Comparative study of five commercial probes for the detection of Epstein-Barr virus (EBV) by <i>in situ</i> hybridization in cases of nodular sclerosis Hodgki's lymphoma

Nonogaki, Suely; Shirata, Neuza K.; Kimura, Lidia M.; Guerra, Juliana M.; Medeiros, Raphael S. S.; Paes, Roberto Antonio P.; Kanamura, Cristina T.; Oliveira, Camila C.; Menezes, Yara de

doi:10.5935/1676-2444.20160065

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PATHOLOGY, Original article • J. Bras. Patol. Med. Lab. 52 (6) • Nov-Dec 2016 • https://doi.org/10.5935/1676-2444.20160065 copy

Comparative study of five commercial probes for the detection of Epstein-Barr virus (EBV) by in situ hybridization in cases of nodular sclerosis Hodgki's lymphoma

Estudo comparativo de cinco sondas comerciais para detecção do vírus Epstein-Barr (EBV) por hibridização in situ em casos de linfoma de Hodgkin esclerose nodular

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Introduction:

Epstein-Barr virus (EBV) may serve as a target in therapeutic treatments, thus reliable diagnostic results are necessary.

Objective:

The aim of this study was to evaluate the accuracy of EBV detection by in situ hybridization (ISH) using five commercial probes in formalin-fixed and paraffin-embedded samples of nodular sclerosis Hodgkin's lymphoma (HL), and to compare the results with immunohistochemistry (IHC) and polymerase chain reaction (PCR).

Material and method:

Thirty samples were selected, 28 were lymph nodes, one bone marrow and one mediastinum. The following parameters were analyzed: signal intensity; proportionality of positive cells; quality of the reaction according to comfort for evaluation, sign quality and homogeneity of labeled cells; background reaction; morphology; presence of artifacts; and positivity in other non-neoplastic cells. All samples were analyzed for EBV detection using the five probes, IHC for latent membrane protein type 1 (LMP1) and PCR for Epstein Barr virus nuclear antigen 1 (EBNA1). Statistical analyses were performed with the R1 software; Fleiss' test and Cohen Kappa index of 5% were considered significant.

Results:

The detection by IHC-LMP1 was 26.7% (8/30) and 66.7% (20/30) by PCR-EBNA1. All probes detected EBV. Positivity was observed in 42/90 (46.7%), 38/90 (42.2%), 45/90 (50%), 27/90 (30%) and 61/90 (67.8%) for probes A, B, C, D and E, respectively.

Discussion:

All five probes demonstrated positivity.

Conclusion:

Probe E showed better rate (67.8%), sensitivity, specificity and accuracy (100%), a very good correlation among the different observers and with PCR, besides great cost-benefits relation.

Key words:
Epstein-Barr virus infections; in situ hybridization; Hodgkin disease

	Probe A	Probe B	Probe C	Probe D	Probe E
Prehybridization	Denaturation at 62ºC for 10 min Deparaffinization in xylene Decreasing concentrations of ethanol Wash. Deionized water Endogenous peroxidase block. Methanol + 6% H₂O₂ v/v. 5 min Nonspecific DNA inhibition. 30 s Deproteinisation. Proteinase K. 5 min. Room temperature Wash. Deionized water Wash. PBS	Deparaffinization in xylene Decreasing concentrations of ethanol Wash. Deionized water Endogenous peroxidase block. 6% H₂O₂ 5 min Deproteinisation. Proteinase K. 10 min. Room temperature Wash. Deionized water Ethanol 95% – Air dry	Deparaffinization in xylene Decreasing concentrations of ethanol Wash. Deionized water Endogenous peroxidase block. 6% H₂O₂ 5 min Deproteinisation. Proteinase K. 10 min. Room temperature Wash. Deionized water Ethanol 95% and 100% – Air dry	Deparaffinization in xylene Decreasing concentrations of ethanol Wash. Deionized water Endogenous peroxidase block. 6% H₂O₂ 5 min Wash. Deionized water Ethanol 95% and 100% – Air dry Desproteinisation. Preheated pepsin. 37ºC for 5 min Wash. Deionized water Ethanol 95% and 100% – Air dry Denaturation. 60ºC for 5 min	Denaturation at 70ºC for 10 min Deparafinization in xylene Decreasing concentrations of ethanol Wash. Deionized water Endogenous peroxidase block. Methanol + 6% H₂O₂ v/v. 5 min Wash. Deionized water Deproteinisation. Pepsin. 3 min for 37ºC Wash. Deionized water Heat Pretreatment Solution EDTA at 95ºC for 15 min Rinse. Deionized water
In situ hybridization	Vortex the vial for 30 s Probe – Single-stranded DNA with 526 nucleotides complementary to EBER (mRNA) labeled with digoxigenin. Hybridization at 62ºC for 1 h.	Vortex the vial for 30 s Probe – PNA fluorescein labeled. Mix of 4 PNA probes complementary to part of both nuclear EBER RNA sequence. Hybridization at 55ºC for 1h30min.	Vortex the vial for 30 s Probe – Mix of oligonucleotides labeled with fluorescein complementary to nuclear mRNA sequence. Hybridization at 37ºC. Overnight.	Vortex the vial for 30 s Probe – Mix of 5 oligonucleotides complementary to the EBER, labeled with digoxigenin. Hybridization at 37ºC. Overnight.	Vortex the vial for 30 s Probe – Mix of 5 oligonucleotides complementary to the EBER, labeled with digoxigenin. Denaturation at 75ºC for 5 min. Hybridization at 55ºC for 1 h.
Posthybridization & detection	Rinse. PBS Mouse conjugated antidigoxigenin. 37ºC for 30min Rinse. PBS Third-generation polimer conjugated to HRP. 37ºC for 30 min Rinse. PBS Substrate solution. DAB. 10 min at room temperature Wash. Deionized water Hematoxylin. 10 s Wash. Deionized water Differentiation. 3 s Wash. Deionized water Ascending concentrations of ethanol Xylene Entellan Neu	Preheated wash solution. 55ºC for 25 min Rinse. TBS Rabbit anti-FITC labeled with alkaline phosphatase. 37ºC for 30 min Rinse. TBS Rinse. Deionized water Substrate solution. NBT/BCIP. 37ºC for 1 h Rinse. Deionized water Hematoxylin. 10 s Wash. Deionized water Differentiation. 3 s Wash. Deionized water Aquatex	Rinse. TBS Wash solution I = TBS + 0.1% Triton X-100 Blocking solution = solution I + 3% BSA + 20% Normal rabbit serum. 10 min. Room temperature Discard the blocking solution Rabbit conjugated F (ab') anti-FITC labered with alkaline phosphatase diluted 1:50 in solution I + 3% BSA. 37ºC for 30 min Rinse. TBS Rinse. Alkaline phosphate buffer pH 9 Substrate diluted 1:50 + 1 μl levamisole for each 1 ml. 37ºC for 1 h Wash. Deionized water Hematoxylin. 10 s Wash. Deionized water Differentiation. 3 s Wash. Deionized water Aquatex	Wash. TBS Anti-digoxigenin labeled with alkaline phosphatase. 37ºC for 30 min Rinse. TBS Rinse. Deionized water Substrate solution. NBT/BCIP. 37ºC for 1 h Wash. Deionized water Nuclear fast red. Room temperature for 5 min Wash. Deionized water Aquatex	Rinse. TBS Rinse. TBS. 55ºC for 5 min Rinse. TBS. Room temperature for 5 min Mouse conjugated anti-digoxigenin. 37ºC for 30 min Rinse. TBS Polimer conjugated to HRP. 37ºC for 30 min Rinse. TBS Substrate solution. DAB. Room temperature for 10 min Wash. Deionized water Hematoxylin. 10 s Wash. Deionized water Differentiation. 3 s Wash. Deionized water Ascending concentrations of ethanol Xylene Entellan Neu

Score	Probe A		Probe B		Probe C		Probe D		Probe E
Score	n	%	n	%	n	%	n	%	n	%
0	48	53.3	52	57.8	45	50	63	70	29	32.2
2-8	42	46.7	38	42.2	45	50	27	30	61	67.8
Total	90	100	90	100	90	100	90	100	90	100

Reaction quality	Probe A		Probe B		Probe C		Probe D		Probe E
Reaction quality	n	%	n	%	n	%	n	%	n	%
3	9	21.4	9	23.7	5	11.1	9	33.3	19	31.1
4	1	2.4	11	28.9	2	4.4	9	33.3	6	9.8
5	6	14.3	9	23.7	8	17.8	7	26	7	11.5
6	6	61.9	9	23.7	30	66.8	2	_.7.4	29	47.6
Total	42	100	38	100	45	100	27	100	64	100

Morphology	Probe A		Probe B		Probe C		Probe D		Probe E
Morphology	n	%	n	%	n	%	n	%	n	%
0	0	0	1	1.1	2	2.2	6	6.7	3	3.3
1	6	6.7	9	10	5	5.6	67	74.4	4	4.5
2	30	33.3	44	48.9	35	38.9	8	8.9	19	21.1
3	54	60	36	40	48	53.3	9	10	64	71.1
Total	90	100	90	100	90	100	90	100	90	100

Background reaction	Probe A		Probe B		Probe C		Probe D		Probe E
Background reaction	n	%	n	%	n	%	n	%	n	%
0	77	85.6	62	68.9	83	92.2	82	91.1	69	76.7
1	13	14.4	28	31.1	7	7.8	8	8.9	21	23.3
Total		90 100		90 100	90	100	90	100	90	100

Artifacts	Probe A		Probe B		Probe C		Probe D		Probe E
Artifacts	n	%	n	%	n	%	n	%	n	%
	63	70	63	70	73	81.4	69	76.7	76	84.4
	27	30	27	30	17	18.2	21	23.3	14	15.6
Total	90	100	90	100	90	100	90	100	90	100

Others cells	Probe A		Probe B		Probe C		Probe D		Probe E
Others cells	n	%	n	%	n	%	n	%	n	%
0	74	82.2	81	90	81	90	83	92.2	62	68.9
1	16	17.8	9	10	9	10	7	7.8	28	31.1
Total	90	100	90	100	90	100	90	100	90	100

Methods	Positive cases/n	%
IHC - LMP1	8/30	26.7
ISH - Probe A	13/30	43.3
ISH - Probe B	12/30	40
ISH - Probe C	15/30	50
ISH - Probe D	9/30	30
ISH - Probe E	20/30	66.7
PCR - EBNA1	20/30	66.7

Probes	Fleiss' Kappa coefficient	z-score	p-value	Interpretation
Probe A	0.777	7.37	< 0.0001	Good
Probe B	0.636	6.03	< 0.0001	Good
Probe C	0.822	7.8	< 0.0001	Very good
Probe D	0.788	7.48	< 0.0001	Good
Probe E	0.900	8.54	0	Very good

Parameter	ISH
Parameter	IHC	Probe A	Probe B	Probe C	Probe D	Probe E
Sensitivity	40	65	60	75	45	100
Specificity	100	100	100	100	100	100
PPV	100	100	100	100	100	100
NPV	45.5	58.8	55.6	66.7	33.3	100
False positive rate	0	0	0	0	0	0
Accuracy	60	76.7	73.3	83.3	63.3	100

	Cohen's Kappa coefficient	z-score	p-value	Interpretation
IHC	0.308	2.34	0.0195	Regular
Probe A	0.553	3.39	0.00070	Moderate
Probe B	0.500	3.16	0.00157	Moderate
Probe C	0.667	3.87	0.000108	Good
Probe D	0.353	2.54	0.0112	Regular
Probe E	1	5.48	< 0.0001	Very good

Probes	Average execution time	Cost - April/2012 US$
Probe A	5h45min	2572
Probe B	6h39min	1685
Probe C	26h37min	2478
Probe D	25h57min	1974
Probe E	6h43min	6 8

References	Probes	Results	Notes
Pinto et al.⁽¹⁹19 Pinto MT, Ferreira FV, Pitombeira MS, et al. Analysis of the association between Epstein Barr virus and classic Hodgkin’s lymphoma in adult patients from Ceará (Brazil) by immunohistochemistry and “in situ” hybridization. J Bras Patol Med Lab. 2006; 42(3): 201-5. DOI: 10.1590/S1676-24442006000300009. https://doi.org/10.1590/S1676-2444200600... ⁾	Probe B	16/23 (69.5%)
Huang et al.⁽²⁰20 Huang X, Nolte I, Gao Z, et al. Epidemiology of classical Hodgkin lymphoma and its association with Epstein Barr virus in Northern China. PLoS One. 2011; 6(6): e21152. PubMed PMID: 21695175. DOI:10.1371/journal.pone.0021152. https://doi.org/10.1371/journal.pone.002... ⁾	Probe B	126/491 (25.7%)
Di Napoli et al.⁽²¹21 di Napoli A, Al-Jadiri MF, Talerico C, et al. Epstein-Barr virus (EBV) positive classical Hodgkin lymphoma of Iraqi children: an immunophenotypic and molecular characterization of Hodgkin/Reed-Sternberg cells. Pediatr Blood Cancer. 2013; 60(12): 2068-72. PubMed PMID: 24000236. DOI: 10.1002/pbc.24654. https://doi.org/10.1002/pbc.24654... ⁾	Probe B	10/16 (62.5%)	ISH-EBER + IHC-LMP1
Palma et al.⁽²²22 Palma I, Sánchez AE, Jiménez-Hernández E, et al. Detection of Epstein-Barr virus and genotyping based on EBNA2 protein in Mexican patients with Hodgkin lymphoma: a comparative study in children and adults. Clin Lymphoma Myeloma Leuk. 2013; 13(3): 266-72. PubMed PMID: 23276887. DOI: 10.1016/j.clml.2012.11.010. https://doi.org/10.1016/j.clml.2012.11.0... ⁾	Probe B	15/33 (45.5%)	ISH-EBER + IHC-LMP1
Tornóczky et al.⁽¹²12 Tornóczky T, Kelényi G, Pajor L. EBER oligonucleotide RNA in situ hybridization in EBV associated neoplasms. Pathol Oncol Res. 1998; 4(3): 201-5. PubMed PMID: 9761938.⁾	Probe C	0/1 (0%)
Vassallo et al.⁽²³23 Vassallo J, Metze K, Traina F, Souza CA, Lorand-Metze I. Expression of Epstein-Barr virus in classical Hodgkin's lymphomas in Brazilian adult patients. Haematologica. 2001; 86(11): 1227-8. PubMed PMID: 11694413.⁾	Probe C	36/61 (59%)
Kwon et al.⁽²⁴24 Kwon JM, Park YH, Kang JH, et al. The effect of Epstein-Barr virus status on clinical outcome in Hodgkin's lymphoma. Ann Hematol. 2006; 85(7): 463-8. PubMed PMID: 16534596. DOI: 10.1007/s00277-006-0081+9. https://doi.org/10.1007/s00277-006-0081+... ⁾	Probe C	8/28 (28.5%)
Abd El All et al.⁽²⁵25 Abd El All HS. Bob-1 is expressed in classic Hodgkin lymphoma. Diagn Pathol. 2007; 2: 10. PubMed PMID: 17346351. DOI:10.1186/1746-1596-2-10. https://doi.org/10.1186/1746-1596-2-10... ⁾	Probe C	6/10 (60%)
Dinand et al.⁽⁴4 Dinand V, Dawar R, Arya LS, Unni R, Mohanty B, Singh R. Hodgkin's lymphoma in Indian children: prevalence and significance of Epstein-Barr virus detection in Hodgkin's and Reed Sternberg cells. Eur J Cancer. 2007; 43(1): 161-8. PubMed PMID: 17113770. DOI:10.1016/j.ejca.2006.08.036. https://doi.org/10.1016/j.ejca.2006.08.0... ⁾	Probe D	32/33 (97%)	ISH-EBER + IHC-LMP1

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[1] Corresponding author: Suely Nonogaki, Núcleo de Patologia Quantitativa; Centro de Patologia; Instituto Adolfo Lutz, Avenida Doutor Arnaldo, 355, 7º andar; Pacaembu, CEP: 01246-000; São Paulo-SP, Brasil, Phone: +55 (11) 3068-2874. e-mail: nonogaki@terra.com.br