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 in the genomic context to design

prevention and therapeutic strategiesmore than 200 distinct types of tumors

Western Countries: 1 out 3 individuals develops cancer1 out 5 individuals dies because of cancer

In most cases somatic alterations Sporadic TumorsSporadic TumorsIn a minority germlinegermline (predisposing) and somatic lesionssomatic lesions

HereditaryHereditary (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: 10-7Probability 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 proliferationinfluence cell proliferation, generating an expanded

cell population target of the second mutation

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

INTERPLAY BETWEEN MUTATIONSAND SELECTIVE FORCES IN MULTISTEP

TUMORIGENESIS

WHICH ARE THE GENES ALTEREDIN 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, 2009Total 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 reportedin the literature

CHROMOSOME ABNORMALITIES IN NEOPLASIA

• PRIMARYPRIMARY abnormalities

important for establishing the tumor

• SECONDARYSECONDARY abnormalities

a) important for tumor progression

b) of no importance for tumor

development - NOISEdevelopment - NOISE

Clinical Significance ofClinical Significance of Cytogenetic FindingsCytogenetic Findings

• • Help to establish a correctHelp to establish a correctDIAGNOSISDIAGNOSIS

• • Help to evaluate Help to evaluate PROGNOSISPROGNOSIS

Frequencies of Frequencies of balanced balanced

chromosomal chromosomal aberrations and aberrations and gene gene fusions in fusions in

cancercancer

Leukemia, Lymphoma and Soft tissue sarcoma Leukemia, Lymphoma and Soft tissue sarcoma

representrepresent only 10% of human tumors: nevertheless they only 10% of human tumors: nevertheless they

account for account for >80% fusion genes>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 PROTOCOLFISH PROTOCOL

MOLECULAR HYBRIDIZATIONMOLECULAR HYBRIDIZATION

Direct labeling

•by incorporation of a modified nucleotide (dUTP) directlydirectlybound to a fluorophorebound 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, Cy5.5Indirect labelingIndirect labeling• by incorporation of a modified nucleotide (dUTP) containing a reporter reporter group such as biotin (BIO) ordigoxigenin (DIG) which are bound by fluorescinatedaffinity 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/DigoxigeninFluorochrome ( +( +) conjugated to a molecule able to bind specifically the reporter molecule: avidin /anti-dig ab

Fluorescence MicroscopyFluorescence 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 differentfilters are separately registered and given a different

pseudocolor by a dedicated software.

PROBESGenomic: 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 (α-sat, β-sat, ribosomal DNA..) and/or locus specific DNA (single sequences).cDNA or RNA: size limit: to be detected probes shouldbe 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 ANOMALIESON INTERPHASE NUCLEI

Go BLOCKED CELLSGo BLOCKED CELLS-Identification of aneuploidies in fresh & paraffin-embedded tumour samples-Identification of aneuploidies in fresh & paraffin-embedded tumour samples

-FISH dual color also evidences:-FISH dual color also evidences:deletions/duplications, inversions, translocationsdeletions/duplications, inversions, translocations

CHROMOSOMALCHROMOSOMAL TRANSLOCATIONSTRANSLOCATIONS

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

Fusion-signal FISHProbes each flanking one of the two bkps

t(9;22)(q34;q11)t(9;22)(q34;q11)

ONCOGENE AMPLIFICATION IN CANCER CELLS

The effect of increased gene dosage through amplificationThe 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 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 complex and discontinuousand 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 CANCER GENES THERAPEUTIC TARGETSTARGETS

RECURRENT ACTIVATED ONCOGENES

DESIGN OF TYROSINEKINASE INHIBITORS

••Herceptin, antibody against HER2/neuHerceptin, antibody against HER2/neu

••ZD 1839 (Iressa) , selective inhibitor inibitore of EGFRZD 1839 (Iressa) , selective inhibitor inibitore of EGFR

••STI57, inhibitor of Bcr-Abl (CML), KIT (GISTs), STI57, inhibitor of Bcr-Abl (CML), KIT (GISTs),

PDGFRPDGFR

nmyc probe normal cells

Cancer derives from the accumulation of SEQUENTIAL MUTATIONS AND

EPIMUTATIONS IN SEVERAL GENES

HOW MANY GENES ARE ALTEREDIN 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 high resolution Resolution limitslimits laboriouslaborious

Resolution limitsResolution limitsdedicated equipmentdedicated equipment

do not providedo not provide provideprovide

a complete view of the genome of the sample under studya complete view of the genome of the sample under study

SIMULTANEOUS IDENTIFICATION OF ALLCHROMOSOMES BY FISH

SCREENING OF THE WHOLE GENOME WHEN A PRELIMINARY CYTOGENETIC CHARACTERIZATION

IS NOT INFORMATIVE

APPLICATIONSAPPLICATIONS-IDENTIFICATION OF CHROMOSOMAL ANOMALIES < 4Mb-IDENTIFICATION OF CHROMOSOMAL ANOMALIES < 4Mb

-CHARACTERIZATION OF MARKER CHROMOSOMES-CHARACTERIZATION OF MARKER CHROMOSOMES

Chromosome-specific libraries labelled differentlyChromosome-specific libraries labelled differentlyfor the 24 chromosomesfor the 24 chromosomes

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

Only 5 Fluorochromes in different combinations allow to have wcps of allOnly 5 Fluorochromes in different combinations allow to have wcps of all24 human chromosomes24 human chromosomes

SKY karyotype

Evaluation of COPY NUMBER CHANGES: Evaluation of COPY NUMBER CHANGES: array CGHarray CGH

METHODSMETHODS

- 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 ofcontrol exps-Experiments in which >5% of the clones show intensity ratio outside the thresholds are ruled out.

Analysis of Analysis of GainsGains and/or and/or LossesLosses

Arraying CapacityArraying Capacity

SPOTTING ROBOTSPOTTING ROBOT

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

APPLICATIONS OF GENOMIC ARRAYSAPPLICATIONS 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 caveatsA 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 factorRecurrent fusion of TMPRSS2 and ETS transcription factorgenes in prostate cancerTomlins SA et al, Science 310:genes in prostate cancerTomlins SA et al, Science 310: 644648, 2005644648, 2005

Bioinformatic approach to look for genes with a very high expression in Bioinformatic approach to look for genes with a very high expression in microarray analyses. Among the top 10 outlier genes were ERG and microarray analyses. Among the top 10 outlier genes were ERG and ETV1 (belonging to the transcription factors ETS family) which wereETV1 (belonging to the transcription factors ETS family) which weresubsequently found to be fused to the 5’ part of the prostate-specificsubsequently found to be fused to the 5’ part of the prostate-specificgene TMPRSS2gene TMPRSS2

Identification of the transforming EML4-ALK fusion geneIdentification of the transforming EML4-ALK fusion genein non-small cell lung cancerin non-small cell lung cancerSoda M et al, Nature 448: 561-566, 2007Soda M et al, Nature 448: 561-566, 2007

Generation of a cDNA expression library from a NSCLC patient andGeneration of a cDNA expression library from a NSCLC patient andfunctional screening (focus formation assay to isolate genes that couldfunctional screening (focus formation assay to isolate genes that couldtransform mouse 3T3 fibroblasts)transform mouse 3T3 fibroblasts)

Recurrent gene fusions in prostate cancerRecurrent gene fusions in prostate cancer

A- Peripheral blood lymphocytes showing two ETV1 (red) and twoTMPRS22 (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 NPLsD- 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 inprostate cancerTomlins SA et al, Science 310: 644648, 2005

TISSUE MICROARRAYS (TMA): miniaturized collections of thousands ofTISSUE MICROARRAYS (TMA): miniaturized collections of thousands ofarrayed tissue spots on a glass slide that provide a template for validationarrayed tissue spots on a glass slide that provide a template for validation

of specific molecular targetsof specific molecular targets

Anatomy of gene fusions in prostate cancer

BIBLIOGRAPHYBIBLIOGRAPHY

• • Volpi EV & Bridger JM: FISH glossary: an overview of the Volpi EV & Bridger JM: FISH glossary: an overview of the

fluorescence in fluorescence in situ hybridization technique. Biotechniques 45: 385-410, situ hybridization technique. Biotechniques 45: 385-410,

20082008

• • Van der Burg M, Poulsen TS et al. Split-signal FISH for detection ofVan der Burg M, Poulsen TS et al. Split-signal FISH for detection of

chromosome aberrations in acute lymphoblastic leukemia.chromosome aberrations in acute lymphoblastic leukemia. Leukemia 18: Leukemia 18:

895-908,2004895-908,2004

• • Pinkel D & Albertson DG: Array comparative hybridization and its Pinkel D & Albertson DG: Array comparative hybridization and its

application application to cancer , Nature Genet 37:11-17, 2005to cancer , Nature Genet 37:11-17, 2005

• • Kumar-Sinha C, Tomlins SA, Chinnaiyan AM: Recurrent gene fusions Kumar-Sinha C, Tomlins SA, Chinnaiyan AM: Recurrent gene fusions

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

• • Mitelman F, Johansson B, Mertens F: The impact of translocations and Mitelman F, Johansson B, Mertens F: The impact of translocations and

gene gene fusions on cancer causation, Nature Rev Cancer 7: 233-245, 2007fusions on cancer causation, Nature Rev Cancer 7: 233-245, 2007