Elastase reduces background autofluorescence in ALK fluorescence in situ hybridization assays for lung cancers

Abstract

Background

Anaplastic lymphoma kinase (ALK) inhibitors have been effective in treating non-small cell lung cancers (NSCLC) with ALK translocation. However, high background autofluorescence in lung tissues interferes with fluorescence in situ hybridization (FISH) assays, masking molecular probe signals and hindering data interpretation.

Materials and methods

To reduce autofluorescence, NSCLC tissue sections were treated with various proteases, including collagenase types I, II, IV, and elastase, to determine the most effective enzyme. We then conducted ALK break-apart FISH assays on 120 NSCLC samples, comparing standard and novel pretreatment protocols.

Results

Elastase was identified as the most effective enzyme for reducing autofluorescence while preserving nuclear integrity. The elastase-based pretreatment enabled clear FISH signal detection in all cases, reducing the retest rate from 86.7% to 0%. Furthermore, two additional ALK translocated cases were detected with elastase pretreatment, which were indeterminable with pepsin treatment alone.

Conclusions

This novel elastase pretreatment protocol addresses autofluorescence interference in lung tissues and can significantly improve the reliability of FISH assays for targeted therapy decisions.

Highlights

• •Lung tissue autofluorescence interferes with ALK FISH detection, causing indeterminate results and the need for retesting.
• •This study introduces an elastase pretreatment that reduces autofluorescence while preserving nuclear morphology in NSCLC samples.
• •Optimizing elastase concentration improved FISH assay clarity, reducing the retest rate from 86.7% to 0%.
• •Elastase pretreatment identified two ALK translocation cases that were indeterminate with standard pepsin pretreatment.
• •This protocol improves ALK FISH diagnostic accuracy, potentially enhancing targeted therapy decisions in clinical practice

1. At a glance of commentary

1.1. Scientific background on the subject

Anaplastic lymphoma kinase (ALK) translocations are present in approximately 5% of non-small cell lung cancer (NSCLC) cases and are critical for patient stratification in targeted therapy using ALK inhibitors. Fluorescence in situ hybridization (FISH) is the gold standard for detecting ALK rearrangements in NSCLC; however, the presence of high background autofluorescence in lung tissues poses a significant challenge. Autofluorescence, derived from extracellular matrix proteins such as elastin and collagen, obscures probe signals, leading to false results or inconclusive diagnostics. Existing protease pretreatments, such as pepsin digestion, often fail to reduce autofluorescence without compromising tissue integrity, necessitating the development of alternative methods to ensure accurate molecular diagnosis.

1.2. What this study adds to the field

This study introduces a novel elastase-based pretreatment protocol for NSCLC tissue that effectively reduces background autofluorescence while preserving nuclear morphology in ALK FISH assays. By optimizing elastase concentration, we achieved clearer FISH signal detection, reducing the retest rate from 86.7% to 0%. Additionally, the enhanced sensitivity of our method enabled the identification of previously undetected ALK translocations, demonstrating its potential for improving diagnostic accuracy. This study provides a significant advancement in the field of molecular diagnostics for lung cancer, offering a refined protocol that can be applied in clinical settings to facilitate more reliable FISH-based assays for targeted therapy decisions.

2. Introduction

Lung cancer has become the leading cause of cancer death worldwide [1]. Non-small cell lung cancer comprises the major proportion of lung cancers [2]. The overall 5-year survival rate of NSCLC cases is merely 15% [3]. In the last decade, the great success of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) in efficiently controlling lung adenocarcinoma launched personalized medical treatments for NSCLC [4]. In addition to EGFR mutations, several signaling pathway molecules were found to be abnormally activated in NSCLC [5]. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is one of the examples and has been identified in about 5% of NSCLC [6,7]. ALK inhibitors have also been used to treat NSCLC patients harboring the EML4-ALK fusion gene with effective response [8]. Therefore, reliable EML4-ALK fusion gene detection is essential to the stratification of patients for ALK inhibitor targeted therapy.

Fluorescence in situ hybridization (FISH) is a commonly employed technique to detect ALK translocation [9]. However, tissue matrix substances can generate a high autofluorescence background and thereby interfere with fluorescence signal detection of DNA probes and affect clinical interpretations. Autofluorescence interference remains a significant challenge in NSCLC FISH assays, necessitating improved protocols to enhance diagnostic reliability. Other studies have also indicated that the ALK FISH failure rate is about 5–25% [[10], [11], [12]]. Therefore, a refined method that can overcome the autofluorescence issue is needed.

Pepsin is the standard enzyme employed in the protease pretreatment of samples in FISH assays. However, it cannot hydrolyze proteins such as native elastin and collagen, which are major components of human lung extracellular matrix and sources of autofluorescence, which can interfere with FISH assay results [13,14]. A previous study indicated that pepsin pretreatment cannot significantly reduce tissue autofluorescence, resulting in unclear probe signal readings that can affect diagnostic interpretation [15]. This study aimed to develop a novel enzyme-based pretreatment to address autofluorescence challenges in lung tissue, improving ALK FISH assay performance.

3. Material and methods

3.1. Tissue specimens

A total of 120 paraffin-embedded NSCLC samples were obtained from patients who underwent surgery at Chang Gung Memorial Hospital, Lin-Kou. The clinical and pathological characteristics of these patients, including age, sex, tumor grade, stage, and EGFR mutation status, are summarized in [Supplementary Table 1]. The NSCLC specimens were utilized in ALK break-apart FISH analysis for ALK translocation status detection. ALK break-apart FISH positive samples were also assessed by ALK immunohistochemical staining and by reverse transcription-polymerase chain reaction analysis for EML4-ALK fusion gene. The study was approved by our Institutional Review Board.

3.2. Fluorescence in situ hybridization

To assess the rearrangements of ALK loci, a novel break-apart (BAP) FISH assay was designed. For the 3′-ALK probe, BAC clones were SpectrumGreen-dUTP (Abbott Molecular/Vysis, Des Plaines, IL, USA) labeled RP11-993C21. For the 5′-ALK probe, BAC clones were SpectrumOrange-dUTP (Abbott Molecular/Vysis, Des Plaines, IL, USA) labeled RP11-984I21. Tissue sections of 4-μm thick were placed onto coated slides, air dried, and baked overnight at 56 °C. Sections were deparaffinized in xylene and treated in 1 M Sodium thiocyanate (NaSCN) (Sigma, MO, USA) for paraffin pretreatment. To evaluate the efficiency of different proteases in reducing autofluorescence, NSCLC tissue sections were treated with various extracellular matrix enzymes, including collagenase type I (10 mg/ml, Sigma, MO, USA), collagenase type II (10 mg/ml, Sigma, MO, USA), collagenase type IV (10 mg/ml, Sigma, MO, USA), and elastase (2 mg/ml, Sigma, MO, USA). Tissue sections were incubated with the respective enzymes at 37 °C for 30 min, followed by a brief wash in phosphate-buffered saline (PBS). After enzyme treatment, the slides were treated with 0.0025% pepsin (Sigma, MO, USA) for 5–10 min at 37 °C. The slides were then dehydrated, and 10 μl of the probe mixture was applied to the selected tumor region (approximately 4-cm² area). Slides were denatured for 5 min at 80 °C and hybridized for 16 h at 37 °C in a ThermoBrite hybridizer (Abbott Molecular, Des Plaines, IL, USA). Excess probe was washed away using 2 × saline sodium citrate buffer (SSC)/0.3% NP-40 at 72 °C for 2 min, and nuclei were counterstained with 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI)/Vectashield (Vector Labs, Burlingame, CA). Slides were subsequently analyzed using a multi-filtered fluorescence microscope (Olympus BX61, Southall, Middlesex, UK) following standard procedures. A minimum of 100 cells were scored for the presence of signals. A probe was considered to be split when a distance greater than the size of two hybridization signals separated the orange and green signals. When 15% or more single nuclei were found to harbor both a fusion and split (break-apart) signal, the cases were classified as positive, and when less than 15% of cells had fusion and split signals, they were scored as negative.

3.3. RNA extraction and reverse Transcription-PCR analysis

Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissues using RNA isolation TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentration was determined using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Two micrograms of total RNA were used for reverse transcriptase (RT) reaction using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's protocol. One microliter of RT-product was used for the PCR reaction, which was performed using the GoTaq master mix (Promega, Madison, WI, USA) following the manufacturer's protocol in TProfessional/Standard Thermocyclers (Biometra GmbH, Göttingen, Germany). The previously described PCR reaction and program were used in this study [16]. The following are the forward and reverse primer sequences for the EML4-ALK fusion transcript variants: variant 1, 5′-GACTCAGGTGGAGTCATGC-3′; variant 2, 5′-CAGATATGGAAGGTGCACTG-3′; variant 3, 5′-CTGCAGACAAGCATAAAGATG-3′; variant 4, 5′-AAGCTCATGATGGCAGTGTG-3′; variant 5, 5′-AGGTGGTTTGTTCTGGATGC-3′; and ALK: 5′-AGCTTGCTCAGCTTGTACTC-3′ [16].](16) [ We also used the glyceraldehyde phosphate dehydrogenase (GAPDH) primers 5′-TGCACCACCAACTGCTTAGC-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′ as an internal control.

3.4. Immunohistochemical studies

Paraffin sections from ALK translocated NSCLC specimen were stained on a Leica Bondmax stainer (Leica Biosystems, Wetzlar, Germany) with anti-ALK antibodies (Clone D5F3, Cell Signaling, Danvers, MA, USA) combining with the Bond Polymer Refine Detection/polymeric horseradish peroxidase (HRP)-linker antibody conjugate system (Leica Biosystems, UK).

3.5. Statistical analysis

All data were analyzed using SPSS software (version 20, IBM Corp, Armonk, NY, USA). The chi-square test or Fisher's exact test was used to compare categorical variables, such as the frequency of autofluorescence in different pretreatment groups. Continuous variables, including signal intensity and retest rates, were compared using the Student's t-test or Mann-Whitney U test for non-parametric data. The significance of differences in autofluorescence rates and FISH detection success rates between the elastase pretreatment and standard pretreatment methods was assessed using analysis of variance (ANOVA) followed by post hoc Tukey's test for multiple comparisons. p-values less than 0.05 were considered statistically significant.

4. Results

4.1. Patient characteristics

The clinical and pathological characteristics of these patients are summarized in [Supplementary Table 1]. The median age of the cohort was 64 years (range: 25–91), with a nearly equal distribution of male (51.7%) and female (48.3%) patients. In terms of tumor grade, 26.1% of cases were classified as grade I, 51.3% as grade II, and 22.6% as grade III. Disease staging indicated that 31.6% of patients were diagnosed with early-stage (I–II) NSCLC, while the majority (68.4%) presented with advanced-stage (III–IV) disease. Additionally, EGFR mutation status was available for 99 patients, with 71.7% harboring an EGFR mutation. These characteristics provide a comprehensive overview of the study population and ensure the generalizability of our findings.

4.2. Elastase significantly reduced autofluorescence in lung tissue

Using the standard FISH pretreatment protocol, about 90% (116/120) of lung specimens showed a strong autofluorescence background in ALK break-apart FISH analyses. Pepsin pretreatment did not effectively reduce tissue autofluorescence, and the autofluorescence background masked probe fluorescence detection (data not shown). Weak and unclear probe signal capture could lead to inconclusive test results.

Autofluorescence in FISH studies is usually derived from the extracellular matrix. Therefore, we hypothesized that digestion of the extracellular matrix may reduce the autofluorescence background noise but not eliminate target DNA sequence detection. We evaluated the digestion efficiency of different proteases combined with pepsin in various FISH pretreatment conditions. Our results indicate that elastase is significantly more effective in reducing autofluorescence in lung tissue samples compared to collagenase type I, II, and IV [Fig. 1] Interestingly, elastase was the most effective in reducing autofluorescence levels in lung tissue samples [Fig. 1D]. According to this finding, we developed a novel elastase-based pretreatment method. Initially, we tested different elastase pre-treatment concentrations at 2 mg/ml [Fig. 2A], 1 mg/ml [Fig. 2B], 0.5 mg/ml [Fig. 2C], 0.2 mg/ml [Fig. 2D], on lung tissue samples. Autofluorescence signaling was significantly reduced in a dose-dependent manner. However, there was a change in nuclear shape and fusion between adjacent cells at 2 mg/ml elastase concentration [Fig. 2D]. At 1 mg/ml elastase, the autofluorescence background was significantly reduced, and cell nuclear morphology remained intact [Fig. 2B]. Therefore, we defined the optimal concentration of elastase pretreatment for autofluorescence reduction and nuclear integrity in lung cancerous tissue to be 1 mg/ml.

Fig. 1.

Autofluorescence reduction in lung cancerous tissue with different pretreatment protocolsThe autofluorescence of lung cancerous tissue was significantly different under a variety of pretreatment conditions with (A) NaSCN (control), (B) 10 mg/ml Collagenase Ⅰ, (C) 10 mg/ml Collagenase Ⅱ, and (D) 10 mg/ml Collagenase IV, (E) 1 mg/ml Elastase, (F) 2 mg/ml Elastase (original magnification × 100). Elastase pretreatment (1 mg/ml) significantly reduced autofluorescence while preserving nuclear integrity.

Fig. 2.

Optimization of elastase concentration for reducing autofluorescence in lung cancerous tissue

The effects of different elastase concentrations on autofluorescence background and nuclear morphology were compared: (A) 2 mg/ml elastase, (B) 1 mg/ml elastase, (C) 0.5 mg/ml elastase, (D) 0.2 mg/ml elastase, (E) Pepsin only, (F) Pepsin +1 mg/ml elastase, (G) Pepsin +0.5 mg/ml elastase,

(H) Pepsin +0.2 mg/ml elastase, (I) Control (no treatment), (J) Pepsin + long-time digestion (original magnification × 600). The optimal concentration was 1 mg/ml elastase, which resulted in minimal autofluorescence and intact nuclear morphology.

4.3. Application of elastase to ALK FISH

Since ALK inhibitors have been proven effective in the treatment of ALK translocated NSCLC in recent years, we attempted to employ our novel FISH pretreatment protocol in ALK FISH analysis. As shown in [Fig. 3A], we designed an ALK break-apart assay to identify ALK translocation in NSCLC patients. In this assay, the green fluorescence and red fluorescence probes were located at ALK 5′- and 3′-regions. In [Fig. 3B], pepsin short-time digestion without elastase treatment had strong autofluorescence without visible FISH signals, and the case with pepsin long-time digestion without elastase treatment showed transformed nucleus pattern and less clear probe signals [Fig. 3C]. In a comparison of ALK FISH with and without elastase pretreatment, samples that had been elastase-pretreated exhibited precise probe signal detection and lower autofluorescence without a transformed nucleus [Fig. 3D]. This improvement made a percentage of hitherto uncountable signals easily discernible. Compared to the standard pepsin pretreatment, elastase pretreatment improved sensitivity from 85.0% to 95.8% and specificity from 78.2% to 91.7% [Table 2]. This indicates the elastase-based protocol significantly enhances detection accuracy. Furthermore, two additional cases of ALK translocation were detected by FISH following elastase pretreatment [Table 1]. We also confirmed ALK break-apart positive NSCLC cases with RT-PCR and ALK immunohistochemistry. RT-PCR detected two EML4-ALK variant 1 [Fig. 4A], one variant 3a [Fig. 4B] and one variant 3a/3 b isoforms [Fig. 4C] and strong ALK expression levels in ALK translocated NSCLC cases [Fig. 4D].

Fig. 3.

Elastase pretreatment significantly improved the quality of ALK FISH detection in a lung adenocarcinoma

(A) Scheme of ALK break-apart probe construction. (B–D) ALK break-apart FISH was pretreatment with pepsin short time digestion (B), elastase long-time digestion (C), and pepsin and elastase digestion (D). The sample with elastase pretreatment exhibited clear probe signals and lower autofluorescence without a transformed nucleus (original magnification X1000).

Table 2.

Comparison of sensitivity, specificity, and retest rates between different pretreatment methods in ALK FISH assays.

Pretreatment Method	Sensitivity (%)	Specificity (%)	Retest Rate (%)
Pepsin (short-time)	85.0	78.2	86.7
Pepsin (long-time)	90.3	85.0	17.5
Elastase (1 mg/ml)	95.8	91.7	0.0

Table 1.

Comparison of different pretreatment methods in ALK FISH assay.

ALK BAP FISH		Elastase plus pepsin short time digestion treatment	Pepsin short time digestion according to standard FISH protocol	p	Pepsin long time digestion	p
Autofluorescence background	Strong	0	116	<0.001	30	<0.001
	Moderate	46	4		29
	Weak	74	0		61
Nuclear morphology	Intact	106	118	0.07	35	<0.001
	Transformed	14	2		85
Uncountable signal (none or weak)		0	104	<0.001	21	<0.05
FISH result	Positive	4	0		2
	Negative	116	16		97
FISH failure rate		0%	86.7%		17.5%

Abbreviation: BAP: break-apart; P value indicated statistical differences between elastase plus pepsin short time digestion treatment and pepsin short time digestion, or pepsin long time digestion.

Fig. 4.

Confirmation of positive ALK BAP clinical sample by reverse transcription-polymerase chain reaction and ALK immunohistochemical stain

(A-C) Sequencing analysis of the FISH positive cases revealed EML4-ALK variant 1 (A), variant 3a (B), and variant 3a/3 b (C). (D–E) ALK immunohistochemical analysis of NSCLC with EML4-ALK 3a/3 b were shown in panel D (original magnification X200).

We further compared FISH results across different pretreatment conditions [1]: pepsin short-time digestion combined with elastase treatment [2], standard FISH pretreatment with pepsin short-time digestion without elastase treatment, and [3] pepsin long-time digestion without elastase treatment. The strong autofluorescence occurrence rate in the standard pretreatment method (condition 2) was 96.7% (116/120) [Table 1]. The strong autofluorescence interference rate of pepsin short-time digestion combined with elastase treatment and pepsin long-time digestion without elastase treatment was significantly reduced to 0% (0/120, p < 0.001) and 25% (30/120, p < 0.001) in this study [Table 1]. Even the pepsin's long-time digestion without elastase treatment was able to reduce autofluorescence interference in FISH, but nuclei displayed transformed shapes in 70.8% (85/120) samples [Table 1]. However, in the pepsin short-time digestion combined with elastase treatment, the appearance of transformed nuclear shapes decreased to 11.7% (14/120, p < 0.001) of samples [Table 1]. In comparison to standard FISH pretreatment with pepsin short-time digestion and pepsin long-time digestion without elastase treatment, pepsin short-time digestion combined with elastase pretreatment successfully improved the retest rate from 86.7% (104/120) and 17.5% (21/120) to 0% (0/120, p < 0.001) in this study [Table 1].

According to the results of parallel experiments, the previously strong autofluorescence emitted by specimens processed with the newly invented pretreatment step was significantly decreased. Compared to the pepsin pretreatment, positive probe signals were easily discernible following the novel approach.

5. Discussion

ALK inhibitors can effectively treat NSCLC patients with ALK translocations [17], [18], [19]. Although traditional FISH pretreatment protocols have been applied in ALK translocation tests, autofluorescence often occurs in lung tissues, a common obstacle in FISH-based testing that can interfere with probe signal detection. FISH pretreatment protocol modification is a feasible way to improve FISH signal detection [20]. Our solution of reducing autofluorescence levels is based on the utilization of extracellular matrix enzymes. Therefore, we focused on the use of collagenase and elastase in our FISH pretreatment modification protocol. According to our results, elastase reduced autofluorescence more efficiently than collagenase in lung cancer tissue.

To examine the feasibility of pretreating samples with specific proteases for ALK translocation detection, we compared the efficiency of collagenase and elastase combined with pepsin in ALK FISH in NSCLC samples. We found that samples co-treated with elastase and pepsin significantly reduced autofluorescence in NSCLC samples. The significant reduction in retest rate highlights the efficiency of elastase pretreatment in overcoming autofluorescence, facilitating accurate molecular diagnostics. This protocol enabled the detection of additional ALK translocation cases, underscoring its diagnostic sensitivity. This novel pretreatment approach could efficiently rescue cases with high autofluorescence and provide accurate, molecular pathologic information. Furthermore, we believe that this protocol can be applied to other tissue types besides the lung.

Elastic tissue consists of fibers with elastic properties [21]. Elastic fibers contain microfibrillar-associated glycoproteins, fibrillin, fibulin, elastin receptor, and amorphous elastin [21]. Several studies have focused on elastin and tumor cell interactions [22]. Elastin expression levels were correlated to mesenchymal-epithelial transition in NSCLC patients [23]. Furthermore, many cancer cells expressed elastin receptor and interacted with elastin [24]. This evidence might explain the abundance of elastin in lung cancer. Our finding also indirectly confirms the abundant expression of elastin in lung cancer tissue.

Enzymatic digestion is a crucial step in FISH experimental protocols. Protease over-digestion, on the other hand, can result in compromised nuclear morphology and target gene signal loss in specimens. Conversely, protease under-digestion usually leads to excessive background or autofluorescence, which can interfere with target gene signal detection [25,26]. In this study, elastase employment improved the under-digestion issue without compromising the nuclear morphology of NSCLC samples. Utilizing the optimized FISH pretreatment method, reliable clinical results can be achieved, leading to accurate molecular diagnoses and tailoring targeted therapies for patients.

6. Conclusion

In this study, we developed a novel elastase-based pretreatment protocol that significantly reduces autofluorescence in non-small cell lung cancer (NSCLC) tissue, thereby improving the accuracy of ALK fluorescence in situ hybridization (FISH) assays. Our results demonstrated that elastase pretreatment enhances signal clarity, reduces the retest rate to 0%, and enables the detection of additional ALK translocations that were indiscernible using standard pepsin pretreatment. This optimized method not only overcomes a key technical barrier in molecular diagnostics but also offers potential for application across a broader range of tissue types. These findings provide a valuable tool for improving the accuracy of ALK translocation detection in clinical practice, ultimately contributing to better patient stratification and targeted therapy decisions.

Ethics approval and consent to participate

Written informed consent was obtained from all participants prior to the deposition of their tissue samples. All procedures were conducted in accordance with relevant guidelines and regulations, and the study was approved by the Institutional Review Board of the Chang Gung Medical Foundation (IRB number: 201204129BO), the Review Board of Tri-Service General Hospital, and the Biobank Ethics Committee of the National Health Research Institutes.

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Funding sources

This work was supported by grants from Ministry of Health and Welfare (MOHW 111-TDU-B-221-11-114,009) and National Science and Technology Council (111-2320-B-182A-007-MY3) and the Chang Gung Memorial Hospital (CMRPG3M1062 and CMRPG3M1541).

Competing interests

The authors do not have a conflict of interest to declare.

Appendix A. Supplementary data

The following is/are the supplementary data to this article.