Clinical versus histological grading in the assessment of cutaneous graft versus host disease.

BACKGROUND: Skin biopsies are often used in daily practice for the diagnosis of acute (aGvHD) or chronic graft versus host disease (cGvHD). With the latest understanding in pathogenesis and new National Institute of Health (NIH) classifications for aGvHD and cGvHD, there is a need to evaluate the current prognostic value of histological grading cutaneous GvHD and its correlation to the clinical grade. METHODS: In a retrospective study with 120 skin biopsies (all taken for suspected GvHD) from 110 patients (all classified according to the NIH), biopsies were revised and graded, blinded for clinical information, for either acute of chronic features. Morphological grades were compared for concordance with the clinical grade and survival analyses were done for clinical and histological grading. RESULTS: Correlation for histologic vs. clinical grading was (very) poor for aGvHD and cGvHD (weighted κ - 0.038 and 0.0009, respectively). Patients with clinical aGvHD had worse prognosis compared to cGvHD. However, at time of biopsy neither clinical nor histological grading predicted the eventual survival for either aGvHD (p = 0.9739 and p = 0.0744, respectively) or cGvHD (p = 0.2149 and p = 0.4465, respectively). CONCLUSIONS: Confirming the diagnosis of GvHD is still a valuable reason for taking a skin biopsy, but this study shows that histologic grading of GvHD in the skin biopsy has no additional value for clinicians in current practice.

Imatinib treatment of poor prognosis mesenchymal-type primary colon cancer: a proof-of-concept study in the preoperative window period (ImPACCT).

BACKGROUND: The identification of four Consensus Molecular Subtypes (CMS1-4) of colorectal cancer forms a new paradigm for the design and evaluation of subtype-directed therapeutic strategies. The most aggressive subtype - CMS4 - has the highest chance of disease recurrence. Novel adjuvant therapies for patients with CMS4 tumours are therefore urgently needed. CMS4 tumours are characterized by expression of mesenchymal and stem-like genes. Previous pre-clinical work has shown that targeting Platelet-Derived Growth Factor Receptors (PDGFRs) and the related KIT receptor with imatinib is potentially effective against mesenchymal-type colon cancer. In the present study we aim to provide proof for the concept that imatinib can reduce the aggressive phenotype of primary CMS4 colon cancer. METHODS: Tumour biopsies from patients with newly diagnosed stage I-III colon cancer will be analysed with a novel RT-qPCR test to pre-select patients with CMS4 tumours. Selected patients (n = 27) will receive treatment with imatinib (400 mg per day) starting two weeks prior to planned tumour resection. To assess treatment-induced changes in the aggressive CMS4 phenotype, RNA sequencing will be performed on pre- and post-treatment tissue samples. DISCUSSION: The development of effective adjuvant therapy for primary colon cancer is hindered by multiple factors. First, new drugs that may have value in the prevention of (early) distant recurrence are almost always first tested in patients with heavily pre-treated metastatic disease. Second, measuring on-target drug effects and biological consequences in tumour tissue is not commonly a part of the study design. Third, due to the lack of patient selection tools, clinical trials in the adjuvant setting require large patient populations. Finally, the evaluation of recurrence-prevention requires a long-term follow-up. In the ImPACCT trial these issues are addressed by including newly diagnosed pre-selected patients with CMS4 tumours prior to primary tumour resection, rather than non-selected patients with late-stage disease. By making use of the pre-operative window period, the biological effect of imatinib treatment on CMS4 tumours can be rapidly assessed. Delivering proof-of-concept for drug action in early stage disease should form the basis for the design of future trials with subtype-targeted therapies in colon cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02685046 . Registration date: February 9, 2016.

Donor-Specific Antibodies Are Produced Locally in Ectopic Lymphoid Structures in Cardiac Allografts.

Cardiac allograft vasculopathy (CAV) is a transplant pathology, limiting graft survival after heart transplantation. CAV arteries are surrounded by ectopic lymphoid structures (ELS) containing B cells and plasma cells. The aim of this study was to characterize the antigenic targets of antibodies produced in ELS. Coronary arteries and surrounding epicardial tissue from 56 transplant recipients were collected during autopsy. Immunofluorescence was used to identify antibody-producing plasma cells. Immunoglobulin levels in tissue lysates were measured by enzyme-linked immunosorbent assay and analyzed for donor-specific HLA antibodies by Luminex assay. Cytokine and receptor expression levels were quantified using quantitative polymerase chain reaction. Plasma cells in ELS were polyclonal and produced IgG and/or IgM antibodies. In epicardial tissue, IgG (p < 0.05) and IgM levels were higher in transplant patients with larger ELS than smaller ELS. In 4 of 21 (19%) patients with ELS, donor-specific HLA type II antibodies were detected locally. Cytokine and receptor expression (CXCR3, interferon γ and TGF-ß) was higher in large ELS in the epicardial tissue than in other vessel wall layers, suggesting active recruitment and proliferation of T and B lymphocytes. ELS exhibited active plasma cells producing locally manufactured antibodies that, in some cases, were directed against the donor HLA, potentially mediating rejection with major consequences for the graft.

Plasma levels of alpha-1-antichymotrypsin are elevated in patients with chronic heart failure, but are of limited prognostic value.

BACKGROUND: There is increasing interest in utilising novel markers of cardiovascular disease risk in patients with chronic heart failure (HF). Recently, it was shown that alpha-1-antichymotrypsin (ACT), an acute-phase protein and major inhibitor of cathpesin G, plays a role in the pathophysiology of HF and may serve as a marker for myocardial distress. OBJECTIVE: To assess whether ACT is independently associated with long-term mortality in chronic HF patients. METHODS: ACT plasma levels were categorised into quartiles. Survival times were analysed using Kaplan-Meier curves and Cox proportional hazards regression, without and with correction for clinically relevant risk factors, including sex, age, duration of HF, kidney function (MDRD), ischaemic HF aetiology and NT-proBNP. RESULTS: Twenty healthy individuals and 224 patients (mean age 71 years, 72 % male, median HF duration 1.6 years) with chronic HF were included. In total, 159 (71 %) patients died. The median survival time was 5.3 (95 % CI 4.5-6.1) years. ACT was significantly elevated in patients (median 433 µg/ml, IQR 279-680) in comparison with controls (median 214 µg/ml, IQR 166-271; p < 0.001). Cox regression analysis demonstrated that ACT was not independently related to long-term mortality in chronic HF patients (crude HR = 1.03, 95 % CI 0.75-1.41, p = 0.871; adjusted HR = 1.12, 95 % CI 0.78-1.60, p = 0.552), which was confirmed by Kaplan-Meier curves. CONCLUSION: ACT levels are elevated in chronic HF patients, but no independent association with long-term mortality can be established.

Current technologies for HER2 testing in breast cancer.

BACKGROUND: Molecular techniques play an increasingly important role in breast cancer detection and help in the prediction of prognosis and treatment response. HER-2/neu predicts the sensitivity of breast tumors to trastuzumab and lapatinib. Presently there are several ways to assess HER2 status at the protein level (e.g. ELISA), at the RNA level (RT-PCR, microarray) and at the DNA level (fluorescence in situ hybridization, chromogenic in situ hybridization (ISH), silver in situ hybridization or multiplex ligation-dependent probe amplification). DESIGN: This paper provides an overview of new developments in HER2 testing. RESULTS: Although these techniques correlate well in comparative studies, discrepancies remain. Each technique has its own (dis)advantages and thus there is no real gold standard. Not surprisingly, there is no consensus at present on which of the protein- or gene-based techniques is superior, on the use of mono- or duo-probe ISH systems, nor on the use of manual or fully-automated staining- and scoring systems. CONCLUSION: Until large clinical trials clearly point out one strategy as the best predictive one for trastuzumab response, the choice for a testing strategy will probably be based on local preferences which consider both practical and economic issues. Standardization, proper internal and external quality control assessment, laboratory accreditation and automation of tissue processing (autostainers) and interpretation methods (image analysis) will play an increasingly important role in HER2 testing.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation.

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level >or=1 x 10(-4) were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1-4). The intervention was associated with graft versus host disease >or=grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0-30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

T cells in cardiac allograft vasculopathy are skewed to memory Th-1 cells in the presence of a distinct Th-2 population.

Cardiac allograft vasculopathy (CAV) in heart transplantation (HTx) patients remains the major complication for long-term survival, due to concentric neointima hyperplasia induced by infiltrating mononuclear cells (MNC). Previously, we showed that activated memory T-helper-1 (Th-1) cells are the major component of infiltrating MNC in coronary arteries with CAV. In this study, a more detailed characterization of the MNC in human coronary arteries with CAV (n = 5) was performed and compared to coronary arteries without CAV (n = 5), by investigating MNC markers (CD1a, DRC-1, CD3, CD20, CD27, CD28, CD56, CD68, CD69, FOXP3 and HLA-DR), cytokines (IL-1A, 2, 4, 10, 12B, IFN-gamma, and TGF-beta1), and chemokine receptors (CCR3, CCR4, CCR5, CCR7, CCR8, CXCR3 and CX3CR1) by immunohistochemical double-labeling and quantitative PCR on mRNA isolated from laser microdissected layers of coronary arteries. T cells in the neointima and adventitia of CAV were skewed toward an activated memory Th-1 phenotype, but in the presence of a distinct Th-2 population. FOXP3 positive T cells were not detected and production of most cytokines was low or absent, except for IFN-gamma, and TGF-beta. This typical composition of T-helper cells and especially production of IFN-gamma and TGF-beta may play an important role in the proliferative CAV reaction.

TNFalpha in patients with end-stage heart failure on medical therapy or supported by a left ventricular assist device.

BACKGROUND: In the heart elevated levels of TNFalpha can cause lethal heart failure, like Dilated Cardiomyopathy (DCM). The level of TNFalpha production is in part determined by promoter gene polymorphisms. We investigated whether the TNFalpha promoter gene polymorphism is in this way involved in the outcome of end-stage heart failure and predicts whether patients require left ventricular assist device (LVAD) support or can be kept on medical therapy (MT)while awaiting heart transplantation (HTx). As most patients in this study received a heart transplant, the role of the TNFalpha polymorphisms in transplant rejection was studied as well. METHODS AND RESULTS: In twenty nine patients with DCM, 35 patients with Ischemic Heart Disease (IHD; both on MT), 26 patients on LVAD support and 61 cardiac transplant donors TNFalpha plasma level was detected by EASIA. In both patients groups high levels of TNFalpha plasma levels was observed however, in patients supported by LVAD this increase was much higher compared to patients on MT. Furthermore, this increase seems to be associated with the TNF 1 allele ('G' at position -308) instead of the TNF2 allele (A at position -308). The promoter polymorphisms at positions -238, -244 and -308 were observed by polymerase chain reaction and sequencing. Polymorphism at positions -238, -244 and -308 did not show any relevant differences between the groups. However, at position -308, a trend of a higher incidence of the TNF2 allele (an "A" at position -308) in DCM patients compared to donors was shown. The distribution of the TNF1 and TNF2 alleles was not different in patients on medical therapy compared to the patients supported by a LVAD. No association was found between patients' TNFalpha promoter gene polymorphism and rejection. However, patients that received a donor heart with the TNF2 allele developed more rejection episodes, compared to patients that received a donor heart with the TNF1 allele. CONCLUSION: TNFalpha levels are high in patients with end-stage heart failure on MT, but even higher in patients on LVAD support. These high TNFalpha plasma levels however, are not correlated with the TNF2 allele but seems to be associated with the TNF1 allele. Furthermore, in HTx the donor TNFalpha gene seem to play a more important role in severity of acute rejection than that of the patient.

Stem cell-derived cardiomyocytes after bone marrow and heart transplantation.

Cardiomyocytes are a stable cell population with only limited potential for renewal after injury. Tissue regeneration may be due to infiltration of stem cells, which differentiate into cardiomyocytes. We have analysed the influx of stem cells in the heart of patients who received either a gender-mismatched BMT (male donor to female recipient) or a gender-mismatched cardiac transplant (HTX; female donor to male recipient). The proportion of infiltrating cells was determined by Y-chromosome in situ hybridization combined with immunohistochemical cell characterization. In BM transplanted patients and in cardiac allotransplant recipients, cardiomyocytes of apparent BM origin were detected. The proportions were similar in both groups and amounted up to 1% of all cardiomyocytes. The number of stem cell-derived cardiomyocytes did not alter significantly in time, but were relatively high in cases where large numbers of BM-derived Y-chromosome-positive infiltrating inflammatory cells were present. The number of Y-chromosome-positive endothelial cells was small and present only in small blood vessels. The number of BM-derived cardiomyocytes in both BMT and HTX is not significantly different between the two types of transplantation and is at most 1%.

Chimerism analysis within 6 months of allogeneic stem cell transplantation predicts relapse in acute myeloid leukemia.

The role of chimerism analysis as a prognostic indicator of relapse after hematopoietic stem cell transplantation (SCT) is controversial. We monitored chimerism status by short tandem repeat-based polymerase chain reaction (PCR) in T- and non-T-cell subsets and retrospectively evaluated clinical outcome in 96 patients with acute myeloid leukemia after myeloablative (MA) or reduced-intensity conditioning SCT. Fifty-six percent of 80 patients in the MA group demonstrated complete donor chimerism (CC) at all time points, whereas 6% had decreasing mixed chimerism (MC), 8% stable MC, 25% increasing MC and 3% increasing and decreasing MC. In 16 RIC patients, these percentages were 12, 50, 6, 6 and 19, respectively, together with 6% nonengraftment. Forty-three out of 96 patients experienced relapse. The last chimerism evaluation before relapse revealed increasing MC in only eight patients. In samples taken between 1 and 6 months post SCT, CC/decreasing MC was significantly related with a lower risk of relapse (31 versus 83%, P<0.000) and mortality (38 versus 83%, P<0.000) than with MC/increasing MC. However, the development of relapse was very rapid. Only very frequent monitoring of chimerism status by highly sensitive methods might identify impending relapse and allow early immunological intervention.

High level of chromosome 15 aneuploidy in head and neck squamous cell carcinoma lesions identified by FISH analysis: limited value of beta2-microglobulin LOH analysis.

In cancer research, loss of heterozygosity (LOH), defined by microsatellite markers, is frequently used in the identification of gene loss. Especially, genomic alterations in the human leukocyte antigen (HLA) genes and the beta2-microglobulin (beta2m) gene on chromosome 15 are of interest regarding their function in the immune system. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in 11 head and neck squamous cell carcinoma (HNSCC) lesions using fluorescence in situ hybridization (FISH). The 11 tumors were selected out of 53 HNSCC lesions based upon beta2m LOH analysis and beta2m expression. Centromere 1 and 15 FISH were developed to determine the chromosome 15 copy number. Sequence-based mutation analysis of beta2m was conducted on tumors without beta2m expression; no mutations in the coding sequences were found. For five HNSCC lesions with LOH and beta2m expression, centromere 15 FISH indicated gain rather than loss. In the majority of the 11 HNSCC lesions, FISH showed centromere 1 and 15 heterogeneity throughout the tumor. Moreover, FISH indicated a more complex chromosome 1 and 15 distribution than could be concluded from microsatellite LOH analysis. Our results show that microsatellite LOH analysis does not represent the beta2m gene copy number and support the results obtained from comparative genomic hybridization (CGH) studies. Conclusions on genomic alterations in tumors cannot be based on LOH data only but depend on the results of immunohistochemical staining, FISH, and CGH.

Apoptotic death of infiltrating cells in human cardiac allografts is regulated by IL-2, FASL, and FLIP.

INTRODUCTION: In vitro studies have shown that apoptotic cell death is triggered by a IL-2-dependent activation of the Fas-FasL pathway and that this pathway can be inhibited by FLIP. METHODS: To define whether FLIP regulates apoptotic death of graft infiltrating T-cells during IL-2-mediated rejection, we analyzed endomyocardial biopsies (EMB) from cardiac allograft recipients for CD3, DNA strand breaks (TUNEL assay), FLIP (mRNA and protein), and FasL mRNA expression. RESULTS: Apoptosis was present in CD3+ T-cell infiltrates. The number of TUNEL-stained mononuclear cells was inversely correlated with FLIP mRNA expression levels (P=.09). FLIP protein was present in 5% to 10% of the infiltrating cells and was constitutively produced by cardiomyocytes irrespective of the rejection grade. Rejection biopsies had elevated IL-2 and FasL mRNA expression levels compared to the expression levels before and after acute rejection (P=.03 and P=.11), while FLIP mRNA expression levels were significantly decreased during rejection (P=.05). CONCLUSION: Our results indicate that during the IL-2-induced rejection process, infiltrated T cells become more sensitive to apoptosis.

Nonlethal transfusion associated graft-versus-host disease in a severe combined immunodeficient patient.

An X-linked severe combined immunodeficient (SCID) patient received a nonirradiated erythrocyte transfusion and developed transfusion-associated graft-versus-host disease (TAGVHD), which was controllable with high-dose corticosteroids. Haplo-identical SCT was performed, after a myeloablative conditioning regimen. At day +26, he developed GVHD. Chimerism studies revealed DNA of the erythrocyte transfusion donor (ETD) and recipient only. Because of early nonengraftment and the presence of alloreactive T cells of ETD origin, the patient was treated with an immunosuppressive conditioning regimen followed by a second SCT from the same donor. While tapering immunosuppression, he again developed mild GVHD, and DNA of ETD and bone marrow donor origin were both present. On cyclosporin, the ETD-DNA signal finally disappeared. High-resolution HLA typing revealed haplo-identity between BMD, ETD and the patient, which might have contributed to the relative mild course of the TAGVHD.

Evidence for heterogeneity of T cell expansion in polymyositis and inclusion body myositis.

Vbeta usage of muscle-infiltrating T lymphocytes in polymyositis (PM) and sporadic inclusion body myositis (s-IBM) was correlated with clinical and histopathological features. Immunohistochemical analysis was combined with complementarity-determining region 3 (CDR3) length analysis in nine muscle biopsies of eight PM patients and six biopsies of five s-IBM patients. Vbeta usage was heterogeneous in seven patients. Four of these patients had definite PM with endomysial located T cell infiltrates, but T cells specifically surrounding and invading individual non-necrotic fibers were not found. In two s-IBM patients, Vbeta 2 usage was increased. In one of them, a repeat biopsy showed a heterogeneous Vbeta usage. We conclude that clonal expansion of muscle-infiltrating T cells could only be detected in part of the patients. Explanations may be that clonal expansion does not take place in all disease phases and that PM is a heterogeneous disease with respect to pathogenesis.

Prognostic factors in localised prostate cancer with emphasis on the application of molecular techniques.

Prostate cancer is the most prevalent malignancy in males in the Western world and the second leading cause of male cancer death. Prostate specific antigen (PSA) based screening and case finding leads to identification of early stage prostate cancer. It is often difficult to discriminate between patients that need curative treatment and those that can be managed conservatively. Prognostic factors are used to make this clinical decision. Based on the classification proposed by the American College of Pathologists and the World Health Organisation, selected prognostic factors in prostate cancer are described. Clinical applicable factors are stage, grade and serum PSA. Prognostic factors that are not routinely used (for various reasons) are ploidy, histological type and cancer volume in needle biopsies. All other factors (including circulating tumour cells, angiogenesis, growth factors, proliferation rate, apoptosis, nuclear morphometry, neuroendocrine differentiation, loss of chromosomal regions, tumour suppresser genes and adhesion molecules) are promising as prognostic factor although currently their use in clinical decisions is not recommended. The role of these factors in prostate cancer growth and their predictive value are discussed. The rapid developments in molecular techniques allow assessment of structure or function of thousands of genes in a prostate biopsy sample. We expect that molecular characterisation of tumour material will become a clinically important tool to predict prognosis in patients with localised prostate cancer.

Identification of human ccn2 (connective tissue growth factor) promoter polymorphisms.

BACKGROUND: Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor beta1 (TGF-beta1), interleukin 10 (IL-10), and tumour necrosis factor alpha (TNF-alpha), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions. AIM: To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression. MATERIALS/METHODS: Seventy seven human DNA samples were sequenced-45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)-using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit. RESULTS: A C to G substitution was identified at position -132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position -447. These substitutions at -132 and -447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position -43 (G), -47 (C), -71 (G) and a C to T substitution at position -198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites. CONCLUSION: Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.

No association between IL-10 promoter gene polymorphism and heart failure or rejection following cardiac transplantation.

Expression of interleukin (IL)-10 influences the frequency of rejection events after organ transplantation. Therefore, 70 heart transplant patients were genotyped for three single nucleotide polymorphisms and a microsatellite polymorphism in the promotor region of the IL-10 gene. The promoter region was amplified by polymerase chain reaction and genotyped by a colorometric oligo ligation assay and gene scan analysis, respectively. Patient groups consisted of patients suffering from dilated cardiomyopathy or ischaemic heart disease. Cardiac donors served as control group. No correlation was found between genotypes and heart failure or rejection after heart transplantation. This may indicate that in heart transplantation, the total balance of cytokine production is more important for post-transplant rejection activities than the levels of IL-10 as such.