Tumour-infiltrating leucocytes as prognostic biomarkers of bevacizumab-treated ovarian cancer patients results from the phase IV MITO16A/MaNGO OV-2 clinical trial

Abstract

The treatment of Epithelial Ovarian cancer (EOC) could benefit from the addition of bevacizumab (BEV) to standard chemotherapy in selected patients. Gene expression (GE) profiling and the evaluation of immune infiltration are used to define patients' prognosis. However, their role as prognostic and/or predictive biomarkers for the efficacy of antiangiogenic therapy efficacy remains uncertain. In this study, we combined GE profiling and multiplex immunofluorescence (MIF) analyses on material from patients enrolled in the phase IV MITO16A/MaNGO OV-2 trial, assessing associations between immune infiltrate and patients' prognosis. Patients were stratified into four molecular subtypes, and CIBERSORTx was applied to infer the cell-type-specific expression pattern of immune populations. MIF evaluated the presence of immune cells in the tumor and stromal compartments. These complementary experimental approaches revealed that immune infiltration is associated with shorter progression-free survival in BEV-treated patients, warranting future investigation to evaluate its use as a viable biomarker for patient stratification. Trial registration: NCT01706120, EudraCT number: 2012-003043-29, Date of registration 24 September 2012.

Fig. 1. Molecular subtypes of samples from the MITO16A case material.

Progression-free survival stratification risk according to TCGA (A) and Tothill (B) classifications of HGSOC cases included in the MITO16A case material. Numbers of patients at risk are reported below each Kaplan–Meier curve. In each panel, pie charts represent cases’ distribution among the different molecular subtypes. C Expression of the CD8 gene in the MITO16A case material. D Distribution of CD8 gene among the molecular subtypes according to TCGA and Tothill classifications, considering all cases or HGSOC only. E Expression of the CD68 gene in the MITO16A case material. F Distribution of CD68 gene among the molecular subtypes according to TCGA and Tothill classifications, considering all cases or HGSOC only. For Tothill classifier: C1 = MES; C2 = IMM; C4 = DIFF; C5 = PRO.

Fig. 2. Multiplex staining evaluating tumor and stroma immune infiltration in samples from the MITO16A case material.

A Scheme of the multiplex analyses using the OPAL Multiplex Assay coupled with multispectral image acquisition. Centralized tumor samples were processed, and single FFPE tissue slides were stained with the indicated antibodies for MIF. Stained sections were acquired with a Nikon microscope coupled with a multispectral camera, and acquired images were analyzed with the InForm software to define the number of immune cells infiltrating the tumors (PanCK-positive areas) and the surrounding stroma. Created with BioRender.com. B On the left, a typical image of immune cells infiltrating the tumor (color code as in A). Scale bar is 100 μm. On the right, pie charts report the number of analyzed slides that proved adequate or not for subsequent analyses after the multi-staining process. C Correlation between tumor and stroma infiltration in the same sample from CD8⁺ (left graph) and CD68⁺ (right graph) immune cells. D Graphs reporting the percentage of CD8 and CD68 immune-excluded (Neg) and immune-infiltrated (Pos) samples, as indicated.

Fig. 3. Prognostic values of CD8 and CD68 immune infiltration in analyzed cases.

A Kaplan–Meier curves evaluating patients’ PFS based on CD8 tumor, stromal, or total (sum of tumor and stromal) infiltration. Patients were stratified using the identified best cutoff values (see Table 2). B Kaplan–Meier curves evaluating patients’ PFS based on CD68 tumor, stromal, or total (sum of tumor and stromal) infiltration using the identified best cutoff values (see Table 2). C Distribution of CD8 and CD68 combining MIF expression and GE data to stratify among the molecular subtypes according to TCGA (left panel) and Tothill (right panel).