Subcutaneous 'lipoma-like' B-cell lymphoma associated with HCV infection: a new presentation of primary extranodal marginal zone B-cell lymphoma of MALT.

BACKGROUND: Hepatitis C virus (HCV) infection has been linked to lymphoproliferative disorders. Marginal zone B-cell lymphoma (MZL) represents one of the most frequent lymphoma subtypes associated with HCV infection. We describe an unusual subset of HCV-associated MZL characterized by subcutaneous presentation. MATERIALS AND METHODS: A series of 12 HCV-positive patients presenting with subcutaneous nodules that revealed lymphoma infiltration at biopsy. Molecular analysis of immunoglobulin heavy chain (IGH) gene rearrangement and FISH investigations for t(11;18)(q21;q21) and t(14;18)(q32;q21) were carried out in nine patients. RESULTS: The 12 patients (median age 69.5 years), all with positive HCV serology, presented with single or multiple subcutaneous nodules resembling lipomas. Histologically the lesions showed lymphoid infiltrates, consistent with extranodal MZL of mucosa-associated lymphoid tissue (MALT). Functional IGH gene rearrangements were identified in nine tested patients, with somatic mutations in 82%, indicating a histogenesis from germinal center-experienced B cells. The t(11;18) was found in two of nine cases. Staging did not show any other lymphoma localization. In two patients, a response was achieved with antiviral treatment. Extracutaneous spread to MALT sites occurred in a case. CONCLUSIONS: Our observations expand the spectrum of HCV-associated lymphomas to include a subset of extranodal MZL characterized by a novel primary 'lipoma-like' subcutaneous presentation and indolent clinical course.

Lymph node reticulum cell neoplasm with progression into cytokeratin-positive interstitial reticulum cell (CIRC) sarcoma: a case study.

AIMS: To detail on sequential biopsies the morphological and immunohistochemical features of a case of primary lymph nodal fibroblastic reticulum cell (FBRC) tumour which progressed into a clinically aggressive cytokeratin-positive interstitial reticulum cell (CIRC) sarcoma. METHODS AND RESULTS: A 70-year-old female underwent surgical excision of an enlarged submandibular lymph node. The nodal architecture was effaced by a neoplastic proliferation of medium to large cells, round to oval to spindle in shape, growing in a storiform pattern. The tumour stained for vimentin, CD68, factor XIIIa, alpha1-antitrypsin, fascin and actin. Dendritic and endothelial cell markers were negative. A diagnosis of FBRC tumour was made by combining pathological and clinical data. The patient received no therapy but 5 months later the tumour relapsed, exhibiting a deceptively pleomorphic cytology, phenotypic changes (strong cytokeratin positivity), intense p53 expression and aggressive clinical course with fatal outcome. In-situ hybridization for Epstein-Barr virus was negative. CONCLUSIONS: We speculate that the morphological changes and p53 expression of the relapsing neoplasm might reflect tumour cell dedifferentiation, in keeping with the aggressive clinical course. The intense p53 expression suggests that this oncoprotein might also play a role in reticulum cell tumorigenesis.

Epstein-Barr virus-positive aggressive lymphoma as a consequence of immunosuppression after multiple salvage treatments for follicular lymphoma.

We report on a patient with follicular non-Hodgkin's lymphoma (NHL) who developed a fatal high-grade Epstein-Barr virus (EBV)-positive NHL after conventional chemotherapies. The sudden onset of the high-grade lymphoma was accompanied by increasing circulating EBV genome copies and was complicated by spontaneous rupture of the spleen. Splenic tissue was diffusely infiltrated by large B cells. In situ hybridization for Epstein-Barr-encoded RNA (EBER) 1-2 was positive in 70% of cells, and molecular analysis revealed the presence of EBV DNA and a monoclonal IgH gene rearrangement. This case shows that the immunosuppression of multiple treatments may induce uncontrolled reactivation of a latent EBV infection, contributing to high-grade transformation in heavily treated lymphoma patients.

Nijmegen breakage syndrome-associated T-cell-rich B-cell lymphoma: case report.

In 1981 Weemaes et al. first described the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by stunted growth, microcephaly, immunodeficiency, spontaneous chromosome instability, and a peculiar predisposition to cancer development. Most NBS-related malignancies are lymphomas, but their pathologic features have rarely been specified. We report here the case of a northern Italian 8-year-old child who, 2 years after the diagnosis of NBS, developed a diffuse large B-cell lymphoma (T cell-rich B-cell lymphoma variant). The histological and immunobiological features of the lymphoma population are analyzed and discussed in detail.

Chromosomal instability and telomere length variations during the life span of human fibroblast clones.

Growth characteristics, karyotype changes, and telomere length variations were analyzed during the life span of 12 anchorage-independent clones isolated from a xeroderma pigmentosum fibroblast strain. After an initial period of comparable active growth, all the clones showed a decline in the growth rate and finally entered a phase of replicative senescence; however, the number of population doublings and the time required to enter senescence varied among the clones. Repeated cytogenetic analyses during culture propagation showed the appearance of chromosome anomalies, mainly telomeric association (tas) and unbalanced translocations. In all the clones the percentage of abnormal mitoses increased with culture passage, but reached different levels (from less than 10% to about 100%). This finding indicates that the replicative block may be associated with differently altered cytogenetic patterns. Specific chromosome arms (5p, 16q, 19q, and 20q) were preferentially involved in tas, suggesting that alterations in chromosome ends may occur which predispose to fusion. In some clones it was possible to demonstrate the origin of marker chromosomes from the evolution of tas. Telomere length analysis by Southern blotting on DNA samples prepared from 7 clones and from the parental cell lines showed that the terminal restriction fragment (TRF) profiles were homogeneous in senescent parental cells and in the clones during the last part of their life in culture, regardless of the degree of karyotype abnormalities. The homogeneity of the TRF profiles supports the hypothesis of a critical telomere length at senescence.

Telomeric fusions in cultured human fibroblasts as a source of genomic instability.

In a human fibroblast clone we studied the evolution, during culture propagation, of a dicentric chromosome consisting of the end-to-end association of the short arm of chromosome 5 and the long arm of chromosome 16. Dual-color fluorescence in situ hybridization (FISH) with painting probes allowed us to define the structure of a variety of derivative chromosomes and to identify the mechanisms by which they originated. Asymmetric interchanges involving the intercentromeric region of the dicentric, bridge-breakage-fusion events, or breaks followed by sister chromatid fusion, originate unstable hetero- or homodicentric chromosomes with deletion or duplication; breakages not followed by reunion, or intradicentric recombination, presumably originate stable rearranged monocentric chromosomes. The variety of the derivatives is extremely large because the observed events may involve any site of the intercentromeric region, although the majority of them occurs after a break in 16qh. The results of this investigation document the evolution through successive steps of a telomeric fusion, a chromosome anomaly frequently observed in tumor and senescent cells. They also demonstrate that in cultured cells of normal origin, starting with this anomaly, various chromosomal mechanisms may produce translocations, duplications, and deletions. The karyotype instability produced by a telomeric fusion can be relevant for carcinogenesis because it may generate genetic changes critical in the multistep process of transformation.

Phenotypic heterogeneity in nucleotide excision repair mutants of rodent complementation groups 1 and 4.

Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.

Condensation anomalies and exclusion in micronuclei of rearranged chromosomes in human fibroblasts cultured in vitro.

Anomalies of chromatin condensation, such as fragmentation, uncoiling and pulverization, were observed in XP9UV25, a xeroderma pigmentosum fibroblast clone in which a high proportion of cells carried an end-to-end dicentric chromosome, dic (5;16) (p15.2;q24), that gives rise during propagation in culture to a variety of dicentric and monocentric derivatives. The coiling anomaly affected exclusively part of a rearranged chromosome, in particular the region previously involved in breakage events. The heterochromatic 16q region, which is a preferential breakpoint in the formation of dicentric and monocentric derivatives, was consistently the limit of the uncoiled or pulverized regions. This observation suggests that the anomalous chromatin behavior could derive from alteration of a region relevant for the correct condensation of the chromosome. In XP9UV25 the frequency of nuclei with associated micronuclei increased with time in culture, in parallel with that of mitoses with dicentric chromosomes. In situ hybridization with DNA probes specific for chromosomes 5 and 16 revealed hybridization signals in about 40% of micronuclei. Since the frequency of micronuclei is about ten times less than that of dicentrics, it is probable that only the rearranged chromosomes undergoing coiling anomalies are excluded in micronuclei.

Gene amplification in Chinese hamster DNA repair deficient mutants.

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2-5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.

Structural instability of a transmissible end-to-end dicentric chromosome in a xeroderma pigmentosum fibroblast clone.

Cytogenetic analysis was performed in a fibroblast clone (XP9UV25) selected for anchorage-independent growth from an XP strain of normal origin and characterized by the presence of clonal chromosome rearrangements. A dicentric chromosome involving the 5p and 16q telomeric regions was observed in XP9UV25 cells at the fifth passage from colony isolation and at successive passages. The specific anomaly was present with increasing frequency (from 22 to 60% of mitoses) during culture propagation, undergoing rearrangements that gave rise to: 1) (5;16) dicentrics with deletions or duplications of the intercentromeric region; 2) homodicentrics for chromosomes 5 or 16, either end-to-end associations or rearranged; and 3) derivative 5p+ and 16q+ monocentric chromosomes. The frequency of other anomalies involving other chromosomes was negligible. These findings represent the first demonstration that a telomeric association leads to a variety of balanced and unbalanced chromosome rearrangements. These rearrangements may result from asymmetric interchanges between sister chromatids, "bridge-breakage-fusion" events during cell division, breakage and reunion of isochromatids, and breakage followed by healing of the ends. The type of anomaly and the sequence of karyotypic changes we observed in the XP9UV25 clone and their mechanisms of origin may be the same as those occurring during transformation from diploidy to aneuploidy in neoplastic cells.

Identification of the eleventh complementation group of UV-sensitive excision repair-defective rodent mutants.

The drug-sensitive mutant UVS1, isolated from the Chinese hamster cell line CHO9, was previously found to complement the UV sensitivity of the excision repair-defective rodent mutants representative of groups 1 to 8 (Hata et al., Cancer Res., 51: 195-198, 1991; M. Numata et al., personal communication). Recently two new complementation groups of UV-sensitive CHO mutants, e.g., groups 9 and 10, have been identified (Stefanini et al., Cancer Res., 51: 3965-3971, 1991). In this paper we demonstrate that the repair defect in UVS1 cells is genetically different from those present in the mutants CHO7PV and CHO4PV, representing groups 9 and 10, respectively. Therefore, UVS1 represents a new complementation group of UV-sensitive rodent cell lines, the eleventh group.

Novel Chinese hamster ultraviolet-sensitive mutants for excision repair form complementation groups 9 and 10.

In this paper we demonstrate that the mutants CHO7PV and CHO4PV isolated by us from the CHO-K1 prol- cell line represent two new complementation groups of UV-sensitive excision repair-defective rodent mutants. We have classified the mutant CHO7PV as representative of Group 9 and CHO4PV as representative of Group 10. Cellular and biochemical characterization of these mutants indicates that they are moderately sensitive to a broad spectrum of mutagens (UV and mono- and bifunctional alkylating agents), partially unable to perform UV-induced DNA repair synthesis, and partially defective in the incision step of the DNA excision repair pathway and in the removal of the two main lesions caused by UV [cyclobutane pyrimidine dimers and (6-4) photo-products]. In terms of UV survival and incision, CHO4PV is apparently more defective than CHO7PV (40% and 50% of wild-type survival, respectively, and 55% and 75% of wild-type incision), whereas when repair DNA synthesis and lesion removal are compared, CHO7PV seems to be more severely affected (30% of wild-type unscheduled DNA synthesis in CHO7PV and 60% in CHO4PV). This suggests a subtlety in the relation between removal of these specific lesions and overall repair capacity and survival.

Cellular and genetic characterization of UV sensitive Chinese hamster mutants.

Results of cellular and genetic characterization of UV sensitive clones (UVs) isolated from CHO-K1 cell line are reported. The cross-sensitivity to agents inducing a variety of DNA lesions, the induction of chromosome aberrations and of 6-thioguanine and ouabain resistant mutants, the occurrence of methotrexate resistant cells were analyzed in clones showing different degrees of UV sensitivity. Genetic analysis was performed by complementation analysis of hybrids obtained by fusion of our mutants with UVs cells belonging to the six complementation groups (c.g.) so far identified. Three clones were assigned to c.g. 2, one clone to c.g. 5. Two clones (CHO7PV and CHO4PV), were able to complement each other and showed complementation after fusion with any of the six c.g.; these clones were considered carriers of two new mutations in genes presumably involved in DNA repair.