Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.

Data on intratumoral heterogeneity of small intestine neuroendocrine tumors (SI-NETs) and related liver metastasis are limited. The aim of this study was to characterize genetic heterogeneity of 5 patients with SI-NETs. Therefore, formalin-fixed, paraffin-embedded tissue samples of primary and metastatic lesions as well as benign liver of five patients with synchronously metastasized, well differentiated SI-NETs were analyzed with whole exome sequencing. For one patient, chip based 850k whole DNA methylome analysis was performed of primary and metastatic tumor tissue as well as control tissue. Thereby, 156 single nucleotide variants (SNVs) in 150 genes were identified and amount of mutations per sample ranged from 9–34 (mean 22). The degree of common (0–94%) and private mutations per sample was strongly varying (6–100%). In all patients, copy number variations (CNV) were found and the degree of intratumoral heterogeneity of CNVs corresponded to SNV analysis. DNA methylation analysis of a patient without common SNVs revealed a large overlap of common methylated CpG sites. In conclusion, SI-NET primary and metastatic lesions show a highly varying degree of intratumoral heterogeneity. Driver events might not be detectable with exome analysis only, and further comprehensive studies including whole genome and epigenetic analyses are warranted.

Intratumoral heterogeneity of intrahepatic cholangiocarcinoma Intratumoral heterogeneity of intrahepatic cholangiocarcinoma

Splenic marginal zone lymphoma (SMZL) is an indolent B-cell non-Hodgkin lymphoma and represents the most common primary malignancy of the spleen. Its precise molecular pathogenesis is still unknown and specific molecular markers for diagnosis or possible targets for causal therapies are lacking. We performed whole exome sequencing (WES) and copy number analysis from laser-microdissected tumor cells of two primary SMZL discovery cases. Selected somatic single nucleotide variants (SNVs) were analyzed using pyrosequencing and Sanger sequencing in an independent validation cohort. Overall, 25 nonsynonymous somatic SNVs were identified, including known mutations in the NOTCH2 and MYD88 genes. Twenty-three of the mutations have not been associated with SMZL before. Many of these seem to be subclonal. Screening of 24 additional SMZL for mutations at the same positions found mutated in the WES approach revealed no recurrence of mutations for ZNF608 and PDE10A, whereas the MYD88 L265P missense mutation was identified in 15 % of cases. An analysis of the NOTCH2 PEST domain and the whole coding region of the transcription factor SMYD1 in eight cases identified no additional case with a NOTCH2 mutation, but two additional cases with SMYD1 alterations. In this first WES approach from microdissected SMZL tissue we confirmed known mutations and discovered new somatic variants. Recurrence of MYD88 mutations in SMZL was validated, but NOTCH2 PEST domain mutations were relatively rare (10 % of cases). Recurrent mutations in the transcription factor SMYD1 have not been described in SMZL before and warrant further investigation.