School of Pathology and Laboratory Medicine: Current and New Research Interests

Transcription

1 School of Pathology and Laboratory Medicine: Current and New Research Interests W/Professor Wendy Erber

2 Current Research Interests Viral immunology and immunogenetics Bone pathology and cell signalling Haemopoiesis, cbl oncogene and myeloid neoplasms Cardiovascular genetics

3 New Research Interests Translational Cancer Pathology Haematological malignancies Non-haemopoietic malignancies New techniques Core Pathology Facility

4 Haematological Malignancies AML ALL AMKL AILT MM CML

5 Haematological Malignancies Proteomics Genomics Single Point Mutations: JAK2 V617F ; KIT D816V MPL W515L ; FLT3 D835Y RAS G12D Translocations: BCR-ABL1 ; PML-RARA ; NPM-ALK E2A-PBX1 ; FIP1L1-PDGFRA Multiple testing modalities Diagnosis, prognosis, MRD Therapeutic prediction Patient focussed Regional / Whole Gene Mutations: JAK2 exon 12 ; MPL exon 10 FLT3 ITD and TKD NPM1 exon 12 ; CEBPA KIT exon 8 ; P53 ; TET2 MLL partial tandem duplication

6 Myeloproliferative Disorders CML CMML EMS CEL SM PV ET PMF BCR-ABL PDGFRB fusions FGFR fusions PDGFRA fusion KIT JAK2 (MPL)

7 EPO J A K 2 JAK2 Pathway JAK2 V617F and MPN Described 2005 Point mutation in JAK2 EPO Receptor 97% Polycythaemia vera 55% ET S T A T 60% Myelofibrosis Cell differentiation Cell proliferation

8 JAK2 V617F + Blank Normal Patient JAK2 V617F-Negative Erythrocytosis 27 year old male Hb: 211 g/l (Hct: 0.63) WCC: 7.3 x10 9 /L Platelets: 286 x10 9 /L JAK2 V617F: negative Epo: < 5.0 IU/L (range = 5-20) Epo-independent erythroid colonies + Epo - Epo

9 Normalised fluoresence minus control PV JAK2 exon12 mutation Morphology Genotype Correlation B H538-K539delinsL -3 E543-D544del -4 Wild type JAK2 exon 12 mutation Temperature ( o C) BMT: erythroid hyperplasia High Resolution Melt analysis

10 Phenotype-Genotype Correlation JAK2 exon 12 N542-E543del (M:E = 1:6) Glycophorin A Myeloperoxidase Percy, Scott et al. 2007

11 JAK2 V617F-Negative Thrombocytosis 36 year female Antenatal FBC Blood count: Hb 130 g/l WBC 7.3 x10 9 /L Plt 916 x10 9 /L CRP 1 ESR 5 JAK2 V617F: Negative Bone marrow A+T Platelet count Apr-02 Aug-05 Oct-08 Jun-09

12 BMA BMT

13 Normalised minus normal MPL exon 10 Analysis HRM analysis W515A Sequence of HRM product G G T G/T G C Normal W515K W515L W515R HRM curve (Case 2) MPL W515L Tryptophan (W) Leucine (L) S505N Temperature o C MPL W515L: Positive ET with MPL mutation

14 MPL Mutations in MPD Tpo S505N Granulocyte G C C T C A N C G C C T-cell G C C T C A G C G C C W515L T G A G G T N G C A G T G A G G T G G C A G W515K T G A G G N N G C A G T G A G G T G G C A G Gain-of-function MPL mutation W515L Thrombocytosis BM fibrosis Splenic infarction MPL compared with both JAK2 V617F and WT: Reduced bone marrow cellularity at diagnosis Reduced erythroid & granulocytic activity No difference in MK cellularity or morphology No difference in reticulin grade

15 JAK2 V617F & MPN Diagnostic Algorithm

16 MPN in situ Phospho- Cell Signalling Analysis JAK2 V617 + JAK2 V617 - Grimwade et al, BJHaem. 2009

17 MPN in situ Genomic Analysis JAK2 V617F mutation in situ PCR In situ genomics Identify individual cells with specific genes / mutation Routine tissue section: DNA amplification in tissue For diagnosis and monitoring Advantage over PCR in solution Numerous applications V617F+ V617F- Gattenlohner et al. Leukaemia. 2009

18 Megakaryocyte Biology in MPN Bone marrow In situ megakaryocyte assessment Immunocytochemistry Cell signalling Apoptosis Laser microdissected megakaryocytes In situ PCR Mutation analysis Sequencing mirna

19 New Techniques in Pathology Imaging flow cytometry Applications: Cell morphology, size, shape Cell cycle, mitosis, signalling, apoptosis, proliferation Molecule localisation Internalisation Spot counting Automated FISH in suspension X / Y chromosomes

20 % Diffuse PML staining Imaging Flow Cytometry: PML Protein A B Immuno-fluorescent microscopy C Wildtype D APML E F G Negative Positive t(15;17) status

21 Translational Cancer Pathology Can technologies used in haematology be applied to non-haemopoietic malignancies? Advanced proteomics Translational genomics In situ genomics (DNA, RNA) Stratification therapeutic prediction Personal cancer genome signature Disease burden

22 Cancer Genome and Pathology Numerous somatic mutations in cancer Driver mutations confer oncogenic properties: Growth advantage, tissue invasion, metastasis, evasion of apoptosis Provide insight into cancer cell biology Identify new drug targets and new diagnostic tests Examples: HER2-positive breast cancers and trastuzumab therapy BCR-ABL1-positive CML and imatinib Passenger mutations: Numerous; DNA damage and repair;?significance

23 In situ Cell Proteomics and Genetics Protein: HER-2/neu Gene Expression: HER-2/neu amplification (Chr 17 centromere: Green / HER-2: red) HER2 amplification, prognosis and treatment response

24 Cancer Genomics and Therapy Colorectal Carcinoma: KRAS mutation and response to EGFR inhibitors BRAF mutation and lack of response to EGFR inhibitors & poor prognosis Non-Small Cell Lung Ca: EGFR mutation predicts response to TKIs

25 GIST: KIT Mutations and TKI Response KIT and PDGFRA mutations TKI inhibitor therapy Imatinib: More effective with exon 11 mutations than KIT exon 9 mutations and wild-type Sunitinib: Small molecule multi-targeted receptor TKI Greater in vitro efficacy with KIT exon 9 mutants and wild-type genotype than exon 11 mutants. CD117 Wozniak A. Cancer Invest

26 Whole Genome Analysis Somatic Rearrangements in Ca Breast Paired-end sequencing strategy Assessed 65 million 500bp DNA fragments in 24 Ca breast lines >2,000 somatic rearrangements Intra-chromosomal translocations (green), tandem duplications (defects in DNA maintenance) Copy number changes (blue) Inter-chromosomal rearrangements (purple) Stephens PJ. Nature 2009;462(7276):1005

27 Cancer Mutations and NGS Cancer gene panel: >700 mutations in 46 genes Massive multiplex PCR and NGS FFPE tissue or cells (10ng DNA) Sensitivity to 5% Proof-of-principle Stratification Tailored-therapy Cancer genome signature Disease monitoring

28 Molecular Genetics and MRD Chronic Myeloid Leukaemia BCR-ABL1 Transcripts E. Gudgin & B. Huntly. Chronic Myeloid Leukemia. In Erber (Ed): Diagnostic Techniques in Hematological Malignancies. 2010

29 Translational Cancer Pathology Bridging the Gap Translating results of research developments into clinical diagnostic pathology Tumour-specific characteristics Personal cancer genome signature Diagnostic precision, prognosis, therapeutic prediction and sensitive disease monitoring For patient benefit and improved health Better insight into disease causation

30 Approaches Neoplasms Platforms MORPHOLOGY Haemopoietic; non-haemopoietic (breast, lung, melanoma etc) Microscopy IHC Routine genetics GENE DNA Chromsomes RNA Transcriptome Translational Cancer Pathology Mutations Translocations Deletions Epigenetics FFPE / cells / LMD PCR (HRM); RT-PCR; qpcr; cell-free DNA NGS / Ion Torrent In situ PCR Imaging flow FISH / immunofish / CISH Digital imaging / Aperio Amplification Dysregulation mirna qrt-pcr In situ RT PCR RNAScope PROTEIN Pathways Regulatory molecules Cell biology Expression -Signalling -Phosphorylation - Apoptosis - Proliferation - Bystander cells Chimeric proteins Blood: - Plasma - Cells - ELISA TMAs: FFPE +/- decal IHC ImmunoFISH Flow cytometry Imaging flow Digital imaging / Aperio

31 UWA School PaLM: Translational Cancer Pathology PathWest: Diagnostic Pathology

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