Introduction 15 18 5 12 20 22 2 3 7 21 25 24 16 8 13 26 28 29 26 29 Overall, this study represents the largest series on hepatic lymphomas to date and due to its restriction to bioptic material reflects the primary diagnostic situation. Materials and methods 16 Overall, 32/205 (16%) cases were reclassified, including eight “low-grade” [5× B-CLL, 2× follicular lymphomas (FL), 1× marginal zone lymphoma (MZL)] and 20 “high-grade” [19× DLBCL, 1× Burkitt lymphoma (BL)] B-cell NHL that were assigned to a specific WHO lymphoma category as well as four cases with a change in the final diagnosis [BL, classical Hodgkin lymphoma (cHL), T-cell-rich B-cell lymphoma (TCRBCL), lymphoplasmacytic lymphoma (LPL) to DLBCL, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma (pTCL) and B-CLL, respectively]. The analyses of the infiltration pattern were based on the assessment of the hepatic architecture. Three main patterns were distinguished: Infiltrates for which an exclusive or predominant association to portal tracts was evident were recorded as portal infiltrates. The second pattern consisted of lymphoma infiltrates which showed a coherent growth pattern thus predominantly resulting in the replacement of the acinar structures. These infiltrates were designated as a nodular growth pattern. Finally, infiltrates that showed prominent intrasinusoidal dispersion of the lymphoma cells were recorded as a sinusoidal growth pattern. Additionally, the density of the infiltrate was semiquantitatively assessed. The presence of scattered neoplastic cells in a background rich in reactive bystander cells (e.g., nonneoplastic T-cells and/or macrophages in T-cell rich B-cell lymphoma) was defined as a loose infiltration pattern, whereas the appearance of coherently appearing neoplastic B- or T-cells was referred to as a dense infiltrate. According to clinical information available, in 76/205 (37%) of the cases the diagnosis of a lymphoma had been previously established from extrahepatic biopsies or peripheral blood indicating secondary involvement of the liver. In four of these cases with known low-grade lymphoma (2× B-CLL, 2× FL) liver biopsy revealed transformation to a high-grade lymphoma (DLBCL). In the remaining cases, the biopsy obtained from the liver represented the site of primary diagnosis. In 26 (13%) patients, additional biopsies from extrahepatic sites were available (13 bone marrow biopsies, 10 lymph node biopsies, and three spleen biopsies). Furthermore, in a few cases clinical data regarding a potential predisposing condition for a primary hepatic lymphoma was available: chronic hepatitis C was reported in four patients (3× DLBCL, 1× B-CLL), HIV infection was present in three patients (2× DLBCL, 1× BL), and one marginal zone lymphoma occurred in a patient after liver transplantation. The frequency of the various lymphoma entities diagnosed in liver biopsy specimen was compared to their frequency and distribution in other extranodal sites (cases from the Consultation and Reference Center for Lymph Node Pathology and Hematopathology, Berlin). Immunohistochemistry 1 14 6 Table 1 Antibodies used in this study Antibody Clone Antigen retrieval Dilution Source ALK1 ALK1 Citrate 1:20 Dako BCL2 124 Citrate 1:25 Dako BCL6 594 Citrate 1:25 Dako CD2 AB75 Citrate 1:50 Novocastra CD3 F7.2.38 Citrate 1:100 Dako CD4 1F6 Citrate 1:25 Novocastra CD5 4C7 Citrate 1:25 Novocastra CD7 CD7-272 EDTA 1:50 Novocastra CD8 C8/144B Citrate 1:100 Dako CD10 56C6 Citrate 1:25 Novocastra CD15 C3D1 Citrate 1:20 Dako CD20 L26 Citrate 1:50 Dako CD21 1F8 Protease 1:50 Dako CD23 1B12 Citrate 1:20 Novocastra CD27 137B4 EDTA 1:100 Novocastra CD30 BerH2 Citrate 1:50 Dako CD43 DF-T1 Citrate 1:50 Dako CD68 PG-M1 Citrate 1:50 Dako CD79a JCB117 Citrate 1:100 Dako CD138 B-B4 Citrate 1:10 Serotec Cyclin D1 P2D11F11 Citrate 1:50 Novocastra EBV-LMP CS1-4 Citrate 1:100 Dako α-heavychain Rabbit polyclonal Citrate 1:40 000 Dako γ-heavy chain Rabbit polyclonal Citrate 1:30 000 Dako δ-heavy chain Rabbit polyclonal Citrate 1:2000 Dako μ-heavy chain Rabbit polyclonal Citrate 1:2000 Dako Ki-67 MIB-1 Citrate 1:2000 Dako κ-light chain Rabbit polyclonal Citrate 1:100 000 Dako λ-light chain HP6054 Citrate 1:16 000 Dako MUM1/IRF4 MUM1p Citrate 1:20 Generously provided by Prof. B. Falini, Perugia, Italy Pax-5 24 Citrate 1:10 Transduction Laboratories TIA-1 2G9 Citrate 1:500 Coulter Molecular pathology analyses Additional molecular pathology analyses were selectively performed for those cases in which immunoprofiling alone was not sufficient to establish a definite diagnosis of a malignant lymphoma. 9 27 Amplification of rearranged IgH genes was independently performed at least twice per case employing three different framework primer sets (BioMed-2 FR1, FR2, and FR3) separately in conjunction with a JH primer (JH22). PCR conditions consisted of 50 cycles of denaturation (95°C, 15 s), primer annealing (60°C, 40 s), and elongation (72°C, 45 s), and the reaction mixture contained 1.5 mM MgCl2, 0.8 mM deoxyribonucleotide triphosphates (dNTPs), 70 pmol VH primers, 30 pmol JH22 primer, and 2 U of AmpliTaq Gold polymerase (Applied Biosystems, Weiterstadt, Germany). Results Characteristics of hepatic lymphomas 11 1 1 Fig. 1 a b c d e f g h i  2 Fig. 2 blue lines red line arrow  2 Table 2 Frequency, age distribution and pattern of infiltration in the different lymphoma entities presenting in the liver (Berlin/Cologne 1994–2003)   Berlin Cologne n Age (range) Age (mean) Male/female Pattern Density of infiltrate Sinusoidal Portal Nodular Dense Loose a 52 28 80(39) 16–87 64 50/30 6 19 72 75 5 T-cell rich B-cell lymphoma (TCRBCL) 9 4 13(6) 35–85 59 9/4 0 11 5 0 13 B-cell chronic lymphocytic leukemia (B-CLL) 13 13 26(13) 44–82 66 16/10 7 25 2 25 1 Classical Hodgkin lymphoma (cHL) 10 13 23(11) 21–91 54 14/9 1 21 10 3 20 Follicular lymphoma (FL) 11 3 14(7) 38–83 62 7/7 0 11 4 14 0 Marginal zone lymphoma (MZL) 6 1 7(3) 49–80 70 6/1 2 7 0 6 1 Leukemic Plasmacytoma (PL) 4 2 6(3) 61–88 70 4/2 4 1 2 5 1 Burkitt lymphoma (BL) 5 – 5(2) 30–70 47 3/2 0 0 5 5 0 Mantle cell lymphoma (MCL) 3 – 3(1) 60–66 63 2/1 1 3 1 3 0 B-lymphoblastic leukaemia (B-ALL) 2 – 2(1) 18–28 22 1/1 2 0 0 2 0 Hairy cell leukaemia (HCL) 1 – 1 42 – 0/1 1 0 0 1 0 Peripheral T-cell lymphoma, unspecified (pTCL) 13 5 18(9) 38–84 62 14/4 6 6 8 11 7 b 5 – 5(2) 24–76 58 5/0 3 3 2 2 3 Hepatosplenic T-cell lymphoma (HSTCL) 1 1 2(1) 38–51 45 2/0 2 0 0 2 0 Total 135 70 205         a n n n b Infiltration patterns of the various lymphoma types 3 1 3 3 3 4 4 4 5 5 3 Table 3 Infiltration pattern, differential diagnosis and characteristic immunohistochemical markers  Fig. 3 a b c d e f g h i  Fig. 4 a b c d e f g h i  Fig. 5 arrows a b c arrows d e f  Comparison of hepatic lymphomas with other extranodal lymphomas The percentage of T-cell lymphomas was considerably higher in the liver (12%, 25/205) when compared to other extranodal sites (5%, 165/3252) except for the skin (50%, mainly mycosis fungoides/Sézary syndrome) and small intestine (34%, mainly enteropathy-type T-cell lymphomas). 4 Table 4 Frequency of extranodal lymphoma at different sites (Berlin 1994–2003)   n n n n n n n n n n DLBCL 25 57 22 66 238 30 32 21 78 52 TCRBCL – – – 7 – – – 3 – 9 FL 1 – 2 168 16 3 3 20 91 11 B-CLL 1 – 2 656 12 – 4 10 7 13 PL 3 – – 473 – – – 1 2 4 MZL – – 40 17 577 4 12 18* 12 6 MCL – – 3 107 7 7 41 19 6 3 LPL – – 3 122 – – 2 13 5 – HCL – – – 112 – – – 6 – 1 B-ALL – 1 – 78 – – – 1 – 2 BL – 1 – 4 – 3 2 – 2 5 cHL – – 3 52 – – – 3 10 10 B-NHL total (%) 30/31 (97%) 59/61 (97%) 75/79 (95%) 1862/1996 (93%) 850/855 (99%) 47/71 (66%) 96/102 (94%) 115/128 (90%) 213/427 (50%) 116/135 (86%) pTCL 1 – 3 93 4 6 6 12 94 13 NK/T – 1 – – – 2 – – 2 – T-ALL – 1 – 29 – – – – 9 – ALCL – – 1 8 1 6 – – 26 5 Enteropathy-type TCL – – – – – 10 – – – – HSTCL – – – 5 – – – 1 – 1 MF – – – – – – – – 83 – T-NHL total (%) 1/31 (3%) 2/61 (3%) 4/79 (5%) 134/1996 (7%) 5/855 (1%) 24/71 (34%) 6/102 (6%) 13/128 (10%) 214/427 (50%) 19/135 (14%) *Including nine cases of splenic marginal zone lymphoma (SMZL) Discussion 16 Many B-cell-derived lymphomas involving the liver revealed a characteristic infiltration pattern, which facilitated the use of a restricted panel of immunohistochemical markers to reach a final diagnosis. This approach is critical in those liver biopsies in which a lymphoma diagnosis has previously not been established, as accurate subtyping of lymphomas is fundamental for the initiation of adequate treatment. 1 11 n 23 15 18 4 31 19 8 17 8 10 5 Similarly, the distinction between a T-cell lymphoma and a drug-induced or viral hepatitis (e.g., due to Epstein-Barr virus) can be very difficult in biopsies containing an increased number of sinusoidal T-cells. Therefore, additional clonality analyses by TCR or IgH PCR were necessary in 5 out of 19 (26%) T-cell lymphomas compared to only 3 out of 116 (3%) B-cell lymphomas to establish a diagnosis. 30 4 In summary, the present study demonstrates the feasibility of subtyping lymphoma infiltrates in liver biopsies according to the WHO classification. The large number of 205 cases provides reliable information regarding the relative frequencies of the different lymphoma entities encountered in the liver and demonstrates the usefulness of infiltration pattern analysis for diagnostic purposes.