Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.     

EBV-determined nuclear antigen (EBNA)-positive cells in the peripheral blood of infectious mononucleosis patients.

After removal of SRBC rosette-forming T-cells from the peripheral blood, the residual, largely B-lymphocyte fraction of five infectious mononucleosis patients was found to contain 0.5-2% blast cells, positive for the EBV-determined nuclear antigen (EBNA). There was a rough parallelism between the presence of large lymphoblasts in the hematological smear, EBNS-positive large blasts in the B-cell fraction and the ability of the T-cell fraction to exert an EBV-specific lymphocytotoxicity on established cell lines in vitro. EBNA-positive B-cells and EBV-specific killer T-cells disappeared after the acute phase of the disease.     

Detection of alphafetoprotein-expressing cells in the blood of patients with hepatoma and hepatitis.

The presence of tumour cells in the blood circulation may predict disease recurrence and metastasis. We have evaluated the specificity and sensitivity of detecting hepatoma cells in blood using nested polymerase chain reaction with primers specific for the alphafetoprotein (AFP) gene. The nested polymerase chain reaction amplified a 270-base pair AFP DNA fragment from cDNA of Hep 3B hepatoma cells. In a reconstitution experiment, AFP mRNA was detected from peripheral mononuclear cells isolated from 10 ml of blood containing as few as ten Hep 3B cells. Peripheral mononuclear cells from the blood of 20 hepatoma patients were analysed, and 19 patients showed positive AFP mRNA expression. Seven of 13 samples from hepatitis patients also showed positive AFP mRNA expression. All five paired samples of peripheral blood or umbilical cord blood from pregnant mothers and their babies, respectively, showed positive AFP expression. None of 22 control samples was positive. The presence of AFP mRNA in the blood of hepatitis or hepatoma patients suggests the presence of circulating hepatoma cells or hepatocytes in the circulation. The high incidence of AFP mRNA in the blood of hepatoma patients supports the notion of early haematogenous spreading of the disease.     

Accuracy of multiplexed Illumina platform-based single-nucleotide polymorphism genotyping compared between genomic and whole genome amplified DNA collected from multiple sources.

Association studies designed to identify the genetic determinants underlying complex disease increasingly require sustainable high-quality DNA resources for large-scale single-nucleotide polymorphism (SNP) genotyping. Recent studies have shown that genomic DNA (gDNA) suitable for SNP genotyping can be obtained from buccal cells and from dried blood spots on Guthrie cards. Further, successful SNP genotyping has been done using the reaction product of multiple displacement amplification of gDNA. We evaluated genotype consistency on the Illumina genotyping platform for 717 to 1,744 SNP loci between replicate samples of gDNA and whole genome amplified DNA (wgaDNA) from a variety of sources. Nine healthy adults provided peripheral blood via venipuncture and buccal cells by mouth rinse. DNA was also obtained from urothelial cells in urine samples from five of the nine subjects. gDNA was extracted from all samples, wgaDNA was generated from each gDNA, and all samples were genotyped. To assess SNP genotyping accuracy of DNA obtained from dried blood spots, gDNA was extracted, amplified, and genotyped from peripheral blood samples and paired Guthrie card samples were obtained from eight childhood leukemia patients. Call rates and replicate concordances for all sample types, regardless of amplification, were >97%, with most sample types having call rates and replicate concordances >99%. Using the gDNA from blood samples as the reference for concordances calculated for all other sample types, we observed concordances >98% regardless of sample type or amplification. We conclude that highly multiplexed Illumina genotyping may be done on gDNA and wgaDNA obtained from whole blood, buccal samples, dried blood spots on Guthrie cards, and possibly even urine samples, with minimal misclassification.     

Demonstration of Epstein-Barr virus-associated nuclear antigen in nasopharyngeal carcinoma cells from fresh biopsies

Fresh nasopharyngeal carcinoma (NPC) biopsies were treated in several ways to yield satisfactory cell preparations for detection of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by anti-complement immunofluorescence tests. If the cells were well dispersed (trypsinization or extensive mechanical dispersal) few or none of them were EBNA-positive. In contrast, if the biopsy preparations contained small to moderately sized tissue fragments (touch preparations or limited mechanical dispersal), nuclear staining was detected in nearly every cell at the margin of the fragments or in cell sheets protruding from them. The stained cells corresponded to the carcinoma cells when compared to histologically stained replicate cell preparations. Nuclear staining was obtaining with anti-EBNA positive sera [healthy donors, NPC patients, convalescents from infectious mononucleosis (IM)], shown to be free of antibodies to other nuclear antigens, but not with anti-EBNA negative sera (healthy donors or patients in the early acute phase of IM). These results confirm and extend previous reports that the carcinoma cells harbor EBV genomes. The implications of these findings are discussed.     

Discrete alterations in the BZLF1 promoter in tumor and non-tumor-associated Epstein-Barr virus

BACKGROUND: Although the Epstein-Barr virus (EBV) is associated with malignant and nonmalignant diseases, its lytic replication is predominantly associated with nonmalignant diseases such as acute infectious mononucleosis (IM) or chronic active EBV infection. Lytic replication is also associated with type B EBV more than with type A EBV. Sustained lytic replication, however, is not compatible with tumor growth. We investigated whether control of an EBV lytic regulatory gene, BZLF1, differed in these diseases. METHODS: Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequence analyses were used to characterize the promoter sequence of BZLF1 (Zp) in 52 tumors (34 non-Hodgkin's lymphomas, 13 post-transplant lymphoproliferative disease samples, and five nasopharyngeal carcinomas), and in peripheral blood lymphocytes from seven patients with chronic active EBV, six with IM, and 40 healthy, EBV-seropositive individuals. All sequences were compared with the prototype EBV strain B95.8 sequence. All statistical tests were two-sided. RESULTS: Three polymorphic Zp sequences were detected. Among the malignant samples, sequence Zp-P, associated with 84% of type A EBV, was identical to that of EBV strain B95.8, whereas a second sequence (Zp-V3), associated exclusively with type B EBV (P<.001), contained three base substitutions. Among the nonmalignant samples, a distinct polymorphism, Zp-V4, containing the substitutions detected in Zp-V3 and an additional base change, was identified in all samples from chronic active EBV, IM, and healthy individuals, but in none of the malignant samples (P<.001). Zp-V4 was independent of the EBV type. CONCLUSIONS: Polymorphisms in the regulatory sequences of BZLF1 are differentially distributed among malignant and nonmalignant cells and may identify EBV subtypes with various lytic activities, including those not associated with malignancies.     

Observations on the type of infection by epstein-barr virus in peripheral lymphoid cells of patients with infectious mononucleosis

Transformation to continuous cell lines has been studied in cultures of peripheral leukocytes from infectious mononucleosis (IM) patients and in co-cultures of IM leukocytes and foetal cord blood leukocytes of opposite sex. The transformed cells in the co-cultures were of mixed origin with foetal cells usually predominating. Neutralizing antisera to EB virus markedly reduced or abolished the incidence of transformation in IM leukocyte cultures. This effect was not due to cytotoxicity and followed the pattern seen with cultures where transformation was known to depend on the intercellular transfer of infectious EB virus. The findings suggest that EB virus is harboured in peripheral lymphocytes of IM patients as a non-productive unexpressed infection which is activated to produce virus in vitro, the particles released then infecting neighbouring cells to give transformed lines. The differences between this mechanism and the one whereby lines arise in culture from the malignant cells of Burkitt's lymphoma are considered, and their significance is discussed.     

Residual normal stem cells can be detected in newly diagnosed chronic myeloid leukemia patients by a new flow cytometric approach and predict for optimal response to imatinib

Insensitivity of chronic myeloid leukemia (CML) hematopoietic stem cells to tyrosine kinase inhibitors (TKIs) prevents eradication of the disease and may be involved in clinical resistance. For improved treatment results more knowledge about CML stem cells is needed. We here present a new flow cytometric approach enabling prospective discrimination of CML stem cells from their normal counterparts within single-patient samples. In 24 of 40 newly diagnosed CML patients residual normal CD34(+)CD38(-) stem cells could be identified by lower CD34 and CD45 expression, lower forward/sideward light scatter and by differences of lineage marker expression (CD7, CD11b and CD56) and of CD90. fluorescent in situ hybridization (FISH) analysis on Fluorescence-activated cell sorting sorted cells proved that populations were BCR-ABL positive or negative and long-term liquid culture assays with subsequent colony forming unit assays and FISH analysis proved their stem cell character. Patients with residual non-leukemic stem cells had lower clinical risk scores (Sokal, Euro), lower hematological toxicity of imatinib (IM) and better molecular responses to IM than patients without. This new approach will expand our possibilities to separate CML and normal stem cells, present in a single bone marrow or peripheral blood sample, thereby offering opportunities to better identify new CML stem-cell-specific targets. Moreover, it may guide optimal clinical CML management.     

Detection of cadherin-6 mRNA by nested RT-PCR as a potential marker for circulating cancer cells in renal cell carcinoma

To detect circulating RCC cells, we established a nested RT-PCR system for cadherin-6 mRNA, which is specifically expressed in RCC. A total of 121 samples of peripheral blood (34 healthy volunteers and 87 patients with RCC) were analyzed in this study. Total RNA of the monocyte fraction of the blood was extracted, then nested RT-PCR using specific primers was performed to detect mRNA of N-cadherin or cadherin-6. Nested RT-PCR revealed that expression of cadherin-6 mRNA was not present in the blood of most healthy volunteers (absent in 32/34), but positive expression was observed in the blood at concentrations of 10 cells/ml or greater of the SKRC-33 RCC cell line, which is a strong expresser of cadherin-6. In peripheral blood from patients with metastatic disease, cadherin-6 mRNA was detected in 70.4% (19/27). Messenger RNA of cadherin-6 was detectable in 45.0% (27/60) of patients with localized tumors. The PCR-based detection system for peripheral blood samples from patients with metastatic disease could reveal the presence of circulating RCC cells in the blood. Detection of cadherin-6 mRNA in non-metastatic presurgical RCC patients suggests that careful follow-up study is necessary in these patients.     

腋淋巴结阴性乳腺癌外周血hMAM检测意义

目的:应用乳腺癌特异性标志物乳腺珠蛋白(hMAM)检测腋淋巴结阴性乳腺癌(axillary lymph node negative breast cancer,ALNNBC)外周血中微转移,并探讨其与临床病理因素的关系.方法:采用巢式RT-PCR方法,分别检测62例ALNNBC、6例乳腺良性疾患和6例健康成人女性外周血中hMAM mRNA的表达,并以MDA-MB415细胞为阳性参考检测该方法的灵敏度.结果:ALNNBC外周血hMAM表达阳性率为32.3%(20/62),乳腺良性疾患组和健康成人女性组全部阴性表达;该方法检测外周血微转移的灵敏度达1/106;C-erbB2基因阳性表达者微转移发生率高(P＜0.01),月经状态、肿瘤大小、组织学类型、ER状态、p53表达等与微转移无关(P＞0.05);复发转移组较无瘤生存组外周血中微转移率高(P＜0.01).结论:hMAM可作为乳腺癌外周血微转移的标志物;ALNNBC微转移与C-erbB2密切相关;外周血微转移可以作为ALNNBC预后指标.     

Detection of additional JH/BCL2 translocations in follicular lymphoma

In vitro enzymatic amplification and direct sequencing were used to detect and characterize t(14;18) recombination junctions in peripheral blood and bone marrow mononuclear cell preparations from patients with follicular lymphoma in remission. Samples from 24/44 patients were found to be positive for translocations involving the major breakpoint region of the BCL2 gene. In samples from seven patients two distinct t(14;18) translocations were shown to be present simultaneously; in one case the second translocation involved the minor cluster region of the BCL2 gene. Biopsy tissue obtained earlier in the course of the disease was available from five of these patients and was shown to contain one of the translocations in each case, but both translocations in only one. Further remission blood and bone marrow samples from the group were also examined. This led to the detection of both translocations in separate samples obtained at different times in a total of four out of the seven cases. In two of the remaining three patients the second translocation could not be amplified from further samples, but in both cases the search led to the identification of a third translocation, again only detectable in a single sample. These findings demonstrate that JH/BCL2 translocations can occur more than once during the course of follicular lymphoma. They suggest that biclonal follicular lymphoma may be more common than has previously been recognized but also raise the possibility that the translocation arises sporadically in the normal lymphoid cells of this group of patients.     

Detection of circulating tumor cells from renal carcinoma patients: experiences of a two-center study

Approximately 30-40% of primary and localized renal cell carcinoma (RCC) will eventually become metastatic disease. Therefore, the detection and molecular characterization of circulating tumor cells (CTC) in RCC may have important prognostic and therapeutic implications. Venous blood samples were obtained from a total of 214 RCC patients before and after nephrectomy or during adjuvant immune chemotherapy in two urological centers. After density gradient centrifugation, the CD45-negative cell population was isolated from peripheral blood samples (BS) by a semi-automated immunomagnetic depletion procedure using the MACS technology. Enriched cell populations potentially containing CTC were stained for cytokeratin and evaluated by a trained pathologist. CTC were found in 105 out of 363 BS (29%) originating from 80 out of 214 patients (37%). The median tumor cell number was five (range 1-51) per BS, i.e. approximately one CTC was detectable per 2-3 ml peripheral blood after tumor cell enrichment. For a subpopulation, follow-up data indicate that 62% of the patients with CTC detection in the blood developed progressive disease with single or multiple distant metastases or died because of RCC within two years. Here we show that the standardized immunomagnetic depletion protocol is a powerful tool for detecting and isolating intact RCC-derived CTC. The occurrence and the quantity of CTC in RCC patients is an early disease event. Furthermore, the occurrence of CTC is correlated with an advanced tumor stage and seems to be associated with a more aggressive tumor phenotype.     

Hematopoietic and gastric uracil-DNA glycosylase activity in megaloblastic anemia and in atrophic gastritis with special reference to pernicious anemia

The activity of the DNA excision repair enzyme uracil-DNA glycosylasewas measured in peripheral blood mononuclear cells and in bonemarrow aspiration samples obtained from patients with perniciousanemia (PA) or other types of megaloblastic anemia (one caseof tapeworm anemia and three cases of myelodysplastic syndromes).In addition, the expression of uracil-DNA glycosylase was investigatedin biopsies from the antrum and body of the stomach obtainedfrom nine PA patients, from five patients having atrophic gastritis(AG) not associated with PA, and from six control patients havingtransient upper abdominal complaints without AG. Our resultsrevealed that there was a considerable interindividual variationin gastric uracil-DNA glycosylase activity. No clear correlationbetween the enzyme level and the level of gastric atrophy wasnoted, although AG is generally regarded as a risk factor ofgastric cancer. Furthermore, uracil-DNA glycosylase activitiesin peripheral blood mononuclear cells and in bone marrow cellsin PA and in myelodysplastic syndromes were similar to the activitiesobserved previously in non-hematological patients and healthypersons. Transient uracil incorporation into DNA may have arole in the cellular abnormalities associated with megaloblastichematopoiesis. The present findings demonstrated that the enzymaticactivity required for rapid removal of uracil from DNA is alsoexpressed in the megaloblastic state.     

Neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA

The presence of tumour cells in peripheral blood of neuroblastoma patients is of considerable clinical importance. Nucleic acid amplification offers an opportunity to detect very small numbers of such cells, but in neuroblastoma a frequent specific abnormality in the tumour DNA suitable for this purpose has yet Co be identified. To facilitate the detection of such cells we have developed RT-PCR using tyrosine hydroxylase (TH) as a tissue-specific target gene. TH mRNA was detected in 3 neuroblastoma cell lines and in all neuroblastoma tumours examined, but was undetectable in peripheral blood from children without neuroblastoma. The method was highly sensitive, detecting 1-10 neuroblastoma cells per 10⁷ blood cells. Thirty blood samples from 24 patients were analysed and results were compared with known disease status. At diagnosis 4/7 patient blood specimens were positive; the four positive samples were from stage-4 patients. In blood samples from these patients 6-8 weeks after the initiation of treatment, TH mRNA was undetectable. Of 7 samples taken at the time of clinical relapse, 5 were positive; 4 of these were from patients with evidence of disseminating disease. Of 16 blood samples from disease-free patients, 14 were negative and 2 were positive. One positive patient in this group subsequently had a clinical relapse. These results show that this technique is of value for detecting neuroblastoma cells in peripheral blood. The significance of these cells at diagnosis, during treatment or on follow-up requires further evaluation.     

Idiopathic myelofibrosis associated with ulcerative colitis

A patient with ulcerative colitis (UC) who developed idiopathic myelofibrosis (IM) is reported. The initial diagnosis of UC was established by colonoscopy and large bowel biopsy, performed after a one-month history of abdominal pain and bloody diarrhea. The patient showed a favorable response to prednisone and mesalamine treatment and six months later he developed a new episode of UC, which was successfully controlled with treatment. However, two years later splenomegaly and anemia were observed, with aniso-poikilocytosis, tear-drop cells, immature myeloid precursors in the peripheral blood, and increased serum LDH, arising the suspicion of IM, a diagnosis that was confirmed by bone marrow biopsy. The present case represents a new association of IM with an autoimmune disease and gives support to the hypothesis of a possible immune basis of some IM cases.     

EB Virus-associated nuclear antigen production and cell proliferation in adult peripheral blood leukocytes inoculated with the QIMR-WIL strain of EB virus.

A nuclear antigen, apparently the Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), was detected by anticomplement immunofluorescence (ACIF) tests in adult peripheral blood leukocytes infected with the QIMR-WIL strain of EBV. EBNA was not detectable at 24 h but appeared in about 11% of the cells by 3 days, and by 5 days up to 64% of the cells were positive. Proliferation of EBNA-positive cells at this stage was confirmed by autoradiography. There was a good correlation between the concentration of virus and the number of EBNA-positive cells in the first 5-7 days. The subsequent course of events was found to be influenced by the initial cell concentration and the time of subculture. EBNA production was delayed in cells infected with higher dilutions of virus but subsequently appeared in a high proportion of cells. Indirect immunofluorescence failed to detect viral capsid antigen (VCA) or early antigen (EA) by 10 days. The results show that EBV infection was abortive and that the critical events of viral transformation occurred within the first few days.