Quantitation of follicular non-Hodgkin's lymphoma cells carrying t(14;18) by competitive polymerase chain reaction

Summary. A competitive polymerase chain reaction (PCR) technique was developed to quantify residual malignant cells in the peripheral blood and bone marrow of patients with low-grade follicular non-Hodgkin's lymphoma carrying a translocation between chromosomes 14 and 18. Artificial segments were constructed imitating a translocation between chromosome 14 and 18. These artificial translocation segments were used as competitor molecules in the quantitative PCR. Serial dilutions of a known amount of patient-derived translocation segments were coamplified with a fixed number of competitor molecules, and a patient specific calibration curve was constructed. Several patient samples were coamplified with an equal number of competitor molecules and the number of t(14;18) translocations within the samples was calculated by comparison with the calibration curve.
The method was demonstrated on samples of four follicular non-Hodgkin's lymphoma (NHL) patients. In a patient transplanted with allogeneic bone marrow declining numbers of residual lymphoma cells were observed.
We conclude that the method is accurate, relatively fast and the general principle of this method can be applied to all malignancies with characteristic abnormalities on DNA or RNA level that are detectable by PCR.     

Quantitative detection of t(14;18)-positive cells in patients with follicular lymphoma before and after autologous bone marrow transplantation

The aim of this study was to evaluate whether a quantitative analysis of circulating t(14;18)-positive cells is of prognostic significance in patients with follicular lymphoma (FL) after myelo-ablative therapy supported by ABMT. We tested DNA from primary lymphoma tissue as well as PBMC before and after ABMT from 15 patients for the presence of the t(14;18) translocation. Nine patients showed a t(14;18) translocation, six patients were t(14;18)-negative. Circulating t(14;18)-positive cells of seven patients were quantitatively determined by limiting dilution assays combined with a two-step PCR and by real-time quantitative PCR. The results of both methods correlate very well. The number of circulating t(14;18)-positive cells decreased significantly in all patients after myeloablative therapy and ABMT, t(14;18)-negative blood samples were found in five of seven patients. In all patients circulating t(14;18)-positive cells reappeared within 2 years after ABMT showing two different patterns. During continuous CR the numbers of circulating t(14;18)-positive cells were found to be stable within one order of magnitude. In contrast, in one patient the relapse was accompanied by a logarithmic increase of t(14;18)-positive cells. In a second patient the enlargement of lymph nodes developing over a period of 12 months was accompanied by very slowly increasing numbers of t(14;18)-positive cells. In all cases where diagnostic lymph node tissue was available, the same t(14;18) translocation was found at first diagnosis and after ABMT as shown by nucleotide sequence analysis. We conclude that the quantitative detection of circulating t(14;18)-positive cells during follow-up of patients with FL after ABMT reflects the clinical course of the disease. Relapses are associated with increasing numbers of circulating t(14;18)-positive cells and continuous complete remissions with stable cell counts.     

An additional segment at 1p36 derived from der(18)t(14;18) in patients with diffuse large B-cell lymphomas transformed from follicular lymphoma

The particular translocation in follicular lymphomas (FLs) is a t(14;18)(q32;q21), recombining the immunoglobulin heavy chain (IgH) gene on chromosome 14 with the B-cell leukemia/lymphoma 2 (BCL2) gene on chromosome 18. Some low-grade FLs are aggressively transformed into diffuse large B-cell lymphomas, presumably by acquisition of secondary chromosomal changes, including chromosomal band 1p36. A common example is add(1)(p36). Because it is difficult to identify the origin of add(1)(p36) even on high-resolution G-banding analysis, we used spectral karyotyping (SKY) and double-color fluorescence in situ hybridization (DC-FISH) to define the t(14;18) and the extra band at 1p36 in two cases of diffuse large B-cell lymphoma (DLBCL). SKY revealed that the extra chromosomal segment on 1p36 was derived from chromosome 18. DC-FISH defined BCL2/IgH fusion signals at 1p36 in addition to t(14;18), suggesting that BCL2/IgH fusion at 1p36 was an evolutionary alteration following the primary BCL2/IgH translocation on der(18) in both cases. Our results indicate that IgH alleles, implicated in chromosomal rearrangement, may themselves frequently be targets for secondary translocations, suggesting that multiple IgH translocations and insertions are associated with the progression of FL.     

Flow cytometry in the bone marrow evaluation of follicular and diffuse large B-cell lymphomas

BACKGROUND AND OBJECTIVES: Bone marrow biopsies are routinely performed in the staging of patients with lymphoma. Despite the lack of evidence for its usefulness, many institutions include flow cytometry (FC) of bone-marrow aspirates in an attempt to increase sensitivity and specificity. The aim of this study is to evaluate the usefulness of FC for the assessment of bone-marrow involvement by lymphoma in follicular (FL) and diffuse large B-cell lymphomas (DLBCL). DESIGN AND METHODS: Seventy-nine bone marrow biopsies from 65 patients diagnosed with FL or DLBCL were examined to compare histology and FC for the assessment of bone-marrow involvement by lymphoma. RESULTS: Bone marrow histology showed involvement (BM+) in 16 cases (20.3%), lack of infiltration (BM(-)) in 52 cases (65.8%) and undetermined or undiagnosed for involvement (BMu) in 11 cases (13.9%). FC was positive for involvement in 28 cases (35.4%) and negative in 51 cases (64.6%). 65 cases (95%) showed concordance between the results of morphology and FC (BM(+)/FC(+) or BM(-)/FC(-)). No BM(+)/FC(-) cases were observed. 3 cases showed discrepant results (BM(-)/FC(+)). In these 3 cases the molecular studies (PCR) demonstrated clonal rearrangement of the heavy immunoglobulin chain (IgH) and/or bcl2-IgH in agreement with the flow results. Among the 11 cases with BMu, all but 2 were FC(+) and concordance with the PCR results was seen in 9 cases (81.9%). INTERPRETATION AND CONCLUSIONS: We conclude that FC is just as sensitive or perhaps slightly more sensitive than histology in the detection of bone marrow involvement in FL and DLBCL. FC studies may be warranted in those cases in which the morphology is not diagnosed. The clinical relevance of the small clonal B-cell population in patients without histologic bone marrow involvement (BM(-)/FC(+) cases) remains an open question.     

Comparison of outcomes after allogeneic hematopoietic stem cell transplantation in patients with follicular lymphoma, diffuse large B-cell lymphoma associated with follicular lymphoma, or de novo diffuse large B-cell lymphoma

The outcome after allogeneic hematopoietic stem cell transplantation (allo-HCT) for diffuse large B-cell lymphoma (DLBCL) associated with follicular lymphoma (FL), which includes DLBCL with pre- or co-existing FL, remains controversial, and few previous reports have compared the outcomes after allo-HCT for FL, DLBCL associated with FL, and de novo DLBCL. We retrospectively analyzed 97 consecutive patients with FL (n = 46), DLBCL associated with FL (n = 22), or de novo DLBCL (n = 29) who received allo-HCT at our institute between 2000 and 2010. With a median follow-up of 53 months, the 5-year overall survival (OS) and progression-free survival (PFS) were, respectively, 77% and 70% for FL, 62% and 57% for DLBCL associated with FL, and 26% and 23% for de novo DLBCL. The 5-year cumulative incidences of non-relapse mortality and disease progression/relapse were, respectively, 16% and 15% for FL, 19% and 24% for DLBCL associated with FL, and 36% and 41% for de novo DLBCL. By a multivariate analysis, the OS and PFS for DLBCL associated with FL were significantly better than those for de novo DLBCL, whereas they were not significantly different from those for FL. These results suggest that allo-HCT may be a promising option for patients with not only advanced FL but also DLBCL associated with FL.     

Prognostic significance of absolute lymphocyte count at diagnosis of diffuse large B-cell lymphoma: a meta-analysis

The prognostic value of absolute lymphocytic count (ALC) has been a recent matter of debate in the study of non-Hodgkin-lymphoma. To evaluate the prognostic value of ALC at diagnosis in patients with diffuse large B-cell lymphoma (DLBCL), we performed a meta-analysis of published studies that provided survival information with reference to ALC at diagnosis. Six studies covering a total of 1,206 subjects were included in this analysis. The summary hazard ratios of low ALC for overall survival were 2.72 (95% confidence interval (CI) 2.15–3.45, P < 0.001) in the entire population, 2.96 (95% CI 2.04–4.29, P < 0.001) in the population that received CHOP, and 2.78 (95% CI 1.87–4.13, P < 0.001) in the population that received R-CHOP. The corresponding ratios for progression-free survival were 2.79 (95% CI 1.90–4.11, P < 0.001) in the entire population, and 2.56 (95% CI 1.66–3.96, P < 0.001) in the population that received R-CHOP. In conclusion, our systematic analysis suggests that low ALC has an adverse effect on outcome in DLBCL. Although it should be borne in mind that this meta-analysis was mainly based on data abstracted from observational studies, these results may justify risk-adapted therapeutic strategies for DLBCL to account for ALC at diagnosis.     

Polymerase chain reaction detection of rearranged immunoglobulin heavy chain DNA in plasma samples is useful in the diagnosis of B-cell lymphoma

Using DNA extracted from plasma samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients, we attempted to detect the monoclonal rearrangement of immunoglobulin heavy chain gene by amplifying complementarity-determining region 3 (CDR3) by semi-nested polymerase chain reaction (PCR) method (plasma PCR). In 19 of 37 (51%) cases, clonal DNA was detected. With the same PCR method using DNA extracted from peripheral blood mononuclear cells, clonal DNA was detected in 8 of the 37 cases (22%). These 8 were in advanced stages with bone marrow (BM) invasion mostly. On the other hand, the 19 positive cases by plasma PCR included those in early stages without BM invasion or with normal soluble interleukin-2 receptor (sIL-2R) and lactate dehydrogenase (LDH) values. In 15 healthy volunteers, plasma PCR showed no clonal DNA. In cases in which tumor biopsy was difficult to perform, plasma PCR was helpful for determining whether or not the tumor was B-NHL. Plasma PCR is simple and has high specificity, although its sensitivity is insufficient.     

The implication of follicular lymphoma patients receiving allogeneic stem cell transplantation from donors carrying t(14;18)-positive cells

We performed real-time quantitative polymerase chain reaction (RQ-PCR) in peripheral blood (PB) and/or bone marrow (BM) samples collected pre- and post transplant from 23 recipient-donor pairs receiving allogeneic stem cell transplantation (allo-SCT) for follicular lymphoma (FL). Of 23 donors, 11 had a PB and/or BM sample positive for t(14;18) (BCL2/IGH fusion) at low levels (相似文献   

Quantitative assessment of disease involvement by follicular lymphoma using real-time polymerase chain reaction measurement of t(14;18)-carrying cells

Chromosomal translocation t(14:18)(q32;q21) is one of the most common karyotypic abnormalities in non-Hodgkin's lymphomas. It occurs in more than 85% of follicular lymphoma (FL) cases. Real-time polymerase chain reaction (Q-Rt-PCR) analysis using double-labeled fluorogenic probes is a new tool in the detection and quantification of t(14;18)-carrying cells. We analyzed 239 specimens with Q-Rt-PCR to detect and quantify t(14;18)-carrying cells. To investigate the clinical usefulness of the quantitative assessment, we analyzed the clinical correlation with 92 FL patients of varying clinical status. Of 59 previously untreated patients, patients with stage IV disease had significantly higher quantities of t(14;18)-carrying cells measurable in the bone marrow or the peripheral blood than patients in clinical stages I to III (P = .003 and .043, respectively). Moreover, of the 33 posttherapy patients. the patients in complete remission appeared to have lower detectable levels of t(14;18)-carrying cells than patients in partial remission or with recurrent disease. Q-Rt-PCR permits a sensitive and quantitative assessment of the extent of disease involvement in patients with t(14;18)-carrying FL. The technique has the potential to be a useful tool in the diagnosis of FL, disease assessment, and prognosticating patients' clinical outcomes.     

The t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile

Recently we have identified subgroups of de novo primary diffuse large B-cell lymphoma (DLBCL) based on complementary DNA microarray-generated gene expression profiles. To correlate the gene expression profiles with cytogenetic abnormalities in these DLBCLs, we examined the occurrence of the t(14;18)(q32;q21) in the 2 distinctive subgroups of DLBCL: one with the germinal center B-cell gene expression signature and the other with the activated B cell-like gene expression signature. The t(14;18) was detected in 7 of 35 cases (20%). All 7 t(14;18)-positive cases had a germinal center B-cell gene expression profile, representing 35% of the cases in this subgroup, and 6 of these 7 cases had very similar gene expression profiles. The expression of bcl-2 and bcl-6 proteins was not significantly different between the t(14;18)-positive and -negative cases, whereas CD10 was detected only in the group with the germinal center B-cell expression profile, and CD10 was most frequently expressed in the t(14;18)-positive cases. This study supports the validity of subdividing DLBCL into 2 major subgroups by gene expression profiling, with the t(14;18) being an important event in the pathogenesis of a subset of DLBCL arising from germinal center B cells. CD10 protein expression is useful in identifying cases of DLBCL with a germinal center B-cell gene expression profile and is often expressed in cases with the t(14;18).     

Low levels of monoclonal small B cells in the bone marrow of patients with diffuse large B-cell lymphoma of activated B-cell type but not of germinal center B-cell type

Background

Multiparameter flow cytometry allows the detection of minor monoclonal B-cell populations. Using this technique combined with morphology, we were struck by the presence of minor populations of small monoclonal B cells in bone marrows of patients with diffuse large B-cell lymphoma in routine diagnostic samples and performed a systematic retrospective study.

Design and Methods

Bone marrows of 165 patients with primary diffuse large B-cell lymphoma without histological evidence of concurrent non-Hodgkin’s lymphoma were studied by routine microscopy of trephines and smears, immunohistochemistry and multiparameter flow cytometry.

Results

Diffuse large B-cell lymphoma infiltration in marrows was documented in 11 of 165 patients. Morphological examination consistently revealed a higher tumor load than evidenced by flow cytometry. Of interest, only 3 of 119 patients with diffuse large B-cell lymphoma not otherwise specified, the largest subtype, showed marrow infiltration. By contrast, flow cytometry revealed a minor monoclonal B-cell population in 24 of 165 patients, none of whom showed diffuse large B-cell lymphoma infiltration by morphology. Of interest, morphological examination revealed the presence of small B cells in the marrows of those patients. Moreover, 11 of 39 (28.2%) of patients with diffuse large B-cell lymphoma not otherwise specified of ABC subtype and only 3 of 80 (3.7%) with the GCB subtype showed these monoclonal small B cells (P=0.0002). In addition 4 of 8 (50%), 4 of 15 (26.7%) and 2 of 3 (66.7%) patients with primary testicular, primary central nervous system and leg-type diffuse large B-cell lymphoma, respectively, showed monoclonal small B cells.

Conclusions

Bone marrow infiltration with diffuse large B-cell lymphoma in patients with diffuse large B-cell lymphoma not otherwise specified is rare at diagnosis. By contrast, a high number of diffuse large B-cell lymphoma not otherwise specified of the ABC subtype but not of GCB subtype is associated with monoclonal small B cells in the marrow. Whether these monoclonal small B cells are precursors of diffuse large B-cell lymphoma of the ABC type or arise in a common background that favors clonal B-cell expansion remains to be demonstrated.     

Interim 18-FDG-PET/CT failed to predict the outcome in diffuse large B-cell lymphoma patients treated at the diagnosis with rituximab-CHOP

Role of interim-PET (I-PET) in diffuse large B-cell Lymphoma (DLBCL) is controversial. To determine predictive value of I-PET on progression-free survival (PFS), we enrolled 88 first-line DLBCL patients treated with 6-8 R-CHOP courses regardless of I-PET. PET/CT were performed at diagnosis, after 2 to 4 courses and at the end of therapy with central reviewing according to visual dichotomous criteria. Results are as follows: I-PET, 72% negative, 28% positive; final-PET (F-PET), 88% negative, 12% positive; clinical complete response 90%. Concordance between clinical response and F-PET negativity was 97% because of 2 false positive. With a median follow-up of 26.2 months, 2-year overall survival and PFS were 91% and 77%, respectively. Two-year PFS for I-PET and F-PET negative versus positive were as follows: I-PET 85% versus 72% (P = .0475); F-PET 83% versus 64% (P < .001). Because of a small number of events, 2 independent bivariate Cox models were tested for PFS. In model 1, F-PET contradicted I-PET (hazard ratio [HR] = 5.03, P = .015 vs 1.27, P = 691); in model 2, F-PET (HR = 4.54) and International propnostic Index score (HR = 5.36, P = .001) remained independent prognostic factors. In conclusion, positive I-PET is not predictive of a worse outcome in DLBCL; larger prospective studies and harmonization of I-PET reading criteria are needed.     

Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma.

We report the results of high-dose chemoradiotherapy and anti-B-cell monoclonal antibody-purged autologous bone marrow transplantation (ABMT) in patients with relapsed indolent follicular lymphoma. Between March 1985 and May 1995, 153 patients underwent ABMT using a uniform ablative regimen with cyclophosphamide and total body irradiation and bone marrow (BM) purging. All patients received multiple chemotherapy regimens before ABMT. At BM harvest, only 30% of patients were in complete remission, and overt BM infiltration was present in 47%. The disease-free survival (DFS) and overall survival (OS) are estimated to be 42% and 66% at 8 years, respectively. Patients whose BM was negative by polymerase chain reaction (PCR) for bcl2/IgH rearrangement after purging experienced longer freedom from recurrence than those whose BM remained PCR positive (P <.0001). Continued PCR negativity in follow-up BM samples was also strongly predictive of continued complete remission (CR). The 12-year survival from diagnosis for these 153 patients is 69%. Considering that the median survival from diagnosis and first recurrence of patients with advanced follicular lymphoma are 8 and 5 years, respectively, our results provide evidence that myeloablative therapy and ABMT may prolong overall survival.     

Role of FDG-PET/CT in detecting lymphomatous bone marrow involvement in patients with newly diagnosed diffuse large B-cell lymphoma

To evaluate the role of FDG-PET/CT in detecting bone marrow (BM) involvement, pre-treatment bilateral bone marrow biopsies (BMBs) and FDG-PET/CT scans of 89 patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-CHOP were reviewed and analyzed. Fourteen patients (15.7%) had lymphomatous involvement based on BMB (BMB+), and 17 patients (19.1%) had the possibility of BM involvement on FDG-PET/CT (FDG-PET/CT+). Seventy-two patients (80.8%) had concordant results between BMB and FDG-PET/CT (seven patients were positive for both, and 65 patients were negative for both), but 17 patients (19.2%) had a discordant interpretation (seven patients were BMB+ and FDG-PET/CT-, and ten were BMB- and FDG-PET/CT+). Although BMB+ patients had an inferior 2-year EFS (37.0% vs. 79.8%, p?相似文献   

Detection of t(14;18) in British follicular lymphoma using cytogenetics,Southern blotting and the polymerase chain reaction

Summary. Cytogenetics, Southern blotting and PCR were used to detect t(14;18) in 72 British patients with follicular lymphoma. The overall incidence of the translocation was 76%. Cytogenetics was the most successful technique, but 10–30% of translocations detected karyotypically were missed by molecular methods, presumably due to breakpoints falling outside the range of probes and primers used here. Reliance on molecular detection alone may considerably underestimate the incidence of t(14;18) and it is therefore essential to use the most comprehensive range of probes and primers available.     

Clinical characteristics and prognostic analysis of Chinese patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma in adults. As it is a highly heterogenous disease, many studies have focused on finding useful prognostic factors to help guide therapy. In this report, we examine several biological markers in 83 patients with DLBCL enrolled in our hospital, including cell origin, serum lactate dehydrogenase (LDH) levels, and international prognostic index (IPI), in order to find the best combination of prognostic factors. We also examined whether DLBCL has a significant geographic difference, since several studies have suggested that the prevalence and potential etiological factors of lymphomas in China may be different from those in other countries. Our results demonstrate that: (1) patients in China have higher extranodal tissue involvement and different extranodal organ distribution than patients reported from other countries; (2) Chinese patients have higher rates of germinal center (GC) cell origin; and (3) among nine prognostic variables, lower IPI scores, GC cell origin determined by immunohistochemical staining, and no more than 1.5 times of normal levels of LDH are statistically significant good prognostic factors in Chinese patients with DLBCL, whereas age at the time of diagnosis, clinical stage, β₂-microglobulin levels, extranodal tissue involvement, and expression levels of Bcl-6 protein were not useful in determining prognosis.