Uncommon RB1 somatic mutations in a unilateral retinoblastoma patient

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a developing retina is a crucial event in the initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed the DNA from tumor tissue and peripheral blood of a unilateral retinoblastoma patient to determine the RB1 mutation status and to provide an accurate genetic counseling. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations. Screening for RB1 mutations was performed by PCR direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and Real Time-PCR analyses. Three different mutations were identified in the tumor DNA, which were absent in blood DNA. The somatic origin of these mutations was vital to rule out the heritable condition in this patient.

El retinoblastoma (RB) es el cáncer ocular más común de la niñez. La inactivación somática de ambos alelos del gen supresor de tumores RB1 en la retina en desarrollo es un evento crucial en la iniciación de la tumorigénesis en la mayoría de los casos de retinoblastoma unilateral. Nosotros analizamos el ADN de tumor y de sangre periférica de un paciente con retinoblastoma unilateral para identificar las mutaciones y así proveer un asesoramiento genético a la familia. Para ello utilizamos un protocolo basado en nuestra previa experiencia para identificar todas las mutaciones en el gen RB1 que causaron el RB. El rastreo de mutaciones se realizó por medio de los siguientes análisis: PCR-secuenciación, amplificación multiplex de sondas ligadas (MLPA) y PCR-Tiempo Real. Se encontraron tres mutaciones diferentes en el ADN del tumor, las cuales estaban ausentes en el ADN de la sangre. El origen somático de estas mutaciones es importante para indicar que la enfermedad no es hereditaria.

Treatment of Retinoblastoma: The Role of External Beam Radiotherapy

The risk of radiotherapy-related secondary cancers in children with constitutional retinoblastoma 1 (RB1) mutations has led to reduced use of external beam radiotherapy (EBRT) for RB. Presently, tumor reduction with chemotherapy with or without focal surgery (chemosurgery) is most commonly undertaken; EBRT is avoided as much as possible and is considered only as the last treatment option prior to enucleation. Nevertheless, approximately 80% of patients are diagnosed at a locally advanced stage, and only 20-25% of early stage RB patients can be cured with a chemosurgery strategy. As a whole, chemotherapy fails in more than two-thirds of eyes with advanced stage disease, requiring EBRT or enucleation. Radiotherapy is still considered necessary for patients with large tumor(s) who are not candidates for chemosurgery but who have visual potential. When radiation therapy is indicated, the lowest possible radiation dose combined with systemic or local chemotherapy and focal surgery may yield the best clinical outcomes in terms of local control and treatment-related toxicity. Proton beam therapy is one EBRT method that can be used for treatment of RB and reduces the radiation dose delivered to the adjacent orbital bone while maintaining an adequate dose to the tumor. To maximize the therapeutic success of treatment of advanced RB, the possibility of integrating radiotherapy at early stages of treatment may need to be discussed by a multidisciplinary team, rather than considering EBRT as only a last treatment option.

Identification of three new mutations in the RB1 gene in patients with sporadic retinoblastoma in Colombia / Identificación de tres nuevas mutaciones en el gen RB1 en pacientes con retinoblastoma esporádico en Colombia

Introduction. Retinoblastoma is a childhood cancer of the retina originated by altered or null retinoblastoma protein (pRb) expression. Genetic alterations in both RB1 alleles in the retinal cells are required for the development of retinoblastoma. In the sporadic form, non-hereditary RB1 gene mutations take place in a single retinoblast cell, and are therefore only present in tumor DNA (somatic mutations). Sporadic retinoblastoma is primarily unilateral, lacks family history and has no risk of transmission to descendants. Genetic tests for detection of RB1 mutation has improved the identification of carriers and facilitated accurate genetic counseling. Objective. To identify mutations in the RB1 gene in Colombian patients with sporadic retinoblastoma by PCR-SSCP followed by sequence. Materials and methods. Four patients with sporadic retinoblastoma were analyzed by PCR-SSCP, followed by DNA sequencing to identify variations in the RB1 gene. Results. We identified five variations in RB1 gene: three new mutations (one germline and two somatic mutations), one new polymorphism and one already reported somatic mutation. Four mutations were found in three patients with unilateral retinoblastoma and one mutation was found in a patient with bilateral retinoblastoma. One of these was a germline mutation in a sporadic unilateral retinoblastoma that was not present in the parents or three siblings analyzed. Conclusions. Our results emphasize the importance of identifying mutations for genetic counseling and clinical management of sporadic retinoblastoma patients. Description of a new RB1 gene variant is interesting since there have been a small number of polymorphisms reported for this gene.

Introducción. El retinoblastoma es un cáncer pediátrico de la retina originado por la expresión alterada o ausente de la proteína del retinoblastoma (pRb). Se requiere la alteración genética de ambos alelos RB1 en las células de la retina para el desarrollo del retinoblastoma. En la forma esporádica, las mutaciones no hereditarias del gen RB1 ocurren en un solo retinoblasto y están presentes sólo en el ADN del tumor (mutaciones somáticas). El retinoblastoma esporádico es generalmente unilateral, no tiene historia familiar y no tiene riesgo de transmisión a la descendencia. Las pruebas genéticas para la detección de mutaciones en RB1 han mejorado la identificación de portadores y han facilitado la precisión de la asesoría genética. Objetivo. Detectar mutaciones en el gen RB1 en pacientes colombianos con retinoblastoma esporádico mediante PCR-SSCP seguido de secuenciación. Materiales y métodos. Se analizaron cuatro pacientes con retinoblastoma esporádico para la detección de variaciones en el gen RB1 mediante PCR-SSCP, seguida de secuenciación. Resultados. Se identificaron cinco variaciones del gen RB1 : tres mutaciones nuevas (una de línea germinal y dos somáticas), un polimorfismo nuevo y una mutación somática ya reportada. Las cuatro mutaciones se encontraron en tres pacientes con retinoblastoma unilateral y uno con bilateral. La mutación germinal se detectó en un paciente con compromiso unilateral y no se encontró en los padres ni en los tres hermanos analizados. Conclusión. Estos resultados enfatizan la importancia, para asesoría genética y manejo clínico, de identificar mutaciones del gen RB1 en pacientes con retinoblastoma esporádico. La descripción de una nueva variante en RB1 es interesante, dado el muy bajo número de polimorfismos reportados para este gen.

Consideraciones clínicas, diagnósticas y de tratamiento en retinoblastoma / Clinical, diagnostic and therapeutic considerations in retinoblastoma

El retinoblastoma es el tumor intraocular primari o más frecuente en l a infancia. Su detección temprana y el inici o del tratamiento adecuado permi te mejorar dramáticamente l a sobrevida en estos niños. En este artícul o se hace una revisión general de l a enfermedad. Se empleó PubMed y se revisaron artículos representativos del tema, que permi tieran dar una idea general de los di ferentes avances alcanzados. Dada su cl ínica característica, el médico de atención primaria, es pieza fundamental en l a captación inicial del paciente. [Vi l lami l JF , Quintero LM, Serrano RA, Moreno IA. Consideraciones cl ínicas, diagnósticas y de tratamiento en retinoblastoma. MedUNAB 201 1; 14:180-187].

Retinoblastoma is the most common primary intraocular tumor in childhood. Its early detection and initiation of appropriate therapy , can dramatically improve the life expectancy in these children. This article is a general review of the disease. PubMed was employed and representative articles about the topic were selected in order to given us a general idea about the advances achieved. Due to clinical features, primary care physician is a fundamental part in the initial catchment of patient. [Villamil JF , Quintero LM, Serrano RA, Moreno IA. Clinical, diagnostic and therapeutic considerations in retinoblastoma. MedUNAB 2011; 14:180-187].

Impact of methylation of the p16INK4a gene on the prognosis ofhead and neck squamous cell carcinoma patients

OBJECTIVES: The inactivation of the tumor suppressor gene p16INK4a plays an important role in the development of malignant tumors, including oral squamous cell carcinoma. The p16 gene is involved in the p16/cyclin-dependent kinase/retinoblastoma (Rb) gene pathway of cell cycle control. The p16 protein is considered a negative regulator of this pathway. The p16 gene encodes an inhibitor of cyclin-dependent kinases 4 and 6 which regulate the phosphorylation of the retinoblastoma gene and G1 to S phase transition in the cell cycle. However, the p16 gene can lose its functionality through point mutations, loss of heterozygosity or methylation of its promoter region. MATERIALS AND METHODS: In this study, the authors analyzed the correlation between various clinicopathological findings-patient age, gender and smoking, disease recurrence, tumor size, stage, and differentiation- and p16 protein expression or p16 promoter hypermethylation in 59 cases of head and neck squamous cell carcinoma. RESULTS: The results revealed p16 protein expression and p16 promoter hypermethylation in 28 cases (47.5%) and 21 cases (35.6%), respectively, of head and neck squamous cell carcinoma. However, neither p16 protein expression nor p16 promoter hypermethylation had any statistical influence on clinicopathological findings or survival rate. CONCLUSION: This data, and a review of the literature, suggest that p16 promoter hypermethylation cannot yet be used as an independent prognostic factor influencing carcinogenesis, but must be considered as an important factor along with other genetic alterations affecting the pRb pathway.

Molecular genetic profiling of Filipino patients with retinoblastoma: A preliminary study

Objective@#To detect and characterize retinoblastoma susceptibility gene (RB1) mutations in tumor samples collected from Filipino patients with retinoblastoma.@*Methods@#Six tumor samples were obtained from Filipino patients diagnosed with retinoblastoma. DNA was extracted from the tumor samples and exons 13-21 of the RB1 gene were amplified by polymerase chain reaction (PCR). PCR amplification products were subsequently purified and sequenced. Mutation detection and characterization were done by alignment of obtained sequences to the RB1 reference sequence from NCBI GenBank using Bioedit® software. The identified mutations were correlated with clinical presentation and family history. These mutations were also compared to known mutations reported in the RB1 Gene Mutation Leiden Open Variation Database (LOVD).@*Results@#Mutations were detected in two out of the six samples. In a patient with unilateral disease and no family history, two mutations were identified: a novel CGT>AGT (Arginine → Serine) missense mutation in position c.1861 of exon 19 and a previously reported CGA>TGA (Arginine → STOP) nonsense mutation in position c. 1735 of exon 18. A possible large exonic deletion was identified in a case of unilateral disease with no family history.@*Conclusion@#We were able to identify both novel and known mutations in the RB1 gene of Filipino retinoblastoma cases using DNA sequencing techniques. These techniques may be applied to further characterize the genetic mutations of Filipino retinoblastoma cases and their families in developing a rational method of genetic testing for early diagnosis and counseling.

RB1 gene mutations in retinoblastoma and its clinical correlation

To find correlation between the type of mutations observed and the severity of the disease using multiple techniques like polymerase chain reactions [PCR], quantitative multiplex PCR, sequencing and RNA analysis. Prospective, observational study. Patients who had been screened for mutations in the RB1 gene were included in the study. Patient details including demographic data; age and sex, laterality, international classification of intraocular retinoblastoma [ICIOR] staging, modality of management, histopathology high risk factors if the eyes were enucleated and metastasis rate were assessed. Seventy four patients were studied. Fifty three patients had bilateral and 21 unilateral disease. Complete genetic data was analyzed for 74 patients and complete clinical correlation was established for all the 49 patients with mutations. Of the total mutations identified, 11/49 [22.4%] of patients had large deletions, 12/49 [24.5%] had small deletions or insertions, 14/49 [28.6%] had nonsense mutations, 7/49 [14.3%] had splice mutations and 5/49 [10.2%] of patients had missense mutations. Four cases were familial. Group E ICIOR stage at presentation was noted in 40% of patients with large deletions, 33% with small deletions whereas 38.5% with splice mutations and 44.4% of patients with missense mutations presented with Group B ICIOR. Twenty five percentages of eyes with large deletions had high risk features on histopathology and one patient among these developed metastasis. Current laboratory testing of RB1 mutations may be feasible in determining the severity of the disease and patient counseling. The study provides a starting point for looking at correlations

Prognostic Role of Rb, p16, Cyclin D1 Proteins in Soft Tissue Sarcomas / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The aim of this study was to determine the expressions of Rb, p16, and cyclin D1 in soft tissue sarcomas, and we also wanted to identify the prognostic factors according to the clinicalpathologic features. MATERIALS AND METHODS: We reviewed the charts and radiographic films of 66 sarcoma patients. Tissue samples were collected from these patients. Immunochemistry was performed using formalin-fixed, paraffin-embedded tissue samples to examine the expressions of p16, Rb, and cyclin D1 proteins. RESULTS: The median duration of overall survival was 47.8 months (range, 20.0 to 70.7 months) and the 5 years survival rate was 39%. As for the correlation between the degree of immunohistochemical staining for Rb protein and the histological tumor grades, there was a significant difference with a p-value of 0.019. However, no significant correlation was shown for p16 and cyclin D1. The overall survival duration of the Rb negative group (staining cell <20%) and the heterogeneous group (cell staining 20 to 80%) was 53.5+/-6.6 months and the overall survival duration of the Rb homogeneous group was 18.3+/-6.4 months, and there was a significant difference with a p-value of 0.016. However, no significant difference was shown between the survival rate according to the p16 and cyclin D1 expressions. On the multivariate analysis that was done with Rb, p16, the tumor size, grade and site, and patient age, the Rb gene expression was the most significant independent prognostic factor with a risk ratio of 3.01 (p=0.04). CONCLUSION: The expression of Rb protein was correlated with the histologic grade and overall survival of patients with soft tissue sarcomas.

Immunohistochemical study of p16[ink4a] and retinoblastoma gene products in different clinicopathological stages of mycosis fungoides

Mycosis fungoides [MF] is usually an indolent disease that, after a variable period of time in a stable phase, evolves into a tumoral form with aggressive behavior. Knowledge about the molecular mechanisms involved in the pathogenesis of tumoral progression in MIF is still scarce. Alterations of p16[ink4a] and retinoblastonia [Rb] have been demonstrated in a wide range of human tumors, p16[ink4a] inhibits Rb protein and thus acts as a negative cell cycle regulator. This prompted us to investigate their hypothetical role in MF progression. Twenty-five patients with MF of different clinicopatholgical stages were studied. p16[ink4a] expression was absent in 28% of the studied cases, where it was more lost in tumoral lesions than in patches and plaques lesions and in higher clinical stages than in low stages and the difference was statistically highly significant [P<0.001]. Therefore, the loss of p16[ink4a] is associated with the aggressive forms of MF. The results of the present study also showed a significant reciprocal relationship between p16[ink4a] and Rb proteins in most MF cases. However, alterations of Rb protein were not correlated with any of the clinicopathological features of the studied cases. In conclusion, this study demonstrated that lack of p16[ink4a], expression is a sensitive and specific marker of advanced cases of MF in comparison to Rb and thus p16[ink4a] could he used as a marker for poor prognostic patients with MF

Loss of Heterozygosity of Major Tumor Suppressor Genes in Invasive Ductal Carcinomas / 한국유방암학회지

PURPOSE: Breast cancer is one of the most frequent malignant tumors in Korea. The major tumor suppressor genes (TSGs) such as p16, Rb, E-cadherin and p53 may play important roles in cell cycle regulation, apoptosis and the regulation of the expression of other genes as well as tumor suppression. Microsatellite alteration such as loss of heterozygosity (LOH) have been reported to be a novel mechanism of carcinogenesis and a useful prognostic factor for many malignant tumors. Also, LOH is also known to be related with allelic loss of various TSGs. This study evaluated LOH of 4 TSGs in invasive ductal carcinomas (IDCs) and we correlated these results with the clinicopathological factors. METHODS: LOH analysis was carried out using a polymerase chain reaction with 12 polymorphic microsatellite markers of 4 TSGs in 50 surgically resected tumors and their non-tumorous counterparts. RESULTS: There was no detectable LOH in the normal tissue. LOH was detected in 86% of the 50 cases of IDCs. LOH was detected on all chromosomes and this showed a statistical difference between benign tumor and malignant tumor. LOH of p16, Rb, E-cadherin and p53 TSGs was detected in 36%, 26%, 54% and 60% of the tumors, respectively. LOH of the p16 and Rb genes was inversely correlated with tumor grade 1. The low rate of detecting LOH on the E-cadherin gene was noted in T1 tumor and stage I disease. LOH of the p53 gene correlated well with the tumor size and stage. The LOH-High results correlate well with the tumor size and stage and the LOH-High results are similar to those of the p53 gene LOH. CONCLUSION: These results suggest that LOH of the 4 major TSGs may contribute to the development and invasion of IDCs. Also, the combined use of various LOH markers may help in deciding the prognosis of IDCs.

Perspectivas en la genómica del retinoblastoma: Implicaciones del gen supresor de tumor RB1 / Genomic retinoblastoma perspectives: Implications of supressor gene of tumor RB1

In order to define the molecular and cellular bases of the development of retinoblastomas it is necessary to know its etiology, and to apply the advances in genome technology to this kind of neoplasia. Retinoblastomas are childhood tumors of the eye with an average incidence of one case in every 15,000-20,000 live births, which occur in sporadic and hereditary forms. The sporadic form appears regularly as a unilateral tumor, while in the familial form of the disease, tumors may be unilateral and bilateral. This neoplasia is characterized by leukocoria, strabism, and heterochromia. The retinoblastoma gene (RBl) is a molecular marker of retinoblastoma tumors. This gene is located in chromosome 13q14.2 and encodes a nuclear phosphoprotein (pRB) of 110 KDa, which plays a major role in cell proliferation control through cell cycle-regulated phosphorylation/dephosphorylation cycles of this protein. The RBl gene is mainly affected by point mutations, which occur most frequently in exons 3, 8, 18 and 20. At the end of the last century, DNA technology has improved notably, allowing for its application to the study of a vast array of diseases. The aim of this work is to show the molecular aspects involved in retinoblastoma which are currently deciphering; this is possible thanks to new technology platforms that have been developed. This will allow us in a near future, to offer tests for the early diagnoses, prognoses, and the determination of individual predisposition towards this neoplasia.

El retinoblastoma es una neoplasia embrionaria que se manifiesta en dos formas: esporádica (no heredada) o familiar (heredada). En los casos esporádicos el tumor es unilateral y en la forma familiar puede presentarse de manera unilateral o bilateral. Esta neoplasia tiene una incidencia promedio de 1/15,000 nacidos vivos, presentando signos y síntomas que incluyen leucocoria, estrabismo, midriasis unilateral y heterocromía. El gen que predispone al desarrollo de retinoblastoma es RBl y se localiza en el cromosoma 13 en la región ql4.2. El gen RBl codifica para una fosfoproteína nuclear que participa de manera importante en la regulación del ciclo celular. De acuerdo con la hipótesis de Knudson, para que se desarrolle la neoplasia se deben presentar dos mutaciones en el gen RBl. Las mutaciones puntuales son las que más frecuentemente se presentan en el gen RBl; la mayoría de los estudios indican que los exones 3, 8, 18, 19 y 20 son las regiones de mutación preferencial. En la áltima década ha habido un gran avance en la tecnología del DNA, lo cual hace posible su aplicación en diferentes enfermedades. Estas herramientas moleculares podrían ser de gran utilidad en el diagnóstico o conocimiento de la predisposición a desarrollar un retinoblastoma. Entre estas valiosas herramientas se cuenta con la hibridación fluorescente realizada in situ, hibridación genómica comparativa, las microhileras y por áltimo la identificación de polimorfismos de un sólo nucleótido. En conclusión, actualmente se están descifrando los aspectos moleculares que están relacionados con el retinoblastoma, gracias a la aplicación de nuevas plataformas tecnológicas. Esto permitirá en un futuro próximo ofrecer pruebas para un diagnóstico temprano o para conocer el pronóstico y la predisposición de individuos a desarrollar esta patología. Con el fin de entender las bases celulares y moleculares del desarrollo del retinoblastoma, el objetivo del presente trabajo es mostrar el estado del arte del conocimiento de esta neoplasia, así como su origen y los avances en la genómica aplicada al retinoblastoma.

Carcinoma de células transicionais da bexiga (CCT): detecção precoce de alterações genéticas como fator diagnóstico e prognostico da progressão tumoral / Transitional cell carcinoma of the bladder (TCC): precocious detection of genetic alterations as diagnosis factor and tumor progression prognosis

Carcinoma de células transicionais (CCT) é o tipo histológico predominante de tumor epitelial maligno da bexiga. Estudos genéticos sobre os processos de iniciação e de progressão tumoral têm contribuído para o melhor entendimento dos mecanismos de carcinogênese urotelial. A ocorrência de alterações genéticas, principalmente em genes supressores de tumor e oncogenes, como CDKN-2, ARF, RB, TP53, H-RAS, BCL-2, c_ERBB-2, cyclin D1 e o receptor EGF-R, tem sido relatada em CCT. A progressão desses tumores parece ocorrer conjuntamente com a aquisição de anomalias cromossômicas com deleções em 9p, 9q e 17p e ganhos em 1q, 5p, 7p, 11q e 17q. Assim, a avaliação genética utilizando marcadores genéticos para o diagnóstico precoce e risco de recorrência e metástases torna-se um método promissor

Dos nuevas mutaciones y tres nuevos polimorfismos en el gen del retinoblastoma en población ecuatoriana / Two new mutations and three new polymorphisms in the gene of retinoblastoma in Ecuadorian population

El cáncer es una enfermedad de origen genético y para comprender mejor los diferentes mecanismos que conllevan a su desarrollo es necesario el estudio de los genes y mutaciones. El retinoblastoma es el tumor de ojo más frecuente de la infancia y se desarrolla a partir de mutaciones en el gen Rb1; estas mutaciones pueden ser de origen hereditario en el 40 por ciento de los casos y espontáneo en el 60 por ciento de los casos. En la actualidad, se dispone de análisis moleculares para determinar origen y así poder brindar un mejor asesoramiento genético. En el presente estudio se analizaron muestras de 31 pacientes de diferentes regiones del Ecuador. Se realizaron 840 reacciones de PCR (reacción en cadena de la polimerasa)...

Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Both tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene.</p><p><b>METHODS</b>After identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry.</p><p><b>RESULTS</b>Neither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene.</p><p><b>CONCLUSION</b>p15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.</p>

Loss of heterozygosity in esophageal squamous cell carcinoma and its precursor lesion / 中华病理学杂志

<p><b>OBJECTIVE</b>To detect the loss of heterozygosity (LOH) in esophageal squamous cell carcinoma and adjacent high-grade squamous dysplasia, and to evaluate possible tumor suppressor genes in the development and progression of invasive malignancy.</p><p><b>METHODS</b>LOH was detected in normal esophageal mucosa, high grade squamous dysplasia and esophageal squamous cell carcinoma using microdissection and polymerase chain reaction technology. The changes of LOH at seven microsatellite markers and the relationship between LOH rate and clinicopathologic parameters were analyzed.</p><p><b>RESULTS</b>In high grade squamous dysplasia, LOH was detected at D13S802 (40%), D13S267 (32%), D13S221 (31%), D9S942 (30%), D17S520 (24%) and D9S171 (33%). However, D17S1798 LOH was not detected. In invasive squamous cell carcinoma, LOH was detected as follows: D13S267 (71%), D13S802 (58%), D17S520 (55%), D13S221 (45%), D9S942 (43%), D9S171 (33%) and D17S1798 (11%). The frequency of LOH in the seven microsatellite markers, the pathologic grade, clinical stage and occurrence of lymph node metastasis did not show any statistically significant correlation (P > 0.05).</p><p><b>CONCLUSIONS</b>The progression from normal squamous epithelium to high grade squamous dysplasia and subsequently to invasive squamous cell carcinoma of the esophagus was associated with accumulation of genetic errors. Possible tumor suppressor genes related to the development of esophageal squamous cell carcinoma may exist near D13S802 (13q12.12). Possible tumor suppressor genes near D13S267 (13q13.1), D17S1798 (17p13.3) and D17S520 (17p13.1) may be related to the progression of esophageal squamous cell carcinoma.</p>

Methylation and expression analysis of p16(INK4a) and RB genes in meningiomas / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the methylation of p16(INK4a) and RB gene, and the expression of p16(INK4a) in meningiomas.</p><p><b>METHODS</b>Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation of p16(INK4a) and RB in 50 cases of meningiomas, and immunostaining was performed to analyze the protein expression of p16(INK4a) in 25 of those cases.</p><p><b>RESULTS</b>No methylation was found in the benign meningiomas, whereas methylation of p16(INK4a)or RB occurred in 6(37.5%) cases of grade II tumors and 4(28.6%) cases of grade III tumors, and among these cases, an atypical meningioma showed methylation of both genes. Thirteen cases showed p16(INK4a) positive expression, but none of them was methylated.</p><p><b>CONCLUSION</b>The methylation of p16(INK4a) or RB is related with the tumorigenesis and progression of atypical and anaplastic meningiomas, and a probable mechanism is that methylation causes the loss of expression and leads to dysfuncation of the p16(INK4a)/cyclin D1/CDK4/RB pathway.</p>

Construction and confirmation of the fused plasmid with Rb bait gene in yeast two-hybrid system / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To screen and clone the novel genes related to cellular proliferation of oral squamous cell carcinoma.</p><p><b>METHODS</b>We selected the Rb gene as the bait protein gene to construct the fusion bait plasmid of yeast two-hybrid. The whole code sequence of Rb gene was acquired by digestion with restricted enzyme EcoRI and BamH1 and reclaimed from its original vector pGBT9-pRb. After being confirmed by electrophoresis, the Rb gene was cloned into the MCS of the plasmid pGBKT7 to construct a recombined plasmid pGBKT7-pRb and the sequence of the recombined plasmid was detected in company. According to the protocol of yeast two hybrid system III, the competent Y187 yeast was prepared, and transformed with recombined plasmid pGBKT7-pRb. Following that, the toxicity and transcriptional activation of this recombined plasmid pGBKT7-pRb in Y187 yeast were tested.</p><p><b>RESULTS</b>The sequence of the recombined plasmid was correct compared with the sequence provided in Genbank. The protein could be correctly synthesized in vitro, and no self-activating transcriptional activation and toxicity was observed in Y187 yeast.</p><p><b>CONCLUSIONS</b>The construction of the recombined plasmid was capable to be used as the fusion bait plasmid in yeast two-hybrid system III, and the recombined Rb-protein could be used as the bait protein successfully.</p>

Correlation of E2F-1 and Rb expression with papillomatosis and ductal carcinoma in situ of the breast / 中华肿瘤杂志

<p><b>OBJECTIVE</b>E2F-1 and Rb are involved in cell cycle regulation. This study was to illustrate the mechanism of transformation from benign papillomatosis to ductal carcinoma in situ (DCIS) of the breast in relation to E2F-1 and Rb expression.</p><p><b>METHODS</b>In situ hybridization (ISH) was used to determinate the expression of E2F-1 and Rb mRNA of mild papillomatosis (MP, n = 40), severe papillomatosis (SP, n = 40) and DCIS (n = 40). Immunohistochemistry (IHC) was used to examine the expression of E2F-1 and Rb protein.</p><p><b>RESULTS</b>The positive rate of E2F-1 mRNA expression in MP, SP and DCIS was 17.5%, 45.0% and 80.0%, and that of E2F-1 protein expression was 20.0%, 47.5% and 77.5%, respectively. There were significant differences among the three groups (P < 0.01), and between any two groups (P < 0.01). The positive rate of Rb mRNA expression in MP, SP and DCIS was 90.0%, 50.5% and 20.0%, and that of Rb protein expression was 85.0%, 52.5% and 22.5%, respectively, with statistically significant difference similar with that of E2F-1. With the progression of papillomatosis to DCIS, the expression of E2F-1 mRNA and protein increased, while that of Rb decreased. The protein expression by IHC was positively correlated with the mRNA expression by ISH. However, that of E2F-1 was negatively correlated with Rb.</p><p><b>CONCLUSION</b>E2F-1 and Rb might provide a valuable basis for screening high risk papillomatosis and new target of gene therapy for pre-cancerous lesions of the breast.</p>

Secuelas tardías de la oncoterapia infantil: las segundas neoplasias malignas / Delayed sequels of the infantile oncotherapy: the second malignant neoplasms

El empleo de modalidades terapéuticas cada vez más efectivas, a la par de la mejoría en los cuidados de soporte, ha pemitido un incremento significativo de los índices de sobrevida del cáncer infantil, de manera que más del 70 por ciento de los niños tratados actualmente pueden sobrevivir al menos 5 años después del diagnóstico. Estos logros en los índices de curación tienen un precio: muy pocos sobrevivientes de cáncer a esta edad estarán libres a largo plazo de problemas relacionados con la terapéutica recibida: cirugía, radioterapia y quimioterapia. Los efectos tardíos comprometen una diversidad de esferas: cardiovascular, endocrinológica, neurológica, gastrointestinal, renal y genitourinaria, músculo esquelético, pulmonar, ocular, etc. Una de las consecuencias más decepcionantes de la curación es la aparición de una segunda neoplasia maligna. Se estima que el 3 por ciento al 12 por ciento de los pacientes tratados por cáncer en la niñez desarrollarán una segunda neoplasia maligna en el transcurso de 20 años depués del diagnóstico. Se presentan 11 casos con diagnóstico de segunda neoplasia maligna evaluados en dos instituciones donde se atienden a niños con cáncer. Se encontró como primera neoplasia más frecuente el retinoblastoma y como segunda neoplasia maligna osteosarcoma, destacándose la relación genética entre ambas entidades

Effects of bcr/abl fusion gene on expression of beta1 integrin and L-selectin in mouse chronic myeloid leukemia cells / 中华血液学杂志

<p><b>OBJECTIVES</b>To explore the effects of p210 bcr/abl fusion gene on expression of beta1 integrin and L-selectin mRNAs in mouse chronic myeloid leukemia (CML) cells.</p><p><b>METHODS</b>Comparisons of beta1 integrin and L-selectin mRNA levels among p210 bcr/abl negative, p210 bcr/abl positive, and p210 bcr/abl-Rb-C-Box positive cells were undertaken by quantity RT-PCR.</p><p><b>RESULTS</b>In p210 bcr/abl positive cells, L-selectin mRNA level was decreased, but beta1 integrin mRNA expression had no change as compared to those in p210 bcr/abl negative cells. When inhibition of bcr-abl tyrosine kinase activity by Rb-C-Box, the L-selectin mRNA expression restored to normal (similar to p210 bcr/abl negative cells).</p><p><b>CONCLUSION</b>p210 bcr/abl oncoprotein inhibits expression of L-selectin mRNA, but not of beta1 integrin mRNA.</p>