Heinz Huber

Predictive Role of Interphase Cytogenetics for Survival of Patients With Multiple Myeloma

PURPOSE Recent metaphase cytogenetic studies suggested that specific chromosomal abnormalities are of prognostic significance in patients with multiple myeloma (MM). Because the true incidence of chromosomal abnormalities in MM is much higher than that detected by metaphase analysis, we used interphase fluorescence in situ hybridization (FISH) to determine the prognostic value of specific chromosomal aberrations. PATIENTS AND METHODS Bone marrow plasma cells from 89 previously untreated patients with MM were studied consecutively by FISH to detect the deletions of 13q14, 17p13, and 11q and the presence of t(11;14)(q13;q32). FISH results were analyzed in the context of clinical parameters (response to treatment and survival after conventional-dose chemotherapy), and a multivariate analysis of prognostic factors was performed. RESULTS By FISH, the deletion of 13q14 occurred in 40 patients (44.9%), deletion of 17p13 in 22 (24.7%), and 11q abnormalities in 14 (15.7%; seven with t(11;14)). Deletions of 13q14 and 17p13 were associated with poor response to induction treatment (46.9% v 77.3% in those without deletions, P =.006 and 40.0% v 73.2%, P =.008, respectively) and short median overall survival (OS) time (24.2 v 88.1 months, P =. 008 and 16.2 v 51.3 months, P =.008, respectively). Short median OS time was also observed for patients with 11q abnormalities (13.1 v 41.6 months, P =.02). According to the number of unfavorable cytogenetic features (deletion of 13q14, deletion of 17p13, and aberrations of 11q) that were present in each patient (0 v 1 v 2 or 3), patients with significantly different OS times could be discriminated from one another (102.4 v 29.6 v 13.9 months, P <.001, respectively). CONCLUSION For patients with MM who were treated with conventional-dose chemotherapy, interphase FISH for 13q14, 17p13, and 11q provides prognostically relevant information in addition to that provided by standard prognostic factors. This observation may be considered for risk-adapted stratifications of MM patients in future clinical trials.

Serum levels of interleukin-6 in multiple myeloma and other hematological disorders: correlation with disease activity and other prognostic parameters

SummaryInterleukin-6 (IL-6) is a multifunctional cytokine involved in the regulation of the terminal differentiation pathway of B lymphocytes. Recent reports revealed its potential role in the in vitro and in vivo growth of human multiple myeloma cells. The mechanism, however, by which IL-6 triggers proliferation of malignant plasma cells remains controversial. Using the very sensitive 7TD1 bioassay we quantified endogenous circulating IL-6 levels in serum samples of 104 patients suffering from monoclonal gammopathies and other hematological disorders [47 with multiple myeloma (MM), 24 with monoclonal gammopathy of unknown significance (MGUS), 8 with myeloproliferative disease, and 25 suffering from lowgrade non-Hodgkins lymphoma (NHL)]. Elevated serum levels of IL-6 (>5 pg/ml) were detected in 42% of the patients with MM, in 13% with MGUS, in 15% with low-grade B-NHL, and in 1 patient with T-NHL. In patients suffering from chronic myeloproliferative diseases, IL-6 levels were within the normal range. In patients with myeloma, IL-6 levels were significantly higher at advanced stages (II/III) or with progressive disease than in patients with MM stage I, MGUS, or at the plateau phase (P<0.01). In patients with monoclonal gammopathies including MGUS, serum IL-6 levels correlated with neopterin, tumor necrosis factor alpha and β2-microglobulin. An inverse correlation was found with hemoglobin levels. From these results, we propose that in myeloma patients serum IL-6 levels may reflect disease activity and tumor cell mass. The correlation with serum neopterin, a macrophage product, also suggests its origin in an activated immune system.

Correlation between neopterin, interferon-gamma and haemoglobin in patients with haematological disorders.

Abstract: In this study, we further investigated a possible link between activation of cell‐mediated immunity and anaemia in patients with haematological neoplasias. We compared serum concentrations of interferon‐gamma and neopterin with haemoglobin levels. Significantly increased interferon‐gamma and neopterin concentrations indicated persistent activation of cell‐mediated immunity. Neopterin levels correlated significantly to interferon‐gamma concentrations and inversely to haemoglobin levels. The data indicate an association between activated macrophages and the development of anaemia in patients with haematological neoplasias.

Urinary neopterine as marker for haematological neoplasias

Urinary neopterine levels were studied in 79 normal subjects and in 112 patients with haematological neoplasias. The mean values in 79 patients with active disease were significantly raised compared to the control group. Results obtained in 79 patients with active disease indicate that 91% had neopterine levels higher than the mean value of 79 normal individuals +3 SD. There is only a little overlap between the range of neopterine levels in cancer patients and the range in healthy subjects. No significant difference was found between the mean urinary neopterine levels of 33 patients with non-Hodgkins or with Hodgkins lymphoma in remission and the healthy group. Only 15% of these patients had elevated neopterine levels. The mean urinary neopterine levels correlated well with the tumor stage in patients with chronic lymphocytic leukaemia and with non-Hodgkins disease. In patients with chronic leukaemia those without hepatosplenomegaly excreted significantly more neopterine than controls, and patients with hepatosplenomegaly significantly more than those without hepatosplenomegaly. It is concluded that urinary neopterine levels are of value for following the progression of haematological neoplasias.

Expression of the neural cell adhesion molecule (CD56) by human myeloma cells.

Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T.B. myelomonocytic, and natural killer (NK)‐cell antigens. Leu‐19/NK.H1 (CD56), a molecule identical to N‐CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual‐parameter flow cytometry was used to correlate N‐CAM positivity with DNA aneuploidy or cytoplasmic immunoglobuiin expression as markers of myeloma cells. When N‐CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N‐CAM frequently exhibited pre‐B cell markers, myeloid antigen, and HLA‐DR, respectively (seven out of eight cases, 88%). Six out of eight N‐CAM‐negative myelomas were of the IgG lamba isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N‐CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N‐CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.

The biology of multiple myeloma

Abstract Multiple myeloma (MM) is a B-cell malignancy originating from pre-switched, follicle center B-lymphocytes which differentiate to plasma cells accumulating in the bone marrow. MM cells are characterized by a profound genetic instability resulting in a complex set of numerical and structural chromosomal abnormalities. Among these abnormalities, translocations involving 14q32, the immunoglobulin heavy-chain locus, are the most frequent aberrations, but translocation partners are remarkably heterogeneous. Chromosome 13q14 may harbor a critical tumor suppressor gene since MM patients with deletion of 13q14 experience short overall survival after conventional-dose and high-dose chemotherapy. Bone marrow stroma cells support growth and survival of MM cells, which in turn influence the bone marrow microenvironment. This is particularly evident by the markedly increased bone marrow vascularization observed in most patients with active MM.

Combined flow cytometric assessment of cell surface antigens and nuclear TdT for the detection of minimal residual disease in acute leukaemia

Summary. To define more precisely the immunophenotype of lymphoid blast cells, a new flow cytometric technique for the simultaneous detection of surface antigens and nuclear terminal deoxynucleotidyl transferase (TdT) was established. After staining for the cell surface marker, mononuclear cells were treated with paraformaldehyde (1 %) and methanol to permeabilize the cell membrane. Then the cells were stained by indirect immunofluorescence using a rabbit anti‐human TdT antibody. Dilution experiments were performed to reveal the sensitivity of the described flow cytometric assay: 0·02% leukaemic cells could reliably be detected above background level among normal peripheral blood lymphocytes. It is concluded that the double‐staining procedure described here is a sensitive tool that contributes to the detection of minimal residual disease in a substantial proportion of acute leukaemias.

Multiple myeloma with deletion of chromosome 13q is characterized by increased bone marrow neovascularization

Anti‐angiogenesis therapy with thalidomide has been reported to have marked activity in multiple myeloma (MM). As cytogenetics is an independent prognostic factor in MM, we analysed bone marrow (BM) angiogenesis and cytogenetic abnormalities in 34 patients with active MM. BM microvessel density (MVD), as determined by staining with anti‐CD34, was significantly higher in MM (MVD: 221 ± 94 per mm2) than in controls (80 ± 36; P < 0·0001). In patients with the presence of at least one unfavourable cytogenetic abnormality (deletion of 13q14, deletion of 17p13, aberrations of 11q), a significantly increased BM MVD was observed (254 ± 93 vs. 160 ± 60 in patients with absence of these abnormalities; P = 0·0035). Further analyses indicated that increased BM MVD was significantly correlated with deletion of 13q14 (259 ± 96 vs. 188 ± 80; P = 0·026), but not with other cytogenetic, clinical and laboratory MM parameters. We conclude that BM neovascularization is particularly high in MM with deletion of 13q14, which provides a rationale for use of anti‐angiogenic strategies in the treatment of MM with high‐risk cytogenetics.

Value of urinary neopterin in the differential diagnosis of bacterial and viral infections.

SummaryNeopterin is released by stimulated macrophages. In this study we analyzed the diagnostic potential of urinary neopterin concentrations in patients with bacterial and viral infection. All but one of 17 patients with viral infection had increased urinary neopterin concentrations. Patients with bacterial urinary tract infection also showed increased neopterin concentrations, whereas patients with bacterial pneumonia had significantly lower neopterin levels. In addition, patients with acute bacterial pneumonia had lower neopterin levels than patients with protracted infection. A significant inverse correlation between urinary neopterin and hemoglobin concentrations was found. Neopterin concentrations could serve as a helpful additional marker of infectious diseases. Combined with other clinical and laboratory parameters it is a useful parameter for distinguishing between viral and bacterial origins of infection, as was shown by multivariate stepwise linear discriminant analysis.

Urinary neopterine in the assessment of lymphoid and myeloid neoplasia, and neopterine levels in haemolytic anaemia and benign monoclonal gammopathy

The urinary neopterine levels were measured by HPLC in 417 normal subjects and in 216 patients with haematological diseases. All patients with active malignancies (multiple myeloma, polycythaemia vera. Hodgkins lymphoma, non-Hodgkins lymphoma, chronic myelocytic and chronic lymphocytic leukaemia) showed highly elevated mean and median values compared to the control groups. Those of patients with multiple myeloma stage I were only raised to near the upper limit of healthy subjects. Of 123 patients with active disease 105 (85%) were above the upper limit. In contrast, the mean and median values of 56 patients with neoplasias in remission (Hodgkins and non-Hodgkins lymphoma, acute leukemia and multiple myeloma) were not different from those of healthy subjects, and only 7 (12.5%) of these patients had levels above the upper limit. In patients with non-malignant diseases (haemolytic anaemia and benign monoclonal paraproteinaemia) the mean and median values were not raised. In patients with non-Hodgkins lymphoma and with chronic lymphocytic leukemia, the neopterine levels corresponded with the tumor stage. The present data suggest tht neopterine assay may supplement laboratory measurements in haematological diseases, providing helpful information.