Immunocytochemical evaluation of the percentage of proliferating cells in pathological bone marrow and peripheral blood samples with the Ki-67 and anti-bromo-deoxyuridine monoclonal antibodies

The monoclonal antibody Ki-67, directed against a nuclear antigen expressed by dividing cells in all the phases of cell cycle except G0 and early G1, was used in combination with an anti-BrdU monoclonal antibody, reacting selectively with cells in S-phase, for assessing the percentage of proliferating cells in bone marrow and peripheral blood samples from patients with lymphoma, leukaemia and multiple myeloma. Immunocytochemical labelling of proliferating cells was performed on marrow frozen sections and/or cytospins using an immunoalkaline phosphatase (APAAP) technique that made it possible to obtain proliferative index measurements in a few hours in contrast to the 3-7 d needed with tritiated thymidine. In the 54 marrow lymphoma cases studied a highly significant correlation was observed between the proportion of Ki-67 (+) cells and the separation into low- and high-grade malignant lymphomas according to the Kiel classification. In patients with multiple myeloma at the first diagnosis, the percentage of Ki-67 (+) cells was low (6-10%). In contrast, a high percentage of Ki-67 (+) cells (40-50%) was observed in a young adult with multiple myeloma, in a patient who first presented at the clinical observation with an extradural mass and in three patients who developed extramedullary masses several years after the initial diagnosis of myeloma. In acute lymphoblastic leukaemias of common type the mean value of Ki-67 labelling was 31.3%. Because of their simplicity and rapidity, immunocytochemical techniques may be expected to replace autoradiography and flow cytometry for the detection of proliferating cells in haematological samples.     

Multiple myeloma: current therapy and a glimpse of the future

Patients with benign monoclonal gammopathy or smouldering multiple myeloma should not be treated. The plasma cell labelling index utilizing tritiated thymidine or a monoclonal antibody to 5-bromo-2-deoxyuridine is helpful in differentiating patients with benign monoclonal gammopathy or smouldering myeloma from those with overt myeloma. Although combinations of chemotherapeutic agents seem to produce a greater number of objective responses than does melphalan-prednisone, a significant difference in survival has not been proved. Possibilities for future treatment include chemotherapy with large intravenous doses of melphalan, very small doses of cyclophosphamide or melphalan, the administration of hydroxyurea before chemotherapy, combination of interferon with alkylating agents, autologous bone marrow transplantation, and improvement of the soft-agar colony-forming assay for myeloma cells. The therapeutic use of monoclonal antibodies to plasma cell antigens may be possible in the future. Much more needs to be learned about the biologic basis of myeloma before real progress can be made.     

Serum beta 2-microglobulin, serum creatinine and bone marrow plasma cells in benign and malignant monoclonal gammopathy

Serum beta 2-microglobulin concentrations were determined in samples of 65 patients with benign or malignant monoclonal gammopathy. In the group of patients suffering from multiple myeloma or Waldenstr?m's macroglobulinemia the mean beta 2-microglobulin level was significantly higher than in the group with benign monoclonal gammopathy. Values above 3 mg/l were highly indicative of malignant disease and observed in 50% of the myeloma patients. Serum creatinine levels were significantly correlated to beta 2-microglobulin levels. However, mean creatinine concentrations did not significantly differ between the two groups of patients. Plasma cells and lymphoplasmocellular elements containing cytoplasmic immunoglobulin were counted in bone marrow samples of all patients. The counts, expressed in percent of nucleated bone marrow cells, allowed a good discrimination between the benign and the malignant group of patients. Bone marrow from patients with multiple myeloma or macroglobulinemia contained more, from patients with benign monoclonal gammopathy less than 17% plasma cells. No significant correlation was noticed between the extent of this plasmocytic bone marrow infiltration and serum beta 2-microglobulin or creatinine levels.     

Monoclonal antibody Ki-67 as a marker of proliferative activity in monoclonal gammopathies

In 16 patients with monoclonal gammopathies of undetermined significance (MGUS) and in 49 with multiple myeloma (MM, 43 untreated and 6 relapsed) we used immunocytochemistry to determine the percentages of bone marrow plasma cells (BMPC) that incorporate bromodeoxyuridine (BUDR-labeling index, BUDR-LI) in vitro and that label with the monoclonal antibody Ki-67 (which recognizes an antigen thought to identify the growth fraction of the population, Ki-67 GF). Both mean and range values were greater for Ki-67 GF than for BUDR-LI. Most patients with high Ki-67 GF also had high BUDR-LI, although a linear correlation was not found between the two parameters. MGUS has lower values than MM, and the difference was much greater for Ki-67 GF than for BUDR-LI (p less than 0.005 vs. p less than 0.05). Differences in Ki-67 GF but not in BUDR-LI were found between MGUS and stage I MM (p less than 0.0005) and between grouped stage I and II MM and stage III MM (p less than 0.025). Both Ki-67 GF and BUDR-LI were significantly (p less than 0.005) greater in relapsed than in untreated MM. Determining Ki-67 GF as a proliferative parameter could be a better way of studying the kinetics of (BMPC) in MGUS and MM than determining the BUDR-LI, since a wider range of values is obtained and this allows patient groups with different clinical characteristics to be separated more easily.     

Ploidy and proliferative characteristics in monoclonal gammopathies

Measurements were performed in 143 patients with monoclonal gammopathy of the cellular substrate in the bone marrow, including analysis of DNA and RNA content and tritiated thymidine labeling index. Aneuploidy by DNA content was present in 80% of 115 patients with active multiple myeloma and in 4 of 9 patients with benign monoclonal gammopathy, but was absent in all 12 patients with myeloma in remission and in 7 individuals with Waldenstrom's macroglobulinemia. With regard to prognosis in multiple myeloma, a low pretreatment plasma cell labeling index of less than or equal to 1% heralded longer survival than that observed in patients with a labeling index less than 1%, independent of myeloma tumor burden and ploidy pattern, except for a subset of 17 patients with a low-degree hyperdiploid abnormality whose survival was not affected by the magnitude of the pretreatment labeling index. Thus, besides tumor burden, tumor proliferative activity and ploidy both appear to have additional prognostic importance for patients with multiple myeloma.     

Detection of monoclonal B lymphocytes in multiple myeloma by immunofluorescence tests of surface immunoglobulins.

Peripheral blood lymphocytes of 16 patients with secretory type of multiple myeloma and 5 with nonmyelomatous monoclonal gammopathy were investigated for the surface immunoglobulins on the cell by immunofluorescence. A low pH shock of cells before staining was applied to dissociate the passively absorbed immunoglobulins present on the cell surface. Increases of B lymphocytes bearing surface immunoglobulins which have the same light chains as those of monoclonal immunoglobulins produced by the plasma cells were found in 5 of 11 common secretory myeloma patients and in all of 6 Bence-Jones myeloma patients. Ratios of cells bearing light chains of kappa- and lambda-types (kappa/lambda) appeared abnormal in almost all with an exception of only 3 cases of myeloma patients, even in the cases where the number of Ig bearing cells did not increase. Increases of possible monoclonal B cells bearing IgG, in addition to IgA cells, were observed in some patients with IgA myeloma. Increases of B cells bearing certain heavy chains were also observed in all 5 patients with Bence-Jones myeloma during the course of disease. No abnormalities of B cells bearing surface immunoglobulin were found in nonmyelomatous monoclonal gammopathy. These results suggest that proliferation of monoclonal B lymphocytes, which may be progenitors to the malignant plasma cells, occurs in a majority of myeloma patients, but not in nonmyelomatous monoclonal gammopathy.     

IgD monoclonal gammopathy with long-term follow-up

Summary. The presence of a serum IgD monoclonal protein (M-protein) is usually indicative of a malignant plasma cell disorder. However, one case of well-documented benign monoclonal gammopathy (BMG) of IgD type has been reported. We describe another patient with IgD monoclonal gammopathy of undetermined significance (MGUS) with long-term follow-up. In this patients an IgD λ M-protein was found on routine serum electrophoresis. She is alive and has no evidence of multiple myeloma or amyloidosis after 8 years of follow-up. Although IgD MGUS is exceedingly rare, this case demonstrates that the presence of a serum IgD M-protein is not necessarily synonymous with a malignant plasma cell process.     

Secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation in multiple myeloma

In the course of multiple myeloma, patients may develop a M-protein band different from the original: secondary monoclonal gammopathy of undetermined significance. In this retrospective single center analysis, we describe the occurrence and clinical relevance of secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation (post-transplant monoclonal gammopathy of undetermined significance). A total of 138 patients who had undergone 139 allogeneic stem cell transplantations (39.6% in the upfront setting and 60.4% for relapsed multiple myeloma) were included in the study. Sixty-seven (48.2%) patients developed secondary monoclonal gammopathy of undetermined significance, after a median latency of 6.9 months. Secondary monoclonal gammopathy of undetermined significance occurred more often in patients who achieved at least very good partial response after allogeneic stem cell transplantation, compared to partial response or less (54.8% vs. 26.5%; P=0.005). The incidence was also higher in the upfront setting as compared to relapsed disease, or with a sibling donor compared to matched unrelated donor, but less often after T-cell depletion. Importantly, development of post-transplant monoclonal gammopathy of undetermined significance as a time-dependent variable independently predicted for superior progression-free and overall survival (median progression-free survival 37.5 vs. 6.3 months, P<0.001; median overall survival 115.3 vs. 31.0 months, P=0.004). Clinicians should be aware of the benign nature of this phenomenon, and secondary monoclonal gammopathy of undetermined significance should not be confused with relapse or progression of disease. (Trial registered with trialregister.nl; HOVON 108: NTR 2958.)     

Measurement of apoptosis and proliferation of bone marrow plasma cells in patients with plasma cell proliferative disorders

The proliferative rate of malignant plasma cells, as measured by the plasma cell labelling index (PCLI), is an important prognostic factor in multiple myeloma (MM); however, the PCLI alone is probably Inadequate to describe tumour growth because it ignores the idea that myeloma cells may have a reduced rate of apoptosis. The aims of this study were to develop a flow cytometric method to measure the apoptosis index of fresh marrow plasma cells and develop a plasma cell growth index (PCGI) that related both proliferation and apoptosis to disease activity. Marrow aspirates were obtained from 91 patients with plasma cell disorders and the plasma cells in apoptosis were identified by either 7-amino actinomycin-D (7-AAD) or annexin V-FITC three-colour flow cytometry. The median plasma cell apoptotic index (PCAI) for patients with monoclonal gammopathy of undetermined significance (MGUS), smouldering or indolent myeloma (SMM/IMM), and new multiple myeloma (MM) was 5.2, 3.4 and 2.4, respectively (P=0.03, MGUS v MM). The median PCLI for these same patient groups was 0.0, 0.2 and 0.6, respectively (P<0.001, MGUS v MM). The paired PCLI and PCAI for each sample were used to derive the PCGI=2 + [PCLI-(O.1)(PCAI)]. The median PCGI for patients with inactive disease (MGUS, SMM/IMM or amyloidosis) was 1.8 compared to 2.4 for those with active disease (new or relapsed MM) (P<0.001). These results suggest that a decrease in the PCAI may be a factor in the progression from MGUS to SMM to overt MM.     

Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67

To assess the prognostic significance of the growth fraction in diffuse large cell lymphoma (DLCL), we studied 105 DLCL patients with the monoclonal antibody Ki-67 applied to frozen tissue sections. Ki-67 detects a nuclear antigen associated with cell proliferation not found in resting cells. Ki-67 findings and other clinical prognostic factors were correlated with outcome using univariate and multivariate analyses in the proportional hazards model. High proliferative activity, defined as nuclear Ki-67 expression in greater than 60% of malignant cells (Ki- 67 greater than 60), was found to be a strong predictor of poor survival among these patients (P = .003, log-rank). The 19 patients with Ki-67 greater than 60% had a median survival of 8 months compared with a median survival of 39 months for the 86 patients with Ki-67 less than or equal to 60%. Examination of pretreatment clinical variables indicated the patient groups were similar with regard to age, sex, stage, B symptoms, tumor bulk, and lactate dehydrogenase (LDH). Both patient groups received comparable curative intent therapy and showed comparable complete response rate precluding treatment differences as modifying outcome. Multivariate analysis indicated Ki-67 is an independent predictor of survival (multivariate P = .006). Further statistical analysis using only B-cell DLCL patients treated with CHOP (63 patients) indicated that Ki-67 greater than 60 retained strong prediction of poor outcome (P = .002, log-rank) among this homogeneous group. We conclude that high proliferative activity (Ki-67 greater than 60) is an independent factor allowing laboratory prediction of probable poor outcome of DLCL.     

Ig VH gene mutational patterns indicate different tumor cell status in human myeloma and monoclonal gammopathy of undetermined significance

Plasma cell tumors display a wide spectrum of clinical progression, ranging from aggressive multiple myeloma to a benign form known as monoclonal gammopathy of undetermined significance (MGUS), which requires no treatment. Because both diseases involve mature Ig- secreting plasma cells, the reason for this variation in malignant behavior is unclear. However, assessment of malignant potential is desirable for choice of treatment protocols. Ig variable (VH) gene sequences analysis has previously shown the tumor cell of multiple myeloma to be postfollicular, with mutated homogeneous clonal sequences indicating no continuing exposure to the somatic hypermutation mechanism, and this was confirmed in 7 of 7 patients. Comparison of the VH gene sequences in the monoclonal cells in MGUS yielded a different result, with 3 of 7 patients demonstrating mutated heterogeneous sequences consistent with the tumor cells remaining under the influence of the mutator. In 1 of 3 of these patients, an IgM-positive precursor cell was identified that expressed heterogeneous VH sequences similar to those of the isotype-switched plasma cell. These results indicate that the clonal cells in MGUS differ from those in myeloma and suggest that the difference may reflect malignant potential.     

Benign monoclonal gammopathy and peripheral neuropathy

Peripheral neuropathy has been described in malignant plasma cell dyscrasias such as multiple myeloma and Waldenst?m's macroglobulinaemia. Since it is not known whether the neuropathy is related to the plasma cell disorder or is a paramalignant phenomenon, 21 consecutive out-patients with benign monoclonal gammopathy (BMG) were analysed for peripheral neuropathies. Eleven patients had noticed slight motor and/or sensory extremity symptoms. Clinical examination, electromyographic and electroneurographic studies of the upper and lower extremities were performed. In five patients all results indicated a neuropathy, six other patients had clinical signs of neuropathy and four additional patients had positive electromyographic and/or electroneurographic results compatible with neuropathy. There were no significant differences in haematological parameters between the group where all results indicated a neuropathy and the totally negative group or between the two groups with and without clinical neuropathy. Thus, the benign form of plasma cell dyscrasias seems also to be associated with mild clinical or subclinical peripheral neuropathy.     

Immunophenotypic aberrations, DNA content, and cell cycle analysis of plasma cells in patients with myeloma and monoclonal gammopathies

We describe the immunophenotypic and gross DNA defects in 55 patients with myeloma and 50 patients with monoclonal gammopathy and review the literature on this subject (MedLine, 1994-2000). Our data confirmed previous reports indicating that in myeloma nearly all marrow plasma cells are abnormal (98.7 +/- 8.1%). In monoclonal gammopathy the fraction of abnormal plasma cells was 35.0 +/- 32.8%. In both myeloma and monoclonal gammopathy, the most frequent aberrant phenotypic features consisted of absence of expression of CD19, strong expression of CD56, and decreased intensity of expression of CD38; aberrant expression of CD10, CD20, CD22, or CD28 was observed in less than one-third of myeloma cases. The vast majority of cases had two or more phenotypic aberrations. In the DNA studies, 7% of myeloma cases were biclonal and 93% of cases were monoclonal. In those studies with only one plasma cell mitotic cycle, 37% had normal DNA content and 63% were aneuploid (hyperploid, 61%; hypoploid, 2%). The mean percentages of plasma cells in S- and G2M phases were 4.9 +/- 8.5 and 4.4 +/- 6.9%, respectively. Thirty-eight percent of cases had more than 3% of plasma cells in S phase. In monoclonal gammopathy, the DNA index of abnormal plasma cells ranged from 0.89 to 1.30 and the percentage of diploid (31%) and aneuploid (69%) cases was not different from the results found in myeloma. The differences in percentage of abnormal plasma cells in S- (7.4 +/- 8.6%) and G2M-phases (2.4 +/- 1.7%) in patients with monoclonal gammopathy were not statistically significant.     

In vitro cytotoxic response to human myeloma plasma cells by peripheral blood leukocytes from patients with multiple myeloma and benign monoclonal gammopathy.

Peripheral blood leukocytes (PBL) from myeloma patients were studied for their capacity to lyse plasma cells from myeloma patients, benign monoclonal gammopathy (BMG) patients, and nonneoplastic disease patients. Plasma cells were isolated from bone marrow, labeled with 51Cr, and cultured with PBL isolated from patients with myeloma, BMG, or nonneoplastic disease, as well as normal individuals. PBL from patients with multiple myeloma demonstrated responses to autologous or allogeneic myeloma plasma cells. Optimum conditions for cytotoxic response included a responder-to-stimulator ratio of 1:1 and an effector-to-target ratio of 20:1. PBL from normal individuals or patients with BMG failed to demonstrate this response. However, PBL from BMG patients, but not normal individuals, could be induced to kill myeloma plasma cells (but not nonmyeloma plasma cells) by simultaneous stimulation with allogeneic lymphocytes and myeloma plasma cells.     

Chromosomal aberrations are shared by malignant plasma cells and a small fraction of circulating CD19+ cells in patients with myeloma and monoclonal gammopathy of undetermined significance

In the present study, we aimed to identify distinct structural and numerical chromosomal aberrations in peripheral blood B cells of patients with myeloma and monoclonal gammopathy of undetermined significance (MGUS), which reflect changes thought to occur at different stages of the disease process. Peripheral blood from 12 patients with multiple myeloma and three patients with MGUS was investigated for the occurrence of retinoblastoma-1 gene deletions, p53 gene deletions and numerical aberrations demonstrated previously to be present in the patients' bone marrow CD138+ cells. By combining immunocytochemical staining for light chains and interphase fluorescence in situ hybridization (FISH), aberrant light-chain +ve cells were detected in the circulating CD19+ cell fraction. Each kind of chromosomal change present in the myeloma tumour cells was found to be shared by a small fraction of CD19+ cells (0.1-1.8%; median 0.36%, n = 6). In one MGUS patient, aberrant cells could be identified with a frequency of 0.34% within the CD19-sorted cell fraction. Clonotypic cells were detected with a frequency of 0.01-0.07% of peripheral blood nucleated cells by m-RNA in situ hybridization with patient-specific probes in three investigated patients. These results provide evidence that the circulating clonotypic B cells are closely related to the malignant plasma cells in myeloma and MGUS.     

The growth fraction of human myeloma cells

Greater reductions of tumor load in patients with multiple myeloma may result from therapeutic strategies that are based on a better knowledge of growth kinetics. We have previously shown that the labeling index of myeloma cells remains unchanged when tumor mass is reduced and that the cells of relapsing patients have differnt biologic properties than the cells present before melphalan-prednisone therapy. This study investigated the growth fraction (GF) of myeloma cells at various disease stages using continuous i.v. infusions of tritiated thymidine. We studied 17 patients on 22 occasions (4 untreated, 2 unresponsive, 6 in remission, and 10 in relapse). All untreated an unresponsive patients and 5 of 6 patients in remission had a GF of less than 4%. GF was defined in these studies as the maximum percentage of labeled plasma cells exposed continuously to tritiated thymidine. Relapsing patients, with the most rapid tumor doubling times, had GF ranging from 14% to 83%. The plasma cell transit time through the proliferative compartment for all of the relapsing patients ranged from 6.6 to 11.9 days and the calculated intrinsic cell loss ranged from 50% to 86%. These findings support our model for the growth kinetics of multiple myeloma that assumes that the entire tumor mass issues from a small proportion of proliferating cells and that the growth kinetics of myeloma cells in relapsing patterns differ from those in untreated and unresponsive patients. Therapeutic trials with cycle-active agents need further investigation in selected relapsing patients who are likely to have a high growth fraction.     

B and T cell markers of bone marrow and peripheral blood lymphoid cells in patients with paraproteinaemia.

Studies have been carried out on B and T cells in bone marrow and peripheral blood from patients with paraproteinaemia. The peripheral blood of patients with multiple myeloma showed a significant increase of B cells, mainly lymphoid cells bearing immunoglobulins corresponding to the paraproteins, while in patients with benign monoclonal gammopathy only a slight increase of B cells and a moderate decrease of T cells have been found. As to the bone marrow, the B cell population was significantly raised in patients with multiple myeloma, but it remained unchanged in patients with benign monoclonal gammopathy. Our findings may offer a new possibility to distinguish between these two diseases and provide further data to their pathogenesis.     

Light chain restriction analysis of bone marrow plasma cells in patients with MGUS or ''solitary'' plasmacytoma: diagnostic value and correlation with clinical course

Twenty-eight patients with a diagnosis of 'solitary' plasmacytoma or with gammopathy but with nondiagnostic morphologic examination of the bone marrow were investigated using a short-term bone marrow culture technique which enriched for the plasma cell fraction. The percentage of monotypic plasma cells in these plasma cell enriched cultures was correlated with the subsequent clinical course. The majority of patients with plasmacytoma and a significant number of those with gammopathy but with otherwise non-diagnostic investigations were found to have a monoclonal plasma cell component of greater than 20%. There was a significant correlation between the percentage of monoclonal plasma cells as detected by bone marrow culture and subsequent progression to disseminated myeloma. These results indicate that early bone marrow involvement can be detected by means of plasma cell culture prior to morphologic identification of marrow plasmacytosis and that short-term plasma cell culture distinguishes patients with early, low bulk myeloma from those with monoclonal gammopathy of uncertain significance (MGUS).     

A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1

We developed a new monoclonal antibody, B-B4, which specifically identifies human plasma cells. It strongly reacts with all multiple myeloma cell lines and with malignant plasma cells of all tumour samples of the multiple myeloma patients tested. B-B4 does not react with any peripheral blood, bone marrow or tonsil cells. Cloning of the B-B4 antigen reveals that the monoclonal antibody recognizes syndecan-1. It appears that the monoclonal antibody B-B4 is a suitable marker for human plasmocyte identification among haemopoietic cells and a useful probe for the diagnosis of haematological malignancies. Furthermore, this monoclonal antibody can be used for depletions prior to CD34 grafting.