Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC) Good afternoon everyone. My talk today is on the work I carried out as part of the development of an EGFR and KRAS testing service for NSCLC at the Aberdeen lab. Joel Tracey1, Caroline Clark1, Christine Bell1, Keith Kerr2, Marianne Nicholson3, Aileen Osborne1, Zosia Miedzybrodzka1, Kevin Kelly1 1Department of Medical Genetics, Polwarth Building, Aberdeen Royal Infirmary, Aberdeen 2Department of Pathology, Aberdeen Royal Infirmary, Aberdeen 3Clinical Oncology, Aberdeen Royal Infirmary, Aberdeen

Non Small Cell Lung Cancer (NSCLC) Lung cancer is one of the most commonly diagnosed types of cancer in the UK Leading cause of cancer-related death in both men and women Non-Small Cell Lung Cancer ~ 80% (3) Adenocarcinoma (ADC) Squamous cell carcinoma (SCC) Large cell carcinoma (LCC) Percentage of NSCLC subtypes in UK In the UK, Lung cancer is the leading cause of cancer related death in both men and women Of the cases diagnosed 80% are NSCLC NSCLC can be divided into 3 histological subtypes: ADC, SCC, LCC

EGFR and KRAS in NSCLC Acquired mutations in the EGFR and KRAS genes are important in the development of NSCLC Mutations result in inappropriately activated proteins – tumour cells become ‘addicted’ to growth signals EGFR and KRAS mutations most common in adenocarcinoma EGFR and KRAS mutations are mutually exclusive EGFR and KRAS are two tyrosine kinases that are important in the development of NSCLC Both are involved in cell signaling pathways that control cell proliferation and survival Acquired mutations in the EGFR and KRAS genes can result in constitutively activated proteins which can lead to uncontrolled cancer cell growth These mutations most commonly occur in adenocarcinomas and are mutually exclusive

Treatment of NSCLC Surgery - possible in about 20% of cases (4) Cytotoxic chemotherapy and/or radiotherapy - mostly ineffective Survival rate poor (7% alive 5 years after diagnosis) (4) Tyrosine Kinase Inhibitors (TKI) – new type of chemotherapeutic agent – fewer side-effects than cytotoxic chemotherapy Target and block growth factor signals – e.g. Epidermal Growth Factor Receptor (EGFR) Current cancer treatments such as surgery, cytotoxic chemotherapy and radiotherapy are largely ineffective in treating NSCLC and as such the survival rate is low TKIs are a new type of treatment which could offer a significant improvement over conventional chemotherapeutics Many TKIs act by targeting and blocking growth factor signals such as those mediated by EGFR

EGFR Tyrosine Kinase Inhibitors Erlotinib (Tarceva) and Gefitinib (Irresa) EGFR targeted TKIs can be used for treatment of NSCLC patients with somatic activating EGFR mutations Mutations within EGFR TK domain enable TKIs to bind with greater affinity Patients with activating EGFR mutations have a better response to TKI therapy and improved survival TKI A number of EGFR TKIs have been developed Clinical trials have shown them to be effective specifically in treating patients with somatic activating mutations in the EGFR gene Mutation positive patients have a better initial response to TKI therapy and improved survival Conversely KRAS mutations are associated with a lack of response to TKI therapy Patients with KRAS mutations show little or no response to TKI treatment

Clinical Trial – IPASS Study EGFR mutation +ve (M+) patients respond better to TKI therapy than chemotherapy but.... EGFR mutation –ve patients (M-) have a poorer response to TKI therapy than chemotherapy!!! Probability of Progression Free Survival Time (months) The results of the recent IPASS clinical trial illustrate well the importance of knowing a patients EGFR mutation status This study compared the response of chemo-naïve EGFR mutation +ve and –ve patients to the EGFR TKI Gefitinib and the chemotherapeutic Carboplatin. The graph shows that EGFR +ve patients (solid lines) responded better to Gefitinib than Carboplatin However, the EGFR –ve patients (dashed lines) had a much poorer response to Gefitinib than Carboplatin This shows that is it important to identify all patients with EGFR mutations however it is equally important to avoid false positive results!

EGFR mutations Mutations in the EGFR gene are present in 10-15% of NSCLC patients And as the diagram on the left shows the mutations mainly occur between Ex 18 to 21 The majority of mutations are associated with sensitivity to TKIs - the two most common mutation types being exon 19 deletions and the L858R mutation in exon 21. Some EGFR mutations, mainly those in exon 20, are associated with resistance to TKIs KRAS mutations occur in about 30% of NSCLC patients and are mainly found in codons 12, 13 and 61 Mutations in the EGFR gene found in 10-15% of NSCLC patients (5, 6) Exon 19 deletions & L858R (Exon 21) make up 85-90% of all mutation +ve cases (7) Exon 20 mutations (e.g. T790M) commonly resistance mutations

KRAS mutations KRAS mutations occur in ~30% of NSCLC tumours (8) 12 13 61 C A G T Codons Wt seq Multi-variable mutations KRAS mutations occur in ~30% of NSCLC tumours (8) Codon 12 most common mutation site

Project Aims Develop and set-up methods for EGFR and KRAS analysis Determine best methods for analysis of EGFR and KRAS mutations Develop and validate appropriate methodologies for testing The aims of this project were to: Initially develop and set-up methods for EGFR and KRAS analysis Determine the best methods for analysis of EGFR and KRAS mutations And further develop and validate the appropriate methods for testing

Samples 48 Adenocarcinoma patient samples (ARI Pathology Dept) 4 EGFR +ve control samples (Holland) All were FFPE lung tumour samples (cores, slides & rolls) 14 DNA samples for KRAS analysis (Transgenomic Inc) Mutations in codon 12, 13 and 61 Varied mutation levels (3% to 33%) During this study we received 48 patient samples from the ARI Path dept as well as 4 EGFR +ve controls All of which were formalin fixed paraffin-embedded lung tumour samples We also received 14 DNA samples specifically for KRAS analysis from Transgenomic

Challenges with NSCLC testing using FFPE samples Frequently low sample quantity = Low DNA yield Variable tumour content within sample (<5% to 100%) Poor DNA quality - Degradation (<300bp), PCR inhibitors Genetic heterogeneity – inter- and intra-tumour variation Pathology departments involvement at this stage important to maximise % tumour – macro-dissection We found there were a number of challenges with NSCLC testing using FFPE samples: Low sample quantity, variable tumour content, poor DNA quality and tumour genetic heterogeneity can all impact on the ability to detect somatic mutations

Methodology Plan Extract DNA from FFPE lung tumour samples (Dewax, phenol/chloroform)   Quantify all DNA samples (Nanodrop) PCR amplification using specific primers (in-house/published) Quantify all DNA samples PCR amplification using specific primers EGFR Direct Sequencing WAVE HS dHPLC + fragment collection WAVE Surveyor Ex19 Fragment Length Analysis Ex21 Pyrosequencing KRAS Direct Sequencing WAVE Surveyor Pyrosequencing EGFR exons 18-21 and KRAS codons 12, 13 and 61 PCR products were subject to various methods of analysis In addition to direct sequencing, high sensitivity WAVE dHPLC and WAVE Surveyor methods, pyrosequencing and fragment length analysis

Principles of Methods Used WAVE HS dHPLC – Partially denaturing, High sensitivity by fluorescent detection (x100), mutation identified by presence of mutant/WT heteroduplex peaks Fragment collection – After passing through detector eluted DNA fragments were collected in vials at 30s intervals WAVE Surveyor – Enzymatic method, detects DNA mismatches, WAVE size separation, mutation identified by presence of cleavage products In the WAVE Surveyor method mismatches in mutant/WT heteroduplexes are cleaved by a restriction enzyme and by using size separation on the WAVE system mutations are identified by the presence of smaller cleavage products The high sensitivity WAVE dHPLC method uses fluorescent labeling and detection of DNA to give greater sensitivity than the standard UV detection. In this partially denaturing method mutants are detected by the presence of heteroduplexes

Principles of Methods Used Fragment Length Analysis – FAM labelled PCR products, size separation on ABI 3130, analysis using Gene Marker software Pyrosequencing – Real-time sequence data, Pyrophosphate (PPi) substrate for reaction cascade, light produced measured – relative to nucleotides incorporated Fragment length analysis was performed by using FAM-labeled primers for PCR. The labeled PCR products were then size separated on the ABI 3130 The pyrosequencing method is a real-time semi-quantitative sequencing method which utilises the pyrophosphate generated during nucleotide incorporation as a substrate for an enzymatic reaction. Light is produced which is relative to the number of nucleotides incorporated.

Summary of KRAS Results Blind study (Transgenomic samples) Pyrosequencer detected all mutations in Transgenomic samples (lowest = 3%) 2 samples not detected by the WAVE Surveyor method (3%) 6 samples were below the detection limit of sequencing (<10%) 33% (16/48) of patient samples positive for KRAS mutations – tested by both pyrosequencing and direct sequencing The results from the KRAS part of this study confirmed that the pyrosequencer is currently the best method available for KRAS analysis…..something which I think many labs have already found It was more sensitive and quicker than both the WAVE surveyor method and direct sequencing 33% of the patients tested were positive for a KRAS mutation which is consistent with published figures Percentage of KRAS mutations identified (by codon)

EGFR Results Sample: EGFR +ve Control Mutation: Exon 19 Del (c.2240-2254del; p.L747 – T751del) +ve control Sequencing WT 267 298 257 Size control +ve control WT 174 Uncleaved product WAVE Surveyor 102 80 This is an example of the EGFR results we obtained with the different methods trialed All three results show the same exon 19 deletion The mutation is indicated by the presence of additional low level sequence in the sequencing result, The heteroduplex peak in the WAVE dHPLC result, And the presence of cleavage products in the WAVE Surveyor result +ve control WT WAVE HS dHPLC

Enrichment of EGFR mutant by WAVE dHPLC + fragment collection Ex 19 WT Ex 19 del (direct seq) The following results we feel show that its possible to enrich for mutant fragments using fragment collection in conjunction with the WAVE dHPLC system The top chromatogram is the wild-type reference sequence. The middle chromatogram is the direct sequencing result for an exon 19 deletion mutant, the mutant sequence is visible as low level peaks The bottom chromatogram is for the same sample sequenced after fragment collection and repeat PCR. We can see the level of the deletion mutant sequence has been increase enough that the mutation surveyor software has identified and indicated the mutation Ex 19 del (enriched by fragment collection + repeat PCR) Sequences analysed with Mutation Surveyor software (Soft Genetics)

Additional methods Ex 19 Fragment length analysis Ex 19 del Ex 19 WT Ex 19 Fragment length analysis G863D L861Q L858R The other methods tested looked specifically at one exon each. Fragment analysis was used to detect exon 19 deletions only. The smaller deletion fragment is indicated by the red arrow in this case. The pyrosequencing method developed was used to detect exon 21 mutations. In this result we can see an L858R mutation at a level of about 28% in the sample Ex 21 Pyro-sequencing L858R mutant

Summary of EGFR Results 12.5% (6/48) of patient samples positive for EGFR mutation (Ex 19 - 3 Deletions; Ex 20 - 1 Insertion; Ex 21 - 2 L858R) All methods detected Ex19 del mutations in 4 EGFR +ve control samples WAVE Surveyor confirmed all mutations found by direct sequence analysis One Ex19 del mutant too low to report by direct sequence analysis but clear by WAVE Surveyor and Fragment length analysis Pyrosequencer – successfully detected Ex21 mutants Confident no false positive results All EGFR +ve samples were KRAS –ve Mutations confirmed by multiple methods To summarise the EGFR results: We found 6 out of 48 patients tested had an EGFR mutation All methods detected the mutations in the positive control samples The WAVE surveyor method successfully detected all mutations found by direct sequencing In addition one mutation missed by direct sequence analysis was detected by WAVE surveyor and fragment analysis methods which would indicate these methods to be more sensitive than direct sequencing The pyrosequencing method successfully detected all exon 21 mutants previously found Overall we feel confident that no false positive results would have been reported as: All EGFR pos samples were confirmed as KRAS neg and the results were confirmed by multiple methods

Comparison of EGFR methods WAVE HS dHPLC SURVEYOR Sequencing Fragment analysis (Ex19 only) Pyrosequencing (Ex 21 only) Hands on time * 2hr 30min 2hr 45min 1hr 30min 2hr 15min Cost (per sample) £16.50 £15.60 £26.70 £0.57 £8.04 Results analysis time* 45min 30min Total Time to result * 40hr 40min 24hr 45min 11hr 15min 6hr 4hr 35min Sample required 120ng 20ng Detection Limit ? ~4-5% ~10% 3-5% A comparison of the performance and costs of each method was also made. The key findings were: There is little difference in hands on times and sample requirements for the various methods However compared to sequencing, the WAVE methods cost less per sample and results analysis time is less Fragment analysis is an inexpensive and quick method of screening EGFR exon 19 Pyrosequencing is more expensive per sample but it is a quick and sensitive method for EGFR exon 21 analysis Times based on analysis of 15 samples Cost per sample does not include staff costs

Conclusions Pick-up rate of EGFR mutations consistent with published data Direct sequencing pick-up rate higher than expected. This likely to be due to enrichment of samples for tumour tissue by macro-dissection WAVE Surveyor, fragment analysis and pyrosequencing methods may be useful as a higher sensitivity screen in conjunction with direct sequencing Fragment collection is a viable method for enrichment of low level mutations To conclude: The overall pick-up rate of EGFR mutations was consistent with published data The direct sequencing pick-up rate was higher than expected as only one mutation was missed but this could likely be due to enrichment of the samples by macro-dissection prior to genetic analysis The WAVE surveyor, fragment analysis and pyrosequencing methods are all promising and may be useful as higher sensitivity screening methods in conjunction with direct sequencing Our results also suggest fragment collection could be a viable method for enrichment of low level mutants

Current Testing Strategy NSCLC Patient (M/F, smoker/non-smoker) SCC / LCC Adenocarcinoma Not Tested Assessment of tumour content and macrodissection Pathology Molecular Genetics EGFR Ex18- 21 PCR KRAS codons 12, 13 and 61 PCR Finally, this chart shows the current EGFR/KRAS testing strategy at the Aberdeen lab. Adenocarcinomas are selected and macro-dissected in pathology and then tested for EGFR and KRAS mutations in parallel by various methods for each gene. Parallel testing by multiple methods is being employed to minimise turn-around times but also for greater confidence in the results Direct Sequencing /Pyrosequencing Direct Sequencing/WAVE Surveyor/Fragment Length Analysis Report

Acknowledgements Aberdeen Lab Clinical/Pathology Caroline Clark Christine Bell Aileen Osborne Louise Carnegie Heather Greig Kevin Kelly Transgenomic Gerald Martin Clinical/Pathology Keith Kerr Marianne Nicholson Zosia Miedzybrodzka Astra Zeneca For providing funding

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