We have analysed the clinical and laboratory features in 544 patients with chronic lymphocytic leukaemia (CLL) with available cytogenetics and fluorescence in-situ hybridization (FISH) analysis for trisomy 12 in half of them, to examine the correlation between chromosome abnormalities and clinical or laboratory parameters. Five chromosome groups were defined: (1) trisomy 12 (18%), detected as the sole abnormality or associated with other changes; (2) del(13)(q12-14) (7%); (3) other abnormal karyotypes (20%); (4) normal karyotype (41%); and (5) no divisions (14%). There were no differences in the age distribution between the five groups. Clinical stages (Binet) were: A (74%), B (12%) and C (14%). Stage A was common in cases with del(13q)(82%), normal (84%) and other abnormal karyotypes (74%), whereas it was less common in trisomy 12 cases (64%) and those with no divisions (48%). Typical CLL morphology was found in 83% of cases; 10% had more than 10% prolymphocytes (CLL/PL) and 7% had other atypical features. CLL with trisomy 12 was the only group with a high frequency of either CLL/PL (31%) or atypical morphology (24%). Atypical morphology and CLL/PL were even more frequent when trisomy 12 was associated with other chromosomal abnormalities (70% v 46%). The incidence of cases with CLL/PL and other atypical morphology was significantly lower in the other chromosome groups (P < 0.001). There were no differences in immunophenotype among the various groups except for a higher frequency of stronger Smlg and FMC7 expression in cases with trisomy 12, particularly those with CLL/PL and other atypical morphology. Our findings confirm that trisomy 12 defines a subgroup of CLL with more frequent atypical morphology, including CLL/PL, stronger SmIg and FMC7 expression, more advanced stages (B and C in 18%) and possibly worse prognosis.

Abnormalities of several cell-cycle regulatory genes includingphoma with villous lymphocytes (SLVL) 4 and in some cases type. 14,17,18 We have shown in two cases that biallelic loss of

The immunological detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia (ALL) has been hampered by the fact that the leukaemic cells represent the malignant counterparts of normal haemopoietic precursors expressing terminal deoxynucleotidyl transferase (TdT), CD10 and CD19. We have used quantitative double-labelling flow cytometry with standard fluorescent beads to convert the mean fluorescence to the number of antigen molecules per cell. The number of TdT, CD10 and CD19 molecules per cell was determined in normal B-cell precursors from 22 healthy donors and eight regenerating marrows from patients with various malignancies and in 20 cases of B-lineage ALL. In normal bone marrow we characterized two different B-cell populations: TdT+/CD10+/CD19+ and TdT-/CD10+/CD19+. We demonstrated a major difference in the level of expression of TdT, CD10 and CD19 between normal bone marrow and B-lineage ALL blasts. Normal TdT+ precursors have significantly higher number of TdT (> 100 x 10(3)) and lower number of CD10 (< 50 x 10(3)) and CD19 (< 10 x 10(3)) molecules per cell than B-lineage ALL blasts (< 100, > 50, > 10 x 10(3) molecules per cell respectively); these differences were statistically highly significant. Furthermore, regenerating marrows had a significantly higher percentage of B-cell precursors than healthy donors. This increase was at the expense of the TdT-/CD10+/CD19+ population which, in the context of B-lineage ALL, could be wrongly interpreted as evidence of relapse if TdT is not included in the analysis. Therefore the quantitative analysis of TdT combined with CD10 and CD19 may allow a clear distinction between normal precursors and minimal residual leukaemia in B-lineage ALL and avoid the pitfall of misinterpreting regenerating B-cells as evidence of relapse.

SummaryDespite the efficacy of current immune-chemotherapy for treatment of Bcell non-Hodgkin lymphoma, a substantial proportion of patients relapse, highlighting the need for new therapeutic modalities. The use of live microorganisms to develop anti-tumoural therapies has evolved since Coley's toxin and is now receiving renewed attention. Salmonella Typhimurium has been shown to be highly effective as an anti-tumour agent in many solid cancer models, but it has not been used in haematooncology. Here, we report that intra-tumoural administration of LVR01 (attenuated S. Typhimurium strain with safety profile) elicits local and systemic anti-tumour immunity, resulting in extended survival in a lymphoma model. LVR01 induces intra-tumoural recruitment of neutrophils and activated CD8 + T cells, as well as increasing the natural killer cell activation status. Furthermore, a systemic specific anti-tumour response with a clear T helper type 1 profile was observed. This approach is an alternative therapeutic strategy for lymphoma patients that could be easily moved into clinical trials.

Summary.To determine the role of the p53 gene in chronic lymphocytic leukaemia (CLL) and its possible involvement in the pathogenesis of a progressive form of CLL characterized by > 10% prolymphocytes (CLL/PL), we selected 32 cases, 17 with typical morphology and 15 CLL/PL. The extent of inactivation of p53 was examined by assessing loss of heterozygosity (LOH) at 17p13.3, by sequencing the highly conserved region (exons 5-9) of the p53 gene and by analysing p53 protein expression. LOH was detected in 8/28 (29%) cases, p53 mutations in 5/32 (16%) cases and p53 expression in 5/27 (19%) cases. Overall 11 cases (30%) had p53 abnormalities of which eight cases had CLL/PL. There was a significant association between CLL/PL and p53 abnormalities (P ¼ 0·05); 75% of cases with LOH, 80% of p53 mutations and 80% of cases positive for p53 protein had CLL/PL. Thus, p53 inactivation is the first gene abnormality identified so far to be involved in the development of CLL/PL.All the cases with typical CLL and p53 abnormalities had only one allele affected whereas 4/6 CLL/PL had both alleles inactivated. This difference in the extent of p53 inactivation suggests that accumulation of p53 abnormalities may be associated with progression of CLL to CLL/PL.CLL cases with p53 abnormalities were characterized by a higher incidence of stage C (P < 0·025), a higher proliferative rate (P ¼ 0·05), short survival (P < 0·005) and resistance to first-line therapy (P < 0·02) but not to nucleoside analogues. Analysis of the correlation between p53 status and incidence of trisomy 12 by fluorescence in situ hybridization (FISH) showed that trisomy 12 was more frequent in cases without p53 abnormalities, suggesting that trisomy 12 and p53 may represent different pathways of transformation in CLL.

BackgroundThere is growing evidence supporting the role of inflammation in aneurysmal subarachnoid hemorrhage (aSAH) pathophysiology and it is of great interest to elucidate which immune mechanisms are involved.Methods12 aSAH patients and 28 healthy controls were enrolled prospectively. We assessed leukocytes subpopulations and their activation status by flow cytometry in cerebrospinal fluid (CSF) and peripheral blood (PB) of SAH patients at the same time and in PB of controls.ResultsMonocytes and neutrophils were activated in CSF of aSAH patients. The percentage of CD14++CD16+ monocytes were higher in CSF than in PB of aSAH patients, and were also increased in PB of aSAH patients compared with controls. An enhanced expression of CD69 was shown in CSF neutrophils compared with PB in aSAH patients. PB of aSAH patients showed lower percentage of total lymphocytes compared with controls PB. Additionally, lymphocytes were activated in CSF and PB of aSAH patients. CD4+ and CD8+ T cells had a decreased expression on CD3 and higher levels of CD69 in CSF compared with PB in aSAH patients. Moreover, PB CD4+ and CD8+ T cells of aSAH patients were activated compared with controls. Additionally, CD28 expression was decreased on CSF T lymphocytes.ConclusionsOur data suggest an important recruitment of leukocytes to the site of injury in aSAH as well as an increased activation at this level. Overall, these results indicate that aSAH probably stimulates both the innate and adaptive immune responses.

The aim of the present study was to analyze BRCA1 and BRCA2 mutations in Uruguayan families with breast and breast/ovarian cancer. Probands from 42 families with at least three cases of female breast cancer (BC) or two cases and subcriteria (paternal transmission, ovarian cancer, bilateral BC, male BC, Ashkenazi Jewish ancestry) in the same lineage, at least one diagnosed before age 50, were screened for germline mutations. PCR amplification of all exons and intron-exon boundaries were performed, followed by protein truncation test, heteroduplex analysis, and direct sequencing. We identified seven different truncating mutations in seven families, five in BRCA2 (three in site-specific BC families and two in breast-ovarian cancer families) and two in BRCA1 (one in a site-specific BC family and the other in a breast-ovarian cancer family). Both BRCA1 mutations (5583insT and 2687T>G) and one of the five BRCA2 mutations (3829insTdel35) were not previously reported. We also detected ten sequence variants of unknown significance, five of them not described before. The low frequency of BRCA1/2 mutations (0.17) is in agreement with that reported in studies which included families with similar selection criteria. However, the observed predominance of BRCA2 (0.12) over BRCA1 mutations (0.05) is in contrast with the higher proportion of BRCA1 mutations communicated for most previous studies, even those with a predominance of site-specific BC families. Meanwhile, it has been described in one Chilean and some Spanish and Italian reports, highlighting the strong dependence between the mutational spectra and the ethnicity of the population analyzed.

B-cell prolymphocytic leukemia (B-PLL) is an aggressive disorder of mature B cells with distinct clinical and pathologic features. To determine the incidence of abnormalities of p53, we analyzed 19 cases of B-PLL by DNA blot to assess loss of heterozygosity (LOH) at 17p13.3, by immunocytochemistry to assess p53 expression, and by direct DNA sequencing of polymerase chain reaction-amplified exons 5 to 9 of the p53 gene. LOH was detected in 10 of 19 (53%) cases, p53 expression was detected in 8 of 17 (47%), and p53 mutations were detected in 10 of 19 (53%) cases. The pattern of mutations was distinct from that observed in other B-cell malignancies. Six cases exhibited missense mutations; 4 were transversions and 2 were transitions. The G:C → A:T transition at cathepsin G dinucleotides commonly reported in p53 mutations in chronic lymphocytic leukemia (CLL) and other hematologic malignancies was observed in only 1 case of B-PLL. Three cases exhibited deletions (ranging from 3 to 35 bp in length) and one case exhibited a 2-bp insertion. In 1 case, a 27-bp deletion resulted in the expression of a p53 protein lacking 9 amino acids from the DNA binding region. All samples with p53 mutation showed loss of germline p53 sequences. However, 3 of 10 showed no LOH by Southern blot, indicating a localized deletion around the p53 locus at 17p13.1. Five of the 10 cases with p53 mutation exhibited detectable p53 expression, including 4 cases with p53 missense mutation and 1 case with deletion. Two of 7 cases with no detectable mutation of p53 nevertheless overexpressed p53. Therefore, there was no correlation between protein expression and p53 mutation in B-PLL. Our data indicate that the overall abnormalities of p53 occurred in 14 of 19 (75%) cases of B-PLL. The frequency of p53 mutation (53%) in B-PLL is the highest reported in B-cell malignancies and may be responsible for the frequent resistance to therapy of this disease. In addition, the pattern of p53 mutation was different from that observed in CLL and other hematologic malignancies and may indicate that a distinct pathogenic mechanism operates in B-PLL.