Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: the role of primary infection

Abstract

Fourteen patients who developed B cell lymphomas or lymphoproliferative lesions after kidney, liver, heart, or heart-lung transplantation in Pittsburgh during 1981-1983 had active infection with Epstein-Barr virus (EBV) of the primary (six patients), reactivated (seven patients), or chronic (one patient) type. In transplant patients without tumors, the incidence of EBV infection was 30% (39 of 128). Only three of these patients had primary infections. Thus the frequency of active infection was significantly higher in patients with tumors, and patients with primary infections were at greater risk of developing tumors. Five of 13 tumors tested contained EBV nuclear antigen (EBNA) and nine of 11 contained EBV genomes detected by DNA-DNA hybridization with BamHI K, BamHI W, or EcoRI B cloned probes. All EBNA-positive tumors, except one, were also positive by hybridization. Only one tumor was negative for both EBNA and EBV DNA. These data suggest that EBV plays an etiologic role in the development of these lesions.

Figure 1

Southern blot analysis of tumor tissue from organ transplant recipients with the EBV probe EcoRI B. Lane A contains the equivalent of 330 copies per cell of probe DNA in a sample of 10⁶ cells. Lane D contains DNA from a lymphoid line containing 40 genome copies per cell; the DNA from 10⁵ cells was loaded in this lane. Lane E contains DNA from an EBV-negative cell line. Lanes F and G contain 1 µg and 500 ng, respectively, of DNA from the lung biopsy of a heart transplant recipient (patient 13); lanes H and I contain 1 µg and 500 ng, respectively, of DNA from a lymph node of a heart-lung transplant recipient (patient 6); lanes J and K contain 1 µg and 500 ng of DNA from one tonsil, and lanes L and M contain 1 µg and 500 ng of DNA from the other tonsil of patient 5, a heart-lung transplant recipient. EBV DNA was found in tissues from patients 13 and 5 but not 6. Lanes G, K, and M each contain the DNA equivalents of 10⁵ cells. The signals corresponding to these three samples represent the equivalent of ~40 copies per cell. Note that all samples contain a group of BamHI fragments expected to be present in EcoRI B probe.

Figure 2

Southern blot analysis with the EBV probe BamHI K. Titration of the probe is found in lanes a–c. Lane a contains 100 pg of the probe. Lane b contains 10 pg of BamHI K, the equivalent of one genome copy per cell in 10⁶ cells. Lane c contains 1 pg. Lane d contains DNA from a lymphoid line containing 40 copies per cell; the DNA from 10⁵ cells was loaded in this lane. Thus, the signal is equivalent to four copies per cell in a sample of 10⁶ cells. Lane e is a negative control. Lanes f–i contain 5 µg of DNA from each of four patients (7, 11, 9, 8); this is ~ 10⁶ cell equivalents of DNA per lane. Thus, samples f, g, and i have 10 copies per cell, and sample h has four copies per cell. Lanes j and k contain DNA from an unrelated patient. The BamHI K fragment is known to vary in size.