Sequence variations of Epstein‐Barr virus‐encoded BARF1 gene in nasopharyngeal carcinomas and healthy donors from southern and northern China

The BamHI A rightward frame 1 (BARF1) gene of the Epstein‐Barr virus (EBV) is involved in carcinogenesis and immunomodulation of EBV‐associated malignancies. The geographical distributions and the disease associations of BARF1 variants remain unclear. In the current study, the BARF1 variants in nasopharyngeal carcinoma (NPC) cases and healthy donors from southern and northern China, the NPC endemic and non‐endemic areas, as well as in 153 sequenced EBV genomes from diseased and normal people from around the world, were determined and compared among areas and populations. Only 1 consistent coding change, V29A, and several consistent silent mutations were identified. Two BARF1 types (B95‐8 and V29A) and 2 B95‐8 subtypes (B95‐8^t165545c and B95‐8^P) were classified. For Chinese isolates, the B95‐8 type was dominant in both southern and northern China, but the isolates from southern China showed a higher frequency of the B95‐8^t165545c subtype than the isolates from northern China (76.0%, 38/50 NPC cases and 50.7%, 37/73 healthy donors vs 26.4%, 24/91 NPC cases and 7.6%, 6/79 healthy donors, P < .0001). Furthermore, the B95‐8^t165545c subtype was more frequent in NPC cases than healthy donors in both southern China (P = .005) and northern China (P = .001). For EBV genomes, the B95‐8^P subtype was dominant in northern China, Europe, America, and Australia, while V29A was dominant in Africa. The B95‐8^t165545c subtype was only identified in Asia and demonstrated high frequency (81.2%, 26/32) in genomes from NPC cases in southern China. These results further reveal conservation and possibly geographically spread variations of BARF1 and may also indicate the preference of EBV strains with the B95‐8^t165545c subtype in NPC cases, without biological or pathogenic implications.     

Role of DNA methylation in the development of Epstein–Barr virus‐associated gastric carcinoma

The frequencies of DNA methylation of certain tumor‐related genes are higher in Epstein–Barr virus (EBV)‐associated gastric carcinomas than in EBV‐negative gastric carcinomas. EBV‐associated gastric carcinomas have distinct clinicopathological features; however, there are no case‐control studies comparing methylation frequency between EBV‐associated gastric carcinomas and controls that have been adjusted according to the clinicopathological features of EBV‐associated gastric carcinomas. This study evaluated 25 EBV‐associated gastric carcinomas that were positive for EBV‐encoded small RNA 1 (EBER‐1) by in situ hybridization and 50 EBV‐negative gastric carcinomas that were matched with the EBV‐associated gastric carcinomas by age, sex, histology, depth of tumor invasion, and stage. Methylation status of 16 loci associated with tumor‐related genes was analyzed by methylation‐specific polymerase chain reaction (PCR) to identify genes in which DNA methylation specifically occurred in EBV‐associated gastric carcinomas. Methylation frequencies of 12 of the 16 genes were higher in EBV‐associated gastric carcinomas than in EBV‐negative controls, and the frequency of methylation of 6 specific loci (MINT2, MINT31, p14, p16, p73, and RUNX3) was significantly higher in EBV‐associated gastric carcinomas than in EBV‐negative controls. There were no significant differences in the methylation frequencies of the other genes. The mean methylation index in EBV‐associated gastric carcinomas was significantly higher than that in EBV‐negative controls. DNA methylation of tumor suppressor genes that regulate the cell cycle and apoptosis specifically occurred in EBV‐associated gastric carcinomas. Aberrant DNA methylation might lead to the development and progression of EBV‐associated gastric carcinoma. J. Med. Virol. 85:121–127, 2012. © 2012 Wiley Periodicals, Inc.     

Inhibition of the LKB1–AMPK pathway by the Epstein–Barr virus‐encoded LMP1 promotes proliferation and transformation of human nasopharyngeal epithelial cells

The association of Epstein–Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1–AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1–AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK–MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1–AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1‐induced cellular transformation (proliferation and anchorage‐independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage‐independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5‐fluorouracil. The current findings demonstrate that LMP1‐mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1–AMPK pathway in the EBV‐driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activation of the FGFR1 signalling pathway by the Epstein–Barr virus‐encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells

Non‐keratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signalling pathways which collectively promote cell proliferation, transformation, angiogenesis, and invasiveness, as well as modulation of energy metabolism. In this study, we report that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentrations. LMP1 also increases the phosphorylation of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c‐Myc, and HIF‐1α, regardless of oxygen availability. Collectively, these findings suggest that LMP1 promotes aerobic glycolysis. With respect to FGFR1 signalling, LMP1 not only increases FGFR1 expression, but also up‐regulates FGF2, leading to constitutive activation of the FGFR1 signalling pathway. Furthermore, two inhibitors of FGFR1 (PD161570 and SU5402) attenuate LMP1‐mediated aerobic glycolysis, cellular transformation (proliferation and anchorage‐independent growth), cell migration, and invasion in nasopharyngeal epithelial cells, identifying FGFR1 signalling as a key pathway in LMP1‐mediated growth transformation. Immunohistochemical staining revealed that high levels of phosphorylated FGFR1 are common in primary NPC specimens and that this correlated with the expression of LMP1. In addition, FGFR1 inhibitors suppress cell proliferation and anchorage‐independent growth of NPC cells. Our current findings demonstrate that LMP1‐mediated FGFR1 activation contributes to aerobic glycolysis and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signalling activation in the EBV‐driven pathogenesis of NPC. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Epstein–Barr virus infection in non-carcinomatous gastric epithelium

Gastric tissue specimens from 20 patients with chronic atrophic gastritis, one of whom also had an early gastric carcinoma, were studied for evidence of Epstein–Barr virus (EBV) infection by Southern blot analysis, DNA and RNA in situ hybridization, and immunohistochemistry for the presence of the EBV-determined nuclear antigen 1 (EBNA-1) and the latent membrane protein 1 (LMP-1). EBV DNA was detected in two cases with chronic atrophic gastritis and in the case with early gastric carcinoma by Southern blot hybridization. DNA in situ hybridization showed EBV genomes in the epithelial cells of two other cases with chronic atrophic gastritis and in non-carcinomatous and carcinomatous epithelial cells of the early gastric carcinoma case. EBNA-1 was detected in all cases. LMP-1 was detected in areas of intestinal metaplasia in eight patients with chronic atrophic gastritis. EBV-encoded small RNA 1 (EBER-1) expression was limited to carcinoma cells. These results show that gastric epithelium is frequently infected with EBV and suggest that prolonged EBV persistence may contribute to the development of gastric carcinoma. © 1997 John Wiley & Sons, Ltd.     

Association of distinctive Epstein–Barr virus variants with gastric carcinoma in Guangzhou,southern China

To investigate the clinicopathologic features, Epstein–Barr virus (EBV) latency pattern and genome polymorphism of EBV‐associated gastric carcinoma (EBVaGC) in Guangzhou, an endemic area of nasopharyngeal carcinoma (NPC), an in situ hybridization assay of EBV‐encoded small RNA‐1 (EBER‐1) was used to identify the presence of EBV in 676 consecutive gastric carcinoma cases. EBV‐encoded proteins EBNA1, EBNA2, LMP1, and ZEBRA were detected by immunohistochemistry. EBV genome polymorphism was also analyzed by PCR and DNA sequencing. Of the 676 cases, 45 EBV‐positive cases (6.7%) were identified, including 37 (8.5%) male and 8 (3.3%) female cases. EBNA1 was detected in 42 cases (93.3%), while EBNA2, LMP1, and ZEBRA were all negative. In the EBV genome polymorphism analysis, type A strain, prototype F, type I, XhoI?, and del‐LMP1 variants were predominant among EBVaGC patients, accounting for 44 (97.8%), 37 (82.2%), 45 (100%), 34 (75.6%), and 42 (93.3%) cases, respectively. Moreover, a new hotspot mutation in the BamHI‐W1/I1 boundary region (148,972 T → C) was found in 39 (86.7%) of the 45 cases. The predominant EBV variants in EBVaGC in Guangzhou are prototype F, type I, and XhoI?, which are different from those in NPC in this area (predominant variant‐type “f”) and in EBVaGC in Latin American countries (predominant type “i” and XhoI+), suggesting that the EBV variants are not only geographically distributed but also disease restricted, and the pathogenic role of EBV in different EBV associated epithelial malignancies in different areas may be distinct. J. Med. Virol. 82:658–667, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of sterol regulatory element‐binding protein 1 (SREBP1)‐mediated lipogenesis by the Epstein–Barr virus‐encoded latent membrane protein 1 (LMP1) promotes cell proliferation and progression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation, and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element‐binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, small interfering RNA (siRNA) knockdown of LMP1 in EBV‐infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mammalian target of rapamycin (mTOR) pathway, through the use of either mTOR inhibitors or siRNAs, significantly reduced LMP1‐mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1‐mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN expression was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells, effects that were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo. Our findings demonstrate that LMP1 activation of SREBP1‐mediated lipogenesis promotes tumor cell growth and is involved in EBV‐driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Characterization of ELISA detection of broad‐spectrum anti‐Epstein–Barr virus antibodies associated with nasopharyngeal carcinoma

Epstein–Barr virus (EBV) infection is associated with undifferentiated nasopharyngeal carcinomas (NPC). A distinct seroreactivity pattern to EBV is predictive of subsequent risk of sporadic and familial nasopharyngeal carcinomas. There are currently no accepted screening tools for guiding the clinical management of individuals at high‐risk for nasopharyngeal carcinomas, particularly unaffected relatives from nasopharyngeal carcinoma multiplex families. Therefore, the reproducibility of a panel of largely synthetic peptide‐based anti‐EBV antibody ELISAs was evaluated and their ability to distinguish nasopharyngeal carcinoma cases from controls was explored. IgG and IgA antibodies against 6 different EBV antigens (10 assays, total) were tested on sera from 97 individuals representing the full spectrum of anti‐EBV seroprevalence (i.e., healthy individuals with no known EBV seroreactivity, healthy individuals with known EBV seroreactivity, and nasopharyngeal carcinoma cases). Each specimen was tested in triplicate to assess within‐batch and across‐batch variation, and the triplicate testing was repeated on two separate days. Reproducibility was assessed by the coefficients of variation (CVs) and intraclass correlation coefficients (ICCs). All markers were detectable in 17% or more of samples. For all but one marker, the overall, within‐batch, and across‐batch CVs were below 15%, and the ICCs were above 70% for all but three markers. Sensitivity of these markers to detect prevalent nasopharyngeal carcinomas ranged from 22% to 100%, and among unaffected controls, most distinguished those with and without known seropositivity. In conclusion, a large number of EBV markers can be measured reliably in serum samples using peptide‐based anti‐EBV ELISAs. J. Med. Virol. 85:524–529, 2013. Puiblished 2012. This is a US government work, and, as such, is in the public domain of The United States of America.     

The dominance of China 1 in the spectrum of Epstein–Barr virus strains from Cantonese patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma is a disease with a remarkable geographic and ethnic distribution, and has a high incidence in southern China. Infection with Epstein–Barr virus (EBV) is an important contributing factor. The profile of EBV strains in Cantonese patients from Guangdong, the nasopharyngeal carcinoma endemic region in southern China, is described on the sequence variations in latent membrane protein 1 carboxyl‐terminus. The results show that China 1 was the dominant EBV strain detected in both the tumor biopsies and samples of throat washings, whereas multiple strains, including China 1, China 2, B95‐8, and Med, were detected in blood samples. In addition, a new strain named China 4 was found in blood samples. These findings suggest that the host population is susceptible to the predominant China 1 strain in the nasopharyngeal carcinoma endemic region of China, but its relationship with the host remains to be characterized further. J. Med. Virol. 81:1253–1260, 2009. © 2009 Wiley‐Liss, Inc.     

The association of genomic variation of Epstein–Barr virus BamHI F fragment with the proliferation of nasopharyngeal carcinoma

Liu Q, Han A, You S, Yang Q, Liang Y, Dong Y. The association of genomic variation of Epstein–Barr virus BamHI F fragment with the proliferation of nasopharyngeal carcinoma. APMIS 2010; 118: 657–64. To investigate the f variant of Epstein–Barr virus (EBV) in nasopharyngeal carcinogenesis, we detected the f variant in primary nasopharyngeal carcinoma (NPC), metastatic carcinoma of the lymph node (LN), and chronic inflammation of the nasopharynx from the Guangdong region. Meanwhile, we analyzed the relationship between the f variant of EBV and LMP1, Fascin, pStat3, p53, Bcl‐2, and Ki‐67 expression in NPC. The results showed that the f variant of EBV was found in 11 cases of primary NPCs with LN metastasis, 12 LN metastases, and 18 primary NPCs without LN metastasis. However, only one demonstrated the F/f variant in 50 cases of chronic inflammation of the nasopharynx. The expression rate of LMP1, Fascin, pStat3, p53, Bcl‐2, and Ki‐67 in NPC with the f or F/f variant was higher than that with the F prototype. Furthermore, there was a significantly positive correlation between the f variant of EBV and Ki‐67 expression (p < 0.05). Our study suggests that the f variant of EBV may be closely related to nasopharyngeal carcinogenesis.     

Epstein–Barr virus in Reed–Sternberg G-like cells in non Hodgkin's lymphomas

In the course of our study on Hodgkin's disease (HD), ten cases of non-Hodgkin's lymphomas (NHL) containing Hodgkin and Reed-Sternberg-like (MRS) cells were encountered. Many of these cases had initially been diagnosed as HD, but on careful review of the histology, with the aid of immunophenotyping studies, they were reclassified as NHL. The presence of Epstein–Barr virus (EBV) in these HRS-like cells was investigated using a combination of EBER in situ hybridization (ISH) and immunostaining for the detection of EBV-encoded latent membrane protein (LMP). HRS-like cells in four cases (two lymphoplasmacytoid lymphomas, one Richter's transformation of lymphoplasmacytoid lymphoma, and one immunoblastic lymphoma of T-cell type) were found to be EBV-positive. In two of these cases, a second biopsy taken up to 10 years later also contained EBV in the HRS-like cells. In three of the four cases, HRS-like cells expressed the activation antigen CD30, but the expression of B- or T-cell antigens was variable. All cases of T-cell-rich B-cell lymphomas were negative for EBV. In conclusion, EBV may play a role in the development of HRS-like cells i some cases of NHL. The relationship of HRS-like cells to HRS cells of HD is discussed.     

Histocompatibility locus antigens regions contribute to the ethnicity bias of Epstein‐Barr virus‐associated nasopharyngeal carcinoma in higher‐incidence populations

Nasopharyngeal carcinoma (NPC) is one the most confusing and rare malignancy in most part of the world with significantly high occurrence in some populations of Southeast Asia, North Africa and Alaska. Apart from the dietary and environmental factors, NPC is well‐associated with Epstein‐Barr virus (EBV) infection in these ethnic groups. However, the internal molecular mechanism(s) for such association in specific populations is not known till date. Polymorphisms in the genes of histocompatibility locus antigens (HLA) are reported in NPC, but association of any particular polymorphism with ethnicity is not established yet. Here, we report a set of HLA polymorphisms in EBV‐infected NPC samples from Northeast Indian population. These polymorphisms might play an important role for the lack of proper immune function against EBV infection and thus, eventually, for NPC generation in endemic populations like those of Northeast India.     

Conservation and mutation of viral interleukin-10 gene in gastric carcinomas and nasopharyngeal carcinomas

The Epstein-Barr virus (EBV) BamHI-C fragment rightward reading frame 1 (BCRF1)-coded viral interleukin-10 (vIL-10), exhibits high homology with human interleukin-10 (hIL-10) gene. The protein product vIL-10, which shares some functional properties with hIL-10, primarily mediates immunosuppressive functions. To characterize the variations of the vIL-10 gene and to explore the association between vIL-10 gene variations and EBV associated diseases, the vIL-10 gene was analyzed (using direct sequencing) in 41 cases of EBV-associated gastric carcinoma (EBVaGC), 83 nasopharyngeal carcinoma biopsies, and 40 throat washing samples from healthy donors in Northern China. One silent mutation (c9980a) was observed in the majority of EBVaGC, nasopharyngeal carcinoma and throat washing samples (134/164, 81.7%). Two consensus mutations (V6M and G23S) were identified in the signal peptide region in some nasopharyngeal carcinoma and throat washing isolates. These results indicate that the pattern B95-8 (identical sequence to B95-8) is the dominant type among the EBV isolates from Northern China, while the pattern SPM (mutation in the signal peptide present only in nasopharyngeal carcinoma and throat washing isolates) seems more relevant with the EBV-positive nasopharyngeal and laryngopharyngeal epithelial cells. The conservation of vIL-10, with a few variations, suggests the critical role of the vIL-10 gene for EBV in gaining an advantage over the host's immune system.     

Cytomegalovirus and Epstein–Barr virus coinfection in three toddlers with prolonged illnesses

Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) usually cause primary and latent infections during childhood; thus, coinfection with these viruses occurs occasionally in children. However, its clinical impact has not been established, and may be underestimated. Three cases of coinfection involving these two viruses in toddlers are described: a 14‐month‐old male with infectious mononucleosis, an 18‐month‐old female with hemophagocytic lymphohistiocytosis, and a 13‐month‐old female with acute hepatitis. All three patients had prolonged illnesses. Serial serological testing and quantitation of viral DNA for CMV and EBV using peripheral blood from the patients suggested primary infections with both viruses. In all three cases, the viral load of EBV and CMV in the early stage of disease exceeded 6.4 × 10³ and 8.8 × 10² copies/ml of whole blood, respectively, suggesting that the viruses were associated with the clinical condition. Recognizing that coinfection with these viruses may modulate the clinical course of disease is important. J. Med. Virol. 81:1399–1402, 2009. © 2009 Wiley‐Liss, Inc.     

Epstein–Barr virus infection induces genome‐wide de novo DNA methylation in non‐neoplastic gastric epithelial cells

Epstein–Barr virus (EBV)‐positive gastric cancer (GC) shows a higher DNA methylation epigenotype. EBV infection can causally induce genome‐wide aberrant DNA methylation, as previously demonstrated by in vitro infection experiments in the low‐methylation GC cell line MKN7. However, whether EBV exerts DNA methylation remodelling properties in non‐neoplastic epithelial cells remains unclear. Here we performed post‐infection time‐series DNA methylation analyses using the immortalized normal gastric epithelial cell line GES1. Genome‐wide analysis using Illumina's Infinium 450 k BeadArray demonstrated global de novo DNA methylation from post‐infection day 17, which was completed by 28 days in a manner similar to that observed in MKN7 cells. De novo methylation of all types of GC‐specific methylation marker genes was observed, indicating that EBV infection is sufficient for gastric epithelial cells to acquire an EBV‐positive GC epigenotype. Pyrosequencing demonstrated that methylation of the viral genome preceded that of the host cellular genome, suggesting the existence of well‐ordered mechanisms that induce methylation. Spatiotemporal representation with differential models revealed dynamic alterations of DNA methylation in promoter regions, occurring from lower‐CpG peripheral regions and extending to higher‐CpG core regions. In summary, EBV infection exerted powerful pressure to induce global de novo DNA methylation in non‐neoplastic cells within a month in a spatiotemporally well‐ordered manner. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The ATM tumour suppressor gene is down‐regulated in EBV‐associated nasopharyngeal carcinoma

A micro‐array analysis using biopsies from patients with EBV‐positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer‐free controls revealed down‐regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q‐PCR confirmed down‐regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down‐regulated only in the EBV‐positive line, C666.1, and in none of five EBV‐negative lines. In vitro infection of EBV‐negative NPC cell lines with a recombinant EBV was followed by the down‐regulation of ATM mRNA and protein, and only EBV‐positive cells showed a defective DNA damage response following γ‐irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Disappearance of the Epstein–Barr virus in a relapse of Hodgkin's disease

Hodgkin's disease (HD) is associated with the Epstein–Barr virus (EBV) in approximately half of cases. This is a report of a case of nodular sclerosing HD of the B-cell type that was associated with EBV in the initial manifestation, but was found to be EBV-negative in the relapse of the tumour. Both tumours displayed similar clinical, pathological, and immunohistochemical features. This finding implies that in a given individual EBV can be lost from malignant tumours and therefore shows that the EBV infection is not required to maintain neoplastic growth of HD tumour cells. © 1997 John Wiley & Sons, Ltd.