Prevalence of Molecular Alterations in a Swiss Cohort of 512 Colorectal Carcinoma Patients by Targeted Next-Generation Sequencing Analysis in Routine Diagnostics.

INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.

Personalized drug testing in human pheochromocytoma/paraganglioma primary cultures.

Aggressive pheochromocytomas and paragangliomas (PPGLs) are difficult to treat, and molecular targeting is being increasingly considered, but with variable results. This study investigates established and novel molecular-targeted drugs and chemotherapeutic agents for the treatment of PPGLs in human primary cultures and murine cell line spheroids. In PPGLs from 33 patients, including 7 metastatic PPGLs, we identified germline or somatic driver mutations in 79% of cases, allowing us to assess potential differences in drug responsivity between pseudohypoxia-associated cluster 1-related (n = 10) and kinase signaling-associated cluster 2-related (n = 14) PPGL primary cultures. Single anti-cancer drugs were either more effective in cluster 1 (cabozantinib, selpercatinib, and 5-FU) or similarly effective in both clusters (everolimus, sunitinib, alpelisib, trametinib, niraparib, entinostat, gemcitabine, AR-A014418, and high-dose zoledronic acid). High-dose estrogen and low-dose zoledronic acid were the only single substances more effective in cluster 2. Neither cluster 1- nor cluster 2-related patient primary cultures responded to HIF-2a inhibitors, temozolomide, dabrafenib, or octreotide. We showed particular efficacy of targeted combination treatments (cabozantinib/everolimus, alpelisib/everolimus, alpelisib/trametinib) in both clusters, with higher efficacy of some targeted combinations in cluster 2 and overall synergistic effects (cabozantinib/everolimus, alpelisib/trametinib) or synergistic effects in cluster 2 (alpelisib/everolimus). Cabozantinib/everolimus combination therapy, gemcitabine, and high-dose zoledronic acid appear to be promising treatment options with particularly high efficacy in SDHB-mutant and metastatic tumors. In conclusion, only minor differences regarding drug responsivity were found between cluster 1 and cluster 2: some single anti-cancer drugs were more effective in cluster 1 and some targeted combination treatments were more effective in cluster 2.

SalvGlandDx - a comprehensive salivary gland neoplasm specific next generation sequencing panel to facilitate diagnosis and identify therapeutic targets.

Diagnosis of salivary gland neoplasms is often challenging due to their high morphological diversity and overlaps. Several recurrent molecular alterations have been described recently, which can serve as powerful diagnostic tools and potential therapeutic targets (e.g. NTRK or RET fusions). However, current sequential molecular testing can be expensive and time consuming. In order to facilitate the diagnosis of salivary gland neoplasms, we designed an all-in-one RNA-based next generation sequencing panel suitable for the detection of mutations, fusions and gene expression levels (including NR4A3) of 27 genes involved in salivary gland neoplasms. Here we present the validation of the "SalvGlandDx" panel on FFPE histological specimen including fine needle aspiration (FNA) cell block material, against the standard methods currently used at our institution. In a second part we describe selected unique cases in which the SalvGlandDx panel allowed proper diagnosis and new insights into special molecular characteristics of selected salivary gland tumors. We characterize a unique salivary gland adenocarcinoma harboring a ZCCHC7-NTRK2 fusion, a highly uncommon spindle cell and pseudoangiomatoid adenoid-cystic carcinoma with MYBL1-NFIB fusion, and a purely oncocytic mucoepidermoid carcinoma, whereas diagnosis could be made by detection of a CRTC3-MAML2 rearrangement on the cell block specimen of the FNA. Further, a rare case of a SS18-ZBTB7A rearranged low-grade adenocarcinoma previously described as potential spectrum of microsecretory adenocarcinoma, is reported. In addition, features of six cases within the spectrum of polymorphous adenocarcinoma / cribriform adenocarcinoma of salivary gland including PRKD1 p.E710D mutations and novel fusions involving PRKAR2A-PRKD1, SNX9-PRKD1 and ATL2-PRKD3, are described.

FOXO1 gene involvement in a non-rhabdomyosarcomatous neoplasm.

Myoepithelial neoplasms of soft tissue are rare tumors with clinical, morphological, immunohistochemical, and genetic heterogeneity. The morphological spectrum of these tumors is broad, and the diagnosis often requires immunostaining to confirm myoepithelial differentiation. Rarely, tumors show a morphology that is typical for myoepithelial neoplasms, while the immunophenotype fails to confirm myoepithelial differentiation. For such lesions, the term "myoepithelioma-like" tumor was introduced. Recently, two cases of myoepithelioma-like tumors of the hands and one case of the foot were described with previously never reported OGT-FOXO gene fusions. Here, we report a 50-year-old woman, with a myoepithelial-like tumor localized in the soft tissue of the forearm and carrying a OGT-FOXO1 fusion gene. Our findings extend the spectrum of mesenchymal tumors involving members of the FOXO family of transcription factors and point to the existence of a family of soft tissue tumors that carry the gene fusion of the OGT-FOXO family.

NUT midline carcinomas and their differentials by a single molecular profiling method: a new promising diagnostic strategy illustrated by a case report.

NUT midline carcinoma is an aggressive neoplasm defined by chromosomal rearrangements of the nuclear protein in testis (NUT) gene (NUTM1). In this article, we present a strategy to detect this rare tumor through a standard DNA methylation array analysis even when occurring in unusual anatomic sites. We illustrate our approach through a case study in which we detected metastatic spread of a NUT midline carcinoma within a bone marrow biopsy that exhibited histological features of a blastoid, undifferentiated neoplasm. Our strategy builds on molecular data derived from The Cancer Genome Atlas and Gene Expression Omnibus as well as computational strategies adopted from the Brain Tumor Methylation Classifier. It is a combined approach that detects the unusual cell lineage of NUT midline carcinomas and makes diagnostic use of the entity-specific copy number alterations.

TP53 and PTEN as driver mutations in Zenker's carcinoma-a clinical presentation.

Zenker carcinoma is still being treated empirically because of the lack of evidence- based guidelines. We report for the first time about the genetic examination of this rare entity. The revealed mutations show genetic similarities with HPV(-)HNSCC which suggests that well-known therapeutic strategies may be applicable for this disease.

Routine next generation sequencing of lymphoid malignancies: clinical utility and challenges from a 3-Year practical experience.

Since 2016, a next-generation sequencing (NGS) panel targeting 68 genes frequently mutated in lymphoid malignancies is an accredited part of routine diagnostics at the Institute of Pathology in Basel, Switzerland. Here, we retrospectively evaluate the feasibility and utility of integrating this NGS platform into routine practice on 80 diagnostic cases of lymphoid proliferations. NGS analysis was useful in most instances, yielding a diagnostically, predictively and/or prognostically meaningful result. In 35 out of the 50 cases, in which conventional histopathological evaluation remained indecisive, molecular subtyping with the NGS panel was helpful to either confirm or support the favored diagnosis, enable a differential diagnosis, or seriously question a suspected diagnosis. A total of 61 actionable or potentially actionable mutations in 34 out of 80 cases that might have enabled patient selection for targeted therapies was detected. NGS panel analysis had implications for prognosis in all 15 cases interrogated for risk assessment.

Tall cell carcinoma of the breast with reversed polarity (TCCRP) with mutations in the IDH2 and PIK3CA genes: a case report.

Tall cell carcinoma with reversed polarity (TCCRP) is a rare breast carcinoma with low malignant potential, initially named "breast tumor resembling the tall cell variant of papillary thyroid carcinoma", which has recently been recognized as a separate entity in the 5th edition of the WHO (World Health Organization) classification of breast tumors. Since the first report of this entity in 2003, more than 40 cases have been reported in the literature. Here, we report another case of this rare tumor in a 60-year-old woman. We performed immunohistochemical analyses and next-generation-sequencing (NGS) using the Oncomine™ Comprehensive DNA Panel (Thermo Fisher Scientific). The tumor showed the typical morphological features of TCCRP and a "triple-negative" phenotype. Moreover, we identified pathogenic mutations in the IDH2 (p.R172G) and PIK3CA (p.H1047R) genes. We report a case of TCCRP of the breast showing the characteristic morphologic, immunohistochemical and molecular features of this entity. There is still a limited number of cases with comprehensive molecular analyses reported in the literature. Therefore, we herewith contribute to a better understanding of the morphological and molecular characteristics as well as the clinical behavior of this rare entity.

Molecular Profile of Gastrointestinal Stromal Tumors in Sixty-Eight Patients from a Single Swiss Institution.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes. OBJECTIVE: We wanted to assess the molecular profile of 68 GIST patients who were sequenced consecutively between 2014 and 2019 at our institute of pathology. METHODS: Our cohort comprised 60 primary and 8 metastatic GIST patients; 43 and 57% of the cases, respectively, were analyzed by Sanger sequencing or next-generation sequencing (NGS). RESULTS: Of the 60 primary GIST patients, 47 (78%) showed a KIT mutation; 2 cases showed a double KIT mutation, and 1 of these was a therapy-naive GIST. Nine (15%) patients harbored a PDGFRA mutation, 2 (3%) had a BRAF mutation, 1 (2%) had a PIK3CA mutation, and 1 (2%) did not show any mutation. One BRAF and the PIK3CA mutation have not been described in GIST before. All metastatic GIST harbored exclusively KIT mutations. CONCLUSION: A retrospective analysis of GIST sequenced at our institute revealed incidences of KIT and PDGFRA mutations comparable to those in other cohorts from Europe. Interestingly, we found 2 previously undescribed mutations in the BRAF and PIK3CA genes as well as 1 treatment-naive case with a double KIT mutation in exon 11.

Nivolumab in chemotherapy-resistant cervical cancer: report of a vulvitis as a novel immune-related adverse event and molecular analysis of a persistent complete response.

BACKGROUND: Treatment options for advanced cervical cancer are limited and patients experiencing recurrence after first-line cisplatin-based chemotherapy and bevacizumab have a poor prognosis. A recent phase II study in advanced cervical cancer has demonstrated a disease control rate of 68.4% with the immune checkpoint inhibitor nivolumab. By blocking immune checkpoints, immunotherapy puts the immune system into a state of hyper-activation that can cause immune-related adverse events. We present the clinical, pathological and molecular data of a patient with metastatic cervical cancer and progressive disease after second-line therapy. We report on the therapeutic response under third-line immunotherapy with nivolumab, the immune-related adverse events (IRAE), and their successful management. CASE PRESENTATION: We report the case of a 62-year-old woman who was diagnosed with advanced squamous cell carcinoma of the cervix with paraaortic lymph node metastases. After an initial combined radio-chemotherapy with cisplatin, she developed local and nodal (supraclavicular) recurrence. Second-line chemotherapy with 6 cycles of carboplatin, paclitaxel, and bevacizumab resulted in a partial response for 6 months. Checkpoint inhibition with nivolumab was started due to progression, leading to persistent complete remission. Immunotherapy was well tolerated for 8 months until the patient presented with an immune-related isolated vulvitis, which was successfully managed with topical corticosteroids. CONCLUSIONS: The persistent complete response after third-line treatment for relapsed chemotherapy-resistant cervical cancer presented in this case highlights the potential of immunotherapy for patients with advanced cervical cancer impressively. To our knowledge, this is the first report of an isolated immune-related vulvitis under nivolumab. This adverse event might be underdiagnosed and mistreated, however, it is of importance due to its impact on quality of life, sexual wellbeing and compliance of patients. Successful IRAE management may enable prolonged immune checkpoint inhibitor therapy. In the future, routine molecular tumour profiling is likely to aid in the stratification of cervical cancer patients for immunotherapy. Here, we provide the methylome data of a case with complete response.

Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens.

BACKGROUND: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. METHODS: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). RESULTS: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. CONCLUSIONS: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Serrated epithelial colorectal polyps (hyperplastic polyps, sessile serrated adenomas) with perineurial stroma: Clinicopathological and molecular analysis of a new series.

Serrated colorectal fibroblastic polyps (FPs) are rare benign mucosal lesions composed of serrated epithelial crypts separated and distorted by intimately associated bland spindle cell proliferations with perineurial-like phenotype. We herein describe 21 new FPs affecting 10 females and 9 males aged 45 to 80 yrs. (mean, 62 yrs). Lesions originated in the sigmoid colon/rectosigmoid junction (n = 16), rectum (n = 2), and other parts of the colon (n = 3). Most patients had additional synchronous or metachronous polyps: classical adenomas (12 patients), sessile serrated adenoma/SSA (1 patient), hyperplastic polyps/HPs (7 patients), both HPs and adenomas (6 patients) and colorectal cancer (2 patients). Size of the lesions varied from 1 to 6 mm (mean: 3 mm). Histologically, all lesions were composed of serrated epithelial crypts that were separated and distorted by spindle cell stromal proliferations (consistently EMA+, claudin-1+ and GLUT-1+). The epithelial component displayed features of HPs (n = 17) and SSA (n = 4). Laser-microdissection-guided molecular testing was successful for 13 epithelial and 9 stromal components (9 paired samples). The BRAF V600E mutation was detected in 54% of the epithelial but in none of the stromal components. In conclusion, colorectal FPs represent genuine serrated epithelial polyps corresponding either to HP or (less frequently) SSA and should be better classified as such with a note on the presence of the stromal component. A more concise terminology reflecting their epithelial nature is needed to fulfill the requirements for colorectal cancer risk assessment and hence adopt appropriate follow-up strategies.

Detection of ROS1-positive non-small cell lung cancer on cytological specimens using immunocytochemistry.

BACKGROUND: Rearrangements of the ROS1 oncogene are found in 1% to 2% of non-small cell lung cancers (NSCLC) and are regarded as mutually exclusive oncogenic driver mutations. Since the approval of targeted therapy for ROS1-positive NSCLC, ROS1 testing has become a part of the diagnostic routine. Fluorescence in situ hybridization (FISH), optionally selected for by immunohistochemistry on histological material, is a common practice for the detection of ROS1 rearrangements. However, NSCLC often is diagnosed by cytology alone, requiring predictive marker testing on cytological specimens. In the current study, the authors explored the accuracy of ROS1 immunocytochemistry (ICC) on non-cell block cytological specimens for the detection of ROS1 rearrangements. METHODS: ICC using the D4D6 antibody on an automated immunostainer was performed prospectively in the routine diagnostic setting on cytological specimens from 295 patients with NSCLC, including adenocarcinoma (241 patients), NSCLC not otherwise specified (50 patients), and other malignancies (4 patients). Any immunostaining was considered positive. RESULTS: ICC was positive in all 13 ROS1-rearranged NSCLC cases confirmed by FISH (12 cases) or next-generation sequencing (1 case). Confirmation of 282 ICC-negative cases was available for 208 patients. The sensitivity, specificity, and positive and negative predictive values for ROS1 ICC compared with the final ROS1 status all were 100%. CONCLUSIONS: ROS1 ICC is an accurate method for the detection of ROS1 rearrangements in NSCLC. Given the high costs and technical challenges of FISH and the rarity of ROS1 rearrangements, ICC is rapid and therefore well suited as a screening method. Cases with equivocal or positive findings on ICC can be confirmed by FISH or molecular tests. Cancer Cytopathol 2018;126:421-9. © 2018 American Cancer Society.

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

BACKGROUND: Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. METHODS: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS: All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS: Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Human Papillomavirus (HPV) Detection in Cytologic Specimens: Similarities and Differences of Available Methodology.

Accumulating evidence regarding the causative role of human papillomavirus (HPV) in a wide range of malignant and nonmalignant diseases highlights the importance of HPV testing. This study describes and discusses the efficacy and characteristics of 4 well-established and commercially available tests. Here, 181 cytologic specimens from cervical smears were analyzed using the HPV SIGN PQ (Diatech) and the Linear Array (Roche) method. Discrepant results were further studied with the Real Time High-Risk HPV (Abbott) method and the INNO-LiPA (Fujirebio) method. Of 181 cytologic specimens, 61 (34%) showed discrepant results. High-risk HPV was not detected in 9 cases by HPV SIGN PQ, in 16 cases by Linear Array, in 10 cases by Real Time High-Risk HPV, and in 6 cases by INNO-LiPA, respectively. Lack of DNA detection or problems in interpreting the result were seen in 9 cases with HPV SIGN PQ, 8 cases with Linear Array, 3 cases with Real Time High-Risk HPV, and 3 cases with INNO-LiPA, respectively. This study indicates that the choice of HPV detection method has a substantial influence on the HPV risk classification of tested PAP smears and clinical follow-up decisions.