Alex P.W.M. Maat

Determination of the Molecular Relationship Between Multiple Tumors Within One Patient Is of Clinical Importance

PURPOSE To determine the molecular relationship between multiple tumors within one patient and to evaluate the impact of this knowledge on clinical management. PATIENTS AND METHODS In 25 consecutive patients with multiple tumors, proven by histology and immunohistochemistry to be identical, molecular aberrations were determined. Each patient had at least one lesion in the lung or head and neck region. Loss of heterozygosity (LOH) and p53 aberration analyses were carried out, and similar aberration profiles suggest clonality and metastasis whereas different profiles suggest independent primary tumors. RESULTS The molecular determinations indicated that 12 patients had a probable second primary tumor and 10 patients had a metastasis of the first lesion. In three patients, both an independent primary tumor and a metastasis were present. The molecular findings determined the course of additional treatment in all 10 patients with metastases, in all three patients with both a second primary tumor and a metastasis, and in seven of 12 patients with a second primary tumor. CONCLUSION By comparing DNA alterations of multiple tumors within one patient, the relationship between the tumors can be assessed. This study shows that in 20 of 25 patients, knowledge of the nature of both lesions was essential in clinical decision making. Furthermore, after thorough analysis of the five cases where clinical decision making was not influenced, there was in retrospect no clear indication for LOH or p53 analysis. Because these molecular analyses can be performed on routine specimens, they can be applied in almost all patients.

The transforming growth factor-beta1 codon 10 gene polymorphism and accelerated graft vascular disease after clinical heart transplantation

Background. The multifunctional cytokine transforming growth factor-(TGF) &bgr;1 is thought to play a role in the pathogenesis of graft vasculardisease (GVD). Polymorphisms at codon 10,(Leu 10 →Pro) and codon 25(Arg 25 →Pro) in the signal sequence of theTGF-&bgr;1 gene regulate the production and secretion of the protein. Weinvestigated whether these polymorphisms are risk factors for the developmentof GVD after clinical hearttransplantation. Method. TGF-&bgr;1 polymorphisms,Leu 10 →Pro andArg 25 →Pro, were determined in DNA from hearttransplant recipients (n=252) and their donors (n=213), usingsequence-specific oligonucleotide probing. GVD was angiographically diagnosed1 year after transplantation. In addition other potential risk factorsincluding underlying disease, recipient and donor age, recipient and donorgender, number of acute rejections in the first year, cold ischemia time, andHLA mismatches were analyzed by univariate and multivariate logisticregressionanalysis. Results. Univariate analysis showed that the recipient TGF-&bgr;1polymorphism Leu 10 →Pro,(P =0.056,&khgr; 2 test), underlying disease(P =0.01,&khgr; 2 test), number of acute rejections in thefirst-year (P =0.03, analysis ofvariance), and donor age (P <0.001,analysis of variance) were risk factors for the development of GVD. TheTGF-&bgr;1 Arg 25 →Pro polymorphism was not arisk factor. Also in the multivariate analysis, the recipient TGF-&bgr;1 codon10 polymorphism was associated with GVD, with patients homozygous for Pro atgreatest risk (odds ratio 7.7, P =0.03). Apart for therecipient TGF-&bgr;1 Leu 10 →Pro polymorphism,donor age appeared to be an independent risk factor for the development of GVDat 1 year. Patients with older donor hearts were at greater risk than patientsreceiving grafts from younger donors (odds ratio 1.1/year, P <0.001). Conclusion. Recipient TGF-&bgr;1Leu 10 →Pro polymorphism and higher donor ageare independent risk factors for the development of GVD after clinical hearttransplantation.

Intragraft FOXP3 mRNA expression reflects antidonor immune reactivity in cardiac allograft patients

Background. Regulatory FOXP3+ T cells control immune responses of effector T cells. However, whether these cells regulate antidonor responses in the graft of cardiac allograft patients is unknown. Therefore, we analyzed the gene expression profiles of regulatory and effector T-cell markers during immunological quiescence and acute rejection. Methods. Quantitative real-time polymerase chain reaction was used to analyze mRNA expression levels in time-zero specimens (n=24) and endomyocardial biopsies (EMB; n=72) of cardiac allograft patients who remained free from rejection (nonrejectors; n=12) and patients with at least one histologically proven acute rejection episode (rejectors; International Society for Heart and Lung Transplantation [ISHLT] rejection grade >2; n=12). Results. For all analyzed regulatory and effector T-cell markers, mRNA expression levels were increased in biopsies taken after heart transplantation compared with those in time-zero specimens. Posttransplantation, the FOXP3 mRNA levels were higher in EMB assigned to a higher ISHLT rejection grade than the biopsies with grade 0: the highest mRNA levels were detected in the rejection biopsies (rejection grade >2; P=0.003). In addition, the mRNA levels of CD25, glucocorticoid-induced TNF receptor family-related gene, cytotoxic T lymphocyte-associated antigen 4, interleukin-2, and granzyme B were also significantly higher in rejecting EMB than in nonrejecting EMB (rejection grade ≤2). This increase in expression levels in relation to the histological rejection grade was only observed in patients who developed an acute rejection episode; the mRNA levels of nonrejectors remained stable irrespective of ISHLT rejection grade. Conclusions. These observations suggest that, after clinical heart transplantation, FOXP3+ T cells do not prevent acute rejection, but rather are a response to antidonor effector T-cell activity.

Donor-Derived Mesenchymal Stem Cells Remain Present and Functional in the Transplanted Human Heart

Mesenchymal stem cells (MSC) are characterized by their multilineage differentiation capacity and immunosuppressive properties. They are resident in virtually all tissues and we have recently characterized MSC from the human heart. Clinical heart transplantation offers a model to study the fate of transplanted human MSC. In this study, we isolated and expanded MSC from heart tissue taken before, and 1 week up to 6 years after heart transplantation. MSC from posttransplantation tissue were all of donor origin, demonstrating the longevity of endogenous MSC and suggesting an absence of immigration of recipient MSC into the heart. MSC isolated from transplanted tissue showed an immunophenotype that was characteristic for MSC and maintained cardiomyogenic and osteogenic differentiation capacity. They furthermore preserved their ability to inhibit the proliferative response of donor‐stimulated recipient peripheral blood mononuclear cells. In conclusion, functional MSC of donor origin remain present in the heart for several years after transplantation.

Preferential depletion of blood myeloid dendritic cells during acute cardiac allograft rejection under controlled immunosuppression.

Allo‐Ag presentation to Ag‐specific T‐lymphocytes by donor or recipient dendritic cells (DCs) induces acute rejection (AR) after solid organ transplantation. It is postulated that myeloid (mDC) and plasmacytoid (pDC) subsets circulate differentially between bone marrow, heart and lymphoid tissues after cardiac transplantation (HTx). We investigated peripheral blood DC subset distribution, maturation and lymphoid homing properties in relation to endomyocardial biopsy (EMB) rejection grade after clinical HTx. Twenty‐one HTx recipients under standard immunosuppression were studied in a 9‐month follow‐up. mDC and pDC numbers were analyzed by flow cytometry in fresh venous whole blood samples collected during the EMB procedures and before histological diagnosis of AR. Subsets were further characterized for maturation marker CD83 and lymphoid homing chemokine receptor CCR7. Although numbers of both DC subsets remained low for the whole post‐HTx period, we observed a negative association of mDCs with rejection grade. Repeated measurements analysis revealed that only mDCs decreased during AR episodes. Rejectors had lower mDC numbers after a 3‐month follow‐up compared to nonrejectors. Furthermore, patients during AR exhibited low proportions of mDCs positive for CD83 or CCR7. These findings suggest peripheral blood mDC depletion in association with selective lymphoid homing of this subset during AR after clinical HTx.

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)‐γ producing cells determined by enzyme‐linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN‐γ producing cells reactive to this pathway increased significantly (P = 0·04) during AR and the number decreased (P = 0·005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN‐γ producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.

Interleukin-21: An interleukin-2 dependent player in rejection processes

Background. Interleukin (IL)-21 is the most recently described cytokine that signals via the common cytokine receptor (γc), is produced by activated CD4+ T-cells, and regulates expansion and effector function of CD8+ T-cells. Materials. To explore the actions of IL-21 with other γc-dependent cytokines in alloreactivity, mRNA expression of IL-21, IL-21R α-chain, and IL-2 proliferation and cytotoxicity was measured after stimulation in mixed lymphocyte reactions. Additionally, IL-21 and IL-21R α-chain expression was studied in biopsies of heart transplant patients. Results. Analysis of mRNA expression levels of allostimulated T-cells showed a 10-fold induction of IL-21 and IL-21R α-chain. Interestingly, induction of IL-21 was highly dependent on IL-2 (as in the presence of anti-IL-2, anti-IL-2R α-chain, and the immunosuppressive drugs cyclosporine A, tacrolimus, and rapamycin) the transcription of IL-21 was almost completely inhibited, whereas in the presence of exogenous IL-2 the mRNA expression of IL-21 was even more upregulated. IL-21 functioned as a costimulator for IL-2 to augment proliferation and cytotoxic responses, while blockade of the IL-2 route abrogated these functions of IL-21. Blockade of the IL-21 route by anti-IL-21R α-chain monoclonal antibodies inhibited the proliferation of alloactivated T-cells. Also, in vivo alloreactivity was associated with IL-21/IL-21R α-chain expression. After heart transplantation, the highest intragraft IL-21, IL-21R α-chain, and IL-2 mRNA expression levels were measured during acute rejection (P<0.001, P=0.01, P=0.03). Conclusion. IL-21 is a critical cytokine for IL-2 dependent immune processes. Blockade of the IL-21 pathway may provide a new perspective for the treatment of allogeneic responses in patients after transplantation.

Inadequate immune regulatory function of CD4+CD25bright+FoxP3+ T cells in heart transplant patients who experience acute cellular rejection.

Background. CD4+CD25bright+FoxP3+ regulatory T cells are suppressors of antigen-activated immune reactivity. Here, we assessed the clinically relevant role of these cells in the control of immune responses directed to a transplanted heart. Methods. We investigated the phenotype and function of peripheral CD4+CD25bright+FoxP3+ T cells in heart transplant patients free from acute rejections (n=9) and in rejectors (n=12) before and during acute cellular rejection. Results. Between rejectors and nonrejectors, the proportion of CD4+CD25bright+ T cells and of FoxP3+ cells within this population was comparable. Yet, CD4+CD25bright+FoxP3+ T cells of rejectors had a higher CD127 expression than those of nonrejectors (P<0.0001). Depletion of CD4+CD25bright+ T cells from peripheral blood mononuclear cells increased the antidonor proliferative response of both nonrejectors (P=0.0005) and rejectors (P=0.03). In rejectors, however, only a 2-fold increase was measured, whereas the nonrejectors’ response became 14 times higher (P=0.002). Reconstitution of CD4+CD25bright+ T cells only suppressed the overall antidonor proliferative response of CD25neg/dim responder cells of nonrejectors significantly (P=0.001). Moreover, the percentage inhibition of the response was higher in nonrejectors than in rejectors (P=0.02). Analyses of pretransplant samples revealed that CD4+CD25bright+ T cells of rejectors already had a lower suppressive capacity than those of nonrejectors before transplantation (P=0.04). Conclusion. CD4+CD25bright+FoxP3+ T cells of heart transplant patients who experience acute rejection had an up-regulated CD127 expression and an inadequate regulatory function compared with those of nonrejecting patients. Our observations suggest that the function of circulating CD4+CD25bright+FoxP3+ regulatory T cells may be pivotal for the prevention of acute cellular rejection after clinical heart transplantation.

New Roads Open Up for Implementing Immunotherapy in Mesothelioma

Treatment options for malignant mesothelioma are limited, and the results with conventional therapies have been rather disappointing to this date. Chemotherapy is the only evidence-based treatment for mesothelioma patients in good clinical condition, with an increase in median survival of only 2 months. Therefore, there is urgent need for a different approach to battle this malignancy. As chronic inflammation precedes mesothelioma, the immune system plays a key role in the initiation of this type of tumour. Also, many immunological cell types can be found within the tumour at different stages of the disease. However, mesothelioma cells can evade the surveillance capacity of the immune system. They build a protective tumour microenvironment to harness themselves against the immune systems attacks, in which they even abuse immune cells to act against the antitumour immune response. In our opinion, modulating the immune system simultaneously with the targeting of mesothelioma tumour cells might prove to be a superior treatment. However, this strategy is challenging since the tumour microenvironment possesses numerous forms of defence strategies. In this paper, we will discuss the interplay between immunological cells that can either inhibit or stimulate tumour growth and the challenges associated with immunotherapy. We will provide possible strategies and discuss opportunities to overcome these problems.

Abdominal aortic aneurysms after heart transplantation

BACKGROUND In recent years abdominal aortic aneurysms were diagnosed in several heart transplant recipients at our center. Only case reports or small series have been reported previously and little is known about abdominal aortic aneurysms after heart transplantation. Therefore, the goals of this study were to estimate the incidence of this condition after heart transplantation, to identify risk factors for its development, and to assess its clinical consequences. METHODS Our investigation was a retrospective, single-center cohort study of 368 consecutive patients transplanted between 1984 and 1999. RESULTS During a mean follow-up of 75 +/- 49 months, 37 of the 368 (10%) transplant recipients and 36 of 202 (18%) of the sub-group with a history of ischemic heart disease were found to have an abdominal aortic aneurysm. All patients were male, and all except 1 had a history of ischemic heart disease. A history of ischemic heart disease prior to heart transplantation was the sole independent risk factor for developing an aneurysm by multivariate analysis. Aneurysm-related events occurred earlier and more frequently in the 7 transplant recipients who already had a dilated abdominal aorta prior to transplantation. The abdominal aortic aneurysm was the direct or indirect cause of death in at least 9 patients. CONCLUSIONS Abdominal aortic aneurysms are relatively frequent after heart transplantation, occur at a younger age than in the general population, and have serious clinical consequences. Close ultrasonographic follow-up of patients with a history of ischemic heart disease or with an abnormal abdominal aorta prior to heart transplantation seems indicated.