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Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections.

Selvi N, Kosova B, Hekimgil M, Gündüz C, Kaymaz BT, Karaca E, Saydam G, Tombuloğlu M, Büyükkeçeci F, Cağırgan S, Ertan Y, Topçuoğlu N - Turk J Haematol (2012)

Bottom Line: 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.Abstract available from the publisher.

View Article: PubMed Central - PubMed

Affiliation: Ege University, School of Medicine, Medical Biology Department, İzmir, Turkey.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: One drop of fixed nuclei suspension was placed onslides and air-dried. Slides were incubated at 37 °C in 2xSSC solution for 30 min. Slides were then washed twicein bidistilled water, and then dehydrated with 70%, 85%,and 100% ethanol for 2 min each. Probe mixture (7 μLof hybridization buffer/1 μL of probe [IgH/bcl2 dualt(14;18)]/2 μL of purified water) was added to each slide and a 22 x 22-mm cover slip was placed over each hybridizationsite and sealed with rubber cement. The slides and probe were co-denatured in a Vysis Hybrite set at a melting temperature of 73 °C and melting time of 5 min, followed by hybridization at 37 °C for 16-20 h. Cover slips were removed and slideswere washed for 5 min in 0.4x SSC/NP40 at 73 °C andtransferred to 2x SSC/NP40 at room temperature for 30 s.Nuclei were counterstained with 5 μL of 40-60-diamidine-2-phenylindole dihydrochloride (DAPI). A Vysis LSI IgH/bcl-2 dual color, dual fusion t(14;18) probe was used for FISH analysis of all nuclei. Cells were visualized with afluorescent microscope (Olympus BX50) and 1000 cells were counted in each slide. Representative cells were photographedusing a computer-based imaging system. Figures 3 and 4 show a positive and negative result for bcl2/IgH rearrangement via FISH, respectively.


Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections.

Selvi N, Kosova B, Hekimgil M, Gündüz C, Kaymaz BT, Karaca E, Saydam G, Tombuloğlu M, Büyükkeçeci F, Cağırgan S, Ertan Y, Topçuoğlu N - Turk J Haematol (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3986950&req=5

f3: 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.
Mentions: One drop of fixed nuclei suspension was placed onslides and air-dried. Slides were incubated at 37 °C in 2xSSC solution for 30 min. Slides were then washed twicein bidistilled water, and then dehydrated with 70%, 85%,and 100% ethanol for 2 min each. Probe mixture (7 μLof hybridization buffer/1 μL of probe [IgH/bcl2 dualt(14;18)]/2 μL of purified water) was added to each slide and a 22 x 22-mm cover slip was placed over each hybridizationsite and sealed with rubber cement. The slides and probe were co-denatured in a Vysis Hybrite set at a melting temperature of 73 °C and melting time of 5 min, followed by hybridization at 37 °C for 16-20 h. Cover slips were removed and slideswere washed for 5 min in 0.4x SSC/NP40 at 73 °C andtransferred to 2x SSC/NP40 at room temperature for 30 s.Nuclei were counterstained with 5 μL of 40-60-diamidine-2-phenylindole dihydrochloride (DAPI). A Vysis LSI IgH/bcl-2 dual color, dual fusion t(14;18) probe was used for FISH analysis of all nuclei. Cells were visualized with afluorescent microscope (Olympus BX50) and 1000 cells were counted in each slide. Representative cells were photographedusing a computer-based imaging system. Figures 3 and 4 show a positive and negative result for bcl2/IgH rearrangement via FISH, respectively.

Bottom Line: 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.Abstract available from the publisher.

View Article: PubMed Central - PubMed

Affiliation: Ege University, School of Medicine, Medical Biology Department, İzmir, Turkey.

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.

No MeSH data available.


Related in: MedlinePlus