4. The light microscope remains the central cancer diagnostic tool for 400 years Zacharias and Hans Jansen (ca 1595) Modern microscope (ca 1995)
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6. Haemato-oncology lab Microscopy Immunophenotyping Cytogenetics Molecular Genetics Sample Integrated report MICROSCOPY: >80% undifferentiated blasts Morphology of acute lymphoblastic leukaemia Eosinophils, basophils and small megakaryocytes suggest blast phase of chronic myeloid leukaemia IMMUNOPHENOTYPE: Blast cells are CD10, CD19, CD79a, CD34, HLA-DR, TdT positive. Weak CD13. They do not express CD33 or myeloperoxidase. DNA index is 1.0 Phenotype of B lymphoblastic leukaemia or B lymphoblastic transformation of CML CYTOGENETICS: Karyotype: 46,XY,t(9;22)(q34;q11) in 10 of 10 metaphases FISH: BCR/ABL 92% positive MOLECULAR GENETICS: BCR-ABL fusion transcript type: p210 e13a2 by RT-PCR OVERALL CONCLUSION: B lymphoblastic blast crisis of chronic myeloid leukaemia Cytology Histology Immunohistochemistry Diagnostic panels Karyotyping FISH Mutational screening RT-PCR qPCR (MRD)
17. Genomic Circos Plot Circos Plot from Pleasance et al, Nature 2010 Deletions/Insertions Substitution density (het) Substitution density (homo) Coding Substitutions Silent Missense Nonsense Splice site Copy number Regions of LOH Structural rearrangements Intrachromosomal Interchromosomal Genomic coordinates
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20. The clinical process in oncology Pre-clinical phase Presentation Diagnosis Treatment Assessment of response Follow-up Relapse
30. Relapsing breast cancer Slide courtesy of Dr Peter Campbell 0.1 0.01 1 0.05 0.5 Undiluted patient plasma 1:10 1:100 1:1000 1:10,000 1:100,000 Normal Water 0 5 10 15 20 25 30 35 40 Cycles of real-time PCR Intensity Non-rearranged genomic region 0.1 0.01 1 0.05 0.5 0 5 10 15 20 25 30 35 40 Cycles of real-time PCR Intensity Tumour-specific rearrangement
31. Serial measurements 150 Months after diagnosis Estimated tumour DNA / mL serum (pg) 25 50 75 100 125 Undetectable Detectable at limit of sensitivity 6 7 8 9 10 11 12 13 14 15 16 17 5 Rearrangement 1 Rearrangement 2 First-line chemotherapy Second-line Paclitaxel CT scan: Localised deposits around T9-10 Chemotherapy: CT scan: Widespread soft-tissue metastases Slide courtesy of Dr Peter Campbell
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35. Genome Campus Data storage & analysis Sulston Building Morgan Building Research Support Facility Data Centre European Bioinformatics Institute
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38. Implications for cancer classification Cellular origin Morphology Differentiation/grading Mutations: Diagnosis Prognosis Treatment Unified Classification?
39. Acute Myeloid Leukaemia – a paradigm of evolving classification Morphology Single entity Various 1950s Morphology & cytochemistry M0-M7 FAB 1976 Morphology, AML with recurrent cytogenetic translocations WHO 2002 Immunophenotyping & AML with multilineage dysplasia Cytogenetics AML, therapy related AML not otherwise categorized Morphology, AML with recurrent genetic abberations WHO 2008 Immunophenotyping , Provisional entity: AML with mutated NPM1 Cytogenetics & Provisional entity: AML with mutated CEBPA Genetics Otherwise as 2002
Haematologist Main interest pathogen. AML Developing interest in molecular diagnostics ... and I will talk to you about the potential impact of advances in DNA sequencing technologies on the field of cancer Δ , albeit with a slight overrepresentation of examples drawn from haematological cancers
1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
As a general indication of how things have been going up until now and as I am sure many of us are aware, the CR-UK str..... recently invited research/Dic centres to apply for funding to offer a service for genotyping a set of mutations deemed to be of clinical relevance. So solid tumours are rapidly going the way of haematological cancers in requiring molecular info as part of their diagnostic work-up.
1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
The magnitude of the change is represented here. This represents a >100 trillion-fold increase in sequencing capacity over 10 years
And to compare this to something more tangible, the white line here shows the rate at which the cost of transistors for computer circuits has been changing over the last decade (halfs every 2 years) – compared to the real cost of sequencing per megabase of DNA sequence
1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
However, other more selective applications are also being developed All of which need to go through a NGS step
The main application of the new technologies is whole matched cancer/constitutional genome sequencing ...
The main application of the new technologies is whole matched cancer/constitutional genome sequencing ...
There are many examples of PCR based approaches including some multiplex PCRs that tackle lists such as the CR-UK. However, these can only be applied to small substitutions and Indels. An example of such an application is a test we are developing that can be used in AML with NK: In this disease, the genome is stable and numbers of coding mutations small. Approximately 20 recurrent mutations have been identified and for some prognostic implications are know. This tool would enable the determination of the effects of different combinations of mutations on prognosis & response to Rx.
1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis