Jana Fassunke

Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

BackgroundThe approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.MethodsSamples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).ResultsThere was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.ConclusionTherefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

Tumor recurrence and malignant progression of gangliogliomas.

Most gangliogliomas (GGs) are benign tumors, but tumor recurrence and malignant progression are observed in some patients.

Heterogeneous Mechanisms of Primary and Acquired Resistance to Third-Generation EGFR Inhibitors.

Purpose: To identify novel mechanisms of resistance to third-generation EGFR inhibitors in patients with lung adenocarcinoma that progressed under therapy with either AZD9291 or rociletinib (CO-1686). Experimental Design: We analyzed tumor biopsies from seven patients obtained before, during, and/or after treatment with AZD9291 or rociletinib (CO-1686). Targeted sequencing and FISH analyses were performed, and the relevance of candidate genes was functionally assessed in in vitro models. Results: We found recurrent amplification of either MET or ERBB2 in tumors that were resistant or developed resistance to third-generation EGFR inhibitors and show that ERBB2 and MET activation can confer resistance to these compounds. Furthermore, we identified a KRASG12S mutation in a patient with acquired resistance to AZD9291 as a potential driver of acquired resistance. Finally, we show that dual inhibition of EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells expressing mutant KRAS. Conclusions: Our data suggest that heterogeneous mechanisms of resistance can drive primary and acquired resistance to third-generation EGFR inhibitors and provide a rationale for potential combination strategies. Clin Cancer Res; 22(19); 4837–47. ©2016 AACR.

Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients

Introduction: The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. Methods: In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. Results: We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. Conclusion: Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

MET amplification status in therapy-naïve adeno- and squamous cell carcinomas of the lung

Purpose: MET is a potential therapeutic target in lung cancer and both MET tyrosine kinase inhibitors and monoclonal antibodies have entered clinical trials. MET signaling can be activated by various mechanisms, including gene amplification. In this study, we aimed to investigate MET amplification status in adeno- and squamous cell carcinomas of the lung. We propose clearly defined amplification scores and provide epidemiologic data on MET amplification in lung cancer. Experimental Design: We evaluated the prevalence of increased MET gene copy numbers in 693 treatment-naïve cancers by FISH, defined clear cutoff criteria, and correlated FISH results to MET IHC. Results: Two thirds (67%) of lung cancers do not have gains in MET gene copy numbers, whereas 3% show a clear-cut high-level amplification (MET/centromer7 ratio ≥2.0 or average gene copy number per nucleus ≥6.0 or ≥10% of tumor cells containing ≥15 MET copies). The remaining cases can be subdivided into intermediate- (6%) and low-level gains (24%). Importantly, MET amplifications occur at equal frequencies in squamous and adenocarcinomas without or with EGFR or KRAS mutations. Conclusion: MET amplification is not a mutually exclusive genetic event in therapy-naïve non–small cell lung cancer. Our data suggest that it might be useful to determine MET amplification (i) before EGFR inhibitor treatment to identify possible primary resistance to anti-EGFR treatment, and (ii) to select cases that harbor KRAS mutations additionally to MET amplification and, thus, may not benefit from MET inhibition. Furthermore, our study provides comprehensive epidemiologic data for upcoming trials with various MET inhibitors. Clin Cancer Res; 21(4); 907–15. ©2014 AACR.

Comprehensive Characterization of Genomic Aberrations in Gangliogliomas by CGH, Array-based CGH and Interphase FISH

Gangliogliomas are generally benign neuroepithelial tumors composed of dysplastic neuronal and neoplastic glial elements. We screened 61 gangliogliomas [World Health Organization (WHO) grade I] for genomic alterations by chromosomal and array‐based comparative genomic hybridization (CGH). Aberrations were detected in 66% of gangliogliomas (mean ± SEM = 2.5 ± 0.5 alterations/tumor). Frequent gains were on chromosomes 7 (21%), 5 (16%), 8 (13%), 12 (12%); frequent losses on 22q (16%), 9 (10%), 10 (8%). Recurrent partial imbalances comprised the minimal overlapping regions dim(10)(q25) and enh(12)(q13.3–q14.1). Unsupervised cluster analysis of genomic profiles detected two major subgroups (group I: complete gain of 7 and additional gains of 5, 8 or 12; group II: no major recurring imbalances, mainly losses). A comparison with low‐grade gliomas (astrocytomas WHO grade II) showed chromosome 5 gain to be significantly more frequent in gangliogliomas. Interphase fluorescence in situ hybridization (FISH) identified the aberrations to be contained in a subpopulation of glial but not in neuronal cells. Two gangliogliomas and their anaplastic recurrences (WHO grade III) were analyzed. Losses of CDKN2A/B and DMBT1 or a gain/amplification of CDK4 found in the anaplastic tumors were already present in the respective gangliogliomas by array CGH and interphase FISH. In summary, genomic profiling in a large series of gangliogliomas could distinguish genetic subgroups even in this low‐grade tumor.

In situ -RT and immunolaser microdissection for mRNA analysis of individual cells isolated from epilepsy-associated glioneuronal tumors

Analysis of gene transcription patterns in complex tissues with multiple cell types is a major challenge. Examination of cellular subpopulations for molecular expression patterns requires their isolation from other surrounding cells. We performed single-cell mRNA analysis to study gangliogliomas obtained from patients with pharmacoresistant epilepsy (n=6), in order to characterize CD34 expressing cells found in these tumors. Fresh-frozen biopsy tissue was analyzed by initial in situ-reverse transcription (in situ-RT) with oligonucleotides, subsequent immunohistochemistry (IHC) to identify specific cell types, and laser-capture microdissection (LCM, herein termed immuno-LCM) to obtain antigen-expressing cell subpopulations. Isolated complementary DNAs (cDNAs) were then quantified by real time-polymerase chain reaction (RT-PCR). We found that short- vs long-term incubation time for the IHC step did not adversely affect cDNA abundance obtained by subsequent RT-PCR, either for high-abundance (glyceraldehyde dehydrogenase; GAPDH), medium-abundance (glial fibrillary acidic protein; GFAP), or low abundance (neurofilament; NFM) gene transcripts. We also determined that the cellular specificity of capture was excellent, as determined by lack of contamination between different immuno-LCM cell isolates. We were therefore able to examine the lineage expression markers of isolated CD34-expressing cells. We observed coexpression of CD34 and NFM, suggesting neuronal differentiation of the CD34 expressing cellular elements in gangliogliomas. Expression markers for other cellular types (myelin basic protein for oligodendroglia; GFAP for astrocytes) were negative. Our findings support the hypothesis that gangiogliomas contain neuronal elements with compromised or atypical differentiation. We consider that this in situ-RT/immuno-LCM protocol is of general applicability, whereby virtually any primary antibody can be used to facilitate capture of individual cells in tissue sections for molecular analysis.

miRNA-221 and miRNA-222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours.

Aberrantly expressed microRNAs (miRNAs) are involved in many diseases including cancer. In gastrointestinal stromal tumours (GISTs) expression of miR‐221 and miR‐222 is reduced compared to control tissue and other sarcomas but the functional effects of this downregulation are not fully understood. This study aimed at evaluating the miR‐221 and miR‐222 expression profiles in different GIST subtypes and the functional role of these miRNAs. Expression of miR‐221 and miR‐222 was analysed in six KIT exon 9 and three KIT exon 11 mutated and nine wildtype GISTs by qPCR. Viability and apoptosis were examined in three different, KIT positive GIST cell lines (GIST882, GIST‐T1 and GIST48) after overexpression of these miRNAs. The modulation of KIT and the PI3K/AKT pathways was determined by Western blot. Wildtype and KIT mutated GISTs revealed reduced miRNA expression compared to adequate control tissue. miRNA expression was lower for wildtype compared to mutated GISTs. Transient transfection of miR‐221 and miR‐222 reduced viability and induced apoptosis by inhibition of KIT expression and its phosphorylation and activation of caspases 3 and 7 in all three GIST cell lines. p‐AKT, AKT and BCL2 expression was reduced after miRNA transfection whereas only slight influence on p‐MTOR, MTOR and BCL2L11 (BIM) was detected. Our results demonstrate that miR‐221 and miR‐222 which are downregulated in wildtype and mutated GISTs, induce apoptosis in vitro by a signalling cascade involving KIT, AKT and BCL2. Therefore, overexpression of these miRNAs seems to functionally counteract oncogenic signalling pathways in GIST.

The reelin pathway components disabled-1 and p35 in gangliogliomas – a mutation and expression analysis

Focal epilepsies in young patients are frequently associated with differentiated glioneuronal tumours. Dysplastic neurones represent a characteristic neuropathological feature of gangliogliomas, the most common entity encountered in this group. Here, we have analysed two major components of the reelin pathway involved in neuronal migration and cortical development, that is, p35 and disabled‐1 (dab1), in gangliogliomas. Genomic structures of human dab1 and p35 were identified ‘in silico’ using the HTGS databank, NCBI BLAST 2.1. DNA sequence analysis was carried out in gangliogliomas obtained from 29 epilepsy patients vs. peripheral blood DNA from non‐affected control individuals (n = 100). Gene expression of dab1 and p35 was determined by real‐time RT‐PCR (reverse transcriptase polymerase chain reaction) in gangliogliomas (n = 14) vs. non‐neoplastic central nervous system tissue (n = 20). The human dab1 gene contains 13 coding exons and is located on chromosome 1p31–32. A single coding exon constitutes the human p35 gene, which is located on chromosome 17q11.2. A novel homologueous genomic region on chromosome 2 has to be taken into account for future studies on p35. One ganglioglioma patient showed a unique polymorphism in the p35 gene. The single base exchange (C to A) at nucleotide 904 of the p35 cDNA (GenBank X80343, start ATG, codon 302) results in a leucine–isoleucine amino acid substitution. No mutations of the dab1 and p35 genes in gangliogliomas were observed. However, significantly lower levels of dab1 and p35 gene transcripts were detected in gangliogliomas compared to controls (dab1 28.24%, t‐test P < 0.001; p35 21.28%, t‐test P < 0.001, in gangliogliomas vs. controls). Our data suggest that mutational events of dab1 and p35 are not involved in the molecular pathogenesis of gangliogliomas. A potential functional role of these developmentally regulated genes for the formation of epileptogenic glioneuronal lesions remains to be elucidated.

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.