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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

Individual CN ratios of selected genes in CUSA and FFPE specimens from all four glioblastomas. A. The CN ratio values for genes with high-level amplifications (CN ratios ≥ 6.0) are shown for individual glioblastomas as indicated along the x-axis. Each gene, except for MDM2, was tested with two probe sets. B. In contrast, the CN ratio values are shown for individual genes that were unaltered within all four glioblastomas. These CN ratios remained in the normal range for all replicates and probe sets in all assays on both types of glioblastoma specimens. Note the marked difference between the ranges of y-axes for A and B. The CN ratios were calculated according to [tumor DNA] / [NB DNA] with averages in 95 % confidence intervals (bars) shown here.
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Figure 6: Individual CN ratios of selected genes in CUSA and FFPE specimens from all four glioblastomas. A. The CN ratio values for genes with high-level amplifications (CN ratios ≥ 6.0) are shown for individual glioblastomas as indicated along the x-axis. Each gene, except for MDM2, was tested with two probe sets. B. In contrast, the CN ratio values are shown for individual genes that were unaltered within all four glioblastomas. These CN ratios remained in the normal range for all replicates and probe sets in all assays on both types of glioblastoma specimens. Note the marked difference between the ranges of y-axes for A and B. The CN ratios were calculated according to [tumor DNA] / [NB DNA] with averages in 95 % confidence intervals (bars) shown here.

Mentions: Normalized CN ratios for specific genes in glioblastomas, including those with high-level amplifications (CN ratio ≥ 6.0) and those with no alterations (all CN ratios ≥0.75 and ≤1.5), are shown in Figure 6 (A & B, respectively). Both the ultrasonic surgical aspiration and corresponding FFPE results from the same tumors are shown. The only high-level gene amplification in the brain metastases was EVI1 (7.02 CN ratio) in LCM2 FFPE but it was not amplified in the corresponding aspiration specimen. The metastases, LCM1 and LCM2, had 2 and 6 mid-level gene amplifications, respectively. The ependymoma (EP1) only had 1 mid-level gene amplification. Among the 10 genes with no alterations in any of the GBMs, including replicates of all probe sets for a specific gene, there were 9 genes that were also unaltered in EP1, and 9 and 6 genes that were unaltered in LCM1 and LCM2, respectively. Totals of all deviations (<0.75 or ≥2.0 in CN ratios) from normal values in ultrasonic aspiration and FFPE specimens are shown in Table 4. Alterations in CN were more frequent in FFPE than in ultrasonic aspiration specimens, 85% and 49% of total changes, respectively. However, FFPE did not completely encompass all of the changes. Restriction of some genomic gains and losses to only one type of specimen from the same tumor is consistent with tumor heterogeneity.


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)

Individual CN ratios of selected genes in CUSA and FFPE specimens from all four glioblastomas. A. The CN ratio values for genes with high-level amplifications (CN ratios ≥ 6.0) are shown for individual glioblastomas as indicated along the x-axis. Each gene, except for MDM2, was tested with two probe sets. B. In contrast, the CN ratio values are shown for individual genes that were unaltered within all four glioblastomas. These CN ratios remained in the normal range for all replicates and probe sets in all assays on both types of glioblastoma specimens. Note the marked difference between the ranges of y-axes for A and B. The CN ratios were calculated according to [tumor DNA] / [NB DNA] with averages in 95 % confidence intervals (bars) shown here.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Individual CN ratios of selected genes in CUSA and FFPE specimens from all four glioblastomas. A. The CN ratio values for genes with high-level amplifications (CN ratios ≥ 6.0) are shown for individual glioblastomas as indicated along the x-axis. Each gene, except for MDM2, was tested with two probe sets. B. In contrast, the CN ratio values are shown for individual genes that were unaltered within all four glioblastomas. These CN ratios remained in the normal range for all replicates and probe sets in all assays on both types of glioblastoma specimens. Note the marked difference between the ranges of y-axes for A and B. The CN ratios were calculated according to [tumor DNA] / [NB DNA] with averages in 95 % confidence intervals (bars) shown here.
Mentions: Normalized CN ratios for specific genes in glioblastomas, including those with high-level amplifications (CN ratio ≥ 6.0) and those with no alterations (all CN ratios ≥0.75 and ≤1.5), are shown in Figure 6 (A & B, respectively). Both the ultrasonic surgical aspiration and corresponding FFPE results from the same tumors are shown. The only high-level gene amplification in the brain metastases was EVI1 (7.02 CN ratio) in LCM2 FFPE but it was not amplified in the corresponding aspiration specimen. The metastases, LCM1 and LCM2, had 2 and 6 mid-level gene amplifications, respectively. The ependymoma (EP1) only had 1 mid-level gene amplification. Among the 10 genes with no alterations in any of the GBMs, including replicates of all probe sets for a specific gene, there were 9 genes that were also unaltered in EP1, and 9 and 6 genes that were unaltered in LCM1 and LCM2, respectively. Totals of all deviations (<0.75 or ≥2.0 in CN ratios) from normal values in ultrasonic aspiration and FFPE specimens are shown in Table 4. Alterations in CN were more frequent in FFPE than in ultrasonic aspiration specimens, 85% and 49% of total changes, respectively. However, FFPE did not completely encompass all of the changes. Restriction of some genomic gains and losses to only one type of specimen from the same tumor is consistent with tumor heterogeneity.

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