Differential expression of c-myc-mRNA and c-MYC-protein during terminal neoplastic B-cell differentiation.
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B-Lymphocytes↗
Biomedical subjects
R Greil
Publications and source records attributed to R Greil.
Investigation of nuclear c-MYC oncoprotein expression in human hematopoiesis: suitability of a rapid and reliable semiquantitative evaluation system.
The biologic functions mediated by the nuclear protooncogene, c-MYC are correlated to gene dosage. Since automated quantification programs are expensive, time-consuming and not easily available, and since analysis by flow cytometry is difficult in the case of nuclear antigens, we examined the suitability and reproducibility of a semiquantitative in situ evaluation system. This system was based on the percentage of nuclear area staining positively, and comprised the following categories: 0: negative, 1+: single scattered grains of the immunocytochemical staining product, 2+: confluence of grains to patches but less than 50% nuclear area positive, and 3+: greater than 50% positive nuclear area. In addition, sensitivity and specificity of two anti-c-MYC antibodies were investigated. Although both antibodies differed slightly in staining pattern and sensitivity, the four quantification categories were applicable for immunostainings of both antisera and highly reproducible when re-evaluated by the same observer (r = 0.98; p = 0.0001) or a second investigator (rAb155 = 0.98, rAb DCPm = 0.96; p = 0.0001), both reading blindly and independently. Comparing our semiquantitative evaluation categories and results of computer-assisted image analysis, the percentage of positive nuclear area (p less than 0.0001), the median staining intensity (p less than 0.0001), and the product of both (p less than 0.0001) differed significantly in the four evaluation categories. This result still held true after correction for nuclear size, which differed appreciably in various cell types (p less than 0.0001). The product of positive nuclear area, staining intensity and nuclear size (microns 2), which best approximates the absolute amount of c-MYC within a certain cell, was clearly different within the four staining categories (p less than 0.0001) and did not depend on cellular morphology within the staining categories 0 to 2. Also, the immunocytochemical technique proved highly reproducible (median day/day variance 0.65% (0-13); r = 0.995). The practicability of this system for semiquantification was demonstrated by (a) correlation of H score values of immunocytochemical stainings with densitometric scans of Western blots and (b) by the fact that peripheral blood lymphocytes, Phytohemagglutinin stimulated blasts, 13 cases of multiple myeloma and HL-60 cells differed concerning their estimated c-MYC amounts (p = 0.0125). This confirms on the effector molecule level results previously reported from mRNA in situ and Northern blotting analyses. We conclude that a simple and highly reproducible evaluation system can be used for in situ comparison of nuclear oncogene dosage.
Gene Expression↗
Expression of the c-myc proto-oncogene in multiple myeloma and chronic lymphocytic leukemia: an in situ analysis.
The c-myc gene plays a pivotal role in mediating the competence state for cell cycle transversion. This biologic role is in contradiction to reports of elevated expression of the gene in multiple myeloma, a tumor with restricted self-renewal capacity. To more clearly define the role of this gene in plasma cells of myeloma patients, c-myc messenger RNA (mRNA) and/or oncoprotein expression were semiquantitatively analyzed on the single cell level in 19 cases of multiple myeloma, among them 1 biclonal case and 1 case with coexistent chronic lymphocytic leukemia (CLL). Performing anti-sense/mRNA in situ hybridization, mature c-myc gene transcripts were detected in 92% (12 of 13) of cases and could definitely be attributed to the plasma cells by our study. The number of Ki 67-positive plasma cells actively passing the cell cycle was less than 1% and independent of c-myc gene expression. However, because the presence of the 152-c-MYC epitope was correlated to extent of marrow plasmacytosis (r = .64; P = .043) and content of plasmablasts (P = .09), the c-myc gene might serve a function different from proliferative activity, but also associated with tumor cell mass. In CLL cells (21 of 22 cases) and their benign counterparts, ie, bone marrow and peripheral blood lymphocytes, the anti-sense/c-myc mRNA hybridization signals remained below the threshold considered as cutpoint between negative and positive. The low amounts of c-myc transcripts were correlated to neither stage of disease (P = .52) nor lymphocyte counts (P = .24). Because the numbers of peripheral blood lymphoma cells were independent of tumor mass and of c-myc gene transcripts expressed, peripheral blood lymphocytosis might more likely reflect homing processes than proliferative activity in CLL.
Antibodies, Monoclonal↗
[Urticarial vasculitis as a symptom of Muckle-Wells syndrome?].
A patient is reported with a history of several years of chronic urticaria, transient fever, arthralgias and secondary systemic amyloidosis. A biopsy of an urticarial lesion showed necrotizing vasculitis and amyloid deposits in the eccrine sweat glands. Amyloid A deposits were also detected in kidney and rectum biopsies. This patient is likely to represent a variant of the Muckle-Wells syndrome (chronic relapsing urticaria, fever, arthralgia, deafness and renal amyloidosis). Hitherto undescribed is the presence of a necrotizing vasculitis as cause of the urticarial rash; further investigation will determine whether or not this finding represents the rule rather than an exception.
Adult↗
A monoclonal antibody directed against the human intercellular adhesion molecule (ICAM-1) modulates the release of tumor necrosis factor-alpha, interferon-gamma and interleukin 1.
ICAM-1 is a cell surface glycoprotein which is one of the ligands for the leukocyte function-associated antigen (LFA-1). It is involved in leukocyte adhesion to endothelial cells as well as in immune functions requiring cell-cell contact. The quantitative expression of ICAM-1 in various cell types can be either induced or enhanced by treatment with cytokines, such as interferon-gamma (IFN-gamma), tumor necrosis factor (TNF)-alpha or interleukin 1 (IL 1), a phenomenon which results in the augmentation of binding to LFA-1-positive cells. In contrast, treatment with anti-ICAM-1 antibodies blocks this binding. A monoclonal antibody (mAb), termed 7F7, which recognizes an epitope on ICAM-1, was used to investigate the role of ICAM-1 in cytokine production by T lymphocytes and monocytes. Production of TNF-alpha. IFN-gamma and IL1 was significantly inhibited (p less than 0.01) by the incubation of mAb 7F7 with phytohemagglutinin-activated blood mononuclear cells (MNC) or isolated E rosette-positive T lymphocytes. The maximal level of inhibition was reached with 1 microgram/ml of purified antibody. A similar inhibition was obtained using saturating concentrations of 400 microliters/ml of mAb 7F7 hybridoma supernatant corresponding to an inhibitory activity of 1 microgram of purified mAb. In contrast, granulocyte/macrophage-colony-stimulating factor release showed a heterogeneous response over five experiments with an increase found in three experiments and a decrease in two experiments. Addition of increasing concentrations of supernatant or purified mAb to unstimulated MNC or T lymphocyte cultures had no effect on cytokine release. The observed inhibition of the production of TNF-alpha. IFN-gamma and IL 1 by antibody-mediated blockade of the ICAM-1 structure probably represents a negative circuit that serves to tune the activation of leukocytes and to avoid an overproduction of cytokines.
Antibodies, Monoclonal↗
[Cytologic molecular detection of oncogene expression: possibilities and prospects in hemato-oncology].
Proto-oncogenes, the normal equivalents to the transforming genes of mammalian tumorigenic retroviruses, are implicated in essential biological processes of normal human cells, like differentiation and proliferation. In vivo and in vitro studies clearly demonstrate that activation of proto-oncogenes with subsequent transformation in tumorigenic oncogenes plays an important role in induction and acceleration of the malignant disease, and also determines metastatic spread and development of resistance to chemotherapy in certain neoplasias. Investigation of these genes and analysis of their activation state should routinely be introduced in staging of hematological neoplasias, thus contributing to refinement of histopathological classification, clearer discrimination between reactive and neoplastic conditions and understanding of pathophysiological processes. Furthermore, impact on definition of high risk patients and novel therapeutic concepts based on a better definition of tumour biology may be expected from these studies. "Molecular cytology" combines detection of oncogenetic mRNA and the relevant oncoprotein on the single cell level by using "mRNA-in situ hybridization" and immuno-histochemistry. Application of this technique will allow to determine the mechanisms regulating the expression of oncogenes to define functional heterogeneity of tumour cell subsets and to clarify the relevance of minimal residual disease.
Cell Transformation, Neoplastic↗
Monoclonal antibody B-ly7: a sensitive marker for detection of minimal residual disease in hairy cell leukemia.
The new monoclonal antibody (MoAb) B-ly7 was tested for its value in bone marrow diagnosis in patients with hairy cell leukemia (HCL). Cryostat sections of bone marrow biopsies were examined by an indirect immunoperoxidase technique. Lymphoma cells from all of 26 HCL cases investigated displayed strong surface membrane staining with the MoAb B-ly7, whereas tumor cells from only one of 63 patients with other lymphoproliferative disorders of B cell type reacted with this antibody. The strong reactivity of hairy cells (HCs) with this marker was not altered after therapy as demonstrated on control biopsies taken from patients treated with interferon(IFN)-alpha-2 or 2'deoxycoformycin(DCF) six-64 weeks after start of treatment. This fact as well as the very low number of B-ly7 positive cells found in a series of 13 normal bone marrow biopsies (mean: 0.3% of bone marrow cells, range: 0.0%-1.0%), which could easily be distinguished from HCs by their lower staining intensity and their morphological appearance, provided the basis for the detection of even single HCs. In our hands, in terms of sensitivity the immunohistological detection of HCs using the MoAb B-ly7 was not only superior to classical morphological techniques but also to other immunohistological parameters usually applied for this purpose. Therefore, this MoAb provides a marker for the identification of HCs, hence monitoring disease activity in HCL, and particularly for a critical response evaluation in patients undergoing treatment with IFN-alpha or DCF.
Antibodies, Monoclonal↗
Immunohistological assessment of bone marrow biopsies from patients with hairy cell leukemia: changes following treatment with alpha-2-interferon and deoxycoformycin.
Forty-six bone marrow biopsies from twelve hairy cell leukemia (HCL) patients, treated with either interferon(IFN)-alpha-2 (n = 8) or 2'deoxycoformycin(DCF) (n = 4), were examined using cryostat sections and an immunoperoxidase technique. Using this sensitive method we were able to demonstrate residual hairy cell (HC) infiltration in five cases, in which evaluation with conventional staining techniques on plastic embedded biopsies revealed complete remission. The amount of HCs in these five samples ranged from 1 to 7% (mean: 3%) of bone marrow cells. Consecutive biopsies in individual HCL patients revealed no changes of the immunological phenotype (CD19, CD22, CD25, CD10, CD11c, FMC7, HLA-DR, surface immunoglobulins) during IFN and DCF treatment. Within the infiltrated bone marrow a considerable number of "reactive" T lymphocytes was identified with prevalence of the T-helper (CD4+) subtype in untreated cases, whereas T-suppressor/cytotoxic (CD8+) cells were within the normal range. IFN treatment resulted in a reduction of CD4+ T lymphocytes (p less than 0.02). Minor alterations of CD8+ T lymphocytes and NK cells (HNK-1 + lymphoid cells) were found in bone marrow during IFN treatment. In DCF-treated patients bone marrow T lymphocytes were markedly reduced below the values of normal bone marrow. This DCF-induced T-cell depression might be related to the clinical observation of persistent cellular immune dysfunctions in HCL patients despite a DCF-induced remission.
Adult↗
Immunohistology for quantification of normal bone marrow lymphocyte subsets.
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Antibodies, Monoclonal↗
In situ hybridization for the detection of low copy numbers of c-abl oncogene mRNA in lymphoma cells: technical approach and comparison with results with anti-oncoprotein antibodies.
We investigated the practical value of antisense RNA/mRNA in situ hybridization for the detection of low level expression of the c-abl oncogene in non-Hodgkin lymphomas. This is of clinical relevance, since we recently showed that low level expression of this proto-oncogene mainly occurs in advanced stage disease of non-Hodgkin lymphomas and in cases of chronic lymphocytic leukemia with progressive course of the disease (Greil R, Gattringer C, Fasching B, Cleveland J, Thaler J, Radaskiewicz T, Gastl G, Huber C, Rapp U, Huber H: Int J Cancer 42:529 1988). When numerous technical parameters were tested for the adaptation of the method, fixation with 4% paraformaldehyde, gelatin coating of the slides, the time concentration product of proteinase K, and the kind of labeling had the greatest impact on results and successful performance of the technique. When the optimized method was applied to the v-abl-transformed NIH 3T3-, the K 562 CML blast cell line and to nine cases of lowly malignant non-Hodgkin lymphomas it semiquantitatively discriminated the varying amounts of v-abl, bcr/c-abl and c-abl mRNA expressed within these cells. Parallel analysis with Northern blotting confirmed the specificity of the method and pointed to a very high sensitivity, including the capacity to detect only few c-abl mRNA molecules/cell. An essential advantage of in situ hybridization was the detection of inhomogeneous expression of the c-abl mRNA within subpopulations of the malignant clone. In addition, this technique might be of particular importance when a gene is only weakly expressed on a small fraction of cells which might easily escape the detection by Northern blotting. Immunocytochemical investigation suggested parallel expression of the oncoprotein in six of seven c-abl mRNA positive cases as well as high specificity and sensitivity for the polyclonal and to a lesser extent for one monoclonal antibody. However, because of the high potential of cross-reactivity of anti-oncoprotein antibodies, parallel investigations on the mRNA level should be performed particularly when new anti-oncoprotein antibodies are applied. Our results demonstrate that this can be performed using in situ hybridization, even when the number of mRNA targets is very low.
Antibodies, Monoclonal↗
Expression of leucocyte function-associated antigen-1 and 7F7-antigen, an adhesion molecule related to intercellular adhesion molecule-1 (ICAM-1) in non-Hodgkin lymphomas and leukaemias: possible influence on growth pattern and leukaemic behaviour.
In 160 non-Hodgkin lymphomas (NHL) of B and T cell origin the expression of leucocyte function-associated antigen-1 (LFA-1) and one of its counter-structures named 7F7-antigen was studied. Functional and structural similarities and the 7F7-reactivity of pICAM-1 transfected COS-cells prove that 7F7-antigen is identical with ICAM-1. The expression of both adhesion structures occurred variably and clearly depended on the maturation stage and on the B and T cell origin of lymphomas but was not associated with the proliferative status of tumour cells. The concomitant expression of both adhesion molecules was a characteristic feature of germinal centre derived NHL, particularly of those with neoplastic follicular structures (corr. contingency coeff. = 0.506; P less than 0.02), whereas both adhesion structures were less often expressed on lymphomas with a more diffuse pattern of tumour infiltration. Most B-NHL of low-grade malignancy expressed LFA-1 while their counterparts of high-grade malignancy often tended to be LFA-1 negative. In B cell neoplasias of low-grade malignancy the lack of ICAM-1 and a leukaemic course of the disease were significantly correlated (P less than 0.0005). The results indicate that the differential expression of both adhesion molecules may account for distinct patterns of growth and spread in subtypes of lymphoid malignancies.
Antigens, Differentiation↗
Importance of an 85 kDa membrane glycoprotein for a variety of cell-cell interactions.
The membrane molecule termed "7F7-antigen" has been found to be involved in several examples of cell-cell interactions. This 85 kDa glycoprotein with a protein core of about 55 kDa contains N-linked and O-linked carbohydrates. It has an isoelectric point of 8.0-8.5 and is expressed on 20% of peripheral blood mononuclear cells, 35% of peripheral blood B-cells, follicular dendritic cells and vascular endothelium. It is also expressed on activated T-cells and its expression on B-cells, fibroblasts and monocytes increases after treatment with PWM, interferon-gamma and after three days culture, respectively. The MAb 7F7 used to define this antigen inhibits the initiation of T-cell proliferation induced by anti-CD3, PHA, ConA and (weakly) allogenic stimulator cells, but does not affect the growth of IL-2 dependent T-cells and does not interfere with the killing of PHA-blasts by allogenic IL-2 dependent T-cells. 7F7 also inhibits the binding of C3-coated sheep erythrocytes to B-cells, the PMA-induced aggregation of U937 and the binding of activated T-cells to fibroblasts. The 7F7-antigen is expressed on some non-Hodgkin lymphomas of B-cell differentiation, particularly those with follicular structure, but not on Burkitt's lymphoma, ALL or carcinomas of various tissues. It is, however, found on fibrous tissue surrounding infiltrating carcinoma cells. The expression of a melanoma antigen, P3.58, which was shown to be identical to 7F7-antigen correlates with stage and spread of invasive melanoma. It was concluded that the 7F7-antigen, which is probably related to a previously described adherence molecule (ICAM-1), is of biological importance for the initiation of T-cell responses. With the possible exception of melanoma its expression on neoplastic cells in vivo is unlikely to be of importance for the spread of malignant disease.
Antigens, Neoplasm↗
In situ quantification of T-cell subsets, NK-like cells and macrophages in Hodgkin's disease: quantity and quality of infiltration density depends on histopathological subtypes.
The present investigation addressed itself to the in situ quantification of reactive cells in tumour tissues affected by Hodgkin's disease. Immunostaining was used for identification and stereology was used for enumeration of T-helper/inducer (CD4+) T-cytotoxic/suppressor (CD8+), NK-like (Leu7+) and cells of macrophage origin (Mono 2+). The evaluation of 50 cases showed that CD4+ cells always outnumbered CD8+ cells but the degree of this predominance varied depending on the histopathological subtypes (n.s. greater than m.c. greater than l.d.). Lymph nodes contained more CD4+ as well as CD8+ cells compared to spleens. Therefore, no changes in the T4:T8 ratio occurred. No significant differences in the densities of NK-like cells were observed, comparing the different histopathological subtypes as well as lymph nodes and spleens. Similarly, macrophage (M phi) density was comparable in all histopathological subtypes. However, lymph nodes contained significantly more M phi compared to spleens. On comparison of reactive cells in Hodgkin's tissues to non-Hodgkin lymphomas (79 cases) and normal controls (7 cases) significantly higher numbers of CD4+, CD8+ and Mono 2+ cells were found in Hodgkin's compared to non-Hodgkin's lymphomas. In contrast, the density of NK-like cells in NHL as well as in normal tissues was fivefold compared to that observed in Hodgkin's tissues.
Hodgkin Disease↗
Growth fraction of tumour cells and infiltration density with natural killer-like (HNK1+) cells in non-Hodgkin lymphomas.
Two markers for cells in the growth fraction, the T9 antigen (i.e. the transferrin receptor) and the T10 antigen were investigated in frozen tissue sections of 105 non-Hodgkin lymphomas (NHL). The results were correlated with the histological subtype and the pattern of tumour infiltration by reactive cells. Special attention was directed to the density of natural killer (NK)-like cells using the anti-HNK1 (Leu7) antibody since the transferrin receptor (tfr) or other growth-associated membrane structures may serve as target for NK cells. Our study confirms a relationship between number of tumour cells with the T9 marker and tissue infiltration by HNK1+ cells in NHL of low (chronic lymphocytic leukaemia, hairy-cell leukaemia, immunocytic lymphoma, centroblastic-centrocytic lymphoma) and intermediate (centrocytic and centroblastic lymphoma) but not in NHL of high malignant grade (immunoblastic and lymphoblastic lymphoma). Comparable results were obtained with the T10 antigen although the correlation was less close. The percentage of cells in the growth fraction, defined by the expression of the T9 and T10 marker, corresponded with prognostically unfavourable subgroups with the remarkable exception of follicular NHL of centroblastic-centrocytic type. This lymphoma showed high numbers of cells with the T9 and the T10 marker in a microenvironment resembling normal germinal centres in many aspects.
Antigens, Surface↗
[Studies of immunologic markers in malignant lymphomas: diagnostic and prognostic relevance].
Immunological markers important for the diagnosis of the following subtypes of Non-Hodgkin lymphomas (NHL) are discussed: lymphocytic lymphomas (i.e.: B-CLL, immunocytic NHL, prolymphocytic- and hairy cell leukemia), NHL derived from follicular center cells (centrocytic, centroblastic/centrocytic and centroblastic NHL), lymphomas with blastoid appearance (various forms of lymphoblastic and "large cell" NHL) and disorders of T lymphocytes (NHL) of peripheral T cells and T gamma lymphocytosis with neutropenia). In these disorders, the patterns of leukocyte surface antigens like T1 (CD5), Tac (CD25), CALLA (CD10), FMC7, Tü1 and Tü33 as well as staining for the terminal transferase (TdT) and of dendritic follicular cells have been examined by investigating specimens of a large group of patients. Apart from the diagnostical value, these evaluations have contributed to a better understanding of normal B cell differentiation whereas the role of marker studies as independent prognostic factors requires additional prognostic studies. Finally, the value of immunoperoxidase or alkaline phosphatase staining of cryostat sections of bone marrow biopsies is emphasized, which allowed the diagnosis and classification of 74% of 92 NHL studied by this technique.
Antibodies, Monoclonal↗
Receptors for the third component of complement: their association with maturation stage in non-Hodgkin lymphomas (NHL) and their possible implication with the development of follicular structures.
In the majority of 188 non-Hodgkin lymphomas (NHL) investigated in this study, we found a simultaneous expression of the receptors for c3b and c3d (CR1 & CR2; cccorr = 0.69, P less than 0.0005). An analysis of the different histological entities of the Kiel classification revealed that this coexpression was most pronounced for germinal centre-derived (cccorr = 0.63, P = 0.0004) and immunocytic NHL (cccorr = 0.86, P = 0.0024), whereas in chronic lymphocytic leukaemias there tended to be a more heterogeneous pattern of complement receptor (CR) expression (cccorr = 0.29, P greater than 0.10). In contrast to these NHL of mid B cell stage, most of the NHL of early (i.e. acute lymphocytic leukaemia, lymphoblastic NHL) and late B cell stage (i.e. hairy cell leukaemia, immunoblastic NHL, multiple myeloma) did not express either of these receptors. CR positive NHL often showed a follicular arrangement of the neoplastic cells and had higher numbers of T helper/inducer (T4) lymphocytes (PCR1 less than 0.00005, PCR2 less than 0.05). Some cases of mid B cell NHL and all cases of hairy cell leukaemia reacted with antibodies against CR3 (i.e. the ic3b receptor).
B-Lymphocytes↗
[Rhabdomyolysis as a late complication of anesthesia--a case of malignant hyperthermia?].
Rhabdomyolysis is usually known to the anaesthetist, who may be confronted with this acute, life-threatening complication during anaesthesia. However, also the medical specialist ought to be familiar with the clinical picture of malignant hyperthermia, since rhabdomyolysis may occur as a late post-anaesthetic complication. Even in oligo-symptomatic and protracted cases of malignant hyperthermia, the case history, electromyogram, exclusion of other possible causes of rhabdomyolysis and, if possible, muscle biopsy contribute to the confirmation of the diagnosis. If a muscle biopsy is not available then a platelet bioassay may substantiate the diagnosis.
Adult↗
[Infiltration pattern of immunocompetent lymphocytes in the tissue of malignant non-Hodgkin's lymphoma: relationship to histological and clinical parameters].
The number and distribution of reactive cells in more than 100 non-Hodgkin lymphomas (NHL) were evaluated in situ in cryostat sections. The results were compared with histological and clinical findings. 40% of the T cell content of normal lymphatic tissues was found in tissues of B-cell NHL. This corresponds to a mean of 2 X 10(4) reactive T-cells/microliter tumour tissue. The numerical density of natural killer (NK) cells within tumours was similar to that of normal lymphatic tissues (0.5 X 10(4) cells/microliter). The distribution of the reactive cells within the tumours was diffuse except in the case of centroblastic/centrocytic lymphomas. On evaluation of the different histological entities a significant correlation was obtained between number of helper/inducer (TH) cells, TH:T-suppressor (Ts) ratio and prognostically favourable histological subgroups. Furthermore, independent of histological criteria, a close correlation was found between a high number of TH-cells, a high TH:Ts ratio and a favourable clinical course (p less than 0.04). NK-cell infiltration was present to a markedly higher extent in tissues of patients with generalized disease as compared with localized disease (p less than 0.03). Low-grade malignant NHL contained significantly more NK cells than high-grade malignant NHL, as was the case also in treated, as opposed to untreated patients. These findings together suggest that reactive cells influence tumour growth via local interactions. However, tissue distribution and infiltration density of T-cell subpopulations and NK cells were clearly different. Thus, in tumour tissues different and independent immunoregulatory mechanisms seem to be associated with these two lymphocyte subsets.
Antibodies, Monoclonal↗