Therapy-orienting testing of EGFR inhibitor-resistant non-small cell lung cancer

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1 226 Therapy-orienting testing of EGFR inhibitor-resistant non-small cell lung cancer C. Dooms, MD, PhD 1, B. Colinet, MD 2, I. Demedts, MD, PhD 3, N. D Haene, MD, PhD 4, V. Ninane, MD, PhD 5, T. Pieters, MD 6, J. Vansteenkiste, MD, PhD 1, B. Weynand, MD 7, P. Pauwels, MD, PhD 8 SUMMARY Somatic sensitising mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are detected in approximately 10% of patients with advanced non-squamous non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (EGFR-TKIs) are the first-line treatment option for patients with an actionable EGFR mutation. Despite initial responses, the majority of patients progress within one to two years after EGFR-TKIs treatment initiation. The most common mechanism of resistance is the development of an additional EGFR-T790M mutation in exon 20, found in 50 60% of EGFR-mutant NSCLC patients who were rebiopsied on EGFR-TKI treatment. Phase II and III trials with osimertinib, a third-generation EGFR-TKI, demonstrated an objective response rate (ORR) of 60 70% and median progression-free survival (mpfs) of months in EGFR-T790M-positive tumours. A tissue biopsy of a progressing lesion for confirmation of histology and molecular characterisation is a critical consideration. However, a repeat tissue biopsy is not possible for every patient. Therefore, a liquid biopsy can be considered for EGFR-T790M mutation testing. Indeed, clinical trials testing osimertinib have shown similar clinical outcomes (ORR and mpfs on osimertinib) in patients with T790M-positive plasma versus T790M-positive tumour tissue. Osimertinib clearly expands relapse treatment options for advanced stage EGFR-mutant NSCLC. Testing for 1 Department of Respiratory Diseases, University Hospitals KU Leuven, Leuven, Belgium, 2 Department of Pneumology, Grand Hôpital de Charleroi, Charleroi, Belgium, 3 Department of Pneumology, Centre for Thoracic Oncology, AZ Delta, Roeselare, Belgium, 4 Pathology Department, Erasme Hospital, Université libre de Bruxelles, Anderlecht, Belgium, 5 Department of Pneumology, Centre Hospitalier Universitaire Saint-Pierre, Brussels, Belgium, 6 Department of Pneumology, Cliniques Universitaires Saint-Luc - Université Catholique de Louvain, Brussels, Belgium, 7 Department of Pathology, University Hospitals KU Leuven, Leuven, Belgium, 8 Department of Pathology, Universitair Ziekenhuis Antwerpen, Edegem, Belgium. Please send all correspondence to: C. Dooms, MD, PhD, Department of Respiratory Diseases, University Hospitals KU Leuven, Herestraat 49, 3000 Leuven, Belgium, tel: , christophe.dooms@uzleuven.be. Conflict of interest: J. Vansteenkiste received an institutional research grant from AstraZeneca and presented lectures for AstraZeneca. B. Colinet received financial compensation for advisory boards and lecture for AstraZeneca. V. Ninane received fees as a speaker from Novartis, AstraZeneca, Pfizer, Boehringer, and MSD and as an advisory board participant from Novartis, AstraZeneca, Boehringer and GSK. P. Pauwels received compensation for the advisory board and research grant from AstraZeneca. I. Demedts presented lectures for AstraZeneca. N. D Haene received fees as a speaker from MSD, Pfizer, Biocartis, and Boehringer and as an advisory board participant from AstraZeneca, Pfizer and Biocartis. B. Weynand received compensation for the advisory board. CD and TP declare no conflict of interest. Funding statement: AstraZeneca was the funding source and funded all costs associated with the development and the publishing of the present manuscript. Acknowledgements: The authors would like to thank U. Miecielica, PhD (XPE Pharma & Science, Wavre, Belgium) for medical writing support. Keywords: EGFR-T790M mutation, EGFR-TKI resistance, liquid biopsy, molecular testing, non-small cell lung cancer, tissue biopsy. VOLUME11SEPTEMBER2017

2 PRACTICE GUIDELINES 227 EGFR-T790M at acquired resistance should become a standard component of patient care in EGFR-mutant tumours. In this manuscript, we propose and discuss two possible clinical diagnostic algorithms that could be used for the therapy-orienting testing of EGFR-TKI-resistant NSCLC patients. Tissue and liquid biopsies involve challenges in terms of specific clinical role, safety, logistics, and cost. (BELG J MED ONCOL 2017;11(5): ) INTRODUCTION Several mutations identified in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene represent actionable alterations in non-squamous non-small cell lung cancer (NSCLC). Somatic sensitising mutations in the gene encoding EGFR are detected in 7-11% of Belgian, Dutch or French patients with advanced stage non-squamous NS- CLC. 1-4 EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib, erlotinib or afatinib are the established first-line treatment option for patients with an actionable EGFR mutation and are associated with an objective response rate (ORR) of 56-74% and a median progression-free survival (mpfs) of ten to fourteen months. 5-7 Despite initial responses to these EGFR-TKIs, the majority of the patients will inevitably demonstrate disease progression within one to two years after treatment initiation. The most common mechanism of resistance is the development of an additional EG- FR-T790M mutation in exon 20, which was found in 50-60% of the EGFR-mutant NSCLC patients who were rebiopsied on EGFR-TKI treatment A drug is now available to overcome this T790M resistance mutation. Osimertinib, a third-generation oral irreversible EGFR-TKI that shows a particular sensitivity for T790M resistance mutations, exhibited a better response rate (confirmed ORR 61 vs. 21%) and mpfs of 9.6 vs. 2.8 months in EGFR-T790M-positive vs. T790M-negative tumour genotyping, respectively. 12 A recently presented update of AURA pooled phase II (n=411) and published AURA phase III reported an ORR of 66% (95% confidence interval [CI] 61 71%) and 71% (95% CI 65 76%), respectively, and PFS of 11.0 months and 10.1 months for T790M-positive tumours treated with osimertinib 80 mg daily. 13,14 Osimertinib has thus expanded the treatment options in advanced stage EGFR-mutant NSCLC, requiring new clinical algorithms that will be discussed in this manuscript, taking into account individual patient molecular profiles. RELEVANCE OF EGFR-T790M TESTING Patients with EGFR-mutant NSCLC may exhibit primary or secondary resistance to EGFR-TKI therapy. 15 Secondary resistance is defined as progressive disease in a patient previously responsive to first- or second-generation EGFR-TKIs. A primary intrinsic resistance is generally defined as the development of progressive disease to EGFR-TKI in two to four months of treatment. This primary resistance is present in around 30% of NSCLC patients with mutant EGFR treated with EGFR-TKI. Inherited de novo T790M mutations, exon 20 insertions, or pro-apoptotic BIM gene deletions have been described in a small group of patients, but the majority of intrinsic EGFR-TKI resistance occurs in otherwise responsive activating EGFR mutations. 16 The mechanistic molecular basis of intrinsic EGFR-TKI resistance in NSCLC patients supposed to be responsive to EGFR-TKI remains largely unknown and specific predictive markers associated with intrinsic resistance to gefitinib, erlotinib or afatinib are lacking. 16 Secondary acquired resistance is defined as progressive disease in a patient previously responsive to first- or second-generation EGFR-TKIs. 17,18 Mechanisms of secondary resistance described to date include: 1) acquisition of a secondary alteration in the EGFR oncogene such as mutation in the kinase domain (e.g. exon 20 T790M gatekeeper mutation) or gene amplification of the kinase itself; 2) activation of downstream signalling or bypass oncogenic mechanism (e.g. PIK3CA mutation, BRAF mutation, HER2 amplification, MET amplification, etc.); 3) phenotypic alteration or transformation (e.g. transformation to small-cell carcinoma histology, or epithelial-mesenchymal transition) (Figure 1). Of these, acquisition of a EGFR-T790M mutation is the most common accounting for >50% of all cases of acquired resistance and can now be effectively targeted by the third-generation EG- FR-TKI osimertinib. 11,15 Although mechanisms of acquired resistance of EGFR-mutant NSCLC to EGFR-TKIs have been identified, little is known about how resistant clones evolve during first-line EG- FR-TKI therapy. Recently, it has been observed that acquired resistance caused by EGFR-T790M gatekeeper mutation can occur either by selection of pre-existing T790M-positive clones or via genetic evolution into de novo T790M acquisition within initially T790M-negative clones; these different paths of resistance might impact on the therapeutic opportunity to overcome resistance in the clinic by third-generation EGFR-TKIs. 19 A gatekeeper mutation deforms the entrance or gate of the ATP binding pocket resulting in a preferen- VOLUME11SEPTEMBER20175

3 228 FIGURE 1. Known mechanisms of EGFR resistance. HER2, human epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor; PIK3CA, phosphatidylinositol 3-kinase catalytic 110-KD alpha; SCLC, small-cell lung cancer; EMT, epithelial-mesenchymal transition. tial binding of ATP compared to the TKI, which induces resistance. OVERVIEW OF THE DIAGNOSTIC SAMPLES THAT CAN OR SHOULD BE USED According to the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC) and Association for Molecular Pathology (AMP) guidelines, EGFR mutation testing can be performed on surgical resection specimens, small biopsy specimens (e.g. bronchoscopic biopsy or percutaneous core needle biopsy) and small cytological specimens (e.g. cell block of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TB- NA) specimen or pleural effusion). 20 Detection of T790M mutation is technically feasible on pleural fluid and cerebrospinal fluid. 21 Testing on cerebrospinal fluid can be done in case of progression/development of brain lesions. However, the fact that brain lesions progress is mostly due to the poor penetrance of first-/second-line EGFR-TKIs in the brain, and more clinical data are required to adequately assess the sensitivity of EGFR-T790M testing on cerebrospinal fluid. 21 Based on literature reports, the majority of specimens taken at disease progression have been tumour tissue biopsies. Two prospective studies assessed the feasibility of a post-progression tumour tissue rebiopsy to genotype, and found a feasibility of an invasive rebiopsy procedure in 50 80% of the patients. 10,22 Indeed, a repeat tissue biopsy is not possible for every patient who develops resistance to EGFR-TKIs due to factors such as disease progression at an inaccessible site (primarily central nervous system) and/or functional decline. Retrospective studies found a rebiopsy feasible in at least 70 80% with complication rates similar to primary sampling and sample adequacy of 80 90%, resulting in a successful adequate tissue biopsy test in 55 75% of patients. 8,11,23 Building effective treatment strategies for EGFR-mutant NS- CLC patients that include successive regimens of tailored therapy will rely on the ability to correctly identify molecular targets over the course of successive regimens. This need, coupled with the limitations of tissue biopsies, heightens the enthusiasm about the recent development of novel bloodbased technologies for genetic testing. The existence of frag- VOLUME11SEPTEMBER2017

4 PRACTICE GUIDELINES 229 FIGURE 2A. Possible clinical algorithms for therapy-oriented testing of EGFR-TKI-resistant non-small cell lung cancer patients: A. Conventional paradigm, a resistance tissue biopsy is preferred whenever feasible. EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; RECIST, Response Evaluation Criteria In Solid Tumours; SRS, stereotactic radiosurgery; SABR, stereotactic ablative body radiotherapy; ctdna, circulating tumour DNA; NGS, next-generation sequencing; FFPE, formalin-fixed paraffin-embedded. * Yu H, et al. J Thorac Oncol 2013;8:346-51; Novello S, et al. Ann Oncol 2016;27:v1-v27. ** use cell stabilisation tube and ship at room temperature to a BELAC accredited laboratory with a validated EGFR-T790M ctdna test. mented DNA in the circulation has already been proven in 1948 by Mandel and Metais. 24 In healthy individuals, the major source of circulating DNA are the apoptotic white blood cells. In cancer patients, also tumour cells can be a source of circulating DNA. As tumours increase in volume, the capacity of phagocytes to eliminate and clear apoptotic and necrotic fragments can be exceeded, leading to passive release of tumour DNA in the circulation (circulating tumour DNA or ctdna). An active release of DNA by cancer cells has also been documented. 25 There is a good correlation between tumour burden and the amount of ctdna. The amount of ctd- NA released in the circulation can go up to 90% of circulating DNA. 26 Thus, blood-based genotyping can serve as a molecular proxy for the corresponding tumour tissue biopsies. VOLUME11SEPTEMBER20175

5 230 FIGURE 2B. Possible clinical algorithms for therapy-oriented testing of EGFR-TKI-resistant non-small cell lung cancer patients: B. Alternate paradigm, plasma genotyping for T790M screening is considered before a biopsy. EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; RECIST, Response Evaluation Criteria In Solid Tumours; SRS, stereotactic radiosurgery; SABR, stereotactic ablative body radiotherapy; ctdna, circulating tumour DNA; NGS, next-generation sequencing; FFPE, formalin-fixed paraffin-embedded. * Yu H, et al. J Thorac Oncol 2013;8:346-51; Novello S, et al. Ann Oncol 2016;27:v1-v27. ** use cell stabilisation tube and ship at room temperature to a BELAC accredited laboratory with a validated EGFR-T790M ctdna test. In a prospective study, the majority (93%) of patients on EG- FR-TKIs were able to undergo a blood draw for the evaluation of ctdna at disease progression what is considered a liquid biopsy, while 80% underwent a tumour tissue biopsy. 10 Overall, 98% were able to undergo either a tumour tissue or liquid biopsy and 77% were able to successfully undergo both a tumour tissue and liquid biopsy at disease progression. Recently, analysis of EGFR mutations from urine specimens (which is also considered a liquid biopsy) has also been demonstrated as feasible with a high concordance to tumour and plasma specimens for diagnostic detection of T790M. 27 To date, literature on urine is scarce, and the conditions and methodology are not yet validated. Therefore, it should not be considered for testing in routine clinical practice today. VOLUME11SEPTEMBER2017

6 PRACTICE GUIDELINES 231 ASSOCIATION OF EGFR-T790M DETECTED IN TISSUE OR LIQUID BIOPSY WITH CLINICAL OUTCOMES TO OSIMERTINIB Clinical outcome is the best reference standard for development of an assay that can be used as a new biomarker for guidance of treatment. A large retrospective cohort of 237 patients with tumour tissue and/or plasma available for a diagnostic genotyping comparison assessed their usefulness as a predictive biomarker for ORR and mpfs. 28 Sensitivity of plasma genotyping by BEAMing (beads, emulsion, amplification, and magnetics) for detection of T790M was 70%. ORR and mpfs with osimertinib were similar in patients with T790M-positive plasma (ORR 63% and mpfs 8.2 months) or T790M-positive tumour tissue (ORR 62% and mpfs 9.7 months). In addition, this study assessed the false-negative and false-positive plasma rates. Patients with T790M-negative plasma had a high ORR at 46%, presumably due to mixture of true-negatives and false-negatives. Therefore, T790M-negative plasma results still need a tumour biopsy because of a negative predictive value of 46% for plasma T790M genotyping, which might suggest a lower disease burden in some patients. It is less clear how much value a biopsy adds in patients with T790M-positive plasma. Of all patients with T790M-positive plasma, 11% had T790M-negative tumour tissue, which yielded an ORR at only 28% presumably reflecting heterogeneity of the T790M resistance mutation as a minor clone at some disease sites. In another study, 23% of T790M-positive plasma specimens had T790M-negative tumour tissue but no association with clinical outcome was reported. 27 Future research should assess whether quantitative plasma genotyping and calculation of relative T790M allele frequency can offer insight into this tumour heterogeneity. Therefore, more data are needed before a conclusion can be drawn whether a negative adequate tissue specimen requires an additional plasma testing. CONCORDANCE RATE BETWEEN MUTATIONS IDENTIFIED IN PLASMA AND TUMOUR TISSUE Concordance rate between plasma and tumour tissue was 88 93% for the known exon 19 or 21 sensitising mutations and only 73% for the exon 20 T790M resistance mutation. 28,29 Indeed, plasma genotyping resulted in rare false-positives for exon 19 deletions and exon 21 L858R compared to tumour tissue genotyping which is concordant with a known absence of heterogeneity for the known sensitising driver mutations. 30 On the other hand, the acquired T790M resistance mutation might be heterogeneously present across different disease sites within one patient. An ongoing clinical trial is validating prospective ctdna testing on plasma in EGFR-mutant NS- CLC treated with EGFR-TKIs as a diagnostic tool based upon the identification of targetable genomic alterations (Clinical- Trials.Gov ID: NCT ). PRE-ANALYTICAL HANDLING OF TUMOUR TISSUE BIOPSIES AND LIQUID BIOPSIES While pre-analytical handling of tissue has been extensively described and documented, handling of liquid biopsies is a work-in-progress. 20 Pathologists should utilise tissue sparing techniques to preserve tumour tissue from formalin-fixed paraffin-embedded specimens for diagnosis and enable subsequent biomarker testing, and determine the adequacy of specimens for EGFR testing by assessing cancer cell content and DNA quantity and quality. Key considerations for liquid biopsy sample processing are to minimise ctdna degradation in the blood collection tube and to prevent the increase of blood cell-derived wild-type genomic DNA released from lysis of nucleated blood cells. Most experience has been gained using EDTA, Streck and Cellsave tubes. These tubes showed similar performance in preserving ctdna for up to six hours before plasma isolation. Streck and Cellsave tubes more constantly stabilised ctdna and wild-type DNA at 48 hours than EDTA tubes. 31 Since in routine practice blood may stay for longer times before it is processed, either as a result of delays at the site of collection, or because blood is mailed to a central processing site, the use of EDTA tubes should be avoided. Storage temperature has also an influence on ctd- NA population. Of note, the tubes should not be stored in a refrigerator. For example, three to five days storage at 4 C resulted in up to 10-fold increase of normal genomic DNA fragments. Storage at room temperature is therefore mandatory. OVERVIEW OF THE DIAGNOSTIC TEST METHODS THAT CAN BE USED PLATFORMS FOR DETECTING EGFR MUTATIONS IN A TISSUE BIOPSY According to the CAP, IASLC and AMP guidelines, consensus opinion suggests that the use of a sensitive test method capable of detecting mutations in histologic or cytologic tissue specimens with as little as 10% tumour cells is strongly encouraged. 20 The minimal amount of tumour required for sensitive detection of EGFR mutations depends on the detection technique and should be established and validated in each testing laboratory. PLATFORMS FOR DETECTING EGFR MUTATIONS IN A LIQUID BIOPSY Several platforms can be used for the sensitive and specific detection of EGFR mutations. Non-digital platforms include VOLUME11SEPTEMBER20175

7 232 KEY MESSAGES FOR CLINICAL PRACTICE 1. Testing for EGFR-T790M at acquired resistance to first-/second-line EGFR-TKIs should now become a standard component of patient care. 2. Although the occurrence of a secondary EGFR-T790M resistance mutation can precede radiological and/ or clinical disease progression, we advise no testing for EGFR-T790M until a clinical judgement and/or presentation conclude that a switch of systemic therapy is required. 3. Tumour tissue biopsies and plasma liquid biopsies involve challenges in terms of specific clinical role, logistics, and cost. We therefore propose a conventional and an alternate clinical algorithm (Figure 2A - conventional algorithm and Figure 2B - alternate algorithm) that could be used. 4. Tumour tissue rebiopsy still has a preferred role as plasma testing could be unreliable for a variety of reasons. EGFR-T790M resistance mutation testing has a higher sensitivity on tumour tissue samples, which brings added value to T790M plasma-negative patients. In addition, tumour tissue rebiopsy can assess alternative resistance mechanisms, which could mandate an alternative therapy. 5. Plasma liquid biopsy has a growing interest. The most relevant limitations for EGFR-T790M testing on plasma liquid biopsy to date are its suboptimal sensitivity and negative predictive value and the requirement of a validated qualified assay with mandatory Belac accreditation. 6. A repetition of testing is encouraged in case of an inconclusive result (e.g. inadequate sample, inadequate tumour tissue DNA, detection of a mutation in the plasma near the detection limit, etc.). 7. In Belgium, EGFR testing (tissue or plasma) is reimbursed once a year per patient when a BELAC accredited test is used. In case more frequent testing is performed, the hospital or patient should pay for the additional tests. the cobas (Roche Molecular Diagnostics) EGFR Mutation Test and the therascreen (Qiagen) EGFR amplification refractory mutation system assay, and digital platforms include the highly sensitive and quantitative droplet digital polymerase chain reaction (ddpcr), as well as the BEAMing digital PCR technique. Next generation sequencing can also be used. T790M detection sensitivity was shown to range between 51% and 73% for the cobas test, between 73% and 81% for BEAMing and between 71% and 77% for the ddpcr platform. 13,32,33 Considering the financial burden, ddpcr could be considered as first choice as it is a simple and inexpensive assay that can be widely adapted. Clinicians should take into account their clinical judgement whenever a T790M mutation is reported around its detection limit of the test method. The turnaround time of tumour tissue EGFR testing should be within ten working days of receiving the specimen in the testing laboratory. 20 The turnaround time of ctdna testing can be faster (three working days) when the testing is centralised but its cost and turnaround time will increase when this is decentralised. To date, in Belgium, EGFR testing (tissue or plasma) is reimbursed once a year per patient when a Belac (the Belgian Accreditation Organisation) accredited test is used. In case more frequent testing is performed, the hospital or patient should pay for the additional tests. CONCLUSIONS Osimertinib clearly expands relapse treatment options for advanced stage EGFR-mutant NSCLC. Testing for EG- FR-T790M should therefore become a standard component of patient care at clinical disease progression in EGFR-mutant tumours on gefitinib, erlotinib or afatinib. A liquid biopsy and/or a tumour tissue biopsy are valid options for genetic retesting, each with their specific strengths and weaknesses. REFERENCES 1. Barlesi F, Mazieres J, Merlio JP, et al. Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT). Lancet. 2016;387(10026): Dooms C, Vliegen L, Vander Borght S, et al. Suitability of small bronchoscop- VOLUME11SEPTEMBER2017

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