Between barriers and inclusion. Multidisciplinary reflections on gender and disability

The European project RISEWISE offered a possible model for a multidisciplinary collaboration. During the four years, innovative goals were pursued for breaking down barriers ranging from creating spaces for dialogue and communication, to exchanging knowledge and experience between disciplines by bringing together academics and associations

Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%-80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis

Validation of an Engineered Cell Model for In Vitro and In Vivo HIF-1 alpha Evaluation by Different Imaging Modalities

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An investigation of trace and isotope light elements in mineral phases from well RN-17 (Reykjanes Peninsula, SW Iceland)

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Reproducibility of the WHO histological criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms.

This study, performed on behalf of the Italian Registry of Thrombocythaemias (Registro Italiano Trombocitemie), aimed to test the inter-observer reproducibility of the histological parameters proposed by the WHO classification for the diagnosis of the Philadelphia chromosome-negative myeloproliferative neoplasms. A series of 103 bone marrow biopsy samples of Philadelphia chromosome-negative myeloproliferative neoplasms consecutively collected in 2004 were classified according to the WHO criteria as follows: essential thrombocythaemia (n=34), primary myelofibrosis (n=44) and polycythaemia vera (n=25). Two independent groups of pathologists reviewed the bone marrow biopsies. The first group was asked to reach a collegial 'consensus' diagnosis. The second group reviewed individually all the cases to recognize the main morphological parameters indicated by the WHO classification and report their results in a database. They were subsequently instructed to individually build a 'personal' diagnosis of myeloproliferative neoplasms subtype just assembling the parameters collected in the database. Our results indicate that high levels of agreement ( 6570%) have been reached for about all of the morphological features. Moreover, among the 18 evaluated histological features, 11 resulted statistically more useful for the differential diagnosis among the different Philadelphia chromosome-negative myeloproliferative neoplasms. Finally, we found a high percentage of agreement (76%) between the 'personal' and 'consensus' diagnosis (Cohen's kappa statistic >0.40). In conclusion, our results support the use of the histological criteria proposed by the WHO classification for the Philadelphia chromosome-negative myeloproliferative neoplasms to ensure a more precise and early diagnosis for these patients

Applicazione ai Tissue Microarray delle tecniche di immunoistochimica e di Ibridazione In Situ Fluorescente per la caratterizzazione immunofenotipica e citogenetica di linfoma a grandi cellule B diffuso

Obbiettivo Lo scopo di questo lavoro \ue8 stato la costruzione di un Tissue Microarray (TMA) pilota per la valutazione immunofenotipica e citogenetica di una casistica di linfoma a grandi cellule B diffuso (DLBCL), tramite analisi immunoistochimiche e di Ibridazione In Situ Fluorescente (FISH). Materiali e Metodi Abbiamo costruito il TMA utilizzando le biopsie linfonodali di 12 pazienti affetti da linfoma a grandi cellule B diffuso; ne abbiamo ottimizzato la costruzione per la lettura al microscopio a fluorescenza distanziando in maniera differenziale i carotaggi dello stesso caso da quelli del casi adiacenti mentre per mantenere la rappresentabilit\ue0 tissutale abbiamo inserito cinque carotaggi da 2 mm per campione. Al TMA abbiamo applicato cinque protocolli immunoistochimici (CD10, BCL6, MUM1, GCET1 e FOXP1) e un protocollo FISH (cMYC). Risultati I dati immunoistochimici sono stati elaborati secondo gli algoritmi di Hans e Choi: secondo il protocollo di Hans sono risultati 8 DLBCL con profilo immunofenotipico centro germinativo simile (GCB) e 4 DLBCL con profilo attivato (ABC); in accordo con l'algoritmo di Choi 7 DLBCL GCB e 5 DLBCL ABC. La conformit\ue0 dei dati immunoistochimici ottenuti \ue8 stata valutata confrontando i risultati con quelli delle indagini immunoistochimiche eseguite su sezione interna, al momento della diagnosi. Abbiamo ottenuto in questo modo una concordanza del 100% con l\u2019algoritmo di Hans e una concordanza del 92% con l\u2019algoritmo di Choi. L\u2019analisi di MYC non ha evidenziato la presenza di traslocazioni ma in tre casi \ue8 stato possibile rilevare polisomie del cromosoma 8. Conclusioni Questo studio ci ha permesso di definire i criteri metodologici per la progettazione e la costruzione di un TMA (con una concordanza del 100% rispetto ai dati ottenuti al momento della diagnosi) che potesse essere letto agevolmente al microscopio a fluorescenza, fornendo cos\uec una piattaforma di analisi ad alta resa per l'esecuzione di indagini immunoistochimiche e citogenetiche FISH

Prognostic relevance of serum beta2 microglobulin in patients with follicular lymphoma treated with anthracycline-containing regimens. A GISL study.

Background and ObjectivesAlthough serum b2 microglobulin (b2M) is an easy parameter to measure, and overexpressedin a large number of lymphoproliferative diseases, its prognostic value hasbeen largely underestimated. The present study examined the influence of b2M levelson overall survival (OS) of patients with follicular lymphoma (FL).Design and MethodsThe prognostic role of b2M was evaluated in 236 patients with FL identified from thedatabases of the Gruppo Italiano per lo Studio dei Linfomi (GISL) and treated withanthracycline-based regimens from 1993 to 2003.ResultsElevated serum b2M levels were found in 82 patients (35%). According to multivariatelogistic regression analysis, elevated b2M levels were associated with elevatedlactate dehydrogenase (LDH) (p=0.021), age (p=0.029), and number of involvednodal areas (p<0.001). The percentage of elevated b2M levels increased progressivelywith increasing FLIPI scores (17%, 38%, and 63% in the low-, intermediate-, andhigh-risk groups, respectively). Five-year OS was 61% (95% CI, 47-73%) and 89% (95%CI, 82-93%) for patients with elevated vs normal b2M levels respectively (p<0.001).Cox regression analysis showed that b2M level had an independent and stable prognosticvalue (HR=3.0; 95%CI, 1.6-5.7). In a multivariate analysis the impact of b2Mlevel on survival was independent of FLIPI score, with a HR of 2.94 (95% CI, 1.54-5.62).Interpretation and ConclusionsOur results demonstrate that in patients treated in the pre-rituximab era, b2M levelwas an independent prognostic marker in addition to FLIPI score. We thus suggestthat b2M be routinely assessed and tested in future prognostic studies of FL patientstreated with combination chemotherapy and anti-CD20 agents

An unusual type of myeloid sarcoma localization following myelofibrosis : a case report and literature review

Myeloid Sarcoma (MS) is a rare malignancy that can present as an isolated disease or more frequently in association with or following acute myeloid leukemia or other myeloid neoplasms and rarely following myelofibrosis. Since molecular pathogenesis and prognostic factors of MS are not well understood, its prognosis remains poor even in the era of novel agents and target therapies. We report the case of a patient with MS following myelofibrosis with multiple subcutaneous, cutaneous and muscle localizations; the latter has been reported in the literature as anecdotal. In this way we aimed to enhance the understanding of this disease

Identification of kitM541l somatic mutation in chronic eosinophilic leukemia, not otherwise specified and its implication in low-dose imatinib response

Activating mutations of KIT receptor tyrosine kinase have been reported in different neoplasms. The M541L KIT substitution (KITM541L) has been described to be associated with pediatric mastocytosis, to enhance growth rate of the affected cells and to confer higher sensitivity to imatinib therapy. We investigated the presence of KITM541L in five males with chronic eosinophilic leukemia, not otherwise specified (CEL, NOS), all negative for Platelet-derived growth factor-alpha (PDGFR) or PDGFRbeta abnormalities, which responded to imatinib therapy. To assess whether the mutation was constitutive or somatic in nature, we evaluated its presence analyzing either the neoplastic or normal cell population (epidermal cells or CD3-positive T lymphocytes). KITM541L substitution was found in 4 out of 5 patients and in all it was somatic in nature. All patients were treated with low dose imatinib (100 mg daily orally), achieving complete and persistent clinical and hematological remission (median follow-up 74 months). One patient relapsed after 50 months. Our study strongly suggests to search for the KITM541L in patients with CEL, NOS, negative for PDGFRalpha and PDGFRbeta abnormalities, to identify a subgroup of cases who may benefit from low dose imatinib therapy