Expression Of Specific Biomarkers Research Articles

Endothelial Progenitor Cells: A Brief Update.

An enormous amount of current data has suggested involvement of endothelial progenitor cells (EPCs) in neovasculogenesis in both human and animal models. EPC level is an indicator of possible cardiovascular risk such as Alzheimer disease. EPC therapeutics requires its identification, isolation, differentiation and thus expansion. We approach here the peculiar techniques through current and previous reports available to find the most plausible and fast way of their expansion to be used in therapeutics. We discuss here the techniques for EPCs isolation from different resources like bone marrow and peripheral blood circulation. EPCs have been isolated by methods which used fibronectin plating and addition of various growth factors to culture media. Particularly, the investigations which tried to enhance EPC differentiation while inducing with growth factors and endothelial nitric oxide synthase are shared. We also include the cryopreservation and other storage methods of EPCs for a longer time. Sufficient amount of EPCs are required in transplantation and other therapeutics which signifies their in vitro expansion. We highlight the role of EPCs in transplantation which improved neurogenesis in animal models of ischemic stroke and human with acute cerebral infarct in the brain. Accumulatively, these data suggest the exhilarating route for enhancing EPC number to make their use in the clinic. Finally, we identify the expression of specific biomarkers in EPCs under the influence of growth factors. This review provides a brief overview of factors involved in EPC expansion and transplantation and raises interesting questions at every stage with constructive suggestions.

Prognostic Significance of Proliferative Markers: Cyclin D1 and CENPF in Oral Squamous Cell Carcinoma Patients-A Cohort Study.

Oral cancer ranks sixth place worldwide among other cancers and is known for its high morbidity and mortality rates. Among oral cancer, oral squamous cell carcinomas (OSCC) remains the most common. Expression of specific biomarkers are known to be related to the prognosis of the OSCC. An immunohistochemistry study was performed to evaluate the expression of Cyclin D1 and CENPF for their prognostic significance in twenty OSCC patients. The follow-up period ranged from 10 to 18months. The expression of these proliferative markers was correlated with demographic, clinical and histopathological grade of the disease. The results revealed 28% mortality rates in relation to OSCC involving retro-molar trigone. Based on the Cyclin D1 and CENPF expression, high mortality rate (Pearson coefficient = 0.800) was seen with grade 3 and 4 expressions of the markers. Decreased survival rates (Pearson coefficient = 0.824) were observed with stage III and IV disease with grade 3 and 4 expressions of the markers. Cyclin D1 and CENPF have shown promising results as proliferative markers which can be linked with prognosis of OSCC in this study. Further studies with larger sample size are needed to extrapolate our findings.

CD146‐targeted nuclear medicine imaging in cancer: State of the art

AbstractThe transmembrane glycoprotein adhesion molecule CD146 is overexpressed in a wide variety of cancers. CD146 expression constitutes a marker of poor prognosis and correlates with cancer progression. Through molecular imaging, a specific biomarker's expression and distribution can be viewed in vivo non‐invasively. Radionuclide‐labeled monoclonal antibodies or relevant fragments that target CD146 may find potential applications in cancer imaging, thereby offering tremendous value in cancer diagnosis, staging, prognosis evaluation, and prediction of drug resistance. This review discusses the recent developments of CD146‐targeted molecular imaging via nuclear medicine, especially in malignant melanoma, brain tumor, lung cancer, liver cancer, breast cancer, and pancreatic cancer. Many studies have proved that CD146 targeting may present a promising strategy for cancer theranostics.

Gastric Metastatic Grade 3 Well-Differentiated Neuroendocrine Tumor: A Case Report

Background: Neuroendocrine neoplasms (NENs) are rare tumors .They are defined by the expression of specific biomarkers. Progress in pathological diagnosis has allowed a better identification and classification of these tumors. The 2019-revised World Health Organization (WHO) classification of tumors of endocrine organs classifies grade 3 gastroenteropancreatic neuroendocrine neoplasm (GEP-NENs) into well-differentiated neuroendocrine tumors (NET G3) and poorly differentiated neuroendocrine carcinomas (NEC G3). There are few reported cases of NET G3 occurring in the stomach. Case presentation: A 63-year- man who suffers from dysphagia and Melaena. Fibroscopy revealed a ulcero-budding process of the cardia. Pathologic examination concluded on NET well-differentiated grade 3. Surgical exploration showed peritoneal carcinomatosis and histologically confirmed liver metastases. He underwent chemotherapy with modified folfox 4 protocol for 3 months with satisfactory clinico-morphological response. Conclusion: A definite diagnosis of NET G3 or NEC G3 must be made to determine the appropriate treatment strategy for patients with GEP-NEN G3. the efficacy of treatments for G3 NETs appear modest, as evidenced by the short PFS observed, therefore, more effective therapies are needed with Further case reports and case series. Keywords: Neuroendocrine tumor G3, stomach, modified FOLFOX 4.

Changes in the microcirculatory bed in patients with post-mastectomy syndrome

The purpose of the study: to determine the features of the state of the microcirculatory bed in women with post-mastectomy syndrome as a cause of fibrosis.Material and methods. The study involved 76 women, of whom two groups were formed. The first group consisted of 46 patients aged 25 to 49 years (mean age – (43.8 ± 3.8) years old) with post-mastectomy syndrome (PMES), the average period after radicle treatment of breast cancer was (4.3 ± 2.1) years. All patients underwent mastectomy, radiation and chemotherapy for the treatment of breast cancer, followed by a comprehensive examination to exclude metastasis and continued tumor growth, as well as to exclude hemodynamically significant violations of venous and arterial blood flow of the upper extremities. The second group was formed of 30 healthy female volunteers aged 25 to 43 years, whose average age was (33.75 ± 10.70) years old. Clinical and instrumental methods of examination were used: anthropometric method, lymphoscintigraphy of the upper extremities, laser Doppler flowmetry, determination of the expression level of intercellular adhesion molecules sICAM-1, sPECAM-1.The results. Gross violations of the function of the microcirculatory bed were revealed: endothelial dysfunction, spasm of precapillary sphincters, decrease in the intensity of blood flow in the metabolic capillaries, the predominance of passive mechanisms of blood flow regulation. These changes lead to tissue hypoxia, which may be the trigger for the development of fibrosis and lymphedema. Endothelial dysfunction, which is confirmed by increased expression of specific biomarkers, may be the basis of these disorders. A complex, long-term change in the work of microvessels leads to a breakdown of compensatory mechanisms.Conclusion. The study of the pathogenetic mechanisms of the development of post-mastectomy syndrome will allow forming preventive strategies in patients after treatment of breast cancer, improving the quality of life of these patients. Further search for promising predictors of the development of fibrosis, the study of changes in the microcirculatory bed will provide additional information about the pathogenesis of PMES, to identify changes in the soft tissues of the upper extremities at an early stage of post-mastectomy syndrome, may be important for the development of more effective methods of treatment of PMES.

High-Plex Spatial RNA Profiling Reveals Cell Type‒Specific Biomarker Expression during Melanoma Development

Early diagnosis of melanoma is critical for improved survival. However, the biomarkers of early melanoma evolution and their origin within the tumor and its microenvironment, including the keratinocytes, are poorly defined. To address this, we used spatial transcript profiling that maintains the morphological tumor context to measure the expression of >1,000 RNAs in situ in patient-derived formalin-fixed, paraffin-embedded tissue sections in primary melanoma and melanocytic nevi. We profiled 134 regions of interest (each 200 μm in diameter) enriched in melanocytes, neighboring keratinocytes, or immune cells. This approach captured distinct expression patterns across cell types and tumor types during melanoma development. Unexpectedly, we discovered that S100A8 is expressed by keratinocytes within the tumor microenvironment during melanoma growth. Immunohistochemistry of 252 tumors showed prominent keratinocyte-derived S100A8 expression in melanoma but not in benign tumors and confirmed the same pattern for S100A8's binding partner S100A9, suggesting that injury to the epidermis may be an early and readily detectable indicator of melanoma development. Together, our results establish a framework for high-plex, spatial, and cell type‒specific resolution of gene expression in archival tissue applicable to the development of biomarkers and characterization of tumor microenvironment interactions in tumor evolution.

Expression Patterns of Microenvironmental Factors and Tenascin-C at the Invasive Front of Stage II and III Colorectal Cancer: Novel Tumor Prognostic Markers.

BackgroundBiological markers expressed in cancer cells and the surrounding cancer-associated fibroblasts (CAF) can be used for prediction of patient prognosis in colorectal cancer (CRC). Here, we used immunohistochemical techniques to evaluate cancer cells’ expression of specific biomarkers that are closely associated with neoplastic progression.MethodsImmunohistochemical markers included Ki-67, p53, β-catenin, MMP7, E-cadherin and HIF1-α. We also characterized microenvironmental markers expressed by CAF, including expression of α-smooth muscle actin, CD10, podoplanin, fibroblast specific protein 1, platelet derived growth factor β, fibroblast association protein, tenascin-C (TNC), ZEB1 and TWIST1. The study population consisted of 286 CRC patients with stage II and III disease. Stage II and III CRC were divided into a first and a second cohort (for validation). The CRCs were stratified using cluster analysis. To identify the utility of prognostic markers in stage II and III CRC, univariate and multivariate analyses were performed in both cohorts.ResultsStage II and III CRCs were stratified into 3 subgroups. Specific subgroups were significantly correlated to disease-free survival using univariate and multivariate analyses in the first cohort. High expression of TNC was identified as a single prognostic marker in both cohorts by univariate and multivariate analyses.ConclusionsWe suggest that the presence of a specific subgroup defined by multiple markers can be used for prediction of CRC outcome in stages II and III. In addition, we showed that high expression of TNC was correlated with a poorer prognosis in stages II and III of CRC.

Studies for Stability of Ex Vivo Produced Megakaryocytes and Their Distribution in Transplanted Mice

▪We previously established an efficient pilot-scale system to generate sufficient megakaryocytes/platelets for potential clinical applications. Here, we present recent studies that demonstrate the feasibility for stable preservation of ex vivo generated megakaryocytes/platelets (MKs/PLTs) and analyze distribution of these cells in transplanted NOD/SCID mice.The 8-day ex vivo culture of megakaryocyte progenitors (MKPs) were harvested and cryopreserved in liquid nitrogen for 1 or 3 months. Cells were then thawed and further cultured for 5 days so that these cells were differentiated toward the megakaryocytic lineage. Meanwhile, matured MKs/PLTs generated from fresh cord blood CD34+ cells on day 13 were collected and stored at 4°C with plasma substitution for 1, 3, 5, and 7 days, respectively. Subsequently, cell viability and specific bio-marker expression were analyzed for their phenotypic stability at various stored conditions. To observe distributions of ex vivo produced MKs/PLTs with transfected lenti-GFP in sub-lethally irradiated NOD/SCID mice (n=5) at the dose of 1.0×107 transfected cells/mouse. After transplantation for 0.5, 3, 6, and 24 h, mouse organs were harvested and images were immediately taken. Furthermore, peripheral blood of each mouse was collected and human GFP+CD41a+ MKs and GFP+CD42b+ PLTs were detected by flow cytometry to evaluate platelet-releasing potential of transplanted MKs in vivo . Mice transplanted with no GFP-labelled cells were used as negative control (n=3). Mice injected with saline alone were used as blank control (n=3).After 5 days' culture of MKPs that had been cryopreserved for 1 or 3 months, cell viability was 79.0%±3.3% or 75.2%±5.6%, with CD41a purity of 85.3%±4.5% or 87.2%±3.9% and CD61 purity of 68.4%±4.1% or 63.2%±2.3%, respectively. Cell viability and bio-marker expression of cryopreserved MKPs after in vitro differentiation were similar with 13-day culture of matured MKs/PLTs from freshly collected cord blood CD34+ cells (Viability: 85.5%±2.7%; CD41a: 82.4%±6.1% and CD61:74.0%±5.5%, respectively). For mature MKs/PLTs stored at 4°C, the cell viability of these four groups was 81.2%±4.5%, 74.3%±5.6%, 73.2%±4.9%, and 71.5%±6.3%, respectively; Percentages of CD41a cells of these four groups were 62.7%±5.1%, 58.3%±4.4%, 59.1%±6.0%, 56.9%±7.7% whereas percentages of CD61 cells were 65.8%±3.9%, 60.7%±2.5%, 45.2%±1.9%, 49.4%±4.4%, respectively. These results strongly suggest that MKPs of 8-day culture can be cryopreserved in liquid nitrogen for 3 months or longer, and matured MKs/PLTs of 13-day culture can be stored at 4°C for 7 days or longer without significantly affecting their viability and immunophenotypic characteristics. It is important to note that none of the groups had any bacterial and fungal contaminations before or after storage and during subsequent culturing processes. The fact that these cells were free of endotoxin and pyrogen strongly suggests that ex vivo generated MKs/PLTs are safe and stable. In mouse studies, no fluorescent signal could be detected in the blank control group, and only a trace amount of auto-fluorescence was detected in lung of the negative mouse group. However, in experimental group strong GFP signals could be observed in lung of each mouse 0.5 h after transplantation with the peak fluorescence between 3 and 6 h. Lung GFP signals remained detectable 24 h post transplantation. In mouse peripheral blood, human GFP+CD41a+ MKs could be detected only in the first 0.5 h after transplantation, and then human CD41a+CD42b+ PLTs emerged at 0.5 h with a low level of 0.5%±0.1%, after which the percentage increased rapidly with a peak level of 3.2%±0.6% at 6 h, indicating that the transplanted human MKs started to release PLTs after they merged into lung. Besides lung, a very weak signal was observed in the kidneys and livers, and completely disappeared at 24 h in mice of the experimental group. These results imply that engrafted mature MKs are capable of migrating into lung for further maturation, releasing platelets in NOD/SCID mice.MKPs of 8-day culture can be cryopreserved in liquid nitrogen for at least 3 months without affecting their viability and phenotypic characteristics. Mature MKs/PLTs of 13-day culture are stable in plasma substitute at 4°C for 7 days. The transplanted human MKs/PLTs appear to primarily home to the lung of NOD/SCID mice to release platelets in vivo . DisclosuresWang:Biopharmagen Corp.: Employment.

Dried Blood Specimens as an Alternative Specimen for Immune Response Monitoring During HIV Infection: A Proof of Concept and Simple Method in a Pediatric Cohort.

Programs to prevent mother-to-child HIV transmission do not reduce the number of infants exposed during pregnancy and breastfeeding. HIV-exposed but uninfected children (HEU) present higher risk of morbidity and mortality than HIV-unexposed and uninfected children (UU). In this line, the study of immune biomarkers in HIV could improve prediction of disease progression, allowing to diminish comorbidity risk. Dried blood specimens (DBS) are an alternative to serum for collecting and transporting samples in countries with limited infrastructure and especially interesting for groups such as pediatrics, where obtaining a high sample volume is challenging. This study explores the usefulness of DBS for immune profile monitoring in samples from 30 children under clinical follow-up in Kinshasa: 10 HIV-infected (HIV+), 10 HEU, and 10 UU. We have measured the gene expression levels of 12 immune and inflammatory markers (CD14, IL-6, TNFα, HVEM, B7.1, HIF-1α, Siglec-10, IRAK-M, CD163, B7H5, PD-L1, and Galectin-9) in DBS samples by reverse transcription of total RNA and RT-qPCR. Principal component analysis, Kruskal–Wallis test, and Mann–Whitney test were performed in order to study group differences. HIV+ children presented significantly higher levels of seven biomarkers (CD14, IL-6 HVEM, B7.1, Siglec-10, HIF-1α, and CD163) than the UU group. In HEU, we found seven biomarkers significantly elevated (CD14, IL-6, HVEM, B7.1, Siglec-10, HIF-1α, and IRAK-M) vs. UU. Six biomarkers (CD14, IL-6, HVEM, B7.1, Siglec-10, and HIF-1α) showed a significantly higher expression in both HIV+ and HEU vs. UU, with HVEM and CD14 being significantly overexpressed among HIV+ vs. HEU. Our data reveal the utility of DBS for immune response monitoring. Moreover, significant differences in specific biomarker expression across groups strongly suggest the effect of HIV infection and/or HIV exposure on these immune biomarkers' expressions.

Single-arm, phase 2 study of regorafenib plus nivolumab in patients with mismatch repair-proficient (pMMR)/microsatellite stable (MSS) colorectal cancer (CRC).

3560 Background: The role of immunotherapy in the treatment of pMMR/MSS metastatic CRC is not established. A Japanese phase 1b trial in this setting showed the combination of regorafenib (multikinase inhibitor with immunomodulatory activity) plus nivolumab (anti PD-1) had encouraging activity and manageable safety (Fukuoka, 2020). This study further assessed the safety and efficacy of this combination. Methods: Patients (pts) from the US aged ≥18 years who progressed on/were intolerant to standard chemotherapy were enrolled. Regorafenib was given orally, once daily in 28-day (D) cycles (21D on/7D off) plus IV nivolumab 480 mg on D1. Regorafenib starting dose was 80 mg; if well tolerated, it could be escalated to 120 mg in Cycle 2. Primary endpoint was overall response rate (ORR; RECIST 1.1); secondary aims included disease control rate (DCR), overall survival (OS), progression-free survival (PFS), and safety (NCI-CTCAE v5.0 grade). Biomarker analysis was exploratory. Results: 70 pts (59% male) started treatment. At baseline, median age was 57 years (range 34–85), ECOG PS 0/1 was 51%/49%, 67% had liver metastases (mets), and the primary tumor site was right-sided colon in 36% and rectum in 17%. Median number of cycles was 3.0 (range 1–13); 41% of pts escalated regorafenib to 120 mg. Five pts (7.1%) had a partial response (PR) lasting ≥16 weeks (wks) and 22 (31.4%) had stable disease (SD); pts without liver mets had a higher ORR (21.7%). In pts with tumor samples (n = 40), higher baseline expression (IHC) of cytotoxic T cells (CD3+/CD8+/GranzymeB+), Tregs (FoxP3+), and macrophages (CD68+) trended with clinical benefit (PR/SD ≥16 wks/PFS); pts with liver mets had lower expression. Lower plasma levels of biomarkers of vascular biology (e.g. VEGF-D, Ang-2, VWF) trended with longer PFS. Grade (Gr) 3 treatment-emergent adverse events (TEAEs) occurred in 53% of pts and Gr 4 in 10%. Three pts had a Gr 5 TEAE: n = 1 related to the combination (sepsis); n = 1 related to nivolumab only by investigator (sepsis); n = 1 unrelated to treatment (respiratory failure). Most common Gr 3/4 TEAEs: maculopapular rash (14%), fatigue (7%), pneumonia (6%), increased bilirubin (6%). Conclusions: Combination treatment with regorafenib (up to 120 mg/day) and nivolumab (480 mg every 28D) has manageable safety. Efficacy of this combination in the North American population did not emulate results in the Japanese population. Absence of liver mets and expression of specific biomarkers indicate a better response and may warrant further analysis. Clinical trial information: NCT04126733. [Table: see text]

Molecular phenotype of high-grade endometrioid carcinoma of the endometrium.

Prognosis of the course of tumor progression is one of urgent problems of clinical oncology. A relevant specificity of endometrial cancer is its clinical polymorphism within the same histological type of the disease. The search for molecular-biological features associated with the aggressive phenotype of endometrioid carcinomas is indisputably urgent. To study molecular-biological features of endometrioid carcinoma of the endometrium (ECE) and to identify the molecular subtype of tumors with high potential of malignancy. Surgical specimens of 127patients with EC, stages I-II, aged 36-72 (the average age- 59.3± 3.2) were studied using morphological and immunohistochemical methods. The multivariant analysis with the Kullback's informative measure and PanelomiX were used to estimate the significance of the expression of specific biomarkers. The expression of a complex of multifunctional markers was evaluated in ECE cells of different malignancy stage: p53, FOXP3, p21WAF1/CIP1, р16INK4a, E2F1, cyclins Е and D1, Her2/neu, с-Myc, Е-cadherin, β-catenin, vimentin, CD44, CD24. A triad of biomarkers with threshold expression levels was determined (р53< 45%; FOXP3> 14%; с-Myc< 10%). The high expression of oncogene c-Myc and oncosuppressor p53along with the low level of FOXP3in tumor cells of ECE was associated with high proliferative potential, low differentiation grade, and deep invasion of a tumor into the myometrium. The molecular phenotype of ECE, most informative in terms of specificity and sensitivity (95%)- р5highFOXP3lowc-Mychigh, was first characterized, which would help identify a high-grade subtype of this cancer form.

Abstract 386: Design and application of an 8-plex multiplex immunofluorescence panel for deep phenotypic profiling of the tumor microenvironment using InSituPlex technology

Abstract Background: The tumor microenvironment is composed of a complicated network of different cell types and activation states, the exact composition of which is important for immuno-oncology research efforts to correlate treatment options and outcome. The number of biomarkers necessary to sufficiently profile the various cellular phenotypes and elucidate contextual relationships in situ is larger than can be accommodated by standard immunohistochemistry (IHC) approaches. Furthermore, conventional IHC requires more sections, potentially exhausting tissue sample availability. To overcome these limitations, Ultivue has developed InSituPlex® technology and UltiMapper™ assays that enable biomarker multiplexing in tissue samples with an automated workflow to support high sample throughput. Methods: An 8-plex immune-profiling panel using the InSituPlex approach was designed and tested to detect and classify T cells, macrophages, and tumor cell populations along with PD-L1 checkpoint expression in multiple tumor types. This assay was designed, developed, and tested using UltiMapper reagents and corresponding protocols. The assay was run on multiple FFPE tissue samples from breast cancer, NSCLC, and colorectal cancer (CRC) cases. Each sample was stained for the following 8 markers: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, and pan-CK. Staining was performed in a single step using the Leica Biosystems BOND RX autostainer. Whole slide imaging (WSI) was performed in two steps on the ZEISS Axio Scan.Z1 slide scanner, and image analysis was performed using HALO software from Indica Labs. Results: The 8-plex immune-profiling panel demonstrated specific biomarker staining and was verified for staining reproducibility. The 8-plex workflow was compatible across multiple tumor tissue types. The samples were characterized by cell analysis and phenotyped based on their expression of specific biomarkers. The following cell phenotypes were detected: T cells (CD3+), cytotoxic T cells (CD3+/CD8+), regulatory T cells (CD4+/FOXP3+), exhausted T cells (CD3+/PD-1+), macrophages (CD68+), and tumor cells (CK+) with a dynamic range of PD-L1 expression levels. Conclusion: The 8-plex panel revealed important immunophenotypes across multiple sample types. The assay was automated and optimized for a high-throughput workflow. By testing across multiple tissue types, this assay showcases the potential for broad application across additional cancer types. The ability to interrogate an 8-plex I/O panel on a single slide with WSI presents an attractive approach for the next generation of IHC, enabling profiling within the tumor microenvironment and sensitive detection of PD-L1 expression in cancer tissues. Funded by NCI Contract No. 75N910D00024 Author affiliations for CGR staff: Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702 Citation Format: Alexis Wong, Mara Koerner, Katir K. Patel, Anna Potier, Stephanie Hennek, Mustapha Abubakar, Mary E. Olanich, Yelena G. Golubeva, Karun Mutreja, Scott Lawrence. Design and application of an 8-plex multiplex immunofluorescence panel for deep phenotypic profiling of the tumor microenvironment using InSituPlex technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 386.

Specific Biomarker Expression Patterns in the Diagnosis of Residual and Recurrent Endometrial Precancers After Progestin Treatment: A Longitudinal Study.

Conservative management with progestin is a treatment option for atypical hyperplasia (AH). However, pathologic diagnosis of residual/recurrent lesions is often problematic because of the profound morphologic changes induced by progestin and the lack of established diagnostic criteria for progestin-treated residual AH. We conducted a longitudinal study of 265 endometrial biopsies from 54 patients with a history of AH on progestin therapy. Patient outcomes were divided into 3 categories after morphologic review and immunohistochemical staining with phosphatase and tensin homolog (PTEN) and paired box 2 (PAX2): (1) persistent or residual disease; (2) recurrent disease; (3) complete response. All specimens were classified into 3 categories based on morphology: (1) persistent/recurrent disease (nonresponse), (2) morphologically uncertain response, (3) optimally treated (complete response). The staining patterns of PTEN/PAX2 were tracked over time in individual patients and correlated with morphologic findings before and after progestin therapy. Our data showed that aberrant expression patterns of PTEN and/or PAX2 were identified in 48 (88.9%) of the 54 primary biopsies and persisted in persistent/recurrent AH across serial endometrial biopsies (n=99, P<0.00001), while normal PTEN and PAX2 expressions were consistently observed in optimally treated cases (n=84, P<0.00001). More importantly, follow-up biopsies that showed a morphologically uncertain response but a PTEN/PAX2 expression pattern identical to the initial biopsy were significantly correlated with persistent or recurrent disease (n=18, P=0.000182), as evidenced by areas with morphologic features diagnostic of AH on subsequent biopsy. Biomarker PTEN/PAX2 signatures offer a valuable diagnostic aid to identify residual AH in progestin-treated endometrial samples for which the biomarker status from preprogestin treated AH is known. The findings of this study are promising for a possible future change of diagnostic practice.

Biomarkers in Tears and Ocular Surface: A Window for Neurodegenerative Diseases.

The purpose of this review is to briefly outline current scientific evidence on the potential role of tear analysis and ocular surface evaluation in diagnosis and monitoring of neurodegenerative diseases, especially Alzheimer disease, Parkinson disease, and glaucoma. A systematic computerized search in the electronic databases PubMed, MEDLINE, and the Cochrane Collaborations was conducted to find eligible articles which their main topic was to investigate the tear and ocular surface in neurodegenerative diseases. After a first screening of titles and abstracts and a full-text review, 26 articles met the inclusion criteria (1 about the neurodegenerative diseases, 3 about the Alzheimer disease, 11 about the Parkinson disease, 11 about glaucoma, and 1 about amyotrophic lateral sclerosis). The ocular surface picture seems to be altered in the setting of neurodegenerative diseases with specific characteristics according to each disease. They seem to be associated with reduced corneal sensitivity and abnormal tear function, and each one presents the expression of specific biomarkers in tears. The study of tears and ocular surface appears to be a new and noninvasive promising way to assist in the diagnosis and monitoring of neurodegenerative diseases.

Inmunoexpresión de biomarcadores Bax, Bcl-2, CD138, H3, Ki-67, MCM3 y p53 en liquen plano oral

This study aims to establish an association of the expression of specific biomarkers in oral lichen planus to understand its biological behavior. Materials and methods: An immunohistochemistry study was conducted in 40 cases of oral lichen planus against BAX, BCL-2, CD138, Histone 3, Ki-67, MCM3 and p53 at the Molecular Pathology Area of the School of Dentistry, UDELAR, Uruguay. Results: A greater expression of BAX was detected compared to BCL-2, suggesting a pro-apoptotic behavior, supported by the absence of p53 expression. MCM3 expression was more sensitive than Ki-67, considering proliferation alterations. CD-138 had a more intense and uniform expression, determining fewer intercellular adhesion alterations. Conclusions: The expression of the proteins studied suggests an alteration in proliferative and apoptotic mechanisms, associated with a pathological behavior of the oral mucosa.

ASIED: a Bayesian adaptive subgroup-identification enrichment design

ABSTRACT Developing targeted therapies based on patients’ baseline characteristics and genomic profiles such as biomarkers has gained growing interests in recent years. Depending on patients’ clinical characteristics, the expression of specific biomarkers or their combinations, different patient subgroups could respond differently to the same treatment. An ideal design, especially at the proof of concept stage, should search for such subgroups and make dynamic adaptation as the trial goes on. When no prior knowledge is available on whether the treatment works on the all-comer population or only works on the subgroup defined by one biomarker or several biomarkers, it is necessary to incorporate the adaptive estimation of the heterogeneous treatment effect to the decision-making at interim analyses. To address this problem, we propose an Adaptive Subgroup-Identification Enrichment Design, ASIED, to simultaneously search for predictive biomarkers, identify the subgroups with differential treatment effects, and modify study entry criteria at interim analyses when justified. More importantly, we construct robust quantitative decision-making rules for population enrichment when the interim outcomes are heterogeneous in the context of a multilevel target product profile, which defines the minimal and targeted levels of treatment effect. Through extensive simulations, the ASIED is demonstrated to achieve desirable operating characteristics and compare favorably against alternatives.

Abstract 4555: Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays

Abstract Background: In recent years, identifying and characterizing biomarkers within the tumor microenvironment (TME) has become a major focus for immune-oncology research. The growing field of immuno-oncology necessitates the identification of more biomarkers that help characterize the TME, and development of complimentary processes to rapidly analyze precious tissue samples. Ultivue® has developed UltiMapper™ assays that provide numerous advantages over alternative multiplexed IHC approaches, including high multiplexing in situ, sample preservation, streamlined workflows with staining completed in the same day, and easy integration into any histology lab. In this study we demonstrate a 10-plex immuno-oncology assay in FFPE tumor tissues by combining markers that profile the T-cell infiltrate, antigen presenting cells (APC), and checkpoint expression along the PD-L1/PD-1 axis. This 10-plex is achieved with a single antibody staining step for a fast workflow that is independent of staining order. The combination of 10 different biomarkers allows for characterization of the TME and investigation of PD-L1 expression on tumor and antigen presenting cells on a single slide. Methods: A 10-plex immunofluorescence assay was carried out using the UltiMapper approach on multiple deidentified FFPE tissue samples, including lung, melanoma, colon, and breast. Each sample was stained for the following 10 markers: CK or SOX10, PD-L1, PD-1, CD3, CD8, CD11c, CD20, CD68, CD163, and MHCII. Staining was performed in a single step using the Leica® Bond® Rx autostainer. Whole slide imaging was performed on various tissue scanners including the Zeiss® Axio Scan.Z1 slide scanner, and image analysis was performed using HALO® software from Indica Labs. Results: Cells were characterized and phenotyped based on their expression of specific biomarkers and the abundance of PD-L1 on macrophages, dendritic cells, B cell, cytotoxic T-cells, and tumor cells as measured. PD-L1 expression was observed on both immune and tumor cells with a range of different expression levels. Spatial analysis was employed to measure the distances between immune cells with different phenotypes and tumor cells to further characterize tumors as hot or cold. Conclusions: The multiplexed UltiMapper IHC approach allows for fast and comprehensive analysis of tumor samples necessary to research complex cancer biology. This approach achieves high quality multiplexed data on various tissue types and preserves precious tissue samples. The ability to interrogate a 10-plex I/O panel on a single slide with whole slide imaging presents a significant leap forward for IHC analysis, providing a faster and more thorough approach to characterize the TME and analyze PD-L1 expression on antigen presenting cells and tumor cells. Citation Format: Heike Boisvert, Alexis Wong, Gourab Chatterjee, Abdul Mohammed, Douglas Wood, Mael Manesse. Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4555.

Possible biomarkers for predicting lymph node metastasis of esophageal squamous cell carcinoma: a review.

Esophageal cancer is the eighth most common form of cancer worldwide, and esophageal squamous cell carcinoma (ESCC) is a major type of esophageal cancer that arises from epithelial cells of the esophagus. Local lymph node metastasis (LNM) is a typical sign of failure for ESCC clinical treatments, and a link has been established between LNM and the aberrant expression of specific biomarkers. In this review, we summarize what is known about nine factors significantly associated with LNM in ESCC patients: phosphatase and tensin homolog (PTEN), mucin 1, vascular endothelial growth factor-C, tumor necrosis factor alpha-induced protein 8 (TNFAIP8), Raf-1 kinase inhibitory protein, stathmin (STMN1), metastasis-associated protein 1, caveolin-1, and interferon-induced transmembrane protein 3. The function of these nine proteins involves four major mechanisms: tumor cell proliferation, tumor cell migration and invasion, epithelium–mesenchymal transition, and chemosensitivity. The roles of PTEN, STMN1, and TNFAIP8 involve at least two of these mechanisms, and we suggest that they are possible biomarkers for predicting LNM in ESCC. However, further retrospective research into PTEN, STMN1, and TNFAIP8 is needed to test their possibilities as indicators.

Long term treatment of metformin impedes development of chemoresistance by regulating cancer stem cell differentiation through taurine generation in ovarian cancer cells.

Development of resistance poses a significant challenge to effective first-line platinum based therapy for epithelial ovarian cancer patients. Cancer Stem Cells are envisaged as a critical underlying factor for therapy resistance. Thus, there is a critical need for developing approaches to diminish the enrichment of cancer stem cells and acquirement of resistance. Administration of metformin, a commonly prescribed drug against Type II diabetes exhibited promising effect in the management of ovarian cancer. However, the effect of long term administration of low dose of metformin as an adjuvant to cisplatin and paclitaxel during acquirement of chemoresistant phenotype has not been investigated so far. Using two isogenic cellular chemoresistant models (A2780 and OAW42) developed in the presence or absence of metformin, we demonstrated the ability of metformin to impede the development of resistance through increased drug sensitivity, increased proliferation, and reduced migratory abilities of the resistant cells. Metformin introduction also decreased the cancer stem cell population, expression of specific biomarkers and pluripotent genes. Further metabolic profiling of these cells using 1H-Nuclear Magnetic Resonance spectroscopy revealed significant modulation in taurine and histidine levels in resistant cells developed in the presence of metformin. Intriguingly, taurine treatment considerably reduced the cancer stem cell population and chemoresistance in resistant cells, indicating a novel role of taurine in differentiation of ovarian cancer stem cells. Altogether this is the first report on the potential role of metformin for targeting the cancer stem cell population via up regulation of taurine, leading to impediment in the acquirement of chemoresistance.

A novel approach to target hypoxic cancer cells via combining β-oxidation inhibitor etomoxir with radiation.

BackgroundHypoxia in tumors is associated with resistance towards various therapies including radiotherapy. In this study, we assessed if hypoxia in cancer spheres could be effectively reduced by adding etomoxir (a β-oxidation inhibitor) immediately after cell irradiation.MethodsWe employed cancer cells’ sphere model to target hypoxia. Confocal imaging was used to analyze hypoxia and expression of specific biomarkers in spheres following various treatments (radiation and/or etomoxir).ResultsEtomoxir (32.5 μM) treatment improved the radiation (2.5 Gy) efficacy against growth of lung adenocarcinoma H460 spheres. More importantly, radiation and etomoxir combination significantly reduced the hypoxic regions (pimonidazole+ areas) in H460 spheres compared to either treatment alone. Also, etomoxir and radiation combination treatment reduced the protein level of biomarkers for proliferation (Ki-67 and cyclin D1), stemness (CD44) and β-oxidation (CPT1A) in H460 spheres. We observed similar efficacy of etomoxir against growth of prostate cancer LNCaP cells’ spheres when combined with radiation. Further, radiation treatment strongly reduced the hypoxic regions (pimonidazole+ areas) in CPT1 knockdown LNCaP cells’ spheres.ConclusionsTogether, these results offer a unique approach to target hypoxia in solid tumors via combining etomoxir with radiation, thereby improving therapeutic efficacy.