Detection of ALK Gene Rearrangements in Non-Small Cell Lung Cancer by Immunocytochemistry and Fluorescence in Situ Hybridization on Cytologic Samples
Suneel RACHAGIRI1, Parikshaa GUPTA2, Nalini GUPTA2, Manish ROHILLA2, Navneet SINGH3, Arvind RAJWANSHI2
1Department of Pathology, Post Graduate Institute of Medical Education and Research, CHANDIGARH, INDIA
2Department of Cytology and Gynaecologic Pathology, Post Graduate Institute of Medical Education and Research, CHANDIGARH, INDIA
3Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research, CHANDIGARH, INDIA
Keywords: Lung adenocarcinoma, Non-small cell lung carcinoma, Anaplastic lymphoma kinase, ALK rearrangement, Fluorescence in situ hybridization, Immunocytochemistry, D5F3
Determination of the molecular status is mandatory for personalized treatment of patients with non-small cell lung carcinoma. The
present study was performed to detect anaplastic lymphoma kinase (ALK) rearrangements in pulmonary adenocarcinoma on cytology samples,
using immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH) on cell-blocks to assess the diagnostic reliability of these two
Material and Method: A total of 50 confirmed lung adenocarcinoma cases were included. In all the 50 cases, ICC was performed for ALK
protein expression by using the D5F3 clone on Ventana platform. On the basis of ALK protein expression on ICC, the cases were categorized as
ALK positive (2+ or 3+ strong cytoplasmic granular positivity) or negative (negative or 1+ cytoplasmic granular positivity). FISH for detection
of ALK gene rearrangement was performed in 7 ALK ICC positive cases and 7 ALK ICC negative cases using the Vysis ALK break apart FISH
Results: Based on ICC, 7(14%) cases were ALK positive and 43(86%) were ALK negative. ALK gene rearrangements in lung adenocarcinoma
were more commonly seen in non-smokers (31.25%) as compared to smokers (6.25%). Among the ALK-ICC positive cases, FISH demonstrated
break apart signal in 5 cases (ALK- ICC positive); however, no break-apart signals were seen in 2 ALK-ICC positive and all the seven ALK-ICC
Conclusion: Immunocytochemistry on cell- blocks using DF53 clone is a highly sensitive and specific method for the detection of ALK gene
rearrangements in lung adenocarcinoma with a greater number of ALK positive cases being detected on ICC as compared to the ALK-FISH.
The treatment for non-small cell lung carcinoma (NSCLC)
has become personalized with the advancements in
molecular pathology and identification of specific
therapeutic target molecules 1
. A variety of molecular
abnormalities have been recognized in lung cancer
including mutations in Kirsten rat sarcoma viral oncogene
homolog (KRAS), epidermal growth factor receptor
(EGFR), BRAF, MEK and HER2 and phosphatidylinositol
3-kinase (PI3K) pathway oncogenes. ALK, ROS1 and RET
showed structural rearrangements which come up with
novel therapeutic targets. MET and fibroblast growth
factor receptor 1 (FGFR1) amplification is noted in
adenocarcinoma and SCC respectively 2
. EGFR mutations
are seen in around 32.3% of lung adenocarcinoma cases
. EGFR mutations, like, point mutations in exons, and
21 and exon 19 deletions, are associated with a dramatic therapeutic response to EGFR tyrosine kinase inhibitors
. The molecular methods used for detection of
EGFR mutations include Sanger sequencing (SS), Next
generation sequencing (NGS) and polymerase chain
reaction-based methods 5
Anaplastic lymphoma kinase (ALK), is a tyrosine
kinase receptor, encoded by the ALK gene. ALK gene
rearrangements are seen in 1.9-6.8% cases of NSCLCs 6.
The most common genetic rearrangement involves the
echinoderm microtubule associated protein-like 4 (EML4)
and ALK leading to formation of the EML4-ALK fusion
gene that encodes for a chimeric protein with intrinsic
tyrosine kinase activity. However, fusion genes involving
other partners have also been detected. Identification of
the ALK gene rearrangement is a mandatory diagnostic
test for NSCLC patients 7,8. This is mainly owing to
the availability of effective ALK-inhibitors like crizotinib, alectinib, and ceritinib, which lead to good therapeutic
response and better five-year survival rates as compared
to the standard chemotherapy regimens 9. Currently,
three methods are available for detecting ALK gene
rearrangements: fluorescence in situ hybridization (FISH),
immunohistochemistry (IHC) and real-time PCR (RTPCR).
FISH has been considered the gold standard method
for detecting ALK gene rearranged NSCLC. However,
the recent guidelines recommend that IHC, using FDA
approved antibodies, is an equivalent alternative for ALK
testing 8. Although ALK testing is frequently performed
on histopathological tissues, testing using cytologic
samples is sparsely documented. The present study was
undertaken to detect ALK gene rearrangements by using
immunocytochemistry (ICC) and the FISH technique on
cell-blocks, in cases diagnosed as lung adenocarcinoma on
This was a one-year prospective study performed on a
total of 50 lung adenocarcinoma (ADC) cases diagnosed
on fine needle aspiration cytology (FNAC) or pleural fluid
cytology. The study was approved by the Institute Ethics
Committee (NK/4423/MD). The objectives were to detect
ALK gene rearrangements by immunocytochemical (ICC)
staining using the D5F3 clone and FISH technique on cellblocks
and to compare the clinicopathologic characteristics
amongst the ALK positive and ALK negative cases.
Direct and/or sediment smears were prepared from
cytologic samples (both air-dried and 95% ethanol-fixed)
and rest of the cytologic material was rinsed into a glass tube containing 1 ml of 1% ammonium oxalate for cellblock
preparation. The air-dried smears were stained with
May Grunwald Giemsa (MGG) and the wet-fixed smears
with haematoxylin and eosin (H&E) and/or Papanicolaou
stain. The cell-blocks were prepared using an already
standardized plasma clot method. ICC was performed on
the cell-blocks, wherever needed, to subtype the tumors
using TTF1, p40, CK7 and Napsin A.
ALK IHC using D5F3 clone
The Ventana ALK (D5F3) CDx assay was used for the
detection of ALK protein expression as a surrogate marker
for ALK gene rearrangements. The strength of cytoplasmic
granular positivity was graded as 3+ (strong positivity in
>90% tumor cells); 2+ (moderate cytoplasmic granular
positivity in 20-90% tumor cells); 1+ faint cytoplasmic
positivity in <20% cells; and 0 (negative for cytoplasmic
ALK gene rearrangement by FISH
FISH was performed for detection of ALK gene rearrangements
in ICC- positive and equal number of randomly
selected ICC- negative cases. FISH was performed
using the Vysis ALK break apart FISH probe kit (Abbott
Molecular). Fluorescence signals (ALK 5’ probe (Spectrum
Green) and the ALK 3’ probe (Spectrum Orange)) were
recorded after viewing under fluorescence microscope
(Olympus WX63 Epi-illumination fluorescence microscope).
At least 50 tumor cell nuclei were evaluated for
each case and the positivity was taken as nuclei showing
split signals or deleted signals (presence of single orange
signal) (Table I). A tumor was interpreted as negative if less than 5 out of 50 tumor cells (<5/50 or <10%) were positive.
A tumor was interpreted as positive if 25 cells out of 50
tumor cells (>25/50 or >50%) were positive. A tumor was
interpreted as equivocal if 5-25 cells (10 to 50%) were positive.
For equivocal cases, a second unbiased evaluation of
slide by another cytopathologist was performed, following
which both the first and second cell counts/readings were
added together and a final percentage was calculated for
100 cells. If the average percentage of the positive cells was
<15% (<15 positive nuclei/100 evaluated tumor nuclei), the
sample was interpreted as negative. However, if the average
percentage of the positive tumor cells was >15% (>15 positive
nuclei/100 tumor cell nuclei evaluated), the sample was
interpreted as positive.
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|Table I: Various signal patterns observed on fluorescence in situ hybridization and their interpretations
For data analysis, SPSS (version 22.0) software was used.
The Shapiro-Wilk test was applied to check the normality
of continuous data like age. For normally distributed
data, mean and SD were reported. Categorical variables
like gender, smoking status, pathologic diagnoses, stage,
etc. were reported as frequency and percentage. The
independent t-test was used to compare the mean of
normally distributed quantitative variables between two groups (ALK IHC positive and negative). The chi square/
Fisher’s Exact test was applied to find out any association
between categorical variables and the study groups. A p
value of < 0.05 was taken as significant.
A total of 50 primary lung adenocarcinoma diagnosed
on the basis of cytomorphology and appropriate panel of
immunocytochemical markers (TTF1, p40, Napsin A and
CK7), were included in the study. Of these, 17 cases were
reported as adenocarcinoma and 10 as NSCLC, favouring
adenocarcinoma on FNAC. Another 23 cases were reported
as metastatic lung adenocarcinoma in pleural effusion
samples (Figure 1A-D
). The age of the patients ranged from
28-82 years with the mean age being 57.5 years (standard
deviation=11.1). The male:female ratio was 1.6:1 with 31
males and 19 females. The lesions were more common
in the right lung (n=36) than the left lung (n=14) and the
upper lobe was more commonly involved as compared to
the lower lobe of the lung. The majority of the cases (n=23;
48%) in the present study were in TNM stage IV with 14
cases having evidence of extra-thoracic metastatic disease,
mostly to the central nervous system and bone.
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|Figure 1: A,B) Pleural fluid smears showing papillary, three dimensional clusters of tumor cells with moderate nuclear pleomorphism,
vesicular chromatin, prominent nucleoli and moderate to abundant amount of vacuolated cytoplasm (a: MGG, 40x; b: Papanicolaou, 20x);
C,D) Fine needle aspiration smears showing loose clusters of tumor cells with moderate nuclear pleomorphism, vesicular chromatin,
prominent nucleoli and moderate cytoplasm (c: MGG, 40x; b: H&E, 40x); E,F) Sections from the cell-blocks prepared from malignant
pleural effusions showing acinar as well as papillary arrangement of the tumor cells (H&E; 40x)
Detection of ALK gene rearrangements by ICC
Seven (14%) cases showed cytoplasmic granular positivity
for ALK antibody (D5F3 clone). Based on the staining
intensity, 5 (71.4%) cases were categorized as 3+ ALK
positive and two (28.6%) cases were categorized as 2+
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|Figure 2: Panel of photomicrographs showing variable intensities of ALK immunocytochemistry (D5F3 clone) on cell-block sections:
A) Weak and fine granular cytoplasmic staining (1+) in the tumor cells; B) Faint, coarse to fine granular cytoplasmic staining (2+) in
the tumor cells; C) Strong, coarse granular cytoplasmic staining (3+) in the tumor cells; D) Section from the appendix (control tissue)
showing coarse granular cytoplasmic staining (3+) in the ganglion cells (20x)
Clinicopathologic parameters were compared amongst
ALK positive and ALK negative groups on ICC. ALK gene
rearrangements were more frequently seen in females
(4/7 (57.1%) cases being ALK positive) as compared to
males (3/7 (42.9%) cases being ALK positive); however,
this difference was not found to be statistically significant
(p=0.08). The mean age of the patients in the ALK positive
group (56 years) as compared to that of ALK negative group
(59 years) was also not statistically significant (p=0.6).
Out of 48 cases with known smoking status, 32 (66.6%)
were smokers and 2/32 (6.25%) cases were ALK positive.
Among smokers, 26 (81.2%) were males and 6 (18.7%) were
females. Among 16 (33.3%) non-smokers, 5/16 (31.25%)
were ALK positive. It was seen that non-smokers were
significantly associated with ALK gene rearrangements
(p=0.03). Furthermore, it was more commonly detected
in females with non-smoking status; however, this was not
statistically significant (p=0.08).
Pleural effusion was noted in 20 (41.7%) cases; however,
the presence of pleural effusion was not found to be
significantly associated with ALK gene rearrangements
(p=0.10). ALK positivity was seen more commonly in cases
reported as adenocarcinoma as compared to cases reported
as NSCLC, favour adenocarcinoma; however, this was not statistically significant (p=0.36) (Table II). Furthermore,
ALK gene rearrangements were seen in cases having focal
solid and acinar (n=5; 71.4%), and papillary (n=2; 28.5%)
architecture (Figure 1E,F).
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|Table II: Distribution of various clinicopathological parameters among the ALK immunocytochemistry (ICC) and fluorescence in situ
hybridization (FISH) positive and negative cases in the present study
In addition, EGFR gene mutational analysis was performed
by real time polymerase chain reaction in 46 (92%) cases
and no case in the ALK positive group showed known
mutations in the exons 18, 19, 20 and 21 of EGFR gene,
reiterating the mutually exclusive existence of these genetic
Two out of seven ALK positive cases received Crizotinib or
Ceritinib. One patient had progression of the disease and
the other one showed a partial response.
Detection of ALK gene rearrangements by FISH
FISH testing for ALK gene rearrangements using the Vysis
ALK break-apart FISH probe kit (Abbott Molecular) was
performed in a total of 14 randomly selected cases (7 ALK
ICC positive cases and 7 ALK ICC negative cases). ALK
rearrangements by the FISH technique could be detected
in 5/7 (71.4%) cases, which were positive on ALK ICC.
Among the ALK-FISH positive cases, the mean percentage
of ALK-FISH positive rearranged nuclei was 79.25% (range
67-91%). FISH positive cases showed presence of split
signal pattern (n=3) and combined 3’ deletion and split
signal pattern (n=2) (Figure 3A-D). In addition, all the 7
ALK ICC negative cases were negative on ALK testing by
FISH, indicating a good concordance between ICC and
FISH (Figure 4A-C).
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|Figure 3: Panel of photomicrographs
showing tumor cells with ALK
rearrangement on FISH: A) Two tumor
nuclei have a single orange signal
(deleted green signal) in addition to
fused signal; B-D) The tumor nuclei
contain rearranged or “broken-apart”
signals and fused signals.
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|Figure 4: Panel of photomicrographs showing tumor cells negative for ALK rearrangement on FISH: A) Three tumor cells with nonrearranged
signals, in the form of fused orange and green signals. Two cells show two fused signals (white arrows) and one cell shows
three fused signals (red arrows), indicating a triploid tumour cell; B) A cell with a single green signal without a corresponding orange
signal, in addition to a fused signal indicates a deletion of the orange portion of the ALK probe (considered negative); C) Non-rearranged
cell with orange and green signals, which are less than two signal diameters apart.
ALK gene rearrangements are seen in 1.9-6.8% cases of
. The most common ALK gene rearrangement in NSCLC is paracentric inversion on the short arm
of chromosome 2, juxtaposing the 5’ end of EML4
(echinoderm microtubule associated protein-like 4) gene to
the 3’ end of the ALK gene 10
. This leads to the formation
of EML4-ALK fusion gene, encoding for a chimeric protein
with intrinsic tyrosine kinase activity. In addition to this,
other break-apart and fusion partners may also be involved
in ALK rearrangements.
There are 4 methods for detecting these genetic
rearrangements including immunohistochemical staining
(IHC), fluorescence in situ hybridization (FISH), reverse
transcriptase‐PCR (RT‐PCR) and next generation
sequencing (NGS). All of these methods have their own
advantages and disadvantages. FISH is considered as the
gold standard for detecting ALK rearrangements, however,
well-validated IHC has been accepted as an equivalent
alternative 7,8. NGS can detect all kinds of fusions,
whereas, FISH and IHC provide no fusion specification
and RT-PCR provides information only regarding EML4-
ALK fusion 11.
In the present study, detection of ALK gene rearrangements
in lung adenocarcinoma cases was carried out using ICC
on cell-blocks. The mean age of the patients in our study
is similar to that observed in the previous studies 12-15.
Lung adenocarcinoma is more common in smokers than
in non-smokers. Similarly, in our study 66.6% patients
were smokers and 33.3% were non-smokers. However,
ALK gene rearrangements were seen more frequently in
non-smokers (31.25%), which correlates well with some
previous studies 10-15. The tumors were more common
in the right lung (n=35) in the present study, which is
similar to a previous study 16,17. Presence of pleural effusion was found to be higher in patients with ALK gene
rearrangement as seen in the previous studies; however,
this was not statistically significant 18,19. There was no
statistically significant difference between lung cancer
stage and ALK gene rearrangements which is in agreement
with the previous studies 20. A thorough comparison of the present study with previously published studies for
detection of ALK rearrangements is shown in the Table III
21-25. The prevalence of ALK rearrangements using FISH
and IHC observed in the present study is in concordance
with other studies wherein the prevalence ranged from 3
to 14.9% and 4 to 15.4%, for FISH and IHC, respectively. The concordance rates of ALK IHC and ALK-FISH in the
published literature are variable and range from 75-100%
(Table III). The concordance rate between ICC and FISH
in the present study was 66.7%.
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|Table III: Comparison of the present study with previously published studies for detection of ALK rearrangements
Higher ALK positivity rates with immunochemistry can be
explained by the fact that ALK IHC detects the ALK protein
expression but not the genetic changes. Similarly, lower
positivity rates of ALK-FISH on cell-blocks, can be due to
the presence of yet unknown type of ALK rearrangement
or genetic abnormalities other than ALK rearrangements,
which may be missed on FISH. As FISH is considered
the gold standard test, the results may indicate that ALKIHC
had false-positive results. Similarly, a few authors
have found that FISH can miss a good number of patients
with ALK-EML4 rearrangements who might benefit from
targeted ALK therapy, so they strongly recommended
ALK-IHC 19,26. When analysed alone with FISH,
their cohort had 4 (7.8%) positive cases whereas the true
incidence was 7 (13.7%) cases 19. This can be because of
extremely minimal splitting of red and green signals giving false negative results. However, rare ALK translocations
that do not cause over expression of ALK protein may lead
to negative IHC and positive FISH results.
ALK gene rearrangements in lung adenocarcinoma are
more commonly seen in females, non-smokers and in
patients having pleural effusions. Among the architectural
patterns, ALK gene rearrangements were common in
cases having focal solid, acinar and papillary architecture.
Immunocytochemistry on cell-blocks using the DF53 clone
is a highly sensitive and specific method for detection of
ALK gene rearrangements in lung adenocarcinoma with
greater number of ALK positive cases being detected on
ICC as compared to ALK-FISH.
CONFLICT of INTEREST
The authors declare no conflict of interest.
Concept: PG, NG, MR, AR, Design: PG, NG, AR, Data
collection or processing: SR, PG, NG, MR, NS, Analysis
or Interpretation: SR, PG, NG, MR, NS, Literature search:
SR, PG, NG, NS, AR, Writing: SR, PG, NG, Approval: SR,
PG, NG, MR, NS, AR.
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