healthmedicinet

Patients and tissues

A total of 222 lung tumor specimens from patients with NSCLC were obtained from the
Biomaterial Bank North after resection from the surgical department of LungenClinic
Grosshansdorf (Table 1), including 110 cases of adenocarcinoma (ADC), 86 cases of squamous cell carcinoma
(SCC), 12 cases of large cell carcinoma (LCC), 6 cases of carcinoids and 2 cases of
adeno-squamous carcinoma. This retrospective study was performed in compliance with
the ethical committee of the University of Lübeck (reference number 12–220). All tumor
samples were histologically classified according to the International Association
for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society
International Multidisciplinary classification of lung adenocarcinoma 2011 25] and WHO guidelines 2010 26]. The lung cancer tissue samples were fixed both with formalin and in parallel using
the alternative HOPE technique and were subsequently embedded in paraffin 27]. Established clinical and histological factors of all tested patients were included
in this study (patient age, gender, histology, TNM classification, smoking status;
Table 1).

Table 1. Characteristics of 222 patients with Non-small cell lung cancer

Tissue microarray (TMAs) construction

Specimens were arranged on TMAs for enhanced comparability of immunohistochemical
stainings as previously described 28]. Appropriate formalin and HOPE-fixed -paraffin embedded A549 cell line known to express
c-MET basal, was used as control. Representative tumor punches (2 mm in diameter)
of two core biopsies were taken from two viable parts of each tumor block using the
Beecher manual arrayer (Beecher instruments, Alpha, Metrix Biotech). This approach
should enhance representative analyzing of immunohistochemical stainings and FISH
analyzes.

Immunohistochemistry (IHC)

MET protein expression was assessed by IHC on 2 ?m thick deparaffinized TMA sections,
using rabbit anti-human c-Met monoclonal antibody (clone SP44) (Spring Biosciences,
Pleasanton, CA, USA) directed against the synthetic peptide derived from C-terminus
of human c-Met protein displaying membranous and/or cytoplasmic epitope. Staining
procedures were conducted according to manufacturer’s protocol in a final 1:1000 dilution.

Phosphorylated MET-expression was assessed by two different primary antibodies purchased
from Cell Signaling (Danvers, MA). Phospho-Met (Y1234/1235) (D26) and Phospho-Met
(Y1349) (130H2) in a final dilution of 1:100.

HOPE-fixed, paraffin-embedded sections were deparaffinized by xylene incubation (two
times 10 min) before SP44 was applied for 30 min at room temperature. Endogenous peroxidases
were blocked by incubation with 3 % H
₂
O
₂
for 10 min. Negative controls omitting the primary antibody were always included.

Formalin-fixed samples from the same tumors were deparaffinized following standard
procedure. Antigen retrieval was achieved by boiling in citrate buffer (10 mM, pH 6.0)
for 10 min (Merck KGaA, Darmstadt, Germany) with subsequent cooling at room temperature
for 20 min. Antigen detection was carried out with DAB substrate kit (DAB chromogen
and DAB substrate), used for 15 min to visualize specific binding. Sections were counterstained
using Meyer’s hemalum and mounted with Pertex (Medite GmbH, Burgdorf, Germany) for
further evaluation.

Evaluation of Met IHC staining

Two observers quantified staining intensities. Samples were evaluated with the MET
scoring algorithm as previously described 18]. MET positivity was defined when scored as 2+ and 3+. 3+ was selected if???50 % of
tumor cells stained with strong intensity; 2+ was defined if???50 % of tumor cells
showed moderate or higher staining but??50 % with strong intensity; 1+ if???50 %
of tumor cells with weak or higher staining but??50 % with moderate or higher intensity;
0 was defined if no staining or 50 % of tumor cells with any intensity.

Phosphorylated MET expression was evaluated according to the intensity of cytoplasmic
staining (no staining?=?0, weak staining?=?1, moderate staining?=?2, strong staining?=?3)
and the percentage of stained tumor cells (0 %?=?0, 1-10 %?=?1, 11-50 %?=?2, 50 %?=?3).
If???50 % of tumor cells showed staining intensity???2, tumor samples were categorized
as positive in this study.

EGFR scoring methodology

EGFR expression is considered as positive, if ?10 % of the tumor cells show membranous
staining of any intensity using x10 and x20 magnification assessed by Dako EGFR PharmDx
data sheet.

Fluorescence in situ hybridization

c-MET gene copy numbers were assessed by fluorescence in situ hybridization (FISH)
using ZytoLight SPEC MET/CEN7 Dual Color Probe (Zytovision, Bremerhafen, Germany).
Before hybridization, sections were deparaffinized, dehydrated and immersed in citrate
buffer (Merck KGaA, Darmstadt, Germany) pH 6 at 98 °C for 15 min, and subsequently
washed twice in distilled water for 2 min. The sections were air dried and pretreated
with pepsin for 5 min before denatured for 10 min at 75 °C. After hybridization at
37 °C for 20 h, slides were washed and counterstained with 1.5 ?g/ml 4’,6’-diamidino-2-phenylindole
(DAPI) mounting medium (Vectashield, Vector laboratories, Burlingame, CA) and coverslips
were fixed with nail polish.

Analysis of FISH signals was performed on an epifluorescence microscope Nikon Eclipse
80i H550L (Nikon) with interference filters (AHF Analysentechnik AG, Tübingen, Germany).

At least 50 non-overlapping interphase nuclei of tumor cells were counted per core.
The nuclei were selected using the DAPI filter under high magnification (x600). For
each probe, the numbers of c-MET and CEN7 per nuclei were separately scored and mean
cMET/CEN7 ratio was determined. FISH MET gene amplification was defined as FISH positive,
when

a) MET/CEP7 ratio was??2.2 or

b) small gene clusters (?4 copies) independent of the MET to CEP 7 ratio

These analysis criteria were adapted to previous publications for the evaluation of
MET amplification 7].

Increased MET gene copy number (GCN) 3 of Chromosome 7 and MET-locus was defined
as polysomy.

Evaluation was performed independently by two scientists well-vised in FISH analysis.

Protein extraction, SDS-PAGE, and western blotting

HOPE-fixed, paraffin-embedded specimens were deparaffinized as previously described
by Olert et al. 27]. Protein extraction was performed using lysis buffer (7 M urea, 2 M thiourea, 100 mM
dithiothreitol (DTT), 4 % CHAPS (3-[3-Cholamidopropyl)-dimethyl-ammonio]-1-propansulfonat),
2 % IGEPAL, 1 % Triton X, 5 mM PMSF, 0.5 mM EDTA, 40 mM Tris). Protein concentration
was determined by Bradford with Coomassie (Bradford) Protein Assay Kit (Pierce, Rockford,
IL, USA). Forty ?g of whole protein lysate was separated by gel electrophoresis and
analyzed by Western Blot using nitrocellulose membrane using iblot gel transfer system
(Invitrogen, Germany). After blocking with 5 % nonfat dry milk (1 h, ambient temperature),
membranes were incubated with Met-specific antibody clone SP44 (1:1000) in TBS-0.05 %
Tween overnight (4 °C). The following day, the membrane was washed three times in
TBS-0.05 % Tween before adding secondary antibody anti rabbit HRP (Cell Signaling)
for 1 h at room temperature. Development was achieved using ECL solution and exposure
to film. Intensities of appropriate MET specific bands were analyzed by band leader.

Quantitative real-time PCR of MET mRNA

Total RNA was isolated from HOPE-fixed tissue samples as described previously 29] using RNeasy minikit (Qiagen) according to the manufactures protocol. RNA integrity
and concentration were determined with the Agilent RNA 6000 Nano Assay Bioanalyzer
(Agilent, Böblingen, Germany). 1 ?g of RNA was transcribed into cDNA with the Maxima
first strand cDNA synthesis kit for RT-qPCR (Thermos Scientific). Genomic DNA was
digested with DNase I within cDNA synthesis. RPL32 was used to normalize differences
in input cDNA amounts. Intron spanning primers were designed with the Universal Probe
Library (Roche Applied Sciences, Germany). Primers were used as follows: Forward primer
5’-TGAAATTCATCCAACCAAATCTT-3’ and reverse primer 5’- AATAGAAAACTGACAATGTTGAGAGG-3’
(probe no. 31) for MET and forward primer 5’- CCACCGTCCCTTCTCTCTT-3’ and reverse primer
5’- GGGCTTCACAAGGGGTCT-3’ (probe no. 10) for RPL32. Amplification was performed with
Light Cycler 480II from Roche Diagnostics. After initial denaturation at 95 °C for
5 min, amplification protocol consisted of 45 cycles (10 s at 95 °C and 30 s at 60 °C)
using Light Cycler 2x Probe Master Mix, 0.4 ?M oligonucleotides and 0.2uM 6-carboxyfluorescein
(FAM)-labeled hydrolysis probes (Roche Applied Science) in a final volume of 10 ?l.
Standard curve was made from serial dilution of template cDNA. Expression levels were
calculated after normalization with RPL32.

Statistical analysis

All statistical analysis in this study were performed with Spearman’s rank nonparametric
correlation test using Prism 6 software. All reported p values are two sided (GraphPad
Prism version 6.0 Software, San Diego, CA).