Immunocytochemistry (ICC):
In all cases with undifferentiated morphology, a suitable panel was
applied for confirmation of neuroblastoma (positivity for neuronal
markers like chromogranin, synaptophysin, and PHOX2B) and its
differentiation from other small round blue cell tumors (negativity for
other markers like CD45, CD99/MIC2, Vimentin, desmin, myogenin). ICC for
ALK protein was performed on cell blocks with adequate cellularity. The
recommended staining protocol for VENTANA anti-ALK (D5F3 clone) CDx
Assay and Rabbit Monoclonal Negative Control Ig with Opti View DAB IHC
Detection Kit and Opti View Amplification Kit on a Benchmark XT
instrument was followed. Positive control for
immunocytochemistry was taken as section of appendix/colon with ganglion
cells. ICC scoring was carried out using H-score wherein the tumor cell
percentage positivity (in increments of 10) with 0, 1+, 2+ and 3+
intensity was evaluated. The percent positivity multiplied by the
intensity was added up to obtain the final H-score as follows: H score=
(0x % positive) + (1+x % positive) + (2+x % positive) + (3+x %
positive). A cut-off of 100 was taken for statistical analysis with a
score>100 being moderate to high ALK expression.