Cancer Cytogenetics

Cancer is a Genomic Disease

Multiple and sequential genetic and epigenetic lesions

Current aim integrate known and to be disclosed changes in the genomic context to design prevention and therapeutic strategies more than 200 distinct types of tumors Western Countries: 1 out 3 individuals develops cancer 1 out 5 individuals dies because of cancer
In most cases somatic alterations Sporadic Tumors In a minority germline (predisposing) and somatic lesions Hereditary (1-2%) and Familial Tumors (up to 10%)

MULTISTEP TUMORIGENESIS (epithelial tumors)

Transformation of a normal epithelial cell Carcinoma requires at least 6 mutational events
Mutation rate per gene per cell generation: 10-7 Probability such no of mutations occur in a single of 1013 cells : 1013 x 10-42 i.e. 1/1029 Tumors are nevertheless observed because : A few mutations influence cell proliferation, generating an expanded cell population target of the second mutation

A few mutations affect the stability of the whole genome at the DNA and the chromosomal level increasing the mutation rate

INTERPLAY BETWEEN MUTATIONS AND SELECTIVE FORCES IN MULTISTEP TUMORIGENESIS

WHICH ARE THE GENES ALTERED IN CANCER?

Proportion of translocated genes in human cancer. Somatically mutated cancer genes with translocations, with mutations other than translocations or with both.

Database last updated on February 18, 2009 Total number of cases = 56,015

CANCER GENES

http://www.ncbi.nlm.nih.gov/ncicgap/ http://cancergenome.nih.gov/ http://www.sanger.ac.uk/genetics/CGP/

The number of cytogenetically abnormal neoplasms reported in the literature

CHROMOSOME ABNORMALITIES IN NEOPLASIA PRIMARY abnormalities

important for establishing the tumor

SECONDARY abnormalities
a) important for tumor progression

b) of no importance for tumor

development - NOISE

Clinical Significance of Cytogenetic Findings

Help to establish a correct DIAGNOSIS Help to evaluate PROGNOSIS

Frequencies of balanced chromosomal aberrations and gene fusions in cancer

Leukemia, Lymphoma and Soft tissue sarcoma represent only 10% of human tumors: nevertheless they account for >80% fusion genes By contrast common Epithelial tumors , responsible for >80% of cancer deaths, account for only 10% of recurrent fusions Recently recurrent fusions have been identified in prostate cancer (accounting for 29% of all cancer in men and 9% of all male cancer deaths) NSCCLC (80% of all lung cancers)

THE TWO MAIN PROTOTYPIC CHROMOSOMAL REARRANGEMENTS

FISH
DENATURATION HYBRIDATION IMMUNOFLUORESCENCE

FISH PROTOCOL

MOLECULAR HYBRIDIZATION

Direct labeling
by incorporation of a modified nucleotide (dUTP) directly bound to a fluorophore (a chemical group which fluoresces when exposed to a specific wavelength of light) such as AMCA; DEAC; CB; FITC; OG; A48; RGr; R6G; TAMRA; TxR; Cy3; Cy3.5; Cy5, Cy5.5 Indirect labeling by incorporation of a modified nucleotide (dUTP) containing a reporter group such as biotin (BIO) or digoxigenin (DIG) which are bound by fluorescinated affinity molecules (streptavidin, anti-dig antibodies)

NON ISOTOPIC MARKERS

3) DETECTION: if probe is not directly labelled the hybridised probe is detected by means of a fluorescinated molecule able to recognise it specifically

Reporter molecule: Biotin/Digoxigenin Fluorochrome ( +) conjugated to a molecule able to bind specifically the reporter molecule: avidin /anti-dig ab

Fluorescence Microscopy
equipped with filters specific to the fluorochromes used to visualise the probe connected to a CCD camera enabling to record the selected image. Images of the same metaphase obtained by using different filters are separately registered and given a different pseudocolor by a dedicated software.

PROBES
Genomic: plasmids (10 Kb) phages (~15-20 Kb) cosmids (40-45 Kb) PACs (~75-100 Kb) BACs (~100-150 Kb) YACs (~0.2-2Mb) The different vectors may contain repetitive DNA (-sat, -sat, ribosomal DNA..) and/or locus specific DNA (single sequences). cDNA or RNA: size limit: to be detected probes should be not < 3 Kb. Whole chromosome painting (wcp) Partial painting libraries

PROBE TYPES

*Contain unique sequences as well as ubiquitary repetitive sequences which should be blocked by Cot-1 DNA to avoid aspecific hybridizations

FISH APPLICATIONS

IDENTIFICATION OF NUMERICAL AND STRUCTURAL ANOMALIES ON INTERPHASE NUCLEI

Go BLOCKED CELLS -Identification of aneuploidies in fresh & paraffin-embedded tumour samples -FISH dual color also evidences: deletions/duplications, inversions, translocations

CHROMOSOMAL TRANSLOCATIONS

The two main approaches of FISH probe design for use on nuclei

Fusion-signal FISH
Probes each flanking one of the two bkps
t(9;22)(q34;q11)

ONCOGENE AMPLIFICATION IN CANCER CELLS

The effect of increased gene dosage through amplification has an important role in tumorigenes, especially of solid tumors ans is used as prognostic marker of tumor aggressivity > 60 genes have been reported to be amplified (and sometimes overexpressed) in different tumors. The architecture of the genomic amplified regions is simple in a few cases (a single gene) or complex and discontinuous (several syntenic and coamplified genes) in other instances
N-myc (2p24) neuroblastoma (20%) HER2/NEU (17q11-12) breast cancer(30%) PDGFRA (5q31)/EGFR (7p12) glioma GLI-MDM2-CDK4-HMGA2 (12q13-15) sarcoma, glioma

CANCER GENES THERAPEUTIC TARGETS

RECURRENT ACTIVATED ONCOGENES DESIGN OF TYROSINEKINASE INHIBITORS Herceptin, antibody against HER2/neu ZD 1839 (Iressa) , selective inhibitor inibitore of EGFR STI57, inhibitor of Bcr-Abl (CML), KIT (GISTs),

PDGFR

nmyc probe normal cells

Cancer derives from the accumulation of SEQUENTIAL MUTATIONS AND EPIMUTATIONS IN SEVERAL GENES

HOW MANY GENES ARE ALTERED IN TUMORS????

LOCUS BY LOCUS ANALYSIS: FISH LOH (loss of heterozygosity) Mutation screening

GENOME WIDE ANALYSIS: SKY (SPECTRAL KARYOTYPING) M-FISH Comparative Genome Hybridization Array-CGH

high resolution Resolution limits laborious

Resolution limits
dedicated equipment

do not provide

provide

a complete view of the genome of the sample under study

SIMULTANEOUS IDENTIFICATION OF ALL CHROMOSOMES BY FISH SCREENING OF THE WHOLE GENOME WHEN A PRELIMINARY CYTOGENETIC CHARACTERIZATION IS NOT INFORMATIVE

APPLICATIONS -IDENTIFICATION OF CHROMOSOMAL ANOMALIES < 4Mb -CHARACTERIZATION OF MARKER CHROMOSOMES

Chromosome-specific libraries labelled differently for the 24 chromosomes

FISH allows the simultaneous use of several probes labeled by fluorochromes at different combinations

Only 5 Fluorochromes in different combinations allow to have wcps of all 24 human chromosomes

SKY karyotype

Evaluation of COPY NUMBER CHANGES: array CGH

METHODS
- DOP-PCR clones spotted by arrayer on coated slides (three spots per each clone) -Dual color labeling of R e T (Cy5 e Cy3) and hybridization on temperature-controlled platform -Image analysis and processing -Informatics (data extraction, stockage and normalization) -Treshold of gain and loss determined based on results of control exps -Experiments in which >5% of the clones show intensity ratio outside the thresholds are ruled out.

Analysis of Gains and/or Losses

Arraying Capacity

SPOTTING ROBOT

Factors influencing the success of array CGH. The difficulty of array-CGH analysis varies among different applications

APPLICATIONS OF GENOMIC ARRAYS

1) Analysis of tumor genomes, especially in solid tumors - RESEARCH: identify amplifications-deletions of novel oncogenes, tumor suppressors involved in neoplastic transformation and progression - DIAGNOSTICS: molecular taxonomy of tumors leading to better prognostics and design of therapeutic strategies

A few caveats
Array-based experiments are very sensitive to

external and internal factors (such as variations in the

spotting, in hybridization..) which may impair the reproducibility of results There are recommendations which permit to verify the reliability of results Standardization is necessary to compare results among different studies

GENOME WIDE APPROACHES FOR IDENTIFICATION OF NEW FUSION GENES

Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancerTomlins SA et al, Science 310: 644648, 2005 Bioinformatic approach to look for genes with a very high expression in microarray analyses. Among the top 10 outlier genes were ERG and ETV1 (belonging to the transcription factors ETS family) which were subsequently found to be fused to the 5 part of the prostate-specific gene TMPRSS2 Identification of the transforming EML4-ALK fusion gene in non-small cell lung cancer Soda M et al, Nature 448: 561-566, 2007 Generation of a cDNA expression library from a NSCLC patient and functional screening (focus formation assay to isolate genes that could transform mouse 3T3 fibroblasts)

Recurrent gene fusions in prostate cancer

A- Peripheral blood lymphocytes showing two ETV1 (red) and two TMPRS22 (green signals) B- Metastatic prostate cancer showing fusion of the signals (yellow signal) C- split-signal approach with two probes spanning the ERG locus shows two yellow signals on NPLs D- metastatic prostate cancer shows ERG rearrangement (split signal of the 5 and 3 probes) E- matrix representation of FISH results on a tissue microarray

Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancerTomlins SA et al, Science 310: 644648, 2005

TISSUE MICROARRAYS (TMA): miniaturized collections of thousands of arrayed tissue spots on a glass slide that provide a template for validation of specific molecular targets

Anatomy of gene fusions in prostate cancer

BIBLIOGRAPHY
Volpi EV & Bridger JM: FISH glossary: an overview of the fluorescence in situ hybridization technique. Biotechniques 45: 385-410, 2008 Van der Burg M, Poulsen TS et al. Split-signal FISH for detection of chromosome aberrations in acute lymphoblastic leukemia. Leukemia 18: 895-908,2004 Pinkel D & Albertson DG: Array comparative hybridization and its application to cancer , Nature Genet 37:11-17, 2005 Kumar-Sinha C, Tomlins SA, Chinnaiyan AM: Recurrent gene fusions

in prostate cancer, Nature Rev Cancer 8: 497-511, 2007

Mitelman F, Johansson B, Mertens F: The impact of translocations and gene fusions on cancer causation, Nature Rev Cancer 7: 233-245, 2007