Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes

Death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of DAP-kinase of 194 bone marrow samples from 160 patients with acute myeloid leukemia (AML) and 34 with a myelodysplastic syndrome (MDS) at the time of initial diagnosis by polymerase chain reaction (PCR). Hypermethylation of DAP-kinase was present in 27.5% (44 of 160) of AML and in 47% (16 of 34) of MDS specimens and significantly correlated to loss of DAP-kinase expression (P =.008). It was significantly more frequent in AML secondary to therapy for other malignancies (s-AML; 14 of 29, 48.3%), as compared to de novo AML (30 of 131, 22.9%, P =.01). DAP-kinase hypermethylation in AML was associated with myelodysplastic changes in the bone marrow at the time of the initial diagnosis (P =.002) and with the presence of cytogenetic abnormalities (P =.02). Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia.     

Aberrant methylation of multiple tumor suppressor genes in acute myeloid leukemia

Hypermethylator phenotype, a propensity of tumors to incur nonrandom concurrent methylation, has been described in several tumors, including acute myeloid leukemia (AML). More recent studies identified methylation of other tumor suppressor genes, DAP-kinase and SOCS1, singly in AML. We therefore assessed the methylation status of these genes concurrently with other known targets of methylation. We used methylation-specific PCR or COBRA to determine the extent of methylation of 10 genes in 28 AML samples from Turkey. In addition to DAP-kinase and SOCS1, we included ER, p15, and E-cadherin (reported to be frequently methylated) as well as p16, GSTP1, and HIC1 (reported as rarely methylated). We also included RARbeta and p73 for which only minimal data in AML is available. All samples were methylated at least in one locus and all except one demonstrated methylation of DAP-kinase, SOCS1, p15, and/or ER. DAP-kinase is the most frequently methylated gene in both pediatric (70%) and adult AML (55%). RARbeta is methylated in 18% and p73 in 10% of AMLs. Methylation of E-cadherin and RARbeta occurs preferentially in AMLs with high methylation index (MI), while epigenetic lesions in SOCS1, DAP-kinase, and p15 appear to be independent. MI may be age-dependent, with a peak in young adults. FAB M3 demonstrated a higher extent of methylation than M2/M4. This study provides an impetus for larger studies to define if the extent and pattern of methylation in subgroups of AML are clinically relevant.     

High-dose therapy and autologous bone marrow transplantation in patients with follicular lymphoma during first remission

We report the results of a study in previously untreated advanced stage patients with follicular lymphoma (FL) who underwent uniform induction chemotherapy with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) followed by myeloablative therapy and anti-B-cell monoclonal antibody purged autologous bone marrow transplantation (ABMT). Eighty-three patients with previously untreated, low-grade FL were enrolled. After CHOP induction, only 36% achieved complete remission (CR) and 77 patients underwent ABMT. Before BM harvest, 70 patients had a known t(14;18), as determined by polymerase chain reaction (PCR), and all remained PCR positive in the BM at harvest. After ABMT, the disease-free survival (DFS) and overall survival are estimated to be 63% and 89% at 3 years, respectively, with a median follow-up of 45 months. Patients whose BM was PCR negative after purging experienced significantly longer freedom from recurrence (FFR) than those whose BM remained PCR positive (P = .0006). Continued PCR negativity in follow-up BM samples was also strongly predictive of continued CR. This study suggests that a subset of patients with advanced FL may experience prolonged clinical and molecular remissions following high-dose ablative therapy, although longer follow-up will be necessary to determine potential impact on overall survival.     

Assessment of bone marrow involvement in non‐Hodgkin’s lymphomas: comparison between histology and flow cytometry

Bone marrow (BM) examination is essential in the staging of non‐Hodgkin’s lymphoma (NHL) patients. Few studies have compared BM histologic findings with results of flow cytometric (FC) analysis. We analyzed the incidence and patterns of histologic BM involvement in a series of 753 patients with NHL. For 498 patients, a concurrent FC analysis on BM was available. Histologic involvement was detected at diagnosis in 311/753 (41%) patients. By FC, BM involvement was clearly detected in 150/498 (30%). After excluding 12 cases with equivocal histology, concordance between the two methods was detected in 411 (85%) cases (27% BMB+/FC+; 58% BMB?/FC?), while discordance was present in 75 (15%) (P < 0.001): 58 cases (12%) were BMB+/FC? and 17 (3%) were BMB?/FC+. Discordance was more frequent in FL and in lymphoplasmacytic lymphoma (LPL). These data demonstrate that the two methods are comparable in qualitative assessment of BM involvement in NHL, with the exception of FL and LPL. In FL, diffuse large B‐cell lymphoma (DLBCL) and LPL, FC underestimates the extent of infiltrate with respect to histology.     

The Value of PET/CT in Detecting Bone Marrow Involvement in Patients With Follicular Lymphoma

Follicular lymphoma (FL) is the 2nd most common type of lymphoma diagnosed in the Western World. Bone marrow (BM) involvement is an adverse prognostic factor in FL, routinely assessed by an arbitrary biopsy of the iliac crest. This study was aimed to investigate the role of positron emission tomography/computed tomography (PET/CT) in identifying BM involvement by FL.In this retrospective, single-center study we reviewed the records of consecutive patients with FL at diagnosis or relapse who underwent staging/restaging workup visual assessment of BM uptake was categorized as either normal, diffusely increased, or focally increased. Quantitative BM fluorine-18-fluro-deoxyglucose (FDG) uptake was measured using mean standardized uptake value (BM-SUV_mean). The diagnosis of BM involvement was based on either BM histological findings or disappearance of increased uptake at end-treatment PET/CT in patients who responded to treatment.Sixty eight cases with FL were included. Sixteen (23.5%) had BM involvement, 13 (19.1%) had a biopsy proven involvement, and 3 (4.4%) had a negative BM biopsy, but increased medullary uptake that normalized post-treatment. BM FDG uptake in these patients was diffuse in 8 (50%) and focal in 8 (50%). Focal increased uptake was indicative of BM involvement; however, diffuse uptake was associated with 17 false positive cases (32.7%). Overall, visual assessment of BM involvement had a negative predictive value (NPV) of 100% and a positive predictive value (PPV) of 48.5%. On a quantitative assessment, BM-SUV_mean was significantly higher in patients with BM involvement (SUV_mean of 3.7 [1.7–6] vs 1.4 [0.4–2.65], P < 0.001). On receiver operator curve (ROC) analysis, BM-SUV_mean > 2.7 had a PPV of 100% for BM involvement (sensitivity of 68%), while BM-SUV_mean < 1.7 had an NPV of 100% (specificity of 73%).Visual assessment of PET/CT is appropriate for ruling out BM involvement by FL. Although focal increased uptake indicates marrow involvement, diffuse uptake is nonspecific. SUV measurement improves PET/CT diagnostic accuracy, identifying additional 19% of patients with BM involvement that would have been otherwise missed.     

A real-time (PCR) for a real life…? Quantitative evaluation of BCL2/IGH in follicular lymphoma and its implications for clinical practice

Follicular lymphoma (FL) is highly associated with the molecular rearrangement BCL2/IGH. Although BCL2/IGH has been studied many times in follicular lymphoma, its real clinical value remains controversial. In this study, we performed quantitative testing by real-time polymerase chain reaction in 56 FL patients with median follow-up of 44 months (range, 9-102 months); chemotherapy was administered in 52 of 56 cases. Pretreatment numbers of BCL2/IGH varied in wide ranges, with a median of 2947 (range, 0-1,261,013) copies/10(6) cellular equivalent in peripheral blood (PB) and 4650 copies/10(6) cellular equivalent (range, 1-1,056,813) in bone marrow (BM), the difference between PB and BM was significant (p?= 0.006). Pretreatment of BCL2/IGH quantities were correlated to clinical parameters (e.g., age, stage, sex, lactate dehydrogenase, B symptoms, grade, bulky disease, chemotherapy regimen) and to progression free-survival. Advanced clinical stage (III and IV) and microscopic BM involvement were significantly associated with higher numbers of BCL2/IGH in PB (p < 0.05) and in BM (p = 0.05), regardless all or newly diagnosed patients were evaluated. High pretreatment burden of BCL2/IGH was associated with significantly shorter progression-free survival; p = 0.003 and p = 0.047 for PB and BM, respectively. In conclusion, pretreatment quantity of BCL2/IGH in PB or BM seems to mirror the extent of disease and can provide an auxiliary prognostic parameter in FL. Our results also support evidence of the negative prognostic value of microscopic BM involvement in FL.     

Comparative analysis between RQ‐PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma

BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ‐PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ‐PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ‐PCR≥10⁻⁵). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR‐negative/minor cluster region‐negative/minor BCL2‐negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression‐free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM−; 6 PB−/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ‐PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized.     

老年肺癌病人痰标本中p16基因异常甲基化的研究

目的分析老年肺癌病人痰标本中p16基因启动子区域异常高甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标记物的价值。方法运用半巢式甲基化特异性聚合酶链反应技术,检测94例老年原发性肺癌病人痰标本和部分对应肿瘤组织,以及10例慢性肺炎病例痰标本中p16基因启动子区域的甲基化改变。结果74%的肺癌病人痰标本中检测到了p16基因异常高甲基化,与传统细胞学(46.8%)相比,痰标本中p16基因异常高甲基化对肿瘤的检出率(74.5%)灵敏度更高(P<0.01);痰细胞中p16甲基化检测和细胞学相结合,对肿瘤的检出率可达86.17%(81/94)。如果痰标本中p16基因启动子区域发生高甲基化,其对应的肿瘤组织中p16基因亦为高甲基化。10例慢性肺炎病人痰标本中仅3例检测出p16基因甲基化。结论痰标本中p16基因甲基化是临床肺癌辅助诊断的有效分子生物标记物之一。     

Influence of cytokines and autologous lymphokine-activated killer cells on leukemic bone marrow cells and colonies in AML

We have already shown that cytokine cocktails (IL-1beta, IL-3, IL-6, SCF, GM-CSF) and/or lymphokine-activated killer (LAK) cells can reduce the amounts of clonal, CD34-positive mononuclear bone marrow cells (BM-MNC) in acute myeloid leukemia (AML). In addition, the influence of those cocktails and/or LAK cells on the clonogenic potential of AML BM-MNC was investigated. BM colonies cultured in agar during different stages of the disease were immunophenotyped in situ: 17 patients at diagnosis, 14 patients in complete remission, 8 patients at relapse, 8 healthy donors. A significant reduction in leukemic cells and colonies positive for CD34 after in vitro culture of BM-MNC with cytokine cocktails was achieved with all samples obtained at diagnosis (n = 8, p < 0.01), in 6 of 8 cases in complete remission but only in 2 of 6 cases at relapse. Cytokine cocktails stimulated granulopoiesis as well as B and T lymphopoiesis. Colonies with leukemic phenotype could never be detected in healthy BM. A significant reduction in leukemic colonies was achieved by coculture of BM-MNC (uncultured or cytokine precultured) with autologous LAK cells in all 4 cases at diagnosis and in 1 case at relapse. An additive effect of in vitro cytokine preincubation of BM samples on the leukemia-reducing effect of LAK cells could be demonstrated in all samples studied (p < 0.001; diagnosis: n = 10, relapse: n = 3, complete remission: n = 7). Patients had a better prognosis if CD34-positive colonies in AML could be reduced by cytokine incubation (p = 0.03) or coculture with autologous LAK cells in vitro (p = 0.04). Our data show that cytokines as well as LAK cells alone and in combination can reduce, however not eliminate clonogenic AML cells. Such mechanisms might be responsible for maintaining stable remissions in AML.     

Hypermethylation of the calcitonin gene in acute lymphoblastic leukaemia is associated with unfavourable clinical outcome

We analysed calcitonin (CALC1) gene hypermethylation using semiquantitative differential polymerase chain reaction in 105 patients with adult (n = 49) and childhood (n = 56) acute lymphoblastic leukaemia (ALL), and studied the association of CALC1 hypermethylation with clinical presentation features and disease outcome. We also investigated the possible relationship between CALC1 methylation status and expression of the cell cycle inhibitor gene p57KIP2. We observed CALC1 hypermethylation in bone marrow cells from 43% (45 out of 105) of ALL patients. Clinical, molecular and laboratory features did not differ significantly between hypermethylated and hypomethylated patients, only T-cell lineage was associated with hypermethylation (14% vs. 47%, P = 0025). Complete remission rate was similar in both groups although hypermethylated patients had a higher relapse rate (68% vs. 19%, P < 0.00001) and mortality rate (55% vs. 36%, P = 0.06) than hypomethylated patients. Estimated disease-free survival (DFS) at 6 years was 66.1% for hypomethylated patients and 5.3% for hypermethylated patients (P < 0,00001). Multivariate analysis from potential prognostic factors demonstrated that CALC1 methylation status was an independent prognostic factor in predicting DFS (P = 0.0001). Separate analysis of adult and childhood ALL patients showed similar results to the whole series. In addition, hypermethylated patients showed downregulation of p57KIP2 expression. Our results suggest that CALC1 gene hypermethylation is associated with an enhanced risk of relapse independently of known poor-prognostic factors and we describe, for the first time, a possible implication of the p57KIP2 gene in the genesis and prognosis of ALL.     

Stage I/II follicular lymphoma: spread of bcl-2/IgH+ cells in blood and bone marrow from primary site of disease and possibility of clearance after involved field radiotherapy

Stage I/IIA follicular lymphoma (FL) is considered a localised disease that can be adequately treated with radiotherapy alone. Bone marrow (BM) and peripheral blood (PB) involvement in FL was investigated by polymerase chain reaction (PCR) in a series of 24 consecutive patients with histologically revised diagnosis and treated with involved field radiotherapy. Despite the limited stage, Bcl-2/IgH+ cells were found at diagnosis in PB and/or BM of 16 patients (66.6%). After treatment, in 9/15 Bcl-2/IgH positive evaluable patients, a disappearance of Bcl-2/IgH+ cells was observed, which persisted after a median follow-up of 43.5 months (range 11-70) in all but one patient. Quantitative PCR demonstrated the feasibility of clearing PB and BM Bcl-2+ cells after local irradiation of the primary site of the disease only when the basal number of lymphoma cells was <1:100 000. Patients with Bcl-2/IgH+ cells at diagnosis or after treatment had a higher likelihood of relapse. Thus, despite a negative BM biopsy, the majority of localised FL Bcl-2/IgH+ cells were found in the PB and BM. Lymphoma cells can reversibly spread from the affected lymph node to PB and BM and, in a proportion of cases, durably disappear after irradiation. The possibility of a persistent lymphoma cell clearance is proportional to the amount of cells detected at presentation by quantitative PCR.     

Polymerase chain reaction detection of cells carrying t(14;18) in bone marrow of patients with follicular and diffuse large B-cell lymphoma: the importance of analysis at diagnosis and significance of long-term follow-up

The t(14;18) is the most frequent chromosomal aberration observed in follicular lymphoma (FL), and is less frequent in diffuse large cell lymphoma (DLCL). The bcl-2/IgH rearrangement constitutes good target for polymerase chain reaction (PCR) detection that allows to find out one tumor cell in 100,000 normal cells. The PCR assay was used to detect bcl-2-rearranged cells in blood and bone marrow (BM) in 63 previously untreated patients with DLCL and in 53 patients with FL. Twenty five FL patients (47%) and 9 DLCL patients (14%) had PCR-detectable lymphoma cells in BM and peripheral blood. Minimal residual disease (MRD) was evaluated in 17 FL and 5 DLCL patients undergoing first-line chemotherapy. Three DLCL patients (60%) but only 1 FL (6%) patient achieved molecular response (PCR-negative status in BM). Two PCR bcl-2/IgH positive patients with FL were treated with rituximab (anti-CD20 antibody) and had no PCR-detectable lymphoma cells in BM after the therapy. Peripheral blood stem cells (PBSC) were harvested in 5 FL (1 PCR-negative) and in 2 DLCL (1 PCR-negative) patients. PCR-positive lymphoma cells contamined PBSC in all patients with BM PCR-positivity before harvesting. Five FL patients underwent autologous transplantation (AT). No bcl-2/IgH positive cells were detected in 4 patients (80%) at any point after AT. One patient achieved molecular response after rituximab treatment. All the patients are in CR 6, 22, 30, 31 and 42 months respectively, after AT. On the other hand, 4 FL patients in clinical complete remission, but with persistent PCR positivity in BM relapsed with median of 21 months (interval, 14-28 months) from the end of a first-line chemotherapy. Thus, the results show that PCR detection of the bcl-2/IgH rearrangement is a very useful method in evaluating the BM infiltration by lymphoma cells especially in the situation of MRD. Conventional chemotherapy did not eradicate bcl-2 positive cells in BM in most of lymphoma patients, but autologous transplantation or rituximab immunotherapy can induce molecular response in a significant proportion of them. Our results support the previous observations of the molecular response importance in view of better disease free and probably also overall survival.     

Promoter Hypermethylation of the DNA-Repair Gene O 6 -Methylguanine—DNA Methyltransferase and p53 Mutation in Diffuse Large B-cell Lymphoma

The gene for the DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which is closely related with cellular sensitivity to alkylating agents, is inactivated by promoter hypermethylation in several human cancers, including malignant lymphoma. Promoter hypermethylation of the MGMT gene is a favorable prognostic factor in diffuse large B-cell lymphoma (DLBCL). Although inactivation of the MGMT gene is closely related to p53 gene mutations in several cancers, the relationship between p53 gene mutation and MGMT inactivation in malignant lymphoma has not been thoroughly examined. We studied the correlation between MGMT hypermethylation and p53 mutation in DLBCL and their impacts on patient prognosis. In a retrospective cohort study, we used a methylation-specific polymerase chain reaction technique to analyze the methylation status of the promoter region of the MGMT gene in 116 DLBCL patients who received cyclophosphamide as part of multidrug combination chemotherapies. Denaturing high-performance liquid chromatography and direct sequencing were used to search for p53 gene mutations in exons 5 through 9 in 96 of the 116 samples. Disease-free survival and overall survival were estimated by the Kaplan-Meier method. Multivariate survival analyses were performed with the Cox proportional hazards model. Forty-five (38.8%) of 116 DLBCL patients showed MGMT promoter hypermethylation. The presence of MGMT hypermethylation was associated with better overall survival (P = .036). MGMT promoter hypermethylation was a prognostic factor that was independent of established prognostic factors, such as age, disease stage, serum lactic dehydrogenase level, and the number of extranodal disease sites (hazard ratio, 2.43; 95% confidence interval, 1.28-4.61; P = .007). p53 mutations were detected in 19 (19.8%) of 96 patients and were identified as a risk factor in the complete remission rate and overall survival (P = .0040, and P = .027, respectively). A correlation between MGMT hypermethylation and p53 mutation or p53 G:C-to-A:T mutation was not observed (P = .88, and P = .31, respectively). MGMT promoter hypermethylation and p53 mutation are useful prognostic markers in DLBCL. The impact of MGMT inactivation on p53 mutation in DLBCL is unclear.     

Autologous stem cell transplantation for follicular lymphoma: no benefit for early transplant?

Autologous stem cell transplantation (SCT) is widely used as salvage treatment for patients with relapsed follicular lymphoma (FL). Although SCT can induce prolonged remissions, it does not appear to be curative in the vast majority of patients. The purpose of this study was to investigate if incorporation of SCT into first-line therapy can improve its efficacy. Fifty-five patients underwent sequential high-dose therapy as up-front (n=33) or salvage treatment (n=22) for advanced stage FL at our institution. Treatment consisted of intensive chemotherapy with dexamethasone, carmustine (BCNU), etoposide, cytarabine, and melphalan (Dexa-BEAM) for mobilization of peripheral stem cells and reduction of tumor load, followed by one of three different myeloablative regimens and SCT. With a median follow-up of 4 years, projected event-free survival (EFS) and overall survival (OS) at 4 years post transplant was 59% and 84%, respectively, with no evidence of plateau in the survival curves. By univariate and multivariate analysis weighing age, sex, stage, BM and extranodal involvement, timing of transplant, ex vivo purging, and conditioning regimen [total body irradiation (TBI) vs non-TBI], the only significant factor predicting for superior EFS and OS was up-front vs salvage transplant (4-year EFS 76% vs 38%, p=0.02; 4-year OS 92% vs 73%, p=0.033). However, when calculated from diagnosis, EFS and OS of the up-front and salvage groups were virtually identical, implying that the longer survival post SCT in the up-front group was completely compensated by the longer interval between diagnosis and transplant in the salvage group. Median OS from diagnosis was 13.5 years. Except for one case of anaplastic large cell lymphoma, secondary neoplasms have not occurred to date. In conclusion, our data indicate that SCT might improve the prognosis of patients with disseminated FL, although it is probably not curative even if applied early during the course of the disease. The optimum timing of SCT remains to be determined by the ongoing randomized multicenter trial of the German Low-grade Lymphoma Study Group. The impact of radiotherapy on the success of SCT does not seem to be as essential as originally believed.     

The effect of human fit- 3 ligand on committed progenitor cell production from normal, aplastic anaemia and Diamond-Blackfan anaemia bone marrow

Summary. We investigated the effect of the human ligand for flt-3 (FL) on the committed progenitor colony formation of normal bone marrow (BM) (n = 9) and BM from four aplastic anaemia (AA) and three Diamond-Blackfan anaemia (DBA) patients. Methylcellulose committed progenitor cell assays were carried out using FL alone and in combinations with granulocyte-macrophage colony-stimulating factor (GM-CSF). interleukin-3 (IL-3) and c-kit ligand (KL). FL alone had a limited, though significant, effect on the production of granulocyte-macrophage colony-forming unit (CFU-GM) colonies from normal BM and showed an additive effect with IL- 3 and GM-CSF separately, but not in combination. FL did not increase the stimulation of KL and did not have an effect on the production of erythroid progenitor colonies. FL had no effect on the AA and DBA BMs studied.     

Improved cytogenetics in multiple myeloma: a study of 151 patients including 117 patients at diagnosis

Between December 1990 and January 1994, bone marrow (BM) samples from 151 patients with multiple myeloma (MM), including 117 patients evaluated at diagnosis, were collected for cytogenetic analysis. A total of 129 patients had assessable metaphases (100 patients at diagnosis). Cytogenetic studies were performed on BM cells after longterm cultures (6 days) with stimulation of cultures by granulocyte- macrophage colony-stimulating factor (GM-CSF), GM-CSF plus interleukin (IL)-6, IL-3 plus IL-6, or GM-CSF plus IL-3 plus IL-6 to improve myeloma cell growth, and 91 patients had an additional unstimulated culture. Sixty-six patients (51%) had cytogenetic abnormalities, including 47 of 100 patients at diagnosis (47%) and 17 of 24 patients at relapse (71%; P = .04). The aberration rate increased with stage (P = .007), BM plasmacytosis (P = .003), beta 2 microglobulin level (P = .001), C-reactive protein (CRP) level (P = .001), and Ki-67 (P = .007). The abnormality detection rate was higher in stimulated than unstimulated cultures, and the difference was statistically significant (P < .01). Hyperdiploidy was observed in 39 patients (30% of patients with an assessable karyotype) and hypodiploidy in 19 patients (15%). Among numeric changes, gains predominantly involved chromosomes 3, 5, 7, 9, 11, 15, 19 and losses, chromosomes 8, 13, 14, and X. The most frequent loss was loss of chromosome 13, observed in 22 patients (15%), including 18 patients at diagnosis (12%). We observed frequent structural changes of chromosomes 1 (15%) and 14 (10%) but also a 5% incidence of 19q13 abnormality and two patients with translocation t(1;16)(p11;p11). By using the proportional hazard univariate model, patients with abnormal karyotypes were demonstrated to have 2.5-fold greater chance of death than patients with normal karyotypes (P < .014). Despite a multivariate approach with the same model, the respective roles of karyotype abnormality, age, stage, and beta 2 microglobulin level could not be clearly ascertained. From these results we conclude that cytogenetic analysis using stimulation of cultures by cytokine(s) may be a promising method to identify about 50% of cytogenetic abnormalities in patients with newly diagnosed MM. Cytogenetic analysis may help to define a high-risk population that would benefit from intensive therapeutic approaches.     

The relative role of peripheral blood and bone marrow for monitoring molecular evidence of disease in follicular lymphoma by quantitative real-time polymerase chain reaction

Peripheral blood (PB) and bone marrow (BM) are used interchangeably for t(14;18) (IgH/BCL-2) molecular monitoring in follicular lymphoma (FL) and detection of rearrangement after treatment has been correlated to increased risk of relapse. To determine the relative value of each tissue, MBR t(14;18) was quantified by real-time polymerase chain reaction in 52 simultaneous paired PB and BM samples from 38 FL patients. In total, 79% of sample pairs taken in remission (n = 19) or when no morphological disease was evident in the BM (n = 29) had t(14;18) copy number within one log difference and the median difference was small. These findings suggest that, in remission, PB may be adequately monitored. In general, however, higher copy number was detected in BM than in the corresponding PB sample.     

High numbers of tumor-infiltrating FOXP3-positive regulatory T cells are associated with improved overall survival in follicular lymphoma

The tumor microenvironment plays an important role in the biologic behavior of follicular lymphoma (FL), but the specific cell subsets involved in this regulation are unknown. To determine the impact of FOXP3-positive regulatory T cells (Tregs) in the progression and outcome of FL patients, we examined samples from 97 patients at diagnosis and 37 at first relapse with an anti-FOXP3 monoclonal antibody. Tregs were quantified using computerized image analysis. The median overall survival (OS) of the series was 9.9 years, and the FL International Prognostic Index (FLIPI) was prognostically significant. The median Treg percentage at diagnosis was 10.5%. Overall, 49 patients had more than 10% Tregs, 30 between 5% to 10%, and 19 less than 5%, with a 5-year OS of 80%, 74%, and 50%, respectively (P = .001). Patients with very low numbers of Tregs (< 5%) presented more frequently with refractory disease (P = .007). The prognostic significance of Treg numbers was independent of the FLIPI. Seven transformed diffuse large B-cell lymphomas (DLBCLs) had lower Treg percentages (mean: 3.3%) than FL grades 1,2 (mean: 12.1%) or 3 (mean: 9%) (P < .02). In conclusion, high Treg numbers predict improved survival of FL patients, while a marked reduction in Tregs is observed on transformation to DLBCL.     

Bcl-6 p53 mutations in lymphomas carrying the bcl-2/Jh rearrangement

BACKGROUND AND OBJECTIVES: The t(14;18)(q32;q21) chromosomal translocation is the hallmark of follicular lymphomas (FL). The translocation induces the overexpression of the Bcl-2 protein and prolongs the survival of clonogenic cells. Tumor cells may acquire additional molecular alterations that may be associated with histologic progression or with chemo-resistance. DESIGN AND METHODS: We analyzed the distribution and association of bcl-6 and p53 mutations in 55 consecutive bcl-2/Jh+ lymphoma samples derived from 43 patients obtained at the time of diagnosis and, in 5 of these patients, during follow-up. A total of 29 bcl-6 point mutations were detected in seventeen patients (40%) associated with major or minor breakpoints of the bcl-2/Jh fusion gene. In seven cases a p53 mutation was detected. Three cases corresponded to FL with the minor breakpoint in the bcl-2 gene and these patients had a favorable clinical evolution, whereas the 4 patients with p53 mutations and the major breakpoint had a bad clinical outcome with morphologic transformation to high-grade lymphoma in three cases. The sequential analysis of 5 patients showed a different timing in the acquisition of mutations: one patient showed bcl-6 and p53 mutations at diagnosis, another patient showed bcl-6 mutations at diagnosis and acquired a p53 mutation later whereas the third patient had a p53 mutation before the appearance of the bcl-6 mutation. RESULTS: We did not find significant differences in survival between patients with FL who showed exclusively bcl-6 mutations and those without bcl-6 mutations, but those patients with a high International Progostic Index score and p53 mutations showed the lowest overall survival (p = 0.002). INTERPRETATION AND CONCLUSIONS: These findings suggest that bcl-2/Jh lymphomas show molecular heterogeneity and that bcl-6 and p53 mutations may be acquired during the evolution of such lymphomas. Bcl-6 mutations, by themselves, do not seem to be associated with a bad prognosis. Rearrangements at the minor bcl-2 locus may have a different molecular evolution.     

Fetal liver generates low CD4 hematopoietic cells in murine stromal cultures

We have demonstrated that 0.2% to 11% of cells from the fetal liver (FL) reacted specifically with high concentrations of anti-CD4 monoclonal antibody (MoAb). CD4+ cells from FL were similar in surface phenotype and fluorescence characteristics to the CD4+ population found previously in adult bone marrow (BM). FL and BM cells were seeded in cultures that allow differentiation to primitive precursors. FL cells released many low CD4+ and low Thy+ cells in the supernatant, while BM cells seeded under the same conditions did not. We studied the nonadherent cells harvested from 10-day FL cultures (greater than 90% low CD4+). In methylcellulose, they were able to produce more colonies that appear to be characteristic of earlier stages in the hierarchy of hematopoietic precursors (especially erythroid bursts and colonies composed of both myeloid and erythroid elements) in comparison with CD4- cells from 10-day BM cultures. CD4+ cells harvested from FL cultures initiated secondary cultures containing both a stromal layer and large hematopoietic colonies when replated under conditions similar to those of primary cultures. Furthermore, a limited number of CD4+ cells from 10-day FL cultures were able to repopulate lethally irradiated mice. Although we cannot formally exclude the possibility that the low CD4 cells produced in FL cultures were derived exclusively from the proliferation of the few CD4 cells found in fresh FL, the dynamic analysis of the development of these cells in culture favors the generation of this important population from a CD4- subset of hematopoietic stem cells (HSCs). We speculate that FL contains a prevalent population of very primitive cells not expressing the CD4 antigen, tentatively called "pre-low CD4 precursors." These primitive cells can differentiate into low CD4+ cells that share many characteristics with pluripotent HSCs of the adult type. These data indicate the possibility of using hematopoietic progenitors obtained by the expansion/differentiation of fetal stem cells in culture for transplantation purposes.