Results
ALK Fluorescence in Situ Hybridization
One hundred and ninety-six lung ADCs were included in this study, consisting of 108 female and 88 male patients. The mean age is 57.4 years (range, 24–81 years). Of these, 63 (32%, of 196) demonstrated an ALK rearrangement (FISH positive), and 133 cases were FISH negative (Figure 1). The FISH-positive cases included 35 women and 28 men with mean age of 52.5 years (range, 24–76 years). On FISH examination, split pattern was observed in 45 cases (71%) and unbalanced rearrangement, characterized by a loss of the 5' probe, was shown in 18 cases (29%). Twelve cases (6%, of 196) showed average green or orange signals of more than three per cell.
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Figure 1.
Detection of ALK fusion in lung cancer patients by immunohistochemistry (IHC), FISH, and real-time RT–PCR assays. (A–D) Semiquantitative IHC staining using an antibody to ALK (D5F3 by Cell signaling [Billerica, MA] with Ventana Untraview DAB detection kit). (A) score 0/negative showing no staining; (B) score 1+ showing faint cytoplasmic reactivity without any background staining; (C) score 2+ showing moderate smooth cytoplasmic staining; and (D) score 3+ showing intense granular cytoplasmic staining in ≥10% of tumor cells. Original magnification ×200. (E–H) Ventana IHC assay revealed strong expression of ALK in all ALK-fusion-positive patients and no expression in ALK-fusion-negative patient. Original magnification ×200. (I–L) FISH carried out with Vysis LSI ALK Dual color Break-Apart FISH probes detected ALK fusion as split red and green signals. (I) FISH-negative case showing intact two fused signals per nucleus. (J and K) FISH-positive cases representing split (red and green) signals. (L) FISH-positive case demonstrating isolated red signal. Original magnification ×1000. (M–P) Graphs from real-time RT–PCR showing change in the normalized reporter signal (delta Rn) against PCR cycle number for reaction 1 of AmoyDx EML4-ALK fusion gene detection kit.
ALK Immunohistochemistry
All patients with CST by Ultraview IHC score of 2+ or 3+ (n = 50) were Ventana IHC positive and all patients with score of 0 (n = 126) were Ventana IHC negative. Among patients with CST IHC score of 1+, 5 (of 20) were Ventana IHC negative.
ALK Real-time RT–PCR
Using real-time RT–PCR, 69 cases (35%, of 196) were identified to have EML4-ALK rearrangement, including 52 cases of EML4-ALK fusion variants 1/2/3a/b, and 2 cases of variants 5a/5b/5'/8. Fifteen cases were both positive for Variants 1/2/3a/b and variants 5a/5b/5'/8.
correlation of ALK FISH, IHC, and Real-time RT–PCR
As shown in supplementary Table S1, available at Annals of Oncology online, of 50 patients with CST by Ultraview IHC score of 2+ or 3+, 48 (96%) were FISH positive and all patients with score of 0 (n = 126) were FISH negative. Among patients with CST IHC score of 1+, 15 (75.0%, 15 of 20) were FISH positive. The sensitivity and specificity of ALK CST IHC with staining intensity score of 1+ or more were 100% and 95%, respectively. The sensitivity and specificity of ALK CST IHC with staining intensity score of 2+ or more were 76% and 98%, respectively.
Of 65 patients with Ventana IHC ALK positive, 63 (97%) were FISH positive and all patients with IHC ALK negative were FISH negative. The sensitivity and specificity of ALK Ventana IHC were 100% and 98%, respectively.
Sixty-two (90%, of 69) patients detected as positive by RT–PCR were FISH positive, while 126 (99%, of 127) patients with ALK negative by RT–PCR were FISH negative. The sensitivity and specificity of RT–PCR for detection of ALK fusion were 98% and 95%, respectively.
Of 69 ALK RT–PCR-positive patients, 64 (93%) were Ventana IHC positive, while 126 (99%, of 127) ALK RT–PCR-negative cases were Ventana IHC negative (supplementary Table S2, available at Annals of Oncology online). The total accordance rate between ALK RT–PCR and Ventana IHC was 97%.
The details of discrepant cases for ALK fusion detection among IHC, FISH, and RT–PCR were shown in supplementary Figure S1 and Table S3, available at Annals of Oncology online).