Predicting response and survival in chemotherapy-treated triple-negative breast cancer.

BACKGROUND: In this study, we evaluated the ability of gene expression profiles to predict chemotherapy response and survival in triple-negative breast cancer (TNBC). METHODS: Gene expression and clinical-pathological data were evaluated in five independent cohorts, including three randomised clinical trials for a total of 1055 patients with TNBC, basal-like disease (BLBC) or both. Previously defined intrinsic molecular subtype and a proliferation signature were determined and tested. Each signature was tested using multivariable logistic regression models (for pCR (pathological complete response)) and Cox models (for survival). Within TNBC, interactions between each signature and the basal-like subtype (vs other subtypes) for predicting either pCR or survival were investigated. RESULTS: Within TNBC, all intrinsic subtypes were identified but BLBC predominated (55-81%). Significant associations between genomic signatures and response and survival after chemotherapy were only identified within BLBC and not within TNBC as a whole. In particular, high expression of a previously identified proliferation signature, or low expression of the luminal A signature, was found independently associated with pCR and improved survival following chemotherapy across different cohorts. Significant interaction tests were only obtained between each signature and the BLBC subtype for prediction of chemotherapy response or survival. CONCLUSIONS: The proliferation signature predicts response and improved survival after chemotherapy, but only within BLBC. This highlights the clinical implications of TNBC heterogeneity, and suggests that future clinical trials focused on this phenotypic subtype should consider stratifying patients as having BLBC or not.

Epstein-Barr virus microRNAs and lung cancer.

BACKGROUND: We conducted the first analysis of viral microRNAs (miRNAs) in lung cancer, with a focus on Epstein-Barr virus (EBV). METHODS: We evaluated viral miRs with a two-channel oligo-array targeting mature, anti-sense miRNAs in 290 cases. In 48 cases, we compared microarray and real-time quantitative PCR (qPCR) expression for three EBV miRNAs. We tested for EBV DNA, RNA, and protein in tumour tissue from six cases with and six cases without strong qPCR-based evidence of EBV miRNAs. RESULTS: The EBV miRNAs strongly differentiated between adenocarcinoma and squamous cell carcinoma using the microarray (P<0.01 for 9 out of 16 EBV miRNAs). However, microarray and qPCR measurements of BART1, BART2, and BHRF1-3 expression were not significantly correlated (P=0.53, 0.94, and 0.47, respectively). Although qPCR provided substantial evidence of EBV miRNAs in 7 out of 48 cases, only 1 of these 7 cases had detectable EBV DNA in tumour tissue. None had detectable EBV RNA or protein by histochemical stains. CONCLUSION: In a comprehensive evaluation of EBV miRNA, DNA, RNA, and protein in lung cancer, we found little evidence of EBV in lung tumour tissue. Discrepancies between microarray- and qPCR-based strategies highlight the difficulty of validating molecular markers of disease. Our results do not support a role of EBV in lung cancer.

Retinoblastoma gene deficiency has mitogenic but not tumorigenic effects on erythropoiesis.

The retinoblastoma protein (Rb), an important ubiquitous cell cycle regulator, was initially identified as the retinoblastoma tumor suppressor. To further address the activities of Rb in proliferation and tumorigenesis in the hematopoietic lineage, we transplanted Rb-/- fetal liver cells into sibling mice and assessed the outcome of Rb-/- hematopoietic cells in both short-term and long-term studies. Rb-/- hematopoietic cells rescued lethally irradiated mice with an efficiency comparable to that of wild-type cells. In spleen colony-forming unit assays, proliferation rates of the Rb-/- cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Rb-/- embryonic livers. Recipients of Rb-/- cells lived for more than 15-18 months, and most blood cell lineages matured normally with the expected switch from fetal to adult hemoglobin. However, the continued presence of nucleated erythrocytes in the peripheral blood and extensive extramedullary erythropoiesis indicated that the Rb-/- erythrocytes were not completely normal. No erythroleukemia developed during the 15-18 month period following transplantation. These results demonstrate the mitogenic effect but not tumorigenic transformation in erythrocyte lineage in the absence of Rb, which is distinct from the effect of Rb deficiency in neuroectodermal cells. The study supports the prevalent model that loss of the ubiquitously expressed tumor suppressor gene predisposes to only a limited spectrum of tumors.

A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer.

An E1B 55 kDa gene-deleted adenovirus, Onyx-015, which reportedly selectively replicates in and lyses p53-deficient cells, was administered by a single intratumoral injection to a total of 22 patients with recurrent head and neck cancer. The objectives of this Phase I study were to determine the safety, feasibility, and efficacy of this therapy and determine any correlation to p53 status. Six cohorts were investigated with a dose escalation from 10(7)-10(11) plaque-forming units. Toxicity was assessed using NCIC criteria. Tumor response was assessed by clinical and radiological measurement. Blood samples were taken to detect adenovirus DNA and neutralizing antibody to adenovirus. Tumor biopsies were taken to detect adenovirus by in situ hybridization. Treatment was well tolerated, with the main toxicity being grade 1/2 flu-like symptoms. Dose-limiting toxicity was not reached at the highest dose of 10(11) plaque-forming units. Twenty-one of the 22 patients treated showed an increase in neutralizing antibody to adenovirus. In situ hybridization showed viral replication in 4 of 22 patients treated, all of whom had mutant p53 tumors. Using conventional response criteria, no objective responses were observed. However, magnetic resonance imaging scans were suggestive of tumor necrosis at the site of viral injection in five patients, three of whom were classified using nonconventional criteria as partial responders, and two of whom were classified using nonconventional criteria as minor responders. Of these five cases, four had mutant p53 tumors. The response duration for the three partial responders was 4, 8, and 12 weeks. An additional eight patients had stable disease in the injected tumors lasting from 4-8 weeks. These preliminary results show that intratumoral administration of Onyx-015 is feasible, well tolerated, and associated with biological activity. Further investigation of Onyx-015, particularly with a more frequent injection protocol and in combination with systemic chemotherapy, is warranted.

Molecular diagnosis of Epstein-Barr virus-related diseases.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis, and it may also be found in a wide variety of benign and malignant lesions including oral hairy leukoplakia, inflammatory pseudotumor, Hodgkin's disease, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. Molecular testing is increasingly important in the diagnosis and monitoring of patients affected by these diseases. In biopsy tissues, molecular detection of EBV-encoded RNA transcripts by in situ hybridization remains the gold standard for proving that a histopathological lesion is EBV-related. EBV-encoded RNA hybridization and EBV LMP1 immunostains are used routinely to detect latent EBV in tissues affected by posttransplant lymphoproliferative disorder (PTLD) or in enlarged nodes from patients with infectious mononucleosis. Traditional serology is the best test for evaluating acute versus remote infection in healthy individuals. High serological titers serve as a tumor marker for some EBV-related malignancies, but titers are not a dependable tumor marker in immunocompromised hosts. EBV viral load testing by quantitative DNA amplification of blood samples is a promising new laboratory test that has proven useful for early diagnosis and monitoring patients with PTLD. Recent studies suggest a role for EBV viral load testing in nasopharyngeal carcinoma, Hodgkin's disease, and AIDS patients with brain lymphoma. Further research is needed to define more fully the clinical utility of viral load tests in the full spectrum of EBV-associated diseases. Gene expression profiling is on the horizon as a means to improve subclassification of EBV-related diseases and to predict response to therapy.

Natural killer-like T-cell lymphoma in the small intestine of a child without evidence of enteropathy.

A previously healthy 6-year-old boy developed symptoms of small intestinal obstruction and was found to have a large intraabdominal mass. At laparotomy the mass involved the jejunum and adjacent mesenteric lymph nodes, requiring resection. Microscopic and immunohistochemical studies demonstrated a T-cell non-Hodgkin's lymphoma, confirmed by finding clonal T-cell receptor-beta and -gamma gene rearrangements by Southern blot analysis. The immunophenotype of this lymphoma-CD3+CD4-CD8-CD56+TIA-1+ beta F1(-)-suggests that the tumor cells are cytotoxic natural killer (NK)-like T cells, probably of CD3+CD4-CD8- intraepithelial cell origin. Examination of the adjacent and distal small intestinal mucosa failed to show any significant pathologic change. This case was unusual because intestinal lymphomas in children are usually of B-cell origin and most commonly have small noncleaved cell morphology. Childhood intestinal T-cell lymphomas have not been the focus of specific study but appear to be rare. In adults, intestinal T-cell lymphomas often arise in the background of gluten-sensitive enteropathy (celiac disease). In contrast, this child had peripheral T-cell lymphoma, with NK-like T-cell features, in the small intestine with no clinical or histologic evidence of enteropathy.

Lymphoepithelioma-like carcinoma of the urinary bladder.

We report the cases of 11 patients who were treated for undifferentiated carcinoma of the urinary bladder with a prominent lymphoid stroma (lymphoepithelioma-like carcinoma [LELC]). The chief complaint of all 11 patients was hematuria. Their ages ranged from 52 to 79 years (mean of 67). All tumors except one invaded the muscle wall and showed the typical syncytial growth pattern of undifferentiated cells with ill-defined cytoplasmic borders, prominent nucleoli, and numerous mitoses. A significant lymphocytic reaction was an essential component of all these tumors. There were three pure LELC tumors without concurrent invasive transitional-cell carcinoma (TCC) or TCC in situ; these cases morphologically simulated large-cell lymphoma. The remainder were mixed TCC and LELC (five predominant and three focal LELC). The tumor cells were immunoreactive for keratin and showed negative results for leukocyte common antigen. The lymphoid population was an admixture of T cells and B cells with a predominance of T cells. Seven patients (four with predominant and three with focal LELC) were treated with various therapeutic methods. Four patients (three with pure and one with predominantly LELC) received only chemotherapy after transurethral resection of the tumor, and follow-up found no evidence of disease for 9-72 months (mean of 38 months). Awareness of an LELC component in a urinary bladder tumor is also important in order to avoid misinterpreting these tumors as malignant lymphoma or severe chronic cystitis. Our data suggest that the pure LELC tumor appears to be morphologically and clinically different from TCC and that it merits recognition as a separate clinicopathologic entity. In addition, there is strong suggestive evidence that it responds to chemotherapy and therefore there is the potential of salvaging bladder function.

Hepatosplenic alphabeta T-cell lymphomas: a report of 14 cases and comparison with hepatosplenic gammadelta T-cell lymphomas.

Hepatosplenic gammadelta T-cell lymphoma is a distinct entity, characterized by occurrence in young adult males with hepatosplenomegaly, B-symptoms, peripheral blood cytopenias, and no lymphadenopathy; lymphomatous infiltrates in the splenic red pulp, hepatic sinusoids, and bone marrow sinuses; T-cell receptor (TCR) gammadelta chains and a cytotoxic T-cell phenotype; isochromosome 7q; and an aggressive clinical course. In comparison, this study describes the clinicopathologic features of 14 hepatosplenic T-cell lymphomas expressing TCR alphabeta chains. They occurred in 11 women and 3 men with a median age of 36 years. Clinical presentation was similar to that described previously for hepatosplenic gammadelta T-cell lymphomas, except for the female preponderance and age distribution (5 patients younger than 13 years of age and 5 patients older than 50 years of age). Disease distribution was primarily in the splenic red pulp and hepatic sinusoids, although liver infiltrates were largely periportal in four cases. Bone marrow involvement, observed in eight patients, was usually interstitial and/or within the sinuses. Lymph nodes were involved in five patients, although lymphadenopathy was demonstrable in only two. Ten cases were composed of intermediate-size tumor cells with round/oval nuclei, slightly dispersed chromatin, inconspicuous nucleoli, and scant to moderate amounts of cytoplasm. Four lymphomas contained primarily large cells with irregular nuclei, dispersed chromatin, discernible nucleoli, and moderate to abundant cytoplasm. Tumor cells in all 14 lymphomas were cytotoxic alphabeta T-cells; 13 co-expressed natural killer cell-associated antigens and showed T-cell clonality. Three lymphomas were associated with Epstein-Barr virus. Two of four cases had an isochromosome 7q. Eleven patients are dead, eight within a year of diagnosis, and two patients have maintained complete remissions after combination chemotherapy. These data show that hepatosplenic T-cell lymphomas include an alphabeta-subtype. This group, along with the previously recognized gammadelta group, should be recognized as phenotypically heterogeneous subtypes of the same disease entity.

Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues.

We evaluated the effect of six different histologic stains on the productivity of PCR amplification of DNA isolated from paraffin-embedded tissue samples. The tissue was collected from glass slides by microdissection techniques, whereby tiny portions of tissue are visually identified through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amplification depended on the type of histologic stain that was used to facilitate microscopic visualization of the undeparaffinized tissue section. The best results were obtained with methyl green and nuclear fast red, while Wright's stain yielded less PCR product. Two other stains, Evans blue and light-green SF yellowish (also known as the counterstain for geomori methenamine silver stain) yielded sufficient PCR products; however, their staining characteristics did not afford satisfactory visualization of nuclear chromatin to discriminate between benign and malignant cells. Our most significant finding was that a commonly used histologic stain, hematoxylin, failed to produce DNA templates that could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting material for PCR. Among the remaining five stains that were evaluated, the best choice depends on the differential staining characteristics of the cells to be dissected.

Neurobehavioral characteristics of CGG amplification status in fragile X females.

Neurobehavioral correlates of CGG amplification were studied in 17 nonretarded adult female carriers of fragile X syndrome. The results revealed a significant relationship between IQ and the number of CGG repeats in the 5' untranslated region of the FMR1 gene. Women with a full mutation (> 200 CGG repeats) scored below average in IQ, visual-spatial perception, visual-spatial organization, and executive function. There were no differences in fine motor dexterity or memory as a function of CGG amplification status. A history of major depressive disorder was identified in 71% of the sample, but incidence of depression was not associated with the degree of CGG amplification. Schizotypal features were noted in 18%. No intellectual or neuropsychological deficit was found in women with a premutation (< 200 CGG repeats). Decrements in IQ, visual-spatial perception, and executive function appear to arise as a consequence of the CGG amplification.

Accumulation of p53 in infectious mononucleosis tissues.

Epstein-Barr virus (EBV) infects lymphocytes, where it persists indefinitely for the life of the host; whether the virus interacts with p53 to maintain itself in these cells is unknown. Lymphoid biopsy samples from 10 patients with infectious mononucleosis (IM) were examined for expression of p53 by immunohistochemistry. Accumulation of p53 was detected in all 10 cases, primarily in large lymphocytes of the expanded paracortex. The presence of EBV was confirmed in all 10 cases by EBER1 (EBV-encoded RNA) in situ hybridization, whereas 11 non-IM control samples lacked significant EBER1 and did not express p53 in paracortical lymphocytes. Interestingly, EBV infection alone does not cause accumulation of intracellular p53, because many more cells expressed EBER1 than p53 in the IM tissues. To determine whether p53 was confined to the subset of infected cells in which viral replication was occurring, BZLF1 immunostains were performed. Viral BZLF1 was detected in 8 of 10 IM tissues; however, the paucity and small size of the BZLF1-expressing lymphocytes suggests that they are not the same cells overexpressing p53. To further examine the relationship between p53 and EBV gene expression, the tissues were studied for latent membrane protein 1 (LMP1) expression by immunohistochemistry. Viral LMP1 was observed in the large paracortical lymphocytes of all 10 cases of IM, indicating co-localization of p53 and LMP1 in these cells. Our findings confirm that p53 overexpression is not specific for nodal malignancy and that p53 accumulation is characteristic of IM. Because p53 was not coexpressed in the same cells as BZLF1, it appears that BZLF1 is not directly responsible for p53 accumulation. Nevertheless, co-localization of p53 and LMP1 in activated-appearing lymphocytes suggests that EBV infection is responsible for p53 accumulation. HUM PATHOL 31:1397-1403.

Loss of p16/CDKN2A tumor suppressor protein in gastric adenocarcinoma is associated with Epstein-Barr virus and anatomic location in the body of the stomach.

Gastric adenocarcinomas (n = 125) were analyzed by immunohistochemistry for the presence of p16, the CDKN2A gene product. This protein was lost in 31 of 125 cases (25%), and loss was associated with location of the tumor in the body of the stomach (P = .001). Loss of p16 was also associated with the presence of Epstein-Barr virus (EBV) in tumor cells as determined by in situ hybridization (P = .022). This effect may relate to anatomic site, because EBV-associated tumors originate more frequently in the body of the stomach. When p16 status was evaluated for ethnic origin of the patient (non-Hispanic white, Hispanic, or black), a strong trend (P = .057) was found for African-American patients to have fewer p16-negative tumors than other patients. This also may relate to anatomic location, because fewer tumors from black patients arose in the body of the stomach (P = .022). No significant associations were detected between p16 status and histological subtype (intestinal v diffuse), the presence of microsatellite instability, grade or stage of the tumor, or age, gender, or survival of the patient. In conclusion, p16 loss is quite common in gastric adenocarcinoma, and such loss is more common in EBV-infected tumors arising in the body of the stomach.

Molecular evidence links lymphomatous polyposis of the gastrointestinal tract with mantle cell lymphoma.

Lymphomatous polyposis (LP) is a subtype of non-Hodgkin's lymphoma manifested by numerous polyps affecting long segments of the gastrointestinal tract. The malignant cells of LP often share morphological and immunophenotypic similarity with cells of nodal-based mantle cell lymphoma. Recent genetic studies have shown that mantle cell lymphomas frequently possess a characteristic translocation of the JH/bcl-1 loci. In this study, polymerase chain reaction (PCR) and Southern blot analysis were used to show the presence of JH/bcl-1 translocation in a typical case of LP of the gastrointestinal tract. This provides strong molecular evidence for a biologic link between LP and mantle cell lymphoma. The findings also imply that detection of this translocation may be useful in the diagnosis of morphologically equivocal gastrointestinal biopsy specimens.

Epstein-Barr virus infection is an early event in gastric carcinogenesis and is independent of bcl-2 expression and p53 accumulation.

Ninety-five cases of adenocarcinoma of the stomach were evaluated for the presence of Epstein-Barr virus (EBV) using a sensitive in situ hybridization assay targeting Epstein-Barr virus-encoded RNA 1 (EBER1) transcripts. EBER1 was detected in 11 of 95 (12%) of cases. When present, the virus was localized to malignant epithelial cells and to dysplastic gastric epithelium, but was not seen in normal-appearing gastric epithelium or intestinal metaplasia. The EBV DNA was monoclonal in all three cases tested by Southern blot analysis of the EBV terminal repeat fragment. These findings suggest that the virus was present before malignant transformation. The presence of EBV was strongly associated with increased numbers of tumor-infiltrating T lymphocytes; however, EBV was not associated with prolonged survival. Neither p53 nor bcl-2 were consistently detected in the EBV-associated tumors. Specifically, 6 of 11 EBV-positive carcinomas had accumulation of p53 protein by immunohistochemical analysis, which was similar to the prevalence of p53 accumulation in EBV-negative specimens and suggests that EBV infection does not substitute for p53 mutations during tumorigenesis. The bcl-2 oncoprotein was expressed in a third of the carcinoma specimens tested, but bcl-2 expression did not correlate with the presence of EBV or with expression of EBV latent membrane protein 1. In conclusion, EBV infection appears to precede malignant transformation in a significant fraction of gastric carcinomas, but neither bcl-2 expression nor p53 accumulation appear to be consistently associated with the presence of the virus.

Recurrent Epstein-Barr virus-associated post-transplant lymphoproliferative disorder: report of a patient with histologically similar but clonally distinct metachronous abdominal and brain lesions.

A liver transplant patient developed a single central nervous system (CNS) intraparenchymal lesion 5 months after the diagnosis of an intraabdominal diffuse large B-cell post-transplant lymphoproliferative disorder (PTLD). Biopsy of the new CNS lesion showed a diffuse large B-cell PTLD morphologically and immunohistochemically indistinguishable from the abdominal lesion. In addition, both lesions were positive for Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR) and for EBV-encoded RNA by in situ hybridization. Although these results were consistent with a metastatic origin for the CNS lesion, the finding of an intraparenchymal lesion without leptomeningeal or dural spread was suggestive of a new primary CNS lymphoma. Proof that the brain lesion was a second primary and not a metastasis was obtained by immunoglobulin gene rearrangement studies and assessment of EBV clonality. Multiple primary lymphoid neoplasms arise at higher frequency in the setting of immunosuppression, and molecular investigations of tumor clonality can provide clinically relevant staging and prognostic information.

Human immunodeficiency virus-associated Hodgkin's disease contains latent, not replicative, Epstein-Barr virus.

Severe immunodeficiency is associated with reactivation of latent Epstein-Barr virus (EBV) that is manifested by virus replication. It is unknown whether EBV replication also occurs in the Hodgkin's disease (HD) tissue of patients infected with the human immunodeficiency virus (HIV). Therefore, we studied paraffin-embedded lymph nodes from 13 cases of HIV-associated HD to determine the latent or replicative state of EBV infection. All patients were seropositive HIV-infected men; additional clinical information was available for 12 patients. The risk factor(s) for HIV infection were homosexuality (n = 7), intravenous drug abuse (n = 2), homosexuality and intravenous drug abuse (n = 1), sexual promiscuity (n = 1), or hemophilia (n = 1). Advanced clinical stage and B symptoms were common at the time of initial diagnosis of HD. The histological subtype of Hodgkin's disease was universally mixed cellularity, except for a single case classified as nodular sclerosis. Seven cases exhibited foci of relative lymphoid depletion. Five cases contained foci of necrosis. Reed-Sternberg (RS) cells and RS cell variants were positive for CD30/BerH2 and negative for CD45/LCA, CD45RO/UCHL1, and CD20/L26 in all cases. Tumor cells were positive for CD15/LeuM1 in seven cases. In all 13 cases, RS cells and RS cell variants were infected by latent EBV as shown by in situ hybridization to EBV-encoded ribonucleic acid (EBER1). In 12 of 13 cases neoplastic cells coexpressed EBV latent membrane protein 1 (LMP1). EBV replication was examined by two different methods: immunohistochemistry to identify EBV-encoded BZLF1 protein and in situ hybridization to detect EBV BHLF1 transcripts. No positivity in RS or RS cell variants was detected with either assay of EBV replication (95% confidence interval [CI] = 0% to 23%). The findings confirm that EBV is detected more frequently in HIV-associated HD when compared with immunocompetent patients with HD. The findings also suggest that EBV is tightly latent within RS and RS cell variants of HIV-associated HD. It appears that factors other than host immune status are important in maintaining EBV latency in HIV-associated HD.

Epstein-Barr virus is detected in undifferentiated nasopharyngeal carcinoma but not in lymphoepithelioma-like carcinoma of the urinary bladder.

The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) and with lymphoepithelioma-like carcinomas developing in certain anatomic sites. In this study, an in situ hybridization was used to identify EBV-encoded ribonucleic acid (RNA) (EBER1) transcripts in 32 of 45 cases of NPC but not in any of the 11 lymphoepithelioma-like carcinomas developing in the urinary bladder. EBER1 was most commonly detected in those NPCs having undifferentiated or nonkeratinizing squamous histology rather than the keratinizing squamous cell subtype of NPC. The EBV-encoded latent membrane protein 1 (LMP1) was expressed focally in only seven of 21 EBER1-positive NPCs by an immunohistochemical technique. These findings imply that EBER1 hybridization is more sensitive than LMP1 immunohistochemistry on paraffin sections in detecting carcinoma-associated virus. Previous in vitro studies have suggested that LMP1 expression might be a function of differentiation, but this study of naturally infected NPCs showed no strong correlation between LMP1 positivity and degree of tumor differentiation, albeit a limited spectrum of differentiation that could be examined. In two cases in which frozen tissue was available, the NPCs were monoclonal with respect to viral DNA structure, implying that the virus was present before malignant transformation. Unlike NPCs, the lymphoepithelioma-like carcinomas of the bladder were uniformly EBV negative, lending further evidence to the growing body of literature linking EBV with lymphoepithelial carcinomas of foregut-derived tissues but not with similar-appearing tumors developing in other anatomic sites.

Epstein-Barr virus infection is associated with p53 accumulation in nasopharyngeal carcinoma.

Eighty-three cases of nasopharyngeal carcinoma were evaluated for the presence of Epstein-Barr virus (EBV) infection in tumor cells by in situ hybridization to EBER1 transcripts, and for p53 expression by immunostains using the D07 antibody which detects native and mutant forms of the p53 protein. A highly significant association was found between EBV infection and p53 overexpression (P = .0004), with 77% of cases coexpressing both markers. This newly discovered association suggests that EBV is not an innocent bystander with respect to p53 accumulation. One possible mediator of the interaction between EBV and p53, viral BZLF1, was not colocalized with p53 in these tumors, suggesting that BZLF1 is not the factor responsible for p53 accumulation. From an epidemiological standpoint, this series of cancers represents an international cohort in which cases from an endemic part of the world (Hong Kong) were examined alongside cases from the United States, where the disease is 50-fold less prevalent. The cancers from Hong Kong tended to be less differentiated and more frequently associated with EBV, suggesting that biological differences might underlie epidemiological variations in tumor prevalence. Finally, we examined 18 potential premalignant lesions of the surface epithelium of the nasopharynx. Although our numbers are small, our data suggest that p53 accumulation might precede EBV infection in the transition from metaplasia to carcinoma in situ. Further studies are needed to dissect the stepwise progression of nasopharyngeal carcinogenesis.

Bcr/abl recombinant DNA analysis versus karyotype in the diagnosis and therapeutic monitoring of chronic myeloid leukemia.

Karyotype and bcr/abl recombinant DNA analyses are two means of detecting the chromosomal aberration in chronic myeloid leukemia. The authors compared these two methods in a retrospective study of 36 patients with CML in which they found the bcr/abl DNA recombinant event in 100% (29 of 29) of those patients who had the Philadelphia chromosome. To achieve this sensitivity, a battery of two bcr probes and three restriction enzymes is necessary. The authors propose a sequential algorithm for efficient use of these probes and enzymes. In 76% of the patients, bcr/abl rearrangement can be detected with a Bgl II digest and a 3' commercial probe. An additional 21% of patients can be detected by a second assay in which the same membrane is rehybridized to a 3' and 5' combination bcr probe. One patient (3%) required an additional restriction enzyme digest with BamH I to detect the recombinant event by the same 3' probe. Karyotype analysis is used to determine cytogenetic remission in patients with CML under therapy. The authors studied the use of DNA analysis by the Southern blot technique to detect a decrease in the relative number of leukemic cells. By dilution studies and densitometric scanning of autoradiographs, the authors were able to detect a 15% decrease in the relative number of cells having the bcr/abl recombinant event. The authors report the preliminary results of three patients in whom they compared the karyotype and recombinant DNA analysis at multiple time points in their clinical course. In conclusion, the bcr/abl recombinant DNA analysis is superior to karyotype for the diagnosis of CML and can be used for monitoring treated patients.

Blast-like cells in cerebrospinal fluid of neonates. Possible germinal matrix origin.

Primitive cell clusters (PCCs) composed of immature blast-like cells were observed in Wright's-stained cerebrospinal fluid (CSF) cytocentrifuge specimens from four infants over an 18-month period. All of these patients had hydrocephalus; in three this was secondary to subependymal germinal matrix intraventricular hemorrhage (IVH) associated with prematurity. The fourth was associated with Arnold-Chiari malformation, Chiari type II. In the CSF samples from the patients with IVH, hemosiderin-laden macrophages were also prominent and, in some cases, were intimately admixed with the PCCs. Immunoperoxidase staining of cytocentrifuge preparations from one of the patients revealed that the PCCs stained with neuron-specific enolase (NSE) but not with pan-leukocyte antibodies. Cells with similar morphologic characteristics to PCCs in the CSF of infants have been infrequently illustrated in the literature and thought to be of hematopoietic origin. However, the immunohistochemical findings and the clinical presentation suggest that PCCs are most likely germinal matrix cells.