Platform Comparison for Evaluation of ALK Protein Immunohistochemical Expression, Genomic Copy Number and Hotspot Mutation Status in Neuroblastomas

<div><p>ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC) protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones), <i>ALK</i> genomic status using single-color chromogenic in situ hybridization (CISH), and <i>ALK</i> hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM)), in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with <i>ALK</i> genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either <i>ALK</i> genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.</p></div

ALK CISH and IHC of an <i>ALK</i> amplified case.

<p>(A) CISH (B) ALK1 IHC (C) 5A4 IHC (D) D5F3 IHC.</p

<i>ALK</i> mutations detected by both Sanger sequencing and IT-PGM.

<p><i>ALK</i> mutations detected by both Sanger sequencing and IT-PGM.</p

Correlation between ALK D5F3 IHC score and mutation status.

<p><i>p</i> (Fisher's exact test)  = 0.004.</p><p>Correlation between ALK D5F3 IHC score and mutation status.</p

Depth of coverage and GC content.

<p>(A) The red dashed line indicates the GC content. The X-axis shows the amplicons ordered with increasing depth of coverage from left to right. (B) There is a negative correlation between depth of coverage and GC content; this is not statistically significant (R² = 0.1).</p

Correlation between D5F3 IHC and <i>ALK</i> CISH (N = 91).

<p><i>p</i> (Fisher's exact test)  = 0.143.</p><p>Correlation between D5F3 IHC and <i>ALK</i> CISH (N = 91).</p

ALK CISH and IHC of an <i>ALK</i> non-amplified case.

<p>(A) CISH (B) ALK1 IHC (C) 5A4 IHC (D) D5F3 IHC.</p

Depth of coverage of amplicons from 5′ to 3′.

<p>The p.F1174 and p.R1275 hotspots are covered by the amplicons marked with a red and purple arrow respectively.</p

Sample type and ALK immunohistochemical expression (N = 105).

<p>Sample type and ALK immunohistochemical expression (N = 105).</p