Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry.

BACKGROUND: Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. METHODS: Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. RESULTS: All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. CONCLUSIONS: The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations

Immunostaining pattern of ALK in NSCLC using Ventana anti-ALK (D5F3) and Novocastra (5A4) antibodies.

<p>ALK IHC reveals variable levels of protein expression: from absent (0) to weak/faint cytoplasmic staining (1+) in negative cases and from moderate (2+) to strong (3+) granular cytoplasmic immunstaining in positive tumors. In ALK IHC-negative cases, the immunoreactivity was always 0 by Novocastra (5A4) IHC, whereas ranged from 0 to 1+ by Ventana antibody. However, in ALK IHC-positive cases, protein expression was always 3+ by Ventana antibody, whereas it ranged from 2+ to 3+ by Novocastra (5A4) IHC. Original magnification: 400×.</p

Study design and specimen selection.

<p>Study design and specimen selection.</p

Box plots for number of <i>ALK</i> positive cells by FISH automatized technique versus intensity of the ALK IHC staining.

<p>With the Ventana anti-ALK antibody (A) and with Novocastra (5A4) antibody (B). Kruskal-Wallis test was performed. The comparisons between the categories in each antibody were statistically significant (p<0,001).</p

Concordance between ALK IHC and <i>ALK</i> FISH.

<p><i>IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.</i></p><p>Concordance between ALK IHC and <i>ALK</i> FISH.</p

Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

BackgroundBased on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples.MethodsForty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system.ResultsAll positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively.ConclusionsThe specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations