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RANK-ligand (RANKL) expression in young breast cancer patients and during pregnancy.

Azim HA, Peccatori FA, Brohée S, Branstetter D, Loi S, Viale G, Piccart M, Dougall WC, Pruneri G, Sotiriou C - Breast Cancer Res. (2015)

Bottom Line: High RANKL expression within primary tumor was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways.At a median follow-up of 65 months, neither RANK nor RANKL expression within tumor was associated with disease free survival in pregnant or non-pregnant group.These results could guide further development of RANKL-targeted therapy.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: RANKL is important in mammary gland development during pregnancy and mediates the initiation and progression of progesterone-induced breast cancer. No clinical data are available on the effect of pregnancy on RANK/RANKL expression in young breast cancer patients.

Methods: We used our previously published dataset of 65 pregnant and 130 matched young breast cancer patients with full clinical, pathological, and survival information. 85% of patients had available transcriptomic data as well. RANK/RANKL expression by immunohistochemistry using H-score on the primary tumor and adjacent normal tissue was performed. We examined the difference in expression of RANK/RANKL between pregnant and non-pregnant patients and their association with clinicopathological features and prognosis. We also evaluated genes and pathways associated with RANK/RANKL expression on primary tumors.

Results: RANKL but not RANK expression was more prevalent in the pregnant group, both on the tumor and adjacent normal tissue, independent of other clinicopathological factors (both P <0.001). 18.7% of pregnant and 5.3% of non-pregnant patients had tumors showing ≥10% of cells with 3+ RANKL expression. RANKL expression was significantly higher in progesterone receptor-positive, and luminal A-like tumors, with negative correlation with Ki-67 (all P <0.001). On the contrary, RANK expression was higher in triple negative tumors (P <0.001). Using false discovery rate <0.05, 151 and 1,207 genes were significantly correlated with tumor-expressed RANKL and RANK expression by immunohistochemistry, respectively. High RANKL expression within primary tumor was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways. At a median follow-up of 65 months, neither RANK nor RANKL expression within tumor was associated with disease free survival in pregnant or non-pregnant group.

Conclusions: Pregnancy increases RANKL expression both in normal breast and primary tumors. These results could guide further development of RANKL-targeted therapy.

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Differences in RANK and RANKL expression between pregnant and nonpregnant breast cancer patients. (a) Expression of RANKL by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANKL H-score was higher in normal breast epithelial cells compared with tumor cells; 50.19 versus 16.13, P <0.001 in all patients; 32.88 versus 8.06, P <0.001 in nonpregnant patients; and 87.29 versus 32.53 in pregnant patients, P <0.001. (b) Expression of RANK by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANK H-score was higher in normal breast epithelial cells compared with tumor cells; 24.65 versus 12.48, P = 0.003 in all patients; 21.72 versus 13.95, P = 0.07 in nonpregnant patients; and 31.52 versus 9.41 in pregnant patients, P = 0.016. RANK, receptor activator for nuclear factor κB; RANKL, receptor activator for nuclear factor κB ligand.
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Fig1: Differences in RANK and RANKL expression between pregnant and nonpregnant breast cancer patients. (a) Expression of RANKL by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANKL H-score was higher in normal breast epithelial cells compared with tumor cells; 50.19 versus 16.13, P <0.001 in all patients; 32.88 versus 8.06, P <0.001 in nonpregnant patients; and 87.29 versus 32.53 in pregnant patients, P <0.001. (b) Expression of RANK by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANK H-score was higher in normal breast epithelial cells compared with tumor cells; 24.65 versus 12.48, P = 0.003 in all patients; 21.72 versus 13.95, P = 0.07 in nonpregnant patients; and 31.52 versus 9.41 in pregnant patients, P = 0.016. RANK, receptor activator for nuclear factor κB; RANKL, receptor activator for nuclear factor κB ligand.

Mentions: RANKL staining was performed on 194 primary tumors (99%) and 176 adjacent normal tissues (90%); the results were positively correlated (r = 0.38, P < 0.001). The mean RANKL expression on normal epithelial cells was higher compared with that on tumor cells (50.19 vs. 16.13, P <0.001) (Figure 1a). Patients diagnosed during pregnancy had significantly higher RANKL expression both on tumor cells (32.53 vs. 8.06, P <0.001) and adjacent normal tissue (87.29 vs. 32.88, P <0.001) (Figure 1a). In total, 18.7% of pregnant patients and 5.3% of nonpregnant patients had ≥10% of cells showing strong RANKL staining (score 3+) on primary tumor, while 55.3% and 29.1% of pregnant and nonpregnant patients had at least 10% of cells 3+ on adjacent normal tissue (Figure 2a,b). Additional file 1 shows examples of different staining intensities of RANKL. Among pregnant patients, a slightly lower expression of RANKL in both tumor and adjacent normal epithelial cells was observed in patients diagnosed in the third trimester of pregnancy (Figure S2a,b in Additional file 2).Figure 1


RANK-ligand (RANKL) expression in young breast cancer patients and during pregnancy.

Azim HA, Peccatori FA, Brohée S, Branstetter D, Loi S, Viale G, Piccart M, Dougall WC, Pruneri G, Sotiriou C - Breast Cancer Res. (2015)

Differences in RANK and RANKL expression between pregnant and nonpregnant breast cancer patients. (a) Expression of RANKL by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANKL H-score was higher in normal breast epithelial cells compared with tumor cells; 50.19 versus 16.13, P <0.001 in all patients; 32.88 versus 8.06, P <0.001 in nonpregnant patients; and 87.29 versus 32.53 in pregnant patients, P <0.001. (b) Expression of RANK by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANK H-score was higher in normal breast epithelial cells compared with tumor cells; 24.65 versus 12.48, P = 0.003 in all patients; 21.72 versus 13.95, P = 0.07 in nonpregnant patients; and 31.52 versus 9.41 in pregnant patients, P = 0.016. RANK, receptor activator for nuclear factor κB; RANKL, receptor activator for nuclear factor κB ligand.
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Related In: Results  -  Collection

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Fig1: Differences in RANK and RANKL expression between pregnant and nonpregnant breast cancer patients. (a) Expression of RANKL by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANKL H-score was higher in normal breast epithelial cells compared with tumor cells; 50.19 versus 16.13, P <0.001 in all patients; 32.88 versus 8.06, P <0.001 in nonpregnant patients; and 87.29 versus 32.53 in pregnant patients, P <0.001. (b) Expression of RANK by immunohistochemistry using the H-score (y axis) in all patients, pregnant patients and nonpregnant patients both in primary tumor (blue) and adjacent normal breast epithelial cells (green). Mean RANK H-score was higher in normal breast epithelial cells compared with tumor cells; 24.65 versus 12.48, P = 0.003 in all patients; 21.72 versus 13.95, P = 0.07 in nonpregnant patients; and 31.52 versus 9.41 in pregnant patients, P = 0.016. RANK, receptor activator for nuclear factor κB; RANKL, receptor activator for nuclear factor κB ligand.
Mentions: RANKL staining was performed on 194 primary tumors (99%) and 176 adjacent normal tissues (90%); the results were positively correlated (r = 0.38, P < 0.001). The mean RANKL expression on normal epithelial cells was higher compared with that on tumor cells (50.19 vs. 16.13, P <0.001) (Figure 1a). Patients diagnosed during pregnancy had significantly higher RANKL expression both on tumor cells (32.53 vs. 8.06, P <0.001) and adjacent normal tissue (87.29 vs. 32.88, P <0.001) (Figure 1a). In total, 18.7% of pregnant patients and 5.3% of nonpregnant patients had ≥10% of cells showing strong RANKL staining (score 3+) on primary tumor, while 55.3% and 29.1% of pregnant and nonpregnant patients had at least 10% of cells 3+ on adjacent normal tissue (Figure 2a,b). Additional file 1 shows examples of different staining intensities of RANKL. Among pregnant patients, a slightly lower expression of RANKL in both tumor and adjacent normal epithelial cells was observed in patients diagnosed in the third trimester of pregnancy (Figure S2a,b in Additional file 2).Figure 1

Bottom Line: High RANKL expression within primary tumor was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways.At a median follow-up of 65 months, neither RANK nor RANKL expression within tumor was associated with disease free survival in pregnant or non-pregnant group.These results could guide further development of RANKL-targeted therapy.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: RANKL is important in mammary gland development during pregnancy and mediates the initiation and progression of progesterone-induced breast cancer. No clinical data are available on the effect of pregnancy on RANK/RANKL expression in young breast cancer patients.

Methods: We used our previously published dataset of 65 pregnant and 130 matched young breast cancer patients with full clinical, pathological, and survival information. 85% of patients had available transcriptomic data as well. RANK/RANKL expression by immunohistochemistry using H-score on the primary tumor and adjacent normal tissue was performed. We examined the difference in expression of RANK/RANKL between pregnant and non-pregnant patients and their association with clinicopathological features and prognosis. We also evaluated genes and pathways associated with RANK/RANKL expression on primary tumors.

Results: RANKL but not RANK expression was more prevalent in the pregnant group, both on the tumor and adjacent normal tissue, independent of other clinicopathological factors (both P <0.001). 18.7% of pregnant and 5.3% of non-pregnant patients had tumors showing ≥10% of cells with 3+ RANKL expression. RANKL expression was significantly higher in progesterone receptor-positive, and luminal A-like tumors, with negative correlation with Ki-67 (all P <0.001). On the contrary, RANK expression was higher in triple negative tumors (P <0.001). Using false discovery rate <0.05, 151 and 1,207 genes were significantly correlated with tumor-expressed RANKL and RANK expression by immunohistochemistry, respectively. High RANKL expression within primary tumor was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways. At a median follow-up of 65 months, neither RANK nor RANKL expression within tumor was associated with disease free survival in pregnant or non-pregnant group.

Conclusions: Pregnancy increases RANKL expression both in normal breast and primary tumors. These results could guide further development of RANKL-targeted therapy.

Show MeSH
Related in: MedlinePlus