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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

Morphology of the cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens from several tumors. Specimens for GBM1, GBM2, GBM4, and LCM1 are shown in the first, second, third, and fourth rows, respectively. The original magnification for the first column (panels A, D, G, and J) was at 100X and the others were at 400X. Comparable morphology of the ultrasonic aspiration and FFPE specimens from each tumor is noted in the high power views shown in the last 2 columns. Cellular pleomorphism and mitoses were prominent. Characteristic regions of necrosis and vascular proliferation were also found in the FFPE sections of the GBMs (not shown). Several clinicopathologic features are listed in Table 2. The portions of the CUSA specimens shown had been processed in the same manner as the FFPE specimens. Hematoxylin and eosin stain. Magnifications bars represent 100 micrometers.
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Figure 2: Morphology of the cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens from several tumors. Specimens for GBM1, GBM2, GBM4, and LCM1 are shown in the first, second, third, and fourth rows, respectively. The original magnification for the first column (panels A, D, G, and J) was at 100X and the others were at 400X. Comparable morphology of the ultrasonic aspiration and FFPE specimens from each tumor is noted in the high power views shown in the last 2 columns. Cellular pleomorphism and mitoses were prominent. Characteristic regions of necrosis and vascular proliferation were also found in the FFPE sections of the GBMs (not shown). Several clinicopathologic features are listed in Table 2. The portions of the CUSA specimens shown had been processed in the same manner as the FFPE specimens. Hematoxylin and eosin stain. Magnifications bars represent 100 micrometers.

Mentions: Features of the tumors were typical for their diagnostic classification as glioblastomas, metastatic tumors, and an ependymoma. Photomicrographs of the ultrasonic surgical aspirations (100X and 400X magnifications) and FFPE sections (400X magnification) illustrates the similarity of tumor cells in the two types of specimens (Figure 2). There were too few cases to evaluate whether characteristic morphologic profiles for amplified gene(s) in these tumors were present. Mitotic figures among the glioblastomas were most numerous in GBM1 and mitoses among all tumors were most numerous in LCM2 (Table 2).


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)

Morphology of the cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens from several tumors. Specimens for GBM1, GBM2, GBM4, and LCM1 are shown in the first, second, third, and fourth rows, respectively. The original magnification for the first column (panels A, D, G, and J) was at 100X and the others were at 400X. Comparable morphology of the ultrasonic aspiration and FFPE specimens from each tumor is noted in the high power views shown in the last 2 columns. Cellular pleomorphism and mitoses were prominent. Characteristic regions of necrosis and vascular proliferation were also found in the FFPE sections of the GBMs (not shown). Several clinicopathologic features are listed in Table 2. The portions of the CUSA specimens shown had been processed in the same manner as the FFPE specimens. Hematoxylin and eosin stain. Magnifications bars represent 100 micrometers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Morphology of the cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens from several tumors. Specimens for GBM1, GBM2, GBM4, and LCM1 are shown in the first, second, third, and fourth rows, respectively. The original magnification for the first column (panels A, D, G, and J) was at 100X and the others were at 400X. Comparable morphology of the ultrasonic aspiration and FFPE specimens from each tumor is noted in the high power views shown in the last 2 columns. Cellular pleomorphism and mitoses were prominent. Characteristic regions of necrosis and vascular proliferation were also found in the FFPE sections of the GBMs (not shown). Several clinicopathologic features are listed in Table 2. The portions of the CUSA specimens shown had been processed in the same manner as the FFPE specimens. Hematoxylin and eosin stain. Magnifications bars represent 100 micrometers.
Mentions: Features of the tumors were typical for their diagnostic classification as glioblastomas, metastatic tumors, and an ependymoma. Photomicrographs of the ultrasonic surgical aspirations (100X and 400X magnifications) and FFPE sections (400X magnification) illustrates the similarity of tumor cells in the two types of specimens (Figure 2). There were too few cases to evaluate whether characteristic morphologic profiles for amplified gene(s) in these tumors were present. Mitotic figures among the glioblastomas were most numerous in GBM1 and mitoses among all tumors were most numerous in LCM2 (Table 2).

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