Attenuated isolated 3 ' signal: a highly challenging therapy relevant ALK FISH pattern in NSCLC

Objectives: Targeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene rearrangement has great predictive value for treatment with small molecule tyrosine kinase inhibitor (crizotinib and alectinib commonly). Fluorescent in situ hybridisation (FISH) assay is a basic diagnostic test designed for detecting ALK gene rearrangements. Although being considered as gold standard method by IASLC's guideline, it is often regarded as difficult and error prone. Our aim was to examine a unique atypical ALK FISH pattern, revealed during a systematic large-scale monitoring, which carries the great risk of misinterpretation, hence may result in loss of patients eligible for targeted therapy.Materials and Methods: Tissue and cytology samples from nearly one thousand patients with advanced stage non-small cell lung cancer (NSCLC, n = 996) were routinely examined by ALK FISH and immunohistochemistry (Ventana ALK-D5F3-CDx assay). Anchored Multiplex PCR based Next Generation Sequencing (AMP-NGS) was used to detect fusion gene transcripts in ambiguous cases.Results: Fifty-nine (5,9%) of the cases were positive with ALK FISH test. Three cases showed atypical pattern with a significantly reduced sized red (3') signal and complete loss of green signals. Digital signal measurement confirmed this finding, showing consistent attenuation of 3' signals throughout the tumours. In all three cases AMP-NGS and ALK IHC verified the presence of a fusion gene and expressed oncoprotein, respectively.Conclusion: Approximately 5% of the 59 ALK positive cases exhibited atypical attenuated isolated 3' signal pattern. The immunohistochemistry and AMP-NGS examinations helped to clarify the presence of oncoprotein and the fusion gene, respectively. Our results emphasize the importance of extensive exploration of the genetic background of any unexpected FISH finding to avoid false diagnosis. This enables clinicians to indicate the adequate therapy with higher efficiency for patients suffering from NSCLC

Primary and secondary clarithromycin resistance in Helicobacter pylori and mathematical modeling of the role of macrolides

Clarithromycin is a macrolide antibiotic widely used for eradication of Helicobacter pylori infection, and thus resistance to this antibiotic is a major cause of treatment failure. Here, we present the results of a retrospective observational study of clarithromycin resistance (Cla-res) in 4744 H. pylori-infected patients from Central Hungary. We use immunohistochemistry and fluorescence in situ hybridization on fixed gastric tissue samples to determine H. pylori infection and to infer Cla-res status, respectively. We correlate this information with macrolide dispensing data for the same patients (available through a prescription database) and develop a mathematical model of the population dynamics of Cla-res H. pylori infections. Cla-res is found in 5.5% of macrolide-naive patients (primary Cla-res), with no significant sex difference. The model predicts that this primary Cla-res originates from transmission of resistant bacteria in 98.7% of cases, and derives from spontaneous mutations in the other 1.3%. We find an age-dependent preponderance of female patients among secondary (macrolide-exposed) clarithromycin-resistant infections, predominantly associated with prior use of macrolides for non-eradication purposes. Our results shed light into the sources of primary resistant cases, and indicate that the growth rate of Cla-res prevalence would likely decrease if macrolides were no longer used for purposes other than H. pylori eradication

Dreidimensionale Rekonstruktion solider Tumoren

In histopathological routine diagnostics, three-dimensional tissue samples are analyzed histologically and/or immunohistochemically in two-dimensional sectional planes due to the high expenditure of time and the lack of digitization possibilities.Here, we demonstrate the application of three-dimensional reconstruction to solid tumors and analyze inter-/intratumoral heterogeneity with respect to epithelial-mesenchymal transition (EMT).Tissue samples from pancreatic, lung, colorectal, and breast cancers as well as colorectal liver metastases were serially processed in 4μm sections. For individual analyses, alternating stains (cytokeratin AE1/3, zinc finger E‑box-binding homeobox 1 (ZEB1), eCadherin) were performed. Subsequently, the tumor cells were analyzed for their morphology (epitheloid amoeboid, mesenchymal) and the expression of ZEB1 and eCadherin. For statistical analysis, all tumor cell aggregates were hierarchically annotated and analyzed.Tumor buds are predominantly associated with the main tumor mass. Furthermore, a shutteling of eCadherin could be observed within tumor cell aggregates smaller than nine cells. ZEB1 is only increasingly expressed in tumor cell groups smaller than five cells.The initial tumor budding and the subsequent decoupling of the tumor bud from the main tumor mass is most likely a two-part process. However, the EMT is not statistically significantly increased within the tumor bud detached from the main tumor mass. It could be shown that the currently valid and known definition of a tumor bud as a cell cluster of less than or equal to five cells cannot be completely classified in the concept of EMT represented by eCadherin and ZEB1