Quantitative analysis of tyrosinase transcripts in blood.

BACKGROUND: Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods. METHODS: Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: Ultraspec(TM)-II RNA isolation system, FastRNA(TM) GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2, 4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5'DNP-GGGGAGCCTTGGGGTTCTGG-3') and internal standard (5'DNP-CGGAGCCCCGAAACCACATC-3') and quantified by ELISA. RESULTS: The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean +/- SD) 1716+/-1341, 2670+/-3174, and 24 320+/-5332 transcripts/mL of blood. Corresponding values were 527+/-497, 2497+/-1033, 14 930+/-1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120-168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found. CONCLUSIONS: The results obtained with different RNA extraction methods illustrate the importance of quantitative methods for validation of methods. The use of QIAamp RNA improved the extraction efficiency considerably. Data from a case study suggest the assay is suitable in the follow-up of patients with high risk of developing metastases.

Automated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV monitor test.

A highly sensitive method of quantitative analysis of hepatitis B virus (HBV) DNA in serum, the Cobas Amplicor HBV Monitor (Cobas-AM) test, was evaluated. Following a manual extraction of viral DNA, amplification, colorimetric detection, and quantitative determination are all automatically performed in the Cobas analyzer. Serially diluted samples with known HBV DNA concentrations were analyzed blindly. All samples with a virus concentration of 400 copies/ml and 83% of samples with a virus concentration of 100 copies/ml could be detected. A linear correlation between input HBV DNA and measured HBV DNA was seen in the range from 100 to 10(5) copies/ml. The mean coefficient of variation was 29.6% for all input levels and 18.9% for HBV DNA concentrations above 400 copies/ml. Samples with an HBV DNA level above 10(9) copies/ml could be reproducibly measured after predilution to 10(-4) or 10(-6) in negative serum; however, the level was underestimated if target DNA after dilution was still above the linear range of the assay. Quantitative results of the Cobas-AM test were interchangeable with measurements by the manual microwell plate version of Amplicor HBV Monitor (MWP-AM); the mean ratio for log Cobas-AM results/log MWP-AM results was 0.97 (standard error of the mean, 0.007) when serum samples from 153 chronic carriers were analyzed. The test should be of value for clinical assessment of chronic carriers and for monitoring the response to antiviral treatment. A limitation is the relatively narrow linear range of the assay, requiring predilution of high-titer (mainly hepatitis B e-antigen-positive) samples.

Tumour necrosis factor-alpha cytokine promoter gene polymorphism in Hodgkin's disease and chronic lymphocytic leukaemia.

Regulation of cytokine levels has been shown to be under genetic control through the coding and promoter sequences of genetic polymorphisms. We elucidated the prevalence of a previously described G to A transition polymorphism at position -308 of the tumour necrosis factor-alpha (TNF-alpha) promoter region in a population of patients with Hodgkin's disease (HD) (n = 36) and chronic lymphocytic leukaemia (CLL) (n = 49) and healthy volunteers (n = 51). The DNA fragment containing this polymorphism was amplified by PCR and sequenced by solid-phase minisequencing. The frequency of the TNF-alpha promoter polymorphism was not significantly different between CLL patients and HD patients compared to controls.

Interleukin-10 mRNA expression in B-cell chronic lymphocytic leukaemia inversely correlates with progression of disease.

Interleukin-10 (IL-10) has been shown in vitro to inhibit survival and spontaneous DNA synthesis in B-cell chronic lymphocytic leukaemia (B-CELL) cells by induction of programmed cell death. We have analysed the presence of mRNA transcripts for IL-10 in purified B-CLL cells from 35 patients by RT-PCR. Transcripts for IL-10 were detected in 11/20 patients with non-progressive disease. In cell preparations from patients with progressive B-CLL IL-10 mRNA were detected in only 2/15 samples (P < or = 0.01). The Epstein-Barr virus status of the cells did not account for the difference in IL-10 mRNA expression observed between the two groups of patients. Thus, IL-10 mRNA expression in leukaemic cells from patients with B-CLL was strongly associated with non-progressive disease. This finding may support other observations suggesting that IL-10 might be a candidate for immune therapy of progressive B-CLL.

Lack of interleukin-2 (IL-2) expression and selective expression of IL-10 mRNA in human renal cell carcinoma.

Freshly isolated tumor-infiltrating lymphocytes (TIL) are often functionally deficient. Since one of the key functional parameters of an immune response is the local production of cytokines, we studied the expression of cytokine genes in freshly isolated renal cancer tissue. Using a PCR-assisted mRNA amplification assay, the constitutive expression of mRNA for 10 different cytokines was assessed in renal cancer tissue. We compared the cytokine mRNA expression in freshly isolated samples of renal carcinomas, renal cancer cell lines established from the tumor samples, peripheral blood mononuclear cells (PBMC) and non-tumor kidney tissue isolated from the same patients. IL-10 mRNA expression was detected only in tumor samples, while renal cancer lines, PBMC and non-tumorous kidney tissues were devoid of this cytokine. One-third of the tumor samples but none of the normal kidney samples also expressed G-CSF mRNA. IL-6, TNF-alpha and IFN-gamma mRNA were expressed non-selectively in tumors, PBMC and normal rental tissue. Expression of IL-2, IL-3 and IL-4 mRNA was not detected in any of the tissues analyzed. Established renal cancer lines exhibited expression of IL-1 alpha, IL-6, TNF-alpha and GM-CSF. Culture of tumor-derived T cells with anti-CD3 monoclonal antibody (MAb) resulted in expression of IL-2, IL-3 and IL-4 mRNA. In contrast, none of these cytokines was detected in culture with recombinant human IL-2 alone. Since IL-10 is known to suppress antigen presentation, these findings have important implications for the possible in vivo role of IL-10 as a suppressor of local anti-tumor response.

Lymphoproliferative disease in SCID mice reconstituted with human Sjögren's syndrome lymphocytes.

Patients with Sjögren's syndrome (SS) have increased frequency of non-Hodgkin's B-cell lymphoma. These lymphomas frequently use a specific subclass of kappa light chain (encoded by variable region gene segment Hum KV325) and exhibit bcl-2 protooncogene translocation t(14;18). In order to determine whether expansion of this B-cell subset could be reproduced in an animal model, immunodeficient SCID (CB-17) mice were reconstituted with lymphocytes from 4 different SS patients at high risk of the development of lymphoma. Tumor-like nodules developed in all 11 SCID mice that received at least 5 x 10(5) lymphocytes from SS salivary glands or peripheral blood samples. However, the tumor-like nodules in the SCID mice differed from SS lymphomas in vivo in that they (1) exhibited multiple immunoglobulin gene rearrangements; (2) did not have expansion of B-cells expressing the Hum KV325 K-light chain; and (3) lacked detectable t(14;18) translocations. Characterization of the SCID tumor-like nodules revealed a high level of Epstein-Barr virus (EBV) DNA, EBV-associated antigens (EA-R, EBNA-2, AND LMP), and the EBV-encoded cytokine BCRF-1 that is structurally similar to IL-10. These results demonstrate that the lymphoproliferation occurring in the salivary glands of SS patients is not reproduced in the SCID/hu chimeric mouse. It is likely that specific factors in the human salivary gland are required for development of lymphoma in SS patients and that such factors are not present in the SCID/hu chimeric mouse. Furthermore, EBV-induced lymphoproliferation, as seen in the SCID/hu chimera, does not lead to expansion of the same lymphoid subsets that occurs in vivo.

OKT3-induced cytokine mRNA expression in human peripheral blood mononuclear cells measured by polymerase chain reaction.

The kinetic profile of cytokine gene expression in normal human peripheral mononuclear cells (MNC) activated by an anti-CD3 monoclonal antibody was studied. The presence or absence of 10 different cytokine mRNA were measured in a polymerase chain reaction (PCR) assisted mRNA amplification assay. After 2 h of stimulation the mRNA for interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), interleukin-3 (IL-3), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were detectable and remained present during the whole time period studied (22 h). Interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were detected after 4 h, while interleukin-10 (IL-10) mRNA did not appear until after 7 h; they all remained expressed at 22 h. A transient expression of interleukin-4 (IL-4) mRNA was observed between 4 and 7 h of stimulation. No gene expression of granulocyte colony stimulating factor (G-CSF) was detected at any time. These results show that anti-CD3 stimulation of MNC leads to a rapid sequential induction of different cytokine mRNA, some with a very transient expression.

Selective expression of interleukin 10, interferon gamma, and granulocyte-macrophage colony-stimulating factor in ovarian cancer biopsies.

The variable clinical response seen with most cancer immunotherapy suggests that there is a large interindividual variation in immunologic response to tumors. One of the key functional parameters of an immune response is the local production of cytokines. As a method to survey the immune status of tumor-infiltrating cells, we have investigated the constitutive expression of cytokine mRNA in biopsies from epithelial ovarian carcinomas by using a PCR-assisted mRNA amplification assay. Using a set of cytokine-specific primers for 10 different cytokines, we have found selective expression of interleukin 10 (IL-10), granulocyte-macrophage colony-stimulating factor, and interferon gamma mRNA in ovarian tumor tissue as compared to normal ovaries and ovarian tumor cell lines. Such differences could not be explained by the extent of T-cell infiltration, since comparing samples with the same intensity of T-cell receptor (TCR) constant region alpha-chain product from the tumor and normal biopsies demonstrated different cytokine patterns. No IL-2 gene expression was detected in the tumor biopsies. IL-2 mRNA, however, became expressed after stimulation of the tumor-derived cells via the CD3 molecule but not after growth in recombinant IL-2 alone. Using the same methodology, we also analyzed the TCR variable region beta-chain gene repertoire. No restriction or biased expression of these genes was observed.

Epstein-Barr virus induced lymphoproliferative tumors in severe combined immunodeficient mice are oligoclonal.

Severe combined immunodeficient (SCID) mice reconstituted with lymphocytes from Epstein-Barr virus (EBV) negative human donors develop aggressive tumors after the chimeric mice are infected with EBV. The tumors were composed of human B cells that expressed EBV encoded antigens (latent membrane protein and EBV nuclear antigen2). Southern blot analysis of DNA from 16 SCID/hu tumors with human Ig gene probes showed that each tumor contained multiple heavy and light chain gene rearrangements. Ig kappa gene rearrangements were frequent, while clonal lambda gene rearrangements were infrequent. Analysis of EBV terminal repeat sequences indicated two or more fused termini in each tumor, consistent with a multiclonal origin. Linear terminal repeat segments and viral antigens (EA-D and EA-R) associated with EBV replication were not detected in the tumors. High levels of human Igs in the SCID/hu serum were oligoclonal and primarily contained kappa light chains. Before the appearance of overt tumors, circulating cells with human and EBV DNA could be detected in the SCID/hu mice by the polymerase chain reaction. We conclude that EBV infection in SCID/hu chimeric mice produces a limited number of transformation events, which give rise to oligoclonal tumors resembling EBV-associated lymphoproliferative disorders in some immune-deficient patients.

High frequency of t(14;18) translocation in salivary gland lymphomas from Sjögren's syndrome patients.

Sjögren's syndrome (SS) is a chronic autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands. These patients have a markedly increased frequency of developing non-Hodgkin's lymphoma in their salivary glands and cervical lymph nodes. Translocations of proto-oncogene bcl-2 t(14;18) were observed in five of seven SS-associated lymphomas by Southern blot analysis. Using primers specific for chromosomes 14 and 18, translocation of the proto-oncogene bcl-2 was detected by polymerase chain reaction (PCR) in all five lymphomas positive by Southern blot analysis. Among SS patients lacking clinical evidence of coexistent lymphoma, no bcl-2 translocations were detected in 50 consecutive salivary gland biopsies. Of particular interest, pre-lymphoma biopsies were available from the seven SS patients who subsequently developed lymphoma and these DNA samples lacked detectable t(14;18) translocations even though they exhibited oligoclonal rearrangements of their immunoglobulin genes. We conclude that the great sensitivity of PCR can help us in detecting early onset of lymphoma in SS patients and aid in understanding the transition from autoimmunity to lymphoma.