The extent of biopsy involvement as an independent predictor of extraprostatic extension and surgical margin status in low risk prostate cancer: implications for treatment selection

PURPOSE: We identify predictors of extraprostatic extension and positive surgical margins in patients with low risk prostate cancer (prostate specific antigen [PSA] 10 ng./ml. or less, biopsy Gleason score 7 or less and clinical stage T1c-2b). MATERIALS AND METHODS: From August 1997 to January 1999, 143 previously untreated patients underwent radical retropubic prostatectomy for clinically localized prostate cancer. A total of 62 patients were low risk, with PSA 10 ng./ml. or less, biopsy Gleason score 7 or less and clinical stage T1c-2b, and had sextant biopsy with separate pathological evaluation of each sextant cores. PSA, clinical stage, biopsy Gleason score, average percentage of cancer in the entire biopsy specimen, maximum percentage of cancer on the most involved core, number of cores involved and bilaterality were evaluated for association with extraprostatic extension, seminal vesicle involvement and positive surgical margins. RESULTS: Of the 62 patients 13 (21%) had extraprostatic extension, 6 (10%) seminal vesicle involvement and 20 (32%) positive surgical margins. Average percentage greater than 10% and maximum percentage greater than 25% were associated with extraprostatic extension (p = 0.01 and 0.004, respectively). Average percentage greater than 10%, maximum percentage greater than 25%, more than 2 cores involved and bilaterality were associated with positive surgical margins (p = 0.007, 0.01, 0.002 and 0.03, respectively). On multivariate analysis maximum percentage remained the only independent predictor of extraprostatic extension (p = 0.03), and the number of cores involved remained an independent predictor of positive surgical margins (p = 0.01). Biopsy Gleason score, PSA and clinical stage did not correlate with extraprostatic extension or positive surgical margins in this patient population. CONCLUSIONS: In low risk prostate cancer the extent of biopsy involvement significantly correlates with the risk of extraprostatic extension and positive surgical margins. Biopsy information should be considered when selecting and modifying treatment modalities.     

Restriction of cell lysis by homologous complement: II. Protection of erythrocytes against lysis by newly activated complement

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1-9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1-9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1-8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1-9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1-9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1-8. The minimum number of 9-sites required to confer a protective effect on EAC1-9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.     

Growth characteristics of circulating hematopoietic progenitor cells from patients with essential thrombocythemia

Peripheral blood mononuclear cells from five patients with essential thrombocythemia (ET) were cultured in vitro to evaluate restricted megakaryocytic (CFU-Meg), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell development. Varying concentrations of aplastic canine serum served as the source of megakaryocyte colony-stimulating activity, and cultured megakaryocyte ploidy distributions were determined by Feulgen staining and microfluorometry. Megakaryocyte colony growth was strikingly abnormal in all five patients evaluated. Four of the 5 had a marked expansion in the concentration of circulating CFU-Meg and 3 of 4 manifested abnormalities in cultured megakaryocyte colony size (2 unusually large and 1 small). Unstimulated megakaryocyte colony growth was substantially increased in three patients. However, the fraction of megakaryocyte progenitors in cell cycle was near or below normal in all instances. Endomitotic megakaryocyte development was disordered in each of the four ET patients in whom it was evaluable. In normal subjects, mean megakaryocyte ploidy values vary biphasically with aplastic canine serum concentration and peak at 13.2 N following 12 to 15 days of culture. In contrast, day 12 mean ploidy values in cultures from the ET patients remained low at all aplastic canine serum concentrations and reached a maximum averaging only 8.4 N. Three patients were evaluated serially at extended culture durations of up to 21 days. The cultured megakaryocyte ploidy was unchanged during this interval for two of the patients. For the third patient, ploidy increased steadily, reaching abnormally high ploidy values by day 21. Progenitor cell expansion was limited to the megakaryocyte line in three patients. However, two patients had substantial increases in CFU-GM, one of whom also had a marked increase in BFU-E. There was no significant unstimulated colony growth by either CFU-GM or BFU-E. These data indicate that ET is usually characterized by an expansion in the concentration of circulating CFU-Meg in vivo which manifest both disordered replication and endoreduplication in vitro.     

Tuberin-heterozygous cell line TSC2ang1 as a model for tuberous sclerosis-associated skin lesions

Tuberous sclerosis (TS), neurological disorder manifesting with the formation of tumors in numerous organ systems, is a disease associated with the upregulation of mammalian target of rapamycin (mTOR) pathway. It has been found that in healthy individuals two tumor suppressor genes, TSC1 and TSC2, encoding proteins called hamartin and tuberin, respectively, are responsible for the control over mTOR kinase. Loss of one of these genes constitutes the genetic background of TS. In the current study, we aimed at evaluating the fitness of the only TS-associated sarcoma cell line deposited in American Tissue Culture Collection, TSC2ang1, for the in vitro studies on TS. We found that the line shows a stable chromosome pattern with typical Robertsonian translocations. Similarly to primary tumors from TS patients, TSC2ang1 cells respond to rapamycin-induced mTOR inhibition. The cells demonstrate activation of both Akt and Erk pathways, but inhibition of neither of them is as effective as mTOR suppression when considering proliferation potential. Based on these results we propose TSC2ang1 as a good and stable model for pathophysiological and pharmacological studies on skin lesions in TS.     

Functional proteomics mapping of a human signaling pathway

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.     

Selective dual nuclear scanning in low-risk patients with chest pain to reliably identify and exclude acute coronary syndromes

STUDY OBJECTIVE: We sought to determine the use in routine clinical practice of selective dual nuclear cardiac scanning (rest and stress) in low-risk patients with chest pain for identifying and excluding acute coronary syndromes (ACSs) during the initial emergency department evaluation. METHODS: A prospective observational study was conducted over 13 months in 1,775 low-risk patients with chest pain who had intermediate- and high-risk ACSs ruled out by means of our 2-hour protocol, which consists of automated serial 12-lead ECG monitoring in conjunction with baseline and 2-hour creatine kinase (CK) MB and troponin I (cTnI) measurements. At the completion of the 2-hour evaluation period, low-risk patients were stratified by means of physician judgment into 1 of 2 categories: category III, possible ACS; and category IV, probable non-ACS chest pain. Level III patients underwent immediate dual nuclear scanning (rest thallium and stress sestamibi scanning), and level IV patients were discharged directly from the ED unless another serious non-ACS medical condition was thought to exist. Rest and stress scans were interpreted by a board-certified radiologist contemporaneous with patient evaluation. All patients were followed up for 30-day ACS, which was defined as acute myocardial infarction, percutaneous transluminal coronary angioplasty/coronary artery bypass grafting, coronary arteriography revealing stenosis of the major coronary artery of 70% or greater not amenable to percutaneous transluminal coronary angioplasty/coronary artery bypass grafting, life-threatening complication, or cardiac death within 30 days of ED presentation. RESULTS: A total of 2,206 ED patients with chest pain were evaluated for ACS during the study period. Four hundred thirty-one patients were excluded for having 1 or more of the following findings: initial ECG diagnostic of injury; baseline CK-MB level, cTnI level, or both diagnostic of acute myocardial infarction; 2-hour DeltaCK-MB level of +1.5 ng/mL or greater; 2-hour DeltacTnI level of +0.2 ng/mL or greater; injury or new or evolving ischemia on serial 12-lead ECG monitoring; or clinical diagnosis of ACS. Of the 1,775 study patients, 805 (45.4%) underwent immediate dual nuclear scanning. A positive stress nuclear scan result was more sensitive (97.3% versus 71.2%, P <.0001) and specific (87.7% versus 72.6%, P <.0001) for 30-day ACS than a positive resting nuclear scan result. The protocol of selective dual nuclear scanning (ie, patients who did not undergo dual nuclear scanning were counted as having a negative test result) had a sensitivity and specificity for 30-day ACS of 93.4% and 94.7%, respectively (positive likelihood ratio 17.6; negative likelihood ratio 0.07). CONCLUSION: Stress nuclear scanning is more sensitive and specific than resting nuclear scanning for identification of ACS in low-risk patients with chest pain. A strategy of using selective dual nuclear scanning once high- and intermediate-risk ACS has been ruled out with our 2-hour evaluation both reliably identifies and reliably excludes 30-day ACS.     

The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor

The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR.     

Carrier detection in hemophilia A: a cooperative international study. I. The carrier phenotype

Eight laboratories in six countries cooperated to clarify several issues concerning the phenotypes of heterozygous carriers of hemophilia "A." Plasma levels of factor VIII (F.VIII:C, formerly VIII:C) and von Willebrand factor (VWF:Ag, formerly VIIIR:Ag) of carriers and normal women were determined by various "in-house" methods; a single lyophilized plasma standard was used for all assays. Analysis of the collated data from 336 carriers (296 obligatory carriers and 40 sporadic carriers) and 137 normal women showed that there was no difference in the F.VIII:C levels of "paternal" carriers (women who had obtained the abnormal gene from their fathers) and "maternal" carriers. Neither was there a difference in the VWF:Ag levels of normal women and either type of carrier. Age was found to have a significant effect on both F.VIII:C and VWF:Ag, values being higher at very young and very old ages, the minima occurring in the 25- to 30-year range. ABO blood type had a striking effect. Women of types A, B, and AB (designated non- O in the study), both normals and carriers, had significantly higher levels of both factors than did women of type O. Analysis by laboratories showed that differences in mean levels of both factors between laboratories were highly significant. It was concluded that age, ABO blood type, and laboratory variation should be taken into account in carrier detection.