We recently showed that the presence of HPV-mRNA in sentinel lymph nodes (SLN) of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival (1). However, for clinical implementation and for the definition of prognostic threshold levels it is essential to expand this qualitative RT-nested-PCR analysis using a quantitative assay. A disadvantage of qPCR is the high standard deviation observed with low template numbers. We aim to circumvent this problem by using digital PCR (dPCR). This strategy allows the analyses of larger volumes by reducing the unspecific background per template molecule due to reaction partitioning.
Serial dilutions of 5 ng – 5 pg RNA (corresponding to 500 – 0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 SLN with varying HPV transcript level. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analysed by qPCR and dPCR. Digital PCR was run in the RainDrop® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template.
Both methods showed comparable sensitivity: In the serial SiHa dilutions, qPCR enabled the reliable detection of 0.5 pg template (0.02-0.05 cells) per reaction. Consistently, in dPCR we achieved a detection of 0.55 pg template per reaction. When analysing the sample specific reproducibility, both methods differed considerably. In qPCR, we detected the 50 pg dilution step reliably with variation coefficients between 0.21 and 0.5 throughout the serial dilution series. Using the same samples, dPCR enabled the detection of the 5 pg dilution step and variation coefficients between 0.04 and 0.5. Generally, we saw with dPCR a substantial reduction of subsampling errors (reduced false negatives). However, in dPCR, the detection of single copies is challenged by the presence of marginal unspecific background signals (1 copy per 275,000 cells).
Compared to real-time PCR, dPCR shows a higher reliably of results while enabling equal sensitivity. The simplicity of the dPCR workflow, with no requirement for a standard curve, and the generation of absolute molecule counts directly from the digital partitions is of particular value for inter and intra- laboratory clinical comparison. These points indicate a high potential of dPCR for the identification and clinical evaluation of occult tumour cells in histologically tumour-free lymph nodes.
M. Dürst et al., Prognostic value of HPV-mRNA in sentinel lymph nodes of cervical cancer patients with pN0-status. Oncotarget 6, 23015 (Sep 8, 2015)