Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparisons between cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens for all normalized CN ratios. A. Bland-Altman analysis demonstrated that normalized CN ratios ([tumor DNA]/[NB DNA]) obtained from either source of DNA corresponded significantly (within 1.96 SD limits, dotted lines) for oncogene amplifications up to at least 15-fold (log value of 1.18). The logarithmic scale was used on the x-axis to separate ratio values for the majority of the data points as much as possible. Note that a normal CN ratio of 1 is equivalent to a log value of 0. The CN ratios for all genes tested with each kit (multiple probes) in all tumors were included, n = 889. B. The CN ratios derived from the two sources of DNA exhibited strong correlations in each tumor with results from each kit shown separately. Assays using the MLPA kits, P171, P172, and P173, had n’s equal to 42, 42 and 43, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475141&req=5

Figure 5: Comparisons between cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens for all normalized CN ratios. A. Bland-Altman analysis demonstrated that normalized CN ratios ([tumor DNA]/[NB DNA]) obtained from either source of DNA corresponded significantly (within 1.96 SD limits, dotted lines) for oncogene amplifications up to at least 15-fold (log value of 1.18). The logarithmic scale was used on the x-axis to separate ratio values for the majority of the data points as much as possible. Note that a normal CN ratio of 1 is equivalent to a log value of 0. The CN ratios for all genes tested with each kit (multiple probes) in all tumors were included, n = 889. B. The CN ratios derived from the two sources of DNA exhibited strong correlations in each tumor with results from each kit shown separately. Assays using the MLPA kits, P171, P172, and P173, had n’s equal to 42, 42 and 43, respectively.

Mentions: Following NB normalization to generate CN ratios, Bland-Altman analysis (n = 889, derived from 127 data points/tumor x 7) found that data in ultrasonic surgical aspiration assays corresponded to data from FFPE assays for the same tumor within 1.96 SD limits, except for some of the amplifications that exceeded CN ratios of 15 (log value = 1.176). A normal CN ratio of 1 corresponds to a log value of 0 (Figure 5A). Correlation coefficients for comparisons ranged from 0.71 to 0.99 in studies of each tumor according to the kits used. Consistency was high with the exception of LCM2 that showed slightly less correspondence, as seen in Figure 5. The n’s were 42, 42, and 43 for P171, P172, and P173 kits, respectively, in each tumor.


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)

Comparisons between cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens for all normalized CN ratios. A. Bland-Altman analysis demonstrated that normalized CN ratios ([tumor DNA]/[NB DNA]) obtained from either source of DNA corresponded significantly (within 1.96 SD limits, dotted lines) for oncogene amplifications up to at least 15-fold (log value of 1.18). The logarithmic scale was used on the x-axis to separate ratio values for the majority of the data points as much as possible. Note that a normal CN ratio of 1 is equivalent to a log value of 0. The CN ratios for all genes tested with each kit (multiple probes) in all tumors were included, n = 889. B. The CN ratios derived from the two sources of DNA exhibited strong correlations in each tumor with results from each kit shown separately. Assays using the MLPA kits, P171, P172, and P173, had n’s equal to 42, 42 and 43, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparisons between cavitronic ultrasonic surgical aspiration (CUSA) and FFPE specimens for all normalized CN ratios. A. Bland-Altman analysis demonstrated that normalized CN ratios ([tumor DNA]/[NB DNA]) obtained from either source of DNA corresponded significantly (within 1.96 SD limits, dotted lines) for oncogene amplifications up to at least 15-fold (log value of 1.18). The logarithmic scale was used on the x-axis to separate ratio values for the majority of the data points as much as possible. Note that a normal CN ratio of 1 is equivalent to a log value of 0. The CN ratios for all genes tested with each kit (multiple probes) in all tumors were included, n = 889. B. The CN ratios derived from the two sources of DNA exhibited strong correlations in each tumor with results from each kit shown separately. Assays using the MLPA kits, P171, P172, and P173, had n’s equal to 42, 42 and 43, respectively.
Mentions: Following NB normalization to generate CN ratios, Bland-Altman analysis (n = 889, derived from 127 data points/tumor x 7) found that data in ultrasonic surgical aspiration assays corresponded to data from FFPE assays for the same tumor within 1.96 SD limits, except for some of the amplifications that exceeded CN ratios of 15 (log value = 1.176). A normal CN ratio of 1 corresponds to a log value of 0 (Figure 5A). Correlation coefficients for comparisons ranged from 0.71 to 0.99 in studies of each tumor according to the kits used. Consistency was high with the exception of LCM2 that showed slightly less correspondence, as seen in Figure 5. The n’s were 42, 42, and 43 for P171, P172, and P173 kits, respectively, in each tumor.

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