Panel Of Normal Human Tissues Research Articles

Generation of a humanized afucosylated BAFF-R antibody withbroad activity against human B-cell malignancies.

B-cell activating factor receptor (BAFF-R) is a mature B-cell survival receptor, which is highly expressed in a wide variety of B-cell malignancies but with minimal expression in immature B cells. These properties make BAFF-R an attractive target for therapy of B-cell lymphomas. We generated a novel humanized anti BAFF-R monoclonal antibody (mAb) with high specificity and potent invitro and invivo activity against B-cell lymphomas and leukemias. The humanized variants of an original chimeric BAFF-R mAb retained BAFF-R binding affinity and antibody-dependent cellular cytotoxicity (ADCC) against a panel of human cell lines and primary lymphoma samples. Furthermore, 1 humanized BAFF-R mAb clone and its afucosylated version, glycoengineered to optimize the primary mechanism of action, prolonged survival of immunodeficient mice bearing human tumor cell lines or patient-derived lymphoma xenografts in 3 separate models, compared with controls. Finally, the tissue specificity of this humanized mAb was confirmed against a broad panel of normal human tissues. Taken together, we have identified a robust lead-candidate BAFF-R mAb for clinical development.

First-in-class monoclonal antibody (mAb) PBP1510 targeting pancreatic adenocarcinoma upregulated factor (PAUF) for pancreatic cancer (PC) treatment: Preclinical perspectives.

e16274 Background: PAUF has been shown to induce PC cell proliferation, angiogenesis and evasion of immune surveillance, thereby promoting tumour progression and metastasis. PBP1510, a novel humanized IgG1 mAb developed by Prestige Biopharma Limited, Singapore, binds to and neutralizes PAUF with high specificity and affinity. With no available targeted therapies against PAUF, the first in its class, PBP1510, has been granted Orphan Drug Designation by EMA, US FDA and Korea MFDS. Methods: To assess inhibition of migration and invasion of PC in vitro, PC cell lines (CFPAC-1 and BxPC-3) with high expression levels of PAUF, and primary PC cells extracted from patients were treated with PBP1510 or human IgG (negative control). Suitability of cynomolgus monkey as animal model for pharmacokinetic (PK) and toxicity studies was verified by comparing binding affinities of PBP1510 to human and monkey PAUF. PK characteristics and immunogenicity of PBP1510 (up to 20 mg/kg), were examined in single dose studies in 6 monkeys. A 4-week repeated dose toxicity study was also conducted in 32 monkeys with PBP1510 (up to 40 mg/kg). An in vitro tissue cross-reactivity study was also conducted in a full panel of normal human tissues, to assess potential toxicity in humans. Results: PBP1510 suppressed the proliferation of the PC cell lines compared to negative control. The migration and invasion of PBP1510 treated PC cell lines as well as the primary tumour cells from patients was significantly reduced compared to the respective control groups in vitro. Similarity in binding affinity of PBP1510 to human PAUF and monkey PAUF, and amino acid sequence homology between human and monkey PAUF, suggest that monkey is an appropriate animal species for toxicity and PK studies of PBP1510. In monkey studies, a dose-proportional increase in systemic exposure was observed at low and high doses. PBP1510 at doses up to 40 mg/kg was well tolerated by study animals and no notable local or systemic toxicity was observed. Low immunogenic profile of PBP1510 was indicated by an absence of anti-PBP1510 antibodies till up to 57 days (8 doses and 28-day recovery period). These data are used to simulate human PK profile and to derive the starting dose and maximum escalated dose for the upcoming Phase 1 clinical studies. Cross-reactivity results did not reveal non-specific binding of PBP1510 in the human tissues except in the cytoplasm of acinar and duct epithelium in the salivary gland which is not expected to be of major toxicologic significance. Conclusions: The efficacy and safety data presented here, along with the data from in vivo mouse studies demonstrating superior anti-tumour activity of PBP1510 treatment, support further clinical development of PBP1510 as a novel anti-cancer agent to treat PAUF-positive PC. The pivotal step of advancing PBP1510 into initial human studies is now in progress.

MDNA55: A locally administered IL4 guided toxin as a targeted treatment for recurrent glioblastoma.

2039 Background: IL4 receptor (IL4R) is frequently and intensely expressed on a variety of human cancers and is associated with poor survival outcomes. Determining the role of the IL4R biomarker in glioblastoma (GBM) will be important for treatment with targeted therapies such as the IL4 fusion toxin MDNA55. Methods: A classification for IL4Rα expression in GBM tissues by H-Score was developed using a validated immunohistochemistry-based approach. MDNA55-05 is an open-label study of MDNA55 administered intratumorally via convection enhanced delivery in recurrent GBM. Levels of IL4Rα expression were assessed retrospectively in 24 subjects in the clinical trial and were correlated with GBM history, imaging responses and survival outcomes following treatment with MDNA55 to explore clinical validation. Results: Range, linearity, specificity and sensitivity testing using a rabbit polyclonal antibody to IL4Rα were performed using normal cortex (negative control) and a panel of normal human tissues and GBM cases from tissue banks. A total of 41 GBM samples were screened and grouped by reactivity thresholds: H-Scores ≥50 were observed in 95% of cases (39/41), H-Scores ≥200 were observed in 51% of cases (21/41), and H-Scores ≥250 were observed in 24% of cases (10/41). GBM tissues obtained at initial diagnosis from subjects enrolled in the trial show that moderate/high IL4R expression (H-Score > 75) was associated with shorter time to first relapse when compared to subjects with low IL4R expression (H-Score ≤ 75) (10.3 mos vs. 16.7 mos, respectively) after upfront standard-of-care treatment, consistent with published findings that IL4R expression is associated with more aggressive disease. Remarkable decreases in tumor size seen in some subjects following MDNA55 treatment were associated only with moderate/high IL4R expression and survival rate at 12 months in this group was also improved (OS12 = 55%) compared to subjects with low IL4R expression (OS12 = 30%). Conclusions: Treatment options for patients with recurrent GBM are very limited and positive outcomes remain rare. Targeting therapies such as MDNA55 by IL4R status may improve patient outcomes and help guide patient selection strategies for future clinical studies. Clinical trial information: NCT02858895.

The long non-coding RNA BC200 (BCYRN1) is critical for cancer cell survival and proliferation

BackgroundBC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized.MethodsDetailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometryResultsBC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest.ConclusionsBC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.

Abstract 581: Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors

Abstract Today antibody therapy is considered to be one of the most important and successful strategies to treat a variety of cancers. For example, the addition of antibodies such as Herceptin and Avastin to a chemotherapy regimen has shown improved survival in the treatment of breast cancer and colorectal cancer, respectively. Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies would greatly contribute to the field of targeted anticancer therapy. This work evaluated the feasibility of using residual clinical material from pediatric neuroblastoma patients to generate antibodies to autologous tumor. Neuroblastoma is the most common extracranial solid tumor in children, accounting for 8-10% of all childhood cancers. Most patients with neuroblastoma are young and commonly present with metastatic disease. Bone marrow aspirate from neuroblatoma patients was the source material for the mononuclear cells and the tumor cells used in present study. Tumor cells were cultured and xenograft tumors were produced in mice. Hybridomas were generated by electrofusion of stimulated bone marrow mononuclear cells with plasmacytoma P3×63.Ag8.653 under hypo-osmolar condition using Eppendorf Multiporator/Helix chamber. Following hypoxanthine-aminopterin-thymidine (HAT) selection and monoclonal distribution, the culture supernatants were assayed for immunoglobulin secretion by ELISA. The supernatants from the positive clones were evaluated by immunofluorescence microscopy for binding to cultured neuroblastoma cells and neuroblastoma xenograft tissue sections derived from the same patient from which the hybridomas were generated. The results demonstrated that multiple hybridomas of bone marrow mononuclear cells secreted monoclonal antibodies that bound autologous neuroblastoma cells. Further evaluation of the tumor-binding antibodies on a panel of normal human tissues showed no binding to most of the tissues in the panel. Successful outcome of these experiments demonstrate the feasibility of generating human monoclonal antibodies from residual marrow specimens that bind autologous neuroblastoma cells. However, it remains to be determined whether these antibodies are bioactive and whether this approach will be generally applicable in more patients with neuroblastoma. It may be concluded that the strategy described here, which exploits the cancer patient's own immune repertoire, has a great potential for neuroblastoma target discovery and developing antibodies with possible therapeutic and/or diagnostic utility in cancer. Citation Format: Girja S. Shukla, Giselle S. Sholler, Yujing Sun, Stephanie C. Pero, Chelsea L. Carman, Ping Zhao, David N. Krag. Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 581.

Abstract 1739: The prevalence of KRAS, PIK3CA, and BRAF mutant subpopulations in tumors may be impacting the success of personalized cancer treatment

Abstract Tumor mutations are being used as predictive biomarkers of response, in order to select the most effective treatment for individual cancer patients. Currently, this is being done without sufficient characterization of relevant oncogene mutations as quantitative biomarkers. The goal of the current study was to define normal and pathological levels of the most prevalent hotspot mutations in the KRAS, PIK3CA, and BRAF genes, to establish the frequency with which the mutations occur as subpopulations, and what diagnostic sensitivity is needed to detect defined percentages of tumors carrying mutant subpopulations. Therefore, the sensitive Allele-specific Competitive Blocker-PCR (ACB-PCR) was used to quantify the levels of specific hotspot point mutations in a panel of normal human tissues and tumors. The mutations examined have established significance in terms of personalized cancer treatment, specifically KRAS G12D, KRAS G12V, BRAF V600E, and PIK3CA H1047R. The tissues examined included lung, colon, pancreas, and thyroid. In colon tumors, the 5th, 25th, 50th, 75th, and 95th percentiles of KRAS G12D mutant fraction (MF) are 1.7 x 10−5, 7.4 x 10−5, 3.0 x 10−4, 2.8 x 10−2, and 8.4 x 10−1, respectively. In lung tumors, the 5th, 25th, 50th, 75th, and 95th percentiles of KRAS G12V MF are 7.0 x 10−6, 1.1 x 10−5, 3.3 x 10−5, 2.3 x 10−2, and 1.2 x 10−1, respectively. Based on the data across these tissue types, 67.5% of tumors carry KRAS G12D or G12V mutation at a subpopulation frequency higher than that observed in normal tissue. Only 18.1% of tumors had a KRAS MF α10−1 (i.e., that detectable by DNA sequencing). From these data it was determined a diagnostic with a sensitivity of 10−2 or 10−3 would detect 27.7% or 43.4% of these tumors, respectively. Surprisingly, analysis of KRAS mutation in papillary thyroid tumors showed KRAS G12V mutations were present above normal thyroid levels, but as subpopulations in 42.1% of papillary thyroid tumors, even though the COSMIC database indicates this mutation occurs in only 0.15% of papillary thyroid tumors. The occurrence of these KRAS G12V mutations was positively correlated with percent tumor necrosis. For PIK3CA H1047R mutation in colon tumors, the 5th, 25th, 50th, 75th, and 95th percentiles are 1.2 x 10−6, 5.3 x 10−4, 7.6 x 10−4, 1.1 x 10−3, and 4.2 x 10−2, respectively. Data on BRAF V600E shows it occurs primarily as large subpopulations in papillary thyroid tumors. For effective development of personalized cancer treatment, quantitative and sensitive analyses of tumor mutations are needed to establish the effect of mutant subpopulations on patient response and/or relapse. Because so many tumors carry KRAS mutation, therapies targeting KRAS mutant cells are needed for use in conjunction with therapies directed against other targets. The views presented do not necessarily reflect those of the US FDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1739. doi:1538-7445.AM2012-1739

Neuropilin-2: a novel biomarker for malignant melanoma?

Neuropilin-2, a cell surface receptor involved in angiogenesis and axonal guidance, has recently been shown to be a critical mediator of tumor-associated lymphangiogenesis. Given that lymphangiogenesis is a major conduit of metastasis in melanomas and that blocking neuropilin-2 function in vivo is effective in inhibiting tumor cell metastasis, we sought to determine the clinical relevance of neuropilin-2 expression in cutaneous melanoma. Immunohistochemical analysis of neuropilin-2 expression was evaluated in nevomelanocytic proliferations that included a tissue microarray and histologic sections from samples of primary melanomas (n = 42; 40 for tissue microarray, 2 for histologic sections), metastatic melanomas (n = 30; 22 for tissue microarray, 8 for histologic sections), and nevi (n = 30; 5 for tissue microarray, 25 for histologic sections), as well as a panel of normal human tissues and select nonmelanocytic tumors. Staining for grading and intensity of neuropilin-2 expression was estimated semiquantitatively as follows for the former: less than 20%, 20% to 60%, and more than 60% of tissue present, and for the latter from 0 to 3, with 3 being the highest and 0 the lowest intensity. In nevomelanocytic proliferations, more than 20% staining for neuropilin-2 was noted in 36 (86%) of 42 cases of primary melanoma, in 27 (90%) of 30 cases of metastatic melanoma, and in 9 (30%) of 30 cases of nevi with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (P < .0001). For staining intensity, an intensity of 2 or more was noted in 36 (86%) of 42 cases of primary melanoma, in 17 (57%) of 30 cases of metastatic melanoma and in 7 (30%) of 23 cases of nevi, with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (P < .0001). In normal human tissue, consistently strong neuropilin-2 staining was noted in kidney (glomerular endothelial cells, collecting tubules, and collecting ducts), skin (epidermal keratinocytes), and testes (epithelium of the seminiferous tubules), whereas in tumoral tissue, consistently strong staining was noted only in renal cell carcinoma but not in any of the other tumors studied. More recently, using a heterotypic coculture methodology with melanoma and endothelial cells, we have demonstrated successful up-regulation of neuropilin-2 and confirmed the critical role of neuropilin-2 in melanoma-endothelial interactions. Because these coculture methods were developed to model melanoma metastasis, the significantly increased and enhanced expression of neuropilin-2 staining in primary and metastatic melanoma versus nevi in the current study suggests that it is also relevant in vivo.

Novel mutations in TTC37 associated with tricho-hepato-enteric syndrome

The Tricho-Hepato-Enteric (THE) syndrome is an autosomal recessive condition marked by early and intractable diarrhea, hair abnormalities, and immune defects. Mutations in TTC37, which encodes the putative protein Thespin, have recently been associated with THE syndrome. In this article, we extend the pattern of TTC37 mutations by the description of 11 novel mutations in 9 patients with a typical THE syndrome. The mutations were spread along the gene sequence, none of themrecurrent. Different types of mutation were observed: frameshift mutations, splice-site altering mutations, or missense mutations, most of them leading to the creation of a premature stop codon. Concurrently, we investigated the pattern of TTC37 expression in a panel of normal human tissues and showed that this gene is widely expressed, with high levels in vascular tissues, lymph node, pituitary, lung, and intestine. In contrast, TTC37 is not expressed in the liver, an organ that is not consistently affected in THE syndrome. Last, we suggested a model for the putative structure of the unknown Thespin protein.

An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing

Death-associated protein kinase 1 (DAPK-1) is a multidomain protein kinase with diverse roles in autophagic, apoptotic and survival pathways. Bioinformatic screens were used to identify a small internal mRNA from the DAPK-1 locus (named s-DAPK-1). This encodes a 295 amino acid polypeptide encompassing part of the ankyrin-repeat domain, the P-loop motifs, part of the cytoskeletal binding domain of DAPK-1, and a unique C-terminal 'tail' extension not present in DAPK-1. Expression of s-DAPK-1 mRNA was detected in a panel of normal human tissues as well as primary colorectal cancers, indicating that its expression occurs in vivo. s-DAPK-1 gene transfection into cells produces two protein products: one with a denatured mass of 44 kDa, and a smaller product of 40 kDa. Double alanine mutation of the C-terminal tail extension of s-DAPK-1 (Gly296/Arg297) prevented production of the 40 kDa fragment, suggesting that the smaller product is generated by in vivo proteolytic processing. The s-DAPK-1 gene cannot substitute for full-length DAPK-1 in an mitogen-activated protein kinase kinase/extracellular signal-regulated kinase-dependent apoptotic transfection assay. However, the transfection of s-DAPK-1 was able to mimic full-length DAPK-1 in the induction of membrane blebbing. The 44 kDa protease-resistant mutant s-DAPK-1G296A/R297A had very low activity in membrane blebbing, whereas the 40 kDa s-DAPK-1Deltatail protein exhibited the highest levels of membrane blebbing. Deletion of the tail extension of s-DAPK-1 increased its half-life, shifted the equilibrium of the protein from cytoskeletal to soluble cytosolic pools, and altered green fluorescent protein-tagged s-DAPK-1 protein localization as observed by confocal microscopy. These data highlight the existence of an alternative product of the DAPK-1 locus, and suggest that proteolytic removal of the C-terminal tail of s-DAPK-1 is required to stimulate maximally its membrane-blebbing function.

Identification of molecular targets for immunotherapy of patients with head and neck squamous cell carcinoma

To identify molecular targets for immunotherapy of head and neck squamous cell carcinoma (HNSCC) patients, we analyzed gene expression profile in matched tumor (HN) and normal fibroblast (FB) cell lines established from a HNSCC patient using microarray technique followed by real-time RT-PCR. Screening against a series of established normal and malignant cell lines followed by screening against a panel of normal human tissues led to the identification of 7 genes (AREG, CDH3, KLK10, NmU, SLPI, ANAX3 and MAL2), which were over-expressed at least 10-fold in tumors over any of the normal tissues. We determined the expression of mRNA encoding these genes against a panel of 15 HNSCC primary tumor samples. Relative expression of these genes was at least 20-fold. Expression of AREG, CDH3, KLK10, NmU and SLPI at the protein level was determined by immunohistochemistry in seven supraglottic laryngeal cancer specimens. All five proteins were expressed in these tumor samples with high intensity. We conclude that these molecules are potential targets for immunotherapy of HNSCC patients.

Expression and localization of inhibitor of apoptosis proteins in normal human tissues

The family of inhibitor of apoptosis (IAP) proteins can suppress apoptosis induced by a variety of triggers. Among the IAPs, cIAP1, cIAP2, and XIAP have been characterized as inhibitors of specific caspases, and their expression, together with that of survivin, has been shown in several studies to play a role as tumor marker and prognostic factor for the survival of patients with cancer. Although survivin is usually not expressed in normal adult tissues, cIAP1, cIAP2, and XIAP have been found broadly expressed at messenger RNA level within normal cells. Here, we report an immunohistochemical study in a comprehensive panel of normal human tissues, and we confirm at the protein level the wide expression of IAPs. These results are consistent with a physiological role of IAPs in normal cells. Moreover, we show that IAPs' expression levels and localization patterns differ depending on the cell lineage. The variable subcellular localization of the IAPs within different cell types suggests that compartmentalization may contribute to regulate their function. The physiological role of these proteins should be further investigated to help tailor IAP-targeted therapeutic strategies for patients with cancer and circumvent possible side effects.

GATA2 May Contribute with EVI1 to the Leukemogenic Mechanism in Patients with 3q21q26 Rearrangements.

Patients with 3q21q26 rearrangements seem to share similar clinicopathologic features and a common molecular mechanism, leading to MDS or AML. The overexpression of EVI1 (3q26) has been implicated in the dysplasia that characterizes this subset of myeloid neoplasias with poor prognosis. However, EVI1 overexpression had been detected in 9% of AML cases without 3q26 abnormalities; and several 3q21q26 cases with no EVI1 overexpression had been reported. Our aim was to characterize different subgroups in 3q rearrangements, and to define the gene expression profile of these groups, specially in the group with 3q21q26 rearrangements. We analyzed 44 samples (6 cell lines and 38 samples of patients) with 3q rearrangements at G-band level, 36 had AML and 8 MDS. As a control, we analyzed 6 samples of patients with 3q rearrangements and lymphoid neoplasias, a cDNA panel of normal human tissues (Clontech), and 10 normal bone marrow samples. FISH analysis was performed using six BACs: PR11-390G14, RP11-475N22 (GATA2), RP11-689D3 (RPN1) on 3q21; RP11-82C9 (EVI1), RP11-115B16 (MDS1) and RP11-196F13 (TRAIL) on 3q26; and a centromere probe. Expression of GATA2, MDS1-EVI1, and EVI1 was measured by a quantitative real-time RT-PCR (Taqman) assay (Applied Biosystems). Samples were first classified attending at the G-banding. FISH analysis showed the heterogeneity of breakpoints in these cases, and the samples were grouped as 3q21 (5 cases), 3q26 (11 cases), 3q21q26 (16 cases) and other 3q rearrangements (12 cases). Real time RT-PCR showed overexpression (OE) of EVI1 and/or GATA2 in 75% of all the cases (33/44), suggesting the important role of these genes in patients with 3q rearrangements and myeloid neoplasias. In patients with lymphoid neoplasias and 3q rearrangements no genes were found to be OE. A different expression profile was found in the groups analyzed (Table 1). In the 3q21 group, 40% of the patients had GATA2 OE, but no EVI1 or MDS1-EVI1 OE was found. Our data show that EVI1 OE could be specific of the groups 3q26 (64%) and 3q21q26 (69%). The number of cases with EVI1 OE was significantly higher in patients with 3q rearrangements than in those with complex karyotype (data not show). In the group with 3q21q26, EVI1 and/or GATA2 were overexpressed in 87.5% of the cases (14/16). In conclusion, we have detected a different expression profile of the genes analyzed among samples with different 3q rearrangements. Our results suggest a more complex mechanism in patients with 3q21q26 rearrangements, in which GATA2 overexpression may contribute with EVI1 to the leukemogenic progress. Further molecular studies are in progress to analyze the different EVI1 transcripts.Table 1:Incidence of the overexpression of EVI1, GATA2 and MDS1-EVI1 in 32 patients with 3q21 and/or 3q26 rearrangements.Frequency & PercentageEVI1GATA2MDS1-EVI13q210% (0/5)40% (2/5)0% (0/5)3q2664% (7/11)73% (8/11)36% (4/11)3q21q2675% (12/16)69% (11/16)13% (2/16)

Differential Distribution of the JC Virus Receptor-Type Sialic Acid in Normal Human Tissues

JC virus (JCV), a member of the polyomavirus family, causes a demyelinating disease of the central nervous system (CNS) in humans known as progressive multifocal leukoencephalopathy. Although glial cells are the principal target of JCV productive infection in progressive multifocal leukoencephalopathy patients, little is known regarding the site of JCV persistence and the mechanisms by which the virus spreads to the CNS to cause disease. Previous work has demonstrated the presence of replicating JCV DNA in B lymphocytes from peripheral blood, tonsil, and spleen and it has been hypothesized that lymphocytes may be one site of JCV persistence. Detection of viral gene products in renal tubules and excretion of JC virions in the urine suggests JCV persistence in the kidney. A respiratory route of viral transmission has also been hypothesized implicating the lung as another possible site of persistent JCV infection. Earlier studies from our laboratory have shown that terminal alpha 2,6-linked sialic acid is a critical component of the JCV receptor. In this report we examined the tissue distribution of this JCV receptor-type sialic acid in a panel of normal human tissues. Our results demonstrate that in normal brain JCV receptor-type sialic acids are expressed on oligodendrocytes and astrocytes, but not on cortical neurons. The receptor-type sialic acid is also more highly expressed on B lymphocytes than on T lymphocytes in normal human spleen and tonsil. In addition, both the kidney and lung express abundant levels of alpha 2-6-linked sialic acids. Our data show a striking correlation between the expression of the JCV receptor-type sialic acid on cells and their susceptibility to infection by the virus. These findings also support the hypothesis of JCV persistence in lymphoid tissue and B-cell-facilitated viral dissemination to the CNS.

Immunohistochemical analysis of Bin1/Amphiphysin II in human tissues: Diverse sites of nuclear expression and losses in prostate cancer

The Bin1/Amphiphysin II gene encodes at least seven alternately spliced adapter proteins that have been implicated in membrane dynamics and nuclear processes. Nuclear localized Bin1 polypeptides have tumor suppressor and proapoptotic activities, suggesting that Bin1 may suppress cancer in tissues where nuclear expression may occur. One question is the extent to which human tissues express nuclear Bin1 isoforms. A secondary issue has been the need for a specific antibody that can detect all the splice isoforms expressed by the human, mouse, and rat Bin1 genes. Using a novel mouse monoclonal antibody with these characteristics, we performed an immunohistochemical analysis of Bin1 expression in a panel of normal human tissues. We also compared the expression profile of Bin1 in normal or malignant tissues derived from human prostate, where Bin1 is a candidate tumor suppressor gene. In brain, a distinct nuclear staining pattern overlapped with a cytosolic staining pattern present in certain layers of the cerebral cortex and cerebellum. Bone marrow cells displayed mainly nuclear localization whereas peripheral lymphoid cells exhibited mainly cytosolic localization. In several epithelial tissues, nuclear or nucleocytosolic staining patterns were displayed by basal cells in skin, breast, or prostate, whereas cytosolic or plasma membrane-associated staining patterns were noted in gastrointestinal cells. Interestingly, a striking gradient of expression was observed in gastrointestinal epithelia, particularly in the large intestine, with the strongest staining displayed by cells destined to undergo apoptosis at the villus tip. In prostate, Bin1 staining was frequently absent in cases of primary prostate adenocarcinoma. This study used a novel reagent to document the extent of expression of nuclear Bin1 isoforms, which exhibit cancer suppression and proapoptotic activity in human cells.

Isolation and tissue profiles of a large panel of phage antibodies binding to the human adipocyte cell surface

Phage display is a powerful technique for the rapid selection and isolation of antibodies to any given target antigen. We have applied this technology to isolate over 100 different human antibodies that bind to antigens expressed in situ on the human adipocyte cell surface. This is a diverse panel of antibodies, as indicated by the V-region sequences. The binding profile of each anti-adipocyte antibody has been characterised using phage antibody immunocytochemistry against a panel of normal human tissues. Although there was some variation in the intensity of the adipocyte staining, each antibody consistently recognised adipocytes, where present, irrespective of the tissue source. In addition, all of the antibodies recognised at least one other cell type other than the adipocyte cell surface. In total, over 50 different tissue-binding profiles were recorded, with the most frequently recognised tissues identified as capillaries or smooth muscle. Extensive tissue binding profiles were generated for some antibodies using a panel of 37 different human tissues. This identified anti-adipocyte antibodies with unexpected profiles, such as FAT.13, which binds only to adipocytes and capillaries in the entire tissue panel. We believe this is the most extensive survey ever undertaken of the human adipocyte cell surface. Moreover, similar methodology could be used to derive complete tissue-binding profiles of antibodies against cell-surface antigens of any cell type. Indeed, by screening antibodies on both normal and diseased tissues, it may be possible to identify antigenic associations between different cell types and the pathologies of many diseases.

Expression of translationally controlled tumor protein mRNA in human colon cancer

The translationally controlled tumor protein (TCTP) was first identified as a tumor-associated protein. Here we described the expression profile of TCTP mRNA using a panel of normal human tissues. We also investigated the cell-cycle-dependent expression of the TCTP mRNA using synchronized cultures of Saccahromyces cerevisiae. The TCTP gene was constantly expressed through the yeast cell cycle. The expression of TCTP mRNA from three human colon carcinoma cell lines and their tumors xenografted in a nude mouse was studied. Our data imply that the TCTP is a housekeeping gene and that its deregulation may be involved in the tumor development of colon carcinoma.

CDw60: an antigen expressed in many normal tissues and in some tumours.

CDw60 is a recently described T-cell antigen, which functionally delivers a costimulatory signal in T-cell activation. In addition, CDw60 has been regarded as a melanoma-associated antigen. To date, only limited information exists on the distribution of CDw60 in other normal and pathologically altered tissues in human. In the present study, the expression of CDw60 was analysed immunohistologically in a large panel of formalin-fixed and paraffin-embedded normal and pathological human tissues. The antigen was detected in several normal tissues, such as epithelia of the reproductive system, exocrine and endocrine glands, glial cells and neurons of the central and peripheral nervous systems, and lymphoid cells. These showed different subcellular distribution patterns, i.e. (1) cell surface labelling of peripheral lymphocytes and lymphocytes of the lymph node and thymus, (2) diffuse cytosolic staining in lymphocytes, subpial glial processes, and the outer plexiform layer of the retina, (3) granular cytoplasmic staining associated with the Golgi apparatus in epithelial cells of certain endocrine and exocrine glands, of the ductus epididymis and deferens, neurons of the peripheral and central nervous system, and lymphocytes and megakaryocytes of the bone marrow. In exocrine glands, e.g. of the prostate and uterine corpus, CDw60-positive Golgi fields were located in the juxtaluminal cell compartment, thus reflecting a polarized distribution. In some malignant tumours, the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were disorderly distributed throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Only in mammary carcinomas was abnormal cell surface labelling detected. A putative de novo expression of CDw60 was observed in pleomorphic adenoma and mucoepidermoid carcinoma of the parotid gland, seminoma, embryonal and teratocarcinoma of the testis, small cell carcinoma of the lung, and malignant melanoma. These results define the CDw60 determinant as a broadly distributed antigen within a large panel of normal human tissues. The antigen is also detectable in some previously undescribed benign and malignant tumours, which may give importance to CDw60 as a possible diagnostic marker.

Monitoring of PPAR alpha protein expression in human tissue by the use of PPAR alpha-specific MAbs.

We report the production and characterization of two PPAR alpha subtype-specific monoclonal antibodies raised against the N-terminal domain of PPAR alpha. P alpha b 11.80A is a Western-reactive antibody, whereas P alpha b 32.51 is useful for immunohistochemistry. Both antibodies exhibited high affinity against the immunogen based on BIAcore analysis, recognized full-length PPAR alpha protein in PPAR alpha-transfected CV-1 cells, and displayed no cross-reactivity against the N-terminal domains of PPAR gamma or PPAR delta proteins as demonstrated by various immunoassays. The application of these antibodies to a panel of normal human tissues revealed that PPAR alpha protein expression is highest in skeletal muscle, liver, and kidney, consistent with previously reported mRNA expression data. These antibodies provide us with valuable tools to further explore the function of PPAR alpha.

The neuronal nuclear antigen recognized by the human anti-Ri autoantibody is expressed in central but not peripheral nervous system neurons

Anti-Ri is a human autoantibody that recognizes a neuronal nuclear antigen (Ri). Biotinylated IgG from serum of two patients with high titers of anti-Ri antibodies was used to study the distribution of the Ri antigen in a panel of normal human tissues. the expression of the Ri antigen was evaluated by an avidin-biotin peroxidase technique and comfirmed by immunoblotting. The Ri antigen was restricted to the neurons of the central nervous system (CNS) and some pituitary cells. Most neurons in dorsal root, Gasserian and sympathetic ganglia, and myenteric plexus were negative or, a few of them, very weakly positive. The functional implication of the different expression of the Ri antigen between neurons of the central and peripheral nervous system is presently unknown.

Mapping of immunogenic regions of human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins with env-encoded synthetic peptides and a monoclonal antibody to gp46.

Antigenic sites on human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins that are immunogenic in man were studied with envelope gene (env)-encoded synthetic peptides and a mAb to HTLV-I gp46 envelope glycoprotein. Antibodies in 78% of sera from HTLV-I seropositive subjects reacted with synthetic peptide 4A (amino acids 190 to 209) from a central region of HTLV-I gp46. Human anti-HTLV-I antibodies also bound to synthetic peptides 6 (29% of sera) and 7 (18% of sera) from a C-terminal region of gp46 (amino acids 296 to 312) and an N-terminal region of gp21 (amino acids 374 to 392), respectively. mAb 1C11 raised to affinity-purified HTLV-I gp46 reacted with gp46 external envelope glycoprotein and gp63 envelope precursor in immunoblot assay and also bound to the surface of HTLV-I+ cells lines HUT-102 and MT-2. Antibody 1C11 did not react with HTLV-II or HIV-infected cells or with a broad panel of normal human tissues or cell lines. In competitive RIA, anti-gp46 antibody 1C11 was inhibited from binding to gp46 either by antibodies from HTLV-I seropositive subjects or by HTLV-I env-encoded synthetic peptide 4A, indicating that 1C11 bound to or near a site on gp46 within amino acids 190 to 209 also recognized by antibodies from HTLV-I-seropositive individuals. When tested in syncytium inhibition assay, mAb 1C11 did not neutralize the infectivity of HTLV-I. Thus, HTLV-I infection in man is associated with a major antibody response to a region of gp46 within amino acids 190 to 209 that is on the surface of virus-infected cells.