CT scan screening for lung cancer: risk factors for nodules and malignancy in a high-risk urban cohort

Background

Low-dose computed tomography (CT) for lung cancer screening can reduce lung cancer mortality. The National Lung Screening Trial reported a 20% reduction in lung cancer mortality in high-risk smokers. However, CT scanning is extremely sensitive and detects non-calcified nodules (NCNs) in 24–50% of subjects, suggesting an unacceptably high false-positive rate. We hypothesized that by reviewing demographic, clinical and nodule characteristics, we could identify risk factors associated with the presence of nodules on screening CT, and with the probability that a NCN was malignant.

Methods

We performed a longitudinal lung cancer biomarker discovery trial (NYU LCBC) that included low-dose CT-screening of high-risk individuals over 50 years of age, with more than 20 pack-year smoking histories, living in an urban setting, and with a potential for asbestos exposure. We used case-control studies to identify risk factors associated with the presence of nodules (n = 625) versus no nodules (n = 557), and lung cancer patients (n = 30) versus benign nodules (n = 128).

Results

The NYU LCBC followed 1182 study subjects prospectively over a 10-year period. We found 52% to have NCNs >4 mm on their baseline screen. Most of the nodules were stable, and 9.7% of solid and 26.2% of sub-solid nodules resolved. We diagnosed 30 lung cancers, 26 stage I. Three patients had synchronous primary lung cancers or multifocal disease. Thus, there were 33 lung cancers: 10 incident, and 23 prevalent. A sub-group of the prevalent group were stable for a prolonged period prior to diagnosis. These were all stage I at diagnosis and 12/13 were adenocarcinomas.

Conclusions

NCNs are common among CT-screened high-risk subjects and can often be managed conservatively. Risk factors for malignancy included increasing age, size and number of nodules, reduced FEV1 and FVC, and increased pack-years smoking. A sub-group of screen-detected cancers are slow-growing and may contribute to over-diagnosis and lead-time biases.     

Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

AIM: To evaluate the ability of anti-ricin A-chain antibodies, delivered intracellularly, to protect against ricin-induced cytotoxicity in RAW264.7 cells.METHODS: Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide. RAW264.7 cells were incubated with these antibodies either before or after ricin exposure. The changes in cytotoxicity were estimated by MTT assay. Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy.RESULTS: Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies. Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells.CONCLUSION: Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for post-exposure treatment of ricin intoxication.     

Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression.

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.     

Overexpression of WISP-1 down-regulated motility and invasion of lung cancer cells through inhibition of Rac activation

Wnt-induced-secreted-protein-1 (WISP-1) is a cysteine-rich, secreted factor belonging to the CCN family. These proteins have been implicated in the inhibition of metastasis; however, the mechanisms involved have not been described. We demonstrated that overexpression of WISP-1 in H460 lung cancer cells inhibited lung metastasis and in vitro cell invasion and motility. We investigated the possibility that WISP-1 may regulate activation of Rac, a small GTPase important for cytoskeletal reorganizations during motility. In an indirect assay, WISP-1-expressing cells exhibited marked reduction in Rac activation compared with control cells. Blocking antibodies to alpha(v)beta(5) and alpha(1) integrins restored Rac activation in WISP-1 cells, suggesting that the inhibitory effect of WISP-1 on Rac lies downstream of integrins. Constitutively activated Rac mutant (RacG12V) was transfected into WISP-1 cells to restore Rac activation and these WISP-1/RacG12V transfectants were used for further studies. We performed microarray and real-time PCR analyses to identify genes involved in invasion that may be differentially regulated by WISP-1. Here, we showed decreased expression of metalloproteinase-1 (MMP-1) in WISP-1 cells compared with controls but increased expression in WISP-1/RacG12V cells. In an invasion assay across collagen I, an MMP-1 target matrix, WISP-1 cells were significantly less invasive compared with controls, whereas WISP-1/RacG12V cells showed elevated invasion levels. This work illustrates a negatively regulated pathway by WISP-1 involving integrins and Rac in the down-regulation of invasion.     

Altered expression of protein kinase C, lck, and CD45 in a 12-O-tetradecanoylphorbol-13-acetate-dependent leukemic T-cell variant that expresses a high level of interleukin-2 receptor.

The compound 12-O-tetradecanoylphorbol-13-acetate (TPA) is extremely toxic to the P13 subclone of the Jurkat human T-cell leukemia line. By selecting for growth in the presence of TPA, we have isolated two TPA-resistant variants of these cells, P13-50 and P13-5/A8. Studies of protein kinase C (PKC) enzyme activity, immunoblot analyses, and assays for PKC mRNAs indicate that both of these variants express lower levels of PKC than do the parental P13 cells. We suggest that this protects them from the toxic effects of TPA. The P13-5/A8 cells are of particular interest because not only are they resistant to TPA toxicity but they actually require TPA for optimal growth. These cells have a more profound decrease in PKC expression that do P13-50 cells. In addition, P13-5/A8 cells display very little, if any, surface expression of CD45, a receptor-linked tyrosine protein phosphatase, and lck, a lymphocyte-specific tyrosine kinase. On the other hand, they express a very high level of interleukin-2 receptor. A model is proposed that suggests that these cells are dependent on TPA because they have defects in both the PKC and tyrosine kinase signal transduction pathways, and that TPA compensates for these defects by providing a strong stimulus to the residual level of PKC. This variant may be useful for studying the interactions between tyrosine kinase and PKC pathways in controlling the various functions of T lymphocytes.