This manuscript summarizes current thinking on the value and promise of evolving circulating tumor cell (CTC) technologies for cancer patient diagnosis, prognosis, and response to therapy, as well as accelerating oncologic drug development. Moving forward requires the application of the classic steps in biomarker development–analytical and clinical validation and clinical qualification for specific contexts of use. To that end, this review describes methods for interactive comparisons of proprietary new technologies, clinical trial designs, a clinical validation qualification strategy, and an approach for effectively carrying out this work through a public-private partnership that includes test developers, drug developers, clinical trialists, the US Food & Drug Administration (FDA) and the US National Cancer Institute (NCI).

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.Sphingosine 1-phosphate (S1P) 2 receptor agonists are likely to be the next generation of pharmacologic agents used to modulate immune system function. The prototype drug of this class is FTY720, which is highly efficacious in prolonging allograft survival and in ameliorating autoimmune disease in a variety of animal models (1-4). FTY720 is being tested in human trials for the indications renal transplantation and multiple sclerosis (5). Further, there is mounting evidence that S1P agonists are efficacious in animal models of atherosclerosis (6), renal ischemia-reperfusion injury (7), and acute lung injury (8).FTY720 is a sphingosine analog that, after activation by phosphorylation (to FTY720-P), disrupts lymphocyte trafficking by decreasing lymphocyte egress from lymph nodes and the thymus (9, 10). Although the precise mechanisms that underlie this phenomenon are uncertain, the profound lymphopenia that is the index of FTY720 action is dependent on agonist action at lymphocyte S1P 1 receptors. Since FTY720-P is also a potent agonist at the S1P 3 , S1P 4 , and S1P 5 receptors (11, 12), it remains unknown whether the multiple therapeutic benefits of the drug correlate with agonist activity at the S1P 1 receptor. The propensity for S1P 1 receptor responses to desensitize (13) and the similar behaviors of S1P 1 receptor null thymocytes and FTY720-treated mouse lymphocytes have led to the suggestion that FTY720-P is a functional antagonist (14). In this scenario, the drug exaggerates S1P tone to the extent that the lymphocyte S1P 1 receptor signaling is chronically down-regulated.The kinase(s) responsible for FTY720 activation is the gateway whereby S1P signaling can be accessed readily with a therapeutic agent. Knowledge of this enzyme is important specifically to guide S1P prodrug design and generally to gain insight into the normal role of S1P in immune function. The identity of the kinase is not known currently; two candidates are sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). These enzymes, which are expressed widely, catalyz...

Introduction Leukocyte recruitment during inflammation is a multistep process involving tethering and rolling of leukocytes on vascular surfaces, followed by firm adhesion and transmigration into the affected tissues (1). Interactions of selectins with glycoconjugate ligands primarily mediate the initial tethering and rolling adhesion of leukocytes (2, 3). L-selectin, expressed on leukocytes, binds to ligands on other leukocytes and on activated endothelial cells. P-selectin, expressed on activated platelets and endothelial cells, and E-selectin, expressed on activated endothelial cells, bind to ligands on leukocytes. Studies with selectin-deficient mice and blocking mAb's indicate that the selectins have both unique and overlapping functions (4, 5). In the microcirculation of the cremaster muscle, P-selectin mediates most tethering and rolling of leukocytes early after trauma-induced inflammation (6), whereas L-selectin supports significant tethering and rolling 6-8 hours after exposure to TNF-α (7). E-selectin supports slow rolling of leukocytes on venules 2-4 hours after stimulation with TNF-α (8). Selectin ligands require α2,3-sialylation and α1,3fucosylation on capping structures such as sialyl Lewis x (sLe x), which bind to the C-type lectin domains of the selectins (9). In addition, sulfation is a critical requirement of ligands that bind to P-and L-selectin. A subset of glycoproteins binds with high affinity or avidity to the selectins (2). Of these, only P-selectin glycoprotein ligand-1 (PSGL-1) has a clearly documented function in mediating selectin-dependent cell adhesion under flow (3, 10). Human and murine PSGL-1 are extended homodimeric sialomucins that are expressed on most leukocytes. PSGL-1 binds to all three selectins in vitro. P-and L-selectin bind to the N-terminal region of PSGL-1 (11-13), whereas E-selectin binds to one or more additional sites on PSGL-1 (14). Human PSGL-1 binds to P-selectin with specific stereochemical requirements that include optimal orientation of three tyrosine sulfates, amino acids, and a core-2 O-glycan capped with sLe x (15-17). PSGL-1 does not require tyrosine sulfation to bind to E-selectin, but expression of sLe x on core-2 O-glycans enhances binding (18). mAb's against the N-terminal region of PSGL-1 markedly inhibit rolling of human and murine leukocytes on P-selectin in vitro (11, 12), rolling of human neutrophils on P-selectin in rat venules in vivo (19), and rolling of murine leukocytes on P-selectin in murine venules in vivo (12, 20). These data demonstrate that