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DR.

MARIE C LINDGREN (Orcid ID : 0000-0002-9757-5243)


Accepted Article DR. BJORN ANDREASSON (Orcid ID : 0000-0003-1525-1331)

Article type : Original Article

Genetic variation in IL28B (IFNL3) and response to

interferon-alpha treatment in myeloproliferative neoplasms.

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.

Correspondence: Marie Lindgren, Department of Medicine, Länssjukhuset i Kalmar, 391 85 Kalmar,


Sweden. Phone: 0046-480-81000. E-mail: marielin@ltkalmar.se

Running title: IL28B and response to IFN-α in MPN .

Key words: Myeloproliferative neoplasms, Essential thrombocythemia, Polycythemia vera

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

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ABSTRACT
Accepted Article
OBJECTIVE:

In myeloproliferative neoplasms (MPN) interferon-alpha (IFN-α) is an effective treatment with

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

treatment response in myeloproliferative neoplasms.

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

IL28B. The genotypes of IL28B were determined by allelic discrimination assays.

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

Myeloproliferative neoplasms, Essential thrombocythemia, Polycythemia Vera

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INTRODUCTION
Accepted Article
Polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are clonal

Philadelphia-negative myeloproliferative neoplasms (MPN) associated with vascular complications

and a long-term risk of evolution to acute leukemia. Myelofibrosis occurs as a primary condition

(PMF) or as a secondary condition to PV (PPV-MF) or ET

(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

prevention of disease evolution.

Interferon-alpha (IFN-α) is a non-leukemogenic drug with a wide range of biological properties

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

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patients of African ancestry led to the first genome-wide association study (GWAS) demonstrating a
Accepted Article genetic polymorphism near the IL28B gene, encoding

interferon-λ 3 (IFNL3), as being highly predictive of response to treatment in HCV genotype 1

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

be associated with higher rates of spontaneous HCV clearance (16).

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

the treatment outcome of IFN-α in a larger setting of MPN patients.

PATIENTS AND METHODS

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

treatment including venesectio and cytoreductive agents, was allowed.

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PATIENT EVALUATION
Accepted Article
Blood samples were acquired from all patients at inclusion visit. Clinical data were recorded

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

the 3 other described criteria.

In MF, we have classified transfusion independency, no palpable splenomegaly and absence of

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

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morphologic assessments during IFN-α treatment. This cohort was for this reason not included in the
Accepted Article statistical analysis on IFN-α treatment response with regard to IL28B.

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

fluorophores to allow allelic discrimination.

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.

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RESULTS
Accepted Article
PATIENT CHARACTERISTICS

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

agent prior to IFN-α treatment.

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

mutations were found in 10/43 ET and 4/10 MF patients.

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

characteristics are summarized in Table 2.

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).

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Side effects noted in small numbers of patients included mild liver function abnormalities, hair loss,
Accepted Article skin reactions (pruritus and erythema), nausea and weight loss. Flare up of autoimmune disorder was

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.

HEMATOLOGIC RESPONSE TO IFN-α

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

13 patients (28%) lacking objective response.

In 9 out of the 10 MF patients, response, as earlier defined, was achieved (6/10) or sustained from

earlier treatment (3/10).

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.

IL28B AND HEMATOLOGIC RESPONSE TO IFN-α IN PV AND ET

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

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groups subdivided by diagnosis and hematologic response, the outcome in PV and ET displayed in
Accepted Article Table 3. CR was compared to the combined group of PR and NR.

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

not reaching statistical significance.

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

cohort alone, no significant difference was found.

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

impact was found in the ET cohort.

IL28B AND HEMATOLOGIC RESPONSE TO HYDROXYUREA

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

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at diagnosis in this cohort was 47,5 years with a male to female ratio of 1:1. The CR rate was 60%
Accepted Article (9/15) in PV, 64% (7/11) in ET and 62% (16/26) in the combined cohort. When comparing the

hematologic response to the genotypes of rs12979860, rs809997 and rs12980275, no associations

between genotypes and response were found. The results are summarized in Table 4.

IL28B AND SIDE EFFECTS IN IFN-α

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

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IFN in non-responders, pointing to a possible association of constitutive germline variants as
Accepted Article determinants of the outcome of IFN-α treatment.

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

described distribution in a northern European population (16, 20).

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

rs12979860 is found a more reliable predictor in a Caucasian population (20, 24).

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

overlap demonstrated by “true” ET mimicking prodromal stages of PV or early – prefibrotic stages of

PMF, which possibly could influence the result (25).

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Regarding the MF cohort in this study, we find a separate analysis impossible as a result of the low
Accepted Article number of MF patients included as well as the lack of response data including sequential evaluation

of bone marrow morphology.

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

se, but of importance only in the setting of IFN-α therapy.

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

increased the production of IFN-α and vice versa (34, 35).

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In HCV infection genotype 1, IL28B genotype is the strongest baseline predictor of response to
Accepted Article pegIFN-α and RBV therapy in previously untreated patients and is used in the clinical decision-making

process for initiating treatment. Despite the advances in the understanding of the cross-talk between

IFN-λ and IFN-α, the underlying mechanism of the genetic

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

expression of the IFN-λ receptor (36, 37).

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

value to validate the findings in this study.

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.

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AUTHOR CONTRIBUTIONS:
Accepted Article ML and BA designed the study and analysed the data. JW performed the genetic analyses. ML, JS, LN,
HK, WG and BA abstracted data from medical charts. ML wrote the first draft of the manuscript. All
the authors received the manuscript draft, provided critical input and approved the final version of
the manuscript.

DISCLOSURES OF CONFLICT OF INTEREST:

The authors have no competing financial interest relevant to this work.

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Table 1. Clinical characteristics of 100 patients with PV, ET and PMF and corresponding peripheral
blood counts at the point of introduction to treatment with IFN-alpha
Accepted Article
PV n=47 ET n=43 MF n=10
Age at diagnosis, years
Median 49 45 51,5
Range 17-66 15-73 36-64
Males/Females 20/27 17/26 4/6
Earlier cytoreductive
treatment 19 17 8
Hydroxyurea 17 12 5
Anagrelide 5 8 6
Other* 2 0 1
Hemoglobin, g/L
Median 133 137 122
Range 104-174 101-163 101-157
Hematocrit, %
Median 44 43 37
Range 36-54 30-49 31-48
Leukocyte count, x 109/L
Median 10,3 8,45 8,5
Range 4-23,6 3,2-17,3 7-39,4
Platelet count, x 109 /L
Median 682 796 651,5
Range 224-1632 243-1814 245-953
JAK2V617F mutation
+/- 33/1 20/12 4/4
% positive of tested 97% 63% 50%
CALR mutation**
+/- - 10/13 4/2
*Busulphan, P32

**Analysed in ET and MF when negative mutation analysis for JAK2V617F

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Table 2. IFN-alpha treatment characteristics.
Accepted Article
Type of Interferon Treatment time Dose per week Treatment response

-Median (months) -Median (range) CR/PR/NR

ET 21 α-2b 73 9 MIE (1.2-20) 16/5/0

5 Peg α-2b 16 50 μg (30-80) 4/1/0

17 Peg α-2a 12 90 μg (67,5-135) 10/7/0

PV 14 α-2b 36 6.3 MIE (4,5-15) 6/1/7

7 Peg α-2b 67 35 μg (30-50) 5/0/2

26 Peg α-2a 12.5 90 μg (45-135) 17/2/7

All* 35 α-2b 58 9 MIE (1,2-20) 22/6/7

12 Peg α-2b 46 40 μg (30-80) 9/1/2

43 Peg α-2a 12.5 90 μg (45-135) 27/9/7

MF 10 Peg α-2a 27 90 μg (30-135) **

*MF not included.** Treatment response in MF is defined as stable disease /clinical improvement20.

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Table 3. IL28B genotype and hematologic response on IFN-α treatment.
Accepted Article
PV n=47 ET n=43 All n=90

CR PR/NR CR PR/NR CR PR/NR


n=28 n=19 n=31 n=12 n=59 n=31

-rs12979860

CC 15 4 p=0,036 16 3 p=0,174 31 7 p=0,007

non-CC 13 15 15 9 28 24

-rs8099917

TT 20 9 p=0,130 19 7 p=1,0 39 16 p=0,255

non-TT 8 10 12 5 20 15

-rs12980275

AA 15 4 p=0,036 17 4 p=0,31 32 8 p=0,014

non-AA 13 15 14 8 27 23

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Table 4. IL28B genotype and hematologic response on hydroxyurea in PV and ET.
Accepted Article
ET n=11 PV n=15 All n=26

CR PR/NR CR PR/NR CR PR/NR


n=7 n=4 n=9 n=6 n=16 n=10

rs12979860

CC 5 2 p=0,576 4 2 p=1,0 9 4 p=0,688

non-CC 2 2 5 4 7 6

rs809997

TT 4 3 p=1,0 6 4 p=1,0 10 7 p=1,0

non-TT 3 1 3 2 6 3

rs12980275

AA 4 3 p=1,0 4 2 p=1,0 8 5 p=1,0

non-AA 3 1 5 4 8 5

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