Search

Background Single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor A (VEGFA) are associated with susceptibility to several diseases including cancer. Correlations between VEGFA rs3025020 genotypes with clinical and laboratory features of primary myelofibrosis (PMF) are unstudied. Methods DNA was analyzed by real-time polymerase chain reaction for VEGFA rs3025020 genotypes in a cohort of 844 subjects with PMF and in two cohorts of normal subjects (N = 247 and N = 107). Results Frequency of rs3025020 minor allele (T) was not significantly different in subjects with PMF compared with normals; however, the T-allele was more frequent in PMF subjects with a calreticulin (CALR)-mutated genotype compared with normals (35 vs. 27%; OR = 1.47 [95% CI, 1.09, 1.98] p = 0.011), especially in subjects with a CALR-type 2/type 2-like mutation (43 vs. 27%; OR = 2.01 [1.25, 3.24] p = 0.004). CALR mutants with the rs3025020 TT genotype had higher CXCR4 expression on CD34-positive blood cells, and those who carried CT/TT genotypes had lower platelet concentrations compared with other genotypes at diagnosis. Overall, subjects with the rs3025020 CT/TT genotype had a lower cumulative incidence of deep vein thrombosis in typical sites (1.6 vs. 4.2%; OR = 0.37 [0.15, 0.90] p = 0.029) and longer interval from diagnosis to first thrombosis (HR = 0.37 [0.14, 0.95] p = 0.039). Conclusion Persons with PMF and the VEGFA rs3025020 minor T-allele are more likely to have a CALR mutation compared with other somatic driver mutations and lower cumulative incidence and hazard for deep vein thrombosis in typical sites.

Introduction: In 1991, we reported 18 persons with a clinical-pathologic entity and termed atypical myeloproliferative disorder because they did not meet the contemporary diagnostic criteria for a myeloproliferative neoplasm. We sought to gain further knowledge on this disease entity. Methods: This retrospective cohort study included consecutive subjects registered in the database of the Center for the Study of Myelofibrosis in Pavia, Italy, from 1998 to 2020 (June), and diagnosed with atypical myeloproliferative disorder according to our adjudicated criteria. We studied clinical, histological, cytogenetic, and molecular covariates and risks of thrombosis, disease progression, and death. Data were compared with those of concurrent subjects with prefibrotic myelofibrosis. Results: Fifteen new subjects with atypical myeloproliferative disorder were identified. Seven were male. Median age was 50 years (IQR, 41–54 years). Thirteen were diagnosed with a synchronous symptomatic or incidentally detected thrombotic event. The bone marrow showed megakaryocyte hyperplasia with dysplasia. JAK2V617F was present in 10 subjects and CALR mutation in one. No other somatic mutations were identified in next generation sequencing. After a median follow-up of 101 months (IQR, 40–160 months), no subject had disease progression or blast transformation. Incidence of post-diagnosis or recurrent thrombosis was 3.9 events (95% confidence interval, 3.5–4.0) and 5.0 events (4.6–5.6) per 100 person-years. Features of subjects with atypical myeloproliferative disorder differed markedly from those of 546 subjects with prefibrotic myelofibrosis. Conclusion: Our data indicate that these 15 persons have a distinct myeloproliferative neoplasm. We propose naming this new disorder clonal megakaryocyte dysplasia with normal blood values.

Introduction Angiotensin-converting-enzyme-2 (ACE-2) was demonstrated to be the receptor for cellular entry of SARS-CoV-2. ACE-2 mRNA was identified in several human tissues and recently also in thyroid cells in vitro. Purpose Aim of the present study was to investigate the effect of pro-inflammatory cytokines on the ACE-2 mRNA levels in human thyroid cells in primary cultures. Methods Primary thyroid cell cultures were treated with IFN-γ and TNF-α alone or in combination for 24 h. ACE-2 mRNA levels were measured by RT-PCR. As a control, the levels of IFN-γ inducible chemokine (CXCL10) were measured in the respective cell culture supernatants. Results The mean levels of ACE-2 mRNA increased after treatment with IFN-γ and TNF-α in all the thyroid cell preparations, while the combination treatment did not consistently synergically increase ACE-2-mRNA. At difference, CXCL10 was consistently increased by IFN-γ and synergically further increased by the combination treatment with IFN-γ + TNF-α, with respect to IFN-γ alone. Conclusions The results of the present study show that IFN-γ and, to a lesser extent TNF-α consistently increase ACE-2 mRNA levels in NHT primary cultures. More interestingly, the combined stimulation (proven to be effective according to the synergic effect registered for CXCL10) produces different responses in terms of ACE-2 mRNA modulation. These results would suggest that elevated levels of pro-inflammatory cytokines could facilitate the entering of the virus in cells by further increasing ACE-2 expression and/or account for the different degree of severity of SARS-COV-2 infection. This hypothesis deserves to be confirmed by further specific studies.

The peculiar combined, or binary involvement of epithelium and stroma makes basal cell carcinoma (BCC) a unique tumour. Nerve fibres have been shown to play an active role in different cancers. A prospective observational study was carried out on punch biopsies harvested within BCC surgical excision specimens. A total of 10 samples of histologically diagnosed BCC, derived from 10 different patients (five females, five males), was included in the study. Within the BCCs, seven different histological sub-types were identified: morphea-like, basosquamous, micronodular, mixed nodular-micronodular, adenoid, nodular and superficial multifocal. Nerve fibres were stained for indirect immunofluorescence targeting protein gene product 9.5. Three different morphological patterns of nerve fibre distribution within the BCCs were identified. Pattern 1 displayed a normal skin nerve pattern, in which the fibres were dislodged by the growing tumour masses. Pattern 2 featured a ball of curved, tangled nerve fibres close to the tumour masses, slightly resembling piloneural collar nerve fibres, wrapped around hair follicles in the normal anatomical setting. Pattern 3 showed nerve fibres crowding in the sub-epidermal layer with focal epidermal hyperinnervation. Such a pattern is reminiscent of the typical anatomical neuro-epithelial interaction in mechanosensory organs. Our study may disclose a hidden third player, of nerves. Thus, tissue involvement of BCCs may be better represented by the triad of epithelium, stroma and nerves, each component retaining some features associated with its developmental setting.

Purpose. This study is aimed at assessing the prevalence of pulmonary artery filling defects (PAFDs) consistent with pulmonary artery embolism (PAE) in patients with SARS-CoV-2 infection and at investigating possible radiological or clinical predictors. Materials and Methods. Computed Tomography Pulmonary Angiographies (CTPAs) from 43 consecutive patients with a confirmed COVID-19 infection were retrospectively reviewed, taking into consideration the revised Geneva score and the D-dimer value for each patient. Filling defects within the pulmonary arteries were recorded along with pleural and parenchymal findings such as ground glass opacities, consolidation, crazy paving, linear consolidation, and pleural effusion. All these variables were compared between patients with and without PAFD. The predictive performance of statistically different parameters was investigated using the receiver operating characteristics (ROC). Results. Pulmonary embolism was diagnosed in 15/43 patients (35%), whereas CTPA and parenchymal changes related to pulmonary COVID-19 disease were evident in 39/43 patients (91%). The revised Geneva score and the mean D-dimer value obtained using two consecutive measurements were significantly higher in patients with PAFD. The ROC analysis demonstrated that a mean D-dimer value is the parameter with the higher predictivity (AUC 0.831) that is acut‐off value>1800 μg/lwhich predicts the probability of PAFD with a sensitivity and specificity of 70% and 78%, respectively. Conclusions. This single centre retrospective report shows a high prevalence of pulmonary artery filling defects revealed using CTPA in COVID-19 patients and demonstrates that the mean value of multiple D-dimer measurements may represent a predicting factor of this complication.

The expression of the CXCR4 chemokine receptor on CD34-positive blood cells is reduced in persons with primary myelofibrosis (PMF). We analyzed the relevance of cytofluorimetric assessment of the percentage of CD34-positive blood cells that had a positive CXCR4 surface expression (CD34/CXCR4-se) in a large cohort of subjects with myeloproliferative neoplasms. Mean CD34/CXCR4-se was lower in subjects with PMF compared with those with essential thrombocythemia (ET) or polycythemia vera (PV). A cutoff value of 39% was associated with a diagnosis of pre-fibrotic PMF vs. ET with a positive predictive value of 97%. In PMF male sex, older age, and MPL mutation were independent correlates of reduced CD34/CXCR4-se and associated with a briefer interval to development of severe anemia, large splenomegaly, thrombocytopenia, leukopenia, elevated CD34-positive blood cells, blast transformation and death. We constructed a prognostic model including age >65 years, hemoglobin 50 × 106/L, and CD34/CXCR4-se

We evaluated the association of VEGFA rs3025039 polymorphism with clinical co-variates and outcomes in 849 subjects with primary myelofibrosis (PMF) and 250 healthy controls. Minor T-allele frequency was higher in subjects with JAK2V617F compared with those without JAK2V617F (18% vs. 13%; p = 0.014). In subjects with JAK2V617F, the TT genotype was associated at diagnosis with lower platelet concentrations (p = 0.033), higher plasma LDH concentration (p = 0.005), higher blood CD34-positive cells (p = 0.027), lower plasma cholesterol concentration (p = 0.046), and higher concentration of high-sensitivity C-reactive protein (p = 0.018). These associations were not found in subjects with PMF without JAK2V617F. In subjects with the TT genotype, risk of death was higher compared with subjects with CC/CT genotypes (HR = 2.12 [1.03, 4.35], p = 0.041). Finally, the TT genotype was associated with higher frequency of deep vein thrombosis in typical sites (12.5% vs. 2.5%; OR = 5.46 [1.51, 19.7], p = 0.009). In conclusion, in subjects with PMF, the VEGFA rs3025039 CT or TT genotypes are more common in those with JAK2V617F than in those without JAK2V67F mutation and are associated with disease severity, poor prognosis, and risk of deep vein thrombosis.

Clinical elements differentiating enteropathy due to angiotensin II-receptor-blockers (ARBs-E) from coeliac disease (CD) are poorly defined. The histopathological features on duodenal and gastric biopsies in these patients still need to be investigated.To describe the clinical phenotype of ARBs-E in comparison to CD, and the histological findings of gastric and duodenal biopsies in ARBs-E.Clinical data of patients with ARBs-E and CD diagnosed between 2013 and 2020 were retrospectively reviewed. Baseline presenting symptoms and demographics were compared (Fisher's exact test and t-test). Gastric and duodenal histology in ARBs-E were revised by two independent pathologists.14 ARBs-E and 112 CD patients were enroled. Weight loss (p 0.01), acute onset of diarrhoea (p 0.01), hospitalization (p 0.01), and older age at diagnosis (p 0.01) were more common in ARBs-E. Duodenal histology in ARBs-E showed intraepithelial lymphocytosis in 71%, increased mucosal eosinophilic count in 57%, with preserved neuroendocrine, Paneth and goblet cells in all patients. Gastric histologic lesions at baseline, including lymphocytic gastritis, eosinophilic gastritis, chronic active gastritis, and metaplastic atrophic gastritis patterns were observed in 73% of patients, without Helicobacter pylori infection.ARBs-E showed a severe clinical phenotype, often requiring hospital admission. Gastric involvement at diagnosis is very common, and this could further support this diagnosis.

To determine the performance of texture analysis and conventional MRI parameters in predicting tumoral response to neoadjuvant chemotherapy and to assess whether a relationship exists between texture tissue heterogeneity and histological type of uterine cervix cancer. Twenty-eight patients with local advanced cervical cancer (FIGO IB2-IIIB), underwent MRI before chemotherapy. Texture analysis parameters were quantified on T2-weighted sequences, as well as the maximum diameter expressed in mm. ADC values were obtained on the ADC map. Statistical analysis included unpaired t test and ROC curve. No statistical correlation was found between conventional parameters and response to NACT. Mean and skewness showed a strong correlation with the histological type: Adenocarcinomas presented higher mean and skewness values (69.8 ± 10.5 and 0.55 ± 0.19) in comparison with squamous cell carcinomas. Using a cutoff value ≥ 29 for mean it was possible to differentiate the two histological types with a sensitivity of 100% and a specificity of 81%. Kurtosis showed a positive correlation with tumor response to NACT resulting higher in responders (v.m. 5.7 ± 1.1) in comparison with non-responders (2.3 ± 0.5). The optimal Kurtosis cutoff value for the identification of non-responders tumors was ≤ 3.7 with a sensitivity of 92% and a specificity of 75%. Texture analysis applied to T2-weighted images of uterine cervical cancer exceeded the role of conventional prognostic factors in predicting tumoral response; moreover, they showed a potential role to differentiate histological tumor types.

Microcalcifications (MCs) are important disease markers for breast cancer. Many studies were conducted on their characterization in female breast cancer (FBC), but no information is available on their composition in male breast cancer (MBC). Raman spectroscopy (RS) is a molecular spectroscopy that can rapidly explore the biochemical composition of MCs without requiring any staining protocol. In this study, we optimized an algorithm to identify the mineral components present in MCs from Raman images. The algorithm was then used to study and compare MCs identified on breast cancer pieces from male and female patients. In total, we analyzed 41 MCs from 5 invasive MBC patients and 149 MCs from 13 invasive FBC patients. Results show that hydroxyapatite is the most abundant type of calcium both in MBC and FBC. However, some differences in the amount and distribution of calcium minerals are present between the two groups. Besides, we observed that MCs in MBC have a higher amount of organic material (collagen) than FBC. To the best of our knowledge, this study provides the first overview of the composition of MCs present in MBC patients; and suggests that these patients have specific features that differentiate them from the previously studied FBC. Our result support thus the need for studies designed explicitly to the understanding of MBC.

Microcalcifications (MCs) are important disease markers for breast cancer. Many studies were conducted on their characterization in female breast cancer (FBC), but no information is available on their composition in male breast cancer (MBC). Raman spectroscopy (RS) is a molecular spectroscopy technique that can explore the biochemical composition of MCs rapidly and without requiring any staining protocol. We here compared, by Raman spectroscopy, the MCs identified on breast cancer pieces from male and female patients. In this study, we used Raman spectroscopy to analyse 41 microcalcifications from 5 invasive MBC patients and 149 MCs from 14 invasive FBC patients. Our findings show that hydroxyapatite is the most abundant type of calcium both in MBC and FBC. However, some differences in the amount and distribution of calcium minerals are present between the two groups. Besides, we observed that MCs in MBC have a higher amount of organic material (collagen) than FBC.This study provides the first overview of the composition of microcalcifications present in MBC and suggests that they have several specific features. Our result support the need for studies specifically designed to the understanding of MBC.

Objective Indeterminate cytological result at fine-needle aspiration cytology (FNAC) remains a clinical challenge for endocrinologists. Aim of the present study was to evaluate whether a coexistent chronic autoimmune thyroiditis (CAT) might affect the diagnostic accuracy of fine-needle aspiration cytology for thyroid nodules. Design and methods A retrospective cohort study was designed including all nodules receiving an indeterminate cytology result (TIR3A or TIR3B) undergoing thyroid surgery and subsequent histological confirmation. Patients were stratified into two groups according to the presence or absence of CAT. The hypothesis to be tested was whether follicular cell alterations induced by CAT might increase the rate of indeterminate cytological results in histologically benign thyroid nodules. Additional control groups were represented by nodules with determinate cytology, either benign (TIR 2) or malignant (TIR5). Results One hundred and eighty-nine indeterminate thyroid nodules were included (67 TIR3A and 122 TIR3B). At post-surgical histology, 46 nodules (24.3%) were malignant. No significant differences were observed in the rate of histologically proven malignancy between patients without CAT and patients with CAT in the TIR3B (29.4% vs 32.4%; P = 0.843) nor TIR3A (13.0% vs 11.4%; P = 1.000) nodules. The rate of coexistent CAT was similar between TIR3B and TIR5 nodules harboring PTC at histology (30.4% vs 39.4%, P = 0.491) and between indeterminate nodules and a control group of TIR2 nodules (39.2% vs 37.0%; P = 0.720). Conclusions The similar rates of histologically proven malignancy found in cytologically indeterminate nodules in the presence or absence of concomitant CAT would not support that CAT itself affects the diagnostic accuracy of fine-needle aspiration cytology.

The impact of reperfusion therapies on cognition has been poorly explored and little knowledge exists. We explored the influence of endovascular treatment (EVT) on cognitive outcome in patients with anterior circulation ischemic stroke. Patients presenting with ischemic stroke due to anterior large vessel occlusion who underwent intravenous thrombolysis (IVT) alone or EVT plus IVT were recruited. Cognitive abilities were evaluated at 6 months from stroke through a neuropsychological test battery. A total of 88 patients with a mean age of 66.3 ± 12.9 years were included, of which 38 treated with IVT alone and 50 with IVT plus EVT. Compared to patients treated with IVT alone, patients who received EVT plus IVT performed significantly better at the neuropsychological tests exploring executive functions, attention, abstract reasoning, visuospatial ability, visual and verbal and memory. At multivariable regression analysis, the EVT was independently associated with the 6-month cognitive performance after the adjustment for age, sex, admission National Institutes of Health Stroke Scale score, systolic blood pressure, glucose level, Alberta Stroke Program Early CT score, side of stroke, site of occlusion, and Back Depression Inventory score [Stroop Test Word Reading: adjβ = 13.99, 95% confidence interval (CI) 8.47–19.50, p adjβ = 6.63, 95% CI 2.46–10.81, p = 0.002; Trail Making Test-A: adjβ = − 92.98, 95% CI − 153.76 to − 32.20, p = 0.003; Trail Making Test-B: adjβ = − 181.12, 95% CI − 266.09 to − 96.15; p adjβ = 1.44, 95% CI 0.77–2.10, p adjβ = 1.10, 95% CI 0.42–1.77, p = 0.002; Coloured Progressive Matrices: adjβ = 5.82, 95% CI 2.71–8.93, p adjβ = 6.02, 95% CI 2.74–9.30, p adjβ = 6.00, 95% CI 2.34–9.66, p = 0.002; Rey Complex Figure Test-Delayed recall: adjβ = 5.73, 95% CI 1.95–9.51, p = 0.003; Rey Auditory Verbal Learning Test-Immediate recall: adjβ = 12.60, 95% CI 6.69–18.52, p adjβ = 1.85, 95% CI 0.24–3.45, p = 0.025]. Patients treated with EVT plus IVT had better cognitive performance than patients treated with IVT alone at 6 months from anterior circulation ischemic stroke.

Supplemental Digital Content is available in the text, The aim of this study was to provide an innovative nomogram to predict the risk of >2 positive nodes in patients fulfilling the Z0011 criteria with 1-2 sentinel lymph nodes (SLNs) only retrieved. From 2007 to 2017, at the Breast Unit of ICS Maugeri Hospital 271 patients with 1-2 macrometastatic SLNs, fulfilling the Z0011 criteria, underwent axillary dissection and were retrospectively reviewed. A mean of 1.5 SLNs per patient were identified and retrieved. One hundred eighty-seven (69.0%) had 1-2 positive nodes, and 84 (31.0%) had >2 metastatic nodes. Independent predictors of axillary status were: positive SLNs/retrieved SLNs ratio (odds ratio [OR] 10.95, P = .001), extranodal extension (OR 5.51, P = .0002), and multifocal disease (OR 2.9, P = .003). A nomogram based on these variables was constructed (area under curve after bootstrap = 0.74). The proposed nomogram might select those patients fulfilling the Z0011 criteria, with 1-2 SLNs harvested, in whom a high axillary tumor burden is expected, aiding to guide adjuvant treatments.

Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment. Significance: Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.

The COVID-19 pandemic is inevitably changing the world in a dramatic way, and the role of computed tomography (CT) scans can be pivotal for the prognosis of COVID-19 patients. Since the start of the pandemic, great care has been given to the relationship between interstitial pneumonia caused by the infection and the onset of thromboembolic phenomena. In this preliminary study, we collected n = 20 CT scans from the Polyclinic of Bari, all from patients positive with COVID-19, nine of which developed pulmonary thromboembolism (PTE). For eight CT scans, we obtained masks of the lesions caused by the infection, annotated by expert radiologists, whereas for the other four CT scans, we obtained masks of the lungs (including both healthy parenchyma and lesions). We developed a deep learning-based segmentation model that utilizes convolutional neural networks (CNNs) in order to accurately segment the lung and lesions. By considering the images from publicly available datasets, we also realized a training set composed of 32 CT scans and a validation set of 10 CT scans. The results obtained from the segmentation task are promising, allowing to reach a Dice coefficient higher than 97%, posing the basis for analysis concerning the assessment of PTE onset. We characterized the segmented region in order to individuate radiomic features that can be useful for the prognosis of PTE. Out of 919 extracted radiomic features, we found that 109 present different distributions according to the Mann–Whitney U test with corrected p-values less than 0.01. Lastly, nine uncorrelated features were retained that can be exploited to realize a prognostic signature.

We measured plasma levels of high-sensitivity C-reactive protein (hs-CRP) in 526 subjects with primary myelofibrosis (PMF). Thirty-eight percent had an elevated hs-CRP level (≥0.3mg/dL). Elevated hs-CRP levels were associated with a progressive disease phenotype, including anemia, high white blood cell count, low platelet count, increased splenomegaly, increased risk of blast transformation, and worse survival. Age≥52years, but no other demographic characteristics, was associated with an elevated hs-CRP level in multivariable logistic regression (odds ratio [OR], 4.29; 95% CI, 2.73-6.77; P

The aim of this study was to demonstrate the correlation between human epidermal growth factor receptor 2 (HER2) overexpression and some poor prognosis factors in patients affected by ductal carcinoma in situ (DCIS). We evaluated 48 cases of DCIS, divided into two groups according to HER2 amplification status. Nuclear grade and "cancerization of lobules" were determined within primary DCIS and Ki67, estrogen receptor (ER), PR, and HER2 expression was established using immunohistochemistry. The histopathological variables in HER2-positive and in HER2-negative patients were compared to determine the recurrence risk. We also considered the median age at the time of surgery according to HER2 status. There were 11 recurrences (23%), 6 DCIS (55%), and 5 invasive cancer (45%). In an 8-year-long median follow-up, we hypothesized high risk of recurrence in HER2-positive DCIS. Patients with HER2-positive DCIS were younger than HER2-negative ones (p = 0.002). HER2-positive DCIS was also related to histopathological predictors of recurrence such as high nuclear grade (p < 0.001), high Ki67 expression (p = 0.003), low ER and PgR levels (p < 0.001), and the presence of "cancerization of lobules" (p < 0.049). Our trial suggests that HER2 amplification in primary DCIS is identified more frequently in younger patients and hypothesizes high risk of recurrence in HER2-positive DCIS related to histopathological predictors of overall relapse as high nuclear grade, high Ki67 expression, low ER and PgR levels, and the presence of "cancerization of lobules." In HER2-positive DCIS, other variables of recurrence risk are compared to HER2-negative lesions, without statistical significance. Our results show that HER2 testing might suggest clinicians the optimal treatment of patients with DCIS.

Background The oncologic benefit of upfront re-excision of involved margins after breast-conserving surgery in the context of current multimodal clinical management of breast cancer is unclear. The aim of the present study was to assess the 5-years locoregional recurrence (LRR)-free and distant metastases (DM)-free survival probabilities in patients not undergoing re-excision of positive margins after lumpectomy for breast cancer. Methods A cohort of 104 patients with positive margins not undergoing re-excision was matched by propensity score with a cohort of 2006 control patients with clear margins after breast-conserving surgery, treated between 2008 and 2018. A multivariate survival analysis was performed accounting for all variables related to LRR and DM, including adjuvant treatments. Results After adjusting for potential confounders, avoiding to re-excise a positive margin after lumpectomy had no effect on 5-years LRR-free survival probability (HR 0.98, 95%CI 0.36–2.67, p = 0.96) or 5-years DM-free survival probability (HR 0.37, 95%CI 0.08–1.61, p = 0.18). No correlation was found between occurrence of LRR and number of involved margins (HR 1.28, 95%CI 0.10–12.4, Log-rank p = 0.83), or extension of infiltrating disease (HR 1.21, 95%CI 0.20–7.40, Log-rank p = 0.83), but a trend toward higher LRR probability was found for invasive ductal (HR 6.92, 95%CI 0.7–68.8, Log-rank p = 0.10) and invasive lobular cancer (HR 12.95, 95%CI 0.79–213.6, Log-rank p = 0.07) on positive margins. Conclusions In the era of multimodal treatment of breast cancer and accurate strategies to reduce the probability of residual disease in the post-lumpectomy cavity, re-excision of positive margins might be omitted in selected patients with low-risk breast cancers.

JAK2 V617F allele burden 50% is associated with response to ruxolitinib in persons with MPN-associated myelofibrosis and splenomegaly requiring therapy

A 35-year-old woman affected by a well-differentiated papillary thyroid carcinoma was referred to our hospital to perform a (131)Iodine ((131)I) whole body scintigraphy for restaging purpose. The patient had been previously treated with total thyroidectomy and three subsequent doses of (131)I for the ablation of a remnant jugular tissue and a suspected metastatic focus at the superior left hemi-thorax. In spite of the previous treatments with (131)I, planar and tomographic images showed the persistence of an area of increased uptake at the superior left hemi-thorax. This finding prompted the surgical resection of the lesion. Histological examination of the surgical specimen showed the presence of a pulmonary tissue consistent with pulmonary sequestration. Even though rare, pulmonary sequestration should be included in the potential causes of false-positive results of radioiodine scans.

Interferons (IFNs) and tumor necrosis factor-α (TNF-α) cooperate in activating several inflammation-related genes, which sustain chronic inflammation in autoimmune thyroid disease (AITD). Much is known about the positive signaling of IFNs to activate gene expression in AITD, while the mechanisms by which IFNs negatively regulate genes remain less studied. While IFNs inhibit CXCL8 secretion in several human cell types, their effects on thyroid cells were not evaluated. Our aim was to study the interplay between TNF-α and type I or type II IFNs on CXCL8 secretion by human thyroid cells. CXCL8 was measured in supernatants of primary cultures of thyroid cells basally and after a 24-h incubation with TNF-α. CXCL8 was detected in thyroid cell supernatants in basal conditions (96.2±23.5 pg/mL) being significantly increased (784.7±217.3 pg/mL; P0.0001 vs. basal) by TNF-α. Twenty-four hour incubation with IFN-γ or IFN-β or IFN-α dose dependently and significantly inhibited both basal and TNF-α-induced CXCL8 secretion. The degree of the inhibitory effect was IFN-γIFN-βIFN-α. This study demonstrates that type I and type II IFNs downregulate both basal and TNF-α-induced CXCL8 secretion by human thyrocytes, IFN-γ being the most powerful inhibitor. Future studies aimed at a better comprehension of the interplay between CXCL8 and thyroid diseases appear worthwhile.

We genotyped 370 subjects with primary myelofibrosis (PMF) and 148 with postpolycythemia vera/postessential thrombocythemia (PPV/PET) MF for mutations of EZH2. Mutational status at diagnosis was correlated with hematologic parameters, clinical manifestations, and outcome. A total of 25 different EZH2 mutations were detected in 5.9% of PMF, 1.2% of PPV-MF, and 9.4% of PET-MF patients; most were exonic heterozygous missense changes. EZH2 mutation coexisted with JAK2V617F or ASXL1 mutation in 12 of 29 (41.4%) and 6 of 27 (22.2%) evaluated patients; TET2 and CBL mutations were found in 2 and 1 patients, respectively. EZH2-mutated PMF patients had significantly higher leukocyte counts, blast-cell counts, and larger spleens at diagnosis, and most of them (52.6%) were in the high-risk International Prognostic Score System (IPSS) category. After a median follow-up of 39 months, 128 patients (25.9%) died, 81 (63.3%) because of leukemia. Leukemia-free survival (LFS) and overall survival (OS) were significantly reduced in EZH2-mutated PMF patients (P = .028 and P < .001, respectively); no such impact was seen for PPV/PET-MF patients, possibly due to the low number of mutated cases. In multivariate analysis, survival of PMF patients was predicted by IPSS high-risk category, a < 25% JAK2V617F allele burden, and EZH2 mutation status. We conclude that EZH2 mutations are independently associated with shorter survival in patients with PMF.

Background. Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph−) myeloproliferative disorder characterized by extramedullary haematopoiesis and abnormal neoangiogenesis in both the bone marrow (BM) and the spleen. We previously provided evidence that endothelial cells (ECs) from either the spleen or the splenic vein of PMF patients frequently share the JAK2V617F mutation with the hematopoietic malignant cells. More recently, we confirmed this observation also in BM-derived ECs of PMF patients. The mechanism underlying this phenomenon remains, however, not yet clarified. RUNX1 is a critical regulator of hematopoiesis, required for hematopoietic stem cell (HSC) generation and function. In human embryo, it is expressed in all emerging HSCs and progenitors and it is a necessary transcription factor for endothelial to hematopoietic transition. In the adult humans it is expressed in all blood cells, in decreasing intensity according to the maturation status, except erythrocytes. In angiogenesis, it induces endothelial differentiation and maturation as well as vascular network formation by promoting expression of VE-cadherin. Finally, it is involved in retinal aberrant neoangiogenesis. Aim. To assess if neoangiogenetic activity observed in spleen and BM of PMF patients is associated with RUNX1 expression in ECs. Patients and Methods. Paraffin-embedded BM sections from patients with MF (n=3), and from patients with lymphomas (named as CTRLs), who underwent BM biopsy for disease staging (n=2), were used for immunostaining analysis. Spleens samples were collected from 3 patients with PMF, who received splenectomy for clinical reasons, and from 2 healthy subjects (HS), who underwent surgery after traumatic damage. Fresh spleen samples were embedded in OCT, and then snap-frozen and stored in liquid nitrogen. Endothelial colony forming cells (ECFCs) were obtained from peripheral blood (n=1 PMF, n=1 HS), PMF spleen tissue (n=1) and cord blood (n=1), according to Ingram et al (Blood 2004;104:2752) and cytospun on slides at confluence. ECFCs, BM and spleen sections were stained with antibodies directed against RUNX1 and VE-cadherin. The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGEJ software. We evaluated 10 fields for each BM and spleen section and measured the number of RUNX1-positive vessels/total vessels. We considered positive a vessel that contained at least one RUNX1-positive cell. The results are shown as mean ± SD. Results. Immunofluorescence staining of spleen and BM sections confirmed the presence of increased neoangiogenetic processes in samples from PMF patients with respect to CTRLs. The staining of BM and spleen sections obtained from PMF patients with antibodies anti-RUNX1 and anti-VE-cadherin (endothelial marker) allowed the detection of RUNX1+VE-cadherin- cells in the parenchima of BM and spleen (Figure 1A, 1C), occasionally in perivascular position (Figure 1A, arrowhead). Interestingly, in all patients analyzed, we could detect RUNX1+VE-cadherin+ cells in both the parenchima and in the vessels both in the BM and the spleen (Figure 1A, 1C, arrow). In BM, 22 ± 12% of the vessels had at least one double positive cell in the microvessel wall; in spleen tissue the percentage of RUNX1+ ECs increased to 60 ± 13%. We detected RUNX1+VE-cadherin- cells only in the BM parenchima of CTRLs but not in the spleen of HS, whereas RUNX1+VE-cadherin+ cells were never observed in the vessels of neither the spleen nor the BM (Figure 1B, 1D) of HS and CTRLs, respectively. The circulating ECFCs obtained from both PMF and HS were, as expected, VE-cadherin+ but did not express RUNX1; however, 30% of spleen-derived-ECFCs of the PMF patient were RUNX1+. In cord blood-derived ECFCs a small percentage (3%) of RUNX1+ cells was observed. Conclusions. Our data show a selective expression of RUNX1 in splenic- and BM-ECs of PMF patients, suggesting that activation of RUNX1 expression could be associated with the neoangiogenetic processes that characterize the disease. The expression of RUNX1 in ECFCs from spleen but not in their circulating counterpart suggests a role for the splenic microenvironment in determining RUNX1 expression in ECs. Further studies are ongoing to assess the genotype of RUNX1+ ECs of PMF patients. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Most solitary hyperfunctiong regions on thyroid scan consist of benign tissue. Here we report a patient with a Burkitt-like lymphoma that was infilterated into a region containing a hyperfunctioning nodule.A 56-year-old man was referred to our Endocrine Unit in May 2009 due to the incidental discovery of a large left thyroid lobe nodule by a computed tomography study. This had been performed to search for a primitive tumor in a patient with bone metastasis. He was clinically and biochemically thyrotoxic with no evidence of humoral thyroid autoimmunity. The nodule had a dyshomogenous appearance at neck ultrasonography, with multiple hypoechogenic areas and calcifications. (99m)-Technetium thyroid scintiscan revealed a hot nodule with suppression of the contralateral lobe. Fine-needle aspiration cytology indicated the presence of neoplastic cells not of thyroid origin. Remission of hyperthyroidism was obtained with methimazole, and the patient was submitted to left lobe thyroidectomy and istmectomy. Histological analysis of the surgical specimen led to a diagnosis of Burkitt-like large B-cell lymphoma harbored within a thyroid adenoma. After further staging, the final diagnosis was stage IV E Burkitt-like lymphoma with the involvement of the bone and the thyroid. This is the first description of an aggressive Burkitt-like lymphoma that infiltrated an hyperfunctioning thyroid adenoma, thus presenting as a hot nodule at thyroid scintiscan. In our patient there was no humoral or histological evidence of thyroid autoimmunity, thus suggesting a metastatic seeding of the lymphoma within the hyperfunctioning thyroid nodule.Involvement of the thyroid gland by Burkitt-like lymphoma is extremely rare as is close localization of malignancy and a hyperfunctioning thyroid nodule. As highlighted by the present report, performing fine-needle aspiration cytology should be always considered in the clinical context of a metastatic disease of unknown origin or when there are ultrasonography signs suggesting malignancy, even when the nodule is hyperfunctioning.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are ubiquitous compounds that may act as endocrine disruptors, neurotoxic agents, and fetal development perturbing substances and may also be carcinogenic, as recently demonstrated in experimental animal models. There is little information on the potential for these compounds to affect the thyroid. Therefore, this study was performed to measure the intrathyroidal levels of PFOA and PFOS in surgical specimens of thyroid glands and to determine if there was a relationship between the concentrations of these substances and the clinical, biochemical, and histologic phenotype of the patients from whom the thyroids were obtained. We also sought to determine if there was a relationship between tissue and serum levels of both PFOA and PFOS.PFOA and PFOS were measured in 28 patients undergoing thyroid surgery for benign (15 multinodular goiters and 7 Graves' disease) and malignant (5 papillary and 1 follicular carcinoma) thyroid disorders.PFOA and PFOS were detectable in all surgical specimens of thyroid tissue. Their median concentrations were 2.0 ng/g (range = 0.4-4.6 ng/g) and 5.3 ng/g (range = 2.1-44.7), respectively. Intrathyroidal concentrations of PFOA and PFOS were similar in the thyroids of patients with thyroid diseases as in thyroid glands obtained at autopsy. There was no relationship between the intrathyroidal concentrations of either PFOA or PFOS and the underlying thyroid disease. A significant correlation between the serum and the tissue levels of PFOS was found in all patients. The serum concentrations of PFOA and PFOS were significantly higher than those in the correspondent surgical specimens.These observations do not support the view that PFOA and PFOS are actively concentrated in the thyroid. PFOA and PFOS, however, are both found in surgical and autopsy thyroid specimens. Therefore, further studies to determine if they have disrupting effects in thyroid cells or tissue, and studies to compare populations with and without these compounds in their thyroid glands, are important.

RET mutations play an important role in the development of human neuroendocrine tumors. The prevalence of the RET polymorphism G691S of exon 11 is higher in patients with medullary thyroid carcinoma (MTC) as compared to the general population. A weak association between RET polymorphisms and sporadic papillary thyroid carcinoma (PTC) has also been described. We hereby describe the association of MTC, bronchial carcinoid tumor, and PTC in a familial setting. A 75-yr-old woman developed MTC 7 yr after successful treatment of a bronchial carcinoid. Serum calcitonin was 12.9 pg/ml with a peak response to pentagastrin (151.0 pg/ml). The patient underwent total thyroidectomy and a genetic mutational analysis of the RET gene. Histological evaluation confirmed MTC with no evidence of lymph nodes involvement. After thyroidectomy serum calcitonin was

A series of 267 gastroenteropancreatic endocrine tumours has been revised from the point of view of histopathologic diagnosis, hormonal profile and clinical behaviour. Results of this investigation, together with revised concepts on the histogenesis of gastroenteropancreatic endocrine growths, allowed to develop detailed classification systems which proved useful for precise tumour diagnosis and for clinicopathologic correlation, with special reference to tumour function, prognosis and therapy. Among 132 pancreatic growths, various types of islet cell tumours (61 cases), with (45 cases) or without (16 cases) hyperfunctional syndrome, were separated from different types of gut-related (38 cases) and 'ectopic' (three cases) tumours, as well as from 25 non-functioning, locally symptomatic tumours, three small cell carcinomas and two mixed endocrine-exocrine tumours. Among 97 intestinal tumours, 39 argentaffin EC cell carcinoids, mostly from the appendix and ileum, were separated from 23 hindgut-type carcinoids, mostly from the rectum, 22 gastrin cell tumours, mainly from the duodenal bulb, five somatostatin cell tumours, mostly from the periampullary region of the duodenum, and two gangliocytic paragangliomas. Among 38 gastric tumours, five small cell 'neuroendocrine' carcinomas were separated from three gastrin cell tumours and 30 argyrophil carcinoids, 27 of which arose in the body fundus, 16 associated with chronic atrophic gastritis and four with combined Zollinger Ellison/Multiple Endocrine Neoplasia Syndrome.

Few investigators have evaluated the usefulness of the JAK2 V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis (PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2 V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2 mutation determined at diagnosis. JAK2 V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2 V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2 V617F genotype should be considered in any future risk stratification of patients with PMF.

Summary The clinical and haematological phenotype of patients with myelofibrosis harbouring MPLW515L/K mutation has not been thoroughly investigated. Of 217 myelofibrosis subjects, 18 (8·2%) had an MPL mutation, four of which (22%) co-existed with JAK2V617F mutation. When compared with MPL wild-type patients, irrespective of JAK2V617F status, those with MPLW515L/K, were more frequently female, were older (61 years vs. 57 years; P = 0·02), presented with more severe anaemia (haemoglobin, 101 g/l vs. 121 g/l; P = 0·002) and were more likely to require regular transfusional support (P = 0·012). These data indicate that MPL mutation in myelofibrosis characterises patients with more severe anaemic phenotype.

In both rodents and humans the development of gastrin-promoted gastric argyrophil enterochromaffin-like cell carcinoids requires the involvement of a genetic factor inherent to multiple endocrine neoplasia syndrome or of type A autoimmune chronic atrophic gastritis. Prolonged severe hypergastrinaemia acting on non-gastritic mucosa, as in Zollinger-Ellison syndrome patients, results in diffuse argyrophil enterochromaffin-like cell hyperplasia but, as a rule, does not produce tumours. Combination of chronic atrophic gastritis (mostly related to Helicobacter pylori infection) with hypergastrinaemia frequently causes linear and micronodular hyperplasia of argyrophil cells, whereas carcinoids are exceptional. No tumours or pre-neoplastic lesions have been observed in patients treated long-term with proton pump inhibitors, apart from rare cases in patients with combined Zollinger-Ellison and multiple endocrine neoplasia syndromes. A moderate increase in the incidence of argyrophil cell clustering, with or without hyperplasia, probably results from the parallel evolution of ulcer-associated Helicobacter gastritis into chronic atrophic gastritis. Eradication of H. pylori with a combination of proton pump inhibitors and antibiotics suppresses gastritis and prevents ulcer recurrence.

Purpose: To elucidate the mechanism by which trastuzumab, a humanized monoclonal antibody against HER2 with proven survival benefit in women with HER2-positive metastatic breast cancer, mediates its antitumor activity. Experimental Design: A pilot study including 11 patients with HER2-positive tumors treated in a neo-adjuvant setting with trastuzumab was performed. Trastuzumab was administered i.v. at a dose of 4 mg/kg followed by three weekly i.v. doses of 2 mg/kg. The primary tumor was surgically removed 7 days after the last treatment. Surgical samples, tumor biopsies, and lymphocytes from these patients were collected for biological studies. Result: Clinical data indicated one complete pathological remission and four partial remissions using RECIST (Response Evaluation Criteria in Solid Tumors). Trastuzumab was well tolerated and neither serious adverse events nor changes in cardiac function were observed during this short-term treatment and after surgery. The biological data showed that, independent of response, (a) all patients showed high levels of circulating trastuzumab; (b) saturating level of trastuzumab was present in all of the tumors; (c) no down-modulation of HER2 was observed in any tumors; (d) no changes in vessel diameter was observed in any tumors; (e) no changes in proliferation was observed in any tumors; and (f) a strong infiltration by lymphoid cells was observed in all cases. Patients with complete remission or partial remission were found to have a higher in situ infiltration of leukocytes and a higher capability to mediate in vitro antibody-dependent cellular cytotoxicity activity. Conclusions: The results of this pilot study argue against trastuzumab activity in patients through down-modulation of HER2 but in favor of antibody-dependent cellular cytotoxicity guiding efforts to optimize the use of trastuzumab in breast cancer patients.

1Unit of Clinical Epidemiology and Center for the Study of Myelofibrosis, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy 2Department of Internal Medicine, Unit of Clinica Medica 1, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy 3Gastroenterology 3 Unit, IRCCS Ca Granda Ospedale Maggiore Policlinico Foundation, Milano, Italy 4Section of Hematology, Department of Critical Care, University of Florence and Istituto Toscano Tumori, Florence, Italy

Thyroid cancer may express estrogen receptors (ERs) and various grades of peri-tumor inflammation. The aim of the study was to evaluate the expression of ERs in relation to the TNM stage and peri-tumor inflammatory infiltrate in differentiated thyroid cancers. 127 patients (109 females, 18 males) with differentiated thyroid cancer (T1 = 91, T2 = 18, T3 = 11, T4 = 7) were evaluated. In tumors and in the correspondent extra-tumor parenchyma, ERs expression was evaluated by immunohistochemistry. In 114 tumors and correspondent peri-tumor tissues, the presence of inflammatory infiltration was also recorded. ER-alpha expression was higher in clinical than in incidental tumors of the T1 subgroup (p = 0.037), and was associated with capsular invasion in T2 tumors (p < 0.0001). ER-beta expression was negatively associated with vascular invasion in T1 (p = 0.005) and T2 tumors (p = 0.015). No significant relationship between ERs expression and tumor phenotype emerged in T3 and T4 subgroups. Tumors without inflammatory cell infiltrate showed a higher expression of both ER-alpha (p = 0.035) and ER-beta (p = 0.026) than the ones with inflammatory infiltrate. The relationship between tumor phenotype and ERs expression did not vary in the presence or absence of peri-tumor inflammatory infiltration. ER-alpha positivity and ER-beta negativity are associated with a more aggressive phenotype in both T1 and T2 thyroid cancers, suggesting that tumor biology may be more relevant than tumor size for cancer risk assessment. Inflammatory status is also associated with ERs expression, but not with tumor growth or phenotype.

Background. Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2R33Q/R33Q (R33Q) mutation. Methods and Results. We engineered an adeno-associated viral vector serotype 9 (AAV9) containing cDNA of CASQ2wild-type (AAV9-CASQ2) plus the green fluorescent protein (GFP) gene to infect newborn R33Q mice studied by in vivo and in vitro protocols at 6, 9, and 12 months to investigate the ability of the infection to prevent the disease and adult R33Q mice studied after 2 months to assess whether the AAV9-CASQ2 delivery could revert the catecholaminergic polymorphic ventricular tachycardia phenotype. In both protocols, we observed the restoration of physiological expression and interaction of CASQ2, junctin, and triadin; the rescue of electrophysiological and ultrastructural abnormalities in calcium release units present in R33Q mice; and the lack of life-threatening arrhythmias. Conclusions. Our data demonstrate that viral gene transfer of wild-type CASQ2 into the heart of R33Q mice prevents and reverts severe manifestations of catecholaminergic polymorphic ventricular tachycardia and that this curative effect lasts for 1 year after a single injection of the vector, thus posing the rationale for the design of a clinical trial., Facultad de Ciencias Médicas, Centro de Investigaciones Cardiovasculares

Functional and genetic aberrations of in vitro -cultured marrow-derived mesenchymal stromal cells of patients with classical Philadelphia-negative myeloproliferative neoplasms

Glucagonomas are pancreatic islet cell tumors arising from the alpha cells which belong to neuroendocrine tumors. They frequently metastasize to the liver. We report the case of a 52- year old man with a pancreatic glucagonoma with synchronous multiple liver metastases treated by surgery, transarterial chemoembolization, percutaneous radiofrequency thermal ablation and long-acting octreotide. Our report confirms that a multimodal approach is very effective in patients with unresectable liver metastases from pancreatic endocrine tumors providing long-lasting palliation and probably prolonging survival.

Gastric cancer shows remarkable heterogeneity in histological pattern, cellular phenotype, and genotype. Tumor subsets identified by varying procedures have shown limited reciprocal correlation and have failed to provide a sound rationale for the characterization and classification of all tumors. Based on a case series of 130 gastric cancers that covered both early (70 cases) and advanced (60 cases) stages and that represented most histological types and structural patterns, this study investigated (1) microsatellite instability and p53 gene mutation by means of PCR-based molecular techniques and (2) p53 protein accumulation or tumor cell immunophenotype by means of immunoperoxidase procedures. It was found that microsatellite instability and p53 gene mutation involve two distinct subsets of both early and advanced-stage glandular (intestinal) cancer, and that, contrastingly, they leave purely diffuse cancers unaffected. Mixed cancers, namely, those in which glandular admixed with diffuse growths, showed scarce microsatellite instability at all stages, whereas prominent p53 gene mutation and p53 protein accumulation was limited to the advanced stage alone. No significant correlation was found between tumor cell immunophenotype and either genotype or histotype, although some correlation with particular structural patterns was detected. Comparison of intramucosal with invasive growths within any given tumor suggested that invasive cancers with diffuse-type growth arise in part from mucosal cancers of glandular or mixed structure through progressive loss of intercellular junctional systems. It is concluded that at least two genetically distinct subsets of glandular cancer, one with microsatellite instability and the other with p53 lesions, should be separated both from purely diffuse cancer and, at least in the advanced stage, from mixed cancer. Available evidence suggests distinct clinicopathologic profiles for such tumor entities.

BACKGROUND Cartilage-forming tumors are benign cartilaginous tumors that rarely affect the spinal canal: they account for 2% of all spinal tumors and 2.6% of all benign bone tumors. Pathologically, they may be classified as chondromas, osteochondromas, chondroblastomas, and chondromyxoid fibromas. This oncotype may remain asymptomatic (it is confined within the vertebral structure) or may present as a hard paravertebral swelling (it invades the paravertebral structures) or more rarely, with a slowly-developing neurologic syndrome (it extends into the vertebral canal). METHODS Thirty-one cases have been reported (including our case) of benign cartilage-forming tumors localized in the lumbar column. Only three cases of chondroma of the lumbar spine presented with lumbar radicular pain. We report a fourth case and review clinical and radiologic characteristics of these lesions. RESULTS Eleven out of the 31 cases were diagnosed as chondromas, 17 as osteochondromas, while in three cases the histopathologic diagnosis was not reported. Seventeen cases originated from the neural arch, seven from the vertebral body, two from the spinous process, and in five cases the exact localization was not reported. This tumor is more frequent in males (21 cases out of 31), than in females (five cases); in five cases the sex was not reported. Mean duration of symptoms was 23 +/- 5.1 months (range: 1-96); chondromas have a short clinical history before diagnosis (13.8 +/- 3.4 months) compared to osteochondromas (28.6 +/- 7.6). Clinical presentation with local swelling is reported in 10 cases, in 10 cases local pain without radicular irradiation, in six cases lumbar pain with sciatica, in two cases signs and symptoms of cord compression, one case of cauda syndrome, while in four cases no clinical details are reported. Among the six cases presenting with sciatica, four were chondromas (in all cases the L4 level was involved), and one osteochondroma, while in one case the histopathologic diagnosis was not reported. CONCLUSION Computed tomography is important and indispensable for preoperative diagnosis, giving a precise indication of tumor extent and location and its relationship to the adjacent structures; while MRI is helpful in detecting patterns related to histologic malignancy. It is important to examine the whole tumor histologically because it is known that there may be small areas that show signs of malignancy; thus is more likely in chondromas than osteochondromas.

Changes in Helicobacter pylori-associated gastritis of the antrum and corpus were investigated in a large number of patients treated with omeprazole, with or without the addition of amoxycillin. To investigate the role of H. pylori-associated gastritis in ulcerogenesis and its interplay with omeprazole, biopsies were taken and evaluated according to the Sydney system. Successful eradication of H. pylori (assessed histologically 4 weeks after the end of therapy) led to prompt and persistent suppression of gastritis activity, slow, partial regression of mononuclear inflammation and an ulcer recurrence rate of only 14% during the 6 months' follow-up. In patients treated with omeprazole and placebo, or where eradication treatment with omeprazole and amoxycillin had failed, transient clearance of H. pylori from the antral (but not oxyntic) mucosa was seen. In both of these groups of patients transient regression in the antrum, and worsening in the corpus, of gastritis activity and mononuclear inflammation were evident, coupled with ulcer recurrence rates of 72 and 46%, respectively. It was concluded that H. pylori colonization and gastritis activity play a crucial role in ulcerogenesis, that acid inhibition treatment improves antral H. pylori gastritis and worsens the oxyntic mucosal gastritis, and that this can be prevented by eradication of the H. pylori infection.

Among endocrine tumors arising in the intestinal tract, midgut argentaffin EC cell carcinoids, duodenal gastrin cell tumors and rectal trabecular L cell carcinoids, in order of decreasing frequency, are those better represented. Together they account for more than 80% of such tumors. Duodenal somatostatin cell tumors, gangliocytic paragangliomas and poorly differentiated neuroendocrine carcinomas, are also well defined tumor entities. The carcinoid syndrome with intermittent flushing, hypotension and diarrhea, and the Zollinger-Ellison syndrome with severe peptic ulcer disease, are the only hyperfunctional syndromes consistently found in association with these tumors. The carcinoid syndrome arises in about 10% of intestinal carcinoids, usually in their advanced metastatic stage. The Zollinger-Ellison syndrome occurs in association with about 40% of gastrin cell tumors, including small intramural growths. Tumor prognosis depends on mode and site of presentation, histology, cell type(s), size, level of invasion, metastases (especially distant metastases) and associated clinical syndrome or background disease.

Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F+ ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.

PurposeIn the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.Patients and methodsWe investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.ResultsAs compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.ConclusionPre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.

Rationale: Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum. Objective: We intended to evaluate whether viral gene transfer of wild-type CASQ2 may rescue the broad spectrum of abnormalities caused by mutant CASQ2. Methods and Results: We used an adeno-associated serotype 9 viral vector to express a green fluorescent protein-tagged CASQ2 construct. Twenty weeks after intraperitoneal injection of the vector in neonate CASQ2 KO mice, we observed normalization of the levels of calsequestrin, triadin, and junctin, rescue of electrophysiological and ultrastructural abnormalities caused by CASQ2 ablation, and lack of life-threatening arrhythmias. Conclusions: We have proven the concept that induction of CASQ2 expression in knockout mice reverts the molecular, structural, and electric abnormalities and prevents life-threatening arrhythmias in CASQ2-defective catecholaminergic polymorphic ventricular tachycardia mice. These data support the view that development of CASQ2 viral gene transfer could have clinical application.

Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cells, characterized by an increased number of circulating CD34+ cells. Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. A subset of patients display immune-related abnormalities that suggest an (auto)immune pathogenesis. In addition, it has been reported a significant association between personal history of a broad span of autoimmune diseases and subsequent risk of myeloproliferative neoplasm (Kristinsson SY Haematologica 2010; 95:1216-1220). CD4+ T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN) (Hasselbalch HC et al. Leuk Res 2013;37:214-20), suggesting that T cells play an important role in their pathogenesis. A recent paper by Keohane C et al (BJH 2015; doi 10.1111/bjh 13519) shows that peripheral blood (PB) T regulatory cells (Tregs) are reduced in MPN patients compared to healthy subjects (CTRLs) and that this decrease is even more pronounced following JAKi therapy (Massa M et al Leukemia 2014;28:449-51). Unpublished data from our group further indicate a significant (p Study design. To investigate the possible factors favouring a decrease of Tregs, we examined in patients with PMF and Results. 1) The percentages of PB CD4+, CD4+CD25+, or CD4+CD25++CD127low/- cells expressing membrane Annexin V were comparable in patients with PMF and CTRLs (data not shown). 2) The phosphorilation of Stat1, Stat3, Stat5, Stat6, and p38-MAPK (expressed as mean fluorescence intensity: MFI) on CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells before and after stimulation with IFNα, IL6, IL4, or PMA was comparable in patients and CTRLs. The Erk1,2 phosphorilation was comparable in patients and CTRLs at baseline condition, while in patients with PMF Erk1,2 phosphorilation following stimulation with PMA was significantly lower in both CD4+CD25+ cells (p=0.039; median MFI 5.4, range 0.3-40) and CD4+CD25++FOXP3+ cells (p=0.01; median MFI 4.3, range 1.4-9.3) than in CTRLs (CD4+CD25+ cells median MFI 8.1, range 4.9-20; CD4+CD25++FOXP3+ cells median MFI 7.3, 3.7-10.8). 3) sIL2rα plasma levels were higher (p=0.01) in patients with PMF (1658 pg/ml, range 476-8968) than in CTRLs (1040 pg/ml, 482-1719); the sIL2rα plasma levels were significantly (R=-0.35, p=0.039) inversely correlated with the percentages of circulating Tregs. No significant correlation was found with the presence (n=13)/absence (n=14) of JAK2V617F mutation. Conclusions. These results indicate that in patients with PMF Tregs present an untuned Erk signaling; moreover, sIL2rα plasma levels are higher in patients than in CTRLs, and significantly inversely correlated with Treg percentages. These findings may partly explain the reduced frequency of Tregs in PMF since Erk signaling is pivotal in T cell differentiation, regulation of T cell homeostasis, and defects in Erk expression have been associated with autoimmunity (Chiung-Fang Chang J Immunol 2012; 189:721-731). Moreover, increased levels of sIL2rα may impact on IL-2-mediated functions and alterate the optimal activation of Erk1/2 that is IL-2 dependent (Lequn Li, Blood. 2005;106:3068-3073), Disclosures No relevant conflicts of interest to declare.

A contribution of Helicobacter pylori gastritis to the pathogenesis of non-ulcer dyspepsia (NUD) remains uncertain.Administration of an appropriate clinical questionnaire followed by endoscopy allowed us to select, among 139 outpatients with dyspepsia, 87 non-ulcer dyspepsia patients with more severe and group-distinctive symptoms, 35 of whom were classified as having ulcer-like (ULD). 38 as dysmotility-like (DLD), and 14 as reflux-like dyspepsia (RLD). Biopsy specimens were evaluated for H. pylori gastritis in accordance with the Sydney system. The 70 H. pylori-positive cases were treated with omeprazole, 20 mg twice daily, and amoxycillin, 1 g three times daily for 2 weeks.Higher rates of H. pylori colonization were found histologically in the gastric mucosa of ULD (91%) and RLD (86%) than in that of DLD (68%) or asymptomatic (42%) patients. ULD differed from RLD patients in their higher score of antritis activity. Three and 6 months after H. pylori eradication ULD (but not DLD) showed significant regression of dypspetic symptoms scores.It seems likely that H. pylori gastritis, with special reference to active antritis, is among causative factors of ULD. Its role in the pathogenesis of RLD and DLD needs further investigation.

To clarify the mechanisms of the gastric mucosal immune—inflammatory response to Helicobacter pylori infection, surgical and biopsy specimens from asymptomatic uninfected, gastritis-free individuals and from H. pylori-positive ulcer patients with chronic gastritis were investigated using light and electron microscopy. Activation of the antigen-transporting endocytic-endosomal system, enhanced expression of the antigen-processing enzyme cathepsin E and de novo expression of antigen-presenting human leukocyte antigen (HLA)-DR molecules have been detected in H. pylori-colonized gastric epithelium. These findings may be crucial in the production of a mucosal immune-inflammatory response to H. pylori infection. Cytoplasmic swelling and vacuolation, micropapillary change, mucin loss, erosion of the juxtaluminal cytoplasm and cell desquamation were the main effects of bacterial cytotoxicity on gastric surface-foveolar epithelium. Activated macrophages and granulocytes (partly linked to the mucosal IgG immune res...