Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies.

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.

Evidence for the significant role of immunoglobulin light chains in antigen recognition and selection in chronic lymphocytic leukemia.

We analyzed somatic hypermutation (SHM) patterns and secondary rearrangements involving the immunoglobulin (IG) light chain (LC) gene loci in 725 patients with chronic lymphocytic leukemia (CLL). Important differences regarding mutational load and targeting were identified in groups of sequences defined by IGKV/IGLV gene usage and/or K/LCDR3 features. Recurrent amino acid (AA) changes in the IGKV/IGLV sequences were observed in subsets of CLL cases with stereotyped B-cell receptors (BCRs), especially those expressing IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 BCRs. Comparison with CLL LC sequences carrying heterogeneous K/LCDR3s or non-CLL LC sequences revealed that distinct amino acid changes appear to be "CLL-biased." Finally, a significant proportion of CLL cases with monotypic LC expression were found to carry multiple potentially functional LC rearrangements, alluding to active, (auto)antigen-driven receptor editing. In conclusion, SHM targeting in CLL LCs is just as precise and, likely, functionally driven as in heavy chains. Secondary LC gene rearrangements and subset-biased mutations in CLL LC genes are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains. Therefore, CLL is characterized not only by stereotyped HCDR3 and heavy chains but, rather, by stereotyped BCRs involving both chains, which generate distinctive antigen-binding grooves.

Toll-like receptor signaling pathway in chronic lymphocytic leukemia: distinct gene expression profiles of potential pathogenic significance in specific subsets of patients.

BACKGROUND: Signaling through the B-cell receptor appears to be a major contributor to the pathogenesis of chronic lymphocytic leukemia. Toll-like receptors bridge the innate and adaptive immune responses by acting as co-stimulatory signals for B cells. The available data on the expression of Toll-like receptors in chronic lymphocytic leukemia are limited and derive from small series of patients. DESIGN AND METHODS: We profiled the expression of genes associated with Toll-like receptor signaling pathways in 192 cases of chronic lymphocytic leukemia and explored potential associations with molecular features of the clonotypic B-cell receptors. RESULTS: Chronic lymphocytic leukemia cells express all Toll-like receptors expressed by normal activated B cells, with high expression of TLR7 and CD180, intermediate expression of TLR1, TLR6, TLR10 and low expression of TLR2 and TLR9. The vast majority of adaptors, effectors and members of the NFKB, JNK/p38, NF/IL6 and IRF pathways are intermediately-to-highly expressed, while inhibitors of Toll-like receptor activity are generally low-to-undetectable, indicating that the Toll-like receptor-signaling framework is competent in chronic lymphocytic leukemia. Significant differences were identified for selected genes between cases carrying mutated or unmutated IGHV genes or assigned to different subsets with stereotyped B-cell receptors. The differentially expressed molecules include receptors, NFκB/MAPK signaling molecules and final targets of the cascade. CONCLUSIONS: The observed variations are suggestive of distinctive activation patterns of the Toll-like receptor signaling pathway in subgroups of cases of chronic lymphocytic leukemia defined by the molecular features of B-cell receptors. Additionally, they indicate that different or concomitant signals acting through receptors other than the B-cell receptor can affect the behavior of the malignant clone.

Activation-induced cytidine deaminase splicing patterns in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) is critically implicated in somatic hypermutation (SHM) and class switch recombination (CSR). AID is expressed as a native transcript and as several splice variants, with as yet undefined roles. Chronic lymphocytic leukemia (CLL) leukemic B cells have also been shown to express AID transcripts, especially in cases with unmutated immunoglobulin (IG) genes. Therefore, AID expression in CLL might potentially be relevant to the disease. The available data on AID-mRNA splicing patterns in CLL are limited and conflicting. Here, we investigated AID-mRNA isoform expression in a series of 195 CLL patients and explored associations with IG gene mutational status and surface immunoglobulin (sIg) isotype expression. Full-length AID transcripts and two splice variants were detected in 110/91/95 cases, respectively. Co-expression of all three AID-mRNA isoforms was significantly more frequent (p<0.001) in cases with unmutated IGHV genes. No significant differences were identified between sIgG vs. sIgMD cases regarding the frequency of AID-mRNA expression. However, expression of at least one AID-mRNA isoform predominated among mutated IgG vs. mutated IgMD cases (p=0.05). These results attest to the biological heterogeneity of CLL and also indicate that AID splice variants may inhibit SHM in CLL cells of the unmutated subtype.

Immunoglobulin kappa gene repertoire and somatic hypermutation patterns in follicular lymphoma.

Immunoglobulin kappa gene usage and somatic mutation patterns were studied in a series of 47 IGKV-J rearrangements amplified in 42 follicular lymphoma (FL) cases. The IGKV1-39/1D-39 gene predominated and was significantly over-represented compared to normal cells, autoreactive cells or other B cell lymphomas. The impact of somatic hypermutation varied significantly; nevertheless, mutation distribution patterns indicated pressure for preservation of the B cell receptor. In conclusion, the present series demonstrates biased usage of IGKV genes in FL and alludes to the important role of immunoglobulin kappa light chains in antigen selection of the clonogenic B cells in FL.

Predominantly post-transcriptional regulation of activation molecules in chronic lymphocytic leukemia: the case of transferrin receptors.

Transcriptional and post-transcriptional control mechanisms have a differential impact on cellular physiology depending on activation status. Several lines of evidence suggest that chronic lymphocytic leukemia (CLL) malignant B cells resemble antigen-experienced and activated B cells. In the present study, we investigated the expression of transferrin receptor 1 (TfR1, CD71), one of the "classical" markers up-regulated upon B-cell activation, and TfR2, a novel receptor for transferrin, in peripheral blood CD19+ B cells from ten healthy individuals and 76 patients with CLL so as to gain insight into potential disease-related differences in underlying regulatory mechanisms. Marked differences in the production and expression of these receptors were detected in malignant but not in normal B cells. Specifically, TfR1 mRNA and protein levels were significantly higher in comparison to TfR2, both in normal and malignant B cells. Furthermore, discrepancies between TfR mRNA and protein expression were observed in CLL; in contrast, mRNA and protein expression levels were generally concordant in normal B cells. Exposure to actinomycin D decreased TfR1 and TfR2 mRNA levels in normal CD19+ B cells but had no effect on CLL malignant cells. The protein synthesis inhibitor cycloheximide had opposing effects in normal vs. CLL malignant B cells: thus, TfR1 and TfR2 mRNA levels were increased in normal B cells, whereas they were unaffected or even suppressed in CLL malignant B cells. These results allude to differential regulation of TfR1 and TfR2 expression in normal B cells vs. CLL. In normal B cells, transcriptional mechanisms exert a critical control over TfR1 and TfR2 expression, whereas in CLL post-transcriptional mechanisms seem to play a complementary and perhaps more important role. This type of control appears to be especially suited for modulation of genes implicated in proliferation of activated cells, like CLL malignant B cells.

Splenic marginal-zone lymphoma: one or more entities? A histologic, immunohistochemical, and molecular study of 42 cases.

We analyzed 42 splenic marginal-zone lymphoma (SMZL) cases diagnosed on splenectomy specimens after established World Health Organization criteria. A predominantly nodular growth pattern was observed in 24 cases; the remainder showed predominantly (11/42) or exclusively (7/42) diffuse infiltration. Twenty-one cases showed the "classic" biphasic appearance; 13 cases exhibited marginal-zone morphology; finally, 8 cases were composed predominantly of small cells. CD21 and CD35 were expressed by 12/42 and 17/38 cases, respectively. DBA.44 was detected in 24/42 cases. Seventeen of 37 cases were surface IgD (SIgD)-positive. Twenty-one of 22 analyzed cases were SIgM-positive (12/21 coexpressed SIgD). Five of 37 cases were SIgG-positive. CD27 staining was observed in 21/35 cases; 7/18 CD27-positive cases coexpressed SIgD; 7/14 CD27-negative cases were SIgD-positive. Forty IGHV-D-J rearrangements were amplified in 34/42 cases: the IGHV4-34 gene predominated, followed by IGHV1-2. Using the 98% homology cut-off, 25/40 (62.5%) IGHV sequences were considered as "mutated": 10/11 cases with monomorphous, marginal-zone morphology were IGHV-mutated; in contrast, 4/6 cases with monomorphous, small-cell morphology were IGHV-unmutated. Five of 7 cases expressing IGHV1 subgroup genes had biphasic morphology, whereas 6/9 IGHV3-expressing cases had monomorphous, marginal-zone morphology. Most IGHV-mutated cases (14/20; 70%) were SIgD-negative; in contrast, 8/11 IGHV-unmutated cases expressed SIgD. CD27 was detected in 10/17 IGHV-mutated and 6/10 IGHV-unmutated cases. Seven of 11 CD27-negative cases were IGHV-mutated; 5/7 CD27-negative/IGHV-mutated cases expressed DBA.44. These results confirm the considerable histologic, immunohistochemical, and molecular heterogeneity of SMZL and indicate an origin from the diverse resident B-cell populations of the normal SMZ.

Transferrin receptor-1 and 2 expression in chronic lymphocytic leukemia.

Transferrin receptor (TfR)-1 and 2 mRNA and CD71 (TfR1) expression was analyzed in 118 CLL patients. Ninety-five out of 109 analyzed cases expressed CD71, mostly at a high level; 60% of CD71 (+) cases were IGH-mutated. All samples were TfR1 mRNA (+); TfR2-alpha/beta mRNA was detected in, respectively, 52/102 and 100/109 cases. Competitive RT-PCR showed widely divergent levels of TfR1 mRNA in cases with high CD71 expression, alluding to post-transcriptional control of TfR1 expression in CLL. The almost uniformly high CD71 expression in CLL is in keeping with the activated status of neoplastic cells, regardless of IGH mutational load.

Immunoglobulin genes in multiple myeloma: expressed and non-expressed repertoires, heavy and light chain pairings and somatic mutation patterns in a series of 101 cases.

BACKGROUND AND OBJECTIVES: The available data on the immunoglobulin gene (IG) repertoire in multiple myeloma (MM) derive mainly from heavy chains; considerably less is known about light chains. We assessed in parallel IGH and IGK/IGL rearrangements in 101 MM patients so as to gain insight into: (i) IG repertoires; (ii) antigen impact; (iii) the role of receptor editing. DESIGN AND METHODS: Bone marrow aspirates were collected from all cases. IGHV-(D)-J and IGLV-J rearrangements were amplified by reverse transcriptase polymerase chain reaction (PCR). In all cases, IGKV-J rearrangements were analyzed in parallel on cDNA/genomic DNA. IGKV-KDE and IGKJ-C-INTRON-KDE were also amplified by DNA-PCR. RT-PCR products were directly sequenced. RESULTS: IGHV3 genes predominated; the IGHV4-34 gene was used in only one case. Five IGKV and five IGLV genes accounted for the majority of in-frame, transcribed IGKV-J or IGLV-J rearrangements. Taking IGKV-J, IGKV-KDE and IGKJ-C-INTRON-KDE rearrangements together, biallelic IGK locus rearrangements were detected in 22/43 k-MM cases. In l-MM, 36/42 cases had at least one rearranged IGK allele; 8/19 IGKV-J rearrangements in l-MM were in-frame. All in-frame, transcribed IGH/IGK/IGL sequences were mutated; parallel heavy/light chain analysis demonstrated a comparable impact of somatic hypermutation. INTERPRETATION AND CONCLUSIONS: Biases in IG repertoire did not seem disease-related but followed a similar pattern to that of the normal repertoire. The under-representation of the IGHV4-34 gene provides an explanation for the paucity of autoimmune phenomena in MM. Somatic mutation patterns indicate the complementary role of MM IGH/IGK/IGL sequences in antigen recognition. Finally, the frequent inactivation of productive IGKV-J joints by secondary rearrangements in MM suggests active receptor editing.

Myocardial infarction under the age of 36: prevalence of thrombophilic disorders.

It has been suggested that thrombotic tendency increases the risk of myocardial infarction (MI). To investigate the association between the risk of MI at a young age and genetic thrombogenic disorders (G20210A mutation in the prothrombin gene, G1691A mutation in the factor V gene and deficiencies of protein C, protein S and antithrombin III) we conducted a case-control study among 70 survivors of MI who had experienced the event before the age of 36 and 260 healthy subjects. The G20210A mutation in the prothrombin gene was found more often in young patients with MI than among controls (11.4 versus 3.1%). The odds ratio (OR) for MI for carriers versus non-carriers was 4 (95% confidence interval [CI], 1.5 to 11.3). The adjusted OR for major cardiovascular risk factors (smoking, hypecholesterolaemia, diabetes mellitus, hypertension and obesity) was 4.3 (95% CI, 1.3 to 14). The simultaneous presence of both G20210A mutation in the prothrombin gene and smoking further increased the risk of MI compared with nonsmokers and non-carriers (OR, 58; 95% CI, 11.4-294). The G1691A mutation in factor V gene was not associated with an increased relative risk for MI (OR, 0.87; 95% CI, 0.26 to 2.5). Finally, there was no significant difference in the prevalence of deficiencies of protein C, protein S and antithrombin III between cases and controls. In conclusion, our data indicate that the G20210A mutation in the prothrombin gene was the only genetic prothrombotic risk factor associated with the risk of developing MI under the age of 36 years.

Prognostic value of C-reactive protein, fibrinogen, interleukin-6, and macrophage colony stimulating factor in severe unstable angina.

BACKGROUND: Inflammatory process plays an important role in the pathogenesis of acute coronary syndromes. HYPOTHESIS: The study was undertaken to evaluate whether admission levels of C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6). and macrophage colony stimulating factor (MCSF) can predict short-term prognosis in patients with unstable angina. METHODS: C-reactive protein, fibrinogen, IL-6, and MCSF were measured on admission in 141 consecutive patients, aged 59 +/- 10 years, with unstable angina (Braunwald class IIIb). Patients were divided into two groups according to their in-hospital outcome: Group 1 comprised 77 patients with a complicated course (2 died, 15 developed nonfatal myocardial infarction, and 60 had recurrence of angina), and Group 2 comprised 64 patients with an uneventful course. RESULTS: Admission median levels of CRP (8.8 vs. 3.1 mg/l, p = 0.0002). fibrinogen (392 vs. 340 mg/dl, p = 0.008), IL-6 (8.8 vs. 4.5 pg/ml, p = 0.03), and MCSF (434 vs. 307 pg/ml, p = 0.0001) were higher in Group I than in Group 2. The MCSF levels were an independent risk factor for in-hospital events, with an adjusted odds ratio for eventful in-hospital outcome of 3.3 (95% confidence interval 1-10.9, p = 0.04), and correlated with levels of IL-6 (r(s) = 0.52, p = 0.0001), CRP (r(s) = 0.43, p = 0.0001), and fibrinogen (r(s) = 0.25, p = 0.004). CONCLUSIONS: These findings suggest that among the studied inflammatory indices only increased admission levels of MCSF are strongly and independently related with adverse short-term prognosis in patients with severe unstable angina.

Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis.

Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, "stereotyped" amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: Pathogenetic implications and clinical correlations.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is biased and characterized by the existence of subsets of cases with closely homologous ("stereotyped") complementarity-determining region 3 (CDR3) sequences. In the present series, 201 (21.9%) of 916 patients with CLL expressed IGHV genes that belonged to 1 of 48 different subsets of sequences with stereotyped heavy chain (H) CDR3. Twenty-six subsets comprised 3 or more sequences and were considered "confirmed." The remaining subsets comprised pairs of sequences and were considered "potential"; public database CLL sequences were found to be members of 9 of 22 "potential" subsets, thereby allowing us to consider them also "confirmed." The chance of belonging to a subset exceeded 35% for unmutated or selected IGHV genes (eg, IGHV1-69/3-21/4-39). Comparison to non-CLL public database sequences showed that HCDR3 restriction is "CLL-related." CLL cases with selected stereotyped immunoglobulins (IGs) were also found to share unique biologic and clinical features. In particular, cases expressing stereotyped IGHV4-39/IGKV1-39-1D-39 and IGHV4-34/IGKV2-30 were always IgG-switched. In addition, IGHV4-34/IGKV2-30 patients were younger and followed a strikingly indolent disease, contrasting other patients (eg, those expressing IGHV3-21/IGLV3-21) who experienced an aggressive disease, regardless of IGHV mutations. These findings suggest that a particular antigen-binding site can be critical in determining the clinical features and outcome for at least some CLL patients.

IGHV gene insertions and deletions in chronic lymphocytic leukemia: "CLL-biased" deletions in a subset of cases with stereotyped receptors.

Nucleotide insertions/duplications or deletions in immunoglobulin heavy chain genes have been found in 24/760 patients (3.15%) with chronic lymphocytic leukemia (CLL). In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. The pattern and location of insertions/duplications or deletions in CLL and their restriction to mutated IGHV rearranged genes strongly suggests that they resulted from somatic hypermutation. Their incidence in CLL is consistent with previous reports in normal, auto-reactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease-specific associations. A striking exception to this rule was identified in CLL IGHV3-21-expressing cases: one amino acid was deleted from the CDR2 region in 16/63 (25.4%) mutated CLL IGHV3-21 sequences (including public database-derived IGHV3-21 CLL cases + the present series) vs. only 2/257 (0.78%) public database-derived mutated non-CLL IGHV3-21 sequences; 15/16 CLL IGHV3-21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3 and light chain CDR3 motifs. This finding further supports the idea of selective antigenic pressures playing a pathogenetic role in some CLL cases.

Analysis of expressed and non-expressed IGK locus rearrangements in chronic lymphocytic leukemia.

Immunoglobulin kappa (IGK) locus rearrangements were analyzed in parallel on cDNA/genomic DNA in 188 kappa- and 103 lambda-chronic lymphocytic leukemia (CLL) cases. IGKV-KDE and IGKJ-C-intron-KDE rearrangements were also analyzed on genomic DNA. In kappa-CLL, only 3 of 188 cases carried double in-frame IGKV-J transcripts: in such cases, the possibility that leukemic cells expressed more than one kappa chain cannot be excluded. Twenty-eight kappa-CLL cases also carried nonexpressed (nontranscribed and/or out-of-frame) IGKV-J rearrangements. Taking IGKV-J, IGKV-KDE, and IGKJ-C-intron-KDE rearrangements together, 38% of kappa-CLL cases carried biallelic IGK locus rearrangements. In lambda-CLL, 69 IGKV-J rearrangements were detected in 64 of 103 cases (62%); 24 rearrangements (38.2%) were in-frame. Four cases carried in-frame IGKV-J transcripts but retained monotypic light-chain expression, suggesting posttranscriptional regulation of allelic exclusion. In all, taking IGKV-J, IGKV-KDE, and IGKJ-C-intron-KDE rearrangements together, 97% of lambda-CLL cases had at least 1 rearranged IGK allele, in keeping with normal cells. IG repertoire comparisons in kappa- versus lambda-CLL revealed that CLL precursor cells tried many rearrangements on the same IGK allele before they became lambda producers. Thirteen of 28 and 26 of 69 non-expressed sequences in, respectively, kappa- or lambda-CLL had < 100% homology to germline. This finding might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in some cases. The inactivation of potentially functional IGKV-J joints by secondary rearrangements indicates active receptor editing in CLL and provides further evidence for the role of antigen in CLL immunopathogenesis.

Immunoglobulin light chain repertoire in chronic lymphocytic leukemia.

Immunoglobulin kappa (IGK) and immunoglobulin lambda (IGL) light chain repertoire was analyzed in 276 chronic lymphocytic leukemia (CLL) cases and compared with the relevant repertoires from normal, autoreactive, and neoplastic cells. Twenty-one functional IGKV genes were used in IGKV-J rearrangements of 179 kappa-CLL cases; the most frequent genes were IGKV3-20(A27), IGKV1-39/1D-39(O2/O12), IGKV1-5(L12), IGKV4-1(B3), and IGKV2-30(A17); 90 (50.3%) of 179 IGK sequences were mutated (similarity < 98%). Twenty functional IGLV genes were used in IGLV-J rearrangements of 97 lambda-CLL cases; the most frequent genes were IGLV3-21(VL2-14), IGLV2-8(VL1-2), and IGLV2-14(VL1-4); 44 of 97 IGL sequences (45.4%) were mutated. Subsets with "CLL-biased" homologous complementarity-determining region 3 (CDR3) were identified: (1) IGKV2-30-IGKJ2, 7 sequences with homologous kappa CDR3 (KCDR3), 5 of 7 associated with homologous IGHV4-34 heavy chains; (2) IGKV1-39/1D-39-IGKJ1/4, 4 unmutated sequences with homologous KCDR3, 2 of 4 associated with homologous IGHV4-39 heavy chains; (3) IGKV1-5-IGKJ1/3, 4 sequences with homologous KCDR3, 2 of 4 associated with unmutated nonhomologous IGHV4-39 heavy chains; (4) IGLV1-44-IGLJ2/3, 2 sequences with homologous lambda CDR3 (LCDR3), associated with homologous IGHV4-b heavy chains; and (5) IGLV3-21-IGLJ2/3, 9 sequences with homologous LCDR3, 3 of 9 associated with homologous IGHV3-21 heavy chains. The existence of subsets that comprise given IGKV-J/IGLV-J domains associated with IGHV-D-J domains that display homologous CDR3 provides further evidence for the role of antigen in CLL pathogenesis.

Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: the lesson of the IGHV3-21 gene.

We studied immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in 553 patients with chronic lymphocytic leukemia (CLL) from the Mediterranean area to gain insight into the potential pathogenetic role of antigenic stimulation. The most commonly represented IGHV genes mirrored the usage of normal B cells, with the exception of IGHV1-18, IGHV3-30.3, and IGHV4-59 that were underrepresented. The IGHV3-21 gene, frequently expressed in Northern European CLL, was present only in 16 cases (2.9%). Based on HCDR3 cluster analysis, cases using IGHV3-21 could be grouped in 2 subsets of similar frequency. The first one (7 of 16 cases) carried a similar HCDR3 amino acid sequence (common-HCDR3 subset), virtually identical to the Scandinavian IGHV3-21 CLL. These cases used the IGHJ6 gene; 4 of 7 were unmutated; 6 of 7 carried the V(lambda)2-14 (IGLV3-21) light-chain gene with a similar LCDR3. All expressed CD38 and had a progressive disease. The second subset (9 of 16) was characterized by heterogeneous HCDR3 rearrangements (nonhomogeneous-HCDR3 subset), diverse IGHJ and IGV light-chain gene usage, variable IGHV mutational status (5 of 9 unmutated), variable CD38 expression, and variable clinical course (4 of 9 progressed). The first subset suggests a potential antigenic element rarely encountered in the Mediterranean area, possibly responsible for a negative outcome. The second subset may reflect the physiologic heterogeneity of expression of IGHV3-21 rearrangements in the normal repertoire and is characterized by a variable clinical outcome.

Prognostic impact of DNA ploidy pattern, S-phase fraction (SPF), and proliferating cell nuclear antigen (PCNA) in patients with primary gastric lymphoma.

BACKGROUND: DNA ploidy, S-phase fraction (SPF), and proliferating cell nuclear antigen (PCNA) are considered to be significant prognostic factors in non-Hodgkin lymphomas. However, reports on their prognostic importance in gastric lymphoma patients are relatively lacking. METHODS: In the present study, we retrospectively studied the above-mentioned parameters in 29 patients with primary gastric lymphoma; 11/29 had B-low grade mucosa associated lymphoid tissue lymphoma (B-MALT), while 18/29 had diffuse large B-cell lymphoma (DLBCL), according to WHO classification. Proliferative activity was studied by staining against PCNA; in addition, the prognostic significance of DNA ploidy and SPF, as determined by flow cytometry, were investigated and compared to the results of the PCNA stainings. RESULTS: Seven out of 29 patients were found to have aneuploid tumors; DNA index values were >1 for all aneuploid lymphomas. There was no difference in DNA aneuploidy in MALT vs. DLBCL. The mean percentage of SPF was 11.4. SPF was found significantly lower in MALT vs. DLBCL (P < 0.05). The mean percentage of PCNA positive tumor cells was 52.6. PCNA protein expression was significantly lower in MALT vs. DLBCL (P < 0.0001). There was a significant positive correlation between PCNA score and SPF (P < 0.01, by Spearman analysis). DNA ploidy had no impact on survival in the present study. Both SPF and PCNA expression were important prognostic factors in the univariate analysis; however, in the multivariate analysis, the only independent prognostic factor for survival was PCNA expression. CONCLUSIONS: These findings indicate that SPF and PCNA are significant prognostic factors in patients with primary gastric lymphomas. However, in the present study, DNA ploidy had no impact on survival in patients with primary gastric lymphomas.

Immunoglobulin heavy- and light-chain repertoire in splenic marginal zone lymphoma.

The considerable heterogeneity in morphology, immunophenotype, genotype, and clinical behavior of splenic marginal zone lymphoma (SMZL) hinders firm conclusions on the origin and differentiation stage of the neoplastic cells. Immunoglobulin (IG) gene usage and somatic mutation patterns were studied in a series of 43 SMZL cases. Clonal IGHV-D-J rearrangements were amplified in 42/43 cases (4 cases carried double rearrangements). Among IGHV-D-J rearrangements, IGHV3 and IGHV4 subgroup genes were used with the highest frequency. Nineteen IGHV genes were unmutated (> 98% homology to the closest germline IGHV gene), whereas 27/46 were mutated. Clonal IGKV-J and IGLV-J gene rearrangements were amplified in 36/43 cases, including 31 IGKV-J (8/31 in lambda light-chain expressing cases) and 12 IGLV-J rearrangements; 9/31 IGKV and 6/12 IGLV sequences were mutated. IGKV-J and IGLV-J rearrangements used 14 IGKV and 9 IGLV different germline genes. Significant evidence for positive selection by classical T-dependent antigen was found in only 5/27 IGHV and 6/15 IGKV+IGLV mutated genes. These results provide evidence for the diverse B-cell subpopulations residing in the SMZ, which could represent physiologic equivalents of distinct SMZL subtypes. Furthermore, they indicate that in SMZL, as in other B cell malignancies, a complementarity imprint of antigen selection might be witnessed either by IGHV, IGKV, or IGLV rearranged sequences.

Prolonged administration of erythropoietin increases erythroid response rate in myelodysplastic syndromes: a phase II trial in 281 patients.

Treatment with recombinant human erythropoietin (rHuEpo) improves anaemia in approximately 20% of patients with myelodysplastic syndromes (MDS). We investigated the potential advantage of a prolonged administration of rHuEpo to achieve higher erythroid response rates (RR) in 281 MDS patients: 118 with refractory anaemia (RA), 77 with refractory anaemia and ringed sideroblasts (RARS), 59 with refractory anaemia with excess of blasts and blast count < 10% (RAEB-I), and 27 with RAEB and blast count between 11-20% (RAEB-II). rHuEpo was given subcutaneously at a dose of 150 U/kg thrice weekly, for a minimum of 26 weeks. Response to treatment was evaluated after 12 and 26 weeks of therapy. The overall RR was 45.1%; the RR for RA, RARS, RAEB-I and RAEB-II were 48.3%, 58.4%, 33.8% and 13% respectively. A significant increase in RR was observed at week 26 in RA, RARS and RAEB-I patients, as the response probability increased with treatment duration. The RR was higher in the good cytogenetic prognostic group and serum Epo level of > 150 U/l at baseline predicted for non-response. The median duration of response was 68 weeks and the overall risk of leukaemic transformation was 21.7%. These results suggest that prolonged administration of rHuEpo produces high and long-lasting erythroid RR in MDS patients with low blast counts, particularly in those with pretreatment serum Epo levels of < 150 U/l and good cytogenetic prognosis.