Genetic diversity within leukemia-associated immunophenotype-defined subclones in AML.

Acute myeloid leukemia (AML) is a highly heterogeneous disease showing dynamic clonal evolution patterns over time. Various subclones may be present simultaneously and subclones may show a different expansion pattern and respond differently to applied therapies. It is already clear that immunophenotyping and genetic analyses may yield overlapping, but also complementary information. Detailed information on the genetic make-up of immunophenotypically defined subclones is however scarce. We performed error-corrected sequencing for 27 myeloid leukemia driver genes in 86, FACS-sorted immunophenotypically characterized normal and aberrant subfractions in 10 AML patients. We identified three main scenarios. In the first group of patients, the two techniques were equally well characterizing the malignancy. In the second group, most of the isolated populations did not express aberrant immunophenotypes but still harbored several genetic aberrancies, indicating that the information obtained only by immunophenotyping would be incomplete. Vice versa, one patient was identified in which genetic mutations were found only in a small fraction of the immunophenotypically defined malignant populations, indicating that the genetic analysis gave an incomplete picture of the disease. We conclude that currently, characterization of leukemic cells in AML by molecular and immunophenotypic techniques is complementary, and infer that both techniques should be used in parallel in order to obtain the most complete view on the disease.

Establishment of harmonization in immunophenotyping: A comparative study of a standardized one-tube lymphocyte-screening panel.

BACKGROUND: Multiparameter flow cytometry has been increasingly used in the identification and characterization of leukemia and lymphoma. However, due to technical complexity, this method still presents some level of variation between laboratories. In an attempt to yield more reproducible results, restrictive, highly standardized procedures have been proposed. The objective of this work was to compare this standardized protocol to a more open and flexible procedure. METHODS: The levels of expression of markers from the Euroflow lymphoid screening tube (LST) panel were evaluated on a population of both healthy and diseased patients using the recommended monoclonal antibody (MoAb) combinations or an alternative combination of either different MoAb clones or different dyes. Results were expressed as the percentages of positive target cells for each marker. RESULTS: Our study shows excellent correlation between the two methods demonstrating that comparable results can be achieved through harmonization of the procedures rather than through the constraints of standardization. CONCLUSION: Our results demonstrate that the harmonization approach is feasible. This frees scientists from the restrictions imposed by a standardization approach.

Intragenic breakpoint within RAD51L1 in a t(6;14)(p21.3;q24) of a pulmonary chondroid hamartoma.

Rearrangements involving chromosome region 14q23-->q24 represent a main cytogenetic subgroup in a variety of benign solid tumors. Recently, in uterine leiomyomas containing the classical t(12;14)(q15;q23-->q24), the primary chromosome 14 target gene was identified as the protein kinase-encoding gene RAD51L1. In this report we show that RAD51L1 is also involved in the frequently occurring t(6;14) (p21;q23-->q24) in pulmonary chondroid hamartomas.

Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library.

Bone marrow plasma cells constitute the bulk of malignant cells in multiple myeloma patients. B-lymphocytes having immunoglobulin heavy chain gene rearrangements identical to those of the malignant clone (clonally related B-lymphocytes) may function as malignant plasma cell precursors. We and others proposed the use of anti-idiotypic antibodies to isolate and study clonally related B-lymphocytes. This strategy failed until now because anti-idiotypic antibodies raised by conventional hybridoma techniques proved to react frequently with epitopes shared by different idiotypes. Recently, we succeeded in selecting specific single chain Fv antibodies from phage libraries. To select single chain Fv bearing phages specifically directed against the immunoglobulin idiotype expressed by myeloma tumor cells we panned a semisynthetic phage library against purified myeloma paraprotein Fab fragments. The selection was performed in the presence of soluble polyclonal immunoglobulin as a competitor. Three independent selections for three myeloma patients yielded 10-26 clones. Between two and seven of the selected clones were reactive with patient Fab and not with polyclonal immunoglobulin in enzyme-linked immunosorbent assays. Five out of six anti-idiotypic single chain Fvs were able to specifically stain fixed monoclonal plasma cells in myeloma bone marrow. Idiotype specificity of these single chain Fvs was confirmed by flow cytometry since they did not react with monoclonal plasma cells of other patients, a panel of nine myeloma cell lines, isolated polyclonal bone marrow plasma cells and cultured B-lymphocytes. Using these anti-idiotypic reagents we were able to detect 25 myeloma plasma cells in a background of 50000 immunoglobulin isotype-matched cells of the myeloma cell line UM-1 or 50000 donor bone marrow cells (sensitivity 0.05%). This paper shows that highly specific anti-idiotypic single chain Fv antibody fragments selected from a phage display library can be used to detect rare idiotypic cells in patient samples.

CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells.

Malignant cells in haemopoietic autografts can contribute to post-transplant relapse. Engraftment of myeloma patients with CD34+ peripheral blood progenitors selected from total autografts reduces the number of tumour cells infused by 2.7-4.5 logs. Residual tumour cells detected in CD34+ selected cells may be due to selection impurity or the existence of malignant CD34+ progenitors. In three patients we evaluated the CD34 purity and tumour load of total autografts, CD34+ progenitors selected with immunomagnetic beads and highly purified CD34+ progenitors obtained in two rounds of selection (combining magnetic with flow cytometry activated cell sorting) to determine the cause of residual tumour cells in CD34 selections. Using allele-specific oligonucleotides (ASO) complementary to the unique Ig heavy chain sequence (CDRIII region) of the malignant clone, semi-quantitative ASO-PCR was capable of detecting one malignant cell in 10(4)-10(5) normal white blood cells. Selection of CD34+ cells from bone marrow (BM) with approximately 20% malignant plasma cells resulted in a 1.4 log reduction of tumour burden. Using two-colour flow-cytometry we observed CD34-, BB4+ malignant plasma cells contaminating this CD34 selection. Prior to sorting, peripheral blood cell autografts (PBCA) contained approximately 0.1% malignant cells. Selection of > 99% pure CD34+ cells using immunomagnetic beads (Dynal) resulted in an approximate 2 log reduction of malignant cells, but residual tumour cells were still detectable. ASO-PCR detected no malignant cells in > 99.9% pure CD34+ peripheral blood progenitors obtained with two rounds of selection (combining magnetic with flow cytometry activated cell sorting). We conclude that CD34+ malignant cells are not detectable in myeloma PBCA and that residual tumour cells in CD34 selections are due to contaminating CD34-negative cells.