ALK, ROS1 and RET fusions in 1139 lung adenocarcinomas: A comprehensive study of common and fusion pattern-specific clinicopathologic, histologic and cytologic features

doi:10.1016/j.lungcan.2014.02.007

Lung Cancer

Volume 84, Issue 2, May 2014, Pages 121-126

https://doi.org/10.1016/j.lungcan.2014.02.007 Get rights and content

Abstract

Background

To have a comprehensive investigation of the clinicopathologic, histologic and cytologic features of fusion-positive lung adenocarcinomas.

Methods

Quantitative real-time reverse transcriptase PCR (qRT-PCR) and reverse transcriptase PCR (RT-PCR) were simultaneously performed to screen ALK, ROS1 and RET fusions in resected tumor samples from 1139 Chinese lung adenocarcinoma patients, with validation of positive results using fluorescent in situ hybridization. Clinicopathologic characteristics, predominant histologic subtype and cytomorphology were assessed in fusion-positive lung adenocarcinomas and compared to those harboring EGFR, KRAS, HER2 or BRAF mutations.

Results

There were 58 (5.1%) ALK fusions, 11 (1.0%) ROS1 fusions and 15 (1.3%) RET fusions. Tumors with ROS1 fusions had significantly larger diameter than ROS1 fusion-negative tumors (P = 0.007), whereas all the 15 tumors harboring RET fusions were ≤3 cm in diameter (P = 0.001). The three fusion genes were all more prevalent in solid-predominant adenocarcinoma. Compared to fusion-negative lung adenocarcinomas, tumors harboring a fusion gene had significantly higher prevalence of extracellular mucin (P < 0.001), cribriform pattern (P < 0.001), signet ring cells (P < 0.001) and hepatoid cytology (P < 0.001). No significant difference in relapse-free survival (P = 0.147) and overall survival (P = 0.444) was observed between fusion-positive and fusion-negative patients.

Conclusions

This study showed fusion-positive lung adenocarcinomas had identifiable common and fusion-pattern specific clinicopathologic, histologic and cytologic features, offering implications for fusion genes screening.

Introduction

Lung adenocarcinoma, which is the most common histologic subtype of lung cancer, is now a disease that could be largely classified into clinically relevant molecular subsets by oncogenic driver mutations, each with unique clinicopathologic features and potential opportunities for targeted therapies.

Recently, gene fusions have been identified as recurrent oncogenic events in lung adenocarcinoma. The first reported fusion oncokinase is ALK rearrangement [1], which occurs in approximately 5% of lung adenocarcinomas [2], [3]. In ALK-positive lung cancer patients, crizotinib has demonstrated its treatment efficacy in terms of a higher response rate and improved progression free survival compared with standard chemotherapy [4]. ROS1 is another identified receptor tyrosine kinase that forms fusions, and is reported to be present in about 1–2% of lung adenocarcinomas [2], [5], [6]. ROS1 fusions also serve as therapeutic targets for crizotinib [6]. RET fusions have been identified as the novel oncogenic drivers in about 1–2% of lung adenocarcinomas [2], [7], [8], [9], [10], and the potential therapeutic targets of multi-targeted kinase inhibitors, vandetanib, sunitinib and sorafenib [2], [7], [8].

As the prevalence of fusion genes in lung adenocarcinoma is comparatively low, identifying the enriched population has definite implications for efficient screening. However, despite their critical importance in targeted therapies, the three fusion genes have not been examined together along with other well-identified driver mutations in a large cohort of lung adenocarcinoma to comprehensively define their common and fusion-pattern specific clinicopathologic, histologic and cytologic features.

In this study, we used a combination strategy of quantitative real-time reverse transcriptase PCR (qRT-PCR) and reverse transcriptase PCR (RT-PCR) for screening of ALK, ROS1 and RET fusion genes in over 1139 lung adenocarcinoma patients, with validation of positive results using fluorescence in situ hybridization (FISH). Mutational analyses of EGFR, KRAS, HER2 and BRAF were also carried out in the same cohort of patients. Clinicopathologic, histologic and cytologic features of fusion-positive lung adenocarcinomas were comprehensively analyzed and compared to those of other well-identified molecular subsets of lung adenocarcinoma.

Section snippets

Patients and samples

From April 2007 to October 2012, we consecutively collected lung tumors resected at the Department of Thoracic Surgery, Fudan University Shanghai Cancer Center. Eligible subjects must have pathologically confirmed lung adenocarcinoma, a minimum of 50% of tumor cells and sufficient tissue for comprehensive mutational analyses. Patients who received neoajuvant chemotherapy were excluded. Total RNA were extracted as per standard protocols (RNeasy Mini Kitt; Qiagen, Hilden, Germany) after frozen

Results

Tumor samples from 1139 lung adenocarcinoma patients were included in the comprehensive mutational analysis. There were 58 (5.1%) ALK fusions, 11 (1.0%) ROS1 fusions and 15 (1.3%) RET fusions. Mutations in EGFR, KRAS, HER2 and BRAF were detected in 735 (64.5%), 86 (7.6%), 27 (2.4%) and 14 (1.2%) patients, respectively. The spectrum of well-identified oncogenic driver mutations in 1139 lung adenocarcinoma was shown in Fig. 1.

Discussion

Our previous studies have investigated ALK and RET fusions in non-small cell lung cancer in relatively smaller samples [9], [12]. In this study, we extended the fusion gene detection to more lung adenocarcinoma samples and also included ROS1 fusions. Some previous studies reported that lung cancers harboring ALK, ROS1 or RET fusions shared some clinical characteristics, such as younger age of onset and never/light smoking history [2], [6], [14]. In this study, we also found some clinical

Conclusion

In conclusion, through comprehensive analysis of ALK, ROS1 and RET fusions along with other well-identified driver mutations in 1139 lung adenocarcinomas, we have comprehensively defined the common and fusion-pattern specific clinicopathologic, histologic and cytologic features of fusion-positive lung adenocarcinomas. Our results have implications for clinical trials seeking to enrich for patients with fusion genes as well as therapeutic strategies.

Conflict of interest

The authors declare no conflicts of interest.

Funding source

This work was supported by the funds from Key Construction Program of the National “985” Project (985III-YFX0102), the National Natural Science Foundation of China (81172218, 81101760, 81101761, and 81372525), the Science and Technology Commission of Shanghai Municipality (Program of Shanghai Subject Chief Scientist; Grant No. 12XD1402000), and the Shanghai Hospital Development Center (No. SHDC12012308).

Acknowledgements

We thank the AstraZeneca Innovation Center China for excellent technical support.

References (19)

W.D. Travis et al.
International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma
J Thorac Oncol
(2011)
H. Li et al.
Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose
Lung Cancer
(2013)
M. Nishino et al.
Histologic and cytomorphologic features of ALK-rearranged lung adenocarcinomas
Mod Pathol
(2012)
M. Soda et al.
Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer
Nature
(2007)
K. Takeuchi et al.
RET, ROS1 and ALK fusions in lung cancer
Nat Med
(2012)
Y. Sun et al.
Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases
J Clin Oncol
(2010)
A.T. Shaw et al.
Crizotinib versus chemotherapy in advanced ALK-positive lung cancer
N Engl J Med
(2013)
C. Li et al.
Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers
PLoS One
(2011)
K. Bergethon et al.
ROS1 rearrangements define a unique molecular class of lung cancers
J Clin Oncol
(2012)

There are more references available in the full text version of this article.

Cited by (194)

Lung adenocarcinoma patients with ROS1-rearranged tumors by sex and smoking intensity
2024, Heliyon
ROS1 rearrangements (ROS1+) define a distinct molecular subset of lung adenocarcinomas. ROS1 + tumors are known to occur more in never-smokers, but the frequency and outcome of ROS1 positivity by sex and smoking intensity are not clearly documented.
This patient cohort study included all never- (<100 cigarettes lifetime) and light- (100 cigarettes-20 pack-years) smokers, and a sample of heavy-smokers. ROS1 + rates by sex and smoking intensity were compared within and beyond our study. Survival outcomes were analyzed using Kaplan-Meier curves and Cox proportional hazards models.
Of the 571 total patients, ROS1 + was detected in 24 (4.2%): 6.4% in men and 3.0% in women; 5.1% in never-, 5.7% in light-, and 1.8% in heavy-smokers (P=0.05). Among the 209 stage IIIB-IV patients, men had much higher ROS1 + rate (11.1%) not only than women (1.7%, P=0.004) in our study, but also than men (0.4%–1.8%) in 8 published studies (Ps = 0.0019–0.0001). ROS1+ rates were similar between never- (9.3%) and light-smokers (8.1%) and significantly lower in heavy-smokers (1.2%, P=0.017), a finding confirmed by 6 published studies (Ps = 0.041–0.0001). Overall survival of ROS1 + patients were significantly better than the ROS1- (P=0.023) mainly due to targeted therapy. Among patients who exhibited resistance to crizotinib, follow-up treatment of entrectinib and lorlatinib showed remarkable survival benefits.
The ROS1 + rates were higher in men than in women, and similar in never- and light-smokers, more pronounced in stage IIIB-IV patients. Newer-generation ALK/ROS1-targeted drugs showed efficacy in a cohort of crizotinib resistant ROS1 + patients. These results, when validated, could assist efficiently accruing ROS1 + patients.
RET Fusion Testing in Patients With NSCLC: The RETING Study
2024, JTO Clinical and Research Reports
RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC.
This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay.
The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively).
In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.
A Detailed Histologic and Molecular Assessment of the Diffuse Sclerosing Variant of Papillary Thyroid Carcinoma
2023, Modern Pathology
Diffuse sclerosing variant papillary thyroid carcinoma (DS-PTC) is characterized clinically by a predilection for children and young adults, bulky neck nodes, and pulmonary metastases. Previous studies have suggested infrequent BRAF^V600E mutation but common RET gene rearrangements. Using strict criteria, we studied 43 DS-PTCs (1.9% of unselected PTCs in our unit). Seventy-nine percent harbored pathogenic gene rearrangements involving RET, NTRK3, NTRK1, ALK, or BRAF; with the remainder driven by BRAF^V600E mutations. All 10 pediatric cases were all gene rearranged (P = .02). Compared with BRAF^V600E-mutated tumors, gene rearrangement was characterized by psammoma bodies involving the entire lobe (P = .038), follicular predominant or mixed follicular architecture (P = .003), pulmonary metastases (24% vs none, P = .04), and absent classical, so-called “BRAF-like” atypia (P = .014). There was no correlation between the presence of gene rearrangement and recurrence-free survival. Features associated with persistent/recurrent disease included pediatric population (P = .030), gene-rearranged tumors (P = .020), microscopic extrathyroidal extension (P = .009), metastases at presentation (P = .007), and stage II disease (P = .015). We conclude that DS-PTC represents 1.9% of papillary thyroid carcinomas and that actionable gene rearrangements are extremely common in DS-PTC. DS-PTC can be divided into 2 distinct molecular subtypes and all BRAF^V600E-negative tumors (1.5% of papillary thyroid carcinomas) are driven by potentially actionable oncogenic fusions.
Histology and molecular testing
2023, Lung Cancer: an Evidence-Based Approach to Multidisciplinary Management
Lung cancer can be broadly classified into non-small cell carcinoma (NSCLC) and small cell carcinoma (SCC), with the former being further subclassified into adenocarcinoma, squamous cell carcinoma, and other less frequent subtypes. Diagnosis of specific lung cancer types are aided by immunohistochemistry markers, including thyroid transcription factor 1 (TTF-1) for adenocarcinoma, p40/p63 for squamous cell carcinoma, and chromogranin, synaptophysin and CD56 for neuroendocrine carcinomas. In lung adenocarcinoma, histologic growth patterns carry prognostic implications, with lepidic predominant tumors being associated with a good prognosis, while micropapillary and solid predominant tumors carrying a worse prognosis. Lepidic pattern is considered non-invasive (in-situ) tumor, and the currently used 8^th edition UICC/AJCC staging system for lung cancer bases the T-stage on size of the invasive component of the tumor. For advanced stage NSCLC patients, testing for predictive biomarkers is essential to guide therapy. These include driver mutations/fusions involving the epidermal growth factor receptor (EGFR), ALK, ROS1, BRAF, KRAS, NTRK1-3, RET and MET genes, as well as aberrant protein expression for Programmed Death Ligand 1 (PD-L1). The list of biomarkers continues to grow as newer therapies are discovered or as they become available. With this ever-increasing number of driver mutations/fusions test requirements, next generation sequencing (NGS) with multiple gene panels is rapidly replacing single gene tests. Moving forward, liquid biopsy to detect circulating tumor DNA is assuming a larger role in molecular diagnostics, monitoring therapeutic response and assessment of minimal residual disease.
Clinical Utility and Performance of an Ultrarapid Multiplex RNA-Based Assay for Detection of ALK, ROS1, RET, and NTRK1/2/3 Rearrangements and MET Exon 14 Skipping Alterations
2022, Journal of Molecular Diagnostics
Several kinase fusions are established targetable drivers in lung cancers. However, rapid and comprehensive detection remains challenging because of diverse partner genes and breakpoints. We assess the clinical utility and performance of a rapid microfluidic multiplex real-time PCR–based assay for simultaneous query of fusions involving ALK, ROS1, RET, and NTRK1/2/3, as well as MET exon 14 skipping, using a 3-hour automated process. Dual analytic strategies were utilized: fusion-specific amplification and 3′ to 5′ expression imbalance. One-hundred and forty-three independent, formalin-fixed, paraffin-embedded tumor samples (112 surgical specimens, 31 cytologic cell blocks) were analyzed: 133 with known kinase gene alterations and 10 negative samples based on clinically validated next-generation sequencing. Testing was successful in 142 (99%) cases. The assay demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27), and 100% (20/20) for ALK, RET, ROS1, and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens aged up to 5 years, with >10% tumor, and inputs of at least 9 mm² (surgical specimens) and 9000 cells (cytologic cell blocks). The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared with immunohistochemistry and molecular methods, while also circumventing the infrastructure dependencies associated with next-generation sequencing and fluorescence in situ hybridization.
Lorlatinib for Previously Treated ALK-Positive Advanced NSCLC: Primary Efficacy and Safety From a Phase 2 Study in People's Republic of China
2022, Journal of Thoracic Oncology
Lorlatinib was found to have activity in ALK-positive NSCLC in a global phase 1 and 2 study. We report an ongoing phase 2 study in Chinese patients with ALK-positive advanced or metastatic NSCLC.
Open-label, dual-cohort study (NCT03909971); patients had progressive disease after ALK tyrosine kinase inhibitor treatment (cohort 1: previous crizotinib; cohort 2: one ALK tyrosine kinase inhibitor other than crizotinib [±prior crizotinib]), more than or equal to one unirradiated extracranial target lesion, and Eastern Cooperative Oncology Group performance status of 0 to 2. Patients received oral lorlatinib 100 mg once daily in continuous 21-day cycles. Primary end point: objective response in cohort 1 by independent central radiology (ICR) according to Response Evaluation Criteria in Solid Tumors version 1.1. Analyses were based on patients receiving more than or equal to one dose.
At data cutoff (August 10, 2020), 109 patients were enrolled (cohort 1: n = 67; cohort 2: n = 42). A total of 47 patients in cohort 1 (70.1%, 95% confidence interval [CI]: 57.7–80.7, p < 0.0001; primary end point) and 20 patients in cohort 2 (47.6%, 95% CI: 32.0–63.6, secondary end point) achieved objective response by ICR. Median progression-free survival was not reached in cohort 1 and was 5.6 months in cohort 2. In patients with brain lesions at baseline, 29 of 36 patients in cohort 1 (80.6%, 95% CI: 64.0–91.8) and 10 of 21 patients in cohort 2 (47.6%, 95% CI: 25.7–70.2) achieved objective intracranial response by ICR. Hypercholesterolemia (92.7%) and hypertriglyceridemia (90.8%) (cluster terms) were common treatment-related adverse events (TRAEs). Nine patients (8.3%) had serious TRAEs; one permanently discontinued from treatment because of TRAEs.
Lorlatinib was found to have a robust and durable response and high intracranial objective response in previously treated Chinese patients with ALK-positive NSCLC.

View all citing articles on Scopus

¹: contributed equally to this work and should be considered co-first authors.

View full text

ALK, ROS1 and RET fusions in 1139 lung adenocarcinomas: A comprehensive study of common and fusion pattern-specific clinicopathologic, histologic and cytologic features

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Patients and samples

Results

Discussion

Conclusion

Conflict of interest

Funding source

Acknowledgements

J Thorac Oncol

Lung Cancer

Mod Pathol

Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer

Nature

RET, ROS1 and ALK fusions in lung cancer

Nat Med

Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases

J Clin Oncol

Crizotinib versus chemotherapy in advanced ALK-positive lung cancer

N Engl J Med

Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers

PLoS One

ROS1 rearrangements define a unique molecular class of lung cancers

J Clin Oncol