Value of Interphase FISH for the Diagnosis of t(11;14)(q13;q32) on Skin Lesions of Mantle Cell Lymphoma

Pierre Dubus, MD, PhD, Paul Young, MD, Marie Beylot-Barry, MD, PhD, Marc A. Belaud-Rotureau, PhD, Philippe Courville, MD, Béatrice Vergier, MD, PhD, Marie Parrens, MD, Bernard Lenormand, MD, PhD, Pascal Joly, MD, PhD, Jean P. Merlio, MD, PhD

Disclosures

Am J Clin Pathol. 2002;118(6) 

In This Article

Materials and Methods

Immunohistochemical analysis was performed on paraffin-embedded sections with the biotin-streptavidin-peroxidase LSAB kit on an automated Chemate (DAKO, Les Ullis, France). Primary antibodies were directed against the following antigens: CD3, CD20, CD43, and MIB-1 (DAKO) and CD5, CD10, and CD23 (Novocastra, Tebu, Le Perray-en-Yvelines, France). The detection of cyclin D1 was performed using P2D11F11 monoclonal antibody (Novocastra) after antigen retrieval in target retrieval solution, high pH (DAKO), and heating for 10 minutes' boiling time in a pressure cooker. For all cases, this procedure was used on formalin-fixed skin specimen sections. For case 1, the procedure was applied to frozen skin sections after 10 minutes postfixation in 4% buffered paraformaldehyde.

DNA was extracted according to a standard phenol chloroform procedure from frozen material or peripheral blood lymphocytes (PBLs) collected after gradient separation. An FR3-JH fragment of the third complementarity determining region of the immunoglobulin heavy chain (IgH) gene was analyzed, as described elsewhere.[13]

Amplification of the t(11;14) breakpoint was performed using the primer pair MCL1 (5'-GATGGGCTTCTCTCACCTACTA-3' and JH (5'-ACCTGAGGAGACGGTGACCAGGGT-3') as described by Molot et al.[25] Amplification of 500 ng of genomic DNA was performed on an automated thermal cycler (Hybaid, Teddington, England) in a final volume of 50 µL with 1.5 U of Taq polymerase (Promega, Madison, WI), 1x buffer (Promega), a 1.5-mmol/L concentration of magnesium chloride, 200-µmol/L concentrations of each deoxynucleotide triphosphate, and 50 pmol of primers MCL1 and JH. After an initial step at 94°C for 5 minutes, 35 cycles were performed; each cycle consisted of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and elongation at 72°C for 1 minute.

Amplicons were electrophoresed on a 1% agarose gel, stained with ethidium bromide and then blotted onto nylon membranes (Hybond N+, Amersham International, Buckinghamshire, England). After prehybridization, membranes were hybridized at 42°C overnight in a solution of 5x standard saline citrate (SSC); 5x Denhardt solution; 0.5% sodium dodecyl sulfate (SDS); 0.2 g/L of salmon testes sonicated, denatured DNA (Sigma, St Louis, MO); and 10 pmol of the 5' digoxigenin-labeled MCL2 internal primer probe 5'-TCAGGCCTTGATAGCTCG-3'. Blots were washed twice in 2x SSC, 0.1% SDS for 15 minutes at room temperature and then twice in 1x SSC and 0.1% SDS for 20 minutes at 55°C.

Membranes were equilibrated for 1 minute in buffer 1 (0.1-mol/L concentration of maleic acid, 0.15-mol/L concentration of sodium chloride, 0.3% polysorbate 20) and incubated for 40 minutes in buffer 1 with 10% blocking reagent (Roche Diagnostics, Meylan, France) and then for 30 minutes with 10 µL of antidigoxigenin alkaline phosphatase-conjugated antibody (Roche Diagnostics) in buffer 1. Blots were washed twice in buffer 1 for 15 minutes at room temperature; equilibrated in a 0.1-mol/L concentration of sodium chloride and a 0.1-mol/L concentration of tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 9.5, for 2 minutes; covered with 1 mL of 1:500 CSPD (Roche Diagnostics) in a 0.1-mol/L concentration of Tris-HCl and a 0.1-mol/L concentration of sodium chloride, pH 9.5; sealed in a plastic bag; and incubated for 15 minutes at 37°C. Blots were exposed to x-ray film (Kodak X-Omat, Rochester, NY) for 16 hours. Negative (reaction mixture without template) and positive (ADN extracted from REC-1 cells provided by T. Al Saati, MD, PhD, CHU Purpan, Toulouse, France) controls were included in each experiment.

The detection of cyclin D1 overexpression by competitive RT-PCR was performed as described by Uchimaru et al,[27] with slight modifications. Briefly, 10 mg of frozen tissue samples or PBLs were homogenized in a 1.5-mL tube with a sterile piston in 1 mL of Trizol reagent (Life Technologies, Cergy-Pontoise, France) for total RNA isolation. Four micrograms of total RNA was incubated with 20 U of RNAse-free DNAse I and then reverse transcribed into complementary DNA (cDNA) using 800 ng of hexamers (pDN6, Roche Diagnostics) and 200 U of Superscript reverse transcriptase (Life Technologies). One tenth of the cDNA was amplified on an automated thermal cycler (Hybaid) in a final volume of 50 µL with 1.5 U of Taq polymerase (Promega), 1x buffer (Promega), a 1.5-mmol/L concentration of magnesium chloride, 200-µmol/L concentrations of each deoxynucleotide triphosphate, and 50 pmol of primers D1S385, D1AS867, D2ASS609, and D3AS649. All primers were preheated for 2 minutes at 95°C before their addition to the reaction mix. The D1S385 upstream primer (5'-CTGGCCATGAACTACCTGGA-3') is consensus to all 3 cyclin D sequences, while D1AS867 (5'-GTCACACTTGATCACTCTGG-3'), D2ASS609 (5'-CATGGCAAACTTAAAGTCGG), and D3AS649 (5'-CCAGGAAATCATGTGCAATC-3') primers are specific to cyclin D1, D2, and D3 respectively. After an initial step at 94°C for 5 minutes, 35 cycles were performed; each cycle consisted of denaturation at 94°C for 1 minute, annealing at 51°C for 1 minute, and elongation at 72°C for 1 minute. RT-PCR amplicons (10 µL) were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed under UV light. Cyclin D1 transcripts were considered overexpressed when the intensity of the 482-base-pair (bp) cyclin D1 band was found to be equal or higher than the sum of the other 2 bands (cyclin D2, 353 bp; cyclin D3, 243 bp). Negative controls (reaction mixture without template and cDNA from a follicular lymphoma) were included in each PCR set.

The LSI IgH/CCND1 Dual Color, Dual Fusion Translocation Probe (Adgenix, Voisins le Bretonneux, France) contains a mixture of an LSI IgH probe set labeled with spectrum green fluorophore and an LSI CCND1 probe labeled with spectrum orange fluorophore. Dual-color FISH was performed on cytologic touch preparations made from frozen skin and lymph node biopsy specimens (case 1) and on 3-µm sections from formalin-fixed, paraffin-embedded material from lymph node (case 1) and skin biopsy specimens (cases 2 and 3). Three reactive lymph node sections were used as control specimens. The FISH procedure was performed according to Remstein et al,[24] with slight modifications. For cytologic touch preparations, the slides were fixed by immersion in methanol (100%, 50%, 5 minutes each at room temperature) and Carnoy (methanol, acetic acid 3/1, 30 minutes at 4°C). Slides were air dried and placed for 1 hour in a 37°C incubator before dehydration with an ethanol series (70%, 80%, 90%, and 100%). Paraffin-embedded tissue sections (3 µm) were deparaffinized by warming for 1 hour at 65°C and xylene immersion (15 minutes at room temperature). After dehydration, slides were treated in a hydrochloride bath (0.2N, 20 minutes at room temperature) followed by a paraffin pretreatment kit (Adgenix). Probe (10 µL) was added to each slide and a coverslip sealed with rubber cement. Codenaturation (1 minute at 85°C for frozen samples; 3 minutes at 85°C for paraffin sections) and hybridization (24 hours at 37°C) were performed on the Hybrite system (Adgenix). Posthybridization washings, dehydration, and counterstaining were performed as described by Remstein et al.[24] Analysis of the signals was performed with a Zeiss-Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany) using a dual or triple bandpass filter set.

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