Spatially resolved, high-dimensional transcriptomics sorts out the evolution of biphasic malignant pleural mesothelioma: new paradigms for immunotherapy.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is a dreadful disease escaping the classical genetic model of cancer evolution and characterized by wide heterogeneity and transcriptional plasticity. Clinical evolution of MPM is marked by a progressive transdifferentiation that converts well differentiated epithelioid (E) cells into undifferentiated and pleomorphic sarcomatoid (S) phenotypes. Catching the way this transition takes place is necessary to understand how MPM develops and progresses and it is mandatory to improve patients' management and life expectancy. Bulk transcriptomic approaches, while providing a significant overview, failed to resolve the timing of this evolution and to identify the hierarchy of molecular events through which this transition takes place. METHODS: We applied a spatially resolved, high-dimensional transcriptomic approach to study MPM morphological evolution. 139 regions across 8 biphasic MPMs (B-MPMs) were profiled using the GeoMx™Digital Spatial Profiler to reconstruct the positional context of transcriptional activities and the spatial topology of MPM cells interactions. Validation was conducted on an independent large cohort of 84 MPMs by targeted digital barcoding analysis. RESULTS: Our results demonstrated the existence of a complex circular ecosystem in which, within a strong asbestos-driven inflammatory environment, MPM and immune cells affect each other to support S-transdifferentiation. We also showed that TGFB1 polarized M2-Tumor Associated Macrophages foster immune evasion and that TGFB1 expression correlates with reduced survival probability. CONCLUSIONS: Besides providing crucial insights into the multidimensional interactions governing MPM clinical evolution, these results open new perspectives to improve the use of immunotherapy in this disease.

From Knowing to Doing: Integrating Systems-Ready Physician Competencies in the Clerkship Setting.

There are increasing calls to graduate physicians with a strong understanding of health systems science (HSS). Many schools have incorporated didactics on health systems science content such as quality improvement, patient safety, or interprofessional education. Creating a systems-ready physician requires more than teaching content in classroom settings. Using Miller's pyramid of assessment of clinical performance, we have developed strategies to move our learners from the cognitive-based "knows" level to the behavior-based "does" level of understanding of the HSS competencies. Our medical students begin learning HSS in classroom settings. Next, the students apply this knowledge during their core clerkships. This gives them an opportunity to get feedback increasingly from high-fidelity clinical settings. We embedded assessment strategies and tools in the clerkship year to facilitate the demonstration, observation, and assessment of HSS competencies in the setting of our core clerkships. We also have students self-assess their competence in our graduation competencies at the end of each year. Student self-assessment from the beginning of the clerkship year to the end showed significant increases in the HSS competencies. Our clerkship student assessment data from our first cohort suggest that faculty had difficulty observing and assessing some of the competencies unique to health systems science. The clerkships have developed multiple projects and assignments to allow students to demonstrate HSS competencies. Faculty and resident training to prompt, observe, and assess these competencies is ongoing to close the assessment gap. In the area of professionalism, student self-assessment and faculty clinical assessment correlate strongly.

Metástasis infrecuente de mesotelioma pleural detectada mediante 18F-FDG PET/TC de cuerpo entero / Uncommon distant metastasis of pleural mesothelioma detected by whole-body 18F-FDG PET/CT

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Bone marrow failure may be caused by chromosome anomalies exerting effects on RUNX1T1 gene.

BACKGROUND: The majority of the cases of bone marrow failure syndromes/aplastic anaemias (BMFS/AA) are non-hereditary and considered idiopathic (80-85%). The peripheral blood picture is variable, with anaemia, neutropenia and/or thrombocytopenia, and the patients with idiopathic BMFS/AA may have a risk of transformation into a myelodysplastic syndrome (MDS) and/or an acute myeloid leukaemia (AML), as ascertained for all inherited BMFS. We already reported four patients with different forms of BMFS/AA with chromosome anomalies as primary etiologic event: the chromosome changes exerted an effect on specific genes, namely RUNX1, MPL, and FLI1, leading to the disease. RESULTS: We report two further patients with non-hereditary BM failure, with diagnosis of severe aplastic anaemia and pancytopenia caused by two different constitutional structural anomalies involving chromosome 8, and possibly leading to the disorder due to effects on the RUNX1T1 gene, which was hypo-expressed and hyper-expressed, respectively, in the two patients. The chromosome change was unbalanced in one patient, and balanced in the other one. CONCLUSIONS: We analyzed the sequence of events in the pathogenesis of the disease in the two patients, including a number of non-haematological signs present in the one with the unbalanced anomaly. We demonstrated that in these two patients the primary event causing BMFS/AA was the constitutional chromosome anomaly. If we take into account the cohort of 219 patients with a similar diagnosis in whom we made cytogenetic studies in the years 2003-2017, we conclude that cytogenetic investigations were instrumental to reach a diagnosis in 52 of them. We postulate that a chromosome change is the primary cause of BMFS/AA in a not negligible proportion of cases, as it was ascertained in 6 of these patients.

Parental versus non-parental-directed donation: an 11-year experience of infectious disease testing at a pediatric tertiary care blood donor center.

BACKGROUND: Directed donation is associated with a higher prevalence of donations that are positive for infectious disease markers; however, little is known about the positive rates among parental-directed, non-parental-directed, and allogeneic donations. STUDY DESIGN AND METHODS: We reviewed blood-collection records from January 1997 through December 2008, including infectious disease results, among parental, non-parental, and community donations. Infectious disease rates were compared by Mann-Whitney U test. RESULTS: In total, 1532 parental, 4910 non-parental, and 17,423 community donations were examined. Among parental donors, the median rate of positive infectious disease testing was 8.66% (interquartile range (IQR), 4.49%) for first-time donors and 1.26% (IQR, 5.86%) for repeat donors; among non-parental donors, the rate was 1.09% (IQR, 0.98%) for first-time donors and 0% (IQR, 0.83%) for repeat donors; and, among community donors, the rate was 2.95% (IQR, 1.50%) for first-time donors and 0.45% (IQR, 0.82%) for repeat donors. The mean rate of positive infectious disease testing for first-time parental donors was significantly higher (7.63%), whereas all repeat donors had similar rates. However, the rate of positive infectious disease testing among first-time non-parental donors was significantly lower than that in the other groups, especially for the period from 2001 through 2008. CONCLUSION: First-time non-parental and community donors had significantly higher infectious disease risk than the respective repeat donors. First-time parental donors had the highest rates of positive infectious disease testing. We suggest that first-time parental blood donation should be discouraged. Repeat community donors or first-time non-parental donors provide a safer alternative. These findings can foster better patient education, donor selection, and possibly a reduced risk of infectious disease.

Lymphomatoid granulomatosis: a practical review for pathologists dealing with this rare pulmonary lymphoproliferative process.

Lymphomatoid granulomatosis (LYG) is a rare B-cell lymphoproliferative disorder predominantly involving the lungs, but poorly-recognized among clinicians and pathologists. It is an Epstein-Barr virus (EBV)-driven disease mimicking several other diseases on clinical and radiological grounds, generally showing multiple, bilateral nodular, ill-defined infiltrates of the lungs tending to coalescence and/or cavitation. LYG often affects middle-aged males with an underlying immunodeficiency and commonly involves skin and central nervous system during disease progression. Diagnosis requires a generous biopsy and careful histologic examination with immunohistochemical staining and molecular demonstration of EBV genome in large atypical B-cells. LYG is graded as I to III based on the number of large EBV-positive B-cells; grades II/III are now considered as a peculiar variant of T-cell rich diffuse large B-cell lymphoma. In this brief review, clinical, radiologic and pathologic features of LYG will be analyzed with focus on differential diagnosis, the most appropriate treatment and prognosis.

Activating c-KIT mutations in a subset of thymic carcinoma and response to different c-KIT inhibitors.

BACKGROUND: To analyze a multi-institutional series of type C thymic carcinomas (TCs) (including neuroendocrine tumors), focusing on the expression and mutations of c-KIT. MATERIALS AND METHODS: Immunohistochemical expression of c-KIT/CD117, p63, CD5 and neuroendocrine markers, as well as mutational analysis of c-KIT exons 9, 11, 13, 14, 17 by direct sequencing of 48 cases of TCs. Immunohistochemical and molecular data were statistically crossed with clinicopathological features. RESULTS: Overall, 29 tumors (60%) expressed CD117, 69% were positive for CD5 and 85% (41 cases) for p63. Neuroendocrine markers stained all six atypical carcinoids and five poorly-differentiated thymic squamous cell carcinomas. Overall, six CD117-positive cases (12.5%) showed c-KIT mutation. No mutation was detected in CD117-negative tumors and carcinoids. All the mutations were found in poorly-differentiated thymic squamous cell carcinomas expressing CD117, CD5, p63 and lacking neuroendocrine markers (6 of 12 cases with these features). Mutations involved exon 11 (four cases: V559A, L576P, Y553N, W557R), exon 9 (E490K) and exon 17 (D820E). CONCLUSIONS: All TCs need an immunohistochemical screening with CD117, while c-KIT mutation analysis is mandatory only in CD117-positive cases, particularly when coexpressing CD5 and p63, lacking neuroendocrine differentiation. The finding of c-KIT mutation can predict efficacy with different c-KIT inhibitors.

Storage of aliquots of apheresis platelets for neonatal use in syringes with and without agitation.

BACKGROUND: To facilitate volume control in neonates, platelets (PLTs) are aliquoted and stored for short periods in non-gas-permeable syringes before infusion. Although agitation of PLTs during storage in gas-permeable bags is performed to maintain their quality, the effect of syringe agitation during storage is unknown. STUDY DESIGN AND METHODS: Double apheresis PLTs (n = 6) were collected and split, providing two identical products. On Days 2 and 4 of storage, aliquots from one bag of each pair were transferred to two syringes and stored for 6 hours on flatbed agitator or were left at 20 to 24 °C without agitation. A series of in vitro tests was performed on Days 0, 2 (Hours 0 and 6), and 4 (Hours 0 and 6). Control samples were obtained from the second matched bag that was stored on the agitator. Data were analyzed by one-way analysis of variance with differences considered significant if the p value was less than 0.05. RESULTS: Comparable results for several PLT variables were obtained with or without agitation of the syringes. On Day 4 Hour 6, pH values were 7.18 ± 0.12 (agitated syringes) and 7.19 ± 0.1 (nonagitated syringes), and extent of shape change and hypotonic shock response measurements were not significantly different between agitated syringes and nonagitated syringes (23.7 ± 6.4 and 74.3 ± 9.8% vs. 23.3 ± 5.4 and 76.0 ± 7.6%), respectively. CONCLUSION: Based on in vitro testing, apheresis PLT aliquots can be stored in syringes for at least 6 hours without agitation before transfusions.

A randomized clinical trial comparing 22G and 25G needles in endoscopic ultrasound-guided fine-needle aspiration of solid lesions.

BACKGROUND AND STUDY AIMS: The study aimed to investigate whether the 25G needle is superior to the 22G needle when used in endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of solid lesions. PATIENTS AND METHODS: The study was a single-center randomized clinical trial. The setting was a tertiary referral hospital, where EUS-FNA of solid lesions was assisted by an on-site cytopathologist, who was blinded to the needle size. The main end point was the number of passes performed to obtain adequate samples. Crossover to the other type of needle was allowed after five passes, or when the gastroenterologist experienced difficulties in puncturing the lesions. RESULTS: A total of 129 solid lesions were randomized and data regarding 127 lesions were analyzed. The mean number of passes was 3.7 (± 1.9) in the 22G needle group and 3.8 (± 2) in the 25G needle groups (difference of means: 0.1; 95% CI: -0.59 to 0.79). Fifty-eight of 63 (92.1%) and 60/64 samples (93.7%) in the 22G and 25G needle groups respectively were adequate (difference: -1.6%; 95%CI: -12.1% to 8.9%). Crossover to the other type of needle was performed in 11/63 (17.5%) and in 12/64 (18.7%) lesions in the two groups respectively (difference: -1.2%; 95%CI: -16.2% to 13.8%). A crossover to the 25G needle was successfully performed in four masses in the uncinate process; these lesions were difficult to puncture using the 22G needle. CONCLUSIONS: Our study failed to demonstrate that the 25G is more effective than the 22G needle in EUS-FNA of solid lesions. However, targeting of lesions in the distal duodenum may be simplified by using the 25G needle.

[Calciphylaxis and penile necrosis: a case report and review of the literature ]. / Calcifilassi e necrosi peniena: case report e revisione della letteratura.

INTRODUCTION: Calciphylaxis is a rare clinic condition characterised by skin necrosis due to medial and intimal calcification of small and medium arteries. It's observed in patients affected by end stage renal disease associated to secondary hyperparathyroidism. Penile involvement has been documented in very few cases. We present both a case of penile calciphylaxis and a review of literature, in order to increase comprehension of pathophysiology, diagnosis and therapy of this rare disease. MATERIALS AND METHODS: A retrospective review of literature was performed after treating a case of penile calciphylaxis. We describe patient characteristics, clinical presentation, laboratory and histo-pathologic findings, therapeutic strategy and outcomes of the case. RESULTS: A 65 year-old man, affected by diabetes, chronic ischemic cardiopathy and chronic renal failure in hemodialytic treatment, was referred to our unit for the presence of increased consistency and significative pain of the distal portion of penis evolving in a complete glans necrosis. Blood levels of parathormone (PTH), calcium (Ca) and phosphorous (P) resulted pathologically elevated, promoting tissutal calcium deposition. The patient was treated with partial penectomy and the histologic findings confirm diagnosis of calciphylaxis, showing an ulcerative necrosis of glans with extensive calcium deposition and luminal narrowing of penile small arteries. CONCLUSIONS: The increase of number of patients with chronic renal failure in hemodialytic treatment could make penile calciphylaxis more prevalent in the future. Early diagnosis, lowering of pathologic blood levels of Ca and P associated to surgical treatment of necrotic lesions of the patient could be fundamental for a better prognosis of this aggressive disease.

Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Activation of the mixed lineage kinase and c-Jun N-terminal kinase (JNK) has been reported in models of PD. Our focus was to discern whether distinct pathways were activated in cell-specific manner within the SNpc. We now demonstrate the selective phosphorylation of p38 MAP kinase within the dopaminergic neurons, whereas JNK activation occurs predominantly in the microglia. p38 activation results in downstream phosphorylation of p53 and increased p53 mediated transcription of Bax and Puma in the ventral midbrain. Treatment with p38 inhibitor, SB239063 protected primary dopaminergic neurons derived from human progenitor cells from MPP(+) mediated cell death and prevented the downstream phosphorylation of p53 and its translocation to the nucleus in vivo, in the ventral midbrain. The increased staining of phosphorylated p38 in the surviving neurons of SNpc in human brain sections from patients with PD and in MPTP treated mice but not in the ventral tegmental area provides further evidence suggesting a role for p38 in the degeneration of dopaminergic neurons of SNpc. We thus demonstrate the cell specific activation of MAP kinase pathways within the SNpc after MPTP treatment emphasizing the role of multiple signaling cascades in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.

Knockdown of cytosolic glutaredoxin 1 leads to loss of mitochondrial membrane potential: implication in neurodegenerative diseases.

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.

Blood irradiator dosimetry with BANG polymer gels.

BACKGROUND: Transfusion-associated graft-versus-host disease is believed to be a preventable complication of blood transfusion in susceptible high-risk patients. Irradiation of allogeneic blood components is considered to be a safe and effective method inhibiting the proliferative capacity of lymphocytes. Physical factors that influence dose delivery include source and sample geometry, homogeneity of the dose distribution within the irradiation volume, and factors that influence dose absorption within the sample. STUDY DESIGN AND METHODS: Polymer gel dosimetry was used to evaluate the three-dimensional dose distribution for blood bags, platelet (PLT) bags, and syringes in typical geometries. RESULTS: The qualitative dose distribution within a cylindrical phantom determined with the polymer gel was in good agreement with the distribution in water determined with a DOSE-MAP film dosimetry system. Dose uniformity in blood bags and syringes was acceptable. Low signal strength and volume averaging hamper determination of the dose in PLT bags. CONCLUSION: Geometrical considerations in routine blood product irradiation may be evaluated with the use of polymer gel dosimetry. The procedures, however, are highly technical and are susceptible to low signal-to-noise and volume averaging considerations under certain geometrical configurations. Under clinical conditions, a reasonably uniform dose can be delivered to blood and blood components in a freestanding irradiator.

The value of c-kit mutational analysis in a cytokeratin positive gastrointestinal stromal tumour.

The expression of cytokeratins in gastrointestinal stromal tumours (GISTs) is rare and may lead to diagnostic confusion when it occurs. This report describes a metastatic GIST that stained strongly for cytokeratins, CD117, and CD34 in a patient who was previously diagnosed with gastric epithelioid angiosarcoma. A review of both tumours showed the same histological and immunohistochemical profiles, and c-kit molecular analysis revealed an insertional mutation at codon 558 of exon 11 in both tumours. Thus, pathologists should be aware that GISTs can occasionally express cytokeratins, and that c-kit mutational investigations may have a key diagnostic role and may prevent diagnostic mistakes that could have important clinical implications.

PDGFR expression in differential diagnosis between KIT-negative gastrointestinal stromal tumours and other primary soft-tissue tumours of the gastrointestinal tract.

AIMS: To investigate the value of platelet-derived growth factor receptors (PDGFRs) by immunohistochemistry in discriminating KIT-negative gastrointestinal stromal tumours (GISTs) from other soft-tissue neoplasms of the digestive tract. METHODS AND RESULTS: One-hundred and sixty-seven primary gastrointestinal mesenchymal tumours (125 GISTs, 15 intra-abdominal desmoids, 12 leiomyomas, eight leiomyosarcomas, three schwannomas, two solitary fibrous tumours, and one case each of inflammatory pseudotumour and fibroid polyp) were reclassified based on morphology and on the immunohistochemical panel recommended by the National Institutes of Health consensus on GIST. All cases were then tested with antibodies specific for PDGFR alpha and beta. Of 125 GISTs, 117 were KIT-positive (93.6%) and eight KIT-negative (6.4%). All the KIT-positive GISTs were negative for both PDGFRs, while all the eight KIT-negative GISTs expressed PDGFR-alpha, with two of them also coexpressing PDGFR-beta. Among the 42 non-GIST tumours, only a small percentage (26.6%) of desmoids immunostained for PDGFR-alpha, two of them coexpressing PDGFR-beta. CONCLUSIONS: Immunostaining with PDGFR-alpha is a helpful marker in discriminating between KIT-negative GISTs and other gastrointestinal mesenchymal lesions: all KIT-negative GISTs were positive for PDFGR-alpha, while none of the other gastrointestinal mesenchymal tumours analysed, except a small subset of desmoids, was reactive with anti-PDGFRs. These preliminary data demonstrate the suitability of commercially available antibodies to detect immunohistochemically the mutually exclusive expression of KIT and PDGFR-alpha previously reported in GISTs by molecular biological techniques. Since PDGFR exists in the form of a homodimer (alphaalpha, betabeta) or heterodimer (alphabeta) and two of the KIT-negative GISTs coexpressed both PDGFR isoforms, further investigations are required to elucidate the role of PDGFR-beta in GISTs.

Feasibility of red blood cell transfusion through small bore central venous catheters used in neonates.

OBJECTIVE: To determine whether packed red blood cell transfusions through small-bore central venous catheters used in critically ill neonates results in significant hemolysis. DESIGN: In vitro experimental study using a mock transfusion setup incorporating a syringe pump, prestorage leukoreduced fresher, and older CPDA-1 red blood cell units and pressure transducer simulating neonatal transfusion through 1.9-Fr NeoPICC central venous catheter. SETTING: Laboratory setting. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Spun hematocrit, plasma free hemoglobin (hemoglobin), lactate dehydrogenase, and potassium were analyzed pretransfusion, at various times during transfusion, and posttransfusion. Intraluminal pressures were measured using a TruWave Disposable Pressure Transducer. Using fresher (5-8 days old) and older (29-30 days old) CPDA-1 red blood cells, we compared 2 and 20 mL/hr flow rates. Statistical analysis was performed using repeated measures of analysis of variance to compare the differences in means between flow rates. Mean intraluminal pressures at the end of each experiment were significantly higher at 20 mL/hr flow rates (>360 mm Hg) in both fresher and older red blood cells than at 2 mL/hr (range, 61-70 mm Hg). Overall, potassium, lactate dehydrogenase, and plasma free hemoglobin concentrations were significantly higher for older red blood cells at either 2 or 20 mL/hr (p<.001). Both fresher and older red blood cells demonstrated higher potassium concentrations at 20 mL/hr (22.4%, p<.001;0.7%, p>.05, respectively); however, these increases were not clinically significant. Furthermore, lactate dehydrogenase, hematocrit, and plasma free hemoglobin differences seen at 2 and 20 mL/hr did not coincide with changes in potassium. CONCLUSIONS: No clinically significant hemolysis was evidenced with red blood cell transfusion through small-bore central venous catheters when using fresher or older CPDA-1 red blood cells at 2 or 20 mL/hr.

Thyroid abscess associated with a substernal goiter. Case report.

An abscess associated with an intrathoracic goiter is an extremely rare condition. The authors report a case of a thyroid abscess complicated by acute dyspnea and asphyxia in a patient of geriatric age with a substernal goiter. Surgical therapy was necessary to obtain a correct diagnosis and an effective treatment.