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EGF Receptor Testing for Non-Small Cell Lung Carcinomas

Juan‐Sebastian Saldivar1,  Zhenbin Chen1,  Steve Sommer1

1City of Hope National Medical Center, Duarte, California

Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
UNIT 10.9
DOI: 
10.1002/0471142905.hg1009s50
Online Posting Date: 
August, 2006
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Abstract

Non-small cell lung cancer (NSCLC) is one of the most common cancers worldwide. An estimated 170,000 new diagnoses are expected this year. Recently, small molecule inhibitors directed at the EGFR kinase domain were approved for the treatment of advanced stages of NSCLC. Genotyping of the EGFR kinase domain has proven to be a useful marker for predicting who will respond to these novel medications. This unit provides a protocol to perform mutation analysis on the EGFR kinase domain where mutations have been associated with significant responsiveness to these EGFR inhibitors. The protocol includes microdissection of tumor tissue from slides, DNA digestion of these cells, amplifying and sequencing pertinent segments of the EGFR gene, and interpretation of the data. The protocol is designed with appropriate redundancy to eliminate allele dropout and to maximize detection of somatic mutations within the tumor.

Keywords: non-small cell lung carcinoma; EGFR; erbB-1; lung cancer

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Preparation of Slides from Paraffin-Embedded Tumor Tissue
  • Support Protocol: Hematoxylin and Eosin (H&E) Staining of Slides
  • Basic Protocol 2: Microdissection and Digestion of Tumor Cells from Slides
  • Basic Protocol 3: Polymerase Chain Reaction (PCR) Amplification of Exons 18-21 of the EGFR Gene and PCR Product Purification
  • Basic Protocol 4: Nested Cycle Sequencing of Purified PCR Products
  • Basic Protocol 5: Sequence Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: Preparation of Slides from Paraffin-Embedded Tumor Tissue

 Materials
  • Paraffin-embedded tumor block
  • Hematoxylin and eosin (see Support Protocol)
  • Microtome
  • Slides and coverslips
  • Microscope
  • Extra-fine-tipped permanent marker
  • Slide holders

Support Protocol: Hematoxylin and Eosin (H&E) Staining of Slides

 Materials
  • Slides containing tumor sections (Basic Protocol 1)
  • Hematoxylin
  • 1% (v/v) alcoholic eosin
  • Xylene
  • 70%, 80%, 95%, and 100% alcohol
  • 1% (v/v) acid alcohol
  • 0.2% (v/v) ammonium hydroxide
  • Coplin jars
  • Slide holder or tweezers

Basic Protocol 2: Microdissection and Digestion of Tumor Cells from Slides

 Materials
  • 10× no. 3 high-fidelity digestion buffer, without MgCl2 (Roche)
  • Proteinase K (Sigma)
  • Tween 20 solution (J.B.T. Baker)
  • EDTA (Sigma)
  • Fish DNA (optional; CeMines Biosystems (http://www.cemines.com))
  • H&E-stained tumor sample slides (see Basic Protocol 1)
  • 100% ethanol
  • 200-µl PCR tubes with caps
  • Inverted microscope with 4×, 10×, and 20× objectives
  • 27 1/2-G needles
  • Thermal cycler (Applied Biosystems 9700 or equivalent) or 50° and 90°C waterbaths

Basic Protocol 3: Polymerase Chain Reaction (PCR) Amplification of Exons 18-21 of the EGFR Gene and PCR Product Purification

 Materials
  • 5 U/µl AmpliTaq gold polymerase and 10× PCR buffer with 15 mM MgCl2 (Roche)
  • 1.25 mM dNTPs
  • 1.0 µg/µl BSA
  • PCR-grade H2O
  • 5 pmol/µl first-round amplification PCR primers (see Table 10.9.1)
  • 1 ng/µl digested DNA (see Basic Protocol 2)
  • 2% (w/v) agarose gel in 1× TAE (unit 7.7)
  • DNA ladder
  • 10 U/µl exonuclease I enzyme (USB)
  • 2 U/µl shrimp alkaline phosphatase enzyme (USB)
  • Thermal cycler (Applied Biosystems 9700 or equivalent)
     
    Table 10.9.1 EGFR External PCR Primers
    ExonPrimerPrimer Sequence 5¢3¢Primer lengthFragment size
    18EGFR18DGCG TGG AAA CAG ACA TAG A19547 bp
    EGFR18UTAA CTT GGG AAA AAC ACT GG20
    19EGFR19DTGT GAT TCG TGG AGC CCA AC20484 bp
    EGFR19UAGG CCA GTG CTG TCT CTA AG20
    20EGFR20DACT TCA CAG CCC TGC GTA A19465 bp
    EGFR20UTAT CCC CAT GGC AAA CTC TT20
    21EGFR21DTGG ATC AGT AGT CAC TAA CGT21413 bp
    EGFR21UGAA TGT CTG GAG AGC ATC CT20

Basic Protocol 4: Nested Cycle Sequencing of Purified PCR Products

 Materials
  • BigDye Terminator v1.1 cycle sequencing kit and 5× sequencing buffer (Applied Biosystems)
  • 20 to 60 ng/µl purified PCR products (see Basic Protocol 3)
  • 1.5 pmol/µl sequencing primers (see Table 10.9.2)
  • CleanSeq magnetic purification beads (Agencourt)
  • 85% ethanol (freshly made)
  • 0.3 mM EDTA (elution buffer)
  • 200-µl PCR tubes
  • Thermal cycler (Applied Biosystems 9700 or equivalent)
  • Magnetic purification tray (Agencourt)
 
Table 10.9.2 EGFR Internal PCR/Sequencing Primers
ExonPrimerPrimer Sequence 5¢3¢Primer lengthFragment size
18EGFR18DNCCA AAT GAG CTG GCA AGT GC20352 bp
EGFR18UNTAT ACA GCT TGC AAG GAC TCT21
19EGFR19DNTGC ATC GCT GGT AAC ATC CA20279 bp
EGFR19UNTCT AGA GCA GAG CAG CTG C19
20EGFR20DNCTC AAG ATC GCA TTC ATG CG20370 bp
EGFR20UNCTC TTG CTA TCC CAG GAG C19
21EGFR21DNATG ACC CTG AAT TCG GAT GC20304 bp
EGFR21UNAAT ACA GCT AGT GGG AAG GC20

Basic Protocol 5: Sequence Analysis

 Materials
  • Purified cycle sequence products (see Basic Protocol 4, step ; use entire 30 µl of eluted sample)
  • Automated sequencer (ABI 3730 from Applied Biosystems or equivalent)
  • Sequencher software (Gene Codes or any other analysis software)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 10.9.1
    Flowchart describing the EGFR testing process.

    View Image
  • Figure 10.9.2
    This image shows the characteristic appearance of adenocarcinoma of the lung at 20× magnification. Note the large size, and the large irregular nuclei of the tumor cells.

    View Image
  • Figure 10.9.3
    Image of an uncoverslipped sample prior to dissection, at 10× magnification. Note the clusters of tumor cells scattered within surrounding lung tissue and inflammatory cells.

    View Image
  • Figure 10.9.4
    Image of the same slide from Figure 10.9.3, post-dissection, at 10× magnification. Note how the clusters of tumor cells were delicately dissected from the surrounding normal tissue.

    View Image
  • Figure 10.9.5
    Schematic diagram of the EGFR gene. Each line represents an exon. The gray lines represent the EGFR kinase domain (exons 18-24), and the numbered lines represent the exons where all somatic mutation in EGFR have been reported thus far (numbered exons 18-21). Cen is the centromeric side of the gene. Tel is the telomeric side of the gene.

    View Image

Literature Cited

Literature Cited
    Fukuoka, M., Yano, S., Giaccone, G., Tamura, T., Nakagawa, K., Douillard, J.Y., Nishiwaki, Y., Vansteenkiste, J., Kudoh, S., Rischin, D., Eek, R., Horai, T., Noda, K., Takata, I., Smit, E., Averbuch, S., Macleod, A., Feyereislova, A., Dong, R.P., and Baselga, J. 2003. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J. Clin. Oncol. 21:2237-2246.
    Kobayashi, S., Boggon, T.J., Dayaram, T., Janne, P.A., Kocher, O., Meyerson, M., Johnson, B.E., Eck, M.J., Tenen, D.G., and Halmos, B. 2005. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. New Engl. J. Med. 352:786-792.
    Kosaka, T., Yatabe, Y., Endoh, H., Kuwano, H., Takahashi, T., and Mitsudomi, T. 2004. Mutations in the epidermal growth factor receptor gene in lung cancer: Biological and clinical implications. Cancer Res. 64:8919-8923.
    Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237-238.
    Lynch, T.J., Bell, D.W., Sordella, R., Gurubhagavatula, S., Okimoto, R.A., Brannigan, B.W., Harris, P.L., Haserlat, S.M., Supko, J.G., Haluska, F.G., Louis, D.N., Christiani, D.C., Settleman, J., and Haber, D.A. 2004. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. New Engl. J. Med. 350:2129-2139.
    Paez, J.G., Janne, P.A., Lee, J.C., Tracy, S., Greulich, H., Gabriel, S., Herman, P., Kaye, F.J., Lindeman, N., Boggon, T.J., Naoki, K., Sasaki, H., Fujii, Y., Eck, M.J., Sellers, W.R., Johnson, B.E., and Meyerson, M. 2004. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497-1500.
    Pao, W., Miller, V., Zakowski, M., Doherty, J., Politi, K., Sarkaria, I., Singh, B., Heelan, R., Rusch, V., Fulton, L., Mardis, E., Kupfer, D., Wilson, R., Kris, M., and Varmus, H. 2004. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. U.S.A. 101:13306-13311.
    Ries, L.A.G., Kosary, C.L., Hankey, B.F., Miller, B.A., and Clegg, L. (eds.) 1999. SEER Cancer Statistics Review, 1973-1996. National Cancer Institute, Bethesda, Md.
    Williams, C., Ponten, F., Moberg, C., Soderkvist, P., Uhlen, M., Ponten, J., Sitbon, G., and Lundeberg, J. 1999. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am. J. Path. 155:1467-1471.
 Internet Resources
    http://www.cityofhope.org/cmdl/egfr_db/index.html

EGFR mutation database.

    http://www.genecodes.com

Sequencher software, demo product available for download.

     
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