Supplementary Data
Comparison of Viral Loads in Cervical Smears with Viral Loads in FFPE Tissues.
Correlation between viral loads in smears andviral loads in FFPE tissues was weak in our series (see Supplementary Figure S1). Discordant results could be due to several reasons. The most probable reason is related to technical issues when processing nucleic acids from FFPE tissues. The most common technical issue isthe nucleic acid degradation or, cross-linking between proteins and nucleic acids caused by formalin fixation[1-3]. This may affect viral load assessment using BSGP5+/6+-PCR/MPGon DNA extracts from FFPE tissues, as viral load was computed by forming the ratio of the relative HPV MFI (%) with the measured β-globin MFI value. Compared to the HPV amplicons with a size of ~150bp, the β-globin amplicons is longer by 38% (208bp). Thus, formalin-induced nucleic acid degradation and fragmentation would affect successful amplification of β-globin stronger than successful amplification of HPV, which is likely to influence the final viral load result from FFPE specimens. An alternative reason is the potential presence of PCR inhibitors in FFPE DNA extracts or, a possibility that viral loads do substantially differ between tissues and smears. Therefore, to this end, BSGP5+/6+-PCR/MPGviral load analysis from FFPE DNA extracts are not considered validated, in opposed to DNA extracts from smears[4] or fresh frozen tissue biopsies [5].
Controls included in the Experimental Design
Each experimental step included “negative controls” to monitorpotential nucleic acid cross-contamination during tissue sectioning, DNA and RNA extractions, (RT)PCR, and hybridization procedures, respectively. The absence of HPV DNA+and/or mRNA+ signals in negative control samples was a confirmation that no cross-contamination with viral nucleic acids occurred. In addition, to monitor assay performance and intra- and inter-plate variability, a sample of HPV16 DNA+/HPV16 mRNA+ cervical cancer was used as a “positive control” on each 96-well plate.
“Negative controls” used were:
a) Tissue-free paraffin block. Ribbons from a tissue-free paraffin blockwere produced during sectioning after each five clinical FFPE specimens. They were usedin DNA and RNA extraction batches, respectively. Each DNA and RNA batch for FFPE specimens included 12 samples (Eppendorf tubes with tissue ribbons) of which 11 containedribbons of clinical specimens, and one tube containedribbons of tissue-free paraffin. DNA and RNA extractions were performed as described [6]. DNA extraction from cervical smears was performedusing a High Pure PCR Product Purification Kit according to the manufacturer’s recommendations (Roche Applied Science), as described [7].
b) Water and MMIX controls. Water controls and (RT)PCR MasterMix (MMIX) controls (MMIX without a DNA or an RNA sample added) were used on 96-well plates (each 7th and each 8th well, respectively) during PCR and RT-PCR setup. A total of six water and six MMIX controls were present on each 96-well plate. These negative controls were used to control for potential cross-contamination during PCR and RT-PCR analysis, respectively.
c) Hybridization buffer. The last well on the 96-well plate in Luminex hybridization included hybridization buffer by omitting the last water control from the PCR plate. This negative control was used to control for potential cross-contamination during hybridization of (RT)PCR products on a Luminex machine (Luminex Corp., Austin, TX).
Suppl FigS1. Correlation of HPV16 loads in cervical smears with HPV16 loads in FFPE tissues.Viral load cut-offs are indicated by dotted lines. Correlation between HPV16 loads in smears and HPV16 loads in FFPE tissues was calculated using Pearson (P) and Spearman (S) correlation coefficients, respectively. Only the Spearman correlation coefficients were significant * (p<0.0001).
Supplementary Table S1. An overview of HPV genotyping, viral load, and HPV mRNA data in 74 smear/tissue pairs.
Supplementary Table S2. List ofunpublished MPG probe sequences used in Luminex hybridization.
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