Differential expression of NF-kappaB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism.

Mucosa-associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly activate the nuclear factor (NF)-kappaB pathway. Gastric MALT lymphomas harboring such translocations usually do not respond to Helicobacter pylori eradication, while most of those without translocation can be cured by antibiotics. To understand the molecular mechanism of these different MALT lymphoma subgroups, we performed gene expression profiling analysis of 21 MALT lymphomas (13 translocation-positive, 8 translocation-negative). Gene set enrichment analysis (GSEA) of the NF-kappaB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions have shown that translocation-positive MALT lymphomas are characterized by an enhanced expression of NF-kappaB target genes, particularly toll like receptor (TLR)6, chemokine, CC motif, receptor (CCR)2, cluster of differentiation (CD)69 and B-cell CLL/lymphoma (BCL)2, while translocation-negative cases were featured by active inflammatory and immune responses, such as interleukin-8, CD86, CD28 and inducible T-cell costimulator (ICOS). Separate analyses of the genes differentially expressed between translocation-positive and -negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. Finally, expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10-mediated NF-kappaB activation in vitro. Our findings provide novel insights into the molecular mechanism of MALT lymphomas with and without translocation, potentially explaining their different clinical behaviors.

Continual monitoring of intraepithelial lymphocyte immunophenotype and clonality is more important than snapshot analysis in the surveillance of refractory coeliac disease.

OBJECTIVE: An aberrant immunophenotype and monoclonality of intraepithelial lymphocytes (IELs) are frequently found in refractory coeliac disease (RCD). However, the utility of continual monitoring of IEL immunophenotype and clonality in the surveillance of RCD remains to be studied. DESIGN: The diagnostic and follow-up biopsies from 33 patients with CD, 7 with suspected RCD, 41 with RCD and 20 with enteropathy-associated T cell lymphoma (EATL) (including 11 evolved from RCD) were investigated by CD3epsilon/CD8 double immunohistochemistry and PCR-based clonality analysis of the rearranged T cell receptor (TCR) genes. RESULTS: An aberrant immunophenotype (CD3epsilon(+)CD8(-) IELs > or =40%) and monoclonality were detected occasionally in CD biopsies, either transiently in patients with CD not compliant with a gluten-free diet or in those who subsequently developed suspected RCD, RCD or EATL. In contrast, the aberrant immunophenotype and monoclonality were found in 30 of 41 (73%) and 24 of 37 (65%) biopsies, respectively, at the time of RCD diagnosis. Among the patients with RCD who did not show these abnormalities in their diagnostic biopsies, 8 of 10 (80%) and 5 of 11 (45%) cases gained an aberrant immunophenotype and monoclonality, respectively, during follow-up. Irrespective of whether detected in diagnostic or follow-up biopsies, persistence of both abnormalities was characteristic of RCD. Importantly, the presence of concurrent persistent monoclonality and aberrant immunophenotype, especially > or =80% CD3epsilon(+)CD8(-) IELs, was a strong predictor of EATL development in patients with RCD (p=0.001). CONCLUSIONS: Continual monitoring of both immunophenotype and clonality of IELs is more important than snapshot analysis for RCD diagnosis and follow-up, and could provide a useful tool for surveillance of patients at risk of EATL.

A20 deletion is associated with copy number gain at the TNFA/B/C locus and occurs preferentially in translocation-negative MALT lymphoma of the ocular adnexa and salivary glands.

The genetic basis of MALT lymphoma is largely unknown. Characteristic chromosomal translocations are frequently associated with gastric and pulmonary cases, but are rare at other sites. We compared the genetic profiles of 33 ocular adnexal and 25 pulmonary MALT lymphomas by 1 Mb array-comparative genomic hybridization (CGH) and revealed recurrent 6q23 losses and 6p21.2-6p22.1 gains exclusive to ocular cases. High-resolution chromosome 6 tile-path array-CGH identified NF-kappaB inhibitor A20 as the target of 6q23.3 deletion and TNFA/B/C locus as a putative target of 6p21.2-22.1 gain. Interphase fluorescence in situ hybridization showed that A20 deletion occurred in MALT lymphoma of the ocular adnexa (8/42=19%), salivary gland (2/24=8%), thyroid (1/9=11%) and liver (1/2), but not in the lung (26), stomach (45) and skin (13). Homozygous deletion was observed in three cases. A20 deletion and TNFA/B/C gain were significantly associated (p<0.001) and exclusively found in cases without characteristic translocation. In ocular cases, A20 deletion was associated with concurrent involvement of different adnexal tissues or extraocular sites at diagnosis (p=0.007), a higher proportion of relapse (67% versus 37%) and a shorter relapse-free survival (p=0.033). A20 deletion and gain at TNFA/B/C locus may thus play an important role in the development of translocation-negative MALT lymphoma.

Chlamydia psittaci is variably associated with ocular adnexal MALT lymphoma in different geographical regions.

Infectious agents play a critical role in MALT lymphoma development. Studies from Italy showed Chlamydia psittaci infection in 87% of ocular adnexal MALT lymphomas and complete or partial regression of the lymphoma after C. psittaci eradication in four of nine cases. However, C. psittaci was not demonstrated in ocular adnexal MALT lymphomas from the USA. This study was thus designed to investigate further the role of C. psittaci, and other infectious agents commonly associated with chronic eye disease, in the development of ocular adnexal MALT lymphoma. The presence of C. psittaci, C. trachomatis, C. pneumoniae, herpes simplex virus 1 and 2 (HSV1, HSV2), and adenovirus 8 and 19 (ADV8, ADV19) was assessed separately by polymerase chain reaction in 142 ocular adnexal MALT lymphomas, 53 non-marginal zone lymphomas, and 51 ocular adnexal biopsies without a lymphoproliferative disorder (LPD), from six geographical regions. C. psittaci was detected at similar low frequencies in non-LPD and non-marginal zone lymphoma groups from different geographical regions (0-14%). Overall, the prevalence of C. psittaci was significantly higher in MALT lymphomas (22%) than in non-LPD (10%, p=0.042) and non-marginal zone lymphoma cases (9%, p=0.033). However, the prevalence of C. psittaci infection in MALT lymphoma showed marked variation among the six geographical regions examined, being most frequent in Germany (47%), followed by the East Coast of the USA (35%) and the Netherlands (29%), but relatively low in Italy (13%), the UK (12%), and Southern China (11%). No significant differences in the detection of C. pneumoniae, C. trachomatis, HSV1, HSV2, ADV8, and ADV19 were found between lymphomas and controls from different geographical regions. In conclusion, our results show that C. psittaci, but not C. pneumoniae, C. trachomatis, HSV1, HSV2, ADV8 or ADV19, is associated with ocular adnexal MALT lymphoma and that this association is variable in different geographical areas.

Loss of heterozygosity at chromosome 9p21 is a frequent finding in enteropathy-type T-cell lymphoma.

Enteropathy-type T-cell lymphoma (ETL) and ulcerative jejunitis (UJ) are rare disorders often occurring in patients with coeliac disease. The genetic events associated with the accumulation of intraepithelial lymphocytes in coeliac disease and tumour development are largely unknown. Deletions at chromosome 9p21, which harbours the tumour suppressor genes p14/ARF, p15/INK4b, and p16/INK4a, and 17p13, where p53 is located, are associated with the development and progression of lymphomas. To examine whether deletions at 9p21 and 17p13 play a role in ETL, 22 cases of ETL and seven cases of UJ were screened for loss of heterozygosity (LOH) by tissue microdissection and polymerase chain reaction (PCR) analysis for microsatellite markers. Furthermore, p53 and p16 protein expression was examined by immunohistochemistry. In addition, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis for detection of mutations in exons 5-8 of the p53 gene was performed in five cases of ETL and three cases of UJ. LOH was found in at least one microsatellite marker at the 9p21 locus in 8 of 22 (36%) ETLs, but not in UJ. Five of nine (56%) tumours composed of large cells showed LOH at 9p21, as opposed to two of eight (25%) tumours with small- or medium-sized cell morphology. The region spanning the p14/p15/p16 gene locus was most frequently affected (five cases); LOH at these markers coincided with loss of p16 protein expression in all of these cases. p53 overexpression was demonstrated in all ETLs examined and in four of seven cases of UJ. However, no alterations of the p53 gene were detected by LOH or PCR-SSCP analysis. The results of this study show that LOH at chromosome 9p21 is frequent in ETL, especially in tumours with large cell morphology; this finding suggests that gene loss at this locus may play a role in the development of ETL.

No germline mutations in CDKN2A (p16) in patients with squamous cell cancer of the head and neck and second primary tumours.

There is increasing evidence that predisposition to some cancers has a genetic component. There is a high incidence of loss of heterozygosity on chromosome 9, in the region of tumour suppressor gene, CDKN2A (also known as p16), in sporadic squamous cell cancer of the head and neck (SCCHN). To investigate the possibility that CDKN2A may be involved in the inherited susceptibility to SCCHN, the 3 coding exons of CDKN2A were sequenced in 40 patients who had developed a second primary cancer after an index squamous cell cancer of the head and neck. No mutations were found and we conclude that CDKN2A mutations do not play a major role in cancer susceptibility in this group.

T(11;18)(q21;q21) is associated with advanced mucosa-associated lymphoid tissue lymphoma that expresses nuclear BCL10.

The development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma is a multistep process and can be clinico-pathologically divided into Helicobacter pylori-associated gastritis, low-grade tumors, and high-grade tumors. The molecular events underlying this progression are largely unknown. However, identification of the genes involved in MALT lymphoma-specific t(11;18)(q21;q21) and t(1;14)(p22;q32) has provided fresh insights into the pathogenesis of this disease. T(11;18)(q21;q21) results in a chimeric transcript between the API2 and the MALT1 genes, whereas t(1;14) (p22;q32) causes aberrant nuclear BCL10 expression. Significantly, nuclear BCL10 expression also occurs frequently in MALT lymphomas without t(1;14)(p22;q32), suggesting an important role for BCL10 in lymphoma development. Thirty-three cases of H pylori gastritis, 72 MALT lymphomas, and 11 mucosal diffuse large B-cell lymphomas (DLBCL) were screened for t(11;18)(q21;q21) by reverse transcription-polymerase chain reaction followed by sequencing. BCL10 expression in lymphoma cases was examined by immunohistochemistry. The API2--MALT1 fusion transcript was not detected in H pylori gastritis and mucosal DLBCL but was found in 25 of 72 (35%) MALT lymphomas of various sites. Nuclear BCL10 expression was seen in 28 of 53 (53%) of MALT lymphomas. Of the gastric cases, the largest group studied, the frequency of both t(11;18)(q21;q21) and nuclear BCL10 expression was significantly higher in tumors that showed dissemination to local lymph nodes or distal sites (14 of 18 = 78% and 14 of 15 = 93%, respectively) than those confined to the stomach (3 of 29 = 10% and 10 of 26 = 38%). Furthermore, t(11;18)(q21;q21) closely correlated with BCL10 nuclear expression. These results indicate that both t(11;18)(q21;q21) and BCL10 nuclear expression are associated with advanced MALT lymphoma and that their oncogenic activities may be related to each other. (Blood. 2001;98:1182-1187)

p53 abnormalities in splenic lymphoma with villous lymphocytes.

The incidence and role of p53 abnormalities have not been reported in splenic lymphoma with villous lymphocytes (SLVL), the leukemic counterpart of splenic marginal zone lymphoma. Because p53 abnormalities correlate with progressive and refractory disease in cancer and isochromosome 17q has been described in SLVL, a low-grade lymphoma that behaves aggressively in a minority of patients, this study investigated p53 changes by molecular and immunophenotypic methods in samples from 59 patients. The p53 deletion was analyzed by fluorescence in situ hybridization, and p53 protein expression was assessed by immunocytochemistry in 35 of 59 cases and by flow cytometry in 20 of 35 patients. Ten patients (17%) had a monoallelic p53 loss, 3 (9%) of 35 nuclear protein expression by immunocytochemistry, and 2 (10%) of 20 by flow cytometry. Two patients had both deletion and protein expression. Direct sequencing of all p53 exons was used to delineate mutations in 9 of 11 patients with an identified abnormality. Mutations, both compromising p53 DNA binding, were identified in the 2 patients with deletion and protein accumulation. Kaplan-Meier analysis revealed a significantly worse survival for patients with p53 abnormalities. Although p53 abnormalities are infrequent in SLVL, they underlie a more aggressive disease course and poor prognosis. (Blood. 2001;97:3552-3558)

Kaposi sarcoma-associated herpesvirus infects monotypic (IgM lambda) but polyclonal naive B cells in Castleman disease and associated lymphoproliferative disorders.

In a previous study, it was shown that the Kaposi sarcoma-associated herpesvirus (KSHV) was specifically associated with monotypic (IgMlambda) plasmablasts in multicentric Castleman disease (MCD). The plasmablasts occur as isolated cells in the mantle zone of B-cell follicles but may form microlymphoma or frank plasmablastic lymphoma. To determine the clonality and cellular origin of the monotypic plasmablasts, the rearranged Ig genes in 13 patients with KSHV-related MCD, including 8 cases with microlymphomas and 2 with frank lymphomas, were studied. To investigate the role of the interleukin 6 (IL-6) receptor signaling in the pathogenesis of MCD and associated lymphoproliferative disorders, viral IL-6 and human IL-6 receptor expression was examined. KSHV-positive plasmablasts were polyclonal in MCD-involved lymphoid tissues in all cases and microlymphomas in 6 of 8 cases. Monoclonal KSHV-positive plasmablasts were seen in microlymphomas of 2 cases and in both frank lymphomas. Despite their mature phenotype, KSHV-positive plasmablasts did not harbor somatic mutations in the rearranged Ig genes, indicating origination from naive B cells. Viral IL-6 was expressed in 10% to 15% of KSHV-positive plasmablasts, whereas the human IL-6 receptor was expressed in most KSHV-positive cells. Thus, KSHV infects monotypic but polyclonal naive B cells and is associated with a range of lymphoproliferative disorders from polyclonal isolated plasmablasts and microlymphomas to monoclonal microlymphoma and frank plasmablastic lymphomas in MCD patients. Activation of the IL-6 receptor signaling pathway may play a role in differentiation of KSHV-infected naive B cells into plasmablasts and development of lymphoproliferative lesions. (Blood. 2001;97:2130-2136)

Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy.

20-30% of gastric mucosa-associated lymphoid tissue (MALT) lymphoma associated with Helicobacter pylori do not regress after antibiotic therapy. Regression can be assessed only by extended follow-up. To assess whether t(11;18, q21;q21), which results in a chimeric transcript between the AP12 and MLT genes, predicts lymphoma resistance to antibiotic therapy, we screened for the fusion transcript with RT-PCR in ten responsive and 12 non-responsive gastric MALT lymphomas. The AP12-MLT transcript was detected in nine (75%) of 12 patients non-responsive to antibiotic therapy but not in responsive patients. Most H pylori-associated gastric MALT lymphomas that do not respond to antibiotic therapy are associated with t(11;18, q21;q21).

Molecular and cytogenetic analysis of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common primary tumor occurring in the central nervous system of adults. Although progress has been made in clinical management of this tumor, little is known about the molecular defects underlying the initiation and progression of GBM. To address these issues, we have characterized five cases of GBM using cytogenetics, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and direct sequencing. All of these tumors were observed to have clonal chromosome aberrations. Complicated chromosome translocations including der(18)t(2;4;12;18), der(X)t(X;10)(q27.1;p12.1) and der(10)t(10;15)(p11.23;q11.2), and der(1) (:1p31-->1q44::7q11. 3-->7qter) were seen in three tumors. Loss of the CDKN2 gene was noted in four tumors. A gain of copy number of the Cathepsin L gene was seen in two tumors. Amplification of the CDK4, MDM2, and GLI/CHOP genes was noted in two tumors, and amplification of the PDGFR gene was detected in one tumor. Mutation of exon 5 of the TP53 gene was found in three tumors. No mutation of the BCL10 gene was detected in five cases of GBM analyzed, although deletion of chromosome 1p was seen in two tumors. These results provide information for further investigation of GBM.

Molecular and cytogenetic analysis of lymphoblastoid and colon cancer cell lines from cotton-top tamarin (Sagiunus oedipus).

The cotton-top tamarin (CTT) (Sagiunus oedipus) has been used as an animal model to investigate the etiology and pathophysiology of several human diseases, including ulcerative colitis and its associated colorectal carcinoma (CRC). Little is known, however, about genetic synteny between CTT and humans, and about chromosome aberrations in CTT CRC. To address these issues, we have analyzed CTT lymphoblastoid and CRC cell lines using cytogenetics, fluorescence in situ hybridization (Zoo-FISH), and direct sequencing. The CTT lymphocytes had pseudodiploid chromosomes of 46. The CTT CRC cells showed near-diploid chromosomes of 45. Several clonal structural aberrations were observed, including der(1), a marker chromosome, and double minutes. Zoo-FISH using human chromosome 2, 3, 5, 6, 9, 11, 13, 15, 16, 17, 19, 22, and X paints identified homologous chromosomes and subchromosomal regions in the CTT genome. Fluorescence in situ hybridization with human telomeric probe also detected a homologous sequence in CTT genome. Direct sequencing of CTT genomic DNA using primers amplifying exons 4 and 15 of the human APC gene identified DNA sequences in CTT genome with 99% and 95% homology, respectively. These results provide a basis for further comparative studies of CTT and human genome.

BCL10 gene mutation in lymphoma.

BCL10 is directly involved in t(1;14)(p22;q32) of mucosa-associated lymphoid tissue (MALT) lymphoma. Wild-type BCL10 promoted apoptosis and suppressed malignant transformation in vitro, whereas truncated mutants lost the pro-apoptotic activity and exhibited gain of function enhancement of transformation. We studied 220 lymphomas for genomic BCL10 mutation by polymerase chain reaction-single-strand conformational polymorphism and DNA sequencing. Nineteen mutations were found in 13 lymphoma specimens, as follows: 8 of 120 (6.7%) mucosa-associated lymphoid tissue (MALT) lymphomas, 4 of 42 (9.5%) follicular lymphomas, and 1 of 23 (4.3%) diffuse large B-cell lymphomas. No mutations were found in 14 mantle cell lymphomas or 21 T-cell lymphomas. High-grade MALT lymphoma tended to show a slightly higher mutation frequency (2 of 25, 8%) than low-grade MALT tumor (6 of 95, 6.3%). Among low-grade gastric MALT lymphoma, mutations were found in 3 of 11 tumors that did not respond to Helicobacter pylori eradication therapy, but none were found in 22 tumors that regressed completely after H pylori eradication. All 14 potentially pathogenic mutations were distributed in the carboxyl terminal domain of BCL10. Deletion accounted for 10 of these mutations; 10 of 14 mutations caused truncated forms of BCL10. Western blot analysis of a mutant case confirmed the presence of truncated BCL10 products of anticipated size. Our results suggest that BCL10 mutation may play a pathogenic role in B-cell lymphoma development, particularly in aggressive and antibiotic unresponsive MALT lymphomas, and may further implicate the biologic importance of the carboxyl terminal of the molecule. (Blood. 2000;95:3885-3890)

Allelotype of uterine leiomyomas.

Uterine leiomyomas are the most common benign tumor that arise from smooth muscle cells of the myometrium. Little is known about the etiology and pathogenesis of this tumor. To investigate the molecular pathogenesis of these tumors, we have conducted an allelotype of 102 leiomyomas from 12 patients, using 67 fluorescently-tagged oligonucleotide primers amplifying microsatellite loci covering all autosomes. No areas of the genome showed frequent loss of heterozygosity (LOH); however, the highest rate of LOH (9%) was observed on 7q, consistent with previous cytogenetic observations. Uterine leiomyomas are sometimes multiple. In general, multiplicity of other types of neoplasm is associated with genetic predisposition to the disease. Because multiple tumors were available from each of the 12 patients studied, we looked for evidence of allele-specific LOH, which might indicate the presence of an underlying predisposition gene. However, no evidence for allele-specific LOH was detected, indicating that if cases of multiple uterine leiomyoma are due to an underlying predisposition gene, it is unlikely to be a recessive oncogene.

Alleletyping of an oligodendrocyte-type-2 astrocyte lineage derive from a human glioblastoma multiforme.

We have conducted alleletyping of two novel cell lines derived from glioblastoma multiforme, which appear to have arisen from different glial lineages, by using 76 fluorescently labeled oligonucleotide primers amplifying microsatellite loci covering the entire human genome. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This cell line generated, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. Hu-O-2A/Gbl cells show allelic losses of loci on chromosomes 2, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 17, 20 and 21. IN1434 cells are likely to have allelic losses of loci on chromosomes 1, 3, 8 and 10, although no control DNA is available for this cell line. These results, for the first time, provide a detailed information of the molecular genetic defects occurring in Hu-O-2A/Gb1 and IN1434.

p53 abnormalities in B-cell prolymphocytic leukemia.

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.

No germline mutations in the dimerization domain of MXI1 in prostate cancer clusters. The CRC/BPG UK Familial Prostate Cancer Study Collaborators. Cancer Research Campaign/British Prostate Group.

There is evidence that predisposition to cancer has a genetic component. Genetic models have suggested that there is at least one highly penetrant gene predisposing to this disease. The oncogene MXI1 on chromosome band 10q24-25 is mutated in a proportion of prostate tumours and loss of heterozygosity occurs at this site, suggesting the location of a tumour suppressor in this region. To investigate the possibility that MXI1 may be involved in inherited susceptibility to prostate cancer, we have sequenced the HLH and ZIP regions of the gene in 38 families with either three cases of prostate cancer or two affected siblings both diagnosed below the age of 67 years. These are the areas within which mutations have been described in some sporadic prostate cancers. No mutations were found in these two important coding regions and we therefore conclude that MXI1 does not make a major contribution to prostate cancer susceptibility.

p53 abnormalities in CLL are associated with excess of prolymphocytes and poor prognosis.

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=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=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.