"Less is More": The Role of Purging in Hematopoietic Stem Cell Transplantation.

The epithet "less is more" is usually applied to the essentials of good design, but it might be equally true of autologous blood or marrow transplantation. Ever since autologous marrow transplantation was first used to reconstitute recipients of high-dose chemotherapy or radiotherapy, there has been much discussion about the relative contribution of residual tumor cells in the graft to the occurrence of subsequent relapse. It was not until the early 1990s that this risk was finally confirmed by the use of gene marking [1]. A retroviral vector was used to mark a proportion of the autologous remission bone marrow from patients with acute myeloid leukemia (AML) before marrow infusion after high-dose therapy. Two recipients relapsed and both had leukemic blasts with the marker, the neomycin-resistance gene. As the safety of autologous hematopoietic stem cell transplantation has increased, the use of high-dose therapy followed by stem cell "rescue" is becoming more widespread. The malignancies treated in this way include leukemia, lymphoma, myeloma, neuroblastoma, breast cancer, and ovarian tumors. In each of these conditions a number of important questions should be addressed: Can we identify and quantitate tumor cells in the grafts and establish their oncogenic potential? If so, how best can we remove them? Can they be removed without compromising the graft, and will such purging produce a clinically significant reduction in relapse risk? Finally, will the procedure be cost-effective?

A novel murine model of allogeneic vaccination against prostate cancer.

Prostate cancer continues to be a major cause of death in men. Surgical and medical treatments of the disease have improved, but metastasic disease remains a significant clinical problem. Novel therapies such as whole cell vaccination offer the potential of treating disease by stimulating the immune system. To study the efficacy of a whole cell vaccine in prostate cancer two strains of mice were used: C57BL/6 (H-2Kb) and C3H/HeJ (H-2K(k)) in combination with four different cell lines. Thus, a model was constructed of allogeneic and syngeneic vaccine, as well as a challenge tumour for each strain. Two novel cell lines were developed during this study. Firstly, the non tumourigeneic PMC-1 was derived from a normal mouse prostate and immortalized with HPV16. Secondly, the tumourigeneic PMC-1 C6ras1p1 was transformed with human ras gene which formed tumours in both SCID and C3H/HeJ mice. Protection, and the nature of the immune response to syngeneic and allogeneic vaccine, in males and females was examined in both strains. Vaccination with both syngeneic and allogeneic irradiated whole cell vaccines induced protection from syngeneic challenge in females. However, no protection was observed when allogeneic vaccine was given to male mice. This correlated with the immune response. Two types of cellular immune responses were generated in females. A NK-mediated response was observed in C57BL/6 mice, whilst C3H/HeJ mice developed a CTL response. Little or no cellular immune response was observed in males. The cytokine profile in C3H/HeJ females was a mixture of Th1 and Th2 whilst a mainly Th1 profile was observed in C57BL/6 mice. Male mice showed a diminished cytokine secretion compared to females which was further depressed after challenge. The difference in immunity was largely as expected, since tolerance to prostate antigens should not normally develop in female mice. However, this makes this model particularly relevant clinically since it directly mimics the human situation and thus may accelerate the development of whole cell vaccines for clinical use.

Baculovirus-mediated expression and characterisation of rat glycogen synthase kinase-3 beta, the mammalian homologue of the Drosophila melanogaster zeste-white 3sgg homeotic gene product.

Molecular cloning of glycogen synthase kinase-3 (GSK-3) has demonstrated the existence of a novel form, termed GSK-3 beta, which is highly related to the well characterised GSK-3 alpha protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK-3 proteins, particularly the beta-form, and the zeste-white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK-3 beta in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK-3 beta protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK-3 alpha. Both GSK-3 proteins activate the MgATP-dependent form of protein phosphatase-1 and thus display 'factor A' activity. Since GSK-3 beta exhibits an identical site specificity to GSK-3 alpha with respect to phosphorylation of the proto-oncogene/transcription factors c-jun and c-myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.

Immunocytochemical detection of breast cancer cells: a comparison of three attachment factors.

The evaluation of contaminating breast cancer cells in hematopoietic grafts is of considerable importance for monitoring the efficiency of purging procedures. We report a comparison of three systems for the in vitro detection and enumeration of metastatic breast cancer cells. Breast cancer cells from established cell lines were mixed with Daudi cells at dilutions ranging from 1:10 to 1:1,000,000, and a predetermined number were fixed in defined areas on microscope slides coated with one of the following attachment factors: (i) Cell-Tak Cell and Tissue Adhesive, (ii) 0.1% solution of Poly-L-Lysine, or (iii) Cel-Line HTC Super Cured slides. We employed a specificity-proven pancytokeratin antibody (A45-B/B3) and the alkaline phosphatase-antialkaline phosphatase (APAAP) staining technique. In multiple experiments, one breast cancer cell in 1,000,000 Daudi cells could reliably be detected in the Cell-Tak and Cel-Line systems and 1 in 100,000, with the Poly-L-Lysine system. The observed number of seeded cells showed a highly significant correlation with the number of cells seeded (p < 0.0001 in all cases). Finally, we used the Cell-Tak method to evaluate clinical material from various sources: from patients with primary carcinomas of the breast, prechemotherapy, and during various chemotherapeutic regimens, as well as from patients with metastatic disease. The system consistently detected tumor cells in bone marrow samples from these patients. All peripheral blood samples from patients with metastatic disease tested positive at incidences ranging from 5 to 19/10(6) peripheral blood mononuclear cells. This is a simple and reliable technique that allows rapid screening of large cell numbers with high resolution of positive cells.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant.

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

Standardization of the immunocytochemical detection of cancer cells in BM and blood: I. establishment of objective criteria for the evaluation of immunostained cells.

BACKGROUND: Detection of isolated tumor cells (TC) in BM from carcinoma patients can predict future relapse. Various molecular and immunocytochemical (ICC) methods have been used to detect these cells, which are present at extremely low frequencies of 10(-5) - 10(-6). The specificity and sensitivity of these techniques may vary widely. In 1996, a European ISHAGE Working Group was founded to standardize and optimize procedures used for the detection of minimal residual disease. We have attempted to develop objective criteria for the evaluation of immunocytochemically identifiable cancer cells. METHODS: An interlaboratory ring experiment was performed, to compare the screening and detection of micrometastasis-positive events between different laboratories. The discrepant results induced us to establish a common consensus on morphological criteria applicable to the identification of immunostained micrometastatic TC. RESULTS: Bared on this consensus evaluation, we propose a classification of stained elements into three groups: (1) 'TC's show pathognomonic signs of epithelial TC-nature, as defined by a clearly enlarged nucleus or clusters of > or = 2 immunopositive cells. (2) 'Probable TC's represent morphological overlap between hematopoietic cells (HC) and TC which lack pathognomonic signs of TC-nature, but do not exhibit clear morphological features of HC. These cells are considered as TC if control staining with an isotype-specific, unrelated Ab is negative. (3) 'TC-negative' cells are defined as 'false positive' HC, skin squamous epithelial cells and artefacts. DISCUSSION: The proposed classification of immunostained events is a first step towards the development of standardized immunocytochemical assays for the detection of occult micrometastatic TC in BM or blood.