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Disappearance of an activated EGFR mutation after treatment with EGFR tyrosine kinase inhibitors
Lung Cancer 78 (2012) 121–124
Contents lists available at SciVerse ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Case report
Disappearance of an activated EGFR mutation after treatment with EGFR tyrosine kinase inhibitors Yoshihiro Honda a , Nagio Takigawa b,∗ , Soichiro Fushimi c , Nobuaki Ochi a,b , Toshio Kubo a , Saeko Ozaki a , Mitsune Tanimoto a , Katsuyuki Kiura a a
Department of Respiratory Medicine, Okayama University Hospital, Japan Department of General Internal Medicine 4, Kawasaki Medical School, Japan c Department of Pathology, Okayama University Hospital, Japan b
a r t i c l e
i n f o
Article history: Received 6 March 2012 Received in revised form 23 June 2012 Accepted 8 July 2012 Keywords: Non-small-cell lung cancer Large-cell carcinoma Epidermal growth factor receptor Cell line
a b s t r a c t A 34-year-old Japanese woman presented with left supraclavicular lymph node swelling. Computed tomography scans revealed a mass on the left lower lobe, pulmonary nodules, and pleural effusion. A lymph node biopsy revealed large-cell carcinoma with an epidermal growth factor receptor (EGFR) deletion mutation, L747–T751 in exon 19. Although malignant pleural effusions carried the same EGFR mutation, progressive pleural effusions after treatment with chemotherapy, gefitinib, and erlotinib did not show any EGFR mutation. A cell line established from the pleural effusion 3 days before the patient expired also did not harbor the EGFR mutation. Histological sections of the lymph node of the patient were similar to those of the xenograft tumor of the cell line. There may be genetic heterogeneity in EGFR mutant tumors. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is the leading cause of cancer deaths worldwide. Although chemotherapy has advanced in the treatment of nonsmall-cell lung cancer (NSCLC), the prognosis is still poor [1]. Over the last decade, molecular-targeted agents have been introduced in the treatment of NSCLC. Gefitinib and erlotinib are active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, which have demonstrated significant activity in NSCLC with somatic mutations within the EGFR tyrosine kinase domain [2,3]. Many new findings have been made using EGFR-mutated lung cancer cell lines [4]. Here, we established a wild-type EGFR lung cancer cell line derived from the patient harboring an activating EGFR mutation and presume that the mutation disappeared after treatment with EGFR tyrosine kinase inhibitors. 2. Case presentation A 34-year-old Japanese woman presented with left supraclavicular lymph node swelling. Her performance status, as defined by Eastern Cooperative Oncology Group criteria, was 1. Computed
∗ Corresponding author at: Department of General Internal Medicine 4, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505, Japan. Tel.: +81 86 225 2111; fax: +81 86 232 8343. E-mail address: [email protected] (N. Takigawa). 0169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2012.07.003
tomography scans showed a mass on the left lower lobe, pulmonary nodules, and bilateral pleural effusion (Fig. 1). The supraclavicular lymph node biopsy revealed a large-cell carcinoma. Diagnosed with the lung cancer (T4N3M1, stage IV), she received a combination of cisplatin (80 mg/m2 , day 1) and docetaxel (60 mg/m2 , day 1), followed by gefitinib (250 mg/body), pemetrexed (500 mg/m2 , day 1), erlotinib (150 mg/body), or a combination of irinotecan (80 mg/m2 , days 1 and 8) and amrubicin (80 mg/m2 , days 1 and 8). Although the combination of irinotecan and amrubicin was our original regimen [5], others were standard first line chemotherapy or salvage treatments. With these treatments, she did not achieve a partial response, although the lymph node specimen carried an EGFR deletion mutation, L747–T751 in exon 19. CT image after gefitinib treatment was shown in Supplementary Fig. 1. Gefitinib and erlotinib were discontinued within 1 month and 2 months, respectively, because of disease progression. Although left pleural effusions carried the same EGFR mutation, the progressive pleural effusion after treatment with gefitinib and erlotinib did not harbor any EGFR mutation. At 3 days before the patient’s death, the left pleural effusion was punctured and cancer cells were obtained. The monoclonal cells were then separated using lymphocyte separation medium (Histopaque, Sigma Chemical Company, St. Louis, MO, USA). The cells were washed, resuspended in ACL-4 culture medium [6], and maintained in the culture medium for over 12 months at 37 ◦ C in 5% CO2 . The culture medium was replenished once per 10–14 days. Then, the cells were cultured in RPMI-1640 medium containing 20% fetal bovine serum, penicillin (100 U/mL),
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(Fig. 3D–F). The tumor had a solid pattern of irregular pleomorphic cells, with abundant compact, eosinophilic cytoplasm. Immunohistochemically, the tumor cells in both specimens were positive for pancytokeratin (AE1/AE3), cytokeratin 7, and vimentin (Fig. 4). Both S-100 and TTF-1 were negative. The LCC-1A and LCC-1B cells did not harbor EGFR mutations in exon 18–21. 3. Discussion
Fig. 1. Computed tomography scan image revealed a mass on the left lower lobe, pulmonary nodules, and bilateral pleural effusion.
and streptomycin (100 g/mL) for 6 months. The culture medium was replenished once per 7–10 days. Then, we did a single-cell cloning and established two cell lines (LCC-1A and LCC-1B). Female 7-week-old athymic mice were purchased from Charles River Laboratories Japan Inc. All mice were provided with sterilized food and water and housed in a barrier facility under a 12/12 h light/dark cycle. Cancer cells (2 × 106 ) were injected subcutaneously into the backs of the mice. EGFR mutations were examined using a commercially available PNA-LNA PCR clamp method [7]. Both LCC-1A and LCC-1B cells grew similarly as adherent cells in the flasks. Fig. 2A (40×) and 2B (100×) show the cytomorphology of LCC-1A. The xenograft tumor of the LCC-1A developed 32 days after injection (Fig. 2C); the indurated milky white section is shown in Fig. 2D. A xenograft tumor also developed from LCC1B cells (data not shown). Histology of the lymph node biopsy (Fig. 3A–C) was reminiscent of that of the LCC-1A xenograft tumor
The majority of EGFR mutant lung cancers initially sensitive to EGFR tyrosine kinase inhibitors become resistant to these agents within 1 year [2,3]. Some possible mechanisms for the acquired resistance have been identified, the most common being the development of an EGFR T790 M ‘gatekeeper’ mutation in around 50% of cases [4]. Other mechanisms of acquired resistance include MET amplification, small-cell transition, and epithelial–mesenchymal transition [4,8]. In approximately 30% of cases, the mechanism of acquired resistance is presently unknown [8]. The cell lines we established harbor neither the T790M mutation nor the original deletion mutation in exon 19. The disappearance of the EGFR mutation might be due to one of the acquired resistance mechanisms. That is, EGFR mutant cells could be excluded by the treatment with gefitinib and erlotinib, and subsequently, only wild-type EGFR cells might be selected. Because of the similarity of the histopathological features of the lymph node biopsy and the xenograft tumor, a contaminating subpopulation seemed unlikely. We need to further characterize this cell line and to clarify the oncogene driver of the cells. In conclusion, there may be genetic heterogeneity in EGFR mutant tumors. Doebele et al. recently described the mechanisms of resistance to crizotinib in patients with ALK gene rearranged NSCLC, showing emergence of an ALK gene fusion–negative tumor [9]. To the best of our knowledge, there have been no reports of disappearance of the EGFR mutation.
Fig. 2. A (40×) and B (100×) show the cytomorphology of LCC-1A. The xenograft tumor of the LCC-1A developed 32 days after injection (C); the indurated milky white section is shown in D.
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Fig. 3. Histology of the lymph node biopsy (A–C) was reminiscent of that of the LCC-1A xenograft tumor (D–F). The tumor had a solid pattern of irregular pleomorphic cells, with abundant compact, eosinophilic cytoplasm.
Fig. 4. The tumor cells in both specimens were positive for pancytokeratin (AE1/AE3), cytokeratin 7, and vimentin. S-100 and TTF-1 were negative.
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Conflict of interest of statement Drs. Takigawa and Kiura have received honoraria for lecturing from AstraZeneca KK and Chugai Pharmaceutical Co. Ltd. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. lungcan.2012.07.003. References [1] Smith W, Khuri FR. The care of the lung cancer patient in the 21st century: a new age. Semin Oncol 2004;31:11–5. [2] Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500.
[3] Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Branningan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39. [4] Gazdar AF. Epidermal growth factor receptor inhibition in lung cancer: the evolving role of individualized therapy. Cancer Metastasis Rev 2010;29:37–48. [5] Nogami N, Hotta K, Segawa Y, Takigawa N, Hosokawa S, Oze I, et al. Phase II study of irinotecan and amrubicin in patients with relapsed non-small cell lung cancer: Okayama Lung Cancer Study Group Trial 0402. Acta Oncol 2012;51:768–73. [6] Gazdar AF, Oie HK. Re: Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium. Cancer Res 1986;46:6011–2. [7] Nagai Y, Miyazawa H, Huqun, Tanaka T, Udagawa K, Kato M, et al. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res 2005;65:7276–82. [8] Oxnard GR, Arcila ME, Chmielecki J, Ladanyi M, Miller VA, Pao W. New strategies in overcoming acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer. Clin Cancer Res 2011;17:5530–7. [9] Doebele RC, Pilling AB, Aisner DL, Kutateladze TG, Le AT, Weickhardt AJ, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res 2012;18:1472–82.
Contents lists available at SciVerse ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Case report
Disappearance of an activated EGFR mutation after treatment with EGFR tyrosine kinase inhibitors Yoshihiro Honda a , Nagio Takigawa b,∗ , Soichiro Fushimi c , Nobuaki Ochi a,b , Toshio Kubo a , Saeko Ozaki a , Mitsune Tanimoto a , Katsuyuki Kiura a a
Department of Respiratory Medicine, Okayama University Hospital, Japan Department of General Internal Medicine 4, Kawasaki Medical School, Japan c Department of Pathology, Okayama University Hospital, Japan b
a r t i c l e
i n f o
Article history: Received 6 March 2012 Received in revised form 23 June 2012 Accepted 8 July 2012 Keywords: Non-small-cell lung cancer Large-cell carcinoma Epidermal growth factor receptor Cell line
a b s t r a c t A 34-year-old Japanese woman presented with left supraclavicular lymph node swelling. Computed tomography scans revealed a mass on the left lower lobe, pulmonary nodules, and pleural effusion. A lymph node biopsy revealed large-cell carcinoma with an epidermal growth factor receptor (EGFR) deletion mutation, L747–T751 in exon 19. Although malignant pleural effusions carried the same EGFR mutation, progressive pleural effusions after treatment with chemotherapy, gefitinib, and erlotinib did not show any EGFR mutation. A cell line established from the pleural effusion 3 days before the patient expired also did not harbor the EGFR mutation. Histological sections of the lymph node of the patient were similar to those of the xenograft tumor of the cell line. There may be genetic heterogeneity in EGFR mutant tumors. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is the leading cause of cancer deaths worldwide. Although chemotherapy has advanced in the treatment of nonsmall-cell lung cancer (NSCLC), the prognosis is still poor [1]. Over the last decade, molecular-targeted agents have been introduced in the treatment of NSCLC. Gefitinib and erlotinib are active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, which have demonstrated significant activity in NSCLC with somatic mutations within the EGFR tyrosine kinase domain [2,3]. Many new findings have been made using EGFR-mutated lung cancer cell lines [4]. Here, we established a wild-type EGFR lung cancer cell line derived from the patient harboring an activating EGFR mutation and presume that the mutation disappeared after treatment with EGFR tyrosine kinase inhibitors. 2. Case presentation A 34-year-old Japanese woman presented with left supraclavicular lymph node swelling. Her performance status, as defined by Eastern Cooperative Oncology Group criteria, was 1. Computed
∗ Corresponding author at: Department of General Internal Medicine 4, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505, Japan. Tel.: +81 86 225 2111; fax: +81 86 232 8343. E-mail address: [email protected] (N. Takigawa). 0169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2012.07.003
tomography scans showed a mass on the left lower lobe, pulmonary nodules, and bilateral pleural effusion (Fig. 1). The supraclavicular lymph node biopsy revealed a large-cell carcinoma. Diagnosed with the lung cancer (T4N3M1, stage IV), she received a combination of cisplatin (80 mg/m2 , day 1) and docetaxel (60 mg/m2 , day 1), followed by gefitinib (250 mg/body), pemetrexed (500 mg/m2 , day 1), erlotinib (150 mg/body), or a combination of irinotecan (80 mg/m2 , days 1 and 8) and amrubicin (80 mg/m2 , days 1 and 8). Although the combination of irinotecan and amrubicin was our original regimen [5], others were standard first line chemotherapy or salvage treatments. With these treatments, she did not achieve a partial response, although the lymph node specimen carried an EGFR deletion mutation, L747–T751 in exon 19. CT image after gefitinib treatment was shown in Supplementary Fig. 1. Gefitinib and erlotinib were discontinued within 1 month and 2 months, respectively, because of disease progression. Although left pleural effusions carried the same EGFR mutation, the progressive pleural effusion after treatment with gefitinib and erlotinib did not harbor any EGFR mutation. At 3 days before the patient’s death, the left pleural effusion was punctured and cancer cells were obtained. The monoclonal cells were then separated using lymphocyte separation medium (Histopaque, Sigma Chemical Company, St. Louis, MO, USA). The cells were washed, resuspended in ACL-4 culture medium [6], and maintained in the culture medium for over 12 months at 37 ◦ C in 5% CO2 . The culture medium was replenished once per 10–14 days. Then, the cells were cultured in RPMI-1640 medium containing 20% fetal bovine serum, penicillin (100 U/mL),
122
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(Fig. 3D–F). The tumor had a solid pattern of irregular pleomorphic cells, with abundant compact, eosinophilic cytoplasm. Immunohistochemically, the tumor cells in both specimens were positive for pancytokeratin (AE1/AE3), cytokeratin 7, and vimentin (Fig. 4). Both S-100 and TTF-1 were negative. The LCC-1A and LCC-1B cells did not harbor EGFR mutations in exon 18–21. 3. Discussion
Fig. 1. Computed tomography scan image revealed a mass on the left lower lobe, pulmonary nodules, and bilateral pleural effusion.
and streptomycin (100 g/mL) for 6 months. The culture medium was replenished once per 7–10 days. Then, we did a single-cell cloning and established two cell lines (LCC-1A and LCC-1B). Female 7-week-old athymic mice were purchased from Charles River Laboratories Japan Inc. All mice were provided with sterilized food and water and housed in a barrier facility under a 12/12 h light/dark cycle. Cancer cells (2 × 106 ) were injected subcutaneously into the backs of the mice. EGFR mutations were examined using a commercially available PNA-LNA PCR clamp method [7]. Both LCC-1A and LCC-1B cells grew similarly as adherent cells in the flasks. Fig. 2A (40×) and 2B (100×) show the cytomorphology of LCC-1A. The xenograft tumor of the LCC-1A developed 32 days after injection (Fig. 2C); the indurated milky white section is shown in Fig. 2D. A xenograft tumor also developed from LCC1B cells (data not shown). Histology of the lymph node biopsy (Fig. 3A–C) was reminiscent of that of the LCC-1A xenograft tumor
The majority of EGFR mutant lung cancers initially sensitive to EGFR tyrosine kinase inhibitors become resistant to these agents within 1 year [2,3]. Some possible mechanisms for the acquired resistance have been identified, the most common being the development of an EGFR T790 M ‘gatekeeper’ mutation in around 50% of cases [4]. Other mechanisms of acquired resistance include MET amplification, small-cell transition, and epithelial–mesenchymal transition [4,8]. In approximately 30% of cases, the mechanism of acquired resistance is presently unknown [8]. The cell lines we established harbor neither the T790M mutation nor the original deletion mutation in exon 19. The disappearance of the EGFR mutation might be due to one of the acquired resistance mechanisms. That is, EGFR mutant cells could be excluded by the treatment with gefitinib and erlotinib, and subsequently, only wild-type EGFR cells might be selected. Because of the similarity of the histopathological features of the lymph node biopsy and the xenograft tumor, a contaminating subpopulation seemed unlikely. We need to further characterize this cell line and to clarify the oncogene driver of the cells. In conclusion, there may be genetic heterogeneity in EGFR mutant tumors. Doebele et al. recently described the mechanisms of resistance to crizotinib in patients with ALK gene rearranged NSCLC, showing emergence of an ALK gene fusion–negative tumor [9]. To the best of our knowledge, there have been no reports of disappearance of the EGFR mutation.
Fig. 2. A (40×) and B (100×) show the cytomorphology of LCC-1A. The xenograft tumor of the LCC-1A developed 32 days after injection (C); the indurated milky white section is shown in D.
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Fig. 3. Histology of the lymph node biopsy (A–C) was reminiscent of that of the LCC-1A xenograft tumor (D–F). The tumor had a solid pattern of irregular pleomorphic cells, with abundant compact, eosinophilic cytoplasm.
Fig. 4. The tumor cells in both specimens were positive for pancytokeratin (AE1/AE3), cytokeratin 7, and vimentin. S-100 and TTF-1 were negative.
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Y. Honda et al. / Lung Cancer 78 (2012) 121–124
Conflict of interest of statement Drs. Takigawa and Kiura have received honoraria for lecturing from AstraZeneca KK and Chugai Pharmaceutical Co. Ltd. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. lungcan.2012.07.003. References [1] Smith W, Khuri FR. The care of the lung cancer patient in the 21st century: a new age. Semin Oncol 2004;31:11–5. [2] Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500.
[3] Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Branningan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39. [4] Gazdar AF. Epidermal growth factor receptor inhibition in lung cancer: the evolving role of individualized therapy. Cancer Metastasis Rev 2010;29:37–48. [5] Nogami N, Hotta K, Segawa Y, Takigawa N, Hosokawa S, Oze I, et al. Phase II study of irinotecan and amrubicin in patients with relapsed non-small cell lung cancer: Okayama Lung Cancer Study Group Trial 0402. Acta Oncol 2012;51:768–73. [6] Gazdar AF, Oie HK. Re: Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium. Cancer Res 1986;46:6011–2. [7] Nagai Y, Miyazawa H, Huqun, Tanaka T, Udagawa K, Kato M, et al. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res 2005;65:7276–82. [8] Oxnard GR, Arcila ME, Chmielecki J, Ladanyi M, Miller VA, Pao W. New strategies in overcoming acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer. Clin Cancer Res 2011;17:5530–7. [9] Doebele RC, Pilling AB, Aisner DL, Kutateladze TG, Le AT, Weickhardt AJ, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res 2012;18:1472–82.