Research ArticleExperimental Studies

Role of FOXO3A in Trastuzumab Combination Chemotherapy in Esophageal Squamous Cell Carcinoma

MASASHI TAKAHASHI, KAZUMASA FUKUDA, YOSHIRO SAIKAWA, RIEKO NAKAMURA, NORIHITO WADA, HIROFUMI KAWAKUBO, HIROYA TAKEUCHI and YUKO KITAGAWA
Anticancer Research April 2020, 40 (4) 1921-1930; DOI: https://doi.org/10.21873/anticanres.14147

Abstract

Background/Aim: Targeting of the human epidermal growth factor receptor 2 (HER2) is suggested to be beneficial for esophageal squamous cell carcinoma (ESCC) patients with HER2 amplification. In this study, we evaluated the effects of combination chemotherapy with HER2-targeted drug trastuzumab in ESCC cells and examined the underlying mechanism contributing to these effects. Materials and Methods: HER2 expression was verified, and the efficacy of chemotherapy with and without trastuzumab was investigated in vitro and in vivo. Results: The combination of trastuzumab and a combined-modality therapy stimulated the PI3K/Akt pathway in ESCC cells overexpressing HER2. Trastuzumab treatment resulted in the intranuclear accumulation of FOXO3A in ESCC xenografts overexpressing HER2. The combination of trastuzumab and a combined-modality therapy enhanced antitumor effects in HER2-overexpressing ESCC xenografts. Conclusion: FOXO3A plays an important role in mediating the effects of trastuzumab, and combination chemotherapy may be a promising treatment for patients with HER2-overexpressing ESCC.

Esophageal cancer has the eighth highest prevalence rate and is the seventh leading cause of death worldwide. Esophageal squamous cell carcinoma (ESCC) typically arises in the upper two-thirds of the intrathoracic esophagus. Recently, the postoperative survival of patients with ESCC has improved; however, the overall ESCC prognosis of patients remains poor. Therefore, the currently available treatment approaches, including surgical intervention, chemotherapy, and radiation therapy, are not effective in treating this devastating cancer. Continual efforts to discover new agents and treatment strategies for ESCC are being made.

In recent years, there has been remarkable progress in molecular targeted therapies for several cancer types. It has been hypothesized that cancer results from a conglomeration of mutations in various genes (1, 2), which are potential therapeutic targets contributing to individualized treatment development. Moreover, there have been efforts to re-classify cancers according to the therapeutic target molecules that they express, as an indication of the appropriate treatment strategy (3).

In recent reports, the high expression of human epidermal growth factor receptor 2 (HER2) has been identified in 13%-41% of ESCC cases (4-8). Thus, targeting HER2 is thought to be useful for ESCC patients with HER2 overexpression. The anti-HER2 antibody trastuzumab is considered an essential therapy for HER2-positive breast cancer (9-17). Combination therapies of trastuzumab with capecitabine or 5-fluorouracil (5FU) and cisplatin (CDDP) have also been used to treat HER2-overexpressing gastric cancer and gastroesophageal junction cancer (18).

Preclinical studies on HER2-targeting agents in HER2-amplified ESCC cells are limited, and more research is required to elucidate the effectiveness and mechanisms of HER2-targeting agents, alone or in combination with other anticancer drugs. Here, we examined the efficacy of a combined-modality therapy involving CDDP and 5FU (CF) with or without the HER2-targeted drug trastuzumab in ESCC cells and determined the underlying mechanisms of the effects. We focused on the forkhead box class O (FOXO) transcription factor FOXO3A. FOXO family members are essential downstream effectors of the phosphoinositide 3 kinase (PI3K)/RAC-α serine/threonine-protein kinase (Akt) pathway (19).

In this investigation, we examined the effects of trastuzumab alone or in combination with CF on HER2-positive ESCC cells in vitro and in vivo to determine its usefulness in clinical practice. By demonstrating that HER2 plays an important role in ESCC, individualized treatments can be designed for patients with ESCC to improve treatment outcomes.

Materials and Methods

Reagents. The anti-HER2 monoclonal antibody trastuzumab (Herceptin™) was obtained from Roche (Basel, Switzerland). CDDP (Briplatin™) was obtained from Bristol-Myers Squibb (New York, NY). 5FU was procured from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan).

Cell culture. ESCC cell lines (TE1, 4, 5, 6, 8, 9, 10, 11, 14, and 15) were provided by the Cell Resource Center for Biomedical Research at Tohoku University (Sendai, Japan). The authentication of each cell line was performed using short tandem repeat analysis. The cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10%-20% fetal bovine serum (FBS; Gibco™/Life Technologies, Carlsbad, CA, USA) and penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan) at 37°C in a humidified environment under 5% CO2/95% air.

Flow cytometry. Flow cytometry was performed to detect the expression of HER2 and Akt phosphorylation in ESCC cells after treatment. ESCC cells were treated with trypsin/ethylenedia-minetetraacetic acid (EDTA), centrifuged, and resuspended in Hanks' balanced salt solution containing 10 mM HEPES (Gibco™, Life Technologies) and 2% FBS. The cells were treated with Blocking One (Nacalai Tesque) for 20 min at room temperature, rinsed, and incubated with an anti-human HER2 antibody or an anti-human p-Akt antibody conjugated to fluorescence isothiocyanate (Pharmingen™, Becton, Dickinson, Franklin Lakes, NJ, USA) for 30 min at 4°C. Dead cells were identified by staining with 1 μg/ml propidium iodide (Sigma-Aldrich). Viable cells (1×106) were examined using the FACSVantage™ SE (Becton, Dickinson). The cells were assigned to the appropriate cell cycle phase using FlowJo software (Tomy Digital Biology, Tokyo, Japan).

Quantitative reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). First-strand cDNA was reverse transcribed from RNA using ReverTra Ace (TOYOBO, Osaka, Japan). PCR reactions containing cDNA samples and EagleTaq Master Mix with the ROX dye (Roche Applied Science, Penzberg, Germany) were run on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems™/Life Technologies) to evaluate the expression of HER2. TaqMan Gene Expression Assay primers and probes were used to amplify glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as the endogenous reference gene, and HER2 (Applied BiosystemsTM/Life Technologies). The PCR reaction parameters involved incubation at 95°C for 20 sec, followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. The results were analyzed using analytical software from Applied Biosystems™ (Life Technologies). The gene expression level was calculated using the 2−ΔΔCT method.

Survival analysis using antitumor agents. ESCC cells were plated into 96-well microplates and cultured for 12 h, followed by incubation with several concentrations of the drugs for 72 h. ESCC cells not exposed to any drug were used as the control group. The proliferation of these cells was evaluated using the water-soluble tetrazolium salt-8 assay (Nacalai Tesque). The optical density (OD) was measured using a Sunrise rainbow absorbance reader (Wako Pure Chemical Industries, Osaka, Japan). The inhibition rate was calculated as follows: Embedded Image

Measurement of antibody-dependent cellular cytotoxicity (ADCC). ESCC cells were plated in sterile 96-well microplates and cultured for 12 h. The cells were exposed to CF, trastuzumab, or the combination of CF and trastuzumab for 48 h, and then the drug-containing medium was replaced with fresh medium. ADCC was evaluated by culturing ESCC cells with peripheral blood mononuclear cells (PBMCs) and quantifying the lactate dehydrogenase (LDH) released from ESCC cells as a result of PBMC-induced ADCC. PBMCs were isolated from heparinized blood from a single healthy donor (MT) using Lymphocyte Separation Solution (Nacalai Tesque). The ESCC cells (target) and PBMCs (effector) were cultured at a 1:40 target:effector ratio in 100 μl DMEM containing 5% fetal calf serum in sterile 96-well microplates in triplicate for 6 h at 37°C, and the cell culture medium was analyzed by the LDH release assay (Roche Applied Science). The target cell spontaneous negative control was prepared in a similar way, i.e., treatment with CF, trastuzumab, or combined CF and trastuzumab, but without the PBMCs. In contrast, the effector cell spontaneous control did not contain the ESCC target cells. ESCC cells frozen at −80°C for 1 h and then exposed to 37°C served as the positive control (target maximum). The absorption at 490 nm was measured using a Sunrise rainbow absorbance reader, and the percentage cytotoxicity was calculated as follows: Embedded Image

In vivo multiple drug assay. Female athymic nude mice were reared under a 12 h:12 h light:dark cycle in an isolated facility with no restrictions on food or water. All animal studies adhered to the guidelines of the Animal Research and Care Committee of the School of Medicine at Keio University. Approximately 1.0×106 cancer cells were subcutaneously implanted into the backs of 6-week-old mice. Drug administration was initiated when the tumor size reached an average volume of 300 mm3. Mice were randomly assigned to control and treatment groups. The treatment groups (n=6/group) were administered CF alone, trastuzumab alone, or a combination of CF and trastuzumab. 5FU (20 μg/mg/day) was intraperitoneally administered on the first 5 days of a 2-week period. CDDP (5 μg/mg/day) and trastuzumab (5 μg/mg/day) were administered intraperitoneally on days 1 and 8. The control group (n=6) received sterile phosphate-buffered saline (PBS) intraperitoneally on days 1-5 and on day 8 of a 2-week period. Tumor volume was determined by vernier caliper measurements of tumor length (L) and width (W) and was calculated as LW2/2. Tumor size and body weight were measured three times a week. The proportion of tumor growth inhibition (TGI %) was evaluated as follows: TGI (%)=[1 − (tumor volume in the treatment group on evaluation day − tumor volume of the treatment group on day 1)/(tumor volume in the control group on evaluation day − tumor volume in the control group on day 1)] ×100. The percentage of body weight change (BWC %) was calculated as follows: BWC (%)=[(BW on evaluation day) - (BW on day 1)] / (BW on day 1) ×100.

Immunohistochemical (IHC) staining. Tumors were fixed in 4% paraformaldehyde for 1 day at room temperature. For HER2 IHC staining, tumor tissues were sectioned at a thickness of 4 μm and placed on aminosilane-coated glass slides (Matsunami Glass, Osaka, Japan). Slides were incubated with the blocking reagent N101 (Wako Pure Chemical Industries) for 20 min. After rinsing with PBS containing 0.05% Tween 20, the blocking of endogenous avidin and biotin was performed by incubating with H2O2 for 15 min. Slides were incubated with an anti-human HER2 monoclonal antibody (Cell Signaling Technology), followed by EnVision™ (Agilent, Santa Clara, CA, USA) as the secondary antibody for 30 min. Diaminobenzidine staining was then performed for 3-5 min. Slides were counterstained with hematoxylin and then cover-slipped using AquatexTM (Merck, Darmstadt, Germany).

Cell staining and counting evaluation. Tissue samples were evaluated under a light microscope. HER2 positivity was defined as strong membrane and cytoplasmic staining in at least 50%-75% of cells. We also evaluated the nuclear translocation of FOXO3A. Intranuclear accumulation was defined as strong nuclear staining compared with cytoplasmic staining in at least 50%-75% of cells. On each slide, the total cell numbers were measured in three selected fields of view using Image J software (National Institute of Health, Bethesda, MD, USA). The number of cells exhibiting FOXO3A nuclear translocation was measured using a combination of visual assessments and Image J software. The evaluation of each slide was performed by two independent investigators who were blinded to the group assignments of the slides.

Statistical analysis. Data were analyzed using SPSS for Windows version 21.0 (SPSS, Chicago, IL, USA). All values are presented as means±standard deviation. Continuous variables were compared using the unpaired Student's t-test or Mann–Whitney U-test. A probability (p) value <0.05 was considered to indicate statistical significance.

Results

Profile and drug sensitivity of high HER2-expressing cells. Various ESCC cell lines (TE1, 4, 5, 6, 8, 9, 10, 11, 14, and 15) were examined for the expression of HER2 by flow cytometry and real-time PCR. Fluorescence-activated cell sorting showed that HER2 expression in TE4 cells was considerably higher than in other cell lines (p<0.05). The HER2 mRNA level in TE4 cells was also higher than in the other cell lines evaluated. Thus, among the ESCC cell lines examined, HER2 was considered to be overexpressed in TE4 cells, which were denoted as HER2 positive (HER2+). In contrast, compared with TE4 cells, HER2 expression was considerably lower in TE11 cells, which were denoted as HER2 negative (HER2−) (Table I). To investigate drug susceptibility, the effects of 5FU, CDDP, and CF on the growth of HER2+ TE4 cells and HER2− TE11 cells were evaluated. HER2+ TE4 and HER2− TE11 cells showed sensitivity to 5FU, with IC50 values of 1.44±0.24 μg/ml and 1.24±0.03 μg/ml, respectively, and sensitivity to CDDP, with IC50 values of 1.72±0.32 and 0.64±0.09, respectively. Therefore, HER2+ TE4 cells showed greater resistance to CF compared with HER2− TE11 cells (Figure 1).

View this table:
Table I.

HER2 status of each line.

Antitumor effects of CF and trastuzumab on HER2-amplified ESCC cell lines. We evaluated ADCC induced by PBMCs after exposure to CF alone, trastuzumab alone, or CF combined with trastuzumab by measuring LDH release from HER2+ TE4 and HER2− TE11 cells (Figure 2). The ADCC activity was markedly higher in HER2+ TE4 cells treated with trastuzumab and CF combined with trastuzumab than in those treated with CF alone. This effect was not detected in HER2− TE11 cells. These data indicate that trastuzumab enhances ADCC in HER2-amplified ESCC cells but not in those without HER2 amplification.

Effect of CF combined with trastuzumab on Akt signaling in ESCC cells. HER2 signaling is mediated by the PI3K/Akt pathway. To investigate the antitumor mechanism of CF and trastuzumab combination therapy, we evaluated the effect of trastuzumab on the Akt signaling pathway. In the HER2+ TE4 cells, CF and trastuzumab treatment decreased Akt phosphorylation compared with the controls (treatment with standalone CF or standalone trastuzumab) (Figure 3). These results indicate that the combination of trastuzumab and CF deactivates the PI3K/Akt pathway.

Effects of trastuzumab and CF combination therapy on HER2-overexpressing human ESCC xenograft models. The anticancer effects of trastuzumab combined with CF therapy were evaluated in high and low HER2-expressing ESCC xenograft models. All treatments were tolerated by both high and low HER2-expressing ESCC xenograft mice during therapy (Figure 4-A, B). In addition, tumor volume was compared among the groups on the final day of the experiment. The tumor size after CF and trastuzumab combination therapy was 0.50±0.25 g, whereas tumor sizes were 1.19±0.28 g, 0.78±0.28 g, and 0.99±0.44 g after the control, CF alone, and trastuzumab alone treatments, respectively. The TGI% was 80.9% after CF and trastuzumab combination treatment and 57.7% and 20.4% after CF alone and trastuzumab alone treatments, respectively. Therefore, CF and trastuzumab combination therapy prevented tumor growth from HER2+ TE4 cells compared with either agent alone (p<0.05) (Figure 4C). In contrast, the additional effect of trastuzumab on CF therapy was not observed in the HER2− TE11 cells (Figure 4D). HER2 and FOXO3A protein expression was measured by IHC in the tumors of each group. HER2 expression in TE4 cells was high, regardless of the therapy (Figure 5A, B). The intranuclear accumulation of FOXO3A in TE4 cells was observed in the trastuzumab alone and CF and trastuzumab combination groups compared with the control and CF alone groups (Figure 6A, B). However, intranuclear accumulation in TE11 cells was not observed in the control, CF alone, trastuzumab alone, or CF and trastuzumab combination groups (Figure 6C, D). Thus, the CF and trastuzumab combination therapy appeared to enhance the anticancer efficacy of either drug alone in HER2-amplified ESCC xenografts.

Figure 1.
Figure 1.

Anti-proliferative effects of 5FU monotherapy, CDDP monotherapy, and CF combination therapy in vitro. HER2-overexpressing TE4 cells or low HER2-expressing TE11 cells were cultured in the supplemented medium for 12 h and then incubated with 5FU (0.1-30 μg/ml), CDDP (0.3-100 μg/ml), or CF (0.3-100 μg/ml) for 72 h. 5FU: 5-fluorouracil; CDDP: cisplatin; CF: cisplatin and 5-fluorouracil.

Figure 2.
Figure 2.

The effect of CF and trastuzumab on ADCC in HER2-amplified ESCC cells. HER2-overexpressing TE4 cells and low HER2-expressing TE11 cells were treated with CF alone, trastuzumab alone, or the combination of CF and trastuzumab for 48 h. ADCC was measured using a 6-h LDH release assay. CF: Cisplatin and 5-fluorouracil; SD: standard deviation.

Figure 3.
Figure 3.

The effect of CF and trastuzumab on cell signaling. Cells were treated with CF alone, trastuzumab alone, or the combination of CF and trastuzumab for 48 h. (A, B) Cells with p-Akt activity were detected by flow cytometry. The frequency of p-Akt activity was lower in HER2-overexpressing cells treated with the combination of CF and trastuzumab than in cells treated with either agent alone. CF: Cisplatin and 5-fluorouracil; Tmab: Trastuzumab; SD: standard deviation.

Discussion

The antitumor efficacy of 5FU was almost equal in HER2+ TE4 cells and HER2− TE11 cells. TE4 cells were less sensitive to CDDP than TE11 cells, suggesting that HER2-overexpressing ESCC cells are less sensitive to platinating agents. This suggests that HER2+ TE4 cells are more resistant to anticancer drugs and may exhibit higher biological malignancy.

The ADCC activity was higher in HER2+ TE4 cells treated with trastuzumab and CF combined with trastuzumab than in those treated with CF alone. This shows that trastuzumab enhances ADCC in HER2+ TE4 cells and that ADCC is not affected by the cytocidal anticancer drugs.

We evaluated the effects of trastuzumab alone or along with CF therapy in ESCC cells expressing high levels of HER2. Trastuzumab and CF inhibited p-Akt levels in HER2-amplified TE4 cells. These data indicate that the combination of trastuzumab and CF can deactivate the PI3K/Akt pathway. Previous studies report that the activation of HER2 may lead to downstream signaling that activates mitogenic and cell survival pathways, such as the PI3K/Akt pathway (20, 21). Inhibition of these pathways by HER2 antagonists such as trastuzumab can lead to inhibition of cell proliferation (22). The results of our study suggest that the combination of trastuzumab and CF could enhance the antitumor effects by inhibition of the PI3K/Akt pathway.

Figure 4.
Figure 4.

Antitumor effects of trastuzumab and CF on tumor growth in high and low HER2-expressing xenograft models. ESCC cells were injected subcutaneously into nude mice, and the mice were randomized into four groups (n=6/group). Treatment with CF, trastuzumab, sterile PBS (control), or combination CF/trastuzumab was started when the tumors in each group reached an average volume of 300 mm3. Tumor size and BW were measured every 2-3 days. (A and B: TE4 cells, C and D: TE11 cells). (A, B) All treatments were tolerated by the high and low HER2-expressing ESCC xenograft mice during therapy. (C) CF and trastuzumab combination therapy prevented tumor growth from high HER2-expressing TE4 cells compared with either agent alone (p<0.05). (D) The additional effect of trastuzumab on CF therapy was not observed in the low HER2-expressing TE11 cells. The results are presented as mean. Student's t-test was used to compare tumor size among the groups at the end of the experiment. Error bars represent standard deviation of the mean (*p<0.05). CF: Cisplatin and 5-fluorouracil; SD: standard deviation.

CF and trastuzumab combination therapy prevented tumor growth from HER2+ TE4 cells compared with either agent alone in ESCC xenograft models. All treatments were tolerable in ESCC xenograft models. This suggests that trastuzumab may be safe and effective in HER2-amplified ESCC patients and patients with breast or gastric cancer.

HER2 expression in HER2+ TE4 cells was high, regardless of the treatment. This indicates that trastuzumab did not decrease HER2 levels in HER2+ TE4 cells.

The intranuclear accumulation of FOXO3A in HER2+ TE4 cells was observed after treatment with trastuzumab regardless of the CF-combined use. The FOXO proteins are important for regulating metabolism, cell proliferation, cell survival, tolerance to stress, and cell lifespan. FOXO3A is an important factor for the growth and survival of malignant cells (23-25). Upon activation, FOXO3A translocates from the cytoplasm into the nucleus, where it activates genes that are responsible for inducing apoptosis, cell cycle arrest, inhibition of DNA repair, and suppression of oxidative stress tolerance. In brief, the activation of FOXO3A promotes tumor control. The results of this study show that the mechanism of trastuzumab's effectiveness may be due to the nuclear accumulation of FOXO3A.

There are very few studies of HER2-targeted agents applied in ESCC models. Furthermore, the signaling mechanism of HER2-targeted agents has not been elucidated. In the present study, we showed that trastuzumab decreased Akt phosphorylation, in addition to ADCC activities, in HER2-overexpressing ESCC cells, but not in low HER2-expressing ESCC cells. In the TE4 cell-derived xenografts, CF and trastuzumab combination therapy substantially inhibited tumor growth. In these xenografts, HER2 expression was not downregulated by trastuzumab monotherapy or CF and trastuzumab combination therapy compared with the control and CF therapy, suggesting that trastuzumab does not downregulate HER2 expression. The intranuclear accumulation of FOXO3A was observed after trastuzumab monotherapy and CF and trastuzumab combination therapy compared with the control and CF therapy, suggesting that trastuzumab increases the nuclear accumulation of FOXO3A. These results indicate that the antitumor activity of trastuzumab in ESCC cells with high HER2 expression is mediated by the intranuclear accumulation of FOXO3A.

Figure 5.
Figure 5.

HER2 expression of tumor after trastuzumab and CF treatment in high and low HER2-expressing xenograft models. Tumor tissues in each treatment group were examined for the expression of HER2 protein by IHC. (A) All TE4 cell groups were strongly positive for expression, and (B) all TE11 cell groups were negative. CF: Cisplatin and 5-fluorouracil.

In terms of biomarkers for gastrointestinal cancers, Ras in colorectal cancer and HER2 in gastric cancer have been used as predictive markers of chemotherapy (18, 26-29). In both colorectal and gastric cancers, a classification system according to biomarker expression has been proposed (30-32). HER2-targeted therapy was first used in breast cancer (9-17). This indicates that treatments targeting a certain molecule may be effective for multiple cancers. For example, anaplastic lymphoma kinase is activated by point mutations and gene fusion and is thought to cause a wide range of cancers in multiple organs, including lung cancer, malignant lymphoma, kidney cancer, neuroblastoma, thyroid cancer, and other organs (3). In the future, cancer classification and treatments may be tailored based on the target molecule. In fact, basket trials, which are clinical trials of multiple carcinomas with gene mutations that are targets for molecular targeted drugs, are underway (33-35), as are umbrella trials, which carry out several treatments targeting various molecules and subgroups for one carcinoma (35, 36). In ESCC, HER2-expressing ESCC cells showed low susceptibility to CDDP and were more susceptible to trastuzumab. HER2 may be an effective predictive marker of the effects of anticancer agents. There is a possibility that ESCC will also be classified into a molecular subgroup based on their HER2 status, as well as breast cancer and gastric cancer. Importantly, FOXO3A may play a role as an effective predictive marker for the action of trastuzumab.

In conclusion, the present study demonstrates that the combination of trastuzumab and CF enhances antitumor activity in HER2-overexpressing ESCC cells and that FOXO3A plays an important role in mediating the effects of trastuzumab. In addition, HER2 amplification is the best predictive marker for the anti-proliferative effects of CF and trastuzumab combination chemotherapy. The combination of trastuzumab and CF may be a promising therapeutic strategy for some patients with HER2-amplified ESCC.

Figure 6.
Figure 6.

FOXO3A expression of tumor after trastuzumab and CF treatment in high and low HER2-expressing xenograft models. Tumor tissues in each treatment group were examined for the expression of FOXO3A protein by IHC. (A, B) Nuclear accumulation of FOXO3A was observed in trastuzumab-treated TE4 cells (C, D) but not in TE11 cells. CF: Cisplatin and 5-fluorouracil.

Acknowledgements

The Authors thank Yumi Yoshimura for technical support with the experiments.

Footnotes

  • Author's Contributions

    MT and KF designed the study. MT carried out all experiments and drafted the manuscript. KF and YS supervised all experiments performed. RN, NW, HK, and HT participated in the data interpretation. As the principal investigator, YK supervised the study. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors report grants from CHUGAI PHARMACEUTICAL CO., LTD., Yakult Honsha Co. Ltd., and Kyouwa Hakkou Kirin Co., Ltd. over the 36 months prior to submission of this work.

  • Received February 11, 2020.
  • Revision received February 19, 2020.
  • Accepted February 20, 2020.

References

  1. ↵
    1. Bailey P,
    2. Chang DK,
    3. Nones K,
    4. Johns AL,
    5. Patch AM,
    6. Gingras MC,
    7. Miller DK,
    8. Christ AN,
    9. Bruxner TJ,
    10. Quinn MC,
    11. Nourse C,
    12. Murtaugh LC,
    13. Harliwong I,
    14. Idrisoglu S,
    15. Manning S,
    16. Nourbakhsh E,
    17. Wani S,
    18. Fink L,
    19. Holmes O,
    20. Chin V,
    21. Anderson MJ,
    22. Kazakoff S,
    23. Leonard C,
    24. Newell F,
    25. Waddell N,
    26. Wood S,
    27. Xu Q,
    28. Wilson PJ,
    29. Cloonan N,
    30. Kassahn KS,
    31. Taylor D,
    32. Quek K,
    33. Robertson A,
    34. Pantano L,
    35. Mincarelli L,
    36. Sanchez LN,
    37. Evers L,
    38. Wu J,
    39. Pinese M,
    40. Cowley MJ,
    41. Jones MD,
    42. Colvin EK,
    43. Nagrial AM,
    44. Humphrey ES,
    45. Chantrill LA,
    46. Mawson A,
    47. Humphris J,
    48. Chou A,
    49. Pajic M,
    50. Scarlett CJ,
    51. Pinho AV,
    52. Giry-Laterriere M,
    53. Rooman I,
    54. Samra JS,
    55. Kench JG,
    56. Lovell JA,
    57. Merrett ND,
    58. Toon CW,
    59. Epari K,
    60. Nguyen NQ,
    61. Barbour A,
    62. Zeps N,
    63. Moran-Jones K,
    64. Jamieson NB,
    65. Graham JS,
    66. Duthie F,
    67. Oien K,
    68. Hair J,
    69. Grutzmann R,
    70. Maitra A,
    71. Iacobuzio-Donahue CA,
    72. Wolfgang CL,
    73. Morgan RA,
    74. Lawlor RT,
    75. Corbo V,
    76. Bassi C,
    77. Rusev B,
    78. Capelli P,
    79. Salvia R,
    80. Tortora G,
    81. Mukhopadhyay D,
    82. Petersen GM,
    83. Munzy DM,
    84. Fisher WE,
    85. Karim SA,
    86. Eshleman JR,
    87. Hruban RH,
    88. Pilarsky C,
    89. Morton JP,
    90. Sansom OJ,
    91. Scarpa A,
    92. Musgrove EA,
    93. Bailey UM,
    94. Hofmann O,
    95. Sutherland RL,
    96. Wheeler DA,
    97. Gill AJ,
    98. Gibbs RA,
    99. Pearson JV,
    100. Biankin AV,
    101. Grimmond SM
    : Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531(7592): 47-52, 2016. PMID: 26909576. DOI: 10.1038/nature16965
    OpenUrlCrossRefPubMed
  2. ↵
    1. Saito M,
    2. Shiraishi K,
    3. Kunitoh H,
    4. Takenoshita S,
    5. Yokota J,
    6. Kohno T
    : Gene aberrations for precision medicine against lung adenocarcinoma. Cancer Sci 107(6): 713-720, 2016. PMID: 4968599. DOI: 10.1111/cas.12941
    OpenUrlCrossRefPubMed
  3. ↵
    1. Mano H
    : ALKoma: a cancer subtype with a shared target. Cancer Discov 2(6): 495-502, 2012. PMID: 22614325. DOI: 10.1158/2159-8290.CD-12-0009
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Zhan N,
    2. Dong WG,
    3. Tang YF,
    4. Wang ZS,
    5. Xiong CL
    : Analysis of HER2 gene amplification and protein expression in esophageal squamous cell carcinoma. Med Oncol 29(2): 933-940, 2012. PMID: 21318736. DOI: 10.1007/s12032-011-9850-y
    OpenUrlCrossRefPubMed
    1. Sato-Kuwabara Y,
    2. Neves JI,
    3. Fregnani JH,
    4. Sallum RA,
    5. Soares FA
    : Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using fluorescence in situ hybridization (fish) and immunohistochemistry. BMC Cancer 9(1): 6, 2009. PMID: 2648997. DOI: 10.1186/1471-2407-9-6
    OpenUrlCrossRefPubMed
    1. Mimura K,
    2. Kono K,
    3. Hanawa M,
    4. Mitsui F,
    5. Sugai H,
    6. Miyagawa N,
    7. Ooi A,
    8. Fujii H
    : Frequencies of HER-2/neu expression and gene amplification in patients with oesophageal squamous cell carcinoma. Br J Cancer 92(7): 1253-1260, 2005. PMID: 2361961. DOI: 10.1038/sj.bjc.6602499
    OpenUrlCrossRefPubMed
    1. Gonzaga IM,
    2. Soares-Lima SC,
    3. de Santos PT,
    4. Blanco TC,
    5. de Reis BS,
    6. Quintella DC,
    7. de Oliveira IM,
    8. de Faria PA,
    9. Kruel CD,
    10. Andreollo NA,
    11. de Simao TA,
    12. Pinto LF
    : Alterations in epidermal growth factor receptors 1 and 2 in esophageal squamous cell carcinomas. BMC Cancer 12(1): 569, 2012. PMID: 3537527. DOI: 10.1186/1471-2407-12-569
    OpenUrlCrossRefPubMed
  8. ↵
    1. Dreilich M,
    2. Wanders A,
    3. Brattstrom D,
    4. Bergstrom S,
    5. Hesselius P,
    6. Wagenius G,
    7. Bergqvist M
    : Her-2 overexpression (3+) in patients with squamous cell esophageal carcinoma correlates with poorer survival. Dis Esophagus 19(4): 224-231, 2006. PMID: 16866851. DOI: 10.1111/j.1442-2050.2006.00570.x
    OpenUrlCrossRefPubMed
  9. ↵
    1. Baselga J,
    2. Bradbury I,
    3. Eidtmann H,
    4. Di Cosimo S,
    5. de Azambuja E,
    6. Aura C,
    7. Gomez H,
    8. Dinh P,
    9. Fauria K,
    10. Van Dooren V,
    11. Aktan G,
    12. Goldhirsch A,
    13. Chang TW,
    14. Horvath Z,
    15. Coccia-Portugal M,
    16. Domont J,
    17. Tseng LM,
    18. Kunz G,
    19. Sohn JH,
    20. Semiglazov V,
    21. Lerzo G,
    22. Palacova M,
    23. Probachai V,
    24. Pusztai L,
    25. Untch M,
    26. Gelber RD,
    27. Piccart-Gebhart M
    : Lapatinib with trastuzumab for HER2-positive early breast cancer (neoaltto): a randomised, open-label, multicentre, phase 3 trial. Lancet 379(9816): 633-640, 2012. PMID: 22257673. DOI: 10.1016/S0140-6736(11)61847-3
    OpenUrlCrossRefPubMed
    1. Dawood S,
    2. Broglio K,
    3. Buzdar AU,
    4. Hortobagyi GN,
    5. Giordano SH
    : Prognosis of women with metastatic breast cancer by HER2 status and trastuzumab treatment: an institutional-based review. J Clin Oncol 28(1): 92-98, 2010. PMID: 2799236. DOI: 10.1200/JCO.2008.19.9844
    OpenUrlAbstract/FREE Full Text
    1. Gianni L,
    2. Eiermann W,
    3. Semiglazov V,
    4. Manikhas A,
    5. Lluch A,
    6. Tjulandin S,
    7. Zambetti M,
    8. Vazquez F,
    9. Byakhow M,
    10. Lichinitser M,
    11. Climent MA,
    12. Ciruelos E,
    13. Ojeda B,
    14. Mansutti M,
    15. Bozhok A,
    16. Baronio R,
    17. Feyereislova A,
    18. Barton C,
    19. Valagussa P,
    20. Baselga J
    : Neoadjuvant chemotherapy with trastuzumab followed by adjuvant trastuzumab versus neoadjuvant chemotherapy alone, in patients with HER2-positive locally advanced breast cancer (the noah trial): a randomised controlled superiority trial with a parallel HER2-negative cohort. Lancet 375(9712): 377-384, 2010. PMID: 20113825. DOI: 10.1016/S0140-6736(09)61964-4
    OpenUrlCrossRefPubMed
    1. Piccart-Gebhart MJ,
    2. Procter M,
    3. Leyland-Jones B,
    4. Goldhirsch A,
    5. Untch M,
    6. Smith I,
    7. Gianni L,
    8. Baselga J,
    9. Bell R,
    10. Jackisch C,
    11. Cameron D,
    12. Dowsett M,
    13. Barrios CH,
    14. Steger G,
    15. Huang CS,
    16. Andersson M,
    17. Inbar M,
    18. Lichinitser M,
    19. Lang I,
    20. Nitz U,
    21. Iwata H,
    22. Thomssen C,
    23. Lohrisch C,
    24. Suter TM,
    25. Ruschoff J,
    26. Suto T,
    27. Greatorex V,
    28. Ward C,
    29. Straehle C,
    30. McFadden E,
    31. Dolci MS,
    32. Gelber RD
    : Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353(16): 1659-1672, 2005. PMID: 16236737. DOI: 10.1056/NEJMoa052306
    OpenUrlCrossRefPubMed
    1. Romond EH,
    2. Perez EA,
    3. Bryant J,
    4. Suman VJ,
    5. Geyer CE Jr..,
    6. Davidson NE,
    7. Tan-Chiu E,
    8. Martino S,
    9. Paik S,
    10. Kaufman PA,
    11. Swain SM,
    12. Pisansky TM,
    13. Fehrenbacher L,
    14. Kutteh LA,
    15. Vogel VG,
    16. Visscher DW,
    17. Yothers G,
    18. Jenkins RB,
    19. Brown AM,
    20. Dakhil SR,
    21. Mamounas EP,
    22. Lingle WL,
    23. Klein PM,
    24. Ingle JN,
    25. Wolmark N
    : Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353(16): 1673-1684, 2005. PMID: 16236738. DOI: 10.1056/NEJMoa052122
    OpenUrlCrossRefPubMed
    1. Slamon D,
    2. Eiermann W,
    3. Robert N,
    4. Pienkowski T,
    5. Martin M,
    6. Press M,
    7. Mackey J,
    8. Glaspy J,
    9. Chan A,
    10. Pawlicki M,
    11. Pinter T,
    12. Valero V,
    13. Liu MC,
    14. Sauter G,
    15. von Minckwitz G,
    16. Visco F,
    17. Bee V,
    18. Buyse M,
    19. Bendahmane B,
    20. Tabah-Fisch I,
    21. Lindsay MA,
    22. Riva A,
    23. Crown J
    : Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med 365(14): 1273-1283, 2011. PMID: 3268553. DOI: 10.1056/NEJMoa0910383
    OpenUrlCrossRefPubMed
    1. Slamon DJ,
    2. Leyland-Jones B,
    3. Shak S,
    4. Fuchs H,
    5. Paton V,
    6. Bajamonde A,
    7. Fleming T,
    8. Eiermann W,
    9. Wolter J,
    10. Pegram M,
    11. Baselga J,
    12. Norton L
    : Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344(11): 783-792, 2001. PMID: 11248153. DOI: 10.1056/NEJM200103153441101
    OpenUrlCrossRefPubMed
    1. Untch M,
    2. Rezai M,
    3. Loibl S,
    4. Fasching PA,
    5. Huober J,
    6. Tesch H,
    7. Bauerfeind I,
    8. Hilfrich J,
    9. Eidtmann H,
    10. Gerber B,
    11. Hanusch C,
    12. Kuhn T,
    13. du Bois A,
    14. Blohmer JU,
    15. Thomssen C,
    16. Dan Costa S,
    17. Jackisch C,
    18. Kaufmann M,
    19. Mehta K,
    20. von Minckwitz G
    : Neoadjuvant treatment with trastuzumab in HER2-positive breast cancer: results from the geparquattro study. J Clin Oncol 28(12): 2024-2031, 2010. PMID: 20308670. DOI: 10.1200/JCO.2009.23.8451
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Vogel CL,
    2. Cobleigh MA,
    3. Tripathy D,
    4. Gutheil JC,
    5. Harris LN,
    6. Fehrenbacher L,
    7. Slamon DJ,
    8. Murphy M,
    9. Novotny WF,
    10. Burchmore M,
    11. Shak S,
    12. Stewart SJ,
    13. Press M
    : Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20(3): 719-726, 2002. PMID: 11821453. DOI: 10.1200/JCO.2002.20.3.719
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Bang YJ,
    2. Van Cutsem E,
    3. Feyereislova A,
    4. Chung HC,
    5. Shen L,
    6. Sawaki A,
    7. Lordick F,
    8. Ohtsu A,
    9. Omuro Y,
    10. Satoh T,
    11. Aprile G,
    12. Kulikov E,
    13. Hill J,
    14. Lehle M,
    15. Ruschoff J,
    16. Kang YK
    : Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (toga): A phase 3, open-label, randomised controlled trial. Lancet 376(9742): 687-697, 2010. PMID: 20728210. DOI: 10.1016/S0140-6736(10)61121-X
    OpenUrlCrossRefPubMed
  19. ↵
    1. Brunet A,
    2. Bonni A,
    3. Zigmond MJ,
    4. Lin MZ,
    5. Juo P,
    6. Hu LS,
    7. Anderson MJ,
    8. Arden KC,
    9. Blenis J,
    10. Greenberg ME
    : Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96(6): 857-868, 1999. PMID: 10102273. DOI: 10.1016/s0092-8674(00)80595-4
    OpenUrlCrossRefPubMed
  20. ↵
    1. Liu W,
    2. Li J,
    3. Roth RA
    : Heregulin regulation of akt/protein kinase B in breast cancer cells. Biochem Biophys Res Commun 261(3): 897-903, 1999. PMID: 10441522. DOI: 10.1006/bbrc.1999.1144
    OpenUrlCrossRefPubMed
  21. ↵
    1. Sliwkowski MX,
    2. Lofgren JA,
    3. Lewis GD,
    4. Hotaling TE,
    5. Fendly BM,
    6. Fox JA
    : Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin Oncol 26: 60-70, 1999. PMID: 10482195.
    OpenUrlPubMed
  22. ↵
    1. Nagata Y,
    2. Lan KH,
    3. Zhou X,
    4. Tan M,
    5. Esteva FJ,
    6. Sahin AA,
    7. Klos KS,
    8. Li P,
    9. Monia BP,
    10. Nguyen NT,
    11. Hortobagyi GN,
    12. Hung MC,
    13. Yu D
    : PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer cell 6(2): 117-127, 2004. PMID: 15324695. DOI: 10.1016/j.ccr.2004.06.022
    OpenUrlCrossRefPubMed
  23. ↵
    1. Tran H,
    2. Brunet A,
    3. Griffith EC,
    4. Greenberg ME
    : The many forks in FOXO's road. Sci STKE 2003(172): RE5, 2003. PMID: 12621150. DOI: 10.1126/stke.2003.172.re5
    OpenUrlCrossRefPubMed
    1. Burgering BM,
    2. Kops GJ
    : Cell cycle and death control: long live Forkheads. Trends Biochem Sci 27(7): 352-360, 2002. PMID: 12114024. DOI: 10.1016/s0968-0004(02)02113-8
    OpenUrlCrossRefPubMed
  25. ↵
    1. Accili D,
    2. Arden KC
    : FOXOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117(4): 421-426, 2004. PMID: 15137936. DOI: 10.1016/s0092-8674(04)00452-0
    OpenUrlCrossRefPubMed
  26. ↵
    1. Amado RG,
    2. Wolf M,
    3. Peeters M,
    4. Van Cutsem E,
    5. Siena S,
    6. Freeman DJ,
    7. Juan T,
    8. Sikorski R,
    9. Suggs S,
    10. Radinsky R,
    11. Patterson SD,
    12. Chang DD
    : Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 26(10): 1626-1634, 2008. PMID: 18316791. DOI: 10.1200/JCO.2007.14.7116
    OpenUrlAbstract/FREE Full Text
    1. Karapetis CS,
    2. Khambata-Ford S,
    3. Jonker DJ,
    4. O'Callaghan CJ,
    5. Tu D,
    6. Tebbutt NC,
    7. Simes RJ,
    8. Chalchal H,
    9. Shapiro JD,
    10. Robitaille S,
    11. Price TJ,
    12. Shepherd L,
    13. Au HJ,
    14. Langer C,
    15. Moore MJ,
    16. Zalcberg JR
    : K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 359(17): 1757-1765, 2008. PMID: 18946061. DOI: 10.1056/NEJMoa0804385
    OpenUrlCrossRefPubMed
    1. Dahabreh IJ,
    2. Terasawa T,
    3. Castaldi PJ,
    4. Trikalinos TA
    : Systematic review: Anti-epidermal growth factor receptor treatment effect modification by KRAS mutations in advanced colorectal cancer. Ann Intern Med 154(1): 37-49, 2011. PMID: 21200037. DOI: 10.7326/0003-4819-154-1-201101040-00006
    OpenUrlCrossRefPubMed
  29. ↵
    1. Douillard JY,
    2. Oliner KS,
    3. Siena S,
    4. Tabernero J,
    5. Burkes R,
    6. Barugel M,
    7. Humblet Y,
    8. Bodoky G,
    9. Cunningham D,
    10. Jassem J,
    11. Rivera F,
    12. Kocakova I,
    13. Ruff P,
    14. Blasinska-Morawiec M,
    15. Smakal M,
    16. Canon JL,
    17. Rother M,
    18. Williams R,
    19. Rong A,
    20. Wiezorek J,
    21. Sidhu R,
    22. Patterson SD
    : Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 369(11): 1023-1034, 2013. PMID: 24024839. DOI: 10.1056/NEJMoa1305275
    OpenUrlCrossRefPubMed
  30. ↵
    1. Guinney J,
    2. Dienstmann R,
    3. Wang X,
    4. de Reynies A,
    5. Schlicker A,
    6. Soneson C,
    7. Marisa L,
    8. Roepman P,
    9. Nyamundanda G,
    10. Angelino P,
    11. Bot BM,
    12. Morris JS,
    13. Simon IM,
    14. Gerster S,
    15. Fessler E,
    16. De Sousa EMF,
    17. Missiaglia E,
    18. Ramay H,
    19. Barras D,
    20. Homicsko K,
    21. Maru D,
    22. Manyam GC,
    23. Broom B,
    24. Boige V,
    25. Perez-Villamil B,
    26. Laderas T,
    27. Salazar R,
    28. Gray JW,
    29. Hanahan D,
    30. Tabernero J,
    31. Bernards R,
    32. Friend SH,
    33. Laurent-Puig P,
    34. Medema JP,
    35. Sadanandam A,
    36. Wessels L,
    37. Delorenzi M,
    38. Kopetz S,
    39. Vermeulen L,
    40. Tejpar S
    : The consensus molecular subtypes of colorectal cancer. Nat Med 21(11): 1350-1356, 2015. PMID: 4636487. DOI: 10.1038/nm.3967
    OpenUrlCrossRefPubMed
    1. Cancer Genome Atlas Research Network
    . Comprehensive molecular characterization of gastric adenocarcinoma. Nature 513(7517): 202-209, 2014. PMID: 4170219. DOI: 10.1038/nature13480
    OpenUrlCrossRefPubMed
  32. ↵
    1. Matboli M,
    2. El-Nakeep S,
    3. Hossam N,
    4. Habieb A,
    5. Azazy AE,
    6. Ebrahim AE,
    7. Nagy Z,
    8. Abdel-Rahman O
    : Exploring the role of molecular biomarkers as a potential weapon against gastric cancer: a review of the literature. World J Gastroenterol 22(26): 5896-5908, 2016. PMID: 4948264. DOI: 10.3748/wjg.v22.i26.5896
    OpenUrlPubMed
  33. ↵
    1. Heinrich MC,
    2. Joensuu H,
    3. Demetri GD,
    4. Corless CL,
    5. Apperley J,
    6. Fletcher JA,
    7. Soulieres D,
    8. Dirnhofer S,
    9. Harlow A,
    10. Town A,
    11. McKinley A,
    12. Supple SG,
    13. Seymour J,
    14. Di Scala L,
    15. van Oosterom A,
    16. Herrmann R,
    17. Nikolova Z,
    18. McArthur AG
    : Phase II, open-label study evaluating the activity of imatinib in treating life-threatening malignancies known to be associated with imatinib-sensitive tyrosine kinases. Clin Cancer Res 14(9): 2717-2725, 2008. PMID: 18451237. DOI: 10.1158/1078-0432.CCR-07-4575
    OpenUrlAbstract/FREE Full Text
    1. Hyman DM,
    2. Puzanov I,
    3. Subbiah V,
    4. Faris JE,
    5. Chau I,
    6. Blay JY,
    7. Wolf J,
    8. Raje NS,
    9. Diamond EL,
    10. Hollebecque A,
    11. Gervais R,
    12. Elez-Fernandez ME,
    13. Italiano A,
    14. Hofheinz RD,
    15. Hidalgo M,
    16. Chan E,
    17. Schuler M,
    18. Lasserre SF,
    19. Makrutzki M,
    20. Sirzen F,
    21. Veronese ML,
    22. Tabernero J,
    23. Baselga J
    : Vemurafenib in multiple nonmelanoma cancers with BRAF V600 mutations. N Engl J Med 373(8): 726-736, 2015. PMID: 4971773. DOI: 10.1056/NEJMoa1502309
    OpenUrlCrossRefPubMed
  35. ↵
    1. Menis J,
    2. Hasan B,
    3. Besse B
    : New clinical research strategies in thoracic oncology: clinical trial design, adaptive, basket and umbrella trials, new end-points and new evaluations of response. Eur Respir Rev 23(133): 367-378, 2014. PMID: 25176973. DOI: 10.1183/09059180.00004214
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Kim ES,
    2. Herbst RS,
    3. Wistuba II,
    4. Lee JJ,
    5. Blumenschein GR Jr..,
    6. Tsao A,
    7. Stewart DJ,
    8. Hicks ME,
    9. Erasmus J Jr..,
    10. Gupta S,
    11. Alden CM,
    12. Liu S,
    13. Tang X,
    14. Khuri FR,
    15. Tran HT,
    16. Johnson BE,
    17. Heymach JV,
    18. Mao L,
    19. Fossella F,
    20. Kies MS,
    21. Papadimitrakopoulou V,
    22. Davis SE,
    23. Lippman SM,
    24. Hong WK
    : The battle trial: personalizing therapy for lung cancer. Cancer Discov 1(1): 44-53, 2011. PMID: 4211116. DOI: 10.1158/2159-8274.CD-10-0010
    OpenUrlAbstract/FREE Full Text
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Role of FOXO3A in Trastuzumab Combination Chemotherapy in Esophageal Squamous Cell Carcinoma
MASASHI TAKAHASHI, KAZUMASA FUKUDA, YOSHIRO SAIKAWA, RIEKO NAKAMURA, NORIHITO WADA, HIROFUMI KAWAKUBO, HIROYA TAKEUCHI, YUKO KITAGAWA
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