1. “Molecular Pathology past present & future”
Thomas Kerr
Principal Healthcare Scientist
Molecular Pathology
Pathology Department
Southern General Hospital

2. “Molecular Pathology past present & future”
Thomas Kerr
Principal Healthcare Scientist
Molecular Diagnostics
Genetics
Southern General Hospital

3. Molecular Pathology ?
The discipline involved in diagnostic, prognostic and predictive testing on samples (usually tumour) removed from the patient in the course of investigation and treatment of disease.

8. Laboratory Facility 8

10. Molecular pathology 1988: Maura Farquharson 1992 MSc project ISH based
J Clin. Path 1996; “Immunoglobulin light chain mRNA detected by in situ hybridisation in diagnostic fine needle aspiration cytology specimens”
First diagnostic molecular pathology technique Kappa & Lambda ISH

11. H&E morphology ICC ISH kappa, lambda & EBV FISH PCR
B & T cell lymphoma
H&E morphology
ICC
ISH kappa, lambda & EBV
FISH
PCR

12. Lambda ICC

13. Lambda ISH

14. IgH clonality by Ceq8000

15. TCRgamma clonality by CEQ8000

16. TCRgamma polyclonal

17. Institute of neurological Science

18. GRI molecular pathology
HER2 ICC & FISH
Sarcoma FISH & RT-PCR
Lymphoma FISH

20. 2002
2003
Resection and Gliadel wafers
No other post-op Rx

21. Normal
1p186
Tumour
Normal
1p226
Tumour

22. Normal
19q112
Tumour
19q219
Normal
Tumour

23. Diagnosis confirmed as GBM grade 4, highly cellular, agressive malignant tumour, necrosis, vascular proliferation, mitotic activity GFAP positive staining ICC.
N040300

24. MGMT promoter status – clinical relevance
valuable for patient counselling
useful in deciding intensity of treatment
can be used clinically to select patients likely to benefit from adjuvant temazolomide treatment.
can potentially save half of patients from receiving chemotherapy.

25. U M
Methylated
N040300

26. Predictive & Prognostic markers in Neuro-oncology
Molecular assays introduced to aid typing & grading of gliomas
Improved understanding of these tumours at molecular level
Especially presence/absence of combined 1p19q loss in oligodendroglial tumours, MGMT promoter gene methylation in glioblastoma, EGFR amplification in grade III astrocytic tumours & glioblastoma & IDH mutation analysis
Now being used to tailor treatment decisions in patients, current therapy combination of surgery, radiotherapy & chemotherapy

27. 49F frontal
60F temp/parietal 1p
19q
Three case reviews to follow.

28. Molecular Haematology 2008
Microsatellites for Chimaerism testing in post-BMT patients
BCRABL (t9;22)
PMLRARA (t15;17) in AML
AML/ETO (t8;21) in AML
Chromosome 16 inversion in AML
JAK 2 status in myeloproliferative diseases
FLT3 mutations

29. Chimaerism

30. Purpose of Chimaerism testing post BMT
To detect an identifiable difference between a patient and their donor
Ability to measure the proportion of donor present in the blood or bone marrow post transplant
Fiona M Reid April 2011

31. Identification of chimaerism
Microsatellites: Short Tandem Repeats
Prepare DNA from blood or marrow
PCR
A number of primer sets in use
Must identify an “informative “ set of primers

32. Preparation of DNA: the EZ1

33. Chimaerism on Lymphoid/Myeloid split
Blood sample post BMT
Use CD3 to enrich for Lymphocyte fraction
Use CD15 to enrich for Myeloid fraction

34. Automacs Proseparator

35. Microsatellites Full donor Chimaerism
Fiona M Reid April 2011

36. Microsatellites Mixed Chimaerism
Fiona M Reid April 2011

37. The Calman review of genetics in relation to healthcare in Scotland
Recommendations from the molecular pathology review group on the future of molecular pathology testing in Scotland. (2010)
The Calman review of genetics in relation to healthcare in Scotland
Better health better care action plan
Better cancer care action plan
Clinical trials
SPAN & Scottish cancer group networked approach to HER2 FISH testing 37

38. Conclusions & recommendations
A molecular pathology service should be developed and run as a NSD supported consortium, mirroring the Scottish genetics laboratory consortium for diagnosis of constitutional genetic abnormalities.
To ensure the stability of the services, equity of access and service development
Linked to the Scottish cancer taskforce, cancer networks and Scottish medicines consortium so that there is early coordinated planning for new testing.

39. Laboratory Facility 39

40. Targeted therapies
Therapeutic monoclonal antibodies
Small molecules

41. Therapeutic monoclonal antibodies
Target specific antigens on proteins-extracellular growth factors eg transmembrane receptors
Monoclonal antibodies end with the stem “mab” e.g. cetuximab which targets EGFR

42. Small molecules
Penetrate the cell membrane to interact with targets inside a cell
Interfere with the enzymatic activity of the target protein
End with the stem “ib”, the agent has inhibitory properties e.g. imatinib in CML

43. Cost Targeted therapies £20000 per patient per year
Molecular test EGFR £50

44. Current Treatment is the Same for Most Patients Outcomes can vary widely=
Patients
Treatment +
Disease
Different
outcomes =
Current treatment generally uses a ‘one size fits all’ approach. However, while a drug may be highly effective in one patient, it may have little or no response in another. Similarly, a drug may be well tolerated or cause severe or even life-threatening side effects depending on the individual.
Different treatment outcomes affect patients’ safety, survival and quality of life 44

45. The Alternative: Personalized Healthcare (PHC) Tailors treatment to the patient
Personalized healthcare is a tailored approach to treatment based on the fact that people are different. Among patients with the same diagnosis, variations in response to treatment can be associated with differences in genetic make-up and/or the molecular nature of their disease.
Personalized healthcare involves stratifying patients into subgroups, based on these differences, enabling the most appropriate known treatment for that subgroup to be chosen.
At least in the foreseeable future, personalized healthcare does not mean developing a unique drug for every patient; rather, rational and optimal use of treatment options.
Molecular diagnostic testing can stratify patients according to their specific genetic makeup and/or the nature of their disease or condition
This approach improves drug safety, may increase patient survival, and may improve quality of life 45

46. Personalized Healthcare Provides benefits for all stakeholders
Patient
Healthcare provider
Payer
Personalized healthcare has potentially far-reaching benefits for all members of the healthcare system.
For patients, quality of care can be improved by selecting the treatment most likely to be effective and minimizing their exposure to adverse effects.
For healthcare professionals, the insight provided through diagnostic tests enables more informed treatment decisions to be made. With personalized healthcare, treatment can be administered with more confidence, ‘trial and error’ prescribing is reduced, and the risk of causing adverse effects is minimized.
Personalized healthcare also has the potential to benefit payers by supporting efficient utilization of healthcare budgets and resources. Cost savings may be made by using expensive therapies for targeted patient groups, minimizing costly adverse events, and potentially reducing long-term expenditure on treating progressive disease.
Improved benefit to risk ratio
Informed treatment decisions
Efficient use of healthcare budgets 46

47. Near to mid-term genomics and oncology portfolio Platform consolidation; Pharma driven focus on oncology
RMD is committed to expanding the oncology menu in order to remain competitive in the oncology IVD market.

48. Current Cobas® Oncology Portfolio Clinically validated biomarker tests on one platform
Disease
Therapeutic Class
Drug(s)
cobas® BRAF Mutation Test
Metastatic Melanoma
BRAF Inhibitors
RG7204 (PLX 4032), Zelboraf
cobas® KRAS Mutation Test
Colorectal Cancer
Anti-EGFR Monoclonal
Antibodies (mAb)
Anti-EGFR mAb
Therapies, cetuximab, panitumumab
cobas® EGFR Mutation Test
Non-Small Cell Lung Cancer (NSCLC)
Anti-EGFR Tyrosine Kinase Inhibitors
Erlotinib, Gefitinib
Speaker Notes
Roche has a growing pipeline of oncology assays in development that are being developed on the cobas 4800 system.
The most advanced of these programs are the BRAF, KRAS and EGFR mutation detection tests.
BRAF V600 mutation assay is being developed in conjunction with the RG7204 (vemurafenib) BRAF inhibitor in metastatic melanoma.
KRAS mutations are associated with lack of response to anti-EGFR mAb therapies.
EGFR mutations are associated with better response to Tyrosine Kinase Inhibitors (TKIs), Tarceva (Roche) and Iressa (AstraZeneca).
PIK3CA mutation assay is being developed in conjunction with the Genentech PI3K inhibitor that is currently in phase 1.
The P53 resequencing assay is an Amplichip test being developed on the Affymetrix platform.

49. cobas® 4800 Oncology Tests Workflow Overview
cobas® 4800 BRAF V600 Mutation Test:
The need for macrodissection must be determined prior to deparaffinization. For each specimen, a pathologist will perform a microscopic assessment of an H&E stained slide and will document the percent tumor cell content. The middle section from a series of slides should be used for the H&E staining. If the percent tumor content is greater than 25%, macrodissection is not required. If the percent tumor content is less than 25%, the section used for DNA Isolation should be macrodissected beforehand.
H&E stain, HE stain or hematoxylin and eosin stain, is a popular staining method in histology. It is the most widely used stain in medical diagnosis; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed H&E section, H+E section, or HE section.
The staining method involves application of hemalum, which is a complex formed from aluminium ions and oxidized haematoxylin. This colors nuclei of cells (and a few other objects, such as keratohyalin granules) blue. The nuclear staining is followed by counterstaining with an aqueous or alcoholic solution of eosin Y, which colors eosinophilic other structures in various shades of red, pink and orange.

50. 197,000 people worldwide are diagnosed with melanoma each year
Melanoma Progression
Melanoma is the most aggressive form of skin cancer originating in the melanocytes.
197,000 people worldwide are diagnosed with melanoma each year

51. BRAF Mutation Test Designed to identify metastatic melanoma patients who may benefit from BRAF inhibitors
The cobas®4800 BRAF V600 Mutation Test
Is designed to determine mutation status of the BRAF gene
Is the companion diagnostic used in the clinical trials for RG7204 (also known as PLX4032), an oral inhibitor of mutated BRAF co-developed by Roche Pharmaceuticals and Plexxikon. BRIM2 +3 clinical trials (>2,000 patients)~ Roche kit is the ONLY clinically validated kit.
RG7204 has shown strong clinical activity in metastatic melanomas harboring the BRAF V600E mutation; clinical data suggests that patients with wild-type BRAF do not respond or respond adversely.
Not all patients with metastatic melanoma respond to BRAF inhibitors.1
The cobas® 4800 BRAF V600 Mutation Test* is designed to determine the BRAF mutation status of patients. Used in conjunction with other clinical data, this information can help physicians select the best treatment options for an individual patient.
1. Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363:
* Product in development 51

52. ” 52

53. Colorectal Cancer Progression
Incidence in the UK: ~70 cases per 100,000
>95% of colorectal cancers arise from the glandular epithelium that lines the gut (adenocarcinomas)

54. cobas® KRAS Mutation Test Designed to help identify colorectal patients who are unlikely to benefit from anti-EGFR mAb therapies
cobas® KRAS Mutation Test:
Is designed to determine mutation status of the KRAS oncogene
Colorectal cancer tumors with KRAS mutations are unresponsive to anti-EGFR therapies1
When combined with other clinical data, this information will help physicians identify colorectal cancer patients who may not benefit from anti-EGFR mAb therapies
1. Amado et al. J Clin Oncol 2009;26:16261634

55. cobas® KRAS Mutation Test Designed to help identify colorectal patients who are unlikely to benefit from anti-EGFR mAb therapies
cobas® KRAS Mutation Test:
Is designed to determine mutation status of the KRAS oncogene
Colorectal cancer tumors with KRAS mutations are unresponsive to anti-EGFR therapies1
When combined with other clinical data, this information will help physicians identify colorectal cancer patients who may not benefit from anti-EGFR mAb therapies
1. Amado et al. J Clin Oncol 2009;26:16261634

56. Non-small cell lung cancer Overview
Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide: ~106,000 new cases of advanced lung cancer per year in the UK, France, Spain, Italy and Germany combined.
NSCLC is the most prevalent and accounts for approximately 85% of all cases.
Prognosis for patients with NSCLC is poor with a 5-year survival rate of only 15%.

57. Non-small Cell Lung Cancer Therapy Options - Overview
Treatment of cancer is guided by disease stage:
early stages: primary treatment is surgical resection
locally advanced or metastatic disease:
platinum-based chemotherapy
targeted therapy
anti-EGFR (cetuximab, panitumumab)
tyrosine kinase inhibitors: erlotinib (Roche Tarceva® and gefitinib (AstraZenaca Iressa®)
anti-VEGF (Roche Avastin®)

58. Non-small Cell Lung Cancer Role of EGFR
EGFR is a receptor tyrosine kinase located on the cell surface of epithelial cells.
EGFR is linked to several signaling pathways including the RAS/MAPK pathway and the PI3K/Akt pathway.
In normal cells, these pathways are tightly regulated and are activated only when a specific ligand (such EGF) binds to the receptor.
In cancer cells, EGFR gene mutations activate the kinase in the absence of a ligand leading to abnormal cell proliferation and tumor formation.

59. cobas® EGFR Mutation Test Designed to help identify NSCLC patients who may benefit from anti-EGFR TKI therapy
cobas® EGFR Mutation Test:
Is designed to detect mutations in the EGFR gene in NSCLC tumor tissue
Test results will help physicians identify NSCLC patients who may benefit from anti- EGFR tyrosine kinase inhibitor therapy

60. Next Generation Sequencing
High throughput, low cost (?) sequencing technology
Human Genome Project started in 1990 and took 13 years to complete
It was an international collaboration which cost $3 billion and used traditional Sanger sequencing
Today you could sequencing the human genome in ~month using NGS
Target is the $1000 genome

61. Next Generation Sequencing
Many NGS techniques, chemistries and instruments now available
Personal genome machines (PGMs) are affordable bench-top instruments e.g. Ion Torrent
NGS allows you to look at many genes or regions of the genome, from many patients, simultaneously
NGS has multiple applications and will replace many of the molecular tests currently available in the lab

62. Ion Torrent

63. The present: small (and a little odd looking)

64. Ion Torrent Personal Genome Machine
Bench-top NGS instrument from Life Technologies.
Purchase the Comprehensive Cancer Panel from Life Technologies which targets exons within 409 oncogenes and tumour suppressor genes.
Requires 10ng of input DNA from FFPE tissue.
Produces amplicons of between bp and has 16,000 primers in 4 tubes.
From DNA extraction to results is less than 24 hours, and the laboratory process is almost fully automated

65. The future: bigger and better
What is bigger and better?
Bigger is looking at more genes, better is improving the patients diagnosis and treatment in doing so
How do we do bigger and better?
By sequencing the entire exome, every single base of each coding exon in the genome
In the future it will be cheaper and easier to sequence the whole exome for every patient

66. NGS: the future

67. NGS: the future

68. NGS: the future
Life Technologies claim the Ion proton will sequence the
entire human genome in 8 hours

70. Acknowledgements Maura Farquharson Willie Stewart
Molecular pathology team SGH
Nicola Williams, Jane Duncan, Rachel Ellis
Aly Grant (Roche)

71. Information CRUK stratified medicine www.mycancergenome.com
Life technologies ion torrent