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Genomic Applications in Pulmonary Malignancies

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Genomic Applications in Pathology

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Abstract

Lung cancer has been the subject of intensive research in the last decade resulting in the translation of basic scientific findings in clinical practice. Several molecular alterations have been defined as “driver mutations” in non-small-cell lung cancer (NSCLC) providing novel targets for lung cancer treatment. This requires, besides a close interaction between researchers, clinicians, and pathologists, the implementation of high-quality molecular diagnostics. Approved personalized therapy approaches include treatment of epidermal growth factor receptor (EGFR) mutant NSCLC with EGFR-directed tyrosine kinase inhibitors, and treatment of NSCLC patients with rearrangements of the anaplastic lymphoma kinase (ALK) oncogene with the ALK inhibitor crizotinib. The clinical relevance of other alterations as mutations and gene amplifications of KRAS, BRAF, HER2, PIK3CA, DDR2, FGFR1, and MET, and chromosomal translocations of RET and ROS1 are currently under investigation in clinical trials. In this context, methods for the detection of these alterations are outlined in this chapter taking into account the suboptimal quality and quantity of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue.

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Authors and Affiliations

  1. Center for Integrated Oncology, Institute of Pathology, University Hospital Cologne, Köln-Bonn, Cologne, Germany

    Reinhard Büttner, Margarete Odenthal & Sabine Merkelbach-Bruse

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  1. Reinhard Büttner
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  2. Margarete Odenthal
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  3. Sabine Merkelbach-Bruse
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Editor information

Editors and Affiliations

  1. Departments of Pathology, Oncology and Urology, Johns Hopkins University, Baltimore, Maryland, USA

    George Jabboure Netto

  2. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Iris Schrijver

Notes

Notes

  1. Note 1.

    Isolating DNA from FFPE tissue

    The amount of tumor cells in a FFPE tissue block may be highly variable. Therefore, an experienced pathologist should review each tumor block, indicate the area of highest proportion of tumor cells, and record his estimate of the percentage of tumor cells prior to analysis. Macrodissection in cases with a larger amount of non-tumorous tissue (more than 20 % of the overall area) may be of use to reduce the amount of wild-type DNA.

  2. Note 2.

    Avoiding contamination during DNA isolation and amplification

    In general, because PCR amplification is a highly sensitive method and susceptible to carryover contamination, the following fundamental rules should be the gold standard in every diagnostic molecular pathology laboratory. The working area should be divided in pre- and post-PCR sections, optimally three independent rooms for extracting DNA, preparing the PCR, and performing the post-PCR analysis. Each section should have specifically assigned equipment and reagents. Plugged pipette tips have to be used throughout and gloves to be changed between the working sections. Also, during cutting the paraffin blocks by microtome, some rules have to be observed to avoid contamination between blocks:

  3. 1.

    Blocks that are proposed for the same analysis should not be cut consecutively.

  4. 2.

    The microtome and the working area should be cleaned from paraffin material after each individual block.

  5. 3.

    Depending on the type of microtome, the knives should be removed or relocated after each block.

  6. 4.

    If the sections are mounted on slides for manual microdissection, the water bath should be cleaned regularly with filter paper and the water should be changed as often as possible.

  7. Note 3.

    Quantity and quality of isolated DNA

    Because the PCR result depends on the number of amplifiable fragments variable amounts of DNA should be used as PCR template, depending on both DNA quantity and extend of DNA fragmentation. It is strongly recommended not to analyze samples with poor DNA quality. In such cases, additional material (e.g., fresh frozen tissue, if available, or another paraffin block) should be used.

  8. Note 4.

    Control reactions

    For each amplification experiment, positive and negative controls should be carried along. A sample with water instead of DNA serves as negative control; a positive control may be DNA extracted from FFPE tissue which was amplified successfully in a previous analysis. The control reactions should be checked by agarose gel electrophoresis. The number of PCR cycles should not exceed 40 cycles. If amplification failed twice, even after sample purification, the analysis may be stopped at this point. The analysis can be retried with another paraffin block.

  9. Note 5.

    Setting up HRM PCR

    The amplicon size (the shorter the better), exclusion of primer dimers, salt concentration, specific melting products with only one single melting domain, and standardized genomic DNA isolation protocols are important points for implementation of highly sensitive HRM assays.

    Each run should include mutated and wild-type DNA as controls. Primers can be designed with the LightCycler Probe Design 2.0 software (Roche Diagnostics) and should be checked for specificity by using the basic local alignment software tool (BLAST) from the National Center for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Performing at least duplicates is necessary to minimize temperature differences on the microtiter plate.

  10. Note 6.

    Evaluation of Fragment Length Analysis

    DNA extracted from the following cell lines can be used as positive controls:

    Human adenocarcinoma cell line PC9, carrying the mutation p.E746_A750 del.

    Human adenocarcinoma cell line NCI-H1975 (ATCC# CRL-5908), wild type for EGFR exon 19

    If wild ty pe, PCR fragments should be 217 bp in length for the first round and 117 bp for the second round.

  11. Note 7.

    Evaluation of ALK, RET, and ROS translocations

    Chromosomal rearrangements with ALK comprise the inversion on chromosome 2 leading to a fusion with EML4 and the translocation affecting ALK, but not EML4, such as ALK-TGF or ALK-KIF5B. Therefore, the use of a break-apart assay is recommended.

    Evaluation is described here for the ZytoLight ® SPEC ALK/EML4 TriCheck™ (Zytovision, Bremerhaven, Germany). This probe system is designed to discriminate between ALK inversions and translocations.

    The assay consists of three differently labelled probes, where two probes hybridize distal and proximal to the ALK gene breakpoint region, respectively, and the third probe binds to the EML4 gene. In an interphase nucleus of a normal cell, two orange/green fusion signals and two blue signals are expected. The EML4-ALK inversion is indicated by one separate green signal, one separate orange signal, and an additional blue signal. The separate green and orange signals each co-localize with a blue signal. During inversion, the 5′ part of ALK can be deleted, so the separate green signal is lost. A signal pattern consisting of one orange/green fusion signal, one orange signal, and a separate green signal as well as two blue signals indicate an ALK translocation without involvement of EML4.

    Break-apart signals have to be detected in at least 15 % of nuclei. Hundred Nuclei are counted to detect the rearrangement. Only nuclei with non-overlapping signals and with the expected number of signals are evaluated. To count signals as two, they have to be separated by at least two signal diameters.

    ROS has several rearrangement partners, and an interstitial deletion as well as a translocation may occur. Therefore, the use of a break-apart assay, which is commercially available, is recommended. Typical break-apart signal patterns as described above for ALK are expected.

    RET inversion on chromosome 10 leads to a fusion between KIF5B and RET. A break-apart assay for both genes can be used but is not yet commercially available for RET. If a KIF5B rearrangement is detected, involvement of ALK has to be ruled out using the ALK probe set described above.

  12. Note 8.

    Evaluation of FGFR1 amplification

    The evaluation of FGFR1 amplification is done according to Schildhaus et al. [117], and is described here with the usage of the ZytoLight SPEC FGFR1/CEN 8 Dual Color Probe (ZytoVision, Bremerhaven, Germany). The probe specific for the centromeric region of chromosome 8 is labelled with an orange fluorochrome, the probe specific for the gene region of FGFR1 is labelled in green.

    Some general points have to be considered when counting fluorescent signals [117]:

    • Scan the entire tumor area for hot spots of increased FGFR1 copy numbers.

    • Count 20 tumor cell nuclei in three areas, either in three hot spots or in three random areas in case of homogeneous signal distribution. Count cohesive tumor cells; do not selectively consider isolated amplified tumor cells from different areas.

    • Count only clearly distinct signals as two separate signals. Count FGFR1 signal doublets and triplets as one signal. In cases of signal clusters give cluster estimation in steps of five signals, for example, 15, 20, or 25 FGFR1 signals. Count micro-clusters as five signals.

    Green FGFR1 and orange centromere 8 signals are counted separately. The FGFR1/CEN8 ratio, the number of cells with ≥5 and ≥15 FGFR1 signals and the average FGFR1 copy number per cell are calculated. Cases are considered as FGFR1 positive (“amplified”) under one of the following conditions:

    1. (1)

      The FGFR1/CEN8 ratio is ≥2.0.

    2. (2)

      The average number of FGFR1 signals per tumor cell nucleus is ≥6.

    3. (3)

      The percentage of tumor cells containing ≥15 FGFR1 signals or large clusters is ≥10 %.

    4. (4)

      The percentage of tumor cells containing ≥5 FGFR1 signals is ≥50 %, with (1–3) representing a high-level and (4) a low-level amplification.

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Büttner, R., Odenthal, M., Merkelbach-Bruse, S. (2015). Genomic Applications in Pulmonary Malignancies. In: Netto, G., Schrijver, I. (eds) Genomic Applications in Pathology. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-0727-4_24

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