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SUPPLEMENTAL MATERIAL
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FGFR1 gene copy number analysis by silver in situ hybridization (SISH)
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Dual-color silver in situ hybridization (SISH) was performed on 4-μm sections of the TMA,
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using a fully automated protocol on the Ventana Bench MarkXT instrument (Ventana, Tucson,
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AZ, US). All reagents, including FGFR1 DNP probe (FGFR1 locus on 8p12) and chromosome 8
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DIG probe (Centromere 8, CEN8) cocktails, ultraView SISH and ultraView Alkaline
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Phosphatase Red ISH detection kits, were obtained from Ventana Medical Systems Inc.
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(Tucson, AZ, US). The slides were deparaffinized, followed by incubation with pH 6 Cell
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Conditioning Buffer (CC2) at 82°C for 36 minutes and by digestion by ISH Protease3 reagent
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for 16 minutes. The probe mixture was put on the slides, then the slides were denatured for
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12 minutes at 85°C, hybridized for 6 hours at 44°C, and washed three times with pH 6.0 Cell
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Conditioning Buffer (CC2) at 68°C. After the application of a horseradish peroxidase–labeled
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rabbit anti-DNP antibody linker, the specific hybridization of the DNP-linked FGFR1 probe to
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its target was visualized by an insoluble precipitate of silver chromogen. After the application
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of an alkaline phosphatase–labeled mouse anti-digoxigenin antibody linker, the visualization
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of digoxigenin-linked CEN8 probe was detected by the soluble precipitate of the alkaline
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phosphatase–based Fast Red chromogenic system. To visualize the complete morphology of
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the tissue, the slides were counterstained with hematoxylin for 8 minutes and
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counterstained with bluing reagent for 8 minutes. After removal from the instrument, slides
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were rinsed with mild detergent, flushed in distilled water, dried for 20 minutes at 60°C, and
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cover slipped.
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1
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The black FGFR1 signals and red CEN8 signals were counted using bright-field microscopy
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under a 40× or 60× objective. FGFR1 and CEN8 signals were counted separately in 50 non-
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overlapping tumor nuclei per core. Fields with higher signal deposits (so called ‘hot-spot’
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areas), were selected for evaluation. If FGFR1 signals showed a homogenous distribution,
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random areas were used for counting the signals. Signals that were physically separated by
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any distance were counted as individual signals. FGFR1 doublets and triplets were counted
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as one signal, but closely spaced groupings of signals consisting of more than three copies
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were considered as clusters of FGFR1 signals. FGFR1 minor signal clusters and major signal
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clusters were counted as 6 and 12 respectively (according to Ventana protocol).
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FGFR1 gene rearrangement and amplification by fluorescence in situ hybridization (FISH)
Slides were subjected to 3 color FISH assay using a FGFR1 break-apart/amplification probe
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set consisting of three reagents: a green probe telomeric to FGFR1, a red probe centromeric to
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FGFR1 and an aqua probe recognizing the centromere of chromosome 8 (Cytocell, Cat. LPS
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018-SA). The slides were deparaffinized, then incubated in 2x SSC at 75˚C for 15 min, and
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digested in Proteinase K solution (0.6mg/ml in 2x SSC) at 45˚C for 20 min. After washing in 2x
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SSC at room temperature for 5 min, slides were dehydrated in ethanol and air dried. The
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probe set was then applied to the selected hybridization areas. DNA codenaturation was
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performed at 85˚C for15 min hybridization occur at 37˚C for 65 h. Post- hybridization washes
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were performed, followed by dehydration. Finally, DAPI was applied to the slide and the area
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covered with coverslip.
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Analysis was performed on epifluorescence microscope using single interference filter
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sets for green (FITC), red (Texas red), aqua and blue (DAPI) as well as dual (red/green) and
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triple (blue, red and green) band pass filters. Fifty nuclei per specimen were analyzed for
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amplification and rearrangement of FGFR1. In the alleles with native status, this FISH probe
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will generate one copy of the red signal (5’FGFR1) fused with one copy of the green signal
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(3’FGFR1) along with one copy of the blue signal (CEN 8). In the case of FGFR1 rearrangement,
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the FISH probe will generate a single red and/or single green signal at least one signal
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diameter apart for each rearranged copy of FGFR1 gene.
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Table 1 FGFR1 postive staining by IHC in the cohort of Poland
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TMA
ID
NSCC3
NSCC2
NSCC4
NSCC4
NSCC1
NSCC1
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FGFR1 IHC
Core 2
Core1
Core 3
average
0
1+
2+
3+
Hscore
0
1+
2+
3+
Hscore
0
1+
2+
3+
Hscore
0
0
60
10
80
60
30
0
40
90
20
40
0
10
0
0
0
0
70
90
0
0
0
0
240
290
40
90
20
40
0
0
60
30
80
40
20
0
10
70
20
60
20
20
20
0
0
0
60
80
10
0
0
0
240
280
80
70
20
60
0
na
na
20
20
30
0
na
na
80
80
70
20
na
na
0
0
0
80
na
na
0
0
0
280
na
na
80
80
70
Na: non-applicable
57
58
59
60
61
3
Hscore
254
285
60
80
40
57
Heterogenity
NO
NO
NO
NO
NO
NO
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Table 2 FGFR1 staining by IHC in the cohort of German
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Patient ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0
40
10
0
80
30
100
100
100
20
100
0
20
40
50
100
0
100
0
0
0
0
0
0
1+
60
90
80
20
70
0
0
0
80
0
100
80
60
50
0
100
0
70
100
100
100
10
100
2+
0
0
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
30
0
0
0
70
0
3+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
0
Total (100)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
64
65
66
67
68
69
70
71
4
H-score
60
90
120
20
70
0
0
0
80
0
100
80
60
50
0
100
0
130
100
100
100
210
100
Heterogenity
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
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Figure 1 The distribution of FGFR1 H-scores
Range of FGFR1 H-scores
300
H-score
250
200
150
100
50
0
0
20
40
60
80
100
Specimen
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FGFR1 H-score ranged from 0 to 285 in SCLC by IHC. There was a separation between the H-
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score for the highest negative case (H-score 5) and the lowest positive case (H-score 40).
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5