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.

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.

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.

Human melanoma metastases demonstrate nonstochastic site-specific antigen heterogeneity that correlates with T-cell infiltration.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

The clinical immunohistochemistry laboratory: regulations and troubleshooting guidelines.

The Clinical Laboratory Improvement Amendments (CLIA) set standards designed to improve the quality of all laboratory testing. In the first portion of this chapter, we discuss the CLIA requirements that apply to most Immunohistochemistry laboratories, and explain topics such as certification, test complexity, patient test management, proficiency testing, personnel, quality control, quality assurance, and compliance. The second portion of this chapter addresses the most common problems encountered in immunohistochemical procedures and the appropriate solutions to correct them.

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.

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.

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.

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.

Proteomic evaluation of archival cytologic material using SELDI affinity mass spectrometry: potential for diagnostic applications.

Proteomic studies of cells via surface-enhanced laser desorption/ionization spectrometry (SELDI) analysis have enabled rapid, reproducible protein profiling directly from crude samples. We applied this technique to archival cytology material to determine whether distinct, reproducible protein fingerprints could be identifiedfor potential diagnostic purposes in blinded specimens. Rapid Romanowsky-stained cytocentrifuged specimens from fine-needle aspirates of metastatic malignant melanoma (with both known cutaneous primary and unknown primary sites), clear cell sarcoma, and renal cell carcinoma and reactive effusions were examined using the SELDI technology. A unique characteristic fingerprint was identified for each disease entity. Fifteen "blinded" unknown samples then were analyzed. When the protein profilefingerprints were plotted against the known fingerprints for the aforementioned diagnoses, the appropriate match or diagnosis was obtained in 13 (87%) of 15 cases. These preliminary findings suggest a substantial potential for SELDI applications to specific pathologic diagnoses.

Comparison of three commonly used cytologic preparations in effusion immunocytochemistry.

Discrepant results in effusion immunocytochemistry are often the result of specimen processing. Smears, cytospins, cell blocks, and monolayer preparations have all been used in various published studies; thus, there is no consistency in the immunostaining process for cytology to compare with the surgical pathology "gold standard" results. We sought to evaluate optimal specimen preparation for the immunostaining of effusion samples. Fourteen reactive and 15 malignant effusion samples (various epithelial/mesothelial neoplasms) were each prepared in three forms: air-dried cytospins (postfixed in ethanol), formalin-fixed, paraffin-embedded cell blocks, and liquid-based thin-layer (ThinPrep, CYTYC, Boxborough, MA) processing. All slides were immunostained with antibodies commonly used in effusion cytology: HBME-1, calretinin, E-cadherin, BerEP4, B72.3, LeuM1, and CA19-9. Cytospin and ThinPrep samples performed in a similar manner: high background staining was encountered in 66% of cases, most evident in three-dimensional clusters of cells. In addition, membrane staining patterns were difficult to interpret. Cell blocks provided the best milieu for morphologic interpretation, with less background staining (only 17% of cases) and results that most closely approximated those reported in the surgical pathology literature. The cost per test for cell block immunocytochemistry was also the most economical for our laboratory.