EGF Receptor Testing for Non‐Small Cell Lung Carcinomas

Juan‐Sebastian Saldivar1, Zhenbin Chen1, Steve Sommer1

1 City 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

  • Basic Protocol 1: Preparation of Slides from Paraffin‐Embedded Tumor Tissue
  • Support Protocol 1: 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 protocol 2)
  • Microtome
  • Slides and coverslips
  • Microscope
  • Extra‐fine‐tipped permanent marker
  • Slide holders

Support Protocol 1: Hematoxylin and Eosin (H&E) Staining of Slides

  Materials
  • Slides containing tumor sections ( 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 MgCl 2 (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 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 MgCl 2 (Roche)
  • 1.25 mM dNTPs
  • 1.0 µg/µl BSA
  • PCR‐grade H 2O
  • 5 pmol/µl first‐round amplification PCR primers (see Table 10.9.1)
  • 1 ng/µl digested DNA (see protocol 3)
  • 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 0.9.1   MaterialsEGFR External PCR Primers

    Exon Primer Primer Sequence 5′→3′ Primer length Fragment size
    18 EGFR18D GCG TGG AAA CAG ACA TAG A 19 547 bp
    EGFR18U TAA CTT GGG AAA AAC ACT GG 20
    19 EGFR19D TGT GAT TCG TGG AGC CCA AC 20 484 bp
    EGFR19U AGG CCA GTG CTG TCT CTA AG 20
    20 EGFR20D ACT TCA CAG CCC TGC GTA A 19 465 bp
    EGFR20U TAT CCC CAT GGC AAA CTC TT 20
    21 EGFR21D TGG ATC AGT AGT CAC TAA CGT 21 413 bp
    EGFR21U GAA TGT CTG GAG AGC ATC CT 20

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 protocol 4)
  • 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)

Basic Protocol 5: Sequence Analysis

  Materials
  • Purified cycle sequence products (see protocol 5, 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)
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Figures

Videos

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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