Am J Pathol. 2002 Aug;161(2):413-20.

Multicolor-FICTION: expanding the possibilities of combined morphologic, immunophenotypic, and genetic single cell analyses.

Martín-Subero JI, Chudoba I, Harder L, Gesk S, Grote W, Novo FJ, Calasanz MJ, Siebert R.

Source

Institute of Human Genetics, University Hospital Kiel, Kiel, Germany. imartin@medgen.uni-kiel.de

Abstract

Phenotypic and genotypic analyses of cells are increasingly essential for understanding pathogenetic mechanisms as well as for diagnosing and classifying malignancies and other diseases. We report a novel multicolor approach based on the FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) technique, which enables the simultaneous detection of morphological, immunophenotypic, and genetic characteristics of single cells. As prerequisite, multicolor interphase fluorescence in situ hybridization assays for B-cell non-Hodgkin's lymphoma and anaplastic large-cell lymphoma have been developed. These assays allow the simultaneous detection of the most frequent primary chromosomal aberrations in these neoplasms, such as t(8;14), t(11;14), t(14;18), and t(3;14), and the various rearrangements of the ALK gene, respectively. To establish the multicolor FICTION technique, these assays were combined with the immunophenotypic detection of lineage- or tumor-specific antigens, namely CD20 and ALK, respectively. For evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes. Thus, three-dimensional information was obtained. The multicolor FICTION assays were applied to well-characterized lymphoma samples, proving the performance, validity, and diagnostic power of the technique. Future multicolor FICTION applications include the detection of preneoplastic lesions, early stage and minimal residual diseases, or micrometastases.

PMID:: 12163366; [PubMed - indexed for MEDLINE]
PMCID:: PMC1850749

Free PMC Article

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Figure 1.

a–j: Composite picture of the MI-FISH and M-FICTION assays for B-NHLs. MI-FISH and M-FICTION images are shown using a false color display. a: Ideograms of chromosomes 3, 8, 11, 14, and 18 showing the BCL6 (3q27), MYC (8q24), BCL1 (11q13), IGH (14q32), and BCL2 (18q21) probes labeled with different dyes. b: Schematic representation of an interphase nucleus containing two isolated copies for each of the genes studied. c: Ideograms of the same chromosomes but with a t(8;14)(q24;q32) translocation. Derivative chromosomes 8 and 14 show co-localization of green and orange signals. d: Schematic representation of an interphase nucleus in which the t(8;14)(q24;q32) translocation is observed as double split of green and orange signals (three signals of each color) and double co-localization of green and orange signals. e–h: MI-FISH in negative and positive controls using the B-NHL multicolor probe set. DAPI is used as nuclear counterstain. e: Intact signal constellation displaying two signals for each locus. f–h: MI-FISH on B-cell lymphomas with IGH translocations affecting MYC, BCL1, and BCL2 are shown, respectively. Notice that a translocation event leads to double split and double co-localization of the signals from the genes involved in the translocation. g: Four co-localizations, reflecting that the captured cell had additional der(11) or der(14) as well as additional signals for BCL6 that might indicate polyploidy. i–j: M-FICTION in a t(8;14)-positive Burkitt’s lymphoma (case 3) using a mAb anti-CD20 and the B-NHL multicolor probe set. i: CD20-negative cell showing a normal pattern of hybridization signals. The blue background staining derives from artificial enhancement necessary to visualize the cell shape. As intact cells were used for the M-FICTION analyses, a pseudo-nuclear CD20 staining would be expected. The lack of such nuclear staining clearly indicates CD20 negativity. j: CD20-positive (intense blue staining) cell displaying a double split and double co-localization of the MYC (orange) and IGH (green) signals, which indicates the presence of a t(8;14). That translocation was detected exclusively in the CD20-positive B-cell subset. k–p: Composite picture of the M-FICTION assay for ALCLs. M-FICTION images are shown using a false color display. k: Ideograms of chromosomes 2 and 5 showing the ALK (2p23), ATIC (2q35), and NPM (5q35) differentially labeled probes. l: Ideograms of the same chromosomes but with a t(2;5)(p23;q35). Derivative chromosome 2 shows a green/pink co-localization and derivative 5 shows an isolated red signal. m: Schematic representation of an ALK-negative interphase cell (gray) containing two co-localizations for ALK (green centromeric/red telomeric) and two isolated signals for the telomeric part of the NPM (pink) and ATIC (orange) breakpoint regions. n: Schematic representation of an ALK-positive interphase cell (blue indicates AMCA) in which the t(2;5)(p23;q35) translocation leads to the dissociation of the green and red probes flanking the ALK gene and furthermore to the co-localization of the ALK centromeric probe (green) with the probe telomeric to the breakpoint in the NPM gene (pink). This co-localization takes place in the der(2) derivative chromosome and an isolated red signal (ALK telomeric) is located in the der(5). o: M-FICTION in a patient with a classical t(2;5) translocation (patient 10). One immunophenotypically positive cell for ALK and two negative cells are shown. The two small ALK-negative (without blue staining) of the upper part of the picture display a normal pattern of hybridization signals: two signals for each probe and co-localization of green and red signals derived from probes flanking the ALK gene (arrowheads). The large ALK-positive (blue staining) cell has a signal pattern indicating the presence of a t(2;5)(p23;q35) translocation. A split of one green and one red signal indicates the break within the ALK locus. The arrows point to a green/pink co-localization derived from the fusion of the ALK and NPM genes. This translocation was found exclusively in those cells expressing ALK. The co-localization of the ALK-tel and ATIC-tel signal (red and orange signals, respectively) was random in this cell and is not indicative for an ATIC/ALK fusion that would result in co-localization of the ATIC-tel (orange) signal with the ALK-cen (green) signal. The arrowhead points to an unrearranged ALK allele, shown as co-localization of red and green signals. p: M-FICTION in patient 9 showing an ALK-positive cell. The arrows point to rearrangements of the ALK gene—displayed as split of red and green probes—without co-localization with NPM or ATIC. The arrowhead points to an unrearranged ALK allele. The co-localization of one green and one orange signal at the top of the figure is because of random overlapping, because it did not appear in the other cells expressing ALK. All of the cells in this patient displayed two couples of isolated red and green signals indicating two rearranged ALK loci. This case was previously shown as bearing an ALK/TPM3 fusion. 30

Multicolor-FICTION

Am J Pathol. 2002 August;161(2):413-420.