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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

FISH results for EGFR are shown. A. Relative frequencies of EGFR/CEP 7 signals (number of cells in intervals/total cells counted multiplied by 100) in FFPE sections of glioblastomas are shown on the y-axis in distributions of signal ratios. Data is skewed to the right due to cells that harboured very high numbers of EGFR signals in each tumor, especially GBM3 and GBM4. Intervals of values for signal ratios along the x-axis are equal except for the last two which are arbitrarily larger (in parentheses). B. Representative tumor cells show EGFR (orange/red) and CEP 7 (green) signals. Nuclei were stained blue with DAPI. All tumors had FFPE sections analyzed and a few also had CUSA specimens analyzed successfully. Others had interference from proteinaceous debris. High power photographs were printed in large format and then digitized.
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Figure 3: FISH results for EGFR are shown. A. Relative frequencies of EGFR/CEP 7 signals (number of cells in intervals/total cells counted multiplied by 100) in FFPE sections of glioblastomas are shown on the y-axis in distributions of signal ratios. Data is skewed to the right due to cells that harboured very high numbers of EGFR signals in each tumor, especially GBM3 and GBM4. Intervals of values for signal ratios along the x-axis are equal except for the last two which are arbitrarily larger (in parentheses). B. Representative tumor cells show EGFR (orange/red) and CEP 7 (green) signals. Nuclei were stained blue with DAPI. All tumors had FFPE sections analyzed and a few also had CUSA specimens analyzed successfully. Others had interference from proteinaceous debris. High power photographs were printed in large format and then digitized.

Mentions: Analysis of EGFR with FISH was used to correlate with MLPA assay results. Signals were counted in the FFPE sections. The CN for EGFR was increased in the four glioblastomas with ratios of EGFR to CEP 7 increased to values of more than 20 in at least some cells of each glioblastoma. In the metastatic tumors averages of CNs for EGFR were 2.5 to 2.8 per cell with EGFR to CEP 7 ratios remaining below 2.0. Distributions of the EGFR/CEP 7 ratios are shown in Figure 3A and illustrate “tailing off” in the data towards high-level amplifications in individual cells. Representative cells with amplified EGFR from the glioblastomas and from one of the metastases lacking amplification are shown in Figure 3B. The EGFR/CEP 7 ratios are listed for all tumors in Table 3. The CN of CEP 7 varied and was increased (averages of 2.7 to 3.8) in glioblastomas. The CN of CEP 7 varied in LCM1 and LCM2 with averages of 2.5 and 2.6, respectively.


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)

FISH results for EGFR are shown. A. Relative frequencies of EGFR/CEP 7 signals (number of cells in intervals/total cells counted multiplied by 100) in FFPE sections of glioblastomas are shown on the y-axis in distributions of signal ratios. Data is skewed to the right due to cells that harboured very high numbers of EGFR signals in each tumor, especially GBM3 and GBM4. Intervals of values for signal ratios along the x-axis are equal except for the last two which are arbitrarily larger (in parentheses). B. Representative tumor cells show EGFR (orange/red) and CEP 7 (green) signals. Nuclei were stained blue with DAPI. All tumors had FFPE sections analyzed and a few also had CUSA specimens analyzed successfully. Others had interference from proteinaceous debris. High power photographs were printed in large format and then digitized.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: FISH results for EGFR are shown. A. Relative frequencies of EGFR/CEP 7 signals (number of cells in intervals/total cells counted multiplied by 100) in FFPE sections of glioblastomas are shown on the y-axis in distributions of signal ratios. Data is skewed to the right due to cells that harboured very high numbers of EGFR signals in each tumor, especially GBM3 and GBM4. Intervals of values for signal ratios along the x-axis are equal except for the last two which are arbitrarily larger (in parentheses). B. Representative tumor cells show EGFR (orange/red) and CEP 7 (green) signals. Nuclei were stained blue with DAPI. All tumors had FFPE sections analyzed and a few also had CUSA specimens analyzed successfully. Others had interference from proteinaceous debris. High power photographs were printed in large format and then digitized.
Mentions: Analysis of EGFR with FISH was used to correlate with MLPA assay results. Signals were counted in the FFPE sections. The CN for EGFR was increased in the four glioblastomas with ratios of EGFR to CEP 7 increased to values of more than 20 in at least some cells of each glioblastoma. In the metastatic tumors averages of CNs for EGFR were 2.5 to 2.8 per cell with EGFR to CEP 7 ratios remaining below 2.0. Distributions of the EGFR/CEP 7 ratios are shown in Figure 3A and illustrate “tailing off” in the data towards high-level amplifications in individual cells. Representative cells with amplified EGFR from the glioblastomas and from one of the metastases lacking amplification are shown in Figure 3B. The EGFR/CEP 7 ratios are listed for all tumors in Table 3. The CN of CEP 7 varied and was increased (averages of 2.7 to 3.8) in glioblastomas. The CN of CEP 7 varied in LCM1 and LCM2 with averages of 2.5 and 2.6, respectively.

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