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Supplemental Material
Biomarker methods
IGF1R gene copy number was determined by both a silver in situ hybridization assay (SISH)
and by fluorescent in situ hybridization assay (FISH). The details of the SISH assay have
been described previously.1 Briefly, the tissue slides were probed with the IGF1R specific
DNA probe obtained from Ventana Medical Systems Inc (Tucson, AZ) according to
manufacturer’s instructions and using the Benchmark XT automated slide stainer with
appropriate secondary and ultraView SISH Detection reagents. Following precipitation of the
silver particles within the nuclei, a single black dot represents a single copy of the IGF1R
gene, whereas a cluster of black dots concentrated so closely together that single dots
cannot be resolved are considered amplified genes. A single black dot was given a score of
one and clusters, if present, were scored as 6 or 12 for small and big clusters, respectively.
A certified pathologist visually scanned the entire tissue specimen and counted signals
within ~15 nuclei in 4 fields with high copy number for a total of 50 nuclei.
For the FISH assay, slides from 12 specimens were subjected to a dual-color FISH assay
using the IGF-1R/CEP15 probe set, following standard protocol in the laboratory. A FISH
probe for the IGF-1R gene (located on 15q26.3) was prepared with the insert from the BAC
clone RP11-262P8 obtained from CHORI (Oakland, CA). DNA was extracted, amplified,
tested for the presence of the gene sequences by PCR-based reactions. DNA was labeled
with SpectrumRed (SR) conjugated dUTPs in aliquots of 1ug using the Vysis Nick Translation
Kit (Des Plaines, IL) according to the manufacturer’s instructions. The labeled DNA was
precipitated with herring sperm and human Cot-1 DNA and the pellet was resuspended in
10 ul of c-DenHyb (Insitus Biotechnologies, Albuquerque, NM). CEP 15 labeled in
SpectrumGreen (SG) was purchased from Abbott Molecular (Des Plaines, IL).
The samples were incubated at 56oC for 4 hours, soaked in CitriSolv wash series 3 times for
10 minutes each and allowed to air dry. The tissue area to be hybridized was marked with a
diamond scriber guided by the marks on the H-E stained slides. The slides were incubated in
2xSSC at 75oC for 5-15 min and in 0.6 mg/ml Proteinase K at 45 oC for 5-15 min. After
washing and dehydrating the specimens in 70% EtOH, 85% EtOH and 100% EtOH for 2 min
each, the slides were allowed to air dry.
The appropriate volume of the probe set probe set was applied to the selected hybridization
areas, which were covered with a coverslip and sealed with rubber cement. DNA
codenaturation was performed at 85oC for 15 min and hybridization allowed to occur at 37 oC
for 16-24 hours.
Post-hybridization washes were performed though incubations in
2xSSC/0.3% NP-40 at 74oC for 2 min and 2xSSC for 2 min each, followed by dehydration.
Finally, 14 ul of DAPI/anti-fade (0.3 ug/ml in Vectashield mounting medium) was applied to
the slide and the area covered with a 22x50 mm coverslip.
Analysis was performed on epifluorescence microscope using a single interference filter sets
for green (FITC), red (Texas red), and blue (DAPI) as well as dual (red/green) and triple
(blue, red, green) band pass filters. Monochromatic images were captured by a CCD camera
(Cool Snap, Photometrics) and merged using dedicated software CytoVision (Genetix).
Slides were scored by one trained reader and independently reviewed
IGF1-R protein expression was evaluated on the tissue specimens by immunohistochemistry
using the G11 clone and ultraView detection kit from Ventana Medical Systems Inc (Tucson,
AZ) according the manufacture’s protocol for the Benchmark XT autostainer. The final HScore was determined by a certified pathologist but summing the products of the five
staining intensity categories (0-4) by the percentage of positive cells (0-100%) for a final
score of 0-400.
Biomarker Results
Figure 1. Hazard ratio and confidence intervals for biomarker association with PFS
Legend: N = number of patients with biomarker measured; HR: Hazard ratio; CI =
confidence interval. A Cox Proportional Hazard model was used regarding the effect of
biomarker status on PFS. Diagnostics for the functional forms of variables were not
performed due to low sample size; free serum IGF, IGFR1 FISH, and IGFR1 SISH values and
IGF IHC H scores were included individually as continuous covariates. No multivariate
analysis was performed. All tests and confidence intervals were two-sided with conclusions
based on a significance level of 0.05. Statistical analyses were performed using SAS/BASE
and SAS/STAT software, Version 9.2 of the SAS System for Windows. Summary: Small
number of patients limit possible conclusions.
Figure 2. Representation of IHC and SISH for IGF1R Gene Copy number
Table 1. Raw Data for Biomarkers
PT
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Dose
cohort
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
3
3
3
IGF IHC H score (up to
400)
IGF
SISH
IGF
FISH
105
2.16
1.98
100
100
75
100
210
160
280
20
90
100
2.24
1.84
2
2.88
2.36
2.14
2.62
3.33
2.16
1.9
5.32
3.58
1.16
1.78
2.44
3.16
2.92
1.6
3.5
PFS
(days)
OS
(days)
22
37
27
52
112
21
217
57
432
34
21
23
20
245
40
40
21
39
31
65
92
135
408
105
541
275
432
56
392
236
44
303
138
70
93
69
Table 2. Free IGF-1 levels and Tumor Characteristics
Number of patients
Male
Median Age
Ever Smokers
EGFR mutation
EGFR wild type
EGFR status not assessed
Quartile 1-3
Quartile 4
10 patients
2 (30%)
66 years
8 (80%)
2 (20%)
7 (70%)
1 (10%)
4 patients
3 (75%)
57 years
3 (75%)
0 (0%)
3 (75%)
1 (25%)
Table 1. Summary Statistics of Erlotinib and OSI-420 Stratified by Course and Day
Drug/ Metabolite
Conc. (ng/ml)
Erlotinib
OSI-420
Course
1
2
1
2
Day
8
1
8
1
N
10
7
9
7
Mean
458.71
489.34
228.01
237.10
Min
17.1
42.4
67.7
5.29
StDev
182.19
230.78
198.86
230.54
Max
680
683
693
553
1.
Badzio A, Wynes MW, Dziadziuszko R, et al. Increased insulin-like growth factor 1 receptor
protein expression and gene copy number in small cell lung cancer. J Thorac Oncol 2010;5:1905-1911.
Med
501.5
601
132
108