Fluorescence In Situ Hybridization Technology Research Articles

Examining Cell-Specific Localization inAedes aegyptiTissues with Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in theAedes aegyptimosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs. The methods presented highlight the synthesis of RNA probes and describe the 2-d process of incubating the tissues/organs with the RNA probes. We also describe tyramide signal amplification for improved signal detection.

FISH painting for chromosome identification of aneuploid cauliflower (Brassica oleracea L. var. botrytis)

A common problem in the cultivation and breeding of cauliflower (Brassica oleracea L. var. botrytis) is the occurrence of aneuploids in offspring families. To reveal the chromosomal cause of such numerical variants, it was necessary to develop karyotype tools with which chromosomes can be easily identified. Since mitotic chromosomes in this crop are morphologically similar and lack differentiating banding patterns, we tested two Fluorescent in situ Hybridization (FISH) procedures for chromosome identification: (1) FISH painting with diagnostic repetitive DNA patterns and (2) cross-species chromosome painting. The first method consists of a five-colour FISH with 5s rDNA, 45S rDNA, and two Brassica rapa centromere-specific repeats, and a B. rapa BAC (KBrH092N02) containing a dispersed repeat of an unknown class. The second method is an advanced FISH technology based on hybridising DNA probes of a related species under adapted stringency conditions to identify their homoeologous loci. To this end, we applied four pools of BACs from Arabidopsis thaliana in a multicolour FISH for a banding pattern on the chromosomes of cauliflower (Brassica oleracea L. var. botrytis). Due to the genome triplication and various chromosome rearrangements of Brassica oleracea compared to Arabidopsis, we used MUMmer whole-genome alignment plot information to select Arabidopsis BAC pools with which all cauliflower chromosomes could be identified. In a sample of 21 plants with aberrant phenotypes, we demonstrated primary trisomy for chromosomes 1–6 and 8, and telo-trisomy for chromosomes 7 and 9. Finally, we discuss the advantages and drawbacks of the two painting methods and eventual alternatives for demonstrating numerical aberrations in the cauliflower populations.Graphical

Application of Fluorescence In Situ Hybridization (FISH) in Oral Microbial Detection.

Varieties of microorganisms reside in the oral cavity contributing to the occurrence and development of microbes associated with oral diseases; however, the distribution and in situ abundance in the biofilm are still unclear. In order to promote the understanding of the ecosystem of oral microbiota and the diagnosis of oral diseases, it is necessary to monitor and compare the oral microorganisms from different niches of the oral cavity in situ. The fluorescence in situ hybridization (FISH) has proven to be a powerful tool for representing the status of oral microorganisms in the oral cavity. FISH is one of the most routinely used cytochemical techniques for genetic detection, identification, and localization by a fluorescently labeled nucleic acid probe, which can hybridize with targeted nucleic acid sequences. It has the advantages of rapidity, safety, high sensitivity, and specificity. FISH allows the identification and quantification of different oral microorganisms simultaneously. It can also visualize microorganisms by combining with other molecular biology technologies to represent the distribution of each microbial community in the oral biofilm. In this review, we summarized and discussed the development of FISH technology and the application of FISH in oral disease diagnosis and oral ecosystem research, highlighted its advantages in oral microbiology, listed the existing problems, and provided suggestions for future development..

Analysis of Fluorescence in Situ Hybridization Detection Results and Clinical Indicators in 51 Patients with Multiple Myeloma

Objective: To investigate the clinical application of fluorescence in situ hybridization (FISH) in multiple myeloma (MM) and the correlation between genetic abnormalities and clinical indicators. Methods: The FISH test results and clinical indicators of 51 newly diagnosed MM patients in our hospital from September 2021 to July 2022 were retrospectively analyzed, and the relationship between test results, genetic abnormalities and clinical indicators was analyzed. The FISH probes included IGH, MAFB/IGH, MAF/IGH, FGFR3/IGH, CCND1/IGH, D13S319/CEP12, P53/CEP17, and 1q21. Results: Genetic abnormalities were detected in 28 of 51 MM patients, with a detection rate of 54.9%. The frequency of the detected FISH probes in genetic abnormalities from high to low was 1q21 amplification in 18 cases (64.29%), IGH rearrangement in 14 cases (50.00%), D13S319 deletion in 12 cases (42.86%) and P53 deletion in 6 cases (21.43%). The results of FISH and the clinical indicators of the patients were analyzed , IGH rearrangement was associated with the elevation of serum creatinine (CRE) and β2-microglobulin (β2-MG), and 1q21 amplification and D13S319 deletion were associated with the elevation of lactate dehydrogenase (LDH). Conclusion: In the cytogenetic examination, FISH is an important detection method, which can quickly detect the genetic abnormalities of MM patients, and these genetic abnormalities are related to the clinical indicators of the disease. FISH technology can play a great role in the diagnosis, the condition assessment and the prognosis judge of MM, and has important clinical significance.

The Independent Adverse Prognostic Significance of 1q21 Gain/Amplification in Newly Diagnosed Multiple Myeloma Patients

Background Multiple myeloma (MM) is a hematological malignancy with proliferation of monoclonal plasma cells which has complex genetics and prognostic heterogeneity. It involves a variety of primary genetic abnormalities, including t (4; 14), t (11; 14), t (14,16) and hyperdiploid karyotypes, as well as secondary genetic abnormalities, including chromosome copy number abnormalities (1q21+, 1p-, 13q-, 17p-, etc) and secondary translocations like MYC. As the most common secondary genetic event, 1q21+ occurs in about 30-40% of NDMM patients while the proportion is especially higher in Chinese population, accounting for 59.7% (96/161) by a domestic study in 2022, it is closely related to the situation that MM patients may have long disease duration and late stage when initial diagnosed in China. Despite its frequency, considerable debate remains regarding to the prognostic impact of 1q21+ in NDMM, 1q21 status hasn't been included among the high-risk markers listed by the IMWG R-ISS stage, and neither in Chinese guidelines. If 1q21+ is included as one of the poor prognostic factors, it will greatly increase the proportion of high-risk patients in NDMM patients. So, our center has begun to take 1q21 seriously as a heterogeneity problem for clinical discussion. Methods 248 NDMM patients admitted in The First Affiliated Hospital of Soochow University from September 01, 2018 to August 31, 2021 of a VRD clinical study, with VRD in combination with ASCT or VRD treatment for 8 cycles, were retrospectively analyzed. 135 cases (54.4%) had 1q21+ by CD38-sorted fluorescence in situ hybridization (FISH), among which 153 patients were compared for the involved genes by CytoScan, focusing on the clinical characteristics, treatment response and prognostic significance by combining FISH and CytoScan technology complementary. Results Among the 248 NDMM patients, 54.4% patients with 1q21+ were more likely to have anemia, hypoalbuminemia, renal insufficiency, high lactate dehydrogenase and high proportion of R-ISS-III stage, and are more likely to be combined with 13q14 deletion, t(4;14), and 1p deletion，and they are all at middle-risk or high-risk groups defined by Prognostic Index score. Multivariate analysis showed that 1q21 gain/amplification was an independent adverse prognostic factor (PFS HR=2.358, 95%CI 1.286-4.324, P=0.006; OS HR=2.598, 95%CI 1.050-6.425, P=0.039). 1q21+ subgroup had an inferior outcome (PFS P=0.0133, OS P=0.0293) (Figure 1a). According to the mSMART3.0 stages, double-hit and triple-hit group had the worst prognosis, with a median PFS of 22 months and a median OS of 32 months. Both PFS and OS were clinically significant (P<0.0001), as shown in Figure 1b. Furthermore 1q21 amplification with more than 4 copy numbers had a shorter PFS than 1q21 gain with 3 copy numbers (24 months vs not reached, P=0.0403), but the OS difference hasn't been not clinically significant (Figure 1c). The positive proportion of 1q21 had no effects on prognosis，while the status of 1q21 in the main clone rather than in the subclone defined by CytoScan when the proportion of abnormal clone populations are differed more than 30%, was an adverse factor affecting the prognosis (PFS P=0.0172, OS P=0.1260). (Figure 1d). ASCT plays an important role in the prognostic survival of 1q21 gain and amplification patients (P<0.05). Outcomes of 1q have been widely discussed in CRD/CTD scheme of Myeloma XI, KRd/KCD scheme of FORTE, VRD/PAD of HOVON65 and so on, our study focusing on the patients with VRD +/- ASCT is also unique, demonstrating that the prognosis of 1q21+ in VRD scheme is also poor, but ASCT can overcome the adverse effects significantly. Conclusions Patients with 1q21+ have clinically significant end-stage organ damage and higher tumor burden, more likely to combine 13q14-, t(4;14), 1p32- and other cytogenetic abnormalities. 1q21+ is an independent high-risk cytogenetic factor for poor prognosis in NDMM patients, of which 4 or more copy numbers and main clone position significantly associated with prognosis results. By combining FISH, CytoScan and other technologies with related abnormalities such as 1q21+，17p-, 1p32-，t (4;14), t(14;16), etc., clinical characteristics, efficacy evaluation, and prognosis can be analyzed more accurately, aiming to identify patients with poor prognosis earlier and enhancing the possibility of moving forward to the treatment strategy based on risk stratification in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of Fluorescence In Situ Hybridization Assisted by Fluorescence Microscope in Detection of Her2 Gene in Breast Cancer Patients.

In order to study the important factors for evaluating the prognosis of breast cancer patients, a fluorescence microscopy-assisted fluorescence in situ hybridization technique was proposed. Compared with other detection techniques, fluorescence in situ hybridization (FISH) technology assisted by a fluorescence microscope has gradually gained favor in related fields due to its advantages of high detection specificity, high sensitivity, and strong experimental period. Combined with the basic overview of fluorescence microscopy and FISH technology, the advantages and application points of FISH technology assisted by fluorescence microscopy in the detection of the Her2 gene in breast cancer patients were studied and discussed. The results show that IHC can be used as the primary screening for HER2 gene status detection; IHC (2+) and IHC (3+) have false positives, which are related to chromosome 17 polysomy, so FISH should be done to confirm the diagnosis.

Clinicopathological and molecular features of small round cell sarcoma of bone and soft tissue: a study of 72 cases

Objective: To investigate the clinicopathological, immunohistochemical and molecular features of small round cell sarcoma (SRCS) of the bone and soft tissue, and to compare the diagnostic value of different techniques. Methods: Seventy-two cases of SRCS of the bone and soft tissue diagnosed at People's Hospital, Peking University from January 2016 to March 2020 were recruited and retrospectively analyzed for pathological morphology, immunophenotype and fluorescence in situ hybridization (FISH) data. Next generation sequencing (NGS) was performed on 13 difficult cases. Results: In the study cohort, the patients ranged in age from 4-55 years, with a male predominance. The most Ewing's sarcomas and osteosarcomas occurred in the bone, while CIC-rearranged sarcomas, BCOR-rearranged sarcoma, synovial sarcoma, extraskeletal myxoid chondrosarcoma and FUS-NFATc2 rearranged sarcoma occurred in soft tissue. Histologically, all cases were composed predominantly of small round cells. Most cases were positive for vimentin and CD99, and showed a variable reactivity for neurogenic markers. Muscle marker and epithelial marker were negative for most cases. Combined with clinical features, histopathologic findings, immunophenotype, FISH and NGS, we diagnosed 46 Ewing sarcomas, 14 osteosarcomas, 3 CIC-rearranged sarcomas, 1 BCOR-rearranged sarcoma, 1 synovial sarcoma, 1 clear cell soft tissue sarcoma, 1 extraskeletal myxoid chondrosarcoma, 1 FUS-NFATc2 rearranged sarcoma, and 4 undifferentiated small round cell sarcomas. Conclusions: SRCS of bone and soft tissue is a group of malignant mesenchymal tumors based on morphological features. Most cases can be diagnosed with a combination of clinical characteristics, morphological features and immunohistochemical phenotype, while some cases require such further tests as FISH and NGS technologies, and NGS can be useful in diagnosing and categorizing SRCS.

Applications of fluorescence in situ hybridization (FISH) in genome research

Fluorescence in situ hybridization (FISH) technique enables the direct detection of DNA sequences inintact cellular materials (e.g. individual chromosomes in metaphase spreads). This review article focuses on theapplications of FISH in genome research, including validation and correction of the genome assembly from thenext-generation sequencing (NGS) projects. DNA probes for specific DNA fragments of the assembly can beobtained from PCR amplicon or cloned products using different vector systems. Localization of these probeson their respective chromosomal regions can be visualized by FISH, providing useful information to crosscheckthe assembly data. Furthermore, the recent refinements in the FISH technology including using smartpooling scheme of differently colored DNA probes, together with consecutive FISH experiments (stripping andreprobing of the same slide) are described. These advances in multicolor FISH can provide crucial linkageinformation on association of linkage groups and assembly scaffolds, resulting in so-called cytogenetic maps.Integration of the cytogenetic maps and assembly sequences assists to resolve the chromosome-level genomeassembly and to reveal new insights in genome architecture and genome evolution. Especially, comparativechromosome painting with pooled DNA probes from one reference species can be used to investigate ancestralrelationships (chromosome homeology and rearrangements) among other not-yet-sequenced species. Inaddition, FISH using DNA probes for certain specific classes of repetitive DNA elements as well as for basicchromosome structures (e.g. centromere or telomere DNA repeats, ribosomal DNA loci) can be used to studythe genome organization and karyotype differentiation. We also discussed about limitations and futureperspectives of the FISH technology.

Detection of Gene Abnormalities in 43 Cases of Chronic Lymphocytic Leukemia by Fluorescence in Situ Hybridization

To explore the value of fluorescence in situ hybridization (FISH) in diagnosis and prognosis evaluation of chronic lymphocytic leukemia (CLL). R banding technique was used for karyotype analysis in 43 cases of CLL, and fluorescence in situ hybridization（FISH） technique was used for analysis of 5 genes in 43 cases of CLL, including D13S25, RB1,ATM, P53 and CEP 12 . The detection rate of chromosome abnormality was 9.3％ for the 43 cases. The chromosome karyotype abnormality was involoved in the number abnormality and the structural abnormality, including No. 2, 6, 14, and sex chromosome. The patients with normal chromosome karyotype were more common（79.1％）. At the same time, mitotic figure in 5 CLL patients did not observed. The positive rate of FISH detection was 55.8％(24/43). Among which, D13S25 deletion was the highest and reached to 37.2％, followed by RB1 deletion（20.9％）, CEP 12 amplification（16.3％）, ATM deletion（9.3％） and P53 deletion（7.0％）. In 24 FISH-positive cases, 20 cases displayed the normal chromosome karyotype, and in 3 cases the mitotic figure was absent. There was only one case of abnormal chromosome, which was not involved in the positive gene tested by FISH. FISH is an important means of CLL genetic detection, which can greatly raise the detection rate of cytogenetics abnormalities in CLL, but the probe quantity is limited, so it is necessary to use FISH technology combined with chromosome karyotype analysis so as to improve the detection rate of abnormal cytogenetics, and provide the basis for the clinical diagnosis and prognosis of CLL.

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications.

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH techniques have visualized intra-nuclear genomic structure and sub-cellular transcriptional dynamics of many genes and revealed their functions in various biological processes.

Genetic abnormality in 101 cases of plasma cell dyscrasias by FISH technology and cytogenetic examination

To investigate several abnormal genes by the fluorescence in situ hybridization (FISH) in multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and reactive plasmacytosis (RP), and to increase the diagnosis and differential diagnosis levels for these common plasma diseases. The clinical manifestations, image and laboratory tests and the FISH detection were retrospectively analyzed in 61 cases of newly diagnosed MM, 20 cases of MGUS and 20 cases of RP from August, 2012 to February, 2015 in the Xiangya Hospital of Central South University. Fifty cases among 61 MM patients showed genetic abnormality by FISH technology. The total positive rate was 81.9%. Among them, 19 cases (31.1%) had 1q21 amplification, 18 cases (29.5%) lacked D13S319, 10 cases (16.4%) missed RB1, 10 cases (16.4%) had IGH translocation and 7 cases (11.4%) lacked p53 gene. The positive rate for two or more genes abnormal was 19.8% in 12 cases. However, in 20 cases of MGUS patients, the positive detection rate was 25%, including 4 cases (20%) of 1q21 augmentation and 2 cases (10%) of IGH translocation. There were not two or more abnormal genes in one case. While in RP cases, only 1 case of patients had D13S319 abnormal gene, and the positive rate was only 5%. There was significant difference (P<0.05) among the 3 groups. The positive detection rate is 81.9% in MM patients by FISH, which is significantly higher than that in patients with MGUS or RP. FISH technology can detect a variety of abnormal genes in MM. It is useful for the differential diagnosis and prognosis for MM, MGUS and RP.

FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe.

Advances in fluorescence in situ hybridization (FISH) make it feasible to detect multiple copy-number changes in hundreds of cells of solid tumors. Studies using FISH, sequencing, and other technologies have revealed substantial intra-tumor heterogeneity. The evolution of subclones in tumors may be modeled by phylogenies. Tumors often harbor aneuploid or polyploid cell populations. Using a FISH probe to estimate changes in ploidy can guide the creation of trees that model changes in ploidy and individual gene copy-number variations. We present FISHtrees 3.0, which implements a ploidy-based tree building method based on mixed integer linear programming (MILP). The ploidy-based modeling in FISHtrees includes a new formulation of the problem of merging trees for changes of a single gene into trees modeling changes in multiple genes and the ploidy. When multiple samples are collected from each patient, varying over time or tumor regions, it is useful to evaluate similarities in tumor progression among the samples. Therefore, we further implemented in FISHtrees 3.0 a new method to build consensus graphs for multiple samples. We validate FISHtrees 3.0 on a simulated data and on FISH data from paired cases of cervical primary and metastatic tumors and on paired breast ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Tests on simulated data show improved accuracy of the ploidy-based approach relative to prior ploidyless methods. Tests on real data further demonstrate novel insights these methods offer into tumor progression processes. Trees for DCIS samples are significantly less complex than trees for paired IDC samples. Consensus graphs show substantial divergence among most paired samples from both sets. Low consensus between DCIS and IDC trees may help explain the difficulty in finding biomarkers that predict which DCIS cases are at most risk to progress to IDC. The FISHtrees software is available at ftp://ftp.ncbi.nih.gov/pub/FISHtrees.

Analysis of laboratory examination, diagnosis and treatment of 57 cases of acute promyelocytic leukemia

Objective To investigate the laboratory examination, diagnosis and treatment of 57 cases of Acute Promyelocytic Leukemia. Methods The clinical manifestation, laboratory morphology-immunology-cytogenetics-molecular (MICM) classification, treatment and follow-up were analyzed retrospectively among 57 newly diagnosed cases of acute promyelocytic leukemia (APL) patients from January 2011 to December 2013 in Xiangya Hospital, Central South University. Results In 57 patients, the first symptom was given priority to with bleeding (73.7%), fever (26.3%), and the organ infiltration accounted for 29.8%. The phenotype of CD13+ CD33+ CD34-HLD-RA- was 59.6% in 57 patients, CD13+ CD33+ CD34+ HLD-RA+ was 8.8%, and the remaining 18 cases were not classic phenotypes. The positive percentage of PML/RARa was 93.0% by fluorescence in situ hybridization (FISH), and the remaining 4 cases were other rare abnormal. Except one case gave up the treatment, the other 56 cases of APL patients reached 100% complete remission rate under early screening and diagnosis, relatively standardized and effective treatment. Conclusions Hemorrhage is the most common clinical symptoms of M3. MICM classification, especially the FISH technology, is the main laboratory basis for diagnosis of M3. After the treatment of early specifications currently, M3 can almost be cured. Key words: Leukemia, promyelocytic, acute/DI/TH

Evaluation of HER2 by automated FISH and IHC in gastric carcinoma biopsies.

The use of trastuzumab (Herceptin) to target HER2 has been applied in breast carcinoma and gastric carcinoma (GC). Previous studies have tested trastuzumab's effectiveness by assessing HER2 expression or HER2 amplification by means of immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). In this work we aimed to evaluate automated FISH and IHC technologies for HER2 detection in GC biopsies to be used in routine pathology practice. The study used an Oracle HER2 IHC System and an LSI HER2/CEP17 Dual Probe on an automated Bond system (Leica Microsystems). One hundred GC biopsies were evaluated including 44 intestinal type, 38 diffuse type and 18 indeterminate type according to Lauren's classification. The overall concordance rate between the automated FISH and IHC methods was 94% (κ = 0.87), as 6 samples were scored as equivocal (4 in IHC and 2 in FISH). Moreover, HER2 positivity was significantly different between the 3 types of GC (p<0.05), being more frequent in intestinal-type GC (23%) than in the other 2 histological types (5% and 0%). Finally, the HER2/CEP17 FISH ratio was significantly different (p<0.01) between disomic and polysomic samples, being higher in polysomic samples (mean 1.633 ± 0.509) than in disomic samples (mean 1.231 ± 0.675). Automated HER2 testing of GC biopsies using the Leica Bond system was useful and efficient. This method allowed us to improve normal routine procedures, minimizing time and costs as well as handling and observation errors.

Detection of Molecular Cytogenetic Aberrations by Fluorescence in Situ Hybridization in Different Bone Marrow Samples of Multiple Myeloma

To detect the molecular cytogenetic abnormalities in different bone marrow samples of multiple myeloma by using fluorescence in situ hybridization (FISH) technology. The bone marrow cells from 48 cases of MM were taken for sorting the plasma cells using CD138 magnetic beads, and the biopsy tissue from 10 cases of MM was taken and embedded in parafin, then the 2 kinds of samples were detected by using FISH. D13s319/RB1, 1q21/P53, IgH, IgH/FGFR3, IgH/MAF probes were detected in 58 patients with new diagnosed multiple myeloma (MM) by FISH technology. Fluorescent signals were both seen in 2 different types of bone marrow samples and cytogenetic aberrations were detected in 30/58 (51.7%) patients, 29.3% (17 out of 58) cases had both D13S319 and RB1 deletion. The positive rates of P53 deletion, 1q21 amplification and IgH rearrangement were 12%, 27.6% and 20.7%, respectively. Only 7 cases (23.3%) had one cytogenetic abnormality, other 23 (76.7%) cases all had 2 to 5 kinds of different abnormalities. More than half of MM patients have cytogenetic change, and most of them are complex chromosomal abnormality. The different kinds of samples can expand the useful extension of FISH technology and acquire more cytogenetic information for clinician.

Analysis of EML4-ALK gene fusion mutation in patients with non-small cell lung cancer

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）是肺癌的主要类型，相关位点突变检测研究已经成为肺癌分子靶向治疗的热门方向，研究NSCLC肿瘤组织中动物微管相关蛋白4与间变性淋巴瘤激酶融合基因（echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase, EML4-ALK）与表皮生长因子受体（epidermal growth factor receptor, EGFR）的基因突变状态，比较免疫组织化学（immunohistochemistry, IHC）与蝎形探针扩增阻滞突变系统（Scorpions amplification refractory mutation system, Scorpions ARMS）荧光定量PCR与荧光原位杂交（fluorescence in situ hybridization, FISH）检测EML4-ALK融合基因与EGFR基因突变的敏感性。方法应用IHC、ARMS荧光定量PCR及FISH技术检测85例NSCLC石蜡包埋肿瘤组织以及癌旁正常肺组织中EML4-ALK融合基因状态，并应用ARMS方法检测EGFR基因第18、19、20和21外显子突变状态。结果115例NSCLC中IHC显示32例有ALK（D5F3）表达，表达率为27.8%;ARMS检测27例存在EML4-ALK融合基因突变，突变检出率为23.5%;53例检出EGFR突变，突变率为46%。而FISH检测23例存在EML4-ALK融合基因突变，检出率为20%，稍低于ARMS检测结果，提示ARMS的敏感度更高。结论联合运用IHC/ARMS荧光定量PCR/FISH技术能够对EML4-ALK融合基因状态做出快速、准确评价。

Diagnosis of bladder cancer from the voided urine specimens using multi-target fluorescence in situ hybridization

The present study aimed to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for bladder cancer in light of the histological diagnosis. Several valuable observations using FISH technologies in voided urine cells were also reported. The multi-target FISH-containing probes for the centromeres of chromosomes 3, 7 and 17 and the 9p21 locus were applied to cytospin specimens prepared from voided urine. Urine samples from 53 bladder cancer patients and 30 patients with benign alterations were used for this study. The histological observations of surgical resection specimens showed that the specificity and sensitivity for the technique were 100.0 and 88.0%, respectively. Statistical analyses showed that there was no significant correlation between FISH-positive rate and the tumor stage/grade (P<0.05). However, the proportion of tumor cells with genetic abnormalities positively correlated with the tumor stage (P<0.01). Furthermore, the number of abnormal cells in muscle-invasive pT2 was significantly higher than that in non-muscle-invasive pTa, pT1 (P<0.01). Of 50 patients with bladder cancer, polysomies of chromosomes 3, 7 and 17 were detected in 84.0, 48.0 and 78.0% of cases, respectively, and loss of the 9p21 gene was detected in 80.0% of cases. In addition, the detailed results from different urine specimens showed that FISH assay was required. FISH assay for chromosomes 3, 7 and 17 and 9p21 has a high specificity and sensitivity in the detection of bladder cancer and may reduce the necessity for cytoscopy treatment.

ALK and ROS1 gene rearrangements detected in colorectal cancer (CRC) by fluorescence in situ hybridization (FISH).

3545 Background: Activation of ROS1 and ALK tyrosine kinases through gene fusions lead to unchecked cell proliferation and transformation. ROS1 and ALK gene fusions were found in about 5% and 2% of lung adenocarcinomas and are highly sensitive to specific tyrosine kinase inhibitors. This study aimed at identifying the presence of ROS1 and ALKrearrangements in CRC using FISH technology. Methods: Arrayed specimens of metastatic CRC (mCRC) patients were tested with a 4-target, 4-color break-apart FISH probe set (Abbott Molecular) designed to simultaneously evaluate the genomic status of ROS1 and ALK. Fused 3’/5’ signals of each gene were considered negative for rearrangement; single 3’/single 5’ (for ROS1) and split 3’-5’ or single 3’ (for ALK) were considered positive for rearrangement. Results: Among 236 mCRC patients tested, two were positive for ROS1 (single 3’ROS1 signals in 39% and 61% of tumor cells) and one was positive for ALK rearrangement (single 3’ALK in 41% of tumor cells). The upper cut-off for positive FISH patterns in the negative specimens was identified as &lt;15% both for ROS1 and ALK. Interestingly, the ALK+ patient displayed intra-tumoral heterogeneity, detected in the tissue cores and confirmed in two resection blocks. The fusion partner for ALK was identified as EML4 by PCR-based tests and sequencing. The fusion partner(s) for ROS1 remains to be identified by other technologies. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. Conclusions: The novel FISH probe set was effective to identify the first cases of ROS1 rearrangements in CRC and re-confirm the occurrence of ALK rearrangements. This supports further evaluation of mCRC cases for ROS1 and ALK gene fusions as these may represent new targets for evaluation in clinical trials. Tumor heterogeneity in the ALK rearrangement must be addressed for screening tests. (Partially supported by research grant from Abbott Molecular and the Colorado CCSG P30CA046934).

What's new in prognostication of melanoma in the dermatopathology laboratory?

With the advent of genetic and epigenetic research, molecular techniques could someday be used to discriminate nevus from melanoma so that ambiguous melanocytic lesions could be more accurately classified or that prognostication could be improved in melanoma patients. That promised day might be closer than realized. The last 20 years of research in cytogenetic and genetic alterations in melanoma have culminated in defined chromosomal lesions discriminating benign from malignant melanocytic tumors. Exploiting these differences, fluorescence in situ hybridization (FISH) can reproducibly discriminate unequivocal melanomas from melanocytic nevi with high sensitivity and specificity. The discriminating power of FISH in melanocytic tumors with ambiguous histopathology is questionable, however, because there is no standard definition of “malignancy.” Additional FISH studies on ambiguous cases are needed through international collaborations where large collections of such cases are shared and the “proof of malignancy” is established by adequate clinical follow-up. This contribution reviews the diagnostic utility of DNA-based FISH technology as it compares the diagnostic accuracy in melanocytic tumors with unambiguous vs ambiguous histopathology. The melanoma epigenome is further characterized through research into various activities of small interfering RNAs, such as microRNAs, providing the pathway for the application of microRNA-based strategies that could be the basis for future diagnostic biomarkers and molecular therapies in melanoma.

Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm.

Archaea are usually minor components of a microbial community and dominated by a large and diverse bacterial population. In contrast, the SM1 Euryarchaeon dominates a sulfidic aquifer by forming subsurface biofilms that contain a very minor bacterial fraction (5%). These unique biofilms are delivered in high biomass to the spring outflow that provides an outstanding window to the subsurface. Despite previous attempts to understand its natural role, the metabolic capacities of the SM1 Euryarchaeon remain mysterious to date. In this study, we focused on the minor bacterial fraction in order to obtain insights into the ecological function of the biofilm. We link phylogenetic diversity information with the spatial distribution of chemical and metabolic compounds by combining three different state-of-the-art methods: PhyloChip G3 DNA microarray technology, fluorescence in situ hybridization (FISH) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy. The results of PhyloChip and FISH technologies provide evidence for selective enrichment of sulfate-reducing bacteria, which was confirmed by the detection of bacterial dissimilatory sulfite reductase subunit B (dsrB) genes via quantitative PCR and sequence-based analyses. We further established a differentiation of archaeal and bacterial cells by SR-FTIR based on typical lipid and carbohydrate signatures, which demonstrated a co-localization of organic sulfate, carbonated mineral and bacterial signatures in the biofilm. All these results strongly indicate an involvement of the SM1 euryarchaeal biofilm in the global cycles of sulfur and carbon and support the hypothesis that sulfidic springs are important habitats for Earth's energy cycles. Moreover, these investigations of a bacterial minority in an Archaea-dominated environment are a remarkable example of the great power of combining highly sensitive microarrays with label-free infrared imaging.