Cristina Pastore

Kaposi's sarcoma‐associated herpesvirus DNA sequences in AIDS‐related and AIDS‐unrelated lymphomatous effusions

Primary effusions presenting as the sole lymphoma localization are also known as body‐cavity‐based‐lymphoma (BCBL), and have been shown to carry Kaposis sarcoma herpesvirus (KSHV) DNA sequences. The aim of this study was a comparative analysis of the clinical, pathologic and molecular features of BCBL and lymphomatous effusions secondary to tissue‐based lymphomas occurring both in the general population and in HIV‐1‐infected individuals. All the lymphomatous effusion samples (seven AIDS‐related and nine AIDS‐unrelated) were subjected to an identical multiparameter investigation, including collection of clinical data, analysis of morphology and immunophenotype, as well as the study of viral sequences and genetic lesions. In six cases defined as BCBL (four AIDS‐related and two AIDS‐unrelated), the patients exhibited exclusive or predominant involvement of the body cavities. BCBL tended to display indeterminate phenotypes (4/6), whereas all AIDS‐related and AIDS‐unrelated lymphomatous effusions secondary to tissue‐based lymphomas consistently expressed B‐cell phenotype. Detection of KSHV DNA sequences was restricted to cases of BCBL (3/4 AIDS‐related and 1/2 AIDS‐unrelated), whereas EBV association (3/4) and expression of EBV‐encoded antigens (LMP‐1, 2/3; EBNA‐2, 1/3) were confined to the AIDS‐related BCBL. Overall, our results confirm that both AIDS‐related and AIDS‐unrelated BCBL preferentially associate with peculiar clinical, immunophenotypic and molecular features among lymphomatous effusions and therefore should be singled out as a specific clinico‐pathologic entity.

Distribution of human herpesvirus-8 sequences throughout the spectrum of AIDS-related neoplasia

Objective:AIDS frequently associates with certain malignancies, including Kaposis sarcoma, non-Hodgkins lymphoma (NHL), and anogenital neoplasia. In this study we aimed to define the frequency of infection by human herpesvirus (HHV)-8 throughout the spectrum of AIDS-related neoplasia in Europe. Design:A tumour panel representative of the distinct types of AIDS-related neoplasms was tested for the presence of HHV-8 DNA sequences. Autologous uninvolved tissues were also tested in selected cases. Methods:The presence of HHV-8 DNA sequences was assayed by a combination of polymerase chain reaction followed by oligohybridization and Southern blot hybridization of genomic DNA with an HHV-8-specific probe. Results:HHV-8 sequences were detected in 100% of AIDS-related Kaposis sarcoma (all 35 cases). Among AIDS-related NHL, HHV-8 sequences selectively clustered with body-cavity-based lymphomas (BCBL; all three cases), although they were consistently negative in small non-cleaved cell lymphomas (none in 18 cases), diffuse large cell lymphomas (none in seven), or anaplastic large cell lymphomas (none in three). No HHV-8 sequences were found in cases of anogenital neoplasia (out of 14) or Hodgkins disease (out of three). HHV-8 DNA sequences were also positive in the uninvolved skin of all six AIDS-related Kaposis sarcoma patients, but not in the circulating lymphocytes of a BCBL patient. Positivity for HHV-8 sequences occurred in patients belonging to all major AIDS risk categories. Conclusions:These data confirm that HHV-8 sequences associate at high frequency with selected types of AIDS-related neoplasia, namely Kaposis sarcoma and BCBL, although they are consistently absent in other types of AIDS-NHL and AIDS-related anogenital neoplasia.

Distribution of Kaposi's sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain

Summary. In this study we have tested the distribution of Kaposis sarcoma herpesvirus (KSHV) DNA sequences throughout the spectrum of lymphoid neoplasia in Italy and Spain. 180 cases of lymphoid malignancies representative of the major histologic and immunophenotypic categories of B‐ and T‐cell tumours were analysed by means of a polymerase chain reaction‐based assay. KSHV sequences were consistently absent in all categories of lymphoid malignancies studied, with the exception of a subset of B‐cell non‐Hodgkins lymphomas localizing in the pleural, pericardial or peritoneal cavities, and fulfilling the diagnostic criteria of body‐cavity‐based lymphoma. The selective and consistent association of KSHV sequences with cases of body‐cavity‐based lymphoma throughout the spectrum of lymphoid neoplasms suggests that KSHV may be involved in the pathogenesis of this peculiar type of lymphoid malignancy.

Establishment of HHV-8-positive and HHV-8-negative lymphoma cell lines from primary lymphomatous effusions.

Primary lymphomatous effusions are represented by cases of non‐Hodgkins lymphoma (NHL) which grow in liquid phase in the serous body cavities in the absence of solid tumour masses. Based on morphologic, immunophenotypic, virologic and genotypic features, primary lymphomatous effusions are distinguished into body cavity–based lymphoma (BCBL), Burkitts lymphoma (BL) and immunoblastic large‐cell lymphoma. The histogenesis and pathogenesis of primary lymphomatous effusions are virtually unclarified. In this study, we have established 2 cell lines (CRO‐AP/1 and CRO‐AP/2) representative of 2 distinct categories of primary lymphomatous effusion, BCBL (CRO‐AP/2) and BL (CRO‐AP/1). Both CRO‐AP/1 and CRO‐AP/2 carry monoclonal re‐arrangements of the immunoglobulin genes which are identical to those of the respective parental tumours. Consistent with its BCBL origin, CRO‐AP/2 is characterised by a non‐B, non‐T phenotype and harbours infection by HHV‐8 (approx. 100 viral copies/cell) and Epstein‐Barr virus. Conversely, CRO‐AP/1 expresses several B cell–associated antigens, lacks HHV‐8 infection and carries the genetic hallmark of BL, i.e., a chromosomal breakpoint of band 8q24. CRO‐AP/1 and CRO‐AP/2 may be valuable for the biologic characterization of primary lymphomatous effusions, particularly since the number of available cell lines derived from such lymphomatous effusions is extremely limited. Int. J. Cancer 73:562–569, 1997.

Molecular heterogeneity of B-lineage diffuse large cell lymphoma

B‐lineage diffuse large cell lymphoma (B‐DLCL) arising de novo is characterized by a marked degree of clinical heterogeneity. To determine whether or not the clinical heterogeneity of de novo B‐DLCL is reflected by heterogeneity in the molecular features of these tumors, we investigated the pattern of distribution of several genetic lesions in 70 cases of de novo B‐DLCL at diagnosis. The panel of genetic lesions tested comprised the molecular alterations most frequently detected in B‐DLCL, including rearrangements of BCL2, BCL6, and MYC as well as deletions of 6q and mutations of TP53. One or more genetic lesions were detected in 39/70 cases of B‐DLCL. Isolated structural alterations of BCL2, BCL6, 6q or TP53 were detected in 8/70, 10/70, 11/70, and 3/70 cases, respectively. No isolated MYC lesions were detected. Six cases carried different combinations of two genetic lesions, including lesions of BCL2 + BCL6 (1 case), BCL2 + MYC (1 case), BCL2 + 6q (2 cases), or BCL6 + 6q (2 cases). One case had accumulated three genetic lesions, namely a rearrangement of BCL2 and BCL6 and a mutation of TP53. Overall, these data show that multiple distinct patterns of genetic lesions may associate with de novo B‐DLCL, indicating that the molecular pathogenesis of this group of lymphomas is characterized by a high degree of molecular heterogeneity. Genes Chromosom Cancer 16:21–30 (1996).

Analysis of microsatellite instability in chronic lymphoproliferative disorders.

Microsatellite instability (MSI) represents one specific pattern of genomic instability and is one of the genetic lesions most frequently detected in human neoplasia. Although MSI has been found to be associated with a wide variety of solid cancers, its involvement in lymphoid malignancies is virtually unexplored. In this study, we have investigated the presence of MSI in chronic lymphoproliferative disorders by comparing the pattern of nine microsatellite repeats (two tetranucleotides, two trinucleotides, and five dinucleotides) on autologous germline and tumor DNA of 23 patients, including 17 with B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), four with hairy cell leukemia, one with lymphoplasmacytoid lymphoma, and one with T-cell chronic lymphocytic leukemia. All samples at diagnosis displayed a germline pattern of the microsatellites examined, thus suggesting that MSI is not involved in the pathogenesis of these lymphoproliferations. Also, no microsatellite alterations were observed in consecutive samples of B-CLL/SLL obtained from the same patient at various stages of the disease both before and after chemotherapy. Conversely, alterations in 3/9 microsatellite repeats were detected in one case of Richters syndrome which had evolved from a pre-existent B-CLL/SLL phase. Overall, the low frequency of MSI among chronic lymphoproliferative disorders adds further weight to the common view that the mechanisms and patterns of genomic instability in lymphoid neoplasia differ markedly from those commonly observed in solid cancers.

Point mutations of the BCL‐6 gene in Burkitt's lymphoma

BCL‐6 codes for a transcription factor implicated in chromosomal translocations of diffuse large cell lymphomas. Recent evidence indicates that BCL‐6 may also be altered by mutations of its 5′ non‐coding regions. In this study we have investigated the distribution of BCL‐6 5′ mutations among 35 Burkitts lymphoma cases representative of the epidemiologic variants of the disease. Mutations were detected in 6/21 (28.6%) sporadic Burkitts lymphomas and 7/14 (50%) endemic Burkitts lymphomas. These data expand the spectrum of B‐cell non‐Hodgkins lymphomas associated with BCL‐6 5′ mutations and have implications for the pathogenesis, histogenesis and clinical monitoring of Burkitts lymphoma.

Human herpesvirus type-8 (HHV-8) in haematopoietic neoplasia.

Human herpesvirus type-8 (HHV-8) is a lymphotropic herpesvirus originally identified in Kaposis sarcoma. Among lymphoproliferative disorders, HHV-8 infection is restricted to body-cavity-based lymphoma (BCBL) and multicentric Castlemans disease (MCD). BCBL are B-cell lymphomas growing in liquid phase in the body cavities and most frequently associated with AIDS. BCBL express indeterminate phenotypes, in all cases are associated with HHV-8 infection, and frequently carry Epstein-Barr virus genomes in the absence of c-MYC rearrangements or other genetic lesions characteristic of B-cell lymphomas. The clinical outcome of BCBL is poor with a median survival of only few months. MCD is an atypical lymphoproliferative disorder which displays marked vascular hyperplasia and is commonly associated with Kaposis sarcoma. HHV-8 infection occurs in 100% of AIDS-related MCD and in approximately 40% of AIDS-unrelated cases. Overall, the consistency of HHV-8 infection in BCBL and MCD, its selectivity throughout the spectrum of lymphoproliferative disorders and the high copy number of HHV-8 DNA sequences in infected cells suggest that the virus plays a pathogenetic role in these disorders.

Analysis of human herpesvirus type 8 infection in AIDS-related and AIDS-unrelated primary central nervous system lymphoma

Human herpesvirus type 8 (HHV-8) has been proposed as a pathogenetic factor for immunosuppression-associated primary central nervous system lymphoma (PCNSL). To verify this hypothesis, HHV-8 infection was investigated in 31 persons with PCNSL (16 AIDS-related, 15 AIDS-unrelated) and in 30 persons with systemic B cell non-Hodgkins lymphomas (B-NHL; 15 AIDS-related, 15 AIDS-unrelated). All subjects with PCNSL scored negative by single-step polymerase chain reaction (PCR), suggesting a tumor virus load of <100 viral copies/200,000 human haploid genome equivalents (HHGE). By applying Poisson assumptions to nested PCR, 16 of 31 persons with PCNSL were devoid of HHV-8 sequences: 1 subject with AIDS and PCNSL had 1-100 viral copies/200,000 HHGE, and 14 with PCNSL had <1 viral copy/200,000 HHGE. Similarly, 10 of 30 persons with systemic B-NHL were devoid of HHV-8 sequences; 20 had <1 viral copy/200,000 HHGE. The extremely low levels of infection rule out a role of HHV-8 in PCNSL pathogenesis and are consistent with HHV-8 infection of bystander cells contaminating the tumor clone.

Microsatellite instability in KSHV/HHV-8 positive body-cavity-based lymphoma

Body-cavity-based lymphoma (BCBL) is a rare non-Hodgkin-s lymphoma (NHL) type growing in liquid phase in the body cavities. Virtually all BCBL associate with Kaposis sarcoma-associated herpesvirus/human herpesvirus type-8 infection in the absence of molecular alterations typical of other NHL categories. Microsatellite instability (MSI), a specific variant of genomic instability frequently associated with human cancers, has not been tested in BCBL thus far. In this report, we have analyzed the presence of MSI in the tumor cells of a male patient affected by AIDS-related BCBL. The genomic configuration of 12 distinct microsatellite loci was investigated by polymerase chain reaction amplification of DNA obtained from the patients tumor cells and from autologous peripheral blood mononuclear cells. MSI was observed at 4/12 microsatellite repeats tested. Absence of the germline allele at one microsatellite locus mapping to chromosome X indicates that, in this BCBL case, MSI represents a clonal genetic lesion affecting virtually all tumor cells. These data suggest that MSI may be potentially involved in the pathogenesis of AIDS-related BCBL.