Recommendations of the Austrian Working Group on ...

Recommendations of the Austrian Working Group on Pulmonary Pathology and Oncology for predictive molecular and immunohistochemical testing in non-small cell lung cancer Helmut H. Popper, Ulrike GruberMösenbacher, Georg Hutarew, Maximilian Hochmair, Gudrun Absenger, Luka Brcic, Leonhard Müllauer, et al. memo - Magazine of European Medical Oncology An International Journal for Oncology and Haematology Professionals ISSN 1865-5041 memo DOI 10.1007/s12254-016-0297-x

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memo DOI 10.1007/s12254-016-0297-x

Recommendations of the Austrian Working Group on Pulmonary Pathology and Oncology for predictive molecular and immunohistochemical testing in non-small cell lung cancer Helmut H. Popper · Ulrike Gruber-Mösenbacher · Georg Hutarew · Maximilian Hochmair · Gudrun Absenger · Luka Brcic · Leonhard Müllauer · Gerhard Dekan · Ulrike Setinek · Dagmar Krenbek · Michael Vesely · Robert Pirker · Wolfgang Hilbe · Rainer Kolb · Gerald Webersinke · Tamara Hernler · Georg Pall · Sigurd Lax · Andrea Mohn-Staudner

Received: 21 July 2016 / Accepted: 8 November 2016 © Springer-Verlag Wien 2016

Summary The introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in the therapy of non-small cell lung cancer (NSCLC) with activating mutations of the EGF receptor has opened a new area of lung cancer treatment strategies and led to an enthusiastic search for additional genetic aberrations. Genetic drivers such as EML4ALK (EML4: echinoderm microtubule-associated protein-like 4; ALK: anaplastic lymphoma kinase) and proto-oncogene tyrosine protein kinase transcribed from the ROS1-gene (ROS1) rearrangements have

been detected and specific treatment options have been developed. A new approach to treatment in lung cancer is immunotherapy by antibodies interfering with immune checkpoint controls. Diagnostic and predictive immunohistochemical staining and molecular tests have to follow specific rules, if applied in daily practice. The Austrian Working Group on Pulmonary Pathology and Oncology (AWGPPO) has presented an updated version of the previous recommendations published in 2013. Questions raised during the past 3 years will be addressed: selection of

H. H. Popper () · L. Brcic Institute of Pathology, Medical University Graz, Graz, Austria [email protected]

R. Pirker Department of Internal Medicine I, Medical University of Vienna – AKH, Vienna, Austria

U. Gruber-Mösenbacher Consultant pathologist, Feldkirch, Austria

W. Hilbe 1st Department of Internal Medicine, Oncology and Haematooncology, Wilhelminen Hospital, Vienna, Austria

G. Hutarew Institute of Pathology, Private University Salzburg, Salzburg, Austria M. Hochmair · A. Mohn-Staudner Department of Pulmonology, Otto Wagner Hospital, Vienna, Austria G. Absenger Department of Oncology, Medical University Graz, Graz, Austria L. Müllauer · G. Dekan Institute of Pathology, Medical University, Vienna, Austria

R. Kolb Department of Pulmonology, Hospital Wels-Grieskirchen, Wels-Grieskirchen, Austria G. Webersinke Laboratory of Molecular Biology and Tumorcytogenetics, Internal Medicine I, Hospital of Barmherzige Schwestern, Linz, Austria T. Hernler Department of Pulmonology, Hospital Hohenems, Hohenems, Austria

U. Setinek · D. Krenbek Institute of Pathology, Otto Wagner Hospital, Vienna, Austria

G. Pall Department of Internal Medicine 5 (Haematology and Oncology), Medical University Innsbruck, Innsbruck, Austria

M. Vesely Institute of Pathology, Hospital Hietzing, Vienna, Austria

S. Lax Department of Pathology, Hospital Graz West, Graz, Austria

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Recommendations of the Austrian Working Group on Pulmonary Pathology and Oncology for predictive. . .


tissue, order of diagnostic immunohistochemical and molecular tests, “reflex” testing, the issue of resistance mechanisms, significance of liquid biopsies, and use and interpretation of antibody reactions for immune checkpoint markers.

Due to improvements in the knowledge of molecular biology of non-small cell lung cancer (NSCLC) and the development and approval of new drugs specifically active in NSCLC subtypes, the Austrian Working Group on Pulmonary Pathology and Oncology (AWGPPO) has presented an updated version of the previous recommendations published in 2011 and 2013 [1, 2]. The working group will address practical questions raised during the past 3 years. The first versions of the recommendations covered EGFR-mutation testing, assessing for ALK and ROS1 rearrangements and contained an algorithm for molecular testing as reflex test at the time of diagnosis. With the publication of the new World Health Organization (WHO) classification of tumors of the lung [3], the algorithm has been refined and clarified by the integration of diagnostic immunohistochemical stains in biopsy diagnosis. The application of new tyrosine kinase inhibitors (TKI), immune checkpoint inhibitors, and new drugs acting to overcome resistance mechanisms against TKI in NSCLC treatment has also influenced the process of molecular testing. Liquid biopsies, a new detection technique for mutated genes in body fluids, and next-generation sequencing techniques allow for extended analyses of lung cancer and will be covered along with remarks on quality assurance for testing methods.

amount might be sufficient, as long as immunohistochemistry for TTF-1 and p40 can be performed. These two markers are recommended to establish the diagnosis in uncertain histology. A general recommendation is to spare as much tissue as possible for molecular tests [5, pp. 823–859] and as a consequence to reduce the number of sections for diagnostic purposes to a minimum according to the WHO classification of 2015 for small biopsies. A low percentage of tumor cells in a specimen, 5% in general, can be sufficient for molecular analysis, depending on the method. For FISH at least 100 viable tumor cells in one fluorescent-stained slide are necessary. It should be noted that the smaller the tissue sample, the less analytical investigation can be done, even with the highly sensitive methods available today. Cell-free tumor DNA can be detected in liquid biopsies. For optimal conservation of biopsies and cytological specimens, some preanalytical rules should be kept in mind: ● Time to fixation (cold ischemia) should not exceed 1 h. ● Standardized fixation medium should be 10% neutrally buffered formalin (4% formaldehyde). ● Fixation time should be between 6 and 48 h. ● Sections for biomarker testing should be prepared timely, close to the analysis [6]. At present there is a debate on the correct site of a biopsy: Should it be from the primary tumor or from the metastasis [7], which nodule of a multifocal tumor should be selected [8]? These questions are driven by the debate on tumor heterogeneity [9, 10], which, however, also exists in primary tumors [11]. As we do not have detailed data about the tumor in biopsies (central portion vs. periphery, invasion front vs. perivascular areas, primary vs. metastatic sites, etc.), we should perform molecular analysis from every tissue that can be biopsied with minimal risk for the patient.

Recommendations for molecular testing

Recommendations for reflex testing

Which type and quantity of tissue are suitable for molecular analysis?

The previous versions of the AWGPPO recommended reflex testing for EGFR mutation as well as for ALK rearrangements in all adenocarcinomas, whereas in all other types of NSCLC, genetic testing was recommended on clinical request. Molecular reflex testing for all forms of neuroendocrine lung carcinomas was declined. Following international recommendations and the 2015 WHO classification [3] of lung cancer including rules for small biopsies, we recommend reflex testing in (a) all adenocarcinomas, (b) NOS carcinomas (TTF1-, p40-), and (c) carcinomas with adenocarcinoma components (TTF1 positive), for EGFR mutations and ALK and ROS1 rearrangement.

Keywords Lung Cancer · Driver genes · Molecular test · Immunohistochemistry · Adenocarcinoma

Introduction

The guidelines for technical and quality issues were published in the previous version, and therefore only a few aspects have to be added. The reader is referred to the previous version [2]. Biopsies Forceps or needle biopsies should contain viable tumor cells for typing and DNA and/or RNA extraction as well as fluorescence in situ hybridization (FISH) analysis. For the differentiation of tumor types, the cells from biopsies or aspiration-cytology specimens (smear or preferred cell block [4]) should be vital. Even a small


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Tests should be performed on biopsies and cytology preparations, as well as on surgically removed lung adenocarcinomas. Most NSCLC in lower stages will recur or set metastases after 1–2 years (approximately 80%), therefore in most cases our analysis will provide the desired information to the clinician before the relapse occurs. Thereby no time is lost for immediate appropriate treatment. In the case of second primaries, biopsies between the first and second primary can be compared for their genetic makeup.

Testing for specific genes EGFR testing EGFR mutation testing includes mutations on exons 18–21 of the EGFR gene, occurring at a frequency of 8–12% in our population [5]. EGFR mutation testing should also be considered for squamous cell and adenosquamous carcinomas in patients with no or minimal smoking history on request [12]. EGFR mutations are rare in invasive mucinous adenocarcinomas and adenocarcinomas with mucin production. These adenocarcinomas are predominantly KRAS mutated [13]. Because they can contain a nonmucin EGFR-mutated component [14], these types should be included in reflex testing. EGFR immunohistochemistry for EGF-antibody treatment Based on a randomized study (FLEX) by Pirker et al. [15], cetuximab, a murine monoclonal antibody (MoAB) against EGFR, showed a significant survival benefit when combined with platinum-based chemotherapy, but this treatment protocol was not approved by the European Medicines Agency (EMA). Recently, necitumumab, a human IgG EGFR antibody, was approved by the EMA for the first-line treatment of NSCLC with EGFR expressing squamous cell histology in combination with chemotherapy [16, 17]. Therefore, immunohistochemical evaluation should be performed upon clinical request. The selection of the antibody should follow the line of testing in the trial by Pirker et al. [15]. The percentage of positively stained tumor cells by an EGFR antibody should be reported. Two tested antibodies were equally sensitive and should be used (pharmDx kit, DAKO; Ventana 3C6 clone). ALK rearrangement The first step in ALK reflex testing should be a test with reliable antibodies for ALK expression by immunohistochemistry (clones D5F3 and 5A4). The VENTANA ALK (D5F3) CDx Assay in FFPE NSCLC tissue stained with a BenchMark XT automated staining instrument is indicated as an aid in identifying patients eligible for treatment with crizotinib (FDA approval 6-12-2015) and regarded as an

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equivalent alternative to FISH for adenocarcinoma [18]. Clone 5A4 can be reported semiquantitatively in intensity grades from 1+ to 3+. Because of the original recommendation of FISH as gold standard method, cases being positive by immunohistochemical stains are still verified by FISH [19]. Other NSCLC can be tested on request following the same step-wise approach as described earlier. ROS1 rearrangement Concerning the ROS1 translocation, crizotinib has been shown to be highly effective [20] and has received approval by the FDA and EMA in ROS1-positive lung cancers [18]. In spite of the fact that no standard method of testing has been published, we recommend ROS1 translocation screening similar to ALK rearrangement by preselection using an immunohistochemical stain (Clone D4D6) and confirmation by FISH in cases with any positive staining. An RT-PCR approach can replace FISH tests for ALK and ROS1 rearrangements. A FISH analysis might be the additional test in cases with conflicting results by immunohistochemistry and RT-PCR. In addition, RTPCR needs to cover at least the most common fusion genes [19, 21–24]. KRAS mutation analysis In some institutions KRAS mutations are performed to reduce EGFR mutation testing. Routine testing for triage purposes is not recommended.

Diagnostics in the case of resistance (EGFR-, ALKand ROS1-TKI) Several studies and presentations at international meetings have highlighted that different types of resistance mechanisms develop in lung carcinomas. Testing for resistance mechanisms is highly recommended, as new drugs have been developed that can overcome resistance mechanisms. To identify the underlying changes it is essential to get a new biopsy for an analysis of the resistance mechanism present in the carcinoma, which may help to adjust the therapy.

Resistance in EGFR-mutated adenocarcinomas T790M is one of the most frequent resistance mutations (up to 50%), which can be treated by third-generation TKIs [25, 26]. Some other common ones on exon 20 are D770_N771 insertions (up to 3%) and the mutations V769L, N771T, and the D761Y mutation on exon 19 [27–29]. Several of these mutations might be targeted by second- and third-generation TKIs [26]. Since a specific treatment option for resistance mutation T790M is available, testing for this mutation is mandatory (see also liquid biopsy).



Furthermore, amplification of MET and HER2 or trans-differentiation into a neuroendocrine carcinoma might be alternative resistance mechanisms [30–32]. Activating mutations in NOTCH1 have recently been identified as the cause of resistance and progression of EGFR-mutated adenocarcinoma [33, 34]. Loss of RB1 and posttranslational modification might underlie the trans-differentiation of EGFRmutated adenocarcinoma into high-grade neuroendocrine carcinomas [35, 36]. Resistance mutations for T790M should be tested, as well as amplification for MET and HER2, as treatment options do exist. Resistance in ALK1-rearranged adenocarcinomas The exact resistance mechanisms in ALK-rearranged lung adenocarcinoma are still under investigation [37]. The most common are secondary mutations in the ALK domain, such as L1196M and G1269A, less common are 1151Tins, L1152R, C1156Y, F1174L, G1202R, and S1206Y [38–40]. Bypass mechanisms such as MET activation occur [41, 42], but also ALK amplification. Interestingly, second- and third-generation ALK inhibitors can target most of the secondary mutations [43]. However, these new-generation ALK inhibitors, too, will induce secondary resistance mutations, for which new drugs are being designed [42, 44, 45]. In case of resistance for ALK inhibitor, resistance mechanisms such as ALK mutations, but also MET activation should be tested. Resistance in ROS1 rearranged adenocarcinomas The most common resistance mutation for CD74ROS1 rearrangement is a glycine-to-arginine substitution at codon G2032R in the ROS1 kinase domain [46, 47]. The ROS1 (G2032R) mutant retains foretinib sensitivity at concentrations below safe, clinically achievable levels [48]. KRAS/NRAS mutations or KRAS amplification induces resistance to ROS1 inhibition by crizotinib [49]. Inhibitors of the RAS signaling pathway can overcome this inhibition. Currently, only phase I trials are available, and thus routine testing is not recommended. The cMET/RET/VEGFR inhibitor cabozantinib (XL184) [50] effectively inhibited the survival of G2032R-mutated crizotinib-resistant adenocarcinoma cells. Cabozantinib but also other newly developed ROS1 inhibitors could overcome all the newly identified secondary resistance mutations; therefore, resistance mutations in the ROS1 gene should be tested.

New analytic techniques

Liquid biopsy Liquid biopsy is a new tool to test genetic abnormalities in circulating tumor cells and circulating DNA. The methods are currently validated in several laboratories. It will be a new tool for testing resistance mechanisms in lung carcinomas with driver genes. At present, circulating tumor cell analysis as a diagnostic tool cannot be recommended as a routine method, because the blood content of circulating tumor cells can be low, and the techniques for harvesting are not standardized. Circulating cell-free (cf)DNA by contrast can be found in a considerable number of patients and can be used for testing resistance mechanisms [51]. In patients with identified resistance mutations, such as T790M on EGFR, liquid biopsy can be used to monitor response to treatment. In addition, as a new resistance mutation, C797S has been described following osimertinib treatment – this might also be evaluated by liquid biopsy. The recommendation is to test for the resistance mutation T790M primarily using biopsy together with cfDNA. Monitoring of the patient can be done using cfDNA only. Different technologies are available for the evaluation of circulating tumor DNA, among which digital systems should be preferred. The most important issue is handling: The plasma sample needs to be transferred quickly into specified tubes such as Streck-tubes or similar. After this step the samples should be processed as soon as possible. Samples can be stored in refrigerators. Because of tumor heterogeneity, discrepancies exist between tissue biopsy and blood samples, which might reflect different tumor cell clones sampled. Therefore, this method should be validated by a rebiopsy.

Next-generation sequencing Next-generation sequencing (NGS) has become a routine tool for the evaluation of mutations and amplification in NSCLC in large institutions or commercial laboratories. It allows us to test for a panel of commonly mutated and amplified genes in lung cancer. Commercially available panels exist and customized panels can be created. As the test will create an enormous amount of data, it is recommended to structure the report in two parts: one describing the findings, and the final diagnosis reporting on those gene abnormalities for which a targeted therapy is available. The interpretation of the data is usually done in tumor boards.

New analytic techniques can be applied on small samples simultaneously, but require complex and costly equipment and produce a large amount of data, which have to be interpreted.


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RT-PCR Commercially available panels of common rearrangements or customized panels may replace FISH analysis for rearrangement testing. RT-PCR has several advantages: ● With one RNA extraction, several rearrangements can be tested simultaneously. ● It saves time for pathologists replacing time-consuming FISH analysis. A disadvantage of RT-PCR is that the amount of ribonucleic acids (RNA) needed is quite high and might not be reached in specimens with low tumor cell content, especially in some needle aspiration samples.

Assessment of copy number variation Beside FISH new technologies such as digital PCR and whole-genome amplification analysis, the nCounter copy number variation (CNV) assay can be used. In addition, array CGH assays with commercially available oligoarrays can be used.

Further markers/targets for adenocarcinomas and other differentiations At present, several new markers are being evaluated for their predictive validity for specific therapies. Whenever possible, patients should be included in such prospective clinical trials.

RET RET translocation with the fusion partners KIF5B, CCDC6, and NCOA4 is rarely a driver in adenocarcinomas, and should be tested on request: Analysis of a rearrangement should be performed by FISH or RTPCR, and histomorphologic (signet ring cell morphology) and clinical criteria could help to find candidates to be tested [52]. RET translocated carcinomas can be treated with multitargeted kinase inhibitors such as vandetanib, sunitinib, and sorafenib.

BRAF Mutations of BRAF occur in about 3% of lung adenocarcinomas [53, 54]. As these are most often V600E mutations, a treatment option can be offered to the patient, based on results of small phase II trials [55]. Therefore testing should be provided on clinical request and patients should be included in clinical trials when possible.

MET MET amplification should routinely be tested by FISH in patients with EGFR-mutated adenocarcinomas

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upon development of resistance (only high amplification will respond to TKI treatment). MET overexpression and MET mutation should be determined with antibodies for MET by immunohistochemistry on clinical request. Exon 14 splice-site mutations occur in 3% of adenocarcinomas, and in 22% of sarcomatoid carcinomas, and therefore MET should be included in carcinoma mutation panels because of the therapeutic consequences [56, 57].

ERBB2/HER2 Mutations of HER2 are rare in lung adenocarcinomas and can be tested upon request. The test for HER2 amplification should be done by immunohistochemistry first, and cases with 2+ and 3+ should be verified by FISH. A treatment option with HER2 inhibitory drugs exists for patients with positive tumors.

KRAS Presently, new drugs are being evaluated for KRASmutated lung cancer patients showing promising results [58–60]. The focus is on inhibition at MEK-ERK and the mTOR signaling cascade. So far no treatment option exists other than clinical studies.

DDR2 DDR2 is mutated in 2% of patients with squamous cell carcinomas. A potential treatment option is available with dasatinib. Owing to limited data and severe side effects, this treatment should be limited to clinical trials in specialized centers. Analysis of DDR2 mutation should be done on clinical request.

FGFR2 FGFR2 amplification and mutations are found in some adenocarcinomas and squamous cell carcinomas. There are multikinase inhibitors available targeting FGFR. To date, no clinical recommendation is available, and therefore testing should only be done on request in specialized centers, where treatment is available.

FGFR1 FGFR1 was reported with enthusiasm and trials with inhibitors were invented. Currently, this therapy is only done in a few centers with questionable results. The main problem is that many of the FGFR1amplified squamous cell carcinomas (diagnosis by FISH) have additional mutations and deletions, such as PTEN deletion and PI3KCA activating mutations, which might overrule the FGFR1 inhibitor therapy. If a test for FGFR1 is requested by clinicians, additional



Fig. 1 Positive staining for PDL1; the tumor cells are completely surrounded by the membranous staining with the antibody. Bar = 50 µm

Fig. 3 Positive staining for PDL1; most tumor cells are weakly stained, some with a complete membranous stain, others are stained focally. Bar = 50 µm

Immune checkpoint modulation

Fig. 2 Negative reaction for PDL1; there are positively stained dendritic cells in the tertiary lymph follicles. Bar = 50 µm

tests for PTEN and PI3K should be performed and reported simultaneously to avoid unnecessary and ineffective treatment (J-C. Soria, AACR Congress, San Diego 2014 and Weeden et al. [61]).

VEGF, VEGFR, PDGFR Antiangiogenic treatment [62, 63] using antibodies for VEGF (bevacizumab) and VEGFR (ramucirumab) is a new option, when combined with chemotherapy. Nintedanib, another antiangiogenic drug, is a TKI targeting VEGFR, PDGFR, FGFR, and RET. In combination with chemotherapy it has shown benefit for lung cancer patients. In both antiangiogenic protocols no tests are recommended.

Immunotherapy is the new field in lung cancer therapy. The system of PD1 (programmed death 1) and PDL1 (progframmed death ligand 1) as well as CTLA4 have been identified as checkpoint controls for immune reactions [17, 62, 64–66]. Pulmonary carcinomas can express PDL1 and PDL2 and by interaction inhibit the activity of cytotoxic lymphocytes against neoantigens expressed by the carcinoma cells. Humanized antibodies have been developed against PD1 and PDL1, the former already released by the FDA and EMA for treatment of squamous and non-squamous lung carcinomas. Nivolumab has been approved without PDL1 test; for pembrolizumab [67, 68] a companion PDL1 test is requested. The FDA has approved the pharmDx-test for PD-L1 (antibody clone 22C3), while in the EMA release no specific antibody is required, but a validated test should be performed. At present we do not recommend a general handling in Austria; however, we recommend that reflex testing should be established in a lung cancer center in agreement with the tumor board of the corresponding hospital. In a recent report concerning the “blueprint project” [69], different antibodies were compared and tested on tissues from patients included in trials. It was reported that three antibodies used in the appropriate test kits show a comparable performance: ● PD-L1 clone 22C3 pharmDx ● PD-L1 clone 28-8 pharmDx ● PD-L1 clone SP263 Ventana Instead of the kits, antibodies can also be used – Abcam: clone 28.8 and Cell Signaling: clone E1L3N; they both showed a similar performance. Positive immunohistochemical staining is reported, if there is any membranous staining, independent of intensity. The membranous staining can be granular


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or form a line between neighboring cells; in strong staining a paranuclear halo should be seen (Figs. 1, 2 and 3). Cut-offs of ≥1% (low expression) and ≥50% (high expression) have been determined for the PD-L1 IHC 22C3 pharmDx companion test. The expression of PDL1 is focally distributed and the test result may be false negative in small biopsies.

Quality control Every single test has to be evaluated for each parameter in the laboratory. Regular controls of test substances and instruments by competent collaborators and clearly defined and understandable SOPs are required. An experienced pathologist, ideally specialized in thoracic pathology, should provide the histological diagnosis, should have an overview over all available samples of one patient, should select tissue for molecular analysis, being familiar with the techniques used, and should provide an integrated report containing histological and molecular findings and diagnoses [5, pp. 828–860]. External quality assessment should be performed, such as interlaboratory ring trials. EGFR, ALK, and ROS1 testing trials are offered in Europe (EQA), and ring trials for PD-L1 immunohistochemistry will be available from the German Quality Control Institution QuiP. Acknowledgements MerckSharpDome (MSD) and Pfizer Austria provided the conference facilities for the above listed experts. The result of this discussion and the resulting manuscript was by no means influenced by the companies. Conflict of interest Several authors have been paid for educational activities invited from pharmaceutical companies, and also act on advisory boards of these companies. H.H. Popper, U. Gruber-Mösenbacher, G. Hutarew, M. Hochmair, G. Absenger, L. Brcic, L. Müllauer, G. Dekan, U. Setinek, D. Krenbek, M. Vesely, R. Pirker, W. Hilbe, R. Kolb, G. Webersinke, T. Hernler, G. Pall, S. Lax, and A. Mohn-Staudner declare that they have no competing interests for this article.

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