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ObjectivesThis study sought to analyze whether the plasmatic level of leukocyte-derived microparticles (LMP) is associated with unstable plaques in patients with high-grade carotid stenosis.BackgroundPreventive carotid surgery in asymptomatic patients is currently debated given the improvement of medical therapy. Therefore, noninvasive biomarkers that can predict plaque instability are needed. The LMPs, originating from activated or apoptotic leukocytes, are the major microparticle (MP) subset in human carotid plaque extracts.MethodsForty-two patients with >70% carotid stenosis were enrolled. Using a new standardized high-sensitivity flow cytometry assay, LMPs were measured before thromboendarterectomy. The removed plaques were characterized as stable or unstable using histological analysis according to the American Heart Association criteria. The LMP levels were analyzed according to the plaque morphology.ResultsThe median LMP levels were significantly higher in patients with unstable plaque (n = 28; CD11bCD66b+ MP/μl 240 [25th to 75th percentile: 147 to 394], and CD15+ MP/μl 147 [60 to 335]) compared to patients with stable plaque (16 [0 to 234] and 55 [36 to 157]; p < 0.001 and p < 0.01, respectively). The increase in LMP levels was also significant when considering only the group of asymptomatic patients with unstable plaque (n = 10; CD11bCD66b+ MP/μl 199 [153 to 410] and CD15+ MP/μl 78 [56 to 258] compared with patients with stable plaque (n = 14; 20 [0 to 251] and 55 [34 to 102]; p < 0.05 and p < 0.05, respectively). After logistic regression, the neurologic symptoms (odds ratio: 48.7, 95% confidence interval: 3.0 to 788, p < 0.01) and the level of CD11bCD66b+ MPs (odds ratio: 24.4, 95% confidence interval: 2.4 to 245, p < 0.01) independently predicted plaque instability.ConclusionsLMP constitute a promising biomarker associated with plaque vulnerability in patients with high-grade carotid stenosis. These data provide clues for identifying asymptomatic subjects that are most at risk of neurologic events.

Embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a recently described embryonal neoplasm of the central nervous system, consisting of a well-circumscribed embryonal tumor of infancy with mixed features of ependymoblastoma (multilayer ependymoblastic rosettes and pseudorosettes) and neuroblastoma (neuroblastic rosettes) in the presence of neuropil-like islands. We present the case of a young child with a very aggressive tumor that rapidly recurred after gross total resection, chemotherapy and radiation. Prominent vascular sclerosis and circumscribed tumor led to the diagnosis of malignant astroblastoma; however, rapid recurrence and progression of this large tumor after gross total resection prompted review of the original pathology. ETANTR is histologically distinct with focal glial fibrillary acid protein (GFAP) and synaptophysin expression in the presence of neuronal and ependymoblastic rosettes with focal neuropil islands. These architectural features, combined with unique chromosome 19q13.42 amplification, confirmed the diagnosis. In this report, we describe tumor stem cell (TSC) marker CD133, CD15 and nestin alterations in ETANTR before and after chemotherapy. We found that TSC marker CD133 was richly expressed after chemotherapy in recurrent ETANTR, while CD15 is depleted compared with that expressed in the original tumor, suggesting that CD133+ cells likely survived initial treatment, further contributing to formation of the recurrent tumor.

Purpose: This study is directed at identifying the cell source(s) of immunomodulatory cytokines in high-grade gliomas and establishing whether the analysis of associated markers has implications for tumor grading. Experimental Design: Glioma specimens classified as WHO grade II–IV by histopathology were assessed by gene expression analysis and immunohistochemistry to identify the cells producing interleukin (IL)-10, which was confirmed by flow cytometry and factor secretion in culture. Finally, principal component analysis (PCA) and mixture discriminant analysis (MDA) were used to investigate associations between expressed genes and glioma grade. Results: The principle source of glioma-associated IL-10 is a cell type that bears phenotype markers consistent with M2 monocytes but does not express all M2-associated genes. Measures of expression of the M2 cell markers CD14, CD68, CD163, and CD204, which are elevated in high-grade gliomas, and the neutrophil/myeloid-derived suppressor cell (MDSC) subset marker CD15, which is reduced, provide the best index of glioma grade. Conclusions: Grade II and IV astrocytomas can be clearly differentiated on the basis of the expression of certain M2 markers in tumor tissues, whereas grade III astrocytomas exhibit a range of expression between the lower and higher grade specimens. The content of CD163+ cells distinguishes grade III astrocytoma subsets with different prognosis. Clin Cancer Res; 19(14); 3776–86. ©2013 AACR.

Castleman's disease (CD) is thought to be related with an initially benign viral disease with cytokine-driven propagation and malignant transformation. This paper reports the first case of a simultaneous discordant lymphoma consisting of lymphocyte-depleted classical Hodgkin's lymphoma (LDCHL) and peripheral T-cell lymphoma (PTCL) arising in a patient with multicentric CD (MCD). PTCL occurred 4 years after the diagnosis of MCD, and LDCHL was developed 6 years after the treatment of PTCL, sequentially. The following year, the patient presented with a relapse of a simultaneous discordant lymphoma. On excisional cervical LN biopsy, immunohistochemical stain pattern was identical with previously diagnosed LDCHL, which expressed CD30, CD15, PAX5, and Epstein-Barr virus (EBV)-encoded RNA. PTCL was positive for CD3, CD4, CD5, CD10, and CD56, and showed identical TCRB and TCRG gene rearrangements to those detected initially. MCD was thought to be the major contributing factor leading to initial PTCL, while EBV-positive LDCHL is thought to have promoted the development of PTCL, as a persistently abnormal immune microenvironment may induce the recurrence of PTCL. MCD runs a more aggressive course and can progress to Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), or combined HL/NHL. Due to its malignant potential, prompt recognition and therapy is critical for these situations, which may be life threatening.

The lymphocyte-depleted type (LD) is a morphological subtype of classical Hodgkin lymphoma (CHL), but its rarity and heterogeneous morphological character makes a definite clinicopathological identification difficult. To characterize this disease, LD cases were compared with other types of CHL. From 1982 to 2006, we collected 310 CHL cases. Among them, 29 cases were diagnosed as LD. We could additionally analyze clinical data of 157 CHL cases (including 28 LD cases) and the immunophenotype of 150 CHL cases (including 28 LD cases). We compared clinicopathological data between LD cases and other types of CHL cases and determined prognostic factors by univariate and multivariate analysis. LD showed a more progressive disease stage (stage 3/4, 64%) than other types of CHL (stage 3/4, 30%; P

Desmoplastic mesothelioma is a rare subtype of diffuse malignant mesothelioma. A 72-year-old woman from East Anatolia presented with chest pain. The images of body positron emission tomography revealed irregular, left pleural thickening involving mediastinal and diaphragmatic surfaces with hypermetabolic characterization. The diagnosis of desmoplastic malignant mesothelioma was confirmed by minithoracotomy and immunohistochemical staining with pan-cytokeratin, cytokeratin 5/6, calretinin, carcinoembryonic antigen, thyroid transcription factor-1, CD15, and HMB-45 on the biopsy specimen. This case is unique in terms of the reporting patient being from a nonendemic area for asbestos-related diseases and in terms of its rare histopathology.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) represents 5% of all Hodgkin lymphoma and has distinct clinico-pathological features. It is typified by the presence of lymphocyte predominant cells, which are CD20(+) , CD15(-) and CD30(-) and are found scattered amongst small B-lymphocytes arranged in a nodular pattern. Patients typically are males presenting with localized, peripheral lymphadenopathy. Despite frequent and often late or multiple relapses the prognosis is favourable. Deaths due to NLPHL are uncommon, but secondary malignancies and other treatment toxicities contribute appreciably to overall mortality. Secondary aggressive non-Hodgkin lymphoma can occur in approximately 7-14% of cases of NLPHL. Given this diseases' rarity, the optimal management is unclear and opinions differ as to whether treatment paradigms should be similar to or differ from those for classical Hodgkin lymphoma. This review provides an overview of the existing literature describing of the outcome and treatment approaches for limited and advanced stage NLPHL.

Myeloid-derived suppressor cells are increased with age and elevated in donors with a history of cancer; an age-related effect has never been shown in humans., As we age, the composition of our peripheral leukocytes changes dramatically. Many of these alterations contribute to the general immune dysfunction that burdens the elderly, which in turn, contributes to increased susceptibility to disease. MDSCs represent a heterogeneous population of immunosuppressive leukocytes that are elevated in the peripheral blood of cancer patients. Given the relation between cancer incidence and age, this study examined the frequency of peripheral blood CD33(+)HLA-DR(−) MDSCs across three cohorts: healthy adults (19–59 years old), community-dwelling seniors (61–76 years old), and frail elderly (67–99 years old). This analysis is the first to demonstrate that MDSCs and specifically the CD11b(+)CD15(+) MDSC subset are increased with age. Proinflammatory cytokines that are required for the differentiation of MDSCs (e.g., TNF-α, IL-6, and IL-1β) were similarly found to be increased in the serum of the frail elderly. Furthermore, the proportion of MDSCs and the CD11b(+)CD15(+) subset were found to be elevated significantly in elderly donors with a history of cancer. This age-related elevation in the frequency of MDSCs may contribute to the increased cancer incidence that occurs with age. Further investigation into the functional consequences of elevated MDSCs will provide valuable insight into the progression of age-related pathologies.

Radial glial cells represent a subpopulation of secondary neural precursor cells that differentiate from neuroepithelial progenitors and are transiently found in the developing CNS of mammals. There is ample evidence for a temporal and spatial arrangement of increasingly committed radial glial cells that is of critical importance for the organisation and specification of different brain regions. For the human ganglionic eminence, recent findings have shown an early molecular specification of this cell type by the CD15 carbohydrate epitope, beginning already at the end of the first trimester. Here we further characterise the CD15+ radial glia cells as bFGF/EGF responsive progenitors allowing its propagation in vitro. By magnet activated cell sorting, its trilineage differentiation potential can be shown by differentiation into (PSANCAM s3 tubulin immunoreactive) neurones, GFAP expressing cells of astrocytic morphology, and O4 positive oligodendrocytes. Subcloning experiments under proliferation conditions reveal ongoing CD15 expression by dividing cells. Although the relative number of CD15+ progenitor cells is found to decrease in favour of CD15- precursor cells during continuous passaging, cell sorting experiments allow the repetitive purification of high numbers of positively selected precursor cells for up to 12 weeks. In conclusion, expression of the cell adhesion molecule CD15 by a subpopulation of proliferative cells from the lateral ganglionic eminence allows easy and reproducible purification of progenitor cells by cell sorting, enabling the generation of a compartment-specific cell pool as a prerequisite for a safe and standardised therapy of neurodegenerative basal forebrain diseases.

Acute coronary syndromes and acute myocardial infarctions are often related to plaque rupture and the formation of thrombi at the site of the rupture. We examined fresh coronary thrombectomy specimens from patients with acute coronary syndromes and assessed their structure and cellularity. The thrombectomy specimens consisted of platelets, erythrocytes and inflammatory cells. Several specimens contained multiple cholesterol crystals. Culture of thrombectomy specimens yielded cells growing in various patterns depending on the culture medium used. Culture in serum-free stem cell enrichment medium yielded cells with features of endothelial progenitor cells which survived in culture for a year. Immunohistochemical analysis of the thrombi revealed cells positive for CD34, cells positive for CD15 and cells positive for desmin in situ, whereas cultured cell from thrombi was desmin positive but pancytokeratin negative. Cells cultured in endothelial cell medium were von Willebrand factor positive. The content of coronary thrombectomy specimens is heterogeneous and consists of blood cells but also possibly cells from the vascular wall and cholesterol crystals. The culture of cells contained in the specimens yielded multiplying cells, some of which demonstrated features of haematopoietic progenitor cells and which differentiated into various cell-types.

Introduction: Metastatic non-small cell lung cancer represents the most common cause of brain metastasis in adults. Interaction between circulating cancer cells and brain endothelial cells play a key role in brain metastasis, but their underlining molecular mechanisms are still not fully understood. The CD15 and CD15s epitopes function as cell adhesion molecules and are known to be correlated with metastasis in non-CNS cancers. This study aims to investigate the role of CD15 and CD15s in cancer cell adhesion and trans-endothelial migration during metastasis to the brain by perturbing the expression of the fucosyltransferases responsible for the synthesis of these epitopes.

Background and aims: Although granulocyte dysfunction is known to occur in cirrhosis, in vivo studies of granulocyte lifespan have not previously been performed. The normal circulating granulocyte survival half-time (G-t½), determined using indium-111 (111In)-radiolabelled granulocytes, is approximately 7 h. In this pilot study we aimed to measure the in vivo G-t½ in compensated alcohol-related cirrhosis.\ud Methods: Sequential venous blood samples were obtained in abstinent subjects with alcohol-related cirrhosis over 24 h post injection (PI) of minimally manipulated 111In-radiolabelled autologous mixed leucocytes. Purified granulocytes were isolated from each sample using a magnetic microbead-antibody technique positively selecting for the marker CD15. Granulocyte-associated radioactivity was expressed relative to peak activity, plotted over time, and G-t½ estimated from data up to 12 h PI. This was compared with normal neutrophil half-time (N-t½), determined using a similar method specifically selecting neutrophils in healthy controls at a collaborating centre.\ud Results: Seven patients with cirrhosis (6 male, aged 57.8 ±9.4yrs, all Child-Pugh class A) and seven normal controls (3 male, 64.4 ±5.6yrs) were studied. Peripheral blood neutrophil counts were similar in both groups (4.6 (3.5-5.5) x 109/l vs 2.8 (2.7-4.4) x 109/l respectively, P=0.277). G-t½ in cirrhosis was significantly lower than N-t½ in controls (2.7 ±0.5h vs 4.4 ±1.0h, P=0.007). Transient rises in granulocyte and neutrophil-associated activities occurred in 4 patients from each group, typically earlier in cirrhosis (4-6 h PI) than in controls (8-10 h), suggesting recirculation of radiolabelled cells released from an unidentified focus.\ud Conclusions: Reduced in vivo granulocyte survival in compensated alcohol-related cirrhosis is a novel finding and potentially another mechanism for immune dysfunction in chronic liver disease. Larger studies are needed to corroborate these pilot data and assess intravascular neutrophil residency in other disease aetiologies.

Background: BAL studies in IPF have shown increased alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) numbers and some lines of evidence suggest a role of these inflammatory cells in the pathogenesis of the disease. Aim of the study: to evaluate the pattern of activation of BAL cells by cytofluorimetric analysis in idiopathic pulmonary fibrosis (IPF) in comparison with other interstitial lung diseases (ILD). Methods: 44 consecutive BAL sample performed at the ILD clinic of the University Hospital of Rome Tor Vergata during standard diagnostic workout were evaluated by cytofluorimetry for expression of the following cell surface markers: CD45, CD71, CD206, HLA-DR, CD15, CD14, CD11c. Diagnosis of the 44 cases were eventually reviewed or based according to the 2013 ATS/ERS guidelines. Results: Final diagnosis was IPF in 16 cases, fibrotic NSIP in 6 cases, cellular NSIP in 3 cases, ILD associated to collagen vascular diseases in 6 cases, Hypersensitivity pneumonia in 5 cases, asbestosis in 2 cases, and unclassifiable ILD in 6 cases. Qualitative cytofluorimetric analysis revealed that IPF BAL samples were characterize by a population of CD206, CD11c and HLA-DR very bright cells, that represented the majority of the cellular population in these samples (74±4%). Quantitative analysis revealed that the sum of the recorded mean intensity fluorescence (MIF) for these 3 surface markers (MIF>250 AU) was associated to the diagnosis of IPF with a sensitivity and specificity greater than 90%. Conclusions: Even with the limitation of the small population, this study reinforce the potential role for BAL cytofluorimetry in the diagnostic workout of ILD patients.

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80 %) and conventional papillary thyroid carcinoma (PTC) (60.7 %) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6 %) and follicular carcinoma (FCA) (32.1 %). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.

An increase in salivary leukocytes may contribute to the exercise-induced increase in salivary antimicrobial proteins (AMPs). However, exercise-induced changes in salivary leukocytes have not been studied. The purpose of the study was to describe salivary leukocyte changes with exercise. Participants (n = 11, 20.3 ± 0.8 years, 57.2 ± 7.6 ml kg−1 min−1 peak oxygen uptake ((VO) 2peak), 11.1 ± 3.9% body fat) ran for 45 min at 75% of VO2peak. Stimulated saliva (12 mL) was collected pre- and immediately post exercise. Saliva was filtered through a 30 µm filter before analysis of leukocytes (CD45+), granulocytes (CD45+CD15+), monocytes (CD45+CD14+), T-cells (CD45+CD3+), and B-cells (CD45+CD20+) using flow cytometry. Saliva was analysed for Lysozyme (Lys) using ELISA. Exercise did not alter any leukocyte subset. The major constituent of leukocytes pre-exercise were granulocytes (57.9 ± 30.3% compared with monocytes: 5.1 ± 2.7%, T-cells: 17.1 ± 8.9%, B-cells: 12.1 ± 10.2%) (P

Hodgkin's lymphoma (HL) is a B cell neoplasm and is classified into two categories: nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL) and classical Hodgkin's lymphoma (CHL). CHL is a clonal lymphoid neoplasm originating from germinal centre B cells; the diagnosis is based on typical Hodgkin and Reed-Sternberg (HRS) giant cells in an inflammatory background. Histologically, four histological subtypes of CHL have been identified: nodular sclerosis, lymphocyte rich, mixed cellularity and lymphocyte-depleted.1 Immunohistochemically, HRS cells in nearly all cases are positive for CD30 and for CD15 in the majority (75–85%) of cases according to the 2008 WHO classification.1 ,2 The histological diagnosis of CHL in most instances is straightforward, particularly with the aid of immunohistochemistry. Other than the prerequisite of CD30 expression by HRS cells, CD15 is frequently referred to as a ‘positive’ marker for diagnosis. Apart from diagnostic importance, CD15 expression is of prognostic value. Lack of CD15 expression in CHD has been reported to be a poor prognostic factor in patients with CHL.3 ,4 In the author's experience in Taiwan, the frequency of CD15 expression in CHL is low, which prompted the author to systemically investigate the prevalence of …

The effect of insulin-like growth factor-1 (IGF-1) on highly enriched human apheresis CD34(+) progenitor cells was investigated in vitro. The progenitor cells were mobilized by treatment with cyclophosphamide + granulocyte - colony stimulating factor (G-CSF) in patients with multiple myeloma. CD34(+) cells were cultured for 7 days in serumfree medium containing stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), and this is referred to as cytokine-dependent proliferation. After 7 days of cytokine-dependent proliferation the total number of viable cells increased 1.6-8.2 times, and subsets of cells expressing the granulocyte marker CD15, the myelomonocytic marker CD64 and the erythrocyte phenotype CD71(high) /CD64(-) were detected among the in vitro cultured cells. Addition of G-CSF together with SCF + IL-3 + GM-CSF increased the number of CD15(+) and CD64(+) cells, but without altering the number of erythroid cells. IGF-1 caused a dose-dependent increase in the number of CD15(+), CD64(+) and CD71(high) /CD64(-) cells, and this increase was detected when cells were cultured in both SCF + IL-3 + GM-CSF alone and G-CSF + SCF + IL-3 + GM-CSF. A minor subset of CD34(+) cells could still be detected among in vitro cultured cells and the number of CD34(+) cells was not altered by adding G-CSF and/or IGF-1. Morphologically recognizable mature granulocytes or erythroid cells could not be detected for any of the combinations investigated. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from apheresis CD34(+) cells.

Objectives: We sought to immunophenotype blasts, monocytes, and granulocytes in chronic myelomonocytic leukemias (CMMLs) and compare CMML subtypes, to identify if significant antigen expression differences existed. Methods: Bone marrow blasts, monocytes, and granulocytes from CMML subgroups (n = 30; World Health Organization types 1/2, proliferative/dysplastic, therapy related/de novo, and low/intermediate/high cytogenetic risk) were immunophenotypically compared by flow cytometry with 10 nonneoplastic control marrows. Results: Aberrancies were present in blasts of 26 (87%) of 30 CMMLs (26 diagnostic; four follow-up) and six (60%) of 10 controls ( P = .089), monocytes of 28 (93%) of 30 CMMLs and six (60%) of 10 controls ( P = .026), and granulocytes of eight (28%) of 29 CMMLs and zero of 10 controls ( P = .166). Underexpression of CD14 and CD15 on monocytes was more common in CMMLs compared with controls ( P = .008 and P = .043). Statistical analysis showed no significant difference in antigen expression between the CMML subgroups on blasts or monocytes; granulocytes demonstrated more common HLA-DR expression in CMML-2 vs CMML-1. Conclusions: These findings confirm heterogeneity within CMML subgroups and find no specific qualitative or quantitative findings characteristic of a subgroup.

B-cell lymphoma (BCL), unclassifiable, with features intermediate between diffuse large BCL (DLBCL) and classical Hodgkin's lymphoma (CHL), is a novel entity to the World Health Organization classification system. These tumors are rare aggressive lymphomas that have a poor prognosis. The present study reports the case of a patient with one such lymphoma that occurred in the spleen, which expressed cluster of differentiation (CD)20, CD79α, melanoma associated antigen (mutated) 1, BCL6, CD15 and CD30. Polymerase chain reaction analysis demonstrated a clonal rearrangement of the genes coding for immunoglobulin heavy chains. The tumor cells demonstrated a negative reaction in the Epstein-Barr virus-encoded small RNA assay. Following the diagnosis of unclassifiable BCL, with intermediate features between DLBCL and CHL, the patient received 7 cycles of the CHOP regimen, and so far, has been in good general condition and tumor-free for 17 months.

Only 5 cases of follicular lymphoma (FL) have been reported in the prostate. A 68-year-old man presented with dysuria. Laboratory data did not indicate malignancy in the prostate with serums PSA of 4.6 ng/ml. Imaging techniques identified no tumors and lympho-adenopathy other than the prostatic enlargement. The patient was clinically diagnosed as prostatic hyperplasia, and trans-urethral resection of prostate (TURP) (65 g) was done. Histologically, the TURP-specimens showed severe nodular proliferation of atypical small lymphocytes consisting of small centrocytic lymphocytes and large centroblastic lymphocytes, the number of the latter being 2/HPF. Immunohistochemically, the tumor cells and nodular areas were positive for CD45, CD20, CD79a, bcl-2, bcl-6, and CD10. They were negative for pan-cytokeratin (AE1/3, CAM5.2), CD3, CD45RO, CD56, PSA, chromogranin-A, synaptophysin, NSE, CD138, CD15, CD30 and cyclin D1. No significant number of plasma cells were seen by immunohistochemistry for light chains and CD138. A pathological diagnosis of primary FL (grade 1) of the prostate was made. No tumors were identified by various imaging techniques, and the prostatic FL was diagnosed as primary. The patient underwent low-dose R-CHOP chemotherapy and focal radiation, probably resulting in complete remission. No recurrence has been found 5 months after the diagnosis.

Summary Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b + CD15 hi CD10 − CD16 low immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor T cell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer.

AimsIn recent years, multiparameter flow cytometry has been increasingly recognised as an important tool in diagnosis of myelodysplastic syndrome and acute myeloid leukaemia (AML). Assessment of myeloid and monocytic ‘immunophenotypic’ dysplasia by flow cytometry in de novo AML has not been evaluated.Methods97 cases of de novo AML cases were identified and reviewed by three hematopathologists. ‘Immunophenotypic’ dysplasia was assessed on blasts, monocytes and granulocytes by mean fluorescence intensity.ResultsUsing the 2008 WHO classification criteria, there were 53 AML-not otherwise specified (NOS) (55%) and 28 AML with myelodysplasia-related changes (AML-MRC) (29%), while 16 cases were ambiguous as to AML-MRC status due to limited maturing cells for morphologic but adequate events number for immunophenotypic evaluation (AML-not evaluable, 16%). Compared with AML-NOS, granulocytic cells in AML-MRC had higher CD33 expression but lower CD45, CD11b and CD15. Monocytes in AML-MRC had lower expression of CD14, CD56 and CD45. Morphologic dysplasia was associated with significantly lower granulocytic forward scatter, side scatter and CD10 but higher CD33 expression.ConclusionsOur results suggest that the workup of AML cases should include flow cytometric assessment of granulocytes and monocytes. This analysis can aid a morphologic impression of multilineage dysplasia in distinguishing AML-MRC from AML-NOS, especially in cases with limited maturing myeloid cells.

Background and aim CD15 is expressed by various cancer types, among these intrahepatic and perihilar cholangiocarcinoma (CCA). Aim of this study was to elucidate CD15 expression in distal CCA as well as in dysplastic biliary tissue and to determine its prognostic significance. Methods Tissue samples from patients with intrahepatic (iCCA, n=22), perihilar (pCCA, n=7) and distal CCA (dCCA, n=15), who underwent surgical resection in the period from 2010 to 2015 were evaluated for CD15 expression. Tissue of synchronous lymph node metastasis (n=13), CCA associated dysplasia (n=20), dysplasia in intraductal biopsies (n=10), benign proliferations (n=13) as well as inflammatory biliary lesions (n=28) and non-inflammatory bile ducts (n=23) were equally evaluated for CD15 expression. Results CD15 was found to be highly expressed in iCCA (81.8%), pCCA (85.7%), dCCA (73.3%), CCA associated dysplasia (70.0%), dysplasia in intraductal biopsies (100%) and metastatic tissue (84.6%). CD15 expression was negative in 58/64 benign bile duct alterations resulting in an overall sensitivity and specificity of CD15 in CCA of 80.7% and 90.6%, respectively. CD15 expression was significantly correlated with a decreased overall survival in patients with CD15-positive CCA associated dysplasia (p=0.003). However, CD15-expression in the invasive tumor component was not correlated with clinical outcome. Conclusion CD15 is a sensitive and specific marker for intraepithelial and invasive neoplasias of the bile duct. Therefore it can be helpful in the delineation of dysplastic and neoplastic biliary cells from non-neoplastic tissue, which frequently causes a diagnostic problem in indeterminate biliary stricture. This article is protected by copyright. All rights reserved.

Backgrounds: Only a few cases of primary classical Hodgkin lymphoma (CHL) are reported previously in stomach. Case: A 77-year-old man presented epigastralgia and received an upper gastrointestinal endoscopy, which revealed multiple polyps. Biopsies (endoscopic mucosal resection) from the lesions showed proliferation of Hodgkin cells (HC) and Reed-Sternberg giant cells (RSC) mixed with non-tumorous inflammatory infiltrates composed of lymphocytes, plasma cells, neutrophils, and histiocytes. Immunohistochemically, the tumor cells (HC and RSC) showed the following immunophenotypes: cytokeratin -, CD45 +++, CD20 +++, CD10 ++. CD79a ++, CD138 +, CD3 -, CD45RO -, CD56 -, CD68 +++, CD30 -, CD15 +++, PAX5 ++, kappa-chain -, lamda-chain -, bcl-2 -, Ki67 20%, p53 +++, vimentin +++, CD68 +++, EMA -, CEA -, S100 protein -, smooth muscle actin -, CD31-, CD34-, factor 8 -, CEA -, EBER -, EBV-latent membrane protein1 -, and EBNA2 -. Immunostainings of endothelial antigens (CD31, CD34, smooth muscle actin, and factor 8-related antigen) showed negative results. A pathological diagnosis of CHL of mixed cellularity type was made, and the patient has first ABVD chemotherapy and then radiation. Conclusion: A very rare case of primary gastric CHL presenting multiple gastric polyps is reported.

CD30 (Ki-1)-positive anaplastic large cell lymphoma (ALCL) has been reported only once in ileum. A 72-year-old presented with melena for 3 weeks duration. Endoscopy identified a large ulcerated tumor (3 cm × 4 cm × 4 cm) in ileum proximal to Bauhin’s valve. Punch biopsies showed broad coagulative necrosis without apparently viable cells, but cell contours were recognizable. Diagnosis was impossible on HE-section, but it suggested malignancy. Since the patient denied second endoscopy, the author managed to make the diagnosis of necrotic punches. Immunohistocheimcally, the coagulative necrosis cells were positive for vimentin, CD45, CD3, CD4, CD5, CD10, CD15, CD30, CD43, CD45RO, p53, and Ki-67 (labeling = 78%). They were negative for cytokeratin (CK) AE 1/3, CK CAM5.2, CK5, CK6, CK7, CK8, CK18, CK19, CK20, EMA, p63, CD8, CD20, CD38, CD138, CD23, CD79α, κ-chain, λ-chain, bcl-2, bcl-6, cyclinD1, CD56, CD57, TdT, chromogranin, synaptophysin, NSE, S100 protein, smooth muscle actin, MUC1, MUC2, MUC5AC, MUC6, KIT and PDGFRA. Since CD30 and CD15 were strongly positive, Hodgkin’s disease and ALCL were considered. However, Hodgkin’s disease was unlikely because no apparent Hodgkin’s or Reed-Sternberg cells were seen. Therefore, CD30-positive ALCL was diagnosed. In addition, because the tumor cells expressed only T-cell markers, the diagnosis of CD30-positive T-cell ALCL was confirmed. The present study could not detect ALK, ALK/NPN, ALK-1, p80, and tissue destructive molecules such as perforins, TIA-1, and granzyme B because tumor cells were totally necrotic. Chromosomal translocation of t (2; 5) and TCR rearrangement could not be performed due to no viable cells. Post-diagnosis whole body examinations using CT, MRI, and PET showed bladder metastasis and multiple lymph node swellings in the para-aortic areas (Ann Arber Stage 4). The patient is now treated by chemotherapy and radiation for 5 months. The present study showed a very rare case of ileal CD30-positive T-cell ALCL and indicated that lymphoma diagnosis is possible even in coagulatively necrotic tissues.

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (-) of β-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.

Classical Hodgkin lymphoma has a characteristic immunophenotype in most cases, with expression of CD30, CD15, and PAX-5, and absence of CD45 and T-lineage markers. However, in a significant subset of cases, atypical staining patterns may be seen for one or more antigens, particularly negative staining for CD15 or staining for one or more B-lineage markers, such as CD20, CD79a, OCT-2, or BOB.1. The greatest pitfall is in the misinterpretation of other cells, such as immunoblasts or histiocytes, as Hodgkin cells.

Glioblastoma multiforme (GBM, WHO grade IV astrocytoma) is the most common and lethal primary brain tumor in adults, with an average survival of slightly more than 1 year after initial diagnosis. GBMs display significant heterogeneity within the tumor mass, among which a subpopulation of cells called glioma-initiating cells (GICs) is responsible for tumorigenesis and resistance to conventional therapies. Therefore, understanding the mechanism underlying the biological properties of GICs would help develop better therapies to target this population for GBM treatment. This protocol provides detailed procedures to isolate GICs and non-GICs from patient's specimen and glioma xenografts, which serves as the first step for the biological studies of GICs. Upon separation of GICs and non-GICs, a series of studies, such as expression profiling and functional screen, etc., can be performed to identify signal pathways responsible for the malignant nature of GICs. Besides, translational studies can also be conducted to examine drug responses of GICs. In sum, isolation of GICs with reliable methods will provide the basis for the further biological studies.

Primary mediastinal (thymic) large B-cell lymphoma (PMLBCL) and nodular sclerosing classical Hodgkin lymphoma (NSCHL) are the major histological types of lymphoma affecting the mediastinum. We reviewed 27 patients with PMLBCL and 14 patients with NSCHL. A poor performance status, high serum lactate dehydrogenase level and strong positivity for PAX5 were all significantly more common in patients with PMLBCL than in those with NSCHL. Severe fibrosis was frequent in NSCHL, but not in PMLBCL. PDL1 was expressed by 11/25 PMLBCLs (44.0%) vs. 1/9 NSCHLs (11.1%). Expression of BCL6 was significantly more frequent in PDL1-positive PMLBCL than in PDL1-negative PMLBCL, but there were no clinical differences between these two groups. Two patients with PMLBCL with a poor prognosis had CD20(-), CD79a(+), CD15(-), and CD30(-), possibly representing a subtype of mediastinal gray zone lymphoma.

Most reports on antibodies that claimed to separate mesothelioma from pulmonary adenocarcinoma originated from academic centers or specialized immunohistochemistry laboratories, but little is known about how such stains perform in general practice laboratories. The Canadian Immunohistochemistry Quality Control program circulates tissue array slides to laboratories across Canada; these are stained and then interpreted by the local laboratory and by a set of experienced reviewers. For Canadian Immunohistochemistry Quality Control run 16, tissue array slides from 16 pulmonary adenocarcinomas and 6 mesotheliomas were stained in 36 different laboratories for CEA, CD15, CK5/6, and calretinin. A total of 736 results (cores) were interpretable. If 3 of 4 staining results concordant with the diagnosis was accepted as definitive, 166/192 (86.4%) mesothelioma cores and 461/544 (84.7%) adenocarcinoma cores were correctly diagnosed. However, if 4 of 4 concordant markers were required, then 93/192 (48.4%) mesothelioma cores and 265/544 (48.7%) adenocarcinoma cores were correctly diagnosed. Only 3/192 (1.6%) mesothelioma cores were incorrectly classified as carcinomas and 8/544 (1.5%) of adenocarcinoma cores incorrectly classified as mesotheliomas on the basis of the immunoprofile (ie, 3 of 4 or 4 of 4 marker results were discordant with the diagnosis). We conclude that, in a study based on results from nonspecialized laboratories, the combination of CEA, CD15, calretinin, and CK5/6, used as a panel, has a very low false-positive rate when separating pulmonary adenocarcinomas from mesotheliomas; however, single negative or incorrect results are common, therefore the panel is only useful diagnostically if 3 of 4 correct results are deemed acceptable for diagnosis.

We report two autopsy cases of primary pulmonary adenocarcinoma associated with unusual pleural spread. Both patients had confirmed history of asbestos exposure. In the first patient the tumor was localized in one pulmonary lobe with marked infiltration into pleura, chest wall and diaphragm. In the second patient the entire right lung was covered by irregularly thickened tumor. Both tumors were mainly located in the extrapulmonary area and the intrapulmonary portions represented only minor components. Histologically, tumor cells demonstrated glandular and papillary growth patterns associated with focal hobnail-like features. Immunohistochemical evaluation revealed diffuse and marked immunoreactivity of TTF-1, CEA, CD15 and MOC31 in both cases, while calretinin, CK5/6, vimentin, thrombomodulin and HBME-1 were broadly positive in one case. D2-40 was not detected in either case. Examination using electron microscopy revealed the presence of sparse and short microvilli in tumor cells. All of the above findings are consistent with adenocarcinoma of the lung. Primary adenocarcinoma with a characteristic pleural extention grossly resembling malignant mesothelioma has been previously reported in the literature as pseudomesotheliomatous adenocarcinoma. This is the first report of pseudomesotheliomatous adenocarcinoma displaying variable immunoprofile with a diagnosis using electron microscopical examination. Additionally, we performed quantitative analysis of asbestos bodies in pseudomesotheliomatous adenocarcinoma.

Aim Immunosuppression and steroid medication have been identified as risk factors for complicated sigmoid diverticulitis. The underlying molecular mechanisms have not yet been elucidated. We hypothesized that glucocorticoid-induced tumour necrosis factor receptor (GITR) and matrix metalloproteinase-9 (MMP-9) might play a role. Method GITR and MMP-9 were analysed at protein [immunohistochemistry/immunofluorescence (IF)] and messenger RNA level (real-time polymerase chain reaction) in surgical specimens with complicated and non-complicated diverticulitis (n = 101). IF double staining and regression analysis were performed for both markers. GITR expression was correlated with clinical data and its usefulness as a diagnostic test was investigated. Results High GITR expression (≥ 41%) was observed in the inflammatory infiltrate in complicated diverticulitis, in contrast to non-complicated diverticulitis where GITR expression was low (P

We recognized a few possible complications of classical Hodgkin lymphoma therapy in a cohort of 209 patients: 8 developed a primary refractory disease (primary progression), 36 showed an early relapse and 21 showed a late relapse. Sialyl-CD15 expression in Hodgkin-Reed-Sternberg cells was significantly more positive in primary refractory Hodgkin lymphoma, which confirms our previously published findings. Bcl-2 showed a significantly lower level of expression in primary refractory disease than in the other follow-up groups. This is in contrast with a previous finding of Bcl-2, associated with a poor prognosis in primary refractory illness. Another category of variables, old age and advanced stages, was significantly different in the various complications but this finding is probably to be expected. We could not demonstrate a difference between the sequels and the control group with regard to several clinical and immunohistochemical markers. Sialyl-CD15 and Bcl-2 expression, in contrast, were confirmed as prognostic factors, mainly of tumor progression into primary refractory disease.

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients.

Background Classical Hodgkin’s lymphoma is characterized by Hodgkin and Reed Sternberg cells, which are of B-cell origin in many cases. We recently highlighted the adverse prognostic significance of cytotoxic molecule expression in patients with classical Hodgkin’s lymphoma. However, the clinical characteristics of cytotoxic molecule-positive classical Hodgkin’s lymphoma remain controversial. Design and Methods We investigated the clinicopathological profiles of 32 patients with cytotoxic molecule-positive Hodgkin’s lymphoma, comprising 23 with nodular sclerosis and 9 with mixed cellularity, and compared these profiles with those of 55 patients with cytotoxic molecule-positive nodal peripheral T-cell lymphoma, not otherwise specified and 439 patients with cytotoxic molecule-negative Hodgkin’s lymphoma. Results The patients with cytotoxic molecule-positive Hodgkin’s lymphoma consisted of 20 men and 12 women with a median age of 50 years (range, 19 to 81). All these patients had lymphadenopathy at presentation, and 14 showed mediastinal involvement. Physical findings included hepatomegaly and splenomegaly in six patients each. Four patients had a bulky mass, and nine showed stage IV disease. The tumor cells of patients with cytotoxic molecule-positive Hodgkin’s lymphoma had a prototypic immunophenotype of CD15+ CD30+ CD45RO− fascin+, with positivity for Epstein-Barr virus in 39% of cases. All patients were negative for Pax5. In comparison with patients with cytotoxic molecule-positive nodal peripheral T-cell lymphomas, not otherwise specified, patients with cytotoxic-positive Hodgkin’s lymphoma had relatively mild clinical symptoms, similar to those of patients with cytotoxic molecule-negative Hodgkin’s lymphoma. Regarding prognosis, the survival of patients with cytotoxic molecule-positive Hodgkin’s lymphoma was worse than that of patients with cytotoxic molecule-negative Hodgkin’s lymphoma ( P =0.0003) but better than that of patients with cytotoxic molecule-positive peripheral T-cell lymphomas, not otherwise specified ( P =0.002). Conclusions Cytotoxic molecule-positive Hodgkin’s lymphoma is characterized by an unfavorable prognosis, even if its clinicopathological features are within the boundaries of classical Hodgkin’s lymphoma. More effective chemotherapy for cytotoxic molecule-positive Hodgkin’s lymphoma is clearly required.

Acute promyelocytic leukaemia (APL) is a unique clinicobiologic entity that can be successfully treated with All-trans Retinoic Acid ATRA-based regimens. Some cases of acute myeloid leukaemia (AML) with nucleophosmin (NPM) mutations have an immunophenotype that is similar to APL. The objective of the study is to compare antigenic expression in a group of APL patients with that in AML patients with NPM mutations and an APL-like immunophenotype (CD15- CD34- HLA-DR-). A consecutive series of 40 APL and 12 NPM patients with an APL-like phenotype were included in the study. Immunophenotypic patterns were investigated by multiparametric flow cytometry. Promyelocytic leukaemia-retinoic acid receptor-α transcript type, NPM and FLT3 mutations were investigated using conventional methods. Statistically significant differences were found between APL and NPM-mutated AML in CD33, CD13, CD2 and CD110 reactivity. CD2 expression was absent in every patient with NPM-mutated AML. In addition, mean fluorescence intensity and the coefficient of variation (cv) of CD33 and CD13 showed statistical differences between the two groups for CD33 (p = 0.007) and a trend to significance for CD13 (p = 0.05). Furthermore, among 45 evaluable patients, CD110 expression statistically differentiates between the two groups: [2/33 (6%) in the APL group and 8/12 (66.6%) in the NPM-mutated AML (p = 0.014)]. However, these traits were subtle, raising the possibility of practical diagnostic challenges. In conclusion, CD110 and CD33 reactivity may be useful to distinguish APL from NPM-mutated AML with CD15, CD34 and HLA-DR negativity. Nevertheless, cytogenetic and molecular characterization is necessary to establish the accurate diagnosis of AML.

This report describes a 60-year-old man with concurrent gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) and classical Hodgkin lymphoma (CHL). Atypical, medium-sized, lymphoid cells proliferated in the mucosa to muscular layer of the stomach showing a lymphoepithelial lesion; admixed with Hodgkin/Reed-Sternberg (HRS) cells and an inflammatory cell background. MALT lymphoma cells expressed CD20, CD79a, PAX5, and BOB.1, and HRS cells expressed CD30, CD15, Epstein–Barr virus–encoded RNA, and EBV-latent membrane protein 1. Only CHL invaded into the regional lymph nodes. Two possibilities of transformation of MALT lymphoma into CHL and de novo CHL within MALT lymphoma are discussed.

Cancer stem cells (CSCs) have been implicated in the maintenance and progression of several types of cancer. The origin and cellular properties of human CSCs are poorly characterized. Here we show that CSC-like cells can be generated in vitro by oncogenic reprogramming of human somatic cells during neoplastic transformation. We find that in vitro transformation confers stem-cell properties to primary differentiated fibroblasts, including the ability to self-renew and to differentiate along multiple lineages. Tumours induced by transformed fibroblasts are hierarchically organized, and the cells that act as CSCs to initiate and maintain tumour growth are marked by the stage-specific embryonic antigen SSEA-1. Heterogeneous lineages of cancer cells in the bulk of the tumour arise through differentiation of SSEA-1(+) fibroblasts, and differentiation is associated with loss of tumorigenic potential. These findings establish an experimental system to characterize cellular and molecular properties of human CSCs and demonstrate that somatic cells have the potential to de-differentiate and acquire properties of CSCs.

A plasmacytoid variant of urothelial carcinoma has been recently recognized in the World Health Organization classification system. This is characterized by a discohesive growth of plasmacytoid cells with eccentric nuclei, extending in the bladder wall and often in the perivesical adipose tissue. Herein, we report the clinicopathologic, immunohistochemical, ultrastructural, and molecular features of a series of plasmacytoid urothelial carcinoma of the urinary bladder. Four bladder carcinomas characterized by epithelial cells with morphologic appearance resembling plasma cells were evaluated at the immunohistochemical, electron microscopic, and molecular genetic levels. Tumor cells stained with cytokeratins, epithelial membrane antigen, GATA-3 (endothelial transcription factor 3), CD15, p53, and p16. In addition, malignant cells strongly stained with CD138 in all the cases, whereas leukocyte common antigen and multiple myeloma 1/interferon regulatory factor 4 were completely negative, nor immunoreactivity was seen for either κ or λ light chains. The electron microscopic examination showed the presence of divergent squamous and glandular differentiation. At variance with conventional urothelial carcinoma, the analysis of exons 4-9 of TP53 gene revealed no alteration in all the 4 tumors tested, and this can be of value in choosing additional chemotherapy after surgery. Plasmacytoid carcinoma of the bladder is a tumor entity, which can be characterized by specific immunohistochemical markers, including positivity for GATA-3, and presents phenotypic and genotypic peculiarities.

The expression of CD10 in tumor cells has been reported to correlate with liver metastasis in colorectal cancer (CRC). However, fibroblasts and immune cells positive for CD10 at the tumor invasion front have not been comprehensively studied. We classified CD10 expression patterns into three types of cells, tumor cells (tCD10), stromal myofibroblasts (sCD10), and immune cells (iCD10), and investigated their correlation with the expression of transforming growth factor-β (TGF-β1) protein and tumor budding grade. Several cell surface markers were stained to detect the phenotype of iCD10(+) cells, including CD3, CD20, CD11b, CD14, CD15, and CD163. Specimens and follow-up data of 206 CRC patients were examined. In multivariate analysis, iCD10 could be an independent prognostic factor for both recurrence-free survival and overall survival in stage I-III CRC (hazard ratio, 2.522 [1.299-4.896], P = 0.006; 2.890 [1.357-6.157], P = 0.006, respectively). The expression of sCD10 and iCD10 was strongly correlated with TGF-β1 expression in tumor cells and tumor budding grade. The phenotype of iCD10(+) cells was CD11b(+) and CD15(+) granulocytes. The infiltration of sCD10(+) fibroblasts and iCD10(+) granulocytes at the tumor invasion front might interact with TGF-β1 protein expression and enhance tumor budding grade. The expression level of iCD10 at the tumor invasion front represented an independent prognostic biomarker in stage I-III CRC and could be integrated into a new staging system.

Summary Aims We previously established the use of a minimal panel of antibodies as sufficient to diagnose most epithelial malignant mesothelioma (MPM). We aimed to validate this approach and investigate the utility of a D2-40 antibody. Methods A series of 80 MPM patients selected for surgery and 21 consecutive patients with pleural metastatic carcinoma were included. A minimal panel of antibodies, consisting of calretinin, BG8 and CD15, and D2-40 was investigated. Results Therewere61 epithelial and 19 biphasic MPM as well as 12 metastatic lung, six breast (5 ductal adenocarcinomas, 1 mixed ductal/lobular adenocarcinoma), two serous papillary ovarian carcinomas and one moderately differentiated colorectal adenocarcinoma. The sensitivity of positive calretinin labelling to confirm the diagnosis of MPM was 97.5%, while the ‘diagnostic sensitivities’ of lack of labelling for BG8 and CD15 were 91.3% and 97.5%, respectively. The use of calretinin, BG8 and CD15 resulted in correct classification in 97.5% of all MPMs. All MPM cases investigated showed at least focal positive D2-40 labelling. Conclusions We have validated the usefulness of a minimal panel of antibodies with calretinin, BG8and CD15as the initial step to the diagnosis of MPM. D2-40 emerged as a helpful diagnostic tool for cases where our initial approach failed to conclusively diagnose MPM.

Schittenhelm J, Simon P, Harter P N, Zachskorn C, Schlaszus H, Rottger F, Winkels M, Weller M, Meyermann R & Mittelbronn M (2011) Histopathology58, 739–749 CD133 expression is associated with small round blue cell tumour morphology in human central nervous system neoplasms Aims: CD133 is considered to be a marker for brain tumour-initiating cells. However, most data on CD133 are derived from animal or in-vitro studies. The aim of this study was to characterize CD133 expression, and the distribution and morphological features of CD133+ cells, in primary and secondary human central nervous system (CNS) neoplasms. Methods and results: Tumours were analysed by real-time reverse transcription polymerase chain reaction, western blot, flow cytometry and immunohistochemistry. Our results show that only small round blue cell tumours (SRBCTs) exhibit strong and consistent CD133 expression. Interestingly, glioblastomas, large-cell carcinomas and sarcomas were negative for CD133. Only glioblastomas with a focal small-cell component exhibited CD133 immunoreactivity in the SRBCT component. In addition, CD133 expression did not correlate with the expression of other markers associated with stem cell differentiation, including CD15 and nestin. Conclusions: CD133 expression in human CNS neoplasms is independent of the grade of malignancy but strongly correlates with SRBCT morphology. Together with recent findings showing that CD133 is quickly upregulated upon hypoxia and that CD133− cells can also exhibit stem cell properties, our data strongly question the suitability of CD133 as a brain tumour stem cell marker in vivo.

Objective: To determine the potential for a novel immunohistochemistry panel to accurately distinguish oncocytoma from other renal tumour subtypes. Material and methods: Forty renal tumours removed by surgery between 2000 and 2006 in a single tertiary referral centre in the UK were studied retrospectively. Paraffin blocks from 10 each of oncocytoma, papillary RCC, clear cell RCC and chromophobe RCC were examined. The tumours were tested using a panel of antibodies comprising CK7, CK18, CD15, N-cadherin, E-cadherin and EpCAM. The primary outcome measure was the number of each tumour type staining positively with each marker. The immunohistochemistry marker was considered to be positive if more than 10% of the tumour cells stained positively. No staining or focal staining (Results: CK7, CD15 and EpCAM were able to distinguish between renal oncocytoma and chromophobe RCC: no oncocytoma stained with either CK7 or EpCAM, however 7/10 (70%) stained positive for CD15. Conversely, 8/10 (80%) chromophobe RCC stained positive with CK7 and EpCAM but none stained for CD15. Conclusions: In this preliminary study the immunohistochemistry panel shows promise in differentiating between renal oncocytoma and chromophobe RCC. The panel deserves prospective evaluation on needle biopsy specimens.

Idiopathic cervical fibrosis is a rare tumefactive inflammatory-sclerosing lesion involving the soft tissues of the head and neck, and a proportion of patients also have synchronous or metachronous inflammatory fibrosclerosing lesions in other anatomic sites. The latter finding suggests that this entity may represent a member of IgG4-related sclerosing diseases. We report 4 cases to support this postulation. The patients were male adults aged 42 to 89 years, who presented with an infiltrative, firm cervical mass. Two patients also had IgG4-related chronic sclerosing sialadenitis of submandibular gland and lymphadenopathy. Histologically, the cervical soft tissue lesions had ill-defined borders, consisting of coalescent nodular lymphoid aggregates accompanied by a sclerotic stroma. Nerve infiltration, skeletal muscle invasion, and phlebitis were present. There was a significant increase in IgG4 + plasma cells (87 to 327 per high-power field, with IgG4/ IgG ratio of 63% to 98%). In the soft tissue lesion of 1 patient, there were expansile foci comprising dense sheets of plasma cells and small lymphoid cells that exhibited κ light chain restriction and clonal immunoglobulin gene rearrangement, consistent with supervening extranodal marginal zone lymphoma. The adjacent lymph node from the same patient showed Epstein-Barr virus (EBV)-positive classical Hodgkin lymphoma with typical morphology and immunophenotype (CD30 + , CD15+, PAX5 + ). Thus lymphoma can supervene in the chronic inflammatory background similar to that recently documented for IgG4-related sclerosing disease of the ocular adnexa.

Aim Renal allografts stored with hypothermic machine perfusion (HPM) have improved outcomes compared to static methods. Passenger immune cells from the allograft have been implicated in acute rejection; improved outcomes with HPM may be due to cell removal by the pump. This study aimed to identify passenger immune cells present in the perfusate of HPM-preserved allografts. Once characterized, future strategies to suppress these cells may prevent acute rejection and improve graft survival. Methods All kidneys placed on HPM prior to transplant from May 2016 to August 2017 were eligible. Perfusate fluid was analyzed using flow cytometry for immune cells and information about the donors was collected. Wilcoxon Scores and Spearman Correlation Coefficient were used in statistical analysis. Results Twenty-seven perfusate specimens were analyzed. Selected donor characteristics: 63.6% male, 36.4% female; 63.6% donation after cardiac death; 54.5% pre-procurement transfusion. The mean HPM and cold ischemia times (CIT) were 9.9 h and 13.9 h respectively, and mean KDPI 36%. Flow cytometry showed a heterogeneous mix of immune cell types in the perfusate, similar to blood. CD15+ granulocytes, CD14+ monocytes, and CD8+ T cells were predominant in the perfusate. Dendritic and natural killer (NK) cells in perfusate were more abundant compared to blood. Conclusions Passenger immune cells, specifically dendritic cells have been implicated in acute rejection. This study showed the perfusate of renal allografts contains an array of immune cells, with a significantly higher percentage of dendritic cells and NK cells, as compared to blood. Future studies would show if HPM removal of these immune cells accounts for improved outcomes. ∗Frequencies of Cell Types in Human Peripheral Blood, www.stemcell.com .

Numerous studies have elucidated that the most pivotal functions of lysine specific demethylase-1 (LSD1) are associated with regulating normal or malignant hematopoiesis by maintaining stem cell self-renewal and regulating myeloid differentiation. In preclinical models, studies with either pharmacological inhibition or genetic knockdown of LSD1 demonstrated that LSD1 is essential for differentiation of progenitor cells during normal hematopoiesis. In the clinic, AML manifests itself via clonal expansion of abnormal differentiation and proliferation of myeloid cells and, therefore, the inhibition of LSD1 activity with small molecule inhibitors could be a promising therapeutic approach for AML. Previously, we reported upon the identification of a flavin adenine dinucleotide (FAD) directed LSD1 specific inhibitor, INCB059872, which is efficacious in preclinical mouse models utilizing human AML cell lines and primary AML cells by inducing cell differentiation as indicated by the induction of CD11b and CD86 markers. Using a larger panel of myeloid and HSC flow cytometry markers, our currents efforts expanded upon these observations to ascertain whether INCB059872 enhanced lineage commitment at hematopoietic stem cell (HSC) and/ or promoted monocytic/granulocytic differentiation of human primary AML cells ex vivo and in human systemic AML PDX models. In both human AML PDX models and human primary AML samples, INCB059872 increased myeloid differentiation with increasing populations of monocytes (CD14+) and granulocytes (CD15+). Furthermore, INCB059872 induced the differentiation of early hematopoietic progenitors, CD34+/CD38- to more committed CD34+/CD38+ multipotent/oligopotent progenitors, which in turn gave rise to lineage specific progenitors in the human AML PDX models. These studies support further exploration of INCB059872 as a promising novel epigenetic agent for AML therapy whose mechanism of action lies in part through the induction of differentiation of leukemic stem/progenitor cells to more committed hematopoietic lineages. Citation Format: Antony Chadderton, Min Ye, Melody Diamond, Valerie Roman, Michael Weber, Chunhong He, Liangxing Wu, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. The FAD-directed LSD1 specific inhibitor, INCB059872, is a promising epigenetic agent for AML therapy by inducing differentiation of leukemic stem/progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1888.

Survival in Hodgkin lymphoma (HL) has significantly increased, and most relapses occur in the first 2 years. We analyzed the proportion of relapses occurring after 5 years, the method of detection, and survival in the database of patients with HL in our hospital (1967-2005). We undertook a retrospective study of clinical and molecular characteristics with the use of 12 different antibodies (Abs): CD30, CD15, CD20, CD3, BCL-2, BCL-6, CD57, EMA, p53, Ki-67, Aurora kinase A, and PAX-5. We studied 534 patients with HD and found 150 relapses (28%), 30 of which occurred 5 years after the end of treatment. The relapses were detected during a scheduled appointment in 16 (54%) asymptomatic patients. Seven of 30 patients relapsed with a different histological type. There were no statistically significant differences in survival between those patients with a late relapse and new remission and those who never relapsed. None of the molecular markers were associated with relapse. Long-term survival was not significantly different in relapsed patients with remission after treatment compared with patients who never relapsed. We need to maintain active follow-up in order to detect late and recoverable relapses.

The multicolor and multiplexing capabilities of semiconductor quantum dots (QDs) are most promising for improving the sensitivity and specificity of in vitro molecular and cellular diagnostics. Here, we report the use of multiplexed QDs and wavelength-resolved imaging to detect and characterize a class of low-abundant tumor cells in Hodgkin’s lymphoma. Known as the Hodgkin’s and Reed-Sternberg (HRS) cells, this class of malignant cells is a pathological hallmark in clinical diagnosis, but it comprises only about 1% of the heterogeneous infiltrating cells in lymph node tissues. To overcome this cellular heterogeneity and rarity problem, we have developed multicolor QD–antibody conjugates to simultaneously detect a panel of four protein biomarkers (CD15, CD30, CD45, and Pax5) directly on human tissue biopsies. This multiplexing approach allows rapid detection and differentiation of rare HRS cells from infiltrating immune cells such as T and B lymphocytes. We have also carried out clinical translation studies involving six confirmed Hodgkin’s lymphoma patients, two suspicious lymphoma cases, and two patients with reactive lymph nodes (but not lymphoma). The results indicate that a distinct QD staining pattern (CD15 positive, CD30 positive, CD45 negative, and Pax5 positive) can be used to not only detect Hodgkin’s lymphoma but also differentiate it from benign lymphoid hyperplasia.