Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing.
Author(s): Zhang J, Fujimoto J, Zhang J, Wedge DC, Song X, Zhang J, Seth S, Chow CW, Cao Y, Gumbs C, Gold KA, Kalhor N, Little L, Mahadeshwar H, Moran C, Protopopov A, Sun H, Tang J, Wu X, Ye Y, William WN, Lee JJ, Heymach JV, Hong WK, Swisher S, Wistuba II, Futreal PA
Publication: Science, 2014, Vol. 346, Page 256-9
PubMed ID: 25301631 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of sequencing multiple regions of the same tumor, smoking status, and patient relapse on the mutational profile of lung adenocarcinomas using next-generation whole exome sequencing and compared the profile with that identified by deep-sequencing.
Conclusion of Paper
Seventy-six percent of the mutations identified were found in all regions of the same tumor but the branch patterns of the shared versus not-shared mutations differed between tumors and these mutations were associated with smoking status. Importantly, deep-sequencing increased the proportion of mutations found in all specimens of a tumor but the different regions still reflected the changing mutational profile of the tumor. Overall, 28% of mutations were consistent with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) activity and generally these mutations were more likely to occur in just a subset of specimens but the difference was significant for only one tumor. The three patients with disease relapse had a larger proportion of mutations not identified in all specimens and a larger subclonal fraction than those without.
Studies
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Study Purpose
This study investigated the effects of sequencing multiple regions of the same tumor, patient characteristics (smoking status, age, and gender), and relapse on the mutational profile of lung adenocarcinomas using next-generation whole exome sequencing and compared the profile with that identified by deep-sequencing. After resection, eight biopsies were obtained using an 18-gauge needle from 11 resected lung adenocarcinoma specimens. From each biopsy an H&E slide was used to determine the percentage of normal and tumor cells. DNA was extracted from the 48 biopsies containing >40% viable tumor cells that were preserved in an unspecified manner (assumed formalin) using an unspecified method. Whole exome sequencing at 138-477X was conducted using the KAPA library kit and Agilent SureSelect Human All Exon V4 kit. Variants were confirmed by deep sequencing of both the tumor and normal tissue.
Summary of Findings:
Although the number of mutations identified varied among tumors, no correlation between the number of mutations and patient age, patient gender, patient smoking status, tumor size, or lymph node status was identified. Of the mutations, 76% and 93% of previously identified cancer gene point mutations were found in all specimens of the same tumor, but the branch patterns of the shared versus not-shared mutations differed between tumors. Interestingly, large copy number variations were not observed in any of the tumors and the deletions and insertions that were found were present in all specimens from the same tumor. When whole exome sequencing (average 277X) and deep sequencing (average 863X) data were compared, the increased sequencing depth of deep-sequencing allowed for identification of the mutations in a higher percentage of specimens. Overall, 28% of mutations identified had a substitution pattern consistent with APOBEC activity and in 7 of 11 patients APOBEC mutations were more likely to occur in just a subset of specimens, but in only one patient was the difference significant.
Shifts in mutation spectrum were observed over time when looking at mutations that were found in all specimens of a tumor versus those found only in some parts of a tumor. Although smokers and never-smokers had different mutations present in all specimens, in two of the former smokers and one smoker there was a shift from the smoking-associated transversion mutations (C>A) that were present in all specimens from that tumor to the non-smoker associated transition mutations (C>T) that were observed in a subset of specimens. The three patients with disease relapse had a larger proportion of mutations not identified in all specimens than those without relapse (40% versus 17%, P=0.006)and had a larger subclonal fraction in their tumor as determined by phylogenetic analysis (41% with relapse versus 24% without, P=0.045) and using a Bayesian Dirichlet process (66% in patients with relapse versus 36% in patients without relapse, p=0.035).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Preaquisition Patient age 50-75 years
Preaquisition Other drugs Smoker
Non-smoker
Prior-smoker
Next generation sequencing Specific Technology platform WES
Deep-sequencing
Biospecimen Aliquots and Components Aliquot size/volume 3 biopsies
4 biopsies
5 biopsies
Biospecimen Aliquots and Components Biospecimen heterogeneity Intratumoral sampling (exact positions not specified)
Preaquisition Prognostic factor 2.0-4.6 cm tumor
Lymph node positive
Lymph node negative