T790M is a mutation of the epidermal growth factor receptor (EGFR), where the amino acid threonine is replaced by methionine at position 790 of exon 20. EGFR is a member of the receptor tyrosine-kinase family and has an important role in the development and progression of non-small cell lung cancer (NSCLC). The overexpression of EGFR may lead to cell proliferation, promotes angiogenesis, tumor invasion and metastasis, and also inhibits cell apoptosis. Acquired T790M mutation is the primary resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs), used as first-line treatment in metastatic non-small cell lung cancer. We present the case of a 63-year-old woman with stage IV NSCLC, with EGFR exon 19 mutation, with TKI in first-line and multiples liquid biopsies performed at progression, who developed the T790M resistance mutation at more than three years after the diagnosis and received treatment with osimertinib.
T790M este o mutaţie a receptorului factorului de creştere epidermic (EGFR), unde aminoacidul treonină este înlocuit cu metionină în poziţia 790 a exonului 20. EGFR este un membru al familiei receptorului tirozin-kinază şi are un rol important în dezvoltarea şi progresia cancerului pulmonar cu celule mici (NSCLC). Supraexprimarea EGFR poate duce la proliferarea celulelor, promovează angiogeneza, invazia tumorală şi metastaza şi, de asemenea, inhibă apoptoza celulară. Mutaţia T790M dobândită este mecanismul principal de rezistenţă la inhibitorii tirozin-kinazei EGFR (TKI), utilizat ca tratament de primă linie în cancerul pulmonar cu celule non-mici metastatice. Prezentăm cazul unei femei de 63 de ani cu NSCLC în stadiul IV, cu mutaţie exon 19 EGFR, cu TKI în prima linie şi multiple biopsii lichide efectuate la progresie, care a dezvoltat mutaţia de rezistenţă T790M la mai mult de trei ani după diagnostic şi a primit tratament cu osimertinib.
Lung cancer, the leading cause of mortality worldwide, is comprised in proportion of 80-85% of non-small cell lung carcinoma (NSCLC). Its five-year survival rate at all stages is 15%. While surgery represents the optimal treatment option for NSCLC in early stages, the majority of patients are diagnosed with advanced, non-operable disease. The standard of care in this case is represented by palliative chemotherapy and/or radiotherapy(1).
Using biomarker testing as a tool to treat NSCLC has brought superior patient outcomes. For example, the identification of epidermal growth factor receptor (EGFR) mutations has led to the development of targeted therapies in NSCLC(2). The incidence is about 10-15% in Caucasian NSCLC patients and 30-40% in Asian patients(3).
EGFR is part of the erbB family of related receptor tyrosine kinases, that includes erbB1 (known as EGFR), erbB2 (HER2), erbB3 (HER3) and erbB4 (HER4).
EGFR represents a transmembrane receptor composed of three regions: the extracellular ligand-binding domain, the transmembrane domain, and the intracellular tyrosine kinase domain(4). Several ligands, such as epidermal growth factor, transforming growth factor and neuregulins, activate EGFR by binding to its extracellular domain; however, the most up-regulated ligand in lung cancer is EGF. The transmembrane domain of EGFR links the ligand-binding region to the intracellular tyrosine kinase signaling domain(5). The binding and the linking lead to receptor dimerization or heterodimerization with other erb family receptors (especially HER2/neu), which are essential steps for EGFR signaling. Ultimately, these signals result in resistance to apoptosis, cellular proliferation, cellular invasion, metastasis and angiogenesis(6).
EGFR is expressed in some normal mesenchymal, epithelial and neurogenic tissues. Its overexpression has been reported to be involved in the pathogenesis of several human malignancies, including NSCLC, cancers of head and neck, cervix, ovaries, prostate, bladder, esophagus, stomach, breast, brain, endometrium and colon(6).
EGFR mutation status is of high value in the screening process of NSCLC and it has been reported that females, non-smokers and patients with adenocarcinoma show a higher prevalence of EGFR mutations(7).
Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor have shown effectiveness as therapy for advanced NSCLC patients with mutation in the EGFR gene(8). Geftinib and erlotinib represent orally active compounds, part of the first generation of inhibitors which act on the intracellular tyrosine kinase domain of EGFR.
Despite an initial treatment response to EGFR-TKI, most of these patients inevitably acquire resistance after a progression-free period of approximately 10 months(9).
Acquired resistance to EGFR-TKIs is defined as the development of disease progression under targeted therapy in an EGFR-mutated NSCLC patient who had achieved a response or stable disease with more than six months of treatment(10). Afatinib and dacomitinib are second-generation TKIs, developed to solve the issue of resistance to the first-generation TKIs and have proven to be more potent against both wild type cells, as well as cells harboring T790M mutation. Resistance eventually develops with second-generation TKIs(11).
There are several mechanisms of resistance based on EGFR mutations which alter drug efficacy, such as T790M mutation in exon 20 of EGFR, overexpression of hepatocyte growth factor (HGF), and MET amplification. The most common, however, is T790M mutation in exon 20, having been detected in approximately 50% of patients treated with first-generation TKIs.
A similar frequency has been identified in patients receiving second-generation EGFR TKIs as first-line treatment(12). The identification of the T790M mutation can be performed from tissue biopsy or by free-circulating plasma DNA analysis(13).
Tissue biopsy – considered the gold standard for cancer detection – represents a biological sample from a mass, whose composition is further analyzed in order to reach a diagnosis. As far as its risks and limitations are concerned, it can be performed only when the mass becomes visible, which means it depends on the advancement of the disease. The inaccessibility of the tumor site at progression is another barrier, which might increase pain and distress for the patient and might add the risk of biopsy complications, since it is an invasive procedure.
The failure to report tumor heterogeneity, as tissue biopsy retrieves only a portion of the tumor, is a limitation, interfering with the possibility of administering targeted therapy(14).
Liquid biopsy is a noninvasive procedure which often consists of blood draw and analysis of cell-free DNA (cfDNA) with the purpose of detecting the presence of cancer(14). However, it can be performed from various biological fluids, such as urine, cerebrospinal fluid, seminal plasma and saliva. There are several molecules which can be measured in body fluids: cfDNA, circulating tumor DNA (ctDNA) – which originates specifically from tumors and whose proportion varies depending on the features of tumor and tumor microenvironment –, circulating RNA classes, circulating tumor cells (CTCs), proteins and exosomes(15).
Consisting more often of a blood sample, liquid biopsy does not raise the problem of difficult-to-reach tumors, nor the distress of the patient even in advanced-stage patients. The main advantage is revealing tumor heterogeneity, which is the key to targeted therapies and the option of early diagnosis, before the mass has become visible, offering increased chances of treatment success(14).
cfDNA – defined as extracellular DNA found in blood or in other body fluids – can be observed in healthy individuals, in patients with malignant diseases, but also in patients with non-malignant pathologies, such as rheumatoid arthritis, lung embolism, erythematic lupus or myocardium infarction. In healthy individuals, cfDNA has been observed in concentrations varying from 0 to 100 ng/ml of blood, while in cancer patients the concentrations in serum or plasma varied between 0 and 1000 ng/ml, having an average of 180 ng/ml, values which can change from one type of cancer to another(16).
The concentration of cfDNA has been studied with the purpose of discriminating between patients with malignant pathology and healthy individuals, and it has been demonstrated that the levels of cfDNA in NSCLC patients are higher in comparison with healthy individuals, a cutoff level of >0.20 mg/ml being able to distinguish lung cancer patients from control cases with a 69-79% sensitivity and a 83-89% specificity(17).
The most common mechanism of resistance to TKIs is T790M mutation. There are two explanations for this resistance: first, threonine 790 is situated in the back of the ATP binding cleft and its substitution with a bulky methionine interferes with the binding of TKIs and, secondly, this mutation strengthens the affinity for adenosine triphosphate (ATP), which reduces the binding of the ATP-competitive TKI(18).
In March 2016, a 63-year-old female, never-smoker, presented to our institution with left posterior thoracic pain and mild dyspnea. She had a personal history of right breast cancer, cT4b cN2 M1OSS (vertebrae L1), stage IV (2000), for which she underwent chemotherapy, surgical treatment, endocrine therapy for five years and radiotherapy for the bone lesion. The clinical examination revealed a good performance status (ECOG=0), and the laboratory analyses were within the limits of normal values.
A whole body computed tomography (CT) scan was performed, which revealed a pulmonary mass in right lower lobe (4x3 cm), osteolysis on the left 9th rib, compression fractures L1 vertebrae, demineralization of L2 vertebrae, and subcarinal and hilar ipsilateral lymphadenopathies.
The histopathological results obtained by bronchial biopsy revealed a pulmonary adenocarcinoma with mutation in exon 19 of EGFR gene and no rearrangements in the ALK gene. Also, multiple secondary bone lesion was seen on bone scan (right clavicle, left parietal bone, the 9th left rib, right zygomatic bone, left acetabulum and left iliac crest). Because of the personal history of neoplasia, in April 2016, it was done a biopsy bone from one suspected lesion (the 9th left rib) which turned out to be a metastasis of lung cancer.
In June 2016, the patient started the treatment with erlotinib 150 mg once a day and bisphosphonate therapy (zoledronic acid) every 4 weeks. During the treatment with erlotinib, she presented several episodes of major bone pain that required palliative radiotherapy, with partial remission of the algal syndrome.
A CT scan performed in October 2018 showed a disease in progression with multiple small nodules in the left lung suggestive for pulmonary metastasis and progression on the bone lesions. The liquid biopsy was collected at that time and was without T790M resistance mutation, thus the patient started chemotherapy with paclitaxel, carboplatin and zoledronic acid.
After nine cycles of chemotherapy, in May 2019, the imaging studies showed stable disease (RECIST 1.1) and the repeated liquid biopsy for T790M resistance mutation was negative (Figure 1).
The patient continued with the same chemotherapy regimen for another six cycles and the CT showed stable disease (RECIST 1.1) – Figure 2. The liquid biopsy collected in November 2019 revealed the presence of T790M resistance mutation in exon 20 and mutation in exon 19 of EGFR gene with no PD-L1 expression. From then on, the patient is on treatment with osimertinib 80 mg once a day and zoledronic acid every 4 weeks, and at the last presentation to the hospital she had clinical improvement, stable disease at CT scan, and did not experience any adverse events during this treatment.
The presented report discusses the case of a patient with NSCLC stage IV who had an EGFR mutation in exon 19 and was diagnosed with T790M mutation after the third liquid biopsy, at more than three years after the onset of the disease.
The use of tissue biopsy to determine T790M mutation status after progressing EGFR-TKI in the first line can be limited by factors such as inaccessible tumor site at progression, variation of T790M status at different tumor sites because of tumor heterogeneity, or risk of biopsy complications(13). On the contrary, liquid biopsy has proven to be an efficient alternative due to its quick turnaround time, minimal invasiveness, convenience, and potential clinical utility(19).
In April 2016, a tissue biopsy was collected from the patient, which has detected EGFR exon 19 mutation and no T790M mutation, using real-time PCR. Exon 19 deletions and point mutations in L858R represent the most frequent somatic activating mutations in the EGFR gene and they offer sensitivity to TKIs(20). It has been proven that first-generation EGFR-TKIs provide superior efficacy compared to platinum-based doublet chemotherapy(21). Consequently, the first choice of treatment was erlotinib.
However, after two years, due to the progression of the disease noticed on the CT scan, a second biopsy was performed. A liquid biopsy was collected at that time and it has shown no T790M mutation. The treatment was switched to chemotherapy, managing to maintain the disease stable. Nevertheless, liquid biopsies have been performed in the meantime, as literature supports this behavior, since the status of T790M is not static, meaning that an initially nondetectable mutation can become detectable upon progression(2).
The presented case report shows a liquid biopsy becoming positive for T790 M mutation at the third testing, being a proof that repeated biopsy is of high importance. The optimal time to repeat liquid biopsy depends, however, on the progression rate, the extent of the disease and the individual features of the patients(2). Consequently, our patient’s therapy was switched to osimertinib.
In AURA3 phase III trial, patients with T790M-positive advanced NSCLC who had progressed after first-line TKI therapy received a third generation of TKI (osimertinib). This drug was associated with a significantly longer progression-free survival than in patients who received treatment with platinum and pemetrexed(22). These results led to the approval by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) of osimertinib for the treatment of patients with positive T790M mutation, EGFR-mutated advanced or metastatic NSCLC after failure of initial treatment with EGFR-TKI.
Osimertinib is an orally available, mono-anilino-pyrimidine compound, which irreversibly binds to the EGFR tyrosine kinase domain by creating a covalent bond with the cysteine residue at codon 797. It has slow absorption and the median time to reach the maximal plasma concentration is approximately 6 hours. Its plasma concentrations lower in time, with an average half-time of 48 hours, and it is eliminated mostly in feces and urine(23).
Osimertinib has proven superior in terms of safety and tolerability compared with first- and second-generation EGFR-TKIs. While the adverse effects of the first- and second-generation EGFR-TKIs are a result of inhibiting wild type EGFR in gastrointestinal tract and skin, osimertinib is an inhibitor of EGFR sensitizing or T790M mutation, sparing wild type EGFR and showing a greater potency against T790M than against wild type EGFR(23).
Its response rate is 61% in patients with T790M positive NSCLC who developed resistance to a prior EGFR TKI and only 21% in those with T790M negative(24).
However, the mechanisms of resistance to osimertinib can occur as well and include: (I) acquired mutations, such as EGFR C797S mutation, which alters osimertinib binding to cysteine at position 797 of EGFR, or wild type EGFR amplification; (II) amplifications of MET, PIK3CA; (III) transformation into small cell histology(25).
In the case of progression under treatment with first-line TKI-EGFR, it is recommended to look for the T790M mutation. The possibility of re-biopsy (of the primary tumor, a lymph node or a secondary lesion) should be considered in order to look for the T790M mutation. If the biopsy cannot be performed or if the T790M mutation is not found on re-biopsy, the liquid biopsy analysis of ctDNA must be collected. A negative result obtained on circulating DNA does not exclude the presence of the T790M mutation at the tumoral level. Repeating the biopsy at the time of disease progression under first-line TKI treatment should be considered as a standard practice in patients with EGFR-mutated NSCLC.
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