Absence of Epstein-Barr Virus in Smooth Muscle Cells of Idiopathic Hypertrophic Pyloric Stenosis.

Idiopathic hypertrophic pyloric stenosis (IHPS) affects approximately 2 infants out of each 1000 live births and usually presents at between 3 and 12 weeks after birth.[1] It is characterized by hypertrophy of the pyloric muscle, causing pyloric channel narrowing and elongation, which results in nonbilious projectile vomiting.[2] Evidence suggests that the pyloric muscle hypertrophy and hyperplasia of IHPS develop postnatally, although the pathogenesis is not fully characterized.[3,4]

Epstein-Barr virus (EBV) is a gammaherpesvirus that infects B lymphocytes and is the principal cause of infectious mononucleosis.[5] Epstein-Barr virus has also been shown to infect smooth muscle cells of leiomyomas and leiomyosarcomas in persons with the acquired immunodeficiency syndrome (AIDS)[6,7] or following organ transplantation.(8) In these cases, the underlying immunosuppression appears to facilitate EBV infection of smooth muscle cells, leading to development of benign leiomyomas, as well as progression to malignant transformation. These lesions are new additions to the list of tumors causally associated with EBV.

The rate of EBV-associated leiomyomas and leiomyosarcomas is higher in children than in adults, which is remarkable considering that many more adults compared to children are infected with human immunodeficiency virus 1 (HIV-1) or undergo organ transplant. After non-Hodgkin lymphoma, leiomyosarcoma is the second leading cancer in children with HIV-1 infection.[9] Children may be more susceptible to developing EBV-associated tumors because primary EBV infection occurs in childhood.[10-12] The younger host may be less well prepared to limit primary EBV infection and the contribution of EBV to malignant transformation than adults, who develop impaired immunosurveillance in the setting of previously established but well-controlled EBV infection.

The ability of EBV to infect smooth muscle cells and its association with leiomyomas in immunocompromised persons, as well as the unique immunologic milieu of the newborn that includes a component of immune compromise,[13] suggested a possible role of EBV in the pathogenesis of IHPS. We investigated EBV infection of smooth muscle cells in the pathogenesis of IHPS by in situ hybridization of smooth muscle biopsy specimens from 10 infants with pyloric stenosis.

MATERIALS AND METHODS

Patient Specimens

Biopsies of the pylorus were obtained from 10 infants with the clinical and anatomic diagnosis of IHPS from 1997 through 1998. The infants included 8 males and 2 females, with a mean age of 47 days (range, 28-78 days) at the time of biopsy. Each infant presented with projectile vomiting and had an ultrasound examination showing pyloric hypertrophy that was confirmed at pyloromyotomy. All 10 patients had marked narrowing of the pylorus.

Tissue Preparation

Full-thickness muscle biopsy specimens of the pylorus were obtained at pyloromyotomy from the 10 infants. The specimens were immediately fixed in 10% formalin and embedded in paraffin. Three-micrometer-thick sections were cut and mounted on polylysine-coated glass slides, and then stained with hematoxylin-eosin using standard methods.

In Situ Hybridization

Epstein-Barr virus was detected by in situ hybridization of EBV-encoded RNA 1 (EBER1) transcripts using complementary probes as previously described.[14] The EBERs are the most abundant transcripts in cells with latent EBV infection. Paraffin tissue sections were placed on silane-coated slides, dewaxed, digested with proteinase K, and then hybridized to digoxigenin-labeled riboprobes. Signal was detected using the Genius system (Boehringer-Mannheim, Indianapolis, Ind). Sections were counterstained with 1% methyl green. To ensure the integrity of template RNA in each tissue section, a control riboprobe targeting ubiquitous cellular U6 RNA was applied in parallel reactions. The U6 RNA transcripts are similar in abundance and nuclear localization to the targeted EBER1 transcripts. Positive tissue controls were sections of EBV-associated Hodgkin disease. Probe controls showed that antisense, but not sense, EBER1 riboprobe stained latently infected cells. This EBER1 assay is expected to detect all EBV-associated lesions containing latently infected cells.[15] Probe templates for EBER1 (RA386) and U6 (RA390) were kindly donated by Richard Ambinder, MD, PhD, of Johns Hopkins University, Baltimore, Md.

RESULTS

The histopathology of the 10 biopsies showed hypertrophied smooth muscle consistent with IHPS (Figure). Cellular U6 RNA was detected in all smooth muscle samples, confirming that the RNA in the specimens was intact and capable of detection by in situ hybridization. All of the 10 smooth muscle biopsies were negative for EBER1.

COMMENT

The EBER1 hybridization demonstrated the absence of EBV in smooth muscle cells of all 10 cases of IHPS that were studied. This finding suggests that IHPS is not an EBV-associated lesion. Serologic studies of newborns were not used because they provide only indirect evidence of EBV infection and are confounded in infants by the presence of maternally acquired antibodies. Detection of EBER1 is the most sensitive method for identification of latent EBV infection in biopsy specimens.[15] The absence of EBER1 in 10 cases of clinically diagnosed and histopathologically confirmed IHPS effectively excludes EBV infection of smooth muscle cells as a likely causal factor in the pathogenesis of IHPS. By comparison, all smooth muscle cells of the EBV-associated leiomyosarcomas harbor EBV, as evidenced by the presence of EBER1 in more than 90% of cells,[16] and therefore it is unlikely that the negative results in these 10 biopsies represented sampling error.

The identification of EBV infection of smooth muscle cells of leiomyosarcoma was first reported in a single case of an adult with HIV-1 infection[17] and in 2 subsequent series published together in early 1995 of 6 HIV-1-infected persons[6] and 3 organ transplant recipients.[8] These reports have been substantiated by numerous additional cases of EBV-associated leiomyomas and leiomyosarcomas in immunocompromised persons, usually secondary to HIV-1 infection or immunosuppression following organ transplantation.[16] However, EBV has not been reported to infect smooth muscle cells of immunocompetent persons and has not been found in leiomyomas or leiomyosarcomas in the absence of HIV-1 infection or other immunocompromised conditions.[16]

The cause of IHPS remains unknown, but treatment with erythromycin during early infancy has recently been identified as a risk factor.[18] These studies show no evidence for a causal role for EBV infection of smooth muscle in the pathogenesis of IHPS.

We thank Phyllis A. Eagan for technical assistance.

References

[1.] Schechter R, Toffs CP, Bateson TE The epidemiology of infantile hypertrophic pyloric stenosis. Paediatr Perinat Epidemiol. 1997;11:407-427.

[2.] Puri P, Lakshmanadass G. Hypertrophic pyloric stenosis. In: Puri P, ed. Newborn Surgery. Oxford, England: Butterworth-Heinemann; 1996:266-271.

[3.] Rollins MD, Shields MD, Quinn RJ, Wooldridge MA. Pyloric stenosis: congenital or acquired? Arch Dis Child. 1989;64:138-139.

[4.] Oue T, Puri P. Smooth muscle cell hypertrophy versus hyperplasia in infantile hypertrophic pyloric stenosis. Pediatr Res. 1999;45:853-857.

[5.] Jenson HB. Infectious mononucleosis. In: Jenson HB, Baltimore RS, eds. Pediatric Infectious Diseases: Principles and Practice. 1st ed. Norwalk, Conn: Appleton & Lange; 1995:565-576.

[6.] McClain KL, Leach CT, Jenson HB, et al. Association of Epstein-Barr virus with leiomyosarcomas in young people with AIDS. N Engl J Med. 1995;332:12-18.

[7.] Jenson HB, Leach CT, McClain KL, et al. Benign and malignant smooth muscle tumors containing Epstein-Barr virus in children with AIDS. Leuk Lymphoma. 1997;27:303-314.

[8.] Lee ES, Locker J, Nalesnik M, et al. The association of Epstein-Barr virus with smooth-muscle tumors occurring after organ transplantation. N Engl J Med. 1995;332:19-25.

[9.] Granovsky MO, Mueller BU, Nicholson HS, Rosenberg PS, Rabkin CS. Cancer in human immunodeficiency virus-infected children: a case series from the Children's Cancer Group and the National Cancer Institute. J Clin Oncol. 1998; 16:1729-1735.

[10.] Evans AS, Niederman JC, McCollum RW. Seroepidemiologic studies of infectious mononucleosis with EB virus. N Engl J Med. 1968;279:1121-1127.

[11.] Evans A, Cox F, Nankervis G, et al. A health and seroepidemiological survey of a community in Barbados. Int J Epidemiol. 1974;3:167-175.

[12.] Wang PS, Evans AS. Prevalence of antibodies to Epstein-Barr virus and cytomegalovirus in sera from a group of children in the People's Republic of China. J Infect Dis. 1986;153:150-152.

[13.] Schelonka RL, Infante AJ. Neonatal immunology. Semin Perinatol. 1998; 22:2-14.

[14.] Gulley ML, Eagan PA, Quintanilla-Martinez L, et al. Epstein-Barr virus DNA is abundant and monoclonal in the Reed-Steinberg cells of Hodgkin's disease: association with mixed cellularity subtype and Hispanic American ethnicity. Blood. 1994;83:1595-1602.

[15.] Barletta JM, Kingma DW, Ling Y, Charache P, Mann RB, Ambinder RF. Rapid in situ hybridization for the diagnosis of latent Epstein-Barr virus infection. Mol Cell Probes. 1993;7:105-109.

[16.] Jenson HB. Leiomyoma and leiomyosarcoma. In: Goedert JJ, ed. Infectious Causes of Cancer: Targets for Intervention. Totowa, NJ: Humana Press; 2000:145-159.

[17.] Prevot S, Neris J, de Saint Maur PP. Detection of Epstein Barr virus in an hepatic leiomyomatous neoplasm in an adult human immunodeficiency virus 1-infected patient. Virchows Archiv. 1994;425:321-325.

[18.] Honein MA, Paulozzi LJ, Himelright IM, et al. Infantile hypertrophic pyloric stenosis after pertussis prophylaxis with erythromycin: a case review and cohort study. Lancet. 1999;354:2101-2105.

Accepted for publication September 20, 2000.

From the Departments of Pediatrics and Microbiology (Dr Jenson) and Pathology (Dr Gulley), The University of Texas Health Science Center at San Antonio; and Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin, Ireland (Dr Puri).

Reprints: Hal B. Jenson, MD, Department of Pediatrics, MSC 7811, University of Texas Health Science Center, 7703 Floyd Curl Dr, San Antonio, TX 78229-3900.