Immunophenotypic Characterization of Canine Splenic Follicular-Derived B-Cell Lymphoma.

Marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL) belong to a subgroup of indolent B-cell lymphomas most commonly reported in the canine spleen. The goal of this study was to characterize the immunophenotype of splenic MZL and MCL in comparison to their human counterparts. Ten MCLs and 28 MZLs were selected based on morphology. A tissue microarray was generated, and expression of CD3, CD5, CD10, CD45, CD20, CD79a, Pax-5, Bcl-2, Bcl-6, cyclin D1, cyclin D3, MCL-1, MUM-1, and Sox-11 was evaluated. Neoplastic cells in all MCLs and MZLs were positive for CD5, CD20, CD45, CD79a, and BCL2 and negative for CD3, CD10, Bcl-6, cyclin D1, and cyclin D3. Positive labeling for Pax-5 was detected in 8 of 10 MCLs and 26 of 28 MZLs. Positive labeling for MUM-1 was detected in 3 of 10 MCLs, and 27 of 28 MZLs were positive for MUM-1. No MCLs but 8 of 24 MZLs were positive for MCL-1. Canine splenic MZL and MCL have a similar immunophenotype as their human counterparts. However, human splenic MCL overexpresses cyclin D1 due to a translocation. A similar genetic alteration has not been reported in dogs. In addition, in contrast to human MZL, canine splenic MZL generally expresses CD5. Following identification of B vs T cells with CD20 and CD3, a panel composed of BCL-2, Bcl-6, MUM-1, and MCL-1 combined with the histomorphological pattern can be used to accurately diagnose MZL and MCL in dogs. Expression of Bcl-2 and lack of MCL-1 expression in MCL may suggest a therapeutic benefit of BCL-2 inhibitors in canine MCL.

Re-evaluating TTF-1 immunohistochemistry in diffuse gliomas: Expression is clone-dependent and associated with tumor location.

TTF-1 is widely used as a marker in routine surgical pathology in the work-up of malignancy. Aberrant expression of TTF-1 in extrapulmonary and extrathyroidal malignancies is a frequently reported phenomenon. In addition to the recently characterized pituicyte-derived tumors of the sella, immunoreactivity has been reported in diffuse gliomas with the SPT24 clone. Here, we sought to evaluate TTF-1 expression with three commercially available clones in a large series of gliomas. Expression was compared across the newly defined diagnostic entities in the 2016 WHO Classification of CNS Tumors. Using tissue microarrays (TMA), 212 diffuse gliomas (WHO grades II - IV) were systematically evaluated with TTF-1 immunohistochemistry using three clones: SPT24, 8G7G3/1, and SP141, and results correlated with clinicopathologic features. 14 high-grade diffuse gliomas demonstrated nuclear staining with the SP141 and SPT24 clones. Two tumors showed weak positivity with the 8G7G3/1 clone. All tumors were high grade by histology (WHO grades III and IV). 86% (12/14) of TTF-1-positive gliomas involved the frontal lobes at diagnosis. No relationship with IDH R132H, ATRX, p53, H3K27M, or EGFR immunohistochemistry was identified. TTF-1 expression in gliomas was not independently prognostic of overall survival. TTF-1 expression in diffuse gliomas is a rare but potentially misleading occurrence. In our cohort, staining occurred with both the SPT24 and SP141 clones at equal intensity and frequency. Clustering of TTF-1-positive tumors in the frontal lobe(s) suggests lineage-specific expression. Due to clone-specific expression in diffuse gliomas, caution must be exercised in the work-up of intracranial tumors with TTF-1.
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Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.

First-in-human phase 1 clinical trial of anti-core 1 O-glycans targeting monoclonal antibody NEO-201 in treatment-refractory solid tumors.

BACKGROUND: NEO201 is a humanized IgG1 monoclonal antibody (mAb) generated against tumor-associated antigens from patients with colorectal cancer. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells. Here, we present outcomes from a phase I trial of NEO-201 in patients with advanced solid tumors that have not responded to standard treatments. METHODS: This was a single site, open label 3 + 3 dose escalation clinical trial. NEO-201 was administered intravenously every two weeks in a 28-day cycle at dose level (DL) 1 (1 mg/kg), DL 1.5 (1.5 mg/kg) and DL 2 (2 mg/kg) until dose limiting toxicity (DLT), disease progression, or patient withdrawal. Disease evaluations were conducted after every 2 cycles. The primary objective was to assess the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of NEO-201. The secondary objective was to assess the antitumor activity by RECIST v1.1. The exploratory objectives assessed pharmacokinetics and the effect of NEO-201 administration on immunologic parameters and their impact on clinical response. RESULTS: Seventeen patients (11 colorectal, 4 pancreatic and 2 breast cancers) were enrolled; 2 patients withdrew after the first dose and were not evaluable for DLT. Twelve of the 15 patients evaluable for safety discontinued due to disease progression and 3 patients discontinued due to DLT (grade 4 febrile neutropenia [1 patient] and prolonged neutropenia [1 patient] at DL 2, and grade 3 prolonged (> 72 h) febrile neutropenia [1 patient] at DL 1.5). A total of 69 doses of NEO-201 were administered (range 1-15, median 4). Common (> 10%) grade 3/4 toxicities occurred as follows: neutropenia (26/69 doses, 17/17 patients), white blood cell decrease (16/69 doses, 12/17 patients), lymphocyte decrease (8/69 doses, 6/17 patients). Thirteen patients were evaluable for disease response; the best response was stable disease (SD) in 4 patients with colorectal cancer. Analysis of soluble factors in serum revealed that a high level of soluble MICA at baseline was correlated with a downregulation of NK cell activation markers and progressive disease. Unexpectedly, flow cytometry showed that NEO-201 also binds to circulating regulatory T cells and reduction of the quantities of these cells was observed especially in patients with SD. CONCLUSIONS: NEO-201 was safe and well tolerated at the MTD of 1.5 mg/kg, with neutropenia being the most common adverse event. Furthermore, a reduction in the percentage of regulatory T cells following NEO-201 treatment supports our ongoing phase II clinical trial evaluating the efficiency of the combination of NEO-201 with the immune checkpoint inhibitor pembrolizumab in adults with treatment-resistant solid tumors. TRIAL REGISTRATION: NCT03476681 . Registered 03/26/2018.

Semiautomated laser capture microdissection of lung adenocarcinoma cytology samples.

OBJECTIVE: In the past decade molecular diagnostics has changed the clinical management of lung adenocarcinoma patients. Molecular diagnostics, however, is largely dependent on the quantity and quality of the tumor DNA that is retrieved from the tissue or cytology samples. Frequently, patients are diagnosed on cytology specimens where the tumor cells are scattered within the cell block, making selecting for tumor enrichment difficult. In the past we have used laser capture microdissection (LCM) to select for pure populations of tumor cells to increase the sensitivity of molecular assays. This study explores several methods for semiautomated computer-guided LCM. STUDY DESIGN: Hematoxylin and eosin- or TTF-1-immunostained slides from a pleural effusion cell block with metastatic lung adenocarcinoma were used for LCM with either AutoScan or a recently described pattern-matching algorithm, spatially invariant vector quantization (SIVQ), to define morphologic predicates (vectors) to select cells of interest. RESULTS: We retrieved pure populations of tumor cells using both algorithm-guided LCM approaches with slight variations in cellular retrievals. Both methods were semiautomated, requiring minimum technical supervision. CONCLUSION: In this study we demonstrate the first semiautomated, computer-guided LCM of a cytology specimen using SIVQ and AutoScan, a first step towards the long-term goal of integrating LCM into the clinical cytology-molecular workflow.

Molecular Subtypes of Primary SCLC Tumors and Their Associations With Neuroendocrine and Therapeutic Markers.

INTRODUCTION: A new molecular subtype classification was recently proposed for SCLC. It is necessary to validate it in primary SCLC tumors by immunohistochemical (IHC) staining and define its clinical relevance. METHODS: We used IHC to assess four subtype markers (ASCL1, NEUROD1, POU2F3, and YAP1) in 194 cores from 146 primary SCLC tumors. The profiles of tumor-associated CD3+ and CD8+ T-cells, MYC paralogs, SLFN11, and SYP were compared among different subtypes. Validation was performed using publicly available RNA sequencing data of SCLC. RESULTS: ASCL1, NEUROD1, POU2F3, and YAP1 were the dominant molecular subtypes in 78.2%, 5.6%, 7%, and 2.8% of the tumors, respectively; 6.3% of the tumors were negative for all four subtype markers. Notably, three cases were uniquely positive for YAP1. Substantial intratumoral heterogeneity was observed, with 17.6% and 2.8% of the tumors being positive for two and three subtype markers, respectively. The non-ASCL1/NEUROD1 tumors had more CD8+ T-cells and manifested more frequently an "inflamed" immunophenotype. L-MYC and MYC were more often associated with ASCL1/NEUROD1 subtypes and non-ASCL1/NEUROD1 subtypes, respectively. SLFN11 expression was absent in 40% of the tumors, especially those negative for the four subtype markers. SYP was often expressed in the ASCL1 and NEUROD1 subtypes and was associated with less tumor-associated CD8+ T-cells and a "desert" immunophenotype. CONCLUSIONS: We validated the new molecular subtype classification in primary SCLC tumors by IHC and identified several intriguing associations between subtypes and therapeutic markers. The new subtype classification may potentially assist treatment decisions in SCLC.

Neutrophil and Eosinophil Extracellular Traps in Hodgkin Lymphoma.

Classic Hodgkin lymphoma (cHL), nodular sclerosis (NS) subtype, is characterized by the presence of Hodgkin/Reed-Sternberg (HRS) cells in an inflammatory background containing neutrophils and/or eosinophils. Both types of granulocytes release extracellular traps (ETs), web-like DNA structures decorated with histones, enzymes, and coagulation factors that promote inflammation, thrombosis, and tumor growth. We investigated whether ETs from neutrophils (NETs) or eosinophils (EETs) are detected in cHL, and evaluated their association with fibrosis. We also studied expression of protease-activated receptor-2 (PAR-2) and phospho-extracellular signal-related kinase (p-ERK), potential targets/effectors of ETs-associated elastase, in HRS cells. Expression of tissue factor (TF) was evaluated, given the procoagulant properties of ETs. We analyzed 32 HL cases, subclassified as 12 NS, 5 mixed-cellularity, 5 lymphocyte-rich, 1 lymphocyte-depleted, 4 nodular lymphocyte-predominant HL (NLPHL), and 5 reactive nodes. Notably, a majority of NS cHL cases exhibited NET formation by immunohistochemistry for citrullinated histones, with 1 case revealing abundant EETs. All other cHL subtypes as well as NLPHL were negative. Immunofluorescence microscopy confirmed NETs with filamentous/delobulated morphology. Moreover, ETs formation correlates with concurrent fibrosis (r = 0.7999; 95% CI, 0.6192-0.9002; P ≤ 0.0001). Results also showed that HRS cells in NS cHL expressed PAR-2 with nuclear p-ERK staining, indicating a neoplastic or inflammatory phenotype. Remarkably, TF was consistently detected in the endothelium of NS cHL cases compared with other subtypes, in keeping with a procoagulant status. A picture emerges whereby the release of ETs and resultant immunothrombosis contribute to the inflammatory tumor microenvironment of NS cHL. This is the first description of NETs in cHL.

Immunohistochemical distinction of paragangliomas from epithelial neuroendocrine tumors-gangliocytic duodenal and cauda equina paragangliomas align with epithelial neuroendocrine tumors.

Distinction of paraganglioma (PGL) from epithelial neuroendocrine tumors (NETs) can be difficult as they can mimic each other by nested architecture and expression of neuroendocrine markers. In this study, we examined differential diagnostic markers in 262 PGLs (142 adrenal pheochromocytomas and 120 extra-adrenal PGLs), 9 duodenal gangliocytic PGLs and 3 cauda equina PGLs, and 286 NETs (81 GI, 78 pancreatic, 42 thoracic, 37 medullary thyroid carcinomas, and 48 high-grade NETs including 32 small cell carcinomas of lung). While keratin expression was nearly uniform in NETs with the exception of few tumors, extensive keratin expression was seen in only one PGL (<1%) and focal expression in 5% PGLs. GATA3 was present in >90% of PGLs but only in 2% of NETs, usually focally. Tyrosine hydroxylase (TH) was expressed in >90% of adrenal, abdominal, and thoracic PGLs but only in 37% of head and neck PGLs, reflecting their variable catecholamine synthesis. Focal or occasional extensive TH-expression was detected in 10% of NETs. CDX2 was a helpful discriminator seen in 28% of pancreatic and most GI NETs but in no PGLs. SOX10 detected sustentacular cells in 85% of PGLs and 7% of NETs, whereas GFAP detected sustentacular cells mainly in PGLs of neck and was absent in NETs. Duodenal gangliocytic PGLs (n = 9) and all cauda equina PGLs (n = 3) expressed keratins, lacked GATA3, showed no or minimal TH expression as some NETs, and contained SOX10 and S100 protein-positive spindle cells negative for GFAP. Ganglion-like epithelioid cells were keratin-positive and negative for TH and SOX10 differing from true ganglion cells. We conclude that duodenal gangliocytic and cauda equina PGLs have a NET-like immunoprofile and differ from ordinary PGLs. NETs can be distinguished from PGLs by their expression of keratins and general lack of GATA3, TH, and GFAP-positive sustentacular cells, and sometimes by expression of CDX2 or TTF1.

Sensitivity of Mesothelioma Cells to PARP Inhibitors Is Not Dependent on BAP1 but Is Enhanced by Temozolomide in Cells With High-Schlafen 11 and Low-O6-methylguanine-DNA Methyltransferase Expression.

INTRODUCTION: BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. METHODS: A total of 10 patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone or in combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. RESULTS: BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 µM to greater than 50 µM and 0.039 µM to greater than 5 µM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. CONCLUSIONS: BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

Low mutation burden and frequent loss of CDKN2A/B and SMARCA2, but not PRC2, define premalignant neurofibromatosis type 1-associated atypical neurofibromas.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a tumor-predisposition disorder caused by germline mutations in NF1. NF1 patients have an 8-16% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft-tissue sarcoma, often arising from preexisting benign plexiform neurofibromas (PNs) and atypical neurofibromas (ANFs). ANFs are distinct from both PN and MPNST, representing an intermediate step in malignant transformation. METHODS: In the first comprehensive genomic analysis of ANF originating from multiple patients, we performed tumor/normal whole-exome sequencing (WES) of 16 ANFs. In addition, we conducted WES of 3 MPNSTs, copy-number meta-analysis of 26 ANFs and 28 MPNSTs, and whole transcriptome sequencing analysis of 5 ANFs and 5 MPNSTs. RESULTS: We identified a low number of mutations (median 1, range 0-5) in the exomes of ANFs (only NF1 somatic mutations were recurrent), and frequent deletions of CDKN2A/B (69%) and SMARCA2 (42%). We determined that polycomb repressor complex 2 (PRC2) genes EED and SUZ12 were frequently mutated, deleted, or downregulated in MPNSTs but not in ANFs. Our pilot gene expression study revealed upregulated NRAS, MDM2, CCND1/2/3, and CDK4/6 in ANFs and MPNSTs, and overexpression of EZH2 in MPNSTs only. CONCLUSIONS: The PN-ANF transition is primarily driven by the deletion of CDKN2A/B. Further progression from ANF to MPNST likely involves broad chromosomal rearrangements and frequent inactivation of the PRC2 genes, loss of the DNA repair genes, and copy-number increase of signal transduction and cell-cycle and pluripotency self-renewal genes.

Evaluation of prime/boost regimens using recombinant poxvirus/tyrosinase vaccines for the treatment of patients with metastatic melanoma.

PURPOSE: Two clinical trials were conducted to evaluate the clinical efficacy and immunologic impact of vaccination against the tyrosinase protein plus systemic interleukin 2 (IL-2) administration in patients with advanced metastatic melanoma. EXPERIMENTAL DESIGN: Full-length tyrosinase was employed as an immunogen to induce diverse immunologic responses against a commonly expressed melanoma antigen. Heterologous prime/boost vaccination with recombinant vaccinia and fowlpox vectors encoding tyrosinase was first explored in a randomized three-arm phase II trial, in which vaccines were administered alone or concurrently with low-dose or high-dose IL-2. In a subsequent single cohort phase II trial, all patients received the same vaccines and high-dose IL-2 sequentially rather than concurrently. RESULTS: Among a total of 64 patients treated on these trials, 8 objective partial responses (12.5%) were observed, all in patients receiving high-dose IL-2. Additional patients showed evidence of lesional regression (mixed tumor response) or overall regression that did not achieve partial response status (minor response). In vitro evidence of enhanced immunity against tyrosinase following protocol treatments was documented in 3 of 49 (6%) patients tested serologically, 3 of 23 (13%) patients tested for T-cell recognition of individual tyrosinase peptides, and 4 of 16 (25%) patients tested for T-cell recognition of full-length tyrosinase protein with real-time reverse transcription-PCR techniques. CONCLUSIONS: Whereas prime/boost immunization with recombinant vaccinia and fowlpox viruses enhanced antityrosinase immunity in some patients with metastatic melanoma, it was ineffective alone in mediating clinical benefit, and in combination with IL-2 did not mediate clinical benefit significantly different from that expected from treatment with IL-2 alone.

The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature.

The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2-3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250.

Diagnostic effects of prolonged storage on fresh effusion samples.

The effects on morphology and diagnostic interpretation of delayed processing of refrigerated effusion samples have not been well documented. The potential for cellular degeneration has led many laboratories to reflexively fix samples rather than submit fresh/refrigerated samples for cytologic examination. We sought to determine if effusion specimens are suitable for morphologic, immunocytochemical, and DNA-based molecular studies after prolonged periods of refrigerated storage time. Ten fresh effusion specimens were refrigerated at 4 degrees C; aliquots were processed at specific points in time (days 0, 3, 5, 7, 10, 14). Specimens evaluated included four pleural (3 benign, 1 breast adenocarcinoma) and six peritoneal (2 ovarian adenocarcinomas, 1 malignant melanoma, 2 mesotheliomas, 1 atypical mesothelial) effusions. The morphology of the cytologic preparations from the 10 effusions was preserved and interpretable after 14 days of storage at 4 degrees C. The immunocytochemical profile of the samples (AE1/AE3, EMA, calretinin, and LCA) was consistent from day 0 to day 14. Amplifiable DNA was present in all samples tested on day 14. We conclude that cytopathologic interpretation of effusion samples remains reliable with refrigeration at 4 degrees C even if processing is delayed.

Expression of CD70 (CD27L) Is Associated With Epithelioid and Sarcomatous Features in IDH-Wild-Type Glioblastoma.

Glioblastoma is an aggressive, often recalcitrant disease. In the majority of cases, prognosis is dismal and current therapies only moderately prolong survival. Immunotherapy is increasingly being recognized as an effective treatment modality. CD70 is a transmembrane protein that shows restricted expression in tissue but has been described in various malignancies. Therapeutic targeting of CD70 has demonstrated antitumor efficacy and is in clinical trials. Here, we sought to characterize CD70 expression in a large cohort of gliomas (n = 205) using tissue microarrays. We identified a subset of tumors (n = 18, 8.8% of high-grade gliomas) exhibiting moderate-to-strong immunoreactivity that enriched for the IDH-wild-type glioblastoma variants gliosarcoma (n = 10) and the newly described epithelioid glioblastoma (n = 4). CD70 expression was associated with prolonged survival in gliosarcoma. Analysis of TCGA datasets showed significantly increased CD70 expression in mesenchymal tumors and prolonged survival in recurrent non-G-CIMP high-expressing tumors. In CD70+ gliomas, there was a significant increase in CD68/CD163/HLA-DR+ tumor-associated macrophages, but not CD27+ TIL. These results confirm prior in vitro studies and demonstrate expression in a clinical cohort. The absence of CD70 expression in the post-treatment setting may portend more clinically aggressive disease in gliosarcoma. However, larger-scale studies will be needed to characterize and validate this relationship.

Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues.

Reduced drug accumulation due to overexpression of individual members of the ATP binding cassette (ABC) superfamily of membrane transporters has been investigated as a cause of multidrug resistance and treatment failure in oncology. This study was designed to develop an immunohistochemical assay to determine the expression and localization of the 72kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a modified avidin-biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3. ABCG2 positivity was consistently found in alveolar pneumocytes, sebaceous glands, transitional epithelium of bladder, interstitial cells of testes, prostate epithelium, endocervical cells of uterus, squamous epithelium of cervix, small and large intestinal mucosa/epithelial cells, islet and acinar cells of pancreas, zona reticularis layer of adrenal gland, kidney cortical tubules and hepatocytes. Placental syncytiotrophoblasts showed both cytoplasmic and surface staining. Our results support a hypothesis concluding that ABCG2 plays a role in the protection of organs from cytotoxins. However, many of the cell types expressing ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates.

The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2.

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the ABCG2-specific inhibitor fumitremorgin C (FTC) in human embryonic kidney cells transfected with full length human ABCG2, while no transport by cells transfected with human ABCB1 was noted. FTC-inhibited transport of phytoporphyrin was also demonstrated in ABCG2-expressing LLC-PK1 pig kidney cells, consistent with the idea that the pig orthologue, like human ABCG2, transports the photosensitizer. ABCG2 expression was confirmed by immunohistochemistry in the hepatocytes of cow, pig and sheep livers. We conclude that phytoporphyrin is a substrate for ABCG2 and that the transporter is likely responsible for its biliary excretion.

Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells.

PURPOSE: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms. EXPERIMENTAL DESIGN: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, ß-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters. RESULTS: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression. CONCLUSIONS: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma.

High-Throughput Microdissection for Next-Generation Sequencing.

Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, in situ hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2.

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.