Ras oncogene mutation in multiple myeloma.

The frequency of ras (H-, K-, and N-ras) and c-myc oncogenes was investigated in multiple myeloma (MM). By means of the polymerase chain reaction (PCR)/oligonucleotide hybridization method, DNA from 56 tumor biopsies was analyzed for the presence of activating mutations involving codons 12 and 61 of the H-, K-, and N-ras genes and codon 13 of the N-ras gene. Mutations, involving the N- or K-ras genes, were detected in 18 of 56 (32%) cases of which 12/43 (27%) were at diagnosis and 6/13 (46%) were after treatment. In some cases, multiple mutations affecting different ras alleles were detected. Direct nucleotide sequence analysis of PCR products indicated that a more heterogeneous nature of the base pair changes than previously shown for other tumors along with a preferential involvement of N-ras codon 61. The heterogeneity of MM cases with respect to the presence of ras oncogenes prompted an analysis of possible correlations with different clinico-pathologic characteristics of MM from which a correlation between the presence of ras oncogenes and a partial or complete lack of response to therapy emerged. The frequency of activating rearrangements or mutations of the c-myc gene were studied by Southern blot analysis and PCR sequencing, respectively. However, contrary to previous reports involving mostly MM cell lines, no structural alterations of the c-myc gene were found. These results indicate that ras, but not c-myc, oncogenes are activated in vivo in MM cells, representing the first oncogene alteration that has been associated at appreciable frequency with this type of malignancy. While the mechanism of occurrence and biological role of ras activation in MM remains to be elucidated, the preliminary correlations observed in this study between the presence of ras oncogenes and poor therapeutic response suggest that further investigations of the possible prognostic significance of these alterations are necessary.

Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.

B cell-derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that >50% of CLLs display hypermutated immunoglobulin variable region (IgV) sequences and a more favorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of IgV hypermutation. To further investigate the phenotype of CLLs, their cellular derivation and their relationship to normal B cells, we have analyzed their gene expression profiles using oligonucleotide-based DNA chip microarrays representative of approximately 12,000 genes. The results show that CLLs display a common and characteristic gene expression profile that is largely independent of their IgV genotype. Nevertheless, a restricted number of genes (<30) have been identified whose differential expression can distinguish IgV mutated versus unmutated cases and identify them in independent panels of cases. Comparison of CLL profiles with those of purified normal B cell subpopulations indicates that the common CLL profile is more related to memory B cells than to those derived from naive B cells, CD5(+) B cells, and GC centroblasts and centrocytes. Finally, this analysis has identified a subset of genes specifically expressed by CLL cells of potential pathogenetic and clinical relevance.

Structural alterations of the NF-kappa B transcription factor lyt-10 in lymphoid malignancies.

We have previously reported the identification of a novel putative proto-oncogene involved in the breakpoint of a t(10;14)(q24;q32) chromosomal translocation in a case of B-cell lymphoma. This gene, called lyt-10 (NFKB2/p52), is a member of the NF-kappa B family of transcription factors and displays a high degree of homology with the NFKB1/p50. Here we describe the genomic organization of the lyt-10 gene based on the restriction analysis of genomic phage clones and the sequence determination of exon-intron boundaries. The lyt-10 gene spans a genomic region of about 8 kb on 10q24, and contains 24 exons, ranging in size between 41 and 258 base pairs. To improve the understanding of the role of lyt-10 in lymphomagenesis, we performed Southern blot analysis to detect alterations of the lyt-10 gene in a large panel of cases representative of different types of lymphoid malignancies. We found rearrangements in 5 of 228 (approximately 2%) cases analysed: two cases of B-cell lymphoma, one case of multiple myeloma and two cases of T-cell lymphoma. The use of various probes specific for different regions of the lyt-10 locus revealed that rearrangements in positive cases lead to the partial or total deletion of the carboxy-terminal region containing the ankyrin domain. Taken together, our results indicate that lyt-10 gene rearrangements represent a recurrent lesion that may be involved in the pathogenesis of both B- and T-cell malignancies, and suggest that truncation of the ankyrin domain may be a common mechanism of lesion leading to abnormal lyt-10 activation in lymphoid neoplasia.

Diagnostic role and prognostic significance of a simplified immunophenotypic classification of mature B cell chronic lymphoid leukemias.

We verified the diagnostic and prognostic role of a simplified immunophenotypic classification (IC) in a series of 258 patients (M/F: 1.4; median age: 64 years; median follow-up: 64 months; 75 deaths) with mature B cell lymphoid leukemias (MBC-LL) for whom no histopathological diagnosis was available because of minimal or no lymph node involvement. The IC was based on the reactivity of three pivotal immunophenotypic markers: CD5, CD23 and SIg intensity. On the basis of different expression patterns, we identified four diagnostic clusters (C) characterized by distinct clinico-biological features and different prognoses: C1 (149 patients) identified most classical B cell chronic lymphocytic leukemias (CLL-type cluster; SIg(dim)/CD5+/CD23+); C2, 38 patients whose clinico-hematological characteristics were intermediate between C1 and C3 (CLL-variant cluster; SIg(bright)/CD5+/CD23+/-or SIg(dim)/CD5-/-/CD23 indifferent); C3 (16 patients) most situations consistent with mantle cell lymphoma in leukemic phase (MCL-type cluster; SIg(bright)/CD5+/CD23-); and C4, 55 cases, most of whom were consistent with leukemic phase lymphoplasmacytic/splenic marginal zone lymphomas (LP/S-type cluster; SIg(bright)/CD5-/+/CD23 indifferent). At univariate survival analysis, prognosis worsened from C1 to C4, C2 and C3 (P = 0.0001), and this was maintained at multivariate analysis (P = 0.006), together with CD11c expression (P = 0.0043), age at diagnosis (cut-off 70 years; P = 0.0008) and platelet count (cut-off 140 x 10(9)/l; P = 0.0034). Besides recognising the two well-known situations of classic B-CLL and MCL, our IC identified situations with distinct prognostic and/or clinical behaviors.

BCL10 gene mutations rarely occur in lymphoid malignancies.

BCL10, a gene involved in apoptosis signalling, has recently been identified through the cloning of chromosomal breakpoints in extranodal (MALT-type) marginal zone lymphomas carrying the t(1;14)(p22;q32) translocation. BCL10 was also found mutated in these cases as well as in other types of lymphoid and solid tumors, suggesting that its inactivation may play an important pathogenetic role; however, this has been questioned by recent studies showing a lack of somatic mutations in human cancers. We report the mutation analysis of exons 1-3 of the BCL10 gene in DNAs from 228 cases of lymphoid malignancies (30 B cell chronic lymphocytic leukemias, 123 B and 45 T non-Hodgkin's lymphomas and 30 multiple myelomas). Somatic mutations were detected in four cases (approximately 2%): one small lymphocytic, one follicular and two diffuse large cell lymphomas. The mutations were all within exon 3 and have not been previously reported. Our data suggest that BCL10 mutations may play only a limited role in the pathogenesis of lymphoid neoplasms.

Scanning and transmission electron microscopy of clonal peripheral blood lymphocytes in low grade non-Hodgkin's B-cell lymphomas with predominant splenomegaly.

Peripheral blood mononuclear cells (PBLs) from 14 patients with low grade non-Hodgkin's B-cell lymphomas with predominant splenomegaly were studied by means of scanning (SEM) and transmission electron microscopy (TEM). All patients had peripheral blood and bone marrow involvement, the absence of lymphoadenopathy, and, except in one case, immunophenotypic features of a malignant proliferation of mature spleen B-cells arising from outside the germinal center, but not consistent with CLL or HCL. Several distinctive cytological features were observed in PBLs of the different subgroups. The SEM surface features of PBLs in patients with intermediate differentiation lymphocytic lymphoma (IDL) (five cases), lymphoplasmacytoid immunocytoma (LP-IC) (two cases), and mixed small and large cells malignant lymphoma (one case) were characterized by the presence of numerous well-developed microvilli. Some distinctive TEM ultrastructural features were also seen in the different cases. In the two cases of splenic lymphoma with villous lymphocytes (SLVL), SEM revealed large and elongated surface microvilli generally arising from two or three poles of the cells. This surface morphology, confirmed by TEM analysis, may be pathognomonic of this disease. Four additional cases, tentatively classified as small lymphocytic lymphoma on the basis of immunophenotypic data, were extremely heterogeneous at both SEM and TEM analysis. The ultrastructural features revealed by SEM and TEM may be useful for the more precise characterization of this heterogeneous group of diseases, which is generally difficult to define even when immunophenotypic and molecular approaches are used.

Structure and function of VLA integrins: differential expression in B-cell leukemia/lymphoma.

The integrin family of adhesion receptors includes at least 11 different alpha subunits and 6 different beta subunits which are associated to form 14 different alpha beta heterodimers, divided into three subfamilies. In particular, beta 1 subfamily integrins (VLA 1-6 proteins) have been found to mediate cell adhesion to extracellular matrix (ECM) component such as fibronectin, collagen, laminin; however, VLA-4 has been found to exhibit both cell-cell and cell-matrix adhesion functions. The reactivity of VLAs is virtually ubiquitous and independent of line or tissue specificity. However, the expression of individual VLAs within single tissues can be modulated according to the type or functional status of the cell. One of the main reasons for interest in these molecules is that they may play a determining role in neoplastic transformation and diffusion; in particular, in lymphoproliferative syndromes, a lack of cell adhesiveness or an abnormal adhesion pattern in neoplastic lymphocytes may free these cells from regulation, thus contributing towards the development of leukemia and/or lymphoma. Studies of VLA expression in B-cell leukemia/lymphomas show a modulation of VLA3 and VLA4 reactivity. The most interesting element is the identification of a VLA3/VLA4 pattern associated with B-cell chronic lymphocytic leukemia (B-CLL) characterised by a reduced expression of VLA4 and the constant expression of VLA3. Although the value of VLA3 as an additional marker for the diagnosis of classical B-CLL is indisputable, the biological/functional significance of this reactivity remains to be confirmed.

Differential expression of very late activation antigen-3 (VLA-3)/VLA-4 in B-cell non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia.

The expression of beta 1 (very late activation antigens, VLA 1-6) and beta 2 integrins (leukocyte adhesion molecules [Leu-CAM]) in cell suspensions from the peripheral blood of 70 patients with B-cell chronic lymphocytic leukemia (B-CLL), 15 patients with leukemic lymphocytic lymphoma of intermediate differentiation (IDL), as well as from the lymph nodes of 20 patients with low/intermediate-grade non-Hodgkin's lymphoma (NHL) was studied with the aim of characterizing their adhesive phenotype and evaluating its relationship to clinical behavior. CD11a(LFA-1) was more expressed in NHL and IDL than in B-CLL (P = .047), although it was demonstrable in 74.2% of cases; CD11c was more expressed in B-CLL (P less than .0001), and its expression was preserved in almost all of the cases of small lymphocytic lymphoma. In NHL patients, including the cases of IDL, VLA-3 expression was observable in 8 of 35 cases (although always at a low level of intensity), while VLA-4 was almost constantly expressed in a way that was similar to its expression in control normal B cells. On the contrary, in B-CLL patients, VLA-3 was expressed (prevalently at high levels) in 87.1% of cases and VLA-4 only in 37.1%. No correlation was found between adhesion molecule patterns and the clinical features of the diseases. The biofunctional significance of the imbalance of VLA-3 and VLA-4 expression in B-CLL is not easy to explain, but it has undoubted intrinsic value as an additional marker for distinguishing B-CLL from, in particular, those B-cell neoplasms (such as IDL) that share many of the immunocytomorphologic characteristics and the putative normal counterpart (the mantle zone) of B-CLL.

Analysis of tumor-specific immunoglobulin gene rearrangement in peripheral blood B-cells of multiple myeloma patients.

The search for bone marrow clone neoplastic precursors in the peripheral blood of multiple myeloma (MM) patients has been the subject of a number of studies which, using different methodological approaches, have led to conflicting results. With the aim of contributing toward resolving this controversy, immunoglobulin (Ig) gene rearrangement in the bone marrow mononuclear cells (BMMC) and B-lymphocyte enriched peripheral cells (BePBC) of 11 appropriately selected advanced MM patients was studied by means of Southern blotting. Peripheral mononuclear cells (PBMC) and BePBC were studied immunophenotypically by evaluating the presence of cytoplasmic Ig positive cells (CyIg+) and CD19/PCA-1 reactivity. A pattern of Ig gene rearrangement identical to that observed in the respective BMMC was found in only two patients who, moreover, had a significant number of CyIg+ cells. Ig gene rearrangement was not found in any of the remaining patients who showed no evidence of the presence of CyIg+ cells, although there were CD19/PCA-1 positive cells. Consequently, the significance of the presence of malignant pre-plasma cell peripheral B-lymphocytes needs to be reconsidered and, in any case, these cells should be looked for in cell populations greatly enriched in B-lymphocytes and, above all, completely lacking in plasma cells or CyIg+ cells.

p53 gene mutations in multiple myeloma are associated with advanced forms of malignancy.

The frequency and type of p53 gene mutations was investigated in a series of 52 cases of multiple myeloma (MM) representative of the different clinical phases and forms of the disease (indolent, 12 cases; chronic, 24 cases; acute/leukemic, 16 cases). DNAs were analyzed for p53 gene mutations in exons 5 to 9 by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing of PCR-amplified fragments. Point mutations were detected in 7 of 52 patients (13%) (5 at exon 8; 1 at exon 6; 1 at exon 7), and were specifically associated with the more advanced and clinically aggressive acute/leukemic forms of MM (7 of 16 [43%].) Three of the mutated cases had been evaluated at clinical presentation in earlier phases of the disease (indolent or chronic) and were found to be negative for p53 mutation. Moreover, three patients with p53 mutation had not received chemotherapy at the time of investigation. These results support the notion that the development of MM is a multistep process and suggest that alterations in the p53 gene may represent an important late event in MM tumor progression.

Poor prognosis in non-villous splenic marginal zone cell lymphoma is associated with p53 mutations.

We have recently reported a series of 15 non-villous splenic marginal zone lymphoma patients, six of whom showed p53 mutations (40%). This molecular alteration did not correlate with any particular clinico-pathologic feature at diagnosis. After a median follow-up of 56 months, four cases evolved into aggressive fatal non-Hodgkin's lymphoma (NHL) and two had refractory progressive disease; interestingly, p53 mutations were demonstrated in five of these patients at diagnosis. As the patients with wild-type p53 presented responsive or indolent disease, this genetic alteration may be an early marker of aggressive transformation or refractoriness. p53 evaluation at diagnosis could be advisable in this particular subset of NHL.

Immunophenotypes in "classical" B-cell chronic lymphocytic leukemia. Correlation with normal cellular counterpart and clinical findings.

This study evaluates the expression of a series of membrane antigens, normally expressed by B-lymphocytes of the lymphocytic mantle and marginal zone, in 90 selected cases of "classical" (mouse red blood cell-receptor+, CD20+, CD5+, surface immunoglobulin +/-) B-chronic lymphocytic leukemia (B-CLL) with the aim of contributing toward identifying the normal counterpart of B-CLL and any correlations between surface antigen pattern and certain clinical characteristics. Clustered (CD23, 25, 39, 40, 27, 1c, w75) and unclustered (NuB1, 7F7, KiB3) monoclonal antibodies (MoAb) were tested. Almost all cases showed high reactivity to CD23, 27, w75, 39, 40, and NuB1: expression of CD1c was very low and that of 7F7, KiB3, and CD25 was variable. The reactivity of 7F7 and KiB3 was strictly correlated, and they correlated individually with CD25. Results show that the most frequent B-CLL phenotype (CD19+, 5+, 23+, 27+, 39+, NuB1+, KiB3 +/-, 7F7 +/-, and CD25 +/-) corresponds to one or more cellular subsets in the mantle zone. No correlation was found between MoAb expression, surface immunoglobulin (SIg) class or type, clinical stage, disease activity, or age at diagnosis. The only difference (statistically borderline) was the expression of 7F7 and KiB3 (in young versus old patients). This suggests that modulations in the expression of surface antigens do not affect the clinical behavior of the disease.

A novel chromosomal translocation t(4; 14)(p16.3; q32) in multiple myeloma involves the fibroblast growth-factor receptor 3 gene.

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), the most consistent chromosomal abnormality is the 14q+ marker, which originates in one third of cases through a t(11; 14)(q13; q32) chromosomal translocation; in the remaining cases, the identity of the partner chromosomes has not been well established. We used a Southern blot approach based on the linkage analysis of the joining (J) and the constant (C) mu, alpha, and gamma regions to detect cases bearing IGH switch-mediated chromosomal translocations. We evaluated DNA of 88 nonkaryotyped patients with MM (78 cases) or plasma cell leukemia (PCL) (10 cases) and found the presence of "illegitimate" rearranged IGH fragments (no comigration between the J and C regions) in 21 cases. To confirm this analysis, we cloned the illegitimate rearranged fragments from three samples, and the molecular and fluorescent in situ hybridization (FISH) analyses indicated the presence of chromosomal translocations juxtaposing a switch IGH region to sequences from chromosomes 11q13 (one PCL case) or 4p16.3 (two MM cases). Interestingly, the breakpoints on 4p16.3 occurred about 14 kb apart in a genomic region located approximately 50 kb centromeric to the fibroblast growth-factor receptor 3 (FGFR3) gene. Moreover, Southern blot analysis using 4p16.3 genomic probes detected a rearrangement in an additional MM tumor. FISH analysis of the MM-derived KMS-11 cell line, reported to be associated with a t(4; 14)(p16.3; q32), showed that the FGFR3 gene was translocated on 14q32. High levels of FGFR3 mRNA expression were observed in the cloned MM tumors and KMS-11 cell line, but not in the cases that were apparently negative for this lesion. Furthermore, a point mutation at codon 373 in the transmembrane domain of the FGFR3 gene resulting in an amino acid substitution (Tyr --> Cys) was detected in the KMS-11 cell line. These findings indicate that the t(4; 14)(p16.3; q32) represents a novel, recurrent chromosomal translocation in MM, and suggest that the FGFR3 gene may be the target of this abnormality and thus contribute to tumorigenesis in MM.

No correlation between response and survival in patients with multiple myeloma treated with vincristine, melphalan, cyclophosphamide, and prednisone.

A vincristine, melphalan, cyclophosphamide, and prednisone (VMCP) multi-drug regimen was used in 85 previously untreated patients with multiple myeloma (MM) (symptomatic Durie Stages II and III) until they became refractory. The prognostic significance of various pretreatment characteristics was evaluated in terms of therapeutic response (according to Southwest Oncology Group [SWOG] and Chronic Leukemia-Myeloma Task Force [TF] criteria) and survival. Therapeutic responses, obtained in 31.2% (SWOG) and 68.7% (TF) of patients, had a significant inverse correlation with myeloma cell mass, serum calcium, and bone status. Median survival time of Stage II and Stage III patients was 39 and 34 months, respectively. Serum B2 microglobulin greater than or equal to 6 micrograms/ml was the only variable correlating unfavorably with survival duration after multi-variate analysis (increased risk = 2.79), although therapeutic response as a time-dependent variable had no effect on survival. These data suggest no correlation between response and survival, partially because of inadequate response assessment criteria and partially because no existing treatment is curative (although current therapeutic approaches may prevent death from complications).

Frequent p53 gene involvement in splenic B-cell leukemia/lymphomas of possible marginal zone origin.

A phenotypic and molecular evaluation was made of 15 patients with mature B-cell leukemia/lymphoma showing exclusive spleen and bone marrow involvement. According to French-American-British criteria, these cases could not be classified as classical B-cell chronic lymphocytic leukemia, hairy cell leukemia and its variant forms, splenic lymphoma with villous lymphocytes, or leukemic phase non-Hodgkin's lymphoma (NHL; follicular or intermediate type). The immunophenotype pattern (high surface Ig and CD25 expression, and little or no reactivity with CD5, CD23, and CD11c) and cytomorphologic features of these neoplasms suggested an origin in the marginal zone of the spleen. Molecular analysis did not show any involvement of the dominantly acting oncogenes generally associated with lymphoid malignancies (c-myc, bcl-2, bcl-1, Ras), but mutations of the p53 tumor suppressor gene involving exons 5, 6, and 8 were found in 6 cases (6 of 15, 40%). In 4 cases, the p53 alterations consisted of a point mutation leading to amino acid substitution. In the remaining 2 cases, an insertion or deletion resulting in a frame-shift of the protein was observed. In all but 1 of the cases, the wild-type sequence at the mutation site was barely visible, implying the loss of the normal p53 allele in leukemic cells. All of the cases showed a clinical course compatible with that of low-grade NHL, regardless of the p53 loss/mutation. Overall, our data suggest the existence of a form of splenic B-cell leukemia/lymphoma of possible marginal zone origin in which p53 inactivation may play an important pathogenetic role.

Analysis of p53 gene mutations in acute myeloid leukemia.

We have previously reported the absence of mutations within exons 5-9 of the p53 gene in a panel of 30 cases of acute promyelocytic leukemia (APL), which represent the M3 FAB type of acute myeloid leukemia (AML). In the present report, we extend our analysis of p53 gene mutations to 70 cases of AML representative of the other FAB types of the disease, including M1 (16 cases), M2 (20 cases), M4 (17 cases), M5 (12 cases), and M6 (5 cases). DNAs were analyzed for p53 gene mutations in exons 5 to 9 by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing of PCR-amplified products. Mutant p53 alleles were detected in 5 of 70 cases; 1 case in exon 5, 2 cases in exon 6, and 2 cases in exon 7. The alterations of the p53 gene were represented by point mutation leading to an amino acid substitution in four cases, and deletion in the remaining case. In four of the five cases, direct sequencing indicated the loss of the normal p53 allele; in the remaining case, two mutations were detected, presumably involving both p53 alleles. Three cases showed mutations at diagnosis; in the remaining two, the mutations were observed in clinical relapse but not at diagnosis. Our results confirm the relatively low incidence of p53 mutations in AML and further support the evidence that p53 plays a role in leukemogenesis through a recessive mechanism (two-hit model) of inactivation of tumor suppressor activity.

Peripheral neuropathy in IgM monoclonal gammopathy and Wäldenstrom's macroglobulinemia: a frequent complication in elderly males with low MAG-reactive serum monoclonal component.

Peripheral neuropathy (PN) is a frequent complication during primary macroglobulinemia (PM), whose immunological genesis has been suggested by various authors. This study involved 65 PM patients (44 men and 21 women aged 35-78), diagnostically divided into MGUS (31 cases), and indolent (IWM, 24 cases) or symptomatic (WM, 10 cases) Waldenstrom macroglobulinemia groups. All patients underwent neurological examination, including electrodiagnostic evaluation and the determination of the serum titre of antimyelin-associated glycoprotein (MAG). An evaluation was made of the prevalence of PN and its correlation with a series of hematological variables. The prevalence of PN was 31.6%: of those with PN, 73.1% manifested both clinical and electrophysiological signs of PN, primarily of the demyelinating type. Significant correlations emerged between the presence of PN and sex (M vs. F P = 0.0001), advanced age (P = 0.049), low MC levels (P = 0.025), high anti-MAG titres (P = 0.001) and high Hb levels (P = 0.001). No significant correlation with the diagnostic definition of PM was found, although the majority of cases with (particularly demyelinating) PN were MGUS or IWM. At multivariate analysis, the presence of PN significantly correlated with sex (P = 0.0001), age (P = 0.019), and anti-MAG titre (P = 0.001). Ten of the 26 PN cases showed no MAG reactivity. Significant correlations between PN and low serum MC levels/high MAG reactivity support the hypothesis of the antibody-mediated origin of many PN, and that the presence of PN depends on the characteristics of the proliferating pathological B clone, rather than on the tumor burden of the form of macroglobulinemia. Clinically, our data reconfirm the frequency of PN during PM and indicate simple clinicohematological variables useful for identifying patients at high neuropathic risk.