Presentation on theme: "MOLECULAR PREDICTIVE MARKERS OF LUNG CARCINOMA: KFSH&RC EXPERIENCE"— Presentation transcript:
1 MOLECULAR PREDICTIVE MARKERS OF LUNG CARCINOMA: KFSH&RC EXPERIENCE 25th IAP-Arab Division Conference07-09 November 2013, Amman, JordanMOLECULAR PREDICTIVEMARKERS OF LUNG CARCINOMA:KFSH&RC EXPERIENCEFouad Al Dayel, MD, FRCPA, FRCPathProfessor and ChairmanDepartment of Pathology and Laboratory MedicineKing Faisal Specialist Hospital and Research CentreRiyadh, Saudi Arabia
2 Lung Cancer Leading cause of cancer mortality 1.4 million death/year worldwide(WHO, 2007) 160,000 death/year in USA(25% of all cancer death in USA) 5-year survival of lung cancer6-15%
7 WHO Classification of Lung Cancer 19671981199920042015Written by pathologistsfor pathologistsGenetic and clinical informationintroduced (not current anymore)5th Edition
8 Lung Carcinoma Small cell carcinoma Non-small cell lung carcinoma (NSCLC)- Adenocarcinoma (includingbronchioalveolar carcinoma BAC)- Adenosquamous carcinoma- Squamous cell carcinoma- Large cell carcinoma- Large cell neuroendocrine carcinoma
9 WHO Classification of Lung Cancer Classification is based on resected specimen. On small biopsy, the differentiation of various subtypes of NSCLC is not reliable in many cases.NSCLC - NOSWHO Classification of Lung Cancer, 2004
10 Revised classification that emphasizes: Integrated multidisciplinary approachfor classification is needed Classification in small biopsies andand cytology specimen (was notaddressed in 2004 WHO Classification) Tissue management by pathologists
11 International Association for the Study of Lung Cancer/American Thoracic Society/EuropeanRespiratory Society International MultidisciplinaryClassification of Lung AdenocarcinomaWilliam D. Travis, MD, Elisabeth Brambilla, MD, Masayuki Noguchi, MD, Andrew G. Nicholson, MD,Kim R. Geisinger, MD, Yasushi Yatabe, MD, David G. Beer, PhD, Charles A. Powell, MD,Gregory J. Riely, MD, Paul E. Van Schil, MD, Kavita Garg, MD, John H. M. Austin, MD,Hisao Asamura, MD, Valerie W. Rusch, MD, Fred R. Hirsch, MD, Giorgio Scagliotti, MD,Tetsuya Mitsudomi, MD, Rudolf M. Huber, MD, Yuichi Ishikawa, MD, James Jett, MD,Montserrat Sanchez-Cespedes, PhD, Jean-Paul Sculier, MD, Takashi Takahashi, MD,Masahiro Tsuboi, MD, Johan Vansteenkiste, MD, Ignacio Wistuba, MD, Pan-Chyr Yang, MD,Denise Aberle, MD, Christian Brambilla, MD, Douglas Flieder, MD, Wilbur Franklin, MD,Adi Gazdar, MD, Michael Gould, MD, MS, Philip Hasleton, MD, Douglas Henderson, MD,Bruce Johnson, MD, David Johnson, MD, Keith Kerr, MD, Keiko Kuriyama, MD, Jin Soo Lee, MD,Vincent A. Miller, MD, Iver Petersen, MD, PhD, Victor Roggli, MD, Rafael Rosell, MD,Nagahiro Saijo, MD, Erik Thunnissen, MD, Ming Tsao, MD, and David Yankelewitz, MDJournal of Thoracic Oncology, Vol. 6, Number 2, February 2011
12 Proposed Classification Major Changes ofProposed Classification Stop usage of “bronchioalveolar carcinoma” Addition of minimally invasive carcinoma Classification of invasive carcinoma according to predominant subtypeJournal of Thoracic Oncology, Vol. 6, Number2, February 2011
13 IASLC/ATS/ERS Classification of Lung Adenocarcinoma in Resection Specimens Preinvasive lesionsAtypical adenomatous hyperplasiaAdenocarcinoma in situ (< 3 cm formerly BAC) Minimally invasive adenocarcinoma (< 3 cmlepidic predominant tumor with < 5 mm invasion) Invasive adenocarcinomaJournal of Thoracic Oncology, Vol. 6, Number2, February 2011
14 Papillary adenocarcinoma Lepidic predominantpatternAcinaradenocarcinomaPapillary adenocarcinomaSolidMicropapillaryadenocarcinomaJournal of Thoracic Oncology, Vol. 6, Number2, February 2011
15 Journal of Thoracic Oncology, Vol. 6, Number2, February 2011
16 Lung Cancer Immunohistochemical stains Differentiate primary pulmonary adenocarcinoma from metastaticcarcinoma Differentiate adenocarcinoma fromsquamous cell carcinoma Distinguish adenocarcinoma from mesothelioma Determine the neuroendocrine statusof the tumorNCCN Guidelines Version 2.20, 2013
17 P40 is superior to P63 for SCC of lung. Am J Surg Pathol, Vol. 35 (1), Jan 2011P40 is superior to P63 for SCC of lung.
19 Chromosomal rearrangement Lung AdenocarcinomaActivating OncogenesDeletion and pointMutationsKRAS (30%)EGFR (15%)Gene AmplificationEGFR (6-9%)Chromosomal rearrangementEML4-ALK (5%)ROS1 (2%)EGFR, EML 4-ALK and KRAS are mutually exclusive
20 Archives of Pathology & Laboratory Medicine Molecular Testing Guideline for EGFR andALK Tyrosine Kinase Inhibitors: Guidelinefrom the College of American Pathologists,International Association for the Study ofLung Cancer, and Association for MolecularPathology.Archives of Pathology & Laboratory MedicineJune 2013, Vol. 137, No. 6, pp
21 NCCN Guidelines Version 2.20, 2013 Testing for EGFR mutations and AlK gene rearrangements is recommended in the NCCN NSCLC guidelines for adenocarcinoma patients.NCCN Guidelines Version 2.20, 2013
22 Lung Carcinoma Distinction is critical between: Adenocarcinoma Pure squamous cell carcinoma Pure small cell carcinoma Pure neuroendocrine carcinomaFor EGFR and Alk testing
23 Lung CarcinomaLung carcinoma with mixed histology (adenosquamous, adeno/small cell)can have EGFR mutation or Alk rearrangement . Testing is requiredIf possibility of adenocarcinoma component cannot be excluded.
24 Lung CancerIt is important to retain sufficient tissue for molecular testingafter establishing diagnosisof adenocarcnoma.
25 Lung CancerMolecular testing results should be available within 2 weeks of receiving samples to molecular labs.
26 EGFR mutations are seen more common (50%) in: WomenNever smokerAsianSelection of patients for EGFR mutation testing is dependent on subtype of lung cancer not on clinical information.
27 Common Mutations Identified in EGFR Gene EGFR transcriptEGFR TK domain(exons 18-21)1818Exons 1–16G719Deletions19Exon 1720D770_N771 insNPGExons 18–24T790ML858R21Exons 25–28L861Riely GJ, et al. Clin Cancer Res 2006;12:839–44
28 EGFR TK Mutations Common Exon 19 in-frame deletion Exon 21 L858R mutation(Lysine to Arginine)Both mutations result in activationof TK domain and associated with sensitivity to TKI.
30 Frequency of EGFR Mutations in Lung Adenocarcinoma32% in East Asia7-15% in Caucasians2% in African AmericaAbout 30% in Saudi population (unpublished data)Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci. 2007;98(12):Suda K, Tomizawa K, Mitsudomi T. Biological and clinical significance of KRAS mutations in lung cancer: an oncogenic driver that contrasts with EGFR mutation. Cancer Metastasis Rev. 2010;29(1):49-60.Reinersman JM, Johnson ML. Riely GJ, et al. Frequency of EGFR and KRAS mutations in lung adenocarcinomas in African Americans. J Thorac Oncol. 2011;6(1):28-31
32 Resistance to EGFR-TKIs Primary resistanceKRAS mutations and Alk gene rearrangementEGFR mutations not sensitive to EGFR TKIs(rare, 2%) – ex 20 insertionBRAF mutations (rare, 3%)Acquired resistanceSecond EGFR mutation: T790M (50% of cases)MET amplification (some)Pi3k mutationsTransformation to small cell lung ca
33 Tissue Sampling Methods in NSCLC Three main methods of obtaining tumour samplesExcised during surgeryBronchoscopic biopsy (for central lesions)Guided needle biopsy (for peripheral lesions)Preservation of DNA is essential (e.g. formalin-fixed, paraffin-embedded tumour sample)Preferably use primary tumour tissuewhen this is not available, may consider metastatic tissue, pleural effusion or blood
34 Tumor Sample Collection Sectioning (at least 50% tumors) Testing for MutationTumor Sample CollectionSectioning (at least 50% tumors)DNA ExtractionK-ras mutations are observed in about 15– 30% of NSCLCthe molecular pathogeneses of adenocarcinomas arising in smokers and never-smokers are distinctAmplificationSequencing
35 EGFR Testing Method Direct (Sanger) sequencing Pyrosequencing High resolution melting analysisPolymerase chain reaction (PCR),allele specific hybridizationReal time PCRWhole exome sequencingWhole genome sequencing
36 Limitations of Mutation Detection by Direct Sequencing Sequencing will not detect proportions of tumor cells below the sensitivity level (25%)Microdissection routinely used to increase tumor content (eliminate non-neoplastic areas)Blocks or unstained sections for DNA extraction should be from the most cellular areas with >50% tumor cellsSelect sections without excessive inflammatory response
37 Adequacy of EGFR Testing Adequacy is determined by malignant cells content and DNA quality and not sample typeSpecimen should be fixed in 10% NBF for 6-48 hoursCell blocks are preferred over smears for cytology samples
38 ALK-rearranged Adenocarcinoma 2-7% of adenocarcinomasSimilar to EGFR mutation positive patient except they are younger and maleYounger patientsNever smokingHigher stageSolid tumor growth, frequent signal cells with abundant intracellular mucin
47 Cancer is a disease of genome Cancer is a disease of genome. Today we have the technology to understand the alterations of these genes using exome sequencing, transcriptome sequencing and whole genome sequencing.