James M. Donahue

Complete Pathologic Response After Neoadjuvant Chemoradiotherapy for Esophageal Cancer Is Associated With Enhanced Survival

BACKGROUND Neoadjuvant chemoradiotherapy followed by esophagogastrectomy has become the standard of care for patients with locally advanced esophageal cancer. This report analyzes our experience with this treatment approach. METHODS From January 1998 through December 2003, all patients from a single institution receiving neoadjuvant chemoradiotherapy followed by esophagogastrectomy were reviewed for operative mortality, morbidity, long-term survival, and factors affecting survival. Only patients preoperatively staged with both computed tomographic scans and endoscopic ultrasound were included. RESULTS There were 162 patients (142 men, 20 women), and the median age was 61 years (range, 22 to 81 years). Histopathology was adenocarcinoma in 143 patients and squamous cell in 19. Pretreatment clinical stage was II in 28 patients (17%), III in 111 (68%), and IV (M1a) in 23 (14%). Ivor Lewis esophagogastrectomy was the most common procedure, occurring in 132 patients. Operative mortality and morbidity was 4.9% and 37%, respectively. Pathologic response was complete in 42 patients (26%), near complete in 27 (17%), partial in 88 (54%), and unresectable in 5 (3%). Five-year survival for overall, complete, near complete, and partial response patients was 34%, 55%, 27%, and 27%, respectively (p = 0.013). Patients whose lymph nodes were rendered free of cancer showed improved overall and disease-free survival compared with patients having persistently positive lymph nodes (p = 0.019). CONCLUSIONS Esophagogastrectomy after neoadjuvant chemoradiotherapy can be performed with low mortality and morbidity. Patients with complete pathologic response have significantly improved long-term survival compared with patients with near complete and partial responses. Future efforts should be directed at understanding determinants of complete responses.

Downregulation of miR-486-5p contributes to tumor progression and metastasis by targeting protumorigenic ARHGAP5 in lung cancer

We have previously shown that miR-486-5p is one of the most downregulated micro RNAs in lung cancer. The objective of the study was to investigate the role of miR-486-5p in the progression and metastasis of non-small-cell lung cancer (NSCLC). We evaluated miR-486-5p expression status on 76 frozen and 33 formalin-fixed paraffin-embedded tissues of NSCLC by quantitative reverse transcriptase PCR to determine its clinicopathologic significance. We then performed function analysis of miR-486-5p to determine its potential roles on cancer cell migration and invasion in vitro and metastasis in vivo. We also investigated the target genes of miR-486-5p in lung tumorigenesis. miR-486-5p expression level was significantly lower in lung tumors compared with their corresponding normal tissues (P<0.0001), and associated with stage (P=0.0001) and lymph node metastasis of NSCLC (P=0.0019). Forced expression of miR-486-5p inhibited NSCLC cell migration and invasion in vitro and metastasis in mice by inhibiting cell proliferation. Furthermore, ectopic expression of miR-486-5p in cancer cells reduced ARHGAP5 expression level, whereas miR-486-5p silencing increased its expression. Luciferase assay demonstrated that miR-486-5p could directly bind to the 3′-untranslated region of ARHGAP5. The expression level of miR-486-5p was inversely correlated with that of ARHGAP5 in lung tumor tissues (P=0.0156). Reduced expression of ARHGAP5 considerably inhibited lung cancer cell migration and invasion, resembling that of miR-486-5p overexpression. miR-486-5p may act as a tumor-suppressor contributing to the progression and metastasis of NSCLC by targeting ARHGAP5. miR-486-5p would provide potential diagnostic and therapeutic targets for the disease.

Regulation of herpes simplex virus γ134.5 expression and oncolysis of diffuse liver metastases by Myb34.5

Myb34.5 is a herpes simplex virus 1 (HSV-1) mutant deleted in the gene for ribonucleotide reductase (ICP6). It also carries a version of γ134.5 (a viral gene product that promotes the dephosphorylation of eIF-2α) that is under control of the E2F-responsive cellular B-myb promoter, rather than of its endogenous promoter. Myb34.5 replication in tumor cells results in their destruction (oncolysis). γ134.5 expression by HSV-1 subverts an important cell defense mechanism against viral replication by preventing shutoff of protein synthesis after viral infection. Infection of colon carcinoma cells with Myb34.5 results in greater eIF-2α dephosphorylation and viral replication compared with infection with HSV-1 mutants completely defective in γ134.5 expression. In contrast, infection of normal hepatocytes with Myb34.5 results in low levels of eIF-2α dephosphorylation and viral replication that are similar to those observed with HSV-1 mutants completely defective in γ134.5 and ICP6. When administered intravascularly into mice with diffuse liver metastases, Myb34.5 has greater antineoplastic activity than HSV-1 mutants with completely defective γ134.5 expression and more restricted biodistribution compared with HSV-1 mutants with wild-type γ134.5 expression. Myb34.5 displays reduced virulence and toxicity compared to HSV-1 mutants with wild-type γ134.5 expression. Portal venous administration of Myb34.5 significantly reduces liver tumor burden in and prolongs the life of mice with diffuse liver metastases. Preexisting Ab’s to HSV-1 do not reduce the antitumor efficacy of Myb34.5 in vivo.

Selectivity of an Oncolytic Herpes Simplex Virus for Cells Expressing the DF3/MUC1 Antigen

Replication-conditional viruses destroy tumors in a process referred to as viral oncolysis. An important prerequisite for this cancer therapy strategy is use of viruses that replicate preferentially in neoplastic cells. In this study the DF3/MUC1 promoter/enhancer sequence is used to regulate expression of γ134.5 to drive replication of a Herpes simplex virus 1 (HSV-1) mutant (DF3γ34.5) preferentially in DF3/MUC1-positive cells. HSV-1 γ134.5 functions to dephosphorylate elongation initiation factor 2α, which is an important step for robust HSV-1 replication. After DF3γ34.5 infection of cells, elongation initiation factor 2α phosphatase activity and viral replication were observed preferentially in DF3/MUC1-positive cells but not in DF3/MUC1-negative cells. Regulation of γ134.5 function results in preferential replication in cancer cells that express DF3/MUC1, restricted biodistribution in vivo, and less toxicity as assessed by LD50. Preferential replication of DF3γ34.5 was observed in DF3/MUC1-positive liver tumors after intravascular perfusion of human liver specimens. DF3γ34.5 was effective against carcinoma xenografts in nude mice. Regulation of γ134.5 by the DF3/MUC1 promoter is a promising strategy for development of HSV-1 mutants for viral oncolysis.

Regulation of Herpes Simplex Virus 1 Replication Using Tumor-Associated Promoters

ObjectiveTo investigate use of transcriptional regulatory elements (promoters) for tumor-associated antigens to achieve HSV-1 replication preferentially in cells that overexpress the tumor-associated antigens. Summary Background DataAn important advantage of replicating viruses for cancer therapy is their ability to simultaneously destroy tumor cells by replication and release progeny virion to infect and destroy adjacent cancer cells. This strategy requires regulation of the viral life cycle to obtain robust replication in neoplastic cells and minimize replication in nonneoplastic cells. MethodsPromoters for the human carcinoembryonic antigen (CEA) and MUC1/DF3 tumor-associated antigens were characterized and cloned into HSV-1 mutants as heterologous promoters regulating expression of two different HSV-1 genes. Viral replication in tumor cells and cytotoxicity was quantified with in vitro assays. Antineoplastic efficacy was characterized in a flank tumor xenograft model. ResultsSeveral CEA promoters were cloned and characterized using luciferase reporter assays. The most specific promoter was used to construct and isolate two different HSV-1 mutants in which critical genes are regulated by this promoter (ICP4 and &ggr;134.5). Similarly, the promoter for the DF3/MUC1 tumor-associated antigen was cloned into a third HSV-1 mutant such that it regulates expression of &ggr;134.5. Regulation of ICP4 expression by the CEA promoter during HSV-1 infection overly attenuates viral replication. Regulation of &ggr;134.5 expression by either the CEA promoter or the MUC1/DF3 promoter during HSV-1 infection modulates viral replication, with preferential replication in cells that overexpress the corresponding tumor-associated antigen. A single intratumoral inoculation of an HSV-1 mutant with the MUC1/DF3 promoter regulating &ggr;134.5 expression results in significant antineoplastic activity in MUC1-positive pancreatic carcinoma xenografts as compared to mock inoculation. ConclusionsPromoters for tumor-associated antigens may be incorporated into the HSV-1 genome to regulate HSV-1 replication. The choices of HSV-1 gene and tumor-associated promoter are important determinants of success of this strategy. Because of its preferential replication in MUC1-positive tumors, an HSV-1 mutant with the MUC1/DF3 promoter regulating &ggr;134.5 expression will undergo further examination as a novel cancer therapy agent.

Competition between RNA-binding proteins CELF1 and HuR modulates MYC translation and intestinal epithelium renewal

ELAV-like family member 1, or CELF1, competes with another RNA-binding protein, HuR, to modulate MYC translation and plays an important role in the regulation of intestinal epithelial renewal.

Oncologic Efficacy of Anatomic Segmentectomy in Stage IA Lung Cancer Patients With T1a Tumors

BACKGROUND Segmentectomy provides an anatomic, parenchymal-sparing strategy for patients with limited lung function. Recently, interest has been renewed in segmentectomy for the treatment of early stage lung cancer. METHODS We reviewed the medical records of all patients undergoing segmentectomy from January 1999 through December 2004. Survival curves were estimated using the Kaplan-Meier method. RESULTS There were 113 consecutive patients (58 men, 55 women); median age was 72.5 years (range, 30 to 94 years). Median forced expiratory volume in 1 second was 1.53 L (range, 0.5 L to 3.27 L). Median diffusion capacity of lung for carbon monoxide was 69% predicted (range, 23% to 129%). Significant comorbidities were present in 62 patients (55%). There was no perioperative mortality. Major morbidity occurred in 28 patients (25%). Mean tumor size was 2.1 cm. Resection margins were negative in all cases. Ninety-two patients (81%) were stage I. Overall 5-year survival was 79% for stage IA patients. Current smoking, diffusion capacity of lung for carbon monoxide less than 69%, tumor size greater than 2 cm, N2 disease, and advanced histology grade were associated with decreased survival by univariate analysis. In a multivariate model, only tumor size greater than 2 cm remained significant. Tumor recurrence was observed in 39 patients (35%): local in 17 patients (15%) and distant only in 22 (20%). For stage IA patients with T1a lesions, local recurrence was 5% and distant recurrence was 13%. Five-year recurrence-free survival of these patients was 69%. CONCLUSIONS Pulmonary segmentectomy can be performed safely in selected patients with preoperative reduced lung function and comorbidities. For stage IA disease, survival approximates that seen after lobectomy, with similar local recurrence rates for patients with T1a tumors.

Overexpression of miR-214-3p in esophageal squamous cancer cells enhances sensitivity to cisplatin by targeting survivin directly and indirectly through CUG-BP1.

Based on its marked overexpression in multiple malignancies and its roles in promoting cell survival and proliferation, survivin is an attractive candidate for targeted therapy. Toward this end, a detailed understanding of the mechanisms regulating survivin expression in different cancer cells will be critical. We have previously shown that the RNA-binding protein (RBP) CUG-BP1 is overexpressed in esophageal cancer cells and post-transcriptionally regulates survivin in these cells. The objective of this study was to investigate the role of microRNAs (miRs) in regulating survivin expression in esophageal cancer cells. Using miR expression profiling analysis, we found that miR-214-3p is one of the most markedly downregulated miRs in two esophageal squamous cancer cell lines compared with esophageal epithelial cells. Interestingly, using miR target prediction programs, both survivin and CUG-BP1 mRNA were found to contain potential binding sites for miR-214-3p. Forced expression of miR-214-3p in esophageal cancer cells leads to a decrease in the mRNA and protein levels of both survivin and CUG-BP1. This effect is due to decreased mRNA stability of both targets. By contrast, silencing miR-214-3p in esophageal epithelial cells leads to an increase in both survivin and CUG-BP1 mRNA and protein. To determine whether the observed effect of miR-214-3p on survivin expression was direct, mediated through CUG-BP1, or both, binding studies utilizing biotin pull-down assays and heterologous luciferase reporter constructs were performed. These demonstrated that the mRNA of survivin and CUG-BP1 each contain two functional miR-214-3p-binding sites as confirmed by mutational analysis. Finally, forced expression of miR-214-3p enhances the sensitivity of esophageal cancer cells to cisplatin-induced apoptosis. This effect is abrogated with rescue expression of survivin or CUG-BP1. These findings suggest that miR-214-3p acts as a tumor suppressor and that its downregulation contributes to chemoresistance in esophageal cancer cells by targeting both survivin and CUG-BP1.

The prognostic importance of pathologically involved celiac node metastases in node‐positive patients with carcinoma of the distal esophagus or gastroesophageal junction: a surgical series from the Mayo Clinic

The management of esophageal cancer with involvement of celiac lymph nodes is controversial. The purpose of this retrospective study was to evaluate the clinical importance of metastases to celiac lymph nodes in patients with carcinoma of the distal esophagus or gastroesophageal junction (GEJ) who undergo surgical treatment with curative intent. We reviewed the medical records of 310 patients who underwent definitive esophagectomy at the Mayo Clinic, Rochester, Minnesota, between 1976 and 1999 for carcinoma of the distal esophagus or GEJ. The disease location was distal esophagus in 163 and GEJ in 147. Fifty-two patients (17%) were found to have celiac node involvement. The survival of these patients was compared with that of 97 N0 patients and 161 N1 patients without celiac node involvement. Squamous cell carcinoma and adenocarcinomas were found in 24% and 76%, respectively. Ivor Lewis esophagectomy was the most common surgical procedure (76%), followed by transhiatal resection (14%) and modified Ivor Lewis procedure (5%). The median number of nodes resected was 15 (range, 2-45). The median survival of the entire group was 18.8 months. The median survival was 48 months (range, 1.6 months-22 years) for N0 patients and 15.9 months (range, 0.03 months-14.4 years) for N1 patients without celiac node disease (P < 0.001). The median survival was 11.7 months (range, 2.2 months-15.7 years) for celiac node-positive patients, and this difference was statistically significant when compared with survival in N0 patients (P= 0.001) but not when compared with that in N1 patients without celiac node disease (P= 0.57). Survival at 3 and 5 years was 61% and 45% for N0 patients, 21% and 9% for N1 patients without celiac node disease, and 18% and 11% for patients with celiac node disease, respectively. At 10 years, 7% of patients with celiac node involvement in their resected specimen were alive. By multivariate analysis, patients with 4 or more positive lymph nodes had the worst prognosis (risk ratio [RR], 2.63; 95% confidence interval [CI], 1.98-3.48), regardless of their location. We concluded that celiac node metastases were not an adverse prognostic indicator in patients with celiac node involvement compared with N1 patients without celiac node disease. Overall, the number of positive nodes, not their location, correlated best with survival. Although median survival was poor, a small number of patients with resected celiac node disease had long-term survival. Patients with undetected celiac node disease at the time of surgical resection who were subsequently found to have celiac node involvement appeared to have a prognosis similar to that of patients with stage III disease. Therefore, treatment with curative intent should be considered for fit patients with celiac node disease.

Src-mediated caveolin-1 phosphorylation regulates intestinal epithelial restitution by altering Ca2+ influx after wounding

Early mucosal restitution occurs as a consequence of intestinal epithelial cell (IEC) migration to reseal superficial wounds, but its exact mechanism remains largely unknown. Caveolin-1 (Cav1), a major component associated with caveolar lipid rafts in the plasma membrane, is implicated in many aspects of cellular functions. This study determined if c-Src kinase (Src)-induced Cav1 phosphorylation promotes intestinal epithelial restitution after wounding by activating Cav1-mediated Ca(2+) signaling. Src directly interacted with Cav1, formed Cav1-Src complexes, and phosphorylated Cav1 in IECs. Inhibition of Src activity by its chemical inhibitor PP2 or suppression of the functional caveolin scaffolding domain by caveolin-scaffolding domain peptides prevented Cav1-Src interaction, reduced Cav1 phosphorylation, decreased Ca(2+) influx, and inhibited cell migration after wounding. Disruption of caveolar lipid raft microdomains by methyl-β-cyclodextrin reduced Cav1-mediated Ca(2+) influx and repressed epithelial restitution. Moreover, Src silencing prevented subcellular redistribution of phosphorylated Cav1 in migrating IECs. These results indicate that Src-induced Cav1 phosphorylation stimulates epithelial restitution by increasing Cav1-mediated Ca(2+) signaling after wounding, thus contributing to the maintenance of gut mucosal integrity under various pathological conditions.