Bean et al. 10.1073/pnas.0710370104.

Supporting Information

Files in this Data Supplement:

SI Figure 4
SI Figure 5
SI Table 3
SI Figure 6
SI Methods
SI Table 4




SI Figure 4

Fig. 4. Copy number alterations on chromosome 7 showing focal peaks spanning regions encoding EGFR (at 55 MB) and MET (at 116 MB). Recurrence plots of a tumor with amplification at the EGFR but not the MET coding region (sample 5) and three samples with amplification at both regions (6, 10a, and 10b). Samples 10a (primary lung lesion) and 10b (metastatic lymph node) are separate tumors from the same patient (only 10a was considered for the comparison shown in Fig. 1). Log2 ratios of copy number for all samples are plotted based on their chromosome position (Mb): green denotes median log2 ratios for each of three neighboring probes; red denotes spans of uniform copy number by segmentation algorithm.





SI Figure 5

Fig. 5. FISH showing polysomy of chromosome 7 and amplification of the MET locus in tumor cells from patient 6. Chromosome 7 copy number was monitored by a centromeric probe (green) while MET amplification was determined using a MET-specific probe (red). DAPI staining of DNA is shown in blue. Representative tumor cells (left and right panels) displayed complete or partial gain of chromosome 7q (~4-6 copies) with additional focal amplifications at loci containing MET in about half of the cells.





SI Figure 6

Fig. 6. ERBB3 signaling in H820 cells is inhibited by siRNA-mediated knockdown of MET. Lysates from H820 and PC-9 cells transfected with either siRNAs against GAPD (G; as a negative control) or MET (M) for 72 h were harvested and subjected to immunoblot analyses with anti-phospho-(p)-MET (Y1234/5), anti-total MET, anti-phospho-(p)-ERBB3 (Y1289), anti-total ERBB3, anti-phospho-(p)-EGFR (Y1092), and anti-actin antibodies as described in Materials and Methods. Total actin is shown as a loading control. For all immunoblots shown, H820 and PC-9 lysates were run together on the same gel and exposed together on the same film with equal exposure times. Results are displayed separated, as intervening lanes of lysates irrelevant to the report presented here are not shown.





Table 3. EGFR mutation and MET status of tumor samples from additional patients with acquired resistance to EGFR inhibitors

Patient

1° EGFR Mutation

T790M

MET

/MTHFR

Drug

Duration, months

20

L858R

Y

1.1 -> 2.3

gefitinib

4

21

L858R

Y

1.1 -> 7.0

gefitinib

11

22

L858R

Y

1.7 -> 2.4

gefitinib

7

23

L858R

Y

1.9 -> 1.7

gefitinib

6

24

L858R

Y

1.8 -> 1.1

gefitinib

7

25

L858R

Y

0.6 -> 0.7

gefitinib

8

26

L858R

Y

1.5 -> 1.5

gefitinib

12

27

L858R

Y

1.1 -> 1.2

gefitinib

11

28

Del E746-A750

Y

1.1 -> 1.1

gefitinib

10

29

Del E746-A750

Y

1.2 -> 1.6

gefitinib

9

30

Del E746-A750

Y

5.6 -> 2.1

gefitinib

8

31

Del E746-A750

Y

0.9 -> 1.0

gefitinib

9

32

Del L747-T751

Y

8.7 -> 7.2

gefitinib

15

33

Del E746-T751 insV

Y

2.4 -> 13.3

gefitinib

14

34

L858R

N

0.6 -> 6.3

gefitinib

6

35

L858R

N

1.4 -> 0.8

gefitinib

11

36

L858R

N

1.6 -> 1.7

gefitinib

8

37

Del E746-A750

N

2.1 -> 1.0

gefitinib

18

38

Del E746-A750

N

0.7 -> 1.4

gefitinib

16

39

Del E746-A750

N

3.0 -> 6.1

gefitinib

19

40

Del E746-A750

N

1.6 -> 2.8

gefitinib

6

41

Del E746-A750 insP

N

0.7 -> 1.0

gefitinib

21

42

Del E746-A750 insP

N

1.1 -> 1.2

gefitinib

6

43

Del L747-P753 insS

N

2.2 -> 2.0

gefitinib

8

For specimens from these patients (numbers 20-43; analyzed at the National Taiwan University Hospital and Graduate Institute of Clinical Medicine, College of Medicine), pre- and posttreatment samples were available for analysis, and both values are shown (pre -> post). See legend for Table 2 for details. MET amplification was assessed as described in the Methods in relation to MTHFR, by qPCR, using standard curves derived from analysis of CL1-5 lung adenocarcinoma cells. Samples were considered to have MET amplification if the MET/MTHFR ratio was >5.0. This more conservative cutoff was used for these samples since no aCGH or FISH was performed to confirm the qPCR. MET/MTHFR ratios for cell lines H820 and PC9 were similar to copy numbers obtained for those cell lines by qPCR at MSKCC (Table 2). Highlighted in green and yellow are samples displaying MET amplification before and/or after treatment, respectively.





Table 4. Primers used to re-sequence exons encoding MET

Exon

Primer

3F

GTTTTCCCAGTCACGACGGTTTCTTACCAGCTTGTTC

3R

AACAGCTATGACCATGGCACAATACCAGATAGAACA

4F

GTTTTCCCAGTCACGACTTTAAACTGAGCTTGTTGGA

4R

AACAGCTATGACCATGTTTCAGAAGATCTCTGGAAT

5F

GTTTTCCCAGTCACGACCATGTACCTTTTGTGTACTTAC

5R

AACAGCTATGACCATGCAAACAAAAACATGTATGCC

6F

GTTTTCCCAGTCACGACGTTCGTTTTCCATATATGTG

6R

AACAGCTATGACCATGAACCTATTTTCCAAGCACAC

7F

GTTTTCCCAGTCACGACTGTCACTTCCTATAAAACAACC

7R

AACAGCTATGACCATGGGGGAGATAAAAACAAAACA

8F

GTTTTCCCAGTCACGACACGGGACAACACAATACAGT

8R

AACAGCTATGACCATGTCAAATTGACAGATGCAACA

9F

GTTTTCCCAGTCACGACCACTTAGGAACCATTGAGTT

9R

AACAGCTATGACCATGCAGCAAAATCATCCTTGTAT

10F

GTTTTCCCAGTCACGACGCAGGTGATTAAATTGAATC

10R

AACAGCTATGACCATGTTAGAGGCAAAGATGCAGAG

11F

GTTTTCCCAGTCACGACATTTTCAATTGATTGGGGTG

11R

AACAGCTATGACCATGGGCCAAGTACAACAATTGTA

12F

GTTTTCCCAGTCACGACATATTCCTTTGCCATTGTTAGC

12R

AACAGCTATGACCATGTGGCTTCCTTATTTACATCA

13F

GTTTTCCCAGTCACGACCAGTTGGTATTTGGGACCCA

13R

AACAGCTATGACCATGCACAAGAATCGACGACAATC

14F

GTTTTCCCAGTCACGACTTACAGTTTAAGATTGTCGTCG

14R

AACAGCTATGACCATGCAACCCACTGAGGTATATGT

15F

GTTTTCCCAGTCACGACAAAAGCTCTTCCTGTTTCAG

15R

AACAG TATGACCATGGCTTACTGGAAAATCGTATT

16F

GTTTTCCCAGTCACGACCACACCTACGTACCTATAGTGGTATTG

16R

AACAGCTATGACCATGTTTCCACAAGGGGAAAGTG

17F

GTTTTCCCAGTCACGACAAACCCTCAGGACAAGATGC

17R

AACAGCTATGACCATGAGGGATGGCTGGCTTACAG

18F

GTTTTCCCAGTCACGACAGGCTTGAGCCATTAAGACC

18R

AACAGCTATGACCATGATCCCCAGGGCTTACACATC

19F

GTTTTCCCAGTCACGACTGGCAATGTCAATGTCAAGC

19R

AACAGCTATGACCATGTGAAGAAAACTGGAATTGGTG

20F

GTTTTCCCAGTCACGACTGTTGCCCAAAACAGAAACC

20R

AACAGCTATGACCATGAAGGCAGGCATTTCTGTAAAAG

21F

GTTTTCCCAGTCACGACTCCTACAACCCGAATACTGC

21R

AACAGCTATGACCATGCCCAGAAGGAGGCTGGTC

Forward primers were tagged with the sequence for M13F1. Reverse primers were tagged with the sequence for M13R1.





SI Methods

Quantitative Real-Time PCR.

 MET and MTHFR (endogenous control) levels were evaluated using the following primers: MET-sense: 5'-CCATCCAGTGTCTCCAGAAGTG-3'; MET-anti-sense: 5'- TTCCCAGTGATAACCAGTGTGTAG-3'; MTHFR-sense: 5'- CCATCTTCCTGCTGCTGTAACTG-3'; MTHFR-anti-sense: 5'-GCCTTCTCTGCCAACTGTCC-3'. For patients nos. 1-16 (analyzed at MSKCC) and associated reference DNA and positive control (H820), for MET, 20 ng of genomic DNA was amplified for 40 cycles (15 sec 95°C, 30 sec 63.3°C) in an IQ5 iCycler (Bio-Rad), using the sybrgreen Supermix (Bio-Rad) and 400 nM primers. Similar conditions were used for amplification of MTHFR, but the 30-sec annealing/extension was carried out at 65°C. For patients nos. 17-19 (analyzed at National Health Research Institutes, Miaoli, Taiwan) and associated reference DNA and positive control (H820), for MET and MTHFR 20 ng of genomic DNA was amplified for 45 cycles (10 sec 95°C, 5 sec 65°C, 5 sec 72°C) in a Light Cycler version 1 (Roche). For patients nos. 20-43 (analyzed at National Taiwan University Hospital and Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan), for MET and MTHFR, 20 ng of genomic DNA was amplified for 40 cycles (15 sec 95°C, 60 sec 60°C) in a ABI 7500 real-time PCR system (ABI), using the QuantiTect SYBR Green PCR kit (Qiagen) and 400 nM primers. For all patients, triplicate cycle time (CT) values were averaged.

For patients nos. 1-19, MET levels were normalized to MTHFR using a reference DNA sample and cutoff values for MET amplification were determined by using positive controls (H820 cell line and patients nos. 6, 10a, and 10b - shown to have MET amplification by aCGH and/or FISH). Fold changes were calculated using the equation 2-DDCT, where DDCT = (CT[MET]sample - CT[MTHFR]sample) - (CT[MET]reference DNA - CT[MTHFR]reference DNA) (1).

For patients nos. 20-43, to calculate the efficiency of the PCR reaction, and to assess the sensitivity of each assay, a 6 point standard curve (160, 80, 40, 20, 4, and 2 ng) was performed using CL1-5 lung adenocarcinoma cells, which do not harbor MET amplification (data not shown). MET amounts were interpolated from the standard curves and normalized to MTHFR amounts. Since no corresponding aCGH or FISH data were available on these samples, we used a more conservative MET/MTHFR ratio cutoff to claim amplification of MET.

aCGH Profiling.

 After washing, hybridized slides were scanned (Agilent) and images quantified using Feature Extraction 8.5 (Agilent). Fluorescence ratios of the scanned images were calculated and the raw aCGH profiles were processed to identify statistically significant transitions in copy number using the circular binary segmentation algorithm (2). Each profile was centered so that log2 ratio of zero is assigned to the predominant copy number, determined by the mode of the distribution of the mean log2 ratio for each segment, weighted by the number of probes per segment. After mode-centering, gains and losses were defined as segment mean log2 ratios of >0.2 or <-0.2 and amplification and deletions as >1 or less than -1, respectively. For analysis of data derived on 244K and 44K platforms, segment mean log2 ratios in each dataset were assessed at each 44K probe location (98% of autosomal probes from the 44K platform are included on the 244K array). Comparison between the two cohorts was restricted to amplified and deleted regions (mean log2 ratios ± 1), excluding regions of published copy number polymorphism (3).

Cell Lines and Viability Assays.

 Cells were seeded into 96-well plates in sextuplicate at a density of 1.2 ´105 cells per ml for H820 and 4 ´ 104 cells per ml for PC-9 and treated with various concentrations of EGFR or MET inhibitors for 72 h. H820 cells were seeded at a higher density than PC-9 to account for the slower rate of growth in H820. Cell viability was calculated according to the CellTiter-Blue-emitted fluorescence at 530 nm (ex)/590 nm (em), using a Fluoroskan Ascent FL plate reader (Thermo Electron Corporation). All curves are normalized to a DMSO-only control. All assays were performed at least three independent times and representative graphs are shown.

FISH.

 H820 cells were cultured in RPMI with 5% FBS until they were »70% confluent. Cells were trypsinized, centrifuged, and then resuspended in 8-ml hypotonic solution (0.075M KCL). After incubation at room temperature for 25 min, 1 ml of Carnoy's fixative (methanol : acetic acid = 3:1) was added. Cells were then centrifuged and resuspended in 10 ml of Carnoy's fixative. This fixation step was repeated twice. Finally, fixed cells were centrifuged and fresh fixative was added to obtain the desired cell concentrations for FISH analysis. Cell suspensions were then dropped on slides. Tumor cells from patient no. 6 were processed similarly without the culture step.

The MET locus-specific FISH probe was made from BAC clone RP11-163C9. BAC DNA was labeled with SpectrumOrange-dUTP using a nick-translation kit (Vysis). Chromosome 7 centromere (CEP7) probe (Vysis) was labeled with SpectrumGreen-dUTP.

Dual-color FISH was performed according to protocols from Vysis with a few modifications. All images were taken using a Nikon Eclipse TE-2000E inverted fluorescent microscope at 100x magnification with a Photometrics CoolSnap HQ2 CCD camera. All images were pseudocolored with NIS Elements and brightness and contrast were adjusted using Adobe Photoshop. No nonlinear manipulations (i.e., gamma function) were performed.

Immunoblotting.

 The following antibodies were used: polyclonal rabbit anti-phospho MET (Y1234/5) and monoclonal mouse anti-total MET (25H2) (both from Cell Signaling; no. 3126 and no. 3127, respectively); monoclonal rabbit anti-phospho-ERBB3 (Y1289) and polyclonal rabbit anti-total ERBB3 (C-17) (from Cell Signaling, no. 4791, and Santa Cruz Biotechnology, no. SC-285, respectively); polyclonal rabbit anti-phospho-EGFR (Y1092) and monoclonal mouse anti-total EGFR (from Cell Signaling, no. 2234, and BD Biosciences Pharmingen, no. 610017, respectively); and rabbit polyclonal anti-total actin (Sigma, no. A2066). Actin is shown as a loading control for all blots. The MET antibodies used detect both the 170-kDa precursor and 140-kDa mature forms of MET. For immunoblot analysis of endogenous MET in H820 and PC-9 cells (Fig. 2B), cells were grown in RPMI with serum and lysates were collected while cells were in log phase growth. For immunoblot analysis of H820 and PC-9 cells treated with XL880 or CL-387,785 (Fig. 3), cells were grown in RPMI with serum until 70-80% confluent and then incubated with either DMSO, XL880, or CL-387,785 for three hours before harvesting lysates. For all immunoblots shown in Fig. 3 and SI Fig. 6, H820 and PC-9 lysates were run together on the same gel and exposed together on the same film with equal exposure times. Results are displayed separated, because intervening lanes of lysates irrelevant to the report presented here are not shown.

siRNA.

 H820 and PC-9 cells were cultured in RPMI with 5% and 10% FBS, respectively. H820 and PC-9 cells were seeded into 6-well plates at 2 ´105 cells per well and 1 ´ 105 cells per well, respectively, and transfected with siRNAs against MET or GAPD after 24-h incubation at 37°C. Briefly, transfections were all carried out with 2 ml of DharmaFECT transfection reagent no. 1 per well and 100 nM siRNA final concentration in a total well volume of 2000 ml of RPMI with 5% and 10% FBS, for H820 and PC-9 cells, respectively. After 24-h incubation, the transfection media was replaced with fresh media. Protein lysates were collected as described after an additional 48 h (72 h posttransfection). In control experiments using fluorescently tagged siRNAs [siGLO Red Transfection Indicator (D-001630; Thermo Fisher Scientific)], we achieved >80% transfection efficiency in target cells (data not shown).

1. Livak KJ, Schmittgen TD (2001) Methods 25:402-408.

2. Olshen AB, Venkatraman ES, Lucito R, Wigler M (2004) Biostatistics (Oxford) 5:557-572.

3. Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, Scherer SW, Lee C (2004) Nat Genet 36:949-951.