Jane Shen-Gunther

HPV molecular assays: Defining analytical and clinical performance characteristics for cervical cytology specimens

OBJECTIVE To compare the analytical and clinical performance characteristics of 3 different primer sets targeting the human papillomavirus (HPV) L1 gene for detection and genotyping of HPV. METHODS Liquid-based cytology was obtained prospectively from 90 colposcopy clinic patients. After automated extraction, cellular DNA was subjected to SYBR Green quantitative polymerase chain reaction (qPCR) using GP5+/6+ primers and conventional PCR with MY09/11 and FAP59/64 primers. The resulting SYBR Green counts and melting temperatures (T(m)) were used for quantification of HPV genomes and discrimination between presence and absence of amplicons. qPCR and PCR products were resolved by gel electrophoresis. HPV+amplicons were sequenced directly and BLAST® aligned for genotype identification. RESULTS Of the 90 samples, 57 (63%) were qPCR+with a range of HPV viral load detected from 191 to 3.4 million genomes (~5 Log(10)). Only HPV-positives exhibited characteristic T(m) (75-80°C). The dynamic range of detection was similar for MY and FAP. Clinical net sensitivity was highest with simultaneous testing using all primer sets (93%) instead of individual primer pairs (GP (67%), MY (62%), FAP (49%)). Of the 27 HPV genotypes identified among 64 sequenced samples, the 3 most prevalent were HPV-16 (20%), -53 (9%), -31 (6%). The 4th rank included HPV-6, -33, -58, -66 (4.7% each). CONCLUSION The performance characteristics of 3 leading PCR-based HPV assays revealed qPCR to be sensitive and specific for HPV detection and quantification. Parallel PCR testing using the 3 primers and direct sequencing offered the greatest clinical sensitivity and breadth of detection for known HPV types.

Laparoscopic paraaortic lymphadenectomy using laparosonic coagulating shears

With marked innovations in endosurgical instrumentation, operative laparoscopy to include lymphadenectomy has become feasible and has a valuable role in the management of gynecologic malignancy. We used laparosonic coagulating shears (LCS) for laparoscopic paraaortic lymphadenectomy in two women with cervical carcinoma. Operating times for the laparoscopic portion were 55 and 65 minutes and blood loss was 20 and 30 ml, respectively. No surgical complications were encountered. Lymphatic tissues were evaluated histologically and no thermal artifacts were identified. The major advantage of the ultrasonically activated scalpel of the LCS is the ability to cut and coagulate tissues simultaneously without electrical current. The LCS may afford the surgeon a greater margin of safety than unipolar electrocoagulation scissors by eliminating potential thermal and electrical injury to vital structures. Ultrasonic-activated technology deserves extended clinical investigation in laparoscopic lymphadenectomy to substantiate our preliminary findings, as well as to explore its potential in gynecologic oncology.