Hyperdiploidy is a common finding in monoclonal gammopathy of undetermined significance and monosomy 13 is restricted to these hyperdiploid patients.

PURPOSE: Two pathways, hyperdiploid and nonhyperdiploid, are proposed for progression to plasma cell neoplasia. Implication of monosomy 13 (Delta13) is unclear in monoclonal gammopathy of undetermined significance (MGUS), and data on DNA content of plasma cells [DNA index (DI)] are rare. EXPERIMENTAL DESIGN: We ascertained DI in 169 multiple myeloma (MM) and 96 MGUS patients. Interphase fluorescence in situ hybridization (FISH) coupled to cytoplasmic staining of specific Ig (cIg-FISH) was done to look for trisomies and to ascertain Delta13. RESULTS: Hyperdiploidy and hypodiploidy were found in 54% and 11.5% of MGUS patients and in 59.5% and 25% of MM patients, respectively. In MGUS patients tested using probes for odd chromosomes, cIg-FISH showed association between trisomies for chromosomes 3, 7, 9, 11, or 15 and hyperdiploidy. Delta13 was found in 45.3% and 24.6% of MM and MGUS patients, respectively. Most Delta13 cases observed in MGUS were found within hyperdiploid clones, 38% versus 11% in hypodiploid cases, in sharp contrast with the occurrence of Delta13 in MM patients, 31.9% and 76.3%, respectively. That peculiar distribution of Delta13 according to DI persisted with other thresholds used to ascertain hyperdiploidy, such as DI >or= 1.05. A strong relationship between IgA peak and hypodiploidy (P = 0.007) was only observed in MM, whereas lambda light chain was significantly associated with hypodiploidy in MGUS (P = 0.001) and MM (P = 0.05). Hyperdiploidy shows similar pattern in MGUS and MM. CONCLUSION: This fits well a hyperdiploid pathway leading to MM after a preceding MGUS stage. Yet-to-be-determined secondary event(s) needs to occur for the transition to MM, unrelated to changes in chromosome number or to loss of chromosome 13. In contrast, the "nonhyperdiploid" pathway needs to be clarified further because hypodiploidy is less common in MGUS than in MM and Delta13 is rare in hypodiploid MGUS patients compared with hypodiploid MM patients.     

Molecular pathogenesis and a consequent classification of multiple myeloma.

There appear to be two pathways involved in the pathogenesis of premalignant non-immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Nearly half of tumors are nonhyperdiploid, and mostly have one of five recurrent IgH translocations: 16% 11q13 (CCN D1), 3% 6p21 (CCN D3), 5% 16q23 (MAF), 2% 20q12 (MAFB), and 15% 4p16 (FGFR3 and MMSET). The remaining hyperdiploid tumors have multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and infrequently one of these five translocations. Although cyclin D1 is not expressed by healthy lymphoid cells, it is bi-allelically dysregulated in a majority of hyperdiploid tumors. Virtually all MM and MGUS tumors have dysregulated and/or increased expression of cyclin D1, D2, or D3, providing an apparent early, unifying event in pathogenesis. The patterns of translocations and cyclin D expression (TC) define a novel classification that includes eight groups: 11q; 6p; MAF; 4p; D1 (34%); D1+D2 (6%); D2 (17%); and none (2%). The hyperdiploid D1 group is virtually absent in extramedullary MM and MM cell lines, suggesting a particularly strong dependence on interaction with the bone marrow microenvironment. Despite shared progression events (RAS mutations, MYC dysregulation, p53 mutations, and additional disruption of the retinoblastoma pathway), the phenotypes of MGUS and MM tumors in the eight TC groups is determined mainly by early oncogenic events. Similar to acute lymphocytic leukemia, MM seems to include several diseases (groups) that have differences in early or initiating events, global gene expression patterns, bone marrow dependence, clinical features, prognosis, and response to therapy.     

Bone marrow angiogenesis in 400 patients with monoclonal gammopathy of undetermined significance, multiple myeloma, and primary amyloidosis.

PURPOSE: To determine whether bone marrow (BM) angiogenesis progressively increases along the spectrum of plasma cell disorders ranging from monoclonal gammopathy of undetermined significance (MGUS) to advanced myeloma. EXPERIMENTAL DESIGN: Four hundred patients with the following disorders were studied: MGUS (76 patients); smoldering (indolent; early-stage) multiple myeloma (SMM; 112 patients); newly diagnosed, active multiple myeloma (MM; 99 patients); relapsed (advanced) multiple myeloma (RMM; 26 patients); and primary amyloidosis (AL; 87 patients). Forty-two normal control BM samples were studied for comparison. BM angiogenesis was studied in a blinded manner by immunohistochemical staining for CD34 to identify microvessels. RESULTS: The median (range) microvessel density (MVD) per x400 high power field was 1.3 (0-11) in the controls, 1.7 (0-10) in AL, 3 (0-23) in MGUS, 4 (1-30) in SMM, 11 (1-48) in newly diagnosed MM, and 20 (6-47) in RMM; P < 0.001. MVD was significantly higher in MGUS, SMM, newly diagnosed MM, and RMM compared with controls and AL; P < 0.001. MVD was not significantly different between controls and AL. By grading, high-grade angiogenesis was present in 0% of controls and AL, 1% of MGUS, 3% of SMM, 29% of newly diagnosed MM, and 42% of RMM; P < 0.001. MVD correlated with the BM plasma cell labeling index (rho = 0.46, P < 0.001) and BM plasma cell percentage (rho 0.5, P < 0.001). Survival was 28 months in SMM and newly diagnosed MM with high-grade angiogenesis, compared with 53 months for those with low- and intermediate-grade angiogenesis; P = 0.02. CONCLUSIONS: BM angiogenesis progressively increases along the spectrum of plasma cell disorders, from the more benign MGUS stage to advanced myeloma, indicating that angiogenesis may be related to disease progression.     

Importance of quantitative histology of bone changes in monoclonal gammopathy

Quantitative histology of bone changes, using undecalcified transiliac bone biopsies (UTBB), was performed blindly in 46 individuals with monoclonal gammopathy (MG), including 17 with MG of undetermined significance (MGUS) and 29 with overt multiple myeloma (MM). Three MGUS presented an excess of osteoclastic resorption (OR) in the vicinity of clusters of tumour cells and developed overt B cell malignancies, chronic lymphocytic leukaemia, Waldenström''s disease and MM respectively. On the other hand, MGUS with normal OR remained stable (median follow-up = 28 months), with one exception who developed a systemic amyloidosis. In MM, excessive OR was only observed in areas invaded by myeloma cells. OR was frequently normal in active MM lacking myeloma cells in UTBB. Active MM without lesions on radiography had excessive OR. IgA and pure Bence Jones MM appeared more osteoclastic than IgG cases (P less than 0.05). Of major interest was the finding that one third of MM presented histological bone changes similar to osteoporosis, osteosclerosis or osteoblastic metastasis. Two major findings must be emphasized from the current data: UTBB could be of major interest for the early detection of a B cell malignancy; heterogeneity of myeloma bone condition is unexpected. If some changes appear directly related to the tumour (i.e. excessive OR or osteoblastic dysfunction), some others are probably accidentally associated with it (i.e. osteoporosis), both needing treatment other than chemotherapy.     

Serum thymidine kinase and beta-2 microglobulin in monoclonal gammopathies

We evaluated the serum thymidine kinase (TK) and beta-2 microglobulin (beta-2) levels of 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and of 29 patients with multiple myeloma (MM). Both parameters were significantly lower in MGUS than in MM patients and in early (stage I + II) than in advanced (stage III) MM. TK was also lower in MGUS than in stage I MM (p less than 0.025). A seven-fold increase of TK level was documented in one patient who developed a full blown picture of MM 6 years after a diagnosis of MGUS. In 3 patients with stage III MM, a sharp decrease in TK (40-77%) and in beta-2 (29-53%) levels at remission was evident with respect to the levels measured at diagnosis. Patients with high levels of TK or beta-2 had a shorter survival than those with low levels; however, this was statistically significant only for beta-2 levels (p less than 0.02). Serum TK as well as beta-2 levels appear to be of clinical value in monoclonal gammopathies and related to the course of the disease.     

Serum thymidine kinase in monoclonal gammopathies. A prospective study. The Cooperative Group for Study and Treatment of Multiple Myeloma.

Between January 1986 and March 1990, the serum levels of thymidine kinase (TK) were evaluated at diagnosis in 97 patients with monoclonal gammopathy of undetermined significance (MGUS) and 149 patients with multiple myeloma (MM) enrolled in a prospective protocol for treatment of MM. At presentation, patients with MGUS had lower TK levels than those with Stage I MM (P less than 0.05) and the overall population of those with MM (P less than 0.0005). TK levels were increased in advanced stages in comparison with earlier ones (P less than 0.01). The TK level was related to survival. With a median follow-up of 29 months, patients with TK levels greater than 7.0 U/microliters had shorter survival times than those with lower levels (medians, 23 and 42 months; P less than 0.0001). In a multivariate analysis, TK explained most of the variability of survival (P less than 0.0001), the remaining being accounted for by serum creatinine and beta-2 microglobulin. No changes in TK levels occurred during follow-up of patients with stable MGUS, whereas TK levels increased in two patients at time of progression to overt MM. In patients with MM, TK levels decreased (P less than 0.01) in those who responded to treatment but increased in those having relapses (P less than 0.03) and those with progressive disease (P less than 0.03). These results indicate that TK has clinical and prognostic relevance in monoclonal gammopathies, and additional investigations are warranted to determine whether it is a useful tool for the clinical evaluation, staging, and follow-up of patients with MM.     

Massive parallel IGHV gene sequencing reveals a germinal center pathway in origins of human multiple myeloma

Human multiple myeloma (MM) is characterized by accumulation of malignant terminally differentiated plasma cells (PCs) in the bone marrow (BM), raising the question when during maturation neoplastic transformation begins. Immunoglobulin IGHV genes carry imprints of clonal tumor history, delineating somatic hypermutation (SHM) events that generally occur in the germinal center (GC). Here, we examine MM-derived IGHV genes using massive parallel deep sequencing, comparing them with profiles in normal BM PCs. In 4/4 presentation IgG MM, monoclonal tumor-derived IGHV sequences revealed significant evidence for intraclonal variation (ICV) in mutation patterns. IGHV sequences of 2/2 normal PC IgG populations revealed dominant oligoclonal expansions, each expansion also displaying mutational ICV. Clonal expansions in MM and in normal BM PCs reveal common IGHV features. In such MM, the data fit a model of tumor origins in which neoplastic transformation is initiated in a GC B-cell committed to terminal differentiation but still targeted by on-going SHM. Strikingly, the data parallel IGHV clonal sequences in some monoclonal gammopathy of undetermined significance (MGUS) known to display on-going SHM imprints. Since MGUS generally precedes MM, these data suggest origins of MGUS and MM with IGHV gene mutational ICV from the same GC B-cell, arising via a distinctive pathway.     

Angiogenesis in multiple myeloma: correlation between in vitro endothelial colonies growth (CFU-En) and clinical-biological features.

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/10(6) BM mononucleated cells in untreated MM samples (2.07 s.d. +/- 1.3) was significantly higher than in normal BM (0.28 +/- 0.48), while no difference was seen between normal BM and MGUS (0.28 +/- 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 +/- 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 +/- 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.     

Expression of p170 protein in multiple myeloma: a clinical study.

The expression of the p170 multidrug resistance protein by bone marrow plasma cells (BMPC) was assessed at clinical presentation in 53 patients with multiple myeloma (MM) using the C219 monoclonal antibody. Twenty-two of the 53 (41 per cent) patients had variable aliquots (1-60 per cent, median = 6 per cent) of p170+ BMPC by immunocytochemistry. Five of 10 patients studied using bivariate flow cytometry had both diploid and hyperdiploid (DNA index ranged from 1.2 to 1.5) BMPC with hyperdiploid clones having significantly greater p170 expression than diploid ones. Of the 37 patients evaluated for a response, 20 (54 per cent) had responded to induction chemotherapy. The presence of p170+ BMPC was a negative indicator for achieving response. The response rate was 75 per cent for p170- and 25 per cent for p170+ cases (p < 0.01), with no difference on the basis of treatment schedule (melphalan and prednisone, 24 patients; peptichemio, vincristine and prednisone, 13 patients). No difference in response and survival duration was found between p170+ and p170- patients. In six of nine patients studied both at diagnosis and following induction chemotherapy the p170+ BMPC% increased irrespective of the type of treatment or outcome.     

Frequent demonstration of human herpesvirus 8 (HHV-8) in bone marrow biopsy samples from Turkish patients with multiple myeloma (MM).

In order to investigate the frequency of HHV-8 in MM patients from another geographic location, we obtained fresh bone marrow (BM) biopsies from Turkish patients with MM (n = 21), monoclonal gammopathy of undetermined significance (MGUS) (n = 2), plasmacytoma (n = 1) with BM plasma cell infiltration, various hematological disorders (n = 6), and five healthy Turkish controls. The frequency of HHV-8 was analyzed by polymerase chain reaction (PCR) in two independent laboratories in the USA and in Turkey. Using fresh BM biopsies, 17/21 MM patients were positive for HHV-8 whereas all five healthy controls, and six patients with other hematological disorders were negative. Two patients with MGUS, and one patient with a solitary plasmacytoma were also negative. The data from the two laboratories were completely concordant. Also using primer pairs for v IRF and v IL-8R confirmed the results observed with the KS330233 primers. Furthermore, sequence analysis demonstrated a C3 strain pattern in the ORF26 region which was also found in MM patients from the US. Thus, HHV-8 is present in the majority of Turkish MM patients, and the absence of the virus in healthy controls further supports its role in the pathogenesis of MM.     

CD44 isoforms are differentially regulated in plasma cell dyscrasias and CD44v9 represents a new independent prognostic parameter in multiple myeloma

To evaluate the role of CD44 variant isoforms (CD44v) in plasma cell dyscrasias, CD44v expression was analysed in bone marrow (BM) biopsies of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) patients, in biopsies of soft tissue infiltration by MM and in extramedullary plasmacytoma samples. Expression of CD44 isoforms containing the 3v, 4v, 6v or 10v domain was observed in 15, 7, 13 and 5% of 87 samples from 49 consecutive MM cases, but could not be detected in ten normal persons or 11 MGUS patients. In contrast, CD44v9 revealed a broader pattern of expression and was observed in plasma cells in three out of ten normal persons and in three out of 11 MGUS cases. In MM, CD44v9 was detected in 32 out of 87 samples (37%) of BM infiltrates and was associated with an advanced Durie and Salmon stage (P<0.03), a progressive disease (P<0.01) and an IgA subtype (P<0.01). Furthermore, CD44v9 expression was observed in three out of five cases of MM soft tissue infiltrates, was often upregulated during disease progression, was significantly correlated with a shorter overall survival (P<0.03) and emerged as an independent prognostic factor in multivariate analysis (stage: relative risk 1.36, P<0.02; CD44v9 expression: relative risk 1.45, P<0.04). These results substantiate the clinical relevance of CD44v domains in plasma cell disorders and establish CD44v9 as a new independent prognostic parameter in MM.     

Immunoglobulin Gene Rearrangement in Plasma Cell Dyscrasias: Detection of Small Clonal Cell Populations in Peripheral Blood and Bone Marrow

The bone marrow (BM) and peripheral blood (PB) samples of 71 patients with plasma cell dyscrasias were analysed by the Southern blot technique for the presence of clonal immunoglobulin (Ig) gene rearrangements. 53% of BM samples examined were archival material such as air dried BM slides or frozen trephine biopsies. The results were related to bone marrow plasmacytosis as determined by cytology and flow cytometry, and other clinical parameters. Clonal Ig gene rearrangements were found in BM samples of 45 (83%) of 54 MM patients and in 3 of 6 patients with monoclonal gammopathy of unknown significance (MGUS). Clonal cell populations in the PB were detected in 11 (30%) of 37 examined MM patients, but in none of the patients with MGUS or solitary plasmacytoma of bone. PB involvement was associated with progressive disease. Circulating monoclonal cells were significantly associated with higher M-protein levels (p 0.05). Thus, circulating clonal precursor cells are encountered more frequently in active MM.     

Characterization of bone marrow T cells in monoclonal gammopathy of undetermined significance, multiple myeloma, and plasma cell leukemia demonstrates increased infiltration by cytotoxic/Th1 T cells demonstrating a squed TCR-Vbeta repertoire

BACKGROUND: The majority of studies published to date regarding the role of the bone marrow (BM) microenvironment in the pathogenesis of monoclonal gammopathies (MG) have focused on the interaction between stroma cells and plasma cells, whereas information concerning the lymphocytes infiltrating the tumor microenvironment is scanty. METHODS: The authors measured the distribution, TCR-Vbeta repertoire, immunophenotype, and functional characteristics of different subsets of BM T lymphocytes from 61 nontreated patients with MG (30 patients with MG of undetermined significance [MGUS], 27 patients with multiple myeloma [MM], and 4 patients with plasma cell leukemia [PCL]). RESULTS: The authors found a significantly increased rate of BM infiltration by T cells in all patient groups, at the expense of CD4+CD8- and CD4-CD8- T lymphocytes and both CD4+CD28- and CD8+CD28- cytotoxic/effector T cell subsets, and associated with TCR-Vbeta expansions in both CD4+ and CD8+ BM T cells in the majority of patients with MGUS, MM, and PCL. Moreover, the percentage of T cells secreting interferon (IFN)-gamma was found to be increased (P < or = 0.05) both in CD4+ and CD8+ T cells in MGUS and MM patients, and a higher plasma concentration of IFN-gamma was found in patients with MM. It is interesting to note that a positive correlation was noted between the proportion of CD28- and both the percentage of IFN-gamma-secreting cells and the proportion of expanded TCR-Vbeta lymphocytes within the total BM CD4+ T cells. CONCLUSIONS: The results of the current study demonstrated an increased infiltration of BM by T cells associated with frequent TCR-Vbeta expansions and a more prominent cytotoxic/Th1 phenotype in all the patient groups studied.     

Fluorescence in situ hybridization analysis of aneuploidization patterns in monoclonal gammopathy of undetermined significance versus multiple myeloma and plasma cell leukemia

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) is a clonal plasma cell (PC) disorder usually characterized by a benign clinical course. However, in approximately 25% of patients, the disorder has been found to evolve into a multiple myeloma (MM). The mechanism leading to the evolution of MGUS remains unknown. The aim of the current study was, first, to assess by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities of chromosomes 6, 9, 13, and 17 in MGUS patients and to compare it with that found in MM and PC leukemia (PCL) patients and, second, to explore the potential heterogeneity of the pathologic PC in MGUS as a way to identify unique cytogenetic patterns different from those frequently observed in MM and PCL. METHODS: Numerical abnormalities of chromosomes 6, 9, 13, and 17 were investigated by dual- and triple-color FISH in bone marrow PC from 208 patients corresponding to MGUS (n = 30), MM (n = 158), and PCL (n = 20) cases. In MGUS and MM patients with < 10% PC, both normal and phenotypically aberrant PC were discriminated by multiparameter flow cytometry, the latter subset being specifically sorted for FISH analysis with a purity of 93% +/- 6%. RESULTS: Overall, 57% of the MGUS patients displayed abnormalities for at least 1 of the 4 chromosomes analyzed compared with 75% of both MM and PCL cases. The most common single chromosome abnormalities detected in MGUS were gains of chromosomes 9 (23%) and/or 6 (21%) and loss of chromosomes 13 (21%) and/or 17 (17%). Compared with MM patients, MGUS patients were found to have both a lower incidence of gains of chromosome 9 (23% vs. 54%, P = 0.002) and monosomy 13/13q(-) deletions (21% vs. 38%, P = 0.07); with respect to PCL cases, MGUS patients were found to have a lower incidence of monosomy 13/13q(-) deletions (21% vs. 75%, P < 0.001) together with a slightly higher frequency of gains of both chromosomes 6 (21% vs. 0%, P = 0.05) and 9 (23% vs. 7%, P = 0.1). The simultaneous use of two or three different chromosome probes showed that within the purified compartment of phenotypically aberrant PC from most MGUS patients (67%), more than 1 PC clone could be identified. In contrast, the incidence of 2 or more PC clones was much lower in MM (19%, P < 0.001) and PCL (15%, P = 0.003). Interestingly, although some FISH patterns were shared by both groups of diseases (i.e., monosomy 13/13q(-) deletions alone, gains of chromosome 9 alone or together with trisomy 6), others were found almost exclusively in either MGUS (i.e., a clone with monosomy 6 and/or 17 together with nuclei displaying a normal chromosome number) or in MM (i.e., monosomy 13/13q(-) deletions together with gains of chromosome 6 and/or 9). CONCLUSIONS: In summary, the results of the current study showed that MGUS patients displayed a high incidence of numerical alterations, which are usually associated with the presence of more than one tumor cell clone. It is interesting to note that the cytogenetic patterns observed in the aneuploid PC clones from MGUS patients were frequently different from those observed in both MM and PCL.     

CD29 expressed on plasma cells is activated by divalent cations and soluble CD106 contained in the bone marrow plasma: refractory activation is associated with enhanced proliferation and exit of clonal plasma cells to circulation in multiple myeloma patients

Malignant plasma cells (PC) in human multiple myeloma (MM) are retained in the bone marrow (BM) microenvironment. Using HUTS21 monoclonal antibody that reacts with active CD29 integrin, we demonstrate that this active form is tightly regulated by divalent cations and soluble CD106 (sCD106) contained in the BM plasma. Moreover, we also show that in vivo expression of the active CD29 on PC was clearly diminished in a minority of MM cases (HUTS21(-) patients). HUTS21(-) cells were refractory to the addition of either normal allogeneic BM plasma or optimal concentrations of exogenous divalent cations and recombinant sCD106. Furthermore, a lower binding to fibronectin was detected in comparison with HUTS21(+) PC. On the other hand, although HUTS21(-) PC showed a reduced amount of total (active+inactive) CD29, western-blot assays demonstrated that these clonal PC contained the two species of CD29, with molecular masses of 110 and 130?kDa, which were expressed on normal or HUTS21(+) PC. Finally, we detected a clear association between the presence of HUTS21(-) PC in the BM and an increased percentage of circulating PC with a high proliferative index, emphasizing the essential role of CD29 in the pathogenesis and progression of this disease.     

A significant association of Ha-ras p21 in neuroblastoma cells with patient prognosis. A retrospective study of 103 cases

To evaluate biologic characteristics of neuroblastoma, the authors examined the expression of Ha-ras gene (Ha-ras p21) in 103 primary tumors obtained at the time of diagnosis. Higher expression of the Ha-ras p21 in tumor cells showed a significant association with lower clinical stage of the tumor at diagnosis (chi-square = 35.418, degrees of freedom [df] = 9, P less than 0.001) and survival of the patients (chi-square = 37.111, df = 3, P less than 0.001). Thirty-six (84%) of 43 patients with decreased Ha-ras p21 expression died of aggressive disease. The Ha-ras DNA was examined in the 32 tumors by Southern blot analysis. Neither augmentation nor deletion of the Ha-ras DNA was observed. Amplification of the N-myc DNA was also examined in 43 cases in comparison with Ha-ras p21 expression. N-myc amplification was detected in 12 (55%) of 22 patients who died, and 19 (86%) of the 22 patients showed a low expression of the Ha-ras p21 in tumor cells. Eighteen (86%) of 21 survivors showed a high expression of the Ha-ras p21. The expression of Ha-ras p21 was thought to be a clinically important marker for prognosis in children with neuroblastoma.     

Ploidy and proliferative activity of human brain tumors. A flow cytofluorometric study

Ploidy and proliferative characteristics were estimated by flow cytometry of the nuclear DNA content of 92 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and single cell suspensions were obtained with a mechanical dissociation technique. Propidium iodide was employed for nuclear DNA staining. Human normal brain tissue was used as internal diploid reference standard. 86% of benign tumors had unimodal DNA distribution with a DNA index (DNA I = modal channel of the G0/1 peak of the studied population/modal channel of the G0/1 peak of the normal brain) usually within the diploid or near-diploid range. 14.0% had aneuploidy, with an additional cell peak having a median DNA I of 1.60. Among malignant tumors, these figures were 61.2 and 38.8% (p less than 0.001). The percentage of S phase cells was higher in malignant (median = 3.6) than in benign tumors (median = 2.0, p less than 0.01), without correlation to histological tumor subtype. Flow cytometry appears to be a useful method for evaluating differences in DNA distribution in tumors of the central nervous system.     

Multiple myeloma cells recruit tumor-supportive macrophages through the CXCR4/CXCL12 axis and promote their polarization toward the M2 phenotype

Multiple myeloma (MM) cells specifically attract peripheral-blood monocytes, while interaction of MM with bone marrow stromal cells (BMSCs) significantly increased monocyte recruitment (p<0.01). The CXCL12 chemokine, produced by both the MM and BMSCs, was found to be a critical regulator of monocyte migration. CXCL12 production was up-regulated under MM-BMSCs co-culture conditions, whereas blockage with anti-CXCR4 antibodies significantly abrogated monocyte recruitment toward a MM-derived conditioned medium (p<0.01). Furthermore, elevated levels of CXCL12 were detected in MM, but not in normal BM samples, whereas malignant MM cells often represented the source of increased CXCL12 in the BM. Blood-derived macrophages effectively supported MM cells proliferation and protected them from chemotherapy-induced apoptosis. Importantly, MM cells affected macrophage polarization, elevating the expression of M2-related scavenger receptor CD206 in macrophages and blocking LPS-induced TNFα secretion (a hallmark of M1 response). Of note, MM-educated macrophages suppressed T-cell proliferation and IFNγ production in response to activation. Finally, increased numbers of CXCR4-expressing CD163+CD206+ macrophages were detected in the BM of MM patients (n=25) in comparison to MGUS (n=11) and normal specimens (n=8).Taken together, these results identify macrophages as important players in MM tumorogenicity, and recognize the CXCR4/CXCL12 axis as a critical regulator of MM-stroma interactions and microenvironment formation.