Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.

Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting.

BACKGROUND: During active transcription, SARS-CoV-2 generates subgenomic regions of viral RNA. While standard SARS-CoV-2 RT-PCR amplifies region(s) of genomic RNA, it cannot distinguish active infection from remnant viral genomic material. However, screening for subgenomic RNA (sgRNA) by RT-PCR may aid in the determination of actively transcribing virus. OBJECTIVES: To evaluate the clinical utility of SARS-CoV-2 sgRNA RT-PCR testing in a pediatric population. STUDY DESIGN: Retrospective analysis was performed on inpatients from February-September 2022 positive for SARS-CoV-2 by RT-PCR with a concomitant order for sgRNA RT-PCR. Chart abstractions were conducted to determine clinical outcomes, management, and infection prevention and control (IPC) practices. RESULTS: Of 95 SARS-CoV-2 positive samples from 75 unique patients, 27 (28.4%) were positive by sgRNA RT-PCR. A negative sgRNA RT-PCR test allowed for de-isolation in 68 (71.6%) patient episodes. Regardless of age or sex, a positive sgRNA RT-PCR result significantly correlated with disease severity (P = 0.007), generalized COVID-19 symptoms (P = 0.012), hospitalization for COVID-19 (P = 0.019), and immune status (P = 0.024). Moreover, sgRNA RT-PCR results prompted changes in management in 28 patients (37.3%); specifically, therapeutic escalation in 13/27 (48.1%) positives and de-escalation in 15/68 (22.1%) negatives. CONCLUSIONS: Taken together, these findings underscore the clinical utility of sgRNA RT-PCR testing in a pediatric population as we report significant associations between sgRNA RT-PCR results and clinical parameters related to COVID-19. These findings align with the proposed use of sgRNA RT-PCR testing to guide patient management and IPC practices in the hospital setting.

A 5-year study of the performance of the Verigene Gram-positive blood culture panel in a pediatric hospital.

High accuracy of direct from positive blood culture molecular panels is imperative, particularly for the detection of resistance determinants as it allows for antimicrobial optimization prior to conventional susceptibility testing. In this study, we provide extensive data since implementation of the Verigene Gram-positive blood culture panel (BC-GP) in 2013. Within 5 years, 1636 blood culture bottles positive for a Gram-positive organism were tested on the BC-GP panel. The BC-GP panel identified 1520 Gram-positive organisms in 1636 (92.9%) blood cultures tested. For positive blood cultures, we observed 96.4% (806/834) concordance to the species level. Compared with conventional antimicrobial susceptibility testing, the positive percent agreement (PPA) of methicillin-resistant SA (MRSA) (50) and methicillin-resistant SE (MRSE) (365) was 100%. The mecA gene was detected in two methicillin-susceptible Staphylococcus aureus (MSSA) and one methicillin-susceptible S. epidermidis (MSSE) with a negative percent agreement (NPA) of 99.1% (221/223) and 99.2% (120/121), respectively. The PPA and NPA for vancomycin-resistant Enterococcus faecium (VRE) was 100%. The BC-GP panel demonstrated excellent performance and clinicians can confidently de-escalate antimicrobial therapy in the absence of mecA and vanA/B gene.

One Year in the Life of a Rapid Syndromic Panel for Meningitis/Encephalitis: a Pediatric Tertiary Care Facility's Experience.

Early establishment of infectious processes allows for expedited clinical management of meningitis and encephalitis. The FilmArray meningitis/encephalitis (FA-M/E) panel provides rapid detection of potential pathogens associated with encephalitis/meningitis in both immunocompetent and compromised patients. Here, we conducted a 1-year review of the performance of the FA-M/E panel at a tertiary care children's hospital. Two hundred sixty-five samples from 251 patients were tested. We found 87.25% (219/251) were negative, 9.96% (25/251) were positive for viral analytes, and 3.19% (8/251) were positive for bacterial analytes. When possible, positive results were confirmed by alternate testing; 4/6 available bacterial positives and 17/20 available viral positives were confirmed by retrospective culture or molecular testing.

Molecular Testing for Detection of Groups A, C, and G ß-Hemolytic Streptococci in Pharyngeal Samples from Children.

BACKGROUND: Group A Streptococcus (GAS) and large colony-forming group C (GCS) and G (GGS) ß-hemolytic streptococci are important causes of acute pharyngitis in children and adults. Rapid and accurate diagnosis of streptococcal pharyngitis can improve patient care and potentially reduce transmission. In this study, we evaluated the performance of the Lyra Direct Strep (LDS) assay for detection of GAS and GCS/GGS compared with traditional culture methods. METHODS: Pharyngeal samples obtained from 278 children presenting to the emergency department with initial negative GAS rapid antigen detection test (RADT) were used. All samples were cultured as part of routine care and tested in batches using the LDS assay. RESULTS: Of 278 pharyngeal samples with negative GAS RADT, 37 (13.3%) and 63 (22.7%) patients were positive for GAS by culture and LDS assay, respectively. Four (1.4%) patients were positive for GCS or GGS by culture or LDS assay. The LDS assay demonstrated sensitivity and specificity of 97.6% and 89.0%, respectively, compared with culture as the gold standard. Repeat culture and an alternate PCR showed that 85.7% (24 of 28) of discrepant samples agreed with findings of the LDS assay. Since implementation, the LDS assay shows a positivity rate of 21.0% (281 of 1340) compared with 11.7% (246 of 2110) by culture in the previous year. CONCLUSIONS: We successfully implemented the LDS assay at our institution and have observed a significant increase in the positivity rate of GAS compared with culture. The LDS assay alone allowed for the elimination of ß-streptococci screening by culture at our institution.

Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens.

Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis. The FilmArray Meningitis/Encephalitis (ME) Panel is a multiplexed in vitro diagnostic test for the simultaneous, rapid (∼1-h) detection of 14 pathogens directly from CSF specimens: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, and Cryptococcus neoformans/Cryptococcus gattii We describe a multicenter evaluation of 1,560 prospectively collected CSF specimens with performance compared to culture (bacterial analytes) and PCR (all other analytes). The FilmArray ME Panel demonstrated a sensitivity or positive percentage of agreement of 100% for 9 of 14 analytes. Enterovirus and human herpesvirus type 6 had agreements of 95.7% and 85.7%, and L. monocytogenes and N. meningitidis were not observed in the study. For S. agalactiae, there was a single false-positive and false-negative result each, for a sensitivity and specificity of 0 and 99.9%, respectively. The specificity or negative percentage of agreement was 99.2% or greater for all other analytes. The FilmArray ME Panel is a sensitive and specific test to aid in diagnosis of ME. With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship are anticipated.

Direct Identification of Aerobic Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Is Accurate and Robust.

BACKGROUND: Bacterial identification in the clinical laboratory can be laborious and expensive. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and cost-effective diagnostic tool for the identification of organisms routinely found in the microbiology laboratory. The objective of this study was to demonstrate that identification of aerobic Gram-positive and Gram-negative organisms could be performed accurately and efficiently by MALDI-TOF MS and the Bruker Biotyper system without the use of time-consuming extraction methodologies. METHODS: Isolates previously recovered by routine culture and workup from clinical specimens were cultured to appropriate media, identified directly by MALDI-TOF MS, and compared to results from various biochemical identification methods. RESULTS: Using the direct-smear method, 99.5% and 98.0% of aerobic Gram-negative and Gram-positive bacteria, respectively, were identified to the genus level. At a score of ≥1.9, 97.6% Gram-negative organisms and 94.6% Gram-positive organisms were correctly identified to the species level by direct-smear method. Only 1.1% of isolates required further reflex to direct-plate extraction. The direct-smear method proved to be robust, as various growth temperatures, media, culture age, and different operators had no notable impact on the bacterial identification rate. CONCLUSION: The direct-smear method is an accurate and time-saving method for routine species-level bacterial identification.

Direct identification of bacteria from positive BacT/ALERT blood culture bottles using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and robust method for the identification of bacteria. In this study, we evaluate the performance of a laboratory-developed lysis method (LDT) for the rapid identification of bacteria from positive BacT/ALERT blood culture bottles. Of the 168 positive bottles tested, 159 were monomicrobial, the majority of which were Gram-positive organisms (61.0% versus 39.0%). Using a cut-off score of ≥1.7, 80.4% of the organisms were correctly identified to the species level, and the identification rate of Gram-negative organisms (90.3%) was found to be significantly greater than that of Gram-positive organisms (78.4%). The simplicity and cost-effectiveness of the LDT enable it to be fully integrated into the routine workflow of the clinical microbiology laboratory, allowing for rapid identification of Gram-positive and Gram-negative bacteria within an hour of blood culture positivity.

Performance of the Verigene Gram-positive blood culture assay for direct detection of Gram-positive organisms and resistance markers in a pediatric hospital.

The performance characteristics of the Verigene Gram-positive blood culture (BC-GP) assay were evaluated in pediatric patients. Concordance of the BC-GP assay was 95.8%, with significant decreases in turnaround time for identification and resistance detection. BC-GP is highly accurate and can be integrated into the routine workflow of the microbiology laboratory.

Mycophenolate mofetil decreases atherosclerotic lesion size by depression of aortic T-lymphocyte and interleukin-17-mediated macrophage accumulation.

OBJECTIVES: This study tested whether immunosuppression with mycophenolate mofetil (MMF) inhibits atherosclerosis development in apolipoprotein-E-deficient (Apoe(-/-)) mice and investigated the mechanism. BACKGROUND: Chronic vascular inflammation involving both innate and adaptive immunity is central in the development of atherosclerosis, but immunosuppressive treatment is not uniformly beneficial. The immunosuppressive MMF targets lymphocyte proliferation by inhibiting inosine-monophosphate dehydrogenase. METHODS: Young and aged Apoe(-/-) mice were treated with 30 mg/kg daily MMF during 12 and 3 weeks of a high-fat diet, respectively. Aortic lesion size and composition was investigated by histology and flow cytometry; soluble inflammatory mediators were investigated by enzyme-linked immunosorbent assay. RESULTS: Macroscopic and histologic aortic atherosclerotic lesions were significantly decreased in both MMF-treated groups. While systemic immunoglobulin G directed against low-density lipoproteins was not significantly altered, the T-cell cytokine interleukin (IL)-17 was significantly reduced in plasma of MMF-treated mice and supernatants from their aortas after T-cell stimulation. The MMF treatment decreased aortic αß T-cell receptor(+) lymphocyte proliferation and cell numbers. Also, aortic contents of CD11b(+)CD11c(+) cells and their proliferation were reduced in MMF-treated Apoe(-/-) mice. The IL-17 supplementation restored the number of proliferating aortic CD11b(+)CD11c(+) cells in MMF-treated mice. The IL-17 receptor A was highly expressed on circulating monocytes that are macrophage progenitors. Genetic deletion of IL-17 receptor A or IL-17A reduced inflammatory peritoneal CD11b(+)CD11c(+) macrophage accumulation. CONCLUSIONS: The lymphocyte-directed immunosuppressant MMF that curbs IL-17 production was a successful antiatherosclerotic treatment. Our data delineate a role for IL-17 in CD11b(+)CD11c(+) cell accumulation.

Chapter 11. Intravital microscopic investigation of leukocyte interactions with the blood vessel wall.

Intravital microscopy is a method to study the microcirculation in living tissues. Transillumination, oblique reflected light illumination, continuous and stroboscopic epifluorescence microscopy can be used to visualized specific cells and molecules. Intravital microscopy is further enhanced by the advent of laser scanning.spinning disk confocal and multi-photon microscopy. Recent advances include blood-perfused flow chambers and microfluidic devises for the study of blood cell interactions with molecularly defined substrates. This chapter focuses on the application of these techniques to study leukocyte interactions with the vascular wall and molecular surfaces.

Monocyte-endothelial cell interactions in the development of atherosclerosis.

The activation of endothelial cells at atherosclerotic lesion-prone sites in the arterial tree results in the up-regulation of cell adhesion molecules and chemokines, which mediate the recruitment of circulating monocytes. Accumulation of monocytes and monocyte-derived phagocytes in the wall of large arteries leads to chronic inflammation and the development and progression of atherosclerosis. This review discusses the nature of these molecules and the mechanisms involved in the early steps of monocyte recruitment into atherosclerotic lesion sites within the vessel wall.

Stromal derived factor-1 (SDF-1/CXCL12) and CXCR4 in renal cell carcinoma metastasis.

Renal cell carcinoma (RCC) is characterized by organ-specific metastases. The chemokine stromal derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 have been suggested to regulate organ-specific metastasis in various other cancers. On this basis, we hypothesized that the biological axis of CXCL12 via interaction with its receptor, CXCR4, is a major mechanism for RCC metastasis. We demonstrated that CXCR4 was significantly expressed on circulating cytokeratin+ RCC cells from patients with known metastatic RCC. We detected up-regulation of CXCR4 mRNA and protein levels on a human RCC cell line by either knockdown of the von Hippel-Lindau (VHL) tumor suppressor protein, or incubating the cells under hypoxic conditions. The enhanced CXCR4 expression was mediated through the interaction of the Hypoxia Inducible Factor-1alpha (HIF-1alpha) with the promoter region of the CXCR4 gene. Furthermore, the expression of CXCR4 on human RCC directly correlated with their metastatic ability in vivo in both heterotopic and orthotopic SCID mouse models of human RCC. Neutralization of CXCL12 in SCID mice abrogated metastasis of RCC to target organs expressing high levels of CXCL12; without altering tumor cell proliferation, apoptosis, or tumor-associated angiogenesis. Therefore, our data suggest that the CXCL12/CXCR4 biological axis plays an important role in regulating the organ-specific metastasis of RCC.

Cancer CXC chemokine networks and tumour angiogenesis.

Chemokines have pleiotropic effects in regulating immunity, angiogenesis, stem cell trafficking, and mediating organ-specific metastases of cancer. In the context of angiogenesis, the CXC chemokine family is a unique group of cytokines known for their ability to behave in a disparate manner in the regulation of angiogenesis. The glutamic acid-leucine-arginine (ELR+) CXC chemokines are potent promoters of angiogenesis, and mediate their angiogenic activity via signal-coupling of CXCR2 on endothelium. By contrast, members of the CXC chemokine family, such as platelet factor-4 (PF4; CXCL4) and interferon-inducible CXC chemokines are potent inhibitors of angiogenesis, and use CXCR3 on endothelium to mediate their angiostatic activity. This review will discuss the biology of CXC chemokines in the context of angiogenesis related to cancer.

CXCR2/CXCR2 ligand biology during lung transplant ischemia-reperfusion injury.

Lung transplantation is a therapeutic option for a number of end-stage pulmonary disorders. Early lung allograft dysfunction (ischemia-reperfusion injury) continues to be the most common cause of early mortality after lung transplantation and a significant risk factor for the development of bronchiolitis obliterans syndrome. Ischemia-reperfusion injury is characterized histopathologically by lung edema and a neutrophil predominate leukocyte extravasation. The specific mechanism(s) that recruit leukocytes to the lung during post-lung transplantation ischemia-reperfusion injury have not been fully elucidated. Because the ELR+ CXC chemokines are potent neutrophil chemoattractants, we investigated their role during post-lung transplantation ischemic-reperfusion injury. We found elevated levels of multiple ELR+ CXC chemokines in human bronchoalveolar lavage fluid from patients with ischemia-reperfusion injury. Proof of concept studies using a rat orthotopic lung transplantation model of "cold" ischemic-reperfusion injury demonstrated an increase in lung graft neutrophil sequestration and injury. In addition, lung expression of CXCL1, CXCL2/3, and their shared receptor CXCR2 paralleled lung neutrophil infiltration and injury. Importantly, inhibition of CXCR2/CXCR2 ligand interactions in vivo led to a marked reduction in lung neutrophil sequestration and graft injury. Taken together these experiments support the notion that increased expression of ELR+ CXC chemokines and their interaction with CXCR2 plays an important role in the pathogenesis of post-lung transplantation cold ischemia-reperfusion injury.

The role of CXCR2/CXCR2 ligand biological axis in renal cell carcinoma.

Renal cell carcinoma (RCC) accounts for 3% of new cancer incidence and mortality in the United States. Studies in RCC have predominantly focused on VEGF in promoting tumor-associated angiogenesis. However, other angiogenic factors may contribute to the overall angiogenic milieu of RCC. We hypothesized that the CXCR2/CXCR2 ligand biological axis represents a mechanism by which RCC cells promote angiogenesis and facilitate tumor growth and metastasis. Therefore, we first examined tumor biopsies and plasma of patients with metastatic RCC for levels of CXCR2 ligands, and RCC tumor biopsies for the expression of CXCR2. The proangiogenic CXCR2 ligands CXCL1, CXCL3, CXCL5, and CXCL8, as well as VEGF were elevated in the plasma of these patients and found to be expressed within the tumors. CXCR2 was found to be expressed on endothelial cells within the tumors. To assess the role of ELR(+) CXC chemokines in RCC, we next used a model of syngeneic RCC (i.e., RENCA) in BALB/c mice. CXCR2 ligand and VEGF expression temporally increased in direct correlation with RENCA growth in CXCR2(+/+) mice. However, there was a marked reduction of RENCA tumor growth in CXCR2(-/-) mice, which correlated with decreased angiogenesis and increased tumor necrosis. Furthermore, in the absence of CXCR2, orthotopic RENCA tumors demonstrated a reduced potential to metastasize to the lungs of CXCR2(-/-) mice. These data support the notion that CXCR2/CXCR2 ligand biology is an important component of RCC tumor-associated angiogenesis and tumorigenesis.

Role of CXCR2/CXCR2 ligands in vascular remodeling during bronchiolitis obliterans syndrome.

Angiogenesis and vascular remodeling support fibroproliferative processes; however, no study has addressed the importance of angiogenesis during fibro-obliteration of the allograft airway during bronchiolitis obliterans syndrome (BOS) that occurs after lung transplantation. The ELR(+) CXC chemokines both mediate neutrophil recruitment and promote angiogenesis. Their shared endothelial cell receptor is the G-coupled protein receptor CXC chemokine receptor 2 (CXCR2). We found that elevated levels of multiple ELR(+) CXC chemokines correlated with the presence of BOS. Proof-of-concept studies using a murine model of BOS not only demonstrated an early neutrophil infiltration but also marked vascular remodeling in the tracheal allografts. In addition, tracheal allograft ELR(+) CXC chemokines were persistently expressed even in the absence of significant neutrophil infiltration and were temporally associated with vascular remodeling during fibro-obliteration of the tracheal allograft. Furthermore, in neutralizing studies, treatment with anti-CXCR2 Abs inhibited early neutrophil infiltration and later vascular remodeling, which resulted in the attenuation of murine BOS. A more profound attenuation of fibro-obliteration was seen when CXCR2(-/-) mice received cyclosporin A. This supports the notion that the CXCR2/CXCR2 ligand biological axis has a bimodal function during the course of BOS: early, it is important for neutrophil recruitment and later, during fibro-obliteration, it is important for vascular remodeling independent of neutrophil recruitment.

Endothelial cell co-stimulation through OX40 augments and prolongs T cell cytokine synthesis by stabilization of cytokine mRNA.

Human endothelial cells (ECs) constitutively express OX40L and co-stimulate memory CD4(+) T cell proliferation that is dependent upon OX40-OX40L interaction. In vivo, OX40 prolongs T cell survival; however, an unanswered question is whether it can also prolong synthesis of proliferation-sustaining cytokines such as IL-2. Here we show that EC co-stimulation results in the secretion of T cell IL-2, IL-3 and IFN-gamma and that in the absence of OX40 signals synthesis largely ceases by 12-18 h, but is prolonged up to 60 h in the presence of OX40 signaling. Blocking OX40-mediated cytokine expression at later times suppresses T cell proliferation and this can be overcome by addition of exogenous IL-2. We find that OX40 signaling has discrete effects on T cell activation as it does not affect expression of IL-10, CD25, CD69 or soluble IL-2R. Also, OX40 does not appear to alter IL-2 transcription, but rather acts to stabilize a subset of cytokine mRNAs, increasing their half-lives by 3-6-fold. We further show that OX40L induces activation of p38 mitogen-activated protein kinase (MAPK) and phosphotidyl-inositol-3-kinase (PI3K) in T cells, and using specific inhibitors, we find that increased mRNA half-life is dependent upon both these pathways but is independent of c-jun-N-terminal kinase (JNK). Thus, EC co-stimulation through OX40 leads to prolonged T cell cytokine synthesis and enhanced proliferation.

Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1alpha.

Non-small cell lung cancer (NSCLC) expresses a particularly aggressive metastatic phenotype, and patients with this disease have a poor prognosis. CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung, breast, kidney, and prostate. In addition, overexpression of the epidermal growth factor receptor (EGFR) is associated with the majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility. Here we show for the first time that activation of the EGFR by EGF increases CXCR4 expression and the migratory capacity of NSCLC cells. Furthermore, many solid tumors are associated with low oxygen tension, and when NSCLC cells were cultured with EGF under hypoxic conditions, CXCR4 expression was dramatically enhanced. A molecular analysis of these events indicated that augmented CXCR4 expression was regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signal transduction pathway, activation of hypoxia inducible factor (HIF) 1alpha, and ultimately HIF-1-dependent transcription of the CXCR4 gene. Thus, a combination of low oxygen tension and overexpression of EGFR within the primary tumor of NSCLC may provide the microenvironmental signals necessary to upregulate CXCR4 expression and promote metastasis.