A 62-year-old man was referred to our university hospital for treatment of advanced adenocarcinoma of the lung after disease progression on two lines of EGFR TKI and one line of chemotherapy in September 2015 (Figure 1).
Four months prior, in April 2015, he was diagnosed with an adenocarcinoma of the left lung with multiple bone metastases in sternum, ribs, and vertebrae. A biopsy from a metastasis in the left femur showed a mutation in the EGFR gene: c.2573T>G; p.(L858R). He was initially treated with gefitinib. After 2 months, the patient showed progression of bone metastases; the same EGFR mutation was found in a biopsy of a rib metastasis, without additional mutations in other mutational hotspots (e.g., BRAF, KRAS, HER2, KIT, ALK, NRAS, PDGFRA, PIK3CA, and MET). Therefore, at that time, carboplatin and pemetrexed were provided and because of pain, local irradiation of a sternal metastasis was applied. After two cycles of chemotherapy, the patient showed disease progression and was referred to our hospital. Because of lack of new treatment options, this patient was discussed in the Groningen MTB consisting of pulmonary oncologists, pathologists, clinical molecular biologists in pathology, general oncologists, and a structural biologist (). The MTB advised to determine the HER2-copy number status as a possible resistant mechanism for EGFR TKI. Fluorescent in situ hybridization (FISH) on a biopsy of a subcutaneous thoracic metastasis revealed HER2 amplification and treatment with afatinib (dual EGFR and HER2 inhibitor) 30 mg QD was started in October 2015. Evaluation by 18-fluorodeoxyglucose positron emission tomography and computer tomography (18-FDG-PET-CT) showed after 6 weeks a significant partial response with disappearance of the FDG activity of the bone metastases and after 4 months in the left upper lobe a single FDG-positive lesion was left. This lesion was irradiated by means of stereotactic ablative radiotherapy (1x 20 Gy), and afatinib was continued. Treatment with afatinib was well tolerated with minor skin rash; patient showed clinical improvement: he had less pain and more energy. Nine months after start of afatinib, progressive disease was again noticed. Growth of the primary tumor in the left upper lobe, a new ipsilateral pulmonary lesion and multiple new bone metastases including the skull, with ingrowth into the brain, causing paralysis of the right facial nerve (Figure 1). Sequence analysis of a new right-sided rib lesion showed the known L858R EGFR mutation and an additional T790M mutation.
Because of the novel T790M, afatinib was discontinued and replaced by osimertinib 80 mg QD. Eight weeks after start of osimertinib a PET-CT showed a response of most lesions except for a growing lesion in the pelvic region and the skull with ingrowth in the brain. Clinically there was, however, temporary improvement of the patient's ability to move his right eyelid and right corner of the mouth, which had been paralyzed due to ingrowth of a skull metastasis into the brain and right facial nerve. A biopsy was performed of a growing FDG-positive lesion in the left pelvic bone that showed adenocarcinoma with the known L858R EGFR mutation, but the previously found T790M mutation was not present in this location (no biopsy of the skull metastasis available). The MTB advised to perform immunohistochemistry on Her2Neu (positive in agreement with HER2 amplification) and to determine MET amplification (negative by FISH). Based on these findings, it was decided to discontinue osimertinib because of the loss of the T790M mutation and to start a combination of paclitaxel 90 mg/m2 on days 1, 8, and 15, and trastuzumab 4 mg/kg on days 1 and 15, in cycles of 4 weeks, because trastuzumab is an HER2 antibody. Radiotherapy 1x 8 Gy was given on the pelvic lesion because of localized pain. 18-FDG-PET-scan after four cycles, paclitaxel and trastuzumab showed again a partial tumor response. No major side effects were observed although symptoms of the paralysis of the right facial nerve did not improve further; it remained stable during the course of therapy. The patient underwent plastic surgery on his right eyelid, which improved the closure of his right eye.
Two months after the fourth and last cycle, the patient presented with a subcutaneous metastasis on his forehead. Afatinib 30 mg QD was started, because this treatment worked before, pending results of a new biopsy. The biopsy, however, yielded insufficient material for mutation analysis, and re-biopsy was scheduled. In the meantime, 18-FDG-PET-scan showed multiple FDG-positive bone lesions (partly new lesions), some close to the myelum, and the patient was admitted to the hospital for radiotherapy on cervical and thoracic vertebrae. Afatinib was discontinued. Biopsy of a new lesion in the thoracic wall showed an EGFR-L858R, T790M mutation, and HER2 amplification. There were no other hotspot mutations in EGFR, BRAF, KRAS, ERBB2 (HER2), ALK, PIK3CA, or MET detected. The case was again reviewed by MTB. It was decided to treat the patient with afatinib 30 mg QD as well as osimertinib 80 mg QD at alternating days, to address the T790M mutation as well as the HER2 amplification resistance mechanism. Since the start of this latest treatment regimen, the subcutaneous skull metastasis disappeared, and the patient experienced less pain, regained his energy, and was able to walk outdoors again. The most recent 18-FDG-PET-CT-scan, 4 months after the start of this latest treatment regimen, showed again a partial response of the bone metastases again. Two months later (December 2017) progression of disease was observed, and the performance status deteriorated. Patient insisted to take a new biopsy from a new thoracic wall metastasis. Mutations analysis showed the known EGFR L858R and T790M mutations together with a new mutation in HER2: L755S. However, his condition got worse in short time, and he died in January 2018. An overview of the clinical findings, the mutational status at different time points and the given treatment regimens is provided in Table 1.
The incidence of EGFR mutations in advanced stage adenocarcinoma of the lung in Caucasian patients is 10-15 and 40-60% in Asian patients. In the north of the Netherlands, the incidence is 9%. L858R mutation in exon 21 of the EGFR kinase domain is the main hotspot mutation in the EGFR gene and accounts for 35-45% of EGFR mutations. L858R mutation increases the kinase activity of EGFR, leading to hyperactivation of downstream signaling pathways improving cell survival and proliferation. An EGFR TKI is the preferred first-line treatment in patients with activating EGFR mutation in non-small-cell lung carcinoma (NSCLC). Gefitinib and erlotinib are first-generation TKI and registered as first-line treatment in patients with metastatic NSCLC with a tumor harboring an activating EGFR mutation within the European Union and according to the Dutch guideline for treatment of NSCLC. These small molecules bind competitively and reversibly to the adenosine triphosphate (ATP) binding site of the tyrosine kinase domain of EGFR. This prevents the autophosphorylation of the TK, blocks the activation of the EGFR signal transduction, inhibits tumor cell proliferation, and induces cell cycle arrest and apoptosis. The majority of patients will progress after 9-12 months of treatment due to various mechanisms of intrinsic or acquired resistance to first-generation EGFR TKIs.
The most common mechanism of acquired TKI resistance is the acquisition of a single recurrent missense mutation within exon 20, the T790M mutation. This mutation leads to the substitution of threonine by methionine at position 790, which encodes part of the kinase domain of the receptor and results in increased affinity for ATP. The T790M mutation can be detected in about 60% of tissue biopsy samples taken after acquired resistance. As residue threonine at position 790 (T790) is located at the entrance in the back of the ATP binding cleft, substitution of residue threonine at position 790 with a bulky methionine (resulting in T790M) may cause steric interference with binding of TKIs. Irreversible inhibitors overcome this resistance simply through covalent binding. Osimertinib is registered for the treatment of NSCLC with an EGFR T790M mutation. It is a selective third-generation TKI which targets the ATP binding site of EGFR via irreversible covalent bond formation. In contrast to many other TKI, osimertinib penetrates the blood-brain barrier. Osimertinib improves overall survival and progression-free survival in T790M-positive NSCLC patients with and without brain metastases. Acquired resistance to osimertinib may be caused by primary coexistence of tumor cell populations with and without T790M mutation due to EGFR C797S mutation. Tumor progression can be explained by growth of the T790M negative population, while the tumor cells expressing T790M mutation are effectively suppressed by osimertinib.
Overexpression of HER2 induces cell transformation and tumorigenic growth and is clinically associated with resistance to erlotinib. HER2 amplification is detected in a subset of EGFR TKI resistant lung tumors. HER2 amplification and T790M mutation are thought to be mutually exclusive. However, in our patient HER2 amplification as well as T790M mutation appeared in the same biopsy of a new lesion in the thorax wall. Afatinib is an ATP-competitive aniline-quinazoline derivate which covalently binds to EGFR, HER2, and HER4 and irreversibly inhibits HER-family phosphorylation and signal transduction. As second generation TKI it is highly potent, irreversible dual EGFR/HER2 tyrosine kinase inhibitor, including the oncogenic EGFR-L858R mutation. Afatinib is registered for advanced NSCLC with EGFR mutations. Clinical benefit of afatinib seems less in patients with de novo T790M mutations. Although afatinib is equally potent against wild-type EGFR and EGFR harboring the T790M mutation, in patients the dose is lower due to toxicity constraints.
Trastuzumab, a humanized monoclonal antibody against HER2, has been reported to be effective in HER2-positive NSCLC in vitro and in case reports.