1. Molecular Path of the GI tract Dr Shaun Walsh

2. Mutation analysis in Tumours A new challenge for oncologists, surgeons pathologists and for patients I’m referring you to the pathologist

3. Rapidly evolving challenge because the basic science of cancer keeps expanding Requiring some pathologists to leave our comfort zone !

4. Today: An introduction The future of pathology Does have current impact on practice Focus on specific examples Stimulate further reading

5. Roles for Molecular Path in GI disease Diagnosis of disease Targetting therapy Screening for disease

6. Topic 1 Diagnosis of disease e.g. Detection of clonality in lymphoma

7. 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

8. 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

9. 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

10. Germline organization of human T cell receptor  and  loci

11. T-Cell Receptor  - and  -Chain Gene Rearrangement and expression

12. The organization of the T-cell receptor γ- and δ-chain loci

13. 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)

14. Clonality Testing Results Clonal peak detected (326bp) Clonal peak detected (267bp) Polyclonal control

15. Interpretation not always straightforward! Polyclonal control ? Possible clonal peak in polyclonal background No clonal peak detected ? No clonal peak detected

16. 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!

17. Topic 2 Targetting therapy e.g. Cetuximab or Panitumumab ( anti-EGFR) in colon cancer

18. 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.

19. Why is KRAS important? It is a downstream signal transducer from many transmembrane growth factor receptors

20. KRAS downstream effects

21. 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

22. 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.

23. Warning: The NCBI web site requires JavaScript to function. more...more... From: Curr Oncol. 2010 July; 17(Supplement 1): S31–S40. Copyright/License ►Request permission to reuseRequest permission to reuse Table III. Methods for analyzing KRAS mutations 22,41 2241 MethodPrincipleSensitiv ity (MT/WT, %) TurnaroundAdvantagesDisadvantages Direct sequencingNon-mutation-specific determination of test case nucleotide sequence and comparison with normal sequence 15–25Slow (4 days to 2 weeks from paraffin) Gold standard Detects all possible mutations Poorly quantitative Insensitive; Labour intensive Open PCR system requires strict control to prevent contamination RFLPMutation presence induces or eliminates specific sites where DNA- targeting enzymes insert cuts in DNA 1Slow (4 days to 2 weeks from paraffin) Requires no specialized equipment, inexpensive Often requires confirmation by sequencing Does not identify specific mutation Non-quantitative Allele-specific probePolymerase chain reaction/selective detection 10Rapid (<2 days from paraffin) Rapid turnaroundRelatively low sensitivity High resolution melting analysis, confirmed by direct sequencing Sequences with mutations hybridize at different, fixed temperatures 5Slow (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 1Rapid (<2 days from paraffin) High sensitivity Rapid 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 sensitivity Rapid turnaround Closed PCR system eliminates risk of contamination Detects only the most common mutations Requires more tissue for analysis than other methods Very Expensive Available as a commercial kit PyrosequencingDetection and measurement of the amount of pyrophosphate released by DNA extension reaction 5–10RapidPrecise 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 MT = Mutant; WT = Wild-type Methods Curr Oncol. 2010 July; 17(Supplement 1): S31–S40.

24. Topic 2 Targetting therapy e.g. Imatinib therapy in Gastrointestinal stromal tumours (GISTs)

25. 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

26. WE KNOW GISTS HAVE A WIDE VARIETY OF GROSS APPEARANCES!

27. WE KNOW GISTS HAVE A VARIETY OF HISTOLOGIC APPEARANCES!

28. Molecular pathway to GIST Dr. Hirota 1998. Most GIST are caused by mutations in the KIT oncogene c-kit mutation GIST KIT positive ‘designer tumour’ Hirota Science 1988

29. 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!

30. New molecular pathways in GIST PDGFRAKITWild Type GIST 85% BRAF NF-1 Carney-Stratakis SDHB Carneys triad

31. Ligand Independent Dimerisation

32. Downstream of KIT

33. KIT and PDGFRA genes have mutation hotspots Imatinib Mutations produce permanently switched on type III tyrosine kinase receptors

34. 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

35. KIT exon 11 mutants (65%) All sites Substitutions, insertions, deletions, ins-del Better respn to imatinib than exon 9

36. 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

37. 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

38. 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!

39. 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

40. 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

41. 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

42. Mechanisms of primary resistance Steric hindrance to TKI binding in KIT exon 9 mutant GISTs Concommitant downstream BRAF, KRAS mutations

43. Topic 3 Screening e.g. Lynch syndrome (Heriditary non polyposis colorectal carcinoma)

44. Risk for Colorectal Carcinoma 020406080100 General population Personal history of colorectal neoplasia Inflammatory bowel disease HNPCC FAP 5% 15%–20% 15%–40% 70%–80% >95% Lifetime risk (%)

45. 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

46. Contribution of Gene Mutations to HNPCC Families MSH2 ~30% MLH1~30% PMS1 (rare) PMS2 (rare) MSH6 (rare) Unknown ~30% SporadicFamilial HNPCC FAP Rare CRC syndromes Liu B et al. Nat Med 2:169, 1996

47. The Pathology of HNPCC Typically less than 100 polyps in the colon

48. The Pathology of HNPCC THE POLYPS ARE STANDARD ADENOMAS

49. 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!

50. Pathology of HNPCC Carcinomas Right sided Multiple Poorly differentiated, Mucin producing Peritumoral lymphocyte infiltration Crohn’s like reaction

51. The Pathology of HNPCC Carcinomas

52. HNPCC Carcinomas OFTEN POORLY DIFFERENTIATED

53. HNPCC Carcinomas BRISK INFLAMMATORY RESPONSE

54. T.I.L’s.

55. 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

56. 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%

57. 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

58. DNA mismatch repair

60. Testing for HNPCC Clinical MSI IHC Gene sequencing

61. MSI Analysis 10%–15% of sporadic tumors have MSI 95% of HNPCC tumors have MSI at multiple loci

62. 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

63. Tag IMMUNOHISTOCHEMISTRY

64. NORMAL COLONIC MUCOSA H&E MLH1 MSH2

65. Strongly positive at crypt base Upper 1/3Lower 1/3

66. MLH1PMS2 MSH2MSH6 Screening Case 1: All four markers present

67. H&E MLH1 NEG MSH2 POS Screening case 2: No MLH1 expression

68. Screening case 3: Loss of MSH2 H&E MSH2 MLH1 + -

69. 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

70. 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

71. The Present

72. The Future