*3.6. Comparison of Tests for the Detection of HR HPV Genotypes and Their Oncogenic Activity*

The comparison of tests for the detection of the most prevalent HR HPV genotypes and their oncogenic activity revealed that the presence of the HR HPV genotypes is higher than the presence of the oncogenic activity of the genotypes in younger women (≤30 years), similar in middle-aged women (31–44 years), and lower in women 45 years and older. The prevalence of oncogenic activity of the HPV genotypes increases with the severity of the cervical intraepithelial lesion. Compared to the prevalence of the examined HR HPV genotypes, the same parameter is lower in women with normal and undefined cytological findings, approximately the same in low-grade lesions, and significantly higher in high-grade lesions (Figure 4). The comparison of tests for the detection of the most prevalent HR HPV genotypes and their oncogenic activity revealed that the presence of the HR HPV genotypes is higher than the presence of the oncogenic activity of the genotypes in younger women (≤30 years), similar in middle‐aged women (31–44 years), and lower in women 45 years and older. The prevalence of oncogenic activity of the HPV genotypes increases with the se‐ verity of the cervical intraepithelial lesion. Compared to the prevalence of the examined HR HPV genotypes, the same parameter is lower in women with normal and undefined cytological findings, approximately the same in low‐grade lesions, and significantly higher in high‐grade lesions (Figure 4).

**Figure 4.** Prevalence of the most prevalent HR HPVs DNA and achieved oncogenic activity accord‐ ing to age (**A**) and cytology (**B**). NILM—negative for an intraepithelial lesion or malignancy; AS‐ CUS—atypical squamous cells of unknown significance; LSIL—low‐grade squamous intraepithelial lesions; HSIL—high‐grade squamous intraepithelial lesions. **Figure 4.** Prevalence of the most prevalent HR HPVs DNA and achieved oncogenic activity according to age (**A**) and cytology (**B**). 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.

The calculated clinical characteristics of the DNA and E6/E7 mRNA HR HPV tests are shown in Table 10. The sensitivity and NPV of both tests were increased with the severity

nificantly higherlevel (67.6–98.2%). The specificity of the E6/E7 mRNA HR HPV test (89.1%) is statistically significantly higher than the HR HPV DNA test (75.6%). The PPV of the HR HPV DNA test is approximately the same for all types of cytological findings (60.3– 64.6%), while for the mRNA HR HPV test, it increases with the degree of the cervical intraepithelial

**(%) (95%) (%) (95%) (%) (95%) (%) (95%)**

\* *p* < 0.05; \*\* *p* < 0.005; \*\*\* *p* < 0.001. CI (95%)—95% confidence interval; PPV—positive predictive value; NPV—negative predictive value; NILM—negative for intraepithelial lesion or malignancy; ASCUS—atypical squamous cells of unknown significance; LSIL—low‐grade squamous intraepi‐

To quantify the diagnostic capabilities of the E6/E7 mRNA HPV test and evaluate its significance, an ROC curve was used to assess the assays for detecting HSIL. The area

lesion (69.8– 78.7%) and it is statistically significantly higher (Table 10).

**Test Cytology Sensitivity CI Specificity CI PPV CI NPV CI**

thelial lesions; HSIL—high‐grade squamous intraepithelial lesions.

**Table 10.** Clinical characteristics of HR HPV DNA and E6/E7 mRNA HPV tests.

ASCUS 67.6 \*\*\* 55.2–78.5 75.6 66.9–83.0 61.3 49.4–72.4 80.4 \* 71.8–87.3 LSIL 88.0 \*\* 75.7–95.5 75.6 66.9–83.0 60.3 48.1–71.6 93.8 \* 86.9–97.7 HSIL 98.2 90.1–100 75.6 66.9–83.0 64.6 53.3–74.9 98.9 94.0–100

ASCUS 29.4 19.0–41.7 89.1 \*\* 82.0–94.0 60.6 42.1–77.1 68.8 60.9–76.0 LSIL 60.0 45.2–73.6 89.1 \*\* 82.0–94.0 69.8 \*\*\* 53.9–82.8 84.1 76.6–90.0 HSIL 88.9 77.4–95.8 89.1 \*\* 82.0–94.0 78.7 \*\*\* 66.3–88.1 94.6 88.7–98.0

The calculated clinical characteristics of the DNA and E6/E7 mRNA HR HPV tests are shown in Table 10. The sensitivity and NPV of both tests were increased with the severity of the cervical intraepithelial lesion, with the HR HPV DNA test showing a statistically significantly higher level (67.6–98.2%). The specificity of the E6/E7 mRNA HR HPV test (89.1%) is statistically significantly higher than the HR HPV DNA test (75.6%). The PPV of the HR HPV DNA test is approximately the same for all types of cytological findings (60.3– 64.6%), while for the mRNA HR HPV test, it increases with the degree of the cervical intraepithelial lesion (69.8– 78.7%) and it is statistically significantly higher (Table 10).


*Diagnostics* **2023**, *13*, x FOR PEER REVIEW 13 of 23

**Table 10.** Clinical characteristics of HR HPV DNA and E6/E7 mRNA HPV tests.

\* *p* < 0.05; \*\* *p* < 0.005; \*\*\* *p* < 0.001. CI (95%)—95% confidence interval; PPV—positive predictive value; NPV negative predictive value; 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.

To quantify the diagnostic capabilities of the E6/E7 mRNA HPV test and evaluate its significance, an ROC curve was used to assess the assays for detecting HSIL. The area determined by the ROC curve (AUC) of E6/E7 mRNA HR HPV is 0.812 (CI (95%): 0.752–0.871), while the area under the ROC curve formed by the parameters of the HR HPV DNA test is 0.740 (CI (95%): 0.680–0.799) (Table 11, Figure 5). determined by the ROC curve (AUC) of E6/E7 mRNA HR HPV is 0.812 (CI (95%): 0.752– 0.871), while the area under the ROC curve formed by the parameters of the HR HPV DNA test is 0.740 (CI (95%): 0.680–0.799) (Table 11, Figure 5).

**Table 11.** Performance of E6/E7 mRNA HR HPV and HR HPV DNA tests in HSIL. **Table 11.** Performance of E6/E7 mRNA HR HPV and HR HPV DNA tests in HSIL.


The relationships between the oncogenic activity of all of the tested HPVs and previ‐ ously singled out HPV 16 vs. the cytological results were analyzed using Spearman's (ρ)

tween the presence of HPV 16 oncogenic activity and cytological status (Spearman's cor‐ relation; ρ = 0.494; *p* < 0.001) (Figure 6A). The oncogenic activity of the tested HPV geno‐ types is associated with a strong statistically significant positive correlation with the de‐ gree of cervical intraepithelial lesion severity (Spearman's correlation; ρ = 0.594; *p* < 0.001)

AUC—area under the ROC curve; SE—standard error; \*\*\* *p* < 0.001; CI (95%)—95% confidence intervals. tervals.

**Figure 5.** ROC curve of HR HPV and E6/E7 HR HPV tests in HSIL. **Figure 5.** ROC curve of HR HPV and E6/E7 HR HPV tests in HSIL.

(Figure 6B).

The relationships between the oncogenic activity of all of the tested HPVs and previously singled out HPV 16 vs. the cytological results were analyzed using Spearman's (ρ) correlation (Figure 6). There is a statistically significant positive moderate correlation between the presence of HPV 16 oncogenic activity and cytological status (Spearman's correlation; ρ = 0.494; *p* < 0.001) (Figure 6A). The oncogenic activity of the tested HPV genotypes is associated with a strong statistically significant positive correlation with the degree of cervical intraepithelial lesion severity (Spearman's correlation; ρ = 0.594; *p* < 0.001) (Figure 6B). *Diagnostics* **2023**, *13*, x FOR PEER REVIEW 14 of 23

**Figure 6.** Correlation between the oncogenic activity of the HPV 16 genotype and cytology (**A**) and overall HPVs' oncogenic activity and cytology (**B**). NILM (1), ASCUS (2), LSIL (3), HSIL (4), and Spearman's correlation coefficient (ρ). **Figure 6.** Correlation between the oncogenic activity of the HPV 16 genotype and cytology (**A**) and overall HPVs' oncogenic activity and cytology (**B**). NILM (1), ASCUS (2), LSIL (3), HSIL (4), and Spearman's correlation coefficient (ρ).

Univariate multinomial logistic regression examined the individual factors (the HR HPV DNA 16, the oncogenic activity of all of the tested HR HPVs, the oncogenic activity of the HR HPV 16 genotype, and the age category) that indicated an increased probability of developing HSIL (Supplementary Materials Tables S1–S4). For the influence of HR HPV 16 on the probability of HSIL, a statistically significant Univariate multinomial logistic regression examined the individual factors (the HR HPV DNA 16, the oncogenic activity of all of the tested HR HPVs, the oncogenic activity of the HR HPV 16 genotype, and the age category) that indicated an increased probability of developing HSIL (Supplementary Materials Tables S1–S4).

predictive value was determined in the NILM and LSIL cytological groups. Patients with the confirmed presence of the HPV 16 genotype through a DNA test have a higher prob‐ ability of being diagnosed with HSIL than NILM (3.8‐fold), while they will have a 2.6‐fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with LSIL (Supplementary Materials Table S1). The influence of the oncogenic activity of all tested of the HR HPV genotypes (E6/E7 For the influence of HR HPV 16 on the probability of HSIL, a statistically significant predictive value was determined in the NILM and LSIL cytological groups. Patients with the confirmed presence of the HPV 16 genotype through a DNA test have a higher probability of being diagnosed with HSIL than NILM (3.8-fold), while they will have a 2.6-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with LSIL (Supplementary Materials Table S1).

mRNA HR HPVs) on diagnosing high‐grade lesions of the cervical epithelium has a statis‐ tically significant prediction found in all cytological groups, NILM, ASCUS, and LSIL. If patients have confirmed indicators of oncogenic activity, they will have an almost seven‐ fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with NILM. The same patients have a 19‐fold higher probability of being diag‐ nosed with HSIL compared to the probability of detecting ASCUS and a 5‐fold higher prob‐ ability of detecting HSIL compared to LSIL (Supplementary Materials Table S2). E6/E7 mRNA HR HPV 16 is an indicator for diagnosing HSIL, and a statistically sig‐ The influence of the oncogenic activity of all tested of the HR HPV genotypes (E6/E7 mRNA HR HPVs) on diagnosing high-grade lesions of the cervical epithelium has a statistically significant prediction found in all cytological groups, NILM, ASCUS, and LSIL. If patients have confirmed indicators of oncogenic activity, they will have an almost seven-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with NILM. The same patients have a 19-fold higher probability of being diagnosed with HSIL compared to the probability of detecting ASCUS and a 5-fold higher probability of detecting HSIL compared to LSIL (Supplementary Materials Table S2).

nificant predictive value was determined for all types of cytological findings, NILM, AS‐ CUS, and LSIL. In a patient with confirmed HPV 16 oncogenic transcripts, the probability of diagnosing HSIL is 50‐fold higher than the probability of diagnosing NILM. Patients with a positive result of E6/E7 mRNAs HR HPV 16 are 12‐fold more likely to be diagnosed with HSIL compared to the detection of ASCUS, while in patients with the same result, the probability of detection of HSIL is 6‐fold higher than the probability of detection of LSIL (Supplementary Materials Table S3). E6/E7 mRNA HR HPV 16 is an indicator for diagnosing HSIL, and a statistically significant predictive value was determined for all types of cytological findings, NILM, ASCUS, and LSIL. In a patient with confirmed HPV 16 oncogenic transcripts, the probability of diagnosing HSIL is 50-fold higher than the probability of diagnosing NILM. Patients with a positive result of E6/E7 mRNAs HR HPV 16 are 12-fold more likely to be diagnosed with HSIL compared to the detection of ASCUS, while in patients with the same result, the

A statistically significant predictive value for the age category was determined in all cytological groups, NILM, ASCUS, and LSIL. The probability of detecting HSIL concern‐ ing normal results in HR‐HPV‐positive patients is 6.3‐fold higher if they are ≥ 45 years of

probability of detecting HSIL concerning LSIL than the youngest (Supplementary Mate‐

rials Table S4).

probability of detection of HSIL is 6-fold higher than the probability of detection of LSIL (Supplementary Materials Table S3).

A statistically significant predictive value for the age category was determined in all cytological groups, NILM, ASCUS, and LSIL. The probability of detecting HSIL concerning normal results in HR-HPV-positive patients is 6.3-fold higher if they are ≥45 years of age than women under 30. A similar prediction for the detection of HSIL was shown concerning ASCUS (6.5-fold). Patients from the oldest age category have a 3.4-fold higher probability of detecting HSIL concerning LSIL than the youngest (Supplementary Materials Table S4).

The predictive model contains four independent variables: HR HPV DNA 16, E6/E7 mRNA HR HPVs, E6/E7 mRNA HR HPV 16, and age category. The HSIL lesion, as a representative of a high degree of cervical atypia, represented a dependent variable concerning all of the analyzed relevant factors. Multivariate logistic regression analysis was applied to all of the analyzed factors to construct a predictive model and named the most relevant predictors for the development of HSIL (Table 12).


**Table 12.** Analysis of the mutual influence of relevant factors for HSIL development.

OR—Odds ratio; <sup>a</sup>—reference; CI (95%)—95% confidence interval; \* *p* < 0.05; \*\* *p* < 0.01; 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.

Analyzing the mutual influence of the examined relevant factors for diagnosing HSIL compared to the probability of diagnosing a normal cytological finding, the strongest statistically significant predictor was determined to be the oncogenic activity of the HPV 16 genotype. If it is detected, the probability of diagnosing HSIL concerning the probability of diagnosing normal results increases 19-fold (OR = 19.10; CI (95%): 1.54–236.98;

*p* = 0.022). A statistically significant but almost three-fold weaker predictor for the detection of HSIL compared to NILM is the patient belonging to the oldest age category (OR = 6.65; CI (95%): 1.66–26.60; *p* = 0.007), while female patients from the age category 31–44 years have a slightly lower statistically significant probability (OR = 5.38; CI (95%): 1.36–21.30; *p* = 0.016) for the detection of the same lesion. The age category was determined as the strongest statistically significant predictor by analyzing the mutual influence of relevant factors for diagnosing HSIL concerning the probability of detecting ASCUS. HR HPV DNA-positive patients belonging to the oldest age category (≥45 years) have an 8.7-fold higher probability of being diagnosed with HSIL compared to the probability of being diagnosed with ASCUS compared to patients belonging to the younger age category (OR = 8.74; CI (95%): 2.15–35.57; *p* = 0.002). Female patients with the proven oncogenic activity of HPV 16 have a probability of detecting HSIL 6.4-fold higher compared to the probability of being diagnosed with ASCUS (OR = 6.38; CI (95%): 1.22–33.54; *p* = 0.029). By analyzing the mutual influence of relevant factors for diagnosing HSIL concerning the probability of detection of LSIL, the strongest statistically significant predictor was determined to be the oncogenic activity of HPV 16 (OR = 5.10; CI (95%): 1.09–23.83; *p* = 0.038), while weaker statistically significant predictability is shown by the patient's belonging to a specific age category. Female patients belonging to the oldest age category (≥45 years) have a 3.7-fold greater probability of detecting an HSIL change compared to the detection of LSIL compared to the younger age category (≤30 years) (OR = 3.72; CI (95 %): 1.16–11.92; *p* = 0.027) (Table 12).
