School of Pathology and Laboratory Medicine: Current and New - PowerPoint PPT Presentation

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

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

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

Haematological Malignancies AML ALL AMKL AILT MM CML

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 Regional / Whole Gene Mutations: – JAK2 exon 12 ; MPL exon 10 Multiple testing modalities – FLT3 ITD and TKD Diagnosis, prognosis, MRD – NPM1 exon 12 ; CEBPA – KIT exon 8 ; P53 ; TET2 Therapeutic prediction – MLL partial tandem duplication Patient focussed

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

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

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

PV JAK2 exon12 mutation Morphology – Genotype Correlation B B 0 0 Normalised fluoresence minus control Normalised fluoresence minus control -1 -1 H538-K539delinsL H538-K539delinsL -2 -2 -3 -3 E543-D544del E543-D544del -4 -4 Wild type Wild type JAK2 exon 12 mutation JAK2 exon 12 mutation -5 -5 72 72 73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84 85 85 86 86 87 87 88 88 Temperature ( o C) Temperature ( o C) BMT: erythroid hyperplasia High Resolution Melt analysis

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

JAK2 V617F-Negative Thrombocytosis • 36 year female • Antenatal FBC 1000 • Blood count: 900 800 – Hb 130 g/L 700 – WBC 7.3 x10 9 /L 600 Apr-02 Aug-05 500 – Plt 916 x10 9 /L Oct-08 400 • CRP 1 Jun-09 300 • ESR 5 200 100 • JAK2 V617F: Negative 0 Platelet count • Bone marrow A+T

BMA BMT

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

MPL Mutations in MPD T-cell Granulocyte Tpo S505N G C C T C A N C G C C 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 • 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 Thrombocytosis – No difference in reticulin grade BM fibrosis Splenic infarction

JAK2 V617F & MPN Diagnostic Algorithm

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

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

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

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

Imaging Flow Cytometry: PML Protein A A B B Immuno-fluorescent microscopy Wildtype APML C C D D E E E F F F G G % Diffuse PML staining % Diffuse PML staining 100 100 80 80 60 60 40 40 20 20 0 0 Negative Negative Positive Positive t(15;17) status t(15;17) status

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

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

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

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

GIST: KIT Mutations and TKI Response • KIT and PDGFRA mutations CD117 • 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. Wozniak A. Cancer Invest . 2010.

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 Stephens PJ. Nature 2009;462(7276):1005 rearrangements (purple)

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

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

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

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

UWA School PaLM: Translational Cancer Pathology PathWest: Diagnostic Pathology