The liver weighed 2000 g. Macroscopically, no mass was identified. There was no evidence of cirrhosis. Histologically, the liver was entirely affected by atypical lymphoid cells that proliferated mainly in the sinusoid (fig 1A). The sinusoids were greatly expanded, hepatic cords were atrophic, and sinusoidal fibrosis was accompanied by an infiltration of lymphoma cells. The lymphoma cells showed pleomorphic nuclei with prominent nucleoli and moderate cytoplasm. In the portal area, lymphoma cells were sparse. Bile stasis was evident in the hepatocytes. No necrotic lesion was seen. The spleen weighed 360 g. Lymphoma cells were diffusely infiltrated in the splenic cord of the red pulp (fig 1B). The white pulp was atrophic. A few lymphoma cells were seen in the sinusoid. In the bone marrow of the vertebral bone, lymphoma cells displayed interstitial or diffuse infiltration with fibrosis. Myeloid and erythroid precursor cells were seen in clusters but megakaryocytes were hardly seen. Sinuses were dilatated but no lymphoma cell cluster was observed.
Figure 1 Histological appearance of our present case. Haematoxylin and eosin staining. (A) Liver: the neoplastic cells diffusely infiltrate the sinusoids; original magnification, ×70. (B) Spleen: the neoplastic cells infiltrate the red pulp cord; sinusal (more ...)
Paraffin wax embedded sections were immunostained by an avidin–biotin horseradish peroxidase complex method. Pretreatment for unmasking of antigens was carried out either by digestion with 0.05% preheated pronase E (type XXIV; Sigma Chemical Co, St Louis, Missouri, USA) in phosphate buffered saline for 20 minutes at 25°C (for βF1 and CD3
), by microwaving in citrate buffer (10 mmol/litre, pH 6.3) twice for five minutes each at 600 W (granzyme B, TIA-1, CD20, CD30, CD8, CD56, and p53), or by autoclaving at 1 bar for 20 minutes, followed by cooling down for 40 minutes (for CD4). The lymphoma cells expressed CD3
, CD8, CD43 and CD45RO. The CD3
staining highlighted sinusoidal infiltration of the liver (fig 2A). TIA-1 (fig 2B), granzyme B, βF1, CD4, CD20, CD30, CD56, CD57, S100, and p53 were not expressed.
Figure 2 Phenotypical characteristics of neoplastic cells. Immunoperoxidase staining. (A) Strong staining for CD3; P, portal vein; original magnification, ×35. (B) No expression of TIA-1 in lymphoma cells; non-neoplastic small lymphocytes (arrows) (more ...)
Epstein-Barr virus (EBV) encoded mRNA 1 (EBER-1) in situ hybridisation was performed on the paraffin wax embedded sections using a fluorescein isothionate labelled EBER-1 30 base oligonucleotide probe under RNase free conditions. The EBER-1 signal was detected on a few lymphoid cells (2%) in the liver sections (not shown).
A polymerase chain reaction study of paraffin wax embedded sections of the liver was performed to detect TCRγ gene rearrangement. DNA was amplified with the following primers: Vγ1, 5′-TCTGG[G/A]GTCTATTACTGTGC-3′; Vγ2, 5′-CTC ACACTCC/TCACTTC-3′; Vγ3, 5′-GAAAGGAATCTGGCATTCCG-3′; Jγ1–2, 5′-CAAGTGTTGTTCCACTGCC-3′; Jpγ1–2, 5′-GTT ACTATGAGC[T/C]TAGTC-3′; and Jpγ, 5′-TGTAATGATAAG CTTTGTTCC-3′. This clearly showed two distinct bands, which suggests clonal rearrangement of two alleles of the TCRγ gene (fig 3).
Figure 3 Polymerase chain reaction for TCRγ chain gene amplified genomic DNA extracted from paraffin wax embedded sections. M, molecular size marker; 1, negative control (blood lymphocytes); P, positive control (intestinal T cell lymphoma); 2, liver of (more ...)
A small number of the lymphoma cells had also infiltrated the kidneys, pancreas, heart, tonsil, stomach, ileum, urinary bladder, prostate, salivary gland, and lymph nodes, including peripancreas, mesentery, and paraaortic lymph nodes.