Introduction

A peptide nucleic acid (PNA)-based mutant mRNA detection system was developed by Nielsen et al., prompting many trials applying PNA probes to the molecular genetics field1,2,3,4,5. In addition to the speed and low-cost, many advantages of the PNA technology have been demonstrated, including high sensitivity and specificity for hybridization to complementary DNA or RNA, single base mismatch discrimination, highly stable binding to DNA or RNA due to the higher melting temperature, and resistance to nuclease digestion and low ionic strength conditions due to the uncharged backbone6,7,8.

Progress in next-generation sequencing technology has enabled us to make genetic diagnostic tools, such as target sequencing panel and cell-free DNA detection methods 9,10,11. The development of genetic diagnostics, including the diagnosis of epidermal growth factor receptor (EGFR), RAS, ROS1, and ALK gene mutations, allows the selection of suitable therapies for patients with non-small cell lung cancers (NSCLC) 12,13,14. Although NGS technology is accurate and covers a wide variety of target genes, the technology is time-consuming and expensive compared to conventional genetic mutation detection technologies15,16,17.

In the present study, we developed a system for detecting EGFR-mutations based on EGFR mutation-specific PNA:DNA probes. This system was successfully applied to the detection of EGFR-mutant tumors in formalin-fixed paraffin-embedded (FFPE) sections of tumors from nude mice transplanted with EGFR-mutant tumor cells18,19. The objective of the present study was to determine the detection efficiency of PNA:DNA probes for EGFR-mutations and the association of the localization of EGFR-mutations with tumor-infiltrating lymphocyte (TIL) status in patients with EGFR-mutant NSCLC.

Since the initiation of the Project High-tech Omics-based Patient Evaluation (HOPE) in 2014, more than 10,000 cancer patients have been enrolled. Cancer genomic profiling was performed in these patients using whole-exome sequencing (WES) and gene expression profiling (GEP). FFPE specimens from patients with EGFR-positive NSCLC in the Project HOPE were included in the present study.

Results

Clinical characteristics of 31 NSCLC patients enrolled in the project HOPE

The characteristics of the 31 NSCLC patients enrolled in the study are shown in Table 1. All patients were enrolled in the HOPE project, and EGFR-mutations were subjected to WES analysis. Twenty cases had EGFR gene mutations and 11 patients had no mutations. The mutations included 11 cases with L858R mutations and 9 cases with exon19 deletion mutations. No significant differences in age, tumor content, variant allele frequency (VAF), and survival time were detected between the mutation types, except that the EGFR gene-mutated cases included more female patients.

Table 1 Clinical characteristics of EGFR-mutation harboring NSCLC patients enrolled in HOPE project.
Full size table

Measuring cytokeratin and EGFR mutation-positive area using PNA:DNA probes

The immunofluorescence staining set included PNA, anti-cytokeratin antibody, and antibodies for TIL-staining, as shown in Table 2. Cytokeratin-positive areas indicate intra-tumoral regions and cytokeratin-negative areas indicate extra-tumoral or stromal regions. No differences in the ratios of cytokeratin-positive areas were detected between the L858R mutation, exon 19 deletion mutation, and no mutation groups (Fig. 1A). EGFR-mutated tumors exhibited positive staining with the PNA:DNA probe (19.5% for L858R mutation probe and 8.6% for exon 19 deletion mutation probe). In contrast, the tumors without EGFR-mutations showed lower positive rate for both PNA:DNA probe staining (Fig. 1B and Fig. 2). The L858R probe exhibited more positive staining than the exon 19 deletion probe.

Table 2 Immunofluorescence stain set using 7-color Opal staining kit.
Full size table
Fig. 1
figure 1

Mutation-specific peptide nucleic acid (PNA)-positive staining in epidermal growth factor receptor (EGFR) mutation-positive lung cancer tissues. Formalin-fixed paraffin-embedded (FFPE) tissues from patients with non-small cell lung cancer were stained with EGFR mutation-specific PNA probes, anti-cytokeratin antibodies, and DAPI. PNA-positive staining rates in cytokeratin-stained cancer tissues were determined. (A) Cytokeratin-positive rate in DAPI-stained cancer tissues from the EGFR-mutated (L858R or exon19-deletion) and no mutation tumor groups. (B) EGFR mutation probe positive rates in cytokeratin-stained cancer tissues from the EGFR-mutated (L858R or exon19-deletion) and no mutation tumor groups. The positive staining rate for each EGFR mutation-specific PNA probe in the EGFR-mutated (L858R or exon19-deletion) and no mutation tumor groups were compared using the Mann–Whitney U test. **P < 0.01.

Full size image
Fig. 2
figure 2

Representative images of PNA:DNA probe and cytokeratin staining in the EGFR-mutated (L858R or exon19-deletion) and no mutation tumor groups. Green: PNA:DNA probe; red: cytokeratin; yellow: positive for PNA:DNA probe and cytokeratin. Magnification: × 200.

Full size image

PNA:DNA probe efficacy for EGFR mutation detection

Receiver operating characteristic (ROC) curve analysis revealed that the L858R probe was more sensitive and specific than the exon 19 deletion probe in detecting tumors with EGFR-mutations (Fig. 3A, B).

Fig. 3
figure 3

A receiver operating characteristic (ROC) curve analysis and comparison of the efficacy of EGFR mutation-specific PNA probes. (A) ROC curve for PNA probe positivity (%) in L858R-mutated and exon19 deletion-mutated tumor groups. The area under the curve for each plot indicates the performance of the L858R-specific (0.97; 95% CI, 0.91–1) and exon19 deletion-specific (0.84; 95% CI, 0.64–1) PNA probes. (B) Comparison of the PNA probe efficacy between the EGFR mutation-positive and EGFR mutation-negative tumor groups. L858R mutation-positive tumor group (n = 11), exon19 deletion mutation-positive tumor group (n = 9), and no EGFR mutation group (n = 11).

Full size image

Comparison of TIL status between L858R mutation-positive tumors and no EGFR mutation tumors

TIL numbers, such as CD4+, CD8+, and CD68+ cells, tended to be lower in the intra-tumoral regions in both the L858R mutation and no mutation group compared to the TIL numbers in the extra-tumoral regions. However, TIL cells were higher in the extra-tumoral region in the L858R mutation group compared with the no mutation group (Fig. 4, Supplementary Fig. S1, S2 and S3). PD-L1+, FoxP3+, and CD204+ cells tended to increase in the intra-tumoral region in the L858R mutation group.

Fig. 4
figure 4

Comparison of the tumor-infiltrating lymphocyte (TIL) status between the L858R mutation-positive and no EGFR mutation tumor groups. Comparison of TIL numbers in cytokeratin-positive intra-tumoral region and cytokeratin-negative extra-tumoral region between L858R mutation-positive tumors and no EGFR mutation tumors. Immune cells, including CD8+PD-1+ T cells, CD4+FoxP3+ T cells, and CD68+ or CD204+ macrophages, were counted using an in-house automatic counting system. The positive immune cell numbers per field in the EGFR-mutated (L858R) and no mutation tumor groups were compared using the Mann–Whitney U test. *P < 0.05, **P < 0.01.

Full size image

Association of TIL status in L858R mutation-positive tumors with overall survival

The higher number of CD204+ immune cells (macrophages) in the extra-tumoral region in the L858R mutated tumors tended to correlate with poor survival (Supplementary Fig. S4) (r =  − 0.519), but no significant correlation was found between CD204+ cell numbers and overall survival. The other TIL numbers did not show any association with overall survival time.

Comparison of TIL status between PNA-positive area and PNA-negative area in the intra-tumoral region of L858R mutated tumors

PD-L1 expression levels tended to decrease in PNA-positive areas compared with PNA-negative areas of the intra-tumoral regions, but no significant difference in PD-L1 expression level was found between PNA-positive areas and PNA-negative areas (Fig. 5). In contrast, CD4+, FoxP3+, and CD204+ cells were more abundant in the L858R-positive tumor areas compared with the negative areas of the intra-tumoral regions in L858R mutated tumors.

Fig. 5
figure 5

Comparison of the tumor-infiltrating lymphocyte (TIL) status between the PNA-positive and PNA-negative areas in the intra-tumoral region of L858R-mutated tumors. The cytokeratin-positive intra-tumoral region (CK +) was classified into PNA-positive (PNA +) area and PNA-negative (PNA −) areas based on the L858R mutation-specific PNA probe staining results. To correctly compare TIL numbers between the PNA-positive and the PNA-negative areas, the immune cell numbers were normalized by dividing the actual immune cell numbers by the ratio of the PNA-positive or PNA-negative stained areas (normalized positive cell number per area). The positive cell numbers per area were compared between the PNA-positive and the PNA-negative areas using the Mann–Whitney U test.

Full size image

Association of intra-tumoral immune cell status between PNA-positive area and PNA-negative area with overall survival

In the intra-tumoral regions of the L858R-mutated tumors, CD204+ cell numbers in PNA-negative areas were significantly and negatively associated with overall survival (r =  − 0.6292) (Fig. 6). In addition, no significant correlation was found between PD-L1+ cell numbers in PNA-positive areas of intra-tumoral regions and overall survival (r =  − 0.5154).

Fig. 6
figure 6

Association of intra-tumoral immune cell status between the PNA-positive and PNA-negative areas and overall survival. Immune or tumor cells, such as PD-L1+ cells, CD68+ cells, and CD204+ cells, were counted using an automatic counting system. The positive cell numbers per field were obtained from the EGFR-mutated (L858R) tumor group and classified into cytokeratin-positive intra-tumoral and cytokeratin-negative extra-tumoral regions. Correlation analysis of the normalized positive cell numbers per area with the overall survival time was performed using Pearson correlation test. *P < 0.05 was considered statistically significant.

Full size image

Discussion

NSCLCs with EGFR-mutations exhibit low PD-L1 expression, TILs, and TMB status. Consequently, these tumors respond poorly to anti-PD-1/PD-L1 blockade and respond better to combination therapy containing cisplatin and EGFR-tyrosine kinase inhibitors (TKIs)20,21,22,23,24. However, the immunological tumor microenvironment (TME) status can change. Dynamic changes in biomarkers and immune cell markers occur after EGFR-TKI treatment. Increased PD-L1 expression and decreased FOXp3+ regulatory T cell number have been detected after EGFR-TKI treatment25,26,27,28. In some cases, the TMB number tended to increase after EGFR-TKI therapy28. Based on these changes in the immunological TME status, anti-PD-1 blockade therapy after EGFR-TKI treatment may be beneficial. Haratani et al. reported that patients with T790M-negative EGFR-mutated NSCLC are more likely to benefit from anti-PD-1 blockade after EGFR-TKI treatment than patients with T790M-positive tumors29.

The recovery of immunological parameters in EGFR-mutated tumors after EGFR-TKI therapy depends on the reduction of EGFR mutation-positive tumor clones. Therefore, rapid and efficient tools such as our PNA probe are very useful. In addition, we demonstrated that heterogeneous immune cells localize between PNA-positive and PNA-negative areas of intra-tumoral regions in L858R mutated tumors. Our PNA probe may be useful for evaluating the TIL status, according to the heterogeneous location of the EGFR mutation in the intra-tumoral region.

The development of other PNA-based EGFR mutation detection methods has been reported, including real-time PCR clamping technology30,31. In a study by Kim et al., genomic EGFR-mutations are detected in 34.6% of non-squamous NSCLC patients using DNA extracted from FFPE specimens, whereas DNA direct sequencing showed a positive rate of 26.3%31. These results indicate that PCR clamping is more effective than DNA sequencing in detecting EGFR-mutations. The sensitivity of our PNA probe for L858R EGFR-mutations was 100% in 11 cases harboring EGFR mutation verified by WES sequencing. This result indicates that the efficiency of the PNA probe is comparable to direct DNA sequencing.

Intra-tumoral genomic heterogeneity is a crucial problem in the clinical field. Various heterogeneous patterns of genomic differences have been detected, including the differences between primary and metastasis lesions32 and the spatiotemporal differences in multiple biopsies33,34. For example, after acquired resistance to the EGFR-TKI against T790M, many genomic mutations, local copy number amplifications, chromosomal instability, and neuroendocrine differentiation are acquired, resulting in the development of osimertinib resistance35,36. We demonstrated that heterogeneous immune cell localization and PD-L1 expression in the PNA-positive and PNA-negative areas of intra-tumoral regions in L858R-mutated tumors can be evaluated. These results suggest that PNA:DNA probes specific for EGFR-mutations can be a useful tool for detecting areas with heterogeneous EGFR-mutations.

Although our results are encouraging, several technical problems and limitations need to be solved. The sensitivity of the PNA probe specific for the L858R mutation is comparable to the sensitivity of direct DNA sequencing, but the PNA probe specific for the exon19-deletion mutation is less sensitive. Thus, the development of a better PNA probe for the exon19-deletion mutation is urgently needed. In addition, the present study had potential biases, including the use of FFPE specimens, the small sample size, and limited data from only one database.

Despite these limitations, we verified the detection efficiency of our PNA:DNA probes and the association of EGFR mutation localization with TIL status and prognosis using FFPE specimens from NSCLC patients with EGFR-mutations. These results suggest that PNA:DNA probes specific for EGFR-mutations are promising tools for detecting tumor heterogeneity and TIL status before and after EGFR-TKI treatment.

Conclusion

In summary, our PNA:DNA probe specific for EGFR L858R mutation detection exhibited a sensitivity of 100% and a specificity of 90% in clinical NSCLC FFPE specimens. Thus, the efficacy of the L858R mutation-specific PNA probe is comparable to the efficacy of direct DNA sequencing. The PNA:DNA probes specific for EGFR-mutations can be useful tools for evaluating tumor heterogeneity and TIL status to facilitate treatment decision-making.

Materials and methods

Ethical statement

All protocols in the study were approved by the Institutional Review Board of the Shizuoka Cancer Center (Authorization Number: 26-62). Written informed consent was obtained from all study participants. All experiments using clinical samples were conducted in accordance with the approved guidelines. This study was conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Human Genome and Genetic Analysis Research.

Patient characteristics

Eighty-one non-small cell lung cancer patients, who were enrolled in the cancer genome project named HOPE at Shizuoka Cancer Center in 2014, were screened for the study. The enrolled patients included 20 cases with EGFR gene mutations (9 Exon19 deletion cases and 11 L858R cases) and 11 cases with no EGFR-mutations. FFPE specimens derived from the 31 NSCLC patients were obtained and used for PNA:DNA probe staining experiments specific for EGFR-mutations.

DNA microarray-based GEP and WES using next-generation sequencing

GEP and WES analyses were described previously37. Briefly, the ratio of the expression intensity between the tumor tissue and the surrounding normal tissue was calculated from normalized values for GEP. In WES, all variants called by the variant caller were filtered using the following parameters: quality < 30, VAF < 10%, and depth of coverage < 20× . WES was performed using a NextSeq 6000 System (Illumina, San Diego, CA, USA), and transcriptome profiling was performed using a SurePrint G3 Human Gene Expression 8 × 60K v2 Microarray (Agilent Technologies, Canta Clara, CA, USA).

Variant calling and annotation

Small variants included small nucleotide variants (SNVs) and indels (≤ 50 bp), and SVs were greater than 50 bp. For SNVs and indel mutations, somatic and germline variants were identified using the DRAGEN Somatic Variant Caller and Small Variant Caller (Illumina), respectively. Variants with a low sequence depth in normal and/or tumor tissues, low variant depth, and/or low VAF in tumors were discarded from the downstream analysis. Variants were annotated using Ensembl Variant Effect Predictor ver. 104. The drivability of the variants was analyzed using an in-house pipeline.

PNA:DNA probes specific for EGFR mutations

The development of PNA:DNA probes specific for the EGFR mutant mRNA sequences were previously reported18. Briefly, for making L858R mutation probe, fluorescein isothiocyanate (FITC)-conjugated PNA (FITC-PNA; fluorescein-O-Linker-PNA (TTTGGCCCGCCCAA)-k-k; Panagene, Daejeon, Korea) and quencher-conjugated DNA (Q-DNA; GCGGGCCA-Dabcyl; Integrated DNA Technologies, Coralville, IA, USA) were prepared. PNA:DNA probes were constructed by hybridizing the synthesized FITC-PNA and Q-DNA. The probes used in the present study were designed specifically for L858R and del E746-A750 EGFR-mutations.

Immunofluorescence staining using a multicolor staining kit

The following antibody were used for immunostaining: anti-human cytokeratin antibody (AE1 and AE3, Nichirei, Tokyo, Japan), anti-human CD4 antibody (4B12, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD8 antibody (C8/144b, GeneTex Inc., Irvine, CA, USA), anti-human PD-1 antibody (NAT105, Abcam, Cambridge, UK), anti-human PD-L1 antibody (28-8, Abcam), anti-human FoxP3 antibody (236A/E7, Abcam), and-human CD68 antibody (3A9A7, Proteintech Inc., Rosemont, IL), and anti-human CD204 antibody (KT118, Transgenic Inc., Kobe, Japan). The immunofluorescence staining set is shown in Table 2. Staining was performed using the Opal 7-color IHC kit (Akoya Biosciences, Inc., Menlo Park, California, USA) and a Zeiss Imager Z1 fluorescence microscope equipped with the ZEN software system (Carl Zeiss, Oberkochen, Germany) as reported previously19. Briefly, after deparaffinization and hydration of FFPE sections, activation by autoclaving 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. Next, PNA:DNA coupling was carried out and used for probe staining.

Establishment of the EGFR mutation detection and TIL automatic counting system

The method for measuring the cytokeratin and PNA probe positive area and TIL numbers was shown previously38. In brief, five of 10 acquired images were selected using a random table. WinROOF (Mitani Shoji, Tokyo, Japan)38, ImageJ (ver.1.53g58), and R software (ver. 3.4.0) were used to measure the fluorescence of positively-stained areas and for the TIL automatic counting system.

Statistical analysis

Positive staining rate and TIL numbers were compared using the Mann–Whitney U test. Correlation analysis of the positive staining rate of the PNA:DNA probe and TIL staining with other parameters was performed using Pearson correlation test. ROC curve analysis was used for a statistical analysis of PNA probe positivity for EGFR mutations. P < 0.05 were considered statistically significant.