A randomized, placebo-controlled trial of aprotinin to reduce primary graft dysfunction following lung transplantation.

PURPOSE: Severe primary graft dysfunction (PGD) is the major early problem following lung transplantation. Aprotinin, a serine protease inhibitor, has many anti-inflammatory properties that might reduce or prevent lung injury. Our hypothesis was that the incidence of PGD could be reduced by a combination of donor lung perfusion and systemic administration of aprotinin to recipients. METHODS AND MATERIALS: The study was randomized and placebo controlled. Donor lungs were perfused during procurement with 4 L Perfadex containing aprotinin (280 mg load + 70 mg/hL) or placebo. Aprotinin or placebo was also administered peri-operatively to the recipients. The study was powered to detect a 10% improvement in the primary endpoint of developing ISHLT grade III PGD anytime within 48 hr following the transplant procedure. RESULTS: There were 48 patients randomized. Diagnosis and the use of bypass were different between groups. The study was stopped prematurely at the planned interim analysis point because of published concerns about renal toxicity of aprotinin. There was no difference in the occurrence of the primary endpoint between groups of patients. The median change from the baseline creatinine level at 24, 48, 72 hr; 7 and 30 d following the transplant was not associated with the administration of aprotinin. CONCLUSIONS: There was no statistically significant difference in the incidence of the primary endpoint between groups in the study. Excess renal failure related to aprotinin administration in a patient population at high risk for the event was not observed.

Early Trends in PaO(2)/fraction of inspired oxygen ratio predict outcome in lung transplant recipients with severe primary graft dysfunction.

BACKGROUND: The development of severe primary graft dysfunction (PGD) is a risk factor for perioperative death following lung transplantation. Our goal is to improve the predictive value of the earliest Pao(2)/fraction of inspired oxygen (P/F) measurements that gauge PGD severity. METHODS: We identified 96 patients with severe PGD (P/F < 200) at ICU arrival through a retrospective review of 431 lung transplants performed at our institution from 1992 to 2005. The P/F trend, represented as quartiles of the 12-h percentage change in P/F, was analyzed using multivariate logistic regression. Study outcomes were 90-day death and long-term survival. RESULTS: The median percentage change in P/F over 12 h was + 52% (interquartile range, +20 to 90%). We observed the highest early mortality among those in the lowest quartile of the P/F trend (an increase in P/F

Evolution of clipping for thoracoscopic sympathectomy in symptomatic hyperhidrosis.

BACKGROUND: Severe hyperhidrosis is treated by thoracic sympathetic chain interrupting through chain transection or clipping. Our study compared the efficacy of these 2 methods. METHODS: Retrospectively, patients who underwent thoracoscopic sympathectomy from 1999 to 2005 had demographic, operative, and postoperative data analyzed. RESULTS: Fifty-four operations were performed for refractory sweating of the palm (72%), axilla (66%), foot (53%), and head/neck (19%). Thirty-seven (69%) underwent clipping; 17 (31%) underwent chain transection. There was no difference in age, sex, or blood loss. One ganglion level was interrupted in 24.1% and 2 levels in 75.4%. Bothersome compensatory hyperhidrosis occurred in 13% (5-clipping and 2-transection). One patient underwent clip removal for debilitating symptoms. Three small pneumothoraces occurred (all in the transection group); all treated expectantly. CONCLUSIONS: Thoracoscopic sympathectomy is a safe outpatient procedure. Both methods yield excellent results with minimal compensatory hyperhidrosis. Thoracoscopic sympathetic chain clipping and transection are equivalent.

Gene expression profiles in esophageal adenocarcinoma.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has risen dramatically in the last two decades. As with other malignancies, changes in gene expression play a key role in the development and progression of these tumors. METHODS: Microarray analysis was used to study gene expression of 12,000 genes in EAC specimens. Adenocarcinoma tissue samples (n = 10) and controls of normal stomach (n = 6) and esophageal (n = 7) mucosa were collected fresh, then rapidly frozen in liquid nitrogen. The messenger ribonucleic acid (mRNA) from the samples was isolated, reverse transcribed, and used to generate biotin-labeled mRNA fragments, which were hybridized to Affymetrix U95 gene chips (AME Bioscience, Norway) for analysis. Additional samples analyzed included tissue containing dysplastic Barrett's epithelium from three patients, metastatic lymph nodes from two patients with EAC, one squamous carcinoma, and two esophageal cancer cell lines. Samples were segregated into groups with similar patterns of gene expression using clustering algorithms and gene sets that differentiated tumors from normal tissue were generated. RESULTS: There were 150 genes that were fourfold up regulated and 183 genes that were fourfold down regulated in the esophageal adenocarcinoma specimens, as compared to normal esophageal mucosa tissue controls. Using paired specimens (n = 5) and the paired t-test (p Value of 0.05) as a filter, only 64 genes were fourfold up regulated and 110 were fourfold down regulated. These groups included cytoskeletal, cell adhesion, tumor suppressor, and signal transduction genes. Hierarchical clustering segregated the samples into the expected divisions. The esophageal cancer cell lines, OE19 and OE33, clustered separately from the EAC specimens. Extremely high gene expression levels of the ERBB2 gene, seen in the microarray analysis of the 2 cell lines, correlated with amplification of the gene determined by Southern blotting. CONCLUSIONS: Gene expression patterns from a small subset of genes distinguish EAC specimens from normal controls. This technique can rapidly identify genes for targeted chemotherapeutic approaches to cancer treatment.

Late outcome of mitral valve surgery for patients with coronary artery disease.

BACKGROUND: We plan to determine whether the cause of mitral valve regurgitation, ischemic or degenerative, affects survival after combined mitral valve repair or replacement and coronary artery bypass grafting (CABG) surgery and to assess the influence of residual mitral regurgitation on late outcome. METHODS: A retrospective study was made of 302 patients having mitral valve repair or replacement and CABG from January 1987 through December 1996. Risk factors for death, for development of New York Heart Association class III or IV congestive heart failure (CHF), and recurrent mitral valve regurgitation were identified by proportional hazards analysis. RESULTS: The cause of mitral regurgitation was ischemic in 137 patients (45%) and degenerative in 165 patients (55%). Valve replacement was performed in 51 patients (17%) and valve repair in 251 patients (83%). Median follow-up was 64 months. Ten-year actuarial survival rates were 33% (95% confidence interval: 22% to 47%) in the ischemic group and 52% (95% confidence interval: 42% to 64%) in the degenerative group. Univariate predictors of death, were entered into a multivariate model. Older age, ejection fraction of 35% or less, three-vessel coronary artery disease, replacement of the mitral valve, and residual mitral regurgitation at dismissal were independent risk factors for death. The cause of mitral valve regurgitation (ischemic or degenerative) was not an independent predictor of long-term survival, class III or IV CHF, or recurrent regurgitation. CONCLUSIONS: Survival after mitral valve surgery and CABG is determined by the extent of coronary disease and ventricular dysfunction and by the success of the valve procedure; etiology of mitral valve regurgitation has relatively little impact on late outcome.

ERBB2 suppression decreases cell growth via apoptosis in gastrointestinal adenocarcinomas.

BACKGROUND: Although the incidence of adenocarcinoma of the esophageal and gastroesophageal junction has increased at an alarming rate in the past 30 years, little improvement has been made in treatment strategies. Previous studies have demonstrated that many upper gastrointestinal (GI) adenocarcinomas exhibit ERBB2 amplification. In cancers proven to have similar amplification, such as breast, ERBB2-targeted therapies have dramatically improved overall survival and disease-free rates of survival. This study uses siRNA to knockdown ERBB2 in GI adenocarcinoma cell lines to evaluate cell viability, apoptosis, and changes in cell cycle. METHODS: A cell line with a baseline amount of ERBB2 (Seg-1) and 2 upper GI adenocarcinoma cell lines with known amplification of ERBB2 (esophageal [OE19] and gastric [MKN45]) were treated with 120 pmol of 1 of 2 independent ERBB2 siRNAs or control siRNA for 6 hours. RESULTS: We demonstrate that knockdown of ERBB2 in esophageal and gastric cancer cell lines with known ERBB2 amplification effectively decreases ERBB2 protein levels and decreases cell viability mainly via apoptotic pathways. CONCLUSION: ERBB-directed therapy may be of benefit in the subset of patients with GI adenocarcinomas exhibiting amplification of ERBB2.

Nontoxic chemical interdiction of the epithelial-to-mesenchymal transition by targeting cap-dependent translation.

Normal growth and development depends upon high fidelity regulation of cap-dependent translation initiation, a process that is usurped and redirected in cancer to mediate acquisition of malignant properties. The epithelial-to-mesenchymal transition (EMT) is a key translationally regulated step in the development of epithelial cancers and pathological tissue fibrosis. To date, no compounds targeting EMT have been developed. Here we report the synthesis of a novel class of histidine triad nucleotide binding protein (HINT)-dependent pronucleotides that interdict EMT by negatively regulating the association of eIF4E with the mRNA cap. Compound eIF4E inhibitor-1 potently inhibited cap-dependent translation in a dose-dependent manner in zebrafish embryos without causing developmental abnormalities and prevented eIF4E from triggering EMT in zebrafish ectoderm explants without toxicity. Metabolism studies with whole cell lysates demonstrated that the prodrug was rapidly converted into 7-BnGMP. Thus we have successfully developed the first nontoxic small molecule able to inhibit EMT, a key process in the development of epithelial cancer and tissue fibrosis, by targeting the interaction of eIF4E with the mRNA cap and demonstrated the tractability of zebrafish as a model organism for studying agents that modulate EMT. Our work provides strong motivation for the continued development of compounds designed to normalize cap-dependent translation as novel chemo-preventive agents and therapeutics for cancer and fibrosis.

Volume-outcome relationship for coronary artery bypass grafting in an era of decreasing volume.

HYPOTHESIS: We hypothesized that the recent reduction in procedure volume for coronary artery bypass grafting (CABG) has led to an increase in the in-hospital mortality rate. DESIGN: Hospital discharge data from the Nationwide Inpatient Sample from January 1, 1988, through December 31, 2003. SETTING: A 20% random sample of patients admitted to US hospitals. PATIENTS: All patients who underwent CABG or percutaneous transluminal coronary interventions. Facilities performing CABG were assigned to standard volume cutoffs. MAIN OUTCOME MEASURES: Rates of cardiac procedures and the proportion of hospitals meeting standard volume cutoffs, as well as the CABG mortality rate. RESULTS: During our 16-year study period, the rate of CABG increased from 7.2 cases per 1000 discharges in 1988 to 12.2 cases in 1997 but then decreased to 9.1 cases in 2003, while the rate of percutaneous interventions tripled. For CABG, the proportion of high-volume hospitals declined from 32.5% in 1997 to 15.5% in 2003. Despite shifts between high- and low-volume hospitals, the CABG mortality rate steadily declined from 5.4% in 1988 to 3.3% in 2003. Hospitals performing the lowest volume of CABG experienced the largest decrease in the in-hospital mortality rate. CONCLUSIONS: Since 1997, CABG volume has declined in the setting of a decrease in in-hospital mortality. A lower mortality rate in the setting of reduced CABG volume is a counterintuitive finding, suggesting that procedure volume is an insufficient predictor of outcome on which to base regionalization strategies.

Ninety-day mortality and major complications are not affected by use of lung allocation score.

BACKGROUND: In May 2005 the Organ Procurement Transplant Network (OPTN) and United Network for Organ Sharing (UNOS) implemented the donor lung allocation score (LAS) system to prioritize organ allocation among prospective transplant recipients. The purpose of our study was to determine the impact of LAS implementation on 90-day survival, early complications and incidence of severe primary graft dysfunction (PGD) after the transplant procedure. METHODS: Early outcomes among 78 patients receiving transplants after the initiation of the scoring system were compared with those of the 78 previous patients. Survival rates at 90 days and 1 year were the primary end-points of the study. Arterial blood-gas measurements were collected for all patients at the time of ICU arrival and at 12, 24 and 48 hours after surgery to determine the distribution of International Society of Heart and Lung Transplant (ISHLT) PGD grade. Major complications within 30 days post-transplant were recorded. RESULTS: We found a small but significant 1-year survival advantage among post-LAS implementation patients, which was largely due to decreased early mortality in comparison to the control cohort. The incidence of ISHLT Grade 3 PGD measured within the first 24 hours after transplant did not differ between groups, nor was there an increase in the rate of major post-operative complications. CONCLUSIONS: Implementation of the LAS system has not been associated with an increase in early mortality, immediate PGD or major complications.

Primary graft dysfunction and long-term pulmonary function after lung transplantation.

BACKGROUND: Severe primary graft dysfunction (PGD) is associated with poor early outcomes after lung transplantation (LTx). Less is known about lingering effects of severe PGD on pulmonary function. The study's aim was to determine whether development of severe primary graft dysfunction in the perioperative period was associated with reduced long term rates of survival or with diminished long term pulmonary function. METHODS: A retrospective review was performed on LTx recipients who received their transplant during the period from 1992 through 2005. PGD severity over the first 48 hours post-transplant was graded using International Society for Heart Lung Transplantation criteria. Pulmonary function was evaluated yearly, and bronchiolitis obliterans syndrome (BOS) was determined from measurements of forced expiratory volume in 1 second (FEV(1)). RESULTS: A total of 374 patients survived at least 90 days post-transplant. Overall survival rates were worse in patients with Grade 3 PGD: 51% at 5 years and 11% at 10 years for patients with Grade 3 PGD; 64% at 5 years and 35% at 10 years for those with Grade 2 PGD; and 66% at 5 years and 38% at 10 years for Grade 0 to 1 PGD (p = 0.001). BOS-free survival rate for patients with Grade 3 PGD was lower compared to those with Grade 0 to 2 for bilateral lung recipients, but not for single-lung recipients. Bilateral lung recipients who developed Grade 3 PGD had a significantly worse mean FEV(1) than those who did not. For single-lung recipients, PGD grade did not correlate with post-transplant pulmonary function. CONCLUSIONS: Development of Grade 3 PGD in the early post-operative period negatively affects long-term survival, BOS-free survival and pulmonary function of bilateral lung transplant recipients who survive the peri-operative period.

Video-assisted thoracoscopic surgery is more favorable than thoracotomy for resection of clinical stage I non-small cell lung cancer.

BACKGROUND: Lobectomy for patients with clinical stage I non-small cell lung cancer (NSCLC) can be performed by thoracotomy or by video-assisted thoracoscopic surgery (VATS). We compared the operative characteristics and postoperative course for patients with clinical stage I NSCLC who underwent lobectomy by VATS or thoracotomy. METHODS: We retrospectively reviewed the charts of all patients undergoing lobectomy for clinical stage I NSCLC from January 1, 1998, through June 30, 2005. RESULTS: We performed 147 lobectomies (88 thoracotomy, 59 VATS) in 147 patients with clinical stage I NSCLC. Patient demographics were similar between groups; however, VATS patients had more hypertension (p = 0.0114), chronic renal insufficiency (p = 0.0479), and previous malignancies (p = 0.0086). The two groups did not differ in pathologic stage, tumor size, histologic results, or number of positive nodes. More total nodes were identified in thoracotomy patients (p = 0.0001), and they had a shorter intensive care unit stay (p = 0.0224). VATS patients had significantly less postoperative pneumonia (p = 0.0023). VATS patients trended toward fewer chest tube days and a shorter hospital length of stay. The two groups did not differ in operative time, blood loss, atrial fibrillation, or number of ventilator days. Median survival between the cohorts was similar (>7.9 years thoracotomy versus >4.6 years VATS, log-rank p = 0.6939). CONCLUSIONS: Patients undergoing VATS lobectomy for clinical stage I NSCLC, despite having more comorbidities, had fewer postoperative complications. The approaches are equivalent in operative time, blood loss, length of stay, and survival rate. Compared with thoracotomy, VATS lobectomy for patients with clinical stage I NSCLC appears to be a less morbid operation.

Wedge gastroplasty and reinforced crural repair: important components of laparoscopic giant or recurrent hiatal hernia repair.

OBJECTIVE: Laparoscopic repair of a giant hiatal hernia (>50% of the stomach above the diaphragm) is associated with short-term recurrence rates of 12% to 42%. Recurrent hiatal hernias often have significantly altered anatomy, making laparoscopic repair challenging. We hypothesized that increasing intra-abdominal esophageal length by means of Collis wedge gastroplasty, complete fat-pad dissection, hernia-sac excision, and primary reinforced crural repair would minimize short-term recurrence and provide adequate symptomatic relief. METHODS: From January 1, 2001, though May 1, 2005, 61 patients underwent laparoscopic repair of a giant or recurrent hiatal hernia with a Collis wedge gastroplasty and Nissen fundoplication. Symptomatic outcomes were assessed with a validated questionnaire (Gastroesophageal Reflux Disease Health-Related Quality of Life). We obtained postoperative radiographic imaging to objectively assess anatomic results at a median of 1.13 years. RESULTS: Of the 61 patients, 12 (20%) were referred to our institution after previous repairs. Operating time averaged 308 +/- 103 minutes. The median hospital stay was 4 days. Postoperative complications occurred in 5 (8.2%) patients. One (1.6%) patient died of cardiac complications. Postoperatively, 52 (85%) patients completed the questionnaire with mean a Gastroesophageal Reflux Disease Health-Related Quality of Life questionnaire score of 1.15 +/- 2.78 (scale, 0-45; 0 = asymptomatic). Overall, 51 (98%) of the 52 respondents were satisfied with their surgical outcome. Postoperative radiographic data were available for 54 (89%) patients. We identified no recurrences at 1-month follow-up, and only 4.7% (2/42) had evidence of radiographic recurrence at 1 year or more. CONCLUSIONS: Consistent use of a Collis wedge gastroplasty with reinforced crural repair minimizes short-term recurrence after minimally invasive giant hiatal hernia repair. Symptomatic results are excellent in most patients.

Risk factors for primary graft dysfunction after lung transplantation.

OBJECTIVE: The International Society for Heart and Lung Transplantation has proposed a new grading system for primary graft dysfunction based on the ratio of arterial oxygen to fraction of inspired oxygen measured within 48 hours after lung transplantation. Worsening primary graft dysfunction grade is associated with increased operative mortality rates and decreased long-term survival. This study evaluated donor and recipient risk factors for postoperative International Society for Heart and Lung Transplantation grade 3 primary graft dysfunction. METHODS: We reviewed donor and recipient medical records of 402 consecutive lung transplantations performed between 1992 and 2004. We calculated a worst International Society for Heart and Lung Transplantation primary graft dysfunction grade in the first 48 hours postoperatively. Severe primary graft dysfunction (International Society for Heart and Lung Transplantation grade 3) was defined by a ratio of arterial oxygen to fraction of inspired oxygen of less than 200. Associations of potential risk factors with grade 3 primary graft dysfunction in the first 48 hours postoperatively were examined through bivariate and multivariate analysis. RESULTS: The 90-day mortality rate associated with the development of International Society for Heart and Lung Transplantation grade 3 primary graft dysfunction in the first 48 hours postoperatively was 17% versus 9% in the group without grade 3 primary graft dysfunction. Significant bivariate risk factors associated with this end point were increasing donor age, donor smoking history of more than 10 pack-years, early transplantation era (1992-1998), increasing preoperative recipient pulmonary artery pressure, and recipient diagnosis. In the multivariate analysis only recipient pulmonary artery pressure, donor age, and transplantation era were associated with grade 3 primary graft dysfunction in the first 48 hours postoperatively at a P value of less than .05. CONCLUSIONS: Our analysis of donor and recipient risk factors for severe primary graft dysfunction identified patient groups at high risk for poor outcomes after lung transplantation that might benefit from treatments aimed at reducing reperfusion injury.

Validation of the proposed International Society for Heart and Lung Transplantation grading system for primary graft dysfunction after lung transplantation.

BACKGROUND: A scoring system was recently proposed to grade the severity of primary graft dysfunction (PGD), a frequent early complication of lung transplantation. The purposes of this study are to: (1) validate the PGD grading system with respect to patient outcomes; and (2) compare the performance of criteria employing the arterial oxygenation to fraction of inspired oxygen (P/F) ratio to an alternative grading system employing the oxygenation index (OI). METHODS: We retrospectively reviewed the medical records of 402 patients having undergone lung transplantation at our institution from 1992 through 2004. The ISHLT PGD grading system was modified and grades were assigned up to 48 hours post-transplantation as follows: Grade 1 PGD, P/F > 300; Grade 2, P/F 200 to 300; and Grade 3, P/F < 200. A worst score T(0-48) was also assigned, which reflects the highest grade recorded between T0 and T48. RESULTS: The prevalence of severe PGD (P/F Grade 3) declined after transplant, from 25% at T0 to 15% at T48. Grouping patients by P/F grade at T48 demonstrated the clearest differentiation of 90-day death rates (Grade 1, 7%; Grade 2, 12%; Grade 3, 33%) (p = 0.0001). T48 OI grade also differentiates 90-day death rates. There was no difference in longer-term survival between patients with PGD Grades 1 and 2. OI grade at T0 qualitatively improved differential mortality between Grades 1 and 2; however, the differences did not reach statistical significance. Patients with a worst score T(0-48) of Grade 3 PGD did have significantly decreased long-term survival, as well as longer ICU and hospital stay, when compared with Grades 1 and 2 PGD. Significant risk factors for short- and long-term mortality in our multivariate model were P/F Grade 3 [worst score T(0-48) as well as T0 grade], single-lung transplant, use of cardiopulmonary bypass and high pre-operative mean pulmonary artery pressure. CONCLUSIONS: There is an increased risk of short- and long-term mortality and length of hospital stay associated with severe (Grade 3) PGD. The proposed ISHLT grading system can rapidly identify patients with poor outcomes who may benefit from early, aggressive treatment. Refinement of the scoring system may further improve patient risk stratification.

Does Perfadex affect outcomes in clinical lung transplantation?

BACKGROUND: The use of a low-potassium-based preservation solution improves gas exchange in experimental models of lung transplantation. However, its efficacy in reducing the incidence of primary graft dysfunction (PGD) and improving patient outcomes in the clinical setting is controversial. METHODS: In this study we measured: oxygenation index (OI); International Society of Heart and Lung Transplantation (ISHLT) PGD grades; extubation times; intensive care unit (ICU) and hospital length of stay; 30-day, 90-day and 1-year survival rates; and bronchiolitis obliterans syndrome (BOS)-free survival. We compared 115 consecutive (2001 to 2004) lung recipients who received allografts preserved with Perfadex, a low-potassium dextran (LPD) solution, and compared the results with the previous 116 consecutive (1999 to 2001) lung recipients who received allografts preserved with modified Euro-Collins (MEC) solution. Recipients were classified as having severe PGD (ISHLT Grade III) if the lowest arterial oxygenation (P) to fraction of inspired oxygen (F) (P/F ratio) within 48 hours post-transplantation was <200. RESULTS: Baseline characteristics of the 2 cohorts were similar except for recipient age (LPD 53.5 vs MEC 49.9 years; p = 0.03). There were no differences in donor age, gender, category of transplant, indication for transplant, use of cardiopulmonary bypass or pre-operative pulmonary artery pressures. When gas-exchange parameters were measured upon arrival to the ICU (T0), at 24 hours post-transplant (T24) and at 48 hours post-transplant (T48), the only significant finding was that the incidence of ISHLT Grade III PGD at T24 was lower in the LPD group compared with the MEC group (8% vs 20%, p = 0.03). The incidence of severe PGD at other timepoints was not statistically different (LPD vs MEC: T0, 17% vs 26%; T0 to T48, 25% vs 31%). Both groups had similar extubation rates at 48 hours post-transplant (LPD 64% vs MEC 67%). The 30-day survival (LPD 93% vs MEC 95%), 90-day survival (LPD 89% vs MEC 89%), 1-year patient survival (LPD 80% vs MEC 77%) and 1-year BOS-free survival (LPD 70% vs MEC 74%) were not statistically different. CONCLUSIONS: Lung preservation with LPD as compared with MEC does not improve early gas exchange or impact 90-day and 1-year mortality. Continued investigation into lung preservation solution composition is necessary to reduce the incidence of PGD.

Recent trends in lung transplantation: the University of Minnesota experience.

The number of transplants performed at our center continues to grow--partly as a result of the use of expanded donors and partly as a result of referrals from programs that have closed. We also anticipate having a more active living-donor lobar transplant program. The major acute problems that we encounter after transplantation are reperfusion injury and pneumonia. Improvements in perioperative mortality and morbidity will come with better lung preservation techniques and with an improved understanding of and an ability to modify the reperfusion process. BOS continues to be a major long-term problem for lung transplant patients. Although we do not understand the underlying pathogenesis of BOS, we are optimistic that BOS-free survival rates will increase with improvements in our ability to detect acute rejection as well as by avoidance of chronic injury to the lung from processes like GERD. Ongoing genetic analysis being conducted at our center will likely provide information about important biomarkers that define these processes.

ERBB2 amplifications in esophageal adenocarcinoma.

BACKGROUND: ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, Her-2-neu) gene amplification and overexpression has been reported in several types of cancer. The purpose of this study was to (1) determine the frequency of ERBB2 amplification (in comparison to other proto-oncogenes) in tumors from patients with esophageal adenocarcinoma, (2) characterize structural details of an ERBB2 amplicon in the esophageal adenocarcinoma cell line OE19 (contains a 100-fold ERBB2 amplification), and (3) test whether growth of the OE19 cell line is sensitive to the ERBB2 inhibitor trastuzumab (Herceptin; Genetech, Inc, San Francisco, CA). METHODS: First, we determined the frequency, by Southern blotting techniques, of amplification of ERBB2 and 13 other proto-oncogenes in a panel of 25 esophageal adenocarcinoma tumors. Then, in a second panel of 10 tumor specimens, expression levels of the ERBB2 gene and of several other genes that flank ERBB2 on chromosome 17 were determined by microarray analysis. Next we characterized the ERBB2 amplicon in the esophageal adenocarcinoma cell line OE19 using cytogenetic methods and a Rec-A protein assisted restriction endonuclease mapping technique. Finally, an in vitro growth inhibition assay was used to measure the sensitivity of OE19 and OE33 cells to treatment with trastuzumab (humanized antibody to ERBB2). RESULTS: ERBB2 was the most frequently amplified proto-oncogene among 25 esophageal adenocarcinoma tumors tested (greater than 10-fold amplification in 3 of 25 (12%) tumors tested). The OE19 cell line contains a 100-fold amplification of the ERBB2 gene, and highly expresses its messenger ribonucleic acid. Transcripts from genes that flank ERBB2 including GRB7, a protein linked to metastasis in esophageal cancer, also showed high levels of expression. In OE19 cells, the ERBB2 amplicon was localized to a homogeneously staining region of chromosome 14. Southern blots from the Rec-A protein assisted restriction endonuclease cleavage mapping experiments in OE19 showed a strong band of 210 kilobases in size, demonstrating that the main amplicon was a tandem repeat. In the in vitro growth inhibition assay, trastuzumab inhibited the OE19 and OE33 cells growth by 49% and 20%, respectively, at a saturating concentration of 20 microg/mL. CONCLUSIONS: ERBB2 is the most frequently amplified proto-oncogene in esophageal adenocarcinoma among the genes that we tested. In the OE19 esophageal adenocarcinoma cell line, the ERBB2 amplicon is translocated onto chromosome 14, is amplified 100-fold at the deoxyribonucleic acid level, and is highly overexpressed at the messenger ribonucleic acid level. Finally, the growth of this cell line was inhibited by incubation with trastuzumab. These results demonstrate that a substantial number of esophageal adenocarcinomas have amplified copies of the ERBB2 gene, and that they may be responsive to ERBB2 targeted therapies such as trastuzumab.