Automating three-dimensional osteoarthritis histopathological grading of human osteochondral tissue using machine learning on contrast-enhanced micro-computed tomography.

OBJECTIVE: To develop and validate a machine learning (ML) approach for automatic three-dimensional (3D) histopathological grading of osteochondral samples imaged with contrast-enhanced micro-computed tomography (CEµCT). DESIGN: A total of 79 osteochondral cores from 24 total knee arthroplasty patients and two asymptomatic donors were imaged using CEµCT with phosphotungstic acid -staining. Volumes-of-interest (VOI) in surface (SZ), deep (DZ) and calcified (CZ) zones were extracted depth-wise and subjected to dimensionally reduced Local Binary Pattern -textural feature analysis. Regularized linear and logistic regression (LR) models were trained zone-wise against the manually assessed semi-quantitative histopathological CEµCT grades (diameter = 2 mm samples). Models were validated using nested leave-one-out cross-validation and an independent test set (4 mm samples). The performance was primarily assessed using Mean Squared Error (MSE) and Average Precision (AP, confidence intervals are given in square brackets). RESULTS: Highest performance on cross-validation was observed for SZ, both on linear regression (MSE = 0.49, 0.69 and 0.71 for SZ, DZ and CZ, respectively) and LR (AP = 0.9 [0.77-0.99], 0.46 [0.28-0.67] and 0.65 [0.41-0.85] for SZ, DZ and CZ, respectively). The test set evaluations yielded increased MSE on all zones. For LR, the performance was also best for the SZ (AP = 0.85 [0.73-0.93], 0.82 [0.70-0.92] and 0.8 [0.67-0.9], for SZ, DZ and CZ, respectively). CONCLUSION: We present the first ML-based automatic 3D histopathological osteoarthritis (OA) grading method which also adequately perform on grading unseen data, especially in SZ. After further development, the method could potentially be applied by OA researchers since the grading software and all source codes are publicly available.

Quantifying Subresolution 3D Morphology of Bone with Clinical Computed Tomography.

The aim of this study was to quantify sub-resolution trabecular bone morphometrics, which are also related to osteoarthritis (OA), from clinical resolution cone beam computed tomography (CBCT). Samples (n = 53) were harvested from human tibiae (N = 4) and femora (N = 7). Grey-level co-occurrence matrix (GLCM) texture and histogram-based parameters were calculated from CBCT imaged trabecular bone data, and compared with the morphometric parameters quantified from micro-computed tomography. As a reference for OA severity, histological sections were subjected to OARSI histopathological grading. GLCM and histogram parameters were correlated to bone morphometrics and OARSI individually. Furthermore, a statistical model of combined GLCM/histogram parameters was generated to estimate the bone morphometrics. Several individual histogram and GLCM parameters had strong associations with various bone morphometrics (|r| > 0.7). The most prominent correlation was observed between the histogram mean and bone volume fraction (r = 0.907). The statistical model combining GLCM and histogram-parameters resulted in even better association with bone volume fraction determined from CBCT data (adjusted R2 change = 0.047). Histopathology showed mainly moderate associations with bone morphometrics (|r| > 0.4). In conclusion, we demonstrated that GLCM- and histogram-based parameters from CBCT imaged trabecular bone (ex vivo) are associated with sub-resolution morphometrics. Our results suggest that sub-resolution morphometrics can be estimated from clinical CBCT images, associations becoming even stronger when combining histogram and GLCM-based parameters.

3D morphometric analysis of calcified cartilage properties using micro-computed tomography.

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (µCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a µ-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA.

In vitro method for 3D morphometry of human articular cartilage chondrons based on micro-computed tomography.

OBJECTIVE: The aims of this study were: to 1) develop a novel sample processing protocol to visualize human articular cartilage (AC) chondrons using micro-computed tomography (µCT), 2) develop and validate an algorithm to quantify the chondron morphology in 3D, and 3) compare the differences in chondron morphology between intact and osteoarthritic AC. METHOD: The developed protocol is based on the dehydration of samples with hexamethyldisilazane (HMDS), followed by imaging with a desktop µCT. Chondron density and depth, as well as volume and sphericity, were calculated in 3D with a custom-made and validated algorithm employing semi-automatic chondron selection and segmentation. The quantitative parameters were analyzed at three AC depth zones (zone 1: 0-10%; zone 2: 10-40%; zone 3: 40-100%) and grouped by the OARSI histological grades (OARSI grades 0-1.0, n = 6; OARSI grades 3.0-3.5, n = 6). RESULTS: After semi-automatic chondron selection and segmentation, 1510 chondrons were approved for 3D morphometric analyses. The chondrons especially in the deeper tissue (zones 2 and 3) were significantly larger (P < 0.001) and less spherical (P < 0.001), respectively, in the OARSI grade 3-3.5 group compared to the OARSI grade 0-1.0 group. No statistically significant difference in chondron density between the OARSI grade groups was observed at different depths. CONCLUSION: We have developed a novel sample processing protocol for chondron imaging in 3D, as well as a high-throughput algorithm to semi-automatically quantify chondron/chondrocyte 3D morphology in AC. Our results also suggest that 3D chondron morphology is affected by the progression of osteoarthritis (OA).

3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography.

OBJECTIVE: Histopathological grading of osteochondral (OC) tissue is widely used in osteoarthritis (OA) research, and it is relatively common in post-surgery in vitro diagnostics. However, relying on thin tissue section, this approach includes a number of limitations, such as: (1) destructiveness, (2) sample processing artefacts, (3) 2D section does not represent spatial 3D structure and composition of the tissue, and (4) the final outcome is subjective. To overcome these limitations, we recently developed a contrast-enhanced µCT (CEµCT) imaging technique to visualize the collagenous extracellular matrix (ECM) of articular cartilage (AC). In the present study, we demonstrate that histopathological scoring of OC tissue from CEµCT is feasible. Moreover, we establish a new, semi-quantitative OA µCT grading system for OC tissue. RESULTS: Pathological features were clearly visualized in AC and subchondral bone (SB) with µCT and verified with histology, as demonstrated with image atlases. Comparison of histopathological grades (OARSI or severity (0-3)) across the characterization approaches, CEµCT and histology, excellent (0.92, 95% CI = [0.84, 0.96], n = 30) or fair (0.50, 95% CI = [0.16, 0.74], n = 27) intra-class correlations (ICC), respectively. A new µCT grading system was successfully established which achieved an excellent cross-method (µCT vs histology) reader-to-reader intra-class correlation (0.78, 95% CI = [0.58, 0.89], n = 27). CONCLUSIONS: We demonstrated that histopathological information relevant to OA can reliably be obtained from CEµCT images. This new grading system could be used as a reference for 3D imaging and analysis techniques intended for volumetric evaluation of OA pathology in research and clinical applications.

Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography.

OBJECTIVE: Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. METHOD: We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland-Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. RESULTS: Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4). CONCLUSIONS: PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

Comparison of cartilage histopathology assessment systems on human knee joints at all stages of osteoarthritis development.

OBJECTIVE: To compare the MANKIN and OARSI cartilage histopathology assessment systems using human articular cartilage from a large number of donors across the adult age spectrum representing all levels of cartilage degradation. DESIGN: Human knees (n=125 from 65 donors; age range 23-92) were obtained from tissue banks. All cartilage surfaces were macroscopically graded. Osteochondral slabs representing the entire central regions of both femoral condyles, tibial plateaus, and the patella were processed for histology and Safranin O - Fast Green staining. Slides representing normal, aged, and osteoarthritis (OA) tissue were scanned and electronic images were scored online by five observers. Statistical analysis was performed for inter- and intra-observer variability, reproducibility and reliability. RESULTS: The inter-observer variability among five observers for the MANKIN system showed a similar good Intra-class correlation coefficient (ICC>0.81) as for the OARSI system (ICC>0.78). Repeat scoring by three of the five readers showed very good agreement (ICC>0.94). Both systems showed a high reproducibility among four of the five readers as indicated by the Spearman's rho value. For the MANKIN system, the surface represented by lesion depth was the parameter where all readers showed an excellent agreement. Other parameters such as cellularity, Safranin O staining intensity and tidemark had greater inter-reader disagreement. CONCLUSION: Both scoring systems were reliable but appeared too complex and time consuming for assessment of lesion severity, the major parameter determined in standardized scoring systems. To rapidly and reproducibly assess severity of cartilage degradation, we propose to develop a simplified system for lesion volume.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rabbit.

AIM: The primary goal of this body of work is to suggest a standardized system for histopathological assessment of experimental surgical instability models of osteoarthritis (OA) in rabbits, building on past experience, to achieve comparability of studies from different centres. An additional objective is to review methodologies that have been employed in the past for assessing OA in rabbits with particular reference to the surgical anterior cruciate ligament transection (ACLT) model. METHODS: A panel of scientists and clinician-scientists with recognized expertise in assessing rabbit models of OA reviewed the literature to provide a critical appraisal of the methods that have been employed to assess both macroscopic and microscopic changes occurring in rabbit joint tissues in experimental OA. In addition, a validation of the proposed histologic histochemical grading system was performed. RESULTS: The ACLT variant of the surgical instability model in skeletally mature rabbits is the variation most capable of reproducing the entire range of cartilage, synovial and bone lesions recognized to be associated with OA. These lesions can be semiquantitatively graded using macroscopic and microscopic techniques. Further, as well as cartilage lesions, this ACLT model can produce synovial and bone lesions similar to that of human OA. CONCLUSIONS: The ACLT variant of the surgical instability model in rabbits is a reproducible and effective model of OA. The cartilage lesions in this model and their response to therapy can be graded according to an adapted histological and histochemical grading system, though also this system is to some extent subjective and, thus, neither objective nor entirely reproducible.

Terminology of osteoarthritis cartilage and bone histopathology - a proposal for a consensus.

Unifying terminology used to describe morphologic features is a very important endeavour to assure comparability of work and papers on osteoarthritis animal models. In this editorial an attempt is presented to define and unify the terminology of the macroscopic and histological description of joint changes in OA for both human OA and the OA animal models.

Total hip replacement in Gaucher's disease: effects of enzyme replacement therapy.

Total hip replacement in patients with Gaucher's disease with symptomatic osteonecrosis of the femoral head is controversial because of the high early failure rates. We describe four patients who had an uncemented total hip replacement following enzyme replacement therapy for a median of two years and one month (1 to 9.8 years) prior to surgery, and who remained on treatment. At operation, the bone had a normal appearance and consistency. Histopathological examination showed that, compared with previous biopsies of untreated Gaucher's disease, the Gaucher cell infiltrate had decreased progressively with therapy, being replaced by normal adipose tissue. The surfaces of viable bone beyond the osteonecrotic areas showed osteoblasts, indicating remodelling. In one case acetabular revision was carried out after 11 years and eight months. The three remaining patients had a mean follow-up of six years and four months (3.3 to 12 years). We recommend initiating enzyme replacement therapy at least one to two years prior to total hip replacement to facilitate bone remodelling and to allow implantation of uncemented components in these young patients.

Fresh osteochondral allografts for posttraumatic knee defects: long-term followup.

Fresh osteochondral allograft transplantation has been an effective treatment option with promising long-term clinical outcomes for focal posttraumatic defects in the knee for young, active individuals. We examined histologic features of 35 fresh osteochondral allograft specimens retrieved at the time of subsequent graft revision, osteotomy, or TKA. Graft survival time ranged from 1 to 25 years based on their time to reoperation. Histologic features of early graft failures were lack of chondrocyte viability and loss of matrix cationic staining. Histologic features of late graft failures were fracture through the graft, active and incomplete remodeling of the graft bone by the host bone, and resorption of the graft tissue by synovial inflammatory activity at graft edges. Histologic features associated with long-term allograft survival included viable chondrocytes, functional preservation of matrix, and complete replacement of the graft bone with the host bone. Given chondrocyte viability, long-term allograft survival depends on graft stability by rigid fixation of host bone to graft bone. With the stable osseous graft base, the hyaline cartilage portion of the allograft can survive and function for 25 years or more.

Osteoarthritis cartilage histopathology: grading and staging.

OBJECTIVE: Current osteoarthritis (OA) histopathology assessment methods have difficulties in their utility for early disease, as well as their reproducibility and validity. Our objective was to devise a more useful method to assess OA histopathology that would have wide application for clinical and experimental OA assessment and would become recognized as the standard method. DESIGN: An OARSI Working Group deliberated on principles, standards and features for an OA cartilage pathology assessment system. Using current knowledge of the pathophysiology of OA morphologic features, a proposed system was presented at OARSI 2000. Subsequently, this was widely circulated for comments amongst experts in OA pathology. RESULTS: An OA cartilage pathology assessment system based on six grades, which reflect depth of the lesion and four stages reflecting extent of OA over the joint surface was developed. CONCLUSIONS: The OARSI cartilage OA histopathology grading system appears consistent and simple to apply. Further studies are required to confirm the system's utility.

The steroidal aromatase inhibitor exemestane prevents bone loss in ovariectomized rats.

The irreversible steroidal aromatase inhibitor exemestane (EXE) is one of three third generation aromatase inhibitors currently prescribed for advanced breast cancer in postmenopausal women. Its principal mechanism of action is to reduce estrogen by inhibiting its synthesis. In addition to its efficacy against breast cancer, its effects on other organs are important, especially when given to women with good-prognosis breast cancer or potentially to healthy women at increased risk of developing breast cancer. The purpose of this study was to evaluate the effects of EXE on bone and lipid metabolism in ovariectomized (OVX) rats. Ten-month-old Sprague-Dawley female rats were sorted into intact controls, intact + EXE, OVX controls, and OVX + EXE groups, and treated by weekly intramuscular injection with vehicle or 100 mg/kg EXE for 16 weeks. The bone mineral density (BMD), mechanical testing, histomorphometry, bone resorption marker-serum pyridinoline (PYD), and bone formation marker-serum osteocalcin (OC) were used to determine the effects of treatment on bone. In addition, total serum cholesterol, triglyceride, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were determined. BMD of the lumbar spine and femur were 11% and 7%, respectively, higher in OVX animals given EXE than in OVX controls (all Ps<0.001). Significant increases in the bending strength and toughness of the femora as well as the compressive strength and elastic modulus of the vertebrae were observed in OVX rats given EXE (all Ps<0.02 vs. OVX controls). Trabecular bone volume (BV) was significantly higher in OVX rats treated with EXE than in OVX controls (P<0.0001). In OVX animals, EXE reduced the OVX-induced increase of serum PYD by 96% (P<0.0001), and the OVX-induced increase of serum OC was completely prevented by treatment with EXE. In OVX animals, EXE resulted in a 28% reduction of serum cholesterol (P<0.0001) and reduced LDL by 64% compared with OVX controls (P<0.0001). The positive results of EXE on bone and lipid metabolism in the OVX rat model merit further investigation of the effects of EXE in postmenopausal women.

High frequency acoustic parameters of human and bovine articular cartilage following experimentally-induced matrix degradation.

Matrix degradation and proteoglycan loss in articular cartilag eare features of early osteoarthritis. To determine the effect of matrix degradation and proteoglycan loss on ultrasound propagation in cartilage, we used papain and interleukin-1alpha to degrade the matrix proteoglycans of human and bovine cartilage samples, respectively. There is also minor collagen alteration associated with these chemical degradation methods. We compared the speed of sound and frequency dependent attenuation (20-40 MHz) of control and experimental paired samples. We found that a loss of matrix proteoglycans and collagen disruption resulted in a 20-30% increase in the frequency dependent attenuation and a 2% decrease in the speed of sound in both human and bovine cartilage. We conclude that the frequency dependent attenuation and speed of sound in articular cartilage are sensitive to experimental modification of the matrix proteoglycans and collagen. These findings suggest that ultrasound can potentially be used to detect morphologic changes in articular cartilage associated with the progression of osteoarthritis.

Experimental study of bone growth around a dental implant after Surgibone grafting.

Histologic and histomorphometric results of bone growth around titanium alloy screw-type implants after Surgibone grafting in New Zealand white rabbits are presented. At 21 days, new bone was formed along the surface of the implant. At 84 days, newly formed bone replaced almost all of the trabecular bone of the graft and reached the shoulder level of the implant. There was a higher percentage of host bone area at 84 days than at any of the earlier experimental periods (P < .01). The average mineral apposition rates ranged from 1.82 to 2.35 microns/day in original bone and 2.55 to 2.80 microns/day in newly formed bone. The results suggest that Surgibone grafting in combination with dental implants can be used to increase the height of the recipient bone and therefore aid in the fixation of the implant in this animal model.

The vascularized pig fibula bone flap model: effects of multiple segmental osteotomies on growth and viability.

Previous work by this laboratory introduced the pig fibula bone flap as a model for the study of the pathophysiology of vascularized bone flaps. Anatomic and hemodynamic studies demonstrated a significant (p < 0.05) decrease in vascular perfusion after a series of segmental osteotomies and rigid fixation (lag screws and miniplates) in the distal end of the flap, suggesting that blood flow to the distal osteotomized segment of the flap may be impaired. Killing the animals after blood flow studies precluded assessment of the effect of these hemodynamic changes on bone healing. Therefore, the aim of this study was to assess the pig fibula bone flap model with respect to viability, healing, and subsequent growth after multiple segmental osteotomies and rigid fixation to contribute to the understanding of vascularized bone flap pathophysiology. Yorkshire pigs (20 to 25 kg) were used for all experiments. Eight pigs underwent unilateral elevation of a vascularized fibula bone flap, which was osteotomized into three segments and orthotopically rigidly fixed using a 2.4-mm mandibular reconstruction plate. The left fibula remained as the control. Fluorochrome labels were injected to assess bone viability and turnover, and both fibulae were assessed for growth radiologically. The fibulae were harvested 21 days postoperatively (when the animals were killed), and bone healing was assessed histologically and clinically. There were no significant differences in preoperative and postoperative lengths of the osteotomized fibulae compared with the controls, suggesting that there was no impairment of growth potential after multiple segmental osteotomies and rigid fixation. Significant (p < 0.05) bony hypertrophy of the osteotomized fibulae was noted when compared with controls. Mobility was observed in 3 of the 32 osteotomies (9 percent), occurring across one proximal and two distal osteotomies in association with failure of fixation. However, histologic and fluorochrome assessment confirmed the viability of all bone segments, as supported by the presence of tetracycline given 2 days postoperatively. The pig fibula bone flap model is well tolerated by the pig. Multiple segmental osteotomies and rigid fixation, previously associated with a significant decrease in blood flow in the distal segment, did not impair either growth potential, viability, or healing ability. It is suggested that the pig fibula is a suitable model for the study of bone flap pathophysiology.

Allotype immunoglobulin enhances alkaline phosphatase activity: implications for the inflammatory response.

To understand the interactions among components of the immune/inflammation response, we studied the effects of immunoglobulins on the phosphatase activity of alkaline phosphatase in vitro. Bovine intestinal alkaline phosphatase was incubated with substrate in the presence of allotypic and xenotypic immunoglobulin. We found that bovine but not rabbit immunoglobulin enhanced the phosphatase activity of bovine intestinal alkaline phosphatase. Similarly, human but not bovine immunoglobulin G enhanced human placental alkaline phosphatase activity. By enhancing alkaline phosphatase activity, immunoglobulins bound to alkaline phosphatase may assist physiologic transport functions and enhance resolution of the inflammatory response. Further, in clinical conditions with high immunoglobulin concentrations, the serum alkaline phosphatase recorded may have spuriously high values.

MRI of the cartilaginous epiphysis of the femoral head in the piglet hip after ischemic damage.

MRI of the cartilaginous epiphysis (CE) of piglet femoral head was performed after ischemic damage to study the changes in the CE found on MRI and to correlate these changes with histologic findings. Avascular necrosis of the femoral head was induced with a suture ligature in nine piglets; one piglet was killed postoperatively on day 3 and on weeks 1, 2, 3, 4, 6, 7, and 8 (two piglets were killed on week 3). MRI of the ischemic and contralateral nonischemic hip joints were obtained with a 60-mm field of view (low resolution MRI). Biopsy cores of the femoral heads were imaged with a 15-mm field of view (high resolution MRI) and correlated with histologic sections. The CE for all operated hips, except for the 3-day postoperative specimen, showed evidence of ischemic changes on histologic assessment; the severity of damage increased with time. The MRI appearance of ischemic and nonischemic CE was clearly different by 2 weeks after the operation. No trilaminar signal pattern was evident in the high resolution T2-weighted (T2W) imaging of the ischemic CE from 2 weeks after the operation. In the 3- to 8-week postoperative specimens, focal areas of low signal intensity on high resolution T1-weighted (T1W) and T2W imaging corresponded to the areas of chondronecrosis found on histologic assessment. The regions of high signal intensity on T2W imaging corresponded to the areas of chondrocyte clusters with increased safranin-O staining. High resolution MRI can demonstrate changes in the CE associated with ischemic injury and may have a role in the assessment of the CE and its development after ischemic injury.

neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group.

PURPOSE: It remains a challenge to predict which women with axillary node-negative (ANN) breast cancer at greatest risk of relapse may benefit most from adjuvant therapy. Increases in neu/erbB-2 have been implicated in breast cancer prognosis. Although overexpression has been investigated extensively, this study represents the first prospective assessment of the prognostic value of neu/erbB-2 DNA amplification in a cohort of women with newly diagnosed ANN. METHODS: A consecutive series of women was monitored for recurrence (median follow-up duration, 36 months) and tumors from 580 individuals were analyzed for amplification. The association of amplification with risk of recurrence was examined in survival analyses with traditional and histologic markers as prognostic factors. RESULTS: Neu/erbB-2 was amplified in 20% of cases. We found an increased risk of disease recurrence when neu/erbB-2 was amplified > or = twofold that persisted with adjustment for other prognostic factors (relative risk, 2.36; P = .002). We found some evidence that amplification was more important in patients who received chemotherapy compared with untreated patients. CONCLUSION: neu/erbB-2 amplification is an independent prognostic factor for risk of recurrence in ANN breast cancer. Women with tumors without neu/erbB-2 amplification have a good prognosis; aggressive therapy in this group is therefore difficult to justify. On the other hand, even with adjuvant chemotherapeutic treatment, women whose tumors exhibit neu/erbB-2 amplification have an increased risk of recurrence. We encourage a randomized trial to compare more aggressive adjuvant chemotherapy versus standard chemotherapy for ANN women whose tumors exhibit neu/erbB-2 amplification.