Background: Immunoglobulin heavy chain (IgH) gene rearrangement has been known to be a useful marker for determining the clonality as well as detecting minimal residual disease in B cell malignancies. This study was performed to establish single polymerase chain reaction (PCR) methods for the detection of IgH gene rearrangements in formalin-fixed, paraffin-embedded tissue of patients with B cell lymphoma and determine the type of JH segments used. Methods: We obtained formalin-fixed, paraffin-embedded tissue sections of 44 patients diagnosed with B cell lymphoma at Ajou University Hospital from January 2005 to January 2007 and reviewed medical records retrospectively. After the extraction of DNA, PCR was performed using VH3 and JHPST primers to detect the third complementarity determining region (CDR3) gene of IgH. Sequence analysis of the PCR products was also done in 23 patients. Results: The CDR3 gene rearrangements were detected in 26 (59%) out of 44 patients with B cell lymphoma. Sequence analysis of the amplified CDR3 gene was successful in 16 (70%) of 23 patients. JH3, JH4, JH5, and JH6 segments were used for CDR3 gene rearrangements in 3 (25%), 4 (33%), 1 (8%), and 4 (33%) patients with diffuse large B cell lymphoma, respectively. Conclusion: Although there are some limitations due to a low sensitivity less than 60%, single PCR using consensus primers could be an effective tool for the detection of CDR3 gene rearrangements in routine laboratory settings. Furthermore, sequence analysis of the CDR3 PCR products will provide basic information necessary for further studies.

The fusion transcript AML1/ETO was detected in the bone marrow of two t(8;21)-negative acute myelogenous leukemia (AML) patients by means of reverse transcription-polymerase chain reaction. This fusion transcript is identical to the one transcribed from the t(8;21) translocation base, as deduced from (a) the size and restriction pattern of the amplified DNA fragment and (b) the DNA sequence analysis of the fusion junction. We also showed that the ETO gene is highly expressed in these patients, much as it is in the t(8;21)-positive AML. Southern blot analysis showed rearrangement of the AML1 gene in one of the patients. Together, our results demonstrate that there is a masked t(8;21) translocation in AML that is not detectable by cytogenetic analysis but is able to transcribe an AML1/ETO fusion transcript similar to that transcribed in t(8;21)-positive AML-M2 patients.

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34(+) CML cells using a strategy that combines a brief exposure to imatinib (0.5-1.0 microM for 72 h) and then mafosfamide (30-90 microg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colony-stimulating factor and thrombopoietin). Treatment with 1.0 microM imatinib, 60 microg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL(+) cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients.

PURPOSE:Advanced-stage follicular lymphoma is considered incurable. The pace of improvements in treatment has been slow. This article analyzes five sequential cohorts of patients with stage IV follicular lymphoma treated between 1972 and 2002.METHODS:Five consecutive studies (two were randomized trials) involving 580 patients were analyzed for overall survival (OS), failure-free survival (FFS), and survival after first relapse. A proportional hazards analysis, and subset analyses using the follicular lymphoma international prognostic index (FLIPI) score were performed. Treatment regimens included: cyclophosphamide, doxorubicin, vincristine, prednisone, bleomycin (CHOP-Bleo); CHOP-Bleo followed by interferon alfa (IFN-alpha); a rotation of three regimens (alternating triple therapy), followed by IFN-alpha; fludarabine, mitoxantrone, dexamethasone (FND) followed by IFN-alpha; and FND plus delayed versus concurrent rituximab followed by IFN-alpha.RESULTS:Improvements in 5-year OS (from 64% to 95%) and FFS (from 29% to 60%) indicate steady progress, perhaps partly due to more effective salvage therapies, but the FFS data also indicate improved front-line therapies; these observations held true after controlling for differences in prognostic factors among the cohorts. The FLIPI model adds rigor to and facilitates comparisons among the different cohorts. An unexpected finding in this study was a trend toward an apparent FFS plateau.CONCLUSION:Evolving therapy, including the incorporation of biologic agents, has led to stepwise significant outcome improvements for patients with advanced-stage follicular lymphoma. The apparent plateau in the FFS curve, starting approximately 8 to 10 years from the beginning of treatment, raises the issue of the potential curability of these patients.

DLI is commonly provided for mixed chimerism post NMT, even in the absence of measurable disease. This is with a high risk for graft-versus-host disease (GVHD) and a major cause of mortality and morbidity after NMT. We hypothesized that this practice may not be routinely needed in pts with indolent diseases, such as FL, and who continue to have stable mixed chimera (SMC) after NMT. We defined SMC as presence 50% to 99% donor cells by PCR analysis, and without any significant decrease of > 20% on two consecutive analysis. We treated prospectively 47 pts with relapsed FL with a NMT after conditioning with fludarabine, cyclophosphamide and high-dose rituximab as previously described (Blood 89:3595, 2001). ATG was added for 2 pts receiving unrelated transplants. Tacrolimus and methotrexate were used for GVHD prophylaxis. All pts received a non-manipulated graft, from peripheral blood (45 pts) or marrow (2 unrelated donors). At the time the first analysis was undertaken post NMT (median 30 days), 13 of 18 pts (72%) in clinical complete remission (CR) and 24 of 29 pts (83%) who had evidence of active disease at study entry had mixed chimera (P = 0.2). The % of donor cells were 93% and 75%, respectively. When the subsequent PCR analysis to assess engraftment was undertaken (median time, 89 days post NMT), median % donor cells were 100% and 95%, respectively, for the pts who were in CR or had active disease at study entry. All pts achieved CR post NST. Median time to achieve CR for pts who had active disease at study entry was 5.5 months after transplant. Twelve pts in CR continue to have SMC at the time of their last PCR analysis, undertaken at a median of 12 months (range, 6 to 55 months) post NMT. Nineteen pts were tested for the amplification of bcl-2 rearrangement from the marrow by PCR. They all had evidence of clonal disease prior study entry. All converted to PCR-negativity. Sixteen of these 19 pts had mixed chimera when they were first tested to be PCR (−) at a median time of 3 mos (range, 1–6 months) post NMT. At a median time follow-up time of 34 months, overall survival and disease-free survival of all 47 pts at 3-year were 88 and 85%, respectively. Only one relapse occurred. This occurred in a pt who had 60% donor at 3 months post NMT; it then decreased to 42% at 9 months. This was followed by graft failure and relapse. DLI was provided to only one pt with decreasing chimerism. This resulted to conversion to full donor after the infusion of to 1 x 105 CD3+ cells/kg, from the HLA-identical sibling donor. The incidence of acute grade II–IV and chronic (extensive and limited) GVHD in the study was 17% and 51%, respectively. These data suggest that DLI may not be needed for SMC after non-T cell depleted NMT for pts with FL who do not have evidence of progression. This strategy resulted in lower than expected acute and chronic GVHD and improved survival.

We have used the combination of fludarabine/cyclophosphamide/rituximab (FCR) as a conditioning regimen for NMT in patients (pts) with relapsed NHL (Blood989:3595, 2001). Seventy-eight consecutive pts were treated. Median age was 53 yrs (range, 21–68). Median # of prior chemoregimens was 3. Twenty-one (27%) had failed a prior autologous. Histologies included follicular (FL)=47 pts (60%), diffuse large cell (DLCL)=16 pts (21%) and mantle cell (MCL)=15 pts (19%). Thirty-nine (50%) were in partial remission (PR), five (6%) had stable or progressive disease (SPD) and 34 (44%) were in complete remission (CR) at study entry. Sixty-nine (88%) had a matched sibling, 6 a matched unrelated, and three a mismatched sibling donor. Ten pts had either PET or Gallium-avid disease at transplant: six were in clinical PR and 4 were in SPD. Peripheral blood was the source of graft in 71 pts (91%). Tacrolimus/Methotrexate were used for GVHD prophylaxis. Median time for ANC >500 was 10 days. Sixty pts did not require any platelet transfusion. All but 2 pts who had SPD, converted to CR post transplant. All pts engrafted donor cells. Six had a secondary graft failure (GF), two of which occurred in the setting of disease progression. With a median follow-up time of 34 months (mos) (range, 3 to 70 mos), only 6 pts (8%) relapsed: two pts were in SPD/PET or Gallium(+), 3 were in PR/PET or Gallium(−) and one was in CR at transplant. None of the 6 pts who were PR/PET or Gallium (+) at study entry relapsed. Overall the risk of relapse in PET/Gallium (+) or (−) disease was not significant ( P =0.15). Five pts received donor lymphocyte infusion (DLI) for disease progression: one achieved CR; the 4 other pts (two were in GF) did not respond. DLI was also given successfully to two other pts because of cytopenia related to a viral infection (1), and because of decreasing chimerism (1). Overall survival (OS) for all pts was 88%, 82% and 74% at 1,2 and 3-yr, respectively. By univariate analysis, OS was 95% at 3-yr for those who had failed a prior autologous transplant ( P = 0.04). Patients with FL histology had also a better 3-yr survival than DLCL [88 % vs 51% ( P = 0.008)]; the difference with MCL (65% at 3-yr) was not statistically significant ( P = 0.2). Non-relapse related mortality was 11%, 13%, and 19% at 1, 2 and 3-yr, respectively. The incidence of acute II-IV GVHD was 17%. Patients with FL disease had lower incidence of acute II-IV and III-IV GVHD than DLCL [11% vs 31%, P = 0.04; and 2% vs 25%, P = 0.02; respectively] which may have contributed to the higher OS rate in FL. Both FL and DLCL pts had similar age and donor type distribution. Two pts developed chronic GVHD post DLI. The incidence of chronic extensive and limited GVHD was 51% and was not statistically different between various histologies. We conclude that NMT with FCR for NHL is an effective treatment even in pts who failed a prior autologous transplantation and is associated with a low incidence of acute GVHD. The lower than expected relapse rate observed may be indicative that NMT could be curative for these diseases.

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.

PURPOSE Existing data suggest that conventional C(H)OP (cyclophosphamide, doxorubin, vincristine, prednisone) regimen may not be intensive enough to achieve molecular response, as measured by polymerase chain reaction (PCR) evidence of translocation (14;18)(q32;21)for follicular lymphoma. This study was undertaken to study the molecular response rate of follicular lymphoma to C(H)OP-based therapy and to achieve prognastic factors for molecular response.PATIENTS AND METHODS Twenty patients with pretreatment PCR evidence of t(14;18)(q32;q21) and at least one posttreatment PCR analysis after the initiation of the treatment with C(H)OP with or without radiation therapy constituted the basis for this analysis. The random effects logistic model was used to analyze the data. the following factors were investigated for their relationship to molecular response: gender, age, beta(2)-microglobulin, use of radiation therapy, Ann Arbor, and International Prognastic Index for malignant lymphoma.RESULTS Median follow-up was 56 months (range, 23-153 months). A total of 135 PCR results were available, 33 from bone marrow and 102 from peripheral blood. Overall, there was a clear and steady decreasing trend toward loss of PCR positivity with increasing time after treatment. By univariate analysis analysis, stage greater than or equal to3, stage = 4, International Prognastic Index greater than or equal to2, and no radiation therapy were adverse factors for molecular response. On multivariate analysis, Ann Arbor stage IV and no radiation therapy were independant risk factors for PCR positivity, both for the peripheral blood data analyzed alone and for all data combined.DISCUSSION It is possible to achieve molecular response with C(H)OP with or without radiation therapy in patients with follicular lymphoma. Response rate depends on the Ann Arbor stage and radiation therapy.

During the last three decades, evidence firmly establishing the close association of nonrandom karyotypic abnormalities with particular types of human cancers has been increasing. The importance of these observations is underscored by the revelation that several oncogenes are mapped to the regions known to be involved in tumor-specific chromosomal aberrations. For lymphoid malignancies in particular, the first consistent karyotypic abnormality was observed in Burkitt’s lymphoma (1). Approximately 75% of Burkitt’s lymphomas display the characteristic t(8;14) translocation and the remaining 25% show either the t(2;8) and the t(8;22). The common feature of these translocations is the involvement of chromosome region 8q24, where the c-myc oncogene resides. Breakpoints on the counterpart chromosomes occur within either the immunoglobulin heavy-chain (IgH) gene locus at 14q32 or one of the immunoglobulin light-chain gene loci at 2p11 or 22g11. Many other cytogenetic aberrations in lymphoid neoplasia appear to follow a similar rule, such as the t(11;14)(q 13;g32) in mantle cell lymphoma and the t(14;18)(q32;q21) in follicular lymphoma.

ObjectiveTo investigate the graft-vs-leukemia effect of allogeneic stem cell transplantation after a nonablative conditioning regimen as treatment for patients with chronic lymphocytic leukemia.Patients and MethodsPatients were eligible to treatment if they were refractory or recurred after a prior response to fludarabine. Seventeen patients were treated. All patients received a preparative regimen of fludarabine (30 mg/m2 daily for 3 days) and intravenous cyclophosphamide (750 mg/m2 daily for 3 days). Ten patients received rituximab in addition to chemotherapy. The median time from diagnosis to transplant was 67 months. Nine of 17 patients had refractory disease.ResultsAll patients had engraftment of donor cells. Eleven (65%) did not require platelet transfusions. Ten patients with persistent disease underwent immunomanipulation to augment GVL effects including immunosuppression withdrawal and donor lymphocyte infusion with or without rituximab treatment. Seven of these 10 patients had a complete response and 2 had a partial response; 8 of these 9 responders had received rituximab with their immunomanipulation process. The final response was complete remission in 12 and partial remission in 4 patients for an overall response rate of 94%. Overall survival was 100% for patients who received the combined chemo-rituximab conditioning regimen, vs 14% for those who received chemotherapy alone (p = 0.03).ConclusionOur results indicate that a pronounced GVL effect occurs after nonmyeloablative allogeneic hematopoietic transplantation for advanced CLL. This activity might be facilitated by rituximab. Prospective controlled trials are needed to define the role of nonablative allogeneic hematopoietic transplantation for treatment of this disease.

BACKGROUNDQuantitative real-time polymerase chain reaction (Q-Rt-PCR) is a new tool in the detection and quantification of the BCR/abl fusion transcripts in chronic myelogenous leukemia (CML). This study investigates its specificity, sensitivity and potential clinical usefulness.PATIENTS AND METHODSParallel analysis of Q-Rt-PCR and the conventional reverse transcription-mediated PCR (RT-PCR) were performed on 567 samples from 481 patients. Treatment response was monitored by Q-Rt-PCR at 6 and 12 months of 61 patients on STI-571 and 103 patients on interferon.RESULTSThe concordance rate between Q-Rt-PCR and RT-PCR was 96.3% (546/567), with 341 positives and 205 negatives. The positive equivalents ranged from 2 x 10(-6) to 1.2 microg of K562 cell RNA. Karyotyping in 372 samples revealed excellent correlation with Q-Rt-PCR measurements (P < 0.001). Setting residual BCR/abl < 0.01 as an early goal of molecular response, we observed that STI-571 induced a better response than interferon: 49% (20 of 41 patients) versus 35% (15 of 62 patients) at 6 months (P = 0.025) and 52% (32 of 61 patients) versus 34% (35 of 103 patients) at 12 months (P = 0.01), respectively.CONCLUSIONSQ-Rt-PCR provides reliable measurements of BCR/abl fusion transcripts. It is potentially useful in assessing molecular residual disease after therapy.

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q2l) chromosomal translocation which juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain locus at 14q32. Quantification of t(14;18) carrying cells in FL patients can be achieved by real-time PCR, a highly sensitive technique for evaluating treatment efficacy and minimal residual disease. Despite the many advantages of real-time technology for this purpose, one disadvantage is that current real-time t(14;18) PCR assays amplify a control gene as a normalizer in a separate reaction. Since each PCR reaction has its own kinetics, separate PCR assays for target and control sequences can potentially result in inaccurate quantification of t(14; 18)-positive cells. In addition, the real-time t(14;18) PCR assays do not determine the size of the amplified fusion sequence, which is helpful for excluding contamination and is commonly used to demonstrate clonal identity between pre- and post-treatment specimens from a patient. To address these limitations, we designed a multiplex real-time PCR protocol that allows amplification of control and target genes in the same reaction and precise size determination of bcl-2/JH fusion sequences by capillary electrophoresis. This multiplex PCR assay is equally sensitive to previous assays, allows more accurate quantification of bcl-2/JH fusion sequences, and is more convenient.

Treatment for patients with stage IV indolent lymphoma ranges from watchful waiting to intensive chemotherapy and stem cell transplantation. In this trial we compared 2 induction regimens followed by 1 year of interferon maintenance therapy. Fludarabine, mitoxantrone (Novantrone), and dexamethasone (FND) were compared with an alternating triple therapy (ATT) regimen (CHOD-Bleo, ESHAP, and NOPP). Maintenance interferon/dexamethasone was given for 1 year in both treatment arms. Endpoints were comparisons of remission rates, survival, failure-free survival (FFS), molecular response rates, and toxicities. One hundred forty-two patients with previously untreated stage IV indolent lymphoma were evaluable (73 on FND; 69 on ATT). The overall response rates were 97% for FND and 97% for ATT (P =.9). The median follow-up is 5.9 years. The 5-year survival rates were 84% with FND and 82% with ATT (P =.9); the 5-year FFS rates were 41% with FND and 50% with ATT (P =.02). In a multivariate analysis, factors predicting for longer FFS were beta(2)-microglobulin less than 3 mg/L (P =.01) and ATT treatment (P =.03). ATT was associated with a substantially higher rate of grade 3-4 toxicities than FND. In conclusion, both regimens were associated with high rates of response and survival. ATT was associated with substantially longer FFS, but it was more toxic than FND.

Summary. The small number of progenitor cells is the major limitation to the use of umbilical cord blood (UCB) for the transplantation of adults. We tested the hypothesis that two units transplanted simultaneously could each contribute to haematopoietic reconstitution. A patient with advanced acute lymphocytic leukaemia received a mismatched, unrelated UCB transplant using units from two donors after conditioning. The recipient achieved a complete remission without graft‐versus‐host disease. Double chimaerism was documented in several leucocyte subpopulations; both units contributed to haematopoiesis until relapse. Triple chimaerism was present from relapse until death due to leukaemia. This approach may potentially improve UCB transplantation outcome for adults lacking a histocompatible donor.

PURPOSE:Aberrant methylation of promoter-associated CpG islands is an epigenetic oncogenic mechanism. The objective of this study was to define the methylation characteristics of patients with acute lymphocytic leukemia (ALL).EXPERIMENTAL DESIGN:Using bisulfite-PCR followed by restriction enzyme digestion (COBRA), we have analyzed the methylation status of 10 promoter-associated CpG islands in 80 untreated adult patients with ALL.RESULTS:Mean methylation density of MDR1, THBS2, MYF3, ER, p15, THBS1, CD10, C-ABL, and p16 was 24.5%, 20.8%, 17.6%, 16.1%, 11.3%, 8.9%, 4.5%, 3.7%, and 1.3% respectively. p73 was methylated in 17 of 80 cases (21.2%). A total of 86.2% of the cases had methylation of at least one gene, and 42.5% of the cases had methylation of three or more genes. MDR1 methylation was inversely correlated with age (P = 0.01). CD10 methylation inversely correlated with CD10 expression (P = 0.0001). Methylation of MDR1 and THBS1 was inversely associated with the presence of the Philadelphia chromosome, whereas C-ABL methylation correlated with the presence of the p210 variant of the Philadelphia chromosome. In univariate analysis, methylation of THBS1 was associated with a favorable outcome (P = 0.02), whereas methylation of p73, p15, and C-ABL was associated with a trend toward worse prognosis.CONCLUSIONS:Aberrant DNA methylation of promoter-associated CpG islands is very common in adult ALL and potentially defines subgroups with distinct clinical and biological characteristics.

This study investigated the use of a nonablative conditioning regimen to decrease toxicity and achieve engraftment of an allogeneic blood stem cell transplant, allowing a graft-versus-malignancy effect to occur. All patients had follicular or small cell lymphocytic lymphoma after relapse from a prior response to conventional chemotherapy. Patients received a preparative regimen of fludarabine (25 mg/m2 given daily for 5 days or 30 mg/m2daily for 3 days) and intravenous cyclophosphamide (1 g/m2given daily for 2 days or 750 mg/m2 daily for 3 days). Nine patients received rituximab in addition to the chemotherapy. Tacrolimus and methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. Twenty patients were studied; their median age was 51 years. Twelve were in complete remission (CR) at transplantation. All patients achieved engraftment of donor cells. The median number of days with severe neutropenia was 6. Only 2 patients required more than one platelet transfusion. The cumulative incidence of acute grade II to IV GVHD was 20%. Only one patient developed acute GVHD of greater than grade II. All patients achieved CR. None have had a relapse of disease, with a median follow-up period of 21 months. The actuarial probability of being alive and in remission at 2 years was 84% (95% confidence interval, 57%-94%). Nonablative chemotherapy with fludarabine/cyclophosphamide followed by allogeneic stem cell transplantation is a promising therapy for indolent lymphoma with minimal toxicity and myelosuppression. Further studies are warranted to compare nonablative allogeneic hematopoietic transplantation with alternative treatment strategies.