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Application of Molecular Pathology in Pathologic Evaluation of Lung Cancer

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Title: Application of Molecular Pathology in Pathologic Evaluation of Lung Cancer


1
Application of Molecular Pathology in Pathologic
Evaluation of Lung Cancer
  • Dr. Aydanur Kargi

2
Problems and Expectations from MP in Surgical
Pathologic Evaluation of LC
  • A.Early detection of malignancy Distinction of
    severe dysplasia from carcinoma.Point of no
    return???
  • B. Prognoses assesment
  • 1. Identification of biomarkers to
    distinguish aggressive tumors
  • with identical morphology in the same
    stage.
  • 2. Histologic typing prognostically more
    relevant classication
  • systems than current histopath.class.,
  • 3. TNMdetection of micrometastasis in
    lymph nodes
  • C. Distinction of primary from metastatic ca?

3
Promises of Molecular Pathology
  • a) Prognostically more relavent classification
    than
  • conventional histopathologic
    classification
  • b) Identification of prognostically relevant
  • genes and protein products in
    morphologically identical LCs
  • c) Identification of genes and their protein
  • products specific to lung tissue and LC
  • in order to detect micrometastasis
  • d) Identification of particular genes and
    their
  • products involved in molecular pathways
    in
  • carcinogenesis to elucidate LC
    pathogenesis
  • e) Identification of genes and their products
    in relation to
  • therapy.

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Carcinogenesis
  • Proliferation Oncogenes and TSG
  • MI (morphology), PCNA,
    Ki-67
  • ApoptosisOncogenes and TSG,
  • AI (morphology,etc.),
  • Bcl-2 and BAX, Survivin,
    COX-2
  • Angiogenesis MVD (f-VIII,CD31, CD34),
  • proangiogenic and
    antiangiogenic proteins (VEGF)
  • Tissue invasion and metastases
  • Morphology,
    E-cadherin/catenin
  • MMP-TIMMP, CD44
  • 1) Early detection
  • 2) Assesment of prognoses

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Detection of molecular changes
  • Identification of specific mutation
  • Single strand conformational polymorphism (SSCP)
  • Rt-PCR, PCR
  • Genomics (cDNA microarray)
  • Detection of abnormal gene products (proteins)
  • IHC
  • FISH
  • Proteomics (microarray)

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A) Early detectionPrecursör Lesions(PL)
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Molecular Changes in PL
  • Multiple genetic and epigenetic changes
    accumulate
  • Allele loss at several loci (3p, 9p, 8p, 17p with
    p53 mutation)
  • Myc and Ras up-regulation, cyclin-D1 expression,
    p53 immunoreactivity, bcl-2 overexpression and
    DNA aneuploidy
  • Microsatellite alterations (MA)
  • Clinics in Chest Medicine. 2002, 23(1)83-101

11
Genetic instability Tumor Supressor
Gene Protooncogene Telomerase Apoptosis Angio
genesis
12
Early LC detection in sputum
  • Cytology14 sensitivite, 99 spesifisite
  • Nuclear image analysis 45-75 sens., 90-98
    spes.
  • RT-PCR abnormal DNA
  • DNA methilation
  • FISH c-myc, EGFR..41sens, 94 spes.
  • K-ras ve p53 mutasyonlari... in pts without ca
  • RNA extraksion studies rapid degradation of RNA
  • Kennedy TC, Hirsch FR. Using molecular
    markers in sputum for the early detection of lung
    cancerA review. Lung Cancer2005 521-27.

13
Problems!!!!
  • Normal epithelium adjacent to cancer,
    preneoplastic and neoplastic epithelium show
    identical genetic changes...
  • 3p loss occurs in about 50 of smokers, MA occur
    in COPD...
  • A marker should be specific for neoplastic
    transformation, not reflect smoking related
    changes, easily detectable and cost effective.

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Result!!!!
  • A prognostically meaningful biomarker could be
    found as a result of prospective studies which
    compare the genetic changes in precursor lesions
    with and without progression into invasive
    carcinoma

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B. Prognoses assesment
  • 1.Identification of biomarkers to distinguish
    aggressive tumors with identical morphology in
    the same stage.
  • 2. Histologic typing prognostically more
  • relevant classication systems than current
  • histopath.class.,
  • 3. TNMdetection of micrometastasis in lymph
  • nodes

22
TSG inactivation p53
  • P53 protein acts as a negative regulator of
    proliferation and as an inducer of apoptosis
    through the transactivation of genes, including
    p21, BAX and GADDA45. Mutant p53 losses these
    functions. Mutation usually prolongs half-life of
    protein and results in nuclear accumulation of
    p53 protein which can be detected by IHC.
  • Meta analysis of 43 studies
  • DNA sequence change 381 of
    1031....37 (25.8 -50.7)
  • Protein overexpression 1725 of
    3579..48 (17.5 - 76.8)
  • Mutation and protein expression in AC
    34 and 36

  • in SCC 52 and 54
  • p53 alteration showed by both DNA and
    protein studies was found to be a significant
    marker of poor prognosis in AC patients, but not
    in SCC patients.
  • P53 Mitsudomi T, et al. Clinic Cancer Research.
    2000 64055-4063.

23
Oncogene activation RAS oncogene
  • RAS genes (HRAS, KRAS, NRAS) code for 4 highly
    homologous 21 kDA proteins called p21.
  • Meta analysis of 43 studies.
  • 9 study IHC 44.6 (of 1548pts.)
  • 34 study PCR 18.4 (of 3779pts.)
  • Survival results .. pejorative 9

  • significantly favourable1
  • not
    significant 31
  • not
    conclusive 2
  • Conclusion KRAS appears to be adverse
    prognostic factor in AC and when studied by
    PCR.
  • RAS Mascaux C, Iannino N, Martin B, et al. Br J
    Cancer. 2005 92131-139

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Oncogene activasion c-erbB family
  • c-erbB-1 (EGFR)
  • c-ErbB-2 (HER2/neu)
  • c-erB-3 /HER3
  • C-erB-4 /HER4

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EGFR (c-erbB-1)
  • 16 study (2810pts), 13 study IHC, 1PCR, 1
    Northern Blot,1
  • IHC 13-80 expression in
    NSCLCs
  • 6 of all
    NSCLCs, 28 of SCC had gene
  • amplification
  • 1 study good prognostic factor
  • 3 study poor pr. factor
  • 12 study NS
  • 11 study evaluated for meta-analysis of
    survival(2185pts) NS
  • Meert AP, Martin B, Delmotte P, et al. Eur
    Respir J. 2002 20975-981

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Oncogene activationHER-2/neu (ErbB2)
  • 345 stage I NSCLC and 207 Stage I-III NE lung
    tumors
  • Protein overexpression (IHC) 80 (23) of
    345 NSCLC, 14(7) of 207 NET
  • Gene amplification (FISH) in 7 of 94
    immunoreactive cases.

  • none in unreactive cases.
  • 1) No significant difference between
    different scoring systems
  • for IHC.
  • 2) No correlation between IHC and FISH
  • 3) Gene ampl. or protein overexpr. were
    not correlated with
  • clinicopathologic variables and
    survival
  • Stage I NSCLC 4 studies (1800 pts)...negative
  • 3 studies
    (1066 pts)...nonsignificant for survival
  • Pelosi et al. Lack of prognostic
    implications of HER-2/neu abnormalities in 345
    stage I NSCLC and 207 stage I-III neuroendocrine
    tumours. Int J Cancer 2005 113101-108.
  • Meert AP, Martin B, Paesmans M, et al. Br J
    Cancer. 2003 89959-965

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Apoptotic Index

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Bcl-2
  • 28 studies( 21NSCLC, 4SCLC, 3 NET)
  • 11 good, 3 poor, 14 NR
  • 25 studies (3370pts.)
  • SCLC(4 st.) 71
  • NET(3 st.)55
  • NSCLC (21 st.) 35
  • All IHC, no genetic change
  • Good prognostic factor (paradoxical!!)
  • Bcl-2 Martin B et al. British J Cancer 2003
    8955-64

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Role of Angiogenesis in Carcinogenesis
  • Tumor growth depends on angiogenesis
  • It plays a role in progression of precursor
    lesion to invasive cancer
  • It is important in metastatic spread and growth
    of metastasized cells in their new soils.

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Tümör hipoksik ortamda 2 mm3 ten büyük ise
anjio-genez olmadan büyüyemez. J.Folkman
1971
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Control of Angiogenesis
-
  • Thrombospondin-1
  • Angiostatin, Endostatin
  • Angiopoietin-2
  • Platelet factor-4
  • TIMP-1,-2
  • IL-1, IL-12
  • Interferon
  • Protamine
  • VEGF/VEGFR sistemi
  • aFGF, bFGF
  • Angiogenin
  • Angiopoietin-1
  • PlGF, PDGF
  • EGF, HGF, TNF, IL-8

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Angiogenesis
42
What is the role of angiogenesis in progression
of insitu ca to invasive ca?
43
The methods to demonstrate angiogenesis and
related proteins
Biochemical monitarization Molecular
techniques IHCangiogenesis (MVD)f VIII, CD31,
CD34), VEGF
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Angiogenesis(MVD)
  • f VIII14 study (1866 pts.), QS52,
    6NS, 8 neg.
  • CD3410 study (1440 pts.), QS59
    4NS, 6 neg.
  • CD31 8 study (1093 pts.), QS59
    3NS, 5 neg.
  • T 32
    13 19
  • MVD Meer AP et al. Br J Cancer 2002 87694-701

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VEGF, bFGF
  • tissue VEGF 15/20 st. poor pr.
  • blood VEGF 10/16 st. poor pr.
  • tissue bFGF poor, controversial
  • blood bFGF 3/5 poor pr.
  • Delmotte P et al. VEGF and survival of patients
    with lung cancer a systematic literature review
    and meta-analysis.Rev Mal Respir 200219577-84
  • Bremnes RM et al. Angiogenesis in NSCLC The
    prognostic impact of neoangiogenesis and the
    cytokines VEGF and bFGF in tumours and blood.
    Lung Cancer 200651143-158

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Multivariate Models
  • DAmico et al.
  • ON erbB-2, TSG RB, p53, Ang. fVIII,
    met CD44
  • substage1, 0-1, 77
  • substage2 2, 62
  • substage3 3-5, 49
  • DAmico et al. A biologic risk model for stage I
    lung cancer immunohistochemical analysis of 408
    patients using 10 molecular markers.J Thorac
    Cardiovasc Surg 1999 117-736-43

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Our Studies
  • Sagol Ö, Kargi A et al. Stereologically
    estimated mean nuclear volume and histopathologic
    malignancy grading as prognostic factors of
    disease extent in non-small cell lung carcinoma.
    Pathology Research and Practise. 2000 196
    683-689.
  • Kargi A et al.Apoptosis, bcl-2 and p53
    expression and their relation to tumour stage in
    non-small cell lung carcinomas (NSCLC). Cancer
    Letters 1997116 185-189
  • Kargi A et al. MUC4 expression and its
    relation to ErbB2 expression, apoptosis, Accepted
    for publication in Pathology Research and
    Practise, March 2006.
  • Tataroglu C, Kargi A et al. Association of
    macrophages, mast cells and eosinophil leukocytes
    with angiogenesis and tumor stage in non-small
    cell lung carcinomas (NSCLC). Lung cancer, 2004
    4347-54.

49
Our Studies
  • Kargi A et al. CD44 expression in metastatic
    and non-metastatic non-small cell lung cancers.
    Cancer Letters 1997 11927-30
  • Ulukus Ç, Kargi A et al. Survivin expression in
    NSCLCs Correlation with apoptosis and other
    apoptotisis related proteins, clinicopathologic
    prognostic factors and prognosis.
    Accepted for publication in
    Appl Immunohistochemistry Mol Morphol, January
    2006.
  • Yaren A, Öztop I, Kargi A et al., Bax, bcl-2 and
    c-kit expression in non-small cell lung cancer
    and their effects on prognosis. In press in Int J
    f Clinical Practise, 2006.

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1. Proliferation and Apoptosis
  • 38 NSCLC (st.I11, stII5, st.III13, st.IV9) AI
    related to TNM
  • 100 NSCLC (early st. , late st. ) AI and
    PCNA not related to

  • TNM
  • 63 NSCLC ..AI not related to survival
  • Ki-67 correlated
    with survival
  • Kargi et al. Apoptosis, bcl-2 and p53 expression
    and their relation to tumour stage in non-small
    cell lung carcinomas (NSCLC). Cancer Letters
    1997116 185-189
  • Kargi et al. MUC4 expression and its relation to
    ErbB2 expression, apoptosis..., Accepted for
    publication in Pathology Research and Practise,
    March 2006.
  • Ulukus Ç, Kargi A et al. Survivin expression in
    NSCLCs Correlation with apoptosis and other
    apoptotisis related proteins, clinicopathologic
    prognostic factors and prognosis. Accepted for
    publication in Appl Immunohistochemistry Mol
    Morphol, 2006

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2. Apoptosis Related Proteinsbcl-2, bax and
survivin(AIP)
  • 63 NSCLC bcl-2.. 22 (34.9) NR to survival
  • bax..25 (39.7) NR to
    survival
  • survivin .. NR to
    survival
  • bcl-2/bax, surv./bax..
    NR to surv.
  • 38 NSCLC bcl-2 , 38, NR to TNM
  • Ulukus Ç, Kargi A et al. Survivin expression in
    NSCLCs Correlation with apoptosis and other
    apoptotisis related proteins, clinicopathologic
    prognostic factors and prognosis. Accepted for
    publication in Appl Immunohistochemistry Mol
    Morphol, 2006
  • Kargi et al. Apoptosis bcl-2 and p53 expression
    and their relation to tumour stage in non-small
    cell lung carcinomas (NSCLC). Cancer Letters
    1997116 185-189

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3. TSG, p53
  • 38 NSCLC 76.3 , NR to TNM
  • 63 NSCLC 66.7 , NR to survival
  • Ulukus Ç, Kargi et al. Survivin expression in
    NSCLCs Correlation with apoptosis and other
    apoptotisis related proteins, clinicopathologic
    prognostic factors and prognosis Accepted for
    publication in Appl Immunohistochemistry Mol
    Morphol, 2006
  • Kargi et al. Apoptosis, bcl-2 and p53 expression
    and their relation to tumour stage in non-small
    cell lung carcinomas (NSCLC). Cancer Letters
    1997116 185-189

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4.Oncogenes
  • MUC-4 and ErbB-2, NR to TNM
  • C-kit, 7, NR to survival
  • COX-2, NR to survival
  • HGF/c-Met, NR to survival
  • Kargi et al. MUC4 expression and its relation to
    ErbB2 expression, apoptosis, Accepted for
    publication in Pathology Research and Practise,
    March 2006.
  • Yaren A, Öztop I, Kargi A et al., Bax, bcl-2 and
    c-kit expression in non-small cell lung cancer
    and their effects on prognosis. In press in Int J
    of Clinical Practise, 2006.

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Invasion and Metastasis
  • CD44s expression of CD44s is inversely
    correlated with metastases.
  • Is loss of CD44s functional equivalent of
    CDv which enhances metastatic potential??
  • Kargi et al. CD44 expression in metastatic and
    non-metastatic non-small cell lung cancers.
    Cancer Letters 199711927-30

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Result!!!
  • MI, Ki-67 is ass. With stage and prognoses
  • Apoptoz, bcl-2, bax, bcl-2/bax, survivin,
  • survivin/bax NR
  • P53 NR
  • ErbB2, MUC4 NR
  • C-kit, COX-2, HGF/c-met NR
  • Angiogenez NR
  • CD44s related to metastasis

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B. Prognoses assesment
  • Identification of biomarkers to distinguish
    aggressive tumors with identical morphology in
    the same stage.
  • 2. Histologic typing prognostically more
    relevant classication systems than current
    histopath.class.,
  • 3. TNMdetection of micrometastasis in lymph
    nodes

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Molecular Pathologic Classification???
  • Concordance among pathologists by using
    histopathologic criteria
  • SCLC X NSCLC.95-100
  • NSCLC well diff.AC X SCC100
  • poor diff.AC X SCC
    50-60???
  • Glandular diff.TTF-1
  • Squamous diff.p63
  • NE diff. NSE,NCAM, synaptophysin,chrom

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Heterogeneity
  • EM Divergent differentiation in even a single
    cell!!!
  • IHC

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Molecular Phenotyping and Classification
  • SCC Charecterized by genes involved with
    detoxification,antioxidant proteins and
    cytokeratins
  • AC Charecterized by surfactant-related and small
    airway-ass. gens(surfactans A2 and B, mucin1,
    pronapsin A), type II pn, Clara cells.
  • LC Epithelial-mesenchymal transition
  • The value of these genes and their products
    in the seperation of prognostically different LC
    types??
  • Neoplasia. 20024141-150.

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Molecular Phenotyping and Classification
  • Bhattacharjee et al.
  • Affimetrix Genechip system, 125 AC pt.
  • 4 subgroup NE, Division and proliferation,
  • surfactant related genes, type II pnomocyte
  • marker
  • Garber et al.
  • cDNA array (23,200cDNAclones), 38 pt.
  • 3 ac subgroups ( metastatic t.in one
    gr.)
  • 2 groups had counterpart in the study of
    Battacharjee.
  • Proc Natl Acad Sci USA 2001 9813790-5
  • Proc Natl Acad Sci USA 20019813784-9

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Gene expression profiling and prognosis
  • Battacharje et.al.186 LC pt.,4 ac subtype.
  • Garber et al. 67 LC pt. 3 ac subtype.
  • Beer et al. 86 LC pt. Survival classifier of
  • 50 genes in
    ac.
  • Wigle et al. 39 LC pt. Survival classifier of
  • 22 genes in
    a.c
  • The gene expression profiles produced by
    different groups had overlaps and distinct
    differences!!!
  • Clinical Cancer Research. 10, 3237-3248,2004.

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3.Detection of Micrometastases
  • CT....13 FN, 50 FP
  • PET....80-90 sens. and specific.
  • Serial sect. and IHC...costly and time consuming
  • RT-PCR.. p53, K-ras better than CK mRNA,
  • sensitivity ()
  • KS1/4.... 93
  • lunx........ 56
  • muc1...... 52
  • CEA........ 44
  • PSE....... 33
  • CK19...... 33
  • Question What is the specificity??? Muc1
    is not specific for ca...Normal LN .
  • Wallace MB et al. Accurate molecular detection
    of non-small cell lung cancer metastases in
    mediastinal lymph nodes by endoscopic
    ultrasound-guided needle aspiration. Chest 2005
    127430-437.

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C) Distinction of Primary from Metastatic
Carcinoma
  • Lung
  • TTF-1, 70AC, 100SCLC,
  • 50 LCNEC.
  • CK7, CK20-
  • CK7-, CK20 Colon
  • PSA, HMB45-Melan-A, LCA..

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Questions
  • Standardisation of speciman collection,
    techniques, statistical methods??
  • Most optimal analysis? Tumor and stroma or only
    tumor cells??
  • What should be analyzed?
  • a)Gene expression profile costly, complex
    data analysis, requires timely coordination of
    several disciplines (biostaticians, clinicians,
    pathologists, cell biologists), ethical
    considerations
  • b)Analysis of gene products by IHC or FISH??

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Conclusion
  • The search for biomarker/biomarkers to seperate
    the precursor lesion with an invasive potential
    is continuing
  • The prognostic value of widely investigated major
    genes and protein products involved in lung
    carcinogenesis is currently mostly controversial.
  • There is a need in prospective studies employing
    standard criteria.
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