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Cancer Commun (Lond)
2018 May 09;381:19. doi: 10.1186/s40880-018-0284-1.
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Xenograft tumors derived from malignant pleural effusion of the patients with non-small-cell lung cancer as models to explore drug resistance.
BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions show dramatic responses to specific tyrosine kinase inhibitors (TKIs); however, after 10-12 months, secondary mutations arise that confer resistance. We generated a murine xenograft model using patient-derived NSCLC cells isolated from the pleural fluid of two patients with NSCLC to investigate the mechanisms of resistance against the ALK- and EGFR-targeted TKIs crizotinib and osimertinib, respectively.
METHODS: Genotypes of patient biopsies and xenograft tumors were determined by whole exome sequencing (WES), and patients and xenograft-bearing mice received targeted treatment (crizotinib or osimertinib) accordingly. Xenograft mice were also treated for prolonged periods to identify whether the development of drug resistance and/or treatment responses were associated with tumor size. Finally, the pathology of patients biopsies and xenograft tumors were compared histologically.
RESULTS: The histological characteristics and chemotherapy responses of xenograft tumors were similar to the actual patients. WES showed that the genotypes of the xenograft and patient tumors were similar (an echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) gene fusion (patient/xenograft: CTC15035EML4-ALK) and EGFR L858R and T790M mutations (patient/xenograft: CTC15063EGFR L858R, T790M)). After continuous crizotinib or osimertinib treatment, WES data suggested that acquired ALK E1210K mutation conferred crizotinib resistance in the CTC15035EML4-ALK xenograft, while decreased frequencies of EGFR L858R and T790M mutations plus the appearance of v-RAF murine sarcoma viral oncogene homolog B (BRAF) G7V mutations and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha (PIK3C2A) A86fs frame shift mutations led to osimertinib resistance in the CTC15063EGFR L858R, T790M xenografts.
CONCLUSIONS: We successfully developed a new method of generating drug resistance xenograft models from liquid biopsies using microfluidic technology, which might be a useful tool to investigate the mechanisms of drug resistance in NSCLC.
2016YFC1303300 National Key R&D Program of China, 81672272 National Natural Science Foundation of China, 201540365 Key Project of Shanghai Health & Family Planning Commission, 14140902800 Science and Technology Commission of Shanghai Municipality (STCSM), 16140902800 Science and Technology Commission of Shanghai Municipality (STCSM)
Fig. 2. Computed tomography (CT) scans and pathology for patient CTC15035EML4–ALK with stage IV bronchogenic carcinoma and the patient-derived xenograft tumors. a Baseline CT chest scan on 2014-11-14 showed there was a nodule in the middle lobe of the right lung and a small amount pleural effusion in the lower lobe of the right lung. b After two gemcitabine plus carboplatin chemotherapy cycles, CT of disease progression on 2014-12-23 showed the pleural effusion increased significantly compared with that of baseline. c After 2 months of crizotinib targeted therapy, CT chest scan on 2015-3-12 showed the disease was stable and the pleural effusion decreased compared with that of pre-crizotinib treatment. Pathology was examined by H&E staining of the tumor biopsy from d patient CTC15035EML4–ALK when pleural effusion increased significantly and, e a xenograft tumor from CTC15035EML4–ALK. Histology of xenograft tumors was well-matched with those of primary tumors
Fig. 3. Computed tomography (CT) scans and pathology for patient CTC15063EGFR L858R, T790M and the patient-derived xenograft tumors. CT chest scans of patient CTC15063EGFR L858R, T790M were recorded 4.5 years after undergoing surgical resection and subsequent adjuvant chemotherapy (navelbine plus cisplatin). a Disease recurrence was found in a CT scan as a right sub-pleural nodule on 2012-3-12; b disease progression 26 months after erlotinib treatment was visible in a CT scan as malignant pleural effusion on 2014-6-27; c CT reexamination after 2 months of osimertinib treatment showed stable disease on 2015-10-8. Pathology (H&E staining) of tumor biopsy from d patient CTC15063EGFR L858R, T790M and e a xenograft CTC15063EGFR L858R, T790M tumor after osimertinib treatment
Fig. 4. The effect of crizotinib on CTC15035EML4–ALK xenografts and their development of crizotinib resistance. a CTC15035EML4–ALK xenograft growth curves for female nu/nu mice (mean ± SEM, n = 6) treated with 50 mg/kg crizotinib or vehicle control for 21 days. b CTC15035EML4–ALK xenograft growth curves in six female nu/nu mice (crizotinib 1–6) for 114 days with 50 mg/kg crizotinib (mean ± SEM, n = 6). c Secondary CTC15035EML4–ALK xenografts derived from crizotinib-6 under 50 mg/kg crizotinib or vehicle control treatments (mean ± SEM, n = 6). d Fusion transcripts of EML4 Exon 18 to ALK Exon 20, which occurred in the tumor biopsy from patient CTC15035EML4–ALK and the CTC15035EML4–ALK xenografts, were identified by RNA amplification sequencing with an ultra-deep sequencing depth of approximately 50,000-fold of the EML4–ALK fusion locus; e the novel acquired ALK E1210K mutation in crizotinib-6 xenografts at a frequency of 9% (d, e are displayed with Integrative Genomics Viewer)
Fig. 5. The effect of osimertinib on CTC15063EGFR L858R, T790M xenografts tumor inhibition or tumor re-development due to osimertinib resistance. a CTC15063EGFR L858R, T790M xenograft growth curves for female nu/nu mice (mean ± SEM, n = 6) treated with 50 mg/kg erlotinib or vehicle control for 35 days. b CTC15063EGFR L858R, T790M xenograft growth curves for female nu/nu mice (mean ± SEM, n = 6) treated with 5 mg/kg osimertinib or vehicle for 24 days. c CTC15063EGFR L858R, T790M xenograft growth curves of six female nu/nu mice (osimertinib 1–6) treated with 5 mg/kg osimertinib for 95 days. d Secondary CTC15063EGFR L858R xenografts derived from osimertinib-3 under 5 mg/kg osimertinib or vehicle control treatments for 21 days (mean ± SEM, n = 6). e Frequencies of the EGFR L858R and T790M mutations in the tumor biopsy from patient CTC15063EGFR L858R, T790M (85.6% and 71.6%, respectively) were similar to those in the initial CTC15063EGFR L858R, T790M xenograft tumors (83.3% and 77.6%, respectively), but were higher than those in osimertinib-3-derived secondary xenografts (53.6% and 41.7%, respectively); f 33 novel secondary mutations were identified in osimertinib-3-derived xenografts, including BRAF (G7V) (11.54%) and PIK3C2A (A86fs) (13.64%)