The SLNs were collected from 100 patients at the Department of Gynecologic Oncology at the Affiliated Tumor Hospital of Guangxi Medical University (Nanning, China) between August 2017 and August 2018. The inclusion criteria met by all patients were as follows: i) Preoperative cervical biopsy pathological diagnosis was confirmed as invasive carcinoma (squamous, adenocarcinoma or adenosquamous carcinoma); ii) International Federation of Gynecology and Obstetrics (FIGO) stage (14) was IIA2-IIA; iii) cervical fluid-based cytology HPV test was performed prior to surgery (HC-2 method), including tests for HR HPV type 17 (HPV16, −18, −31, −33, −35, −39, −45, −51, −52, −53, −56, −58, −59, −66, −68, −82 and −83); and iv) the complete data of clinical cases were collected. The exclusion criteria were as follows: i) Patients who underwent previous cervical treatment (Loop Electrosurgical Excision Procedure and cone excision); ii) the imaging findings indicated marked retroperitoneal lymph node metastasis or distant metastasis; and iii) patients who received preoperative radiotherapy. The present study was approved by The Medical Ethics Committee of Guangxi Medical University Affiliated Tumor Hospital (registration number, LW2018028). Written consent was obtained from all patients prior to sample collection.

Prior to surgery, the patients were placed in the lithotomy position and were anesthetized. The normal cervix around the tumor was injected with 1 ml of carbon nanoparticles (CNP) at 3 and 9 o'clock, with a deep (0.3±0.5 cm) cervical injection. The injection process lasted for at least 3 min, following which pressure was applied locally to prevent the CNP extravasation after the injection. During surgery, lymph nodes staining in the drainage field of the pelvic lymph system were identified. The lymph nodes that stained within 15 min of injection were defined as SLNs, whilst those remaining were classed as non-SLNs (nSLNs). Subsequently, the SLNs were removed and the tumor position was recorded and counted. Following detection of the SLNs, patients underwent laparoscopic radical hysterectomy + pelvic lymphadenectomy (with or without para-aortic lymph node sampling). The same surgical group of physicians performed the surgery. These physicians were gynecologists of the affiliated tumor hospital of Guangxi medical university, and were from the same operation group of the surgeon who operated.

Following collection of the SLNs, the fresh tissue was cut into 3 mm thick sections. The samples were then transferred into tagged sterile EP tubes without RNA enzymes, and stored at −80°C. Other specimens were labeled and sent for routine pathological examination.

The following reagents were purchased from Takara Biotechnology Co., Ltd. Tissue RNA extraction kits (Takara MiniBEST Universal RNA Extraction Kit PrimeScript™), transcriptional Master Mix (Perfect Real Time, SYBR^®) and RT-PCR reagents (Premix Ex Taq TM II Perfect Real Time).

The following primer pairs were used for the RT-qPCR: HPV16-mRNA: Forward: 5′-AAGGGCGTAACCGAAATCGGT-3′ and reverse: 5′-GTTTGCAGCTCTGTGCATA-3′; amplified fragments 141 bp. HPV18-mRNA: Forward, 5′-CATTTTGTGAACAGGCAGAGC-3′ and reverse: 5′-ACTTGTGCATCATTGTGGACC-3′; and GAPDH: Forward: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse: 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′.

HPV-DNA was selected for extraction from SLNs under sterile RNase-free conditions to prevent RNA degradation, according to the manufacturer's protocol. The total RNA was measured using a nucleic acid protein analyzer (NanoDropND-2000 micrometer; NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Specimens with A_260/280 values between 1.8–2.1 were of good purity. Specimens with high integrity and purity were subjected to RT in order to synthesize cDNA. cDNA reaction system for reverse transcription synthesis was as follows: 5× PrimeScript RTMaster Mix ×1(for RealTime) 4.0 µl + RNA 4.0–12.0 µl (depending on RNA concentration) + RNaseFree dH₂O to 20.0 µl at 37°C for 15 min (reverse transcription reaction), 85°C for 5 sec (deactivation reaction of reverse transcriptase), at 4°C for 4–5 min, and finally stored at −20°C.

The total reaction system was 20 µl, which included 2.0 µl cDNA, 0.8 µl sense and anti-sense primers, 10.0 µl SYBR^®Premix Ex Taq™ II, 0.4 µl ROX Reference Dye (50×) and 20.0 µl distilled H₂O. The follow PCR cycle was applied: Stage 1, predenaturation at 95°C for 30 sec and for 1 cycle; stage 2, PCR reaction at 95°C for 5 sec, 60°C for 30–34 sec, and for 40 cycles in total. The target genome, the internal reference genome and the 2-tube negative control (with sterile distilled water instead of cDNA) were set for each amplification. Each experiment was performed in triplicate.

Agarose (1 g) was mixed with 50 ml 0.5X TBE buffer to make 5% agarose gel. DNA marker and PCR products were loaded into the wells and electrophoresis was performed at 100V for 50 min. Following electrophoresis, the gel was removed and the results were recorded using a color analyzer. Each experiment was performed in triplicate. The expression of HPV-DNA in SLNs was analyzed, using 50–500 bp DNA markers as a reference standard. The DNA sequence amplified by PCR was compared with the HPV16 and HPV18 DNA in the gene bank (HPV 16 sequence, https://www.ncbi.nlm.nih.gov/nucleotide/KY994539.1?report=genbank&log$=nuclalign&blast_rank=94&RID=XRTDTSNB016; HPV18 sequence, http://www.ncbi.nlm.nih.gov/nucleotide/MF288722.1?report=genbank&log$=nuclalign&blast_rank=3&RID=XRS4T07H014) for Blast sequence alignment.

The reaction system and reaction conditions were as follows: 20 µl of total reaction system containing 1.0 µl of cDNA template, 0.8 µl HPV16 and HPV18 gene sense and anti-sense primers each, 10.0 µl of SYBR^®Premix Ex Taq™ II, 0.4 µl of ROX Reference Dye (50×) and 20.0 µl of distilled H₂O. The reaction conditions were as follows: Stage 1, predenaturation at 95°C for 30 sec and for 1 cycle; stage 2, PCR reaction at 95°C for 5 sec, 60°C for 30 sec, and for 40 cycles in total; stage 3, 95°C for 15 sec and for 1 cycle; predenaturation at 94°C for 5 min, followed by 30 sec at 94°C, 1 min at 55°C, and 1 min at 72°C for a total of 40 cycles, and finally extended at 72°C for 10 min. Samples were stored at 4°C. Following the reaction, the fluorescence reaction curve was automatically obtained by software TOWER3G 3.4 (Analytik Jena AG), and the amplification efficiency and cycle threshold value (15) of each reaction system were calculated. Each experiment was performed in triplicate.

SLNs were fixed in 10% formaldehyde for 24 h at room temperature, washed and dehydrated with increasing gradient of ethanol (70% for 2–4 h, 80% for 2–4 h, 95% for 2–4 h and 100% for 1–2 h), and washed with xylene for 30 min to 2 h. Samples (3 µm thick) were then embedded in paraffin and the lymph node slices were stained with hematoxylin and eosin (H&E) for 50 min at room temperature prior to IHC analysis. SLNs were incubated with the wide-spectrum horn cytokeratin AEl/AE3 (no. M351501-2; Dako; Agilent Technologies, Inc.) for expression in the cytoplasm, which was detected using the two-step anti-rabbit/mouse universal IHC kit (no. GK500705; Dako Agilent Technologies, Inc.). The criteria for the tumor cells were as follows: Large nucleus, deep staining, an imbalance of nucleo-cytoplasm ratio and AE1/AE3 is positive. The SLNs that were negative in pathology but HPV-DNA positive were further examined using an ultrastaging protocol (16). Two adjacent 5-µm sections were cut at each of two levels 200 µm apart. At each level, one slide was stained with H&E (20 min at room temperature) and the other was used for immunohistochemistry (IHC) using the anti-cytokeratin AE1:AE3 (1 h at room temperature). Sections were imaged using an optical microscope (OlympusBX45; Olympus Corporation; magnification, ×100).

Statistical analysis was performed using SPSS software (IBM Corp.; version 22.0;). Standard summary statistics were used to describe primary data: Data were presented by the median values. The positive rate of lymph node metastasis was compared by χ² test. The Mann-Whitney rank sum test of non-parametric data was used to compare the mean independent values of continuous variables. The association between discrete variables was determined by Fisher's exact test. P<0.05 was considered to indicate a statistically significant difference.

The present study included 100 patients with CC. The median age was 52 years (range 33–68 years) and the median body mass index was 22.42 kg/m² (range 15.60–27.64 kg/m²). There were a total of 78 cases of squamous cell carcinoma, 20 cases of adenocarcinoma, two cases of adenosquamous carcinoma, 51 cases of grade 3 (G3), 27 cases of moderate differentiation, eight cases of high differentiation and 14 unclassified cases. Additionally, there were zero cases with IA2 stage, 32 cases with IB1 stage, 24 cases with IB2 stage, 21 cases with stage IIA1 and 23 cases with stage IIA2 CC. The tumor diameter was ≤2 cm in 19 cases, 2–4 cm in 45 cases and >4 cm in 36 cases. In the 36 cases where the tumor lesions were >4 cm, paclitaxel + oxaliplatin neoadjuvant chemotherapy was administrated over 1–2 courses. Furthermore, there were 28 cases of cervical stromal invasion <1/3, 30 cases of 1/3-2/3, 42 cases of >2/3, 30 cases of Lymphovascular Space Invasion (LVSI) positive, 92 cases of HPV positive and 8 cases of HPV negative. There were 1,680 lymph node dissections, including 345 SLNs and 1,335 nSLNs (Table I).

The results of agarose gel electrophoresis in the present study demonstrated that the HPV16 and HPV18 genes had a size of 141 and 80 bp, respectively (Fig. 1). The sequencing of HPV16 and HPV18 was a part of the whole sequencing presented in Fig. 2. The results of the present study demonstrated that the amplified DNA sequence was the same when compared with GenBank HPV16 and HPV18. Sequencing was conducted by Sangon Biotech Co., Ltd. (Fig. 3), which further confirmed that the obtained target gene sequence was correct.

Of the 100 patients, 92 cases were diagnosed HPV-DNA positive by cervical liquid-based cytology, whereby the positive rate was 92%. HPV16 was the most common type, which was detected in 71 cases. Of these, one case was mixed with HPV66, one case was mixed with HPV35 and one case was mixed with both HPV52 and HPV42. HPV18, HPV51, HPV33 and HPV59-DNA were detected in 13, four, two and two cases, respectively. Furthermore, two cases were mixed with HPV16 and HPV18. The lymph node metastasis was detected in 14 patients via routine pathological examination. In addition, the RT-qPCR demonstrated that 28 patients had HPV-DNA expression in SLNs, whereas 72 patients did not. Of those positive for HPV-DNA expression, 13 were HPV16 positive, nine were HPV18 positive and six were other positive (HPV66, 35, 52 and 45; Table II).

IHC analysis demonstrated positive staining of AEl/AE3 in the cytoplasm of cancer cells (Fig. 4B), which presented with a characteristic yellow-brown color, while no signal was observed in the SLN lymph nodes (Fig. 4A).

The expression of HPV16/18 was positive in 109 SLNs in 28 patients; [positive rate was 31.6% (109/345)]. The histopathological examination demonstrated that there were 44 SLNs with metastasis in 14 cases [positive rate was 12.8% (44/345)]; and the pathologically positive lymph nodes were also all positive for HPV16/18. Of the 301 negative lymph nodes confirmed by pathological histology, the expression of HPV16/18 was positive in 65 SLNs [positive rate was 21.6% (65/301)] (P<0.001). Of the 14 patients with positive histopathology, the detection rate of HPV16/18 DNA was 100% (14/14), of which, HPV16 accounted for 71.4% (10/14) and HPV18 for 28.6% (4/14). In the 86 patients (301 lymph nodes) with negative histopathology, the detection rate of HPV16/18 DNA was 21.6% (65/301), while the detection rates of HPV16 and HPV18 were 20.6% (62/301) and 1.7% (5/301), respectively. HPV16 and HPV18 were simultaneously positive in five (1.7%) lymph nodes. In addition, ultrastaging was performed on all SLNs negative with H&E staining, which detected micrometastases in 13 of the 14 patients (Table III).

A total of 345 SLNs were surgically removed, of which 44 were positive and 301 were negative. The median detection of HPV-DNA of the negative SLNs was 2.50 (1.90–2.90) copies/ng, while the median examination of HPV-DNA of the positive SLNs were 8.55 (6.20–9.80) copies/ng (Fig. 5; Z=−5.823, P<0.001).

According to the clinicopathological features of the patients, numerous factors were selected to evaluate the association between positive expression of HPV-DNA and SLNs. The results of the present study demonstrated that the positive expression of HPV-DNA in SLN was significantly associated with clinical stage (P<0.001). Progression in the clinical stage was associated with an increase in tumor size and a greater possibility of metastasis (P=0.010). Nevertheless, the positive expression of HPV-DNA in SLN was not associated with age of patient, depth of cervical invasion, histological grade, lymphatic or LVSI, SCCAg (P>0.05; Table IV).