Plasma levels and tissue expression of soluble TGFßrIII receptor in women with early-stage breast cancer and in healthy women: a prospective observational study.

BACKGROUND: Mammary carcinogenesis is partly regulated by the transforming growth factor beta (TGFß) signaling pathway. Its function in cancer progression and metastasis is highly dependent on disease stage, and it is likely modulated by the ratio of membrane-bound vs. soluble TGFßrIII (sTGFßrIII). In this prospective observational study, we assessed tissue expression and plasma levels of sTGFßrIII in healthy women, women with benign breast lesions and in early-stage breast cancer patients. METHODS: In a preliminary study, plasma sTGFßrIII levels were determined in 13 healthy women (age 19-40 years) at different phases of the ovarian cycle, and in 15 patients (age 35-75 years) at different times of the day. The main study assessed plasma concentrations of sTGFßrIII in: (i) 158 healthy women in whom breast lesions were excluded; (ii) 65 women with benign breast lesions; (iii) 147 women with newly diagnosed breast cancer classified as American Joint Committee on Cancer (AJCC) stages 0 to IIB. Completers provided blood samples before surgery and at 10-30 and 160-180 days after surgery. Plasma sTGFßrIII concentrations were determined using an indirect ELISA kit. Part of the removed tissues underwent immunohistochemical (IHC) staining and analysis of tissue TGFßrIII expression. RESULTS: There appeared no relevant variations in plasma sTGFßrIII levels at different times of the day or different ovarian cycle phases. Before surgery, breast cancer patients had somewhat higher sTGFßrIII than healthy women, or those with benign breast lesions (by 14.5 and 26 ng/mL, respectively), with a tendency of larger differences at higher age. This correlated with lower expression of TGFßrIII in breast cancer vs. healthy tissue samples. At 160-180 days after surgery, plasma sTGFßrIII levels in breast cancer patients declined by 23-26 ng/mL. CONCLUSIONS: Plasma sTGFßrIII levels do not seem to relevantly vary during the day or the ovarian cycle. The coinciding higher plasma levels in newly diagnosed cancer patients than in healthy subjects and lower TGFßrIII expression in the malignant than in healthy breast tissue suggest ectodomain shedding as a source of circulating sTGFßrIII. Decline in plasma levels after tumor removal supports such a view.

The level of serum pro-matrix metalloproteinase-2 as a prognostic factor in patients with invasive ductal breast cancer.

This paper analyses data of 150 female patients undergoing surgical treatment for invasive ductal breast cancer at the University Hospital for Tumors from January 2006 to January 2007. The control group consisted of 50 healthy women. The patients were classified into three groups, depending on their tumor differentiation, i.e. grade I, II and III tumor groups. Each group consisted of 50 patients. Traditional prognostic factors including: age, tumor size and differentiation grade, axillary lymph node status, presence of distant metastases, steroid receptor findings, vascular invasion of the primary tumor, presence of an extensive intraductal component (EIC) in the primary tumor, HER-2 protein expression were evaluated. Both the patients' and controls' serum levels of proMMP-2 (pro-matrix metalloproteinase-2) were assessed using the ELISA method. The aim of the study was to assess pathohistological prognostic factors and the level of serum proMMP-2 in the three patient groups and the controls, compare the relationship between the prognostic factors and the level of serum proMMP-2 in the patient groups, and upon the results, determine possible features of proMMP-2 as a prognostic factor in breast cancer patients. The study results showed no difference in proMMP-2 concentrations between the three patient groups and the controls. No statistically significant difference in the serum proMMP-2 concentration was found between the patient groups, although the grade III group values were the highest showing a trend toward statistical significance. Comparison of proMMP-2 and prognostic factors revealed a statistically significant correlation between proMMP-2 and age in patients with histologic grade I tumors. There was no statistically significant correlation between circulating proMMP-2 and other pathohistological prognostic factors.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies.

BACKGROUND: Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. METHODS: After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. RESULTS: Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. CONCLUSION: This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

Prognostic significance of matrix metalloproteinases 2 and 9 in endometrial cancer.

We investigated the prognostic significance of matrix metalloproteinases 2 (MMP 2) and 9 (MMP 9) in endometrial cancer (EC). The expression of MMP 2 and MMP 9 was analyzed immunohistochemically in 73 primary EC patients. In most cases, the gelatinases were predominantly localized to epithelial cell of tumor origin. In univariate analysis histological type, tumor grade, FIGO (1988) surgical stage and high stromal MMP 2 expression were identified as a significant determinant for EC recurrence, while epithelial MMP 2 expression and epithelial and stromal MMP 9 expression were not. Multivariate analysis revealed a subgroup of patient age > or = 63.6 years with endometrioid adenocarcinoma and papillary serous carcinoma, all FIGO (2009) stage I disease where strong staining of stromal MMP 2 increase risk of EC recurrence (p = 0.037).

Prognostic value of lymphovascular space invasion in endometrial cancer.

AIM: To evaluate the prognostic significance of lymphovascular space invasion (LVSI) in endometrial cancer (EC) patients. METHODS: A total of 73 patients who underwent surgery for endometrial cancer at the University Hospital for Tumors, Zagreb, Croatia and University Hospital Centre Split, Croatia, in the period from 1999 to 2008 were evaluated. RESULTS: Lymphovascular space invasion was identified in 58 cases (79.5%). Univariate analysis and multivariate analysis demonstrated a significant association between LVSI and EC recurrence (p=0.004; HR 0.241, CI 0.114-0.511). The median time from diagnosis to EC recurrence of patients with positive LVSI was 20 months, and the median time from diagnosis to EC recurrence of patients with negative LVSI was 86 months (relative risk of recurrence 0.24). CONCLUSION: Lymphovascular space invasion identified on routine haematoxylin and eosin stained sections, is a significant independent predictor of endometrial cancer recurrence.