Ependimoma intracraneal: apuntes clínicos, moleculares y anatomopatológicos / Intracranial ependymoma: clinical, molecular and pathological notes

RESUMEN Los ependimomas surgen de las células ependimarias que revisten los ventrículos y los pasajes en el encéfalo y el centro de la médula espinal. Las células ependimarias producen líquido cefalorraquídeo. Se decidió la realización de una revisión acerca del ependimoma intracraneal teniendo en cuenta que no existe artículo nacional que trate este tema, siendo la mayoría de los trabajos consultados referentes a la misma variante histológica pero en localización espinal, cuyo objetivo es describir la características clínicas, moleculares y anatomopatológicas del ependimoma intracraneal. Se realizó la búsqueda de artículos en revistas de las bases de datos: PubMed, Scielo y EBSCO. La búsqueda se limitó a artículos con el texto completo, publicados fundamentalmente en los últimos cinco años. El ependimoma intracraneal es un tumor frecuente en la edad pediátrica, sus manifestaciones clínicas dependen de su localización, presenta una gran diversidad molecular y anatomoptológica (AU).

SUMMARY Ependymomas arise from ependymal cells that line the ventricles and passages in the brain and center of the spinal cord. Ependymal cells produce cerebrospinal fluid. It was decided to conduct a review about intracranial ependymoma taking into account that there is no national article dealing with this issue, with most of the works consulted referring to the same histological variant but in spinal location, whose objective is to describe the clinical characteristics, Molecular and pathological pathways of intracranial ependymoma. We searched articles in journals of the databases: PubMed, Scielo and EBSCO. The search was limited to articles with the full text, published mainly in the last five years. Intracranial ependymoma is a frequent tumor in the pediatric age, its clinical manifestations depend on its location, it has a great molecular and anatomoptological diversity (AU).

Cost analysis of smear microscopy and the Xpert assay for tuberculosis diagnosis: average turnaround time

Abstract INTRODUCTION: Rapid and accurate tuberculosis detection is critical for improving patient diagnosis and decreasing tuberculosis transmission. Molecular assays can significantly increase laboratory costs; therefore, the average time and economic impact should be evaluated before implementing a new technology. The aim of this study was to evaluate the cost and average turnaround time of smear microscopy and Xpert assay at a university hospital. METHODS: The turnaround time and cost of the laboratory diagnosis of tuberculosis were calculated based on the mean cost and activity based costing (ABC). RESULTS: The average turnaround time for smear microscopy was 16.6 hours while that for Xpert was 24.1 hours. The Xpert had a mean cost of USD 17.37 with an ABC of USD 10.86, while smear microscopy had a mean cost of USD 13.31 with an ABC of USD 6.01. The sensitivity of smear microscopy was 42.9% and its specificity was 99.1%, while the Xpert assay had a sensitivity of 100% and a specificity of 96.7%. CONCLUSIONS: The Xpert assay has high accuracy; however, the turnaround time and cost of smear microscopy were lower than those of Xpert.

Caracterización del gen de fusión RUNX1-RUNX1T1 en pacientes cubanos con leucemia mieloide aguda, 2000-2016 / Characterization of RUNX1-RUNX1T1 fusion gene in Cuban patients with acute myeloid leukemia, 2000-2016

Introducción: el gen de fusión RUNX1-RUNX1T codifica para una proteína quimérica con múltiples efectos en la proliferación, diferenciación y viabilidad de las células leucémicas. Objetivo: describir el comportamiento del RUNX1-RUNX1T1 en pacientes cubanos con dicha enfermedad. Método: Para ello se estudió el gen de fusión RUNX1-RUNX1T1 en 251 pacientes con leucemia mieloide aguda, mediante la reacción en cadena de la polimerasa, en el Instituto de Hematología e Inmunología de La Habana, entre los años 2000 y 2016. Resultados: El 20,3 por ciento (51 pacientes) fue positivo para el gen de fusión RUNX1-RUNX1T1, con una edad comprendida entre los 11 meses y los 80 años, media de 26 años. En los pacientes pediátricos la frecuencia del transcrito fue casi el doble de la de los adultos (29,2 por ciento y 15,3 por ciento, respectivamente) (p= 0,009). Mayor cantidad de pacientes masculinos presentaron el gen quimérico. En menores de 25 años hubo una mayor frecuencia del transcrito (p=0,019) con predominio significativo de la mutación en los adolescentes (p=0,027). Cinco pacientes fueron positivos al RUNX1-RUNX1T1 y a la duplicación interna en tándem del gen FLT3 (12,2 por ciento). Ningún paciente positivo al RUNX1-RUNX1T1 presentó el gen de fusión CBFB-MYH11. La mayor asociación estuvo con la mutación A del gen NPM1 para un 25 por ciento. El debut de la enfermedad se caracterizó por anemia moderada (p= 0,024), trombocitopenia severa (p= 0,004) y gran infiltración medular. La mayor discrepancia entre diagnósticos se concentró entre las variantes morfológicas M2 y M3 (p= 0,000). Conclusiones: En pacientes cubanos la leucemia mieloide aguda con gen de fusión RUNX1-RUNX1T1 positivo, tiene un comportamiento similar a lo descrito internacionalmente con algunas particularidadesen las características hematológicas de presentación de la enfermedad. El estudio molecular es imprescindible para definir el diagnóstico, y la estrategia terapéutica en estos pacientes(AU)

Introduction: The RUNX1-RUNX1T fusion gene codes for a chimeric protein with multiple effects on the proliferation, differentiation and viability of leukemic cells. Objective: To describe the behavior of RUNX1-RUNX1T1 in Cuban patients with this disease. Method: The RUNX1-RUNX1T1 fusion gene was studied in 251 patients with acute myeloid leukemia, through the polymerase chain reaction, at the Institute of Hematology and Immunology of Havana, between 2000 and 2016. Results: The 20.3 percent (51 patients) were positive for the RUNX1-RUNX1T1 fusion gene, with an age between 11 months and 80 years, average of 26 years.In pediatric patients, the transcript frequency was almost twice that of adults (29.2 percent and 15.3 percent , respectively) (p= 0.009). More male patients presented the chimeric gene. There was a higher frequency of the transcript in children under 25 years of age (p= 0.019) with a significant predominance of the mutation in adolescents (p= 0.027).Five patients were positive for RUNX1-RUNX1T1 and for internal tandem duplication of the FLT3 gene (12.2 percent ).No patient positive for RUNX1-RUNX1T1 presented the CBFB-MYH11 fusion gene. The greatest association was with the A mutation of the NPM1 gene for 25 percent . The onset of the disease was characterized by moderate anemia (p= 0.024), severe thrombocytopenia (p= 0.004) and extensive bone marrow infiltration. The greatest discrepancy between diagnoses was concentrated between the morphological variants M2 and M3 (p= 0.000). Conclusions: In Cuban patients, acute myeloid leukemia with a positive RUNX1-RUNX1T1 fusion gene has a behavior similar to that described internationally with some peculiarities in the hematological characteristics of the disease presentation.The molecular study is essential to define the diagnosis, and the therapeutic strategy in these patients(AU)

Análisis de los polimorfismos p. K832R y p. T991T en pacientes cubanos con diagnóstico clínico de la enfermedad de Wilson / p. K832R and p. T991T polymorphisms’ analysis in Cuban patients with clinical diagnosis of Wilson's disease

Introducción: La Enfermedad de Wilson se caracteriza por la acumulación de cobre en hígado, cerebro, riñones y cornea. Se transmite con un patrón de herencia autosómico recesivo. La causa molecular que la provoca son las mutaciones en el gen ATP7B. Se han informado en la literatura más de 139 polimorfismos en el gen ATP7B. Objetivo: Identificar los cambios conformacionales en los exones 10 y 13 y detectar los polimorfismos p.K832R y p.T991T en el gen ATP7B en pacientes cubanos con diagnóstico clínico de Enfermedad de Wilson. Material y Métodos: Se realizó un estudio descriptivo, durante el período 2012 al 2013, que incluyó 27 pacientes con diagnóstico clínico de Enfermedad de Wilson. Para la amplificación del fragmento de interés, se utilizó la técnica de Reacción en Cadena de la Polimerasa y para identificar los cambios conformacionales se aplicó la técnica de Polimorfismo Conformacional de Simple Cadena, en el exón 10 y 13 del gen ATP7B. La presencia de los polimorfismos p.K832R y p.T991T fueron identificados por secuenciación. Resultados: Se detectaron tres cambios conformacionales diferentes denominados: (a, b y c) en el exón 10 y (a y b) en el exón 13 del gen ATP7B. La frecuencia alélica de los polimorfismos p. K832R y p.T991T en 27 pacientes cubanos con diagnóstico clínico de la Enfermedad de Wilson es 35,2 por ciento y 5,6 por ciento respectivamente. Conclusiones: Se analizó por primera vez en Cuba la combinación de los polimorfismos p. K832R y p. T991T que posibilitará hacer estudios moleculares por métodos indirectos(AU)

Introduction: Wilson's disease is characterized by accumulation of copper in the liver, brain and cornea. It is transmitted with an autosomal recessive inherited disorder. The molecular causes are mutations in the ATP7B gene. It has been reported in the literature more than 139polymorphisms of the ATP7B gene. Objective: Identify the conformational changes in exons 10 and 13 and detect the polymorphisms p.K832R and p.T991T in the ATP7B gene in Cuban patients with clinical diagnosis of Wilson's disease. Material and Methods: Was performed a descriptive study including 27 patients with Wilson’s disease ranging in the time from 2012 to 2013. Were applied the polymerase chain reaction to amplify the fragment of interest and the Conformation Polymorphism Single-Chain procedures in the exon 10 and 13 of the ATP7B gene. The p. K832R and p. T991T polymorphisms were detected by sequencing this fragment. Results: Three different conformational changes were identified: (a, b and c) in exon 10 and (a and b) in exon 13 of the ATP7B gene. The allelic frequency of polymorphisms p. K832R and p. T991T in 27 Cuban patients with clinical diagnosis of Wilson's disease is 35.2 percent and 5.6 percent, respectively. Conclusions: It is the first time in Cuba that a combination of the polymorphisms p. K832R and p. T991T were identified which will allow to make possible molecular studies by indirect methods(AU)

Utilidad de las herramientas moleculares para la identificación de Leptospira spp. en muestras humanas, animales y ambientales / Usefulness of molecular tools to identify Leptospira spp. in human, animal and environmental samples

Leptospirosis es una enfermedad zoonótica endémica de potencial epidémico que afecta la salud pública y la producción pecuaria alrededor del mundo. Su agente etiológico es una espiroqueta del género Leptospira, con 20 especies reportadas hasta el momento, son las más importantes Leptospira interrogans (patógena) y Leptospirabiflexa (saprófita). Esta bacteria se transmite mediante contacto directo o indirecto en especial con orinade animales infectados, es la transmisión por medio del agua una de las más importantes. En cuanto al diagnóstico se ha evidenciado que diversas pruebas moleculares tienen una alta especificidad y sensibilidad; sin embargo, el conocimiento de la epidemiología de la leptospirosis se ha basado principalmente en estudios serológicos que han utilizado la prueba de aglutinación microscópica que presenta debilidades en sus resultados e interpretación. El objetivo del presente artículo es presentar una revisión actualizada sobre la utilidad de las herramientas moleculares para la identificación de Leptospira spp. en muestras humanas, animales y ambientales. Se llevó a cabo una búsqueda de literaturaen diferentes bases de datos como Pubmed, Science Direct, SciELO, Scopus y Redalyc.Las publicaciones encontradas fueron artículos originales y de revisión, entre otros, publicados entre 1965 y 2014. Se determinó que las herramientas moleculares permiten una identificación directa, rápida, definitivay precisa del agente etiológico, apoyan el diagnóstico, aportan al conocimiento real de laprevalencia e incidencia de la enfermedad. Las herramientas moleculares permiten la identificación de nuevas especies a partir de aislamientos obtenidos de diversas fuentes y ayudan a orientar los programas de prevención y control de esta zoonosis(AU)

Leptospirosis is an endemic and potentially epidemic zoonosis affecting public health and livestock production worldwide. Its etiological agent is a spirochaete of the genus Leptospira, with 20 species reported to date, of which Leptospira interrogans (pathogenic) and Leptospira biflexa (saprophyte) are the most important. This bacterium is transmitted by direct or indirect contact with urine from infected animals, so water is one of the major transmission ways. Regarding diagnosis, many molecular tests have been evinced to have high specificity and sensitivity; however, knowledge on the epidemiology of leptospirosis has been based mainly on serological studies using the microscopic agglutination test, which has weaknesses in its results and interpretation. The aim of this article is to present an update review on the usefulness of molecular tools in the identification of Leptospira spp. in human, animal and environmental samples. A literature search was conducted in different databases such as PubMed, ScienceDirect, SciELO, Scopus and Redalyc. The publications found were original and review articles, among others, published between 1965 and 2014. It was found that the molecular tools allow direct, quick, definitive and precise identification of the etiologic agent, support the diagnosis, and contribute to real knowledge on the disease prevalence and incidence. Molecular tools enable the identification of new species isolates obtained from various sources and help guide prevention programs and control of this zoonosis(AU)

Evaluación de diferentes muestras para la detección molecular de Mycobacterium leprae en Cuba / Evaluation of different samples for Mycobacterium leprae molecular detection in Cuba

Introducción: en la actualidad, la principal estrategia para controlar la infección por Mycobacterium leprae es la detección precoz y la multiterapia. El objetivo del presente estudio fue evaluar la utilidad del empleo de diferentes muestras clínicas en el diagnóstico molecular de la infección por M. leprae. Métodos: se evaluaron 22 pacientes con clínica sugestiva de lepra. Se tomaron muestras de linfa de cuatro puntos (ambos lóbulos auriculares y ambos codos) recogidas en una lámina porta objetos (lámina de baciloscopía) y en un hisopo, además muestra de hisopado nasal, muestra de sangre total y de tejido de la lesión. Las muestras se analizaron mediante baciloscopía, histología y PCR según correspondió en cada caso. Por otro lado, para determinar la sensibilidad y especificidad del método, se realizó un estudio de casos y controles en el que se emplearon 40 láminas de baciloscopías negativas de pacientes con lepra paucibacilar y 40 láminas negativas de personas sin lepra. Resultados: el 54,5 por ciento de los pacientes resultó positivo por baciloscopía. Solo en el 41 por ciento de los pacientes la histología tuvo resultados concluyentes de lepra. En el 100 por ciento de los pacientes se detectó la presencia de ADN de M. leprae a partir de la lámina de baciloscopía y el hisopado de linfa. En el 95,45 por ciento de los pacientes se pudo amplificar la secuencia diana a partir de la sangre total y solo en el 31,8 por ciento de los pacientes el hisopado nasal resultó positivo. El estudio de casos y controles mostró que la sensibilidad y especificidad de la PCR respecto al diagnóstico convencional fue de 100 por ciento. Conclusión: el diagnóstico de M. leprae mediante PCR, es de gran utilidad cuando las técnicas convencionales no son concluyentes. La lámina de baciloscopía y el hisopado de linfa constituyen las muestras clínicas más útiles para la confirmación molecular de la infección por M. leprae(AU)

Introduction: Currently, the early detection and treatment with multitherapy are the main strategy to control the infection by Mycobacterium leprae. The objective of the present study was to evaluate the usefulness of different clinical samples for molecular diagnosis of M. leprae infection. Methods: Twenty two patients with suggestive clinical leprosy were analyzed. Different clinical samples were taken by slit skin smear, histopathology and PCR. To determine the sensitivity and specificity of the PCR method, a case-control study was also performed using 40 slides from negative smears of patients with leprosy paucibacillary and 40 from individuals without leprosy. Results: From all patient studied fifty-four percent were positive by slit skin smear and 41 percent were conclusive of leprosy by histopathology. M. leprae DNA was detected in slit skin smears and lymph swabs in 100 percent of patients. In 95,45 percent of patients were detected M. leprae DNA in whole blood and in 31,8 percent of them in nasal swab. The sensitivity of PCR respect to conventional diagnostic was 100 percent, the specificity was 97.5 percent, and the positive predictive value was 97.56 and the negative predictive value was100 percent. Conclusion: The diagnosis of M. leprae by PCR is very useful when conventional techniques are inconclusive. The slit skin smears and lymph swabs are the most useful clinical samples for molecular confirmation of infection with M. leprae(AU)

Marcadores moleculares en el cáncer de tiroides / Molecular markers for thyroid cancer

La importancia del estudio del nódulo tiroideo es excluir una lesión maligna, ya que, aunque la mayoría son lesiones benignas, existe un riesgo de malignidad de un 5-10 por ciento. La mayoría de estos son carcinomas bien diferenciados, que se originan del epitelio folicular. A pesar de que la mayoría de las lesiones son benignas, la distinción entre estas y los carcinomas, es crucial para un tratamiento y seguimiento apropiado. La biopsia por punción con aguja fina permite realizar el diagnóstico en la mayoría de los casos, sin embargo, esta presenta limitaciones, particularmente referidas al diagnóstico de las lesiones foliculares. En un esfuerzo por mejorar la precisión diagnóstica de la biopsia y ofrecer nuevos criterios para el diagnóstico, múltiples marcadores moleculares han sido propuestos, algunos de los cuales presentan gran aprobación, mientras que otros requieren aún validación para su implementación. En este artículo se realiza una revisión actualizada de los marcadores moleculares que presentan mayor número de evidencias, los que son metodológicamente más asequibles y potencialmente utilizables para el diagnóstico prequirúrgico del nódulo tiroideo(AU)

The importance of the study of the thyroid nodule lies in excluding the possibility of a malignant lesion because the majority of lesions are benign but there is a malignancy risk of 5 to 10 percent. Most of them are well differentiated carcinomas originating in the follicular epithelium. In spite of the fact that the majority are benign lesions, distinguishing them from carcinomas is crucial to treatment and adequate follow-up. Fine-needle biopsy allows making the diagnosis in most of cases. However, this method is restricted, particularly when diagnosing follicular lesions. In an effort to improve the diagnostic accuracy of biopsy and to provide new diagnosing criteria, a number of molecular markers have been put forward, some of which has wide range of approval whereas others still awaits to be validated for further implementation. This article presented an updated review of molecular markers with higher number of evidence, more accessible and potentially usable from a methodological viewpoint for diagnosis of the thyroid nodule before surgery(AU)

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Fast and robust protocol for prenatal diagnosis of mucopolysaccharidosis type II

Introduction: Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal disorder caused by deficiency of iduronate-2-sulfatase (IDS). In this study, we proposed a new protocol for prenatal diagnosis, using DNA obtained from amniotic fluid cells that did not attach to the bottom of the culture flask after the first medium change. Methods: Four pregnant MPS II carriers were referred to the Medical Genetics Service of Hospital de Clinicas de Porto Alegre for a prenatal diagnosis and identification of the disease, which were performed by polymerase chain reaction (PCR) amplification, restriction fragment length polymorphism, and sequencing according to the mutation previously found in the family. Results: The analysis indicated the absence of mutation in three fetal materials and the presence of mutation in one case. Concomitantly, cytogenetic and biochemical analyses were performed after 12 days of cell culture, and only one case showed absence of enzyme activity, confirming the molecular analysis. Conclusions: This diagnostic protocol designed to provide more robust results and safer genetic counseling suggests that DNA obtained from floating amniotic fluid cells can be used as a source of fetal material to allow a faster alternative for prenatal care through molecular analysis. Determination of IDS gene mutation in fetal amniotic fluid cells together with IDS enzyme activity testing is a rapid, sensitive and accurate method for prenatal diagnosis of MPS II for high-risk pregnant women.

A novel tissue microarray instrumentation:The HT-1 tissue microarrayer.

Background: Tissue microarray (TMA) is a novel and useful tool to efficiently analyze gene expression in histological tissues. Aim: Cost-efficient and easy to use automated tissue arrayers will provide a better instrumentation to generate TMAs. Thus, we designed and produced our tissue microarrayer to meet these needs. Materials and Methods: The HT-1 tissue microarrayer we designed and manufactured consists primarily of four parts, including an instrument to make array pores for the recipient paraffin blocks, a punch needle, an instrument for negative-pressure embedding, and a special manipulator. By using the HT-1, 14 different TMAs were made to accommodate 312 cases of tissues and TMA sections were tested by hematoxylin-eosin (H&E) staining, in situ hybridization, and immunohistochemistry. Results: Expand: Hematoxylin and eosin staining showed that the tissue cylinders were similar, even, and in order on the slides. Most importantly, the HT-1 microarrayer can make array pores in the recipient paraffin block with a single application in seconds. The HT-1 also contains a unique negative pressure system for embedding TMA blocks. In addition, HT-1 can make tissue cylinders with the same levels and depth for equally embedded and sectioning. Conclusions: The HT-1 tissue microarrayer is a device that is simple, economical and easy to use.

Correlation of p53 and KI-67 expression with grade and subtype of ependymoma.

Introduction: The morphological criteria for grading ependymomas were always felt subjective. Recently some studies have showed that Ki-67 and p53 immunolabeling are important prognostic markers in ependymomas. Materials and Methods: All the cases of ependymomas diagnosed from 2005 to 2010 were graded according to WHO classification for central nervous system (CNS) tumors 2007. Two tissue microarray (TMA) blocks were prepared. Immunohistochemical analysis with glial fibrillary acidic protein (GFAP), epithelial membrane antigen (EMA), Ki-67 and p53 was performed. The difference in expression of p53 and Ki-67 in various tumor grades and subtypes was evaluated using Student's t test. Results: There were 54 cases with a M: F ratio of 1.34 : 1, age ranging from 7 years to 65 years (mean 29.35 years). There were 33 intracranial and 21 spinal cases. There were 9 grade I ependymomas, 32 grade II ependymomas and 13 grade III ependymomas. GFAP immunopositivity was seen in all the cases and EMA was positive in 49% cases. The mean p53 indices were higher in grade III and grade II tumors (26.26% and 26.08%) as compared to subependymomas (7.25%). But these values did not show statistical significance (P = 0.2). The Ki-67 labeling index increased from grade I to grade III tumors. The difference was highly significant between grade II and grade III (0.5% vs. 2.75, P = 0.016). Conclusion: Ki-67 labeling index correlates with grade of ependymoma (P = 0.016). There is no correlation between p53 expression and grade of ependymomas.

Discriminating thyroid cancers from benign lesions based on differential expression of a limited set of miRNA using paraffin embedded tissues.

Background : Micro-RNAs (miRNAs) are expressed in a tissue-specific manner and are known to demonstrate differential expression even among the various subtypes of a given tumor. This differential expression has been harnessed successfully in the development of diagnostic assays for various malignant tumors. These assays have been found to be relevant and of value as additional diagnostic tools even among thyroid tumors, particularly with regard to thyroid carcinomas of follicular morphology. Materials and Methods : A limited set of miRNA have been assessed as part of this study in an effort to use minimal number of miRNA markers (miR-187, miR-221, miR-222, and miR-224) to differentiate the benign from the malignant thyroid tumors using miRNA derived from paraffin embedded material. Results : While miR-221 and miR-222 were found to provide good accuracy as individual markers (86% and 84%), a combination of the two provided slightly better accuracy (91%). Both miR-221 and 222 were able to significantly differentiate malignant tumors from the benign samples (P< 0.001) individually and as a combination of markers. However, inclusion of miR-187 and miR-224 in the panel did not provide any additional benefit. Conclusion : While a combination of miR-221 and 222 when used in a diagnostic panel could provide fairly good accuracy additional markers may need to be investigated to augment their diagnostic utility.

Comparative profiling of plasma proteome from breast cancer patients reveals thrombospondin-1 and BRWD3 as serological biomarkers

Breast cancer is the most common cancer in women worldwide. It is necessary to identify biomarkers for early detection, to make accurate prognoses, and to monitor for any recurrence of the cancer. In order to identify potential breast cancer biomarkers, we analyzed the plasma samples of women diagnosed with breast cancer and age-matched normal healthy women by mTRAQ-based stable isotope-labeling mass spectrometry. We identified and quantified 204 proteins including thrombospondin-1 (THBS1) and bromodomain and WD repeat-containing protein 3 (BRWD3) which were increased by more than 5-fold in breast cancer plasma. The plasma levels of the two proteins were evaluated by Western blot assay to confirm for their diagnostic value as serum markers. A 1.8-fold increase in BRWD3 was observed while comparing the plasma levels of breast cancer patients (n = 54) with age-matched normal healthy controls (n = 30), and the area under the receiver operating characteristic curve (AUC) was 0.917. THBS1 was detected in pooled breast cancer plasma at the ratio similar to mTRAQ ratio (> 5-fold). The AUC value for THBS1 was 0.875. The increase of THBS1 was more prominent in estrogen receptor negative and progesterone receptor negative patients than receptor-positive patients. Our results are evidence of the diagnostic value of THBS1 in detecting breast cancer. Based on our findings, we suggest a proteomic method for protein identification and quantification lead to effective biomarker discovery.

Assessment of HER2/Neu status by fluorescence in situ hybridization in immunohistochemistry-equivocal cases of invasive ductal carcinoma and aberrant signal patterns: A study at a tertiary cancer center.

Introduction: HER-2/neu status determines the eligibility for targeted therapy with trastuzumab in breast carcinoma. Evaluation for HER-2/neu protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in situ hybridization (FISH) has become the gold standard. Aims: Since data on HER-2/neu assessment by IHC and FISH and studies regarding concordance between the results of the two techniques are limited, especially from India, we sought to study HER-2 gene amplification status by FISH in equivocal (2+) cases by IHC and also study aberrant signal patterns. Settings and Design: Mastectomies and breast core biopsies, equivocal for HER-2/neu protein expression, were analyzed for HER-2 amplification by FISH. Materials and Methods: IHC (DAKO) and FISH (PathVysion dual-probe system) tests were performed on 68 of 112 (after exclusion) 10% neutral buffered formalin (NBF)-fixed paraffin-embedded tissues and evaluated according to American Society of Clinical Oncology ASCO guidelines. Statistical Analysis Used: Chi-square (χ2 ) test and the two-tailed P value were applied using Graphpad Quickcels software, version 2006. Results: It was found that 73.5% of the IHC 2+ patients were negative for HER-2/neu amplification, 25% were positive (ratios ranging from 2.3 to 5.6) and 1 patient was equivocal (2.2). Retesting FISH HER-2 equivocal case on another tumor block by IHC demonstrated HER-2 overexpression of protein 3+, thus resolving the equivocal status. Polysomy and HER-2 genetic heterogeneity were seen frequently. Conclusions: The findings reiterate that IHC HER-2 equivocal cases are a heterogenous group and need FISH for further categorization. Low concurrence (25%) rate between both IHC and FISH results in the equivocal scenario can be attributed to tumors with polysomy 17 and HER-2/neu genetic heterogeneity.

Clonality assessment of lymphoproliferative lesions using the polymerase chain reaction: An analysis of two methods.

Background: Lymphoid malignancies are a heterogeneous group of disorders which may be difficult to differentiate from reactive proliferations even after immunohistochemistry. Polymerase chain reaction (PCR) is believed to be a good adjunct tool for diagnosis. Materials and Methods: We examined 24 cases of neoplastic and non-neoplastic lymphoproliferative lesions in this study and evaluated the PCR as an additional tool in the confirmation of the diagnosis. Two different PCR methodologies were evaluated. Results: In the evaluation of the T-cell PCR, it was seen that the correlation using both the commercial kits and the custom-synthesized primers was highly significant at a P value of <0.05. In the evaluation of the B-cell PCR, it was seen that the correlation using both the commercial kits and the custom-synthesized primers was not significant using either method (P > 0.05). Conclusions: Both the methods showed an excellent concordance for T-cell γ gene rearrangements, However, the same was not seen in the B-cell receptor rearrangements. This may be because of the small sample size or the inability of consensus V primers to recognize complementary DNA sequences in all of the V segments.