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Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections
OBJECTIVE: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18) (q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based on the detection of mbr or mcr in paraffin-embedded tissue samples.METHODS: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint was identified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminested PCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNA Master SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embedded tissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2 (B-cell leukemia-lymphoma 2) (spectrum orange) probes.RESULTS: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12) was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all, 24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangement in any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to be t(14;18) positive via the FISH method.CONCLUSION: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation.

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Authors: Nur Selvi, Buket Kosova, Mine Hekimgil, Cumhur Gündüz, Burçin Tezcanlı Kaymaz, Emin Karaca, Güray Saydam, Murat Tombuloğlu, Filiz Büyükkeçeci, Seçkin Çağırgan, Yeşim Ertan, Nejat Topçuoğlu

Keywords: Foliküler lenfoma, FISH, Multiplex PCR, Semi-nested PCR

  • Year: 2012
  • Number: 2
  • Referance No: 93898

  • Figure 1: Representative gel photo of multiplex PCR results.Minor band shows PCR amplification products (120-200 bp). The band above shows the multiplex PCR strategy in which control primers were used to detect IgH/bcl-2 rearrangement. The first line represents the 50-bp DNA ladder [PUC 19 DNA/MspI (HpaII)]. Interpretation is as follows: Sample 1: MBR-negative case; samples 2 and 3: MBR-positive cases; sample 4: negative control.
  • Figure 2: Representative gel photo of semi-nested PCR results. The first line on the left represents the 50-bp DNA ladder (Fermentas). Sample 1: MBR-positive control (Roche Diagnostic); samples 2-9: MBR-positive samples (each sample shows the PCR amplification products [50 bp] using primers specific to the bcl-2 MBR and JH consensus; sample 10: negative control.
  • Figure 3: FISH analysis of an abnormal nucleus from FL patient 10. The interphase FISH shows a 1R1G2F signal pattern. R: Red bcl-2 signals; G: green IgH signals; F: IgH/bcl-2 fusion signals.
  • Figure 4: FISH analysis of an abnormal nucleus from FL patient 17. The interphase FISH shows a 2R2G signal pattern. R: Red bcl-2 signals; G: green IgH signals.

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