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Emerging evidence indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in oesophageal adenocarcinoma (EAC) patients, particularly as novel non-invasive, early detection, surveillance and prognostic classifiers.Metagenome sequencing was performed on 81 serum specimens collected across EAC spectrum, with sequencing reads classified using Bracken and MetaPhlAn3. Followed by the Linear Discriminant Analysis effect size (LEfSe) method to identify microbial profiles between groups. Logistic regression and Kaplan-Meier analyses were used to build classifiers.A significant loss of alpha and beta diversity was identified in serum specimens from EAC patients. We observed a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 (95% CI: 0.78-0.95; P 0.001) and this panel in conjunction with the TNM stage was a robust predictor of overall survival (≥24 months; AUC = 0.84 (95% CI: 0.66-0.92; P = 0.006)).This study firstly describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.Accumulating data indicates the clinical relevance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in patients with oesophageal adenocarcinoma (EAC). Herein, we performed metagenome sequencing in serum specimens from EAC patients 81 collected across EAC spectrum and observed a significant loss of alpha and beta diversity, with a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 and this panel, in conjunction with the TNM stage, was a robust predictor of overall survival. This study for the first time describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.

Esophageal adenocarcinoma (EAC) is a leading cause of cancer deaths. Pexidartinib, a multi-gene tyrosine kinase inhibitor, through targeting colony-stimulating factor 1 (CSF-1) receptor (CSF-1R), down modulates macrophage-mediated pro-survival tumor signaling. Previously, CSF-1R inhibitors have successfully shown to enhance antitumor activity of PD-1/PD-L1 inhibitors by suppressing tumor immune evasion, in solid tumors. In this study, we investigated the antitumor activity of pexidartinib alone or in combination with blockade of PD-1 in a de novo EAC rat model. Here, we showed limited toxicity with significant tumor shrinkage in pexidartinib treated animals compared to controls, single agent and in combination with a PD-1 inhibitor, AUNP-12. Suppression of CSF-1/CSF-1R axis resulted in enhanced infiltration of CD3 + CD8 + T cells with reduced M2 macrophage polarization, in the tumor microenvironment (TME). Endpoint tissue gene expression in pexidartinib treated animals demonstrated upregulation of BAX, Cas3, TNFα, IFNγ and IL6 and downregulation of Ki67, IL13, IL10, TGFβ and Arg1 (P

Recently, we characterized fibroblast growth factor receptor 1 amplification as a target for a rational therapy in lung squamous cell carcinoma. Patients harboring this genetic event are currently eligible for treatment with antifibroblast growth factor receptor small-molecule inhibitors in phase I clinical trials. This has the potential to significantly improve standard therapy for lung squamous cell carcinoma patients. The aim of this study was to elucidate whether fibroblast growth factor receptor 1 amplification is also a common genetic event in head and neck squamous cell carcinoma. For this purpose, we assembled a cohort of 555 patients, including 264 with metastatic disease and 147 with recurrent disease. Formalin-fixed, paraffin-embedded material of primary tumors, metastases and recurrences were assessed for fibroblast growth factor receptor 1 copy number status using fluorescence in situ hybridization. Human papilloma virus status was detected by p16 immunohistochemistry staining and PCR-ELISA. Molecular parameters were correlated with each other and with clinicopathological data. We found 15% of primary head and neck squamous cell carcinoma to display a fibroblast growth factor receptor 1 amplification. In nearly all cases, metastatic and recurrent tumor samples shared the same amplification status as the corresponding primary tumor. Fibroblast growth factor receptor 1 amplification was associated with nicotine and alcohol consumption, but was mutually exclusive with human papilloma virus infection. Amplification of the gene was associated with parameters of worse outcome. Our data identify fibroblast growth factor receptor 1 amplification as a frequent event in primary and metastatic head and neck squamous cell carcinoma and represents a potential biomarker for more aggressive disease. Fibroblast growth factor receptor 1-amplified tumors could potentially comprise a subset of head and neck squamous cell carcinoma against which targeted small-molecule inhibitors hold therapeutic efficacy.

Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemoperfusion (HIPEC) are frequently used to treat appendiceal carcinomatosis. Some patients require multivisceral resection because of the volume of disease. It is unclear whether extent of CRS impacts survival in appendiceal carcinomatosis.We analyzed 282 patients undergoing attempted CRS/HIPEC for appendiceal carcinomatosis. Patients were defined as having undergone Extensive CRS (n = 60) if they had3 organ resections or2 anastomoses; a subgroup of Extreme CRS patients (n = 10) had ≥5 organ resections and ≥3 anastomoses. Kaplan-Meier survival curves and multivariate Cox-regression models were used to identify prognostic factors affecting outcomes.Relative to the comparison group, patients undergoing Extensive CRS had a higher median peritoneal carcinomatosis index, operative duration, blood loss, and length of stay. No difference in completeness of cytoreduction, severe morbidity, or 60-day mortality was evident. Subgroup analysis of 10 patients undergoing extreme CRS likewise revealed no increase in severe morbidity or mortality. Median progression-free (PFS) and overall survival (OS) were 23.5 and 74 months in the comparison group; 18.5 (p = 0.086) and 51 (p = 0.85) months in the Extensive CRS group; and 40 months and not reached in the Extreme CRS subgroup. In a multivariable analysis, extent of CRS was not independently associated with PFS or OS.Extensive CRS is associated with greater OR time, blood loss, and length of stay, but is not associated with higher morbidity, mortality, or inferior oncologic outcomes in patients with appendiceal carcinomatosis.

Since first being described in 2009, single-incision laparoscopic splenectomy has been described in a limited number of case reports and small case series. No studies have evaluated single-incision splenectomy in unselected patients, and outcomes of the procedure have not previously been compared to standard laparoscopy.A retrospective review was conducted to evaluate all single-incision splenectomies performed by a single surgeon between June 2010 and June 2011. Additionally, patients who underwent standard laparoscopic splenectomy by surgeons in the same tertiary referral surgical oncology group were evaluated to serve as a control group. Demographic data, operative parameters, and postoperative outcomes were assessed.Eight patients underwent successful single-incision splenectomy during the study period without conversion to an open procedure or requiring additional ports. The median operative time was 92.5 min. There was 25 % morbidity and no mortality in the study group. Median length of stay was 4 days. Additionally, 18 patients who underwent standard laparoscopic splenectomy were evaluated for comparison. No significant differences were identified in the preoperative patient characteristics between the two groups. Single-incision splenectomy was associated with a shorter operative time (92.5 vs. 172 min, p = 0.003), lower conversion rate, equivalent length of stay, reduced mortality, similar morbidity, and comparable postoperative narcotic requirements.Single-incision splenectomy is feasible, safe, and efficient in an unselected patient population in the hands of an experienced laparoscopic surgeon. The single-incision technique is comparable to standard laparoscopic splenectomy in terms of operative time and perioperative outcomes.

Tumors systemically initiate metastatic niches in distant target metastatic organs. These niches, composed of bone marrow–derived hematopoietic cells, provide permissive conditions for future metastases. However, the mechanisms by which these cells mediate outgrowth of metastatic tumor cells are not completely known. Using mouse models of spontaneous breast cancer, we show enhanced recruitment of bone marrow–derived CD11b+Gr1+ myeloid progenitor cells in the premetastatic lungs. Gene expression profiling revealed that the myeloid cells from metastatic lungs express versican, an extracellular matrix proteoglycan. Notably, versican in metastatic lungs was mainly contributed by the CD11b+Ly6Chigh monocytic fraction of the myeloid cells and not the tumor cells or other stromal cells. Versican knockdown in the bone marrow significantly impaired lung metastases in vivo, without impacting their recruitment to the lungs or altering the immune microenvironment. Versican stimulated mesenchymal to epithelial transition of metastatic tumor cells by attenuating phospho-Smad2 levels, which resulted in elevated cell proliferation and accelerated metastases. Analysis of clinical specimens showed elevated versican expression within the metastatic lung of patients with breast cancer. Together, our findings suggest that selectively targeting tumor-elicited myeloid cells or versican represents a potential therapeutic strategy for combating metastatic disease. Cancer Res; 72(6); 1384–94. ©2012 AACR.

In cancer, proto-oncogenes are often altered by genomic amplification. Here we report recurrent focal amplifications of chromosomal segment 4q12 overlapping the proto-oncogenes PDGFRA and KIT in non-small cell lung cancer (NSCLC). Single nucleotide polymorphism (SNP) array and fluorescent in situ hybridization (FISH) analysis indicate that 4q12 is amplified in 3-7% of lung adenocarcinomas and 8-10% of lung squamous cell carcinomas. In addition, we demonstrate that the NSCLC cell line NCI-H1703 exhibits focal amplification of PDGFRA and is dependent on PDGFRalpha activity for cell growth. Treatment of NCI-H1703 cells with PDGFRA-specific shRNAs or with the PDGFRalpha/KIT small molecule inhibitors imatinib or sunitinib leads to cell growth inhibition. However, these observations do not extend to NSCLC cell lines with lower-amplitude and broader gains of chromosome 4q. Together these observations implicate PDGFRA and KIT as potential oncogenes in NSCLC, but further study is needed to define the specific characteristics of those tumors that could respond to PDGFRalpha/KIT inhibitors.

Recent advances in the characterization of the lung cancer genome have suggested that KRAS may frequently be amplified, although little is known regarding the significance of this finding. This is in contrast with activating mutations of KRAS, which occur in approximately 20% of non–small cell lung carcinomas (NSCLCs). We used fluorescence in situ hybridization to provide direct evidence of KRAS amplification for the first time in clinical specimens. We detected amplification in 7 of 100 consecutive NSCLCs, with a concurrent activating KRAS mutation in 4 cases. KRAS amplification was associated with greater expression of p21 as assessed by quantitative immunohistochemical analysis (P = .015). Our data indicate that a sizable subgroup of NSCLCs harbor KRAS amplification, some of which also contain point mutations, and suggest that an increased KRAS copy number may drive p21 overexpression. KRAS amplification may define a unique clinicopathologic subset of NSCLCs with potentially altered responsiveness to targeted therapies.

Combined small cell lung carcinomas (CSCLCs) are small cell lung carcinomas (SCLCs) containing discrete areas of non–small cell morphologic components, found in up to 30% of SCLCs. We assessed immunophenotypic and genotypic similarity between the distinct morphologic constituents in 7 CSCLCs. Each was stained for synaptophysin, CD56, chromogranin, bcl-2, thyroid transcription factor (TTF)-1, cytokeratin 7, and PAX-5. Loss of heterozygosity (LOH) analysis was performed using markers on 3p (2 loci), 17p (3 loci), 17q (1 locus), and 22q (2 loci). The distinct components shared an identical immunophenotype in 6 cases; all were positive for synaptophysin and CD56 in both components. LOH analysis revealed shared loss of least 1 marker in every lesion, including LOH at 22q13 (characteristic of SCLC) in 5 tumors. The individual elements constituting CSCLCs are closely related in most cases despite their distinct morphologic appearances. The prevalent expression of synaptophysin and CD56 and loss of 22q13 suggest that these tumors are biologically closer to SCLC.

Context.—Lung and breast carcinomas are among the most prevalent cancers. Advances in cancer therapies can provide survival benefit and be potentially curative, even in metastatic disease. Due to the high prevalence of these carcinomas, it is not unusual to encounter lung nodule(s) in a patient with breast carcinoma, and distinguishing between primary and metastatic disease is critical for management/treatment. Occasionally neuroendocrine differentiation is present in breast carcinoma, making its distinction from pulmonary/nonpulmonary neuroendocrine tumors in the lung difficult. Objective.—To assess estrogen and progesterone receptor expression in the entire spectrum of pulmonary neuroendocrine tumors. Design.—Seventy-one neuroendocrine neoplasms including typical carcinoids (42), atypical carcinoids (7), small cell carcinomas (14), large cell neuroendocrine carcinomas (2), and combined small cell carcinomas (6) were evaluated for estrogen and progesterone receptors. Mammary and non–small cell lung carcinomas were also stained for comparison. Results.—The entire spectrum of neuroendocrine neoplasms demonstrated focal to diffuse estrogen (typical carcinoid, 23; atypical carcinoid, 6; small cell carcinoma, 8; large cell neuroendocrine carcinoma, 2; combined small cell carcinoma, 4) and progesterone (typical carcinoid, 11; atypical carcinoid, 2; small cell carcinoma, 7; large cell neuroendocrine carcinoma, 0; combined small cell carcinoma, 2) expression. There was no correlation between sex and estrogen/progesterone status. Estrogen and progesterone staining were also noted in endothelial cells. Relative to neuroendocrine carcinomas, mammary carcinomas expressed estrogen and progesterone more frequently. Non–small cell carcinomas had greater and similar immunoreactivity for estrogen and progesterone, respectively. Conclusions.—Although estrogen and progesterone receptor staining is frequently associated with breast and gynecologic primaries, it can also be observed in “nontarget” organs. Therefore, presence of estrogen and/or progesterone expression in neuroendocrine tumors involving the lung should not exclude a primary pulmonary neoplasm.

There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.

The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

Shiga toxins (Stxs), encoded by the stxA and stxB genes, are important contributors to the virulence of Escherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxB genes in STEC strains are located on the genomes of resident prophages of the λ family immediately downstream of the phage late promoters ( p R′ ). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate p R′ promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p R′ as well as terminator kilobases distal to p R′ . To test if this Q-directed processive transcription plays a role in stx 2 AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which p R′ and part of Q were deleted but which has an intact p Stx, the previously described stx 2 AB -associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p R′ promoter. Since transcription initiating at p R′ ultimately requires activation of the phage lytic cascade, expression of stx 2 AB in STEC depends primarily on prophage induction. By showing this central role for the prophage in stx 2 AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.

The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H 2 O 2 ), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H 2 O 2 and also human neutrophils, an important endogenous source of H 2 O 2 , induced Stx2 expression by an EHEC clinical isolate. Both H 2 O 2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.

ObjectivesThe transcription factor SOX2 (3q26.3-q27) is an embryonic stem cell factor contributing to the induction of pluripotency in terminally differentiated somatic cells. Recently, amplification of the SOX2 gene locus has been described in squamous cell carcinoma (SCC) of different organ sites. Aim of this study was to investigate amplification and expression status of SOX2 in sinonasal carcinomas and to correlate the results with clinico-pathological data.Materials and methodsA total of 119 primary tumor samples from the sinonasal region were assessed by fluorescence in-situ hybridization and immunohistochemistry for SOX2 gene amplification and protein expression, respectively. Of these, 59 were SSCs, 18 sinonasal undifferentiated carcinomas (SNUC), 10 carcinomas associated with an inverted papilloma (INVC), 19 adenocarcinomas (AD) and 13 adenoid cystic carcinomas (ACC).ResultsSOX2 amplifications were found in subsets of SCCs (37.5%), SNUCs (35.3%), INVCs (37.5%) and ADs (8.3%) but not in ACCs. SOX2 amplification resulted in increased protein expression. Patients with SOX2-amplified sinonasal carcinomas showed a significantly higher rate of tumor recurrences than SOX2 non-amplified tumors.ConclusionThis is the first study assessing SOX2 amplification and expression in a large cohort of sinonasal carcinomas. As opposed to AD and ACC, SOX2 amplifications were detected in more than 1/3 of all SCCs, SNUCs and INVCs. We therefore suggest that SNUCs are molecularly closely related to SCCs and INVCs and that these entities represent a subgroup of sinonasal carcinomas relying on SOX2 acquisition during oncogenesis. SOX2 amplification appears to identify sinonasal carcinomas that are more likely to relapse after primary therapy, suggesting that these patients might benefit from a more aggressive therapy regime.

The significance of tumor markers in patients with appendiceal carcinomatosis is poorly defined. We determined preoperative and postoperative tumor marker levels in patients undergoing cytoreductive surgery (CRS) and heated intraperitoneal chemoperfusion (HIPEC) and examined their association with clinicopathologic features and survival.A total of 176 patients undergoing attempted CRS/HIPEC for appendiceal carcinomatosis had at least 1 tumor marker measured. Marker levels were correlated with tumor characteristics and oncologic outcomes. Kaplan-Meier curves and multivariate Cox regression models were used to identify prognostic factors affecting progression and survival.At least 1 marker was elevated prior to CRS/HIPEC in 70 % of patients (CEA, 54.1 %; CA19-9, 47.7 %; CA-125, 47.2 %). Among patients with elevated preoperative marker levels, normalization occurred postoperatively in 79.4 % for CEA, 92.3 % for CA19-9, and 60 % for CA-125. Absolute preoperative tumor marker levels correlated with peritoneal carcinomatosis index (PCI) (p.0002), and the number of elevated markers was associated with PCI and progression-free survival (PFS). Elevated postoperative CEA level was associated with decreased PFS (median, 13 vs 36 months, p = .0008). On multivariate Cox regression analysis, elevated preoperative CA19-9 was associated with shorter PFS (hazard ratio [HR] 2.9, 95 % confidence interval [95 % CI] 1.5-5.3, p = .0008), whereas elevated CA-125 was associated with shorter overall survival (HR 2.6, 95 % CI 1.3-5.4, p = .01).Most patients with appendiceal carcinomatosis will have at least 1 elevated tumor marker and will normalize following CRS/HIPEC, allowing for ongoing surveillance. CA19-9 is a promising biomarker for early progression following CRS/HIPEC, whereas CA-125 is associated with shorter survival.

Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.

Acquired chromosomal aberrations play an important role in tumour development and progression. Such genetic alterations occur in a significant proportion of non-small cell lung carcinomas (NSCLCs) and include amplification of 14q13.3, which contains the TTF1 gene. We asked whether TTF1 amplification is associated with increased TTF1 protein expression in NSCLCs, and whether TTF1 is associated with clinicopathological features, including patient survival. We used a FISH assay and quantitative immunohistochemical staining to interrogate a population-based cohort of 538 NSCLCs from Swiss patients for TTF1 amplification and protein expression. We found TTF1 amplification in approximately 13% of adenocarcinomas (ACs) and in approximately 9% of squamous cell carcinomas (SCCs) and TTF1 amplification was associated with increased TTF1 protein expression. High-level TTF1 expression was significantly associated with smaller tumour size, female gender and longer overall survival only among ACs (median survival 82 versus 28 months; p = 0.002). On multivariate analysis, high TTF1 expression was an independent predictor of favourable prognosis in patients with AC [hazard ratio, 0.56 (95% CI 0.38-0.83); p = 0.008]. We conclude that TTF1 amplification is a mechanism of high-level TTF1 expression in a subset of NSCLCs. When expressed at high levels, this routinely used diagnostic marker is also an independent biomarker of favourable prognosis in AC.

Hypothermia is an independent risk factor for mortality in trauma patients. We describe the use of continuous venovenous haemodialysis (CVVHD) as a rewarming method in a hypothermic, multiply injured patient. CVVHD achieved rapid rewarming and holds advantages over established rewarming methods in the trauma setting.

Objectives Although the chemokine CXCL12 and its receptor CXCR4 have been implicated in metastasis of non–small cell lung carcinoma, the prognostic significance of these molecules is poorly defined. This study aimed to determine whether expression of these molecules is associated with clinicopathologic features and disease-free survival in non–small cell lung carcinoma. Methods Immunohistochemical staining for CXCL12 and CXCR4 was performed on 154 primary non–small cell lung carcinomas. Staining intensity was compared with tumor histotype, TNM stage, and disease-free survival; correlation was assessed by using the Fisher's exact test, and Kaplan–Meier and Cox multivariate proportional hazards regression analysis. Results Intense CXCL12 immunostaining was associated with nodal metastasis, although no difference in survival was observed. The prognostic relevance of CXCR4 was dependent on its subcellular location: in univariate analysis intense nuclear staining was significantly associated with lower T classification and improved disease-free survival in patients with adenocarcinoma, whereas cytomembranous staining was associated with distant metastasis and decreased disease-free survival. On multivariate analysis, cytomembranous CXCR4 expression conferred a significantly worse disease-free survival (relative risk, 2.8; 95% confidence interval, 1.4–5.7; P = .004). Conclusions Cytomembranous expression of the chemokine receptor CXCR4 in adenocarcinoma of the lung is an independent risk factor associated with worse disease-free survival, whereas nuclear staining confers a survival benefit. These findings are consistent with a model in which CXCR4 promotes tumor cell proliferation and metastasis when present in the cytoplasm or cell membrane, whereas localization of this molecule in the nucleus prevents it from exerting these effects.

A recurrent gene fusion between EML4 and ALK in 6.7% of non-small cell lung cancers (NSCLCs) and NKX2-1 (TTF1, TITF1) high-level amplifications in 12% of adenocarcinomas of the lung were independently reported recently. Because the EML4-ALK fusion was only shown by a reverse transcription-polymerase chain reaction approach, we developed fluorescent in situ hybridization assays to interrogate more than 600 NSCLCs using break-apart probes for EML4 and ALK. We found that EML4-ALK fusions occur in less than 3% of NSCLC samples and that EML4 and/or ALK amplifications also occur. We also observed that, in most cases in which an EML4/ALK alteration is detected, not all of the tumor cells harbor the lesion. By using a detailed multi-fluorescent in situ hybridization probe assay and reverse transcription-polymerase chain reaction, we have evidence that other, more common mechanisms besides gene inversion exist including the possibility of other fusion partners for ALK and EML4. Furthermore, we confirmed the NKX2-1 high-level amplification in a significant subset of NSCLC and found this amplification to be mutually exclusive to ALK and EML4 rearrangements.

The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

ObjectiveIn the cancer population, pericardial effusions are a common and potentially life-threatening occurrence. Although decompression benefits most patients, paradoxical hemodynamic instability (PHI) develops in some, with hypotension and shock in the immediate postoperative period. This study examines paradoxical hemodynamic instability after pericardial window and identifies prognostic factors in patients with cancer who are treated for pericardial effusion.MethodsRetrospective review of 179 consecutive pericardial windows performed for pericardial effusion in a tertiary cancer center over a 5-year period (January 2004 through March 2009). Demographic, surgical, pathologic, and echocardiographic data were analyzed for the end points of paradoxical hemodynamic instability (pressor-dependent hypotension requiring intensive care unit admission) and overall survival.ResultsThe most common malignancies were lung (44%), breast (20%), hematologic (10%), and gastrointestinal (7%). Overall survival for the group was poor (median, 5 months); patients with hematologic malignant disease fared significantly better than the others (median survival 36 months; P = .008). Paradoxical hemodynamic instability occurred in 19 (11%) patients. These patients were more likely to have evidence of tamponade on echocardiogram (89% vs 56%; P = .005), positive cytology/pathology (68% vs 41%; P = .03), and higher volume drained (674 mL vs 495 mL; P = .003). Overall survival was significantly shorter in those in whom paradoxical hemodynamic instability developed (median survival 35 vs 189 days; hazard ratio = 3; P