Concurrent chemotherapy inhibits herpes simplex virus-1 replication and oncolysis.

Herpes simplex virus-1 (HSV-1) replication in cancer cells leads to their destruction (viral oncolysis) and has been under investigation as an experimental cancer therapy in clinical trials as single agents, and as combinations with chemotherapy. Cellular responses to chemotherapy modulate viral replication, but these interactions are poorly understood. To investigate the effect of chemotherapy on HSV-1 oncolysis, viral replication in cells exposed to 5-fluorouracil (5-FU), irinotecan (CPT-11), methotrexate (MTX) or a cytokine (tumor necrosis factor-α (TNF-α)) was examined. Exposure of colon and pancreatic cancer cells to 5-FU, CPT-11 or MTX in vitro significantly antagonizes both HSV-1 replication and lytic oncolysis. Nuclear factor-κB (NF-κB) activation is required for efficient viral replication, and experimental inhibition of this response with an IκBα dominant-negative repressor significantly antagonizes HSV-1 replication. Nonetheless, cells exposed to 5-FU, CPT-11, TNF-α or HSV-1 activate NF-κB. Cells exposed to MTX do not activate NF-κB, suggesting a possible role for NF-κB inhibition in the decreased viral replication observed following exposure to MTX. The role of eukaryotic initiation factor 2α (eIF-2α) dephosphorylation was examined; HSV-1-mediated eIF-2α dephosphorylation proceeds normally in HT29 cells exposed to 5-FU, CPT-11 or MTX. This report demonstrates that cellular responses to chemotherapeutic agents provide an unfavorable environment for HSV-1-mediated oncolysis, and these observations are relevant to the design of both preclinical and clinical studies of HSV-1 oncolysis.

Active bleeding in acute subarachnoid hemorrhage observed by multiphase dynamic-enhanced CT.

BACKGROUND AND PURPOSE: Acute SAH is reportedly associated with rebleeding from aneurysms, and recent advances in imaging technology allow us to visualize active bleeding in SAH cases. This study aimed to retrospectively investigate the incidence and characteristics of active bleeding in patients with spontaneous SAH by using multiphase dynamic-enhanced CT. MATERIALS AND METHODS: We retrospectively surveyed a series of patients with SAH who underwent CTP with 18-phase dynamic enhancement and confirmed the presence of extravasated contrast medium in the source image. We compared clinical features between 2 groups of patients with and without extravasation. RESULTS: Active bleeding was observed with increasing enhancement in 25.5% (13/51) of patients. All patients with extravasation were in Claassen grade 3 or 4 and WFNS grades 3, 4, or 5. The other group without extravasation included patients in all grades. A significant difference was observed in Claassen grade, WFNS grade, and increase of hematomas in follow-up CT (P < .05, for each) between the 2 groups. All CTP results of patients with extravasation were obtained within 2 hours of the onset of symptoms of SAH (P < .05). There was no significant difference in mortality at 14 days between the 2 groups (P = .128). CONCLUSIONS: A high incidence of active bleeding (25.5%) was detected by multiphase dynamic-enhanced CT in patients with acute SAH. These results indicate that an awareness of active bleeding in patients with SAH has the potential to affect the treatment strategy.

Antitumor efficacy of oncolytic herpes simplex virus adsorbed onto antigen-specific lymphocytes.

Although several studies have reported that locally administering oncolytic viruses effectively targets malignancies, the efficacy of systemically administered oncolytic viruses is restricted. Recently, however, it was reported that systemic administration of oncolytic vesicular stomatitis virus adsorbed onto antigen-specific lymphocytes was effective against malignancies. We hypothesized that intravenously administering such virus might have significant potential in treatment of the malignant tumors. We adsorbed oncolytic herpes simplex virus-1 mutant R3616 onto lymphocytes harvested from mice with acquired antitumor immunity. We administered adsorbed R3616 to peritoneally disseminated tumors and analyzed the efficacy of this treatment. Mice administered adsorbed R3616 survived significantly longer than mice administered R3616 adsorbed onto non-specific lymphocytes, or mice administered either virus or tumor antigen-specific lymphocytes alone. In this context, herpes oncolytic virus is a promising treatment not only for primary lesions, but also for multiple metastasizing lesions. This treatment strategy may become one of the most effective methods for systemic virus delivery.

Impact of novel oncolytic virus HF10 on cellular components of the tumor microenviroment in patients with recurrent breast cancer.

Oncolytic viruses are a promising method of cancer therapy, even for advanced malignancies. HF10, a spontaneously mutated herpes simplex type 1, is a potent oncolytic agent. The interaction of oncolytic herpes viruses with the tumor microenvironment has not been well characterized. We injected HF10 into tumors of patients with recurrent breast carcinoma, and sought to determine its effects on the tumor microenvironment. Six patients with recurrent breast cancer were recruited to the study. Tumors were divided into two groups: saline-injected (control) and HF10-injected (treatment). We investigated several parameters including neovascularization (CD31) and tumor lymphocyte infiltration (CD8, CD4), determined by immunohistochemistry, and apoptosis, determined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Median apoptotic cell count was lower in the treatment group (P=0.016). Angiogenesis was significantly higher in treatment group (P=0.032). Count of CD8-positive lymphocytes infiltrating the tumors was higher in the treatment group (P=0.008). We were unable to determine CD4-positive lymphocyte infiltration. An effective oncolytic viral agent must replicate efficiently in tumor cells, leading to higher viral counts, in order to aid viral penetration. HF10 seems to meet this criterion; furthermore, it induces potent antitumor immunity. The increase in angiogenesis may be due to either viral replication or the inflammatory response.

Meta-analysis of microarray gene expression studies on intracranial aneurysms.

The rupture of intracranial aneurysms (IAs) is one of the most devastating neurological conditions known to date. Although treatment has changed dramatically throughout the last decades, the outcome of patients still has a poor prognosis. Besides environmental factors, genomics seem to be a very important factor in the genesis of this disease. Different approaches to decrypt genomic causes were pursued throughout the last years. Microarray gene expression studies comparing aneurysmal and healthy tissue seem to be one of the most promising approaches. However, large amounts of data created with each study, make a comparison or interpretation of results difficult. We analyzed microarray gene expression studies on IAs (vs. control tissue) and compared lists of genes with altered expression provided by the authors. Additionally functional pathway analysis was performed. We identified five microarray gene expression studies analyzing a total of 60 samples of IA tissue (30 ruptured IA, 30 unruptured IA). A total of 507 genes with altered expression were listed, of which 57 showed differences in more than two studies and seven in more than three studies (BCL2, COL1A2, COL3A1, COL5A2, CXCL12, TIMP4, TNC). The meta-analysis of five microarray gene expression studies on IAs revealed seven genes that are very likely to be involved in the genesis of IAs. Further analysis of these genes might provide valuable information on mechanisms causing this disease.

Clinical trial of nicardipine prolonged-release implants for preventing cerebral vasospasm: multicenter cooperative study in Tokyo.

BACKGROUND: since October 1999, nicardipine pellets (NP) have been used to prevent vasospasm in patients with subarachnoid hemorrhage (SAH). We started a multicenter cooperative study on Jan 1, 2007, and 136 patients in six hospitals were enrolled to this trial in 2 years. The incidence of cerebral vasospasm and outcome were examined in these patients. METHODS: the patients with SAH were treated with NP during surgery after clipping of their aneurysms. FINDINGS: the study included 87 female patients, 38 over 70 years old, 34 in grades 4 and 5, and 46 of Fisher group 2 or 4. Aneurysms were located on anterior circulation in 133, posterior in 3. All patients were treated with Fasudil hydrochloride except for 3. Two to twelve pellets were implanted in the cistern where thick clots existed and vasospasm was highly likely. Delayed ischemic neurological deficits (DIND), angiographical vasospasm and cerebral infarctions were seen in 11 of 134 (8.2%), 32 of 130 patients (24.6%), and 16 of 129 (12.4%), respectively. No complications were experienced. Independent rate at 3 months was 78%. CONCLUSIONS: the incidence of cerebral vasospasm in this multicenter trial is similar to that of our first trial performed in a single center.

A phase I dose-escalation clinical trial of intraoperative direct intratumoral injection of HF10 oncolytic virus in non-resectable patients with advanced pancreatic cancer.

In 2005, we initiated a clinical trial that examined the efficacy of the oncolytic virus HF10 to treat pancreatic cancer. Pancreatic cancer continues to have a high mortality rate, despite multimodal treatments for patients, and new therapeutic methods are greatly needed. The current mainstream methods for cancer treatment include biological therapeutics such as trastuzumab (Herceptin) for breast cancer or erlotinib (Tarceva) for non-small cell lung cancer. Oncolytic virus therapy is a new and promising treatment strategy for cancer. Oncolytic viruses are novel biological therapeutics for advanced cancer that appear to have a wide spectrum of anticancer activity with minimal human toxicity. To examine the efficacy of oncolytic virus therapy for pancreatic cancer, we initiated pilot studies by injecting six patients with non-resectable pancreatic cancer with three doses of HF10. All patients were monitored for 30 days for local and systemic adverse effects and were not administered any other therapeutics during this period. There were no adverse side-effects, and we observed some therapeutic potential based on tumor marker levels, survival, pathological findings and diagnostic radiography. The tumors were classified as stable disease in three patients, partial response in one patient and progressive disease in two patients.

Association of the Jun dimerization protein 2 gene with intracranial aneurysms in Japanese and Korean cohorts as compared to a Dutch cohort.

In a previous study a linkage region for association to IA patients was found on chromosome 14q22. In this study, we report the findings of a positional candidate gene, Jun dimerization Protein 2 (JDP2), and single nucleotide polymorphisms (SNP) of that gene that are associated with intracranial aneurysms in different ethnic populations. We screened the linkage region around chromosome 14q22 and narrowed it down to JDP2. We then genotyped case and control groups of three different ethnic populations: 403 Japanese intracranial aneurysm (IA) cases and 412 controls, 181 Korean IA cases and 181 controls, 379 Dutch cases and 642 Dutch controls. Genotyping was performed using polymerase chain reaction and direct sequencing technology. The allele distribution of three SNPs (two intronic: rs741846; P=0.0041 and rs175646; P=0.0014, and one in the untranslated region: rs8215; P=0.019) and their genotype distribution showed significant association in the Japanese IA patients. The allelic and genotypic frequency of one intronic SNP (rs175646; P=0.0135 and P=0.0137, respectively) and the genotypic frequency for the SNP in the UTR region (rs8215; P=0.049) was also significantly different between cases and controls of the Korean cohort. There was no difference in allelic or genotypic frequencies in the Dutch population. These SNPs in JDP2 are associated with intracranial aneurysms, suggesting that variation in or near JDP2 play a role in susceptibility to IAs in East Asian populations.

Risk factors for regrowth of intracranial meningiomas after gamma knife radiosurgery: importance of the histopathological grade and MIB-1 index.

INTRODUCTION: The influence of histopathological grade and MIB-1 index of intracranial meningioma on the results of its radiosurgical management is not clear. The objective of the present retrospective study was to make an evaluation of these factors along with an analysis of other variables associated with progression-free survival after gamma knife radiosurgery (GKR). PATIENTS AND METHODS: Thirty-four intracranial meningiomas with known detailed histopathological diagnosis were analyzed. Tumors of WHO histopathological grades I, II, and III were diagnosed in 24, 3, and 7 cases, respectively. The median MIB-1 index was 1.3% (range: 0-31.9%). In 14 cases the MIB-1 index was 3.0% and more. In 26 cases the treatment was done at the time of tumor recurrence. Median volume of the neoplasm at the time of GKR was 4.1 mL (range: 0.4-43.1 mL). Median marginal dose was 12 Gy (range: 8-19 Gy). Median length of follow-up constituted 63 months (range: 19-132 months). RESULTS: Actuarial progression-free survival at 1, 3, 5, and 10 years constituted 100, 94, 83, and 58%, respectively. Histopathological grade II or III (p<0.0001), MIB-1 index 3% and more (p=0.0004), and non-skull base location (p=0.0026) of the tumor showed negative associations with progression-free survival in multivariate analyses. Actuarial progression-free survival at 5 years after GKR for benign and non-benign meningiomas constituted 100 and 45%, respectively (p<0.0001). CONCLUSION: Radiosurgery is a highly effective management option for benign intracranial meningiomas, but growth control of non-benign ones is significantly worse. It requires close neuroradiological follow-up and necessitates the search for modified treatment strategies.

Follow-up of Large Aneurysms Treated with Coil Embolization at an Acute Stage in Patients with Poor-Grade Subarachnoid Hemorrhage.

SUMMARY: This study evaluated the clinical and angiographic outcome of large aneurysms treated with coil embolization at an acute stage in patients with poor-grade subarachnoid hemorrhage (SAH). Between July 1, 2001 and June 30, 2004, eight consecutive WFNS grade 5 patients with large aneurysms (15~23 mm) were treated with endovascular coil embolization within two days and followed for at least 30 months. There were three middle cerebral and five internal carotid artery aneurysms. No patients were treated by craniotomy and none survived without treatment. Two patients died of primary brain damage or cerebral vasospasm within one month. One patient died of pneumonia at 24 months. Four patients were alive with good recovery or moderate disability at the time of final follow-up (30~66 months). Angiography immediately after the procedure showed complete occlusion in three, neck remnant in four, and body filling in one patient. No complication was seen related to the procedure. Three aneurysms that were initially neck remnant developed body filling due to coil compaction. Two were re-treated with coils at six and 12 months and resulted in neck remnant. One patient refused re-treatment and died of re-bleeding. Endovascular coil embolization can be selected at an acute stage for the treatment of aneurysms in patients with poor-grade SAH without intraparenchymal hematoma even if the aneur-ysm is large. Serial follow up by MRA/angiography is necessary for at least 12 months.

Ossified frontosphenoorbital meningioma en plaque, mimicking extensive hyperostosis.

Small intratumoral calcifications are often seen within meningioma, but ossification of the whole neoplasm is very rare. The case of an ossified frontosphenoorbital meningioma en plaque is presented. The radiological appearance resembled extensive hyperostosis extending from the anterior clinoid process to the cerebral convexity and falx cerebri. It is possible that, in some cases of meningioma, the identified "hyperostosis" represents partial ossification of the tumor itself.

Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells.

Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery. Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool. Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation. Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16). Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway "Antigen Presentation" to have the highest upregulation in IA tissue (P=7.3E-10). Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody. Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation.

Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is highly malignant and prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumour appearing is of clinical importance. At this point, no convenient method is available to determine the origin of these HCCs. Tissue samples were obtained from 19 patients and analysed for the promoter hypermethylation status of multiple tumour suppressor genes (p16, DAP-Kinase, MGMT, GSTP1, APC, RIZ1, SFRP1, SFRP2, SFRP5, RUNX3, and SOCS1) using methylation-specific PCR (MSP). Methylation status was used to determine tumour clonality. In each of the 19 cases, at least one tumour was recognised as having an aberrantly methylated gene. The frequency of the methylation in tumour tissue was 57.1% in p16, 2.4% in DAP-kinase, 23.8% in GSTP1, 90.5% in APC, 45.2% in RIZ1, 64.3% in SFRP1, 59.5% in SFRP2, 28.6% in SFRP5, 47.6% in RUNX3, and 54.8% in SOCS1, while in MGMT, no aberrant methylation was detected. The methylation status of these genes was assessed using MSP as being either positive or negative, and was used to determine the tumour clonality. The clonality of every tumour could be decided even with lesions that could not be judged by clinical diagnosis or by another molecular method (mt DNA mutation). Determining the methylation status of multiple genes in multicentric HCC was useful as a clonal marker and provided useful information for characterising the tumour. From our findings, multicentric HCCs tend to occur more independently than metastatically from the original tumour. Expanded study should be pursued further for a better understanding of the molecular mechanism of hepatocarcinogenesis.

Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models.

BACKGROUND/AIMS: Oncolytic viral therapy is used worldwide. Many genetically engineered viruses have been evaluated for their potential as a new therapeutic agent for cancer. HF10, herpes simplex virus (HSV) type-1 clone, has remarkable anti-tumor effects, based on our previous research. In this study, we investigated the ability of HF10 to infect and lyse murine colon cancer cells, CT26, in vitro, and tested its efficacy in an immuno-competent animal model of colorectal cancer. Further, we attempted to evaluate HF10/paclitaxel combination therapy. METHODOLOGY: In vitro, viral replication and cytotoxicity of HF10 against CT26 was observed. In vivo, BALB/c mice harboring carcinomatous peritonitis of CT26 cells were treated with HF10, paclitaxel or HF10 combined with paclitaxel. RESULTS: HF10 is effective for peritoneal dissemination without ascites. The combination of HF10 and paclitaxel prolonged survival of mice bearing carcinomatous dissemination of CT26 compared with the controls, HF10 alone and paclitaxel alone. Paclitaxel did not suppress viral replication and cytotoxicity of HF10. CONCLUSIONS: These results indicate that the combination of HF10 and paclitaxel had a remarkable effect as a cancer therapy and this method is applicable to almost all advanced cancers. This new combination therapy is a potentially epoch-making cancer therapy.

Suitability of a US3-inactivated HSV mutant (L1BR1) as an oncolytic virus for pancreatic cancer therapy.

Recently, the use of oncolytic viruses against cancer has attracted considerable attention. We studied the potential of the US3 locus-deficient herpes simplex virus (HSV), L1BR1, for oncolytic virus therapy. Its high specificity and potency indicate that L1BR1 is a promising candidate as a new oncolytic virus against pancreatic cancer. Moreover, the virus exhibited the unique characteristic of increasing apoptosis when used in combination with anticancer drugs. We assessed the feasibility of using the US3 locus-deficient HSV named L1BR1 as a new replication-competent oncolytic virus for the treatment of pancreatic cancer. The US3 locus of HSV has been shown to be a key gene in producing a multifunctional protein kinase that inhibits apoptosis induced by viral infections, chemicals and ultraviolet (UV) light. L1BR1 has been reported to be more than 10 000-fold less virulent than the parental virus in mice. In this study, we examined the tumor specificity and oncolytic effect of this attenuated replication-competent virus, L1BR1, in pancreatic cancers derived from SW1990, Capan2 and Bxpc-3cells compared with the parent virus and other well-known oncolytic herpes viruses (R3616 and hrR3). We also studied the efficacy of L1BR1 for the induction of apoptosis as an attribute of this virus in combination with the anticancer drugs 5FU and cisplatin. The combined treatment of the pancreatic cancer cells with L1BR1 and these anticancer drugs enhanced apoptosis significantly. More importantly, L1BR1 showed the lowest replication capacity in normal human hepatocytes, but the highest tumor-reducing effect in vivo among the oncolytic herpes viruses tested. In addition, L1BR1 significantly increased the induction of apoptosis of cancer cells when treated in combination with anticancer drugs although the parental virus inhibited the induction of apoptosis. These results suggest that L1BR1 is promising as a new anticancer oncolytic virus.

Oncolytic virus therapy--foreword.

We are very pleased and proud to be able to publish this special issue of Current Cancer Drug Targets devoted to oncolytic virus therapy covering basic and clinical research on adenovirus, vaccinia virus, herpes virus, and Newcastle disease virus. In these papers, we welcome the world's top authorities in the field who have generously contributed their latest review articles for exclusive publication in this special issue. Moreover, this issue also includes a range of opinion from government drug organizations. Here we simply wish to bring together the newest knowledge and experience in the field of cutting-edge oncolytic virus therapy for researchers and every kind of cancer therapist. The Foreword presents a historical perspective on the development of oncolytic virus together with the encouraging results of recent clinical trials (e.g., H101 has been tested in clinical trial of nearly 250 patients and approved for human use by the Chinese FDA, while PV701 has been tried in over 110 patients, as described in our special issue).

Clinical experiment of mutant herpes simplex virus HF10 therapy for cancer.

We reviewed our clinical trial using mutant herpes simplex virus "HF10". We have evaluated the safety and effect of HF10 against recurrent breast cancer since 2003 and also applied HF10 to non-resectable pancreatic cancer since 2005. An oncolytic herpes simplex virus type 1, mutant HF10, has been isolated and evaluated for anti-tumor efficacy in syngeneic immunocompetent mouse models. From long time before clinical trial, we have found that the mutant virus can have remarkable potential to effectively treat cancer in experimental studies using animals, and that all of the surviving mice acquire resistance to rechallenge of the tumor cells. A number of studies have shown that HF10 is effective and safe for use in localized or peritoneally disseminated malignant tumors of non-neuronal origin in animals. Pilot studies using HF10 have been initiated in patients with metastatic breast cancer. For each patient, 0.5 ml HF10 diluents at various doses were injected into test nodule, and 0.5 ml sterile saline was injected into a second nodule. All patients were monitored for local and systemic adverse effects, and the nodules were excised 14 days after viral injection for histopathological studies. All patients tolerated the clinical trial well. While no adverse effects occurred, there was cancer cell death and 30-100% regression histopathologically in recurrent breast cancer. As mentioned above, intratumoral injection of mutant herpes simplex virus HF10 for recurrent metastatic breast cancer was safe and effective. Also a trial for non-resectable pancreatic cancer being carried out on the basis of the above result has proved to be innocuous and has been in progress to assess the clinical benefit and enhance the potentiality of HF10 against cancer.

Fatal intratumoral hemorrhage immediately after gamma knife radiosurgery for brain metastases: case report.

Radiosurgical treatment of brain tumors is sometimes considered to be free from significant acute complications or adverse effects. A rare case of fatal intratumoral hemorrhage immediately after gamma knife radiosurgery (GKR) for brain metastasis is reported. A 46-year-old woman with lung cancer complicated by systemic dissemination experienced an acute episode of headache, speech disturbances, and right-side hemiparesis. She had no history of arterial hypertension or coagulation disorders. CT and MRI disclosed multiple brain metastases. The largest tumor, which was located in the left frontal lobe and caused a significant mass effect, was removed microsurgically without any complications. GKR for nine residual metastases was done on the fourth postoperative day. The marginal dose, which corresponded to the 50% prescription isodose line, constituted 20 Gy. No complications were noticed during frame fixation, treatment itself, or frame removal. Fifteen minutes after the end of the GKR session the patient acutely fell into a deep coma. Urgent CT disclosed a massive hemorrhage in the left cerebellar hemisphere in the vicinity of the radiosurgically treated lesion. The patient died 4 days later and autopsy confirmed the presence of intratumoral hemorrhage. In conclusion, GKR for metastatic brain tumors should not be considered as a risk-free procedure and, while extremely rare, even fatal complications can occur after treatment.

Genomewide-linkage and haplotype-association studies map intracranial aneurysm to chromosome 7q11.

Rupture of intracranial aneurysms (IAs) causes subarachnoid hemorrhage, a devastating condition with high morbidity and mortality. Angiographic and autopsy studies show that IA is a common disorder, with a prevalence of 3%-6%. Although IA has a substantial genetic component, little attention has been given to the genetic determinants. We report here a genomewide linkage study of IA in 104 Japanese affected sib pairs in which positive evidence of linkage on chromosomes 5q22-31 (maximum LOD score [MLS] 2.24), 7q11 (MLS 3.22), and 14q22 (MLS 2.31) were found. The best evidence of linkage is detected at D7S2472, in the vicinity of the elastin gene (ELN), a candidate gene for IA. Fourteen distinct single-nucleotide polymorphisms (SNPs) were identified in ELN, and no obvious allelic association between IA and each SNP was observed. The haplotype between the intron-20/intron-23 polymorphism of ELN is strongly associated with IA (P=3.81x10-6), and homozygous patients are at high risk (P=.002), with an odds ratio of 4.39. These findings suggest that a genetic locus for IA lies within or close to the ELN locus on chromosome 7.