c-Cbl RING finger mutant mice: A preclinical model for human myeloid leukemias

5% to 20% incidence of c-Cbl RING finger mutations in patients with:

myelodysplastic syndromes (MDS)

chronic myelomonocytic leukemia (CMML)

juvenile myelomonocytic leukemia (JMML)

atypical chronic myeloid leukema (aCML)

secondary acute myeloid leukemia (sAML)

School of Pathology and Laboratory Medicine Wally Langdon et al

Summary:

• Mice with a c-Cbl RING finger mutation develop a myeloproliferative disease that progresses to leukemia.

• The c-Cbl RING finger mutation promotes enhances FLT3, c-Kit and Akt signalling in hematopoietic progenitors.

•Knockout of FLT3 ligand prevents leukemia development in these mice.

[Rathinam, Thien, Flavell and Langdon, Cancer Cell 18, 341-352 (2010)]

Current and future studies:

Treatment of pre-leukemic and leukemic mice with inhibitors of FLT3, c-Kit, c-Src and PI 3-kinase.

Initial studies with FLT3 inhibitor AC220 are encouraging.

Anatomical PathologyRoyal Perth Hospital

• Dr Jacqueline Bentel• Dr Marc Thomas• PhD Students

– Jasmine Tay– Alison Louw– Ebony Rouse

• Research performed in Anatomical Pathology, PathWest Laboratory Medicine, Royal Perth Hospital in collaboration with Winthrop Professor Jennet Harvey

• Breast cancer– Effects of androgen receptor and

MAPK signalling on p27 and the regulation of breast cancer growth

– Interaction between the androgen receptor and ETS1 in breast cancer cells

• Prostate cancer– Characterisation of the E3

ubiquitin ligases, RMND5A and RMND5B

– ETS1 effects on the androgen responsiveness of prostate cancer cells

Adrian Charles• Wilms tumour

– Identifying chromosome 22 anomalies– Undersanding genetic progression from precursor

lesions

• The role of stroma in acute childhood leukaemia with Ursula Kees

• Genetic changes in NUT carcinoma, a rare tumour of children with Ursula Kees

• Stillbirth– Producing and introducing national guidelines– Epidemiological research

Paediatric pathology – Adrian Charles

Using Alloreactive NK cells to treat leukaemiaCampbell Witt, Frank Christiansen, Pathol & Lab Med.

NK cells from allogeneic donors kill leukaemia cells without causing graft-versus-host disease.

•Understand immunogenetic genetic difference required between donor and patient (HLA, KIR genes).

•Find best clinical scenario in which to harness allogeneic NK cell infusions.

•Developing TGA compliant methods for in vitro expansion of allogeneic NK cells.

•Currently designing phase I trial.

FOLEY BA, …..CHRISTIANSEN FT, WITT CS. The Reactivity of Bw4-positive HLA-B and HLA-A Alleles with KIR3DL1: Implicationsfor Patient and Donor Suitability for Haploidentical Stem Cell Transplants. Blood, 2008,112:435-43

DE SANTIS D, FOLEY BA, JOHN E, SENITZER D, CHRISTIANSEN FT, WITT CS. Rapid, flow cytometric assay for NK alloreactivity reveals exceptions to rules governing alloreactivity. Biol Blood Marrow Transplantation 2010, 16, 179-91.

How does the “9A” Allele of the NK cell receptor KIR2DL4 predispose to asthma?

C Witt, F Christiansen, P Holt. Pathol & Lab Med

Copies of 9A allele

GeometricMean IgE(kU/L)+/-2SEM

0

20

40

60

80

100

0 1 26 mths

24 mths

60 mthsp < 0.01

0

1

2

0 1 2

Year 1p < 0.01

Year 2Year 5

IncidenceRelativeRisk

(%) Asthma Prevalenceat 2 years

0

p = 0.02

0 1 2

10

20

30

Serum IgE Severe RTIs Asthma

•9A allele encodes non-functional receptor preventing INFg response to ligand HLA-G•Predisposition to Th2 response?•IFNg important in defense against viruses in early life?•IFNg important in generation of Treg cells?

GOODRIDGE, JP, WITT CS, CHRISTIANSEN, FT, WARREN HS. KIR2DL4 (CD158d) genotype influences expressionand function in NK cells. J Immunol (2003) 171: 1768-74.GOODRIDGE JP …., WlTT CS.. Three Common Alleles of KIR2DL4 (CD158d) Encode Constitutively Expressed, Inducible and Secreted Receptors in NK Cells. Eur J Immunol 37 (2007), 199-211.

The Role of Anti-HLA Antibodies in Renal Allograft RejectionS Fidler, C Witt, A Irish, F Christiansen. Pathol & Lab Med

High titre, pre-Tx, anti-donor antibodies are deterimental to graft outcome.

• Role of low titre Abs pre and post Tx unclear.– Titre, antibody class, complement fixing, locus

(HLA-A,B,C,DR,DQ,DP,MICA,MICB)– Interaction with transfusions, solCD30– Predicting permissible HLA mismatches

LANGAN LL, IRISH A, WITT CS, CHRISTIANSEN FT (2007) Post transplant HLA class II antibodies and high soluble CD30 levels are independently associated with poor kidney graft survival. American Journal of Transplantation. 7, (2007), 847.

School of Pathology and Laboratory Medicine – RPH Unit

Patricia Price et alSensory neuropathy in HIV patientsOur studies of patients treated in Melbourne, Jakarta, Kuala Lumpur and Johannesburg show neuropathy is more common in - taller and older patients- African patients- patients carrying the genotype TNF-1031 (and we have characterised the TNF haplotypes around this)

Current studies address the role of chemokines in the accumulation of inflammatory cells around the neurons and the experience of pain

Specific CMV and HIV proteins may modulate natural killer cell responses and affect the long-term outcome for HIV patients

Patricia Price (UWA), Kate Cherry (Alfred Hosp), Peter Kamermans (Witts, J’burg)PhD Students: Constance Chew (UWA), Toni Wadley (Witts)

Immune determinants of the outcome of Hepatitis C virus infection and its treatment with interferon- /ribavirin

•An algorithm (“TIPscore”) based on TGF and IP-10 adjusted for age, sex and HCV genotype optimally predicted virological response to therapy.

•Low IFNg responses to HCV antigens IFNa / RBV therapy associated with impaired cytokine responses to dendritic cells

•Propagation of HCV in culture and assessment of neutralizing antibodies.

Cohort studies of patients treated at RPH include……..

Patricia Price, Silvia Lee (UWA)Jim Flexman (RPH)

UNDERSTANDING IMMUNOLOGICAL SUSCEPTIBILITY TONON-TUBERCULOUS MYCOBACTERIAL (NTM) LUNG DISEASE

Dr Andrew Lim | Prof. Patricia Price | Prof. Grant Waterer

Lung disease due to NTM is a growing health problem. It remains unclear why some individuals are susceptible to NTM infection. Current antibiotic treatment is problematic, so immune-based therapies may be a better alternative.

Aims: (A) To elucidate whether defects exist in innate and adaptive immune responses in patients with NTM lung disease; and (B) To determine whether these immune responses associate with treatment outcome.

Immune cells and pathways we are targeting:• T cells (Th1, Th2, Th17 and IL-10-producing subsets) – are there deficiencies in the

abilities of T cells to produce IFNγ or other proinflammatory cytokines in response to mitogenic or antigenic stimulation?

• Macrophages (IFNγR, IL-1R and TLR pathways) – are there abnormalities in the abilities of host macrophages (blood and airway) to mature/activate, to produce inflammatory molecules or to phagocytose NTM?

Our study of HCV IRD in HCV co-infected patients showed that IRD was most common in patients with low antibody levels pre-ART. T-cell studies ongoing

In KL, we characterized T-cell, dendritic cell and monocyte responses during cryptococcal & TB IRD

Immune Restoration Disease

Some HIV patients responding to ART become sick following an excessive immune response to antigens of persistent 2o infections.

IRD are most common in patients starting ART with opportunistic infections and low CD4 T-cell counts so we have set up projects in Asia

We demonstrated the importance of mycobacterial burden in the development of TB IRD in a TB clinic in Delhi

Patricia Price (UWA), Evy Yunihastuti (UI), Adeeba K (UM)…….PhD Students: Dino Tan & Ben Oliver (UWA)……..

School of Pathology and Laboratory Medicine – RPH Unit

Martyn French et al• NH&MRC Program Grant group - HIV and HCV

immunopathogenesis and vaccines

• National Centre in HIV Epidemiology and Clinical Research

• International Network for Strategic Initiatives in Global HIV Trials (INSIGHT)

• HIV Pathogenesis Program of the University of KwaZulu Natal and the Ragon Institute of MIT, MGH and Harvard

• International Network for the Study of HIV-associated immune reconstitution inflammatory syndrome (INSHI)

School of Pathology and Laboratory Medicine – RPH Unit

Martyn French et al

Oliver BG, Elliott JH, Price P, Phillips M, Saphonn V, Vun MC, Kaldor JM, Cooper DA, French MA. Mediators of Innate and Adaptive Immune Responses Differentially Affect Immune Restoration Disease Associated with Mycobacterium tuberculosis in HIV Patients Beginning Antiretroviral Therapy. J Infect Dis. 2010;202:1728-1737.

Haddow LJ, Colebunders R, Meintjes G, Lawn SD, Elliott JH, Manabe YC, Bohjanen PR, Sungkanuparph S, Easterbrook PJ, French MA, Boulware DR; International Network for the Study of HIV-associated IRIS (INSHI). Cryptococcal immune reconstitution inflammatory syndrome in HIV-1-infected individuals: proposed clinical case definitions. Lancet Infect Dis. 2010;10:791-802.

French MA, Tanaskovic S, Law MG, Lim A, Fernandez S, Ward LD, Kelleher A, Emery S. Vaccine-induced IgG2 anti-HIV p24 is associated with control of HIV in patients with a 'high-affinity' FcgammaRIIa genotype. AIDS. 2010;24:1983-90.

School of Pathology and Laboratory Medicine – RPH Unit

Martyn French et al

Cozzi-Lepri A, French MA, Baxter J, Okhuysen P, Plana M, Nehaus J, Landay A for the INSIGHT SMART study group. Resumption of HIV replication in SMART Study patients is associated with monocyte/macrophage derived cytokine and chemokine changes in addition to CD4+ T cell decline. To be submitted

Fernandez S, Tanaskovic S, Helbig K, Beard M, Purcell D, Murray J, Lewin S, Price P, French MA. Naive CD4+ T cell deficiency in HIV patients responding to long-term ART is associated with increased interferon-stimulated gene expression in CD4+ T cells. To be submitted

Byakagwa H, French MA, Lewin SR, Kelleher AD, Amin J, Garsia R, Haskelberg H, Kelly M, Boyd MA, Cooper DA, Purcell DFJ, Emery S on behalf of the CORAL Study Group. Antiretroviral therapy intensification with raltegravir or anti-lipopolysaccharide immunoglobulin from hyper-immune bovine colostrum in antiretroviral-treated patients exhibiting a suboptimal CD4+ T-cell response: the CORAL study. To be submitted