Effect on DNA Stability of a Novel Pellet Storing Method for Liquid-Based Cytology Specimens Fixed Using an Alcohol-Based Preservation Solution: Studies Using EGFR Mutation Detection on a Lung Adenocarcinoma Cell Line

Lung cancer is a major global healthcare challenge and is associated with a high morbidity and mortality rate [1]. Recently, molecular therapeutics for lung cancer has improved treatment results [2, 3]. Molecular therapeutics is effective for patients with advanced lung adenocarcinoma who harbor a specific active cancer-driving mutation. However, in spite of these advances, lung cancer remains the leading cause of cancer-related mortality worldwide [1]. Gene testing for the selection of therapeutics has been standardized, and the number of target genes for active cancer-driving mutations and rearrangements (fusions) in non-small cell lung cancer (NSCLC) has increased in recent years. Epidermal growth factor receptor (EGFR) mutations have a high frequency of occurrence, and they are present in 30–50% of patients with lung adenocarcinoma in East Asian countries and 10–20% in America and Europe [4]. Although it is reasonable to conduct sequential screening for mutations or fusion (beginning with high-frequency target genes), this approach would require substantial investment of time and quantity of specimens. To overcome this, multiplex testing using next-generation sequencing is beneficial for patients, and it is currently replacing single-plex testing [5, 6]. Conventional specimens for multiplex testing are tumor tissues (i.e., formalin-fixed paraffin-embedded [FFPE]); however, cytology specimens (i.e., pleural effusion, bronchoalveolar lavage fluid, and cells obtained using needle aspiration or scratching) have been added to this list and are approved by insurance agencies in Japan in 2023 [7].

The liquid-based cytology (LBC) method began to be widely used in the early 2000s for the clinical diagnosis of gynecological malignancies. The advantages of this method over the conventional Papanicolaou smear method include uniform distribution of cells, elimination of sample drying, and reduced loss of specimens, which results in a decrease in unsatisfactory specimens [8‒10]. Additionally, residual LBC specimens can be used for genetic analysis, and promising results have been reported in the analyses using LBC specimens from patients with lung cancer [11‒14].

There are two types of LBC preservation solutions: those that contain formaldehyde and those that do not. Formaldehyde is known to improve the imaging of specimens for diagnosis by preserving cellular morphology and antigenicity [15, 16]. However, formaldehyde is also well-known to cause DNA degradation [17, 18]. In our previous study, we showed DNA fragmentation in adenocarcinoma cell lines after 1 day of fixation in 10% formalin [19]. Additionally, in our previous study, H1975 cells fixed in CytoRich™ Red (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 0.4% formaldehyde were used to analyze whether the preservation of the cell pellets (CPs) improved DNA stability. We reported that the pellet storage method for LBC specimens improved DNA stability and reduced DNA degradation by avoiding over-fixation compared with the conventional method of storing cell suspensions in a preservation solution [20]. Specifically, DNA specimens extracted from cells that were directly suspended in CytoRich Red™ preservation solution showed DNA degradation after 5 days of preservation. However, DNA specimens extracted from cells stored in the form of pellets, after removal of the preservation solution, retained high-quality DNA for up to 28 days. The results showed that the novel method reduced DNA degradation by avoiding over-fixation compared with the conventional method of using cell suspensions containing formaldehyde. Therefore, this novel preservation method is promising for obtaining high-quality DNA from residual LBC specimens for genomic analyses. However, the use of alcohol-based LBC preservation solutions without formalin has not been demonstrated. As ethanol maintains higher nucleic acid stability than formalin [19, 21‒23], we designed this study to demonstrate whether this novel method can further improve DNA stability. Residual cytological specimens of NSCLC fixed with PreservCyt^® solution and preserved at room temperature are suitable for EGFR mutation detection [24]. Therefore, in this study, we used an alcohol-based LBC preservation solution (PreservCyt^® for non-gynecologic samples; Hologic, Marlborough, MA, USA) to determine whether this novel preservation method improves DNA stability. In particular, we used human lung adenocarcinoma cell lines to examine the quality of DNA obtained with this method and performed EGFR mutation analyses, which are representative mutation genes in NSCLC.

The human lung adenocarcinoma cell line H1975 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640; Merck, Darmstadt, Germany) medium supplemented with 10% fetal calf serum (Nichirei Bioscience Inc., Tokyo, Japan), 2 mml-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37°C in 5% CO₂/95% air. H1975 cells contain the EGFR double mutation: T790M in exon 20 and L858R in exon 21.

The collected H1975 cells were immersed in PreservCyt^® solution (for non-gynecologic samples) for 15 min at 1 × 10⁶ cells/mL. The fixed cells were split into two groups: one group that was stored as a suspension in PreservCyt^® (Susp group) and the other group stored as a CP group. The cells in the Susp group were kept immersed in the preservation solution and stored. The cells in CP group were centrifuged, and the preservation solution was removed by aspiration. The cells were washed once with phosphate-buffered saline (PBS) and centrifuged. After removing PBS, CPs were stored. Each group was stored for 7, 14, or 28 days at ambient temperature. DNA was extracted from control (day 0) samples immediately after 15 min of fixation and used as a common control in both groups. In addition, the fixed cells were further split into two groups: wet CP and dry CP (d-CP). The d-CP group was stored in tubes with open lids. Each group was stored for 7 days at ambient temperature. Figure 1 provides a schematic illustration of the specimen processing.

Genomic DNA was purified using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The manufacturer’s extraction protocol “for cultured cells” was followed. In the Susp group, cells were washed once with PBS after centrifugation, and the extraction procedure was initiated. For the CP and d-CP groups, the CPs were directly subjected to the extraction procedure. The absorbance of the extracted DNA was measured at 260 nm (A260) using a DeNovix DS-11 UV-Vis Spectrophotometer (DeNovix Inc., Wilmington, DE, USA), and the amount of double-stranded (ds) DNA was measured using a Qubit Fluorometer (Thermo Fisher Scientific). The DNA integrity number (DIN) was measured using a 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA).

Genomic DNA extracted from the cells of each group was analyzed for mutations in exons 20 (T790M) and 21 (L858R) of EGFR by Cycleave PCR using a C1000 Touch thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Genomic DNA samples (5 ng, quantified by Qubit) were amplified using Cycleave PCR Reaction Mix (Takara Bio, Shiga, Japan). DNA fragmentation was evaluated by comparing the threshold cycle (Ct) values for each preservation period in each group.

Slides were prepared using cells from the Susp and CP groups. The slides were fixed with 95% ethanol and stained using the Papanicolaou method.

All data are expressed as the mean ± standard error of the mean. Statistical analyses were performed using JMP Pro 17 software (SAS Institute Inc., Cary, NC, USA). Student’s t test and Welch’s t test were used to compare groups. p value <0.05 was considered to be statistically significant. Statistical analyses were double-checked by Ace Statistics (Osaka, Japan) using IBM SPSS Statistics 24.0 for Windows (IBM Corporation, Chicago, IL, USA).

Absorbance (260 nm) of the DNA obtained from the Susp group was significantly higher than that of DNA from the CP group at each time point (p < 0.001). These values decreased with increasing storage duration in both groups (Fig. 2a). The total amount of dsDNA extracted from the cells in the CP group was significantly higher than that extracted from the Susp group at each time point (day 7: p < 0.001, days 14 and 28: p < 0.05). The amount of dsDNA decreased by day 28 in both groups (Fig. 2b). The DIN values of DNA from the CP group were significantly higher than those of DNA from the Susp group (p < 0.001). These values decreased slightly with increasing storage duration in both groups (Fig. 2c).

Absorbance (260 nm) of DNA from the d-CP group was significantly higher than that of DNA from the CP group (p < 0.0001) (Fig. 3a). The total amount of dsDNA extracted from the cells in the CP group was significantly higher than that obtained from the d-CP group (p < 0.0001). The yields of dsDNA from the d-CP group were reduced to less than half of those in the CP group (Fig. 3b). The average DIN value of DNA from the CP group was significantly higher than that of DNA from the d-CP group (p < 0.0001) (Fig. 3c).

To assess the efficiency of mutation detection at the indicated time points, we compared the Ct values detected using the Cycleave PCR method. In both Susp and CP groups, EGFR exon 20 (T790M) and exon 21 (L858R) mutations were detected in all specimens. The Ct value of exon 20 (T790M) from specimens in the CP group was significantly lower than that of specimens in the Susp group on day 14 but not on days 7 and 28 (p < 0.05) (Fig. 4a). The Ct value of exon 21 (L858R) from specimens in the CP group was significantly lower than that of specimens in the Susp group at all indicated time points (days 7 and 14: p < 0.001, days 28: p < 0.05) (Fig. 4b). In both the CP and d-CP groups, EGFR exon 20 (T790M) and exon 21 (L858R) mutations were detected in all specimens. The respective Ct values were similar between the two groups and did not differ significantly (Fig. 5a, b).

To assess the cell morphology of cells stored as a CP, we compared the cytomorphologic features of the CP group and the Susp group at the indicated time points. Malignant findings, such as high nuclear-to-cytoplasmic ratio, vesicular chromatin, and enlarged nuclei with prominent nucleoli, were observed in both groups. However, in the CP group, slight cell shrinkage and aggregation were noted at 28 days (Fig. 6).

In the present study, we demonstrated that a novel method of storing LBC specimens fixed in an alcohol-based preservation solution in the pellet state improved DNA stability for up to 28 days after preservation. Formalin and ethanol are commonly used to fix cells and tissues. Formalin is known to cause DNA degradation [17, 18], and the quality of DNA extracted from fixed cells and tissues declines over time. Ethanol maintains higher nucleic acid stability than formalin [19, 21‒23]. Most LBC preservation solutions contain approximately 30–50% alcohol for fixation. The alcohol component in these solutions likely contributes to the good stability of nucleic acids extracted from cells, which may enhance the success of genetic analyses [11‒14]. The applicability of LBC specimens for genetic analysis is a notable advantage. However, the results of this study showed that the intact DNA extracted from cells in the CP group was significantly higher and of better quality than that extracted from the Susp group. The result indicates that DNA stability remained when cells were preserved in PreservCyt^® solution, but DNA was degraded by DNase in preservation solution in the Susp group due to DNase activity in the preservation solution. PreservCyt^® solution consists of 35–55% methanol, with the remainder primarily water, which is not nuclease-free. This water content may have contributed to DNA degradation. In contrast, DNA extracted from the cells in the CP group that were stored after removal of PreservCyt^® solution was less affected by DNase than the Susp group, suggesting that DNA degradation was suppressed. The A260 values in the Susp group were higher than those in the CP group, which can be explained by the hyperchromic effect, which refers to an increase in absorbance owing to DNA degradation. These findings suggest that, over prolonged storage periods, the negative impact of DNase activity in the LBC preservation solution outweighs the stabilizing effect of alcohol on DNA quality. The Ct values for EGFR mutation detection in the CP group were significantly lower than those in the Susp group on days 14. Lower Ct values indicate higher sensitivity of mutation detection, confirming that DNA from the CP group was of superior quality. This difference in Ct values may be explained by the lower amount of denatured DNA or RNA in the DNA solution extracted from the CP group, which does not interfere with PCR efficiency. In addition, the higher DIN values in the CP group further support the conclusion that DNA quality in the CP group was superior to that in the Susp group. This supports the suitability of our proposed method for the long-term storage of residual LBC specimens for DNA extraction; however, it should be noted that genetic analysis may fail if the DNA yields are measured using absorbance alone.

When we preserved some CP specimens with the proposed novel method, one specimen was unexpectedly found to be completely dried out using PreservCyt^®, whereas this was not observed in the previous study with CytoRich Red™ [20]. As PreservCyt^® solution has a higher methanol content than CytoRich Red™ (PreservCyt^® solution consisted mainly of methanol 35–55%, whereas CytoRich Red™ consisted mainly of 23.3% isopropanol, 10% methanol, 6.7% ethylene glycol, and 0.4% formaldehyde), we hypothesized that it would be easier to dry, although specimens were washed once with PBS. We were aware of the possibility that the specimens could dry out, and we attempted to extract DNA from the dried specimens. The amount of intact DNA extracted from cells in the CP group was significantly higher and of superior quality than that extracted from cells in the d-CP group. However, the A260 values in the d-CP group were higher than those in the CP group. This result can be explained by the hyperchromic effect, which refers to an increase in absorbance owing to DNA degradation resulting from a decrease in dsDNA. The Ct values for EGFR mutation detection were almost the same between the CP and d-CP groups, with no significant difference. Therefore, DNA extraction should be performed even if the pellets are unintentionally dried, as DNA quality is maintained even when the CPs are dried. Furthermore, this study demonstrated that DNA can be successfully extracted from completely dried CPs and used for mutation detection. In future studies, we aim to investigate whether storing pellets at ambient temperature after they have been rapidly dried using an oven could allow for even longer storage periods while maintaining DNA quality.

This study has a few limitations. First, the specimens were not refrigerated and were stored at ambient temperature. In our previous study using CytoRich Red™, specimens stored using the pellet method for 3 days were of nearly identical DNA quality, whether stored at ambient temperature, refrigerated, or frozen. However, specimens stored as suspension in CytoRich Red™ showed DNA fragmentation only at ambient temperature, whereas the DNA quality of the specimens was maintained when they were refrigerated or frozen [20]. The same is true for the storage temperature of FFPE tissue specimens, where refrigeration preserves the nucleic acid quality [25], which also applies to the temperature during the process of fixing FFPE [26]. Therefore, cells of the Susp or CP groups might have improved DNA quality in the case of refrigeration. However, the method for storing CPs at ambient temperature is convenient, simple, and economical, and we adopted it in our proposed pellet method. Second, in this study, the quality of RNA was not evaluated. For multiplex testing using DNA and RNA, it is necessary to evaluate the quality of RNA extracted from LBC specimens to include this specimen type for genetic testing in the future. Furthermore, we only evaluated a single cell line with a sufficient number of cells. Future studies should test clinical LBC specimens, such as EBUS-TBN specimens, particularly from lung adenocarcinoma, including the wild-type or those containing several mutations. Expanding the study to include clinical specimens, which are often available only in small quantities, will provide more clinically relevant data and ultimately benefit patients. In addition, we detected EGFR mutations using the Cycleave PCR method, which is highly specific for mutation detection. The observed differences between the Susp and CP groups were more pronounced for L858R than for T790M. These results suggest that even in the CP group, which demonstrated higher DIN values than the Susp group, testing with different cell lines containing other mutations or clinical specimens is necessary, as results can vary depending on the primers and probes used. Importantly, the CP group consistently exhibited a significantly higher amount of intact DNA than the Susp group, indicating that this method is well-suited for the storage of LBC specimens for genetic analysis. Although our primary focus was on DNA quality for genetic analysis; however, we also evaluated cell morphology, the original purpose of LBC. No significant differences in cell morphology were observed between the CP and Susp groups after storage, suggesting that our proposed novel method supports the replication of LBC specimens. However, since this study was conducted using a single cell line, future studies should evaluate clinical specimens to confirm these findings.

In conclusion, when an alcohol-based preservation solution was used for fixing LBC specimens, DNA stability was higher in samples stored as wet pellets through the prompt removal of the preservation solution post-fixation compared with samples stored as suspensions. Even when the stored specimens were totally dried, the DNA quality was maintained in the CP, although the yield was reduced. Therefore, storage of LBC specimens as CPs after fixation in alcohol-based preservation solutions shows potential as a viable strategy for genetic analyses.

We thank Editage (https://www.editage.com) for English language editing and Ace Statistics (http://www.ace-stat.co.jp/) for double-checking the statistical analyses.

The current study was decided not subject to ethics review by the Ethics Committee of Kitasato University Medical Ethics Organization because this study was performed using only cell line.

The authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

Y.M., T.Y., K.Y., and Y.S. performed study concept and design, interpretation of data. Y.M. performed material preparation, data collection and analysis. Y.M. wrote the first draft of the manuscript and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.