PMC5602471_01
Histology of the left supraclavicular lymph node biopsy, August 2014.. Notes: Adenocarcinoma (A, hematoxylin & eosin stain, magnification, x200) harboring EGFR 19 exon deletion (B, ARMS). Strong expression of ALK protein with the monoclonal ALK antibody (C, clone D5F3, IHC, magnification, x200). Head MRI (D) and isotope bone scans (E) revealed metastatic mass lesions in the brain and bones. The first and third panels in E show images captured from the front, and the second and fourth panels show images captured from back. The first and second images were taken at a different time than the third and fourth images. CT scans of the thorax prior to and following treatment with gefitinib: (F) prior to treatment, (G) 7 months, and (H) 8 months after treatment with gefitinib. The red arrows indicate the tumor lesions. Rn is the ratio of the fluorescence emission intensity of target reporter genes to that of the standard dyes.  Rn=Rn-baseline. All image scans were captured in the Affiliated Cancer Hospital of Xiangya School of Medicine.. Abbreviations: ARMS, amplication-refractory mutation system assay; CT, computed tomography; EGFR, epidermal growth factor receptor; IHC, immunohistochemistry; MRI, magnetic resonance imaging; Rn, normalized reporter.
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A left supraclavicular lymph node surgical biopsy was conducted (size 1.5x1.5 cm), and the pathological diagnosis was adenocarcinoma (cT4N3M1ab according to the 7th AJCC/UICC tumor staging system) (Figure 1A), with EGFR exon 19 deletion proven using amplication-refractory mutation system assay (Figure 1B) and ALK showing up as positive using Ventana immunohistochemistry (IHC) (Figure 1C). The metastatic lesions in the brain and bones are shown in Figure 1D and E. The main target lesion in the left lung prior to the treatment is shown in Figure 1F. Evaluation performed at 1 month revealed partial response across all lesions, with minimal disease burden at 7 months (Figure 1G). However, CT scan showed progression of the lung lesions after 8 months, because of several new lesions in both lungs (Figure 1H).
CT scans of the thorax prior to and following treatment with gefitinib and crizotinib.. Notes: (A) Prior to crizotinib treatment. (B) 10 months after treatment with gefitinib and crizotinib. NGS results of metastatic lymph node biopsy in R4, April 2016 (red arrows indicate the tumor lesions): (C) deletion in EGFR exon 19 (p.747_750del); (D) mTOR mutation.. Abbreviations: CT, computed tomography; EGFR, epidermal growth factor receptor; mTOR, mammalian target of rapamycin; NGS, next-generation sequencing.
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During the treatment, the patient showed no major adverse events; however, CT scan still showed slow progression (Figure 2A and B). NGS results showed EGFR exon 19 deletion (allele frequency [AF] 36.2%, Figure 2C) with PIK3CA/Akt/mammalian target of rapamycin (mTOR) pathway amplifications (PIK3CA, Akt1, mTOR copy number of 3.46, 3.51, 3.09, respectively) and mTOR mutation (AF 68.4%, Figure 2D).
Histology of the metastatic lymph node biopsy in R4, April 2016.. Notes: (A) ALK-negative result on IHC (D5F3, magnification, x100). (B) ALK-negative result in FISH. A cutoff of >15% tumor cells harboring split signals was used to determine ALK-positive tumors. The results of the left supraclavicular lymph node biopsy, August 2014: (C) deletion in EGFR exon 19 (p.747_750del) as seen in NGS; FISH reveals a split of red and green probes; (D) ALK showed no signal in FISH.. Abbreviations: EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing.
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Due to the presence of ALK overexpression at initial diagnosis, we assessed ALK status using Ventana IHC (Figure 3A) and fluorescence in situ hybridization (FISH) (Figure 3B), both of which were negative. NGS (the supraclavicular lymph node biopsy specimen in August 2014, when NGS was not popular). NGS showed that the tissue biopsy obtained at initial diagnosis only harbored EGFR mutation in exon 19 deletion (AF 34%, Figure 3C), and ALK FISH also showed no signal (Figure 3D).
CT scans of the thorax prior to and following treatment with chemotherapy, everolimus and AZD9291 and NGS results of plasma ctDNA.. Notes: (A) After 2 cycles of chemotherapy treatment and prior to everolimus, the tumor progressed to the liver and contralateral lung (B) 1.5 months after everolimus treatment and prior to AZD9291 (red arrows indicate the tumor lesions). (C) Deletion in EGFR exon 19 (p.747_750del); (D) T790M in EGFR exon 20. (E) 1.4 months, and (F) 1.9 months after treatment with AZD9291.. Abbreviations: CT, computed tomography; ctDNA, circulating tumor DNA; EGFR, epidermal growth factor receptor; NGS, next-generation sequencing.
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After 2 cycles, CT showed that the disease had progressed to liver and both lungs (Figure 4A). Evaluation was performed after 1.5 months of treatment with everolimus, and CT showed stable disease (Figure 4B). T790M mutation (allele frequency 7.88%, Figure 4D). The patient was administered AZD 9291 at a dose of 80 mg/d and achieved partial response with significant reduction in the bilateral pulmonary and liver nodules after 1.4 months (Figure 4E) accompanied with the amelioration of left shoulder pain. AZD9291 for 1.9 months. A CT scan showed dramatic bulky lung disease (Figure 4F), and the patient passed away on September 30, 2016; the time to progression or death from the initial administration of AZD9291 for the patient was 1.9 months.