Rapid Detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors

Long N Truong,Jay F Leblanc,Marie E Beckner,Shashikant Patil,Anil Nanda,Mary L Nordberg,Sherry S Martin

doi:10.1186/1746-1596-7-66

Abstract

BackgroundGenomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes.MethodsSurgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, [tumor DNA]/[NB DNA]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers.ResultsPurification of DNA from ultrasonic surgical aspirations was rapid (<3 days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level (≥ 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level (≥3.0 & <6.0 CN ratio) amplifications detected in FFPE were also detected as being increased (≥ 2.0 CN ratio) in the aspirations.ConclusionsBuffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE.Virtual slideshttp://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466

Highlights

Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment
Brain tumor specimens for molecular studies include ultrasonic surgical aspirations available at the time of surgery and small, diagnostic biopsies that are processed as formalin-fixed, paraffin-embedded (FFPE) samples
Tumor genomes are being evaluated to varying extents by fluorescence in situ hybridizaiton (FISH), array comparative genomic hybridization, single nucleotide polymorphism (SNP) arrays, specific mutation detection with various PCR methods, methylation studies, targeted sequencing of selected genes, and whole genome sequencing

Summary

Introduction

Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes. Assays to identify amplified genes are proposed to become critical in patient care as monoclonal antibodies and small molecules that inhibit proteins encoded by oncogenes become available. Brain tumor specimens for molecular studies include ultrasonic surgical aspirations available at the time of surgery and small, diagnostic biopsies that are processed as formalin-fixed, paraffin-embedded (FFPE) samples. Tissue frozen for storage at the time of surgery can be released for DNA studies, it is not usually available outside of research settings. Surgical aspirations can quickly provide DNA to speed up turnaround times and produce higher yields than FFPE sections of small biopsies

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Conclusion