Rapid detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors.

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.

EGFR expression as an ancillary tool for diagnosing lung cancer in cytology specimens.

Lung cancer evolves in a multistep process, and its early detection portends a better prognosis. Bronchial washings/brushings and fine-needle aspirations are often used as early screening and cytological diagnosis of lung cancer. In some cases, it is difficult to differentiate morphologically malignant from reactive cells. Epidermal growth factor receptor (EGFR) is a transmembrane receptor overexpressed in high percentage lung cancers, and contributes to tumor growth. Assessing EGFR expression levels by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) may provide critical information of tumor marker abnormalities, assist in the cytological diagnosis, and stratify patients for EGFR inhibitor therapy. Fifty patients with bronchial washings/brushings or fine-needle aspiration specimens, and corresponding histologically confirmed lung biopsies, were studied for EGFR expression with FISH and IHC. Copy numbers of the EGFR gene locus were analyzed with those of chromosome 7 by FISH. EGFR and FISH results were compared to our FISH data with combined EGFR, c-myc, 5p15.2, and chromosome 6 probes in selected cases. Cell blocks, if available, and tissue biopsy sections were used for EGFR IHC. The intensity of IHC was scored, and quantified. Only balanced aneuploidy of EGFR was identified by FISH. Gene amplification was not detected. The chromosomal abnormalities of EGFR were often accompanied by other chromosomal aneuploidies demonstrated in c-myc (8q24), 5p15.2 or 6p, indicating a general genomic instability. About half of the specimens with confirmed malignancy showed EGFR balanced aneuploidy by FISH, and gene copy number was not coupled with protein expression in many cases. The benign or reactive cytology specimens confirmed by biopsies had high specificity by FISH (96%) and IHC (88%). FISH and IHC analysis of EGFR, possibly along with other tumor markers, may be a useful ancillary tool to classify difficult cytology cases and inform clinicians arranging targeted chemotherapy.