Detection of ALK Gene Rearrangements in Formalin-Fixed, Paraffin-Embedded Tissue Using a Fluorescence In Situ Hybridization (FISH) Probe

Tan, Louis; Do, Elaine; Chong, Siew Meng; Koay, Evelyn Siew Chuan

doi:10.2165/00066982-200307010-00005

Cited by 15 publications

(4 citation statements)

References 8 publications

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“…Small biopsies and aspirate specimens are particularly difficult for interpretation because of the possibility of dilution with normal cells . It is known that, in ALK FISH testing, the use of archival tissue is associated with an increase in the rate of failed tests due to lack of hybridization or interpretable signals . The use of archival tissue might have made scoring of these cases even more technically challenging.…”

Section: Discussionmentioning

confidence: 99%

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis

et al. 2017

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Section: Discussionmentioning

confidence: 99%

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis

et al. 2017

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“…Published studies using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis or fluorescent in situ hybridization (FISH) to characterize EML4 - ALK fusions in lung cancer have revealed frequencies ranging from 0.5% to 7.5% in general lung cancer patients and a frequency of 13.5% in clinical factor-enriched cases [23], [27,28]. Here, we report the development of a technology based on rapid amplification of cDNA ends (RACE)-coupled PCR and sequencing to analyze expression of ALK fusion genes.…”

Section: Introductionmentioning

confidence: 99%

Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression

et al. 2010

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BackgroundThe anaplastic lymphoma kinase (ALK) gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer.ResultsRACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC) patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%). Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62) in patients with adenocarcinomas, 19.23% (10/52) in never-smokers, and 42.80% (9/21) in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03), younger age of onset (P = 0.03), and adenocarcinomas without EGFR/KRAS mutations (P = 0.04). A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20). Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018). However, expression of EML4 did not differ between the groups.ConclusionsThe EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

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“…Successful adaptation of such protocols, initially developed for whole cell applications, to conventionally processed tissue using FFPET sections that can be prepared in a histology laboratory using a conventional microtome, has enabled integration of molecular pathology practice into the routine diagnosis and study of lymphomas, leukemias, and sarcomas (Barrans et al 2002(Barrans et al , 2003Frater et al 2001;Cataldo et al 1999;Shaminie et al 2003;Tan et al 2003;Bijwaard et al 2002;Kumar et al 1999;Lee et al 1993;Misra et al 1995;Haralambieva et al 2002Haralambieva et al , 2003Cook 2004;Takada et al 2003). Validation of reagents used for FISH testing is an essential step prior to productive translational research and clinical testing implementation.…”

Section: Discussionmentioning

confidence: 99%

“…Interphase FISH assays do not require dividing cells, can usually be performed within 24-48 h of biopsy, and now provide robust molecular tools ancillary to conventional morphologic and immunophenotypic diagnosis. Originally developed for gravity or sedimentation fresh whole cellular preparations, or nuclei extracted from paraffin (Paternoster et al 2002;Wolfe and Herrington 1997;Schurter et al 2002;Barrans et al 2002), interphase FISH assays have recently been successfully adapted to formalin fixed paraffin embedded tissue sections (FFPET) for both structural deletions and translocations (Barrans et al 2002(Barrans et al , 2003Frater et al 2001;Cataldo et al 1999;Shaminie et al 2003;Tan et al 2003;Bijwaard et al 2002;Kumar et al 1999;Lee et al 1993;Misra et al 1995;Haralambieva et al 2002Haralambieva et al , 2003Cook 2004). Appropriate FFPE tissues for interphase analysis include lymph node or other extranodal tissue containing non-Hodgkin lymphoma, clot sections prepared from aspirated bone marrow samples, or sarcomas and malignant small round cell tumors.…”

Section: Introductionmentioning

confidence: 99%

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts

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et al. 2006

J Mol Hist

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Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.

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Detection of ALK Gene Rearrangements in Formalin-Fixed, Paraffin-Embedded Tissue Using a Fluorescence In Situ Hybridization (FISH) Probe

Abstract: The difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

Cited by 15 publications

References 8 publications

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis

Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts

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