Early-stage cervical cancer: Tumor delineation by magnetic resonance imaging and ultrasound — A European multicenter trial

ObjectiveTo compare the diagnostic accuracy of ultrasound (US) and magnetic resonance imaging (MRI) in the preoperative assessment of early-stage cervical cancer using pathologic findings as the reference standard.Patients and methodsProspective multi-center trial enrolling 209 consecutive women with early-stage cervical cancer (FIGO IA2–IIA) scheduled for surgery. The following parameters were assessed on US and MRI and compared to pathology: remaining tumor, size, tumor stromal invasion < 2/3 (superficial) or ≥ 2/3 (deep), and parametrial invasion.ResultsComplete data were available for 182 patients. The agreement between US and pathology was excellent for detecting tumors, correctly classifying bulky tumors (> 4 cm), and detecting deep stromal invasion (kappa values 0.84, 0.82, and 0.81 respectively); and good for classifying small tumors (< 2 cm) and detecting parametrial invasion (kappa values 0.78 and 0.75, respectively). The agreement between MRI and histology was good for classifying tumors as < 2 cm, or > 4 cm, and detecting deep stromal invasion (kappa values 0.71, 0.76, and 0.77, respectively). It was moderately accurate in tumor detection, and in assessing parametrial invasion (kappa values 0.52 and 0.45, respectively).The agreement between histology and US was significantly better in assessing residual tumor (p < 0.001) and parametrial invasion (p < 0.001) than the results obtained by MRI. Imaging methods were not significantly influenced by previous cone biopsy.ConclusionUS and MRI are highly accurate for the preoperative assessment of women with early-stage cervical cancer, although US may be more accurate in detecting residual tumors and assessing parametrial invasion.     

Dystrophin and utrophin “double knockout” dystrophic mice exhibit a spectrum of degenerative musculoskeletal abnormalities

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. In addition, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that a deficiency in skeletal homeostasis coexists but little is known about the effects of DMD on bone and other connective tissues within the musculoskeletal system. Recent evidence has emerged implicating adult stem cell dysfunction in DMD myopathogenesis. Given the common mesenchymal origin of muscle and bone, we sought to investigate bone and other musculoskeletal tissues in a DMD mouse model. Here, we report that dystrophin–utrophin double knockout (dko) mice exhibit a spectrum of degenerative changes, outside skeletal muscle, in bone, articular cartilage, and intervertebral discs, in addition to reduced lifespan, muscle degeneration, spinal deformity, and cardiomyopathy previously reported. We also report these mice to have a reduced capacity for bone healing and exhibit spontaneous heterotopic ossification in the hind limb muscles. Therefore, we propose the dko mouse as a model for premature musculoskeletal aging and posit that a similar phenomenon may occur in patients with DMD. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 343–349, 2013     

A methodology to ensure and improve accuracy of Ki67 labelling index estimation by automated digital image analysis in breast cancer tissue

Introduction

Immunohistochemical Ki67 labelling index (Ki67 LI) reflects proliferative activity and is a potential prognostic/predictive marker of breast cancer. However, its clinical utility is hindered by the lack of standardized measurement methodologies. Besides tissue heterogeneity aspects, the key element of methodology remains accurate estimation of Ki67-stained/counterstained tumour cell profiles. We aimed to develop a methodology to ensure and improve accuracy of the digital image analysis (DIA) approach.

Methods

Tissue microarrays (one 1-mm spot per patient, n = 164) from invasive ductal breast carcinoma were stained for Ki67 and scanned. Criterion standard (Ki67-Count) was obtained by counting positive and negative tumour cell profiles using a stereology grid overlaid on a spot image. DIA was performed with Aperio Genie/Nuclear algorithms. A bias was estimated by ANOVA, correlation and regression analyses. Calibration steps of the DIA by adjusting the algorithm settings were performed: first, by subjective DIA quality assessment (DIA-1), and second, to compensate the bias established (DIA-2). Visual estimate (Ki67-VE) on the same images was performed by five pathologists independently.

Results

ANOVA revealed significant underestimation bias (P < 0.05) for DIA-0, DIA-1 and two pathologists’ VE, while DIA-2, VE-median and three other VEs were within the same range. Regression analyses revealed best accuracy for the DIA-2 (R-square = 0.90) exceeding that of VE-median, individual VEs and other DIA settings. Bidirectional bias for the DIA-2 with overestimation at low, and underestimation at high ends of the scale was detected. Measurement error correction by inverse regression was applied to improve DIA-2-based prediction of the Ki67-Count, in particular for the clinically relevant interval of Ki67-Count < 40%. Potential clinical impact of the prediction was tested by dichotomising the cases at the cut-off values of 10, 15, and 20%. Misclassification rate of 5-7% was achieved, compared to that of 11-18% for the VE-median-based prediction.

Conclusions

Our experiments provide methodology to achieve accurate Ki67-LI estimation by DIA, based on proper validation, calibration, and measurement error correction procedures, guided by quantified bias from reference values obtained by stereology grid count. This basic validation step is an important prerequisite for high-throughput automated DIA applications to investigate tissue heterogeneity and clinical utility aspects of Ki67 and other immunohistochemistry (IHC) biomarkers.     

Development of a self-inactivating tet-on retroviral vector expressing bone morphogenetic protein 4 to achieve regulated bone formation.

The aims of this study were to explore the possibility of improving the design of self-inactivating (SI) retroviral vectors and to develop an SI vector that would allow optimal tet-on-regulated therapeutic gene expression. To minimize any interference between the viral promoter and the inducible promoter, we deleted different regulatory elements in the 3'LTR and examined their effects on transgene expression in transfected or transduced cells. In transfected cells, such deletions reduced the transgene expression. The insertion of a polyadenylation sequence could not completely compensate for this effect. We observed three patterns of transgene expression in cells transduced with these tet-on retroviral vectors: (1) high levels of both basal and inducible expression, (2) low levels of both basal and inducible expression, and (3) low levels of basal and high levels of inducible expression. After using the optimal vector to transduce muscle-derived stem cells, we were able to regulate the strong in vitro expression of transgenes-including enhanced green fluorescent protein and bone morphogenetic protein 4-via the addition or withdrawal of doxycycline (Dox). Implantation of the transduced cells and subsequent Dox-dependent induction of gene expression resulted in bone formation in vivo. Thus, we have developed an optimal SI retroviral vector that maintains a high titer, efficiently transduces muscle-derived stem cells, and enables both high levels of inducible gene expression in vitro and robust regulated bone formation in vivo.     

The safety and efficacy of multiple consecutive cryo lesions in canine pulmonary veins-left atrial junction

OBJECTIVE: The purpose of the study is to evaluate the safety and efficacy of multiple cryo lesions in canine pulmonary veins-left atrial junction. BACKGROUND: The use of radiofrequency to achieve electrical isolation of the pulmonary veins (PVs) has been associated with PV stenosis. No information is currently available concerning the safety and the electrophysiological effects of multiple and consecutive cryo applications at the PV-left atrial junction. METHODS: Liquid N(2)O was delivered into semi-compliant 15 to 22-mm-diameter balloons. In 13 dogs weighing 34 +/- 2 kg, one to four consecutive cryo lesions were randomly applied to each PV for 3 minutes in 6-minute intervals. The pre- and post-PV sizes were recorded by angiography. Electrogram activity and pacing thresholds were recorded before and after cryo. PV patency and the PV-atrial tissue characteristics were evaluated grossly and histologically. RESULTS: Pacing capture was not possible with 10 mA postablation in 26/46 (57%) electrodes, and in 20 (43%) electrodes pacing threshold increased from 1.6 +/- 1.7 mA to 7.8 +/- 3.2 mA. The total elimination of recorded electrograms was noted in 22%, 29%, and 18% following 1, 2, and 3 cryo lesions respectively. After 4 lesions this value increased to 53%. No significant changes in PV diameter were recorded in any of the veins pre vs the terminal study. The PVs and PV-atrial interface tissue were soft, compliant, and without collagen or cartilaginous tissue. There was no hemoptysis in any of the dogs. CONCLUSION: In this study, cryo balloon technology is effective and safe regardless of the number of lesions applied and the freezing temperatures achieved. Four consecutive cryo applications result in a significant increase in pacing threshold and a decrease in activity of local atrial electrograms.     

Accuracy of endoscopic ultrasound for restaging rectal cancer following neoadjuvant chemoradiation therapy

OBJECTIVES: Endoscopic ultrasound (EUS) provides important information in the initial staging of patients with rectal cancer. Preoperative combined modality chemotherapy and radiation (neoadjuvant therapy) for patients with locally advanced rectal cancer may reduce local recurrence and improve survival. The accuracy of EUS restaging of rectal cancer after chemoradiation has not been extensively studied and its usefulness is unclear. The aim of this study was to verify the accuracy of EUS in staging rectal cancer after neoadjuvant chemoradiation in a large cohort of patients. METHODS: EUS staging was performed before and after concurrent 5-fluorouracil and hyperfractionated radiotherapy in 82 patients with recently diagnosed locally advanced rectal cancer. All patients underwent subsequent surgical resection and complete pathologic staging. RESULTS: After chemoradiation, 16 patients (20%) had no residual disease at pathologic staging. (T0N0). However, EUS correctly predicted complete response to chemoradiation in only 10 of 16 patients (63%). Overall accuracy of EUS post chemoradiation for pathologic T-stage was only 48%. Fourteen percent were understaged and 38% overstaged. EUS accuracy for N-stage was 77%. The T-category was correctly staged before surgery in 23 of the 56 responders (41%) and in 16 of 24 nonresponders (67%). EUS was unable to accurately distinguish postradiation changes from residual tumor. CONCLUSION: EUS staging of rectal cancer after chemoradiation is inaccurate, especially in the group of patients with visual and EUS evidence of response. Its routine use for staging purposes after neoadjuvant chemoradiation for rectal cancer should be discouraged.     

Converse relationship between in vitro osteogenic differentiation and in vivo bone healing elicited by different populations of muscle-derived cells genetically engineered to express BMP4.

In this study, we compared the use of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve bone healing to that of muscle-derived non-osteoprogenitor cells (PP1 cells). Surprisingly, the use of PP1 cells resulted in an improved outcome because of the lack of adverse responses to BMP4 involving cell differentiation, proliferation, and apoptosis. INTRODUCTION: Although researchers frequently opt to use osteogenic cells for osteogenic bone morphogenetic protein (BMP)-based ex vivo gene therapy to improve bone healing, it remains unclear whether the osteogenic potential of a cellular vehicle affects the outcome of bone healing applications. Here we compared the use of muscle-derived non-osteoprogenitor cells (PP1 cells) to that of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve the healing of bone defects. MATERIALS AND METHODS: Two distinct populations of primary rat muscle-derived cells--PP1 and PP6--were selected, transduced with retroviral vectors to express BMP4 or a marker gene (LacZ), and implanted into critical-sized calvarial defects created in syngeneic rats. The bone healing was monitored radiographically and histologically at 7 and 14 weeks after implantation. Cellular responses to BMP4 were evaluated by alkaline phosphatase histochemical staining and RT-PCR of another osteogenic marker to indicate osteogenic differentiation, a cell proliferation assay and BrdU (bromodeoxyuridine) labeling to assess cell proliferation, and the TUNEL assay to determine apoptosis. RESULTS AND CONCLUSIONS: In all animals (nine rats per group), transduced PP1 cells expressing BMP4 demonstrated significantly advanced healing compared with PP6 cells expressing BMP4 and control cells expressing LacZ. We found that constitutive BMP4 expression negatively impacted the in vitro proliferation and in vivo survival rates of PP6 cells, but not PP1 cells. BMP4 exposure also directly inhibited the proliferation and induced the apoptosis of PP6 cells, but not PP1 cells. The impairment in PP6 cell proliferation was directly associated with the osteogenic differentiation of these cells. These results indicate that PP1 cells are better suited than osteoprogenitor cells for use as cellular vehicles to deliver osteogenic BMP4 to improve bone healing and that cellular behavior in response to a particular gene can be used to predict the cells' performance as delivery vehicles in ex vivo gene therapy.