Application of a novel microscopic technique for quantifying CA125 binding to circulating mononuclear cells in longitudinal specimens during treatment for ovarian cancer.

BACKGROUND: Measurement of serum CA125, an antigenic fragment of human mucin 16 (MUC16), is used to monitor the clinical progression of epithelial ovarian cancer (EOC). However, rather than simply a passive marker reflecting tumor burden, MUC16 may have a more active role by binding to immune cells and altering their tumor response. We developed a research tool to measure MUC16-binding to the surfaces of peripheral blood mononuclear cell (PBMC) subtypes and tested its research value using specimens collected serially from a woman being treated for high grade serous EOC. METHODS: Cryopreserved PBMCs were mixed with anti-CA125 antibody-labeled plasmonic gold nanoparticles (PNPs) to detect cell surface MUC16-binding along with fluorescent stains to identify B cells, NK cells, NK-T cells, T cells, and monocytes. From 3D darkfield images, a computer algorithm was applied to enumerate PNP-binding and fluorescence microscopy to identify cell lineage. Average MUC16-binding was determined by fitting a Poisson distribution to PNP-counts across similar cell types. MUC16-binding to cell types was correlated with treatment details, CA125 levels, and complete blood count (CBC) data. RESULTS: Over a 21-month period, monocytes had the highest level of MUC16-binding which was positively correlated with serum CA125 and inversely correlated with circulating monocyte and lymphocyte counts. Fluctuations of PNP-binding to NK cells were associated temporally with types of chemotherapy and surgical events. Levels of MUC16 bound to NK cells were positively correlated with levels of MUC16 bound to T and NK-T cells and inversely correlated with circulating platelets. CONCLUSIONS: Assessment of MUC16-binding among cryopreserved PBMC cell types can be accomplished using darkfield and fluorescence microscopy. Correlations observed between level of binding by cell type with serum CA125, CBC data, and treatment details suggest that the new techniques may offer novel insights into EOC's clinical course.

Multi-modal nonlinear optical and thermal imaging platform for label-free characterization of biological tissue.

The ability to characterize the combined structural, functional, and thermal properties of biophysically dynamic samples is needed to address critical questions related to tissue structure, physiological dynamics, and disease progression. Towards this, we have developed an imaging platform that enables multiple nonlinear imaging modalities to be combined with thermal imaging on a common sample. Here we demonstrate label-free multimodal imaging of live cells, excised tissues, and live rodent brain models. While potential applications of this technology are wide-ranging, we expect it to be especially useful in addressing biomedical research questions aimed at the biomolecular and biophysical properties of tissue and their physiology.

Characterization of Cell-Bound CA125 on Immune Cell Subtypes of Ovarian Cancer Patients Using a Novel Imaging Platform.

MUC16, a sialomucin that contains the ovarian cancer biomarker CA125, binds at low abundance to leucocytes via the immune receptor, Siglec-9. Conventional fluorescence-based imaging techniques lack the sensitivity to assess this low-abundance event, prompting us to develop a novel "digital" optical cytometry technique for qualitative and quantitative assessment of CA125 binding to peripheral blood mononuclear cells (PBMC). Plasmonic nanoparticle labeled detection antibody allows assessment of CA125 at the near-single molecule level when bound to specific immune cell lineages that are simultaneously identified using multiparameter fluorescence imaging. Image analysis and deep learning were used to quantify CA125 per each cell lineage. PBMC from treatment naïve ovarian cancer patients (N = 14) showed higher cell surface abundance of CA125 on the aggregate PBMC population as well as on NK (p = 0.013), T (p < 0.001) and B cells (p = 0.024) compared to circulating lymphocytes of healthy donors (N = 7). Differences in CA125 binding to monocytes or NK-T cells between the two cohorts were not significant. There was no correlation between the PBMC-bound and serum levels of CA125, suggesting that these two compartments are not in stoichiometric equilibrium. Understanding where and how subset-specific cell-bound surface CA125 takes place may provide guidance towards a new diagnostic biomarker in ovarian cancer.

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins.

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.

Are disease mortality rates and survival rates complementary? If not, can they be related? Which is more relevant?

The relationship of mortality to survival is complicated and this paper suggests the two rates should both be defined for each condition or illness in order better to appreciate the projected real impact of the illness in question on patients. And if available conventional mortality data are "old" or not yet available, the above derived formula offers an alternate method of determining the mortality rate, provided one knows or can learn the prevalence of the illness and already has the more often published survival data. Physician lead discussions with patients today regarding such issues of survival and mortality often lead to the consideration of whether to undertake increasingly more invasive and/or expensive and complicated early diagnostic studies and/or interventions. The concerned patients and their families are many times relatively medically unsophisticated and not unexpectedly fearful. Both states of mind render them less able to understand complicated and, often to them, abstruse sounding explanations of their options. They most often default to the recommendation of their physician(s) and rely ultimately on sound analysis of the risks and probabilities of the alternatives by their doctor(s) to craft a course of investigation and therapy for them that is most likely to result in their improved outcome based on his or her judgment and experience. They trust the physician to optimize a plan for them. Therefore, in light of misleading conclusions a physician might draw by considering changes in survival statistics alone, it is worthwhile that he or she also understand and factor in mortality data that would follow in the wake of pursuing a recommended strategy. It is offered that use of the above derived formula: M = (1-S)*P provides a relatively simple tool for retrieving this more defining mortality statistic that is otherwise frequently more difficult to locate and is often not included in published reports of promising new investigative or therapeutic strategies. I propose, having personally accumulated more than desired patient perspective, it is essential for South Carolina physicians charged with responsibility of advising their patients on major and difficult choices understand the importance of and difference between these two outcome rates derived from both diagnostic studies and therapeutic interventions. The significance of these ratios or rates to and for the patient, especially if they conflict, should be conveyed to patients at the appropriate conversational level based on the education and understanding of the patient and his/her family. By comprehending and explaining both these ratios in layman's terms appropriate to each individual patient, a clearer understanding of the merit or lack thereof of the available options for that individual can be attained.

A century of cardiology as reported in The Journal of the South Carolina Medical Association.

Through the years, numerous articles and symposium issues in The Journal have provided South Carolina physicians with up-to-date information on strategies and technologies for diagnosis and management of heart diseases. The Journal has also featured articles on the epidemiology and prevention of heart disease, and on two occasions published the proceedings of national workshops on heart disease. During this interval heart disease crested as the most frequent cause of death in South Carolina and indeed in the United States. However, in the last three years, due to reduced cigarette consumption and other preventive and therapeutic advances, heart disease mortality has declined faster than cancer related deaths. As a result, since 2002, malignancies now kill more Americans than does heart disease. This victory, of sorts, creates an optimistic beginning of the second century of The Journal of the South Carolina Medical Association.