Pattern Of Antigen Expression Research Articles

Different Growth Patterns of Canine Prostatic Carcinoma Suggests Different Models of Tumor-Initiating Cells.

Controversies remain regarding the cell type from which human prostate cancer originates, and many attempts have been made to identify the cellular origin of canine prostate cancer but without definitive proof. This study aims to evaluate the expression of luminal (androgen receptor [AR], cytokeratin [CK]8/18) and basal (CK14, CK5) cell markers in different histologic subtypes of canine prostatic carcinoma (PC) and to suggest the most likely tumor-initiating cells. Normal prostates (n = 8) were characterized by AR+CK8/18+ luminal cells and few CK5+ basal cells, while CK14 was absent. Similar pattern was observed in all 35 prostates with benign prostatic hyperplasia, except few scattered CK14+ basal cells in 13 samples (37.14%). AR was localized in the nucleus of both normal and hyperplastic cells. In 34 samples of PC, the following growth patterns were identified: cribriform (44.12%), solid (32.35%), small acinar/ductal (20.59%), and micropapillary (2.94%). Most PCs expressed AR and CK8/18, while CK5 and CK14 expression was observed in 25% and 20% of cases, respectively. AR revealed a variable intracellular distribution, both nuclear and cytoplasmic. Solid PC was characterized by an undifferentiated or aberrant phenotype with a reduced expression of AR and CK8/18, increased number of CK14+ cells, and 7 antigen expression patterns. This study demonstrated a predominance of differentiated luminal cell types in canine prostatic tumors, although the role of basal cells in prostate carcinogenesis should also be considered. Moreover, few scattered CK5+ cells in AR+CK8/18+ tumors identified the existence of intermediate cells, from which neoplastic transformation may alternatively commence.

Nanoscopic Localization of Surface-Exposed Antigens of Borrelia burgdorferi.

Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates' immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas.

DEVELOPMENTAL RELATIONSHIP AMONG CD56BRIGHT, CD56DIM NK CELLS AND TYPE-2 INNATE LYMPHOID CELLS (ILC2)

Innate lymphoid cells (ILCs) are immune effector cells which, differently from T and B lymphocytes, do not express rearranged receptors. Besides NK cells that belong to the ILC1 group, the novel lineages have raised a question regarding their developmental relationship. To this regard, the ILC groups differ not only for their effector functions but also for their anatomical localization, suggesting the possibility that a common progenitor may migrate and differentiate in different ILCs, depending on the tissue microenvironment. On the other hand, we have recently indicated that CD56dim and CD56bright NK cells, both considered in the ILC1 group, originate from two distinct precursors which generate cells with convergent phenotypes and functions. CD56bright /CD117+ NK cells, differently from CD56dim/CD117neg NK cells, early express natural cytotoxicity receptors (NCRs), while lately upregulate granzyme- B, perforin, LFA-1, and CD94-CD159a heterodimer (1). Within the ILC family, the recently defined type-2 ILCs (ILC2), characterized by CD161+/CD117+/NKp44neg phenotype, typically produces IL-13 and IL-5 (2). Interestingly, a similar subset had been described several years ago (3). At that time, these cells had been considered an immature stage of NK cell differentiation which, under opportune cytokine stimulation, upregulated CD56 and CD94 antigens, produced IFN-gamma and became cytotoxic. New evidences suggest that, rather than an immature NK cell stage, these cells represent a mature lineage which, similar to CD56bright NK cells, early expresses CD117 and CD161 antigens. However, during their differentiation, ILC-2 upregulate granzyme B and CD94 in absence of NCRs, thus resembling the CD56dim NK cell lineage in their developmental pattern of antigen expression.

Flow Cytometric Immunophenotyping in Myelodysplasia: Discovery and Diagnosis

Diagnosis of myelodysplastic syndromes (MDS) is based on the integration of results from diagnostic tools. Initial laboratory assessments in patients with suspected MDS comprise analysis of peripheral blood and bone marrow with the gold standard of cytomorphology, conventional cytogenetics and interphase fluorescence-in-situ hybridization (FISH). Single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) are emerging techniques. The pathological hallmark of MDS is dysplasia. Flow cytometry (FC) can identify aberrancies in antigen expression and differentiation patterns that are indicative of dysplasia. FC is regarded instrumental and now even recommended in the diagnostic work-up of (suspected) MDS, especially when dysplasia by cytomorphology is minimal and cytogenetics shows no (typical) abnormalities. No single FC marker has been identified that is specific for MDS. The WHO-2008 classification recommends the presence of three or more FC aberrancies as highly suggestive for MDS. Minimal requirements to analyze dysplasia by FC have been proposed by the European LeukemiaNet (ELNet) working group (ELNet iMDS-Flow. ELNet recommendations should enable a categorization of FC results in cytopenic patients as "normal", "suggestive of", or “high probability of” MDS. FC as a single technique is not sufficient for the diagnosis of MDS and should be part of an integrated diagnostic work-up. Within the ELNet-iMDS-Flow group, a score based on four cardinal parameters was validated in MDS patients with <5% blasts and non-clonal cytopenic controls. This diagnostic score comprises: a) SSC of granulocytes (reflecting granularity) as ratio to lymphocytes; b) percentage of CD34+ myeloid progenitors of nucleated cells; c) percentage of B cell progenitors within the total CD34+ compartment; and d) expression of CD45 on CD34+ myeloid progenitors as ratio to lymphocytes. An abnormal value for every single parameter is assigned 1 point; MDS is highly suggestive when a score of 2 or more is obtained. Sensitivity of this diagnostic score was 70% and specificity 92%. It was demonstrated that this score also separates distinct subgroups with respect to prognosis within IPSS-R risk groups. Most flow scores mainly incorporate markers that cover the myelomonocytic lineage, e.g. the flow cytometric scoring system (FCSS). The latter separates patients with no and mild-to-moderate dysplasia from those with severe dysplasia. The FCSS has recently been shown to add significantly in separating patients into low or high risk disease in the revised IPSS subgroups. Finally, FCSS originally designed as a prognostic score, but can also be applied as a diagnostic score as combined with the 4-parameter FC diagnostic score to further improve sensitivity and specificity. Flow profiles based on the myelomonocytic lineage may fail to recognize MDS patients that exclusively show erythroid and/or megakaryocytic dysplasia. Analysis of megakaryocytes is hampered by their paucity. Recent advances shows that when adding erythroid markers such as CD71, CD36 and CD117/CD105 may add significantly to the diagnostic score of MDS by FC. Interestingly, aberrancies in the myelomonocytic lineage have been shown in patients with solely erythroid dysplasia with impact on prognosis. In addition, aberrant FC profile of myeloid progenitors has been associated with high transfusion requirements and disease progression as well as with a short duration of response or even lack of response to growth factor and azacitidine treatment. In conclusion, FC is a useful tool in the diagnostic work-up of MDS. Further studies on the value of FC in diagnosis, prognosis and predicting response to treatment are ongoing, as well as correlation of FC results with data obtained by SNP and NGS within prospective multicenter clinical trials in low and high risk MDS. DisclosuresVan de Loosdrecht:celgene: Honoraria, Research Funding; alexion: Research Funding.

Diagnosis of Blastic Plasmacytoid Dendritic Cell Neoplasm By Using 10-Colour Flow Cytometry and Achievement of Remission By Hypercvad Therapy

Background: Although classified by WHO 2008 as belonging to the category “Acute myeloid leukemia and related precursor neoplasms”, Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) presents as an acute leukemia (AL) only in a minority of cases. There are only few studies describing the comprehensive immunophenotypic pattern of BPDCN in the bone marrow. Furthermore, given the rarity of this hematologic malignancy optimal frontline therapy is unclear.Patients and Methods: This retrospective analysis evaluates the diagnostic flow cytometry pattern and outcome of 9 patients who were diagnosed with BPDCN at the Princess Margaret Cancer Centre between December 2008 and June 2014. A four tube 10-color flow cytometry (FCM) panel has been used to correctly make the diagnosis of BPDCN in 6 patients, whereas a 5-colour panel was used in the remaining patients in conjunction with immunohistochemistry. The following markers were included in the10-color panel: Tube 1: CD65 FITC, CD13 PE, CD14 ECD, CD33 PC5.5, CD34 PC7, CD117 APC, CD7 A700, CD11b A750, CD16 PB, and CD45 KO; Tube 2: CD36 FITC, CD64 PE, CD56 ECD, CD33 PC5.5, CD34 PC7, CD123 APC, CD19 A700, CD38 A750, HLA-DR PB, and CD45 KO; Tube 3: CD71 FITC, CD11c PE, CD4 ECD, CD33 PC5.5, CD34 PC7, CD2 APC, CD10 A700, CD235a A750, CD15 PB, and CD45 KO; Tube 4:nuclear (n) TdT FITC, cytoplasmic (cyt.) MPO PE, CD14 ECD, CD33 PC5.5, CD34 PC7, cyt.CD79a APC, cyt.CD22 A700, CD19 A750, cyt.CD3 PB, and CD45 KO.Results: Median age was 66 years (range, 25 to 91 years); 3 patients were over the age of 70 years. Fifty-six percent were males. All presented with skin lesions and 78% presented each with lymphadenopathy and bone marrow involvement. Cytogenetics were poor-risk in 2 patients, intermediate-risk in 3 and unknown or inconclusive in 4. By 10-color FCM, leukemic cells were in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA-DR, CD71, but negative for CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD34, CD64, CD65, CD235a. Other markers, such as cyt.MPO, cyt.CD3, cyt.CD22 and nTdT were negative, while dim cyt.CD79a was seen in 3 cases. CD7 expression was found in 5 cases, whereas CD2 and CD117 were found in single cases only. BM involvement by BPDCN leukemic cells ranged from 27% to 92% of the marrow cellularity. Skin involvement showed dense infiltrate of cells with blastoid morphology and characteristic grenz zone.Seven patients received front-line induction therapy with HyperCVAD with an overall response rate of 86% (4 complete remissions (CR), 2 unconfirmed CRs). One patient died of multi-organ failure during induction. Three of 6 responders underwent planned allogeneic hematopoietic cell transplantation (HCT); 1 patient has since died of acute graft versus host disease (GVHD), whereas 2 are alive in remission with chronic GVHD, 12 and 14 months post transplant with complete donor chimerism. One transplant ineligible patient relapsed 22 months after achievement of CR1. Median follow-up of all patients was 12 months with a overall survival at 1 year of 59.3% for the entire group. Patients who underwent allogeneic HCT had overall survival at 1 year of 66.7% and for the chemotherapy group was 27.8% at 1 year.(p=0.34).Conclusion: An accurate diagnosis of BPDCN can be made by 10-colour FCM using a 4-tube acute leukemia panel. BPDCN demonstrates a characteristic pattern of antigen expression . Although front-line induction chemotherapy with HyperCVAD can yield high CR rates, allogeneic HCT should be performed in first CR for transplant eligible patients, as this appears to be required for long term durable remissions. For transplant ineligible or relapsed BPDCN patients, there is an unmet need for novel therapeutic agents. DisclosuresPorwit:Beckman-Coulter: Speakers Bureau. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.

Antigen Expression Patterns of Plasma Cell Myeloma: An Association of Cytogenetic Abnormality and International Staging System (ISS) for Myeloma.

Immunophenotyping of plasma cell has become an important diagnostic tool for plasma cell myeloma. There have been a few studies for association of antigen expression and cytogenetic abnormality of plasma cell myeloma. A total of 68 symptomatic/smoldering plasma cell myeloma case were analyzed by multicolor flow cytometry using CD38 and CD138 for primary gating of plasma cells. A conventional cytogenetics and fluorescence in situ hybridization (FISH) studies for detection of del(13q) or aneuploidy, del(17p), and IGH/FGFR translocation were done. We statistically analyzed the association of antigen expression and cytogenetic abnormality/myeloma stage (international staging system for multiple myeloma). Positive expression of CD19, CD28, CD45, CD56, CD117, and CD274 was detected in 8.8%, 50.0%, 50.0%, 75.0%, 39.7%, and 2.9% of cases, respectively. CD117-negative cases were associated with hypodiploidy (P = 0.017). CD45-negative cases were associated with deletion 13 or aneuploidy (P < 0.001) and del(17p)(P = 0.011) by FISH. CD45-negativity or CD117-negativity was associated with advanced stage (P = 0.012 and P = 0.016, respectively). The antigen expression patterns of myeloma plasma cell were associated with cytogenetic abnormality and stage.

Abstract 2027: Novel 3D tumor-immune cell spheroid models for assessment of cancer immunotherapy agents

Abstract The pre-clinical development of anticancer immunotherapeutics is based on 2D co-culture systems that rely on use of allogeneic immune effector cells and established tumor cell lines. These are, however, suboptimal models unable to resemble the complex histopathological, morphological and immunosuppressive features present within tumor microenvironment and devoid of nutritional, metabolic and oxygen gradients established in primary tumors. In addition, when maintained in 2D, tumor cells undergo exponential growth and lack formation of proliferating, quiescent and necrotic cores established in tumors. Due to absence of cell polarity, the level and pattern of tumor antigen expression is different between 2D and 3D cultures and interactions between tumor cells and immune cells in 2D sub optimally represent those occurring in vivo. To overcome these limitations, we established novel types of 3D models that more closely mimic the histological complexity of tumors and better resemble cell-cell, cell-matrix and tumor-stroma interactions, i.e. 1) the heterotypic 3D spheroids composed of tumor cells, fibroblasts, endothelial cells, matrix components and immune effector cells, 2) 3D ex vivo thick tumor sections offering an intact tumor microenvironment with autologous tumor-infiltrating lymphocytes (TILs). In the first model, lymphocytes were either admixed with tumor cells, fibroblasts, endothelial cells and matrix components (collagen, Matrigel or both) giving rise to heterotypic 3D co-cultures containing low levels of immune effector cells (resembling poorly-infiltrated tumors), or were stimulated with low dose of antibody-conjugated immunocytokine (DP47-IL2v, 10 nM, overnight), which promoted their infiltration into pre-formed tumor/fibroblast spheroids (resembling highly-infiltrated tumors). In the second model, primary tumors collected from naïve or DP47-IL2v-treated mice were sliced ex vivo to obtain 400 μm-thick tumor sections that were maintained in culture for up to 7 days. 3D models were characterized by quantification of size and IHC staining of tumor, stromal and endothelial cell antigens and immune effector cells. We further validated our models by testing the activity of tumor- or stroma-targeted immunotherapies. Incubation of spheroids with 50 or 100 nM of tumor-targeted T Cell Bispecific (TCB) antibody led to crosslinking of T cells to tumor cells resulting in elimination of tumor areas (but not of the fibroblast core) and overall reduction in spheroid size. On the contrary, spheroids incubated with untargeted TCB remained intact. Taken together, the novel models presented here provide the first example of heterotypic 3D microtissues containing the immune effector cells and offer a versatile system to characterize the activity of tumor- and stroma-targeted immunotherapies and explore their combinations. Citation Format: Laura Morra, Inja Waldhauer, Christian Klein, Pablo Umana, Marina Bacac. Novel 3D tumor-immune cell spheroid models for assessment of cancer immunotherapy agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2014-2027

High placenta-specific 1/low prostate-specific antigen expression pattern in high-grade prostate adenocarcinoma.

The scarcity of effective therapeutic approaches for prostate cancer (PCa) has encouraged steadily growing interest for the identification of novel antigenic targets. Placenta-specific 1 (PLAC1) is a novel cancer-testis antigen with reported ectopic expression in a variety of tumors and cancer cell lines. The purpose of the present study was to investigate for the first time the differential expression of PLAC1 in PCa tissues. We investigated the differential expression of PLAC1 in PCa, high-grade prostatic intraepithelial neoplasia (HPIN), benign prostatic hyperplasia (BPH), and nonneoplastic/nonhyperplastic prostate tissues using microarray-based immunohistochemistry (n=227). The correlation of PLAC1 expression with certain clinicopathological parameters and expression of prostate-specific antigen (PSA), as a prostate epithelial cell differentiation marker, were investigated. Placenta-specific 1 (PLAC1) expression was increased in a stepwise manner from BPH to PCa, which expressed highest levels of this molecule, while in a majority of normal tissues, PLAC1 expression was not detected. Moreover, PLAC1 expression was positively associated with Gleason score (p≤0.001). Interestingly, there was a negative correlation between PLAC1 and PSA expression in patients with PCa and HPIN (p≤0.01). Increment of PLAC1 expression increased the odds of PCa and HPIN diagnosis (OR 49.45, 95% CI for OR 16.17-151.25). Our findings on differential expression of PLAC1 in PCa plus its positive association with Gleason score and negative correlation with PSA expression highlight the potential usefulness of PLAC1 for targeted PC therapy especially for patients with advanced disease.

Astrocytoma-associated antigens - IL13Rα2, Fra-1, and EphA2 as potential markers to monitor the status of tumour-derived cell cultures in vitro.

BackgroundThe molecular heterogeneity of high-grade astrocytomas underlies the difficulties in the development of representative and valuable in vitro experimental models for their studies.The purpose of our study was to estimate the value of astrocytoma-associated antigens (AAAs) - IL13Rα2, Fra-1, EphA2 - and the most common molecular aberrations typical for astrocytomas as potential markers to screen the status of tumour-derived cell cultures in vitro.MethodsThe tumour-derived cell cultures were established from high-grade astrocytomas. The expression analyses of the tested genes were performed via semi-quantitative real-time PCR and subsequently verified by immunohistochemical and immunocytochemical technique. The analyses of molecular aberrations at DNA level included gene dosage status evaluation based on real-time PCR, sequencing analysis, and loss of heterozygosity (LOH) assay.ResultsThe expression analyses based on semi-quantitative real-time PCR showed that in the final stage of culture the expression level of all tested AAAs was significantly higher or at least comparable to that of primary tumours; however, two expression patterns were observed during cell culture establishment. Analysis at the single cell level via immunocytochemistry also demonstrated an increase of the level of tested proteins and/or selection of tumour cell populations strongly positive for AAAs vs. other cell types including admixed non-tumoural cells. Confrontation of AAA expression data with the results of molecular analyses at DNA level seems to support the latter, revealing that the expression pattern of astrocytoma-associated antigens in tumour-derived cells in subsequent stages of culture is convergent with changes in the molecular profile of examined cell populations.ConclusionsThe consistency of the obtained results seems to support the use of the selected AAAs, in particular IL13Rα2 and Fra-1, as tools facilitating the establishment of tumour-derived cultures. However, the intratumoural heterogeneity of high-grade astrocytomas may require further detailed characterisation of the molecular profile of a tumour in order to evaluate the value of the experimental model in relation to the individual context of particular studies.

OMIP-023: 10-color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes.

THIS panel was developed and optimized to determine the phenotype and activation of 15 different leukocyte subtypes in one run. Leukocytes are identified by expression of CD45 (leu-1) pan leukocyte antigen. Neutrophil (CD16), monocyte (CD14), T(CD3), B-lymphocyte (CD19), and NK-cell (CD16 and CD56) markers are employed. Special gating strategy is used for subtyping of granulocytes (e.g., eosinophils, neutrophils, basophils). For further T-cell phenotyping, CD4/CD8 markers are used for differentiation of four T-cell subtypes, CD25/CD127 for regulatory T cell identification and CD3/CD16/CD56 for NKT-cells, additionally. Special gating strategies have been developed for B-cell, NK-cell, and monocyte subtyping. For detection and analysis of activation also further activation markers (HLA-DR, CD38, CD25, CD127, and CD69) are analyzed. This panel has been established for analysis of human RBC-lysed EDTAtreated whole blood samples and for cord blood (Table 1). Since the starting material is fresh EDTA-treated blood, dead cells are probably not an issue. Thus to save one channel for specific staining the vitality staining was not used in the panel.

Comparison of cancer stem cell antigen expression by tumor cell lines and by tumor biopsies from dogs with melanoma and osteosarcoma

Cancer stem cells (CSCs) represent a small subpopulation of tumor cells that play a critical role in initiating and sustaining tumor growth. However, we currently have an incomplete understanding of the expression patterns of CSC antigens in tumors of dogs, nor do we understand how expression of these antigens vary between tumor cell lines and tumor biopsy specimens. Therefore, we used flow cytometry and commonly reported CSC surface and intracellular markers to evaluate the phenotype and overall frequency of CSC subpopulations in tumor cell lines and primary tumor biopsy samples from dogs with melanoma and osteosarcoma. We found that cells expressing common CSC antigens were rare in tumor cell lines, with the exception of tumor cells expressing CD44 and CD90. In contrast, tumor cells expressing conventional CSC antigens such as CD133, CD34, CD44, CD24 and Oct3/4 were much more common in tumor biopsy samples. Notably, the frequency and types of putative CSC subpopulations were very similar in biopsy samples from dogs with either melanoma or osteosarcoma. Our results suggest that the tumor microenvironment significantly influences CSC subpopulations within tumors and that tumor cell lines may not accurately reflect the actual frequency or types of CSC subpopulations present in tumor tissues in vivo.

Cancer testis antigen expression in testicular germ cell tumorigenesis

Cancer testis antigens are encoded by germ line-associated genes that are present in normal germ cells of testis and ovary but not in differentiated tissues. Their expression in various human cancer types has been interpreted as ‘re-expression' or as intratumoral progenitor cell signature. Cancer testis antigen expression patterns have not yet been studied in germ cell tumorigenesis with specific emphasis on intratubular germ cell neoplasia unclassified as a precursor lesion for testicular germ cell tumors. Immunohistochemistry was used to study MAGEA3, MAGEA4, MAGEC1, GAGE1 and CTAG1B expression in 325 primary testicular germ cell tumors, including 94 mixed germ cell tumors. Seminomatous and non-seminomatous components were separately arranged and evaluated on tissue microarrays. Spermatogonia in the normal testis were positive, whereas intratubular germ cell neoplasia unclassified was negative for all five CT antigens. Cancer testis antigen expression was only found in 3% (CTAG1B), 10% (GAGE1, MAGEA4), 33% (MAGEA3) and 40% (MAGEC1) of classic seminoma but not in non-seminomatous testicular germ cell tumors. In contrast, all spermatocytic seminomas were positive for cancer testis antigens. These data are consistent with a different cell origin in spermatocytic seminoma compared with classic seminoma and support a progression model with loss of cancer testis antigens in early tumorigenesis of testicular germ cell tumors and later re-expression in a subset of seminomas.

Human Melanoma Metastases Demonstrate Nonstochastic Site-Specific Antigen Heterogeneity That Correlates with T-cell Infiltration

Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies. From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4(+) and CD8(+) T-cell infiltrate. Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8(+) and CD4(+) T-cell infiltrates. Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies.

Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens

The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range-equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody-based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a melanoma patient cohort to an experimental therapeutic NY-ESO-1-based cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT antigen expression and epitope usage.

Antigen expression patterns of MYC-rearranged versus non-MYC-rearranged B-cell lymphomas by flow cytometry

B-cell lymphomas with MYC rearrangement are a heterogeneous and unique group of neoplasms that may behave aggressively, especially in conjunction with other oncogenic alterations that cooperate with MYC, warranting early and intensive treatment in a subset of patients. Identification of MYC-rearranged B-cell lymphomas by immunophenotypic analysis could help guide cytogenetic work-up, as well as expedite therapeutic interventions. Using flow cytometry we analyzed the expression of CD10, CD19, CD20, CD38 and CD45 in 53 patients with three distinct neoplasms with MYC rearrangements: Burkitt lymphoma (n = 12), double hit lymphoma (n = 17) and diffuse large B-cell lymphoma (DLBCL) with MYC rearrangement (n = 8). The expression profile of these commonly used antibodies was similar among these three groups. The antigenic pattern of bright CD38 and dim CD45 was unique to the MYC-rearranged neoplasms when compared to non-MYC-rearranged aggressive DLBCL (n = 16), with a combined sensitivity of 67% and specificity of 100%, suggesting a shared underlying biology among these MYC rearranged B-cell lymphomas.

The role of microparticles in chronic obstructive pulmonary disease.

Accumulating evidence suggests that cell injury in lung tissues is closely connected to disease progression in chronic obstructive pulmonary disease (COPD). Microparticles (MPs) are shed membrane vesicles that are released from platelets, leukocytes, red blood cells, and endothelial cells when these cells are activated or undergo apoptosis under inflammatory conditions. Based on increasing evidence that endothelial injury in the pulmonary capillary vasculature leads to lung destruction, and because cardiovascular diseases are the main cause of death among individuals with COPD, endothelial MPs (EMPs) are now receiving attention as potential biomarkers for COPD. There are eight types of EMPs which are defined by the presence of different endothelial markers on the cell membrane: vascular endothelial-cadherin; platelet endothelial cell adhesion molecule; melanoma cell adhesion molecule; E-selectin; CD51; CD105; von Willebrand factor; and CD143 EMPs. Vascular endothelial-cadherin, platelet endothelial cell adhesion molecule, and E-selectin EMPs are increased in patients with stable COPD and are further increased in patients with exacerbated COPD compared to non-COPD patients. In addition, the levels of these three EMPs in patients with stable COPD are significantly correlated with lung destruction and airflow limitation. These results indicate that endothelial injury is closely connected to the pathophysiology of COPD. Interestingly, the variations in the levels of the eight EMP subtypes were not identical with changes in patient condition. Although the clinical significance of the differences in these eight EMP subtypes remains unclear, evaluating the expression pattern of endothelial antigens on circulating MPs might predict the presence and degree of endothelial injury in COPD patients. In addition, circulating MPs are proposed to have several physiological functions in vivo, such as intercellular crosstalk; the increase in EMPs in COPD seems to play a role in the pathophysiology of this disease.

Abstract B24: A systematic search for cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets.

Abstract Background: A wide range of therapeutic cancer vaccines that target diverse tumor-associated antigens are currently been evaluated in early phase clinical trials. However the effectiveness of cancer vaccines is so far limited, potentially due in part to the limited availability of candidate targets with broad expression in a tumor type. Cancer/testis (CT) antigens are a major target of cancer vaccines, because CT antigens are specifically expressed in immune-privileged testis and placenta, and exhibit aberrant expression in various types of cancer, resulting in high immunogenicity with no self-tolerance and reduced adverse effects. However expression of particular antigens is limited to a subset of tumors of a given type. Thus there is a need to identify antigens with complementary expression patterns for more effective therapeutic intervention. Methods: Gene expression data of BioGPS, and DNA methylation and RNA-seq data of TCGA lung cancer samples were downloaded. All lung cancer cell lines were cultured in RPMI1640 containing 10% FBS. A total of 83 pairs of treatment naive lung adenocarcinoma tissues and adjacent non-tumor lung tissues had been obtained earlier with informed consent. Gene expression data for cell lines and tissues were obtained using the Illumina Human WG-6 v3.0 Expression BeadChips. For mass spectrometric analysis, 38 non-small cell lung cancer (NSCLC) cell lines were cultured according to the standard SILAC protocol. The same batch of cells was used for analysis of whole cell extract, conditioned media and cell surface proteins. Tissue microarrays comprised 463 surgically resected NSCLC tumor specimens collected under an IRB protocol. Monoclonal anti-mouse VCX3A antibody was used for Western blot and immunohistochemistry analysis. Results: With mining the public database BioGPS, we identified VCX/Y gene family as novel CT antigens in lung cancer. Our findings also suggested epigenetic regulation of VCX/Y gene expression, and potential oncogenic roles of VCX/Y genes. Expression of one of the VCX/Y family genes, VCX3A, was further confirmed at the protein level in ~20% of lung adenocarcinoma and ~35% of squamous cell carcinoma tumors. We further examined gene expression patterns of CT antigens to determine whether a panel could be assembled with broad representation in lung adenocarcinoma. Of 255 known CT antigens, 109 CT antigens were profiled at both mRNA and protein levels in 38 NSCLC cell lines. mRNA expression levels of 10 CT antigens were significantly and positively correlated with protein expression levels. We examined mRNA expression profiles of these 10 CT antigens, together with VCX and MAGEA3 which is a current target for vaccine immunotherapy, in 83 lung adenocarcinoma tumors. We selected XAGE1, VCX, IL13RA2, and SYCE1 to form a CT antigen panel and compared mRNA expression levels of these four genes between tumor and adjacent non-tumor tissue. One or more of the CT antigens in the panel were overexpressed in 66 (79.5%) of lung adenocarcinomas. Conclusions: The present study identifies for the first time a novel CT antigen VCX/Y gene family in lung cancer. A CT antigen panel was found to provide a broad coverage of lung adenocarcinoma tumors tested, suggestive of a potential for more effective immunotherapy through targeting the combined antigen panel compared to a single antigen target. Citation Format: Ayumu Taguchi, Allen D. Taylor, Jaime Rodriguez, Muge Celiktas, Hui Liu, Xiaotu Ma, Qing Zhang, Chee-Hong Wong, Alice Chin, Luc Girard, Carmen Behrens, Wan Lam, Stephen Lam, John D. Minna, Ignacio I. Wistuba, Adi F. Gazdar, Samir M. Hanash. A systematic search for cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B24.

Descriptive analysis of antigen expression pattern in refractory anemia, refractory anemia with ringed sideroblasts and refractory anemia with excess blast cases using flow cytometry

Descriptive analysis of antigen expression pattern in refractory anemia, refractory anemia with ringed sideroblasts and refractory anemia with excess blast cases using flow cytometry

New Therapies for Hairy Cell Leukemia

Copyright: © 2014 Robak T. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Hairy Cell Leukemia (HCL) is a B-cell chronic lymphoproliferative disorder characterized by marked splenomegaly, progressive pancytopenia and reactive marrow fibrosis [1]. The annual incidence of HCL is estimated to be 0.3 cases per 100 000 [2]. Hairy cells characteristically stain strongly positive for tartrate-resistant acid phosphatase, a typical pattern of B-cell antigen expression (CD19, CD20) and coexpression of CD11c, CD25 and CD103. Cell surface markers which are associated with normal B-cell activation, especially CD22, CD25, CD72 and CD40, are strongly expressed by leukemic cells [3]. Recently, the BRAF V600E mutation has been found in all evaluated patients with HCL and has been described as a disease-defining genetic event [4]. Hairy Cell Leukemia Variant (HCL-V) is a rare and indolent form of small, mature, B-cell leukemia comprising about 2% of leukemias [5]. The World Health Organization (WHO) now recognizes HCL-V as a provisional entity distinct from classic HCL.

Flow cytometry (FCM) in myeloid malignancies

Myelodysplasic syndromes (MDS) and acute myeloid leukaemia (AML) are the predominant myeloid neoplasms where FCM has both a diagnostic and prognostic role in addition to minimal residual disease (MRD) testing in AML. Criteria for the diagnosis and classification of MDS have been refined and proposed in a consensus International MDS Working Group publication (2007) with immunophenotyping recommended as a co-criterion for diagnosis. Utilising our understanding and delineation of normal antigen maturation and expression patterns of myelomonocytic precursors enables the detection of aberrancy in mature and immature populations. This forms the basis of reporting MDS and AML MRD FCM. There is a large body of literature supporting the routine utility of immunophenotyping in the diagnostic workup of MDS and provocative data in patients treated serially with hypomethy-lating agents. However, further validation and definitions of aberrancy and flow scoring systems are required to enable the introduction of immunophenotyping into mainstream diagnostic laboratories. Standardisation efforts are underway within the European LeukemiaNet. FCM is routinely used for the diagnosis of AML, however MRD testing requires significant expertise. Despite numerous publications demonstrating prognostic significance in MRD at both post induction and post consolidation time points, most are single institution studies highlighting the sophistication required by laboratories.