Application of Molecular Pathology in Pathologic Evaluation of Lung Cancer Dr. Aydanur Kargı
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. TNM…detection of micrometastasis in lymph nodes C. Distinction of primary from metastatic ca?
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.
Carcinogenesis • Proliferation: Oncogenes and TSG MI (morphology), PCNA, Ki-67 • Apoptosis:Oncogenes 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-TİMMP, CD44 1) Early detection 2) Assesment of prognoses
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 • FİSH • Proteomics (microarray)
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 Molecular Changes in PL
Genetic instability Tumor Supressor Gene Protooncogene Telomerase Apoptosis Angiogenesis
Early LC detection in sputum • Cytology:%14 sensitivite, %99 spesifisite • Nuclear image analysis: %45-75 sens., %90-98 spes. • RT-PCR :abnormal DNA • DNA methilation • FİSH: c-myc, EGFR..%41sens, %94 spes. • K-ras ve p53 mutasyonları...+ 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 cancer:A review. Lung Cancer2005; 521-27.
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….
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
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. TNM…detection of micrometastasis in lymph nodes
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; 6:4055-4063.
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 favourable:1 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; 92:131-139
Oncogene activasion: c-erbB family • c-erbB-1 (EGFR) • c-ErbB-2 (HER2/neu) • c-erB-3 /HER3 • C-erB-4 /HER4
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; 20:975-981
Oncogene activation:HER-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 (FİSH): 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 FİSH 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; 113:101-108. **Meert AP, Martin B, Paesmans M, et al. Br J Cancer. 2003; 89:959-965
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; 89;55-64
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.
Tümör hipoksik ortamda 2 mm3 ten büyük ise anjio-genez olmadan büyüyemez.J.Folkman 1971
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
What is the role of angiogenesis in progression of insitu ca to invasive ca?
The methods to demonstrate angiogenesis and related proteins *Biochemical monitarization *Molecular techniques *IHC…angiogenesis (MVD)…f VIII, CD31, CD34), VEGF
Angiogenesis(MVD) • f VIII:14 study (1866 pts.), QS:52%, 6NS, 8 neg. • CD34:10 study (1440 pts.), QS:59% 4NS, 6 neg. • CD31: 8 study (1093 pts.), QS:59% 3NS, 5 neg. T: 32 13 19 • MVD: Meer AP et al. Br J Cancer 2002; 87:694-701
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 2002;19:577-84 • Bremnes RM et al. Angiogenesis in NSCLC: The prognostic impact of neoangiogenesis and the cytokines VEGF and bFGF in tumours and blood. Lung Cancer 2006;51:143-158
Multivariate Models • D’Amico et al. ON: erbB-2, TSG: RB, p53, Ang.: fVIII, met: CD44 substage:1, 0-1, 77% substage2: 2, 62% substage3: 3-5, 49% D‘Amico 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
Our Studies * Sağol Ö, Kargı 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. * Kargı A et al.Apoptosis, bcl-2 and p53 expression and their relation to tumour stage in non-small cell lung carcinomas (NSCLC). Cancer Letters 1997;116: 185-189 * Kargı A et al. MUC4 expression and its relation to ErbB2 expression, apoptosis, Accepted for publication in Pathology Research and Practise, March 2006. * Tataroğlu C, Kargı 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; 43:47-54.
Our Studies * Kargı A et al. CD44 expression in metastatic and non-metastatic non-small cell lung cancers. Cancer Letters 1997; 119:27-30 • Ulukuş Ç, Kargı 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, Kargı 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.
1. Proliferation and Apoptosis • 38 NSCLC (st.I:11, stII5, st.III:13, st.IV:9): 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 1997;116: 185-189 • Kargi et al. MUC4 expression and its relation to ErbB2 expression, apoptosis..., Accepted for publication in Pathology Research and Practise, March 2006. • Ulukuş Ç, Kargı 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