Molecular Path of the GI tract

Dr Shaun Walsh

Mutation analysis in Tumours

• A new challenge for oncologists, surgeons pathologists and for patients

I’m referring you to the pathologist

Rapidly evolving challenge because the basic science of cancer keeps expanding

Requiring some pathologists to leave our comfort zone !

Today: An introduction

• The future of pathology

• Does have current impact on practice

• Focus on specific examples

• Stimulate further reading

Roles for Molecular Path in GI disease

• Diagnosis of disease

• Targetting therapy

• Screening for disease

Topic 1

• Diagnosis of disease

• e.g. Detection of clonality in lymphoma

Lymphoma Diagnosis

• Most lymphomas are diagnosed using routine H&E microscopy and immunohistochemistry

• Diagnosis and classification of malignant lymphomas can be problematic area, especially early stage disease

• In majority of lymphoma - antigen receptor (Ig or TCR) gene rearrangement occurs before transformation

• Monoclonality of tumour cells; all tumour cells progeny of single malignantly transformed cell (unique Ig or TCR gene rearrangement)

• Clonality testing can be valuable test to confirm diagnosis of lymphoma

• Discrimination between polyclonal reactive processes and monoclonal malignant tumours e.g. Follicular hyperplasia vs lymphoma

Clonality

• Cancer cells are the progeny of a single malignantly transformed cell

• Monoclonality key feature of malignant tumour cell populations

• Identically rearranged Ig/TCR genes

• Discriminates from oligoclonal or polyclonal reactive processes

Ig/TCR gene rearrangement

• Occurs during early differentiation• Results in enormous diversity antigen receptors• Genes encoding Ig & TCR molecules formed by

stepwise rearrangement of V, D and J gene segments (V(D)J recombination)

• Nucleotides randomly deleted and inserted joining sites• Formation of CDR3 (antigen binding part of antibodies,

highly variable, unique length and sequence)• Standardized BIOMED2 multiplex PCR assays• Increased number of targets for clonality detection

Germline organization of human T cell receptor and loci

T-Cell Receptor - and -Chain Gene Rearrangement and expression

The organization of the T-cell receptor γ-

and δ-chain loci

Clonality Testing Method

Fresh, frozen and FFPE samples

Extract DNA of medium quality/quantity (>200-300bp)

PCR using consensus primers for rearranged IgH and TCRG genes

Fluorescent fragment analysis (GeneScan)

Clonality Testing Results

Clonal peak detected (326bp)

Clonal peak detected (267bp)

Polyclonal control

Polyclonal control

Interpretation not always straightforward!

Polyclonal control

? Possible clonal peak in polyclonal background

No clonal peak detected

? No clonal peak detected

Limitations/Pitfalls

False negatives (sensitivity)• lack of primer annealing• somatic hypermutation (germinal centres), primer binding sites altered• poor quality DNA (FFPE)

False positives (specificity)• Pseudoclonality (esp small tissue samples, needle core biopsies - scant

cellularity) • Minimised by analysis of duplicate samples; more than one sample from

same patient

Not always markers of lineage

Results must be interpreted in the appropriate clinical and pathological context!

Topic 2

• Targetting therapy e.g.

• Cetuximab or Panitumumab ( anti-EGFR) in colon cancer

KRAS and Colon ca• KRAS mutations can be detected in approximately 30-40% of all

CRC.

• Patients with KRAS mutations in codons 12 or 13 do not benefit from anti-EGFR therapy with cetuximab or panitumumab.

• In contrast, about 40% of patients with metastatic colorectal cancer unresponsive to other therapies, and who lack a KRAS mutation, show a partial response with these agents.

• These findings suggest that only patients without KRAS mutations should be eligible to receive these therapies.

Why is KRAS important?

It is a downstream signal transducer from many transmembrane growth factor receptors

KRAS downstream effects

Why is KRAS important?

• If KRAS is mutated and constituently activated then blocking a receptor upstream will make no difference

• Avoid unnecessary and expensive therapy

KRAS and Colon Ca

• Identification of the right cells for assay analysis.

• KRAS mutations are detected on DNA from tumour sections.

• A pathologist’s evaluation of the tissue section used for DNA extraction is required to ensure that tumour cells are present in the specimen and that tumour cells are present in adequate quantity/concentration for the KRAS test that is utilized by the lab.

Method Principle Sensitivity

(MT/WT, %)

Turnaround Advantages Disadvantages

Direct sequencing Non-mutation-specific determination of test case nucleotide sequence and

comparison with normal sequence

15–25 Slow (4 days to 2 weeks

from paraffin)

Gold standardDetects all possible mutations

Poorly quantitativeInsensitive; Labour

intensiveOpen PCR system requires

strict control to prevent contamination

RFLP Mutation presence induces or eliminates specific sites where DNA-targeting enzymes insert cuts in DNA

1 Slow (4 days to 2 weeks

from paraffin)

Requires no specialized equipment, inexpensive

Often requires confirmation by

sequencingDoes not identify specific

mutationNon-quantitative

Allele-specific probe Polymerase chain reaction/selective detection

10 Rapid (<2 days from paraffin)

Rapid turnaround Relatively low sensitivity

High resolution melting analysis, confirmed by direct sequencing

Sequences with mutations hybridize at different, fixed temperatures

5 Slow (4 days to 2 weeks

from paraffin)

Can screen for mutations prior to sequencing

Complicated Requires sequencing confirmation

Considerable manual input required

Amplification refractory mutation system (ARMS)

Mutation specific polymerase chain reaction/detection

1 Rapid (<2 days from paraffin)

High sensitivityRapid turnaround

Detects only single specific mutation per reaction

Requires specially engineered primer/probe

TheraScreen™ KRAS testing kit (DxS, Manchester, United Kingdom)

Combination of ARMS and real-time PCR technology

1–5% Rapid (2 days) High sensitivityRapid turnaround

Closed PCR system eliminates risk of contamination

Detects only the most common mutations

Requires more tissue for analysis than other

methodsVery Expensive

Available as a commercial kit

Pyrosequencing Detection and measurement of the amount of pyrophosphate released by

DNA extension reaction

5–10 Rapid Precise and reproducible allele quantification

Allows sequencing of relatively small PCR products (useful for degraded

DNA samples).

Short reading length for sequences used

Open PCR system requires strict control to prevent

contamination

Methods

Curr Oncol. 2010 July; 17(Supplement 1): S31–S40.

Topic 2

• Targetting therapy e.g.

• Imatinib therapy in Gastrointestinal stromal tumours (GISTs)

What are GISTs?

• Gastrointestinal stromal tumours

• Rare, but in the past, largely lethal GI tract sarcomas

• First example of a sarcoma with effective designer therapy

WE KNOW GISTS HAVE A WIDE VARIETY OF GROSS APPEARANCES!

WE KNOW GISTS HAVE A VARIETY OF HISTOLOGIC APPEARANCES!

Molecular pathway to GISTDr. Hirota 1998. Most GIST are caused by

mutations in the KIT oncogene

c-kit mutation

GISTKIT positive

‘designer tumour’Hirota Science 1988

KIT

DOG-1 (98%)

KIT (95%)

CD34 (66%)

GISTS can have a fairly predictable immunoprofile

Very helpful in confirming the diagnosis

Doesn’t tell you prognosis or how to treat!

New molecular pathways in GIST

PDGFRA KIT Wild Type

GIST

85%

BRAF

NF-1Carney-Stratakis

SDHB

Carneys triad

Ligand Independent Dimerisation

Downstream of KIT

KIT and PDGFRA genes have mutation hotspots

Imatinib

Mutations produce permanently switched on type III tyrosine kinase receptors

Mutation analysis

• At the least very helpful to know whether you are dealing with a KIT exon 9 or 11 mutation

• Is the tumour primarily resistant?

• More information is emerging about many other mutations

KIT exon 11 mutants (65%)

• All sites

• Substitutions, insertions, deletions, ins-del

• Better respn to imatinib than exon 9

KIT exon 11 mutants

• Upstream 5’ short deletions and substitutions less aggressive than downstream 3’ deletions (557-558)

• Still controversial about TKI response

• LoH Chromosome 4q or ‘homozygous’ exon 11 mutation are aggressive

KIT exon 9 mutants (9%)• More commonly small bowel and high risk

• Most have insertion of 6 base pairs

• KIT exon 9 more imatinib resistant and may need higher dose imatinib

• May respond to sunitinib

• Prob. due to steric

hindrance of TKI binding

PDGFRA exon 18 mutants

• Rare (6-7%)

• Missense

• Stomach,omentum

• Epithelioid on H&E

• May be KIT neg by IHC

• Benign course

• Affects kinase activation loop domain

• Most are TKI sensitive ....... BUT!

PDGFRA exon 18 mutants

• Some are primarily resistant to TKI’s

• D842V (commonest)

• RD841-842KI

• DI842-843IM

• All affect codon 842 in the kinase domain

TKI resistance in GIST

• Primary

- tumour progression in first 6/12 of TKI rx

• Secondary (acquired)

- tumour progression after initially good respn/stable disease usually 12-36months

Primary Resistance

• Occurs in GISTs of all mutation types

• More frequent in KIT exon 9, PDGFRA and wild type GISTs than in KIT exon 11 GISTs

• Commonest is D842V mutant in PDGFRA exon 18

Mechanisms of primary resistance

• Steric hindrance to TKI binding in KIT exon 9 mutant GISTs

• Concommitant downstream BRAF, KRAS mutations

Topic 3

• Screening e.g.

• Lynch syndrome (Heriditary non polyposis colorectal carcinoma)

Risk for Colorectal Carcinoma

0 20 40 60 80 100

General populationGeneral population

Personal history of Personal history of colorectal neoplasiacolorectal neoplasia

Inflammatory Inflammatory bowel diseasebowel disease

HNPCCHNPCC

FAPFAP

5%5%

15%–20%15%–20%

15%–40%15%–40%

70%–80%70%–80%

>95%>95%

Lifetime risk (%)Lifetime risk (%)

Lynch syndrome• Hereditary non-polyposis colorectal cancer

• Autosomal dominant (80%pen)

• Onset CRC aged 45-55yrs

• Mutated genes which effect DNA mismatch repair

• MLH1, MSH2, MSH6, PMS1, PMS2

• Microsatellite instability

• Accumulation of genetic damage

Contribution of Gene Mutations to HNPCC Families

MSH2 MSH2 ~30%~30%

MLH1MLH1~30%~30%

PMS1 PMS1 (rare)(rare)

PMS2PMS2 (rare) (rare)

MSH6 MSH6 (rare)(rare)

Unknown Unknown ~30%~30%

SporadicSporadic FamilialFamilial

HNPCCHNPCC

FAPFAP

Rare CRC Rare CRC syndromessyndromes

Liu B et al. Liu B et al. Nat MedNat Med 2:169, 1996 2:169, 1996

The Pathology of HNPCC

Typically less than 100 polyps in the colon

The Pathology of HNPCC

THE POLYPS ARE STANDARD ADENOMAS

The Pathology of HNPCC

• Histopathology report documents number of adenomas

• Under 100 considered typical in the past

• Search for supervening carcinomas

• Staging if necessary

• HNPCC carcinomas look a little different!

Pathology of HNPCC Carcinomas• Right sided

• Multiple

• Poorly differentiated,

• Mucin producing

• Peritumoral lymphocyte infiltration

• Crohn’s like reaction

The Pathology of HNPCC Carcinomas

HNPCC Carcinomas

OFTEN POORLY DIFFERENTIATED

HNPCC Carcinomas

BRISK INFLAMMATORY RESPONSE

T.I.L’s.

Problems

• Sporadic cancers can and often do look identical

• Adenomas are identical

• You cannot distinguish between heriditary and sporadic patients by simple methods

• You can miss the oppurtunity to intervene in whole kindreds

Risk of cancer in HNPCC

• Lifetime cancer risks:• – Colorectal 80%• – Endometrial 20-60%• – Gastric 13-19%• – Ovarian 9-12%• – Biliary tract 2%• – Urinary tract 4%• – Small bowel 1-4%• – Brain/CNS 1-3%

Role of Pathologist in heriditary CRC

Historical:

Diagnosis of heriditary CRC was based on history, family history, macroscopic and microscopic pathologic features

Present day:

Screening for gene mutations

DNA mismatch repair

Testing for HNPCC

• Clinical

• MSI

• IHC

• Gene sequencing

MSI Analysis

10%–15% of sporadic tumors have MSI95% of HNPCC tumors have MSI at multiple loci

MSI genetic testing

• Performed on FFPE tissue.

• NCI panel

• – 5 Markers

• – >2 show MSI then MSI – High

• – 1 shows MSI then MSI – Low

• – None show MSI then MSS

Tag

Tag

IMMUNOHISTOCHEMISTRY

NORMAL COLONIC MUCOSA

H&E

MLH1

MSH2

MSH2

Strongly positive at crypt base

Upper 1/3 Lower 1/3

MLH1 PMS2

MSH2 MSH6

Screening Case 1: All four markers present

H&E

MLH1NEG

MSH2POS

Screening case 2:No MLH1 expression

Screening case 3: Loss of MSH2

H&E

MSH2

MLH1+

-

Joint approach

• If immunohistochemistry identifies a missing repair protein

• And MSI is also detected

• The candidate gene is sequenced

• If a mutation is found

• The entire family can be screened with a blood test instead of colonoscopy

Purpose of Today

• Cancer diagnosis, screening and treatment is changing

• You need to get with the program

• A sound knowledge of Molecular biology is required

The Present

The Future