Alexandre Moulin

Intravitreal chemotherapy for vitreous disease in retinoblastoma revisited: from prohibition to conditional indications

Background Tumour control of vitreous seeds remains challenging owing to their resistance to radiation and systemic chemotherapy. Objective To describe the short-term efficacy of intravitreal melphalan for vitreous disease in retinoblastoma using a new injection technique and dose. Methods This study is a retrospective non-comparative review of 23 consecutive heavily pretreated patients (23 eyes) with active vitreous seeding and eligible for intravitreous chemotherapy (IViC). They received a total of 122 intravitreal injections of melphalan (20–30 μg) given every 7–10 days. The ocular status was objectively monitored under anaesthesia with fundus photography. Results All patients are alive without evidence of extraocular spread (95% CI 82.19% to 100%). Concomitant treatments, including other chemotherapeutic modalities, were used until complete sterilisation of the retinal seeding source and subretinal seeds. Globe retention was achieved in 87% (20/23) of cases. All retained eyes were in complete remission after a median follow-up period of 22 months (range 9–31 months). The Kaplan–Meier estimate of ocular survival rates at 2 years was 84.14% (95% CI 62.48% to 95.28%). A localised peripheral salt-and-pepper retinopathy was noted in 10 eyes (43%) at the site of injection. Conclusions This study reports the first clinically documented case series of patients with retinoblastoma treated with IViC. Despite a possible confounding effect of concomitant chemotherapy prescription using other routes of administration in four of the successfully treated eyes (20%), IViC achieved an unprecedented success rate of tumour control in the presence of vitreous seeding. Of note, none of the treated eyes required external beam irradiation to control the vitreous seeding. Further studies are required to assess IViC retinal toxicity and to better delineate its role in the management of retinoblastoma.

Profiling safety of intravitreal injections for retinoblastoma using an anti-reflux procedure and sterilisation of the needle track

Background The preservation of globe integrity has always been a major concern during the treatment of retinoblastoma for fear of extraocular or metastatic spread. Intravitreal chemotherapy has been attempted as a desperate salvage therapy only for eyes with refractory retinoblastoma. Published data on the safety and efficacy of this route are, however, limited. Methods A modified technique of intravitreal injection in eyes with retinoblastoma is described. All children with retinoblastoma who received one or more intravitreal injections using this technique were retrospectively reviewed concerning ocular complications of the injection procedure as well as clinical or histopathological evidence of tumour spread. Results 30 eyes of 30 children with retinoblastoma received a total of 135 intravitreal injections, with a median follw-up duration of 13.5 months. No extraocular spread was seen on clinical follow-up in any patients and there was no tumour contamination of the retrieved entry sites histopathologically analysed among the five enucleated eyes. No significant ocular side effects were observed except transient localised vitreous haemorrhage (3/135). Conclusion This technique is potentially safe and effective at a low cost and may play a promising role, especially in the treatment of recurrent and/or resistant vitreous disease in retinoblastoma, as an alternative to enucleation and/or external beam radiotherapy. However, this treatment should not replace the primary standard of care of retinoblastoma and should not be considered in group E eyes. Its application should be approved by an ophthalmological-oncological team and it should be performed by an experienced eye surgeon in a tertiary referral centre after careful selection of a tumour-free injection site.

Melanocytes and Pigmentation Are Affected in Dopachrome Tautomerase Knockout Mice

ABSTRACT The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dcttm1(Cre)Bee ] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dctslt /Dctslt ) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.

A Novel Approach to Characterize Clonality and Differentiation of Human Melanoma-Specific T Cell Responses: Spontaneous Priming and Efficient Boosting by Vaccination

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8+ T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells.

Summary Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.

Comprehensive Genetic Landscape of Uveal Melanoma by Whole-Genome Sequencing

Uveal melanoma (UM) is a rare intraocular tumor that, similar to cutaneous melanoma, originates from melanocytes. To gain insights into its genetics, we performed whole-genome sequencing at very deep coverage of tumor-control pairs in 33 samples (24 primary and 9 metastases). Genome-wide, the number of coding mutations was rather low (only 17 variants per tumor on average; range 7-28), thus radically different from cutaneous melanoma, where hundreds of exonic DNA insults are usually detected. Furthermore, no UV light-induced mutational signature was identified. Recurrent coding mutations were found in the known UM drivers GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. Other genes, i.e., TP53BP1, CSMD1, TTC28, DLK2, and KTN1, were also found to harbor somatic mutations in more than one individual, possibly indicating a previously undescribed association with UM pathogenesis. De novo assembly of unmatched reads from non-coding DNA revealed peculiar copy-number variations defining specific UM subtypes, which in turn could be associated with metastatic transformation. Mutational-driven comparison with other tumor types showed that UM is very similar to pediatric tumors, characterized by very few somatic insults and, possibly, important epigenetic changes. Through the analysis of whole-genome sequencing data, our findings shed new light on the molecular genetics of uveal melanoma, delineating it as an atypical tumor of the adult for which somatic events other than mutations in exonic DNA shape its genetic landscape and define its metastatic potential.

Methylation of CpG island promoters in uveal melanoma

Background: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. Aim: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. Methods: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. Results: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. Conclusions: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma.

Treatment Strategies in Primary Vitreoretinal Lymphoma: A 17-Center European Collaborative Study

IMPORTANCE The best treatment option for primary vitreoretinal lymphoma (PVRL) without signs of central nervous system lymphoma (CNSL) involvement determined on magnetic resonance imaging or in cerebrospinal fluid is unknown. OBJECTIVE To evaluate the outcomes of treatment regimens used for PVRL in the prevention of subsequent CNSL. DESIGN, SETTING, AND PARTICIPANTS A retrospective cohort study was conducted at 17 referral ophthalmologic centers in Europe. We reviewed clinical, laboratory, and imaging data on 78 patients with PVRL who did not have CNSL on presentation between January 1, 1991, and December 31, 2012, with a focus on the incidence of CNS manifestations during the follow-up period. INTERVENTIONS The term extensive treatment was used for various combinations of systemic and intrathecal chemotherapy, whole-brain radiotherapy, and peripheral blood stem cell transplantation. Therapy to prevent CNSL included ocular radiotherapy and/or ocular chemotherapy (group A, 31 patients), extensive systemic treatment (group B, 21 patients), and a combination of ocular and extensive treatment (group C, 23 patients); 3 patients did not receive treatment. A total of 40 patients received systemic chemotherapy. MAIN OUTCOMES AND MEASURES Development of CNSL following the diagnosis of PVRL relative to the use or nonuse of systemic chemotherapy and other treatment regimens. RESULTS Overall, CNSL developed in 28 of 78 patients (36%) at a median follow-up of 49 months. Specifically, CNSL developed in 10 of 31 (32%) in group A, 9 of 21 (43%) in group B, and 9 of 23 (39%) in group C. The 5-year cumulative survival rate was lower in patients with CNSL (35% [95% CI, 50% to 86%]) than in patients without CNSL (68% [95% CI, 19% to 51%]; P = .003) and was similar among all treatment groups (P = .10). Adverse systemic effects occurred in 9 of 40 (23%) patients receiving systemic chemotherapy; the most common of these effects was acute renal failure. CONCLUSIONS AND RELEVANCE In the present series of patients with isolated PVRL, the use of systemic chemotherapy was not proven to prevent CNSL and was associated with more severe adverse effects compared with local treatment.

Infrequent promoter methylation of the MGMT gene in liver metastases from uveal melanoma.

Uveal melanoma is associated with a high mortality rate once metastases occur, with over >90% of metastatic patients dying within less than 1 year from metastases to the liver. The intraarterial hepatic (iah) administration of the alkylating agent fotemustine holds some promise with response rates of 36% and median survival of 15 months. Here, we investigated whether the DNA‐repair‐protein MGMT may be involved in the variability of response to fotemustine and temozolomide in uveal melanoma. Epigenetic inactivation of MGMT has been demonstrated to be a predictive marker for benefit from alkylating agent therapy in glioblastoma. We found a methylated MGMT promoter in 6% of liver metastases from 34 uveal melanoma patients. The mean MGMT activity measured in liver metastases with negligible liver tissue content was significantly lower than in liver tissue (146 versus 523 fmol/mg protein, p = 0.002). Expression of the MGMT protein was detectable in 50% of 88 metastases by immunohistochemistry on a tissue microarray. Expression was heterogeneous, and in accordance with MGMT activity data, usually lower than in the surrounding liver. Differential MGMT activity/expression between metastasis and liver tissue and more efficient depletion of MGMT with higher doses of alkylating agent therapy using iah delivery may provide the pharmacologic window for the higher response rate. However, these results do not support MGMT methylation status or protein expression as predictive markers for treatment outcome to iah chemotherapy with alkylating agents.

Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation

The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair.