Background: Digital droplet PCR (ddPCR) is a relatively new technique used to detect molecular alterations with unprecedented precision and accuracy. It is particularly useful for detecting point mutations and copy number variation (CNV) in samples with small amounts of target DNA. This proof of principle study was conducted to see if ddPCR technology could be applied to cytology specimens to detect molecular alterations which may influence diagnostic decision making.
Methods: A range of cytological specimens derived from bladder or pancreaticobiliary origin, with varying diagnoses but with an emphasis on abnormality, underwent ddPCR testing. DNA was manually extracted from Thinprep vials, cell blocks or direct fine needle aspiration smears. ddPCR was performed using two somatic point mutations TP53 R248W and TP53 R273H assays for both urine and pancreaticobiliary cytology specimens. Three CNV assays; CDKN2A, E2F3 and YWHAZ were applied to urine samples. SMAD4 and CDKN2A CNVs were applied to the pancreaticobiliary samples.
Results: 16 of 21 urine specimens showed molecular alterations using ddPCR testing. 12 of those 16 cases were associated with malignant outcomes. The pancreaticobiliary specimens showed 14 of 37 specimens with molecular alterations, all of which were associated with carcinoma. We demonstrated an increasing percentage of molecular aberrations associated with increasing severity of cytological results.
Conclusion: Our results have shown that ddPCR can identify both mutations and CNVs in urine and pancreaticobiliary cytology derived samples. Being able to detect these molecular alterations may reduce the number of equivocal results leading to more timely and informed patient management decisions.
Keywords: copy number variation; ddPCR; diagnostic enhancement; pancreaticobiliary cytology; point mutation; urine cytology.
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