Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Molecular study of signaling-pathway genes in experimental rat thyroid carcinoma.

INTRODUCTION: A study was conducted on histological patterns and biomolecular changes in Goitrogen-induced experimental rat thyroid tumors. The link between the histological types observed and N-ras, B-raf, and PI3KCA gene mutations widely reported in human thyroid cancers was explored. MATERIAL AND METHODS: An analysis was done on paraffin-embedded tumor tissue sections from Wistar rats receiving 1% potassium perchlorate (KClO(4)) added to the ad libitum drinking-water supply over an 18-month period. Three experimental subgroups were formed, each comprising 10 thyroids: subgroup I (control) consisted of thyroids from untreated controls; subgroups II and III (experimental) consisted of thyroids from KClO(4)-treated rats, displaying capsular, vascular, or both invasion but no metastasis (II), or distant metastasis (III). DNA was extracted from paraffin-embedded tissues. To test for the genetic mutations most widely reported in human thyroid cancers, exon 1 of the N-ras gene, exons 9 and 20 of the PI3KCA gene, and exon 15 of the B-raf gene were amplified and sequenced. RESULTS: All tumors were of the follicular type. None of the 20 experimental rat thyroids displayed the expected gene mutations reported in humans. However, 90% of them contained four new B-raf gene mutations and all were silent and did not cause an amino acid substitution in the protein chain. CONCLUSIONS: Biomolecular analysis suggested that N-ras, PI3KCA, and B-raf gene mutations may not be involved in thyroid tumor formation using the experimental procedure applied in this study. But the four mutations in B-raf, though without functional repercussions, may be a specific marker for this tumor type.

Gastrointestinal stromal tumors (GISTs): role of CD 117 and PDGFRA Golgi-like staining pattern in the recognition of mutational status.

AIMS: to determine whether potential correlations between CD117 and PDGFRA might serve as an indication for targeted therapies. MATERIAL AND METHODS: immunohistochemical expression of CD117 and PDGFRA was evaluated in 99 paraffin-embedded GISTs in conjunction with KIT and PDGFRA mutational status. RESULTS: CD117-positive staining was noted in 93 out of 99 cases. The predominant staining pattern was cytoplasmic, either with or without membrane accentuation; in 44.5% of cases, a clear Golgi-like pattern was evident. Correlations were found between KIT mutation and both CD117 expression (p = 0.006) and Golgi-like pattern (p = 0.026). Cytoplasmic PDGFRA-positive staining was detected in 87% of cases, both with and without membrane accentuation; in 8% cases an evident Golgi-like staining pattern was observed. A significant correlation was noted between PDGFRA mutations and Golgi-like staining pattern (p = 0.001). Moreover, 95% of PDGFRA-positive GISTs were also CD117-positive, suggesting that expression of the two markers is not mutually exclusive; most of these had mutations in KIT exon 11. PDGFRA-positive/CD117-negative tumors had mutations in PDGFRA, mainly in exon 18. PDGFRA-negative/CD117-negative staining was observed in 15% of cases, all of which displayed mutations in KIT exon 11. CD117-positive/PDGFRA-negative cases were characterized by mutations in KIT, mainly in exon 11. CONCLUSIONS: CD117 and PDGFRA staining are not exclusive, and the presence of a Golgi-like staining pattern for either, whilst not pathognomonic, is highly suggestive of KIT and PDGFRA mutated GISTs, respectively, and may be used with some reservations as an alternative indication for prescribing targeted therapies.

Inmunohistochemical profile of solid cell nest of thyroid gland.

It is widely held that solid cell nests (SCN) of the thyroid are ultimobranchial body remnants. SCNs are composed of main cells and C cells. It has been suggested that main cells might be pluripotent cells contributing to the histogenesis of C cells and follicular cells, as well as to the formation of certain thyroid tumors. The present study sought to analyze the immunohistochemical profile of SCN and to investigate the potential stem cell role of SCN main cells. Tissue sections from ten cases of nodular hyperplasia (non-tumor goiter) with SCNs were retrieved from the files of the Hospital Infanta Luisa (Seville, Spain). Parathormone (PTH), calcitonin (CT), thyroglobulin (TG), thyroid transcription factor (TTF-1), galectin 3 (GAL3), cytokeratin 19 (CK 19), p63, bcl-2, OCT4, and SALL4 expression were evaluated by immunohistochemistry. Patient clinical data were collected, and tissue sections were stained with hematoxylin-eosin for histological examination. Most cells stained negative for PTH, CT, TG, and TTF-1. Some cells staining positive for TTF-1 and CT required discussion. However, bcl-2, p63, GAL3, and CK 19 protein expression was detected in main cells. OCT4 protein expression was detected in only two cases, and SALL4 expression in none. Positive staining for bcl-2 and p63, and negative staining for PTH, CT, and TG in SCN main cells are both consistent with the widely accepted minimalist definition of stem cells, thus supporting the hypothesis that they may play a stem cell role in the thyroid gland, although further research will be required into stem cell markers. Furthermore, p63 and GAL-3 staining provides a much more sensitive means of detecting SCNs than staining for carcinoembryonic antigen, calcitonin, or other markers; this may help to distinguish SCNs from their mimics.

Molecular findings of nasopharyngeal carcinoma in a European population.

OBJECTIVE: To explore biomolecular characteristics of a group of patients with nasopharyngeal carcinoma from European (Spanish) hospitals, addressing the pathogenesis of the tumor and the response to treatment. STUDY DESIGN: Cyclin D1 and p16 expression were evaluated immunohistochemically in 33 tissue samples of nasopharyngeal carcinoma. CCDN1 gene amplification and p16 gene deletion were studied by fluorescence in situ hybridization. Patient clinical data were examined, and tissues were evaluated histologically using hematoxylin-eosin staining. RESULTS: Cyclin D1 overexpression was found in 19 cases, and p16 expression was undetected in 30 cases. An association was observed between impaired p16 expression and cyclin D1 overexpression (p = 0.034). Eleven patients displayed p16 gene deletion and CCDN1 gene amplification. CONCLUSION: Cyclin D1 overexpression and CCDN1 amplification, loss of p16 expression and p16 deletion may be among the genetic alterations involved in the pathogenesis of nasopharyngeal carcinoma.

Tumores del estroma gastrointestinal (GISTs): patrón de tinción tipo Golgi de CD 117 Y PDGFRA en el reconocimiento del estado mutacional / Gastrointestinal stromal tumors (GISTs): role of CD 117 and PDGFRA Golgi-like staining pattern in the recognition of mutational status

Objetivo: determinar si las posibles correlaciones entre CD117 y PDGFRA podrían servir como una indicación de terapias dirigidas. Material y métodos: la expresión inmunohistoquímica de CD117 y PDGFRA se evaluó en 99 GIST incluidos en parafina en conjunción con el estado mutacional de KIT y PDGFRA Resultados: se observó tinción CD117-positivo en 93 de los 99 casos. El patrón de tinción predominante fue citoplasmático o de membrana; en el 44,5% de los casos, se evidencio patrón de tipo Golgi. Se encontraron correlaciones entre la mutación KIT tanto con la expresión de CD117 (p = 0,006) y con el patrón tipo Golgi (p = 0,026). Se detectó tinción citoplasmática PDGFRA-positiva en el 87% de los casos, con y sin acentuación de membrana, en el 8% se observó patrón de tinción tipo Golgi. Se observó una correlación significativa entre las mutaciones PDGFRA y el patrón de tinción tipo Golgi (p = 0,001). Por otra parte, el 95% de los GIST PDGFRA positivos también fueron CD117-positivo, lo que sugiere que la expresión de los dos marcadores no se excluyen mutuamente, la mayoría de ellos tenían mutaciones en el exón 11 de KIT. Los tumores PDGFRA-positivo/CD117-negativo tenían mutaciones en PDGFRA, principalmente en el exón 18. Se observó tinción PDGFRA-negativo/CD117-negativo en el 15% de los casos, todos los cuales revelaban mutaciones en el exón 11 de KIT. Los casos CD117-positivo/PDGFRA-negativo casos se caracteriza por mutaciones en KIT, principalmente en el exón 11. Conclusiones: las tinciones CD117 y PDGFRA no son excluyentes, y la presencia de un patrón de tinción de Golgi, aunque no es patognomónica, es altamente sugestiva de GIST mutado en KIT y PDGFRA, respectivamente, y se puede utilizar con algunas reservas, como una indicación alternativa para la prescripción de terapias dirigidas(AU)

Aims: determine whether potential correlations between CD117 to and PDGFRA might serve as an indication for targeted therapies. Material and methods: immunohistochemical expression of CD117 and PDGFRA was evaluated in 99 paraffin-embedded GISTs in conjunction with KIT and PDGFRA mutational status. Results: CD117-positive staining was noted in 93 out of 99 cases. The predominant staining pattern was cytoplasmic, either with or without membrane accentuation; in 44.5% of cases, a clear Golgi-like pattern was evident. Correlations were found be - tween KIT mutation and both CD117 expression (p = 0.006) and Golgi-like pattern (p = 0.026). Cytoplasmic PDGFRA-positive staining was detected in 87% of cases, both with and without membrane accentuation; in 8% cases an evident Golgi-like staining pattern was observed. A significant correlation was noted between PDGFRA mutations and Golgi-like staining pattern (p = 0.001). Moreover, 95% of PDGFRA-positive GISTs were also CD117- positive, suggesting that expression of the two markers is not mutually exclusive; most of these had mutations in KIT exon 11. PDGFRA-positive/CD117-negative tumors had mutations in PDGFRA, mainly in exon 18. PDGFRA-negative/CD117-negative staining was observed in 15% of cases, all of which displayed mutations in KIT exon 11. CD117-positive/PDGFRA-negative cases were characterized by mutations in KIT, mainly in exon 11. Conclusions: CD117 and PDGFRA staining are not exclusive, and the presence of a Golgi-like staining pattern for either, whilst not pathognomonic, is highly suggestive of KIT and PDGFRA mutated GISTs, respectively, and may be used with some reservations as an alternative indication for prescribing targeted therapies(AU)