Carsten Hirt

Treatment Results in Localized Primary Gastric Lymphoma: Data of Patients Registered Within the German Multicenter Study (GIT NHL 02/96)

PURPOSE In the prospective study 02/96 on primary GI lymphoma, we have collected data on histology, clinical features, and treatment results. In particular, in stages I and II localized primary gastric lymphoma (PGL), our objectives were to reduce treatment intensity and to confirm our hypothesis from study 01/92, which maintained that an organ-preserving approach is not inferior to primary surgery. PATIENTS AND METHODS Patients receiving radiotherapy and/or chemotherapy were stratified for histologic grade, stage, and whether surgery had been carried out or not (as decided by each participating center). Patients with aggressive PGL received six cycles of CHOP-14 (cyclophosphamide, doxorubicin, vincristine, and prednisone) followed by involved-field radiotherapy (40 Gy). Patients with indolent PGL (including patients experiencing treatment failure with antibiotic therapy for Helicobacter pylori) were treated with extended-field radiotherapy. The volume depended on stage. The irradiation dose was 30 Gy, followed by a boost of 10 Gy (the latter omitted after complete resection) to the tumor region. RESULTS Seven hundred forty-seven patients were accrued. Of these patients, 393 with localized PGL were treated with radiotherapy and/or chemotherapy only or additional surgery between December 1996 and December 2003. The survival rate at 42 months for patients treated with surgery was 86% compared with 91.0% for patients without surgery. CONCLUSION In this nonrandomized study (02/96), we reproduced the previous results of study 01/92 showing no disadvantage for an organ-preserving treatment. Therefore, primary stomach resection should be questioned.

BCL-2/JH rearrangements in circulating B cells of healthy blood donors and patients with nonmalignant diseases.

PURPOSE To answer the question whether t(14;18)-positive cells can be detected by polymerase chain reaction (PCR) in the peripheral blood of healthy blood donors and patients with nonmalignant diseases. PATIENTS AND METHODS Peripheral-blood mononuclear cells (PBMNC) from healthy donors (n = 36) and patients with nonmalignant diseases (n = 21) were examined by two-step PCR for the detection of t(14;18)-positive cells with a breakpoint within the major breakpoint region (MBR). Approximate numbers of t(14;18)-positive cells were determined using limiting dilution assays, as well as the stochastic multiple-tube approach. RESULTS We were able to detect t(14;18)-positive cells in PBMNC of approximately 50% of healthy donors and patients with nonmalignant diseases if DNA amounts up to 10 microg were tested. Compared with 17 t(14;18)-positive patients being in complete remission after radiotherapy for low-stage malignant follicular lymphoma, the majority of 26 healthy donors were found to have significantly lower numbers of t(14;18)-positive cells circulating in the peripheral blood. In the case of six healthy donors, more than one t(14;18) DNA fragment based on size and nucleotide sequence analysis was detected. In one healthy individual, four different t(14;18)-positive cell clones were found in nine samples found over 5 years. CONCLUSION The occurrence of the t(14;18) translocation is not restricted to follicular lymphoma cells. In healthy donors, long-lived t(14;18)-positive cells can be detected by PCR if the sensitivity is high enough. Based on nucleotide sequence analysis, the t(14;18) DNA fragments detected in healthy donors cannot be distinguished from those found in follicular lymphomas.

Chromosomal translocation t(14;18) in healthy individuals.

The t(14;18)-translocation can frequently detected in the peripheral blood and tissue samples from healthy individuals. If the sensitivity of the assay used for the detection is high enough in almost all healthy individuals one or multiple cell clones carrying the t(14;18)-translocation can be found with a frequency of 1-100 rearranged cells in 10(6) normal cells. The frequency of these cells seems to be increased with age and smoking habits measured in pack years. The prevalence in Asian (Japanese) individuals appears to be lower than in Caucasians. It has been postulated that this translocation is a primary event for the subsequent development of a follicular lymphoma.

Prevalence and frequency of circulating t(14;18)-MBR translocation carrying cells in healthy individuals

The t(14;18) translocation is a common genetic aberration that can be seen as an early step in pathogenesis of follicular lymphoma (FL). The significance of low level circulating t(14;18)‐positive cells in healthy individuals as clonal lymphoma precursors or indicators of risk is still unclear. We determined the age dependent prevalence and frequency of BCL2/IgH rearrangements in 715 healthy individuals ranging from newborns to octo‐ and nonagenarians. These results were compared with number of circulating t(14;18)‐positive cells in 108 FL patients at initial presentation. The overall prevalence of BCL2/IgH junctions in this large sample was 46% (327/715). However, there was a striking dependence upon age. Specifically, among individuals up to 10 years old, none had detectable circulating t(14;18)‐positive cells. In the age groups representing 10–50 years old, we found a steady elevation in the prevalence of BCL2/IgH junctions up to a prevalence of 66%. Further increases of the prevalence in individuals older than 50 years were not seen. The mean frequency of BCL2/IgH junctions in healthy individuals ≥40 years (18–26 × 10−6) was significantly higher than in younger subjects (7–9 × 10−6). Four percent (31/715) of individuals carried more than one t(14;18)‐positive cell per 25,000 peripheral blood mononuclear cells (PBMNCs). In comparison, 108 stage III/IV FL patients had a median number of circulating t(14;18)‐positive malignant FL cells of about 9200/1 million PBMNCs (range 7–1,000,000). These findings will further improve the understanding of the relevance of t(14;18)‐positive cells in healthy individuals as a risk marker toward the development into lymphoma precursors.

AID-deficient Bcl-xL transgenic mice develop delayed atypical plasma cell tumors with unusual Ig/Myc chromosomal rearrangements

Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) class switch recombination and somatic hypermutation, and has also been implicated in translocations between Ig switch regions and c-Myc in plasma cell tumors in mice. We asked if AID is required for accelerated tumor development in pristane-treated Bcl-xL transgenic BALB/c mice deficient in AID (pBxAicda−/−). pBxAicda −/− mice developed tumors with a lower frequency (24 vs. 62%) and a longer mean latency (108 vs. 36 d) than AID-sufficient mice. The tumors appeared in oil granuloma tissue and did not form ascites. By interphase fluorescence in situ hybridization, six out of nine pBxAicda −/− primary tumors had T(12;15) and one had T(6;15) chromosomal translocations. Two tumors were transplantable and established as stable cell lines. Molecular and cytogenetic analyses showed that one had an unusual unbalanced T(12;15) translocation, with IgH Cμ and Pvt-1 oriented head to tail at the breakpoint, resulting in an elevated expression of c-Myc. In contrast, the second was T(12;15) negative, but had an elevated N-Myc expression caused by a paracentric inversion of chromosome 12. Thus, novel mechanisms juxtapose Ig and Myc-family genes in AID-deficient plasma cell tumors.

Distribution of t(14;18)-positive, putative lymphoma precursor cells among B-cell subsets in healthy individuals.

The t(14;18)(q32;q21) is the characteristic chromosomal translocation of follicular lymphoma (FL). Highly sensitive polymerase chain reaction (PCR) techniques can also detect t(14;18)‐sequences in the blood and lymphoid tissues of healthy individuals (HI). The aim of this study was to determine the immunophenotypic markers of t(14;18)‐positive cells in HI and to relate these features to lymphocyte maturation. B cells from 10 subjects with t(14;18)‐positive and three subjects with t(14;18)‐negative peripheral blood mononuclear cells (PBMC) were fluorescence‐activated cell sorted for antigen‐naïve (CD27−), immunoglobulin M (IgM) memory (IgM+CD27+) and switched memory (IgM− CD27+) cells. t(14;18)‐recombinations were detected by quantitative PCR. Among PBMC‐positive subjects, t(14;18)‐frequency was significantly higher in IgM memory (median: 380/106) than in antigen‐naïve (median: 16/106) or switched memory (median: 5/106) B cells. All PBMC‐negative subjects nevertheless had detectable t(14;18) in sorted B cells; levels were lower than in PBMC‐positive subjects, but had the same relative predominance. These results suggest that t(14;18) is generated during early B‐cell development in the bone marrow and that affected cells may mature and expand in germinal centres. t(14;18)‐frequency was highest in IgM memory cells, a B‐cell subset that shares immunophenotypic similarities with FL. The significance of these cells as lymphoma precursors or indicators of lymphoma risk remains to be established.

The cell fate determinant Llgl1 influences HSC fitness and prognosis in AML.

Inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.

Rapid and sustained clearance of circulating lymphoma cells after chemotherapy plus rituximab: clinical significance of quantitative t(14;18) PCR monitoring in advanced stage follicular lymphoma patients†

This study of first‐line treatment in advanced‐stage follicular lymphoma patients analysed the effects of MCP (mitoxantrone, chlorambucil and prednisolone) chemotherapy alone or in combination with rituximab (R‐MCP) on circulating lymphoma cells (CLC) and assessed the prognostic value of a quantitative monitoring of CLC. CLC numbers were determined by quantitative polymerase chain reaction (PCR) for the t(14;18)‐translocation or by allele‐specific PCR for rearranged immunoglobulin heavy chain genes. We analysed blood samples from 43 patients treated in a randomized trial comparing eight cycles of MCP versus R‐MCP. Clearance of CLC at the end of therapy was achieved in 21/25 patients (84%) treated with R‐MCP compared with 0/18 after MCP alone (P < 0·0001). A ≥2 log CLC reduction was associated with a favourable clinical response (P = 0·0004) and prolonged event‐free survival (P = 0·02). In R‐MCP patients, stable CLC numbers or consistently PCR‐negative blood samples were associated with a continuing clinical remission whereas in two patients a relapse was preceded by a ≥2 log CLC increase. This study demonstrated that R‐MCP led to a rapid and sustained eradication of CLC and a ≥2 log CLC reduction was associated with a superior quality and duration of the clinical response.

T(14;18) Translocations and Risk of Follicular Lymphoma

The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and a frequent abnormality in other types of non-Hodgkin lymphoma (NHL). In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, the biological significance of which is beginning to be explored. Translocation prevalence and frequency are potential risk factors for developing NHL. Here, we review the published data and describe recent and ongoing work on this promising biomarker. We have a series of studies in four major areas: 1) t(14;18) prevalence and frequency in healthy individuals; 2) maturation of translocation-harboring cells; 3) effect of rituximab treatment on t(14;18) carriage; and 4) predictive and clonotypic relationship between t(14;18) and follicular lymphoma or other NHL. Further studies are warranted to increase understanding of this crucial molecular event in the development of hematopoietic malignancies. Potential applications include determination of elevated risk for lymphoma, early detection of disease, and identification of molecular targets for preventive interventions.

High-dose methotrexate-based immuno-chemotherapy for elderly primary CNS lymphoma patients (PRIMAIN study)

To investigate immuno-chemotherapy for elderly immuno-competent patients (⩾65 years) with newly diagnosed primary central nervous system lymphoma, we conducted a multicentre single-arm trial. One cycle consisted of rituximab (375 mg/m2, days 1, 15, 29), high-dose methotrexate (3 g/m2 days 2, 16, 30), procarbazine (60 mg/m2 days 2–11) and lomustine (110 mg/m2, day 2)—R-MPL protocol. Owing to infectious complications, we omitted lomustine during the study and consecutive patients were treated with the R-MP protocol. Three cycles were scheduled and repeated on day 43. Subsequently, patients commenced 4 weekly maintenance treatment with procarbazine (100 mg for 5 days). Primary end point was complete remission (CR) after 3 cycles. We included 107 patients (69 treated with R-MPL and 38 with R-MP). In all, 38/107 patients achieved CR (35.5%) and 15 (14.0%) achieved partial remission. R-MP was associated with a lower CR rate (31.6%) compared with R-MPL (37.7%), but respective 2-year progression-free survival (All 37.3%; R-MP 34.9%; R-MPL 38.8%) and overall survival (All 47.0%; R-MP 47.7%; R-MPL 46.0%) rates were similar. R-MP was associated with less ⩾grade 3 toxicities compared with R-MPL (71.1% vs 87.0%). R-MP is more feasible while still associated with similar efficacy compared with R-MPL and warrants further improvement in future studies.