Frequency of HLA-A*02:01 in the Brazilian population and its impact on uveal melanoma systemic treatment.

Uveal melanoma is a rare malignancy originating from extracutaneous melanocytes on the uveal layer of the eyes. The incidence varies depending on the ethnic and racial global distribution, as uveal melanoma is more frequently diagnosed in non-Hispanic White subjects when compared with Hispanic, Asian, or Black individuals. Despite all the local effective management of uveal melanoma, roughly 50% of the cases will develop distant metastases. For these cases, the historical median overall survival is around 12 months. Recently, tebentafusp became the first therapy to receive Food and Drug Administration approval following a phase 3 trial demonstrating a continued long-term benefit for overall survival among adult HLA-A*02:01-positive patients with previously untreated metastatic uveal melanoma. Since 2021, high-resolution sequence-based HLA typing has been considered the gold standard for determining HLA alleles and haplotypes for the Brazilian Bone Marrow Donor Registry (REDOME) donors. To depict the HLA-A*02:01-positivity in Brazilian individuals, the REDOME database was queried out for the donors included from 2021 to 2023 and tested for HLA in high-resolution platforms. A total of 203,  44 donors were included and the frequency of the HLA-A*02:01 was 21.01%, much lower compared to the frequency in North Americans and Europeans (around 45%). Despite tebentafusp has demonstrated promising results in the treatment of uveal melanoma, the number of patients to benefit from this new approach can strongly vary by ethnic and racial issues. New strategies for the systemic treatment of advanced uveal melanoma have to be developed and tested as this disease still represents an unmet medical need.

Global RNAseq of ocular cells reveals gene dysregulation in both asymptomatic and with Congenital Zika Syndrome infants exposed prenatally to Zika virus.

In 2015, Brazil reported an outbreak identified as Zika virus (ZIKV) infection associated with congenital abnormalities. To date, a total of 86 countries and territories have described evidence of Zika infection and recently the appearance of the African ZIKV lineage in Brazil highlights the risk of a new epidemic. The spectrum of ZIKV infection-induced alterations at both cellular and molecular levels is not completely elucidated. Here, we present for the first time the gene expression responses associated with prenatal ZIKV infection from ocular cells. We applied a recently developed non-invasive method (impression cytology) which use eye cells as a model for ZIKV studies. The ocular profiling revealed significant differences between exposed and control groups, as well as a different pattern in ocular transcripts from Congenital Zika Syndrome (CZS) compared to ZIKV-exposed but asymptomatic infants. Our data showed pathways related to mismatch repair, cancer, and PI3K/AKT/mTOR signaling and genes probably causative or protective in the modulation of ZIKV infection. Ocular cells revealed the effects of ZIKV infection on primordial neuronal cell genes, evidenced by changes in genes associated with embryonic cells. The changes in gene expression support an association with the gestational period of the infection and provide evidence for the resulting clinical and ophthalmological pathologies. Additionally, the findings of cell death- and cancer-associated deregulated genes raise concerns about the early onset of other potential pathologies including the need for tumor surveillance. Our results thus provide direct evidence that infants exposed prenatally to the Zika virus, not only with CZS but also without clinical signs (asymptomatic) express cellular and molecular changes with potential clinical implications.

Molecular alterations in retinoblastoma beyond RB1.

Retinoblastoma is the most common malignant ocular tumor in children. Although RB1 alterations are most frequently involved in the etiology of retinoblastoma, candidate driver events and somatic alterations leading to cell transformation, tumor onset and progression remain poorly understood. In this study, we identified novel genomic alterations in tumors with a panel of 160 genes. Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) were initially performed for identifying patients without apparent RB1 alterations in blood DNA. Subsequently, NGS analyses of 24 paired (blood/tumor) samples of these patients were carried out for identifying somatic mutations and copy number variation in RB1 and other 159 genes. One novel pathogenic RB1 mutation and seven novel VUS were identified as well as 90 novel pathogenic mutations in 61 other genes. Twenty-three genes appeared exclusively mutated in tumors without altered RB1 alleles and three frequently affected biological pathways while five other tumors did not show pathogenic RB1 alterations or SNV/indels in 159 other genes. Curiously, deletion of GATA2, AKT1, ARID1A, DNMT3A, MAP2K2, MEN1, MTOR, PTCH1 and SUFU (in homo- or heterozygosity) were exclusively found in these tumors when compared to those with any pathogenic alterations, probably indicating genes that might be essential for the development of retinoblastoma regardless of a functional RB1. Identification of genes associated with retinoblastoma will contribute to understanding presently unknown aspects of this malignancy, which might be essential for its initiation and progression, as well as providing valuable molecular markers.

Epidemiology of uveal melanoma in Brazil.

PURPOSE: To report the prevalence of uveal melanoma in a Hospital database in Brazil over the period of 16 years (2000 to 2016). DESIGN: Descriptive epidemiological study evaluating the Brazilian Hospital Based Cancer Registries. PARTICIPANTS/METHODS: Uveal melanomas were identified based on ICD-O-3 codes C69.3 [choroid], C69.4 [ciliary body and iris], and C69.2 [retina]) derived from the Integrator Registry database. Kolmogorov-Smirnov Test was used for evaluation of normality of data, t-test and Chi square were used for categorical and continuous variables respectively using SPSS Software. MAIN OUTCOME MEASURES: Age, sex, education, regional distribution, clinical staging at the diagnosis, time from diagnosis to treatment (≤ 60 days versus > 60 days) and first-course therapy (surgery, chemotherapy, radiotherapy or a combination of such). RESULTS: There were 2166 cases of uveal melanoma representing 5.4% of all cases of melanoma. Histological confirmation of uveal melanoma was available in all cases. Higher prevalence of 1139 cases (52.6%) in women than 1027 cases (47.4%) in men was observed. Age distribution revealed 1411 cases (65.1%) in the group between 41 and 69 years old. A total of 429 (19.8%) patients were classified as initial disease and 334 (15.4%) as advanced (regional or distant metastases). Staging as initial disease was more frequent (113-24.8%) in patients with > 8 school years than in patients with < 8 school years (179-17.6%) reflecting disparities in healthcare access between those two populations. No difference was noticed in terms of diagnosis, staging and treatment after the Brazilian "60 days law" (Federal Law 12.732/12) came into effect in 2013 regulating the maximum period that a patient with cancer has to wait until start the treatment. CONCLUSION: Epidemiological data is critical for planning early treatment strategies and allocating medical resources. This study intended to understand the characteristics of uveal melanoma in Brazil.

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

IMPORTANCE: Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined. OBJECTIVES: The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches. DESIGN SETTINGS AND PARTICIPANTS: Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments. RESULTS: Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies. CONCLUSIONS AND RELEVANCE: Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.

Prediagnostic Intervals in Retinoblastoma: Experience at an Oncology Center in Brazil.

PURPOSE: Retinoblastoma is the most common intraocular malignancy of childhood. In most cases, parents are the first to notice leukocoria and other symptoms before undergoing a prolonged period of stress before diagnosis. The purpose of this study was to determine prediagnostic intervals of patients with retinoblastoma at an oncology tertiary center (Instituto Nacional de Cancer) in Rio de Janeiro, Brazil, and relate them to stage at diagnosis, eye salvage, and survival. METHODS: Parents or caregivers of children with retinoblastoma registered between January 2006 and September 2013 were interviewed using a semistructured individually applied questionnaire, concerning their trajectory before registration. RESULTS: Out of 76 patients, 39 (51%) were girls, 52 (68%) had unilateral retinoblastoma, and 24 (32%) had bilateral retinoblastoma, totaling 100 affected eyes. The most common stage of diagnosis was the intraocular group, with 63 (83%) patients; nine (12%) were extraocular, and four (5%) had metastatic disease. During the follow-up time of 37 ± 24.5 months, 10 (13%) patients died and 70 (70%) eyes were enucleated. Mean family interval was 1.6 ± 2.6 months, mean medical interval was 5.0 ± 6.2 months, mean referral interval was 0.2 ± 1.4 months, and mean overall interval was 7.1 ± 6.9 months. In univariate analysis, age at diagnosis, maternal education, medical interval, and overall interval were significantly related to advanced stage at diagnosis and survival. In multivariate analysis, maternal education and medical interval were significantly related to advanced stage at diagnosis and survival. No variables affected eye salvage. CONCLUSION: Medical interval was responsible for 70% of the overall interval; therefore, programs or campaigns targeting retinoblastoma early diagnosis should focus emphasize in medical awareness.

An international survey of classification and treatment choices for group D retinoblastoma.

AIM: To determine which IIRC scheme was used by retinoblastoma centers worldwide and the percentage of D eyes treated primarily with enucleation versus globe salvaging therapies as well as to correlate trends in treatment choice to IIRC version used and geographic region. METHODS: An anonymized electronic survey was offered to 115 physicians at 39 retinoblastoma centers worldwide asking about IIRC classification schemes and treatment patterns used between 2008 and 2012. Participants were asked to record which version of the IIRC was used for classification, how many group D eyes were diagnosed, and how many eyes were treated with enucleation versus globe salvaging therapies. Averages of eyes per treatment modality were calculated and stratified by both IIRC version and geographic region. Statistical significance was determined by Chi-square, ANOVA and Kruskal-Wallis tests using Prism. RESULTS: The survey was completed by 29% of physicians invited to participate. Totally 1807 D eyes were diagnosed. Regarding IIRC system, 27% of centers used the Children's Hospital of Los Angeles (CHLA) version, 33% used the Children's Oncology Group (COG) version, 23% used the Philadelphia version, and 17% were unsure. The rate for primary enucleation varied between 0 and 100% and the mean was 29%. By IIRC version, primary enucleation rates were: Philadelphia, 8%; COG, 34%; and CHLA, 37%. By geographic region, primary enucleation rates were: Latin America, 57%; Asia, 40%; Europe, 36%; Africa, 10%, US, 8%; and Middle East, 8%. However, systemic chemoreduction was used more often than enucleation in all regions except Latin America with a mean of 57% per center (P<0.0001). CONCLUSION: Worldwide there is no consensus on which IIRC version is used, systemic chemoreduction was the most frequently used initial treatment during the study period followed by enucleation and primary treatment modality, especially enucleation, varied greatly with regards to IIRC version used and geographic region.

Use of intra-arterial chemotherapy for retinoblastoma: results of a survey.

AIM: To obtain baseline knowledge about the current use of intra-arterial chemotherapy (SSOAIC) in centers worldwide. METHODS: A survey including questions about the use of SSOAIC was emailed to retinoblastoma experts. RESULTS: Seventy-nine (response rate 69.9%) doctors from 63 centers in 35 countries responded. Thirty-one centers from 19 countries use SSOAIC. Twelve performed more than 50 procedures. Melphalan is the most commonly used drug but 15 centers use more than one drug. First line therapy for advanced unilateral disease is the most common use of SSOAIC (74.2%). Centers with larger experience (>50 applications) were less likely using melphalan alone (P=0.06) and significantly more likely using SSOAIC in more situations such as second line in preference to radiotherapy P=0.05. Nineteen (61.2%) stated that SSOAIC improved their results and 21 (77.8%) reported less toxicity compared to other treatments. Three centers reported that SSOAIC did not improve their results. There were regional variations in the use of SSOAIC which is used more frequently as secondary treatment in Europe compared to the USA and Japan. Ten centers identified cost is the major limiting factor for SSOAIC. CONCLUSION: SSOAIC is used in an increasing number of centers worldwide with regional variations. Centers with more experience in SSOAIC use it in more situations including other drugs than melphalan. The majority of the centers using this technique reported improved results and few complications.

Clinical pharmacokinetics of intra-arterial melphalan and topotecan combination in patients with retinoblastoma.

PURPOSE: To assess the antitumor activity, toxicity, and plasma pharmacokinetics of the combination of melphalan and topotecan for superselective ophthalmic artery infusion (SSOAI) treatment of children with retinoblastoma. DESIGN: Single-center, prospective, clinical pharmacokinetic study. PARTICIPANTS: Twenty-six patients (27 eyes) with intraocular retinoblastoma. METHODS: Patients with an indication for SSOAI received melphalan (3-6 mg) and topotecan (0.5-1 mg; doses calculated by age and weight). Plasma samples were obtained for pharmacokinetic studies, and a population approach via nonlinear mixed effects modeling was used. Safety and efficacy were assessed and compared with historical cohorts of patients treated with melphalan single-agent SSOAI. MAIN OUTCOME MEASURES: Melphalan and topotecan pharmacokinetic parameters and efficacy and safety parameters. RESULTS: Twenty-seven eyes from 26 consecutive patients received 66 cycles of SSOAI melphalan and topotecan in combination. All 5 eyes treated as primary therapy responded to the combination chemotherapy and were preserved. Sixteen of the 22 eyes with relapsed or resistant tumors responded, but 3 of them ultimately underwent enucleation at a median of 8 months (range, 7.9-9.1 months). The incidence of grade III and IV neutropenia was 10.6% and 1.5%, respectively, which was comparable with historical controls of single-agent SSOAI melphalan. No episode of fever neutropenia was observed, and no patient required transfusion of blood products. The large variability in melphalan pharmacokinetics was explained by body weight (P <0.05). Concomitant topotecan administration did not influence melphalan pharmacokinetic parameters. There was no effect of the sequence of melphalan and topotecan administration in plasma pharmacokinetics. CONCLUSIONS: A regimen combining melphalan and topotecan for SSOAI treatment of retinoblastoma is active and well tolerated. This combination chemotherapy previously showed synergistic pharmacologic activity, and we herein provide evidence of not increasing the hematologic toxicity compared with single-agent melphalan.

Screening of RB1 alterations in Brazilian patients with retinoblastoma and relatives with retinoma: phenotypic and genotypic associations.

PURPOSE: To identify constitutional alterations of the retinoblastoma 1 gene (RB1) in two cohorts of Brazilian patients with retinoblastoma and to analyze genotype-phenotype associations. METHODS: Molecular screening was carried out by direct sequencing of the 27 RB1 exons and flanking regions in blood DNA of 71 patients with retinoblastoma and 4 relatives with retinoma, and with multiplex ligation-dependent probe amplification (MLPA) in 21 patients. The presumed impact of nucleotide substitutions on the structure of the retinoblastoma protein (pRB) was predicted by Polymorphism Phenotyping-2 (PolyPhen-2). Kaplan-Meier and log-rank test were used for estimating 60-month survival rates. RESULTS: One hundred two nucleotide substitutions were detected, 92 substitutions in 59 patients with retinoblastoma and 10 substitutions in 4 individuals with retinoma. Eight substitutions were novel. The majority of substitutions were intronic (86.2%). More than one substitution was present in 37.3% of patients. Twenty-one duplications and 11 deletions were found in 12 patients; some of which with both types of alterations. Duplications/deletions were found in four patients lacking constitutional alterations when analyzed by sequencing, and in eight patients carrying one or more polymorphic intronic substitutions. The global 60-month survival rate in patients was 91.8% (Confidence Interval95% = 85.0 - 99.1). Significant, lower survival rates were found in extraocular presentation (81.0%) versus intraocular tumors (P = 0.014), first enucleation after 1 month following diagnosis (80.9%) versus earlier first enucleation (P = 0.020), and relapse (100.0%) versus absence of relapse (P = 0.0005). CONCLUSIONS: Fifteen substitutions (4 intronic and 11 exonic) were identified as probably or likely pathogenic. Four of these 11 exonic substitutions were novel. Survival rates, however, were not affected by presence of these probably or likely pathogenic alterations, most of which not found in patients with retinoblastoma from other Latin American countries. These differences might be related to the different ethnic composition of the Latin American cohorts. Portuguese Abstract.

Influence of MDM2 and MDM4 on development and survival in hereditary retinoblastoma.

BACKGROUND: Retinoblastoma (RB) accounts for 3% of all childhood malignancies, with different incidences around the world. This malignancy results from loss-of-function of both RB1 alleles although other genes, like MDM2 and MDM4, have been proposed to be involved in tumor development. PROCEDURE: We genotyped rs2279744T>G and rs937283A>G in MDM2, and rs4252668T>C and rs116197192G>A in MDM4, in 104 unrelated RB patients and 104 controls. Sixty-month survival Kaplan-Meier curves and χ(2)-tests were performed for estimating the putative effect of MDM2 and MDM4 alleles on disease progression and survival of RB patients. RESULTS: MDM2 rs2279744G was significantly more frequent in controls, indicating an apparently protective effect on RB development. However, survival of patients who carried a constitutional RB1 mutation was significantly lower with rs2279744TG or GG than with rs2279744TT. Presence of rs2279744G and a constitutional RB1 mutation was sixfold more frequent in the 0-12 month age group than other age groups at onset of symptoms (P = 0.0401). MDM4 rs4252668C was present at a significantly higher frequency in controls while the frequency of MDM4 rs116197192G was significantly higher in RB patients, suggesting that this allele might increase the risk of developing RB. CONCLUSION: Our results indicate that MDM2 and MDM4 polymorphisms may influence development and/or survival in RB.

Constitutive RB1 mutation in a child conceived by in vitro fertilization: implications for genetic counseling.

BACKGROUND: The purpose of this study was to identify mutations associated with bilateral retinoblastoma in a quadruplet conceived by in vitro fertilization, and to trace the parental origin of mutations in the four quadruplets and their father. METHODS: Mutational screening was carried out by sequencing. Genotyping was carried out for determining quadruplet zygosity. RESULTS: The proband was a carrier of a novel RB1 constitutive mutation (g.2056C>G) which was not detected in her father or her unaffected sisters, and of two other mutations (g.39606 C>T and g.174351T>A) also present in two monozygotic sisters. The novel mutation probably occurred de novo while the others were of likely maternal origin. The novel mutation, affecting the Kozak consensus at the 5'UTR of RB1 and g.174351T>A were likely associated to retinoblastoma in the proband. CONCLUSION: Molecular diagnosis of retinoblastoma requires genotypic data of the family for determining hereditary transmission. In the case of children generated by IVF with oocytes from an anonymous donor which had been stored in a cell repository, this might not be successfully accomplished, making precise diagnosis impracticable for genetic counseling.

Hereditary retinoblastoma transmitted by maternal germline mosaicism.

BACKGROUND: Investigating transmission of a constitutive, g78238C > T (R552X), RB1 mutation in four affected children descended from three different unaffected fathers and an unaffected mother. PROCEDURES: Sequence data analyses and allele-specific PCR assays were used to investigate the presence of the mutation in four affected children, five unaffected sibs (or half-sibs), and the unaffected mother. Haplotyping was carried out for confirming that the children descended from different fathers. RESULTS: Haplotyping excluded the possibility of paternal transmission of a de novo mutation and provided evidence of maternal germline mosaicism. The mutation was apparently absent in blood- and buccal cell-DNA of the mother who also showed a normal fundoscopy. CONCLUSIONS: Our findings indicated that mosaicism was restricted to the maternal germline. The mutational event must have occurred at least 4 weeks post-conception, unlike the early mutational events of most mosaics, occurring between fertilization and the 8th day of conception. The implications of these findings are discussed in view that genetic counselling should discriminate between germline mosaicism and de novo events in pseudo-low-penetrant hereditary retinoblastoma.

Benzoporphyrin (verteporfin) photodynamic therapy for choroidal neovascularization age-related macular degeneration

Purpose: To review and discuss the available treatment for choroidal neovascularization (CNV) due to age-related macular degeneration (AMD) emphasizing to photodynamic therapy (PDT). Methods: Published papers from 1974 until 1999 related to AMD, its available treatments and PDT were reviewed. Results: The most used effective treatment for CNV is laser photocoagulation which does not have a major impact on the blindness due to AMD. Alternative techniques of prevention and treatment are under investigation including surgery, pharmacological antiangiogenic treatment and prevention with photocoagulation. PDT using verteporfin demonstrated effectiveness in closing CNV membranes in an experimental model. A clinical trials showed that a single course treatment could stabilize the area and extend of leakage from subfoveal CNV with classic component in the majority of patients for up 3 months (Phase I/II). A 12-month data for quarterly treatments was recently reported and showed that vision stabilization, meaning vision improvement, no change, or loss of < 3 lines, occurred in 61,4 per cent of the verteporfin-treated eyes and verteporfin-treated eyes are more likely to avoid a six-line or greater loss (85 per cent) compared to placebo (76 per cent). Another clinical trial (Phase III B) is under way to analize the role of PDT in the occult type of CNV. Conclusion: Treatment for this devastating condition remains a challenge. These clinical studies have shown an early closure of the CNV following PDT with exciting results in terms of visual acuity, although on a long-term basis these results are yet to be proven. Photodynamic therapy is one of the most promising new technologies to treat CNV.