Background KRAS mutations in colorectal cancer primary tumors predict resistance to anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody therapy in patients with metastatic colorectal cancer, and thus represent a true indicator of EGFR pathway activation status.Methodology/Principal Findings KRAS mutations were retrospectively studied using polymerase chain reactions and subsequent sequencing of codons 12 and 13 (exon 2) in 110 patients with metastatic colorectal tumors. These studies were performed using tissue samples from both the primary tumor and their related metastases (93 liver, 84%; 17 lung, 16%). All patients received adjuvant 5-Fluorouracil-based polychemotherapy after resection of metastases. None received anti-EGFR therapy. Mutations in KRAS were observed in 37 (34%) of primary tumors and in 40 (36%) of related metastases, yielding a 94% level of concordance (kappa index 0.86). Patients with primary tumors possessing KRAS mutations had a shorter disease-free survival period after metastasis resection (12.0 vs 18.0 months; P = 0.035) than those who did not. A higher percentage of KRAS mutations was detected in primary tumors of patiens with lung metastases than in patients with liver metastases (59% vs 32%; p = 0.054). To further evaluate this finding we analyzed 120 additional patients with unresectable metastatic colorectal cancer who previously had their primary tumors evaluated for KRAS mutational status for clinical purposes. Separately, the analysis of these 120 patients showed a tendency towards a higher degree of KRAS mutations in primary tumors of patients with lung metastases, although it did not reach statistical significance. Taken together the group of 230 patients showed that KRAS was mutated significantly more often in the primary tumors of patients with lung metastases (57% vs 35%; P = 0.006).Conclusions/SignificanceOur results suggest a role for KRAS mutations in the propensity of primary colorectal tumors to metastasize to the lung.

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human ␤-globin locus construct results in delayed human ␥-to ␤-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the ␤-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress ␥-globin gene expression and facilitate ␥-to ␤-globin switching.

No abstract

We have previously reported the structure of a chromatin remodeling complex (PYR complex) with Ikaros as its DNA binding subunit that is specifically present in adult murine and human hematopoietic cells. We now show that homozygous Ikaros ''knockout'' (null) mice lack the PYR complex, demonstrating the requirement for Ikaros in the formation of the complex on DNA. Heterozygous Ikaros null mice have about half as much PYR complex, indicating a dosage effect for both Ikaros and PYR complex. We also show that Ikaros null mice have multiple hematopoietic cell defects including anemia and megakaryocytic abnormalities, in addition to previously reported lymphoid and stem cell defects. The null mice also have a delay in murine embryonic to adult ␤-globin switching and a delay in human ␥ to ␤ switching, consistent with a previously suggested role for PYR complex in this process. Lastly, cDNA array analyses indicate that several hematopoietic cell-specific genes in all blood lineages are either up-or down-regulated in 14-day embryos from Ikaros null as compared with wild-type mice. These results indicate that Ikaros and PYR complex function together in vivo at many adult hematopoietic cell-specific genes and at intergenic sites, affecting their expression and leading to pleiotropic hematopoietic defects. C hromatin remodeling complexes are associated with both activation and repression of expression of specific eukaryotic genes. SWI͞SNF containing ATPase͞helicase subunits (e.g., BRG1) have been shown to remodel chromatin and activate gene transcription at the ␤-globin locus as well as elsewhere (1, 2). Similarly, NuRD complexes using other ATPase͞helicase subunits (e.g.Mi-2␤) together with histone deacetylases have been associated with chromatin deacetylation and repression of specific gene expression (3). We have recently described a SWI͞SNF and NuRD-containing complex associated with the DNA binding transcription factor Ikaros that is present only in adult hematopoietic cells (4,5). This complex binds to Ikaros-like DNA binding sites including a long polypyrimidine-rich sequence upstream of the human ␦-globin gene and was thus called PYR complex. Deletion of this sequence in a human ␤-locus containing cosmid (carrying sequences from the human A␥ through the adult ␤-globin gene) in transgenic mice results in delayed human ␥-to ␤-globin switching (4).We now have studied Ikaros null mice whose Ikaros gene has been truncated by deletion of the two C-terminal zinc fingers required for Ikaros protein dimerization and function (6, 7). We show that Ikaros null mice completely lack PYR complex, indicating that Ikaros is required for PYR complex formation. In addition, heterozygous null mice have about half as much PYR complex as wild-type mice, indicating a dosage effect for both Ikaros and PYR complex. In this model for evaluating Ikaros and PYR complex function, we find multiple hematopoietic defects including anemia and thrombocytosis, along with previously reported lymphoid and stem cell abnormalities (8). We also show tha...

Recent reports demonstrate the feasibility of quantifying gene expression by using RNA isolated from blocks of formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The development of molecular tests for clinical use based on archival materials would be of great utility in the search for and validation of important genes or gene expression profiles. In this study, we compared the performance of different normalization strategies in the correlation of quantitative data between fresh frozen (FF) and FFPE samples and analyzed the parameters that characterize such correlation for each gene. Total RNA extracted from FFPE samples presented a shift in raw cycle threshold (Cq) values that can be explained by its extensive degradation. Proper normalization can compensate for the effects of RNA degradation in gene expression measurements. We show that correlation between normalized expression values is better for moderately to highly expressed genes whose expression varies significantly between samples. Nevertheless, some genes had no correlation. These genes should not be included in molecular tests for clinical use based on FFPE samples. Our results could serve as a guide when developing clinical diagnostic tests based on RT-qPCR analyses of FFPE tissues in the coming era of treatment decision-making based on gene expression profiling.

Summary A third component of complement (C3) capture enzyme‐linked immunosorbent assay was used to determine the concentration of IgG circulating immune complexes (CIC) in 91 dogs with naturally acquired leishmania infection and in a control group of 24 healthy dogs. Results were expressed as a percentage of a reference standard. Mean concentrations of CIC were significantly (P < 0.001) higher in leishmania‐infected dogs (228.725 ± 14.283 %) than in controls (74.542 ± 12.614 %). An increase in CIC concentration was found in 57.1 % of the leishmania‐infected dogs. No significant differences could be recorded in CIC levels between males and females in either group. Infected dogs showing hypercreatininemia rendered a statistically significant (P < 0.030) higher serum CIC concentration than sick dogs with normal creatininemia. When hypercreatininemia (≥ 1.30 mg/dl) was used as an indicator for CIC increase, the positive predictive value obtained was 0.9 indicating that renal function impairment was associated with high serum CIC concentration in 90 % of the infected dogs.

Co-expression networks may provide insights into the patterns of molecular interactions that underlie cellular processes. To obtain a better understanding of miRNA expression patterns in gastric adenocarcinoma and to provide markers that can be associated with histopathological findings, we performed weighted gene correlation network analysis (WGCNA) and compare it with a supervised analysis. Integrative analysis of target predictions and miRNA expression profiles in gastric cancer samples was also performed. WGCNA identified a module of co-expressed miRNAs that were associated with histological traits and tumor condition. Hub genes were identified based on statistical analysis and network centrality. The miRNAs 100, let-7c, 125b and 99a stood out for their association with the diffuse histological subtype. The 181 miRNA family and miRNA 21 highlighted for their association with the tumoral phenotype. The integrated analysis of miRNA and gene expression profiles showed the let-7 miRNA family playing a central role in the regulatory relationships.

Protein phosphorylation affects most eukaryotic cellular processes and its deregulation is considered a hallmark of cancer and other diseases. Phosphoproteomics may enable monitoring of altered signaling pathways as a means of stratifying tumors and facilitating the discovery of new drugs. Unfortunately, the development of molecular tests for clinical use is constrained by the limited availability of fresh frozen, clinically annotated samples. Here we report phosphopeptide analysis in human archival formalin-fixed, paraffin-embedded (FFPE) cancer samples based on immobilized metal affinity chromatography followed by liquid chromatography coupled with tandem mass spectrometry and selected reaction monitoring techniques. Our results indicate the equivalence of detectable phosphorylation rates in archival FFPE and fresh frozen tissues. Moreover, we demonstrate the applicability of targeted assays for phosphopeptide analysis in clinical archival FFPE samples, using an experimental workflow suitable for processing and analyzing large sample series. This work paves the way for the application of shotgun and targeted phosphoproteomics approaches in clinically relevant studies using archival clinical samples.