Concomitant Rare KRAS and BRAF Mutations in Lung Adenocarcinoma: A Case Report

: In July 2020, an active smoker, 63-year old man was admitted to the oncology unit of A.O.R.N. Sant’Anna e San Sebastiano (Caserta, Italy). Chest radiology highlighted right pleural e ﬀ usion. Total-body CT scanning revealed a solid lesion with lobulated contours in the apical segment of the upper right lobe. The patient’s oncologist requested a molecular assessment of EGFR, ALK, ROS1, BRAF, and KRAS, as well as an evaluation of PD-L1 expression level. To this end, we carried out NGS analysis, on DNA extracted from cytospins, by adopting a custom-designed NGS panel (SiRe ® ). Overall, no actionable mutations in the tested genes were identiﬁed. Conversely, concomitant BRAF exon 11 p.G469A and a KRAS exon 4 p.A146T mutations were detected. Owing to the limited data on the presence of KRAS exon 4 p.A146T point mutation in lung adenocarcinoma patients, a further molecular conﬁrmatory analysis was carried out with a dedicated KRAS cartridge on a fully automated real time polymerase chain reaction. When DNA was extracted from the TTF-1 positive tumor cell slide, the same KRAS alteration was observed. Unfortunately, the patient died in August 2020 before having the chance to start any type of treatment. automated real time polymerase chain reaction platform (Idylla TM , Biocartis, Mechelen, Belgium), conﬁrming the same KRAS alteration.

(e.g., AMG510, Amgen, Thousand Oaks, CA) [6]. Hence, to optimize the limited amount of starting tissue material for the detection of a plethora of available biomarkers, laboratories have adopted next generation sequencing (NGS), a complex yet useful tool to assess several biomarkers for different patients, simultaneously [7].
Here, we report a rare case of NSCLC featuring an unusual association of BRAF and KRAS point mutations.

Case Report
In July 2020, an active smoker, 63-year old man was admitted to the oncology unit of A.O.R.N. Sant'Anna e San Sebastiano (Caserta, Italy). The patient reported severe dyspnoea and 30 days of persistent dry cough. Chest radiology highlighted right pleural effusion. Total-body CT scanning revealed a solid lesion with lobulated contours in the apical segment of the upper right lobe and great post-contrastographic enhancement with a large implantation base on the anterior mediastinal pleura indissociable from its bronco-vascular structures. The solid lesion measured 36 × 22 mm. The CT analysis also confirmed a concomitant apical-basal pleural effusion measuring up to 60 mm in diameter. Such lesion caused subtotal atelectasis of the middle and right lower lobe ( Figure 1A,B). In addition, pathological pericentimetric para-tracheal, pre-and sub-carinal homolateral, aortic-pulmonary, and Barety window lymphadenopathies, measuring up to 2 cm in maximum diameter, were also observed. (Figure 1C,D). No other lesions were identified in the other examined body regions.
J. Mol. Pathol. 2020, 1, FOR PEER REVIEW 2 also been paid to novel predictive biomarkers, such as Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) exon 2 p.G12C, for the administration of specific TKIs (e.g., AMG510, Amgen, Thousand Oaks, CA) [6]. Hence, to optimize the limited amount of starting tissue material for the detection of a plethora of available biomarkers, laboratories have adopted next generation sequencing (NGS), a complex yet useful tool to assess several biomarkers for different patients, simultaneously [7].
Here, we report a rare case of NSCLC featuring an unusual association of BRAF and KRAS point mutations.

Case Report
In July 2020, an active smoker, 63-year old man was admitted to the oncology unit of A.O.R.N. Sant'Anna e San Sebastiano (Caserta, Italy). The patient reported severe dyspnoea and 30 days of persistent dry cough. Chest radiology highlighted right pleural effusion. Total-body CT scanning revealed a solid lesion with lobulated contours in the apical segment of the upper right lobe and great post-contrastographic enhancement with a large implantation base on the anterior mediastinal pleura indissociable from its bronco-vascular structures. The solid lesion measured 36 × 22 mm. The CT analysis also confirmed a concomitant apical-basal pleural effusion measuring up to 60 mm in diameter. Such lesion caused subtotal atelectasis of the middle and right lower lobe ( Figure 1A,B). In addition, pathological pericentimetric para-tracheal, pre-and sub-carinal homolateral, aorticpulmonary, and Barety window lymphadenopathies, measuring up to 2 cm in maximum diameter, were also observed. (Figure 1C   Evacuative palliative thoracentesis (about 1600 mL of bloody pleural fluid) and diagnostic bronchoscopy with biopsy of lymph node station number seven were performed. The cytological picture displayed atypical epithelial cells with severe nuclear abnormalities organized in glandular structures, as evidenced in H&E stained cytospins (Figure 2A,B). Immunocytochemical (ICC) analysis highlighted a strong positivity for thyroid transcription factor 1 (TTF-1) and negativity for calretinin and Wilms tumor protein (WT1); cytokeratin 7 (CK7) staining was not contributive ( Figure 2C,D). Overall, on the basis of these clinical, radiological, morphological, and immunocytochemical features, a diagnosis of NSCLC favor adenocarcinoma was made. middle and right lower lobe was observed. (C,D) Pathological pericentimetric para-tracheal, pre-and sub-carinal homolateral, aortic-pulmonary and Barety window lymphadenopathies, measuring up to 2 cm in maximum diameter, were observed.
Evacuative palliative thoracentesis (about 1600 mL of bloody pleural fluid) and diagnostic bronchoscopy with biopsy of lymph node station number seven were performed. The cytological picture displayed atypical epithelial cells with severe nuclear abnormalities organized in glandular structures, as evidenced in H&E stained cytospins (Figure 2A,B). Immunocytochemical (ICC) analysis highlighted a strong positivity for thyroid transcription factor 1 (TTF-1) and negativity for calretinin and Wilms tumor protein (WT1); cytokeratin 7 (CK7) staining was not contributive ( Figure  2C,D). Overall, on the basis of these clinical, radiological, morphological, and immunocytochemical features, a diagnosis of NSCLC favor adenocarcinoma was made.  Owing to the limited data on the presence of KRAS exon 4 p.A146T point mutation in lung adenocarcinoma patients, a further molecular confirmatory analysis was carried out with a dedicated KRAS cartridge on a fully automated real time polymerase chain reaction (RT-PCR) platform (Idylla TM , Biocartis, Mechelen, Belgium) [14]. When DNA was extracted from the TTF-1 positive tumor cell slide, the same KRAS alteration was observed ( Figure 3C). Unfortunately, the patient died in August 2020 before having the chance to start any type of treatment.

Ethical Statement
Written informed consent was obtained from the patient and documented in accordance with the general authorization to process personal data for scientific research purposes from 'The Italian Data Protection Authority' (http://www.garanteprivacy.it/web/guest/home/docweb/-/docwebdisplay/export/2485392).
All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Declaration of Helsinki  [8][9][10][11][12][13]. The analysis was performed on the Ion Torrent S5 (ThermoFisher Scientific, Waltham, MA) platform. Overall, no actionable mutations in the tested genes were identified. Conversely, concomitant BRAF exon 11 p.G469A (allelic frequency 42.9%) and a KRAS exon 4 p.A146T (allelic frequency 31.9%) mutations were detected ( Figure 3A,B). Owing to the limited data on the presence of KRAS exon 4 p.A146T point mutation in lung adenocarcinoma patients, a further molecular confirmatory analysis was carried out with a dedicated KRAS cartridge on a fully automated real time polymerase chain reaction (RT-PCR) platform (Idylla TM , Biocartis, Mechelen, Belgium) [14]. When DNA was extracted from the TTF-1 positive tumor cell slide, the same KRAS alteration was observed ( Figure 3C). Unfortunately, the patient died in August 2020 before having the chance to start any type of treatment.

Ethical Statement
Written informed consent was obtained from the patient and documented in accordance with the general authorization to process personal data for scientific research purposes from 'The Italian Data Protection Authority' (http://www.garanteprivacy.it/web/guest/home/docweb/-/docwebdisplay/export/ 2485392). All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Declaration of Helsinki (http://www.wma.net/en/ 30publications/10policies/b3/).

Discussion
Here, we report a rare case of lung adenocarcinoma harboring concomitant KRAS exon 4 p.A146T (COSM19404) and BRAF exon 11 p.G469A (COSM460) point mutations. To the best of our knowledge, although KRAS and BRAF co-occurring mutations have already been described in the literature, no study has ever reported the co-occurrence of these two rare genomic events in NSCLC patients.
Typically, KRAS mutations are found in 25-30% of NSCLC patients, in particular in Western countries and in smokers [15]. Up to 90% of KRAS mutations are found in codon 12 (exon 2), and, in particular, in p.G12C and p.G12V, due to G:C to T:A transversion in smokers [16]. Preliminary studies from early clinical trials have indicated that although these mutations are associated with negative prognoses, KRAS exon 2 p.G12C mutations, which account for about 13% of NSCLC patients, have acquired a relevant positive predictive significance as actionable biomarker [17]. On the other hand, KRAS exon 4 p.A146T is a very rare event in NSCLC adenocarcinoma patients (0.08%) [18]. This mutation seems to be associated with elevated expression levels of both RAS-GTP and extracellular signal-regulated kinase (ERK) compared with wild-type RAS cells. Therefore, KRAS exon 4 p.A146T is considered an activating missense mutation [19,20]. Recently, Wang et al. further confirmed the very rare occurrence of KRAS exon 4 p.A146T (0.41%) in the metastatic lung adenocarcinoma setting [21]. Not surprisingly, KRAS exon 4 p.A146T point mutation plays a negative predictive role when EGFR TKIs are administered [22].
As opposed to KRAS mutations, BRAF mutations have a lower incidence, occurring in only 1.5-3.5% to 7-8% of NSCLC patients. They are typically associated with micropapillary adenocarcinoma histotype, and with current and former smokers [23]. Discordant results have however been reported on the prevalence of BRAF exon 15 p.V600E and non-p.V600E alterations [24][25][26]. Similar to KRAS exon 4 p.A146T point mutation, the occurrence of BRAF exon 11 p.G469A is reported in a low percentage of lung adenocarcinomas (0.42%) [18]. To date, some studies indicate that only BRAF exon 15 p.V600E point mutation may predict the response to the combination of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) [27,28]. By contrast, other studies have highlighted the sensitivity of BRAF non-p.V600 point mutations, including BRAF exon 11 p.G469A, to this drug combination [26].
As of today, the co-occurrence of KRAS and BRAF mutations in lung adenocarcinoma is not well established. In a large experience, the Lung Cancer Mutation Consortium evidenced the occurrence of BRAF mutations with other concomitant oncogenic drivers (including KRAS) in a limited number of patients (5%) [29]. Salimian et al. reported concomitant KRAS and BRAF alterations in only 11% of analyzed cases [25]. Interestingly, in our present case and in a previous experience [24], we found that all cases with a concomitant KRAS mutation harbored a BRAF non-p.V600E alteration [25]. Similarly, another study has reported activating KRAS mutations in 11 (18%) out of 63 BRAF mutated patients [30]. Of note, all cases featured a BRAF non-p.V600E point mutation. Furthermore, two harbored a KRAS exon 4 p.A146T with a BRAF exon 11 p.G466V or BRAF exon 15 p.K601N [30]. As far as treatment is concerned, Gautschi et al. highlighted that the co-existence of KRAS exon 2 p.G12V with a BRAF exon 15 p.V600K determined the resistance to BRAF TKI treatments [31]. However, novel therapeutic approaches are under investigation for these patients. Interestingly, promising results have been reported in both RAS and BRAF mutated cell lines and in phase I dose-escalation and expansion studies of two novel molecules, ulixertinib (ERK1/2 kinase inhibitor) and LY3009120 (pan-RAF inhibitor) [32,33].
In conclusion, this is the first report describing the association of two rare mutations in KRAS and BRAF (KRAS exon 4 p.A146T and BRAF exon 11 p.G469A) in a lung adenocarcinoma patient. In this experience, our custom-designed NGS panel enabled us to run a successful genetic testing despite having scant cytological material. Nonetheless, even though custom-designed NGS yield accurate data, we recommend that orthogonal methodologies should be implemented to guarantee the accuracy of very rare molecular events, such as the KRAS exon 4 p.A146T point mutation. Finally, one limitation of this case report is that, unfortunately, our patient died before he could benefit from a personalized treatment plan. Consequently, further studies will be needed to better define the role of these co-occurring mutations in the hope of designing personalized treatment strategies for advanced stage NSCLC patients. Funding: The authors have not declared a specific grant for this research from any funding agency in the public, commercial, or not-for-profit sectors.