Ricardo Romero
email: romeromartinricardo@hotmail.com

MD Ananda Castano

MD Jose Abelairas

MD, PhD Jesus Peralta

MD, PhD Margarita Sanchez-Orgaz

PhD Alvaro Arbizu

MD Jose Vallejo-Garcia

Keywords: Peripheral primitive neuroectodermal tumor of the orbit, orbital primitive neuroectodermal tumor, orbital pPNET

Peripheral primitive neuroectodermal tumors (pPNETs) are a group of soft tissue tumors of neuroepithelial origin, that arise outside the central and sympathetic nervous system. Orbital location is infrequent, to the best of our knowledge only sixteen cases have been reported in the literature. With this article we would like to report the demographics and clinical characteristics, diagnostic features, differential diagnosis, prognosis and therapeutic options of primary orbital peripheral primitive neuroectodermal tumor, based on our patients and on the cases reported in the literature up to now. Differential diagnosis should be made with other small round cells tumors, immunohistochemical and ultrastructural techniques are essential for this purpose.

Although bone invasion and extraorbital extension are possible, systemic metastases are uncommon in the cases of orbital pPNETs. Surgery has been the initial treatment in most cases; chemotherapy +/-radiotherapy is considered the best additional treatment.

The orbital variety seems to be less aggressive than other forms of pPNETs since most of the patients reported were alive after the follow up period (at least 6 months).

INTRODUCTION

Peripheral primitive neuroectodermal tumors (pPNETs) are a group of malignant soft tissue tumors of neuroepithelial origin that arise outside the central and sympathetic nervous system, and occur predominantly in children and young adults.

Histopathologically, pPNET consists of small round cells with hyperchromatic nuclei, high nuclear-cytoplasmic ratio and varying degrees of neural differentiation detected by immunohistochemical and/or ultrastructural techniques.

The first case of pPNET was reported by Stout in 1918 located in the ulnar nerve [START_REF] Stout | A tumor of the ulnar nerve[END_REF] . In 1973, Hart and Earle introduced the term primitive neuroectodermal tumor (PNET) to describe embryonal neoplasms of the central nervous system that did not fulfil the diagnostic criteria for other entities [START_REF] Hart | Primitive neuroectodermal tumors of the brain in children[END_REF] , and it was not until the late 1970s that pPNET was admitted as an own entity.

Orbital location is unusual, to the best of our knowledge only 16 cases have been reported in the literature up to April'10 [START_REF] Kim | Multimodality treatment approach in management of primary peripheral primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Das | Primary primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Romero | Primary peripheral primitive neuroectodermal tumour of the orbit: case report[END_REF][START_REF] Tamer | Primary peripheral primitive neuroectodermal tumour of the orbit[END_REF][START_REF] Romero | Recurrence of peripheral primitive neuroectodermal tumor of the orbit with systemic metastases[END_REF][START_REF] Lezrek | Orbital primitive neuroectodermal tumor with intracranial extension. A case report[END_REF][START_REF] Sen | Primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Hyun | Metastatic peripheral primitive neuroectodermal tumor (PNET) masquerading as liver abscess: a case report of liver metastasis in orbital PNET[END_REF][START_REF] Alyahya | Primitive neuroectodermal tumor of the orbit in a 5-year old girl with microphthalmia[END_REF][START_REF] Kiratli | Primitive neuroectodermal tumor of the orbit in an adult. A case report and literature review[END_REF][START_REF] Singh | Primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Arora | Primary orbital primitive neuroectodermal tumour with immunohistochemical and electron microscopic confirmation[END_REF][START_REF] Wilson | Primary peripheral neuroepithelioma of the orbit with intracranial extension[END_REF][START_REF] Shuangshoti | Primary intraorbital extraocular primitive neuroectodermal (neuroepithelial) tumour[END_REF][START_REF] Howard | Neuroepithelioma of the orbit[END_REF] . We describe one case of primary orbital pPNET and discuss its clinical, histopathologic, radiologic, and therapeutic features based on our patients and on the cases reported in the literature.

MATERIALS AND METHODS

We reviewed the clinical record of one patient diagnosed with orbital pPNET in the Department of Paediatric Ophthalmology of University Hospital La Paz, Madrid, a tertiary referral centre in Spain. Recorded data included the following characteristics: demographic (age, gender), clinical (signs and symptoms, orbital location, recurrences, metastases, follow up), histopathologic, radiologic, and therapeutic.

An intensive online literature search was made in order to find all the orbital pPNET cases reported up-to-date. The sources used for the literature search included PubMed (http://www.pubmed.com) and The Cochrane Library (http://cochranelibrary.com) and the terms utilized for the search were: "Orbital peripheral primitive neuroectodermal tumor", "Orbit peripheral primitive neuroectodermal tumor.", "Orbital primitive neuroectodermic tumor", "Orbit primitive neuroectodermic tumor", "Orbital peripheral primitive neuroectodermal tumour", "Orbit peripheral primitive neuroectodermal tumour.", "Orbital primitive neuroectodermic tumour", "Orbit primitive neuroectodermic tumour", "Orbit neuroepithelioma", "Orbital neuroepithelioma".

CASE REPORT

A previously healthy 22-month old boy with proptosis of the right eye for the last week was referred to our hospital in February 2006.

External ocular examination showed mild eyelid inflammation and proptosis with diminished retropulsion of the right eye. The mentioned eye was displaced laterally and inferiorly with restriction of ocular movements superiorly. Apparently there was no pain. Visual acuity could not be measured because of lack of collaboration.

Biomicroscopy found a 2 milimetres (mm), round, superficial, inferior ulcer in the right eye. Funduscopy of both eyes was unremarkable and intraocular pressures were normal.

A computed tomography (CT) scan of the right orbit found a poorly-defined, extraconal orbital mass, superomedially located, with lateral and inferior displacement of the globe.

The mass enhanced after contrast administration and measured 15x18x18 mm. There was no evidence of extraorbital extension or bone erosion (Figures 1A,1B). A nonhomogeneous lesion with some cystic spaces was described in the B-scan orbital ultrasonography performed.

A few days after the first examination, the patient was seen in the emergency department of our hospital because of acute worsening of proptosis and periorbital hematoma. An excisional biopsy of the mass, via transconjunctival orbitotomy, was then performed removing most of the tumor. The tissue was formalin fixed, processed for paraffin embedding and subsequently stained with hematoxylin-eosin and periodic acid-Schiff (PAS). Microscopically the tumor had several hemorrhagic and necrotic areas and was composed of small round blue cells with scant clear cytoplasm, inconspicuous nucleoli and high mitotic activity (Figure 1C). No rosettes or pseudorosettes were found. The PAS reaction was positive (Figure 1D). Sections were studied immunohistochemically using the following antibodies: neuron-specific enolase (code no. IR612, Dako), synaptophysin (code no. IR776, Dako), CD99 (MIC2 gene) (code no. IR057, Dako), glial fibrillary acid protein (code no. IR524, Dako), neurofilaments (code no. IR607, Dako), chromogranin (code no. IR502, Dako), S100 protein (code no. IR504, Dako), vimentin (code no. IR630, Dako), CD57 (LEU-7/HNK-1) (code no. IR647, Dako), common actin (clone HHF35, Dako), desmin (code no. IR606, Dako), CD45 (code no. IR751, Dako) and low molecular weight cytokeratin (NCL-L-CK8-TS1, Novocastra). The antibodies were applied synchronously with appropriate positive control slides. The tumor cells were positive for synaptophysin, neurofilaments (Figure 1E), CD99 (Figure 1F), chromogranin, S100 protein, vimentin and CD57. No cells were stained with antibodies against neuron-specific enolase, glial fibrillary acidic protein, desmin, common actin, CD45 and low molecular weight cytokeratin. After these histopathological and immunohistochemical findings the diagnosis of primitive neuroectodermal tumor was made.

The extensive systemic evaluation (bone marrow biopsy, cranial magnetic resonance imaging (MRI), and CT scans of thorax, abdomen, pelvis and extremities) was negative and the diagnosis of orbital pPNET was established.

The patient was successfully treated with additional radiotherapy and chemotherapy (vincristine, ifosfamide, doxorubicin, etoposide, cyclophosphamide, actinomicine), according to the Spanish Society of Paediatric Oncology (SEOP) 2001 recommendations for Ewing's sarcoma [START_REF] Huerta | High Risk Ewing's sarcomas. Effectiveness and toxicity of a regimen designed for children and used in adults[END_REF] , and he is in remission up to February'10.

Consecutive orbital imaging techniques performed over these years have not detected tumoral pathology (Figures 2A,2B).

Although very infrequent, another case of orbital pPNET was diagnosed in our department a few years ago [START_REF] Romero | Recurrence of peripheral primitive neuroectodermal tumor of the orbit with systemic metastases[END_REF] . While reviewing this case, the tumor samples were recovered and new and more complete histopathological studies, including inmunohistochemical stains, were performed obtaining new results. Paraffin sections of the tumor were stained with hematoxylin-eosin and PAS. Histopathologic examination demonstrated a proliferation of small, round blue cells with scant clear cytoplasm and without nucleoli. There were some foci of necrosis and frequent mitotic figures. No rosettes or pseudorosettes were identified. The PAS stain was negative. Sections were studied by immunohistochemistry for the list of antibodies mentioned in the previous case. These antibodies were applied synchronously with appropriate positive control slides. Immunohistochemically the tumor cells expressed synaptophysin, neurofilaments, CD99 and vimentin. All other stains (neuron-specific enolase, gilal fibrillary acid protein, chromogranin, S-100 protein, CD57, common actin, desmin, CD45, low molecular weight cytokeratin) were negative.

RESULTS

PPNETs located in the orbit are extremely rare, only sixteen cases were found after an intensive online literature search. The most important data of these sixteen cases are summarized in table 1. Orbital pPNETS mainly affect patients in paediatric ages, thirteen of the sixteen reviewed cases were younger than sixteen years of age. No sex predilection was detected.

Proptosis was the initial sign reported in the majority of cases. The most frequent orbital locations were the lateral wall (seven cases) and the inferior wall (four cases). In more than two thirds of the patients the tumor appeared in the right orbit.

Immunohistochemical techniques are the histopathologic diagnostic tool more often used to establish the diagnosis of pPNET. Neuron-specific enolase and CD99 are the immunohistochemical markers more commonly detected in the published cases, followed by S100 protein, synaptophysin, and vimentin. Ultrastructural techniques were used in eight patients, in two cases to make the histopathologic diagnosis and in the rest to confirm the results obtained by immunohistochemistry.

Bone invasion +/-extraorbital extension was detected in five cases but only three patients developed metastases (liver, lungs and lymph node). Just one of these three patients, the one with lymph node involvement, showed bone invasion or extraorbital extension.

Multi-discipline treatment, including different combinations of surgery, chemotherapy and radiotherapy, has been used to treat most of the orbital pPNETs reported. Four patients were managed only with surgery without additional radiotherapy or chemotherapy.

Survival time was longer than thirty months in seven of the seventeen patients. Five of these seven patients with longer survival time were treated with surgery, chemotherapy and radiotherapy, the remaining two patients received chemotherapy and radiotherapy without surgery.

DISCUSSION

PNETs are a group of heterogeneous malignant neuroepithelial tumors that presumably origin from the neural crest [START_REF] Sen | Primitive neuroectodermal tumor of the orbit[END_REF] . According to the third edition of International Classification of Childhood Cancer [START_REF] Steliarova-Foucher | International Classification of Childhood. Cancer, Third Edition[END_REF] and to the 2007 World Health Organization [START_REF] Rousseau | The 2007 WHO classification of tumors of the central nervous system -what has changed?[END_REF] , PNETs are embryonal neoplasms that occur predominantly in children and young adults.

The majority of PNETs arise in the central nervous system and in the sympathetic nervous system. When they occur outside these locations they are called pPNETs being the thoracopulmonary region the most common primary site of pPNET, followed by the head and neck [START_REF] Miller | Primitive neuroectodermal tumour of the chest wall (Askin tumour): CT and roentgenographic findings in a 51-year-old male[END_REF][START_REF] Jones | Peripheral primitive neuroectodermal tumors of the head and neck[END_REF][START_REF] Jurgens | Malignant peripheral neuroectodermal tumors. A retrospective analysis of 42 patients[END_REF] .

There is considerable controversy concerning the histogenesis of pPNET. Different hypotheses have been postulated to explain the presence of PNET out of the central nervous system and the sympathetic nervous system: spread out of undifferentiated neuroepithelial cells to the peripheral tissues from the neural tube; migration of neuroepithelial cells in association with peripheral nerves [START_REF] Shuangshoti | Primary intraorbital extraocular primitive neuroectodermal (neuroepithelial) tumour[END_REF][START_REF] Howard | Neuroepithelioma of the orbit[END_REF] .

PPNET represents 4-17% of all paediatric soft tissue tumors and appears more often in adolescents and young adults with no sex predilection [START_REF] Dehner | Primitive neuroectodermal tumor and Ewing's sarcoma[END_REF][START_REF] Kushner | Extracranial primitive neuroectodermal tumors: the Memorial Sloan Kettering Cancer Center experience[END_REF] .

Primary orbital pPNET is very infrequent, only 16 cases have been reported in the literature. There is an additional article published as an orbital pPNET [START_REF] Bansal | Primitive neuroectodermal tumour of the orbit: a case report[END_REF] Ultrastructural studies by electron microscopy show the presence of neurosecretory granules, cytoplasmic filaments, cytoplasmic microtubules and synaptic-like junctions.

Electron microscopy is a very useful technique but it is not available in many centers.

Although in the case we present, no rosettes were identified on light microscopy, neural differentiation was established immunohistochemically. Unluckily we could not access to studies with electron microscopy in order to confirm ultrastructurally the diagnosis.

With the improvement of the diagnostic techniques, probably more cases of pPNET will be reported in the future and it is possible that some so-called extraosseous Ewing's sarcoma, today would have been diagnosed as pPNET.

PPNET are aggressive tumors. Bone invasion and extraorbital extension have been described in the published cases of orbital pPNET [START_REF] Lezrek | Orbital primitive neuroectodermal tumor with intracranial extension. A case report[END_REF][START_REF] Sen | Primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Singh | Primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Wilson | Primary peripheral neuroepithelioma of the orbit with intracranial extension[END_REF][START_REF] Howard | Neuroepithelioma of the orbit[END_REF] , but only two cases showed alive for at least 30 months. Longer follow-up of the patients is needed to evaluate the long-term prognosis of orbital pPNET.

In conclusion, management of these tumors must be aggressive using multi-discipline treatment and a regular follow-up is mandatory to rule out recurrences and metastases.

Further studies are necessary to determinate the best therapeutic guideline.

  . -: absent. N/A: non available. NSE: neuron-specific enolase. S100: S100 protein. NF: neurofilaments. SYTPH: Synaptophysin. GFAP: glial fibrillary acid protein. CHT: chemotherapy. RT: radiotherapy.

  ), and vimentin. Positive staining for the cited neural markers is very suggestive of pPNET, but it is important to consider the lack of specificity of most neural markers and the variable immunohistochemical features of pPNET.

	neural differentiation. Immunohistochemical and/or ultrastructural techniques are
	essential for this purpose. Ewing's sarcoma has a better prognosis than pPNET, making
	necessary the distinction between these two entities. Schmidt et al reported a disease-
	free survival rate in Ewing's sarcoma patients at 7.5 years follow-up of 60% compared
	with 45% in pPNET patients 33 .
	but this case As mentioned above, histopathologically, pPNET demonstrates varying grades of neural
	corresponds to a melanotic neuroectodermal tumor with distinct clinical and differentiation. Microscopically we can see Homer-Wright rosette formation;
	histopathological features. identification of Homer-Wright rosettes is associated with these tumors but it is not
	Orbital pPNET tend to occur in younger patients in comparison with other pPNET, only diagnostic of pPNET. Immnunohistochemical studies can be positive for one or several
	3 of the 16 published cases had more than 13 years of age at the diagnosis. neural markers: neuron-specific enolase, synaptophysin, CD99 (MIC2 gene), glial
	Histopathologically pPNETs are composed of small, round, hyperchromatic cells with fibrillary acid protein, neurofilaments, chromogranin, S100 protein, CD57 (LEU-
	varying degrees of neural differentiation. Differential diagnosis should be made with 7/HNK-1
	other small round cell tumors as: Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma,
	lymphoma, osteogenic sarcoma and mesenchymal chondrosarcoma. In the past,
	pPNETs have been included within Ewing's sarcomas because they share microscopic,
	molecular and genetic abnormalities. Many authors consider that pPNET and
	extraosseous Ewing's sarcoma represent opposite ends of the same spectrum of
	malignancy 25 . Both tumors present the same genetic aberrations, that is, translocation
	(11;22)(q24;q12) 26,27,28 and less common, translocation (21;22)(q22;q12) 29,30 .
	Translocation (11;22)(q24;q12) results in the fusion of EWSR1 gene (Ewing's sarcoma
	gene) with the FL1 gene while in translocation (21;22)(q22;q12) the fusion occurs
	between EWSR1 gene and ERG gene. Other chromosomal rearrangements:
	translocation (7;22)(p22;q12) 31 , translocation (2;22)(q33;q12) 31 , translocation
	(17;22)(q12;q12) 29 , and translocation (4;19)(q35;q13) 32 are rare. PPNET can be
	distinguished from Ewing's sarcoma and other small round cell tumors by establishing

months

systemic metastases [START_REF] Romero | Recurrence of peripheral primitive neuroectodermal tumor of the orbit with systemic metastases[END_REF][START_REF] Hyun | Metastatic peripheral primitive neuroectodermal tumor (PNET) masquerading as liver abscess: a case report of liver metastasis in orbital PNET[END_REF] , and regional lymph node involvement was found in another case [START_REF] Howard | Neuroepithelioma of the orbit[END_REF] . None of the two cases with systemic metastases showed local bone invasion or extraorbital extension. Common locations for metastases from pPNETs include bone marrow, bone, brain, and lungs. The lungs [START_REF] Romero | Recurrence of peripheral primitive neuroectodermal tumor of the orbit with systemic metastases[END_REF] and the liver [START_REF] Hyun | Metastatic peripheral primitive neuroectodermal tumor (PNET) masquerading as liver abscess: a case report of liver metastasis in orbital PNET[END_REF] are the sites of the metastases reported in the two cases of orbital pPNET. The lack of metastases in the orbital form of pPNET could be explained by the poor lymphatic system of the orbit.

There is no general consensus for the best therapeutic strategy for pPNET and a therapeutic approach similar to that utilized for Ewing's sarcoma has been used because of similarities between the two tumors [START_REF] Huerta | High Risk Ewing's sarcomas. Effectiveness and toxicity of a regimen designed for children and used in adults[END_REF][START_REF] Thacker | Current treatment for Ewing's sarcoma[END_REF][START_REF] Elomaa | Five-year results in Ewing's sarcoma. The Scandinavian Sarcoma Group experience with the SSG IX protocol[END_REF][START_REF] Iwamotoy | Diagnosis and treatment of Ewing's sarcoma[END_REF] . Surgery has been the initial treatment for orbital pPNET in most cases and has been the initial therapeutic approach used in our patients, althoug some of the published cases have been treated with chemoteraphy and radiotherapy without surgery, with fairly good results [START_REF] Kim | Multimodality treatment approach in management of primary peripheral primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Lezrek | Orbital primitive neuroectodermal tumor with intracranial extension. A case report[END_REF][START_REF] Singh | Primitive neuroectodermal tumor of the orbit[END_REF] . When performed, surgery should remove the tumor completely but this might be technically difficult.

Chemotherapy +/-radiotherapy is considered by many authors as the best additional treatment [START_REF] Kim | Multimodality treatment approach in management of primary peripheral primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Romero | Primary peripheral primitive neuroectodermal tumour of the orbit: case report[END_REF][START_REF] Lezrek | Orbital primitive neuroectodermal tumor with intracranial extension. A case report[END_REF][START_REF] Hyun | Metastatic peripheral primitive neuroectodermal tumor (PNET) masquerading as liver abscess: a case report of liver metastasis in orbital PNET[END_REF][START_REF] Alyahya | Primitive neuroectodermal tumor of the orbit in a 5-year old girl with microphthalmia[END_REF][START_REF] Singh | Primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Wilson | Primary peripheral neuroepithelioma of the orbit with intracranial extension[END_REF][START_REF] Kushner | Very-high-dose short-term chemotherapy for poor-risk peripheral primitive neuroectodemal tumors, including Ewing's sarcoma, in children and young adults[END_REF] . Recent studies suggest that multi-discipline treatment including systemic chemotherapy and surgery could prolong survival in patients with Ewing's sarcoma family tumors [START_REF] Peng | Efficacy and survival of 92 cases of Ewing's sarcoma family of tumor initially treated with multidisciplinary therapy[END_REF] . The mentioned study reports that CAV/IE (Cyclophosphamide, Adriamycin, Vincristine/ Ifosfamide, Etoposide) scheme might be better than other chemotherapy schemes. This chemotherapy modality has been used with good results in some of the latest cases of orbital pPNET published [START_REF] Kim | Multimodality treatment approach in management of primary peripheral primitive neuroectodermal tumor of the orbit[END_REF][START_REF] Romero | Primary peripheral primitive neuroectodermal tumour of the orbit: case report[END_REF] , and it is very similar to the chemotherapy protocol used in our patient following the Spanish Society of Paediatric Oncology (SEOP) 2001 recommendations for Ewing's sarcoma [START_REF] Huerta | High Risk Ewing's sarcomas. Effectiveness and toxicity of a regimen designed for children and used in adults[END_REF] . Other authors defend the role of radiotherapy in the treatment of these tumors. Laskar et al suggest that radiation remains an important component in the multimodality management of Ewing's sarcoma family tumors, recommending the use of post-operative radiotherapy when gross or microscopic positive margins are reported and when there are clear margins but there is a poor histopahtological response to chemotherapy [START_REF] Laskar | Post-operative radiotherapy for Ewing sarcoma: when, how and how much?[END_REF] ; according to the Cooperative Ewing Sarcoma Study (CESS) and to the European Intergroup Cooperative Ewing Sarcoma Study (EICESS) a poor histopathological response is considered when necrosis is less than 90% 40 however other authors show better results when the necrosis threshold is 95% or even 99% [START_REF] Elomaa | Five-year results in Ewing's sarcoma. The Scandinavian Sarcoma Group experience with the SSG IX protocol[END_REF][START_REF] Oberlin | Prognostic factors in localized Ewing's tumours and peripheral neuroectodermal tumours: the third study of the French Society of Paediatric Oncology (EW88 study)[END_REF] .

The timing of post-operative radiation is an issue to be solved, some authors recommend the radiation after surgery within 6-10 weeks of surgery [START_REF] Laskar | Post-operative radiotherapy for Ewing sarcoma: when, how and how much?[END_REF][START_REF] Dunst | Radiation therapy as local treatment in Ewing´s sarcoma. Results of the Cooperative Ewing´s Sarcoma Studies CESS 81 and CESS 86[END_REF] , while others report no significant difference with further delay [START_REF] Schuck | Postoperative radiotherapy in the treatment of Ewing tumors: Influence of the interval between surgery and radiotherapy[END_REF] .

The best survival results in the orbital pPNET patients, have been obtained in those treated with surgery, chemotherapy and radiotherapy. Five of the published patients, and the present case, have been treated with such therapeutic combination and the survival time has been longer than thirty months for all of them [START_REF] Romero | Recurrence of peripheral primitive neuroectodermal tumor of the orbit with systemic metastases[END_REF][START_REF] Hyun | Metastatic peripheral primitive neuroectodermal tumor (PNET) masquerading as liver abscess: a case report of liver metastasis in orbital PNET[END_REF][START_REF] Alyahya | Primitive neuroectodermal tumor of the orbit in a 5-year old girl with microphthalmia[END_REF][START_REF] Wilson | Primary peripheral neuroepithelioma of the orbit with intracranial extension[END_REF] , except for one patient that was seventeen months [START_REF] Romero | Primary peripheral primitive neuroectodermal tumour of the orbit: case report[END_REF] .

New therapeutic approaches based on Ewing's sarcoma biology, such as anti-vascular endothelial growth factors [START_REF] Adamo | Phase II study of doxorubicin and bevacizumab for patients with metastatic soft-tissue sarcomas[END_REF][START_REF] Dalal | Vascular endothelial growth factor: a therapeutic target for tumors of the Ewing's sarcorma family[END_REF] , anti-insluin-like growth factors [START_REF] Benini | Inhibition of insulin-like growth factor I receptor increases the antitumor activity of doxorubicin and vincristine against Ewing´s sarcoma cells[END_REF][START_REF] Benini | Insulin-like growth factor binding protein 3 as an anticancer molecule in Ewing's sarcoma[END_REF] retinoic acid derivatives [START_REF] Myatt | The sensitivity of the Ewing´s sarcoma family of tumours to fenretinide-induced cell death is increased by EWS-Fli-dependent modulation of p38(MAPK) activity[END_REF][START_REF] Myatt | p38MAPK-Dependent sensitivity of Ewing´s sarcoma family of tumours to fenretinide-induced cell death[END_REF] , and agents that interfere with the mTOR (mammalian target of rapamycin) [START_REF] Mateo-Lonzano | Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation[END_REF] are on study and represent promising future therapeutic options.

PPNETs have rapid progression and bad prognosis, metastases and local extension are often found during initial diagnosis. The overall survival rate of pPNET is poor, Kushner et al documented a progression-free survival of localized tumors greater then 5 centimetres of 25% at 24 months 51 . Orbital variety seems to be less aggressive than other forms of pPNET since most of the patients reported were alive after the follow up period (at least 6 months), and since seven of the seventeen patients (41'2%) have been


MD Stefan Reuter
email: stefan.reuter@med.uni-duesseldorf.de

Mark Oette

Nadine Sichtig

Rolf Kaiser

Melanie Balduin

Bjoern Jensen

Dieter Häussinger

Björn Jensen

Changes in the HIV-1 Mutational Profile Before First-line HAART in the RESINA Cohort

Keywords: resistance, selection pressure, naïve, transmission, mutational pattern resistance, selection pressure, naïve, transmission, mutational pattern

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

INTRODUCTION

Transmitted drug resistance mutations (TDRMs) affect significantly the success of firstline antiretroviral treatment [START_REF] Johnson | Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy[END_REF][START_REF] Poggensee | Impact of transmission of drug-resistant HIV on the course of infection and the treatment success. Data from the German HIV-1 Seroconverter Study[END_REF][START_REF] Von Hentig | Predictive factors for response to a boosted dual HIV-protease inhibitor therapy with saquinavir and lopinavir in extensively pre-treated patients[END_REF][START_REF] Ghosn | HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time[END_REF][START_REF] Little | Antiretroviral-drug resistance among patients recently infected with HIV[END_REF][START_REF] Harzic | Genotypic drug resistance during HIV-1 primary infection in France (1996-1999): frequency and response to treatment[END_REF], Tang and Pillay, 2004[START_REF] Simen | Low-abundance drug-resistant viral variants in chronically HIVinfected, antiretroviral treatment-naive patients significantly impact treatment outcomes[END_REF]. TDRMs in primary HIV infection have been recognized as a relevant problem for patient management [START_REF] Little | Antiretroviral-drug resistance among patients recently infected with HIV[END_REF][START_REF] Yerly | Transmission of HIV-1 drug resistance in Switzerland: a 10-year molecular epidemiology survey[END_REF], driving the World Health Organization (WHO) to recommend continued surveillance of primary resistance through national HIV/AIDS control programs [START_REF] Lazzari | HIV drug resistance surveillance: summary of an April 2003 WHO consultation[END_REF]. The prevalence of primary resistance varies significantly between studies and between geographical locations, ranging from 2% to 51% [START_REF] Varella | Human immunodeficiency virus type 1 protease and reverse transcriptase mutation patterns among treatment-naive patients in different stages of infection in Rio de Janeiro, Brazil[END_REF][START_REF] Dilernia | Drugresistance surveillance among newly HIV-1 diagnosed individuals in Buenos Aires, Argentina[END_REF][START_REF] Han | Genotypic resistance mutations to antiretroviral drugs in treatment-naive HIV/AIDS patients living in Liaoning Province, China: baseline prevalence and subtype-specific difference[END_REF][START_REF] Chang | Trends of antiretroviral drug resistance in treatment-naive patients with human immunodeficiency virus type 1 infection in Taiwan[END_REF][START_REF] Oette | Primary drug-resistance in HIV-positive patients on initiation of first-line antiretroviral therapy in Germany[END_REF][START_REF] Palacios | Prevalence of primary resistance mutations in patients with newly diagnosed HIV infection in the province of Malaga (Spain).][END_REF][START_REF] Sanchez | Primary antiretroviral drug resistance among patients diagnosed with HIV infection in Gran Canaria (Spain) between 2002 and 2005[END_REF][START_REF] Horban | High prevalence of genotypic resistance to nucleoside reverse transcriptase inhibitors among therapy-naive individuals from the Warsaw cohort[END_REF]. In Germany, the prevalence of primary resistance has ranged above 10% in various studies, and thus has an impact on the management of HIV infection [START_REF] Sagir | Trends of prevalence of primary HIV drug resistance in Germany[END_REF][START_REF] Oette | Primary HIV drug resistance and efficacy of first-line antiretroviral therapy guided by resistance testing[END_REF][START_REF] Poggensee | Impact of transmission of drug-resistant HIV on the course of infection and the treatment success. Data from the German HIV-1 Seroconverter Study[END_REF][START_REF] Duwe | Frequency of genotypic and phenotypic drug-resistant HIV-1 among therapynaive patients of the German Seroconverter Study[END_REF].

The consideration of TDRMs for tailoring first-line treatment has led to improved outcomes, with similar treatment efficacy as compared to patients without primary resistance [START_REF] Oette | Primary HIV drug resistance and efficacy of first-line antiretroviral therapy guided by resistance testing[END_REF][START_REF] Shet | Tracking the prevalence of transmitted antiretroviral drug-resistant HIV-1: a decade of experience[END_REF]. The determination of the resistance profile in treatment-naïve patients infected by HIV has now become standard of care and is recommended in major guidelines (German-Austrian guidelineset al., 2009[START_REF] Clumeck | European AIDS Clinical Society (EACS) guidelines for the clinical management and treatment of HIV-infected adults[END_REF][START_REF] Hirsch | Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 recommendations of an International AIDS Society-USA panel[END_REF].

Sporadic observations have been made about changing patterns of TDRMs even without selective pressure by antiretroviral treatment. For example, higher mutation rates have been described immediately after seroconversion as compared to a further progressed time point during chronic infection [START_REF] Sanchez | Primary antiretroviral drug resistance among patients diagnosed with HIV infection in Gran Canaria (Spain) between 2002 and 2005[END_REF]. In other cases, persistence of resistant

Selection of patients

Written informed consent was obtained from each patient prior to inclusion into the study.

According to the protocol, testing for primary resistance in the German RESINA cohort should be performed uniformly at the time when initiation of antiretroviral treatment (ART) is imminent. In some patients, however, testing for primary resistance had been performed without subsequent initiation of ART, because the responsible physician had ultimately decided to postpone the start of treatment. Review of the patient chart and interviews with the responsible physicians revealed that retesting was performed because the first analysis had been premature without the necessity to immediately start treatment.

As a consequence, resistance testing was repeated in these cases immediately before the beginning of ART. In the present study, all of these longitudinal resistance analyses were studied, no patient was excluded.

Genotypic resistance testing

Viral RNA was isolated from patient plasma using QIAamp viral RNA mini-kit (Qiagen, Hilden, Germany). Reverse transcription and polymerase chain reaction were performed HotStarTaq kit (Qiagen, Hilden, Germany) and the primer pair Res3 und Res4 (3RES:5´ T CTT GAT AAA TTT GAT ATG TCC 3´ 4RES:5´ AGA CAG GCT AAT TTT TTA GGG A 3´).

PCR products were purified using the QIAquick spin PCR purification kit (Qiagen, Hilden, Germany) and extended using the ViroSeq HIV-1 Genotyping System sequencing module (Applied Biosystems, Foster City, CA, USA). Extension products were purified using MultiScreen purification plates (Millipore, Bedford, MA, USA) and Sephadex G-50 superfine (Amersham Biosciences, Upsala, Sweden) and were run on an ABI PRISM 3100

Avant . Sequence data were generated by using Sequencing Analysis v3.4 (Applied Biosystems, Foster City, CA, USA). The sequences were edited using the ViroSeq HIV-1

Genotyping software v2.6 (Applied Biosystems, Foster City, CA, USA).

Multidrug resistance

Multidrug resistance (MDR) was defined as concurrent major resistance mutations against the drug classes NRTIs (= nucleoside reverse transcriptase inhibitors), NNRTIs (= nonnucleoside reverse transcriptase inhibitors) and PIs (= protease inhibitors).

Phylogenetic Analysis

The Arevir database was searched for similar sequences and the resulting set of 54 sequences, together with the HxB2 reference strain and 8 unrelated sequences, were analyzed with (Publication PDF at http://www.kumarlab.net/publications). The phylogenetic tree was constructed using the neighbor-joining method. A bootstrap test with 1000 replications was used to obtain the confidence values.

Statistical analysis

In this pilot study descriptive statistics were calculated, such as frequencies of categorical outcomes, median and range of continuous variables. with decreasing mutations).

Several new mutations were found in the follow-up analysis (table 2, group B1). The median time between both resistance analyses was 335 days (average 299, range 17-491).

These increasing mutations were most prevalent in the RT gene (K65R, V75I, K103N, Y181C, M184I, L210W, T215I), while only one of the emerging mutations was found in the PR gene (M46I). Homologies between sequences for patients 1 to 5 (table 2) were as follows: 99.07%, 99.05%, 97.65%, 99.54% and 99.46%, the close relationship between samples can be evaluated additionally in figure 1.

In 10 out of 18 cases with TDRMs persisting mutations were found over time (

DISCUSSION

The RESINA project is a prospective multicenter study on the epidemiology of primary drug resistance and treatment outcome in North Rhine-Westphalia, Germany. In this project, ongoing surveillance of transmitted resistance mutations has been conducted since 2001 [START_REF] Sagir | Trends of prevalence of primary HIV drug resistance in Germany[END_REF][START_REF] Oette | Primary HIV drug resistance and efficacy of first-line antiretroviral therapy guided by resistance testing[END_REF], with more than 2000 patients included to date.

Primary drug resistance was determined in patients infected chronically with unknown date of infection. The present study comprised 49 treatment-naïve patients infected chronically with HIV. These patients received two resistance tests before initiation of antiretroviral therapy. TDRMs were detected in 18 patients and a changing mutational pattern was found in 8 out of 18 patients with TDRMs over time.

The TDRM rate of 36.7% (18/49) of our patients is much higher than the estimated rate of 10% in Germany [START_REF] Sagir | Trends of prevalence of primary HIV drug resistance in Germany[END_REF][START_REF] Oette | Primary HIV drug resistance and efficacy of first-line antiretroviral therapy guided by resistance testing[END_REF][START_REF] Poggensee | Impact of transmission of drug-resistant HIV on the course of infection and the treatment success. Data from the German HIV-1 Seroconverter Study[END_REF][START_REF] Duwe | Frequency of genotypic and phenotypic drug-resistant HIV-1 among therapynaive patients of the German Seroconverter Study[END_REF]. Due to the small number of patients, the number of patients with TDRM cannot be considered representative in this sub analysis of the RESINA cohort. Furthermore, due to the retrospective nature of this study it cannot be excluded that physicians performed repeated testing more readily in patients with known TDRM.

Several new mutations were found in the follow-up analysis in 5 patients (table 2).

To date, accumulation of resistance mutations in treatment-naïve patients have been described rarely. Despite persistence of mutations, no increase of mutations was observed in several other studies [START_REF] Fox | Transmitted drug-resistant HIV-1 in primary HIV-1 infection; incidence, evolution and impact on response to antiretroviral therapy[END_REF][START_REF] Ghosn | HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time[END_REF][START_REF] Pao | Long-term persistence of primary genotypic resistance after HIV-1 seroconversion[END_REF][START_REF] Barbour | Persistence of primary drug resistance among recently HIV-1 infected adults[END_REF], Cane et al., 2009). In analogy to the present results, in one case of primary infection development of 2 additional RT mutations (M184V and V75M) was noted after 5 months of infection [START_REF] Chan | Prolonged retention of drug resistance mutations and rapid disease progression in the absence of therapy after primary HIV infection[END_REF].

Mutations may be acquired newly through superinfection with a second strain [START_REF] Allen | HIV-1 superinfection[END_REF]. Homologies between sequences for our patients with increasing mutations were above 97.6% in all 5 cases, and therefore the hypothesis of superinfection was unlikely in these cases. The phylogenetic tree (figure 1) confirms the very close relationships between the respective sample pairs. An alternative explanation is the emergence of new resistance mutations through selection pressure exerted by the immune system of the host. Some mutations which confer drug resistance incidentally can also enable the virus to evade recognition by the cytotoxic T-lymphocytes (CTL) and are therefore selected even in the absence of drugs as an immune-escape mutant [START_REF] Mueller | Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease[END_REF]. An increasing body of evidence suggests that a considerable proportion of transmitted HIV-1 drug resistance is not detected by conventional genotyping and that minority strains harbouring resistance mutations can have clinical consequences [START_REF] Johnson | Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy[END_REF][START_REF] Metzner | Minority quasispecies of drug-resistant HIV-1 that lead to early therapy failure in treatment-naive and -adherent patients[END_REF][START_REF] Simen | Low-abundance drug-resistant viral variants in chronically HIVinfected, antiretroviral treatment-naive patients significantly impact treatment outcomes[END_REF]. The dynamics of HIV quasispecies with changing proportions of the population can result in the detection of minority strains in a repeated standard resistance testing. Furthermore, a genetic viral diversity in different sites of the body has been observed, and from these reservoirs the re-emergence of resistant viral strains appears plausible [START_REF] Lambotte | The lymphocyte HIV reservoir in patients on long-term HAART is a memory of virus evolution[END_REF].

In 10 out of 18 cases with TDRMs persisting mutations were found over time (table 2). It must be noted that in the group with persisting TDRMs (B2) the interval between genotypic resistance analyses was shorter as compared to the groups with changes in the mutational pattern over time (B1 and B3). The frequency of mutational changes over time may therefore have been underestimated. In accordance with the present findings, [START_REF] Fox | Transmitted drug-resistant HIV-1 in primary HIV-1 infection; incidence, evolution and impact on response to antiretroviral therapy[END_REF] had observed persisting TDRMs in 8 out of 9 patients. Seven patients showed single-class mutations and in 2 cases multiple mutations were found. In contrast to the rapid disappearance of secondary mutations in patients with failure of ART [START_REF] Deeks | Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIVinfected patients with detectable viremia[END_REF], primary mutations in treatment-naïve patients may persist for long periods of time [START_REF] Chan | Prolonged retention of drug resistance mutations and rapid disease progression in the absence of therapy after primary HIV infection[END_REF]. [START_REF] Ghosn | HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time[END_REF] also found that acquired resistant viruses establish themselves as the dominant viral population at primary infection. A large diversity of RT mutations without PR mutations was found among the 7 cases with persisting single-class resistance patterns: 3x M41L, Y188L, 2x K219Q, T215A, T215CS, 2x T215D and P225H. In the present analysis, persistence of TDRMs in the PR gene was not observed in patients with single class mutations. This finding is supported by a previous study [START_REF] Pao | Long-term persistence of primary genotypic resistance after HIV-1 seroconversion[END_REF]. As an explanation for this finding, [START_REF] Wirden | Comparison of the dynamics of resistance-associated mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors after cessation of antiretroviral combination therapy[END_REF] observed that the kinetics of the shift to wild-type amino acid residues were significantly faster for PR mutations than for mutations in the RT gene.

Some mutant viruses may possess a better replicative capacity in comparison to wild-type virus. It can therefore be hypothesized that in contrast to wild-type-virus, long-term persistence of certain mutations is advantageous for a viral population. Several of the TDRMs found have been shown previously to persist after transmission even in the absence of antiretroviral therapy, including D67N, K70R, M41L, T69N and some T215 variants, [START_REF] Barbour | Persistence of primary drug resistance among recently HIV-1 infected adults[END_REF][START_REF] Pao | Long-term persistence of primary genotypic resistance after HIV-1 seroconversion[END_REF] as well as the K103N mutation [START_REF] Ghosn | HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time[END_REF][START_REF] Capetti | Can a K103N HIV strain stably overcome the wild type in the absence of non-nucleoside reverse transcriptase inhibitor selective pressure?[END_REF], Cane et al., 2009). Interestingly, in some cases persistence of TDRMs was documented for up to 5 years [START_REF] Brenner | Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection[END_REF].

Two multi-resistant virus strains were detected, each with identical resistance patterns over time (table 2, samples 12 and 14). Long-term persistence of MDR virus over several years in the treatment-naive setting has been observed [START_REF] Pao | Long-term persistence of primary genotypic resistance after HIV-1 seroconversion[END_REF][START_REF] Brenner | Persistence of multidrug-resistant HIV-1 in primary infection leading to superinfection[END_REF][START_REF] Brenner | Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection[END_REF], Delaugerre et al., 2004) and was not outgrown by a virus strain with wild-type sequence. As an explanation for this finding, it has been hypothesized that primary mutations can be transmitted in a homogeneous viral species without coexisting wild-type virus [START_REF] Simon | Evolving patterns of HIV-1 resistance to antiretroviral agents in newly infected individuals[END_REF]. In these cases, there would be no competition with a wild-type virus.

While a primary transmitted M184V mutation persisted over a 9-month observation period in one study [START_REF] Barbour | Persistence of primary drug resistance among recently HIV-1 infected adults[END_REF], mutations such as K65R and M184V in the RT gene are known to cause a considerable reduction in viral fitness. They may therefore disappear more rapidly but remain within the viral archive and may be detectable using more sensitive assays than those in current routine use (Caneet al., 2005). This concurs with the present observation that neither the K65R nor the M184V mutation was observed to persist over extended periods of time (table 2). Various other TDRMs, such as Y181C, K219Q [START_REF] Barbour | Persistence of primary drug resistance among recently HIV-1 infected adults[END_REF][START_REF] Pao | Long-term persistence of primary genotypic resistance after HIV-1 seroconversion[END_REF] and D67N [START_REF] Barbour | Persistence of primary drug resistance among recently HIV-1 infected adults[END_REF] have been described as reverting to wild type. In addition, there is evidence for a reversion of the T215Y mutation to a fitter revertant virus, such as T215S/D/C [START_REF] Fox | Transmitted drug-resistant HIV-1 in primary HIV-1 infection; incidence, evolution and impact on response to antiretroviral therapy[END_REF][START_REF] Ghosn | HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time[END_REF][START_REF] Pao | Long-term persistence of primary genotypic resistance after HIV-1 seroconversion[END_REF][START_REF] Barbour | Persistence of primary drug resistance among recently HIV-1 infected adults[END_REF][START_REF] Brenner | Persistence of multidrug-resistant HIV-1 in primary infection leading to superinfection[END_REF], Caneet al., 2005). Two out of 3 of our patients with decreasing mutations over time (patients 17 and 18, table 2)

showed high homologies between sequences. However, in patient 16 it remains unclear whether the markedly lower similarity between the sequences (93.01%) is the result of superinfection or a laboratory error (sample swapping, contamination).

It may be concluded from these data that in order to detect as many TDRMs as possible, resistance testing should be performed soon after establishing the diagnosis.

The above results demonstrate that the resistance pattern can change without selection pressure by antiretroviral drugs, but may be driven by viral dynamics of strains with different resistance patterns and the varying ability of the immune system to display and recognize the different epitopes of the various viral gene products with resistance mutations (so called "escape mutants"). Another important factor for the persistence or reversion of resistance mutations in the absence of selective drug pressure is their impact on the viral replicative capacity or "viral fitness".

While genotypic resistance testing is now recommended uniformly in all major treatment guidelines for treatment-naive patients, the recommended timing for genotypic resistance testing varies. In Germany and Austria resistance testing is recommended prior to start of [START_REF] Guidelines | Antiretroviral therapy of HIV infection (position in September 2008)][END_REF][START_REF] Clumeck | European AIDS Clinical Society (EACS) guidelines for the clinical management and treatment of HIV-infected adults[END_REF][START_REF] Hirsch | Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 recommendations of an International AIDS Society-USA panel[END_REF].

Without the knowledge of the individual infection chain, our findings suggest an important role for drug resistance testing both at baseline and before the start of antiretroviral treatment in naïve patients.

Without antiretroviral treatment, a trend toward more pronounced progression of disease was observed in the group without TDRMs (group A): CD4 cells decreased by a median of 91cells /µl between the two resistance analyses. In contrast, in the group with detected TDRMs (group B) CD4 cells did not change significantly over time (median of 1 cell /µl).

In analogy to the more pronounced CD4 cell decrease in group A, an increase in viral load by a median of 11,798 copies /ml was observed in this group, while in group B the viral load did not increase over time (-10,392 copies /ml). A subgroup analysis in group B revealed that the improved immune status over time was found in patients with an unchanged mutational pattern (B2 in table 3), while increases or decreases in mutations tended to be associated with a stable or a deteriorating immune function.

Conflicting results are found in the literature with regard to alterations of immunological parameters. In contrast to the above observations, a faster rate of CD4 decline was observed in TDRMs by [START_REF] Fox | Transmitted drug-resistant HIV-1 in primary HIV-1 infection; incidence, evolution and impact on response to antiretroviral therapy[END_REF] and other investigators found comparable rates of CD4 decline between TDRMs and wild-type HIV [START_REF] Bhaskaran | Do patients who are infected with drug-resistant HIV have a different CD4 cell decline after seroconversion? An exploratory analysis in the UK Register of HIV Seroconverters[END_REF].

In support of the present findings, Cane et al. (2009) showed that TDRMs remained stable for up to two years and that this stable situation was associated with low viral loads. The finding of low viral loads in TDRMs was first described by [START_REF] Salomon | Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study[END_REF] and may be attributable to a compromised replicative capacity of some resistant HIV strains F o r P e e r R e v i e w [START_REF] Brenner | Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection[END_REF], hypothetically enabling the host immune system to exert greater control than has been seen in most patients infected with wild-type virus.

Several limitations of the present analysis must be acknowledged, as the small number of patients may imply inherent bias. Rates of disease progression may have been influenced

by the retrospective study approach and the clinician-driven selection of patients.

Furthermore, the prevalence of transmitted drug resistance may have been underestimated, as population sequencing frequently fails to detect low-frequency variants. This problem could be solved by "deep sequencing", but these techniques are currently not established as routine methods. A prospective study in a larger, random group of patients will be indispensable to confirm the present data regarding the clinically-relevant question of changing mutational patterns and their influence on disease progression.

Conclusions

In this longitudinal analysis of primary HIV resistance mutations, a high frequency of

  the study population Between the years 2001 and 2008 a longitudinal analysis of resistance mutations was performed in HIV-positive treatment-naïve patients aged >18 years. Two blood samples were analyzed at different time-points by population sequencing for resistance mutations in the protease (PR) and reverse transcriptase (RT) of the HIV-1 pol-gene. Only major mutations were scored as resistance-associated according to the current Stanford HIV Drug Resistance Database (November 6, 2009), (available at http://hivdb.stanford.edu).

  Clustal and Mega4 (Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.

  analyzed in 49 HIV-positive treatment-naïve patients. Group A consisted of patients with wild-type virus at the baseline and during follow-up. Group B comprised patients with detection of resistance mutations either at the baseline or during follow-up. Median time between resistance tests in group A (n=31 patients) were 146 days (average 391 days, range 17-2002 days) and 297 days (average 302 days, range 9-2066 days) in group B (n=18 patients).

Figure 1 :

 1 Figure 1: Phylogenetic relationships between the HIV-1 protease/reverse transcriptase

Table 1

 1

depicts the homogeneous distribution of baseline characteristics between group A (without TDRMs) and group B (with TDRMs). No major differences between groups were noted with regard to age, gender, mode of transmission, CD4 count, viral load and HIVsubtype.

TDRMs were detected in 18 out of 49 patients (36.78%) (group B). A changing mutational profile in the major viral population was found in 8 out of these 18 patients between the two resistance analyses over time (table 2, groups B1 with increasing mutations and B3

  In 3 patients decreasing mutations were found over time(table 2, group B3). The median time between both resistance analyses was 759 days (average 713, range 458-921). Single mutations lost were the RT mutation M184V and the revertant T215S. In the third case, multiple TDRMs in the RT gene reverted to wild type in the second analysis after 759 days,

	Reuter et al.	Changes in transmitted drug resistance	9/25
	including M184V, K103N, V108I and G190A. Homologies between sequences for patients
	16, 17 and 18 (table 2) were 93.01%, 99.33% and 98.29%, respectively.
	Transmitted MDR virus was found in 2 cases and both showed identical resistance patterns
	F o r over time (table 2, patients 12 and 14). They harboured the following mutations: Patient 12: PR: F53L, V82A, L90M, RT: K103N, Y181C, L210W, T215D Patient 14: PR: L24I, M46I, V82A, I84V, RT: D67G, K103N, T215V, K219Q F o r
		P P
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		v i e v i e
		w w

table 2, group B2). The median time between both resistance analyses was 46 days (average 181, range 10-610). Single class mutations were found in 8 cases, while multidrug TDRMs were detected in 2 cases.

CD4 cells decreased by a median of 91 cells /µl between the two resistance analyses in group A (table

3

). In contrast, CD4 cells in the group with detected resistance mutations (group B) remained unchanged, with a median change of 1 cell /µl. In analogy to the more pronounced CD4 cell decrease in group A, an increase in viral load by a median of 11,798 copies /ml was observed in this group, while in group B the viral load decreased over time (-10,392 copies /ml). A subgroup analysis in group B revealed that the improved immune situation over time was found in patients with an unchanged mutational pattern (B2), while increases or decreases in mutations did not reveal this trend (B1 and B3).

  In contrast, European and U.S.Guidelines both recommend testing as part of the initial patient assessment, even if treatment is not initiated (German-Austrian guidelineset

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	treatment.
		F o r
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		e
		e r
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		v i e
		w

John Wiley & SonsJournal of Medical Virology

Journal of Medical Virology

ACKNOWLEDGEMENTS

We would like to thank Eugen Schülter, Claudia Müller and Angelika Hergesell for valuable help in data acquisition. We are indebted to Ms. Sandra Lessmann for reading the manuscript as a native speaker and for correcting grammatical errors. Furthermore, we are grateful for the valuable contributions from collaborating study centers: P. Arbter, Krefeld; R. Baumann, Neuss; I. Becker-Boost, Duisburg; A. Busch, S. Christensen, Münster; H. Carls, Düsseldorf; G. Fätkenheuer, Köln; B. Gantke, Düsseldorf; R. Gippert, P. Hartmann, H. Quaing, Münster; I. Greiffendorf, Krefeld; M. Grüneberg, Münster; M. Hower, Dortmund; K. Isernhagen, K. Römer, Köln; H. Knechten, P. Braun, R. Ehret, Aachen; W. Köthemann, A. Neuwirth, Köln; F. Kwirant, Duisburg; U. Marder, D. Petry, A. Strehlow, Düsseldorf; S. Mauruschat, Wuppertal; S. Mauss, G. Schmutz, Düsseldorf; T. Schumacher, A. Mutz, Osnabrück; M. Paffenholz, Köln; M. Radecki, Köln; M. Reith, Gottstein, Düsseldorf; K. Remberg, Krefeld; A. Rieke, Koblenz; J. K. Rockstroh, Bonn; M. Sandmann, J. Velardi, Wuppertal; M. Schäfer, Bielefeld; S. Schoelzel, Troisdorf; S. Scholten, Köln; T. Scholten, Hagen; S. Schons, Düsseldorf; T. Schumacher, Köln; D. Schuster, K. Schuster, Wuppertal; J. Stechel, Köln; M. Wichmann, Köln;W. Wiesel, A. Theisen, Köln.

FUNDING

The RESINA study is supported by a grant from the German Ministry of Health and Social Security (grant no. AZ 319-4476-02/3). Additional financial support was granted by the Heinz-Ansmann-Foundation for HIV-research. Page 16 of 25 John Wiley & Sons Journal of Medical Virology

Ethical approval

The present study was approved by the ethical committee of the University of Düsseldorf.

TABLES


MD Giuseppe Querques
email: giuseppe.querques@hotmail.it.

MD Elya Berboucha

MD Nicolas Leveziel

MD Alfredo Pece

MD Eric H Souied

Preferential Hyperacuity Perimeter in

Keywords: Age-related macular degeneration, choroidal neovascularization, metamorphopsia, optical coherence tomography, preferential hyperacuity perimeter, ranibizumab

come

Introduction

Visual acuity, the most widely diffuse measure of visual function, has shown poor degree of correlation with both qualitative (fluorescein angiography) and quantitative (optical coherence tomography [OCT]) assessments of macular morphology [START_REF] Doris | Relation between macular morphology and visual function in patients with choroidal neovascularisation of age-related macular degeneration[END_REF][START_REF] Hogg | Macular lesion composition and location influence visual function in AMD[END_REF][START_REF] Querques | Correlation of visual function impairment and optical coherence tomography findings in patients with adult-onset foveomacular vitelliform macular dystrophy[END_REF] . Also other less commonly used measures of function, such as contrast sensitivity, microperimetry, near acuity and reading speed, did not show better relationships with morphological features compared with visual acuity [START_REF] Mcclure | Correlations between visual function and performance of daily living tasks[END_REF][START_REF] Mitchell | The MacDQoL Individualized Measure of the Impact of Macular Degeneration on Quality of Life: Reliability and Responsiveness[END_REF][START_REF] Moutray | Relationships between clinical measures of visual function, fluorescein angiographic and optical coherence tomography features in patients with subfoveal choroidal neovascularisation[END_REF][START_REF] Browning | Relationship between optical coherence tomography-measured central retinal thickness and visual acuity in diabetic macular edema[END_REF] . Therefore, to date, there are still no simple and reproducible functional tests that reflect the morphological state of the macula in patients affected with diseases like exudative age-related macular degeneration (AMD).

The Prospective OCT Imaging of Patients with Neovascular AMD Treated with intraOcular Ranibizumab (PrONTO) Study [START_REF] Lalwani | A variable-dosing regimen with intravitreal ranibizumab for neovascular age-related macular degeneration: year 2 of the PrONTO Study[END_REF] showed that monthly visits, for both functional and morphological monitoring, are necessary in patients undergoing intravitreal anti-VEGF treatment for AMD, in order to take optimal re-treatment decisions [START_REF] Querques | Ranibizumab for exudative age-related macular degeneration: 24-month outcomes from a single-centre institutional setting[END_REF] . However, based on clinical experience, it seems that in such patients, visual acuity changes would lag behind morphological worsening. Therefore, availability of an objective and easily performed functional test, which well correlates with macular morphology, would be extremely helpful in the clinic setting.

Preferential Hyperacuity Perimetry (Foresee PHP®, Notal Vision, Israel) has recently gained popularity as a device for monitoring visual distortion, and has been used to localize functional deficits in the central 14 degrees of visual field in patients with AMD [START_REF] Alster | Preferential Hyperacuity Perimeter (PreView PHP) for detecting choroidal neovascularization study[END_REF][START_REF] Goldstein | Results of a multicenter clinical trial to evaluate the preferential hyperacuity perimeter for detection of agerelated macular degeneration[END_REF] .

Since 2005 Preferential Hyperacuity Perimeter has been proposed as a tool to detect CNV [START_REF] Alster | Preferential Hyperacuity Perimeter (PreView PHP) for detecting choroidal neovascularization study[END_REF] . Results of a multicenter clinical trial confirmed the usefulness of Preferential Hyperacuity Perimeter for detection of AMD [START_REF] Goldstein | Results of a multicenter clinical trial to evaluate the preferential hyperacuity perimeter for detection of agerelated macular degeneration[END_REF] . Recently, the home version of the Preferential Hyperacuity Perimeter, the ForeseeHome, which is based on a technology that is similar to the Foresee PHP, has shown a good sensitivity and specificity in discriminating between patients with newly diagnosed CNV and intermediate AMD [START_REF] Loewenstein | Toward earlier detection of choroidal neovascularization secondary to age-related macular degeneration: multicenter evaluation of a preferential hyperacuity perimeter designed as a home device[END_REF] .

Preferential Hyperacuity Perimeter has also been suggested as a tool for monitoring the therapeutic response to photodynamic therapy and anti-VEGF treatment in neovascular AMD [START_REF] Trevino | Recent progress in macular function self assessment[END_REF][START_REF] Das | Distortion maps from preferential hyperacuity perimetry are helpful in monitoring functional response to Lucentis therapy[END_REF] .

In this pilot study we investigated the ability of Foresee Preferential Hyperacuity Perimetry (PHP) in assessing responsiveness to ranibizumab therapy. With this aim, we analyzed the short-term functional and morphological fluctuations after intraviteal ranibizumab injection for exudative AMD.

Methods

Fourteen consecutive patients with exudative AMD in one eye received an intravitreal injection of ranibizumab (0.05 ml/0.5mg) at the University Eye Clinic of Creteil. In this prospective pilot study, we investigated the ability of PHP in assessing responsiveness to ranibizumab therapy. Before administration of ranibizumab, patients were informed about the experimental aspects of the study and the need for repeating several examinations at each scheduled follow-up visit. Criteria for inclusion were: 1) age ≥ 50 years old, 2) bestcorrected visual acuity (BCVA) between 20/200 and 20/40 as evaluated by Early Treatment Diabetic Retinopathy Study (ETDRS) charts, 3) presence of newly diagnosed subfoveal CNV secondary to AMD showing at least 50% active lesion as evaluated by fundus biomicroscopy, fluorescein angiography (FA) and infracyanine green angiography (ICGA), 4) subretinal fluid, cystic maculopathy, or 1-mm central macular thickness of at least 250 µm on OCT. Patients with previous treatment for exudative AMD in the study eye, previous surgery in the study eye in the last 6 months before intravitreal injection of ranibizumab (potentially influencing the macular status), refractive error of more than -6 diopters, CNV attributable to other causes than AMD, active intraocular inflammation or any other retinopathy (such as diabetic retinopathy, retinal venous occlusion or epiretinal membrane) in the study eye, were excluded.

Foresee PHP was performed 1 hour before the treatment (baseline) under the supervision of a trained physician (GQ). During the test, the patient's visual attention is drawn to a fixation cue displayed at the center of a touch screen. When the device automatically recognizes that the patient is ready to respond, a stimulus is briefly presented on the display. Each stimulus consists of a dotted line, of which a few dots are misaligned relative to the main axis of the stimulus. If the degree of misalignment exceeds a personal threshold, the patient may perceive an artificial distortion. The patient's task is to identify where the distortion appears, and to mark this location on the display. A succession of such stimuli is flashed on various locations of the display, such as to cover the entire macular field. When a stimulus is flashed on a location that corresponds to a healthy portion of the retina, the artificial distortion is readily detected. If the stimulus is flashed on a location that corresponds to a CNV lesion, RPE elevation may create the perception of a pathological distortion. In the patient's perception, competition for visual attention takes place between the artificial and pathological distortions. If the pathological distortion is more prominent than the artificial distortion, the patient is more likely to perceive the former and ignore the latter. During the test, varying sizes of artificial distortions are displayed, allowing quantification of the degree of the pathological distortion. A compilation of the errors performed during the test enables location of the visual distortions as well as estimation of their severity. For severity measurements we have used the "PHP test score" (a continuous, global score, between -30 and +600).

One hour before performing intravitreal ranibizumab injection, each patient also underwent a baseline assessment of BCVA measured at 4 m with standard ETDRS charts, and a complete ophthalmic examination that included slit-lamp biomicroscopy, intraocular pressure measurement and spectral domain OCT utilizing the Spectralis Heidelberg Retinal Angiograph OCT (Spectralis HRA-OCT Heidelberg Engineering, Heidelberg, Germany). In this study we used, at each follow-up visit, the "follow-up" protocol of Spectralis HRA-OCT, which grants reacquisition of each OCT line actually referenced with the baseline acquisition.

The retinal thickness of the 1-mm central retina (central macular thickness) was evaluated using a 19 horizontal lines protocol. Several OCT parameters, reflecting the disease activity, were also evaluated on a single (for each eye) spectral domain OCT scan. For Since the normality assumption was not satisfied for the measurement variables, we have used nonparametric tests: the Spearman correlation was evaluated, in order to examine the degree of correlation between OCT average parameters change, LogMAR BCVA average change and PHP test score average change. Differences between baseline and 1 month visits were compared using Wilcoxon signed rank test.

For the question of whether an increase in PHP test score within the individual was associated with an increase in OCT combined parameter, we had removed the differences between subjects and look only at changes within by multiple regression method, using the OCT data as the outcome variable and the PHP test score and the subject as the predictor variables. Subject is treated as a categorical factor using dummy variables [START_REF] Bland | Statistics notes: Calculating correlation coefficients with repeated observations: Part 1-correlation within subjects[END_REF] .

In order to investigate the relative change vs baseline examination, all parameters were collected to a table and classified as "better", "worse" or "same" with respect to BCVA (LogMAR change >0.1), "combined OCT parameter" (change > 10 microns) and PHP test score (change > 15). An analysis of variance for repeated measures (ANOVA) was performed in order to determine the effect of time on the different measurements. The chosen level of statistical significance was p < 0.05.

Results

Fourteen treatment naive eyes of 14 consecutive Patients with exudative AMD met the inclusion criteria and were included in the analysis. Demographic and clinical data for these patients are summarized in table 1. The mean age was 83±6.2 years, and the gender distribution was 78% women. All included patients presented at each scheduled follow-up visit, and underwent all examinations except for 3 patients who missed the PHP examination at the last follow-up visit (1 month after the intravitreal ranibizumab injection).

PHP in exudative AMD 10 Therefore, 67 out of the 70 scheduled PHP tests were performed at 1 month. All PHP tests made in this study except 3 (4%) showed a good a reliability score.

In It is notable that there was an improvement in all parameters, and that for BCVA and OCT the majority of significant changes happened within 1 day to 1 week after treatment (p<0.05). Interestingly, there was an increase in the metamorphopsia score one hour after the injection. The formal analysis and the quantification of the relationships between functional and morphological changes are presented in figures 2-4. There was a significant correlation between the PHP metamorphopsia changes and the OCT anatomical changes (0.81 Spearman correlation, p<0.05). A significant correlation was found between the PHP metamorphopsia changes and the mean BCVA changes (0.95 Spearman correlation, p<0.05). There was also a significant correlation between the mean BCVA changes and the OCT anatomical changes (0.95 Spearman correlation, p<0.05).

The correlation coefficient between OCT measurements and PHP score within subjects was 0.51 (p<0.05) (which indicates that an increase in OCT combined parameter was associated with an increase in PHP test score within the individual).

An analysis of variance for repeated measures (ANOVA) showed a negative significant impact of treatment (improvement over time) on BCVA logMAR, OCT combined parameter, and PHP test score (Type III tests of fixed effects, p<0.05). The time points with the highest impact on the model were 1 week and 1month after treatment for both BCVA and OCT, and the 1 week time point for the PHP (p<0.05 for these time points).

Categorized functional and morphological changes ("better", "worse" or "same"), for each eye, at each time-point relative to baseline, are reported in table 3.

Discussion

In this prospective study we investigated the ability of Foresee PHP in assessing responsiveness to intravitreal ranibizumab injection, in 14 consecutive patients affected with newly diagnosed exudative AMD. After a single intravitreal ranibizumab injection, we found mean PHP metamorphopsia test score significantly improved from 20.4±35 to 9.2±23. Moreover, in order to test if PHP functional measures correlate with macular morphology measures, we also investigated the significance of correlation existing between PHP test score, and several OCT parameters, during the duration of the study.

Interestingly, the functional improvements as evaluated by PHP well reflected the mean reduction in all OCT parameters (central macular thickness, maximal retinal thickness at the fovea, maximal height of subretinal fluid, maximal diameter of the largest retinal cyst and maximal height of pigment epithelium detachment) showing a significant correlation with metamorphopsia changes (0.81 Spearman correlation, p<0.05).

In our series, also mean BCVA significantly improved from 20/80 to 20/60, after the single intravitreal ranibizumab injection, and a significant correlation was found between BCVA changes and the PHP metamorphopsia changes (2 different functional measures) during the duration of the study (0.95 Pearson correlation, p<0.05).

It is noteworthy that both a significant functional (PHP and BCVA), and morphological improvement (OCT parameters) were recorded as soon as 1 day after the intravitreal ranibizumab injection. The exceptional increase in the metamorphopsia score one hour after the injection, may be attribute to the emotional state and maybe some local disturbances due to the injection, which, all may affect a psychophysical test.

Das et al [START_REF] Das | Distortion maps from preferential hyperacuity perimetry are helpful in monitoring functional response to Lucentis therapy[END_REF] have recently published a retrospective analysis (lacking of a standardized interval between treatment and reexamination) of usually collected clinical data (BCVA and OCT) and PHP from patients undergoing treatment with ranibizumab. The authors found that changes in PHP distortion maps did not correlate with maximal retinal thickness, and with BCVA (a total of 34 BCVA-PHP tests-retests). Also, BCVA changes (less than half of one ETDRS line) did not correlate with OCT changes. However, it is worth noting that the authors did not set out to specifically test whether PHP could help with assessing responsiveness to ranibizumab therapy. Conversely, in the current study, we included only treatment naive eyes, which were regularly followed (prospectively) and re-examined at several fixed time-points (67 BCVA-PHP tests-retests), during a limited 1month period.

The strong correlation here reported between functional PHP metamorphopsia changes and morphological OCT changes is even more important considering the population PHP in exudative AMD 13 included in this study: patients with exudative AMD undergoing intravitreal anti-VEGF injections. Given that there is universal acceptance amongst clinicians that monthly visits, for both functional and morphological monitoring, are necessary in patients undergoing intravitreal anti-VEGF treatment for AMD, the availability of an objective and easily performed functional test (as the PHP), which correlates well with macular morphology, would be extremely helpful in the clinic setting, in order to take optimal re-treatment decisions. For this reason, a home version of the PHP, the ForeseeHome, is currently under development. The ForeseeHome is based on a technology that is similar to the Foresee PHP. This home-based test has been designed for self-monitoring AMD patients (Loewenstein A. Poster presented at: AAO Annual Meeting, November 8-11 2008;

Atlanta). As with Foresee PHP, the ForeseeHome seems to be superior to the Amsler grid in terms of accuracy of CNV detection (Loewenstein A. Poster presented at ASRS Annual Meeting, October 10-15 2008; Maui) [START_REF] Loewenstein | Toward earlier detection of choroidal neovascularization secondary to age-related macular degeneration: multicenter evaluation of a preferential hyperacuity perimeter designed as a home device[END_REF] . It is expected that the test will serve as a follow- up tool for patients to self-test with the ForeseeHome on a frequent basis [START_REF] Loewenstein | Toward earlier detection of choroidal neovascularization secondary to age-related macular degeneration: multicenter evaluation of a preferential hyperacuity perimeter designed as a home device[END_REF] .

Our study has several limitations. All patients underwent a single injection of ranibizumab.

The sample size was small, and 3 patients missed the PHP examination at 1 month; also, despite 67 PHP tests were performed over 1 month, follow-up is short, and no definitive conclusion can be drawn. In addition, even though we found a significant correlation between progressive reduction of morphological signs of disease activity (OCT parameters), and functional improvements as evaluated by PHP, this applied to newly diagnosed active CNVs; therefore, we actually have no data on such a correlation, in eyes with CNV evolving towards quiescent inactive fibro-atrophic lesions, as it may happen following a certain number of repeated anti-VEGF treatments. Finally, we did not perform other tests (such as NEI VFQ -25 and microperimetry), which would have been useful to PHP in exudative AMD 14 better characterize the short-term functional and morphological fluctuations in these patients.

In conclusion, we found that functional improvement in metamorphopsia test score after intravitreal ranibizumab injection, well correlates with improvement in several OCT parameters reflecting the disease activity. Our findings suggest that PHP may be

  selection, scans had to show the lesion and the presumed fovea. Parameters measured in the selected scan included: maximal retinal thickness at the fovea, maximal height of subretinal fluid, maximal diameter of the largest retinal cyst and maximal height of pigment epithelium detachment. PHP test (performed under the supervision of a trained physician [GQ]), as well as assessment of BCVA measured at 4 m with standard ETDRS charts, and Spectralis HRA-OCT utilizing the "follow-up" protocol, were performed 1 hour, 1 day, 1 week and 1 month after the intravitreal injection of ranibizumab. Descriptive statistics were performed to present, by graphs, the trends of change for the different features (BCVA converted to the logarithm of the minimum angle of resolution [LogMAR], the OCT parameters investigated [central macular thickness, maximal retinal thickness at the fovea, maximal height of subretinal fluid, maximal diameter of the largest retinal cyst and maximal height of pigment epithelium detachment], the "combined parameter" [sum the last former four parameters] and PHP measurements represented as "test score" (Test score = log [Probability(Responses pattern\CNV) / Probability(Responses pattern\Intermediate)]).

Figure 1 .

 1 Figure 1. Example of functional and morphological fluctuation, as evaluated by

Figure 2 .

 2 Figure 2. Relationship between spectral domain optical coherence tomography (SD OCT)

Figure 3 .

 3 Figure 3. Relationship between best-corrected visual acuity (BCVA) and Preferential

Figure 4 .

 4 Figure 4. Relationship between spectral domain optical coherence tomography (SD OCT)

  table 2 are summarized the functional changes (as evaluated by PHP metamorphopsia test score and BCVA) and the morphological changes (as evaluated by Spectralis spectral domain OCT, "follow-up protocol") within the first month from intravitreal ranibizumab injection. Mean baseline PHP metamorphopsia test score was 20.4 ±35, and there was a significant improvement to 9.2 ±23, at 1 month (Wilcoxon signed rank test, p < 0.05). Mean baseline BCVA was 0.6 LogMAR (20/80), and there was a significant improvement to 0.5 LogMAR (20/60), at 1 month (Wilcoxon signed rank test, p < 0.05).

The mean baseline central macular thickness was 413 ± 142 μm. It significantly reduced to 303 ± 58 μm at 1 month (Wilcoxon signed rank test, p < 0.05). Mean reduction in "combined OCT parameter" (central macular thickness, maximal retinal thickness at the fovea, maximal height of subretinal fluid, maximal diameter of the largest retinal cyst and maximal height of pigment epithelium detachment) was also recorded at 1 month (Wilcoxon signed rank test, p < 0.05).

The natural dynamics, both anatomical and functional, within the first month following intravitreal ranibizumab injection are presented in table 2 and figure 1 (case presentation).

Table 1 .

 1 Demographic and clinical data of patients treated by intravitreal ranibizumab injection for exudative macular degeneration.

	Patient (eye) #	GENDER	AGE	EYE	CNV SUBTYPE
	1	M	89	LE	Occult
	2	F	81	RE	Predominantly Classic
	3	F	74	RE	Retinal Angiomatous Proliferation
	4	F	74	LE	Occul
	5	F	84	RE	Minimally Classic
	6	F	89	LE	Minimally Classic
	7	F	84	LE	Occul
	8	F	82	RE	Occul
	9	F	76	LE	Occul
	10	F	81	RE	Occul
	11	M	77	LE	Occul
	12	F	96	LE	Occul
	13	M	80	RE	Occul
	14	F	86	RE	Occul

M: male; F: female; RE: right eye; LE: left eye; CNV: choroidal neovasularization.

Table 2 .

 2 Functional and morphological characteristics of patients with exudative macular degeneration, at baseline (1 hour befo re intravitral ranibizumab injection), and 1 hour, 1 day, 1 week and 1 month after intravitral ranibizumab injection.

									Combined
									OCT
		Mean BCVA	PHP		Mean		Mean		parameters
		(LogMAR)	Test Score	Mean CMT	MHSRF	Mean MRTF	MHPED	Mean MDRC	(Mean)
	Baseline	0.6 ±0.3	20.4 ±35	413 ± 142μm	79 ± 93μm	384 ±109μm	192 ±137μm	102 ±151μm	757 ±298μm
	1 hour	0.6 ±0.3	24 ±33	410 ± 142 μm	77 ± 91μm	381 ±108μm	192 ±136μm	102 ±151μm	752 ±296μm
	1 day	0.59 ±0.34	13 ±23	393 ± 149 μm	66 ± 89μm	357 ±101μm	178 ±138μm	73 ±142μm	675 ±258μm
	1 week	0.52 ±0.32	12.7 ±21	318 ± 65μm	21 ± 63μm	318 ±85μm	152 ±85μm	20 ±43μm	511 ±128μm
	1 month	0.5 ±0.32	9.2 ±23 *	303 ± 58μm	4 ± 13μm	297 ±79μm	134 ± 66μm	15 ±39μm	448 ±107 μm

BCVA: best corrected visual acuity -logarithm of the minimum angle of resolution (LogMAR); CMT: central macular thickness; HSR F: maximal height of subretinal fluid; MRTF: maximal retinal thickness at the fovea; HPED maximal height of pigment epithelium detachment; MDRC: max imal diameter of the largest retinal cyst; combined optical coherence tomography (OCT) parameters investigated (CMT, SRTF, MRTF, HPED, MDRC); Prefer ential Hyperacuity Perimetry (PHP) test score.

Acknowledgements

This study was supported thorough a research fellowship by the Retina 3000 Foundation.

The principal investigator had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

"The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in British Journal of Ophthalmology and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms)." Giuseppe Querques: Competing Interest: None declared. Elya Berboucha: Competing Interest: None declared. Nicolas Leveziel: Competing Interest: None declared. Alfredo Pece: Competing Interest: None declared. Eric H Souied: Competing Interest: None declared.

This study was supported thorough a research fellowship by the Retina 3000 Foundation.