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)

A) Early detection:Precursör Lesions(PL)

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

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

Apoptotic İndex

*

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

VEGF/VEGFR sistemiaFGF, bFGFAngiogeninAngiopoietin-1PlGF, PDGFEGF, HGF, TNF, IL-8

Thrombospondin-1Angiostatin, EndostatinAngiopoietin-2Platelet factor-4TIMP-1,-2IL-1, IL-12InterferonProtamine

+ -

Angiogenesis

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

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

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

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

3. TSG, p53 38 NSCLC: 76.3% +, NR to TNM 63 NSCLC: 66.7% +, NR to survival

Ulukuş 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 1997;116: 185-189

4.Oncogenes

MUC-4 and ErbB-2, NR to TNMC-kit, 7%, NR to survivalCOX-2, NR to survivalHGF/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, 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 of Clinical Practise, 2006.

Invasion and Metastasis

CD44s : expression of CD44s is inversely correlated with metastases.

Is loss of CD44s functional equivalent of CDv which enhances metastatic potential??

* Kargı et al. CD44 expression in metastatic and non-metastatic non-small cell lung cancers. Cancer Letters 1997;119:27-30

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

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

Molecular Pathologic Classification???

Concordance among pathologists by using histopathologic criteria :

SCLC X NSCLC….95-100% NSCLC; well diff.AC X SCC:100% poor diff.AC X SCC: 50-60%??? Glandular diff.:TTF-1 Squamous diff.:p63 NE diff.: NSE,NCAM, synaptophysin,chrom…

Heterogeneity

EM: Divergent differentiation in even a single cell!!!

IHC

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. 2002;4:141-150.

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; 98:13790-5**Proc Natl Acad Sci USA 2001;98:13784-9

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.

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; 127:430-437.

C) Distinction of Primary from Metastatic Carcinoma

Lung:

TTF-1+, 70%AC, 100%SCLC,

50% LCNEC.

CK7+, CK20- CK7-, CK20+: Colon PSA, HMB45-Melan-A, LCA…..

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 FİSH??

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.