Judit Zubovits

Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.

Understanding and quantifying the mechanical properties of breast tissues has been a subject of interest for the past two decades. This has been motivated in part by interest in modelling soft tissue response for surgery planning and virtual-reality-based surgical training. Interpreting elastography images for diagnostic purposes also requires a sound understanding of normal and pathological tissue mechanical properties. Reliable data on tissue elastic properties are very limited and those which are available tend to be inconsistent, in part as a result of measurement methodology. We have developed specialized techniques to measure tissue elasticity of breast normal tissues and tumour specimens and applied them to 169 fresh ex vivo breast tissue samples including fat and fibroglandular tissue as well as a range of benign and malignant breast tumour types. Results show that, under small deformation conditions, the elastic modulus of normal breast fat and fibroglandular tissues are similar while fibroadenomas were approximately twice the stiffness. Fibrocystic disease and malignant tumours exhibited a 3-6-fold increased stiffness with high-grade invasive ductal carcinoma exhibiting up to a 13-fold increase in stiffness compared to fibrogalndular tissue. A statistical analysis showed that differences between the elastic modulus of the majority of those tissues were statistically significant. Implications for the specificity advantages of elastography are reviewed.

Quantitative Ultrasound Evaluation of Tumor Cell Death Response in Locally Advanced Breast Cancer Patients Receiving Chemotherapy

Purpose: Quantitative ultrasound techniques have been recently shown to be capable of detecting cell death through studies conducted on in vitro and in vivo models. This study investigates for the first time the potential of early detection of tumor cell death in response to clinical cancer therapy administration in patients using quantitative ultrasound spectroscopic methods. Experimental Design: Patients (n = 24) with locally advanced breast cancer received neoadjuvant chemotherapy treatments. Ultrasound data were collected before treatment onset and at 4 times during treatment (weeks 1, 4, and 8, and preoperatively). Quantitative ultrasound parameters were evaluated for clinically responsive and nonresponding patients. Results: Results indicated that quantitative ultrasound parameters showed significant changes for patients who responded to treatment, and no similar alteration was observed in treatment-refractory patients. Such differences between clinically and pathologically determined responding and nonresponding patients were statistically significant (P < 0.05) after 4 weeks of chemotherapy. Responding patients showed changes in parameters related to cell death with, on average, an increase in mid-band fit and 0-MHz intercept of 9.1 ± 1.2 dBr and 8.9 ± 1.9 dBr, respectively, whereas spectral slope was invariant. Linear discriminant analysis revealed a sensitivity of 100% and a specificity of 83.3% for distinguishing nonresponding patients by the fourth week into a course of chemotherapy lasting several months. Conclusion: This study reports for the first time that quantitative ultrasound spectroscopic methods can be applied clinically to evaluate cancer treatment responses noninvasively. The results form a basis for monitoring chemotherapy effects and facilitating the personalization of cancer treatment. Clin Cancer Res; 19(8); 2163–74. ©2013 AACR.

Whole-specimen histopathology: a method to produce whole-mount breast serial sections for 3-D digital histopathology imaging

Aims:  To develop a method for preparing diagnostic‐quality, whole‐mount serial sections of breast specimens while preserving 3‐D conformation. This required supporting the fresh specimen prior to breadloafing and refining the conventional tissue processing method. The overall goal is to use digital images of whole‐specimen histopathology to improve the estimation of extent of disease.

D2-40, a novel immunohistochemical marker in differentiating dermatofibroma from dermatofibrosarcoma protuberans

The distinction between dermatofibroma, particularly cellular variant, and dermatofibrosarcoma protuberans in excisional biopsies is usually straightforward. However, a separation between the two may be sometimes challenging, especially in superficial biopsies. Although factor XIIIa and CD34 immunostains are useful in differentiating dermatofibroma and dermatofibrosarcoma protuberans in most instances, focal CD34 positivity may be seen in cellular fibrous histiocytoma. Some cases reveal overlapping immunostain results. D2-40 identifies a 40-kDa O-linked sialoglycoprotein present on a variety of tissues including testicular germ cell tumors as well as lymphatic endothelium. In this study, we investigated the utility of D2-40 in separating dermatofibroma from dermatofibrosarcoma protuberans and compared the results with other commonly used immunostains. Fifty-six cases of dermatofibroma (including six cellular variant) and 29 cases of dermatofibrosarcoma protuberans were retrieved from the archives of Department of Anatomic Pathology at Sunnybrook Health Sciences Center in University of Toronto. We applied factor XIIIa, CD34, and monoclonal mouse anti-D2-40 immunostains to formalin-fixed, paraffin-embedded tissue sections. All 56 (100%) cases of dermatofibroma demonstrated strong and diffuse immunoreactivity to D2-40 in the spindle cells and stroma. Similarly, factor XIIIa showed strong and diffuse positivity in the spindle cells. Nearly all dermatofibromas were negative for CD34 except one case revealing focal positivity. None of dermatofibrosarcoma protuberans cases were labeled by D2-40, although four cases showed weak and patchy background staining in contrary to diffuse, strong, and crisp staining seen in dermatofibromas. Our results indicate that D2-40 seems to be a sensitive immunohistochemical marker for dermatofibromas, including cellular variant. Focal and faint D2-40 staining may be seen in the stroma of dermatofibrosarcoma protuberans. Our findings suggest that D2-40 can be used as a complementary immunostain to factor XIIIa and CD34 in problematic and challenging cases on superficial biopsies.

Low-frequency quantitative ultrasound imaging of cell death in vivo.

PURPOSE Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models. METHODS Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy. RESULTS Ultrasound-based spectroscopic biomarkers indicated significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r2=0.71, 0.82; p<0.001). CONCLUSIONS In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency, in addition to high-frequency ultrasound, can detect tissue changes associated with cell death in vivo in response to cancer treatments.

Effects of partner proteins on BCA2 RING ligase activity

BackgroundBCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.MethodsHere, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.ResultsTen unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.ConclusionsThe interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.

An Improved Processing Method for Breast Whole-Mount Serial Sections for Three-Dimensional Histopathology Imaging

To develop and validate improved processing methods for producing diagnostic-quality, whole-mount serial sections for 3-dimensional imaging of whole-breast histopathologic studies, we subjected 4-mm-thick whole-specimen slices to a 38-hour microwave-assisted protocol. Morphologic features, antigenicity, and tissue shrinkage were evaluated. A schedule using the tissue processor was optimized by evaluating the serial section yield for 3 schedules. The microwave-based processing schedule is adequate for producing diagnostic-quality whole-mount breast serial sections of an area up to 6,000 mm(2) and is compatible with a variety of immunohistochemical stains. A mean +/- SE total tissue shrinkage of 8.4% +/- 0.2% resulted. For the tissue processor, optimal results are obtained using a 59-hour schedule. Total fixation and processing time for whole-mount serial breast sections has been reduced from 21 days to 38 hours, with microwave assistance, and to 59 hours without. No adverse effects of microwaves on morphologic features, antigenicity, or gross tissue dimensions were observed.

Three-dimensional digital breast histopathology imaging

We have developed a digital histology imaging system that has the potential to improve the accuracy of surgical margin assessment in the treatment of breast cancer by providing finer sampling and 3D visualization. The system is capable of producing a 3D representation of histopathology from an entire lumpectomy specimen. We acquire digital photomicrographs of a stack of large (120 x 170 mm) histology slides cut serially through the entire specimen. The images are then registered and displayed in 2D and 3D. This approach dramatically improves sampling and can improve visualization of tissue structures compared to current, small-format histology. The system consists of a brightfield microscope, adapted with a freeze-frame digital video camera and a large, motorized translation stage. The image of each slide is acquired as a mosaic of adjacent tiles, each tile representing one field-of-view of the microscope, and the mosaic is assembled into a seamless composite image. The assembly is done by a program developed to build image sets at six different levels within a multiresolution pyramid. A database-linked viewing program has been created to efficiently register and display the animated stack of images, which occupies about 80 GB of disk space per lumpectomy at full resolution, on a high-resolution (3840 x 2400 pixels) colour monitor. The scanning or tiling approach to digitization is inherently susceptible to two artefacts which disrupt the composite image, and which impose more stringent requirements on system performance. Although non-uniform illumination across any one isolated tile may not be discernible, the eye readily detects this non-uniformity when the entire assembly of tiles is viewed. The pattern is caused by deficiencies in optical alignment, spectrum of the light source, or camera corrections. The imaging task requires that features as small as 3.2 μm in extent be seamlessly preserved. However, inadequate accuracy in positioning of the translation stage produces visible discontinuities between adjacent features. Both of these effects can distract the viewer from the perception of diagnostically important features. Here we describe the system design and discuss methods for the correction of these artefacts. In addition, we outline our approach to rendering the processing and display of these large images computationally feasible.