Pathological Examination and Genetic Mutation Testing: Bridging Their Best Practices Using <scp>MINtS</scp>

Fresh and cytology specimen suitability: Fresh specimens preserved in nucleic acid preservation solution help prevent DNA damage and RNA degradation. Cytology specimens, particularly valuable for patients without tissue biopsies, not only confirm the presence of tumour cells but also provide high-quality DNA and RNA. Recommended collection techniques: Clinical trials have established methods to obtain optimal cytology specimens, and optimal specimens are defined as those containing > 100 ng DNA and > 50 ng RNA, which ensures mutation detection rates comparable to or better than tissue-based tests. Intraoperative specimen collection: During surgery, small tissue samples can be collected at the time of rapid pathological diagnosis and preserved in RNA stabilisation solutions, minimising nucleic acid degradation and enabling high-quality mutation testing. In lung cancer, identifying oncogenic driver gene mutations, such as EGFR mutations and ALK fusion genes, and administering targeted inhibitors have been shown to significantly prolong survival in both advanced cancer treatment [1, 2] and adjuvant therapy after surgery [3, 4]. Therefore, screening for the presence of gene mutations at the time of diagnosis or at surgery has become crucial for determining treatment strategies. Pathological diagnosis is primarily performed using FFPE (formalin-fixed paraffin-embedded) tissue. However, it is known that the FFPE preparation process has a significant impact on the performance of genetic testing [5]. Formalin damages DNA, and if there is a delay before formalin fixation, RNA degradation can occur. The ideal material for genetic mutation testing is fresh specimens directly placed into nucleic acids preservation solution, for example, RNAlater (Thermo-Fisher Scientific, MA, USA). In nucleic acids preservation solution, nucleic acids remain stable for 24 h at room temperature, 1 month at 4°C and semi-permanently at −20°C [6]. Since genetic mutation testing significantly affects patient prognosis, it must be conducted with high accuracy. Therefore, in addition to formalin-fixed specimens, it is desirable to prepare a separate specimen placed in nucleic acids preservation solution immediately after collection for genetic mutation testing. At the time of cancer diagnosis, some patients are only able to provide cytology specimens. In Japan, many cases of lung cancer are diagnosed through bronchoscopy [7], but in 20%–30% of patients, tissue specimens cannot be obtained, and only cytology specimens are collected (Hagiwara K. personal communication). To perform genetic mutation testing for all patients, it is essential that such testing can also be conducted using cytology specimens [7]. Cytology specimens are sometimes treated as a form of liquid biopsy, but they are fundamentally different from liquid biopsies using serum or urine. Cytology specimens not only allow for confirmation of the presence of tumour cells but also ensure that the DNA and RNA being tested include those originating from tumour cells. In contrast, serum and urine do not offer this capability. As a result, tests using cytology specimens can clearly determine the mutation profile of tumour cells. Cytology specimens offer these advantages, but they often show significant variability in quality. To effectively utilise cytology specimens, a clear definition of appropriate cytology specimens and clinical procedures for their collection is necessary. We developed MINtS (Mutation Investigator using the next-era sequencer) [8] as a genetic mutation testing method that meets all these conditions and established appropriate specimen collection procedures. Based on the fact that most specimens judged as cancerous by pathologists have a cancer cell content of greater than 1% [9], MINtS was designed as a genetic mutation test capable of detecting mutations in both tissue and cytology specimens with a cancer cell content of 1% or higher, enabling us to test all specimens diagnosed by pathologists as containing cancer. In late 2024, MINtS received regulatory approval from the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan as a companion diagnostic for EGFR, ALK, and BRAF and was included in the national health insurance coverage. Figure 1A illustrates the procedure for performing MINtS using specimens collected at the time of diagnosis. Since gene mutation testing is conducted on the cytology specimen, the tissue specimen remains available for comprehensive pathological examination without being consumed by genetic testing. In a clinical trial conducted under the Advanced Medicine A Program of Japan's Ministry of Health, Labour and Welfare, criteria were established for identifying cytology specimens suitable for gene mutation testing (optimal specimens) [10]. Optimal specimens are defined by the quantity of extracted nucleic acids, specifically requiring more than 100 ng of DNA and 50 ng of RNA. Additionally, recommended collection techniques for obtaining optimal specimens were identified. The gene mutation detection rate using optimal specimens was found to be comparable to, or even superior to, that of conventional companion diagnostics utilising tissue specimens. Figure 1B illustrates the procedure for conducting MINtS using surgical specimens. Prolonged delays in fixation after surgery can result in significant RNA degradation [11]. To address this, regardless of whether an intraoperative rapid pathological diagnosis is performed, collecting a small tissue sample at the timing when a rapid pathological diagnosis would typically be performed and preserving it in a nucleic acids preservation solution ensures the freshest specimen for high-quality gene mutation testing. This approach is expected to be effective not only for companion diagnostics but also for comprehensive genome profiling (CGP). The clinical utility of this procedure is currently being evaluated in an ongoing clinical trial. In summary, MINtS is a multi-gene companion diagnostic test based on NGS. To ensure the highest quality in both pathological examination and genetic mutation testing, it is essential to: (1) actively utilise cytology specimens, (2) collect specimens in a nucleic acids preservation solution, and (3) modify the clinical procedure to enable the collection of fresh specimens. By adopting MINtS, more patients are expected to benefit from high-quality genetic mutation testing. We would like to express our gratitude for all the support provided by NEJSG (North-East Japan Study Group) in the development of MINtS. Koichi Hagiwara serves as a supervising physician at Eiken Clinical Laboratory, Eiken Chemical Co. Ltd., and receives compensation for this role. He has received royalties from LSI Medience Co. Ltd. and honoraria from Chugai Pharmaceutical Co. Ltd. and AstraZeneca plc. He is currently a director of the North-East Japan Study Group (NEJSG) and has previously served as a director of the Japan Lung Cancer Society and the Japan Respiratory Society. Hiroyoshi Tsubochi has nothing to declare.