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

Viral immunology and immunogenetics Bone pathology and cell signalling Haemopoiesis, cbl

• ncogene and

myeloid neoplasms Cardiovascular genetics

New Research Interests

Translational Cancer Pathology

• Haematological malignancies
• Non-haemopoietic malignancies
• New techniques
• Core Pathology Facility

Haematological Malignancies

AILT MM AMKL ALL AML 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 – FLT3 ITD and TKD – NPM1 exon 12 ; CEBPA – KIT exon 8 ; P53 ; TET2 – MLL partial tandem duplication

Multiple testing modalities Diagnosis, prognosis, MRD Therapeutic prediction Patient focussed

Myeloproliferative Disorders

CML BCR-ABL CMML PDGFRB fusions EMS FGFR fusions CEL PDGFRA fusion SM KIT PV ET PMF

JAK2 (MPL)

JAK2 Pathway

EPO Receptor

S T A T J A K 2

Cell proliferation

EPO

Cell differentiation

• JAK2 V617F and MPN
• Described 2005
• Point mutation in JAK2
• 97% Polycythaemia vera
• 55% ET
• 60% Myelofibrosis

• 27 year old male

Hb: 211 g/L (Hct: 0.63) WCC: 7.3 x109/L Platelets: 286 x109/L

• JAK2 V617F: negative
• Epo: < 5.0 IU/L (range = 5-20)
• Epo-independent erythroid colonies

+ Epo

• Epo

JAK2 V617F-Negative Erythrocytosis

JAK2 V617F + Blank Normal Patient

PV JAK2 exon12 mutation

Morphology – Genotype Correlation

B

72 88

• 1
• 4
• 3
• 2
• 5

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Temperature (oC) Normalised fluoresence minus control H538-K539delinsL E543-D544del Wild type JAK2 exon 12 mutation

B

72 88

• 1
• 4
• 3
• 2
• 5

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Temperature (oC) Normalised fluoresence minus control H538-K539delinsL E543-D544del Wild type JAK2 exon 12 mutation

High Resolution Melt analysis BMT: erythroid hyperplasia

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
• Blood count:

– Hb 130 g/L – WBC 7.3 x109/L – Plt 916 x109/L

• CRP

1

• ESR 5
• JAK2 V617F: Negative
• Bone marrow A+T

100 200 300 400 500 600 700 800 900 1000 Platelet count Apr-02 Aug-05 Oct-08 Jun-09

BMA BMT

Temperature oC Normalised minus normal

W515A

W515R

W515K W515L S505N

HRM curve (Case 2)

G G T G/T G C

MPL W515L

Tryptophan (W) Leucine (L)

HRM analysis Sequence of HRM product

Normal

MPL exon 10 Analysis

MPL W515L: Positive ET with MPL mutation

MPL Mutations in MPD

Granulocyte T-cell

Tpo

W515L W515K

T G A G G T N G C A G T G A G G T G G C A G T G A G G N N G C A G T G A G G T G G C A G

S505N

G C C T C A N C G C C G C C T C A G C G C C

• 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

Thrombocytosis BM fibrosis Splenic infarction

W515L

Gain-of-function MPL mutation

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

• 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

Gattenlohner et al. Leukaemia. 2009

V617F+ V617F-

Mutation analysis Sequencing miRNA In situ PCR Immunocytochemistry Cell signalling Apoptosis

In situ megakaryocyte assessment Laser microdissected megakaryocytes Bone marrow

Megakaryocyte Biology in MPN

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 B C D E F G

Negative Positive 20 40 60 80 100

t(15;17) status % Diffuse PML staining

A B C D E F E F G

Negative Positive 20 40 60 80 100

t(15;17) status % Diffuse PML staining

Wildtype APML Immuno-fluorescent microscopy

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
• 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.

CD117

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

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

Approaches

Neoplasms

Platforms

MORPHOLOGY Haemopoietic; non-haemopoietic (breast, lung , melanoma etc) Microscopy IHC Routine genetics GENE DNA Chromsomes 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 RNA Transcriptome 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

Translational Cancer Pathology

UWA School PaLM:

Translational Cancer Pathology

PathWest:

Diagnostic Pathology