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Practical Molecular Testing in Surgical Pathology

Martin P Powers, MDUCSF Pathology and Laboratory

Medicine28 May 2011

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Disclaimer

• I am one of the directors of the Molecular Diagnostics Lab at UCSF

• The lab and medical center may receive revenue from some of the testing hereafter described.

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Some assumptions

• Know that DNA, RNA and protein exist• Know that DNA is transcribed into RNA,

which is then translated into protein• Know that techniques like PCR, DNA

sequencing and FISH exist– To be explained in more detail later

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Outline (approximate)

• Oncogenes and Tumor Suppressors• Detecting clinically relevant mutations in

oncogenes and tumor suppressors– Large DNA rearrangements

• FISH, RT-PCR, LOH, etc

– Small DNA mutations• PCR, sequencing, etc.

• Technical issues• We can only scratch the surface in 45 min

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Introduction to oncogenes and tumor suppressors

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Hallmarks of Cancer

• Genes that promote these hallmarks– ONCOGENES– accelerators– Mutations “switch them

on”

• Genes that inhibit these hallmarks– TUMOR

SUPPRESSORS– “brakes”– Mutations “knock them

out”

Cell. 2000 Jan 7;100(1):57-70

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Oncogenes and tumor suppressors

• Oncogenes– Promote cell division/invasion or Inhibit

apoptosis (cell death)

– Gain of function mutation

– Dominant mutation• Only one allele mutated (usually) to promote

tumorigenesis• Wild type copy can still be present

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Somatic mutations and oncogenes

• Mutation in oncogene is only in tumor cells– (sporadic tumors)

• Surrounding stroma, inflammatory and all other cells are wild-type.

Cells of body don’t have mutation

Cells of tumor only have mutation

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Somatic mutations and oncogenes

Chromosome with oncogenic mutation

Wild-type (normal) chromosome

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Oncogenes

– Mechanisms• Amplification• Overexpression by translocation• Mutation, hyperactivation

– Translocation– Point mutation

– Types of Genes• Growth Factors• Growth Factor Receptors• Signal Transducers• Transcription factors• Cyclins and Cyclin dependant kinases• Anti-apoptotic molecules

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Growth factors, signal transduction and the cell cycle

Robbins & Cotran Pathologic Basis of Disease Online12

Tumor suppressors

• Tumor suppressors– Gatekeepers (direct)

• Inhibit cell division• Promote apoptosis

– Caretakers• Protect the genome (DNA

repair)• Indirect: Increase

mutations in oncogenes and gatekeepers

Robbins & Cotran Pathologic Basis of Disease Online

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Tumor suppressors• Mechanisms

– Point mutations– Deletions – silencing of gene expression (or combo for each

copy)

• Types of genes– Inhibitory growth receptors/ligands– Pro-apoptotic molecules– Cell adhesion molecules– Inhibitors of signal transduction– Cell cycle inhibitors– Sensors or enzymes for DNA repair

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Growth factors, signal transduction and the cell cycle

Robbins & Cotran Pathologic Basis of Disease Online

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Tumor suppressor genes

• Must be inactive to promote tumorigenesis (lose your brakes)

• Recessive mutations• Must lose both copies in the tumor

– Knudson’s two-hit hypothesis– Inherit the first mutation in inherited cancer

syndromes• Often one of the copies is lost through a “loss of

heterozygozity” whereby the entire chromosome, or chromosome arm, or large deletion deletes one of the copies, while a point mutation (or other small mutation) was on the other copy.

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Knudson’s two hit hypothesis

Human Molecular Genetics 2Strachan, Tom and Read, Andrew P.New York and London: Garland Science; c1999

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Somatic vs inherited genetics (for tumor suppressor genes)

• Inherited • Somatic (sporadic)Rb mutation

All cells of body inherit one mutation-Predisposition to RB = familial retinoblastoma- dominant inheritance

Two Mutations acquired in postnatal life in same cell;two mutations required for tumor;- Cellular recessive

Some cells get second hit (somatic)- Retinoblastoma tumor

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Detecting clinically relevant oncogene mutations

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Amplification

• Examples– ERBB2 (HER2/neu)

• Worse prognosis in breast cancer• Candidate for anti-ERBB2 therapy (such as

trastuzumab) for breast, GU, stomach/GE cancer

– EGFR• Worse prognosis and diagnostic for GBM

– MYC (or MYCN)• Worse prognosis in neuroblastoma and

medulloblastoma

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Mechanism of oncogene activation: amplification

OR HER2OR EGFR

Robbins & Cotran Pathologic Basis of Disease, 9th ed.

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HER2(ERBB2) fish

• 17q11.2-12• Often amplifies a

homogenously staining region

• FISH– HER2 = orange

– CEP17 = green

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HER2(ERBB2) fish

• 17q11.2-12• Often amplifies as a

homogenously staining region

• FISH– HER2 = orange– CEP17 = green

• HER2/CEP17 ratio– <1.8 = not amplified– >2.2 = amplified– 1.8-2.2 = equivocal

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Example photograph: ERBB2

Positive for Amplification Negative for

Amplification 24

Example photograph: EGFR

Positive for Amplification

Negative for Amplification

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Over-expression by translocation

• Common in lymphomas– IGH-BCL2

• Follicular lymphoma

– IGH-MYC• Burkitt lymphoma

– IGH-CCND1• Mantle cell lymphoma

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Mechanisms of oncogene activiation: translocation to active chromatin

Human Molecular Genetics 2Strachan, Tom and Read, Andrew P.New York and London: Garland Science; c1999

Robbins & Cotran Pathologic Basis of Disease, 9th ed.

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Translocation to create a fusion gene

• BCR-ABL– CML and AML

• EWSR1-FLI1– Ewing Sarcoma

• NPM-ALK– Anaplastic large cell lymphoma

• EML4-ALK– Non-small cell lung cancer

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Mechanisms of oncogene activation: translocation- novel chimeric gene

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FISH for translocations

• Breakapart – (one partner of translocation)

MYCIGK@ IGH@

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FISH for translocations

• Breakapart – (one partner of translocation)

MYCIGK@ IGH@

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FISH for translocations

• Fusion – (both partners partner of translocation)

IGH@ MYC

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FISH for translocations

• Fusion – (both partners partner of translocation)

IGH@IGK@

MYC

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FISH advantages

• Visualize tumor cells separate from normal cells

• Small tumor samples can be studied• Visualize any heterogeneity in the tumor

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PCR for translocations

• Over-expression translocations– No fusion transcript

– Must do DNA PCR• Sensitivity depends on spacing of breakpoints

• Fusion translocations– Fusion transcript– Allows for RT-PCR

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What is PCR

• Exponential amplification of DNA based on sequence specific primers– Amplify specific sequence between the

primers

• If you start with RNA– RT-PCR– RT (Reverse Transcription) step first converts

RNA to cDNA and then you amplify like PCR

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2 copies

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4 copies

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8 copies

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PCR = exponential target amplification

• Final copies = n2c

• C = cycles

• N = starting number of copies

• If n = 1– After 1 cycle = 2, after 2 cycles = 4, after 3 cycles = 8,

after 4 cycles = 16, after 5 cycles = 32– After 10 cycles, 1024– After 20 cycles, ~1 million

– After 30 cycles, ~1 billion

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Translocations on DNA

• Need the breakpoints to be close together• IGH-BCL2• 70% sensitive

Leuk Res. 2008 May;32(5):737-42. Epub 2007 Oct 26

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Translocations on RNA

• Advantage of exon specific primers• BCR-ABL

Adapted from Genes Chromosomes Cancer. 2001 Oct;32(2):97-111 50

Activating point mutations in oncogenes

• KRAS– Resistance to EGFR therapy in colorectal

carcinoma

• BRAF– Resistance to EGFR therapy (colon),

candidate for BRAF therapy (melanoma)

• EGFR– Response to EGFR therapy in lung cancer

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How many mutations are there

• COSMIC– Catalogue of somatic mutations in cancer

– Database of literature reported mutations in cancer in different genes

– http://www.sanger.ac.uk/genetics/CGP/cosmic/

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BRAF in COSMIC

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KRAS in COSMIC

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EGFR in lung

• Mutations mostly in 4 different exons (18-21)

• 2 hotspots• L858 point mutation

(exon 21)• Indels near amino

acid 746-747 (exon 19)

55J Natl Cancer Inst. 2005 Mar 2;97(5):339-46 56

Techniques for mutation detection

• Sequencing• Allele specific PCR• Real-time PCR• Allele specific extension (post-PCR)• others

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Issues to consider for mutation testing

• Analytical sensitivity– How much DNA is needed?

• Total amount of DNA, and therefore specimen required

• 1ng of DNA represents about 330 diploid cells• 1 diploid genome is about 6.6 pg of DNA

– How many % mutant copies need to be present to detect a mutant signal?

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% mutant burden

• DNA Sequencing• Analytic Sensitivity ~20%

•50% mutant alleles•20% mutant allele

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To dissect or not to dissect

12 tumor cells, 8 lymphocytes: 30% mutant allele burden 60

To dissect or not to dissect

12 tumor cells, 8 lymphocytes, 50 fibroblasts and endothelial cells: 8.5% mutant burden, but can dissect a discreet tumor mass with less normal cells

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To dissect or not to dissect

12 tumor cells, 8 lymphocytes, 50 fibroblasts and endothelial cells: 8.5% mutant burden, but no discreet tumor mass

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Important things to think about

• Analytical sensitivity and whether or not your lab dissects

• Choice of mutations that will be tested for– Is the panel comprehensive enough for your

clinicians need

• Fixative

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Effects of Fixative on DNA

• Buffered formalin– Damages DNA, but not as bad as unbuffered formalin

which will nearly totatlly degrade DNA

• Alcohol based fixatives– Good preservation of DNA

• B5, Bouin, Zenkers– Very bad for DNA

• Decalcification– Strong acids are very bad for DNA

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Testing for tumor suppressors

• DNA sequencing• Loss of functions mutations• May be anywhere in gene

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Test for loss of other copy of gene

• Deletion/Absence of other copy by FISH• LOH of polymorphic markers in

neighborhood of gene

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Deletion testing by FISH: 1p/19q

• Good prognosis and prediction of chemotherapeutic response

• Actual tumor suppressors not cloned yet• Tend to delete the entire 1p and 19q

chromosome arms

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1p/19q FISH

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1p/19q FISH

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LOH studies

D1S

407D

1S199

D1S

1161D

1S162

Marker Tumor Normal

D1S407 4 4,5

D1S199 6 6,8

D1S1161 3 3,5

D1S162 7 5,7

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To dissect or not to dissect

12 tumor cells, 8 lymphocytes, 50 fibroblasts and endothelial cells: 8.5% mutant burden, but no discreet tumor mass

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Testing for tumor suppressor loss: Testing a phenotype: MSI

• Microsatellite instability– A molecular phenotype

– Results from genetic lesions in the MMR machinery (mismatch repair)

– Inherited mutations in MMR• Lynch, Turcot, Muir-Torre Syndromes

– Sporadic loss of MMR• Sporadic MSI-high tumors

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Microsatellite instability

• Microsatellites– Oligonucleotide repeats

• Single nucleotide, dinucleotide, trinucleotide, etc

– Slippage during replication– Repaired by Mismatch repair machinery

– Absence of MMR - unstable

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Figure 12.8a The Biology of Cancer (© Garland Science 2007)

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Mismatch repair

• “Caretakers”• MSH6 and MSH2• MLH1and PMS2• Excise and repair mismatches and looped

out bases in DNA

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In the figure below, patients 1 and 3 show instability

Patient 1

Patient 2

Patient 3

Normal

Tumor

Normal

Tumor

Normal

Tumor

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Lynch vs. Sporadic MSI-high tumors

• 15% of colorectal carcinoma: MSI-high• Lynch(3-5%) and Sporadic(10-12%)• Sporadic

– MLH1 hypermethylation (CIMP-high)• Sometimes seen in Lynch (as a mechanism of

LOH)

– BRAF mutations• Mutated in sporadic (~50%)• Wt in lynch (almost always)

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Thank you for listening

Any Questions?

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References• "Catalogue of Somatic Mutations in Cancer." Retrieved April

25, 2011, from http://www.sanger.ac.uk/genetics/CGP/cosmic/.

• Coleman, W. B. and G. J. Tsongalis (2006). Molecular diagnostics : for the clinical laboratorian. Humana Press, Totowa, N. J.

• Espinet, B., B. Bellosillo, et al. (2008). "FISH is better than BIOMED-2 PCR to detect IgH/BCL2 translocation in follicular lymphoma at diagnosis using paraffin-embedded tissue sections." Leuk Res 32(5): 737-742.

• Hanahan, D. and R. A. Weinberg (2000). "The hallmarks of cancer." Cell 100(1): 57-70.

• Hunt JL (2008) “Molecular Pathology in Anatomic Pathology Practice: A Review of Basic Principles.” Arch Pathol Lab Med 132(2): 248-260

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References

• Kumar, V., A. K. Abbas, et al., Eds. (2010). Robbins and Cotran Pathologic Basis of Disease, Saunders Elsevier.

• Shigematsu, H., L. Lin, et al. (2005). "Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers." J Natl Cancer Inst 97(5): 339-346.

• Strachan, T. and A. P. Read (1999). Human Molecular Genetics, Wiley.

• Wang, Y. L., A. Bagg, et al. (2001). "Chronic myelogenous leukemia: laboratory diagnosis and monitoring." Genes Chromosomes Cancer 32(2): 97-111.

• Weinberg, R. A. (2007). The biology of cancer. New York, Garland Science.