck dissection. HGT of salivary gland carcinoma can occur either at initial presentation or less commonly at the time of recurrence, sometimes following postoperative radiotherapy. The potential for HGT in almost any type of salivary gland carcinoma warrants a thorough sampling of all salivary gland malignancies to prevent oversight of a HG component.

Nationwide evaluation of mutation-tailored treatment of gastrointestinal stromal tumors in daily clinical practice.

BACKGROUND: Molecular analysis of KIT and PDGFRA is critical for tyrosine kinase inhibitor treatment selection of gastrointestinal stromal tumors (GISTs) and hence recommended by international guidelines. We performed a nationwide study into the application of predictive mutation testing in GIST patients and its impact on targeted treatment decisions in clinical practice. METHODS: Real-world clinical and pathology information was obtained from GIST patients with initial diagnosis in 2017-2018 through database linkage between the Netherlands Cancer Registry and the nationwide Dutch Pathology Registry. RESULTS: Predictive mutation analysis was performed in 89% of the patients with high risk or metastatic disease. Molecular testing rates were higher for patients treated in expertise centers (96%) compared to non-expertise centers (75%, P < 0.01). Imatinib therapy was applied in 81% of the patients with high risk or metastatic disease without patient's refusal or adverse characteristics, e.g., comorbidities or resistance mutations. Mutation analysis that was performed in 97% of these imatinib-treated cases, did not guarantee mutation-tailored treatment: 2% of these patients had the PDGFRA p.D842V resistance mutation and 7% initiated imatinib therapy at the normal instead of high dose despite of having a KIT exon 9 mutation. CONCLUSION: In conclusion, nationwide real-world data show that over 81% of the eligible high risk or metastatic disease patients receive targeted therapy, which was tailored to the mutation status as recommended in guidelines in 88% of cases. Therefore, still 27% of these GIST patients misses out on mutation-tailored treatment. The reasons for suboptimal uptake of testing and treatment require further study.

The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use.

Biobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation-a nationwide network and registry of histo- and cytopathology in the Netherlands-was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA's nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA's nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (- 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA's national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

Significant inter- and intra-laboratory variation in grading of invasive breast cancer: A nationwide study of 33,043 patients in the Netherlands.

Accurate, consistent and reproducible grading by pathologists is of key-importance for identification of individual patients with invasive breast cancer (IBC) that will or will not benefit from adjuvant systemic treatment. We studied the laboratory-specific grading variation using nationwide real-life data to create insight and awareness in grading variation. Synoptic pathology reports of all IBC resection-specimens, obtained between 2013 and 2016, were retrieved from the nationwide Dutch Pathology Registry (PALGA). Absolute differences in laboratory-proportions of Grades I-III were compared to the national reference. Multivariable logistic regression provided laboratory-specific odds ratios (ORs) for high- vs. low-grade IBC. 33,792 IBC pathology reports of 33,043 patients from 39 laboratories were included, of which 28.1% were reported as Grade I (range between laboratories 16.3-43.3%), 47.6% as Grade II (38.4-57.8%), and 24.3% as Grade III (15.5-34.3%). Based on national guidelines, the indication for adjuvant chemotherapy was dependent on histologic grade in 29.9% of patients. After case-mix correction, 20 laboratories (51.3%) showed a significantly deviant OR. Significant grading differences were also observed among pathologists within laboratories. In this cohort of 33,043 breast cancer patients, we observed substantial inter- and intra-laboratory variation in histologic grading. It can be anticipated that this has influenced outcome including exposure to unnecessary toxicity, since choice of adjuvant chemotherapy was dependent on grade in nearly a third of patients. Better standardization and training seems warranted.

Digital pathology-aided assessment of tumor-infiltrating T lymphocytes in advanced stage, HPV-negative head and neck tumors.

AIM: This study aimed to evaluate the presence and prognostic value of tumor-infiltrating T cells in the tumor epithelium in advanced stage, HPV-negative head and neck squamous cell carcinoma (HNSCC) patients treated with primary chemoradiotherapy using digital pathology. METHODS: Pre-treatment biopsies from 80 oropharyngeal, 52 hypopharyngeal, and 29 laryngeal cancer patients were collected in a tissue microarray (TMA) and immunohistochemically stained for T-cell markers CD3, CD4, CD8, FoxP3, and PD1, and for immune checkpoint PD-L1. For each marker, the number of positive tumor-infiltrating lymphocytes (TILs) per mm2 tumor epithelium was digitally quantified and correlated to overall survival (OS), disease-free survival (DFS), and locoregional control (LRC), as well as to clinicopathological characteristics. Differences in clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan-Meier curves. RESULTS: The patient cohort had a 3-year OS of 58%, with a median follow-up of 53 months. None of the T-cell markers showed a correlation with OS, DFS or LRC. A low N stage was correlated to a better prognosis (OS: HR 0.39, p = 0.0028, DFS: HR 0.34, p = < 0.001, LRC: HR 0.24, p = 0.008). High TIL counts were more often observed in PD-L1-positive tumors (p < 0.05). CONCLUSION: This study showed an objective, digital pathology-aided method to assess TILs in the tumor epithelium. However, it did not provide evidence for a prognostic role of the presence of CD3 + , CD4 + , CD8 + , FoxP3 + , and PD1 + TILs in the tumor epithelium of advanced stage, HPV-negative HNSCC patients treated with primary chemoradiotherapy.

MET ectodomain shedding is associated with poor disease-free survival of patients diagnosed with oral squamous cell carcinoma.

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35-4.30 and P = 0.003)-though not overall survival (HR = 1.64; 95% CI, 0.92-2.94 and P = 0.095)-after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET.

Comparability of PD-L1 immunohistochemistry assays for non-small-cell lung cancer: a systematic review.

Programmed cell death ligand 1 (PD-L1) immunohistochemistry is used to determine which patients with advanced non-small-cell lung cancer (NSCLC) respond best to treatment with PD-L1 inhibitors. For each inhibitor, a unique immunohistochemical assay was developed. This systematic review gives an up-to-date insight into the comparability of standardised immunohistochemical assays and laboratory-developed tests (LDTs), focusing specifically on tumour cell (TC) staining and scoring. A systematic search was performed identifying publications that assessed interassay, interobserver and/or interlaboratory concordance of PD-L1 assays and LDTs in tissue of NSCLC patients. Of 4294 publications identified through the systematic search, 27 fulfilled the inclusion criteria and were of sufficient methodological quality. Studies assessing interassay concordance found high agreement between assays 22C3, 28-8 and SP263 and properly validated LDTs, and lower concordance for comparisons involving SP142. A decrease in concordance, however, is seen with use of cut-offs, which hampers interchangeability of PD-L1 immunohistochemistry assays and LDTs. Studies assessing interobserver concordance found high agreement for all assays and LDTs, but lower agreement with use of a 1% cut-off. This may be problematic in clinical practice, as discordance between pathologists at this cut-off may result in some patients being denied valuable treatment options. Finally, five studies assessed interlaboratory concordance and found moderate to high agreement levels for various assays and LDTs. However, to assess the actual existence of interlaboratory variation in PD-L1 testing and PD-L1 positivity in clinical practice, studies using real-world clinical pathology data are needed.

Prostate-specific membrane antigen (PSMA) expression in adenoid cystic carcinoma of the head and neck.

BACKGROUND: Treatment options for advanced head and neck adenoid cystic carcinoma (AdCC) are limited. Prostate-Specific Membrane Antigen (PSMA), a transmembrane protein that is known for its use in diagnostics and targeted therapy in prostate cancer, is also expressed by AdCC. This study aimed to analyse PSMA expression in a large cohort of primary, recurrent and metastasized AdCC of the head and neck. METHODS: One hundred ten consecutive patients with histologically confirmed AdCC in the period 1990-2017 were included. An analysis was made of clinical details, revised pathology and semiquantitative immunohistochemical expression of PSMA on tissue microarray and whole slides. Associations of PSMA expression with clinicopathological parameters were explored and survival was analysed by multivariate Cox-proportional Hazard analysis. RESULTS: PSMA expression was present in 94% of the 110 primary tumours, with a median of 31% positive cells (IQR 15-60%). Primary tumours (n = 18) that recurred (n = 15) and/or had metastases (n = 10) demonstrated 40, 60 and 23% expression respectively. Expression was not independently related to increased pathological stage, tumour grade, and the occurrence of locoregional recurrence or metastasis. After dichotomization, only primary tumour PSMA expression ≤10% appeared to be associated with reduced 10-years recurrence-free survival (HR 3.0, 95% CI 1.1-8.5, p = .04). CONCLUSIONS: PSMA is highly expressed in primary, recurrent and metastatic AdCC of the salivary and seromucous glands. PSMA expression has no value in predicting clinical behaviour of AdCC although low expression may indicate a reduced recurrence-free survival. This study provides supporting results to consider using PSMA as target for imaging and therapy when other diagnostic and palliative treatment options fail.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-ß signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-ß (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-ß results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-ß becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-ß signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a 'drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Significant inter- and intra-laboratory variation in grading of ductal carcinoma in situ of the breast: a nationwide study of 4901 patients in the Netherlands.

PURPOSE: A considerable part of ductal carcinoma in situ (DCIS) lesions may never progress into invasive breast cancer. However, standard treatment consists of surgical excision. Trials aim to identify a subgroup of low-risk DCIS patients that can safely forgo surgical treatment based on histologic grade, which highlights the importance of accurate grading. Using real-life nationwide data, we aimed to create insight and awareness in grading variation of DCIS in daily clinical practice. METHODS: All synoptic pathology reports of pure DCIS resection specimens between 2013 and 2016 were retrieved from PALGA, the nationwide Dutch Pathology Registry. Absolute differences in proportions of grade I-III were visualized using funnel plots. Multivariable analysis was performed by logistic regression to correct for case-mix, providing odds ratios and 95% confidence intervals for high-grade (III) versus low-grade (I-II) DCIS. RESULTS: 4952 DCIS reports from 36 laboratories were included, of which 12.5% were reported as grade I (range 6.1-24.4%), 39.5% as grade II (18.2-57.6%), and 48.0% as grade III (30.2-72.7%). After correction for case-mix, 14 laboratories (38.9%) reported a significantly lower (n = 4) or higher (n = 10) proportion of high-grade DCIS than the reference laboratory. Adjusted ORs (95%CI) ranged from 0.52 (0.31-0.87) to 3.83 (1.42-10.39). Significant grading differences were also observed among pathologists within laboratories. CONCLUSION: In this cohort of 4901 patients, we observed substantial inter- and intra-laboratory variation in DCIS grading, not explained by differences in case-mix. Therefore, there is an urgent need for nationwide standardization of grading practices, especially since the future management of DCIS may alter significantly depending on histologic grade.

Well-differentiated Neuroendocrine Carcinoma of the Larynx: Confusion of Terminology and Uncertainty of Early Studies.

Well-differentiated neuroendocrine carcinoma (also known as "carcinoid") of the larynx is an exceedingly rare tumor that has an epithelial origin. These tumors are malignant and have a low, but definite, risk of metastasis. Although it can be challenging, this tumor should be differentiated from moderately differentiated neuroendocrine carcinoma (also known as "atypical carcinoid"). The clinical and pathologic features of this tumor, as well as treatment and prognosis, are reviewed in detail.

Tumor classification with MALDI-MSI data of tissue microarrays: A case study.

With mass spectrometry imaging (MSI) on tissue microarrays (TMAs) a large number of biomolecules can be studied for many patients at the same time, making it an attractive tool for biomarker discovery. Here we investigate whether lymph node metastasis can be predicted from MALDI-MSI data. Measurements are performed on TMAs and then filtered based on spectral intensity and the percentage of tumor cells, after which the resulting data for 122 patients is further preprocessed. We assume differences between patients with and without metastasis are expressed in a limited number of features. Two univariate feature selection methods are applied to reduce the dimensionality of the MALDI-MSI data. The selected features are then used in combination with three classifiers. The best classification scores are obtained with a decision tree classifier, which classifies about 72% of patients correctly. Almost all the predictive power comes from a single peak (m/z 718.4). The sensitivity of our classification approach, which can be generically used to search for biomarkers, is investigated using artificially modified data.