An odd dancing couple. Non‐small cell lung carcinoma with coexisting EGFR mutation and NTRK‐1 translocation: A case report

In the 21st century, there has been a dramatic shift in the diagnosis and management of non‐small cell lung carcinoma (NSCLC), with an increasing use of minimally invasive tissue acquisition methods. Current treatments require morphologic subtyping and biomarker information in all cases. Determining such biomarkers is a continuously evolving field; current guidelines state that the determination of mutations on the Epidermal Growth Factor (EFGR), Kirsten Rat Sarcoma viral oncogene homolog (KRAS), Proto‐oncogene B‐Raf (BRAF), Human epidermal growth factor receptor 2 (HER2) and Anaplastic Lymphoma Kinase (ALK), genes as well as fusions on genes such as ROS Proto‐Oncogene 1, Receptor Tyrosine Kinase (ROS1), MET proto‐oncogene, receptor tyrosine kinase (MET), RET proto‐oncogene (RET), and the Neurotrophic Tyrosine Receptor Kinase (NTRK) family is mandatory. While analyzing such alterations, some of them were first reported to be mutually exclusive, although in recent years, it has been shown otherwise in some of these cases. Moreover, so was the case with the concomitant expression of NTRK fusions and EGFR mutations. We present a case report of a patient with concomitant EGFR mutation and NTRK1 fusion.


| INTRODUCTION
In the 21st century, there has been a dramatic shift in the diagnosis and management of non-small cell lung carcinoma (NSCLC), with an increasing use of minimally invasive tissue acquisition methods.Current treatments require morphologic subtyping with/without immunohistochemistry (IHC), and biomarker information in all cases.
Determining such biomarkers is a continuously evolving field; current guidelines state that the determination of EFGR, KRAS, BRAF, HER2, ALK, ROS1, MET, RET, and the NTRK family is mandatory. 1ile analyzing such alterations, some of them were first reported to be mutually exclusive, 2,3 although in recent years, it has been shown otherwise in some of these cases. 4Moreover, so was the case with the concomitant expression of NTRK fusions and EGFR mutations. 5,6| CASE REPORT

| Clinical and radiological findings
A 76-year-old male former smoker with a medical history of type 2 diabetes underwent radical prostatectomy for prostate adenocarcinoma 3 years ago.A CT scan performed in his referral hospital at that time revealed a mass in the right superior lobe of the lung.A close follow-up was recommended.
The patient came to our Institution in May 2022 seeking a second opinion.A whole-body CT-PET Scan showed a lung mass as well as an enlarged lymph node (LN) on the inferior right paratracheal level.

| Cytological findings
Aspirate smears and cell block (CB) were obtained from the LN.Six passes were performed and assessed by a cytopathologist during the procedure.For each pass, the samples were alcohol-fixed and Papanicolau stained, as well as air-dried and Diff-Quik stained.The CB was immediately fixed in formalin, paraffin-embedded, and hematoxylineosin (H&E) stained.
The smears were highly cellular, showing an hematic background in which both isolated atypical epithelioid cells, three-dimensional cohesive groups, and intermixed anthracotic macrophages.Scattered calcifications were seen.Atypical epithelial cells showed cohesive basophilic cytoplasm, cytoplasmic vacuoles, and moderate nuclear atypia.The nuclei were round and hyperchromatic, with a thickened nuclear membrane and prominent nucleoli.Nuclear inclusions were also noted, as well as a moderate mitotic count.Figure 1.
On CB, back-to-back glands and cribriform architecture with atypical cells with the same characteristics as in the smears.
On the immunohistochemical analysis performed on CB, the cells showed diffuse membrane positivity with CK7 and nuclear positivity for TTF-1.PD-L1 IHQ (Roche, VENTANA PD-L1, SP263) showed membranous cytoplasmic staining in 10% of the neoplastic cells.

| Molecular findings
As requested by the oncologist, a quick monogenic analysis was performed to study mutations on EGFR, BRAF, KRAS, and fusions in ALK, ROS1, RET, MET exon 14, and NTRK with a real-time PCR technology using the Idylla™ platform.DNA and RNA were extracted from Papanicolaou Stained smears.This study revealed the presence of an EGFR Exon 19 deletion and an "expression imbalance" for NTRK1 fusion.
The result of "expression imbalance" is not clinically relevant; however, a FISH analysis confirmed the presence of NTRK1 fusion.

| Macroscopic and microscopic findings
The patient subsequently underwent a lobectomy with hilar and mediastinal lymphadenectomy.The resected specimen showed a tan,

| DISCUSSION
Lung cancer is one of the most frequent neoplastic diseases worldwide.Out of which adenocarcinoma comprises the majority. 7Nowadays, due to the presence of targetable therapies, mutations on EGFR, BRAF, KRAS, rearrangements of the ALK, ROS1, NTRK, and RET genes, Her-2 and PD-L1 analysis need to be tested [8][9][10] on those patients.Since many of these patients are diagnosed at an advanced stage, cytological samples are frequently the only sample available for morphological diagnosis and biomarker analysis.The adequacy of such samples for those purposes has been proven repeatedly. 11,124]13 In recent years, increasing reports have been published showing some concomitant alterations previously deemed incompatible. 2,4,14arrangements in NTRK1 are truly uncommon.They have been identified in up to 0.1%-0.2% of all NSCLCs, 15 and they were thought to be mutually exclusive from other common mutations. 5,6It has been reported that concurrent mutation in EGFR with NTRK1 fusions may occur.This information could prove useful in further research and understanding of these genetic abnormalities. 3,14This is of special value, not only because NTRK family fusions may be subject to targeted therapies, but because concomitant alterations of both genes has been proven of prognostic value, for NTRK1 may be involved as a resistance mechanism to EGFR TK Inhibitors. 3

| CONCLUSIONS
Biomarker analysis for NSCLC is mandatory regardless of the available sample type, according to current guidelines.Some of these genetical alterations were thought to be mutually exclusive.However, recent reports show this not to be the case.As our knowledge of these alterations continues to grow, more of these conundrums may be more evident.
Second, cytological samples, recognized as suitable samples for molecular testing, prove to be not only adequate but often the only viable option for many NSCLC patients.
Highly cellular smears, showing an hematic background in which both isolated atypical epithelioid cells.(B) Three-dimensional cohesive groups, and intermixed anthracotic macrophages.(C) Scattered calcifications were seen.(D) Atypical epithelial cells showed cohesive basophilic cytoplasm, cytoplasmic vacuoles, and moderate nuclear atypia.Mitotic figures were present.[Color figure can be viewed at wileyonlinelibrary.com] ill-defined 43 mm mass that was 40 mm away from the bronchial margin.Ten lymph nodes were found from mediastinal lymph node stations 8, 4R, 7, and 2R.Histologically, an adenocarcinoma with different growth patterns (60% acinar, 30% papillary, and 10% solid) was seen.No lymphovascular invasion was noted, and neither was Spreading through Air Spaces (STAS).Both surgical and pleural margins were not affected.Two lymph nodes from stations 4R and 2R were positive for metastasis.Immunohistochemically, tumor cells showed nuclear positivity with TTF-1 and membranous positivity with 22C3 PD-L1 antibody (Dako PharmDx for Autostainer Link 48) in less than 1% of the overall tumor cells.

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I G U R E 2 FISH analysis for NTRK1 fusion using ZytoLight SPEC NTRK1 Dual Color Break apart Probe (PL123).Arrowheads pointing at break apart signals.[Color figure can be viewed at wileyonlinelibrary.com]