All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.

Polymerase chain reaction (PCR) of bcl-2 provides an extremely sensitive method to detect minimal disease in approximately 50% of patients with non-Hodgkin's lymphomas (NHL). In an attempt to determine the clinical usefulness of this technique, we examined the bone marrow (BM) of 152 patients with advanced-stage NHL at the time of evaluation and after induction or salvage chemotherapy before autologous BM transplantation. The BM proved to be an accessible and reproducible tissue source to determine PCR positivity because all of the 102 patients examined had the same PCR-amplifiable breakpoint in their BM and lymph node. At the time of evaluation, PCR analysis in advanced-stage NHL patients added little additional information to morphologic analysis because each technique identified BM infiltration in approximately 70% of patients. PCR was significantly more useful in determining BM infiltration after induction or salvage therapy. At that time, approximately 50% of patients had morphologically normal BM, whereas PCR analysis remained positive in 100% of those with an amplifiable breakpoint. These observations were confirmed in a clinical trial attempting to induce remission in previously untreated low-grade advanced-stage NHL patients. In this series, PCR was positive in all patients after treatment although the BM was histologically uninvolved in 50% of cases, showing that conventional therapy did not eradicate bcl-2-positive cells.     

Transplantation of enriched and purged peripheral blood progenitor cells from a single apheresis product in patients with non-Hodgkin's lymphoma

High-dose chemotherapy with or without radiotherapy followed by autologous transplantation of hematopoietic progenitor cells is an effective treatment for patients with high-risk or relapsed non- Hodgkin's lymphoma. Chemotherapy and/or hematopoietic growth factors have been used to mobilize progenitor cells in the peripheral blood for transplantation. However, the mobilized blood cell products have been found to be frequently contaminated with tumor cells, and techniques have not been developed to purge tumor cells from these products. In addition, the minimum number of hematopoietic progenitor cells required for engraftment has not yet been fully elucidated. We treated 21 patients with a single infusion of cyclophosphamide (4 g/m2) followed by daily administration of granulocyte colony-stimulating factor (G- CSF). After recovery of the white blood cell count, a single 3-hour apheresis collection was performed. The apheresis product was then applied to a discontinuous Percoll gradient. The low-density fractions resulting from this separation procedure were enriched for CD34+ progenitor cells (total cell yield, 19.5%; CD34+ cell recovery, 81.2%). These enriched cellular products were treated with a panel of anti-B cell or anti-T cell monoclonal antibodies and complement in an effort to remove residual tumor cells. After treatment of the patient with myeloablative therapies, the enriched and purged cells were reinfused. Hematologic recovery was rapid, with median neutrophil engraftment in 10 days [absolute neutrophil count (ANC), greater than 0.5 x 10(9)/L] and 11 days (ANC, greater than 1.0 x 10(9)/L). Median platelet transfusion independence required 13 days. The rapidity of multilineage engraftment correlated with the number of CD34+ cells per kilogram that were infused. Patients who received more than 2 x 10(6) CD34+ cells per kilogram had rapid hematologic engraftment, whereas those patients transplanted with less than 2 x 10(6) CD34+ cells per kilogram had slower platelet recovery. Modeling studies using a lymphoma cell line with a t(14; 18) chromosomal translocation demonstrated the successful removal of tumor cells assayed using the polymerase chain reaction (PCR) after the processing and purging. Four of the 21 patients had PCR- detectable lymphoma cells in the bone marrow and peripheral blood; however, the enriched and purged blood products reinfused in all four did not contain detectable tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl- 2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR- detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR- detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.     

In vivo depletion of B cells using a combination of high-dose cytosine arabinoside/mitoxantrone and rituximab for autografting in patients with non-Hodgkin's lymphoma

We performed a pilot study including rituximab (Mabthera; IDEC-C2B8, Hoffmann-La Roche) with a sequential high-dose therapy protocol in 15 patients with follicular and three patients with mantle cell lymphoma and studied the potential of the chemoimmunotherapy to induce depletion of malignant B cells in vivo. Our treatment protocol included induction with three cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy, followed by peripheral blood stem cell (PBSC) mobilization using high-dose cytosine arabinoside (2 g/m2 every 12 h, days 1 and 2) and mitoxantrone (10 mg/m2, days 2 and 3) (HAM), preceeded by rituximab (375 mg/m2). The proportion of CD19+ B cells in blood and bone marrow decreased from 1.2 +/- 0.4% to 0.13 +/- 0. 1% (P = 0.01) and from 2.7 +/- 0.8% to 0.8 +/- 0.5% (P = 0.03) respectively. The number of t(14;18)-positive cells in blood and bone marrow progressively decreased with treatment, as assessed by the quantitative real-time PCR assay in four patients. Conversion to PCR-negativity was achieved in the peripheral blood (PB) of seven informative patients. Leucaphereses were performed during the granulocyte colony-stimulating factor (G-CSF)-supported leucocyte recovery phase. In 17 of 18 patients, a median of 15.1 x 106 CD34+ cells/kg body weight (BW) could be harvested by a single procedure for enrichment by an immunomagnetic method. Leucapheresis products contained 51.3 +/- 28.8 x 104 CD19+ B cells/kg BW (mean) and were t(14;18) PCR negative in all seven informative patients. These data compare favourably with results obtained in patients treated with the same regimen without rituximab. The high-dose therapy (n = 12 patients), including total body irradiation (14.4 Gy) and cyclophosphamide (200 mg/kg BW), was also preceeded by rituximab. Recovery of neutrophils to > 0.5 x 109/l and of platelets to > 20 x 109/l required a median of 13.5 and 11.5 d (range 11-24 and 9-24 d) respectively. In conclusion, the addition of the CD20 antibody to chemotherapy ensured tumour depletion in vivo and allowed the collection of PBSCs devoid of tumour cells and with conserved engraftment capability.     

Characterization of the interleukin 2 receptor beta chain using three distinct monoclonal antibodies.

The human high-affinity receptor for interleukin 2 (IL-2) has been proposed as being a membrane complex composed of at least two distinct polypeptide chains: p55 (alpha chain), recognized by the anti-Tac monoclonal antibody (mAb), and p75 (beta chain), both of which are capable of binding IL-2. Whereas the alpha chain itself has been shown to be nonfunctional, the beta chain appears to be pivotal in the IL-2 signal transduction, although the beta chain is otherwise poorly characterized. Three beta chain-specific mAbs, designated Mik-beta 1, -beta 2, and -beta 3, were developed. Mik-beta 1 and -beta 2 completely inhibited the IL-2 binding to the beta chain, whereas Mik-beta 3 immunoprecipitated the beta chain crosslinked with 125I-labeled IL-2. The beta chain immunoprecipitated by these mAbs was revealed to have a Mr of 68,000-72,000. High-affinity IL-2 binding was completely abolished by Mik-beta 1. Although IL-2-dependent T-cell growth at high IL-2 concentrations was not inhibited by the anti-Tac, it was almost completely inhibited by Mik-beta 1 in the presence of the anti-Tac. These results clearly indicate that the beta chain is an indispensable component to the high-affinity IL-2 receptor and is responsible for the IL-2 signal transduction. The beta chain was found to be constitutively expressed without the alpha chain on the surface of peripheral blood Leu-19+ natural killer cells.     

Recurrence of Bcl-2/IgH polymerase chain reaction positivity following a prolonged molecular remission can be unrelated to the original follicular lymphoma clone

OBJECTIVE: The aim of this study was to evaluate whether reappearance of polymerase chain reaction (PCR) positivity for the Bcl-2/IgH translocation following a phase of molecular remission in autografted follicular lymphoma (FL) patients is always associated with reappearance of the original neoplastic clone. PATIENTS AND METHODS: The molecular follow-up of 119 autografted Bcl-2/IgH positive patients was evaluated by nested PCR. In case of molecular recurrence, direct sequencing of involved rearrangements has been performed both at diagnosis and at the time of recurrence. The two sequences then were compared in terms of breakpoints, N insertions, and JH usage. RESULTS: Seventy-five patients achieving molecular remission were identified in our patient sample (63%). Of these patients, eight (10.6%) experienced molecular recurrence. Direct sequencing of the Bcl-2/IgH translocation performed at diagnosis and recurrence showed identical rearrangements in six subjects and unrelated rearrangements in two. As opposed to most true molecular relapses, unrelated rearrangements always occurred several years after transplantation. To date, the two subjects carrying unrelated rearrangements show no signs of active lymphoproliferative disease. CONCLUSIONS: This report is the first evidence that Bcl-2/IgH rearrangements unrelated to the original tumor clone can lead to false-positive results during the molecular follow-up of autografted FL patients. Based on these results, we recommend confirmation by direct sequencing, at least for patients experiencing molecular relapse 2 or more years after the end of treatment. This will be particularly important for patients enrolled in clinical trials that schedule additional treatment in case of molecular evidence of persistent disease activity.     

Prevalence and clinical significance of anticardiolipin and anti-beta2-glycoprotein-I antibodies in patients with non-Hodgkin's lymphoma

The association of antiphospholipid antibodies (APA) has been reported in several cases of patients with non-Hodgkin's lymphoma (NHL) with or without thromboembolic complications. The purpose of this study was to analyse systematically the prevalence of APA and its clinical significance in lymphoma patients. Sera of 90 consecutive unselected patients with NHL were tested for the presence of anticardiolipin (aCL) antibodies and anti-beta2-glycoprotein-I (anti-beta2-GPI) antibodies. The patients were followed up over a median period of 14 months to note the occurrence of thromboembolism. We found APA in 24 out of 90 NHL patients (26.6%). Elevated APA were more often detected in women and in the elderly. The presence of elevated APA was not correlated with the histology and the stage of the lymphoma. None of the 24 patients with elevated APA developed a thromboembolic event in the follow-up period. Thromboembolic events were observed in 12 patients (13.3%), all with negative APA. High APA titres and the combination of positive aCL- and anti-beta2-GPI antibodies, features which are known to be more strongly correlated with thrombosis among patients with antiphospholipid syndrome and systemic lupus erythematous (SLE), were very uncommon in our cohort of NHL patients (3.3%). Vessel compression by lymphoma but not elevated APA remains the main cause of thrombosis in NHL patients.     

Geographical aspects of bcl-2 gene involvement in Japanese patients with non-Hodgkin's B-cell lymphomas.

Using three separate bcl-2 probes, we examined involvement of the bcl-2 gene in Japanese patients with non-Hodgkin's B-cell lymphomas. Of 52 patients with follicular lymphoma (FL), 24 had rearrangements. In a group of 50 patients with diffuse lymphoma, three of 32 patients with diffuse large cell lymphoma had rearrangements. The frequency of rearrangements in each of these groups, as detected by both major and minor breakpoint cluster region probes, was compatible with that found in other Far Eastern studies. However, the difference in frequency between the groups studied in the Far East and the West was significant, and these two geographically distinct populations also displayed a difference in the breakpoint distribution. In the immunophenotype study of 33 patients with FL, the expression of CD10 antigen correlated with bcl-2 involvement, whereas none of the other B markers emerged as parameters to distinguish between the two lymphoma groups; those with, and without, the rearrangements.     

Arterial thrombosis associated with anticardiolipin and anti-beta2-glycoprotein-I antibodies in patients with non-Hodgkin's lymphoma: a report of two cases

Autoimmune phenomena in lymphoid malignancies are often observed. However, clinical manifestations such as a secondary antiphospholipid syndrome in the presence of antiphospholipid antibodies are rarely reported. Furthermore, in the few cases of lymphomas so far reported with thrombosis associated with elevated antiphospholipid antibodies, the anti-beta2-glycoprotein-I antibodies have not been studied. We report on two cases of arterial thrombosis occuring in patients with B-cell lymphoma who presented with positive anticardiolipin and anti-beta2-glycoprotein-I antibodies. Our observation suggests that patients with non-Hodgkin's lymphoma and both anticardiolipin and anti-beta2-glycoprotein-I antibodies may be, similar to lupus patients, at considerable risk towards thrombosis, especially towards arterial thrombosis.     

Burden of Mycobacterium tuberculosis in sputum samples can be reliably determined using a quantitative,non-radioactive polymerase chain reaction assay

Objective: To develop a rapid assay for quantitation of Mycobacterium tuberculosis in sputum samples using the competitive polymerase chain reaction (PCR) and a colorimetric microtiter well detection format.Design: The assay relies on the co-amplification of a 419 base pair (bp) pab fragment of M. tuberculosis together with a target template (pab/tet) made by splicing a fragment of tet excised from pbr322 between the 5′ and 3′ ends of the pab fragment to create a 380 bp hybrid template amplified with the same primers but readily distinguishable using probes specific for either pab or tet.Results: We demonstrate a good correlation between the results obtained using this assay and the results of quantitative culture.Conclusion: This assay provides quantitative information regarding M. tuberculosis burden in samples containing between 10³ and 10⁸ colony forming units/milliliter (CFU/ml).     

IgG2a monoclonal antibodies inhibit human tumor growth through interaction with effector cells.

Monoclonal antibodies of IgG2a isotype specifically inhibited growth of human tumors in nude mice. Twenty-three monoclonal antibodies of other isotypes showed no tumoricidal reactivity. Complement depletion of nude mice had no effect on tumor suppression by monoclonal antibody. The role of T and killer cells as mediators of the monoclonal antibody effect in nude mice was virtually excluded. On the other hand, macrophages were strongly incriminated as effector cells because silica treatment of nude mice abolished the tumoricidal effect of monoclonal antibody. IgG2a monoclonal antibody-dependent macrophage-mediated cytotoxicity assays with human tumor cells in culture resulted in specific lysis of tumor cells.     

Elevation of activated platelet-dependent chemokines in patients with anti-CD20 monoclonal antibody (rituximab)-treated non-Hodgkin's lymphoma.

This study measured and compared levels of some chemokines in patients with rituximab-treated non-Hodgkin lymphoma because they may participate in the mechanism of efficacy of rituximab in non-Hodgkin lymphoma patients. Monocytic chemotactant protein-1, RANTES (regulated on activation, normally T-cell expressed and secreted), eotaxin, interleukin-8, neutrophil-activating protein-78, stromal cell-derived factor-1, and growth-regulating oncogene-alpha in patients with rituximab-treated non-Hodgkin lymphoma were measured by enzyme-linked immunosorbent assay. Levels of RANTES were higher in non-Hodgkin lymphoma patients than in controls. Levels of monocytic chemotactant protein-1, RANTES, and neutrophil-activating protein-78 were significantly elevated before and after chemotherapy with rituximab treatment. However, the level of stromal cell-derived factor-1 did not exhibit a significant change. Before to after chemotherapy without rituximab treatment, all chemokine levels did not exhibit significant changes. These findings suggest that activated platelet-dependent chemokines such as RANTES and neutrophil-activating protein-78 may modulate the efficacy of rituximab in antibody-dependent cellular cytotoxity.     

Immunodiagnosis of chronic lymphatic leukemia and leukemic non-Hodgkin's lymphoma with the monoclonal antibodies BL-Ig-L/1, BL-DR/1 and BL-T2

3 self made monoclonal antibodies are tested with the indirect immunofluorescence method for the immunologic diagnostic of the circulating lymphocytes from 24 chronic lymphatic leukaemia (CLL) and 14 leukaemic Non-Hodgkin lymphoma (NHL) patients. In comparison, investigations were done with various specific rabbit antibody F(ab)2 fragments and antisera. The monoclonal antibody BL-Ig-L/1, which is directed to the human Ig L-chains, marked the neoplastic lymphocytes from 12 of 24 CLL and from 9 of 14 leukaemic NHL as membrane Ig+. The monoclonal antibody BL-DR/1, which reacts with a HLA-DR, binds to the blood lymphocytes of 23 CLL and 11 NHL patients. By this BL-DR/1 is superior to BL-Ig-L/1 for the immunologic diagnostic of the non-T-cell neoplasia. The with normal peripheral T-cells reactive monoclonal antibody BL-T2 reacts with malignant B lymphocytes on an equal scale as BL-DR/1. It is not qualified for the differentiation of malignant blood lymphocytes.     

Selective purging by human interleukin-2 activated lymphocytes of bone marrows contaminated with a lymphoma line or autologous leukaemic cells

The ability of recombinant interleukin 2 (rIL2) activated lymphocytes (LAK) to purge BM samples contaminated by tumour cells was evaluated. Human BM mononuclear cells were contaminated with 10% of the lymphoma line CA46 and then cultured in liquid medium containing 1000 U/ml of rIL2 and/or LAK autologous to the used BM. At the end of coculture the growth of residual tumour cells and of CFU-GM were evaluated by clonogenic assay. No tumour cell growth was observed in 5/5 independent experiments after 18 h of coculture with LAK. No significant inhibition of CFU-GM growth was also noted. Subsequently, the effect of LAK on BM obtained from four leukaemic patients and contaminated with 20-50% of their own AML and ALL cells was studied using MAb as a tool for identifying leukaemic cells. LAK eliminated 24-78% of contaminating cryopreserved uncultured autologous leukaemic cells. In five cases the BM was contaminated by a low (2%) amount of ALL cells. In these patients the monoclonal heavy chain rearrangement typical of ALL was no longer visible after coculture with LAK. Evidence for selective tumour cytotoxicity by LAK was confirmed by using autologous BM cells as hot and cold targets in a 51Cr release assay. Finally, successful haematologic reconstitution of lethally irradiated BALB/c mice was obtained using syngeneic BM cocultured with LAK. These results support the investigational use of rIL2 and LAK in the treatment of human leukaemia.     

Cell surface characterization of malignant T cells from lymphoblastic lymphoma using monoclonal antibodies: evidence for phenotypic differences between malignant T cells from patients with acute lymphoblastic leukemia and lymphoblastic lymphoma

A series of monoclonal antibodies was used for the characterization of malignant T cells from 21 patients with lymphoblastic lymphoma (LL). The tumor population from these patients showed a marked degree of phenotypic heterogeneity and a proportion (one-third) of patients had tumor cells that did not conform exactly with the cells normally detected in the thymus. However, these cell populations could be related to the early or common or late thymocyte population (about one- third of the patients in each category). This contrast, with the characterization of malignant T cells from 43 patients with acute lymphoblastic leukemia (ALL) that could be related to either early or common thymocytes, with an exception of two patients categorized as having a tumor population related to late thymocytes. Further phenotypic differences between cells from ALL and LL could be demonstrated by investigation with two additional monoclonal antibodies, A50 and U4. Among patients with malignant T cells related to common thymocyte, 0/12 patients with ALL had cells recognized by A50, where 5/8 patients with LL had A50+ cells. Among patients with early thymocytes, only patients with ALL had cells recognized by U4. In addition, 5 LL patients had cells reactive with J5, a monoclonal antibody recognizing the common ALL antigen (CALLA). Since CALLA was found on cells related to common and late thymocytes, CALLA is neither lineage specific, nor can it be viewed as being peculiar to malignant lymphoid cells arrested at very immature stages of differentiation.