Nicole LaPlante

Identification of the Protein Target of Myelin-Binding Ligands by Immunohistochemistry and Biochemical Analyses

The ability to visualize myelin is important in the diagnosis of demyelinating disorders and the detection of myelin-containing nerves during surgery. The development of myelin-selective imaging agents requires that a defined target for these agents be identified and that a robust assay against the target be developed to allow for assessment of structure-activity relationships. We describe an immunohistochemical analysis and a fluorescence polarization binding assay using purified myelin basic protein (MBP) that provides quantitative evidence that MBP is the molecular binding partner of previously described myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111.

Single-cell heterogeneity in ductal carcinoma in situ of breast.

Heterogeneous patterns of mutations and RNA expression have been well documented in invasive cancers. However, technological challenges have limited the ability to study heterogeneity of protein expression. This is particularly true for pre-invasive lesions such as ductal carcinoma in situ of the breast. Cell-level heterogeneity in ductal carcinoma in situ was analyzed in a single 5 μm tissue section using a multiplexed immunofluorescence analysis of 11 disease-related markers (EGFR, HER2, HER4, S6, pmTOR, CD44v6, SLC7A5 and CD10, CD4, CD8 and CD20, plus pan-cytokeratin, pan-cadherin, DAPI, and Na+K+ATPase for cell segmentation). Expression was quantified at cell level using a single-cell segmentation algorithm. K-means clustering was used to determine co-expression patterns of epithelial cell markers and immune markers. We document for the first time the presence of epithelial cell heterogeneity within ducts, between ducts and between patients with ductal carcinoma in situ. There was moderate heterogeneity in a distribution of eight clusters within each duct (average Shannon index 0.76; range 0–1.61). Furthermore, within each patient, the average Shannon index across all ducts ranged from 0.33 to 1.02 (s.d. 0.09–0.38). As the distribution of clusters within ducts was uneven, the analysis of eight ducts might be sufficient to represent all the clusters ie within- and between-duct heterogeneity. The pattern of epithelial cell clustering was associated with the presence and type of immune infiltrates, indicating a complex interaction between the epithelial tumor and immune system for each patient. This analysis also provides the first evidence that simultaneous analysis of both the epithelial and immune/stromal components might be necessary to understand the complex milieu in ductal carcinoma in situ lesions.

Compact solid state pulsed power architecture for biomedical workflows: Modular topology, programmable pulse output and experimental validation on Ex vivo platelet activation

We describe a system based on a Marx generator approach using all solid state components in a modular topology that enables higher voltages by simply adding more modules. This novel system maximizes parameter flexibility, including electric pulse amplitude, duration and repetition rate. Example of pulses generated by this all solid state generator of ~20 kV/cm, ~ 600 ns pulse width, and more than 300 A per pulse are shown to activate platelets by stimulating human PRP (platelet rich plasma) in a 2 mm cuvette. Two growth factors released during platelet activation were identical whether nsPEFs (nanosecond pulsed electric fields) or bovine thrombin, the standard clinical platelet activator, was used. Future in vivo studies will assess the effectiveness of nsPEFs activated platelet gels compared to those activated by bovine thrombin.

Robust single cell quantification of immune cell subtypes in histological samples

Due to the rapid increase in immunotherapies there is an urgent need to develop new tools for robust in situ immune cell-typing and quantification to understand disease mechanisms and therapeutic responses. In this paper, we present a new machine-learning based method for classifying immune cell types in human tissue from highly multiplexed data. The proposed method is based on: i) identifying the most representative cell clusters across multiple slides by performing hierarchical multi-channel and multi-slide clustering; ii) from the clusters of interest, we then learn a biological-phenotypical-taxonomical cell model by solving a multi-class and multi-label classification problem. We have applied this methodology for the simultaneous classification of T and B cells using CD3 and CD20 markers and further sub-classification of T cells (CD3+) into CD4+ and CD8+, and FoxP3+ cells (within CD3+ and CD4+ cells). The method allows estimating statistical measurements used for correlation analysis with clinical data. Our method is generic and can be applied for any cell type classification problem. We obtain an average accuracy of ∼95% across six immune cell types/subtypes following simultaneous classification with this approach.

Abstract P2-04-06: Demonstration of immune cell and pathway heterogeneity in Singapore DCIS samples using novel hyperplexing method (MultiOmyx®)

Breast cancer is the most common malignancy in Singapore women with rising incidence across all ethnic groups (Chinese, Malays and Indians). Ductal carcinoma in situ (DCIS) is the earliest non-invasive stage of disease and has been shown to account for approximately 26% of diagnoses in all women participating in the Breast-Screen Singapore program. Despite availability of breast screening, there are still Singapore women presenting with locally advanced breast cancer. The goal of the current study was to investigate pathway and immune cell heterogeneity in low, intermediate and high nuclear grade DCIS using a newly developed method for in situ hyperplexed analysis of multiple proteins in a single FFPE tissue (MultiOmyx). FFPE samples from patients (n= 15) diagnosed with DCIS were provided by Singapore General Hospital. Patients were of Chinese origin, ranged from 50-59 years, were all post-menopausal, and included low (n=5), intermediate (n=5) and high grade (n=5) samples. All histological diagnoses were reviewed by a single pathologist. Following a single antigen retrieval step, DAPI and cytokeratin staining was conducted and imaged at 10X. Based on DAPI, cytokeratin and autofluorescence, an HE San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-06.

Abstract P4-05-12: MultiOmyx™ screening for prognostic indicators of breast cancer

Over the last three decades, the incidence of early-stage breast cancer has doubled while the incidence of late stage breast cancer has slightly decreased; however, the standard treatment for both has not changed significantly in the last 10 years. Of those diagnosed with early-stage breast cancer, 30% will develop recurrent disease, though there is currently no diagnostic available to identify these cases. Conversely, identifying late stage patients with a low- risk for recurrence could spare them from post-surgical chemotherapy. We have developed a technology that allows in situ staining and analysis of up to 60 proteins on a single tissue slide. In this exploratory study we evaluated 25 known biomarkers on a cohort of more than 800 breast cancer cases assembled onto three tissue arrays. The cohort spans clinical grades and breast cancer subtypes, and is thus useful in novel associations of biomarker expression across a variety of clinical features. Twenty-five markers were chosen based on literature reports for marker association with disease recurrence and represent a range of biological features including signaling pathways and cell phenotypes. The slides were sequentially stained with antibodies that were directly conjugated to Cy3 and Cy5 dyes, imaged, evaluated for quality and subject inclusion, and subsequently analyzed for statistical correlations of expression values to clinical features. The images were then processed using a single cell analysis algorithm which allows for quantification of individual tumor cells and signal intensities for the markers to be extracted from sub-cellular domains (nucleus, cytoplasm, and membrane of each cell) allowing unique patterns of protein expression to be determined. Univariate Cox proportional hazards analysis was first applied to determine those features with strongest association with ‘death due to disease’. Of the 25 markers analyzed, several markers (e.g. API3, AMPH2, beta-catenin, CD44, CEACAM5, CK15, CK19, cMET, Her2, Ki67, TRIM29) were found to have strong association with poor outcome (FDR TM methodology enabled us to verify a subset of biomarkers that are clinically relevant to breast cancer outcome. Using this technology could lead to identification of novel biosignatures that stratify patients and enable precision medicine that results in better treatment decisions and prognosis for patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-12.

Soluble beta-amyloid oligomer-selective fluorophore: histological characterization of binding to the PDAPP model and human brain

placed in holders to immobilize the heads. A 7-cm inner volume coil was used as a transmitter coil and a 1.5-cm inner diameter surface coil was used as a receiver to collect data in aBruker 4.7TBioSpec small animalMRI instrument. Imageswith slice thickness 0.5mm, field of view of 2cm x 2cm andmatrix 128 x 128 (zero filling to 256 x 256) was collected to cover the visual system from eyes to Superior Colliculus. Spin-echo Diffusion Tensor Imaging (DTI)was performedwithTR3 s, TE29ms, duration between a diffusion gradient pair1⁄4 20 ms, diffusion gradient duration1⁄4 3 ms, and six-direction diffusion schemewith b-values of 0 and 0.85 ms/mm2. Using software written in Matlab (MathWorks, Natick, MA, USA), the eigenvalues derived from diffusion tensor were used to calculate axial diffusivity, radial diffusivity, relative anisotropy (RA), and trace of the diffusion tensor (Tr). To evaluate the axonal damages in optic nerves and optic tracts, animals were sacrificed in 3 months after AB treatment. The integrity of axons was evaluated using a primary antibody against phosphorylated neurofilament (pNF, SMI-31), and myelin integrity was assessed with a primary antibody against myelin basic protein (MBP). Histological sections were examined using an Olympus Fluoview Confocal Microscope equipped with a 60x oil objective for further analysis. Results: Typical DTI maps from normal and AB-treated mice were shown in Figure 1, in which the optic nerves and tracts were pointed by arrows. Among the measured white matter tracts, only optic tracts, and optic nerves showed significantDTI changes causedbyAB. In optic tract, the right side (ipsilateral side) of the tracts showed a 12-16% decrease of axial diffusivity in 1 and 3 months after AB injection while the left side of the tracts remained normal (Figure 2). As for optic nerves significant changes were found in Trmaps, which appeared a 13% reduction in the left nerves but appeared normal in the right nerves (Figure 3). Such a change of Tr in the left nerves closely related to the decrease of axial but the change of axial diffusivity did not reach a significant level. Immunohistochemistry showed results inconsistent with the DTI findings. SMI-31 staining was used to detect axonal integrity, and MBP stainingwas used to characterizemyelin loss. Comparing the left and right optic tract (Figure 4), right optic tract showed severe loss of axons (Figure 5). Conclusions: The importance of this study can be summarized in two aspects. First, this is the first study that demonstrated a potential pathway that axonal damagewhich can be induced by and exposure of axonal terminal to ABwithout first affecting the neuronal cell bodies.We used the unique anatomical feature of retinal ganglion cells with their cell bodies in the eye but the elongated axons reaching to themiddle of the brain.Micro-injection of AB1-42 in the axonal terminals of optic tract causedoptic tract axonal damage in 1month (based on DTI). Three months after AB treatment, the histological examination showed severe axonal loss in ipsilateral optic tracts (Figure 4). Given the monocular nature of the mouse visual pathway, i.e. 96% RGC axons projecting to the opposite hemisphere, the damage to the ipsilateral optic tract would cause more damage in the contralateral than the ipsilateral optic nerves. In line with our prediction, severe damage was seen in the contralateral but not ipsilateral optic nerves (Figure 5). Using the mouse visual system, we demonstrated a potential mechanism of axonal degeneration of the central nervous system in the brains of AD.Another important finding of this study is to demonstrate the feasibility of using noninvasive DTI imaging markers to evaluate axonal damage progress inAD. DTI is a powerfulMRI imagingmodal, which is very sensitive to detect changes of tissue microstructure in white matter. Studies have found changes of DTI in patients in association with cognitive impairment. DTI is a clinical available imaging modality. This finding has significant clinical impact as to use of DTI for diagnosis of AD and evaluation of the treatment.