molecular path of the gi tract dr shaun walsh. mutation analysis in tumours a new challenge for...
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Molecular Path of the GI tract
Dr Shaun Walsh
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Mutation analysis in Tumours
• A new challenge for oncologists, surgeons pathologists and for patients
I’m referring you to the pathologist
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Rapidly evolving challenge because the basic science of cancer keeps expanding
Requiring some pathologists to leave our comfort zone !
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Today: An introduction
• The future of pathology
• Does have current impact on practice
• Focus on specific examples
• Stimulate further reading
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Roles for Molecular Path in GI disease
• Diagnosis of disease
• Targetting therapy
• Screening for disease
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Topic 1
• Diagnosis of disease
• e.g. Detection of clonality in lymphoma
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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
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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
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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
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Germline organization of human T cell receptor and loci
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T-Cell Receptor - and -Chain Gene Rearrangement and expression
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The organization of the T-cell receptor γ-
and δ-chain loci
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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)
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Clonality Testing Results
Clonal peak detected (326bp)
Clonal peak detected (267bp)
Polyclonal control
Polyclonal control
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Interpretation not always straightforward!
Polyclonal control
? Possible clonal peak in polyclonal background
No clonal peak detected
? No clonal peak detected
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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!
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Topic 2
• Targetting therapy e.g.
• Cetuximab or Panitumumab ( anti-EGFR) in colon cancer
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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.
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Why is KRAS important?
It is a downstream signal transducer from many transmembrane growth factor receptors
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KRAS downstream effects
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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
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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.
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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.
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Topic 2
• Targetting therapy e.g.
• Imatinib therapy in Gastrointestinal stromal tumours (GISTs)
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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
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WE KNOW GISTS HAVE A WIDE VARIETY OF GROSS APPEARANCES!
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WE KNOW GISTS HAVE A VARIETY OF HISTOLOGIC APPEARANCES!
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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
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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!
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New molecular pathways in GIST
PDGFRA KIT Wild Type
GIST
85%
BRAF
NF-1Carney-Stratakis
SDHB
Carneys triad
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Ligand Independent Dimerisation
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Downstream of KIT
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KIT and PDGFRA genes have mutation hotspots
Imatinib
Mutations produce permanently switched on type III tyrosine kinase receptors
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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
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KIT exon 11 mutants (65%)
• All sites
• Substitutions, insertions, deletions, ins-del
• Better respn to imatinib than exon 9
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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
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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
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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!
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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
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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
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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
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Mechanisms of primary resistance
• Steric hindrance to TKI binding in KIT exon 9 mutant GISTs
• Concommitant downstream BRAF, KRAS mutations
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Topic 3
• Screening e.g.
• Lynch syndrome (Heriditary non polyposis colorectal carcinoma)
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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 (%)
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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
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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
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The Pathology of HNPCC
Typically less than 100 polyps in the colon
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The Pathology of HNPCC
THE POLYPS ARE STANDARD ADENOMAS
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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!
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Pathology of HNPCC Carcinomas• Right sided
• Multiple
• Poorly differentiated,
• Mucin producing
• Peritumoral lymphocyte infiltration
• Crohn’s like reaction
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The Pathology of HNPCC Carcinomas
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HNPCC Carcinomas
OFTEN POORLY DIFFERENTIATED
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HNPCC Carcinomas
BRISK INFLAMMATORY RESPONSE
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T.I.L’s.
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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
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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%
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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
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DNA mismatch repair
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Testing for HNPCC
• Clinical
• MSI
• IHC
• Gene sequencing
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MSI Analysis
10%–15% of sporadic tumors have MSI95% of HNPCC tumors have MSI at multiple loci
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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
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Tag
Tag
IMMUNOHISTOCHEMISTRY
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NORMAL COLONIC MUCOSA
H&E
MLH1
MSH2
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MSH2
Strongly positive at crypt base
Upper 1/3 Lower 1/3
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MLH1 PMS2
MSH2 MSH6
Screening Case 1: All four markers present
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H&E
MLH1NEG
MSH2POS
Screening case 2:No MLH1 expression
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Screening case 3: Loss of MSH2
H&E
MSH2
MLH1+
-
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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
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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
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The Present
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The Future