Ulf Thunberg

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis.

Diffuse large B-cell lymphoma (DLBCL) has been shown to be comprised of at least two prognostic entities, depending on its resemblance to normal germinal center or activated B cells, using global gene expression profiling. Also, the expression patterns of bcl-6, CD10 and IRF-4 (also known as MUM1) have been suggested as alternative means of identifying the germinal- and nongerminal center (activated B-cell like) groups. In the present study, we evaluated by immunohistochemistry the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 in a large material of 161 DLBCL patients. Using two different approaches, patients with germinal center phenotype displayed a significantly better survival than the nongerminal center group. Positive staining for bcl-6 or CD10 predicted for superior survival, while expression of IRF-4 alone showed no association with prognosis. Furthermore, expression of bcl-2 was associated with worse event-free survival and overall survival. In a multivariate analysis, a high international prognostic index score (3–5), non-GC phenotype and bcl-2 were independent adverse prognostic factors. Here we confirm the prognostic importance of determining the germinal- or nongerminal center phenotype in patients with DLBCL.

Somatic hypermutation and VH gene usage in mantle cell lymphoma

Abstract:  Mantle cell lymphoma (MCL) is considered to derive from naïve, pregerminal center (GC) CD5+ B‐cells. However, the cell of origin has been questioned in recent studies that showed somatic hypermutations in the immunoglobulin (Ig) variable heavy chain (VH) genes in subsets of MCL. To clarify this issue, we analyzed the IgVH genes for the presence of somatic hypermutations in 51 MCL cases. Twenty percent of the MCL cases displayed somatically mutated VH genes (defined as >2% mutated), whereas 80% showed unmutated VH genes. This finding suggests that MCL is a genetically heterogeneous disease, with the majority of cases originating from unmutated pre‐GC B‐cells and a subset deriving from more mature B‐cells which have been exposed to the GC environment and have undergone somatic hypermutation. A biased VH gene usage has been demonstrated in several B‐cell malignancies; however, this has not yet been investigated in MCL, although VH4‐34 overusage has been indicated by small studies. Interestingly, we found a restricted usage of three individual VH genes in our MCL material; VH4‐34 (22%), VH3‐21 (16%) and VH5‐51 (12%). This novel finding of preferential VH gene usage in half of the MCL cases may suggest an antigen driven process occurring in B‐cells expressing specific VH genes, thus implicating that Ig specificity could be involved in mantle cell lymphoma development.

Chromosomal Imbalances in Diffuse Large B-Cell Lymphoma Detected by Comparative Genomic Hybridization

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma. In contrast to many other hematological malignancies, no chromosomal abnormalities with a diagnostic or prognostic value have been identified in DLBCL. Numerical chromosomal imbalances were characterized by comparative genomic hybridization (CGH) performed on 54 DLBCL tumors from a total of 40 patients. The clonal relatedness was demonstrated in 9 of 11 pairs of matched diagnostic tumors and their relapses as determined by IGH gene rearrangement analysis and/or the CGH profiles. Furthermore, immunohistochemical expression analyses of BCL2 and BCL6/LAZ3 were performed on all cases. Copy number changes were detected in 94% of the diagnostic tumor samples and in all of the relapses. Chromosomal losses in diagnostic tumors were preferentially observed at 8p22-pter (29%), 1p34-pter (26%), 6q23-qter (20%), 17p12-pter (17%) and 22q (17%), 9p23-pter (14%), whereas gains were mainly seen in Xq25–26 (43%), 13q22 (26%), 12cen-q14 (20%), 3q24–25 (11%), 7 (11%), and 18q12–21 (11%). Loss of 22q was significantly more commonly seen in the diagnostic tumor samples with more advanced clinical stage in other words, Stage III–IV compared with Stage I–II, and band 18q21 was significantly more often gained in relapses as compared to diagnostic tumors. None of the recurrent alterations were detected as a single abnormality, suggesting that other genetic lesions below the detection level of CGH may be the initiating event in the tumorigenesis of DLBCL. However, the distribution of CGH alterations support the idea of a progression of genetic events where loss of 8p and 9p and gain of 3q, 13q, and 18q would represent relatively early events because they were distributed in tumors with only two abnormalities.

Association between telomere length and V H gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications

The immunoglobulin VH gene mutation status can divide B-cell chronic lymphocytic leukaemia (CLL) into two entities with a different clinical course. Cases with unmutated VH genes, considered to evolve from pregerminal centre (GC) cells, have a worse outcome compared to cases showing mutated VH genes, that is, post-GC derived. Also, telomere length has been reported to be of prognostic significance in CLL. Interestingly, telomerase becomes activated during the GC reaction and an elongation of the telomeres occurs in GC B cells. We performed telomere length and VH gene analysis in a series of 61 CLL cases, in order to investigate if the unique telomere lengthening shown in GC B cells could reflect the telomere status in the two subsets of mutated and unmutated CLL. A novel association was found between VH gene mutation status and telomere length, since significantly shorter telomeres were demonstrated in the unmutated group compared to the mutated group (mean length 4.3 vs 6.3 kbp). Shorter telomeres also constituted a subgroup with a worse prognosis than cases with longer telomeres (median survival 59 vs 159 months). Furthermore, the Ig gene sequence data revealed that samples with high mutations frequency (>6%) had long telomeres (∼8 kbp). Thus, both the telomere and VH gene mutation status in CLL appear linked, which may reflect the proliferative history of the clonal cells with regard to the GC reaction.

Clonal evolution as judged by immunoglobulin heavy chain gene rearrangements in relapsing precursor-B acute lymphoblastic leukemia.

Abstract:  Oligoclonality and ongoing clonal evolution are common features in patients with precursor‐B (pre‐B) acute lymphoblastic leukemia (ALL), as judged by immunoglobulin heavy chain (IgH) gene rearrangement analysis. These features are considered to be results of secondary rearrangements after malignant transformation or emergence of new tumor clones. In the present study we analyzed the IgH gene rearrangement status in 18 cases with relapsing pre‐B ALL using variable heavy chain (VH) gene family specific polymerase chain reaction (PCR) amplification and single stranded conformation polymorphism (SSCP) analysis. Clonal IgH rearrangements were displayed in all leukemias but one, and altered rearrangement patterns occurred in five cases (29%), which were selected for detailed nucleotide sequence analysis. In one case, multiple subclones at diagnosis were suggested to be derived from a progenitor clone through joining of different VH germline gene segments to a pre‐existing D–JH complex (VH to D–JH joining). Evidence for VH gene replacement with identical N‐sequences at the VH‐D junction and a common D–JH region was observed in one case. Diversification at the VH–D junction consisting of heterogeneous N‐sequences were observed in one case. This molecular modification of the VH‐D region could fit a hypothesized “open‐and‐shut” mechanism. Nevertheless, despite these ongoing events at least one IgH rearrangement remained unchanged throughout the disease in most patients, indicating that the immunoglobulin heavy chain locus can be a suitable marker for detection of minimal residual disease (MRD).

Mast cell infiltration is a favourable prognostic factor in diffuse large B-cell lymphoma

Previous studies indicate that the inflammatory response in diffuse large B‐cell lymphomas (DLBCL) is important for the clinical outcome. Mast cells are key regulators in this response; we investigated whether the number of tryptase‐positive mast cells is correlated with clinical outcome. Patients with many mast cells had a significantly better event‐free survival (EFS) compared to those with few mast cells (P < 0·03 in both germinal centre (GC) and non‐GC DLBCL. This supports the idea that the infiltration of mast cells is a reflection of the host inflammatory response and is related to a favourable outcome.

VH3-21 gene usage in chronic lymphocytic leukemia: Characterization of a new subgroup with distinct molecular features and poor survival

During recent years it has become evident that lymphoproliferative diseases of B-cell origin display preferential immunoglobulin (Ig) variable heavy chain (VH) gene usage. For instance, the VH1-69 and VH4-34 genes were early found to be overexpressed in B-cell chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. The implications of biased VH gene usage have been speculated to be a result of stimulation of unknown antigens, which gives increased proliferation of B-cells with certain VH gene configuration and consequently higher probability to undergo transformation. Thus, VH gene usage may play a role in development of leukemias and lymphomas. Recently, we could confirm the over usage of the VH1-69 and VH4-34 genes in CLL, but a novel finding was that the VH3-21 gene was preferentially utilized in CLL patients with mutated VH genes. These VH3-21+ Ig rearrangements showed molecular peculiarities such as shorter lengths of the third complementarity determining region (CDR) and had similar amino acid composition of their CDR3s, implicating recognition of the same antigen in individual tumors. Most of the VH3-21+ patients also showed a predominance of λ chain expression and biased usage of 1 specific Vλ gene, V2-14. Furthermore, overall survival appeared to correlate with VH3-21 usage and, regardless of VH gene mutation status, VH3-21+ patients had a poor outcome. All in all, it appears that VH3-21 gene usage define a new entity of CLL. The remaining question now to be clarified is if antigen(s) actually are involved in the pathogenesis of VH3-21+ CLL.

Association of the 1513C polymorphism in the P2X7 gene with familial forms of chronic lymphocytic leukaemia

Association of the 1513C polymorphism in the P2X7 gene with familial forms of chronic lymphocytic leukaemia.

The G(-248)A polymorphism in the promoter region of the Bax gene does not correlate with prognostic markers or overall survival in chronic lymphocytic leukemia

The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.