Aberrant FHIT protein expression in classical Hodgkin’s lymphoma: a potential marker

Pathology

ISSN: 0031-3025 (Print) 1465-3931 (Online) Journal homepage: http://www.tandfonline.com/loi/ipat20

Aberrant FHIT protein expression in classical Hodgkin's lymphoma: a potential marker Po Zhao, Alfred King Yin Lam, Ya‐Li Lu, Mei Zhong, Ling‐Hong Chen & Xiao‐Lu Pu To cite this article: Po Zhao, Alfred King Yin Lam, Ya‐Li Lu, Mei Zhong, Ling‐Hong Chen & Xiao‐Lu Pu (2006) Aberrant FHIT protein expression in classical Hodgkin's lymphoma: a potential marker, Pathology, 38:5, 399-402 To link to this article: https://doi.org/10.1080/00313020600922512

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Pathology (October 2006) 38(5), pp. 399–402

ANATOMICAL PATHOLOGY

Aberrant FHIT protein expression in classical Hodgkin’s lymphoma: a potential marker PO ZHAO*, ALFRED KING YIN LAM{, YA-LI LU*, MEI ZHONG*, LING-HONG CHEN* XIAO-LU PU*

AND

*Department of Pathology, General Hospital of Chinese People’s Liberation Army, Beijing, People’s Republic of China, and {Discipline of Pathology, Griffith Medical School, Gold Coast, Queensland, Australia

Summary Aims: The fragile histidine triad (FHIT) gene is frequently inactivated in human cancers; however, the FHIT gene remains unexplored in Hodgkin’s lymphoma. The aim of this study was to investigate the role of FHIT expression in classical Hodgkin’s lymphoma. Methods: Classical Hodgkin’s lymphomas were analysed for FHIT gene expression by two-step non-biotin immunohistochemical method and Western blotting. Results: Thirty of the 33 (91%) cases of Hodgkin’s lymphoma tested were positive for FHIT protein by immuohistochemistry. The expression of FHIT was mainly located in cytoplasm of Reed–Sternberg (HRS) cells. The protein expression was also documented by Western blotting. The non-Hodgkin’s lymphomas were negative for FHIT protein. Conclusions: The results indicate that abnormal FHIT expression is noted frequently in classical Hodgkin’s lymphoma and the expression can give insight into the pathogenesis of the disease. The protein may serve as a marker to localise HRS cells in classical Hodgkin’s lymphoma. Key words: Hodgkin’s lymphoma, FHIT, Reed–Sternberg cells. Received 31 March, revised and accepted 5 June 2006

INTRODUCTION Hodgkin’s lymphoma comprises two distinct entities; namely, nodular lymphocyte predominant Hodgkin’s lymphoma and classical Hodgkin’s lymphoma. Classical Hodgkin’s lymphoma is a monoclonal lymphoid neoplasm composed of mononucleate Hodgkin’s cells and multinucleated Reed–Sternberg (HRS) cells residing in an infiltrate containing a variable mixture of non-neoplastic small lymphocytes, eosinophils, neutrophils, histiocytes, plasma cells fibroblasts and collagen fibres.1 There are four morphological variants of classical Hodgkin’s lymphoma: nodular sclerosis, mixed cellularity, lymphocyte-rich and lymphocyte-depleted. HRS cells are pathognomonic in Hodgkin’s lymphoma. However, HRS cells may be scanty and difficult to identify in sections. The pathogenesis of classical Hodgkin’s lymphoma remains unknown. The nature of the Reed–Sternberg cell has been one of the most controversial issues in pathology. Practically all the cells present in the normal lymph node have been proposed at one time or another as possible

progenitors of HRS cells, including B cells, T cells, histiocytes, follicular dendritic cells, and interdigitating dendritic cells.2–5 The current literature favours that neoplastic cells in Hodgkin’s lymphoma are derived from mature B cells at the germinal stage of differentiation. Also, Epstein–Barr virus (EBV) is suggested to play a certain role in the pathogenesis of Hodgkin’s lymphoma;6–8 however, the virus is noted in only a small portion of Hodgkin’s lymphomas. Thus, it is worth searching for other factors involved in the pathogenesis of Hodgkin’s lymphoma. A growing body of evidence supports the concept that certain human chromosomal fragile sites have roles to play in cancer.9 Research in this area has focused on the fragile histidine triad gene (FHIT) located in chromosome 3 at 3p14.2. FHIT is altered in many kinds of primary or cultured carcinoma in the form of deletion within both FHIT alleles, resulting in loss of exons and concomitant absence of full-length FHIT transcript and protein.10 Alterations of the FHIT gene are common and have been found in primary tumours and cell lines of lung, breast, head and neck, oesophagus, stomach, colon and rectum, pancreas, kidney, cervix, and liver.9–12 In haematological malignancies, genetic alternations or decreased FHIT protein expression is noted in leukaemias, myelodysplastic/myeloproliferative diseases, multiple myleoma and non-Hodgkin’s lymphomas.13–20 The role of FHIT in Hodgkin’s lymphoma has not been reported in the literature. In this study, we investigate the pathogenetic role of the expression of FHIT protein in classical Hodgkin’s lymphoma. It is hoped that this study will give information on the pathogenesis of this lymphoma.

MATERIALS AND METHODS Patients Thirty-three patients (20 men, 13 women) with histologically confirmed classical Hodgkin’s lymphoma were recruited into the study. Consent to the research was given by the standard human ethics committee approval. The Hodgkin’s lymphomas were classified as nodular sclerosis (n513), mixed cellularity (n55), lymphocyte-rich (n511) and lymphocyte-depleted (n54) variants according to the World Health Organization recommendations.1 Immunohistochemistry A formalin-fixed, paraffin-embedded block was selected from each patient with Hodgkin’s lymphoma for immunohistochemical study. Paraffin sections 4 mm thick were cut, dewaxed in xylene and rehydrated in a

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graded ethanol series. The sections were immersed in 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity and rinsed in running water. They were then immersed in boiling 0.01 M citrate buffer (pH 6.0) in a pressure cooker. The pressure cooker was sealed and brought to full pressure. The heating time was 2 min. After that, the pressure cooker was de-pressured and cooled under running water. The lid was then removed, and the hot buffer was flushed out with cold water from a running tap. The cooled sections were washed twice in phosphate buffered saline (PBS) before immunocytochemical staining. The primary antibodies were then added to the sections. The polyclonal rabbit antibody against human FHIT protein (Zymed, USA) was diluted 1:100 for 1 h. After exposure to primary antibody overnight, the sections were allowed to react with the polyperoxidase-anti-mouse/rabbit IgG for 20 min using the standard PV-6000 Polymer Detection System (Zymed). This is a non-biotin detection system; thus, non-specific staining due to endogenous biotin was avoided. The sections were then washed in water, counterstained with Mayer’s haematoxylin for 1 min at room temperature, dehydrated, cleaned and mounted. Paraffin blocks of human liver, lung spleen and breast ductal carcinoma tissues were used as the positive controls. Negative controls were sections treated the same as above but with omission of the primary antibody and replaced by 0.01 M PBS. In addition, 10 B-cell lymphomas and 8 T-cell lymphomas and 10 reactive lymph nodes (4 lymph nodes sampled during resection of carcinomas and 6 lymphadenitis) were used for comparison. The B-cell lymphomas comprised five diffuse large B-cell lymphomas, four extranodal marginal zone B-cell lymphomas (MALT lymphomas) and one Burkitt’s lymphoma. The T-cell lymphomas were two anaplastic large cell lymphomas, two extranodal NK/T-cell lymphomas of nasal type, two angioimmunoblastic T-cell lymphomas and two peripheral T-cell lymphomas, unspecified. The histiocytes in the background of lymphomas were used as internal positive control. Western blotting Five classical Hodgkin’s lymphomas (two nodular sclerosis, two mixed cellularity and one lymphocyte depleted) were prospectively recruited into the study. The tumour samples were collected within 30 min of surgery and were immediately frozen in liquid nitrogen and stored at 280uC. The tissues were homogenised in lysis buffer (20 mmol/L Tris-HCL at pH 7.5, 1 mmol/L EDTA, 1 mmol/L EGTA, 150 mmol/L sodium chloride, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L b-glycerolphophate, 1 mg/mL leupeptin, 1 mol/L phenylmethylsulfonyl fluoride). After removing cellular debris by centrifugation, the protein content was quantitated by BCA protein assay kit (Pierce Biotech, USA). The samples were fractionated on SDSpolyacrylamide and electrotransferred to nitrocellulose membrane. The membrane was incubated in a blocking buffer (1 g/L Tween-20 with 50 g/L non-fat dry milk in Tris-buffered saline) for 3 h. The polyclonal rabbit antibody against human FHIT protein (Zymed) was diluted by primary antibody dilution buffer (1 g/L Tween-20, 50 g/L BSA in Tris-buffered saline) at 1:2000. The membrane stripe was then incubated with FHIT antibody overnight at 4uC. The expression of FHIT protein was detected using Phototope-HRP (horseradish peroxidase) Western Blot Detection Kit (Cell Signaling Technology, USA).

RESULTS FHIT protein was noted in 91% (30/33) of the classical Hodgkin’s lymphomas by immunohistochemistry (Fig. 1, 2). The three Hodgkin’s diseases negative for FHIT were two mixed cellularity type and one lymphocyte-rich type. Western blotting analysis also confirmed the presence of FHIT protein in five cases in which the protein was detected by immunohistochemistry. The FHIT protein was noted mainly in the cytoplasm and infrequently in the nucleus of Hodgkin’s and Reed–Sternberg (HRS) cells. Some Hodgkin’s cells also showed membranous staining of FHIT protein. The mummified HRS cells were often

Pathology (2006), 38(5), October

negative for FHIT protein. Many of the HRS cells with very prominent nucleoli could be found negative for FHIT staining. The histiocytes in the background of the lymphoma or other tissues showed strong nuclear staining for the FHIT protein. No FHIT staining was noted in the lymphocytes, plasma cells or eosinophils. In the control tissues and reactive lymph nodes, histiocytic lineage cells including macrophage, Langerhans, and dendritic cells (interdigitating and follicular dendritic cells) were positive for FHIT. The staining was noted in the nuclei of the histiocytes. In the non-Hodgkin’s lymphomas (B-cell and T-cell lymphomas), FHIT protein was not found.

DISCUSSION Aberrant expression of FHIT was noted in a few series studying non-Hodgkin’s lymphoma. Hussain et al. reported loss of FHIT in 50% of cell lines from Burkitt’s lymphoma, whereas Ferrer et al. found expression of aberrant and nonfunctional transcripts of the FHIT gene in 80% of cell lines.16,17 In situ location of FHIT protein was performed by Chen et al. in 31 diffuse large B-cell lymphomas15 and they noted that less than half showed strong FHIT expression as detected by immuohistochemical method. In this study, we found that all 18 non-Hodgkin’s lymphomas (including B-cell lymphomas and T-cell lymphomas) lacked FHIT protein expression. The difference in prevalence of aberrations of FHIT in different series may be related to different experimental conditions. Nevertheless, all the studies suggest that alterations of FHIT gene are important in non-Hodgkin’s lymphoma. The present study is the first to analyse FHIT protein expression in classical Hodgkin’s lymphoma. FHIT protein was noted in 91% of the classical Hodgkin’s lymphoma samples. Thus, unlike other malignancies, loss of the tumour suppressor functions of FHIT is not important in classical Hodgkin’s lymphoma. This supports the notion that the pathogenesis of Hodgkin’s lymphoma is different from non-Hodgkin’s lymphomas. Also, FHIT staining highlighted the Reed–Sternberg cells from the background lymphocytes in the lymphoma. Thus, FHIT can be used an adjuvant marker to identify Reed–Sternberg cells in cases when the diagnosis of Hodgkin’s lymphoma is in doubt. The current literature suggests that the neoplastic cells in Hodgkin’s lymphoma are derived from mature B cells at the germinal stage of differentiation.5 However, there is evidence of histiocytic differentiation in Hodgkin’s lymphoma. Pinkus et al. found that fascin, a marker for dendritic cells, was strongly expressed in 92.5% (173/187) of HRS cells in Hodgkin’s lymphoma.21 Nakamura et al. showed that HRS cells expressed the follicular dendritic cell marker, CD21.22 In the present study, FHIT staining was confined to cells of histiocytic lineage, such as macrophage, Langerhans, and dendritic cells (interdigitating and follicular dendritic cells). Lymphocytes and malignancies of lymphoid origin (T-cell and B-cell lymphomas) were negative for FHIT. Thus, the neoplastic cells of Hodgkin’s lymphoma, HRS cells, may be derived from germinal B cells with lymphohistiocytic phenotype that can express FHIT. The alteration of cellular localisation may be important in the mechanism of carcinogenesis. In fact, changes in

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Fig. 1 FHIT protein expression status. FHIT protein is expressed in the nucleus of histiocytic lineage cells including (A) foamy cells, (B) dust cells and (C) Kuffer’s cells but is negative in (D) the polymorphs. Lymphocytes both in (E) B and (F) T regions of lymph nodes are negative, but macrophages and dendritic cells are positive for FHIT expression. FHIT staining is negative in the tumour cells of (G) B-cell lymphoma and (H) T-cell lymphoma.

cellular localisation of oncoproteins during pathogenesis have been reported in cell lines and fresh samples of leukaemia.23 Golebiowski et al. first reported the results of distribution of FHIT protein and its intracellular localisation in rat cells.24 They showed that FHIT protein was localised exclusively in the nucleus and the plasma membrane, supporting the hypothesis that FHIT is a signalling molecule. In this study, FHIT protein was expressed in the nucleus in the histiocytes in non-neoplastic tissues. On the other hand, FHIT protein was noted mostly in the cytoplasm of HRS cells. FHIT may be a stronger

tumour suppressor in the nucleus than in the cytoplasm. The transitional FHIT protein from nucleus to cytoplasm and membrane, and finally to the loss of expression can be found in the HRS cells. Thus, similar to the finding in leukaemia, the change in cellular location of FHIT may be related to the evolution of classical Hodgkin’s lymphoma. Address for correspondence: Dr P. Zhao, Department of Pathology, General Hospital of Chinese People’s Liberation Army, 28 Fuxing Road, Beijing 100853, People’s Republic of China. E-mail: zhaopo@plagh. com.cn. Professor A. K. Y. Lam, Griffith Medical School, Medicine and Oral Health Centre, PMB 50 GCMC Bundall, Gold Coast, Qld 9726, Australia. E-mail: [email protected]

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Fig. 2 FHIT protein expression in Hodgkin’s lymphoma. HRS cells in the Hodgkin’s lymphoma are positive for the expression of FHIT protein; the positive staining can be found in the (A) membrane, (A–D) cytoplasm and (D) nucleus.

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13. Ishii H, Furukawa Y. Alterations of common chromosome fragile sites in hematopoietic malignancies. Int J Hematol 2004; 79: 238–42. 14. Kameoka Y, Tagawa H, Tsuzuki S, et al. Contig array CGH at 3p14.2 points to the FRA3B/FHIT common fragile region as the target gene in diffuse large B-cell lymphoma. Oncogene 2004; 23: 9148–54. 15. Chen PM, Yang MH, Hsiao LT, et al. Decreased FHIT protein expression correlates with a worse prognosis in patients with diffuse large B-cell lymphoma. Oncol Rep 2004; 11: 349–56. 16. Ferrer M, Lopez-Borges S, Lazo PA. Expression of aberrant functional and nonfunctional transcripts of the FHIT gene in Burkitt’s lymphomas. Mol Carcinog 1999; 25: 55–63. 17. Hussain A, Gutierrez MI, Timson G, et al. Frequent silencing of fragile histidine triad gene (FHIT) in Burkitt’s lymphoma is associated with aberrant hypermethylation. Genes Chromosomes Cancer 2004; 41: 321–9. 18. Takada S, Morita K, Hayashi K, et al. Methylation status of fragile histidine triad (FHIT) gene and its clinical impact on prognosis of patients with multiple myeloma. Eur J Haematol 2005; 75: 505–10. 19. Hallas C, Albitar M, Letofsky J, Keating MJ, Huebner K, Croce CM. Loss of FHIT expression in acute lymphoblastic leukemia. Clin Cancer Res 1999; 5: 2409–14. 20. Luan X, Ramesh KH, Cannizzaro LA. FHIT gene transcript alterations occur frequently in myeloproliferative and myelodysplastic diseases. Cytogenet Cell Genet 1998; 81: 183–8. 21. Pinkus GS, Pinkus JL, Langhoff E, et al. Fascin, a sensitive new marker for Reed-Sternberg cells of Hodgkin’s desease: evidence for dendritic or B cell derivation? Am J Pathol 1997; 150: 543–62. 22. Nakamura S, Nagahama M, Kagami Y, et al. Hodgkin’s disease expressing follicular dendritic cell marker CD21 without any other Bcell marker; a clinicopathologic study of nine cases. Am J Surg Pathol 1999; 23: 363–76. 23. Wetzler M, Talpaz M, Van Etten RA, Hirsh-Ginsberg C, Beran M, Kurzrock R. Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation. J Clin Invest 1993; 92: 1925–39. 24. Golebiowski F, Kowara R, Pawelczyk T. Distribution of Fhit protein in rat tissues and its intracellular localization. Mol Cell Biochem 2001; 226: 49–55.