Analysis of the progression of cervical cancer in a low-and-middle-income country: From pre-malignancy to invasive disease

Abstract

To better understand cervical cancer progression, we analyzed RNA from 262 biopsies from women referred for colposcopy. We determined the HPV type and analyzed the expression of 51 genes. HPV31 was significantly more prevalent in precancer than stage 1 cancer and invasive cancer (p < 0.0001), and HPV16 increased in invasive disease (p < 0.0001). CCNE1, MELTF, and ULBP2 were significantly increased in HPV16-positive compared to HPV31 precancers, while NECTIN2 and HLA-E expression decreased. Markers of the innate immune system, DNA repair genes, and cell cycle genes are significantly increased during cancer progression (p = 0.0001). In contrast, the TP53 and RB1 tumor suppressor gene expression is significantly decreased in cancer cells. The T cell markers CD28 and FLT3LG expression decreased in cancer while FOXP3, IDO1, and ULBP2 expression increased. There is a significantly higher survival rate in individuals with increased expression of CD28 (p = 0.0005), FOXP3 (p = 0.0002), IDO1 (p = 0.038), FLT3LG (p = 0.026), APOBEC3B (p = 0.0011), and RUNX3 (p = 0.019), and a significantly lower survival rate in individuals with increased expression of ULBP2 (p = 0.035). These results will help us elucidate the molecular factors influencing the progression of cervical precancer to cancer. Understanding the risk of progression of specific HPV types and sublineages may aid in the triage of positive patients, and better knowledge of the immune response may aid in developing and applying immunotherapies.

Keywords: Cancer progression; Cervical cancer; Gene expression; Human papillomavirus; Immune surveillance; Precancer; Tumor microenvironment.

Fig. 1

Distribution of HPV type during cervical cancer progression. The percentage of samples in precancer, stage 1 cancer, and invasive cancer are shown by HPV type. HPV68 is included in the panel but was not detected in any precancer or stage 1 cancer; therefore, it is not shown on this graph.

Fig. 2

Analysis of gene expression differences in HPV16 and HPV31-positive precancers. These box and whisker plots represent the expression level of various genes in HPV16 and HPV31-positive precancers. Gene expression is shown on the y-axis, while HPV type is on the x-axis. The expression of cell proliferation promoters CCNE1 and MELTF, the NK-cell regulators ULBP2, and the DNA-repair gene PALB2 are increased in HPV16-positive precancers. The expression of the NK-cell co-stimulator and co-inhibitor NECTIN2, the immune gene IRF-1, and the NK-cell educator HLA-E is decreased in HPV16-positive precancers. ∗∗∗ indicates significance of p < 0.001, ∗∗ indicates significance of p < 0.01, and ∗ indicates significance of p < 0.05.

Fig. 3

Analysis of individual gene expression in controls, precancer, and cancer. These box and whisker plots represent the expression level of various genes corresponding to their histological category, and gene expression is shown on the y-axis. In contrast, the histological type is shown on the x-axis. Expression of innate immune system genes (APOBEC3A and APOBEC3B), cell cycle genes (CCNA2 and CCNE1), and immune cell markers (CD28 and FLT3LG) is decreased in individuals with cancer. In contrast, expression of FOXP3, IDO1, and ULBP2, markers of immune system suppression and evasion, is increased. The expression of tumor suppressors (TP53, RB1, RUNX2, and RUNX3) is decreased in individuals with cancer. The expression of the NK-cell co-stimulator and co-inhibitor NECTIN2 does not significantly change between controls, precancer, and cancer. ∗∗∗ indicates significance of p < 0.001, ∗∗ indicates significance of p < 0.01, and ∗ indicates significance of p < 0.05.

Fig. 4

Survival of patients by expression of selected genes. These Kaplan-Meier curves show how expression levels of selected genes correspond with survival time. Time in months is shown on the x-axis, and the probability of survival is shown on the y-axis. The red line represents patients with lower expression, while the teal line represents patients with higher expression. Individuals with higher expression of APOBEC3B, CD28, FLTLG3, FOXP3, IDO1, and RUNX3 had significantly better survival than individuals with lower expression. In patients with SCC, higher expression of APOBEC3A was associated with improved survival, while lower expression of ULBP2 was associated with enhanced survival.

Fig. 5

Changes in the expression of adaptive immune genes drive cervical cancer cell proliferation. A. HPV31 and HPV33 are more prevalent in precancer, while HPV16 and HPV18 are more prevalent in cancer. Genes listed under Infection (Controls) have significantly higher expression prior to the development of precancer. Genes listed under precancer increase in expression between controls and precancer cases. Genes listed under cancer increase in expression between precancer cases and cancer cases. Increased expression of genes listed in blue is associated with improved survival, while increased expression of genes listed in red is associated with worse survival. B. Increased expression of IDO1 promotes FOXP3 expression, which promotes proliferation of T-regulatory cells. Decreased expression of FLT3LG prevents the maturation of dendritic cells. T-cell suppression allows cervical tumor cells to proliferate. Expression of soluble ULBP2 by cervical tumor cells inhibits NK cells, enabling immune evasion by tumor cells.