Colorectal cancer

Molecular diagnostics

Rachel Butler

All Wales Genetics Lab

• Jass model (Histopathology, 2007)

• Current molecular markers

• Future molecular markers and analytical

tools

Molecular diagnostics

2. CIMP-high, partial

methylation of MLH1,

BRAF mutation,

chromosomally stable,

MSS or MSI-L, origin

in serrated polyps

(8%).

Pathways of Colorectal Carcinogenesis (Jass)

5. Lynch syndrome, MSI-H,

CIMP-negative, BRAF

mutation negative,

chromosomally stable, origin

in adenomas (3%).

3. CIMP-low, KRAS

mutation, MGMT

methylation,

chromosomal

instability, MSS or

MSI-L, origin in

adenomas or serrated

polyps (20%).

1. CIMP-high, MSI-H, methylation of

MLH1, BRAF mutation, chromosomally

stable, origin in serrated polyps, known

generally as sporadic MSI-H (12%).

4. Chromosomal

instability, MSS,

CIMP-negative,

origin in

adenomas (57%).

Epigenetic DNA Methylation

CpG Island Methylator Phenotype: CIMP

• coordinated methylation of genes:

MLH1, CDKN2A/p16, MGMT, IGF2, RUNX3, SOCS1, MINTs and others

• associated with BRAF mutation.

• 10 – 20% colon cancers.

2. CIMP-high, partial

methylation of MLH1,

BRAF mutation,

chromosomally stable,

MSS or MSI-L, origin

in serrated polyps

(8%).

Pathways of Colorectal Carcinogenesis (Jass)

5. Lynch syndrome, MSI-H,

CIMP-negative, BRAF

mutation negative,

chromosomally stable, origin

in adenomas (3%).

3. CIMP-low, KRAS

mutation, MGMT

methylation,

chromosomal

instability, MSS or

MSI-L, origin in

adenomas or serrated

polyps (20%).

1. CIMP-high, MSI-H, methylation of

MLH1, BRAF mutation, chromosomally

stable, origin in serrated polyps, known

generally as sporadic MSI-H (12%).

4. Chromosomal

instability, MSS,

CIMP-negative,

origin in

adenomas (57%).

Colorectal cancer pathways

Noffsinger AE. Ann. Rev Pathol. 2009;4:343-64.

Predictive and prognostic markers

• MSI – Lynch syndrome

– No benefit with chemo in sporadic tumours

• KRAS – don’t respond to EGFR MAb – p.G13D ??

• BRAF – poor prognosis

Microsatellite instability • Lynch syndrome

– MSI analysis or MMR IHC

– BRAF p.V600E or MLH1 methylation

– Determines diagnosis and management

Or

• Sporadic CRC – MSI analysis or MMR IHC

– BRAF p.V600E or MLH1 methylation and/or FH (i.e. Not Lynch!)

– Determines treatment

KRAS: -ve predictive marker

• Monoclonal antibody to EGFR (Cetuximab) only effective in patients whose tumours are KRAS wild-type

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12 14 16 18

Time from Randomisation (Months)

Pro

port

ion A

live

Cetuximab

BSC

Cetuximab

BSC

117 108 95 81 52 34 20 9 6 2113 92 69 36 24 17 12 5 3 3

Study arm MS

(months)

95% CI

Cetuximab +

BSC

9.5 7.7 –

10.3

BSC alone 4.8 4.2 –

5.5

Tejpar et al, JCO 2012

The EGFR

MAb story

The EGFR

MAb story

Response to EGFR

MAb also reduced by

tumours with

mutations in NRAS,

BRAF and PIK3CA,

and EREG low

expressors

FOCUS4

• Biomarker trial to identify and validate

treatments for:

– BRAF mutations

– PTEN loss and PIK3CA mutations

– KRAS and NRAS mutations

– EGFR pathway dependent (i.e. WT for

everything including MMR IHC)

• Determine EREG expression status

Parallel testing for multiple

mutations….

Many molecular tools available…….

Cost

Sensitivity and

specificity

Availability

of

equipment

How many

markers

required…now

and in the

future

Ability to

work on FFPE

Flexibility to

add to panel Availability of

commercial (CE-

marked) test

Mutation type

analysed

Batching

and RTs

Molecular tools

KRAS, NRAS, PIK3CA, (PIK3R1), BRAF

KRAS c.35G>A

(p.Gly12Asp)

My prediction…. We will test diagnostically for

• KRAS, NRAS, BRAF and PIK3CA

• MSI (or MMR IHC) in Lynch and sporadic tumours

• Possibly start to use gene expression markers

• Start using multi-gene testing platforms [cost vs required markers]

• All will require EQA

Many thanks

FOCUS4

UK NEQAS