Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

Over 30% of patients with splenic marginal zone lymphoma express the same immunoglobulin heavy variable gene: ontogenetic implications.

We performed an immunogenetic analysis of 345 IGHV-IGHD-IGHJ rearrangements from 337 cases with primary splenic small B-cell lymphomas of marginal-zone origin. Three immunoglobulin (IG) heavy variable (IGHV) genes accounted for 45.8% of the cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23, 8.1%). Particularly for the IGHV1-2 gene, strong biases were evident regarding utilization of different alleles, with 79/86 rearrangements (92%) using allele (*)04. Among cases more stringently classified as splenic marginal-zone lymphoma (SMZL) thanks to the availability of splenic histopathological specimens, the frequency of IGHV1-2(*)04 peaked at 31%. The IGHV1-2(*)04 rearrangements carried significantly longer complementarity-determining region-3 (CDR3) than all other cases and showed biased IGHD gene usage, leading to CDR3s with common motifs. The great majority of analyzed rearrangements (299/345, 86.7%) carried IGHV genes with some impact of somatic hypermutation, from minimal to pronounced. Noticeably, 75/79 (95%) IGHV1-2(*)04 rearrangements were mutated; however, they mostly (56/75 cases; 74.6%) carried few mutations (97-99.9% germline identity) of conservative nature and restricted distribution. These distinctive features of the IG receptors indicate selection by (super)antigenic element(s) in the pathogenesis of SMZL. Furthermore, they raise the possibility that certain SMZL subtypes could derive from progenitor populations adapted to particular antigenic challenges through selection of VH domain specificities, in particular the IGHV1-2(*)04 allele.

The prognostic impact of clinical and molecular features in hairy cell leukaemia variant and splenic marginal zone lymphoma.

Hairy cell leukaemia variant (HCL-variant) and splenic marginal zone lymphoma (SMZL) are disorders with overlapping features. We investigated the prognostic impact in these disorders of clinical and molecular features including IGH VDJ rearrangements, IGHV gene usage and TP 53 mutations. Clinical and laboratory data were collected before therapy from 35 HCL-variant and 68 SMZL cases. End-points were the need for treatment and overall survival. 97% of HCL-variant and 77% of SMZL cases required treatment (P = 0·009). Survival at 5 years was significantly worse in HCL-variant [57% (95% confidence interval 38-73%)] compared with SMZL [84% (71-91%); Hazard Ratio 2·25 (1·20-4·25), P = 0·01]. In HCL-variant, adverse prognostic factors for survival were older age (P = 0·04), anaemia (P = 0·01) and TP 53 mutations (P = 0·02). In SMZL, splenomegaly, anaemia and IGHV genes with >98% homology to the germline predicted the need for treatment; older age, anaemia and IGHV unmutated genes (100% homology) predicted shorter survival. IGHV gene usage had no impact on clinical outcome in either disease. The combination of unfavourable factors allowed patients to be stratified into risk groups with significant differences in survival. Although HCL-variant and SMZL share some features, they have different outcomes, influenced by clinical and biological factors.

Abnormalities on 1q and 7q are associated with poor outcome in sporadic Burkitt's lymphoma. A cytogenetic and comparative genomic hybridization study.

Comparative genomic hybridization (CGH) studies have demonstrated a high incidence of chromosomal imbalances in non-Hodgkin's lymphoma. However, the information on the genomic imbalances in Burkitt's Lymphoma (BL) is scanty. Conventional cytogenetics was performed in 34 cases, and long-distance PCR for t(8;14) was performed in 18 cases. A total of 170 changes were present with a median of four changes per case (range 1-22). Gains of chromosomal material (143) were more frequent than amplifications (5) or losses (22). The most frequent aberrations were gains on chromosomes 12q (26%), Xq (22%), 22q (20%), 20q (17%) and 9q (15%). Losses predominantly involved chromosomes 13q (17%) and 4q (9%). High-level amplifications were present in the regions 1q23-31 (three cases), 6p12-p25 and 8p22-p23. Upon comparing BL vs Burkitt's cell leukemia (BCL), the latter had more changes (mean 4.3 +/- 2.2) than BL (mean 2.7 +/- 3.2). In addition, BCL cases showed more frequently gains on 8q, 9q, 14q, 20q, and 20q, 9q, 8q and 14q, as well as losses on 13q and 4q. Concerning outcome, the presence of abnormalities on 1q (ascertained either by cytogenetics or by CGH), and imbalances on 7q (P=0.01) were associated with a short survival.

Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation.

The c-kit proto-oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK-cells, and to early T-cell precursors and it is not expressed by normal B-cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkin's disease and anaplastic large-cell lymphoma. In the present paper we describe a case of a B-cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B-cell population. Further studies are necessary to explore both the functional role of c-kit expression in the neoplastic B-cells from this patient and its potential utility for the diagnosis and follow-up of patients with B-cell non-Hodgkin's lymphoma.

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

Daunorubicin and gambogic acid co-loaded cysteamine-CdTe quantum dots minimizing the multidrug resistance of lymphoma in vitro and in vivo

To minimize the side effects and the multidrug resistance (MDR) arising from daunorubicin (DNR) treatment of malignant lymphoma, a chemotherapy formulation of cysteamine-modified cadmium tellurium (Cys-CdTe) quantum dots coloaded with DNR and gambogic acid (GA) nanoparticles (DNR-GA-Cys-CdTe NPs) was developed. The physical property, drug-loading efficiency and drug release behavior of these DNR-GA-Cys-CdTe NPs were evaluated, and their cytotoxicity was explored by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide assay. These DNR-GA-Cys-CdTe NPs possessed a pH-responsive behavior, and displayed a dose-dependent antiproliferative activity on multidrug-resistant lymphoma Raji/DNR cells. The accumulation of DNR inside the cells, revealed by flow cytometry assay, and the down-regulated expression of P-glycoprotein inside the Raji/DNR cells measured by Western blotting assay indicated that these DNR-GA-Cys-CdTe NPs could minimize the MDR of Raji/DNR cells. This multidrug delivery system would be a promising strategy for minimizing MDR against the lymphoma.

Parotid gland oncocytoma in HCV-positive patient with non-Hodgkin's lymphoma. Case report

We report a case of parotid gland oncocytoma in a patient with chronic infection from hepatitis C virus (HCV) and associated non-Hodgkin's lymphoma and xerophthalmia. Our case confirms the triple tropism of the HCV: hepatotropism, lymphotropism and sialotropism.

Leishmaniasis Mimicking Lymphoma and/or Liver Cirrhosis

A 76-year-old man was admitted to hospital with fever, weight loss, pancytopenia, hepatosplenomegaly and a double monoclonal component IgM-IgG-k, suggesting a diagnosis of myeloma. Bone marrow and liver biopsies disclosed the presence of Donovan bodies, and the titre of anti-Leishmania antibodies was extremely high. After treatment with liposomal amphotericin B, the titre of antibodies fell considerably, while monoclonal components, pancytopenia and clinical symptoms slowly disappeared. Polyclonal γ-globulins are made of innumerable monoclonal components, one of which can appear as a recognizable band and be misdiagnosed as myeloma when representing the high titre of an antibody directed towards a specific antigen.

Intravascular Lymphoma as an Uncommon Cause of Anasarca

Objectives: To report a case of intravascular lymphoma (IVL) in a Caucasian patient who presented with anasarca as his sole clinical sign. Material and Methods: A man presented with anasarca-type oedema and fatigue. After excluding heart failure, hepatic cirrhosis, nephrotic syndrome, hypothyroidism, AL-amyloidosis and adverse drug reaction which can all cause oedema, we turned our attention to capillary permeability disorders. Results: Closer review of the bone marrow aspirate demonstrated haemophagocytic histiocytosis, while core, renal and duodenal biopsies showed a B-cell IVL. Conclusion: The differential diagnosis of anasarca, a relatively common clinical sign, should include IVL although the diagnosis may still be challenging.

Long-term consequences of Hodgkin lymphoma therapy on T-cell lymphopoiesis

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