Marny Fedrigo

The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation

During the last 25 years, antibody-mediated rejection of the cardiac allograft has evolved from a relatively obscure concept to a recognized clinical complication in the management of heart transplant patients. Herein we report the consensus findings from a series of meetings held between 2010-2012 to develop a Working Formulation for the pathologic diagnosis, grading, and reporting of cardiac antibody-mediated rejection. The diagnostic criteria for its morphologic and immunopathologic components are enumerated, illustrated, and described in detail. Numerous challenges and unresolved clinical, immunologic, and pathologic questions remain to which a Working Formulation may facilitate answers.

Can C4d immunostaining on endomyocardial biopsies be considered a prognostic biomarker in heart transplant recipients

Background. The aim of this study was to assess the significance of positive C4d capillary immunostaining of endomyocardial biopsies and its correlation to clinical outcome in adult heart transplant recipients. Methods. Nine hundred eighty-five endomyocardial biopsies from 107 heart transplant recipients were evaluated. Immunostaining for detection of intragraft C4d capillary deposition was performed on paraffin-embedded tissue using anti-human C4d polyclonal antibody. Results. Positive staining of C4d was present in 36 patients (34%) and antibody-mediated rejection in eight patients (7%). The patients were subdivided into four groups on the basis of their C4d, circulating antidonor antibodies (donor-specific antibodies [DSAs]), and graft function: group 1=C4d positive, DSA negative, and no graft dysfunction; group 2=C4d positive, DSA positive, and no graft dysfunction; group 3=C4d positive, DSA positive, and signs of graft dysfunction, and group 0 (control)=all negative. An higher mortality risk was found in C4d-positive patients, when compared with negative ones (unadjusted hazard ratios: group 1: 18, group 2: 61, and group 3: 32-fold risk; P<0.0001). Conclusions. Antibody-mediated rejection is a complex and ongoing phenomenon with different phenotypic features. C4d positive predicts worse prognosis. C4d negative and DSA can be used as early mortality predictors in patients without signs of graft dysfunction.

Gastrointestinal stromal tumors: Report of an audit and review of the literature

Gastrointestinal stromal tumors (GISTs), tumors characterized by c-KIT mutations, are the most frequent mesenchymal tumors of the digestive tract. The stomach is the most commonly involved site. Localization, size and mitotic rate are reliable predictors of survival and the two milestones of GISTs treatment are surgery and imatinib. This article is aimed to report the data of an audit, carried out on the morphological and clinical aspects of the disease and to review the present knowledge on GISTs. A total of 172 patients with GISTs (M : F=1 : 1; mean age 65 years) were recruited. The stomach was the most frequently involved site. In 50% of the cases the tumor was smaller than 5 cm, whereas major symptoms were observed in 43% of the cases. Predictors of progressive disease were present only in a small percentage of cases but the disease was in the metastatic phase in over 25% of the cases at diagnosis. Familial aggregation was rare but a consistent share of the patients (21%) had other synchronous or metachronous cancers. The most frequent mutations were in-frame deletions and point mutations of c-KIT exon 11. This report confirms in part the available data on GIST in a consecutive series of patients recruited in Italy and shows that only large collaborative multicenter studies provide data sound enough to enable making reasonable clinical and therapeutic choices, and suggests that, as a measure of secondary prevention, a diagnostic definition should be obtained in all submucosal lesions of the GI tract and that GIST patients should be screened for second tumors.

Role of morphologic parameters on endomyocardial biopsy to detect sub-clinical antibody-mediated rejection in heart transplantation.

BACKGROUND The present study evaluated if morphologic parameters detect signs of early sub-clinical or latent stages of antibody-mediated rejection (AMR) and their correlation with C4d staining in cardiac transplants recipients. METHODS The study reviewed 1,270 endomyocardial biopsies (EMB) from 131 patients. Of these, 61 stained positive for C4d in the absence of acute cellular rejection >2R. Sixty-six EMB specimens negative for C4d were matched for pre-transplant diagnosis, time after transplantation, age, and acute cellular rejection (ACR) grading. Histopathologic evaluation and C4d staining were performed on formalin-fixed, paraffin-embedded sections using the C4d polyclonal antibody. RESULTS Of the 8 histologic characteristics evaluated, only endothelial swelling (78.7% sensitivity, 28.8% specificity; positive likelihood ratio, 1.10) and interstitial edema (77% sensitivity, 31.8% specificity; positive likelihood ratio, 1.13) could be considered fair predictors of C4d capillary positivity. The presence of mononuclear cells in capillaries in relation to C4d positivity showed 39.3% sensitivity and 68.2% specificity. Combining the parameters endothelial swelling and mononuclear cells in capillaries, sensitivity was 31.1% (95% confidence interval [CI] 19.9-44.3) and specificity was 71.2% (95 CI, 58.8-81.7), with a positive likelihood ratio of 1.08 (95% CI, 0.68-1.84). CONCLUSIONS Our results showed that histologic parameters did not always detect signs of early sub-clinical or latent stages of AMR. Combining the parameters of endothelial swelling and intracapillary mononuclear cells did not significantly improve the sensitivity or specificity. Screening recommendations should, therefore, be modified to include more sensitive tests such as C4d staining in the routine protocol to improve patient risk stratification.

Intravascular macrophages in cardiac allograft biopsies for diagnosis of early and late antibody-mediated rejection

BACKGROUND The aim of our study was to evaluate the role of intravascular macrophages in the diagnosis of early and late antibody-mediated rejection (AMR) on endomyocardial biopsies (EMBs). METHODS We reviewed 1,420 consecutive EMBs from 131 patients and selected 75 C4d+ EMBs. The C4d+ group was compared with a control group (66 patients) matched for age, gender, date of transplantation, follow-up, immunosuppressive regimen and primary heart disease. A total of 141 EMBs were evaluated. Immunoperoxidase staining for C4d and CD68 were performed. Post-transplant IgG anti-HLA reactivity was investigated by Luminex technology. Clinical data were also collected. Fourteen EMBs were available from 11 symptomatic AMR patients. RESULTS Of the 141 EMBs evaluated, 53 were positive for intravascular macrophages (CD68); among them, 32 were also positive for C4d (32 of 53, 60.4%). Of the 88 CD68- EMBs, 43 were also C4d+ (43 of 88, 48.9%). Of the 53 CD68+ EMBs, 30 EMBs were within the first year since transplantation (30 of 53, 57.8%), and among these 21 were also positive for C4d (21 of 30, 70.0%). In the late period, among the 23 CD68+ EMBs (23 of 53, 42.2%) 11 were also positive for C4d (11 of 23, 47.8%). In the early period, intravascular macrophages were more common in symptomatic (3 of 3, 100%) than asymptomatic (3 of 11, 27.3%) patients. Sensitivity and specificity of intravascular macrophages in predicting donor-specific antibodies (DSA) within the first year were 50.0% and 100.0%, respectively. CONCLUSIONS Intravascular macrophages predict C4d, DSA and symptoms early after transplantation; however, in the late period, they are unable to identify patients with circulating DSA, C4d and/or symptoms.

Bone-marrow–derived CXCR4-positive tissue-committed stem cell recruitment in human right ventricular remodeling

The epicardium contributes to cardiac formation, particularly during embryogenesis. It remains to be seen if it is also involved in postnatal myocardial homeostasis. This study evaluates the topographic distribution of stem cells (c-Kit) and extracardiac progenitor cells (CXCR4+) and their contribution to ventricular remodeling in a model of pressure volume overload leading to right ventricle hypertrophy. Eleven specimens with hypoplastic left heart syndrome were evaluated and compared with 6 normal hearts from subjects matched for age and weight. All underwent Norwood procedure with the right ventricle becoming a systemic one, with pressure and volume overload leading to right ventricle remodeling. Transmural cardiac tissue samples from the right ventricle were analyzed by immunohistochemistry and morphometry. This is the first study to demonstrate that c-Kit-positive progenitor cells and tissue-committed stem cells (CXCR4+/CD45-) are higher in children with systemic right ventricle remodeling. We also show that the localization of cardiac progenitor and recruited CXCR4+ stem cells in the myocardium is site specific in hearts with right ventricle hypertrophy. These cells are mainly scattered in the interstitium of the epicardial layer. In contrast, myocyte proliferation is not a key process in right ventricular hypertrophy. Induced by the overexpression of SDF-1α by the myocardium, CXCR4 cell mobilization resembles SDF-1 homing factor distribution, showing transmural enhanced expression from the endocardium toward the epicardium. The study provides evidences of the site-specific epicardial localization of stem cells in a model of pressure/volume overload and suggests that the epicardium acts as a permissive niche in normal and pathologic conditions.

The clinical performance of a chemiluminescent immunoassay in detecting anti-cardiolipin and anti-β2 glycoprotein I antibodies. A comparison with a homemade ELISA method

Abstract Background: Fully automated chemiluminescence immunoassays (CLIAs) are emerging technologies for the detection of anti-cardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies for anti-phospholipid syndrome (APS) classification, which is commonly based on an enzyme-linked immunosorbent assay (ELISA) test result. CLIA and a homemade ELISA were used in this study to detect these antibodies, and their performances were compared. Methods: Sera were collected from 104 patients with primary APS, 88 seronegative subjects who met the clinical but not the laboratory criteria for APS, and 150 control subjects. IgG/IgM aCL and IgG/IgM anti-β2GPI antibodies were determined in the sera using a CLIA (HemosIL AcuStar®) and a homemade ELISA. Results: CLIA had a significantly lower comparative sensitivity for IgM aCL and IgG/IgM IgG anti-β2GPI antibodies; its comparative specificity was higher with respect to ELISA for IgM aCL and IgM anti-β2GPI antibodies. The two techniques showed a high, significant agreement (p<0.001) and a significant titer correlation (p<0.001). CLIA also detected IgG/IgM aCL and IgG anti-β2GPI antibodies in the seronegative patients. There was a significantly higher prevalence of IgG aCL and IgG anti-β2GPI antibodies (p<0.001 and p=0.01, respectively) in those patients with respect to that in the control population. Conclusions: Despite a lower comparative sensitivity, CLIA showed a higher comparative specificity for some aPL and a good level of agreement and correlation with a homemade ELISA. CLIA also detected some aCL and anti-β2GPI antibodies in the seronegative patients not usually identified by homemade ELISA.

Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging

Cardiovascular diseases are the world’s number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na+, K+, and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.FigureWorkflow of the 3D reconstruction. A Tissue section, B gold deposition is done by sputter coating, C, C1 SIMS-ToF mass analyzer, C, C2 mass spectral peaks, C, C3 datacube images; D, E Reconstruction of the heart showing 3D-spatial distributions of three different ions 145 m/z (red), 23 m/z (green), and 39 m/z (blue); F coregistration of 40 individual MS imaging

Coronary cardiac allograft vasculopathy versus native atherosclerosis: difficulties in classification

Cardiac allograft vasculopathy is regarded as a progressive and diffuse intimal hyperplastic lesion of arteries and veins that leads to insidious vessel narrowing and to allograft ischemic disease, such as acute myocardial infarction or sudden cardiac death. The coronary lesions in transplanted hearts are considered as a particular type of arteriosclerosis with many similarities but also significant differences compared to native coronary atherosclerosis. It is particularly difficult for pathologists to systematically classify the lesions and to elucidate their origins, since over time, the allograft immune responses cause vascular pathology characterized by not only the onset of de novo fibrocellular lesions but also remodeling of already-existing native atherosclerotic lesions in the donor heart. Intraplaque hemorrhages, which result from newly formed leaky microvessels, may cause rapid increase of stenosis and generate a substrate for plaque destabilization. Comparing cardiac allograft vasculopathy from explanted hearts at autopsy with native coronary atherosclerosis from hearts removed at transplantation has revealed that ongoing intraplaque hemorrhages are also an important feature of cardiac allograft vasculopathy and may be important factors in the rapid progression of cardiac allograft vasculopathy.

Recent Developments on Coronary Microvasculopathy after Heart Transplantation:A New Target in the Therapy of Cardiac Allograft Vasculopathy

Heart transplantation (HTx) is the treatment of choice for patients with refractory end-stage heart diseases. Although the procedure is considered effective in extending and improving quality of life, the onset of cardiac allograft vasculopathy (CAV) continues to limit the long-term success of HTx. Emerging data indicate that the endothelium plays a significant role in the onset, progression and complication of this multifactorial disease, with both immunologic and nonimmunologic risk factors contributing to its development. Improving our understanding of the integral role of the coronary microcirculation in CAV is of crucial clinical interest since it could provide further insights into the related pathophysiological mechanisms and possible new strategies for CAV prevention and therapy. Assessment of coronary microvasculopathy has been shown to be of predictive value after HTx. Predominant allograft microvascular dysfunction is detectable in 15-20% of patients after HTx. Very recently, stenotic microvasculopathy (detected in biopsy samples) has been characterized as a prognostic factor for long-term survival after HTx. The ability to detect and distinguish changes in epicardial and microvascular function may aid in identifying modifiable factors that lead to CAV. Improved immunosuppressive drugs, including mycophenolate mofetil and proliferation signal inhibitors, as well as statins (in part via immunomodulation), may have a beneficial effect on coronary microcirculation after HTx, although there is still a need to confirm the impact of vasodilators in improving the prognosis of HTx patients. We review the role of coronary microvasculopathy in HTx, its prevention and new potential pharmacological interventions.