New monoclonal antibodies (mAbs) of the 9th HLDA Workshop: an immunohistochemical analysis of the FACS and IHC panel mAbs on normal and activated skin and tonsil

Cross reactivity and multiple reactivities are very frequently detected in tissue studies; this study summarize the pattern of tissue staining of normal and pathological skin (affected from cutaneous lymphomas) and normal tonsil observed by using FACS and IHC panels of the 9th HLDA Workshop. FACS panel: cryostat sections of frozen tissues were dried for 12 hours, fixed 10" in aceton and stained by an avidin-biotin immunoalkaline phosphatase method (DAKO). IHC panel: formalin fixed paraffin-embedded sections were dewaxed, heated in EDTA buffer (PH-8) for 15 min in a pressure cooker and immunostained by an avidin-biotin immunoalkaline-phosphatase method (DAKO). A) Skin reactivity results: N\ub0 7 (FcRLB) and N\ub08 (CD20L) labelled epithelial cells (EP) and polymophonuclear leucocytes (PMN); N\ub0 16 (XBP-1s) smooth muscle (SM), EP, dendritic cells (DR) and melanocytes (MEL); N\ub0 18 DC, SM and EP; N\ub0 20/21 (FCRL1-2) EP, collagen fibers (COLL) and nerves (N); N\ub0 24 EP, COLL fibers, macrophages (MO) and fibroblasts (Fb); N\ub0 26 (SC3) hair and eccrine gland (ADN); N\ub0 30(CD210) EP; N\ub0 32(CDW329) DR, MO, EP; N\ub0 33 DC, EP and ADN; N\ub0 34 MO, EP and AND; N\ub0 35 MO; N\ub0 39 (CD124) EP; N\ub0 40(CD200) vessels (Vs) and ADN; N\ub0 41(CD130) Vs and EP; N\ub0 48(CD196) EP and ADN; 49(CD267) ADN; N\ub0 52(CD191) EP and AND; 53(Int.beta5) and 54 (CD320) Vs, EP, AND, Fb; N\ub0 55(DR4), N\ub0 56(CMKLR1) EP and AND; N\ub0 57(DR5) MO, Fb, EP, and AND; N\ub0 58(Notch-1) Vs, EP and AND; N\ub0 59 (4IgB7-H3) EP, Vs, striated muscle; N\ub0 74(Int.beta6) EP and AND; N\ub0 75(Dectin-1) Vs, EP and AND; N\ub0 76-101(TREM-1) Vs, EP, MO and PMN; N\ub0 77(Siglec-5/14) PMN and MO; N\ub0 78(AMICA) Epand AND; N\ub0 80(Siglec-9) MO, DR and PMN; N\ub0 82(int.alfaVbeta5) MO, DR, EP, COLL, Fb, Vs; N\ub0 84(DCIR) PMN; N\ub0 85/99(GITR) PMN, EP and AND; N\ub0 86(CD229) MO; N\ub0 87(CD319) MO and plasmocytes (PC); N\ub0 88(CD48) MO, DR and EP; N\ub0 84(CD84) MO, DR and basal membrane (BM); N\ub0 93(NTBA) MO, DR and PC; N\ub0 94(HVEM) MO, Vs and EP; N\ub0 95(TSLPR) nerves (Nv), EP and ADN; N\ub0 98 (Galectin-3/Mac-2) MO, PMN, TBM, VS,COLL and EP; N\ub0 100(Lymphotoxin betaR) EP; N\ub0 106(CD150) MO-DR; N\ub0 115(BLK 10B/2) Vs; N\ub0 116(Bclx/8H) SM, EP, ADN, COLL, Fb; N\ub0 128(CD38) EP; N\ub0 223(AID) EP, ADN and MEL; N\ub0 263(EVI2) MO and PC. B) Tonsil reactivity with mononuclear cells and resident cells were detected in all mAbs, also if some reagents showed in our method high background (n\ub0 6, 16, 17, 19, 24, 26, 27, 59, 69, 72, 81, 115, 116) or weak staining (n\ub0 7, 8, 9, 10, 29, 32, 62, 67, 71, 78, 90, 91, 92). Results: N\ub0 11(TRAIL-R2/DR5), 223 (AID), 263 (EVI2b) and 268 (LSP1) showed a pan leukocyte reaction; interestingly BLIMP1 specific N\ub0 6/19 showed a cytoplasmic wide reactivity with FOL, PC, MO and T cell areas, whereas N\ub0103/105 showed a strong nuclear staining of the PC and of the EP cells; N\ub013/107(BTLA) and 48(CD196-CCR6) stained PC and MG/MT zone of the FOL; N\ub0 12/37/66 (PD1) and CD152 intrafollicular activated cells (IAC), whereas N\ub050(CXCR5) stained IAC and MG and MT zone of the FOL; N\ub0 14(GCET-1), 17(LMO2), 18 (KLHL-6), N\ub070 (B7H4) stained GC; N\ub0 29 GC and DR; N\ub0 20/108(FCRL1), 21/110(FCRL2) MG/MT/GC and PC; N\ub0 94 (HVEM), 114 (BLK154), 235 (BAFF-R) MG/MT zone; N\ub0 97 (TCL1) nucleus of the FOL/MT zone; N\ub0 27 (weak), 128-CD38 PC. We hope that our efforts may be useful to better understanding the tissue distribution of the analyzed reagents

Linear IgA bullous dermatosis in adults and children: A clinical and immunopathological study of 38 patients

Background: Linear IgA bullous dermatosis (LABD) is a rare autoimmune subepithelial vesiculobullous disease due to IgA autoantibodies directed against different antigens of the basement membrane zone (BMZ) of the skin and/or mucosae. It affects mainly preschool-aged children and adults, with only few studies on large series. The aim of this study was to assess possible differences between adults and children regarding clinical presentation, immunopathologic features, management and course of the disease. Methods: A retrospective review of 38 LABD patients, followed-up from November 2006 to September 2018, was performed. Results: Of 38 patients, 27 were adults and 11 children. Mean age at diagnosis was 5.4 years and 60.6 years in the pediatric and adult group, respectively. Considering both groups, limbs were the most commonly involved site (73.7%), followed by trunk (55.3%), head (36.8%) and buttocks (13.2%). Interestingly, head (p = 0.008), particularly perioral (p = 0.001), involvement, as well as "string of pearls" arrangement (p = 0.03), were more prevalent in children. Mucosal involvement was seen in 9 (23.7%) patients and was more frequent in children than adults (45.5% vs 14.8%, respectively, p = 0.09). Linear IgA deposits along the BMZ were observed in 30 patients (78.9%), while linear/granular IgA deposits in 8 patients (21.1%). Dapsone was the most commonly used drug (78.9%) and complete remission was achieved in most cases (81.6%). Conclusions: Our epidemiological and clinicopathological findings relative to a large cohort of LABD patients are mostly consistent with the literature data. Interestingly, head, notably perioral, involvement and "string of pearls" arrangement occurred more frequently in the paediatric than adult group. The above clinical parameters may be regarded as diagnostic tools for LABD in children

New monoclonal antibodies (mAbs) against B-cells antigens: immunohistochemical analysis of cryostat sections of primary cutaneous B lymphomas (PC-BL)

To define specific reactivities of PC-BL (WHO/EORTC 2008 classification), we analyzed, by using an immunohistochemical method, the mAbs of the FACS panel of the 9th HLDA Workshop on tissue sections of the different cutaneous lymphomas entities: follicular centre B-cell lymphomas (FCL), marginal zone lymphomas (MZL) and diffuse large B-cells lymphomas (DLBL) leg-type. Cryostat sections of frozen tissues were dried for 12 hours, fixed 10" in aceton and stained by an avidin-biotin immunoalkaline phosphatase method (DAKO). Tissue reactivities of lymphocytes and/or other resident cells were detected by using all mAbs, except for n\ub0 49-51-81-90-91-109. Specific lymphoma reactivities were detected on PC-BL: Group 1 mAbs stained all PC-BL: n\ub0 88 (CD48). Group 2 mAbs stained PC-FCL: n\ub020(hFCRL1); 34(CD152); 35 (CD80), 36(CD126); 37(PD1-CD279); 38(B7-DC-CD273); 40 (CD200); 41(CD130); 46 (CD184-CXCR4); 47 (BTLA-CD272); 50(CD185-CXCR5); 56(CMKLR1); 57 (DR5); 77(Siglec-5/14); 83 (CRTAM); 86(CD229); 89 (CD84); 93(NTBA); 94(HVEM); 96(TWEAK); 97(TCL1); 100(LT-betaR); 106(CD150). Group 3 mAbs stained PC-MZL: n\ub0 36(CD126); 47(BTLA); 50(CXCR5); 55(DR4); 77(Siglec-5/14); 83(CRTAM); 84(DCIR/CLEC4A); 86(CD229); 87(CD319); 93(NTBA). Group 4 mAbs stained PC-DLBL: n\ub0 35(CD80); 36(CD126); 39(CD124); 50(CD185); 55(DR4); 56(CMKLR1); 57(DR5); 75(Dectin-1); 76-101(TREM-1); 82(Integrin alfaVbeta5); 83(CRTAM); 85-99(GITR); 86(CD229); 87(CD319); 89(CD84); 92(Dr3- TRAMP); 93(NTBA); 94(HVEM-CD258); 97(TCL-1); 100(Lymphotoxin betaR). Furthermore, variable mixed reactivities with infiltrating or neoplastic cells were observed by using several mAbs and other Mabs showed many tissue cross reactivities and were not easy to evaluate by IHC (further investigations may be done on "pure" infiltrates, cell suspensions of neoplastic cells or by using double labeling techniques-confocal laser microscopy). Finally, mAbs n\ub0 21-22-23-30-31-32-33-42-44-45-48-49-51-52-53-54-58-74-78-79-80-81-82-84-90-91-98-109 were negative in all lymphoma cases. This study may be useful to better define specific profile of different PC-BL entities

Studio array-based comparative genomic hybridization dei linfomi primitivi cutanei epidermotropi aggressivi CD8+

Il linfoma cutaneo a cellule T citotossico CD8+ epidermotropo aggressivo (AeCD8+cx) \ue8 una forma molto rara di linfoma cutaneo a cellule T, appartenente al gruppo dei linfomi a cellule T primitivi cutanei periferici non altrimenti specificati (PC-PTL/NOS), secondo la classificazione WHO/EORTC del 2005, ed \ue8 stato descritto per la prima volta da Berti et al. nel 1999. Questo tipo di neoplasia presenta una proliferazione epidermotropa di linfociti T CD8+ citotossici (TIA-1+, GrB+, Perforin+), a decorso clinico aggressivo non responsivi alla polichemioterapia e al trapianto. Abbiamo condotto un'analisi di Array-based Comparative Genomic Hybridization (arrayCGH) su DNA estratto dalle lesioni cutanee di 13 pazienti affetti da AeCD8+cx al momento della diagnosi. Dai risultati \ue8 emersa la presenza di alcune regioni cromosomiche alterate in un numero significativo di pazienti. Tra queste le pi\uf9 interessanti risultano essere le amplificazioni del 3p21, 7q, 8q24, 11q12-q13, 16p, 17q, trisomia19, e 22q, oltre alla delezione del 9p21. All'interno di queste regioni sono contenuti molti geni coinvolti nella regolazione di processi biologici che possono giocare un ruolo fondamentale nella patogenesi o nello sviluppo di questo linfoma. Nella delezione del 9p21 emerge la nullisomia dei geni CDKN2A e CDKN2B che sono inibitori specifici delle chinasi ciclina-dipendente che si legano alla ciclina D. Risulta quindi che l'impossibilit\ue0 di inibire l'attivazione di questa ciclina possa portare ad una iperattivit\ue0 di quest'ultima con conseguente aumentata proliferazione linfocitaria. Per quanto riguarda le regioni amplificate sembrerebbero essere importanti alcuni geni che portano ad una aumentata attivazione del JAK/STAT signaling pathway. In particolare le duplicazioni di JAK3, STAT3 e STAT5 potrebbero giustificare il fenotipo a cellule CD8 e il comportamento aggressivo dei linfociti neoplastici. L'amplificazione di JAK3 \ue8 dovuta alla presenza di trisomia 19 totale o parziale. In questo cromosoma sembrano essere contenuti diversi geni che se overespressi portano ad una proliferazione incontrollata di linfociti come ad esempio JUNB, JUND, KIR3DL2, AKT2, LYL1 e RELB. Questi dati costituiscono la base per l'interpretazione molecolare di questa patologia e andranno corredati con le analisi di espressione genica e RT-PCR al fine di determinare un miglior approccio terapeutico

Cutaneous extranodal NK/T-cell lymphoma: a clinicopathologic study of 5 patients with array-based comparative genomic hybridization

Extranodal natural killer/T-cell (ENK/T) lymphoma is a rare neoplasm, subcategorized into ENKITnasal ENK/T-N) and ENK/T-nasal type (ENK/T-NT) lymphomas. ENK/T-NT lymphoma with initial presentation in the skin is known as primary cutaneous (PC) ENK/T-NT lymphoma. Patients and methods: The aim ofthis study was to investigate pathogenesis, genomic alterations, and prognosis ofcutaneous ENK/T lymphomas to provide further insights into clinicopathologic features and genetic mechanism of lymphomagenesis. A retrospective case study of 5 white patients affected by ENK/T lymphoma (4 PC-ENK/T-NT and l ENKIT-N with cutaneous involvement) was performed. Results: Most ofthe cases presented with multiple nodular and ulcerated lesions localized on the extremities. A considerable percentage had disease in advanced stage at diagnosis with a 12-month survival rate of 40%. Genomic alterations were detected by array-based comparative genomic hybridization that showed gains of 1q, 7q and loss of 17p in the cases of PC-ENK/T-NT Iymphomas and gain of 7q and 10ss of 9p, 12p, 12q in the case of ENK/T-N lymphoma. Conclusion: ENK/T lymphoma is a very aggressive entity, and, in our cases, tbe exc1usively cutaneous presentation was not associated with a better prognosis. The results of our array comparative genomic hybridization analysis could be useful to better define the diffcrent ENK/T lymphoma subgroups with cutaneous involvement and new protocols of treatment

Cutaneous T-cell lymphomas CD8+

Recently the expression ofcytotoxic CD8+ immunophenotype in several cutaneous T-cell lymphomas entities was identified. Around 5% of mycosis fungoides (MF) may express a CD8+, CD45RA+, TIA-1+, CD5-(intraepitelial-pagetoid variant) or a CD8+, CD45RO+, CD5+, TIA-1 (dermal, band-like lichenoid variant) immunophenotype and usually present large erythemato-squamous, hycthyosiform plaques in the gluteal area or arms, showing a very indolent course as classical MF. Annular or erythemato-squamous psoriasiform lesions in the abdominal area or arms, with an hypochromic evolution and an indolent course, was characteristic of MF in children and adolescent. In the group of CD30+ lymphoproliferative disorders CD8+ lymphomatoid papulosis cases were associated witb a PLEVA type clinical presentation, with papulo-necrotic or papulo-haemorragic blistering skin lesions and strong epidermotropism. Cases of CD30+, CD8+ cutaneous anaplastic large cell lymphoma were also observed with frequent systemic involvement and fatal outcome in 50% of our cases. Subcutaneous panniculitis-like T-cell lymphoma in the classical cytotoxic (CD45RO+, TIA-1+) alpha-beta (BF-1+) variant showed an intermediate prognosis characterized by cutaneous relapses and possible haemophagocytic syndrome. Aggressive epidermotropic cytotoxic CD8+ T-cell lymphoma (AeCxCD8+L) showed multiple hyperkeratotic or ulcerated nodules and plaques, with a typical CD8+, TIA-1, CD45RA+, CD5-/+, CD2+/-phenotype and a very aggressive fatal course. Cases of pleomorphic small-medium or medium-large lymphoma with an aggressive course (4 cases) or with a benign evolutions may be also CD8+, interestingly three of these aggressive cases were CD8- at presentation and positive in subsequent biopsies. Finally, very rare cases of NK/T nasal type extranodal lymphomas or cases of hydroa vacciniforme-like T ce1l lymphoma showing a very aggressive course may express CD8+, but in association with EBV strong positivity. Cytomorphology, immunohistochemistry and genomics must be used for the diagnosis and in the future they should be useful to better classify CD8+ CTCLs variants

Seriousness, preventability, and burden impact of reported adverse drug reactions in Lombardy emergency departments : a retrospective 2-year characterization

OBJECTIVE: The purpose of this study was to determine the prevalence of adverse drug reactions (ADRs) reported in emergency departments (EDs) and carry out a thorough characterization of these to assess preventability, seriousness that required hospitalization, subsequent 30-day mortality, and economic burden. METHODS: This was a retrospective cohort study of data from an active pharmacovigilance project at 32 EDs in the Lombardy region collected between January 1, 2010 and December 31, 2011. Demographic, clinical, and pharmacological data on patients admitted to EDs were collected by trained and qualified monitors, and deterministic record linkage was performed to estimate hospitalizations. Pharmacoeconomic analyses were based on Diagnosis-Related Group reimbursement. RESULTS: 8,862 ADRs collected with an overall prevalence rate of 3.5 per 1,000 visits. Of all ADRs, 42% were probably/definitely preventable and 46.4% were serious, 15% required hospitalization, and 1.5% resulted in death. The System Organ Classes most frequently associated with ADRs were: skin and subcutaneous tissue, gastrointestinal, respiratory thoracic and mediastinal, and nervous system disorders. The most common Anatomical Therapeutic Chemical classes involved in admissions were J (anti-infectives and immunomodulating agents), B (blood and blood-forming organs), and N (nervous system). Older age, yellow and red triage, higher number of concomitantly taken drugs, and previous attendance in ED for the same ADR were significantly associated with an increased risk of hospitalization. The total cost associated with ADR management was 5,184,270, with a mean cost per patient of 585. Fifty-eight percent of the economic burden was defined as probably/definitely preventable. CONCLUSION: ADRs are a serious health/economic issue in EDs. This assessment provides a thorough estimation of their seriousness, preventability, and burden impact in a large population from a representative European region

Comparative Safety of Originator and Biosimilar Epoetin Alfa Drugs: An Observational Prospective Multicenter Study

Background: Erythropoiesis-stimulating agents (ESAs) are biological molecules approved for the treatment of anemia associated with chronic renal failure. Biosimilars were licensed for use in Europe in 2007. Aim: This study aimed to compare the safety profile of biosimilars with respect to the reference product in a nephrology setting. Methods: A prospective study was conducted in four Italian regions between 1 October 2013 and 30 June 2015. The study population included patients aged 65 18 years undergoing hemodialysis and treated with epoetins as per the clinical practice of the participating centers. The two comparison cohorts included patients treated with either an originator or a biosimilar epoetin alfa. Each patient was followed up until occurrence of any safety outcome of interest (grouped into three major categories), switch to a different ESA product, transplant or peritoneal dialysis, death, or end of the study period, whichever came first. Results: Overall, 867 subjects were included in the study (originator: N = 423; biosimilar: N = 444). Biosimilar users were older than originator users (median age of 76 vs 64 years, respectively), more frequently affected by arrhythmia (29.3 vs 22.5%), and less frequently candidates for transplantation (3.8 vs 18.2%). Cox-regression analysis showed no increase in risk of safety outcomes in biosimilar users, even after adjusting for confounding factors: 1.0 (95% confidence interval [CI] 0.7\u20131.3) for any outcomes; 1.1 (95% CI 0.7\u20131.8) for problems related to dialysis device; 0.9 (95% CI 0.6\u20131.5) for cardio- and cerebro-vascular conditions; 0.9 (95% CI 0.6\u20131.5) for infections. Conclusion: This study confirms the comparable safety profiles of originator and biosimilar epoetin alfa drugs when used in patients receiving dialysis

Array-based comparative genomic hybridization analysis of aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma (AECD8+L), extranodal NK/T nasal type lymphoma (ENK/T-NT) and blastic plasmocytoid dendritic cell neoplasia (BPDCN)

To better define molecular alterations involved in these proliferations, we performed an arraybased high resolution comparative genomic hybridization (aCGH) analysis on DNA extracted from skin lesions of 13 patients affected from AECD8+L, 5 patients from ENK/T-NT and 21 patients from BPDCN. In AECD8+ lymphoma, our results showed recurrent alterations of chromosomal regions found also in other CTCL, such as amplification of 3p21 (46% of patients), 7q (54%), 8q24 (54%), 16p(77%), 17q (92%), and the deletion of 9p21 (69%), and several alterations seemingly typical for AECD8+L: i.e. amplification of 11q12-q13 (69%), 22q (69%) and trisomy of 19 (69%). Within these amplified regions, the combination of duplication of JAK3 (chr. 19p13.11) and STAT3/STAT5B (chr. 17q21) might explain the hyper-activation of JAK / STAT signaling pathway, with an increased proliferation and an increased anti apoptotic activity. Interestingly, constitutive Jak3 signaling in murine lymphopoiesis, in a bone marrow transplantation model, induces an aggressive lymphoproliferative disorder characterized by the expansion of CD8+, TCR\u3b1\u3b2+ T cells. In addition chromosome 19 contains several genes that can lead to uncontrolled proliferation of cells if overexpressed, such as JUNB, JUND, KIR3DL2, AKT2, LYL1, BCL3 and RELB, alone or in combination with the proto-oncogene RELA, present in the amplified region 11q12-q13. A retrospective case study of 5 white patients affected by ENK/T lymphoma (4 PC-ENK/T-NT and 1 ENK/T-N with cutaneous involvement) was also performed. Genomic alterations were detected by aCGH hybridization that showed gains of 1q, 7q and loss of 17p in the cases of PC-ENK/T-NT lymphomas and gain of 7q and loss of 9p, 12p, 12q in the case of ENK/T-N lymphoma. In our cases, the exclusively cutaneous presentation was not associated with a better prognosis. BPDCN is a rare, often fatal disease: all patients had skin lesions, 12 with extracutaneous disease at diagnosis. By aCGH there were chromosomal imbalances in all biopsies, with an average of 7 copy number alterations/case and losses more frequent than gains (141 vs 18); large interstitial/telomeric imbalances prevailing over the entire chromosome losses/gains (127 vs 32). Common deleted regions (CDR) were found on chromosomes 5, 7, 9, 12, 13 and 14. A CDR at 9p21.3, hosting CDKN2A suppressor gene (P16INK4a, p14ARF), was present in 15 cases; 6 in biallelic status. Chromosome 13 monosomy was found in 11 cases and we identified a minimal CDR on 13q13.1-q14.3, including RB1, CCNA1 and KPNAP3. In 12 cases a monoallelic CDR encompassed 12p13.2-p13.1, hosting CDKN1B (p27/KIP1). Additionally, 4 patients had del(7)(p12), a region harbouring IKZF/Ikaros, defective in cases of acute lymphoblastic leukaemia with poor prognosis. In conclusion, AECD8+L, PC_ENK/T-NT and BPDCN are aggressive neoplastic diseases showing complex genetic alterations, involving activation, proliferations and apoptosis, that may explain the poor response to therapy. These data, complemented with gene expression analysis and immunohistochemical evaluation should help us in deciphering biologic and molecular mechanisms of these disease entities and may become important tools in diagnosis and classification or to find new therapeutic approaches

Antipsychotic drug exposure and risk of pulmonary embolism: a population-based, nested case–control study

Only three observational studies investigated whether exposure to antipsychotics is associated with an increased risk of pulmonary embolism, with conflicting results. This study was therefore carried out to establish the risk of pulmonary embolism associated with antipsychotic drugs, and to ascertain the risk associated with first- and second-generation antipsychotic drugs, and with exposure to individual drugs