Kristen rat sarcoma virus ( KRAS ) G12F‐positive non‐small cell lung cancer mimicking KRAS G12C positivity: A case report

Abstract Searching for driver gene alteration is a prerequisite for chemotherapy of non‐small cell lung cancer. Due to its high sensitivity and concordance rate, the Amoy Dx Pan Lung Cancer PCR panel has been approved and is widely used in Japan. In this report, we describe a case in which a positive result for Kristen rat sarcoma virus (KRAS) exon2 p.G12F, a rare KRAS mutation, may have led to a false‐positive result for KRAS exon2 p.G12C on AMOY. Genetic analysis in this case was performed by LC‐SCRUM‐Asia.


INTRODUCTION
2][3][4] The Oncomine Dx Target Test Multi-CDx System and the Amoy Dx Pan Lung Cancer PCR panel are widely used as multiplex tests in Japan, but there are some differences, such as the former is next-generation sequencing (NGS) and the latter is real-time polymerase chain reaction (PCR)-based, as well as in detectable gene mutations and the turnaround time (TAT) from specimen submission to the final test result.
Kristen rat sarcoma virus (KRAS) is the most common genetic mutation in NSCLC in the U.S. and Europe, along with epidermal growth factor receptor, and is found in approximately 30% of Western lung adenocarcinomas 5 and 10%-14% of Japanese lung adenocarcinomas. 6,7KRAS exon2 p.G12C (KRAS G12C) mutation is now an important test target because a molecularly targeted drug called sotorasib has shown efficacy 8 and been approved for KRAS G12Cpositive lung cancer.KRAS exon2 p.G12F (KRAS G12F), which is also a type of KRAS mutation, but is considered a rare genetic mutation, accounting for only 1% of KRASpositive non-small cell lung cancer. 9In this study, we report a case in which KRAS G12C was falsely positive on AMOY which was actually showing KRAS G12F positive.

CASE REPORT
A 67-year-old male presented with dyspnea and was referred to our institution after the discovery of right pleural effusion during a previous medical examination (Figure 1A,B).Thoracentesis was performed, and cytology revealed BerEP4 positivity, thyroid transcription factor-1 positivity and adenocarcinoma of the lung in the pleural fluid cell block (Figure 1C1-3).Pleural fluid was submitted to LC-SCRUM-Asia, where both AMOY and Oncomine Precision Assy (Oncomine) (Thermo Fisher Scientific) testing were performed.LC-SCRUM-Asia, previously named LC-SCRUM--Japan, is a prospective, nationwide, clinical, and genomic screening program for lung cancer (UMIN ID: UMIN000010234).AMOY showed positivity for all KRAS G12C, G12D/S, and G12A/V/R, G13C mutations, while Oncomine indicated positivity for KRAS exon 2 p.G12D (KRAS G12D) and G12F mutations.The therascreen KRAS PCR kit (Qiagen) confirmed the absence of the G12C mutation.The false-positive G12C result in AMOY due to the presence of G12F positivity was diagnosed.The patient had spinal canal extension of bone metastases, for which radiation therapy was initiated prior to starting chemotherapy with pembrolizumab.

DISCUSSION
AMOY is a PCR method that measures the presence or absence of each gene mutation by observing the nucleotide sequence.AMOY has an excellent sensitivity and short TAT, 10 and is currently approved in Japan as a companion diagnostic function for advanced stage lung cancer.The corresponding c.34G > T sequence is used to confirm the presence or absence of KRAS G12C in AMOY.However, KRAS G12F has also been found to exhibit c.34G > T, 9 although it is not subject to AMOY detection.The amino acid substitutions and corresponding base substitutions in KRAS G12 are shown in Table 1. 11KRAS G12F also has c.35G > T, 9 but it is not subject to detection, leading to a false positive result for KRAS G12C in AMOY.Furthermore, the coexistence of C.34G > T and c.35G > T was reported in 6% of c.34G > T cases, 70% of which were G12F, but other patterns such as concomitant G12C and G12V or G12C and G12F were also observed; 9,12 therefore caution is required in interpreting the results of this study because only one of the panels can be used under the Japanese insurance system.Fortunately, the design of the LC-SCRUM-Asia study allowed for simultaneous testing of Oncomine and AMOY in the present case, and thus we could confirm a positive result for G12F and a negative result for G12C.It is desirable to take necessary measures such as the addition of a therascreen that detects G12C with GGT > TGT (c.34G > T) as appropriate.
Additionally, the simultaneous mutation of G12D (c.35G > A) and G12F detected in this study has not been previously reported, 9,[12][13][14] and is considered to be a very rare duplication.It has been suggested that when KRAS G12C and G12F coexist, G12C may be the preceding genetic mutation, 9 but the clinical significance of the mutation demonstrated in this case is unknown.

F I G U R E 1
Radiological findings and cytology.The patient had left pleural effusion, and a tumor was found in the left upper lobe.Cytology of pleural fluid revealed malignant cells and thyroid transcription factor-1 (TTF-1) stain of the cell block was positive, indicating adenocarcinoma.(a) Chest X-ray.(b) Chest computed tomography (CT) scan.(c1) Cytology.(c2) Hematoxylin-eosin staining of cell block prepared from pleural effusion, Â400.(c3) TTF-1 staining of cell block prepared from pleural effusion, Â400.T A B L E 1 The pattern of substitution in KRAS G12-positive lung cancer.