Assessing neoplastic lesions for malignancy is typically done by cytologic evaluation of cells obtained from a biopsy or captured by other methods such as mechanical exfoliation. This type of examination is used to detect cell atypia, or changes in nuclear and cytoplasmic morphology, indicative of malignancy. While cytology is highly specific, the sensitivity is only moderate, resulting in decreased detection accuracy. This risk of producing a false-negative result necessitates the use of adjunctive testing to improve detection sensitivity and overall accuracy of results.
“While cytology is highly specific, the sensitivity is only moderate, resulting in decreased detection accuracy.”
Fluorescence in situ hybridization (FISH) is a technique that uses fluorescent DNA probes to detect copy number changes of specific genes or large-scale genomic rearrangements. FISH is commonly used alongside cytology to increase the sensitivity of detecting malignancies in cytologic specimens and to provide gene-level information on the cells in the lesion. However, this increased sensitivity comes at the expense of decreased specificity, and therefore adjunctive FISH and cytologic testing is about as accurate as cytologic testing alone. For example, J.C. Dudley and colleagues recently showed that the addition of FISH to cytology increased detection sensitivity of pancreaticobiliary malignancy from 67% to 76%, although the overall accuracy remained unchanged at 85%. Thus, alternative types of adjunctive assays must be considered to improve the accuracy of malignancy detection in cytologic specimens.
“…adjunctive FISH and cytologic testing is about as accurate as cytologic testing alone.”
As a powerful alternative to FISH, next-generation sequencing (NGS) can simultaneously detect copy number changes in large panels of specific genes, thereby overcoming the limited multiplexing capability of FISH. Sequence information provided by NGS can also be used to identify mutations and genomic rearrangements known to be associated with malignancy.
As conventional NGS methods produce enormous amounts of sequencing data that can be difficult to analyze, recently developed methods to generate target-enriched DNA libraries for NGS may prove to be particularly useful for improved variant detection. Anchored multiplex PCR (AMP) is one such enrichment strategy that can generate targeted gene panels for NGS from cytologic specimens. AMP is particularly versatile in that it produces target-enriched libraries that simultaneously provide accurate information on gene-level copy numbers, genomic rearrangements, and mutations associated with malignancy. Replacing FISH with AMP-based NGS should greatly enhance the accuracy of detecting malignancies in cytologic specimens.
“…the overall accuracy of results improved from 85% to 91% when AMP-based NGS was added to cytology.”
For pancreaticobiliary malignancy, Dudley and colleagues demonstrated an increase in detection sensitivity from 67% to 85% when AMP-based NGS was combined with cytology, a markedly more sensitive adjunctive assay compared to FISH and cytology. Importantly, the overall accuracy of results improved from 85% to 91% when AMP-based NGS was added to cytology. Further addition of FISH to AMP-based NGS and cytology did not further enhance sensitivity and even resulted in decreased accuracy due to the low specificity inherent to FISH. These recent findings demonstrate that AMP-based NGS provides greater sensitivity and overall detection accuracy than FISH.J.C. Dudley et al., Next-Generation Sequencing and Fluorescence in Situ Hybridization Have Comparable Performance Characteristics in the Analysis of Pancreaticobiliary Brushings for Malignancy. J Mol Diagn. 18(1):124-30 (2015).
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