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2.
Acta Neuropathol ; 147(1): 44, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38386085

RESUMO

The development of brain metastases hallmarks disease progression in 20-40% of melanoma patients and is a serious obstacle to therapy. Understanding the processes involved in the development and maintenance of melanoma brain metastases (MBM) is critical for the discovery of novel therapeutic strategies. Here, we generated transcriptome and methylome profiles of MBM showing high or low abundance of infiltrated Iba1high tumor-associated microglia and macrophages (TAMs). Our survey identified potential prognostic markers of favorable disease course and response to immune checkpoint inhibitor (ICi) therapy, among them APBB1IP and the interferon-responsive gene ITGB7. In MBM with high ITGB7/APBB1IP levels, the accumulation of TAMs correlated significantly with the immune score. Signature-based deconvolution of MBM via single sample GSEA revealed enrichment of interferon-response and immune signatures and revealed inflammation, stress and MET receptor signaling. MET receptor phosphorylation/activation maybe elicited by inflammatory processes in brain metastatic melanoma cells via stroma cell-released HGF. We found phospho-METY1234/1235 in a subset of MBM and observed a marked response of brain metastasis-derived cell lines (BMCs) that lacked druggable BRAF mutations or developed resistance to BRAF inhibitors (BRAFi) in vivo to MET inhibitors PHA-665752 and ARQ197 (tivantinib). In summary, the activation of MET receptor in brain colonizing melanoma cells by stromal cell-released HGF may promote tumor self-maintenance and expansion and might counteract ICi therapy. Therefore, therapeutic targeting of MET possibly serves as a promising strategy to control intracranial progressive disease and improve patient survival.


Assuntos
Neoplasias Encefálicas , Melanoma , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Progressão da Doença , Interferons
3.
J Mol Med (Berl) ; 102(2): 247-255, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38127137

RESUMO

Objective parameters to quantify psoriatic inflammation are needed for interdisciplinary patient care, as well as preclinical experimental models. This study evaluates neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in psoriasis patients and five murine models of psoriasis-like skin disease based on topical imiquimod application and overexpression of IL-17A under different promotors. We performed a single-center prospective observational study in a German population, investigating psoriasis patients prior to, 4 weeks, and 16 weeks post begin of systemic anti-inflammatory therapy. Psoriasis area and severity index (PASI), blood count, and C-reactive protein (CRP) levels were attained at each timepoint. Additionally, five murine models of psoriasis-like skin disease involving five distinct experimental procedures differing in time of disease-onset and severity were investigated regarding PLR and NLR. Of 43 recruited psoriasis patients, 34 patients were followed up to 16 weeks. The cohort was 69.77% male, showing a median age of 32.0 years (range 19.0-67.0; IQR 26). The median PASI decreased from 16.35 (8.0-50.0; 10.20) to 1.6 (0-10.3; 2.56) after 16 weeks of systemic therapy. Spearman's correlation showed statistically significant positive correlation for NLR with PASI (rs = 0.27, p = 0.006), however not for PLR. NLR, but not PLR, was significantly associated with PASI in a multiple linear regression analysis including age, sex, psoriasis arthritis, and smoking. In the murine models of psoriasis-like skin disease, both NLR and PLR were significantly increased in the acute-severe models compared to controls (p < 0.001, p = 0.005, and p = 0.02, respectively), demonstrating gradually less increased values from severe-acute to mild-late-onset psoriatic phenotype. NLR was significantly associated with PASI in psoriatic patients as well as psoriatic phenotype in different murine psoriasis models. Our data warrants investigation of NLR in psoriasis patients and preclinical psoriasis models as an objective biomarker of psoriatic skin inflammation. KEY MESSAGES : NLR, but not PLR, showed a statistically significant positive correlation with Psoriasis Area and Severity Index (PASI) in our human psoriasis cohort. Both NLR and PLR were significantly increased in murine psoriasis models compared to matched controls, with gradually less increased values from severe-acute to mild-late-onset psoriatic phenotype. NLR may represent an easily available, cheap, and objective parameter to monitor psoriatic inflammation in both clinical patient routine, as well as preclinical experimental murine models.


Assuntos
Neutrófilos , Psoríase , Humanos , Masculino , Animais , Camundongos , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Feminino , Modelos Animais de Doenças , Linfócitos , Inflamação
4.
J Cancer Res Clin Oncol ; 149(3): 1049-1061, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35246724

RESUMO

OBJECTIVES: Perioperative chemo-(radio-) therapy is the accepted standard in European patients with locally advanced adenocarcinoma of the esophagogastric junction or stomach (AEG/AS). However, 30-85% of patients do not respond to this treatment. The aim of our study was the identification of predictive biomarkers in pre-therapeutic endoscopic tumor biopsies from patients with histopathologic response (Becker-1) versus non-response (Becker-2/3) to preoperative chemotherapy. METHODS: Formalin-fixed paraffin-embedded biopsies from 36 Caucasian patients (Becker-1 n = 11, Becker-2 n = 7, Becker-3 n = 18) with AEG/AS, taken prior to neoadjuvant chemotherapy were selected. For RNA expression analysis, we employed the NanoString nCounter System. To identify genomic alterations like single nucleotide variants (SNV), copy number variation (CNV) and fusion events, we used Illumina TST170 gene panel. For HER2 and FGFR2 protein expression, immunostaining was performed. Furthermore, we analyzed the microsatellite instability (MSI) and Epstein-Barr virus (EBV) infection status by EBER in situ hybridization. RESULTS: Heat map and principal component analyses showed no clustering by means of gene expression according to regression grade. Concerning two recently proposed predictive markers, our data showed equal distribution for MSI (Becker-1: 2; Becker-2: 1; Becker-3: 3; out of 29 tested) and EBV infection was rare (1/32). We could not reveal discriminating target genes concerning SNV, but found a higher mutational burden in non-responders versus responders and fusion (in 6/14) and CNV events (in 5/14) exclusively in Becker-3. CONCLUSIONS: Although we could not identify discriminating target genes, our data suggest that molecular alterations are in general more prevalent in patients with AEG/AS belonging to the non-responding Becker group 3.


Assuntos
Adenocarcinoma , Infecções por Vírus Epstein-Barr , Neoplasias Esofágicas , Neoplasias Gástricas , Humanos , Terapia Neoadjuvante , Neoplasias Gástricas/patologia , Infecções por Vírus Epstein-Barr/patologia , Variações do Número de Cópias de DNA , Herpesvirus Humano 4/genética , Junção Esofagogástrica/patologia , Instabilidade de Microssatélites , Perfilação da Expressão Gênica , Adenocarcinoma/patologia , Biópsia , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Esofágicas/patologia
5.
Virchows Arch ; 482(4): 697-706, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36367572

RESUMO

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries-including Germany-assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Mutação/genética , Medicina de Precisão , Classe I de Fosfatidilinositol 3-Quinases/genética , Europa (Continente)
6.
Nat Commun ; 13(1): 7148, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-36443295

RESUMO

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs.


Assuntos
Carcinoma , Metilação de DNA , Humanos , Metilação de DNA/genética , Proteômica , Reprodutibilidade dos Testes , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição
7.
Anticancer Res ; 42(5): 2405-2413, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35489745

RESUMO

BACKGROUND/AIM: This study analyzed the expression of p16 in a large cohort of patients suffering from oral squamous cell carcinoma (OSCC) who received initial surgical therapy in order to evaluate the prognostic significance of p16 expression and to analyze its value as a surrogate marker to determine human papilloma virus (HPV) status. MATERIALS AND METHODS: Immunohistochemical staining of p16 was performed on tissue microarrays. Different expression levels of p16 (>25%; >50%; ≥70%) with a moderate to strong intensity were correlated with the clinical outcome. HPV DNA was analyzed by polymerase chain reaction (PCR). RESULTS: A total of 281 patients were included in this study. The p16 expression obtained using the abovementioned three different cutoffs did not significantly influence 5-year overall survival (OS) (p=0.23; p=0.45; p=0.23) nor recurrence-free survival (RFS) (p=0.79; p=0.45; p=0.142). In univariate Cox regression analysis, the p16 expression level was not a risk factor for OS (HR=0.637; 95%CI=0.271-1.5; p=0.300) and RFS (HR=0.74; 95%CI=0.339-1.61; p=0.449). A total of 17 patients (6.0%) were p16 positive with a cutoff ≥70%. HPV DNA was found in 4/11 of these cases by PCR, resulting in a positive predictive value of 0.36. In patients receiving adjuvant radio(chemo)therapy, a significantly (p=0.042) longer OS was observed in patients with p16 expression greater than 25% vs. ≤25%. CONCLUSION: In comparison with OPSCC, (strong) p16 positivity is rare in OSCC; however, in patients receiving primary surgery with adjuvant radio(chemo)therapy, p16 expression is associated with a higher survival rate. In conjunction with prior studies, p16 does not seem to be a reliable surrogate marker for HPV infection in OSCC.


Assuntos
Alphapapillomavirus , Inibidor p16 de Quinase Dependente de Ciclina , Neoplasias Bucais , Infecções por Papillomavirus , Biomarcadores Tumorais/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Neoplasias Bucais/complicações , Neoplasias Bucais/metabolismo , Papillomaviridae , Infecções por Papillomavirus/complicações , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/complicações , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo
8.
Breast Cancer Res Treat ; 191(2): 327-333, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34783927

RESUMO

BACKGROUND: Risk assessment on the molecular level is important in predictive pathology to determine the risk of metastatic disease for ERpos, HER2neg breast cancer. The gene expression test EndoPredict (EP) was trained and validated for prediction of a 10-year risk of distant recurrence to support therapy decisions regarding endocrine therapy alone or in combination with chemotherapy. The EP test provides the 12-gene Molecular Score (MS) and the EPclin-Score (EPclin), which combines the molecular score with tumor size and nodal status. In this project we investigated the correlation of 12-gene MS and EPclin scores with classical pathological markers. METHODS: EndoPredict-based gene expression profiling was performed prospectively in a total of 1652 patients between 2017 and 2020. We investigated tumor grading and Ki67 cut-offs of 20% for binary classification as well as 10% and 30% for three classes (low, intermediate, high), based on national and international guidelines. RESULTS: 410 (24.8%) of 1652 patients were classified as 12-gene MS low risk and 626 (37.9%) as EPclin low risk. We found significant positive associations between 12-gene MS and grading (p < 0.001), EPclin and grading (p = 0.001), 12-gene MS and Ki67 (p < 0.001), and EPclin and Ki67 (p < 0.001). However, clinically relevant differences between EP test results, Ki67 and tumor grading were observed. For example, 118 (26.3%) of 449 patients with Ki67 > 20% were classified as low risk by EPclin. Same differences were seen comparing EP test results and tumor grading. CONCLUSION: In this study we could show that EP risk scores are distributed differentially among Ki67 expression groups, especially in Ki67 low and high tumors with a substantial proportion of patients with EPclin high risk results in Ki67 low tumors and vice versa. This suggests that classical pathological parameters and gene expression parameters are not interchangeable, but should be used in combination for risk assessment.


Assuntos
Neoplasias da Mama , Neoplasias da Mama/genética , Feminino , Humanos , Prognóstico , Receptor ErbB-2/genética , Receptores de Estrogênio , Medição de Risco
9.
J Pathol ; 256(4): 378-387, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34878655

RESUMO

In head and neck squamous cell cancers (HNSCs) that present as metastases with an unknown primary (HNSC-CUPs), the identification of a primary tumor improves therapy options and increases patient survival. However, the currently available diagnostic methods are laborious and do not offer a sufficient detection rate. Predictive machine learning models based on DNA methylation profiles have recently emerged as a promising technique for tumor classification. We applied this technique to HNSC to develop a tool that can improve the diagnostic work-up for HNSC-CUPs. On a reference cohort of 405 primary HNSC samples, we developed four classifiers based on different machine learning models [random forest (RF), neural network (NN), elastic net penalized logistic regression (LOGREG), and support vector machine (SVM)] that predict the primary site of HNSC tumors from their DNA methylation profile. The classifiers achieved high classification accuracies (RF = 83%, NN = 88%, LOGREG = SVM = 89%) on an independent cohort of 64 HNSC metastases. Further, the NN, LOGREG, and SVM models significantly outperformed p16 status as a marker for an origin in the oropharynx. In conclusion, the DNA methylation profiles of HNSC metastases are characteristic for their primary sites, and the classifiers developed in this study, which are made available to the scientific community, can provide valuable information to guide the diagnostic work-up of HNSC-CUP. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Metilação de DNA , Neoplasias de Cabeça e Pescoço , Neoplasias de Cabeça e Pescoço/genética , Humanos , Aprendizado de Máquina , Redes Neurais de Computação , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética
10.
Oncogene ; 40(50): 6748-6758, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34663877

RESUMO

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also the tumor microenvironment can guide precision medicine. A comprehensive and in-depth characterization of the tumor microenvironment is challenging since its cell populations are diverse and can be important even if scarce. To identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to ten human lung adenocarcinomas and ten normal control tissues. Our analyses revealed heterogeneous carcinoma cell transcriptomes reflecting histological grade and oncogenic pathway activities, and two distinct microenvironmental patterns. The immune-activated CP²E microenvironment was composed of cancer-associated myofibroblasts, proinflammatory monocyte-derived macrophages, plasmacytoid dendritic cells and exhausted CD8+ T cells, and was prognostically unfavorable. In contrast, the inert N³MC microenvironment was characterized by normal-like myofibroblasts, non-inflammatory monocyte-derived macrophages, NK cells, myeloid dendritic cells and conventional T cells, and was associated with a favorable prognosis. Microenvironmental marker genes and signatures identified in single-cell profiles had progonostic value in bulk tumor profiles. In summary, single-cell RNA profiling of lung adenocarcinoma provides additional prognostic information based on the microenvironment, and may help to predict therapy response and to reveal possible target cell populations for future therapeutic approaches.


Assuntos
Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Análise de Célula Única/métodos , Transcriptoma , Microambiente Tumoral , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/imunologia , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Prognóstico , Taxa de Sobrevida
11.
Int J Infect Dis ; 103: 628-635, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33401036

RESUMO

OBJECTIVES: In coronavirus disease 2019 (COVID-19), the adaptive immune response is of considerable importance, and detailed cellular immune reactions in the hematological system of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are yet to be clarified. METHODS: This study reports the morphological characterization of both bone marrow and spleen in 11 COVID-19 decedents with respect to findings in the peripheral blood and pulmonary SARS-CoV-2 burden. RESULTS: In the bone marrow, activation and left shift were found in at least 55% of patients, which was mirrored by peripheral anaemia, granulocytic immaturity and multiple thromboembolic events. Signs of sepsis-acquired immunodeficiency were found in the setting of an abscess-forming superinfection of viral COVID-19 pneumonia. Furthermore, a severe B cell loss was observed in the bone marrow and/or spleen in 64% of COVID-19 patients. This was reflected by lymphocytopenia in the peripheral blood. As compared to B cell preservation, B cell loss was associated with a higher pulmonary SARS-CoV-2 burden and only a marginal decrease of of T cell counts. CONCLUSIONS: The results of this study suggest the presence of sepsis-related immunodeficiency in severe COVID-19 pneumonia with superinfection. Furthermore, our findings indicate that lymphocytopenia in COVID-19 is accompanied by B cell depletion in hematopoietic tissue, which might impede the durability of the humoral immune response to SARS-CoV-2.


Assuntos
Linfócitos B/imunologia , Medula Óssea/imunologia , COVID-19/imunologia , Linfopenia/etiologia , SARS-CoV-2 , Sepse/imunologia , Baço/imunologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
J Am Chem Soc ; 142(49): 20554-20559, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33226797

RESUMO

Reduction of the aluminum iodide AlI2AriPr8 (1; AriPr8 = C6H-2,6-(C6H2-2,4,6-Pri3)2-3,5-Pri2) with 5% w/w Na/NaCl in hexanes gave a dark red solution from which the monomeric alanediyl :AlAriPr8 (2) was isolated in ca. 28% yield as yellow-orange crystals. Compounds 1 and 2 were characterized by X-ray crystallography, electronic and NMR spectroscopy, and theoretical calculations. The Al atom in 2 is one-coordinate, and the compound displays two absorptions in its electronic spectrum at 354 and 455 nm. It reacts with H2 under ambient conditions to give the aluminum hydride {AlH(µ-H)AriPr8}2, probably via a weakly bound dimer of 2 as an intermediate.

13.
Genes Chromosomes Cancer ; 59(8): 445-453, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32319699

RESUMO

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH- and tNGS-based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels.


Assuntos
Biomarcadores Tumorais/genética , Testes Genéticos/métodos , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , RNA-Seq/métodos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Testes Genéticos/normas , Humanos , Hibridização in Situ Fluorescente/métodos , Neoplasias/diagnóstico , RNA-Seq/normas , Sensibilidade e Especificidade , Preservação de Tecido/métodos
14.
Genes Chromosomes Cancer ; 59(3): 178-188, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31652375

RESUMO

NTRK fusions involving three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, and NTRK3 and a variety of fusion partners were identified as oncogenic drivers across many cancer types. Drugs that target the chimeric protein product require the identification of the underlying gene fusion. This advocates the diagnostic use of molecular assays ranging from fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR)/Sanger approaches to targeted next-generation sequencing (NGS). Immunohistochemistry may be used as a screening tool and adjunct diagnostic assay in this context. Although FISH and RT-PCR/Sanger approaches are widely adopted in routine diagnostics, current experience with targeted RNA-based NGS is limited. Here, we report on the analysis of major assays (TruSight TST170 and TruSight RNA Fusion [Illumina]; Archer FusionPlex Solid Tumor, Archer FusionPlex Lung, and Archer FusionPlex Oncology [Archer]; Oncomine Comprehensive Assay v3 RNA and Oncomine Focus RNA [Thermo Fisher Scientific]) that are commercially available. The data set includes performance results of a multicentric comparative wet-lab study as well as an in silico analysis on the ability to detect the broad range of NTRK fusions reported until now. A test algorithm that reflects assay methodology is provided. This data will support implementation of targeted RNA sequencing in routine diagnostics and inform screening and testing strategies that have been brought forward.


Assuntos
Biomarcadores Tumorais , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Fator de Crescimento Neural/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Tomada de Decisão Clínica , Gerenciamento Clínico , Feminino , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lactente , Masculino , Pessoa de Meia-Idade , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Reprodutibilidade dos Testes , Fluxo de Trabalho , Adulto Jovem
15.
Clin Lung Cancer ; 20(5): 350-362.e4, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31175009

RESUMO

BACKGROUND: Standard therapy of advanced non-small-cell lung cancer harboring an activating mutation in the epidermal growth factor receptor (EGFR) gene is treatment with tyrosine kinase inhibitors (TKI). However, for rare and compound mutations of the EGFR gene, the clinical evidence of TKI therapy is still unclear. PATIENTS AND METHODS: A total of 2906 lung cancer samples were analyzed for EGFR mutations during routine analysis between 2010 and 2017. The samples have been investigated by Sanger sequencing and since 2014 by next-generation sequencing. RESULTS: We detected EGFR mutations in 408 specimens (14%). Among these, we found 41 samples with rare and 22 with compound mutations. In these 63 samples, 56 different rare EGFR mutations occurred. Information about the clinical outcome was available for 37. Among those with rare mutations, only one patient harboring the mutation p.G874D had disease that responded to first-generation TKI therapy. In contrast, the disease of all patients with compound mutations responded to first- or second-generation TKI therapy. Furthermore, we collected data on clinical relevance regarding TKI therapy from different databases and from an additional literature search, and only found data for 36 of the 56 detected rare mutations. CONCLUSION: Information about the clinical outcome of patients with rare and compound EGFR mutations remains limited. At present, second- and third-generation TKIs are available, which may represent new treatment strategies for these patients. Therefore, it is becoming increasingly important to maintain databases concerning rare EGFR mutations.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/terapia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
16.
BMC Cancer ; 18(1): 1158, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30466405

RESUMO

BACKGROUND: Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. METHODS: We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. RESULTS: Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. CONCLUSIONS: The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Quinase do Linfoma Anaplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/metabolismo , Sensibilidade e Especificidade , Transcriptoma
17.
Oncotarget ; 9(26): 18529-18539, 2018 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-29719623

RESUMO

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

18.
Exp Mol Pathol ; 104(1): 76-81, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29337243

RESUMO

Anti-EGFR-targeted therapy is used to treat metastatic colorectal cancers with RAS wild-type. However, resistance to targeted therapy is often observed and can be primary or acquired. One reason for primary resistance is the presence of mutations that are undetected due to genetic heterogeneity, which can be expressed by differences present in primary tumor and distant metastasis or recurrence or by an intratumoral heterogeneity (presence of different subclones in the investigated tumor sample). The aim of our study was to investigate if morphological heterogeneity can be an indicator of intratumoral heterogeneity. We analysed 13 samples with homogeneous and six samples with heterogeneous morphology with NGS. We were able to demonstrate that intratumoral genetic heterogeneity is present in all studied tumor samples, independent of homogeneous or heterogeneous morphology. Moreover, one sample of our cohort with morphological and genetic heterogeneity had a genetic wild-type profile in one tumor component. Therefore, we recommend to include each morphologically identifiable tumor component in the mutational analysis to not overlook resistance-inducing or potentially targetable mutations.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Receptores ErbB/genética , Genes erbB-1 , Genes ras , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Estudos Retrospectivos
19.
Virchows Arch ; 471(4): 509-520, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28884371

RESUMO

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.


Assuntos
DNA Tumoral Circulante/análise , Análise Mutacional de DNA/normas , Receptores ErbB/análise , Técnicas de Genotipagem/normas , Garantia da Qualidade dos Cuidados de Saúde , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Alemanha , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética
20.
Pathol Res Pract ; 213(6): 606-611, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28551386

RESUMO

Evaluation of the RAS mutation status is necessary for patients with advanced colorectal cancer to predict the response to anti-EGFR therapy. In routine diagnostics, FFPE tissue samples are tested by sequencing (amplicon-based NGS and Sanger) to obtain the RAS status of the patient. Samples that are collected after chemotherapy occasionally contain necrotic tissue. Furthermore, colorectal cancer tissue sometimes has mucinous components. This may pose a challenge to molecular analysis because mucinous tumor samples often contain only few tumor cells compared to solid tumor samples. Therefore, the aim of this study was to explore if mucin or necrosis affect mutation analysis and if mucinous tumor samples contain enough tumor cells for reliable mutation detection. To this end, we analyzed KRAS status in 10 samples showing mucin production and 10 samples with necrosis. In all 20 samples the tissue areas with the highest amount of mucin or necrosis were used for re-evaluation. These results show no differences with those obtained during routine diagnostics, where analysis of mucinous or necrotic areas was tried to avoid. Our study thus shows that mucin and necrosis have no influence on KRAS mutation analysis. Furthermore we were able to demonstrate that mucinous adenocarcinoma contains enough tumor cells for a valid mutation analysis. In addition, we also observed only minor differences in KRAS status results when comparing Sanger sequencing with NGS. Both methods detected the KRAS mutation in 19 of 20 tested samples, even for mutated samples with low allele-frequencies.


Assuntos
Adenocarcinoma Mucinoso/patologia , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma Mucinoso/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Humanos , Mutação , Necrose/genética , Necrose/patologia , Estudos Retrospectivos
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