Sabato 27 ottobre 2012 - Pacini Editore

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ALK (A)) and E6;A20, which also referred to as variants 1 and
3, respectively. These have been respectively detected in 33%
and 29% of NSCLC patients with EML4-ALK rearrangement 3 4 .
Consequent to the EML4-ALK rearrangement, the encoded protein
contains the N-terminal part of EML4 and the intracellular
catalytic domain of ALK. Replacement of the extracellular and
transmembrane domain of ALK with a region of EML4 results
in constitutive dimerization of the kinase domain and thereby a
consequent increase in its catalytic activity 3 . Detection of ALK
gene fusion is currently indicated as a predictive marker of treatment
response to small molecule inibitors of ALK in NSCLC:
crizotinib. Crizotinib, a new and selective TKI targeting ALK
and MET has been approved on August 26, 2011, by the FDA
to treat locally advanced or metastatic NSCLC that harbor the
abnormal fusion gene. In Phase I and Phase II trials, crizotinib
was shown to be highly active in patients with advanced ALKpositive
NSCLC, with overall response rates of 50–60%. EML4-
ALK is also associated with anti-EGFR TKI resistance. The gold
standard method to detect ALK rearrangement is fluorescent in
situ hybridization (FISH). However many published studies using
reverse transcritase-polymerase chain reaction analysis to characterize
EML4-ALK. FISH detects any rearrangement involving
ALK, including potentially rare uncharacterized rearrangement.
PCR identifies specific fusion variants. Although both/these
techniques are sensitive and specific for detecting EML4-ALK,
we want to compare the data obtain through the two different
approaches. We aimed to estabilish if PCR can be used to take
additional advantage of genetic analyses, especially in case were
the FISH was not applicable.
Matherials and method. This study comprised a series of 46
patients with NSCLC (39 adenocarcinoma, 5 metastasis of lung
adenocarcinoma, and 2 Squamous cell lung cancer) who underwent
surgery in the Department of Surgery at the University of
Pisa from 2007 to 2011. From each paraffin block, 4 consecutive
10µm sections were cut to extract the DNA (for EGFR and KRAS
mutation analysis) and the RNA (for EML4-ALK analysis).
An adjacent 5µm section was used for FISH (for EML4-ALK
analysis). All samples were analyzed using both FISH and PCR
approach in parallel. Nucleic acid was isolated using a commercially
kit specific to formalin-fixed and paraffin-embedded
(FFPE) sample. The quality and quantitation of RNA and DNA
were verified by Agilent 2100 bioanalyzer. RNA (500ng) from
each sample was reverse transcribed using random hexamer primers
to produce cDNA from all RNAs. For detection of EML4-
ALK fusion cDNAs, we have carried out three independent PCR.
The first one, was performed using a set of primers to identify the
variant 1; forward primer is located at exon 13 of EML4, whereas
the reverse primer is located at exon 20 of ALK, resulting in a
170-base pair PCR product. To search for other variants of rearrangement,
as described in the study of Soda et al. 3 , we used
primers that target fusion variant 1 (247-bp).
These primers could also detect variant 2 which would have
yielded a considerebly longer amplication (≈1Kb); the forward
primer Fusion-RT-S is located in exon 13 of EML4, while the
reverse primer Fusion-RT-AS, in exon 20 of ALK. The latter
variant may have been expressen in the fusion positive cases
but missed in our assay because of the difficulties in amplifying
long amplicons from RNA extracted from FFPE. To analyze
the shorter variant of EML4-ALK transcript (155/188-bp), the
variant 3, the ALK Fusion- RT-AS primer was combined with a
forward primer located in exon 6 of EML4: EML4-ex6F. PCR
primers GAPDH-S and GAPDH-AS for glyceraldehyde-3phosphate
dehydrogenase cDNA (452bp) were used as control
for cDNA integrity. Polymerase chain reaction products were
assessed and visualized by 1,5% agarose gel electrophoresis.
PCRs were performed in triplicate, each one in 25µl reactions
containing 100 ng cDNA, 12,5µl of Taq PCR Master Mix and
0.5 µl of each primer (20µM).
CONGRESSO aNNualE di aNatOmia patOlOGiCa SiapEC – iap • fiRENzE, 25-27 OttOBRE 2012
To asses for rearrangement of the ALK locus on 2p23 using commercially
available ALK (Vysis ALK Break Apart FISH Probe
Kit, Abbott Laboratories) probes we performed on 4µm paraffinembedded
thin sections. The test employs two differently labeled
probes on opposite sides of the breakpoint of the ALK gene.
When hybridized with the Vysis ALK Break Apart FISH Probes,
the 2p23 ALK region in its native state will be seen as two immediately
adjacent or fused orange/green (yellow) signals. However,
if a chromosome rearrangement at the 2p23 ALK breakpoint region
has occurred, one orange and one green signal separated by
at least two signal diameters will be seen. Alternatively, a single
orange signal(deletion of green signal) in addition to a fused or
broken apart signal may be seen.
A minimum of 50 tumor cells must be scored and a specimen
is considered positive for ALK rearrangement when > 15% of
the cells show split signals. All experiments were done without
knowledge of the RT-PCR results for EML4-ALK. Mutation
analysis of EGFR (exons 18-21) and KRAS (codons 12, 13 and
61) were performed using the HotStarTaq Master Mix Kit, as
per manufacturer’s instructions. The presence of an appropriate
PCR product was confirmed by resolving the PCR products on
a 1.5% agarose gel. PCR products were purified using the PCR
Purification Kit and sequenced using fluorescent dye-terminator
chemistry. Mutations were identified by visual analysis of the
sequence chromatograms using SeqScape.
Results. Among 46 NSCLC analyzed 6 (13%) showed EML4-
ALK rearrangement by FISH analysis; while, EML4-ALK transcript,
detected by RT-PCR, were found in 9 cases of 46 (21.7%).
Of 9 positive-PCR cases 6 (13%) showed EML4-ALK fusion
variant 1 and 3 (6.5%) showed EML4-ALK fusion variant 3.
We identified only one (2.17%) in-frame deletion in EGFR
exon 19 (E746-A750del); while, single amino acid substitution
involving codons 12 and 13 of KRAS, were identified in 7 of 46
NSCLC (15%). In particular, we found that 3 patients had G12C,
3 patients had G12D and 1 patient had G12S.
Discussion. An overall concordance of FISH and RT-PCR (being
either negative or positive in both tests) was found in 39 cases
(84.7%) and a discrepancy was identified in 7 cases (15.3%).
In detail, 4 samples resulted positive in both FISH and RT-PCR
analysis, 2 FISH positive samples were RT-PCR negative, 5
samples showing transcript expression were FISH negative (or
below the cut-off value) and 35 samples were negative for EML4-
ALK rearrangement detection.
ALK fusion and EGFR and KRAS mutations appears to be mutually
exclusive. Although, we identified one patient who had both
EML4-ALK rearrangement and KRAS mutation (G12D); while,
all positive cases for ALK rearrangement did not show EGFR
mutations.
In conclusion, the usefulness of RT-PCR analysis for EML4-
ALK rearrangement detection is still not fully satisfactory due to
the fact that either alternative EML4 exons were involved or that
EML4 was not the ALK fusion partner in all the FISH positive
patients. As a consequence a multiplex RT-PCR reaction should
be performed to fully cover the fusion variants but this approach
is difficult to apply in routine condition poor quality samples.
Finally FISH analysis remains the gold standard methods for
EML4-ALK rearrangement detection.
references
1 Chiarle R, et al. The anaplastic lymphoma kinase in the pathogenesis
of cancer. Nat Rev Cancer 2008;8:11-23.
2 Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements:
a new therapeutic target in a molecularly defined subset of
non-small cell lung cancer. J Thorac Oncol 2009;4:1450-4.
3 Soda M, et al. Identification of the transforming EML4-ALK fusion
gene in non-small-cell lung cancer. Nature 2007;448:561-6.
4 Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement
in non-small cell lung cancer and non-tumor lung tissues. The American
Journal of Pathology 2009;174:661-670.