Case report: EGFR-mutant lung adenocarcinoma with the TP53 and RB1 mutations showed resistance to TKI therapy

Introduction

Lung cancer is the most common cause of cancer-related death worldwide and is classified into 2 broad histological subtypes: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) (1,2). Over the past decades, genomic alterations in cancer driver genes have been identified in NSCLC, and targeted therapies have become the standard care for NSCLC patients. Epidermal growth factor receptor (EGFR) gene mutations can be detected in approximately 40–50% of Chinese patients with NSCLC (3), and EGFR tyrosine kinase inhibitors (TKIs) therapy has been recommended as the first-line treatment for EGFR-mutated advanced NSCLC (4). Many patients respond well to EGFR-TKIs and enter prolonged remission; however, some experience disease progression due to acquired resistance. Several mechanisms of acquired resistance to EGFR-TKIs have been identified, such as the acquired T790M mutation, which is the primary mechanism of resistance to first-generation EGFR-TKIs, mesenchymal epithelial transition factor receptor (MET) amplification, the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation, the B-Raf proto-oncogene (BRAF) mutation, and the transformation of adenocarcinoma with high-grade neuroendocrine carcinoma to large cell neuroendocrine carcinoma (LCNEC), SCLC, and their combined type (5). Studies have shown that TP53 inactivation and retinoblastoma protein (RB1) loss might be the potential mechanisms underlying SCLC phenotype conversion after TKI resistance (6). However, the prognosis after SCLC diagnosis remains poor and current treatment strategies derived from primary SCLC seem to be ineffective.

In this article, we present 2 cases of patients who were initially diagnosed with lung adenocarcinoma harboring the EGFR mutation. Following the emergence of EGFR-TKI resistance, the TP53 and RB1 mutations were revealed, and a transformation from lung adenocarcinoma to SCLC was observed in 1 patient. Such findings indicate the limited benefit of EGFR-TKIs to patients with the EGFR, TP53, and RB1 methanations. We present the following article in accordance with the CARE reporting checklist (available at https://dx.doi.org/10.21037/apm-21-2016).

Case presentation

Case 1

A 57-year-old man who had never smoked presented at our hospital with cough and fever in 2016. His performance status was 0 at diagnosis. A computed tomography (CT) scan of the chest revealed a mass in the dorsal segment and posterior basal segment and effusion in the left thoracic cavity. Positron emission tomography (PET)/CT scans further revealed the metastasis of multiple lymph nodes in the bilateral subclavian fossa, mediastinum, the left lung hilar and para-aortic lymph nodes, and the liver (S6). The lung biopsy pathology result showed that the tumor cells were positive for carcinoembryonic antigen (CEA), cytokeratin (CK)18, CK7, epithelial membrane antigen, napsin A, synaptophysin, thyroid transcription factor 1, and Ki-67 (80%), but negative for chromogranin A, CK14, CK20, CK5/6, tumor protein (p)40, and p63 (see Figure 1). This patient was diagnosed with poorly differentiated lung adenocarcinoma with moderately differentiated neuroendocrine tumors. The clinical tumor (T), nodes (N), and metastases (M) stage was T2bN2M1b, stage IV. To determine the potential therapeutic regimens, a tumor sample was sent for a next-generation sequencing (NGS) analysis using a panel of 450 cancer-related genes. The results revealed the EGFR L858R, RB1 R445*, and TP53 Y205C mutations. Informed consent was obtained from the patient.

Figure 1 Lung and liver specimens from Case 1. Photomicrograph of lung biopsy with H&E staining. Immunohistochemical analysis showing tumor cells positive for synaptophysin (+) and napsin-A (+).

The timeline of the progress of the disease is shown in Figure 2. In April 2016, the patient underwent 6 cycles of pemetrexed (800 mg) plus cisplatin (400 mg) chemotherapy. 6 months later, a PET/CT scan showed a good partial response (PR). During re-examination in January 2017, a CT scan showed an enlargement of the mass in the left lower lobe and a doubling of the extent of lung metastasis, suggesting progressive disease (PD). The patient was then treated with erlotinib (150 mg once daily) targeting the EGFR L858R mutation. However, after 3 months, CT scan showed new lesions in the liver (S6). A blood sample was subjected to NGS analysis in April 2017, which revealed an EGFR T790M mutation in addition to the existing EGFR L858R, RB1 R445*, and TP53 Y205C mutations. Osimertinib therapy (80 mg once daily) was commenced immediately; however, the disease progressed rapidly, with enlarged lesions presenting in the lung and liver. The patient was then treated with docetaxel monotherapy, but the disease progressed, and multiple brain metastases and enlarged lesions in the lung and liver were observed. A liver biopsy showed metastases of lung adenocarcinoma, and an NGS analysis of the liver specimen showed MET amplification in addition to the existing EGFR L858R, TP53, and RB1 mutations. The patient was then treated with crizotinib (250 mg once daily) plus erlotinib (125 mg once daily). Unfortunately, he died 1 month after starting this therapy.

Figure 2 The treatment history of Case 1. NGS results, medical information, and a CT scan are shown. NGS, next-generation sequencing.

Case 2

A 66-year-old female patient underwent surgery 6 years ago for a benign bone tumor of the left lower extremity. On April 05, 2018, she attended another hospital for treatment due to repeated fever. A chest CT scan showed a left lung mass with pleural effusion, and after a bronchoscopy pathological examination, she was diagnosed with lung cancer. The patient was treated with oral Chinese medicine (and did not receive standard treatment). On August 2, 2018, the patient was admitted to our hospital, as she had been experiencing left lower limb pain for more than 1 month. Thoracic and abdominal CT scans showed a hilus pulmonis mass in the inferior lobe of the left lung, multiple pulmonary nodules, an enlarged lymph node in the left hilus pulmonis, and a bone change in the left iliac crest and left 6th anterior rib, indicating a high possibility of lung cancer with metastasis (see Figure 3). A pathological examination of the bronchoscopy on August 3, 2018 revealed moderately differentiated lung adenocarcinoma at T4N1M1 stage IV (see Figure 4). The patient’s immunohistochemistry were as follows: TTF-1(+), Napsin-A(+), CK7(+), CK5/6(−). On August 7, 2018, whole-body bone imaging showed abnormal bone metabolism in the left 7th rib, the left iliac wing, and medial iliac crest, and the upper right femur, indicating bone metastasis.

Figure 3 The treatment history of Case 2. NGS results, medical information, and a CT scan are shown. NGS, next-generation sequencing.

Figure 4 H&E staining of bronchoscopy on August 3, 2018 showed lung adenocarcinoma, and H&E staining of the left lung tissue on July 23, 2019 revealed SCLC. SCLC, small-cell lung cancer.

On August 3, 2018, the lesion in the inferior lobe of the left lung was examined using NGS, and EGFR L747 and TP53 R342* mutations were detected. To target the EGFR mutation, the patient began oral icotinib (125 mg, tid) treatment on August 20, 2018, after which the lesions continued to shrink, and the efficacy evaluation was PR. The results of a NGS analysis of a blood sample on January 2, 2019 showed that the EGFR L747 mutation had disappeared, which indicated that the EGFR-TKI treatment of had been effective. However, upon being re-examined on May 21, 2019, an enlarged tumor in the inferior lobe of the left lung and lymph node enlargement in the right hilum and mediastinum were observed (see Figure 3), and the efficacy evaluation was progressed disease (PD). On May 21, 2019, another NGS analysis of a blood sample was conducted, and EML4 exon6-ALK EXON20 fusion, EGFR L747, PIK3CA, TP53 R342*, and RB1 mutations were revealed. Oral icotinib (125 mg, qd) plus crizotinib (200 mg, bid) was administered from June 6, 2019 to July 22, 2019. However, a CT re-examination showed increased tumor volume and lymph node, demonstrating PD.

On July 23, 2019, left lung tissue was taken for a puncture biopsy and the patient was diagnosed with SCLC (see Figure 4). The immunohistochemical results showed CK(+), CK7(−), TTF-1(+), Syn(+), CD56(−), CgA(+), and Ki-67 (>95%). The puncture biopsy tissue was also sent for a NGS analysis, and EGFR L747, TP53, and RB1 mutations were found. On July 27, 2019, 1 course of EP chemotherapy (etoposide 0.1 g d1–3+ cisplatin 30 mg d1–2) was started, and the patient’s condition improved and the chemotherapy was well tolerated. Due to the existence of the EGFR mutation, EP+EGFR-TKI therapy was subsequently applied. A chest CT re-examination on August 5, 2019 showed that the tumor size was smaller than before. From September 2019 to December 2019, 4 courses of EP+EGFR-TKI were continued, and the re-examination in November 2019 indicated stable disease (SD). However, PD was observed.

On December 23, 2019, a puncture biopsy of the left lung mass was performed, which showed a complex of neuroendocrine carcinoma, SCLC, and lung adenocarcinoma, and a NGS analysis was performed, which revealed EGFR L747, TP53 R342*, and RB1 mutations. Icotinib treatment was stopped. On January 7, 2020, bevacizumab (400 mg) plus albumin paclitaxel (400 mg) were administered, and on February 18, 2020, anvetin plus albumin paclitaxel were administered for 1 course. A re-examination in March 2020 showed PD, and oral anlotinib (12 mg) treatment began on March 20, 2020. Unfortunately, the patient died in April 2020.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

The transformation of NSCLC to SCLC is observed in approximately 3–14% of EGFR0-mutated NSCLC patients with acquired EGFR-TKI resistance (7), and the co-mutation of RB1 and TP53 is virtually a universal feature of SCLC. In Case 2, the patient was initially diagnosed with lung adenocarcinoma with EGFR and TP53 mutations. After the EGFR-TKI (icotinib) therapy, the EGFR mutation disappeared, and PR was achieved with progression free survival (PFS) for 9 months. However, PD was observed and the patient was diagnosed with SCLC with EGFR, TP53, and RB1 mutations. In relation to the mechanism of transformation in this case, it may be that both lung adenocarcinoma and SCLC were present at the initial diagnosis due to the tumor heterogeneity; however, the adenocarcinoma was only revealed by the pathological biopsy. After EGFR-TKI therapy, the adenocarcinoma cells were successfully killed, and the SCLC component survived and became dominant (8). Additionally, the emergence of the TP53 and RB1 mutations are likely to be an important driver of SCLC transformation, given that TP53 inactivation and RB1 loss are common in transformed SCLC (9).

In Case 1, the patient was initially diagnosed with lung adenocarcinoma with neuroendocrine differentiation harboring the EGFR, TP5, and RB1 mutations. NSCLC with uncertain neuroendocrine differentiation (3%) can be further classified as LCNEC (3%) (10), which harbors components of adenocarcinoma, squamous cell carcinoma, giant cell carcinoma, and spindle cell carcinoma (11). In another study, 45 paired samples of tumor and normal tissue from LCNEC patients were subjected to the targeted sequencing of 241 cancer-related genes, and divided into the following 3 subsets: (I) the SCLC-like subset, which was characterized by co-altered TP53 and RB1; (II) the NSCLC-like subset, which was characterized by lack of co-altered TP53 and RB1; and (III) the carcinoid-like subset, which was characterized by the mutations of STK11, KRAS, KEAP1, and NFE2L2 (12). In addition, Miyoshi et al. (13) observed inactivating mutations in TP53 (71%) and RB1 (26%) in samples from 78 LCNEC patients. Based on the molecular characteristics, our patient in Case 1 is more similar to the LCNEC-SCLC-like subset than the other subsets. It may be that in the middle or late stage, the disease is very likely to transform into SCLC or mixed lung cancer. However, as only limited tissue was available for biopsy, further differential diagnoses could not be performed.

Transformed SCLC has many of the characteristics of classical SCLC (14). The most effective treatment strategies of transformed SCLC should refer to the standard treatment of classic SCLC. As SCLCs with EGFR mutations were reported to be sensitive to EGFR-TKIs (15), it is speculated that EGFR-TKIs may also be effective in treating NSCLC-transformed SCLC. Chen et al. reported on a patient with adenocarcinoma that transformed to SCLC after treatment with a 3rd-generation-TKI for 4.3 months. After developing a resistance to EGFR-TKI, the patient was treated with a variety of regimens, including etoposide combined with carboplatin (EC), irinotecan combined with oxaliplatin (IO), Abraxane, and Apatinib. However, quick progression was observed (16). Lai et al. presented 2 cases in which the patients diagnoses transformed to SCLC after 1st- and 3rd-generation EGFR-TKI resistance and response to EP regimen and erlotinib; these patients passed away due to acute pulmonary embolism or severe pneumonia (17).

A recent study demonstrated that the median PFS (mPFS) for 1st-/2nd-generation TKIs in NSCLC patients with EGFR mutations was 14.0 months, and the most common mutations identified in samples with transformation to SCLC were TP53 (17/25, 68.0%), RB1 (9/25, 36.0%), and PIK3CA (3/25, 12.0%). After SCLC transformation, platinum-etoposide was the most common treatment regimen. The earlier occurrence of SCLC transformation after EGFR-TKI resistance was associated with poorer prognosis (18). Similarly, in Case 1, the patient was not responsive to erlotinib or osimertinib even in the presence of L858R and T790M mutations. MET amplification, which has long been known to be an important mechanism of resistance to EGFR-TKI therapy in NSCLC, was later identified in the liver specimen. Combining crizotinib with EGFR-TKI may achieve a better clinical response (19). However, this patient was also resistant to crizotinib plus erlotinib therapy. The patient achieved PR after paclitaxel-cisplatin chemotherapy treatment, but died 1 month later. In Case 2, the patient achieved PR after icotinib and had a PFS period of 9 months. However, EGFR-TKI resistance was observed and SCLC transformation was identified. Subsequent treatment, including EP, EP + icotinib, bevacizumab + albumin paclitaxel, anvetin + albumin paclitaxel, and anlotinib, did not have good efficacy.

In summary, we reported on the cases of 2 patients with lung adenocarcinoma with EGFR mutations who developed a resistance to EGFR-TKI. One patient transformed to SCLC after EGFR-TKI resistance and additional TP53, and RB1 mutations were identified. Subsequent treatments did not have satisfactory efficacy, and the patients’ prognoses were poor. Repeated biopsies and broader molecular analyses, such as NGS, are necessary to identify changes in the histological type and describe the genetic characteristics of intricate cancers, after which appropriate treatment strategies can be identified.

Acknowledgments

We would like to thank the patients for providing samples for this study.

Funding: This research was supported by the Foshan Science and Technology Innovation Project (Medical Science and Technology Innovation Platform) (Grant No.: FS0AA-KJ218-1301-0036).

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://dx.doi.org/10.21037/apm-21-2016

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/apm-21-2016). JZ and MX received personal fees from Shanghai OrigiMed Co., Ltd. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Ethics Committee of the Nanhai People’s Hospital. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)