Genetic variants of GPX1 and SOD2 and breast cancer risk at the Ontario site of the Breast Cancer Family Registry.

There are several genes that code for enzymes, including various forms of superoxide dismutase and glutathione peroxidase, that protect the cell against oxidative damage that, in turn, can lead to carcinogenesis. There are a few common genetic polymorphisms in these genes that lead to altered proteins. Three that have been identified are SOD2 Val-9Ala, GPX1 Pro198Leu, and the GPX1 GCG repeat (three alleles with four, five, or six repeats). The SOD2 variant has been associated with increased breast cancer risk in two studies. The GPX1 variants have not been studied with respect to breast cancer, but Pro198Leu has been associated with lung cancer. We conducted a case-control study of these three polymorphisms in incident, invasive breast cancer in Caucasian women under 55. There were 399 cases and 372 controls genotyped, of whom 488 were premenopausal, 208 postmenopausal, and 75 of unknown menopausal status. We were unable to replicate the previously observed association with SOD2 Val-9Ala and also found no association between breast cancer and GPX1 Pro198Leu. However, the allele of GPX1 containing four GCG repeats was significantly associated with breast cancer risk in premenopausal women (odds ratio, 1.55; 95% confidence interval, 1.04-2.30 for carriers versus noncarriers). There is a significant trend of increasing risk with increasing number of alleles with four GCG repeats (P = 0.03). This variant has not previously been reported to be associated with breast cancer.     

Expression of constitutively active Notch4 (Int-3) modulates myeloid proliferation and differentiation and promotes expansion of hematopoietic progenitors.

The Notch family of transmembrane receptors has been implicated in the regulation of many developmental processes. In this study, we evaluated the role of Notch4 in immature hematopoietic progenitors by inducing, with retroviral transduction, enforced expression of Int-3, the oncogenic and constitutively active form of mouse Notch4. Int-3-transduced human myeloid leukemia (HL-60) cells demonstrated significantly delayed expression of differentiation markers following retinoic acid and 12-0-tetradecanoylphorbol 13-acetate treatment. Furthermore, HL-60 cells expressing Int-3 displayed a slower growth rate than cells infected with void virus, and accumulation in the G0/G1 phases of cell cycle. Transduction with deletion mutants of Int-3 defined the importance of individual domains of the protein (in particular, the ANK domain and the C-terminal domain) in the inhibition of differentiation and growth arrest of HL-60 cells. When mouse bone marrow enriched for stem cells (5-fluorouracil-resistant, lineage negative) was transduced and cultured for two weeks, the Int-3-transduced population displayed a lower expression of differentiation markers and a three- to five-fold higher frequency of colony-forming cells (CFU-GM/BFU-E) than control cultures. These results strongly support the notion that Notch signaling inhibits differentiation and promotes expansion of hematopoietic stem/progenitor cells.     

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.     

Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, in combination with gemcitabine for advanced pancreatic cancer: a multicenter phase II Trial.

PURPOSE: To determine the response rate, time to disease progression, survival duration and rate, and toxicity with the combination of cetuximab and gemcitabine in patients with epidermal growth factor receptor (EGFR)-expressing advanced pancreatic cancer. PATIENTS AND METHODS: Patients with measurable locally advanced or metastatic pancreatic cancer who had never received chemotherapy for their advanced disease and had immunohistochemical evidence of EGFR expression were eligible for the multicenter phase II trial. Patients were treated with cetuximab at an initial dose of 400 mg/m(2), followed by 250 mg/m(2) weekly for 7 weeks. Gemcitabine was administered at 1,000 mg/m(2) for 7 weeks, followed by 1 week of rest. In subsequent cycles, cetuximab was administered weekly, and gemcitabine was administered weekly for 3 weeks every 4 weeks. RESULTS: Sixty-one patients were screened for EGFR expression, 58 patients (95%) had at least 1+ staining, and 41 were enrolled onto the trial. Five patients (12.2%) achieved a partial response, and 26 (63.4%) had stable disease. The median time to disease progression was 3.8 months, and the median overall survival duration was 7.1 months. One-year progression-free survival and overall survival rates were 12% and 31.7%, respectively. The most frequently reported grade 3 or 4 adverse events were neutropenia (39.0%), asthenia (22.0%), abdominal pain (22.0%), and thrombocytopenia (17.1%). CONCLUSION: Cetuximab in combination with gemcitabine showed promising activity against advanced pancreatic cancer. Further clinical investigation is warranted.     

Immunomodulation for intestinal transplantation by allograft irradiation, adjunct donor bone marrow infusion, or both

BACKGROUND: The passenger leukocytes in the intestine have a lineage profile that predisposes to graft-versus-host disease (GVHD) in some animal models and have inferior tolerogenic qualities compared with the leukocytes in the liver, other solid organs, and bone marrow. Elimination by ex vivo irradiation of mature lymphoid elements from the bowel allografts is known to eliminate the GVHD risk. We hypothesized that infusion of donor bone marrow cells (BMC) in recipients of irradiated intestine would improve tolerogenesis without increasing the risk of GVHD. METHODS: Orthotopic small intestine transplantation was performed with the GVHD-prone Lewis (LEW)-to-Brown Norway (BN) combination and the reverse GVHD-resistant BN-to-LEW model under a short course of tacrolimus treatment (1 mg/kg/day, days 0-13, 20, 27). Grafts were irradiated ex vivo, using a 137Cs source. In selected experimental groups, donor BMC (2.5 x 10(8)) were infused on the day of small intestine transplantation. RESULTS: The unmodified LEW intestine remained intact, whether transplanted alone or with adjunct donor BMC infusion, but all of the BN recipients died of GVHD after approximately 2 months. Intestinal graft irradiation (10 Gy) effectively prevented the GVHD and prolonged survival to 92.5 days, but all of the BN recipients died with chronic rejection of the LEW grafts, which was prevented by infusion of adjunct donor BMC without causing GVHD. In the GVHD-resistant reverse strain direction (BN-->LEW), all intestinal recipients treated for 27 days with tacrolimus survived > or =150 days without regard for graft irradiation or adjunct BMC, but chronic rejection was severe in the irradiated intestine, moderate in the unaltered graft, and least in the irradiated intestine transplanted with adjunct BMC. Mild arteritis in the 150 day allografts of both strain combinations (i.e., LEW--> BN and BN-->LEW) may have been irradiation associated, but this was prevented when weekly doses of tacrolimus were continued for the duration of the experiment rather than being stopped at 27 days. CONCLUSIONS: Recipients are protected from GVHD by irradiating intestinal allografts, but the resulting leukocyte depletion leads to chronic rejection of the transplanted bowel. The chronic rejection is prevented with adjunct donor BMC without causing GVHD. Although application of the strategy may be limited by the possibility of radiation injury, the results are consistent with the paradigm that we have proposed to explain organ-induced graft acceptance, tolerance, and chronic rejection.     

Morphologic changes in efferent ductules and epididymis in estrogen receptor-alpha knockout mice

Estrogen has been shown to have an important role in fluid reabsorption in efferent ductules of the testis. Our previous study of the estrogen receptor-alpha knockout mouse (ERKO) showed that the efferent ductules and rete testis were primary targets of estrogen receptor function. In the present study, a more comprehensive evaluation of the ERKO male reproductive tract was performed to determine the severity of effects in efferent ductules as well as the epididymis. The following observations were found in ERKO males: 1) blind-ending efferent ductules were more prevalent in ERKO than in wild type (WT) tissues; 2) glycogen-containing cells were observed at the rete testis-efferent ductule junction; 3) the tubular diameters of the efferent ductules and initial segment epididymides were dilated; 4) efferent ductules were dilated between 130 to 300% over wild type ductules; 5) efferent ductule epithelial height was reduced nearly 50%; 6) microvilli of nonciliated cells of efferent ductules were 64% shorter in length; 7) cilia were reduced in number; 8) initial segment epithelium was displaced into regions adjacent to the rete testis and in short segments of the common region of efferent ductule; 9) apical, narrow, and clear cells of the epididymis also were abnormal in some regions; 10) in the corpus and cauda regions, sperm granulomas were noted in one third of the ERKO males. In conclusion, the entire reproductive tract is affected in ERKO males. The cells showing the greatest effects were estrogen receptor-positive cells. It appears that in the ERKO mouse there are developmental anomalies that must be considered separately from adult dysfunctional changes in the male reproductive tract.