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Assessment of retinoblastoma RNA reflux after intravitreal injection of melphalan
  1. Ursula Winter1,2,
  2. Michael Nicolas3,
  3. Mariana Sgroi4,
  4. Claudia Sampor5,
  5. Ana Torbidoni2,
  6. Adriana Fandiño4,
  7. Guillermo L Chantada2,5,
  8. Francis L Munier3,
  9. Paula Schaiquevich1,2
  1. 1Unit of Clinical Pharmacokinetics, Hospital de Pediatría JP Garrahan, Buenos Aires, Argentina
  2. 2National Scientific and Technical Research Council, CONICET, Buenos Aires, Argentina
  3. 3Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Lausanne, Switzerland
  4. 4Department of Ophthalmology, Hospital de Pediatría JP Garrahan, Buenos Aires, Argentina
  5. 5Department of Hematology-Oncology, Hospital de Pediatría JP Garrahan, Buenos Aires, Argentina
  1. Correspondence to Dr Paula Schaiquevich, Unit of Clinical Pharmacokinetics, Hospital de Pediatría J.P. Garrahan, Combate de los Pozos 1881, C1245AAL, Buenos Aires, Argentina; paula.schaiquevich{at}


Background Intravitreal injection of chemotherapy in retinoblastoma eyes with vitreous seeds may lead to a risk of extraocular tumour dissemination that has not been assessed so far.

Aims To develop a sensitive and clinically feasible technique to assess for potential retinoblastoma cell reflux after intravitreal injection of melphalan.

Methods Filter papers were cut in 6 mm diameter circles and sterilised before use. Eyes with retinoblastoma vitreous seeds (group D, International Classification) received weekly intravitreal melphalan injections (20 µg or 30 µg/dose) followed by cryotherapy as part of local treatment. Immediately after finishing the injection and cryotherapy, filter papers were placed on the injection site and on the cryoprobe tip to assess for the expression of the cone-rod homeobox gene (CRX) by real-time qPCR as a surrogate of retinoblastoma RNA. The assay was developed and validated to determine sensitivity, linearity, recovery, repeatability and reproducibility.

Results The assay for quantitation of CRX expression was linear in the range of 1 to 1000 cells. The lowest limit of detection was one retinoblastoma cell and allowed to recover 100% of the cell load in external supplementation. A total of 14 eyes received 22 cycles of intravitreal melphalan and were evaluated for potential extraocular tumour cell dissemination using the developed technique. None of the cycles were positive for CRX in samples from the scar or from the cryoprobe tip.

Conclusions A sensitive and simple method of tumour cell assessment has been developed that can be used in the clinics to assess for potential extraocular dissemination after intravitreal injections to assure its performance.

  • experimental laboratory
  • retina
  • treatment medical
  • vitreous

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  • Contributors UW and PS conceived and designed the study. UW and MN developed and validated the molecular biology technique. UW, AT and MN processed the samples. MS, AF, CS and FM participated in subject enrolment and sample collection. GLC, PS and FM designed the study and wrote the paper. All authors contributed to the review of the literature, in drafting the manuscript and in approving the final manuscript.

  • Funding This work was funded by the National Institute of Cancer, Ministry of Health of Argentina, Fund for Ophthalmic Knowledge, New York, USA; Fundación Natalie D Flexer de Ayuda al Niño con Cáncer.

  • Competing interests None declared.

  • Patient consent Guardian consent obtained.

  • Ethics approval Institutional Review Boards of both Hospital de Pediatria JP Garrahan, Argentina (Protocol # 857) and Jules-Gonin Eye Hospital, Switzerland (authorisation #2016-00149)

  • Provenance and peer review Not commissioned; externally peer reviewed.

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