Detecting EML4-ALK Fusion Gene Mutation in Endobronchial Aspiration Specimens
The October 15, 2010 issue of Clinical Cancer Research has an intriguing article that could have major implications for staging and prognostication in lung cancer. Sakairi and colleagues from Chiba Cancer Center in Japan show the feasibility of using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to obtain specimens suitable for testing for EML4-ALK fusion gene in NSCLC lung cancer patients with proven hilar and/or mediastinal lymph node metastasis.
The protocol for this study involved chest CT followed by EBUS-TBNA for lymph nodes >5 mm. They obtained a histologic core using a 22-gauge needle and divided the specimen into two parts with one part placed in 20% (!) formalin and the other half placed in a cryopreservative solution and then frozen at −80℃. Histological examination was performed in all cases. The authors present a triage for EML4-ALK fusion gene mutation including screening by IHC, followed by FISH, and then final confirmation with RT-PCR. They also tested for EGFR gene mutation in all samples.
The IHC procedure is of interest to pathologists because they used a intercalated antibody-enhanced polymer (iAEP) technique (Takeuchi K et al. Clin Cancer Res 2009;15: 3143–9) which incorporates an intercalating antibody between the primary antibody to ALK and dextran polymer–based detection reagents. They used a primary antibody specific for the intracellular region of ALK (5A4, Abcam) which is not a ALK fusion mutation specific antibody.
Adenocarcinoma was identified in 75% of cases and 20% of patients were never smokers. A mean of 2.1 lymph nodes were sampled per patient and the median size of the sampled nodes was 12 mm. Six out of 109 cases examined by IHC were ALK immunopositive; 17 were categorized as "suspicious," including 1 "probably positive" case. FISH confirmed the presence of the ALK fusion gene in all size cases but the "probably positive" case had too few cells for FISH analysis. However, RT-PCR confirmed the presence of EML4-ALK in all six IHC-positive cases as well as the "probably positive" case for a total of 7 cases positive for the ALK fusion gene (6.4% of all cases). EGFR mutation was detected in 25 cases (22.4%).
All ALK-positive cases had an adenocarcinoma histology and lacked EGFR gene mutations. Six out of the seven ALK-positive cases were either never-smokers or light smokers. No significant difference in gender was observed between ALK-positive and ALK-negative patients. Interestingly, the mean primary tumor diameter of ALK-positive cases was significantly smaller than that of ALK-negative cases (28.6 mm versus 41.9 mm; P < 0.05).
If these methods and findings can be replicated, EBUS-TBNA with multiplex gene mutation testing could have a major effect on the diagnosis, staging and management of lung cancer. In the more short-term future, the use of immunohistochemistry with iAEP technique that would include using a highly sensitive mutation-specific antibody would have a major impact on testing for ALK since FISH testing is technically difficult due to the close proximity of the two fusion gene components.
http://pathlabmed.typepad.com/surgical_pathology_and_la/2010/11/detecting-alk-in-endobronchial.html
The protocol for this study involved chest CT followed by EBUS-TBNA for lymph nodes >5 mm. They obtained a histologic core using a 22-gauge needle and divided the specimen into two parts with one part placed in 20% (!) formalin and the other half placed in a cryopreservative solution and then frozen at −80℃. Histological examination was performed in all cases. The authors present a triage for EML4-ALK fusion gene mutation including screening by IHC, followed by FISH, and then final confirmation with RT-PCR. They also tested for EGFR gene mutation in all samples.
The IHC procedure is of interest to pathologists because they used a intercalated antibody-enhanced polymer (iAEP) technique (Takeuchi K et al. Clin Cancer Res 2009;15: 3143–9) which incorporates an intercalating antibody between the primary antibody to ALK and dextran polymer–based detection reagents. They used a primary antibody specific for the intracellular region of ALK (5A4, Abcam) which is not a ALK fusion mutation specific antibody.
Adenocarcinoma was identified in 75% of cases and 20% of patients were never smokers. A mean of 2.1 lymph nodes were sampled per patient and the median size of the sampled nodes was 12 mm. Six out of 109 cases examined by IHC were ALK immunopositive; 17 were categorized as "suspicious," including 1 "probably positive" case. FISH confirmed the presence of the ALK fusion gene in all size cases but the "probably positive" case had too few cells for FISH analysis. However, RT-PCR confirmed the presence of EML4-ALK in all six IHC-positive cases as well as the "probably positive" case for a total of 7 cases positive for the ALK fusion gene (6.4% of all cases). EGFR mutation was detected in 25 cases (22.4%).
All ALK-positive cases had an adenocarcinoma histology and lacked EGFR gene mutations. Six out of the seven ALK-positive cases were either never-smokers or light smokers. No significant difference in gender was observed between ALK-positive and ALK-negative patients. Interestingly, the mean primary tumor diameter of ALK-positive cases was significantly smaller than that of ALK-negative cases (28.6 mm versus 41.9 mm; P < 0.05).
If these methods and findings can be replicated, EBUS-TBNA with multiplex gene mutation testing could have a major effect on the diagnosis, staging and management of lung cancer. In the more short-term future, the use of immunohistochemistry with iAEP technique that would include using a highly sensitive mutation-specific antibody would have a major impact on testing for ALK since FISH testing is technically difficult due to the close proximity of the two fusion gene components.
http://pathlabmed.typepad.com/surgical_pathology_and_la/2010/11/detecting-alk-in-endobronchial.html
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