Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Marie Lindgren1, Jan Samuelsson2, Lars Nilsson3, Håvar Knutsen4, Waleed Ghanima5, Johan Westin6,
Peter L Johansson7,8, Björn Andréasson7,8.
1
Department of Medicine, Kalmar County Hospital, Kalmar, Sweden.
2
Department of Medicine, Stockholm South Hospital, Stockholm, Sweden.
3
Department of Hematology, Skåne Universtity Hospital, Lund, Sweden.
4
Department of Hematology, Ullevål University Hospital, Oslo, Norway.
5
Department of Medicine, Östfold Hospital, Fredrikstad, Norway.
6
Department of Infectious Diseases, Sahlgrenska University Hospital, Gothenburg, Sweden.
7
Department of Hematology and Coagulation, Sahlgrenska University Hospital, Gothenburg, Sweden.
8
Department of Medicine, Section of Hematology, NU Hospital, Uddevalla, Sweden.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/ejh.13034
disease-modifying properties but currently with no clear predictors of treatment outcome. Recent
genome-wide association studies in chronic hepatitis C, have found a strong influence of genetic
polymorphism near the IL28B (IFNL3) gene on response to IFN-α treatment. In this study we
sought to evaluate the prognostic impact of IL28B rs12979860, rs8099917 and rs12980275 on IFN-α
METHOD:
We retrospectively evaluated 100 patients with MPN treated with IFN-α. The hematologic treatment
response on IFN-α was compared between patients and correlated to host genetic variations in
RESULTS:
The CC genotype of rs12979860 was found significantly associated with hematologic response in
polycythemia vera (PV) with a complete response (CR) in 79% (CC) compared to 48% (non-CC),
(p=0,036). No association between the genotypes and treatment response on hydroxyurea was
found.
CONCLUSION:
These results imply an effect of IL28B genotype on the outcome of IFN-α treatment in MPN.
KEYWORDS
and a long-term risk of evolution to acute leukemia. Myelofibrosis occurs as a primary condition
(PET-MF) (1). Current treatment strategies are largely based on risk stratification according to the risk
of thrombotic and hemorrhagic events (2). An expansion of our understanding of the pathogenesis of
MPNs, including the recent description of Calreticulin (CALR) mutations (3, 4), as well as an increasing
knowledge of the clinical and biological effects of the growing range of treatment options, will
hopefully lead to treatment recommendations with regard to disease modifying options including
including antiproliferative, immunomodulatory and antiangiogenic activities (5). In MPNs, IFN-α has a
potential disease modifying effect, shown to be effective in inducing hematologic and molecular
responses and to reduce vascular events (6, 7). In addition, recent studies indicate a possibility of
reversing bone marrow histopathologic abnormalities in early PMF (8, 9). However, a widespread use
of IFN-α in MPNs is limited because of concerns of patient tolerability and the need of parenteral
administration (10).
For antiviral purposes, pegylated (peg) IFN-α, in combination with ribavirin (RBV), has been the
standard-of-care treatment in chronic Hepatitis C virus (HCV) infection (11). The discrepancy
between sustained viral response (SVR) rates in patients of European ancestry as compared to
infection (12). Subsequent GWAS confirm this finding with identification of several single nucleotide
polymorphisms (SNPs) in the same region, including the CC genotype at rs12979860, the TT genotype
at rs8099917 and the AA genotype at rs12980275(13-15). Moreover, the favorable SNPs are found to
The modifying effect on the treatment response on IFN-α in HCV, with regard to the polymorphisms
in IL28B, indicate that inborn variations in genes involved in inflammation are related to the outcome
of IFN-α treatment. In a recent pilot study involving 20 patients with PV and ET exposed to IFN-α, we
find a significant difference in the rates of complete hematologic remissions (CR) with a higher
degree of CR in patients with the IL28B rs12979860 CC gt compared to the non-CC gt (17). In this
retrospective study we seek to evaluate the potential impact of host genetic variations near IL28B on
INCLUSION CRITERIA
Patients with a diagnosis of PV, ET and MF according to WHO 2008 criteria were identified and
recruited from 9 centers in Sweden and Norway. Patients on current or previous treatment with IFN-
α, were eligible. Inclusion required a minimum of IFN-α treatment duration of 3 months. Prior
respectively. Medical records were reviewed to collect laboratory and clinical data covering the time
period of IFN-α treatment. Peripheral blood values were recorded at initiation of IFN-α and at the
time of best clinical sustained response. Data on dosage of IFN-α, adverse events, causes of
termination of therapy and vascular complications were collected. Palpable splenomegaly was
recorded, if apparent, at the time of diagnosis and the time of clinical evaluations. JAK2 V617F
mutation status and changes in allelic burden were recorded when available. In JAK2 V617F negative
patients with ET or MF, CALR mutations were analysed at the time of the IL28B SNP analyses.
RESPONSE CRITERIA
Hematologic response in ET and PV was evaluated using the European Leukemia Net (ELN) criteria
from 2009 (18). Complete response (CR) in patients with ET was defined as normalization of platelet
count (≤400 x 109/L), white blood cell (WBC) count (≤ 10 x 109/L) and absence of disease related
symptoms (microvascular disturbances, pruritus and headache). Partial response (PR) required a
platelet count ≤600 x 109/L or at least a 50% reduction (but still > 400x109/L). CR in patients with PV
was defined as hematocrit < 45% without phlebotomy, normalization of WBC and platelet counts,
and absence of disease related symptoms. PR required a hematocrit less than 45 % or response in
constitutional symptoms as clinical improvement (CI), in concordance with IWG-MRT and ELN
criteria(19). The MF cohort was not evaluated as CR or PR due to lack of data on bone marrow
GENETIC ANALYSES
A 5 mL peripheral blood sample was collected from participating patients. DNA from whole blood
samples was isolated using MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics, Mannheim,
Germany) for IL28B genotyping. The variability at rs12979860, rs12980275 and rs8099917 was
determined by allelic discrimination assays using Taqman minor groove binding probes, using
primers and probes described in The Supplementary Index. Briefly, the analysis was performed by
running a two-step PCR (15 s at 95_C; 60 s at 60_C) on an ABI 7300 instrument (Applied Biosystems,
Foster City, CA, USA), followed by a post-PCR read of fluorescence intensity from the two
ETHICAL CONSIDERATIONS
In accordance to the Helsinki declaration, the Regional Ethical Committees in Gothenburg, Sweden
and South East, Norway approved the study. All patients gave their written informed consent.
STATISTICAL METHODS
Standard descriptive statistics including mean, median and range were used. Log Rank test was used
when two groups were compared. Fisher´s exact test was used to compare response rates.
Baseline characteristics are summarized in Table 1. One hundred patients (PV n=47, ET n=43, MF
n=10) diagnosed between 1987 and 2012 were recruited. Myelosuppressive treatment prior to IFN-α
was recorded in 44 patients and included hydroxyurea (HU) (n=34), anagrelide (n=19), busulphan
(n=2) and radioactive phosphorus (n=1). Ten patients had received more than one cytoreductive
Records on JAK2V617F mutational status were available in 74 patients (74%). JAK2V617F was found
positive in 33 out of 34 in PV (97%), 20 out of 32 in ET (63%) and 4 out of 8 (50%) in MF. Data on
baseline JAK2V617F quantification and continued follow up were available in 17 patients (17%). CALR
IFN-α TREATMENT
The median treatment duration for IFN-α was 34 months. Most patients (n=53) received pegIFN α-2a
at a median dose of 90 mcg/w and a median treatment time of 15 months. Corresponding values for
pegIFN-α-2b were 12 patients, median dose 40 mcg/w, median treatment time 46 months and for
IFN-α-2b, 35 patients, median dose 9 MIU/w and median treatment time 58 months. The treatment
Hematologic and non-hematologic side effects were recorded in 76 patients (76%). Hematologic
toxicity included anemia (n=4), thrombocytopenia (n=3) and leuko-/neutropenia (n=4). The most
common non-hematologic side effects were fatigue (n=30), myalgia (n=28) and depression (n=21).
seen in two patients with psoriasis and rheumatoid arthritis, respectively. A stillbirth was recorded in
a patient treated with pegIFN-α-2a during pregnancy. Notably, only one vascular event was recorded
during IFN-α treatment. A 64-year old woman with PV, in CR since 92 months, developed a
myocardial infarction after 94 months of IFN-α-2b treatment. Side effects requiring discontinuation
of treatment occurred in 34 patients (34%). 19 of these patients were in CR when IFN-α treatment
was withdrawn.
Similar rates of complete hematologic response on IFN-α treatment were observed in patients with
PV and ET. CR or PR was observed in 28 (60%) and 2 (4%) PV patients. In ET, 30 patients (70 %)
achieved CR while 13 (30%) reached PR. No response (NR) was only recorded in the PV cohort, with
In 9 out of the 10 MF patients, response, as earlier defined, was achieved (6/10) or sustained from
Most responses were achieved within the first five months of treatment but in PV up to 64 months of
treatment were recorded until CR was reached, due to a need of supplemental phlebotomies.
The genotypes of IL28B rs12979860, rs8099917 and rs12980275 were determined in all patients. The
distribution was as expected in a European study setting (16, 20). We compared genotypes within
A significant association between the CC genotype of rs12979860 and hematologic response was
found in the combined cohort of PV and ET, with a greater rate of CR (31/38; 82%) when compared
to the non-CC genotype (28/52; 46%), (p=0,007). Divided by diagnosis, the association was significant
in PV with CR in 79% (15/19) compared to CR in 48% (13/15), (p=0,036) in the non-CC genotype.
Corresponding data in ET were CR in 84% (16/20), compared to 63% (15/24), (p=0,174), and thereby
The AA genotype of rs12980275 did show the same level of association with CR in PV (p=0,036) and
in the combined PV and ET cohort (p=0.014) when compared to the non-AA genotype. In the ET
Regarding the TT genotype of rs8099917, less impact was found in PV with CR in 69% (20/29)
compared to the non-TT with a CR in 44% (8/18), and in the combined cohort of PV and ET with CR in
71 % (39/55), compared to 57% (20/35), the differences not reaching statistical significance. No
With regard to a possible influence of IL28B polymorphism on other cytoreductive treatment than
IFN-α, the included patients with a prior treatment with HU were analysed. Twenty-six patients, 15
PV and 11 ET with a treatment of HU prior to IFN-α, had sufficient data for analysis. The median age
between genotypes and response were found. The results are summarized in Table 4.
No associations between side effects on IFN-α and the studied IL28B genotypes were found in this
cohort.
DISCUSSION
IFN-α is a potential disease modifying treatment option in MPN but its clinical use limited mainly by
concerns about patient tolerability. In contrast to most reports on pegIFN-α-2a, a recent study from
our group do not show a superior tolerability for pegylated forms when compared to conventional
interferon, suggestive of a poor tolerance to low-grade toxicity in the long term (10). The difficulty to
evaluate treatment response in the short term, the long treatment duration, as well as the large
burden of side effects, highlights the need for identification of determinants of treatment response.
The involvement of constitutive or acquired genetic changes on the magnitude of IFN-α response is
to this date unclear. In the PEGINVERA study, Them et al report high response rates on peg-proline-
IFN-α-2b treatment. In a further analysis of the PEGINVERA cohort including high resolution SNP
microarrays, the responses to peg-proline IFN-alpha-2b are found independent of the numbers and
types of chromosomal aberrations(21). The lack of clear evidence of the involvement of somatic
aberrations in the non-responding patients thereby support the theory of an intrinsic resistance to
In this study, 100 patients exposed to treatment with IFN-α, due to a diagnosis of MPN, are
evaluated with regard to hematologic response and a possible association with the genetic
polymorphism in IL28B. In PV and ET the rates of complete hematologic response on IFN-α therapy
are in line with earlier phase II studies as well as retrospective analyses of IFN treatment outside of a
clinical trial (7, 22, 23). The frequencies of the analysed SNPs in this study cohort correspond with the
In PV and in the combined cohort of PV and ET, we find a strong influence of the CC genotype
rs1297986 (12) on hematologic response to IFN-α. The favourable AA gt of rs12980275 follows the
same pattern, explained by a strong linkage disequilibrium (11). The favourable TT of rs8099917 has
only a slight impact. These findings correspond with earlier studies on IFN-α and HCV, where the
In ET, the rs12979860 shows an impact on hematologic response but less than in PV. The rs8099917
does not show any impact. The lower degree of impact of the IL28B SNPs in ET in comparison to PV,
may reflect the difference in the set response criteria in ET and PV with fewer goals to be met in ET
to determine a complete hematologic response and thereby less distinctions between the different
grades of response (18). There is also a considerable heterogeneity within ET, with phenotypic
Notably, treatment response on HU does not show any correlation to IL28B polymorphism. This
finding implies that the polymorphism in IL28B is not a predictor of treatment response in MPN per
The interferon-lambdas (IFN-λ), IL28B, IL28A and IL29, also known as IFNL3, IFNL2 and IFNL1, where
initially described in 2003 (26, 27). These 3 cytokine genes, that comprise the type III subset of IFNs,
are closely related and form a cluster on a chromosome region mapped 19q13. The IFN-λs resemble
the IL10 family in genomic structure but on a functional level they are more related to the type I
IFNs, including IFN-α and IFN-β (28, 29). The functional similarity results from a common signalling
pathway that is activated by both type I and type III IFNs; signalling through either the IFN-α or IFN-λ
receptor result in the induction of a common set of IFN-stimulated genes (ISGs) through the JAK-
STAT pathways (30, 31). However, type III IFNs act through a different heterodimeric receptor
complex, composed of the IL-10 receptor 2 chain (IL10R2) and the unique IFN-λ receptor 1 (IFNλR1).
In contrast to the ubiquitous expression of the IFN-α receptor (IFNAR), the expression of the IFN-λ
receptor has been found to be more limited. Early reports stated mainly an expression on epithelial
cells and hepatocytes (32, 33) but recent studies on human peripheral blood mononuclear cells have
shown that human dendritic cells, which are the main producers of IFN-α, both produces IFN-λ as
well as expresses its receptor. The production of IFN-α and IFN-λ, was also found to be linked; IFN-λ
process for initiating treatment. Despite the advances in the understanding of the cross-talk between
polymorphism in IL28B and its highly predictive value on response to IFN-α treatment in HCV
genotype 1, is still unclear. Peg-IFN-λ-1a, is currently under development for the treatment of
chronic HCV, under the hypothesis of improved treatment tolerability as a result of the more limited
Major limitations of this study are its retrospective nature, possible selection bias and foremost the
lack of sufficient data on JAK2V617F allelic burden and subsequent monitoring because of a diagnosis
and initiation of IFN-α treatment in several patients before the discovery of the JAK2V617F mutation.
An evaluation of the molecular response on IFN-α with regard to IL28B polymorphism would be of
In conclusion, our data shows a significant association between rs12979860 polymorphism and
hematologic response to IFN-α in PV and in the combined cohort of PV and ET. These findings imply
that inborn variations in the genes involved in inflammation are related to the outcome of IFN-α
treatment in MPNs. The genetic polymorphism of IL28B may be a valuable predictor for response to
IFN-α treatment in MPN patients, but our findings has to be validated in further studies.
References
1. Campbell PJ, Green AR. The myeloproliferative disorders. The New England journal of
medicine. 2006; 355(23): 2452-66.
3. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, Avezov E, Li J,
Kollmann K, Kent DG, Aziz A, Godfrey AL, Hinton J, Martincorena I, Van Loo P, Jones AV, Guglielmelli
P, Tarpey P, Harding HP, Fitzpatrick JD, Goudie CT, Ortmann CA, Loughran SJ, Raine K, Jones DR,
Butler AP, Teague JW, O'Meara S, McLaren S, Bianchi M, Silber Y, Dimitropoulou D, Bloxham D,
Mudie L, Maddison M, Robinson B, Keohane C, Maclean C, Hill K, Orchard K, Tauro S, Du MQ, Greaves
M, Bowen D, Huntly BJ, Harrison CN, Cross NC, Ron D, Vannucchi AM, Papaemmanuil E, Campbell PJ,
Green AR. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. The New
England journal of medicine. 2013; 369(25): 2391-405.
4. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, Them NC,
Berg T, Gisslinger B, Pietra D, Chen D, Vladimer GI, Bagienski K, Milanesi C, Casetti IC, Sant'Antonio E,
Ferretti V, Elena C, Schischlik F, Cleary C, Six M, Schalling M, Schonegger A, Bock C, Malcovati L,
Pascutto C, Superti-Furga G, Cazzola M, Kralovics R. Somatic mutations of calreticulin in
myeloproliferative neoplasms. The New England journal of medicine. 2013; 369(25): 2379-90.
5. Stein BL, Tiu RV. Biological rationale and clinical use of interferon in the classical BCR-
ABL-negative myeloproliferative neoplasms. Journal of interferon & cytokine research : the official
journal of the International Society for Interferon and Cytokine Research. 2013; 33(4): 145-53.
9. Silver RT, Kiladjian JJ, Hasselbalch HC. Interferon and the treatment of polycythemia
vera, essential thrombocythemia and myelofibrosis. Expert review of hematology. 2013; 6(1): 49-58.
11. Afdhal NH, McHutchison JG, Zeuzem S, Mangia A, Pawlotsky JM, Murray JS, Shianna
KV, Tanaka Y, Thomas DL, Booth DR, Goldstein DB. Hepatitis C pharmacogenetics: state of the art in
2010. Hepatology (Baltimore, Md). 2011; 53(1): 336-45.
12. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, Heinzen EL, Qiu P,
Bertelsen AH, Muir AJ, Sulkowski M, McHutchison JG, Goldstein DB. Genetic variation in IL28B
predicts hepatitis C treatment-induced viral clearance. Nature. 2009; 461(7262): 399-401.
16. Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O'Huigin C, Kidd J, Kidd K, Khakoo SI,
Alexander G, Goedert JJ, Kirk GD, Donfield SM, Rosen HR, Tobler LH, Busch MP, McHutchison JG,
Goldstein DB, Carrington M. Genetic variation in IL28B and spontaneous clearance of hepatitis C
virus. Nature. 2009; 461(7265): 798-801.
24. Muir AJ, Gong L, Johnson SG, Lee MT, Williams MS, Klein TE, Caudle KE, Nelson DR.
Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for IFNL3 (IL28B) genotype
and PEG interferon-alpha-based regimens. Clinical pharmacology and therapeutics. 2014; 95(2): 141-
6.
26. Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, Shah NK, Langer JA,
Sheikh F, Dickensheets H, Donnelly RP. IFN-lambdas mediate antiviral protection through a distinct
class II cytokine receptor complex. Nature immunology. 2003; 4(1): 69-77.
29. Kotenko SV. IFN-lambdas. Current opinion in immunology. 2011; 23(5): 583-90.
30. Stark GR, Darnell JE, Jr. The JAK-STAT pathway at twenty. Immunity. 2012; 36(4): 503-
14.
31. Kotredes KP, Gamero AM. Interferons as inducers of apoptosis in malignant cells.
Journal of interferon & cytokine research : the official journal of the International Society for
Interferon and Cytokine Research. 2013; 33(4): 162-70.
32. Lasfar A, Abushahba W, Balan M, Cohen-Solal KA. Interferon lambda: a new sword in
cancer immunotherapy. Clinical & developmental immunology. 2011; 2011: 349575.
33. Olagnier D, Hiscott J. Type I and type III interferon-induced immune response: it's a
matter of kinetics and magnitude. Hepatology (Baltimore, Md). 2014; 59(4): 1225-8.
34. Yin Z, Dai J, Deng J, Sheikh F, Natalia M, Shih T, Lewis-Antes A, Amrute SB, Garrigues U,
Doyle S, Donnelly RP, Kotenko SV, Fitzgerald-Bocarsly P. Type III IFNs are produced by and stimulate
human plasmacytoid dendritic cells. Journal of immunology (Baltimore, Md : 1950). 2012; 189(6):
2735-45.
35. Zhang S, Kodys K, Li K, Szabo G. Human type 2 myeloid dendritic cells produce
interferon-lambda and amplify interferon-alpha in response to hepatitis C virus infection.
Gastroenterology. 2013; 144(2): 414-25.e7.
36. Muir AJ, Shiffman ML, Zaman A, Yoffe B, de la Torre A, Flamm S, Gordon SC, Marotta
P, Vierling JM, Lopez-Talavera JC, Byrnes-Blake K, Fontana D, Freeman J, Gray T, Hausman D, Hunder
NN, Lawitz E. Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients
with chronic genotype 1 hepatitis C virus infection. Hepatology (Baltimore, Md). 2010; 52(3): 822-32.
37. Muir AJ, Arora S, Everson G, Flisiak R, George J, Ghalib R, Gordon SC, Gray T,
Greenbloom S, Hassanein T, Hillson J, Horga MA, Jacobson IM, Jeffers L, Kowdley KV, Lawitz E, Lueth
S, Rodriguez-Torres M, Rustgi V, Shemanski L, Shiffman ML, Srinivasan S, Vargas HE, Vierling JM, Xu
D, Lopez-Talavera JC, Zeuzem S. A randomized phase 2b study of peginterferon lambda-1a for the
treatment of chronic HCV infection. Journal of hepatology. 2014; 61(6): 1238-46.
*MF not included.** Treatment response in MF is defined as stable disease /clinical improvement20.
-rs12979860
non-CC 13 15 15 9 28 24
-rs8099917
non-TT 8 10 12 5 20 15
-rs12980275
non-AA 13 15 14 8 27 23
rs12979860
non-CC 2 2 5 4 7 6
rs809997
non-TT 3 1 3 2 6 3
rs12980275
non-AA 3 1 5 4 8 5