Cellular immune responses against the tumour associated polymorphic epithelial mucin MUC1

The human epithelial mucin MUC1 is expressed on the apical surface of a large number of normal epithelial tissues, including the ducts of the breast, ovary, pancreas, lung and colon. Adenocarcinomata that arise from these tissues can show marked over expression of MUC1, as well as aberrant glycosylation and loss of apical distribution of this mucin. These observations led to the attempt to use MUC1 a potential target for tumour immunotherapy. The work described in this thesis aims to evaluate cellular immune responses directed against the MUC1 antigen, using mouse model systems. The project addresses fundamental questions relating to the feasibility of using MUC1-based antigens for tumour immunotherapy, and has led to the identification of MHC class I-restricted epitopes both in mice and in humans; that may have clinical relevance. Firstly, the induction of MUC1-specific CTL lines and their characterisation in a C57BL/6 mouse model is described. This includes mapping of the dominant CTL epitope recognised by MUC1-specific CTL and analysis of anti-tumour effects of these CTL in vivo. Adoptive transfer of CTL cultured in vitro into MUC1 transgenic mice, where human MUC1 is expressed as a self antigen, has allowed evaluation of immunogenicity and tumour protection in a setting in which autoimmunity may occur. The results demonstrate that the host can tolerate effective anti-tumour immune responses to MUC1 without induction of autoimmunity. The second major part of the work aimed to identify human MHC class I-restricted MUC1-derived epitopes. The merits of the different possible approaches for epitope identification are discussed and the identification of HLA A*0201-restricted epitopes is described. Potential MHC binding peptides were identified by motif scoring. These peptides were then analysed for binding and complex stability with HLA-A*0201. The immunogenicity and physiological relevance of these peptides is shown by induction of CTL responses and tumour protection in a transgenic mouse model expressing HLA A*0201-Kb

Effectiveness of crizotinib in a patient with ALK IHC-positive/FISH-negative metastatic lung adenocarcinoma.

We report a case of crizotinib effectiveness in a heavily pretreated patient with a metastatic NSCLC initially considered IHC-positive and FISH-negative for ALK rearrangement. After repeated analyses of tumor samples, borderline ALK FISH-positivity (18.5% positive cells) was demonstrated

Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics

Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting.Here, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling.High-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry.In summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis

Impact of DNA quantification methods on massively parallel sequencing.

<p>(A) Mean amplicon coverage determined by in-house software for each sample, quantification method and amount of DNA used for multiplex PCR amplification. (B) Number of all variants called by in-house software for each sample, quantification method and starting material used.</p

Comparison of five automated DNA extraction systems.

<p>Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.</p

Impact of DNA quantification methods on downstream applications.

<p>(A) Electrophoretic pattern of 13 DNA extracts on a 1% agarose gel. (B) Amplified 275 bp fragment of the <i>EGFR</i> gene. 20 ng of sample DNA determined by each quantification method was used for PCR amplification. + indicates a positive control, - a negative control.</p

Assessment of DNA quality obtained from each extraction method and impact on downstream applications.

<p>(A) Electrophoretic pattern of DNA extracts 1, 3, 4 and 5 of each extraction method on a 1% agarose gel. Ladder indicates a 1 kb DNA ladder as molecular weight size marker (B) 201 bp, 404 bp and 614 bp amplified DNA fragments of the <i>GAPDH</i> gene for sample 1, 3, 4, 5, of each extraction method. + indicates a positive control and – a negative control. (C) 125–175 bp multiplex PCR product for sample 1, 3, 4 and 5 of each extraction method. + indicates a positive control and – a negative control.</p

Sequences and product sizes of PCR and qPCR primers.

<p>F: Forward.</p><p>R: Reverse.</p