ALBERT

Description: 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.

Description: Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony- forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU- S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine- induced pathways for mobilization of primitive stem cells.

Description: A technique of irradiating the entire mouse except for one hind limb was developed to provide repeated proliferative demand on the stem cell pool. Animals received 200 cGY weekly for a total dose of 3,400 to 4,000 cGy. During irradiation, shielded bone marrow cellularity was similar to that of unirradiated controls. Shielded marrow colony- forming unit (CFUs) content increased while marrow CFUs self renewal capacity decreased as compared with unirradiated age-matched controls. Following irradiation experimental animals were monitored monthly for 10 to 12 months for marrow cellularity, CFUs content, and self renewal capacity. Shielded marrow cellularity and CFUs content remained elevated over age-matched controls throughout the period of observation. These findings are compatible with the requirement of the shielded hind limb to provide hematopoietic support for the remainder of the animal. Shielded marrow self renewal capacity, a measurement reflecting primitive hematopoietic stem cell function, remained depressed and did not recover with time. These experiments provide evidence for there being limitations on the self renewal capacity of the stem cell compartment. While the small amount of shielded marrow had sufficient capacity to support the animal its average self renewal capacity was permanently reduced.

Description: Murine bone marrow chimera models were used to assess the efficacy of host total body irradiation (TBI) given at different doses, dose rates, and fractionation schemes in providing for engraftment of syngeneic and allogeneic bone marrow. B6-Hbbd congenic and LP mice, respectively, were used as donors (10(7) bone marrow cells) for syngeneic and allogenic (H-2 compatible) transplantation in standard B6 recipients. Stable marrow chimerism was determined from host and donor stem cell- derived hemoglobin phenotypes (Hbbs and Hbbd) on gel electrophoresis at 3 months posttransplant. Partial engraftment of syngeneic marrow was seen at single doses as low as 2 Gy, with the donor component increasing steadily with increasing TBI dose to a level of 100% at 7 Gy. Immunologic resistance of the host appeared to prevent allogeneic engraftment until 5.5 Gy. A very steep radiation dose response was then observed so that the level of chimerism with 6 Gy and above became comparable with syngeneic engraftment. Low dose rate (5 cGy minute-1) and fractionated TBI required higher total doses for equivalent engraftment (radiation dose-sparing) in both syngeneic and allogenic bone marrow transplantation. This displacement in the dose-response curve on fractionation was seen with interfraction intervals of 3 and 6 hours. A further dose-sparing effect was observed on extending the interval to 18 and 24 hours, but only for allogeneic transplantation, and may therefore be related to recovery of immune-mediated graft resistance. The involvement of multiple target cell populations in determining allogenic engraftment rendered the application of the linear-quadratic model for radiation cell survival problematic in this case. The recovery in dose when low dose rate and 6-hour interfraction intervals were applied in either syngeneic or allogeneic BMT is consistent with appreciable sub-lethal damage repair in the primitive self-renewing stem cell population of the host marrow. These results contrast with the poor repair capacity of the 11-day spleen colony- forming units (CFUs) population after fractionated irradiation and support the notion that ablation of early stem cells in the pre-CFUs compartment is essential for long-term marrow engraftment.

Description: Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 μg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 107 congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 × 107 or 3 × 107 donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 ± 2.7), compared with 5-FU alone (18.5 ± 2.6, P 〈 .0005), or saline controls (17.8 ± 1.7, P 〈 .0001). In an additional experiment, granulocyte colony-stimulating factor (100 μg/dose) was added to a reduced dose of KL (12.5 μg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

Description: The spatial organization of hematopoietic cell subsets of differing proliferative potential within the bone marrow microenvironment has come under increasing interest. In particular, it has been suggested that hematopoietic stem cells (HSCs) normally reside in a regulatory niche situated at the endosteal bone surface and in close proximity to osteoblasts. In the present study we have investigated how different hematopoietic cell subsets are distributed along a Hoechst dye perfusion gradient that may reflect the distance from marrow blood vessels and the level of oxygenation. C57BL/6J mice were intravenously injected with two doses of Hoechst 33342 at 10 and 5 min before marrow cell harvesting, a period that we determined was insufficient for active dye exclusion in vitro. Flow cytometric analysis revealed a wide distribution of Hoechst staining over 3 logs fluorescence intensity. The cells were then sorted from 6 different regions of the Hoechst gradient and evaluated for short- and long-term in vitro repopulating cells in the cobblestone area-forming cell (CAFC) assay. The primitive CAFC subset appearing at day 28 in culture was shown to be progressively enriched with decreasing Hoechst fluorescence while the short-term repopulating day 7 CAFC subset frequencies was the highest at an intermediate level of Hoechst staining. We further investigated whether primitive HSCs exist in a very low oxygen tension by administering the reductive 2-nitroimidazole compound pimonidazole in vivo and performed flow cytometric analysis on sorted primitive HSCs residing in high Hoechst dye effluxing side population (SP). In comparison to whole bone marrow or non-SP cells, the SP fraction showed increased intracellular staining with an anti-pimonidazole antibody that recognizes pimonidazole adducts formed only under hypoxic conditions (less than pO2 of 10 mm Hg). Comparison with thymocytes that are already known to be hypoxic in vivo (Hale et al. Am J Physiol Heart Circ Physiol282: H1467–H1477, 2002) showed both low Hoechst dye perfusion and positive anti-pimonidazole antibody staining. These results represent the first direct evidence that the hematopoietic cell hierarchy is spatially organized in relation to blood vessels and that the stem cell niche exists at the lowest end of an oxygen gradient. These findings have important implications in hematopoiesis and stem cell lodgement, and suggest that the location of hematopoietic stem cells in situ may render them more resistant to oxygen-dependent mutagenic and cytotoxic agents.

Description: High dose therapy and autologous stem cell transplantation (ASCT) has been employed as salvage therapy for patients (pts) with relapsed follicular non-Hodgkin’s lymphoma (FL). Recently, the randomized European CUP trial demonstrated that following 3 cycles of CHOP, ASCT significantly improved both disease free and overall survival when compared to continued CHOP in pts with relapsed FL. In an attempt to improve the results of high dose therapy in FL, we undertook 2 sequential studies for pts with FL in first remission. In the first study, 77 pts with previously untreated advanced stage FL (median age 43), received 6–8 cycles of standard dose CHOP (SD-CHOP) + involved field radiotherapy followed by high dose chemoradiotherapy and anti-B cell purged autologous bone marrow transplantation (ABMT). Following SD-CHOP induction, 27 of 77 pts (36%) were in CR and 50 in a minimal disease state (〈 2 cm masses, 〈 20% BM involvement). At BM harvest, 36 pts had histologic evidence of BM involvement. In the second trial, 19 pts with advanced stage disease (median age 44) were treated with 4 cycles of dose intensified CHOP (HD-CHOP) (cyclophosphamide 1.5 g/m2 day 1 and 2) with G-CSF support followed by high dose chemoradiotherapy and anti-B cell purged autologous ABMT. Following HD-CHOP, 13 of 19 pts were in CR. At BM harvest, 7 of the 19 HD-CHOP pts had histologic BM involvement. Following ABMT, there were 2 acute in-hospital deaths in the pts receiving SD-CHOP induction and none in the pts who received HD-CHOP induction. Nine late deaths in remission were observed in the SD-CHOP pts including 6 from MDS/AML, and 2 late deaths in the HD-CHOP pts. Thirty-three pts who received SD-CHOP remain alive without relapse, with a median follow up of 12 years. Ten pts who received HD-CHOP induction remain alive without relapse, with a median follow up of 9.1 years. For pts receiving SD-CHOP induction, the DFS and overall survival at 10 years are 42% (90% CI: 33%–52%) and 64% (90% CI: 55%–73%), respectively. For pts who received HD-CHOP induction, the DFS and overall survival at 10 years are 59% (90% CI: 38%–79%) and 75% (90% CI: 57%–93%), respectively. The impact of BM treatment and DFS was examined. Pts with known bcl-2 translocations for whom post-BM purging samples were available for pcr examination were analyzed. Following ABMT, pts who were reinfused with pcr negative BM had a significantly better DFS than the pts who were reinfused with pcr positive BM. This study suggests that a subset of pts with FL experience long term remission following ABMT in first remission.

Description: Purpose: To describe changes in pulmonary function and subjective dyspnea over time in patients who received bleomycin-based chemotherapy for Hodgkin’s disease (HD) and to identify predictors for decline in pulmonary function and subjective dyspnea after HD therapy Methods: 52 patients with newly-diagnosed classical HD receiving bleomycin-based chemotherapy with or without mediastinal radiation therapy were enrolled. Patients underwent baseline pulmonary function tests (PFTs), and completed the Pulmonary Functional Status and Dyspnea Questionnaire (PFSDQ-M) pre-treatment. The PFSDQ-M is a 40-item, validated questionnaire measuring the 3 areas of dyspnea, fatigue and activity. The scores range from 0–10, with higher scores representing worse health status. The PFTs and PFSDQ-M were repeated at 1 month, 6 months, 1 year and 2 years post-treatment. Other information collected were number of cycles of bleomycin, radiation dosimetric parameters, and smoking history. This report is based on the 33 patients who had completed treatment and had at least follow-up at 1 month post-treatment. 19 patients received chemotherapy alone and 14 received combined modality therapy. Three-dimensional radiation planning was used and lung dose-volume histograms were generated. Wilcoxon rank sum tests were used to compare the baseline and 1-month objective and subjective lung function results, and to determine whether the addition of radiation therapy to chemotherapy, and among patients who received combined modality therapy, whether the percentage of lung volume receiving 20 Gray (V20), contribute to the changes. A general linear model was used to identify predictors for decline in percentage of predicted carbon monoxide diffusing capacity (%DLCO) at 1 month. Results: At baseline, the median %DLCO was 94% (range: 53–139%). At 1 month post treatment, it was significantly lower at 82% (range: 46–133%) (p=0.0003). Decline in %DLCO at 1 month was not significantly predicted by the addition of mediastinal irradiation (p=0.84), number of cycles of bleomycin (p=0.6), and smoking history (p=0.14), but it was significantly predicted by the baseline %DLCO, with a lower baseline %DLCO associating with less decline in %DLCO (p=0.03). However, the baseline %DLCO also significantly correlated with treatment, with 72% of patients who received chemotherapy alone having a baseline %DLCO =〈 94% (median), compared with 23% of patients treated with combined modality therapy (p=0.011). The median general dyspnea and fatigue scores pre-treatment were 0 (range: 0–6) and 2 (range: 0–7), respectively, and remained the same at 1 month post-treatment. For the 14 patients treated with combined modality therapy, the median V20 was 31.5 % (range: 12–36%). There was no significant effect of V20 on the decline in %DLCO or changes in general dyspnea and general fatigue scores at 1 month. Conclusions: Preliminary results of this study showed significant decline of %DLCO at 1 month post-treatment, but no significant changes in subjective dyspnea and fatigue. None of the treatment-related variables significantly predicted for changes in the pulmonary function at 1 month. The baseline %DLCO was negatively associated with decline in %DLCO, which may be due to response of the mediastinal disease to treatment. However, this association may be confounded by the type of treatment received. Further follow-up is needed to determine the long-term effect of the treatment on lung function.

Description: The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta- positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90- day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences.

Description: ASCT improves progression-free and likely overall survival in patients with relapsed follicular lymphoma. Randomized trials of ASCT as upfront therapy have generally also found an improvement in progression-free survival, but this benefit has been counterbalanced by an increase in second malignancies. We report the long-term outcome of two sequential prospective clinical trials of autologous bone marrow transplantation for advanced stage follicular lymphoma patients under 60 in first remission. In the 1st study 83 previously untreated patients received 6–8 cycles of CHOP induction and in the 2nd study 20 patients received 4 cycles of high-dose CHOP induction (cyclophosphamide 1.5 g/m2 d1–2 with g-csf support). The median age of the patients was 42 (range 19–57). 17% had high FLIPI scores, 58% intermediate and 25% low; all patients were under 60 and 21% had missing data required in the FLIPI. 96% had stage III or IV disease. 77 of 83 patients on the 1st study, and 19 of 20 on the 2nd study (N=96 total), achieved a protocol-defined minimal disease state after CHOP induction (〈 2 cm masses,