Allelic genotyping reveals a hierarchy of genomic alterations in mantle cell lymphoma associated to cell proliferation.

Mantle cell lymphoma (MCL) is a distinct subentity of non-Hodgkin lymphoma, characterized by the chromosomal translocation t(11;14)(q13;q32) leading to an overexpression of cyclin D1 in virtually all cases. However, additional cytogenetic aberrations are apparent in the vast majority of MCL. Applying LOH analysis in 52 MCL patient samples, we confirmed frequent alterations in 9p21 (28.6%) and p53 (28.9%) but also detected allelic losses in 1p21, 9q21, 13q13-14, 13q31-32, 17p13.1, and 17p13.3 in 28-45% of cases and allelic gains in 3q27-28 and 19p13.3 in 14-22% of cases. In addition, losses in the 2p23 and 7q22-35 genomic regions not previously described to be altered in MCL were identified in up to 20% of cases. Applying multivariate analysis, a cluster of genomic aberrations including 1p21, 3q27, 7q22-36, 6p24, 9p21, 9q31, and 16p12 alterations was identified which was closely associated to cell proliferation as determined by Ki67 immunostaining. This proliferation-dependent network of oncogenic alterations complements the previously identified proliferation expression signature described by RNA expression profiling in MCL.

Detailed mapping of chromosome 17p deletions reveals HIC1 as a novel tumor suppressor gene candidate telomeric to TP53 in diffuse large B-cell lymphoma.

Deletions in the short arm of chromosome 17 (17p) involving the tumor suppressor TP53 occur in up to 20% of diffuse large B-cell lymphomas (DLBCLs). Although inactivation of both alleles of a tumor suppressor gene is usually required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in DLBCLs, suggesting the possible existence of additional tumor suppressor genes in 17p. Using a bacterial artificial chromosome (BAC) and Phage 1 artificial chromosome (PAC) contig, we here define a minimally deleted region in DLBCLs encompassing approximately 0.8 MB telomeric to the TP53 locus. This genomic region harbors the tumor suppressor Hypermethylated in Cancer 1 (HIC1). Methylation-specific PCR demonstrated hypermethylation of HIC1 exon 1a in a substantial subset of DLBCLs, which is accompanied by simultaneous HIC1 deletion of the second allele in 90% of cases. In contrast, HIC1 inactivation by hypermethylation was rarely encountered in DLBCLs without concomitant loss of the second allele. DLBCL patients with complete inactivation of both HIC1 and TP53 may be characterized by an even inferior clinical course than patients with inactivation of TP53 alone, suggesting a functional cooperation between these two proteins. These findings strongly imply HIC1 as a novel tumor suppressor in a subset of DLBCLs.

[Severe foetal growth retardation in a patient with uterus bicornis, velamentous insertion and partial placental abruption in the 26th week of gestation--a case report]. / Schwere intrauterine Wachstumsretardierung bei Uterus bicornis, Insertio velamentosa und vorzeitiger partieller Plazentalösung in der 26. Schwangerschaftswoche - eine Kasuistik.

Foetal growth retardation (IUGR) occurs in approximately 3-10 % of all pregnancies and may result from foetal, maternal or placenta-related conditions. In IUGR, the placental weight is often reduced and the placental capacity, reflected by the organ's weight, is impaired. Uterine malformations have an incidence of 3-4 % and may be the cause of placental abruptions occurring in 0.4-1.3 % of all pregnancies. We report on a patient in the 26 (th) week of pregnancy who was admitted with vaginal bleeding. A uterus bicornis had been found previously. Sonography showed severe foetal growth retardation and a pathological foetal Doppler signal. A haematoma located cranial of the os uteri was sonographically diagnosed, and a partial placental abruption was suspected. Due to a pathological cardiotocography, a primary Caesarean section was performed. Intraoperative evaluation confirmed the presence of a uterus bicornis. In addition, the placenta showed an insertio velamentosa. The growth retarded foetus - 490 g birth weight - was anaemic. Respiratory therapy and surfactant substitution were performed because of a respiratory distress syndrome. At a corrected age of 8 weeks the boy was sent home without neurological sequelae. In the case reported, a malformation of the uterus was the cause of a pathologically altered placenta. The multiple factors responsible for the described severe intrauterine growth retardation were a low placental weight and thus a reduced placental capacity, an impaired foetal circulation caused by the velamentous insertion, as well as a partial placental abruption. In normotensive pregnancies with IUGR, macroscopic and histopathological examinations of the placenta are therefore strongly recommended. Prior to getting pregnant, the therapeutic options should be explained to women with uterine malformations.

Centrosome aberrations as a possible mechanism for chromosomal instability in non-Hodgkin's lymphoma.

Recently, centrosome aberrations have been described as a possible cause of aneuploidy in many solid tumors. To investigate whether centrosome aberrations occur in non-Hodgkin's lymphoma (NHL) and correlate with histologic subtype, karyotype, and other biological disease features, we examined 24 follicular lymphomas (FL), 18 diffuse large-B-cell lymphomas (DLCL), 33 mantle cell lymphomas (MCL), and 17 extranodal marginal zone B-cell lymphomas (MZBCL), using antibodies to centrosomal proteins. All 92 NHL displayed numerical and structural centrosome aberrations as compared to nonmalignant lymphoid tissue. Centrosome abnormalities were detectable in 32.3% of the cells in NHL, but in only 5.5% of lymphoid cells from 30 control individuals (P<0.0001). Indolent FL and MZBCL contained only 25.8 and 28.8% cells with abnormal centrosomes. In contrast, aggressive DLCL and MCL harbored centrosome aberrations in 41.8 and 35.0% of the cells, respectively (P<0.0001). Centrosomal aberrations correlated to lymphoma grade, mitotic, and proliferation indices, but not to the p53 labeling index. Importantly, diploid MCL contained 31.2% cells with abnormal centrosomes, while tetraploid samples harbored centrosome aberrations in 55.6% of the cells (P<0.0001). These results indicate that centrosome defects are common in NHL and suggest that they may contribute to the acquisition of chromosomal instability typically seen in NHL.

Immunohistochemical analysis of B-cell lymphoma using tissue microarrays identifies particular phenotypic profiles of B-cell lymphomas.

AIMS: To validate the applicability of tissue microarray (TM) in immunohistochemical profiling of B-cell lymphoma and to identify particular phenotypic profiles of B-cell neoplasms. METHODS AND RESULTS: Eighty-two diffuse large B-cell lymphomas (DLBL), 54 follicular lymphomas (FL) and 74 mantle cell lymphomas (MCL) were arrayed. Immunohistochemical stains of TM were compared with immunostains of conventional, formalin-fixed and frozen material sections. Concordant staining results were obtained in more than 88% of cases for CD20, CD3, CD5, CD10, CD23, Bcl-2, IgD, secretory differentiation, p53 and p21 expression. Prognostically relevant hot-spot expression of Ki67 yielded concordant results in 71%. Applying TM for characterization of p27KIP1 expression, both typical and blastoid MCL only rarely showed p27KIP1 expression (9% and 15%), whereas 32% of nodal DLBL were p27KIP1-positive, irrespective of high proliferative activity. Among 22 B-cell lymphomas investigated genetically, a p53 + p21- immunophenotype in >20% of tumour cells correlated with p53 locus deletion. CONCLUSIONS: Lymphoma TM allows for immunohistochemical profiling of human B-cell lymphoma with a comparable accuracy to immunohistochemical studies performed on conventional tissue sections. Nodal DLBLs showed significantly more frequent expression of IgD and p27KIP1 than extranodal DLBL. MCL and DLBL frequently showed aberrant p27KIP1 expression. A p53 + p21- immunophenotype in >20% of tumour cells in B-cell non-Hodgkin's lymphoma correlates with p53 gene deletion.

Loss of Fas (CD95/APO-1) regulatory function is an important step in early MALT-type lymphoma development.

Fas (CD95, APO-1) mutations were found in autoimmune diseases and some lymphomas, suggesting impairment of Fas-mediated cell death signaling that may cause tumor development. Because mucosa-associated lymphoid tissue (MALT)-type lymphoma B cells recognize autoantigens and proliferate in response to antigen and T cell-mediated signals, it is suggestive that autoreactive B cell lymphoma precursor cells may have escaped the Fas-mediated checkpoint that normally operates in healthy individuals. Using different biochemical, molecular, and functional approaches, we analyzed the Fas signaling in malignant B cells from seven MALT-type lymphomas that were additionally characterized for the t(11;18)(q21;q21) and four gastric diffuse large B cell lymphomas (DLBL). All DLBLs and three of seven MALT-type lymphomas were resistant to Fas-mediated apoptosis in vitro. Moreover, four of five MALT-type lymphomas analyzed and one of three DLBLs analyzed showed mutations in Fas mRNA transcripts but no loss of heterozygosity in the Fas promotor region. Alternative mechanisms of resistance to apoptosis, such as decreased expression of Fas or production of soluble Fas were not operative. Therefore, it is suggestive that a subgroup of MALT-type lymphoma B cells, irrespective of t(11;18)(q21;q21), escape the censoring Fas pathway by mutating and inactivating Fas. This identifies a key regulatory step in early MALT-type lymphomagenesis.

Heterogeneity of the API2-MALT1 gene rearrangement in MALT-type lymphoma.

The translocation t(11;18)(q21;q21), which is the most frequent chromosomal aberration in extranodal marginal zone B cell lymphomas of MALT-type, was characterised in a series of 34 biopsies, including 18 gastric non-Hodgkin's lymphomas (NHL) of MALT-type, six MALT-type NHL of extragastral origin and 10 extranodal large B cell lymphomas (LBL). Based on fluorescence in situ hybridisation, STS-PCR analysis and screening of genomic PAC libraries, a physical map of contiguous DNA probes on chromosome 11 was constructed containing the anti-apoptotic genes API2 and API1 adjacent to the translocation breakpoint. RACE-PCR experiments revealed MALT1 the chromosome 18-derived fusion partner of API2, which has also been reported recently by other groups. RT-PCR analysis and DNA sequencing demonstrated the expression of an API2-MALT1 fusion transcript in 18/24 gastral and extragastral MALT-type lymphomas. In none of 10 LBLs was a translocation specific RT-PCR product detected. Five variants of the fusion transcript were identified and in all instances the open reading frame of the fused portion of the MALT1 gene was maintained. The molecular analysis of these variants allowed the design of optimised assays for the diagnosis of the API2-MALT1 gene rearrangement.

Marginal zone B-cell lymphomas (MZBL) arising at different sites represent different biological entities.

Most entities of B-cell malignant non-Hodgkin's lymphomas (NHL) are characterized by typical primary chromosomal changes such as the t(14;18) in follicular lymphoma or the t(11;14) in mantle cell lymphoma. In contrast, marginal zone B-cell lymphomas (MZBL), arising at different nodal and extranodal sites, are poorly characterized on the genetic level. We performed cytogenetic investigations in 20 splenic and in 10 nodal MZBL and analyzed 52 MZBL (including 12 MALT-type lymphomas) for deletions of TP53, D13S25, and RB1 loci by fluorescence in situ hybridization. A new nonrandom chromosomal aberration, del(10)(q22q24), was found as a clonal anomaly in 3 out of 20 cases of splenic MZBL. Further recurring abnormalities such as del(7q) or trisomy 3 were found to be characteristic chromosomal changes in a subset of splenic MZBL. TP53 was deleted in 5/25 cases of splenic MZBL. Deletions involving band 13q14 were only rarely encountered, challenging a previous report that stated a dissociated D13S25-RB1 status as characteristic in splenic MZBL. There are fundamental differences between the different subtypes of marginal zone lymphomas as defined with current classification schemes. Splenic MZBL, in contrast to most other entities of B-cell NHL, seems to constitute a heterogeneous disease especially with regard to genetic alterations. del(10)(q22q24) could be of importance at least in a subset of this lymphoma entity.

[Genetic and biological features define two types of follicular non-Hodgkin grade 3 lymphoma]. / Genetische und biologische Unterschiede definieren zwei Varianten follikulärer Non-Hodgkin-Lymphome Grad 3.

In the REAL classification system, follicular lymphomas (FL) were subdivided into three grades depending on the number of blasts (6). In this study, we were interested in defining biological parameters possibly being important in the delineation of subgroups. Between 1990 and 1998, biological and cytogenetic investigations were performed on 91 FL. Clonal aberrations were found in all cases. The tumours were subclassified according to the blast content and the morphology of the centrocytes into 29 FL 1, 33 FL 2, 15 FL 3, and 14 FL 3 with a diffuse large B-cell lymphoma component (FL 3 + DLBL). They were characterised by classical cytogenetics, for their mitotic (MI) and proliferative (PI) indices, and CD10, bcl-2, and p53-expression. In contrast to FL 1 and FL 2, which showed a common genetic background with t(14;18), and only differed by their blast content and MI/PI, FL 3 (with or without associated DLBL) turned out to be an inhomogeneous group. 11 follicular lymphomas (with > 150 blasts/10HPF) still showed maturation to centrocytes. They were positive for CD10 and harboured the t(14;18) in 73%. These cases correspond to a "high grade" variant of centroblastic-centrocytic lymphoma according to the Kiel classification (FL 3a). In 18 cases with a follicular or follicular and diffuse growth pattern, the infiltrate consisted of centroblasts exclusively. These tumours were CD10+ in only 50% and were t(14;18)+ in only 22%. Secretory differentiation (clg+) was found in 44%. They were--with respect to primary and secondary chromosome aberrations--more comparable to a follicular variant of DLBL and hence, correspond to centroblastic lymphoma, follicular or centroblastic lymphoma, follicular and diffuse according to the Kiel classification (FL 3b). By histomorphological, biological and cytogenetic investigations, therefore, FL 3 can be delineated into two different biological subgroups with obviously different transformation pathways.

Exclusive detection of the t(11;18)(q21;q21) in extranodal marginal zone B cell lymphomas (MZBL) of MALT type in contrast to other MZBL and extranodal large B cell lymphomas.

Extranodal mucosa-associated lymphoid tissue (MALT)-type lymphomas and nodal and splenic marginal zone B cell lymphomas (MZBL) share morphological and immunophenotypic features with marginal zone B cells of reactive lymphoid tissues. Although displaying a similar immunophenotype, recent investigations suggest fundamental genetic differences among these subgroups. To determine the prevalence of the t(11;18) in a larger series of MALT-type lymphomas and to investigate a possible occurrence in other lymphomas, we screened 106 non-Hodgkin's lymphomas (NHL) by interphase cytogenetics using yeast artificial chromosome (YAC) probes flanking the breakpoint at 11q21. A signal constellation indicating a disruption in 11q21 and thus pointing to the presence of the t(11;18) was observed in 9 of 33 (27%) low-grade lymphomas of MALT type. The complete absence of t(11;18)-positive cells in 32 primary and secondary extranodal high-grade lymphomas suggests that low-grade lymphomas of MALT type characterized by the t(11;18) are unlikely to transform into high-grade tumors. The absence of tumor cells carrying the t(11;18) in nodal MZBL challenges the assumption that most, if not all, of these tumors represent the nodal manifestation of a so far undetected low-grade lymphoma of MALT type. The t(11;18) was not detected in a single case of 29 splenic MZBL investigated. This observation strengthens the view that splenic MZBL are biologically different from extranodal MZBL of MALT type.

A biological role for deletions in chromosomal band 13q14 in mantle cell and peripheral t-cell lymphomas?

Structural aberrations of chromosomal band 13q14 are frequent in B-cell chronic lymphocytic leukemia (B-CLL) and target a putative tumor suppressor gene in the genomic region between the RB1 gene and the genetic marker D13S25. Recently, it has been suggested that alterations of this particular region might also be of relevance for the pathogenesis of mantle cell lymphomas (MCL). We applied dual-color fluorescence in situ hybridization (FISH) using probes for the RB1 and/or D13S25 loci and screened a total of 236 B- and T-cell non-Hodgkin's lymphomas (NHL) for deletions occurring in this genomic region. In MCL, the high rate (12/32; 38%) of hemizygous deletions and especially a deletion pattern similar to B-CLL in four of the cases provide further evidence that a substantial proportion of MCL cases may share a common way of pathogenesis with B-CLL. In other B-cell NHL, the frequency of allelic loss affecting 13q14 was overall low. However, the finding of 13q14 microdeletions in seven cases without detectable alterations of chromosome 13 at G-banding analysis might indicate a possible involvement of this genetic region also for the lymphomagenesis of single cases of B-cell NHL other than B-CLL and MCL. In T-cell NHL, allelic loss at 13q14 was encountered in three of 13 peripheral T-NHL, NOS. Taking into account the very limited cytogenetic data yet available in this entity, our series provides further evidence that 13q14 changes might represent one of the most frequent genetic abnormalities in T-cell NHL.

t(1;2)(q21;p23) and t(2;3)(p23;q21): two novel variant translocations of the t(2;5)(p23;q35) in anaplastic large cell lymphoma.

Cytogenetic investigations in two cases of anaplastic large cell lymphoma (ALCL) showed novel variants of the classical (2;5)(p23;q35) translocation, namely a t(1;2)(q21;p23) and a t(2;3)(p23;q21). The tumor cells in both cases gave positive immunohistochemical labeling for ALK protein (with both monoclonal and polyclonal antibodies), demonstrating that these translocations induce aberrant expression of this kinase and suggesting that genes other than NPM can activate the ALK gene in ALCL. These two cases were shown by an in vitro kinase assay to express ALK kinases (104 kD and 97 kD, respectively), which differed in size from the classical NPM-ALK fusion product (80 kD). Moreover, ALK expression was confined to the cytoplasm of the tumor cells in each case, supporting the hypothesis that the observed nuclear localization of NPM-ALK in classical ALCL is not the site of oncogenic activity of the ALK kinase.

Jumping translocation of 1q as the sole aberration in a case of follicular lymphoma.

Cytogenetic and fluorescence in situ hybridization (FISH) studies in a case of follicular lymphoma grade III showed a "jumping translocation" of chromosome 1q21-qter to chromosomes Xq28 and 18q23, which resulted in a partial trisomy 1q as the only chromosome aberration. This case represents, to the best of our knowledge, the first report of a jumping translocation in a malignant lymphoma occurring as the sole aberration.

Trisomy 3 is not a common feature in malignant lymphomas of mucosa-associated lymphoid tissue type.

The genetic background of extranodal marginal zone B-cell non-Hodgkin's lymphoma (NHL) of mucosa-associated lymphoid tissue (MALT) type is poorly understood. In contrast to most entities of primary nodal lymphomas, few cytogenetic data are available, and gene rearrangements frequently encountered in and highly characteristic of certain entities of systemic NHL are absent in this type of lymphoma. Recently, it was suggested that MALT-type NHLs are associated with certain numerical chromosome aberrations and especially with trisomy 3. We performed an extensive study using a sensitive double (bicolor) fluorescence in situ hybridization technique for the analysis of trisomies for chromosomes 3, 7, 12, and 18 in 60 samples of low-grade and 45 high-grade MALT-type tumors. In the low-grade cases, trisomy 3 was found in a frequency of only 20%. High-grade lymphomas of MALT type were associated with trisomies 3, 7, 12, and 18 in 36, 20, 18, and 13% of the cases, respectively. Whereas no difference was encountered for trisomy 3 in primary and secondary/simultaneous high-grade lymphomas, +7 and +12 were associated with primary lymphomas, and a +18 was predominantly found in secondary/simultaneous high-grade NHL. These results challenge earlier reports describing a high frequency of +3 in low-grade MALT-type NHL and indicate a possibly different genetic evolution pattern of primary and secondary/simultaneous high-grade lymphomas of primary mucosal origin.

Chromosomal abnormalities in nodal and extranodal CD30+ anaplastic large cell lymphomas: infrequent detection of the t(2;5) in extranodal lymphomas.

To determine the significance of the t(2;5)(p23;q35) translocation in nodal and extranodal anaplastic large cell lymphoma (ALCL), we performed cytogenetic, molecular genetic, and immunohistochemical analyses of tumor tissues from 11 patients with CD30+ ALCL. Three of five patients with nodal ALCL had additional infiltration of the skin. Six patients had extranodal ALCL, two had primary intestinal ALCL, three had a primary cutaneous ALCL, and one had osseous ALCL. Cytogenetic investigation detected the t(2;5) in all patients with nodal ALCL but not extranodal ALCL. Tumor cells in t(2;5)+ lesions also stained immunohistochemically for p80NPM/ALK, whereas no staining for p80NPM/ALK was detected in extranodal ALCL. Two extranodal lesions had NPM/ALK fusion transcripts detected by nested reverse transcriptase-polymerase chain reaction. Fluorescence in situ hybridization analysis of these two lymphomas showed in one case a significant number (4%) of cells with a split hybridization signal, indicative of disruption of the NPM gene. Additional recurrent breakpoints observed in extranodal ALCL were 1p36, 6p25, and 8q24. Loss of genetic material occurred at 6q in one extranodal ALCL. Our results suggest that the t(2;5) more frequently plays a pathogenetic role in primary nodal than in extranodal ALCL and that this translocation may not be the primary event in some CD30+ ALCL.

A lymphohistiocytic variant of anaplastic large cell lymphoma with demonstration of the t(2;5)(p23;q35) chromosome translocation.

Cytogenetic investigations were performed in a case of a nodal malignant non-Hodgkin's lymphoma. Histopathological analysis from an involved lymph node as well as from a skin biopsy revealed a lymphohistiocytic variant of CD30-positive anaplastic large cell lymphoma (ALCL). A t(2;5)(p23;q35) chromosome translocation could be observed in all metaphases analysed. This finding was confirmed both by RT-PCR analysis of the NPM/ALK fusion protein and by positive staining with the p80(NPM/ALK) antibody. To the best of our knowledge, this is the first report of a t(2;5) documented by classic cytogenetics in the lymphohistiocytic variant of ALCL.

The cytomorphological spectrum of mantle cell lymphoma is reflected by distinct biological features.

Mantle cell (centrocytic) non-Hodgkin's lymphoma (MCL) is a malignant tumour with unique biological features. The pathogenesis of MCL seems to be strongly associated with aberrant function of the cell cycle. 110 cases of MCL have been analysed for their cytomorphological features, mitotic and proliferation indices, bcl-1 rearrangements, p53 expression patterns and DNA content by both interphase cytogenetic as well as DNA flow cytometric analyses. According to cytomorphology, three subtypes were recognized: a common, a lymphoblastoid and a pleomorphic variant of MCL. Blastic MCL subtypes were characterized by distinctly elevated mitotic and proliferation indices, frequent bcl-1 rearrangements at the MTC locus, and overexpression of p53. The most interesting finding, however, was a striking tendency of blastoid MCL subtypes to harbour chromosome numbers in the tetraploid range, a feature clearly separating these neoplasms from other types of B-cell NHL and possibly being related to its unphysiological expression of cyclin D1. Although characterised by a uniform immunophenotype and common biological background, MCL shows a broad spectrum of morphological features ranging from small cell to blastic types, and this spectrum is mirrored by distinct biological features.