FISH Is Superior to PCR in Detecting t(14;18)(q32;q21)-IgH/bcl-2 in Follicular Lymphoma Using Paraffin-Embedded Tissue Samples

Division of Anatomic Pathology, Mayo Clinic, Rochester, MN 55905, USA.
American Journal of Clinical Pathology (Impact Factor: 2.51). 10/2005; 124(3):421-9. DOI: 10.1309/BLH8-MMK8-5UBQ-4K6R
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Detection of t(14;18)(q32;q21)-IgH/bcl-2, which is present in 70% to 95% of follicular lymphomas (FLs), might aid in diagnosing FL. The efficacy of routine polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques in detecting t(14;18) in paraffin-embedded tissue samples was compared on 5 normal tonsils and 28 FLs demonstrated to be t(14;18)+ by previous karyotyping. There was technical failure in 14 (50%) of the FLs by PCR, likely due to B-5 fixation, and 4 (14%) of FLs by FISH, likely due to advanced specimen age. In the remaining successful cases, 5 (36%) of 14 were positive by PCR and 24 (100%) of 24 were positive by FISH. All 5 normal tonsils were negative by both methods. FISH is superior to PCR for detecting t(14;18) from paraffin-embedded tissue samples because it is more sensitive and equally specific.

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    • "FISH for t(14;18) (q32;q21) was performed on formalin-fixed, paraffin-embedded tissue sections as previously described [9] using a dual-fusion LSI IGH/BCL2 probe (08L60-020, Vysis, Downers Grove, IL). FISH signals were analyzed using a fluorescence microscope (Olympus BX51, Tokyo, Japan) equipped with a DP72 camera and DP2-BSW software (Olympus, Tokyo, Japan). "
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    ABSTRACT: The revised 2008 World Health Organization classification maintains a histological grading system (grades 1-3) for follicular lymphoma (FL). The value of grading FL has been debated. This study will yield deeper insights into the morphologic, immunophenotypic characterization and t(14;18) translocation in FL and explore their significance of diagnosisof Chinese FL subgroups.Methods; We retrospectively reviewed the FL diagnoses according to the 2008 WHO classification in all diagnostic specimens from a multicentric cohort of 122 Chinese patients. Upon review, 115 cases proved to be truly FL. CD10, BCL6, MUM1, BCL2 and t(14;18) (q32;q21) translocation were detected by Envision immunostaining technique and fluorescence in situ hybridization. FL1 has larger proportion of follicular pattern (93.0%) than that of FL2 (73.7%, P = 0.036), FL3B (63.6%, P = 0.003) and FL3A (77.4%, P = 0.053), although the last P value was more than 0.05 (Pearson's chi-squared test). Areas of DLBCL were present in 25.8% (8/31) of FL3A and more frequent in FL3B (59.1%, 13/22; P = 0.015). The positivity of CD10 andBCL2 in FL1-2 were significantly higher than those in FL3 (P < 0.001, P = 0.043, respectively). The positivity of MUM1 in FL1-2 was significantly lower than that in FL3 (10.2% vs. 51.0%; P < 0.001). Furthermore the positivity of MUM1 in FL3A was significantly lower than that in FL3B (37.9% vs. 68.2%; P = 0.032). The positivity of t(14;18) was higher in FL1-2 than in FL3 (73.5% vs. 35.6%, P < 0.001), and was higher in FL3A than in FL3B (51.9% vs. 11.1%, P = 0.005). t(14;18) was significantly correlated with CD10+ (R = 0.453, P < 0.001) and MUM1+ (R = 0.482, P < 0.001). FL1 and FL2 were immunophenotypically and genomically similar, while FL3A and FL3B were partly immunophenotypically similar but morphologically, genomically distinct. FL3A was genomically closer to FL1-2, whereas FL3A was genomically closer DLBCL. Thus wehypothesize that FL may in fact be a heterogeneous indolent lymphoma encompassing entities with distinct molecular pathogenesis and genetic characteristics. Immunohistochemical and genetic characterization helps to distinguish subgroups of FLs.Virtual slides: The virtual slide(s) for this article can be found here:
    Diagnostic Pathology 09/2013; 8(1):154. DOI:10.1186/1746-1596-8-154 · 2.60 Impact Factor
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    • "All 26 DNA samples were tested for the IgH/bcl-2 rearrangement using a multiplex PCR protocol, and specific primers for the MBR and jH consensus regions (Table 1), as previously described [21]. Primer sequences and PCR conditions were based on previously described protocols optimized for genomic DNA, and were nearly identical to those used in recent studies [13,14]. "
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    ABSTRACT: 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 onthe detection of mbr or mcr in paraffin-embedded tissue samples. Material and 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 wasidentified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminestedPCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNAMaster SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embeddedtissue 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 rearrangementin any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to bet(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.
    Turkish Journal of Haematology 06/2012; 29(2):126-34. DOI:10.5505/tjh.2012.93898 · 0.36 Impact Factor
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    • "Molecular analyses were performed on genomic DNA extracted by paraffin blocks. DNA samples were amplified by PCR according to the already published methods for the FR3 and FR2 immunoglobulin heavy chain gene, for the beta-globin gene and MBR-BCL2/JH rearrangement [18-20]. PCR products were run on polyacrilamide gels and visualized by ethidium bromide staining. "
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    ABSTRACT: Recently, human germinal center-associated lymphoma (HGAL) gene protein has been proposed as an adjunctive follicular marker to CD10 and BCL6. Our aim was to evaluate immunoreactivity for HGAL in 82 cases of follicular lymphomas (FLs)--67 nodal, 5 cutaneous and 10 transformed--which were all analysed histologically, by immunohistochemistry and PCR. Immunostaining for HGAL was more frequently positive (97.6%) than that for BCL6 (92.7%) and CD10 (90.2%) in FLs; the cases negative for bcl6 and/or for CD10 were all positive for HGAL, whereas the two cases negative for HGAL were positive with BCL6; no difference in HGAL immunostaining was found among different malignant subtypes or grades. Therefore, HGAL can be used in the immunostaining of FLs as the most sensitive germinal center (GC)-marker; when applied alone, it would half the immunostaining costs, reserving the use of the other two markers only to HGAL-negative cases.
    Diagnostic Pathology 10/2011; 6(1):97. DOI:10.1186/1746-1596-6-97 · 2.60 Impact Factor
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