Testing for Oncogenic Abnormalities

Introduction

Introduction

Rapid diagnostic procedures and treatment decisions are essential for patients with advanced non-small cell lung cancer (NSCLC); therefore, effective collaboration between oncologists and pathologists is key to ensuring all patients are tested at initial diagnosis for all relevant molecular markers. This section will provide evidence-based guidance on who should be tested, when they should be tested, and how to perform the tests.

According to current guidelines from the European Society for Medical Oncology (ESMO)1,2 and from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP):3

  • ALK testing should be carried out systematically in advanced non-squamous NSCLC
  • All advanced NSCLC patients should be tested for ALK at initial diagnosis, allowing patients to receive the most appropriate treatment as early as possible
  • It is preferable to test for different molecular markers in parallel as sequential testing may delay treatment and is a less efficient use of limited tissue samples
  • Molecular test results should be available within 10 working days of receiving the specimen in the testing laboratory.

US National Comprehensive Cancer Network (NCCN) clinical guidelines recommend upfront, parallel testing for EGFR, ALK and ROS1 in patients with non-squamous NSCLC at diagnosis of advanced disease.4 Parallel testing for EGFR, ALK and ROS1 is also supported by an EU expert working group of pathologists.5 Testing is also recommended for patients with squamous cell carcinoma and a never-/light-smoking history.

Click below to read published guidance on molecular testing in NSCLC:

Due to the increasing number of molecular tests that should be performed on NSCLC specimens, tissue sample size should be maximised whenever possible. Tissue handling, processing and sectioning should be standardised to minimise wastage and optimised for the staining procedures and molecular tests required for NSCLC. Histological and cytological specimens are both potentially suitable for ALK and ROS1 testing. If the initial tissue sample is small, three to four spare sections should be cut upfront to avoid tissue loss from recutting.6

Click below to access the IASLC Atlas of ALK and ROS1 Testing in Lung Cancer:

References

  1. Novello S, Barlesi F, Califano R, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2016;27(Suppl. 5):v1–v27
  2. Kerr KM, Bubendorf L, Edelman MJ, et al. Second ESMO consensus conference on lung cancer: pathology and molecular biomarkers for non-small-cell lung cancer. Ann Oncol 2014;25:1681–1690
  3. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors. J Mol Diagn 2013;15:415–453
  4. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: non-small cell lung cancer – Version 2.2017. 2016
  5. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503
  6. Thunnissen E, Bubendorf L, Dietel M, et al. EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations. Virchows Arch 2012;461:245–57

TESTING FOR ALK

Fluorescence in-situ hybridisation

Fluorescence in-situ hybridisation (FISH) involves the hybridisation of a fluorophore-labelled single-stranded DNA probe which is complementary in sequence to the genetic region of interest. The fluorophore signal can be visualised using a fluorescence microscope.

In non-small cell lung cancer (NSCLC) specimens, ALK gene rearrangements can be detected using a break-apart FISH probe assay. The assay contains two fluorophore-labelled probes that flank the break point of the ALK gene, one on the 3’ segment (orange) and one on the 5’ segment (green). Once the FISH test has been performed on a lung cancer sample, the resulting slide is viewed under the microscope.

If rearrangement has occurred, nuclei will contain ‘broken apart’ orange and green signals, which appear separated by at least two signal diameters. If deletion has occurred, nuclei will contain single orange signals. Both instances are an ALK positive result. If no activating rearrangement or deletion in the ALK gene locus has occurred, nuclei will contain fused orange and green signals (either overlapping, adjacent, or less than two signal diameters apart) or nuclei will contain single green signals, in addition to fused signals. This is an ALK negative result.

A specimen is positive for ALK if >25/50 cells are judged positive. A specimen is negative for ALK if <5/50 cells are judged positive. If there is uncertainty, a second count should be conducted and an average calculated. If the average percentage of positive cells is ≥15% (of 100 cells) the sample is considered positive.

Watch the video clips below to see Professor Lukas Bubendorf of the Institute of Pathology, University Hospital Basel, Switzerland, explaining some of the critical steps in the ALK FISH test.

Introduction to testing for ALK and guidance on specimen preparation and processing (https://www.youtube.com/watch?v=LxG1LCqE4gg)

Demonstration of the key steps in the ALK FISH assay (https://www.youtube.com/watch?v=ucUV97PwSgA)

Overview of how to assess ALK FISH slides (https://www.youtube.com/watch?v=xzV326ikei4)

The Vysis ALK Break Apart FISH assay is optimised for lung cancer specimens and identifies multiple types of rearrangements involving the ALK gene locus. Click below to view the package insert.

Although not validated for use with cytological specimens, some laboratories have experienced success with their own modified protocols. Click below to view a protocol for cytological specimens from Professor Bubendorf’s laboratory.

Immunohistochemistry

Dr Erik Thunnissen, Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands, discusses testing for ALK using IHC:

“Immunohistochemical detection of the ALK protein is a valuable screening tool to test NSCLC samples for ALK rearrangements and there have been reports of good concordance between IHC and FISH for the detection of aberrant ALK.1–7 One of the particular challenges with IHC is that even in ALK-rearranged NSCLC, ALK protein expression is relatively low. Standard detection methodology, as used in the identification of ALK-rearranged anaplastic lymphomas, is inadequate for the detection of all cases of ALK-rearranged NSCLC. Currently, there are three primary antibodies commonly referred to in the published literature; clone 5A4 (Novocastra, Leica, but also available pre-diluted from Abcam), ALK1 (Dako) and D5F3 (Ventana/Cell Signaling Technology), which are often used in conjunction with enhanced detection systems for signal amplification (Leica/Novocastra Novolink, Dako Advance, Tyramide, EnVision+, Ventana OptiView).”

[Personal communication from Dr Erik Thunnissen, Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands, and adapted from Thunnissen E, et al. Virchows Arch 2012;461(3):245–257 http://www.ncbi.nlm.nih.gov/pubmed/22825000]

The VENTANA ALK (D5F3) CDx assay is an FDA-approved companion diagnostic and CE-IVD-labelled ALK IHC assay. According to the package insert:

  • A specimen is positive for ALK if there is strong, granular, cytoplasmic, brown staining in the tumour cells (any percentage of positive tumour cells); staining is usually homogenous, with a uniform level of intensity throughout the neoplastic portions of the tumour
  • A specimen is negative for ALK in the absence of strong, granular, cytoplasmic staining in the tumour cells.

Watch the video clip below to see Dr Spasenija Savic Prince of the Institute of Pathology, University Hospital Basel, Switzerland, discuss immunohistochemical detection of ALK in NSCLC. (https://www.youtube.com/watch?v=MmyvLDnkZog)

A positive control slide should be included with every test staining run to confirm reagents are functioning properly and guard against false-negative results. A negative control slide should also be included, to check for background staining and confirm the absence of target antigen labelling. Pathologists should be aware of various artefacts that may lead to false-positive staining, including:

  • Light cytoplasmic stippling in alveolar macrophages
  • Cells of neuronal origin
  • Glandular epithelial staining
  • Cells with lymphocytic infiltrate
  • Normal mucosa in NSCLC (including mucin)
  • Necrotic tumour areas.

Click below to access resources on ALK IHC:

References

  1. Yi ES, Boland JM, Maleszewski JJ, et al. Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: IHC score algorithm for FISH. J Thorac Oncol 2011;6:459–465
  2. Yang P, Kulig K, Boland JM, et al. Worse disease-free survival in never-smokers with ALK+ lung adenocarcinoma. J Thorac Oncol 2012;7:90–97
  3. Paik JH, Choe G, Kim H, et al. Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small cell lung cancer: correlation with fluorescence in situ hybridization. J Thorac Oncol 2011;6:466–472
  4. McLeer-Florin A, Moro-Sibilot D, Melis A, et al. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol 2012;7(2):348–354
  5. Hofman P, Ilie M, Hofman V, et al. Immunohistochemistry to identify EGFR mutations or ALK rearrangements in patients with lung adenocarcinoma. Ann Oncol 2012;23(7):1738–1743
  6. Kim H, Yoo SB, Choe JY, et al. Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression. J Thorac Oncol 2011;6(8):1359–1366
  7. Mino-Kenudson M, Chirieac LR, Law K, et al. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clin Cancer Res 2010;16(5):1561–1571

Reverse Transcription-Polymerase Chain Reaction

A number of different reverse transcription-polymerase chain reaction (RT-PCR)-based approaches can be used to detect the presence of aberrant ALK mRNA transcripts. These methods are highly sensitive and specific, rapid and require relatively little sample material.

RT-PCR-based approaches for identifying ALK+ tumours include:

  • Assays to detect unbalanced expression of the 5’ and 3’ ALK mRNA regions1,2
  • Multiplex PCR assays using primer pairs specific to known ALK fusion variants3

There are many EML4-ALK fusion variants and while the breakpoint of ALK is constantly located before the 5' end of exon 20 where the coding sequence of the kinase domain starts, the breakpoint of EML4 has been observed in various exons. Therefore, multiplex primer sets should be designed to detect all the possible EML4-ALK variants.

The Amoy EML4-ALK Fusion Gene Detection Kit is a CE-IVD-labelled RT-PCR assay to detect EML4-ALK fusion. Click below to view further information:

Watch the video clip below to see Dr Karen Zwaenepoel of University Hospital Antwerp, Belgium, explain the principles of RT-PCR for detecting ALK rearrangements. (https://www.youtube.com/watch?v=qbFKok6HN5o)

Watch the video clip below to see Dr Karen Zwaenepoel of University Hospital Antwerp, Belgium, discuss the advantages and disadvantages of RT-PCR for detecting ALK rearrangements. (https://www.youtube.com/watch?v=BpNLVle6XNk)

References

  1. Wang R, Pan Y, Li C, et al. The use of quantitative real-time reverse transcriptase PCR for 5' and 3' portions of ALK transcripts to detect ALK rearrangements in lung cancers. Clin Cancer Res 2012;18:4725–4732
  2. Gruber K, Horn H, Kalla J, et al. Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay. J Thorac Oncol 2014;9:307–315
  3. Wallander ML, Geiersbach KB, Tripp SR, et al. Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing. Arch Pathol Lab Med 2012;136:796–803

Testing for ROS1

Fluorescence in-situ hybridisation

Fluorescence in-situ hybridisation (FISH) testing for ROS1 gene rearrangements shares the same principles as testing for ALK gene rearrangements, with ROS1 FISH assays generally using a dual-colour break-apart probe design:1

  • Two different coloured fluorophore probes are used to label the 3’ (green) and 5’ (orange) segments of the fusion breakpoint
  • A normal (ROS1 negative) sample will show nuclei with two fused signals
  • ROS1 rearrangement can be visualised as ‘broken apart’ coloured signals:
    • ‘classic’ pattern: one fusion signal (ROS1 negative) and two separated 3’ and 5’ signals
    • ‘atypical’ pattern: one fusion signal (ROS1 negative) and one isolated 3’ signal without the corresponding 5’ signal.

Figure: Example of FISH signal patterns using ROS1 break-apart assay. Vysis LSI ROS1 (Cen) SpectrumGreen Probe and Vysis LSI ROS1 (Tel) SpectrumOrange Probe (Abbott Molecular, IL, USA) on histological specimens.
Taken from Bubendorf L, et al. Virchows Arch 2016.1

For a sample to be judged positive for ROS1 rearrangement, at least 25/50 tumour cells must show positive signals. Samples with <5/50 positive tumour cells are negative for ROS1 rearrangement. If there is uncertainty, a second count should be conducted and an average calculated. If the average proportion of positive cells is ≥15% (of 100 cells) the sample is considered positive.1,2

As with ALK, only intact cells with non-overlapping nuclei should be scored during analysis. To optimise FISH results, use of sections older than 6 months is not advised as this may result in poor hybridisation.1

References

  1. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503
  2. Thunnissen E, Bubendorf L, Dietel M, et al. EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations. Virchows Arch 2012;461:245–257

Immunohistochemistry

Immunohistochemistry (IHC) is an effective screening tool for ROS1-positive NSCLC, with sensitivity of 100% in most studies and specificity of 92–100% using the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA).1

Screening tumours by IHC can avoid unnecessary FISH analysis in ROS1-negative tumours, thereby reducing the cost of testing. It is essential that appropriate positive and negative control slides are included to ensure the IHC test is functioning appropriately. A cell block of the HCC78 cell line which contains the SLC34A2–ROS1 fusion gene can serve as a positive control.1

Positive ROS1 IHC typically reveals finely granular cytoplasmic staining. However, the staining pattern may vary depending on the function and subcellular location of the gene fusion partner. For example:2,3

  • CD74–ROS1 fusion is associated with globular staining
  • EZR–ROS1 fusion is associated with membranous staining.

Figure: Examples of positive ROS1 IHC in histological NSCLC specimens (D4D6 antibody, Ventana BenchMark XT; DAB chromogen).
Taken from Bubendorf L, et al. Virchows Arch 2016.1

Pathologists should be aware of artefacts that may lead to false-positive staining, which may occur in:1

  • Non-neoplastic hyperplastic type II pneumocytes (where weak ROS1 expression can occasionally occur)
  • Alveolar macrophages
  • Osteoclast-type giant cells in bone metastases, where strong, granular cytoplasmic staining has been detected.

As with ALK, a positive control slide should be included with every ROS1 test staining run to confirm reagents are functioning properly and guard against false-negative results. A negative control slide should also be included, to check for background staining and confirm the absence of target antigen labelling.

References

  1. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503
  2. Boyle TA, Masago K, Ellison KE, et al. ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer. Clin Lung Cancer 2015;16:106–111
  3. Yoshida A, Tsuta K, Wakai S, et al. Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers. Mod Pathol 2014;27:711–720

Reverse Transcription-Polymerase Chain Reaction

While FISH and IHC currently form the cornerstone for ROS1 testing, other methodologies are available. Non-in situ approaches which can be used to detect ROS1 gene rearrangements include RT-PCR and next-generation sequencing (NGS). RT-PCR has been successful utilised to identify aberrant ROS1 mRNA transcripts with a sensitivity of 100% and a specificity of 85–100%, using FISH as the reference standard method.1–3

RT-PCR-based-approaches for identifying ROS1+ tumours include:

  • Multiplex PCR assays using primer sets specific to known ROS1 fusion variants4,5
  • Assays which utilise a dual capture and reported probe system to detect known ROS1 fusion gene transcripts.4,6

The AmoyDx ROS1 Gene Fusions Detection Kit is a CE- IVD-labelled RT-PCR assay to detect 14 ROS1 gene fusions.

References

  1. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503
  2. Shan L, Lian F, Guo L, et al. Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR. PLoS One 2015;10:e0120422
  3. Cao B, Wei P, Liu Z, et al. Detection of lung adenocarcinoma with ROS1 rearrangement by IHC, FISH, and RT-PCR and analysis of its clinicopathologic features. Onco Targets Ther 2016;9:131–138
  4. Lee SE, Lee B, Hong M, et al. Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma. Mod Pathol 2015;28:468–479
  5. Ribeiro-Silva A, Zhang H, Jeffrey SS. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol Biol 2007;8:118
  6. Lira ME, Choi YL, Lim SM, et al. A single-tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer. J Mol Diagn 2014;16:229–243

ROS1 Testing Algorithm

Integrating ROS1 into current testing algorithms

In order to achieve efficient use of tissue and quick turn-around of testing results, this algorithm suggests testing for EGFR, ALK and ROS1 in parallel.

Figure: Algorithm for predictive genetic testing in advanced NSCLC in routine practice.
Taken from Bubendorf L, et al. Virchows Arch 2016.1

Watch the video clip below to see Dr Sanjay Popat from The Royal Marsden, London, UK, give an overview of how, when and who to test for ROS1 fusions. (https://www.youtube.com/watch?v=vz4e0uqt5qc)

External quality assessment

Laboratories conducting molecular testing of NSCLC specimens should consider participating in EQA programmes, which can help to ensure and enhance proficiency in molecular testing. Two pathology societies are listed below which are conducting EQA programmes for ROS1 testing.

European Society of Pathology EQA

http://lung.eqascheme.org

UK NEQAS

https://www.ukneqas-molgen.org.uk/molecular-pathology

Reference

  1. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503

Testing for EGFR

DNA Sequencing

The most common method for testing non-small cell lung cancer (NSCLC) tissue samples for epidermal growth factor receptor (EGFR) mutations is direct DNA sequencing.1 Historically, this has meant Sanger sequencing, although newer methods and testing kits are available.1,2 Immunohistochemistry (IHC) and fluorescence in-situ hybridisation (FISH) are not recommended for EGFR testing.2

Sanger sequencing gives a direct read out of the DNA sequence from a DNA sample. Reading the sequence can confirm the presence or absence of an EGFR mutation and the type of mutation present. EGFR mutations are well characterised with the two most common, representing 90% of all EGFR mutations, occurring in short in-frame deletions in exon 19 and the L858R point mutation in exon 21.2,3 Patients harbouring these common mutations tend to respond better to EGFR inhibitors than patients with other, less common mutations.3

The main drawback of Sanger sequencing is sensitivity. A given mutation will not be detected unless it is present in at least 25% of the DNA sample.1 In cases where the mutation is heterozygous, this would mean that at least 50% of the targeted cells need to harbour the mutation for Sanger sequencing to detect it. As a result, some guidelines only recommend Sanger sequencing if >30% of the sample cells are tumour cells.4

For this reason, test selection and sample selection are important in EGFR testing. Due to the prevalence of the more common mutations, targeted kits that detect only these mutations have been developed. These kits, commonly based on PCR amplification of the target regions, are able to detect EGFR mutations present in as low as 2.5% of the sample DNA.1 These targeted kits offer a higher throughput and good sensitivity, but their inability to detect uncommon EGFR mutations means that results often have to be verified by Sanger sequencing.1

Given the preference for high concentrations of tumour DNA in EGFR mutation testing, the tissue sample should be enriched in tumour cells. A larger tumour sample, such as a resection, is preferred as manual dissection can increase the tumour cell concentration.2 Cytological samples, where tumour DNA is at a lower concentration, can be tested using more sensitive test kits e.g. PCR-based methods.1

References

  1. Ellison G, Zhu G, Moulis A, et al. EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples. J Clin Pathol 2013;66:79–89
  2. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol 2013;8:823–859
  3. Wu JY, Yu CJ, Change YC, et al. Effectiveness of Tyrosine Kinase Inhibitors on “Uncommon” Epidermal Growth Factor Receptor Mutations of Unknown Clinical Significance in Non–Small Cell Lung Cancer. Clin Cancer Res 2011;7:3812–3821
  4. NICE. EGFR-TK mutation testing in adults with locally advanced or metastatic non-small cell lung cancer. Diagnostic guidance 2013. Available at: https://www.nice.org.uk/guidance/dg9/chapter/1-Recommendations (accessed March 2017).

Liquid Biopsy

This is a blood test that detects evidence of cancer cells or tumour DNA in the circulation. This technique has generated a great deal of interest due to its ability to detect driver oncogenes, such as epidermal growth factor receptor (EGFR) mutants, in non-small cell lung cancer (NSCLC). Other biological fluids such as saliva and urine are also used to detect EGFR mutations as they contain a variety of biomolecules, such as DNA, mRNA, miRNA, proteins and metabolites that can be used to detect mutations missed in biopsies because of tumour heterogeneity or inadequate sample quality.1 In September 2014, the Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion for circulating tumour DNA based-assessment of EGFR mutation to be used when prescribing an EGFR TKI.2 Following this, in September 2015, the cobas® EGFR Mutation Test v2 was launched in Europe in countries that accept the CE mark.3 This device is validated for use with either plasma or tumour tissue samples to identify EGFR mutations, including T790M. These advances mean patients who have locally advanced or metastatic NSCLC, without available or evaluable tumour samples for EGFR mutation analysis, could benefit from this non-invasive testing.

 

References

  1. Huang W, Chen Y, Yang S, et al. Liquid biopsy genotyping in lung cancer: ready for clinical utility? Oncotarget 2017;Epub ahead of print
  2. AstraZeneca. Press Release. September 2014. https://www.astrazeneca.com/media-centre/press-releases/2014/iressa-chmp-positive-opinion-blood-based-diagnostic-testing-european-label-26092014.html#! (Accessed March 2017)
  3. Roche. Press Release. September 2015. http://www.roche.com/media/store/releases/med-cor-2015-09-28.htm (Accessed March 2017)

Next-generation Sequencing (NGS)

Next-Generation Sequencing

The rapid development of technologies for large-scale sequencing (NGS) has facilitated high-throughput molecular analysis that offers various advantages over traditional sequencing, including the ability to fully sequence large numbers of genes in a single test and simultaneously detect deletions, insertions, copy number alterations, rearrangements and exome-wide base substitutions (including known hot-spot mutations) in all known cancer-related genes.1

Currently, NGS platforms, including whole genome, whole exome, and targeted gene sequencing, represent emerging diagnostic methodologies for the detection of oncogene fusions and mutations in tumour tissue specimens, including formalin-fixed paraffin-embedded tissue samples.2,3

Several NGS strategies for detecting gene fusions have been developed, including hybrid-capture-based target enrichment, multiplex amplicon RNA massive parallel sequencing, personalised analysis of rearranged ends (PARE) and anchored multiplex PCR (AMP).4–7 A number of commercial NGS panels covering ALK and ROS1 gene fusions are available, including the Thermo Fisher Oncomine Fusion panel and the ArcherDx FusionPlex™ v2 panel.

Watch the video clip below to see Dr Karen Zwaenepoel of University Hospital Antwerp, Belgium, explain the principles of NGS for detecting ALK rearrangements. (https://www.youtube.com/watch?v=MMe1rG8vfZ0)

References

  1. Ross J, Cronin M. Whole cancer genome sequencing by next-generation methods. Am J Clin Pathol 2011;136:527–539
  2. Lipson D, Capelletti M, Yelensky R, et al. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nature Med 2012;18:382–384
  3. Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nature Med 2012;18:378–381
  4. Drilon A, Wang L, Arcila ME, et al. Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in lung adenocarcinomas otherwise negative for such alterations by other genomic testing approaches. Clin Cancer Res 2015;21(16):3631–3639
  5. Moskalev EA, Frohnauer J, Merkelbach-Bruse S, et al. Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing. Lung Cancer 2014;84(3):215–221
  6. Zheng Z, Liebers M, Zhelyazkova B, et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med 2014;20(12):1479–1484
  7. Leary RJ, Kinde I, Diehl F, et al. Development of personalized tumor biomarkers using massively parallel sequencing. Sci Transl Med 2010;2(20):20ra14

Testing for PD-L1

Testing for PD-L1

Programmed death receptor 1 (PD-1) is expressed on surfaces of immune cells, particularly activated lymphocytes, with a normal function to down-modulate unwanted or excessive immune responses. Cancer cells can express high levels of PD-1 ligand (PD-L1) on their surface and thus bind the PD-1 receptor on activated immune cells within the tumour microenvironment, effectively switching them off. In non-small cell lung cancer (NSCLC), the binding of PD-1 and PD-L1 protects the tumour from immune attack, and inhibition of either of these by monoclonal antibodies removes this protective effect resulting in exposure of the tumour to immune destruction.1,2

Various monoclonal antibodies which block the interaction between checkpoint molecules, PD-1 on immune cells and PD-L1 on cancer cells, have been used to treat NSCLC and there is considerable interest in using PD-L1 immunohistochemical staining to guide the use of these targeted treatments in patients. Several PD-L1 immunohistochemistry (IHC) antibodies and assays are available or in development, which have been established with corresponding staining methodologies and automated platforms. Laboratories should adhere to the recommended staining protocols of the manufacturer, as protocol modifications might lead to false-positive or false-negative PD-L1 results.3–5

PD-L1 kits and platforms available3,5

Methodology

Kit assay

Automated platform

Definition of positive test

Kit-based assay*

Dako 22C3 pharmDx

Dako Autostainer
Link 48

≥1% membranous staining of TC or IC that are intercalating or at the tumour interface

 

Dako PD-L1 IHC 28-8 pharmDx

Dako Autostainer
Link 48

≥5% membranous staining of TC (minimum 100 cells evaluated)

 

VENTANA PD-L1 (SP142)

VENTANA BenchMark (GX, XT and Ultra)

Each specimen assigned a score based on both tumour and immune cell PD-L1:

  • TC3/IC3 PD-L1 ≥50%
  • TC2/IC2 PD-L1 5-49%
  • TC1/IC1 PD-L1 1-4%
  • TC0/IC0 PD-L1 <1%

 

VENTANA PD-L1 (SP263)

Ventana Benchmark platforms (GX, XT
and Ultra)

≥25% membranous staining of TC

Standalone PD-L1 antibodies

28-8 (RabMAb): Bristol Myers Squibb clone available from Abcam

E1L3N (RabMAb): Cell Signaling Technology

SP142 (RabMAb): Spring Bioscience

*CE-IVD-marked predictive assay; Research use only
IC, immune cells; TC, tumour cells

It should be noted that to date, different approaches have been taken when assessing PD-L1 IHC, using different diagnostic antibodies to assess PD-L1 expression, different technical staining platforms and different definitions of a “positive” predictive IHC stain. In some cases, expression of PD-L1 on immune cells as opposed to, or in combination with, expression in tumour cells, has been chosen as the biomarker. To try and enable a better understanding of the similarities and differences between the four PD-L1 assays, the Blueprint PD-L1 IHC Assay Comparison Project was founded. This is an industrial-academic collaborative partnership to provide information on the analytical and clinical comparability between the PD-L1 assays to better understand their technical performance.6,7

The ESP Lung EQA Scheme 2017 is running a pilot for PD-L1 which provides information on how well a laboratory’s assay has worked, and any potential staining issues that may lead to false-positive or false-negative results.

References

  1. Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy--inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res 2012;18:6580–6587
  2. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207–212
  3. Cree IA, Booton R, Cane P, et al. PD-L1 testing for lung cancer in the UK: recognizing the challenges for implementation. Histopathology 2016;69:177–186
  4. Chen YB, Mu CY, Huang JA. Clinical significance of programmed death-1 ligand-1 expression in patients with non-small cell lung cancer: a 5-year-follow-up study. Tumori 2012;98:751–755
  5. Grigg C and Rizvi NA. PD-L1 biomarker testing for non-small cell lung cancer: truth or fiction? J Immunother Cancer 2016;4:48
  6. Kerr KM, Tsao MS, Nicholson AG, et al. Programmed Death-Ligand 1 Immunohistochemistry in Lung Cancer: In what state is this art? J Thorac Oncol 2015;10:985–899
  7. Hirsch FR, McElhinny A, Stanforth D, et al. PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project. J Thorac Oncol 2017;12:208–222

Practical Considerations

Practical Considerations

Oncogenic drivers are generally mutually exclusive and patients with ROS1 rearrangements tend to share similar characteristics to those commonly associated with ALK-positive non-small cell lung cancer (NSCLC).1–3 Consequently, when ROS1 testing is required, it is reasonable to test the same tumours currently being selected for EGFR mutation and ALK gene rearrangement. If ROS1 testing is performed in parallel rather than delayed until ALK rearrangements and EGFR mutations are proven negative, this will save tissue and time.4 If this is not possible, extra blank sections can be obtained when the tumour sample is first cut.2,4

It is essential that validated tests are used for routine testing with appropriate controls and participation in external quality assessment schemes is advised.


Figure: Preparation of tissue sections during diagnostic workup.
Current routine practice involves making additional sections for IHC assay and/or molecular testing after the initial sectioning and hematoxylin and eosin (H & E) staining for histologic diagnosis. Multiple sequential sectioning may deplete the tumor volume each time block trimming is necessary. In the era of molecularly targeted therapies, the preparation of additional unstained sections for possible IHC analysis and/or molecular testing may significantly reduce the amount of tissue sample lost and improve turnaround time.

External quality assessment

It is essential that molecular test results are accurate, reliable, reported in a timely manner and can be clearly understood by the clinician. Laboratories conducting molecular testing of NSCLC specimens should consider participating in external quality assessment (EQA) programmes as they can help to ensure and enhance proficiency in molecular testing.

Watch the video clip below to see Professor Els Dequeker of the University of Leuven, Belgium, give an overview of the European Society of Pathology’s EQA scheme. (https://www.youtube.com/watch?v=eVM7BhlZElI)

References

  1. Gainor JF, Varghese AM, Ou SH, et al. ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer. Clin Cancer Res 2013;19:4273–4281
  2. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469(5):489–503
  3. Manning G, Whyte DB, Martinez R, et al. The protein kinase complement of the human genome. Science 2002;298(5600):1912–1934
  4. Tsao MS, Hirsch FR, Yatabe Y, eds. IASLC Atlas of ALK and ROS1 testing in lung cancer. International Association for the Study of Lung Cancer. Editorial Rx Press. 2016
Further Information
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