Myc Translocation In Burkitt Lymphoma

The translocation of c-MYC gene on 8q24 is a hallmark of Burkitt lymphoma. Two yeast artificial chromosomes

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Fig. 1 CISH of MYC on a sample with normal chromosome 8. Majority of red and brown signals are adjacent to each other. The red and brown signals could be clearly differentiated. (View this art in color at www.dekker.com.)

(YACs) on the centromeric side of c-MYC gene and two YACs on the telomeric side of c-MYC gene were selected. The centromeric probe was labeled with digoxigenin and the telomeric probe was labeled with biotin. On the normal chromosome, the two probes were close together. On the abnormal chromosome, the signals of the two probes were apart.

Four-micrometer sections were cut from FFPE tissues and placed on Superfrost Plus slides (Fisher Scientific). The slides were dried in an oven at 37°C overnight. After dewaxing and dehydration, the slides were placed in a preheated pretreatment solution (100 mM Tris-base, 50 mM EDTA, pH 7) in a microwave oven (GE Appliances, model JE1390GA 001) at 199°F for 15 min. After pretreatment, the slides were rinsed with PBS and digested with Digest-All 3 (Zymed) at 37°C for 2 min. The slides were rinsed in PBS and postfixed in 10% buffered formalin for 1 min, dehydrated in graded ethanol, and air dried.

Ten microliters (50 ng) of MYC probe (Zymed) was added over the section and covered with a coverslip. The edges of the coverslip were sealed with rubber cement. The slides were placed in HYBrite slide warmer (Vysis) at 94°C for 3 min and incubated in a humidified chamber at 37°C overnight. The stringency wash was carried out in 0.5 x SSC at 72°C for 5 min. For detection, the sections were preblocked with CAS-Block (Zymed) for 10 min and incubated with enzyme conjugate containing alkaline phosphatase (AP)-labeled antidigoxigenin (Roche Applied Science) and horseradish peroxidase (HRP)-labeled streptavidin (Zymed) at 1:500 dilution in antibody diluent

(10% normal goat serum in CAS-Block) for 60 min. After washing in PBS/T (0.025% Tween-20 in PBS), the color for HRP was developed using DAB (Zymed) and the color for AP was developed with Fast Red (Zymed) according to the manufacturer's instruction. The slides were counterstained with hematoxylin and mounted in Glycer-ogel (DAKO).

CISH allowed the morphology of the tissue sections to be easily examined. The area of tumor cells could be identified at low magnification; chromosome ISH results on these tumor cells were then examined using 100 x objective. The microscope must be of sufficiently high quality to allow the brown color of DAB and red color of Fast Red be clearly differentiated (Figs. 1 and 2) at high magnification.

In the ideal condition, signals on a tissue section that do not have chromosome translocation at 8q24 should all be shown as adjacent red/brown signals. However, often, only part of each nucleus was present on the section. If the sectioning cut through chromosome 8 at area close to 8q24, then individual red or brown signal may be observed on normal tissue samples. In a 4-p.m-thick section of normal lymph node, up to 5% of the signals may be shown as single red or single brown signal.

Because of sectioning effect, many nuclei will have incomplete signal profiles. The traditional way of reading FISH results is to look for a nucleus containing all four of the expected red and brown signals. However, a simpler approach is to identify regions predominantly composed of neoplastic cells, then simply record the total number of red, brown, and red/brown signals in that region. The tumor with the translocation should have about 1/3 of all

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Fig. 2 CISH of MYC on a sample with 8q24 translocation. The image shows individual red, individual brown, and red/brown adjacent signals. (View this art in color at www.dekker.com.)
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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