Purpose: The epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated with different frequencies in non^small cell lung cancer and mutations predict clinical response to EGFR inhibitors. The present study compared the mutational status of EGFR, KRAS, and BRAF in primary tumors with the one in corresponding lymph node metastases. Experimental Design: Direct bidirectional sequencing of EGFR gene exons 18 to 21, KRAS gene codons 12/13 and 61to 68, and BRAF exon 15 was done on 96 paired samples of primary lung adenocarcinomas and corresponding locoregional lymph node metastases. In addition, comparative genomic hybridization analyses in two pairs of corresponding primary and metastatic tumor samples with discordant EGFR mutation status were done. Results: Mutations in EGFR, KRAS, and BRAF were observed in 7 (7%), 36 (38%), and 2 (2%) patients, respectively. Interestingly, KRAS mutations were observed in two patients with an EGFR mutation. Mutations in primary tumors and lymph node metastases were identical in 1of 7 (14%) patients in case of EGFR and 11 of 36 (31%) patients in case of KRAS. One patient harbored different KRAS mutations in primary and corresponding metastatic tumors. Comparative genomic hybridization analysis revealed similar patterns of chromosomal changes, strongly supporting a common clonal origin of primary tumors and metastases. Conclusions:The possibilityofdifferences inthe mutationalstatus of EGFR, KRAS, BRAF between primary tumors and corresponding lymph node metastases should be considered whenever these mutations are used for the selection of patients for EGFR-directed tyrosine kinase inhibitor therapy.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-β induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

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Reactivation of persistent human adenoviruses (HAdVs) is associated with high morbidity and mortality in paediatric haematopoietic stem cell transplant (HSCT) recipients. Although invasive HAdV infections mainly arise from the gastrointestinal (GI) tract, the specific sites of HAdV persistence are not well characterised. We prospectively screened biopsies from 143 non-HSCT paediatric patients undergoing GI endoscopy and monitored serial stool specimens from 148 paediatric HSCT recipients for the presence of HAdV by real-time PCR. Persistence of HAdV in the GI tract was identified in 31% of children, with the highest prevalence in the terminal ileum. In situ hybridisation and immunohistochemistry identified HAdV persistence in lymphoid cells of the lamina propria, whereas biopsies from five transplant recipients revealed high numbers of replicating HAdV in intestinal epithelial cells. The prevalence of HAdV species, the frequencies of persistence in the GI tract and reactivations post transplant indicated a correlation of intestinal HAdV shedding pre-transplant with high risk of invasive infection. HAdV persistence in the GI tract is a likely origin of infectious complications in immunocompromised children. Intestinal lymphocytes represent a reservoir for HAdV persistence and reactivation, whereas the intestinal epithelium is the main site of viral proliferation preceding dissemination. The findings have important implications for assessing the risk of life-threatening invasive HAdV infections.

Fibroblast growth factors (FGFs) and their high-affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several nonliver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up-regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC-1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia-mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum-starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal-regulated kinase 1/2 and ribosomal protein S6. In contrast, down-modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells' potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC-derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. Conclusion: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy. (HEPATOLOGY 2011;53:854-864) H epatocellular carcinoma (HCC) is the thirdleading cause of cancer deaths worldwide. 1 Important risk factors for this disease are persistent infections with hepatitis viruses and chronic steatohepatitis due to ethanol abuse and obesity, which contribute to the increasing incidence of HCC in Abbreviations: AHR, aryl hydrocarbon receptor; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; ETS, E twenty-six; FACS, fluorescenceactivated cell sorting; FBS, fetal bovine serum; FCS, fetal colf serum; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GSK3b, glycogen synthase kinase 3b; HCC, hepatocellular carcinoma; HIF, hypoxia inducible factor; MAP, mitogen-activated protein; MF, myofibroblast; mRNA, messenger RNA; MTF, metal-responsive transcription factor; pERK, phosphorylated extracellular signal-regulated kinase; pGSK3b, phosphorylated glycogen synthase kinase 3b; pS6, phosphorylated S6; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; siFGF18, small interfering RNA targeting fibroblast growth factor 18; siRNA, small interfering RNA;...

Noninvasive liver imaging has developed rapidly resulting in increased accuracy for detecting primary and secondary hepatic tumors. Intraoperative ultrasonography (IOUS) was commonly considered to be the gold standard for liver staging, but the current value of IOUS is unknown in view of more sophisticated radiologic tools. The purpose of this prospective study was to evaluate the impact of IOUS on the treatment of 149 patients undergoing liver surgery for malignant disease (colorectal metastasis, 61 patients; hepatoma, 52 patients; other hepatic malignant tumors, 36 patients). The sensitivities of computed tomography (CT), helical CT, magnetic resonance imaging, and IOUS in patients with colorectal metastases were 69.2%, 82.5%, 84.9%, and 95.2% in a segment-by-segment analysis; in patients with hepatoma their sensitivities were 76.9%, 90.9%, 93.0%, and 99.3%; and in patients with other hepatic malignancies they were 66.7%, 89.6%, 93.3%, and 98.9%, respectively. Additional malignant lesions (AMLs) were first detected by inspection and palpation in 20 patients (13.4%). In another 18 patients (12.1%) IOUS revealed at least one AML. Overall, the findings obtained only by IOUS changed the surgical strategy in 34 cases (22.8%). It was concluded that IOUS, having undergone some refinement as well, still has immense diagnostic value in hepatectomy candidates. Frequently avoiding palliative liver resection and occasionally disproving unresectability as assessed by preoperative imaging, IOUS still has a significant impact on surgical decision making and should still be considered the gold standard.