Elsevier

Lung Cancer

Volume 126, December 2018, Pages 80-88
Lung Cancer

Subclonal evolution of pulmonary adenocarcinomas delineated by spatially distributed somatic mitochondrial mutations

https://doi.org/10.1016/j.lungcan.2018.10.024Get rights and content

Highlights

  • Somatic mtDNA mutations could be detected in 96% (18/19) of the analyzed cases.

  • mtDNA mutations were distributed ubiquitously or regionally restricted.

  • Spatial mapping of the mutations indicated subclones and phylogenetic relations.

  • Histological and mtDNA mutation based differentiation was not concurrent.

  • Ubiquitous mtDNA mutations were associated with shorter disease-free survival.

Abstract

Objectives

The potential role of cancer associated somatic mutations of the mitochondrial genome (mtDNA) is controversial and still poorly understood. Our group and others recently challenged a direct tumorigenic impact and suggested a passenger-like character. In combination with the known increased mutation rate, somatic mtDNA mutations account for an interesting tool to delineate tumor evolution. Here, we comprehensively analyzed the spatial distribution of somatic mtDNA mutations throughout whole tumor sections of pulmonary adenocarcinoma (ADC).

Materials and methods

Central sections of 19 ADC were analyzed in a segmented manner (11–34 segments/tumor) together with non-neoplastic tissue samples and lymph node metastasis, if present. We performed whole mtDNA sequencing and real-time PCR based quantification of mtDNA copy numbers for all samples. Further, histological growth patterns were determined on H&E sections and the tumor cell content was quantified by digital pathology analyses.

Results

Somatic mtDNA mutations were present in 96% (18/19) of the analyzed tumors, either ubiquitously or restricted to specific tumor regions. Spatial and histological mapping of the mutations enabled the identification of subclonal structures and phylogenetic relations within a tumor section indicating different progression levels. In this regard, lymph node metastases seem to be related to early events in ADC development. There was no concurrence between histological and mtDNA mutation based clusters. However, micropapillary patterns occurred only in tumors with ubiquitous mutations. ADC with more than two ubiquitous mutations were associated with shorter disease-free survival (p < 0.01).

Conclusion

Cancer related mtDNA mutations are interesting candidates for the understanding of subclonal ADC evolution and perspectively for monitoring tumor progression. Our data reveal a potential prognostic relevance of somatic mtDNA mutations.

Introduction

Despite great improvements through the implementation of personalized therapeutic strategies for the treatment of lung adenocarcinoma (ADC) [1] targeting e.g. EGFR mutations, ALK and ROS1 rearrangements or PD/PDL-1, prognosis remains limited. The development of resistance mechanisms [2] is a major cause for the still low 5-year overall survival rate of less than 20% [3]. A key factor of therapy resistance is intratumor heterogeneity (ITH) [4,5], mainly due to genomic and epigenomic instability as well as to a branched subclonal evolution caused by differing selective conditions, like microenvironmental stress [6]. To further improve clinical outcome, better understanding of the mechanisms of tumor evolution resulting in multi-level tumor heterogeneity is of great importance. Employing comprehensive sequencing approaches, several studies have meticulously analyzed the temporal and spatial genetic heterogeneity in nuclear DNA and identified the dynamics of subclonal lesions during tumor progression and therapeutic treatment [2,[7], [8], [9], [10]], some of which are already clinically relevant for prediction of therapy resistance [11] and recurrence [12].

Addressing the role of mitochondrial alterations in tumorigenesis [[13], [14], [15]], we comprehensively analyzed the prevalence of somatic mutations of the mitochondrial genome (mtDNA) in non-small cell lung cancer recently [16]. Our findings suggest that mtDNA mutations are passenger mutations rather than directly oncogenic. However, with a 10-fold higher mutation rate compared to genomic mutations, somatic mtDNA mutations might be a sensitive tool to identify different subclonal lineages within a tumor and thus support deciphering spatial tumor evolution [17].

In order to shed more light on the potential role of mitochondria in this scenario, we comprehensively investigated the spatial distribution of somatic mtDNA mutations in ADC. We demonstrate that the identification and differentiation of ubiquitous and regional restricted mutations is a promising tool to identify spatial tumor evolution by detecting subclonal structures and the correlation with disease-free survival (DFS).

Section snippets

Samples

All tumor specimens used in the present study were resected at the Thoraxklinik at University Hospital Heidelberg and diagnosed according to the 2015 World Health Organization Classification criteria for lung cancer [18] at the Institute of Pathology at Heidelberg University.

Formalin-fixed and paraffin embedded tumor samples were processed with the support of the tissue bank of the National Center for Tumor Diseases (NCT; project: # 1746, # 2015, # 2577) and used in accordance with the ethical

Differentiation of ubiquitous and regional restricted somatic mitochondrial mutations

In total, we analyzed 392 samples from 19 ADC (Table 1). Heteroplasmic mutations were identified in four cases (#1, #7, #8, #19) which were present in the corresponding non-neoplastic tissues with an allele frequency of less than 100%, as well as throughout all tumor samples of a respective case.

Somatic mtDNA mutations could be detected in at least one segment in 18 (96%) of the analyzed cases, with a range of 0–7 mutations per segment and 1–17 different mutations per tumor (Fig. 1A). A total

Discussion

In this study, we comprehensively analyzed the spatial distribution of somatic mtDNA mutations throughout a complete 2-dimensional expansion of 19 ADC. We demonstrated that multi-regional analyses identify subclonal structures, which help to build oncogenic models for a better understanding of spatial morpho-molecular ADC evolution. Further, we shed more light on the potential tumorigenic impact of mtDNA mutations as well as their prognostic relevance.

We detected somatic mtDNA mutations in all

Funding

AW was supported by an excellence grant from the Else Kröner-Fresenius Foundation [2014_EKES.05].

Acknowledgements

We thank Veronika Geißler and Christiane Zgorzelski for excellent technical assistance and the Tissue Bank of the National Centre for Tumor Diseases, Heidelberg.

References (38)

  • A. Warth et al.

    Genetic changes of non-small cell lung cancer under neoadjuvant therapy

    Oncotarget

    (2016)
  • A.H. Noone et al.

    SEER Cancer Statistics Review 1975-2015

    (2018)
  • I. Dagogo-Jack et al.

    Tumour heterogeneity and resistance to cancer therapies

    Nat. Rev. Clin. Oncol.

    (2018)
  • C.M. Lovly et al.

    Tumor heterogeneity and therapeutic resistance

    Am. Soc. Clin. Oncol. Educ. Book

    (2016)
  • A.A. Alizadeh et al.

    Toward understanding and exploiting tumor heterogeneity

    Nat. Med.

    (2015)
  • E.C. de Bruin et al.

    Spatial and temporal diversity in genomic instability processes defines lung cancer evolution

    Science

    (2014)
  • M. Jamal-Hanjani et al.

    Tracking the evolution of non-small-Cell lung Cancer

    N. Engl. J. Med.

    (2017)
  • N. McGranahan et al.

    Clonal status of actionable driver events and the timing of mutational processes in cancer evolution

    Sci. Transl. Med.

    (2015)
  • J. Zhang et al.

    Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing

    Science

    (2014)
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    Present address: Institute of Pathology, Cytopathology, and Molecular Pathology UEGP MVZ Giessen / Wetzlar / Limburg, Germany.

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