Mutational analysis of pulmonary tumours with neuroendocrine features using targeted massive parallel sequencing: a comparison of a neglected tumour group

Abstract

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows.

Methods: A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes.

Results: After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020).

Conclusions: The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

Figure 1

Genetic profile of four analysed neuroendocrine lung tumour subtypes carrying gene mutations determined by targeted massive parallel sequencing are shown. Columns represent mutations per patient; rows summarise mutations occurring in a particular gene. Dark blue boxes indicate more than one mutation per patient in the corresponding gene.

Figure 2

(A) Genes within entity-specific mutation frequency are shown. The JAK3, NRAS, RB1 and VHL variants were just detected in SCLCs, whereas ATM, TP53, ALK and BRAF mutations were found in both high-grade tumour entities. The FGFR2 mutation occurred exclusively in LCNEC. The KIT, PTEN, HNF1A and SMO variants were only found in ACs, and the SMAD4 variant was only present in TC. The GNAS mutations were limited to carcinoids. (B) Specific genes showing a higher mutation frequency with increasing tumour malignancy. The bars indicate the distribution of the mutation between the different tumour entities. The TP53 and ATM variants were just detected in high-grade tumours with ATM variants mostly in SCLCs; PIK3CA and ERBB4 showed additional variants in AC; RET mutations were present in all four entities, but mainly in SCLCs, whereas ERBB2 showed an increasing mutation frequency with an increase of the tumour malignancy.

Figure 3

Kaplan–Meier curves for progression-free survival (PFS) (A) and overall survival (OS) (B) differentiated by the patients' mutational status are shown. Because of the excellent survival rate of pulmonary carcinoids, only high-grade cases are depicted. Of note, samples had mutations in both ATM and APC, showing an association to PFS (P=0.020). The relation of this double mutation has to be clarified. The PIK3CA mutated patients with high-grade tumours showed a reduced OS compared with their wild-type counterparts (P=0.040).