Genomic markers for malignant progression in pulmonary adenocarcinoma with bronchioloalveolar features

Abstract

Bronchioloalveolar carcinoma (BAC), a subtype of lung adenocarcinoma (ADC) without stromal, vascular, or pleural invasion, is considered an in situ tumor with a 100% survival rate. However, the histological criteria for invasion remain controversial. BAC-like areas may accompany otherwise invasive adenocarcinoma, referred to as mixed type adenocarcinoma with BAC features (AWBF). AWBF are considered to evolve from BAC, representing a paradigm for malignant progression in ADC. However, the supporting molecular evidence remains forthcoming. Here, we have studied the genomic changes of BAC and AWBF by array comparative genomic hybridization (CGH). We used submegabase-resolution tiling set array CGH to compare the genomic profiles of 14 BAC or BAC with focal area suspicious for invasion with those of 15 AWBF. Threshold-filtering and frequency-scoring analysis found that genomic profiles of noninvasive and focally invasive BAC are indistinguishable and show fewer aberrations than tumor cells in BAC-like areas of AWBF. These aberrations occurred mainly at the subtelomeric chromosomal regions. Increased genomic alterations were noted between BAC-like and invasive areas of AWBF. We identified 113 genes that best differentiated BAC from AWBF and were considered candidate marker genes for tumor invasion and progression. Correlative gene expression analyses demonstrated a high percentage of them to be poor prognosis markers in early stage ADC. Quantitative PCR also validated the amplification and overexpression of PDCD6 and TERT on chromosome 5p and the prognostic significance of PDCD6 in early stage ADC patients. We identified candidate genes that may be responsible for and are potential markers for malignant progression in AWBF.

Fig. 1.

BAC compared with AWBF by histology, frequency scoring, and threshold filtering. (A1) BAC showing typical growth pattern of tumoral cells along the preexisting alveolar scaffold without evidence of invasion. (H&E staining; ×100.) (A2) AWBF has both BAC-like and invasive areas. (H&E staining; ×16.) (Inset) High-power view of the invasive component. (H&E staining; ×100.) (B) Frequency scoring of BAC (green) compared with AWBF (red) illustrates the percentage of cases at which a change in genomic content has occurred in each of the study groups. Some changes were shared by BAC and AWBF (yellow). The presentation is per array loci; gains are represented by the colors on the right, and deletions are represented by the colors on the left. Vertical black thick and thin lines represent 100% and 50% of the samples, respectively. Blue arrows highlight the chromosomal areas of most frequent changes in BAC. (C) Unsupervised hierarchical clustering (Genesis software) of 119 clones selected by threshold filtering shows complete segregation of the two study groups: BAC (green rectangle on the right) and AWBF (red rectangle on the left). The color code of data corresponds to log₂ ratio of array CGH signals.

Fig. 2.

Increase in genomic instability. Shown is a karyotypic presentation of the log₂ ratio of array CGH signals (SeeGH software). Normal genomic content is represented by the midline (blue); clonal gains deviate to the right (green lines) and deletions to the left (red lines). The lower images show progression of genomic instability represented by more chromosomal aberrations than the upper images (purple brackets). (A) Synchronous BAC and AWBF in the same patient. (B) AWBF sampled in BAC and invasive areas.

Fig. 3.

PDCD6 validation and markers of poor prognosis. (A) QPCR performed on genomic DNA showed statistically significant differences in PDCD6 gene copy number between BAC and AWBF (P = 0.01), confirming the array CGH analysis that identified its amplification. (B) QPCR performed on 10 paired cDNAs of ADC-normal samples. PDCD6 was significantly overexpressed in tumor compared with normal lung tissue (P < 0.01), with a mean 3-fold higher expression. (C) Multivariate analysis adjusting for stage, histology, and differentiation that relied on QPCR of cDNA from 85 NSCLC samples found that PDCD6 was an independent poor prognostic factor for overall survival in stage I–II ADC patients. (D–F) Kaplan-Meier survival curves of SERPINE1 (D), GNB2 (E), and ST13 (F), based on gene expression data from the Duke database. Expression data were dichotomized at the median.

Fig. 4.

TERT validation by QPCR and FISH. (A) TERT content measured by array CGH and QPCR on genomic DNA (relative to normal control) and FISH (mean gene copy number per nucleus) show high correlation between the different methods. The black lines connecting copy number of TERT (filled circles) and 5q (open squares) are drawn to highlight the difference in copy number between the two probes. The blue boxes mark the AWBF sampled in invasive areas. Samples T41A, T44A, and T46A represent the BAC-like area, and T41B, T44B, and T46B represent the invasive area of AWBF. (B) FISH performed on AWBF in BAC area (sample T195) using the dual-color FISH probe mix that contains the hTERT locus (5p15, green signal) and the control D5S89 probe (5q31, red signal). Gain of TERT is reflected by the increased number of green compared with red foci.