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Mutations of NFKBIA in biopsy specimens from Hodgkin lymphoma
Cancer Genetics and Cytogenetics 197 (2010) 152e157
Mutations of NFKBIA in biopsy specimens from Hodgkin lymphoma Xiaojian Liua, Hong Yub, Wentao Yangb, Xiaoyan Zhoub, Hongfeng Lub, Daren Shib,* a
Department of Chemotherapy, Cancer Hospital, Department of Oncology, Shanghai Medical Collage, Fudan University, 270 Dongan Road, Shanghai 200032, P.R. China b Department of Pathology, Cancer Hospital, Department of Oncology, Shanghai Medical Collage, Fudan University, 270 Dongan Road, Shanghai 200032, P.R. China Received 5 July 2009; received in revised form 5 October 2009; accepted 3 November 2009
Abstract
To determine the possible alteration of the inhibitor kBa (HUGO-approved symbol, NFKBIA) gene in Chinese Hodgkin lymphoma (HL) patients, NFKBIA mRNA and protein expression in 22 primary HL patients were examined. Individual tumor cells were used for amplification to obtain the NFKBIA gene, and the polymerase chain reaction products were sequenced. Compared with reactive surrounding lymphocytes, inhibitor kBa protein (IkBa) expression was weaker in the cytoplasm of H-RS (Hodgkin and ReedeSternberg) cells. NFKBIA mRNA was strongly expressed in HRS cells from HL sections, and little was detected in the reactive surrounding lymphocytes. A total of 37.5% of the patients with HL had mutations in the NFKBIA gene. Some mutations possibly resulted in C-terminally truncated form of the IkBa. These data suggest that the impairment of the IkBa functions was produced during the pathogenesis of the tumor cell clone in Chinese HL patients. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Hodgkin lymphoma (HL) is a unique malignancy pathologically characterized by a minority of the cell population called H-RS (Hodgkin and ReedeSternberg) cells surrounded by normal inflammatory cells, which are the major cell component. Researchers have confirmed the germinal center B-cell origin of the H-RS cells in most HL. At present, the molecular events leading to transformation of the malignant cells remain to be elucidated. In about 40% of cases of classical HL, the H-RS cells are infected by EpsteineBarr virus (EBV), indicating that EBV plays an important role in HL pathogenesis. Also, most cases of HL are EBV negative [1]. Even though studies on HLderived tissue or cell lines suggested that p53 mutations, bcl-2 translocations, and Fas mutations might play a role in the pathogenesis of HL, neither of these ideas could be validated when primary H-RS cells were investigated [2,3]. Studies in several HL-derived cell lines identified constitutive RelA/p50 activity as their characteristics, suggesting a role of RELA/NFKB1 (v-rel reticuloendotheliosis viral oncogene homolog A/nuclear factor of k light polypeptide gene enhancer in B-cells 1) in the pathogenesis of HL [4]. * Corresponding author. Tel.: þ86021 64175590-5100; fax: 86 (021)64036901. E-mail address: [email protected] (D. Shi). 0165-4608/10/$ e see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.11.005
RelA/p50 is mainly regulated by inhibitor kB family proteins, primarily by IkBa. After being stimulated by a number of agents, including cytokines and phorbol esters, and cross-linking of surface IgM and viral infection, signalinduced activation of the inhibitor kB kinases leads to phosphorylation of the IkBa on serine 32 and 36, which are subsequently degraded by the ubiquitin proteosome pathway. As a consequence of rapid IkBa degradation, RelA/p50 is allowed to translocate into the nucleus and activate kB site dependent genes [5]. A reduction of IkBa was observed in some adult T-cell leukemia cases in spite of the increased levels of NFKBIA mRNA. Hence, constitutive activity of RelA/p50 could be caused by aberrant of IkBa in this setting. Previous studies have found that mutations of NFKBIA play a crucial role in RelA/p50 activation in primary HL patients [6]. However, the data are still scarce, and most results were based on HL cell lines. We need a larger data set. Further, even though the similar incidence of EBV infection was found, HL in Eastern countries is characterized by a lower incidence rate compared with the West. This difference could be attributed to genetic factors between the two groups [7]. We preliminarily focus on aberrant IkBa and elucidate its role in pathogenesis of Chinese patients with primary HL. Further studies will be needed to explore other inhibitor kB family members or other mechanisms involved in activation of RelA/p50 in HL patients.
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2. Materials and methods 2.1. HL samples All cases of classic HL were selected from the files of the Department of Pathology, Shanghai Cancer Hospital, Fudan University, after obtaining informed consent. All the included samples represent at-diagnosis biopsy samples. Any biopsy samples from relapsed disease were discarded. The histologic confirmation of HL and subtype was achieved in 22 cases by central review by means of standard tissue sections, and diagnoses were made according to the criteria of the 2008 World Health Organization classification [8]. Among these diagnoses, nodular sclerosing HL accounts for 40.9% (9 of 22) and mixed cellularity for 50.0% (11 of 22). One lymphocyte depletion case and one lymphocyte-rich case in patients with HL were included. 2.2. Immunohistochemical staining for IkBa Immunohistochemistry (IHC) for IkBa was performed by the EnVision System (Dako, Glostrup, Denmark). Paraffin blocks were sectioned at a thickness of 4 mm and dried for 16 hours at 56 C before being dewaxed in xylene and rehydrated through a graded ethanol series and washed with phosphate-buffered saline. Antigen retrieval was achieved by heat treatment in a pressure cooker for 2 minutes in 10 mM citrate buffer (pH 6.5). Before staining the sections, endogenous peroxidase was blocked. IHC staining was performed on these sections by using the antibodies C-21 (sc-37) from Santa Cruz Biotechnology (Santa Cruz, CA), a polyclonal affinity-purified antibody raised against carboxy terminus of human IkBa. After incubation with C-21 (1:200), immunodetection was performed with the LSAB (Labeled Streptavidin Biotin) Visualization System (Dako) with diaminobenzidine chromogen used as substrate. Sections were counterstained with hematoxylin. 2.3. In situ hybridization for NFKBIA mRNA NFKBIA mRNA was detected by in situ hybridization (ISH) with digoxin-conjugated oligonucleotide probe (Wuhan Boster Biological Technology, Wuhan, China). Briefly, sections were pretreated with pepsin for 5 seconds at 37 C, then incubated with a NFKBIA oligonucleotide probe overnight at 40 C in a humid chamber and afterward washed with trisodium citrate solution for 45 minutes at 37 C with shaking. Detection was performed with biotinylated antidigoxin antibody for 60 minutes at 37 C, followed by peroxidase-labeled streptavidin with diaminobenzidine chromogen as substrate. Sections were counterstained with hematoxylin. 2.4. Interpretation of IHC and ISH sections To guarantee the reproducibility of the method for IHC or ISH, and to ease statistical analyses, we used clear-cut
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criteria [9]. Ten high-power fields in each slide were viewed. Thirty H-RS cells or surrounding reactive lymphocytes in each case should be counted. In brief, the intensity of staining for IkBa was scored in each specimen on a scale of 0 to 3 (0 5 negative staining, 1 5 weakly positive staining, 2 5 moderately positive staining, 3 5 strongly positive staining). The extent to which positive cells were seen in each specimendthat is, the extent of distribution of positive cellsdwas estimated and scored on a scale of 0 to 4 (0 5 negative; 1 5 positive staining in 1 to 25% of cells; 2 5 positive staining in 26 to 50%; 3 5 positive staining in 51 to75%; 4 5 positive staining in 76 to 100%). Each patient was evaluated for the sum of the two parameters. A sum of scores of more than 4 was considered positive staining. 2.5. Laser microdissection Fresh frozen tissue was cut at 6-mm thickness in a cryostat. Sections were applied on Leica membrane-coated slides (Leica Microsystems, Wetzlar, Germany). A routine method for CD30 IHC detection on the slides has been described elsewhere [12]. CD30-positive single H-RS cells were microdissected from the sections by the Leica laser microdissection (LMD) system and pooled in 20e100 mL of lysis buffer (10 mM Tris-HCl, pH 8.0, 1% Tween 20) and stored at 20 C. 2.6. Polymerase chain reaction amplification DNA of fresh frozen tissue was extracted from thawed samples by Qiagen Pico Pure DNA Extraction Kit according to the manufacturer’s protocol (Qiagen, Shanghai, China). Details (including primers) for polymerase chain reaction (PCR) amplification of exons 1e6 were as previously published [6]. Seminested PCR was performed in a PE9700 Hybaid (Perkin-Elmer, Waltham, MA) thermal reactor, and the amplified products were analyzed by electrophoresis in 2% agarose gels. 2.7. Direct sequence analysis The PCR products obtained from the HL-derived cell lines or from individual H-RS cells were sequenced by an automated sequencer ABI377 (BioAsia, Shanghai, China). The resulting sequences were compared with the sequences of the NFKBIA gene. 2.8. Statistical analysis The statistical analyses were performed by SPSS for Windows version 13.0 (SPSS, Chicago, IL). All cases were first grouped as H-RS positive or negative (1 5 positive, 2 5 negative). Variables were compared by McNemar tests to determine the difference between protein and mRNA expression. The Wilcoxon signed rank test was used for comparing immunostaining or ISH labeling between
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H-RS cells and surrounding reactive cells. P ! 0.05 was considered statistically significant (two-tailed).
generated a preterminal stop codon. One with a deletion of one nucleotide led to frameshift. These three mutations all presumedly resulted in a C-terminally truncated form of the IkBa (Table 2; Fig. 2).
3. Results 3.1. IHC staining and ISH for IkBa 4. Discussion A total of 36.4% patients with HL showed positive IkBa expression in H-RS cells by IHC. However, 90.9% of the reactive surrounding lymphocytes showed positive expression. The average scores representing IkBa expression in cytoplasm of H-RS cells by IHC showed lower expression than that of reactive surrounding lymphocytes (Fig. 1). A total of 86.4% patients with HL showed positive NFKBIA mRNA expression in H-RS cells by ISH, however, no patients showed positive NFKBIA mRNA expression in reactive surrounding lymphocytes. NFKBIA mRNA expression by ISH was highly intensive in H-RS cells, whereas just a little was detected in reactive surrounding lymphocytes, showing a lower hybridized signal in comparison with H-RS cells (Fig. 1). The average scores representing NFKBIA mRNA expression in cytoplasm of H-RS cells by ISH were higher than those of reactive surrounding lymphocytes. The scores for the IkBa protein and NFKBIA mRNA expression in H-RS cells or in reactive surrounding cells are summarized in Table 1. There were statistically significant differences between IkBa protein and NFKBIA mRNA expression in H-RS cells (P 5 0.003). (P values were calculated results of McNemar tests by SPSS.) 3.2. Analysis of the NFKBIA in H-RS cells from patients with primary HL Sixteen CD30-positive patients with primary HL were included. Several single H-RS cell portions were collected from each lymph node biopsy sample of each patient using the Leica LMD system. Each aliquot was analyzed by amplification of exons IeVI of NFKBIA mRNA. Among them, several products from each patient with PCR positive were sequenced. The mutations were all unique nucleotide exchanges or deletions. A total of 37.5% of the patients with HL had mutations in the NFKBIA located in exons 1, 4, 5, and 6. Two patients with a point mutation in exon 5
By a single-cell microdissection technique, Ku¨ppers et al. [10] showed that H-RS cells arise from monoclonal B lymphocytes in the germinal centers of lymphoid tissue and that they carry clone-specific rearrangements of IgG (immunoglobulin G) genes. Apoptosis of these cells was found to be inhibited by the constitutively expressed RelA/p50. These results end the long controversy about the nature of Hodgkin disease: is it an inflammatory or infectious disease, or a true malignant process? HL occurs less commonly in Chinese (2.3 cases per 100,000 persons) than in white patients. The difference in incidence rate is caused by environment and genetics [7]. EBV has been implicated in the etiology of HL. EBVencoded small nuclear RNAs status is positive in approximately 40% in primary HL patients in China, similar to that in the Western world. Studies on HL-derived tissue or cell lines suggested that HL is a high level of constitutive nuclear RelA/p50 activity, which stimulates proliferation and confers resistance to apoptosis. Other genetic defects like p53 mutations or bcl-2 translocations may play a role in the pathogenesis of HL. Neither of these ideas could be validated when primary H-RS cells were investigated. Low, frequent Fas mutations or NFKBIE (nuclear factor of k light polypeptide gene enhancer in B-cells inhibitor, 3) mutations in HL cells might explain the constitutive activity of RelA/p50. The aim of this work was to identify molecular defects of IkBa of HL patients in the Chinese population. In NFKBIA / mice, constitutive nuclear factor kB activation could be observed in lymphoid cells [11]. Hence, IkBa seems to play a major role in regulation of nuclear factor kB activity in lymphoid system. By ISH, Emmerich et al. [12] found that NFKBIA mRNAs were strongly overexpressed in 20 HL patients. In contrast to the H-RS cells, only some reactive lymphocytes expressed low to moderate
Fig. 1. Immunohistochemical staining (arrows) of IkBa by C-21 (A) and in situ hybridization of NFKBIA mRNA by NFKBIA antisense probe (B) or by sense probe (C). Compared with background lymphocytes, the IkBa expression in cytoplasm of Hodgkin and ReedeSternberg cells was weak, and NFKBIA mRNA expression was strong (400).
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Table 1 Protein and mRNA expression in H-RS cells in Hodgkin lymphoma* NFKBIA mRNA by ISH
IkBa by IHC Cell
No. of cases
Positive, no. (%)
Average score (mean 6 SD)
Positive, no. (%)
Average score (mean 6 SD)
H-RS RS
22 22
8 (36.4)a 20 (90.9)
3.41 6 1.50b 5.77 6 1.11b
19 (86.4)a 0 (0)
5.27 6 1.08c 2.82 6 0.59c
Abbreviations: H-RS, Hodgkin and ReedeSternberg cells; NFKBIA, nuclear factor of k light polypeptide gene enhancer in B-cells inhibitor, a; IkBa, inhibitor kBa protein; IHC, immunohistochemistry; ISH, in situ hybridization; RS, reactive surrounding cells. * Variables were compared by McNemar tests to determine the difference between protein and mRNA expression. The Wilcoxon signed rank test was used for comparing immunostaining or ISH labeling between H-RS cells and surrounding reactive cells. P values were calculated results by SPSS. a The difference between IkBa and NFKBIA mRNA expression in H-RS cells (P 5 0.003). b The IkBa expression of H-RS cells was lower than that of RS (P ! 0.0001). c The NFKBIA mRNA expression of H-RS cells was higher than that of RS (P ! 0.0001).
amounts of NFKBIA-specific transcripts. To test whether this is also valid for primary H-RS cells from HL patients in the Chinese population, we examined 22 cases of classic HL by a highly sensitive ISH with an NFKBIA mRNA specific cDNA probe. A total of 86.4% patients with HL showed positive NFKBIA mRNA expression in H-RS cells by ISH. In most cases, we detected overexpression of NFKBIA mRNA in the H-RS cells, whereas we detected no or very little amounts of NFKBIA mRNA in reactive surrounding lymphoid tissues (P ! 0.0001). (P values were calculated results of Wilcoxon signed rank test by SPSS.) Therefore, we confirmed that the high level of NFKBIA mRNA was a characteristic feature of primary H-RS cells. On the other hand, 36.4% patients with HL showed positive IkBa expression in H-RS cells by IHC. We found that the scores representing IkBa expression in cytoplasm of H-RS cells was lower than that in reactive surrounding lymphoid tissues by IHC (P ! 0.0001). (P values were calculated results of Wilcoxon signed rank test by SPSS.) Little literature has documented the use of the IHC method to present the expression of IkBa protein in paraffin-embedded sample blocks of HL patients. Also, there were significant differences between IkBa and NFKBIA mRNA expression (P 5 0.003). (P values were calculated results of McNemar tests by SPSS.) Obviously, a reduction of IkBa expression
was observed in H-RS cells in spite of the increased levels of NFKBIA mRNA. Because RelA/p50 activates transcription of its own inhibitor, overexpression of NFKBIA mRNA reflects the high transcriptional activity of RELA/NFKB1 in HL [13]. It has been demonstrated that in normal cells, enforced expression of NFKBIA leads to accumulation of RelA/p50 protein and involves the export of p50 into the cytoplasm, thus contributing to the termination of RelA/p50 activity. The paradoxical finding of high RelA/p50 activity despite the strong expression of its inhibitor indicates that the nuclear factor kB/inhibitor kB system is greatly deregulated in H-RS cells. Cabannes et al. [14] established that the mutations in the NFKBIA gene in HL suggest a tumor suppressor role for NFKBIA in HL patients and related HL cell lines. Obviously, the ubiquitin proteosome pathway may play a role in the process. In addition, a defect of the IkBa molecule could be the mechanism for this loss of control of nuclear factor kB/inhibitor kB system. Researchers have analyzed IkBa in HL-derived cell lines by Western blot test and confirmed a disruption of the coding sequence by a stop codon in some of these cell lines. To analyze whether comparable NFKBIA mutations also occur in primary H-RS cells, we isolated single H-RS cells from CD30-immunostained tissue sections of 16 HL
Table 2 Detection of mutations in IeVI exon regions of the NFKBIA in H-RS cellsa Case
Type
Cells/aliquot (no.)
No. of PCR-positive aliquots
No. of aliquots sequenced
Mutations (exon)
Type
1 2 3 8 13 14
NS MC MC NS NS NS
30 35 40 45 25 35
5 5 6 5 4 6
3 2 2 3 2 3
2692b T-A (5) Delb2538 G (4) 2692b T-A (5) 3584 G-A (6) 3584 G-A (6) 52C-T (1)
nonsense frameshift nonsense trailer trailer trailer
These sequence data are available from National Center for Biotechnology Information, GenBank (NT_026437, NG_007571, with 50 untranslated region assembled another 592 bp). Abbreviations: NFKBIA, nuclear factor of k light polypeptide gene enhancer in B-cells inhibitor, a; H-RS, Hodgkin and ReedeSternberg cells; PCR, polymerase chain reaction; NS, nodular sclerosing Hodgkin lymphoma; MC, mixed-cellularity Hodgkin lymphoma. a No mutations were found in surrounding reactive cells. b In most cases, two or more PCR-positive aliquots along with each exon were sequenced; all products has the same mutations.
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Fig. 2. Chromatographs of DNA sequence for exon 5 (A) and exon 4 (B). (A) Arrow points to site 2692, where the peak shown indicates a T-A mutation, which formed preterminal TGA stop codon. (top, sense and antisense). No mutations were found in the surrounding reactive cells (bottom, sense and antisense). (B) Arrow points to site 2538, where it indicates a G deletion (top). No mutations were found in surrounding reactive cells (bottom). National Center for Biotechnology Information, GenBank, NT_026437, NG_007571, with 50 untranslated region assembled another 592 bp.
patients from the previously mentioned 22 cases of classic HL. The NFKBIA gene was analyzed by single-cell PCR. Three mutations all presumably resulted in a C-terminally truncated form of the IkBa. EBV has been implicated in the etiology of HL by both epidemiologic and serologic studies. IkBa expression level in HL-derived cell lines has been well established by Western blot test in the literature. In Jungnickel’s study, clonal deleterious somatic mutations in the NFKBIA gene were detected in the H-RS cells in two of three EBV-negative and none of two EBV-positive cases of HL [6]. This indicates that the pathogenesis of HL was due to EBV infection or mutations of NFKBIA, and that the two are mutually independent. These results could not be confirmed in our experiments. We tested the IkBa expression level in 22 primary HL patients by IHC. The EBV infection was investigated by testing latent membrane protein 1 (LMP1) expression by IHC. Although LMP-1 status was not detected in all our cases, the correlation of IkBa protein expression levels with EBV status seems variable. NFKBIA aberrant expression was supposed to occur in EBV-positive casesda finding different than the Jungnickel’s conclusions. The exact correlation of IkBa protein expression levels with EBV status is an interesting aspect that should be further investigated. The NFKBIA of two cases contained a mutation generating a preterminal stop of the translational machinery (Table 2). The stop codon was not found in the surrounding nonmalignant lymphoid cells of the same patient, indicating that this mutation was specific for the H-RS cells. One patient had deletion of one nucleotide, which led to a preterminal stop-producing frameshift. Overexpression of NFKBIA mRNA and low intensive staining of IkBa protein in the H-RS cells have occurred in these cases. It is conceivable that the C-terminally truncated protein can block the wild-type protein. Also, aside from its function of reserved RelA/p50 complex in the cytoplasm, IkBa can localize in the nucleus and dissociate the RelA/p50e DNA complex [13]. A prerequisite for effective inhibition of RelA/p50eDNA binding are the ankyrin repeats and an intact C terminus [4]. A role of the C-terminal end for
the inhibition of RelA/p50eDNA binding has recently been suggested by an x-ray structure analysis of the RelA/p50/ IkBa complex [15]. Furthermore, IkBa contains a nuclear export signal at amino acids 264e281, which confers active shuttling of the RelA/p50/IkBa complex to the cytosol. Therefore, the truncated IkBa may protect DNA-bound RelA/p50 from dissociation by wild-type IkBa and interfere with the nuclear export pathway [16]. Consequently, RelA/p50eDNA binding activity would be maintained in H-RS cells. The truncated IkBa and its impact on normal IkBa led to the IHC detection insensitivity. Although the observed mutations were found only in 3 of 16 cases, our data provide evidence of gene aberrations in a large number of Chinese HL patients. Permanently activated RelA/p50 in primary H-RS cells might be a consequence of gene mutation of one of its inhibitors. It is therefore tempting to speculate that defects of additional members of the inhibitor kB family contribute to the functional blockage of RelA/p50 in the remaining cases. Indepth studies are required to investigate this possibility.
References [1] Brauninger A, Schmitz R, Bechtel D, Renne C, Hansmann ML, Kuppers R. Molecular biology of Hodgkin’s and Reed/Sternberg cells in Hodgkin’s lymphoma. Int J Cancer 2006;118:1853e61. [2] Montesinos-Rongen M, Roers A, Ku¨ppers R, Rajewsky K, Hansmann ML. Mutation of the p53 gene is not a typical feature of Hodgkin and ReedeSternberg cells in Hodgkin’s disease. Blood 1999;94:1755e60. [3] Maggio EM, Van Den Berg A, de Jong D, Diepstra A, Poppema S. Low frequency of FAS mutations in ReedeSternberg cells of Hodgkin’s lymphoma. Am J Pathol 2003;162:29e35. [4] Younes A, Garg A, Aggarwal BB. Nuclear transcription factorkappaB in Hodgkin’s disease. Leuk Lymphoma 2003;44:929e35. [5] Bargou RC, Emmerich F, Krappmann D, Bommert K, Mapara MY, Arnold W, Royer HD, Grinstein E, Greiner A, Scheidereit C, Dorken B. Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin’s disease tumor cells. J Clin Invest 1997;100:2961e9. [6] Jungnickel B, Staratschek-Jox A, Brauninger A, Spieker T, Wolf J, Diehl V, Hansmann ML, Rajewsky K, Kuppers R. Clonal deleterious mutations in the IkappaBalpha gene in the malignant cells in Hodgkin’s lymphoma. J Exp Med 2000;191:395e402.
X. Liu et al. / Cancer Genetics and Cytogenetics 197 (2010) 152e157 [7] Au WY, Gascoyne RD, Gallagher RE, Le N, Klasa RD, Liang RH, Choy C, Foo W, Connors JM. Hodgkin’s lymphoma in Chinese migrants to British Columbia: a 25-year survey. Ann Oncol 2004; 15:626e30. [8] Swerdlow SH, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. Pathology and genetics of tumours of hematopoietic and lymphoid tissues. In: World Health Organization classification of tumours. Lyon: International Agency for Research on Cancer Press, 2008; pp. 326e9. [9] Koga H, Sakisaka S, Ohishi M, Kawaguchi T, Taniguchi E, Sasatomi K, Harada M, Kusaba T, Tanaka M, Kimura R, Nakashima Y, Nakashima O, Kojiro M, Kurohiji T, Sata M. Expression of cyclooxygenase-2 in human hepatocellular carcinoma: relevance to tumor dedifferentiation. Hepatology 1999;29:688e96. [10] Ku¨ppers R, Rajewsky K, Zhao M, Simons G, Laumann R, Fischer R, Hansmann ML. Hodgkin disease: Hodgkin and Reede Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci USA 1994;91:10962e6. [11] Hinz M, Lemke P, Anagnostopoulos I, Hacker C, Krappmann D, Mathas S, Do¨rken B, Zenke M, Stein H, Scheidereit C. Nuclear factor
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kappaB-dependent gene expression profiling of Hodgkin’s disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity. J Exp Med 2002; 196:605e17. Emmerich F, Meiser M, Hummel M, Demel G, Foss HD, Jundt F, Mathas S, Krappmann D, Scheidereit C, Stein H, Do¨rken B. Overexpression of I kappa B alpha without inhibition of NF-kappaB activity and mutations in the I kappa B alpha gene in ReedeSternberg cells. Blood 1999;94:3129e34. Bargou RC, Leng C, Krappmann D, Emmerich F, Mapara MY, Bommert K, Royer HD, Scheidereit C, Do¨rken B. High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/ ReedeSternberg cells. Blood 1996;87:4340e7. Cabannes E, Khan G, Aillet F, Jarrett RF, Hay RT. Mutations in the IkBa gene in Hodgkin’s disease suggest a tumour suppressor role for IkappaBalpha. Oncogene 1999;18:3063e70. Truhlar SM, Torpey JW, Komives EA. Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB. DNA interaction fold upon binding to NF-kappaB. Proc Natl Acad Sci USA 2006;103:18951e6. Huxford T, Huang DB, Malek S, Ghosh G. The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NFkappaB inactivation. Cell 1998;95:759e70.
Mutations of NFKBIA in biopsy specimens from Hodgkin lymphoma Xiaojian Liua, Hong Yub, Wentao Yangb, Xiaoyan Zhoub, Hongfeng Lub, Daren Shib,* a
Department of Chemotherapy, Cancer Hospital, Department of Oncology, Shanghai Medical Collage, Fudan University, 270 Dongan Road, Shanghai 200032, P.R. China b Department of Pathology, Cancer Hospital, Department of Oncology, Shanghai Medical Collage, Fudan University, 270 Dongan Road, Shanghai 200032, P.R. China Received 5 July 2009; received in revised form 5 October 2009; accepted 3 November 2009
Abstract
To determine the possible alteration of the inhibitor kBa (HUGO-approved symbol, NFKBIA) gene in Chinese Hodgkin lymphoma (HL) patients, NFKBIA mRNA and protein expression in 22 primary HL patients were examined. Individual tumor cells were used for amplification to obtain the NFKBIA gene, and the polymerase chain reaction products were sequenced. Compared with reactive surrounding lymphocytes, inhibitor kBa protein (IkBa) expression was weaker in the cytoplasm of H-RS (Hodgkin and ReedeSternberg) cells. NFKBIA mRNA was strongly expressed in HRS cells from HL sections, and little was detected in the reactive surrounding lymphocytes. A total of 37.5% of the patients with HL had mutations in the NFKBIA gene. Some mutations possibly resulted in C-terminally truncated form of the IkBa. These data suggest that the impairment of the IkBa functions was produced during the pathogenesis of the tumor cell clone in Chinese HL patients. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Hodgkin lymphoma (HL) is a unique malignancy pathologically characterized by a minority of the cell population called H-RS (Hodgkin and ReedeSternberg) cells surrounded by normal inflammatory cells, which are the major cell component. Researchers have confirmed the germinal center B-cell origin of the H-RS cells in most HL. At present, the molecular events leading to transformation of the malignant cells remain to be elucidated. In about 40% of cases of classical HL, the H-RS cells are infected by EpsteineBarr virus (EBV), indicating that EBV plays an important role in HL pathogenesis. Also, most cases of HL are EBV negative [1]. Even though studies on HLderived tissue or cell lines suggested that p53 mutations, bcl-2 translocations, and Fas mutations might play a role in the pathogenesis of HL, neither of these ideas could be validated when primary H-RS cells were investigated [2,3]. Studies in several HL-derived cell lines identified constitutive RelA/p50 activity as their characteristics, suggesting a role of RELA/NFKB1 (v-rel reticuloendotheliosis viral oncogene homolog A/nuclear factor of k light polypeptide gene enhancer in B-cells 1) in the pathogenesis of HL [4]. * Corresponding author. Tel.: þ86021 64175590-5100; fax: 86 (021)64036901. E-mail address: [email protected] (D. Shi). 0165-4608/10/$ e see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.11.005
RelA/p50 is mainly regulated by inhibitor kB family proteins, primarily by IkBa. After being stimulated by a number of agents, including cytokines and phorbol esters, and cross-linking of surface IgM and viral infection, signalinduced activation of the inhibitor kB kinases leads to phosphorylation of the IkBa on serine 32 and 36, which are subsequently degraded by the ubiquitin proteosome pathway. As a consequence of rapid IkBa degradation, RelA/p50 is allowed to translocate into the nucleus and activate kB site dependent genes [5]. A reduction of IkBa was observed in some adult T-cell leukemia cases in spite of the increased levels of NFKBIA mRNA. Hence, constitutive activity of RelA/p50 could be caused by aberrant of IkBa in this setting. Previous studies have found that mutations of NFKBIA play a crucial role in RelA/p50 activation in primary HL patients [6]. However, the data are still scarce, and most results were based on HL cell lines. We need a larger data set. Further, even though the similar incidence of EBV infection was found, HL in Eastern countries is characterized by a lower incidence rate compared with the West. This difference could be attributed to genetic factors between the two groups [7]. We preliminarily focus on aberrant IkBa and elucidate its role in pathogenesis of Chinese patients with primary HL. Further studies will be needed to explore other inhibitor kB family members or other mechanisms involved in activation of RelA/p50 in HL patients.
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2. Materials and methods 2.1. HL samples All cases of classic HL were selected from the files of the Department of Pathology, Shanghai Cancer Hospital, Fudan University, after obtaining informed consent. All the included samples represent at-diagnosis biopsy samples. Any biopsy samples from relapsed disease were discarded. The histologic confirmation of HL and subtype was achieved in 22 cases by central review by means of standard tissue sections, and diagnoses were made according to the criteria of the 2008 World Health Organization classification [8]. Among these diagnoses, nodular sclerosing HL accounts for 40.9% (9 of 22) and mixed cellularity for 50.0% (11 of 22). One lymphocyte depletion case and one lymphocyte-rich case in patients with HL were included. 2.2. Immunohistochemical staining for IkBa Immunohistochemistry (IHC) for IkBa was performed by the EnVision System (Dako, Glostrup, Denmark). Paraffin blocks were sectioned at a thickness of 4 mm and dried for 16 hours at 56 C before being dewaxed in xylene and rehydrated through a graded ethanol series and washed with phosphate-buffered saline. Antigen retrieval was achieved by heat treatment in a pressure cooker for 2 minutes in 10 mM citrate buffer (pH 6.5). Before staining the sections, endogenous peroxidase was blocked. IHC staining was performed on these sections by using the antibodies C-21 (sc-37) from Santa Cruz Biotechnology (Santa Cruz, CA), a polyclonal affinity-purified antibody raised against carboxy terminus of human IkBa. After incubation with C-21 (1:200), immunodetection was performed with the LSAB (Labeled Streptavidin Biotin) Visualization System (Dako) with diaminobenzidine chromogen used as substrate. Sections were counterstained with hematoxylin. 2.3. In situ hybridization for NFKBIA mRNA NFKBIA mRNA was detected by in situ hybridization (ISH) with digoxin-conjugated oligonucleotide probe (Wuhan Boster Biological Technology, Wuhan, China). Briefly, sections were pretreated with pepsin for 5 seconds at 37 C, then incubated with a NFKBIA oligonucleotide probe overnight at 40 C in a humid chamber and afterward washed with trisodium citrate solution for 45 minutes at 37 C with shaking. Detection was performed with biotinylated antidigoxin antibody for 60 minutes at 37 C, followed by peroxidase-labeled streptavidin with diaminobenzidine chromogen as substrate. Sections were counterstained with hematoxylin. 2.4. Interpretation of IHC and ISH sections To guarantee the reproducibility of the method for IHC or ISH, and to ease statistical analyses, we used clear-cut
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criteria [9]. Ten high-power fields in each slide were viewed. Thirty H-RS cells or surrounding reactive lymphocytes in each case should be counted. In brief, the intensity of staining for IkBa was scored in each specimen on a scale of 0 to 3 (0 5 negative staining, 1 5 weakly positive staining, 2 5 moderately positive staining, 3 5 strongly positive staining). The extent to which positive cells were seen in each specimendthat is, the extent of distribution of positive cellsdwas estimated and scored on a scale of 0 to 4 (0 5 negative; 1 5 positive staining in 1 to 25% of cells; 2 5 positive staining in 26 to 50%; 3 5 positive staining in 51 to75%; 4 5 positive staining in 76 to 100%). Each patient was evaluated for the sum of the two parameters. A sum of scores of more than 4 was considered positive staining. 2.5. Laser microdissection Fresh frozen tissue was cut at 6-mm thickness in a cryostat. Sections were applied on Leica membrane-coated slides (Leica Microsystems, Wetzlar, Germany). A routine method for CD30 IHC detection on the slides has been described elsewhere [12]. CD30-positive single H-RS cells were microdissected from the sections by the Leica laser microdissection (LMD) system and pooled in 20e100 mL of lysis buffer (10 mM Tris-HCl, pH 8.0, 1% Tween 20) and stored at 20 C. 2.6. Polymerase chain reaction amplification DNA of fresh frozen tissue was extracted from thawed samples by Qiagen Pico Pure DNA Extraction Kit according to the manufacturer’s protocol (Qiagen, Shanghai, China). Details (including primers) for polymerase chain reaction (PCR) amplification of exons 1e6 were as previously published [6]. Seminested PCR was performed in a PE9700 Hybaid (Perkin-Elmer, Waltham, MA) thermal reactor, and the amplified products were analyzed by electrophoresis in 2% agarose gels. 2.7. Direct sequence analysis The PCR products obtained from the HL-derived cell lines or from individual H-RS cells were sequenced by an automated sequencer ABI377 (BioAsia, Shanghai, China). The resulting sequences were compared with the sequences of the NFKBIA gene. 2.8. Statistical analysis The statistical analyses were performed by SPSS for Windows version 13.0 (SPSS, Chicago, IL). All cases were first grouped as H-RS positive or negative (1 5 positive, 2 5 negative). Variables were compared by McNemar tests to determine the difference between protein and mRNA expression. The Wilcoxon signed rank test was used for comparing immunostaining or ISH labeling between
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H-RS cells and surrounding reactive cells. P ! 0.05 was considered statistically significant (two-tailed).
generated a preterminal stop codon. One with a deletion of one nucleotide led to frameshift. These three mutations all presumedly resulted in a C-terminally truncated form of the IkBa (Table 2; Fig. 2).
3. Results 3.1. IHC staining and ISH for IkBa 4. Discussion A total of 36.4% patients with HL showed positive IkBa expression in H-RS cells by IHC. However, 90.9% of the reactive surrounding lymphocytes showed positive expression. The average scores representing IkBa expression in cytoplasm of H-RS cells by IHC showed lower expression than that of reactive surrounding lymphocytes (Fig. 1). A total of 86.4% patients with HL showed positive NFKBIA mRNA expression in H-RS cells by ISH, however, no patients showed positive NFKBIA mRNA expression in reactive surrounding lymphocytes. NFKBIA mRNA expression by ISH was highly intensive in H-RS cells, whereas just a little was detected in reactive surrounding lymphocytes, showing a lower hybridized signal in comparison with H-RS cells (Fig. 1). The average scores representing NFKBIA mRNA expression in cytoplasm of H-RS cells by ISH were higher than those of reactive surrounding lymphocytes. The scores for the IkBa protein and NFKBIA mRNA expression in H-RS cells or in reactive surrounding cells are summarized in Table 1. There were statistically significant differences between IkBa protein and NFKBIA mRNA expression in H-RS cells (P 5 0.003). (P values were calculated results of McNemar tests by SPSS.) 3.2. Analysis of the NFKBIA in H-RS cells from patients with primary HL Sixteen CD30-positive patients with primary HL were included. Several single H-RS cell portions were collected from each lymph node biopsy sample of each patient using the Leica LMD system. Each aliquot was analyzed by amplification of exons IeVI of NFKBIA mRNA. Among them, several products from each patient with PCR positive were sequenced. The mutations were all unique nucleotide exchanges or deletions. A total of 37.5% of the patients with HL had mutations in the NFKBIA located in exons 1, 4, 5, and 6. Two patients with a point mutation in exon 5
By a single-cell microdissection technique, Ku¨ppers et al. [10] showed that H-RS cells arise from monoclonal B lymphocytes in the germinal centers of lymphoid tissue and that they carry clone-specific rearrangements of IgG (immunoglobulin G) genes. Apoptosis of these cells was found to be inhibited by the constitutively expressed RelA/p50. These results end the long controversy about the nature of Hodgkin disease: is it an inflammatory or infectious disease, or a true malignant process? HL occurs less commonly in Chinese (2.3 cases per 100,000 persons) than in white patients. The difference in incidence rate is caused by environment and genetics [7]. EBV has been implicated in the etiology of HL. EBVencoded small nuclear RNAs status is positive in approximately 40% in primary HL patients in China, similar to that in the Western world. Studies on HL-derived tissue or cell lines suggested that HL is a high level of constitutive nuclear RelA/p50 activity, which stimulates proliferation and confers resistance to apoptosis. Other genetic defects like p53 mutations or bcl-2 translocations may play a role in the pathogenesis of HL. Neither of these ideas could be validated when primary H-RS cells were investigated. Low, frequent Fas mutations or NFKBIE (nuclear factor of k light polypeptide gene enhancer in B-cells inhibitor, 3) mutations in HL cells might explain the constitutive activity of RelA/p50. The aim of this work was to identify molecular defects of IkBa of HL patients in the Chinese population. In NFKBIA / mice, constitutive nuclear factor kB activation could be observed in lymphoid cells [11]. Hence, IkBa seems to play a major role in regulation of nuclear factor kB activity in lymphoid system. By ISH, Emmerich et al. [12] found that NFKBIA mRNAs were strongly overexpressed in 20 HL patients. In contrast to the H-RS cells, only some reactive lymphocytes expressed low to moderate
Fig. 1. Immunohistochemical staining (arrows) of IkBa by C-21 (A) and in situ hybridization of NFKBIA mRNA by NFKBIA antisense probe (B) or by sense probe (C). Compared with background lymphocytes, the IkBa expression in cytoplasm of Hodgkin and ReedeSternberg cells was weak, and NFKBIA mRNA expression was strong (400).
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Table 1 Protein and mRNA expression in H-RS cells in Hodgkin lymphoma* NFKBIA mRNA by ISH
IkBa by IHC Cell
No. of cases
Positive, no. (%)
Average score (mean 6 SD)
Positive, no. (%)
Average score (mean 6 SD)
H-RS RS
22 22
8 (36.4)a 20 (90.9)
3.41 6 1.50b 5.77 6 1.11b
19 (86.4)a 0 (0)
5.27 6 1.08c 2.82 6 0.59c
Abbreviations: H-RS, Hodgkin and ReedeSternberg cells; NFKBIA, nuclear factor of k light polypeptide gene enhancer in B-cells inhibitor, a; IkBa, inhibitor kBa protein; IHC, immunohistochemistry; ISH, in situ hybridization; RS, reactive surrounding cells. * Variables were compared by McNemar tests to determine the difference between protein and mRNA expression. The Wilcoxon signed rank test was used for comparing immunostaining or ISH labeling between H-RS cells and surrounding reactive cells. P values were calculated results by SPSS. a The difference between IkBa and NFKBIA mRNA expression in H-RS cells (P 5 0.003). b The IkBa expression of H-RS cells was lower than that of RS (P ! 0.0001). c The NFKBIA mRNA expression of H-RS cells was higher than that of RS (P ! 0.0001).
amounts of NFKBIA-specific transcripts. To test whether this is also valid for primary H-RS cells from HL patients in the Chinese population, we examined 22 cases of classic HL by a highly sensitive ISH with an NFKBIA mRNA specific cDNA probe. A total of 86.4% patients with HL showed positive NFKBIA mRNA expression in H-RS cells by ISH. In most cases, we detected overexpression of NFKBIA mRNA in the H-RS cells, whereas we detected no or very little amounts of NFKBIA mRNA in reactive surrounding lymphoid tissues (P ! 0.0001). (P values were calculated results of Wilcoxon signed rank test by SPSS.) Therefore, we confirmed that the high level of NFKBIA mRNA was a characteristic feature of primary H-RS cells. On the other hand, 36.4% patients with HL showed positive IkBa expression in H-RS cells by IHC. We found that the scores representing IkBa expression in cytoplasm of H-RS cells was lower than that in reactive surrounding lymphoid tissues by IHC (P ! 0.0001). (P values were calculated results of Wilcoxon signed rank test by SPSS.) Little literature has documented the use of the IHC method to present the expression of IkBa protein in paraffin-embedded sample blocks of HL patients. Also, there were significant differences between IkBa and NFKBIA mRNA expression (P 5 0.003). (P values were calculated results of McNemar tests by SPSS.) Obviously, a reduction of IkBa expression
was observed in H-RS cells in spite of the increased levels of NFKBIA mRNA. Because RelA/p50 activates transcription of its own inhibitor, overexpression of NFKBIA mRNA reflects the high transcriptional activity of RELA/NFKB1 in HL [13]. It has been demonstrated that in normal cells, enforced expression of NFKBIA leads to accumulation of RelA/p50 protein and involves the export of p50 into the cytoplasm, thus contributing to the termination of RelA/p50 activity. The paradoxical finding of high RelA/p50 activity despite the strong expression of its inhibitor indicates that the nuclear factor kB/inhibitor kB system is greatly deregulated in H-RS cells. Cabannes et al. [14] established that the mutations in the NFKBIA gene in HL suggest a tumor suppressor role for NFKBIA in HL patients and related HL cell lines. Obviously, the ubiquitin proteosome pathway may play a role in the process. In addition, a defect of the IkBa molecule could be the mechanism for this loss of control of nuclear factor kB/inhibitor kB system. Researchers have analyzed IkBa in HL-derived cell lines by Western blot test and confirmed a disruption of the coding sequence by a stop codon in some of these cell lines. To analyze whether comparable NFKBIA mutations also occur in primary H-RS cells, we isolated single H-RS cells from CD30-immunostained tissue sections of 16 HL
Table 2 Detection of mutations in IeVI exon regions of the NFKBIA in H-RS cellsa Case
Type
Cells/aliquot (no.)
No. of PCR-positive aliquots
No. of aliquots sequenced
Mutations (exon)
Type
1 2 3 8 13 14
NS MC MC NS NS NS
30 35 40 45 25 35
5 5 6 5 4 6
3 2 2 3 2 3
2692b T-A (5) Delb2538 G (4) 2692b T-A (5) 3584 G-A (6) 3584 G-A (6) 52C-T (1)
nonsense frameshift nonsense trailer trailer trailer
These sequence data are available from National Center for Biotechnology Information, GenBank (NT_026437, NG_007571, with 50 untranslated region assembled another 592 bp). Abbreviations: NFKBIA, nuclear factor of k light polypeptide gene enhancer in B-cells inhibitor, a; H-RS, Hodgkin and ReedeSternberg cells; PCR, polymerase chain reaction; NS, nodular sclerosing Hodgkin lymphoma; MC, mixed-cellularity Hodgkin lymphoma. a No mutations were found in surrounding reactive cells. b In most cases, two or more PCR-positive aliquots along with each exon were sequenced; all products has the same mutations.
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Fig. 2. Chromatographs of DNA sequence for exon 5 (A) and exon 4 (B). (A) Arrow points to site 2692, where the peak shown indicates a T-A mutation, which formed preterminal TGA stop codon. (top, sense and antisense). No mutations were found in the surrounding reactive cells (bottom, sense and antisense). (B) Arrow points to site 2538, where it indicates a G deletion (top). No mutations were found in surrounding reactive cells (bottom). National Center for Biotechnology Information, GenBank, NT_026437, NG_007571, with 50 untranslated region assembled another 592 bp.
patients from the previously mentioned 22 cases of classic HL. The NFKBIA gene was analyzed by single-cell PCR. Three mutations all presumably resulted in a C-terminally truncated form of the IkBa. EBV has been implicated in the etiology of HL by both epidemiologic and serologic studies. IkBa expression level in HL-derived cell lines has been well established by Western blot test in the literature. In Jungnickel’s study, clonal deleterious somatic mutations in the NFKBIA gene were detected in the H-RS cells in two of three EBV-negative and none of two EBV-positive cases of HL [6]. This indicates that the pathogenesis of HL was due to EBV infection or mutations of NFKBIA, and that the two are mutually independent. These results could not be confirmed in our experiments. We tested the IkBa expression level in 22 primary HL patients by IHC. The EBV infection was investigated by testing latent membrane protein 1 (LMP1) expression by IHC. Although LMP-1 status was not detected in all our cases, the correlation of IkBa protein expression levels with EBV status seems variable. NFKBIA aberrant expression was supposed to occur in EBV-positive casesda finding different than the Jungnickel’s conclusions. The exact correlation of IkBa protein expression levels with EBV status is an interesting aspect that should be further investigated. The NFKBIA of two cases contained a mutation generating a preterminal stop of the translational machinery (Table 2). The stop codon was not found in the surrounding nonmalignant lymphoid cells of the same patient, indicating that this mutation was specific for the H-RS cells. One patient had deletion of one nucleotide, which led to a preterminal stop-producing frameshift. Overexpression of NFKBIA mRNA and low intensive staining of IkBa protein in the H-RS cells have occurred in these cases. It is conceivable that the C-terminally truncated protein can block the wild-type protein. Also, aside from its function of reserved RelA/p50 complex in the cytoplasm, IkBa can localize in the nucleus and dissociate the RelA/p50e DNA complex [13]. A prerequisite for effective inhibition of RelA/p50eDNA binding are the ankyrin repeats and an intact C terminus [4]. A role of the C-terminal end for
the inhibition of RelA/p50eDNA binding has recently been suggested by an x-ray structure analysis of the RelA/p50/ IkBa complex [15]. Furthermore, IkBa contains a nuclear export signal at amino acids 264e281, which confers active shuttling of the RelA/p50/IkBa complex to the cytosol. Therefore, the truncated IkBa may protect DNA-bound RelA/p50 from dissociation by wild-type IkBa and interfere with the nuclear export pathway [16]. Consequently, RelA/p50eDNA binding activity would be maintained in H-RS cells. The truncated IkBa and its impact on normal IkBa led to the IHC detection insensitivity. Although the observed mutations were found only in 3 of 16 cases, our data provide evidence of gene aberrations in a large number of Chinese HL patients. Permanently activated RelA/p50 in primary H-RS cells might be a consequence of gene mutation of one of its inhibitors. It is therefore tempting to speculate that defects of additional members of the inhibitor kB family contribute to the functional blockage of RelA/p50 in the remaining cases. Indepth studies are required to investigate this possibility.
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