Assay for ALK Rearrangement in Primary Lung Adenocarcinoma
Assay for ALK Rearrangement in Primary Lung Adenocarcinoma
All included patients had received curative surgery at the Cancer Hospital, Chinese Academy of Medical Sciences (CAMS), Beijing, China, between August 2011 and July 2012. All these tumor samples were fixed in 10% neutral buffered formalin for 24–48 h and embedded in paraffin and routinely diagnosed as primary lung ADC. All these samples were previously screened for ALK fusion by FISH, including 63 ALK-positive and 133 ALK-negative cases. The study protocol was approved by the Institute Review Board of the Cancer Hospital, CAMS. Tissue microarray blocks were built to perform IHC and FISH assays (for more details, see the supplementary Appendix, available at Annals of Oncology online).
FISH analysis was carried out with the Vysis LSI ALK Dual color, Break Apart Rearrangement Pobe (Vysis/Abbott, Abbott Park, IL) according to the manufacturer's instructions. Four cases that failed to get satisfactory signals on TMA were tested further on whole sections of the corresponding tumor blocks by FISH.
Two automated IHC assays were carried out in this study.
One assay (CST IHC) used Rabbit monoclonal ALK antibody (clone D5F3, Cell Signaling Technology) diluted 1 : 50 using a Ventana Ultraview DAB detection kit in a Ventana Benchmark XT stainer (Ventana Medical Systems, Tucson, AZ). ALK IHC was scored using the scoring scheme proposed as follows: 0, no staining; 1+, faint cytoplasmic reactivity without any background staining; 2+, moderate cytoplasmic reactivity; and 3+, granular cytoplasmic reactivity of strong intensity in ≥10% of tumor cells.
The other assay (Ventana IHC) was a fully automated IHC assay developed by Ventana recently, using the pre-diluted Ventana anti-ALK (D5F3) Rabbit monoclonal primary antibody, together with the Optiview DAB IHC detection kit and Optiview Amplification kit on the Benchmark XT stainer. Each case was also stained with a matched Rabbit Monoclonal Negative Control Ig antibody. According to the manufacture's scoring algorithm, binary scoring system (positive or negative for ALK status) was adopted for evaluating the staining results. Neoplastic cells labeled with the ALK IHC assay are evaluated for presence or absence of the DAB signal. Presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) was deemed to be ALK positive, while absence of strong granular cytoplasmic staining in tumor cells was deemed to be ALK negative.
The EML4-ALK fusion mRNA was readily detected by PCR using AmoyDx EML4-ALK Fusion Gene Detection Kit (Amoy Diagnostics, Xiamen, China) according to manufacturer's instruction (see the supplementary Appendix, available at Annals of Oncology online, for more details). The amplified PCR products from some samples were subjected to direct sequencing, using AB3500xl DNA Sequencer (Applied Biosystems).
Patients and Methods
Patients
All included patients had received curative surgery at the Cancer Hospital, Chinese Academy of Medical Sciences (CAMS), Beijing, China, between August 2011 and July 2012. All these tumor samples were fixed in 10% neutral buffered formalin for 24–48 h and embedded in paraffin and routinely diagnosed as primary lung ADC. All these samples were previously screened for ALK fusion by FISH, including 63 ALK-positive and 133 ALK-negative cases. The study protocol was approved by the Institute Review Board of the Cancer Hospital, CAMS. Tissue microarray blocks were built to perform IHC and FISH assays (for more details, see the supplementary Appendix, available at Annals of Oncology online).
Fluorescence in Situ Hybridization
FISH analysis was carried out with the Vysis LSI ALK Dual color, Break Apart Rearrangement Pobe (Vysis/Abbott, Abbott Park, IL) according to the manufacturer's instructions. Four cases that failed to get satisfactory signals on TMA were tested further on whole sections of the corresponding tumor blocks by FISH.
Immunohistochemistry
Two automated IHC assays were carried out in this study.
One assay (CST IHC) used Rabbit monoclonal ALK antibody (clone D5F3, Cell Signaling Technology) diluted 1 : 50 using a Ventana Ultraview DAB detection kit in a Ventana Benchmark XT stainer (Ventana Medical Systems, Tucson, AZ). ALK IHC was scored using the scoring scheme proposed as follows: 0, no staining; 1+, faint cytoplasmic reactivity without any background staining; 2+, moderate cytoplasmic reactivity; and 3+, granular cytoplasmic reactivity of strong intensity in ≥10% of tumor cells.
The other assay (Ventana IHC) was a fully automated IHC assay developed by Ventana recently, using the pre-diluted Ventana anti-ALK (D5F3) Rabbit monoclonal primary antibody, together with the Optiview DAB IHC detection kit and Optiview Amplification kit on the Benchmark XT stainer. Each case was also stained with a matched Rabbit Monoclonal Negative Control Ig antibody. According to the manufacture's scoring algorithm, binary scoring system (positive or negative for ALK status) was adopted for evaluating the staining results. Neoplastic cells labeled with the ALK IHC assay are evaluated for presence or absence of the DAB signal. Presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) was deemed to be ALK positive, while absence of strong granular cytoplasmic staining in tumor cells was deemed to be ALK negative.
Real-time RT–PCR
The EML4-ALK fusion mRNA was readily detected by PCR using AmoyDx EML4-ALK Fusion Gene Detection Kit (Amoy Diagnostics, Xiamen, China) according to manufacturer's instruction (see the supplementary Appendix, available at Annals of Oncology online, for more details). The amplified PCR products from some samples were subjected to direct sequencing, using AB3500xl DNA Sequencer (Applied Biosystems).
