Miravirsen dosing in chronic hepatitis C patients results in decreased microRNA-122 levels without affecting other microRNAs in plasma.

BACKGROUND: MicroRNA-122 (miR-122) is an important host factor for hepatitis C virus replication. Administration of miravirsen, an anti-miR-122 oligonucleotide, resulted in a dose dependent and prolonged decrease in HCV RNA levels in chronic hepatitis C patients. AIM: To assess the plasma level of various miRNAs in patients dosed with miravirsen. METHODS: We included 16 of 36 chronic hepatitis C patients who received five injections of either 3 mg/kg (n = 4), 5 mg/kg (n = 4), 7 mg/kg (n = 4) miravirsen or placebo (n = 4) over a 4-week period in a double-blind, randomised phase 2a study. Plasma levels of 179 miRNAs were determined by qPCR and compared between patients dosed with miravirsen or placebo. RESULTS: Median plasma miR-122 level at baseline in patients receiving miravirsen was 3.9 × 10(3) compared to 1.3 × 10(4) copies/4 µL in placebo-dosed patients (P = 0.68). At week 1, 4, 6 and 10/12, patients dosed with miravirsen had respectively a median 72-fold, 174-fold, 1109-fold and 552-fold lower expression of miR-122 than at baseline (P = 0.001, as compared to patients receiving placebo). At week 4 of dosing, miRNA-profiling demonstrated a significant lower expression of miR-210 and miR-532-5p compared to baseline (3.0 and 4.7-fold lower respectively). However, subsequent longitudinal analysis showed no significant differences in miR-210 and miR-532-5p plasma levels throughout the study period. CONCLUSIONS: We demonstrated a substantial and prolonged decrease in plasma miR-122 levels in patients dosed with miravirsen. Plasma levels of other miRNAs were not significantly affected by antagonising miR-122.

A comparison of instrumentation using Naviflex and Profile nickel-titanium engine-driven rotary instruments.

This study was designed to compare the changes in canal configuration resulting from instrumentation by either Profile or Naviflex instruments. Forty mesial canals in extracted human molar teeth were embedded and sectioned at two root levels. Reassembled teeth were instrumented with a modified crown-down technique as described in the Profile training video for Profile files and in a similar manner for Naviflex instruments. Superimposed pre- and postinstrumented cross-sectional root images were projected, traced, and scanned into a computer for analysis. Canal movement, in relation to the furca, and canal area change were recorded. The results showed no significant difference in canal center movement or canal area change between the Profile or Naviflex groups. The degree of canal curvature had no effect on canal center movement or canal area change.

Geographic distribution of co-dominant DNA stemlines in breast carcinoma.

Breast carcinomas often contain multiple DNA stemlines in flow cytometric DNA histograms. However, due to mixing during tissue disaggregation the microanatomical relationship between the cells which comprise distinct stemlines is unclear. We performed image cytophotometric DNA analysis (IA) on two separate areas of intact tissue sections of 19 breast carcinomas which were selected on the basis of flow cytometric (FCM) DNA content heterogeneity (i.e., multiple stemlines). For comparison, similar analyses were performed on seven tumors with unimodal FCM DNA histograms. Six of the 7 tumors (86%) with unimodal FCM histograms were also unimodal in both IA DNA histograms. Among tumors with heterogeneous FCM DNA histograms, the presence of multiple stemlines was confirmed in IA DNA histograms in 16/19. In nine of these 16 cases, multiple DNA stemlines having similar DNA indices were present in both areas of neoplasm examined with IA. The remaining seven cases displayed unimodal IA histograms in both areas, however DNA indices differed between the two histograms. These findings imply that cell populations corresponding to flow cytometrically detected DNA stemlines are often intimately admixed, even within geographically separated portions of breast tumors. This pattern suggests that productive interactions between genetically distinct tumor populations may lead to stable co-dominance of ancestral clones during progression of some breast carcinomas.

Biologic and clinical significance of basic fibroblast growth factor immunostaining in breast carcinoma.

Acetone-fixed cryostat sections of 79 breast carcinomas were immunostained with antibodies to basic fibroblast growth factor (bFGF) and urokinase-type plasminogen activator (uPA). Staining intensity was then compared with microvessel density assessed by manually counting vascular spaces highlighted by immunostaining vascular basal lamina (Type IV) collagen. Extensive (2+) bFGF immunoreactivity was present in neoplastic cells of 30 tumors (38%) and in host-derived stromal cells of 29 cases (37%). Disease recurrence correlated with bFGF staining: 0 to 1+ stromal staining, 30% recurred versus 2+ stromal staining, 73% recurred (P = 0.001) (54-mo median follow-up). Neither stromal nor epithelial bFGF staining correlated significantly with microvessel count; however, there was a statistically significant association between stromal cell bFGF staining and uPA staining of peritumor host cells: absent bFGF--0% 2+ uPA versus weak bFGF--9% 2+ uPA versus 2+ bFGF--29% uPA (P = 0.01). We conclude that elevated expression of bFGF in breast carcinomas is associated with aggressive clinical behavior. Its biologic significance, however, appears more closely related to extracellular matrix remodeling than to induction of prominent neovascularization per se.

Assessment and significance of diploid-range epithelial populations in DNA aneuploid breast carcinomas using multi-parametric flow cytometry.

A 2-color (PI, cytokeratin--FITC) multi-parametric analysis of intact cells was used to reveal diploid-range epithelial populations by flow cytometry in 108 consecutive DNA aneuploid breast carcinomas. Thirty-eight tumors (35%) contained a significant diploid range epithelial population, defined as cytokeratin-positive cells having a DNA content indistinguishable from that of endogenous lymphocytes and comprising at least 20% of all cytokeratin-positive cells. An additional 23 cases (21%) contained a minor diploid range epithelial population having a normal DNA content and comprising only 5-20% of all cytokeratin-positive cells. Multiple DNA aneuploid stemlines were present in 24 cases (22%). Diploid-range populations were more frequent (91%) in tetraploid cases than in hyperdiploid (32%), hypodiploid (17%) or hypertetraploid cases (20%). The presence of diploid epithelial populations and/or multiple aneuploid stemlines correlated with histologic parameters, including an extensive intraductal component (unimodal--4% vs. multi-modal--57%, P = 0.001), heterogeneous differentiation (unimodal--0% vs. multi-modal--52%, P = 0.001), and multi-focal growth with residual interspersed benign tissue (unimodal--8% vs. multimodal--57%, P = 0.01). These data show that diploid-range epithelial cells are frequent in aneuploid breast carcinomas analyzed by flow cytometry. In some tumors, these populations undoubtedly reflect the presence of residual benign epithelium. The numerical dominance of other histograms by near-diploid measurements suggests the presence of diploid-range neoplastic stemlines which would be 'hidden' by contaminating host-derived cells in single parameter DNA histograms. Finally, the correlation of DNA content heterogeneity with distinctive histologic patterns of breast neoplasia implies that co-existing stemlines may have biological significance in the progression of some tumors.

Enhanced expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the stroma of breast carcinomas correlates with tumor recurrence.

The 72-kDa (MMP-2, gelatinase A) and the 92-kDa (MMP-9, gelatinase B) matrix metalloproteinases have been associated with tumor cell invasion and metastasis. Immunohistological staining of MMP-2 and MMP-9, basal lamina collagen IV and TIMP-2 were performed on frozen sections of 83 invasive breast carcinomas. MMP-2 and MMP-9 were associated with neoplastic cell plasma membrane in 72% of cases and exhibited inter-tumoral variability of staining intensity. MMP-2 and MMP-9 staining was not correlated with presence of metastases at time of diagnosis or with disease outcome. TIMP-2 was detected in the peri-tumoral stroma and was present in 87% of cases. Residual benign breast tissue was negative for TIMP-2 staining. Neoplasms with diffuse TIMP-2 staining (24%) recurred significantly more frequently (75% recurred) than cases with focal (42% recurred) or absent (27% recurred) TIMP-2. Presence of collagen IV was negatively correlated with gelatinase staining. We conclude that up-regulation of MMP-2 and MMP-9 expression in breast tumor cells is reciprocally correlated to collagen IV staining. Clinical outcome, however, is more closely related to the presence of TIMP-2 than the corresponding MMPs. Enhanced TIMP-2 expression, therefore, may denote a stromal response to tumor invasion, indicative of aggressive behavior in a subset of breast carcinomas.

Flow cytometric enumeration and kinetic analysis of inflammatory cell populations in breast carcinomas.

Inflammatory cell populations were quantitated in 76 consecutive mechanically dissociated clinical breast carcinoma specimens using multiparameter, two-color (PI-cytokeratin/FITC, PI-LCA/FITC) flow cytometric analysis. The percent LCA-positive events varied from 1.3-62.5% (mean = 11%) and correlated with degree of histologic inflammatory cell infiltrate (mild-5.8% LCA+events vs. marked-35.9% LCA + events, P < 0.001), abnormal DNA content (diploid range--4.6% LCA + events vs. aneuploid--13.3% LCA + events, P = 0.003) and poor tumor differentiation (well-moderate-6.5% LCA+events vs. poor--19.9% LCA + events, P = 0.001). Synthesis phase fractions in LCA-positive populations were uniformly less than the corresponding cytokeratin (CK)-positive cells (mean LCA + SPF = 4.4% vs. mean CK + SPF = 15.5%) and varied from 3-11%. Proliferation among inflammatory populations did not correlate statistically with the total percent of LCA or CK-positive events, nor with the SPF in epithelial populations. However, proliferative activity of inflammatory components was greater in tumors with predominately intratumoral, vs. peritumoral, inflammatory cell distribution (4.6% vs. 3.3%, P = 0.03) and in tumors with greater numbers of large, 'transformed' lymphocytes (few = 3.25% vs. many = 6.5% LCA + SPF, P = 0.001). We conclude multiparameter flow cytometric analysis of mechanically-dissociated breast tumors is representative of tumor infiltrating lymphoid populations in breast tumors and provides a potentially useful means of studying biologically relevant tumor-host interactions.

Immunohistologic evaluation of invasion-associated proteases in breast carcinoma.

Immunostaining of two invasion-associated proteolytic enzymes, cathepsin D (CD) and urokinase-type plasminogen activator (uPA), was assessed in cryostat sections of 86 stage-heterogeneous breast carcinomas using monoclonal antibodies. Most tumors displayed a focal and/or heterogeneous staining pattern. Overall, staining was more frequent in host-derived stromal and inflammatory cells (uPA 54%, CD 89%) than neoplastic epithelium per se (uPA 24%, CD 70%). Intense (i.e., 2+) stromal, but not neoplastic, CD was significantly correlated with nodal or systematic metastases (node negative--10% versus node positive/systemic--33%, p = 0.04). Further, cumulative staining of more than one enzyme (CD + uPA) or more than one tumor component (stroma + epithelium) correlated with metastatic disease (no metastases--35% versus metastatic--72%, p = 0.005). Neither stromal nor epithelial CD alone was significantly correlated with short-term recurrence free survival, however additive CD staining (i.e., stromal + epithelial) was strongly predictive, overall (both + -75% recurred versus both weak/negative--16% recurred, p = 0.0004) and in node positive patients (p = 0.02). We conclude that (a) enzymes putatively mediating extracellular matrix dissolution may be derived from multiple sources and (b) the metastatic capacity and/or clinical aggressiveness of breast carcinomas may reflect overall proteolytic enzyme expression, suggesting that cooperative enzyme interaction may be required for invasive growth and/or metastasis.