Molecular diagnostic studies permit the detection of tumor cell contamination of HPC grafts with a sensitivity that is 3 to 4 logs greater than histologic methods or flow cytometry.[2] Assay sensitivity may be as great as 10-6 when disease-specific oligonucleotide probes are used for a polymerase chain reaction (PCR) analysis.[3,4] With these tools, a wide variety of tumor cell types may be detected in the HPC grafts of patients undergoing autologous transplantation, including those with multiple myeloma,[5] NHL,[6] AML,[7] neuroblastoma, Ewing's sarcoma,[8] chronic myelogenous leukemia,[9] acute lymphoblastic leukemia,[9] breast cancer,[10] and germ cell tumors.[11] Graft contamination by tumor cells may be easily detected in patients with multiple myeloma. In an analysis of blood samples from 152 patients with multiple myeloma, phenotypically abnormal CD19+ cells were identified in a majority. These cells could be detected following treatment with conventional- dose therapy without any correlation between the tumor cell number and the paraprotein level; however, they rose in number in the setting of progressive disease. In eight patients for whom PCR analysis could be used to detect disease-specific rearrangement in the complementary determining region III (CDRIII) gene, clonal rearrangements were detected in the peripheral blood B cells of all patients.[12] In a separate study, PCR analysis demonstrated contamination by CDRIII-rearranged cells in 90% of leukopheresis products from patients with multiple myeloma.[13] PCR studies have gone even further to permit quantification of tumor cell- contamination of HPC grafts. In a study that used semiquantitative PCR to assess HPC grafts from 14 patients with multiple myeloma, the median level of graft contamination by PCRpositive cells was 10-4 (range: 10-3 to 10-5).[14] There may be differential contamination between bone marrow and blood HPC. In 13 patients with multiple myeloma who underwent both bone marrow and chemotherapy/ granulocyte colony-stimulating factor (G-CSF, Neupogen)-mobilized blood HPC collection, the median percentage of clonal cells in blood HPC products was 0.0024% (range: < 0.0002% to 0.3520%). In bone marrow products, the median was 0.74% (range: 0.20% to 6.98%).[15] Similar analysis for rearrangements in bcl-2, bcl-1, immunoglobulin heavy-chain, and T-cell receptor genes commonly detects tumor cell contamination in HPC products from patients with NHL. PCR analysis for the presence of t(14;18) in 52 patients with NHL demonstrated that 65% of the patients had evidence of the rearrangement in either their blood or bone marrow prior to blood HPC collection. HPC products from these patients were PCR-positive in 29 of 52 patients (including 4 patients whose blood and bone marrow were PCR-negative).[ 13] Rearranged cells may also be detected in HPC products from patients with intermediate- and highgrade NHL. Among 20 such patients, PCR analysis detected an abnormal molecular marker in HPC products from 17 of 20 patients.[6] Quantitative PCR studies demonstrate that significant numbers of clonal cells may be present in the peripheral blood and HPC products of patients with NHL. In one study, 15 of 37 patients with diffuse large-cell NHL had PCR-detectable rearrangements of CDRIII. At the time of potential blood HPC collection, the level of peripheral blood tumor contamination ranged from 10-2 to < 10-5 (median: 10-2).[16] In a separate quantification study, 26 of 28 evaluable patients had evidence of bcl-2/IgH rearrangements in either blood or bone marrow at the time of HPC collection. The number of rearranged cells ranged from 1 to approximately 105 rear- ranged cells per million mononuclear cells.[17] Graft contamination also occurs in patients with AML.[9] In a representative study, HPC products from all 15 patients with AML1/ETO-positive AML, were PCR-positive on the first day of autologous HPC collection. Of the 11 patients who underwent a second collection procedure, all products were PCR-positive.[7] Gene-Marking Studies
In the absence of direct evidence that HPC contaminants might contribute to systemic relapse, the aforementioned data would be a mere curiosity. Gene-marking techniques using transfection of HPC grafts with the neomycin(Drug information on neomycin)- resistance gene, however, provide direct evidence that contaminating tumor cells may contribute directly to systemic relapse. Brenner and colleagues showed that the neomycin-resistance marker could be detected in leukemic blasts at the time of relapse by PCR analysis in two children who had undergone prior autologous transplantation for AML.[18] Similar studies have demonstrated that the neomycin-resistance marker gene may be detected at the time of systemic relapse in bcr-abl- positive cells in patients who have undergone autologous transplantation for chronic myelogenous leukemia[ 19] and in neuroblasts in patients with neuroblastoma undergoing autologous HPC transplantation.[20] The primary shortcoming of these data is that they do not indicate the frequency with which graft contamination constitutes the principle source for systemic relapse. Methods of Graft Purging In Vivo Purging
Purging technologies attempt to eliminate tumor cell contamination of HPC through direct or indirect manipulation of the graft. Patients may be treated in vivo with systemic chemotherapy and/or monoclonal antibody (MoAb)-based therapies in the hope of reducing the whole-body and circulating tumor cell burden. Thus, in vivo purging methods attempt to both improve systemic control of the ma- lignant disease and alter the kinetics of tumor cell mobilization. The paradigm for in vivo purging is derived from data suggesting that there may be differential hematopoietic recovery with preferential mobilization of normal vs malignant hematopoiesis following intensive chemotherapy. Carella and colleagues treated 30 consecutive chronic myelogenous leukemia patients with either ICE (idarubicin [Idamycin], 8 mg/m2/d for 5 days; cytarabine(Drug information on cytarabine), 800 mg/m2/d for 5 days; and etoposide(Drug information on etoposide), 150 mg/m2/d for 3 days) or mini-ICE (the same agents, but with idarubicin(Drug information on idarubicin) and cytarabine administered for only 3 days) mobilization chemotherapy. Philadelphia chromosome-negative HPC products were collected in 22 patients.[ 21] Other groups have confirmed there results.
- Rituximab-The recent availability of therapeutic MoAbs provides another potential mechanism for in vivo purging. Rituximab (Rituxan)-a humanized, chimeric MoAb directed against the B-cell surface antigen CD20-has considerable antidisease activity in patients with B-cell NHL and may produce molecular remissions in some patients with bcl-2- positive follicular NHL when used in combination with the CHOP regimen (cyclophosphamide [Cytoxan, Neosar], doxorubicin(Drug information on doxorubicin) HCl, vincristine [Oncovin], prednisone(Drug information on prednisone)).[22] Alone or in combination with systemic chemotherapy, this agent may reduce levels of circulating bcl-2-positive cells in patients with follicular NHL.[23] In a group of 23 patients with follicular NHL, rituximab(Drug information on rituximab) was administered to 11 patients in standard doses 1 week and 2 days, respectively, prior to autologous HPC collection. By realtime PCR analysis of bcl-2 gene rearrangements, 6 of 10 HPC products collected from patients who received rituximab were PCR-negative. In contrast, only 1 of 9 products from patients mobilized without rituximab was PCR-negative.[24] In a separate study, 28 patients with mantle cell lymphoma underwent blood HPC collection after each of two cycles of mobilization therapy combined with rituximab. Real-time PCR analysis of products obtained from 17 patients after the first cycle (cyclophosphamide, 7 g/m2, and rituximab, 375 mg/m2) demonstrated that 42% of products were PCR-negative for bcl-1 gene rearrangements. Analysis of 19 patients after the second cycle of therapy (cytarabine, 2 g/m2 every 12 hours for 6 days, and rituximab, 375 mg/m2) demonstrated that all products were PCR-negative.[25] When mobilization kinetics from patients with NHL who receive rituximab as an in vivo purging regimen are compared to historical controls who did not receive rituximab, the median CD34+ cell yield, colonyforming unit-granulocyte/monocyte (CFU-GM) and burst-forming unit- erythrocyte (BFU-E) appear to be comparable.[26] Other investigators, however, find a trend toward poorer mobilization and engraftment kinetics in patients who receive rituximab.[27]
- Imatinib-Novel agents such as imatinib(Drug information on imatinib) mesylate (Gleevec) show promise as in vivo purging agents. In a phase II trial of autologous blood HPC collection in patients with chronic myelogenous leukemia, imatinib was administered prior to G-CSF-stimulated cell collection. All 10 patients achieved a complete cytogenetic response and bcr-abl rearranged metaphases were suppressed below the level of detection by fluorescence in situ hybridization (FISH). Nine patients achieved their collection goal, and HPC products from eight patients had a normal karyotype and negative FISH studies.[28]
HPC grafts may also be manipulated ex vivo through laboratory procedures performed after collection. These ex vivo purging methods may be used to directly remove or destroy contaminating tumor cells (negative selection), or conversely, hematopoietic progenitors cells may be selected from the graft (positive selection) and the remainder of the graft (including tumor cells) discarded.
- Chemotherapeutic Agents-One of the best-studied negative-selection methods involves ex vivo incubation of the HPC graft with cytotoxic chemotherapeutic drugs such as the cyclophosphamide(Drug information on cyclophosphamide) cogeners 4-hydroperoxycyclophosphamide (4-HC) and mafosfamide or with etoposide and corticosteroids. The former two drugs are analogous agents, with mafosfamide having been used more extensively in Europe and 4-HC having been the subject of considerable study in the United States (including use in over 700 patients at Johns Hopkins Medical Center). While 4-HC largely spares primitive hematopoietic stem cells, it produces dose-dependent toxicity against tumor cells and may reduce committed progenitors such as CFU-GM by ≥ 99%.[29] 4-HC purging may therefore cause significantly delayed neutrophil engraftment times.[30] Because direct evidence of its effectiveness is limited and was never confirmed in a prospective randomized trial, the US Food and Drug Administration (FDA) chose not to license 4-HC.[29]
- Monoclonal Antibodies-Antibodies-MoAbs may be used for ex vivo purging. One method involves incubation of mononuclear cellenriched HPC product with one or more disease-appropriate MoAbs and exogenous (typically rabbit) complement. Use of a single MoAb and complement may achieve approximately 3 logs of tumor cell depletion, and use of multiple antibodies may result in up to 6 logs of depletion following three cycles of purging.[31] Ball and colleagues used a similar method for purging HPC in patients with AML. HPC are concomitantly incubated with the MoAbs PM-81 (anti-CD15) with or without the addition of AML-2-23 (anti-CD14) and complement. In 138 patients transplanted between 1984 and 1997, median engraftment times for neutrophils and platelets were comparable to those seen using 4-HC-or mafosfamidepurged grafts.[32] MoAbs directed against specific malignancy-associated surface proteins may also be conjugated directed to a magnetic particle or, following incubation with the target cell, may be subsequently bound by species-specific antibodies that are in turn conjugated to a magnetic particle. Bound cells may be thereafter removed by passage through a magnetic cell separator. In 38 patients with B-cell NHL whose bone marrow products underwent two cycles of immunomagnetic separation, median CD34+ cell recovery was 57% (range: 38% to 80%), whereas CD19+ cells were reduced by a median of 1.8 logs (range: 0.1 to 4.7 logs).[33] In a separate study, bone marrow and blood HPC grafts from 40 patients with bcr-abl-positive acute lymphoblastic leukemia were treated ex vivo with immunomagnetic bead separation. The median recovery of CD34+ cells from both HPC sources was comparable to that in the prior study. PCR analysis of HPC grafts following pur- ging demonstrated a median bcr-abl depletion of 2.3 logs and 1 log, respectively. At the conclusion of purging, 0 of 19 bone marrow HPC grafts and 4 of 17 blood HPC grafts previously PCR-positive for bcr-abl became negative.[34] Alternative forms of MoAb-based negative selection include the use of MoAbs conjugated to immunotoxins.[ 35] Other novel means of ex vivo negative-selection include photodynamic therapy,[36] Adenoviral vector-based methods of tumor cell cytolysis,[37] antisense therapy,[38] incubation with lymphokine-activated killer cells,[39] and incubation with interleukin (IL)-2 (Proleukin).[40]
- Cell Selection Devices-Positive selection alternatively exploits progenitor cell expression of the cell surface marker CD34. CD34+ cells may be bound by MoAbs that are conjugated to a magnetic bead and the bound cells captured by an immunomagnetic cell collection device such as the Baxter Isolex 300i or the Miltenyi Clini- MACS. CD34+ cells may be similarly bound by biotinylated MoAbs that may be captured on an avidin column; this was the basis for the CellPro CEPRATE device. Cells that do not express CD34 antigen are discarded. Alternatively, CD34+ cells may be sorted using ultra-high-speed fluorescence- activated cell sorting.[41] Among 51 patients or donors undergoing blood HPC mobilization for either autologous or allogeneic transplantation, products processed with Isolex 300i achieved a median CD34+ purity of 88.9% (range: 47.8% to 98.3%). The median CD34+ cell recovery was 45.1% (range: 13.8% to 76.2%). CD3+ T cells and CD19+ B cells were depleted by 3.7 and 2.8 logs, respectively.[42] A separate study evaluated the processing of 71 autologous and allogeneic blood and bone marrow HPC grafts with the Clini- MACS; the median CD34+ cell purity and CD34+ cell recovery was 97.02% (range: 68.3% to 99.7%) and 71% (range: 24% to 105%), respectively.[ 43] The Isolex 300i is thus far the only device in the United States that is approved by the FDA for CD34+ cell selection.
Interest in graft purging was dramatically stimulated by the 1991 publication of a Dana-Farber Cancer Institute trial that evaluated the course of 114 patients with follicular NHL who underwent assessment for graft contamination by malignant cells prior to autologous transplantation.[31] PCR analysis detected disease-specific gene rearrangements in bone marrow samples from all patients prior to bone marrow purging. Bone marrow HPC from patients was purged with three cycles of incubation with MoAbs and rabbit complement. Bone marrow HPC from 57 patients remained PCRpositive for clonal gene rearrangements following purging, whereas products from 57 patients were rendered PCR-negative. Patients underwent autologous transplantation following a preparative regimen that consisted of total-body irradiation and cyclophosphamide (60 mg/kg IV daily for 2 days). At a median follow-up of 23 months, 53 of 57 patients who received PCR-negative products remained alive and free of disease, compared with only 21 of 57 patients who received PCR-positive products. The difference between groups was statistically significant. This effect was seen in patients transplanted in complete and partial remission. Using a Cox proportional-hazards regression analysis, the risk of relapse was 9.9 times greater for patients who received PCRpositive products.[31] These data were updated in a 1999 review of outcomes for 153 patients with follicular NHL in first relapse or incomplete initial remission who underwent transplantation with the aforementioned regimen. The projected disease- free survival at 8 years was 47%, and the overall survival was 66%. A subgroup of 113 patients was evaluated by PCR for evidence of bcl-2 rearrangements. The projected freedom from relapse was 89% for patients who received PCR-negative products vs 19% for patients who received PCRpositive products.[44] This difference was statistically significant, and in a univariate analysis, use of PCR-posi- tive HPCs was associated with a worse rate of freedom from relapse.
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