Multiparametric evaluation of flow cytometric synthesis phase fraction determination in dual-labelled breast carcinomas.

Multiparametric, two-color DNA and cell cycle analyses were performed on 112 consecutive mechanically dissociated, ethanol-fixed breast carcinomas using a dual-label method with monoclonal antibodies (CAM 5.2) to cytokeratin (CK) and leukocyte common antigen (LCA) with propidium iodide (PI) staining. There was marked intertumoral variation of CK-positive (range, 3-87%; mean, 40%) and LCA-positive (range, 1-28%; mean, 6.5%) events in DNA histograms. Approximately 70% of DNA aneuploid cells were CK positive. CAM 5.2-stained (avidin-biotin technique) Cytospin preparations correlated with flow cytometric (FCM) detection of CK-positive cells in 15/21 (71%) cases. In each discrepant case, FCM detected greater numbers of CK-positive cells. Cytospin controls of tumor suspensions revealed that cytoplasmic loss was the major cause of decreased CK staining. Synthesis phase fraction (SPF) calculation from CK-gated histograms resulted in kinetic indices (mean ungated, 12.3%, vs. mean CK-gated, 16.8%; P less than .01) with improved statistical correlations with tumor grade and estrogen receptor (ER) status. Differences between ungated vs. CK-gated SPF were greatest in cases having less than 20% CK-positive events (P less than .05). Cases with lower CK staining events generally had higher SPF and were more often high grade (below median CK staining, 61% high grade, vs. above median CK staining, 31% high grade) and ER-negative (below median CK staining, 55% ER negative, vs. above median CK staining, 12% ER negative).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a linkage map and mapping of phenotypic polymorphisms in a free-living population of Soay sheep (Ovis aries)

An understanding of the determinants of trait variation and the selective forces acting on it in natural populations would give insights into the process of evolution. The combination of long-term studies of individuals living in the wild and better genomic resources for nonmodel organisms makes achieving this goal feasible. This article reports the development of a complete linkage map in a pedigree of free-living Soay sheep on St. Kilda and its application to mapping the loci responsible for three morphological polymorphisms for which the maintenance of variation demands explanation. The map was derived from 251 microsatellite and four allozyme markers and covers 3350 cM (approximately 90% of the sheep genome) at approximately 15-cM intervals. Marker order was consistent with the published sheep map with the exception of one region on chromosome 1 and one on chromosome 12. Coat color maps to chromosome 2 where a strong candidate gene, tyrosinase-related protein 1 (TYRP1), has also been mapped. Coat pattern maps to chromosome 13, close to the candidate locus Agouti. Horn type maps to chromosome 10, a location similar to that previously identified in domestic sheep. These findings represent an advance in the dissection of the genetic diversity in the wild and provide the foundation for QTL analyses in the study population.     

Biogeochemical cycling of methylmercury at Barn Island Salt Marsh,Stonington, CT,USA

Monomethylmercury (MMHg) is toxic, and is the primary form of Hg thatbioaccumulates in the food web. An understanding of its distribution,production, and transport is needed. Prior investigations indicate thatmethylation is mediated by sulfate-reducing bacteria, yet limited in highsulfate environments. High rates of microbial respiration and strong oxygengradients are found in salt marshes. It is hypothesized that significant in situ methylation takes place in the redox transition zone of sulfate rich( 28 mM) salt marsh sediment. Results from a water column surveyof Barn Island Salt Marsh in October 1996 showed that ca. 61pmol m^-2 d^-1 of dissolved MMHg were discharged toadjacent coastal waters, while 16 pmol m^-2 d^-1 ofparticulate MMHg were entrained in the marsh, implying an in situsource. In-sediment MMHg production rates were determined by²⁰³Hg radiotracer studies. At the surface, methylation rates variedover both long (i.e., 100's m; 11–1120 pmol m^-2 d^-1) andshort (i.e., 10 cm; 11–108 pmol m^-2 d^-1) spatial scales. Methylation rate profiles from both low and high MMHg production sitesexhibited an exponential decrease below the redox transition zone. Porewater was collected with multi-chambered in situ dialysis (30 kDa)samplers [Peepers] and analyzed for MMHg. Temporal differences in porewater MMHg accumulation (i.e., May > September > November)were found. Results from May showed a significant gradient at thesediment water interface. The transport out of the sediments estimated byFick's Law (ca. 390 pmol MMHg m-2 d^-1) suggeststhat MMHg entered the marsh water by diffusion. This workdemonstrates the potential for elevated in situ Hg methylation in highsulfate environments.     

Histopathologic and flow-cytometric analysis of neoplastic and benign "background" tissue in breast carcinoma resections.

Two-color, multiparametric synthesis phase fraction (SPF) analysis of cytokeratin-labeled epithelial cells was flow cytometrically performed on both benign (SPFb) and malignant tissue samples (if available, SPFt) from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1) tumor differentiation, (2) the proportion of tumor comprised of duct carcinoma-in situ (DCIS), and (3) the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34% of cases), atrophic (AT, 33% of cases), proliferative fibrocystic (PFC, 26% of cases), and non-proliferative fibrocystic (NPFC, 7% of cases). SPFt was inversely correlated with extent of DCIS (DCIS = 0-20% tumor volume - 12.7% mean SPFt, vs. DCIS >20% tumor volume - 6.4% mean SPFt, p = 0.001). SPFt also correlated with the histology of background benign breast tissue (NTDLU - 14.8% mean SPFt vs. AT - 6.9% mean SPFt vs. PFC - 12.7% mean SPFt, p = 0.05) but it did not correlate with patient age or SPFb (overall mean = 0.73%). SPFb was correlated with patient age (>56 yr - 0.59% mean SPFb vs. <56 yr - 0.84% mean SPFb, p = 0.02), with background histology (NTDLU - 1.1% mean SPFb vs. AT - 0.43% mean SPFb vs. PFC - 0.70% mean SPFb, p < 0.02) and with the grade of the neoplasm (well/moderate - 0.58% mean vs. poorly differentiated - 0.85% mean, p = 0.04). Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01). We conclude: (1) breast carcinomas arising from a background of more actively cycling pre-involutional or proliferative fibrocystic epithelium have a greater proliferative fraction than tumors arising from atrophic epithelium, implying that the differentiation status of target cells may impact the effect(s) of tumorigenic events; (2) PFC may represent delayed, abnormal or interrupted involution rather than a hyperproliferative state relative to NTDLU, suggesting that it facilitates neoplasia by extending the period of exposure to promoter agents such as endogenous hormones, and (3) lower SPFt in breast neoplasia with more abundant "residual" DCIS may reflect a lengthier pre-invasive disease interval due to intrinsically less aggressive phenotype.