Usefulness of the human papillomavirus DNA chip test as a complementary method for cervical cytology

Pap smear

Using a cervical brush, cells were collected from the transformation zone of cervix, and they were fixed in a PreservCyt solution bottle. Then, slides were prepared using the ThinPrep 2000 system. The prepared slides were smeared in 20 mm diameter circles before Papanicolaou staining was conducted. The 2014 Bethesda System was used for the cytologic reading.

Cervical biopsy

A pathologist received the biopsy specimen and cut it into appropriately sized pieces while it was still fresh. Then, the specimen was fixed using a 10% formalin solution. Hematoxylin and eosin staining is representative methods of preparing tissues specimens.

HPV DNA chip test

Using a sterilized brush for cell collection, the exfoliated cells were collected from the cervical membrane, fixed in a vessel that contained a buffer solution, and washed twice using a 10-time diluted washer solution that was included in the MyGene HPV DNA chip. After the supernatant was removed, 200 µl of a buffer solution for DNA extraction was added to the cells, their reaction was allowed for more than 3 h in a 50°C water bath and the solution was boiled at 100°C in a heating block for 20 min and then centrifuged at 14,000 rpm for 10 min to allow the use of the supernatant as a DNA.

Polymerase chain reaction (PCR) is a nested PCR. Using MY09/MY11 primer (5’-GCM CAG GGW CAT AAY AAT GG-3’ 5’-CGT CCM ARR GGA WAC TGA TC-3’), the L1 region that was an HPV target DNA was amplified, and then, the L1 region was amplified one more time using GP5+/ GP6+ primer (5’-TTT GTT ACT GTG GTA GAT ACT AC-3’, 5’Cy5-GAA AAA TAA ACT GTA AAT CAT ATT C-3’) that was attached with fluorescent materials. As a DNA control factor, β-globin primer (5’-GGT TGG CCA ATC TAC TCC CAG G-3’, 5’-TGG TCT CCT TAA ACC TGT CTT G-3’) was used for the first PCR amplification. The 25 µl DNA recombinant composition for the first amplification of each specimen included: 7.0 µl each of Premix I and Premix III, which were included in 12.7 µl of the third distilled water and the kit; 0.3 µl of Taq polymerase; and 5.0 µl of DNA. The reaction was performed at 50°C for 3 min for one cycle, at 95°C for 30 s, at 55°C for 30 s, at 72°C for 30 s for 35 cycles, and at 72°C for 3 min for one cycle, and then finished at 4°C.

The 25 µl DNA recombinant composition for the second amplification of each specimen included: 16.5 µl of the third distilled water; 6.5 µl of Premix II, which was included in the kit; 0.3 µl of Taq polymerase; and 2.0 µl of the first DNA. The reaction was performed at 95°C for 5 min for one cycle, at 95°C for 30 s, at 50°C for 40 s, at 72°C for 30 s for 20 cycles, and at 72°C for 3 min for one cycle, and then finished at 4°C.

After the DNA amplifications were completed, 10 µl of DNA was denatured at 95°C for 5 min; mixed with the 30 µl buffer solution that was heated at 43°C; and divided into the holes of the high integration HPV DNA chip microarray to which were attached the oligonucleotide probes that were specific to the 24 subtypes of the HPV genotype in appropriate concentrations. Then, a hybridization reaction was performed in a dark, moist box at 43°C for 1 h. The remaining amplified DNA was mixed with 2.5% agarose gel and 5 µl of bromophenol blue and divided into the holes with a TBE buffer (×0.5), after which a 100 bp DNA-sized marker was divided before electrophoresis was conducted at 150 V for 15 min. After the electrophoresis was completed, the DNA of the agarose was stained with 0.5 µg/ml of ethidium bromide, and the DNA band was observed under UV. In the HPV-positive case at the first PCR reaction, the DNA band was observed at 450 bp; and at the second PCR reaction, at 150 bp. In terms of β-globin, the DNA band was observed at 250 bp.

After the hybridization reaction was completed, the DNA chip slides were washed twice for 5 min each in a horizontal shaker at 300 rpm using washer buffer Solution I (2 × SSC, 0.1% SDS), twice for 5 min each using washer buffer Solution II (0.2 × SSC), and once for 5 min using washer buffer Solution III (0.1 × SSC). Then, the washer solution was removed and the slides were analyzed with a microarray scanner to confirm the presence of 20 HPV genotypes according to two subtypes: The high-risk and low-risk groups. Types 6, 11, 40, 43, 54, and 70 were classified under the low-risk group, and Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 66, and 68 under the high-risk group.

High-risk HPV-positive rate and high-risk HPV subtype distribution according to the results of the follow-up cytology

Of the 1780 Pap normal group, 176 (9.9%) were high-risk HPV positive; of the 70 Pap low-grade lesion group, 53 (75.7%); and of the 47 Pap high-grade lesion group, 46 (97.9%). The Pap low-grade lesion group showed a higher high-risk HPV-positive rate (P < 0.001) than the Pap normal group, and the Pap high-grade lesion group showed a significantly higher high-risk HPV-positive rate than the Pap low-grade lesion group (P = 0.002) [Table 3].

In all the groups, the most common high-risk HPV subtype was HPV 16, which was observed in 35 subjects (2.0% or 20.6% of the 170 high-risk HPV-positive subjects) of the 1780 Pap normal group subjects, 14 (20.0% or 26.4% of the 60 high-risk HPV-positive subjects) of the 70 Pap low-grade lesion subjects, and 24 (51.1% or 52.1% of the 46 HPV-positive subjects) of the 47 Pap high-risk lesion subjects. In the PAP high-grade lesion group, HPV 16 was significantly more frequently observed than the other high-risk HPV subtypes (P = 0.004). However, HPV 53 was less frequently observed than other high-risk HPV subtypes in the PAP high-grade lesion group (P = 0.004) [Table 4].

High-risk HPV-positive rate and high-risk HPV subtype distribution according to the results of the follow-up biopsy

Of the 281 biopsy subjects, 159 (56.6%) were high-risk HPV positive; of the 140 biopsy normal subjects, 31 (22.1%); of the 67 biopsy low-grade lesion group, 55 (82.1%); and of the 74 biopsy high-grade lesion group, 73 (98.6%). The biopsy low-grade lesion group showed a higher high-risk HPV-positive rate than the biopsy normal group (P < 0.001) and a significantly lower high-risk HPV-positive rate than the biopsy high-grade lesion group (P = 0.001) [Table 3]. The most common HPV subtype in the biopsy high-grade lesion group was also HPV-16 [27 of the 74 (36.5%) or 37.0% of the 73 HPV-positive subjects], which showed a significantly higher frequency than the other subtypes (P = 0.023). However, HPV 53 was less frequently observed than other high-risk HPV subtypes in the biopsy high-grade lesion group (P = 0.004) [Table 5].