Quantitative Biophotonics for Tissue Characterization and Function

肿瘤生物学的进展为针对恶性转化的特定分子机制失活的靶向治疗奠定了基础。该方法最突出的例子是使用单克隆抗体（mAb）曲妥珠单抗抗体-药物偶联物T-DM1治疗人表皮生长因子受体2 （HER2）阳性癌症。HER2表达升高与癌细胞增殖和存活增加有关，因此导致治疗结果不佳。鉴于单克隆抗体的疗效取决于其靶点在肿瘤细胞上的过表达，开发评估受体表达的技术对于监测治疗效果非常重要，从而使人们能够优化治疗。目前临床评价HER2表达是基于免疫组织化学（IHC）或荧光原位杂交（FISH）染色活检组织。这两种方法都是离体技术，由于肿瘤的异质性，可能会产生假阳性或阴性结果，很难用于监测治疗。另一方面，使用HER2特异性荧光标记探针的分子成像，可以实时评估体内HER2受体的状态和治疗后的状态。注射后，这种探针优先集中在感兴趣的部位（例如，肿瘤中），提供体内HER2表达的定量信息。在将一些广泛使用的HER2特异性药物（如曲妥珠单抗）递送到病变部位的同时，肿瘤细胞中的探针积累也可能发生。因此，可以在不干扰治疗本身的情况下监测HER2表达，使这种造影剂适合于成像和治疗范例。

Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for malignant transformation. The most prominent example of this approach is use of a monoclonal antibody (mAb) trastuzumab antibody-drug conjugate, T-DM1 for treatment of Human Epidermal Growth Factor Receptor 2 (HER2) positive cancers. Elevated HER2 expression is associated with increased proliferation and survival of cancer cells and, thereby, contributes to poor therapy outcomes. Given that the efficacy of the mAb depends on overexpression of its target on tumor cells, development of techniques to assess the receptor expression is extremely important for monitoring the efficacy of therapy, allowing one to optimize the treatment. Current clinical evaluation of HER2 expression is based on Immunohistochemistry (IHC) or Fluorescence in situ hybridization (FISH) staining of biopsied tissue. Both methodologies are ex vivo techniques and, due to tumor heterogeneity, may deliver false-positive or -negative results and can hardly be used to monitor therapy. On the other hand, molecular imaging, using HER2-specific fluorescently-labeled probes, allows assessing the status of HER2 receptors in vivo and following treatment in real time. After injection, such probes concentrate preferentially at the sites of interest (e.g., in the tumor), providing quantitative information on HER2 expression in vivo. Probe accumulation at the tumor cells can occur in parallel to the delivery of some widely used HER2 - specific drugs, such as, Trastuzumab, to the diseased site. As a result, HER2 expression can be monitored without interference with the treatment itself, making this contrast agent suitable for an image and treat paradigm. According to our compartmental kinetic model, the quantitative information about HER2 expression level can be extracted from the time dependence of fluorescence intensity, evaluated from the time series of images. Normalized rate of accumulation (NRA) of HER2 specific ligands in the tumor that takes into account the initial concentrations of the free probe ligands in blood is an indicator of the total concentration of HER2 receptors in the tumor. This assumption is supported by our observations that obtained values of NRA are proportional to overexpression of HER2 of the corresponding tumor type, as measured by ELISA and other methods. We have also showed that ABD-Affibody based probe, asymptotic values of fluorescence intensities after similar normalization are proportional to average HER2 expression for different types of breast carcinomas. However, for probes with much faster kinetics, as those based on monomer Affibody, the relationship between concentration of available HER2 receptors and asymptotic fluorescence intensities is not that straightforward, since a large fraction of receptors can be left unbound for the lack of free fluorescent ligands at longer times , where  is washout time. We found that for probes with fast washout time, such as HER2-specific Affibody-DyLight conjugates, fluorescence imaging based on repeated injections can provide quantitative assessment on HER2 expression level in HER2-positive carcinoma xenografts. This information is extracted from time series of fluorescent images, analyzed in the framework of kinetic model, developed to describe probe binding after each injection. The results show linear correlations between our reconstructed values for HER2 expression with HER2 expression measured by ELISA ex vivo assays in the same tumors. Additional information about the tumor characteristics can be extracted from the lifetime fluorescence measurements, including binding affinity of the probe to cancer cells and environmental conditions (e.g., pH) at a given site, based on local variations in the lifetimes for specially designed probes. We have continued our studies on in-vivo fluorescence lifetime imaging to monitor the efficacy of treatment. Our preliminary results indicate significant correlation between measured fluorescence lifetimes and the outcome of the treatment in the mouse model of human carcinoma. We started a new project for detection of basal cell carcinoma (BCC), the most common skin cancer in human in collaboration with dermatology department at Memorial Sloan Kettering Cancer Center. In the first phase, the affinity and sensitivity of targeted NIR fluorescence probe was tested in-vitro on cancer cell lines with high and no- expression of BerEP4, which is the most expressed biomarker in BCC. In collaboration with NICHD (Dr. Stratakis), we are recruiting Cushings patients for optical imaging before and after surgery. Preliminary results based on five patients with Cushing diagnostics were presented in Annual Meeting of Endocrinology (ENDO) 2013, San Francisco. Four of the 5 patients were surgically cured of disease with post-operative measurements of plasma cortisol. The early results by multispectral imaging (MSI) on the right cheek of the cured patients prior to surgery and a mean of 7 3 days after surgery showed a significant decrease in total blood volume when measured before and after surgery. In contrast, for the only patient in our sample, who was not surgically cured, the fraction of blood volume increased from pre to post-operative measurements. In this pilot study, preliminary data indicate that a decrease in facial plethora after surgery correlates with cure of Cushings. We are pursuing the Kaposi Sarcoma (KS) studies in ongoing clinical trials under four different NCI protocols and therapeutic agents. The goal is to further evaluate diffuse multispectral imaging as a potential supplement to existing response assessment in KS, providing an early non-invasive marker of treatment efficacy. In our preliminary results, multi-spectral images of Kaposi Sarcoma skin lesions were taken over the course of treatment, and blood volume and oxygenation concentration maps were obtained through Principal Component Analysis (PCA) of the data. Corresponding images were compared with clinical and pathological assessment, provided by conventional means. In agreement with our hypothesis that successful treatment would decrease the blood volume in the lesions, the normalized standard deviation for blood volume decreased in each of the 5 patients whose lesion responded to treatment, while the normalized standard deviation for blood volume increased in the 2 patients, whose lesion did not respond to therapy. These initial results confirm that concentrations of oxygenated hemoglobin in the tumor can become a quantitative marker of tumor response to the therapy. We have also continued our work on using polarization as a marker of structural changes. Polarization imaging allows one to separate contributions from surface specular reflection of the tissues and back scattered light from the deeper layers. As a result, hidden subsurface structures can be visualized to assess the transition of tissue from the healthy to diseased state. Correlation and filtering algorithms have been designed to improve image quality in the presence of noise and uncover regions of high statistical similarity, related, for example to the tissue collagen structure. To improve our previous setup, the polarization imaging system was upgraded to make possible automatic changes in focal length (stack of several co- and cross-polarized images at different focal planes). The system was tested on two healthy female volunteers over four weeks to observe the changes in the cervical structure at different time points of the menstrual cycle.