A Novel LOC101927967 Intergenic Region ALK Fusion Identified by NGS and Validated by IHC and FISH in a Patient with Early Stage Adenocarcinoma of Lung.

Anaplastic lymphoma kinase (ALK) gene rearrangement is an essential driver mutation identified in approximately 5% of non-small cell lung cancers (NSCLCs). The results of clinical trials have demonstrated the impressive efficacy of ALK tyrosine kinase inhibitors (ALK-TKIs). Besides the classic EML4-ALK fusions, a growing list of gene fusion partners for ALK in NSCLC have been identified with heterogeneous clinical responses to ALK-TKIs. However, a LOC101927967-ALK fusion has not been reported in NSCLC. Herein, a novel LOC101927967 downstream intergenic region ALK fusion in an early-stage patient with lung adenocarcinoma was first identified by next-generation sequencing (NGS) and verified by immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH), which might provide a treatment option for postoperative recurrence.

Introduction

Anaplastic lymphoma kinase (ALK) gene rearrangements appeared in approximately 5% of lung adenocarcinomas, which define a distinct molecular subgroup of non-small cell lung cancers (NSCLCs).1 Clinical management ALK tyrosine kinase inhibitors (ALK-TKIs) have achieved impressive clinical outcomes.2 Besides the classic EML4-ALK fusions, emerging fusion partners for ALK in NSCLCs have been identified, including intergenic-breakpoint fusions, which might also generate functional fusion protein by chromothripsis or alternative splicing and respond to crizotinib or alectinib.3 Herein, we reported a novel LOC101927967 downstream intergenic region ALK fusion in an early-stage patient with lung adenocarcinoma by next-generation sequencing (NGS) and was verified by immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH).

Case Presentation

A 61-year-old Chinese man went to our hospital with cough, fever, and chest tightness for one month in April 2020. A chest computed tomography (CT) scan showed 8mm frosted glass in the left superior lobe. The edge was not smooth and had a burr (Figure 1A). According to the examinations related to admission, the preliminary diagnosis was a pulmonary malignancy. After the exclusion of surgical contraindications, the left upper lobectomy was performed on April 15, 2020. Postoperative pathology released invasive mucinous adenocarcinoma with a predominant lepidic growth consisting of a less micropapillary pattern. The patient was diagnosed with stage IA (pT1aN0M0) lung adenocarcinoma. CT scan was performed after the surgery in June 2020 (Figure 1B). For precision therapy, NGS analysis was carried out using a commercially available capture-based targeted sequencing panel, targeting 11 driver genes (EGFR, ALK, ROS1, BRAF, ERBB2, MET, RET, KRAS, and NTRK1-3) (Simceredx, CAP certified Laboratory) to provide a treatment option for postoperative recurrence. A novel LOC101927967(Intergenic)-ALK fusion was detected (Figure 2A and B). This fusion was generated by chr2:78561496 (intergenic region between LOC101927967 and REG3G) and exons 20–29 of ALK, with the abundance of 13.35%. No other mutations were discovered. The result was confirmed by IHC staining (Ventana with D5F3 clone) and FISH (ZytoLight ALK Break Apart FISH Probe)(MEDx (Suzhou) Translational Medicine Co. Ltd) (Figure 3).

Figure 1

Chest computed tomography revealed the different stages of the patient’s lung tumor. (A) Initial diagnosis revealed a nodular high-density shadow in the posterior segment of the upper lobe tip of the left lung in April 2020. (B) CT scan after the surgery in June 2020.

Figure 2

Next-generation sequencing findings of LOC101927967-ALK fusion. (A) The Integrative Genomics Viewer snapshot of LOC101927967-ALK. (B) Schematic representation of the LOC101927967 intergenic region ALK fusion, this variant was generated by the fusion of intergenic region between LOC101927967 and REG3G with exons 20–29 of ALK.

Figure 3

LOC101927967-ALK fusion in the patient with lung adenocarcinoma. (A) A split signal of ALK was observed with a frequency of 66% in the fluorescence in situ hybridization image. (B) The immunohistochemistry staining indicated strong positive ALK protein expression.

To date, there is no information about LOC101927967(Intergenic)-ALK fusion and validation assay. This LOC101927967(Intergenic)-ALK fusion was generated by the downstream intergenic region of LOC101927967 with exons 19–29 of ALK, retaining the complete kinase domain of ALK. In addition, the results of IHC and FISH confirmed ALK fusion existed, which might drive oncogenesis. It is typically challenging to identify ALK fusion variants, incredibly complex rearrangements with intergenic breakpoints.

Discussion

To our knowledge, this is the first report describing the fusion of the intergenic region between LOC101927967 intergenic region and ALK exon 20 in lung adenocarcinoma. The verification results of IHC and FISH indicated this novel fusion could be considered a sensitive variant, which may benefit from ALK-TKIs such as crizotinib or alectinib. ALK rearrangements generate an oncogenic ALK tyrosine kinase, which could activate downstream signaling pathways involving MAPK and PIK3/AKT/mTOR pathways.4 Multiple fusion partners have been described in lung cancer, but the response magnitude and duration are heterogeneous. To date, FISH and IHC methods are commonly used in clinical practice,5–7 although neither can identify a specific type of ALK fusion partners. FISH, the only FDA-approved companion diagnostic test for crizotinib treatment, is recognized as the standard gold method for clinical testing of ALK status. However, it is fraught with technical and financial problems and has a potential for a false-negative rate.8 IHC is widely implemented in pathology laboratories, is relatively inexpensive, faster, and easy to use. But its interpretation remains challenging to standardize and time-consuming.9 NGS, a high-throughput molecular analysis, can simultaneously detect single nucleotide variants (SNVs), gene copy number changes, deletions, insertions, and fusions, which can provide a wealth of information for clinical treatment. Thus NGS can be used as a screening method for ALK status and other driver genes,10,11 and FISH and/or IHC test can be a reasonable verification.12

In resected early-stage NSCLC, reports of the prognostic significance of ALK-rearrangement have been contradictory. A European Project reported ALK-positive early-stage lung adenocarcinomas was related to better overall survival (OS).13 In contrast, ALK rearrangements were associated with worse recurrence-free survival (RFS) than other driver genes.14 However, Liu et al reported that ALK rearrangement was not an independent prognostic factor in patients with surgically resected lung adenocarcinoma.15 Due to the early stage of lung cancer and financial considerations, the patient chooses to regularly surveillance instead of adjuvant therapy. ALK-TKIs can be considered after the patient progresses in our case. Our case report expands the spectrum of ALK arrangement types and provides possible ALK-TKI for consideration. Furthermore, more research is needed better to understand intergenic ALK fusion and ALK-positive early-stage NSCLC.

Conclusion

In conclusion, a novel LOC101927967(Intergenic)-ALK oncogenic fusion in a patient with early-stage adenocarcinoma of the lung was identified for the first time, which expanded the ALK fusion spectrum, and the patient in our case might benefit from the ALK-TKIs in the future.