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A method of high-throughput functional evaluation of EGFR gene variants of unknown significance in cancer

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Science Translational Medicine  15 Nov 2017:
Vol. 9, Issue 416, eaan6566
DOI: 10.1126/scitranslmed.aan6566
  • Fig. 1. Schematic representation of the MANO method.

    Mouse 3T3 or Ba/F3 cells were infected with recombinant retrovirus expressing oncoproteins with corresponding 6-bp bar codes. Equal numbers of the stably transduced cells were mixed and cultured with different types of medium and/or treated with TKIs or vehicle. gDNA was harvested from the mixture of the remaining viable cells at the appropriate periods for each assay. The bar code sequences were PCR-amplified and subjected to deep sequencing on MiSeq sequencers to quantitate their relative abundance (a direct reflection of the cell number). To evaluate the transforming potential of each oncoprotein, the read number for each bar code was normalized to that of day 0. To evaluate the inhibition profile of test compounds across transduced clones in the mixture, the read number for each bar code was normalized to that of the vehicle-treated control. WT, wild-type.

  • Fig. 2. The MANO method in vitro and in vivo.

    (A) Mouse 3T3 cells were infected with a retrovirus expressing the oncoprotein shown at the top. The indicated number of cells was mixed together in the wells of a six-well tissue culture plate, and gDNA was prepared from the mixture the next day. The 6-bp bar codes were PCR-amplified and subjected to deep sequencing. The relative read numbers of bar codes in the mixtures were compared to those of the initial input of cells infected with a retrovirus for EGFR(L858R) (n = 4). (B) Mouse 3T3 cells were infected with a retrovirus expressing an oncogene or the corresponding wild-type gene (indicated on the right) and subjected to a focus-formation assay. On day 14, the cells were stained with Giemsa solution. A retrovirus expressing GFP was used as a negative control. (C) Temporal changes in the proportion of 3T3 or Ba/F3 cells expressing each oncoprotein, the corresponding wild-type protein, or GFP are shown with different colors as indicated in the lower right panel. Mouse 3T3 cells were either cultured in DMEM containing 10% FBS (upper left) or subcutaneously injected into nu/nu mice (upper right), and the relative proportion of cell clones was assessed at the indicated times using the MANO method. The read number for each cell clone was normalized to that of day 0. Ba/F3 cell clones expressing oncoproteins were cultured in vitro in RPMI 1640 without IL-3 and similarly analyzed using the MANO method (lower left). The yellow arrowhead indicates the complete depletion of several cell clones at day 6. (D) The correlation between the focus-formation assay and the MANO method. The Giemsa-stained area (%) in the 3T3 focus-formation assay in (B) was compared to the fold change of the read number on day 8 relative to day 0 in 3T3 cells cultured in vitro using the MANO method in (C). The Pearson’s correlation coefficient (r) was calculated.

  • Fig. 3. TKI sensitivity assessed by the MANO method.

    (A) Ba/F3 cells expressing each of the 16 genes shown on the right were mixed and cultured in the presence of different concentrations of gefitinib, erlotinib, afatinib, osimertinib, rociletinib, crizotinib, alectinib, or puromycin. Bar code read numbers of the compound-treated cells were normalized to those of the dimethyl sulfoxide (DMSO)–treated mixture, and the relative viability (%) of each cell clone on day 5 is color-coded according to the indicated scheme. (B) Comparison of cell viability measured with alamarBlue cell viability assay and the MANO method for Ba/F3 cells with 10 EGFR mutants in (A). Each data point was normalized to vehicle-treated cells. Pearson’s correlation coefficient (r) was calculated as 0.89 (P < 0.0001). The low ratio area is magnified in the right panel. (C) Changes in the relative cell populations in the tumors of mice treated with TKIs. The MANO method was used to quantify the bar code read numbers of tumors in 10 erlotinib-treated and 10 vehicle-treated mice. The bar code number for each cell line was normalized to the total bar code numbers of the tumor on day 18, and the calculated number was subsequently used to determine the percentage contribution of each cell line to the tumors. The mean relative cell population (%) within the tumors is shown for mice treated with either vehicle (green circle) or erlotinib (orange circle). The blue and pink arrows represent decrease and increase in the relative cell populations, respectively, in the tumors treated with erlotinib compared to those treated with vehicle. Error bars denote SD.

  • Fig. 4. Functional annotation of VUS in EGFR.

    (A) Venn diagram revealing the numbers of oncogenic EGFR mutants seen in 3T3 cells using a focus-formation assay (3T3 FF), 3T3 cells by the MANO method (3T3 MANO), Ba/F3 cells using the MANO method (Ba/F3 MANO), or BA/F3 cells individually examined for IL-3–independent growth (Ba/F3 individual). The method for the evaluation of the transforming activity in each assay is described in detail in the legend for fig. S6. (B) Ba/F3 cells expressing the L858R, A839T, or T790M_C797S_Cis mutants of EGFR were treated with the indicated concentrations of gefitinib, erlotinib, afatinib, or osimertinib for 72 hours. Viable cells (%) were measured using the alamarBlue cell viability assay, and the results are shown as the means of five independent experiments. (C) Ba/F3 cells expressing 86 EGFR mutants (indicated at top) were treated with either DMSO or EGFR TKIs (gefitinib, erlotinib, afatinib, osimertinib, or rociletinib) at 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM, and the relative viability of TKI-treated cells relative to the corresponding DMSO-treated cells is color-coded according to the scheme indicated at the top left. Missense, deletion, and insertion mutations are shown in green, brown, and red, respectively.

  • Fig. 5. Compound mutations in EGFR.

    (A) The frequency and patterns of EGFR compound mutations. Three hundred ninety cases formerly identified as positive for EGFR mutations (L858R, exon 19 deletion, G719 mutations, and L861Q) were examined for EGFR target sequence to investigate the frequency and the patterns of EGFR compound mutations. (B) The fold change in the ratio of 3T3 cell number with each EGFR compound mutation in cis (x axis) or trans (y axis) on day 12 compared with that of the corresponding EGFR single mutation is plotted. (C) Ba/F3 cells expressing 106 EGFR mutants (indicated at top) were treated with either DMSO or EGFR TKIs (gefitinib, erlotinib, afatinib, or osimertinib) at 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM, and the viability of TKI-treated cells relative to the corresponding DMSO-treated cells is color-coded according to the scheme indicated at the top left. (D) Ba/F3 cells expressing single or compound EGFR mutations (indicated at the right) were treated with the indicated concentrations of gefitinib, erlotinib, afatinib, or osimertinib for 72 hours without IL-3. The percentage of viable cells relative to that of parental Ba/F3 cells similarly treated in the presence of IL-3 was measured using the alamarBlue cell viability assay (n = 5). The mean median inhibitory concentration (IC50) values of the EGFR mutants for each TKI were calculated from five independent experiments.

  • Fig. 6. Cetuximab sensitivity of EGFR mutations assessed by the MANO method.

    (A) Ba/F3 cells expressing 158 EGFR mutants (indicated at top) were treated with either DMSO or cetuximab at 0.001, 0.01, 0.1, 1, 10, or 100 μg/ml, and the viability of cetuximab-treated cells relative to that of the corresponding DMSO-treated cells is color-coded according to the scheme indicated at the top right. (B) Ba/F3 cells expressing the indicated mutants of EGFR were treated with the indicated concentrations of cetuximab for 72 hours. Viable cells (%) were measured using the alamarBlue cell viability assay, and the results are shown as the means of five independent experiments. (C) Evaluation of cetuximab sensitivity in vivo. Mouse 3T3 cells (1 × 106) transfected with the indicated expression constructs were injected into the subcutaneous tissue of mice (n = 5 per group). Tumor volumes were calculated as described in Materials and Methods. Error bars, SD; *P < 0.01, the tumor volume of the cells expressing R108K_L858R_Cis (left) or L718Q_L858R_Cis (right) treated with vehicle is compared with that of the corresponding cells treated with cetuximab at day 20. P < 0.01, the tumor volume of the cells expressing R108K (left) or L718Q (right) treated with vehicle is compared with that of the corresponding cells treated with cetuximab at day 20 (left) or day 38 (right).

  • Fig. 7. Assessment of drug sensitivity for EGFR mutants.

    The drug sensitivities of the indicated EGFR mutants were evaluated using the MANO method in Ba/F3 cells. The drug sensitivity was categorized as sensitive, partially sensitive, or resistant based on the IC90 of each mutant with each drug.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/416/eaan6566/DC1

    Fig. S1. Quantification of focus-formation assay.

    Fig. S2. Temporal changes in the proportion of 3T3 cells expressing 25 different genes.

    Fig. S3. Transforming activity evaluation using the MANO method in vitro.

    Fig. S4. AlamarBlue cell viability assay in Ba/F3 cells expressing EGFR mutants.

    Fig. S5. In vivo evaluation of sensitivity to afatinib using the MANO method.

    Fig. S6. In vitro transforming activity of 101 EGFR mutants evaluated by the MANO method.

    Fig. S7. Immunoblot analysis of Ba/F3 cells expressing EGFR mutants.

    Fig. S8. The droplet digital PCR assay for the detection of EGFR E709A and L858R.

    Fig. S9. Focus-formation assay of 3T3 cells with EGFR L858R compound mutations.

    Fig. S10. Focus-formation assay of 3T3 cells expressing EGFR G719 compound mutations or exon 19 deletion compound mutations.

    Fig. S11. The sensitivity of EGFR mutants to dacomitinib, nazartinib, and neratinib.

    Table S1. The raw data of Fig. 3A (provided as an Excel file).

    Table S2. One hundred one recurrent EGFR mutations in COSMIC database analyzed with the MANO method (provided as an Excel file).

    Table S3. Barcode sequences used in the MANO method for EGFR compound mutations (provided as an Excel file).

    Table S4. The raw data of Fig. 4C (provided as an Excel file).

    Table S5. Recurrent NSCLC cases after gefitinib treatment without the EGFR T790M mutation.

    Table S6. Clinical and molecular characteristics of surgically resected lung adenocarcinoma with EGFR compound mutations (provided as an Excel file).

    Table S7. The frequency and pattern of E709 compound mutations.

    Table S8. The raw data of Fig. 5C (provided as an Excel file).

    Table S9. The raw data of Fig. 6A (provided as an Excel file).

    Table S10. Candidate drug for each EGFR mutation (provided as an Excel file).

  • Supplementary Material for:

    A method of high-throughput functional evaluation of EGFR gene variants of unknown significance in cancer

    Shinji Kohsaka,* Masaaki Nagano, Toshihide Ueno, Yoshiyuki Suehara, Takuo Hayashi, Naoko Shimada, Kazuhisa Takahashi, Kenji Suzuki, Kazuya Takamochi, Fumiyuki Takahashi, Hiroyuki Mano*

    *Corresponding author. Email: kohsakas{at}m.u-tokyo.ac.jp (S.K.); hmano{at}m.u-tokyo.ac.jp (H.M.)

    Published 15 November 2017, Sci. Transl. Med. 9, eaan6566 (2017)
    DOI: 10.1126/scitranslmed.aan6566

    This PDF file includes:

    • Fig. S1. Quantification of focus-formation assay.
    • Fig. S2. Temporal changes in the proportion of 3T3 cells expressing 25 different genes.
    • Fig. S3. Transforming activity evaluation using the MANO method in vitro.
    • Fig. S4. AlamarBlue cell viability assay in Ba/F3 cells expressing EGFR mutants.
    • Fig. S5. In vivo evaluation of sensitivity to afatinib using the MANO method.
    • Fig. S6. In vitro transforming activity of 101 EGFR mutants evaluated by the MANO method.
    • Fig. S7. Immunoblot analysis of Ba/F3 cells expressing EGFR mutants.
    • Fig. S8. The droplet digital PCR assay for the detection of EGFR E709A and L858R.
    • Fig. S9. Focus-formation assay of 3T3 cells with EGFR L858R compound mutations.
    • Fig. S10. Focus-formation assay of 3T3 cells expressing EGFR G719 compound mutations or exon 19 deletion compound mutations.
    • Fig. S11. The sensitivity of EGFR mutants to dacomitinib, nazartinib, and neratinib.
    • Table S5. Recurrent NSCLC cases after gefitinib treatment without the EGFR T790M mutation.
    • Table S7. The frequency and pattern of E709 compound mutations.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. The raw data of Fig. 3A (provided as an Excel file).
    • Table S2. One hundred one recurrent EGFR mutations in COSMIC database analyzed with the MANO method (provided as an Excel file).
    • Table S3. Barcode sequences used in the MANO method for EGFR compound mutations (provided as an Excel file).
    • Table S4. The raw data of Fig. 4C (provided as an Excel file).
    • Table S6. Clinical and molecular characteristics of surgically resected lung adenocarcinoma with EGFR compound mutations (provided as an Excel file).
    • Table S8. The raw data of Fig. 5C (provided as an Excel file).
    • Table S9. The raw data of Fig. 6A (provided as an Excel file).
    • Table S10. Candidate drug for each EGFR mutation (provided as an Excel file).

    [Download Supplementary Tables]

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