Discussion
Neuroblastic tumors are subjected to risk stratification for management
purposes and have an overall poor prognosis despite the extensive
multimodal therapies highlighting the need for discovery of alternative
therapeutic targets. 4,5,18 One such target is the
Anaplastic Lymphoma Kinase or ALK gene which has been the subject
of intense research in neuroblastomas and reviewed by several authors
recently7,19 This study was a small step to establish
the expression of ALK protein in samples obtained by fine needle
aspiration biopsy and evaluate its role in prognostication and risk
stratification. We further evaluated ALK gene mutations in a subset of
cases by NGS on cell scrapes.
ALK protein is a receptor tyrosine kinase that has an expression
restricted to the developing central and peripheral nervous system and
is critical for embryonal neuronogenesis in the sympathetic ganglia and
tumors showing ALK overexpression referred to as ALKomas might benefit
from directed therapies.20 ALK protein expression in
tissues may be evaluated by immunohistochemistry. There are many
antibodies commercially available and a comparative analysis of various
antibody clones revealed the high sensitivity of D5F3 clone, justifying
its choice in this study.13 In this study, ALK
expression was evaluated on adequately cellular cell blocks from FNA
samples and 65% (35/54) cases showed positivity. The heterogeneity of
ALK expression in most cases necessitated H-scoring which allowed the
selection of strongly positive cases even if heterogenous and excluded
the weak positives. There are a few studies that have evaluated ALK
protein in histopathology sections including tissue microarrays with a
frequency of 41-66% which is in agreement with our
observations. 11,13,14 Our results on FNA cell blocks
are comparable to studies on conventional histopathology specimens and
thus it may be concluded that cell blocks are an acceptable substitute
for evaluating ALK expression by immunocytochemistry.
ALK gene mutations in neuroblastoma is well
studied.19,21,22 There is however no report from India
and from cytology samples. Tumour DNA was extracted from MGG stained
representative and cellular smears, a novel aspect of the study. The use
of smears for molecular studies including NGS has been highlighted
previously.23 Out of 21 cases evaluated, 3
pathogenic/likely pathogenic mutations were observed, two in ALKgene and one in the IDH1 gene. Both ALK mutations resulted
in F1174L, which is the most frequent oncogenic driver mutation seen in
12.4% cases of sporadic neuroblastomas.8,24 Bresler
et al in their comprehensive genomic study of ALK mutations in
1596 samples revealed tyrosine kinase domain mutations in 8% cases
involving 3 hotspots R1275Q, F1174 and F1245 accounting for 43%, 30%
and 12% of cases respectively.21 The F1174 was
associated with an aggressive tumor phenotype as was observed in our
cases too.
An interesting aspect of neuroblastoma is the chromosome 2p, which is
home to two important oncogenes, MYCN at 2p24 and ALK at
2p23.2. In our study, MYCN amplification by FISH did not
correlate with ALK protein expression (p=0.35). However,
immunohistochemistry for MYCN protein was significantly associated with
ALK protein expression.14 A meta-analysis of
neuroblastomas comparing mutations in the ALK gene mutation
spectrum with MYCN amplification status found ALKmutations occur in equal frequencies across all genomic subtypes, but
the F1174L mutants were observed in a higher frequency inMYCN -amplified tumors, demonstrated higher transforming capacity
resulting in more aggressive tumors.25 Interestingly
the ALK F1178L knock-in mice (corresponding to
F1174L in humans) resulted in enhanced proliferation and extended
neurogenesis but no tumor formation in the absence ofMYCN co-expression.26 In another study on a
mouse model, a co-operative interaction ofALKF1174L/MYCN led to aggressive and lethal
tumors with activation of the PI3K/AKT/mTOR and MAPK
pathways.27
Out of the 50 patients with complete follow-up in this study, 28
patients (56%) died due to disease progression and 22 patients (44%)
were alive without disease. In spite of this, in this small cohort of 60
patients, INRG stratification into high, intermediate, and low-risk
groups had a significant effect on outcome (p=0.02) and overall survival
(p=0.0001), similar to previous reports.28,29 ALK
expression did not correlate with the age of patient, morphological
type, stage (stage 4 vs non-stage 4), MYCN status, and INRG
group. We chose a cut-off of an H-score of 100 for statistical analysis.
Those less than 100 represented negative or with low levels of
expression. ALK protein expression independently had no correlation with
outcome and overall survival which is in concordance with a previous
report by Lee et al11 and De Brouwer et
al25. Hence, we evaluated the effect of various
parameters in the ALK positive cases wherein morphology correlated well
with outcome (p=0.01) and overall survival (p=0.02) with poorly
differentiated having a better overall survival than undifferentiated
neuroblastoma. Our pilot observations need confirmation in larger study
cohorts. When risk stratification was applied only to ALK positive
tumors, significant results towards overall survival were observed
(p=0.03). Weiser et al found a significantly higher number ofALK mutations in high-risk versus low-risk cases (p=0.01) but
there were no significant results with age or histology similar to our
study.30 On the other hand, Carpenter et al reported a
correlation of increased ALK mRNA with poor
prognosis.19 In vitro studies of ALK
signaling in cultured neuroblasts resulted in transient proliferation
followed by differentiation and long-term survival of post-mitotic
neurons.26 A novel IDH1 gene missense mutation
was observed in a single case. IDH1 gene mutations are common in
gliomas and other tumors of the CNS and hemato-lymphoid
malignancies.31 Functional in vitro studies on cell
lines or mouse models are required to elucidate its role in
neuroblastoma.
In view of the complex interplay of so many variables for risk
stratification in neuroblastoma, all parameters which significantly
correlated with outcome such as age, stage, cytomorphology were
evaluated along with ALK expression in a multivariate Cox proportional
hazard model which gave significant results (p= 0.04). A similar
analysis substituting ALK with MYCN was performed which was not
statistically significant. Our pilot study data suggests that while ALK
is not an independent prognostic factor, that it may have a role along
with other traditional prognostic factors with ALK negativity predicting
a worse outcome (Hazard Ratio, 2.3). A subset of poorly differentiated
neuroblastomas who are ALK positive have a good survival and would
otherwise would have been categorized as having a poor prognosis based
on traditional parameters. Our data is limited due to the small cohort
size and a potential inclusion bias of advanced cases which are
subjected to FNAB. However, these observations need validation on
studies with larger number of cases with better statistical power.
On the other hand, ALK has emerged as a potential predictive therapeutic
biomarker and an ideal candidate for targeted therapy in neuroblastoma
due to the dismal prognostic scenario.32 Several ALK
inhibitors are in various phases of drug development and scrutiny in
neuroblastoma and have shown promising results. Apart from crizotinib,
other ALK inhibitors like lorlatinib, brigatinib, ceritinib, alectinib,
entrectinib are undergoing clinical trials along with a combination of
TRKA/B/C, MDM2, and CDK4/6 inhibitors and these are reviewed by Pacenta
and Macy.33 These trials offer a ray of hope in
neuroblastoma, a tumor that remains a challenge for the pediatric
oncologist. Hence detection of ALK protein expression levels in any
sample of neuroblastoma assumes significance.