Evaluation of Circulating Tumor DNA for Methylated BCAT1 and IKZF1 to Detect Recurrence of Stage II/Stage III Colorectal Cancer (CRC).

BACKGROUND: Most recurrences of early-stage colorectal cancer detected with current surveillance measures are widespread and incurable. Circulating tumor DNA (ctDNA) may facilitate earlier diagnosis of recurrent colorectal cancer and improve cancer-related outcomes. METHODS: Plasma from patients undergoing standard surveillance after definitive treatment for stage II/III colorectal cancer was assayed with COLVERA and carcinoembryonic antigen (CEA) at a single time point. Results were correlated with radiographic imaging. Assay performance, including sensitivity and specificity for recurrence, were compared. Impact of potentially confounding variables was also explored. RESULTS: 322 patients were included in the final analysis, and 27 recurrences were documented over a median follow-up period of 15 months. Sensitivity for recurrence was 63% [confidence interval (CI), 42.4-80.6] and 48% (CI, 28.7-68.1) for COLVERA and CEA (≥5 ng/mL), respectively (P = 0.046), while specificity was 91.5% (CI, 87.7-94.4) and 96.3% (CI, 93.4-98.1), respectively (P = 0.016). Smoking and age were independent predictors of CEA but not COLVERA positivity. CONCLUSIONS: COLVERA was more sensitive but less specific than CEA in detecting recurrent colorectal cancer. Short median follow-up may have been responsible for apparent false positives in COLVERA. Studies with serial sampling and longer follow-up are needed to assess whether earlier detection of colorectal cancer recurrence translates into clinical benefit. IMPACT: This prospective study showed that COLVERA (a two-gene ctDNA assay) was more sensitive for detection of recurrence in a cohort of patients undergoing surveillance after definitive therapy for stages II and III colorectal cancer.

The predictive role of plasma TGF-beta1 during radiation therapy for radiation-induced lung toxicity deserves further study in patients with non-small cell lung cancer.

BACKGROUND: This study aimed to further investigate the role of circulating TGF-beta1 during radiation therapy (RT) in predicting radiation-induced lung toxicity (RILT). METHODS AND MATERIALS: Patients with stages I-III non-small cell lung cancer treated with RT based therapy were included in this study. Platelet poor plasma was obtained pre-RT, at 2 and 4 weeks during-RT, and at the end of RT. TGF-beta1 was measured using an enzyme-linked immunosorbent assay. The primary endpoint for RILT was >or=grade 2 radiation pneumonitis or fibrosis. RESULTS: Twenty-six patients with a minimum follow-up of 12 months were included. Six patients (23.1%) experienced >or=grade 2 RILT. There was no significant difference in absolute TGF-beta1 levels pre-RT, at 2 and 4 weeks during-RT, or at the end of RT between patients with and without RILT. The TGF-beta1 ratios (over the pre-RT levels) for patients with and without RILT at 2, 4 weeks during-, and the end of RT were 2.8+/-2.2 and 1.0+/-0.6 (P=0.123), 2.3+/-1.3 and 0.8+/-0.5 (P=0.001), 1.5+/-0.9 and 0.8+/-0.5 (P=0.098), respectively. Using 2.0 as a cut-off, the TGF-beta1 ratio at 4 weeks during-RT predicted RILT with a sensitivity and specificity of 66.7% and 95.0%, respectively. CONCLUSION: Elevation of plasma TGF-beta1 level 4 weeks during-RT is significantly predictive of RILT. The role of plasma TGF-beta1 in predicting RILT deserves further study.

Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish.

In tuberculosis, infecting mycobacteria are phagocytosed by macrophages, which then migrate into deeper tissue and recruit additional cells to form the granulomas that eventually contain infection. Mycobacteria are exquisitely adapted macrophage pathogens, and observations in the mouse model of tuberculosis have suggested that mycobacterial growth is not inhibited in macrophages until adaptive immunity is induced. Using the optically transparent and genetically tractable zebrafish embryo-Mycobacterium marinum model of tuberculosis, we have directly examined early infection in the presence and absence of macrophages. The absence of macrophages led rapidly to higher bacterial burdens, suggesting that macrophages control infection early and are not an optimal growth niche. However, we show that macrophages play a critical role in tissue dissemination of mycobacteria. We propose that residence within macrophages represents an evolutionary trade-off for pathogenic mycobacteria that slows their early growth but provides a mechanism for tissue dissemination.

Differential regulation of primitive myelopoiesis in the zebrafish by Spi-1/Pu.1 and C/ebp1.

The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.

pak2a mutations cause cerebral hemorrhage in redhead zebrafish.

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd(mi149)), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.

Obscurin is required for the lateral alignment of striated myofibrils in zebrafish.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.

Distinct roles for SCL in erythroid specification and maturation in zebrafish.

The stem cell leukemia (SCL) transcription factor is essential for vertebrate hematopoiesis. Using the powerful zebrafish model for embryonic analysis, we compared the effects of either reducing or ablating Scl using morpholino-modified antisense RNAs. Ablation of Scl resulted in the loss of primitive and definitive hematopoiesis, consistent with its essential role in these processes. Interestingly, in embryos with severely reduced Scl levels, erythroid progenitors expressing gata1 and embryonic globin developed. Erythroid maturation was deficient in these Scl hypomorphs, supporting that Scl was required both for the erythroid specification and for the maturation steps, with maturation requiring higher Scl levels than specification. Although all hematopoietic functions were rescued by wild-type Scl mRNA, an Scl DNA binding mutant rescued primitive and definitive hematopoiesis but did not rescue primitive erythroid maturation. Together, we showed that there is a distinct Scl hypomorphic phenotype and demonstrated that distinct functions are required for the roles of Scl in the specification and differentiation of primitive and definitive hematopoietic lineages. Our results revealed that Scl participates in multiple processes requiring different levels and functions. Further, we identified an Scl hypomorphic phenotype distinct from the null state.

Self-association of Gata1 enhances transcriptional activity in vivo in zebra fish embryos.

Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3'-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.

A nonsense mutation in zebrafish gata1 causes the bloodless phenotype in vlad tepes.

Vlad tepes (vlt(m651)) is one of only five "bloodless" zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in vlt(m651). Embryos homozygous for the vlt(m651) mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the gata1 gene, 1015C --> T (Arg-339 --> Stop), in vlt(m651). The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish gata1 gene was able to rescue the bloodless phenotype in vlt(m651). These results show that the vlt(m651) mutation is a previously uncharacterized gata1 allele in the zebrafish. The vlt(m651) mutation sheds new light on Gata1 structure and function in vivo, demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.