Research ArticleArticles
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Localization of EGFR Mutations in Non-small-cell Lung Cancer Tissues Using Mutation-specific PNA-DNA Probes

HAJIME SHIGETO, HARUO MIYATA, TADASHI ASHIZAWA, AKIRA IIZUKA, YASUFUMI KIKUCHI, CHIKAKO HOZUMI, CHIE MAEDA, KEN YAMAGUCHI, SHOHEI YAMAMURA and YASUTO AKIYAMA
Cancer Genomics & Proteomics July 2023, 20 (4) 375-382; DOI: https://doi.org/10.21873/cgp.20389

Abstract

Background/Aim: Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed. Materials and Methods: Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations. Results: The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein. Conclusion: PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.

Key Words

Since the advent of the first generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, many efficient EGFR signaling inhibitors have been developed, and these agents have improved the overall survival of patients with advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR mutations (1-3). In addition, survival of patients with NSCLC without EGFR mutations has benefited from immune checkpoint blockade therapy, which has shown therapeutic advantages over conventional chemotherapies (4, 5).

However, it is generally accepted that up-regulation of EGFR signaling as a result of EGFR mutations may be an immunosuppressive event which can contribute to down-regulation of programmed death-1, a low tumor mutation burden, and a low number of tumor-infiltrating lymphocytes within the tumor (6-8). However, the dynamic changes in the tumor-infiltrating lymphocyte status or other events in the NSCLC tumor microenvironment after treatment with such inhibitors have not been elucidated precisely, except for a few studies (9, 10). Furthermore, the effect of EGFR signaling inhibitors on mutation status in tumor tissues should be investigated to develop improved inhibitors for tumors with heterogenous mutations. To evaluate the localization of EGFR mutation-positive areas in tumors, a simple and efficient mutation detection technology needs to be developed, but such technology remains lacking to date.

Recently, our group developed peptide nucleic acid (PNA)-DNA probes specific for the mRNA sequences for mutant EGFR and verified detection of EGFR mutations of lung cancer cell lines on single-cell microarray chip (11). These PNA-DNA probes function by hybridization of synthesized fluorescein isothiocyanate (FITC)-labelled PNA and EGFR-mutant mRNA.

In the present study, using EGFR mutation-specific PNA-DNA probes, we successfully developed a system for detecting EGFR mutations using multicolor-based immunofluorescence staining in formalin-fixed paraffin-embedded (FFPE) sections from tumors derived from EGFR-mutant NSCLC cells.

Materials and Methods

PNA-DNA probes specific for EGFR mutations. The development of PNA-DNA probes specific for the EGFR mutant mRNA sequences were previously reported by our group (11). Briefly, fluorescein isothiocyanate (FITC)-conjugated PNA (FITC-PNA; Panagene, Daejeon, Republic of Korea) and quencher-conjugated DNA (Q-DNA; Integrated DNA Technologies, Coralville, IA, USA) were prepared. PNA-DNA probes were constructed by hybridization of the synthesized FITC-PNA and Q-DNA. These probes were designed to be specific for each of the following EGFR mutations: L858R, del E746-A750 and T790M. For immunofluorescence staining using an antibody to mutant EGFR, anti-EGFR L858R (clone SP125, Abcam, Cambridge, UK) was purchased.

NSCLC cell lines and xenografts. Four human NSCLC cell lines were purchased: A549, NCI-H1975 and HCC827 from the American Type Culture Collection (Manassas, VA, USA) and PC-9 from Japanese Collection of Research Bioresources cell bank (Saito, Osaka, Japan). A549 cells harbor the wild type EGFR gene. NCI-H1975 cells harbor the L858R and T790M EGFR mutations, and HCC827 and PC-9 harbor deletions in exon 19 (Table I). These cell lines were cultured in RPMI1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Life Technology, Carlsbad, CA, USA), penicillin, and streptomycin. Male nude mice (BALB/cA-nu/nu, 5-6 weeks old) were obtained from Nippon Clea (Tokyo, Japan). In vivo tumors were obtained 2-3 weeks after subcutaneous transplantation of 1×106 cells into three nude mice per cell line. All animals were cared for and treated according to the guidelines for the welfare and use of animals in cancer research, and the experimental procedures were approved by the Animal Care and Use Committee of Shizuoka Cancer Center Research Institute (approval number: 2020-5). Tumor tissues were obtained from duplicate experiments. FFPE sections from each tumor were prepared by the Bozo Research Center (Gotemba, Shizuoka, Japan) and used for multicolor immunofluorescence staining. For immunofluorescence staining using normal human lung tissues, FFPE sections were purchased from Biochain Institute (Newark, CA, USA) and the effect of EGFR mutation-specific probes on staining of normal lung tissues was investigated.

View this table:
Table I.

Non-small-cell lung cancer cell lines with epidermal growth factor receptor (EGFR) mutations.

Immunofluorescence staining using a multicolor staining kit. For multicolor immunofluorescence staining, the following antibody was used: anti-human cytokeratin antibody purchased from Nichirei Bio (AE1 and AE3; Tokyo, Japan). Immunofluorescence staining was performed using an Opal 4-color IHC kit (Akoya Biosciences, Inc., Menlo Park, CA, USA), and images were acquired with a Zeiss Imager Z1 fluorescence microscope equipped with the ZEN software system (Carl Zeiss, Oberkochen, Germany). Three fluorescent reagents, 4′,6-diamidino-2-phenylindole (DAPI) and cytokeratin antibodies and FITC-conjugated PNA-DNA probes, were used with the Opal kit. Fluorescence quantification was performed by image analysis based on an in-house-developed algorithm using WinROOF (Mitani Shoji, Tokyo, Japan) (12), ImageJ (ver.1.53g58) and R software (ver. 3.4.0).

After deparaffinization and hydration of FFPE sections, activation by autoclaving at 95°C for 20 min in citrate buffer (pH 6.0) was performed. After refixation with formaldehyde, sections were blocked with serum-free DAKO protein block reagent (Agilent Technologies, Santa Clara, CA, USA). Endogenous peroxidase was quenched with 3% H2O2/MeOH. After incubation with primary and secondary antibodies, fluorescence reaction was carried out using an Opal IHC kit (Akoya Biosciences). Next, PNA-DNA coupling (80°C for 10 min followed by 37°C for 16 h) was carried out, and subsequently PNA and DAPI reactions were performed.

Image analysis and EGFR mutation detection. A flow chart showing the processes for measuring the positive areas is shown in Figure 1. Ten images from 20 images acquired were selected using a random table. Firstly, we identified areas positive for DAPI or cytokeratin and measured these to obtain area A. Secondly, within those areas, we identified areas staining positively with the EGFR mutation-specific PNA-DNA probes and measured these to obtain area B. Subsequently, the ratio of B to A (percentage area) was calculated, and PNA-DNA staining was rated as positive when significantly higher than the ratio in EGFR wild-type A549 tumor.

Figure 1.
Figure 1.

Schematic of the procedure for measuring positive staining based on our in-house developed algorithm using the Winroof imaging software. Area A was rated as the area staining positively with 4′,6-diamidino-2-phenylindole (DAPI) or antibody to cytokeratin. Area B was rated as the area staining positively for mutation-specific peptide nucleic acid (PNA)-DNA probes. Finally, the ratio of B to A was calculated. The difference in the ratio between DAPI staining and anti-cytokeratin antibody staining was statistically evaluated.

Statistical analysis. Comparison of the percentage of positive area between mutation-positive and mutation-negative tumors was performed using the Mann–Whitney U-test. Correlation analysis of the positive staining rate of the PNA-DNA probe and the EGFR L858R antibody was performed by Pearson correlation test using R software (13). Values of p<0.05 were considered statistically significant.

Results

Immunofluorescence staining using PNA-DNA probes and a multicolor staining kit. In the comparison of the between DAPI and cytokeratin percentage of positive area, the latter was higher for all mutant EGFR-specific PNA-DNA probes (Figure 2 and Figure 3). Specifically, only the NCI-H1975 cell line with L858R mutation showed positive staining with the L858 probe, and in both DAPI-stained and cytokeratin-stained areas. In addition, both the HCC827 and PC-9 cell lines were positively stained with the probe for del E746-A750 in exon 19. The NCI-H1975 cell line exhibited positive staining with the T790M probe, but the PC-9 cell line also showed strong, non-specific probe staining in both DAPI-stained and cytokeratin-stained areas. In summary, all three EGFR mutation probes were able to detect the corresponding EGFR mutation in truly mutation-positive tumors; however, the T790M probe exhibited strong nonspecific staining. Moreover, the A549 cell line, with wild-type EGFR gene, did not show any staining with the three EGFR mutation-specific probes.

Figure 2.
Figure 2.

Comparison of mutation-specific peptide nucleic acid (PNA)-positive rate among epidermal growth factor receptor (EGFR) mutation-positive lung cancer tissues in 4′,6-diamidino-2-phenylindole (DAPI)-positive area. Tissues from tumors developing from four non-small-cell lung cancer cell lines were stained with each EGFR mutation-specific PNA probe and DAPI. The PNA-positive rate in DAPI-stained cancer tissues was determined. The positive rate of staining for each PNA probe in three EGFR mutation-positive lung cancer tissues was compared with that in A549 tumors without EGFR mutation and statistically evaluated using the Mann–Whitney U-test. Statistically significantly different at: *p<0.05 and **p<0.001. Magnification: 200×. Green color in images: PNA-DNA probe; cyan color: DAPI.

Figure 3.
Figure 3.

Comparison of mutation-specific peptide nucleic acid (PNA)-positive rate among epidermal growth factor receptor (EGFR) mutation-positive lung cancer tissues in cytokeratin-positive area. Tissues from tumors developing from four non-small-cell lung cancer cell lines were stained with each EGFR mutation-specific PNA probe and anti-cytokeratin. The PNA-positive rate in cytokeratin-stained cancer tissues was determined. The positive rate of staining for each PNA probe in three EGFR mutation-positive lung cancer tissues was compared with that in A549 tumors without EGFR mutations and statistically evaluated by the Mann-Whitney U-test. **Statistically significantly different at p<0.001. Magnification: 200×. Green color in images: PNA-DNA probe; orange color: cytokeratin.

Additionally, the percentage of cytokeratin-positive area was not significantly different among the EGFR-mutant NSCLC cell lines for any PNA-DNA probe staining (Figure 4).

Figure 4.
Figure 4.

Comparison of the cytokeratin-positive rate among non-small-cell lung cancer cell lines in the peptide nucleic acid (PNA)-DNA probe and cytokeratin staining experiments. Each panel shows the cytokeratin-positive rate in four lung cancer tissues stained with each PNA-DNA probe. Each column shows the mean±standard deviation from images of 10 areas in the stained slides. The positive rate of cytokeratin staining was compared among tissues from tumors developing from four non-small-cell lung cancer cell lines using the Mann-Whitney U-test. Magnification: 200×.

Immunofluorescence staining of normal human lung tissue using PNA-DNA probes. Normal human lung tissues did not stain positively with any of the three EGFR mutation probes (Figure 5A). The FFPE sections derived from normal lung tissue contained alveolar epithelial and stromal tissues, as shown in the hematoxylin-eosin-stained specimen.

Figure 5.
Figure 5.

Staining with peptide nucleic acid (PNA)-DNA probe in normal human lung tissues and comparison of PNA-DNA probe staining with epidermal growth factor receptor (EGFR) mutation-specific antibody (Ab) staining in four lung cancer tissues. A: Images of hematoxylin and eosin (HE) staining and EGFR mutation-specific PNA probe staining in normal human lung tissues. Magnification: 200×. B: Comparison of EGFR mutation (L858R)-specific PNA probe staining of non-small-cell lung cancer tissues with EGFR L858R monoclonal antibody staining. Green color in images: PNA-DNA probe; orange color: cytokeratin. Magnification: 200×.

Correlation of EGFR mutation staining between the PNA-DNA probe and-EGFR L858R antibody. There was similar localization of positive staining for the PNA-DNA probe and for the antibody to EGFR L858R. Comparison indicated that the positive staining rate with the EGFR mutation probe was comparable to that of anti-EGFR L858R in the NCI-H1975 cell line (Figure 5B). Moreover, the two staining agents exhibited a high positive correlation in the NCI-H1975 cell line (r=0.913, Figure 6).

Figure 6.
Figure 6.

Correlation analysis of the positive rates of peptide nucleic acid (PNA)-DNA probe staining and epidermal growth factor receptor (EGFR) mutation (L858R)-specific antibody staining in NCI-H1975 tumor tissue. The positive rates of PNA-DNA probe and anti-EGFR L858R from 10 images were plotted. Correlation between the staining ability of the PNA-DNA probe and anti-EGFR L858R was statistically evaluated using Pearson correlation analysis. Magnification: 200×.

Discussion

To date, numerous advantages of PNA technology have been verified: i) High sensitivity and specificity for hybridization to complementary DNA or RNA; ii) single-base mismatch discrimination; iii) highly stable binding to DNA or RNA because of the higher melting temperature; and iv) resistance to nuclease digestion and low ionic strength conditions because of the uncharged backbone (14-20).

Since the development of PNA probes by Nielsen et al., many applications in molecular genetics and cytogenetics have been attempted (14-18). Specifically, the application of PNA probes in fluorescence in situ hybridization (FISH) using FFPE specimens from driver mutation-positive cancer tissues has not yet been performed as far as we are aware. Conventional DNA- or RNA-FISH methods are frequently feasible for cytogenetic analysis aiming to investigate the structural features of chromosomes (21, 22). Considering the many advantageous features of PNA probes, the potential for the application of PNAs for the detection of driver mutations in various cancer tissues as sensitive ISH probes is very valuable (23, 24).

In the present study, we focused on the use of EGFR mutation-specific PNA-DNA probes, which can efficiently detect single-base mismatch mutations, instead of conventional DNA or RNA-FISH for detection of mutant mRNA in NSCLC tissues known to harbor EGFR mutations. Importantly, the main characteristics of our method were to visualize and localize EGFR-mutant areas efficiently in NSCLC tissue sections.

Fontenete et al. reported that the effectiveness of FISH in discriminating mismatched base pairs was improved by the use of locked nucleic acid or PNA probes compared to DNA probes (25). There are several reasons why PNA probes have a better hybridization ability than other agents, as described above: their high melting point, their stability because of the uncharged backbone, and their resistance to degradation by nucleases. Furthermore, considering the lack of significant staining observed in A549 tumors with the wild-type EGFR gene and normal human lung tissues, PNA-DNA probes can be developed for specific mutant mRNA detection.

Additionally, in the comparison study, the PNA-DNA probe was able to detect L858R mutant tumors with a sensitivity comparable to that of the EGFR (L858R) antibody, as shown in Figure 5. Seo et al., demonstrated that when the ISH-positive staining score was greater than 3+, the sensitivity of antibody to EGFR-mutated (L858R) protein was low (41.3%) (26). The difference in the target molecule of the PNA probe, i.e., mRNA, and the antibody, i.e., mutant peptide, might be responsible for that phenomenon. However, in our study, there was no significant difference in sensitivity for the L858R mutation between the PNA probe and the antibody.

With regard to PNA probe-based technology enabling discrimination between mismatched base pairs, PNA-mediated PCR clamping or PNA-locked nucleic acid (LNA)-mediated loop-mediated isothermal amplification (LAMP) are reported to be novel and rapid methods for detecting KRAS proto-oncogene, GTPase (KRAS) mutations (27, 28), but these are not available for FFPE evaluation yet.

By means of preparing EGFR mutation-specific PNA-DNA probes, it becomes possible to evaluate the effect of EGFR signaling inhibitors more specifically in terms of EGFR mutation localization. However, the present PNA-DNA probe method still has a limitation in regard to clinical specimens because there is no significant evidence of its advantages over other methods of mutation detection; more staining data using clinical specimens is therefore required.

Finally, there is an intriguing feature regarding PNA function. Based on previous reports that PNAs inhibit the transcription and translation of genes by tightly binding to DNA or mRNA (29-31), it is possible that PNAs may have an effect on tumor growth by targeting missense driver mutations responsible for cancer progression and metastasis, but this remains to be elucidated.

In the current study, using EGFR mutation-specific PNA-DNA probes, we successfully developed a multicolor-based immunofluorescence staining system for detecting EGFR mutations in FFPE. Our results suggest that PNA-DNA probes may have potential as universal and efficient tools to detect various types of cancer-driver gene mutations at the single-cell or tissue level.

Acknowledgements

This study was supported by a grant to Shohei Yamamura from JSPS KAKENHI (grant no. 22H03983), Japan.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest.

  • Authors’ Contributions

    HS and HM participated equally in the design of the study and drafting of the article and were responsible for completing the study. YA supervised all procedures of the study and strongly supported the completion of the article. AI, YK, CH, and CM performed the in vitro immunological experiments. TA performed the in vivo experiments. KY and SY reviewed the article. All Authors read and approved the final draft.

  • Received April 11, 2023.
  • Revision received June 9, 2023.
  • Accepted June 19, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Paez J,
    2. Jänne P,
    3. Lee J,
    4. Tracy S,
    5. Greulich H,
    6. Gabriel S,
    7. Herman P,
    8. Kaye F,
    9. Lindeman N,
    10. Boggon T,
    11. Naoki K,
    12. Sasaki H,
    13. Fujii Y,
    14. Eck M,
    15. Sellers W,
    16. Johnson B,
    17. Meyerson M
    : EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304(5676): 1497-1500, 2004. DOI: 10.1126/science.1099314
    OpenUrlAbstract/FREE Full Text
    1. Yang Z,
    2. Hackshaw A,
    3. Feng Q,
    4. Fu X,
    5. Zhang Y,
    6. Mao C,
    7. Tang J
    : Comparison of gefitinib, erlotinib and afatinib in non-small cell lung cancer: A meta-analysis. Int J Cancer 140(12): 2805-2819, 2017. DOI: 10.1002/ijc.30691
    OpenUrlCrossRefPubMed
  3. ↵
    1. Lamb Y
    : Osimertinib: a review in previously untreated, EGFR mutation-positive, advanced NSCLC. Target Oncol 16(5): 687-695, 2021. DOI: 10.1007/s11523-021-00839-w
    OpenUrlCrossRef
  4. ↵
    1. Borghaei H,
    2. Paz-Ares L,
    3. Horn L,
    4. Spigel D,
    5. Steins M,
    6. Ready N,
    7. Chow L,
    8. Vokes E,
    9. Felip E,
    10. Holgado E,
    11. Barlesi F,
    12. Kohlhäufl M,
    13. Arrieta O,
    14. Burgio M,
    15. Fayette J,
    16. Lena H,
    17. Poddubskaya E,
    18. Gerber D,
    19. Gettinger S,
    20. Rudin C,
    21. Rizvi N,
    22. Crinò L,
    23. Blumenschein G,
    24. Antonia S,
    25. Dorange C,
    26. Harbison C,
    27. Graf Finckenstein F,
    28. Brahmer J
    : Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. N Engl J Med 373(17): 1627-1639, 2015. DOI: 10.1056/NEJMoa1507643
    OpenUrlCrossRefPubMed
  5. ↵
    1. Rizvi N,
    2. Hellmann M,
    3. Snyder A,
    4. Kvistborg P,
    5. Makarov V,
    6. Havel J,
    7. Lee W,
    8. Yuan J,
    9. Wong P,
    10. Ho T,
    11. Miller M,
    12. Rekhtman N,
    13. Moreira A,
    14. Ibrahim F,
    15. Bruggeman C,
    16. Gasmi B,
    17. Zappasodi R,
    18. Maeda Y,
    19. Sander C,
    20. Garon E,
    21. Merghoub T,
    22. Wolchok J,
    23. Schumacher T,
    24. Chan T
    : Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348(6230): 124-128, 2015. DOI: 10.1126/science.aaa1348
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Lee C,
    2. Man J,
    3. Lord S,
    4. Links M,
    5. Gebski V,
    6. Mok T,
    7. Yang J
    : Checkpoint inhibitors in metastatic EGFR- mutated non-small cell lung cancer-a meta-analysis. J Thorac Oncol 12(2): 403-407, 2017. DOI: 10.1016/j.jtho.2016.10.007
    OpenUrlCrossRefPubMed
    1. Hayashi H,
    2. Sugawara S,
    3. Fukuda Y,
    4. Fujimoto D,
    5. Miura S,
    6. Ota K,
    7. Ozawa Y,
    8. Hara S,
    9. Tanizaki J,
    10. Azuma K,
    11. Omori S,
    12. Tachihara M,
    13. Nishino K,
    14. Bessho A,
    15. Chiba Y,
    16. Haratani K,
    17. Sakai K,
    18. Nishio K,
    19. Yamamoto N,
    20. Nakagawa K
    : A randomized Phase II study comparing nivolumab with carboplatin-pemetrexed for EGFR-mutated NSCLC with resistance to EGFR tyrosine kinase inhibitors (WJOG8515L). Clin Cancer Res 28(5): 893-902, 2022. DOI: 10.1158/1078-0432.CCR-21-3194
    OpenUrlAbstract
  8. ↵
    1. Chen N,
    2. Fang W,
    3. Zhan J,
    4. Hong S,
    5. Tang Y,
    6. Kang S,
    7. Zhang Y,
    8. He X,
    9. Zhou T,
    10. Qin T,
    11. Huang Y,
    12. Yi X,
    13. Zhang L
    : Upregulation of PD-L1 by EGFR activation mediates the immune escape in EGFR-driven NSCLC: implication for optional immune targeted therapy for NSCLC patients with EGFR mutation. J Thorac Oncol 10(6): 910-923, 2015. DOI: 10.1097/JTO.0000000000000500
    OpenUrlCrossRefPubMed
  9. ↵
    1. Takashima Y,
    2. Sakakibara-Konishi J,
    3. Hatanaka Y,
    4. Hatanaka K,
    5. Ohhara Y,
    6. Oizumi S,
    7. Hida Y,
    8. Kaga K,
    9. Kinoshita I,
    10. Dosaka-Akita H,
    11. Matsuno Y,
    12. Nishimura M
    : Clinicopathologic features and immune microenvironment of non-small-cell lung cancer with primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Clin Lung Cancer 19(4): 352-359.e1, 2018. DOI: 10.1016/j.cllc.2018.02.004
    OpenUrlCrossRef
  10. ↵
    1. Isomoto K,
    2. Haratani K,
    3. Hayashi H,
    4. Shimizu S,
    5. Tomida S,
    6. Niwa T,
    7. Yokoyama T,
    8. Fukuda Y,
    9. Chiba Y,
    10. Kato R,
    11. Tanizaki J,
    12. Tanaka K,
    13. Takeda M,
    14. Ogura T,
    15. Ishida T,
    16. Ito A,
    17. Nakagawa K
    : Impact of EGFR-TKI treatment on the tumor immune microenvironment in EGFR mutation-positive non-small cell lung cancer. Clin Cancer Res 26(8): 2037-2046, 2020. DOI: 10.1158/1078-0432.CCR-19-2027
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Shigeto H,
    2. Ohtsuki T,
    3. Iizuka A,
    4. Akiyama Y,
    5. Yamamura S
    : Imaging analysis of EGFR mutated cancer cells using peptide nucleic acid (PNA)-DNA probes. Analyst 144(15): 4613-4621, 2019. DOI: 10.1039/c9an00725c
    OpenUrlCrossRef
  12. ↵
    1. Yasui K,
    2. Kondou R,
    3. Miyata H,
    4. Iizuka A,
    5. Ashizawa T,
    6. Nagashima T,
    7. Ohshima K,
    8. Urakami K,
    9. Muramatsu K,
    10. Sugino T,
    11. Yamaguchi K,
    12. Ogawa H,
    13. Onoe T,
    14. Harada H,
    15. Asakura H,
    16. Murayama S,
    17. Nishimura T,
    18. Goto S,
    19. Okada S,
    20. Mukaigawa T,
    21. Hamauchi S,
    22. Yokota T,
    23. Onozawa Y,
    24. Akiyama Y
    : Immunological and genetic characterization of patients with head and neck cancer who developed recurrence. Anticancer Res 42(9): 4417-4428, 2022. DOI: 10.21873/anticanres.15942
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Kanda Y
    : Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplantation 48(3): 452-458, 2013. DOI: 10.1038/bmt.2012.244
    OpenUrlCrossRefPubMed
  14. ↵
    1. Nielsen P,
    2. Egholm M,
    3. Berg R,
    4. Buchardt O
    : Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254(5037): 1497-1500, 1991. DOI: 10.1126/science.1962210
    OpenUrlAbstract/FREE Full Text
    1. Nielsen PE,
    2. Egholm M
    : An introduction to peptide nucleic acid. Curr Issues Mol Biol 1(1-2): 89-104, 1999.
    OpenUrlPubMed
    1. Pellestor F,
    2. Paulasova P
    : The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics. Eur J Hum Genet 12(9): 694-700, 2004. DOI: 10.1038/sj.ejhg.5201226
    OpenUrlCrossRefPubMed
    1. Vilaivan T
    : Fluorogenic PNA probes. Beilstein J Org Chem 14: 253-281, 2018. DOI: 10.3762/bjoc.14.17
    OpenUrlCrossRef
  18. ↵
    1. Sadamoto S,
    2. Shinozaki M,
    3. Nagi M,
    4. Nihonyanagi Y,
    5. Ejima K,
    6. Mitsuda A,
    7. Wakayama M,
    8. Tochigi N,
    9. Murakami Y,
    10. Hishima T,
    11. Nemoto T,
    12. Nakamura S,
    13. Miyazaki Y,
    14. Shibuya K
    : Histopathological study on the prevalence of trichosporonosis in formalin-fixed and paraffin-embedded tissue autopsy sections by in situ hybridization with peptide nucleic acid probe. Med Mycol 58(4): 460-468, 2020. DOI: 10.1093/mmy/myz096
    OpenUrlCrossRef
    1. Nielsen P
    : PNA Technology. Mol Biotechnol 26(3): 233-248, 2004. DOI: 10.1385/MB:26:3:233
    OpenUrlCrossRefPubMed
  20. ↵
    1. Pellestor F,
    2. Paulasova P
    : The peptide nucleic acids, efficient tools for molecular diagnosis (review). Int J Mol Med 13(4): 521-525, 2004.
    OpenUrlPubMed
  21. ↵
    1. Gelali E,
    2. Custodio J,
    3. Girelli G,
    4. Wernersson E,
    5. Crosetto N,
    6. Bienko M
    : An application-directed, versatile DNA FISH platform for research and diagnostics. Methods Mol Biol 1766: 303-333, 2018. DOI: 10.1007/978-1-4939-7768-0_17
    OpenUrlCrossRef
  22. ↵
    1. Xiao L,
    2. Labaer J,
    3. Guo J
    : Highly sensitive and multiplexed in situ RNA profiling with cleavable fluorescent tyramide. Cells 10(6): 1277, 2021. DOI: 10.3390/cells10061277
    OpenUrlCrossRef
  23. ↵
    1. Shigeto H,
    2. Yamada E,
    3. Kitamatsu M,
    4. Ohtsuki T,
    5. Iizuka A,
    6. Akiyama Y,
    7. Yamamura S
    : Analysis of single nucleotide-mutated single-cancer cells using the combined technologies of single-cell microarray chips and peptide nucleic acid-DNA probes. Micromachines 11(7): 628, 2020. DOI: 10.3390/mi11070628
    OpenUrlCrossRef
  24. ↵
    1. Chen C,
    2. Hong Y,
    3. Ontiveros S,
    4. Egholm M,
    5. Strauss W
    : Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH. Mamm Genome 10(1): 13-18, 1999. DOI: 10.1007/s003359900934
    OpenUrlCrossRefPubMed
  25. ↵
    1. Fontenete S,
    2. Barros J,
    3. Madureira P,
    4. Figueiredo C,
    5. Wengel J,
    6. Azevedo NV
    : Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids. Appl Microbiol Biotechnol 99(9): 3961-3969, 2015. DOI: 10.1007/s00253-015-6389-4
    OpenUrlCrossRef
  26. ↵
    1. Seo A,
    2. Park T,
    3. Jin Y,
    4. Sun P,
    5. Kim H,
    6. Chang H,
    7. Chung J
    : Novel EGFR mutation-specific antibodies for lung adenocarcinoma: Highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry. Lung Cancer 83(3): 316-323, 2014. DOI: 10.1016/j.lungcan.2013.12.008
    OpenUrlCrossRefPubMed
  27. ↵
    1. Beau-Faller M,
    2. Legrain M,
    3. Voegeli A,
    4. Guérin E,
    5. Lavaux T,
    6. Ruppert A,
    7. Neuville A,
    8. Massard G,
    9. Wihlm J,
    10. Quoix E,
    11. Oudet P,
    12. Gaub M
    : Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping. Br J Cancer 100(6): 985-992, 2009. DOI: 10.1038/sj.bjc.6604925
    OpenUrlCrossRefPubMed
  28. ↵
    1. Itonaga M,
    2. Matsuzaki I,
    3. Warigaya K,
    4. Tamura T,
    5. Shimizu Y,
    6. Fujimoto M,
    7. Kojima F,
    8. Ichinose M,
    9. Murata S
    : Novel methodology for rapid detection of KRAS mutation using PNA-LNA mediated loop-mediated isothermal amplification. PLOS ONE 11(3): e0151654, 2016. DOI: 10.1371/journal.pone.0151654
    OpenUrlCrossRef
  29. ↵
    1. Nielsen P,
    2. Egholm M,
    3. Buchardt O
    : Sequence-specific transcription arrest by peptide nucleic acid bound to the DNA template strand. Gene 149(1): 139-145, 1994. DOI: 10.1016/0378-1119(94)90422-7
    OpenUrlCrossRefPubMed
    1. Rothman J,
    2. Surriga O,
    3. De Stanchina E,
    4. Vasudeva S,
    5. Schwartz G
    : Obstruction of BRAFV600E transcription by complementary PNA oligomers as a means to inhibit BRAF-mutant melanoma growth. Cancer Gene Ther 24(9): 401-408, 2017. DOI: 10.1038/cgt.2017.34
    OpenUrlCrossRef
  31. ↵
    1. Gasparello J,
    2. Gambari L,
    3. Papi C,
    4. Rozzi A,
    5. Manicardi A,
    6. Corradini R,
    7. Gambari R,
    8. Finotti A
    : High levels of apoptosis are induced in the human colon cancer HT-29 cell line by co-administration of sulforaphane and a peptide nucleic acid targeting miR-15b-5p. Nucleic Acid Ther 30(3): 164-174, 2020. DOI: 10.1089/nat.2019.0825
    OpenUrlCrossRefPubMed
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Localization of EGFR Mutations in Non-small-cell Lung Cancer Tissues Using Mutation-specific PNA-DNA Probes
HAJIME SHIGETO, HARUO MIYATA, TADASHI ASHIZAWA, AKIRA IIZUKA, YASUFUMI KIKUCHI, CHIKAKO HOZUMI, CHIE MAEDA, KEN YAMAGUCHI, SHOHEI YAMAMURA, YASUTO AKIYAMA
Cancer Genomics & Proteomics Jul 2023, 20 (4) 375-382; DOI: 10.21873/cgp.20389
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