Elisabeth Huber

Regenerating islet-derived 3-alpha is a biomarker of gastrointestinal graft-versus-host disease

There are no plasma biomarkers specific for GVHD of the gastrointestinal (GI) tract, the GVHD target organ most associated with nonrelapse mortality (NRM) following hematopoietic cell transplantation (HCT). Using an unbiased, large-scale, quantitative proteomic discovery approach to identify candidate biomarkers that were increased in plasma from HCT patients with GI GVHD, 74 proteins were increased at least 2-fold; 5 were of GI origin. We validated the lead candidate, REG3α, by ELISA in samples from 1014 HCT patients from 3 transplantation centers. Plasma REG3α concentrations were 3-fold higher in patients at GI GVHD onset than in all other patients and correlated most closely with lower GI GVHD. REG3α concentrations at GVHD onset predicted response to therapy at 4 weeks, 1-year NRM, and 1-year survival (P ≤ .001). In a multivariate analysis, advanced clinical stage, severe histologic damage, and high REG3α concentrations at GVHD diagnosis independently predicted 1-year NRM, which progressively increased with higher numbers of onset risk factors present: 25% for patients with 0 risk factors to 86% with 3 risk factors present (P < .001). REG3α is a plasma biomarker of GI GVHD that can be combined with clinical stage and histologic grade to improve risk stratification of patients.

A Reliable Tool to Determine Cell Viability in Complex 3-D Culture: The Acid Phosphatase Assay

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 µm and 900 µm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario. (Journal of Biomolecular Screening 2007:925-937)

Low Paneth cell numbers at onset of gastrointestinal graft-versus-host disease identify patients at high risk for nonrelapse mortality.

Acute graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is an often lethal complication of allogeneic hematopoietic cell transplant. Clinical severity correlates with outcomes, but histopathologic grading is primarily used to confirm the clinical diagnosis. One barrier to using histopathologic grading to predict clinical outcomes is inter-grader variability among transplant centers. Recent experimental models have shown that the loss of Paneth cells, which are located in the small intestine and help regulate the GI microbiome by secreting antimicrobial peptides, correlates with clinical GVHD severity. Because Paneth cells are easy to identify and quantify by light microscopy, we evaluated the mean number of Paneth cells per high-powered field (hpf) in 116 duodenal biopsies obtained at diagnosis of GI GVHD at 2 different centers with their clinical outcomes. Paneth cell counts were reproducible between centers (r(2) = 0.81; P < .0001). Lower numbers of Paneth cells at diagnosis correlated with clinically more severe GI GVHD (P < .0001) and less likelihood of response to GVHD treatment (P < .0001). A threshold of 4 Paneth cells per hpf stratified patients according to nonrelapse mortality (28% vs 56%; P = .004). We conclude that the enumeration of duodenal Paneth cells is a readily available index of disease severity that provides important information regarding GVHD prognosis.

Staged surgery with neoadjuvant 90Y-DOTATOC therapy for down-sizing synchronous bilobular hepatic metastases from a neuroendocrine pancreatic tumor

PurposeTreatment with DOTA-d-Phe(1)-Tyr(3)-octreotide (DOTATOC), labeled with beta-emitting radioisotope yttrium-90 (90Y-DOTATOC), has successfully been used for the palliative treatment of patients with advanced somatostatin receptor-expressing neuroendocrine tumors (NETs). However, controversy persists as to whether patients with metastatic NETs of the pancreas should undergo radical (salvage) surgery or receive palliative therapy. We proposed that 90Y-DOTATOC could be used in a neoadjuvant intention for improving therapy of hepatic NET metastases.Materials and methodsWe investigated a novel therapy concept in a 49-year-old patient presenting with a neuroendocrine tumor of the pancreatic tail and synchronous multiple bilobular hepatic metastases. After surgical removal of the large primary tumor by extended left en bloc resection of the pancreatic tail, the patient received neoadjuvant 90Y-DOTATOC for therapy of primarily non-resectable bilobular hepatic metastases.ResultsThe 90Y-DOTATOC therapy resulted in an impressive regression of hepatic lesions, thus facilitating surgical removal of all remaining liver metastases in a second operation (staged surgery). In addition, one lesion was ablated using radiofrequency ablation (RFA). At 1-year of follow-up after hepatic R0 resection/RFA, there was no evidence of tumor recurrence or extrahepatic metastasis.ConclusionsThe neoadjuvant use of 90Y-DOTATOC therapy could prove valuable for treatment of advanced pancreatic NETs metastatic to the liver in terms of facilitating R0 resection by applying staged surgery concepts.

Comparison between quantitative assessment of bowel wall vascularization by contrast-enhanced ultrasound and results of histopathological scoring in ulcerative colitis.

PurposeIn ulcerative colitis (UC), endoscopic methods are preferred for assessment of extent and activity of disease. Due to the invasive nature of endoscopical examinations, replacement by other, reliable imaging procedures would be helpful. Contrast-enhanced ultrasound (CEUS) in combination with perfusion assessment using a specific quantification software might be such a new diagnostic tool. Thus, we compared the findings of CEUS with the results of endoscopically taken specimens applying a histopathological scoring system.MethodsWe prospectively evaluated 15 patients with proven UC undergoing endoscopy. CEUS was performed and the quantification software Qontrast® applied to obtain contrast-enhanced sonographic perfusion maps. Moreover, in each patient C-reactive protein (CRP) was measured and taken biopsies were assessed using an advanced scoring system. Four patients had to be excluded from final analysis.ResultsThere was a trend to higher Peak (%) values with increasing histological inflammation. Furthermore, a strong negative correlation between the ratio TTP (s)/Peak (%) (Spearman’s correlation r = −0.761, p < 0.01) was found. There was no significant relationship between CRP and histopathological scoring or CEUS parameters, respectively.ConclusionQuantitative evaluation with CEUS, particularly the calculation of the ratio TTP (s)/Peak (%), provides a simple method for assessment of inflammatory activity in UC.

Consensus on the histopathological evaluation of liver biopsies from patients following allogeneic hematopoietic cell transplantation

After allogeneic hematopoietic cell transplantation (alloHCT) liver biopsy is performed for enigmatic liver disorders when noninvasive diagnostic steps have failed in establishing a definitive diagnosis. This document provides an updated consensus on the prerequisites for proper evaluation of liver biopsies in alloHCT patients and the histological diagnostic criteria for liver graft-versus-host disease (GvHD). The Working Group’s recommendations for the histological diagnosis of liver GvHD were derived from the peer-reviewed literature and from the consensus diagnosis of a total of 30 coded liver biopsies. Acceptance of the recommendations was tested by a survey distributed to all HCT centers in Austria, Germany and Switzerland. Consensus was achieved for biopsy indications, methods of sample acquisition and processing, reporting and interpretation of biopsy findings. As GvHD is variably treated and the treatment modalities have changed over time, the panel endorses the use of more frequent biopsies in clinical studies in order to improve the present challenging clinical and diagnostic situation.

Steroid treatment alters adhesion molecule and chemokine expression in experimental acute graft-vs.-host disease of the intestinal tract

OBJECTIVE Acute graft-vs.-host disease (aGVHD) is a major complication after allogeneic bone marrow transplantation (allo-BMT) that is characterized by high morbidity and mortality. Systemic treatment with steroids has been the mainstay of first-line therapy of aGVHD, although controlled experimental data in this context are limited. MATERIALS AND METHODS Using a haploidentical murine BMT model, steroid effects on hepatic and intestinal inflammation during aGVHD have been investigated. Lethally irradiated B6D2F1 mice received bone marrow cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. RESULTS Intraperitoneal administration of prednisolone (2 mg/kg body weight every day) early after onset of GVHD from day +10 until day +42 resulted in reduced clinical GVHD severity and improved survival of allogeneic recipients. Although the liver was barely affected by prednisolone treatment, aGVHD-related histopathologic injury of the gastrointestinal tract was strongly reduced in association with diminished expression of interferon-γ, tumor necrosis factor, CXCL 9-11, CCL2-3, mucosal addressin cell adhesion molecule-1, and intercellular adhesion molecule-1. Prednisolone-induced reduction of adhesion molecule expression in the gut manifested earlier than seen for cytokines or chemokines. Interestingly, when starting steroid treatment on day +28, the course of GVHD was unchanged and no major differences in cyto- or chemokine expression in gastrointestinal tract or liver on day +42 were seen. CONCLUSIONS When started early after GVHD onset, prednisolone-related beneficial effects can affect aGVHD target organs differently, involving divergent regulation of inflammation and leukocyte migration. Specifically, a change in adhesion properties between leukocytes and endothelial cells in the gastrointestinal tract may be one of the initial steps in a cascade of steroid-related aGVHD-attenuating events.

Tumor-Induced Osteomalacia: Increased Level of FGF-23 in a Patient with a Phosphaturic Mesenchymal Tumor at the Tibia Expressing Periostin.

In our case, a 45-year-old male patient had multiple fractures accompanied by hypophosphatemia. FGF-23 levels were significantly increased, and total body magnetic resonance imaging (MRI) revealed a tumor mass located at the distal tibia leading to the diagnosis of tumor-induced osteomalacia (TIO). After resection of the tumor, hypophosphatemia and the increased levels of FGF-23 normalized within a few days. Subsequent microscopic examination and immunohistochemical analysis revealed a phosphaturic mesenchymal tumor mixed connective tissue variant (PMTMCT) showing a positive expression of somatostatin receptor 2A (SSTR2A), CD68, and Periostin. Electron microscopy demonstrated a poorly differentiated mesenchymal tumor with a multifocal giant cell component and evidence of neurosecretory-granules. However, the resected margins showed no tumor-free tissue, and therefore a subsequent postoperative radiotherapy was performed. The patient is still in complete remission after 34 months. Tumor resection of PMTMCTs is the therapy of choice. Subsequent radiotherapy in case of incompletely resected tumors can be an important option to avoid recurrence or metastasis even though this occurs rarely. The prognostic value of expression of Periostin has to be evaluated more precisely in a larger series of patients with TIO.

Consensus diagnostic histopathological criteria for acute gastrointestinal graft versus host disease improve interobserver reproducibility.

Graft versus host disease (GvHD) is a clinically important complication after allogeneic hematopoietic stem cell transplantation (HSCT). Its diagnosis relies on clinical and histopathological findings. In order to evaluate and improve inter-institutional diagnostic agreement on histological diagnosis and grading of acute gastrointestinal GvHD, we conducted a round robin test, which included 33 biopsies from 23 patients after HSCT. Five pathologists from different institutions independently evaluated the original sections from the biopsies submitted for diagnosis. Based on their results, consensus qualitative criteria for the assessment of typical histological features of GvHD (e.g., apoptosis, crypt destruction, mucosa denudation) were proposed, including detailed descriptions as well as histological images. In a second round robin test with involvement of the same pathologists, the reproducibility of both diagnosis and grading had improved. Remaining differences were mostly related to differential diagnostic considerations, including viral infection or toxic side effects of medication, which should be resolved by integrating histopathological findings with proper clinical information.

Preventive Azithromycin Treatment Reduces Noninfectious Lung Injury and Acute Graft-versus-Host Disease in a Murine Model of Allogeneic Hematopoietic Cell Transplantation

Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)- dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c(+) dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.