Julia Beretov

Prediction of Local Recurrence, Distant Metastases, and Death After Breast-Conserving Therapy in Early-Stage Invasive Breast Cancer Using a Five-Biomarker Panel

PURPOSE To determine the clinical utility of intrinsic molecular phenotype after breast-conserving therapy (BCT) with lumpectomy and whole-breast irradiation with or without a cavity boost. PATIENTS AND METHODS Four hundred ninety-eight patients with invasive breast cancer were enrolled into a randomized trial of BCT with or without a tumor bed radiation boost. Tumors were classified by intrinsic molecular phenotype as luminal A or B, HER-2, basal-like, or unclassified using a five-biomarker panel: estrogen receptor, progesterone receptor, HER-2, CK5/6, and epidermal growth factor receptor. Kaplan-Meier and Cox proportional hazards methodology were used to ascertain relationships to ipsilateral breast tumor recurrence (IBTR), locoregional recurrence (LRR), distant disease-free survival (DDFS), and death from breast cancer. RESULTS Median follow-up was 84 months. Three hundred ninety-four patients were classified as luminal A, 23 were luminal B, 52 were basal, 13 were HER-2, and 16 were unclassified. There were 24 IBTR (4.8%), 35 LRR (7%), 47 distant metastases (9.4%), and 37 breast cancer deaths (7.4%). The overall 5-year disease-free rates for the whole cohort were: IBTR 97.4%, LRR 95.6%, DDFS 92.9%, and breast cancer-specific death 96.3%. A significant difference was observed for survival between subtypes for LRR (P = .012), DDFS (P = .0035), and breast cancer-specific death (P = .0482), but not for IBTR (P = .346). CONCLUSION The 5-year and 10-year survival rates varied according to molecular subtype. Although this approach provides additional information to predict time to IBTR, LRR, DDFS, and death from breast cancer, its predictive power is less than that of traditional pathologic indices. This information may be useful in discussing outcomes and planning management with patients after BCT.

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression

Background:The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP).Methods:We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC50) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined.Results:CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC50) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression.Conclusions:Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.

Malignant phyllodes tumours of the breast display increased stromal p53 protein expression

To determine the variation in p53 protein expression in phyllodes tumours and fibroadenomas of the breast.

CD44 variant 6 is associated with prostate cancer metastasis and chemo‐/radioresistance

Prostate cancer (CaP) is the second leading malignancy in older men in Western countries. The role of CD44 variant 6 (CD44v6) in CaP progression and therapeutic resistance is still uncertain. Here, we investigated the roles of CD44v6 in CaP metastasis and chemo/radioresistance. Expression of CD44v6 in metastatic CaP cell lines, human primary CaP tissues and lymph node metastases was assessed using immunofluorescence and immunohistochemistry, respectively.

In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.

Bismuth-213 radioimmunotherapy with C595 anti–MUC1 monoclonal antibody in an ovarian cancer ascites model

Purpose: Control of ovarian cancer (OC) ascites remains a major objective in post-surgical treatment. The aim of this study was to investigate the effect of targeted alpha therapy (TAT) for the control of ascites in an OC ascites mouse model; the biodistribution of 213Bi-C595 and its long term toxicity. Methods: The expression of tumor-associated antigen mucin-1 (MUC-1) in OVCAR3 ascites cells in mice and OC cancer tissues in patients was detected by indirect immmunostaining. The monoclonal antibody (MAb) C595 was labeled with 213Bi using the chelator cDTPA to form the alpha-immunoconjugate (AIC). Mice were injected with different concentrations of AIC by i.p administration. Changes in tumor progression were assessed by measurement of the circumference of the abdomen. Results: MUC-1 is strongly expressed in 73% of OC tissues. At 9 days post-cell inoculation in mice, a single injection of 355 MBq/kg of 213Bi-C595 can prolong survival by 25 days. A high tumor: blood ratio (5.8) was found in biodistribution study. The maximum tolerance dose (MTD) was more than 1180 MBq/kg up to 21 weeks. Conclusions: C595 is a specific targeting vector for ovarian cancer cells, which show a high percentage of expression of MUC1. 213Bi-C595 can effectively target and kill ovarian cancer cells in vitro and in vivo. 213Bi-C595 is the recommended alpha conjugate for a Phase I clinical trial for ovarian cancer.

Expression of urokinase plasminogen activator and its receptor in advanced epithelial ovarian cancer patients

BACKGROUND The urokinase plasminogen activator (uPA) system has been implicated in progression and poor prognosis in epithelial ovarian cancer (EOC) patients. The present study investigated the distribution of uPA and its receptor (uPAR) in EOC cell lines, primary and metastatic tumors, and the relationship between uPA/uPAR and matrix metalloproteinase (MMP) expression using immunohistochemistry. We also studied the association between uPA/uPAR expression and clinical and pathological parameters including disease progression free survival (PFS). METHODS The expression of uPA/uPAR was examined on paraffin-embedded tissue sections from primary EOC (n=100), and matched metastatic lesions (n=30) of untreated patients, normal ovarian tissues (n=20) as well as 8 primary and metastatic EOC cell lines by immunohistochemistry. Co-immunolabeling of uPA and MMP-1, -2, -9 or MT1-MMP was examined using confocal microscopy. RESULTS The expression of uPA/uPAR was found in most primary (92% and 88% positive, respectively), metastatic ovarian tumors (93% and 90% positive, respectively), and all of examined EOC cell lines. The majority of specimens showed moderate to strong immunostaining of tumor and stromal cells; for primary specimens, this was significantly associated with tumor stage, grade and time to relapse (P<0.01). Overexpression of uPA/uPAR was found to be associated with an unfavorable prognosis with significantly reduced median disease PFS of 16 vs. 33 months for uPA (P<0.001), and 15 vs. 28 months for uPAR (P<0.001). Co-localization of uPA with MMP-1, -2, -9 or MT1-MMP was also seen in primary tumors and metastatic lesions. CONCLUSIONS The expression of uPA/uPAR was associated with EOC progression. uPA/uPAR are useful markers for EOC prognosis and could be promising therapeutic targets for treating incurable, recurrent EOC.

Anti-MUC1 Monoclonal Antibody (C595) and Docetaxel Markedly Reduce Tumor Burden and Ascites, and Prolong Survival in an in vivo Ovarian Cancer Model

MUC1 is associated with cellular transformation and tumorigenicity and is considered as an important tumor-associated antigen (TAA) for cancer therapy. We previously reported that anti-MUC1 monoclonal antibody C595 (MAb C595) plus docetaxel (DTX) increased efficacy of DTX alone and caused cultured human epithelial ovarian cancer (EOC) cells to undergo apoptosis. To further study the mechanisms of this combination-mediated apoptosis, we investigated the effectiveness of this combination therapy in vivo in an intraperitoneal (i.p.) EOC mouse model. OVCAR-3 cells were implanted intraperitoneally in female athymic nude mice and allowed to grow tumor and ascites. Mice were then treated with single MAb C595, DTX, combination test (MAb C595 and DTX), combination control (negative MAb IgG3 and DTX) or vehicle control i.p for 3 weeks. Treated mice were killed 4 weeks post-treatment. Ascites volume, tumor weight, CA125 levels from ascites and survival of animals were assessed. The expression of MUC1, CD31, Ki-67, TUNEL and apoptotic proteins in tumor xenografts was evaluated by immunohistochemistry. MAb C595 alone inhibited i.p. tumor growth and ascites production in a dose-dependent manner but did not obviously prevent tumor development. However, combination test significantly reduced ascites volume, tumor growth and metastases, CA125 levels in ascites and improved survival of treated mice compared with single agent-treated mice, combination control or vehicle control-treated mice (P<0.05). The data was in a good agreement with that from cultured cells in vitro. The mechanisms behind the observed effects could be through targeting MUC1 antigens, inhibition of tumor angiogenesis, and induction of apoptosis. Our results suggest that this combination approach can effectively reduce tumor burden and ascites, prolong survival of animals through induction of tumor apoptosis and necrosis, and may provide a potential therapy for advanced metastatic EOC.

Preparation and testing of bevacizumab radioimmunoconjugates with Bismuth-213 and Bismuth-205/Bismuth-206.

Bevacizumab, a humanized anti-VEGF monoclonal antibody has shown promise in various clinical trials. We report the development and testing of Bi-213 (an alpha-emitting radionuclide) labeled bevacizumab for in vitro and in vivo studies using two different chelators viz cDTPA and CHX-A″. The developed labeling method showed high labeling yields of 93.6% and 89.7% for cDTPA and CHX-A″ respectively and the results were reproducible. The in vitro and in vivo stability tests were carried out using Bi-213 and long half-life Bismuth isotope (Bi-205 / Bi-206) for pharmacokinetics. The in vitro results showed remarkable stability of the radiolabeled bevacizumab regardless of the chelator. The in vivo pharmacokinetics studies however, showed that the uptake and retention of cDTPA- bevacizumab was significantly higher in kidneys (p-value 0.02) and lower in liver and spleen (p-value