The oncolytic effect in vivo of reovirus on tumour cells that have survived reovirus cell killing in vitro.

The use of oncolytic viruses has received considerable attention in recent years and many viruses have proved to be effective against a variety of cancer models and a few are currently being used in clinical trials. However, the possible emergence and outcome of virus-resistant tumour cells has not been addressed. We previously reported the effective use of reovirus against lymphoid malignancies, including the Burkitt's lymphoma cell line Raji. Here we isolated in vitro persistently infected (PI) Raji cells, and cells 'cured' of persistent reovirus infection ('cured' cells). Both PI and cured Raji cells resisted reovirus infection and cell killing in vitro. In vivo, the PI cells were non-tumorigenic in SCID mice, but cured cells regained the parental cells' ability to form tumours. Tumour xenografts from the cured cells, however, were highly susceptible to reovirus oncolysis in vivo. This susceptibility was due to the proteolytic environment within tumours that facilitates reovirus infection and cell killing. Our results show that persistent infection by reovirus impedes tumour development and that although PI cells cleared of reovirus are tumorigenic, they are killed upon rechallenge with reovirus. Both the PI and cured states are therefore not likely to be significant barriers to reovirus oncolytic therapy.

Leukocyte elastase in murine and human non-Hodgkin lymphomas.

Extracellular proteases play a crucial role in the invasive behavior of normal and transformed leukocytes. Thus far, however, most of the attention has been focused on members of the family of matrix metalloproteinases. In this work, we show that lymphoma cells can express leukocyte elastase (LE) and recruit the enzyme at their surface via ICAM-1. The expression of LE by lymphoma cells was augmented significantly by stimulation with IL-6 and IL-13, both of which also induced the expression of MMP-9. Although LE and IL-13 transcripts were detected in several non-Hodgkin's lymphomas, immunohistochemical analysis of lymphoma tissues also showed that LE was strongly expressed in infiltrating leukocytes. Given the spectrum of key molecules that can be cleaved by LE and that LE and MMP-9 are involved in the invasive behavior of normal or transformed leukocytes, our results raise the hypothesis that LE plays a crucial role in the multistep processes of inflammation and lymphoma metastasis.

Bone marrow pathology after bone marrow transplantation.

Trephine biopsy evaluation in patients who have undergone high dose chemotherapy with stem cell infusion performed by an experienced pathologist is important in evaluation of hematopoietical restitution, evaluation of complications such as graft rejection, infections, relapse or secondary neoplasms. In the first week after high dose chemotherapy or radiotherapy bone marrow aplasia is evident. In the case of accompanying myelofibrosis, fragmentation of reticular and collagen fibers is noticeable only following stem cell transplantation. The first precursors, which appear after the second or third week after transplantation are erythroid precursors found in small groups. Promyelocytes and myelocytes are observed between the second and fourth week, the ration off erythroid to myeloid populations at that time is 1:1. Megakaryocytes ate the last precursors to appear. Trephine biopsy evaluation is useful in diagnosis of graft rejection, especially in case of a discrepancy between the blood morphology examination and bone marrow cellularity. Granuloma formation is an unspecific finding, which requires diagnosis of infections. Residual disease can be recognized in histopathologic examination, or sooner, with immunophenotyping, cytogenetic examination using FISH or PCR. Secondary neoplasms to high dose radiotherapy or chemotherapy are usually lymphoproliferative processes connected with latent EBV infections, myelodysplastic syndromes and acute myeloblastic leukaemia.

Matrix metalloproteinases and their tissue inhibitors - expression, role and regulation in human malignant non-Hodgkin's lymphomas.

Human malignant non-Hodgkin's lymphomas (NHL) represent a heterogeneous group of neoplasms, which vary in their clinical behavior and pathophysiology. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been shown to play a role in the pathophysiology and clinical aggressiveness of human NHL. In this setting, MMP-9 and TIMP-1 appear to be the most important members of the MMP and TIMP families, and overexpression of both correlates with a poor clinical outcome of patients with NHL. MMP-9 and TIMP-1, however, act through different mechanisms and are produced by different cell types. Expression of both is upregulated by interleukin-6 (IL-6), a cytokine that is known as one of the factors involved in the pathophysiology of human NHL. In this review we summarize the complex regulation of MMP and TIMP expression in human NHL and propose a mechanism by which MMP-9, TIMP-1 and IL-6 may influence the biology of these tumors.

Interleukin-6 regulation of matrix metalloproteinase (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) expression in malignant non-Hodgkin's lymphomas.

We showed previously that human malignant non-Hodgkin's lymphomas (NHL) degrade extracellular matrix (ECM) components through the action of metalloproteinases and that elevated expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) correlated with a poor clinical outcome in patients with NHL. In the present study we sought to investigate whether there is any correlation between the expression of gelatinases (MMP-2 and MMP-9), TIMP-1, and the expression of cytokines and growth factors such as interleukin-1beta (IL-1beta), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGFbeta), and basic fibroblast growth factor (bFGF) in human NHL. In lymphoma tissues obtained from 32 patients, elevated expression of IL-6 correlated significantly with elevated messenger RNA (mRNA) levels of MMP-9, MMP-2, and TIMP-1. Moreover, in human lymphoid cell lines of B- and T-cell origin (Raji, Jurkat, and NC-37), IL-6 stimulated production of MMP-9 and MMP-2 but not TIMP-1. In the Matrigel invasion assay IL-6 significantly upregulated transmigration of Raji and Jurkat cells, which in turn was inhibited by recombinant human TIMP-1 and anti-MMP-9 and MMP-2 antibodies. We postulate that IL-6 may play a role in the clinical aggressiveness of human NHL by stimulating MMP production.

Expression of matrix metalloproteinases (MMP-2 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in acute myelogenous leukaemia blasts: comparison with normal bone marrow cells.

We compared the expression of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL-60, K-562 and KG-1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP-9 and/or MMP-2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP-1 and TIMP-2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP-1, the HL-60 cells also expressed TIMP-2. In contrast, normal steady-state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP-2 or MMP-9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP-9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP-1 and TIMP-2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP-2, the activated form of this enzyme was found in media conditioned by cells obtained from long-term cultures of normal and AML bone marrow adherent layers. Our finding of up-regulated production of gelatinases, TIMP-1 and TIMP-2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.

Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis.

A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. No corresponding increase in TIMP protein activity was detected by reverse zymography. This appears to result from the formation of TIMP/MMP complexes. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis.

Proteolytic activity of human non-Hodgkin's lymphomas.

This study was conducted to assess the net proteolytic activity of human non-Hodgkin's lymphomas (NHLs). We have compared the extracellular matrix (ECM)-degradative abilities of human NHLs, reactive lymphoid hyperplasias, and established lymphoid cell lines using Matrigel invasion and elastin degradation assays. The inhibition studies allowed identification of the classes of proteinases involved in ECM degradation. Our results indicate that lymphocytes and other leukocytes derived from both human NHLs and reactive lymphoid hyperplasias are capable of Matrigel penetration, but only cells derived from the high-grade human NHLs degrade elastin in vitro. Established lymphoid cell lines (both malignant and Epstein-Barr virus immortalized) do not produce MMP-9, do not penetrate the Matrigel, and do not degrade elastin. Moreover, in human NHLs, elastolytic activity is blocked by metalloproteinase inhibitors, while inhibitors of the other classes of proteolytic enzymes have only minor effects. This study identifies metalloproteinases as the most important class of proteinases involved in ECM degradation by NHLs. The previous studies suggest that, within this class, MMP-9 represents the key enzyme that plays a role in the biological aggressiveness of human NHLs.

Comparative analysis of the expression patterns of metalloproteinases and their inhibitors in breast neoplasia, sporadic colorectal neoplasia, pulmonary carcinomas and malignant non-Hodgkin's lymphomas in humans.

Matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play essential roles in the remodelling of the extracellular matrix (ECM). Results of in vivo and in vitro studies suggest that the balance between MMPs and TIMPs is altered in neoplasia, contributing to the invasive and metastatic properties of malignant tumours. In this study we have analysed the expression of five MMP genes and TIMP-1 and TIMP-2 in 37 benign and malignant lesions of human breast using Northern blot analysis. MMP-9 (92 kDa gelatinase) and MMP-11 (stromelysin 3) were most consistently expressed by carcinomas. Based on detection of either MMP-9 or MMP-11 mRNAs, we were able to distinguish between malignant and benign disease with a predictive accuracy of 90% with 94% sensitivity and 85% specificity. Subsequently, these results were compared with results for carcinomas of colon and lung and malignant non-Hodgkin's lymphomas (NHL). Elevated MMP-9 and TIMP-1 expression was observed in all four systems. MMP-11 characterised all carcinomas as well as carcinomas in situ but was not detectable in NHL. Our data therefore argue that there are remarkably similar patterns of specific functions involved in ECM remodelling that correlate with malignancy in different human tumours of different histogenesis. However, MMP-11 expression is a characteristic of tumours of epithelial origin that is not found in lymphoid neoplasia. Thus it suggests that MMP-11 may play a regulatory role in the invasion and metastasis of carcinomas.

Molecular detection of the (2;5) translocation of non-Hodgkin's lymphoma by reverse transcriptase-polymerase chain reaction.

The t(2;5)(p23;q35) translocation was initially identified in cases of anaplastic large-cell lymphoma (ALCL) that expressed the Ki-1 (CD30) antigen. We have recently cloned this translocation and shown it to encode a chimeric product consisting of the N-terminal portion of a nonribosomal nucleolar phosphoprotein, nucleophosmin (NPM), from chromosome 5, fused to the kinase domain of a novel transmembrane tyrosine-specific protein kinase, anaplastic lymphoma kinase (ALK), from chromosome 2. To better define the spectrum of lymphomas that contain this translocation, we have analyzed 70 cases of non-Hodgkin's lymphoma (NHL) for expression of the t(2;5)-derived NPM/ALK chimeric message by reverse transcriptase-polymerase chain reaction (RT-PCR). Using a previously described set of oligonucleotide primers, NPM/ALK chimeric transcripts were detected in 21 of 22 cases that contained the t(2;5) by cytogenetic analysis and in 10 of 48 cases that either lacked evidence of the t(2;5) or had unsuccessful cytogenetics. In all but 1 case, the NPM/ALK PCR products were of identical size and sequence, suggesting that the genomic chromosome breaks are clustered in a single intron in both NPM and ALK. The NPM/ALK-expressing cases were not confined to NHLs with anaplastic morphology and included 15 ALCLs, 6 immunoblastic lymphomas, and 10 diffuse large-cell lymphomas. Moreover, only slightly greater than half of the cases with anaplastic morphology and 59% of CD30-expressing cases were NPM/ALK positive. Thus, neither anaplastic morphology nor the expression of CD30 accurately predicted the presence of this molecular genetic subtype of lymphoma.

Immunoglobulin and T-cell receptor gene rearrangements in lesions of mucosa-associated lymphoid tissue.

Twenty-one cases of mucosa-associated lymphoid tissue (MALT) lesions have been analyzed by Southern blot for immunoglobulin heavy chain (IgH) and T-cell receptor beta chain (TcR beta) gene rearrangements (GR). The sites included colon, stomach, liver, nasopharynx, salivary and lacrimal gland, conjunctiva, tonsil, breast, and lung. Two of the lesions (parotid and conjunctiva) were malignant lymphomas and one case showed lymphoproliferative disorder. In the cases of malignant lymphomas, IgH GR were detected, and in the case of lymphoproliferative disorder, both IgH and TcR beta genes were rearranged. Among the remaining 18 cases, 9 showed inflammatory infiltrate, 3 lymphoid hyperplasia, 3 atypical lymphoid hyperplasia, 1 carcinoma of the tonsil, 1 breast carcinoma, and one was a sample of normal Peyer's patches. Among these 18 cases, 3 showed TcR beta GR, 6 showed double IgH and TcR beta GR, and 4 IgH GR. Often multiple rearranged bands were observed, composing 10-30% of the total DNA analyzed. The control tissue (Peyer's patches) showed no GR. Because IgH and TcR beta GR are used to determine monoclonal proliferations of T and B lymphocytes, which occur in malignant lymphomas, it is vital to determine the specificity of such a test. This report stresses the fact that in MALT lesions false-positive results are not uncommon and therefore the results of IgH and TcR beta GR studies have to be interpreted with caution. The presence of multiple GR in the inflammatory lesions indicates proliferation of minor monoclonal populations that can be detected with the use of Southern blot technology.

Expression pattern of metalloproteinases and their inhibitors changes with the progression of human sporadic colorectal neoplasia.

Several studies have implicated the extracellular matrix-degrading metalloproteinases (MMPs) as essential agents in tumor cell invasion and metastasis. In the present study, we have investigated the patterns of expression of a number of MMPs and their specific tissue inhibitors (TIMP-1 and TIMP-2) in human colonic tissue samples that represent various stages of progression from adenomas showing different degrees of dysplasia to adenocarcinomas. We assessed levels of mRNA by Northern blot analysis and the results were measured semiquantitatively by densitometry. In total, we analyzed nine adenomas of varying size and with varying degrees of dysplasia, three adenomas with adenocarcinoma (malignant polyps), and five adenocarcinomas. Although expression of MMP and TIMP mRNA was highly intercorrelated, transcripts for stromelysin 3 and TIMP-2 (high) showed the strongest relation to the neoplastic process. Detection of stromelysin 3 mRNA accompanied a diagnosis of severe dysplasia or malignancy, whereas levels of TIMP-2 (high) mRNA transcripts permitted finer distinctions on the neoplastic continuum. These data indicate changes within extracellular matrix acquired during the process of malignant transformation of human sporadic colorectal neoplasia.

Patterns of expression of metalloproteinases and their inhibitors in human malignant lymphomas.

Forty-two cases of malignant lymphomas were studied for the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) by Northern blot and in situ hybridization. The lymphomas were classified according to Working Formulation classification (25 high grade, 15 low grade, and 2 intermediate grade). The MMPs studied included: 72-kDa and 92-kDa gelatinases (type IV collagenases), interstitial collagenase, PUMP-1 (MMP-7), and stromelysins 1 and 3. TIMPs included TIMP-1 and TIMP-2. All but one case expressed TIMP-1, and TIMP-2 transcripts were detected in 35 cases. Among the MMPs, 92-kDa gelatinase was expressed most consistently (35 cases), whereas mRNA transcripts of 72-kDa gelatinase, interstitial collagenase, and PUMP-1 were detected in only a few cases. Stromelysins 1 and 3 mRNAs were not detected in any of the tumors studied. However, marked differences in the level of expression of certain MMPs and TIMPs were found among different grades of malignant lymphomas. The low grade tumors expressed low and relatively constant levels of TIMP-1, TIMP-2, and 92-kDa gelatinase transcripts, whereas high grade lymphomas displayed variable amounts of mRNAs for TIMPs and MMPs, with a trend toward elevated TIMP-1 and 92-kDa gelatinase mRNA levels. In situ hybridization localized TIMP-1 transcripts to stromal cells, while 92-kDa gelatinase transcripts were most abundant in "starry sky" macrophages and large lymphoma cells. Zymography showed that active 92-kDa gelatinase is present in tumor protein extracts and differences in the level of the enzymatic activity were seen between low and high grade lymphomas. Our data indicate that 92-kDa gelatinase and TIMP-1 expression by human malignant lymphomas may play an important role in controlling their biologic aggressiveness.

Expression of metalloproteinases and their inhibitors in primary pulmonary carcinomas.

Nine primary pulmonary carcinomas, one metastatic carcinoma, and two malignant pleural mesotheliomas have been analysed for the expression at the mRNA level of metalloproteinases (MPs) and tissue inhibitors of MPs (TIMPs). In situ hybridisation showed TIMP-1 and TIMP-2 transcripts predominantly over tumour stroma and gelatinases evenly distributed over both stromal and tumour cells. While both TIMP-1 and TIMP-2 were expressed in non-neoplastic lungs (NNL) as well as in carcinomas, stromelysin 3 (ST3), 92 kDa gelatinase and interstitial collagenase were expressed only by carcinomas. Expression of these MPs by carcinomas was independent of histologic type and such tumour features as fibrosis or necrosis. The consistent expression of ST3 by all of the carcinomas examined and absence of its expression in NNL indicates that ST3 production is likely associated with the malignant phenotype. However, since 92 kDa gelatinase and interstitial collagenase transcripts were found in some but not all tumour samples, their expression is not a uniform feature of pulmonary carcinomas. The possible prognostic significance of the expression of the latter two enzymes by carcinomas remains to be established.

Relationship between the clinical aggressiveness of large cell immunoblastic lymphomas and expression of 92 kDa gelatinase (type IV collagenase) and tissue inhibitor of metalloproteinases-1 (TIMP-1) RNAs.

Aberrant expression of secreted proteinases and their specific inhibitors is believed to represent an important factor in the pathogenesis of invasion and metastases of malignant neoplasms. Our previous data indicated a link between elevated expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and the clinical aggressiveness of malignant non-Hodgkin's lymphomas. Further studies are presented on eighteen cases of high grade, large cell immunoblastic lymphoma in which expression at the RNA level of TIMP-1 and the metalloproteinase, 92 kDa gelatinase, were analyzed. Factors that may influence production of 92 kDa gelatinase, such as necrosis, vascularity, proliferative activity, and extranodal extension, as well as clinical parameters, such as age and sex, stage, location, and survival were assessed. Statistical analysis showed that, although clinical stage was the most important predictor of survival, after controlling for age at diagnosis, levels of 92 kDa gelatinase transcripts added to the ability to predict survival.

Differentiation of human B-cell malignant lymphomas is independent of the octamer lymphoid specific binding factor (Oct-2).

We have shown previously that the presence and action of immunoglobulin gene promoter specific trans-acting factors correlates with the stages of 'differentiation' of human lymphoid neoplasms. The regulatory sequence described by us was located upstream of the octamer motif which is known to bind lymphoid specific trans-acting factor Oct-2. In the present study we attempted to establish if the Oct-2 factor was present in fresh human tissue of B-cell origin and if the levels of Oct-2 also correlated with the stages of human lymphoid differentiation. We applied DNA mobility shift assay using the same cases which we utilized in our previous work. We compared the levels of Oct-2 with the levels of ubiquitous octamer binding factor Oct-1. Oct-2 was present in all lymphoid cells of B-cell origin (from fresh surgical specimens and in long-term tissue cultured cells) with the exception of a pre-B-cell line NALM-6. The relative abundance of Oct-2 varied, however, and the ratio of Oct-2 to Oct-1 was variable in different types of B cells. This phenomenon did not correlate with the stages of differentiation of human lymphoid neoplasms. There was also no correlation between the expression of Oct-2 and levels of immunoglobulin-specific messenger RNAs. These findings indicate that the control of, immunoglobulin expression in relation to the differentiation of human B-cell neoplasms requires factors other than Oct-2.

Granulocytic sarcoma of the ovary. An unusual case presentation.

We report an unusual occurrence of granulocytic sarcoma presenting as an ovarian mass in a 46-year-old woman with a history of dysplastic nevus syndrome. During workup of the ovarian mass, the diagnosis of acute myelogenous leukemia was made. A fine-needle aspirate of the ovarian mass showed granulocytic sarcoma. The patient died of complications of the acute leukemia a short time later. A postmortem examination was performed, which confirmed the nature of the ovarian mass as a granulocytic sarcoma. Material was obtained for flow cytometry, immunohistochemical and histochemical studies, and electron microscopy. Ploidy analysis of the tumor showed it to be diploid with an S phase of 4.8% and a G2 + M ratio of 0.5%. To our knowledge, there is only one previous report of a primary ovarian presentation of granulocytic sarcoma, and only four cases in which granulocytic sarcoma was diagnosed by fine-needle aspiration cytology. The association between dysplastic nevus syndrome and acute myeloid leukemia in this case is discussed with reference to a review of the metachronous association between melanoma and leukemia as described in the literature.