Community emergence of CTX-M type extended-spectrum beta-lactamases among urinary Escherichia coli from women.

OBJECTIVES: To conduct a territory-wide study of extended-spectrum beta-lactamases (ESBLs) among community isolates of urinary Escherichia coli from women in Hong Kong. METHODS: Up to 50 consecutive single-patient E. coli isolates, collected from 13 laboratories in 2004, were studied. The ESBLs were characterized by PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by PFGE and phylogenetic group PCRs. RESULTS: Forty-two ESBL producers were found among 600 consecutive isolates tested. The ESBL prevalence was 7.3% (15/205) for women aged 18-35 years, 5% (11/219) for women aged 36-50 years, 6.3% (4/63) for women aged 51-64 years and 10.6% (12/113) for women aged >or=65 years (P=0.3). The ESBL-producing isolates were often multidrug-resistant and CTX-M-14 was found in 37 isolates, CTX-M-15 in 3 isolates and CTX-M-3 in 2 isolates. PFGE revealed no significant clusters among the ESBL producers. Overall, CTX-M-14 producers were significantly more likely to belong to group D than non-ESBL producers [18/37 (48.6%) versus 13/57 (22.8%), P=0.009]. However, 7 of 13 (53.8%) CTX-M-14 producers from women aged 18-35 years represented phylogenetic group B2, compared with 7 of 24 (29.2%) for women of all other ages (P=0.1). CONCLUSIONS: The study documented the community emergence of CTX-M as the predominant ESBL type among urinary isolates from women. The spread of CTX-M enzymes among isolates from young women is concerning and deserves close monitoring.

Double sarcomatoid carcinomas of the oesophagus.

Sarcomatoid carcinoma of the oesophagus is uncommon. A case of double sarcomatoid carcinomas was identified in the oesophagus of a 48-year-old man. This is the fourth case of multiple primary sarcomatoid carcinomas of the oesophagus and the first case with detailed pathological features presented. The clinicopathological features of multiple primary tumours and sarcomatoid carcinoma of the oesophagus are also reviewed.

Expression of p53 in oesophageal squamous cell carcinoma in Hong Kong Chinese.

We studied p53 overexpression in a series of 99 primary oesophageal squamous cell carcinomas (28 well-differentiated, 42 moderately-differentiated and 29 poorly-differentiated squamous cell carcinomas) from Chinese patients using the p53 protein specific mouse monoclonal antibody DO-7 on paraffin sections. The p53 protein was detected in 30% (30 cases) of the tumours. A significantly higher positive rate was noted in the poorly-differentiated tumours (11% for the well-differentiated, 31% for the moderately-differentiated and 48% for the poorly-differentiated tumours). In addition, strong positive p53 staining was identified only in the less differentiated tumour cells in the periphery of the tumour cell nests in all the cases and the expression was weaker in the better differentiated foci. The central keratinizing areas and the immediately adjacent tumour cells were always negative for p53. The adjacent normal oesophageal mucosa was all negative for p53 protein but the non-invasive dysplastic epithelium next to the tumours could also be strongly positive for p53 protein (four out of 14 cases in which the dysplastic epithelium adjacent to the tumour was adequately sampled). In two out of these four cases, the dysplastic epithelium showed staining for p53; even the adjacent invasive tumour was negative for p53. It is concluded that there is a strong relationship between p53 overexpression and tumour cell differentiation in oesophageal squamous carcinoma and overexpression of p53 can occur in non-invasive tumour cells.

Epstein-Barr virus is localized in the tumour cells of nasal lymphomas of NK, T or B cell type.

Seven cases of nasal lymphoma were studied to identify the lineage of Epstein-Barr virus (EBV)+ cells using dual-labelling methods. Five cases were phenotypically and genotypically of natural killer cell (NK) type with germ-line configuration of T-cell receptor (TcR) beta-chain gene and immunoglobulin heavy-chain joining region (IgJH) gene, with one case each of T- and B-cell type showing rearranged TcR beta or IgJH and lambda-light chain genes respectively. EBV genome was clonal in all these cases except in the B-cell case where its clonality was undeterminable. Using in situ hybridization (ISH) for EBV-encoded small nuclear RNA 1 and 2 (EBER), signal was detected in 45% to 88% of nucleated cells in the tumours. Immunostaining for EBV latent membrane protein-I (LMP) also revealed numerous LMP+ cells in 3/5 NK-type cases and the T- and B-cell cases. Using ISH for EBER combined with immunostaining for CD markers and double immunohistochemistry for LMP and CD markers, the predominant lineage of the EBV+ cells was identified as: CD2+CD3-CD19-CD20- CD45R0 +/- CD56+CD68- in the NK-type cases, CD2+CD3 +/- CD19-CD20- CD45R0+CD56-CD68- in the T-cell case and CD20+CD45R0-CD68- in the B-cell case, in agreement with the genotype and phenotype of each tumour. These results show that, in EBV+ nasal lymphomas of NK, T- or B-cell lineage, EBV was consistently associated with the tumour-cell population and support the view that EBV serves a promoting role in the pathogenesis of different types of EBV+ nasal lymphoma.

Cytokeratin expression in non-neoplastic oesophageal epithelium and squamous cell carcinoma of the oesophagus.

The expression of cytokeratins (CK) 19, 8, 18, 13, 10 and 7 was examined in 35 cases of squamous cell carcinomas of the oesophagus (10 well-differentiated, 13 moderately-differentiated, and 12 poorly-differentiated) and the adjacent mucosa by means of a panel of monoclonal antibodies on frozen sections. The study was undertaken to assess the pattern of expression of these keratins in oesophageal tumours and its relation to the degree of differentiation. The normal oesophageal epithelia expressed CK19 in 86%, CK18 in 17% and CK13 in 14% of cases. CK8, CK10 and CK7 immunoreactivity was not observed. The tumours expressed CK19 in 86%, CK8 in 46%, CK18 in 97%, CK13 in 83%, CK10 in 34% and CK7 in 29% of cases. Thus, the so-called simple epithelial markers CK18 and CK19 occurred in the majority of oesophageal squamous cell carcinomas. CK13 (the so-called non-keratinizing squamous epithelial marker) was only infrequently demonstrated in the non-neoplastic oesophageal mucosa, and its expression was more frequent in carcinomas. CK10 was not demonstrated in non-neoplastic mucosa, but was mostly associated with well-differentiated carcinomas. We therefore conclude that the pattern of expression of cytokeratins in oesophageal carcinomas is different from that in normal oesophageal epithelia and varies with differentiation.

Evidence for lytic infection by Epstein-Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals.

Normal nasopharyngeal tissues from 23 individuals who died of causes unrelated to the upper respiratory system and had no evidence of Epstein-Barr virus (EBV)-related diseases were studied using in situ hybridisation (ISH) and immunohistochemistry for the detection of EBV RNA and expression of EBV proteins, respectively. ISH using 35S-labelled riboprobe for EBV EBER RNA showed occasional to a few EBER+ lymphocytes in the stroma of nasopharyngeal mucosa in 14/16 cases with available paraffin-embedded tissues. In addition, very rare intraepithelial EBER+ lymphocytes were also detected in 3/16 cases. However, in none of these cases was EBER detected in the epithelial cells. Similar results were obtained using a nonradioactive ISH method for EBER (Dako). In 3/23 cases, immunostaining using monoclonal antibodies for EBV proteins on cryostat sections showed occasional cells in the stroma expressing EBV nuclear antigen 2 (EBNA2), latent membrane protein-1 (LMP), and switch protein encoded by BZLF1 gene (ZEBRA) in two cases and only very rare LMP+ and ZEBRA+ cells in one other case. Double immunostaining combining alkaline phosphatase anti-alkaline phosphatase (APAAP) for CD markers and indirect immunofluorescence for LMP showed that the LMP+ cells were either CD19+ or less frequently CD3+, but none were CD68+. These results show that both B and T lymphocytes harbouring EBV can be found in the normal nasopharyngeal tissues. Interestingly, EBV proteins associated with lytic viral replication--diffuse early antigen (EA-D), viral capsid antigen (VCA), or membrane antigen (MA)--were also detected in rare cells in the stroma in one case, and in another case only one MA+ cell was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of correlation between expression of Epstein-Barr virus (EBV) latent membrane protein and bcl-2 oncoprotein in vivo.

AIMS: To evaluate whether there is any correlation between the expression of Epstein-Barr virus (EBV) latent membrane protein (LMP) and oncoprotein bcl-2 in the lymph node biopsy specimens of a Chinese patient with EBV-related reactive lymphoproliferation who later developed T cell lymphoma after a short period of time. METHODS: Immunohistochemistry, with a standard alkaline phosphatase antialkaline phosphatase (APAAP) method and New Fuchsin as a chromogen, was used for single staining of bcl-2 or LMP. Double immunostaining combining APAAP and indirect immunofluorescence was performed for dual labelling of LMP and bcl-2. RESULTS: bcl-2 was expressed in 10-30% of cells in the first lymph node biopsy specimen (EBV-associated lymphoproliferative disorder) and 30-50% of cells in the second lymph node biopsy specimen (T cell lymphoma). LMP was expressed in the first biopsy specimen but not in the second. Double immunostaining results showed that around 78% of LMP positive cells were bcl-2 negative and 94% bcl-2 positive cells were LMP negative. Among the very small fraction of LMP and bcl-2 double positive cells, the intensity of bcl-2 staining was heterogeneous and was not always stronger than that observed in LMP negative bcl-2 positive cells. CONCLUSIONS: The expression of bcl-2 protein is independent of LMP protein status in vivo. Several mechanisms may be involved in EBV associated lymphomagenesis, and bcl-2 induction may occur independently of LMP expression.

Improved double immunohistochemical staining method for cryostat and paraffin wax sections, combining alkaline phosphatase anti-alkaline phosphatase and indirect immunofluorescence.

AIMS: To develop and immunohistochemical staining method for cryostat and paraffin wax sections so that two different antigens in the same section of tissues could be detected by combining immunoenzyme and immunofluorescence techniques. METHODS: This double immunohistochemical staining method combines alkaline phosphatase-anti-alkaline phosphatase (APAAP) using New Fuchsin as a chromogen and indirect immunofluorescence. RESULTS: APAAP staining for one antigen of this double immunohistochemical staining method was observed under bright field conditions alternating with immunofluorescence for another antigen under ultraviolet light. The double exposed photograph of both easily identified the two signals within the same cell. CONCLUSIONS: This double immunohistochemical staining method can overcome the disadvantages of any masking effect of the double immunoenzymatic methods and the background problems of double immunofluorescence method especially when applied to paraffin wax sections. It also permits good morphological identification of the doubly stained cells which may be of crucial importance in studies on pathology specimens.

Epstein-Barr virus (EBV)-related lymphoproliferative disorder with subsequent EBV-negative T-cell lymphoma.

A 58-year-old Chinese man presented initially with generalized lymphadenopathy, and lymph-node biopsy showed disturbed architecture with preponderance of large B-blasts mixed with numerous CD8+ T lymphocytes, consistent with an acute Epstein-Barr virus (EBV) infection. Immunohistological and gene rearrangement studies confirmed the absence of clonal T or B cells. Polyclonal EBV with lytic infection was detected by Southern blot hybridization (SoBH). Expression of EBV proteins (EBNA2, LMP and ZEBRA) was detected in a proportion of cells by immunostaining. EBV-lytic proteins EA-D, VCA, MA were also detected in rare scattered cells. Double immunostaining showed that the LMP-positive cells were of B and of T phenotype: 73% CD19+, 26% CD2+, 23% CD3+, 8% CD4+, 17% CD8+. After biopsy, there was spontaneous regression of lymph-node enlargement, but lymphadenopathy recurred 8 months later, and the second lymph-node biopsy showed T-cell lymphoma, confirmed by detection of clonally rearranged T-cell-receptor beta-chain gene. However, EBV genome could not be detected in the second biopsy by SoBH, in situ hybridization for EBV-encoded EBER RNA, and immunostaining for EBNA2, LMP and ZEBRA was also negative. This case is of special interest because an EBV-negative T-cell lymphoma developed shortly after an acute episode of EBV-related lymphoproliferation, even though many EBV-positive T cells were detected during the acute episode. EBV was apparently not a direct cause of the lymphoma, but the close temporal association of the 2 lesions supports the hypothesis that EBV can act as a co-factor in lymphomagenesis.

Small cell carcinoma of the esophagus.

BACKGROUND: Small cell carcinoma of the esophagus is regarded as having a poor prognosis with frequent systemic dissemination. METHODS: A review of the records and histologic sections of 11 patients with primary small cell carcinoma of the esophagus seen in 11 years was undertaken. They were analyzed and compared with the more common squamous cell carcinomas and adenocarcinomas. RESULTS: Small cell carcinoma of the esophagus constituted 1% of all esophageal tumors and was mainly located at the middle and lower thirds (90%) of the esophagus. Primary treatment consisted of tumor resection in five patients (46%), chemotherapy and radiotherapy in two (18%); surgical bypass in one (9%), radiotherapy after exploratory laparotomy in one (9%), intubation in one (9%), and no active intervention in one (9%). Two of the five resected tumors were Stage IIB disease, and three were Stage III disease. Five of the six patients in the non-resection group had distant metastases at presentation (45% of all patients). The median survival of patients who had chemotherapy (three of whom also had radiotherapy) was 16.7 months (range, 2.8-72 months) and was 2.2 months (range, 4 days to 9.1 months) for those with no chemotherapy. The overall median survival was 3.1 months for all patients. The prognosis was not significantly different from those with squamous cell carcinomas or adenocarcinomas. CONCLUSIONS: Small cell carcinoma of the esophagus should be regarded as a systemic disease, and multimodality treatment, including chemotherapy, should be used. Surgery may be offered in selected patients to manage local disease as part of a chemotherapy-based treatment program.

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS: To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS: Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS: In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS: These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.

Squamous cell carcinoma of the oesophagus with mucin-secreting component (muco-epidermoid carcinoma and adenosquamous carcinoma): a clinicopathologic study and a review of literature.

The clinicopathological features of 11 cases of primary oesophageal squamous cell carcinomas with mucin-secreting component (eight muco-epidermoid and three adenosquamous carcinomas) are presented. The incidence was 2.2% of all resected primary oesophageal tumors. The mean age was 64 years and the male to female ratio was 4.5:1. The mean diameter of these tumours was 4 cm. Twenty-seven per cent of these tumours were in the upper, 46% in the middle and 27% in the lower portion of the oesophagus. The age, sex and site distribution of these tumors were similar to those of squamous cell carcinomas. The medium survival of these patients was 15 months. The world literature concerning this rare entity was reviewed and compared with the findings of the present study. No major racial differences were noted. In addition, the classification of this group of tumours was also discussed.

Squamous cell carcinoma of the esophagus with mucin-secreting component. Mucoepidermoid carcinoma.

Among 1058 patients with cancer of the esophagus, 20 patients with mucoepidermoid or adenosquamous cell carcinoma of the esophagus and cardia, together defined as squamous cell carcinoma with a mucin-secreting component, were seen over a 10-year period. Their records were reviewed and appropriate comparisons were also made with the more common squamous cell carcinomas and adenocarcinomas. Squamous cell carcinoma with mucin-secreting component comprised 1.9% of all tumors encountered. Clinical features including age, male predominance, symptoms at presentation, length of tumor, and appearance of tumor did not differ from those of squamous cell and adenocarcinoma. The location of these tumors, however, followed that of squamous cell carcinomas, with 55% in the middle third and 25% in the lower third. Adenocarcinomas were found predominantly at the cardia (83%). Operability and resectability rates were higher than those of squamous cell and adenocarcinomas. Primary treatment consisted of resection in 19 of the 20 patients (95%); 18 of them had a one-stage resection and 1 patient had a two-stage resection. Postresection staging showed that 5% had stage I disease, 16% had stage II, and 79% had stage III disease. None of the patients who underwent resection died within 30 days of the operation. The mortality after 30 days was 10.5%. The 1 patient in whom intubation was the primary treatment had distant metastases at the time of presentation (stage IV). The overall median survival was 9.2 months. The median survival for patients who had their tumors resected was 9.5 months. The survival improved to 33 months for curative resection but was only 8.7 months for palliative resection. The 1-, 2-, and 5-year survivals were 46%, 39%, and 0%, respectively. This prognosis was not significantly different from that of patients with squamous cell carcinoma or adenocarcinoma.

Latent sites of Epstein-Barr virus infection.

Epstein-Barr virus (EBV) has been detected in a wide spectrum of tumors. This study investigates the detection rate of EBV-DNA by Southern blot hybridization analysis (SOBH) and polymerase chain reaction (PCR) in different tissues from persons without apparent EBV-related diseases. Of 20 tonsillectomy specimens studied, SOBH indicated positivity for EBV-DNA in 1 case, and PCR indicated positivity in 10. In autopsies performed on patients with no apparent evidence of EBV-related diseases, the viral DNA was only detected by PCR in the following: parotid gland (7/15), submandibular gland (8/20), nasopharynx (8/10), tonsil (8/10), larynx (5/6), lung (5/9), cervical lymph node (7/10), mediastinal lymph node (7/10), abdominal lymph node (4/10), spleen (6/10), thyroid (5/10), liver (1/10), pancreas (1/4), kidney (4/10), uterine cervix (1/4), ovary (1/5) and testis (1/3). These results provide a baseline for interpreting the role of EBV in carcinogenesis.

Usefulness of follicular dendritic cell pattern in classification of peripheral T-cell lymphomas.

Classification of peripheral T-cell lymphomas based on morphological criteria can present problems due to overlap in histological features amongst the subtypes. An immunohistochemical study was designed to study the follicular dendritic cell patterns in 21 cases of peripheral T-cell lymphoma which had been classified using the updated Kiel classification. Three patterns of distribution were observed: 1 follicular dendritic cells not detected (3 cases); 2 follicular dendritic cells restricted to remnant follicle centres (7); 3 follicular dendritic cells present as an expanded network of cells exceeding the confines of germinal centres (11). Ten out of 11 angioimmunoblastic lymphomas showed an expanded network of follicular dendritic cells. The only negative case showed features which, on review, were in keeping with a pleomorphic, medium and large cell lymphoma showing an unusual proliferation of small venules. Other than angioimmunoblastic lymphomas, only one other case showed follicular dendritic cell hyperplasia. This was an unclassified peripheral T-cell lymphoma. We conclude that follicular dendritic cell hyperplasia may be used an an aid to diagnosis of the angioimmunoblastic type of peripheral T-cell lymphoma, and we recommend the routine staining of these cells in typing of T-cell lymphomas to facilitate comparison between centres.

Histochemistry of mucin secreting components in mucoepidermoid and adenosquamous carcinoma of the oesophagus.

AIMS: To determine the direction of differentiation of the mucin secreting components in a rare group of oesophageal tumours--oesophageal squamous cell carcinomas with prominent mucin secreting components (mucoepidermoid carcinomas and adenosquamous carcinomas). METHODS: In a review of 617 cases of primary carcinoma of the oesophagus, 16 cases of squamous cell carcinoma with prominent mucin secreting components were studied using a battery of histochemical techniques. RESULTS: The mucin produced by these tumours was mixed and included a variable content of enzyme labile sialomucin (positive for mucicarmine, periodic acid Schiff, and alcian blue, and sensitive to sialidase digestion and negative for high iron diamine-alcian blue). Retrospective analysis of endoscopic biopsy specimens taken from these tumours showed that mucin was present in five (42%) cases. CONCLUSIONS: The glandular component of this group of tumours histochemically differentiated in the direction of oesophageal glands: examination of the mucin secreting component in squamous cell carcinoma in resected specimens is therefore required for recording the true incidence of this type of tumour.

Clonal Epstein-Barr virus in lymphoepithelioma-like carcinoma of the lung.

Lymphoepithelioma of the nasopharynx is an undifferentiated carcinoma with a prominent lymphoid infiltrate. A consistent association with Epstein-Barr virus (EBV) has been demonstrated over the years by a variety of methods. More recently, undifferentiated carcinomas with a similar morphology have been described in other anatomical locations, often of foregut origin. However, for lymphoepithelioma-like carcinoma, the association with EBV has been more tenuous, especially in Western countries. Interestingly, these tumors have shown a geographic distribution similar to nasopharyngeal lymphoepithelioma, with relatively high frequency of EBV positive cases in Asian patients. We report five cases of lymphoepithelioma-like carcinoma arising in the lung. In situ hybridization revealed the presence of EBV in all cases with localization to the epithelial cells only. Southern-blot analysis from two cases revealed the presence of clonal episomal EBV in the tumor tissue. These findings further support the hypothesis that EBV is also associated with lymphoepithelioma-like lung carcinomas and suggest that EBV infection has preceded the clonal expansion of the tumor.

The significance of proliferating cell nuclear antigen in human trophoblastic disease: an immunohistochemical study.

The expression of proliferating cell nuclear antigen (PCNA) in human trophoblastic disease was assessed immunohistochemically in tissue from 29 spontaneous abortions, 33 partial moles, 40 complete moles and 23 choriocarcinomas using the monoclonal antibody PC10. PCNA immunoreactivity occurred predominantly in the cytotrophoblasts in each of the four types of tissues. Quantitative analysis showed that the choriocarcinoma group gave a statistically significant higher PCNA index than the other three. There was no significant difference between the groups of spontaneous abortion, partial or complete mole. Sixteen of the 73 patients with partial and complete moles developed persistent gestational trophoblastic disease and there was no significant difference between the patients requiring chemotherapy and those who did not. We conclude that choriocarcinoma has a significantly higher PCNA proliferative index whilst hydatidiform moles cannot be distinguished from abortions by such analysis. The PCNA index does not appear to be useful in predicting the progression of molar pregnancies to persistent trophoblastic diseases.

Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations.

We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.