Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas.

Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.

Proliferation-dependent expression and phosphorylation of pRB in B cell non-Hodgkin's lymphomas: dependence on RB1 copy number.

Some studies have suggested that a significant fraction of non-Hodgkin's lymphomas (NHL) do not express pRB protein, possibly due to deletions of RB1. We examined RB1/centromere 17 copy number by fluorescent in situ hybridisation, and pRB expression/phosphorylation by immunohistochemistry (IHC) and immunoblotting (IB) in 66 cases of B cell NHL. Thirteen cases had lost one RB1 copy relative to centromere 17 copy number and total DNA content. Case 458/88 had no RB1 copies. pRB levels were heterogeneous as assessed by IB (0.04-1.12 relative units), but all tumours, except for case 458/88, expressed pRB localised to the nucleus in >75% of the tumour cells by IHC. The fraction of phosphorylated pRB was correlated with pRB expression (r(2)= 0.56, P < 0.001). The 14 cases with loss of RB1 had lower pRB expression (median 0.25) than those without (median 0.48, P < 0.001), but a correlation with S phase fraction (r(2) = 0.43, P < 0.001; previously published data for tumour-specific S phase and apoptotic fractions) indicated that the variation in pRB expression was due to differences in proliferative activity. Furthermore, the regression lines for pRB expression vs S phase fraction were not different for the cases with or without loss of one RB1 copy (P = 0.5). Cases 154/88 (one RB1 copy) and 258/88 (two RB1 copies), in addition to case 458/88, had low expression of (hypophosphorylated) pRB (0.04, 0.08 and 0.04), despite their high S phase fractions (21%, 17% and 21%). There was no association between pRB expression/RB1 copy number and apoptotic fraction. Neither pRB expression nor loss of RB1 had prognostic value, but cases 154/88, 258/88, and 458/88 had short survival times (5, 3 and 46 months, respectively) compared to the others (median survival: 44 months, P = 0.03). It is suggested that pRB expression and function are normal in 63 of 66 NHL cases, including 12 of 13 lymphomas with loss of one RB1 allele.

c-MYC, RB-1, TP53, and centromere 8 and 17 copy number in B-cell non-Hodgkin's lymphomas assessed by dual-color fluorescence in situ hybridization.

Dual-color interphase FISH was performed to B-cell Non-Hodgkin's lymphomas to detect numerical genetic alterations in c-MYC, RB-1, TP53 and centromere 8 and 17. The probe combinations c-MYC/centromere 8, RB-1/centromere 8 and TP53/centromere 17 were applied, and the hybridization signals scored in a correlated fashion. Copy number aberrations was found in 24 of 45 lymphomas examined (53%). Nine tumors (20%) had increased c-MYC gene copy number (three to 8 copies). Seven of these nine had increase in c-MYC copies relative to centromere 8 copy number. Allelic loss of RB-1 was found in 9 tumors (20%), one tumor lacked both alleles. Nine cases (20%) showed hemizygous TP53 deletion. TP53 deletion was significantly associated with high-grade histology (P = 0.02). Numerical alterations in c-MYC and RB-1 were not associated with prognosis. Patients with hemizygous TP53 deletion had shorter survival (relative risk = 6.1, P<0.001) than the ones with two or more alleles.

[Primary non-Hodgkin's lymphoma of the brain]. / Primaert non-Hodgkins lymfom i hjernen.

Between 1980 and 1993, 26 patients were treated for primary lymphoma in the central nervous system at the Norwegian Radium Hospital. This is a rare disease with poor prognosis and thus represents a great therapeutic challenge. Immunocompromised, e.g. AIDS patients, are a group at high risk, but the incidence has increased among immunocompetent patients as well. Median patient age was 64 years; and none of the patients had any signs of immunodeficiency. 23 of the patients received radiation therapy. 13 of the patients received some form of chemotherapy. The overall median survival was 19 months. WHO performance status 0-2, unifocal lesion, absence of steroid dependency and normal serum levels of LDH were all associated with longer survival. Although complete remissions were achieved in most patients, relapses in the central nervous system were frequent.

Loss of chromosome 11q21-23.1 and 17p and gain of chromosome 6p are independent prognostic indicators in B-cell non-Hodgkin's lymphoma.

Comparative genomic hybridization (CGH) was employed to study chromosomal aberrations in relation to cell proliferation, apoptosis, and patient survival in 94 cases of B-cell non-Hodgkin's lymphoma diagnosed between 1983 and 1993. Eighty cases had aberrations by CGH. Chromosomal regions 1p21-31.1 (10%), 6cen-q24 (12%), 8p (11%), 9p21-ter (14%), 11q21-23.1 (11%), 13q13-21.1 (12%), and 17p (15%) were frequently lost. Gains were found at 3q21-ter (22%), 6p (11%), 7p (12%), 8q23-ter (13%), 12cen-q15 (17%), 17q24-ter (13%), and 18q13.3-21 (20%). A high number of aberrations (> or = 4, 33 cases) was associated (P < or = 0.001) with the mantle cell and diffuse large B-cell lymphoma subtypes, a high fraction of tumour cells in S phase, and short survival (RR (relative risk) = 3.7). Loss of 1p21-31.1, 8p, 9p21-ter, 11q21-23.1, and 13q13-21.1 were associated with mantle cell lymphoma (P < or = 0.03), while gain of 6p and 12cen-q15 were more frequent in diffuse large B-cell and small lymphocytic lymphoma, respectively (P = 0.04). Loss of 8p and 17p, and gain of 3q21-ter, 6p, 7p, and 8q23-ter were associated with a high S phase fraction (P < or = 0.03), but none of the aberrations were associated with tumour apoptotic fraction (P > or = 0.13). The most important prognostic CGH parameters (P < 0.001) were losses of 11q21-23.1 (RR = 3.8) and 17p (RR = 4.4), and gain of 6p (RR = 4.2). The latter parameters and IPI were the only ones with independent prognostic value (RR = 10, 5.0, 6.7, and 3.7, respectively; P < 0.001) when assessed together with lymphoma sub-type, primary versus relapse cases, treatment, B symptoms, S phase fraction, and presence of BCL1 and BCL2 translocations. A combined CGH/IPI binary parameter had high prognostic value for patients receiving different treatments, with various lymphoma sub-types, and for primary as well as relapse cases.

Oncogenic aberrations in the p53 pathway are associated with a high S phase fraction and poor patient survival in B-cell Non-Hodgkin's lymphoma.

The implications of aberrations in the p53 pathway for induction of apoptosis and regulation of S phase entry, and for patient survival, were investigated in 83 B-cell Non-Hodgkin's lymphomas. Eight cases had missense mutations in exons 5, 7, 8 and 9 as revealed by constant denaturant gel electrophoresis and sequencing. Fifteen cases had lost 1 TP53 allele as revealed by fluorescent in situ hybridization and comparative genomic hybridization. Ten cases expressed high levels of p53 as assessed by immunoblotting and immunohistochemistry. S phase fractions were higher, apoptotic fractions were the same and survival times were shorter in all aberration groups compared with the cases with no TP53/p53 aberrations. Since many tumors had more than one TP53/p53 aberration, the tumors were divided into groups with the following characteristics: no TP53/p53 aberrations; loss of one TP53 allele only (9 cases), TP53 point mutation (8 cases), high-level p53 expression and no TP53 mutation (3 cases). Tumors from the 3 latter groups had higher median S phase fractions (5%, 7.6%, and 5%, respectively, p<0.02) than the cases without any aberrations (1.1%), and survival time for these patients was much shorter (relative risks of 5.9, 8.9, and 6.6, respectively, p<0.003). Apoptotic fractions were similar in all these groups (p=0.09). Multivariate analysis showed that the presence of TP53/p53 aberrations is a strong and independent prognostic parameter in B-cell Non-Hodgkin's lymphoma.