qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

<p>Abstract</p> <p>Background</p> <p>Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the <it>KIT </it>or <it>PDGFRA </it>gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of <it>KIT </it>as well as of the alternative receptor tyrosine kinase genes <it>FLT3</it>, <it>CSF1-R</it>, <it>PDGFRB</it>, <it>AXL </it>and <it>MET </it>by qPCR. wt-GIST were compared to samples with mutations in <it>KIT </it>exon 9 and 11 and <it>PDGFRA </it>exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST.</p> <p>Results</p> <p>Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes <it>POLR2A</it>, <it>PPIA</it>, <it>RPLPO </it>and <it>TFRC </it>were chosen for further analysis of the GIST samples. Overexpression of <it>KIT </it>compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with <it>PDGFRA </it>exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of <it>FLT3</it>, <it>CSF1R </it>and <it>AXL </it>were determined. An exception was the sample group with <it>KIT </it>exon 9 mutation. A significantly reduced expression of <it>CSF1R</it>, <it>FLT3 </it>and <it>PDGFRB </it>compared to the normal tissue was detected. GIST with mutations in <it>KIT </it>exon 9 and 11 and in <it>PDGFRA </it>exon 18 showed a significant <it>PDGFRB </it>downregulation.</p> <p>Conclusions</p> <p>As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST.</p

Expression of cell cycle regulators and frequency of TP53 mutations in high risk gastrointestinal stromal tumors prior to adjuvant imatinib treatment

Despite of multitude investigations no reliable prognostic immunohistochemical biomarkers in GIST have been established so far with added value to predict the recurrence risk of high risk GIST besides mitotic count, primary location and size. In this study, we analyzed the prognostic relevance of eight cell cycle and apoptosis modulators and of TP53 mutations for prognosis in GIST with high risk of recurrence prior to adjuvant treatment with imatinib. In total, 400 patients with high risk for GIST recurrence were randomly assigned for adjuvant imatinib either for one or for three years following laparotomy. 320 primary tumor samples with available tumor tissue were immunohistochemically analyzed prior to treatment for the expression of cell cycle regulators and apoptosis modulators cyclin D1, p21, p16, CDK4, E2F1, MDM2, p53 and p-RB1. TP53 mutational analysis was possible in 245 cases. A high expression of CDK4 was observed in 32.8% of all cases and was associated with a favorable recurrence free survival (RFS), whereas high expression of MDM2 (12.2%) or p53 (35.3%) was associated with a shorter RFS. These results were independent from the primary KIT or PDGFRA mutation. In GISTs with higher mitotic counts was a significantly increased expression of cyclin D1, p53 and E2F1. The expression of p16 and E2F1 significantly correlated to a non-gastric localization. Furthermore, we observed a significant higher expression of p21 and E2F1 in KIT mutant GISTs compared to PDGFRA mutant and wt GISTs. The overall frequency of TP53 mutations was low (n = 8; 3.5%) and could not be predicted by the immunohistochemical expression of p53. In summary, mutation analysis in TP53 plays a minor role in the subgroup of high-risk GIST before adjuvant treatment with imatinib. Strong expression of MDM2 and p53 correlated with a shorter recurrence free survival, whereas a strong expression of CDK4 correlated to a better recurrence free survival.Peer reviewe

Testing for deficient mismatch repair and microsatellite instability : A focused update

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021). Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education. // Der Nachweis der Mismatch-Reparatur-Defizienz (dMMR) mit konsekutiver hochgradiger Mikrosatelliteninstabilität (MSI-H) ist inzwischen fester Bestandteil der Diagnostik des kolorektalen Karzinoms (KRK). Galt MSI anfänglich als möglicher Indikator einer erblichen Krebsdisposition (Lynch-Syndrom, LS) steht heute die Vorhersage des Therapieansprechens auf Immuncheckpoint-Inhibitoren (ICI) im Vordergrund. Entsprechende Empfehlungen und Testalgorithmen liegen für den Einsatz in der Primärdiagnostik vor (Übersicht in: Rüschoff et al. 2021). Aufgrund des damit verbundenen routinemäßigen Einsatzes und des sich erweiternden Indikationsspektrums von ICI-Therapien für Nicht-KRK wie Endometrium‑, Dünndarm‑, Magen- und Gallenwegskarzinome wird eine aktualisierte Übersicht zur dMMR/MSI-Testung vorgelegt. Fokus sind die Herausforderungen bei der Beurteilung immunhistochemischer Färbungen und die Wertigkeit PCR-basierter Verfahren unter Berücksichtigung des erweiterten ICI-Indikationsspektrums. Anhand neuer Daten zur Häufigkeit und Art von Diskordanzen zwischen dMMR- und MSI-Befund und der möglichen Rolle von Next Generation Sequencing zu deren Aufklärung wird ein praxisorientiertes Diagramm zur Entscheidungsfindung im diagnostischen Alltag vorgestellt. Wir weisen zudem auf die Bedeutung systematischer Qualitätssicherungsmaßnahmen (z. B. QuIP MSI-Portal und Ringversuche) einschließlich einer regelmäßigen Fortbildung hin

Lung cancer as a paradigm for precision oncology in solid tumours

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the western world. However, the combination of molecular genotyping and subsequent systematic treatment of decoded target structures is a prime example of precision oncology in solid tumours. In this review, current targets of approved therapeutics and potential targets in clinical and preclinical trials are outlined. Furthermore, immune checkpoint inhibitors, as promising new therapeutic options, which have already been applied successfully in cases of lung cancer, are introduced. A major issue of targeted treatment of lung tumours is the persistent development of resistance. The underlying mechanisms and established and potentially applicable alternative therapeutic approaches are described. In this process of precision oncology, immunohistochemistry, fluorescence in situ hybridization, and parallel sequencing are crucial diagnostic tools

Therapeutic strategies in male breast cancer: Clinical implications of chromosome 17 gene alterations and molecular subtypes

Male breast cancer (MBC) is a rare disease. To date, therapy is mainly based on studies and clinical experiences with breast cancer in women. Only little is known about molecular typing of MBC, particularly with regard to potential biological predictors for adjuvant therapy. In female breast cancer tumors with chromosome 17 centromere (CEP17) duplication, HER2 and/or Topoisomerase II alpha (Topo II-alpha) gene alterations have been suggested to be associated with poor prognosis and increased sensitivity to anthracycline-containing regimens. In a well characterized cohort of 96 primary invasive MBC, we studied CEP17, HER2 and Topo II-alpha alterations by fluorescence in-situ hybridization (FISH), and expression of hormone receptors (HR), HER2 and Ki67 by immunohistochemistry to define molecular subtypes. Tumor characteristics and follow-up data were available and correlated with molecular findings. HER2 amplification and Topo II-alpha amplification/deletion were exceptionally rare in MBC (6.3% and 3.1%, respectively). CEP17 polysomy were found in 9.4% of tumors. HER2, Topo II-alpha and CEP17 gene alterations were not correlated to patients outcome. 96.9% of our cases were HR positive. Triple negative tumors were found in only 3.1% of the cases. In nodal negative tumors luminal A subtypes were significantly associated with better overall survival. Our results provide evidence for a predominant male breast cancer phenotype, characterized by HR expression and a lack of HER2/Topo II-alpha alterations and CEP17 duplicates. Therefore, the impact of anthracycline sensitivity linked to HER2/Topo II-alpha alterations as found in female breast cancer has low clinical significance for this specific male breast cancer phenotype. (C) 2013 Elsevier Ltd. All rights reserved