Loredana Villari

CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia

Chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share many features and their differential diagnosis may be challenging, especially when a leukemic picture alone is present. Monoclonal antibody panels are often useful, with CD23 being the most reliable. However, MCL diagnosis should be confirmed by immunohistochemical cyclin D1 detection, sometimes with equivocal or even negative results. Other cytofluorimetric, cytogenetics or molecular techniques are reliable but not widely available. B-CLL leukemic cells express CD200, a membrane glycoprotein belonging to the immunoglobulin superfamily. We investigated its expression on fresh neoplastic cells of 93 patients with a CD5+ lymphoproliferative disease (79 selected B-CLL and 14 MCL in leukemic phase). Although these data cannot be generalized, all B-CLL samples we examined were positive, with CD200 present on the vast majority of the cells while, in MCL patients, CD200 was expressed by a small minority of CD5+ cells in three subjects and totally absent in the remaining 11. We then examined CD200 expression on paraffin-embedded lymphoid tissues and bone marrow (BM) trephine biopsies from 23 B-CLL and 44 MCL patients. Again, all B-CLL cells were CD200+ both in lymph nodes and in BM while all MCL cells were negative. Adding CD200 in routine panels could be of diagnostic utility in excluding MCL diagnosis.

Heat Shock Protein 27 Expression in the Epithelium of Periapical Lesions

Heat shock protein (HSP) 27 is a member of the small HSP family that plays a part in the regulation of epithelial cell growth and differentiation, wound healing, apoptosis and cell protection against inflammatory cytotoxicity mediators. Thus the expression of HSP27 was investigated immunohistochemically in periapical granulomas with epithelial rests of Malassez and in radicular cysts. Anti-HSP27 mouse monoclonal antibody and peroxidase-labeled streptavidin-biotin standard technique were used to study the expression of HSP27. Proliferating epithelial cell rests, and islands of epithelium and epithelial lining of microcysts strongly reacted throughout all layers, whereas radicular cysts epithelial lining presented mainly a moderate suprabasal staining pattern. However both the proliferating epithelial cell rests and the radicular cysts shared an over-expression of HSP27 immunostaining intensity in coincidence with the presence of local infiltration of immune cells. HSP27 may play several roles in periapical lesions that include contributing to the migration of epithelial cell rests and an increased resistance both to necrotic and apoptotic cell deaths.

THYMIC CARCINOMA, SYSTEMIC LUPUS ERYTHEMATOSUS, AND HYPERTROPHIC PULMONARY OSTEOARTHROPATHY IN AN 11-YEAR-OLD BOY: A Novel Association

Thymic carcinoma is exceptionally rare in children and it has never previously been associated with autoimmune disorders. The authors report the case of an 11-year-old boy with thymic carcinoma, hypertrophic pulmonary osteoarthropathy, and an autoimmune disease that resembled systemic lupus erythematosus. To their knowledge, this is the first case of such complex clinical findings. The tumor was of high grade histologically and the boy died after 1 year, in spite of chemotherapy and radiotherapy. A review is presented of the available medical literature on thymic malignancy in childhood.

Effect of prolonged incubation with copper on endothelium-dependent relaxation in rat isolated aorta.

We investigated the effects of prolonged exposure to copper (Cu2+) on vascular functioning of isolated rat aorta. Aortic rings were exposed to CuSO4 (3–24 h) in Dulbeccos modified Eagle medium with or without 10% foetal bovine serum (FBS) and then challenged with vasoconstrictors or vasodilators in the absence of Cu2+. Exposure to 2 μM Cu2+ in the absence of FBS did not modify the response to phenylephrine (PE) or acetylcholine (ACh) in aortic rings incubated for 24 h. Identical exposure in the presence of FBS increased the contractile response to 1 μM PE by 30% (P<0.05) and impaired the relaxant response to 3 μM ACh or 1 μM A23187 (ACh, from 65.7±7.1 to 6.2±1.1%, n=8; A23187, from 74.6±8.2 to 12.0±0.8%, n=6; P<0.01 for both). Cu2+ exposure did not affect the relaxant response to NO‐donors. Impairment of vasorelaxation appeared 3 h after incubation with 2 μM Cu2+ and required 12 h to attain a steady state. Vasorelaxation to ACh was partially restored by 1 mM tiron (intracellular scavenger of superoxide ions; maximum relaxation 34.2±6.4%, n=10, P<0.01 vs Cu2+ alone), whereas catalase, superoxide dismutase or cycloheximide were ineffective. Twenty‐four hour‐exposure to 2 μM Cu2+ did not affect endothelium integrity or eNOS expression, and increased the Cu content in arterial rings from 6.8±1.1 to 18.9±2.9 ng mg−1 wet weight, n=8; P<0.01. Our results show that, in the presence of FBS, prolonged exposure to submicromolar concentrations of Cu2+ impaired endothelium‐dependent vasorelaxation in aortic rings, probably through an intracellular generation of superoxide ions.

Increased phospho-mTOR expression in megakaryocytic cells derived from CD34+ progenitors of essential thrombocythaemia and myelofibrosis patients.

The mammalian target of rapamycin (mTOR) is a serine/ threonine kinase that forms different multiprotein complexes with diverse subunit compositions, downstream substrates and biological effects (Ma & Blenis, 2009). As mTOR is a key regulator of cell growth and metabolism, its improper activation has been previously linked to carcinogenesis (Meric-Bernstam & Gonzalez-Angulo, 2009). Polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are BCR-ABL1-negative myeloproliferative neoplasms (MPNs). ET is characterized by megakaryocytic bone marrow hyperplasia and a sustained elevation of platelet count with a tendency for thrombosis and haemorrhage (Levine & Gilliland, 2008). PMF can arise ‘de novo’ or evolve from PV or ET. In PMF, the abnormal proliferation of an aberrant clone eventually leads to the replacement of the normal bone marrow with connective tissue fibres (Tefferi & Vainchenker, 2011). As the molecular culprit(s) of these disorders still need(s) to be elucidated, current treatment options are not curative and have yet to produce clear benefits in terms of increasing overall survival (Tefferi & Vainchenker, 2011). Mammalian target of rapamycin is a pivotal downstream target of the thrombopoietin receptor (Drayer et al, 2006). Thus, it is not surprising that several studies have previously demonstrated critical contributions of mTOR signalling in the proliferation and differentiation of normal megakaryocyte (MK) cultures (Drayer et al, 2006; Raslova et al, 2006). However, the role of mTOR in the megakaryopoiesis of ET and PMF cells remains to be investigated. As thrombocytosis is a common clinical feature of both ET and PMF, we wanted to establish whether mTOR activation was involved in the increased platelet counts observed in these MPNs. To this end, CD34-positive (CD34+) progenitors were employed to reproduce the different stages of human MK differentiation that generate morphologically and functionally mature platelets (Guerriero et al, 1995; Deutsch & Tomer, 2006). After acquiring informed consent, human CD34+ cells were isolated from healthy donors, ET and PMF patients using the midi-MACS immunomagnetic separation system (Miltenyi Biotec, Auburn, CA, USA) obtaining a >90% pure cell population. Megakaryocytic differentiation was then induced by growing these cells for 14 d in serum-free medium in the presence of bovine serum albumin, insulin, human transferrin, human low-density lipoprotein, and 100 ng/ml thrombopoietin (PeproTech, London, UK). The expression of the CD34 and CD61 surface markers was evaluated by flow cytometry, collecting cells at different time-points (days 0, 3, 12) and using a fluorescein isothiocyanate-labelled anti-CD34 antibody and an anti-CD61 antibody bound to phycoerythrin (Miltenyi Biotec). A gradual decrease in the CD34 signal associated with increased expression of the MK-specific CD61 marker confirmed the purity of the initial population and its progressive megakaryocytic differentiation (Figure S1A). Morphological observations after May–Grunwald Giemsa staining (Sigma, St Louis, MO, USA) correlated with the flow cytometry data, showing large polynucleated MK cells after 12 d of culture (Figure S1B). A unilineage system was subsequently employed to establish if MK cells isolated from patients with ET or PMF expressed higher levels of phosphorylated mTOR (p-mTOR) when compared to healthy individuals. We initially performed an immunofluorescence (IF) analysis in which cells were stained with an anti-p-mTOR (recognizing serine 2448) antibody (Cell Signaling, Danvers, MA, USA) after 3 and 12 d of culture. While we found no differences in mTOR phosphorylation after 3 d (Fig 1A, top panels), p-mTOR was increased in MKs from ET and PMF patients obtained after 12 d of culture (Fig 1A, bottom panels), but not in cells derived from healthy donors. To quantify the qualitative differences detected by IF, we analysed mTOR phosphorylation by flow cytometry, evaluating the xfold increase in median fluorescence intensity over the isotype control (D’Asaro et al, 2010). This analysis confirmed higher p-mTOR expression in ET and PMF-derived MKs generated after 12 d of culture (Fig 1B). To further confirm these data, we performed an anti-pmTOR immunohistochemical analysis on 35 bone marrow specimens derived from patients diagnosed with ET (n = 14) or PMF (n = 21). Three micrometre-thick sections were incubated with the same anti-p-mTOR antibody employed for the IF analyses. We found higher p-mTOR expression in 34 of 35 bone marrow samples as compared to the mostly negative staining observed in healthy individuals (Fig 2). The present study provides the first comparison of p-mTOR expression in MK cells derived from the CD34+ progenitors of healthy donors, ET and PMF patients. Recent evidence suggests that targeting mTOR with the oral inhibitor everolimus may be clinically useful for patients

Distribution of extracellular matrix glycoproteins in the human mesonephros

We analyzed the expression and distribution of collagen types IV and VI, laminin and fibronectin during the development and regression of the mesonephros in human embryos and fetuses ranging from 6 to 12 weeks of gestation by indirect immunoperoxidase methods. Type IV collagen, laminin and fibronectin were detected along the glomerular, tubular and capsular basement membranes of developing and mature nephrons. Only type IV collagen and fibronectin were found in the mesangium. Type VI collagen formed a delicate interstitial fibrillar network and a continuous basement membrane-like structure along the mesonephric nephrons. Basement membranes (GBM) of developing and mature glomeruli showed a distinct continuous staining for this collagen. The mesangial matrix was rich in type VI collagen. Mesonephric involution started during the 8th week of gestation and coincided with a moderate expansion of mesangial matrix and progressive collapse of the capillary walls, while the tubules became thinner and shorter. Staining for all extracellular matrix glycoproteins studied showed GBM wrinkling, gradual disintegration of some capillary loops and glomerulosclerosis. The sclerotic glomeruli were strongly positive for type IV collagen and less positive for type VI collagen and fibronectin. Laminin was absent. Our results indicate that collagen types IV, VI, laminin and fibronectin may be involved in the development and regression of the human mesonephros.

Deciduoid-like stromal cells in a diabetic patient with bilateral gynecomastia: a potential diagnostic pitfall

Sir, Epithelioid stromal cells (ESCs) were originally described as a typical feature of diabetic mastopathy, along with lymphocytic mastitis [5]. As these cells have also been reported in the breast stroma of patients with hypothyroidism and systemic lupus erythematous, it has been postulated that they may represent a reliable morphological marker of autoimmune disorders [1]. Diabetic mastopathy may be clinically and histologically confused with carcinoma [2], especially in those cases where there is a presence of large-sized ESCs [1]. We report the first case of gynecomastia with extensive deciduoid-like stromal changes in a patient affected by long-standing insulin-dependent diabetes mellitus (IDDM). A 44-year-old man presented with a retro-areolar nodule in the left breast. Ultrasound analysis revealed an irregular outlined, non-homogeneous hypoechoic mass. The lesion was surgically excised. After a 1-year clinical follow-up, a similar lesion was noted and excised from the contralateral breast. The patient referred to had a 31-year history of IDDM, and he had been undergoing hemodialysis for diabetic nephropathy for 2 years. Grossly, both surgical samples consisted of unencapsulated fibrous nodular masses, measuring 3 cm and 4 cm in greatest diameter, respectively, with a firm, grayish-white cut surface. Histologically, both lesions exhibited the typical features of florid gynecomastia: increased number of ducts with epithelial proliferation and periductal myxoid stroma containing slight lymphocytic infiltration (Fig. 1A). An unexpected feature was the presence in the interlobular stroma of numerous, large cells with deciduoid-like features, arranged in solid sheets and embedded in fibrous tissue (Fig. 1A, B). These cells were round, ovoid, or polygonal in shape, with an abundant pale-to-eosinophilic cytoplasm, well-defined cellular borders and large, round vesicular nuclei containing one or more prominent nucleoli (Fig. 1B). A low number of cells were binucleate and showed a granular eosinophilic cytoplasm. A mild nuclear pleomorphism was focally seen. Mitotic figures were seen infrequently (2–3 50 high-power field). No atypical mitosis or necrosis was identified. A moderate stromal perivascular lymphocytic infiltrate was observed. Immunohistochemical analyses, revealing reactivity for vimentin, a-smooth-muscle actin (20–30% of cells) (Fig. 1C) and no staining with pancytokeratins, epithelial membrane antigen, desmin, h-caldesmon, calponin, CD34, S100 protein, HMB45, CD68 and p63 were consistent with a fibro/myofibroblastic nature of deciduoid-like stromal cells. The association of diabetes mellitus with gynecomastia has only rarely been reported [3]. As plump stromal cells are lacking in common gynecomastia [3], we think that the deciduoid-like stromal cells of the present case represent a morphological variant of ESCs, typically occurring in diabetic mastopathy [1, 5]. It is likely that the deciduoid-like morphology is the result of reactive stromal changes to a hormonally related proliferative disease (gynecomastia) and to hemodialytic treatment, which may play a role through tissue micro-environmental modifications. In a large series of lymphocytic mastitis due to different basic diseases, the two most florid examples of epithelioid stromal changes were seen in the only diabetic male patient and in a woman undergoing hemodialysis by systemic lupus erythematosus [1]. Interestingly, our patient was a diabetic man undergoing hemodialysis. Awareness of deciduoid-like stromal changes in patients with the association diabetes mellitus/gynecomastia is crucial for pathologist to avoid confusion with benign and malignant tumors. Differential diagnosis mainly reG. Magro ()) Dipartimento G.F. Ingrassia, Anatomia Patologica, Universit di Catania, Via S. Sofia 87, 95123 Catania, Italia e-mail: g.magro@unict.it Tel.: +39-95-3782024 Fax: +39-95-3782023

Hodgkin's disease as a second malignant neoplasm in childhood: report of a case and review of the literature.

There is a known association between lymphoid malignancy and Hodgkins disease(HD), but the development of HD in children who have been treated for leukemia or lymphoma is very uncommon. Hodgkins disease is, after retinoblastoma, the most common primary tumor that is associated with development of second malignant neoplasm. For reasons that remain to be determined, HD is very rare as a second malignancy [1, 2, 3]. We report the caseofa eight-year-old girl whodevelopedHD 6 years aftertreatment forcommon acute lymphoblastic leukemia (ALL). This case promptedus to review the published literature for cases of secondary HD in childhood. Our experience suggests that we should follow strictly our patients with ALL and be ready to intervene with invasive diagnostic procedures at the least suspicion of a second or recurrent neoplasm. The most frequent causes of second tumors are radiotherapy, genetic susceptibility and prior treatment with certain chemotherapeutic agents, suchas nitrogen mustards. It is likely that any typeof immunodeficiency, even without symptoms, might play a role in the development of second tumors in childhood.

The prognostic value of the myeloid-mediated immunosuppression marker Arginase-1 in classic Hodgkin lymphoma

Purpose Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL. Experimental design First, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N = 40) and validation (N = 78) sets. Results s-Arg-1 was higher in patients with advanced stage (p = 0.045), B-symptoms (p = 0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p = 0.012). Baseline levels of s-Arg-1 > 200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2. Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p = 0.0004 and p = 0.012). Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N = 63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N = 46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N = 9) with a 3-Y PFS of 37% (p = 0.0004). Conclusions We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a novel potential biomarker for HL prognosis.

Oral Lesion as Unusual First Manifestation of Multiple Myeloma: Case Reports and Review of the Literature

Extramedullary plasmacytoma (EMP) and solitary bone plasmacytoma (SBP) represent a disease continuum through a multistage process of cell differentiation, survival, proliferation, and dissemination, strictly related to multiple myeloma (MM), the second most common hematological malignancy. Herein, we report two cases of recurrent oral plasmacytoma progressed to MM, in which the first clinical sign of a more widespread disease was limited to the mouth. Based on our experience, we recommend a strict workup for the differential diagnosis between EMP, SBP, and MM for patients with oral plasmacytoma, including radiological exam of the skeleton, magnetic resonance imaging (MRI) of the bone, and positive emission tomography (FDG-PET). MRI and possibly PET can all be used to more sensitively detect EM plasmacytoma sites.