Synaptophysin-Ki67 double stain: a novel technique that improves interobserver agreement in the grading of well-differentiated gastrointestinal neuroendocrine tumors.

A common problem in the assessment of Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors is distinguishing tumor from non-tumor. This is because background stromal lymphocytes, entrapped non-neoplastic glands, and the delicate vascular network characteristic of neuroendocrine tumors frequently contain a subset of proliferating cells. Furthermore, in small biopsies, crush and cautery artifact can alter the morphologic appearance of tumor cells, making the Ki67 proliferative index more difficult to assess. To address these issues, we developed a synaptophysin-Ki67 double stain using a commercially available immunohistochemistry kit, allowing simultaneous visualization of tumor and proliferating nuclei. To test this method, three gastrointestinal pathologists individually graded 50 gastrointestinal neuroendocrine tumors first using synaptophysin-Ki67 double-stained slides and then, after a washout period, using Ki67-only stained slides (along with routine hematoxylin- and eosin-stained slides). Interobserver agreement on Ki67 proliferative index was moderate using the Ki67-only stained slides (intraclass correlation 0.51, 95% confidence interval: 0.35-0.66) and improved using the synaptophysin-Ki67 double stain (intraclass correlation 0.79, 95% confidence interval: 0.69-0.86). Similarly, interobserver agreement on tumor grade was fair with Ki67-only stained slides (κ=0.39, P<0.001) and improved with the double stain (κ=0.58, P<0.001). Analysis of individual pathologists' scores revealed that fewer total number of tumor cells counted correlated with higher grade designation and appeared to contribute to grade discordance. In tumors cited as particularly challenging to assess by the pathologists, three of four tumors were grade discordant with the Ki67-only stain, whereas all four tumors were grade concordant with the synaptophysin-Ki67 stain. The synaptophysin-Ki67 double stain is the first technique to address specifically the histomorphologic challenges of evaluating Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors. Although further validation is needed, this study provides evidence that the synaptophysin-Ki67 double stain can improve interobserver agreement

Autofluorescence lifetime augmented reality as a means for real-time robotic surgery guidance in human patients.

Due to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols

XPO1 inhibition by selinexor induces potent cytotoxicity against high grade bladder malignancies.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies

Autofluorescence lifetime augmented reality as a means for real-time robotic surgery guidance in human patients

Due to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols

Expression of interleukin 6 (IL-6) correlates with oestrogen receptor in human breast carcinoma

Multifunctional cytokines play important and only partially defined roles in mammary tumour development and progression. Normal human mammary epithelial cells constitutively produce interleukin 6 (IL-6), IL-8 and a non-secreted form of tumour necrosis factor. Transformation of mammary epithelial cells by different oncogenes is frequently associated with alterations of cytokine/growth factor production and responsiveness. In the present study we analysed the expression of IL-6 in 149 cases of invasive breast carcinoma and the data have been correlated with clinico-pathological variables including tumour size, histological grade, nodal status, and oestrogen and progesterone receptors, Ki67 and p53, protein expression. Though the majority of breast carcinomas expressed at least low levels of immunoreactive IL-6, we found that expression of this cytokine was inversely associated with histological tumour grade (P = 0.0017), but not with tumour size and nodal status. Ki67 positivity was inversely correlated with IL-6 expression (P = 0.027). Among biological parameters analysed, a direct association was found between the percentage of IL-6-positive cells and that of oestrogen (P = 0.00005) and progesterone (P = 0.025) receptor-positive cells. No correlation was observed between IL-6 and p53 protein expression. These data indicate that down regulation of IL-6 is associated with highly malignant mammary carcinomas. It will be of interest to evaluate whether alterations of cytokines that are constitutively produced by mammary cells are also associated with high-grade tumours

Deep Functional and Molecular Characterization of a High-Risk Undifferentiated Pleomorphic Sarcoma.

Nonrhabdomyosarcoma soft-tissue sarcomas (STSs) are a class of 50+ cancers arising in muscle and soft tissues of children, adolescents, and adults. Rarity of each subtype often precludes subtype-specific preclinical research, leaving many STS patients with limited treatment options should frontline therapy be insufficient. When clinical options are exhausted, personalized therapy assignment approaches may help direct patient care. Here, we report the results of an adult female STS patient with relapsed undifferentiated pleomorphic sarcoma (UPS) who self-drove exploration of a wide array of personalized Clinical Laboratory Improvement Amendments (CLIAs) level and research-level diagnostics, including state of the art genomic, proteomic

Lin28 Promotes Growth of Prostate Cancer Cells and Activates the Androgen Receptor

Prostate cancer (CaP) progresses to a castration-resistant state assisted by multifold molecular changes, most of which involve activation of the androgen receptor (AR). Having previously demonstrated the importance of the Lin28/let-7/Myc axis in CaP, we tested the hypothesis that Lin28 is overexpressed in CaP and that it activates AR and promotes growth of CaP cells. We analyzed human clinical CaP samples for the expression of Lin28 by quantitative real-time RT-PCR, Western blot analysis, and IHC. Growth characteristics of CaP cell lines transiently and stably expressing Lin28 were examined. The clonogenic ability of CaP cells expressing Lin28 was determined by colony formation and soft agar assays. Increase in expression of AR and subsequent increase in transcription of AR-target genes were analyzed by quantitative real-time RT-PCR, luciferase assays, and ELISA. LNCaP cells stably expressing Lin28 were injected into nude mice, and tumorigenesis was monitored. We found that Lin28 is overexpressed in clinical CaP compared to benign prostates. Overexpression of Lin28 enhanced, while down-regulation reduced, growth of CaP cells. Lin28 enhanced the ability of CaP cells to form colonies in anchorage-dependent and anchorage-independent conditions. LNCaP cells stably expressing Lin28 exhibited significantly higher tumorigenic ability in vivo. Lin28 induced expression of the AR and its target genes such as PSA and NKX3.1. Collectively, our findings demonstrate a novel role for Lin28 in CaP development and activation of the AR axis

Lin28 Promotes Growth of Prostate Cancer Cells and Activates the Androgen Receptor

Prostate cancer (CaP) progresses to a castration-resistant state assisted by multifold molecular changes, most of which involve activation of the androgen receptor (AR). Having previously demonstrated the importance of the Lin28/let-7/Myc axis in CaP, we tested the hypothesis that Lin28 is overexpressed in CaP and that it activates AR and promotes growth of CaP cells. We analyzed human clinical CaP samples for the expression of Lin28 by quantitative real-time RT-PCR, Western blot analysis, and IHC. Growth characteristics of CaP cell lines transiently and stably expressing Lin28 were examined. The clonogenic ability of CaP cells expressing Lin28 was determined by colony formation and soft agar assays. Increase in expression of AR and subsequent increase in transcription of AR-target genes were analyzed by quantitative real-time RT-PCR, luciferase assays, and ELISA. LNCaP cells stably expressing Lin28 were injected into nude mice, and tumorigenesis was monitored. We found that Lin28 is overexpressed in clinical CaP compared to benign prostates. Overexpression of Lin28 enhanced, while down-regulation reduced, growth of CaP cells. Lin28 enhanced the ability of CaP cells to form colonies in anchorage-dependent and anchorage-independent conditions. LNCaP cells stably expressing Lin28 exhibited significantly higher tumorigenic ability in vivo. Lin28 induced expression of the AR and its target genes such as PSA and NKX3.1. Collectively, our findings demonstrate a novel role for Lin28 in CaP development and activation of the AR axis