Acta Neuropathol (1992) 84:331 - 334 HeAu mpaihologica (~) Springer-Verlag1992 Three cases of primary cerebral lymphoma in AIDS patients: detection of Epstein-Barr virus by in situ hybridization and Southern blot technique C.VitaP, J. P. Merlio 2, J. RiveP, A.VitaP, P. Gautris 1, M. Beylot z, A. de MascareP, and B. Bloch 2 Department of INeuropathology, and 2Histology, Bordeaux II University, France Received October 11, 1991/Revised, accepted February 24, 1992 Summary. Three cases of primaEr cerebral lymphoma in acquired immunodeficiency syndrome were studied. Tumoral fragments taken at autopsy were frozen and studied by the Southern blot technique (SBT). Other tumoral fragments were fixed in formalin, embedded in paraffin and used for in situ hybridization (ISH) with biotinylated probes for DNA of Epstein-Barr virus (EBV). ISH was positive in each case with a spotty nuclear labelling of certain tumoral cells. SBTevidenced a clonal rearrangement of the immunoglobulin heavy chain gene in each case. In addition, EBV DNA was detected in each frozen fragment with only one restric- tion pattern, indicating that the EBV- infected cell population was a clonal expansion of a progenitor cell. Key words: Primary cerebral lymphoma - AIDS - Epstein-Barr virus - In situ hybridization - Southern Blot serially sectioned brains were embedded in paraffin and stained with hematoxylin-eosin, Giemsa and periodic acid-Schiff stains. In situ hybridization ISH for EBV detection was performed using a commercially available biotinylated cDNA probe (Enzo Biochem, New York, N.Y.). Tissues were small paraffin-embedded fragments cut into 10-~tm-thick sections that were deposited on gelatinized slides. Paraffin was removed in toluene baths, and sections were dehy- drated in ethanol and rinsed in tap water.They were then incubated in 0.4 % pepsin for 15 rain at room temperature, rinsed in distilled water, and dehydrated in ethanol.The cDNA probe, diluted 1 : 10 in the hybridization buffer [50 % formamide, 4 x standard sodium citrate (SSC), 10 % dextran sulfate, salmon sperm DNA], was deposited and the slides were coverslipped. Sections were incu- bated for 15 rain at 95 ~ for DNA denaturation, then over-night at 42 ~ for hybridization. Sections were then rinsed in SSC, incu- bated with avidin alkaline phosphatase (Dako, Santa Barbara, Calif.) for 30 rain at 37~ then in Nitroblue Tetrazolium (NBT) Bromochloroindolylphosphate (BCIP) was described for detection of colored reaction [4]. Recently attention has focused on primary cerebral lymphomas (PCL) in AIDS patients, who are prone to develop lymphomas in extranodal localizations. These are B cell lymphomas [5], and the presence of Epstein- Barr virus (EBV) has recently been demonstrated in certain cases [1, 5, 10, 15].We have had the opportunity of studying three recent cases by in situ hybridization (ISH) and Southern blot technique (SBT). Material and methods All three patients had confirmed premortem diagnoses of AIDS and their clinical features are summarized in Table 1. In each case small tumoral fragments from brains removed at autopsy were frozen at -80 ~ The brains were then fixed by immersion in 10 % formaldehyde solution for 3 weeks. Numerous fragments from Correspondence to: C.Vital, Anatomic Pathologique, CHR Pelle- grin, F-33076 Bordeaux, Cedex, France DNA probes Fragments purified from plasmids were labeled by random priming according to the manufacturer's procedure (Boehringer-Mann- Table 1. Summary of main data in three primarycerebral lympho- mas Case Age Sex Location EBV EBV (years) (ISH) (SBT) 1 40 M Parietal lobe + + cerebellum 2 39 M Thalamus + + cerebellum 3 43 M Thalamus + + cerebellum M, male; EBV, Epstein-Barr virus; ISH, in situ hybridization; SBT, Southern blot technique