Annette Schmitt-Gräff

Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: A multicenter survey

Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1–7) and SR-cGVHD (1–10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3–90.7%, 95% confidence interval (CI)) and 97.4% (92.3–100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

The Nlrp3 inflammasome regulates acute graft-versus-host disease.

Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease.

Fibrogenesis and Fibrolysis in Collagenous Colitis Patterns of Procollagen Types I and IV, Matrix- Metalloproteinase-1 and -13, and TIMP-1 Gene Expression

Collagenous colitis is characterized by the deposition of a superficial subepithelial collagenous layer, the pathogenesis of which is unknown. Because the excess matrix deposition is potentially reversible, a labile imbalance between fibrogenesis and fibrolysis may be suspected. Expression of procollagen alpha1(I) and alpha1(IV), matrix-metalloproteinase (MMP)-1 and -13, and tissue inhibitor of metalloproteinase (TIMP)-1 genes was semiquantitated by in situ hybridization on serial biopsies of 12 patients with collagenous colitis and compared to controls. Collagen types I, III, IV, and VI, tenascin, undulin/collagen XIV, and alpha-actin were localized by immunohistology. The superficial collagen layer stained strongly for collagen types I, III, and VI, and particularly for tenascin, but not for undulin. Elevated procollagen alpha1(I), procollagen alpha1(IV), and TIMP-1 transcript levels were found in alpha-actin-positive cells with linear distribution underneath the superficial collagenous layer, whereas MMP-1 RNA expression was variable and restricted to cell clusters. MMP-13 expression was undetectable. The patterns of procollagen alpha1(I)- and alpha1(IV)-specific labeling, combined with an intense tenascin- but absent undulin-specific staining, indicate deposition of an immature interstitial matrix that may be susceptible to degradation. The restricted MMP-1 RNA expression, counteracted by increased TIMP-1 expression, suggests locally impaired fibrolysis as a relevant factor in the pathogenesis of collagenous colitis.

Epithelial Tissue Chimerism after Human Hematopoietic Cell Transplantation Is a Real Phenomenon

Bone marrow transplantation in animals has been shown to generate epithelial populations, a phenomenon that has also recently been suggested to take place after human hematopoietic cell transplantation. However, reports in humans are not conclusive because they still leave open the possibility that the identified donor-derived cells are not epithelial cells but intraepithelial lymphocytes. Here, we demonstrate that donor-derived CD45(+) hematopoietic cells in close contact with epithelial tissue may be falsely characterized as donor-derived epithelial cells if the three-dimensional structure of the tissue is not considered and the hematopoietic markers are not examined. By using a rigorous three-dimensional analysis on single sections of colon biopsies triple stained with donor-specific, epithelial-specific, and hematopoietic-specific markers we demonstrate that chimerism of colon epithelium is a real phenomenon occurring constantly after human hematopoietic cell transplantation. We exclude horizontal DNA transfer or cell fusion as the underlying mechanism of our findings. Tissue damage enhances the engraftment of the donor-derived epithelial cells. The physiological and therapeutical role of the donor-derived epithelial cells after human hematopoietic cell transplantation needs further investigation. However, their identification requires stringent and unequivocal detection systems.

Analysis of molecular pathways in sporadic neuroendocrine tumors of the gastro-entero-pancreatic system

Little is known about the molecular pathogenesis of neuroendocrine tumors (NET) of the gastro‐entero‐pancreatic (GEP) system. We analyzed genetic and epigenetic alterations as well as the CpG island methylator phenotype (CIMP). The study comprised 118 well‐differentiated fore‐ and mid‐gut GEP‐NET from 71 patients. In addition to loss of heterozygosity (LOH), microsatellite instability (MSI) and the methylation status of various tumor associated genes were examined. The expression profile of p16, APC and MENIN was investigated by immunohistochemistry. None of the tumors was highly microsatellite unstable, LOH was found in 22.2%. Significant differences in promoter hypermethylation were identified in the RUNX3 and the O6‐MGMT genes. We found a significant loss of p16 expression in insulinomas (p = 0.05) and functional NET (p = 0.01), respectively. APC was expressed less in gastrinomas (p = 0.01) and functional GEP‐NET (p = 0.05) vs. nonfunctional tumors. MENIN expression was reduced in pancreatic vs. extrapancreatic NET (p = 0.008) and in insulinomas vs. nonfunctional GEP‐NET (p = 0.019) and NET associated with the carcinoid syndrome (p = 0.029). Further CIMP and a Ki‐67 index >10% showed a close correlation. Outcome analysis of 19 patients showed a better survival for CIMP‐negative patients. The analyses identified significant genetic and epigenetic alterations in well‐differentiated fore‐ and mid‐gut NET. CIMP, similar to Ki‐67, might turn out to be of prognostic relevance.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvβ3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

Imaging anti-angiogenic treatment response with DCE-VCT, DCE-MRI and DWI in an animal model of breast cancer bone metastasis

As current classification systems for the assessment of treatment response in bone metastasis do not meet the needs of oncologists, new imaging biomarkers are desirable. Therefore, the diagnostic impact of dynamic contrast enhanced (DCE)-volumetric computed tomography (VCT) (descriptive analysis), DCE-MRI (two-compartment model) and diffusion weighted imaging (DWI) for monitoring anti-angiogenic therapy effects of the VEGF antibody bevacizumab in breast cancer bone metastases in rats was studied. Nude rats (n=8 animals treated with bevacizumab and n=9 untreated control rats) with site-specific osteolytic bone metastasis of the hind leg were imaged with a 1.5T clinical MRI-scanner in an animal coil as well as in a volumetric CT-scanner at days 30, 40, 50 and 60 after inoculation of MDA-MB-231 human breast cancer cells. From these data, osteolytic lesion size (OLS), peak enhancement (PE), area under the curve (AUC), amplitude (A), exchange rate constant (k(ep)) and apparent diffusion coefficient (ADC) were determined in bone metastases. Prior to changes in OLS (p< or =0.05 at days 50 and 60) there was already a significant decrease in PE, AUC and A (p< or =0.05 at days 40-60) in treated animals compared to controls. However, for k(ep) and ADC there were no significant differences between the groups at any time point (p>0.05 at days 40-60). In conclusion, anti-angiogenic treatment response in osteolytic breast cancer bone metastases can be assessed early with surrogate markers of vascularization, while DWI appears to be insensitive.

Spleen tyrosine kinase (Syk) is a potent target for GvHD prevention at different cellular levels.

Acute graft-versus-host disease (GvHD) limits the applicability of allogeneic hematopoietic cell transplantation for the treatment of leukemia. GvHD occurs as a consequence of multiple activating events in antigen-presenting cells (APCs) and T cells (Tcs). Spleen tyrosine kinase (Syk) is an intracellular non-receptor tyrosine kinase involved in multiple signaling events of immune cells. Therefore, we hypothesized that Syk may be a promising target to inhibit GvHD, which involves activation of different immune cell populations. In vivo expansion of luciferase+ donor Tcs in mice developing GvHD was reduced by treatment with the Syk inhibitor Fostamatinib, which led to increased survival and reduced histologically confirmed GvHD severity. Importantly, in vivo and in vitro cytotoxicity against leukemia target cells and anti-murine cytomegalovirus immune responses were not impacted by Fostamatinib. In APCs Syk inhibition reduced the expression of costimulatory molecules and disrupted cytoskeletal organization with consecutive APC migratory defects in vitro and in vivo while phagocytic activity remained intact. On the basis of these immunomodulatory effects on different cell populations, we conclude that Syk targeting in alloantigen-activated Tcs and APCs with pharmacologic inhibitors, already applied successfully in anti-lymphoma therapy, has clinical potential to reduce GvHD, especially as anti-leukemia and anti-viral immunity were preserved.

Molecular characteristics and predictors of survival in patients with malignant neuroendocrine tumors

To better understand the molecular pathogenesis of neuroendocrine tumors (NET), we investigated the molecular and clinical characteristics of malignant poorly differentiated colorectal NET and compared these findings with sporadic CRC and well‐differentiated benign and malignant fore‐/midgut NET. Tumors were analyzed and correlated for microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP). NET were scored for proliferation using Ki‐67. A total of 34 malignant poorly differentiated colorectal NET, 38 well‐differentiated benign and malignant fore‐/midgut‐NET and 150 sporadic colorectal cancers (CRC) with known MSI status were investigated. Among the sporadic CRC, CIMP was significantly correlated with MSI‐high (MSI‐H) (p < 0.001). Of the 34 colorectal NET, 0/1 of the MSI‐H, 3/5 (60%) of the MSI‐L and 13/19 (68%) of the MSS tumors were CIMP+ (p = 0.17). Of the fore‐/midgut‐NET, none was MSI‐H. 20/34 (59%) colorectal NET vs. 11/38 (29%) fore‐/midgut‐NET were CIMP+ (p = 0.01). The Ki‐67 index was significantly higher in poorly differentiated colorectal NET compared to the less malignant fore‐/midgut‐NET (p < 0.0001). Besides the location in the colon, Ki‐67 predicted poor outcome in NET (p < 0.0001). CIMP status did not affect survival. In NET, p16 methylation predicted a poor outcome (p = 0.0004). We conclude that molecular pathogenesis in sporadic CRC and poorly differentiated colorectal NET is different despite some similarities. Main differences between malignant well‐differentiated and poorly differentiated NET are the Ki‐67 proliferation rate and differential methylation in tumor‐associated genes. Predictors of a poor outcome in patients with NET are poor differentiation, a high Ki‐67 index and p16 methylation.

Role of T Cells in Virus Control and Disease after Infection with Pneumonia Virus of Mice

ABSTRACT Infection of mice with pneumonia virus of mice (PVM) is used as a natural host experimental model for studying the pathogenesis of infection with the closely related human respiratory syncytial virus. We analyzed the contribution of T cells to virus control and pathology after PVM infection. Control of a sublethal infection with PVM strain 15 in C57BL/6 mice was accompanied by a 100-fold increase in pulmonary cytotoxic T lymphocytes, 20% of which were specific for PVM. T-cell-deficient mice failed to eliminate PVM and became virus carriers in the absence of the clinical or histopathological signs of pneumonia that occurred after infection of control mice. Mice with limited T-cell numbers did not achieve virus control without weight loss, indicating that T-cell-mediated virus control was closely linked to immunopathology. Both CD4 and CD8 T cells independently contributed to virus elimination and disease. Virus control and disease were similar in the absence of perforin, gamma interferon, or tumor necrosis factor alpha. Interestingly, disease and mortality after lethal high-dose PVM infection were independent of T cells. These data illustrate a key role for T cells in control of PVM infection and demonstrate that both T-cell-dependent and -independent pathways contribute to disease in a viral dose-dependent fashion.