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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

Multiple gene amplifications detected with MLPA in GBM1-4 shown as line graphs of peak heights. MLPA results for genes in tumors were compared with non-neoplastic (or normal) brain DNA. Heights of peaks reflected amounts of genes, or their CN. Mid to high-level gene amplifications were obvious by visual inspection of peak heights (mm) for at least one gene in each glioblastoma. These are labelled and also asterisks indicate these genes along the x-axes. Line graphs connecting the peak heights are colored according to the legends. Tumor and normal brain (NB) were tested concurrently.
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Figure 4: Multiple gene amplifications detected with MLPA in GBM1-4 shown as line graphs of peak heights. MLPA results for genes in tumors were compared with non-neoplastic (or normal) brain DNA. Heights of peaks reflected amounts of genes, or their CN. Mid to high-level gene amplifications were obvious by visual inspection of peak heights (mm) for at least one gene in each glioblastoma. These are labelled and also asterisks indicate these genes along the x-axes. Line graphs connecting the peak heights are colored according to the legends. Tumor and normal brain (NB) were tested concurrently.

Mentions: Productive CE runs were obtained for all tumors. Prior to normalization of tumor CN, the output of CE data from all four glioblastomas and one of two metastases revealed obvious amplifications in at least one gene for each tumor. Measurements of the peaks for each gene were connected to create line graphs. The obvious peaks representing high-level and some mid-level gene amplifications in the glioblastomas are shown in Figure 4. Genes are listed along the x-axes according to amplicon size within each kit (P171, 172 and 173). Easily recognized amplifications occurred for EGFR, CDK4, MDM2, CYP27B1, and PDGFRA in one or more GBMs, and also for CCNE1 in one of the brain metastases (not shown) using DNA from ultrasonic surgical aspirations. Comparable amplifications for the same genes were also detected in corresponding FFPE specimens (not shown). Results for non-amplified genes in the tumors and in normal brain (assayed concurrently) constitute the baselines of graphs in Figure 4. The EGFR data obtained with MLPA is included in Table 3 along with FISH data.


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)

Multiple gene amplifications detected with MLPA in GBM1-4 shown as line graphs of peak heights. MLPA results for genes in tumors were compared with non-neoplastic (or normal) brain DNA. Heights of peaks reflected amounts of genes, or their CN. Mid to high-level gene amplifications were obvious by visual inspection of peak heights (mm) for at least one gene in each glioblastoma. These are labelled and also asterisks indicate these genes along the x-axes. Line graphs connecting the peak heights are colored according to the legends. Tumor and normal brain (NB) were tested concurrently.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Multiple gene amplifications detected with MLPA in GBM1-4 shown as line graphs of peak heights. MLPA results for genes in tumors were compared with non-neoplastic (or normal) brain DNA. Heights of peaks reflected amounts of genes, or their CN. Mid to high-level gene amplifications were obvious by visual inspection of peak heights (mm) for at least one gene in each glioblastoma. These are labelled and also asterisks indicate these genes along the x-axes. Line graphs connecting the peak heights are colored according to the legends. Tumor and normal brain (NB) were tested concurrently.
Mentions: Productive CE runs were obtained for all tumors. Prior to normalization of tumor CN, the output of CE data from all four glioblastomas and one of two metastases revealed obvious amplifications in at least one gene for each tumor. Measurements of the peaks for each gene were connected to create line graphs. The obvious peaks representing high-level and some mid-level gene amplifications in the glioblastomas are shown in Figure 4. Genes are listed along the x-axes according to amplicon size within each kit (P171, 172 and 173). Easily recognized amplifications occurred for EGFR, CDK4, MDM2, CYP27B1, and PDGFRA in one or more GBMs, and also for CCNE1 in one of the brain metastases (not shown) using DNA from ultrasonic surgical aspirations. Comparable amplifications for the same genes were also detected in corresponding FFPE specimens (not shown). Results for non-amplified genes in the tumors and in normal brain (assayed concurrently) constitute the baselines of graphs in Figure 4. The EGFR data obtained with MLPA is included in Table 3 along with FISH data.

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