In this study the factors that enable consistent, sensitive but background free in situ hybridization for DNA sequences using biotinyl-tyramide reagent have been investigated. Different protocols were compared and assessed according to two criteria. Firstly whether the protocol was sufficiently sensitive to routinely detect single copy (~8 kb) HPV-16 in SiHa cells/chromosome spreads, and, secondly, whether the protocol routinely gave a clean background on archival FFPE tissues. According to these criteria a protocol has been optimized for routine use (Table 1) that allows detection of the 1–2 copies of HPV 16 DNA integrated into the genome of SiHa cells utilizing a chromogenic end-point. This protocol has been successfully applied to cell-line models (Figures 1A,1B,1C) and to examine HPV infections amongst archival FFPE cervical carcinoma (Figure 1D) and CIN lesions (Figures 2A,2B,2C). Integration of HPV16 in metaphase chromosome spreads prepared from the SiHa cell-line has also been demonstrated using a fluorescent reporter molecule (Figures 1E and 1F).
The most important factors for high sensitivity were the use a sodium citrate step followed by the use of pepsin/HCl for tissue unmasking, the use of probes at a concentration of > 0.5 ng/μl hybridization mix, and, the concentration of primary streptavidin-peroxidase. That HPV DNA integrated into metaphase chromosomes was detectable by fluorescence but not chromogenic stains indicates an additional order of sensitivity is possible by combination of biotinyl-tyramide with reagents such as FITC.
A sodium citrate tissue-unmasking step was also of key importance for the elimination of background staining. AEC was the chromogen of choice for clean background. Certain probes were more "dirty" than others. Composition and pH of hybridization mix and post-hybridization wash buffer pH were important in these instances.
The optimized protocol was applied to archival FFPE cervical carcinomas and CIN lesions for the detection of HPV DNA. "Punctate" signals were detected amongst carcinomas and high-grade CIN lesions but were rare amongst (non-conylomatous) CIN I lesions, which were characterized by "diffuse" signals occupying entire nuclei and detectable in the mid/superficial layers. These observations are consistent with earlier studies that a punctate signal is representative of integration and a diffuse signal of episomal HPV, and with the theory that integration is an important factor in lesion progression [2, 12]. The data suggest the optimized method may be useful in screening CIN lesions for integration. However, further studies are required to confirm a punctate signal is only representative of integration. The demonstration of 1–2 copies of integrated HPV DNA detected as punctate signal begs the question as to why single/low-copy episomal HPV is not also detectable as punctate signal. Initial infection with HPV is understood to occur in the basal layer of the epithelium, and productive replication is tied to cell differentiation/maturation (Figure 2A.) . HPV was not detected by the assay in basal/parabasal cells of (non-condylomatous) CIN I lesions (> 30 examined; unless HPV-16 positive ) suggesting low-copy number episomal HPV was not detectable and indicating a limit to the sensitivity of the assay.
A possible explanation maybe that that ~8 kilo-base single/low-copy episomal HPV sequences may be subject to relatively more degradation and loss from tissues during routine processing than HPV DNA that is physically integrated into the human chromosome (~50 – 250 mega-base pairs). Excessive degradation may affect retention of DNA sequences during in situ hybridization assay as well as hybridization efficiency. Additionally/alternatively, the sensitivity of the assay on archival tissues may be less than that on the SiHa cell-line model despite attempting equivalent procedures through formalin-fixation and routine paraffin embedding. If this is the case, then punctate signal detected in carcinomas and high-grade CIN lesions may only represent integration of multiple copies of HPV at sites within the genome, and low-copy episomal (or integrated) DNA may remain sub-threshold on archival FFPE samples. Episomal HPV can also occur in the form of concatamers comprising multiple copies of the HPV genome compounded into 'super-molecules'. It can be speculated that these forms could also give rise to punctate signals.
Centromeric probe signal was readily demonstrable using biotinyl-tyramide based in situ hybridization (Figure 2D). Currently numerical chromosome count is generally determined by hybridization with directly labeled fluorescent probes. This has the advantages of allowing simultaneous assay of two or more differently labeled probes and detection directly after post-hybridization washes. The disadvantage is the requirement for specialized microscopy, visualization of tissue specimens through multiple filters, multiple image capture, and the relatively short half-life during which signal is visible. Biotinyl-tyramide assay has the disadvantage of requiring several incubations to detect biotin label. It has the advantages of allowing hematoxylin counterstaining that allows easier determination of cell/tissue morphology, visualization by bright field microscopy, and signal that is available for counting more than one year after test application (slides have been kept in the dark). Unequivocal signal was also demonstrated (to the authors' knowledge for first time by a tyramide based assay) of the 1–2 copies of HPV16 integrated at chromosome 13q21-31 (Figure 1E and 1F) on metaphase spreads prepared from SiHa cells.
There are many variables associated with tyramide-ISH. In this study a number of these have been systematically examined and a sensitive background-free assay has been demonstrated. In this study biotin-labeled probes and biotinylated-tyramide were utilized. Background may also be reduced by using digoxigenin-labeled probe and anti-digoxigenin antibody conjugated with horseradish peroxidase before the amplification step of biotinyl-tyramide (Dr. Chao-Cheng Huang, personal communication). Possibly oligonucleotide probes may also give less background than full-length sequences. Non-biotin-based-tyramide reagents, including fluorescent reagents are available from PerkinElmer Life Sciences and from DAKO. These reagents (used in conjunction with non-biotin-labeled probes) may be particularly beneficial for applications on tissues where endogenous biotin is problematic. An additional level of sensitivity may be possible on tissues using fluorescene given the finding that HPV integrated on chromosome spreads could be demonstrated with FITC but not AEC or DAB.