PD-L1 mRNA Detection in Immunohistochemically Negative Patients: A Complementary Method for a Better Treatment Selection?

BACKGROUND/AIM: PD-L1 inhibitors have been approved for cisplatin-ineligible urothelial cancer patients relapsing after radical cystectomy. A prerequisite for therapy is a positive PD-L1 expression in the tumor tissue, whereas no options are available for patients with negative PD-L1 status. However, studies revealed that many PD-L1-negative patients also responded to PD-L1 therapy. This study investigated the feasibility of PD-L1 mRNA complementary RNA in situ hybridization (RNAish) analysis to detect PD-L1-responders independent of PD-L1 protein status. MATERIALS AND METHODS: Immunohistochemistry and RNA in situ hybridization were used to assess PD-L1 protein and mRNA in radical cystectomy tissue from patients with advanced and metastasized urothelial cancer. RESULTS: In this study, PD-L1 protein and mRNA were detected in ≥90% of the examined tissue. Positive PD-L1 mRNA expression (≥1%) on TC and IC could be evaluated in 77% and 31% of the cases, respectively. Moreover, scatterplot analysis revealed a PD-L1 mRNA positive and PD-L1 protein negative subpopulation. According to the CPS score, positive PD-L1 protein expression could be evaluated in 88% and positive PD-L1 mRNA expression in 71% of the cases. Scatterplot analysis of the CPS scores revealed a CPS protein negative but CPS mRNA positive small subpopulation. CONCLUSION: The feasibility of RNAish on formalin-fixed tissue could be proven. Moreover, complementary PD-L1 RNAish identified a sub-population of PD-L1 protein-negative and PD-L1 mRNA-positive patients, which may benefit from PD-L1 therapy.

Characteristics of Clinically Classified Oral Lichen Planus in Optical Coherence Tomography: A Descriptive Case-Series Study.

Malignant transformation of oral lichen planus (OLP) into oral squamous cell carcinoma is considered as one of the most serious complications of OLP. For the early detection of oral cancer in OLP follow-up, accurate localization of the OLP center is still difficult but often required for confirmatory biopsy with histopathological examination. Optical coherence tomography (OCT) offers the potential for more reliable biopsy sampling in the oral cavity as it is capable of non-invasively imaging the degenerated oral layer structure. In this case-series study with 15 patients, features of clinically classified forms of OLP in OCT cross-sections were registered and correlated with available histologic sections. Besides patients with reticular, atrophic, erosive and plaque-like OLP, two patients with leukoplakia were included for differentiation. The results show that OCT yields information about the epithelial surface, thickness and reflectivity, as well as the identifiability of the basement membrane and the vessel network, which could be used to complement the visual clinical appearance of OLP variants and allow a more accurate localization of the OLP center. This forms the basis for further studies on OCT-assisted non-invasive clinical classification of OLP, with the aim of enabling decision support for biopsy sampling in the future.

A Handheld Fiber-Optic Probe to Enable Optical Coherence Tomography of Oral Soft Tissue.

This study presents a highly miniaturized, handheld probe developed for rapid assessment of soft tissue using optical coherencetomography (OCT). OCT is a non-invasive optical technology capable of visualizing the sub-surface structural changes that occur in soft tissue disease such as oral lichen planus. However, usage of OCT in the oral cavity has been limited, as the requirements for high-quality optical scanning have often resulted in probes that are heavy, unwieldy and clinically impractical. In this paper, we present a novel probe that combines an all-fiber optical design with a light-weight magnetic scanning mechanism to provide easy access to the oral cavity. The resulting probe is approximately the size of a pen (10 mm × 140 mm) and weighs only 10 grams. To demonstrate the feasibility and high image quality achieved with the probe, imaging was performed on the buccal mucosa and alveolar mucosa during routine clinical assessment of six patients diagnosed with oral lichen planus. Results show the loss of normal tissue structure within the lesion, and contrast this with the clear delineation of tissue layers in adjacent inconspicuous regions. The results also demonstrate the ability of the probe to acquire a three-dimensional data volume by manually sweeping across the surface of the mucosa. The findings of this study show the feasibility of using a small, lightweight probe to identify pathological features in oral soft tissue.

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions.

Background: Within the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown. Methods: We studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease. Results: CD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression. Conclusions: CD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

A Complex Network of Tumor Microenvironment in Human High-Grade Serous Ovarian Cancer.

Purpose: Most high-grade serous ovarian cancer (HGSOC) patients develop recurrent disease after first-line treatment, frequently with fatal outcome. This work aims at studying the molecular biology of both primary and recurrent HGSOC.Experimental Design: Gene expression profiles of matched primary and recurrent fresh-frozen tumor tissues from 66 HGSOC patients were obtained by RNA sequencing. Clustering analyses and pairwise comparison of the profiles between matched samples and subsequent functional alignment were used for the identification of molecular characteristics of HGSOC.Results: Both primary and recurrent HGSOC samples presented predominant gene expression differences in their microenvironment, determined by a panel of genes covering all major pathways of immune activation together with a number of genes involved in the remodeling of extracellular matrix and adipose tissues. Stratifying tumor tissues into immune active and silent groups, we further discovered that although some recurrent tumors shared the same immune status as their primary counterparts, others switched the immune status, either from silent to active or active to silent. Interestingly, genes belonging to the B7-CD28 immune checkpoint family, known for their major role as negative regulators of the immune response, were overexpressed in the immune active tumors. Searching for potential tumor antigens, CEACAM21, a member of the carcinoembryonic antigen family, was found to be significantly overexpressed in immune active tissues in comparison with the silent ones.Conclusions: The results illustrate the complexity of the tumor microenvironment in HGSOC and reveal the molecular relationship between primary and recurrent tumors, which have multiple therapeutic implications. Clin Cancer Res; 23(24); 7621-32. ©2017 AACR.

Porcine liver decellularization under oscillating pressure conditions: a technical refinement to improve the homogeneity of the decellularization process.

Decellularization and recellularization of parenchymal organs may facilitate the generation of autologous functional liver organoids by repopulation of decellularized porcine liver matrices with induced liver cells. We present an accelerated (7 h overall perfusion time) and effective protocol for human-scale liver decellularization by pressure-controlled perfusion with 1% Triton X-100 and 1% sodium dodecyl sulfate via the hepatic artery (120 mmHg) and portal vein (60 mmHg). In addition, we analyzed the effect of oscillating pressure conditions on pig liver decellularization (n=19). The proprietary perfusion device used to generate these pressure conditions mimics intra-abdominal conditions during respiration to optimize microperfusion within livers and thus optimize the homogeneity of the decellularization process. The efficiency of perfusion decellularization was analyzed by macroscopic observation, histological staining (hematoxylin and eosin [H&E], Sirius red, and alcian blue), immunohistochemical staining (collagen IV, laminin, and fibronectin), and biochemical assessment (DNA, collagen, and glycosaminoglycans) of decellularized liver matrices. The integrity of the extracellular matrix (ECM) postdecellularization was visualized by corrosion casting and three-dimensional computed tomography scanning. We found that livers perfused under oscillating pressure conditions (P(+)) showed a more homogenous course of decellularization and contained less DNA compared with livers perfused without oscillating pressure conditions (P(-)). Microscopically, livers from the (P(-)) group showed remnant cell clusters, while no cells were found in livers from the (P(+)) group. The grade of disruption of the ECM was higher in livers from the (P(-)) group, although the perfusion rates and pressure did not significantly differ. Immunohistochemical staining revealed that important matrix components were still present after decellularization. Corrosion casting showed an intact vascular (portal vein and hepatic artery) and biliary framework. In summary, the presented protocol for pig liver decellularization is quick (7 h) and effective. The application of oscillating pressure conditions improves the homogeneity of perfusion and thus the outcome of the decellularization process.

A comprehensively characterized large panel of head and neck cancer patient-derived xenografts identifies the mTOR inhibitor everolimus as potential new treatment option.

Patient-derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5-fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5-fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.

Evidence of haematopoiesis within the developing human diencephalon. Correlations with vertebrate phylogeny.

AIM: The sites of haematopoiesis during human ontogeny can be correlated to the sites where haematopoiesis occurs in vertebrate phylogeny. As haematopoiesis has been described in the diencephalon and pituitary gland of water-inhabiting vertebrates we wanted to find out whether such a phenomenon also occurs in human embryos. MATERIAL AND METHODS: Paraffin-embedded specimens from the diencephalon and pituitary gland of human embryos at the 7th to 22nd gestational week and from adults were investigated by conventional histology and immunohistology for the presence of haematopoietic cells. RESULTS: Cellular accumulations predominantly of erythroid and megakaryocytic lineage were identified in the floor of the developing diencephalon of the 7th/8th gestational week. At the older developmental stages of the 18th to 22nd gestational week loose aggregates of haematopoietic cells within the leptomeningeal spaces adjacent to the hypophyseal infundibulum were detected in 2 out of 7 cases analyzed. CONCLUSIONS: As it has been proposed that lymphohaematopoietic clusters occasionally occur within the brain in bone marrow-less vertebrates as a response to noxious agents, we speculate that this temporal appearance of haematopoietic cell clusters within the diencephalon floor in early human ontogeny could also be due to fetal immunomodulations.

Epstein-barr virus infection of monocytoid B-cell proliferates: an early feature of primary viral infection?

Monocytoid cells are a subset of B lymphocytes with characteristic morphology and immunophenotype, which proliferate in a broad variety of reactive lymph node conditions. Up to now, no direct infection of these cells by a pathogenic organism has been identified. We found in a series of lymph node specimens from 13 patients having in common a prominent monocytoid B (MCB)-cell reaction in the absence of epithelioid cells or necrosis evidence that Epstein-Barr virus (EBV) can infect MCB cells. Clinical and serologic findings indicative for an acute EBV infection were reported only in 2 cases. By in situ hybridization and immunohistology, a number of MCB cells in all cases was found to contain EBV-encoded small nuclear RNAs and to express the EBV-encoded nuclear antigen (EBNA)-2 and, in lesser extent, the latent membrane protein-1. EBV-transcripts and proteins were also detectable in a number of extrafollicular B blasts and germinal center B cells in 10 of 13 cases. Our findings imply that an EBV infection of MCB cells associated with predominant EBNA-2 expression represents an early sign of primary infection and that it should be included in the differential diagnosis of activated lymph nodes with prominent MCB-cell reaction in the absence of epithelioid cells.