Theodora Hadjistilianou

Pathogenesis of posterior capsular opacification Part II: Histopathological and in vitro culture findings

ABSTRACT The most interesting sources of information about the pathogenesis of posterior capsular opacification seem to be histopathological studies and in vitro tissue cultures. Since our surgical technique is extracapsular cataract extraction, the explants we used for tissue culture consisted of the anterior capsule epithelial sheet without the equatorial germinative zone. We successfully overcame several problems by using the autologous plasma clot culture method. This medium, considered the optimal one for this type of culture, allowed us to study the heterogeneous behavior of the epithelial cells in culture. Using the plasma clot culture method, we were able to demonstrate in vitro fibroblastic transformation of the epithelial cells. Histopathological findings of particular cases of posterior capsule opacification and immunohistochemistry of the human lens are also reported.

Superselective ophthalmic artery infusion of melphalan for intraocular retinoblastoma: preliminary results from 140 treatments

Purpose:  To report our experience in superselective ophthalmic artery infusion of melphalan (SOAIM) for intraocular retinoblastoma.

Is CT still useful in the study protocol of retinoblastoma

BACKGROUND AND PURPOSE: Intralesional calcium deposition is considered a key element for differentiating retinoblastoma from simulating lesions. Our aim was to assess whether MR imaging associated with ophthalmologic investigations (ophthalmoscopy and ultrasonography) could replace CT in the detection of diagnostic intralesional calcifications in retinoblastoma. MATERIALS AND METHODS: Ophthalmoscopic findings, MR images, CT scans, and histologic examination of 28 retinoblastomas from 23 consecutive children (11 males, 12 females; age range at admission, 1–35 months; mean age, 11 months; median age, 9 months) were retrospectively evaluated. Ultrasonography was performed in 18 patients with 21 retinoblastomas. MR imaging included T2-weighted spin-echo and gradient-echo images, fluid-attenuated inversion recovery images, and T1-weighted spin-echo images with and without contrast enhancement. Clinical data were integrated with MR imaging data to evaluate the utility of both approaches to discover calcifications; particularly, a correlation between intralesional signal-intensity void spots on MR imaging and hyperattenuating areas on CT scans was performed. RESULTS: Ophthalmoscopy detected calcifications in 12 of 28 eyes (42.85%). Ultrasonography detected calcifications in 20 of 21 eyes (95.23%). CT showed hyperattenuating intralesional areas consistent with calcifications in 27 of 28 eyes (96.42%). MR imaging showed intralesional signal-intensity void spots in 25 of 28 eyes (89.28%). All spots detected with MR imaging matched the presence of calcifications on CT scans. Gradient-echo T2*-weighted and fast spin-echo T2-weighted images showed the highest degree of correlation with CT. When we put together ophthalmoscopy, ultrasonography, and MR imaging data, no calcifications detected on CT were missed, and the differential diagnosis was thorough. CONCLUSIONS: A combination of clinical data and MR images may remove potentially harmful ionizing radiation from the study protocol of retinoblastoma.

Genomic differences between retinoma and retinoblastoma

Introduction. Genomic copy number changes are involved in the multi-step process transforming normal retina in retinoblastoma after RB1 mutational events. Previous studies on retinoblastoma samples led to a multi-step model in which after two successive RB1 mutations, further genomic changes accompany malignancy: 1q32.1 gain is followed by 6p22 gain, that in turn is followed by 16q22 loss and 2p24.1 gain. Retinoma is a benign variant of retinoblastoma that was initially considered a tumor regression, but recent evidences suggest that it rather represents a pre-malignant lesion. Genetic studies on retinoma tissue have rarely been performed. Materials and methods. We investigated by Real-Time qPCR, copy number changes of candidate genes located within the 4 hot-spot regions (MDM4 at 1q32.1, MYCN at 2p24.1, E2F3 at 6p22 and CDH11 at 16q22) in retina, retinoma and retinoblastoma tissues from two different patients. Results. Our results demonstrated that some copy number changes thought to belong to early (MDM4 gain) or late stage (MYCN and E2F3 gain) of retinoblastoma are already present in retinoma at the same (for MDM4) or at lower (for MYCN and E2F3) copy number variation respect to retinoblastoma. CDH11 copy number is not altered in the two retinoma samples, but gain is present in one of the two retinoblastomas. Discussion. Our results suggest that MDM4 gain may be involved in the early transition from normal retina to retinoma, while MYCN and E2F3 progressive gain may represent driving factors of tumor progression. These results also confirm the pre-malignant nature of retinoma.

Array comparative genomic hybridization in retinoma and retinoblastoma tissues

In retinoblastoma, two RB1 mutations are necessary for tumor development. Recurrent genomic rearrangements may represent subsequent events required for retinoblastoma progression. Array‐comparative genomic hybridization was carried out in 18 eye samples, 10 from bilateral and eight from unilateral retinoblastoma patients. Two unilateral cases also showed areas of retinoma. The most frequent imbalance in retinoblastomas was 6p gain (40%), followed by gains at 1q12‐q25.3, 2p24.3‐p24.2, 9q22.2, and 9q33.1 and losses at 11q24.3, 13q13.2‐q22.3, and 16q12.1‐q21. Bilateral cases showed a lower number of imbalances than unilateral cases (P = 0.002). Unilateral cases were divided into low‐level (≤4) and high‐level (÷7) chromosomal instability groups. The first group presented with younger age at diagnosis (mean 511 days) compared with the second group (mean 1606 days). In one retinoma case ophthalmoscopically diagnosed as a benign lesion no rearrangements were detected, whereas the adjacent retinoblastoma displayed seven aberrations. The other retinoma case identified by retrospective histopathological examination shared three rearrangements with the adjacent retinoblastoma. Two other gene‐free rearrangements were retinoma specific. One rearrangement, dup5p, was retinoblastoma specific and included the SKP2 gene. Genomic profiling indicated that the first retinoma was a pretumoral lesion, whereas the other represents a subclone of cells bearing ‘benign’ rearrangements overwhelmed by another subclone presenting aberrations with higher ‘oncogenic’ potential. In summary, the present study shows that bilateral and unilateral retinoblastoma have different chromosomal instability that correlates with the age of tumor onset in unilateral cases. This is the first report of genomic profiling in retinoma tissue, shedding light on the different nature of lesions named ‘retinoma’. (Cancer Sci 2009; 100: 465–471)

Epigenetic and copy number variation analysis in retinoblastoma by MS-MLPA.

Retinoblastoma is the most common primary intraocular malignancy in children. Two step inactivation of RB1 (M1-M2) represents the key event in the pathogenesis of retinoblastoma but additional genetic and epigenetic events (M3-Mn) are required for tumor development. In the present study, we employed Methylation Specific Multiplex Ligation Probe Assay to investigate methylation status and copy number changes of 25 and 39 oncosuppressor genes, respectively. This technique was applied to analyse 12 retinoblastomas (5 bilateral and 7 unilateral) and results were compared to corresponding normal retina. We identified hypermethylation in seven new genes: MSH6 (50%), CD44 (42%), PAX5 (42%), GATA5 (25%), TP53 (8%), VHL (8%) and GSTP1 (8%) and we confirmed the previously reported hypermethylation of MGMT (58%), RB1 (17%) and CDKN2 (8%). These genes belong to key pathways including DNA repair, pRB and p53 signalling, transcriptional regulation, protein degradation, cell-cell interaction, cellular adhesion and migration. In the same group of retinoblastomas, a total of 29 copy number changes (19 duplications and 10 deletions) have been identified. Interestingly, we found deletions of the following oncosuppressor genes that might contribute to drive retinoblastoma tumorigenesis: TP53, CDH13, GATA5, CHFR, TP73 and IGSF4. The present data highlight the importance of epigenetic changes in retinoblastoma and indicate seven hypermethylated oncosuppressors never associated before to retinoblastoma pathogenesis. This study also confirms the presence of copy number variations in retinoblastoma, expecially in unilateral cases (mean 3 ±1.3) where these changes were found more frequently respect to bilateral cases (mean 1.4 ± 1.1).

Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations

AbstractRetinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.

Does the evidence matter in medicine? The retinoblastoma paradigm

Retinoblastoma (Rb) is the most common intraocular malignant tumour in childhood, with an incidence of 1 in 15,000 live births. Complete information on this rare tumour can be easily accessed through the internet, although many aspect concerning the aetiology and pathogenesis of the disease, are still controversial. The “two hit” theory, formulated in 1971 to explain the variegated clinical expression of the disease, is based on the idea that single gene mutation may determine the development of cancer. However, this view does not take into account the most recent evidences showing the role of aneuploidy and chromosome instability in cancer. Also, a number of other genes and epigenetic mechanisms are involved in the genesis of retinoblastoma. More importantly, the “two hit” theory makes predictions, concerning the age distribution of the tumour, its mode of “transmission” (hereditary retinoblastoma), and its pathogenesis, which are not fulfilled by the clinical reality. Overall, the “two hit” theory represents a rather simplistic and outdated model to explain tumour development and clinical evolution of retinoblastoma.

Histopathological evaluation for the prognosis of retinoblastoma

In the present study histologic, clinical and follow-up data of 37 cases of retinoblastoma were statistically analyzed by means of a multivariate method. The evaluated histological parameters were: growth pattern, degree of differentiation, number of mitoses, presence of necrosis, pseudorosettes, blood vessel basophilia, calcification, extension to anterior chamber, invasion of optic nerve and ocular coats (choroid and sclera). The analysis was based upon the Cox proportional hazards regression model. Invasion of ocular coats proved to be highly correlated to the prognosis (p less than 0.0002). None of the other parameters showed a significant relation to survival.

A case of coloboma in a newborn to a woman taking mycophenolate mofetil in pregnancy after kidney transplantation.

Recently, mycophenolate mofetil (MMF) has been introduced in the immunosuppressive strategy after kidney transplantation. Recently, the existence of a MMF associated embriopathy has been hypothesized, namely, multiple craniofacial malformations. Only 1 report has described chorioretinal coloboma. We report a case of woman who used MMF throughout pregnancy after kidney transplantation. Her newborn developed coloboma of the right eye associated with an ocular cyst without any other malformation. The other drugs used by our patient are not considered teratogenic. Therefore, it seems reasonable to conclude a causal relationship between MMF and the malformation observed in this newborn.