The measurement of circulating nucleic acids has transformed the management of chronic viral infections such as HIV. The development of analogous markers for individuals with cancer could similarly enhance the management of their disease. DNA containing somatic mutations is highly tumor specific and thus, in theory, can provide optimum markers. However, the number of circulating mutant gene fragments is small compared to the number of normal circulating DNA fragments, making it difficult to detect and quantify them with the sensitivity required for meaningful clinical use. In this study, we applied a highly sensitive approach to quantify circulating tumor DNA (ctDNA) in 162 plasma samples from 18 subjects undergoing multimodality therapy for colorectal cancer. We found that ctDNA measurements could be used to reliably monitor tumor dynamics in subjects with cancer who were undergoing surgery or chemotherapy. We suggest that this personalized genetic approach could be generally applied to individuals with other types of cancer.

The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

Post‐translational activation of the higher eukaryotic transcription factor NF‐kappa B requires both phosphorylation and proteolytic degradation of the inhibitory subunit I kappa B‐alpha. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of I kappa B‐alpha in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, we have identified serines 32 and 36 in human I kappa B‐alpha as essential for the control of I kappa B‐alpha stability and the activation of NF‐kappa B in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative I kappa B proteins attenuating kappa B‐dependent transactivation in response to OA, phorbol 12‐myristate 13‐acetate (PMA) and tumor necrosis factor‐alpha (TNF). While both endogenous and transiently expressed wild‐type I kappa B‐alpha were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of I kappa B‐alpha remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize I kappa B‐alpha, leading to NF‐kappa B activation.

Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently upregulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1 and 4% of these survivors had acquired new KRAS mutations. The glycolysis inhibitor, 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.Mutations in oncogenes and tumor suppressor genes endow cancer cells with the ability to outgrow their neighboring cells in situ (1). Though numerous studies have identified the downstream effects of such mutations and their biochemical mediators, there is relatively little known about the microenvironmental conditions that provide the selective advantage that allows cells with such mutations to clonally expand. Mutations in KRAS commonly occur in colorectal, pancreatic, and some forms of lung cancer, while BRAF mutations occur commonly in melanomas as well as in colorectal tumors without KRAS mutations (2-4). BRAF and KRAS mutations are mutually exclusive, that is, do not occur in the same tumor, suggesting a common origin and effect. Indeed, KRAS binds to and activates BRAF, thereby activating MAPK signaling pathways (5,6). Despite advances in the molecular delineation of the RAS/ RAF pathway, the specific environmental pressures that drive KRAS and BRAF mutations and how KRAS and BRAF mutations alleviate these pressures are unknown.

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) α/β lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTα- and LTβ-deficient mice. Treatment of adult mice with antagonists of LTα1β2 also leads to decreased BLC expression. These findings indicate that LTα1β2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTα-, and LTβ-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus–induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTα1β2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen.

Cep164 provides a molecular link between the mother centriole and the ciliary membrane biogenesis machinery by interacting with the GEF Rabin8 and the GTPase Rab8.

Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.

Our data show that one ROI species, H2O2 acts as a messenger in the TNF- and okadaic acid-induced post-translational activation of NF-kappa B. Superoxide is only indirectly involved, as a source for H2O2. These data explain the inhibitory effects of many antioxidative compounds on the activation of NF-kappa B and its target genes. H2O2 is overproduced in response to various stimuli, and normal levels of catalase appear insufficient to remove it completely. H2O2 can therefore accumulate and act as an intracellular messenger molecule in the response to pathogens.