Rapid detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors.
BACKGROUND
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. 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.


METHODS
Surgical 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.


RESULTS
Purification 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.


CONCLUSIONS
Buffy 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.
http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466.
Department of Biological Sciences, Louisiana State University - Shreveport, One University Place, Shreveport, LA 71115, USA.
Long N
LN
Truong
Shashikant
S
Patil
Sherry S
SS
Martin
Jay F
JF
LeBlanc
Anil
A
Nanda
Mary L
ML
Nordberg
Marie E
ME
Beckner
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D001707:Biopsy, Needle; D001932:Brain Neoplasms / Q000235:genetics* / Q000556:secondary / Q000601:surgery; D002498:Centrifugation; D004806:Ependymoma / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D005784:Gene Amplification; D005909:Glioblastoma / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000235:genetics* / Q000556:secondary; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009857:Oncogenes; D016612:Paraffin Embedding; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013997:Time Factors; D016707:Tissue Fixation; D059708:Ultrasonic Surgical Procedures
D014408:Biomarkers, Tumor; D005404:Fixatives; D005557:Formaldehyde; C512478:EGFR protein, human; D066246:ErbB Receptors