AAC Accepts, published online ahead of print on 19 March 2012 Antimicrob. Agents Chemother. doi:10.1128/AAC.06473-11 Copyright © 2012, American Society for Microbiology. All Rights Reserved.

1

1

Association of ITPA gene polymorphisms and the risk of

2

ribavirin-induced anemia in HIV/HCV co-infected

3

patients on HCV combination therapy

4 5

Pere Domingo1*, Josep Mª Guardiola1*, Juliana Salazar2, Ferran Torres3, Mª Gracia

6

Mateo1, Cristina Pacho1, Mª del Mar Gutierrez1, Karuna Lamarca1, Angels Fontanet1,

7

Jordi Martin1, Jessica Muñoz1, Francesc Vidal4, and Montserrat Baiget5

8 9

1.- Infectious Diseases Unit. Hospital de la Santa Creu i Sant Pau. Universitat

10

Autònoma de Barcelona, Barcelona, Spain. Red de Investigación en SIDA (RIS).

11

Instituto de Salud Carlos III, Madrid.

12

2.- CIBERER (U-705), Barcelona, Spain.

13

3.- Statistics & Methodology Support Unit (USEM), IDIBAPS, (Hospital Clinic);

14

Laboratory of Biostatistics & Epidemiology (Universitat Autònoma de Barcelona),

15

Barcelona, Spain.

16

4.- Hospital Universitari Joan XXIII. Universitat Rovira I Virgili. Tarragona, Spain. Red de

17

Investigación en SIDA (RIS). Instituto de Salud Carlos III, Madrid.

18

5.- Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat

19

Autònoma de Barcelona, Barcelona, Spain.

20 21

Word count: 3580

22 23

Running headline: ITPA and RBV-induced anemia in HIV/HCV co-infection

24 25 26 27

*Both authors contributed equally to the manuscript

2

28

Address correspondence to:

29

Dr. Pere Domingo

30

Infectious Diseases Unit

31

Hospital de la Santa Creu i Sant Pau

32

Av. Sant Antoni Mª Claret, 167

33

08025 Barcelona

34

Spain

35

Phone : +34935565624

36

Fax : +34935565938

37

E-mail : [email protected]/[email protected]

38 39 40 41

3

42

Abstract

43

Background

44

Polymorphisms of the ITPA gene have been associated with anemia during

45

combination therapy in HCV mono-infected patients. Our aim was to confirm this

46

association in HIV/HCV co-infected patients

47

Methods

48

In this prospective, observational study, 73 HIV/HCV co-infected patients treated with

49

Peg-IFN plus RBV were enrolled. Two SNPs within or adjacent to ITPA gene (rs1127354

50

and rs7270101) were genotyped. The associations between the ITPA genotype and

51

anemia or treatment outcome were examined.

52

Results

53

Fifty-nine patients (80.8%) had CC at rs 1127354 whereas 14 (19.2%) had a CA/AA

54

ITPA genotype. Percent decreases from baseline hemoglobin level were significantly

55

greater in patients with CC than in those with CA/AA genotype at week 4 (P = 0.0003),

56

week 12 (P < 0.0001), week 36 (P = 0.0102), but not at the end of treatment. RBV dose

57

reduction was more often needed in patients with CC than in those with CA/AA

58

genotype (OR = 11.81; 95%CI: 1.45-256.17, P = 0.0039) as was erythropoietin therapy

59

(OR = 8.28; 95%CI: 1.04-371.12, P = 0.0057). Risk factors independently associated

60

with percent hemoglobin nadir decrease were: RBV dose reduction (OR = 11.72; 95CI:

61

6.82-16.63, P < 0.001), baseline hemoglobin (OR = 1.69; 95%CI: 0.23-3.15, P = 0.024),

62

and BMI (OR = -0.7, 95%CI: -1.43-0.03, P = 0.061). ITPA polymorphism was not an

63

independent predictor of sustained virological response.

64

Conclusions

65

Polymorphisms at rs 1127354 in the ITPA gene influence hemoglobin levels during

66

combination HCV therapy and the need for RBV dose reduction and erythropoietin

67

use in HIV/HCV co-infected patients.

68 69 70

Word count: 250

4

71

Key words: Inosine triphosphatase, Inosine triphosphate, pegylated interferon,

72

ribavirin, anemia, hemolysis, erythropoietin, polymorphism, HIV/HCV co-infection,

73

sustained virological response.

74 75 76

5

77

Introduction

78

Hepatitis C virus (HCV) infection is one of the most common co-morbid conditions in

79

patients with human immunodeficiency virus (HIV) infection [25]. Its prevalence is

80

especially high in Southern European countries, and its importance is highlighted by

81

the fact that end-stage liver disease is an important cause of death in HCV/HIV co-

82

infected patients [2, 25]. The standard of care in HCV infection in HIV/HCV co-infected

83

patients is combined therapy with pegylated interferon (PEG-IFN) plus ribavirin (RBV),

84

although the success rate of such combination antiviral therapy is around 30% in

85

patients with genotype 1 HCV infection [5, 33]. Furthermore, HCV therapy is difficult

86

to tolerate with significant associated morbidity. Among the important adverse effects

87

that may compromise the effectiveness of HCV therapy, hematologic toxicity is

88

common and may lead to RBV dose reductions that may affect treatment efficacy [5,

89

33]. Among HCV therapy-associated hematologic adverse effects, anemia is

90

particularly important because of its implications for treatment outcomes and its

91

frequency.

92

Anemia in the setting of HCV therapy may be caused by both components of

93

treatment: a bone marrow blockade by PEG-IFN and hemolytic anemia induced by

94

RBV [3, 8]. Recently, two functional variants in the inosine triphosphatase (ITPA) gene,

95

which encodes for ITPA on chromosome 20, have been associated with RBV-induced

96

anemia in HCV mono-infected patients treated with PEG-IFN plus RBV [9]. We sought

97

to confirm such an association in a cohort of HIV/HCV co-infected patients treated

98

with PEG-IFN plus RBV, trying to replicate the association between the functional ITPA

99

variants and hemoglobin decrease over the course of HCV therapy. Our working

100

hypothesis was that ITPA gene polymorphisms are associated with RBV-induced

101

anemia in HCV/HIV co-infected patients treated with PEGIFN + RBV.

102 103

Patients and Methods

104

Study Population. The cohort of patients in the present study is derived from a well-

105

characterized cohort of 389 HIV/HCV co-infected patients on active follow-up at the

6

106

Hospital de la Santa Creu i Sant Pau in Barcelona. All the patients consented to the

107

provision of genetic material as part of their co-infections assessment. To be included

108

in the study all the patients had to be stable, either treated or untreated, with respect

109

to HIV infection. Non-responders to previous IFN-based therapies were not included in

110

the study. Patients with chronic renal disease or creatinine clearance ≤50 mL/min,

111

hemoglobin ≤11.5 g/dL, neutrophil ≤1,500/mm3 or platelet ≤70,000/mm3 at baseline

112

were also excluded. Subjects who were hospitalized or had a frank cognitive

113

impairment such as delirium or dementia on enrolment were not eligible. Patients

114

with opportunistic infections, neoplasms or fever of undetermined origin were not

115

considered for HCV therapy. The diagnosis of AIDS was based on the 1993 revised case

116

definition of the Centers for Disease Control and Prevention [6]. All the patients were

117

negative for hepatitis B surface antigen, did not have evidence of other liver diseases,

118

and had not received other therapies, except for combined antiretroviral therapy. All

119

patients had had abnormal levels of serum alanine amino transferase (ALT) for more

120

than 6 months and were positive for anti-HCV antibody and serum HCV-RNA. The

121

study was approved by the Ethics Committee of the Hospital de la Santa Creu i Sant

122

Pau.

123

HCV RNA levels

124

Plasma HCV-RNA was measured using a real-time PCR assay (COBAS TaqMan, Roche,

125

Barcelona, Spain), which has a detection limit below 15 IU/ml. HCV genotyping was

126

performed using a commercial real-time PCR hybridization assay (Versant HCV

127

Genotype v2.0 LiPA; Siemens, Barcelona, Spain).

128

Liver fibrosis staging

129

The extent of liver fibrosis was measured using transient elastography by FibroScan

130

(Echosens, Paris, France). The median value of all tests per patient is expressed in

131

kiloPascal (kPa) units. Cirrhosis, corresponding to METAVIR score F4, was defined for

132

liver stiffness values of 14 kPa or higher.

133

HCV combination therapy

134

Treatment regimens included PEG-IFN alpha 2α at standard doses (180 µg per week)

135

plus weight-adjusted RBV (1000 mg/day for patients weighting 75 kg). Patients with HCV genotypes 1 or 4 received

137

either 48 or 72 weeks of treatment; patients with HCV genotype 3 received 24 or 48

138

weeks of treatment, according to virological response at week 4 (patients with

139

positive HCV viral load at week 4 had six months more of treatment). Therapy was

140

stopped in patients with a suboptimal virological response at weeks 12 (HCV viral load

141

decrease under two logs with respect to baseline values) and 24 (positive HCV viral

142

load). Sustained virological response (SVR) was defined as undetectable HCV RNA in

143

serum at the end of follow-up (24 weeks after cessation of treatment). Patients in

144

whom qualitative serum HCV RNA test result was positive at 24 weeks were

145

considered non responders, and therapy was stopped.

146

PEG-IFN and RBV dose modification followed the standard criteria and procedures

147

[16]. Specifically, RBV dose was cut by 200 mg in patients receiving 1000 mg or 1200

148

mg, when hemoglobin decreased < 12 g/dL, and by another 200 mg when it was

149

below < 10 g/dL. RBV treatment was stopped when hemoglobin decreased to 3 g/dl;

171

4) Hb reductions over the course of therapy, both defined quantitatively (absolute and

172

percent decreases) and qualitatively (> 3 g/dl reduction); 5) The need for RBV dose

173

reduction, 6) The need for r-huEPO therapy, and 7) Rate of SVR.

174

Statistical analyses. Data are expressed as mean and standard deviation (SD) or as

175

otherwise specified. Continuous variables were assessed with the Mann–Whitney test

176

for two groups or a nonparametric analysis of variance by applying a rank

177

transformation on the dependent variable (Rank-ANOVA) for more than two groups

178

with Bonferroni alpha-adjustment for post-hoc comparisons. Categorical data such as

179

genotype and allele frequencies were compared by use of the Fisher’s exact test. The

180

level of significance was established at 0.05 and all reported P values are two-sided.

181

All analyses were performed with the SAS version 9.1.3 software (SAS Institute Inc.,

182

Cary, NC). Stepwise logistic regression analysis was used to examine the association of

183

SVR, anemia, and other parameters with polymorphisms in the ITPA gene. The

184

variables selected to enter into stepwise regression were those that correlated

185

significantly with ITPA gene polymorphisms (after Bonferroni correction for multiple

186

testing).

187 188

Results

189

Study population. Among 389 patients with HIV/HCV co-infection, 73 received

190

therapy with PEG-IFN) plus RBV for a median time of 9.6 ± 3.7 months (range: 3-18

191

months). Fifty-six patients (76%) had a complete HCV treatment course. The mean

192

duration of treatment in the 17 patients (24%) who did not complete treatment was

193

5.8 ± 1.1 months (range: 3-8 months). Baseline characteristics of the 73 patients and

9

194

their genotypes at rs 1127354 in the ITPA gene are shown in Table 1. There were 22

195

cirrhotic patients without differences between groups (OR = 1.10 [95%CI: 0.27-5.43], P

196

= 0.8566).

197

There were no statistically significant differences between treated and not treated for

198

HCV co-infected patients in terms of genotypes at rs 1127354 in the ITPA gene (OR =

199

1.55; 95%IC: 0.80-3.17, P = 0.2240). There were no differences either between both

200

groups in terms of genotypes at rs 7270101 in the ITPA gene (OR = 1.25; 95%IC: 0.28-

201

7.88, P = 0.9736).

202

Duration of HCV infection and HCV genotypes are shown in Table 1. There were no

203

significant differences between treated patients according to genotype at rs 1127354

204

in the ITPA gene and HCV genotype (Table 1). There were no differences either

205

between groups according to genotype at rs 1127354 in the ITPA gene and genotype

206

of the IL28 gene (44.1% vs. 28.6% for CC genotype, OR = 0.51; 95%CI: 0.11-2.04, P =

207

0.4488), and genotype at rs 7270101 in the ITPA gene (27.2% vs. 14.3% for AC/CC

208

genotype, OR = 0.45; 95%CI: 0.04-2.39, P = 4938) (Table 1).

209

Most of the patients were virologically well-controlled in terms of HIV infection (79.4%

210

undetectable HIV-1 RNA). There were no differences between both groups of patients

211

in terms of antiretroviral drug exposure (Table 2). There were not differences between

212

HCV-treated patients according to genotype at rs 1127354 in the ITPA gene and ABC-

213

based cART (38.9% vs. 37.7%, P = 0.8205). Twelve patients had low hemoglobin levels

214

at baseline and were given a RBV dose decreased by 200 mg. There were 8 patients

215

with CC and 4 with CA + AA genotypes of the ITPA gene (P = 0.2271) (Table 1).

216

Sustained virological response and IL28B and ITPA polymorphism. Thirty-nine

217

patients (53.4%) achieved SVR. SVR was associated with HCV genotype (88.9% for

218

genotype 3 and 41.8% for genotypes 1 & 4; OR = 11.13; 95%CI: 2.20-105.90 , P =

219

0.0013), IL28B genotype at rs 12979860 (70.0% for CC and 41.9% for CT/TT genotypes,

220

respectively; OR = 3.24; 95%CI: 1.09-9.92, P = 0.0311), and with genotype at rs

221

1127354 in the ITPA gene (78.6% for CA+AA and 47.4% for CC genotypes, respectively;

222

OR = 4.06; 95%CI: 0.92-24.52, P = 0.0371). SVR was not associated with genotypes at

223

rs 7270101 in the ITPA gene (OR = 1.52, 95%CI: 0.45-5.32, P = 0.6305). A multivariable

10

224

analysis was performed taking SVR as the dependent variable and age, sex, BMI,

225

baseline HCV RNA, HCV genotype, RBV dose reduction, platelet count, baseline

226

fibrosis, IL28B genotype, and ITPA genotype as independent variables. Independent

227

predictors of SVR were: HCV genotype, age, baseline HCV RNA, and RBV dose

228

reduction (Table 3).

229

Decrease in hemoglobin levels during PEG-IFN + RBV therapy. Figure 1 shows the

230

percent decreases in Hb levels between 59 patients with CC and 14 with CA/AA

231

genotypes of the ITPA gene. Hb decreased more in patients with CC than CA/AA

232

genotypes at week 4 (-2.6 ± 1.3 vs. -0.90 ± 0.9 , P = 0.0002) and week 12 (-3.9 ± 1.8 vs.

233

-1.7 ± 0.7, P = 0.0003). The Hb nadir was reached earlier in patients with CC genotype

234

(13.7 ± 9.9 weeks) than in patients with CA/AA genotype (25.0 ± 10.5 weeks) (P =

235

0.0004). The percent decrease in Hb level at week 4 was -17.4 ± 10.3% for patients

236

with CC genotype whereas it was -6.1 ± 6.9% for those with CA/AA genotype (P =

237

0.0003). At week 12, the percent decrease in Hb was -23.4 ± 10.9% for patients with

238

CC genotype whereas it was -11.9 ± 5.2% for those with CA/AA genotype (P = 0.0003).

239

At week 36, the percent decrease in Hb levels was -18.6 ± 15.3% for patients with CC

240

genotype whereas it was -7.0 ± 11.7% for those with CA/AA genotype (P = 0.0192),

241

whereas at week 48 the respective decreases were -15.4 ± 17.5% and –12.7 ± 10.9% (P

242

= 0.6605). The percentage of patients who presented a decrease of Hb ≥ 3 g/dl from

243

baseline at each time point is shown in Figure 2. Genotypes at rs 7270101 in the ITPA

244

gene were not associated with Hb decrease measured in any form. Genotypes at rs

245

1127354 in the ITPA gene were not associated with maximal white blood cell count

246

decrease (-3.48 ± 1.69 vs. -2.86 ± 1.19 x 103/mm3, respectively for CC and CA/AA

247

genotypes, P = 0.2910).

248

Modification of RBV during PEG-IFN + RBV therapy. RBV dose was reduced ≥ 200 mg

249

in 15 patients (34.2%), because of Hb decrease. During the first 12 weeks of therapy,

250

the proportion of patients receiving the full dose of RBV was higher for patients with

251

the CA/AA than for those with the CC genotype (100% vs. 54.2%, OR = 11.81; 95%CI:

252

1.45-256.17, P = 0.0039). Therefore, none of CA/AA carriers needed to reduce RBV

253

dosage. Patients who needed RBV dosage modification showed a significant decrease

11

254

in percentages of SVR (32.0% vs. 64.6%, OR: 3.88; IC95% 1.25-12.50; P = 0.0163).

255

Among the 48 patients who did not require a reduction of RBV dosage, 64% (31/48)

256

had SVR. Although no significant differences were observed between the CA/AA and

257

CC genotypes (78.6% vs. 58.8%, OR 2.57; IC95%: 0.57-16.64, P = 0.3201), a higher

258

percentage of SVR was observed in patients with CA/AA genotype. Genotypes at rs

259

7270101 in the ITPA gene were not associated with modification of RBV dose.

260

Administration of erythropoietin (r-huEPO) during PEG-IFN + RBV therapy. Twenty

261

patients (27.4%) needed administration of r-huEPO because of anemia. The Hb level at

262

baseline (15.4 ± 1.5 vs. 14.4 ± 1.5 g/dl, P = 0.0189), and the percent decrease at week

263

4 (-20.3 ± 11.3% vs. -12.6 ± 9.3%, P = 0.0064) and at week 12 (-32.1 ± 10.7% vs. -16.1 ±

264

7.4%, P < 0.0001) of patients who did or did not receive r-huEPO was statistically

265

different. Forty-one percent of patients with CC genotype needed r-huEPO, whereas

266

none of the patients with CA/AA genotype needed r-huEPO (OR = 8.28; 95%CI: 1.04-

267

371.12, P = 0.0057).

268

Factors influencing decrease in hemoglobin levels during PEG-IFN + RBV therapy.

269

To determine the factors associated with Hb decrease during PEG-IFN plus RBV

270

therapy in HIV/HCV co-infected patients, a logistic regression analysis was performed

271

taking maximum percent Hb decrease as the dependent variable and age, sex, BMI,

272

baseline Hb level, baseline platelet count, RBV dose (reduced vs. not reduced), and

273

genotypes at rs 1127354 in the ITPA gene as independent variables. Independent

274

predictors of Hb decrease were: RBV dose reduction, baseline hemoglobin, and BMI

275

(Table 4).

276 277

Discussion

278

Our study shows a strong association of the development of RBV-induced anemia,

279

measured in any form, with polymorphisms in the rs 1127354 of the ITPA gene in

280

HIV/HCV co-infected patients treated with PEG-IFN plus RBV. This finding is similar to

281

the associations found in clinical trials of treated HCV mono-infected patients [20, 31,

282

32]. This finding suggests that, whatever the operating protective mechanism is, the

12

283

toxic effects of RBV triphosphate on the red blood cells may be modulated by

284

functional polymorphisms in the ITPA gene. Moreover, this is in agreement with the

285

dose-dependent mechanism through which RBV causes its toxic effects on red blood

286

cell membrane [24]. Although the effect was most evident early in treatment, it

287

persisted throughout. The protective effect of polymorphisms in ITPA resulted in no

288

need for RBV dose reductions and thus a greater cumulative RBV exposure.

289

Additionally, patients with the protective genotype had no need for r-huEPO.

290

Adverse effects of combination antiviral therapy for HCV infection are the most

291

common cause of treatment discontinuation and can jeopardize treatment adherence,

292

thus compromising the effectiveness of treatment. The rates of treatment

293

discontinuation in mono-infected patients range from 24.5% to 27%, and

294

discontinuation usually occurs within the first 6 months of treatment, anemia being

295

the cause of discontinuation in one third of these patients [11]. Therefore, anemia is

296

not only highly incidental, but is also of significant magnitude, and in two studies of

297

combination HCV antiviral therapy, Hb decreased by at least 3 g/dl in 54% of the

298

patient population and by more than 25% from baseline in approximately 28% of

299

patients [29]. Furthermore, in HIV/HCV co-infected patients, combination therapy for

300

HCV infection is associated with more profound anemia than seen in mono-infected

301

patients [29]. In fact, the Hb decreased by at least 3 g/dl in 50.6% of patients in the

302

present study, and a decrease of ≥ 25% of baseline Hb level was observed in 67.1%,

303

figures similar to those reported by others [12]. This is most likely due to a higher

304

prevalence of pre-treatment anemia in co-infected patients as well as to the potential

305

need for treatment with antiretrovirals or other medications that may cause anemia.

306

Apart from its effects on therapy discontinuation and adherence to HCV combination

307

therapy, anemia is the main cause of RBV dose reduction. RBV-induced anemia

308

requires RBV dose modification in 9% to 22% of HCV mono-infected patients [10, 17].

309

Usually, dose reduction is needed early in treatment (during the first 12 weeks) and

310

this reduction in RBV dose appears to be critical to achieve SVR [10, 28]. It is

311

recommended to avoid dose reduction in patients in whom the response rate is lower

312

and in whom a maximal effort is required to increase the individual’s chance of

13

313

response. This group includes, among others, patients with HIV/HCV co-infection [10,

314

33]. Among co-infected patients, RBV dose reduction because of anemia is needed in

315

around a third of patients [4, 12], figures similar to the 34.2% found in our study. The

316

association of RBV dose reduction and anemia is so strong that it prevented ITPA

317

being and independent predictor of anemia in our multivariate model, because of co-

318

linearity between both variables.

319

To avoid both discontinuation and RBV dose reduction, r-huEPO has been successfully

320

used both in mono-infected and co-infected patients to stimulate erythropoiesis,

321

otherwise compromised in HCV-infected patients [1, 21]. As r-huEPO therapy, which is

322

needed in about a third of treated co-infected patients (27% among our patients) [21],

323

is a surrogate marker of RBV-induced anemia in treated HIV/HCV co-infected patients,

324

the difference in its use with respect to polymorphism at rs 1127354 in the ITPA gene

325

found in our work was expectable. However, even with these correcting measures,

326

the Hb level between CC and CA/AA patients was significantly different until week 48.

327

Although anemia complicating combination HCV therapy may be caused by PEG-IFN or

328

RBV, the driving force is hemolytic anemia caused by RBV triphosphate accumulation

329

in erythrocytes, which in turn induces oxidative damage to membranes, eventually

330

leading to extravascular hemolysis [8, 24]. The mechanism of protection from

331

hemolysis by increased ITP intra-erythrocytic levels is poorly understood, but it has

332

been suggested that increase in intracellular ITP may in turn cause a decrease in

333

intracellular phosphate concentration, which may prevent the conversion of RBV into

334

RBV triphosphate or, alternatively, that ITP complexes with RBV triphosphate thus

335

conferring protection against hemolysis [10, 31]. Recent evidence indicates that ITP

336

protects against RBV-induced anemia by substituting for GTP (depleted by RBV) in the

337

biosynthesis of ATP [13]. Since ITP intracellular levels are dependent on ITPA activity,

338

which in turn is modulated by functional polymorphisms in the ITPA gene [26], these

339

polymorphisms may in the end determine the degree of protection against RBV-

340

induced hemolytic anemia in HIV/HCV co-infected patients, as has been recently

341

reported [19, 23], and our work suggests.

14

342

Data regarding the association of ITPA polymorphism and combination HCV treatment

343

outcome are quite controversial, some studies showing such an association [14],

344

whereas others [7, 19, 23, 31, 32] do not see any association between ITPA gene,

345

anemia and SVR. This association may only be the reflection of decreased treatment

346

efficacy due to dose reduction of RBV in patients with severe anemia, because the

347

potential of RBV dose reduction to limit treatment efficacy is well known [28]. This is

348

probably the case in our study since ITPA polymorphism was not an independent

349

predictor of SVR.

350

Treatment with inhibitors of the HCV serine protease together with peg-interferon

351

plus ribavirin is associated with anemia beyond that seen with peg-interferon plus

352

ribavirin therapy [18, 22]. In this setting, two Japanese studies have shown that ITPA

353

polymorphisms are still able to influence hemoglobin levels during triple HCV therapy

354

[7, 30].

355

However, the present results have inherent limitations. Firstly, this is a cross-sectional

356

study and, therefore, no causal relationships can or should be drawn. Second, we have

357

not measured ITPA expression, nor have we looked at ITP intracellular levels in

358

erythrocytes. Notwithstanding that, several mutations leading to ITPA deficiency,

359

which is a benign cell enzymopathy characterized by accumulation of ITP in

360

erythrocytes, and increased toxicity of purine analogue drugs have been well

361

characterized [27]. Among them, mutation at rs 1127354 of the ITPA gene causes a

362

substantial reduction in ITPA activity, and homozygosity for P32T mutation causes

363

non-detectable ITPA activity [9]. Altogether these data indicate that there is a good

364

correlation between ITPA gene polymorphisms, ITPA functional activity and, therefore,

365

ITP accumulation in red blood cells [15, 26]. Third, the number of patients included in

366

the present study is relatively small.

367

In summary, polymorphism at rs 1127354 in the ITPA gene is strongly associated with

368

RBV-induced anemia in HIV/HCV-co-infected patients treated with PEG-IFN plus RBV.

369

This finding has the potential to inform clinical decision-making, especially in patients

370

who need aggressive dose escalation strategies with RBV or those who are at high risk

15

371

of anemia or related morbidity, such as older patients, patients with chronic renal

372

dysfunction or hemoglobinopathies.

373

16

374 375 376

Financial support

377

Red de Investigación en SIDA (RIS RD06/006/0022, RD06/0006/1004).

378

CIBERER (U-705), Barcelona, Spain

379 380 381

Conflict of interest

382 383

No author declared any conflict of interest.

384 385 386

Contributorship statement

387

PD, JMG, and MB conceived the research, designed the database and wrote the article.

388

JS performed the genetics for the study. AF and JM retrieved and processed the blood

389

samples. GM, MMG, CP, JM and KL enrolled the patients and monitored them

390

throughout HCV therapy.

17

391

Figure legends

392 393

Figure 1.

Percent decrease in hemoglobin levels according to ITPA genotype over

394

the course of HCV combination therapy

395

CCr = Patients with CC genotype who had RBV dose reduced and/or r-huEPO

396

administered

397

CCf = Patients with CC genotype who did not have RBV dose reduced and/or r-huEPO

398

administered

399

Error bars express standard error of the mean. Hb level at each time point was

400

measure twice with a 24 hr. interval.

401

* P < 0.05 between CA/AA and CC genotypes

402 403 404

Figure 2.

Percentage of patients with a decrease of ≥ 3 g/dl of hemoglobin

405

according to genotype at rs 1127354 in the ITPA gene per week of

406

treatment. Patients in the CC group include all patients irrespective of

407

RBV dose reduction or r-huEPO administration.

408 409 410

18

411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462

References [1]

Afdhal NH, Dieterich DT, Pockros PJ, Schiff ER, Shiffman ML, Sulkowski MS, Wright T, Younossi Z, Goon BL, Tang KL, Bowers PJ; Proactive Study Group. 2004. Epoetin alfa maintains ribavirin dose in HCV-infected patients: A prospective, double-blind, randomized controlled study. Gastroenterology. 126: 1302-1311.

[2]

Antiretroviral Therapy Cohort Collaboration. 2010. Causes of death in HIV-1-infected patients treated with antiretroviral therapy, 1996-2006: collaborative analysis of 13 HIV cohort studies. Clin. Infect. Dis. 50: 1387-1396.

[3]

Bani-Sadr F, Goderel I, Penalba C, Billaud E, Doll J, Welker Y, Cacoub P, Pol S, Perronne C, Carrat F; ANRS HC02 - Ribavic Study team. 2007. Risk factors for anaemia in human immunodeficiency virus/hepatitis C virus-coinfected patients treated with interferon plus ribavirin. J. Viral. Hepat. 14: 639-444.

[4]

Bräu N, Rodriguez-Torres M, Prokupek D, Bonacini M, Giffen CA, Smith JJ, Frost KR, Kostman JR. 2004. Treatment of chronic hepatitis C in HIV/HCV-coinfection with interferon a-2b + fullcourse vs. 16-week delayed ribavirin. Hepatology. 39: 989-998.

[5]

Carrat F, Bani-Sadr F, Pol S, Rosenthal E, Lunel-Fabiani F, Benzekri A, Morand P, Goujard C, Pialoux G, Piroth L, Salmon-Céron D, Degott C, Cacoub P, Perronne C, ANRS HCO2 RIBAVIC Study Team. 2004. Pegylated interferon alfa-2b vs. standard interferon alfa-2b, plus ribavirin, for chronic hepatitis C in HIV-infected patients: a randomized controlled trial. JAMA. 292: 2839-2848.

[6]

Centers for Disease Control. 1993. 1993 revised classification system for HIV infection and expanded surveillance for case definition for AIDS among adolescents and adults. MMWR. 41 (RR-17): 1-13.

[7]

Chayama K, Hayes CN, Abe H, Miki D, Ochi H, Karino Y, Toyota J, Nakamura Y, Kamatani N, Sezaki H, Kobayashi M, Akuta N, Suzuki F, Kumada H. 2011. IL28B but not ITPA polymorphism is predictive of response to pegylated interferon, ribavirin, and telaprevir triple therapy in patients with genotype 1 hepatitis C. J Infect Dis. 204: 84-93.

[8]

De Franceschi L, Fattovich G, Turrini F, Ayi K, Brugnara C, Manzato F, Noventa F, Stanzial AM, Solero P, Corrocher R. 2000. Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage. Hepatology. 31: 9971004.

[9]

Fellay J, Thompson AJ, Ge D, Gumbs CE, Urban TJ, Shianna KV, Little LD, Qiu P, Bertelsen AH, Watson M, Warner A, Muir AJ, Brass C, Albrecht J, Sulkowski M, McHutchison JG, Goldstein DB. 2010. ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C. Nature. 464: 405-408.

[10] Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Gonçales FL Jr, Häussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. 2002. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med. 347: 975-82. [11] Gaeta GB, Precone DF, Felaco FM, Bruno R, Spadaro A, Stornaiuolo G, Stanzione M, Ascione T, De Sena R, Campanone A, Filice G, Piccinino F. 2002. Premature discontinuation of interferon plus ribavirin for adverse effects: A multicentre survey in ‘real world’ patients with chronic hepatitis C. Aliment. Pharmacol. Ther. 16: 1633-1639.

19

463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515

[12] Henry DH, Slim J, Lamarca A, Bowers P, Leitz G; HIV/HCV Coinfection Natural History Study Group. 2007. Natural History of Anemia Associated with Interferon/Ribavirin Therapy for Patients with HIV/HCV Coinfection. AIDS. Res. Hum. Retrovir. 23: 1-9. [13] Hitomi Y, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ, Gumbs CE, Shianna KV, Urban TJ, Goldstein DB. 2011. Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function. Gastroenterology. 140: 1314-1321. [14] Kurosaki M, Tanaka Y, Tanaka K, Suzuki Y, Hoshioka Y, Tamaki N, Kato T, Yasui Y, Hosokawa T, Ueda K, Tsuchiya K, Kuzuya T, Nakanishi H, Itakura J, Takahashi Y, Asahina Y, Matsuura K, Sugauchi F, Enomoto N, Nishida N, Tokunaga K, Mizokami M, Izumi N. 2011. Relationship between polymorphisms of the inosine triphosphatase gene and anaemia or outcome after treatment with pegylated interferon and ribavirin. Antivir. Ther. 16: 685-694. [15] Maeda T, Sumi S, Ueta A, Ohkubo Y, Ito T, Marinaki AM, Kurono Y, Hasegawa S, Togari H. 2005. Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency in the Japanese population. Mol. Genet. Metab. 85: 271-279. [16] Mangia A, Santoro R, Minerva N, Ricci GL, Carretta V, Persico M, Vinelli F, Scotto G, Bacca D, Annese M, Romano M, Zechini F, Sogari F, Spirito F, Andriulli A. 2005. Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N. Engl. J. Med. 352: 2609-2617. [17] Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet. 358: 958-965. [18] McHutchison JG, Everson GT, Gordon SC, Jacobson IM, Sulkowski M, Kauffman R, McNair L, Alam J, Muir AJ; PROVE1 Study Team. 2009. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med. 360: 1827-1838. [19] Naggie S, Rallon NI, Benito JM, Morello J, Rodriguez-Novoa S, Clark PJ, Thompson AJ, Shianna KV, Vispo E, McHutchison JG, Goldstein DB, Soriano V. 2012.Variants in the ITPA gene protect against ribavirin-induced hemolytic anemia in HIV/HCV-coinfected patients with all HCV genotypes. J. Infect. Dis. 205: 376-383. [20] Ochi H, Maekawa T, Abe H, Hayashida Y, Nakano R, Kubo M, Tsunoda T, Hayes CN, Kumada H, Nakamura Y, Chayama K. 2010. ITPA polymorphism affects ribavirin-induced anemia and outcomes of therapy- A genome-wide study of Japanese HCV virus patients. Gastroenterology. 139: 1190-1197. [21] Pau AK, McLaughlin MM, Hu Z, Agyemang AF, Polis MA, Kottilil S. 2006. Predictors for hematopoietic growth factors use in HIV/HCV-coinfected patients treated with peginterferon alfa 2b and ribavirin. AIDS. Patient. Care. STDS. 20: 612-619. [22] Poordad F, McCone J Jr, Bacon BR, Bruno S, Manns MP, Sulkowski MS, Jacobson IM, Reddy KR, Goodman ZD, Boparai N, DiNubile MJ, Sniukiene V, Brass CA, Albrecht JK, Bronowicki JP; SPRINT-2 Investigators. 2011. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med. 364: 1195-1206. [23] Rallón NI, Morello J, Labarga P, Benito JM, Rodríguez-Nóvoa S, Vispo E, Barreiro P, Castro MÁ, Aguirrebengoa K, Pineda JA, Miralles P, Tellez MJ, Portu J, Miralles C, Ocampo A, Soriano V;

20

516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568

Peginterferon Ribavirin Coinfection Team. 2011. Impact of Inosine Triphosphatase Gene Variants on the Risk of Anemia in HIV/Hepatitis C Virus–Coinfected Patients Treated for Chronic Hepatitis C. Clin. Infect. Dis. 53: 1291-1295. [24] Reddy KR, Nelson DR, Zeuzem S. 2009. Ribavirin: current role in the optimal clinical management of chronic hepatitis C. J. Hepatol. 50: 402-411. [25] Roca B, Suarez I, Gonzalez J, Garrido M, de la Fuente B, Teira R, Geijo P, Cosin J, Perez-Cortes S, Galindo MJ, Lozano F, Domingo P, Viciana P, Ribera E, Vergara A, Sánchez T. 2003. Hepatitis C virus and human immunodeficiency virus co-infection in Spain. J. Infect. 47: 117124. [26] Shipkova M, Lorenz K, Oellerich M, Wieland E, von Ahsen N. 2006. Measurement of erythrocyte inosine triphosphate pyrophosphohydrolase (ITPA) activity by HPLC and correlation of ITPA genotype-phenotype in a Caucasian population. Clin. Chem. 52: 240-247. [27] Stocco G, Cheok MH, Crews KR, Dervieux T, French D, Pei D, Yang W, Cheng C, Pui CH, Relling MV, Evans WE. 2009. Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for acute lymphoblastic leukemia. Clin. Pharmacol. Ther. 85: 164-172. [28] Sulkowski MS, Wasserman R, Brooks L, Ball L, Gish R. 2004. Changes in haemoglobin during interferon alpha-2b pus ribavirin combination therapy for chronic hepatitis C virus infection. J. Viral. Hepat. 11: 243-250. [29] Sulkowski MS. 2004. Defining the standard of care: Randomized controlled trials for the treatment of hepatitis C in the HIV-infected person. Hepatology. 39: 906-908. [30] Suzuki F, Suzuki Y, Akuta N, Sezaki H, Hirakawa M, Kawamura Y, Hosaka T, Kobayashi M, Saito S, Arase Y, Ikeda K, Kobayashi M, Chayama K, Kamatani N, Nakamura Y, Miyakawa Y, Kumada H. 2011. Influence of ITPA polymorphisms on decreases of hemoglobin during treatment with pegylated interferon, ribavirin, and telaprevir. Hepatology. 53: 415-421. [31] Thompson AJ, Fellay J, Patel K, Tillmann HL, Naggie S, Ge D, Urban TJ, Shianna KV, Muir AJ, Fried MW, Afdhal NH, Goldstein DB, McHutchison JG. 2010. Variants in the ITPA gene protect against tibavirin-induced hemolytic anemia and decrease the need for ribavirin dose reduction. Gastroenterology. 139: 1181-1189. [32] Thompson AJ, Santoro R, Piazzolla V, Clark PJ, Naggie S, Tillmann HL, Patel K, Muir AJ, Shianna KV, Mottola L, Petruzzellis D, Romano M, Sogari F, Facciorusso D, Goldstein DB, McHutchison JG, Mangia A. 2011. Inosine triphosphatase genetic variants are protective against anemia during antiviral therapy for HCV2/3 but do not decrease dose reductions of RBV or increase SVR. Hepatology. 53: 389-395. [33] Torriani FJ, Rodriguez-Torres M, Rockstroh JK, Lissen E, Gonzalez-García J, Lazzarin A, Carosi G, Sasadeusz J, Katlama C, Montaner J, Sette H Jr, Passe S, De Pamphilis J, Duff F, Schrenk UM, Dieterich DT, APRICOT Study Group.0 2004. Peginterferon Alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients. N. Engl. J. Med. 351: 438-450.

21

569 Table 1. Baseline characteristics of HIV-HCV co-infected patients treated with peg-interferon and ribavirin Total

ITPA genotypes at rs 1127354 CC

Number

P value

CA + AA

73

59

14

43/30

35/24

8/6

0.8816

Age, years

46.8 ± 5.5

46.7 ± 5.2

47.1 ± 6.6

0.8091

Body weight, kg

68.1 ± 8.5

68.6 ± 8.5

66.3 ± 8.3

0.3664

23.2 ± 3.2

23.0 ± 3.0

23.9 ± 3.6

0.3648

MsM, %

11 (15.1)

8 (13.6)

3 (21.4)

0.4015

HTSX, %

20 (27.4)

18 (30.5)

2 (14.3)

IDU, %

42 (57.5)

33 (55.9)

9 (64.3)

Duration of HCV infection, years

15.9 ± 6.2

15.9 ± 6.5

15.5 ± 4.8

0.8122

Cirrhosis, %

22 (30.1)

18 (30.5)

4 (28.6)

0.8566

0.4488

Sex, male/female

2

BMI, kg/m

Means of acquiring HIV

Genotypes of the IL28 gene CC, %

30 (41.1)

26 (44.1)

4 (28.6)

CT + TT, %

43 (58.9)

33 (55.9)

10 (71.4)

AA, %

55 (75.3)

43 (72.8)

12 (85.7)

AC/CC, %

18 (24.6)

16 (27.2)

2 (14.3)

Baseline hemoglobin, g/dl

14.5± 1.6

14.6 ± 1.6

13.9 ± 1.5

0.1268

Baseline hemoglobin < 13 g/dl, %

12 (16.4)

8 (13.6)

4 (28.6)

0.2271

Baseline platelets, x 104/mm3

16.3±4.4

16.4 ± 4.7

15.4 ± 3.4

0.4357

5.41 ±1.4

5.52 ± 1.46

4.95 ± 1.08

0.1751

43.4

44.0

41.2

0.0534

AST, IU/l

61.9 ± 27.8

62.3 ± 29.8

60.3 ± 17.7

0.8118

ALT, IU/l

74.6 ± 33.4

75.0 ± 35.8

72.6 ± 21.3

0.8118

6.2 ± 0.7

6.2 ± 0.7

6.1 ± 0.5

0.5735

55 (75.3)

46 (77.9)

9 (64.3)

Genotypes at rs 7270101

3

3

Baseline WBC, x 10 /mm Albumin, g/l

HCV RNA, log10IU/ml HCV genotype

0.4669 1&4, (%) 3, (%)

Fibrosis, kPa

0.4938

18 (24.7)

13 (22.1)

5 (35.7)

10.7 ± 11.8

10.5 ± 11.5

11.8 ± 13.5

0.7180

All parameters in mean ± standard deviation unless otherwise specified. Kg = kilogram, m2 = squared meters, g = grams, dl = deciliter, l = liter, IU = international units, kPa = kilopascals, ITPA = inosine triphosphatase, BMI = body mass index, MsM = Men who have sex with men, HTSX = heterosexuals, IDU = Intravenous drug users, WBC = white blood cell, g = grams, l = liter, dl = deciliter, AST = aspartate aminotransferase, ALT = alanine aminotransferase, HCV = hepatitis C virus

22

570 Table 2. HIV infection parameters and antiretroviral drug exposure in HIV-HCV co-infected patients treated with peg-interferon and ribavirin Total ITPA genotypes at rs 1127354 P value CC CA + AA Number 73 59 14 ART concomitant to HCV therapy 0.6385 PI-based, % 29 (39.7) 24 (40.7) 5 (35.7) NNRTI-based. % 34 (46.6) 26 (44.1) 8 (57.1) None, % 6 (8.2) 6 (10.2) 0 (0) 3 NRTIs, % 3 (4.1) 2 (3.4) 1 (7.1) INsTI-based, % 1 (1.4) 1 (1.7) 0 (0) NRTI backbone 0.0835 TDF + TDF, % 34 (46.6) 23 (38.9) 11 (78.6) ABC + 3TC, % 18 (24.6) 18 (30.5) 0 (0) None, % 14 (19.2) 12 (20.3) 2 (14.3) ABC + TDF, % 1 (1.4) 2 (3.4) 0 (0) AZT + 3TC, % 1 (1.4) 1 (1.7) 0 (0) ddI + 3TC, % 1 (1.4) 0 (0) 1 (7.1) ddI + ABC, % 1 (1.4) 1 (1.7) 0 (0) 3TC alone, % 1 (1.4) 1 (1.7) 0 (0) ABC alone, % 1 (1.4) 1 (1.7) 0 (0) AZT-based, % 1 (1.4) 1 (1.7) 0 (0) 0.9999 ABC-based, % 29 (39.7) 23 (38.9) 6 (42.8) 0.9701 Individual antiretroviral exposure AZT, m 30.1 ± 43.9 27.4 ± 36.9 41.0 ± 66.6 0.3421 3TC/FTC, m 72.3 ± 52.9 69.4 ± 54.9 84.0 ± 44.0 0.3976 d4T, m 36.9 ± 40.5 38.2 ± 41.1 31.7 ± 39.2 0.6240 ddI, m 27.3 ± 38.7 26.0 ±39.0 32.6 ± 38.5 0.6037 ddC, m 3.8 ± 10.8 4.6 ± 11.9 0.7 ± 2.3 0.2617 ABC, m 26.9 ± 42.7 29.7 ± 43.4 15.6 ± 39.6 0.3078 TDF, m 32.1 ± 35.7 27.2 ± 34.5 51.9 ± 35.3 0.0308 EFV, m 34.6 ± 48.8 31.8 ± 48.9 46.2 ± 48.9 0.3661 NVP, m 8.9 ± 22.7 9.6 ± 24.9 5.6 ± 7.9 0.5777 NRTIs, m 229.6 ± 135.8 222.7 ± 141.7 257.5 ± 109.4 0.4312 PIs, m 46.9 ± 51.1 44.3 ± 51.9 57.4 ± 48.6 0.4320 RAL, m 2.5 ± 9.2 2.9 ± 10.2 0.7 ± 2.3 0.4526 HIV RNA,log10 copies/ml 1.6 ± 0.7 (1.3-1.3) 1.6 ± 0.7 1.5 ± 0.8 0.7340 Current CD4 count, cells/mm3 635 ± 314 629 ± 340 660 ± 190 0.7603 Undetectable HIV RNA, % 58 (79.4) 46 (77.9) 12 (85.7) 0.7778 CD4 nadir, mean ± SD, cells/mm3 229 ± 162 219 ± 144 271 ± 224 0.3227 CD4 nadir < 100 cells/mm3, % 16 (27.2) 13 (27.7) 3 (25.0) 0.8523 All parameters in mean ± standard deviation unless otherwise specified. HIV = human immunodeficiency virus , ART = antiretroviral therapy, PIs = protease inhibitors , NNRTIs = non-nucleoside reverse transcriptase inhibitor, NRTIs = nucleoside reverse transcriptase inhibitors, INsTI = integrase strand transfer inhibitor, AZT = zidovudine, 3TC = lamivudine, FTC = emtricitabine, d4T = stavudine, ddI = didanosine, ddC = zalcitabine, ABC = abacavir, TDF = tenofovir, EFV = efavirenz, NVP = nevirapine, RAL = raltegravir, m = months. ml = milliliters.

571 572

23

573 Table 3. Independent predictors of sustained virologial response in 73 HIV/HCV co-infected patients treated with peg-interferon plus ribavirin Variable

Odds ratio

95% Confidence intervals

P value

HCV genotype

24.83

2.64-233-07

< 0.001

Age

9.02

1.49-54.61

0.03

Baseline HCV RNA

0.35

0.12-0.98

0.05

RBV dose reduction

0.10

0.02-0.54

0.01

HIV = human immunodeficiency virus, HCV = Hepatitis C virus, RNA = ribonucleic acid, RBV = ribavirin

574 575 576 577 578 Table 4. Independent predictors of maximum percent decreas in Hb in 73 HIV/HCV co-infected patients treated with peg-interferon plus ribavirin Variable

Odds ratio

95% Confidence intervals

P value

RBV dose reduction

11.72

6.82-16.63

< 0.001

Baseline Hb level

1.69

0.23-3.15

0.024

BMI

0.7

-1.43-0.03

0.061

HIV = human immunodeficiency virus, HCV = Hepatitis C virus, RNA = ribonucleic acid, RBV = ribavirin, Hb = hemoglobin, BMI = body mass index

579 580 581 582

CCr Bl

w44

CCf w12 12

CA/AA w36 36

w48 48

0

Percent deccrease of Hbb level

-55 -10 -15 -20

*

*

*

*

-25

* -30

*

-35 -40 Week

*

CC P=0 0.0001 0001

Hb redduction ≥ 3 g/l ((%)

60 50

CA/AA P=0 0.0225 0225 P = 0.1265

P=0 0.0015 0015

40 30 20 10 0

w4

w12

w36 Week

w48