Mapping of MYC breakpoints in 8q24 rearrangements involving non-immunoglobulin partners in B-cell lymphomas.

Chromosomal translocations joining the immunoglobulin (IG) and MYC genes have been extensively reported in Burkitt's and non-Burkitt's lymphomas but data concerning MYC rearrangements with non-IG partners are scarce. In this study, 8q24 breakpoints from 17 B-cell lymphomas involving non-IG loci were mapped by fluorescence in situ hybridization (FISH). In seven cases the breakpoint was inside a small region encompassing MYC: in one t(7;8)(p12;q24) and two t(3;8)(q27;q24), it was telomeric to MYC whereas in four cases, one t(2;8)(p15;q24) and three t(8;9)(q24;p13) it was located in a 85 kb region encompassing MYC. In these seven cases, partner regions identified by FISH contained genes known to be involved in lymphomagenesis, namely BCL6, BCL11A, PAX5 and IKAROS. Breakpoints were cloned in two t(8;9)(q24;p13), 2.5 and 7 kb downstream from MYC and several hundred kb 5' to PAX5 on chromosome 9, joining MYC to ZCCHC7 and to ZBTB5 exon 2, two genes encoding zinc-finger proteins. In these seven cases, MYC expression measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) was significantly higher when compared to that of patients without 8q24 rearrangement (P=0.006). These results suggest that these rearrangements are the consequence of a non-random process targeting MYC together with non-IG genes involved in lymphocyte differentiation and lymphoma progression.

Clinical and biological relevance of single-nucleotide polymorphisms and acquired somatic mutations of the BCL6 first intron in follicular lymphoma.

Genetic modifications of the BCL6 gene in lymphoma include translocations, deletions, and somatic mutations (SM) of the 5' noncoding region. Three single-nucleotide polymorphisms (SNPs) of the major mutation cluster region (MMC) have been reported, including two substitutions (397G/C, 502G/A) and one deletion (520DeltaT). Clinical and biological relevance of these SNPs are unknown. Based on a case-control study, BCL6 SNPs frequencies were assessed in 97 t(14;18) follicular lymphomas (FL) and in 54 lymphomas with 3q27 rearrangement. Allele frequencies were similar in the FL and controls groups. The 397 G/C genotype was correlated to a higher-grade transformation risk (P=0.02). SM were observed in 39.1% of FL and were characterized by a clustering distribution (hot spots spanning position 420-435, 106-127, and 590-600). No correlation between genotypes or acquired mutational status and BCL6 expression was demonstrated. However, gel mobility-shift assays, using SNPs containing probes show results representative for protein/DNA complexes. This study demonstrates that the first BCL6 intron is a highly variable region as a consequence of both SNP and SM, which may contribute to biology and outcome of FL.

Establishment and characterisation of a human glioma cell line.

A new cell line, CB109, has been established from a human glioblastoma multiforme. The cytoskeleton was positive for glial fibrillary acidic protein, vimentin and fibronectin. Hyaluronan (HA) and the HA-binding protein hyaluronectin (HN) were expressed in the cell cytoplasm and in the extracellular matrix of spheroids and plated cells. Hyaluronidase did not prevent spheroid formation suggesting that HA was not involved in the cell-cell adhesion. HA precoating prevented cell adherence to the plates and favoured spheroid formation. HA was secreted in relatively large amounts into the culture medium. High performance liquid chromatography demonstrated that HA was in the high molecular weight form. The rate of HN secretion by cells was very low. Basic fibroblast growth factor significantly increased the proliferation in vitro and tumour growth after grafting into nude mice. The epidermal growth factor receptor was not expressed on cultivated CB109 cells. Cytogenetic analysis showed polysomy 7, structural rearrangement of chromosome 10 short arm and a translocation 13q13-q14 without detectable alteration of the RB gene.

Effect of aging on neointima formation and hyaluronan, hyaluronidase and hyaluronectin production in injured rat aorta.

Few studies have examined the effect of aging on arterial wall response to injury, and the results are discordant. Moreover, the effect of aging on hyaluronan synthesis in injured vessels is unknown. The aim of this present study was to determine the effect of aging on neointima formation and hyaluronan (HA), hyaluronidase and hyaluronectin production in injured rat aorta. Aorta was analysed in sham-operated rats (group D0) and 14 (D14) and 28 (D28) days after injury using biochemical and immunohistochemical techniques. Uninjured aorta of old rats was more thickened than that of young rats; it showed a decreased number of arterial smooth muscle cells (ASMC) and was characterized by HA accumulation in the intima and increased hyaluronidase activity. Intima-media wet weight was significantly increased in young rats at D14 and D28 but remained unchanged in old rats. DNA content was significantly enhanced at D14 in both young and old rats. DNA decreased slightly in young rats at D28 but significantly in old rats to return to control level. HA content and hyaluronidase activity in the intima-media were markedly increased in young rats at D14 (+148% and +116% respectively) but slightly in old rats (+23% and +15% respectively). Both HA and hyaluronidase activity continued to increase at D28, but remained more produced in young rats. The immunohistochemical analysis showed the formation of a thickened neointima in young rats, which was associated with strong expression of HA and HN. Neointima of old rats was reduced; it also showed strong expression of HA and HN but their distributions were different from those observed in neointima of young rats. In conclusion, aorta of old rats showed an increased amount of HA in the intima and elevated activity of hyaluronidase. Injury induced formation of a significant neointima in young rats but not in old rats. This was correlated with more HA and hyaluronidase production in injured aorta of young rats. As HA is considered to increase extracellular matrix space and to promote ASMC proliferation and migration, our findings suggest that HA may be implicated in intima thickening with age and after injury.

Hyaluronan and hyaluronectin production in injured rat thoracic aorta.

The aim of our study was to investigate the production of hyaluronan (HA) by the intima-media during the sclerotic response to aortic injury with a catheter balloon in the rat. In addition we analyzed, for the first time in this model, the production of a glycoprotein (hyaluronectin, HN) which binds specifically to HA. HA and HN were analyzed in control (D0), 14 (D14) and 28 (D28) days after injury using biochemical and immunohistochemical techniques. Intima-media DNA content and wet weight increased significantly on D14 and declined on D28 (but remained significantly increased in comparison to controls). HA content (median in D0 = 448 ng) increased significantly on D14 (2P < 0.04) and on D28 (2P < 0.02). HN content (median in D0 = 920 ng) increased significantly on D14 (2P < 0.05) but decreased on D28 to return to the control level. On D0 the amount of HN was about 3 times higher than that of HA (median ratio HA/HN = 0.34). The ratio remained unchanged on D14 but significantly increased on D28 (2P < 0.02). HPLC and Western blotting showed no difference between HN extracted from normal aorta and HN extracted from injured aorta at D14. Different isoforms of HN were present in both cases, ranging from 400 to 45 kDa. The HA increase on D14 and D28 was not related to a change in hyaluronidase activity of aortic tissue. Immunohistochemical analysis showed at D0 a small amount of HA around arterial smooth muscle cells (ASMC) in media, at D14 more HA was localized around and between ASMC in media and neointima but at D28 it was localized mainly near the vessel lumen. HN formed all the time (D0, D14 and D28) a continuous layer localized near the vessel lumen. In vitro studies showed that production of HA and HN was stimulated when ASMC proliferate and HA at high concentrations (1-100 micrograms/ml) reduced, in a dose dependent manner, ASMC growth. In conclusion our results show that both neointima formation in vivo and ASMC proliferation in vitro correlated with increased HA and HN production. This suggests that HA and HN are probably involved in the formation of neointima. On the other hand, the finding that HA continued to increase in the aorta when neointima decreased and that high concentrations of HA reduce ASMC proliferation in culture suggest that HA might be involved in the regression of neointima.

Immunoreactivity of boronated antibodies.

Boronated antibodies have already been evaluated as agents in neutron capture therapy. Because the boronation procedure may alter the properties of the antibody it is important to study the immunoreactivity of the conjugated antibody before in vivo use. In our studies of two dextran-boronated monoclonal antibodies, anti-glial fibrillary acidic protein antibody, and anti-hyaluronectin antibody, we have used ELISA and immunohistological methods to determine antibody activity and specificity. A ten-fold decrease in activity was observed for both antibodies in ELISA, and non-specific interactions were seen in both immunohistological and ELISA procedures. The boron compound used was shown to be at least partly responsible for these non-specific interactions.

Specific monoclonal antibodies to glial fibrillary acidic protein (GFAP).

Anti-glial fibrillary acidic protein (GFAP) monoclonal antibody has been obtained by fusing SP2/O myeloma cells with splenic cells from mice immunized with human GFAP. It belongs to the IgG1 class and it recognizes an epitope on GFAP which is shared by each fragment of the protein. Immunohistological studies show that the epitope characterized is absolutely specific for GFAP.

Hyaluronectin: detection with monoclonal antibodies in human tumors.

Two monoclonal IgG1 antibodies (MAbs) were raised against human brain hyaluronectin (HN) and used to characterize tumor HN. They were screened using an enzyme immunological technique (ELISA) combined with the HN property of specific binding to hyaluronic acid. They were shown to detect two different epitopes (HN1 and HN2) in human normal brain as well as in most tumors. Both HN1 and HN2 epitopes were found associated with mesenchymal benign or neoplastic proliferations (e.g. connective areas of fibroadenomas, extracellular matrix of fibrosarcomas) and with reactive connective tissue (e.g. stroma reaction of carcinomas, ground substance of gliomas). The results corresponded with those previously obtained with polyclonal rabbit antibodies and confirmed that HN is a constant marker of desmoplasia. Thus anti-HN MAbs recognize an antigen that is associated with tumor development and will be suitable for targeting.

[Glial fibrillary acidic protein and central nervous system tumors. Immunohistochemical study of a series of 207 cases. 2: Medulloblastomas. Hemangioblastomas. Other tumors. Discussion]. / Protéine gliofibrillaire acide (GFA) et tumeurs nerveuses centrales. Etude immunohistochimique d'une série de 207 cas. IIe Partie: Médulloblastomes. Hémangioblastomes. Autres tumeurs. Discussion.

The presence of glial fibrillary acidic protein (GFA) was tested in biopsy or autopsy specimens of 207 human central nervous system tumors. Samples were fixed, paraffin-embedded and GFA was detected using the peroxidase-antiperoxidase technique. The second part of this study dealt mainly with medulloblastomas and hemangioblastomas. Of the 17 cerebellar medulloblastomas, 15 contained less than 5% GFA positive cells. These were classified according to the Mannoji et al., 1981, criteria. Ten medulloblastomas contained type 1, 2 and 3 cells; 1 was composed of only type 1 cells, and 4 were composed of only type 3 cells. Of the 8 cerebellar or medullary hemangioblastomas, 3 contained only a few GFA positive cells which were, in two cases stroma cells and in another, astrocytes. GFA positive cells were found in 7 oligoastrocytomas, 3 gangliogliomas and 1 hamartoma of the hypothalamus. Similarly, a GFA positive astrocytic differentiation was found in a pineocytoma. In a case of tuberous sclerosis no GFA was found in the giant cells of cortical tubers. Nor was GFA observed in one intracerebral tumor of tuberous sclerosis. GFA was not found in 5 meningiomas, 4 oligodendrogliomas, 4 pituitary adenomas, 3 neurinomas of the VIII cranial nerve, 1 primary cerebellar malignant lymphoma, 1 familial lymphohistiocytosis with cerebellar involvement and 4 brain metastases. The authors also include diagnostic, histogenetic and nosological comments -about neuroglial tumors. The findings in the two parts of this study fail to establish a correlation between GFA positivity and histological grade of astrocytomas and glioblastomas.

[Glial fibrillary acidic protein and central nervous tumors. Immunohistochemical study of a series of 207 cases. 1: Astrocytomas. Glioblastomas. Ependymomas. Papillomas of the choroid plexus]. / Protéine gliofibrillaire acide (GFA) et tumeurs nerveuses centrales. Etude immunohistochimique d'une série de 207 cas. 1re partie: Astrocytomes. Glioblastomes. Ependymomes. Papillomes des plexus choroïdes.

The presence of glial fibrillary acidic protein (GFA) was tested in biopsy or autopsy specimens of 207 human central tumors. Samples were fixed, paraffin-embedded and GFA was detected using the peroxidase-antiperoxidase technic. The first part of this study dealt mainly with glioblastomas (36 cases) and astrocytomas (87 cases). Tumors were classified in three categories according to their histological grade: 24 astrocytomas grade I and II, 63 astrocytomas grade III and IV and 36 glioblastomas (grade IV). For each of these tumors GFA positive cells were counted in 5 different fields using objectives of 2,5, 10 and 25. The results were as follows: less than 50% GFA positive cells were found in 22 out of 24 low grade astrocytomas, 49 out of 63 high grade astrocytomas and 32 out of 36 glioblastomas. Conversely, over 50% GFA positivity was found in 2 benign astrocytomas, 14 malignant astrocytomas and 4 glioblastomas. In 3 high grade tumors (2 astrocytomas, 1 glioblastoma) GFA positivity was found to be over 75%. Of the 17 ependymomas, 14 were GFA positive; however, no correlation could be established between the degree of GFA positivity and histological grade. The three papillomas of the choroid plexus of the lateral ventricles found in children were GFA negative. In adults, a GFA positive focus of ependymal differentiation was found in a papilloma of the choroid plexus in the posterior cerebral fossa.

[Spontaneous bone marrow micrometastasis of a cerebral glioma. Immunohistochemical diagnosis in a biopsy sample and review of the literature]. / Micrométastase ostéomédullaire spontanée d'un gliome cérébral. Diagnostic immunohistochimique sur un prélèvement biopsique et revue de la littérature.

A 55 year-old woman was admitted to hospital in January 1981 with transient expressive dysphasia. Past personal history was unremarkable except for a six-month history of renal colic and thrombophlebitis in the veins of the right leg. Computed tomographic scan of the head and carotid angiogram revealed a left calcified temporoparietal tumor. Because of pulmonary embolism it was decided to refute a cerebral biopsy. The patient also declined radiotherapy. In May 1983, a thorough workup revealed an incomplete fracture of the first lumbar vertebra and a diffuse demineralization of the rachis and pelvis. Four weeks later she developed temporal epilepsy and pulmonary embolism. A whole brain irradiation (60 Gy) was performed in August 1983. The patient's condition remained clinically stable until December 1984 when she was readmitted to hospital with a severe weight loss, diffuse osseous pain and pancytopenia. A bone marrow biopsy from the iliac crest showed a diffuse tumor involvement. Peroxidase-antiperoxidase staining using monoclonal antiserum to glial fibrillary acidic protein was strongly positive in numerous tumors cells. The pathological diagnosis was bone marrow metastasis by glioma. She died in March 1985, 4 years and 3 months after the first admission to hospital. Autopsy was not performed. A literature search reveals only 9 cases of extraneural spreading of astrocytomas and glioblastomas in the absence of previous craniotomy with post-mortem examination. The authors also comment on the clinical, pathological and histogenic aspects of extraneural metastasis of gliomas.

[Nervous system tumour proteins. G.F.A. and hyaluronectin (author's transl)]. / Les proteéines des tumeurs du système nerveux. G.F.A. et hyaluronectine.

G.F.A. (gliofibrillary acid protein) and hyaluronectin studied here are two antigens defined by the heteroantibodies which appear in the serum of the immunised animal. G.F.A. is associated with gliofilaments and is considered as an astrocyte marker, either normal or tumoral. Hyaluronectin, is found in the Ranvier nodes and at the periphery of neurones. It is not specific to the nervous system. It is found in all tissues and tumors, and in particular marks the intercellular spaces of gliomas, where it is associated with hyaluronic acid for which it has a great affinity. Serum estimation of these Proteins is being undertaken with the view to a clinical application.

[Production of monoclonal antibodies against gliofibrillary acid protein (GFAP) and their specificity]. / Obtention d'anticorps monoclonaux anti-protéine gliofibrillaire acide (GFAP) et étude de leur spécificité.

Splenic Mice cells immunized with glial fibrillary acidic protein (GFAP) were fused with SP 2/0 myeloma cells. After screening and cloning we obtained two types of hybridomas. Some of them secrete IgG class antibodies, the others IgM class antibodies. The specificity of these antibodies has been tested by three immunoenzymatic methods. The results are that IgG monoclonal antibodies identify an astrocyte-GFAP specific epitope and IgM monoclonal antibodies cross-react with a common epitope to GFAP and vimentin.

Therapeutic efficacy of intralesional 131I-labelled hyaluronectin in grafted human glioblastoma.

The grafted human glioblastoma cell CB109 was used as a model for intralesional therapy with 131I-labelled hyaluronectin glycoprotein (131I-HN).131I-HN bound specifically to in situ hyaluronic acid (HA), a main component of the extracellular matrix which is involved in tumour invasion. Labelling experimental conditions were determined and, finally, 25 microCi/microgHN, 1 microg chloramine-T/microgHN and a 60-s stirring period provided a 131I-HN preparation with an optimal affinity for HA (64% compared to unlabelled HN). Following intratumoral injection, 131I-HN was retained with a limited diffusion outside the tumour. On day 4 the radioactivity concentrated in the tumour was still 25 times greater than that in the liver, spleen and kidneys combined. For therapeutic assays, 65 microCi 131I-HN was injected into the tumour, resulting in a delivery of 6.8 Gy over a 7-day period. Controls received unlabelled HN, heat-inactivated HN, a mixture of inactivated HN plus free 131I or no treatment (six animals per group). Tumour volumes were evaluated every second day from treatment day and the rate of tumour growth was expressed as a ratio of tumour size at time intervals to the tumour size at the time of injection. Growth curves were compared: heat-inactivated with or without free 131I had no anti-tumour effect. Unlabelled HN-injected tumours had a slightly slower growth rate than untreated tumours (p < 0.02) and growth rate of 131I-HN-injected tumours was much lower (p < 0.00002). A pronounced inhibitory effect with intralesional 131I-labelled HN injection resulted from a combination of a) blockage of HA, a proliferation facilitating factor, and b) local irradiation of tumoral tissue, while uptake in normal tissues was minimized.

Staining of hyaluronan in rat cerebellum with a hyaluronectin-antihyaluronectin immune complex.

The presence of hyaluronan was studied histochemically in the adult rat cerebellum. We used the hyaluronectin--antihyaluronectin immune complex technique based on the high affinity of hyaluronectin for hyaluronan. The immune complex was prepared with hyaluronectin from a human brain extract and an anti-hyaluronectin monoclonal antibody, which does not react with rat hyaluronectin. This is a specific probe for detecting hyaluronan in rat tissues without any reaction for tissue hyaluronectin. Hyaluronan was found at the nodes of Ranvier, in the perineuronal microenvironment of the deep nuclei and at the Purkinje cells surrounding the initial segment of the axon. It was located at the same places as hyaluronectin, in areas specialized in ion exchanges and neurotransmission. This suggests that the hyaluronectin-hyaluronan complex could be involved in these processes. The immune complex technique with anti-hyaluronectin monoclonal antibody thus seems to be a specific and valuable tool for investigations of the distribution of hyaluronan in the rat cerebellum.

Hyaluronan digestion and synthesis in an experimental model of metastatic tumour.

To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse. H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan. Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography. Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique. Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH. Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium. The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion. These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.

Hyaluronan: fundamental principles and applications in cancer.

Hyaluronan (HYA) plays a particular role in cancer cell microenvironment. A component of the desmoplasia. HYA is associated to other macromolecules and contributes to the net structure of the matrix. Cancer cells exhibit binding sites (CD44, RHAMM) for HYA. The cell adhesion to HYA can influence the cell motility and different factors could interplay to facilitate cell detachment from HYA. HYA protects cancer cells against immune cell attack. Serum HYA is often increased in metastatic patients.

111In-hyaluronectin, a new probe for radiodetection of tumours: biodistribution and imaging in grafted mice.

Hyaluronectin (HN), a glycoprotein which shows a strong specific affinity for Hyaluronic Acid (HA), a very high molecular weight glycosaminoglycan expressed in the stroma reaction of all tumours, was used to radiolocalize grafted autologous carcinomas (CB33 and CB03) in mice. After conjugation to DTPA (0.5-3.7 DTPA/HN) and radiolabelling with 111In (5 microCi/micrograms HN) HN retained 93% of its affinity for HA. Different preparations of HN purified from lamb brain were assayed. The best results were obtained when the HN molecules reactive with HA were selected by gel permeation to discard unreactive molecules which were unable to complex with HA. At 48 hours the tumour to blood ratio was 7.7 with a localization index of 2.2 in CB33; there was 2.8% uptake of the injected dose (ID) into CB33 and 1.6% into CB03. 111In-HN uptake per gram of tumour was tumour-size dependent: the smaller masses had the higher uptake. Tumours were visualized and either early images of 111In-HN distribution or simultaneous distribution images of 99mTc-phytate or 201Tl-DTPA were used for the subtraction treatments.

Activation and inhibition of human cancer cell hyaluronidase by proteins.

Results regarding hyaluronidase activity in tumor extracts or cell lines are subject to variations according to the method used for the assay and, sometimes, within an assay. Hyaluronidase was assayed at pH 3.8 in the culture medium of the human cancer-derived cell lines SA87 and H460M by several techniques: HPLC, Reissig technique, ELSA technique, and zymographic analysis. The optimal pH was between 3.3 and 4 in solutions at constant 150 mM sodium concentration. The enzyme was reversibly inhibited by sodium concentration over 200 mM. The activity of purified hyaluronidase increased in the presence of low concentrations of the specific HA-binding glycoprotein hyaluronectin, or of bovine serum albumin or immunoglobulins, or of human albumin, transferrin, or hemoglobin, showing that proteins cooperate in enzyme activity. The ELSA technique showed that optimal pH was slightly lower in the presence of HN than that with BSA. The optimal BSA concentration was determined with the ELSA technique at 0.1 g/liter, and excess of either protein inhibited hyaluronidase. When measured with the Reissig technique, the activity of purified enzyme in the presence of 0.1 g/liter BSA was up to fourfold that without BSA. The cooperative effect of BSA was visualized by zymography. We conclude that the total protein content of hyaluronidase solutions must be considered to correctly interpret quantitation of the enzyme in sera or tissue extracts because protein concentrations above 200 microg/liter lead to underestimation of the enzyme.