Effect of thrombin peptide 508 (TP508) on bone healing during distraction osteogenesis in rabbit tibia

Thrombin-related peptide 508 (TP508) accelerates bone regeneration during distraction osteogenesis (DO). We have examined the effect of TP508 on bone regeneration during DO by immunolocalization of Runx2 protein, a marker of osteoblast differentiation, and of osteopontin (OPN) and bone sialoprotein (BSP), two late markers of the osteoblast lineage. Distraction was performed in tibiae of rabbits over a period of 6 days. TP508 (30 or 300 μg) or vehicle was injected into the distraction gap at the beginning and end of the distraction period. Two weeks after active distraction, tissue samples were harvested and processed for immunohistochemical analysis. We also tested the in vitro effect of TP508 on Runx2 mRNA expression in osteoblast-like (MC3T3-E1) cells by polymerase chain reaction analysis. Runx2 and OPN protein were observed in preosteoblasts, osteoblasts, osteocytes of newly formed bone, blood vessel cells and many fibroblast-like cells of the soft connective tissue. Immunostaining for BSP was more restricted to osteoblasts and osteocytes. Significantly more Runx2- and OPN-expressing cells were seen in the group treated with 300 μg TP508 than in the control group injected with saline or with 30 μg TP508. However, TP508 failed to increase Runx2 mRNA levels significantly in MC3T3-E1 cells after 2–3 days of exposure. Our data suggest that TP508 enhances bone regeneration during DO by increasing the proportion of cells of the osteoblastic lineage. Clinically, TP508 may shorten the healing time during DO; this might be of benefit when bone regeneration is slow.
Introduction
Distraction osteogenesis (DO) is a well-established technique, originally developed in orthopedic surgery for bone lengthening (Kojimoto et al. 1988) and later also used to treat hereditary malformations in the craniomaxillofacial region (Amir et al. 2006). DO is characterized by the formation of new bone between two osteotomized bone segments, which are separated by gradual traction. One of the clinical phases of the distraction technique is the bone consolidation period, defined as the time between the end of active distraction and the removal of the distraction device. It represents the time needed for a complete bridging of the distraction gap by bone and a further maturation of this bone. Typically, the bone consolidation phase takes approximately 6–12 weeks in the craniomaxillofacial region and 3–6 months in long bones (Fischgrund et al. 1994; Smith et al. 1999; Felemovicius et al. 2000; Amir et al. 2006). Although DO is believed to be superior to other bone augmentation techniques, many attempts have been made to improve the technique to enhance the bone regeneration process in the distraction gap (Hagiwara and Bell 2000; Li et al. 2002; Mofid et al. 2002; Takamine et al. 2002; Schortinghuis et al. 2005). Such improvements would provide the opportunity to shorten the bone consolidation period and hence minimize complications such as the development of non-union, infection or fracture. Various methods have been tested to promote bone formation in the distraction gap, e.g. electrical and mechanical stimulation (Hagiwara and Bell 2000; Mofid et al. 2002), transplantation of osteoblast-like cells (Takamine et al. 2002) or administration of growth factors such as bone morphogenetic proteins (Li et al. 2002) or fibroblast growth factor 2 (FGF-2; Okazaki et al. 1999). Another procedure to accelerate bone regeneration involves the application of thrombin-related peptide 508 (TP508; Ryaby et al. 2000; Sheller et al. 2004; Li et al. 2005a,b; Wang et al. 2005).
TP508 is a synthetic peptide consisting of 23 amino acids, which represent the natural amino acid sequence of the receptor-binding domain of human thrombin (pro-thrombin amino acids 508–530; Sower et al. 1999; Stiernberg et al. 2000). Thrombin is an important factor in blood homeostasis, inflammation and wound healing (Coughlin et al. 1992). It plays a role in the formation of fibrin clots and platelet activation. Thrombin also stimulates the cell proliferation, migration and/or differentiation of various cell types, such as fibroblasts, endothelial cells and lymphocytes. All these cell types contain one or more thrombin receptors (Chen and Buchanan 1975; Belloni et al. 1992). TP508 appears to mimic and accelerate many of the effects of native thrombin in initiating wound healing through a non-proteolytic pathway (Sower et al. 1999). Histological studies have indicated that TP508 is chemotactic for neutrophils, lymphocytes and monocytic cells; it enhances the formation of granulation tissue, neovascularization and other wound-healing events (Sower et al. 1999; Ryaby et al. 2000; Naldini et al. 2004).
TP508 has also been tested for its potential to enhance bone formation (Ryaby et al. 2000; Sheller et al. 2004; Li et al. 2005a,b; Wang et al. 2005). A single injection of 1 μg TP508 accelerates fracture healing in rat long bone fracture. The breaking strength of bones of young and aged rats injected with TP508 has been enhanced over that of controls (Ryaby et al. 2000). One recent study of TP508 treatment during DO has revealed a significantly greater bone mineral density in TP508-treated groups than in saline-treated controls (Li et al. 2005a,b). Histological analysis has also shown advanced bone consolidation and remodeling after TP508 treatment.
In the present study, we report the effect of TP508 in the regulation of bone regeneration during DO in more detail. We have examined Runx2/Cbfa1, osteopontin (OPN) and bone sialoprotein (BSP) protein expression in tissue sections of rabbit long bones undergoing DO. Runx2 is an essential transcription factor for osteoblast differentiation and for extracellular matrix gene expression (Komori et al. 1997; Karsenty et al. 1999). In Runx2 knockout mice, both intramembranous and endochondral ossification are blocked because of the maturational arrest of osteoblasts (Komori et al. 1997). Runx2 can directly stimulate the transcription of the collagen type I and osteoblast-related genes, such as those for OPN and BSP, both major non-collagenous proteins found in the extracellular matrix of bone (Karsenty et al. 1999). We have hypothesized that TP508 treatment during DO enhances the expression of Runx2, OPN and BSP.
Materials and methods
Animal model of DO
The paraffin blocks containing distracted rabbit long bones were those prepared previously (Li et al. 2005a,b) from 30 adult male New Zealand White rabbits (age 24 weeks, body weight 2.6–3.5 kg). External fixators were applied to stabilize the osteotomized mid-tibia for 7 days of the latency period and were activated for 6 days, once a day at a rate of 1.4 mm. This distraction rate was intentionally chosen in order to copy the poor bone healing condition as previously described (Li et al. 1997).
During the experiment, seven animals were excluded in the study because of soft tissue complications (n = 2), pinhole fracture (n = 3) and anaesthetic death (n = 2). The remaining 23 rabbits were randomly divided into three experimental groups, each group consisting of minimally seven rabbits. TP508 (OrthoLogic, Tempe, USA) at two different concentrations (30 and 300 μg TP508) or saline were injected percutanously at the beginning and at the end of the lengthening phase. The first group (n = 8) received injections of 300 μl saline containing 300 μg TP508, the second group (n = 8) received injections of 300 μl saline containing 30 μg TP508 and the third group (n = 7) received injections of 300 μl saline alone and served as a control group. The injections were given in three different areas (100 μl in each area): the proximal, central and distal area of the distraction gap. Two weeks after the lengthening phase, all animals were killed. Tissue samples consisting of the distraction gap and 5 mm proximal and distal to the pre-existing bone were then harvested and immediately fixed in 95% ethanol for further examination. Tissues were decalcified with EDTA and embedded in paraffin. Serial sections (5 μm thick) were cut and mounted on poly-lysine-coated glass slides. The peripheral quantitative computed tomography and histological examination of these bones has been published previously (Li et al. 2005a,b).
All animal experimental procedures were approved and performed following the guidelines for animal scientific procedures (Animal Scientific Procedures Act 1986, British Home Office).
Antibodies
The mouse recombinant monoclonal antibody Pebp2alphaA was kindly donated by Dr. K. Sasaguri, Kanawaga Dental School, Japan. This antibody has been used and characterized previously and reacts with all isotypes of Runx2 (Bronckers et al. 2001, 2005). The mouse anti-rat OPN monoclonal antibody (MPIIIB101) was obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences Iowa City, IA 52242, USA). The mouse anti-human BSP (monoclonal antibody A4232.1) was purchased from Immunodiagnostik, Bensheim, Germany. Both anti-OPN and anti-BSP antibodies have been used and specified previously (Ibrahim et al. 2000; Bronckers et al. 2005; Zerbo et al. 2005).
Immunohistochemical procedures
Tissue sections were de-paraffinized in xylene and rehydrated through a graded series of aqueous ethanol solutions. Endogenous peroxidase was quenched by incubation in 3% H2O2 in 100% ethanol (5 min). A pre-incubation in hot citrate buffer (0.01 M, pH 6.0, 95°C, 20 min) was performed for antigen retrieval prior to incubation with 30% normal horse serum (30 min). Sections were then incubated overnight with primary monoclonal anti-Runx2 antibody (1:200), anti-BSP antibody (1:400) or anti-OPN antibody (1:600) at 4°C in a humidified chamber. After washes with phosphate-buffered saline (PBS), biotinylated horse anti-mouse IgG (ABC-peroxidase Elite kit; Vector Laboratories, Burlingame, Calif., USA) was added at a dilution of 1:200 and the sections were incubated at room temperature for a minimum of 60 min. The sections were washed again with PBS, incubated with ABC peroxidase complex for another 60 min followed by a maximum of 20 min in 3,3′ diaminobenzidine substrate solution (Vectastain kit) to visualize the immunoreaction, counterstained with methyl green for 2–3 min, mounted in 50% glycerol and covered with a glass coverslip. As negative controls, sections were incubated with non-immune mouse IgG in place of the primary antibody.
Scoring
Sections were examined by using a Leica DM RA microscope equipped with a Leica DC 200 digital camera. The central areas of the gap were selected as the region of interest (ROI). First, the boundaries of the distraction gap (indicated by the osteotomy lines) were determined and subsequently a grid was positioned over the ROI halfway between the two osteotomy lines. The grid was 609 μm in width and 479 μm in length. The ROI consisted of some newly formed bone (mostly in the group treated with 300 μg TP508) and fibrous tissue (Fig. 1a). Counting was performed by using the grid at a magnification of 200×, with a minimum of three sections, at least 30 μm apart, per animal. Some tissue blocks were of poor quality or no longer contained a complete intact gap area with an ROI; this reduced the number of animals per group to 5–6 animals. The total number of cells was counted (immunopositive and immunonegative cells). The sections were coded and randomized. Quantification was performed by one investigator (L.A.) who was unaware of the origin of the sections. Numbers of immunopositive cells were noted in absolute terms and were expressed as the percentage of all (immunopositive and negative) cells present in the ROI.
Fig. 1Immunolocalization of Runx2 and osteopontin proteins in TP508-treated groups (a-c, e 300 μg TP508 group, d 30 μg TP508 group). a Overview of immunoreactions in a biopsy taken from rabbit long bone after distraction osteogenesis Runx2 staining (FT fibrous tissue, NB new bone, P periosteum, PC periosteal callus). A white grid was used to count the number of immunopositive and negative cells in the region of interest. ×50. b Runx2 staining in the nuclei and cytoplasm of the cells. Runx2 was strongly expressed in osteoblasts (Ob). Note that young osteocytes (Ocy), which had recently embedded in the bone matrix, expressed Runx2 protein. Cells adjacent to the osteoblast layer, presumably pre-osteoblasts (Pre-Ob), also expressed Runx2, but with lower intensity (B bone). ×400. c Immunostaining for osteopontin. Positive staining was found in the cytoplasm of the cells (white arrows). Note that expression was stronger towards the newly formed bone (NB). ×200. d Runx2 immunostaining in tissue of a rabbit injected with 30 μg TP508. Expression was much lower than that after injection with 300 μg TP508 and was similar to saline-injected control tissue (not shown). Fewer fibrocartilage-like cells (FCC) were stained than after injection with 300 μg TP508 (HC hypertrophic cartilage-like cells). ×200. e Negative control stained with non-immune IgG instead of primary antibodies to Runx2. Note the lack of positive staining. Counter-staining with methyl green. ×200. Bars 20 μm (b, d), 50 μm (c, e), 300 μm (a)
Cell cultures
The mouse osteoblast-like MC3T3-E1 cell line, derived from newborn mouse calvaria, was routinely maintained in α-MEM (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (HyClone), 120 μg/ml penicillin (Sigma, St Louis, Mo., USA), 100 μg/ml streptomycin sulphate (Sigma), 1.25 μg/ml fungizone (Gibco), 50 μg/ml sodium ascorbate (Merck, Darmstadt, Germany), 10 mM β-glycerophosphate (Sigma) and 300 μg/ml glutamine (Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air. This cell line is capable of expressing Runx2 mRNA (Tsuji et al. 1998). To stimulate cells to express osteoblastic markers, they were first exposed to 10 nM dexamethasone in 25 cm2 flasks (Greiner BioOne, Solingen, Germany) for 1 week. Upon confluency (typically after 5 days), cells were harvested by using 0.25% trypsin and 0.1% EDTA in PBS, centrifuged at 600g for 10 min, washed and plated in 24-well culture dishes (Greiner BioOne) at three different cell densities (3×104, 1.5×104 and 7.5×103 cells/well) in order to obtain a comparable number of cells at the end of each experiment. After 1 day of culture, the medium was replaced by media containing various concentrations of TP508 (0, 10 or 100 μg/ml). Throughout the experiment, all media were supplemented with 10 nM dexamethasone. Cells were exposed to TP508 for 24, 48 and 72 h, after which times, the cells were collected. The experiment was carried out in quadruplicate and repeated once.
RNA analysis and quantitative real-time polymerase chain reaction
Total RNA from cultured cells was isolated by using TRIZOL reagent (Gibco) according to the manufacturer’s instructions. The RNA content was determined by measuring the absorbance in water at 260 nm by means of an Ultrospec III spectrophotometer (Amersham, Buckinghamshire, England). cDNA synthesis was performed by using 750 ng total RNA in a final reaction volume of 20 μl containing 5 U transcriptor (Roche Applied Science), 5× polymerase chain reaction (PCR) buffer, 4 U random primers (Roche), 20 U protector RNase inhibitor (Roche), 1 mmol each dNTP and 10 μl template. The reverse transcription step was performed on a Gene Amp 9700 Thermocycler (Applied Biosystems, Foster City, Calif., USA) at 55°C for 30 min followed by 85°C for 5 min.
Real-time PCR was performed on the ABI PRISM 7700 sequence detection system (Applied Biosystems). The phosphobilinogen deaminase gene (PBGD) served as the endogenous reference (de Vries et al. 1999) to normalize Runx2 expression. The primers for the amplification of Runx2 mRNA were 5′-ATGCTTCATTCGCCTCAC-3′ and 5′-ACTGCTTGCAGCCTTAAAT-3′ (GenBank database, accession no. NM 001024630). The PCR primers for PBGD, amplified as the internal reference, were 5′-AGTGATGAAAGATGGGCAACT-3′ and 5′-TCTGGACCATCTTCTTGCTGA-3′ (accession no. BC 003861). For the amplification of the Runx2 and PBGD products, 37.5 ng cDNA was added to the PCR mixture containing SYBR Green PCR Master Mix consisting of SYBR Green I Dye, AmpliTaq Gold DNA polymerase, dNTPs with dUTP, a passive reference and buffer (Applied Biosystems) and 300 nM of each primer, in a final volume of 25 μl. The enzyme was activated by preheating the samples for 10 min at 95°C, followed by a two-step PCR procedure consisting of a denaturation step at 95°C for 15 s and an annealing and extension step at 60°C for 1 min for 45 cycles. Relative expression was calculated by using the comparative Ct method. Samples were normalized for the expression of PBGD by calculating ΔCt (CtRunx2−CtPBGD); subsequently, the ΔΔCt values were calculated as ΔCtsample−ΔCtcalibrator , where the calibrator was the control sample (without TP508 incubation). Relative expression of the Runx2 gene was expressed as 2-(ΔΔ Ct) (Livak and Schmittgen 2001).
Statistical analysis
Values obtained from the scoring of tissue sections and normalized mRNA levels determined by PCR analysis (presented as means and standard deviations) were analysed by using GraphPad Prism 4 for Windows. For the analysis of the immunostaining, an average value was calculated per animal for each protein and treatment; these average values were used to determine the mean and standard deviation for each treatment group (5–6 animals/group). Analysis of variance (ANOVA) was used to test for statistical significance, which was accepted when P<0.05 (two-tailed). Tukey’s post test was performed to compare individual pairs of groups if P<0.05. Linear regression analysis was used to examine the correlation between Runx2 mRNA expression and culture time.
Results
Immunohistochemistry demonstrated different localization patterns for Runx2, OPN and BSP proteins. Cells expressing these proteins were detected throughout the distraction gap and in the periosteum, the periosteal callus and the old (predistraction) bone (Fig. 1a). Runx2 staining was located in nuclei and cytoplasm, whereas strong staining for OPN and BSP was localized in the cytoplasm (Figs. 1b,c,2i,3c); weak staining for BSP and OPN was also seen in the extracellular bone matrix. Figures 1, 2, 3 present the different staining patterns and Table 1 summarizes the average intensity of immunostaining in the ROI.
Fig. 2Runx2 (a, d, g), OPN (b, e, h) and BSP (c, f, i) expression in the distraction gap (B bone, FT fibrous tissue, FC fibrous cartilage-like tissue, asterisks central area of distraction gap, hatch layers of cells adjacent to the newly formed bone, arrows osteoblasts, arrowheads osteocytes) of the various groups (a-c 0 μg TP508 group, d-f 30 μg TP508 group, g-i 300 μg TP508 group). Inset (i): Newly formed bone in the gap. Original magnifications: a 50×; d, g 100×; b, e, h 100×; c, f, i, inset 200×. Bars 20 μm (c, f, i), 50 μm (b, d, e, g, h), 100 μm (a)Fig. 3Immunolocalization of Runx2, OPN and BSP in the 300 μg TP508 group. a Runx2 staining of a vessel wall (EC endothelial cells, SMC smooth muscle cells). Note that some cells near the blood vessel weakly express Runx2 (white arrows). ×400. b Osteopontin staining of a vessel wall. Note that the cells surrounding the blood vessel are immunonegative and only stain weakly for methyl green (white arrows). ×400. c BSP staining is restricted to the layer of osteoblasts (Ob) and hardly occurs in fibrous tissue × 400. d OPN staining is expressed in osteoblasts (Ob) and osteocytes of new bone (NB) and some fibrous tissue cells close to osteoblasts, presumably pre-osteoblasts (Pre-Ob). ×200. Bars 20 μm (b), 50 μm (c, d), 100 μm (a)Table 1Intensity scores of immunostaining in region of interest (± weak, + moderate, ++ strong, +++ very strong). The intensity of immunostaining was scored in the central area of the gap (region of interest) at 40× final magnification. Variation exists between sections in each animal and between animals within groupsMarkerGroup with 0 μg TP508Group with 30 μg TP508Group with 300 μg TP508RUNX2+ to +++ to ++++ to +++OPN± to ++± to ++++BSP± to +++±+ to +++
Expression of Runx2, OPN and BSP in the group injected with saline (control group) was similar to that in the group treated with 30 μg TP508 (Figs. 1d, 2a–f). The intensity of the staining in both groups was however much lower than that in the 300 μg TP508 group (Figs. 1c,2g,h,3c,d). Runx2 and OPN were detected in active osteoblasts, in fibroblasts next to the osteoblastic layer (presumably pre-osteoblasts), in osteocytes in the old (predistraction) and newly formed bone, in fibroblasts of the periosteum, in the central area of the gap, in endothelial and smooth muscle cells of the vessels and in chondrocyte-like cells next to new bone. We found two types of cartilage-like tissue in the group treated with 30 μg TP508 and in control group, i.e. fibrocartilage-like (collagen fibres between lacunae) and hypertrophic-like (evenly dispersed chondrocytes, without clear fibres; Fig. 1d). Higher numbers of cells associated with these two tissues were found in the group treated with 30 μg TP508 and in the control group than in the group treated with 300 μg TP508 (Fig. 2a,d,g). More fibrocartilage-like cells than hypertrophic cartilage-like cells were positive for Runx2 (Fig. 1d). Sections in which primary antibodies had been replaced with non-immune antibodies were negative (Fig. 1e).
The Runx2- and OPN-positive blood vessel cells (both the endothelial and smooth muscle cells) were more evident in the group with 300 μg TP508 (Fig. 3a,b). They appeared to occur at locations with high bone formation activity. Connective tissue cells in close vicinity to Runx2-positive blood vessels also stained faintly for Runx2 but not for OPN. Immunostaining for BSP was more restricted than that for Runx2 and OPN and was seen in osteoblasts and young osteocytes (Fig. 3c,d). Weak expression was found in some of the cells considered to be pre-osteoblasts. Fibroblast-like cells in the central fibrous area of the distraction gap expressed Runx2 (Fig. 2a,d,g) and OPN (Fig. 2b,e,h) but were negative for BSP. The number and the intensity of Runx2 and OPN in the gap increased from fibrous tissue towards the newly formed bone (Fig. 1c). Weak BSP staining was seen in the area within the gap in which bone trabecules started to be formed (Fig. 2c,f,i).
In the group injected with 300 μg TP508, immunostaining for Runx2, OPN and BSP was distributed throughout the regenerating areas. The immunostaining for Runx2, OPN and BSP in the connective tissue in the gap (endosteal area) was relatively high and as intense as in the periosteal callus tissue. In the 30 μg TP508 group and in the control group, staining was less intense in the central gap area than in the periosteal callus area.
Quantitative immunohistochemistry was performed to investigate the effect of TP508 injection on the total number of cells and on the number of differentiated bone cells in the central area of the gap. The total number of cells (immunostained and unstained) in the ROI was not significantly different between the three groups. Mean values and the standard deviation of the total number of cells in the tissues treated with 0, 30 μg TP508 and 300 μg TP508 were 958±217, 1140±137 and 1161±282, respectively (ANOVA, P=0.5). This indicated that TP508 injection had no overall effect on total number of cells in the distraction gap. Next, we counted the number of cells, in the ROI, expressing each of the three antibodies tested. Absolute values of immunopositive cells in the 0, 30 μg TP508 and 300 μg TP508 groups were as follows: for Runx2, 263±61, 288±171, 560±115, respectively; for OPN, 299±201, 244±40, 677±216, respectively; for BSP, 255±111, 252±73, 480±31, respectively. We also expressed the number of immunopositive cells as a percentage of the total number of cells (Fig. 4). The number of Runx2- and OPN-expressing cells was significantly higher in the group treated with 300 μg TP508 than in the control group that received only saline (P<0.05; Fig. 4). The number of BSP-expressing cells was also higher in the 300 μg TP508 group than in the 30 μg TP508 group (P<0.05). No significant differences were found between the 30 μg TP508 group and the control group receiving saline (P>0.05).
Fig. 4Effect of TP508 injection on the number of immunopositive cells in the central portion of the distraction gap (black bars Runx2-positive cells, grey bars OPN-positive cells, white bars BSP-positive cells). Data are presented as percentage cells immunopositive for each of the three proteins examined (means±SD). *The percentages of Runx2-, OPN- and BSP-positive cells in the 300 μg TP508 group were significantly higher than for cells with the corresponding proteins in the saline-treated control group; the percentage of BSP-positive cells in the 300 μg group was also significantly higher than that in the 30 μg group (ANOVA, P<0.05)
The previous data suggested that TP508 increased the differentiation of osteogenic cells into osteoblasts in situ. To determine whether TP508 had any direct effect on osteogenic cells that were in the process of turning into osteoblasts, we performed in vitro experiments with the (mouse) osteoblast-cell line MC3T3-E1 after pre-culture in the presence of dexamethasone. After a 24-h stimulation with TP508, we found a significant (but slight) downregulation of Runx2 mRNA at 10 μg/ml TP508 (P=0.008) but no change at 100 μg/ml TP508 (Fig. 5).
Fig. 5Quantitative reverse transcription/polymerase chain reaction analysis of Runx2 mRNA expression in mouse osteoblast-like (MC3T3-E1) cells cultured for 24, 48 and 72 h in the presence of TP508 (black bars control group with 0 μg/ml TP508, hatched bars 10 μg/ml TP508, white bars 100 μg/ml TP508). Data are presented as means±SD (n=8) and are representative of two experiments. *The 10 μg/ml TP508 group at 24 h of incubation was significantly lower than the control group (one-way ANOVA; Tukey’s multiple comparison test, P<0.01). Correlation analysis between mRNA and culture time: r2=0.94, P=0.06 for 10 μg/ml TP508; r2=0.99, P=0.06 for 100 μg/ml TP508
Discussion
This study demonstrates a positive effect of TP508 on the synthetic activity of bone-forming cells in the distraction gap in rabbit long bones. After injection of a high dose of TP508, significantly more cells expressing Runx2, OPN and BSP are located in the central part of the distraction gap, predominantly consisting of soft connective tissue. We have also found that these cells stain more intensely for Runx2, OPN and BSP suggesting that TP508 not only proportionally increases the number of osteogenic cells in the distraction gap but also their expression level. The finding that TP508 enhances bone-forming activity in the soft tissue gap area is in agreement with previous morphometric data indicating that TP508 stimulates bone regeneration in the distraction gap (Li et al. 2005a,b).
The underlying molecular mechanism of the stimulation of Runx2 expression in the distraction gap after TP508 treatment is not clear. Our in vitro experiment with osteoblast-like MC3T3-cells suggests that TP508 does not have a significant positive effect on Runx2 mRNA. However, we cannot rule out that the MCT3C3 cells are too far advanced along the osteoblast differentiation pathway to respond to TP508 for they had been pre-cultured in dexamethasone-containing medium before they were exposed to TP508. No definitive conclusions can therefore be drawn from these in vitro data. In vivo, TP508 may have stimulated the differentiation of osteogenic cells though the activity of other non-osteogenic cell types. Injection of TP508 into a bone fracture model upregulates FGF-2 mRNA in (not further specified) tissues at the fracture site (Wang et al. 2005). Since FGF-2 is known to be an upstream regulator of Runx2 (Franceschi and Xiao 2003), one explanation for the increased Runx2 expression in the gap is via an enhanced cytokine release by inflammatory cells, which in turn stimulates osteoblast differentiation (Okazaki et al. 1999; Stiernberg et al. 2000; Franceschi and Xiao 2003; Naldini et al. 2004). Alternatively, TP508 stimulates blood vessel growth in vivo (Norfleet et al. 2000; Li et al. 2005a,b; Vartanian et al. 2005; Wang et al. 2005) and this subsequently enhances bone formation. TP508 increases recruitment of human aortic and microvascular endothelial cells through chemotaxis (Norfleet et al. 2000; Li et al. 2002), upregulates angiogenesis-related genes (Wang et al. 2005) and acts directly on microvascular cells to accelerate sprouting (Vartanian et al. 2005). Vascularization provides the oxygen that is required for the metabolic activity of the osteoblasts (Tuncay et al. 1994; Salim et al. 2004); it provides homes for circulating stem cells that, under proper stimulation, differentiate into osteoblastic cells (Kuznetsov et al. 2001). In addition, vessel cells secrete paracrine factors that can regulate bone cell metabolism (Villars et al. 2000; Street et al. 2002). Thus, the angiogenic properties of TP508 might help to enhance bone growth.
In the present study, we have also found Runx2 protein expression in endothelial cells, vascular smooth muscle cells and loose connective cells surrounding developing blood vessels at sites of active bone and tissue formation. Recent studies have presented evidence for transient Runx2 mRNA and protein expression during angiogenesis in embryonic human orofacial tissues (Bronckers et al. 2005), in human primary bone marrow endothelial cells and in endothelial cell lines (Namba et al. 2000; Sun et al. 2001). Thus, our data suggest that, with respect to Runx2 expression, the formation of blood vessels in regenerating adult tissues during DO resembles vessel formation in embryonic tissues. Of note, under certain conditions such as inflammation, cells associated with vessels (e.g. pericytes, endothelial cells and smooth muscle cells) or their precursors are capable of differentiating into osteoblast-like cells (Schor et al. 1995; Reilly et al. 1998; Kinner and Spector 2002) suggesting that the forming microvasculature is a potential source for osteogenic cells.
The distribution and staining intensity of Runx2, OPN and BSP varies between the different groups. In the control group and in the group treated with a low dose of TP508, staining is more pronounced in the periosteal callus than in the tissue in the distraction gap. However, in the group treated with a high dose of TP508, we have found the same high staining in both periosteal callus and gap tissue. Periosteal callus formation is a common finding during fracture healing (Gerstenfeld et al. 2003). New woven bone is formed rapidly around the fractured segments, because of stimulation of the periosteum that contains many osteoprogenitor cells. In the same section, a high staining intensity in the periosteal callus can therefore be considered as an internal reference. Hence, as the staining for Runx2, OPN and BSP in the distraction gap is as intense as that in the periosteal callus at the high dose of TP508, this is a clear indication that the high dose of TP508 stimulates bone formation in the distraction gap. This observation is in accordance to a previous histological study that has demonstrated advanced bone consolidation in a group treated with a high dose of TP508 (Li et al. 2005b). TP508 might stimulate the migration of osteoblast precursors to the distraction gap (Li et al. 2005a). Li and colleagues have shown that human periosteal-derived osteoblasts and human microvascular endothelial cells are chemotactic to TP508 (Li et al. 2005a). Therefore, the presence of TP508 in the gap might attract osteoprogenitor cells in the periosteum to migrate into the distraction gap.
Runx2 is a critical gene for osteoblast differentiation and for the function of the fully differentiated osteoblast (Komori et al. 1997; Karsenty et al. 1999). The Runx2 protein binds to the osteoblast-specific element 2 that is present in the promoter regions of all major extracellular matrix genes expressed in osteoblasts, including the genes for osteocalcin (OC), OPN and BSP and it regulates the expression of these proteins (Karsenty et al. 1999). OPN and BSP belong to the Small Integrin-Binding LIgand, N-linked Glycoprotein (or SIBLING) family (Fisher et al. 2001). Among other functions, members of this family are known to have a role in mineralization, as they bind strongly to hydroxyapatite (Fisher et al. 2001).
In conclusion, we have demonstrated that the increase of bone regeneration by thrombin-related peptide TP508 is associated with an increase in the immunostaining for Runx2, an essential transcription factor of the osteoblastic lineage, and for the bone matrix proteins BSP and OPN. TP508 may thus be a candidate for enhancing bone regeneration when bone regeneration is slow, as occurs in elderly patients.

thrombin peptide
distraction osteogenesis
bone regeneration
runx2
osteopontin
bone sialoprotein
immunohistochemistry
rabbit (new zealand white, adult, male)


A Case Study of a Retracted Systematic Review on Interactive Health Communication Applications: Impact on Media, Scientists, and Patients

Background In October 2004, a flawed systematic review entitled “Interactive Health Communication Applications for People with Chronic Disease” was published in the Cochrane Library, accompanied by several press releases in which authors warned the public of the negative health consequences of interactive health communication applications, including the Internet. Within days of the review's publication, scientists identified major coding errors and other methodological problems that invalidated the principal conclusions of the study and led to a retraction. While the original study results and their negative conclusions were widely publicized in the media, the retraction seemed to go unnoticed.
Introduction
Publication of the Review
On October 18, 2004, the Cochrane Collaboration, a organization which produces and disseminates systematic reviews of health care interventions [1], published a review entitled “Interactive Health Communication Applications for People with Chronic Disease” [2], which from this point on will be referred to as the “IHCA review.” The IHCA review was edited by the Cochrane Consumers and Communication Review Group [3]. Those who prepare reviews volunteer to work in one of many Collaborative Review Groups, with editorial teams overseeing the preparation and maintenance of the reviews.
Interactive health communication applications (IHCAs) were defined in the IHCA review as “computer-based, usually Web-based, health information packages for patients that combine information with social-, decision-, or ‘behavior change'-support” [2]. The results of the IHCA review showed that IHCAs had a positive effect on knowledge and on social support, no effect on behavioral outcomes, and a negative effect on clinical outcomes.
The principal conclusion of the review was “consumers whose primary aim is to achieve optimal clinical outcomes should not use an IHCA” [2]. This conclusion was the focus of a press release which the mass media widely circulated (as will be documented later). However, only days later, the IHCA review was found to be flawed and was retracted.
Retractions
The National Library of Medicine (NLM) is a leader in the bibliographic handling of retractions. The Medical Subject Headings (MeSH) contain the concept “retracted publication,” which identifies a citation previously published and now retracted through a formal issuance from the author, publisher, or other authorized agent. In January 2005, the PubMed query “Retracted Publication[Publication Type] AND 1971:2004[edat]” retrieved 619 retracted citations that entered PubMed between 1971 and 2004. Since the query “1971:2004[edat]” retrieves approximately 12.5 million citations, less than 1 in 10000 publications have been retracted.
Friedman [4] studied 60 fraudulent articles by one scientist. Journals in which the scientist had published were notified of the fraud. Only 7 articles were subsequently tagged in PubMed with “Retracted Publication.” The delay between publication of a paper and its retraction often has deleterious effects [5]. Furthermore, journals and institutions are hesitant to issue a statement of errors in published work unless the author of the work confesses to the error, which authors may resist doing because such an admission can be career-damaging.
While very few publications are officially retracted, the concern about factors related to retractions is substantial. The study of retractions itself might be indexed with MeSH concepts such as “scientific misconduct,” although the fraction of retractions that stem from error as opposed to scientific misconduct is not known. The query “Scientific Misconduct[majr] AND 1971:2004[edat]” in PubMed retrieved 1840 citations. This body of literature recommends that medical researchers constructively criticize the research practices of others in their institution to reduce the likelihood of misconduct [6].
The objective of this paper is to document the IHCA review as an event in the history of medical publishing, to identify the factors leading to the publicizing of a retracted publication, and to assess the implications.
Methods
The objectives of this research called for various study methods. The author employed the following three methods: (1) historical processes of collecting documents about a contemporary event and organizing them thematically; (2) ethnographic processes of author participation in the event, personal communication with other participants in the event, interpretation of communications, and construction of models; (3) content analyses based on bibliographic database and Internet searches, coding of the retrieved documents, and tallying of the code frequencies.
The ethnographic method employs the extended case method, and the extended case method applies reflexive science to ethnography. Buroway describes reflexive science as follows: “Reflexive science starts out from dialogue, virtual or real, between observer and participants, embeds such dialogue within a second dialogue between local processes and extralocal forces that in turn can only be comprehended through a third, expanding dialogue of theory with itself” [7].
Various database and Internet searches were employed to study the impact of the review and to quantify the difference between mass media coverage of the original publication and its retraction. LexisNexis Academic databases of health news and general news were searched, as was Google. The queries were designed in an iterative process that began with keywords from the question to be addressed but refined the query based on study of the query retrieval results. The retrieved results were coded, and the coding language was also developed in an iterative process. First, the obvious codes “about the review” and “about the retraction” were introduced. Each retrieved document was classified into a single code by the author. If the retrieved document was not appropriately described by an existing code, then the coding language was augmented. The Web of Science was also queried to identify academic citations, but no citations were identified (data not shown). Most database and Internet searches were conducted in May 2005.
To better understand how special the publicity accorded the IHCA review was, this study was extended to three other publications: 2 of these were retracted publications tagged as “Retracted Publication” (1 Cochrane review, but not eHealth related, and 1 non-Cochrane review, but eHealth related), and 1 was a meta-analysis with a scope similar to that of the IHCA review. These 3 reports were identified through PubMed searches.
Results
The following qualitative results on the impact of the IHCA review are organized into three main sections: scientist reaction, mass media reaction, and patient reaction.
The section on scientist reaction considers Cochrane reviewers' reactions and how eHealth scientists responded to the IHCA review in the comment section of the Cochrane database. The mass media section provides the Cochrane retraction and then explores, via LexisNexis and Google results, the reaction of the mass media to the IHCA review. The patient reaction section shares dialogue from patient-patient online discussions that reveals the reactions of patients to the IHCA review.
Scientist Reaction
The Cochrane Collaboration allows anyone to submit comments to the published reviews. Two scientists' comments on the IHCA review appeared independently on October 28, 2004. Kummervold and Eysenbach criticized the IHCA review for both its protocol and its coding.
Kummervold explained in detail how the coding of the meta-analysis was incorrect: “We can't get the numbers to add up, it looks like they are reversed in 8 of the 11 studies...” [8]. He delineated the facts and the interpretation for each of the 8 studies at issue; for example, regarding the HbA1c measurement in the Lehmann 2003 paper, he stated that Lehmann reported a reduction in HbA1c of 0.8 for the intervention group, and 0.1 for the control group, which should be interpreted as a positive result for the intervention group. Kummervold added: “We also find it strange that you focus so much on the overall estimates when there is so much heterogeneity in the material. The conclusion seems to be overstated” [8].
Eysenbach had similar comments, stressing that a formal meta-analysis of these heterogeneous studies was problematic, and that the three studies which contributed most to the “negative” result were in fact positive: “Apart from the fact that I do not think that it is legitimate to do a formal meta-analysis using papers measuring totally heterogeneous outcomes with different types of interventions, I also notice that the overall effect estimate is ‘negative' (eg, ‘favoring control') because of three studies…. However, when I read these three studies I cannot find that their result[s] are negative…. If my suspicion is correct, then this is quite a catastrophic error, and quite an embarrassment for Cochrane to let such an error slip through peer-review” [9].
On November 10, 2004, the Cochrane Consumers and Communication Review Group reacted to the discovered errors [10] with a notice that included the following: “The review will be withdrawn as soon as possible…. As the corrections to the review have not been completed yet, it would be premature to announce any reversal of the review's findings at this stage.… The original press releases regarding this review were made not by the Cochrane Collaboration itself but by University College London….”
John Wiley & Sons (the publisher of the Cochrane Database) released to EurekAlert a retraction on December 6, 2004: “The review originally determined that…chronically ill people using interactive programmes had worse clinical outcomes than those who did not. Regrettably, errors in data analysis meant that these outcomes were reported incorrectly.... It is expected that the revised results will be published in April 2005” [11].
The April 2005 edition of the Cochrane Systematic Reviews did not mention the IHCA review. Royle, the chief executive officer of the Cochrane Collaboration, said that further review of the revised report was ongoing and no date could be given as to when the review might be published (personal communication, April 25, 2005).
Mass Media Reaction
The Cochrane Database of Systematic Reviews is not read by the typical consumer. However, Murray's employer, the University College London (UCL), worked with Murray to widely publicize the result. UCL posted a news bulletin on its website on October 18, 2004 that remained there as of May 25, 2005. The bulletin was titled “Knowledge may be hazardous to web consumers' health” and stated the following: “People who use their computers to find information about their chronic disease often wind up in worse condition than if they had listened to their doctor, according to a UCL review of studies on internet health.… One reason…might be because knowledge-seekers become so steeped in information from the Internet they make treatment choices on their own, contradicting advice from their doctors” [12].
Most significantly, the UCL bulletin was circulated to information intermediaries that are considered the main entrance to the world's mass media, including AlphaGalileo and EurekAlert.
A search on LexisNexis Academic with the query “Elizabeth Murray AND health” for the period June 2004 to May 2005 revealed a total of 15 relevant press reports, in the following categories:
Medical and Health News: There were 9 publications with titles such as UCL's press release title of “Knowledge may be hazardous to web consumers' health.” The publications appeared in places like Life Science Weekly, Law and Health Weekly, and Health and Medicine Week.
General News–Major Papers: There were 5 relevant articles, such as one entitled “Why medical advice from the internet can be bad for your health” in the British The Daily Telegraph and another entitled “Medical Web sites may be unhealthy places to learn about ills” in the Omaha World Herald. Only 1 article was about the retraction, published in the Ottawa Citizen on October 18, 2004.
Time Incorporated Publications: There was 1 article in the November 1, 2004 issue of Time entitled “Click to Get Sick?” [13].
Among the 15 results from the LexisNexis Academic database, only 1 newspaper report, authored by Tom Spears, dealt specifically with the retraction [14]. Spears, in personal communication with this author (November 18, 2005), said, “I was fairly stunned today to learn that it [IHCA review] has been withdrawn; I found out only because I was looking up the study for my daughter, a science student. Now I'm covering the sequel for tomorrow's paper.… I scan EurekAlert faithfully, as many reporters do, and never saw a hint of anything there.”
To further test whether the media emphasized the false negative result but minimally covered the retraction, a content analysis on Google was performed on May 24, 2005. The query was “health AND Cochrane AND Murray AND (interactive OR web OR internet)” for English pages, within the past year. Of the first 200 retrieved hits, 170 pages were not related to the IHCA review. Of the remaining 30 pages, 23 (77%) were reports about the original publication that did not mention the retraction, and an additional page was a bibliography (at a UCL site) that included a citation to the IHCA review, again without mentioning the retraction. All reports (except the bibliography) used a title such as “Click to Get Sick?” and emphasized the negative impact on clinical outcomes of using the Web. The reports came from such reputable sources as the British Broadcasting Corporation and US News and World Report. In contrast, only 6 pages (20%) addressed the retraction: 2 were the original press releases now marked with “retraction” but still emphasizing in their particulars the negative health impact, 3 were Web pages at Cochrane sites, and 1 was an announcement from MedicalNews entitled “Updated press release to October 2004 Cochrane Review.” The latter was the only non-Cochrane–related page primarily addressing the retraction.
The grey literature reported on the mass media. For example, The Neuroscience for Kids Newsletter summarized [15] the “Click to Get Sick?” Time article by Sanjay Gupta, and a Web archive for patient education at the Samaritan Health Center pointed patients to Gupta's article. This author wrote to Gupta and asked him to write about the retraction, but Gupta did not reply.
NLM indexed the IHCA review and entered the citation for it (including its abstract) in PubMed on October 21, 2004. The “Retracted Publication” tag did not, however, appear in PubMed until March 24, 2005.
Patient Reaction
Some patients reported the news about the IHCA review to their patient-patient online discussion groups. In a neurology patient discussion group [16], a patient posted the entire BBC news story. Patients responded in two ways. Some rejected the IHCA review result and added strong comments, such as “I have gotten more help and answers for problems from knowledgeable people on this Internet Forum than I have from any of the multitude of doctors I have seen over the last 12 years.” Others accepted the conclusion but insisted that patients could filter bad information from good and benefit in the end from the web. These patients were not aware of the retraction of the IHCA review.
This author reported the Time “Click to Get Sick?” article to two head-and-neck cancer patient discussion groups to which he belongs. A day later he reported the retraction from the Cochrane Database. One member of the discussion group replied: “Thanks for the update–the negative findings seemed odd to me when I read it, so I'm glad it's being revised.” This author, in his role as a cancer patient, also formally commented on the IHCA review at the Cochrane Database site [17].
The typical patient with a chronic disease has no formal medical training and is ill prepared to critique a meta-analysis of clinical trials. However, the typical patient is vulnerable to cultural pressures, as they are partially shaped by and reflected in the mass media.
Comparison With Another Cochrane Retraction
For comparison, a search for further retracted Cochrane reviews using the PubMed query “Cochrane Database Syst Rev[TA] AND Retracted Publication[PT] AND 1971:2005/5/25[edat]” was conducted. One reference, in addition to the IHCA review already discussed, was identified, which was a retracted review by Brewster et al [18] about antihypertensives. The retraction for the Brewster et al review is explained on the Cochrane website as follows: “This systematic review has been withdrawn temporarily because its contents are potentially misleading.”
A search on LexisNexis with the query “Brewster AND antihypertensive” for the period November 2004 to May 2005 retrieved no articles in either the “General News–Major Papers” category or the “Medical and Health News” category.
A search on Google for “Brewster antihypertensive” followed by an examination of the first 100 retrieved pages identified 23 relevant pages, which had a very different content pattern than the hits for the IHCA review. They all contained citations of papers from Brewster et al, who have published elsewhere on the same subject as in their review. The Brewster et al publication attracting the most attention was an article [19] in the Annals of Internal Medicine that was not retracted but has the same title as the Cochrane review. Thus, the only other retracted Cochrane review had a very different mass media, scientific, and web impact than the IHCA review.
Comparison With Other Retracted Articles Related to eHealth
To determine whether other articles on a similar topic to the IHCA review have been retracted, a search was first made for articles on a similar subject that had been MeSH indexed in PubMed. The article by Demiris [20] seemed relevant, and its two MeSH index terms were “Disease Management” and “Internet.” A search on PubMed for “Retracted Publication[PT] AND Disease Management[majr] AND Internet[majr] AND 1995:2005/5/25[edat]” returned no citations. When the search was broadened by removing the term “Disease Management,” 1 retracted reference was retrieved, entitled “The quality of surgical information on the Internet” [21]. As previously described in the Journal of Medical Internet Research, this article was retracted due to a case of cyberplagiarism, with large sections of the paper having been lifted from different websites [22].
A search on LexisNexis Academic with the query “McKinley and surgical and Internet” for the period 1995 to May 2005 revealed no relevant press reports, neither in the “General News–Major Papers” category (three hits were all not relevant to the McKinley article) or in the “Medical and Health News” category.
A search on Google for English pages with the query “McKinley surgical Internet” revealed 96 irrelevant pointers in the first 100 results. Of the remaining 4 relevant hits, 1 was the article about the plagiarism [22], which precipitated the retraction of the McKinley et al manuscript, and 3 were academic references to the McKinley et al article, which did not note it being retracted.
Thus, the only other retraction of a published article appearing in PubMed similar in topic (the Internet) to the IHCA review had a very different pattern of reactions than the IHCA review.
A Similar Meta-Analysis on eHealth
The IHCA review addressed a topic that the mass media found interesting. Have any other recent publications also been a meta-analysis on the impact of interactive applications on health, and, if yes, what was the mass media reaction? Using the query “Meta-analysis AND Web AND Chronic Illness” in PubMed, we found only 1 citation: Wantland et al [23] did a meta-analysis on Web-based health interventions that was published (in the Journal of Medical Internet Research) about the same time as the IHCA review. The paper concluded that “the effect size comparisons in the use of Web-based interventions compared to non-Web-based interventions showed an improvement in outcomes for individuals using Web-based interventions to achieve the specified knowledge and/or behavior change for the studied outcome variables.”
What has been the impact of the Wantland et al paper and how does that compare to the impact of the IHCA review? The Wantland et al paper was not announced with a press release in EurekAlert. A search on LexisNexis Academic for newspaper articles about the Wantland et al paper retrieves no articles. The queries performed were similar to those performed for the IHCA review and included “Wantland AND health” for 2004 through 2005 in General News/Major Papers.
A search was done on Google for “Wantland health Web” on May 24, 2005. Of the first 200 returns, 182 were not relevant. Of the remaining 18 hits, 15 pages contained academic citations to Wantland et al, 2 announced the appearance of the article, and 1 was a personal blog that commented on the article.
Thus, most of the Google returns that gave Wantland et al citations are academic in character and very different from the mass media coverage afforded the IHCA review.
Discussion
As shown, the IHCA review provides a perhaps unprecedented case from which lessons should be drawn. Only one other Cochrane review (about antihypertensives) has been retracted, and that one received negligible mass media attention. The only retracted publication in PubMed that is indexed under the MeSH concept of “Internet” (the IHCA review did not have time to get indexed before it was withdrawn) received no newspaper coverage. The paper most similar to the IHCA review in topic and method (the Wantland et al report [23]) received considerable academic attention but no newspaper coverage. In other words, special circumstances must have come together for the IHCA review situation.
This section next presents a framework based on tiers of response. The first tier is medical scientists. The second tier is the mass media spreading medical press releases. The third tier is the patient community reacting to the mass media and the scientists.
First-Order Problem
In an effort to critique the problem that occurred, one might build on the analysis of misconduct in toxicology by Purchase. Purchase [24] identified four roots of misconduct:
Intention of the work
Conduct of the studies
Design and interpretation of studies
Bias from conflict of interest
In the case of the IHCA review, the intention was scientifically appropriate, namely to gain further insight about IHCAs through a systematic review. In the other three categories, fault can be found:
The errors in the coding of data should not have been made. The coauthors Nazareth and Tai, who are credited with doing the coding, have good enough credentials to not lay the blame on lack of experience: Nazareth is a Professor at UCL and is Scientific Director of the British Medical Research Council's General Practice and Research Framework, and Tai has coauthored several articles over the past two decades that appeared in refereed medical journals. An explanation for the miscoding in terms of experience of the coders is not apparent.
The design of the study has been criticized as lumping together studies which are too heterogeneous in their design, interventions, and outcomes [8,9]. The protocol might have been more rigorously vetted by the Cochrane Review Group, and the authors should have been more cautious in their interpretation of results and emphasized the weakness of the design in their publicity.
The reporting of the work suggests possible bias. The authors and their employers have sensationalized a result that catches the media's attention. For some observers, the review appeared biased in that the authors, who are affiliated with medical institutions, concluded that patients should listen to their doctor, instead of seeking help on the Internet.
Purchase [24] claims that a partial solution to this first-order problem is the institutionalization of quality controls. In the 1970s, good laboratory practice regulations were introduced, but comparable regulations do not exist for meta-analyses. For quality control of a meta-analysis the scientific community relies on the research team, the researchers' institution, and the referees. A medical research institution, such as the UCL Medical School, presumably embraces results from its researchers that can earn mass media coverage and is not the appropriate institution to prevent sensationalizing. Referees can not be expected to detect when laboratory data are intentionally modified [25]; however, in this case they could have been expected to detect when data available to them are miscoded. Problems with refereeing have been frequently noted and in particular for the Cochrane Database [26].
Open commentary, as exists for the Cochrane Database after a publication, is one way to identify flaws. Extending the open commentary to the refereeing phase might reduce the likelihood of something going to press with errors. A submitted article might be available to the public and a community of hundreds of registered scientists could be invited to make anonymous comment. Submissions online would require extensive online commenting that reached a consensus before a submission could be considered “published.” Other approaches to increase the commentary on the research process include refereeing the protocol phase [27], which is done by the Cochrane Collaboration but apparently not with the necessary rigor or topic expertise.
Second-Order Problem
The second-order problem is a press release and subsequent mass media coverage of the release. Winsten's classic study of science and the media shows how the truth is repeatedly misrepresented by journalists and researchers: “The most striking finding which emerged from the interviews [of medical journalists] is the dominant distorting influence of the competitive force in journalism.… As economic competition among hospitals has intensified, they have begun to compete aggressively for publicity.… With increasing frequency…scientists…are using the media to attach their names to important findings before their competitors do.… The result has been a spiraling competition, sometimes characterized by exaggerated claims” [28].
Online media have stimulated further competition [29]. The case of the IHCA review reflects these pressures. The UCL press release contained inaccuracies, even if the review would have been scientifically sound, in order to gain mass media attention. For instance, the subtitle of the press release was “Knowledge may be hazardous to web consumers' health.” In truth, the IHCA review was not about Web applications, per se, but about IHCAs, which are defined more broadly than “Web applications.” However, writing a news article about IHCAs is less likely to catch attention than an article about the Web. The UCL press release did not introduce and define the term IHCA, and Murray issued statements that implied the Web was the issue. By the time the information from the press release made it into the mass media, the material had been modified enough to lose any mention of IHCAs. For instance, the Time article said, “People who use the Web to get information about their chronic diseases often wind up in worse shape than before they logged on.”
One way for researchers to prevent the mass media from misrepresenting the truth is for researchers to understand how the media work and to interact with the media accordingly [28]. Murray should have known that her words might be twisted to emphasize what would sell newspaper space and should not have wildly speculated. The reputations of the Cochrane Collaboration and UCL partially account for the wide dissemination of the original press release. Yet, neither organization has taken adequate steps to undo the impact of the media reporting on the IHCA review.
The honesty of the press could be improved with the Internet [30]. Online health care mass media publications could allow the public to make comments on news articles. Rating techniques, such as employed at eBay and Slashdot, might be used to give prominence to quality feedback [31].
Third-Order Problem
The third-order problem concerns the long-term impact of the mass media. While electronic publications might be erased from a computer or marked as retracted, this does not consistently happen. Furthermore, some of the mass media coverage of the IHCA review is on paper and sits on people's bedside tables with no practical way to be retracted [32].
Although this author did not (yet) find any citations to the IHCA review in Web of Science, previous studies have confirmed that a retracted scientific publication may continue to have impact without readers recognizing its retracted status. For instance, one study [33] tracking the citation pattern of 82 retracted articles revealed that, together, they were cited 733 times after their retraction, but only a small fraction of the citations referred to the retraction. In the case of the mass media, retracted publications might be read by people without them seeing the separate retraction notice.
If and when the revised IHCA review is published, what could it say that would undo the effect of the original publication? If the conclusion is that IHCAs result in improved clinical outcomes, then the medical profession will want to closely study the protocol and might have grounds to discredit the conclusion. The media trumpeted the IHCA review conclusion partly because it was counterintuitive but was backed by top-notch institutions. If the conclusion becomes intuitive, then the media are unlikely to be interested in it.
The reactions to the IHCA review in patient online discussions highlight the importance of virtual communities in helping patients deal with published information. Simple extensions to Web-based, patient, discussion systems could help patients connect to Web-based publications. For instance, when a patient posts a message to a Web-based discussion board, the Web system could parse the message and provide links from the message to relevant articles on the Web. Patients might follow the links and engage in discourse about the validity and implications of the literature. This might lessen the potential ill effects of publications that are wrong or misleading.
Conclusions
This special medical publishing event was marked by incorrect coding and a desire for maximum publicity. The IHCA review authors, their employers, and the Cochrane Collaboration were responsible for quality control, and failed. The mass media played their part by widely publicizing a sensational message but not reacting to the notice that that sensational message was false. The false result that patients are clinically harmed by interactive applications was very strongly delivered to patients worldwide. The broad lesson to be re-learned is that potentially sensational results should be carefully scrutinized before being sensationalized.

patients
mass media
retraction of publication
online systems
medical errors
editorial policies


The significance of lobular neoplasia on needle core biopsy of the breast

The management of a core biopsy diagnosis of lobular neoplasia is controversial. Detailed radiological–pathological review of 47 patients with cores showing classical lobular neoplasia was performed (patients with pleomorphic lobular carcinoma in situ (LCIS) or associated risk lesions were considered separately). Immediate surgical excision in 25 patients showed invasive carcinoma in 7, ductal carcinoma in situ (DCIS) in 1 and pleomorphic LCIS in 1; radiological–pathological review showed that the core biopsy missed a mass in 5, missed calcification in 2 and that calcification appeared adequately sampled in 2. Nineteen patients had follow-up of at least 2 years. Four patients developed malignancy at the site of the core biopsy (invasive carcinoma in three, DCIS in one); one carcinoma was mammographically occult, one patient had dense original mammograms and two had calcifications apparently adequately sampled by the core. In conclusion, most carcinomas identified at the site of core biopsy showing lobular neoplasia were the result of the core missing the radiological lesion, emphasising the importance of multidisciplinary review and investigation of any discordance. Some carcinomas were found after apparently adequate core biopsy, raising the question of whether excision biopsy should be considered after all core biopsy diagnoses of lobular neoplasia.
Introduction
Lobular carcinoma in situ (LCIS) was described by Foot and Stewart [25]. The term atypical lobular hyperplasia (ALH) was later introduced to describe a similar lesion, but with less marked changes. We prefer the term lobular neoplasia [30], which includes both LCIS and ALH, particularly for core biopsies in which there is limited tissue, making distinction between ALH and LCIS difficult.
The relationship between lobular neoplasia and invasive carcinoma of the breast is controversial [28]. Lobular neoplasia is often multifocal and bilateral [4]. There is evidence that it is a risk factor for invasive carcinoma in both breasts: after a diagnosis of lobular neoplasia, there is an increased risk of subsequent invasive carcinoma in both breasts [30], and the majority are ductal in type. Recent evidence suggests that lobular neoplasia may, like ductal carcinoma in situ (DCIS), be a non-obligate precursor for invasive carcinoma at the same site. There is a higher risk of invasive carcinoma in the ipsilateral than in the contralateral breast. The proportion of subsequent carcinomas that are of lobular type is higher than in unselected series of carcinomas [45]. The morphology of the cells in lobular neoplasia and invasive lobular carcinoma is similar. The same truncating mutation in the E-cadherin gene has been found in invasive lobular carcinoma and adjacent LCIS [57].
The significance of lobular neoplasia in breast needle core biopsies is uncertain. Invasive carcinomas are sometimes identified after a core biopsy diagnosis of lobular neoplasia. Some studies suggest this occurs because the core biopsy missed the lesion and so does not explain the clinical or radiological abnormality [41]. Other studies suggest that carcinomas may be identified after the diagnosis of lobular neoplasia on core biopsy even if the core biopsy explains the clinical or radiological abnormality [20]. An important consideration in radiological–pathological correlation is whether classical lobular neoplasia is associated with calcification. The traditional view is that lobular neoplasia does not have a radiological correlate [4], but recent reports have described an association between classical lobular neoplasia and calcification. We also wished to investigate our impression that, in core biopsies containing classical lobular neoplasia, calcification is often associated with columnar cell change.
Pleomorphic LCIS is a recently described variant of lobular neoplasia. The growth pattern and absence of E-cadherin expression resemble classical LCIS. However, the marked nuclear pleomorphism, frequent presence of central necrosis and calcification and expression of c-erbB-2 are similar to high-grade DCIS [50]. In the past, pleomorphic LCIS would usually have been diagnosed as high-grade DCIS.
The main aim of this study was to assess the risk of invasive carcinoma and DCIS at the site of classical lobular neoplasia diagnosed on breast needle core biopsies. In view of the clear morphological differences, pleomorphic LCIS was considered separately. A second aim was to investigate the localisation of calcification in core biopsies containing lobular neoplasia.
Materials and methods
This project was discussed with the chair of the Nottingham University Hospitals Research Ethics Committee who considered that it was a service evaluation and, therefore, did not require formal ethical approval. A multidisciplinary review of the pathology and radiology of patients with a core biopsy diagnosis of lobular neoplasia from July 1998 to June 2006 was performed. The biopsies from July 1998 to June 2000 were included in a previous study [35]. The diagnosis of lobular neoplasia was made on haematoxylin and eosin sections. Immunohistochemistry for E-cadherin was performed for lesions with a differential diagnosis including DCIS or pleomorphic LCIS. Lobular neoplasia was subdivided into ALH and LCIS using the criteria described by Page et al. [44]. Pleomorphic LCIS was defined as a high-grade dyscohesive intra-acinar proliferation that was E-cadherin negative.
Patients were excluded if they had synchronous or previous invasive carcinoma or DCIS in the same breast. Two patients with contralateral invasive carcinoma were included. Core biopsies containing invasive carcinoma, DCIS or an area suspicious of either diagnosis were excluded. Cores containing pleomorphic LCIS, an atypical intraductal epithelial proliferation (including atypical ductal hyperplasia), radial scar or a papillary lesion were considered separately. Thus, the main group of core biopsies studied contained classical lobular neoplasia with no other risk lesions: we termed this group “simple” classical lobular neoplasia.
The core biopsies were reviewed for the following features: location of any calcification and presence of columnar cell change. Six cores were not available, so could not be reviewed. The frequency of columnar cell change was compared with 87 consecutive core biopsies reported by one observer (AHSL) as normal or benign.
Results
Simple classical lobular neoplasia on core biopsy
Forty-nine core biopsies with lobular neoplasia from 47 women satisfied the entry criteria (0.3% of the 14,597 diagnostic core biopsies performed during this period). Two patients had two core biopsies from the same area of the breast containing lobular neoplasia: both are only counted once in the following results. The median age was 52 years (range 33 to 81).
Twenty-six patients presented with mammographic screening abnormalities: 20 with calcification, 3 with a mass, 2 with calcification and distortion and 1 with a mass and calcification. Nineteen patients presented symptomatically: ten with a mass, seven with a thickening and two with a thickening and calcification. Two women had calcification identified by mammography performed in one patient as part of the investigation of a contralateral carcinoma and, in the other, as follow-up of a contralateral carcinoma. Eight core biopsies were freehand, 14 were ultrasound guided and 25 were performed under stereotactic guidance.
There was an association between lobular neoplasia and columnar cell change (see Table 1). Calcification was seen in 25 of 41 cores with slides available for review. It was seen in lobular neoplasia in six biopsies (15%); in one the calcification was only seen in the lobular neoplasia, but in the other five, it was also present in other changes (columnar cell change in three, sclerosing adenosis in one, fibroadenoma in one). Of the other 19 biopsies with calcification, columnar cell change was the most commonly associated pathology (10 biopsies).
Table 1Extent of columnar cell change in cores with lobular neoplasia and controlsColumnar cell changeCore biopsy with lobular neoplasiaControls (normal or benign core biopsy)None12 (29%)58 (67%)One lobule or duct9 (22%)14 (16%)At least two lobules or ducts20 (49%)15 (17%)χ2 = 18, P = 0.0001
Surgical excision within 2 months of core biopsy was performed in 25 patients. Excision showed malignancy in nine patients (see Table 2). In seven patients, pathology–radiology correlation showed that the core biopsy did not explain the radiological abnormality (a mass in five and calcification in two). Five of these seven patients were seen in the first 2 years of this study. Excision showed benign changes in 16, although one woman had invasive carcinoma of mixed ductal and lobular type diagnosed 33 months after the core biopsy in a separate quadrant of the same breast.
Table 2Details of patients with invasive carcinoma, DCIS or pleomorphic LCIS at the site of the core biopsy showing simple lobular neoplasiaAgePresentationCore pathologyTime to excision/monthsFinal diagnosis (surgical procedure)Pathology–radiology correlation60Screen-detected massALH, CCCh, calcs113 mm IDC, G3, LN 0/4 (Mx)Core missed mass75Symptomatic massLCIS, UEH, CCCh26 mm IDC, G2, LN not examined (Mx)Core missed mass63Screen-detected distortion and calcification. Mass on USALH114 mm ILC, G2, LN 0/5 (WLE)Core missed mass54Screen-detected calcificationALH14 mm tubulobular carcinoma G1 + 50 mm DCIS, LN 0/6 (Mx)Core missed calcs43Symptomatic massLCIS19 mm IDLC, G2, LN 0/7 (Mx)Core missed mass68Screen-detected massALH112 mm ILC, G2, LN 0/6 (Mx)Core missed mass64Symptomatic mass, no mass on US. Mammographic calcificationALH, cyst, calcs17 mm DCIS (WLE)Calcs sampled by core54Screen-detected calcificationALH112 mm tubular carcinoma + 35 mm DCIS, LN 0/4 (WLE)Core missed calcs45Symptomatic cysts. Mammographic calcificationLCIS, calcs15 mm pleomorphic LCIS (WLE)Calcs sampled by core52Screen-detected calcificationALH, UEH, CCCh, calcs319 mm ILC, G2, LN 1/6 (Mx)Mammographically occult mass70Screen-detected calcificationALH, calcs2936 mm IDC, G3, LN 6/16 (Mx)Dense original mammogram51Screen-detected calcificationALH, CCCh, calcs2630 mm DCIS + extensive LCIS (Mx)Calcs sampled by core55Screen-detected calcificationALH, CCCh, calcs29IDC 8 mm, G3, 45 mm DCIS, LN 1/4 (Mx)Calcs sampled by coreUS ultrasound, ALH atypical lobular hyperplasia, LCIS lobular carcinoma in situ, UEH epithelial hyperplasia of usual type, CCCh columnar cell change, calcs calcification, IDC invasive ductal carcinoma, ILC invasive lobular carcinoma, IDLC invasive ductal and lobular carcinoma, DCIS ductal carcinoma in situ, G histological grade, LN axillary lymph nodes, Mx mastectomy, WLE wide local excision
Nineteen patients who had not had a diagnostic surgical excision had follow-up of at least 2 years (range 25 to 105 months). None of these patients received radiotherapy to the breast. Malignancy was identified in seven women. In four, the malignancy was at the same site as the core biopsy (see Table 2), in one DCIS was found in a different quadrant of the same breast and two developed contralateral invasive carcinoma. Three women had no surgical excision and follow-up of less than 2 years. Neither of the two women with a previous history of contralateral invasive carcinoma developed carcinoma at the site of the core biopsy showing lobular neoplasia.
No significant relationships were found between the diagnosis of malignancy (DCIS, pleomorphic LCIS or invasive carcinoma) at the site of the core biopsy and the following features: age of the woman, diagnosis of ALH or LCIS on the core biopsy, whether core biopsy or vacuum-assisted biopsy was performed and the method of guidance (freehand, ultrasound or stereotactic).
Pleomorphic LCIS on core biopsy
Both patients with pleomorphic LCIS on core biopsy presented with calcification detected by mammographic screening. In both, calcification was seen histologically in association with the pleomorphic LCIS. Both had a diagnostic surgical excision, which showed cribriform DCIS in one and further LCIS in the other.
Atypical intraductal epithelial proliferation/radial scar/papillary lesion
Eight patients had atypical intraductal epithelial proliferation, radial scar or a papillary lesion in addition to lobular neoplasia on the core biopsy. All eight presented with abnormalities detected by mammographic screening: calcification in five, calcification and distortion in one, mass in one and distortion in one. Seven patients had a diagnostic surgical excision, and one patient had her lesion removed with vacuum-assisted mammotomy. DCIS was found in one of three women with core diagnosis of atypical intraductal epithelial atypia and in one of two with core diagnosis of radial scar. Papillary DCIS with a 3-mm focus of invasion was found after a core diagnosis of papillary lesion with atypical intraductal epithelial proliferation. One patient with a core diagnosis of radial scar with atypical intraductal epithelial proliferation and one with a papillary lesion had benign findings at excision.
Discussion
Relation between lobular neoplasia and calcification
An important question in the management of lobular neoplasia on core biopsy is the relationship between lobular neoplasia and calcification. Lobular neoplasia is not typically associated with any specific clinical abnormality, and there are no characteristic macroscopic features. Early papers suggested that classical lobular neoplasia does not have any diagnostic mammographic features [4]. In the past, surgical biopsies containing lobular neoplasia were frequently performed for mammographic calcification, but pathologically, the calcification was mostly associated with fibrocystic change and identified within the lobular neoplasia in about 20% [4, 11, 24, 55]. Calcification is associated with the lobular neoplasia in between 8% and 53% of core biopsies containing classical lobular neoplasia in different studies [1, 3, 14, 20, 41]. Benign calcification is frequently seen close to invasive carcinomas in surgical specimens [52]. It is well recognised that malignancy may be an incidental discovery on a biopsy for calcification that is associated only with benign disease [49].
The present study confirms the association between lobular neoplasia and columnar cell change [8, 41]. In older studies, the majority of surgical biopsies containing lobular neoplasia were performed for fibrocystic change [30]. In the present study, calcification was associated with lobular neoplasia in 15% of core biopsies, but commonly was also seen in fibrocystic change as well. Calcification was associated with columnar cell change in 32% of cores with lobular neoplasia. The association of calcification with columnar cell change is well recognised [27]. Our results support the view that, in biopsies containing lobular neoplasia, most of the calcification is associated with fibrocystic change, particularly columnar cell change. Importantly, the lobular neoplasia is incidental to the calcification in the majority of biopsies.
A recent study of collagenous spherulosis found that 15 of 59 patients had associated LCIS [48]. In contrast, only one of the patients in the present study with lobular neoplasia had associated collagenous spherulosis.
Relationship between simple classical lobular neoplasia and carcinoma
In this study, 44 women with simple classical lobular neoplasia on core biopsy had surgical excision or follow-up of at least 2 years. Twenty-five women had immediate surgical excision, which showed invasive carcinoma, DCIS or pleomorphic LCIS in nine (36%). In the majority, the core biopsy was judged to have missed the clinical/radiological lesion. This emphasises the importance of clinico-pathological review and further investigation of any discordance. Most of the missed lesions occurred in the early part of the study, suggesting that the accuracy of radiological localisation of core biopsies may have improved later in the series. A smaller number of carcinomas occurred after lobular neoplasia on core biopsy apparently incidental to the clinical or radiological lesion. This raises the question of whether excision biopsy should be considered after all core biopsy diagnoses of lobular neoplasia. The frequency of malignancy if the patients with radiological–pathological discordance are excluded is 11% (2/18).
Four of 19 patients with at least 2 years follow-up developed carcinoma at the site of the core biopsy. In one, the carcinoma was mammographically occult, although it was clinically palpable, in one the original mammogram was dense and two women had calcification apparently adequately sampled by the core biopsy. Definite comment cannot be made, but the clinical histories suggest that it is possible that the carcinoma may have been identified earlier in some of these patients if they had had an immediate surgical biopsy. If these women are added to those without radiological–pathological discordance in the above paragraph, the frequency of malignancy is 16% (6/37).
Previous published studies of simple lobular neoplasia on core biopsy were reviewed [1–3, 6, 9, 10, 12, 14, 16–18, 20, 21, 23, 25, 26, 31, 33, 36, 37, 39, 41–43, 46, 47, 51, 54, 58, 59]. The data in most studies are not representative. Probably the major bias is that only about half of the lesions had a surgical biopsy and it is not clear how patients were selected for excision biopsy. Furthermore, most studies only report image-guided or stereotactic biopsies. A surgical biopsy was performed in 561 patients with a core biopsy diagnosis of lobular neoplasia with invasive carcinoma found in 50 or 49 and DCIS in 32 or 33 (82 [15%] in total). It is ‘impossible to know if some of the reported cases are those in which the targeted lesion has been missed and re-excision was done for failure to successfully sample the target’ [15]. In reviewing the previous studies, we have assumed that sampling was adequate for core biopsy performed for calcification if calcification was present in the core biopsy. Of those 52 malignancies with details available, 25 (48%) had a radiological–pathological discordance (missed mass in 22 and missed calcification in 3), and 27 had apparently adequately sampled calcification.
One hundred four patients who did not have immediate surgical excision had reported follow-up. The only four patients (4%) with invasive carcinoma or DCIS on follow-up at the site of core biopsy are the four patients described in the present study. The absence of subsequent malignancy in other studies is very surprising given that lobular neoplasia is a well-established risk factor for later carcinoma. Nevertheless, the low rate suggests that the chance of invasive carcinoma or DCIS is lower in those not having immediate surgical excision.
It is difficult to estimate the chance of finding invasive carcinoma or DCIS if all patients had an excision biopsy. The 15% rate found in the subset of patients who had immediate excision biopsy is probably an overestimate. If the cases with radiological–pathological discordance are excluded, the risk is probably about half of this, approximately 8%. If one assumes that half of the patients who did not have an immediate excision did not have invasive carcinoma or DCIS at the core site, this gives an estimate of about 4%. This represents an estimate of the lower limit of risk.
Some studies suggest that the risk of finding invasive carcinoma or DCIS is a little higher if the core biopsy shows LCIS rather than ALH [13]. In view of the subjectivity of this distinction on core biopsy, it is probably of limited use in routine practice.
It is clearly appropriate to investigate patients with simple classical lobular neoplasia on core biopsy that does not explain the clinical or radiological abnormality. If a definitive diagnosis cannot be made with further core biopsy, then an excision biopsy is indicated.
The management of simple classical lobular neoplasia on core biopsy with no radiological–pathological discordance is less straightforward. As discussed above, the literature suggests that the risk of malignancy is in the range of 4% to 8%. This risk is comparable to that associated with radial scar or papillary lesion with no epithelial atypia [7, 34, 35, 40, 53]. Both radial scars and papillary lesions are excised in many centres according to current guidance [19]. The major difference with lobular neoplasia, however, is that both are usually clearly defined lesions on radiological examination and therefore easy to excise.
There is a need for prospective studies with surgical excision of all lesions so that an unbiased assessment of the risk of simple classical lobular neoplasia on core biopsy can be made. Careful radiology–pathology correlation is essential. It may be possible to stratify the risk within this group using clinical, radiological or pathological features.
Lobular neoplasia associated with radial scars, papillary lesions and atypical intraductal epithelial proliferations
Most centres excise radial scars, papillary lesions and atypical intraductal epithelial proliferations diagnosed on core biopsy. In the present study, carcinoma was found on excision of three of the eight lesions with these lesions in combination with lobular neoplasia. This supports the practice of excising such lesions.
Pleomorphic LCIS
In the present study, both cores with pleomorphic LCIS had associated calcification. In contrast to the controversial nature of the association between calcification and classical LCIS, the literature suggests that there is a clear relationship between calcification and pleomorphic LCIS [29, 50]. There are limited data on the significance of pleomorphic LCIS on core biopsy. One of the two patients in the present study had DCIS in the excision specimen. Three of the six patients described in the literature [5, 20, 37] had invasive carcinoma on excision, but radiology showed a density in one and an architectural distortion in another. Although the follow-up data are limited, we believe that, until there is further evidence, it is prudent to recommend that pleomorphic LCIS is managed as DCIS because of the morphological similarity of pleomorphic LCIS to high-grade DCIS. The present study may underestimate the frequency of pleomorphic LCIS, as E-cadherin immunohistochemistry was infrequently performed early in the study period and some cases may have been diagnosed as high-grade DCIS.
Some lobular neoplasia shows features overlapping with DCIS and is difficult to classify even with E-cadherin immunohistochemistry [32, 38]. Lobular neoplasia with comedo necrosis, but without marked nuclear pleomorphism, has recently been described [22]. The clinical significance of these rare clinico-pathological scenarios is uncertain. Until there is more evidence, it would be prudent to recommend surgical biopsy of such lesions because of the overlap of features with DCIS. Mass forming lobular neoplasia can occur rarely [56], but is an example of clinico-pathological discordance, so surgical biopsy is prudent.
In conclusion, we recommend excision after a core biopsy diagnosis of lobular neoplasia in the following circumstances:
Discordance of clinical or radiological findings with the pathological changes (this includes mass-forming lobular neoplasia)Lobular neoplasia with atypical histological features including pleomorphic LCIS, lobular neoplasia with necrosis, and when it is not possible to exclude DCIS despite E-cadherin immunohistochemistryIf there is an associated risk lesion, such as atypical intraductal epithelial proliferation, radial scar or papillary lesion.
We suggest that further studies are needed to guide the management of simple classical lobular neoplasia, as the data on the risk of associated carcinoma remain unclear in this group of patients.

lobular neoplasia
breast
pleomorphic lobular carcinoma in situ
lobular carcinoma in situ
atypical lobular neoplasia
needle biopsy