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Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

Stimmer L, Dehay S, Nemati F, Massonnet G, Richon S, Decaudin D, Klijanienko J, Johannes L - BMC Cancer (2014)

Bottom Line: Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04).Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates.

View Article: PubMed Central - PubMed

Affiliation: Endocytic Trafficking and Therapeutic Delivery Group, UMR3666 CNRS - U1143 INSERM, Institut Curie-Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France. ludger.johannes@curie.fr.

ABSTRACT

Background: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.

Methods: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.

Results: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.

Conclusion: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

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Gb3 expression in primary breast tumors and lymph node metastases. A: Estrogen receptor and Gb3 expression in primary breast tumors. Note: Gb3 positivity (Gb3 + group) is correlated with estrogen receptor expression. **: p ≤ 0.01, ER: estrogen receptor. B: Presence of axillary lymph node metastases in patients with and without Gb3 expression in primary breast cancer. Note a higher number of patients with lymph node metastases in the Gb3 negative group. *: p ≤ 0.03, LN: lymph node. C: Relative numbers of Gb3 expressing cells. D: Change in Gb3 expression between primary and metastatic tumors. Note: 40% of patients showed relative increase, whereas 50% had no change, and 10% lost Gb3 expression in the lymph node metastases compared to the primary tumor.
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Fig2: Gb3 expression in primary breast tumors and lymph node metastases. A: Estrogen receptor and Gb3 expression in primary breast tumors. Note: Gb3 positivity (Gb3 + group) is correlated with estrogen receptor expression. **: p ≤ 0.01, ER: estrogen receptor. B: Presence of axillary lymph node metastases in patients with and without Gb3 expression in primary breast cancer. Note a higher number of patients with lymph node metastases in the Gb3 negative group. *: p ≤ 0.03, LN: lymph node. C: Relative numbers of Gb3 expressing cells. D: Change in Gb3 expression between primary and metastatic tumors. Note: 40% of patients showed relative increase, whereas 50% had no change, and 10% lost Gb3 expression in the lymph node metastases compared to the primary tumor.

Mentions: Fine-needle aspiration (FNA) is a standard cytological tool for diagnostics of suspected primary or secondary malignancies [24]. In order to evaluate Gb3 expression on epithelial cancer cells, we developed a novel technique for the ex vivo STxB labeling (ESL) in which live cells from dissociated tumors are incubated with STxB prior to microscopical viewing (see Methods section). We combined the FNA and ESL techniques on cytological specimens of primary breast cancers of 107 patients. Roughly 100 to 500 cells with epithelial morphology per patient were subjected to ESL. Cell density and morphology were judged on MGG stained slides and on ESL slides using DAPI nuclear stain. Examined patients were separated in two groups according to Gb3 expression in primary tumors. 62 patients (57.9%) expressed Gb3 in primary tumor (Gb3 positive group). Regarding the receptor status of Gb3 positive patients, 48/62 (77.4%) were positive for estrogen receptor (ER), 35/62 (56.5%) for progesterone receptor (PR), and 40/62 (64.5%) for HER2. 5/62 (8.1%) were triple negative (Table 1). Using Pearson’s chi-squared test, Gb3 expression was significantly correlated to estrogen receptor expression (p ≤ 0.01; Figure 2A), whereas no correlation was found with age, histological type, grade, molecular classification, mitotic index, progesterone receptor or HER2 expression. Furthermore, no significant relationship was found between Gb3 positivity and the presence of lymph node metastases. 33 of 62 Gb3 positive patients (53.2%) had lymphatic extension, whereas 29/62 patients (46.8%) had no lymph node metastases.Absence of Gb3 expression (Gb3 negative group) was observed in 45 patients (42.1%). Gb3 negativity was significantly correlated (p ≤ 0.03) with higher number of lymph node metastases in this group (Figure 2B); including 34/45 lymph node positive (75.6%) and 11/45 lymph node negative (24.4%) patients. No correlation was found in this group with age, histological type, grade, molecular classification, mitotic index, estrogen and progesterone receptor or HER2 expression.Table 1


Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

Stimmer L, Dehay S, Nemati F, Massonnet G, Richon S, Decaudin D, Klijanienko J, Johannes L - BMC Cancer (2014)

Gb3 expression in primary breast tumors and lymph node metastases. A: Estrogen receptor and Gb3 expression in primary breast tumors. Note: Gb3 positivity (Gb3 + group) is correlated with estrogen receptor expression. **: p ≤ 0.01, ER: estrogen receptor. B: Presence of axillary lymph node metastases in patients with and without Gb3 expression in primary breast cancer. Note a higher number of patients with lymph node metastases in the Gb3 negative group. *: p ≤ 0.03, LN: lymph node. C: Relative numbers of Gb3 expressing cells. D: Change in Gb3 expression between primary and metastatic tumors. Note: 40% of patients showed relative increase, whereas 50% had no change, and 10% lost Gb3 expression in the lymph node metastases compared to the primary tumor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig2: Gb3 expression in primary breast tumors and lymph node metastases. A: Estrogen receptor and Gb3 expression in primary breast tumors. Note: Gb3 positivity (Gb3 + group) is correlated with estrogen receptor expression. **: p ≤ 0.01, ER: estrogen receptor. B: Presence of axillary lymph node metastases in patients with and without Gb3 expression in primary breast cancer. Note a higher number of patients with lymph node metastases in the Gb3 negative group. *: p ≤ 0.03, LN: lymph node. C: Relative numbers of Gb3 expressing cells. D: Change in Gb3 expression between primary and metastatic tumors. Note: 40% of patients showed relative increase, whereas 50% had no change, and 10% lost Gb3 expression in the lymph node metastases compared to the primary tumor.
Mentions: Fine-needle aspiration (FNA) is a standard cytological tool for diagnostics of suspected primary or secondary malignancies [24]. In order to evaluate Gb3 expression on epithelial cancer cells, we developed a novel technique for the ex vivo STxB labeling (ESL) in which live cells from dissociated tumors are incubated with STxB prior to microscopical viewing (see Methods section). We combined the FNA and ESL techniques on cytological specimens of primary breast cancers of 107 patients. Roughly 100 to 500 cells with epithelial morphology per patient were subjected to ESL. Cell density and morphology were judged on MGG stained slides and on ESL slides using DAPI nuclear stain. Examined patients were separated in two groups according to Gb3 expression in primary tumors. 62 patients (57.9%) expressed Gb3 in primary tumor (Gb3 positive group). Regarding the receptor status of Gb3 positive patients, 48/62 (77.4%) were positive for estrogen receptor (ER), 35/62 (56.5%) for progesterone receptor (PR), and 40/62 (64.5%) for HER2. 5/62 (8.1%) were triple negative (Table 1). Using Pearson’s chi-squared test, Gb3 expression was significantly correlated to estrogen receptor expression (p ≤ 0.01; Figure 2A), whereas no correlation was found with age, histological type, grade, molecular classification, mitotic index, progesterone receptor or HER2 expression. Furthermore, no significant relationship was found between Gb3 positivity and the presence of lymph node metastases. 33 of 62 Gb3 positive patients (53.2%) had lymphatic extension, whereas 29/62 patients (46.8%) had no lymph node metastases.Absence of Gb3 expression (Gb3 negative group) was observed in 45 patients (42.1%). Gb3 negativity was significantly correlated (p ≤ 0.03) with higher number of lymph node metastases in this group (Figure 2B); including 34/45 lymph node positive (75.6%) and 11/45 lymph node negative (24.4%) patients. No correlation was found in this group with age, histological type, grade, molecular classification, mitotic index, estrogen and progesterone receptor or HER2 expression.Table 1

Bottom Line: Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04).Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates.

View Article: PubMed Central - PubMed

Affiliation: Endocytic Trafficking and Therapeutic Delivery Group, UMR3666 CNRS - U1143 INSERM, Institut Curie-Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France. ludger.johannes@curie.fr.

ABSTRACT

Background: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.

Methods: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.

Results: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.

Conclusion: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

Show MeSH
Related in: MedlinePlus