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KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffin-embedded tissue only.

Togashi Y, Soda M, Sakata S, Sugawara E, Hatano S, Asaka R, Nakajima T, Mano H, Takeuchi K - PLoS ONE (2012)

Bottom Line: These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients.The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization.This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Pathology Project for Molecular Targets, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.

ABSTRACT
The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5'-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

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ALK-rearranged lung adenocarcinoma without EML4-ALK and KIF5B-ALK.Panel A shows the results of anti-ALK immunohistochemistry with the iAEP method on pulmonary adenocarcinoma in situ, nonmucinous. The staining pattern was diffusely cytoplasmic. The basal side of tumor cells was more strongly stained, indicating an uneven subcellular localization of KLC1-ALK protein. FISH analyses revealed that this case was positive in the split assay for ALK (Panel B: individual 5′- and 3′-signals are observed) and negative in EML4-ALK and KIF5B-ALK fusion assays (Panel C: EML4, red; ALK, green; Panel D: KIF5B, green; ALK, red).
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pone-0031323-g001: ALK-rearranged lung adenocarcinoma without EML4-ALK and KIF5B-ALK.Panel A shows the results of anti-ALK immunohistochemistry with the iAEP method on pulmonary adenocarcinoma in situ, nonmucinous. The staining pattern was diffusely cytoplasmic. The basal side of tumor cells was more strongly stained, indicating an uneven subcellular localization of KLC1-ALK protein. FISH analyses revealed that this case was positive in the split assay for ALK (Panel B: individual 5′- and 3′-signals are observed) and negative in EML4-ALK and KIF5B-ALK fusion assays (Panel C: EML4, red; ALK, green; Panel D: KIF5B, green; ALK, red).

Mentions: A FFPE tissue block of pulmonary adenocarcinoma in situ, nonmucinous (formerly called bronchioloalveolar carcinoma) [36], which was excised from a 47-year-old female patient was used [37]. This carcinoma was negative for EML4-ALK and KIF5B-ALK, although the presence of ALK rearrangement was confirmed by anti-ALK iAEP immunohistochemistry and a split fluorescence in situ hybridization (FISH) assay for ALK (hereafter referred to as the unknown ALK fusion-positive case) (Figure 1) [37]. Two FFPE tissue blocks of ALK-positive tumor cases were also employed, for which the presence of EML4-ALK or KIF5B-ALK had already been confirmed. Total RNA was extracted from each FFPE tissue with the use of the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Applied Biosystems Japan, Tokyo, Japan). The ages of the 3 FFPE blocks used (time from FFPE tissue production to RNA extraction) were 65, 40, and 51 months for the unknown ALK fusion-positive case, EML4-ALK, and KIF5B-ALK, respectively. Written informed consent was obtained from each patient. The study was approved by the institutional review board of the Shizuoka Cancer Center (approval ID 22-J132-22-1) and the Japanese Foundation for Cancer Research (approval ID 2010-1011).


KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffin-embedded tissue only.

Togashi Y, Soda M, Sakata S, Sugawara E, Hatano S, Asaka R, Nakajima T, Mano H, Takeuchi K - PLoS ONE (2012)

ALK-rearranged lung adenocarcinoma without EML4-ALK and KIF5B-ALK.Panel A shows the results of anti-ALK immunohistochemistry with the iAEP method on pulmonary adenocarcinoma in situ, nonmucinous. The staining pattern was diffusely cytoplasmic. The basal side of tumor cells was more strongly stained, indicating an uneven subcellular localization of KLC1-ALK protein. FISH analyses revealed that this case was positive in the split assay for ALK (Panel B: individual 5′- and 3′-signals are observed) and negative in EML4-ALK and KIF5B-ALK fusion assays (Panel C: EML4, red; ALK, green; Panel D: KIF5B, green; ALK, red).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275577&req=5

pone-0031323-g001: ALK-rearranged lung adenocarcinoma without EML4-ALK and KIF5B-ALK.Panel A shows the results of anti-ALK immunohistochemistry with the iAEP method on pulmonary adenocarcinoma in situ, nonmucinous. The staining pattern was diffusely cytoplasmic. The basal side of tumor cells was more strongly stained, indicating an uneven subcellular localization of KLC1-ALK protein. FISH analyses revealed that this case was positive in the split assay for ALK (Panel B: individual 5′- and 3′-signals are observed) and negative in EML4-ALK and KIF5B-ALK fusion assays (Panel C: EML4, red; ALK, green; Panel D: KIF5B, green; ALK, red).
Mentions: A FFPE tissue block of pulmonary adenocarcinoma in situ, nonmucinous (formerly called bronchioloalveolar carcinoma) [36], which was excised from a 47-year-old female patient was used [37]. This carcinoma was negative for EML4-ALK and KIF5B-ALK, although the presence of ALK rearrangement was confirmed by anti-ALK iAEP immunohistochemistry and a split fluorescence in situ hybridization (FISH) assay for ALK (hereafter referred to as the unknown ALK fusion-positive case) (Figure 1) [37]. Two FFPE tissue blocks of ALK-positive tumor cases were also employed, for which the presence of EML4-ALK or KIF5B-ALK had already been confirmed. Total RNA was extracted from each FFPE tissue with the use of the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Applied Biosystems Japan, Tokyo, Japan). The ages of the 3 FFPE blocks used (time from FFPE tissue production to RNA extraction) were 65, 40, and 51 months for the unknown ALK fusion-positive case, EML4-ALK, and KIF5B-ALK, respectively. Written informed consent was obtained from each patient. The study was approved by the institutional review board of the Shizuoka Cancer Center (approval ID 22-J132-22-1) and the Japanese Foundation for Cancer Research (approval ID 2010-1011).

Bottom Line: These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients.The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization.This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Pathology Project for Molecular Targets, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.

ABSTRACT
The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5'-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

Show MeSH
Related in: MedlinePlus