Figure 1
Figure 1. Immunophenotypic, molecular, and molecular cytogenetic findings in a synchronous, clonally related in situ and manifest follicular lymphoma. Immunohistochemical analysis of BCL2. (A) BCL2 expression in some germinal centers of a morphologically reactive lymph node. Inset: higher magnification of a BCL2 positive germinal center. (Labvision, BCL2 clone 100/D5). (B) Lymph node biopsy with the manifest follicular lymphoma, BCL2 negative. Insert: Interphase FISH analysis using double-color BCL2 break-apart probe (Abbott, Vysis; LSI BCL2 BAP) shows one allele with a normal co-localized signal (yellow arrow) and the second allele with a split of the red and green signal (arrows red and green), indicating a BCL2 break. (C-D) Clonality analysis of the IGH framework 2 (FR2) region shows an identical monoclonal peak of 256 base pairs in both lesions. (E) Sequence analysis of the major BCL2 breakpoint region showed an identical de novo sequence (+D) in both lesions. (F-G) Sequence analysis of BCL2 gene, exon 1 shows a wild-type sequence in the FLIS lesion (left), whereas the mFL shows a point mutation in residue 48 resulting in amino acid substitution (c.144 C > G, p.I48M ATC-ATG). (H) Array-CGH profile of chromosome 6 ideogram reveals loss in 6q14-qter (red label) and gain in 6p22.2-p12.3 (blue label) in the mFL. (I-J) Interphase FISH analysis using FISH probes for 6q21 (RP3–515A4 Spectrum green), 6q27 (RP3–450D5 Spectrum red), and a centromeric probe for chromosome 6 (CEP6 Spectrum aqua; Abbott, Vysis) False color display of the same area in panels I and J. Blue channel in panel I is DAPI counterstain whereas in panel J is Spectrum Aqua (CEP6). Nuclei contain only 1 6q21 and 6q27 signal (green, red) but 2 blue signals (CEP6) indicating deletion in 6q21 and q27 in line with the array CGH. FISH images were acquired with a 100×/1.40 oil-immersion objective in a Zeiss Axioskop fluorescence microscope (Zeiss) equipped with the appropriate filter sets and were documented and processed using the ISIS imaging system (MetaSystems). PCR analysis for IGH gene rearrangements was performed with BIOMED-2 primers for FR2 and FR3 using Phusion hot start DNA polymerase (Finnzymes) with adequate amplification conditions and D4-fluorescent dye primer modification (Sigma-Aldrich). The products were separated by capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System and analyzed with GenomeLab GeXP 10.2 software (Beckman Coulter). Genomic DNA from residue 29-96 of the BCL2 gene was amplified using AmpliTaq Gold DNA Polymerase. PCR products were cloned into the pGEM-T easy vector and transfected into JM109 competent cells. Bacterial plasmids were processed according to standard procedures, and mFL and FLIS clones were subjected to dye terminator cycle sequencing (DTCS-Quick Start Master Mix) using M13 primers and capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System 10.2 software. Array CGH of mFL and FLIS samples was performed on a Human Genome CGH Microarray 244K platform (Agilent Technologies) using Bio Prime Array-CGH Genomic Labeling Kit and hybridized using the manufacturer's protocol (Invitrogen). Slides were scanned with a GenePix 4000B microarray reader (Molecular Devices Corporation). Signal intensities from the generated images were measured and evaluated with Feature Extraction 9.5.3 and DNA Analytics v4.0.81 software packages, respectively (Agilent Technologies). Array CGH gains and losses were defined by use of a trial version of Nexus 6.0 β Discovery Edition.

Immunophenotypic, molecular, and molecular cytogenetic findings in a synchronous, clonally related in situ and manifest follicular lymphoma. Immunohistochemical analysis of BCL2. (A) BCL2 expression in some germinal centers of a morphologically reactive lymph node. Inset: higher magnification of a BCL2 positive germinal center. (Labvision, BCL2 clone 100/D5). (B) Lymph node biopsy with the manifest follicular lymphoma, BCL2 negative. Insert: Interphase FISH analysis using double-color BCL2 break-apart probe (Abbott, Vysis; LSI BCL2 BAP) shows one allele with a normal co-localized signal (yellow arrow) and the second allele with a split of the red and green signal (arrows red and green), indicating a BCL2 break. (C-D) Clonality analysis of the IGH framework 2 (FR2) region shows an identical monoclonal peak of 256 base pairs in both lesions. (E) Sequence analysis of the major BCL2 breakpoint region showed an identical de novo sequence (+D) in both lesions. (F-G) Sequence analysis of BCL2 gene, exon 1 shows a wild-type sequence in the FLIS lesion (left), whereas the mFL shows a point mutation in residue 48 resulting in amino acid substitution (c.144 C > G, p.I48M ATC-ATG). (H) Array-CGH profile of chromosome 6 ideogram reveals loss in 6q14-qter (red label) and gain in 6p22.2-p12.3 (blue label) in the mFL. (I-J) Interphase FISH analysis using FISH probes for 6q21 (RP3–515A4 Spectrum green), 6q27 (RP3–450D5 Spectrum red), and a centromeric probe for chromosome 6 (CEP6 Spectrum aqua; Abbott, Vysis) False color display of the same area in panels I and J. Blue channel in panel I is DAPI counterstain whereas in panel J is Spectrum Aqua (CEP6). Nuclei contain only 1 6q21 and 6q27 signal (green, red) but 2 blue signals (CEP6) indicating deletion in 6q21 and q27 in line with the array CGH. FISH images were acquired with a 100×/1.40 oil-immersion objective in a Zeiss Axioskop fluorescence microscope (Zeiss) equipped with the appropriate filter sets and were documented and processed using the ISIS imaging system (MetaSystems). PCR analysis for IGH gene rearrangements was performed with BIOMED-2 primers for FR2 and FR3 using Phusion hot start DNA polymerase (Finnzymes) with adequate amplification conditions and D4-fluorescent dye primer modification (Sigma-Aldrich). The products were separated by capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System and analyzed with GenomeLab GeXP 10.2 software (Beckman Coulter). Genomic DNA from residue 29-96 of the BCL2 gene was amplified using AmpliTaq Gold DNA Polymerase. PCR products were cloned into the pGEM-T easy vector and transfected into JM109 competent cells. Bacterial plasmids were processed according to standard procedures, and mFL and FLIS clones were subjected to dye terminator cycle sequencing (DTCS-Quick Start Master Mix) using M13 primers and capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System 10.2 software. Array CGH of mFL and FLIS samples was performed on a Human Genome CGH Microarray 244K platform (Agilent Technologies) using Bio Prime Array-CGH Genomic Labeling Kit and hybridized using the manufacturer's protocol (Invitrogen). Slides were scanned with a GenePix 4000B microarray reader (Molecular Devices Corporation). Signal intensities from the generated images were measured and evaluated with Feature Extraction 9.5.3 and DNA Analytics v4.0.81 software packages, respectively (Agilent Technologies). Array CGH gains and losses were defined by use of a trial version of Nexus 6.0 β Discovery Edition.

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