Longitudinal analysis of circulating myeloma cells detected by allele specific mRNA in situ hybridization.

Individual myeloma cells in the peripheral blood of 7 patients with multiple myeloma were detected by mRNA in situ hybridization (ISH) using biotinylated antisense oligonucleotide probes to non-germline sequences of the CDR3 region of the immunoglobulin heavy chain gene. Peripheral blood samples from these patients were studied over a period of 2-3 years. The number of circulating myeloma cells varied from 0.

Induction of somatic intrachromosomal recombination inversion events by cyclophosphamide in a transgenic mouse model.

Somatic intrachromosomal recombination (SICR) can result in chromosomal inversion and deletion, mechanisms which are important in carcinogenesis. We have utilised a transgenic mouse model to study SICR inversion events in spleen cells. The transgenic construct is designed so that expression of an Escherichia coli lacZ transgene only occurs in a cell when an SICR inversion event occurs in the region of the transgene.

Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia.

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.

Leukaemia presenting as marrow hypoplasia: molecular detection of the leukaemic clone at the time of initial presentation.

Occasional cases of transient marrow hypoplasia in childhood evolve into acute leukaemia. We studied two children who presented with marrow hypoplasia following infection and who developed acute lymphoblastic leukaemia 2-3 months later. A simple polymerase-chain-reaction (PCR) test for monoclonality showed that immunoglobulin heavy-chain gene rearrangements of the same size were present at the times of both hypoplasia and leukaemia, and DNA sequencing confirmed identity of these rearrangements.

Effect of the Philadelphia chromosome on minimal residual disease in acute lymphoblastic leukemia.

The Philadelphia translocation is associated with a poor prognosis in adults and children with acute lymphoblastic leukemia, even though the majority of patients achieve remission. To test the hypothesis that the translocation leads to drug resistance in vivo, we studied 61 children and 20 adults with acute lymphoblastic leukemia and used the level of minimal residual disease at the end of induction as the measure of drug resistance in vivo. In children the presence of the translocation was associated with a significant increase in residual disease, indicating higher drug resistance in vivo; five of seven Philadelphia-positive children but only five of 54 Philadelphia-negative children had a minimal residual disease level >10(-3), a level which is associated with a high risk of relapse in childhood acute lymphoblastic leukemia of standard risk.

The use of monoclonal gene rearrangement for detection of minimal residual disease in acute lymphoblastic leukemia of childhood.

Sensitive quantification of minimal residual disease (MRD) using the polymerase chain reaction (PCR) is strongly predictive of outcome in childhood acute lymphoblastic leukemia (ALL), with MRD levels at the end of induction therapy of >10(-3) predicting a poor outcome. Methods for sensitive quantification are, however, complicated and time-consuming. Detection by PCR of monoclonal immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements is simple and can be used in routine laboratories but is non-quantitative and of lower but uncertain sensitivity.

The estimation of in vivo mutation rate and frequency from samples of human lymphocytes.

In vivo mutation assays usually involve enumeration of mutant cells in samples drawn from individuals or populations, and inferences concerning quantitative aspects of mutation are made from the results. Individual cells within the samples may show clonal relationships with each other. For populations of constant size, which do not obey Luria-Delbruck kinetics, mutation frequency should be calculated from the number of mutant cells and not from the number of mutant clones.

Myeloma stem cells in autografting in multiple myeloma.

Autologous transplantation has been used increasingly over the last 10 years for the treatment of multiple myeloma. As is the case in other cancers treated by high-dose therapy and stem cell rescue, the contribution of occult tumor cells in the graft to relapse posttransplant remains to be resolved. In this report, we review the biology and differentiation of plasma cells in the context of their significance as an origin of relapse in multiple myeloma.

Relationship between minimal residual disease and outcome in adult acute lymphoblastic leukemia.

In children with acute lymphoblastic leukemia (ALL), the level of minimal residual disease (MRD) at the end of induction strongly predicts outcome, presumably because it measures both drug sensitivity and the number of leukemic cells requiring elimination. Children with high levels (> 10(-3) leukemic cells per marrow cell) nearly always relapse, whereas those with low levels (<2 x 10(-5)) seldom do. However, the importance of MRD in adult ALL is unclear.

Renal allograft rejection--in situ demonstration of cytotoxic intratubular cells.

A nonisotopic in situ hybridization method to detect perforin mRNA was developed in cytospin preparations of IL-2-stimulated normal human lymphocytes and applied to formalin-fixed acutely rejected renal transplant material. Individual cells expressing perforin mRNA were localized in severely damaged tubular areas, and a number of these cells appeared to be located inside the tubular basement membrane in close association with tubular epithelial cells. Immunoperoxidase staining in acetone-fixed cryostat sections of acutely rejected kidney confirmed that a considerable proportion of infiltrating cells was CD8+; many of these were in an intratubular location.

Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests.

lt could be speculated for patients with myeloma and other lymphoproliferative disorders that peripheral blood stem cells may be preferable to bone marrow for autologous transplantation because they may be less contaminated by neoplastic cells. To test this possibility, the immunoglobulin heavy chain gene rearrangement and limiting dilution polymerase chain reaction were used to sensitively quantify myeloma cells in bone marrow and peripheral blood stem cell collections, taken at a similar time, from eight patients with multiple myeloma. Levels of residual disease in the peripheral blood stem cell harvests were variable and did not reflect the tumour burden in the marrow.

Characterization of clonal immunoglobulin heavy chain and I cell receptor gamma gene rearrangements during progression of childhood acute lymphoblastic leukemia.

Instability of antigen receptor gene rearrangements during progression of acute lymphoblastic leukemia (ALL) has important implications for polymerase chain reaction (PCR)-based techniques using these genes for the detection of minimal residual disease (MRD). Antigen receptor gene instability may lead to false negative results in bone marrow samples taken during remission. Utilizing the PCR and consensus primers for rearranged immunoglobulin heavy chain (IgH) and T cell receptor gamma (TCR gamma) gene sequences, we analyzed the bone marrow samples at diagnosis and first relapse for 37 children with ALL.

In situ lymphoproliferation in renal transplant biopsies.

A double immunohistochemical labelling procedure in paraffin-embedded renal tissue is reported in which CD3 was targeted as a T cell marker and Ki67 as a marker of cell proliferation. Proliferating and quiescent T cells were unequivocally identified in situ, and their precise location within the kidney was clarified by the use of periodic acid-Schiff counterstaining to outline the basement membranes. Proliferating tubular epithelial cells were also clearly identified.