Introduction

Lung cancer causes the largest number of cancer related deaths worldwide. Currently, more than 85% of lung cancer cases are classified as non-small-cell-lung cancer (NSCLC). Adenocarcinoma is the most common histological type of NSCLC1. Although conventional chemotherapy is the main treatment for the majority of advanced NSCLC patients, new targeted therapies have been developed for a subset of patients harboring key oncogenic alterations2. For example, drugs targeting epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations have been successfully used in clinical medicine2, 3. ALK gene translocation was first discovered in NSCLC in 20074. This translocation results from a small inversion in chromosome 2p leading to aberrant ALK gene translocation expression in the cytoplasm and uncontrolled cellular proliferation and survival4, 5. The US Food and Drug Administration (FDA) have approved crizotinib and ceritinib, the first and second-generation ALK inhibitors respectively6. The identification of ALK gene translocation in NSCLC is the basis of targeted therapy with ALK inhibitors. Three methods have been utilized to detect ALK gene translocations: fluorescent in situ hybridisation (FISH), real-time reverse transcription-PCR (RT-PCR), and novel fully automated Ventana ALK D5F3 immunohistochemistry (IHC). Previous study by Ying et al. has demonstrated the 100% sensitivity and 98% specificity of the Ventana ALK assay, respectively7.

The morphology and genetics of tumour heterogeneity are topics of great interest in cancer research8. Recently, several solid malignant tumours have been found to be genetically heterogeneous8,9,10. For example, IHC analysis showed that EGFR expression in primary NSCLC tumour/metastasis had a discordance rate of 33.3%10. In this study, we applied Ventana ALK D5F3 IHC to investigate the heterogeneity of ALK gene translocations in excision specimens and compared the ALK status between primary tumours and their corresponding metastatic lymph nodes.

ALK gene translocation was previously found to be mutually exclusive with other driver gene mutations11. However, several recent reports have identified an overlap between ALK translocation and other driver gene mutations12,13,14,15,16,17. Here, we analyze the association of ALK gene translocation with the occurrence of other driver gene mutations by directly sequencing the EGFR, KRAS, BRAF, and HER2 gene mutations and with clinicopathological characteristics.

Results

Characteristics of ALK-positive lung adenocarcinoma samples

Clinicopathological features of 106 ALK-immunopositive cases were compared with 90 ALK-immunonegative cases (Table 1). The median age of patients with ALK-positive was 52.5 years (28–77 years) among 51 men and 55 women. At the time of resection, 45 (42.5%), 10 (9.4%) and 51 (48.1%) patients with ALK-positive were in Stages I, II and III, respectively. Among these cases, 54.7% (58/106) had lymph node metastasis. ALK-positive cases occurred in younger patients (p = 0.020), and they presented at a higher clinical stage (p < 0.001). Compared with ALK-negative patients, ALK-positive tumours were more likely to show lymph node metastases (p < 0.001). There was no significant difference in smoking history and family history of cancer among these two groups of patients (p > 0.05).

Table 1 Clinicopathologic Characteristics of ALK-positive and ALK-negative Lung adenocarcinoma samples.
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Histological characteristics of ALK-positive lung adenocarcinoma samples

The histological characteristics of ALK-positive tumours are illustrated in Table 2. Although ALK-positive cases were not significantly associated with acinar predominant growth pattern (p = 0.373), they were positively associated with micropapillary (p < 0.001) and solid predominant growth pattern (p = 0.005).

Table 2 Histologic characteristics of ALK-positive lung adenocarcinoma samples.
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ALK gene translocation status in primary tumour cells

All 106 ALK-positive cases were successfully examined by ALK D5F3 IHC. All cases showed diffused cytoplasmic staining pattern in the section of FFPE tissue tumour samples, without diverse signal intensities (Fig. 1B). All ALK-positive cases in this study showed a homogeneous strong ALK-expression of all neoplastic cells.

Figure 1
figure 1

ALK gene status determined by the Ventana D5F3 IHC in primary tumour and corresponding lymph node metastasis. A representative case is shown: graph of the primary tumour under light microscopy (hematoxylin and eosin, ×200) (A) and strong immunoreactivity of ALK-D5F3 in the primary tumour (B) (×200); Graph of the corresponding lymph node metastasis under light microscopy (hematoxylin and eosin, ×200) on TMA (C) and strong immunoreactivity of ALK-D5F3 in the corresponding lymph node metastasis (D) (×200).

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ALK gene translocation status in the lymph node metastases

Fifty three patients (53/196, 27%), including 37 ALK-positive cases and 16 ALK-negative cases, with paired primary tumours and lymph node metastases samples were further investigated for concordance of ALK status. Among these 53 paired sample patients, pathological N1 disease was confirmed in 10 (18.9%) patients, N2 disease in 42 (79.2%) patients and N3 disease in 1 (1.9%) patient. The characteristics of these 53 paired cases are illustrated in Table 3. Lymph node metastases from the 37 ALK positive cases were examined and ALK expression with diffused cytoplasmic staining pattern exhibited in all lymph node metastases (Fig. 1D). ALK expression was absent in the metastases of the 16 patients with no ALK expression in their primary tumours. No discordant case of ALK expression was observed between the primary tumours and their corresponding lymph node metastases (Table 4).

Table 3 Clinicopathologic characteristics of 53 paired cases.
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Table 4 ALK translocation in primary lung adenocarcinoma samples and their lymph node metastases by the Ventana D5F3 IHC.
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Analysis of EGFR, KRAS, BRAF and HER2 genetic alterations in 106 ALK-positive cases

Genotyping for EGFR, KRAS, BRAF and HER2 mutations was conducted with direct sequencing in the ALK-positive 106 cases. EGFR mutations were detected in 5 (4.7%) cases. However, KRAS, BRAF and HER2 mutations were not detected in any specimens. EGFR mutation types and locations are shown in Table 5. Three cases with deletions in exon 19, whereas two had mutations in exon 21 (L858R).

Table 5 Clinicopathological features of patients with concomitant EGFR and ALK alterations.
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Discussion

Tumour heterogeneity refers to the existence of subpopulations of cells with distinct genotypes and phenotypes that may harbour divergent biological behaviors, within a primary tumour and its metastases; alternatively the divergence is between tumours of the same histopathological subtype, even between spatially separated regions within single biopsies (intra- and inter- tumour, respectively)8. Investigation of tumour heterogeneity will provide valuable information for diagnostic and/or therapeutic procedures.

The selection of patients for crizotinib therapy relies on the ALK status of the tumour18. Thus, accurate determination of ALK status is crucial to ensure the potential clinical benefit of ALK inhibitors and to avoid the toxic effects in inappropriately selected patients3. The most commonly treatment-related adverse reactions in crizotinib therapy were vision disorder, gastrointestinal disorders and oedema19. Majority of the ALK gene translocation status are evaluated in primary tumours of NSCLC and in practice, most clinical decision-making for patients with advanced NSCLC depends on single-tumour biopsy samples from primary or metastatic sites, even from cytological specimens20, 21. Therefore, the biomarker status is assumed to remain unchanged during metastatic progression.

In the present study, we evaluated the intratumour heterogeneity of ALK gene translocation in 106 ALK-positive excision specimens by the Ventana ALK D5F3 IHC and also investigated whether the ALK status changed during disease progression in 53 pairs of primary tumours and corresponding lymph node metastases. We found the ALK expression was homogenous in lung adenocarcinoma samples and there was no discordant case of ALK status between primary tumours and corresponding lymph node metastases. Therefore, primary tumours can clearly represent the ALK status of the metastatic tumours. The ALK status remained unchanged during metastasis.

Metastatic advanced lung cancer continues to represent a major health problem worldwide. Targeted therapies are the standard therapeutic options for these patients. Approximately 70–85% of new lung cancers are unsuitable for surgical therapy; thus, only small biopsy specimens are available for diagnosis or further genetic detection20. By examining the ALK status in the primary tumours and corresponding metastatic tumours in our series, we propose that ALK gene translocation is a stable genetic alteration once it has occurred. This hypothesis has important clinical implications. Firstly, fine needle biopsy or aspiration of primary and/or metastatic tumours and pleural effusions are feasible for the detection of ALK gene translocations. Secondly, despite the marked antitumour activity of crizotinib, some ALK-positive patients developed resistant to crizotinib on average within the first year or two of TKI therapy6. In general, crizotinib resistance in ALK-positive NSCLC occurs through secondary resistance mutations located in the ALK TK domain, amplification of the ALK fusion gene, and activation of bypass tracks6, 22. Therefore, we suggested that a change in ALK status between the primary tumour and metastasis was not the cause of crizotinib resistance.

Previous studies have demonstrated the intratumour heterogeneity of ALK gene translocation in lung adenocarcinoma samples. Cai et al.23 acquired 45 spatially separated tumour cell subpopulations using laser-capture microdissection from 20 patients with ALK-FISH positivity. A total of 45 lesions were tested for ALK translocation by RT-PCR and 34 lesions were positive for ALK translocation, but 11 lesions were ALK wild-type regions. Abe et al.24 observed a total of 64 tumour areas in nine ALK-FISH positive cases using darkfield fluorescence microscopy; positive ALK was found in 50 areas. However, in the current study with the Ventana ALK- D5F3 IHC, we did not detect intratumour heterogeneity of ALK status because ALK protein expression was diffusely positive in all neoplastic cells. Several studies considered that the intratumoural ALK gene translocation heterogeneity may be associated with technical problems. Camidge et al.25 recorded the percentage of positive cells, pattern of positivity (split, single red, or both), and copy number of fused, isolated red and green signals by evaluating 90 ALK FISH positive NSCLC cases. They concluded that the semivertical orientation of tumour cells and/or nuclear truncation in paraffin-embedded tissue sections may make precise identification of break-apart signals difficult. Proietti et al.21 reported that FISH on conventional cytology, in which tumour cells are arranged in a single layer, have much higher positive rates for FISH than histology specimens. Moreover, ALK FISH using small biopsy sections cannot examine as many fields and cells as is the case with excision samples. RT-PCR, proposed as an alternative screening method for ALK gene translocation, is also not recommended for the detection of ALK translocation. RT-PCR can not be able to detect all fusion transcript variants. Additionally, RT-PCR is more stringent requirements for the sample preparation due to the rapid degradation of RNA.

The Ventana ALK D5F3 IHC detection system, measuring the expression of the ALK protein containing the C-terminal active kinase domain, is a sensitive method for detecting ALK gene translocation, especially in paraffin-embedded tissues, which was approved as a CE-IVD (in vitro diagnostic products) in Europe, China, and US since 2012, 2013 and 2015, respectively7, 26,27,28,29,30. We described a case with a negative FISH result that was later identified as ALK-positive by Ventana IHC and responded well to crizotinib. Targeted next generation sequencing revealed a new ALK partner gene (BIRC6) and the paracentric inversion for generating this fusion gene31. Wekken A. et al.30 evaluated tumour response rate and survival after crizotinib treatment of 29 consecutive patients with ALK-positive advanced NSCLC diagnosed by FISH and/or Ventana ALK D5F3 IHC on small biopsies or fine needle aspirations (FNA). They concluded that Ventana ALK D5F3 IHC was superior to ALK-FISH on small biopsies and FNA to predict tumour response and survival to crizotinib for advanced NSCLC patients. Therefore, the Ventana IHC assay may be more sensitive than FISH for detecting ALK status in metastases and/or small biopsies which often contain only few tumour cells.

In this study, we found that a portion of lung adenocarcinoma patients with concomitant EGFR and ALK alterations. The dual-positive cases accounted for 4.7% (5/106) of ALK-positive lung adenocarcinoma samples using direct sequencing, which was similar to the ratio of 4.4% (4/91) reported by Won et al.13 but lower than the ratio of 18.6% (12/70) reported by Yang et al.17; both studies used the same detection method. Won et al.13 reported that the frequency of co-alteration detection increase in the same cohort when sensitive detection methods for EGFR mutation are applied, such as real-time PCR, targeted NGS, and mutant-enriched NGS. We reviewed the literatures and summarized the relevant clinicopathological and molecular characteristics of ALK/EGFR dual-positive cases; majority of cases were adenocarcinomas in the advanced stage and mainly involved Asian patients, most EGFR mutations were deletions in exon 19 and point mutations in exon 21, and patients showed differential sensitivities to EGFR-TKI and/or ALK-TKI13, 14, 16, 17.

Intratumour heterogeneity (ITH) has recently been elucidated in several cancer types with the use of next-generation sequencing (NGS) approaches23, 32,33,34,35. Clonal analyses by de Bruin et al.34, and Zhang et al.33 reported substantial intratumour heterogeneity (ITH) within lung adenocarcinoma samples, by using multiregion whole-exome sequencing (WES) and/or whole genome sequencing (WGS). Recent evidence supports a model of trunk-branched clonal evolution leading to variable ITH and complex clonal architecture of tumours33, 35, 36. The clonal structure of a tumour is visually represented as a phylogenetic tree, the ubiquitous alterations present in all tumour regions map to the trunk, whereas heterogeneous events present in only some regions of the tumour map to the branches. The findings of homogeneous ALK gene translocation in lung adenocarcinoma samples, and concordant ALK status between primary and lymph node metastasis in this study, suggested that ALK gene translocation is a ubiquitous event, must occur very early in lung adenocarcinoma pathogenesis33. The finding of ALK/EGFR coaltered lung adenocarcinoma cases may be explained by clonal evolutionary dynamics and the resulting complex clonal architecture of lung adenocarcinoma samples. In addition, in this study, we also demonstrated that ALK-positive cases more commonly showed lymph node metastasis and presented at higher clinical stage, and ALK gene translocations were significantly more common in lung adenocarcinoma samples with micropapillary and solid predominant patterns which associated with poorer prognosis37. We confirmed that tumour cells with ALK gene translocations contained enhanced metastatic potential and acquired the metastatic phenotype.

Conclusion

In summary, using the Ventana (D5F3) IHC, we found the homogeneity of ALK expression in lung adenocarcinoma samples and concordance in ALK status between primary tumours and corresponding lymph node metastases. Molecular testing for ALK translocation may be performed in either primary tumour or lymph node metastasis samples from the same patient. However, the main limitation in our study is that the metastases did not include different metastatic sites, especially distant metastases. Loco-regional or concurrent regional lymph node metastases are not considered to be biologically equivalent to distant metastases9. We also found a small portion of lung adenocarcinoma samples have concomitant EGFR and ALK alterations by using direct sequencing. The clinical relevance of these concurrent alterations remains to be elucidated in future studies.

Methods

Patients and tumour samples

All formalin-fixed and paraffin-embedded (FFPE) tissue sections were obtained from patients with histologically confirmed primary lung adenocarcinoma. The patients previously underwent curative surgery at the Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, China, between February 2013 and February 2015. Written informed contents were obtained from all subjects before collecting the samples. All the methods were carried out in accordance with the institutional guidelines and approved by the Ethical Review Committee of the Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, China. Tumours were immunoassayed with ALK D5F3 antibody as described below. A total of 106 ALK- immunopositive cases were collected. Ninety ALK-immunonegative lung adenocarcinoma cases were randomly selected and included in the study as controls.

The ALK status between primary tumours and their metastases was compared in 37 ALK-immunopositive and 16 ALK-immunonegative cases, as well as their corresponding metastatic lymph nodes. Thirty-seven ALK-positive cases with their corresponding metastatic lymph nodes were selected among 106 ALK-positive cases, meanwhile, 16 ALK-negative cases with their corresponding metastatic lymph nodes were selected among 90 ALK-negative cases. Our study included only metastatic tumour tissues with diameters greater than 0.5 cm from lymph nodes. The lymph node metastases of 53 cases were conducted into three tissue microarrays (TMA). Lymph node metastases were sampled by collecting 2.0 mm-diameter cores from two different representative sites based on hematoxylin and eosin (HE) stained sections.

Age, gender, cancer stage, smoking status and treatment of all patients were recorded. All cases had one slide stained for hematoxylin and eosin to confirm the presence of adequate tumour tissue. For each case, multiple slides corresponding to whole tissue sections were reviewed by two pathologists according to the 2011 International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) International Multidisciplinary Classification of Lung Adenocarcinoma and the American Joint Committee on Cancer (7th edition) of tumour, node and metastasis staging criteria38, 39. In mixed-subtype adenocarcinoma samples, we assessed the percentage of each histological pattern (Lepidic, Acinar, Papillary, Micropapillary, Solid) in 5% increments and recorded the predominant histological pattern38. Cases with differences between the two reviewers were reevaluated and a consensus interpretation was rendered. None of patients had received prior ALK or EGFR TKI therapy.

Ventana IHC staining and scoring

ALK-IHC was performed on 4 μm-thick formalin fixed, paraffin-embedded tissue sections or TMA slides using the Ventana ALK D5F3 CDx assay on a Ventana Benchmark XT automated slide-processing system (Ventana Medical Systems Inc., Tucson, AZ). Briefly, slides of lung adenocarcinoma tumours were subjected to deparaffinization using EZ Prep (Ventana Medical Systems Inc.). Tissue sections were incubated with anti-ALK antibody (clone D5F3, Ventana Medical Systems Inc.) for 20 min. Optiview DAB IHC detection kit (Ventana Medical Systems Inc.) and Optiview amplification kit (Ventana Medical Systems Inc.) were used according to the manufacturer’s recommendations for the visualization of the bound primary antibody27. Tissue slides were counterstained with hematoxylin II and Bluing Reagent (Ventana Medical Systems Inc.). An ALK-positive cell lines embedded in agar/FFPE or sections of normal appendix containing ALK-positive ganglion cells, were used as ALK-IHC external controls in each run (Ventana Medical Systems Inc.). Two consecutive 4μm-thick FFPE tissue slides was cut, one slide was used in ALK-D5F3 IHC analysis, and the other for routine negative control staining for a matched rabbit monoclonal negative antibody (Ventana Medical Systems Inc.). For evaluating the staining results, a binary scoring system (positive or negative for ALK status) was used (package insert for VENTANA anti-ALK D5F3 Rabbit Monoclonal Primary Antibody, Cat.N0.790-4794/06679072001). Tumour cells with strong granular cytoplasmic staining (any percentage of positive tumour cells) were designated as ALK-positive, whereas tumour cells without strong granular cytoplasmic staining were designated as ALK-negative. Negative quality control sections were first evaluated for lack of staining27. Considering that Ventana ALK D5F3 IHC produced more intense cytoplasmic signals because of excessive chromogen deposition, which resulted in false-positive staining40, the ALK expression in our study was assessed independently by one trained scientist (J.Y) and one pathologist (N.L).

Direct sequencing of EGFR, KRAS, BRAF and HER2

Direct Sanger sequencing for EGFR, KRAS, BRAF and HER2 was conducted. Briefly, genomic DNA was extracted from 106 ALK-positive FFPE tissues. Mutations in exons 18–21 of EGFR, V600E of BRAF, exon 2 of KRAS, and exon 20 of HER2 were amplified by PCR. PCR products were purified with QIA quick PCR purification kits (Qiagen, Hilden, Germany) and submitted for sequencing with an ABI 3500XL analyser (Applied Biosystems, Caarlsbad, CA, USA) with the POP7 polymer according to the manufacturer’s protocol. Details of the methodology were described in a previous study41.

Statistical analysis

Fisher’s exact test was used to compare categorical data for clinicopathological characteristics between ALK-immunopositive and ALK-immunonegative subgroups. All p values are based on two-sided hypothesis test. The statistical analysis were conducted using SPSS version 17.0 software (SPSS, Chicago, IL, USA), and statistical significance was set as p < 0.05.