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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37-99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment.

Mantle cell lymphomas (MCLs) are characterized by 11q13 chromosomal translocations and cyclin D1 overexpression. The secondary genetic and molecular events involved in the progression of these tumors are not well known. In this study, we have analyzed 45 MCLs (32 typical and 13 blastoid variants) by comparative genomic hybridization (CGH). To identify the possible genes included in the abnormal chromosome regions, selected cases were analyzed for P53, P16(INK4a), RB, C-MYC, N-MYC, BCL2, BCL6, CDK4, and BMI-1 gene alterations. The most frequent imbalances detected by CGH were gains of chromosomes 3q (49%), 7p (27%), 8q (22%), 12q (20%), 18q (18%), and 9q34 (16%) and losses of chromosomes 13 (44%), 6q (27%), 1p (24%), 11q14-q23 (22%), 10p14-p15 (18%), 17p (16%), and 9p (16%). High-level DNA amplifications were identified in 11 different regions of the genome, predominantly in 3q27-q29 (13%), 18q23 (9%), and Xq28 (7%). The CGH analysis allowed the identification of regional consensus areas in most of the frequently involved chromosomes. Chromosome gains (P =. 02) and losses (P =.01) and DNA amplifications (P =.015) were significantly higher in blastoid variants. The significant differences between blastoid and typical tumors were gains of 3q, 7p, and 12q, and losses of 17p. CGH losses of 17p correlated with P53 gene deletions and mutations. Similarly, gains of 12q and high-level DNA amplifications of 10p12-p13 were associated with CDK4 and BMI-1 gene amplifications, respectively. One of 2 cases with 8q24 amplification showed C-MYC amplification by Southern blot. Alterations in 2p, 3q, 13, and 18q were not associated with N-MYC, BCL6, RB, or BCL2 alterations, respectively, suggesting that other genes may be the targets of these genetic abnormalities in MCLs. Increased number of gains (0 v 1-4 v >4 gains per case) (P =.002), gains of 3q (P =.02), gains of 12q (P =.03), and losses of 9p (P =. 003) were significantly associated with a shorter survival of the patients. These results indicate that an increased number of chromosome imbalances are associated with blastoid variants of MCLs and may have prognostic significance.

Aims: Splenic marginal zone lymphoma (SMZL) is characterized by a micronodular infiltrate of the splenic white pulp, centred on pre-existing follicles, with a peripheral rim of 'marginal' zone B-cells, always accompanied by a variable degree of red pulp infiltration. These histological features can be closely mimicked by a variety of other small B-cell lymphomas when they involve the spleen, which makes recognition of SMZL difficult. We therefore have compared the histopathological and immunohistochemical features of other non-Hodgkin's lymphoma (NHL) types with those of SMZL., Methods and Results: We selected cases of splenic involvement by different types of B-cell lymphoma, including mantle cell lymphoma (MCL), follicular lymphoma (FL), immunocytoma (IM) and lymphocytic lymphoma (B-CLL). A micronodular pattern and marginal zone differentiation were both found to be frequently present in FL and MCL, and with lesser frequency in IM and B-CLL. The main morphological feature useful for differential diagnosis was the cytological composition of the white pulp tumoral nodules. SMZL is distinguished by characteristic dimorphic cytology, different from the monomorphic cytology of MCL, and the distinctive mixture of centroblasts and centrocytes which is the rule in FL. B-CLL could also be identified on the basis of the polymorphic cytology including small lymphocytes and prolymphocytes, whereas cases diagnosed as IM show prominent plasmacytic differentiation, lacking the features of the other lymphoma types. Immunohistochemistry was particularly useful for the differential diagnosis. Thus the recognition of MCL was facilitated by the identification of cyclin D1 and CD43 reactivity, while FL could be recognized by the lack of IgD expression or the distinctive pattern of Ki67 staining found in SMZL. B-CLL cells were CD23+, CD43+., Conclusion: The results of this study provide morphological and immunohistological information useful in the recognition of the different varieties of NHLs when involving the spleen and the differential diagnosis of SMZL.

The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.

Background: The goal of this study was to analyze the presenting features, natural history, and prognostic factors in 59 patients with well characterized mantle cell lymphoma (MCL)., Methods: Cases were classified as nodular or diffuse and as typical or blastic variants. Age, performance status (PS), histologic variants, mitotic index (MI), hematologic parameters, tumor extension data, and International Prognostic Index (IPI) were recorded and evaluated for prognosis., Results: The median age of the patients was 63 years (range, 39-83 years), and the male to female ratio was 3:1. Fifty-three patients had typical histology (3 nodular and 50 diffuse), and 6 had the blastic variant. Approximately 95% of patients presented with advanced stage disease (Ann Arbor Stage III-IV). Leukemic expression was observed in 58%. Complete and partial response rates were 19% and 46%, respectively. Parameters associated with lower response rate were Stage IV, high/intermediate or high risk IPI, and increased lactate dehydrogenase (LDH) level. In the logistic regression analysis, high LDH level and Stage IV disease were associated independently with lower response rate. Median survival was 49 months. Parameters associated with a short survival were: poor PS, splenomegaly, B-symptoms, MI > 2.5, leukocyte count > 10 x 10(9)/L, high LDH level, blastic variant, and high/intermediate or high risk IPI. In the Cox proportional hazards regression model, only poor PS (relative risk [RR] = 3.3; P = 0.002), splenomegaly (RR = 2.8; P = 0.007), and MI > 2.5 (RR = 2.4; P = 0.012) were associated with short survival., Conclusions: In this series, patients with MCL presented with advanced stage and extranodal involvement. Only a minority of patients achieved a complete response. The median survival was 4 years, with PS, splenomegaly, and MI being the most important factors predicting survival. These results show clearly that more effective therapies for MCL are needed.

Splenic marginal zone lymphoma (SMZL) has recently been proposed as a distinctive type of low-grade B-cell lymphoma. Although there is general agreement that this entity exists, its precise definition is blurred by uncertainty in differential diagnosis from other low-grade B-cell lymphomas. There is even more uncertainty as to the histology of splenic hilar and peripheral lymph nodes involved by SMZL. We therefore reviewed the histological and immunohistochemical features of 19 of these lymph nodes (14 hilar and five peripheral) from 14 cases of classical SMZL and compared them with the features of lymph nodes involved by other B-cell lymphomas. The morphology and immunohistology of the lymph nodes resemble those found in the white pulp of the spleen, showing a distinctive pattern, different from that which is observed in other B-cell lymphomas. In these cases, the overall architecture of the lymph nodes is effaced and replaced by a nodular infiltrate, although the sinuses are preserved in most hilar lymph nodes. Some of the nodules contain a central reactive follicular center, around which there is a broad zone of small lymphocytes. In other cases, the central area is partially infiltrated or, more commonly, totally replaced by these small lymphocytes, which in the periphery of the nodules showed a pale, slightly larger cytoplasm. Scattered nucleolated blasts are present, largely confined to the periphery of the nodules. The tumoral cells express immunoglobulin (Ig)D, IgM, and Ig light chain restriction and show a low proliferation fraction. These findings confirm that SMZL is a real entity, and not merely a morphological pattern of splenic infiltration by different types of low-grade B-cell lymphoma.

p53 is a tumour suppressor gene which is often found to be inactivated in most types of human cancer. p53 is a transcription factor, the inactivation of which may lead to significant variations in the levels of p53 downstream proteins, such as p21WAF1/CIP1 and MDM2. In view of the significance of p21WAF1/CIP1 and MDM2 as wild-type (wt) p53 targets, this study was undertaken to monitor the varying expression of these proteins in non-Hodgkin's lymphomas (NHLs) in relation to p53 gene status. A total of 57 cases of different histological types of NHL were included in this study. Proteins p53, p21WAF1/CIP1, and MDM2 were analysed by immunohistochemical techniques, taking the levels expressed in reactive lymphoid tissues as reference points. p53 gene point mutations (exons 5-8) were looked for using the PCR-SSCP technique and direct sequencing. Fifteen of the 57 cases studied showed 16 mutations at the p53 gene: 12 missense, one nonsense, two silent mutations, and one frameshift deletion. Most missense mutations were associated with high levels of p53 protein, while the nonsense mutations and frameshift deletion did not induce detectable levels of p53. All cases with mutation at the p53 gene (15) showed null or low levels of p21WAF1/CIP1 and MDM2 proteins, suggesting that null or missense mutations at this gene give rise to a protein that is unable to transactivate the p21WAF1/CIP1 and MDM2 genes. The association between missense p53 mutation and dissociate immunophenotype (p53+, MDM2-, p21-) was statistically significant (Fisher's exact test, P = 0.0024). This anomalous p53+, MDM2-, p21- phenotype was also found in a small group of five cases with wt p53; this could indicate that in these cases p53 transactivation capacity has been abrogated by a mechanism other than p53 mutation. Most cases with the wt p53 gene show simultaneous immunohistochemical expression of all three proteins and often display higher levels than those found in reactive lymphoid tissue. There is a tendency for EBV-positive cases to harbour high levels of p53+ and p21+, suggesting that EBV could be involved in the nuclear accumulation of p53 and p21WAF1/CIP1 in NHL.

We report the first case of Epstein Barr virus (EBV) positive non-Hodgkin's lymphoma occurring 20 years after continuous phenytoin therapy for idiopathic epilepsy. This malignant lymphoproliferative disease was preceded by an phenytoin-associated benign reactive lymphadenopathy. Serological findings suggested a chronic active EBV infection. Immunohistochemistry showed that neoplastic cells expressed B antigens (CD20, lambda) and the bcl-2 protein. The presence of EBV-encoded latent membrane protein 1 (LMP 1) was also expressed on neoplastic cells. Cytotoxic chemotherapy (CHOP) reversed the non-Hodgkin's lymphoma.

Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb.

The t(11;14)(q13;q32) translocation and its molecular counterpart bcl-1 rearrangement are highly characteristic of mantle cell lymphomas (MCLs). Most of these translocations occur at the major translocation cluster (MTC) in a tight area that makes this rearrangement identifiable by the polymerase chain reaction (PCR). In this study, the specificity and sensitivity of the PCR technique in the identification of bcl-1 rearrangement and its suitability to amplify the t(11;14) MTC in fixed, paraffin-embedded tissues were analyzed. Genomic DNA was obtained from 21 MCLs and 1 chronic lymphocytic leukemia (CLL) with the t(11;14) translocation. The bcl-1 rearrangement was studied by Southern blot with the MTC, p94PS, and PRAD-1 probes. Polymerase chain reaction was performed using a JH consensus primer and specific primers for chromosome II in the MTC region. bcl-1 rearrangement was identified by Southern blot in the MTC in nine (43%) MCLs and in the p94PS region in the CLL. Polymerase chain reaction analysis of genomic DNA showed that the nine MCLs with MTC rearrangement also had an amplifiable band of the expected size (100%). No amplifiable products were detected in the negative MCLs or in the CLL. The specificity of the PCR products was confirmed by hybridization with an internal MTC oligonucleotide probe. Amplifiable DNA was obtained from the paraffin blocks of 7 cases with MTC rearrangement and 11 negative tumors. bcl-1 rearrangement was detected in this DNA of 6 positive MCLs (86%) by PCR and in none of the negative cases. In conclusion, this study demonstrates that the PCR technique is highly sensitive and specific for the detection of the bcl-1 rearrangement at the MTC. It can be used with both high molecular weight DNA and DNA obtained from formalin-fixed, paraffin-embedded tissues.

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD-1 gene overexpression. Some aggressive variants have been recognized with a blastic or large cell morphology, higher proliferative activity, and shorter survival. p53 gene mutations in lymphoid neoplasms have been detected mainly in high grade lymphomas and have been associated with tumor progression in follicular and small lymphocytic lymphomas. To determine the role of p53 alterations in MCL, we examined 35 typical and 8 aggressive variants (5 blastic and 3 large cell) of MCLs by a combination of immunohistochemistry, single-strand conformational polymorphism analysis of genomic DNA and/or cDNA obtained by reverse transcriptase-polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. Of the 8 aggressive MCLs, 3 (38%) contained missense point mutations in axon 8 codon 278 (Pro --> Leu), exon 8 codon 273 Arg --> His), and exon 5 codon 151 (Pro --> Ser), respectively. A diffuse p53 protein overexpression was observed in more than 50% of the tumor cells in these 3 cases. A fourth blastic MCL also showed strong p53 immunoreactivity. However, no mutations were detected in exons 5-9 in this case. p53 expression was also detected in 10% of the cells in an additional large cell type of MCL and in less than 1% of the cells in 6 typical cases. No mutations were detected in any of these cases or in the remaining cases with no expression of the protein. Four nucleotide changes were observed by single-strand conformational polymorphism analysis in 4 typical MCLs with no overexpression of the protein. Direct sequencing showed that these nucleotide changes were located at exon 6 (1 case), intron 7 (2 cases), and intron 8 (1 case). The changes in exon 6 and intron 7 were known polymorphisms. The nucleotide change in intron 8 was outside splicing sites of the neighboring exons. The overall survival of the 3 patients with p53 mutations (median, 18.3 months) was significantly shorter than that of patients with the nonmutated MCLs (median, 49 months; P < .01). These findings indicate that p53 gene mutations are an infrequent phenomenon in MCLs and are associated with a subset of aggressive variants.

The recognition and classification of the different varieties of splenic low-grade B-cell lymphomas have been hampered by the rarity of histological studies of surgical splenectomy specimens of B-cell lymphoma. In an effort to characterize the recently described splenic marginal zone lymphoma (SMZL), we conducted a survey of 13 patients with this type of tumor using the criteria defined by Schmid for its recognition (Schmid et al., Am J Surg Pathol 1992;16:455-66). Primary splenic high-grade lymphomas, T-cell lymphomas, and secondary infiltration by other recognized low-grade B-cell lymphomas, with the exception of splenic lymphoma with villous lymphocytes, were excluded. This selection gave rise to a homogeneous group of tumors with similar clinical, histological, immunohistochemical, and molecular features. Our study showed the critical parameters for their recognition to be morphological, including macroscopic micronodularity and the constant presence of white- and red-pulp infiltration, marginal zone pattern, and plasmacytic differentiation. No t(14;18) or PRAD-1/cyclin D1 overexpression was detect able in any case. Clinically, the tumors were widespread with a protracted evolution. Nodal infiltration by SMZL in our cases was morphologically similar to monocytoid B-cell lymphoma. SMZL could constitute the largest group of primary splenic malignant lymphomas, partially overlapping with splenic lymphoma with villous lymphocytes. Specific molecular markers for SMZL have yet to be defined. Because of the limited number of cases, the question of therapy for this group of lymphomas must remain open for the future.

The morphology, phenotype, genotype and clinical behaviour of four cases of mantle cell lymphoma (centrocytic lymphoma) presenting primarily in mucosa (two gastric, one in large bowel and one tonsillar) are reviewed. Their relationship with the broader group of mantle cell and mucosa-associated lymphoid tissue (MALT) lymphomas is also discussed. All four tumours showed a monomorphic picture of mantle cells (centrocytes) arranged in a diffuse, or vaguely nodular, pattern. Scattered non-neoplastic germinal centres were entrapped within the tumour cells, although there was no follicular colonization. In two cases distinct epithelial infiltration by tumour cells was observed. All four tumours had a CD19, CD20, CD5, IgD, Leu8 immunophenotype, whereas KiM1P and CD10 expression were absent. DRC antibody showed loose aggregates of dendritic cells in three of four cases. Three cases showed PRAD-1/Cyclin D1 overexpression by Northern blot analysis. Although we were not able to detect bcl-1 rearrangement in the major translocation cluster (MTC) breakpoint, the possibility of bcl-1 rearrangement involving other cluster breakpoints cannot be ruled out. The four cases evolved as a disseminated disease, involving either peripheral lymph nodes, spleen or bone marrow. The biological behaviour of mantle cell lymphoma presenting in mucosa appears, irrespective of localization or macroscopic presentation, similar to that of nodal mantle cell lymphoma. Their tendency to dissemination contrasts with MALT lymphomas, which tend to remain localized, and from which mucosa mantle cell lymphoma must be distinguished. The presence of lymphoepithelial lesions does not seem to be a useful differential feature, since occasional epithelial infiltration was seen in two cases. Reactivity with CD5 appears to be especially useful in distinguishing these, since all four cases were clearly positive, in contrast with what is usually found in MALT lymphomas.

The p53 gene located in the short arm of chromosome 17 at position 17p13, is involved in the negative regulation of cellular growth. p53 mutation seems to be the most frequent genetic alteration found in human cancer. Mutant conformation of the p53 gene is associated with cell proliferation and tumour progression, and in most cases implies p53 stabilization, which renders the p53 protein detectable through the use of immunohistochemical techniques. p53 expression is a frequent finding in high grade lymphomas of either B or T cell lineage, having been detected in 30% of cases in our series. The focal presence of p53+ cells was seen in a wide range of low and high grade lymphomas, including lymphadenitis and reactive tonsils. In 37.5% of cases this increased expression of p53 was secondary to mutation in highly conserved regions (exons 5-8). Unlike findings reported in other tumours, in lymphomas, p53 expression seems to be secondary to genetic alterations other than p53 mutation. Initial data suggest that the MDM2 protein could be involved in inactivating p53 protein in most of these cases. Finally, p53 expression has been found to be a poor prognostic marker in high grade B-cell lymphomas in a large series of cases. High p53 expression was associated with a short survival, this relation being stronger in cases with simultaneous bcl2 expression.

Neoplastic monocytoid B-cells (MBCs) are present in different amounts in several types of non-Hodgkin's lymphomas (NHLs), including monocytoid B-cell lymphoma (MBCL), splenic marginal zone lymphoma (SMZL), mucosa-associated lymphoid tissue (MALT) low-grade B-cell lymphomas, and follicular centroblastic-centrocytic (CB-CC) lymphomas. In an attempt to clarify the relationships between different groups of tumors with a significant monocytoid component, we studied six primary lymph node MBCL, three SMZL, seven MALT lymphomas, and four CB-CC with monocytoid differentiation. Their clinical, morphological, immunohistochemical and molecular features were compared. The results show wide overlapping between MALT and MBCL in terms of morphology, immunophenotype, and molecular features. Follicular colonization was a characteristic finding in both groups. Some MBCL revealed mucosal involvement during the course of the disease, suggesting a possible MALT origin. Our data support the suggestion that the use of the term MBCL should be discontinued in cases with mucosal involvement, as they are probably examples of lymph node involvement brought about by MALT lymphomas. Although SMZL have some overlapping features with MBCL and MALT lymphomas, some of the clinical and morphological specific findings justify their distinction from the other groups. The CB-CCs with monocytoid differentiation frequently harbored t(14;18), lacking any significant differentiating features from conventional follicular CB-CC lymphomas. Additional studies are needed to define the molecular features of MBCL and other marginal zone tumors.

p53 overexpression has been found to be a fairly common feature in high grade lymphomas in the majority of tumoral cells. The results vary from series to series, from 25% to 33% of cases. To assess whether immunohistochemical positivity for p53 correlated with the presence of structural gene abnormalities, DNA from 16 non-Hodgkin's lymphomas with high and low p53 values was amplified and sequenced to determine the existence of point mutations in the highly conserved regions of the p53 gene. In the group of 8 cases containing high levels of protein, 3 cases showed missense point mutations at the codons mapping between exons 5 through 8. Of the 8 cases of tumors containing undetectable or low levels of p53 protein, 1 case presented a nonsense point mutation giving a stop codon. No missense mutations were detected in this group. The finding of p53 mutations in 4 of 16 cases confirms the presence of p53 gene mutations in high grade lymphomas distributed over different histologic groups. These include Burkitt's lymphoma, together with centroblastic, immunoblastic, and large cell lymphoma of mucosa origin. Nevertheless, the absence of mutations in 5 of the 8 cases that overexpressed p53 suggests that the nuclear or cytoplasmic stabilization of p53 protein could also depend on other factors. The absence of detectable levels of p53 protein cannot discount the existence of p53 mutations, as is shown by a case of Burkitt's lymphoma in which a nonsense mutation was detected. The impact of this range of p53 alterations on clinical course and treatment response of the patients deserves to be explored, in an attempt to differentiate the specific consequences of each one.

Gastric low-grade B cell lymphomas originating in mucosa-associated lymphoid tissues are composed of reactive hyperplastic germinal centers and interfollicular centrocyte-like cells. Their polymorphic, histologic composition and infrequent dissemination beyond the gastric wall led to the denomination of these tumors as "pseudolymphomas." To elucidate their clonal character, a Southern blot study of DNA and immunohistological study was carried out on 14 cases of surgical specimens from gastrectomy (11 low-grade and three high-grade tumors). Monoclonal and polyclonal antibodies were used on frozen and paraffin sections. A mucosa-associated lymphoid tissue origin for the tumors was assigned due to their centrocyte-like morphology and the presence of lymphoepithelial lesions. Southern blot analysis of DNA and immunohistological results confirm the monoclonal composition of gastric low-grade lymphomas in all cases. Although both types of technique correlated well on the predominant light-chain, Southern blot DNA study was nevertheless more sensitive than the immunohistochemical techniques. Surprisingly, in two cases of gastric low-grade lymphoma, Southern blot DNA analysis of histologically reactive regional lymph nodes showed an unexpected immunoglobulin heavy chain gene rearrangement. This coincided with that found in the gastric wall. Results confirm the monoclonal nature of the low-grade gastric lymphoma. This supports consideration of this tumor as an indolent primary lymphoma of the stomach, confirming the suitability of excluding the term "pseudolymphoma." Involvement of regional lymph nodes may be a more frequent occurrence than previously detected through morphological study.