Michaela Angelika Ihle

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.

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.

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.

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.

Clinicopathological Characteristics of RET Rearranged Lung Cancer in European Patients

Introduction Rearrangements of RET are rare oncogenic events in patients with non–small cell lung cancer (NSCLC). While the characterization of Asian patients suggests a predominance of nonsmokers of young age in this genetically defined lung cancer subgroup, little is known about the characteristics of non‐Asian patients. We present the results of an analysis of a European cohort of patients with RET rearranged NSCLC. Methods Nine hundred ninety‐seven patients with KRAS/EGFR/ALK wildtype lung adenocarcinomas were analyzed using fluorescence in situ hybridization for RET fusions. Tumor specimens were molecularly profiled and clinicopathological characteristics of the patients were collected. Results Rearrangements of RET were identified in 22 patients, with a prevalence of 2.2% in the KRAS/EGFR/ALK wildtype subgroup. Co‐occurring genetic aberrations were detected in 10 patients, and the majority had mutations in TP53. The median age at diagnosis was 62 years (range, 39–80 years; mean ± SD, 61 ± 11.7 years) with a higher proportion of men (59% versus 41%). There was only a slight predominance of nonsmokers (54.5%) compared to current or former smokers (45.5%). Conclusions Patients with RET rearranged adenocarcinomas represent a rare and heterogeneous NSCLC subgroup. In some contrast to published data, we see a high prevalence of current and former smokers in our white RET cohort. The significance of co‐occurring aberrations, so far, is unclear.

MET Gene Copy Number Alterations and Expression of MET and Hepatocyte Growth Factor Are Potential Biomarkers in Angiosarcomas and Undifferentiated Pleomorphic Sarcomas

Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry.

Oncogene and therapeutic target analyses in atypical fibroxanthomas and pleomorphic dermal sarcomas.

Background Until now, almost nothing is known about the tumorigenesis of atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS). Our hypothesis is that AFX is the non-infiltrating precursor lesion of PDS. Materials and Methods We performed the world-wide most comprehensive immunohistochemical and mutational analysis in well-defined AFX (n=5) and PDS (n=5). Results In NGS-based mutation analyses of selected regions by a 17 hotspot gene panel of 102 amplicons we could detect TP53 mutations in all PDS as well as in the only analyzed AFX and PDS of the same patient. Besides, we detected mutations in the CDKN2A, HRAS, KNSTRN and PIK3CA genes. Performing immunohistochemistry for CTNNB1, KIT, CDK4, c-MYC, CTLA-4, CCND1, EGFR, EPCAM, ERBB2, IMP3, INI-1, MKI67, MDM2, MET, p40, TP53, PD-L1 and SOX2 overexpression of TP53, CCND1 and CDK4 was seen in AFX as well as in PDS. IMP3 was upregulated in 2 AFX (weak staining) and 4 PDS (strong staining). FISH analyses for the genes FGFR1, FGFR2 and FGFR3 revealed negative results in all tumors. Conclusions UV-induced TP53 mutations as well as CCND1/CDK4 changes seem to play essential roles in tumorigenesis of PDS. Furthermore, we found some more interesting mutated genes in other oncogene pathways (activating mutations of HRAS and PIK3CA). All AFX and PDS investigated immunohistochemically presented with similar oncogene expression profiles (TP53, CCND1, CDK4 overexpression) and the single case with an AFX and PDS showed complete identical TP53 and PIK3CA mutation profiles in both tumors. This reinforces our hypothesis that AFX is the non-infiltrating precursor lesion of PDS.

Clinicopathological and molecular features of a large cohort of gastrointestinal stromal tumors (GISTs) and review of the literature: BRAF mutations in KIT/PDGFRA wild-type GISTs are rare events

In KIT/PDGFRA wild-type gastrointestinal stromal tumors (wt-GISTs), BRAF mutations are regarded as alternative pathogenic events driving tumorigenesis. In our study, we aimed at analyzing a large cohort (n=444) of GISTs for BRAF mutations using molecular and immunohistochemical methods. More than 3000 GIST samples from caucasian patients were available in our GIST and Sarcoma Registry NRW. Of these, we selected 172 wt-GISTs to evaluate the frequency of BRAF mutations. Furthermore, 272 GISTs with a representative KIT and PDGFRA mutational status were selected. BRAF mutational status was evaluated by high-resolution melting analysis, Sanger sequencing, and VE1 immunohistochemistry. A BRAF mutation (p.V600E) was found in 7 cases (3.9%) of the wt-GIST cohort. In 2 cases, multiple synchronous tumors harbored the same somatic BRAF mutation. VE1 immunohistochemical staining had a sensitivity of 81.8% and a specificity of 97.5% to detect BRAF p.V600E mutations. Analyzing our cases and the cases reported in the literature (n=37), the percentage of intermediate and high-risk BRAF-mutated wt-GISTs (17/31; 54.8%) was comparable to that recorded for large GIST cohorts irrespective of the mutational status. BRAF mutations are rare events in wt-GISTs, and VE1 immunohistochemistry appears to be a valuable pre-screening tool for the detection of BRAF p.V600E mutations. BRAF mutations in GISTs do not seem to have a prognostic value per se. However, as BRAF inhibition represents a therapeutic option to control disease, we suggest the assessment of the BRAF mutational status, especially in the setting of advanced GIST disease.

EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.