Roberta Assante

Diagnostics of Primary Immunodeficiencies through Next-Generation Sequencing

Background Recently, a growing number of novel genetic defects underlying primary immunodeficiencies (PIDs) have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS) technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome. Objective To evaluate the role of targeted NGS and whole exome sequencing (WES) in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures. Methods We retrospectively analyzed genetic variants identified through targeted NGS or WES in 45 patients with complex PID of unknown etiology. Results Forty-seven variants were identified using targeted NGS, while 5 were identified using WES. Newly identified genetic variants were classified into four groups: (I) variations associated with a well-defined PID, (II) variations associated with atypical features of a well-defined PID, (III) functionally relevant variations potentially involved in the immunological features, and (IV) non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%). Among them, four patients presented with a typical well-defined PID. In the remaining three cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causative per se of the disorder. Conclusion NGS technologies represent a cost-effective and rapid first-line genetic approach for the evaluation of complex PIDs. WES, despite a moderate higher cost compared to targeted, is emerging as a valuable tool to reach in a timely manner, a PID diagnosis with a considerable potential to draw genotype–phenotype correlation. Nevertheless, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants might be proven informative in future studies with larger cohorts of patients.

Incremental prognostic value of stress myocardial perfusion imaging in asymptomatic diabetic patients

OBJECTIVE Stress myocardial perfusion single-photon emission computed tomography (MPS) variables are robust estimators of prognosis. No data are available on the comparative ability of stress MPS risk markers using varied iterative and risk classification approaches in asymptomatic diabetic patients. We compared analytical approaches to estimate the added value of MPS variables in estimating coronary artery disease (CAD) outcomes in asymptomatic diabetic patients. We also evaluated the temporal characteristics of cardiac risk according to MPS findings. METHODS A total of 436 consecutive asymptomatic diabetic patients who underwent stress-rest gated MPS were prospectively enrolled. Multivariable Cox proportional hazards model was employed to estimate cardiac death and nonfatal myocardial infarction (MI). Risk reclassification was calculated and parametric survival analysis was used to predict time to events. RESULTS At multivariable analysis, post-stress left ventricular ejection fraction (LVEF) and stress MPS ischemia were independent predictors of CAD death or MI (both p < 0.01). The net reclassification improvement by adding MPS results to a model including pre-test CAD likelihood was 0.25 (95% confidence interval 0.06-0.44; p < 0.01). Parametric survival analysis showed the highest probability of CAD death or MI and the major risk acceleration in time in patients with stress MPS ischemia and post-stress LVEF ≤45%. CONCLUSION In asymptomatic diabetic patients, analytical approaches that establish the reclassification of events may serve for estimation of improved outcomes for stress MPS. Post-stress LVEF and stress-induced ischemia by gated MPS influence the temporal characteristic of the patients risk at long-term follow-up.

APECED: A Paradigm of Complex Interactions between Genetic Background and Susceptibility Factors

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disease, caused by mutations of a single gene named Autoimmune regulator gene (AIRE) which results in a failure of T-cell tolerance. Central tolerance takes place within the thymus and represents the mechanism by which potentially auto-reactive T-cells are eliminated through the negative selection process. The expression of tissue-specific antigens (TSAs) by medullary thymic epithelial cells (mTECs) in the thymus is a key process in the central tolerance and is driven by the protein encoded by AIRE gene, the transcription factor autoimmune regulator (AIRE). A failure in this process caused by AIRE mutations is thought to be responsible of the systemic autoimmune reactions of APECED. APECED is characterized by several autoimmune endocrine and non-endocrine manifestations and the phenotype is often complex. Although APECED is the paradigm of a monogenic autoimmune disorder, it is characterized by a wide variability of the clinical expression even between siblings with the same genotype, thus implying that additional mechanisms, other than the failure of Aire function, are involved in the pathogenesis of the disease. Unraveling open issues of the molecular basis of APECED, will help improve diagnosis, management, and therapeutical strategies of this complex disease.

Quantitative relationship between coronary artery calcium and myocardial blood flow by hybrid rubidium-82 PET/CT imaging in patients with suspected coronary artery disease

BackgroundWe assessed the relationship between coronary artery calcium (CAC) score, myocardial blood flow (MBF) and coronary flow reserve (CFR) in patients undergoing hybrid 82Rb positron emission tomography (PET)/computed tomography (CT) imaging for suspected CAD. We also evaluated if CAC score is able to predict a reduced CFR independently from conventional coronary risk factors.MethodsA total of 637 (mean age 58 ± 13 years) consecutive patients were studied. CAC score was measured according to the Agatston method and patients were categorized into 4 groups (0, 0.01-99.9, 100-399.9, and ≥400). Baseline and hyperemic MBF were automatically quantified. CFR was calculated as the ratio of hyperemic to baseline MBF and it was considered reduced when <2.ResultsGlobal CAC score showed a significant inverse correlation with hyperemic MBF and CFR (both P < .001), while no correlation between CAC score and baseline MBF was found. At multivariable logistic regression analysis age, diabetes and CAC score were independently associated with reduced CFR (all P < .001). The addition of CAC score to clinical data increased the global chi-square value for predicting reduced CFR from 81.01 to 91.13 (P < .01). Continuous net reclassification improvement, obtained by adding CAC score to clinical data, was 0.36.ConclusionsCAC score provides incremental information about coronary vascular function over established CAD risk factors in patients with suspected CAD and it might be helpful for identifying those with a reduced CFR.

Prognostic role of 18F-FDG PET/CT in the postoperative evaluation of differentiated thyroid cancer patients

Purpose The aim of this study was to evaluate the role of 18F-FDG PET/CT performed after surgery but before radioiodine therapy in patients with differentiated thyroid cancer. Procedures FDG PET/CT was performed off l-thyroxine in 60 newly diagnosed differentiated thyroid cancer patients. Clinical and hematological evaluation as well as high-resolution neck ultrasound were performed. All patients underwent a complete follow-up (range, 6-67 months; mean [SD], 31.7 [20.6] months). The date of recurrence or the most recent office visit was recorded. Progression-free survival (PFS) is the primary end point of this study. Analysis was performed by Cox proportional hazards model. Survival curves were generated using Kaplan-Meier estimates, and the log-rank test was used to assess significance. Results FDG PET/CT was negative in 63% of patients, 20% had FDG thyroid bed uptake, 5% distant metastases, and 12% lymph node FDG uptake. In patients with positive FDG PET/CT scan (ie, those with distant metastases or lymph node uptake), a higher rate of recurrence was observed (50% vs 6%, P < 0.05). Thyroglobulin, neck ultrasound, stage, and FDG PET/CT correlated with PFS at univariate analysis. At multivariate analysis, only thyroglobulin and FDG PET/CT continued to be predictors of PFS. Patients with a negative FDG PET/CT scan have a better PFS either in the whole group or in those with elevated thyroglobulin level (both >2 ng/mL and >10 ng/mL). Conclusions FDG PET/CT was abnormal in 17% of patients. Moreover, FDG PET/CT has an independent prognostic role, with a better PFS in patients with a negative scan.

Long-Term Survival Benefit of Coronary Revascularization in Patients Undergoing Stress Myocardial Perfusion Imaging

BACKGROUND We assessed the relationship between clinical outcome and coronary revascularization according to stress-gated myocardial perfusion single-photon emission computed tomography (MPS) in an observational series of patients with suspected or known coronary artery disease (CAD), on long-term follow-up. METHODSANDRESULTS The study group consisted of 2,059 patients. During a median follow-up of 61 months, 184 events occurred (126 cardiac deaths and 58 non-fatal MI). The impact of revascularization during follow-up on event-free survival was evaluated using an extended Cox regression model, adjusting for potential clinical and MPS confounders. Revascularization was treated as a binary non-reversible time-dependent covariate. Predefined interactions tested were: (1) revascularization and summed difference score (SDS); (2) revascularization and post-stress left ventricular (LV) ejection fraction (EF); and (3) SDS and post-stress LVEF. Revascularization had a significant effect on event-free survival (adjusted HR, 0.19; P<0.001). Significant interactions were found between revascularization and SDS (P=0.045), and between LVEF and SDS (P=0.015). The protective effect of revascularization increased as SDS increased. For SDS <6 the reduction in HR was detectable only for reduced LVEF. CONCLUSIONS Both the degree of stress-induced ischemia and LVEF predict the effect of revascularization on outcome in patients with suspected or known CAD. The protective effect of revascularization appears to be greater in patients with severe ischemia and preserved LVEF.

MIBG molecular imaging for evaluating response to chemotherapy in patients with malignant pheochromocytoma: preliminary results.

Abstract Malignant pheochromocytomas respond to chemotherapy with a reduction in tumor size and catecholamine secretion. We investigated the usefulness of molecular imaging with meta-iodobenzylguanidine (MIBG) for evaluating the effects of chemotherapy in patients with malignant pheochromocytoma. Six patients were studied before and after 6 ± 4 months of combination chemotherapy with cyclophosphamide, vincristine, and dacarbazine. Urinary catecholamines, metanephrines, and vanillylmandelic acid (VMA) levels were measured before and after chemotherapy. [131I]MIBG uptake was calculated for each tumor lesion on images before and after chemotherapy. An intensity ratio (IR) of abnormal to normal tissue count density was used to evaluate the change in lesion activity with therapy. Urinary catecholamines, metanephrines, and VMA significantly decreased with chemotherapy. MIBG uptake decreased in most lesions and the reduction in overall IR correlated with the reduction in urinary VMA. However, the change in individual lesions was variable and MIBG IR did not change or increased in a number of lesions. In conclusion, MIBG imaging is useful in the evaluation of patients with malignant pheochromocytoma who are receiving chemotherapy. It can provide not only a measure of overall effectiveness of treatment but also allows a lesion-by-lesion evaluation of the heterogeneity of response to chemotherapy.

Molecular evidence for a thymus-independent partial T cell development in a FOXN1-/- athymic human fetus.

The thymus is the primary organ able to support T cell ontogeny, abrogated in FOXN1−/− human athymia. Although evidence indicates that in animal models T lymphocytes may differentiate at extrathymic sites, whether this process is really thymus-independent has still to be clarified. In an athymic FOXN1−/− fetus, in which we previously described a total blockage of CD4+ and partial blockage of CD8+ cell development, we investigated whether intestine could play a role as extrathymic site of T-lymphopoiesis in humans. We document the presence of few extrathymically developed T lymphocytes and the presence in the intestine of CD3+ and CD8+, but not of CD4+ cells, a few of them exhibiting a CD45RA+ naïve phenotype. The expression of CD3εεpTα, RAG1 and RAG2 transcripts in the intestine and TCR gene rearrangement was also documented, thus indicating that in humans the partial T cell ontogeny occurring at extrathymic sites is a thymus- and FOXN1-independent process.

Real-time gated-SPECT myocardial perfusion imaging with CZT detectors: A promising tool for monitoring left ventricular function

The use of stress single-photon emission computed tomography (SPECT) cardiac imaging for the assessment of myocardial perfusion has become one of the most used noninvasive approaches for the management of patients with known or suspected coronary artery disease (CAD). The possibility to perform gatedSPECT as a routine part of clinical protocols offers the advantage to add to perfusion data functional parameters, such as left ventricular (LV) volumes, ejection fraction (EF), and regional wall motion, increasing the diagnostic accuracy and clinical utility of cardiac SPECT imaging. The assessment of LV volumes and EF represents a powerful and reliable method for the prediction of long-term prognosis and for clinical decision making in patients with heart diseases. There are many automated algorithms for quantification of SPECT parameters of myocardial perfusion and LV function, which have demonstrated accuracy and reproducibility. For the assessment of LV volumes and function, cardiac magnetic resonance (CMR) is considered the reference standard noninvasive method for validation of other imaging techniques. It is due to the characteristics of CMR imaging, a method that does not depend on geometric assumptions of LV shape. This approach is characterized by high reproducibility, excellent temporal and spatial resolution, and low interobserver and intraobserver variability. A good agreement of LV functional parameters between gated-SPECT by conventional Anger camera and CMR imaging has been demonstrated both at rest and post-stress. However, it has also been shown that gated-SPECT LV volumes and functional parameters may be underestimated by using standard cameras compared to CMR, showing high variability. Probably, the underestimation of end-diastolic and end-systolic volumes may be mainly explained by the limited SPECT temporal and spatial resolution. Other factors that may contribute to the discrepancies between these two modalities include the use of different algorithms and the possibility that in patients with large perfusion defects, severe reduction or absence of photon counts in a myocardial region may lead to an underestimation of regional wall motion, due to inadequate visualization. Using conventional Anger camera with waiting periods after stress tracer injection of at least 15 minutes, but sometimes also up to 60 minutes, post-stress LV function and volumes may be underestimated and early ischemic functional changes after stress test might be not identifiable. In addition, acquisition time with conventional cameras is very long and this may lead to a resolution of the LV function during this long time. All these characteristics do not allow to identifying post-stress LV function and wall motion abnormalities, which have been demonstrated to be correlated with severe obstructive CAD. The introduction of new dedicated cardiac cameras based on cadmium-zinc-telluride (CZT) semiconductor technology, characterized by a higher photon sensitivity and higher temporal and spatial resolution than standard systems, could overcome most of the limitations of conventional Anger cameras. The use of CZT camera technology enables a significant reduction in both radiation exposure and acquisition time without loss of image quality. Previous studies have demonstrated excellent agreement between CZT SPECT and CMR imaging for the measurement of LVEF, although significant differences were found in the measurement of Reprint requests: Alberto Cuocolo, MD, Department of Advanced Biomedical Sciences, University Federico II, Via Pansini 5, 80131 Naples, Italy; cuocolo@unina.it J Nucl Cardiol 2019;26:1743–5. 1071-3581/

Coronary atherosclerotic burden vs. coronary vascular function in diabetic and nondiabetic patients with normal myocardial perfusion: a propensity score analysis

34.00 Copyright 2018 American Society of Nuclear Cardiology.