*3.3. E6/E7 mRNA in Cervical Samples*

Figure 3 shows the study design with sample processing to analyze the expression of the E6/E7 mRNA of the most prevalent HR HPV in cervical samples from Serbian women. A total of 291 cervical samples, which include HPV 16-, 31-, 33- and 51-positive (*n* = 172) and HR-HPV-negative samples (*n* = 119), were tested by the HPV mRNA test. The E6/E7 mRNA HPV was detected exclusively in HR-HPV-DNA-positive samples (Table 6). E6 and E7 transcripts of the four most frequent HR HPVs were detected in 57.5% (115/200) of the HR HPV DNA confirmed genotypes. Accordingly, the distribution of E6/E7 mRNA HR HPV 16, 31, 33, and 51 are shown in Table 6. The E6/E7 mRNA HR HPV 16 was the most abundant, which accounted for 25.5% (51/200) of HR HPV genotypes. Next in frequency was the E6/E7 mRNA HR HPV 31 in 16.5% (33/200), while the E6/E7 mRNAs HR HPV 33 and 51 were equally represented in 8% (16/200) and 7.5% (15/200), respectively. Almost

HPV 16

HPV 31

HPV 33

HPV 51

every second HPV 16 genotype is oncogenically expressed (48.6%; 51/105), and it was detected in 29.7% (51/172) HPV-DNA-positive samples. The oncogenic activity of HPV 31 was detected in approximately every fifth (19.2%; 33/172) HPV-DNA-positive sample. Regarding the oncogenic activity of the remaining tested genotypes, HPV 33 and HPV 51 were detected in roughly every tenth HPV-DNA-positive sample (Table 6). The results of expressing E6 and E7 HR HPV oncogenes were expressed through the dispersion of the obtained Ct values. The oncogenic activity of HPV 16 is detected by the lowest registered value (Ct = 16) (Supplementary Materials Figure S1). *Diagnostics* **2023**, *13*, x FOR PEER REVIEW 8 of 23

**Figure 2.** Age‐specific analyses of the most prevalent HR HPV DNA in different cytological groups. NILM—negative for an intraepithelial lesion or malignancy; ASCUS—atypical squamous cells of unknown significance; LSIL—low‐grade squamous intraepithelial lesions; HSIL—high‐grade squa‐ mous intraepithelial lesions. **Figure 2.** Age-specific analyses of the most prevalent HR HPV DNA in different cytological groups. NILM—negative for an intraepithelial lesion or malignancy; ASCUS—atypical squamous cells of unknown significance; LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions. *Diagnostics* **2023**, *13*, x FOR PEER REVIEW 9 of 23

sample. Regarding the oncogenic activity of the remaining tested genotypes, HPV 33 and HPV 51 were detected in roughly every tenth HPV‐DNA‐positive sample (Table 6). The **Figure 3.** Flowchart presenting the study design. HR—high risk; HPV—human papillomavirus. **Figure 3.** Flowchart presenting the study design. HR—high risk; HPV—human papillomavirus.

#### results of expressing E6 and E7 HR HPV oncogenes were expressed through the disper‐ sion of the obtained Ct values. The oncogenic activity of HPV 16 is detected by the lowest **Table 6.** Analyses of E6/E7 mRNA HPV 16, 31, 33, and 51 in cervical samples. *3.4. Prevalence of HR HPV Based on E6/E7 mRNA HPV Expression in Different Cytological Groups*

− 167 (83.5) 14 (29.8) 244 (83.8) 19.2 (33/172) 70.2 (33/47)

− 185 (92.5) 9 (37.5) 267 (91.8) 8.7 (15/172) 62.5 (15/24)

*3.4. Prevalence of HR HPV Based on E6/E7 mRNA HPV Expression in Different Cytological*

The expression of the E6 and E7 genes as indicators of the oncogenic activity of HR HPV 16, 31, 33, and 51 was analyzed concerning cytological findings. The oncogenic ac‐ tivity of the tested genotypes increases with the severity of the cervical intraepithelial le‐ sion. A statistically significant difference in E6/E7 mRNA HPV expression among the var‐ ious cytological groups was observed (χ<sup>2</sup> test; χ<sup>2</sup> = 108.623; *p* < 0.001). E6/E7 mRNA HPVs are the most prevalent in patients with HSIL cytological findings (88.9%). In the group of patients with LSIL cytological findings, it was demonstrated in a lower percentage (60%). A two‐fold lower prevalence is observed in patients with ASCUS (29.4%). Oncogene ac‐ tivity in women with normal cytological findings is present in 10.9% of samples (Table 7). Subsequently, the E6/E7 mRNA distribution of HR‐HPV‐DNA‐positive samples accord‐ ing to genotype and cytological groups was analyzed. E6/E7 mRNA HR HPV 16 is the most represented in patients with HSIL cytological findings (64.2%), while in the other groups of cytological findings, it was demonstrated in a lower percentage (3.4–22.7%). A statistically significant difference was found in the number of positive findings of E6/E7

registered value (Ct = 16) (Supplementary Materials Figure S1). **E6/E7 mRNA HPV Genotypes Genotypes** *<sup>n</sup>* **(%) HR HPV 16, 31, 33, 51 Cervical Samples (***n* **= 291) E6/E7 mRNA HR HPV/Most Prevalent HR‐HPV‐DNA‐Posi‐ tive Samples (%) E6/E7 mRNA HR HPV Positive/HR‐ HPV‐DNA‐Positive (%) Positive** *<sup>n</sup>* **(%) Negative** *<sup>n</sup>* **(%)** + 51 (25.5) 51 (48.6) 0 (0.0) − 149 (74.5) 54 (51.4) 186 (63.9) 29.7 (51/172) 48.5 (51/105) Total: 200 (100) 105 (36.1) 186 (63.9) + 33 (16.5) 33 (70.2) 0 (0.0) The expression of the E6 and E7 genes as indicators of the oncogenic activity of HR HPV 16, 31, 33, and 51 was analyzed concerning cytological findings. The oncogenic activity of the tested genotypes increases with the severity of the cervical intraepithelial lesion. A statistically significant difference in E6/E7 mRNA HPV expression among the various cytological groups was observed (χ 2 test; χ <sup>2</sup> = 108.623; *p* < 0.001). E6/E7 mRNA HPVs are the most prevalent in patients with HSIL cytological findings (88.9%). In the group of patients with LSIL cytological findings, it was demonstrated in a lower percentage

Total E6/E7 mRNA genotypes <sup>+</sup> <sup>115</sup> (57.5) <sup>0</sup> (0.0) 66.9 (115/172) 57.5 (115/200) ‐ <sup>85</sup> (42.5) <sup>119</sup> (100)

Total: 200 (100) 47 (16.2) 244 (83.8)

Total: 200 (100) 24 (8.2) 267 (91.8)

+ 15 (7.5) 15 (62.5) 0 (0.0)

Total: 200 (100) 24 (8.2) 267 (91.8)

*Groups*

(60%). A two-fold lower prevalence is observed in patients with ASCUS (29.4%). Oncogene activity in women with normal cytological findings is present in 10.9% of samples (Table 7). Subsequently, the E6/E7 mRNA distribution of HR-HPV-DNA-positive samples according to genotype and cytological groups was analyzed. E6/E7 mRNA HR HPV 16 is the most represented in patients with HSIL cytological findings (64.2%), while in the other groups of cytological findings, it was demonstrated in a lower percentage (3.4–22.7%). A statistically significant difference was found in the number of positive findings of E6/E7 mRNA HR HPV 16 concerning the cytological status (χ 2 test; χ <sup>2</sup> = 46.881; *p* < 0.001). This result singled out the HR HPV 16 genotype for further analyses. The presence of E6/E7 mRNA HR HPV 31 was the least detected in HSIL (7.5%) compared to other cytological groups (22.7–26.1%). The distribution of the oncogenic activity of the remaining genotypes (HR HPV 33 and 51) is approximately the same across different cytological groups and remains at a low level (2.2–11.4%) (Table 7).

**Table 6.** Analyses of E6/E7 mRNA HPV 16, 31, 33, and 51 in cervical samples.


**Table 7.** Distribution of E6/E7 mRNA HR HPV according to cytology.



\*\*\* *p* < 0.001. NILM—negative for intraepithelial lesion or malignancy; ASCUS—atypical squamous cells of unknown significance; LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions.

The analyses of the oncogenic activity of multiple HR HPV infections are presented by frequencies. The overall oncogenic activity, including both single and multiple HR HPV infections detected using the E6/E7 mRNA HR HPV test, increases with the degree of cervical lesion severity (60–100%). The oncogenic activity detected in single-genotype infections (20.0–85.7%) is higher compared to the oncogenic activity of multiple genotypes (0–40%) (Table 8).

**Table 8.** Analysis of the oncogenic activity of multiple infections of the most prevalent HR HPV.


\* (f2/f1) × 100; \*\* (f3/f1) × 100; NILM—negative for an intraepithelial lesion or malignancy; ASCUS—atypical squamous cells of unknown significance; LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions.
