Treatment of primary mediastinal large B cell lymphoma with an alternating chemotherapy regimen based on high-dose methotrexate.

Primary mediastinal large B cell lymphomas (MLCL) differ from other diffuse large cell lymphomas, leading to a description as a separate entity in the current World Health Organization classification. Dose intensification improves long-term results, but no standard therapy has been established so far. We investigated the use of a high-dose methotrexate-based alternating chemotherapy regimen (B-ALL protocol of the German ALL study group) followed by consolidative mediastinal radiotherapy first as a single-center trial, then later as a prospective multicenter trial in 44 patients with a median age of 33 years. Response rates exceeded 90% with an overall survival rate of 80% in the single-center group (8.6 years median follow-up) and 82% in the multicenter group (2.5 years follow-up).Short-term toxicity was manageable, but required hospitalization: the rates of grade 3 or 4 toxicity were 20% (for mucositis), 42% (for neutropenia), 29% (for thrombocytopenia), and 9% (for neutropenic fever). No relapse occurred more than 2 years after diagnosis and initiation of treatment, but unfortunately, no patient with overt progression or relapse within these 2 years could be salvaged. Future directions in the treatment of MLCL will not focus on further dose intensification, but rather on the incorporation of (radio)immunotherapy as a therapeutic tool and gene expression profiling as well as positron emission tomography-computed tomography as stratifying tools.

Stem cell mobilization in multiple myeloma patients: do we need an age-adjusted regimen for the elderly?

The upper age limit for autologous progenitor cell transplantation in multiple myeloma patients is increasing continuously. We examined whether this shift in the age of pretreated myeloma patients requires modification of mobilization regimen. We compared retrospectively 21 consecutive progenitor cell mobilizations in 15 pts < 60 years (median age 56, range 37-59) with 33 consecutive mobilizations in 23 pts > 60 years (median age 65, range 60-73) of age. The number of CD34 positive circulating cells before scheduled leukapheresis was a mean of 67,935 cells/mL (SEM +/- 17,614) in the younger population and a mean of 19,069 (SEM +/- 5,396) for older pts (P = 0.0027). In patients >60 years, 13/33 mobilizations (including 2 patients with 2 failing attempts) were not successful (39%), compared to 6/21 mobilizations (29%, including 1 patient with 3 failing attempts) in the younger population. The increased number of progenitor cells in the grafts of younger patients led to a more rapid regeneration of leukocytes and platelets after stem cell infusion. Our data show that stem cell mobilization in older multiple myeloma patients is inferior compared to a younger patient population. There is a trend towards more leukapheresis until the target stem cell dose has been collected, and the decreased number of progenitor cells in the actual graft delays engraftment of leukocytes and platelets. The overall number of unsuccessful mobilization attempts, however, did not differ significantly between both age groups. A special "age-adjusted" increase in the dose of growth factors seems unjustified. Improvements in timing of leukapheresis, growth factor application, and mobilizing chemotherapy regimen as well as the use of alternative cytokines should be investigated for both age groups.

Culture of haematopoietic cells in a 3-D bioreactor made of Al2O3 or apatite foam.

Foams of Al2O3 and apatite ceramics with interconnecting pores were produced using a new technique. The surfaces of the ceramics served as substrates for the culture of human peripheral and bone marrow derived stem cells. Up to 27 days the cells were kept in culture where they proliferated and developed into different morphologies consistent with bone marrow cell lines.

Successful treatment of extracranially metastasized pineal gland germinoma with high-dose methotrexate.

Germinoma of the pineal gland is a rare disease usually confined to the brain which responds well to radiotherapy. Spinal seeding occurs in approximately 4% of cases and distant metastases are extremely rare. We report on a 27-year-old female with an intracranially metastasized pineal gland germinoma, meningeal carcinomatosis and distant bone metastases. Treatment was initiated with intrathecal methotrexate (MTX) and continued with high-dose intravenous MTX. The therapy was very well tolerated apart from reversible hepatic toxicity requiring a dose reduction. The patient was in complete remission after three courses followed by two consolidation cycles; the patient has now been in continuous complete remission for more than 22 months. This is the first report to show that MTX is a potent drug in treating pineal gland germinoma. Long-term side effects of radiotherapy such as reduced mental function or hypopituitarism can probably be avoided. Single-agent high-dose MTX may provide high efficacy with limited adverse effects, especially at a more advanced tumor stage with spinal seeding and extracranial disease.

Flow cytometric CD34+ determination in stem cell transplantation: before or after cryopreservation of grafts?

Various attempts have been made to standardize and improve the reproducibility of flow cytometric determination of CD34+ hematopoietic progenitor cells. It is still not clear, however, whether the quantification of CD34+ cells in a stem cell graft should be done before or after cryopreservation. To address this issue, we investigated 78 unselected and 32 immunomagnetically selected autologous and allogeneic leukapheresis products (LA) before and after cryopreservation using pilot vials. Cell numbers were quantified within a Neubauer chamber, and CD34+ content was determined by flow cytometry; propidium iodide staining was used to exclude dead cells from analysis. Before freezing, the mean viable CD34 cell content in the unselected samples was 1.22% and increased after thawing to a mean of 2.16% of viable cells. Taking into account cell loss and cell death, the overall recovery of viable cells was 64.5%; all CD34+ cells could be recovered. Mean purity in the CD34-selected cell fraction was 85% (48-97) before and 91.3% (67-99) after thawing. The number of viable cells was 86.8% before and 86.1% after freezing with a 93.9% recovery of total cells. This leads to a mean 93.7% (SD +/- 23.1) recovery of viable cells and 100% (SD +/- 22.3) recovery of viable CD34+ cells. There was no significant difference in tolerance to freeze/thaw stress between cells from heavily pretreated autologous patients and healthy allogeneic donors. Our data show that freezing significantly increases the percentage of CD34(+) cells in unmanipulated LA, probably due to the death of granulocytes and mononuclear cells (MNCs). Nevertheless, the overall number of viable CD34+ cells in unselected as well as selected samples remains unchanged. Thus, CD34 data from different laboratories, for example, within multicenter trials, should be comparable independent of the different time points of acquisition.

Chromosomal aberrations characteristic for sAML/sMDS are not detectable by random screening using FISH in peripheral blood-derived grafts used for autologous transplantation.

Acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) is observed in 5 to 10% of patients treated with high-dose chemotherapies followed by autologous stem cell and bone marrow transplantation. Both diseases are frequently associated with monosomy 7 (-7), trisomy 8 (+8), loss of the long arm of chromosome 5 (-5q), and deletions including the TP53-gene region according to del(17)(p13). In this study, we examined whether these chromosomal aberrations are already detectable in blood stem cells from patients who have all been treated with standard chemotherapies prior to peripheral blood stem cell transplantation (PBSCT). Therefore, we screened peripheral blood derived stem cells obtained at the time of stem cell harvest for the presence of -7, +8, -5q, and del(17)(p13) by fluorescence in situ hybridization (FISH). Our series included 40 patients: 4 patients with Hodgkin's disease, 6 patients with non-Hodgkin-lymphoma (NHL), 1 patient with ALL, 4 patients with plasmocytoma, and 25 patients with solid tumors. Peripheral blood mononuclear cells (PBMC) from eight healthy blood donors served as controls. Assuming a hybridization efficiency of >98%, the cut-off level of non diploid cells was determined for each DNA-probe. None of the stem cell preparations exhibited chromosomal damage. Our findings indicate that chromosomal damage is a rare event in stem cell autografts.

Efficacy and safety of simultaneous immunomagnetic CD34+ cell selection and breast cancer cell purging in peripheral blood progenitor cell samples used for hematopoietic rescue after high-dose therapy.

We have established a new simultaneous positive/negative selection procedure using the Baxter Isolex 300i system. We tested its tumor cell (TC) purging efficacy by tumor contamination tests ex vivo and its safety in a group of 17 breast cancer (BC) patients by measuring hematopoietic recovery after high-dose (HD) therapy and autologous stem cell rescue with the selected cells. Tumor contamination tests resulted in a TC depletion of 4.1-6.0 log steps. The CD34+ cell yield in this experimental setting was 38.9-91.5%, and the CD34+ cell purity was 86.0-96.0%. In a group of 17 BC patients (5 high-risk adjuvant, > or = 10 lymph nodes positive, and 12 metastatic), we processed leukapheresis products (LPs) by simultaneous positive/negative selection. In these clinical samples, the mean CD34+ cell yield was 56.2% (range, 14.0-80.1%), and the CD34+ cell purity was 94.5% (range, 69.0-99.8%). Additionally, we screened samples of the patients' LPs before and after the purging procedure for contaminating TC by immunocytochemistry. In 15 of 17 tested cases, TCs were detectable prior to the purging procedure. After the procedure, we could not detect residual TCs in 16 of 17 cases. In one case, we found a highly reduced number of TCs. Furthermore, we evaluated the times for hematopoietic reconstitution in a group of five BC patients in the high-risk adjuvant situation who underwent HD chemotherapy and hematopoietic rescue with positive/negative selected stem cells and compared it with our own data from 10 BC patients who, after identical HD therapy, received only positively selected CD34+ cells and 14 patients who, after identical HD therapy, received autografts purged by incubation with toxic ether lipids (ET-18-OCH3). In all groups, a leukocyte count of >2000 cells/microl was reached at day +10. A platelet count of > 50,000 cells/microl was reached at day +12 in the ET-18-OCH3 group and at day +14 in the other two groups. Furthermore, 12 patients with metastatic disease rescued with positive/negative selected stem cells after HD therapy also showed fast and comparable hematopoietic recovery. The new simultaneous immunomagnetic positive/negative selection using a closed system is effective and safe. Processing LPs leads to a similar CD34+ cell yield, a higher TC depletion compared to standard CD34+ cell selection, and no delay in hematopoietic recovery.

Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells.

We compared UCB mononuclear cells (MNC) with CD34+ selected cells in a serum-free static culture system. Cell number proliferation of MNCs was inferior to CD34+ selected cells. MNCs, however, showed a substantial increase from 0.94% CD34+ cells on day 0 to 5.8% on day 7, whereas in the CD34+ selected samples the CD34+ cell content declined continously from 62.2% on day 0 to 27.7% on day 7. The number of CFU-GM increased during culture of both cell fractions. Here, only the MNCs showed a substantial increase in clonogenicity on day 7 and day 14 to 11.1- and 4.1-fold input, respectively. This expansion of the CD34+ progenitor cell pool in the MNCs fraction was at least in part attributable to T cells, since the physical abrogation of T cells blocked this effect. Refeeding and reseeding of cells on day 7 had stimulating effects especially on the CD34+ cells, where cell number proliferation increased from 16.3-fold without to 58.1-fold on day 14. Also, we could find sporadic chromosomal aberrations in four of 100 metaphases examined after 7-20 days of ex vivo expansion. The significance of this observation needs to be clarified in a larger series.

[Clubbed fingers and arthralgia as a reversible paraneoplastic syndrome (Pierre-Marie-Bamberger syndrome) in non-small-cell bronchial carcinoma]. / Trommelschlegelfinger und Arthralgien als reversibles paraneoplastisches Syndrom (Pierre-Marie-Bamberger-Syndrom) beim nicht-kleinzelligen Bronchialkarzinom.

HISTORY AND CLINICAL FINDINGS: During chemotherapy for a non-small-cell bronchial carcinoma with metastasis to the right femur (previously locally excised), a 34-year-old man suddenly developed severe, lasting joint pain in the ankle, knee, elbow and wrist without signs of increased warmth or swelling of these joints. At the time of diagnosis clubbed fingers had been noted. INVESTIGATIONS: Radiography of the hands showed bilateral periosteal hyperostoses. Computed tomography of the thorax revealed tumor progression. DIAGNOSIS, TREATMENT AND COURSE: The triad of clubbed fingers, periosteal hyperostoses and arthralgia/arthritis with the pulmonary findings established the diagnosis of hypertrophic pulmonary osteoarthritis (HPO; Marie-Bamberger syndrome). Initially there had merely been the signs of the much more frequent incomplete form with only the clubbed fingers, the complete form developing with progression of the disease during chemotherapy, joint pains dominating the symptoms. After right upper lobectomy (primary adenocarcinoma) the joint pains ceased and the finger clubbing regressed. CONCLUSIONS: Only 10% of non-small-cell bronchial carcinomas are associated with HPO. Conversely, such a tumor is found in 90% of HPO of recent onset and should therefore be sought of when searching for the primary tumor. The signs of HPO are reversible if the underlying disease is adequately treated.

Is there a place for 2-CDA in the treatment of B-CLL?

There is no doubt about 2-CDA being a very potent lymphotoxic agent that displays high efficacy in the treatment of CLL. It interferes with the intranuclear machinery of DNA regulation, and causes death to proliferative active, as well as resting lymphocytes. Interruption of crucial pathways that are evident for cell survival translates into high clinical response rates in CLL. CR and PR rates comparable to those reported on fludarabine are achieved in relapsed or refractory CLL. Even though trials on previously untreated CLL are still ongoing, a consistent trend towards durable, high CR rates becomes apparent. The toxicity is comparable to that of fludarabine and consists of infections, as well as thrombocytopenia. Clinical as well as in vitro studies suggest a crossresistance between the two purine analogues, indicating that sequential treatment is not useful. Given these data, although preliminary in case of de novo CLL, 2-CDA has to be recognized as one of the most effective cytostatic drugs currently available for CLL treatment. Large prospective trials (in comparison with fludarabine) will assess the role of 2-CDA as standard treatment. Such trials should also have the aim to substantiate the potential of 2-CDA as induction treatment followed by high-dose consolidation.

Serum levels of soluble CD23, but not soluble CD25, predict disease progression in early stage B-cell chronic lymphocytic leukemia.

Serum levels of the soluble forms of CD23 (sCD23) and CD25 (sCD25) were prospectively analyzed with respect to their prognostic relevance in early stage B-cell chronic lymphocytic leukemia (B-CLL). SCD23 and sCD25 levels were determined in 105 patients with newly diagnosed B-CLL (Binet stage A). In 93 of the patients, these levels were correlated with other already established indicators for risk of disease progression, including the histologic pattern of bone marrow infiltration, lymphocyte doubling time (LDT), and the serum level of thymidine kinase (TK). High serum levels of both sCD23 and of sCD25 were associated with a diffuse bone marrow infiltration, a LDT < or = 12 months, and elevated (>5 U/L) serum TK, respectively. Moreover, examination of the clinical course of 76 untreated patients showed that high levels of sCD23, but not of sCD25, at initial diagnosis were linked with disease progression. Furthermore, in a stepwise Cox regression model, high levels of sCD23 and a short LDT were shown to be strong predictors of progressive disease within the first year of disease presentation. Therefore, it appears to be justified to incorporate sCD23 levels into the risk profile of early stage B-CLL and to take them into account for stratification in risk-adapted treatment strategies.

Ex vivo expansion of human umbilical cord blood does not lead to co-expansion of contaminating maternal mononuclear cells.

One of the main limiting factors for increased use of human umbilical cord blood (UCB) in adult allogeneic transplantation is the small number of progenitor cells that can be collected and infused. Ex vivo expansion of UCB might help to overcome this limitation. Whether an expansion of UCB cells will also lead to co-expansion of contaminating maternal cells, and thus may alter graft characteristics and lead to an increased incidence of GVHD, has not been looked at so far. We initiated cultures with UCB mononuclear cells (MNC) in a standard medium containing stem cell factor (SCF), flt-3L, II-3, IL-6, EPO and G-CSF. To address the question of contaminating maternal cells we performed interphase FISH analysis of the X and Y chromosome simultaneously. Male (XY) cord blood samples were investigated for maternal (XX) cells at day 0 and at several time points during culture. We could not detect maternal cells in any of the nine samples studied when cultures were started at day 0. Culturing did not expand previously undetected maternal cells into a range that could be seen with FISH technology, as all samples remained negative for maternal cells throughout culture periods of 14 days. We then artificially contaminated male UCB with maternal mononuclear cells at concentrations of 5 and 15% at day 0. After 14 days, maternal MNC were still detectable, but the percentage was reduced to 1.7% and 6%, respectively. During culturing of CD34+-selected UCB the content of maternal cells also declined from a mean of 1.6% after contamination to 0.4% on day 7. Taken together we could show that maternal cells co-cultured with UCB do not co-expand and thus do not interfere with ex vivo expansion of UCB for adult allogeneic transplantation.

Peripheral blood progenitor cell mobilization with Dexa-Beam/G-CSF, ether lipid purging, and autologous transplantation after high-dose CBV treatment: a safe and effective regimen in patients with poor risk malignant lymphomas.

High-dose chemotherapy followed by autologous peripheral blood progenitor cell transplantation (PBPCT) is increasingly applied in patients with relapsed, poor risk malignant lymphomas. Different strategies for progenitor cell mobilization using cytoreductive chemotherapy, hematopoietic growth factors, or both have been described. We studied the safety and efficacy of a modified DexaBEAM regimen (dexamethasone, BCNU [carmustine], etoposide, ara-C, melphalan) followed by granulocyte-colony stimulating factor (G-CSF) that was administered in order to minimize any residual disease and to obtain a sufficient amount of progenitor cells in the autografts. Until now, 16 patients at poor risk (8 with Hodgkin's disease, 8 with non-Hodgkin's lymphoma) entered the study. All the 12 patients with measurable disease at study entry responded to DexaBEAM. Median time of subsequent leukopenia (leukocytes < 1.000/microL) was 6 days (range 5-8 days). Peak numbers of CD34+ hematopoietic progenitor cells appeared in the peripheral blood after a median of 20 days (range 18-22 days) after onset of therapy. At that time, peripheral mononuclear cells were collected for autografting. Thereafter, the leukapheresis products were frozen until the day of transplantation, either unpurged in the case of Hodgkin's disease or purged with the ether lipid edelfosine in cases of non-Hodgkin's lymphoma. After high-dose chemotherapy with the CBV regimen (cyclophosphamide, BCNU, etoposide) the patients received their autografts, followed again by G-CSF treatment. A stable hematopoietic recovery was reached with granulocytes > 2.000/muL within 11 days (range 8-17 days), and platelets > 50.000/microL within 15 days (range 10-31 days), respectively, without significant differences between the purged and unpurged transplants. After a median follow-up of 28 months (range 1-40 months) 7 patients are alive without signs of recurrent disease, while 1 patient has died due to acute treatment related toxicity. Three patients had refractory disease, and 5 have relapsed of whom 4 have died. In summary, the DexaBEAM/G-CSF/CBV strategy appears to be safe and effective for salvage treatment in patients with poor risk malignant lymphomas.

Chemopurging of peripheral blood-derived progenitor cells by alkyl-lysophospholipid and its effect on haematopoietic rescue after high-dose therapy.

One reason for relapse after high-dose tumor therapy with subsequent autologous stem cell transplantation is tumor cell contamination of the graft. Removal of tumor cells from bone marrow grafts by chemopurging with the ether lipid edelfosine has been established as an effective and simple method. When compared with bone marrow derived grafts, progenitor cells from peripheral blood have considerably reduced the haematological recovery times. However, this advantage is put at risk by the nonspecific haematotoxic activity of the purging agent. We therefore compared the in vitro recovery of peripheral blood derived progenitor cells (PBPC) from either non-purged (n = 41) or purged (75 micrograms/ml of ether lipid for 4 h at 37 degrees C, n = 48) leukapheresis products. The recovery of CFU-GM after cryopreservation was 63 +/- 4% without and 48 +/- 3% with purging (P = 0.007). After high-dose therapy, patients (n = 37) received similar amounts of either non-purged (n = 17) or purged (n = 20) autologous PBPC. The median haematological recovery times (non-purged vs purged) to > 500 WBC/microlitres were 9.0 vs 8.5 days after transplantation, to > 2000 PMN/microlitres 10.5 vs 10.0 days, and to > 50,000 PLT/microlitres 15.5 vs 14.0 days. All differences were statistically not significant. We conclude that ether lipid purging of PBPC leads to a significant, however tolerable loss of progenitor cells in vitro, and that haematological recovery times after high-dose therapy are identically short, provided similar amounts of PBPC are reinfused.

Trisomy 12 in B-cell chronic lymphocytic leukemia: correlation with advanced disease, atypical morphology, high levels of sCD25, and with refractoriness to treatment.

In situ hybridization was performed to study the clinical significance of trisomy 12 in fifty patients with B-cell chronic lymphocytic leukemia at various stages of disease. Trisomy 12 was detected in 12%-65% (median 53%) of the circulating neoplastic cells in seven out of 20 patients with advanced Binet stage C disease. In contrast, 22 patients with Binet stage A and eight patients with Binet stage B disease were found to be negative for trisomy 12. As occurrence of trisomy 12 was associated with the presence of B-symptoms and hepatosplenomegaly, its association with advanced disease was further considered. In addition, atypical morphology was a common finding in trisomic patients who also displayed higher serum levels of soluble CD25 than patients without trisomy at Binet stage C. No significant differences were detected in serum levels of soluble CD8 and of soluble CD23. No correlation with a lymphocyte doubling time of < 12 months, marked lymphadenopathy, or prior treatment was apparent. However, refractoriness to treatment was evident more frequently in trisomic than in non-trisomic patients (p < .05). In conclusion, trisomy 12 in B-cell chronic lymphocytic leukemia appears to occur predominantly in advanced and symptomatic disease with atypical morphology. It could indicate a high risk for treatment failure thus serving as a marker of poor prognosis in this disease.

Combination of mitoxantrone and etoposide in the treatment of myelodysplastic syndromes transformed into acute myeloid leukemia.

Mitoxantrone and etoposide have both been shown to be effective in de novo acute myeloid leukemia. Therefore, the combination of mitoxantrone and etoposide, the NOVE regimen, was examined in the treatment of myelodysplastic syndromes (MDS) transformed into acute myeloid leukemia (AML). Twenty-one previously untreated patients (eight females, thirteen males) with a median age of 56 years (range 28-67 years) were studied. Diagnosis of MDS was made within the range of six months to three years before transformation into AML occurred. The NOVE regimen consisted of mitoxantrone 10 mg/m2 day 1-5, and of etoposide 100 mg/m2 day 1-5. After one single course of therapy eleven patients achieved a complete remission (CR) and three patients a partial remission (PR). Nine patients (six in CR and three in PR) received a second course, and the PR was completed to a CR in one patient. Thus, the overall response rate was 66% (14/21 patients), and the CR rate was 57% (12/21 patients). Median duration of CR was 7 months (range 2-10 months). Median survival was 10 months (range 3-20 months) for complete responders and 3 months (range 1-10 months) for patients who did not achieve a CR. For patients with subsequent CR, median time of granulocytopenia (< 500/microliters) and thrombocytopenia (< 20.000/microliters) was 20 days and 18 days, respectively. In conclusion, the NOVE regimen appears to be effective in AML secondary to MDS. However, strategies for remission maintenance are warranted.

Detection of circulating monoclonal lymphocytes in multiple myeloma patients by analysis of gene rearrangements: correlation with progressive disease.

Multiple myeloma is characterized by the proliferation of a monoclonal plasma cell population that essentially spreads in the bone marrow. However, immunological data and studies on clonal gene rearrangements have provided evidence for circulating malignant B-lymphocytes. Herein, we report on the detection and clinical significance of monoclonal lymphocytes in the peripheral blood of multiple myeloma patients. Applying the analysis of immunoglobulin gene rearrangements, clonal circulating lymphocytes were found in 8 out of 37 patients. In three of these eight patients, DNA from the bone marrow could be examined as well, and the same gene rearrangement as in peripheral blood was detected. This observation indicates that the monoclonal lymphocytes in the peripheral blood are part of the malignant plasma cell clone in the bone marrow. There was a strong correlation between progressive stage III disease and the detection of circulating tumour cells, whereas neither in stable stage III nor stage I disease could clonal gene rearrangements be detected. Our findings indicate the high incidence of monoclonal lymphocytes in the peripheral blood from patients with progressive multiple myeloma. This may be of importance with respect to cell harvesting strategies for autologous peripheral stem cell transplantation in multiple myeloma.