PhD Mattia Cf Prosperi

Maurizio Zazzi

Grazia Punzi

Laura Monno

Grazia Colao

Paola Corsi

Simona Di Giambenedetto

Genny Meini

Valeria Ghisetti

Stefano Bonora

Low Rate of Virological Failure and Maintenance of Susceptibility to HIV-1 Protease Inhibitors with First-Line Lopinavir/Ritonavir-Based Antiretroviral Treatment in Clinical Practice

Keywords: human immunodeficiency virus type 1, lopinavir/ritonavir, first-line antiretroviral therapy, genotypic resistance, virologic failure ritonavir, first-line 55 antiretroviral therapy, antiretroviral drug resistance, virologic failure

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

F o r P e e r R e v i e w

survival analysis, 39 (7.1%) and 43 (7.8%) of the 548 examined patient cases had an HIV-1 RNA 40 >500 and >50 copies/ml, respectively, after 6 months of treatment. Cox proportional hazard 41 models detected baseline HIV-1 RNA (RH 1.79, 95%CI 1.10-2.92 per 1-log 10 increase, P = 0.02) and 42 resistance to the nucleoside backbone (RH 1.04, 95%CI 1.02-1.06 per 10-point increase using the 43

Stanford HIVdb algorithm, P < 0.001) as independent predictors of HIV-1 RNA at >500 copies/ml, 44 but not at the >50 copies/ml cutoff criteria. Higher baseline viral load, older patient age, Introduction 59 60 Ritonavir-boosted protease inhibitors (PIs) have been used successfully for years and still 61 remain a key component of highly active antiretroviral therapy (HAART). A major advantage of 62 boosted PIs in first-line therapy is the very low risk of selecting drug-resistant HIV-1 variants at 63 virological failure, as shown in multiple clinical trials [START_REF] Bartlett | 291 Minimizing resistance consequences after virologic failure on initial combination therapy: a 292 systematic overview[END_REF][START_REF] Gupta | Emergence of drug resistance in HIV type 1-infected 307 patients after receipt of first-line highly active antiretroviral therapy: a systematic review of clinical 308 trials[END_REF]Lima 64 et al., 2008;[START_REF] Molina | Once-daily 330 atazanavir/ritonavir versus twice-daily lopinavir/ritonavir, each in combination with tenofovir and 331 emtricitabine, for management of antiretroviral-naive HIV-1-infected patients: 48 week efficacy 332 and safety results of the CASTLE study[END_REF][START_REF] Ortiz | Efficacy and safety of once-339 daily darunavir/ritonavir versus lopinavir/ritonavir in treatment-naive HIV-1-infected patients at 340 week 48[END_REF][START_REF] Mills | Once-daily darunavir/ritonavir vs. lopinavir/ritonavir in 326 treatment-naive, HIV-1-infected patients: 96-week analysis[END_REF]. This feature is, in fact, the 65 main argument made by experts favouring boosted PI over non-nucleoside reverse transcriptase 66 inhibitor (NNRTI)-based first-line therapy because NNRTIs select typically for NNRTI-resistant virus 67 at failure [Riddler et al., 2008]. In addition, resistance to the backbone nucleoside reverse 68 transcriptase inhibitors (NRTIs) occurs less frequently with a boosted PI compared to an NNRTI-69 based therapy [START_REF] Soulié | Nucleoside reverse transcriptase inhibitor-sparing 355 regimen (nonnucleoside reverse transcriptase inhibitor + protease inhibitor) was more likely 356 associated with resistance comparing to nonnucleoside reverse transcriptase inhibitor or protease 357 inhibitor + nucleoside reverse transcriptase inhibitor in the randomized ANRS 121 trial[END_REF]. 70

While the latest treatment guidelines recommend other boosted PIs for first-line HAART 71 [DHHS, 2009], lopinavir/ritonavir (LPV/rtv) has been the most widely used PI owing to its long-72 standing availability, unique heat-stable co-formulation, efficacy and safety profiles [Oldfield et al., 73 2006]. In order to evaluate virological response to first-line LPV/rtv-based therapy and confirm its 74 low propensity for selecting resistance mutations in HIV-1 protease in clinical practice, a targeted 75 analysis was carried out using the Italian Antiretroviral Resistance Cohort Analysis (ARCA) 76 observational database. ARCA (www.hivarca.net) is a nationwide initiative started in 2002 which 77 retrospectively and prospectively collates clinical and virological data from HIV-1-infected patients 78 followed at 105 centres. The data include demographics, hepatitis B and C status, AIDS-defining 79 events, antiretroviral treatment, viral load, CD4+ T cell counts, HIV-1 genotype. First-line treatments comprised of LPV/rtv plus two NRTIs were extracted from the ARCA 87 database. All patients with available HIV-1 genotype and HIV-1 RNA load at baseline were included 88 and all HIV-1 RNA load data available from the treatment start date to the latest follow-up time 89 were collected. The rate of treatment failure and development of drug resistance were evaluated 90 by survival analyses using the cases with at least one follow-up HIV-1 RNA load measurement 91 obtained after 3 months and after 6 months of uninterrupted treatment. Information on HIV-1 92 genotype obtained between 30 days after treatment start and 90 days after LPV/rtv 93 discontinuation was also included, if no other PI was administered after LPV/rtv. 94

The Stanford HIVdb version 6.0.1 (http://hivdb.stanford.edu/) algorithm was used to 95 calculate genotypic resistance scores (GRS). HIVdb scores below 10 are classified as full drug 96 susceptibility, from 10 to 14 as potential low-level resistance, from 15 to 29 as low-level 97 resistance, from 30 to 59 as intermediate resistance and >59 as high-level resistance. This five-98 level system was converted to a three-category classification by grouping scores up to 14 as 99 susceptibility, 15 to 59 as intermediate resistance and >59 as complete resistance. A continuous 100 numeric GRS scale was used in the multivariable analysis in order to minimise the number of 101 parameters to be optimised: results are showed per 10 points higher, which roughly corresponds 102 to a one-category increase in the Stanford HIVdb categorisation. 103

Survival analysis using the Kaplan-Meier method and Cox proportional hazard models was 104 performed using the following outcome measures: having an HIV-1 RNA load >500 and >50 105 copies/ml at 3 months after treatment initiation; an HIV-1 RNA load >500 and >50 copies/ml at 6 106 months after treatment initiation; intermediate or full LPV/rtv resistance defined as a GRS >14; 107 and the presence of at least one major LPV/rtv resistance mutation according to the 2009 IAS-USA 108 list (i.e. V32I, I47A/V or V82A/F/S/T) [START_REF] Johnson | Update of the drug resistance mutations in HIV-1: December 2009[END_REF]. Cases not reaching the specific 109 The ARCA database contained 1,141 patients who were treated with first line LPV/rtv-119 containing HAART regimens. Of these, 549 (48%) did not have a baseline genotype and were 120 excluded from the analysis. The remaining 592 patients had baseline median (IQR) CD4+ T cell 121 counts and HIV-1 RNA load of 127 (48-255) cells/mm 3 and 5.20 (4.80-5.60) log 10 copies/ml, 122 respectively. The median (IQR) calendar year of LPV/rtv initiation was 2006 (2004-2007). The NRTI 123 backbones employed most frequently were tenofovir plus emtricitabine (37.5%), zidovudine plus 124 lamivudine (34.3%), abacavir plus lamivudine (12.2%), and tenofovir plus lamivudine (10.6%). 125

At baseline, 15 (2.5%) and 1 (0.2%) patients presented with a LPV/rtv intermediate and 126

resistant GRS, respectively. The prevalence of genotypes carrying at least one major IAS mutation 127 associated to LPV/rtv resistance was 7.3% (n=43). Intermediate and full resistance to at least one 128 NRTI were detected in 5.4% and 2.5% of the cases, with 1.8% showing intermediate and 5.7% full 129 resistance to at least one NNRTI; and 1.8% and 2.0% to at least one PI, respectively. When looking 130 at the GRS of the treatment provided, the prevalence of regimens with all fully active drugs (i.e. no 131 drug with a GRS >14) was 93%, while 2.9% of regimen were composed by either at least one drug 132 with full resistance or two drugs with intermediate resistance. Ten (1.7%) genotypes were found 133 with both intermediate-level resistance to LPV/rtv and to at least one of the other drugs in the 134

backbone. 135

Forty-four (7.4%) of the 592 patients had not have a follow-up HIV-1 RNA measurement 136 after 3 or 6 months hence were excluded from subsequent incidence and survival analyses. 137 Following 3-and 6 months therapy, 74 (13.5%) and 39 (7.1%) patients had an HIV-1 RNA >500 138 copies/ml, respectively. Using the more stringent criterion, the number of cases with a confirmed 139 HIV-1 RNA >50 copies/ml after 3 and 6 months were 77 (14%) and 43 (7.8%), respectively. 140 2). There were no changes in Stanford 167

HIVdb LPV/rtv susceptibility category in any case. Among the 30 patients with a LPV/rtv-sensitive 168 baseline genotype, only one was found with more than one additional minor PI mutation (patient 169 25: L10F, I13V, L63P, V77I, V82I) but this did not have any impact on the predicted LPV/rtv 170 susceptibility. In some other cases, the baseline and follow-up genotype differed for one minor 171 mutation, either acquired or lost. Resistance mutation I54V, contributing to resistance to LPV/rtv, 172 was observed in three cases with pre-existing LPV/rtv resistance. In one of the four cases 173 harboring a virus populations with intermediate resistance to LPV/rtv at baseline (patient 20), the 174 major PI mutation M46I was identified at the follow-up stage. Moreover, major LPV/rtv mutations 175 M46I and I47V were found in one of the two patients (patient 8) with a virus that was already 176 highly resistant to LPV/rtv at baseline. There were four cases with NRTI resistance mutations, 177 namely M184I/V with or without a more extensive set of NRTI resistance mutations. In one of 178 these patients, the follow-up HIV-1 genotype also contained NNRTI resistance mutations as a 179 result of NNRTI therapy following discontinuation of LPV/rtv. Overall, 30 of the 36 (83.3%) follow-180 up genotypes were susceptible to LPV/rtv and to at least another PI,33 (91.7%) [START_REF] Geretti | Epidemiology of antiretroviral drug resistance in drug-naïve persons[END_REF] and may benefit from boosted PI therapy. Interestingly, it was observed that 209 decreased susceptibility to the NRTI backbone appeared to compromise treatment more than 210 decreased susceptibility to LPV/rtv. This highlights the potency of this high-genetic barrier PI, 211 although the low number of cases with resistance at baseline may have hampered the detection of 212 minor effects. 213

According to the genotypic susceptibility profile at failure, for the vast majority of patients 214 a fully active regimen can be still based on a boosted PI or consisting of two NRTI plus one NNRTI. 215

While novel drug classes are being considered for convenient and potentially less toxic first-line 216 treatment, boosted PIs, particularly LPV/rtv, maintain an excellent and hardly achievable efficacy 217 record of in terms of suppression of virus replication and limitation of drug resistance evolution. In 218 addition, recent trial data suggest better tolerability and/or higher potency for the currently 219 recommended boosted PIs compared to LPV/rtv [START_REF] Madruga | Efficacy and safety of darunavir-ritonavir compared with that of lopinavir-ritonavir at 48 321 weeks in treatment-experienced, HIV-infected patients in TITAN: a randomised controlled phase III 322 trial[END_REF][START_REF] Molina | Once-daily 330 atazanavir/ritonavir versus twice-daily lopinavir/ritonavir, each in combination with tenofovir and 331 emtricitabine, for management of antiretroviral-naive HIV-1-infected patients: 48 week efficacy 332 and safety results of the CASTLE study[END_REF]

  PI)-resistant HIV-1 has hardly ever been detected at failed boosted PI-based 34 first-line antiretroviral regimens in clinical trials. However, this phenomenon has not been 35 investigated in clinical practice. To address this gap, data from patients starting a first-line 36 lopinavir/ritonavir (LPV/rtv)-based therapy with available baseline HIV-1 RNA load, a viral 37 genotype and follow-up viral load after 3 months and 6 months of treatment were extracted from 38 the Italian Antiretroviral Resistance Cohort Analysis (ARCA) observational database. Based on 39

  at the regimen discontinuation date or at the latest available HIV-1 RNA 110 or HIV-1 genotype if the regimen was not discontinued. After adjusting for the other explanatory 111 variables, the Cox model provides an estimate of the treatment effect on the remaining data with 112 no outcome event. In addition, the model allows estimating the outcome event hazard for each 113 individual, based on his/her baseline variables.

  According to Kaplan-Meier analysis (Fig.1) the estimated proportion of patients with an /rtv discontinuation. Paired analysis of the baseline and follow-up genotypes 166 revealed very limited selection of protease mutations (Table

  . While 220 this strengthens the importance of the PI class as a cornerstone in HAART, future analysis of large 221 cohort data will be required to confirm such advantages in clinical practice. Infettive e Tropicali La Sapienza -Umberto I), Ombretta Turriziani (ROMA -Medicina Sperimentale e Patologia -280 Sezione Virologia -La Sapienza), Marco Montano (ROMA -Virologia per Malattie Infettive Tor Vergata), Giovanni 281 Cenderello (SAN REMO -Malattie Infettive), Angela Gonnelli (SIENA -Malattie Infettive), Maurizio Zazzi (SIENA -282 Virologia), Michele Palumbo (TERNI -Malattie Infettive), Valeria Ghisetti (TORINO -Laboratorio di Virologia, Ospedale 283 Amedeo di Savoia), Stefano Bonora (TORINO -Malattie Infettive Amedeo di Savoia), Palma Delle Foglie (TRENTO -284 Malattie Infettive), Cristina Rossi (TREVISO -Malattie Infettive), Federica Poletti (VERBANIA -Malattie Infettive 285 VERBANIA), Vincenzo Mondino (VERBANIA -Virologia), Marina Malena (VERONA -Centro di Medicina Preventiva-ULSS 286 20), Emanuela Lattuada (VERONA -Malattie Infettive).

Table 1a .

 1a Multivariable Cox regression analysis: predictors of virological failure beginning from month 6 after treatment initiation (n=548).

	HIV-RNA measured at or after 6 months
				>50 copies/ml		>500 copies/ml
	factor			RH	95% CI	p-value	RH	95% CI	p-value
	year of birth (per 1-year increase)	0.994 (0.958-1.03)	0.725	1.024 (0.983-1.066)	0.264
			male vs. female	0.649 (0.298-1.415)	0.277	0.575 (0.248-1.336)	0.198
	gender		unknown vs. female	1.041 (0.128-8.463)	0.970	1.374 (0.17-11.079)	0.765
			homo/bisexual vs. heterosexual	1.264 (0.524-3.046)	0.602	1.368 (0.53-3.534)	0.517
			intravenous drug user vs. heterosexual	0.512 (0.105-2.498)	0.408	1.519 (0.432-5.346)	0.515
	mode of HIV transmission	other/unknown vs. heterosexual	1.236 (0.558-2.737)	0.602	1.156 (0.477-2.802)	0.748
	calendar year of HAART initiation (per more recent)	0.919 (0.719-1.174)	0.497	0.978 (0.733-1.304)	0.878
		abacavir+lamivudine vs. tenofovir+emtricitabine	0.362 (0.079-1.65)	0.189	0.197 (0.025-1.533)	0.121
		zidovudine+lamivudine vs. tenofovir+emtricitabine	1.142 (0.47-2.773)	0.769	0.991 (0.375-2.619)	0.986
	backbone	other backbone vs. tenofovir+emtricitabine	1.592 (0.403-6.299)	0.507	1.936 (0.402-9.323)	0.410
	HAART	tenofovir+lamivudine vs. tenofovir+emtricitabine	0.275 (0.056-1.361)	0.114	0.754 (0.206-2.767)	0.671
	baseline CD4+ cell count cells/mm3 (per log higher)	0.844 (0.656-1.087)	0.190	0.892 (0.679-1.173)	0.414
	baseline HIV-RNA load cp/ml (per log10 higher)	0.844 (0.529-1.348)	0.479	1.790 (1.097-2.921)	0.020
	lopinavir GRS (per 10 points higher)	0.975 (0.912-1.042)	0.450	0.945 (0.89-1.003)	0.064
	backbone GRS (per 10 points higher)	1.002 (0.979-1.026)	0.849	1.038 (1.017-1.06)	0.0003

Table 1b .

 1b Multivariable Cox regression analysis: predictors of virological failure beginning from month 3 after treatment initiation (n=548).

	HIV-RNA measured at or after 3 months
				>50 copies/ml		>500 copies/ml
	Factor			RH	95% CI	p-value	RH	95% CI	p-value
	year of birth (per 1 year increase)	1.018 (0.991-1.045)	0.2012	1.032 (1.002-1.063)	0.0365
			male vs. female	1.233 (0.679-2.239)	0.4915	0.918 (0.504-1.67)	0.7784
	gender		unknown vs. female	1.497 (0.339-6.605)	0.5941	0.468 (0.06-3.63)	0.4677
			homo/bisexual vs. heterosexual	0.875 (0.468-1.634)	0.6751	0.827 (0.407-1.678)	0.5984
			intravenous drug user vs. heterosexual	0.202 (0.046-0.892)	0.0348	1.065 (0.433-2.621)	0.8909
	mode of HIV transmission	other/unknown vs. heterosexual	0.935 (0.517-1.69)	0.8228	1.223 (0.673-2.225)	0.5091
	calendar year of HAART initiation (per more recent)	0.974 (0.805-1.178)	0.7859	1.101 (0.903-1.343)	0.3402
		abacavir+lamivudine vs. tenofovir+emtricitabine	0.255 (0.077-0.849)	0.0259	0.111 (0.015-0.824)	0.0316
		zidovudine+lamivudine vs. tenofovir+emtricitabine	0.895 (0.467-1.716)	0.7386	1.138 (0.585-2.212)	0.7041
	backbone	other backbone vs. tenofovir+emtricitabine	1.546 (0.505-4.737)	0.4457	2.015 (0.646-6.291)	0.2275
	HAART	tenofovir+lamivudine vs. tenofovir+emtricitabine	0.897 (0.397-2.029)	0.7948	1.199 (0.485-2.967)	0.6938
	baseline CD4+ cell count cells/mm3 (per log higher)	0.875 (0.732-1.047)	0.145	0.967 (0.795-1.176)	0.7382
	baseline HIV-RNA load cp/ml (per log10 higher)	1.127 (0.799-1.589)	0.4968	1.901 (1.312-2.754)	0.0007
	lopinavir GRS (per 10 points higher)	0.997 (0.958-1.039)	0.9007	0.999 (0.966-1.032)	0.9361
	backbone GRS (per 10 points higher)	1.001 (0.983-1.02)	0.8793	1.021 (1.005-1.037)	0.0101

F o r P e e r R e v i e w

Table 2 .

 2 Evolution of protease and reverse transcriptase resistance mutations under lopinavir/ritonavir plus 2 nucleoside/tide reverse transcriptase inhibitors pressure in 36 previously naive patients with available baseline and follow-up HIV-1 genotype. Substitutions emerging during treatment are in bold.Figure 1.Kaplan-Meier curves of survival rates with respect to a virological failure at or after 3 or 6 months using different HIV-RNA thresholds (>1000, >500 and >50 cp/ml), to a Stanford's lopinavir genotypic resistance score >14 (intermediate/full resistance), to an accumulation of at least one major IAS resistance mutation to lopinavir/ritonavir, and to a therapy discontinuation (at any time for any reason) end-point.Low Rate of Virological Failure and Maintenance of Susceptibility to HIV-Mattia CF Prosperi 1 , Maurizio Zazzi 2 , Grazia Punzi 3 , Laura Monno 4 , Grazia Colao 5 , Paola Corsi 6 , Simona Di Gismondo 10 , Patrizia Bagnarelli 11 , Tiziana Carli 12 , and Andrea De Luca 1,13 , for the ARCA Collaborative Group.

		Baseline
		HIV-RNA
	patient nrti a	load	Baseline genotype b	Follow-up genotype b
		Log10
		copies/ml

PR: 10F 20I 36I 46I 54V 60E 63P 73T 84V 90M 93L RT: 41L 67N 101E 108I 118I 181C 190A 210W 215Y 219R 221Y I/I F o r P e e r R e v i e w Figure 1. Kaplan-Meier curves of survival rates with respect to a virological failure at or after 3 or 6 months using different HIV-RNA thresholds (>1000, >500 and >50 cp/ml), to a Stanford's lopinavir genotypic resistance score >14 (intermediate/full resistance), to an accumulation of at least one major IAS resistance mutation to lopinavir/ritonavir, and to a therapy discontinuation (at any time for any reason) end-point. 370x244mm (96 x 96 DPI)

  The oldest HIV-1 80 genotype entry is dated back to 1991. At the time of this study, data from 19,984 patients were 81

	84 85	Patients and Methods
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John Wiley & SonsJournal of Medical Virology

Acknowledgements 224


Jiad N Mcheik

Isabelle Dichamp

Guillaume Levard

Stéphanie Ragot

Agnès Beby-Defaux

Céline Grosos

Véronique Couvrat

Gérard ; Agius
email: g.agius@chu-poitiers.fr

Infantile Hypertrophic Pyloric Stenosis: Are Viruses Involved? 5 6 Running head: Hypertrophic Pyloric Stenosis and Viruses 7 8 9 10

Keywords: cell culture, PCR, adenovirus, enterovirus cell culture, PCR, adenovirus, enterovirus 53

30 31 32 Infantile hypertrophic pyloric stenosis (IHPS) is characterized by abnormal thickening of the internal 33 circular muscle layer. IHPS is known to be due to a combination of genetic and environmental 34 factors, but its precise causes and pathophysiology are poorly understood. The objective of the 35 study is to determine the prevalence of the principal viruses targeting the respiratory and digestive 36 tracts in children with IHPS. Nasopharyngeal fluids, stools, vomit, and surgical pyloric muscle 37 fragments and swabs were tested by cell culture, viral antigen assay and PCR. IHPS was 38 diagnosed in 23 boys and 8 girls with a mean (± SD) age of 42 ± 15 days (range 20-88 days).

39

There was no seasonal pattern of diagnosis. Twenty-two children (71%) lost weight (mean 246 ± 40 164 g, range 30-600 g) after the onset of vomiting, and five (16.1%) were dehydrated. Seven

41

(22.6%) infants had been exposed to an infectious contact within 15 days before admission, and 42 one on the day of admission (3.2%). Ear, nose and throat samples and pyloric muscle specimens

43

were negative for all the viruses tested. An adenovirus type 3 was recovered from one stool 44 sample, and RT-PCR was positive for an enterovirus on one vomit sample. This study suggests 45 that the principal viruses targeting the respiratory and digestive tracts are not responsible for IHPS.

INTRODUCTION

58

Infantile hypertrophic pyloric stenosis (IHPS) is characterized by abnormal thickening of the 59 internal circular muscle layer [Jablonski et al., 2006]. The clinical signs are fairly typical, with 60 vomiting after feeding (sometimes projectile) and weight loss. The infants most frequently affected 61 are first male children about 6 weeks old [START_REF] Murtagh | Infantile hypertrophic pyloric stenosis[END_REF][START_REF] Hernanz-Schulman | Infantile hypertrophic pyloric stenosis[END_REF].

62

Abdominal ultrasonography confirms the diagnosis and reveals the pyloric olive [Hernanz-63 Schulman, 2003]. Extramucosal myotomy, after rehydration and correction of biological disorders,

64

gives excellent results with few if any complications.

65

IHPS is thought to result from a complex interplay of genetic and environmental factors 66 [START_REF] Velaoras | Hypertrophic pyloric stenosis in twins: same 277 genes or same environments?[END_REF]. Since the principal description [START_REF] Hirschprung | Falle Von Angeborener Pylorustenose, Beobachtet bei Sauglingen[END_REF] of primary muscle 67 hypertrophy, many possible causes have been forwarded (motor, endocrine, pharmacological, 68 constitutional and genetic), but none has been confirmed [Pantelli, 2009]. However, the possible 69 role of infections has been examined rarely [START_REF] Paulozzi | Is Helicobacter pylori a cause of infantile hypertrophic pyloric stenosis?[END_REF][START_REF] Jenson | Absence of Epstein-Barr virus in smooth muscle cells of 244 idiopathic hypertrophic pyloric stenosis[END_REF]Dahshan et al., 70 2006;Sherwood et al., 2007]. Yet the seasonal pattern of IHPS [START_REF] Webb | Infantile hypertrophic pyloric stenosis in South Glamorgan 281 1970-9. Effects of changes in feeding practice[END_REF]

79

The following data were recorded: age, sex, infectious status, drug treatment, family history,

112

Multiplex PCR was carried out as described elsewhere [START_REF] Stockton | Multiplex PCR for typing and 269 subtyping influenza and respiratory syncytial viruses[END_REF]]. This nested 113 PCR approach detects RSV A and B and circulating influenza viruses A(H1N1), A(H3N2) and B.

114

The two PCR rounds were done with primer pairs targeting the hemagglutinin (H) gene of influenza

148

Seventeen infants had plain abdominal radiography, which revealed abnormalities in eight 149 cases (47%), with two cases of gastric distension, five cases of gastric stasis, and one case of 150 small bowel air-fluid levels. Sonographic examination of the pyloric region always confirmed IHPS,

151

showing a mean pyloric muscle thickness of 4.9 ± 1.1 mm and a mean pyloric length of 20.4 ± 4.6 152 mm on longitudinal sections. Thickness and length of the pyloric muscle correlated weakly 153 (rho=0.54; p=0.003). Gestation was significantly shorter among infants with a pyloric thickness of 154 less than 5 mm on sonography than in those with a thickness of 5 mm or more (37.9 ± 2.1 versus 155 39.2 ± 1.7 weeks; p=0.03).

156

The main characteristics of the 31 patients and their mothers are shown in Table I.

157

The mean blood electrolyte levels were as follows: potassium 4.4 ± 0.8 mmol/l [2.5 -5.5];

159

The parents were interviewed and asked about recent exposure to infectious contacts (Table The precise frequency of IHPS is poorly documented [START_REF] Wang | Prevalence of infantile hypertrophic 279 pyloric stenosis in Texas, 1999-2002[END_REF]. In Europe, an overall 167 incidence of 2.0 per 1000 live births has been reported, with a range of 0.86 to 3.96 per 1000 168 [START_REF] Pedersen | Infantile 259 hypertrophic pyloric stenosis: a comparative study of incidence and other epidemiological 260 characteristics in seven European regions[END_REF].

169

The clinical and epidemiological characteristics of the patients were in keeping with those 170 reported in the literature [START_REF] Murtagh | Infantile hypertrophic pyloric stenosis[END_REF][START_REF] Hernanz-Schulman | Infantile hypertrophic pyloric stenosis[END_REF][START_REF] Wang | Prevalence of infantile hypertrophic 279 pyloric stenosis in Texas, 1999-2002[END_REF], 171 including some cases with a positive family history. A positive maternal history has been linked to a 172 higher risk of IHPS in infants [START_REF] Panteli | New insights into the pathogenesis of infantile pyloric stenosis[END_REF][START_REF] Carter | Inheritance of congenital pyloric stenosis[END_REF]. The only noteworthy 173 difference with other series is the lack of a seasonal pattern of IHPS in the patients in the present 174 study [START_REF] Webb | Infantile hypertrophic pyloric stenosis in South Glamorgan 281 1970-9. Effects of changes in feeding practice[END_REF]. Sonographic examination showed a larger muscle thickness and pyloric 175 F o r P e e r R e v i e w [START_REF] Panteli | New insights into the pathogenesis of infantile pyloric stenosis[END_REF]. The main potential contributing factors are maternal treatment in early pregnancy 179 with an anti-emetic [START_REF] Aselton | Pyloric stenosis and 216 maternal Bendectin exposure[END_REF] or macrolide [Sørensen et al., 2003], pyloric muscle 180 defects, such as smooth muscle cells [START_REF] Guarino | Structural immaturity of the pylorus muscle in infantile 228 hypertrophic pyloric stenosis[END_REF], increased growth factor expression 181 [Jablonski et al., 2006], abnormal distribution and organization of extracellular matrix proteins [Oue 182 and Puri, 1999], defective pyloric innervation [START_REF] Kobayashi | Pyloric stenosis: 246 new histopathologic perspective using confocal laser scanning[END_REF], absence or rarity of 183 interstitial cells of Cajal [START_REF] Vanderwinden | Study of the interstitial cells of 275 Cajal in infantile hypertrophic pyloric stenosis[END_REF], and low nitric oxide synthase expression in 184 fibers innervating the pyloric circular muscle, leading to decreased plasma nitrile levels [Huang et 185 al., 2006]. However, to our knowledge, the only microbial pathogens to have been sought in this 186 setting are Helicobacter pylori [START_REF] Dahshan | Helicobacter pylori and 222 infantile hypertrophic pyloric stenosis: is there a possible relationship[END_REF]Sherwood et al., 2007] and Epstein-Barr virus 187 [START_REF] Jenson | Absence of Epstein-Barr virus in smooth muscle cells of 244 idiopathic hypertrophic pyloric stenosis[END_REF]. H. pylori antigens were not detected by an enzyme immunoassay technique 188 in series of 39 stool specimens from children with IHPS [Sherwood et al., 2007]. A recent study of 189 gastric biopsies from 16 patients showed three cases of chronic active gastritis and six cases of 190 mild focal gastritis [START_REF] Dahshan | Helicobacter pylori and 222 infantile hypertrophic pyloric stenosis: is there a possible relationship[END_REF]. However, histopathological examination and not by itself suggest a role in IHPS, as ADV can be excreted for very long periods in the stools of 203 newborns with no other signs of infection [START_REF] Madeley | Viruses in the stools[END_REF]. This finding also appears to be previously healthy children [START_REF] Munoz | Disseminated adenovirus disease in 249 immunocompromised and immunocompetent children[END_REF]]. Thus, the negative results of all virological 206 investigations of pyloric samples from the patients exclude the involvement of the viruses 207 examined.

208

This study suggests that the principal viruses targeting the respiratory and digestive tracts are

  and its frequent 71 association with a viral syndrome raises the possibility of a viral etiology. The prevalence of the 72 principal viruses targeting the respiratory and digestive tracts was examined in children with IHPS. took place between May, 2007 and December, 2008 in the pediatric 77 surgery department of Poitiers University hospital in France. All children admitted consecutively for 78 IHPS confirmed sonographically were included.

805

  clinical data (date of onset of vomiting, weight changes, presence of peristaltic waves, palpability of 81 the pyloric olive, and signs of dehydration), plain abdominal radiography and pyloric sonography, 82 and blood electrolyte levels. Peristaltic waves of gastric contraction indicate hypertrophy of the 83 gastric muscle secondary to slowly progressive obstruction: their observation makes pyloric 84 stenosis likely. Information on the pregnancy and delivery, gestational age, breast-feeding and 85 possible neonatal disorders was also collected. Informed consent was granted by the parents of 86 Nasopharyngeal aspirates were tested for the principal viruses targeting the respiratory tract, 88 including influenza type A and B viruses circulating at the time of the study, respiratory syncytial 89 virus (RSV) type A and B, metapneumovirus (MPV), Paramyxovirus parainfluenza (PIV) type 1, 2 90 and 3, rhinovirus (RHV), coronaviruses OC43 and 229E, and adenovirus (ADV). Stools or rectal 91 swabs, vomit samples, intra-operative pyloric swabs, and biopsy specimens of the pyloric muscle 92 fragment were tested for viruses targeting the digestive tract, including ADV, enterovirus (EV) and 93 rotavirus (RV).

94

  This study was approved by the Institutional Ethics Committee of Poitiers University Hospital,95and informed consent of the parents of the infants was obtained in all cases. 96 97 Direct Immunofluorescence and Cell Culture 98 Direct immunofluorescence was performed with fluorescein-labeled monoclonal antibodies 99 against RSV A+B and influenza A and B (Monofluo Screen RSV, Monofluo Kit Influenza; Biorad). 100 Nasopharyngeal aspirates and rectal, vomit and stool samples were inoculated on MRC-5 101 human diploid cells, LLCMK-2 heteroploid simian cells, HeLa 229 cells, and MDCK heteroploid 102 canine cells. Cell cultures were maintained for at least three weeks, and were passaged blindly samples were tested for RV and ADV antigens with an immunochromatograpic 107 method (SD Rota/Adeno BioLine; Standard diagnostics), as recommended by the manufacturer. 108 Colored lines appearing after sample migration indicate positivity. 109 110 Multiplex PCR for Influenza A and B, and RSV A and B; PCR for ADV, MPV, PIV type 1, 2 and 111 3, RHV, and Coronaviruses OC43 and 229E

6

  115viruses A(H1), A(H3) and B, and a sequence overlapping the RSV N (capsid) and P116(phosphoprotein) genes.The other PCR assays were carried out as reported previously, using the following targets: a 118 161-bp region of the ADV-2 hexon gene sharing a high degree of nucleotide homology with 47 119 human ADV serotypes[Hierholzer et al., 1993]; the MPV matrix gene, with an amplicon size of 416 in 5 cases (16.1%). Symptoms lasted an average of 19.0 ± 9.4 days in 147 dehydrated children and 11.0 ± 9.4 days in the other children (p=0.04).

  158 chloride 96.1 ± 10.0 mmol/l [66 -108]; and bicarbonate 28.8 ± 7.8 mmol/l [16 -49].

  160

  II). A history of antibiotic therapy was only mentioned in one case. All virologic tests of ENT 161 samples and pyloric specimens (pyloric muscle fragments and intra-operative swabs) were 162 negative. The only positive samples were a stool specimen (ADV 3 detected by cell culture) and a 163 vomit sample (EV detected by RT-PCR) (TableII).

191

  immunohistochemical staining revealed no bacteria, and the urease test was negative in all 192 samples.[START_REF] Jenson | Absence of Epstein-Barr virus in smooth muscle cells of 244 idiopathic hypertrophic pyloric stenosis[END_REF] failed to detect EBV in smooth muscle cells from 10 patients with 193 IHPS diagnosed clinically and histopathologically, by means of in situ hybridization of EBV-194 encoded RNA1 (EBER1). 195 The results described above argue against the involvement of a virus in IHPS. The case in 196 which an enterovirus was detected by RT-PCR in a vomit sample which was negative by cell 197 culture could be explained either by a small inoculum or by a non-culturable enterovirus. After oral 198 infection, enteroviruses multiply with the oropharyngeal and intestinal mucosae, leading to 199 abundant orofecal excretion of virions. Our case of PCR positivity was therefore probably 200 fortuitous, especially as asymptomatic enterovirus infection is frequent in newborns [Tebruegge 201 and Curtis, 2009]. Likewise, detection of an ADV 3 strain in the stools of one of the patients does

  202

  204incidental, although strains of serotype 3 can cause frequently fatal disseminated disease in 205

209

  not responsible for IHPS in infants. However, larger studies including other viruses are needed to 210 rule out definitively viral involvement in IHPS.

[START_REF] Hernanz-Schulman | Infantile hypertrophic pyloric stenosis[END_REF]].

The risk of IHPS has been linked to a variety of factors, with sometimes contradictory results

adenovirus in clinical specimens by polymerase chain reaction and liquid-phase hybridization

quantitated by time-resolved fluorometry. J Clin Microbiol 31:1886-1891.

cause ? Pediatr Surg Int 23:61-63.

Sørensen HT, Skriver MV,Pedersen L, Larsen H, Ebbesen F, Schønheyder HC. 2003. Risk of

F o r P e e r R e v i e w


Eugeni Lopez-Bonet

Maria Carmen Pérez-Martínez

Begoña Martin-Castillo

Miguel Alonso-Ruano

Francesc Tuca

Cristina Oliveras-Ferraros

Sílvia Cufí

Alejandro Vazquez-Martin

Miquel Beltràn

Luis Bernadó

Ph.D Javier A Menendez
email: jmenendez@iconcologia.net

Diagnostic utility of mammaglobin and GCDFP-15 in the identification of primary neuroendocrine carcinomas of the breast

Keywords: Neuroendocrine tumors, Mammaglobin, GCDFP-15, breast cancer

In addition to the presence of morphological neuroendocrine (NE) features, expression of the NE markers chromogranin A and synaptophysin has been considered the most sensitive and specific immunohistochemical feature in the diagnosis of pure, solid NE breast carcinomas [NEBC ; 1]. Following these criteria, the existence of NEBC can be accurately confirmed in <2% of all primary breast cancers when using adequate cytological specimens including fine needle aspiration smears [START_REF] Who | World Health Organization classification of tumors. Pathology and genetics of tumours of the breast and female genital organs[END_REF][START_REF] Günhan-Bilgen | Neuroendocrine differentiated breast carcinoma: imaging features correlated with clinical and histopathological findings[END_REF][START_REF] Kawasaki | Neuroendocrine ductal carcinoma in situ of the breast: cytological features in 32 cases[END_REF][START_REF] Righi | Neuroendocrine differentiation in breast cancer: established facts and unresolved problems[END_REF]. Within a working model for a breast cancer molecular taxonomy [START_REF] Perou | Molecular portraits of human breast tumours[END_REF], recent gene expression profiling studies have confirmed a distinct nature of NEBC from conventional invasive ductal carcinomas as well as their belonging to the goodprognosis (being positive for hormone receptors and HER2-negative) subgroup of luminal A breast tumors [START_REF] Weigelt | Mucinous and neuroendocrine breast carcinomas are transcriptionally distinct from invasive ductal carcinomas of no special type[END_REF]. NEBC may occur, however, as a first presentation of an occult metastatic non-mammary neuroendocrine tumor or as a bona fide primary breast tumor. Therefore, the clinical effect of the occurrence of neuroendocrine differentiation in breast cancer should be carefully examined to reduce or avoid the risk of a misdiagnosis or under-diagnosis. In this regard, Richter-Ehrenstein et al.

have recently evaluated a new diagnostic tool to distinguish between primary NEBC from a metastatic disease of other sites [START_REF] Richter-Ehrenstein | Solid neuroendocrine carcinomas of the breast: metastases or primary tumors?[END_REF]. Using the immunohistochemical markers for epithelial of breast origin mammaglobin and Gross Cystic Disease Fluid Protein (GCDFP-15), they compared a subset of pure NEBC (n=9) with a cohort of nonmammary neuroendocrine tumors. In their hands, mammaglobin was positive in 4 out of 9 tumors while GCDFP-15 was expressed in 6 out of 9 cases. In three patients, both markers were negative. Remarkably, they did not find any expression of mammaglobin or GCFFP-15 in immunohistochemical evaluation of chromogranin A/synaptophysin-positive non-mammary neuroendocrine tumors [START_REF] Richter-Ehrenstein | Solid neuroendocrine carcinomas of the breast: metastases or primary tumors?[END_REF].

We have recently determined the incidence, clinico-pathological features and immunohistochemical profiling of primary pure NEBC in a large series of infiltrating breast tumors collected from 1989 to 2010 in our institution (Dr. Josep Trueta University Hospital, Girona, Catalonia). From the entire series of more than 1500 carcinomas, only 8 cases fully satisfied the primary pure NEBC criteria by the WHO (i.e. the presence of > 50% tumor immunoreactivity for one of neuroendocrine markers including chromogranin, synaptophysin and CD56), which constitutes an incidence of solid NEBC as low as 0.5% [data updated from ref. 8]. To definitely confirm the primary nature of these endocrine tumors, we recently tested the hypothesis that mammaglobin stating can help, in combination with GCDFP-15 (a sensitive and specific marker of breast carcinoma), to establish the correct diagnosis of breast carcinomas [START_REF] Bhargava | Mammaglobin vs GCDFP-15: an immunohistologic validation survey for sensitivity and specificity[END_REF]. Table 1 summarizes the immunohistochemical profile for the expression of estrogen receptor (ER), progesterone receptor (PR), HER2, mammaglobin, and GCDFP-15 in tumor specimens from NEBC patients. Our findings largely recapitulate those presented by Richter-Ehrenstein et al [START_REF] Richter-Ehrenstein | Solid neuroendocrine carcinomas of the breast: metastases or primary tumors?[END_REF]. All NEBC expressed high levels of estrogen and progesterone receptors. Mammaglobin was positive in 4 out of 9 samples. In contrast, GCDFP-15 was expressed in 7 out of 9 specimens. We did not observe discordant expression of mammaglobin and GCDFP-15 markers between primary and metastatic tissues in a patient that debuted with metastasis in the soft tissue of the check prior to diagnosis of primary NEBC. It was intriguing that HER2 2+ staining (but absence of gene amplification) was found in the sole NEBC that was negative for both markers. Richter-Ehrenstein et al similarly reported that HER2 overexpression was found in only one NEBC, which was negative for both markers [START_REF] Richter-Ehrenstein | Solid neuroendocrine carcinomas of the breast: metastases or primary tumors?[END_REF]. Although the very small number of cases obviously limits the relevance of the correlation between HER2 positivity and mammaglobin/GCDFP-15 double-negative NEBC patients, additional studies with longer follow-up and more cases need to be further pursued to establish whether this "combimarker" (i.e. detecting HER2 overexpression in the absence of mammaglobin/GCDFP-15 markers) may delineate a distinctive subgroup of luminal B-like (i.e. ER-positive or PR-positive and HER2-positive [START_REF] Carey | Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study[END_REF]) among the well-established ER/PR-positive HER2-negative status of NEBC. Although GCDFP-15 has classically been considered a secretary marker of apocrine differentiation in breast cancer [START_REF] Caputo | A novel aspartyl proteinase from apocrine epithelia and breast tumors[END_REF][START_REF] Sapino | Expression of apocrine differentiation markers in neuroendocrine breast carcinomas of aged women[END_REF], the fact that the GCDFP-15 positivity is transient (i.e. breast cancer cases which have apocrine features but lack GCDFP-15 expression suggest advanced apocrine carcinomas [START_REF] Honma | Expression of GCDFP-15 and AR decreases in larger or node-positive apocrine carcinomas of the breast[END_REF]) together with the pre-clinical finding that the prolactin-inducible protein PIP/GCDFP-15 gene was the mostly up-regulated mRNA in luminal B (ER-positive/HER2-positive) BT-474 breast cancer cells treated with the insulin-like growth factor-I receptor (IGF-IR) antagonist NVP-AEW541 and the anti-HER2 monoclonal antibody trastuzumab [START_REF] Honma | Expression of GCDFP-15 and AR decreases in larger or node-positive apocrine carcinomas of the breast[END_REF],

reinforce the suggestion that yet to be established regulatory relationship between GCDFP-15, HER2, and apocrine differentiation would take place in subsets of NEBC [START_REF] Esparís-Ogando | Synergic antitumoral effect of an IGF-IR inhibitor and trastuzumab on HER2-overexpressing breast cancer cells[END_REF][START_REF] Bhargava | Breast cancer molecular class ERBB2: preponderance of tumors with apocrine differentiation and expression of basal phenotype markers CK5, CK5/6, and EGFR[END_REF]. In summary, our findings strongly support Richter-Ehrenstein's conclusion that mammaglobin and GCDFP-15 as markers of epithelial breast origin may efficiently work as a new reliable diagnostic tool to distinguish endocrine tumors of the breast from a metastatic non-mammary neuroendocrine disease. Given that the diagnosis of NEBC cases has been made mostly following surgical intervention including mastectomy and that most studies have confirmed that NEBC show a less aggressive clinical behavior when compared with unselected breast cancers, these findings strongly suggest that when suspecting neuroendocrine differentiation, preoperative pathological diagnosis by core biopsies can be employed to immunohistochemically establish the primary nature of mammary NEBC to guide in their conservative medical and surgical management. Further research effort using large clinically annotated series of breast tumors.is warranted to definitely establish whether NEBC defined by two-(chromogranin A and synaptophysin) or five (chromogranin A, synaptophysin, HER2, mammaglobin and GCDFP-15)-biomarker surrogate panels might explain a recent suggestion of invasive NEBC as a distinctive subtype of biologically aggressive breast cancer [START_REF] Wei | Invasive neuroendocrine carcinoma of the breast: a distinctive subtype of aggressive mammary carcinoma[END_REF]

FIGURE LEGEND

Figure 1 .

 1 Figure 1. Immunohistochemical staining of different NEBC tissues for mammaglobin and GCDFP-15. Representative slides of primary (and metastatic) lesions of solid NEBC including surgical specimens and trucut core biopsies were analyzed for mammaglobin (FLEX Monoclonal Mouse Anti-Human Mammaglobin Clone 304-1A5, Ready-to-Use [Link]. DAKO. Code IR074) .and GCDFP-15 FLEX Monoclonal Mouse Anti-Human Gross Cystic Disease Fluid Protein-15 Clone 23A3 Ready-to-Use [Link]. DAKO. Code IR077) using standardized immunohistochemical techniques and robotic autostainers (Dako Autostainer Plus system; DAKO, DakoCytomation, Inc. Glostrup, Denmark). Binding results were visualized with the Envision ® System (DakoCytomation, Inc.), which uses horseradish peroxidase-labelled polymer that is conjugated with secondary antibodies. 3,3'-diaminobenzidine tetrahydrochloride (DAB) was used for the visualization of the antibody/enzyme complex. Slides were counterstained for nuclei with Meyer's hematoxylin and examined by light microscopy. Tumor immunoreactivity (negative, weak positive, moderate/strong positive) was scored in combination with the percent of cells showing positive staining. Figure shows typical examples of negative (Neg) or positive (Pos) strong staining of some tumor cells for mammaglobin (A, B, C) and moderate to strong cytoplasmic expression for GCDFP-15 (D, E, F)

Table 1 .

 1 Immunohistochemical expression profiles of primary pure neuroendocrine breast carcinomas This woman debuted with metastasis in the soft tissue of the check (6M) b Extensive staging revealed a primary neuroendocrine breast carcinoma (6P) c HER2 immunohistochemistry was performed using a Dako HercepTest (Dako) kit according to the designated procedure

	Patient	Estrogen	Progesterone	HER2	Mammaglobin GCDFP-
	Number	Receptor	Receptor			15
	1	Positive	Positive	-	Positive	Positive
	2	Positive	Positive	-	Negative	Positive
	3	Positive	Positive	-	Positive	Positive
	4	Positive	Positive	-	Negative	Positive
	5	Positive	Positive	-	Positive	Positive
	6M a	Positive	Positive	-	Negative	Positive
	6P b	Positive	Positive	-	Negative	Positive
	7	Positive	Positive	2+	Negative	Negative
				(HercepTest ) c
	8	Positive	Positive	-	Positive	Negative

a

Acknowledgements: This work was supported in part by the Xarxa de Banc de Tumors de Catalunya (XBTC) sponsored by the Pla Director d'Oncologia de Catalunya. Alejandro Vazquez-Martin is the recipient of a "Sara Borrell" post-doctoral contract (CD08/00283, Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria -FIS-, Spain). Work at Menendez' laboratory is supported in part by the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria -FIS-, Spain, Grants CP05-00090 and PI06-0778 and RD06-0020-0028), the Fundación Científica de la Asociación Española Contra el Cáncer (AECC, Spain), and by the Ministerio de Ciencia e Innovación (SAF2009-11579, Plan Nacional de I+D+ I, MICINN, Spain).