Pierre Brousset

High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

Hodgkins disease is commonly associated with EBV latent infection. The incidence of EBV reactivation (active infection or EBV infection with replicative cycle) was evaluated in a series of 30 patients with untreated Hodgkins disease (except for one case with chronic lymphocytic leukemia) by quantitation of EBV DNA and titration of anti‐ZEBRA antibodies in serum samples. DNA was detected in serum (>2.5 × 102 genomes/ml) in 15 of 30 patients and was more frequent in Hodgkins disease with EBV‐positive Reed‐Sternberg cells (10/12) than in EBV‐negative cases (5/18), (P < 0.01). Of interest was the demonstration that viremia correlated well with increased titers of anti‐ZEBRA IgG and/or standard serological profiles of EBV reactivation (12/15), (P < 0.05). However the lack of EBV replicative cycle in Reed‐Sternberg cells (negative for ZEBRA antigen and early antigen BHLF1) suggests that the viral replication occurs in a nonneoplastic cell compartment rather than in tumor cells. The measurement of EBV DNA loads and the titration of anti‐ZEBRA antibodies shed new lights on the link between activation of EBV replication and Hodgkins disease: these serological markers together with the determination of the EBV status of the tumor suggest that replication of the viral genome occurs with a decreased efficiency of the immune system, thus allowing progression of the tumor. J. Med. Virol. 57:383–389, 1999.© 1999 Wiley‐Liss, Inc.

In vivo patterns of bcl-2 family protein expression in breast carcinomas in relation to apoptosis

The expression of the pro‐apoptotic proteins (Bax, Bak) and anti‐apoptotic proteins (Bcl‐2, Bcl‐X, Mcl‐1) was studied by immunohistochemistry in 110 invasive ductal breast carcinomas. The results were correlated with tumour grade, expression of oestrogen receptor (ER) and p53 protein, and the apoptotic index by combined morphology, immunohistochemistry, and a terminal UTP nick end labelling (TUNEL) procedure. Overall, Bcl‐2, Bcl‐X, Mcl‐1, Bax, Bak, ER, and p53 were detected in 62, 75, 68, 75, 60, 68 and 26 per cent of the cases respectively, but at different levels in each case. A high apoptotic index was correlated with high tumour grade (pu2005<u20050·001), overexpression of p53 (pu2005<u20050·001), Bak expression (pu2005<u20050·001), and low expression of Bcl‐2 (pu2005<u20050·001) and ER (pu2005<u20050·001). No correlation was found between the apoptotic index and Bax, Bcl‐X, and Mcl‐1 immunostaining results. The expression of Bcl‐2 and Bcl‐X was correlated to that of ER. Overall, the results of this study strongly suggest that Bcl‐2 and Bak expression is critical in regulating apoptosis in breast carcinomas. Copyright

ASSOCIATION OF THE EPSTEIN-BARR VIRUS WITH HODGKIN’S DISEASE IN SOUTHERN ISRAEL

Epstein‐Barr virus (EBV) has been frequently documented in the putative neoplastic Hodgkin‐Reed‐Sternberg (HRS) cells, in lymph nodes from patients with Hodgkins disease (HD). This association varies in different geographic areas and between industrialized and developing countries, as does the epidemiological pattern of the disease. In the present study of 106 cases of HD from the Soroka Medical Center in Beer‐Sheva, which serves as the only hospital for most of the southern part of Israel, we found an association with EBV expression in only 30% of the patients; 45% of mixed cellularity (MC) cases compared with 21% of nodular sclerosis (NS) cases were positive for EBV. The number of patients in the 0‐14‐year‐old age group was limited; however, 8 of these 11 children were EBV positive. This low association rate of HD with the presence of EBV sequences is probably related to the small number of children in our series. A low proportion of EBV‐associated disease in older adults may be contributory. Other factors may be involved. Int. J. Cancer, 71:138–141, 1997.

BCL-X GENE EXPRESSION IN HODGKIN'S DISEASE

Bcl-x is a Bcl-2-family protein that has been previously detected in cortical thymocytes, plasma cells, and activated lymphocytes. We report here on the high detection rate of the Bcl-x protein found in 86% of Hodgkins disease samples and on the significance regarding its complex role among the Bcl-2-family of proteins: Bcl-x is known to heterodimerize with Bcl-2 (an anti-apoptosis protein) and with Bax, a potent inducer of cell death. Moreover, recent evidences show that Bcl-x may induce multiple drug resistance in vitro, suggesting that chemical or biological interactions with this protein may have potential therapeutic value in Hodgkins disease.

A Comparison of Distinct Modes of Tumor Cell Death in Hodgkin's Disease Using Morphology and in Situ DNA Fragmentation

The study examined the morphology and frequency of cell death occurring spontaneously in lymph nodes from patients with Hodgkins disease. In addition to necrosis, which was infrequent and usually in patches, we document two cell types showing features of individual cell death: mummy cells end apoptotic cells. Mummy cells present no evidence of DNA fragmentation, but show electron microscopic features of dark cells. Apoptotic Hodgkin-Reed-Sternberg cells are found frequently and are easier to demonstrate by in situ and labeling of fragmented DNA than by light microscopy only. In many cases phagocytosis of apoptotic cells is also documented. The significance of these findings to the limited number of Hodgkin-Reed-Sternberg cells in most cases of Hodgkins disease is discussed.

Kaposi's sarcoma-associated herpesvirus (KSHV) in bone marrow biopsies of patients with Waldenstrom's macroglobulinaemia.

Kaposis sarcoma‐associated herpesvirus (KSHV) is suspected to play a role in the aetiology of multiple myeloma. Because of similarities in the pathophysiology of multiple myeloma and Waldenstroms macroglobulinaemia (WM), we investigated DNA samples from 20 bone marrow biopsies with WM for the detection of KSHV by PCR (KS330/ORF26). We performed two rounds of amplification and found that only 1/20 of the DNA samples obtained from biopsies had a detectable KSHV sequence. The positive patient was also infected by the human immunodeficiency virus (HIV). Our data provide evidence that KSHV cannot be implicated in the pathogenesis of WM.

KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS (KSHV) IN BONE MARROW BIOPSY FROM PATIENTS WITH MULTIPLE MYELOMA: PCR AMPLIFICATION OF ORF26 BUT NOT ORF72 AND ORF75 SEQUENCES

We read with interest the paper on itraconazole versus ̄uconazole in neutropenic patients (Morgenstern et al, 1999). Morgenstern et al (1999) stated that `no direct comparisons have been published. In fact, we published in June 1998 the ®rst report of a randomized trial comparing both drugs in neutropenic patients with haematological malignancies as an abstract of the 1998 EHA meeting (Huijgens et al, 1998). The full report was published very recently (Huijgens et al, 1999). In contrast to the Morgenstern trial, ours is a doubleblind trial in which 202 patients received either itraconazole 200 mg or ̄uconazole 100 mg daily, in capsules. We found no differences in overall mortality or motality due to suspected or proven fungal infections. In both the itraconazole and ̄uconazole group, we found four cases of Aspergillus and/or Mucor infection. The trough plasma levels of itraconazole were well within the published range and almost equal to the minimal inhibitory concentration (MIC) breakpoint for itraconazole in candidal disease. There are no published data on itraconazole plasma levels and clinical ef®cacy against Aspergillus and Mucor species. However, the levels we found were in the range of the MICs of Aspergillus strains found in two recent studies (Rath, 1998; Verweij et al, 1998). We concluded that there was little to choose between the drugs, except for the easier handling of ̄uconazole. Morgenstern et al (1999) explained their unblinded design by pointing to the need for a double-placebo technique in a blinded study. This is not a very strong reason, and an unblinded design calls for rigid de®nitions of end-points, standardized antibacterial strategies and very precise explanations for exclusions and drop-outs, especially when one or two patient outcomes may make a difference. The de®nition of suspected fungal infection is given twice, in rather different terms. Furthermore, the antibacterial regimens do not seem to be standardized and the duration of therapy before going to empirical antifungal therapy ranges between 72 and 96 h, which in an unblinded study suggests bias. Furthermore, analysing oral infections is very dif®cult as many patients harbour Candida species in their mouth during azole therapy and clinical diagnosis is therefore unreliable. The number of withdrawals was considerable. Even a single case of proven fungal infection would substantially in ̄uence statistical analysis and outcome. The question remains why an intention-to-treat analysis of data has not been performed. This would have provided more insight in the occurrence of fungal infections in the two treatment groups as a whole and would de®nitely have better re ̄ected daily clinical practice, in which therapy failure due to noncompliance or adverse events is a frequent and signi®cant problem. Since small numbers have major effects on statistical outcome, there should be a critical note on the number of deaths due to Aspergillus. Of the ®ve cases of fatal aspergillosis in the ̄uconazole group, including those observed after the of®cial study period, three occurred at one centre. As a consequence, the most important risk factor for death due to invasive aspergillosis seems to be not the use of either itraconazole or ̄uconazole but admission to that particular clinic. The authors concluded that `presumably physicians will choose between ̄uconazole and itraconazole depending on their views of the risk of aspergillus in their patient population. This advice is rather vague, probably re ̄ecting their awareness that their trial has some important ̄aws, making clearer conclusions impossible. The jury on which type of prophylaxis against fungal disease in neutropenic patients is preferable is still out! Department of Haematology, P. C. H U I J G E N S 1 , Department of Clinical G. J. T I M M E R S 1 , Microbiology and Infection A. M. S I M O O N S -S M I T 2 , Control and A.C. VA N L O E N E N 3

Detection of the Cell Death-Inducing Protein BAK in Reed-Sternberg Cells of Hodgkin's Disease

The expression of the cell death-inducing protein, Bak, was investigated in 41 cases of Hodgkins disease and was correlated with Epstein-Barr virus (EBV) status. Overall, Bak immunostaining was observed in 35/41 cases (85%). Among the 22 EBV-positive cases, 20 cases (91%) expressed Bak while 15/19 EBV-negative cases (79%) contained Bak-positive Reed-Sternberg cells. The expression of Bak, as assessed by the staining intensity and the numbers of positive tumor cells, varied greatly from case to case but was high in 6 cases (15%). Our findings show that, similar to Bax, a second apoptosis-inducing gene Bak is frequently expressed in Hodgkins disease. Whilst Bak is suspected to protect cells immortalized by EBV from apoptosis, its expression in Hodgkins disease appears to be unrelated to the EBV status of Reed-Sternberg cells. Moreover, the potential pro-apoptotic functions related to Bak and Bax in Hodgkins disease might be surpassed by a stronger expression of anti-apoptotic molecules thus explaining tumor progression.