Genomic markers for malignant progression in pulmonary adenocarcinoma with bronchioloalveolar features Sarit Aviel-Ronen*, Bradley P. Coe†‡, Suzanne K. Lau*§, Gilda da Cunha Santos*¶, Chang-Qi Zhu*, Dan Strumpf*, Igor Jurisica*§ʈ, Wan L. Lam†‡, and Ming-Sound Tsao*§¶** *University Health Network, Ontario Cancer Institute and Princess Margaret Hospital Site, Toronto, ON, Canada M5G 2M9; †Department of Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, Vancouver, BC, Canada V5Z 1L3; Departments of §Medical Biophysics, ¶Laboratory Medicine and Pathobiology, and ʈComputer Science, University of Toronto, Toronto, ON, Canada M5G 2C1; and ‡University of British Columbia, Vancouver, BC, Canada V6T 289 Edited by John D. Minna, University of Texas Southwestern Medical Center, Dallas, TX, and accepted by the Editorial Board April 30, 2008 (received for review October 9, 2007) Bronchioloalveolar carcinoma (BAC), a subtype of lung adenocar- epidermal growth factor receptor (EGFR) inhibitors (8). The initial cinoma (ADC) without stromal, vascular, or pleural invasion, is studies that recognized BAC as a distinct entity reported 5-year considered an in situ tumor with a 100% survival rate. However, survival rates of 100% (9–11), but more recent studies have the histological criteria for invasion remain controversial. BAC-like reported lower 5-year survival rates of 83–86% for resected stage areas may accompany otherwise invasive adenocarcinoma, re- I BAC patients (12–14). These rates possibly reflect difficulties in ferred to as mixed type adenocarcinoma with BAC features applying the histological criteria of invasion in BAC or AWBF. (AWBF). AWBF are considered to evolve from BAC, representing a Some studies have also reported that BAC with focal areas of paradigm for malignant progression in ADC. However, the sup- microinvasion may also have excellent prognosis similar to nonin- porting molecular evidence remains forthcoming. Here, we have vasive BAC (11, 15). The identification of genes/proteins that may studied the genomic changes of BAC and AWBF by array com- distinguish BAC from AWBF and are predictors of ADC with poor parative genomic hybridization (CGH). We used submegabase- prognosis could be useful for the establishment of molecular resolution tiling set array CGH to compare the genomic profiles of pathological classification of lung ADC. In this study, we have used 14 BAC or BAC with focal area suspicious for invasion with those array comparative genomic hybridization (CGH) to test our hy- of 15 AWBF. Threshold-filtering and frequency-scoring analysis pothesis that BAC is molecularly distinguishable from AWBF by found that genomic profiles of noninvasive and focally invasive their differential genomic profiles and that marker genes for BAC are indistinguishable and show fewer aberrations than tumor invasion and/or poor prognosis may be identified. cells in BAC-like areas of AWBF. These aberrations occurred mainly at the subtelomeric chromosomal regions. Increased genomic al- Results terations were noted between BAC-like and invasive areas of Most chromosomal changes in both BAC and AWBF were subtle, AWBF. We identified 113 genes that best differentiated BAC from indicating low levels of genomic alteration as well as partial atten- AWBF and were considered candidate marker genes for tumor uation by contaminating nonneoplastic host cells. The profiles of invasion and progression. Correlative gene expression analyses BAC and BAC with focal areas suspicious for invasion were demonstrated a high percentage of them to be poor prognosis indistinguishable and showed low copy gains at 1p, 2q, 5p, 7p, 11p, markers in early stage ADC. Quantitative PCR also validated the 11q, 12q, 16p, 16q, 17q, 20q, and 21q (Fig. 1B). Copy gains typically amplification and overexpression of PDCD6 and TERT on chromo- occurred at the subtelomeric regions. AWBF had similar chromo- some 5p and the prognostic significance of PDCD6 in early stage somal changes but with greater variability and frequency and longer ADC patients. We identified candidate genes that may be respon- segmental alterations. Deletions were also more common in AWBF sible for and are potential markers for malignant progression and were observed mainly on 3p and 5q and to a lesser extent on in AWBF. 4q and 6q. In two patients with synchronous BAC and invasive AWBF, the BAC-like area of the latter showed greater aberrations ͉ ͉ array comparative genomic hybridization bronchioloalveolar carcinoma than the BAC (Fig. 2A). In two other AWBF, greater alterations ͉ ͉ microarray non-small-cell lung carcinoma prognostic markers were also noted in BAC-like areas compared with invasive areas (Fig. 2B). Normal lung samples showed no alteration of these ung adenocarcinoma (ADC) accounts for Ϸ35% of all lung regions. Lcancers and has an overall 5-year survival of 17% (1). The recent World Health Organization (WHO) classification recognized a particular subtype, bronchioloalveolar carcinoma (BAC), for its Author contributions: W.L.L. and M.-S.T. designed research; S.A.-R., B.P.C., S.K.L., and noninvasive features and excellent prognosis (2). BAC has a distinct G.d.C.S. performed research; S.A.-R., B.P.C., S.K.L., and I.J. contributed new reagents/ analytic tools; S.A.-R., B.P.C., S.K.L., G.d.C.S., C.-Q.Z., and D.S. analyzed data; and S.A.-R., histological pattern of tumor cells growing along preexisting alve- B.P.C., S.K.L., C.-Q.Z., D.S., and M.-S.T. wrote the paper. olar framework without evidence of stromal, pleural, or vascular The authors declare no conflict of interest. invasion. Yet, some invasive ADC, classified as mixed type, may This article is a PNAS Direct Submission. J.D.M. is a guest editor invited by the Editorial have components or large areas of BAC-like pattern. Multistage Board. development of adenocarcinoma putatively involves progression Freely available online through the PNAS open access option. from atypical adenomatous hyperplasia (AAH) through BAC to Data deposition: The data reported in this paper have been deposited in the Gene invasive mixed type ADC with BAC features (AWBF) (3–5). Mice Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE11945). that express oncogenic KRAS develop histological changes that **To whom correspondence should be addressed at: Ontario Cancer Institute, Toronto, ON, range from mild hyperplasia/dysplasia analogous to atypical adeno- Canada M5G 2M9. E-mail: [email protected]. matous hyperplasia to alveolar adenomas and ultimately display This article contains supporting information online at www.pnas.org/cgi/content/full/ overt ADC (6, 7). BAC-associated tumors have gained significant 0709618105/DCSupplemental. MEDICAL SCIENCES attention for their potentially greater sensitivity to treatment by © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709618105 PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10155–10160 Downloaded by guest on September 25, 2021 A C AWBF BAC A BAC B AWBF-BAC area Chr 5 Chr 8 Chr 7 Chr 19 1 AWBF AWBF-invasive area 2 Fig. 2. Increase in genomic instability. Shown is a karyotypic presentation of the log2 ratio of array CGH signals (SeeGH software). Normal genomic content -0.5 +0.5 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 B of genomic instability represented by more chromosomal aberrations than -100% +100% the upper images (purple brackets). (A) Synchronous BAC and AWBF in the same patient. (B) AWBF sampled in BAC and invasive areas. Using threshold-filtering, we identified 119 clones that distin- guished BAC from AWBF. Hierarchical clustering of all cases using these clones separated BAC from AWBF samples (Fig. 1C). In addition, a Fisher’s Exact Test comparing the frequency of genomic changes between the BAC and AWBF groups yielded a list of 517 clones that best differentiated the two lesions. Integrating these two analyses was accomplished by applying a 10-clone ‘‘window’’ to identify shared regions [supporting information (SI) Text]. The result was a list of 256 candidate clones of high interest, from which a shorter list of 58 clones with gains in AWBF compared with BAC was selected. These clones included 113 unique amplified genes (Table S1) that could represent invasion and tumor progression markers for AWBF. Quantitative polymerase chain reaction (QPCR) validated the gene content changes in 33 of the 113 candidate marker genes; 25 genes (75.8%) (Table S2) showed significantly higher gene copy number in AWBF compared with BAC. Among the evaluated genes were TERT and PDCD6, which we selected for further validation by QPCR and/or FISH based on their location on chromosome 5p, which showed prominent genomic changes (Fig. 1B). Measurement of both genes by QPCR demonstrated signifi- cant differences in gene copy number between BAC and AWBF (P ϭ 0.03 for TERT and P ϭ 0.02 for PDCD6), consistent with the array CGH results (Fig. 3A). Using FISH, we also studied the gene copy of TERT and chromosome 5 in 21 tumors. The correlation coefficients were 0.76 between array CGH and QPCR, 0.50 be- tween QPCR and FISH, and 0.53 between array CGH and FISH (Fig. 4). FISH appears the most sensitive in detecting the amplifi- cation levels and revealed the existence of chromosome 5 polysomy, especially in AWBF. Furthermore, FISH showed increased signal in the invasive area of AWBF compared with the BAC-like area in two samples, T41 and T46 (Fig. 4A). The coefficient of correlation Fig. 1. BAC compared with AWBF by histology, frequency scoring, and thresh- for PDCD6 amplification between array CGH and QPCR was 0.94. old filtering. (A1) BAC showing typical growth pattern of tumoral cells along the Using real-time QPCR (RT-QPCR), we showed that in 10 preexisting alveolar scaffold without evidence of invasion.