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

Truong LN, Patil S, Martin SS, LeBlanc JF, Nanda A, Nordberg ML, Beckner ME - Diagn Pathol (2012)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University - Shreveport, One University Place, Shreveport, LA 71115, USA.

ABSTRACT

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.

Virtual slides: http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466.

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Related in: MedlinePlus

Scatterplots of DNA values for genes in non-neoplastic brain versus normal values provided in MLPA kits. The amounts of DNA represent copy number (CN) for each gene assayed. The non-neoplastic (or normal) brain DNA (NB) was evaluated by comparing its values with expected values provided by the manufacturer. Regression analysis of NB values, that were obtained when FFPE-GBM1 was tested with the P171 kit, identified ERBB4 at the lower limit of the 95 % confidence interval as an outlier. The overlays of observed NB values with normal values included in the P171, P172, and P173 kits demonstrated overall correspondence for both sources of non-neoplastic DNA in the data distributions. Their trendlines are also shown.
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Figure 1: Scatterplots of DNA values for genes in non-neoplastic brain versus normal values provided in MLPA kits. The amounts of DNA represent copy number (CN) for each gene assayed. The non-neoplastic (or normal) brain DNA (NB) was evaluated by comparing its values with expected values provided by the manufacturer. Regression analysis of NB values, that were obtained when FFPE-GBM1 was tested with the P171 kit, identified ERBB4 at the lower limit of the 95 % confidence interval as an outlier. The overlays of observed NB values with normal values included in the P171, P172, and P173 kits demonstrated overall correspondence for both sources of non-neoplastic DNA in the data distributions. Their trendlines are also shown.

Mentions: The CN ratios, or fold-differences from normal for each gene, are represented by the ratio, [tumor DNA] / [NB DNA], derived from measurements of the CE peaks. The CN ratio was calculated after peaks of non-amplified genes on CE graphs, representing relative amounts of DNA, were matched with the average of two NB samples analyzed in the same assay run. Peak heights of non-amplified genes in tumor samples and NB were adjusted to approximately the same scale as graphical printouts from CE were produced. Final, finer adjustments were made in spreadsheets by maximizing alignments of trendlines for non-amplified genes. Scatterplots were evaluated to check the reactions of NB DNA with the MLPA probes compared to expected values provided by the manufacturer. Overlays of NB results obtained in GBM1’s assays of FFPE, using P171, P172, and P173 kits, with graphs of normal values provided in the MLPA kits’ literature, demonstrated comparable overall detection of the two populations of normal DNA data points (Figure 1).


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

Truong LN, Patil S, Martin SS, LeBlanc JF, Nanda A, Nordberg ML, Beckner ME - Diagn Pathol (2012)

Scatterplots of DNA values for genes in non-neoplastic brain versus normal values provided in MLPA kits. The amounts of DNA represent copy number (CN) for each gene assayed. The non-neoplastic (or normal) brain DNA (NB) was evaluated by comparing its values with expected values provided by the manufacturer. Regression analysis of NB values, that were obtained when FFPE-GBM1 was tested with the P171 kit, identified ERBB4 at the lower limit of the 95 % confidence interval as an outlier. The overlays of observed NB values with normal values included in the P171, P172, and P173 kits demonstrated overall correspondence for both sources of non-neoplastic DNA in the data distributions. Their trendlines are also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475141&req=5

Figure 1: Scatterplots of DNA values for genes in non-neoplastic brain versus normal values provided in MLPA kits. The amounts of DNA represent copy number (CN) for each gene assayed. The non-neoplastic (or normal) brain DNA (NB) was evaluated by comparing its values with expected values provided by the manufacturer. Regression analysis of NB values, that were obtained when FFPE-GBM1 was tested with the P171 kit, identified ERBB4 at the lower limit of the 95 % confidence interval as an outlier. The overlays of observed NB values with normal values included in the P171, P172, and P173 kits demonstrated overall correspondence for both sources of non-neoplastic DNA in the data distributions. Their trendlines are also shown.
Mentions: The CN ratios, or fold-differences from normal for each gene, are represented by the ratio, [tumor DNA] / [NB DNA], derived from measurements of the CE peaks. The CN ratio was calculated after peaks of non-amplified genes on CE graphs, representing relative amounts of DNA, were matched with the average of two NB samples analyzed in the same assay run. Peak heights of non-amplified genes in tumor samples and NB were adjusted to approximately the same scale as graphical printouts from CE were produced. Final, finer adjustments were made in spreadsheets by maximizing alignments of trendlines for non-amplified genes. Scatterplots were evaluated to check the reactions of NB DNA with the MLPA probes compared to expected values provided by the manufacturer. Overlays of NB results obtained in GBM1’s assays of FFPE, using P171, P172, and P173 kits, with graphs of normal values provided in the MLPA kits’ literature, demonstrated comparable overall detection of the two populations of normal DNA data points (Figure 1).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University - Shreveport, One University Place, Shreveport, LA 71115, USA.

ABSTRACT

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.

Virtual slides: http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466.

Show MeSH
Related in: MedlinePlus