CXCL12 expression by healthy and malignant ovarian epithelial cells

Véronique Machelon, Françoise Gaudin, Sophie Camilleri-Broët, Salam Nasreddine, Laurence Bouchet-Delbos, Eric Pujade-Lauraine, Jerôme Alexandre, Laurence Gladieff, Fernando Arenzana-Seisdedos, Dominique Emilie, Sophie Prévot, Philippe Broët, Karl Balabanian, Véronique Machelon, Françoise Gaudin, Sophie Camilleri-Broët, Salam Nasreddine, Laurence Bouchet-Delbos, Eric Pujade-Lauraine, Jerôme Alexandre, Laurence Gladieff, Fernando Arenzana-Seisdedos, Dominique Emilie, Sophie Prévot, Philippe Broët, Karl Balabanian

Abstract

Background: CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value.

Methods: Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves.

Results: Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival.

Conclusion: Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself.

Trial registration: ClinicalTrials.gov: NCT00052468.

Figures

Figure 1
Figure 1
CXCL12 expression in healthy and malignant ovaries. (A) Healthy ovary (i), CXCL12 immunoreactivity in OSE (inset is the outlined region on the tissue specimen containing surface epithelium, × 40), faint staining in the stroma and no signal in follicles and oocytes (arrow) (× 20); fallopian tube (ii), CXCL12 immunoreactivity in cells of the epithelium (× 40). (B) Serous (i) and mucinous (ii) benign epithelial ovarian tumors, CXCL12 immunoreactivity in proliferating epithelial cells (× 40). (C) Serous (i and iii) and mucinous (ii and iv) borderline epithelial ovarian tumors with low (CXCL12low, i and ii) or high (CXCL12high, iii and iv) levels of CXCL12 staining (× 40). (D) Malignant epithelial ovarian tumors: serous (i), mucinous (ii), clear-cell (iii) and endometrioid (iv), CXCL12 immunoreactivity in epithelial cells is confined to the cytoplasm, with frequent strong staining of the membrane (arrows), no staining in the nuclei of tumor cells or in the stroma (× 40). (E) Cytocentrifuged CD326+ epithelial (i) and CD326- non epithelial (ii) cells isolated from malignant ascites collected from a patient diagnosed with invasive EOC, CXCL12 is detected only in CD326+ cells (× 40). (F) Non epithelial ovarian tumors: granulosa tumor (i) and dysgerminoma with characteristic morphological features, i.e. Exner bodies (arrow) (ii), absence of CXCL12 immunostaining from both tumors (× 40). No labeling was detected when the K15C anti-CXCL12 mAb was omitted or a 100-fold molar excess of recombinant CXCL12 was added to the mAb before incubation with tissues.
Figure 2
Figure 2
Steady-state levels of CXCL12 transcripts in healthy and malignant ovarian tissues. (A) CXCL12 mRNA was detected by conventional PCR and was of the expected size (417 bp) in healthy ovaries (faint signal), benign and invasive ovarian tumors (strong signal) and in CD326+ epithelial cell-enriched malignant ascites samples. By contrast, CXCL12 transcripts were absent from CD326- non epithelial cells. The results presented are from one experiment representative of three carried out. The white vertical line separates lanes not run on the same gel. (B) CXCL12 mRNA levels were quantified by real-time PCR and are expressed as CXCL12/β-actin ratios. The diagram shows the distribution of values and means for EOC samples identified as CXCL12high and CXCL12low/moderate. Each symbol represents an individual sample run in duplicate. The P value presented is that for a two-tailed Student's t test.
Figure 3
Figure 3
Overall survival and progression-free survival as a function of CXCL12 expression. Plots of Kaplan-Meier estimates for overall survival (A) and progression-free survival (B) of EOC patients with tumor tissues identified as CXCL12low/moderate (n = 97, solid blue line) or CXCL12high (n = 86, dotted red line). P values are those obtained in log-rank tests.

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