Prolonged Response to Afatinib and Crizotinib in a Rare Case of EGFR-, HER2-, MET- and ROS1-Alterated Lung Adenocarcinoma

We present the case of a 70-year-old never-smoking female patient with epidermal growth factor receptor (EGFR) p.L858R-mutated metastatic non-small cell lung cancer (NSCLC). After three months of first-line treatment with erlotinib, progression occurred and platinum/pemetrexed was initiated, followed by a response for more than two years. After the progression, the molecular testing of a vertebral metastasis revealed a ROS proto-oncogene 1 (ROS1) translocation and a human epidermal growth factor receptor 2 (HER2) p.S310F mutation, in addition to the known EGFR p.L858R mutation. Crizotinib then led to a durable response of 17 months. The molecular retesting of the tumour cells obtained from the recurrent pleural effusion revealed the absence of the ROS1 translocation, whereas the EGFR and HER2 mutations were still present. Afatinib was added to the crizotinib, and the combination treatment resulted in another durable response of more than two years. The patient died more than 7 years after the initial diagnosis of metastatic NSCLC. This case demonstrates that the repeated molecular testing of metastatic NSCLC may identify new druggable genomic alterations that can impact the patient management and improve the patient outcome.


Introduction
The guidelines recommend repeated genomic tumour testing in metastatic non-small cell lung cancer (NSCLC) to guide the treatment decisions during the course of the disease [1,2].The detection of targetable driver mutations ideally results in the treatment with targeted therapies.There is limited knowledge regarding the impact of the co-alterations that may alter the response to targeted therapies.Therefore, careful decision-making is crucial in this setting.

Case Presentation
A 70-year-old never-smoking female presented with a cough and dyspnoea during exercise in October 2015.Computed tomography (CT) and 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) showed a spiculated nodule in the lingula of the lung, additional bilateral pulmonary nodules and enlarged locoregional lymph nodes.Primary adenocarcinoma of the lung was diagnosed by bronchoalveolar lavage (BAL) and biopsy of a bronchial mass (TNM staging (7th edition): cT4, cN3, cM1a, stage IV).The nextgeneration sequencing (NGS; Ion AmpliSeq Colon and Lung Cancer Research Panel v2) of the tumour DNA revealed a classical epidermal growth factor receptor (EGFR) mutation in exon 21 (p.L858R; c.2573T > G).Additionally, a MET amplification (mean gene copy number 9; MET/CEN7-ratio 2.4) was detected by fluorescence in situ hybridization (FISH).
The patient was started on first-line erlotinib (150 mg daily) in November 2015, which led to a stable disease.In February 2016, the patient complained about increasing cough and dyspnoea due to pulmonary progression.The molecular testing of the carcinoma cells obtained by BAL showed no EGFR T790M mutation, and she was started on carboplatin and pemetrexed followed by pemetrexed maintenance, resulting in a partial remission for 32 months.In November 2018, the patient underwent spinal surgery for a pathological fracture of the 12th thoracic vertebra.Molecular testing (Oncomine Focus Assay) was performed on the epidural tumour tissue that was removed at the time of surgery.In addition to the initial EGFR p.L858R mutation, a human epidermal growth factor receptor 2 (HER2) mutation (p.S310F, c.929C > T) was discovered.Break-apart ROS proto-oncogene 1 (ROS1) FISH was positive for ROS1 translocation (split signals in 39% of tumour cell nuclei, Figure 1).Furthermore, FISH revealed persistence of the MET amplification (mean gene copy number 4.6; MET/CEN7 ratio 2.2).
next-generation sequencing (NGS; Ion AmpliSeq Colon and Lung Cancer Resear v2) of the tumour DNA revealed a classical epidermal growth factor receptor (EGFR tion in exon 21 (p.L858R; c.2573T > G).Additionally, a MET amplification (mean ge number 9; MET/CEN7-ratio 2.4) was detected by fluorescence in situ hybridization The patient was started on first-line erlotinib (150 mg daily) in November 201 led to a stable disease.In February 2016, the patient complained about increasin and dyspnoea due to pulmonary progression.The molecular testing of the carcino obtained by BAL showed no EGFR T790M mutation, and she was started on car and pemetrexed followed by pemetrexed maintenance, resulting in a partial remi 32 months.In November 2018, the patient underwent spinal surgery for a path fracture of the 12th thoracic vertebra.Molecular testing (Oncomine Focus Assay) formed on the epidural tumour tissue that was removed at the time of surgery.In to the initial EGFR p.L858R mutation, a human epidermal growth factor receptor 2 mutation (p.S310F, c.929C > T) was discovered.Break-apart ROS proto-oncogene 1 FISH was positive for ROS1 translocation (split signals in 39% of tumour cell nu ure 1).Furthermore, FISH revealed persistence of the MET amplification (mean ge number 4.6; MET/CEN7 ratio 2.2).Third-line crizotinib monotherapy (250 mg twice per day) was started in F 2019, resulting in a partial remission lasting 17 months until July 2020, when the again progressed in the lungs and pleura.The molecular testing (Oncomine Focu of the tumour cells from the pleural effusion showed persistent EGFR p.L858R an p.S310F mutations, but no ROS1 fusion transcripts.In addition, the ROS1 FISH also negative.Due to the stable lesions interpreted as a response to the treatment of the thorax, crizotinib was continued and the pan-HER-inhibitor afatinib 30 m was added to target EGFR and HER2 in October 2020.Crizotinib subsequently h reduced to 200 mg every other day due to oedema and afatinib to 20 mg at 2 out o due to skin toxicity.This time, a partial response with a clinical benefit could be o for two years (Figure 2).Third-line crizotinib monotherapy (250 mg twice per day) was started in February 2019, resulting in a partial remission lasting 17 months until July 2020, when the disease again progressed in the lungs and pleura.The molecular testing (Oncomine Focus Assay) of the tumour cells from the pleural effusion showed persistent EGFR p.L858R and HER2 p.S310F mutations, but no ROS1 fusion transcripts.In addition, the ROS1 FISH test was also negative.Due to the stable lesions interpreted as a response to the treatment outside of the thorax, crizotinib was continued and the pan-HER-inhibitor afatinib 30 mg daily was added to target EGFR and HER2 in October 2020.Crizotinib subsequently had to be reduced to 200 mg every other day due to oedema and afatinib to 20 mg at 2 out of 3 days due to skin toxicity.This time, a partial response with a clinical benefit could be observed for two years (Figure 2).
Unfortunately, the patient deteriorated in November 2022 due to progressive disease and confusion as a result of likely paraneoplastic multiple infarct dementia and died in December 2022, more than seven years after the initial diagnosis of metastatic NSCLC.The molecular findings in the course of the disease are summarized in Figure 3.

Discussion
In this case report, we describe the history of a never-smoking female patient with metastatic adenocarcinoma of the lung with an EGFR p.L858R mutation, a HER2 p.S310F mutation and a MET amplification developing a ROS1 translocation during the course of her disease.Our case illustrates the importance of repeated molecular testing upon disease progression in NSCLC patients treated with targeted therapy, which is also recommended in the international guidelines [1,2].
EGFR mutations occur in 10-20% of the lung adenocarcinomas in Western populations [3][4][5].In our patient, the duration of disease control was rather short with erlotinib, a selective EGFR tyrosine kinase inhibitor (TKI).Although the outcome of the patients with the EGFR p.L858R mutation is inferior compared with EGFR exon 19 deletions, firstline therapy with erlotinib typically results in a progression-free survival in the range of 12 months [6][7][8].One reason for the short duration of the response to erlotinib may have been the de novo MET amplification at the first diagnosis.MET amplifications are reported in approximately 5% of the patients with acquired resistance to first-generation EGFR TKIs [9][10][11] but are also rarely encountered as co-alterations in EGFR TKI-naïve patients [12,13].The HER2 p.S310F mutation may also have contributed to the reduced response to erlotinib in our patient [14].This mutation had already been present at the time of diagnosis, as demonstrated by the post hoc NGS analysis using a larger gene panel.
After the progression on EGFR TKI, the patient had a prolonged response to pemetrexed-based chemotherapy for more than two years.Surprisingly, at the time of the progression on pemetrexed, a ROS1 translocation was found by FISH (39% of the tumour cells with a split signal) upon molecular NSCLC retesting.However, ROS1 fusion transcripts could not be detected in a post hoc analysis of the tumour RNA using the FusionPlex Expanded Sarcoma Assay.The ROS1 translocation had not been present upon the initial diagnosis, which has been confirmed by a recently performed post hoc test using the tumour cells obtained at the time of the first diagnosis.More favourable outcomes with pemetrexed have been observed in ROS1-altered NSCLC compared to ALKor EGFR-positive disease [15].Co-alterations of the ROS1 and EGFR genes are exquisitely rare [16][17][18][19].
Based on the positive ROS1 FISH result, a systemic therapy with crizotinib, a TKI inhibiting ROS1, MET and ALK, was started [20][21][22][23].Crizotinib has been investigated in the PROFILE 1001 trial and shown to be effective in ROS1-altered NSCLC with durable responses (median duration of response 24.7 months; 95% confidence interval (CI) 15.2-45.3)[21].Our patient was able to clinically benefit for 2 years as well, suggesting that, indeed, ROS1, rather than MET, was likely the driver of the tumour progression.The patient developed new pleural effusions under the systemic treatment with crizotinib after approximately 15 months.Interestingly, the ROS1 translocation disappeared, whereas the known EGFR and HER2 mutations were still present.The addition of afatinib, a pan-HER-inhibitor, to crizotinib with the intention to target the HER2 and EGFR mutation led to another prolonged response of over 2 years [24].Afatinib shows some activity not only against classical EGFR but also HER2-mutated tumours [25,26].In addition, afatinib has been used successfully in a patient with metastatic lung adenocarcinoma harbouring an EGFR p.L858R mutation co-occurring with the same HER2 p.S310F mutation [27].The preferred approach of the concurrent inhibition of all the detected alterations with a combination therapy rather than a single-agent therapy against the postulated resistance mutation has been recently shown in the INSIGHT 2 study [28,29].In this study, patients with EGFR-mutated NSCLC and a MET amplification as the resistance mechanism showed a much higher response rate (ORR) with the combination of osimertinib and tepotinib compared to tepotinib alone (54.5% [95% CI 32.2-75.6]vs. 8.3% [95% CI 0.2-38.5])[28].
According to this observation, nowadays, one would have preferably treated this patient with the combination of afatinib and crizotinib from the beginning of the detection of the resistance mutations.
Our case underlines the importance of repeated molecular testing in NSCLC treated with targeted therapies in order to individualise the systemic treatment in the course of the disease [1,2].Due to tumour heterogeneity, mixed responses and oligoprogression on the EGFR TKIs may occur when subclonal tumour cells harbouring different EGFR or other gene alterations co-exist with tumour cells still harbouring sensitive EGFR mutations [30][31][32].
With broader and deeper molecular testing, the presence of co-mutations and the emergence of novel resistance mutations during TKI therapy are becoming increasingly challenging in clinical practice.Nowadays, the first-line treatment of patients with lung cancer harbouring a classical EGFR mutation (exon 19 deletion or L858R mutation in exon 21) is usually osimertinib, a third-generation irreversible EGFR TKI, with or without chemotherapy as the first-line therapy [33,34].The emergence of ROS1 rearrangement as a resistance mechanism is extremely rare, although it has also been reported in this context [35].

Conclusions
Our case presentation underlines the importance of repeated testing for molecular alterations in the case of disease progression.The combination of afatinib and crizotinib in a patient with EGFR-, HER2-, ROS1and MET-altered NSCLC led to a durable response and was well-tolerated after dose modification.

Figure 1 .
Figure 1.FISH analysis of NSCLC cells using ROS1 dual colour break-apart probe.Nucl signal pattern characterized by separate red and green signals indicative of ROS1 transloc

Figure 1 .
Figure 1.FISH analysis of NSCLC cells using ROS1 dual colour break-apart probe.Nuclei show a signal pattern characterized by separate red and green signals indicative of ROS1 translocation.

Figure 2 .
Figure 2. Computed tomography of the thorax with contrast before (A) and three months after the addition (B) of crizotinib to afatinib in October 2020.The mass (thick arrow) in the lingula responded to therapy.Incidental findings include subpleural consolidations (thin arrow) and dystelectasis (arrowheads) and slight interlobar effusion (*) on the right side.

Figure 2 .
Figure 2. Computed tomography of the thorax with contrast before (A) and three months after the addition (B) of crizotinib to afatinib in October 2020.The mass (thick arrow) in the lingula responded to therapy.Incidental findings include subpleural consolidations (thin arrow) and dystelectasis (arrowheads) and slight interlobar effusion (*) on the right side.

Figure 3 .
Figure 3. Overview of the molecular testing results and treatments during the course of disease (including important results from retrospective testing).The hatchings indicate the treatment with a drug combination.

Figure 3 .
Figure 3. Overview of the molecular testing results and treatments during the course of disease (including important results from retrospective testing).The hatchings indicate the treatment with a drug combination.