BACKGROUND

Genomic intra- and intertumor heterogeneity is one main reason for relapse and resistance to therapy. There is a shortage of studies characterizing the level of genomic intertumoral heterogeneity of known cancer genes in multiple longitudinal biopsies of individual patients. Additionally, a workflow to deconvulate the intermixture of tumor and stromal components is missing. To overcome these limitations and to decipher the genomic heterogeneity and clonal evolution, we developed a method and applied it on matched (primary-recurrence/metastasis) non-squamous, non-small cell lung cancers (NSCLC).

METHODS

Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin and paraffin embedded (FFPE) tissues, followed by multiparamter flow sorting. Sorted populations were subjected to genomic profiling by high resolution array comparative genomic hybridization (aCGH) and massively parallel sequencing (MPS). DAPI allowed to seperate populations by ploidy and anti-TTF1 antibodies was used to control for tumor nuclei. Array-CGH, combined with ploidy, was used to retrieve genome wide copy numbers. The Comprehensive Cancer Panel that covers all-exons of 409 cancer genes was applied on all sorted tumor and stromal populations to detect somatic mutations and their variant allelic frequency (VAF).

RESULTS

MPP was successfully applied to 44 frozen or FFPE tissue specimens from 19 patients. Array-CGH and MPS of TTF1-negative, normal cells were concordant to germline controls. Sequencing revealed that 50% of mutations are shared between primary tumors and metastases. Except for one patient, mutations with VAF>0.3 are shared between primary and metastasis. The variant allelic frequency was significantly higher in shared mutations than mutations that were private to one tumor. Besides common activating mutations in EGFR and KRAS we found biallelic inactivation in tumor suppressor genes like KEAP1, NF1, STK11 and TP53. Two clonal evolutionary patterns were found: 1) early and 2) late divergence. Matched tumors without any shared mutations were classified as unrelated primary tumors.

CONCLUSION

The power of MPP is to increase the precision of downstream analysis, due to the sorting of pure populations of tumor cells. It permitted to infer the clonal evolution of tumor populations with unprecedented confidence. The low level of genomic heterogeneity of mutations with VAF>0.3 in this study is in line with recent data from Zhang et al., who showed that the poor precision of low depth sequencing (<277x) contributes to an overestimation of genomic heterogeneity (Zhang et al., Science 2014). Integrational analysis of ploidy, chromosomal aberrations and mutations in 409 cancer genes allowed to draw the evolutionary picture of each patient's disease. MPP appears as a promising tool for identification of genomic vulnerabilities that could be exploited for tailored treatment.

Citation Format: Thomas Lorber, Tanja Dietsche, Valeria Perrina, Darius Juskevicius, Arthur Krause, David Mueller, Michael Barrett, Christian Ruiz, Lukas Bubendorf. Clonal evolution and genomic tumor heterogeneity in non-small cell lung cancer deciphered by multiparameter ploidy profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 158.