Katy Meunier

Marianne Ferron

Claire Calmel

Jean-François Fléjou

Marc Pocard
email: marc.pocard@gmail.com

Françoise Praz
email: francoise.praz@upmc.fr

Impact of MLH1 expression on tumour evolution after curative surgical tumour resection in a murine orthotopic xenograft model for human MSI colon cancer

Colorectal cancers (CRCs) displaying microsatellite instability (MSI) most often result from MLH1 deficiency. The aim of this study was to assess the impact of MLH1 expression per se on tumour evolution after curative surgical resection using a xenograft tumour model.

Transplantable tumours established with the human MLH1-deficient HCT116 cell line and its MLH1-complemented isogenic clone, mlh1-3, were implanted onto the caecum of NOD/SCID mice. Curative surgical resection was performed at day 10 in half of the animals. The HCT116-derived tumours were more voluminous compared to the mlh1-3 ones (P = .001).

Lymph node metastases and peritoneal carcinomatosis occurred significantly more often in the group of mice grafted with HCT116 (P = .007 and P = .035, respectively). Mlh1-3-grafted mice did not develop peritoneal carcinomatosis or liver metastasis. After surgical resection, lymph node metastases only arose in the group of mice implanted with HCT116 and the rate of cure was significantly lower than in the mlh1-3 group (P = .047). The murine orthotopic xenograft model based on isogenic human CRC cell lines allowed us to reveal the impact of MLH1 expression on tumour evolution in mice who underwent curative surgical resection and in mice whose tumour was left in situ. Our data indicate that the behaviour of MLH1-deficient CRC is not only governed by mutations arising in genes harbouring microsatellite repeated sequences, but also from their defect in MLH1 per se.

INTRODUCTION

Colorectal carcinoma (CRC) is the third most common type of cancer and the second leading cause of cancer-related death. [START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF] Approximately one-fourth of patients can be cured by surgery alone, whereas the remaining need to be treated with chemotherapy due to metastasis at the time of diagnosis, development of metastasis or recurrence of disease. The development of CRC involves at least three partially overlapping oncogenic pathways displaying characteristic genetic or epigenetic alterations: microsatellite instability (MSI), CpG island methylator phenotype, and chromosome instability. [START_REF] Pino | The chromosomal instability pathway in colon cancer[END_REF][START_REF] Zaanan | Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications[END_REF][START_REF] Hughes | The CpG island methylator phenotype in colorectal cancer: progress and problems[END_REF] MSI CRCs represent about 15% of all CRC and arise due to mismatch repair (MMR) deficiency, either as a result of inherited mutations in one of the MMR genes, most often MSH2 or MLH1, or, more often, through somatic epigenetic methylation of the MLH1 promoter. [START_REF] Zaanan | Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications[END_REF][START_REF] Kane | Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repairdefective human tumor cell lines[END_REF][START_REF] Boland | Microsatellite instability in colorectal cancer[END_REF] Patients with MSI CRC have a better stage-adjusted prognosis than those with CRC displaying chromosome instability, [START_REF] Walther | Genetic prognostic and predictive markers in colorectal cancer[END_REF][START_REF] Roth | Integrated analysis of molecular and clinical prognostic factors in stage II/III colon cancer[END_REF] and there is increasing evidence showing that MSI influences the response to chemotherapy, [START_REF] Zaanan | Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications[END_REF][START_REF] Fallik | Microsatellite instability is a predictive factor of the tumor response to irinotecan in patients with advanced colorectal cancer[END_REF][START_REF] Sinicrope | Clinical implications of microsatellite instability in sporadic colon cancers[END_REF][START_REF] Hewish | Mismatch repair deficient colorectal cancer in the era of personalized treatment[END_REF][START_REF] Vilar | Microsatellite instability in colorectal cancer-the stable evidence[END_REF][START_REF] Zaanan | Impact of p53 expression and microsatellite instability on stage III colon cancer disease-free survival in patients treated by 5-fluorouracil and leucovorin with or without oxaliplatin[END_REF][START_REF] Pritchard | Colorectal cancer molecular biology moves into clinical practice[END_REF][START_REF] Zaanan | Defective mismatch repair status as a prognostic biomarker of disease-free survival in stage III colon cancer patients treated with adjuvant FOLFOX chemotherapy[END_REF] to ionizing radiation, [START_REF] Robinson | Enhanced radiosensitization with gemcitabine in mismatch repair-deficient HCT116 cells[END_REF] and to immunotherapy. [START_REF] Le | PD-1 blockade in tumors with mismatch-repair deficiency[END_REF][START_REF] Dunne | Immune-derived PD-L1 gene expression defines a subgroup of stage II/III colorectal cancer patients with favorable prognosis who may be harmed by adjuvant chemotherapy[END_REF] It is widely accepted that MMR deficiency is not a transforming event per se, but that it promotes oncogenesis through accumulation of mutations arising during replication as they are left uncorrected due to MMR deficiency. [START_REF] Calabrese | Colorectal pretumor progression before and after loss of DNA mismatch repair[END_REF][START_REF] Shibata | When does MMR loss occur during HNPCC progression?[END_REF] Simple sequence repeats, also known as microsatellites, are particularly prone to insertion and deletion mutations resulting from polymerase slippage. Several genes involved in the control of cell proliferation and differentiation, and DNA damage signalling and repair carry a mononucleotide repeat, [START_REF] Loire | Hypermutability of genes in Homo sapiens due to the hosting of long mono-SSR[END_REF][START_REF] Shah | Defective mismatch repair, microsatellite mutation bias, and variability in clinical cancer phenotypes[END_REF] which is mutated in MSI CRC at a frequency higher than expected from their length, suggesting that their inactivation may participate in tumour initiation and/or progression. [START_REF] Miquel | Frequent alteration of DNA damage signalling and repair pathways in human colorectal cancers with microsatellite instability[END_REF][START_REF] Woerner | SelTarbase, a database of human mononucleotide-microsatellite mutations and their potential impact to tumorigenesis and immunology[END_REF] In addition to the numerous MSI-driven mutations inactivating target genes that may be considered as putative novel oncogenes, [START_REF] Gylfe | Identification of candidate oncogenes in human colorectal cancers with microsatellite instability[END_REF] MLH1 deficiency itself may further contribute to tumour progression owing to its role in recombination [START_REF] Evans | Roles for mismatch repair factors in regulating genetic recombination[END_REF][START_REF] Harfe | DNA mismatch repair and genetic instability[END_REF] and apoptosis. [START_REF] Hawn | Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint[END_REF][START_REF] D'atri | Involvement of the mismatch repair system in temozolomide-induced apoptosis[END_REF][START_REF] Davis | Defective expression of the DNA mismatch repair protein, MLH1, alters G2-M cell cycle checkpoint arrest following ionizing radiation[END_REF][START_REF] Hickman | Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents[END_REF] Metastasis is a multistep process that requires the ability of tumour cells to invade the underlying tissues, survive in the circulation and colonize various organs, mostly lymph nodes, liver and lung in the case of CRC. Most of the mutations found in CRC metastases are present in the paired primary lesion, suggesting that dissemination is an early event during cancer development. [START_REF] Jones | Comparative lesion sequencing provides insights into tumor evolution[END_REF] Hence, the potential for metastatic development of CRC possibly relies on intrinsic properties of the primary tumour. [START_REF] Jones | Comparative lesion sequencing provides insights into tumor evolution[END_REF] Orthotopic CRC xenograft mouse models have been established by transplanting human colon cancers onto the caecum of deficient mice. [START_REF] Bresalier | A new animal model for human colon cancer metastasis[END_REF][START_REF] Fidler | Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis[END_REF][START_REF] Kuo | Early resection of primary orthotopicallygrowing human colon tumor in nude mouse prevents liver metastasis: further evidence for patient-like hematogenous metastatic route[END_REF][START_REF] Pocard | Efficiency of orthotopic xenograft models for human colon cancers[END_REF] Orthotopic xenografts are clinically more relevant than subcutaneous tumours as they may give rise to local or distant metastasis formation, in lymph nodes, in the peritoneum (carcinomatosis), or in the liver. 34,36- 41 Furthermore, orthotopic xenografts are valuable models to evaluate tumour progression after curative surgical tumour resection since they represent the possible fate of tumours arising in humans, i.e. cure (when there is no evidence of disease), local recurrence or distant metastases. [START_REF] Pocard | Single alteration of p53 or E-cadherin genes can alter the surgical resection benefit in an experimental model of colon cancer[END_REF][START_REF] Flatmark | Twelve colorectal cancer cell lines exhibit highly variable growth and metastatic capacities in an orthotopic model in nude mice[END_REF] As an example, we previously reported that specific alterations of Ecadherin expression or TP53 in LoVo, an MSI colon cancer cell line, dramatically changed the rate of cure and metastatic behaviour after surgical resection of the tumour. [START_REF] Pocard | Single alteration of p53 or E-cadherin genes can alter the surgical resection benefit in an experimental model of colon cancer[END_REF] The purpose of the present study was to investigate whether MLH1 expression per se had an impact on the evolution of the tumour after curative surgical resection or when left in situ, using a murine orthotopic xenograft model based on the MLH1-deficient HCT116 cell line and an isogenic MLH1-complemented clone.

MATERIALS AND METHODS

Cell lines

Experiments were performed on the MLH1-deficient colorectal cancer cell line HCT116, kindly provided by Peter Karran in june 1995 (Imperial Cancer Research Fund, Clare Hall, United Kingdom), and its MLH1-complemented counterpart cell line mlh1-3, obtained by transfecting the HCT116 cell line with an expression vector containing the wild-type MLH1 cDNA, as described. [START_REF] Jacob | The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorectal cancer cells[END_REF] Cells were regularly authenticated using the short tandem repeat (STR) panel recommended by the International Cell Line Authentication Committee, as described in detail below. In particular, cells were typed at the beginning and at the end of the experimental procedures; the genotypes of the parental HCT116 and its MLH1complemented counterpart cell line are shown in Supporting Information Table 1. Compared with the HCT116 genotype published by the American Type Cell Culture (ATCC), there are 2 minor differences, consisting of the addition of 1 bp at the longer vWA allele (22.1 instead of 22) and the partial (in HCT116) or complete (in mlh1-3) loss of the chromosome Y-specific amelogenin allele. As discussed in the Results section, such differences do not challenge the identity of the HCT116 cells used in this study, but rather underline that novel indel mutations are highly susceptible to arise during replication of the mismatch repair-deficient HCT116 cells. Cells were grown in monolayer cultures in DMEM (Dulbecco's Modified Eagle's Medium, Sigma, France), supplemented with 10% of heat-inactivated foetal bovine serum, Lglutamine (2 mM), penicillin (100 g/mL), and streptomycin (100 UI/mL), in a humidified incubator at 5% CO2. The mlh1-3 cell line was maintained under selective pressure by adding 100 µg/mL hygromycin, until injection into mice.

Genomic DNA extraction and STR fingerprinting

The DNeasy tissue kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from cell pellets, according to the manufacturer's instructions. STR genotypes were established using eight highly polymorphic tetranucleotide STR markers (D5S818, D7S820, D13S317, D16S539, CSF1PO, THO1, TPOX, and vWA) and the Amelogenin marker that discriminates the X from the Y chromosome due to a 6-bp insertion. PCR were carried out using 0.2 mM dNTP, 0.15 mM primers (0.2 mM for D7S820), 0.5U KAPA2G Robust HotStartTaq polymerase (Kapa Biosystems, Wilmington, MA), and sense primers labeled with 6-FAM or HEX. After a 3-min step at 95°C, 35 cycles of 15 sec at 95°C, 15 sec at 55°C and 15 sec at 72°C, followed by a 1 min final extension at 72°C were performed. PCR products were diluted in formamide containing ROX-labelled 400 HD size markers (PE Applied Biosystems, Foster City, CA) and electrophoresed in 36-cm capillaries containing POP-7 on an ABI PRISM® 3130 Genetic Analyzer (PE Applied Biosystems). The apparent sizes of the alleles were determined using the GeneMapper Analysis software (PE Applied Biosystems).

MSI analysis

MSI was investigated using four highly polymorphic CAn dinucleotide microsatellite markers located on three different chromosome arms (5q, 17p, and 18q). PCR was carried out in 20 μL using 0.2 mM dNTP, 0.5 U HotStarTaq polymerase (Qiagen), and sense primers labelled with 6-FAM (D5S107), HEX (D18S474) or NED (D17S1791, D18S1127). The primer sequences are from the Ensembl Genome System website. After a 15-min step at 95°C, 35 cycles of 30 sec at 94°C, 30 sec at 55°C and 30 sec at 72°C, followed by a 6 min final extension at 72°C, were performed. PCR products were analysed as described above for STR fingerprinting.

Detection of frameshift mutations

The regions encompassing the repeated mononucleotide sequences contained in 26 selected genes (Table 1) were amplified either in monoplex or in multiplex PCR, as previously described. [START_REF] Miquel | Frequent alteration of DNA damage signalling and repair pathways in human colorectal cancers with microsatellite instability[END_REF] Oligonucleotide sequences and protocol details are available upon request. The electrophoresis profiles of the PCR products obtained with mlh1-3 were compared to the HCT116 reference profiles.

MLH1 protein expression

MLH1 protein expression in HCT116 and mlh1-3 cell lines was detected by Western blot using a rabbit polyclonal antibody raised against MLH1 (PC56, 1 µg/mL, Oncogene Research Products, Cambridge, MA) followed by subsequent incubation with sheep antirabbit antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech, Uppsala, Sweden). Analysis of tumour tissues obtained from mice was performed by immunohistochemistry on 4-µm slides incubated with an MLH1-specific mouse monoclonal antibody (dilution 1:400, clone G168-728, BD Biosciences, East Rutherford, NJ) after antigen retrieval with buffer, pH 6.0.

Animal experiments

Locally bred female NOD/SCID mice of 6-8 weeks of age were used for implantation. The animals were maintained under specific pathogen-free conditions, and food and water were supplied ad libitum. Housing and all procedures involving animals were conducted in accordance with the European Communities Council Directive (2010/63/UE) for the care and use of animals for experimental procedures and complied with the regulations of the French Ethics Committee in Animal Experiment "Charles Darwin" registered at the "Comité National de Réflexion Ethique sur l'Expérimentation Animale" (Ile-de-France, Paris, no5). Pr Marc Pocard supervised all experiments (agreement no. 75-1229 approved by the "Direction Départementale de la Protection des Populations", Paris, France). All efforts have been made to protect mice from pain, suffering, injury and disease.

Tumour models

Subcutaneous tumours were established by injecting 5 x 10 6 cells into the flank of 5 mice per cell line. When subcutaneous tumours were exponentially growing, mice were sacrificed to remove the tumours, which were then cut into fragments. The tumour fragments were subsequently implanted subcutaneously into the lateral flank of 5 other mice under inhaled anaesthesia and the incision was closed with a metal wound clip. When tumours reached a sufficient volume, they were resected and cut in 2 x 2 x 2 mm fragments for further intracaecal grafts.

Transplantation procedure

For each cell line, 30 mice were anaesthetized by intraperitoneal injection of 0.2 mL solution of ketamine (100 mg/kg body weight, Ketalar, Parapharm, Chinon, France) and xylazine (40 mg/kg body weight, Rompun, Bayer, La Garenne-Colombes, France). The abdomen was prepared for sterile surgery, as previously described. [START_REF] Pocard | Efficiency of orthotopic xenograft models for human colon cancers[END_REF] A median incision was performed to exteriorize the colon. A 2 x 2 x 2 mm fragment of solid tumour was then deposited at the surface of the serous membrane of the caecum, fixed with a thread (vicryl 7/0, Ethicon, France) and covered with 0.5 mL of biological glue (Beriplast, Aventis, Gentilly, France). The caecum was finally replaced in the abdominal cavity and the abdominal wall was closed in two layers: the aponevrose with thread (prolene 5/0, Ethicon, Issy-les-Moulineaux, France) and the skin with a metal wound clip. The group of 30 mice transplanted with either HCT116 or mlh1-3 cells were randomly divided into 2 groups of 15 mice: one in which the tumours were left in situ to evaluate the natural tumour history and one that underwent surgical resection; mice were caught randomly and were assigned alternately to each group.

Surgical resection

Surgical resection was performed 10 days after orthotopic transplantation. Mice were anesthetized, abdomen was prepared for sterile surgery and a median incision was made, using the above-described protocol. The colon was exposed after careful dissection and haemostasis of peritoneal adhesions. The caecum was completely resected using a surgical clip, as described. [START_REF] Kuo | Early resection of primary orthotopicallygrowing human colon tumor in nude mouse prevents liver metastasis: further evidence for patient-like hematogenous metastatic route[END_REF][START_REF] Pocard | Efficiency of orthotopic xenograft models for human colon cancers[END_REF] The clip was covered for final haemostasis and asepsis with 0.5 mL of biological glue. The bowel was replaced in the abdominal cavity and the wall was closed in two layers.

Tumour growth and metastasis evaluation

For each animal, an autopsy was performed at day 45 after tumour implantation. Tumour volumes were calculated using the formula V = a x b 2 / 2, where a is the length of the tumour (largest diameter) and b is the perpendicular width of the tumour (smallest diameter), both measured with a calliper. For each animal, local recurrence and distant metastases (lymph node, liver and lung metastases, peritoneal carcinomatosis) were searched macroscopically at autopsy with histologic confirmation and immunohistochemical labelling of MLH1, as described above. The animals were considered cured if no tumour could be detected in any of the above-mentioned organs when inspected at autopsy.

Statistical analyses

On the basis of a unilateral alpha type one error of 5%, it was necessary to include 10 mice per group to achieve a 90% statistical power if the expected event ratio (local or distant recurrence) was 4, and 8 mice per group if the mean tumour volume ratio was expected to decrease 4-fold upon expression of MLH1. Two-sided Fisher's exact test was used to compare the proportion of mice with recurrence, lymph node metastases, peritoneal carcinomatosis and liver metastases. Tumour volumes were compared using a one-tailed Mann-Whitney U test. Analyses were performed using the Prism 5 software (GraphPad Software Inc, La Jolla, CA).

RESULTS

Feasibility of orthotopic intracaecal xenografts and tumour resection

Among the 60 NOD/SCID mice that had been grafted on the caecum, one died during surgery, one the following day and one at day 8 (1/30 and 2/30 for HCT116 and mlh1-3, respectively), giving a 5% graft-related mortality. Among the 27 animals that did not undergo surgery, tumour graft implantation was successful in 21 animals (10/14 and 11/13, for HCT116 and mlh1-3, respectively), giving an overall tumour take rate of 78%. Among the 30 animals that underwent surgical resection at day 10, five of them (16%) died during the first 8 days after surgical tumour resection (2/15 and 3/15 for HCT116-R and mlh1-3-R, respectively), with one dying the next day.

Microscopy of haematoxylin-eosin (HE) sections showed that macroscopic tumours from both HCT116 and mlh1-3 exhibited invasive growth in the murine caecum towards the intestinal lumen (Figure 1A andB); both HCT116 and mlh1-3 tumours were able to grow through the serosa and invade the muscular layer and the mucosa. Visual examination at autopsy and microscopic examination of HE-stained sections showed that tumours also gave rise to lymph node metastases (Figure 1C andD), liver metastases in one case (Figure 1E and F), and peritoneal carcinomatosis (Figure 1G andH). HCT116-derived tumours cells were more frequently poorly differentiated, with increased necrosis, than mlh1-3; in addition, HCT116 cells were smaller and less cohesive.

Molecular characteristics of the HCT116 and mlh1-3 cell lines and their counterpart tumours

The expression of MLH1 was investigated in the cell lines and their derived tumours by Western blot and immunohistochemical staining with an anti-MLH1 antibody (Figure 2A-C).

As expected, the expression of MLH1 was undetectable in the HCT116 cell line and in the derived HCT116 tumour tissue (Figure 2A andB). In the mlh1-3 cell line obtained by transfecting the MLH1-deficient HCT116 cell line with a wild type MLH1-expressing vector, MLH1 was expressed at normal control levels (Figure 2A) and sustained throughout the course of the experiments (not shown). MLH1 expression in grafted mlh1-3 tumours was maintained in spite of discontinued antibiotic selection pressure for the in vivo experiments (Figure 2C). Interestingly, HE staining showed that tumours derived from HCT116 or mlh1-3 cells displayed similar morphological features (Figure 2B andC). Figure 2 shows the MSI phenotype of the HCT116 and mlh1-3 cell lines, as well as their corresponding tumour tissues, determined using 3 highly polymorphic dinucleotide microsatellite markers. As expected, the HCT116 cell line (Figure 2D) displayed a typical high MSI profile, with numerous peaks corresponding to the amplification of alleles derived through deletion or insertion of CA motifs in the parental alleles. Conversely, no microsatellite instability could be detected in the mlh1-3 cell line (Figure 2E), or its derived subcutaneous (Figure 2F) or intracaecal (Figure 2G) tumour tissues obtained from the 23 mice analysed at autopsy (Figure 2 and data not shown).

We further compared the mutational status of 26 genes harbouring a mononucleotide repeat in HCT116 and mlh1-3 cells (Table 1). No difference was found between the two cell lines: the coding repeated sequences were wild-type in sixteen genes, while seven genes displayed a monoallelic 1 bp deletion (BAX, MBD4, POLD3, RAD50, RECQL, TOPBP1 and TOPORS) together with a wild-type allele; the other three genes had 1-or 2-bp biallelic deletions (MRE11, MSH3 and TGFBR2) (Table 1).

DNA fingerprinting of the two cell lines and their derived intracaecal tumours was performed using 8 STR markers that enables a 1 in 10 8 discrimination rate for unrelated individuals (Supporting Information Table 1). Alleles are referred to by their number of repeats determined using a reference panel of six cell lines of known genotype. Profiles were identical for all samples tested confirming that mlh1-3 and intracaecal tumours derived from HCT116. Yet, there were minor differences between the HCT116 and mlh1-3 profiles and the one available at the ATCC website. The first difference was located at the vWA marker and consisted of an addition of a single nucleotide that was detected in HCT116 and its mlh1-3 derived cell line, indicating that this mutation occurred in the HCT116 parental cell line before it was complemented with the MLH1-expressing vector; this observation was not unexpected since HCT116 is deficient in mismatch repair. The other difference is that the intensity of the Y-chromosome-specific amelogenin allele is below expected suggesting that part of the cells lost their Y-chromosome, or that a 6-bp deletion occurred in the Y-chromosome, generating a size that is identical to that of the X-chromosome.

Spontaneous evolution of the HCT116 and mlh1-3 intracaecal tumour xenografts

As shown in Table 2 and Figure 3, the mean tumour volume in the group of mice implanted with mlh1-3-derived tumours was significantly smaller than in those with HCT116 (P = .001), despite large variations within each group. Lymph node metastases and peritoneal carcinomatosis occurred significantly more often in the group of mice grafted with HCT116, compared to the group of mlh1-3 tumour-bearing animals (P = .007 and P = .035, respectively) (Table 2). Mice grafted with mlh1-3 tumours did not develop peritoneal carcinomatosis or liver metastasis; the only liver metastasis was observed in the group of mice grafted with the HCT116 tumour (Table 2).

Evolution of the HCT116 and mlh1-3 intracaecal tumour xenografts after surgical resection

In order to study tumour evolution after curative surgery, HCT116 and mlh1-3 intracaecal tumour xenografts were resected at day 10. The rate of local recurrence after surgical resection did not differ significantly between the two groups, varying from 38% (5/13) for HCT116-R to 25% (3/12) for mlh1-3-R (Table 2); tumour volumes varied widely in both groups and were not significantly different. Lymph node metastases occurred only in the group of mice initially implanted with the HCT116 tumour, but the difference failed to reach the level of significance (P = .096). Moreover, a single mouse of the mlh1-3 group displayed peritoneal carcinomatosis, whereas it occurred at a significantly higher frequency in mice initially grafted with the HCT116 tumour (P = .011). No liver metastases were detected in either group. The rate of cure was significantly higher in the mlh1-3 group in which only 3/12 animals relapsed, while 9/13 animals of the HCT116 group relapsed (P = .047) (Table 2).

DISCUSSION

CRCs are characterized by accumulation of genetic alterations that drive tumour progression from non-invasive adenomas to invasive adenocarcinomas. High-throughput arrays have been extensively used to uncover transcriptional gene expression or genomic aberration profiles in primary CRCs that might predict prognosis of patients with stage II [START_REF] Agesen | ColoGuideEx: a robust gene classifier specific for stage II colorectal cancer prognosis[END_REF][START_REF] Maak | Independent validation of a prognostic genomic signature (ColoPrint) for patients with stage II colon cancer[END_REF][START_REF] Kopetz | Genomic classifier ColoPrint predicts recurrence in stage II colorectal cancer patients more accurately than clinical factors[END_REF] and/or stage III [START_REF] Salazar | Gene expression signature to improve prognosis prediction of stage II and III colorectal cancer[END_REF][START_REF] Wang | A molecular signature for the prediction of recurrence in colorectal cancer[END_REF] CRC, or the site of metastases. [START_REF] Bruin | Specific genomic aberrations in primary colorectal cancer are associated with liver metastases[END_REF][START_REF] Watanabe | Prediction of liver metastasis after colorectal cancer using reverse transcription-polymerase chain reaction analysis of 10 genes[END_REF] Despite considerable efforts, no signature has been translated into the clinical management of patients due to low prediction accuracy. [START_REF] Sanz-Pamplona | Clinical value of prognosis gene expression signatures in colorectal cancer: a systematic review[END_REF] Patient-derived xenograft models have been developed to address specifically the role of putative oncogenes or tumour suppressor genes in CRC progression, or to predict drug sensitivity. [START_REF] Dangles-Marie | Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features[END_REF][START_REF] Bertotti | A molecularly annotated platform of patientderived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer[END_REF][START_REF] Julien | Characterization of a large panel of patientderived tumor xenografts representing the clinical heterogeneity of human colorectal cancer[END_REF][START_REF] Tentler | Patient-derived tumour xenografts as models for oncology drug development[END_REF][START_REF] Nunes | Evaluating patient-derived colorectal cancer xenografts as preclinical models by comparison with patient clinical data[END_REF] For instance, orthotopic xenograft tumour models in immunodeficient mice have been successfully used to assess the function of TP53 and E-cadherin, [START_REF] Pocard | Single alteration of p53 or E-cadherin genes can alter the surgical resection benefit in an experimental model of colon cancer[END_REF] and more recently that of TGFRII 41 in CRC progression. Interestingly, intracaecal xenografted tumours may be subsequently resected, mimicking the surgical resection of primary CRC in humans, thus providing an opportunity to evaluate the benefit of surgery in well-defined tumour models.

Our orthotopic xenograft model recapitulates all aspects of human CRC progression, i.e. lymph node invasion, peritoneal carcinomatosis, liver metastasis and local recurrence after tumour surgery, with the exception of lung metastasis. Importantly, we were able to show that the MLH1 cDNA remained stably expressed throughout the experiments, even when selection pressure by hygromycin had been removed for in vivo experiments. All cell lines and tumours also shared the spectrum of mutations in the 26 mononucleotide coding repeats analysed and an identical profile at the 8 tetranucleotide STR loci, supporting that mlh1-3 is genetically close to the parental HCT116. In addition, all cell lines gave rise to tumours displaying similar morphological features. Thus, our model based on the isogenic HCT116 and mlh1-3 cell lines seems to be appropriate to investigate the impact of MLH1 expression on tumour progression independently of the MSI-driven mutations that previously arose in target genes.

We here show that the growth of tumours derived from the MLH1-complemented mlh1-3 cell line was slower compared to HCT116, a finding that is in keeping with the fact that MSI CRC tend to be larger than MSS CRC, possibly reflecting the role of MLH1 in cell cycle regulation and apoptosis. [START_REF] O'brien | Signalling cell cycle arrest and cell death through the MMR System[END_REF] Interestingly, restoring MLH1 expression significantly decreased the rate of peritoneal carcinomatosis regardless of whether the tumour was resected or not. This is in line with a recent publication reporting that as many as 25% of the tumours arising in patients undergoing cytoreductive surgery for peritoneal carcinomatosis from CRC displayed MSI, a frequency that is higher than in unselected stage IV CRC, suggesting that MSI might favour peritoneal metastases. [START_REF] Massalou | Peritoneal carcinomatosis of colorectal cancer: novel clinical and molecular outcomes[END_REF] The decreased incidence of lymph node invasion was statistically significant when comparing mice that did not undergo surgery, but not for mice that underwent tumour resection, possibly due to lack of power since the number of mice was rather low. Yet, ethical considerations led us to reduce the number of animals used to achieve our aim, which was to document substantial differences in tumour progression. MSI CRCs are considered to have a better prognosis compared to MSS CRCs, but the underlying molecular alterations have not been identified yet. [START_REF] Popat | Systematic review of microsatellite instability and colorectal cancer prognosis[END_REF] In particular, the impact of MMR deficiency per se on tumour progression, independently of MSI-driven mutations, remained to be investigated. The possibility that mutations in one of the genes most frequently mutated in MSI CRC, represent prognostic biomarkers has been investigated in several studies with conflicting results. [START_REF] Watanabe | Molecular predictors of survival after adjuvant chemotherapy for colon cancer[END_REF][START_REF] Kim | Prognostic and predictive roles of highdegree microsatellite instability in colon cancer: a National Cancer Institute-National Surgical Adjuvant Breast and Bowel Project Collaborative Study[END_REF][START_REF] Shima | TGFBR2 and BAX mononucleotide tract mutations, microsatellite instability, and prognosis in 1072 colorectal cancers[END_REF] Interestingly, a recent study reported that reexpressing TGFBR2 in the HCT116 cell line that carries a biallelic TGFBR2 frameshift mutation, increased cell survival and motility in vitro and enhanced their in vivo metastatic potential in an orthotopic model in nude mice, indicating that TGF signalling may indeed modulate the metastatic potential of MSI CRC. [START_REF] Liu | Restoration of transforming growth factor-beta receptor II expression in colon cancer cells with microsatellite instability increases metastatic potential in vivo[END_REF] Experiments in which expression of wildtype MLH1 is obtained by introducing a normal human chromosome 3 also provide a normal TGFBR2 allele, because the TGFBR2 gene is located in the close vicinity of MLH1 on chromosome 3, 62 thus hampering the investigation of the respective contribution of MLH1 and TGFBR2 per se. The HCT116 / mlh1-3 model that we established overcomes this problem since MLH1 is restored by transfecting a vector expressing the wild-type MLH1 cDNA, which does not change the TGFBR2 status of HCT116 cells. [START_REF] Jacob | The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorectal cancer cells[END_REF] Our results show that the clinical status of mice xenografted with the MLH1-expressing tumour was significantly improved compared to those with the parental HCT116-derived tumour, underlying the role of MLH1 deficiency in itself in tumour progression. Expression of MLH1 was notably associated with a significantly lower chance of recurrence after curative surgery, as well as a decreased incidence of lymph node invasion and peritoneal carcinomatosis in animals, regardless of whether the tumour was resected or left in situ.

In our xenograft experiments, the absence of MLH1 expression tended to be associated with a worse evolution, while patients with MSI CRC had a better stage-adjusted prognosis than those with CRC displaying chromosome instability. [START_REF] Walther | Genetic prognostic and predictive markers in colorectal cancer[END_REF][START_REF] Roth | Integrated analysis of molecular and clinical prognostic factors in stage II/III colon cancer[END_REF] The major distinction between these apparently contradictory findings is that, in humans, the differences between MSI and non-MSI tumours reside not only in the fact that one of the MMR genes (most often MLH1 or MSH2) is not functional, but also in the genome-wide accumulation of de novo mutations due to inefficient post-replicative repair, while in the animal model that we used, the only difference that we could detect was MLH1 expression. Although such animal models may help deciphering the impact of specific genetic alterations, such as MLH1 deficiency, on cancer progression, there are several limitations. Tumour xenograft models are not wellsuited to study interactions between cancer cells and tumour environment because interspecies molecular communications, including ligand-receptor interactions, are likely to differ. [START_REF] Tentler | Patient-derived tumour xenografts as models for oncology drug development[END_REF] In addition, being severely immuno-compromised, NOD/SCID mice obviate assessing the well-recognized role of the immune system, in the metastatic process in CRC. [START_REF] Kirilovsky | Rational bases for the use of the Immunoscore in routine clinical settings as a prognostic and predictive biomarker in cancer patients[END_REF] Addressing this question would be extremely hard as reconstitution of a human immune system in immunodeficient mice should be done with patient-matched immune cells, which greatly limits the feasibility.

FIGURES LEGENDS FIGURE 1.

Examples of tumours and metastases obtained with the HCT116 cell line.

Macroscopic examination and hematoxylin-eosin-safran stained slides of tumours developing in the caecum (A and B), lymph node (C and D), liver (E and F) and peritoneum (G and H). Gene symbols are according to HUGO gene nomenclature committee. Gene ID is from the NCBI Entrez gene database. The alleles detected were either wild-type (wt), or carry a 1 bp or a 2 bp deletion (noted as "-1" or "-2", respectively); wt : only wild-type alleles were detected; -1/wt : both wt alleles and alleles carrying a 1 bp deletion were detected; -1: only alleles with a 1 bp deletion were detected; -2/-1: alleles carrying a 2 bp deletion and alleles with a 1 bp deletion were detected. All repeats are located in the coding sequences, except for MRE11A whose T11 repeat is located in an intron and whose shortening induces exon skipping; thus, a 1 or 2 bp deletion results in a frameshift mutation. Fifteen mice were initially grafted in each group; the number of mice analysed decreased due to early death after tumour graft (n = 3) or surgical resection (n = 5), and to the absence of tumour take (n = 6). The volumes of tumours growing on the caecum were compared using a one-tailed Mann-Whitney U test. To compare the number of mice with recurrence, lymph node metastases, peritoneal carcinomatosis and liver metastases, P values were calculated using two-sided Fisher's exact tests.

a not analyzed (na): no P value could be calculated to compare the number of mice with liver metastases after tumour resection since there was no event in either group.

FIGURE 2 .

 2 FIGURE 2.MLH1 expression and MSI phenotyping in HCT116 and mlh1-3 cell lines, and

FIGURE 3 .

 3 FIGURE 3.Growth of intracaecal HCT116 and mlh1-3 derived tumours. Volumes of intracaecal tumours derived from HCT116 (HCT116 ) and mlh1-3 (mlh1-3 ) when left in

TABLE 1 .

 1 Analysis of the mutation status of genes harbouring a mononucleotide repeat sequence in HCT116 and mlh1-3 cell lines

	Gene symbol	Gene ID	Repeat	Location	HCT116	mlh1-3
	ATR	545	A10	3q23	wt	wt
	BAX	581	G8	19q13.3-q13.4	-1/wt	-1/wt
	BLM	641	A9	15q26.1	wt	wt
	BRCA1	672	A8	17q21	wt	wt
	CDC25C	995	A8	5q31	wt	wt
	CHEK1	1111	A9	11q24-q24	wt	wt
	ERCC5	2073	A9	13q33	wt	wt
	LIG3	3980	A8	17q11.2-q12	wt	wt
	MBD4	8930	A10	3q21-q22	-1/wt	-1/wt
	MRE11A	4361	T11	11q21	-2/-1	-2/-1
	MSH3	4437	A8	5q11-q12	-1	-1
	MSH6	2956	C8	2p16	wt	wt
	POLA1	5422	A8	Xp22.1-p21.3	wt	wt
	POLD3	10714	A9	11q14	-1/wt	-1/wt
	PRKDC	5591	A10	8q11	wt	wt
	RAD50	10111	A9	5q31	-1/wt	-1/wt
	RBBP8	5932	A9	18q11.2	wt	wt
	RECQL	5965	A9	12p12	-1/wt	-1/wt
	RIF1	55183	A8	2q23.3	wt	wt
	SMC1B	27127	A8	22q13.21	wt	wt
	TCF7L2	6934	A9	10q25.3	wt	wt
	TGFBR2	7048	A10	3p22	-1	-1
	TOPBP1	11073	A8	3q22.1	-1/wt	-1/wt
	TOPORS	10210	A8	9p21	-1/wt	-1/wt
	WRN	7486	A8	8p12-p11.2	wt	wt
	XRCC2	7516	T8	7q36.1	wt	wt

TABLE 2 .

 2 Clinical status of mice with intracaecal HCT116 and mlh1-3 tumour xenografts left in situ or after surgical resection

			Spontaneous			Evolution
		tumour evolution	after tumour resection
		HCT116	mlh1-3	P	HCT116-R mlh1-3-R	P
	Number of mice	10	11		13	12
	Tumour volume (mm 3 )
	Mean SEM	1483 242	431 215	.001	686 526	1787 843	.125
	Recurrence after surgery				9	3	.047
	Local recurrence after surgery				5	3	.673
	Distant recurrence	7	1	.007	8	1	.011
	Lymph node metastases	7	1	.007	4	0	.096
	Liver metastases	1	0	.476	0	0	na a
	Peritoneal carcinomatosis	4	0	.035	8	1	.011

ACKNOWLEDGMENTS

We thank Tatiana Ledent for her help in managing the animal house and Dr Erik Ruuth for critical reading of the manuscript.

Funding information. The authors thank the Fondation ARC pour la Recherche sur le

Cancer for financial support to KM and a grant to MP (#3395).

Supporting Information Table 1. DNA fingerprinting of HCT116 and mlh1-3 cell lines and their derived intracaecal tumours Profiling was performed using the eight short tandem repeat (STR) markers recommended by the American Type Cell Culture (ATCC), which enables a 1 in 10 8 discrimination rate for unrelated individuals. Multiplex PCR was carried and analyzed as in Figure 2D-G. Alleles are referred to by their number of repeats defined using a panel of six cell lines used as reference. The Y chromosome specific amelogenin allele shown in parentheses indicate that its intensity is far below expected. The size of the vWA 22.1 allele is 1 bp longer than the allele with 22 repeats reported for the HCT116 cell line on the ATCC website.


Josephine Brice
email: josephine.brice@gmail.com

Mariam Sylla

Sophie Sayon

Fatoumata Telly

Djeneba Bocar-Fofana

Robert Murphy

Sidonie Lambert-Niclot

Eve Todesco

Maxime Grude

Francis Barin

Souleymane Diallo

Deenan Pillay

Anne Derache

Vincent Calvez

Anne-Geneviève Marcelin

Almoustapha Issiaka Maiga

Qualitative and quantitative HIV antibodies and viral reservoir size characterization in vertically infected children with virological suppression

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

INTRODUCTION

Pediatric HIV infection remains a major public health issue despite large implementation of prevention of mother-to-child transmission programmes. 1 It requires early and lifelong HAART to control the viral replication with a risk of accumulating toxicity and viral drug resistance. International guidelines are now recommending initiation of HAART in all vertically HIV-1-infected (VHIV) children regardless of clinical and immunological conditions [START_REF]Panel on Antiretroviral Therapy and Medical Management of HIV-Infected Children. Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection[END_REF] as early treatment reduces mortality and improves immune recovery. [START_REF] Cotton | Early time-limited antiretroviral therapy versus deferred therapy in South African infants infected with HIV: results from the children with HIV early antiretroviral (CHER) randomised trial[END_REF] Proviral DNA HIV-1 reservoirs are established early during infection and represent a barrier to functional cure. [START_REF] Ananworanich | Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection[END_REF] A low HIV viral reservoir is associated with a lower risk of disease progression. Despite prolonged HAART, HIV-1 persists as transcriptionally inactive proviruses in long half-life memory resting CD4 T cells. [START_REF] Archin | Immediate antiviral therapy appears to restrict resting CD4+ cell HIV-1 infection without accelerating the decay of latent infection[END_REF] The lowest reservoir has been described in elite controllers (EC) in whom HIV-1 replication is controlled without treatment. [START_REF] Lambotte | HIV controllers: a homogeneous group of HIV-1-infected patients with spontaneous control of viral replication[END_REF] However, in post-treatment controllers (PTC), HIV-1 remission and low reservoir are observed after HAART interruption mainly when treatment was initiated during primary HIV infection (PHI). [START_REF] Sáez-Cirión | Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study[END_REF] The relation between the size of the viral reservoir, and the precocity and duration of HAART has been previously described in children. [START_REF] Persaud | Influence of age at virologic control on peripheral blood human immunodeficiency virus reservoir size and serostatus in perinatally infected adolescents[END_REF][START_REF] Luzuriaga | HIV Type 1 (HIV-1) Proviral Reservoirs Decay Continuously Under Sustained Virologic Control in HIV-1-Infected Children Who Received Early Treatment[END_REF][START_REF] Ananworanich | Reduced markers of HIV persistence and restricted HIV-specific immune responses after early antiretroviral therapy in children[END_REF] However, more studies are required to identify simple predictors of the reservoir size in VHIV children, in real world conditions within high HIV prevalence settings, especially in VHIV children.

The absence of detectable viremia 12 years after treatment cessation in one VHIV child has suggested that early HAART initiation could lead to functional cure. [START_REF] Frange | HIV-1 virological remission lasting more than 12 years after interruption of early antiretroviral therapy in a perinatally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report[END_REF] HIV infected people first develop anti-gp41 antibodies and only several weeks later, anti-gp120 antibodies. [START_REF] Overbaugh | The Antibody Response against HIV-1[END_REF] The Berlin patient, who was cured of HIV following a stem cell transplant, dysplayed a complete loss of anti-p24 antibodies and a low but still detectable response to gp41. [START_REF] Burbelo | HIV antibody characterization as a method to quantify reservoir size during curative interventions[END_REF] Only few EC subpopulations show this HIV antibody profile. Thus, monitoring the response to p24 and gp41 may be useful in HIV cure studies.

Several HIV-1 seroreversions have been observed in VHIV children who initiated HAART within the first months of life. [START_REF] Hainaut | Effectiveness of antiretroviral therapy initiated before the age of 2 months in infants vertically infected with human immunodeficiency virus type 1[END_REF][START_REF] Zanchetta | Early therapy in HIV-1-infected children: effect on HIV-1 dynamics and HIV-1-specific immune response[END_REF] By blocking viral replication, the early virostatic treatment might prevent the development of the HIV-1 specific antibody response, either quantitatively (antibody level) or qualitatively (antibody activity), thus leading to HIV seroreversion or substantially lower anti-HIV antibody levels. One analysis demonstrated that age of HAART initiation and plasma viral load were strong predictors of serostatus, and both were independently associated. [START_REF] Payne | Reactivity of routine HIV antibody tests in children who initiated antiretroviral therapy in early infancy as part of the Children with HIV Early Antiretroviral Therapy (CHER) trial: a retrospective analysis[END_REF] Kuhn et al showed that the absence of HIV antibody response indicated a smaller HIV-1 viral reservoir, and HAART initiated at 3 months of age was the upper limit to see the benefits of early HAART. [START_REF] Kuhn | Young age at start of antiretroviral therapy and negative HIV antibody results in HIV-infected children when suppressed[END_REF] Only one study has desmontrated the utility of HIV serostatus as a surrogate marker of the reservoir size. [START_REF] Persaud | Influence of age at virologic control on peripheral blood human immunodeficiency virus reservoir size and serostatus in perinatally infected adolescents[END_REF] Here we describe the factors associated with HIV antibody activity or level and the viral reservoir size in HAART-treated VHIV children with heterogeneity of age, time of therapy, and duration of virological suppression.

METHODS

This cross-sectional study conducted within a prospective cohort included HAART-treated VHIV children followed at Gabriel Touré Hospital (Bamako, Mali) with sustained virological suppression (HIV-1 RNA plasma ≤ 50 copies/mL). All participants were known virologically suppressed at their previous visit (six months) and confirmed during the study. Participants older than 20 years old, those with HIV-2 infection, treated for less than three months, or without any data recorded were excluded from the study. After obtaining the parent's written or oral informed consent from all participants, five millilitres of extra blood samples were collected in EDTA tubes during the routine follow up visit. Assent was obtained from child participants according to local institutional review board guidelines. The study was approved by the National AIDS program at the Ministry Of Health in Mali (CSLS/MS) in collaboration with the Malian Institutional Ethics Committee at the Faculty of Medicine, Pharmacy and Odontostomatolgy of health and life sciences in Bamako under the reference number N°10-05-FMPOS.

In order to evaluate the size of the HIV-1 viral reservoir, total DNA was extracted from PBMC's derived from whole blood using an automated technique (MagNA Pure, Roche, Manheim, Germany). The cellassociated HIV-1 DNA level was quantified using a real-time PCR method which amplifying a region in the LTR gene, as previously described. [START_REF] Avettand-Fènoël | LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01)[END_REF] Proviral burden was expressed as HIV DNA copies per 1 million cells (quantification limit: 10 copies/PCR, i.e. 66 copies/10 6 cells considered as undetectable).

Dried serum spots (DSS) were used to evaluate the HIV-specific antibody response. The level of antibodies targeting the gp41 immunodominant epitope (IDE) was measured following a previously described protocol. [START_REF] Barin | Development and validation of an immunoassay for identification of recent human immunodeficiency virus type 1 infections and its use on dried serum spots[END_REF] An equimolar mixture of two 30 amino-acids oligopeptides was used, representing the IDE consensus sequences of HIV-1 group M and subtype D, respectively. A low mixture concentration allowed the binding of late antibodies that had acquired sufficient avidity and then semi-quantitative detection by spectrophotometry. The result was expressed as an optical density (OD). The activity of anti-gp41 antibodies was systematically tested in quadruplicate.

The 4 th generation ARCHITECT HIV Ag/Ab Combo assay (Abbott Laboratories, Wiesbaden, Germany) was performed as previously described to quantify the humoral response in DSS. The result was defined as relative light units, then compared to a cut-off signal. Samples with signal-to-cut-off (S/CO) values ≥ 1.00 were considered reactive and those < 1.00 non-reactive.

We used univariate association between HIV DNA level, anti-gp41 antibody activities or HIV-antibody levels and variables defining current and past HIV disease (age, sex, WHO stage at study, HAART type at study, CD4 cell count at study, HAART duration, maternal prophylaxis type, age at HAART initiation), with the Spearman rank correlation coefficient for continuous variables and the Fisher's exact test for categorical variables.

RESULTS

From August 2013 to April 2014, 97 VHIV children with virological suppression were enrolled. Their median age was 9.8 years old at time of inclusion (IQR = 7 -13.1), they started HAART at a median age of 3.3 years (IQR = 1.9 -7), and were receiving HAART for a median 5.4 years (IQR = 3.5 -7). Table 1 summarizes the demographic, immunologic and virologic characteristics of the children.

The median anti-gp41 antibodies activity was 0.29 OD (IQR = 0.18 -0.75). A low activity of anti-gp41 antibodies was associated with both a younger age at treatment initiation (p = 0.01; Figure 1) and with a lower level of anti-HIV antibodies (p = 0.0015; Figure 1). Overall, eight seroreversions were identified (negative ELISA Architect) in which 2 children had an HIV DNA under the threshold (1 detectable and 1 undetectable) and a low anti-gp41 antibodies activity. All the seroreverted children started HAART before two years of age, at a median age of 1.1 years, and were on HAART for the past 7.3 years in median.

The median level of total HIV DNA was 445 copies/10 6 cells (IQR = 187 -914). No correlation was found between anti-gp41 antibodies activity or age at treatment initiation and HIV DNA (p = 0.27; Figure 1). The 9 children with an HIV DNA level under the threshold tended to have a lower anti-gp41 antibodies activity compared to children with an HIV DNA > 66 copies//10 6 cells (p = 0.11).

DISCUSSION

This study indicates that a significant proportion of virologically suppressed VHIV children who initiated HAART before the age of two years stopped to produce and/or progressively lost the HIV antibodies. This is consistent with the idea that early HAART halts the antigenic stimulation which is necessary to sustain an HIV-specific antibody response. [START_REF] Persaud | Influence of age at virologic control on peripheral blood human immunodeficiency virus reservoir size and serostatus in perinatally infected adolescents[END_REF][START_REF] Luzuriaga | HIV Type 1 (HIV-1) Proviral Reservoirs Decay Continuously Under Sustained Virologic Control in HIV-1-Infected Children Who Received Early Treatment[END_REF][START_REF] Ananworanich | Reduced markers of HIV persistence and restricted HIV-specific immune responses after early antiretroviral therapy in children[END_REF][START_REF] Luzuriaga | Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses[END_REF] In addition, some of these children with seroreversion had a very low HIV reservoir, at least identified in the peripheral blood, and could therefore represent an ideal population for studies investigating novel immunotherapeutic strategies aiming to achieve HAART-free remission.

Although current HAART can effectively control HIV replication to clinically undetectable levels for years, existing strategies do not eradicate HIV-1 reservoirs in VHIV children. [START_REF] Frange | HIV-1 virological remission lasting more than 12 years after interruption of early antiretroviral therapy in a perinatally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report[END_REF] One of the limitation of our study was that our cohort did not include children who started HAART before five months of life, and therefore we were not able to identify more seroreversions. Nonetheless, we found 50% of seroreversion in VHIV children who initiated treatment before two or one years old (8/16 and 4/8 respectively), consistently with other studies in occidental settings that showed 50% to 94% of seroreversion when treatment was initiated before 3 months of life. [START_REF] Hainaut | Effectiveness of antiretroviral therapy initiated before the age of 2 months in infants vertically infected with human immunodeficiency virus type 1[END_REF][START_REF] Zanchetta | Early therapy in HIV-1-infected children: effect on HIV-1 dynamics and HIV-1-specific immune response[END_REF][START_REF] Luzuriaga | Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses[END_REF] Children can acquire HIV-1 in utero, during delivery or breastfeeding. 1 In our study, the time of HIV transmission was not known. But, we assumed that seroreversion probably occured in children who had treatment initiation soon after the HIV infection acquisition. Indeed, when using one of the most sensitive assays available, we were able to find several seroreversions. In addition, we found an association between a low anti-gp41 antibodies activity and a younger age at treatment initiation.

Early effective control of HIV replication has been associated with incomplete development of HIVspecific immune responses in children. [START_REF] Hainaut | Effectiveness of antiretroviral therapy initiated before the age of 2 months in infants vertically infected with human immunodeficiency virus type 1[END_REF][START_REF] Zanchetta | Early therapy in HIV-1-infected children: effect on HIV-1 dynamics and HIV-1-specific immune response[END_REF][START_REF] Luzuriaga | Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses[END_REF] It would be of interest to study the initial development of anti-HIV antibodies (like activity and quantification of anti-gp41 antibodies ) in early treated infants to determine whether the primary responses is affected or the influence occurs later on, leading to a decrease of responses over time. The decrease of antibody production and/or their avidity against some epitopes should reflect the absence of circulating antigenic viral particles, showing the absence of residual viral replication.

In conclusion, the results of this study show that HIV-1 seroreversion and low anti-gp41 activity in VHIV children with early HAART initiation happened and should be considered as a proof-of-concept study to evaluate strategies targeting HAART-free remission (i.e long-term undetectable viremia for an undefined period, in the absence of HAART). [START_REF] Deeks | International AIDS Society global scientific strategy: towards an HIV cure 2016[END_REF] This study identified potential candidates with low viral reservoir and low antibody levels or activity. Defining the immunologic and virologic end-points after HAART in VHIV children and identifying factors and biomarkers associated with limited proviral reservoir size are essential to define therapeutic strategies, in order to achieve HIV remission or cure in this population.

Figure 1 :

 1 Figure 1: Distribution of anti-gp41 antibodies activity by age at HAART initiation (A) and by anti-HIV antibodies level (B) using Spearman correlation. Distribution of HIV DNA level by age at HAART initiation (C) and by anti-gp41 antibodies activity (D) using Spearman correlation.

ACKNOWLEDGEMENTS

Global Health Center, Department of Infectious Diseases, Northwestern University, Chicago IL, USA. FUNDING This study was funded by Agence Nationale de Recherche sur le Sida et les Hépatites virales. TRANSPARENCY DECLARATION None to declare.