Search

According to expert guidelines, lymph node surgical excision is the standard of care for lymphoma diagnosis. However, core needle biopsy (CNB) has become widely accepted as part of the lymphoma diagnostic workup over the past decades. The aim of this study was to present the largest multicenter inventory of lymph nodes sampled either by CNB or surgical excision in patients with suspected lymphoma and to compare their diagnostic performance in routine pathologic practice. We reviewed 32 285 cases registered in the French Lymphopath network, which provides a systematic expert review of all lymphoma diagnoses in France, and evaluated the percentage of CNB and surgical excision cases accurately diagnosed according to the World Health Organization classification. Although CNB provided a definitive diagnosis in 92.3% and seemed to be a reliable method of investigation for most patients with suspected lymphoma, it remained less conclusive than surgical excision, which provided a definitive diagnosis in 98.1%. Discordance rates between referral and expert diagnoses were higher on CNB (23.1%) than on surgical excision (21.2%; P = .004), and referral pathologists provided more cases with unclassified lymphoma or equivocal lesion through CNB. In such cases, expert review improved the diagnostic workup by classifying ∼90% of cases, with higher efficacy on surgical excision (93.3%) than CNB (81.4%; P < 10−6). Moreover, diagnostic concordance for reactive lesions was higher on surgical excision than CNB (P = .009). Overall, although CNB accurately diagnoses lymphoma in most instances, it increases the risk of erroneous or nondefinitive conclusions. This large-scale survey also emphasizes the need for systematic expert review in cases of lymphoma suspicion, especially in those sampled by using CNB.

Key Points Diagnosis of TCL often relies on PCR-based TCR clonality testing, which displays low specificity and requires high-quality DNA.Targeted NGS and T-cell clonality assessment are complementary techniques that should be applied in hard front-line diagnosis., Immunomorphological diagnosis of T-cell lymphoma (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays detect only 80% of TCL, and clonal lymphocyte populations may also appear in nonneoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques’ performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 nonneoplastic T-cell infiltrates were divided into 2 cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7%-45.5%), whereas no differences were observed in terms of sensitivity (95.1%-97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

Classical Hodgkin's Lymphoma (cHL) has been well studied over the past few decades. Newly diagnosed patients are treated with combinations of chemotherapeutics, the most common of which includes adriamycin, bleomycin, vinblastine, dacarbazine (ABVD). Approximately 75-85% of newly diagnosed cHL patients show favourable outcomes in response to ABVD treatment resulting in long lasting remission. The remaining 15-25% of diagnosed cHL patients will either show primary resistance to ABVD or relapse during or after the completion of therapy. Despite the fact that cHL biology has been extensively investigated, a robust biomarker to predict initial anthracycline based treatment failure remains an unresolved challenge in the management of the disease. Refractory and relapsing HL patients can be treated with several other treatment modalities including the recently developed brentuximab vedotin, and the PD-1 inhibitors nivolumab and pembrolizumab. The improvement in the outcomes of refractory and relapsed cHL patients treated with such advanced therapeutics presents a rational to advance such therapeutics to first line therapy for all newly diagnosed cHL patients. However, these therapeutics are accompanied with more adverse side effects compared to ABVD, and they present a significantly higher cost burden on the healthcare systems. In addition, clinical studies have shown that not all patients do respond favourably to these therapeutics. In spite of the development of the advanced therapies, the absence of a tool to stratify cHL patients at point of diagnosis remains to be a critical unmet clinical need. In previous reports, the 3-dimensional analysis of telomeres using TeloView® technology conducted on a proof of concept cohort of 27 HL patients revealed that TeloView® analysis, on the group patient level, showed distinct telomere profiles for HL patients who relapsed versus patients who remained stable. In this report, we conducted a retrospective clinical study including a multi-centre cohort of 156 HL patients, 125 remained stable for over 5 years, and 31 patients relapsed within 12 months. We targeted to identify, on the individual patient's level, significant telomeres parameters suitable to predict individual patient outcome at point of diagnosis. General univariate analysis including t-test and Anova identified derived telomeres parameters suitable for predictive modelling analysis including: total and average telomeres length, telomeric aggregates, nuclear volume, quartile nuclear volume and very short telomeres (t-stumps). Multivariate analysis using logistic regression procedures allowed for developing of significant predictive models using combinations of 2, 3 & 4 predictors. The highest predictive power was attained by a 4-predictors model with a predictive power of 0.76 presented as area under the curve (AUC) in receiver operating characteristic (ROC) curve illustration, achieving 75% positive predictive value, and 71% negative predictive value. The results of this study offer a long awaited path to a precision medicine approach for the treatment of cHL patients. The ready-to-use predictive model will further be validated retrospectively on an independent patient cohort, and can further be validated prospectively in longitudinal studies once the test is adopted in the clinic. Disclosures Louis: Telo Genomics Corp.: Consultancy, Current equity holder in publicly-traded company. Johnson:Roche/Genentech, Merck, Bristol-Myers Squibb, AbbVie: Consultancy; AbbVie: Research Funding; Roche/Genentech, Merck: Honoraria. Ludkovski:Telo Genomics Corp.: Current Employment. Shifrin:Telo Genomics Corp.: Current Employment. Mai:Telo Genomics Corp.: Consultancy, Current equity holder in publicly-traded company. Knecht:Telo Genomics Corp.: Consultancy, Current equity holder in publicly-traded company.

Abstract 2678 Introduction: Peripheral T-cell Lymphomas (PTCL) are types of rare and heterogeneous Non-Hodgkin's Lymphoma (NHL) that, in general, are associated with a poor clinical outcome. Discovery of new prognostic tools is thus a current and major challenge. In a cohort of 122 cases of PTCL collected from a multicentric T-cell lymphoma consortium (TENOMIC), we analyzed the expression of 80 non-coding small nucleolar RNAs (snoRNA) using high-throughput quantitative PCR (Fluidigm). SnoRNAs belong to the non-coding RNA family, and participate to diverse biological processes, most importantly ribosomal RNA maturation. Patients and methods: PTCL samples (n=122, 32 ALCL (22 ALK+, 10 ALK-) and 90 non-ALCL cases) were collected from TENOMIC. For each case, a consensus diagnosis was made by a panel of expert hemopathologists (some of the patients participated in a GELA-sponsored clinical trial, the others benefited from a national review protocol for T-cell lymphomas). Total RNA from sorted healthy donor-derived T-lymphocytes (n=35) and tumor samples were extracted (Trizol), integrity was verified using Agilent NanoChip, and reverse transcribed and assessed for snoRNA expression levels using The Fluidigm high-throughput quantitative PCR method. Medical cases were used to calculate progression-free and overall survival (PFS and OS) in 78 non-ALCL patients (26 PTCL-NOS, 46 AITL, 6 other rare entities) who received CHOP or CHOP-like regimen first-line. Results: First, we used unsupervised hierarchical clustering to show that, irrespectively from cell of the origin of the T cells, PTCL cells had a significant down-regulation of snoRNA expression in 63% of the snoRNA tested. In particular, there was a clear delineation between AITL, a tumor of T-follicular Helper (TFH) origin, and normal TFH cells from healthy donors. Among PTCL entities, ALCL had a specific snoRNA profile, but unsupervised clustering was not able to distinguish ALK- from ALK+ patients. However, a supervised comparison identified snoRNA U3 as a discriminant marker that sorted ALK+ from ALK- ALCL samples. Unsupervised or supervised snoRNA clustering failed to distinguish AITL from the other subgroups of PTCL. Second, we assessed prognostic impact of snoRNA expression in 78 non-ALCL patients. Characteristics of the cohorts were as follows: median age 65y/74y, Ann-Arbor stage III-IV in 94%/88%, elevated LDH in 76.7%/73.1%, IPI score 3–5 in 70%/61%, respectively. Although the snoRNA expression profiles of AITL and other PTCL subtypes appeared very similar, unsupervised clustering revealed the over-expression of 8 snoRNA in a subgroup of 31 patients with 45% OS at 5y (vs 47 patients with 18% OS at 5y). In AITL and PTCL-NOS cases, median OS was 26mo and 13mo, respectively. These thresholds were used in a supervised analysis to try to identify a specific prognostic snoRNA signature. A three snoRNA set was found significantly over-expressed in patients with the best prognosis, both in AITL patients (HBII-239, U59B, U90 impacted both on OS and PFS), and in PTCL-NOS patients (HBII-239, HBII-438A, U80 impacted on OS). Very interestingly, these signatures were not correlated neither with IPI or IPI-T scores, nor with overall response rates after CHOP. Lastly, we investigated the prognostic impact of HBII-239 snoRNA as a single marker. HBII-239 over-expression was statistically associated with prolonged PFS and OS in the entire cohort of 78 patients. Third, we investigated the impact of HBII-239 over-expression on cellular pathways in the FEPD cell line. This snoRNA is a precursor for microRNA-768, which is processed from the 3p strand of HBII-239. The FEPD cell line had a weaker expression of miR-768-3p as compared to patients' samples. Transfection of a miR-768-3p microRNAmimic induced a significant reduction of proliferation rate (as assessed by an MTS assay), due to a block at the G1/S checkpoint. In patients with low HBII-239 level, it is therefore expected that proliferation rate of lymphomatous cells would be increased, translating into decreased OS. Conclusions: Our study showed that snoRNAs are differentially regulated in normal compared to malignant T-cell populations. Besides a global down-regulation of these molecules, specific signatures may have a prognostic significance in PTCL. The different snoRNAs that can be regarded as potential biomarkers in these tumors may play a direct role in different cellular pathways. Disclosures: No relevant conflicts of interest to declare.

Seven cases of large B-cell lymphoma which define a previously unrecognized subgroup are reported. Morphologically they are comprised of monomorphic large immunoblast-like cells, containing large central nucleoli, which tend to invade lymphatic sinuses. Superficially they resemble anaplastic large cell lymphoma (ALCL) but they lack CD30. These lymphomas express epithelial membrane antigen (as do ALCL), but also contain intracytoplasmic IgA of a single light chain type (five cases) and an endoplasmic reticulum–associated marker detected by antibody VS38. They lack lineage-associated leukocyte antigens with the exception of CD4 (5 of 5 cases) and CD57 (5 of 7 cases). They are labeled by antibodies detecting both the intracytoplasmic and extracellular regions of the ALK receptor kinase, suggesting that they express the full-length form of this molecule. This was confirmed by Western blotting (in the one case tested) which showed a band of 200 kD in tumor cell lysates, and by polymerase chain reaction (PCR) amplification of mRNA encoding intracellular and extracellular ALK sequences (in the two cases tested). There was no evidence by cytogenetics (one case analyzed) or reverse transcriptase-PCR (three cases tested) of the 2; 5 translocation or the resultant NPM-ALK gene, as is commonly found in ALCL. All but one of the patients were male and all but one were adults, and in all but the latter case the disease followed an aggressive course.

We used the recently described sensitive and rapid detection assay called telomeric repeat amplification protocol (TRAP) to detect telomerase activity in lymphoblastoid (n = 5) and lymphoma cell lines (n = 7), hyperplastic lymph nodes (n = 6) and tonsils (n = 5), and tissues involved by non-Hodgkin's lymphoma (NHL) (n = 43) and Hodgkin's disease (HD) (n = 14). Clearly evident telomerase activity was found in all lymphoblastoid and lymphoma cell lines, and in 34 of 43 cases (80%) of NHL. These results were expected because of the proliferative and immortal nature of the cell lines and most malignant cells. However, positive results were obtained with the TRAP assay in all hyperplastic lymph nodes and tonsils, which raises the issue of derepression of telomerase activity during an immune response. Telomerase activity alone therefore does not distinguish malignant lymphoid proliferations from reactive states. Telomerase activity was detected in only 1 of 14 cases (7%) of lymphoid tissues involved by HD. Eight of the 13 negative cases were considered to be interpretable because of the lack (3 of 13 cases) or low level (5 of 13 cases) of telomerase inhibition. The five remaining cases could not be evaluated because of their telomerase inhibitor content. The findings imply either transient or very low levels of telomerase activity in HD or that HD for the greater part is a telomerase-independent neoplasm. Microdissection studies are needed to identify subsets of cells carrying telomerase activity in both reactive and neoplastic lymphoid tissues.

The expression of bcl-x protein, a bcl-2-related protein present in cortical thymocytes, activated lymphocytes, and plasma cells of reactive lymph nodes, was investigated in 44 cases of Hodgkin's disease (HD) in parallel with bcl-2 and Epstein-Barr virus (EBV) status. Eighty-six percent of the cases were positive for bcl-x, among them 27% with a strong signal in more than 75% of the Reed-Sternberg cells. Positivity for bcl-x was found in, respectively, 100% and 92% of the nodular sclerosis and mixed cellularity subtypes, although 4 cases of lymphocyte predominance subtype were negative. This finding was in contrast with the weaker positivity for bcl-2 staining in 44% of the cases. EBV small RNAs were detected in 43% of the cases by using in situ hybridization. Of interest, 100% of the EBV-positive samples were positive for bcl-x, whereas only 38% of these cases were bcl-2 positive. Our findings show that the bcl-x gene expression is high in HD, suggesting that bcl-x may have a role in the pathogenesis of at least some cases of HD via apoptosis regulation.

Twelve cases of relapsing Hodgkin's disease were investigated for the presence of Epstein-Barr virus (EBV). Of these, 7 cases contained EBV gene products (LMP1, EBER RNA) in the diagnostic Reed-Sternberg cells and variants at first presentation and at relapse(s), whereas 5 cases were negative at both first diagnosis and relapse. Among the 7 EBV- positive cases, material for DNA extraction was available in 2 cases at both diagnosis and relapse(s). Ig and T-cell receptor gene rearrangements displayed a germline configuration in the 2 cases. However, Southern blot analysis of the terminal repeats (TR) of EBV genome showed that, in 1 of the 2 cases, the fragment was of the same size at diagnosis and in the subsequent two relapses (1 early and 1 late). The second case contained monoclonal EBV genome at diagnosis, but the Southern analysis of the TR was negative at relapse. The latent membrane protein (LMP1) sequence analysis confirmed the persistence of a distinctive viral strain in each of the 2 cases with individual abnormalities within the carboxy terminal region (5 point mutations and a 30-bp deletion for the first case and 6 point mutations for the second case). The persistence of a given strain in early and late relapses is evidence towards the view that in Hodgkin's disease such relapses are related to a single residual tumor cell clone.

This study of 52 European patients with Hodgkin's disease (HD) expressing the latent membrane protein 1 (LMP1) oncogene within diagnostic Hodgkin and Reed-Sternberg (HRS) cells was performed to detect LMP1 isolates carrying deletions and to characterize them at a molecular and histologic level. Deletions were identified in 5 cases, clustered near the 3′ end of the LMP1 gene, and histologically associated with numerous HRS cells. DNA sequencing showed homology with the deletions seen in the Asian nasopharyngeal carcinoma (NPC) isolates CAO and 1510. Our findings suggest that partial deletions of the LMP1 oncogene, associated with aggressive behavior in NPC CAO and NPC 1510, occur at a particular localization and confer a proliferative phenotype to lymphoid cells in HD.

Microscopic intracellular detection of Epstein-Barr virus (EBV) messenger RNA in Reed-Sternberg cells of Hodgkin's disease (HD) was possible by in situ hybridization, in tissue sections prepared by a method termed modified acetone methyl benzoate xylene (ModAMeX). The ModAMeX method was initially developed for simultaneous optimal preservation of leucocyte differentiation antigens and morphology. Two biotinylated DNA probes, corresponding to the same BamHI-W (internal repeat) of the EBV genome were used. EBV mRNA was detected in neoplastic cells in 16 of 54 (30%) lymph node biopsy specimens from usual subtypes of HD (lymphocyte predominance, 0/5; nodular sclerosis, 4/22; mixed cellularity, 12/26; unclassified, 0/1). EBV mRNA was also detected in the lymph node biopsy of 1 additional human immunodeficiency virus (HIV)-related case of HD (mixed cellularity) and in 2 of 4 cases of B-cell lymphomas occurring in patients with acquired immunodeficiency syndrome (AIDS). In other non-Hodgkin's lymphomas, EBV mRNA was detected in only 1 of 41 cases. Cases of HD positive for EBV mRNA were immunostained by CD30 and CD15 antibodies. The hybridization signals were exclusively restricted to Reed-Sternberg cells and variants. When analyzed retrospectively, no statistically significant correlation emerged between hybridization findings, EBV serology, or disease outcome over the 3 years of the availability of ModAMeX technique. The findings support the contention of a direct role of EBV in the pathogenesis of HD, at least in some cases.

Abstract 5067 Background: Hematologic neoplasms associated with the chromosomal translocation t(2;11)(p21;q23) form a distinct genetic entity with diverse manifestations, and have been strongly linked with up-regulation of the microRNA miR-125b-1 (Bousquet et al, J Exp Med, 2008). Of 41 patients with myeloid malignancies and t(2;11)(p21;23) reported in the world's literature, all but 2 were cases of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), the latter cases frequently high-grade MDS transforming to AML. In 20 of 41 cases deletion of 5q was detected as a secondary cytogenetic abnormality. Only two cases of chronic myeloproliferative disorders associated with t(2;11)(p21;23) have been reported: one of chronic myeloid leukemia with a secondary t(9;22) (Ph) translocation (Royer-Pokora et al, Leukemia, 2003) and one of polycythemia vera (Acar et al, Amer J Hematol, 2006). We encountered a patient with a mixed myelodysplastic/myeloproliferative neoplasm clinically resembling polycythemia vera that was associated with t(2;11)(p21;q23) who was found to have persistent elevation in the blood of the microRNA miR-125b-1. Case Report: A 52 year-old male presented with upper extremity pain and headache. A CBC revealed the white blood cell count was 12.5 ×103/uL (55% neutrophils, 12% lymphocytes, 8% monocytes, 5% eosinophils, 21% basophils), red blood cell count 6.62 × 106/uL, hemoglobin 22.4 g/dL, hematocrit 68.1%, MCV 103 fL, and platelets 112 × 103/uL. Bone marrow aspiration and biopsy revealed a markedly hypercellular marrow (100%) with 1+ reticulin fibrosis, mild trilineal dysplastic morphology, and a blast count of 0.8%. Cytogenetic studies disclosed 46, XY, t(2;11)(p21;q23) [4]/46, idem, del (5)(q15q31) [4]/46, XY, del (5) (q13q31) [2]/46, XY[10]. FISH studies were negative for BCR/ABL1 fusion and MLL rearrangement. Molecular analysis for the JAK2V617F allele was negative. A diagnosis of mixed myelodysplastic/myeloproliferative neoplasm, unclassified was made (WHO 2008). The patient was begun on hydroxyurea, 500 mg daily. Four months after diagnosis the blood counts were within normal range, at which time quantitative real-time PCR (qRT-PCR; Bousquet et al, J Exp Med, 2008) performed on a sample of whole blood revealed a twenty-fold elevation of the microRNA species miR-125b-1, compared to healthy donors (n=3), and JAK2V617F-negative (n=3) and JAK2V617F-positive (n=2) polycythemia vera controls. Repeat qRT-PCR performed on whole blood 15 months after diagnosis confirmed persistently elevated miR-125b-1, nearly 200-fold above healthy donor controls (n=3). 18 months after diagnosis the patient remains healthy, with normal blood counts on 500 mg of hydroxyurea daily (white blood cell count 5.54 ×103/uL, red cell count 5.35 × 106/uL, hemoglobin 13.7 g/dL, hematocrit 47.2%, MCV 88.2, platelets 132.0 × 103/uL). Conclusion: This case expands the clinical-pathologic spectrum of hematologic malignancies associated with t(2;11)(p21;23), and confirms the link between t(2;11)(p21;23) and up-regulation of miR-125b-1. Translocation t(2;11)(p21;23) with miR-125b-1 up-regulation should be considered as a rare possibility in the differential diagnosis of JAK2V617F-negative polycythemia vera. Disclosures: No relevant conflicts of interest to declare.

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes which are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is responsible for a strong up-regulation of miR-125b (6 to 90 fold). In vitro experiments revealed that miR-125b was able to block monocytic and granulocytic differentiation of leukemic cells and primary CD34+ human blasts. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation and myeloid neoplasms carrying the t(2;11) translocation define a new clinico-pathological entity.

Hemophagocytic syndrome (HPS) is characterized by an uncontrolled proliferation of macrophages displaying extensive phagocytosis of hematopoietic cells that can be associated with non Hodgkin lymphomas. However, Hodgkin lymphoma associated with hemophagocytic syndrome (HL-HPS) has rarely been published. We report here a large series of 34 patients presenting HL-HPS. Histological analysis and Epstein-Barr Virus (EBV) studies were performed. The 34 patients were enrolled from 1992 to 2006. HPS diagnosis was based on usual clinico-biological criteria. Bone marrow aspiration or biopsy were performed in all cases and confirmed HPS, showing hemophagocytic features in scattered macrophages. HL was documented on histological material in all 34 patients. The biopsies were performed on different localizations: lymph nodes (n=18), bone marrow (n=27), liver (n=2) and tonsil (n=1). All histological slides were reviewed and classified according to the World Health Organization (WHO) Lymphoma classification. The presence of EBV in the tumour cells was detected using EBER RNA in situ hybridization and the expression of viral proteins LMP-1, EBNA-2 by immunoperoxidase. Patients were 26 men and 8 women (sex ratio M/F = 3.3). The median age was 45 years (range 19–84). HIV status was negative in 26 patients and positive in 8 patients. All patients were in clinical stage IVB. HL subtypes (18 lymph nodes) were Mixed Cellularity (n=12, 67%), Nodular Sclerosis (n=2, 11%) and Lymphocyte Depleted (n=4, 22%). Extra nodal tissues were highly infiltrated by tumour cells in all cases. The presence of EBV in tumour cells was detected in 32 out of the 34 patients (94%). In all EBV positive cases, high levels of LMP-1 without EBNA2 expression were detected, defining a latency II. This study reports the largest series to date of HL-HPS. The features of HL-HPS are particular by the high proportion of mixed cellularity subtypes (67%) in contrast to the frequency of nodular sclerosis subtypes observed in the general population of non overt immunosuppressed patients, and by the striking high proportion of EBV associated HL (32 out of the 34 patients, 94%). Our findings suggest not only a pathogenic role of EBV, but also a defective immune system in the control of EBV infection in HL-HPS patients.The high predominance of male in our population of HPS-HL (sex ratio M/F = 4.3 after exclusion of HIV positive patients) and the strong association with EBV could suggest X-linked immune defect in these patients.

Allogeneic stem cell transplantation (SCT) can induce long-term disease-free survival in refractory follicular lymphomas (FL) but is impaired by high rate of transplant-related mortality (TRM). Reduced conditioning followed by allogeneic SCT is a promising concept in FL, allowing a graft-versus-lymphoma (GVL) effect while reducing the TRM. We piloted a clinical trial using this approach in relapsed/primary refractory low-grade lymphomas. From 1998 to 2003, 31 patients (pts) with an HLA-identical sibling have been enrolled. Main pts characteristics were : age = 49 years (range, 32–61 y); grade : I/II FL = 26; FL with histologic transformation = 5; gender = 16 male and 15 female; ≥ 2 prior therapies before transplantation = 23; primary refractory = 8; stage IV = 22; Bulky disease = 16; mean LDH rate before transplantation = 335 UI/l (range, 205–858); residual disease at transplantation (partial response (PR) + progression) = 20. Previous treatments were as follow : anthracyclins = 26, alkylants = 21, anti-CD20 = 15, purine analogs = 7, autologous SCT = 6, radiotherapy = 5. The conditioning regimen was IV fludarabine 30 mg/m²/day (from day −5 to −2); PO busulfan 2 mg/kg/day (from day −7 to −6); antithymocyte globulin (ATG) 2.5 mg/kg/day (from day −4 to −3). 14/31 (45%) and 17/31 (55%) pts received peripheral blood and bone marrow stem cells respectively with a median number of CD34+ cells of 5 x 106/kg (range, 1.8–11.9). Graft-versus-host-disease (GVHD) prophylaxis consisted in cyclosporine A alone. All patients achieved complete donor (> 95%) chimerism after allogeneic SCT. Median duration of neutropenia (< 0.5 G/L) and thrombocytopenia (< 25 G/L) were 3 days (range, 0–16) and 3 days (range, 0–29) respectively. 8/31 pts (26%) had CMV reactivation but no CMV disease. 3/31 (10%) developed grade II–IV acute GVHD. 10/31 (32%) developed chronic GVHD (5 moderate and 5 extensive). The median follow-up time was 20 months. The TRM was 19% (6/31). Causes of death were aGVHD = 1, auto-immune hepatitis = 1, bacteraemia = 2, pulmonary toxoplasmosis = 1 and Lyell syndrome = 1. Overall response rate was 97% (21 CR + 9 PR) at day 100 and at 1 year (24 CR + 6 PR). The estimated 2-year overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) rate were 75%, 65% and 85% respectively. 2 relapses occurred. Both were treated by anti-CD20, achieved CR and are still alive. Donor age, Bulky disease and LDH were significant for EFS. Altogether, these data suggest that non-myeloablative SCT can induce a high rate of durable remissions with reduced toxicity in this poor risk population.

A new B-lymphoma cell line (DEAU-cell line) was established from a diffuse large-cell lymphoma (centroblastic type) and was successfully grafted in athymic nude mice. Monoclonal antibodies (MoAbs) were generated using splenocytes of DEAU-tumor bearing mice. Before the fusion experiments, cellular immunity of the mice bearing growing DEAU tumors was restored by injection of spleen cells from conventional Balb/C mice. Spleen cells from conventional Balb/C mice immunized with DEAU-cell line were also used for the generation of MoAbs. Four MoAbs (DBB.42 and DBA.44 from normal Balb/C mice, and DNA.7 and DND.53 from athymic nude mice) were investigated because they identified B-cell- associated antigens not destroyed by fixatives. DBB.42 recognized a pan- B cell-associated antigen (molecular weight (mol wt) = 45 Kd). DBA.44 detected a B-cell antigen (mol wt not determined) expressed on a subpopulation of B lymphocytes in the mantle zone of lymphoid follicles. DNA.7 also defined a B-cell antigen (43 Kd) mainly expressed on germinal center cells. Similarly, DND.53 recognized a B-cell antigen (two bands of mol wt 20 Kd and 35 Kd, respectively) mainly expressed on germinal center cells and mantle zone lymphocytes and interdigitating reticulum cells in the paracortical area. Major differences were found in the reactivities of these MoAbs on malignant lymphomas. DBB.42 was positive with almost all B-cell lymphomas and some T-cell lymphomas. Within the group of low-grade B-cell lymphomas, DBA.44 reacted principally with hairy-cell leukemia. DNA.7 reacted mainly with high- grade B-cell lymphomas with a weak positivity in low-grade B-cell lymphomas. DND.53 reacted with all but one B-cell lymphoma, cells of histiocytosis X, and Reed-Sternberg cells. These findings indicate that new MoAbs can be generated by using spleen cells from athymic mice bearing human tumors as well as by new lymphoid cell lines. The MoAbs so generated, as in the present study, are deemed potentially useful for the recognition of B-cell lymphomas in routine diagnostic histopathology. In addition, DND.53 could be of value for the diagnosis of histiocytosis X and the detection of Reed-Sternberg cells in Hodgkin's disease.