Identification of human lymphoma cells by antisera to malignancy-associated nucleolar antigens

The non-Hodgkin's lymphomas (NHL) are a diverse group of human lymphoid neoplasms that have long presented pathologists with formidable diagnostic challenges. These tumors of the immune system are thought to represent neoplastic transformations of most of the recognized stages in T and B lymphocyte ontogeny. Lymphoma cells, however, often simulate their normal lymphocytic counterparts both morphologically and cell surface phenotypically, creating difficulties in discriminating normal from neoplastic lymphocytes. We have used heteroantisera to the human malignancy-associated nucleolar antigen (HMNA) to prospectively evaluate its efficacy in identifying the morphologically neoplastic cells in NHL lesions. In 65 cases of T and B cell histopathologic types of NHL, the antisera reacted with nucleoli in the morphologically and cytogenetically neoplastic lymphoma cells, but not with normal- appearing lymphoid and other cell types present in the lesions. Control specimens from normal and hyperplastic lymphoid tissue also failed to react with anti-HMNA antibodies. Normal activated lymphoid cells in vitro and growth-factor-dependent normal lymphoid cell lines also failed to express the nucleolar antigen(s). These data suggest that the HMNA is a valuable tumor cell marker for neoplastic human lymphoid cell populations and can be used with other types of cell markers for a better definition of the neoplastic cells in NHL.     

Lymphocyte surface characteristics in malignant lymphoma

The surface of lymphocytes obtained from fresh biopsy specimens from 41 patients with malignant lymphoma and from 30 normal subjects or patients with non-neoplastic lymphadenopathy were investigated. Immunoglobulin on the cell surface was used to identify B cells, whereas T cells were recognized by their reactivity with an antithymocyte antiserum and their ability to form rosettes with sheep erythrocytes. Normal and inflammatory lymph nodes were composed predominantly of T lymphocytes, as were nodes from 14 patients with Hodgkin's disease. Two thymomas were T cell proliferations, whereas a node from a patient with ataxia-telanglectasia was devoid of T lymphocytes. The presence of immunoglobulin on the cell surface indicated that 19 of 21 lymphocytic lymphomas were B cell proliferations, whereas the cells from 3 histiocytic lymphomas (reticulum cell sarcomas) and 1 mixed histiocytic and lymphocytic lymphoma were devoid of surface immunoglobulin. In immunoglobulin-positive tumors, one predominant heavy chain and one predominant light chain could usually be identified, thus establishing the clonal character of the neoplastic proliferation. Ten of 11 diffuse poorly differentiated lymphocytic lymphomas were composed of cells with large amounts of surface immunoglobulin, whereas only 1 of 5 diffuse well differentiated lymphocytic tumors contained such abundant surface immunoglobulin. The surface immunoglobulin data indicate the existence of at least two subspecies of B cell neoplasms. A small lymphocyte with sparse surface immunoglobulin proliferates as diffuse well differentiated lymphocytic lymphoma and chronic lymphocytic leukemia, whereas a larger lymphocyte with abundant surface immunoglobulin proliferates as diffuse poorly differentiated lymphocytic lymphoma and lymphosarcoma cell leukemia.     

Growth of primary T-cell non-Hodgkin's lymphomata in SCID-hu mice: requirement for a human lymphoid microenvironment.

We reasoned that the SCID-hu mouse could provide an appropriate lymphoid or stromal microenvironment to support the growth of primary human lymphoma. Heterotransplantation of nine cases of primary T-cell non-Hodgkin's lymphoma (NHL) into untreated SCID mice and SCID mice reconstituted with human fetal thymus, spleen, and liver (SCID-hu) resulted in the development of lymphoid tumors in five (56%) cases. Two clonal T-cell NHL grew after a mean of 90 days after injection of primary lymphoma cell suspensions into the thymus xenografts in SCID-hu mice and failed to grow in a variety of sites in SCID mice, except for small tumors that developed after a long (157-day) latency period after intracranial injection of tumor cell suspensions into weanling SCID mice. Successful serial transplantation of NHL in SCID and SCID-hu mice required the presence of a human lymphoid or tumor microenvironment, and was enhanced by pretreating the SCID mice with 175 rad radiation and antiasialo antisera. Analysis of the primary and transplanted T-cell tumors showed identical patterns of T-cell surface markers by flow cytometry and immunophenotyping of fixed tissue sections, and, in one case, reactivity with a specific monoclonal antibody to V beta 5.1. Genotyping of the transplanted tumors showed T-cell receptor gene rearrangements identical to those present in the primary tumors. In one case, the presence of Epstein-Barr virus-positive B cells in association with the primary tumor resulted in the growth of a lymphoblastoid B-cell neoplasm in addition to the malignant T-cell lymphoma after transplantation of tumor fragments to SCID mice. The data support the hypothesis that a human lymphoid microenvironment enhances the growth of T-cell NHL in SCID mice. The SCID-hu thymus graft provides an apparently unique microenvironment that supports the growth of primary T-cell NHL, and can be used to study the interaction between lymphoma cells, nontransformed lymphoid cells, and the surrounding stromal microenvironment in vivo.     

Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B-lineage non-Hodgkin's lymphoma cells.

Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B-lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence-activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS-sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.     

Cell surface phenotype in lymphoproliferative disease

The surface phenotype of peripheral blood lymphocytes from 45 patients with chronic lymphocytic leukemia (CLL) and of lymphoid tissue from 100 patients with other lymphoproliferative disease was determined. Surface immunoglobulin (SIg) and complement receptor were employed as B cell markers, and reactivity with sheep erythrocytes (E rosettes) and antithymocyte globulin was used to identify T cells. Of these markers, only SIg and E rosettes reliably identified cells of the respective lineages. SIg identification with fluorescent antiserums had the added advantages of allowing the discrimination between different B cell subsets on the basis of staining intensity, and establishing the clonal character of a proliferation by the presence of predominant light and heavy chains.A uniform surface phenotype was observed in CLL, characterized by faintly staining SIgM with or without SIgD (one sixth of the patients were SIgG-positive instead), the presence of complement receptor, and the absence of reactivity with either sheep erythrocytes or antithymocyte globulin. Nodular (poorly differentiated lymphocytic) lymphoma was also uniformly of B cell lineage, but staining for SIgM was brighter (one third of these tumors were instead SIgG-bearing): Although cell suspensions of these neoplasms were uniformly E rosette-negative, the presence of complement receptor and reactivity with antithymocyte globulin were variable. Diffuse poorly differentiated lymphocytic lymphoma and two cases of Burkitt's lymphoma were very bright SIgM-positive, E rosette-negative neoplasms. Sixty per cent of the diffuse histiocytic lymphomas exhibited either SIgM- or SIgG-positive B cells which stained brightly, but the other 40 per cent comprised tumors either of T cell lineage or of a phenotype which could not be conclusively identified. The mixed histiocytic and lymphocytic lymphomas, which occupy an awkward place in pathology, could not be defined by surface markers. Surface markers should not replace routine pathologic criteria as the basis of classifying lymphoma, but cell phenotype is proving an increasingly useful added dimension for understanding and classifying this complex group of diseases.     

Lymphosarcoma cell leukemia: the contribution of cell surface study to diagnosis

Cell surface immunoglobulin, complement receptor, and spontaneous rosette formation with sheep erythrocytes were investigated in 43 patients with malignant lymphoma, including 13 with lymphosarcoma cell leukemia, and in 59 patients with chronic lymphocytic leukemia. The quantity of immunoglobulin on the lymphocyte surface was estimated from the intensity of fluorescent staining with fluorescein-conjugated anti- immunoglobulin antisera. At least two, and probably three, B cell species could be recognized by cell surface study. Cells from chronic lymphocytic leukemia and diffuse well-differentiated lymphocytic lymphoma had sparse amounts of surface immunoglobulin, while the cells of diffuse poorly differentiated lymphocytic lymphoma had large quantities of this material. Nodular lymphoma probably represented a third B-cell subtype with intermediate amounts of surface immunoglobulin. The lymphocytes of chronic lymphosarcoma cell leukemia exhibited the intense surface staining, which was characteristic of the underlying poorly differentiated lymphocytic lymphoma (diffuse or nodular), and could be readily distinguished from the faint-staining chronic lymphocytic leukemia cells.     

Impaired expression of cell surface receptors for B cell growth factor by chronic lymphocytic leukemia B cells

Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). The role of BCGF in the regulation of malignant B cell proliferation is unclear. Therefore, we studied the proliferative response of purified chronic lymphocytic leukemia (CLL) B cells to BCGF. For all CLL patients studied, CLL B cells showed a decreased proliferative response as compared with control B cells for BCGF- induced B cell proliferation (patient 291 +/- 59 cpm v control 3,942 +/- 622, mean +/- SEM). This impaired proliferative response appeared to be intrinsic to CLL B cells since it was not corrected by incubation with increasing concentrations of BCGF. Attainment of normal B cell responsiveness to BCGF requires the processing of an initial activation signal which results in the expression of cell surface receptors for BCGF. Increasing concentrations of the B cell activation signal (the F(ab')2 fragment of goat anti-human mu chain) did not improve CLL B cell responsiveness to BCGF. Three-day activated CLL B cells compared with activated control B cells demonstrated a marked impairment in their ability to absorb out the BCGF activity present in the BCGF preparation (BCGF activity absorbed out, patient 12.8% v control 53%). Pretreatment of CLL B cells with neuraminidase failed to improve either the proliferative response to BCGF or the expression of cell surface receptors for BCGF by the CLL B cells. This study suggests that the impaired responsiveness to BCGF by CLL B cells is the result of impaired expression of cell surface receptors for BCGF when CLL B cells are exposed to activation signals.     

Autocrine expression of platelet-derived growth factor B in B cell chronic lymphocytic leukemia

Platelet-derived growth factor (PDGF) regulates clonal proliferation of malignant pre-B cell lines, but little is known about its role in normal B lymphocyte differentiation and malignant transformation. To understand the expression of PDGF-A, PDGF-B and the beta-receptor (PDGF-Rbeta) in B cell lymphoproliferative disorders, we used an immunohistochemical method to stain formalin-fixed, paraffin-embedded tissues in 5 patients with reactive lymphoid hyperplasia, 15 with non-Hodgkin's lymphoma and 23 with B cell chronic lymphocytic leukemia (B-CLL). Abundant PDGF-A, rather than PDGF-B, was expressed in normal B cell differentiation. There was no difference in the expression of PDGF-A and PDGF-B between patients with reactive lymphoid hyperplasia and patients with malignant lymphoproliferative disorders. Among the patients with B-CLL, the expression of PDGF-B was much stronger than the expression of PDGF-A, and 18 of the patients had coexpression of PDGF-B and PDGF-Rbeta. A larger proportion of patients with B-CLL than with non-Hodgkin's lymphoma had expression of PDGF-B and PDGF-Rbeta. In conclusion, PDGF-A expression in all stages of B lymphocyte differentiation suggests that it is important in B cell differentiation and proliferation. Expression of PDGF-B and PDGF-Rbeta suggests that autocrine signaling of PDGF may be important in malignant transformation of B-CLL. However, further studies are necessary to confirm these conclusions.     

Establishment and characterization of human B-cell lymphoma cell lines using B-cell growth factor

B-cell non-Hodgkin's lymphomas (NHL-B) have been difficult to establish in long-term cell culture using standard techniques. We report the establishment of five representative cell lines from high grade NHL-B using B-cell growth factor (BCGF). The five NHL-B cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. The cell lines showed at least a sevenfold dose-dependent increase in proliferation in vitro over background in the presence of BCGF. Other putative B-cell growth-stimulating cytokines showed no significant proliferative activity or were inhibitory in some cases. NHL-B cell lines secreted growth factor(s) into culture supernatants that mediated at least a fivefold dose-dependent increase in cell proliferation in autochthonous lymphoma cells and a 10-fold or greater stimulation in growth factor-dependent normal B cell lines in vitro. The cell lines show monoclonal rearrangements of IgH genes and nonrandom chromosomal abnormalities characteristic of NHL-B, while the expression of Epstein-Barr virus associated antigen (EBNA-I) is present in two of the five cell lines. The studies show that lineage-specific growth factors may be used to establish neoplastic B cell lines in vitro, which are important experimental systems for cellular and molecular studies in the NHL-B.     

Proliferation of B cells from chronic lymphocytic leukemia is selectively promoted by B cell growth factor

B cell activation was studied in B cells from B cell chronic lymphocytic leukemia (B-CLL) patients. After in vitro stimulation, these B cells showed extensive proliferation in the presence of high-molecular-weight B cell growth factor (BCGF). In contrast, this effect was not observed upon addition of recombinant interleukin-2 (IL-2). In agreement, upon stimulation, B cells expressed Bac-1 antigen but failed to acquire the IL-2 receptor. These results demonstrate that the utilization of the BCGF pathway can be segregated from that of IL-2 in B cells from B-CLL patients.     

Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.

Follicular lymphomas recapitulate the architecture of germinal centers (GCs) of normal secondary lymphoid follicles. Using an in vitro binding assay, it has recently been demonstrated that the normal B lymphocytes bind to GCs. This interaction is mediated by a receptor-ligand pair consisting of the beta 1 integrin very late antigen 4 (VLA-4) on the B cell, and the vascular cell adhesion molecule-1 (VCAM-1) expressed on follicular dendritic cells (FDC). Considering the similarities between follicular lymphomas and normal GCs, the adhesive interaction of follicular non-Hodgkin's lymphoma (NHL) cells and GCs was examined. Cells isolated from 16 of 24 cases of follicular NHL bound to normal GCs. Neoplastic follicles could similarly support the binding of follicular NHL cells. This adhesion was inhibited by monoclonal antibodies (MoAbs) directed against VLA-4 and VCAM-1. This supports the hypothesis that the neoplastic follicles used the identical adhesive interactions responsible, at least in part, for the localization of normal B cells to GCs. Adhesion receptors have an important role in the regulation of normal lymphoid cell proliferation, differentiation, and localization. Therefore, an understanding of the adhesive interaction of follicular NHL cells with GCs may provide insight into the clinical and biologic behavior of these diseases.     

B8.7 antigen is present on B-cell precursor acute lymphocytic leukemia. Correlation with the low molecular weight B-cell growth factor responsiveness of these cells

The purpose of this study was to analyze the expression of B8.7 antigen and its implication in the low molecular weight B-Cell growth factor (LMW BCGF) proliferative pathway at the early stages of the human B-cell differentiation. After an overnight incubation in culture medium of B-cell precursor acute lymphoblastic leukemias (ALL), we demonstrated the presence of B8.7 antigen in 18 of 25 cases (72%). Such an incubation also induced a significant increase in the LMW BCGF responsiveness of ALL cells (P less than 0.03). In addition, we showed a significant correlation between B8.7 expression and the ability of pre-B ALL cells to respond to LMW BCGF. As previously described for normal B cells, the anti-B8.7 monoclonal antibody inhibited the LMW BCGF-dependent proliferation of pre-B ALL cells in a dose-dependent manner. These data indicate that B8.7 antigen is expressed and may be functionally related to the LMW BCGF pathway at the pre-B cell stages of differentiation. These results also suggest that human B-cell precursor ALL are not only phenotypically similar to their normal B lymphocyte counterparts, but are also sensitive to the same immunoregulatory cytokines that control normal cell growth.     

Adhesion to high endothelial venules: a model for dissemination mechanisms in non-Hodgkin's lymphoma

The interaction of human lymphoma cells with high endothelial venules (HEVs) on sections of lymphatic tissues was studied in 44 cases of non- Hodgkin's lymphoma (NHL) with the in vitro HEV binding assay. The relative adherence ratio (RAR) of lymphoma cells to HEVs as related to that of reactive lymphocytes was 0.29 to 4.64 in 38 cases of B chronic lymphocytic leukemia (CLL), 1.15 and 1.54 in two cases of immunocytic NHL, 1.12 and 0.70 in two cases of centrocytic NHL, 1.98 in one case of a peripheral T-NHL, whereas plasma cell leukemia cells adhered very weakly (RAR 0.1). Among the patients suffering from CLL a pronounced HEV binding ability of tumor cells correlated significantly with the more unfavorable Binet stages B and C (median 1.32) as well as with a widespread lymphatic dissemination, which strongly indicates a hematogenous, HEV-mediated spread (median 1.34). In contrast, weak adherence to HEVs was associated with Binet stage A (median 0.85; P < .05) and with a lacking or only localized clinical involvement of lymph nodes (median 0.84; P < .01). Thus, specific HEV recognition processes even operate in lymphoid neoplasms and via this mechanism seem to influence the dissemination of tumors.     

B and T cell lymphomas: Analysis of blood and lymph nodes in 87 patients

B and T cell populations were studied in blood and neoplastic tissues from 64 untreated and 23 treated patients with non-Hodgkin's lymphoma. This study was undertaken primarily to evaluate the relation of B and T cell markers in various lymphomas to the currently accepted morphologic classifications and to determine the utility of various tissues in defining the cell of origin of a lymphoma. When histologically involved blood, bone marrow, lymph nodes or body fluids were studied, a B or T cell origin of the lymphoma was identified in 26 of 28 (68 per cent) patients. A B cell origin was found in 17 adults classified as having nodular (N) or diffuse (D) poorly differentiated lymphocytic lymphoma (PDLL). One lymphoma of T cell origin was observed in an adult with poorly differentiated lymphocytic lymphoma-diffuse (PDLL-D). In contrast, all cases of PDLL-D in children were T cell in origin. The origin of American Burkitt's (stem cell) lymphoma in two children was the B cell. When histologically involved blood was studied, a B or T cell origin was demonstrated in 10 of 21 (48 per cent) adults. Evidence of a monoclonal proliferation of B lymphocytes in the blood was found two adults with more than 7 per cent lymphoma cells in Wright-Giemsa stained blood smears. When neoplastic lymph nodes were studied, the diagnosis of a B cell lymphoma was made in 8 of 12 (67 per cent) adults. Study of surface markers on malignant cells in cerebrospinal or serosal fluids frequently revealed a B or T cell origin of the lymphoma. B and T lymphocyte numbers in the blood did not correlate with immunoglobulin or skin test abnormalities. Abnormalities in circulating B or T cell percentages at diagnosis were a poor prognostic sign in patients with PDLL-D.     

Differential response of malignant human B-cells to anti-IgM immunoglobulin (anti-mu) and B-cell growth factor: unique direct cytotoxicity of anti-mu on prolymphocytic leukemia cells

We examined in vitro tritiated thymidine uptake by B cells from seven prolymphocytic leukemia (PLL), seven chronic lymphocytic leukemia (CLL), and four plasma cell leukemia (PCL) patients in response to culture with anti-human IgM antibody (anti-mu) and B-cell growth factor (BCGF). In contrast to the stimulatory effect observed in normal B-cell cultures, the divalent F(ab')2 anti-mu antibody uniquely inhibited the autonomous proliferation and induced marked cytotoxicity in six of seven PLL cell cultures independent of complement or Fc-receptor-mediated mechanisms. There was neither stimulation or inhibition of the slgM+ CLL or the slgM- PCL cells. The inhibitory effect on the PLL cells was observed at an anti-mu concentration below the stimulatory threshold for normal B cells. Significant impairment of trypan blue exclusion was delayed until 48 hours, with morphological cellular changes suggestive of a programmed cell death mechanism or apoptosis. The cytotoxicity was independent of the slgM-staining intensity or the autonomous and BCGF-augmented DNA synthetic activity of the PLL cells and was similar in patients with de novo PLL or with prolymphocytic transformation of CLL. Cells from a PLL patient were separated by elutriation into two fractions, a BCGF-unresponsive large "transformed" cell fraction with marked autonomous DNA synthesis and a smaller lymphoid cell subset with low 3H-thymidine uptake that could be augmented by BCGF. Both fractions were equally susceptible to the cytotoxic effect of anti-mu. These data suggest that certain slgM-bearing malignant B cells are susceptible to anti-mu-triggered cytotoxicity. They may represent the malignant counterpart of a "tolerogenic" normal B-cell subset, and the unique direct cytotoxicity of anti-mu may provide a therapeutic strategy specifically for PLL.     

A prospective study of 728 cases of non-Hodgkin lymphoma from a single laboratory in Shanghai,China

The frequency of subtypes of lymphoid neoplasms was determined in a prospective series of 831 patients presenting at 29 Shanghai hospitals over a 4-year period. Diagnosis and classification was established in a single laboratory according to the 2001 WHO classification system. The frequency of non-Hodgkin lymphoma was 87.6% (n = 728) and Hodgkin lymphoma was 12.4% (n = 103). The most prevalent NHL subtypes diagnosed using WHO criteria were diffuse large B cell lymphoma (DLBCL), precursor B lymphoblastic leukemia/lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Although a low incidence has been reported in some Asian populations, CLL/SLL was commonly encountered, indicating that chronic lymphoid neoplasms are not rare in Shanghai. Consistent with previous reports, our findings indicate a decrease in the frequency of follicular lymphoma and an increase in T cell neoplasms compared to the West. Precursor T lymphoblastic leukemia/lymphoma, anaplastic large T cell lymphoma, aggressive NK cell leukemia, angioimmunoblastic T cell lymphoma and peripheral T cell lymphoma were prominent subtypes of T cell NHL.     

Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B cell differentiation

A series of monoclonal antibodies that define B cell restricted and associated antigens was utilized in an attempt to characterize tumors of B lineage and to relate these tumors to B cell differentiative stages. Antigens that were previously shown to be B cell restricted on normal B lymphocytes were similarly expressed only on B cell malignancies. In contrast, antigens that were B cell associated were also found on tumors of other lineages. Moreover, on the basis of cell surface phenotypes, tumors of B cell origin were divided into three major subgroups, which corresponded to the level of differentiation of the malignant tumor cell: pre-B cell stage (non-T acute lymphoblastic leukemia and chronic myelocytic leukemia in lymphoid blast crisis); the mid-B cell stage (chronic lymphocytic leukemia, poorly differentiated lymphomas); and secretory B cell stage (large cell lymphomas and plasma cell tumors). A hypothetical model is derived that relates the malignant B cell to its normal cellular counterpart on the basis of cell surface expression of this panel of B cell-restricted and B cell- associated antigens.     

Mutator phenotype in human hematopoietic neoplasms and its association with deletions disabling DNA repair genes and bcl-2 rearrangements.

As mice carrying mutations of the DNA mismatch repair genes MSH2 and MSH6 often develop lymphoid neoplasms, we addressed the prevalence of the replication error (RER(+)) phenotype, a manifestation of an underlying defect of DNA mismatch repair genes, in human lymphoid tumors. We compared microsatellite instability (MSI) at 10 loci in 37 lymphoid tumors, including 16 acute lymphoid leukemias (ALL) and 21 non-Hodgkin's lymphomas (NHL), and in 29 acute myeloid leukemias (AML). Significant differences in MSI prevalence between AMLs and ALLs emerged, and MSI occurrence was more frequent in the NHLs versus AMLs. Indeed, only 3 of 29 (10%) AMLs exhibited MSI, thus confirming its paucity in myeloid tumors, while 10 of 37 (27%) lymphoid tumors, 6 ALLs and 4 NHLs, disclosed an RER(+) phenotype. In 1 ALL patient, the same molecular alterations were observed in correspondence with a relapse, but were not detected during remission over a 14-month follow-up; in another ALL patient, findings correlated with impending clinical relapse. These results suggest that the study of MSI in lymphoid tumors might provide a useful molecular tool to monitor disease progression in a subset of ALLs. To correlate MSI with other known genetic abnormalities, we investigated the status of the proto-oncogene, bcl-2, in the lymphoma patients and found that 4 of 4 NHL patients with MSI carried bcl-2 rearrangements, thus linking genomic instability to enhanced cell survival in NHL; moreover, no p53 mutations were found in these patients. Finally, we addressed the putative cause of MSI in hematopoietic tumors by searching for both mutations and deletions affecting DNA repair genes. A limited genetic analysis did not show any tumor-specific mutation in MLH1 exons 9 and 16 and in MSH2 exons 5 and 13. However, loss of heterozygosity (LOH) of markers closely linked to mismatch repair genes MLH1, MSH2, and PMS2 was demonstrated in 4 of 6 ALLs and 1 of 3 AMLs with MSI. These observations indicate that chromosomal deletions might represent a mechanism of inactivation of DNA repair genes in acute leukemia.     

Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients

Recent studies have shown that normal human T cells contain a high- molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.     

Identification of B-cell growth factors (interleukin-14; high molecular weight-B-cell growth factors) in effusion fluids from patients with aggressive B-cell lymphomas

The molecular basis of neoplastic B-cell growth is complex and poorly understood. Cytokines have been postulated to contribute to neoplastic cell growth, and many in vitro studies have confirmed this prediction, but little is known about the in vivo role of these growth factors. We have examined the production of interleukin-14 (IL-14) (high molecular weight [HMW], B-cell growth factor [BCGF]) by aggressive intermediate (diffuse large cell) lymphomas of the B-cell type non-Hodgkin's lymphoma (NHL-B) in four patients with lymphomatous effusions. In these studies, IL-14 was detected in the effusion fluids by Western blots and IL-14 mRNA was constitutively expressed in the freshly isolated lymphoma cells that also expressed the receptor for IL-14 (IL14R). Lymphoma B cells placed at low serum and cell density proliferated in vitro to either purified IL-14 or IL-14 derived from effusion fluids. Antibodies to IL-14 removed the growth-stimulating cytokine(s) from the effusions. Cell lines developed from these patients produced IL-14 in vitro and antisense oligos to IL-14 blocked their growth in vitro. Thus, autocrine or paracrine production of IL-14 may play a significant role in the rapid proliferation of aggressive NHL-B. Interrupting this pathway could be a useful goal of therapy for patients resistant to conventional chemotherapy.