High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment

Genomic abnormalties in leiomyosarcoma and gastrointestinal stromal tumour

Leiomyosarcoma and GISTs are malignant tumours arising from the mesenchyme. Until relatively recently GISTs were classified as smooth muscle tumours such as leiomyosarcoma, however they are now recognised as a distinct entity arising from the interstitial cells of the Cajal.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Correlation of Probe log₂ ratios of paired FF and FFPE samples of 3 leiomyosarcoma cases.

<p>Pearson’s Correlation, r of log₂ ratio values of all probes on tumour DNA samples was calculated using GraphPad Prism software and statistically significant (p<0.001).</p

Comparison of Array CGH results in paired Fresh Frozen and Formalin-fixed Paraffin -Embedded samples from LMS 9.

<p><b>Panel A:</b> Graphical whole-genome views of copy number aberrations (CNAs) identified in both sample types showing close similarities on most chromosomes. <b>Panel B:</b> Higher resolution graphical views of Chromosome 11 showing the close similarity in gain and loss patterns detected in both sample types. <b>Panel C:</b> High-resolution views showing the most dissimilar CNA pattern detected between both sample types on chromosome 4. On Panel A, aberrations called by FASST2 algorithm are represented by blue triangles to the right (amplifications) and red triangles to the left (deletions) of the chromosomes. Double blue and red triangles/lines represent high-level amplifications and two-copy deletion, respectively. On Panels B and C, dots represent individual probe log₂ ratios plotted as a function of their chromosomal position with a moving average of probe log₂ ratios (wavy dark blue line). Aberration calls are represented by thick black lines with corresponding shaded blue areas above (amplifications) and red areas below (deletions) the zero line.</p

Comparison of Array CGH results in paired Fresh Frozen and Formalin-fixed Paraffin -Embedded samples from LMS 11.

<p><b>Panel A:</b> Graphical whole-genome views of both sample types showing that majority of the copy number aberrations (CNAs) identified in the macro-dissected FFPE sample were not detected in the FF sample. Deletions on the long arms of chromosomes 9, 14 and 15 as well as the short arm of chromosome 16 were the called on both sample types. <b>Panel B:</b> High resolution graphical views of a 6 Mb region along on Chromosome 14 (14q24.1) showing a group of probes with an average log₂ ratio of approximately 0.6 and the corresponding single copy amplification detected in the FFPE sample but no aberrations detected in the FF sample. <b>Panel C:</b> High-resolution graphical views showing a closely similar copy number aberration detected on Chromosome 15 (15q11.2) in both sample types with similar probe log₂ ratios. On Panel A, aberrations called by FASST2 algorithm are represented by blue triangles to the right (amplifications) and red triangles to the left (deletions) of the chromosomes. Double blue and red triangles/lines represent high-level amplifications and two-copy deletion, respectively. On Panels B and C, dots represent individual probe log₂ ratios plotted as a function of their chromosomal position with a moving average of probe log₂ ratios (wavy dark blue line). Aberration calls are represented by thick black lines with corresponding shaded blue areas above (amplifications) and red areas below (deletions) the zero line.</p

Two-colour Interphase Fluorescence in situ Hybridisation (FISH) Images of nuclei of cultured leiomyosarcoma cells.

<p>Most cells have five or more chromosome 11 centromere (green signals), but relatively fewer copies of the ATM region 11q22 (red signals) representing copy number deletion. Nuclei are stained with DAPI (blue). Cells were derived from short-term cultures from fresh tissue (LMS 9).</p

Frequency Plot of Common Genomic Copy Number Aberrations among 22 FFPE Leiomyosarcomas.

<p>Commonly aberrant regions are plotted as a function of their chromosomal position. Red bars to the left of the chromosome represent frequency of deletions and blue bars to the right of the chromosome represent amplifications. The heights of the bars correspond to the relative frequency of aberrations among the cases. All CNAs are detected using the FASST2 algorithm.</p

A summary of FFPE leiomyosarcoma cases included in this study.

<p>DLR Spread – Derivative Log Ratio Spread of Array Data.</p>§ -<p>Additional fresh samples obtained and frozen before fixing in formalin.</p

Optimisation of DNA Labelling Protocol.

*<p>NP – Array CGH <b>not performed</b> on sample (if sample failed on spectrophotometry). Spectrophotometry for DNA concentration and Degree of Labelling (DoL) done using Nanodrop® ND-2000 and optimal DoL = 0.75–2.5% (for ULS- Cy5); or 1.75–3.5% (for ULS Cy3). Dye Signal Intensity calculated as part of Quality Control Metrics by Agilent Feature Extraction Software (v10.7.3).</p