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Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.

Murphy SJ, Cheville JC, Zarei S, Johnson SH, Sikkink RA, Kosari F, Feldman AL, Eckloff BW, Karnes RJ, Vasmatzis G - DNA Res. (2012)

Bottom Line: Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols.Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation.Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Medical Sciences Building 2, 200 First St., SW, Rochester, MN 55905, USA. murphy.stephen@mayo.edu

ABSTRACT
High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

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LCM and WGA of prostate cancers. (a) H&E stained frozen section of prostate tissues. The central panel highlights the adjacent G3 and G4 cancers, with the individual G3 and G4 regions magnified further in the left and right panels, respectively. (b) LCM images before (PreLCM) and after (PostLCM) capture and the G3 and G4 cancer cells engrafted on the LCM caps. Products of direct in situ WGA of LCM-isolated cells (c) from two adjacent prostate lesions from six parallel tissue sections (1–6) and (d) from LCM of additional prostate (12 cases) and lung (4 cases) tissues [upper panels, 1% agarose gels, 1 kb ladders (L)]. Multiplex PCR from the corresponding tissues (lower panels, 4% agarose gels). The corresponding multiplex PCR products are shown in the lower panels (4% agarose gel with 100 bp ladders). Controls utilized gDNA (10 ng) in the WGA reaction or unamplified gDNA (unA) in the multiplex PCR.
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DSS021F1: LCM and WGA of prostate cancers. (a) H&E stained frozen section of prostate tissues. The central panel highlights the adjacent G3 and G4 cancers, with the individual G3 and G4 regions magnified further in the left and right panels, respectively. (b) LCM images before (PreLCM) and after (PostLCM) capture and the G3 and G4 cancer cells engrafted on the LCM caps. Products of direct in situ WGA of LCM-isolated cells (c) from two adjacent prostate lesions from six parallel tissue sections (1–6) and (d) from LCM of additional prostate (12 cases) and lung (4 cases) tissues [upper panels, 1% agarose gels, 1 kb ladders (L)]. Multiplex PCR from the corresponding tissues (lower panels, 4% agarose gels). The corresponding multiplex PCR products are shown in the lower panels (4% agarose gel with 100 bp ladders). Controls utilized gDNA (10 ng) in the WGA reaction or unamplified gDNA (unA) in the multiplex PCR.

Mentions: A prostate cancer case containing two regions of adjacent but distinct GP3 and GP4 tumours (Fig. 1a, central panel) was selected for the study. For the GP3 tumour (Fig. 1a, left panel), the infiltrative glands are well formed, each with a discernible round to oval shape. The GP4 tumour (Fig. 1a, right panel) shows a loss of distinct glandular differentiation in comparison with GP3, and cells are arranged in cribriform structures as well as sheets of tumour cells. LCM was used to isolate cells specific to the GP3 and GP4 populations minimizing the contamination of adjacent cells. Figure 1b demonstrates the power of the LCM technique, showing the section images before and after the laser-mediated extraction, as well as the cellular population of the GP3 and GP4 isolated on the LCM caps.Figure 1.


Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.

Murphy SJ, Cheville JC, Zarei S, Johnson SH, Sikkink RA, Kosari F, Feldman AL, Eckloff BW, Karnes RJ, Vasmatzis G - DNA Res. (2012)

LCM and WGA of prostate cancers. (a) H&E stained frozen section of prostate tissues. The central panel highlights the adjacent G3 and G4 cancers, with the individual G3 and G4 regions magnified further in the left and right panels, respectively. (b) LCM images before (PreLCM) and after (PostLCM) capture and the G3 and G4 cancer cells engrafted on the LCM caps. Products of direct in situ WGA of LCM-isolated cells (c) from two adjacent prostate lesions from six parallel tissue sections (1–6) and (d) from LCM of additional prostate (12 cases) and lung (4 cases) tissues [upper panels, 1% agarose gels, 1 kb ladders (L)]. Multiplex PCR from the corresponding tissues (lower panels, 4% agarose gels). The corresponding multiplex PCR products are shown in the lower panels (4% agarose gel with 100 bp ladders). Controls utilized gDNA (10 ng) in the WGA reaction or unamplified gDNA (unA) in the multiplex PCR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473372&req=5

DSS021F1: LCM and WGA of prostate cancers. (a) H&E stained frozen section of prostate tissues. The central panel highlights the adjacent G3 and G4 cancers, with the individual G3 and G4 regions magnified further in the left and right panels, respectively. (b) LCM images before (PreLCM) and after (PostLCM) capture and the G3 and G4 cancer cells engrafted on the LCM caps. Products of direct in situ WGA of LCM-isolated cells (c) from two adjacent prostate lesions from six parallel tissue sections (1–6) and (d) from LCM of additional prostate (12 cases) and lung (4 cases) tissues [upper panels, 1% agarose gels, 1 kb ladders (L)]. Multiplex PCR from the corresponding tissues (lower panels, 4% agarose gels). The corresponding multiplex PCR products are shown in the lower panels (4% agarose gel with 100 bp ladders). Controls utilized gDNA (10 ng) in the WGA reaction or unamplified gDNA (unA) in the multiplex PCR.
Mentions: A prostate cancer case containing two regions of adjacent but distinct GP3 and GP4 tumours (Fig. 1a, central panel) was selected for the study. For the GP3 tumour (Fig. 1a, left panel), the infiltrative glands are well formed, each with a discernible round to oval shape. The GP4 tumour (Fig. 1a, right panel) shows a loss of distinct glandular differentiation in comparison with GP3, and cells are arranged in cribriform structures as well as sheets of tumour cells. LCM was used to isolate cells specific to the GP3 and GP4 populations minimizing the contamination of adjacent cells. Figure 1b demonstrates the power of the LCM technique, showing the section images before and after the laser-mediated extraction, as well as the cellular population of the GP3 and GP4 isolated on the LCM caps.Figure 1.

Bottom Line: Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols.Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation.Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Medical Sciences Building 2, 200 First St., SW, Rochester, MN 55905, USA. murphy.stephen@mayo.edu

ABSTRACT
High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

Show MeSH
Related in: MedlinePlus