High-resolution array comparative genomic hybridization of single micrometastatic tumor cells

Fuhrmann, Christine; Schmidt‐Kittler, Oleg; Stoecklein, Nikolas H.; Petat-Dutter, Karina; Vay, Christian; Bockler, Kerstin; Reinhardt, Richard; Ragg, Thomas; Klein, Christoph

doi:10.1093/nar/gkn101

Cited by 56 publications

(53 citation statements)

References 25 publications

(45 reference statements)

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“…For mutational profiling, we used a robust adapter-linker PCR for WGA that allows comprehensive genomic profiling of fixed and unfixed single cells (12,16,(28)(29)(30)(31)(32). As expected, our data show that CTC are a profoundly heterogeneous cell population.…”

Section: Discussionsupporting

confidence: 72%

Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer

et al. 2014

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The prognosis of colorectal cancer is closely linked to the occurrence of distant metastases. Systemic dissemination is most likely caused by circulating tumor cells (CTC). Despite the fundamental role of CTC within the metastatic cascade, technical obstacles have so far prevented detailed genomic and, in particular, phenotypic analyses of CTC, which may provide molecular targets to delay or prevent distant metastases. We show here a detailed genomic analysis of single colorectal cancer-derived CTC by array comparative genomic hybridization (aCGH), mutational profiling, and microsatellite instability (MSI) analysis. Furthermore, we report the first gene expression analysis of manually selected colorectal cancer-derived CTC by quantitative real-time PCR (qRT-PCR) to investigate transcriptional changes, enabling CTC to survive in circulation and form distant metastases. aCGH confirmed the tumor cell identity of CellSearch-isolated colorectal cancer-derived CTC. Mutational and MSI analyses revealed mutational profiles of CTC to be similar, but not identical to the corresponding tumor tissue. Several CTC exhibited mutations in key genes such as KRAS or TP53 that could not be detected in the tumor. Gene expression analyses revealed both a pronounced upregulation of CD47 as a potential immune-escape mechanism and a significant downregulation of several other pathways, suggesting a dormant state of viable CTC. Our results suggest mutational heterogeneity between tumor tissue and CTC that should be considered in future trials on targeted therapy and monitoring of response. The finding of upregulated immune-escape pathways, which may be responsible for survival of CTC in circulation, could provide a promising target to disrupt the metastatic cascade in colorectal cancer. Cancer Res; 74(6); 1694-704. Ó2014 AACR.

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Section: Discussionsupporting

confidence: 72%

Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer

et al. 2014

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“…This was achieved by discarding spots with a reference signal lower than the background level plus 2 standard deviations of the background level. Signal median intensities were corrected by subtracting the local background level and the log 2 (test/reference) ratios were globally normalized (16,27). Distribution of M values (log 2 ch1/ch2) corresponding to majority of genes was adjusted to zero by using a normalization factor as follows: MЈ ϭ M Ϫ log 2 N, where MЈ is the normalized log 2 ratio and N is a normalization factor (calculated by summing the measured intensities in both channels (channel 1 [ch1] and channel 2[ch2]) (40).…”

Section: Methodsmentioning

confidence: 99%

Crucial Role for Insertion Sequence Elements in Lactobacillus helveticus Evolution as Revealed by Interstrain Genomic Comparison

Kaleta

O’Callaghan

Fitzgerald

et al. 2010

Appl Environ Microbiol

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Lactobacillus helveticus is a versatile dairy bacterium found to possess heterogeneous genotypes depending on the ecosystem from which it was isolated. The recently published genome sequence showed the remarkable flexibility of its structure, demonstrated by a substantial level of insertion sequence (IS) element expansion in association with massive gene decay. To assess this diversity and examine the level of genome plasticity within the L. helveticus species, an array-based comparative genome hybridization (aCGH) experiment was designed in which 10 strains were analyzed. The aCGH experiment revealed 16 clusters of open reading frames (ORFs) flanked by IS elements. Four of these ORFs are associated with restriction/modification which may have played a role in accelerated evolution of strains in a commercially intensive ecosystem undoubtedly challenged through successive phage attack. Furthermore, analysis of the IS-flanked clusters demonstrated that the most frequently encountered ISs were also those most abundant in the genome (IS1201, ISL2, ISLhe1, ISLhe2, ISLhe65, and ISLhe63). These findings contribute to the overall viewpoint of the versatile character of IS elements and the role they may play in bacterial genome plasticity.Lactobacillus helveticus is a gram-positive, homofermentative lactic acid bacterium which is widely used in the manufacture of cheeses, such as Swiss cheese and some Cheddar-type cheeses (22,25). It is also commonly used in the production of different types of Italian cheeses, such as Parmigiano Reggiano (18) and Grana Padano, where it contributes to the formation of specific flavor compounds (42).Phylogenetic analysis of ribosomal protein sequences derived from lactobacilli and streptococci classified L. helveticus in the same group along with both gastrointestinal (GI) tract and dairy-specific species (14). Comparative analysis of the 16S rRNA of L. helveticus DPC4571 revealed 98.4% identity with Lactobacillus acidophilus NCFM and indicated that this probiotic strain was closely related to strain DPC4571, despite the different environments these two lactobacilli inhabit (4). The results of genomic analysis of L. helveticus suggested that two major events have occurred in the diversification process of L. helveticus from a common ancestor with L. acidophilus, selective gene loss and acquisition of a large number of insertion sequence (IS) elements (4). IS elements are DNA sequences capable of independent transposition within and between bacterial genomes (31). Their capacity for independent mobility demonstrates the parasitic nature of these elements (11); however, they can also be regarded as having a positive influence, as they assist in promoting genetic variation (1). Thus, even though the primal character of these elements remains unclear in that they may be considered simply as selfish DNA elements, their impact on the architecture of microbial genomes is undeniable. It has already been demonstrated that IS-related mutations occur in Escherichia coli (44), Lactococcus lactis (10)...

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“…Intratumor heterogeneity studies profiling or sequencing DNA from individual tumor cells require whole-genome amplification (WGA). By using commercially available methods for WGA, it is possible to amplify DNA from a single cell to a level where it can be profiled by microarrays, but these studies have been challenged by technical difficulty and limited reproducibility (75)(76)(77)(78). Analyzing WGA fragments from single cells using targeted approaches such as DNA microarrays is problematic because fragments are randomly amplified from a small fraction (<10%) of the genome, and thus many fail to hybridize to their target probes.…”

Section: Figurementioning

confidence: 99%

Insight into the heterogeneity of breast cancer through next-generation sequencing

Russnes¹,

Navin²,

Hicks³

et al. 2011

J. Clin. Invest.

216

154

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Rapid and sophisticated improvements in molecular analysis have allowed us to sequence whole human genomes as well as cancer genomes, and the findings suggest that we may be approaching the ability to individualize the diagnosis and treatment of cancer. This paradigmatic shift in approach will require clinicians and researchers to overcome several challenges including the huge spectrum of tumor types within a given cancer, as well as the cellto-cell variations observed within tumors. This review discusses how next-generation sequencing of breast cancer genomes already reveals insight into tumor heterogeneity and how it can contribute to future breast cancer classification and management.

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High-resolution array comparative genomic hybridization of single micrometastatic tumor cells

Cited by 56 publications

References 25 publications

Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer

Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer

Crucial Role for Insertion Sequence Elements in Lactobacillus helveticus Evolution as Revealed by Interstrain Genomic Comparison

Insight into the heterogeneity of breast cancer through next-generation sequencing

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