Virchows Archiv

, Volume 468, Issue 6, pp 651–662 | Cite as

Deciphering intra-tumor heterogeneity of lung adenocarcinoma confirms that dominant, branching, and private gene mutations occur within individual tumor nodules

  • Giuseppe Pelosi
  • Alessio Pellegrinelli
  • Alessandra Fabbri
  • Elena Tamborini
  • Federica Perrone
  • Giulio Settanni
  • Adele Busico
  • Benedetta Picciani
  • Maria Adele Testi
  • Lucia Militti
  • Patrick Maisonneuve
  • Barbara Valeri
  • Angelica Sonzogni
  • Claudia Proto
  • Marina Garassino
  • Filippo De Braud
  • Ugo Pastorino
Original Article

Abstract

While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR, 3 with a KRAS, and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2, PIK3CA, TP53, and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1, ALK, BRAF, HER2, KDR, LKB1, PTEN, MET, SMAD4, SMARCB1, and SRC) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance.

Keywords

Mutation Gene Next-generation sequencing Intra-tumor heterogeneity EGFR KRAS ALK Dominant Branching Private/private/hitchhiker 

Supplementary material

428_2016_1931_Fig4_ESM.gif (8 kb)
S1_Figure 1S

Fluorescence in situ hybridization analysis for ALK in the case #2 ALK-trans-2 showed break-apart signal splitting in most (90 %) tumor cells, indicative of early driver molecular events. (GIF 8 kb)

428_2016_1931_MOESM1_ESM.tiff (798 kb)
High resolution image (TIFF 798 kb)
428_2016_1931_Fig5_ESM.gif (21 kb)
S2_Figure 2S

Alignment outputs of T-NGS analysis for EGFR (case #1 EGFR-1) showed a complex deletion of 15 bp (DEL aa L747 - A 750) co-existent with E746A point (GAA > GCA) mutation in exon 19 (A) and for PIK3CA (case #17 Wild Type-2) with E547K point (GAA/AAA) mutation in exon 10 (B). (GIF 21 kb)

428_2016_1931_MOESM2_ESM.tiff (2.8 mb)
High resolution image (TIFF 2832 kb)
428_2016_1931_Fig6_ESM.gif (3 kb)
S3_Figure 3S

General overall (OS) and disease-free survival (DFS) curves are shown in the entire series of 20 ADC patients. At the 48 month-follow-up most patients died of disease (left) or experienced tumor relapse (right). (GIF 3 kb)

428_2016_1931_MOESM3_ESM.tiff (1.2 mb)
High resolution image (TIFF 1248 kb)
428_2016_1931_MOESM4_ESM.doc (450 kb)
ESM 4(DOC 450 kb)
428_2016_1931_MOESM5_ESM.doc (924 kb)
ESM 5(DOC 924 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Giuseppe Pelosi
    • 1
    • 2
  • Alessio Pellegrinelli
    • 1
  • Alessandra Fabbri
    • 1
  • Elena Tamborini
    • 1
  • Federica Perrone
    • 1
  • Giulio Settanni
    • 1
  • Adele Busico
    • 1
  • Benedetta Picciani
    • 1
  • Maria Adele Testi
    • 1
  • Lucia Militti
    • 1
  • Patrick Maisonneuve
    • 3
  • Barbara Valeri
    • 1
  • Angelica Sonzogni
    • 1
  • Claudia Proto
    • 4
  • Marina Garassino
    • 4
  • Filippo De Braud
    • 4
  • Ugo Pastorino
    • 5
  1. 1.Department of Pathology and Laboratory MedicineFondazione IRCCS Istituto Nazionale dei TumoriMilanItaly
  2. 2.Department of Biomedical and Clinical Sciences “Luigi Sacco”Università degli StudiMilanItaly
  3. 3.Division of Epidemiology and BiostatisticsEuropean Institute of OncologyMilanItaly
  4. 4.Department of Medical OncologyFondazione IRCCS Istituto Nazionale dei TumoriMilanItaly
  5. 5.Division of Thoracic SurgeryFondazione IRCCS Istituto Nazionale dei TumoriMilanItaly

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