Cytokeratin-positive Cells Research Articles

Characterization of Gastrospheres Using 3D Coculture System.

To understand the molecular mechanisms involved in gastric disorders and regeneration, we need an in vitro tridimensional (3D) culture model, which can mimic the in vivo gastric microenvironment. A 3D coculture system named gastrosphere is proposed herein, composed of primary human gastric epithelial and stromal cells. The primary cultures were obtained from endoscopic gastric biopsies, and after mechanical and enzymatic dispersion, epithelial (HGE3) and stromal (HGS12) cells were expanded. After extensive immunocytochemical characterization, cells were seeded onto 96-well round bottom plates previously covered with 1% agarose. Cells were cultured in KM-F12 culture medium with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, in humidified air at 37°C and atmosphere containing 5% CO2 for 72h or until spheres formation. Then gastrospheres were carefully transferred to a rotary cell culture system (RCCS-4), and maintained for 07, 14, 21, and 28days. Gastrospheres were morphologically characterized by immunocytochemistry [cytokeratins (CK), vimentin, α-smooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and type IV collagen (CIV), proliferating cell nuclear antigen (PCNA)], and electron microscopy. In gastrospheres, the cytokeratin-positive epithelial cells were found in the outer layer, while vimentin-positive stromal cells were localized in the center of the gastrospheres. PCNA+ cells were mainly seen at the peripheral and in the intermediary region while nestin+ cells were also depicted in the latter zone. Scanning electron microscopy revealed groups of cohesive gastric cells at the periphery, while transmission electron microscopy demonstrated some differentiated mucous-like or zymogenic-like cells in the periphery and stromal structures located at the center of the 3D structures. Extracellular matrix was deposed between cells. Our data suggest that in vitro gastrospheres recapitulate the in vivo gastric microenvironment.

Lymphoepithelioma-like carcinoma of the skin

Primary cutaneous lymphoepithelioma-like carcinoma is a rare disease with low metastatic potential. Its morphologic and pathological features are similar to those of nasopharyngeal lymphoepithelial carcinoma. We report the case of a 60-year-old man with an infrapalpebral pearly papule, measuring 0.6 cm in diameter. The lesion was excised with a clinical hypothesis of basal cell carcinoma or squamous cell carcinoma. Histopathological analysis revealed a malignant neoplasm with syncytial arrangement of cells with vesicular nuclei, associated with dense lymphocytic infiltrate. Immunohistochemistry revealed cytokeratin-positive cells (AE1/AE3) and p63 protein, indicating epithelial histogenesis and squamous differentiation. A negative Epstein-Barr virus test result was achieved by immunohistochemistry. Primary lymphoepithelioma-like carcinoma of the skin is a differential diagnosis of lesions with prominent inflammatory infiltrates.

Prognostic significance of Cytokeratin 20-positive lymph node vascular endothelial growth factor A mRNA and chromodomain helicase DNA binding protein 4 in pN0 colorectal cancer patients.

BACKGROUNDCytokeratin 20-positive cells in lymph nodes from pN0 colorectal cancer (CRC) patients were detected previously by us. The aims of this study were to investigate which tumor metastasis-related genes were involved and their potential clinical significance.RESULTSFourteen of 84 (17%) genes were differentially expressed by at least 2-fold. Among them, 10 genes were up-regulated whereas 4 genes were down-regulated. Those differential expressed genes were validated in the second cohort of specimens. Follow-up analysis for 60 months showed that patients with lymph node vascular endothelial growth factor A (VEGF-A) mRNA and chromodomain helicase DNA binding protein 4 (CHD4) mRNA expression higher than the median copies had significantly shorter time to recurrence than those with lower than the median copies. Multivariate analysis showed that VEGF-A mRNA, CHD4 mRNA and lymphatic vessel involvement were independent prognostic factors for disease recurrence.CONCLUSIONSVEGF-A mRNA and CHD4 mRNA were up-regulated in CK20-positive pN0 lymph nodes and they may have prognostic significance in pN0 CRC patients.METHODSTwo cohorts of lymph node specimens from pN0 CRC patients of each with and without CK20-positive cells were recruited. In the first cohort, tumor metastasis genes were profiled using gene expression arrays. Differential expressed genes were validated in the second cohort. Moreover, their prognostic significance was examined by following-up the second cohort of patients with CK20-positive cells for 60 months and all histopathological findings were correlated to recurrence.

Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay.

- As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. - To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). - Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. - We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. - Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load.

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary-infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3-21days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r=0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin-positive cells in the milk from the infused udders (r=0.734, P<0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.

Abstract 3791: Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma

Abstract &amp;lt;Background&amp;gt; It has been reported that the examination of circulating tumor cells (CTCs) is beneficial for predicting tumor stage or treatment response. The epitherial cell adhesion molecule (EpCAM) and cytokeratin has been used as epithelial markers for the identification of CTCs. Since the epithelial-mesenchymal transition is one of malignant phenotype of cancer cells, it is necessary to investigate other markers which might detect mesenchymal CTCs. &amp;lt;Aim&amp;gt; This study aims to clarify the clinical significance of EpCAM negative and CEA positive CTCs in patients with gastric cancer. &amp;lt;Methods&amp;gt; Forty patients who underwent operation for gastric cancer were enrolled in this study. A total of 10ml peripheral blood samples were obtained preoperatively. Informed consent to participate in this study was obtained from all of the patients before their surgery. This study was approved by the Human Ethics Review Committee of our University. After collection of mononuclear cell fraction, these cells were stained by PAPI, anti-CD45, anti-EpCAM, anti-cytokeratin, and anti-CEA (CD66e) antibody. We enumerated the number of cells which showed EpCAM, cytokeratin, and CEA positive expression among CD45 negative cells. ROC analyses were performed for investigating predictive value of these cell counts for distant metastasis. We examined association between these cell counts and clinicopathological features. &amp;lt;Results&amp;gt; ROC analyses revealed that the AUC for predicting distant metastasis using EpCAM, Cytokeratin, and CEA positive cell counts were 0.583, 0.638, and 0.769, respectively. The median number of EpCAM negative and CEA positive cells was 231 (range: 0-61897). The cell counts of EpCAM negative and CEA positive cells were significantly associated with Tumor depth (p=0.0084), lymph node metastasis (p=0.0107), and tumor size (p=0.023). &amp;lt;Conclusions&amp;gt; CEA might be useful marker for detecting high risk of recurrence in patients with gastric cancer. Citation Format: Yuichiro Miki, Masakazu Yashiro, Tomohisa Okuno, Kishu Kitayama, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2017-3791

Cellular Expression of PD-L1 in the Peripheral Blood of Lung Cancer Patients is Associated with Worse Survival.

Background: Lung cancer treatment has become increasingly dependent upon invasive biopsies to profile tumors for personalized therapy. Recently, tumor expression of programmed death-ligand 1 (PD-L1) has gained interest as a potential predictor of response to immunotherapy. Circulating biomarkers present an opportunity for tumor profiling without the risks of invasive procedures. We characterized PD-L1 expression within populations of nucleated cells in the peripheral blood of lung cancer patients in hopes of expanding the role of liquid biopsy in this setting.Methods: Peripheral blood samples from a multi-institutional prospective study of patients with clinical diagnosis of lung cancer were subjected to cytomorphometric and immunohistochemical evaluation using single-cell, automated slide-based, digital pathology. PD-L1 expression was determined by immunofluorescence.Results: PD-L1 expression was detected within peripheral circulating cells associated with malignancy (CCAM) in 26 of 112 (23%) non-small cell lung cancer patients. Two distinct populations of nucleated, nonhematolymphoid, PD-L1-expressing cells were identified; cytokeratin positive (CK+, PD-L1+, CD45-) and cytokeratin negative (CK-, PD-L1+, CD45-) cells, both with cytomorphometric features (size, nuclear-to-cytoplasm ratio) consistent with tumor cells. Patients with >1.1 PD-L1(+) cell/mL (n = 14/112) experienced worse overall survival than patients with ≤1.1 PD-L1(+) cell/mL (2-year OS: 31.2% vs. 78.8%, P = 0.00159). In a Cox model adjusting for stage, high PD-L1(+) cell burden remained a significant predictor of mortality (HR = 3.85; 95% confidence interval, 1.64-9.09; P = 0.002).Conclusions: PD-L1 expression is detectable in two distinct cell populations in the peripheral blood of lung cancer patients and is associated with worse survival.Impact: These findings could represent a step forward in the development of minimally invasive liquid biopsies for the profiling of tumors. Cancer Epidemiol Biomarkers Prev; 26(7); 1139-45. ©2017 AACR.

Abstract 1635: Multiplexed analysis of fixed tumor tissues using imaging mass cytometry

Abstract Introduction: Comprehensive evaluation of the tumor microenvironment with preservation of spatial context requires simultaneous in situ detection of multiple targets. Current fluorescence-based methods can accommodate up to 5-6 distinct markers and can be applied to formalin fixed, paraffin embedded tissue (FFPE). Imaging mass cytometry uses metal-conjugated antibodies and mass spectrometry to perform highly multiparametric, quantitative measurement of protein targets within tissue sections providing unprecedented detail of quantitative tissue labeling. To date, the IMC technology has not been widely available and its performance has not yet been compared with other validated methods. Methods: Using multiplexed quantitative immunofluorescence (QIF, AQUA/Genoptix) and imaging mass cytometry (IMC, Fluidigm) we validated and optimized metal-conjugated assays to detect HER2 (29D8-CST, 176Yb), CD3 (DAKO, 170Er), Ki-67 (B56-BD biosciences, 168Er), Histone H3 (D1H2-CST, 176Yb), pancytokeratin (C11-Biorad, 162Dy), Vimentin (RV202-BD Pharmingen, 156Gd), LipoR (Fluidigm, 115In) and DNA/nuclei (DNA intercalator, Fluidigm, 191/193Ir). The markers were applied to FFPE samples from tumor cell lines, human tonsil, lymph node; and breast and lung carcinomas represented in tissue microarray format. Data was visualized using MCD viewer and ImageJ software and results from QIF and IMC were compared. Results: Metal conjugation did not affect the performance of the primary antibodies studied here. The staining patterns of the epithelial/tumor and stromal compartments evaluated using the IMC platform and QIF were comparable. HER2 signal was higher in tumors and cell lines harboring HER2 amplification by FISH/IHC. The CD3 signal was higher in T-cell lymphoma cells and in the inter-follicular areas of human tonsil and lymph nodes. Ki-67 signal was nuclear, co-localized with Histone H3 and showed positivity in lymphoid germinal centers, cytokeratin-positive carcinoma cells and CD3+ tumor infiltrating lymphocytes. Results obtained using the IMC at different time points in serial tissue sections were comparable (P&amp;lt;0.05). Conclusion: Quantitative and reproducible measurement of immune and non-immune targets with spatial resolution using the IMC platform is feasible in FFPE breast and lung carcinomas and the results are comparable to multiplexed QIF. Expansion of the current panel to include &amp;gt;30 markers for immune cell phenotypes/function and tumor markers is ongoing. Citation Format: Franz Villarroel-Espindola, Daniel Carvajal-Hausdorf, Ila Datar, Amanda Esch, Narges Rashidi, Ala Nassar, Shelly Ren, Ruth R. Montgomery, Roy S. Herbst, David L. Rimm, Kurt A. Schalper. Multiplexed analysis of fixed tumor tissues using imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1635. doi:10.1158/1538-7445.AM2017-1635

Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients.

Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48–85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.

Morphological and immunohistochemical characterization of spontaneous endometriosis in rhesus macaques (Macaca mulatta).

Several cases of spontaneous endometriosis in middle-aged to old rhesus macaques (Macaca mulatta) from the breeding colony of the German Primate Center were thoroughly characterized with regards to anatomical distribution and macroscopic appearance, histological differentiation and immunohistochemical profile including somatic markers, hormonal receptors, and proliferation indices. More than half of the examined animals (five of nine) were directly related to one breeding male, supporting a strong genetic predisposition. Histologically, four different types of endometriotic lesions, depending on the degree of ectopic endometrial gland and stromal differentiation (well differentiated, purely stromal, mixed differentiation, poorly differentiated), could be constantly identified within all animals. Immunohistochemistry (IHC) of cytokeratin (CK), vimentin, smooth muscle actin (SMA), desmin, estrogen (ER), and progesterone (PR) receptors as well as of the nuclear proteins Ki67 and p53 revealed varying staining patterns in the four different types of endometriosis differentiation and compared to normal endometrium. Purely stromal, mixed, or poorly differentiated lesions, especially, showed additional cytokeratin-positive stromal cells, whereas epithelial cells of endometriosis with mixed or poor differentiation increasingly expressed mesenchymal markers (vimentin, SMA). Hormonal receptor and Ki67 expression in well-differentiated endometriotic lesions mostly reflected that of normal endometrial tissue according to the cyclic phase of the animal, while the expression gradually diminished with decreasing grade of differentiation. However, increased nuclear accumulations of p53 antigen could only be continuously detected in epithelial cells of mixed or poorly differentiated endometriosis. Altogether, these findings support the pathogenetic theory of coelomic metaplasia, since the expression profiles of somatic markers in less differentiated forms closely resembled that of mesothelial cells. Thus, the four different histological types of endometriosis might display subsequent grades of differentiation in the course of time, with poorly differentiated types representing newly formed, immature lesions and well-differentiated types being older, fully differentiated forms, rather than being the outcome of dedifferentiation processes.

Cytokeratin 19 promoter directs the expression of Cre recombinase in various epithelia of transgenic mice

Cytokeratin 19 (K19) is expressed in various differentiated cells, including gastric, intestinal and bronchial epithelial cells, and liver duct cells. Here, we generated a transgenic mouse line, K19-Cre, in which the expression of Cre recombinase was controlled by the promoter of K19. To test the tissue distribution and excision activity of Cre recombinase, K19-Cre transgenic mice were bred with Rosa26 reporter strain and a mouse strain that carries PTEN conditional alleles (PTENLoxp/Loxp). At mRNA level, Cre was strongly expressed in the stomach, lung and intestine, while in stomach, lung, and liver at protein level. The immunoreactivity to Cre was strongly observed the cytoplasm of gastric, bronchial and intestinal epithelial cells. Cre activity was detectable in gastric, bronchial and intestinal epithelial cells, according to LacZ staining. In K19-Cre/PTEN Loxp/Loxp mice, PTEN was abrogated in stomach, intestine, lung, liver and breast, the former two of which were verified by in situ PCR. There appeared breast cancer with PTEN loss. These data suggest that K19 promoter may be a useful tool to study the pathophysiological functions of cytokeratin 19-positive cells, especially gastrointestinal epithelial cells. Cell specificity of neoplasia is not completely attributable to the cell-specific expression of oncogenes and cell-specific loss of tumor suppressor genes.

Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition.

Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR.

Effects of an immunosuppressive treatment on the rat prostate.

The aim of this study was to determine the influence of different combinations of immunosuppressive drugs on the morphology, ultrastructure, and expression of proliferating cell nuclear antigen and cytoskeleton proteins in the rat dorsolateral prostate. The studies were conducted on 48 male Wistar rats. The animals were divided into eight groups: a control group and seven experimental groups. For 6 months, the animals in the experimental groups were administered a combination of drugs including rapamycin (Rapa), cyclosporin A, tacrolimus (Tac), mycophenolate mofetil, and prednisone (Pred), according to the standard three-drug regimens for immunosuppressive therapy used in clinical practice. An evaluation of the morphology and ultrastructure was conducted, and a quantitative evaluation of the expression of proliferating cell nuclear antigen and desmin- and cytokeratin-positive cells with weak, moderate, and strong expression was performed. The combination of Rapa, Tac, and Pred caused the smallest morphological and ultrastructural changes in the rat prostate cells. In the case of rats whose treatment was switched to Rapa monotherapy, a decreased percentage of proliferating cells of both the glandular epithelium and the stroma was found. Decreases in body weight and changes in the expression of cytokeratin and desmin were observed in all the experimental rats. The combination of Rapa, Tac, and Pred would seem to be the most beneficial for patients who do not suffer from prostate diseases. Our results justify the use of inhibitors of the mammalian target of Rapa in the treatment of patients with prostate cancer. The changes in the expression of cytoskeleton proteins may be the result of direct adverse effects of the immunosuppressive drugs, which are studied in this article, on the structure and organization of intermediate filament proteins.

Sentinel lymph node detection rates using indocyanine green in women with early-stage cervical cancer

ObjectiveOur study objective was to determine feasibility and mapping rates using indocyanine green (ICG) for sentinel lymph node (SLN) mapping in early-stage cervical cancer. MethodsWe performed a retrospective review of all women who underwent SLN mapping with ICG during primary surgical management of early-stage cervical cancer by robotic-assisted radical hysterectomy (RA-RH) or fertility-sparing surgery. Patients were treated at two high-volume centers from 10/2012 to 02/2016. Completion pelvic lymphadenectomy was performed after SLN biopsy; additionally, removal of clinically enlarged/suspicious nodes was part of the SLN treatment algorithm. ResultsThirty women with a median age of 42.5 and BMI of 26.5 were included. Most (90%) had stage IB disease, and 67% had squamous histology. RA-RH was performed in 86.7% of cases. One patient underwent fertility-sparing surgery. Median cervical tumor size was 2.0cm. At least one SLN was detected in all cases (100%), with bilateral mapping achieved in 87%. SLN detection was not impacted by tumor size and was most commonly identified in the hypogastric (40.3%), obturator (26.0%), and external iliac (20.8%) regions. Five cases of lymphatic metastasis were identified (16.7%): three in clinically enlarged SLNs, one in a clinically enlarged non-SLN, and one case with cytokeratin positive cells in an SLN. All metastatic disease would have been detected even if full lymphadenectomy had been omitted from our treatment algorithm, ConclusionsSLN mapping with ICG is feasible and results in high detection rates in women with early-stage cervical cancer. Prospective studies are needed to determine if SLN mapping can replace lymphadenectomy in this setting.

Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer.

BackgroundTumor-infiltrating lymphocyte (TIL) count in breast cancer carries prognostic information and represents a potential predictive marker for emerging immunotherapies. However, the distribution of the lymphocyte subpopulations is not well defined. The goals of this study were to examine intratumor heterogeneity in TIL subpopulation counts in different fields of view (FOV) within each section, in different sections from the same biopsy, and between biopsies from different regions of the same cancer using quantitative immunofluorescence (QIF).MethodsWe used multiplexed QIF to quantify cytokeratin-positive epithelial cells, and CD3-positive, CD8-positive and CD20-positive lymphocytes in tissue sections from multiple biopsies obtained from different areas of 31 surgically resected primary breast carcinomas (93 samples total). Log2-transformed QIF scores or concordance and variance component analyses with linear mixed-effects models were used. Cohen’s kappa index [k] of high versus low scores, defined as above and below the median, was used to measure sample similarity between areas.ResultsWe found a strong positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC] = 0.827). CD3 and CD8 showed a weaker but significant association with CD20 (CC = 0.446 and 0.363, respectively). For each marker, the variation between different FOVs in the same section was higher than the variation between sections or between biopsies of the same cancer. The intraclass correlation coefficients (ICC) were 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In component analysis, 66–69 % of the variance was attributable to differences between FOVs in the same section and 30–33 % was due to differences between biopsies from different areas of the same cancer. Section to section differences were negligible. Concordance for low versus high marker status assignment in single biopsies compared to all three biopsies combined yielded k = 0.705 for CD3, k = 0.655 for CD8, and k = 0.603 for CD20.ConclusionsT and B lymphocytes show more heterogeneity across the dimensions of a single section than between different sections or regions of a given breast tumor. This observation suggests that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0737-x) contains supplementary material, which is available to authorized users.

Abstract 4990: Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy

Abstract Background: PD-L1 expression can be induced by radiotherapy (RT) and may be an immune escape mechanism after RT, and has the potential to predict for responsiveness to immune checkpoint blockade immunotherapy. However, repetitive biopsies for monitoring PD-L1 expression in tumor and/or stroma are not feasible. Sequential assessment of PD-L1 expression on cancer associated circulating cells during treatment may be a way to measure the efficacy of combining RT and immunotherapy. RAD50 is a DNA repair gene that can be used in tracking tumor cell response to radiation. We therefore evaluated PD-L1 expression and RAD50 induction in CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during RT to track expression changes of these markers. Methods: Twenty-nine pts with stage I-IV lung cancer were included in this prospective pilot study. Four pts received radiation therapy for stage I disease and 25 other pts received chemoradiation for stage II-IV disease. A baseline blood sample (7.5 ml) was drawn prior to the start of RT (T0), and a second blood sample was drawn at a treatment visit during RT (T1) for a total of 58 samples. Blood was processed using CellSieve™ microfiltration (Creatv MicroTech), stained for cytokeratin 8, 18 &amp; 19 and CD45, and imaged. Using the QUASR (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci were quantified within nuclear regions and PD-L1 pixel intensity measured and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+). Results: There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in all but 2 samples (97%). CTCs were found in 69% of T0 and 72% of T1 samples, and CAMLs in 79% of T0 and 97% of T1 samples. PD-L1 expression ranged from 34-2552 pixel intensity, with an average of 285 at T0 and 525 at T1 (p = 0.07). Interestingly, 9 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 12 pts with low/no PD-L1 expression remained low at T1, and 8 pts had high PD-L1 expression that remained high or decreased at T1. RAD50 foci ranged from 0-16 per cell, with an average of 0.56 at T0 that increased to 4.68 at T1 (p&amp;lt;0.001) during radiotherapy. There was no correlation between RAD50 induction and PD-L1 expression. Conclusions PD-L1 expression and RAD 50 foci were quantifiable in both CTCs and CAMLs, and a correlative response to radiotherapy +/- chemotherapy was quantified. This data suggests that sequential tracking of CTCs and immune-related cells from the primary lung tumor is feasible using microfiltration and may potentially serve as a predictive biomarker for cancer therapy sensitivity. Citation Format: Daniel L. Adams, Martin J. Edelman, Penny Fang, Wen Jiang, Jianzhong He, Ting Xu, Hui Gao, James M. Reuben, Yawei Qiao, Steven Hahn, Ritsuko Komaki, Zhongxing Liao, Cha-Mei Tang, Steven H. Lin. Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4990.

Abstract 2244: Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients

Abstract Background and Purpose: Circulating tumor cells (CTCs) are relatively rare cells defined as tumor cells circulating in the peripheral blood of patients with solid tumors. Diagnosis utilizing CTCs is expected to help guide decision-making for precision cancer medicine. We developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Here we evaluated its performance using preclinical spike-in model and blood samples from non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients. Material and method: The automated MCA system consists of components such as chambered cartridge containing micro metal filter, reagent and waste reservoirs, and peristaltic pump. To evaluate the recovery of CTCs, preclinical experiments using NSCLC cells, NCI-H820, A549, NCI-H441 and NCI-H23 spiked into peripheral whole blood from healthy volunteers were performed. For clinical evaluation, 6 mL of peripheral whole blood was collected from 50 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Samples were collected in an EDTA-containing tube and were processed within 3 hours of blood draw. Recovered cells on the filter were then fixed, permeabilized, and stained automatically and high-resolution fluorescent images were obtained using fluorescence microscope. We defined CTC as DAPI-positive, cytokeratin-positive and CD45-negative cell. Results: Results of the preclinical study showed that up to 90% of spiked-in tumor cells were recovered, confirming that the detection sensitivity by this automated device is on par with that by previous manual detection procedure. Demographics of 50 lung cancer patients enrolled in clinical study were as follows: median age 72 (range, 48 to 85); male 66%; stage III/IV 12/88%; NSCLC/SCLC 78/22%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 / 6 mL, respectively. Three or more CTCs were detected in 71% of patients with advanced lung cancer (39 out of 50) and five or more CTCs were detected in 52% of patients (26 out of 50) (median CTC count 13.5). Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those with intrathoracic metasitasis (median CTC count, 8 versus 4, p = 0.058). A head-to-head comparison between CellSearch system and our system was conducted in NSCLC patients, showing the superiority of our system (median CTC count, 0 versus 11.25, p = 0.0001, n = 17). Conclusions Our results suggest that the automated MCA device has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This device also enables higher throughput owing to its automated procedure. Further clinical evaluation including the detection of PD-L1 expression will be performed in an expansion cohort. Citation Format: Satomi Yagi, Yasuhiro Koh, Hiroaki Akamatsu, Woong Kim, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2244.

Metastatic Malignant Melanoma With Complete Loss of Differentiation Markers (Undifferentiated/Dedifferentiated Melanoma)

Metastatic malignant melanoma is notorious for its phenotypic diversity and loss of differentiation markers. We herein summarized our experience with 14 metastatic melanomas showing complete loss of immunohistochemical melanocytic markers (with or without heterologous differentiation). Patients included 11 men and 3 women aged 24 to 78 years (median, 67 y). Thirteen patients had histologically confirmed primary skin melanoma, and 1 had metastatic melanoma of unknown primary. Undifferentiated metastasis was diagnosed synchronous to primary tumor (n=1), following skin melanoma by 3 months to 9 years (n=11) and preceding it by 1 year (n=1). Sites of undifferentiated metastases were axillary (3), inguinal (1), or submandibular (1) lymph nodes, digestive tract (2), bone/soft tissue (2), lung/pleura (2), and disseminated (n=3). Histology of metastases mimicked undifferentiated pleomorphic or spindle cell sarcoma with variable myxoid and giant cell areas (n=10) and cytokeratin-positive undifferentiated small cell sarcoma (n=1). Three cases showed heterologous dedifferentiation: pleomorphic rhabdomyosarcoma (n=1), teratocarcinosarcoma-like with prominent rhabdomyoblasts (n=1), and adenocarcinoma-like with metaplastic bone (n=1). All cases were negative for S100, melanoma cocktail, HMB45, Melan A, and SOX10. Other markers showed following results: smooth muscle actin (1/14), p16 (1/14), TP53 (2/12), pancytokeratin (4/14), desmin (5/14), h-caldesmon (0/9), and MDM2/CDK4 (0/5). SMARCB1 was intact in 8/8 cases. Genotyping showed BRAF(V600E) mutation (5/14), NRAS mutation (5/14), and BRAF/NRAS wild-type (4/14). In conclusion, undifferentiated/dedifferentiated metastatic melanoma is likely underrecognized and frequently mistaken for undifferentiated sarcoma or other neoplasms. Diagnosis of undifferentiated sarcoma at sites where melanoma metastasis are frequent (eg, inguinal and axillary region) should be made with great caution and warrants exploration of the remote history. Genotyping is a helpful surrogate marker in classifying such difficult cases. In the light of available targeted therapies, recognition of undifferentiated/dedifferentiated metastatic melanoma is mandatory for appropriate treatment.

Transdifferentiation of mouse visceral yolk sac cells into parietal yolk sac cells in vitro

The mouse embryonic yolk sac is an extraembryonic membrane that consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and functions in hematopoietic-circulation in the fetal stage. The present study was undertaken to examine the normal development of both murine VYS and PYS tissues using various molecular markers, and to establish a novel VYS cell culture system in vitro for analyzing differentiation potentials of VYS cells. RT-PCR and immunohistochemical analyses of gene expression in VYS and PYS tissues during development revealed several useful markers for their identification: HNF1β, HNF4α, Cdh1 (E-cadherin), Krt8 and Krt18 for VYS epithelial cells, and Stra6, Snail1, Thbd and vimentin for PYS cells. PYS cells exhibited mesenchymal characteristics in gene expression and morphology. When VYS cells at 11.5 days of gestation were cultured in vitro for 7 days, the number of HNF1β-, HNF4α-, E-cadherin- and cytokeratin-positive VYS epithelial cells was significantly reduced and, instead, Stra6-and vimentin-positive PYS-like cells increased with culture. RT-PCR analyses also demonstrated that gene expression of VYS markers decreased, whereas that of PYS markers increased in the primary culture of VYS cells. These data indicate that VYS epithelial cells rapidly transdifferentiate into PYS cells having mesenchymal characteristics in vitro, which may provide a culture system suitable for studying molecular mechanisms of VYS transdifferentiation into PYS cells and also epithelial-mesenchymal transition.

Ex Vivo Stromal Cell-Derived Factor 1-Mediated Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Hepatocytes Is Enhanced by Chinese Medicine Yiguanjian Drug-Containing Serum.

Yiguanjian is administered in traditional Chinese medicine for liver diseases and has been demonstrated to reduce liver fibrosis. This study investigated the effect of Yiguanjian drug-containing serum (YGJ) with Stromal Cell-Derived Factor 1 (SDF-1) and Hepatocyte Growth Factor (HGF) on the differentiation of murine bone-marrow-derived mesenchymal cells (BM-MSCs) into hepatocytes in vitro. Adherent MSCs were isolated from murine bone marrow. Differentiation was induced by 20 ng/mL HGF, 50 ng/mL SDF-1, and 20% Yiguanjian drug-containing serum for 7 to 28 days, and mature hepatocytes' marker albumin (ALB) and cholangiocytes' marker cytokeratin-18 (CK-18) were assessed by immunocytochemistry and western blot. BM-MSCs exhibited homogeneous spindle shape growth after subculture and stained positive for CD90 and negative for CD34. After induction with HGF + normal serum or YGJ for 14 days, HGF + SDF-1 + normal serum for 7 days, or HGF + SDF-1 + YGJ for 5 days, MSCs' morphology changed gradually and begun to resemble hepatocyte-like cells. Cultures supplemented with HGF + SDF-1 + YGJ contained significantly higher proportions of ALB and CK-18 positive cells than cultures supplemented with HGF + SDF-1 + normal serum at day 7. These observations corroborated the results of western blot. In conclusion, Yiguanjian drug-containing serum could facilitate the differentiation of murine BM-MSCs into hepatocytes in vitro and has a synergistic effect with SDF-1 and HGF.