Association of transcobalamin c. 776C>G with overall survival in patients with primary central nervous system lymphoma.

BACKGROUND: Chemotherapy for primary central nervous system lymphoma (PCNSL) is based on methotrexate (MTX), which interferes with both nucleic acid synthesis and methionine metabolism. We have reported previously that genetic variants with influence on methionine metabolism are associated with MTX side effects, that is, the occurrence of white matter lesions as a sign of MTX neurotoxicity. Here, we investigated whether such variants are associated with MTX efficacy in terms of overall survival in MTX-treated PCNSL patients. METHODS: We analysed seven genetic variants influencing methionine metabolism in 68 PCNSL patients treated with systemic and facultative intraventricular MTX-based polychemotherapy (Bonn protocol). RESULTS: Median age at diagnosis was 59 years (range: 28-77), 32 patients were female. Younger age (Wald=8.9; P=0.003) and the wild-type C (CC) allele of the genotype transcobalamin c (Tc2). 776C>G (Wald=6.7; P=0.010) were associated with longer overall survival in a multivariate COX regression analysis. CONCLUSION: This observation suggests that the missense variant Tc2. 776C>G influences both neurotoxicity and efficacy of MTX in the Bonn PCNSL protocol.

[MR perfusion and spectroscopic imaging in WHO grade II astrocytomas]. / MR-Perfusions- und spektroskopische Bildgebung bei WHO-Grad-II-Astrozytomen.

BACKGROUND: This study evaluates whether MR perfusion imaging and spectroscopic imaging (MRSI) can depict anaplastic areas in WHO grade II astrocytomas, whether these areas are co-localized, and whether the prognosis can be better predicted. MATERIAL AND METHODS: Fifteen patients (nine female, six male, aged 42+/-14 years) with WHO grade II astrocytomas but without preceding radio- or chemotherapy were examined every 3 months with MR perfusion imaging and MRSI (mean follow-up 18 months). Using a region of interest analysis, the regional relative cerebral blood volume (rrCBV) and blood flow (rrCBF) were measured in tumor tissue. In the same areas, choline/creatine (Cho/Cr) and choline/N-acetyl-aspartate (Cho/NAA) ratios were quantified. RESULTS: During follow-up, nine patients had stable disease. In six patients, the tumor showed progression and contrast-enhancement. The progressing tumors had already had higher perfusion (rrCBF 2.1+/-1.4; rrCBV 1.9+/-1.1) parameters than the stable astrocytomas (rrCBF 1.2+/-0.6, p=0.01; rrCBV 1.4+/-0.8, p=0.05) at first examination. However, the Cho/NAA and Cho/Cr ratios only tended to be higher than in stable astrocytomas (Cho/NAA 2.4+/-1.0 vs. 2.0+/-1.5, p=0.23; Cho/Cr 1.7+/-0.6 vs. 1.4+/-0.5, p=0.06). In all six progressing tumors, areas of maximum perfusion and maximum Cho/NAA and Cho/Cr ratio were co-localized. During follow-up, contrast-enhancement was observed in these areas. CONCLUSIONS: MR perfusion imaging can depict anaplastic areas in WHO grade II astrocytomas earlier than conventional MRI and thus enables a better prediction of prognosis.

Rapid distinction of acute demyelinating disorders and central nervous system lymphoma by molecular analysis of cerebrospinal fluid cells.

Polymerase chain reaction (PCR) based automated high-resolution fragment analysis of rearranged immunoglobulin heavy-chain genes is a highly sensitive means for identifying clonal B-cell responses. We used this technique to distinguish polyclonal inflammatory from monoclonal neoplastic B-cell populations in the cerebrospinal fluid (CSF) of three patients with acute demyelinating disorders of the central nervous system whose clinical, magnetic resonance imaging (MRI) and CSF features did not permit unequivocal exclusion of primary central nervous system lymphoma (pC-NSL). This approach is highly suitable for detecting CNS inflammation particularly when lymphomatous involvement cannot be ruled out by noninvasive diagnostic procedures alone.

Molecular analysis of the CDR3 encoding region of the immunoglobulin heavy chain locus in cerebrospinal fluid cells as a diagnostic tool in lymphomatous meningitis.

The diagnosis of leptomeningeal B-cell lymphoma is based on the identification of malignant B cells in the cerebrospinal fluid (CSF). Frequently, cytology does not allow clear distinction between neoplastic lymphoid cells and reactively transformed mononuclear cells. Individual B-cell clones can be identified on the basis of DNA sequences that encode the highly diverse complementarity-determining region (CDR3) of the immunoglobulin heavy chain locus (IgH). We studied CSF samples from 5 patients with B-cell malignancies and cytological evidence of leptomeningeal involvement, using polymerase chain reaction (PCR)-based high-resolution capillary electrophoresis and automated fluorescence analysis to detect PCR fragments. As controls, we assessed CSF specimens from 7 patients with inflammatory neurological diseases and three samples without pathological findings. In all patients with B-cell malignancies, a single PCR product was generated, indicating that CDR3-specific fragments were derived from monoclonal cell populations. CSF samples from patients with inflammatory diseases yielded multiple CDR3 amplicons, suggesting the presence of a polyclonal B-cell activation. No PCR product could be amplified in normal CSF samples. Automated fluorescence detection of CDR3 fragments is a highly sensitive and rapid method to distinguish neoplastic monoclonal and reactive polyclonal B-cell populations in the CSF.

[Leptomeningeal dissemination of chronic lymphatic leukemia. Molecular genetic detection in cerebrospinal fluid]. / Leptomeningeale Aussaat einer chronisch lymphatischen Leukämie. Molekulargenetischer Nachweis im Liquor.

The diagnosis of leptomeningeal dissemination of chronic lymphatic leukemia (CLL) by conventional cytology is unreliable because cytomorphologic criteria of malignancy are often lacking. Immunophenotyping of leukocyte differentiation antigens may also be of limited diagnostic value due to the small number of cells in cerebrospinal fluid (CSF) samples. Molecular methods may support the specific diagnosis of leptomeningeal infiltration of CLL. We present an 54 old patient who was diagnosed with CLL five years ago. Despite clinical signs of leptomeningeal involvement neither magnetic resonance imaging (MRI) nor conventional CSF analysis were suggestive of lymphomatous meningitis. Using PCR we selectively amplified the highly variable and clone-specific CDR3 region of the locus encoding the immunoglobulin heavy chain (IgH) in DNA obtained from both CSF and peripheral blood cells. Analysis of PCR products by high resolution gel electrophoresis revealed a single DNA fragment respectively indicating the presence of a monoclonal cell population in both compartments. DNA sequence analysis of the amplified CDR3 segments confirmed the clonal identity of cells and the leptomeningeal dissemination of CLL.

Degradation of porcine brain natriuretic peptide (pBNP-26) by endoprotease-24.11 from kidney cortical membranes.

Porcine brain natriuretic peptide of 26 amino acid residues (pBNP-26) is inactivated by endoprotease-24.11 (EC 3.4.24.11) of kidney cortical membranes. In contrast to human alpha atrial natriuretic peptide/cardiodilatin (ANP/CDD) showing a single major cleavage within the disulfide-linked loop between Cys and Phe in position 7 and 8, pBNP-26 is cleaved at several sites. Although both pBNP-26 and ANP/CDD exhibit Cys-Phe peptide bonds at the corresponding positions this bond is not cleaved in BNP-26.

Initial and early effects of adriamycin in murine sarcoma 180 cannot be restored in a resistant subline by increasing the uptake and external concentration of the drug.

We demonstrate the early effects (1 day) of Adriamycin (ADM) on proliferation-stimulated and quiescent sensitive, and ADM-resistant cells of the murine tumor sarcoma 180 (S 180). By investigating cell-cycle distribution and thymidine labeling, it can be shown that sensitive cells are strongly affected by the drug, even in a proliferationarrested state. A remarkable but slow DNA synthesis is the prominent effect of this drug treatment on sensitive cells, even under nonstimulating conditions. In resistant cells, neither an increase in concentration nor a variation in drug uptake can induce effects that could be compared with those observed in the sensitive line. From these results we conclude that the early effects of ADM are not modulated by drug uptake.

Flow cytometric analysis of primary lung carcinomas and their lymph node metastases.

Specimens of primary lung carcinomas and lymph node metastases from the same 18 patients were investigated by means of flow cytometry. The number of DNA stemlines, DNA indices, the proportion of diploid cells in the tumors and the distribution of the cell cycle phases were compared. In 10 patients DNA stemlines and DNA indices were identical in primary tumors and metastases. In two cases the DNA indices were doubled in metastases. In 6 cases the primary tumors contained two abnormal DNA stemlines and their metastases contained only one aneuploid stemline. Gross differences between primary tumors and lymph node metastases with regard to the proportion of cell cycle phases could not be found. The large variation between primary tumors and lymph node metastases with regard to DNA stemlines indicates that flow cytometric analysis of lymph nodes gives only limited information about the primary tumors.

Prognostic relevance of ploidy, proliferation, and resistance-predictive tests in ovarian carcinoma.

In a cooperative study specimens of 37 patients with stage III and IV ovarian carcinomas who had been treated with chemotherapy were investigated utilizing flow cytometry and an in vitro short-term test for predicting resistance. Patients with aneuploid tumors had significantly shorter survival rates than did those with diploid tumors. Patients whose tumors showed a low G0/G1 cell proportion or a high proliferation pool (S- and G2/M cell-proportion) seemed to die earlier. There was also a tendency for patients with in vitro resistant tumors to die earlier under chemotherapy than those with sensitive tumors.

Anthracycline resistance and consequences of the in situ-in vitro transfer.

Adriamycin-resistant and normal cells of the sarcoma 180 of the mouse undergo qualitatively different deflections from the in situ state when prepared for an experiment. Resistant cells perform a fast reactive decline in the proliferative activity. They are capable of quiescence as defined by the time needed for the induction of the proliferation. Sensitive cells seem to be unable to quiesce and are only slowed down. These facts must be taken into account in interpretation of similar results. Differences in experiments need not necessarily imply differences in situ. Such in vitro appearing differences between sensitive and adriamycin-resistant cells of the murine sarcoma 180 include the retention of the mitochondria-specific stain rhodamine 123 and the uptake of anthracyclines, both being reduced in resistant cells. After labeling sensitive cells with thymidine in vivo and sorting them according to their rhodamine 123-derived fluorescence, the label was only found in the major, highly fluorescing fraction. A small low-fluorescing fraction remained unlabeled. We were able to demonstrate similar results with labeled anthracyclines applied to both the sensitive and the resistant cells in a short period between the removal of the cells from the ascites and the cell sorting. The adriamycin resistance seems to be joined with the ability of the cells to reduce their proliferative activity following changes to unfavorable conditions in vitro. Quiescent cells of the resistant line demonstrate the "anthracycline pump." Substances which are known to increase the sensitivity of anthracycline-resistant cells (TWEEN, verapamil) also shift the cells from low to high rhodamine 123-fluorescence.

DNA distribution in non-small-cell lung carcinomas and its relationship to clinical behavior.

A study of 187 surgical specimens of tumors of patients with non-small-cell lung carcinomas was carried out by means of flow cytometry. Eighty-four percent of the tumors were classified as tumors with abnormal DNA stemlines (DNA aneuploidy). Patients with tumors demonstrating DNA aneuploidy had significantly shorter survival times than those with tumors demonstrating DNA diploidy (p = .009). Cell cycle analysis was possible in 122 tumors. Patients whose tumors had 0-8% S-phase cells died later than patients whose tumors had 9-16% S-phase cells (p = .018). In addition, patients with tumors with a low fraction of labeled S-phase cells (autoradiography) had a better prognosis than patients with tumors with a high proportion of labeled S-phase cells (p = .041).

Isopycnic density-gradient centrifugation: a separation parameter which improves flow cytometric measurements on heterogeneous tumors.

The analytic and prognostic value of flow cytometric measurements can generally be improved by preseparation of cells. The present data demonstrate clearly the distinct character of bands obtained in density-gradient separation. Density-gradient centrifugation in Percoll after extensive Ca2+ + Mg2+ elimination helps to resolve more detail in flow cytometric measurements (e.g., additional DNA stem lines in human tumors) and can be used in studies of tumor heterogeneity. The significance of investigations of tumor heterogeneity is demonstrated by the different reactivity of subpopulations of the experimental murine tumor S 180 to vincristine.

Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.

Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluorometrically: propidium iodide (PI) with fluoresceinisothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPII) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitrobenzodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combination revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest. With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.

The diagnosis of endometrial carcinoma by means of jet wash and DNA flow-through cytophotometry.

Flow-through cytophotometric determination of nuclear DNA content on jet wash material from the endometrium was employed in the diagnosis of endometrial carcinoma. One hundred and thirty cases were studied. In comparison to results from cytodiagnosis, flow-through photometry yielded a false negative rate of 31.6% and a false positive rate of 42.2%. The false negative findings resulted in part from the small relative frequency of atypical cells in a mixed population of normal and atypical cells in certain cases. Besides this, we often found carcinomas with a diploid DNA stem line, which could not be distinguished cytophotometrically from normal corpus endometrium (Sandritter, 1952; Atkin et al., 1959; Hustin, 1976). The flow-through photometrically false positive findings may have resulted either from cell aggregates or from a nuclear DNA content elevated over the diploid value in proliferating cells (D. Wagner et al., 1968; D. Wagner and Richard, 1968). The observed false negative and false positive rates demonstrate that flow-through photometric determination of nuclear DNA content is unsuitable for the diagnosis of endometrial carcinoma.

Differential diagnosis between follicular adenoma and follicular carcinoma of the thyroid by nuclear DNA determination.

Distinction between follicular adenomas and follicular carcinomas of the thyroid using fine needle aspiration cytology presents a problem. We investigated whether Feulgen photometric nucear DNA determination could offer additional, usable information for differential diagnosis. Since both diploid carcinomas and polyploid, widely scattered adenomas and nodular colloid goiter are seen, we found that nuclear DNA determinations proved useful in differential diagnosis only for occasional special cases.

Extended application of flow microfluorometry by means of dual laser excitation.

A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region, DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specificity combined with a great stability under UV illumination. By separating two focussed laser beams at their intersecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.

The significance of DNA flow-through fluorescence cytophotometry for the diagnosis of prostate carcinoma.

Flow-through fluorescence cytophotometric determination of nuclear DNA content was employed for the diagnosis of prostate carcinoma. Fine needle aspiration biopsy material from the prostate of 220 patients was used for study. A false negative rate of 11.4% and a false positive rate of 29.7% were obtained when the results of flow-through photometry were compared with those of traditional cytodiagnosis. It was found that 4.5% of the specimens were unsuitable for cytologic diagnosis and 10.9% for flow-through cytophotometry. False negative DNA histograms may be due to two factors: either the number of tumor cells is small or there are tumor cells whose nuclear DNA content does not differ from that of a normal cell population. False positive findings result from proliferating cells in inflammatory activation. Errors in preparation of the material and mechanical mistakes, such as cellular clumping and coincidences, are less likely causes. The greater percentage of specimens which were inadequate for cytophotometry was due to the large number of cells needed for a utilizable flow-through photometric histogram. The high rate of false negative and false positive results (11.4% and 29.7%, respectively) argues against using flow-through photometric nuclear DNA determination for the diagnosis of prostate carcinoma.