Breast cancer expression of YKL-40 correlates with tumour grade, poor differentiation, and other cancer markers.

BACKGROUND: Serum levels of a secreted glycoprotein YKL-40 are elevated in patients with a wide range of cancers including breast, colorectal, and ovarian cancers. Furthermore, these increased levels correlate with poorer survival of cancer patients, suggesting that serum levels of YKL-40 might be a prognostic biomarker. However, the tissue expression of YKL-40 and its relationship with clinical outcomes and other potential markers are poorly understood. METHODS: Tissue samples from invasive breast cancers, breast ductal carcinoma in situ (DCIS), and cancer-free reduction mammoplasty were enrolled. YKL-40 expression was measured using immunohistochemistry and evaluated by a semi-quantification assay. Statistical analyses explored the relationship of YKL-40 with clinical outcome and other breast cancer biomarkers. RESULTS: Breast ductal carcinoma in situ expressed low and moderate levels of YKL-40. In the subset of 203 patients with invasive cancer, YKL-40 levels were positively correlated with tumour grade (P<0.0001) and Her2/neu (P<0.01), but negatively correlated with oestrogen (P<0.0001) and progesterone receptor (P<0.0001). YKL-40 levels were inversely correlated with expressions of GATA3 (P=0.0137) and E-cadherin (P=0.0417). CONCLUSION: These data demonstrate that expression levels of YKL-40 are associated with tumour grade, poor differentiation, and other breast cancer markers, highlighting that tissue levels of YKL-40 serve as a valuable biomarker for breast cancer diagnosis and prognosis.

Mechanistic modelling of dynamic MRI data predicts that tumour heterogeneity decreases therapeutic response.

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) contains crucial information about tumour heterogeneity and the transport limitations that reduce drug efficacy. Mathematical modelling of drug delivery and cellular responsiveness based on underutilised DCE-MRI data has the unique potential to predict therapeutic responsiveness for individual patients. METHODS: To interpret DCE-MRI data, we created a modelling framework that operates over multiple time and length scales and incorporates intracellular metabolism, nutrient and drug diffusion, trans-vascular permeability, and angiogenesis. The computational methodology was used to analyse DCE-MR images collected from eight breast cancer patients at Baystate Medical Center in Springfield, MA. RESULTS: Computer simulations showed that trans-vascular transport was correlated with tumour aggressiveness because increased vessel growth and permeability provided more nutrients for cell proliferation. Model simulations also indicate that vessel density minimally affects tissue growth and drug response, and nutrient availability promotes growth. Finally, the simulations indicate that increased transport heterogeneity is coupled with increased tumour growth and poor drug response. CONCLUSION: Mathematical modelling based on DCE-MRI has the potential to aid treatment decisions and improve overall cancer care. This model is the critical first step in the creation of a comprehensive and predictive computational method.

Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice.

BACKGROUND: An effective cancer therapeutic must selectively target tumours with minimal systemic toxicity. Expression of a cytotoxic protein using Salmonella typhimurium would enable spatial and temporal control of delivery because these bacteria preferentially target tumours over normal tissue. METHODS: We engineered non-pathogenic S. typhimurium to secrete murine TNF-related apoptosis-inducing ligand (TRAIL) under the control of the prokaryotic radiation-inducible RecA promoter. The response of the RecA promoter to radiation was measured using fluorometry and immunoblotting. TRAIL toxicity was determined using flow cytometry and by measuring caspase-3 activation. A syngeneic murine tumour model was used to determine bacterial accumulation and the response to expressed TRAIL. RESULTS: After irradiation, engineered S. typhimurium secreted TRAIL, which caused caspase-3-mediated apoptosis and death in 4T1 mammary carcinoma cells in culture. Systemic injection of Salmonella and induction of TRAIL expression using 2 Gy gamma-irradiation caused a significant delay in mammary tumour growth and reduced the risk of death by 76% when compared with irradiated controls. Repeated dosing with TRAIL-bearing Salmonella in conjunction with radiation improved the 30-day survival from 0 to 100%. CONCLUSION: These results show the pre-clinical utility of S. typhimurium as a TRAIL expression vector that effectively reduces tumour growth and extends host survival.

Incidence and therapeutic implications of synchronous colonic pathology in colorectal adenocarcinoma.

BACKGROUND: The presence of synchronous benign and malignant colonic pathology may influence the magnitude of surgery for colorectal adenocarcinoma. The aim of this prospective study was to quantitate the need for a more extensive surgical procedure because of synchronous pathology in colorectal cancer patients. METHODS: Between 1984 and 1996, 235 consecutive patients were treated for colorectal adenocarcinoma. Preoperative survey of the colon in 228 patients included colonoscopy (91%) and double contrast barium enema (35.7%). Seven patients were excluded for incomplete preoperative survey because of perforating or obstructing colon carcinoma or acute ulcerative colitis. RESULTS: One hundred four patients (45.6%) had the following synchronous colonic lesions: benign polyps (68 patients, 29.8%), diverticular disease (30, 13.1%), ulcerative colitis (10, 4.4%), synchronous adenocarcinoma (8, 3.5%), and Crohn's colitis (3, 1.3%). Pathologic examination demonstrated three additional synchronous adenocarcinomas for a total of 11 patients (4.9%). Twenty-five (11%) required more extensive surgery than dictated by the primary cancer. Of these 25 patients, 17 had a benign or premalignant condition associated with their carcinoma and 8 had a synchronous carcinoma. Seventeen patients underwent a sphincter-saving procedure. Of the remaining eight patients requiring sphincter ablation, seven were needed because of a synchronous nonmalignant lesion, rather than because of the primary tumor. CONCLUSIONS: In our patient population, the incidence of synchronous colorectal lesions was 45.6%. Synchronous colorectal cancer occurred in 4.9%. In 11%, the presence of synchronous colorectal lesions made the surgical procedure more extensive than that dictated by the primary cancer, and in 3%, the need for a sphincter ablating procedure was dictated by a synchronous nonmalignant lesion.

Introduction of human adenomatous polyposis coli gene into Min mice via cationic liposomes.

BACKGROUND: The adenomatous polyposis coli (APC) gene is a tumor-suppressor gene involved in familial polyposis coli (FAP), a hereditary disease heralded by the development of hundreds of colorectal adenomas. A mouse model for FAP, the multiple intestinal neoplasia (Min) mouse, develops multiple adenomatous polyps of the large and small intestine similar to their human counterparts. To test the feasibility of introducing normal human APC as a means of either preventing or reversing polyp formation, we describe a method of in vivo transfection of APC into colonic epithelium of the Min mouse. METHODS: Anesthetized young (4 weeks) Min mice were treated with enemas containing lipofectant and a normal human APC cDNA plasmid every 72 hours for 2 months and then euthanized at 24, 48, and 72 hours after the last treatment. Polymerase chain reaction (PCR) was used to detect the presence of the plasmid DNA. RESULTS: PCR on the extracted colonic epithelial DNA showed the presence of plasmid DNA up to 72 hours after the last treatment. Expression of the plasmid construct was confirmed by reverse transcriptase-PCR. CONCLUSIONS: We have demonstrated the repeated introduction and detection of normal human APC in the colonic epithelium of the Min Mouse in vivo during an extended period of time with no toxic side effects by means of our prolonged therapy.

Total mesenteric excision in the surgical treatment of rectal cancer: a prospective study.

BACKGROUND: Total mesorectal excision has been advocated in conjunction with low anterior or abdominoperineal resection as the optimal surgical treatment for rectal cancer. It involves removal of the entire rectal mesentery as an intact unit and maximizes the likelihood of obtaining a negative circumferential margin. OBJECTIVES: To prospectively validate the efficacy of total mesorectal excision in obtaining locoregional control, to identify the perioperative factors influencing the selection of either a sphincter sparing or a sphincter ablating procedure, and to identify independent factors that may influence long-term prognosis in rectal cancers. SETTINGS: Tertiary referral center. PATIENTS: Seventy-three consecutive patients with rectal cancer located within 10 cm of the anal verge were treated from 1984 to 1997 by the senior author (F.M.). Sixty-five patients form the basis of our analysis after the exclusion of 7 patients who had their cancer removed transanally and 1 patient who had a permanent diverting stoma as the only procedure. RESULTS: Twenty-six patients underwent a sphincter ablating procedure; 39 underwent a sphincter sparing procedure. Operative mortality was 1.5%. Follow-up was complete in 64 patients (39+/-30 months; range, 3-126 months). Five-year actuarial survival rates were 88% for the 34 patients with stage I and II adenocarcinoma and 65% for the 22 patients with stage III adenocarcinoma. The local recurrence rate was 6.2% overall, but only 3.1% in the potentially curable group (stages I-III). When only patients who did not receive adjuvant chemoradiation therapy were considered (n=23), local recurrence rate was 8.3% overall and 0% in the potentially curable group. Tumor stage (P=.04) and vascular and/or lymphatic invasion (P=.002) were statistically significant in their association with survival. Circumferential lesions (P<.001), gross invasion of contiguous organs (P<.001) and distance from the anal verge of less than 5 cm (P=.01) were statistically significant in their association with the choice of a sphincter ablating procedure. CONCLUSIONS: This study confirms the efficacy of total mesorectal excision in minimizing locoregional recurrence rates and confirms the well-established prognostic value of stage and microinvasion. Moreover, it indicates that circumferential lesions, distance from anal verge, and gross invasion of contiguous organs are significant perioperative factors in the selection of the type of surgical procedure.

In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis.

Motile bacteria can overcome diffusion resistances that substantially reduce the efficacy of standard cancer therapies. Many reports have also recently described the ability of Salmonella to deliver therapeutic molecules to tumors. Despite this potential, little is known about the spatiotemporal dynamics of bacterial accumulation in solid tumors. Ultimately this timing will affect how these microbes are used therapeutically. To determine how bacteria localize, we intravenously injected Salmonella typhimurium into BALB/c mice with 4T1 mammary carcinoma and measured the average bacterial content as a function of time. Immunohistochemistry was used to measure the extent of apoptosis, the average distance of bacteria from tumor vasculature and the location of bacteria in four different regions: the core, transition, body and edge. Bacteria accumulation was also measured in pulmonary and hepatic metastases. The doubling time of bacterial colonies in tumors was measured to be 16.8 h, and colonization was determined to delay tumor growth by 48 h. From 12 and 48 h after injection, the average distance between bacterial colonies and functional vasculature significantly increased from 130 to 310 µm. After 48 h, bacteria migrated away from the tumor edge toward the central core and induced apoptosis. After 96 h, bacteria began to marginate to the tumor transition zone. All observed metastases contained Salmonella and the extent of bacterial colocalization with metastatic tissue was 44% compared with 0.5% with normal liver parenchyma. These results demonstrate that Salmonella can penetrate tumor tissue and can selectively target metastases, two critical characteristics of a targeted cancer therapeutic.

YKL-40, a secreted glycoprotein, promotes tumor angiogenesis.

Tumor angiogenesis is of paramount importance in solid tumor development. Elevated serum levels of YKL-40, which is a secreted heparin-binding glycoprotein, have been associated with a worse prognosis from various advanced human cancers. Yet the role of YKL-40 activity in these cancers is still missing. In this study, we showed that ectopic expression of YKL-40 in MDA-MB-231 breast cancer cells and in HCT-116 colon cancer cells led to larger tumor formation with an extensive angiogenic phenotype than did control cancer cells in mice. Affinity-purified recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis in vitro, the effects of which are similar to the activities observed using MDA-MB-231 and HCT-116 cell-conditioned medium after transfection with YKL-40. Furthermore, YKL-40 was found to induce coordination of membrane-bound receptor syndecan-1 and integrin alpha(v)beta(3) and to activate an intracellular signaling cascade, including focal adhesion kinase and mitogen-activated protein kinase extracellular signal-related kinase1/2 in endothelial cells. Moreover, blockade of YKL-40 using small-interfering RNA gene knockdown suppressed tumor angiogenesis in vitro and in vivo. Immunohistochemical analysis of human breast cancer showed a correlation between YKL-40 expression and blood vessel density. These findings provide novel insights into angiogenic activities and molecular mechanisms of YKL-40 in cancer development.

Selective expression of carcinoembryonic antigen promoter in cancer cell lines: targeting strategy for gene therapy in colorectal cancer.

PURPOSE: This study was designed to characterize the mechanisms regulating the expression of the human carcinoembryonic antigen promoter (pCEA), in terms of tissue-specific targeting for gene therapy. The promoter was subcloned to a luciferase reporter gene (pCEA/Luc) in our laboratory and compared with a virally controlled luciferase vector (pSV40/Luc). METHODS: Four human cancer cell lines (HeLa, SW480, Caco2, and SW1116) were transfected with either pCEA/Luc or pSV40/Luc. Cells were treated with interferon-gamma and assayed at 72 hours after treatment. Carcinoembryonic antigen level was measured by enzyme immunoassay. Luciferase expression was measured at 48 hours and one week after transfection by luminometry. RESULTS: Luciferase activity after transfection with pCEA/Luc was higher in CEA-positive cells than in CEA-negative cells (P < 0.0001). pCEA/Luc demonstrated higher activity than pSV40/Luc in CEA-positive cells (P < 0.0001), but not in CEA-negative cells. In Caco2 cells, which before confluence are CEA-negative, luciferase expression increased on reaching confluence (P < 0.0001). Well to moderately differentiated cells responded to the interferon-gamma treatment, but the increase in CEA secretion did not correspond to an increase in pCEA/Luc expression. CONCLUSIONS: The expression of pCEA correlates well with the CEA production by the specific cell line offering a potential tissue-specific targeting strategy for colon cancer gene therapy. Furthermore, the tissue-specific CEA promoter has a higher and more persistent activity in CEA-positive human cancer cells than a viral promoter. The lack of response to interferon-gamma treatment suggests a different mechanism of action for interferon-gamma other than directly interacting with the promoter.

Quantitation of in vivo gene delivery by restriction enzyme PCR generated polymorphism.

The Multiple intestinal neoplasia (Min) mouse develops multiple polyps in the intestine, due to a heterozygous mutation of the Apc locus. Our laboratory has been introducing normal human adenomatous polyposis coli (APC) gene into the Min mouse through liposome enema to prevent or reverse polyp formation. We have quantitated the amount of normal human APC gene delivered in vivo by a restriction enzyme site specific quantitative PCR. Adult Min and BALB/C mice were treated with lipofectant and human APC complementary DNA (cDNA) plasmid. Min colonic DNA was amplified using primers for Apc nucleotide 2524F (5'2524-TCTCGTTCTGAGAAAGACAGAAGCT) and 2679R (5"2679-TGATACTTCTTCCAAAGCTTTGGCTAT). Highlighted primer sequences were purposely different so as to generate two HindIII restriction enzyme sites in the presence of normal mouse Apc (Apc+). Genomic DNA from untreated Min colonic epithelium revealed two bands: 144 bp for ApcMin and 123 bp for Apc+. BALB/C DNA was amplified using primers flanking a region within the APC gene containing a HindIII site on the human APC, which is absent in the murine APC (Apc). Min's DNA extracted 24 hr after treatment demonstrated a plasmid content of 3% due to a relative increase in the Apc+ (123 bp) band. Six weeks of treatments increased delivery to 10%. APC gene therapy of colonic epithelium can be quantitatively measured through restriction enzyme quantitative PCR. Long-term treatment further increases gene delivery. PCR generated polymorphism is a reliable and reproducible technique to initially optimize transfection conditions and ultimately quantitate efficacy in an in vivo gene delivery model.

Human APC gene expression in rodent colonic epithelium in vivo using liposomal gene delivery.

Mutations or loss of the APC tumor-suppressor gene is important for the development of colorectal polyps and cancers, but little is known about the function of this gene in normal tissue. To study the role of APC and other genes in colonocytes in vivo, a system was developed whereby transient expression of genes is established in normal rodent colonic epithelium, using liposomal gene delivery by rectal catheter infusion. Expression of a beta-galactosidase reporter gene and of the human APC gene under a constitutive promoter is demonstrated. A high efficiency of transfection is maintained, with close to 100% of epithelial cells expressing the introduced gene. Expression is transient and does not persist beyond 4 days, consistent with the normal turnover time of gut epithelium, but it can be maintained by repeated treatments. Human APC was expressed for three weeks under these conditions at approximately one-tenth the level of the endogenous APC gene, and no toxicity was observed beyond that attributed to repeated rectal enemas. These results reveal that in vivo expression of exogenous gene is feasible using a liposomal delivery system and suggest a method to further study the physiologic role of APC or other genes in the interrelated process of colonic epithelial proliferation and differentiation.