1. Pegylated interferon for PV & ET
Josef Prchal, University of Utah, Salt Lake City
Educated at School of Medicine Karlova Universita, Praha

2. Studies of X-inactivation: MPNs are clonal disorders

3. Clonal development in polycythemia vera
JAK2V617F

4. Beneficial effect of Peg-IFNα
in PV and ET patients
Cytoreductive Rx treatment in PV/ET can:
hematological remission & decrease thromboses
but clonal hematopoiesis persists (i.e. normal HSCs still suppressed)
Only IFNα can induce:
hematological remission
reduction of JAK2 V617F allelic burden
return of polyclonal hematopoiesis (suppression of the pre-JAK2V617F PV clone) Liu at all, Blood 2003
Kiladjian, Blood, 2008, Quintas, Cardama, JCO, 2009
Liu, Blood, 2003

5. Is this salutary effect of Peg-IFNα achieved by
stimulation of an immune response against the PV clone ?
preferential activation of normal dormant stem cells (HSCs) ?
suppression of the PV clone by Peg-IFNα? Via TNFa?

6. Studied patients (MPD-RC 111 and MPD-RC 112)
Phase 3 randomized trial high-risk polycythemia vera (PV) and essential thrombocythemia (ET): Peg-IFNα versus hydroxyurea
MPD-RC 111
PV and ET patients intolerant or refractory to hydroxyurea and patients with Budd Chiari syndrome
Patients who do not participate in the protocols

7. Effect of PegasysTM in PV and ET research methods
Determination of JAK2V617F allelic burden in clonal granulocytes and CD34+ stem cells
Response of PV/ET clones to Peg-IFNα in informative females (q-TCA clonality assay based on X-chromosomal inactivation)
Correlation with circulating regulatory T cells
Correlation with HSC cell cycle status
Correlation with HSC TNFa levels

8. Molecular response to Peg-IFNα
We studied 23 patients (11 females and 12 males) with allelic burden of JAK2V617F at enrollment (1.2% %)
Following treatment at 6 mo, 14 patients (60.8%) demonstrated a decrease in JAK2V617F allelic burden
Females patients with reversion from monoclonal to polyclonal hematopoiesis
JAK2617F allelic burden in %
P1-PV
P2-PV
P3-PV
Females patients with persistence of clonal hematopoiesis after treatment with Peg-IFNα
JAK2617F allelic burden in %
P4-PV
P5-PV
P6-PV
P7-PV
P8-PV
P9-ET
P10-PV
P11-PV
Before treatment GNC
Before treatment CD34+
After Peg-IFNα GNC
After Peg-IFNα CD34+

9. Correlation between the absolute number of peripheral blood Tregs and the JAK2V617F allelic burden in PV patients during the treatment with Peg-IFNα
JAK2V617F
Treqs
Peripheral blood Tregs (fold difference)
JAK2 V617F (%)
Peripheral blood Tregs (fold difference)
JAK2 V617F (%)
Treqs
JAK2V617F
Days
Days
Peg-IFNα:
mobilizes marrow Tregs to the periphery, decreasing their immunosuppressive and tumor promoting influence in marrow microenvironment?
effects on Tregs may be associated with suppression of pre-JAK2V617F and JAK2V617F –positive clonal hematopoiesis?

10. Peg-IFNα and HSC in PV ET
Peggy Goodell
Katherine King

11. Total HSC number and proliferation
Peg-IFNα:
decreases the percentage of HSCs in G0 (quiescence) and increases the number of HSCs in G2/MS-phase- suggesting that Peg-IFNα therapy promotes normal HSC proliferation/differentiation
n=8
n=13

12. Myeloid Differentiation
Cyclin genes Expression
Peg-IFNα:
increases cell cycle associated genes expression
Myeloid Differentiation
Peg-IFNα:
increases colony-forming units in methylcellulose, reflecting a greater number of functional progenitor cells –correlation with decreased CBC??
n=10
N=19

13. TNFα transcript before and after Peg-IFNα in BM CD34+ cells
Relative expression of TNFα
Relative expression of TNFα

14. Interaction plot for treatment modality and time on log (TNF-relative expression) and JAK2V617F mutation

15. define the immune response to PV and ET
Ongoing Studies
define the immune response to PV and ET
The humoral response induced by Peg-IFNα to Tumor Associated Ag (TAA) expressed by Hematopoeitic Stem Cells (HSC).
T-Cell response:multi color flow cytometry effector CD4+ T cells, CD4+Foxp3+ Treg and CD8+ T cells. Using surface markers, (CD3, CD4, CD8, CD25, CD38, CD39, CD45RO, CD197, CTLA-4 and HLA-DR) and intracellular markers (Foxp3, Helios and Ki-67) we will be able to determine the frequency as well as the phenotype of different T cells in the PB and BM and compare them pre and post Peg-IFNα treatment
Quantitate the expression of immune response regulatory checkpoint molecules (PD-1, CTLA-4, OX40 and 4-1BB) at the surface of T cells
Correlate with clonality, JAK2V617F allelic burden, and TNFa before and after Peg-IFNα (with Vladimir Divoky)

16. Stem cells components in PV and ET patients
Prchal, Blood 2011

17. Summary
We conclude that Peg-IFNα can suppress both pre-JAK2V617F and JAK2V617F PV/ET clones in some PV/ET patients, as demonstrated by evaluation of HSCs, granulocytes and platelets, but this often occurs asynchronously
In some patients hematological and molecular response delayed as long as 2 years, as yet in study of >30 patients none of the patients with dramatic decrease of JAK2V617F allelic burden (<3 %) achieved full molecular remission
Our data demonstrate that Peg-IFNα therapy is associated with an increase in HSCs cycling and mobilization of Tregs to the circulation
After Peg-IFNα therapy dramatic decrease of TNF-α in stem cells of responders
Is benefit of Peg-IFNα justified by >side-effects and increased costs compared to hydoxyurea Rx?