MicroRNA expression profiling of sputum for the detection of early and locally advanced non-small-cell lung cancer: a prospective case-control study.

BACKGROUND: Non-small-cell lung cancer (nsclc) is associated with very poor overall survival because 70% of patients present with locally advanced or metastatic disease at the time of diagnosis. Micrornas (mirnas) are a class of short, noncoding rna molecules whose presence in samples of biologic fluids such as sputum has demonstrated promise as a potential means of detecting nsclc. We investigated the stage-specific nsclc detection potential of an efficient panel of 3 mirnas (mir-21, mir-210, mir-372) using a single sputum sample. METHODS: A single spontaneously expectorated sputum sample was prospectively collected from 21 early nsclc (≤stage ii) patients, 22 advanced nsclc (≥stage iii) patients, and 10 control subjects. Mirna expression profiles were determined by quantitative real-time polymerase chain reaction and were analyzed by unsupervised hierarchical cluster analysis. RESULTS: Mean tumour size (±95% confidence interval) in the early and advanced nsclc patients was 3.3 cm ± 0.9 cm and 4.8 cm ± 0.7 cm respectively. Adenocarcinoma constituted 61.9% of the early and 45.5% of the advanced nsclc cases respectively. In comparing the early nsclc group with the control group, the mirna panel yielded a diagnostic sensitivity of 67% and a specificity of 90.0%. For the advanced nsclc group, the mirna panel detected nsclc with a sensitivity and specificity of 64% and 100% respectively. CONCLUSIONS: A sputum mir-21, mir-210, and mir-372 expression profile might provide a sensitive and highly specific means for detecting nsclc. Sputum mirna analysis demonstrates promise as a potential complementary screening tool.

Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells.

Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by cancer type. The factors that influence the relative utilization of gene inactivation pathways are poorly understood. In this study, we describe a detailed quantitative analysis of the three major gene inactivation mechanisms for a model gene at two different genomic integration sites in mouse embryonic stem (ES) cells. In addition, we targeted the major DNA methyltransferase gene, Dnmt1, to investigate the relative contribution of DNA methylation to these various competing gene inactivation pathways. Our data show that gene loss is the predominant mode of inactivation of a herpes simplex virus thymidine kinase neomycin phosphotransferase reporter gene (HSV-TKNeo) at the two integration sites tested and that this event is significantly reduced in Dnmt1-deficient cells. Gene silencing by promoter methylation requires Dnmt1, suggesting that the expression of Dnmt3a and Dnmt3b alone in ES cells is insufficient to achieve effective gene silencing. We used a novel assay to show that missense mutation rates are also substantially reduced in Dnmt1-deficient cells. This is the first direct demonstration that DNA methylation affects point mutation rates in mammalian cells. Surprisingly, the fraction of CpG transition mutations was not reduced in Dnmt1-deficient cells. Finally, we show that methyl group-deficient growth conditions do not cause an increase in missense mutation rates in Dnmt1-proficient cells, as predicted by methyltransferase-mediated mutagenesis models. We conclude that Dnmt1 deficiency and the accompanying genomic DNA hypomethylation result in a reduction of three major pathways of gene inactivation in our model system.

p57KIP2 expression and loss of heterozygosity during immortal conversion of cultured human mammary epithelial cells.

We have uncovered a novel role for the cyclin-dependent kinase inhibitor, p57KIP2, during the immortalization of cultured human mammary epithelial cells (HMECs). HMECs immortalized after chemical carcinogen exposure initially expressed little or no telomerase activity, and their telomeres continued to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined to < or = 3 kb exhibited slow heterogeneous growth and contained many nonproliferative cells. These conditionally immortal HMEC cultures accumulated large quantities of p57 protein. With continued passage, the conditionally immortal cell populations very gradually converted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerase activity, and stabilization of telomere length. The fully immortal HMECs that grew well did not accumulate p57 in G0 or during the cell cycle. DNA and RNA analysis of mass populations and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically short telomeres was repeatedly accompanied by loss of the expressed p57 allele and transient expression of the allele imprinted previously. Conditionally immortal 184A1 with mean TRF > 3 kb, infected with retroviruses containing the p57 gene, exhibited premature slow heterogeneous growth. Conversely, exogenous expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, in 184A1 with mean TRF > 3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling populations. These data indicate that in HMECs that have overcome replicative senescence, p57 may provide an additional barrier against indefinite proliferation. Overcoming p57-mediated growth inhibition in these cells may be crucial for acquisition of the unlimited growth potential thought to be critical for malignant progression.

Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents.

Immunoisolation of allogeneic cells within a membrane-bound device is a unique approach for gene therapy. We employed an immunoisolation device that protects allograft, but not xenograft, cells from destruction, to implant a human fibroblast line (MSU 1.2) in athymic rodents. Cells, transduced with the MFG-human factor IX retroviral vector, and expressing 0.9 microg/10(6) cells/day in vitro, were implanted in rats (four 40-microl devices, each containing 2 x 10(7) cells, two subcutaneously, two in epididymal fat) and in mice (two 20-microl devices, each containing 2 x 10(6) cells, subcutaneously). Plasma factor IX levels increased for 50 days, reaching maxima of 203 ng/ml (rat) and 597 ng/ml (mouse), and both continued at greater than 100 ng/ml for more than 140 days. A clone derived from the transduced cells, making 5 microg of factor IX/10(6) cells/day, was implanted within a device (one 20-microl device containing 2.5 x 10(6) cells), or without a device (1 x 10(7) cells implanted freely), either subcutaneously or in epididymal fat. The freely implanted cells expressed transiently, reaching more than 100 ng/ml in each site by day 4, but dropped to zero by day 20 (subcutaneous) or day 90 (epididymal fat). In devices, levels gradually increased to 100 ng/ml (subcutaneous) or 300 ng/ml (epididymal fat), remaining high for more than 100 days. These results show long-term, high-level expression of a human protein: (1) when cells are implanted within a cell transplantation device, but not when the cells are freely implanted, and (2) from a transgene driven by a viral promoter. An alloprotective device will enable the use of cloned cell lines that can be subjected to stringent quality control assessment that is impossible to achieve with autologous approaches.

Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice.

Continuous delivery of factor VIII (FVIII) protein in hemophiliacs by gene therapy will represent a major clinical advance over the current practice of infrequent administration of purified FVIII. Conceptually, retroviral vectors that can permanently insert the FVIII gene into the DNA of the host cell appear the most suitable vehicles for this specific purpose. However, most retroviral vector systems have shown a poor performance in the production of FVIII from primary cells in vitro and in vivo. Here we report the retroviral-mediated gene delivery of a B-domain-deleted human FVIII by using the MFG vector system. This vector permitted efficient transduction of the majority of the primary cells in culture without the use of a selectable marker. High levels of FVIII were produced by various transduced primary cells in vitro. Upon transplantation of primary fibroblasts into mice, therapeutic levels of FVIII in the circulation were obtained for > 1 week. The capacity of primary cells to deliver the FVIII into the circulation was strongly dependent on the site of implantation. These results represent a major step forward in development of gene therapy for treating hemophilia A.

Oligodendroglioma. The Princess Margaret Hospital experience (1958-1984).

BACKGROUND: Oligodendrogliomas are rare central nervous system (CNS) tumors. Although surgery remains the primary treatment, the role of postoperative radiation treatment remains a matter of controversy. This study assesses whether postoperative radiation improves survival and local tumor control when compared with surgical treatment alone. METHODS: The medical records of 72 patients with the diagnosis of oligodendroglioma, registered at the Princess Margaret Hospital (PMH) between 1958 and 1984, were reviewed retrospectively. The data of 68 patients were evaluated, but 4 patients were excluded from the analysis because of inadequate follow-up. The median follow-up period at the time of analysis was 7.5 years. Tumors were classified as oligodendrogliomas, malignant oligodendrogliomas, or mixed oligoastrocytomas. Fifty-eight (85%) patients received postoperative irradiation at initial diagnosis, and 10 (15%) patients were treated with surgery alone. In most patients (76%), the radiation dose was 5000 cGy in 25 fractions given during a period of 5 weeks. RESULTS: Five- and 10-year overall survival was 66% and 30%, respectively. Forty-two (62.0%) patients experienced disease relapse during the follow-up period. The median time to relapse from initial diagnosis was 2.6 years. Most tumor recurrences were at the primary site. Two patients developed distant metastasis within the CNS axis. Histologic subtype was the only significant prognostic factor for survival. The patients with oligodendroglioma and malignant oligodendroglioma had 5-year survival rates of 73% and 32%, respectively (P = 0.033). CONCLUSIONS: The authors drew three conclusions. (1) Most patients eventually died with this tumor from local recurrence without distant metastasis. Local tumor control remains the major problem. (2) Tumor histologic subtype is a significant prognostic factor. (3) No statistically significant benefit can be attributed to postoperative irradiation; however, the number of patients not receiving radiation therapy was small (10). Thus, the role of radiation therapy remains a matter of controversy.

Severe acute thrombocytopenia following infusion of plasma containing anti P1A1.

An 18-year-old girl with a preoperative platelet count of 216,000/cmm received 3 units of stored plasma at the time of surgery. Within 6 hours her platelet count had fallen to 5,000/mm3 and hemorrhagic manifestations appeared. One of the plasma donors was found to be P1A1 negative with an anti P1A1 antibody. The recipient, who was P1A1 positive, recovered normal platelet counts by day 8 post plasma infusion.