Chromosomal Abnormalities and Cancer

Outline
Improved resolution: from chromosomes to genes Cytogenetics history AML = Somatic translocation (gene:gene) MDS = Somatic non-translocation (deletion) Sarcoma somatic translocations (ETS genes) Prostate cancer translocation = Paper

liquid

solid

Single Gene Probes( deletion or amplification)

P58 CLK-1 Locus (1p36). D7S486 (7q31). Retinoblastoma (13q14). P53 (17p13.1). Her-2/ neu (17q11.2-q12).

Enumeration probes for all chromosomes

Dual Color Translocation Probes.
bcr/abl translocation t(9;22)(q34;q11.2). M-bcr/abl translocation t(9;22)(q34;q11.2). IGH/CCND1 translocation t(11;14)(q13;q32). PML/RARA translocation t(15;17)(q22;q21.1). TEL/AML1 translocation t(12;21)(p13;q22).

Cancer Cytogenetics

Other cytogenetic abnormalities in cancer

Other translocations associated with leukemias include t(8;21)(q22;q22) producing Acute myeloblastic leukemia. t(15;17)(q22;q11-12) producing Acute promyelocytic leukemia t(8;14)(q24;q32) Burkett lymphoma t(11;22)(q24;q12) Ewing Sarcoma Multiple anomalies common Correlation between cytogenetic change and outcome is variable

Advance in Cancer Cytogenetics

Conventional cytogenetics Molecular cytogenetics FISH (Fluorescence In Situ Hybridization) CGH (Comparative Genomic Hybridization) M-FISH(Multi-FISH, Spectral karyotyping)

Techniques in Solid Tumor Cytogenetics

1. Preparation of tumor sample 2. Tumor cell culture Culture Media FBS (11-20%) Antibiotics Insulin Additives Transferrin Tumor cells Hydrocortisone 2. Harvest EDGF 1) Colcemid (0.01-0.1mcg/ml) treatment 2) Hypotonic (0.050-0.075M KCl) treatment 3) Fixation 4) Slide preparation 3. Staining and analysis

25000 20000 15000 10000 5000 0

Solid Tumors Malig Lymphoma Hemat Malignancy

1983 1985 1988 1991 1994

Catalog of Chromosome Aberrations in Cancer

Genomic screens for mutations

Resolution

*
Low

FISH Array CGH (BACs)

* *

RNA profiling Array CGH (oligos)

High
DNA sequence

* *

Cytogenetics SNPs Spectral karyotyping (SKY) Comparative Genomic Hybridization (CGH)

Flemming draws first chromosome Waldeyer coins chromosome Painter reports 48 chromosomes = normal

Tjio reports 46 chromosomes = normal

syndromes

Down +21 Turner XO Klinefelter XXY

1890 von Hansemann observes mitotic aberrations in tumor cells

dark ages Technical problems

1960 Nowell & Hungerford = Ph chromosome & CML

1. better cultures 2. hypotonic solution 3. colchicine

The findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemia.

Mutation types in human cancer

Nat Rev Cancer 4, 177 (2004)

Chromosome

Gene

Drug

1960

1973

1984 Heisterkamp & Groffen clone genes at breakpoints (BCR, ABL)

1998-2001 Druker uses Gleevec to treat CML

Nowell & Hungerford = Ph chromosome & CML Rowley reports Ph chr. = t(9;22)

Drug

Gene

ATRA and acute promyelocytic leukemia (APL)

1977 Rowley reports t(15;17) and APL

1988

1990

1993-1999

Huang reports the use of All-Trans Retinoic Acid (ATRA) for APL

Randomized trials use ATRA for APL Cure = 70-80% at 5 years Borrow & de The report cloning PML (chr.15) RAR (chr.17)

Most fusion proteins are necessary but NOT sufficient to cause cancer
Cooperating mutations!
Reciprocal fusion genes. Other clonal cytogenetic changes genes.

Cytogenetics to discover other changes:

Mouse cytogenetics difficult due to acrocentric chromosomes of similar size Spectral karyotyping (SKY)

Spectral Karyotyping (SKY)

Spectral Karyotyping (SKY) and Multiple Fluorescence In Situ Hybridization(M-FISH)

Simultaneous visualization of all human (or mouse) Chromosomes in different colors. A combination of five fluorochromes to paint all 22 autosomes,and the X and the Y. then analyzed based on their particular emission spectra.

Spectral Karyotype of human chromosomes

Ewing Sarcoma

del(2) identified by Spectral Karyotyping (SKY)

PR = 1/5 del(2)
1.0
WT (n=33) RP (n=31)

Leukemia-Free Survival Probability

0.8 0.6 0.4

PR (n=27)

PR+RP (n=28)

0.2 0.0 0 100 200 300 400 500 600 700 800 900 1000

Time (days)

PR+RP = 11/13 del(2)

PNAS 97, 13306 (2000)

del(2) occurs in several murine AML models & is found in human AML Human chr. 11

Mouse chr. 2

Radiation-induced AML = 21 megabases

540 genes

Berger (1991), Silver (1997), Genes Chrom Cancer

Deleted in up to 14% of relapsed APL patients

SKY-cont.
Advantages:
Mapping of chromosomal breakpoints. Detection of subtle translocations. Identification of marker chromosomes,homogeneously staining regions,and double minute chromosomes. Characterization of complex rearrangements.

Disadvantages:

Very expensive equipments. The technique is labor intensive. Dose not detect structural rearrangements within a single chromosome. Low resolution (up to 15 mb ). Specific, not a screening method.

Nontranslocations in human cancer

Amplifications Deletions
Many potential candidate genes compared to translocations

Nat Rev Cancer 4, 177 (2004)

Genomic screens for mutations

Resolution
FISH Array CGH (BACs) RNA profiling Array CGH (oligos)

Low
cytogenetics SNPs Spectral karyotyping (SKY) Comparative Genomic Hybridization (CGH)

High
DNA sequence

Genomic Comparative Hybridization(CGH)

Fluorescent molecular tech. that identifies DNA gains,losses,and amplifications (mapping to)metaphase chromosomes. Based on quantitative two-color FISH (FITC for tumor DNA and TRITC for the normal DNA).

Advantages:
Require only genomic tumor DNA. Can be applied to fresh or frozen tissues,cell lines, and archival formalin-fixed paraffin-embedded samples.

Comparative Genomic Hybridization(CGH)

Comparative Genomic Hybridization(CGH)

Genomic Comparative Hybridization(CGH)

Comparative Genomic Hybridization(CGH)

Genomic Comparative Hybridization(CGH)

Disadvantages:
Cannot detect balanced abnormalities. Chromosomal copy changes < 10 mb. are not resolved. Copy no. changes < of the analyzed cells are not detected.

Array CGH (aCGH)

A B
repeat sequences masked

GC-rich AT-rich

Maskless Array Synthesis (MAS) Digital Micromirror Device (DMD)

Isothermal probes probe Tm = 76 C variable length 45mer to 85mer

Genome scanning using aCGH

Normal DNA Tumor DNA
Long oligos
Decreased Tumor DNA copy Increased Tumor DNA copy

Green:red ratio Log2 Ratio

Chr. 1

Chr. 22/X

NORMAL CYTOGENETICS

Normal cytogenetics AML (37kb deletion)

How do you know if this deletion is important for AML in this patient?

Cancer genes with somatic mutations

Nat Rev Cancer 4, 177 (2004)

Ewings sarcoma
First solid tumor associated with recurrent chromosomal abnormality.

1984 Turc-Carel & Aurias report t(11;22) in Ewings sarcoma

1992

1993 Zucman & Sorensen report t(21;22) EWSERG (ETS gene)

1995

Delattre reports EWS & FLI1 (ETS gene) are fused in t(11;22)

Jeon report t(7;22) EWS-ETV1 (ETS gene)

Eur J Cancer 41, 2513 (2005)

EWS-ETS fusions are oncogenic

EWS-FLI1 transduced NIH-3T3 cells (fibroblasts):
Form colonies in soft agar & foci in cell culture Serum independent growth in bulk culture Form round cell tumors in nude mice

Ablation of either the EWS or FLI-1 domains abolish the transforming activity RNAi to fusion abolishes transforming activity

EWS-ETS fusions are necessary but NOT sufficient

EWS-FLI1 fusion cannot transform PRIMARY mouse or human cells. Causes cell growth arrest or apoptosis. 30% of EWS show biallelic loss of cell cycle gene p16. EWS-FLI1 transduced p16-/- cells show less growth arrest, but only limited tumors in nude mice. Other cooperating pathways needed.

Summary
Some genes have multiple fusion partners (RAR, EWS, many others) Fusion proteins are often necessary but NOT sufficient to cause cancer (PMLRAR, EWS-FLI1) Role of reciprocal fusion partners (RARPML) Other cooperating mutations are important

TMPRSS2
Transmembrane serine protease Prostate localized (colon, stomach, salivary glands) Androgen regulated Tmprss2 -/- mice are normal No over-expression models

ERG1
Most consistently over-expressed gene in 14/18 patients (78%) using paired normal and malignant tissue.

ETV1
Sarcoma = EWS-ETV1 fusion expression is oncogenic in a nude mouse model. ETV1 -/- mice display severe motor discoordination due to loss of establishment of functional sensory-motor circuitry in the developing spinal cord.

Conclusions
TMPRSS2-ERG ETV1 fusions may have important implications for prostate cancer. TMPRSS2 fusion in 20/22 cases overexpressing ERG or ETV1, suggesting this is the most likely explaination. Findings may overestimate prevelance. Findings suggests that causal gene rearrangements may exist in common epithelial tumors but are masked by mutator phenotype.