BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis.

In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 µmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.

[New diagnostic methods in non-Hodgkin lymphoma]. / Nye diagnostiske metoder ved non-Hodgkins lymfom.

We describe five patients with non-Hodgkin's lymphoma (NHL) in which immunohistochemical investigation (IH) and polymerase chain reaction (PCR) were important for diagnosis and choice of treatment. One patient got the diagnosis follicular NHL after PCR showed t(14;18) in a fine needle biopsy from a tumour in the abdomen. Two small and partly traumatized biopsies from two patients were diagnosed as low grade NHL of B-cell type after PCR showed monoclonality for B-lymphocytes and IH showed B-cell phenotype and low proliferation rate. Biopsy from another patient showed positive immunohistochemical reaction for cyclin D1 and t(11;14) by PCR compatible with mantel cell lymphoma. One patient was diagnosed with high grade NHL of T-cell type after IH showed positive reaction for T-cell markers and high proliferation rate, and PCR showed monoclonality for T-lymphocytes. These cases demonstrate that IH and PCR can often benefit patients with NHL. The new methods contribute to enhanced diagnostic security and precision in subclassification and make possible the use of small biopsies which save the patients from major surgery for diagnostic purposes.

[Telepathology in Mid-Norway]. / Telepatologi i Midt-Norge.

Telepathology can provide frozen section service to hospitals without a pathology service of their own. Using a dynamic-robotic system with 6 ISDN B-channels (Telemed A200, AM Elektronikk A/S, Oslo, Norway), the pathologists at the University Hospital in Trondheim, Norway during a three-year period from 1995 successfully performed 116 diagnostic sessions for two rural Norwegian hospitals. In 90% of the cases a diagnosis was provided. There was no false positive diagnosis and only 3% false negative cases without clinical consequence. Deferred cases (10%) were mainly due to poor quality of the frozen sections and a conservative attitude among the pathologists. It is concluded that the diagnostic accuracy is good, but for safety reasons telepathology should be offered on a regular basis, so that the skills of the technicians and pathologists involved are kept up. In Mid-Norway, five more hospitals will be included in a telemedicine network comprising eight hospitals in the region.

Marked osteoblastopenia and reduced bone formation in a model of multiple myeloma bone disease in severe combined immunodeficiency mice.

We report on an in vivo model of human myeloma producing bone disease in irradiated severe combined immunodeficiency disease mice using the human myeloma cell line JJN-3 and its subline JJN-3 T1. The cell lines are not Epstein-Barr virus transformed and produce large amounts of hepatocyte growth factor (HGF). Mice had radiological signs of osteolysis and mild hypercalcemia. Xenografted cells were predominantly found in bone marrow and brown adipose tissue, but also in meninges and liver. Take was documented by histopathological examination, immunophenotyping of cultured bone marrow, and radiography. HGF was detected in serum and bone marrow plasma. Disease generally occurred within 45 days of intravenous inoculation and was signaled by paraparesis or signs of intracranial neoplasia. More than 90% of the mice had take of xenografts. The subline JJN-3 T1 gave more reproducible bone marrow take than the native cell line. Bone histomorphometric examination revealed a 99% reduction in osteoblast counts and a 33% reduction in osteoclast counts in areas of tumor growth. Bone formation rates were reduced by 53%. The results suggest that osteoblastopenia and reduced bone formation is of importance for the occurrence of osteolytic lesions in this model.

Diver's hand: a skin disorder common in occupational saturation diving.

Saturation divers regularly inspect North Sea installations, working at depth for periods of 12-16 days. Diver's hand is a particular problem in saturation diving, and there is no effective protection or treatment available. This paper presents the occurrence of diver's hand and describes the disease in clinical and epidemiological terms. Three studies of diver's hand have been carried out, in 1990, 1994, and 1995. Most long term saturation divers have had diver's hand at some time in their professional career. Diver's hand seems able to occur without any previous skin symptoms, and divers without diver's hand can have several other skin symptoms during a saturation period. It is likely that diver's hand is a specific phenomenon associated with saturation diving.

[Low grade non-Hodgkin lymphoma of B-cell type. What is the benefit of polymerase chain reaction and immunohistochemical examination?]. / Lavgradige non-Hodgkins lymfomer av B-celletype. Hvilken nytte har vi av polymerasekjedereaksjon og immunhistokjemisk undersøkelse?

Subclassification of malignant non-Hodgkin's lymphoma is important to be able to choose the right treatment. However, this subclassification can be difficult, and immunohistochemistry and polymerase chain reaction are used to improve diagnostic safety. We have investigated the value of immunohistochemistry and polymerase chain reaction in the subclassification of 49 low grade non-Hodgkin's lymphomas of B-cell type. Immunohistochemistry showed B-cell phenotype in all biopsies, and 23 (85%) of 27 follicular lymphomas showed positive reaction for bcl-2 antibodies in follicles. Polymerase chain reaction demonstrated a monoclonal pattern for IgH in 36 (73%) of 49 biopsies, and 10 (37%) of 27 follicular lymphomas demonstrated translocation (14; 18). Immunohistochemistry and polymerase chain reaction contributed to subclassification or confirmation of the diagnosis of 46 (94%) of 49 biopsies. Our conclusion is that immunohistochemistry and polymerase chain reaction have a natural place in the diagnosis of lymphomas.

Identification of genetic determinants responsible for the rapid immunosuppressive activity and the low leukemogenic potential of a variant of Friend leukemia virus, FIS-2.

An immunosuppressive variant of Friend murine leukemia virus (F-MuLV), FIS-2, induces suppression of the primary antibody response against sheep erythrocytes (SRBC) in adult NMRI mice more efficiently than the prototype F-MuLV clone 57 (cl.57). It is, however, less potent than F-MuLV cl.57 in inducing erythroleukemia upon inoculation into newborn NMRI mice. Nucleotide sequence analysis shows a high degree of homology between the two viruses. Single point mutations are scattered over both the gag and the env encoding regions. The most notable mutations are the deletion of one direct repeat and a few single point mutations occurring in the binding sites for cellular transcriptional factors in the FIS-2 long terminal repeat region (LTR). To define the genetic determinants responsible for the pathogenic properties of FIS-2, we constructed six chimeras between FIS-2 and F-MuLV cl.57. Adult mice were infected with the chimeras, and their primary antibody responses against SRBC were investigated. The results showed that the fragment encompassing the FIS-2 env encoding region SU is responsible for the increased immunosuppressive activity in adult mice. A leukemogenicity assay was also performed by infecting newborn mice with the chimeras. Consistent with the previous studies, it showed that the deletion of one direct repeat in the FIS-2 LTR is responsible for the long latent period of erythroleukemia induced by FIS-2 in newborn-inoculated mice. However, studies of cell type-specific transcriptional activities of FIS-2 and F-MuLV cl.57 LTRs using LTR-chloramphenicol acetyltransferase constructs showed that the deletion of one direct repeat does not reduce the transcriptional activity of the FIS-2 LTR. The activity is either comparable to or higher than the transcriptional activity of the F-MuLV cl.57 LTR in the different cell lines that we used, even in an erythroleukemia cell line. It seems that the high transcriptional strength of the FIS-2 LTR is not sufficient to give FIS-2 a high leukemogenic effect. This suggestion is inconsistent with the previous suggestion that the transcriptional strength of an LTR in a given cell type is correlated with the leukemogenic potential in the corresponding tissue. In other words, these data indicate that the direct repeats in the F-MuLV LTR may play other roles besides transcriptional enhancer in the leukemogenesis of F-MuLV.

[Mycosis fungoides. Early diagnosis and evaluation of treatment by polymerase chain reaction]. / Mycosis fungoides. Tidligdiagnostikk og evaluering av behandling ved hjelp av polymerasekjedereaksjon.

We describe the case of a 60-year-old man who, after seven years with eczematous changes, developed erythrodermia, clinically suspect of mycosis fungoides. T-cell receptor gamma (TCR-gamma) gene rearrangements were retrospectively investigated by polymerase chain reaction (PCR). DNA from a formalin-fixed, paraffin-embedded punch biopsy showed a monoclonal pattern compatible with mycosis fungoides two years before the diagnosis was confirmed histopathologically. PCR was also used to evaluate the effect of different treatments. This case demonstrates the value of PCR for early diagnosis and evaluation of treatment of cutaneous T-cell lymphoma.

Molecular cloning and characterization of an immunosuppressive and weakly oncogenic variant of Friend murine leukemia virus, FIS-2.

The FIS variant is a weakly leukemogenic, relatively strong immunosuppressive murine retrovirus which was isolated from the T helper cells of adult NMRI mice infected with Friend murine leukemia virus (F-MuLV) complex (FV). Unlike FV, it does not induce acute erythroleukemia but retains the immunosuppressive property of FV and induces suppression of the primary antibody response rapidly and persistently in adult mice. A previous study showed that the FIS variant contains two viral components, a replication-competent virus and a defective virus. In this study, we have biologically purified the FIS variant by end point dilution and we show that the replication-competent virus FIS-2 alone can induce immunosuppression as the parental FIS variant. Most newborn mice infected with FIS-2 developed erythroleukemia, but with an increased latency period compared with that of F-MuLV clone 57. In contrast, FIS-2 induced suppression of the primary antibody response and disease more rapidly than F-MuLV clone 57 in immunocompetent, adult mice. FIS-2 was further molecularly cloned and characterized. Restriction mapping and nucleotide sequence analysis of FIS-2 showed a high degree of homology between FIS-2 and F-MuLV clone 57, suggesting that FIS-2 is a variant of F-MuLV. The striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat and the single point mutation in the binding sites for core-binding protein and FVa compared with the long terminal repeat of F-MuLV clone 57. Two single point mutations led to the appearance of two extra potential N glycosylation sites in the FIS-2 gag-encoded glycoprotein. Together, the results suggest that FIS-2 represents an interesting murine model to study retrovirus-induced immunosuppression on the basis of its unique combined property of low leukemogenicity and relatively strong and persistent immunosuppressive activity in adult mice.

Cytokine-associated tissue injury and lethality in mice: a comparative study.

A comparative study was performed to examine the lethal effects of several cytokines injected into mice sensitized with actinomycin D (Act-D). Consistent with published data, human tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) (0.2-5 micrograms) caused the death of the animals within 8-12 hr after injection. Human interleukin-6 (IL-6) and interleukin-8 (IL-8) (0.6-6 micrograms) known to be induced by TNF-alpha did not show any lethal effects, indicating that TNF-alpha-associated lethality is not mediated by IL-6 or IL-8. Human tumor necrosis factor-beta (TNF-beta) (also called lymphotoxin), which shares structural and functional properties with TNF-alpha, was as potent as TNF-alpha in its lethal effects. Murine interferon-gamma (IFN-gamma) (0.04-5 micrograms) was also tested and showed no lethal effects in this model. In addition, a synthetic peptide corresponding to amino acid residues 163-171 of IL-1 beta, and which has been shown to lack the inflammatory effects of IL-1 beta, also caused no lethality among Act-D sensitized mice. The pretreatment of mice with IL-6, IL-8, or IFN-gamma had no protective effects on TNF-alpha or IL-1 beta-induced lethality in contrast to the protection observed by a pretreatment with TNF-alpha/IL-1 beta themselves or with endotoxin. Histopathologic data showed that severe tissue injury in vital organs is associated with the rapid lethality among sensitized mice.

Toxicity of n-C9 to n-C13 alkanes in the rat on short term inhalation.

Male Sprague Dawley rats were exposed to inhalation of n-C9 to n-C13 alkanes close to air saturation at 20 degrees (4438, 1369, 442, 142 and 41 p.p.m., respectively) for 8 hours and observed for the following 14 days. In addition, exposure to higher and lower concentrations of n-C9 was performed. The concentration of alkane in the brain after exposure exceeded that of blood for the lower alkanes, while the higher alkanes possessed a brain/blood ratio equal to or less than unity. Gross ataxia, general and focal seizure and spasms were observed in animals exposed to n-C9 in the range from 5280 to 3560 p.p.m. No toxic effects were observed in animals exposed to 2414 p.p.m. of n-C9 or to the other alkanes. An LC50 value for n-C9 of 4467 +/- 189 p.p.m. was estimated. Despite the clinical improvement in animals surviving the n-C9 exposure of 4438 p.p.m. (6/10), severe cerebellar damages were found at autopsy at the end of the observation period, with a loss of Purkinje cells as the most prominent feature. Immediate post mortem examination (4/10) showed marked vascular congestion of the liver as well as slight fatty degeneration but no cerebellar damage. No abnormalities were observed in animals exposed to the other alkanes. The significant distribution in the brain of the n-C9 alkane, the clinical signs of cerebellar dysfunction and the damage of cerebellar neurons would suggest CNS to be a possible target organ for the toxic effects of the n-C9 alkane.