Fluorescence in situ hybridization (FISH) as an irreplaceable diagnostic tool for Williams-Beuren syndrome in developing countries: a literature review / Hibridização in situ fluorescente (FISH) como ferramenta diagnóstica insubstituível para a síndrome de Williams-Beuren em países em desenvolvimento: uma revisão de literatura

ABSTRACT Objective: The aim of this study was to sum up and characterize all Williams-Beuren syndrome cases diagnosed by fluorescence in situ hybridization (FISH) since its implementation, as well as to discuss FISH as a cost-effective methodology in developing countries. Data source: From January 1986 to January 2022, articles were selected using the databases in PubMed (Medline) and SciELO. The following terms were used: Williams syndrome and In Situ Hybridization, Fluorescence. Inclusion criteria included Williams-Beuren syndrome cases diagnosed by FISH with a stratified phenotype of each patient. Only studies written in English, Spanish, and Portuguese were included. Studies with overlapping syndromes or genetic conditions were excluded. Data synthesis After screening, 64 articles were included. A total of 205 individuals with Williams-Beuren syndrome diagnosed by FISH were included and further analyzed. Cardiovascular malformations were the most frequent finding (85.4%). Supravalvular aortic stenosis (62.4%) and pulmonary stenosis (30.7%) were the main cardiac alterations described. Conclusions: Our literature review reinforces that cardiac features may be the key to early diagnosis in Williams-Beuren syndrome patients. In addition, FISH may be the best diagnostic tool for developing nations that have limited access to new technologic resources.

RESUMO Objetivo: Caracterizar todos os casos de síndrome de Williams-Beuren (SWB) diagnosticados por hibridização in situ fluorescente (FISH) desde sua implementação, assim como discutir a relação custo-benefício da metodologia de FISH em países em desenvolvimento. Fontes de dados: Entre janeiro de 1986 e janeiro de 2022 foi realizada uma busca nas bases de dados PubMed (Medical Literature Analysis and Retrieval System Online — Medline) e Scientific Electronic Library Online (SciELO) usando os seguintes termos: síndrome de Williams e hibridização in situ fluorescente. O critério de inclusão utilizado foi conter a descrição detalhada de caso(s) de SWB por FISH. Apenas estudos escritos em inglês, espanhol e português foram incluídos. Trabalhos que apresentavam sobreposição de síndromes/condições genéticas foram excluídos. Síntese dos dados: Após os processos de inclusão, 64 artigos e 205 indivíduos com SWB diagnosticados por meio do método de FISH foram incluídos. O achado mais frequente entre os indivíduos foi a presença de algum tipo de malformação cardíaca (85,4%). A estenose aórtica supravalvar (62,4%) e a estenose pulmonar (30,7%) foram as alterações cardíacas mais descritas. A maioria dos estudos era proveniente dos continentes Europa, Ásia e América do Norte. Conclusões: A presente revisão de literatura reitera que as malformações cardíacas podem ser a chave para o diagnóstico precoce em pacientes com SWB. Ainda, a técnica de FISH parece ser a melhor ferramenta de diagnóstico para os países em desenvolvimento, cujo acesso às novas tecnologias ainda é escasso.

Correlation of clinicopathologic features of Filipino primary breast cancer patients with HER2 Subgroups classified according to the ASCO/CAP 2018 breast cancer HER2 testing guidelines

Background@#Guidelines for testing human epidermal growth factor receptor 2 (HER2) in breast cancer using fluorescence in situ hybridization (FISH) were released in 2018. These guidelines were jointly developed by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) to achieve a clearer designation of breast cancer HER2 status. Clinical correlation with other factors was also considered appropriate, especially for those cases classified under ISH groups 2, 3, and 4.@*Objective@#In this study, we examined clinicopathologic features among Filipino breast cancer patients whose HER2 status was reclassified based on the 2018 ASCO/CAP guidelines.@*Methodology@#One hundred and thirty-two (132) breast cancer cases with immunohistochemistry (IHC) equivocal results in the Medical City were enrolled from January 2017 up to December 2020. HER2 FISH results classified under groups 2, 3, and 4 were then re-evaluated for HER2-IHC status in accordance with the 2018 ASCO/CAP guidelines. The relationship between clinicopathologic features and HER2 status was analyzed using the Fisher exact test.@*Results@#Significant differences were found in histologic type, nuclear pleomorphism, mitotic rate, progesterone receptor (PR) status, and regional lymph node involvement among the reclassified ISH groups. In the conv+ group, the tumor cells did not involve the regional lymph nodes as compared to group 5, where the tumor cells were involved. The conv- group had a higher grade for nuclear pleomorphism, mitotic count, and overall Nottingham Histologic Grade than group 5. There was a significant association between progesterone receptors among the conv- group and group 1.@*Conclusion@#Filipino breast cancer cases whose HER2 status was reclassified to negative following the 2018 ASCO/CAP guidelines had statistically different clinicopathologic features from those classified as group 5.

Clinical value of different genetic testing methods for detection of true fetal chromosome mosaicism / 中华围产医学杂志

Objective:To investigate the performance of chromosome karyotype, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) in prenatal diagnosis of true fetal chromosome mosaicism. Methods:This retrospective study enrolled 40 women with true fetal chromosome mosaicism from 4 071 singleton pregnant women who were indicated for and underwent amniocentesis or/and cordocentesis in the the First Affiliated Hospital of Sun Yat-sen University from April 2018 to August 2021. The results of chromosome karyotyping, CMA and FISH, the types of chromosomal mosaicism, mosaicism ratio and pregnancy outcomes were analyzed using Chi-square test. Results:(1) The detection rate of true fetal mosaicism was 0.98% (40/4 071). (2) Sex chromosome mosaicism accounted for 42.5% (17/40). Other chromosomal mosaicism involved chromosomes 21, 22, 18, 16, 7, 12, 15, 17 and 20, as well as balanced chromosomal translocation. (3) The detection rate of true fetal mosaicism by chromosome karyotyping was 77.4% (24/31) from amniotic fluid samples and 10/19 from umbilical cord blood samples, while that data by CMA was 76.7% (23/30) and 7/11,respectively. (4) Of the 40 pregnant women with fetal chromosome mosaicism, FISH test was performed on 20 cases (14 cases were verified with both amniotic fluid and umbilical cord blood samples, five with amniotic fluid samples and one with umbilical cord blood sample), and all of the diagnosis of mosaicism were confirmed. For those with mosaicism ratio <30%, the detection rate by FISH was higher than that by CMA among amniotic fluid samples [14/19 vs 43.5% (10/23), χ2=3.88, P=0.049]. (5) Among the 40 pregnant women, five were lost to follow-up; 18 chose to terminate the pregnancy; and 17 continued the pregnancy to delivery. No abnormalities in mental or physical development were reported in the 17 neonates after birth or during on-line follow-up between 6 to 24 months old. Of the 14 pregnant women with mosaicism ratio <30% which confirmed by FISH, eight chose to continue the pregnancy, and no abnormalities in mental development or growth were found in the neonates. Conclusions:In prenatal diagnosis of true fetal choromosome mosaicism, the incidence of sex chromosome mosaicism is the highest. FISH may improve the prenatal diagnosis rate of mosaicism and is more accurate in determining the mosaicism ratio. The combination of FISH, CMA and chromosome karyotyping would significantly improve the detection rate of chromosomal mosaicism and assess the mosaicism ratio more accurately, which is of great value in clinical consultation and evaluation of fetal prognosis.

Potential unreliability of ALK variant allele frequency in the efficacy prediction of targeted therapy in NSCLC / 医学前沿

Anaplastic lymphoma kinase (ALK) is the most common fusion gene involved in non-small cell lung cancer (NSCLC), and remarkable response has been achieved with the use of ALK tyrosine kinase inhibitors (ALK-TKIs). However, the clinical efficacy is highly variable. Pre-existing intratumoral heterogeneity (ITH) has been proven to contribute to the poor treatment response and the resistance to targeted therapies. In this work, we investigated whether the variant allele frequencies (VAFs) of ALK fusions can help assess ITH and predict targeted therapy efficacy. Through the application of next-generation sequencing (NGS), 7.2% (326/4548) of patients were detected to be ALK positive. On the basis of the adjusted VAF (adjVAF, VAF normalization for tumor purity) of four different threshold values (adjVAF < 50%, 40%, 30%, or 20%), the association of ALK subclonality with crizotinib efficacy was assessed. Nonetheless, no statistical association was observed between median progression-free survival (PFS) and ALK subclonality assessed by adjVAF, and a poor correlation of adjVAF with PFS was found among the 85 patients who received first-line crizotinib. Results suggest that the ALK VAF determined by hybrid capture-based NGS is probably unreliable for ITH assessment and targeted therapy efficacy prediction in NSCLC.

FISH patterns of ROS1, MET, and ALK with a correlation of ALK immunohistochemistry in lung cancer: a case for introducing ALK immunohistochemistry ‘Equivocal’ interpretation category in the Ventana anti?ALK (D5F3) CDx assay ? A tertiary cancer center experience

Background: Mutations in ROS1, ALK, and MET genes are targetable alterations in non-small cell lung cancer (NSCLC). They can be evaluated by different techniques, most commonly fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Methods: We explored the prevalence of ROS1, ALK, MET mutations, discuss clinicopathological associations and FISH signal patterns on 413 consecutive cases of EGFR negative lung carcinoma from March 2016 to April 2017 using FISH for ALK, ROS1, and MET along with ALK (D5F3) IHC. Results: ROS1 gene rearrangement, ALK positivity (IHC and/or FISH), and MET amplification were seen in 18/358 (5%) cases, 76/392 cases (19.4%), and 10/370 (2.7%) cases, respectively. ALK FISH and ALK IHC were positive in 51/300 (17%) and 58/330 cases (17.57%), respectively, while 8/330 (2.4%) cases were ALK IHC “equivocal” of which 3/8 (37.5%) were ALK FISH positive. Of ALK FISH and IHC co-tested cases, 43/238 (18.07%) cases were positive by both techniques, while 15/43 (34.88%) of ALK positive cases showed discordant ALK FISH and IHC results. All ROS1 rearranged and MET amplified cases were adenocarcinoma. Signet ring cell histology was associated with 78.57% likelihood of being either ALK or ROS1 positive. Genomic heterogeneity was seen in 30% of MET amplified cases. Conclusions: ALK/ROS1/MET gene alterations were found in 25.18% of NSCLC cases. An ALK IHC “equivocal” interpretation category should be incorporated into practice. Atypical patterns of ROS1 and genomic heterogeneity need to be evaluated further for any clinical relevance.

Pattern of Cytogenetic Abnormality by Fluorescence in Situ Hybridization (FISH) in Multiple Myeloma Patients in a Tertiary Hospital

Background: Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance, with survival duration ranging from a few months to more than 10 years. Cytogenetic abnormalities (CA) detected by fluorescence in situ hybridization (FISH) are of major prognostic significance since e.g. patients with del(17p), t(4;14) or gain 1q21 show dismal outcome. Objective: To evaluate the cytogenetic patterns by fluorescence in situ hybridization (FISH) of clinically diagnosed cases of multiple myeloma.Methods:This cross-sectional study was conducted in Department of Haematology, Dhaka Medical College Hospital, Dhaka, from January 2018 to December 2018. A total number of 30 patients with multiple myeloma were analyzed cytogenetically by interphase fluorescence in situ hybridization (iFISH). The collected data were analyzed by using the Statistical Package for Social Science (SPSS-24) for windows version 10.0.Results:Out of 30 diagnosed Multiple Myeloma cases the mean age was 56.37�.38 years and male to female ratio was almost 3:1. Sixteen (56.7%) of 30 patients. Among 30 cases of 8 cases were thyrogenicity positive of 7(23.3%) patients was detected del 13q positive. Isolated del 13q was found in 4 cases. 2 cases were found coexistence of del 13q and del 17p positive ;1 case was found coexistence of del 13q and t(4;14) positive and rest of 1 case had del 17 p positive. There was no detectable t (11; 14) and t(14;16) in any of 30 cases.Conclusion:FISH panel for Multiple Myeloma including del (13q); t(11;14); t(4;14), del(17p), t(14;16) is very important molecular test for the prognosis , risk stratification, treatment modality of the patient. On the basis of cytogenetic abnormality Multiple Myeloma risk stratification is modified now a day. This Revised International Staging system R-ISS is a simple and powerful prognostic staging system.

Influence of the number of high-risk cytogenetic abnormalities on the clinical characteristics and prognosis in 360 patients with newly diagnosed multiple myeloma / 中华血液学杂志

Objective: To investigate the influence of the number of high-risk cytogenetic abnormalities (HRCA) on the clinical characteristics and prognosis of patients with newly diagnosed multiple myeloma (MM) . Methods: A total of 360 patients with newly diagnosed MM admitted to Jiangsu Province Hospital between November 2013 and September 2020 were included in this study. Cytoplasmic light chain immunofluorescence with fluorescence in situ hybridization (cIg-FISH) was used to detect HRCA. Cytogenetic abnormalities were combined with clinical characteristics and outcomes for further analysis. Results: Among the 360 patients, 120 patients (33.3%) presented with no HRCAs, and 175 (48.6%) , 61 (16.9%) , and four (1.1%) patients had one, two, and three HRCA (s) , respectively. Patients were divided into three groups, including the no-HRCA group, one-HRCA group, and ≥two-HRCA group, according to the number of HRCAs. There were significant differences in the R-ISS stage, hemoglobin level, albumin level, and the proportion of bone marrow plasma cells among the three groups (P<0.05) . The COX proportional-hazards model identified extramedullary disease (P=0.018) , HRCA ≥ 2 (P=0.001) , and absence of autologous hematopoietic stem cell transplantation (P<0.001) as independent risk factors for progression free survival (PFS) and identified lactate dehydrogenase (LDH) level ≥ 220 U/L (P<0.001) , HRCA ≥2 (P=0.001) , and absence of autologous hematopoietic stem cell transplantation (P=0.005) as independent risk factors for overall survival (OS) . The median PFS was 28 months, 22 months, and 14 months (P=0.005) for the three cohorts, and their OS was not reached,60 months, and 30 months (P=0.001) , respectively. Conclusions: HRCA ≥ 2 is an independent risk factor for decreased survival in patients with newly diagnosed MM. More HRCAs result in heavier tumor burden, as well as a higher risk of disease progression and death.

Granulocytic sarcoma of the breast: A case report in Lao PDR / Lao Medical Journal

@#Granulocytic sarcoma (GS) also known as myeloid sarcoma, chloroma, myeloblastoma, or extramedullary myeloid tumor is a neoplasm composed of immature myeloid cells. The common sites of involvement include bone, central nervous system, soft tissue, lymph nodes, and skin. The involvement of GS in breast tissue is very rare. The incidence of breast GS is 2/1,000,000 in adults. Those affected range in age from 16 to 72 years, with the mean age of 31 years. Primary, isolated, or non-leukemic GS of the breast is defined when bone marrow biopsy confirms the absence of other hematologic malignancy. We here report a case of granulocytic sarcoma of the left breast in a 33 year-old woman who presented with a breast mass. She was initially diagnosed as having diffuse lymphoma, large cell type on Hematoxylin and eosin stain (H&E) histopathology. The tumor cells were, however, strongly positive for myeloperoxidase (MPO), CD117, CD34, and CD43 but negative for CD45, CD20, CD3, or cytokeratin. Although further clinical information, such as complete blood count or aspiration biopsy of bone marrow tissue, was absent, we finally diagnosed this case as GS by additional immunohistochemical study. What happened to the patient?

Clinicopathological analysis of children with Xp11.2 translocation/ TFE3 gene fusion associated renal cell carcinoma / 中华实用儿科临床杂志

Objective:To summary the problems that may be encountered in the diagnosis of Xp11.2 translocation/TFE3 gene fusion associated renal cell carcinomas (Xp11 RCC) and to improve the understanding and diagnostic level.Methods:The clinical and pathological data of 5 children with Xp11 RCC pathologically diagnosed in Children′s Hospital of Capital Institute of Pediatrics from January 2015 to December 2019 were collected for retrospective analysis.Results:The 5 cases included 2 males and 3 females with the age of 4-8 years old.All cases presented with abdominal mass.Four cases received radical nephrectomy and radical tumor resection, and 1 case received simple tumor resection after related examination.Routine HE staining, immunohistochemical staining and fluorescence in situ hybridi-zation (FISH) were performed after surgery.The histological morphology of tumor was varied, and the tumor cells were arranged in nest flake, acinar or papillary pattern, with abundant cytoplasm form completely transparent to eosinophilic staining (pink), and gravel-like calcification was visible.Micropapillary arranged tumor cells appeared in 1 case besides classic pattern; in another case, the tumor cells were highly eosinophilic with abundant cytoplasm and visible round or elliptic eosinophilic bodies.The tumor cells in 5 cases showed diffuse and strong expression of TFE3, and FISH assay showed abnormal separation signal.Conclusions:Xp11 RCC is a relatively rare renal malignant tumor with diverse histological morphology, which should be distinguished from other common renal tumors in children.Its immunohistochemical expression and molecular detection are of specificity, and it should be diagnosed based on clinical incidence.

Citogenética de las hemopatías malignas en la era de la secuenciación / Cytogenetics of malignant hemopathies in the sequencing era

Las neoplasias hematológicas se caracterizan por un gran número y complejidad de alteraciones genéticas, desde la formación de genes de fusión a partir de translocaciones e inversiones cromosómicas hasta mutaciones génicas y alteraciones epigenéticas que han permitido la identificación de nuevos oncogenes y genes supresores de tumores responsables de su etiología. Al abordar el estudio genético de las leucemias se utilizan múltiples técnicas como la citogenética convencional, citogenética molecular (hibridaciónin situ por fluorescencia (FISH), esta última con una mayor sensibilidad, especificidad y rapidez que permiten el diagnóstico, la estratificación pronóstica y seguimiento de la enfermedad. Las técnicas anteriores se integran con técnicas de biología molecular, secuenciación génica, entre otras, que permiten el hallazgo de nuevos marcadores genéticos con una mejor caracterización de las hemopatías malignas y la posibilidad del desarrollo de nuevos fármacos específicos que actúen sobre la diana molecular. El objetivo fue revisar la utilidad de la citogenética y la secuenciación génica en el estudio de la leucemia mieloide aguda y la leucemia linfocítica crónica. Ante las ventajas, desventajas y limitaciones de estas técnicas genéticas es necesario utilizarlas de forma complementaria y nunca excluyente(AU)

Hematological neoplasms are characterized by a large number and great complexity of genetic disorders, from the formation of fusion genes after chromosomal translocations and inversions to gene mutation and epigenetic disorders that have permitted the identification of new oncogenes and tumor-suppressing genes responsible for their etiology. When addressing the genetic study of leukemias, multiple techniques are used, such as conventional cytogenetics, molecular cytogenetics, and fluorescence in situ hybridization (FISH), the latter having the higher degree of sensitivity, specificity and speed, which allow diagnosis, prognostic stratification and follow-up of the disease. The previous techniques are integrated with molecular biology techniques, gene sequencing, among others, which allow discovery of new genetic markers with better characterization of malignant hemopathies and the possibility of developing new specific drugs against the molecular target. The objective was to review the usefulness of cytogenetics and gene sequencing in the study of acute myeloid leukemia and chronic lymphocytic leukemia. Given the advantages, disadvantages and limitations of these genetic techniques, it is necessary to use them in as complementary but never exclusive management ways(AU)

A de novo marker chromosome 15 in a child with isolated developmental delay

We report a rare case of a 14-month-old male child who was referred for developmental delay. Clinical examination revealed a hypotonic infant with speech delay and no dysmorphic features. The banding cytogenetics revealed a small supernumerary marker chromosome. Upon silver staining, the marker showed the presence of satellite regions on either ends. Further, analysis using fluorescence in situ hybridization on marker chromosome revealed its origin from chromosome 15.

Características del diagnóstico prenatal por hibridación fluorescente in situ en Cuba / Features of the prenatal diagnosis by fluorescence in situ hybridization in Cuba

Introducción: El diagnóstico prenatal mediante la hibridación fluorescente in situ disminuye el tiempo de diagnóstico al no ser necesario el cultivo celular. Objetivo: Describir las características y experiencias del diagnóstico prenatal por hibridación fluorescente in situ en Cuba. Método: En amniocitos in situ se aplicaron sondas CEP y LSI para la detección de aneuploidías de los cromosomas 21,18,13, X y Y y sondas LSI para la detección de deleciones asociadas a síndromes de microdeleción. Resultados: Se remitieron al Centro Nacional de Genética Médica 629 casos de alto riesgo genético. Prevaleció la indicación de alteraciones fetales detectadas por ecografía. En 612 (97 por ciento) casos se obtuvo un diagnóstico satisfactorio, entre ellos, 50 (8,1 por ciento) casos positivos, con predominio del síndrome Down en 26 casos. Se corroboraron por citogenética convencional 312 casos con 98 por ciento de concordancia con los resultados obtenidos por hibridación fluorescente in situ. Se utilizó el líquido amniótico refrigerado para corroborar casos de diagnóstico dudoso obtenido por citogenética y se detectaron 3 fetos con mosaicos cromosómicos, el origen de un cromosoma marcador y la definición del sexo fetal en un caso. Conclusiones: Con la tecnología por hibridación fluorescente in situ el diagnóstico prenatal logra una segura opción de análisis en aquellos casos de embarazos de alto riesgo genético. Debido a limitaciones tecnológicas, la prueba por hibridación fluorescente in situ en células amnióticas en interfase, se ha adaptado a nuestras condiciones, para lograr siempre un diagnóstico seguro con el menor perjuicio posible a la embarazada, el feto y su familia(AU)

Introduction: Prenatal diagnosis by fluorescent in situ hybridization decreases the time of diagnosis not being necessary the cell culture. Objective: To describe the characteristics and experiences of prenatal diagnosis by fluorescent in situ hybridization in Cuba. Method: In in situ amniocytes CEP catheters were applied and LSI for the detection of aneuploidies of the 21,18,13, X and Y chromosomes, and LSI catheters for the detection of deletions associated with microdeletion syndromes. Results: 629 cases of high genetic risk were referred to the National Center of Medical Genetics. There was a prevalence of the indication of fetal abnormalities detected by ultrasound. In 612 (97 percent) cases the diagnosis was achieved in a satisfactory form, among them 50 (8.1 percent) positive cases, with predominance of Down syndrome in 26 cases. There were corroborated 312 cases by conventional cytogenetics with 98 percent of agreement with the results obtained by fluorescent in situ hybridization. It was used the cooled amniotic fluid to corroborate cases of uncertain diagnosis obtained by cytogenetics and there were detected 3 fetuses with chromosomal mosaics, the origin of a marker chromosome and the definition of fetal sex in one case. Conclusions: With the technology by fluorescent in situ hybridization, the prenatal diagnosis achieved a safe analysis option in cases of genetic high-risk pregnancies. Due to technological limitations, the test by fluorescent in situ hybridization in amniotic cells in interphase has adapted to the conditions in order to always achieve a safe diagnosis with the less possible damage to the pregnant women, the fetus and its family(AU)

Cytogenetic and molecular aberrations in childhood B lineage acute lymphoblastic leukaemia

Background: Acute Lymphoblastic Leukemia (ALL) is a common hematological malignancy in children and is characterized by genetic changes such as mutations and chromosomal translocations. These cytogenetic and molecular abnormalities have got diagnostic and prognostic significance. Identification of these abnormalities helps in risk categorization and appropriate therapy. Aim of the study was to assess the cytogenetic/molecular abnormalities associated with B Lineage ALL in children.Methods: It was a hospital based retrospective observational study of 79 children diagnosed with B Lineage ALL by Bone marrow aspirate morphology and flow cytometry.' Bone marrow samples or Peripheral blood were sent for cytogenetic/molecular analysis by Fluorescent in situ Hybridization technique. Descriptive data analysis was done using SPSS software.Results: Out of 199 cases 163(82%) were B Lineage ALL. 79(48%) undergone molecular analysis. Out of 79 cases of B lineage ALL, Translocation t(9;22) BCR-ABL1' was positive in 2(2.5%) cases , Translocation t(12;21) TEL/AML1' was positive 9(11%) cases and MLL (KMT2A) Gene Rearrangements was seen in 6(7.6%) children. Out of 79 cases of B lineage ALL, 6(7.6%) were Infantile ALL (Males 1(17%); Females 5(83%)).' 4(67%) cases were positive for MLL (KMT2A) Gene Rearrangement, all of them were female children. Over all 17(22%) cases (Males 4(24%); Females 13(76%)) were positive for molecular abnormalities.Conclusions: Many children with ALL have got Cytogenetic and Molecular abnormalities. The highest percentage of cytogenetic and molecular genetic abnormalities was related to t(12;21)TEL/AML1 in B Lineage ALL children, if present confer favourable prognosis. MLL (KMT2A) Gene Rearrangement was the common molecular abnormality in Infantile B ALL, presence of it leads to high risk categorization and confer poor prognosis. The evaluation of cytogenetic and molecular genetic abnormalities in children is essential in estimating the prognosis in B Lineage ALL children, which will be a great contribution to offer appropriate therapeutic approaches.

Comparison between Fluorescence in-situ Hybridization (FISH), Reverse Transcriptase PCR (RT-PCR) and fragment analysis, for detection of t (X; 18) (p11; q11) translocation in synovial sarcomas

Background: Synovial sarcoma (SS) is an aggressive, but a relatively chemosensitive soft tissue sarcoma, characterized by a specific, t (X;18)(p11;q11) translocation, leading to formation of SS18–SSX chimeric transcript. This translocation can be detected by various techniques, such as fluorescence in-situ hybridization (FISH), reverse transcriptase PCR (RT-PCR) and fragment analysis. Objectives: To compare the results of detection of t (X;18)(p11;q11) translocation, across three different platforms, in order to determine the most optimal and sensitive technique. Methods: Formalin-fixed paraffin embedded (FFPE) tissue sections of 45 soft tissue sarcomas were analyzed, including 16 cases of SS confirmed by histopathology, immunohistochemistry and molecular technique (s)(Group 1); 13 cases, wherein SS was one of the differential diagnosis, preceding molecular testing (Group 2) and 16 cases of various other sarcomas (Group 3). Various immunohistochemical (IHC) markers studied, including INI1/SMARCB1. All cases were tested for t (X;18) translocation, by fragment Analysis, FISH and RT-PCR. Results: There were 23 cases of SS, including 16 of group 1 and 7 of group 2. By fragment analysis, t (X;18)(p11;q11) translocation was detected in 22/23 cases (95.6%). By FISH, SS18 gene rearrangement was detected in 18/22 cases (78.2%), whereas by RT-PCR, SS18-SSX transcripts were detected in 15/23 cases (65.2%). Immunohistochemically, a unique “weak to absent”/reduced INI1 immunostaining pattern was exclusively observed in 12/13 cases of SS (92.3%). Fragment analysis and FISH were relatively more sensitive techniques. Unique “weak to absent”INI1 immunoexpression significantly correlated with positive t (X;18) translocation results (P = 0.0001). Conclusion: The present study constitutes first such study from our subcontinent. Fragment analysis is a promising technique for detection of t (X;18)(p11;q11) translocation. FISH and INI1 immunostaining pattern were also relatively more sensitive, over RT-PCR.

Application of environment-friendly bio-tissue sample preparation kit in fluorescence in situ hybridization detection of HER2 protein 2-positive invasive breast cancer / 中国组织工程研究

BACKGROUND: HER2 status assessment is an Important biological Index for the treatment and prognosis of Invasive breast cancer. Pre-treatment of tissues such as Immobilization, dehydration, transparency and dewaxlng Is a necessary procedure for HER2 protein and gene detection after pathological paraffin sections, and also an important factor affecting Immunohlstochemlstry and fluorescence in situ hybridization. OBJECTIVE: To explore the application value of environment-friendly biological tissue sample preparation kit In fluorescence in situ hybridization detection of HER2 protein 2-positive Invasive breast cancer. METHODS: 402 Invasive breast cancer specimens were collected from Shantou Central Hospital from January 2015 to March 2019. The same specimens were semi-dissected and randomly divided Into two groups. The control group was treated by traditional reagent formaldehyde immobilization-ethanol dehydratlon-xylene transparency and dewaxlng, and paraffin sections were made. The experimental group was treated with formaldehyde immobilization-ethanol dehydratlon-xylene transparent dewaxlng. Environment-friendly biological tissue sample preparation kit (Including environmentally friendly stationary fluid, dehydration fluid, transparent liquid, dewaxing fluid) was used to make slices. The expression of HER2 protein was detected by Immunohlstochemlstry. The amplification of HER2 gene was detected by fluorescence in situ hybridization In 131 Invasive breast cancer specimens with positive HER2 protein expression. RESULTS AND CONCLUSION: The expression of HER2 protein In both experimental and control groups was specific and cell localization was correct. There were no significant differences In HER2 protein positive rate, uncertainty rate, and negative rate between the two groups (P > 0.05).The coincidence rate of HER2 protein expression between the two groups was 99.00%. The background of HER2 gene was clear In both groups, and the signals of HER2 and CM 7 double probes were clear. There was no cross-reaction and the double probe signal was precisely located In the nucleus of cancer cells. There was no significant difference In the number of successful cells between the two groups (P > 0.05). There was no significant difference in the positive rate and negative rate of HER2 gene amplification between the two groups (P > 0.05). The coincidence rate of HER2 gene amplification between the two groups was 97.71%. The average signal number of HER2 gene and the ratio of HER2/cells In both groups were all equal. There was no significant difference in the mean number of Ch17 signal, Ch17/cell ratio and HER2/CM7 ratio between the two groups (P > 0.05). There was no significant difference in the total positive rate of HER2 between the two groups (P > 0.05). The results showed that compared with the traditional reagents, the invasive breast cancer samples prepared by environment-friendly bio-tissue sample preparation kit had no effect on HER2 protein expression. The expression of HER2 protein does not affect the amplification of HER2 gene, which can meet the needs of clinical detection.

Frequent loss of CD10 expression in follicular lymphoma with leukaemic presentation

@# Follicular lymphoma (FL) is usually a nodal lymphoma expressing CD10, rarely with leukaemic presentation (FL-LP). Materials and Methods: We searched for FL-LP in our institution from 2000 to 2018 and characterised the neoplastic cells by flow cytometry, immunohistochemistry and fluorescence in situ hybridization. Thirteen (6.1%) of 212 FL cases were FL-LP, all de novo neoplasms. The leukaemic cells were small in 12 cases and large in one. All had concurrent FL, mostly (92%; 12/13) low-grade. The single case with large leukaemic cells had a concurrent primary splenic low-grade FL and a double-hit large B-cell lymphoma in the marrow. Results: CD10 was expressed in the leukaemic cells in 38% (5/13) cases by flow cytometry and in 77% (10/13) cases in tumours (p= 0.0471). IGH/BCL2 reciprocal translocation was identified in 85% (11/13) cases. Most patients were treated with chemotherapy. In a median follow-up time of 36 months, nine patients were in complete remission. The 2- and 5-year survival rates were at 100% and 83%, respectively. In this study, we characterised a series of de novo FL-LP in Taiwan. All patients had concurrent nodal and/or tissue tumours, which might suggest that these patients seek medical help too late. Conclusion: The lower CD10 expression rate by flow cytometry than by immunohistochemistry might be due to different epitopes for these assays. Alternatively, loss of CD10 expression might play a role in the pathogenesis of leukaemic change. The clinical course of FL-LP could be aggressive, but a significant proportion of the patients obtained complete remission with chemotherapy.

An acute myeloid leukemia case with concurrent 11q23 anomaly and D13S319 deficiency diagnosed by combined inter- and metaphase fluorescence in situ hybridization / 中华医学遗传学杂志

Objective@#To carry out multipath cytogenetic analysis of a rare case of acute myeloid leukemia (AML) with 11q23 aberration and D13S319 deletion.@*Methods@#G+ R banding technique was used to analyze the chromosomal karyotype of the patient after 24 h of cell culture. Combined interphase and metaphase fluorescence in situ hybridization (FISH) was used to detect specific chromosomal sites for complex translocations and minor missing fragments.@*Results@#The patient was found to harbor MLL-AF10 fusion gene due to rearrangement of the mixed lineage leukemia (MLL) gene in conjunct with deletion of the D13S319 locus on chromosome 13.@*Conclusion@#Whether MLL gene rearrangement and absence of D13S319 locus has a double impact on AML should attract more attention. For AML patient with clonal abnormalities such as 13q-, del (13)(q14), -13 or der (13), FISH assay should be proof and considered to determine the size of missing fragment so as targeted therapy may be implemented.

Developmental retardation due to paternal 5q/11q translocation in a Chinese infant: clinical, chromosomal and microarray characterization

Although it is known that the parental carriers of chromosomal translocation are considered to be at high risk for spontaneous abortion and embryonic death, normal gestation and delivery remain possible. This study aims to investigate the genetic factors of a Chinese infant with multiple malformations and severe postnatal development retardation. In this study, the routine cytogenetic analysis, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) analysis were performed. Conventional karyotype analyses revealed normal karyotypes of all family members. CMA of the DNA of the proband revealed a 8.3Mb duplication of 5q35.1-qter and a 6.9Mb deletion of 11q24.3-qter. FISH analyses verified a paternal tiny translocation between the long arm of chromosomes 5 and 11. Our investigation serves to provide important information on genetic counselling for the patient and future pregnancies in this family. Moreover, the combined use ofCMAand FISH is effective for clarifying pathogenically submicroscopic copy number variants.

Prognostic impact of BCL2, BCL6 and MYC status in de novo diffuse large B-cell lymphoma: a regional study of 43 patients

Background: Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma with marked biologic heterogeneity. We aimed to evaluate the status of MYC, BCL2, BCL6 in patients with DLBCL.Methods: Herein, we have investigated the prognostic relevance of MYC, BCL2 and BCL6 from 43 de novo DLBCL patients.Results: In this study, protein overexpression of BCL2 and BCL6 was encountered in 46.5% (n=20) and 27.9% (n=12) of the tumors, respectively.  Rearrangements in MYC, BCL6, and BCL2 were detected in 9.3% (n=4), 25.6% (n=11), and 4.7% (n=2) of the cases, respectively. Any statistically significant difference could not be found between Bcl-2, Bcl-6 expression, C-MYC rearrangement and the survival.Conclusions: We concluded that C-MYC and BCL2 may contribute to aggressive transformation, so more mechanism-based therapy should be explored. A larger study is warranted to better understand the immunophenotypic and molecular features of DLBCL and their respective impact on patient survival.

Williams–Beuren syndrome in Mexican patients confirmed by FISH and assessed by aCGH

Williams–Beuren syndrome (WBS) has a prevalence of 1/7500–20000 live births and results principally from a de novo deletion in 7q11.23 with a length of 1.5 Mb or 1.8 Mb. This study aimed to determine the frequency of 7q11.23 deletion, size of the segment lost, and involved genes in 47 patients with a clinical diagnosis of WBS and analysed by fluorescence in situ hybridization (FISH); among them, 31 had the expected deletion. Micro-array comparative genomic hybridization (aCGH) confirmed the loss in all 18 positive-patients tested: 14 patients had a 1.5 Mb deletion with the same breakpoints at 7q11.23 (hg19: 72726578–74139390) and comprising 24 coding genes from TRIM50 to GTF2I. Four patients showed an atypical deletion: two had a 1.6Mb loss encompassing 27 coding genes, from NSUN5 to GTF2IRD2; another had a 1.7 Mb deletion involving 27 coding genes, from POM121 to GTF2I; the remaining patient presented a deletion of 1.2 Mb that included 21 coding genes from POM121 to LIMK1. aCGH confirmed the lack of deletion in 5/16 negative-patients by FISH. All 47 patients had the characteristic facial phenotype of WBS and 45 of 47 had the typical behavioural and developmental abnormalities. Our observations further confirm that patients with a classical deletion present a typical WBS phenotype, whereas those with a high (criteria of the American Association of Pediatrics, APP) clinical scorebut lacking the expected deletion may harbour an ELN point mutation. Overall, the concomitant CNVs appeared to be incidental findings.