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Development of Molecular Methodologies for the Enhanced Detection of Tumour Biomarkers. Michelle Wood , Hood Mugalassi, Justyna Tull, Linda Meredith, Rachel Butler Institute of Medical Genetics, University Hospital Wales, Cardiff. Lung cancer is the leading cause of cancer deaths.

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Development of molecular methodologies for the enhanced detection of tumour biomarkers

Development of Molecular Methodologies for the Enhanced Detection of Tumour Biomarkers

Michelle Wood, Hood Mugalassi, Justyna Tull, Linda Meredith, Rachel Butler

Institute of Medical Genetics, University Hospital Wales, Cardiff


Non small cell lung cancer and metastatic colorectal cancer

Lung cancer is the leading cause of cancer deaths. Detection of Tumour Biomarkers

NSCLC major subtype.

>70% diagnosed with advanced disease.

5 year survival <15%.

EGFR tyrosine kinase inhibitors (i.e.Gefitinib) used to treat patients refractory to first line chemotherapy.

Response in 10% unselected patients.

Patients with metastic colorectal cancer – 5 yr survival <10%.

Monoclonal antibody therapy targeted to EGF receptor approved for irinotecan resistant mCRC (i.e. Cetuximab).

Response in 10-20% unselected patients.

Non Small Cell Lung Cancer and metastatic Colorectal cancer


Egfr and kras mutations

Mutations in tyrosine kinase domain of EGFR– predict those patients likely to respond to TKI therapy.

90% patients with mutation -15bp deletion in exon 19 or single point mutation in exon 21.

Select patients that will benefit from TKI therapy.

EGFR and KRAS mutations


Development of molecular methodologies for the enhanced detection of tumour biomarkers

EGFR and KRAS mutations patients likely to respond to TKI therapy.

  • Mutations in tyrosine kinase domain of EGFR– predict those patients likely to respond to TKI therapy.

  • 90% patients with mutation -15bp deletion in exon 19 or single point mutation in exon 21.

  • Select patients that will benefit from TKI therapy.


Egfr and kras mutations1
EGFR and KRAS mutations patients likely to respond to TKI therapy.

  • KRAS mutations occur in 20-30% NSCLC and 30-40% mCRC.

  • Predict lack of response to TKIs and cetuximab.

  • Most frequent mutations occur in codons 12&13.

  • Select patients that will not benefit from TKI or Cetuximab therapy.


Egfr and kras mutations2
EGFR and KRAS mutations patients likely to respond to TKI therapy.

  • KRAS mutations occur in 20-30% NSCLC and 30-40% mCRC.

  • Predict lack of response to TKIs and cetuximab.

  • Most frequent mutations occur in codons 12&13.

  • Select patients that will not benefit from TKI or Cetuximab therapy.


Aims of project
Aims of project patients likely to respond to TKI therapy.

  • To investigate the use of alternative, more sensitive and cheaper technologies for the detection of EGFR mutations.

  • To develop Co-amplification at low denaturation temperatures PCR (COLD-PCR) to improve detection of low frequency KRAS and EGFR mutations.

  • To investigate the use of cell free DNA as an alternative source of tumour DNA.


Alternative methodologies
Alternative methodologies patients likely to respond to TKI therapy.

  • EGFR mutations detected by sequencing – not sensitive enough, nested PCR, > 5 days, higher cost.

  • Designed assays for MALDI-TOF, High resolution melt analysis, pyrosequencing and fluorescent PCR.

  • Looked at sensitivity, number of mutations detected, efficiency and cost.


Maldi tof
MALDI-TOF patients likely to respond to TKI therapy.

  • Multiplex PCR – screen several mutations within a single reaction.

  • 96 well format – multiple patient samples.

  • Quick assay can be done cheaply.

  • Assay designed for EGFR – problems with clustered mutations.


High resolution melt analysis
High resolution melt analysis patients likely to respond to TKI therapy.

  • Assay designed to cover the four exons, potential to detect all possible mutations.

  • Cheap and quick assay, but needs additional sequencing reaction.

Nomoto et al. 2006 Am J Clin Pathol126:608-615


Pyrosequencing
Pyrosequencing patients likely to respond to TKI therapy.

Exon 18

  • Designed assays for exons 18, 20 and 21.

  • Detects 7 sensitising mutations, 3 resistance mutations.

  • Sensitive down to 5% mutant in wild type background.

  • Result in <5 days.

  • Validated on multiple patient samples – introduced as a new diagnostic service.

Exon 21

Exon 20


Fluorescent pcr
Fluorescent PCR patients likely to respond to TKI therapy.

Exon 19 Normal

15bp deletion

15bp deletion

15bp deletion

9bp

deletion

Exon 20 insertions Normal

  • Fluorescently labelled primers – amplify exons 19 or 20.

  • Detect deletions or insertions based on fragment size.

  • Exon 19 deletion – 45% patients with TKI sensitising mutations

  • Exon 20 insertions – resistance to tyrosine kinase inhibitors

  • Along with pyrosequencing will detect 95% of patients with TKI sensitising mutations.

12bp insertion


Development of molecular methodologies for the enhanced detection of tumour biomarkers

Comparison of technologies patients likely to respond to TKI therapy.


Co amplification at l ow d enaturation temperature pcr cold pcr
Co patients likely to respond to TKI therapy.-amplification at Low Denaturation temperature-PCR (COLD-PCR)

  • Alternative PCR method -preferentially enriches minority alleles from mixture of wild type and mutant sequences.

  • For samples with very low tumour content.

  • For short sequences <200bp

Li et al. 2008, Nature Medicine 14: 579-584


Development of molecular methodologies for the enhanced detection of tumour biomarkers

KRAS mutation patients likely to respond to TKI therapy.

EGFR mutation


Cell free dna

Tumour DNA extracted from fixed biopsy samples or tumour resections

Problems with quality of DNA due to fixation

Mixture of normal and tumour DNA

Long time to process by histopathologists.

Macrodissected to enrich tumour content

Some patients have no tumour sample available

Cell free DNA shed directly from tumour

Extracted from the plasma component of whole blood

Large fragment sizes of tumour DNA possible

Small quantities extracted ~ 100ng/ ml plasma

Cell free DNA


Use of cell free dna as an alternative source of tumour dna
Use of cell free DNA as an alternative source of tumour DNA resections

Whole blood

  • Stability of cell free DNA in whole blood and in plasma component

  • 5 normal and 6 NSCLC patient samples left as plasma or whole blood for 0, 1, 2 days.

  • Look at quantity and quality of the DNA by ALU-PCR.

Plasma


Dna integrity in whole blood and plasma

Whole blood resections

Plasma

DNA integrity in whole blood and plasma

  • DNA concentration drops dramatically after one day left as blood or plasma.

  • DNA integrity unaffected.

  • Tested 5 NSCLC patient samples for mutations found in tumours.

  • 3/5 patients positive for tumour mutation.


Summary
Summary resections

  • Pyrosequencing assay and fluorescent PCR fragment analysis successfully validated and now used in the lab – combined detects ~ 95% of patients with EGFR TKI sensitising mutations plus 55% of resistance mutations.

  • COLD-PCR assay designed for NSCLC and mCRC samples with low tumour content.

  • Plasma needs to be extracted from blood on day of collection.

  • Tumour mutation could be detected in 3/5 (60%) plasma samples.


Acknowledgements

All Wales medical genetics service resections

Justyna Tull

Hood Mugalassi

Linda Meredith

Rachel Butler

Cardiff University

Peter Davies

Kiran Mantriparagada

Lyudmila Georgieva

CMFT Manchester

Emma Howard

Llandoch and Velindra Hospitals, Cardiff

Dr Jason Lester

Dr Mick Button

University Hospital Birmingham

Brendan O’Sullivan

Phillip Taniere

Institute of Molecular Medicine, St James Hospital, Ireland

Kathy Gately

Acknowledgements