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The immunoglobulin heavy chain variable gene (IGHV) mutational status has been recognized as an important predictor of prognosis in chronic lymphocytic leukaemia (CLL) since 1999. More recently, other features of the B-cell receptor, such as stereotypy, have been identified as capable of refining the prognostic potential of IGHV status in the clinical assessment of CLL patients. In this context, different genes belonging to the IGHV3 subgroup, the most frequently used subgroup in CLL, have been shown to denote disease subsets that either display a bad prognosis (i.e. IGHV3-21, IGHV3-23) or are associated with particularly good clinical outcomes, including a highly stable/indolent clinical course, even prone to spontaneous regression (i.e. IGHV3-72, IGHV3-30). The present review focuses on the molecular and biological features of CLL-expressing specific genes belonging to the IGHV3 subgroup that are known to mark disease subsets with completely different clinical courses, and may be possibly related to CLL pathogenesis via antigen and/or superantigen involvement., (© 2011 Blackwell Publishing Ltd.)

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.

Post-transplant lymphoproliferative disorders (PTLD) are complications of solid organ transplantation associated with severe morbidity and mortality. Diffuse large B-cell lymphoma (DLBCL) represents the most common form of monomorphic PTLD. We studied 44 cases of post-transplant DLBCL (PT-DLBCL) with high-density genome wide single nucleotide polymorphism-based arrays, and compared them with 105 cases of immunocompetent DLBCL (IC-DLBCL) and 28 cases of Human Immunodeficiency Virus-associated DLBCL (HIV-DLBCL). PT-DLBCL showed a genomic profile with specific features, although their genomic complexity was overall similar to that observed in IC- and HIV-DLBCL. Among the loci more frequently deleted in PT-DLBCL there were small interstitial deletions targeting known fragile sites, such as FRA1B, FRA2E and FRA3B. Deletions at 2p16.1 (FRA2E) were the most common lesions in PT-DLBCL, occurring at a frequency that was significantly higher than in IC-DLBCL. Genetic lesions that characterized post-germinal center IC-DLBCL were under-represented in our series of PT-DLBCL. Two other differences between IC-DLBCL and PT-DLBCL were the lack of del(13q14.3) (MIR15/MIR16) and of copy neutral LOH affecting 6p [major histocompatibility complex (MHC) locus] in the latter group. In conclusion, PT-DLBCL presented unique features when compared with IC-DLBCL. Changes in PT-DLBCL were partially different to those in HIV-DLBCL, suggesting different pathogenetic mechanisms in the two conditions linked to immunodeficiency.

A fraction of chronic lymphocytic leukemia (CLL) carries highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity-determining region-3 (HCDR3), often associated with a restricted selection of IG(K/L)V light chains. We analyzed the features of CLL expressing homologous HCDR3 in a cohort of 264 Ukrainian patients by merging them with a recently published reference series of 1426 cases. This approach allowed us to identify 96/264 (36%) cases as expressing homologous HCDR3, subdivided into 47 subsets. Among these, 27 apparently novel subsets were identified, although most of them were composed of two sequences per subset ('potential subsets'). CLL cases belonging to several stereotyped subsets showed HCDR3 homologies with various autoreactive clones. Our analysis identified molecular and clinical features of a Ukrainian cohort of patients with CLL.

Purpose: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL., Experimental Design: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23-expressing CLL., Results: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non-IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non-IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non-IGHV3-23 CLL., Conclusions: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features.

Non-Hodgkin lymphomas (NHL) represent a frequent complication of human immunodeficiency virus (HIV) infection. To elucidate HIV-NHL pathogenesis, we performed a genome-wide DNA profiling based on a single nucleotide polymorphism-based microarray comparative genomic hybridization in 57 HIV-lymphomas and, for comparison, in 105 immunocompetent diffuse large B-cell lymphomas (IC-DLBCL). Genomic complexity varied across HIV-NHL subtypes. HIV-Burkitt lymphoma showed a significantly lower number of lesions than HIV-DLBCL (P = 0.032), whereas the median number of copy number changes was significantly higher in Epstein-Barr virus negative (EBV-) HIV-DLBCL (42.5, range 8-153) compared to EBV+ cases (22; range 3-41; P = 0.029). Compared to IC-DLBCL, HIV-DLBCL displayed a distinct genomic profile with no gains of 18q and specific genetic lesions. Fragile sites-associated genes, including FHIT (FRA3B), WWOX (FRA16D), DCC (FRA18B) and PARK2 (FRA6E) were frequently inactivated in HIV-NHL by interstitial deletions, and a significantly higher prevalence of FHIT alterations was observed in HIV-DLBCL compared to IC-DLBCL. The same genes involved by fragile site deletions were also frequently affected by aberrant methylation of regulative regions.

Post-transplant lymphoproliferative disorders (PTLD) are a life-threatening complication of solid organ transplantation or, more rarely, hematopoietic stem cell transplantation. The majority of PTLD is of B-cell origin and associated with Epstein-Barr virus (EBV) infection. PTLD generally display involvement of extranodal sites, aggressive histology and aggressive clinical behavior. The molecular pathogenesis of PTLD involves infection by oncogenic viruses, namely EBV, as well as genetic or epigenetic alterations of several cellular genes. At variance with lymphoma arising in immunocompetent hosts, whose genome is relatively stable, a fraction of PTLD are characterized by microsatellite instability as a consequence of defects in the DNA mismatch repair mechanism. Apart from microsatellite instability, molecular alterations of cellular genes recognized in PTLD include alterations of cMYC, BCL6, TP53, DNA hypermethylation, and aberrant somatic hypermutation of protooncogenes. The occurrence of IGV mutations in the overwhelming majority of PTLD documents that malignant transformation targets germinal centre (GC) B-cells and their descendants both in EBV-positive and EBV-negative cases. Analysis of phenotypic markers of B-cell histogenesis, namely BCL6, MUM1 and CD138, allows further distinction of PTLD histogenetic categories. PTLD expressing the BCL6+/MUM1+/-/CD138- profile reflect B-cells actively experiencing the GC reaction, and comprise diffuse large B-cell lymphoma (DLBCL) centroblastic and Burkitt lymphoma. PTLD expressing the BCL6-/MUM1+/CD138- phenotype putatively derive from B-cells that have concluded the GC reaction, and comprise the majority of polymorphic PTLD and a fraction of DLBCL immunoblastic. A third group of PTLD is reminiscent of post-GC and preterminally differentiated B-cells that show the BCL6-/MUM1+/CD138+ phenotype, and are morphologically represented by either polymorphic PTLD or DLBCL immunoblastic.

Background: To analyze clinical features and to discuss the modality of investigation and treatment of a series of mucosa-associated lymphoid tissue (MALT) lymphomas. To investigate the prevalence of aberrant promoter methylation, responsible for gene inactivation, in a selected panel of genes potentially involved in the pathogenesis of B-cell malignancies as O6-methylguanine-DNA methyltransferase (MGMT), p73, death-associated protein kinase (DAP-k)., Methods: Nine patients with primary MALT lymphoma of the salivary glands were retrospectively reviewed. MGMT, p73, DAP-k apoptotic pathways were tested., Results: Methylation of DAP-k was common (5/8; 63%). Histological examination ensured diagnostic confirmation, whereas fine-needle aspiration cytology was not definitively diagnostic., Conclusion: Histological assessment is the gold standard in the diagnosis of salivary gland lesions. Parotidectomy represents a safe and reliable diagnostic tool leading to a definite diagnosis of MALT lymphomas in all cases and curative without other treatment in early-stage MALT lymphoma.

Post-transplant lymphoproliferative disorders (PTLDs) represent a frequent complication of solid organ transplantation. Although most PTLDs arise from recipient lymphoid cells, a considerable fraction of cases may arise from donor B-cells. In an attempt to clarify the histogenesis and pathogenesis of PTLDs derived from donor B-cells, monoclonal PTLDs occurring in liver transplant recipients were chosen as a model to compare donor (D-PTLDs) versus recipient PTLDs (R-PTLDs). The tumour panel included nine D-PTLDs and six R-PTLDs. D-PTLDs were early-onset, EBV-infected lymphoproliferations classified as polymorphic PTLD (P-PTLD; n = 7) or diffuse large B-cell lymphoma (DLBCL; n = 2) with tumour localization confined to the hepatic hilum. All R-PTLDs were late-onset DLBCLs and showed extrahepatic localization. A BCL-6(-)/MUM1(+)/CD138(+/-) phenotype, consistent with a post-germinal centre (GC) stage of pre-terminal B-cell differentiation, was observed in all D-PTLDs and in 2/6 R-PTLDs, whereas a BCL6(+)/MUM1(-)/CD138(-) profile, reminiscent of GC B-cells, was detected in 4/6 R-PTLDs. The presence of somatic IGHV hypermutation was observed in 6/9 D-PTLDs and in 4/6 R-PTLDs, suggesting derivation from antigen-experienced B-cells. IGHV4-39 was the IGHV gene most frequently encountered, being rearranged in 3/9 D-PTLDs. Among IGHV-mutated PTLDs, a mutational profile suggesting antigen stimulation and/or selection was observed in 4/6 D-s and in 2/4 R-PTLDs. The presence of ongoing IGHV mutations was detected in 2/4 D-PTLDs. Aberrant SHM was detected in 10/15 (66.7%) PTLDs, including 6/9 D-PTLDs and 4/6 R-PTLDs. Our findings suggest that (i) D-PTLDs show a clinical presentation distinct from R-PTLDs; (ii) immunophenotypic and genetic features of D-PTLDs are consistent with mature, GC-experienced B-cells; (iii) transformed donor-derived B-cells may experience antigen-driven stimulation and selection, and may acquire genetic lesions during neoplastic expansion in the recipient environment; and (iv) EBV infection and expression of viral oncoproteins may be relevant in the pathogenesis of D-PTLDs., ((c) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.

Purpose: Del17p13 predicts poor outcome and chemorefractoriness in chronic lymphocytic leukemia (CLL). Conversely, it is unknown whether TP53 mutations carry any prognostic value independent of del17p13. We tested the independent prognostic value of TP53 mutations in CLL., Experimental Design: The study was based on a consecutive series of 308 CLL. DNA sequencing of TP53 exons 2 to 10 and del17p13 interphase fluorescence in situ hybridization were done at CLL diagnosis. Study end points were survival and chemorefractoriness., Results: At diagnosis, TP53 mutations (n = 32) occurred in 31 of 308 (10.0%) patients. Of all CLL showing TP53 disruption by either mutation and/or deletion (n = 44), 10 cases (22.7%) showed TP53 mutations in the absence of del17p13. Multivariate analysis selected TP53 mutations (hazard ratio, 3.20; P = 0.002) as an independent predictor of overall survival after adjustment for del17p13. Also, multivariate analysis selected TP53 mutations (hazard ratio, 3.97; P < 0.001) as an independent predictor of chemorefractoriness after adjustment for del17p13. Compared with cases without TP53 alterations, CLL harboring any type of TP53 disruption (mutation only, del17p13 only, or both mutation and del17p13) uniformly displayed a high prevalence of unfavorable prognosticators and poor outcome. Analysis of sequential CLL samples showed the acquisition of new or additional TP53 alterations at the time of chemorefractoriness., Conclusions: These data show that (a) TP53 mutations are an independent predictor of short survival and chemorefractoriness, and (b) that CLL presenting with TP53 mutations without del17p13 fare as poorly as CLL carrying del17p13. Because CLL harboring TP53 mutations without del17p13 are currently not recognized by conventional diagnostic strategies, these results may be relevant for a comprehensive prognostic characterization of CLL.

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such 'stereotyped' BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22.4%) with stereotyped BCR were identified. Among 30 confirmed clusters (>or=3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these 'M clusters'. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.

Deletion of 17p (TP53) identifies a rare subset of chronic lymphocytic leukaemia (17p- CLL) with aggressive behaviour. Genome-wide DNA-profiling was performed to investigate 18 patients with 17p- CLL. All cases had multiple copy-number (CN) changes. Among the several recurrent CN changes identified, 8q24.13-q24.1-gain (MYC), 8p-loss (TNFRSF10A/B, also known as TRAIL1/2) and 2p16.1-p14-gain (REL/BCL11A) appeared frequently represented. 8p-loss and 2p16.1-p14-gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p-loss) and overall survival (8p-loss and 2p16.1-p14-gain, P < 0.05). These observations document a highly unstable genome in 17p- CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.

Background and Purpose: Functional neuroimaging and electrophysiological studies suggest that dysplastic neural tissue in malformations of cortical development may participate in task performance, and that functional organization can be altered beyond visible lesion boundaries. The aim of this work was to investigate cortical function in a heterogeneous group of patients with malformations of cortical development., Methods: Twelve patients participated in the study, 2 for each of the following categories: subcortical, periventricular, and band heterotopia, unilateral and bilateral polymicrogyria, and focal cortical dysplasia. Functional magnetic resonance imaging was performed with finger tapping, somatosensory and visual stimulation, and language-related tasks., Results: We found activations within the dysplastic tissue in subcortical heterotopia, band heterotopia, and polymicrogyria, but not in periventricular heterotopic nodules. In one of the patients with focal cortical dysplasia, language-related activation involved part of the lesion. Functional reorganization beyond visible lesion boundaries was seen, with different patterns, in 4 patients., Conclusions: In accordance with previous reports, our findings indicate that dysplastic neural tissue can be activated during task performance, and that in some patients, extensive functional reorganization occurs, highlighting the importance of functional magnetic resonance imaging in presurgical planning in those patients for whom epilepsy surgery is considered as an option.

Predictors of chronic lymphocytic leukaemia (CLL) transformation to Richter syndrome (RS) are not established and were investigated in 185 consecutive CLL cases. Actuarial incidence of RS (n = 17; all diffuse large B-cell lymphomas) at 10 years was 16.2% (95% confidence interval: 8.0-24.4%). At CLL diagnosis, prognosticators of RS by univariate analysis were IGHV homology >/=98% (P = 0.006), IGHV4-39 usage (P < 0.001), del13q14 absence (P = 0.004), expression of CD38 (P < 0.001) and ZAP70 (P = 0.004), size (P < 0.001) and number (P < 0.001) of lymph nodes, advanced Binet stage (P = 0.002), and lactate dehydrogenase (P < 0.001). Multivariate analysis, performed separately for biological and clinical variables, identified CD38 expression [Hazard ratio (HR) = 4.26; P = 0.018], IGHV4-39 usage (HR = 4.29; P = 0.018), and lymph node size >/=3 cm (HR = 9.07; P < 0.001) as independent RS prognosticators. A multivariate model simultaneously analysing biological and clinical variables identified lymph node size >/=3 cm (HR = 6.51; P = 0.001) and del13q14 absence (HR = 4.08; P = 0.031) as independent RS prognosticators. Risk factors of CLL transformation differed from risk factors of CLL progression. These results suggest that CD38 and del13q14 may identify biological subsets of CLL with different RS predisposition. Predominant nodal disease, CD38 expression, IGHV4-39 usage, and absence of del13q14 may help in predicting RS at CLL diagnosis. Close monitoring and a careful biopsy policy are needed in patients carrying transformation risk factors.

Hairy cell leukaemia (HCL) is a rare B-cell neoplasm for which the molecular mechanisms are largely unknown. High-density genome-wide DNA profiling was performed with Affymetrix 250K arrays to analyse copy number (CN) changes and loss of heterozygosity (LOH) in 16 cases of HCL. Four of 16 cases (25%) demonstrated gross non-recurrent CN deletions. Within the affected regions, we identified genes involved in bone marrow fibrosis (FGF12) and response to treatment (TP53) in individual cases. Large regions (> 5 Mb) of LOH without any concomitant DNA CN changes were identified in 5/16 (31%) HCL and were indicative of uniparental disomy UD. The germline origin of UD was demonstrated in one case for which a matched normal sample was available. Overall analysis of LOH showed that identical loci were recurrently targeted in chromosomes 1, 2 and 6. As a whole, however, HCL showed a remarkably stable genome. This finding adds to several other features that are unique to HCL among mature B-cell tumours.

Ph-negative chronic myeloproliferative disorders (CMPD) are characterized by constitutive Janus kinase-signal transducer and activator of transcription (JAK-STAT) activation. SOCS3, SOCS1 and PTPN6 (SHP1) are negative regulators of the JAK-STAT pathway. We investigated epigenetic and genetic inactivation of SOCS3, SOCS1 and PTPN6 in 112 CMPD and 20 acute myeloid leukaemia (AML) post-CMPD. SOCS3 methylation occurred at high frequency in both CMPD (46/112; 41.1%) and AML post-CMPD (10/17; 58.8%) and was associated with transcriptional silencing. In contrast, methylation of SOCS1 and PTPN6 was observed in only a fraction of CMPD (15/112, 13.4% for SOCS1; and 8/112, 7.1% for PTPN6) and AML post-CMPD (3/20, 15% for SOCS1; and 1/20, 5% for PTPN6). No somatic mutations of SOCS1 were found in CMPD. SOCS3, SOCS1 and PTPN6 methylation occurred in both JAK2V617F-positive (35.1% for SOCS3; 14.9% for SOCS1; 8.1% for PTPN6) and JAK2V617F-negative (57.1% for SOCS3; 14.3% for SOCS1; and 9.5% for PTPN6) CMPD. These data indicate that methylation of SOCS3 and, to a lesser extent, SOCS1 and PTPN6 is a frequent event in both JAK2V617F-positive and -negative CMPD and may act as an alternative or complementary mechanism to JAK2 mutations, enhancing cytokine signal transduction. The frequent inactivation of SOCS3 is a novel finding in CMPD with potential implications for the molecular pathology of these disorders.

Lymphocytosis associated with viral infection is generally polyclonal or oligoclonal. In this article, we describe a case of transient monoclonal CD8+/CD57+ T-cell lymphocytosis with large granular lymphocyte (LGL) morphology occurring after primary CMV infection and review cases of virus-associated monoclonal CD8+ T-cell expansions reported in the literature. Several clinical features shared by virus-associated monoclonal CD8+ T-cell expansions suggest the reactive nature of the lymphocytosis. Based on this, our case report and those reported in the literature support the notion that T-cell receptor clonality per se is not necessarily indicative of malignancy. These observations further corroborate the need for a close follow-up before assigning the diagnosis of LGL leukemia to individuals developing monoclonal CD8+ T-cell expansions.

We report the case of an Epstein-Barr virus (EBV)- and human immunodeficiency virus-serum negative patient suffering from repeatedly relapsing classical Hodgkin's Lymphoma (cHL) associated with a histological picture of plasma cell-hyaline vascular (PC-HV) form of Castleman's disease (CD). The CD30- and CD15-positive, Reed-Sternberg/Hodgkin cells, only occasionally expressed the CD20 molecule, but not leukocyte common antigen and latent membrane protein-1. Single-strand polymerase chain reaction failed to detect human herpesvirus 8 or EBV in the involved tissues. At the time of second relapse in July 2005, the clinical picture was characterized by a palpable right hypogastric mass, disclosed at physical exam, in the absence of other enlarged peripheral lymph nodes, subjective symptoms or laboratory profile alterations. Combined hybrid-(18)F-fluorodeoxyglucose positron emission-computerized tomography (18F-FDG PET/CT) showed increased radionuclide uptake in multiple external iliac lymph nodes [standardized uptake value (SUV) of 7.4] and non-palpable left supraclavicular lymph nodes (SUV of 5.8). Relapsing cHL in the context of mixed PC-HV CD was documented in two of three surgically excised abdominal lymph nodes never previously enlarged or involved by any lymphoproliferative disease. Because of the limited disease extension and failure to induce continuous remission with previous conventional chemoradiotherapy, the patient was treated with six rituximab injections. This immunotherapy induced significant reduction in size of supraclavicular lymph nodes as evident at ultrasound (US) scan (<1 vs. 2.5 cm, post- vs. pretherapy), which was confirmed by the 18F-FDG PET/CT in October 2005, despite no modification in SUV of 4.2. 18F-FDG PET/CT also disclosed no radionuclide uptake by abdominal lymph nodes. Thus, a second course of four additional rituximab injections was given and subsequent 18F-FDG PET/CT indicated persistent, but reduced incorporation of radionuclide compared to the pretherapy value (SUV of 2.7) in the supraclavicular area and confirmed a normal metabolic activity in the iliac external lymph nodes. Because of uncertain persistent disease in the supraclavicular nodal site, involved-field radiotherapy (RT) was delivered in that area as consolidation treatment. After completion of rituximab and RT for 16 and 14 months respectively, US and 18F-FDG PET/CT exams were indicative of complete remission. This case is in concordance with previously published data suggesting that rituximab immunotherapy might be a valid option in the treatment of CD and also have a role in the management of relapsing cHL.

Monoclonal gammopathy of uncertain significance (MGUS) may become symptomatic for autoimmune manifestations. We report on the prevalence and clinical course of immune thrombocytopenic purpura (ITP) observed in a consecutive series of 228 MGUS patients. At MGUS diagnosis, ITP was determined in 6/228 cases, accounting for a prevalence of 2630/100 000 [95% confidence interval (CI): 1210-5620]. One incidental ITP case occurred after 21 months of follow-up. After a follow-up of 681.3 patient-years, the crude incidence of ITP in MGUS was 146.8 per 100 000 patient-year (95% CI: 3.7-817.8). Overall, these observations point to an association between MGUS and ITP.

CD3+ T-cell large granular lymphocyte (LGL) leukaemia is a mature T-cell neoplasm of cytotoxic T-lymphocytes. Neutropenia represents the most frequent peripheral blood cytopenia associated with CD3+ T-cell LGL leukaemia. A wide variety of diseases associated with LGL leukaemia have been reported, both autoimmune and neoplastic. We describe for the first time the association of true thymic hyperplasia with CD3+ T-cell line LGL leukaemia. The patient presented with severe symptomatic neutropenia. Complete and persistent haematological and molecular remission was induced with an association of low-dose methotrexate and cyclosporin A, followed by thymectomy.

Hairy cell leukaemia (HCL) occasionally displays a monoclonal gammopathy, yet the association of HCL with paraproteinemic demyelinating neuropathy (PDN) has not been reported. We describe a HCL case complicated by PDN and high titers of monoclonal IgM against myelin associated glycoprotein (MAG). Heavy and light chains of the patient's anti-MAG monoclonal protein were consistent with those expressed by HCL cells. After treatment with cladribrine, remission of HCL strictly paralleled disappearance of the IgM monoclonal protein and of the serum anti-MAG activity, and led to PDN clinical and electrophysiological improvement. Purine analogs may represent a choice in IgM PDN associated with lymphoproliferative disorders.

IGHV3-21-using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3-IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3-IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3-IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3-IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non-IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3-IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3-IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

Objective: The aim of this study was to determine the occurrence of polymorphic variants of EBV BamHI fragment Z (BZLF1) promoter zone Zp in tumor and non-tumor-associated EBV. We characterized the Zp region in type A and type B EBV, infecting AIDS-related non-Hodgkin's lymphomas (AIDS-NHL) and non-malignant lymphoid tissues derived from HIV-positive patients and from healthy individuals., Methods: The Zp region was directly sequenced in 133 EBV-positive DNA samples: 63 AIDS-NHL (32 systemic AIDS-NHL and 31 AIDS-primary central nervous system lymphoma [AIDS-PCNSL]), 30 lymphoid tissues derived from HIV-positive individuals and 40 lymphoid samples derived from healthy individuals. The chi square test was used to assess for statistically significant differences among proportions, and a two-tailed P value

Idiopathic erythrocytosis (IE) is a primary erythrocytosis not fulfilling the criteria for polycythemia vera (PV) diagnosis. In order to verify the relationship between IE and PV, we screened JAK2V617F mutation in a consecutive series of 11 IE and, for comparison, in 15 PV. JAK2V617F mutation was screened by both cDNA sequencing and mutation specific PCR in both peripheral blood and bone marrow samples. All 11 IE tested negative for JAK2V617F mutation, which, conversely, occurred in 11/15 (73.3%) PV. Our results demonstrate that JAK2V617F is absent in IE and may represent a useful molecular marker for distinguishing IE from PV.

Post-transplant lymphoproliferative disorders (PTLD) derive from antigen-experienced B-cells and represent a major complication of solid organ transplantation. We characterized usage, mutation frequency and mutation pattern of immunoglobulin variable (IGV) gene rearrangements in 50 PTLD (polymorphic PTLD, n=10; diffuse large B-cell lymphoma, n=35; and Burkitt/Burkitt-like lymphoma, n=5). Among PTLD yielding clonal IGV amplimers, a functional IGV heavy chain (IGHV) rearrangement was found in 40/50 (80.0%) cases, whereas a potentially functional IGV light chain rearrangement was identified in 36/46 (78.3%) PTLD. By combining IGHV and IGV light chain rearrangements, 10/50 (20.0%) PTLD carried crippling mutations, precluding expression of a functional B-cell receptor (BCR). Immunohistochemistry showed detectable expression of IG light chains in only 18/43 (41.9%) PTLD. Failure to detect a functional IGV rearrangement associated with lack of IGV expression. Our data suggest that a large fraction of PTLD arise from germinal centre (GC)-experienced B-cells that display impaired BCR. Since a functional BCR is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC reaction. The high frequency of IGV loci inactivation appears to be a peculiar feature of PTLD among immunodeficiency-associated lymphoproliferations.

Background: Posttransplantation lymphoproliferative disorders (PTLDs) are heterogeneous lymphoid proliferations representing a major complication of solid organ transplant. This study details the clinicopathological and molecular features of 17 B-cell PTLDs observed in a single center series of 988 heart and/or lung transplant recipients., Methods: Cases were classified according to World Health Organization lymphoma classification and tested for Epstein-Barr Virus (EBV), clonality, histogenetic phenotypic (CD10, Bcl-6, MUM1, CD138), and genotypic (immunoglobulin and BCL-6 genes somatic hypermutation) markers., Results: This series of 17 PTLDs included: two B-cell monoclonal polymorphic PTLDs and 15 B-cell monomorphic PTLDs (13 diffuse large B-cell lymphomas [DLBCL] and 2 Burkitt lymphomas [BL]). EBV was detected in 9/17 cases. A monoclonal immunoglobulin variable (IGV) genes rearrangement was documented in 17/17 cases; IGV somatic hypermutation was found in 88% of cases, indicating a prevalent origin from germinal center (GC)-experienced B cells. Using immunophenotypic markers, three histogenetic profiles were identified: a) CD10/bcl-6/MUM1/CD138, mimicking GC B-cells; b) CD10-/bcl-6+/MUM1+/CD138-, reminiscent of B-cells at the latest phases of GC reaction; and c) CD10-/bcl-6-/MUM1+/CD138+/-, consistent with preterminally differentiated B-cells., Conclusions: Correlation between morphology, histogenesis, and EBV status demonstrated a high degree of homogeneity in the two GC-related groups, mostly including EBV-negative cases with BL and DLBCL-centroblastic features; the third group, consisting of post GC EBV-positive cases, was histologically less homogeneous, as it included polymorphic PTLDs and DLBCL with immunoblastic and anaplastic features. The EBV-negative cases with GC histogenetic phenotype showed a slightly better outcome; however, such less aggressive prognostic trend was not confirmed by statistical analysis.

The molecular mechanisms involved in histologic transformation of follicular lymphoma (FL) and B-chronic lymphocytic leukemia (B-CLL) to diffuse large B-cell lymphoma (DLBCL) are heterogeneous and largely unknown. Here we explored whether aberrant somatic hypermutation, leading to the acquisition of new mutations in PIM-1, PAX-5, RhoH/TTF and c-MYC genes, is involved in transformation from FL or B-CLL to DLBCL. Eighteen sequential pairs of FL/DLBCL (n=9) and B-CLL/DLBCL (n=9) were investigated. Our findings demonstrate that acquisition of novel mutations due to aberrant somatic hypermutation was associated with DLBCL transformation in 5/9 (55.5%) cases of FL and 2/9 (22.2%) cases of B-CLL.

The frequent expression of the otherwise rare IgVH3-72 gene was demonstrated in highly stable B-cell chronic lymphocytic leukaemias (B-CLLs). Here we describe the immunophenotypic profiles of eleven IgVH3-72 B-CLLs, by investigating expression of ZAP-70 and of a set of surface antigens previously demonstrated to represent the signature of distinct B-CLL prognostic groups. All IgVH3-72 B-CLLs revealed a homogeneous immunophenotypic profile, expressing all the markers associated with good prognosis, but not those representing the signature of bad prognosis B-CLLs, including ZAP-70. Our results corroborate the notion that IgVH3-72 B-CLLs represent a molecularly and immunophenotypically homogeneous disease group with a good prognosis.

Aberrant somatic hypermutation (SHM) has been identified as a mechanism for genomewide instability in diffuse large B-cell lymphoma (DLBCL). To assess whether aberrant SHM plays a role in the molecular pathogenesis of Hodgkin lymphoma (HL), we investigated microdissected neoplastic cells of nodular lymphocyte-predominant HL (NLPHL; n = 10) and classic HL (cHL; n = 9) for the presence of mutations in the 5' sequences of 4 previously identified aberrant SHM targets (PIM1, PAX5, RhoH/TTF, c-MYC). Mutations in one or more genes were detected in 80% of NLPHLs and 55% of cHLs, with 50% and 30% of patients carrying mutations in 2 or more genes, respectively. The most frequently involved protooncogene was PAX5, mutated in 7 of 9 patients with NLPHL and 2 of 9 patients with cHL. In total, 34 mutations were detected in NLPHL (frequency, 1.04/1,000 bp) and 35 were detected in patients with cHL (frequency, 1.92/1,000 bp). Mutations were of somatic origin because they were absent in control T cells and shared most of the features of the immunoglobulin variable (IGV) gene-associated SHM mechanism-ie, single nucleotide substitutions (n = 63) with rare deletions/insertions (n = 6) and a predominance of transitions over transversions with preferential targeting motifs. Our finding that NLPHL and cHL are targeted by aberrant SHM, as is DLBCL, suggests that these lymphomas may share common molecular pathogenetic events.

Post-transplant lymphoproliferative disorders (PTLD) are a major complication of solid organ transplantation, representing a cause of severe morbidity and mortality. Apart from Epstein-Barr virus infection, knowledge of the pathogenesis of monoclonal PTLD is limited. Powerful analysis techniques, such as whole genomic DNA profiling (array comparative genomic hybridisation), can improve our understanding of PTLD pathogenesis. Whole genome profiling using the Affymetrix GeneChip Human Mapping 10 k 2.0 was performed on 20 PTLD cases and 25 cases of diffuse large B-cell lymphoma (DLBCL) from immunocompetent patients as a control group. Recurrent lesions were detected among all the samples. Chromosome 18q, 7q, 3q and 12 were the most common gains in the control group. Chromosomes 5p and 11p were commonly gained in PTLD-DLBCL. The latter had frequent losses of 6q, 17p, 1p and 9p. Chromosome 12p was the most frequent target of deletions among PTLD-DLBCL cases. Loss of heterozygosity (LOH) did not always match DNA loss: chromosome 10 seemed to be targeted by uniparental disomy in PTLD. Small deletions and gains, involving both known (BCL2 and PAX5) and unknown genes (ZDHHC14), were identified. These data suggest that PTLD share, at a lower frequency, common genetic aberrations with DLBCL from immunocompetent patients. The demonstration of 9p13 amplification emphasises the importance of PAX5 in PTLD. The combination of DNA copy number and LOH assessment lead to the hypothesis that uniparental disomy may be a potential mechanism in B-cell lymphomagenesis.

Primary effusion lymphomas (PELs) are invariably infected by human herpesvirus type 8 (HHV8) and often co-infected by Epstein-Barr virus (EBV). We found that expression of major histocompatibility complex class I (MHC-I) surface molecules was significantly decreased in PEL cells when compared with HHV8 negative lymphomas, irrespective of EBV infection. MHC-I downregulation rendered PEL cells sensitive to recognition and killing by natural killer (NK) cells. Intriguingly, analysis of MHC-I non-restricted cytotoxicity in two PEL patients indicated a reduced NK cell activity when compared with healthy individuals. These data suggest that PEL outgrowth may require an impaired NK cell function.

Post-transplant lymphoproliferative disorders (PTLD) represent a serious complication of solid organ and allogeneic bone marrow transplantation. PTLD generally display B-cell lineage derivation, involvement of extranodal sites, aggressive histology and clinical behaviour, and frequent association with EBV infection. The occurrence of IgV mutations in the overwhelming majority of PTLD documents that malignant transformation targets germinal centre (GC) B-cells and their descendants both in EBV-positive and EBV-negative cases. Analysis of phenotypic markers of B-cell histogenesis, namely BCL6, MUM-1 and CD138, allows further distinction of PTLD histogenetic categories. PTLD expressing the BCL6(+)/MUM1(+/-)/CD138(-) profile reflect B-cells actively experiencing the GC reaction and comprise diffuse large B-cell lymphoma (DLBCL) centroblastic and Burkitt lymphoma. PTLD expressing the BCL6(-)/MUM1(+)/CD138(-) phenotype putatively derive from B-cells that have concluded the GC reaction and comprise the majority of polymorphic PTLD and a fraction of DLBCL. A third group of PTLD is reminiscent of post-GC and pre- terminally differentiated B-cells that show the BCL6(-)/MUM1(+)/CD138(+) phenotype and are morphologically represented by either polymorphic PTLD or DLBCL immunoblastic. The molecular pathogenesis of PTLD involves infection by oncogenic viruses, namely Epstein-Barr virus, as well as genetic or epigenetic alterations of several cellular genes. At variance with lymphoma arising in immunocompetent hosts, whose genome is relatively stable, a fraction of PTLD are characterized by microsatellite instability as a consequence of defects in the DNA mismatch repair mechanism. Apart from microsatellite instability, molecular alterations of cellular genes recognized in PTLD include alterations of c-MYC, BCL-6, p53, DNA hypermethylation, and aberrant somatic hypermutation of proto-oncogenes.

Hepatitis C virus (HCV) and aberrant somatic hypermutation (SHM) have each been suggested to contribute to the development of B-cell non-Hodgkin's lymphoma (NHL). The incidence of PIM-1, PAX-5, RhoH/TTF, and c-MYC mutations in tumour biopsy specimens from 32 HCV-infected B-cell NHL patients was analysed to determine whether the extent of aberrant SHM among these patients differed from that previously reported for HCV-negative B-cell NHL patients. Mutation of PIM-1, PAX-5, RhoH/TTF, and c-MYC was detected in 4 (13%), 5 (16%), 4 (13%), and 4 (13%) of 32 samples, respectively. In HCV-positive B-cell NHL patients, the frequency of aberrant SHM was lower than that already found in HCV-negative B-cell NHL patients. This indicates that, unlike B-cell lymphomas from HCV-negative patients, aberrant SHM may not contribute significantly to malignant transformation in HCV-associated B-cell lymphomas., (2005 Pathological Society of Great Britain and Ireland)

We investigated the prevalence and prognostic role of CpG island methylation of the reduced folate carrier (RFC) gene promoter region in primary central nervous system lymphoma (PCNSL) in immunocompetent patients. Genomic DNA from 40 PCNSL was used for methylation-specific polymerase chain reaction and bisulphite genomic sequencing of the RFC promoter region. Human immunodeficiency virus-negative systemic diffuse large B-cell lymphomas (DLBCL) were used as controls (n = 50). The impact on outcome of RFC promoter methylation was assessed in 37 PCNSL patients treated with high-dose methotrexate (HD-MTX)-based chemotherapy +/- radiotherapy. RFC promoter methylation occurred in 12 of 40 (30%) PCNSL and in four of 50 (8%) DLBCL (P = 0.01). Of 37 PCNSL treated with HD-MTX-based chemotherapy, methylation occurred in nine cases (24%, M-PCNSL), while 28 cases (76%, U-PCNSL) were negative. Three M-PCNSL (33%) and 15 U-PCNSL (54%) achieved complete remission (CR) after primary chemotherapy. Logistic regression confirmed the independent association between CR rate and International Extranodal Lymphoma Study Group score (P = 0.03), RFC promoter methylation (P = 0.07) and use of cytarabine (P = 0.08). The 3-year failure-free survival (FFS) and overall survival for M-PCNSL and U-PCNSL was 0% vs. 31 +/- 9% (P = 0.34) and 0% vs. 31 +/- 9% (P = 0.35) respectively. This is the first study to assess the methylation status of the RFC promoter in human tumour samples. RFC methylation is more common in PCNSL compared with systemic DLBCL, and is associated with a lower CR rate to HD-MTX-based chemotherapy. If confirmed in prospective trials on PCNSL treated with HD-MTX alone, these data may suggest the necessity for alternative strategies in M-PCNSL considering the increased risk of MTX resistance by tumour cells.

The mutator phenotype caused by defects in the mismatch repair system is observed in a subset of solid neoplasms characterized by widespread microsatellite instability-high (MSI-H). It is known to be very rare in non-Hodgkin lymphomas (NHL), whereas mutator NHL is the most frequent tumor subtype in mismatch repair-deficient mice. By screening a series of 603 human NHL with specific markers of the mutator phenotype, we found here 12 MSI-H cases (12/603, 2%). Of interest, we demonstrated that this phenotype was specifically associated with immunodeficiency-related lymphomas (ID-RL), because it was observed in both posttransplant lymphoproliferative disorders (9/111, 8.1%) and HIV infection-related lymphomas (3/128, 2.3%) but not in a large series of NHL arising in the general population (0/364) (P < 0.0001). The MSI pathway is known to lead to the production of hundreds of abnormal protein neoantigens that are generated in MSI-H neoplasms by frameshift mutations of a number of genes containing coding microsatellite sequences. As expected, MSI-H ID-RL were found to harbor such genetic alterations in 12 target genes with a putative role in lymphomagenesis. The observation that the MSI-H phenotype was restricted to HIV infection-related lymphomas and posttransplant lymphoproliferative disorders suggests the existence of the highly immunogenic mutator pathway as a novel oncogenic process in lymphomagenesis whose role is favored when host immunosurveillance is reduced. Because MSI-H-positive cases were found to be either Epstein-Barr virus-positive or -negative, the mutator pathway should act synergistically or not with this other oncogenic factor, playing an important role in ID-RL.

Novel N-glycosylation sites are introduced by somatic mutation into the V genes of the majority of follicular lymphomas. Sites are positively selected and rare in normal memory B cells, indicating a potential role in tumour survival in the germinal centre (GC). The incidence of c. 40% in diffuse large B-cell lymphomas (DLBCL) parallels the known heterogeneity of the disease. Immunodeficiency-related non-Hodgkin's lymphomas (NHL) include post-transplant lymphoproliferative disorders (PTLD) and acquired immunodeficiency syndrome-related NHL (AIDS-NHL). Most PTLD derive from B cells that carry mutated VH genes and that have completed the GC reaction. All AIDS-NHL carry mutated VH genes and variable features of GC or post-GC cells. To determine if N-glycosylation is a feature of immunodeficiency-related lymphomas, we analysed the VH genes of 19 PTLD and 36 AIDS-NHL. Novel sites were rare in PTLD (4/19), similar to memory B cells (P = 0.15). AIDS-NHL, including DLBCL and Burkitt's lymphomas (BL), showed heterogeneity with 16 of 36 (44%) having novel sites. The findings indicate no selection of N-glycosylation sites in PTLD, consistent with post-GC features. The variable incidence of N-glycosylation sites in AIDS-NHL mirrors that in DLBCL and sporadic BL of immunocompetent hosts, supporting the known heterogeneity of these disorders, and possibly pointing to distinct routes of tumour development.

Background and Objectives: Aberrant promoter methylation targets CpG islands causing gene silencing. We explored aberrant promoter methylation of genes potentially involved in B-cell malignancies and encoding proteins implicated in DNA repair (O6-methylguanine-DNA methyltransferase, MGMT), detoxification of environmental xenobiotics (glutathione S-transferase P1, GSTP1), apoptosis regulation (death associated protein kinase, DAP-k and caspase 8, CASP8) and cell cycle control (p73)., Design and Methods: Three hundred and seventeen B-cell malignancies were investigated by methylation-specific polymerase chain reaction (MSP) of MGMT, GSTP1, DAP-k, CASP8 and p73 genes. In selected cases, MSP results were matched to protein expression studies by immunohistochemistry or Western blotting., Results: DAP-k promoter methylation occurred at highest frequency in follicular lymphoma (85.0%) and MALT-lymphoma (72.2%). MGMT methylation targeted both precursor B-cell neoplasia (23.8%) and mature B-cell tumors (27.6%). GSTP1 methylation was commonest in hairy cell leukemia (75.0%), follicular lymphoma (55.5%), Burkitt s lymphoma (52.0%), and MALT lymphoma (50.0%). Methylation of p73 and CASP8 was rare or absent. DAP-k and MGMT methylation caused absent protein expression., Interpretation and Conclusions: Methylation of MGMT, DAP-k and GSTP1 represents a major pathogenetic event in several B-cell malignancies. In follicular lymphoma and MALT lymphoma, frequent inactivation of the apoptosis extrinsic pathway through DAP-k methylation may reinforce the survival advantage already conferred by deregulation of the intrinsic apoptotic pathway. Inactivation of GSTP1 in gastric MALT lymphoma represents an additional mechanism favoring accumulation of reactive oxygen species and lymphomagenesis. Finally, the frequency of GSTP1 aberrant methylation in diffuse large B-cell lymphoma prompts studies aimed at verifying the prognostic impact of this epigenetic lesion in these lymphomas.

Aim: Post-transplant lymphoproliferative disorders (PTLD) represent a serious complication of solid organ transplantation. Despite several advances in the biological categorization of PTLD, current classifications are not fully predictive of the clinical behavior of the disease. This study assessed a comprehensive molecular analysis of the clinico-pathologic spectrum of PTLD in order to better clarify the physiopathology of these disorders., Methods: Fifty-two monoclonal PTLD were investigated for: 1). somatic hypermutation of IgV genes by direct sequencing of IgV rearrangements; 2). expression of BCL6, MUM1 and CD138 proteins by immunohistochemistry; 3). aberrant hypermethylation of DAP-kinase gene by methylation-specific polymerase chain reaction (PCR); 4). genotypic characterization of Epstein Barr virus (EBV) in EBV infected PTLD by PCR analysis of the prevalence of deletions in the carboxyterminal portion of the LMP1 gene and for the definition of type-1/type-2 EBV infection., Results: We report that virtually all monoclonal PTLD originate from B cells that have experienced the germinal center (GC) reaction reflecting different stages of mature B cell differentiation and that tumor development seems frequently associated with EBV and/or other molecular lesions preventing apoptosis of cells that have failed the physiological process of germinal center reaction., Conclusion: To date, classification of PTLD is mainly based on morphology and conventional immunophenotyping. Because current classification schemes are not fully predictive of prognosis, knowledge of PTLD histogenesis and pathogenesis may potentially contribute to refine the distinction of PTLD into more homogeneous categories with prognostic relevance.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a profound dysregulation of the host's immune system at both the humoral and cellular level. We investigated to see if this dysregulation could be due partly to thymus dysfunction by quantifying the number of signal joint T-cell receptor excision circles (sjTRECs) in peripheral blood mononuclear cells of 30 untreated B-CLL patients at diagnosis and in age-matched healthy controls. sjTRECs were found decreased, normal and elevated in 19, 9 and 2 patients, respectively, in comparison to age-matched controls. We next speculated that sjTREC levels might be related to an accredited B-CLL prognostic marker represented by the somatic hypermutation (SHM) status of the variable heavy chain (V(H)) genes. Eight of 17 patients with SHMs had sjTREC levels in the range of or higher than normal donors, whereas only 3 of the 13 patients lacking SHMs had normal sjTREC levels. After a 5-year observation period in 16 patients for whom a clinical follow-up was available, only 2 of 10 patients with SHMs progressed vs. 5 of 6 patients without SHMs and 3 of 7 patients with normal or higher sjTREC levels progressed vs. 4 of 9 with low sjTREC levels. Our study demonstrates that sjTREC levels are decreased in >60% of B-CLL patients and suggests a potential role of thymus in the immune dysfunction of these patients., (Copyright 2003 Wiley-Liss, Inc.)

Aberrant promoter hypermethylation is a mechanism of tumour suppressor gene inactivation. We explored aberrant promoter hypermethylation of multiple genes in 88 human immunodeficiency virus (HIV)-non Hodgkin lymphomas (NHL), 25 post-transplant lymphoproliferative disorders (PTLD) and five common variable immunodeficiency (CVI)-related NHL. Twenty-six of 79 (32.9%) HIV-NHL, eight of 14 (57.1%) PTLD and two of five (40.0%) CVI-NHL showed aberrant hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT). Aberrant hypermethylation of death-associated protein-kinase (DAP-K) occurred in 70 of 84 (83.3%) HIV-NHL, 19 of 25 (72.0%) PTLD and three of five (60.0%) CVI-NHL. These data implicate MGMT and DAP-K hypermethylation in lymphomagenesis of immunodeficient hosts. In particular, promoter hypermethylation of DAP-K represents the most frequent molecular alteration yet identified in immunodeficiency-related lymphomas.

The pathogenesis of AIDS-related non-Hodgkin lymphomas (AIDS-NHLs) is associated with chromosomal translocations that deregulate the expression of various oncogenes. Recently, a novel mechanism of genetic lesion, termed aberrant hypermutation, has been identified in diffuse large B-cell lymphoma (DLBCL) of immunocompetent hosts. In these tumors, the somatic hypermutation (SHM) process that normally targets immunoglobulin V (IgV) genes in B cells appears to misfire and causes mutations in the 5' sequences of multiple proto-oncogenes, including PIM-1, PAX-5, RhoH/TTF, and c-MYC. To investigate whether aberrant hypermutation occurs also in AIDS-NHL, we studied the mutation profile of these 4 genes in various histologic subtypes. Mutations in 1 gene or more were detected in 19 of 39 (48.7%) AIDS-NHL cases (10 of 18 AIDS-diffuse large B-cell lymphoma; 4 of 11 AIDS-Burkitt lymphoma; 4 of 6 AIDS-primary effusion lymphoma; 1 of 4 AIDS-primary central nervous system lymphoma), with 9 of 39 (23.1%) cases carrying mutations in 2 or more genes. Overall, PIM-1 was mutated in 5 of 39 (12.8%), PAX-5 in 8 of 39 (20.5%), RhoH/TTF in 9 of 39 (23.1%), and c-MYC in 7 of 27 (25.9%) AIDS-NHL cases. Mutations were represented mainly by single base pair substitutions (n = 63) with rare deletions/insertions (n = 5) and displayed features typical of the IgV-associated SHM process. In addition, a number of mutations in PIM-1 and c-MYC were found to affect coding exons, leading to amino acid substitutions with likely functional consequences. Analysis of intraclonal heterogeneity documented that the aberrant hypermutation activity may be ongoing in at least some cases. These data indicate that aberrant hypermutation is associated with various subtypes of AIDS-NHL and may represent a major contributor to their pathogenesis.

Different biologic features have been associated with a more or less aggressive clinical course in chronic lymphocytic leukemia (CLL). In the present study, 20 patients with highly stable CLL observed at a single institution over a period of 10 to 23 years and who never required treatment were extensively characterized. The aim was to identify a distinct and reproducible biologic profile associated with disease stability that may be used to recognize at presentation CLL patients who are likely to have a very benign clinical course and for whom treatment is not indicated. The results obtained indicate that numerous parameters are closely associated with disease stability: a typical CLL morphology and immunophenotype, the lack of expression of the CD38 antigen, the mutated immunoglobulin (Ig) heavy (H) chain variable (V) pattern, the absence of p53 mutations, a CD4/CD8 ratio more than 1, the lack of 17p and 11q deletions and of complex karyotypic aberrations, and the occurrence of the 13q14 deletion. No case displayed the VH3-21 gene, linked in mutated CLL with a poor outcome. In addition, the VH1-69 gene associated with unmutated CLL cases was never detected. These biologic features were coupled with an indolent clinical course characterized by an unmodified clinical stage over time, and by lack of autoimmune phenomena and of major infections requiring parental antibiotics. At a time when aggressive therapeutic strategies are always more frequently used in the management of CLL, the distinctive features of patients with long-lived stable disease should be prospectively identified at presentation.

The transduction of Notch signal plays an intricate role in cell differentiation and pathogenesis of haematological malignancies as well as in certain congenital conditions. We found no genomic changes in either gene in 34 leukaemic samples and 25 leukaemia and lymphoma cell lines. The functionality of Notch signalling was tested using HES1 gene activation. We show that Notch signalling is differentially regulated in T-acute lymphoblastic leukaemia (ALL) and B-lymphoma cells. The Notch pathway is intact in a majority of B-lymphoma cell lines, but EBNA2, which mimics notch function, can occasionally activate the pathway. In contrast, the Notch pathway is constitutively active in T-ALL. This is the first demonstration of a distinction between B-lymphomas and T-cell leukaemias in the functioning of the Notch-signalling pathway. This might be related to their pathogenesis., (Copyright 2002 Wiley-Liss, Inc.)