Speicheldrüsenkarzinome – ein aktueller Überblick : Fortschritte in der molekularen Typisierung: Teil I

Zusammenfassung In den letzten Jahren hat die Charakterisierung der Speicheldrüsenkarzinome einen großen Wandel durchlebt. Morphologisch definierte Entitäten konnten zu einem Großteil auch molekular mit einem oftmals distinkten Genotyp charakterisiert werden. Der erste Teil des Artikels gibt einen Überblick über die Fortschritte der molekularen Charakteristiken des Mukoepidermoidkarzinoms, adenoid-zystischen Karzinoms, Azinuszellkarzinoms, des sekretorischen und intraduktalen Karzinoms sowie des hyalinisierenden klarzelligen Karzinoms. Der molekulare Genotyp kann dabei insbesondere bei der Klassifizierung ungewöhnlicher morphologischer Varianten von großem Nutzen sein. Rekurrente NTRK- oder RET-Genfusionen können dabei nicht nur als diagnostisches Hilfsmittel, sondern auch für eine potenzielle gezielte Therapie genutzt werden. Abstract In recent years, the characterization of salivary gland tumors has undergone a major transformation. Morphologically defined entities could to a large extent also be characterized molecularly with an often distinct genotype. The first part of this article reviews the advances in the molecular characteristics of mucoepidermoid carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, secretory carcinoma, intraductal carcinoma, and hyalinizing clear cell carcinoma. The molecular genotype can be particularly useful in classifying unusual morphologic variants. Recurrent NTRK or RET gene fusions can be used not only as a diagnostic tool but also for potential targeted therapy

Challenging Tumor Heterogeneity with HER2, p16 and Somatostatin Receptor 2 Expression in a Case of EBV-Associated Lymphoepithelial Carcinoma of the Salivary Gland

BACKGROUND Lymphoepithelial carcinoma of the salivary glands (LECSG) is a rare disease in the Western hemisphere that is typically associated with an EBV infection. The molecular mechanisms of LECSG tumorigenesis are poorly understood. RESULTS Here we report a case of EBV-associated LECSG with an unusual immunophenotype. The tumor exhibited bi-morphic histological features with a mutually exclusive expression of HER2 and p16. The p16-positive domain of the tumor immunohistochemically co-expressed late membrane protein 1 (LMP-1), while the HER2 positive domain did not. Both tumor regions expressed SSTR2. METHODS In situ hybridization confirmed the EBV origin of the tumor while extensive immunohistochemical characterization and the recently established RNA-based next generation sequencing panel ("SalvGlandDx" panel) did not reveal evidence for another salivary gland neoplasm. No HPV co-infection was detected by in situ hybridization or PCR-based screenings and no ERBB2 gene amplification was detected by fluorescence in situ hybridization. CONCLUSION These findings suggest tumor heterogeneity and lack of genomic aberrations in EBV-associated LECSGs. The heterogenous and unusual immunohistochemical features explain the diagnostic difficulties and simultaneously extend the immunophenotype spectrum of this tumor entity

Combinational expression of tumor testis antigens NY-ESO-1, MAGE-A3, and MAGE-A4 predicts response to immunotherapy in mucosal melanoma patients

PURPOSE: Immunotherapy using immune checkpoint inhibitors (ICI) has revolutionized cancer treatment in recent years, particularly in melanoma. While response to immunotherapy is associated with high tumor mutational burden (TMB), PD-L1 expression, and microsatellite instability in several cancers, tumors lacking these biomarkers can still respond to this treatment. Especially, mucosal melanoma, commonly exhibiting low TMB compared to cutaneous melanoma, may respond to immunotherapy with immune checkpoint inhibitors. Therefore, the aim of our study was to investigate novel biomarkers in mucosal melanoma that predict response to combined ipilimumab and nivolumab. METHODS: We investigated 10 tumor samples from 10 patients (three responders, seven non-responders) before treatment and six tumor samples from five patients after progression using a targeted Next Generation Sequencing (NGS) gene expression panel. The findings were corroborated with an independent method (i.e., immunohistochemical staining) on the same 10 tumor samples before treatment and, to increase the cohort, in addition on three tumor samples before treatment of more recent patients (one responder, two non-responders). RESULTS: With the targeted gene expression panel, we found the three tumor testis antigens CTAG1B (NY-ESO-1), MAGE-A3, and MAGE-A4 to be predominantly expressed in responding tumors. This marker panel was either not or not completely expressed in non-responders (p < 0.01). Using immunohistochemistry for all three markers, we could confirm the elevated expression in tumors responding to the ipilimumab/nivolumab combination therapy. CONCLUSION: In conclusion, these three biomarkers await validation in a larger patient cohort and could be easily used in future routine diagnostics to predict the outcome of ipilimumab/nivolumab combination therapy in mucosal melanoma patients

Acquired resistance to anti-PD1 therapy in patients with NSCLC associates with immunosuppressive T cell phenotype

Immune checkpoint inhibitor treatment has the potential to prolong survival in non-small cell lung cancer (NSCLC), however, some of the patients develop resistance following initial response. Here, we analyze the immune phenotype of matching tumor samples from a cohort of NSCLC patients showing good initial response to immune checkpoint inhibitors, followed by acquired resistance at later time points. By using imaging mass cytometry and whole exome and RNA sequencing, we detect two patterns of resistance¨: One group of patients is characterized by reduced numbers of tumor-infiltrating CD8$^{+}$ T cells and reduced expression of PD-L1 after development of resistance, whereas the other group shows high CD8$^{+}$ T cell infiltration and high expression of PD-L1 in addition to markedly elevated expression of other immune-inhibitory molecules. In two cases, we detect downregulation of type I and II IFN pathways following progression to resistance, which could lead to an impaired anti-tumor immune response. This study thus captures the development of immune checkpoint inhibitor resistance as it progresses and deepens our mechanistic understanding of immunotherapy response in NSCLC

Multicenter Evaluation of the Fully Automated PCR-Based Idylla EGFR Mutation Assay on Forman-Fixed, Paraffin-Embedded Tissue of Human Lung Cancer

Before initiating treatment of advanced non-small-cell lung cancer with tyrosine kinase inhibitors (eg, erlotinib, gefitinib, osimertinib, and afatinib), which inhibit the catalytic activity of epidermal growth factor receptor (EGFR), clinical guidelines require determining the EGFR mutational status for activating (EGFR exons 18, 19, 20, or 21) and resistance (EGFR exon 20) mutations. The EGFR resistance mutation T790M should be monitored at cancer progression. The Idylla EGFR Mutation Assay, performed on the Idylla molecular diagnostics platform, is a fully automated (<2.5 hours turnaround time) sample-to-result molecular test to qualitatively detect 51 EGFR oncogene point mutations, deletions, or insertions. In a 15-center evaluation, Idylla results on 449 archived formalin-fixed, paraffin-embedded tissue sections, originating from non-small-cell lung cancer biopsies and resection specimens, were compared with data obtained earlier with routine reference methods, including next-generation sequencing, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays. When results were discordant, a third method of analysis was performed, when possible, to confirm test results. After confirmation testing and excluding invalids/errors and discordant results by design, a concordance of 97.6% was obtained between Idylla and routine test results. Even with <10 mm(2) of tissue area, a valid Idylla result was obtained in 98.9% of the cases. The Idylla EGFR Mutation Assay enables sensitive detection of most relevant EGFR mutations in concordance with current guidelines, with minimal molecular expertise or infrastructure

Clinical and Molecular Features of Skin Malignancies in Muir-Torre Syndrome

Background: We investigated the mutational landscape of skin tumors in patients with Muir-Torre Syndrome (MTS) a hereditary autosomal dominant mismatch repair disorder of increased cancer susceptibility, and examined mutations other than in the DNA mismatch repair (MMR) genes. Methods: This retrospective single-center case series included seven patients with the diagnosis of Muir-Torre Syndrome with precise medical history and family history. Mutational analysis of tumor samples Formalin-fixed paraffin-embedded tissue blocks of skin lesions associated with Muir-Torre Syndrome were used for further analysis. All skin tumors were analyzed with the Oncomine Comprehensive Assay v3 (Life Technologies), which includes 161 of the most relevant cancer driver genes. Results: Eleven skin neoplasms (nine sebaceous tumors, one melanoma, one cutaneous squamous cell carcinoma) were diagnosed in seven patients. In two patients, visceral malignancies preceded the diagnosis of the skin tumors and one patient was diagnosed with a visceral malignancy after a sebaceous tumor. History of familial cancer of Lynch Syndrome (LS) was reported in three patients. The most frequently detected mutation was in the MSH2 gene, followed by mutations in the NOTCH1/2 and TP53 gene. Conclusion, this study provides a molecular analysis of Muir-Torre Syndrome associated and non-associated skin tumors in patients with Muir-Torre Syndrome. Patients with sebaceous lesions should undergo microsatellite instability analysis and accurate evaluation of personal and family history to detect a possible Muir-Torre syndrome. As secondary malignancies may appear years after the first occurrence of sebaceous tumors, lifelong screening is mandatory

Standardized diagnostic algorithm for spitzoid lesions aids clinical decision-making and management: a case series from a Swiss reference center

Importance: Spitzoid lesions are a group of melanocytic tumors characterized by spindle-like or epithelioid cells with variable malignant potential. While some spitzoid lesions are classified as evidently benign or malignant by clinic and histology, others present with unclear clinical and histological characteristics and are categorized as lesions of intermediate biologic potential. These lesions represent a challenge for pathologists and clinicians alike. No consensus on ancillary diagnostics and clinical management exists. Prediction of their clinical course is difficult. The implementation of ancillary diagnostics is currently subject of extensive discussions. Observations: We report three cases of spitzoid lesions in three young female patients (3-, 15- and 17 years old) from a single reference center with different clinical and histological manifestations. In each case, uncertain clinical and histological presentation led to the stepwise application of additional diagnostics using immunohistochemistry and a custom next generation sequencing panel optimized for melanocytic lesions (MelArray). Combining ancillary diagnostics helped determine clinical management in all cases by characterizing the biology of these lesions. Conclusions and relevance: We illustrate how clinical, histological and molecular features contribute to an optimized management plan in these critical situations and present a possible algorithm for the assessment of spitzoid neoplasms. Keywords: dermatopathology; diagnostic algorithm; melanocytic lesion; pediatric dermatology; spitzoid nevi

Who’s Driving? Switch of Drivers in Immunotherapy-Treated Progressing Sinonasal Melanoma

Mucosal melanoma can be driven by various driver mutations in genes such as NRAS, KIT, or KRAS. However, some cases present with only weak drivers, or lacking known oncogenic drivers, suggesting immunotherapy over targeted therapy. While resistance mechanisms to immunotherapy in cutaneous melanoma have been uncovered, including alterations in JAK1/2, B2M, or STK11, a switch of oncogenic drivers under immunotherapy has not yet been observed. We report three cases of metastatic sinonasal melanoma that switched oncogenic drivers from KRAS, KIT, or no driver to NRAS during or after immunotherapy, thereby showing progressive disease. One of the cases presented with three spatially separate driver mutations in the primary tumor, whereas the NRAS clone persisted under immunotherapy. In comparison, three different control cases receiving radiotherapy only did not show a change of the detectable molecular drivers in their respective recurrences or metastases. In summary, these data provide an important rationale for longitudinal molecular testing, based on evidence for an unforeseen recurrent event of molecular driver switch to NRAS in progressing sinonasal melanoma. These findings provide the basis for further studies on a potential causal relation of emerging NRAS mutant clones and immunotherapy

Setting a diagnostic benchmark for tumor BRCA testing: Detection of BRCA1 and BRCA2 large genomic rearrangements in FFPE tissue - A pilot study

PARP inhibitors are used for treatment of tumors lacking function of the double-strand DNA break repair proteins BRCA1 or BRCA2 and are already approved for several cancer types. Thus, it is clinically crucial to determine germline as well as somatic BRCA1/2 mutations in those patients. The amplicon-based Oncomine BRCA1 and BRCA2 Assay is a test routinely used in diagnostics with FFPE specimens. The assay is validated for the detection of mutations, however, data on its performance in detecting large genomic rearrangements in FFPE tissue, is scarce. We cross-validated Oncomine BRCA1 and BRCA2 Assay in blood samples and/or FFPE tissue with multiplex ligation-dependent probe amplification (MLPA) for exon deletions and with OncoScan and an in-house hybridization-based target capture assay (MelArray) with a customized pipeline for the detection of loss of heterozygosity (LOH) and heterozygous versus complete gene loss. The Oncomine BRCA1 and BRCA2 Assay could detect both exon deletion and mono- and bi-allelic losses of the BRCA1/2 genes. We show that the therapeutically relevant large genomic rearrangements are reliably detected with the amplicon-based Oncomine BRCA1 and BRCA2 Assay in FFPE tumor tissue. Based on our data, we suggest tumor BRCA testing as standard diagnostic prescreening prior to germline BRCA testing